Metabolising herbicide protein, gene and application

FIELD: medicine.

SUBSTANCE: there is provided DNA that codes protein able to transform a compound of formula (II) specified in description of invention into a compound of formula (III) specified in description of invention with an electron transport system containing an electron donor. Protein is able to metabolise herbicides.

EFFECT: introduction of DNA to plants with an expression of the specified protein provides herbicide resistance thereto.

26 cl, 66 dwg, 35 tbl, 75 ex

 

The technical FIELD

This invention relates to a protein capable of metabolizing the herbicide compound (herbicide metabolizing protein, its gene and their use.

BACKGROUND of the INVENTION

Herbicides when they are applied put in the required amount of diluted solution. However, there are situations in which there are excessive. There are also situations in which the applied herbicide after application for some time remains in the soil or in the rest of the plant. Initially, provided that the security of the herbicides tested, such a small amount of residual solvents or residues had little effect on the environment or on cultivated after this crop. However, if there is a way in which is contained the herbicide compound is transformed into the combination of lower herbicide activity, for example, can be processed to inactivate residual solvents or residues described above, as necessary.

In addition, in the case of application of the herbicide, there are situations in which it is difficult to distinguish cultivated plants from weeds those species that have common features, for selective destruction only weeds. Therefore, it is desirable to develop a new way DL is giving the herbicide-resistance to a plant of the target.

DISCLOSURE of INVENTIONS

In these circumstances, the authors of this invention have conducted intensive research and as a result have found that inhibitory protoporphyrinogen (hereinafter sometimes referred to as “PPO”) herbicide compound can be converted by reaction with a specific protein in the combination of lower herbicide activity, which led to completion of this invention.

Thus, in this invention include:

1. DNA encoding a herbicide metabolizing protein, where this protein is selected from the group consisting of:

(A1) a protein containing the amino acid sequence shown in SEQ ID NO:1;

(A2) a protein containing the amino acid sequence shown in SEQ ID NO:2;

(A3) a protein containing the amino acid sequence shown in SEQ ID NO:3;

(A4) a protein containing the amino acid sequence shown in SEQ ID NO:108;

(A5) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II):

in the compound of formula (III):

and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A6) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 80% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A11) a protein containing the amino acid sequence shown in SEQ ID NO:159;

(A12) a protein containing the amino acid sequence shown in SEQ ID NO:160;

(A13) a protein containing the amino acid sequence shown in SEQ ID NO:136;

(A14) a protein containing the amino acid sequence shown in SEQ ID NO:137;

(A15) a protein containing the amino acid sequence shown in SEQ ID NO:138;

(A16) a protein containing the amino acid sequence shown in SEQ ID NO:215;

(A17) a protein containing the amino acid sequence shown in SEQ ID NO:216;

(A18) a protein containing the amino acid sequence shown in SEQ ID NO:217;

(A19) a protein containing the amino acid sequence shown in SEQ ID NO:218;

(A20) protein containing amino acids is th sequence, shown in SEQ ID NO:219;

(A21) a protein containing the amino acid sequence shown in SEQ ID NO:220;

(A22) a protein containing the amino acid sequence shown in SEQ ID NO:221;

(A23) a protein containing the amino acid sequence shown in SEQ ID NO:222;

(A24) a protein containing the amino acid sequence shown in SEQ ID NO:223;

(A25) a protein containing the amino acid sequence shown in SEQ ID NO:224;

(A26) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221 or SEQ ID NO:223 or amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:222, or SEQ ID NO:224;

(A27) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing amino acids follow etelnost, encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223 or SEQ ID NO:224; and

(A28) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, amplificatoare polymerase chain reaction with a primer containing the nucleotide sequence shown in any of SEQ ID NO:124 and SEQ ID NO:128, a primer containing the nucleotide sequence shown in SEQ ID NO:129, and using as the template the chromosomal DNA of Streptomyces phaeochromogenes, Streptomyces testaceus, Streptomyces achromogenes, Streptomyces griseofuscus, Streptomyces thermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus, Streptomyces roseorubens, Streptomyces rutgersensis, Streptomyces steffisburgensis or Saccharopolyspora taberi;

2. DNA containing the nucleotide sequence selected from the group consisting of:

(A1) the nucleotide sequence shown in SEQ ID NO:6;

(A2) a nucleotide in which sledovatelnot, shown in SEQ ID NO:7;

(A3) the nucleotide sequence shown in SEQ ID NO:8;

(A4) the nucleotide sequence shown in SEQ ID NO:109;

(A5) the nucleotide sequence shown in SEQ ID NO:139;

(A6) the nucleotide sequence shown in SEQ ID NO:140;

(A7) the nucleotide sequence shown in SEQ ID NO:141;

(A8) the nucleotide sequence shown in SEQ ID NO:142;

(A9) the nucleotide sequence shown in SEQ ID NO:143;

(a10) the nucleotide sequence shown in SEQ ID NO:225;

(A11) the nucleotide sequence shown in SEQ ID NO:226;

(A12) the nucleotide sequence shown in SEQ ID NO:227;

(A13) the nucleotide sequence shown in SEQ ID NO:228;

(A14) the nucleotide sequence shown in SEQ ID NO:229;

(A15) the nucleotide sequence shown in SEQ ID NO:230;

(A16) the nucleotide sequence shown in SEQ ID NO:231;

(A17) the nucleotide sequence shown in SEQ ID NO:232;

(A18) the nucleotide sequence shown in SEQ ID NO:233;

(A19) the nucleotide sequence shown in SEQ ID NO:234;

(A20) a nucleotide sequence that encodes the amino acid sequence of the protein capable of transforming in the presence of a system of electron transport containing electron donor, a compound of four the uly (II) in the compound of formula (III), moreover, the specified nucleotide sequence has at least 80% sequence identity with the nucleotide sequence shown in any of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:109; and

(A21) a nucleotide sequence that encodes the amino acid sequence of the protein capable of transforming in the presence of a system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III), and the specified nucleotide sequence has at least 90% sequence identity with the nucleotide sequence shown in any of SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, or SEQ ID NO:234;

3. The DNA according to claim 1, containing the nucleotide sequence encoding the amino acid sequence of the specified protein, where the use of codons in the specified nucleotide sequence is in the range of plus or minus 4% usage codons in the genes of the species of the host cell in which to enter this DNA, and GC-content of the specified nucleotide sequence is at least 40% and maximum 60%;

4. DNA containing the nucleotide sequence shown in SEQ ID NO:214;

5. DNA containing the nucleotide sequence shown is SEQ ID NO:368;

6. DNA containing the nucleotide sequence shown in SEQ ID NO:393;

7. DNA in which a DNA having a nucleotide sequence encoding a transit signal sequence of intracellular organelles attached "to the left" from the DNA according to claim 1 in reading frame;

8. DNA in which the DNA according to claim 1 and a promoter functional in the cell-master, functionally linked;

9. A vector containing the DNA according to claim 1;

10. A method of obtaining a vector to which the stages of embedding the DNA according to claim 1 in a vector that replicates in the cell-host;

11. The transformant in which the DNA according to claim 1 is introduced into the cell-host;

12. The transformant according to claim 11, where the host-cell is a cell of a microorganism or plant cell;

13. The method of producing transformant which the stages of introduction into the cell of the host DNA according to claim 1;

14. A method of obtaining a protein capable of converting the compound of formula (II) in the compound of formula (III), and specified the method comprises the stage of culturing transformant according to claim 11 and extraction produced the specified protein;

15. The use of DNA according to claim 1 for obtaining a protein capable of converting the compound of formula (II) in the compound of formula (III);

16. The method of imparting to a plant resistance to a herbicide which the stages of introducing into a plant cell and expression in the plant cell DNA pop;

17. Polynucleotide having a partial nucleotide sequence of the DNA according to claim 1 or a nucleotide sequence complementary to the specified partial nucleotide sequence;

18. The method of detecting DNA that encodes a protein capable of converting the compound of formula (II) in the compound of formula (III)providing phase detection of DNA, which hybridized probe by hybridization using as a probe the DNA according to claim 1 or polynucleotide at 17;

19. The method of detecting DNA that encodes a protein capable of converting the compound of formula (II) in the compound of formula (III)providing phase detection of DNA amplified in polymerase chain reaction with polynucleotide on 17 as primers;

20. The method according to claim 19, where at least one of the primers selected from the group consisting of polynucleotide containing the nucleotide sequence shown in any of SEQ ID NO:124 and SEQ ID NO:128, and polynucleotide containing the nucleotide sequence shown in SEQ ID NO:129;

21. A method of obtaining a DNA that encodes a protein capable of converting the compound of formula (II) in the compound of formula (III)which the stages of DNA extraction, detektirovanii the method according to p or 19;

22. Method of screening cells with DNA encoding a protein capable of converting the compound of formula (II) is a compound of formula (III), providing phase detection of the indicated DNA from the test cells according to the method according to p or 19;

23. Metabolizing the herbicide protein selected from the group consisting of:

(A1) a protein containing the amino acid sequence shown in SEQ ID NO:1;

(A2) a protein containing the amino acid sequence shown in SEQ ID NO:2;

(A3) a protein containing the amino acid sequence shown in SEQ ID NO:3;

(A4) a protein containing the amino acid sequence shown in SEQ ID NO:108;

(A5) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A6) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 80% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in lubies sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A11) a protein containing the amino acid sequence shown in SEQ ID NO:159;

(A12) a protein containing the amino acid sequence shown in SEQ ID NO:160;

(A13) a protein containing the amino acid sequence shown in SEQ ID NO:136;

(A14) a protein containing the amino acid sequence shown in SEQ ID NO:137;

(A15) a protein containing the amino acid sequence shown in SEQ ID NO:138;

(A16) a protein containing the amino acid sequence shown in SEQ ID NO:215;

(A17) a protein containing the amino acid sequence shown in SEQ ID NO:216;

(A18) a protein containing the amino acid sequence shown in SEQ ID NO:217;

(A19) a protein containing the amino acid sequence shown in SEQ ID NO:218;

(A20) a protein containing the amino acid sequence shown in SEQ ID NO:219;

(A21) a protein containing the amino acid sequence shown in SEQ ID NO:220;

(A22) a protein containing the amino acid sequence shown in SEQ ID NO:221;

(A23) a protein containing the amino acid sequence shown in SEQ ID NO:222;

(A24) a protein containing the amino acid sequence shown in SEQ ID NO:223;

(A25) a protein containing the amino acid sequence shown in SEQ ID NO:224;

(A26 protein, able to turn in the presence of a system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221 or SEQ ID NO:223 or amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:222, or SEQ ID NO:224;

(A27) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223 or SEQ ID NO:224; and

(A28) protein, is able to transform into the presence of the system of transport of electrons, with the holding electron donor, the compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, amplificatoare polymerase chain reaction with a primer containing the nucleotide sequence shown in any of SEQ ID NO:124 and SEQ ID NO:128, a primer containing the nucleotide sequence shown in SEQ ID NO:129, and using as the template the chromosomal DNA of Streptomyces phaeochromogenes, Streptomyces testaceus, Streptomyces achromogenes, Streptomyces griseofuscus, Streptomyces thermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus, Streptomyces roseorubens, Streptomyces rutgersensis, Streptomyces steffisburgensis or Saccharopolyspora taberi;

24. The antibody recognizing herbicide metabolizing protein selected from the group consisting of:

(A1) a protein containing the amino acid sequence shown in SEQ ID NO:1;

(A2) a protein containing the amino acid sequence shown in SEQ ID NO:2;

(A3) a protein containing the amino acid sequence shown in SEQ ID NO:3;

(A4) a protein containing the amino acid sequence shown in SEQ ID NO:108;

(A5) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having, for men is our least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A6) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 80% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A11) a protein containing the amino acid sequence shown in SEQ ID NO:159;

(A12) a protein containing the amino acid sequence shown in SEQ ID NO:160;

(A13) a protein containing the amino acid sequence shown in SEQ ID NO:136;

(A14) a protein containing the amino acid sequence shown in SEQ ID NO:137;

(A15) a protein containing the amino acid sequence shown in SEQ ID NO:138;

(A16) a protein containing the amino acid sequence shown in SEQ ID NO:215;

(A17) a protein containing the amino acid sequence shown in SEQ ID NO:216;

(A18) a protein containing the amino acid sequence shown in SEQ ID NO:217;/p>

(A19) a protein containing the amino acid sequence shown in SEQ ID NO:218;

(A20) a protein containing the amino acid sequence shown in SEQ ID NO:219;

(A21) a protein containing the amino acid sequence shown in SEQ ID NO:220;

(A22) a protein containing the amino acid sequence shown in SEQ ID NO:221;

(A23) a protein containing the amino acid sequence shown in SEQ ID NO:222;

(A24) a protein containing the amino acid sequence shown in SEQ ID NO:223;

(A25) a protein containing the amino acid sequence shown in SEQ ID NO:224;

(A26) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221 or SEQ ID NO:223 or amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:222, or SEQ ID NO:224;

(A27) protein, is able to transform into prisutstvie and transport system of electrons, containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223 or SEQ ID NO:224; and

(A28) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, amplificatoare polymerase chain reaction with a primer containing the nucleotide sequence shown in any of SEQ ID NO:124 and SEQ ID NO:128, a primer containing the nucleotide sequence shown in SEQ ID NO:129, and using as the template the chromosomal DNA of Streptomyces phaeochromogenes, Streptomyces testaceus, Streptomyces achromogenes, Streptomyces griseofuscus, Streptomyces thermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus, Streptomyces roseorubens, Streptomyces rutgersensis, Streptomyces steffisburgensis or Saccharopolyspora taberi;

25. Method detection metabolizing Gerbi the ID protein, providing:

(1) the stage of contacting the test compound with the antibody that recognizes this protein, and

(2) the stage of detection of the complex specified protein and the indicated antibodies arising as a result of this contact,

where this protein is selected from the group consisting of:

(A1) a protein containing the amino acid sequence shown in SEQ ID NO:1;

(A2) a protein containing the amino acid sequence shown in SEQ ID NO:2;

(A3) a protein containing the amino acid sequence shown in SEQ ID NO:3;

(A4) a protein containing the amino acid sequence shown in SEQ ID NO:108;

(A5) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A6) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 80% identity p is coherence with nucleotide sequence, the coding amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A11) a protein containing the amino acid sequence shown in SEQ ID NO:159;

(A12) a protein containing the amino acid sequence shown in SEQ ID NO:160;

(A13) a protein containing the amino acid sequence shown in SEQ ID NO:136;

(A14) a protein containing the amino acid sequence shown in SEQ ID NO:137;

(A15) a protein containing the amino acid sequence shown in SEQ ID NO:138;

(A16) a protein containing the amino acid sequence shown in SEQ ID NO:215;

(A17) a protein containing the amino acid sequence shown in SEQ ID NO:216;

(A18) a protein containing the amino acid sequence shown in SEQ ID NO:217;

(A19) a protein containing the amino acid sequence shown in SEQ ID NO:218;

(A20) a protein containing the amino acid sequence shown in SEQ ID NO:219;

(A21) a protein containing the amino acid sequence shown in SEQ ID NO:220;

(A22) a protein containing the amino acid sequence shown in SEQ ID NO:221;

(A23) a protein containing the amino acid sequence shown in SEQ ID NO:222;

(A24) a protein containing the amino acid sequence shown in SEQ D NO:223;

(A25) a protein containing the amino acid sequence shown in SEQ ID NO:224;

(A26) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221 or SEQ ID NO:223 or amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:222, or SEQ ID NO:224;

(A27) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:23 or SEQ ID NO:224; and

(A28) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, amplificatoare polymerase chain reaction with a primer containing the nucleotide sequence shown in any of SEQ ID NO:124 and SEQ ID NO:128, a primer containing the nucleotide sequence shown in SEQ ID NO:129, and using as the template the chromosomal DNA of Streptomyces phaeochromogenes, Streptomyces testaceus, Streptomyces achromogenes, Streptomyces griseofuscus, Streptomyces thermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus, Streptomyces roseorubens, Streptomyces rutgersensis, Streptomyces steffisburgensis or Saccharopolyspora taberi;

26. Set for analysis or detection of containing the antibody according to paragraph 24;

27. DNA encoding ferredoxin selected from the group consisting of:

(B1) a protein containing the amino acid sequence shown in SEQ ID NO:12;

(B2) a protein containing the amino acid sequence shown in SEQ ID NO:13;

(B3) a protein containing the amino acid sequence shown in SEQ ID NO:14;

(B4) a protein containing the amino acid sequence shown in SEQ ID NO:111;

(B5) ferredoxin containing the amino acid sequence of alnost, having at least 80% sequence identity with the amino acid sequence shown in any of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:111;

(B6) ferredoxin containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:111;

(B7) a protein containing the amino acid sequence shown in SEQ ID NO:149;

(B8) a protein containing the amino acid sequence shown in SEQ ID NO:150;

(B9) a protein containing the amino acid sequence shown in SEQ ID NO:151;

(10) a protein containing the amino acid sequence shown in SEQ ID NO:152;

(B11) a protein containing the amino acid sequence shown in SEQ ID NO:153;

(B12) ferredoxin containing amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251 or SEQ ID NO:253, or an amino acid sequence having, in least 90% sequence identity with the amino acid in what sledovatelnot, shown in any of SEQ ID NO:150, SEQ ID NO:252 or SEQ ID NO:254;

(B13) ferredoxin containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253 or SEQ ID NO:254;

(B14) a protein containing the amino acid sequence shown in SEQ ID NO:245;

(B15) a protein containing the amino acid sequence shown in SEQ ID NO:247;

(B16) a protein containing the amino acid sequence shown in SEQ ID NO:248;

(B17) a protein containing the amino acid sequence shown in SEQ ID NO:249;

(B18) a protein containing the amino acid sequence shown in SEQ ID NO:250;

(B19) protein containing the amino acid sequence shown in SEQ ID NO:251;

(B20) a protein containing the amino acid sequence shown in SEQ ID NO:252;

(B21) a protein containing the amino acid sequence shown in SEQ ID NO:253; and

(B22) a protein containing the amino acid sequence shown in SEQ ID NO:254;

28. DNA containing the nucleotide sequence selected from the group consisting of the C:

(b1) the nucleotide sequence shown in SEQ ID NO:15;

(b2) the nucleotide sequence shown in SEQ ID NO:16;

(b3) the nucleotide sequence shown in SEQ ID NO:17;

(b4) the nucleotide sequence shown in SEQ ID NO:112;

(b5) the nucleotide sequence shown in SEQ ID NO:154;

(b6) the nucleotide sequence shown in SEQ ID NO:155;

(b7) the nucleotide sequence shown in SEQ ID NO:156;

(b8) the nucleotide sequence shown in SEQ ID NO:157;

(b9) the nucleotide sequence shown in SEQ ID NO:158;

(b10) the nucleotide sequence shown in SEQ ID NO:255;

(b11) the nucleotide sequence shown in SEQ ID NO:257;

(b12) the nucleotide sequence shown in SEQ ID NO:258;

(b13) the nucleotide sequence shown in SEQ ID NO:259;

(b14) the nucleotide sequence shown in SEQ ID NO:260;

(b15) the nucleotide sequence shown in SEQ ID NO:261;

(b16) the nucleotide sequence shown in SEQ ID NO:262;

(b17) the nucleotide sequence shown in SEQ ID NO:263;

(b18) the nucleotide sequence shown in SEQ ID NO:264; and

(b19) a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown in any of SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:112, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263 or SEQ ID NO:264;

29. A vector containing the DNA according p;

30. The transformant, in which DNA on p introduced into the cell-host;

31. Ferredoxin selected from the group consisting of:

(B1) a protein containing the amino acid sequence shown in SEQ ID NO:12;

(B2) a protein containing the amino acid sequence shown in SEQ ID NO:13;

(B3) a protein containing the amino acid sequence shown in SEQ ID NO:14;

(B4) a protein containing the amino acid sequence shown in SEQ ID NO:111;

(B5) ferredoxin containing amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:111;

(B6) ferredoxin containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:111;

(B7) a protein containing the amino acid sequence shown in SEQ ID NO:149;

(B8) a protein containing the amino acid series is here, shown in SEQ ID NO:150;

(B9) a protein containing the amino acid sequence shown in SEQ ID NO:151;

(10) a protein containing the amino acid sequence shown in SEQ ID NO:152;

(B11) a protein containing the amino acid sequence shown in SEQ ID NO:153;

(B12) ferredoxin containing amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251 or SEQ ID NO:253, or an amino acid sequence having, in least 90% sequence identity with the amino acid sequence shown in any of SEQ ID NO:150, SEQ ID NO:252 or SEQ ID NO:254;

(B13) ferredoxin containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253 or SEQ ID NO:254;

(B14) a protein containing the amino acid sequence shown in SEQ ID NO:245;

(B15) a protein containing the amino acid sequence, azanuy in SEQ ID NO:247;

(B16) a protein containing the amino acid sequence shown in SEQ ID NO:248;

(B17) a protein containing the amino acid sequence shown in SEQ ID NO:249;

(B18) a protein containing the amino acid sequence shown in SEQ ID NO:250;

(B19) protein containing the amino acid sequence shown in SEQ ID NO:251;

(B20) a protein containing the amino acid sequence shown in SEQ ID NO:252;

(B21) a protein containing the amino acid sequence shown in SEQ ID NO:253; and

(B22) a protein containing the amino acid sequence shown in SEQ ID NO:254;

32. DNA containing the nucleotide sequence selected from the group consisting of:

(ab1) the nucleotide sequence shown in SEQ ID NO:9;

(ab2) the nucleotide sequence shown in SEQ ID NO:10;

(ab3) the nucleotide sequence shown in SEQ ID NO:11;

(ab4) the nucleotide sequence shown in SEQ ID NO:110;

(ab5) the nucleotide sequence shown in SEQ ID NO:144;

(ab6) the nucleotide sequence shown in SEQ ID NO:145;

(ab7) the nucleotide sequence shown in SEQ ID NO:146;

(ab8) the nucleotide sequence shown in SEQ ID NO:147;

(ab9) the nucleotide sequence shown in SEQ ID NO:148;

(ab10) the nucleotide sequence shown is in SEQ ID NO:235;

(ab11) the nucleotide sequence shown in SEQ ID NO:236;

(ab12) the nucleotide sequence shown in SEQ ID NO:237;

(ab13) the nucleotide sequence shown in SEQ ID NO:238;

(ab14) the nucleotide sequence shown in SEQ ID NO:239;

(ab15) the nucleotide sequence shown in SEQ ID NO:240;

(ab16) the nucleotide sequence shown in SEQ ID NO:241;

(ab17) the nucleotide sequence shown in SEQ ID NO:242;

(ab18) the nucleotide sequence shown in SEQ ID NO:243; and

(ab19) the nucleotide sequence shown in SEQ ID NO:244;

33. A vector containing the DNA according p;

34. The transformant, in which DNA on p introduced into the cell-host;

35. The transformant according to clause 34, where the host-cell is a cell of a microorganism or plant cell;

36. The method of producing transformant which the stages of introduction into the cell of the host DNA by p;

37. A method of obtaining a protein capable of converting the compound of formula (II) in the compound of formula (III)which the stages of cultivation transformant at 34 and extraction produced the specified protein;

38. Method for killing weeds which the stages of applying the compound to the area of cultivation of the plant expressing at least one herbicide metabolizing protein selected from the group, with the standing of:

(A1) a protein containing the amino acid sequence shown in SEQ ID NO:1;

(A2) a protein containing the amino acid sequence shown in SEQ ID NO:2;

(A3) a protein containing the amino acid sequence shown in SEQ ID NO:3;

(A4) a protein containing the amino acid sequence shown in SEQ ID NO:108;

(A5) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A6) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 80% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A7) protein, is able to transform into the presence system of electron transport containing electron donor, with the unity of formula (II) in the compound of formula (III) and containing the amino acid sequence, encoded by DNA that hybridizes in stringent conditions with a DNA containing the nucleotide sequence encoding the amino acid sequence shown in any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A8) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, amplificatoare polymerase chain reaction with a primer containing the nucleotide sequence shown in SEQ ID NO:129, primer containing the nucleotide sequence shown in any of SEQ ID NO:124 and SEQ ID NO:128, and using as matrix the chromosome of the microorganism belonging to Streptomyces or Saccharopolyspora;

(A9) a protein containing the amino acid sequence shown in SEQ ID NO:4;

(A11) a protein containing the amino acid sequence shown in SEQ ID NO:159;

(A12) a protein containing the amino acid sequence shown in SEQ ID NO:160;

(A13) a protein containing the amino acid sequence shown in SEQ ID NO:136;

(A14) a protein containing the amino acid sequence shown in SEQ ID NO:137;

(A15) a protein containing the amino acid sequence shown in SEQ ID NO:138;

(A16) a protein containing the amino acid is tnou sequence, shown in SEQ ID NO:215;

(A17) a protein containing the amino acid sequence shown in SEQ ID NO:216;

(A18) a protein containing the amino acid sequence shown in SEQ ID NO:217;

(A19) a protein containing the amino acid sequence shown in SEQ ID NO:218;

(A20) a protein containing the amino acid sequence shown in SEQ ID NO:219;

(A21) a protein containing the amino acid sequence shown in SEQ ID NO:220;

(A22) a protein containing the amino acid sequence shown in SEQ ID NO:221;

(A23) a protein containing the amino acid sequence shown in SEQ ID NO:222;

(A24) a protein containing the amino acid sequence shown in SEQ ID NO:223;

(A25) a protein containing the amino acid sequence shown in SEQ ID NO:224;

(A26) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221 or SEQ ID NO:223 or amino acid sequence having at least 90% sequence identity is lnasty with the amino acid sequence, shown in any of the sequences SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:222, or SEQ ID NO:224; and

(A27) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223 or SEQ ID NO:224;

where the specified compound is a compound of formula (I):

where in the formula (I), G represents a group shown in any of the following formulas (G-1 - G-9:

where G-1 - G-9

X represents an oxygen atom or a sulfur atom;

Y represents an oxygen atom or a sulfur atom;

R1represents a hydrogen atom or a halogen atom;

R2represents a hydrogen atom, a C1-C8alkyl group, a C1-C8halogenating group, halogen atom, hydroxyl group, a group-OR9group-SH, a group-S(O)pR9group-COR9group-CO2R9/sup> , group-C(O)SR9, group-C(O)NR11R12group-CONH2group-SNO group-CR9=NOR18the group-CH=CR19CO2R9group-CH2R19CO2R9group-CO2N=CR13R14the nitro-group, a cyano group,- NHSO2R15group-NHSO2Other15the group-NR9R20the group-NH2or phenyl group which may be substituted by one or more1-C4alkyl groups which may be the same or different;

p is 0, 1 or 2;

R3is1-C2alkyl group, a C1-C2halogenating group, a group-och3group-SCH3group-OCHF2, a halogen atom, a cyano, a nitro-group or

With1-C3alkoxygroup, substituted phenyl group which may be substituted on the ring, at least one Deputy, selected from a halogen atom, a C1-C3alkyl group, a C1-C3halogenoalkanes groups, OR28group

NR11R28group SR28, ceanography, group CO2R28and nitro;

R4represents a hydrogen atom, a C1-C3alkyl group or a C1-C3halogenating group;

R5represents a hydrogen atom, a C1-C3alkiline the group, With1-C3halogenating group, cyclopropyl group, vinyl group, With2alkylamino group, a cyano group,- C(O)R20group-CO2R20, group-C(O)NR20R21group

-R16R17CN, the group- (CR16R17C(O)R20the group With16R17CO2R20group- (CR16R17C(O)NR20R21group-CHR16OH, a group-CHR16OC(O)R20or group-OCHR16OC(O)NR20R21or, when G is G-2 or G-6, R4and R5can represent the group C=O together with the carbon atom to which they are attached;

R6is1-C6alkyl group, a C1-C6halogenating group2-C6alkoxyalkyl group3-C6alkenylphenol group or3-C6alkylamino group;

R7represents a hydrogen atom, a C1-C6alkyl group, a C1-C6halogenating group, a halogen atom, a group-S(O)2(C1-C6alkyl) or a group-C(=O)R22;

R8represents a hydrogen atom, a C1-C8alkyl group, a C3-C8cycloalkyl group3-C8alkenylphenol group3-C8alkylamino group1-C8halogenating group2-C8alkoxyalkyl, With3-C8alkoxylalkyl group3-C8halogenalkyls group3-C8halogenalkyls group1-C8alkylsulfonyl group1-C8halogenated.sulphonated group3-C8alkoxycarbonylmethyl group, a group-S(O)2NH(C1-C8alkyl), group-C(O)R23or benzyl group which may be substituted, R24on the phenyl ring;

R9is1-C8alkyl group, a C3-C8cycloalkyl group3-C8alkenylphenol group3-C8alkylamino group1-C8halogenating group2-C8alkoxyalkyl group2-C8alkylthiomethyl group2-C8alkylsulfonyl group2-C8alkylsulfonyl group,

With4-C8alkoxylalkyl group4-C8cycloalkylcarbonyl group4-C8cycloalkenyl group,

With4-C8alkenylacyl group4-C8alkyloxyalkyl group3-C8halogenoacetyl group,

With4-C8halogenocarboxylic group,

With4-C8halogenallylacetic group,

With4-C8cyclol intialising group, With4-C8alkenylacyl group4-C8alkynylamino group1-C4alkyl group, substituted phenoxypropane, which may be substituted on the ring, at least one Deputy, selected from a halogen atom, a C1-C3alkyl groups and C1-C3halogenoalkanes group1-C4alkyl group, substituted benzyloxycarbonyl, which may be substituted on the ring, at least one Deputy, selected from a halogen atom, a C1-C3alkyl groups and C1-C3halogenoalkanes group4-C8trialkylsilyl group2-C8cyanoaniline group3-C8halogencontaining group3-C8halogenalkyls group5-C8alkoxyalkyl group5-C8halogenoacetyl group5-C8alkylthiomethyl group3-C8halogenalkyls group5-C8alkoxyalkyl group5-C8halogenoacetyl group5-C8alkylthiomethyl group2-C8alkylcarboxylic group, benzyl group which may be substituted on the ring, at least one Deputy, selected from a halogen atom, a C1-C3Ala the school group, With1-C3halogenoalkanes groups,- OR28group-NR11R28group-SR28, ceanography, group-CO2R28and nitro-group, a group

- (CR16R17R10group CR16R17CO2R20group- (CR16R17P(O)(OR10)2group

- (CR16R17P(S)(OR10)2group- (CR16R17C(O)NR11R12group- (CR16R17C(O)NH2group

-C(=CR26R27)COR10, group-C(=CR26R27)CO2R20, group-C(=CR26R27)P(O)(OR10)2, group-C(=CR26R27)P(S)(OR10)2, group-C(=CR26R27)C(O)NR11R12, group-C(=CR26R27)C(O)NH2or any of the rings shown in Q-1 - Q-7:

which may be substituted on the ring, at least one Deputy, selected from a halogen atom, a C1-C6alkyl group, a C1-C6halogenoalkanes group2-C6alkenylphenol group2-C6halogenoalkanes group2-C6alkenylphenol group3-C6halogenosilanes group2-C8alkoxyalkyl groups,- OR28group-SR28group-NR11R28With3-C8alkoxycarbonylmethyl group2 -C4carboxialkilnuyu groups,- CO2R28and cyanopropyl;

R10is1-C6alkyl group, a C2-C6alkenylphenol group3-C6alkylamino group or tetrahydrofuranyl group;

R11and R13independently represent a hydrogen atom or a C1-C4alkyl group;

R12is1-C6alkyl group, a C3-C6cycloalkyl group3-C6alkenylphenol group3-C6alkylamino group2-C6alkoxyalkyl group1-C6halogenating group3-C6halogenalkyls group3-C6halogenalkyls group, phenyl group which may be substituted on the ring, at least one Deputy, selected from a halogen atom, a C1-C4alkyl groups and C1-C4alkoxygroup, or a group- (CR16R17CO2R25; or

R11and R12together may represent -(CH2)5-, -(CH2)4- or-CH2CH2Och2CH2or in this case, the ring may be substituted by the Deputy, is selected from C1-C3alkyl groups, phenyl groups and benzyl groups;

R14is1-C4alkylen the Yu group or phenyl group, which may be substituted on the ring by the Deputy selected from a halogen atom, a C1-C3alkyl groups and C1-C3halogenoalkanes group; or

R13and R14may present With3-C8cycloalkyl group together with the carbon atom to which they are attached;

R15is1-C4alkyl group, a C1-C4halogenating group or

With3-C6alkenylphenol group;

R16and R17independently represent a hydrogen atom or a C1-C4alkyl group, a C1-C4halogenating group2-C4alkenylphenol group2-C4halogenalkyls group2-C4alkylamino group3-C4halogenalkyls group; or

R16and R17may present With3-C6cycloalkyl group with the carbon atom to which they are attached, or the thus formed ring may be substituted by at least one Deputy, selected from a halogen atom, a C1-C3alkyl groups and C1-C3halogenoalkanes group;

R18represents a halogen atom, a C1-C6alkyl group, a C3-C6alkenylphenol group or3-C6alkylamino group;

R19represents the atom of water is kind, With1-C4alkyl group or a halogen atom,

R20represents a hydrogen atom, a C1-C6alkyl group, a C3-C6cycloalkyl group3-C6alkenylphenol group3-C6alkylamino group2-C6alkoxyalkyl group1-C6halogenating group3-C6halogenalkyls group3-C6halogenalkyls group, phenyl group which may be substituted on the ring, at least one Deputy, selected from a halogen atom, a C1-C4alkyl groups and groups OR28or a group- (CR16R17CO2R25;

R21represents a hydrogen atom, a C1-C2alkyl group or a group-CO2(C1-C4alkyl);

R22represents a hydrogen atom, a C1-C6alkyl group, a C1-C6alkoxygroup or the group NH(C1-C6alkyl);

R23is1-C6alkyl group, a C1-C6halogenating group1-C6alkoxygroup, the group NH(C1-C6alkyl), benzyl group, With2-C8dialkylamino or phenyl group which may be substituted, R24;

R24is1-C6alkyl group, 1-2 halogen atom, a C1With 6alkoxygroup or group CF3;

R25is1-C6alkyl group, a C1-C6halogenating group3-C6alkenylphenol group3-C6halogenalkyls group3-C6alkylamino group or3-C6halogenalkyls group;

R26and R27represent, each independently, a hydrogen atom, a C1-C4alkyl group, a C1-C4halogenating group2-C4alkenylphenol group2-C4halogenalkyls group2-C4alkylamino group3-C4halogenalkyls group, group-OR28group-other28or group-SR28; or

R26and R27may present With3-C8cycloalkyl group with the carbon atom to which they are attached, or are each formed such rings may be substituted by at least one Deputy, selected from a halogen atom, a C1-C3alkyl groups and C1-C3halogenoalkanes group; and

R28represents a hydrogen atom, a C1-C6alkyl group, a C1-C6halogenating group3-C6alkenylphenol group3-C6halogenalkyls group3-C6alkylamino group3-C62-C4carboxialkilnuyu group, With3-C8alkoxycarbonylmethyl group,

With3-C8halogenocarboxylic group,

With5-C9alkenylcarbazoles group,

With5-C9halogenocarboxylic group,

With5-C9alkyloxyalkyl group,

With5-C9halogenlithiumcarbenoid group,

With5-C9cycloalkylcarbonyl group or

With5-C9halogenlithiumcarbenoid group;

39. Method for killing weeds which the stages of applying the compounds in the area of plant cultivation, expressing at least one protein selected from the group consisting of:

(A1) a protein containing the amino acid sequence shown in SEQ ID NO:1;

(A2) a protein containing the amino acid sequence shown in SEQ ID NO:2;

(A3) a protein containing the amino acid sequence shown in SEQ ID NO:3;

(A4) a protein containing the amino acid sequence shown in SEQ ID NO:108;

(A5) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having, by at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A6) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 80% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A11) a protein containing the amino acid sequence shown in SEQ ID NO:159;

(A12) a protein containing the amino acid sequence shown in SEQ ID NO:160;

(A13) a protein containing the amino acid sequence shown in SEQ ID NO:136;

(A14) a protein containing the amino acid sequence shown in SEQ ID NO:137;

(A15) a protein containing the amino acid sequence shown in SEQ ID NO:138;

(A16) a protein containing the amino acid sequence shown in SEQ ID NO:215;

(A17) a protein containing the amino acid sequence shown in SEQ ID NO:216;

(A18) a protein containing the amino acid sequence shown in SEQ ID NO:217;/p>

(A19) a protein containing the amino acid sequence shown in SEQ ID NO:218;

(A20) a protein containing the amino acid sequence shown in SEQ ID NO:219;

(A21) a protein containing the amino acid sequence shown in SEQ ID NO:220;

(A22) a protein containing the amino acid sequence shown in SEQ ID NO:221;

(A23) a protein containing the amino acid sequence shown in SEQ ID NO:222;

(A24) a protein containing the amino acid sequence shown in SEQ ID NO:223;

(A25) a protein containing the amino acid sequence shown in SEQ ID NO:224;

(A26) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221 or SEQ ID NO:223 or amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:222, or SEQ ID NO:224;

(A27) protein, is able to transform into prisutstvie and transport system of electrons, containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223 or SEQ ID NO:224; and

(A28) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, amplificatoare polymerase chain reaction with a primer containing the nucleotide sequence shown in any of SEQ ID NO:124 and SEQ ID NO:128, a primer containing the nucleotide sequence shown in SEQ ID NO:129, and using as the template the chromosomal DNA of Streptomyces phaeochromogenes, Streptomyces testaceus, Streptomyces achromogenes, Streptomyces griseofuscus, Streptomyces thermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus, Streptomyces roseorubens, Streptomyces rutgersensis, Streptomyces steffisburgensis or Saccharopolyspora taberi;

40. A method of evaluating the resistance of the cell to connect the structure of formula (I), providing:

(1) the stage of contacting the compounds with a cell expressing at least one herbicide metabolizing protein selected from the group consisting of:

(A1) a protein containing the amino acid sequence shown in SEQ ID NO:1;

(A2) a protein containing the amino acid sequence shown in SEQ ID NO:2;

(A3) a protein containing the amino acid sequence shown in SEQ ID NO:3;

(A4) a protein containing the amino acid sequence shown in SEQ ID NO:108;

(A5) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A6) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 80% sequence identity with a nucleotide sequence that encodes the amino acid posledovatelno the ü, shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A7) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, which hybridizes in stringent conditions with a DNA containing the nucleotide sequence encoding the amino acid sequence shown in any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A8) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, amplificatoare polymerase chain reaction with a primer containing the nucleotide sequence shown in SEQ ID NO:129, primer containing the nucleotide sequence shown in any of SEQ ID NO:124 and SEQ ID NO:128, and using as matrix the chromosome of the microorganism belonging to Streptomyces or Saccharopolyspora;

(A9) a protein containing the amino acid sequence shown in SEQ ID NO:4;

(A11) a protein containing the amino acid sequence shown in SEQ ID NO:159;

(A12) a protein containing the amino acid sequence shown in SEQ ID NO:160;

(A13) the tree, containing the amino acid sequence shown in SEQ ID NO:136;

(A14) a protein containing the amino acid sequence shown in SEQ ID NO:137;

(A15) a protein containing the amino acid sequence shown in SEQ ID NO:138;

(A16) a protein containing the amino acid sequence shown in SEQ ID NO:215;

(A17) a protein containing the amino acid sequence shown in SEQ ID NO:216;

(A18) a protein containing the amino acid sequence shown in SEQ ID NO:217;

(A19) a protein containing the amino acid sequence shown in SEQ ID NO:218;

(A20) a protein containing the amino acid sequence shown in SEQ ID NO:219;

(A21) a protein containing the amino acid sequence shown in SEQ ID NO:220;

(A22) a protein containing the amino acid sequence shown in SEQ ID NO:221;

(A23) a protein containing the amino acid sequence shown in SEQ ID NO:222;

(A24) a protein containing the amino acid sequence shown in SEQ ID NO:223;

(A25) a protein containing the amino acid sequence shown in SEQ ID NO:224;

(A26) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221 or SEQ ID NO:223 or amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:222, or SEQ ID NO:224; and

(A27) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223 or SEQ ID NO:224; and

(2) the stage of assessing the degree of damage to the cells which were contacted with the connection on the specified stage (1);

41. The method according to p, where this cell is a cell of a microorganism or plant cell;

42. The method of selection of cells resistant to the compound of formula (I)in which stage of the cell selection is based on sustainability, evaluated in the method according to p;

43. The cell is resistant to a herbicide selected using the method according to § 42, or its culture;

44. A method of evaluating the resistance of plants to the compound of formula (I), including:

(1) the stage of contacting the compounds with a plant expressing at least one herbicide metabolizing protein selected from the group consisting of:

(A1) a protein containing the amino acid sequence shown in SEQ ID NO:1;

(A2) a protein containing the amino acid sequence shown in SEQ ID NO:2;

(A3) a protein containing the amino acid sequence shown in SEQ ID NO:3;

(A4) a protein containing the amino acid sequence shown in SEQ ID NO:108;

(A5) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A6) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid posledovatel the face, encoded by a nucleotide sequence having at least 80% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A7) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, which hybridizes in stringent conditions with a DNA containing the nucleotide sequence encoding the amino acid sequence shown in any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A8) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, amplificatoare polymerase chain reaction with a primer containing the nucleotide sequence shown in SEQ ID NO:129, primer containing the nucleotide sequence shown in any of SEQ ID NO:124 and SEQ ID NO:128, and using as matrix the chromosome of the microorganism belonging to Streptomyces or Saccharopolyspora;

(A9) a protein containing the amino acid sequence, showing what nnuu in SEQ ID NO:4;

(A11) a protein containing the amino acid sequence shown in SEQ ID NO:159;

(A12) a protein containing the amino acid sequence shown in SEQ ID NO:160;

(A13) a protein containing the amino acid sequence shown in SEQ ID NO:136;

(A14) a protein containing the amino acid sequence shown in SEQ ID NO:137;

(A15) a protein containing the amino acid sequence shown in SEQ ID NO:138;

(A16) a protein containing the amino acid sequence shown in SEQ ID NO:215;

(A17) a protein containing the amino acid sequence shown in SEQ ID NO:216;

(A18) a protein containing the amino acid sequence shown in SEQ ID NO:217;

(A19) a protein containing the amino acid sequence shown in SEQ ID NO:218;

(A20) a protein containing the amino acid sequence shown in SEQ ID NO:219;

(A21) a protein containing the amino acid sequence shown in SEQ ID NO:220;

(A22) a protein containing the amino acid sequence shown in SEQ ID NO:221;

(A23) a protein containing the amino acid sequence shown in SEQ ID NO:222;

(A24) a protein containing the amino acid sequence shown in SEQ ID NO:223;

(A25) a protein containing the amino acid sequence shown in SEQ ID NO:224;

(A26) a protein capable pravr which exists in the presence of a system of electron transport, containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221 or SEQ ID NO:223 or amino acid sequence with least 90% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:222, or SEQ ID NO:224; and

(A27) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223 or SEQ ID NO:224; and

(2) the stage of assessing the degree of damage to the plants, which is contacted with the compound described in stage (1);

45. The method of selection of plants resistant is go to the compound of formula (I), providing the stage of selection of plants based on the resistance measured in the method according to item 44.

46. Herbicide tolerant plant that is selected on the basis of the method according to item 45, or his posterity.

47. The processing method of the compounds of formula (I), involving the reaction of the compounds in the presence of a system of electron transport containing electron donor with at least one herbicide metabolizing protein selected from the group consisting of:

(A1) a protein containing the amino acid sequence shown in SEQ ID NO:1;

(A2) a protein containing the amino acid sequence shown in SEQ ID NO:2;

(A3) a protein containing the amino acid sequence shown in SEQ ID NO:3;

(A4) a protein containing the amino acid sequence shown in SEQ ID NO:108;

(A5) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A6) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the unity of the formula (III) and containing the amino acid sequence, encoded by a nucleotide sequence having at least 80% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A7) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, which hybridizes in stringent conditions with a DNA containing the nucleotide sequence encoding the amino acid sequence shown in any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A8) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, amplificatoare polymerase chain reaction with a primer containing the nucleotide sequence shown in SEQ ID NO:129, primer containing the nucleotide sequence shown in any of SEQ ID NO:124 and SEQ ID NO:128, and using as matrix the chromosome of the microorganism belonging to Streptomyces or Saccharopolyspora;

(A9) a protein containing the amino acid sequence, showing what nnuu in SEQ ID NO:4;

(A11) a protein containing the amino acid sequence shown in SEQ ID NO:159;

(A12) a protein containing the amino acid sequence shown in SEQ ID NO:160;

(A13) a protein containing the amino acid sequence shown in SEQ ID NO:136;

(A14) a protein containing the amino acid sequence shown in SEQ ID NO:137;

(A15) a protein containing the amino acid sequence shown in SEQ ID NO:138;

(A16) a protein containing the amino acid sequence shown in SEQ ID NO:215;

(A17) a protein containing the amino acid sequence shown in SEQ ID NO:216;

(A18) a protein containing the amino acid sequence shown in SEQ ID NO:217;

(A19) a protein containing the amino acid sequence shown in SEQ ID NO:218;

(A20) a protein containing the amino acid sequence shown in SEQ ID NO:219;

(A21) a protein containing the amino acid sequence shown in SEQ ID NO:220;

(A22) a protein containing the amino acid sequence shown in SEQ ID NO:221;

(A23) a protein containing the amino acid sequence shown in SEQ ID NO:222;

(A24) a protein containing the amino acid sequence shown in SEQ ID NO:223;

(A25) a protein containing the amino acid sequence shown in SEQ ID NO:224;

(A26) a protein capable pravr which exists in the presence of a system of electron transport, containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221 or SEQ ID NO:223 or amino acid sequence with least 90% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:222, or SEQ ID NO:224;

(A27) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223 or SEQ ID NO:224;

48. The method according to p, where the reaction of the compound with a herbicide metabolizing protein occurs through contact connection transformants, where DN is, encoding a herbicide metabolizing protein, is introduced into the cell-master in position, allowing its expression in a given cell;

49. Application for treatment of compounds of formula (I) herbicide metabolizing protein selected from the group consisting of:

(A1) a protein containing the amino acid sequence shown in SEQ ID NO:1;

(A2) a protein containing the amino acid sequence shown in SEQ ID NO:2;

(A3) a protein containing the amino acid sequence shown in SEQ ID NO:3;

(A4) a protein containing the amino acid sequence shown in SEQ ID NO:108;

(A5) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A6) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 80% sequence identity with the nucleotide PEFC is a sequence, the coding amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A7) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, which hybridizes in stringent conditions with a DNA containing the nucleotide sequence encoding the amino acid sequence shown in any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A8) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, amplificatoare polymerase chain reaction with a primer containing the nucleotide sequence shown in SEQ ID NO:129, primer containing the nucleotide sequence shown in any of SEQ ID NO:124 and SEQ ID NO:128, and using as matrix the chromosome of the microorganism belonging to Streptomyces or Saccharopolyspora;

(A9) a protein containing the amino acid sequence shown in SEQ ID NO:4;

(A11) a protein containing the amino acid sequence shown in SEQ ID NO:159;

(A12) a protein containing the amino acid on sledovatelnot, shown in SEQ ID NO:160;

(A13) a protein containing the amino acid sequence shown in SEQ ID NO:136;

(A14) a protein containing the amino acid sequence shown in SEQ ID NO:137;

(A15) a protein containing the amino acid sequence shown in SEQ ID NO:138;

(A16) a protein containing the amino acid sequence shown in SEQ ID NO:215;

(A17) a protein containing the amino acid sequence shown in SEQ ID NO:216;

(A18) a protein containing the amino acid sequence shown in SEQ ID NO:217;

(A19) a protein containing the amino acid sequence shown in SEQ ID NO:218;

(A20) a protein containing the amino acid sequence shown in SEQ ID NO:219;

(A21) a protein containing the amino acid sequence shown in SEQ ID NO:220;

(A22) a protein containing the amino acid sequence shown in SEQ ID NO:221;

(A23) a protein containing the amino acid sequence shown in SEQ ID NO:222;

(A24) a protein containing the amino acid sequence shown in SEQ ID NO:223;

(A25) a protein containing the amino acid sequence shown in SEQ ID NO:224;

(A26) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing linakis is now the sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221 or SEQ ID NO:223 or amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:222, or SEQ ID NO:224; and

(A27) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223 or SEQ ID NO:224; and

50. Application for treatment of compounds of formula (I) polynucleotide encoding herbicide metabolizing protein selected from the group consisting of:

(A1) a protein containing the amino acid sequence shown in SEQ ID NO:1;

(A2) a protein containing the amino acid is the selected, shown in SEQ ID NO:2;

(A3) a protein containing the amino acid sequence shown in SEQ ID NO:3;

(A4) a protein containing the amino acid sequence shown in SEQ ID NO:108;

(A5) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A6) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 80% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A7) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, which hybridizes in hard the conditions with DNA containing the nucleotide sequence encoding the amino acid sequence shown in any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A8) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, amplificatoare polymerase chain reaction with a primer containing the nucleotide sequence shown in SEQ ID NO:129, primer containing the nucleotide sequence shown in any of SEQ ID NO:124 and SEQ ID NO:128, and using as matrix the chromosome of the microorganism belonging to Streptomyces or Saccharopolyspora;

(A9) a protein containing the amino acid sequence shown in SEQ ID NO:4;

(A11) a protein containing the amino acid sequence shown in SEQ ID NO:159;

(A12) a protein containing the amino acid sequence shown in SEQ ID NO:160;

(A13) a protein containing the amino acid sequence shown in SEQ ID NO:136;

(A14) a protein containing the amino acid sequence shown in SEQ ID NO:137;

(A15) a protein containing the amino acid sequence shown in SEQ ID NO:138;

(A16) a protein containing the amino acid sequence shown in SEQ ID NO:215;

(A17) protein, with the holding amino acid sequence, shown in SEQ ID NO:216;

(A18) a protein containing the amino acid sequence shown in SEQ ID NO:217;

(A19) a protein containing the amino acid sequence shown in SEQ ID NO:218;

(A20) a protein containing the amino acid sequence shown in SEQ ID NO:219;

(A21) a protein containing the amino acid sequence shown in SEQ ID NO:220;

(A22) a protein containing the amino acid sequence shown in SEQ ID NO:221;

(A23) a protein containing the amino acid sequence shown in SEQ ID NO:222;

(A24) a protein containing the amino acid sequence shown in SEQ ID NO:223;

(A25) a protein containing the amino acid sequence shown in SEQ ID NO:224;

(A26) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221 or SEQ ID NO:223 or amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in any of the follower is awn SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:222, or SEQ ID NO:224; and

(A27) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223 or SEQ ID NO:224.

BRIEF DESCRIPTION of DRAWINGS

Fig. 1 shows the site of annealing of PCR primers used to obtain the DNA of the present invention (A1) and the DNA of the present invention (B1). Each of the numbers refers to the identification number of the sequence, SEQ ID showing the nucleotide sequence of these primers. Arrows indicate the site of annealing of oligonucleotide primers having the nucleotide sequence shown SEQ ID number, and the direction of elongation of DNA polymerase reactions with these primers. Dotted lines represent DNA amplified using PCR using these primers. The thick line represents the area adjacent to the site of injection of DNA vecto is a, used to obtain a library of chromosomal DNA.

Fig. 2 shows the site of annealing of PCR primers used to obtain the DNA of the present invention (A2) and the DNA of the present invention (B2). Each of the numbers refers to the identification number of the sequence, SEQ ID showing the nucleotide sequence of these primers. Arrows indicate the site of annealing of oligonucleotide primers having the nucleotide sequence shown SEQ ID number, and the direction of elongation of DNA polymerase reactions with these primers. Dotted lines represent DNA amplified using PCR using these primers. The thick line represents the area adjacent to the site of injection of the DNA vector used to obtain a library of chromosomal DNA.

Fig. 3 shows the site of annealing of PCR primers used to obtain the DNA of the present invention (A4) and the DNA of the present invention (B4). Each of the numbers refers to the identification number of the sequence, SEQ ID showing the nucleotide sequence of these primers. Arrows indicate the site of annealing of oligonucleotide primers having the nucleotide sequence shown SEQ ID number, and the direction of elongation of DNA polymerase reactions with these primers. Dotted lines represent DNA, amplifica is consistent through PCR using these primers. The thick line represents the area adjacent to the site of injection of the DNA vector, to obtain a library of chromosomal DNA. However oligonucleotide primer represented by number 57, is a primer that annealed to the area adjacent to the site of injection of the DNA vector used to obtain a library of chromosomal DNA, and is not able to otjihase with the DNA of the present invention (A4).

Fig. 4 shows the restriction map of plasmid pKSN2.

Fig. 5 shows the restriction map of plasmid pCRrSt12.

Fig. 6 shows the restriction map of plasmid pCR657ET.

Fig. 7 shows a restriction map of plasmid pCR657FET.

Fig. 8 shows a restriction map of plasmid pCR657Bs.

Fig. 9 shows a restriction map of plasmid pCR657FBs.

Fig. 10 shows a restriction map of plasmid pUCrSt12.

Fig. 11 shows the restriction map of plasmid pUCrSt657.

Fig. 12 shows a restriction map of plasmid pUCrSt657F.

Fig. 13 shows a restriction map of plasmid pUCCR16G6-p/t.

Fig. 14 shows the structure of the linker NotI-EcoRI obtained by annealing of the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:89, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:90.

Fig. 15 shows a restriction map of plasmid pUCCR16G6-p/tΔ.

Fig. 16 shows the structure of the Lin the EPA HindI-NotI, obtained by the annealing of the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:91, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:92.

Fig. 17 shows a restriction map of plasmid pNdC6-ΔT.

Fig. 18 shows a restriction map of plasmid

pSUM-NdG6-rSt657.

Fig. 19 shows a restriction map of plasmid

pSUM-NdG6-rSt657F.

Fig. 20 shows a restriction map of plasmid pKFrSt12.

Fig. 21 shows a restriction map of plasmid

pKFrSt12-657.

Fig. 22 shows a restriction map of plasmid

pKFrSt12-657F.

Fig. 23 shows a restriction map of plasmid

pSUM-NdG6-rSt12-657.

Fig. 24 shows a restriction map of plasmid

pSUM-NdG6-rSt12-657F.

Fig. 25 shows the structure of the linker HindI-NotI-EcoRI obtained by annealing of the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:98, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:99.

Fig. 26 shows a restriction map of plasmid pBI121S.

Fig. 27 shows a restriction map of plasmid

pBI-NdG6-rSt-657.

Fig. 28 shows a restriction map of plasmid

pBI-NdG6-rSt-657F.

Fig. 29 shows a restriction map of plasmid

pBI-NdG6-rSt12-657.

Fig. 30 shows a restriction map of plasmid

pBI-NdG6-rSt12-657F.

Fig. 31 shows the AET restriction map of plasmid pCR923Sp.

Fig. 32 shows a restriction map of plasmid

pNdG6-rSt12.

Fig. 33 shows a restriction map of plasmid

pSUM-NdG6-rSt-923.

Fig. 34 shows a restriction map of plasmid

pKFrSt12-923.

Fig. 35 shows a restriction map of plasmid

pSUM-NdG6-rSt12-923.

Fig. 36 shows a restriction map of plasmid

pBI-NdG6-rSt-923.

Fig. 37 shows a restriction map of plasmid

pBI-NdG6-rSt12-923.

Fig. 38 shows the restriction map of plasmid pCR671ET.

Fig. 39 shows a restriction map of plasmid pCR671Bs.

Fig. 40 shows a restriction map of plasmid pUCrSt671.

Fig. 41 shows a restriction map of plasmid

pSUM-NdG6-rSt-671.

Fig. 42 shows a restriction map of plasmid

pKFrSt12-671.

Fig. 43 shows a restriction map of plasmid

pSUM-NdG6-rSt12-671.

Fig. 44 shows a restriction map of plasmid

pBI-NdG6-rSt-671.

Fig. 45 shows the restriction map of plasmid

pBI-NdG6-rSt12-671.

Fig. 46 shows the results obtained by detection with the use of agarose gel electrophoresis of DNA amplified using PCR using as primers the oligonucleotide having a partial nucleotide sequence of DNA (A) of the present invention. Track 1, 7, 8, 12, 19, 26, 27, 32, 37, 42 and 47 represent electrophoresis DNA marker (product splitting ϕ 174/HaeIII). Other dorok the present electrophoresis of samples shown in tables 20 and 21.

Fig. 47 shows the structure of the linker obtained by annealing of the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:134, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:135.

Fig. 48 shows a restriction map of plasmid pUCrSt657soy.

Fig. 49 shows a restriction map of plasmid

pSUM-NdG6-rSt-657soy.

Fig. 50 shows a restriction map of plasmid

pKFrSt12-657soy.

Fig. 51 shows the restriction map of plasmid

pSUM-NdG6-rSt12-657soy.

Fig. 52 shows a restriction map of plasmid

pBI-NdG6-rSt-657soy.

Fig. 53 shows a restriction map of plasmid

pBI-NdG6-rSt12-657soy.

Fig. 54 shows a restriction map of plasmid pUCrSt1584soy.

Fig. 55 shows a restriction map of plasmid

pSUM-NdG6-rSt-1584soy.

Fig. 56 shows a restriction map of plasmid

pKFrSt12-1584soy.

Fig. 57 shows a restriction map of plasmid

pSUM-NdG6-rSt12-1584soy.

Fig. 58 shows a restriction map of plasmid

pBI-NdG6-rSt-1584soy.

Fig. 59 shows a restriction map of plasmid

pBI-NdG6-rSt12-1584soy.

Fig. 60 shows a restriction map of plasmid pUCrSt1609soy.

Fig. 61 shows a restriction map of plasmid

pSUM-NdG6-rSt-1609soy.

Fig. 62 shows the structure of the linker Not-a-ASOT obtained by annealing of the oligonucleotide, with the present of the nucleotide sequence, shown in SEQ ID NO:402, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:403.

Fig. 63 shows a restriction map of plasmid

pUCrSt12-1609soy.

Fig. 64 shows a restriction map of plasmid

pSUM-NdG6-rSt12-1609soy.

Fig. 65 shows a restriction map of plasmid

pBI-NdG6-rSt-1609soy.

Fig. 66 shows a restriction map of plasmid

pBI-NdG6-rSt12-1609soy.

Abbreviations used in the above figures, are explained below.

DNA A1: DNA (A1) of the present invention

DNA A2: DNA (A2) of the present invention

DNA A3: DNA (A3) of the present invention

DNA A4: DNA (A4) this invention

DNA B1: DNA (B1) of the present invention

DNA B2: DNA (B2) of the present invention

DNA B4: DNA (B4) this invention

DNA A1S: DNA (A1)S of this invention

DNA 23S: DNA (A23)S of this invention

DNA 25S: DNA (A25)S of this invention

tac p: tac promoter

rrnB t: terminator rrnB

ColE1 ori: the site of initiation of replication of plasmid ColE1

Ampr: resistance gene ampicillin

RuBPCssCTP: nucleotide sequence encoding a chloroplast transit peptide of the small subunit of ribulose-1,5-bestofferbuy of the soybean cultivar Jack)

A: nucleotide sequence encoding the 12 amino acids of the Mature protein, after the chloroplast transit peptide of the small subunit is ribulose-1,5-bestofferbuy of the soybean cultivar Jack).

Kmr: gene kanamycin-resistant

F1 ori: the site of initiation of replication of plasmid F1

R16G6p: promoter CRl6G6

CR16t: terminator CR16

CR16tΔ: DNA in which the nucleotide sequence right from the restriction site of restrictase ScaI removed from terminator CR16

CR16G6pΔ: DNA in which the nucleotide sequence left from the restriction site of restrictase NdeI removed from terminator CR16G6

NOSp: the promoter of the gene nepalensis

NPTII: gene kanamycin-resistant

NOSt: terminator gene nepalensis

GUS: gene β-glucuronidase

RB: right border sequence of T-DNA

LB: left border sequence of T-DNA

NdeI, HindIII, BspHI, EcoRI, BamHI, Eat, SphI, and Cloned, SacI, BglII, NotI, ScaI: restriction sites appropriate restriction enzymes (restricted).

The BEST WAY of carrying out the INVENTION

Below the invention is explained in detail.

Metabolizing the herbicide protein, selected from the following group of proteins (hereinafter sometimes called "protein (a) of this invention"), capable of converting the compound of formula (II) (hereinafter sometimes referred to as "compound (II)") in the compound of formula (III) (hereinafter sometimes referred to as "compound (III)"):

<groups of proteins>

(A1) a protein containing the amino acid sequence shown in SEQ ID NO:1;

(A2) a protein containing the amino acid sequence is lnost, shown in SEQ ID NO:2;

(A3) a protein containing the amino acid sequence shown in SEQ ID NO:3;

(A4) a protein containing the amino acid sequence shown in SEQ ID NO:108;

(A5) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A6) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 80% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A11) a protein containing the amino acid sequence shown in SEQ ID NO:159;

(A12) a protein containing the amino acid sequence shown in SEQ ID NO:160;

(A13) a protein containing the amino acid sequence shown in SEQ ID NO:136;

<> (A14) a protein containing the amino acid sequence shown in SEQ ID NO:137;

(A15) a protein containing the amino acid sequence shown in SEQ ID NO:138;

(A16) a protein containing the amino acid sequence shown in SEQ ID NO:215;

(A17) a protein containing the amino acid sequence shown in SEQ ID NO:216;

(A18) a protein containing the amino acid sequence shown in SEQ ID NO:217;

(A19) a protein containing the amino acid sequence shown in SEQ ID NO:218;

(A20) a protein containing the amino acid sequence shown in SEQ ID NO:219;

(A21) a protein containing the amino acid sequence shown in SEQ ID NO:220;

(A22) a protein containing the amino acid sequence shown in SEQ ID NO:221;

(A23) a protein containing the amino acid sequence shown in SEQ ID NO:222;

(A24) a protein containing the amino acid sequence shown in SEQ ID NO:223;

(A25) a protein containing the amino acid sequence shown in SEQ ID NO:224;

(A26) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid PEFC is a sequence, shown in any of the sequences SEQ ID NO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221 or SEQ ID NO:223 or amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:222, or SEQ ID NO:224;

(A27) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223 or SEQ ID NO:224; and

(A28) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, amplificatoare polymerase chain reaction with a primer containing the nucleotide sequence shown in any of SEQ ID NO:124 and SEQ ID NO:128, a primer containing the nucleotide sequence, shown in SEQ ID NO:129, and using as the template the chromosomal DNA of Streptomyces phaeochromogenes, Streptomyces testaceus, Streptomyces achromogenes, Streptomyces griseofuscus, Streptomyces thermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus, Streptomyces roseorubens, Streptomyces rutgersensis, Streptomyces steffisburgensis or Saccharopolyspora taberi.

As typical examples of the protein (A) of the present invention are mentioned:

a protein containing the amino acid sequence shown in SEQ ID NO:1 (hereinafter sometimes referred to as “protein (A1) of the present invention”);

a protein containing the amino acid sequence shown in SEQ ID NO:2 (hereinafter sometimes referred to as “protein (A2) of the present invention”);

a protein containing the amino acid sequence shown in SEQ ID NO:3 (hereinafter sometimes referred to as “protein (A3) of the present invention”);

a protein containing the amino acid sequence shown in SEQ ID NO:108 (hereinafter sometimes referred to as “protein (A4) of this invention”);

a protein containing the amino acid sequence shown in SEQ ID NO:159 (hereinafter sometimes referred to as “protein (A11) of this invention”);

a protein containing the amino acid sequence shown in SEQ ID NO:160 (hereinafter sometimes referred to as “protein (A12) of this invention”);

a protein containing the amino acid sequence of alnost, shown in SEQ ID NO:136 (hereinafter sometimes referred to as “protein (A13) of this invention”);

a protein containing the amino acid sequence shown in SEQ ID NO:137 (hereinafter sometimes referred to as “protein (A14) of this invention”);

a protein containing the amino acid sequence shown in SEQ ID NO:138 (hereinafter sometimes referred to as “protein (A15) of this invention”);

a protein containing the amino acid sequence shown in SEQ ID NO:215 (hereinafter sometimes referred to as “protein (A16) of this invention”);

a protein containing the amino acid sequence shown in SEQ ID NO:216 (hereinafter sometimes referred to as “protein (A17) of this invention”);

a protein containing the amino acid sequence shown in SEQ ID NO:217 (hereinafter sometimes referred to as “protein (A18) of this invention”);

a protein containing the amino acid sequence shown in SEQ ID NO:218 (hereinafter sometimes referred to as “protein (A19) this invention”);

a protein containing the amino acid sequence shown in SEQ ID NO:219 (hereinafter sometimes referred to as “protein (A20) of this invention”);

a protein containing the amino acid sequence shown in SEQ ID NO:220 (hereinafter sometimes referred to as “protein (A21) of this invention”);

a protein containing the amino acid sequence shown in SEQ ID NO:221 (hereinafter sometimes called the first “protein (A22) of this invention”);

a protein containing the amino acid sequence shown in SEQ ID NO:222 (hereinafter sometimes referred to as “protein (A23) of this invention”);

a protein containing the amino acid sequence shown in SEQ ID NO:223 (hereinafter sometimes referred to as “protein (A24) of this invention”); and

a protein containing the amino acid sequence shown in SEQ ID NO:224 (hereinafter sometimes referred to as “protein (A25) the present invention”).

For example, the reaction of the herbicide compounds of formula (I) PPO-inhibitor type (hereinafter sometimes referred to as "compound (I)) with the protein (A) of the present invention is able to make the connection specified in the connection with lower herbicide activity.

Further, in the processing for converting the compound (I) in the combination of lower herbicide activity can also be used herbicide metabolizing protein selected from the following group (hereinafter sometimes called "data protein (A)"):

<groups of proteins>

(A1) a protein containing the amino acid sequence shown in SEQ ID NO:1;

(A2) a protein containing the amino acid sequence shown in SEQ ID NO:2;

(A3) a protein containing the amino acid sequence shown in SEQ ID NO:3;

(A4) a protein containing the amino acid sequence shown in SEQ ID NO:108;

(A5) protein, is able to build the AMB in the presence of a system of electron transport, containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A6) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence having at least 80% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A7) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, which hybridizes in stringent conditions with a DNA containing the nucleotide sequence encoding the amino acid sequence shown in any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108;

(A8) protein, is able to transform into the presence system of electron transport containing donor power is and, the compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by the DNA, amplificatoare polymerase chain reaction with a primer containing the nucleotide sequence shown in SEQ ID NO:129, primer containing the nucleotide sequence shown in any of SEQ ID NO:124 and SEQ ID NO:128, and using as matrix the chromosome of the microorganism belonging to Streptomyces or Saccharopolyspora;

(A9) a protein containing the amino acid sequence shown in SEQ ID NO:4;

(A11) a protein containing the amino acid sequence shown in SEQ ID NO:159;

(A12) a protein containing the amino acid sequence shown in SEQ ID NO:160;

(A13) a protein containing the amino acid sequence shown in SEQ ID NO:136;

(A14) a protein containing the amino acid sequence shown in SEQ ID NO:137;

(A15) a protein containing the amino acid sequence shown in SEQ ID NO:138;

(A16) a protein containing the amino acid sequence shown in SEQ ID NO:215;

(A17) a protein containing the amino acid sequence shown in SEQ ID NO:216;

(A18) a protein containing the amino acid sequence shown in SEQ ID NO:217;

(A19) a protein containing the amino acid sequence shown in SEQ ID NO:218;

(A20) the protein content of asego amino acid sequence, shown in SEQ ID NO:219;

(A21) a protein containing the amino acid sequence shown in SEQ ID NO:220;

(A22) a protein containing the amino acid sequence shown in SEQ ID NO:221;

(A23) a protein containing the amino acid sequence shown in SEQ ID NO:222;

(A24) a protein containing the amino acid sequence shown in SEQ ID NO:223;

(A25) a protein containing the amino acid sequence shown in SEQ ID NO:224;

(A26) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221 or SEQ ID NO:223 or amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in any of the sequences SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:222, or SEQ ID NO:224; and

(A27) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing amino acid after outermost, encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of the sequences SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223 or SEQ ID NO:224.

As examples of this protein (A) can be mentioned protein a of the present invention described above. In addition, as other examples can be mentioned

a protein containing the amino acid sequence shown in SEQ ID NO:4, (hereinafter sometimes called "data protein (A9)") and

a protein containing the amino acid sequence shown in SEQ ID NO:5, (hereinafter sometimes called "data protein (a10)").

In the amino acid sequence of the protein shown in (A5), (A6), (A7), (A8), (A26), (A27) and (A28) in the above groups of proteins, differences that can be observed from the amino acid sequence shown in SEQ ID NO:1, 2, 3, 108, 159, 160, 136, 137, 138, 215, 216, 217, 218, 219, 220, 221, 222, 223, or 224 are such as deletion, replacement and addition of certain amino acids. Such differences include, for example, a deletion in the processing that the above protein containing the amino acid sequence shown in SEQ ID NO:1, 2, 3, 108, 159, 60, 136, 137, 138, 215, 216, 217, 218, 219, 220, 221, 222, 223 or 224, gets in the cage. Further, they include polymorphic variation which occurs in nature, originating from differences of view, the individual or the like, of the organism from which the protein; amino acid deletions, substitutions and additions resulting from genetic mutations introduced by artificial means, for example by way of site-directed mutagenesis by the method of random mutagenesis treatment with mutagens, etc.

The number of amino acids exposed to such deletions, substitutions and additions may be in the range in which the protein (A) can develop the ability to turn the compound (II) into the compound (III). In addition, as a replacement of amino acids can be mentioned, for example, replacement of the amino acid, which is similar in hydrophobicity, charge, RK, stereostructures the sign or the like, as such substitutions specifically mentioned, for example, substitutions within groups: (1) glycine and alanine; (2) valine, isoleucine and leucine; (3) aspartic acid, glutamic acid, asparagine and glutamine; (4) serine and threonine; (5) lysine and arginine; (6) phenylalanine and tyrosine; and so on

Further, in the present protein (A) preferably, the cysteine present at position matching when matching with cysteine to amino acid number 357 in aminodinitrotoluenes, shown in SEQ ID NO:1 has been saved (not subjected to deletion or substitution): examples of this include cysteine cysteine to amino acid number 350 in the amino acid sequence shown in SEQ ID NO:2, with cysteine amino acid number 344 in the amino acid sequence shown in SEQ ID NO:3, a cysteine to amino acid number 360 in the amino acid sequence shown in SEQ ID NO:108; cysteine to amino acid number 359 in the amino acid sequence shown in SEQ ID NO:4, with cysteine amino acid number 355 in the amino acid sequence shown in SEQ ID NO:5, cysteine to amino acid number 358 in the amino acid sequence shown in SEQ ID NO:159, cysteine to amino acid number 374 in the amino acid sequence shown in SEQ ID NO:160, cysteine to amino acid number 351 in the amino acid sequence shown in SEQ ID NO:136, cysteine to amino acid number 358 in the amino acid sequence shown in SEQ ID NO:137, cysteine to amino acid number 358 in the amino acid sequence shown in SEQ ID NO:138, cysteine to amino acid number 347 in the amino acid sequence shown in SEQ ID NO:222, with cysteine amino acid number 347 in the amino acid sequence shown in SEQ ID NO:224, etc.

As ways of artificially causing such cases the tion, add or replace amino acids (hereinafter referred to in General as “a modification of amino acids”), is mentioned, for example, a method comprising the stage of carrying out site-directed mutagenesis of DNA that encodes the amino acid sequence shown in any of SEQ ID NO:1, 2, 3, 108, 159, 160, 136, 137, 138, 215, 216, 217, 218, 219, 220, 221, 222, 223, or 224, with subsequent expression of such DNA using the conventional method. As a method of site-directed mutagenesis, for example, be mentioned a method which uses amber-mutations (Gapped Duplex method, Nucleic Acids Res., 12, 9441-9456 (1984)), the PCR method using primers for introducing mutations, etc. in Addition, as an artificial amino acid modifications mentioned, for example, a method comprising the stage of carrying out random mutagenesis of DNA that encodes any of the amino acid sequence shown in SEQ ID NO:1, 2, 3, 108, 159, 160, 136, 137, 138, 215, 216, 217, 218, 219, 220, 221, 222, 223 or 224, with subsequent expression of such DNA using the conventional way. As a method of random mutagenesis may be mentioned, for example, a method of performing PCR using DNA that encodes any of the above amino acid sequences, as a matrix and using a pair of primers that can amplify the full length of each of these DNA under conditions in which the concentration of each of dATP, dTTP, dGTP and dCTP, used as substrate, different from the ordinary, or in conditions in which the concentration of Mg2+which stimulates the polymerase reaction is increased to a higher concentration than the commonly used concentration. As such PCR methods mentioned, for example, the method described in Method in Molecular Biology, (31), 1994, 97-112. In addition, there may be mentioned the method described in Patent PCT publication WO 00/09682.

In this invention, "sequence identity" refers to the homology and identity between two nucleotide sequences or two amino acid sequences. This "sequence identity" may be determined by comparing two sequences, each of which is aligned in an optimal state, throughout the district for these test sequences. Thus, under optimal alignment of the test sequences of nucleic acids or amino acid sequences can be used to add or deletions (e.g., gaps, i.e. gaps) as such. Such sequence identity can be calculated by means of the stage of obtaining the alignment conducted by homology analysis using programs such as FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 4, 2444-2448 (1988)), BLAST (Altschul et al., Journal of Molcular Biology, 215, 403-410 (1990)), CLUSTAL W (Thompson, Higgins and Gibson, Nucleic Acid Research, 22, 4673-4680 (1994a)), etc. Such programs may be, for example, is usually used on a web page (http://www.ddbj.nig.ac.jp) DNA data Bank of Japan (international data Bank, working in the center for information biology, and a data Bank of DNA, Japan). In addition, the sequence identity can be determined using commercially available software for sequence analysis. For example, specifically, it can be calculated by obtaining alignment conducted by homology analysis according to the method of Lipman-Pearson (Lipman, D.J. and Pearson, W.R., Science, 227, 1435-1441, (1985)) using GENETYX-WIN Ver.5 Software Development Company, Ltd.).

As "stringent conditions"described in (A7)can be mentioned, for example, conditions in which the hybrid is formed at 45°C in a solution containing 6SSC (with the assumption 10SSC solution containing 1.5 M NaCl and 0.15 M trinatriytsitrat), and then this hybrid washed at 50°C 2SSC (Molecular Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6) for hybridization carried out in accordance with the conventional method described, for example, in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning, 2nd Edition, Cold Spring Harbor Press. The concentration of salt in the stage of washing can be selected, for example, from the range of conditions of 2 x SSC (low hardness) to conditions of 0.2 x SSC (high stiffness). The temperature in the stage of washing can be selected, for example, in the range from room temperature (low stringency) to 65°C (high stiffness). Alternatively, as salt concentration and temperature can be changed.

As the DNA that "hybridizes in stringent conditions with a DNA containing the nucleotide sequence encoding the amino acid sequence shown in any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:108, specifically, for example, may be mentioned a DNA containing the nucleotide sequence encoding the amino acid sequence shown in any of SEQ ID NO:1, 2, 3, 4, 5, 108, 159, 160, 136, 137, 138, 215, 216, 217, 218, 219, 220, 221, 222, 223, or 224, DNA containing the nucleotide sequence shown in any of SEQ ID NO:6, 7, 8, 78, 84, 109, 139, 140, 141, 142, 143, 225, 226, 227, 228, 229, 230, 231, 232, 233 or 234, etc. May be mentioned a DNA containing a nucleotide sequence having at least about 60% identity with the nucleotide sequence shown in any of SEQ ID NO:6, 7, 8, 78, 84, 109, 139, 140, 141, 142, 143, 225, 226, 227, 228, 229, 230, 231, 232, 233 or 234.

The molecular mass of this protein (A) is equal to approximately 30000-60000 and usually equal to about 40000-50000 (compared, for example, a protein consisting of the amino acid sequence shown in any of SEQ ID NO:1, 2, 3, 108, 159, 160, 136, 137, 138, 215, 216, 217, 218, 219, 220, 221, 222, 223, or 224), when identification is the molecular weight by electrophoresis containing sodium dodecyl sulphate polyacrylamide gel (hereinafter called "electrophoresis LTO-PAG"). In addition, the present protein (A), while its ability to convert compound (I) in the compound (III) is not eliminated, can be used as a protein, to which was added the amino acid sequence of the left from its amino end or right from its carboxy-end.

As a marker of the ability of this protein to metabolize the herbicide connection PPO-inhibitor type formula (I) may be mentioned the ability to convert compound (II) into the compound (III). This ability may be, for example, confirmed by the reaction of the compound (II) with the protein (A) in the presence of a system of electron transport containing electron donor, such as coenzyme NADPH, and detection of the resulting compound (III).

The term "system of electron transport containing electron donor"refers to a system in which there is a redox reaction and the electron is transferred from an electron donor to this protein (And). As an electron donor can be, for example, mentioned coenzymes NADPH, NADH, etc. Such as proteins, which may represent a system of electron transport from NADPH to this protein (A)can be mentioned ferredoxin and ferredoxin-NADP+-reductase, NADPH-cytochrome P-450-reductase, etc.

For example, to confirm the ability to turn compounds the s (II) in the compound (III), the reaction solution is about pH 7, containing the present protein (A), β-NADPH, ferredoxin, ferredoxin-NADP+-reductase and the compound (II)labeled with a radioactive isotope, incubated at about 30°C for approximately 10 minutes - 1 hour. Then after adjusting pH of the reaction solution to an acidic pH of hydrochloric acid, it is extracted with ethyl acetate. After exposure to the extracted an ethyl acetate layer thin-layer chromatography (“TLC”) are autoradiography, and the ability to turn the compound (II) into the compound (III) can be confirmed by detection of the labeled compound (III).

To obtain this protein (A), for example, first get a DNA encoding the present protein (A) (hereinafter sometimes referred to collectively in this DNA (A)"), in accordance with generally accepted methods of genetic engineering (for example, the methods described in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning, 2nd Edition, Cold Spring Harbor Press).

As examples of this DNA (A) can be mentioned a DNA encoding a protein (A) of the present invention (hereinafter sometimes referred to as "DNA (a) of this invention"). As typical examples of the DNA (a) of this invention may be mentioned:

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:1 (hereinafter sometimes referred to as "DNA (A1) of the present invention");

DNA encoding the protein content is of ASI amino acid sequence, shown in SEQ ID NO:2 (hereinafter sometimes referred to as "DNA (A2) of the present invention");

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:3 (hereinafter sometimes called "DNA (A3) of the present invention");

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:108 (hereinafter sometimes called "DNA (A4) of this invention");

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:159 (hereinafter sometimes called "DNA (A11) of this invention");

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:160 (hereinafter sometimes called "DNA (A12) of this invention");

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:136 (hereinafter sometimes called "DNA (A13) of this invention");

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:137 (hereinafter sometimes called "DNA (A14) of this invention");

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:138 (hereinafter sometimes called "DNA (A15) of this invention");

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:215 (hereinafter sometimes called "DNA (A16) of this invention");

DNA coderush what I protein containing the amino acid sequence shown in SEQ ID NO:216 (hereinafter sometimes called "DNA (A17) of this invention");

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:217 (hereinafter sometimes called "DNA (A18) of this invention");

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:218 (hereinafter sometimes called "DNA (A19) this invention");

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:219 (hereinafter sometimes called "DNA (A20) of this invention");

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:220 (hereinafter sometimes called "DNA (A21) of this invention");

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:221 (hereinafter sometimes called "DNA (A22) of this invention");

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:222 (hereinafter sometimes called "DNA (A23) of this invention");

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:223 (hereinafter sometimes called "DNA (A24) of this invention");

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:224 (hereinafter called inog is a "DNA (A25) of this invention"); etc.

Further, as more specific examples of the DNA (a) of this invention may be mentioned:

DNA containing the nucleotide sequence shown in SEQ ID NO:6;

DNA containing the nucleotide sequence shown in SEQ ID NO:9;

DNA containing the nucleotide sequence shown in SEQ ID NO:7;

DNA containing the nucleotide sequence shown in SEQ ID NO:10;

DNA containing the nucleotide sequence shown in SEQ ID NO:8;

DNA containing the nucleotide sequence shown in SEQ ID NO:11;

DNA containing the nucleotide sequence shown in SEQ ID NO:109;

DNA containing the nucleotide sequence shown in SEQ ID NO:110;

DNA containing the nucleotide sequence shown in SEQ ID NO:139;

DNA containing the nucleotide sequence shown in SEQ ID NO:144;

DNA containing the nucleotide sequence shown in SEQ ID NO:140;

DNA containing the nucleotide sequence shown in SEQ ID NO:145;

DNA containing the nucleotide sequence shown in SEQ ID NO:141;

DNA containing the nucleotide sequence shown in SEQ ID NO:146;

DNA containing the nucleotide sequence shown in SEQ ID NO:142;

DNA containing the nucleotide sequence shown in SEQ ID NO147;

DNA containing the nucleotide sequence shown in SEQ ID NO:143;

DNA containing the nucleotide sequence shown in SEQ ID NO:148;

DNA containing the nucleotide sequence shown in SEQ ID NO:225;

DNA containing the nucleotide sequence shown in SEQ ID NO:235;

DNA containing the nucleotide sequence shown in SEQ ID NO:226;

DNA containing the nucleotide sequence shown in SEQ ID NO:236;

DNA containing the nucleotide sequence shown in SEQ ID NO:227;

DNA containing the nucleotide sequence shown in SEQ ID NO:237;

DNA containing the nucleotide sequence shown in SEQ ID NO:228;

DNA containing the nucleotide sequence shown in SEQ ID NO:238;

DNA containing the nucleotide sequence shown in SEQ ID NO:229;

DNA containing the nucleotide sequence shown in SEQ ID NO:239;

DNA containing the nucleotide sequence shown in SEQ ID NO:230;

DNA containing the nucleotide sequence shown in SEQ ID NO:240;

DNA containing the nucleotide sequence shown in SEQ ID NO:231;

DNA containing the nucleotide sequence shown in SEQ ID NO:241;

DNA containing the nucleotide sequence shown in SEQ ID NO:232;

DNA starasiantattoo sequence, shown in SEQ ID NO:242;

DNA containing the nucleotide sequence shown in SEQ ID NO:233;

DNA containing the nucleotide sequence shown in SEQ ID NO:243;

DNA containing the nucleotide sequence shown in SEQ ID NO:234;

DNA containing the nucleotide sequence shown in SEQ ID NO:244;

DNA containing the nucleotide sequence shown in SEQ ID NO:214;

DNA containing the nucleotide sequence shown in SEQ ID NO:368;

DNA containing the nucleotide sequence shown in SEQ ID NO:393;

DNA encoding a protein capable of converting in the presence of a system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III), and having at least 80% sequence identity with the nucleotide sequence shown in any of SEQ ID nos:6, 7, 8 or 109;

DNA encoding a protein capable of converting in the presence of a system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III), and having at least 90% sequence identity with the nucleotide sequences shown in any of SEQ ID NO:139, 140, 141, 142, 143, 225, 226, 227, 228, 229, 230, 231, 232, 233 or 234; etc.

Further, as examples of this DNA (A), in addition to DNA (A) of the present invention described above, minutse:

DNA containing the nucleotide sequence encoding a protein containing the amino acid sequence shown in SEQ ID NO:4 (hereinafter sometimes referred to as "this DNA (A9)");

DNA containing the nucleotide sequence shown in SEQ ID NO:78;

DNA containing the nucleotide sequence encoding a protein containing the amino acid sequence shown in SEQ ID NO:5 (hereinafter sometimes referred to as "this DNA (a10)");

DNA containing the nucleotide sequence shown in SEQ ID NO:84;

DNA containing the nucleotide sequence shown in SEQ ID NO:85; and so on

This DNA may be, for example, DNA, cloned from a natural source, and can be DNA, obtained by introducing a deletion, substitution or addition of nucleotide (nucleotide) DNA cloned from a natural source, for example, by the method of site-directed mutagenesis by the method of random mutagenesis, and can be artificially synthesized DNA. Then the protein (A) can be produced or obtained by the expression obtained in this DNA in accordance with conventional methods of genetic engineering. Such paths can be obtained this protein (A).

This DNA (A) can be obtained, for example, in the following ways. First get the chromosomal DNA by using common methods of genetic engineering, and as described in Molecular Cloning: A Laboratory Manual 2nd Edition (1989), Cold Spring Harbor Laboratory Press; Current Protocols in Molecular Biology (1987), John Wiley and Sons, Incorporated, from microorganisms belonging to Streptomyces, such as Streptomyces phaeochromogenes, Streptomyces testaceus, Streptomyces achromogenes, Streptomyces griseolus, Streptomyces carbophilus, Streptomyces griseofuscus, Streptomyces thermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus, Streptomyces roseorubens, Streptomyces rutgersensis and Streptomyces steffisburgensis, and more specifically, Streptomyces phaeochromogenes IFO12898, Streptomyces testaceus ATCC21469, Streptomyces achromogenes IFO 12735, Streptomyces griseolus ATCC11796, Streptomyces carbophilus SANK62585, Streptomyces griseofuscus IFO 12870t, Streptomyces thermocoerulescens IFO 14273t, Streptomyces nogalater IFO 13445, Streptomyces tsusimaensis IFO 13782, Streptomyces glomerochromogenes IFO 13673t, Streptomyces olivochromogenes IFO 12444, Streptomyces ornatus IFO 13069t, Streptomyces griseus ATCC 10137, Streptomyces griseus IFO 13849T, Streptomyces lanatus IFO 12787T, Streptomyces misawanensis IFO 13855T, Streptomyces pallidus IFO 13434T, Streptomyces roseorubens IFO 13682T, Streptomyces rutgersensis IFO 15875T and Streptomyces steffisburgensis IFO 13446T and the like, or from microorganisms belonging to Saccharopolyspora, such as Saccharopolyspora taberi, more specifically Saccharopolyspora taberi JCM 9383t, etc. Then, after partial cleavage of the chromosomal DNA restriction enzyme, such as Sau3AI, extract DNA, approximately 2 TPN Obtained DNA clone into the vector in accordance with conventional methods of genetic engineering, are described in Molecular Cloning: A Laboratory Manual 2nd Edition (1989), Cold Spring Harbor Laboratory Press; Current Protocols in Molecular Biology (1987), John Wiley and Sons, Incorporated. As a vector, for example, can is to be used plasmid vector pUC 119 (TaKaRa Shuzo Company), pTVA 118N (Takara Shuzo Compani), pBlueScript II (Toyobo Company), pCR2.1-TOPO (Invitrogen), pTrc99A (Amersham Pharmacia Biotech Company), pKK331-1A (Amersham Pharmacia Biotech Company), etc. Library of chromosomal DNA can be obtained by extraction of plasmids from the obtained clone.

This DNA (A) can be obtained by hybridization of the probe with the resulting library of chromosomal DNA under the conditions described below, and the detection and extraction of DNA, which is associated specifically with this probe. The probe may be DNA, consisting of approximately at least 20 nucleotides, containing the nucleotide sequence encoding the amino acid sequence shown in one of SEQ ID NO:1, 2, 3, or 108. As typical examples of the DNA that can be used as probes, referred to DNA containing the nucleic acid shown in any of SEQ ID nos:6, 7, 8 or 109; DNA containing a partial nucleotide sequence of the nucleic acid sequence shown in any of SEQ ID nos:6, 7, 8 or 109; DNA containing a nucleotide sequence complementary to the specified partial nucleotide sequence; and so on

DNA used as a probe mark a radioactive isotope, fluorescent staining, etc. For labeling DNA with radioactive isotope, can be, for example, used set for random labelling Boehringer or Takara Shuzo Company. In addition to the, DNA labeled with32R, can be obtained by carrying out PCR. DNA to be sensing, are used as matrix. dCTP, usually used in the PCR reaction solution, replace (α32P)dCTP. In addition, when tagging DNA fluorescent staining, for example, can be used set for labeling and detection of DIG-High Prime DNA Labelling and Detection Starter Kit II (Roche Company).

Next explained is a typical example of the receiving probe. For example, DNA labeled with digoxigenin containing the full nucleotide sequence shown in SEQ ID NO:6, can be obtained using the chromosomal DNA obtained from Streptomyces phaeochromogenes IFO 12898, as described above, or library of chromosomal DNA as the template, using as primers the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:93, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:94, and PCR, as described in the examples below, for example, set to synthesis of probes PCR DIG Probe Synthesis Kit (Roche Diagnostics GmbH) in accordance with the attached manual. Similarly, DNA labeled with digoxigenin containing the nucleotide sequence from nucleotide 57 to nucleotide 730 SEQ ID NO:6, can be obtained using the chromosomal DNA obtained from Streptomyces phaeochromogenes IO 12898, described above, or library of chromosomal DNA as template. As for primers, this PCR is carried out with the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:130, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:131. In addition, DNA labeled with digoxigenin containing the full nucleotide sequence shown in SEQ ID NO:7, can be obtained using chromosomal DNA derived from Saccharopolyspora taberi JCM 9383t described above, or library of chromosomal DNA as template. As for primers, this PCR is carried out with the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:61, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:62. In addition, DNA labeled with digoxigenin containing the full nucleotide sequence shown in SEQ ID NO:8, can be obtained using the chromosomal DNA obtained from Streptomyces testaceus ATCC21469 described above, or library of chromosomal DNA as template. As for primers, this PCR is carried out with the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:70, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:71. In addition, DNA, labeled Digox Genina, containing the nucleotide sequence from nucleotide 21 to nucleotide 691 SEQ ID NO:8, can be obtained using the chromosomal DNA obtained from Streptomyces testaceus ATCC21469 described above, or library of chromosomal DNA as template. As for primers, this PCR is carried out with the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:132, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:133.

The ways in which the probe allow to gibridizatsiya with a library of chromosomal DNA, can include hybridization colony and plaque hybridization, and can be selected in a suitable way that is compatible with the type of vector used for preparation of the library. When designing libraries using plasmid vectors are conducting the hybridization of colonies. Specifically, first obtained transformants by introduction of the DNA of this library in the microorganism, in which the plasmid vector used to construct this library is replicated. The resulting transformants are bred and distribute the Cup with agar and grown until colonies. When using a phage vector to construct the libraries hybridization plaques. Specifically, first, the microorganism which is used to obtain library phage vector is a replicable, mixed with phage from this library in the context in which is it possible infection. Then this mixture is mixed with soft agar. The mixture is then spread on agar plate was assessed. After that, the mixture was cultivated before the appearance of plaques.

Then, if any of the above hybridisable place the membrane on the surface of the plates with agar, which conducted cultivation, and colonies of transformants or phage particles in the plaques are transferred to the membrane. After the alkali treatment of the membrane spend neutralizing treatment. Then DNA elyuirovaniya from the transformants or phage particles fixed on the membrane. More specifically, for example, in the event of hybridizing plaques of phage particles adsorb on the membrane location nitrocellulose membrane or a nylon membrane, in particular, for example, Hybond-N+((Amersham Pharmacia Biotech Company), a Cup of agar and wait for 1 minute. The membrane is immersed in an alkali solution (1.5 M NaCl and 0.5 N. NaOH) for about 3 minutes to dissolve the phage particles, and the elution ragovoy DNA on the membrane. Then the membrane is soaked in neutralizing solution (1.5 M NaCl and 0.5 m Tris-HCl buffer pH 7.5) for about 5 minutes. After washing this membrane in washing solution (0.3 M NaCl, 30 mm sodium citrate, 0.2 M Tris-HCl buffer pH 7.5) for, for example, approximately 5 minutes DNA phage fixed n the membrane by incubation at approximately 80°C for approximately 90 minutes.

Using the thus prepared membrane spend hybridization with the above-described DNA as a probe. Hybridization can be performed, for example, in accordance with the description in Molecular Cloning, A Laboratory Manual 2nd Edition (1989), Cold Spring Harbor Laboratory Press, etc.

Although for conducting hybridization fit different temperature conditions and reagents, membrane obtained as described above, impregnate and incubated for 1 to 4 hours at 42-65°C prehybridization solution, which is prepared at a ratio of 50 ál - 200 ál 1 cm2the membranes. For example, prehybridization solution may contain 450 - 900 mm NaCl and 45 - 90 mm sodium citrate, may contain sodium dodecyl sulphate (hereinafter called “LTOs”) in a concentration of 0.1-1.0% and may contain denatured nonspecific DNA at a concentration of from 0 to 200 μg/ml and can sometimes contain albumin, fecal and polyvinylpyrrolidone, each at a concentration of 0-0,2%. Then the membrane, for example, impregnated and incubated for 12 to 20 hours at 42-65°C in hybridization solution, which is prepared at a ratio of from 50 to 200 μl of 1 cm2the membranes. Hybridization solution is, for example, a mixture of prehybridization solution, which may contain 450 - 900 mm NaCl and 45 - 90 mm sodium citrate, may contain LTOs in a concentration of 0.1-1.0% and may contain denatured especificas the th DNA in concentrations from 0 to 200 μg/ml and can sometimes contain albumin, ficol and polyvinylpyrrolidone, each at a concentration of 0-0,2%, with a probe obtained by the production method, described above (in the relative number 1,h4pulse/min - 2,h6pulse/min 1 cm2membrane). Then the membrane is removed and washed for 5 to 15 minutes approximately 2-4 times using a wash solution 42-65°C, which contains 15 - 300 mm NaCl, 1.5 to 30 mm sodium citrate and 0.1 to 1.0% - ordinator. Then, after a light rinse 2SSC-solution (300 mm NaCl and 30 mm sodium citrate) the membrane is dried. By detecting the position of the probe on the membrane by placing the membrane autoradiography identify the position of DNA hybridization with the probe on the membrane. Alternatively, prehybridization and hybridization can be performed using a commercially available kit for hybridization, such as hybridization solution contained in the set of DIG-High Prime DNA Labelling and Detection Starter Kit II (Roche). After hybridization, the membrane is washed, for example, twice for 5 minutes at room temperature in 2SSC containing 0.1% LTOs, with subsequent laundering twice for 15 minutes at 65°C in 0,5SSC containing 0.1% ordinator. The provisions of the DNA on the membrane, hybridization with the probe, detects alternately processing the washed membrane with a solution for detecting contained in the specified collection, and det is the titration of probe position on the membrane.

Clones corresponding to the positions of the detected DNA on the membrane, identify the source agar medium, and they can be removed by scratching for selection of clones carrying these DNA.

This DNA (A), obtained as described above can be cloned into a vector in accordance with conventional methods of genetic engineering, are described in Molecular Cloning: A Laboratory Manual 2nd Edition (1989), Cold Spring Harbor Laboratory Press; Current Protocols in Molecular Biology (1987), John Wiley and Sons, Incorporated, etc. as a vector, for example, can be used, specifically, the plasmid vector pUC119 (Takara Shuzo Company), pTVA118N (Takara Shuzo Compani), pBlueScript II (Toyobo Company), pCR2.1-TOPO (Invitrogen Company), pTrc99A (Pharmacia Company), pKK331-1A (Pharmacia Company), etc.

Further, the nucleotide sequence of this DNA (A), obtained in accordance with the description above, can be analyzed using the method that uses dimethoxytrityl described in F. Sanger, S. Nicklen, A.R. Coulson, Proc. Natl. Acad. Sci. USA (1977) 74:5463-5467. To prepare the samples for analysis of the nucleotide sequence may be used commercially available reagent, such as the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Company).

This DNA (A) can also be obtained in the following way. This DNA may be amplified by PCR. PCR can be used as the template the chromosomal DNA or chromosomal DNA library, polucen the Yu, as described above, from microorganisms belonging to Streptomyces, such as Streptomyces phaeochromogenes, Streptomyces testaceus, Streptomyces achromogenes, Streptomyces griseolus, Streptomyces carbophilus, Streptomyces griseofuscus, Streptomyces thermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus, Streptomyces roseorubens, Streptomyces rutgersensis and Streptomyces steffisburgensis, and more specifically, Streptomyces phaeochromogenes IFO12898, Streptomyces testaceus ATCC21469, Streptomyces achromogenes IFO 12735, Streptomyces griseolus ATCC11796, Streptomyces carbophilus SANK62585, Streptomyces griseofuscus IFO 12870t, Streptomyces thermocoerulescens IFO 14273t, Streptomyces nogalater IFO 13445, Streptomyces tsusimaensis IFO 13782, Streptomyces glomerochromogenes IFO 13673t, Streptomyces olivochromogenes IFO 12444, Streptomyces ornatus IFO 13069t, Streptomyces griseus ATCC 10137, Streptomyces griseus IFO 13849T, Streptomyces lanatus IFO 12787T, Streptomyces misawanensis IFO 13855T, Streptomyces pallidus IFO 13434T, Streptomyces roseorubens IFO 13682T, Streptomyces rutgersensis IFO 15875T and Streptomyces steffisburgensis IFO 13446T and the like, or from microorganisms belonging to Saccharopolyspora, such as Saccharopolyspora taberi, more specifically Saccharopolyspora taberi JCM 9383t etc. PCR can also be used oligonucleotide containing, least about 20 nucleotides 5'-end a nucleotide sequence that encodes the amino acid sequence shown in any of SEQ ID NO: 1, 2, 3, 4, 5, 108, 159, 160, 136, 137, 138, 215, 216, 217, 218, 219, 220, 221, 222, 223 or 224, with oligonucleotide containing a nucleotide sequence complementary to at least about 20 nucleotides adjacent to C3'-end or "right" from the 3'-end nucleotide sequence, encoding any of the amino acid sequences given above. PCR can be carried out under the conditions described below. On the 5'-terminal side of the primer used for PCR as described above, may be added to the sequence recognition by the restriction enzyme.

More specifically, for example, DNA containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1, a DNA containing the nucleotide sequence shown in SEQ ID NO:6, or the like can be obtained by PCR using as a template the chromosomal DNA or chromosomal DNA library derived from Streptomyces phaeochromogenes IFO 12898, and using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:51, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:52. Alternatively, DNA containing the nucleotide sequence shown in SEQ ID NO:9 (containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1)may be amplified by PCR using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:51, and the oligonucleotide containing the nucleotide sequence shown is SEQ ID NO:53.

For example, DNA containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2, a DNA containing the nucleotide sequence shown in SEQ ID NO:7, or the like can be obtained by PCR using as a template the chromosomal DNA or chromosomal DNA library derived from Saccharopolyspora taberi JCM 9383t, and using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:61, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:62. Alternatively, DNA containing the nucleotide sequence shown in SEQ ID NO:10 (containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2)may be amplified by PCR using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:61, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:63.

For example, DNA containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:108, a DNA containing the nucleotide sequence shown in SEQ ID NO:109, or the like can be obtained by carrying out PCR using catastematic chromosomal DNA or chromosomal DNA library, derived from Streptomyces achromogenes IFO 12735, and using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:119, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:120. Alternatively, DNA containing the nucleotide sequence shown in SEQ ID NO:110 (containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:108), may be amplified by PCR using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:119, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:121.

For example, DNA containing the nucleotide sequence shown in SEQ ID NO:144 (containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:159), can be obtained by PCR using as a template the chromosomal DNA or chromosomal DNA library derived from Streptomyces nogalater IFO 13445, and using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:165, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:166.

For example, DNA containing Amu is amidou sequence, shown in SEQ ID NO:145 (containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:160), can be obtained by PCR using as a template the chromosomal DNA or chromosomal DNA library derived from Streptomyces tsusimaensis IFO 13782, and using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:171, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:172.

For example, DNA containing the nucleotide sequence shown in SEQ ID NO:146 (containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:136), can be obtained by PCR using as a template the chromosomal DNA or chromosomal DNA library derived from Streptomyces thermocoerulescens IFO 14273t, and using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:177, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:178.

For example, DNA containing the nucleotide sequence shown in SEQ ID NO:147 (containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:137), can be obtained the rst is by PCR using as a template the chromosomal DNA or chromosomal DNA library, derived from Streptomyces glomerochromogenes IFO 13673t, and using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:183, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:184.

For example, DNA containing the nucleotide sequence shown in SEQ ID NO:148 (containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:138), can be obtained by PCR using as a template the chromosomal DNA or chromosomal DNA library derived from Streptomyces olivochromogenes IFO 12444, and using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:184, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:185.

When used as a matrix library DNA, in which chromosomal DNA is introduced, for example, in the vector, the DNA may be either amplified by PCR using as primers the oligonucleotide containing a nucleotide sequence selected from the nucleotide sequence that encodes any of the amino acid sequence shown in SEQ ID NO:1, 2, 3, 4, 5, 108, 159, 160, 136, 137 or 138 (for example, the oligonucleotide containing nucleotide PEFC is the sequence of at least about 20 nucleotides 5'-terminal side a nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO:1), and the oligonucleotide of at least about 20 nucleotides, containing a nucleotide sequence complementary to the nucleotide sequence adjacent to the site of DNA integration vector used to construct the library. On the side of the 5'-end primer used for PCR as described above, may be added to the sequence recognition by the restriction enzyme.

As conditions for the above-described PCR, may be mentioned the condition of maintaining 97°C for 2 minutes, then repeat 10 cycles, where each cycle includes maintaining 97°C for 15 seconds, then 65°C for 30 seconds, and then 72°C for 2 minutes; then 15 cycles, where each cycle includes maintaining 97°C for 15 seconds, and 68°C for 30 seconds, and then 72°C for 2 minutes (with the addition of 20 seconds for each cycle alternately); and then maintaining 72°C for 7 minutes. This PCR can use the reaction solution 50 µl containing 50 ng of chromosomal DNA, containing 300 nm of each of the 2 primers in such pairs as described above, containing 5.0 μl of dNTP mixture (a mixture of 2.0 mm each of the four types of dNTP), containing 5.0 μl 10Expand HF buffer(containing MgCl 2Roche Molecular Biochemicals Company) and containing a 0.75 μl of a mixture of enzymes Expand HiFi (Roche Molecular Biochemicals Company).

Alternatively, it may be referred to the condition of maintaining 97°C for 2 minutes, then repeating 30 cycles, where each cycle includes maintaining 97°C for 15 seconds, then 60°C for 30 seconds, and then 72°C for 90 seconds; and then maintaining the reaction solution at 72°C for 4 minutes. This PCR can use the reaction solution 50 μl containing 250 ng of chromosomal DNA containing 200 nm of each of the 2 primers in such pairs as described above, containing 5.0 μl dNTP-mix (a mixture of 2.5 mm each of the 4 types of dNTP), 5 μl 10ExTaq buffer (containing MgCl2, Takara Shuzo Company) and containing 0.5 μl of ExTaq polymerase (Takara Shuzo Company).

Alternatively, for example, oligonucleotides can be designed and prepared for use as primers based on the nucleotide sequence of the area in respect of which the sequence identity is particularly high in the nucleotide sequence shown in SEQ ID NO:6, 7, 8 or 109. This DNA (A) can also be obtained by carrying out PCR using the oligonucleotides as primers and the chromosomal DNA or chromosomal DNA library. Chromosomal DNA or chromosomal DNA library can be obtained, as described above, microorganis is s, belonging to Streptomyces, such as Streptomyces phaeochromogenes, Streptomyces testaceus, Streptomyces achromogenes, Streptomyces griseolus, Streptomyces carbophilus, Streptomyces griseofuscus, Streptomyces thermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus, Streptomyces roseorubens, Streptomyces rutgersensis and Streptomyces steffisburgensis, and more specifically, Streptomyces phaeochromogenes IFO12898, Streptomyces testaceus ATCC21469, Streptomyces achromogenes IFO 12735, Streptomyces griseolus ATCC11796, Streptomyces carbophilus SANK62585, Streptomyces griseofuscus IFO 12870t, Streptomyces thermocoerulescens IFO 14273t, Streptomyces nogalater IFO 13445, Streptomyces tsusimaensis IFO 13782, Streptomyces glomerochromogenes IFO 13673t, Streptomyces olivochromogenes IFO 12444, Streptomyces ornatus IFO 13069t, Streptomyces griseus ATCC 10137, Streptomyces griseus IFO 13849T, Streptomyces lanatus IFO 12787T, Streptomyces misawanensis IFO 13855T, Streptomyces pallidus IFO 13434T, Streptomyces roseorubens IFO 13682T, Streptomyces rutgersensis IFO 15875T and Streptomyces steffisburgensis IFO 13446T and the like, or from microorganisms belonging to Saccharopolyspora, such as Saccharopolyspora taberi, more specifically Saccharopolyspora taberi JCM 9383t, etc. as a "district in respect of which the sequence identity is particularly high in the nucleotide the sequence shown in SEQ ID NO:6, 7, 8 or 109", is mentioned, for example, the area corresponding to the region consisting of nucleotides 290-315, 458-485, 496-525 or 1046-1073 in the nucleotide sequence shown in SEQ ID NO:6. As primers, designed on the basis of such areas of this nucleotide sequence, can be mentioned, which, for example, primer containing the nucleotide sequence shown in any of SEQ ID NO:124-129.

SEQ ID NO:124 based on the nucleotide sequence of the region corresponding to the region consisting of the above-mentioned nucleotides 290-315;

SEQ ID NO:125 based on the nucleotide sequence of the region corresponding to the region consisting of the above-mentioned nucleotides 458-485;

SEQ ID NO:126 based on the nucleotide sequence of the region corresponding to the region consisting of the above-mentioned nucleotides 458-485;

SEQ ID NO:127 based on the nucleotide sequence of the region corresponding to the region consisting of the above-mentioned nucleotides 496-525;

SEQ ID NO:128 based on the nucleotide sequence of the region corresponding to the region consisting of the above-mentioned nucleotides 496-525; and

SEQ ID NO:129 based on the nucleotide sequence of the region corresponding to the region consisting of the above-mentioned nucleotides 1046-1073.

For example, DNA from approximately 800 BP amplified using as primers a pair of the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:124, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:129. DNA from approximately 600 BP amplified using as primers a pair of the oligonucleotide, with the holding nucleotide sequence, shown in SEQ ID NO:125, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:129. DNA from approximately 600 BP amplified using as primers a pair of the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:126, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:129. DNA from approximately 580 BP amplified using as primers a pair of the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:127, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:129. In addition, DNA from approximately 580 BP amplified using as primers a pair of the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:128, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:129.

As conditions for PCR, in particular, for example, may be mentioned the condition of the maintenance of 95°C for 1 minute; repeat for 30 cycles, where a cycle includes the maintenance of 94°C for 15 seconds, then 65°C for 30 seconds, and then 72°C for 1 minute; and then maintaining 72°C for 5 minutes. Can be used the reaction solution 25 μl containing 10 ng of chromosomal DNA containing 200 nm is of each of the 2 primers, containing 0.5 μl of dNTP mixture (a mixture of 10 mm each of the 4 types of dNTP)containing 5 ál of 5x reaction buffer for GC genomic PCR, containing 5 µl of 5 M GC-melt and containing 0.5 μl of the mixture of genomic polymerase Advantage-GC genomic polymerase mix (Clontech Company).

By extracting DNA, amplified, as described above, can be obtained DNA containing a partial nucleotide sequence of this DNA (A). Then, based on the nucleotide sequence, which is obtained DNA containing a partial nucleotide sequence of this DNA (A), design and get oligonucleotide containing a partial nucleotide sequence of at least about 20 nucleotides of the specified nucleotide sequence, or an oligonucleotide containing a nucleotide sequence complementary to a partial nucleotide sequence of at least about 20 nucleotides of the specified nucleotide sequence. DNA containing a partial nucleotide sequence of this DNA (A), elongated right from the 3'-end or left from the 5'-end of DNA containing a partial nucleotide sequence of this DNA (A), obtained as described above, can be obtained by carrying out PCR. This PCR can be used as a primer pair of the oligonucleotide obtained as described above on the basis of the nucleotide sequence of the DNA, containing a partial nucleotide sequence of this DNA (A), and the oligonucleotide of at least about 20 nucleotides, containing the nucleotide sequence of the area adjacent to the site of DNA integration vector used to construct the above library, or the oligonucleotide of at least about 20 BP, containing a nucleotide sequence complementary to such a nucleotide sequence of this region. This PCR can, for example, be used as a matrix library of chromosomal DNA derived from microorganisms that are capable of converting the compound (II) into the compound (III)as described above. The connection of such DNA containing a partial nucleotide sequence of this DNA (A), you can get this DNA (A). In this method of obtaining can be used commercially available kit, such as the Universal Genome Walker (Clontech Company). Alternatively, this DNA (A) can be obtained by conducting PCR by primers based on the full nucleotide sequence of this DNA (A), obtained by attaching a partial nucleotide sequence of this DNA (A), as described above, by using such primers, using as the template the chromosomal DNA library as described above.

For example the EP, DNA containing a nucleotide sequence consisting of nucleotides 316-1048 SEQ ID NO:139 (partial nucleotide sequence of the nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO:159), can be obtained by PCR using as a template the chromosomal DNA or chromosomal DNA library derived from Streptomyces nogalater IFO 13445, and using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:124, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:129. DNA containing the nucleotide sequence, extended to the right from its 3'-end or the left of the 5'-end, receive in accordance with the above description based on the nucleotide sequence of the obtained DNA. DNA containing the nucleotide sequence shown in SEQ ID NO:144 (containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:159, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:149), can be obtained by attaching the DNA.

For example, DNA containing a nucleotide sequence consisting of nucleotides 364-1096 SEQ ID NO:140 (partial nucleotide posledovatelno and nucleotide sequence, the coding amino acid sequence shown in SEQ ID NO:160), can be obtained by PCR using as a template the chromosomal DNA or chromosomal DNA library derived from Streptomyces tsusimaensis IFO 13782, and using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:124, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:129. DNA containing the nucleotide sequence, extended to the right from its 3'-end or the left of the 5'-end, receive in accordance with the above description based on the nucleotide sequence of the obtained DNA. DNA containing the nucleotide sequence shown in SEQ ID NO:145 (containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:150, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:160), can be obtained by attaching the DNA.

For example, DNA containing a nucleotide sequence consisting of nucleotides 295-1027 SEQ ID NO:141 (partial nucleotide sequence of the nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO:136), can be obtained by PCR using as a researcher is as a matrix of chromosomal DNA or chromosomal DNA library, derived from Streptomyces thermocoerulensis IFO 14273t, and using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:124, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:129. DNA containing the nucleotide sequence, extended to the right from its 3'-end or the left of the 5'-end, receive in accordance with the above description based on the nucleotide sequence of the obtained DNA. DNA containing the nucleotide sequence shown in SEQ ID NO:146 (containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:136, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:151), can be obtained by attaching the DNA.

For example, DNA containing a nucleotide sequence consisting of nucleotides 316-1048 SEQ ID NO:142 (partial nucleotide sequence of the nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO:137), can be obtained by PCR using as a template the chromosomal DNA or chromosomal DNA library derived from Streptomyces glomerochromogenes IFO 13673t, and using as primers the oligonucleotide containing nucleotide pic is egovernance, shown in SEQ ID NO:124, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:129. DNA containing the nucleotide sequence, extended to the right from its 3'-end or the left of the 5'-end, receive in accordance with the above description based on the nucleotide sequence of the obtained DNA. DNA containing the nucleotide sequence shown in SEQ ID NO:147 (containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:137, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:152), can be obtained by attaching the DNA.

For example, DNA containing a nucleotide sequence consisting of nucleotides 316-1048 SEQ ID NO:143 (partial nucleotide sequence of the nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO:138), can be obtained by PCR using as a template the chromosomal DNA or chromosomal DNA library derived from Streptomyces olivochromogenes IFO 12444, and using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:124, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:129. DNA containing well is leonidou sequence, extended to the right from its 3'-end or the left of the 5'-end, receive in accordance with the above description based on the nucleotide sequence of the obtained DNA. DNA containing the nucleotide sequence shown in SEQ ID NO:148 (containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:138, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:153), can be obtained by attaching the DNA.

For example, DNA containing a nucleotide sequence consisting of nucleotides 289-1015 SEQ ID NO:232 (partial nucleotide sequence of the nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO:222), can be obtained by PCR using as a template the chromosomal DNA or chromosomal DNA library derived from Streptomyces roseorubens IFO 13682t, and using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:124, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:129. DNA containing the nucleotide sequence, extended to the right from its 3'-end or the left of the 5'-end, receive in accordance with the description above on the basis of the nucleotide is the first sequence of the obtained DNA. DNA containing the nucleotide sequence shown in SEQ ID NO:242 (containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:232, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:252), can be obtained by attaching the DNA.

For example, DNA containing a nucleotide sequence consisting of nucleotides 289-1015 SEQ ID NO:234 (partial nucleotide sequence of the nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO:224), can be obtained by PCR using as a template the chromosomal DNA or chromosomal DNA library derived from Streptomyces steffisburgensis IFO 13446t, and using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:124, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:129. DNA containing the nucleotide sequence, extended to the right from its 3'-end or the left of the 5'-end, receive in accordance with the above description based on the nucleotide sequence of the obtained DNA. DNA containing the nucleotide sequence shown in SEQ ID NO:244 (containing the nucleotide sequence, coderush the Yu amino acid sequence, shown in SEQ ID NO:234, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:254), can be obtained by attaching the DNA.

This DNA (A), obtained using PCR, as described above, may be cloned into a vector in accordance with conventional methods of genetic engineering, are described in Molecular Cloning: A Laboratory Manual 2nd Edition (1989), Cold Spring Harbor Laboratory Press; Current Protocols in Molecular Biology (1987), John Wiley and Sons, Incorporated, etc. for Example, specifically, cloning can be carried out using plasmid vectors such as pBlueScript II Stratagene Company, or plasmid vector contained in the kit TA cloning Invitrogen Company.

Next, this DNA (A) can be obtained, for example, as described below. First construct the nucleotide sequence. This nucleotide sequence encodes the amino acid sequence of the protein encoded by this DNA (A). This nucleotide sequence has a GC content of maximum 60% minimum 40%, preferably a maximum of 55% and a minimum of 45%. The use of codons in the nucleotide sequence that encodes the amino acid sequence of the above protein is in the range of plus or minus 4% usage codons in the genes of the species of the host cell in which to enter this DNA (A). Through polucheniya, having constructed a nucleotide sequence in accordance with generally accepted methods of genetic engineering can be obtained this DNA (A).

For example, can be constructed as described below the nucleotide sequence encoding the amino acid sequence (SEQ ID NO:1) protein of this invention (A1) and having a GC content of a maximum of 55% and a minimum of 45%where the use of codons in this nucleotide sequence that encodes the amino acid sequence of the above protein is in the range of plus or minus 4% usage codons in genes from soybean. First, for example, compare the use of codons (table 22 and table 23) in the nucleotide sequence (SEQ ID NO:6)that encodes the amino acid sequence of the protein (A1) of the present invention, which can be obtained from Streptomyces phaeochromogenes IFO12898, and the use of codons in soybean (table 24 and table 25). On the basis of the result of this comparison adds nucleotide substitutions in the nucleotide sequence shown in SEQ ID NO:6, so that the GC content is a maximum of 55% and a minimum of 45% and the use of codons is in the range of plus or minus 4% usage codons soybeans. As such nucleotide substitutions select nucleotide substitution, which does not lead to the replacement of amino acids. E.g. the measures the use of the CTG codon encoding leucine is 1,22% of the genes in soybean and to 7.09% in the nucleotide sequence shown in SEQ ID NO:6. As a result, for example, each of the codons CTG, starting from nucleotide 106, 163, 181, 226, 289, 292, 544, 1111 and 1210 of the nucleotide sequence shown in SEQ ID NO:6 is replaced by codons CTT; each of the codons CTG, starting at nucleotide 211, 547 and 1084, replace the codons of a HUNDRED; the codon CTG, starting at nucleotide 334, replace the codon TTA; each of the codons CTG, starting from nucleotide 664, 718, 733, 772, 835, 1120 and 1141, replace the codon TTG; and the codon CTG, starting from nucleotide 787, replace the codon TTA. In SEQ ID NO:214 shows a nucleotide sequence designed using codons which are shown in table 26 and table 27. In the nucleotide sequence shown in SEQ ID NO:214, for example, the use of the CTG codon encoding leucine, is 1,71% and is within the range of plus or minus 4% usage codons (1,22%) of soybean. DNA containing the nucleotide sequence shown in SEQ ID NO:214, can be obtained by introduction of nucleotide substitutions in the DNA having the nucleotide sequence shown in SEQ ID NO:6, in accordance with methods site-directed mutagenesis described, e.g., in Sambrook, J., Fritsch, E.F. and Maniatis, T.; Molecular Cloning, 2nd Edition, Cold Spring Harbor Press. Alternatively,DNA, having the nucleotide sequence shown in SEQ ID NO:214, can be obtained by the method of synthesis of DNA using PCR as described in example 46 below.

Similarly, the nucleotide sequence shown in SEQ ID NO:368, is an example of constructing a nucleotide sequence that encodes the amino acid sequence (SEQ ID NO:222) protein (A23) this invention and having a GC content of a maximum of 55% and a minimum of 45%where the use of codons in the nucleotide sequence that encodes the amino acid sequence of the above protein is in the range of plus or minus 4% usage codons for genes from soybean. Further, the nucleotide sequence shown in SEQ ID NO:393, is an example of constructing a nucleotide sequence that encodes the amino acid sequence (SEQ ID NO:224) protein (A25) this invention and having a GC content of a maximum of 55% and a minimum of 45%where the use of codons in the nucleotide sequence that encodes the amino acid sequence of the above protein is in the range of plus or minus 4% usage codons for genes from soybean.

This DNA (A), thus obtained, may be cloned into a vector in accordance with conventional methods of genetic engineering, as described in Sambrook, J., Fritsch, E.F. and Maniatis, T.; “olecular Cloning, 2nd Edition (1989) Cold Spring Harbor Press; and “Current Protocols in Molecular Biology” (1987), John Wiley and Sons, Incorporated, etc. as a vector, for example, can be used, specifically, the plasmid vector pUC 119 (Takara Shuzo Company), pTVA 118N (Takara Shuzo Compani), pBlueScript II (Toyobo Company), pCR2.1-TOPO (Invitrogen Company), pTrc99A (Pharmacia Company), pKK331-1A (Pharmacia Company), etc.

Further, the nucleotide sequence of this DNA (A), thus obtained, can be analyzed using the method that uses dimethoxytrityl described in F. Sanger, S. Nicklen, A.R. Coulson, Proceedings of National Academy of Science USA (1977) 74:5463-5467.

The ability to metabolize the herbicide connection PPO-inhibitor type formula (I) of this protein (A), which is encoded by the DNA of (A)obtained by the method described above can be confirmed by the ability to convert compound (II) into the compound (III) as a marker in the manner described below. First, as described below, the specified DNA inserted into the vector so that it is attached to the right of the promoter that can function in this cell the owner and this vector is introduced into a cell of the host to receive transformant. Then the culture of transformant or extract, obtained as a result of the destruction of this culture, is subjected to the interaction with the compound (II) in the presence of a system of electron transport containing electron donor, such as the coenzyme NADPH. Formed as a result of the reaction products analyzed for detection of the compound (III). In this way can be detected in the transformant capable of metabolizing the compound (II) to produce the compound (III), and may be determined that such transformant carries this DNA (S)encoding a protein having such ability. More specifically, for example, prepare 30 ál of reaction solution consisting of 0.1 M potassium phosphate buffer (pH 7.0)containing culture or extract above transformant, electron donor, such as β-NADPH, at a final concentration of approximately 2 mm, ferredoxin obtained from spinach, in a final concentration of approximately 2 mg/ml, ferredoxins at a final concentration of approximately 0.1 u/ml and 3 ppm of compound (II)labeled with a radioactive isotope. This reaction solution was incubated at approximately 30 - 40°C for 10 minutes to 1 hour. After this incubation, add 3 ál 2 N. HCl and 90 μl of ethyl acetate, stirred and centrifuged at 8000 g to obtain the supernatant. After drying of the supernatant in a vacuum the residue is dissolved in ethyl acetate and the resulting solution show on the TLC plate of silica gel. The TLC plate analyze using autoradiography. Through the identification of spots corresponding to compound (III), radiolabelled can be confirmed by the ability to turn the compound (II) into the compound (III).

On the IC DNA encodes a protein capable of converting the compound (II) into the compound (III), or a microorganism having such DNA may be further carried out by hybridization or PCR as described above using the DNA of (a) of this invention or polynucleotide containing a partial nucleotide sequence of the specified DNA or nucleotide sequence complementary to this partial nucleotide sequence.

In particular, carried out, for example, hybridization, as described above, and identify DNA from which hybridized probe. Hybridization is carried out with the use of DNA (a) of this invention or polynucleotide containing a partial nucleotide sequence of DNA (a) of this invention, or a nucleotide sequence complementary to this partial nucleotide sequence as a probe and genomic DNA derived from natural organisms, such as microorganisms belonging to Streptomyces, such as Streptomyces phaeochromogenes, Streptomyces testaceus, Streptomyces achromogenes, Streptomyces griseolus, Streptomyces carbophilus, Streptomyces griseofuscus, Streptomyces thermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus, Streptomyces roseorubens and Streptomyces rutgersensis and Streptomyces steffisburgensis; microorganisms belonging to Saccharopolyspora, such as Saccharopolyspora taberi; and so on as typical examples of the DNA which can be used as a probe, can be mentioned DNA containing the full nucleotide sequence shown in any of SEQ ID NO:6, 7, 8, 109, 139, 140, 141, 142, 143, 225, 226, 227, 228, 229, 230, 231, 232, 233 or 234; DNA containing a nucleotide sequence consisting of nucleotides 57-730 the nucleotide sequence shown in SEQ ID NO:6; a DNA containing a nucleotide sequence consisting of nucleotides 21-691 the nucleotide sequence shown in SEQ ID NO:8; and so on

Alternatively, PCR may be performed, as described above, and can be detected amplificatory DNA. PCR uses polynucleotide containing a partial nucleotide sequence of DNA (a) of this invention, or a nucleotide sequence complementary to this partial nucleotide sequence. PCR uses as template genomic DNA, obtained from natural microorganism, for example, from microorganisms belonging to Streptomyces, such as Streptomyces phaeochromogenes, Streptomyces testaceus, Streptomyces achromogenes, Streptomyces griseolus, Streptomyces carbophilus, Streptomyces griseofuscus, Streptomyces thermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus, Streptomyces roseorubens, Streptomyces rutgersensis and Streptomyces steffisburgensis; microorganisms belonging to Saccharopolyspora, for example Saccharopolyspoa taberi; etc. as primers can be mentioned primers that were designed based on the nucleotide sequence of area identity sequence in respect of which is particularly high in the nucleotide sequence shown in SEQ ID NO:6, 7, 8 or 109, as described above. As more specific examples of these primers are referred to a pair of primer containing the nucleotide sequence shown in any of SEQ ID nos:124 to 128, and a primer containing the nucleotide sequence shown in SEQ ID NO:129.

Detektirovaniya thus DNA isolated. When selected DNA does not contain the full nucleotide sequence of this DNA (A), this DNA is used to transform the DNA corresponding to a full-sized nucleotide sequence, as described above. The resulting DNA is injected into the cell host to obtain transformant. The ability to turn the compound (II) into the compound (III) of the proteins encoded by DNA that is introduced into the transformant can be evaluated using cultures obtained transformant and measures the ability of transformation of compound (II) into the compound (III) as described above.

For the expression of this DNA in the cell is the owner of this DNA (A) introducing into the cell-master in position, allowing its expression in the specified glue the ke. Under the introduction of this DNA (A) in position, allowing the expression" mean that this DNA (A) introducing into the cell host such that it is placed in position adjacent to the nucleotide sequence controlling the transcription and translation of this nucleotide sequence (i.e., for example, a nucleotide sequence that controls the production of this protein and RNA that encodes the protein (A)).

For the introduction of this DNA in the cell host such that it is placed in position, allowing its expression, for example, DNA in which the present DNA (a) and the promoter, functional in this cell the owner, are functionally related, is injected into the cell host. The term “functionally linked” means here that the state in which this DNA (A) is associated with the promoter in such a way that it is expressed under the control of this promoter in the introduction of this DNA in a cell of the host.

When a host cell is a cell of a microorganism, as a functional promoter can be, for example, the promoter of the lactose operon of E. coli, the promoter of the tryptophan operon of E. coli, the promoter of T7 phage or artificial promoters that are functional in E. coli, such as the promoter tac or the trc promoter and so Forth, can be used by the promoter, the source is present to the left of this DNA (is) in the chromosome of the microorganism, belonging to Streptomyces or Saccharopolyspora.

When a host cell is a plant cell as a functional promoter can be mentioned, for example, derived from T-DNA of constitutive promoters such as the promoter of the gene nepalensis and the promoter of the gene octopunctata; derived from viruses of plant promoters, such as promoters 19S and 35S of cauliflower mosaic virus; inducible promoters such as the promoter of the gene of phenylalaninamide, the promoter of the gene malcontents and the promoter of the gene associated with the pathogenesis of protein; the promoter of the plants described in the Japanese publication has not yet passed the examination of the patent application number 2000-166577. Next, the terminator functional in plant cells, may be associated with DNA, which is functionally linked to a promoter functional in a plant cell, and this DNA (A). In this case, is usually preferred that the terminator was connected right from this DNA (A). As a functional terminator mentioned, for example, derived from T-DNA constitutive terminators, such as the terminator gene napadisylate (NOS); derived from plant viruses terminators, such as the termination of the virus in onion (Allium) GV1 or GV2; terminator plants described in the Japanese publication has not yet passed the examination of the patent application number 2000-166577, etc.

With the introduction of Yes is Noah DNA (A) thus, this DNA is placed in a position that allows her expression, for example, can be used with DNA having a nucleotide sequence encoding a signal (transfer) transit in intracellular organelle attached "to the left" from this DNA (A) so that the reading frames are in the same reading frame. The term " attached “so that the reading frames are in the same reading frame”, we mean that the reading frame sequence signal (transfer) transit in intracellular organelle and reading frame of the DNA of (A) are connected to form a single continuous reading frame. As a transit signal sequence that provides transition and localization of the protein in the intracellular organelle in the plant cell, may be mentioned, for example, transit signal sequence derived from the cytoplasmic precursor protein, localized in the chloroplast of the plant, as described in U.S. Patent number 5717084, chimeric sequences, formed from various transit signal sequences described in U.S. Patent number RE36449. More specifically, mentioned chloroplast transit peptide derived from the small subunit of ribulose-1,5-bestofferbuy soy, which can b shall be obtained in accordance with the method, described in example 15 below.

Usually, this DNA (A), this DNA (A), is attached to the DNA having the nucleotide sequence encoding the signal (transfer) transit in intracellular organelle, as described above, or DNA, in which such DNA is functionally linked to a promoter functional in the cell host, can be embedded in a vector, applicable in the cell host, and this vector is introduced into a cell of the host. When applying the vector already has a promoter functional in the cell host, this DNA (A) can be embedded right from the promoter present in the vector, so that the specified promoter and the DNA (A) can be functionally linked.

As a vector, in particular, when using E. coli as the host cell can be, for example, the pUC 119 (Takara Shuzo Company), pTVA 118N (Takara Shuzo Company), pBlueScript II (Stratagene Company), pCR2.1-TOPO (Invitrogen), pTrc99A (Amersham Pharmacia Biotech Company), pKK331-1A (Amersham Pharmacia Biotech Company), 11d (Novagen), etc. by using a vector containing a selective marker (e.g., genes that give resistance to antibiotics, such as the gene for resistance to kanamycin, the gene of resistance to neomycin, and the like), the advantage is that the transformant, which introduced this DNA can be selected by phenotype selective marker as an indicator.

As a way of introducing this D Is To (A) or vector, contains the DNA (A), the cell host may be mentioned the method described in Shin Seikagaku Zikken Kouza (Nippon-Seikagaku-Kai eds., Tokyo Called Dozin), Vol. 17, Biseibutu-Zikken-Hou, when the host-cell is a microorganism, such as E. coli, Bacillus subtilis, Bacillus brevis, Pseudomonas sp., Zymomonas sp., lactic acid bacteria, acetic acid bacteria, Staphylococcus sp., Streptomyces sp., Saccharopolyspora sp. or yeast, such as Saccharomyces cerevisiae, Schizosaccharomyces ponmbe, fungus, such as Aspergillus, etc. Alternative, for example, can be used a calcium chloride method described in Sambrook, J., Fritsch, E.F. and Maniatis, T.; “Molecular Cloning, 2nd Edition”, Cold Spring Harbor Press (Molecular Biology, John Wiley and Sons, N.Y. (1989); “Current Protocols in Molecular Biology” (1987), John Wiley and Sons, Incorporated, or method of electroporation, as described in “Methods in Electroporation: Gene Pulser/E. coli Pulser System”, Bio-Rad Laboratories (1993).

The transformant that was introduced this DNA (A) or the vector containing this DNA (a)may be, for example, selected on phenotype selective marker contained in the vector in which the DNA of (A) was built, as described above, as an indicator. Further, the content transformance this DNA (A) or vector containing this DNA (A)can be confirmed by obtaining DNA from this transformant and then conducting with the obtained DNA analysis methods of genetic engineering are described, for example, in “Molecular Cloning, 2nd Edition”, Cold Spring Harbor Press (Molecular Biology, John Wiley and Sons, N.Y. (1989) (such as supporting the giving sites restricts, DNA sequencing, hybridization to Southern, PCR and the like).

When the host-cell is a plant cell, can be mentioned, for example, dicotyledonous plants, such as tobacco, cotton, rapeseed, sugar beet and Arabidopsis, canola, flax, sunflower, potato, alfalfa, lettuce, banana, soybean, pea, plant of the legume family, pine, poplar, Apple, grape, orange, lemon, other citrus fruits, almonds, hazelnuts, other nuts; monocots, such as maize, rice, wheat, barley, rye, oats, sorghum, sugar cane and turf; etc. as cells that enter this DNA (A)can be referred to the tissue of the plant, the whole plant, cultivated cells, seeds, etc.

As a way of introducing this DNA (A) or vector containing this DNA (A), in the cell-master mentioned methods such as Agrobacterium infection (publication of the examined application to the Japan patent No. 2-58917 and publication has not yet passed the examination, the Japan patent No. 60-70080), electroporation into protoplasts (publication not yet passed the examination, the Japan patent No. 60-251887 and publication has not yet passed the examination, the Japan patent No. 5-68575) or the way of the shotgun (or rifle) for particles (publication not yet passed the examination of the patent application Japan No. 5-508316 and publication has not yet been vetted by the expertise of the application on p the tent Japan No. 63-258525).

In such cases, for example, the transformant that was introduced this DNA can be selected based on the phenotype of breeding marker as an indicator of the introduction into a plant cell simultaneously with the vector containing this DNA (A), the selective marker gene selected from GigabitEthernet, neomycinphosphotransferase gene and gene chloramphenicolchloramphenicol. Gene selective marker and the DNA (A) can be embedded in the same vector and introduced into a cell of the host. A vector containing the gene selective marker, and a vector containing this DNA (A)can also be entered at the same time. The transformant that was introduced this DNA (A)can also be selected by culturing in a medium containing herbicide connection PPO-inhibitor type formula (I), and clone selection, are able to multiply in this environment. Does this this transformant DNA (A)can be confirmed by obtaining DNA from transformant and then conducting with the obtained DNA analysis methods of genetic engineering are described, for example, in “Molecular Cloning, 2nd Edition”, Cold Spring Harbor Press (Molecular Biology, John Wiley and Sons, N.Y. (1989) (such as confirmation of sites restricts, DNA sequencing, hybridization to Southern, PCR and the like). This DNA (A)entered in cell host may be stored in locations in the cell, other than DNA, the soda is jamaca in the kernel, being insertional in the DNA contained in the intracellular organelles such as the chloroplast.

From the transformed plant cells obtained in this way can be obtained transgenic plant, which was introduced this DNA (A), the regeneration of whole plants according to the method of cultivation of plant cells, described in Shokubutu-Idenshi-Sosa-Manual: Transgenic-Shokubutu-No-Tukurikata (Uchimiya, Kodansha-Scientific, 1990), pp. 27-55. In addition, the desired type of plants that entered this DNA (A)can be obtained by crossing the type of plant from the transgenic plant, which introduced this DNA (A), so that this DNA (A) is introduced into the chromosome of a desired type of plant.

In particular, for example, rice or Arabidopsis, which has put them in this DNA (a) and expressing the present protein (A)can be obtained according to the method described in Model-Shokubutu-No-Jikken-Protocol: Ine, Shiroinunazuna-Hen (Supervisors: Koh SHIMAMOTO and Kiyotaka OKADA, Shujun-sha, 1996), Fourth chapter. In addition, can be obtained from soy, has put it this DNA (a) and expressing the present protein (A), an introduction to somatic embryo of soybean using guns for particles in accordance with the method described in the publication has not yet been vetted by the expertise of the patent application of Japan No. 3-291501. Similarly, corn, having entered into it this DNA (a) and expressing the present protein (A)can be obtained by introducing in ematichesky the germ of corn using guns for particles in accordance with the method, described Fromm, M.E., et al., Bio/Technology, 8; p 838 (1990). Wheat, having entered into it this DNA (a) and expressing the present protein (A)can be obtained by introducing this gene into sterile cultured immature scutellum wheat using guns for particles in accordance with generally accepted method described Takumi et al., Journal of Breeding Society (1995), 44; Extra Vol. 1, p 57. Similarly, barley, having entered into it this DNA (a) and expressing the present protein (A)can be obtained by the introduction of sterile cultured immature scutellum barley using guns for particles in accordance with generally accepted method described Hagio, et al., Journal of Breeding Society (1995), 44; Extra Vol. 1, p 67.

The transformant having entered into it this DNA (a) and expressing the present protein (A), can reduce the damage of the plant compound (I) by conversion of the specified herbicide connection in the connection less herbicide activity in cells. In particular, for example, the distribution of the microorganism expressing the present protein (A), the area of cultivation of the desired cultivated plants before sowing of the seeds of this plant herbicide compound remaining in the soil, can be metabolized and damage desirable plants may be reduced. In addition, as a result of obtaining the desired varieties, exp is esteroideo this protein (A), the ability to metabolize the herbicide connection PPO-inhibitor type formulas (I) to compounds of lower activity is transferred to the specified plant. As a result, damage to plants this herbicide compound in this plant is reduced and given stability to the specified connection.

This protein may be obtained, for example, by culturing cells containing this DNA (A). As such cells can be mentioned microorganism expressing this DNA (A) and having the ability to produce the protein (S), such as a strain of microorganism isolated from a natural source that contains the DNA (A), mutant strains derived from this natural strain processing agents or ultraviolet rays or the like, More specifically, for example, can be mentioned microorganisms belonging to Streptomyces, such as Streptomyces phaeochromogenes IFO12898, Streptomyces testaceus ATCC21469, Streptomyces achromogenes IFO 12735, Streptomyces griseolus ATCC11796, Streptomyces carbophilus SANK62585, Streptomyces griseofuscus IFO 12870t, Streptomyces thermocoerulescens IFO 14273t, Streptomyces nogalater IFO 13445, Streptomyces tsusimaensis IFO 13782, Streptomyces glomerochromogenes IFO 13673t, Streptomyces olivochromogenes IFO 12444, Streptomyces ornatus IFO 13069t, Streptomyces griseus ATCC 10137, Streptomyces griseus IFO 13849T, Streptomyces lanatus IFO 12787T, Streptomyces misawanensis IFO 13855T, Streptomyces pallidus IFO 13434T, Streptomyces roseorubens IFO 13682T, Streptomyces rutgersensis IFO 15875T and Streptomyces steffisburgensis IFO 13446T etc., or microorganisms belonging to Saccharpolyspora, such as Saccharopolyspora taberi JCM 9383t and so Forth, may be mentioned the transformant that was introduced this DNA (A) or the vector containing this DNA (A). Specifically, for example, is mentioned transformant in which this DNA (A), functionally associated with the tac promoter, trc promoter, the lac promoter or the promoter of phage T7, introduced in E. coli. As more specific examples mentioned E. coli JM109/pKSN657, E. coli JM109/pKSN657F, E. coli JM109/pKSN923, E. coli JM109/pKSN923F, E. coli JM109/pKSN11796, E. coli JM109/pKSN11796F, E. coli JM109/pKSN671, E. coli JM109/pKSN671F, E. coli JM109/pKSNSCA, E. coli JM109/pKSN646, E. coli JM109/pKSN646F, E. coli JM109/pKSN849AF, E. coli JM109/pKSN1618F, E. coli JM109/pKSN474F, E. coli JM109/pKSN1491AF, E.coli JMl09/pKSN1555AF, E. coli JM109/pKSN1584F, E. coli JM109/pKSN1609F, etc. described in the examples below.

As the medium for culturing the aforementioned microorganisms containing this DNA (A), may be used any of the media, commonly used for culturing a microorganism which contains sources of carbon and nitrogen sources, organic and inorganic salts as needed. May be added a compound which is a precursor of heme, such as aminolevulinic acid.

As a carbon source are mentioned, for example, saccharides such as glucose, fructose, sucrose and dextrin; polyalcohols, xylytol, such as glycerol and sorbitol; and organic acids such as fumaric acid, citric acid and Pirovano is if acid; etc. the Number of carbon sources listed above, which must be added to the environment, usually equal to approximately 0.1% (wt./about.) - 10% (wt./about.) in the calculation of the total number of this environment.

As the nitrogen source are mentioned, for example, ammonium salts of inorganic acids such as ammonium chloride, ammonium sulfate and ammonium phosphate; ammonium salts of organic acids such as ammonium fumarate and ammonium citrate; organic nitrogen sources such as meat extract, yeast extract, malt extract powder, soy, steeping water corn powder, cotton seeds, dried yeast, casein hydrolysate; and amino acids. Among the above nitrogen sources most frequently used can be ammonium salts of organic acids, organic sources of nitrogen and amino acids as sources of carbon. The sources of nitrogen that should be added to the environment, usually equal to approximately 0.1% (wt./about.) - 10% (wt./about.) in the calculation of the total number of this environment.

As the inorganic salts are mentioned, for example, phosphates such as potassium phosphate, Dikili-phosphate, sodium phosphate, disodium phosphate; chlorides such as potassium chloride, sodium chloride, uranyl cobalt chloride; sulfates such as magnesium sulfate heptahydrate ferric sulfate, heptahydrate is of Ulfat zinc, trihydrate manganese sulfate, etc. the Amount that must be added, usually approximately equal to 0.0001% (wt./about.) - 1% (wt./about.) in the calculation of the total number of this environment.

In the case of cultivation of transformant holding this DNA (A), attached right from the promoter of T7 phage and DNA in which the nucleotide sequence encoding a RNA polymerase T7 (lysogen λ DE3)attached right from the promoter lac UV5, can usually be added to a small amount, for example, isopropyl-β-D-thiogalactoside (hereinafter called "IPTG") as an inductor for induction of the production of this protein (And). IPTG can also be added to the environment in case of cultivation of transformant, having entered into his DNA, in which the present DNA (A) is functionally connected with the type of promoter, which is induced by lactose, such as the tac promoter, trc promoter and lac promoter.

The microorganism containing the DNA (A), can be cultured in accordance with the method, usually used for culturing a microorganism, including a liquid-phase cultivation, such as cultivation with rotary shaking cultivation with vibrating reciprocating shaking fermentation containers (cultivation using fermentors containers) and cultivation in tanks (tank is); or solid-phase cultivation. When applying the fermenter container is usually in the fermenter must be entered aseptic air at the speed of aeration approximately 0.1 to approximately 2 volume of the culture fluid per minute. The temperature at which carry out the cultivation can vary in the range, allowing the microorganism to grow, and is typically in the range from approximately 15°to approximately 40°C, and pH is usually in the range from approximately 6 to approximately 8. Time of cultivation may vary depending on cultivation conditions and is usually about 1 day, about 10 days.

This protein is (A)produced by a microorganism containing the DNA (a)may be used, for example, in various forms during processing herbicide compounds PPO-inhibitor type formula (I), such as culture of a microorganism producing the protein (A), the cell of the microorganism producing the protein (A), the material obtained by the processing of such cells, cell-free extract of a microorganism, roughly purified protein purified protein and other Material, obtained by processing cells, described above, includes, for example, freeze-dried cell, dried acetone cage pounded the cell autolysate cells treated with ultrasound is tapped, processed with alkali cell, treated with an organic solvent cage etc. Alternative, the protein (S) in any of the various forms described above, can be immobilized in accordance with known methods, such as the method of binding media using adsorption on inorganic carrier such as silica gel or a ceramic material, a derivative of a polysaccharide, such as DEAE-cellulose, a synthetic polymer, such as Amberite IRA-935 (trade name, manufactured Rohm and Haas) and the like, and method of use, using the inclusion of spatial polymeric matrix, such as polyacrylamide, structurai polysaccharide gel (for example, carrageenophyte gel), alginic acid gel, agar gel, etc. and then used in the processing described above herbicide connection.

As the cleaning methods of this protein (S) from a culture of the microorganism containing the DNA (A), can be used conventional methods used in protein purification. For example, there may be mentioned the following method.

First, cells harvested from a culture of the microorganism by centrifugation or similar way and then physically destroy sonification, processing DYNOMILL, processing FRENCH press, etc. or destroy chemically with the use of surface-active in the society or lyse cells enzyme, such as lysozyme. From the thus obtained lysate insoluble matter is removed by centrifugation, filtration through a membrane or the like to obtain a cell-free extract, which is then fractionary any suitable means for separation and purification, such as cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, gel filtration chromatography and the like, obtaining thus purified this protein (And). Supporting materials used in this chromatography include, for example, a resin carrier, such as cellulose, dextran and agarose, United with carboxymethyl (KM) group, diethylaminoethyl (DEAE) group, phenyl group or butilkoi group. Can also be used commercially available column, already Packed in any media, such as Q-Sepharose FF, Phenyl-Sepharose HP, PD-10 and HiLoad 26/10 Q-Sepharose HP (trade name, from Amersham Pharmacia Biotech), TSK-gel G3000SW (trade name, TOSOH CORPORATION).

Provides one example of purification of this protein (A).

The cells of the microorganism producing the protein (A), collected by centrifugation and then suspended in a buffer such as 0.1 M potassium phosphate (pH 7.0). This suspension is treated with ultrasound for cell disruption and the resulting lysate centrifuged at approximately 40000 g for the roughly 30 minutes to obtain a supernatant, which then centrifuged at 150000 g for about 1 hour to extract supernatant (cell free extract). The obtained cell-free extract is subjected to fractionation with ammonium sulfate to obtain a fraction which is soluble in the presence of 45%-saturated ammonium sulfate and precipitated with 55%-saturated ammonium sulfate. After replacing the solvent of this fraction with buffer not containing ammonium sulfate, for example, 1 M potassium phosphate, using a PD10 column (Amersham Pharmacia Biotech Company), the resulting fraction is applied, for example, on a column HiLoad 26/10 Q-Sepharose HP (Amersham Pharmacia Biotech Company). Column elute 20 mm istripovana with a linear gradient of NaCl to obtain a series of fractions of the eluate. Faction detecting activity in the conversion of compound (II) into the compound (III) in the presence of a system of electron transport containing electron donor, such as coenzyme NADPH, extract. Then, after replacing the buffer in these fractions using, for example, PD10 column (Amersham Pharmacia Biotech Company), the fractions obtained is applied on a column Bio-Scale Ceramic such as hydroxyapatite column SNT-type I I (Bio-Rad Compamy). After washing the column with buffer a (2 mm potassium phosphate buffer containing 1.5 mm CaCl2; pH 7.0), column elute buffer And a linear gradient of buffer B (100 mm potassium phosphate buffer containing 0.03 mm CaCl 2) to obtain a series of fractions of the eluate. Faction detecting activity in the conversion of compound (II) into the compound (III) in the presence of a system of electron transport containing electron donor, such as coenzyme NADPH, extract. Then, after replacing the buffer in these fractions using, for example, PD10 column (Amersham Pharmacia Biotech Company), the obtained fractions are concentrated, for example, using ultrafiltration (microcon filter unit mickrocon-30; Millipore Company). The resulting fraction is injected, for example, in column 75 PG HiLoad 16/60 Superdex (Amersham Pharmacia Biotech Company) and elute 0.05 M potassium phosphate buffer, containing 0.15 M NaCl (pH 7.0), with a series of fractions of the eluate. Faction detecting activity in the conversion of compound (II) into the compound (III) in the presence of a system of electron transport containing electron donor, such as coenzyme NADPH, extract. This protein may be purified by separation by electrophoresis in LTO-SDS page, if required.

Purification of the protein (A) of the present invention as described above, with the subsequent use of the obtained protein (a) of this invention as an immune antigen, may be obtained antibody that recognizes the protein (A) of the present invention (hereinafter sometimes referred to as "antibody (a) of this invention")).

Specifically, for example, the animal is subjected to immunization data protein (A), purified as described above, in which the quality of the antigen. For example, to immunize an animal, such as a mouse, hamster, Guinea pig, chicken, rat, rabbit, dog or the like, the antigen is administered at least once using the conventional method of immunization, as described, for example, in W.H. Newsome, J. Assoc. Off. Anal. Chem. 70(6) 1025-1027 (1987). As the schemes mentioned, for example, the introduction of 2 or 3 times at 7-30-day intervals, preferably with 12 to 16-day intervals. Dose of its introduction is equal to, for example, from about 0.05 mg to 2 mg of antigen for each injection. The route of administration may be selected from subcutaneous, intradermal, intraperitoneal administration, intravenous and intramuscular injection, and the injection provided intravenously, intraabdominal or subcutaneously, is a typical form of introduction. This antigen is used usually after dissolution in a suitable buffer such as sodium phosphate buffer, or physiological saline solution containing at least one type of commonly used adjuvant, such as complete beta-blockers (mixture Aracel A, Bayol F and the dead tubercle bacilli), system adjuvants RAS [MPL (monophosphorylated A) + TDM (synthetic dikarenakan trehalose) + CWS (cell wall skeleton)] or aluminum hydroxide. However, depending on the method or conditions of introduction described adjuvants can't and what to use. The term "adjuvant" means a substance that, when introduced to the antigen enhances nonspecific immune response against this antigen. After the animal, which is injected antigen, for 0.5-4 months take a small amount of blood, for example, from the ear vein of the animal and measure the antibody titer. When the antibody titer increases, the antigen is administered additionally during the appropriate number of injections, depending on the specific cases. For example, the antigen may be administered once in a dose of approximately 100 to 1000 micrograms. After one or two months after the last injection, the blood is collected in the usual way from the immunized animal. The fractionation of the blood of the conventional methods such as precipitation by centrifugation or ammonium sulfate or polyethylene glycol, chromatography, such as gel-filtration chromatography, ion exchange chromatography and affinity chromatography, and the like, antibody (a) of this invention can be obtained as polyclonal antisera. Further, this anticavity can be incubated, for example at 56°C for 30 minutes to inactivate complement system.

Alternatively, the polypeptide containing a partial amino acid sequence of the protein (a) of this invention are synthesized chemically and is introduced as the immune Antiga is but an animal with obtaining thus antibodies (A) of the present invention. As the amino acid sequence of the polypeptide used as an immune antigen, select amino acid sequence, which has possibly the most low homology with the amino acid sequences of other proteins from the amino acid sequence of the protein (A) of the present invention. The polypeptide having a length of 10 to 15 amino acids comprising the selected amino acid sequence, synthesize chemically accepted way and sew, for example, with protein carrier, such as hemocyanin Limulus plyhemus, using MBS, etc. and then used to immunize an animal, such as rabbit, as described above.

Then the derived antibody (A) of the present invention is brought into contact with the test sample, and then the complex of this protein in the test sample with the antibody described above, detects the conventional immunological method, with detection thus protein (a) of this invention or of the polypeptide, containing a partial amino acid sequence in the test sample. Specifically, for example, it is possible to evaluate the presence of protein (a) of this invention or to determine the amount of protein (a) of this invention tested in the test sample by Western blot analysis using antibodies (A) of the present invention, as shown in examples 45 or 73 below.

Further, for example, a cell expressing the present protein (A), can be detected by contacting the antibody (a) of this invention with the test cell or test sample obtained from the test cells, with subsequent detection of the complex described above antibody and protein in the test cell or test sample obtained from this test cell, in accordance with conventional immunological methods. Detection of cells expressing the protein (a) of this invention, therefore you can also select from a variety of cells of the cell expressing the protein (A) of the present invention. You can also clone or to select a clone containing protein (a) of this invention, with the use of antibodies (A) of the present invention. For example, can be obtained genomic library extraction of genomic DNA from a cell that expresses the protein (a) of this invention, with the subsequent incorporation of this genomic DNA expressing vector. This genomic library is injected into the cell. From the obtained group of cells, select the cell expressing the protein (a) of this invention, with the use of antibodies (A) of the present invention, as described above.

The set containing the antibody (a) of this invention may be used to detect the protein (A) of the present invention, as described above, or for analysis, detection or POI is ka cells, expressing protein (S) of the present invention. This set of this invention may contain the reagents required for the above-described methods of analysis, other than the antibody (a) of this invention and may be of such a reagent used in conjunction with the antibody (a) of this invention.

Through reaction herbicide compounds PPO-type inhibitor of formula (I) in the presence of a system of electron transport containing electron donor, such as coenzyme NADPH, with the protein (A) above compound is metabolized and turned into a combination of lower herbicide activity. Specifically, for example, by reaction of compound (II) in the presence of a system of electron transport containing electron donor, such as coenzyme NADPH, with the protein (A), the compound (II) is converted to the compound (III), which essentially does not detect herbicide activity. An example of a protein (A) in such cases is a protein (A) of the present invention. Can be used a variation of this protein (a) and can be used together multiple variations.

The compound of formula (I) is a compound having the structure of uracil. As typical examples may be mentioned the compound (II) or the compound of any of formulas (IV) to (IX) (hereinafter referred to, respectively, the compound (IV) with compound (X)). The compound (II) and compound (IX) can be synthesized according to the method described in the publication has not yet been vetted by the expertise of the patent application of Japan with the number 2000-319264, the compound (IV) and the compound (V) in accordance with the method described in U.S. Patent No. 5183492, the compound (VI) in accordance with the method described in U.S. patent No. 5674810, the compound (VII) in accordance with the method described in the publication has not yet been vetted by the expertise of the patent application of Japan with the number 3-204865, the compound (VIII) in accordance with the method described in the publication has not yet been vetted by the expertise of the patent application of Japan with the number 6-321941.

Further, as typical examples of the compounds of formula (I) may be mentioned a compound of any of formulas (X) to (XVII) (hereinafter respectively called the compound (X) is the compound (XVII)).

Compounds that can be a substrate metabolizing reactions promoted by this protein (A)can be selected using the compounds present in the reaction in which the compound (II)labeled with a radioactive isotope, interacts with the protein (A) in the presence of a system of electron transport containing electron donor, such as coenzyme NADPH, and detection as a marker for competitive inhibition Rea is the conversion of this protein (A) labeled compound (II) labeled compound (III). When analyzing the presence of competitive inhibition by test compound, this is a test connection is usually added in an amount up to 1-100 times the molar concentration of the labeled compound (II).

A reaction in which compound (I) reacts with the protein (A)can be carried out, for example, in an aqueous buffer containing inorganic salts, such as, for example, a phosphate of an alkali metal such as sodium phosphate and potassium phosphate; or organic acid salts, such as alkali metal acetate such as sodium acetate and potassium acetate; or the like, the Concentration of the compounds of formula (I) in solution metabolizing reaction is usually equal to a maximum of approximately 30% (weight/volume) and preferably approximately 0,001% (weight/volume) - 20% (weight/volume). The number of systems of transport of electrons containing electron donor, such as NADPH, or a given protein (A) can vary, for example, depending on the time period of the reaction. The reaction temperature usually selected from a range from approximately 10 to 70°C, and it is usually equal to about 20-50°C. the pH of the reaction is chosen from the range of usually from about 4 to 12 and preferably from about 5 to 10. The period of the reaction time may vary and is usually from about 1 hour to 10 days.

Further, the reaction in which compound (I) usaimage which corresponds with the protein (A), can be performed in the cell that contains the DNA (A). As the cell that contains the DNA (A)can be, for example, be mentioned microorganism that is able to Express this DNA (a) and to produce the protein (S), such as a strain of microorganisms isolated from a natural source that contains the DNA (A), mutant strain derived from this strain of microorganism treatment by chemicals or ultraviolet radiation, the transformed cell is a microorganism in which the present DNA (A) or the vector containing this DNA (A)introduced into the cell host. Further, there may be mentioned transformed plant cell in which you entered this DNA (a)or cell transformed plant in which introduced this DNA (A). In such cases, the compound of formula (I) can be directly applied to the cell that contains the DNA (A), or may be added to the culture medium of this cell or in the soil in contact with the cell, so that it could enter into this cell. System transport of electrons containing electron donor, such as NADPH, can be system, the source is present in the cell, or it can be added from outside the cell.

Metabolism of compound (I) of this protein (A) can be confirmed, for example, detection of compounds produced by metabolism of the compound (I). To the specific, for example, the compound (III)produced in the metabolism of compounds (II)can be detected HPLC or TLC analysis as described above.

Next, the metabolism of compound (I) of this protein (A) can be confirmed on the basis that herbicide activity in the reaction solution after the reaction of the compound (I) with the protein (A) is relatively lower than in the case where the compound (I) does not interact with the protein (A). As a way of testing herbicide activity is mentioned, for example, the way in which the above reaction solutions are applied to weeds, such as plushie millet (Echinochloa crus-galli), Alopecurus myservername (Alopercurus myosuroides), bindweed plyuschevidnaya (Ipomoea hederacea) and canetic Theophrastus (Abutilon theophrasti), and experience herbicide effects; or the way in which the plants were grown on soil samples, to which add the above reaction solutions, and experience herbicide effects; and so Forth, may be mentioned a method in which the above reaction solutions can be applied in the form of spots on the leaf disk taken from a plant, and then examine the presence of damaged plants (bleaching), the resulting reaction solution.

In addition, the metabolism of compound (I) of this protein (A) can be confirmed by detection as the token PPO-inhibiting activity in the reaction solution, after interaction of the compound (I) with the protein (A), which is relatively lower than the activity in the reaction solution in which the compound (I) does not interact with the protein (A). PPO is an enzyme that catalyzes the conversion of protoporphyrinogen IX to protoporphyrin IX (hereinafter called “PPIX”). For example, after adding the above reaction solution to the reaction system PPO, add protoporphyrinogen IX, which is the substrate of PPO, and incubated for approximately 1-2 hours at 30°C in the dark. Then measure the amount of PPIX in each of preincubating solutions using HPLC or similar methods. When the amount of PPIX in the system, which is added to the reaction solution after the interaction of the compound (I) with the protein (A), is greater than the amount of PPIX in the system, which is added to the reaction solution in which the compound (I) does not interact with the protein (A), it determines that the compound (I) was metabolized by this protein (And). As a PPO can be used a protein purified from plants, etc. or can be used fraction of chloroplasts extracted from plants. When using fractions of chloroplasts in the reaction system PPO instead of protoporphyrinogen IX can be used aminolevulinate acid. Am malevolencia acid is a precursor of protoporphyrinogen IX in the biosynthesis pathway of chlorophyll-heme. A more specific example is shown in example 42 below.

By reaction of this protein (A) can thus be carried out processing of herbicide compounds PPO-inhibitor type formula (I), which leads to metabolize and transform this compound in the combination of lower herbicide activity. Damage to plants specified connection can be reduced by processing, in which the specified connection, which was spattered on the area of cultivation of plants, in particular, for example, the connection that was spattered on the area of plant cultivation and remains in the residue of the plant or in the soil or the like, interacts with the protein (A).

As a "system of electron transport containing electron donor, which can be used for the reaction of the compound (I) with the protein (a)may be, for example, the system containing NADPH, ferredoxin and ferredoxin-NADP+-reductase.

As a way to provide a "system of electron transport containing electron donor", in the system for the reaction of the compound (I) with the protein (a)may be, for example, mentioned a method of adding to the above reaction system of NADPH, ferredoxin derived from plants, such as spinach, and ferredoxin-NADP+-reductase derived from plants, such as spinach. Yes is it to this reaction system may be added to the fraction containing the component, functional system for transport of electrons in the reaction system of this protein (A), which can be obtained from a microorganism, such as E. coli. To obtain this fraction, for example, after the cells are harvested from a culture of the microorganism by centrifugation or the like, these cells destroy physically sonification, processing DYNOMILL, FRENCH PRESS treatment and the like, or chemically destroy with the use of surfactants or lyse cells enzyme such as lysozyme. From the thus obtained lysate insoluble matter is removed by centrifugation, filtration through a membrane or the like to obtain a cell-free extract. This cell-free extract can be used instead of ferredoxin in the form of a fraction containing a component that is functional for a system of transport of electrons in the reaction system of this protein (And). In addition, when the system which can transport an electron from an electron donor to this protein (A), is present in such a cell, as is the case when the reaction of this protein (A) with compound (I) is carried out in the cell such as the cell of a microorganism or plant cell, the transport of electrons can not be added from the outside.

As f is redoxide can be used for example, ferredoxin derived from microorganisms belonging to Streptomyces, such as Streptomyces phaeochromogenes, Streptomyces testaceus, Streptomyces achromogenes, Streptomyces griseolus, Streptomyces thermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus, Streptomyces roseorubens, Streptomyces rutgersensis and Streptomyces steffisburgensis, and more specifically, Streptomyces phaeochromogenes IFO12898, Streptomyces testaceus ATCC21469, Streptomyces achromogenes IFO 12735, Streptomyces griseolus ATCC11796, Streptomyces thermocoerulescens IFO 14273t, Streptomyces nogalater IFO 13445, Streptomyces tsusimaensis IFO 13782, Streptomyces glomerochromogenes IFO 13673t, Streptomyces olivochromogenes IFO 12444, Streptomyces ornatus IFO 13069t, Streptomyces griseus ATCC 10137, Streptomyces griseus IFO 13849T, Streptomyces lanatus IFO 12787T, Streptomyces misawanensis IFO 13855T, Streptomyces pallidus IFO 13434T, Streptomyces roseorubens IFO 13682T, Streptomyces rutgersensis IFO 15875T and Streptomyces steffisburgensis IFO 13446T and the like, or from microorganisms belonging to Saccharopolyspora, such as Saccharopolyspora taberi, more specifically Saccharopolyspora taberi JCM 9383t and the like (hereinafter sometimes collectively called "data protein (In)"). Specifically, for example, may be mentioned ferredoxin selected from the group of proteins below (hereinafter sometimes referred to as "protein (C) of this invention").

<a group of proteins>

(B1) a protein containing the amino acid sequence shown in SEQ ID NO:12 (hereinafter sometimes referred to as "protein (B1) of the present invention");

(B2) a protein containing the amino acid sequence shown in SEQ ID NO:13 (hereinafter and is then called "protein (B2) of the present invention");

(B3) a protein containing the amino acid sequence shown in SEQ ID NO:14 (hereinafter sometimes referred to as "protein (B3) of the present invention");

(B4) a protein containing the amino acid sequence shown in SEQ ID NO:111 (hereinafter sometimes referred to as "protein (B4) of this invention");

(B5) ferredoxin containing amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:111;

(B6) ferredoxin containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:111;

(B7) a protein containing the amino acid sequence shown in SEQ ID NO:149 (hereinafter sometimes referred to as "protein (V7) of this invention");

(B8) a protein containing the amino acid sequence shown in SEQ ID NO:150 (hereinafter sometimes referred to as "protein (B8) of this invention");

(B9) a protein containing the amino acid sequence shown in SEQ ID NO:151 (hereinafter sometimes referred to as "protein (B9) of this invention");

(10) a protein containing the amino acid serial is lnost, shown in SEQ ID NO:152 (hereinafter sometimes referred to as "protein (10) of this invention");

(B11) a protein containing the amino acid sequence shown in SEQ ID NO:153 (hereinafter sometimes referred to as "protein (B11) of this invention");

(B12) ferredoxin containing amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any one of the amino acid sequence shown in SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251 or SEQ ID NO:253, or amino acid sequence having at least 90% sequence identity with any of the amino acid sequence shown in SEQ ID NO:150, SEQ ID NO:252 or SEQ ID NO:254;

(B13) ferredoxin containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with any of the nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253 or SEQ ID NO:254;

(B14) a protein containing the amino acid sequence shown in SEQ ID NO:245;

(B15) a protein containing the amino acid sequence, pokazanno is in SEQ ID NO:247;

(B16) a protein containing the amino acid sequence shown in SEQ ID NO:248;

(B17) a protein containing the amino acid sequence shown in SEQ ID NO:249;

(B18) a protein containing the amino acid sequence shown in SEQ ID NO:250;

(B19) protein containing the amino acid sequence shown in SEQ ID NO:251;

(B20) a protein containing the amino acid sequence shown in SEQ ID NO:252;

(B21) a protein containing the amino acid sequence shown in SEQ ID NO:253; and

(B22) a protein containing the amino acid sequence shown in SEQ ID NO:254.

DNA encoding this protein (In) (hereinafter sometimes called “the DNA of (C)”)can be obtained in accordance with conventional methods of genetic engineering, are described in Molecular Cloning: A Laboratory Manual 2nd Edition (1989), Cold Spring Harbor Laboratory Press; Current Protocols in Molecular Biology (1987), John Wiley and Sons, Incorporated, etc. on the basis of nucleotide sequences encoding amino acid sequences (B) of the present invention, shown in SEQ ID NO:12, 13, 14, 111, 149, 150, 151, 152, 153, 245, 247, 248, 249, 250, 251, 252, 253 or 254.

As the DNA that encodes a protein (C) of the present invention (sometimes referred to collectively DNA (C) of the present invention”)can be mentioned

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:12 (hereinafter sometimes the so-called “DNA (B1) of the present invention);

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:13 (hereinafter sometimes referred to as “DNA (B2) of the present invention);

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:14 (hereinafter sometimes referred to as “DNA (B3) of the present invention);

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:111 (hereinafter sometimes referred to as “DNA (B4) of this invention”);

DNA encoding ferredoxin containing amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:111;

DNA encoding ferredoxin containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:111;

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:149 (hereinafter sometimes referred to as “DNA (V7) of the present invention);

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:150 (hereinafter sometimes referred to as “DNA (B8) this is about the invention);

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:151 (hereinafter sometimes referred to as “DNA (B9) of the present invention);

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:152 (hereinafter sometimes referred to as “DNA (10) of the present invention);

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:153 (hereinafter sometimes referred to as “DNA (B11) of the present invention);

DNA encoding ferredoxin containing amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any of SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251 or SEQ ID NO:253, or an amino acid sequence having, in least 90% sequence identity with the amino acid sequence shown in any of SEQ ID NO:150, SEQ ID NO:252 or SEQ ID NO:254;

DNA encoding ferredoxin containing the amino acid sequence encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence that encodes the amino acid sequence shown in any of SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:49, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253 or SEQ ID NO:254;

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:245;

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:247;

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:248;

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:249;

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:250;

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:251;

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:252;

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:253, and

DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO:254.

As more specific examples of DNA (C) of this invention may be mentioned a DNA containing the nucleotide sequence shown in any of SEQ ID NO:15, 16, 17, 112, 154, 155, 156, 157, 158, 255, 257, 258, 259, 260, 261, 262, 263 or 264, or a DNA containing a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown in any of SEQ ID NO:15 16, 17, 112, 154, 155, 156, 157, 158, 255, 257, 258, 259, 260, 261, 262, 263 or 264.

Such DNA can be obtained by carrying out the ways in which PCR is carried out with the use of DNA containing a partial nucleotide sequence of these nucleotide sequences as primers, or methods of hybridization, in which such DNA used as probes, in accordance with the conditions described above in the methods of the DNA (A).

For example, specifically, a DNA containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:12, or a DNA containing the nucleotide sequence shown in SEQ ID NO:15 can be obtained by PCR using as a template the chromosomal DNA or chromosomal DNA library derived from Streptomyces phaeochromogenes IFO 12898, and using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:105, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:53.

Next, a DNA containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:13, or a DNA containing the nucleotide sequence shown in SEQ ID NO:16, can be obtained by PCR using as a template the chromosomal DNA of elibility chromosomal DNA, derived from Saccharopolyspora taberi JCM 9383t, and using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:106, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:63.

Next, a DNA containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:14, or a DNA containing the nucleotide sequence shown in SEQ ID NO:17, can be obtained by PCR using as a template the chromosomal DNA or chromosomal DNA library derived from Streptomyces testaceus ATCC21469, and using as primers the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:107, and the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO:72.

Further, for example, DNA (B) of this invention can be obtained by hybridization with chromosomal DNA library, DNA, consisting of at least about 20 nucleotides, containing the nucleotide sequence encoding the amino acid sequence shown in any of SEQ ID NO:12, 13, 14, 111, 149, 150, 151, 152 or 153, as probe under the conditions described above, followed by detection and extraction of DNA, specifically associated with the specified probe. Library of chromosomal Ddcmode to be obtained, as described above, from microorganisms belonging to Streptomyces, such as Streptomyces phaeochromogenes, Streptomyces testaceus, Streptomyces achromogenes, Streptomyces thermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus, Streptomyces roseorubens, Streptomyces rutgersensis and Streptomyces steffisburgensis, and more specifically, Streptomyces phaeochromogenes IFO12898, Streptomyces testaceus ATCC21469, Streptomyces achromogenes IFO 12735, Streptomyces thermocoerulescens IFO 14273t, Streptomyces nogalater IFO 13445, Streptomyces tsusimaensis IFO 13782, Streptomyces glomerochromogenes IFO 13673t, Streptomyces olivochromogenes IFO 12444, Streptomyces ornatus IFO 13069t, Streptomyces griseus ATCC 10137, Streptomyces griseus IFO 13849T, Streptomyces lanatus IFO 12787T, Streptomyces misawanensis IFO 13855T, Streptomyces pallidus IFO 13434T, Streptomyces roseorubens IFO 13682T, Streptomyces rutgersensis IFO 15875T and Streptomyces steffisburgensis IFO 13446T, and the like, or from microorganisms belonging to Saccharopolyspora, such as Saccharopolyspora taberi, more specifically Saccharopolyspora taberi JCM 9383t etc. as specific examples of the DNA that can be used as such probes may be mentioned a DNA containing the nucleotide sequence shown in any of SEQ ID NO:15, 16, 17, 112, 154, 155, 156, 157, 158, 255, 257, 258, 259, 260, 261, 262, 263 or 264; DNA containing a partial nucleotide sequence of such nucleotide sequences; or a DNA containing a nucleotide sequence complementary to the specified partial nucleotide sequences.

For the expression of this DNA (C) cell-chose the nom, for example, DNA in which the present DNA (A) and promoter functional in the cell-master, functionally connected, receive in accordance with accepted methods of genetic engineering, as described in “Molecular Cloning: A Laboratory Manual 2nd Edition (1989)”, Cold Spring Harbor Laboratory Press; Current Protocols in Molecular Biology (1987)”, John Wiley and Sons, Incorporated, and the like, and is introduced into a cell of the host. Does the obtained transformant this DNA (I), can be checked by obtaining DNA from transformant and then conducting with the obtained DNA analysis methods of genetic engineering are described, for example, in “Molecular Cloning 2nd Edition, Cold Spring Harbor Laboratory Press (Molecular Biology, John Wiley and Sons, N.Y. (1989) (such as confirmation of sites restricts, DNA sequencing, hybridization to Southern, PCR and the like).

This DNA (b) and this DNA (A) can be expressed in the same cell by introducing into the cell that contains the DNA (A), DNA in which the present DNA (A) and promoter functional in the cell-master, are functionally related.

This protein (In) can be obtained, for example, by culturing cells containing this DNA (). As such cells can be mentioned microorganism expressing this DNA (b) and capable of producing the protein (In), such as a strain of microorganism isolated from a natural source that contains the DNA (B), mutant strains derived from the specified natural what about the strain of processing agents or ultraviolet rays, or the like, for Example, can be mentioned microorganisms belonging to Streptomyces, such as Streptomyces phaeochromogenes, Streptomyces testaceus, Streptomyces achromogenes, Streptomyces griseolus, Streptomyces thermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus, Streptomyces roseorubens, Streptomyces rutgersensis and Streptomyces steffisburgensis, and more specifically, Streptomyces phaeochromogenes IFO12898, Streptomyces testaceus ATCC21469, Streptomyces achromogenes IFO 12735, Streptomyces griseolus ATCC11796, Streptomyces thermocoerulescens IFO 14273t, Streptomyces nogalater IFO 13445, Streptomyces tsusimaensis IFO 13782, Streptomyces glomerochromogenes IFO 13673t, Streptomyces olivochromogenes IFO 12444, Streptomyces ornatus IFO 13069t, Streptomyces griseus ATCC 10137, Streptomyces griseus IFO 13849T, Streptomyces lanatus IFO 12787T, Streptomyces misawanensis IFO 13855T, Streptomyces pallidus IFO 13434T, Streptomyces roseorubens IFO 13682T, Streptomyces rutgersensis IFO 15875T and Streptomyces steffisburgensis IFO 13446T and the like, or from microorganisms belonging to Saccharopolyspora, such as Saccharopolyspora taberi, more specifically Saccharopolyspora taberi JCM 9383t and so Forth, may be mentioned the transformant that was introduced this DNA (). For example, specifically, may be mentioned the transformant in which this DNA (B)functionally associated with the tac promoter, trc promoter, the lac promoter or the promoter of T7 phage, was introduced into E. coli. As more specific examples may be mentioned E. coli JM109/pKSN657FD, E. coli JM109/pKSN923FD, E. coli JM109/pKSN671FD, etc. described in the examples below.

The microorganism containing the DNA (In), Kul is to Virovitica in accordance with the method, commonly used for cultivation of the microorganism and, more specifically, carried out in accordance with the conditions described above in the methods of cultivation of the microorganism containing the DNA (A).

This protein (B)produced by a microorganism containing the DNA (I), can be used, for example, in various forms in the reaction system of this protein (A), such as culture of a microorganism producing the protein (C), the cell of the microorganism producing the protein (C), the material obtained by the processing of such cells, cell-free extract of a microorganism, roughly purified protein purified protein and other Material, obtained by processing cells, described above, includes, for example, freeze-dried cell, dried acetone cage pounded the cell, the cell autolysate ultrasonic assisted cell processed with alkali cell, treated with an organic solvent cage etc. Alternative, the protein (C) in any of the various forms described above, can be immobilized in accordance with known methods, such as the method of binding to a carrier that uses the adsorption on the synthesized polymer and the like, and method of use, using the inclusion of spatial polymer matrix, and then used in the reaction system of this protein (A).

As the means of purification of this protein (In) from a culture of the microorganism, contains the DNA (b)may be used conventional methods used in protein purification. For example, there may be mentioned the following method.

First, cells harvested from a culture of the microorganism by centrifugation or similar way and then physically destroy sonification, etc. or destroy chemically with the use of surfactants or lyse cells enzyme such as lysozyme. From the thus obtained lysate insoluble matter is removed by centrifugation, filtration through a membrane or the like to obtain a cell-free extract, which is then fractionary any suitable means for separation and purification, such as cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, gel filtration chromatography and the like, obtaining thus purified this protein (In). By separating the thus obtained fractions by electrophoresis in LTO-page the protein (In) can be dopolnitelnoe cleared.

The function of this protein (In) as ferredoxin can be confirmed as a feature vector of an electron from ferredoxin-NADP+-reductase to this protein (A) in the reaction system, in which the compound (I) interacts with the protein (A). For example, specifically, confirmed the e can be done by adding this protein (b) with NADPH, ferredoxin-NADP+-reductase and protein (A) to a reaction system, in which the compound (I) interacts with the protein (A), followed by detection of the conversion of compound (II) into the compound (III).

In the method of controlling weeds of the present invention the compound (I) applied to the area of cultivation of plants expressing the present protein (A). This plant can Express one variation of this protein (A) or may Express multiple variations of this protein (And). As this protein may be, for example, mentioned protein (A) of the present invention. Plants expressing the present protein (A)can be obtained in the form of transgenic plants, which was introduced this DNA (A). This introduction includes the introduction of this DNA in a plant cell, as described above, so that this DNA is placed in a position allowing its expression, with subsequent regeneration of plants from the obtained transformed cells. This DNA (A)introduced into the plant may have an associated left her the nucleotide sequence encoding the signal transfer in the intracellular organelle so that the reading frames of these sequences are in the same reading frame.

The plant, having entered into it this DNA (a) and expressing the present protein (A), metaboliser the t connection (I), in his cell, the combination of lower herbicide activity. As a result, the damage to plants from herbicide compounds in the plant is reduced and given stability to the specified connection. Consequently, this plant, having entered into it this DNA (a) and expressing the present protein (A), can grow well even in the case where the compound (I) applied to the area of its cultivation. Weeds, other than a plant that has entered into it this DNA (a) and expressing the present protein (A), can be effectively removed by cultivation of specified plants and applying the specified herbicide composition to the area of cultivation. It is possible to improve the yield of the above plants, improvement of quality, reduction of use of the herbicide and saving labor costs.

Assessment of the sustainability of cells expressing this protein (A)to the compound of formula (I) or herbicide compositions containing the specified connection, can be carried out by contacting cells expressing the gene encoding this protein (S)with specified connection or specified herbicide composition and assessment of the damage to this cell.

Specifically, to assess the stability of the cells of the microorganism expressing the present protein (A)to the compound of formula (I) or herbicide compositions containing the the group of (I), can be obtained from transformed E. coli expressing PPO plants and the protein (A). This includes obtaining additional introduction of this DNA (A), for example, transformed in E. coli, which can be used to assess the inhibition of PPO activity was described in the patent application of Japan No. 11-102534, more specifically, the transformed E. coli in which the gene PPO plants described in U.S. patent number 5939602 or other functionally introduced into the E. coli strain VT3 and receive the expression of this gene PPO. The strain of E. coli VT3 has the defective gene PPO and not capable of proliferation, as described F. Yamamoto, H. Inokuti, H. Ozaki, (1988) Japanese Journal of Genetics, Vol. 63, pg. 237-249. Resistance connection or herbicide composition can be assessed by culturing the obtained transformed E. coli with shaking for approximately 18-24 hours at 37°C in liquid culture medium containing the compound (I) or herbicide composition comprising the specified connection, the number of 0-1,0 ppm, and measurement of proliferation specified transformed E. coli by optical density at 600 nm. As this protein may be, for example, mentioned protein (a) of this invention.

As a way of assessing the sustainability of plants expressing the present protein (A)to the compound of formula (I) or herbicide compositions containing the decrees of the data connection, may be mentioned the method of application of the herbicide composition to the plant and measuring the growth of this plant. For a more quantitative test, for example, the first pieces of the leaves of this plant are immersed in aqueous solutions containing compound (I) at various concentrations, or aqueous solutions of the compound (I) sprinkle the pieces of the leaves of this plant, then allow them to be on the agar medium in the light at room temperature. A few days later, the chlorophyll is extracted from the leaves of a plant in accordance with the method described Mackenney, G., J. Biol. Chem., 140; p 315 (1941), for the determination of chlorophyll. For example, specifically, take the leaves of the plant and cut them to equal 2 parts along the main vein. Herbicide composition is distributed over the entire surface of one of the halves of the sheet. The other half of the sheet is left untreated. These pieces of sheet placed on MS medium containing 0.8% agar, and incubated in the light at room temperature for 7 days. Then, each piece pound with the pestle in the mortar in 5 ml of 80% aqueous acetone for extraction of chlorophyll. The liquid extract was diluted 10 times with 80% aqueous acetone solution and the optical density was measured at 750 nm, 663 nm and 645 nm to calculate the total chlorophyll content according to the method described Mackenney, G., J. Biol. Chem. (1941) 140; p 315. CTE is Yan stability to the compound (I) can be estimated comparatively expression in the percentiles of the content of total chlorophyll processed piece of the total chlorophyll-a raw piece. As this protein may be, for example, mentioned protein (a) of this invention.

On the basis of the above-described method of evaluating degree of resistance to the compound (I) or herbicide composition containing the compound (I)can be selected plant or plant cell that detects the resistance to the compound (I) or herbicide composition containing the compound (I). For example, select a plant where there is no damage from spillage of compound (I) or herbicide compositions containing this compound, the area of cultivation of this plant, or select a plant cell, which continuously grows during cultivation in the presence of compound (I). For example, specifically, the ground stuff in a plastic vessel having, for example, a diameter of 10 cm and a depth of 10 see the Seeds of plants are sown and cultivated in the greenhouse. Preparing an emulsion by mixing 5 parts of herbicide compositions containing the compound (I), 6 parts sorpol3005X (Toho chemicals) and 89 parts of xylene. A certain amount of this emulsion is diluted with water containing 0.1% (vol./about.) providing an adhesion agent, in a ratio of 1000 liters per 1 hectare, and distribute uniformly gun for spraying on all sides of the foliage from the top of the plants cultivated in the above-described vessel. After cultivation of these plants for 16 days in the greenhouse studies the blow damage these plants. Plants that are not observed damage, or plants, in which damage is reduced, can be selected. Hereinafter, can be obtained progeny plant by crossing these selected plants.

EXAMPLES

Below the invention is explained in more detail by examples, but is not limited to these examples.

HPLC analysis of the content in examples 1, 41 and 42 and clearing fractions of this compound was carried out under the conditions shown below.

(Condition 1 HPLC analysis)

Column: SUMIPAX ODS211 (Sumika Chemical Analysis Service)

The column temperature: 35°C

The flow speed: 1 ml/min

The wavelength of detection: UV nm

Eluent A: 0,01% aqueous solution TFU

Eluent b: acetonitrile

The elution conditions: the Sample is injected into the column

balanced mixture of solvents 90% solvent a and 10% solvent C. Then the mixture solvent of 90% solvent a and 10% eluent To flow for 5 minutes. This is followed by a flow of solvent mixture of eluent a and eluent b Within 20 minutes, while the proportion of eluent In 10% to 90%. Then the mixture solvent of 10% of eluent a and 90% eluent flows In for 8 minutes.

Example 1. The conversion of compound (II) organism

(1) the Utilization of compound (II)

Various microorganisms are shown in tables 1 and 2, were grown in medium with ISP2 agar (1,0% (wt./about.) extract of malt is, of 0.4% (wt./about.) yeast extract, and 0.4% (wt./about.) glucose, and 2.0% (wt./about.) agar, pH of 7.3). "Full loop" of each of the microorganisms was added to the TGY medium (0,5% (wt./about.) tripton, a 0.5% (wt./about.) yeast extract, 0.1% (wt./about.) glucose, of 0.01% (wt./about.) KN2RHO4, pH 7.0) and incubated with shaking at 30°C for 2-4 days. One tenth of a milliliter (0.1 ml) of the obtained culture was incubated with shaking in 3 ml of medium for sporoobrazovanie (0,1% (wt./about.) meat extract, and 0.2% (wt./about.) tryptose, 1% (wt./about.) glucose, a pH of 7.1)containing the compound (II) at 100 ppm, for 7-8 days at 30°C. Fifty microliters (50 ál) 2 N. HCl was added to the resulting culture was extracted with 3 ml ethyl acetate. Received an ethyl acetate layer was analyzed using HPLC. The concentration of compound (II) decreased (retention time column 23.9 minutes) and new peaks detected for compounds with retention times of 21.6 minutes and 22.2 minutes (each referred to as metabolite (I) and metabolite (II)). The results are shown in tables 1 and 2.

Table 1
the strain of microorganismthe concentration of compound (II) (ppm)the peak area of the metabolite (I) (X104)the peak area of the metabolite (II) (x1 4)
Streptomyces cacaoiasoensis IFO1381377,83,433,57
Streptomyces griseofuscus IFO12870t49,5of 7.969,86
Streptomyces ornatus IFO13069t65,34,305,00
Streptomyces thermocoerulescens IFO14273t51,77,479,16
Streptomyces roseochromogenes ATCC1340081,90,710,82
Streptomyces lavendulae ATCC1192489,61,021,50
Streptomyces griseus ATCC1013765,6to 6.191,30
Streptomyces griseus ATCC1142930,312,8156
Streptomyces griseus ATCC1247551,10,522,27
Streptomyces griseus ATCC15395 to 75.21,91of 2.26
Streptomyces erythreus ATCC1163554,64,946,05
Streptomyces scabies IFO311188,33,284,40
Streptomyces griseus IFO310222,614.4V18,5
Streptomyces catenulae IFO1284885,33,811,59
Streptomyces kasugaensis ATCC1571492,41,080,91
Streptomyces rimosus ATCC 1097070,92,302,87
Streptomyces achromogenes IFO127350,015,921,8
Streptomyces lydicus IFO1305862,05,486,69

Table 2
strain ICRI is the body the concentration of compound (II) (ppm)the peak area of the metabolite (I) (X104)the peak area of the metabolite (II) (X104)
Streptomyces phaeochromogenes IFO1289846,48,2810,5
Streptomyces afghaniensis IFO1283180,62,543,59
Streptomyces hachijoensis IFO1278283,94,992,91
Streptomyces argenteolus var. toyonakensis ATCC2146813,014,919,2
Streptomyces testaceus ATCC2146918,4the 11.614.4V
Streptomyces purpurascens ATCC2548970,9lower than the 5.376,11
Streptomyces griseochromogenes ATCC1451153,93,003,97
Streptomyces kasugaensis IFO13851to 66.312,1 12,6
Streptomyces argenteolus var. toyonakensis ATCC21468t90,12,753,01
Streptomyces roseochromogenes ATCC13400t71,8of 4.664,00
Streptomyces nogalater IFO1344512,8of 21.924,9
Streptomyces roseochromogenus ATCC2189574,24,14by 5.87
Streptomyces fimicarius ATCC2190046,58,3311,3
Streptomyces chartreusis ATCC2190161,13,703,94
Streptomyces globisporus subsp. globisporus ATCC2190379,92,862,52
Streptomyces griseolus ATCC11796014.4Vto 19.9
Saccharopolyspora taberi JCM9383T82,9of 5.837,71
Strptomyces sp. SANK6258554,62,303,44

(2) determination of the structure of the metabolite (I) and metabolite (II)

Frozen original material Streptomyces griseus ATCC11429 was added to 3 ml of culture medium for the microorganism (0,7% (wt./about.) polypeptid, a 0.5% (wt./about.) yeast extract, and 1.0% (wt./about.) glucose and 0.5% (wt./about.) To2NRA4pH of 7.2) and incubated with shaking in a test tube overnight to obtain a pre-culture. This pre-culture was added to 300 ml of medium for microorganism containing the compound (II) at a concentration of 100 ppm, This volume is divided into 100 small test-tubes in 3 ml each and incubated with shaking at 30°C for 6 days. After adjusting 250 ml of this culture to pH 2 by addition of HCl and it was extracted with 250 ml ethyl acetate. The solvent was removed from an ethyl acetate layer. The residue was dissolved in 3 ml of acetone and the sample was applied on the TLC plate silica gel (silica gel TLC plates 60F25420 cm x 20 cm, 0.25 mm thick, Merck Company). The TLC plate showed a mixture of 5:7:1 (vol./about./about.) toluene:formic acid:ethyl formate. Took a Rf value of about 0.58 silica gel. Such content is the TLC plates were extracted with acetone. From the layer extraction with acetone was removed. The residue was dissolved in 10 ml of acetonitrile and fractionarea is using HPLC. The fractions containing only the metabolite (I) and metabolite (II), extracted with obtaining 3.7 mg metabolites (hereinafter called "metabolite").

Conducted mass spectrometry metabolite a Metabolite And had a mass that was 14 less than the mass of the compound (II). Next, from the H-NMR analysis determined that the metabolite is a compound having the structure shown in formula (III).

(3) testing of the herbicide activity of the compound (III)

The soil was filled in a round plastic vessel having a diameter of 10 cm and a depth of 10 cm Plushie millet, Alopecurus myservername and bindweed plyuschevidnaya were sown and cultivated in a greenhouse for 10 days. Five (5) parts of the test compounds, 6 parts sorpol3005X (Toho Chemical Company) and 89 parts of xylene are well mixed to obtain an emulsion. A certain amount of this emulsion was diluted with water containing 0.1% (vol./about.) providing an adhesion agent, in a ratio of 1000 liters per 1 hectare, and distributed uniformly gun for spraying on all sides of the foliage from the top of the plants cultivated in the above-described vessel. After cultivation of these plants for 16 days in the greenhouse were investigated herbicide activity of the test compounds. The results are shown in table 3.

Table 3
Test-connectionConcentration (g/ha)Herbicide activity
plushie milletAlopecurus myservernamebindweed plyuschevidnaya
The compound (II)500101010
125101010
The compound (III)500000
125000

The soil was filled in a round plastic vessel having a diameter of 10 cm and a depth of 10 cm Plushie millet, Alopecurus myservername and bindweed plyuschevidnaya were sown. Five (5) parts of the test compounds, 6 parts sorpol3005X (Toho Chemical Company) and 89 parts of xylene are well mixed to obtain an emulsion. A certain amount of this emulsion was diluted with water containing 0.1% (vol./about.) providing an adhesion agent, in a ratio of 1000 liters per 1 hectare, and distributed odero is but a gun for spraying on the soil surface. After cultivation of these plants for 19 days in the greenhouse were investigated herbicide activity of the test compounds. The results are shown in table 4.

Table 4
Test-connectionConcentration (g/ha)Herbicide activity
plushie milletAlopecurus myservernamebindweed plyuschevidnaya
The compound (II)500101010
The compound (III)500000

In the above tables 3 and 4 strength herbicide activity shows step by step how 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. The number "0" represents a situation in which the state of germination or vegetation during the test plants used for this test was comparable and generally had no or essentially had no differences with the state of the untreated control. The number "10" represents the situation, to the which the plant was fully savalalo or germination or vegetation were completely overwhelmed.

Example 2. Obtaining protein (A1) of the present invention

(1) preparation of the crude extract of cells

Frozen original material Streptomyces phaeochromogenes IFO 12898 was added to 100 ml of medium A (0.1% (wt./about.) glucose and 0.5% (wt./about.) tripton, a 0.5% (wt./about.) yeast extract, of 0.1 (wt./about.) dicale-phosphate, pH 7.0) in a triangular flask of 500 ml, and incubated with rotary swing at 30°C for 1 day to obtain a pre-culture. Eight milliliters (8 ml) pre-culture was added to 200 ml of medium a and incubated with rotary swing in the flask with septum 500 ml at 30°C for 2 days. Precipitation cells were obtained by centrifugation (3000 g, 5 min) obtained culture. The yeast cells suspended in 100 ml of medium (1% (wt./about.) glucose, 0.1% of meat extract, and 0.2% (wt./about.) tryptose)containing the compound (II) at 100 ppm, and incubated with the reciprocating swing in a Sakaguchi flask of 500 ml for 16 hours at 30°C. Precipitation cells were obtained by centrifugation (3000 g, 5 min), 10 l of the resulting culture. The obtained precipitation cells were washed twice in 1 l of 0.1 M potassium phosphate buffer (pH 7.0) to give 162 g of precipitation cells.

The yeast cells suspended in 0.1 M potassium phosphate buffer (pH 7.0) at 2 ml per 1 g of precipitation cells and added 1 mm PMSF, 5 mm benzamidine·HCl, 1 mm EDTA and 1 mm dithiothreitol. The solution cellular Lisa is and has been the destruction twice this suspension using a French press (1000 kg/cm 2) (Ohtake Seisakusho). After centrifugation of the solution in the cell lysate (40000 g, 30 min) the supernatant was removed and centrifuged for 1 hour at 150000 g for extraction of the supernatant (hereinafter called "the crude cell extract").

(2) Determination of the ability to turn the compound (II) into the compound (III)

Prepared 30 μl of the reaction solution of 0.1 M potassium phosphate buffer (pH 7.0)containing 3 ppm of compound (II)labeled14S, 2.4 mm β-NADPH (hereinafter referred to as "component a") (Oriental Yeast Company), 0.5 mg/ml ferredoxin obtained from spinach (hereinafter referred to as "component B") (Sigma Company), 1 u/ml paradoxicality (hereinafter referred to as "component C") (Sigma Company) and 18 μl of crude cell extract obtained in example 2(1). The reaction solution was kept at 30°C for one hour. In addition, prepared and handled in the same way, the reaction solution without adding at least one component used in the above reaction solution, selected from the component A, component b and component C. was Added three microliters (3 ál) 2 N. HCl and 90 μl of ethyl acetate in each of these reaction solutions after the specified curing and stirred. The resulting reaction solution was centrifuged at 8000 g for extraction of 75 μl of an ethyl acetate layer. After drying, an ethyl acetate SL is s under reduced pressure the residue was dissolved in 6.0 ál of ethyl acetate. Five microlitres (5,0 µl) of this solution was applied on TLC plate (silica gel TLC plates 60F25420 cm x 20 cm, 0.25 mm thick, Merck Company). The TLC plate showed a mixture of 6:1:2 chloroform, acetic acid and ethyl acetate for about 1 hour. Then the solvent was allowed to evaporate. The TLC plate was exposed during the night on the imaging plate (Fuji Film Company). Then, the imaging plate was analyzed by the image Analyzer BAS2000 (Fuji Film Company). The presence of spots corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). These results are shown in table 5.

Table 5
Components of the reactionspot compound (III)
component acomponentcomponentthe crude cell extractthe compound (II)labeled14
+++-+-
++ ++++
-++++-
+--++-

(3) Fractionation of the crude cell extract

To the crude cell extract obtained in example 2(1), was added ammonium sulfate to 45% saturation. After stirring cooled in ice conditions were obtained supernatant by centrifugation for 10 minutes at 12000 g. After adding ammonium sulfate to the supernatant up to 55% saturation and mixing cooled in ice conditions the precipitate was obtained by centrifugation for 10 minutes at 12000 g. The residue was dissolved in 27.5 ml of 20 mm bestreplibng buffer (pH 7.0). This solution was subjected to chromatography on a PD10 column (Amersham Pharmacia Company) and was suirable 20 mm mistresspopular buffer (pH 7.0) to obtain a 38.5 ml fractions containing proteins (hereinafter referred to as "fraction 45-55% of ammonium sulfate").

(4) Isolation of the protein (A1) of the present invention

Fraction 45-55% of ammonium sulfate obtained when the ore 2(3), was injected into the column HiLoad 26/10 Q-Sepharose HP (Amersham Pharmacia Company). Then after passing 106 ml of 20 mm bestreplibng buffer (pH 7.0) through the column, 20 mm listresponse buffer proceeded with a linear gradient of NaCl (NaCl gradient was 0,001415 M/min, the concentration range of NaCl was from 0 M to 0,375 M, the flow rate was 3 ml/min) for fractional extraction fractions of 25 ml, eluruumiks when the concentration of NaCl from of 0.21 to 0.22 M Then extracted fraction was subjected to chromatography on a PD10 column (Amersham Pharmacia Biotech Company) and was suirable 20 mm mistresspopular buffer (pH 7.0) to obtain fractions containing protein.

The extracted fraction was applied to a PD10 column (Amersham Pharmacia Biotech Company) with elution buffer (2 mm potassium phosphate buffer containing 1.5 mm NaCl, pH 7.0) to obtain fractions containing protein. Then these fractions were injected into the column SNT-I Bio-Scale Ceramic Hydroxyapatite Type I (Bio-Rad Company). Thirty milliliters (30 ml) buffer And passed through the column. Then the buffer And proceeded with a linear gradient of buffer B (100 mm potassium phosphate buffer containing 0.03 M NaCl; linear gradient started at 100% buffer And up to 50% buffer over a period of 100 minutes, the flow rate was 2 ml/min) for fractional extraction fractions, eluruumiks when the concentration of the buffer 17 to 20%. Then, the obtained fraction was applied to a PD10 column (Amersham Pharmacia Biotech Company) and was suirable 0.05 M potassium phosphate buffer(pH 7.0) to obtain fractions containing protein.

The fractions obtained were concentrated 20-fold using ultrafiltration membrane (Microcon YM-30, Millipore Company) and was injected into the column 75 PG HiLoad 16/60 Superdex (Amersham Pharmacia Biotech Company). 50 mm potassium phosphate buffer, containing 0.15 M NaCl (pH 7.0), proceeded (flow rate 1 ml/min) through the column. The eluate was fractionally 2 ml in each tube. Faction, eluruumina when the elution volumes 56 - 66 ml fraction was extracted separately. The protein contained in each of these fractions were analyzed by electrophoresis in 10-20% of the LTO-PAG.

Instead of the crude cell extract in the reaction solution described in example 2(2), the fractions obtained were added and maintained in the presence of component A, component B, component C and the compound (II)labeled14With, like in example 2(2). The reaction solutions after aging were analyzed using TLC to study the intensity of the spots corresponding to compound (III)labeled14C. was Observed that the protein moves to the position 47 kDa in the above-described electrophoresis LTO-PAG, had fluctuations in the concentrations of these bands fractions, add one by one, which were parallel with the fluctuations of the intensity of the spots corresponding to compound (III). This protein was extracted from the gel LTO-SDS page and subjected to amino acid analysis using sequ is of General proteins (Applied Biosystems Company, Procise 494HT, pulsed liquid method). The result was obtained amino acid sequence shown in SEQ ID NO:18. Then, after cleavage of this protein by trypsin derived cleaved material was analyzed on a mass spectrometer (ThermoQuest Company, Ion Trap Mass Spectrometer LCQ, column: LC Packings Company PepMap C18, 75 µm x 150 mm, solvent A: 0.1% Of HOAc-H2O, solvent B: 0.1% of SPLA-methanol, gradient: a linear gradient, starting with elution with a mixture of 95% solvent a and 5% solvent and increasing concentrations up to 100% of the solvent for 30 minutes, the flow rate of 0.2 µl/min). The result was obtained sequence shown in SEQ ID NO:19.

Example 3. Obtaining DNA (A1) of the present invention

(1) preparation of chromosomal DNA of Streptomyces phaeochromogenes IFO12898

Streptomyces phaeochromogenes IFO12898 incubated with shaking at 30°C for 1 to 3 days in 50 ml of YEME medium (0,3% (wt./about.) yeast extract and 0.5% (wt./about.) bactopeptone, of 0.3% (wt./about.) the malt extract, and 1.0% (wt./about.) glucose, 34% (wt./about.) sucrose and 0.2% (vol./about.) 2.5 M MgCl2.6N2About). The cells were collected. The obtained cells suspended in YEME medium containing 1,4% (wt./about.) glycine and 60 mm EDTA, and additionally incubated with shaking for one day. Cells were removed from culture medium. After washing once with distilled water them resuspendable the buffer (100 mm Tris-HCl (pH 8.0), 100 mm EDTA, 10 mm NaCl) at 1 ml per 200 mg of cells. Added two hundred micrograms per milliliter (200 μg/ml) of lysozyme egg white. This cell suspension was incubated with shaking at 30°C for one hour. Then added 0.5% of LTOs and 1 mg/ml proteinase K. cell Suspension was incubated at 55°C for 3 hours. Cell suspension was extracted twice with a mixture of phenol, chloroform and isoamyl alcohol to extract each of the water layers. Then there was one extraction with a mixture of chloroform and isoamyl alcohol to extract the water layer. Chromosomal DNA was obtained by precipitation with ethanol from the aqueous layer.

(2) obtaining a library of chromosomal DNA of Streptomycesphaeochromogenes IFO12898

943 ng of chromosomal DNA obtained in example 3(1), split 1 restrictase Sau3AI at 37°C for 60 minutes. The obtained split the solution was separated by electrophoresis in a 0.7% agarose gel. DNA size approximately 2.0 TPN extracted from the gel. This DNA was purified using the kit for purification of DNA Prep-A-GeneR(Bio-Rad Company) in accordance with the instructions attached to the specified set, with 10 µl of a solution containing the desired DNA. Microliter (1 μl) of the DNA solution, 98 ng plasmid vector pUC118 cleaved by the restriction enzyme BamHI and treated with dephosphorylation, and 11 μl of solution I of a set of ligation Ver. 2 (Takara huzo Company) were mixed and incubated over night at 16°C. E. coli DH5α was transformed using 5 ál of the solution for ligating. E. coli was cultured with shaking overnight at 30°C. From the obtained culture medium was extracted E. coli. The plasmids were extracted with obtaining a library of chromosomal DNA.

(3) Isolation of DNA (A1) of the present invention

PCR was performed using as a template the chromosomal DNA obtained in example 3(1) (Fig. 1). As primers used a couple of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:35, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:36 (hereinafter referred to as "pair 1 primers"). The nucleotide sequence shown in SEQ ID NO:35, designed based on the nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO:18. Further, the nucleotide sequence shown in SEQ ID NO:36, designed based on the nucleotide sequence complementary to the nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO:19. The reaction solution for PCR was made up to 25 μl by adding each of the 2 primers, brought to 200 nm, 250 ng of the above chromosomal DNA and 0.5 μl of dNTP mixture (a mixture of 10 mm each of the 4 types of dNTP; Clontech Company), 5 μl 5GC-genomic reaction b is Fehr for PCR (Clontech Company), 1,1 ál of 25 mm Mg(OAc)25 µl 5 M GC-melt (Clontech Company) and 0.5 μl of a mixture of genomic polymerase Advantage-GC (Clontech Company) and distilled water. The PCR reaction conditions were as follows: after the maintenance of 95°C for 1 minute, repeating 30 cycles, where each cycle consisted of maintaining 94°C for 15 seconds, then 60°C for 30 seconds, then 72°C for 1 minute, and then maintaining at 72°C for 5 minutes. After maintaining the reaction solution was applied to a 4% agarose gel for electrophoresis. Area of the gel containing DNA approximately 159 BP were removed. DNA was purified from the extracted gel using a set of extraction from gels QIAquick (Qiagen Company) in accordance with the attached instructions. The resulting DNA ligated to the cloning vector TA pCR2.1 (Invitrogen Company) in accordance with the instructions attached to the specified vector, and introduced into E. coli 10F'. Plasmid DNA was obtained from the received transformant E. coli using the kit QIAprep Spin Miniprep Kit (Qiagen Company). The sequencing reaction was performed with a set of Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company) in accordance with the instructions attached to the specified set, using as primers the primer-M (Applied Biosystems Japan Company) and primer 13Rev (Applied Biosystems Japan Company). In this reaction sequence was obtained using plasmid DNA as template. The product of the s reactions were analyzed using DNA sequencing machine A (Applied Biosystems Japan Company). In the received nucleotide sequence consisting of nucleotides 36-132 the nucleotide sequence shown in SEQ ID NO:9. The specified nucleotide sequence encodes the amino acid sequence consisting of amino acids 12-23 amino acid sequence shown in SEQ ID NO:18. In this regard, it was expected that the DNA encodes a protein (A1) of the present invention.

Then PCR was performed, similar to that described above, with a mixture of genomic polymerase Advantage-GC (Clontech Company) and using the chromosomal DNA obtained in example 3(2), as a matrix. As primers used a couple of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:37, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:38 (hereinafter referred to as "pair of 2 primers) or a pair of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:39, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:40 (hereinafter referred to as "a pair of 3 primers").

Then amplified using PCR DNA having a nucleotide sequence in which the 3'end extends after the nucleotide is shown as nucleotides 132 to the nucleotide sequence shown in SEQ ID NO:9. PCR was performed using as the atrice reaction solution, obtained using 2 pairs of primers, and using as primers a pair of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:41, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:38 (hereinafter referred to as "a pair of 4 primers"). Similarly, amplified using PCR DNA having a nucleotide sequence in which the 5'-end stretches after nucleotide is shown as a 36 nucleotide of the nucleotide sequence shown in SEQ ID NO:9. PCR was performed using as template the reaction solution obtained using 3 pairs of primers, and using as primers a pair of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:42, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:40 (hereinafter referred to as "pair of primers 5"). DNA size 2 TPN, amplified using 4 pairs of primers, and DNA size of 150 BP, amplified using 5 pairs of primers, cloned into the cloning vector TA pCR2.1 similar to that described above. Plasmid DNA was obtained from the received transformant E. coli using the kit QIAprep Spin Miniprep Kit (Qiagen Company). The sequencing reaction was performed with a set of Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company)in accordance with the instructions, attached to the specified set, using as primers the primer-M (Applied Biosystems Japan Company) and primer 13Rev (Applied Biosystems Japan Company) and the oligonucleotides shown in SEQ ID NO:43-50. In this reaction sequence was obtained using plasmid DNA as template. The reaction products were analyzed using DNA sequencing machine A (Applied Biosystems Japan Company). In the sequencing of the nucleotide sequence of the DNA size 2 TPN amplified using 4 pairs of primers, got nucleotide sequence consisting of nucleotides 133-1439 the nucleotide sequence shown in SEQ ID NO:9. Further, as a result of the sequencing of the nucleotide sequence of the DNA size of 150 BP amplified using 5 pairs of primers, got nucleotide sequence consisting of nucleotides 1-35 nucleotide sequence shown in SEQ ID NO:9. In the compounds obtained nucleotide sequences obtained nucleotide sequence shown in SEQ ID NO:9. In the specified nucleotide sequence was attended by two open reading frames. Thus, it contained the nucleotide sequence (SEQ ID NO:6), consisting of 1227 nucleotides including the stop codon) and encoding a 408 amino acid residues, as well as nucleotide is the selected (SEQ ID NO:15), consisting of nucleotide 201 (including the stop codon) and encoding a 66 amino acid residues. It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:1)encoded by the nucleotide sequence shown in SEQ ID NO:6, equal 45213 Yes. Further, the amino acid sequence encoded by the specified nucleotide sequence contained the amino acid sequence (SEQ ID NO:18), some of the sequencing of amino acids from the N-Terminus of the protein (A1) of the present invention, and amino acid sequence (SEQ ID NO:19), some of sequencing amino acids cleaved by trypsin fragments using mass spectrometric analysis. It was calculated that the molecular mass of this protein consisting of the amino acid sequence (SEQ ID NO:12)encoded by the nucleotide sequence shown in SEQ ID NO:15, equal 6818 Yes.

Example 4. Expression of the protein (A1) of the present invention in E. coli

(1) Obtaining a transformed E. coli with protein (A1) of the present invention

PCR was performed using as a template the chromosomal DNA obtained from Streptomyces phaeochromogenes IFO12898 in example 3(1), and using PCR system Expand High Fidelity (Roche Molecular Biochemicals Company). As primers used a couple of the oligonucleotide having the nucleotide sequence is lnost, shown in SEQ ID NO:51, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:52 (hereinafter referred to as "a pair of 19 primers"), or a pair of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:51, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:53 (hereinafter referred to as "a pair of 20 primers"). The reaction solution for PCR was brought up to 50 μl by adding each of the two primers, brought to 300 nm, 50 ng of the above chromosomal DNA, 5,0 ál dNTP mixture (a mixture of 2.0 mm each of the 4 types of dNTP), 5 μl 10x buffer Expand HF (containing MgCl2) and 0.75 μl of a mixture of enzymes Expand HiFi and distilled water. The PCR reaction conditions were as follows: after maintaining 97°C for 2 minutes, repeating 10 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 65°C for 30 seconds, then 72°C for 2 minutes, then spent 15 cycles, where each cycle consisted of maintaining 97°C for 15 sec, then 68°C for 30 s, and then 72°C for 2 minutes (which was added 20 seconds to maintain at 72°C for each cycle); then maintain 72°C for 7 minutes. After maintaining the reaction solution was applied on a 1% agarose gel electrophoresis. The area of the gel containing DNA with a size of approximately 1.2 TPN, extracted from the gel, which caused the reaction the solution, using a pair of 19 primers. The area of the gel containing DNA size approximately 1.5 TPN, extracted from the gel, which caused the reaction solution using a pair of 20 primers. DNA was purified from each of the extracted gels using a set of extraction from gels QIAquick (Qiagen Company) in accordance with the attached instructions. The resulting DNA ligated to the cloning vector TA pCR2.1 (Invitrogen Company) in accordance with the instructions attached to the specified vector, and introduced into E. coli 10F'. Plasmid DNA was obtained from the obtained transformants of E. coli using the kit QIAprep Spin Miniprep Kit (Qiagen Company). The sequencing reaction was performed with a set of Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company) in accordance with the instructions attached to the specified set, using as primers the primer-M (Applied Biosystems Japan Company) and primer 13Rev (Applied Biosystems Japan Company), the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:43, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:46. In this reaction sequence was obtained using plasmid DNA as template. The reaction products were analyzed using DNA sequencing machine A (Applied Biosystems Japan Company). On the basis of these results, the plasmid having the nucleotide sequence shown in SEQ ID NO:6, was n the induced pCR657, and a plasmid having the nucleotide sequence shown in SEQ ID NO:9, was named pCR657F.

In addition, the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:134, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:135, annealed together with the receipt of the linker (Fig. 47). Plasmid pKSN24R2 (Akiyoshi-ShibaTa, M. et al., Eur. J. Biochem. 224: P335 (1994)) were digested HindIII and XmnI. This linker was incorporated into the resulting DNA approximately 3 TPN Obtained plasmid was named pKSN2 (Fig. 4).

Then each of the plasmids pCR657 and pCR657F were digested with restrictase NdeI and HindIII. The cleavage products were subjected to agarose gel electrophoresis. The area of the gel containing DNA with a size of approximately 1.2 TPN, cut out of the gel, which was applied cleavage products pCR657. The area of the gel containing DNA size approximately 1.5 TPN, cut out of the gel, which was applied cleavage products pCR657F. These DNA was purified from each of the extracted gels using a set of extraction from gels QIAquick (Qiagen Company) in accordance with the attached instructions. Each of the obtained DNA and plasmid pKSN2, split NdeI and HindIII, ligated using set for ligating Ver. 1 (Takara Shuzo Company) in accordance with the instructions attached to the specified collection, and introduced into E. coli JM109. From the obtained transformants of E. coli were obtained plasma the major DNA. Their structure was analyzed. A plasmid containing the nucleotide sequence shown in SEQ ID NO:6, in which DNA size approximately 1.2 TPN encoding a protein (A1) of the present invention, inserted between the NdeI site and the HindIII site pKSN2, was named pKSN657. Next, a plasmid containing the nucleotide sequence shown in SEQ ID NO:9, in which DNA size approximately 1.5 TPN encoding a protein (A1) of the present invention, inserted between the NdeI site and the HindIII site pKSN2, was named pKSN657F. Each of the above plasmids pKSN657 and pKSN657F was introduced into E. coli JM109. The resulting E. coli transformants were named respectively JM109/pKSN657 and JM109/pKSN657F. Next, plasmid pKSN2 was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN2.

(2) protein Expression (A1) of the present invention in E. coli and the selection of the specified protein

Each of E. coli JM109/pKSN657, JM109/pKSN657F and JM109/pKSN2 were cultured over night at 37°C in 10 ml of medium TV (1,2% (wt./about.) tripton, 2,4% (wt./about.) yeast extract, and 0.4% (wt./about.) glycerol, 17 mm potassium dihydrophosphate, 72 mm dicale-phosphate)containing 50 μg/ml ampicillin. A milliliter (1 ml) of the obtained culture medium was transferred into 100 ml of medium TV containing 50 μg/ml of ampicillin, and cultured at 26°C. After reaching the OD660 approximately 0.5 was added 5-aminolevulinate acid to a final concentration of 500 μm, kultivirovanie is continued. After 30 minutes, then IPTG was added to a final concentration of 1 mm and were additionally cultured for 17 hours.

Cells were removed from each culture medium, washed with 0.1 M Tris-HCl-buffer (pH 7.5) and suspended in 10 ml of the above buffer containing 1 mm PMSF. The obtained cell suspension was subjected to 6 times the processing on the ultrasonic disintegrator (Sonifier (Branson Sonic Power Company)) for 3 minutes each time in terms of radiated power 3, load cycle 30%, to obtain solutions of cell lysates. After centrifugation of the solutions of cell lysates (1200 g, 5 minutes) supernatant were removed and centrifuged (150000 g, 70 minutes) to obtain a supernatant fractions (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN657, called "extract of E. coli pKSN657", the supernatant fraction obtained from E. coli JM109/pKSN657F, called "extract of E. coli pKSN657F", and the supernatant fraction obtained from E. coli JM109/pKSN2, called "extract of E. coli pKSN2"). One microliter (1 μl) of the above fractions supernatants were analyzed by electrophoresis in 15% -25% of the LTO-SDS page and stained Kumasi blue (hereinafter referred to as “STS”). The result is clearly more intense bands were detected in the extract of E. coli pKSN657 and in the extract of E. coli pKSN657F than in the extract of E. coli pKSN2, electrophoresis, corresponding to molecular mass of 47 kDa. In the extract of E. coli pKSN657F about what narazili more intense band, than in the extract of E. coli pKSN657. It was shown that E. coli JM109/pKSN657F expressed protein (A1) of the present invention to a higher extent than E. coli JM109/pKSN657.

(3) the Discovery of an ability to convert compound (II) into the compound (III)

Preparing the reaction solution, 30 μl and kept them in for 1 hour at 30°C. These reaction solutions consisted of 0.1 M potassium phosphate buffer (pH 7.0)containing 3 ppm of compound (II)labeled14S, 2 mm β-NADPH (hereinafter referred to as "component a") (Oriental Yeast Company), 0.2 mg/ml ferredoxin obtained from spinach (hereinafter referred to as "component B") (Sigma Company), 1 u/ml paradoxicality (hereinafter referred to as "component C") (Sigma Company) and 18 μl of the supernatant fraction obtained in example 4(2). Then I prepared and kept in this way the reaction solution without adding at least one component used in the above reaction solution, selected from the component A, component b and component C. was Added three microliters (3 ál) 2 N. HCl and 90 μl of ethyl acetate in each of these reaction solutions after the specified curing and stirred. The resulting reaction solution was centrifuged at 8000 g for extraction of 75 μl of an ethyl acetate layer. After drying, an ethyl acetate layer under reduced pressure, the residue was dissolved in 6.0 ál of ethyl acetate. Five microlitres(5,0 µl) of this solution was applied on TLC plate (silica gel TLC plates 60F 25420 cm x 20 cm, 0.25 mm thick, Merck Company). The TLC plate showed a mixture of 6:1:2 chloroform, acetic acid and ethyl acetate for about 1 hour. Then the solvent was allowed to evaporate. The TLC plate was exposed during the night on the imaging plate (Fuji Film Company). Then, the imaging plate was analyzed by the image Analyzer BAS2000 (Fuji Film Company). The presence of spots corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). These results are shown in table 6.

Table 6
Components of the reactionspot compound (III)
component acomponentcomponentextract
E. coli
the compound (II)labeled14
+++-+-
+++pKSN2+-
+++pKSN657++
-++pKSN657+-
+-+pKSN657+-
++-pKSN657++
+++pKSN657F++
-++pKSN657F+-
+-+pKSN657F+-
++- pKSN657F++

Example 5. Obtaining protein (A2) of the present invention

(1) preparation of the crude extract of cells

Frozen original material Saccharopolyspora taberi JCM 9383t was added to 10 ml of medium A (0.1% (wt./about.) glucose and 0.5% (wt./about.) tripton, a 0.5% (wt./about.) yeast extract, 0.1% (wt./about.) dicale-phosphate, pH 7.0) in a test tube of 10 ml and incubated with shaking at 30°C for 1 day to obtain a pre-culture. Eight milliliters (8 ml) pre-culture was added to 200 ml of medium and incubated while rotating the flask with septum 500 ml at 30°C for 2 days. Precipitation cells were obtained by centrifugation (3000 g, 10 min) 10 l of the resulting culture. The yeast cells suspended in 100 ml of medium (1% (wt./about.) glucose, 0.1% of meat extract, and 0.2% (wt./about.) tryptose)containing the compound (II) at 100 ppm, and incubated with the reciprocating swing in a Sakaguchi flask of 500 ml for 20 hours at 30°C. Precipitation cells were obtained by centrifugation (3000 g, 10 min) 10 l mixture. The obtained precipitation cells were washed twice in 1 l of 0.1 M potassium phosphate buffer (pH 7.0) to obtain 119 g of precipitation cells.

The yeast cells suspended in 0.1 M potassium phosphate buffer (pH 7.0) at 2 ml per 1 g of precipitation cells. Added 1 mm PMSF, 5 mm benzamidine·HCl, 1 m is EDTA, 3 µg/ml leupeptin, 3 μg/ml of pepstatin and 1 mm dithiothreitol. The solution of the cell lysate was obtained by destruction twice this suspension using a French press (1000 kg/cm2) (Ohtake Seisakusho). After centrifugation of the solution in the cell lysate (40000 g, 30 min) the supernatant was removed and centrifuged for 1 hour at 150000 g for extraction of the supernatant (hereinafter called "the crude cell extract").

(2) determination of the ability to convert compound (II) into the compound (III)

Prepared 30 μl of the reaction solution of 0.1 M potassium phosphate buffer (pH 7.0)containing 3 ppm of compound (II)labeled14S, 2.4 mm β-NADPH (hereinafter referred to as "component a") (Oriental Yeast Company), 0.5 mg/ml ferredoxin obtained from spinach (hereinafter referred to as "component B") (Sigma Company), 1 u/ml paradoxicality (hereinafter referred to as "component C") (Sigma Company) and 18 μl of crude cell extract obtained in example 5(1). The reaction solution was kept at 30°C for one hour. In addition, prepared and handled in the same way, the reaction solution without adding at least one component used in the above reaction solution, selected from the component A, component b and component C. was Added three microliters (3 ál) 2 N. HCl and 90 μl of ethyl acetate in each of these reaction solutions of th the specified curing and stirred. The resulting reaction solution was centrifuged at 8000 g for extraction of 75 μl of an ethyl acetate layer. After drying, an ethyl acetate layer under reduced pressure, the residue was dissolved in 6.0 ál of ethyl acetate. Five microlitres (5,0 µl) of this solution was applied on TLC plate (silica gel TLC plates 60F25420 cm x 20 cm, 0.25 mm thick, Merck Company). The TLC plate showed a mixture of 6:1:2 chloroform, acetic acid and ethyl acetate for about 1 hour. Then the solvent was allowed to evaporate. The TLC plate was exposed during the night on the imaging plate (Fuji Film Company). Then, the imaging plate was analyzed by the image Analyzer BAS2000 (Fuji Film Company). The presence of spots corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). These results are shown in table 7.

Table 7
Components of the reactionspot compound (III)
component acomponentcomponentthe crude cell extractthe compound (II)labeled14
+ ++-+-
++++++
-++++-
+--++-

(3) Fractionation of the crude cell extract

To the crude cell extract obtained in example 5(1), was added ammonium sulfate to 45% saturation. After stirring cooled in ice conditions were obtained supernatant by centrifugation for 10 minutes at 12000 g. After adding ammonium sulfate to the supernatant up to 55% saturation and mixing cooled in ice conditions the precipitate was obtained by centrifugation for 10 minutes at 12000 g. The residue was dissolved 32.5 ml of 20 mm bestreplibng buffer (pH 7.0). This solution was subjected to chromatography on a PD10 column (Amersham Pharmacia Company) and was suirable 20 mm mistresspopular buffer (pH 7) with the receipt of 45.5 ml fractions, containing proteins (hereinafter referred to as "fraction 45-55% of ammonium sulfate").

(4) Isolation of the protein (A2) of the present invention

Fraction 45-55% of ammonium sulfate obtained in example 5(3), was injected into the column HiLoad 26/10 Q-Sepharose HP (Amersham Pharmacia Company). Then after passing 100 ml of 20 mm bestreplibng buffer (pH 7.0) through the column, 20 mm listresponse buffer proceeded with a linear gradient of NaCl (NaCl gradient was 0,004 M/min, the concentration range of NaCl was from 0 M to 0.5 M, the flow rate was 8 ml/min) for fractional extraction with 30 ml fractions, eluruumiks when NaCl concentration from 0.25 to 0.26 M Then extracted fraction was subjected to chromatography on a PD10 column (Amersham Pharmacia Biotech Company) and was suirable 20 mm mistresspopular buffer (pH 7.0) to obtain fractions containing protein.

The extracted fraction was applied to a PD10 column (Amersham Pharmacia Biotech Company) with elution buffer (2 mm potassium phosphate buffer containing 1.5 mm NaCl, pH 7.0) to obtain fractions containing protein. Then these fractions were injected into the column SNT-I Bio-Scale Ceramic Hydroxyapatite Type I (Bio-Rad Company). Twenty milliliters (20 ml) buffer And passed through the column. Then the buffer And proceeded with a linear gradient of buffer B (100 mm potassium phosphate buffer containing 0.03 mm NaCl; linear gradient started at 100% buffer And up to 50% buffer over a period of 100 minutes, the flow rate was 2 ml/min) for fractional retrieve the ing 10 ml fractions, eluruumiks when the concentration of the buffer 23 to 25%. Then, the obtained fraction was applied to a PD10 column (Amersham Pharmacia Biotech Company) and was suirable 0.05 M potassium phosphate buffer (pH 7.0) to obtain fractions containing protein.

The fractions obtained were concentrated to approximately 770 μl using ultrafiltration membrane (Microcon YM-30, Millipore Company) and was injected into the column 75 PG HiLoad 16/60 Superdex (Amersham Pharmacia Biotech Company). 50 mm potassium phosphate buffer, containing 0.15 M NaCl (pH 7.0), proceeded (flow rate 1 ml/min) through the column. The eluate was fractionally 2 ml in each tube. Faction, eluruumina when the elution volumes more or less 61 ml fraction was extracted separately. The protein contained in each of these fractions were analyzed by electrophoresis in 10-20% of the LTO-PAG.

Instead of the crude cell extract in the reaction solution described in example 5(2), the fractions obtained were added and maintained in the presence of component A, component B, component C and the compound (II)labeled14With, like in example 5(2). The reaction solutions after aging were analyzed using TLC to study the intensity of the spots corresponding to compound (III)labeled14C. was Observed that the protein moves to the position 47 kDa in the above-described electrophoresis LTO-PAG, had fluctuations in the concentrations of these bands fractions, doba is mined in turn, which are parallel with the fluctuations of the intensity of the spots corresponding to compound (III). This protein was extracted from the gel LTO-SDS page and subjected to amino acid analysis using protein sequencing machine (Applied Biosystems Company, Procise 494HT, pulsed liquid method) for sequencing the N-terminal amino acid sequence. The result was obtained amino acid sequence shown in SEQ ID NO:20. Then, after cleavage of this protein by trypsin derived cleaved material was analyzed on a mass spectrometer (ThermoQuest Company, Ion Trap Mass Spectrometer LCQ, column: LC Packings Company PepMap C18, 75 µm x 150 mm, solvent A: 0.1% Of HOAc-H2O, solvent B: 0.1% of SPLA-methanol, gradient: a linear gradient, starting with elution with a mixture of 95% solvent a and 5% solvent and increasing concentrations up to 100% of the solvent for 30 minutes, the flow rate of 0.2 µl/min). The result was obtained sequence shown in SEQ ID NO:21.

Example 6. Obtaining DNA (A2) of the present invention

(1) preparation of chromosomal DNA Saccharopolyspora taberi JCM 9383t

Saccharopolyspora taberi JCM 9383t were cultured with shaking at 30°C for 1 to 3 days in 50 ml of YEME medium (0,3% (wt./about.) yeast extract and 0.5% (wt./about.) bactopeptone, of 0.3% (wt./about.) the malt extract, and 1.0% (wt./about.) glucose, 34% (wt./about.) sucrose and 0.2% (vol./about.) 2.5 M MgCl2N2About). CL is TCI collected. The obtained cells suspended in YEME medium containing 1,4% (wt./about.) glycine and 60 mm EDTA, and additionally incubated with shaking for one day. Cells were removed from culture medium. After washing once with distilled water them resuspendable buffer (100 mm Tris-HCl (pH 8.0), 100 mm EDTA, 10 mm NaCl) at 1 ml per 200 mg of cells. Added two hundred micrograms per milliliter (200 μg/ml) of lysozyme egg white. This cell suspension was shaken at 30°C for one hour. Then added 0.5% of LTOs and 1 mg/ml proteinase K. cell Suspension was incubated at 55°C for 3 hours. Cell suspension was extracted twice with a mixture of phenol, chloroform and isoamyl alcohol to extract each of the water layers. Then there was one extraction with a mixture of chloroform and isoamyl alcohol to extract the water layer. Chromosomal DNA was obtained by precipitation with ethanol from the aqueous layer.

(2) obtaining a library of chromosomal DNA Saccharopolyspora taberi JCM 9383t

Nineteen hundred micrograms (19 g) of the chromosomal DNA obtained in example 5(1), was digested 0,78 units restrictase Sau3AI at 37°C for 60 minutes. The obtained split the solution was separated by electrophoresis in 1% agarose gel. DNA size approximately 2.0 TPN extracted from the gel. This DNA was purified using a kit for extraction from gels QIAquick Gel Extraction (Qiagen Company)in accordance with the instructions, attached to the specified collection, and was concentrated by ethanol precipitation with 10 μl of solution containing the desired DNA. 8 microlitres (8 μl) of the DNA solution, 100 ng of plasmid vector pUC118 cleaved by the restriction enzyme BamHI and treated with dephosphorylation, and 12 μl of solution I of a set of ligation Ver. 2 (Takara Shuzo Company) were mixed and incubated for 3 hours at 16°C. E. coli DH5α was transformed using the solution for ligating. Transformants of E. coli were cultured over night at 37°C in an environment with LB-agar containing 50 mg/l ampicillin. The obtained colony was removed from the agar medium. Plasmids were extracted and they were called by the library of chromosomal DNA.

(3) Isolation of DNA (A2) of the present invention

PCR was performed using as a template the chromosomal DNA obtained in example 6(1) with the PCR system Expand HiFi (Boehringer Manheim Company) (Fig. 2). As primers used a couple of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:54, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:55 (hereinafter referred to as "a pair of 6 primers"). The nucleotide sequence shown in SEQ ID NO:54, designed based on the nucleotide sequence that encodes a N-terminal amino acid sequence shown in SEQ ID NO:20. Next, well leonidou sequence, shown in SEQ ID NO:55, designed based on the nucleotide sequence complementary to the nucleotide sequence that encodes the internal amino acid sequence shown in SEQ ID NO:21. The reaction solution for PCR was made up to 25 μl by the addition of 300 ng of the above chromosomal DNA of each of these 2 primers, brought to 7.5 pmol, 0.2 µl dNTP mixture (mixture 2 mm each of the 4 types of dNTP), and 2.5 µl of 10x buffer (containing MgCl2), 0,19 μl of a mixture of enzymes Expand HiFi and distilled water. The PCR reaction conditions were as follows: after maintaining 97°C for 2 minutes, repeating 10 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 65°C for 30 seconds, and then 72°C for 1 minute; then 15 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 65°C for 30 seconds, and then 72°C for 1 minute (20 seconds added to the conditioning at 72°C for each cycle); and then maintaining 72°C for 7 minutes. After this incubation, the reaction solution was applied on a 2% agarose gel for electrophoresis. The area of the gel containing DNA of approximately 800 BP were removed. DNA was purified from the extracted gel using a set of extraction from gels Qiagenquick (Qiagen Company) in accordance with the attached instructions. The resulting DNA ligated with cloning is m THE vector pCRII-TOPO (Invitrogen Company) in accordance with the instructions, attached to the specified vector, and introduced into E. coli 10F'. Plasmid DNA was obtained from the received transformant E. coli using a set of Qiagen Tip20 (Qiagen Company). The sequencing reaction was performed with a set of Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company) in accordance with the instructions attached to the specified set, using as primers the primer-M (Applied Biosystems Japan Company) and primer 13Rev (Applied Biosystems Japan Company). The reaction products were analyzed using DNA sequencing machine A (Applied Biosystems Japan Company). In the received nucleotide sequence consisting of nucleotides 36-819 the nucleotide sequence shown in SEQ ID NO:10. Nucleotides 37-60 the nucleotide sequence shown in SEQ ID NO:10, encodes part of amino acid sequence shown in SEQ ID NO:20. In this regard, it was expected that the DNA encodes a protein (A2) of the present invention.

Then PCR was performed using the chromosomal DNA obtained in example 6(2), as a matrix, similar to that described above with PCR system Expand HiFi. As primers used a couple of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:56, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:57 (hereinafter referred to as "a pair of 7 primers"). By carrying out PCR with such the primers amplified DNA having a nucleotide sequence in which the 5'-end extended after the nucleotide is shown as a 36 nucleotide of the nucleotide sequence shown in SEQ ID NO:10. Further, as the primers used a couple of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:58, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:59 (hereinafter referred to as "a pair of 8 primers"). By carrying out PCR with these primers amplified DNA having a nucleotide sequence in which the 3'end is extended after the nucleotide is shown as nucleotide 819 nucleotide sequence shown in SEQ ID NO:10. Each of the DNA size 1.3 TPN amplified using 7 pairs of primers, and DNA size of 0.4 TPN amplified using a pair of 8 primers, cloned into the cloning vector pCRII TA-TOPO. From the received transformant E. coli was obtained plasmid DNA using Qiagen Tip20 (Qiagen Company). The sequencing reaction was performed with a set of Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company) in accordance with the instructions attached to the specified set, using as primers the primer-M (Applied Biosystems Japan Company) and primer 13Rev (Applied Biosystems Japan Company) and the oligonucleotide shown in SEQ ID NO:60. The reaction products were analyzed with the use of the eat DNA sequencing machine A (Applied Biosystems Japan Company). In the sequencing of the nucleotide sequence of the DNA size 1.3 TPN amplified using 7 pairs of primers, got nucleotide sequence consisting of nucleotides 1-35 nucleotide sequence shown in SEQ ID NO:10. Further, in the sequencing of the nucleotide sequence of DNA with a size of 0.4 TPN amplified using primer pair 8, got a nucleotide sequence consisting of nucleotides 819-1415 the nucleotide sequence shown in SEQ ID NO:10. In the connection of the received sequences received the nucleotide sequence shown in SEQ ID NO:10. In the specified nucleotide sequence was attended by two open reading frames (ORF). Thus, it contained the nucleotide sequence (SEQ ID NO:7), consisting of 1206 nucleotides including the stop codon) and encoding a 401 amino acid residue, and the nucleotide sequence (SEQ ID NO:16), consisting of 198 nucleotides including the stop codon) and encoding a 65 amino acid residues. It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:2)encoded by the nucleotide sequence shown in SEQ ID NO:7, equal 43983 Yes. Further, the amino acid sequence encoded by the specified n is cleotide sequence, contained the amino acid sequence (SEQ ID NO:20), some of the sequencing of amino acids from the N-Terminus of the protein (A2) of the present invention, and amino acid sequence (SEQ ID NO:21), some of sequencing amino acids cleaved by trypsin fragments using mass spectrometric analysis. It was calculated that the molecular mass of this protein consisting of the amino acid sequence of SEQ ID NO:13, encoded by the nucleotide sequence shown in SEQ ID NO:16, equal 6707 Yes.

Example 7. Expression of the protein (A2) of the present invention in E. coli

(1) Obtaining a transformed E. coli with protein (A1) of the present invention

PCR was performed using as a template the chromosomal DNA derived from Saccharopolyspora taberi JCM 9383t in example 6(1), and using PCR system Expand HiFi (Boehringer Manheim Company). As primers used a couple of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:61, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:62 (hereinafter referred to as "a pair of 21 primers"), or a pair of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:61, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:63 (hereinafter referred to as "a pair of 22 primers"). The reaction process is for PCR was brought up to 50 μl by adding each of the two primers, brought to 300 nm, 50 ng of the above chromosomal DNA, 5,0 ál dNTP mixture (a mixture of 2.0 mm each of the 4 types of dNTP), 5 μl 10x buffer Expand HF (containing MgCl2) and 0.75 μl of a mixture of enzymes Expand HiFi and distilled water. The PCR reaction conditions were as follows: after maintaining 97°C for 2 minutes, repeating 10 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 60°C for 30 seconds, then 72°C for 1 minute; then 15 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 60°C for 30 seconds, and then 72°C for 1 minute (20 seconds added to maintain 72°C for each cycle); and then maintaining 72°C for 7 minutes. After this incubation, the reaction solution was applied on a 1% agarose gel electrophoresis. The area of the gel containing DNA with a size of approximately 1.2 TPN, extracted from the gel, which caused the reaction solution using a couple of 21 primers. The area of the gel containing DNA size approximately 1.4 TPN, extracted from the gel, which caused the reaction solution using a couple of 22 primers. DNA was purified from each of the extracted gels using a set of extraction from gels QIAquick (Qiagen Company) in accordance with the attached instructions. The resulting DNA ligated to the cloning vector pCRII-TOPO (Invitrogen Company) in accordance with the instructions is, included in the specified vector, and introduced into E. coli 10F'. Plasmid DNA was obtained from the obtained transformants of E. coli using a set of Qiagen Tip20 (Qiagen Company). Then the sequencing reaction was performed with a set of Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company) in accordance with the instructions attached to the specified set, using as primers the primer-M (Applied Biosystems Japan Company) and primer 13Rev (Applied Biosystems Japan Company), the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:56, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:64. The reaction products were analyzed using DNA sequencing machine A (Applied Biosystems Japan Company). On the basis of these results, the plasmid having the nucleotide sequence shown in SEQ ID NO:7, was named pCR923, and the plasmid having the nucleotide sequence shown in SEQ ID NO:10, was named pCR923F.

Then each of the plasmids pCR923 and pCR923F were digested with restrictase NdeI and HindIII. The cleavage products were subjected to agarose gel electrophoresis. The area of the gel containing DNA with a size of approximately 1.2 TPN, cut out of the gel, which was applied cleavage products pCR923. The area of the gel containing DNA size approximately 1.4 TPN, cut out of the gel, which was applied cleavage products pCR923F. These DNA was purified from each of the extraction is i.i.d. gels using a set of extraction from gels QIAquick (Qiagen Company) in accordance with the attached instructions. Each of the obtained DNA and plasmid pKSN2, split NdeI and HindIII, ligated using set for ligating Ver. 1 (Takara Shuzo Company) in accordance with the instructions attached to the specified collection, and introduced into E. coli JM109. From the obtained transformants of E. coli were obtained plasmid DNA. Their structures were analyzed. A plasmid containing the nucleotide sequence shown in SEQ ID NO:7, in which DNA size approximately 1.2 TPN encoding a protein (A2) of the present invention, inserted between the NdeI site and the HindIII site pKSN2, was named pKSN923. Next, a plasmid containing the nucleotide sequence shown in SEQ ID NO:10, in which DNA is approximately 1,4 TPN encoding a protein (A2) of the present invention, inserted between the NdeI site and the HindIII site pKSN2, was named pKSN923F. Each of the above plasmids pKSN923 and pKSN923F was introduced into E. coli JM109. The resulting E. coli transformants were named respectively JM109/pKSN923 and JM109/pKSN923F. Next, plasmid pKSN2 was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN2.

(2) the expression of the protein (A2) of the present invention in E. coli and the selection of the specified protein

Each of E. coli JM109/pKSN923 and JM109/pKSN923F and JM109/pKSN2 were cultured over night at 37°C in 10 ml of medium TV (1,2% (wt./about.) tripton, 2,4% (wt./about.) yeast extract, and 0.4% (wt./about.) glycerol, 17 mm potassium dihydrophosphate, 72 mm dicale-phosphate)containing 50 is kg/ml ampicillin. A milliliter (1 ml) of the obtained culture medium was transferred into 100 ml of medium TV containing 50 μg/ml of ampicillin, and cultured at 26°C. After reaching the OD660 approximately 0.5 was added 5-aminolevulinate acid to a final concentration of 500 μm, and the cultivation was continued. After 30 minutes, then IPTG was added to a final concentration of 1 mm and were additionally cultured for 17 hours.

Cells were removed from each culture medium, washed with 0.1 M Tris-HCl-buffer (pH 7.5) and suspended in 10 ml of the above buffer containing 1 mm PMSF. The obtained cell suspension was subjected to 6 times the processing on the ultrasonic disintegrator (Sonifier (Branson Sonic Power Company)) for 3 minutes each time in terms of radiated power 3, load cycle 30%, to obtain solutions of cell lysates. After centrifugation of the solutions of cell lysates (HD, 5 minutes) supernatant were removed and centrifuged (HD, 70 minutes) to obtain a supernatant fractions (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN923, called "extract of E. coli pKSN923", the supernatant fraction obtained from E. coli JM109/pKSN923F, called "extract of E. coli pKSN923F", and the supernatant fraction obtained from E. coli JM109/pKSN2, called "extract of E. coli pKSN2"). One microliter (1 μl) of the above fractions supernatants were analyzed by electrophoresis in 15%-25% of the LTO-PAG and krasivo STS. The result is clearly more intense bands were detected in the extract of E. coli pKSN923 and in the extract of E. coli pKSN923F than in the extract of E. coli pKSN2, electrophoresis, corresponding to molecular mass of 47 kDa. It was confirmed that E. coli JM109/pKSN923 and E. coli JM109/pKSN923F expressed protein (A2) of the present invention.

(3) the Discovery of an ability to convert compound (II) into the compound (III)

Preparing the reaction solution, 30 ml and kept for 10 minutes at 30°C. These reaction solutions consisted of 0.1 M potassium phosphate buffer (pH 7.0)containing 3 ppm of compound (II)labeled14S, 2 mm β-NADPH (hereinafter referred to as "component a") (Oriental Yeast Company), 0.2 mg/ml ferredoxin obtained from spinach (hereinafter referred to as "component B") (Sigma Company), 1 u/ml paradoxicality (hereinafter referred to as "component C") (Sigma Company) and 18 μl of the supernatant fraction obtained in example 7(2). Then I prepared and kept in this way the reaction solution without adding at least one component used in the above reaction solution, selected from the component A, component b and component C. was Added three microliters (3 ál) 2 N. HCl and 90 μl of ethyl acetate in each of these reaction solutions after the specified curing and stirred. The resulting reaction solution was centrifuged at 8000 g leisurecare 75 μl of an ethyl acetate layer. After drying, an ethyl acetate layer under reduced pressure, the residue was dissolved in 6.0 ál of ethyl acetate. Five microlitres (5,0 µl) of this solution was applied on TLC plate (silica gel TLC plates 60F25420 cm x 20 cm, 0.25 mm thick, Merck Company). The TLC plate showed a mixture of 6:1:2 chloroform, acetic acid and ethyl acetate for about 1 hour. Then the solvent was allowed to evaporate. The TLC plate was exposed during the night on the imaging plate (Fuji Film Company). Then, the imaging plate was analyzed by the image Analyzer BAS2000 (Fuji Film Company). The presence of spots corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). These results are shown in table 8.

Table 8
Components of the reactionspot compound (III)
component acomponentcomponentextract
E. coli
the compound (II)labeled14
+++-+ -
+++pKSN2+-
+++pKSN923++
-++pKSN923+-
+-+pKSN923+-
++-pKSN9231+
+++pKSN923F++
-++pKSN923F+-
+- +pKSN923F+-
++-pKSN923F++

Example 8. Obtaining protein (a10) this invention

(1) preparation of the crude extract of cells

Frozen original material Streptomyces griseolus ATCC 11796 was added to 250 ml of medium In (0,1% (wt./about.) glucose, 0.1% (wt./about.) meat extract, and 0.2% (wt./about.) tryptase) in a flask with septum 500 ml, and incubated with rotary swing at 30°C for 3 days to obtain a pre-culture. Forty milliliters (40 ml) pre-culture was added to 400 ml of medium and incubated with rotary swing triangular flask of 1 l at 30°C for 24 hours. After the termination of the cultivation, the culture was allowed to settle. Removed only 220 ml of supernatant. Similarly prepared 220 ml of fresh medium was added to the remaining 220 ml of culture medium to a volume of 440 ml of the Compound (II) was added to this amount to the number of 100 ppm, Cells were incubated with rotary shaking triangular flask of 1 l at 30°C for 40 hours. Precipitation cells were obtained by centrifugation (3000 g, 5 min) 2.6 liters of the obtained culture. The obtained precipitation cells PR is mawali 1 l of 0.1 M PIPES-NaOH buffer (pH 6.8) to obtain 26 g of precipitation cells.

The yeast cells suspended in 0.1 M PIPES-NaOH buffer (pH 6.8) at 3 ml per 1 g of cell precipitation and added 1 mm PMSF, 5 mm .HCl, 1 mm EDTA, 3 mg/ml leupeptin, 3 μg/ml of pepstatin and 1 mm dithiothreitol. The solution of the cell lysate was obtained by destruction twice this suspension using a French press (1000 kg/cm2) (Ohtake Seisakusho). After centrifugation of the solution in the cell lysate (40000 g, 30 min) the supernatant was removed and centrifuged for 1 hour at 150000 g for extraction of the supernatant (hereinafter called "the crude cell extract").

(2) determination of the ability to convert compound (II) into the compound (III)

Prepared 30 μl of the reaction solution of 0.1 M potassium phosphate buffer (pH 7.0)containing 3 ppm of compound (II)labeled14S, 2.4 mm β-NADPH (hereinafter referred to as "component a") (Oriental Yeast Company), 0.5 mg/ml ferredoxin obtained from spinach (hereinafter referred to as "component B") (Sigma Company), 1 u/ml paradoxicality (hereinafter referred to as "component C") (Sigma Company) and 18 μl of crude cell extract obtained in example 8(1). The reaction solution was kept at 30°C for one hour. In addition, prepared and handled in the same way, the reaction solution without adding at least one component used in the above reaction solution, selected from opponent And, component b and component C. was Added three microliters (3 ál) 2 N. HCl and 90 μl of ethyl acetate in each of these reaction solutions after the specified curing and stirred. The resulting reaction solution was centrifuged at 8000 g for extraction of 75 μl of an ethyl acetate layer. After drying, an ethyl acetate layer under reduced pressure, the residue was dissolved in 6.0 ál of ethyl acetate. Five microlitres (5,0 µl) of this solution was applied on TLC plate (silica gel TLC plates 60F25420 cm x 20 cm, 0.25 mm thick, Merck Company). The TLC plate showed a mixture of 6:1:2 chloroform, acetic acid and ethyl acetate for about 1 hour. Then the solvent was allowed to evaporate. The TLC plate was exposed during the night on the imaging plate (Fuji Film Company). Then, the imaging plate was analyzed by the image Analyzer BAS2000 (Fuji Film Company). The presence of spots corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). These results are shown in table 9.

Table 9
Components of the reactionspot compound (III)
component acomponentthe components is t the crude cell extractthe compound (II)labeled14
+++-+-
++++++
-++++-
+--++-

(3) Fractionation of the crude cell extract

To the crude cell extract obtained in example 8(1), was added ammonium sulfate to 45% saturation. After stirring cooled in ice conditions were obtained supernatant by centrifugation for 10 minutes at 12000 g. After adding ammonium sulfate to the supernatant up to 55% saturation and mixing cooled in ice conditions the precipitate was obtained by centrifugation for 10 minutes at 12000 g. This sieges which it was dissolved in 20 mm mistresspopular buffer (pH 7.0) to a quantity of 10 ml This solution was subjected to chromatography on a PD10 column (Amersham Pharmacia Company) and was suirable 20 mm mistresspopular buffer (pH 7.0) to obtain 14 ml fractions containing proteins (hereinafter referred to as "fraction 45-55% of ammonium sulfate").

(4) Isolation of the protein (a10) this invention

Fraction 45-55% of ammonium sulfate obtained in example 8(3), was injected into the column MonoQ HR 10/10 (Amersham Pharmacia Company). Then after flow 16 ml of 20 mm bestreplibng buffer (pH 7.0) through the column, 20 mm listresponse buffer proceeded with a linear gradient of NaCl (NaCl gradient was 0,00625 M/min, the concentration range of NaCl was from 0 M to 0.5 M, the flow rate was 4 ml/min) for fractional extract 15 ml fractions, eluruumiks when the concentration of NaCl from 0.28 to 0.31 M Then extracted fraction was subjected to chromatography on a PD10 column (Amersham Pharmacia Biotech Company) and was suirable 20 mm mistresspopular buffer (pH 7.0) to obtain fractions containing protein.

The extracted fraction was applied to a PD10 column (Amersham Pharmacia Biotech Company) with elution buffer (2 mm potassium phosphate buffer containing 1.5 mm NaCl, pH 7.0) to obtain fractions containing protein. Then these fractions were injected into the column SNT-I Bio-Scale Ceramic Hydroxyapatite Type I (BioRad Company). Fifty milliliters (50 ml) buffer And passed through the column. Then the buffer And proceeded with a linear gradient of buffer B (100 mm potassium phosphate buffer containing 0.03 mm NaCl; linear Gras is ient started at 100% buffer And up to 50% buffer over a period of 40 minutes, the flow rate was 5 ml/min) for fractional extraction fractions, eluruumiks when the concentration of the buffer from 16 to 31%. Then, the obtained fraction was applied to a PD10 column (Amersham Pharmacia Biotech Company) and was suirable 0.05 M potassium phosphate buffer (pH 7.0) to obtain fractions containing protein. The protein contained in each of these fractions were analyzed by electrophoresis in 10-20% of the LTO-PAG.

Instead of the crude cell extract in the reaction solution described in example 8(2), the fractions obtained were added and maintained in the presence of component A, component B, component C and the compound (II)labeled14With, like in example 8(2). The reaction solutions after aging were analyzed using TLC to study the intensity of the spots corresponding to compound (III)labeled14C. was Observed that the protein moves to the position 47 kDa in the above-described electrophoresis LTO-PAG, had fluctuations in the concentrations of these bands fractions, add one by one, which were parallel with the fluctuations of the intensity of the spots corresponding to compound (III). This protein was extracted from the gel LTO-page and subjected to cleavage by trypsin. Received cleaved material was analyzed on a mass spectrometer (ThermoQuest Company, Ion Trap Mass Spectrometer LCQ, column: LC Packings Company PepMap C18, 75 µm x 150 mm, solvent A: 0.1% Of HOAc-H2O, dissolve the spruce In: 0.1% of SPLA-methanol, gradient: linear gradient, starting with elution with a mixture of 95% solvent a and 5% solvent and increasing concentrations up to 100% of the solvent for 30 minutes, the flow rate of 0.2 µl/min). This has resulted in the sequence shown in SEQ ID NO:22-34.

Example 9. Obtaining chromosomal DNA of Streptomyces griseolus ATCC 11796

Streptomyces griseolus ATCC 11796 incubated with shaking at 30°C for 1 to 3 days in 50 ml of YEME medium (0,3% (wt./about.) yeast extract and 0.5% (wt./about.) bactopeptone, of 0.3% (wt./about.) the malt extract, and 1.0% (wt./about.) glucose, 34% (wt./about.) sucrose and 0.2% (vol./about.) 2.5 M MgCl2·6N2About). The cells were collected. The obtained cells suspended in YEME medium containing 1,4% (wt./about.) glycine and 60 mm EDTA, and additionally incubated with shaking for one day. Cells were removed from culture medium. After washing once with distilled water them resuspendable buffer (100 mm Tris-HCl (pH 8.0), 100 mm EDTA, 10 mm NaCl) at 1 ml per 200 mg of cells. Added two hundred micrograms per milliliter (200 μg/ml) of lysozyme egg white. This cell suspension was shaken at 30°C for one hour. Then added 0.5% of LTOs and 1 mg/ml proteinase K. cell Suspension were incubated at C for 3 hours. Cell suspension was extracted twice with a mixture of phenol, chloroform and isoamyl alcohol to extract each of Vodnikov. Then there was one extraction with a mixture of chloroform and isoamyl alcohol to extract the water layer. Chromosomal DNA was obtained by precipitation with ethanol from the aqueous layer.

Example 10. Obtaining DNA encoding this protein (a10), and expression in E. coli

(1) Obtaining a transformed E. coli having this DNA

PCR was performed using as a template the chromosomal DNA obtained from Streptomyces griseolus ATCC 11796 in example 9, and using PCR system Expand High Fidelity (Roche Molecular Biochemicals Company). As primers used a couple of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:79, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:80 (hereinafter referred to as "a pair of 23 primers"), or a pair of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:79, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:81 (hereinafter referred to as "a pair of 24 primers"). The reaction solution for PCR was brought up to 50 μl by adding these 2 primers, brought to 300 nm, 50 ng of the above chromosomal DNA, 5,0 ál dNTP mixture (a mixture of 2.0 mm each of the 4 types of dNTP), 5 μl 10x buffer Expand HF (containing MgCl2) and 0.75 μl of a mixture of enzymes Expand HiFi and distilled water. The PCR reaction conditions were as follows: after maintaining 97°C. in the course is 2 minutes repeat 10 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 65°C for 30 seconds, then 72°C for 2 minutes; then 15 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, and 68°C for 30 seconds, then 72°C for 2 minutes; (added 20 seconds to maintain 72°C for each cycle); and then maintaining 72°C for 7 minutes. After this incubation, each of the reaction solutions were applied to 1% agarose gel electrophoresis. The area of the gel containing DNA with a size of approximately 1.2 TPN, extracted from the gel, which caused the reaction solution using a pair of 23 primers. The area of the gel containing DNA size approximately 1.5 TPN, extracted from the gel, which caused the reaction solution using a pair of 24 primers. DNA was purified from each of the extracted gels using a set of extraction from gels QIAquick (Qiagen Company) in accordance with the attached instructions. The resulting DNA ligated to the cloning vector pCR2.1-TOPO (Invitrogen Company) in accordance with the instructions attached to the specified vector, and introduced into E. coli 10F'. Plasmid DNA was obtained from the obtained transformants of E. coli using the kit Qiaprep Spin Miniprep Kit (Qiagen Company). Then the sequencing reaction was performed with a set of Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company) in accordance with the instructions, p is imagemme to the specified set, using as primers primer-M (Applied Biosystems Japan Company) and primer 13Rev (Applied Biosystems Japan Company), the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:82, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:83. In the sequencing reactions used the obtained plasmid DNA as template. The reaction products were analyzed using DNA sequencing machine A (Applied Biosystems Japan Company). On the basis of these results, the plasmid having the nucleotide sequence shown in SEQ ID NO:84, was named pCR11796, and the plasmid having the nucleotide sequence shown in SEQ ID NO:85, was named pCR11796F. Two open reading frames (ORFS) present in the nucleotide sequence shown in SEQ ID NO:85. Thus, it contained the nucleotide sequence (SEQ ID NO:84), comprising 1221 nucleotides including the stop codon) and encoding a 406 amino acid residues (amino acid sequence shown in SEQ ID NO:5), and the nucleotide sequence consisting of 210 nucleotides (including the stop codon) and encoding a 69 amino acid residues.

Then each of the plasmids pCR11796 and pCR11796F were digested with restrictase NdeI and HindIII. The cleavage products were subjected to agarose gel electrophoresis. The area of the gel containing DNA size priblizitel is but 1,2 TPN, cut out of the gel, which was applied cleavage products pCR11796. The area of the gel containing DNA size approximately 1.5 TPN, cut out of the gel, which was applied cleavage products pCR11796F. These DNA was purified from each of the extracted gels using a set of extraction from gels Qiagen quick (Qiagen Company) in accordance with the attached instructions. Each of the obtained DNA and plasmid pKSN2, split NdeI and HindIII, ligated using set for ligating Ver. 1 (Takara Shuzo Company) in accordance with the instructions attached to the specified collection, and introduced into E. coli JM109. From the obtained transformants of E. coli were obtained plasmid DNA. Their structures were analyzed. A plasmid containing the nucleotide sequence shown in SEQ ID NO:84, in which DNA size approximately 1.2 TPN encoding a protein (a10) of the present invention, inserted between the NdeI site and the HindIII site pKSN2, was named pKSN11796. Next, a plasmid containing the nucleotide sequence shown in SEQ ID NO:85, in which DNA size approximately 1.5 TPN encoding a protein (a10) of the present invention, inserted between the NdeI site and the HindIII site pKSN2, was named pKSN11796F. Each of the above plasmids pKSN11796 and pKSN11796F was introduced into E. coli JM109. The resulting E. coli transformants were named respectively JM109/pKSN11796 and JM109/pKSN11796F. Next, plasmid pKSN2 was introduced into E. coli JM109. Received Tran the formant E. coli was named JM109/pKSN2.

(2) the Expression of this protein (a10) in E. coli and the selection of the specified protein

Each of E. coli JM109/pKSN11796, JM109/pKSN11796F and JM109/pKSN2 were cultured over night at 37°C in 10 ml of medium TV (1,2% (wt./about.) tripton, 2,4% (wt./about.) yeast extract, and 0.4% (wt./about.) glycerol, 17 mm potassium dihydrophosphate, 72 mm dicale-phosphate)containing 50 μg/ml ampicillin. A milliliter (1 ml) of the obtained culture medium was transferred into 100 ml of medium TV containing 50 μg/ml of ampicillin, and cultured at 26°C. After reaching the OD660 approximately 0.5 was added 5-aminolevulinate acid to a final concentration of 500 μm, and the cultivation was continued. After 30 minutes, then IPTG was added to a final concentration of 1 mm and were additionally cultured for 17 hours.

Cells were removed from each culture medium, washed with 0.1 M Tris-HCl-buffer (pH 7.5) and suspended in 10 ml of the above buffer containing 1 mm PMSF. The obtained cell suspension was subjected to 6 times the processing on the ultrasonic disintegrator (Sonifier (Branson Sonic Power Company)) for 3 minutes each time in terms of radiated power 3, load cycle 30%, to obtain solutions of cell lysates. After centrifugation of the solutions of cell lysates (1200 g, 5 minutes) supernatant were removed and centrifuged (150000 g, 70 minutes) to obtain a supernatant fractions (next, f is the share of the supernatant, obtained from E. coli JM109/pKSN11796, called "extract of E. coli pKSN11796", the supernatant fraction obtained from E. coli JM109/pKSN11796F, called "extract of E. coli pKSN11796F", and the supernatant fraction obtained from E. coli JM109/pKSN2, called "extract of E. coli pKSN2"). One microliter (1 μl) of the above fractions supernatants were analyzed by electrophoresis in 15% -25% of the LTO-SDS page and stained Kumasi blue (hereinafter referred to as “STS”). The result is clearly more intense bands were detected in the extract of E. coli pKSN11796 and in the extract of E. coli /pKSN11796F than in the extract of E. coli pKSN2, electrophoresis, corresponding to molecular mass of 45 kDa. In the extract of E. coli pKSN11796F found more intense band than in the extract of E. coli pKSN11796. It was shown that E. coli JM109/pKSN11796F expressed this protein (a10) to a higher extent than E. coli JM109/pKSN11796.

(3) the Discovery of an ability to convert compound (II) into the compound (III)

Preparing the reaction solution, 30 μl and kept them in for 1 hour at 30°C. These reaction solutions consisted of 0.1 M potassium phosphate buffer (pH 7.0)containing 3 ppm of compound (II)labeled14S, 2 mm β-NADPH (hereinafter referred to as "component a") (Oriental Yeast Company), 2 mg/ml ferredoxin obtained from spinach (hereinafter referred to as "component B") (Sigma Company), 0.1 u/ml paradoxicality (hereinafter referred to as "component C") (Sigma Company) and 18 μl fracc and supernatant, obtained in example 10(2). Then I prepared and kept in this way the reaction solution without adding at least one component used in the above reaction solution, selected from the component A, component b and component C. was Added three microliters (3 ál) 2 N. HCl and 90 μl of ethyl acetate in each of these reaction solutions after the specified curing and stirred. The resulting reaction solution was centrifuged at 8000 g for extraction of 75 μl of an ethyl acetate layer. After drying, an ethyl acetate layer under reduced pressure, the residue was dissolved in 6.0 ál of ethyl acetate. Five microlitres (5,0 µl) of this solution was applied on TLC plate (silica gel TLC plates 60F25420 cm x 20 cm, 0.25 mm thick, Merck Company). The TLC plate showed a mixture of 6:1:2 chloroform, acetic acid and ethyl acetate for about 1 hour. Then the solvent was allowed to evaporate. The TLC plate was exposed during the night on the imaging plate (Fuji Film Company). Then, the imaging plate was analyzed by the image Analyzer BAS2000 (Fuji Film Company). The presence of spots corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). These results are shown in table 10.

Table 10
Components of the reactionspot compound (III)
component acomponentcomponentextract
E. coli
the compound (II)labeled14
+++-+-
+++pKSN2+-
+++pKSN11796++
-++pKSN11796+-
+-+pKSN11796+-
++ -pKSN11796++
+++pKSN11796F++
-++pKSN11796F+-
+-+pKSN11796F+-
++-pKSN11796F++

Example 11. Obtaining DNA (A3) of the present invention

(1) preparation of chromosomal DNA of Streptomyces testaceus ATCC21469

Streptomyces testaceus ATCC21469 incubated with shaking at 30°C for 1 to 3 days in 50 ml of YEME medium (0,3% (wt./about.) yeast extract and 0.5% (wt./about.) bactopeptone, of 0.3% (wt./about.) the malt extract, and 1.0% (wt./about.) glucose, 34% (wt./about.) sucrose and 0.2% (vol./about.) 2.5 M MgCl2·6N2About). The cells were collected. The obtained cells suspended in YEME medium containing 1,4% (wt./about.) glycine and 60 mm EDTA, and advanced, and who was kopirovali with shaking for one day. Cells were removed from culture medium. After washing once with distilled water them resuspendable buffer (100 mm Tris-HCl (pH 8.0), 100 mm EDTA, 10 mm NaCl) at 1 ml per 200 mg of cells. Added two hundred micrograms per milliliter (200 μg/ml) of lysozyme egg white. This cell suspension was shaken at 30°C for one hour. Then added 0.5% of LTOs and 1 mg/ml proteinase K. cell Suspension was incubated at 55°C for 3 hours. Cell suspension was extracted twice with a mixture of phenol, chloroform and isoamyl alcohol to extract each of the water layers. Then there was one extraction with a mixture of chloroform and isoamyl alcohol to extract the water layer. Chromosomal DNA was obtained by precipitation with ethanol from the aqueous layer.

(2) Isolation of DNA (A3) of the present invention

PCR was performed using as a template the chromosomal DNA obtained in example 11(1). As primers used a couple of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:65, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:66 (hereinafter referred to as "a pair of 9 primers"). The reaction solution for PCR was brought up to 50 μl by adding 250 ng of the above chromosomal DNA of each of these 2 primers, brought to 200 nm, 4 μl of dNTP mixture (a mixture of 2.5 mm each of the 4 types of dNTP), 5 μl 10x ETaq buffer, a 0.5 ál of polymerase ExTaq (Takara Shuzo Company) and distilled water. The PCR reaction conditions were as follows: after maintaining 97°C for 2 minutes, repeating 30 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 60°C for 30 seconds, then 72°C for 90 seconds, and then maintain 72°C for 4 minutes. After this incubation, the reaction solution was applied to 0.8% agarose gel electrophoresis. The area of the gel containing DNA size approximately 1.4 TPN, was removed. DNA was purified from the extracted gel using a set of extraction from gels QIAquick (Qiagen Company) in accordance with the attached instructions. The resulting DNA ligated to the cloning vector TA pCR2.1 (Invitrogen Company) in accordance with the instructions attached to the specified vector, and introduced into E. coli 10F'. Plasmid DNA was obtained from the received transformant E. coli using the kit QIAprep Spin Miniprep Kit (Qiagen Company). The sequencing reaction was performed with a set of Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company) in accordance with the instructions attached to the specified set, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:67, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:68. In the sequencing reactions used the obtained plasmid in the quality of the ve matrices. The reaction products were analyzed using DNA sequencing machine A (Applied Biosystems Japan Company). He got the nucleotide sequence shown in SEQ ID NO:69. Two open reading frames (ORF) were present in the specified nucleotide sequence. Thus, it contained the nucleotide sequence consisting of 1188 nucleotides including the stop codon) and encoding a 395 amino acid residues, and the nucleotide sequence (SEQ ID NO:17), consisting of 195 nucleotides including the stop codon) and 64 encoding amino acid residue. It was calculated that the molecular mass of the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:17, equal 6666 Yes.

Example 12. The protein expression (A3) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (A3) of the present invention

PCR was performed using as a template the chromosomal DNA obtained in example 11(1), and using ExTaq polymerase (Takara Shuzo Company) in conditions similar to the conditions described above. As primers used a couple of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:70, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:71 (hereinafter referred to as "a pair of 10 primers"), or a couple of ol is gonucleotide, having the nucleotide sequence shown in SEQ ID NO:70, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:72 (hereinafter referred to as "a pair of 11 primers"). DNA size 1.2 TPN, amplified using 10 pairs of primers, and DNA 1.5 TPN, amplified using a pair of 11 primers, cloned into the cloning vector TA pCR2.1 according to the above described ways. Plasmid DNA was obtained from the obtained transformants of E. coli using the kit QIAprep Spin Miniprep Kit (Qiagen Company). The sequencing reaction was performed with a set of Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company) in accordance with the instructions attached to the specified set, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:67, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:68. In the sequencing reactions used the obtained plasmid DNA as template. The reaction products were analyzed using DNA sequencing machine A (Applied Biosystems Japan Company). In the result, it was shown that plasmid, cloned DNA, amplified by a pair of 10 primer has the nucleotide sequence shown in SEQ ID NO:8. It has been shown that plasmid, cloned DNA, amplified by a pair of 11 primers, who meet the nucleotide sequence, shown in SEQ ID NO:11. Two open reading frames (ORF) were present in the specified nucleotide sequence shown in SEQ ID NO:11. Thus, it contained the nucleotide sequence (SEQ ID NO:8), consisting of 1188 nucleotides including the stop codon) and encoding a 395 amino acid residues, and a nucleotide sequence consisting of 195 nucleotides including the stop codon) and 64 encoding amino acid residue. It was estimated that the molecular weight of the protein consisting of the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:8, equal 43752 Yes. With regard to the obtained plasmid, the plasmid having the nucleotide sequence shown in SEQ ID NO:8, was named pCR671, and the plasmid having the nucleotide sequence shown in SEQ ID NO:11, was named pCR671F.

Then each of the plasmids pCR671 and pCR671F were digested with restrictase NdeI and HindIII. The cleavage products were subjected to agarose gel electrophoresis. The area of the gel containing DNA with a size of approximately 1.2 TPN, cut out of the gel, which was applied cleavage products pCR671. The area of the gel containing DNA size approximately 1.5 TPN, cut out of the gel, which was applied cleavage products pCR671F. These DNA was purified from each of the extracted gels using a set of extraction from gels Qiagen quick (Qiaen Company) in accordance with the attached instructions. Each of the obtained DNA and plasmid pKSN2, split NdeI and HindIII, ligated using set for ligating Ver. 1 (Takara Shuzo Company) in accordance with the instructions attached to the specified collection, and introduced into E. coli JM109. From the obtained transformants of E. coli were obtained plasmid DNA. Their structures were analyzed. A plasmid containing the nucleotide sequence shown in SEQ ID NO:8, in which DNA is approximately 1200 BP, encoding a protein (A3) of the present invention, inserted between the NdeI site and the HindIII site pKSN2, was named pKSN671. Next, a plasmid containing the nucleotide sequence shown in SEQ ID NO:11, in which DNA is approximately 1400 BP, encoding a protein (A3) of the present invention, inserted between the NdeI site and the HindIII site pKSN2, was named pKSN671F. Each of the above plasmids pKSN671 and pKSN671F was introduced into E. coli JM109. The resulting E. coli transformants were named respectively JM109/pKSN671 and JM109/pKSN671F. Next, plasmid pKSN2 was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN2.

(2) protein Expression (A3) of the present invention in E. coli and the selection of the specified protein

Each of E. coli JM109/pKSN671, JM109/pKSN671F and JM109/pKSN2 were cultured over night at 37°C in 10 ml of medium TV (1,2% (wt./about.) tripton, 2,4% (wt./about.) yeast extract, and 0.4% (wt./about.) glycerol, 17 mm potassium dihydrophosphate, 72 mm dicale-phosphate)containing 50 mcgml ampicillin. A milliliter (1 ml) of the obtained culture medium was transferred into 100 ml of medium TV containing 50 μg/ml of ampicillin, and cultured at 26°C. After reaching the OD660 approximately 0.5 was added 5-aminolevulinate acid to a final concentration of 500 μm, and the cultivation was continued. After 30 minutes, then IPTG was added to a final concentration of 1 mm and were additionally cultured for 17 hours.

Cells were removed from each culture medium, washed with 0.1 M Tris-HCl-buffer (pH 7.5) and suspended in 10 ml of the above buffer containing 1 mm PMSF. The obtained cell suspension was subjected to 6 times the processing on the ultrasonic disintegrator (Sonifier (Branson Sonic Power Company)) for 3 minutes each time in terms of radiated power 3, load cycle 30%, to obtain solutions of cell lysates. After centrifugation of the solutions of cell lysates (1200 g, 5 minutes) supernatant were removed and centrifuged (150000 g, 70 minutes) to obtain a supernatant fractions (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN671, called "extract of E. coli pKSN671", the supernatant fraction obtained from E. coli JM109/pKSN671F, called "extract of E. coli pKSN671F", and the supernatant fraction obtained from E. coli JM109/pKSN2, called "extract of E. coli pKSN2").

(3) the Discovery of an ability to convert compound (II) into the compound (III)

Was prepared by the reaction of RA who works in 30 μl and kept them in for 1 hour at 30°C. These reaction solutions consisted of 0.1 M potassium phosphate buffer (pH 7.0)containing 3 ppm of compound (II)labeled14S, 2 mm β-NADPH (hereinafter referred to as "component a") (Oriental Yeast Company), 2 mg/ml ferredoxin obtained from spinach (hereinafter referred to as "component B") (Sigma Company), 0.1 u/ml paradoxicality (hereinafter referred to as "component C") (Sigma Company) and 18 μl of the supernatant fraction obtained in example 12(2). Then I prepared and kept in this way the reaction solution without adding at least one component used in the above reaction solution, selected from the component A, component b and component C. was Added three microliters (3 ál) 2 N. HCl and 90 μl of ethyl acetate in each of these reaction solutions after the specified curing and stirred. The resulting reaction solution was centrifuged at 8000 g for extraction of 75 μl of an ethyl acetate layer. After drying, an ethyl acetate layer under reduced pressure, the residue was dissolved in 6.0 ál of ethyl acetate. Five microlitres (5,0 µl) of this solution was applied on TLC plate (silica gel TLC plates 60F25420 cm x 20 cm, 0.25 mm thick, Merck Company). The TLC plate showed a mixture of 6:1:2 chloroform, acetic acid and ethyl acetate for about 1 hour. Then the solvent was allowed to evaporate. The TLC plate was exposed in the Techa is their night on the imaging plate (Fuji Film Company). Then, the imaging plate was analyzed by the image Analyzer BAS2000 (Fuji Film Company). The presence of spots corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). These results are shown in table 11.

Table 11
Components of the reactionspot compound (III)
component acomponentcomponentextract
E. coli
the compound (II)labeled14
++--+-
+++pKSN2+-
+++pKSN671++
-++ pKSN671+-
+-+pKSN671+-
++-pKSN671++
+++pKSN671F++
-++pKSN671F+-
+-+pKSN671F+-
++-pKSN671F++

Example 13. Getting this DNA (A9)

(1) preparation of chromosomal DNA of Streptomyces carbophilus SANK62585

Streptomyces carbophilus SANK62585 (FERM BP-1145) were incubated with shaking at 30°C in the course is 1 day in 50 ml of YEME medium (0,3% (wt./about.) yeast extract, of 0.5% (wt./about.) bactopeptone, of 0.3% (wt./about.) the malt extract, and 1.0% (wt./about.) glucose, 34% (wt./about.) sucrose and 0.2% (vol./about.) 2.5 M MgCl2N2About). Then cells were collected. The obtained cells suspended in YEME medium containing 1,4% (wt./about.) glycine and 60 mm EDTA, and additionally incubated with shaking for one day. Cells were removed from culture medium. After washing once with distilled water them resuspendable buffer (100 mm Tris-HCl (pH 8.0), 100 mm EDTA, 10 mm NaCl) at 1 ml per 200 mg of cells. Added two hundred micrograms per milliliter (200 μg/ml) of lysozyme egg white. This cell suspension was shaken at 30°C for one hour. Then added 0.5% of LTOs and 1 mg/ml proteinase K. cell Suspension was incubated at 55°C for 3 hours. Cell suspension was extracted twice with a mixture of phenol, chloroform and isoamyl alcohol to extract each of the water layers. Then there was one extraction with a mixture of chloroform and isoamyl alcohol to extract the water layer. Chromosomal DNA was obtained by precipitation with ethanol from the aqueous layer.

(2) DNA isolation (A9) this invention

PCR was performed using as a template the chromosomal DNA obtained in example 13(1). As primers used a couple of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:74, and oligonu is leotide, having the nucleotide sequence shown in SEQ ID NO:75 (hereinafter referred to as "a pair of 12 primers"), or a pair of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:76, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:77 (hereinafter referred to as "a pair of 13 primers"). The reaction solution for PCR was brought up to 50 μl by adding each of the 2 primers, brought to 200 nm, 250 ng of the above chromosomal DNA, 4 μl of dNTP mixture (a mixture of 2.5 mm each of the 4 types of dNTP), 5 μl 10x ExTaq buffer and 0.5 ál of polymerase ExTaq (Takara Shuzo Company) and distilled water. The PCR reaction conditions were as follows: after the maintenance of 95°C for 2 minutes, repeating 30 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 60°C for 30 seconds, then 72°C for 90 seconds, and then maintaining at 72°C for 4 minutes. After this incubation, the reaction solution was applied to 0.8% agarose gel electrophoresis. The area of the gel containing DNA with a size of approximately 500 BP were extracted from the gel, which caused the reaction solution for PCR, using a pair of 12 primers. The area of the gel containing DNA of approximately 800 BP was extracted from the gel, which caused the reaction solution for PCR, using a pair of 13 primers. DNA was purified from each of the extracted gel using the m kit for extraction from gels QIAquick (Qiagen Company) in accordance with the attached instructions. The resulting DNA ligated to the cloning vector TA pCR2.1 (Invitrogen Company) in accordance with the instructions attached to the specified vector, and introduced into E. coli 10F'. Plasmid DNA was obtained from the obtained transformants of E. coli using the kit QIAprep Spin Miniprep Kit (Qiagen Company). The sequencing reaction was performed with a set of Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company) in accordance with the instructions attached to the specified set, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:67, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:68. In the sequencing reactions used the obtained plasmid DNA as matrix. The reaction products were analyzed using DNA sequencing machine A (Applied Biosystems Japan Company). In the nucleotide sequence consisting of nucleotides 1-498 the nucleotide sequence shown in SEQ ID NO:78, obtained using DNA obtained from PCR using a pair of 12 primers. The nucleotide sequence consisting of nucleotides 469-1233 the nucleotide sequence shown in SEQ ID NO:78, obtained using DNA obtained from PCR using a pair of 13 primers. A plasmid having the nucleotide sequence of nucleotides 1-498 shown in SEQ ID NO:78, was named pCRSCA1. lasmea, having the nucleotide sequence of nucleotides 469-1233 shown in SEQ ID NO:78, was named pCRSCA2.

Example 14. Expression of this protein (A9) in E. coli

(1) Obtaining a transformed E. coli having this DNA (A9)

After receiving the plasmid in example 13(2) above plasmid pCRSCA1 were digested NdeI and NcoI and pCRSCA2 were digested NdeI and NcoI. The cleavage products were subjected to agarose gel electrophoresis. The area of the gel containing DNA with a size of approximately 500 BP were excised from the gel, which was applied cleavage products pCRSCA1. The area of the gel containing DNA of approximately 800 BP was cut out of the gel, which was applied cleavage products pCRSCA2. DNA was purified from each of the extracted gels using a set of extraction from gels QIAquick (Qiagen Company) in accordance with the attached instructions. Two types of DNA ligated with plasmid pKSN2, split NdeI and HindIII, using the set for ligating Ver. 1 (Takara Shuzo Company) in accordance with the instructions attached to the specified collection, and introduced into E. coli JM109. From the obtained transformants of E. coli were obtained plasmid DNA. Their structures were analyzed. A plasmid containing the nucleotide sequence shown in SEQ ID NO:78, in which DNA encoding a protein (A9) of the present invention, inserted between the NdeI site and the HindIII site pKSN2, was named pKSNSCA.

(2) xpressia this protein (A9) in E. coli and the selection of the specified protein

E. coli JM109/pKSNSCA were cultured over night at 37°C in 10 ml of medium TV (1,2% (wt./about.) tripton, 2,4% (wt./about.) yeast extract, and 0.4% (wt./about.) glycerol, 17 mm potassium dihydrophosphate, 72 mm dicale-phosphate)containing 50 μg/ml ampicillin. The obtained culture medium was transferred into 100 ml of medium TV containing 50 μg/ml ampicillin, so that OD660 was 0,2, and cultivated at 26°C. After reaching the OD660 approximately 2.0 was added 5-aminolevulinate acid to a final concentration of 500 μm, and the cultivation was continued. After 30 minutes, then IPTG was added to final concentration of 200 mm and was further cultured for 5 hours.

Cells were removed from each of the culture media, washed with 0.1 M Tris-HCl-buffer (pH 7.5) and suspended in 10 ml of the above buffer containing 1 mm PMSF. The obtained cell suspension was subjected to 6 times the processing on the ultrasonic disintegrator (Sonifier (Branson Sonic Power Company)) for 3 minutes each time in terms of radiated power 3, load cycle 30%, to obtain solutions of cell lysates. After centrifugation of the solutions of cell lysates (1200 g, 5 minutes) supernatant were removed and centrifuged (150000 g, 70 minutes) to obtain a supernatant fractions (hereinafter the supernatant fraction obtained from E. coli JM109/pKSNSCA, called “extract of E. coli pKSNSCA”).

b> (3) the Discovery of an ability to convert compound (II) into the compound (III)

Preparing the reaction solution, 30 ml and kept for 10 minutes at 30°C. These reaction solutions consisted of 0.1 M potassium phosphate buffer (pH 7.0)containing 3 ppm of compound (II)labeled14S, 2 mm β-NADPH (hereinafter referred to as "component a") (Oriental Yeast Company), 2 mg/ml ferredoxin obtained from spinach (hereinafter referred to as "component B") (Sigma Company), 0.1 u/ml paradoxicality (hereinafter referred to as "component C") (Sigma Company) and 18 μl of the supernatant fraction obtained in example 14(2). Then I prepared and kept in this way the reaction solution without adding at least one component used in the above reaction solution, selected from the component A, component b and component C. was Added three microliters (3 ál) 2 N. HCl and 90 μl of ethyl acetate in each of these reaction solutions after the specified curing and stirred. The resulting reaction solution was centrifuged at 8000 g for extraction of 75 μl of an ethyl acetate layer. After drying, an ethyl acetate layer under reduced pressure, the residue was dissolved in 6.0 ál of ethyl acetate. Five microlitres (5,0 µl) of this solution was applied on TLC plate (silica gel TLC plates 60F25420 cm x 20 cm, 0.25 mm thick, Merck Company). The TLC plate showed the shift of the d 6:1:2 chloroform, acetic acid and ethyl acetate for about 1 hour. Then the solvent was allowed to evaporate. The TLC plate was exposed during the night on the imaging plate (Fuji Film Company). Then, the imaging plate was analyzed by the image Analyzer BAS2000 (Fuji Film Company). The presence of spots corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). These results are shown in table 12.

Table 12
Components of the reactionspot compound (III)
component acomponentcomponentextract
E. coli
the compound (II)labeled14
+++-+-
+++pKSNSCA++

Example 15. The selection gene RuBPC soybean

After inoculation of the soybean cultivar Jack) soy cultivar the Wali at 27°C for 30 days and collected leaves. Two-tenths grams (0.2 g) - 0.3 g of the collected leaves were frozen with liquid nitrogen and ground in a pestle in a mortar. Then from the pulverized product was extracted total RNA in accordance with the instruction manual supplied with the solvent for the extraction of RNA ISOGEN (Nippon Gene Company). Further, the synthesized cDNA using the synthesis system of the first circuit Superscript for RT-PCR (Invitrogen Company), by performing procedures in accordance with the attached manual. Specifically, the first chain cDNA was synthesized using primer Oligo(dT)12-18provided this set, as a primer and total RNA soy as a matrix and addition of reverse transcriptase provided by this kit. Then using PCR amplified DNA encoding the chloroplast transit peptide of the small subunit of ribulose-1,5-bisphosphatase (hereinafter ribulose-1,5-bisphosphatase called "RuBPC") of the soybean cultivar Jack), followed by 12 amino acids of the Mature protein (hereinafter chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), sometimes referred to as "rSt"; and DNA encoding the chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), followed by 12 amino acids of the Mature protein, referred to as "this DNA rSt12"). In this PCR used the obtained cDNA as template and as primers the oligonucleotide having nucleotide the second sequence, shown in SEQ ID NO:86, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:87. In PCR used Taq polymerase LA ((Takara Shuzo Company). PCR was performed maintenance once 94°C for 3 minutes; 30 cycles, where each cycle consisted of maintaining 98°C for 25 seconds and then 68°C for 1 minute; and maintenance once 72°C for 10 minutes. Plasmid pCRrSt12 (Fig. 5) was obtained by embedding the amplified DNA at the site of the cloned PCR product from plasmid pCR2.1 (Invitrogen Company). Then the plasmid was introduced into competent cells of E. coli strain JM109 and selected resistant to ampicillin strains. Then, the nucleotide sequence of the plasmid contained in the selected resistant to ampicillin strains was determined using a set of Dye terminator cycle sequencing FS ready (PE Applied Biosystems Company) and DNA sequencing machine 373S (PE Applied Biosystems Company). The result has been the nucleotide sequence shown in SEQ ID NO:88. It was confirmed that the plasmid pCRrSt12 contain this DNA rSt12.

Example 16. Design chloroplast expression plasmids containing DNA (A1) of the present invention, for direct introduction

(1) Isolation of DNA (A1) of the present invention

DNA containing the nucleotide sequence shown in SEQ ID NO:6, amplified using PCR. PCR was performed using as template the genome of the th DNA of Streptomyces phaeochromogenes IFO12898 and using as primers the oligonucleotide, consisting of the nucleotide sequence shown in SEQ ID NO:93, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:94. Next, a DNA containing the nucleotide sequence shown in SEQ ID NO:9, amplified using PCR. This PCR was performed using as primers the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:93, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:95. In the specified PCR used PCR system Expand High Fidelity (Boehringer Company). After maintaining once 97°C for 2 minutes was performed 10 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 60°C for 30 seconds, and then 72°C for 1 minute; then spent 15 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 60°C for 30 seconds, and then 72°C for 1 minute (20 seconds added to maintain 72°C for each cycle); and then spent maintaining 72°C for 7 minutes. Plasmids pCR657ET (Fig. 6) and pCR657FET (Fig. 7) was obtained by integration of amplified DNA in the area of cloning of PCR product pCR2.1 (Invitrogen Company). In addition, other than when using the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:96, and the oligonucleotide consisting of nucleotidyltransferase, shown in SEQ ID NO:94, plasmid pCR657Bs (Fig. 8) was obtained using procedures similar to those described above. In addition, other than when using the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:96, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:97, plasmid pCR657FBs (Fig. 9) was obtained using procedures similar to those described above. Then the plasmid was introduced into competent cells of E. coli DH5α (Takara Shuzo Company) and selected resistant to ampicillin cells. Further, the nucleotide sequences of these plasmids contained in resistant to ampicillin strains was determined using kit BigDye terminator cycle sequencing Ready Reaction kit v.2.0 (PE Applied Biosystems Company) and DNA sequencing machine 3100 (PE Applied Biosystems Company). In the result, it was confirmed that the plasmid pCR657ET and pCR657Bs have the nucleotide sequence shown in SEQ ID NO:6. It was confirmed that the plasmid pCR657FET and pCR657FBs have the nucleotide sequence shown in SEQ ID NO:9.

(2) Construction of chloroplast expression plasmids with DNA (A1) of the present invention, for direct introduction - part (1)

A plasmid containing a chimeric DNA in which DNA (A1) of the present invention is attached immediately after the nucleotide sequence encoding the chloroplast trinitybaptist small subunit RuBPC of the soybean cultivar Jack) (hereinafter sometimes referred to as "sequence, encodes a chloroplast transit peptide"), without changing the reading frames in the codons, was designed as a plasmid for introduction of DNA (A1) of the present invention in a plant using a method with a gun for particles.

First, pCRrSt12 were digested with restrictase HindIII and kpni restriction sites. Was isolated DNA containing this DNA rSt12 invention. Then received DNA approximately 2640 P.N. removing about DNA size 40 BP of the plasmid vector pUC19 (Takara Shuzo Company) splitting restrictase HindIII and kpni restriction sites. Then, the 5'end of this DNA was dephosphorylated alkaline phosphatase calf intestine (Takara Shuzo Company). DNA containing this DNA rSt12 obtained from pCRrSt12, built into it by getting pUCrSt12 (Fig. 10). Then DNA containing DNA (A1) of the present invention, was isolated by splitting each of the plasmids pCR657ET and pCR657FET restrictase Eat and SacI. Each of the obtained DNA was embedded between the restriction site Esot and restriction site SacI pUCrSt12 to obtain plasmid pUCrSt657 (Fig. 11) and pUCrSt657F (Fig. 12)containing a chimeric DNA in which DNA (A1) of the present invention is attached immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons.

pBICR16G6PT (described in the previous examination of the patent application 2000-166577 Japan) Rassel is whether the restriction enzyme EcoRI for DNA extraction approximately 3 TPN (Hereinafter, the promoter contained in the DNA described in the above has not yet passed the examination of the patent application of Japan, referred to as the “promoter CR16G6”. In addition, the terminator contained in the DNA described in the above has not yet passed the examination of the patent application of Japan, also known as “terminator CR16”). After cleavage of the plasmid vector pUC19 (Takara Shuzo Company) by the restriction enzyme EcoRI, 5'-end of the specified DNA was dephosphorylated alkaline phosphatase calf intestine (Takara Shuzo Company). DNA size 3 TPN obtained from pBICR16G6PT, built into it to obtain plasmid pUCCR16G6-p/t (Fig. 13). pUCCR16G6-p/t was digested with restrictase HindIII and ScaI for DNA containing the promoter CR16G6. Then the splitting of the plasmid vector pUC19 (Takara Shuzo Company) restrictase HindIII and EcoRI DNA size 51 gel was removed and received the remaining DNA, consisting of 2635 P.N. Then 5'-end of the specified DNA was dephosphorylated alkaline phosphatase calf intestine (Takara Shuzo Company). The above DNA containing the promoter CR16G6 obtained from pUCCR16G6-p/t, and the linker NotI-EcoRI (Fig. 14), obtained from annealing of the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:89, with the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:90, built into it by getting pUCCR12G6-p/tΔ (Fig. 15). pUCCR12G6-p/tΔ were digested with restrictase NdeI and EcoRI for DNA extraction, having the th partial nucleotide sequence of the terminator CR16t. Next, the plasmid vector pUC19 (Takara Shuzo Company) was digested with restrictase HindIII and EcoRI to obtain a DNA size 2635 BP 5'-end of the specified DNA was dephosphorylated alkaline phosphatase calf intestine (Takara Shuzo Company). The above DNA having a partial nucleotide sequence of the terminator CR16t obtained from pUCCR12G6-p/tΔ, and the linker HindIII-NotI (Fig. 16)obtained by annealing of the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:91, with the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:92, was built in her getting pNdG6-ΔT (Fig. 17).

Then by splitting each of the plasmids pUCrSt657 and pUCrSt657F restrictase BamHI and SacI were isolated DNA containing the chimeric DNA in which DNA (A1) of the present invention is attached immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons. This DNA was built between the site restrictase BglII site of restrictase SacI plasmids pNdG6-ΔT to obtain plasmid pSUM-NdG6-rSt-657 (Fig. 18) and the plasmid pSUM-NdG6-rSt-657F (Fig. 19).

(3) the Design of a chloroplast expression plasmids with DNA (A1) of the present invention, for direct introduction - part (2)

A plasmid containing a chimeric DNA in which DNA (A1) of the present invention process is Diana immediately after the nucleotide sequence, encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without altering the reading frames in the codons, was designed as a plasmid for introduction of DNA (A1) of the present invention in a plant using a method with a gun for particles. First, after cleavage of the plasmid vector pKF19 (Takara Shuzo Company) by the restriction enzyme BspHI, the ends of the DNA was a small mistake by adding nucleotides to the double-stranded gap using DNA polymerase KOD (Toyobo Corporation). Plasmid pKF19ΔBs received by semiclassical obtained DNA DNA ligase T4. pUCrSt12 obtained in example 1 was digested with restrictase HindIII and kpni restriction sites. DNA containing this DNA rSt12, were isolated. Plasmid pKF19ΔBs were digested with restrictase HindIII and kpni restriction sites with obtaining DNA approximately 2160 BP 5'-end of the specified DNA was dephosphorylated alkaline phosphatase calf intestine (Takara Shuzo Company). DNA containing this DNA rSt12 obtained from pUCrSt12, built into it by getting pKFrSt12 (Fig. 20). Then the plasmids pCR657Bs and pCR657FBs obtained in example 16(1), uncoupled, each, restrictase BspHI and SacI emitting DNA containing DNA (A1) of the present invention. Each of these DNA was embedded between the BspHI restriction site and a restriction site SacI plasmids pKFrSt12 obtaining plasmids pKFrSt12-657 (Fig. 21) and the plasmid pKFrSt12-657F (Fig. 22), which contained the chimeric DNA, which DNA (A1) of the present invention was added immediately after the nucleotide sequence, encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without altering the reading frames in the codons.

Then each of the plasmids pKFrSt12-657 and pKFrSt12-657F were digested with restrictase BamHI and SacI to obtain DNA containing DNA (A1) of the present invention. Each of these DNA was embedded between the restriction site BglII and restriction site SacI plasmids pNdG6-ΔT obtained in example 16(2), to obtain plasmid pSUM-NdG6-rSt12-657 (Fig. 23) and pSUM-NdG6-rSt12-657F (Fig. 24), where the chimeric DNA in which DNA (A1) of the present invention was added immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without altering the reading frames in the codons, was connected right from the promoter CR16G6.

Example 17. Introduction DNA (A1) of the present invention in soybean

(1) Obtaining proliferative somatic embryos

After immersion pods of the soybean varieties: Fayette and Jack) in 1% solution of sodium hypochlorite for sterilization of extracted immature seeds. Shell seeds otslushival and pulled immature embryo, which has a diameter of 2-5 mm Embryonic axis derived immature embryo was cut with a scalpel to obtain immature cotyledon. This immature cotyledon divided the and 2 parts of cotyledon (cotyledons). Each part of cotyledon (each sameday) were placed in an environment for the development of somatic embryos, respectively. Environment for the development of somatic embryos was curing environment, where the 0.2% (wt./about.) Gerrit added to the environment Murashige-Skoog (described in Murashige T. and Skoog F., Physiol. Plant (1962) 15, p473; hereinafter referred to as "medium MS"), which was established pH 7.0 and which had added to her 180 μm 2,4-D and 30 g/l sucrose. 1 month after this room on Wednesday formed globular embryo was transplanted into the environment for the cultivation of somatic embryos. Environment for the cultivation of somatic embryos was curing environment, where the 0.2% (wt./about.) Gelrite was added to the medium MS, which was brought to pH 5.8 and which had 90 μm 2,4-D and 30 g/l sucrose, added to it. After this globular embryos were transferred into fresh medium for the cultivation of somatic embryos 5-8 times at intervals of 2-3 weeks. Each of the culture conditions, using the above environment for the development of somatic embryos and the environment for the cultivation of somatic embryos was carried out at 23 hours of light with 1 hour of darkness and at 23-25°C during the day.

(2) the introduction of the gene proliferative somatic embryos

After transplantation globular embryo obtained in example 17(1), on a fresh environment for the cultivation of somatic embryos and the cult is growing in 2-3 days globular embryo used for the introduction of a gene. The plasmid pSUM-NdG6-rSt657, pSUM-NdG6-rSt657F, pSUM-NdG6-rSt12657 and pSUM-NdG6-rSt12657F was applied in the form of a coating on gold particles with a diameter of 1.0 μm for introduction of a gene from using guns for particles. The number of plasmids was of 1.66 μg to 1 mg of gold particles. After the introduction of a gene germ were additionally cultured for 2-3 days. Each of the culture conditions were performed for 23 hours of light with 1 hour of darkness and at 23-25°C during the day.

(3) the Selection of somatic embryos with hygromycin

Globular embryos after injection of the gene obtained in example 17(2), were transferred to selective medium for somatic embryos. Selective medium for somatic embryos was cured, and 0.2% (wt./about.) Gerrit and 15 mg/l of hygromycin was added to the medium MS, which was brought to pH 5.8 and which had added to her 2,4-D and 30 g/l sucrose. Survivors of globular embryos were then transplanted to fresh selective medium for somatic embryo 5-8 times at intervals of 2-3 weeks. At this time, selective medium for somatic embryos was curing environment, where the 0.2% (wt./about.) Gelrite and 30 mg/l of hygromycin was added to the medium MS, which was brought to pH 5.8 and which had added to her 90 μm 2,4-D and 30 g/l sucrose. Each of the culture conditions when using the above selective environment for soma is practical embryos was carried out at 23 hours of light with 1 hour of darkness and at 23-25°C during the day.

(4) the Selection of somatic embryos with compound (II)

Globular embryos after injection of the gene obtained in example 17(2), were transferred to selective medium for somatic embryos. Selective medium for somatic embryos was cured, and 0.2% (wt./about.) Gerrit and 0.1 mg/l of compound (II) was added to the medium MS, which was brought to pH 5.8 and which had added to her 90 μm 2,4-D and 30 g/l sucrose. Survivors of globular embryos were then transplanted to fresh selective medium for somatic embryo 5-8 times at intervals of 2-3 weeks. At this time, selective medium for somatic embryos was curing environment, where the 0.2% (wt./about.) Gelrite and 0.3-1 mg/l of compound (II) was added to the medium MS, which was brought to pH 5.8 and which had added to her 90 μm 2,4-D and 30 g/l sucrose. Each of the culture conditions when using the above selective medium for somatic embryos was carried out at 23 hours of light with 1 hour of darkness and at 23-25°C during the day.

(5) Regeneration of plants from somatic embryo

Globular embryos, selected in example 17(3) or 17(4), transplanted into an environment for the development and cultured for 4 weeks at 23 hours light and 1 hour dark and at 23-25°C during the day. Environment for development is curable environment where 0,8% (wt./about.) agar (Wak Pure Chemical Industries, Lts., application for plant tissue culture) are added to the medium MS, which is brought to pH 5.8 and which has added 60 g/l maltose. After 6-8 weeks, then get the type embryos cotyledons from white to yellow. These type embryos cotyledons are transplanted into the environment for germination and cultured for 2 weeks. Environment for germination is curable environment, where the 0.2% (wt./about.) Gerrit added to the medium MS, which is brought to pH 5.8 and has added thereto at a concentration of 30 g/l sucrose. The result can be obtained from soy, which develops leaves and has roots.

(6) Acclimate and culturing the regenerated plants

Soy, obtained in example 17(5), are transplanted into the garden soil and accelerate in the incubation chamber at 23 hours light and 1 hour dark and 23-25°C during the day. After 2 weeks of this rooted plant is transferred into a vessel having a diameter of 9 cm, and cultured at room temperature. The cultivation conditions at room temperature using natural light at 23-25°C during the day. After two to four months after that, collect seeds of soybean.

(7) Evaluation of resistance to the herbicide compound (II)

The leaves of the regenerated plants are harvested and divided equally into 2 piece along the main vein. The compound (II) spray the whole surface of one of the pieces of sheet. Another piece left untreated. These pieces listw place on Wednesday, MS, containing 0.8% agar, and allowed to stand at room temperature for 7 days in a bright place. Then, each piece pound with the pestle in the mortar in 5 ml of 80% aqueous acetone for extraction of chlorophyll. The liquid extract was diluted 10 times with 80% aqueous acetone solution and measure the optical density at 750 nm, 663 nm and 645 nm to calculate the total chlorophyll content according to the method Mackenney G., J. Biol. Chem. (1941) 140, p 315. The degree of resistance to the compound (II) can relatively be evaluated expression in the percentiles of the total chlorophyll-a processed piece of the total chlorophyll-a raw piece of paper.

Further, the ground stuff in a plastic vessel having a diameter of 10 cm and a depth of 10 see the Seeds of the above plants are sown and cultivated in the greenhouse. Preparing an emulsion by mixing 5 parts of compound (II), 6 parts sorpol3005X (Toho chemicals) and 89 parts of xylene. A certain amount of this emulsion is diluted with water containing 0.1% (vol./about.) providing an adhesion agent, in a ratio of 1000 liters per 1 hectare, and distribute uniformly gun for spraying on all sides of the foliage from the top of the plants cultivated in the above-described vessel. After cultivation of these plants for 16 days in the greenhouse is slidout damage to these plants and to assess the sustainability of the compound (II).

Example 18. Design chloroplast expression plasmids with DNA (A1) of the present invention, for introduction using Agrobacterium

Designed plasmid for introduction of DNA (A1) of the present invention in a plant using the method using Agrobacterium. First, after splitting the binary plasmid vector pBI121 (Clontech Company) by the restriction enzyme NotI ends of DNA was a small mistake by adding nucleotides to docebocore the gap using DNA polymerase I (Takara Shuzo Company). For simocyclinone used DNA ligase T4. After splitting the resulting plasmid with the restriction enzyme EcoRI, the DNA ends were a small mistake by adding nucleotides to the double-stranded gap using DNA polymerase I (Takara Shuzo Company). For simocyclinone used DNA T4 ligase to obtain plasmid pBI121Δ NotIEcoRI. After splitting this plasmid HindIII 5'-end of the DNA was dephosphorylated alkaline phosphatase calf intestine (Takara Shuzo Company). The linker HindIII-NotI-EcoRI (Fig. 25)obtained by the annealing of the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:98, the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:99, embedded in it. A binary plasmid vector pBI121S (Fig. 26) was obtained by semiclassical. The indicated plasmid has a structure in which the linker HindIII-NotI-EcoRI was built in the direction in cat the rum restriction site HindIII, the restriction site NotI and the restriction site EcoRI are arranged in order from the location adjacent to the gene of β-glucuronidase.

Then each of the plasmid pSUM-NdG6-rSt-657 and pSUM-NdG6-rSt-657F were digested with restrictase HindIII and EcoRI to obtain from each of them chimeric DNA in which DNA (A1) of the present invention is attached immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons. These DNA was embedded between the restriction site HindIII and the restriction site EcoRI above binary plasmid vector pBI121S with obtaining the plasmid pBI-NdG6-rSt-657 (Fig. 27) and pBI-NdG6-rSt-657F (Fig. 28). Further, each of the above plasmid pSUM-NdG6-rSt12-657 and pSUM-NdG6-rSt12-657F were digested with restrictase HindIII and EcoRI to obtain from each of them chimeric DNA in which DNA (A1) of the present invention is attached immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without altering the reading frames in the codons. These DNA was embedded between the restriction site HindIII and the restriction site EcoRI above binary plasmid vector pBI121S with obtaining the plasmid pBI-NdG6-rSt12-657 (Fig. 29) and pBI-NdG6-rSt12-657F (Fig. 30).

Example 19. Introduction DNA (A1) of this is bretania in tobacco

DNA (A1) of the present invention was introduced into tobacco using the method using Agrobacterium, using the plasmid pBI-NdG6-rSt-657, plasmid pBI-NdG6-rSt-657F, the plasmid pBI-NdG6-rSt12-657 and the plasmid pBI-NdG6-rSt12-657F obtained in example 18.

First, the plasmid pBI-NdG6-rSt-657, pBI-NdG6-rSt-657F, pBI-NdG6-rSt12-657 and pBI-NdG6-rSt12-657F was introduced into Agrobacterium tumefaciens LBA4404 (Clontech Company) respectively. The transformed Agrobacterium strains carrying pBI-NdG6-rSt-657, pBI-NdG6-rSt-657F, pBI-NdG6-rSt12-657 or pBI-NdG6-rSt12-657F, were isolated by culturing the obtained transformants in a medium with LB-agar (0.5% of yeast extract, 1.0% lactotropes and 0.5% NaCl)containing 300 mg/l streptomycin, 100 mg/l rifampicin and 25 mg/l kanamycin, and the selection of resistant colonies.

Then, in accordance with the method described in the Manual for Gene Manipulation of Plant (Hirofumi UCHIMIYA, Kodan-sha Scientific, 1992), this gene was introduced into tobacco. The Agrobacterium strains carrying the above plasmids, cultured, each at 28°C overnight in LB-medium containing 300 mg/l streptomycin, 100 mg/l rifampicin and 25 mg/l kanamycin, and then pieces of tobacco leaves (Nicotiana tabacum strain SR1), cultivated sterile, immersed in liquid culture medium. Pieces of leaves were planted and cultivated at room temperature for 2 days on light in agar medium MS (MS inorganic salts, MS vitamins, 3% sucrose and 0.8% agar; described in Murashige T. and Skoog F., Physiol. Plant. (1962) 15, p 473)containing 0.1 mg/l naphthyloxy the Oh of the acid and 1.0 mg/l of benzylaminopurine. Then the pieces of leaves were washed in sterilized water and were cultured for 7 days with medium MS agar containing 0.1 mg/l naphthylacetic acid, 1.0 mg/l of benzylaminopurine and 500 mg/l Cefotaxime. Then, these pieces of leaves were transplanted and cultivated in the medium MS with agar containing 0.1 mg/l naphthylacetic acid, 1.0 mg/l benzylaminopurine, 500 mg/l Cefotaxime and 100 mg/l kanamycin. Cultivation was performed continuously for 4 months when transplanting pieces of leaves on fresh medium of the same composition at intervals of 4 weeks. At this time unattached buds developing from these pieces of leaves were transplanted and were rooted on medium MS agar containing 300 mg/l Cefotaxime and 50 mg/l kanamycin to obtain regenerated rastignac. The regenerated plant was transplanted and cultivated in an environment with MS agar containing 50 mg/l kanamycin, obtaining transgenic tobacco, which was introduced to the area of the T-DNA pBI-NdG6-rSt-657, pBI-NdG6-rSt-657F, pBI-NdG6-rSt12-657 or pBI-NdG6-rSt12-657F, respectively.

Next, plasmid PBI121S obtained in example 18, was introduced into tobacco by the method using Agrobacterium. The transformed Agrobacterium strain carrying pBI121S, was isolated as described above, except that the used plasmid pBI121S instead of pBI-NdG6-rSt-657, pBI-NdG6-rSt-657F, pBI-NdG6-rSt12-657 and pBI-NdG6-rSt12-657F. Then transgenic tobacco, which was rst is Yong district T-DNA plasmids pBI121S, received, similar to that described above, using a given transformed Agrobacterium.

Three leaves were taken from transgenic tobacco. Each sheet was divided into 4 pieces, and each piece had a width of 5-7 mm. Each piece of the sheet was dropped off on Wednesday with MS agar containing 0.1 mg/l of compound (II), and cultured in the light at room temperature. On the 7th day of cultivation was observed herbicide damage to each of these pieces of paper. Pieces of leaves obtained from tobacco, which was introduced control DNA (area T-DNA plasmids pBI121S), became white and was savagely. In contrast, pieces of leaves, obtained from tobacco, which was introduced DNA (A1) of the present invention (the area of the T-DNA plasmid pBI-NdG6-rSt-657, plasmid pBI-NdG6-rSt12-657, plasmid pBI-NdG6-rSt-657F or plasmid pBI-NdG6-rSt12-657F), grew continuously.

Example 20. Introduction DNA of the present invention in a plant

Designed plasmid for introduction of DNA (A2) of the present invention in a plant using guns for particles and using the method using Agrobacterium. First, DNA (A2) of the present invention having the nucleotide sequence shown in SEQ ID NO:7, amplified using PCR. PCR was performed using as template genomic DNA Saccharopolyspora taberi JCM9383t and using as primers the oligonucleotide consisting of the Amu is ateneu sequence, shown in SEQ ID NO:100, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:101. In the specified PCR used PCR system Expand High Fidelity (Boehringer Company). After maintaining once 97°C for 2 minutes was performed repeating 10 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 60°C for 30 seconds, and then 72°C for 60 seconds; then 15 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 60°C for 30 seconds, and then 72°C for 1 minute (20 seconds added to maintain 72°C for each cycle); and then maintaining 72°C for 7 minutes. Plasmid pCR923Sp (Fig. 31) was obtained by integration of amplified DNA in the area of cloning of PCR product pCR2.1-TOPO (Invitrogen Company). Then, this plasmid was introduced into competent cells of E. coli JM109 (Takara Shuzo Company) and selected resistant to ampicillin cells. Further, the nucleotide sequence of the plasmid contained in resistant to ampicillin strains was determined using kit BigDye terminator cycle sequencing Ready Reaction kit v.2.0 (PE Applied Biosystems Company) and DNA sequencing machine 373S (PE Applied Biosystems Company). In the result, it was confirmed that the plasmid pCR923Sp has the nucleotide sequence shown in SEQ ID NO:7.

Plasmid pKFrSt12 constructed in example 16(3), were digested with restrictase BamHI and SacI for DNA containing this D Is To rSt12. Specified DNA was embedded between the restriction site BglII and restriction site SacI pNdG6-ΔT obtained in example 16(2), to obtain plasmid pNdG6-rSt12 (Fig. 32). Plasmid pCR923Sp were digested with restrictase SphI and kpni restriction sites with obtaining DNA containing DNA (A2) of the present invention. Plasmid pNdG6-rSt12 were digested with restrictase SphI and kpni restriction sites to remove DNA that encodes 12 amino acids of the Mature protein of the small subunit of RuBPC of the soybean cultivar Jack). In their place were built above DNA containing DNA (A2) of the present invention, obtained from a plasmid pCR923Sp, obtaining pSUM-NdG6-rSt-923 (Fig. 33), where the promoter CR16G6 was attached right from the chimeric DNA in which the DNA was added directly after the sequence encoding the chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frame at codon.

Then plasmid pCR923Sp were digested with restriction enzyme SphI. After blunting the ends of the resulting DNA DNA polymerase KOD specified DNA was further digested with restriction enzyme kpni restriction sites for DNA containing DNA (A2) of the present invention. Plasmid pKFrSt12 obtained in example 16(3), were digested with restriction enzyme BspHI. After blunting the ends of the resulting DNA DNA polymerase KOD specified DNA was further digested with restriction enzyme kpni restriction sites to remove DNA approximately 20 BP In its place was built the above DNA, containing the th DNA (A2) of the present invention, derived from plasmids pCR923Sp, obtaining the plasmid pKFrSt12-923 (Fig. 34)containing a chimeric DNA in which DNA (A2) of the present invention was added immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without altering the reading frames in the codons. pKFrSt12-923 was digested with restrictase SphI and kpni restriction sites with obtaining chimeric DNA in which the joined DNA (A2) of the present invention and DNA encoding the first 12 amino acids of the Mature protein of the small subunit of RuBPC of the soybean cultivar Jack). Plasmid pNdG6-rSt12 were digested with restrictase SphI and kpni restriction sites to remove DNA that encodes 12 amino acids of the Mature protein of the small subunit of RuBPC soybean (Jack). In its place was built the above chimeric DNA derived from the plasmid pKFrSt12-923, obtaining the plasmid pSUM-NdG6-rSt12-923 (Fig. 35), in which the promoter CR16G6 attached right from the chimeric DNA in which the above DNA containing DNA (A2) of the present invention, attached directly after the sequence encoding the chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without altering the reading frame at codon.

DNA (A2) of the present invention was introduced into soybean way, using the gun for particles, procedures, and anticrime procedures of the method, described in example 17, using the obtained plasmid pSUM-NdG6-rSt-923 and pSUM-NdG6-rSt12-923.

The above plasmid pSUM-NdG6-rSt-923 was digested with restrictase HindIII and EcoRI for DNA containing a chimeric DNA in which the DNA containing DNA (A2) of the present invention, attached directly after the sequence encoding the chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frame at codon. As and when receiving pBI-NdG6-rSt657 in example 18, the above-mentioned DNA containing the chimeric DNA derived from the plasmid pSUM-NdG6-rSt-923, was built between the restriction site HindIII and the restriction site EcoRI binary vector pBI121S obtaining pBI-NdG6-rSt-923 (Fig. 36). Further, the above plasmid pSUM-NdG6-rSt12-923 was digested with restrictase HindIII and EcoRI for DNA containing a chimeric DNA in which the DNA containing DNA (A2) of the present invention, attached directly after the sequence encoding the chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without altering the reading frames in the codons. This chimeric DNA obtained from pSUM-NdG6-rSt12-923, was built between the restriction site HindIII and the restriction site EcoRI binary vector pBI121S obtaining pBI-NdG6-rSt12-923 (Fig. 37).

Each of the plasmid pBI-NdG6-rSt-923 and pBI-NdG6-rSt12-923 enter and in Agrobacterium tumefaciens LBA4404. The obtained transformants were cultured in LB-medium containing 300 μg/ml of streptomycin, 100 μg/ml rifampicin and 25 μg/ml kanamycin. Transformants were selected for isolation of Agrobacterium strains carrying pBI-NdG6-rSt-923 or pBI-NdG6-rSt12-923.

Pieces of sterile leaves of cultivated tobacco were injected with each of the Agrobacterium strain carrying pBI-NdG6-rSt-923, and Agrobacterium strain carrying pBI-NdG6-rSt12-923. Tobacco, which was introduced DNA (A2) of the present invention, was obtained by procedures similar to the methods described in example 19.

Three (3) sheet was taken from transgenic tobacco. Each sheet was divided into 4 pieces, and each piece had a width of 5-7 mm. Each piece of the sheet was dropped off on Wednesday with MS agar containing 0.1 mg/l of compound (II), and cultured in the light at room temperature. On the 7th day of cultivation was observed herbicide damage to each of these pieces of paper. Pieces of leaves obtained from tobacco, which was introduced control DNA (area T-DNA plasmids pBI121S), became white and was savagely. In contrast, pieces of leaves, obtained from tobacco, which was introduced DNA (A2) of the present invention (the area of the T-DNA plasmid pBI-NdG6-rSt-923 or plasmid pBI-NdG6-rSt12-923), grew continuously.

Example 21. Introduction DNA (A3) of the present invention in tobacco

Designed plasmid for introduction of DNA (A3) of the present invention in which asthenia using guns for particles and using the method using Agrobacterium.

First, DNA (A3) of the present invention having the nucleotide sequence shown in SEQ ID NO:8, amplified using PCR. PCR was performed using as template genomic DNA of Streptomyces testaceus ATCC21469 and using as primers the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:102, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:103. In the specified PCR used PCR system Expand High Fidelity (Boehringer Company). After maintaining once 97°C for 2 minutes was performed repeating 10 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 60°C for 30 seconds, and then 72°C for 1 minute; then 15 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 60°C for 30 seconds, and then 72°C for 1 minute (20 seconds added to maintain 72°C for each cycle); and then there was maintenance once 72°C for 7 minutes. Plasmid pCR671 (Fig. 38) was obtained by integration of amplified DNA in the area of cloning of PCR product pCR2.1 (Invitrogen Company). Then plasmid pCR671Bs (Fig. 39) was obtained by procedures similar to the method described above, except that used as primers for PCR oligonucleotide consisting of the nucleotide sequence shown in SEQ IDNO:104, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:103. Then the plasmid was introduced into competent cells of E. coli JM109 (Takara Shuzo Company) and selected resistant to ampicillin cells. Further, the nucleotide sequence of the plasmid contained in resistant to ampicillin strains was determined using kit BigDye terminator cycle sequencing Ready Reaction kit v.2.0 (PE Applied Biosystems Company) and DNA sequencing machine 3100 (PE Applied Biosystems Company). In the result, it was confirmed that the plasmid pCR671 and pCR671Bs have the nucleotide sequence shown in SEQ ID NO:8.

Plasmid pCR671ET were digested with restrictase EcoT221 and kpni restriction sites for DNA containing DNA (A3) of the present invention. Specified DNA was built between EcoT22I restriction site and a restriction site kpni restriction sites to obtain plasmid pUCrSt671 (Fig. 40)containing a chimeric DNA in which DNA (A3) of the present invention is attached directly after the sequence encoding the chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frame at codon. Plasmid pUCrSt671 were digested with restrictase NheI and kpni restriction sites for DNA containing DNA (A3) of the present invention. Plasmid pNdG6-rSt12 obtained in example 16(2), were digested with restrictase NheI and kpni restriction sites to remove DNA approximately 80 BP In its place was built the above DNA containing DNA (A3) this is subramania, derived from plasmids pUCrSt671, obtaining pSUM-NdG6-rSt-671 (Fig. 41), where the promoter CR16G6 attached right from the chimeric DNA in which DNA (A3) of the present invention is attached directly after the sequence encoding the chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frame at codon.

Plasmid pCR671Bs were digested with restrictase BspHI and kpni restriction sites for DNA containing DNA (A3) of the present invention. Specified DNA was embedded between the BspHI restriction site and a restriction site kpni restriction sites pKFrSt12 obtained in example 16(3), to obtain plasmid pKFrSt12-671 (Fig. 42)containing a chimeric DNA in which DNA (A3) of the present invention is attached directly after the sequence encoding the chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without altering the reading frame at codon. Plasmid pNdG6-rSt12 obtained in example 20 was digested with restrictase NheI and kpni restriction sites to remove DNA approximately 80 BP In its place was built the above DNA containing DNA (A3) of the present invention, obtained from a plasmid pKFrSt12-671, obtaining pSUM-NdG6-rSt12-671 (Fig. 43), where the promoter CR16G6 attached right from the chimeric DNA in which DNA (A3) of the present invention is attached directly after the sequence, codereuse the chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without changing the reading frame at codon.

DNA (A3) of the present invention was introduced into soybean way, using the gun for particles, the procedures are identical to the procedures described in example 17, using the obtained plasmid pSUM-NdG6-rSt-671 and pSUM-NdG6-rSt12-671.

The above plasmid pSUM-NdG6-rSt-671 were digested with restrictase HindIII and EcoRI to highlight the chimeric DNA in which the DNA (A3) of the present invention is attached directly after the sequence encoding the chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frame at codon. The above DNA containing the chimeric DNA derived from the plasmid pSUM-NdG6-rSt-671, built between the restriction site HindIII and the restriction site EcoRI above binary plasmid vector pBI121S obtained in example 18, to obtain pBI-NdG6-rSt-671 (Fig. 44). Further, the above plasmid pSUM-NdG6-rSt12-671 were digested with restrictase HindIII and EcoRI for DNA containing a chimeric DNA in which the DNA containing DNA (A3) of the present invention, attached directly after the sequence encoding the chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without altering the reading frames in the codons. Chimeric DNA obtained from pSUM-NdG6-rSt12-671, built between the restriction site HindIII and the restriction site EcoRI binary plasmid vector pBI121S obtaining pBI-NdG6-rSt12-671 (Fig. 45).

Each of the plasmid pBI-NdG6-rSt-671 and pBI-NdG6-rSt12-671 was introduced into Agrobacterium tumefaciens LBA4404. The obtained transformants were cultured in LB-medium containing 300 μg/ml of streptomycin, 100 μg/ml rifampicin and 25 μg/ml kanamycin. Transformants were selected for isolation of Agrobacterium strains carrying pBI-NdG6-rSt-671 and pBI-NdG6-rSt12-671.

Pieces of sterile leaves of cultivated tobacco were injected with each of the Agrobacterium strain carrying pBI-NdG6-rSt-671, and Agrobacterium strain carrying pBI-NdG6-rSt12-671. Tobacco, which was introduced DNA (A3) of the present invention, was obtained by procedures similar to the methods described in example 19.

Three (3) sheet was taken from transgenic tobacco. Each sheet was divided into 4 pieces,and each piece had a width of 5-7 mm. Each piece of the sheet was dropped off on Wednesday with MS agar containing 0.1 mg/l of compound (II), and cultured in the light at room temperature. On the 7th day of cultivation was observed herbicide damage to each of these pieces of paper.

Example 22. Expression of the protein (B1) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (B1) of the present invention

PCR was performed using as a template the chromosomal DNA obtained from Streptomyces phaeochromogenes IFO 12898 in example 3(1). The reaction solution for PCR was brought up to 50 μl by the addition of 300 ng of the above chromosomal DNA, 4 μl of dNTP mixture (a mixture of 2.5 mm to the each of the 4 types of dNTP), 5 ál of 10x ExTaq buffer and 0.5 ál of polymerase ExTaq (Takara Shuzo Company), distilled water and 200 nm of each oligonucleotide having the nucleotide sequence shown in SEQ ID NO:105, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:53. The PCR reaction conditions were as follows: after maintaining 97°C for 2 minutes, repeating 25 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 60°C for 30 seconds, and then 72°C for 90 seconds; and then maintaining 72°C for 4 minutes. After keeping the reaction solution and the vector pCR2.1-TOPO (Invitrogen Company) ligated in accordance with the instructions attached to the specified vector, and introduced into E. coli 10F'. Plasmid DNA was obtained from the obtained transformants of E. coli using the kit QIAprep Spin Miniprep Kit (Qiagen Company). The sequencing reaction was performed with a set of Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company) in accordance with the instructions attached to the specified set, using as primers the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:67, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:68. In the sequencing reactions used the obtained plasmid DNA as template. The reaction products were analyzed using sequencing machine is DNA A (Applied Biosystems Japan Company). On the basis of these results, the plasmid having the nucleotide sequence shown in SEQ ID NO:15, was named pCR657FD.

Then pCR657FD were digested with restrictase NdeI and HindIII. The cleavage products were subjected to agarose gel electrophoresis. The area of the gel containing DNA with a size of approximately 200 BP were excised from the gel. This DNA was purified from the extracted gels using a set of extraction from gels QIAquick (Qiagen Company) in accordance with the attached instructions. The resulting DNA and plasmid pKSN2, split NdeI and HindIII, ligated using set for ligating Ver. 1 (Takara Shuzo Company) in accordance with the instructions attached to the specified collection, and introduced into E. coli JM109. From the obtained transformants of E. coli were obtained plasmid DNA. Their structures were analyzed. A plasmid containing the nucleotide sequence shown in SEQ ID NO:15, in which the DNA with a size of approximately 200 BP, encoding a protein (B1) of the present invention, inserted between the NdeI site and the HindIII site pKSN2, was named pKSN657FD. Plasmid pKSN657FD was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN657FD. Next, plasmid pKSN2 was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN2.

(2) the expression of the protein (B1) of the present invention in E. coli and the selection of the specified protein

Each of E. coli JM109/pKSN657FD and E. coli JM109/pKSN2 were cultured over night at 37°the 10 ml of medium TV (1,2% (wt./about.) tripton, 2,4% (wt./about.) yeast extract, and 0.4% (wt./about.) glycerol, 17 mm potassium dihydrophosphate, 72 mm dicale-phosphate)containing 50 μg/ml ampicillin. A milliliter (1 ml) of the obtained culture medium was transferred into 100 ml of medium TV containing 50 μg/ml of ampicillin, and cultured at 26°C. After 30 minutes after reaching the OD660 of approximately of 0.5, IPTG was added to a final concentration of 1 mm and were additionally cultured for 20 hours.

Cells were removed from each culture medium, washed with 0.1 M Tris-HCl-buffer (pH 7.5) and suspended in 10 ml of the above buffer containing 1 mm PMSF. The obtained cell suspension was subjected to 6 times the processing on the ultrasonic disintegrator (Sonifier (Branson Sonic Power Company)) for 3 minutes each time in terms of radiated power 3, load cycle 30%, to obtain solutions of cell lysates. After centrifugation of the solutions of cell lysates (1200 g, 5 minutes) supernatant were removed and centrifuged (150000 g, 70 minutes) to obtain fractions of supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN657FD, called “extract of E. coli pKSN657FD”, and the supernatant fraction obtained from E. coli JM109/pKSN2, called “extract of E. coli pKSN2”). One microliter (1 μl) of the above fractions supernatants were analyzed by electrophoresis in 15% -25% of the LTO-SDS page and stained STS. The result is clearly more intense in the wasps were detected in the extract of E. coli pKSN657FD than in the extract of E. coli pKSN2, electrophoresis, corresponding to molecular mass of 7 kDa. It was shown that E. coli JM109/pKSN657FD expressed protein (B1) of the present invention.

(3) Application of protein (B1) of the present invention to the reaction system for the conversion of compound (II) into the compound (III)

Preparing the reaction solution, 30 ml and kept for 10 minutes at 30°C. These reaction solutions consisted of 0.1 M potassium phosphate buffer (pH 7.0)containing 3 ppm of compound (II)labeled14S, 2 mm β-NADPH (hereinafter referred to as "component a") (Oriental Yeast Company), 9 μl of the extract of E. coli pKSN657FD obtained in example 22(2), 0.1 u/ml paradoxicality (hereinafter referred to as "component C") (Sigma Company) and 15 µl of the extract of E. coli pKSN657F obtained in example 4(2) (hereinafter referred to as "component D"). Next, preparing the reaction solution, to which was added 2 mg/ml ferredoxin obtained from spinach (hereinafter referred to as "component B") (Sigma Company) instead of the extract of E. coli pKSN657FD, and the reaction solution, in which nothing was added instead of the extract of E. coli pKSN657FD. These reaction solutions were passed in the same way. Added three microliters (3 ál) 2 N. HCl and 90 μl of ethyl acetate in each of these reaction solutions after the specified curing and stirred. The resulting reaction solution was centrifuged at 8000 g is La extract 75 μl of an ethyl acetate layer. After drying, an ethyl acetate layer under reduced pressure, the residue was dissolved in 6.0 ál of ethyl acetate. Five microlitres (5,0 µl) of this solution was applied on TLC plate (silica gel TLC plates 60F25420 cm x 20 cm, 0.25 mm thick, Merck Company). The TLC plate showed a mixture of 6:1:2 chloroform, acetic acid and ethyl acetate for about 1 hour. Then the solvent was allowed to evaporate. The TLC plate was exposed during the night on the imaging plate (Fuji Film Company). Then, the imaging plate was analyzed by the image Analyzer BAS2000 (Fuji Film Company). The presence of spots corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). These results are shown in table 13.

Table 13
Components of the reactionspot compound (III)
component aextract
E. coli
componentcomponentcomponent Dthe compound (II),
labeled14
+pKSN657FD- ++++
+--+++-
+-+++++

Example 23. Expression of the protein (B2) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (B2) of the present invention

PCR was performed using as a template the chromosomal DNA derived from Saccharopolyspora taberi JCM9383t in example 6(1). The reaction solution for PCR was brought up to 50 μl by the addition of 300 ng of the above chromosomal DNA, 4 μl of dNTP mixture (a mixture of 2.5 mm each of the 4 types of dNTP), 5 μl 10x ExTaq buffer and 0.5 ál of polymerase ExTaq (Takara Shuzo Company), distilled water and 200 nm of each oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:106, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:63. The PCR reaction conditions were as follows: after maintaining 97°C for 2 minutes, repeating 25 cycles, where each cycle consisted of maintaining 9°C for 15 seconds, then 60°C for 30 seconds, and then 72°C for 90 seconds; and then maintaining 72°C for 4 minutes. After keeping the reaction solution and the vector pCR2.1-TOPO (Invitrogen Company) ligated in accordance with the instructions supplied in the specified vector, and introduced into E. coli 10F'. Plasmid DNA was obtained from the obtained transformants of E. coli using the kit QIAprep Spin Miniprep Kit (Qiagen Company). The sequencing reaction was performed with a set of Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company) in accordance with the instructions attached to the specified set, using as primers the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:67, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:68. In the sequencing reactions used the obtained plasmid DNA as template. The reaction products were analyzed using DNA sequencing machine A (Applied Biosystems Japan Company). On the basis of these results, the plasmid having the nucleotide sequence shown in SEQ ID NO:16, was named pCR923FD.

Then plasmid pCR923FD were digested with restrictase NdeI and HindIII. The cleavage products were subjected to agarose gel electrophoresis. The area of the gel containing DNA with a size of approximately 200 BP were excised from the gel. This DNA was purified from the extracted gels using a set of Clextral from gels QIAquick (Qiagen Company) in accordance with the attached instructions. The resulting DNA and plasmid pKSN2, split NdeI and HindIII, ligated using set for ligating Ver. 1 (Takara Shuzo Company) in accordance with the instructions attached to the specified collection, and introduced into E. coli JM109. From the obtained transformants of E. coli were obtained plasmid DNA. Their structures were analyzed. A plasmid containing the nucleotide sequence shown in SEQ ID NO:16, in which the DNA with a size of approximately 200 BP, encoding a protein (B2) of the present invention, inserted between the NdeI site and the HindIII site pKSN2, was named pKSN923FD. Plasmid pKSN923FD was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN923FD. Next, plasmid pKSN2 was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN2.

(2) the expression of the protein (B2) of the present invention in E. coli and the selection of the specified protein

Each of E. coli JM109/pKSN923FD and E. coli JM109/pKSN2 were cultured over night at 37°C in 10 ml of medium TV (1,2% (wt./about.) tripton, 2,4% (wt./about.) yeast extract, and 0.4% (wt./about.) glycerol, 17 mm potassium dihydrophosphate, 72 mm dicale-phosphate)containing 50 μg/ml ampicillin. A milliliter (1 ml) of the obtained culture medium was transferred into 100 ml of medium TV containing 50 μg/ml of ampicillin, and cultured at 26°C. After 30 minutes after reaching the OD660 approximately 0.5 was added IPTG to a final concentration of 1 mm and were additionally cultured for 20 hours.

the entrances were removed from each culture medium, were washed in 0.1 M Tris-HCl-buffer (pH 7.5) and suspended in 10 ml of the above buffer containing 1 mm PMSF. The obtained cell suspension was subjected to 6 times the processing on the ultrasonic disintegrator (Sonifier (Branson Sonic Power Company)) for 3 minutes each time in terms of radiated power 3, load cycle 30%, to obtain solutions of cell lysates. After centrifugation of the solutions of cell lysates (1200 g, 5 minutes) supernatant were removed and centrifuged (150000 g, 70 minutes) to obtain fractions of supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN923FD, called “extract of E. coli pKSN923FD”, and the supernatant fraction obtained from E. coli JM109/pKSN2, called “extract of E. coli pKSN2”). One microliter (1 μl) of the above fractions supernatants were analyzed by electrophoresis in 15% -25% of the LTO-SDS page and stained STS. As a result, detection is clearly more intense bands in the extract of E. coli pKSN923FD than in the extract of E. coli pKSN2, electrophoresis, corresponding to molecular mass of 7 kDa, it is possible to confirm the expression of the protein (B2) of the present invention in E. coli.

(3) Application of protein (B2) of the present invention to the reaction system for the conversion of compound (II) into the compound (III)

Preparing the reaction solution, 30 ml and kept for 10 minutes at 30°C. These reaction solutions consisted of 0.1 M potassium phosphate is the buffer (pH 7.0), containing 3 ppm of compound (II)labeled14S, 2 mm β-NADPH (hereinafter referred to as "component a") (Oriental Yeast Company), 9 μl of the extract of E. coli pKSN923FD obtained in example 23(2), 0.1 u/ml paradoxicality (hereinafter referred to as "component C") (Sigma Company) and 15 µl of the extract of E. coli pKSN657F obtained in example 4(2) (hereinafter referred to as "component D"). Next, preparing the reaction solution, to which was added 2 mg/ml ferredoxin obtained from spinach (hereinafter referred to as "component B") (Sigma Company) instead of the extract of E. coli pKSN923FD, and the reaction solution, in which nothing was added instead of the extract of E. coli pKSN923FD. These reaction solutions were passed in the same way. Added three microliters (3 ál) 2 N. HCl and 90 μl of ethyl acetate in each of these reaction solutions after the specified curing and stirred. The resulting reaction solution was centrifuged at 8000 g for extraction of 75 μl of an ethyl acetate layer. After drying, an ethyl acetate layer under reduced pressure, the residue was dissolved in 6.0 ál of ethyl acetate. Five microlitres (5,0 µl) of this solution was applied on TLC plate (silica gel TLC plates 60F25420 cm x 20 cm, 0.25 mm thick, Merck Company). The TLC plate showed a mixture of 6:1:2 chloroform, acetic acid and ethyl acetate for about 1 hour. Then the solvent was allowed to evaporate. The TLC plate was exposed for what of the night on the imaging plate (Fuji Film Company). Then, the imaging plate was analyzed by the image Analyzer BAS2000 (Fuji Film Company). The presence of spots corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). By confirming that the compound (III) is formed in the reaction containing the component a, an extract of E. coli pKSN923FD, component C and component D, it can be proven that protein (B2) of the present invention may be used in place of ferredoxin obtained from spinach, into the reaction system, the conversion of compound (II) into the compound (III).

Example 24. Expression of the protein (B3) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (B3) of the present invention

PCR was carried out similarly to the methods described in example 23(1), except for use as matrix chromosomal DNA obtained from Streptomyces testaceus ATCC 21469 in example 11(1), and using as primers the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:107, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:72. Plasmid pCR671FD having the nucleotide sequence shown in SEQ ID NO:17, was obtained similarly to the method described in example 23(1), using the obtained reaction solution.

Then using the indicated plasmids, plasmid pKSN671F, in which DNA (B3) of the present invention inserted between the NdeI site and the HindIII site pKSN2 get similar to the method described in example 23(1). The introduction of this plasmid into E. coli JM109 can be obtained from E. coli JM109/pKSN671FD with DNA (B3) of the present invention.

(2) the expression of the protein (B3) of the present invention in E. coli and the selection of the specified protein

Using E. coli JM109/pKSN671FD fraction of the supernatant (hereinafter called “extract of E. coli pKSN671FD”) is extracted similarly to the method described in example 23(2). One microliter (1 μl) of the above fractions supernatants analyzed by electrophoresis in 15% -25% of the LTO-page and stained STS. In the result of detection is clearly more intense bands in the extract of E. coli pKSN671FD than in the extract of E. coli pKSN2, electrophoresis, corresponding to molecular mass of 7 kDa, it is possible to confirm the expression of the protein (B3) of the present invention in E. coli.

(3) Application of protein (B3) of the present invention to the reaction system for the conversion of compound (II) into the compound (III)

Except for the use of the extract of E. coli pKSN671FD obtained in example 24(2), the spot corresponding to the compound (III)labeled14(Rf values of 0.24 and 0.29 to), is confirmed by the similar method described in example 23(3). Confirmation that the compound (III) is formed in the reaction, And includes the extract of E. coli pKSN671FD component and comp the element D, it can be proven that protein (B3) of the present invention may be used in place of ferredoxin obtained from spinach, into the reaction system, the conversion of compound (II) into the compound (III).

Example 25. Obtaining protein (A4) this invention

(1) preparation of the crude extract of cells

Frozen original material Streptomyces achromogenes IFO12735 was added to 10 ml of medium A (0.1% (wt./about.) glucose and 0.5% (wt./about.) tripton, a 0.5% (wt./about.) yeast extract, 0.1% (wt./about.) dicale-phosphate, pH 7.0) in a large test tube and incubated with shaking at 30°C for 1 day to obtain a pre-culture. Eight milliliters (8 ml) pre-culture was added to 200 ml of medium a and incubated with rotary shaking in a flask with septum 500 ml at 30°C for 2 days. Precipitation cells were obtained by centrifugation (3000 g, 10 min) obtained culture. The yeast cells suspended in 100 ml of medium (1% (wt./about.) glucose, 0.1% of meat extract, and 0.2% (wt./about.) tryptose)containing the compound (II) at 100 ppm, and incubated with the reciprocating swing in a Sakaguchi flask of 500 ml for 20 hours at 30°C. Precipitation cells were obtained by centrifugation (3000 g, 10 min) 2 l of the resulting culture. The obtained precipitation cells were washed twice in 1 l of 0.1 M potassium phosphate buffer (pH 7.0) to give 136 g of precipitation cells.

This OS is DKI cells suspended in 0.1 M potassium phosphate buffer (pH 7.0) at 1 - 2 ml per 1 g of precipitation cells. To the cell suspension was added to 1 mm PMSF, 5 mm benzamidine·HCl, 1 mm EDTA, 3 mg/ml leupeptin, 3 μg/ml of pepstatin and 1 mm dicitrate(ol). The solution of the cell lysate was obtained by destruction twice this suspension using a French press (1000 kg/cm2) (Ohtake Seisakusho). After centrifugation of the solution in the cell lysate (40000 g, 30 min) the supernatant was removed and centrifuged for 1 hour at 150000 g for extraction of the supernatant (hereinafter called "the crude cell extract").

(2) Determination of the ability to turn the compound (II) into the compound (III)

Prepared 30 μl of the reaction solution of 0.1 M potassium phosphate buffer (pH 7.0)containing 3 ppm of compound (II)labeled14S, 2.4 mm β-NADPH (hereinafter referred to as "component a") (Oriental Yeast Company), 0.5 mg/ml ferredoxin obtained from spinach (hereinafter referred to as "component B") (Sigma Company), 1 u/ml paradoxicality (hereinafter referred to as "component C") (Sigma Company) and 15 μl of crude cell extract obtained in example 25(1). The reaction solution was kept at 30°C for 1 hour. In addition, prepared and handled in the same way, the reaction solution without adding at least one component used in the above reaction solution, selected from the component A, component b and component C. EXT is ulali three microliters (3 ál) 2 N. HCl and 90 μl of ethyl acetate in each of these reaction solutions after the specified curing and stirred. The resulting reaction solution was centrifuged at 8000 g for extraction of 75 μl of an ethyl acetate layer. After drying, an ethyl acetate layer under reduced pressure, the residue was dissolved in 6.0 ál of ethyl acetate. Five microlitres (5,0 µl) of this solution was applied on TLC plate (silica gel TLC plates 60F25420 cm x 20 cm, 0.25 mm thick, Merck Company). The TLC plate showed a mixture of 6:1:2 chloroform, acetic acid and ethyl acetate for about 1 hour. Then the solvent was allowed to evaporate. The TLC plate was exposed during the night on the imaging plate (Fuji Film Company). Then, the imaging plate was analyzed by the image Analyzer BAS2000 (Fuji Film Company). The presence of spots corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). These results are shown in table 14.

Table 14
Components of the reactionspot compound (III)
component acomponentcomponentthe crude cell extract/td> the compound (II)labeled14
+++-+-
++++++
-++++-
+--++-

(3) Fractionation of the crude cell extract

To the crude cell extract obtained in example 25(1), was added ammonium sulfate to 45% saturation. After stirring cooled in ice conditions were obtained supernatant by centrifugation for 30 minutes at 12000 g. After adding ammonium sulfate to the supernatant up to 55% saturation and mixing cooled in ice conditions the precipitate was obtained by centrifugation for 10 minutes at 12000 g. The residue was dissolved in 12.5 ml of 20 mm bestreplibng buffer (pH 7.0).This solution was subjected to chromatography on a PD10 column (Amersham Pharmacia Company) and was suirable 20 mm mistresspopular buffer (pH 7.0) to obtain a 17.5 ml fractions, containing proteins (hereinafter referred to as "fraction 45-55% of ammonium sulfate").

(4) Isolation of the protein (A4) this invention

Fraction 45-55% of ammonium sulfate obtained in example 25(3), was injected into the column HiLoad 26/10 Q-Sepharose HP (Amersham Pharmacia Company). Then after passing 100 ml of 20 mm bestreplibng buffer (pH 7.0) through the column, 20 mm listresponse buffer proceeded with a linear gradient of NaCl (NaCl gradient was 0,004 M/min, the concentration range of NaCl was from 0 M to 1 M, the flow rate was 4 ml/min) for fractional extraction with 30 ml fractions, eluruumiks when NaCl concentration from 0.12 M to 0,165 M Then extracted fraction was subjected to chromatography on a PD10 column (Amersham Pharmacia Biotech Company) and was suirable 20 mm mistresspopular buffer (pH 7.0) to obtain fractions containing protein.

The extracted fraction was applied to a PD10 column (Amersham Pharmacia Biotech Company) with elution buffer (2 mm potassium phosphate buffer containing 1.5 mm NaCl, pH 7.0) to obtain fractions containing protein. Then these fractions were injected into the column SNT-I Bio-Scale Ceramic Hydroxyapatite Type I (Bio-Rad Company). Twenty milliliters (20 ml) buffer And passed through the column. Then the buffer And proceeded with a linear gradient of buffer B (100 mm potassium phosphate buffer containing 0.03 mm NaCl; linear gradient started at 100% buffer And up to 50% buffer over a period of 100 minutes, the flow rate was 2 ml/min) for fractional WPI is ecene fractions, eluruumiks when the concentration of the buffer from 4 to 6%. Then, the obtained fraction was applied to a PD10 column (Amersham Pharmacia Biotech Company) and was suirable 0.05 M potassium phosphate buffer (pH 7.0) to obtain fractions containing protein.

A similar amount of 0.05 M potassium phosphate buffer (pH 7.0)containing 2.0 M ammonium sulfate was added to the extracted fractions and mixed. Then izvlechenie fractions were injected with column 1 ml RESOURSE PHE (Amersham Pharmacia Biotech Company). After passing 5 ml of 0.05 M potassium phosphate buffer (pH 7.0)containing 1.0 M ammonium sulfate, missed 0.05 M potassium phosphate buffer (pH 7.0) with a linear gradient of ammonium sulfate (concentration gradient of ammonium sulfate was 1 - 0 M, the flow rate was 2 ml/min) for fractional extraction fractions, eluruumiks when the ammonium sulfate concentration from about 0.4 M to 0.5 M. the Protein contained in these fractions were analyzed by electrophoresis in 10-20% of the LTO-PAG.

Instead of the crude extract of the cells in the reaction solution described in example 25(2), was added to the extracted fractions and stood in the presence of component A, component B, component C and the compound (II)labeled14Since, analogously to example 25(2). The reaction solutions after incubation were analyzed by TLC for the study of the intensity of the spots corresponding to compound (III)labeled14C. this protein that moves the I in a position approximately 45 kDa at the specified electrophoresis LTO-SDS page, extracted from the gel and subjected to analysis of amino acid sequences using protein sequencing machine (Applied Biosystems Company, Procise NT, pulsed liquid method) for sequencing N-terminal amino acid sequence. In the received amino acid sequence shown in SEQ ID NO:113.

Example 26. Obtaining DNA (A4) this invention

(1) preparation of chromosomal DNA of Streptomyces achromogenes IFO 12735

Streptomyces achromogenes IFO 12735 were cultured with shaking at 30°C for 1 to 3 days in 50 ml of YEME medium (0,3% (wt./about.) yeast extract and 0.5% (wt./about.) bactopeptone, of 0.3% (wt./about.) the malt extract, and 1.0% (wt./about.) glucose, 34% (wt./about.) sucrose and 0.2% (vol./about.) 2.5 M MgCl2·6N2About). The cells were collected. The obtained cells suspended in YEME medium containing 1,4% (wt./about.) glycine and 60 mm EDTA, and additionally incubated with shaking for one day. Cells were removed from culture medium. After washing once with distilled water them resuspendable buffer (100 mm Tris-HCl (pH 8.0), 100 mm EDTA, 10 mm NaCl) at 1 ml per 200 mg of cells. Added two hundred micrograms per milliliter (200 μg/ml) of lysozyme egg white. This cell suspension was shaken at 30°C for one hour. Then added 0.5% of LTOs and 1 mg/ml proteinase K. cell Suspension was incubated at 55°C for 3 hours. Cell suspension extragere the Ali twice with a mixture of phenol, chloroform and isoamyl alcohol to extract each of the water layers. Then there was one extraction with a mixture of chloroform and isoamyl alcohol to extract the water layer. Chromosomal DNA was obtained by precipitation with ethanol from the aqueous layer.

(2) obtaining a library of chromosomal DNA of Streptomyces achromogenes IFO 12735

Thirty-eight micrograms (38 g) of the chromosomal DNA obtained in example 26(1), was digested 3.2 units restrictase Sau3AI at 37°C for 60 minutes. The obtained split the solution was separated by electrophoresis in 1% agarose gel. DNA size approximately 2.0 TPN extracted from the gel. This DNA was purified using a kit for extraction from gels QIAquick Gel Extraction (Qiagen Company) in accordance with the instructions attached to the specified collection, and was concentrated by ethanol precipitation with 20 μl of solution containing the desired DNA. 8 microlitres (8 μl) of the DNA solution, 100 ng of plasmid vector pUC118 cleaved by the restriction enzyme BamHI and treated with dephosphorylation, and 16 μl of solution I of a set of ligation Ver. 2 (Takara Shuzo Company) were mixed and incubated for 3 hours at 16°C. E. coli DH5α was transformed using the solution for ligating and distributed on medium containing LB-agar containing 50 mg/l ampicillin, for culturing overnight at 37°C. resulting colonies were removed from yeah the new environment. The plasmids were extracted. The resulting plasmids were named by the library of chromosomal DNA.

(3) Isolation of DNA (A4) this invention

PCR was performed using as a template the chromosomal DNA obtained in example 26(2). As primers used a couple of the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:114, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:57. The nucleotide sequence shown in SEQ ID NO:114, designed based on the amino acid sequence shown in SEQ ID NO:113. For the preparation of the reaction solution used PCR system Expand HiFi (Boehringer Manheim Company). The reaction solution for PCR was made up to 25 μl by the addition of 2.5 μl of the above chromosomal DNA library, each of the 2 primers, brought to 7.5 pmol, 0.2 µl dNTP mixture (mixture 2 mm each of the 4 types of dNTP), and 2.5 µl of 10x buffer (containing MgCl2), 0,38 μl of a mixture of enzymes Expand HiFi and distilled water. The PCR reaction conditions were as follows: after maintaining 97°C for 2 minutes, repeating 10 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 65°C for 30 seconds, then 72°C for 1 minute; then 15 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 65°C for 30 Sekou is d, then 72°C for 1 minute (20 seconds added for incubation at 72°C for each cycle); and then maintaining 72°C for 7 minutes. After this incubation of 2.5 μl of the reaction solution is used as a solution matrix for the second time PCR. As primers used a couple of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:115, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:57. The nucleotide sequence shown in SEQ ID NO:115, designed based on the amino acid sequence shown in SEQ ID NO:113. Like the above-described method for PCR used PCR system Expand HiFi (Boehringer Manheim Company). The reaction solution after aging was applied on a 2% agarose gel for electrophoresis. The area of the gel containing DNA of approximately 800 BP were removed. DNA was purified from the extracted gel using a set of extraction from gels QIAquick (Qiagen Company) in accordance with the attached instructions. The resulting DNA ligated to the cloning vector pCRII TA-TOPO (Invitrogen Company) in accordance with the instructions attached to the specified vector, and introduced into E. coli 10F'. Plasmid DNA was obtained from the received transformant E. coli using a set of Qiagen Tip20 (Qiagen Company). The sequencing reaction was performed with nab the rum Big Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company) in accordance with the instructions, attached to the specified set, using a primer having the nucleotide sequence shown in SEQ ID NO:67, and a primer having the nucleotide sequence shown in SEQ ID NO:68. The obtained plasmid was used as template in sequencing reactions. The reaction products were analyzed using DNA sequencing machine 3100 (Applied Biosystems Japan Company). In the received nucleotide sequence consisting of nucleotides 57-832 the nucleotide sequence shown in SEQ ID NO:110. In the obtained nucleotide sequence of nucleotides 58-60 nucleotide sequence shown in SEQ ID NO:110, encode the amino acid 20 to amino acid sequence shown in SEQ ID NO:113.

Then PCR was performed with PCR system Expand HiFi (Boehringer Manheim Company) under the above conditions, using the library of chromosomal DNA obtained in example 26(2), as a matrix. As primers used a couple of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:116, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:59. Amplified DNA size approximately 1.4 TPN cloned in a cloning vector pCRII-TOPO. Plasmid DNA was obtained from the resulting E. coli transformants using the Qiagen Tip20 (Qiagen Company). The reaction sequen the simulation was performed using a set of Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company) in accordance with the instructions, attached to the specified set, using a primer having the nucleotide sequence shown in SEQ ID NO:67, and a primer having the nucleotide sequence shown in SEQ ID NO:68. The obtained plasmid was used as template in sequencing reactions. The reaction products were analyzed using DNA sequencing machine 3100 (Applied Biosystems Japan Company). The result was obtained nucleotide sequence consisting of nucleotides 1-58 nucleotide sequence shown in SEQ ID NO:110.

Conducted DNA cloning, stretching right from the 3'-end nucleotide is shown as nucleotide 832 nucleotide sequence shown in SEQ ID NO:110. Specifically, 13 μg of the chromosomal DNA of Streptomyces achromogenes IFO 12735, obtained in example 26(1), were digested overnight 200 s restrictase HincII at 37°C. After phenol extraction, the DNA was purified by ethanol precipitation. The resulting DNA was used to obtain 20 μl of an aqueous solution. Four microliters (4 μl) of this solution, 1,9 ál of 15 ám adaptor Walker Adaptor (Genome walking on the genome), and 1.6 μl of 10x buffer for ligation and 0.5 ál 6E/μl of T4 ligase were mixed and kept overnight at 16°C. then kept at 70°C for 5 minutes and added 72 ál of distilled water to provide library Genome Walker. PCR was performed using pointed to by the th library as a matrix. The reaction solution for PCR, brought to 50 μl, was provided by the addition of 1 ál of library Genome Walker and primer API (provide a universal set of Genome Walker) and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:117, each to the number of 200 nm, the addition of 1 μl of dNTP mixture (a mixture of 10 mm each of the 4 types of dNTP), 10 ál of 5x buffer for GC genomic PCR and 2.2 μl of 25 mm Mg(OAc)210 μl of 5 M GC-melt and 1 μl of a mixture of genomic polymerase Advantage-GC and by adding distilled water. Reaction conditions PCR after incubation at 95°C for 1 minute were as follows: carrying 7 cycles, where each cycle consisted of maintaining 94°C for 10 seconds, and then 72°C for 3 min; 36 cycles, where each cycle consisted of maintaining 94°C for 10 seconds and then 68°C for 3 minutes; and maintaining 68°C for 7 minutes. The reaction solution after the incubation were diluted 50 times with distilled water. These PCR products were named products of the first PCR were used as template for the second PCR. PCR, brought to 50 μl, was provided by the addition of 1 µl of the PCR products and primer AR (provide a universal set of Genome Walker) and the oligonucleotide shown in SEQ ID NO:118, each to the number of 200 nm, the addition of 1 μl of dNTP mixture (a mixture of 10 mm each of the 4 types of dNTP), 10 ál of 5x buffer for GC genomic PCR ,2 μl of 25 mm Mg(OAc) 210 μl of 5 M GC-melt and 1 μl of a mixture of genomic polymerase Advantage-GC and by adding distilled water. Reaction conditions PCR after maintaining 95°C for 1 minute were as follows: 5 cycles, where each cycle consisted of maintaining 94°C for 10 seconds, and then 72°C for 3 min; 20 cycles, where each cycle consisted of maintaining 94°C for 10 seconds and then 68°C for 3 minutes; and maintaining 68°C for 7 minutes. The reaction solution after the incubation were subjected to electrophoresis in 1% agarose gel. The area of the gel containing DNA of approximately 1300 BP were removed. DNA was purified from the extracted gel using a set of extraction from gels QIAquick (Qiagen Company) in accordance with the attached instructions. The resulting DNA ligated to the cloning vector pCRII-TOPO (Invitrogen Company) in accordance with the instructions supplied in the specified vector, and introduced into E. coli 10F'. Plasmid DNA was obtained from the received transformant E. coli using a set of Qiagen Tip20 (Qiagen Company). The sequencing reaction was performed with a set of Big Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company) in accordance with the instructions attached to the specified set, using as primers the oligonucleotide shown in SEQ ID NO:67, and the oligonucleotide shown in SEQ ID NO:68. The obtained plasmid was used as a matrix in Rea is the sequencing. The reaction products were analyzed using DNA sequencing machine 3100 (Applied Biosystems Japan Company). In the received nucleotide sequence consisting of nucleotides 644-1454 the nucleotide sequence shown in SEQ ID NO:110. In the connection of all the analyzed nucleotide sequences obtained nucleotide sequence shown in SEQ ID NO:110. Two open reading frames (ORF) were present in the specified nucleotide sequence. Thus, it contained the nucleotide sequence (SEQ ID NO:109), consisting of 1236 nucleotides including the stop codon) and encoding a 411 amino acid residues (SEQ ID NO:108), and the nucleotide sequence (SEQ ID NO:112), consisting of 192 nucleotides including the stop codon) and encoding a 63 amino acid residues (SEQ ID NO:111). It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:108), encoded by the nucleotide sequence shown in SEQ ID NO:109, equal 45465 Yes. Further, the amino acid sequence encoded by the specified nucleotide sequence contained the amino acid sequence (SEQ ID NO:113), certain of the sequence of amino acids from the N-Terminus of the protein (A4) of the present invention. It was estimated that the molecular weight of the protein consisting of the amino acid sequence (EQ ID NO:111), encoded by the nucleotide sequence shown in SEQ ID NO:112, equal 6871 Yes.

Example 27. Expression of the protein (A4) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (A4) this invention

PCR was performed using as a template the chromosomal DNA obtained from Streptomyces achromogenes IFO 12735 in example 26(1), and using PCR system Expand HiFi (Boehringer Manheim Company). As primers used a couple of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:119, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:120 (hereinafter referred to as "a pair of 25 primers"), or a pair of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:119, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:121 (hereinafter referred to as "a pair of 26 primers"). The reaction solution for PCR was brought up to 50 μl by adding each of the 2 primers, brought to 300 nm, 50 ng of the above chromosomal DNA, 5,0 ál dNTP mixture (a mixture of 2.0 mm each of the 4 types of dNTP), 5,0 ál 10x buffer Expand HF (containing MgCl2) and 0.75 μl of a mixture of enzymes Expand HiFi and distilled water. The PCR reaction conditions were as follows: after maintaining 97°C for 2 minutes, repeating 10 cycles, where each cycle consisted of holding at 97°C during the 15 seconds, then 60°C for 30 seconds, then 72°C for 1 minute; then 15 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 60°C for 30 seconds, and then 72°C for 1 minute (20 seconds added to maintain 72°C for each cycle); and then maintaining 72°C for 7 minutes. After this incubation, the reaction solution was applied on a 1% agarose gel electrophoresis. The area of the gel containing DNA size approximately 1.3 TPN, extracted from the gel, which caused the reaction solution using a couple of 25 primers. The area of the gel containing DNA approximately 1,6 TPN, extracted from the gel, which caused the reaction solution using a pair of 26 primers. DNA was purified from each of the extracted gels using a set of extraction from gels QIAquick (Qiagen Company) in accordance with the attached instructions. The resulting DNA ligated to the cloning vector pCRII-TOPO (Invitrogen Company) in accordance with the instructions supplied in the specified vector, and introduced into E. coli 10F'. Plasmid DNA was obtained from the obtained transformants of E. coli using a set of Qiagen Tip20 (Qiagen Company). Then the sequencing reaction was performed with a set of Big Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company) in accordance with the instructions attached to the specified set, using as primers ol is gonucleotide, shown in SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:122 and SEQ ID NO:123. In the sequencing reactions used the obtained plasmid DNA as template. The reaction products were analyzed using DNA sequencing machine 3100 (Applied Biosystems Japan Company). On the basis of these results, the plasmid having the nucleotide sequence shown in SEQ ID NO:109, was named pCR646, and the plasmid having the nucleotide sequence shown in SEQ ID NO:110, was named pCR646F.

Then each of the plasmids pCR646 and pCR646F were digested with restrictase NdeI and HindIII. The cleavage products were subjected to agarose gel electrophoresis. The area of the gel containing DNA size approximately 1.3 TPN, cut out of the gel, which was applied cleavage products pCR646. The area of the gel containing DNA approximately 1,6 TPN, cut out of the gel, which was applied cleavage products pCR646F. These DNA was purified from each of the extracted gels using a set of extraction from gels QIAquick (Qiagen Company) in accordance with the attached instructions. Each of the obtained DNA and plasmid pKSN2, split NdeI and HindIII, ligated using set for ligating Ver. 1 (Takara Shuzo Company) in accordance with the instructions attached to the specified collection, and introduced into E. coli JM109. From the obtained transformants of E. coli were obtained plasmid DNA. Their structures were analyzed. A plasmid containing the nucleus is IGNOU sequence, shown in SEQ ID NO:109, in which DNA size approximately 1.3 TPN encoding a protein (A4) of the present invention, inserted between the NdeI site and the HindIII site pKSN2, was named pKSN646. Next, a plasmid containing the nucleotide sequence shown in SEQ ID NO:110, in which DNA is approximately 1,6 TPN encoding a protein (A4) of the present invention, inserted between the NdeI site and the HindIII site pKSN2, was named pKSN646F. Each of the above plasmids pKSN646 and pKSN646F was introduced into E. coli JM109. The resulting E. coli transformants were named respectively JM109/pKSN646 and JM109/pKSN646F. Next, plasmid pKSN2 was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN2.

(2) the expression of the protein (A4) of the present invention in E. coli and the selection of the specified protein

Each of E. coli JM109/pKSN646, JM109/pKSN646F and JM109/pKSN2 were cultured over night at 37°C in 10 ml of medium TV (1,2% (wt./about.) tripton, 2,4% (wt./about.) yeast extract, and 0.4% (wt./about.) glycerol, 17 mm potassium dihydrophosphate, 72 mm dicale-phosphate)containing 50 μg/ml ampicillin. A milliliter (1 ml) of the obtained culture medium was transferred into 100 ml of medium TV containing 50 μg/ml of ampicillin, and cultured at 26°C. After reaching the OD660 approximately 0.5 was added 5-aminolevulinate acid to a final concentration of 500 μm, and the cultivation was continued. After 30 minutes, then IPTG was added to a final concentration of 1 m and were additionally cultured for 17 hours.

Cells were removed from each culture medium, washed with 0.1 M Tris-HCl-buffer (pH 7.5) and suspended in 10 ml of the above buffer containing 1 mm PMSF. The obtained cell suspension was subjected to 6 times the processing on the ultrasonic disintegrator (Sonifier (Branson Sonic Power Company)) for 3 minutes each time in terms of radiated power 3, load cycle 30%, to obtain solutions of cell lysates. After centrifugation of the solutions of cell lysates (HD, 5 minutes) supernatant were removed and centrifuged (HD, 70 minutes) to obtain fractions of supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN646, called "extract of E. coli pKSN646", the supernatant fraction obtained from E. coli JM109/pKSN646F, called "extract of E. coli pKSN646F", and the supernatant fraction obtained from E. coli JM109/pKSN2, called "extract of E. coli pKSN2"). One microliter (1 μl) of the above fractions supernatants were analyzed by electrophoresis in 15% -25% of the LTO-SDS page and stained STS. In the result by the detecting is clearly more intense bands in the extract of E. coli pKSN646 and in the extract of E. coli pKSN646F than in the extract of E. coli pKSN2, electrophoresis, corresponding to the molecular mass CD, you can confirm that the protein (A4) of the present invention is expressed in E. coli.

(3) the Discovery of an ability to convert compound (II) into the compound (III)

Goth is twisted to the reaction solution, 30 ml and kept for 10 minutes at 30°C. These reaction solutions consisted of 0.1 M potassium phosphate buffer (pH 7.0)containing 3 ppm of compound (II)labeled14S, 2 mm β-NADPH (hereinafter referred to as "component a") (Oriental Yeast Company), 2 mg/ml ferredoxin obtained from spinach (hereinafter referred to as "component B") (Sigma Company), 0.1 u/ml paradoxicality (hereinafter referred to as "component C") (Sigma Company) and 18 μl of the supernatant fraction obtained in example 27(2). Then I prepared and kept in this way the reaction solution without adding at least one component used in the above reaction solution, selected from the component A, component b and component C. was Added three microliters (3 ál) 2 N. HCl and 90 μl of ethyl acetate in each of these reaction solutions after the specified curing and stirred. The resulting reaction solution was centrifuged at 8000 g for extraction of 75 μl of an ethyl acetate layer. After drying, an ethyl acetate layer under reduced pressure, the residue was dissolved in 6.0 ál of ethyl acetate. Five microlitres (5,0 µl) of this solution was applied on TLC plate (silica gel TLC plates 60F25420 cm x 20 cm, 0.25 mm thick, Merck Company). The TLC plate showed a mixture of 6:1:2 chloroform, acetic acid and ethyl acetate for about 1 hour. Then the solvent was allowed to evaporate. The TLC plate was exposed in the Techa is their night on the imaging plate (Fuji Film Company). Then, the imaging plate was analyzed by the image Analyzer BAS2000 (Fuji Film Company). The presence of spots corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). It can be confirmed the formation of compound (III) in the reaction solutions containing component a, component B, component C and the extract of E. coli pKSN646, or in reaction solutions containing component a, component B, component C and the extract of E. coli pKSN646F.

Example 28. The sequence identity relative to the protein of the present invention

Identity sequence relative to the proteins of the present invention and the DNA of the present invention were analyzed using the program GENETIX-WIN Ver. 5 (Software Development Company). Mapping the building was obtained by analysis of homology way of Lipmans-Pearson (Lipman, D.J. and Pearson, W.R., Science, 227, 1435-1441 (1989)).

In relation to the amino acid sequence of proteins (A1)-(A4) of this invention was determined by the identity of the sequence relative to each other and relative to the known protein with the highest homology. These results are shown in table 15.

Table 15
protein (A1) of the present inventionBel is to (A2) of the present invention protein (A3) of the present inventionprotein (A4) this inventionknown protein with the highest homology*
protein (A1) of the present invention100%47%64%48%73%
AAC25766
protein (A2) of the present invention47%100%48%51%52%
CAB46536
protein (A3) of the present invention64%48%100%46%67%
AAC25766
protein (A4) this invention48%51%46%100%50%
CAB46536
* The identity of the sequence shown at the top, and the access number of the specified protein in the Entrez database (provided by Center for biotechnology information, http://www.3.ncbi.nlm.nih.gov/Entrez/), shown at the bottom.

In relation to the nucleotide sequence that is tednik DNA sequences (A1) of the present invention, having the nucleotide sequence shown in SEQ ID NO:6, DNA (A2) of the present invention having the nucleotide sequence shown in SEQ ID NO:7, DNA (A3) of the present invention having the nucleotide sequence shown in SEQ ID NO:8, and DNA (A4) of the present invention having the nucleotide sequence shown in SEQ ID NO:109, were determined identity of the sequences relative to each other and relative to known genes with the highest homology. These results are shown in table 16.

64%
Table 16
SEQ ID NO:6 [DNA (A1) of the present invention]SEQ ID NO:7
[DNA (A2) of the present invention]
SEQ ID NO:8
[DNA (A3) of this invention]
SEQ ID NO:109
[DNA (A4) of this invention]
known genes with the highest homology*
SEQ ID NO:6 [DNA (A1) of the present invention]100%61%74%62%77%
AF072709
SEQ ID NO:7 [DNA (A2) of the present invention]61%100%65%66%
Y18574
SEQ ID NO:8 [DNA (A3) of this invention]74%64%100%63%75%
AF072709
SEQ ID NO:109
[DNA (A4) of this invention]
62%65%63%100%64%
Y18574
* The identity of the sequence shown at the top, and the access number of the specified gene in the Entrez database (provided by Center for biotechnology information, http://www.3.ncbi.nlm.nih.gov/Entrez/), shown at the bottom.

In relation to the amino acid sequence of proteins (B1)-(B4) of this invention was determined by the identity of the sequence relative to each other and relative to the known protein with the highest homology. These results are shown in table 17.

Table 17
protein (B1) of the present inventionprotein (B2) of the present inventionprotein (B3) this izobreteny the protein (B4) this inventionknown protein with the highest homology*
protein (B1) of the present invention100%45%78%41%76%
AAC25765
protein (B2) of the present invention45%100%40%41%60%
AAF71770
protein (B3) of the present invention78%40%100%40%73%
AAC25765
protein (B4) this invention41%41%40%100%55%
AAA26824
* The identity of the sequence shown at the top, and the access number of the specified protein in the Entrez database (provided by Center for biotechnology information, http://www.3.ncbi.nlm.nih.gov/Entrez/), shown at the bottom.

In relation to the nucleotide sequences of DNA (B1) of the present invention having the nucleotide sequence shown in SEQ ID NO:15, DNA (B2) of the present invention having the nucleotide sequence shown in SEQ ID NO:16, DNA (B3) of the present invention having the nucleotide sequence shown in SEQ ID NO:17, and DNA (B4) of the present invention having the nucleotide sequence shown in SEQ ID NO:112, were determined identity of the sequences relative to each other and relative to known genes with the highest homology. These results are shown in table 18.

Table 18
SEQ ID NO:15 [DNA (B1) of this invention]SEQ ID NO:16
[DNA (B2) of this invention]
SEQ ID NO:17
[DNA (B3) of this invention]
SEQ ID NO:112
[DNA (B4) of this invention]
known genes with the highest homology*
SEQ ID NO:15 [DNA (B1) of this invention]100%60%80%59%84%
AF072709
SEQ ID NO:16 [DNA (B2) of this invention]60%100%60% 59%66%
M32238
SEQ ID NO:17 [DNA (B3) of this invention]80%60%100%65%79%
AF072709
SEQ ID NO:112
[DNA (B4) of this invention]
59%59%65%100%66%
M32239
* The identity of the sequence shown at the top, and the access number of the specified gene in the Entrez database (provided by Center for biotechnology information, http://www.3.ncbi.nlm.nih.gov/Entrez/), shown at the bottom.

Example 29. PCR using oligonucleotide having a partial nucleotide sequence of DNA (a) of this invention as a primer

PCR was performed using as template each: chromosomal DNA of Streptomyces phaeochromogenes IFO 12898, obtained in example 2; the chromosomal DNA Saccharopolyspora taberi JCM 9383t obtained in example 5; chromosomal DNA of Streptomyces griseolus ATCC 11796 obtained in example 9; the chromosomal DNA of Streptomyces testaceus ATCC 21469 obtained in example 11; the chromosomal DNA of Streptomyces achromogenes IFO 12735, obtained in example 26; and each of the chromosomal DNA of Streptomyces griseofuscus IFO 12870t, Streptomyces thermocoeruescens IFO 14273t and Streptomyces nogalater IFO 13445, is obtained analogously to the method described in example 2. As primers used 5 pairs of primers shown in table 19. The predicted size of the DNA amplified using PCR using each of the pairs of primers based on the nucleotide sequence shown in SEQ ID NO:6, is shown as table 19.

The reaction solution for PCR was made up to 25 μl by the addition of 200 nm of each of the 2 primer pairs shown in table 19, by adding 10 ng of chromosomal DNA, and 0.5 μl of dNTP mixture (a mixture of 10 mm each of the 4 types of dNTP), 5 μl of 5x buffer for GC genomic PCR, 1,1 ál of 25 mm Mg(OAc)25 µl 5 M GC-melt and 0.5 μl of a mixture of genomic polymerase Advantage-GC and the addition of water. Reaction conditions PCR after maintaining 95°C for 1 minute were as follows: repeating 30 cycles, where each cycle consisted of maintaining 94°C for 15 seconds, then 60°C for 30 seconds, and then 72°C for 1 minute; and maintain 72°C for 5 minutes. Each of these reaction solutions after the specified incubation were analyzed by electrophoresis in 3% agarose gel. The results are shown in Fig. 46 and table 20 and table 21. Amplification of the predicted size of the DNA was observed in each case or in all cases with the pairs of primers 14, 15, 16, 17 and 18, and also in cases of using chromosomal DNA from any of these strains as a matrix.

Table 19
primer pairprimerprimeramplificatory DNA
14SEQ ID NO:124SEQ ID NO:129about 800 BP
15SEQ ID NO:125SEQ ID NO:129about 600 BP
16SEQ ID NO:126SEQ ID NO:129about 600 BP
17SEQ ID NO:127SEQ ID NO:129approximately 580 BP
18SEQ ID NO:128SEQ ID NO:129approximately 580 BP
Table 20
TrackReagentsamplification of DNA*
the origin matrix of chromosomal DNA primer pair
2Streptomyces phaeochromogenes IFO 1289814+
3Streptomyces phaeochromogenes IFO 1289815+
4Streptomyces phaeochromogenes IFO 1289816+
5Streptomyces phaeochromogenes IFO 1289817+
6Streptomyces phaeochromogenes IFO 1289818+
9Streptomyces testaceus ATCC2146914+
10Saccharopolyspora taberi
JCM 9393t
14+
11Streptomyces griseolus ATCC1179614+
13Streptomyces testaceus ATCC2146915+
14Saccharopolypora taberi JCM9393t 15+
15Streptomyces griseolus
ATCC 11796
15+
16Streptomyces testaceus
ATCC 21469
16+
17Saccharopolyspora taberi
JCM 9393t
16+
18Streptomyces griseolus
ATCC 11796
16+
20Streptomyces testaceus
ATCC 21469
17+
21Saccharopolyspora taberi
JCM 9393t
17+
22Streptomyces griseolus
ATCC 11796
17+
23Streptomyces testaceus
ATCC 21469
18+
24Saccharopolyspora taberi
JCM 9393t
18+
25Streptomyces griseolus
ATCC 11796
18+
* “+” means that there was detected the predicted size of the DNA, and “-“ means that it has not been detected.

Table 21
TrackReagentsamplification of DNA*
the origin matrix of chromosomal DNAprimer pair
28Streptomyces griseofuscus
IFO 12870t
14+
29Streptomyces thermocoerulescens IFO 14273t14+
30Streptomyces achromogenes
IFO 12735
14-
31Streptomyces nogalater
IFO 13445
14+
33Streptomyces griseofuscus IFO 12870t15 +
34Streptomyces thermocoerulescens IFO 14273t15+
35Streptomyces achromogenes
IFO 12735
15-
36Streptomyces nogalater
IFO 13445
15+
38Streptomyces griseofuscus
IFO 12870t
16+
39Streptomyces thermocoerulescens IFO 14273t16+
40Streptomyces achromogenes
IFO 12735
16+
41Streptomyces nogalater
IFO 13445
16+
43Streptomyces griseofuscus
IFO 12870t
17+
44Streptomyces thermocoerulescens IFO 14273t17+
45treptomyces achromogenes IFO 12735 17+
46Streptomyces nogalater
IFO 13445
17+
48Streptomyces griseofuscus
IFO 12870t
18-
49Streptomyces thermocoerulescens IFO 14273t18+
50Streptomyces achromogenes
IFO 12735
18-
51Streptomyces nogalater
IFO 13445
18+
* “+” means that there was detected the predicted size of the DNA, and “-“ means that it has not been detected.

Example 30. Hybridization using as a probe a DNA comprising a partial nucleotide sequence of this DNA (a) and DNA (a) of this invention

(1) Receiving probe

DNA comprising a partial nucleotide sequence of DNA (A1) of the present invention or the partial nucleotide sequence of DNA (A1) of the present invention, was obtained in the form of a probe, labeled d is gokigenyou (DIG-labeled probe). PCR was performed with a kit for the synthesis of DIG-probe for PCR (Roche Diagnostics GmbH Company) in accordance with the attached manual using as the template the chromosomal DNA of Streptomyces phaeochromogenes IFO 12898, obtained in example 3, and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:93, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:94. The reaction solution for PCR was brought up to 50 μl by adding each of the 2 primers, brought to 200 nm, by adding 50 ng of chromosomal DNA, and 2.5 μl of dNTP mixture (a mixture of 2.0 mm each of the 4 types of dNTP), and 2.5 ál of DIG-mixture for PCR (mixture of 2.0 mm each of the 4 types of dNTP labeled with DIG), 5 ál 10x PCR buffer and 0.75 μl of a mixture of enzymes Expand HiFi and by adding distilled water. The PCR reaction conditions were as follows: after incubation at 95°C for 2 minutes, repeating 10 cycles, where each cycle consisted of maintaining 95°C for 10 seconds, then 60°C for 30 seconds, and then 72°C for 2 minutes; then 15 cycles, where each cycle consisted of maintaining 95°C for 10 seconds, then 60°C for 30 seconds, and then 72°C for 2 minutes (20 seconds added to maintain 72°C for each cycle); and then maintaining 72°C for 7 minutes. The reaction solution was then subjected to electrophoresis in 1% agarose gel. As a result, the e was confirmed by amplification of DNA by size of approximately 1.3 TPN This amplified DNA was extracted with obtaining DNA labeled with digoxigenin and having the nucleotide sequence shown in SEQ ID NO:6. Using a similar method, PCR was performed using as a template the chromosomal DNA of Streptomyces phaeochromogenes IFO 12898 and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:130, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:131. DNA amplified specified PCR, extracted with obtaining DNA labeled with digoxigenin and having a nucleotide sequence consisting of nucleotides 57-730 the nucleotide sequence shown in SEQ ID NO:6.

In a similar way PCR was performed using as a template the chromosomal DNA Saccharopolyspora taberi JCM 9383t obtained in example 6, and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:61, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:62. DNA amplified specified PCR, extracted with obtaining DNA labeled with digoxigenin and having the nucleotide sequence shown in SEQ ID NO:7.

Next, using a similar method, PCR was performed using as a template the chromosomal the NC Streptomyces testaceus ATCC 21469, obtained in example 11 and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:70, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:71. DNA amplified specified PCR, extracted with obtaining DNA labeled with digoxigenin and having the nucleotide sequence shown in SEQ ID NO:8. Next, PCR was performed using the above chromosomal DNA as template and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:132, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:133. DNA amplified specified PCR, extracted with obtaining DNA labeled with digoxigenin and having a nucleotide sequence consisting of nucleotides 21-691 the nucleotide sequence shown in SEQ ID NO:8.

(2) Dot-blot-hybridization

Each of the DNA pKSN657 obtained in example 4 (containing DNA DNA (A1) of the present invention), the DNA pKSN923 obtained in example 7 (containing DNA DNA (A2) of the present invention), the DNA pKSN671 obtained in example 12 (containing DNA DNA (A3) of the present invention), the DNA pKSNSCA obtained in example 14 (DNA containing this DNA (A9), and DNA pKSN11796 obtained in example 10 (DNA containing this DNA (a10), nano is or blotting on nylon the membrane Hybond N+ (Amersham Pharmacia Company) to the number of 100 ng and 10 ng. Ultraviolet light was directed on the obtained membrane using transilluminator within 5 minutes.

The set of DIG-High Prime DNA Labeling and Detector Starter Kit II (Roche Diagnostics GmbH Company) was used for hybridization and detection in accordance with the attached manual. As the probes used each of the DNA labeled with digoxigenin and obtained in example 30(1), which was kept at 100°C for 5 minutes and then quickly cooled in ice (hereinafter called “DIG-labeled probe”). Above the membrane with printed spots (fortified areas) kept at 42°C for 30 minutes in 2.0 ml DIGEasyHyb, provide the specified collection. Then 2.0 ml DIG Easy Hyb, 5,0 ál of DIG-labeled probes and the membrane was concluded in a plastic bag for hybridization and kept at 42°C for 18 hours. The membrane was removed, washed twice in 2SSC containing 0.1% LTOs, for 5 minutes at room temperature and then washed twice in 0,5SSC containing 0.1% LTOs at 65°C for 15 minutes. Then the membrane was washed in 50 ml otmennogo buffer for 2 minutes, then kept in 50 ml of blocking solution at room temperature for 30 minutes, then kept in a 2.0 ml solution of antibody for 30 minutes and then washed twice in 50 ml otmennogo buffer for 15 minutes. Then, after keeping in 50 ml of detection buffer for 5 minutes, e is the membrane was concluded in the bag for hybridization with 2.0 ml of color substrate and kept at room temperature for 18 hours. The signal was detected in each of the cases of hybridization with each of the reagents 10 ng and 100 ng each of pKSN657, pKSN923, pKSN671, pKSNSCA and pKSN11796.

Example 31. Obtaining DNA (A11) this invention

(1) preparation of chromosomal DNA of Streptomyces nogalator IFO13445

Streptomyces nogalator IFO13445 were cultured with shaking at 30°C for 3 days in 50 ml of medium YGY (0,5% (wt./about.) yeast extract and 0.5% (wt./about.) tripton, 0.1% (wt./about.) glucose and 0.1% (wt./about.) K2HPO4, pH 7.0). The cells were collected. The obtained cells suspended in the medium YGY containing 1,4% (wt./about.) glycine and 60 mm EDTA, and additionally incubated with shaking for one day. Cells were collected from culture medium. After washing once with distilled water them suspended in 3.5 ml of buffer 1 (50 mm Tris-HCl (pH 8.0), 50 mm EDTA, 0.5% tween-20 and 0.5% Triton X-100). Eighty microlitres (80 μl) of a solution of lysozyme 100 μg/ml and 100 μl of Qiagen protease (600 may/ml, Qiagen Company) was added to this suspension and kept at 37°C for one hour. Then add 1.2 ml of buffer B2 (3 M guanidine·HCl and 20% tween-20), were mixed and kept at 50°C for 30 minutes. The resulting solution of cell lysate was added to the genomic chip Qiagen 100G (Qiagen Company), brings in the buffer QBT (750 mm NaCl, 50 mm MOPS (pH 7.0), 15% isopropanol and 0.15% Triton X-100). Then, after washing of the chip twice with 7.5 ml of buffer QC (50 mm MOPS (pH 7.0) and 15% and propanol), DNA was suirable by passing 5 ml of buffer QF (1.25 M NaCl, 50 mm Tris-HCl (pH 8.5), 15% isopropanol). 3.5 ml of isopropanol was mixed with the obtained DNA solution for deposition and extraction of chromosomal DNA. After washing with 70% ethanol extracted chromosomal DNA was dissolved in 1 ml of buffer live TV.

(2) Isolation of DNA having a partial nucleotide sequence of DNA (A11) this invention

PCR was performed using as a template the chromosomal DNA obtained in example 31(1), and using a pair of 14 primers in accordance with the method described in example 29. Amplified DNA ligated to the cloning vector pCRII-TOPO (Invitrogen Company) in accordance with the instructions attached to the specified vector, and then introduced into E. coli TOP10F'. Plasmid DNA was obtained from the received transformant E. coli using a set of Qiagen Tip20 (Qiagen Company). The sequencing reaction was performed with a set of Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company) in accordance with the instructions attached to the specified collection, using a primer having the nucleotide sequence shown in SEQ ID NO:57, and a primer having the nucleotide sequence shown in SEQ ID NO:59. In sequencing reactions used the obtained plasmid as a template. The reaction products were analyzed using DNA sequencing machine 3100 (Applied Biosystems Japan ompany has). The result has been a nucleotide sequence consisting of nucleotides 316-1048 the nucleotide sequence shown in SEQ ID NO:139.

Next, the chromosomal DNA obtained in example 31(1), was digested with restriction enzyme PvuII. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:161, and primer API (universal Genome Walker (Clontech Company)). Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:162, and primer AP2 (universal Genome Walker (Clontech Company)). The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1-330 the nucleotide sequence shown in SEQ ID NO:144.

Next, the chromosomal DNA obtained in example 31(1), was digested with restriction enzyme HincII. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR carried out and the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:163, and primer API (universal Genome Walker (Clontech Company)). Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:164, and primer AP2 (universal Genome Walker (Clontech Company)). The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 983-1449 the nucleotide sequence shown in SEQ ID NO:144.

(3) Analysis of DNA sequences (A11) this invention

The nucleotide sequence shown in SEQ ID NO:144, received by the connection of the nucleotide sequences provided DNA obtained in example 31(2). Attended two open reading frames (ORF). Thus, it contained the nucleotide sequence (SEQ ID NO:139), consisting of 1230 nucleotides including the stop codon) and encoding a 409 amino acid residues (SEQ ID NO:159), and the nucleotide sequence (SEQ ID NO:154), consisting of 207 nucleotides including the stop codon) and encoding 68 aminokislot the x residues (SEQ ID NO:149). It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:159), encoded by the nucleotide sequence shown in SEQ ID NO:139, equal 45177 Yes. Further, it was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:149), encoded by the nucleotide sequence shown in SEQ ID NO:154, equal 7147 Yes.

Example 32. Expression of the protein (A11) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (A11) this invention

PCR was performed using as a template the chromosomal DNA obtained from Streptomyces nogalator IFO 13445 in example 31(1), and using PCR system Expand HiFi (Boehringer Manheim Company). As primers used a couple of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:165, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:166. The composition of the reaction solution and ageing were similar to conditions described in example 27(1). After this incubation, the reaction solution was subjected to electrophoresis in 1% agarose gel. The area of the gel containing DNA size approximately 1.5 TPN, was removed. DNA was purified from the extracted gel using a set of extraction from gels QIAquick (Qiagen Company) in accordance with the attached instructions Obtained DNA ligated to the cloning vector pCRII-TOPO (Invitrogen Company) in accordance with the instructions, attached to the specified vector, and introduced into E. coli 10F'. Plasmid DNA was obtained from the obtained transformants of E. coli using a set of Qiagen Tip20 (Qiagen Company). The sequencing reaction was performed with a set of Dye terminator cycle sequencing FS ready (Applied Biosystems Japan Company) in accordance with the instructions attached to the specified set, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:57, 59 and 186. In the sequencing reactions used the obtained plasmid DNA as template. The reaction products were analyzed using DNA sequencing machine 3100 (Applied Biosystems Japan Company). On the basis of these results, the plasmid having the nucleotide sequence shown in SEQ ID NO:144, was named pCR849AF.

Then pCR849F were digested with restrictase NdeI and HindIII. The cleavage products were subjected to agarose gel electrophoresis. The area of the gel containing DNA size approximately 1.5 TPN, cut out of the gel. DNA was purified from the extracted gel using a set of extraction from gels QIAquick (Qiagen Company) in accordance with the attached instructions. The resulting DNA and plasmid pKSN2, split NdeI and HindIII, ligated using set for ligating Ver. 2 (Takara Shuzo Company) in accordance with the instructions attached to the specified collection, and introduced into E. coli JM109. From the obtained transformants of E. coli p. who were given plasmid DNA. Their structures were analyzed. A plasmid containing the nucleotide sequence shown in SEQ ID NO:144, in which DNA size approximately 1.5 TPN encoding a protein (A4) of the present invention, inserted between the NdeI site and the HindIII site pKSN2, was named pKSN849AF. Plasmid pKSN849F was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN849F. Next, plasmid pKSN2 was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN2.

(2) protein Expression (A11) of the present invention in E. coli and the selection of the specified protein

Like in example 4(2) cultivated each of E. coli JM109/pKSN849AF and JM109/pKSN2. The cells were extracted. Prepared solutions of cell lysates. According to the method described in example 4(2)of these solutions cell lysates were obtained fractions supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN849AF, called “extract of E. coli pKSN849AF”, and the supernatant fraction obtained from E. coli JM109/pKSN2, called “extract of E. coli pKSN2”).

(3) the Discovery of an ability to convert compound (II) into the compound (III)

The reaction solution, 30 μl were prepared and kept for 10 minutes at 30°C. These reaction solutions consisted of 0.1 M potassium phosphate buffer (pH 7.0)containing 3 ppm of compound (II)labeled14S, 2 mm β-NADPH (hereinafter referred to as "component a") (Oriental Yeast Company), 2 mg/ml ferredoxin obtained from spinach is (hereinafter referred to as "component B") (Sigma Company), of 0.1 u/ml paradoxicality (hereinafter referred to as "component C") (Sigma Company) and 23 μl of the supernatant fraction obtained in example 32(2). Like in example 4(3), these reaction solutions after aging were extracted with ethyl acetate and extracted layers were analyzed using TLC. After developing TLC plates the presence of stains on it, corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). The spot corresponding to the compound (III), were detected from the reaction solution containing the extract of E. coli pKSN849F. In contrast, this spot is not detected from the reaction solution containing the extract of E. coli pKSN2.

Example 33. Obtaining DNA (A12) this invention

(1) preparation of chromosomal DNA of Streptomyces tsusimaensis IFO 13782

Chromosomal DNA of Streptomyces tsusimaensis IFO 13782 was obtained according to the method described in example 31(1).

(2) Isolation of DNA having a partial nucleotide sequence of DNA (A12) this invention

PCR was performed using as a template the chromosomal DNA of Streptomyces tsusimaensis IFO 13782 obtained in example 33(1), and using a pair of 14 primers in accordance with the method described in example 29. Similarly to example 31(2), amplified DNA ligated to the cloning vector pCRII-TOPO (Invitrogen Company). Its nucleotide sequence was analyzed. In d is the query result was obtained nucleotide sequence, consisting of nucleotides 364-1096 the nucleotide sequence shown in SEQ ID NO:140.

Next, the chromosomal DNA obtained in example 33(1), was digested with restriction enzyme SmaI. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:167, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:168, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1-392 the nucleotide sequence shown in SEQ ID NO:145.

Next, the chromosomal DNA obtained in example 33(1), was digested with restriction enzyme PvuII. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as a matrix and use what Lovanium of the oligonucleotide, having the nucleotide sequence shown in SEQ ID NO:169, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:170, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1048-1480 the nucleotide sequence shown in SEQ ID NO:145.

(3) Analysis of DNA sequences (A12) this invention

The nucleotide sequence shown in SEQ ID NO:145, received by the connection of the nucleotide sequences provided DNA obtained in example 33(2). In the specified nucleotide sequence was attended by two open reading frames (ORF). Thus, it contained the nucleotide sequence (SEQ ID NO:140), comprising 1278 nucleotides including the stop codon) and encoding a 425 amino acid residues (SEQ ID NO:160), and the nucleotide sequence (SEQ ID NO:155), consisting of 198 nucleotides including the stop codon) and encoding a 65 amino acid residues (SEQ ID NO:150). It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:160), encoded by the nucleotide sequence shown in SEQ IDNO:140, equal 46549 Yes. Further, it was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:150), encoded by the nucleotide sequence shown in SEQ ID NO:155, equal 6510 Yes.

Example 34. Expression of DNA (A12) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (A12) this invention

PCR was performed similarly to example 32(1), except for use as matrix chromosomal DNA obtained from Streptomyces tsusimaensis IFO 13782 in example 33(1) and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:171, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:172. Similarly to example 32(1), DNA was purified from the reaction solution for PCR and cloned into the cloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequence of the obtained plasmid DNA was analyzed using oligonucleotides having the nucleotide sequences shown, respectively, in SEQ ID nos:57, 59, 171, 172 and 187. On the basis of the results obtained plasmid having the nucleotide sequence shown in SEQ ID NO:145, was named pCR1618F. Similarly to example 32(1), pCR1618F were digested with restrictase NdeI and HindIII. From the products of cleavage of the purified DNA size approximately 1.5 TPN Obtained DNA is plasmid pKSN2, split NdeI and HindIII, ligated to obtain plasmid containing the nucleotide sequence shown in SEQ ID NO:145, in which DNA encoding a protein (A12) of the present invention, inserted between the NdeI site and the HindIII site pKSN2 (hereinafter called “pKSN1618F”). The indicated plasmid was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN1618F.

(2) protein Expression (A12) of the present invention in E. coli and the selection of the specified protein

Like in example 4(2) cultivated each of E. coli JM109/pKSN1618F and JM109/pKSN2. The cells were extracted. Prepared solutions of cell lysates. According to the method described in example 4(2)of these solutions cell lysates were obtained fractions supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN1618F, called “extract of E. coli pKSN1618F”, and the supernatant fraction obtained from E. coli JM109/pKSN2, called “extract of E. coli pKSN2”).

(3) the Discovery of an ability to convert compound (II) into the compound (III)

The reaction solution, 30 μl were prepared and kept for 10 minutes at 30°C. with the exception of the use of fractions supernatants obtained in example 34(2) (extract of E. coli pKSN1618F or extract of E. coli pKSN2), the reaction solutions were prepared similarly to example 32(3). These reaction solutions after aging were extracted with ethyl acetate and extracted layers were analyzed using TLC. After expression the Oia TLC plates the presence of stains on it, the corresponding compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). The spot corresponding to the compound (III), were detected from the reaction solution containing the extract of E. coli pKSN1618F. In contrast, this spot is not detected from the reaction solution containing the extract of E. coli pKSN2.

Example 35. Obtaining DNA (A13) this invention

(1) preparation of chromosomal DNA of Streptomyces thermocoerulesces IFO 14273t

Chromosomal DNA of Streptomyces thermocoerulesces IFO 14273t was obtained according to the method described in example 31(1).

(2) Isolation of DNA having a partial nucleotide sequence of DNA (A13) this invention

PCR was performed using as a template the chromosomal DNA of Streptomyces thermocoerulesces IFO 14273t obtained in example 35(1), and using a pair of 14 primers in accordance with the method described in example 29. Similarly to example 31(2), amplified DNA ligated to the cloning vector pCRII-TOPO (Invitrogen Company). Its nucleotide sequence was analyzed. The result was obtained nucleotide sequence consisting of nucleotides 295-1027 the nucleotide sequence shown in SEQ ID NO:141.

Next, the chromosomal DNA obtained in example 35(1), was digested with restriction enzyme HincII. Library genome walker was obtained using the obtained DNA in accordance with the method described is in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:173, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:174, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1-370 the nucleotide sequence shown in SEQ ID NO:146.

Next, the chromosomal DNA obtained in example 35(1), was digested with restriction enzyme SmaI. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:175, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having nucleotidyltransferase, shown in SEQ ID NO:176, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 960-1473 the nucleotide sequence shown in SEQ ID NO:146.

(3) Analysis of DNA sequences (A13) this invention

The nucleotide sequence shown in SEQ ID NO:146, received by the connection of the nucleotide sequences provided DNA obtained in example 35(2). In the specified nucleotide sequence was attended by two open reading frames (ORF). Thus, it contained the nucleotide sequence (SEQ ID NO:141), consisting of 1209 nucleotides including the stop codon) and encoding a 402 amino acid residue (SEQ ID NO:136), and the nucleotide sequence (SEQ ID NO:156), consisting of 252 nucleotides including the stop codon) and 83 encoding amino acid residues (SEQ ID NO:151). It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:136), encoded by the nucleotide sequence shown in SEQ ID NO:141, equal 44629 Yes. Further, it was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:151), encoded by the nucleotide sequence shown in SEQ ID NO:156, equal 8635 Yes.

Example 36. Expression of DNA (A13) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (A13) this invention

PCR was performed similarly to example 32(1), except for use as matrix chromosomal DNA obtained from Streptomyces thermocoerulesces IFO 14273t in example 35(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:177, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:178. Similarly to example 32(1), DNA was purified from the reaction solution for PCR and cloned into the cloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequence of the obtained plasmid DNA was analyzed using oligonucleotides having the nucleotide sequences shown, respectively, in SEQ ID nos:57, 59, 173, 175 and 188. On the basis of the results obtained plasmid having the nucleotide sequence shown in SEQ ID NO:146, was named pCR474F. Similarly to example 32(1), pCR474F were digested with restrictase NdeI and HindIII. From the products of cleavage of the purified DNA size approximately 1.5 TPN Obtained DNA and plasmid pKSN2, split NdeI and HindIII, ligated to obtain plasmid containing the nucleotide sequence shown in SEQ ID NO:146, in which DNA encoding a protein (A13) of the present invention, inserted between the NdeI site and the HindIII site pKSN2 (hereinafter called “pKSN474F”). The decree is ing plasmid was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN474F.

(2) protein Expression (A13) of the present invention in E. coli and the selection of the specified protein

Like in example 4(2) cultivated each of E. coli JM109/pKSN474F and JM109/pKSN2. The cells were extracted. Prepared solutions of cell lysates. According to the method described in example 4(2)of these solutions cell lysates were obtained fractions supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN474F, called “extract of E. coli pKSN474F”, and the supernatant fraction obtained from E. coli JM109/pKSN2, called “extract of E. coli pKSN2”).

(3) the Discovery of an ability to convert compound (II) into the compound (III)

The reaction solution, 30 μl were prepared and kept for 10 minutes at 30°C. with the exception of the use of fractions supernatants obtained in example 36(2) (extract of E. coli pKSN474F or extract of E. coli pKSN2), the reaction solutions were prepared similarly to example 32(3). These reaction solutions after aging were extracted with ethyl acetate and extracted layers were analyzed using TLC. After developing TLC plates the presence of stains on it, corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). The spot corresponding to the compound (III), were detected from the reaction solution containing the extract of E. coli pKSN474F. In contrast, this spot is not de who was intervalos from the reaction solution, containing extract of E. coli pKSN2.

Example 37. Obtaining DNA (A14) this invention

(1) preparation of chromosomal DNA of Streptomyces glomerochromogenes IFO 13673t

Chromosomal DNA of Streptomyces glomerochromogenes IFO 13673t was obtained according to the method described in example 31(1).

(2) Isolation of DNA having a partial nucleotide sequence of DNA (A14) this invention

PCR was performed using as a template the chromosomal DNA of Streptomyces glomerochromogenes IFO 13673t obtained in example 37(1), and using a pair of 14 primers in accordance with the method described in example 29. Similarly to example 31(2), amplified DNA was cloned into the cloning vector pCRII-TOPO (Invitrogen Company). Its nucleotide sequence was analyzed. The result was obtained nucleotide sequence consisting of nucleotides 316-1048 the nucleotide sequence shown in SEQ ID NO:142.

Next, the chromosomal DNA obtained in example 37(1), was digested with restriction enzyme SmaI. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:179, and primer AP1. Then P Is P performed under conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:180 and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1-330 the nucleotide sequence shown in SEQ ID NO:147.

Next, the chromosomal DNA obtained in example 37(1), was digested with restriction enzyme HincII. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:181, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:182, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 982-1449 the nucleotide sequence shown in SEQ ID NO:147.

(3) Analysis of DNA sequences(A14) this invention

The nucleotide sequence shown in SEQ ID NO:147, received by the connection of the nucleotide sequences provided DNA obtained in example 37(2). In the specified nucleotide sequence was attended by two open reading frames (ORF). Thus, it contained the nucleotide sequence (SEQ ID NO:142), consisting of 1230 nucleotides including the stop codon) and 409 encoding amino acid residue (SEQ ID NO:137), and the nucleotide sequence (SEQ ID NO:157), consisting of 207 nucleotides including the stop codon) and encoding a 68 amino acid residues (SEQ ID NO:152). It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:137), encoded by the nucleotide sequence shown in SEQ ID NO:142, equal 45089 Yes. Further, it was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:152), encoded by the nucleotide sequence shown in SEQ ID NO:157, equal 7174 Yes.

Example 38. Expression of DNA (A14) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (A14) this invention

PCR was performed similarly to example 32(1), except for use as matrix chromosomal DNA obtained from Streptomyces glomerochromogenes IFO 13673t in example 37(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:183, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:184. Similarly to example 32(1), DNA was purified from the reaction solution for PCR and cloned into the cloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequence of the obtained plasmid DNA was analyzed using oligonucleotides having the nucleotide sequences shown, respectively, in SEQ ID nos:57, 59 and 189. On the basis of the results obtained plasmid having the nucleotide sequence shown in SEQ ID NO:147, was named pCR1491F. Similarly to example 32(1), pCR1491F were digested with restrictase NdeI and HindIII. From the products of cleavage of the purified DNA size approximately 1.5 TPN Obtained DNA and plasmid pKSN2, split NdeI and HindIII, ligated to obtain plasmid containing the nucleotide sequence shown in SEQ ID NO:147, in which DNA encoding a protein (A14) of the present invention, inserted between the NdeI site and the HindIII site pKSN2 (hereinafter called “pKSN1491F”). The indicated plasmid was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN1491F.

(2) protein Expression (A14) of the present invention in E. coli and the selection of the specified protein

Like in example 4(2) cultivated each of E. coli JM109/pKSN1491F and JM109/pKSN2. The cells were extracted. Prepared solutions of cell lysates. According to the method described in example 4(2), from this the x solutions cell lysates were obtained fractions supernatant (hereinafter fraction of the supernatant, obtained from E. coli JM109/pKSN1491F, called “extract of E. coli pKSN1491F”, and the supernatant fraction obtained from E. coli JM109/pKSN2, called “extract of E. coli pKSN2”).

(3) the Discovery of an ability to convert compound (II) into the compound (III)

The reaction solution, 30 μl were prepared and kept for 10 minutes at 30°C. with the exception of the use of fractions supernatants obtained in example 38(2) (extract of E. coli pKSN1491F or extract of E. coli pKSN2), the reaction solutions were prepared similarly to example 32(3). These reaction solutions after aging were extracted with ethyl acetate and extracted layers were analyzed using TLC. After developing TLC plates the presence of stains on it, corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). The spot corresponding to the compound (III), were detected from the reaction solution containing the extract of E. coli pKSN1491F. In contrast, this spot is not detected from the reaction solution containing the extract of E. coli pKSN2.

Example 39. Obtaining DNA (A15) this invention

(1) preparation of chromosomal DNA of Streptomyces olivochromogenes IFO 12444

Chromosomal DNA of Streptomyces olivochromogenes IFO 12444 was obtained according to the method described in example 31(1).

(2) Isolation of DNA having a partial nucleotide sequence of DNA (A15) this invention

PCR Provo is or using as template the chromosomal DNA of Streptomyces olivochromogenes IFO 12444, obtained in example 39(1), and using a pair of 14 primers in accordance with the method described in example 29. Similarly to example 31(2), amplified DNA ligated to the cloning vector pCRII-TOPO (Invitrogen Company). Its nucleotide sequence was analyzed. The result was obtained nucleotide sequence consisting of nucleotides 316-1048 the nucleotide sequence shown in SEQ ID NO:143.

Next, the chromosomal DNA obtained in example 37(1), was digested with restriction enzyme SmaI. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using DNA as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:179, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:180 and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1-330 the nucleotide sequence shown in SEQ ID NO:148.

Next, the chromosomal DNA, recip is nnow in example 39(1), were digested with restriction enzyme SmaI. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:181, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:182, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 982-1449 the nucleotide sequence shown in SEQ ID NO:148.

(3) Analysis of DNA sequences (A15) this invention

The nucleotide sequence shown in SEQ ID NO:148, received by the connection of the nucleotide sequences provided DNA obtained in example 39(2). In the specified nucleotide sequence was attended by two open reading frames (ORF). Thus, it contained the nucleotide sequence (SEQ ID NO:143), comprising 1230 nucleotides including the stop codon) and encoding a 409 amino acid residues(SEQ ID NO:138), and the nucleotide sequence (SEQ ID NO:158), consisting of 207 nucleotides including the stop codon) and encoding a 68 amino acid residues (SEQ ID NO:153). It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:138), encoded by the nucleotide sequence shown in SEQ ID NO:143, equal 45116 Yes. Further, it was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:153), encoded by the nucleotide sequence shown in SEQ ID NO:158, equal 7179 Yes.

Example 40. Expression of DNA (A15) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (A15) this invention

PCR was performed similarly to example 32(1), except for use as matrix chromosomal DNA obtained from Streptomyces olivochromogenes IFO 12444 in example 39(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:184, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:185. Similarly to example 32(1), DNA was purified from the reaction solution for PCR and cloned into the cloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequence of the obtained plasmid DNA was analyzed with the use of oligonucleotides having nucleotide sequence, the display is installed, respectively, in SEQ ID nos:57, 59 and 189. On the basis of the results obtained plasmid having the nucleotide sequence shown in SEQ ID NO:148, was named pCR1555F. Similarly to example 32(1), pCR1555AF were digested with restrictase NdeI and HindIII. From the products of cleavage of the purified DNA size approximately 1.5 TPN Obtained DNA and plasmid pKSN2, split NdeI and HindIII, ligated to obtain plasmid containing the nucleotide sequence shown in SEQ ID NO:148, in which DNA encoding a protein (A15) of the present invention, inserted between the NdeI site and the HindIII site pKSN2 (hereinafter called “pKSN1555F”). The indicated plasmid was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN1555F.

(2) protein Expression (A15) of the present invention in E. coli and the selection of the specified protein

Like in example 4(2) cultivated each of E. coli JM109/pKSN1555F and JM109/pKSN2. The cells were extracted. Prepared solutions of cell lysates. According to the method described in example 4(2)of these solutions cell lysates were obtained fractions supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN1555F, called “extract of E. coli pKSN1555F”, and the supernatant fraction obtained from E. coli JM109/pKSN2, called “extract of E. coli pKSN2”).

(3) the Discovery of an ability to convert compound (II) into the compound (III)

The reaction solution, 30 μl were prepared and kept for 10 m is the gram at 30°C. Except fractions supernatants obtained in example 40(2) (extract of E. coli pKSN1555F or extract of E. coli pKSN2), the reaction solutions were prepared similarly to example 32(3). These reaction solutions after aging were extracted with ethyl acetate and extracted layers were analyzed using TLC. After developing TLC plates the presence of stains on it, corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). The spot corresponding to the compound (III), were detected from the reaction solution containing the extract of E. coli pKSN1555F. In contrast, this spot is not detected from the reaction solution containing the extract of E. coli pKSN2.

Example 41. The conversion of compounds protein (A1) of the present invention

(1) Obtaining fractions plastids

One hundred grams (100 g) seed radish field (Takii seeds) were sown in moistened paper laboratory towel in the tray, and cultivated at 25°C for 6 days in the dark and then were cultured for 4 hours under a fluorescent lamp. Thirty grams (30 g) green cotyledons were ground using a homogenizer Nissei AM-8 (Nihonseiki Seisakusho; 18000-20000 rpm, 4°C, 5 seconds) in the buffer for destruction (1 mm magnesium chloride, 20 mm N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic, 10 mm N-2-hydroxyethylpiperazine-N'-2-econsultant, 0.5 mm e is THE 5 mm cysteine, 0.5 M sucrose; pH of 7.7). The resulting solution of the cell lysate was passed through 4 layers of nylon gauze. The resulting solution was centrifuged (13170 g, 4°C, 1 minute). The fractions obtained residue suspended in 60 ml of buffer for destruction and centrifuged (2640 g, 4°C, 2 minutes). Faction balance resuspendable in 10 ml of buffer for destruction, was layered on the buffer high density (1 mm magnesium chloride, 20 mm N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic, 30 mm N-2-hydroxyethylpiperazine-N'-2-econsultant, 0.5 mm EDTA, 5 mm cysteine, 0.6 M sucrose; pH 7,7) in a centrifuge tube and centrifuged (675 g, 4°C, 15 minutes). The remains suspended in 3 ml of buffer for suspension (1 mm magnesium chloride, 20 mm N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic, 30 mm N-2-hydroxyethylpiperazine-N'-2-econsultant, 0.5 mm EDTA; pH of 7.7), and these suspensions were called faction plastid.

(2) Conversion of the compound (XII) protein (A1) of the present invention

Received 100 μl of reaction solution of 50 mm potassium phosphate buffer (pH 7.0)containing 5 ppm of compound (XII), 3 mm β-NADPH (hereinafter referred to as "component a") (Oriental Yeast Company), 1 mg/ml ferredoxin obtained from spinach (hereinafter referred to as "component B") (Sigma Company), and 0.15 u/ml paradoxicality (hereinafter referred to as "component C") (Sigma Company) and the supernatant fraction obtained in example 4(2). Reactio the hydrated solution was kept at 30°C for 10 minutes. In addition, prepared and handled in the same way, 100 μl of reaction solution of 50 mm potassium phosphate buffer (pH 7.0) without adding at least one component used in the above reaction solution, selected from the component A, component B, component C and the supernatant fraction obtained in example 4(2). Added ten microlitres (10 ál) 2 N. hydrochloric acid and 500 μl of ethyl acetate in each of these reaction solutions after the specified curing and stirred. The resulting reaction solution was centrifuged at 8000 g for extracting 490 μl of an ethyl acetate layer. After drying, an ethyl acetate layer under reduced pressure, the residue was dissolved in 100 μl of 50 mm potassium phosphate buffer (pH 7.0). Forty microliters (40 μl) of the solutions of the fractions (the solution fraction obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 4(2), called "the solution to (XII)-convert (A1)"; then, the solution fraction obtained from the reaction solution containing no component A, component B, component C and the supernatant fraction obtained in example 4(2), referred to as "solution (XII)control (A1)") were analyzed using HPLC. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A1), the concentration of the link is (XII), detected from the solution to (XII)-convert (A1)was lower. Then, from the solution to (XII)-convert (A1) was detected peak, which is not detected from the solution (XII)control (A1). Conducted mass spectrometry for the compounds contained in this peak. The mass of the compounds contained in this peak was 14 less than the mass of the compounds (XII).

Twenty microliters (20 µl) 32-fold dilution of the above solution to (XII)-convert (A1) and 60 μl of the plastid fraction obtained in example 41(1), was mixed. In dark conditions was added 20 μl of substrate solution (10 mm ATP, 5 mm aminolevulinate acid, 4 mm glutathion reductase and 0.6 mm+; pH 6.5; next, this substrate solution is called the "substrate solution PPO") and kept at 30°C for 1.5 hours. Further, instead of the above 20 μl of a 32-fold dilution of the above solution to (XII)-convert (A1) was prepared by the reaction solution, to which was added 20 μg 32-fold dilution solution (XII)control (A1) and added the substrate solution PPO and kept the same way. After conditioning in each reaction solution was added three hundred microlitres (300 μl) mixture of dimethyl sulfoxide-methanol (dimethylsulfoxide:methanol = 7:3) and centrifuged (8000 g, 4°C, 10 minutes). Supernatant was extracted and subjected to analysis addressed the o-phase HPLC under the conditions of analysis, below, to measure the amount of PPIX. The amount of PPIX in the reaction solution, which was added to the solution to (XII)-convert (A1), was greater than the amount of PPIX in the reaction solution, to which was added a solution of (XII)control (A1).

(Condition 2 analysis HPLC)

column: SUMIPAX ODS212 (Sumika Chemical Analysis Service)

the flow rate: 2 ml/min

the wavelength of detection: fluorescence excitation: 410 nm, emission: 630 nm

eluent: a mixture of 95:5 methanol and 1 M ammonium acetate (pH 5,7).

(3) Conversion of the compound (XIII) protein (A1) of the present invention

With the exception of using 5 ppm of compound (XIII) instead of 5 ppm of the compound (XII), the reaction solutions were obtained and maintained is similar to the method described in example 41(2). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 100 μl of dimethyl sulfoxide. The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 4(2), called "the solution to (XIII)-convert (A1)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 4(2), referred to as "solution (XIII)control (A1)") of the Academy of Sciences who was literally by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XIII)detected from the solution (XIII)control (A1), the concentration of the compound (XIII)detected from the solution to (XIII)-convert (A1)was lower. Then, from the solution to (XIII)-convert (A1) was detected peak, which is not detected from the solution (XIII)control (A1). Performed mass spectrometry on the connection contained in this peak. The mass of the compounds contained in this peak was 14 less than the mass of compound (XIII).

Twenty microliters (20 µl) 128-fold dilution of the above solution to (XIII)-convert (A1) and 60 μl fractions plastids were mixed. In dark conditions was added 20 μl of substrate solution PPO and kept at 30°C for 1.5 hours. Further, instead of the above 20 µl 128-fold dilution of the above solution to (XIII)-convert (A1)preparing a reaction solution to which was added 20 μl of 128-fold dilution solution (XIII)control (A1), and added the substrate solution PPO and kept the same way. Analogously to example 41(2), after keeping prepared each of the reaction solutions was subjected to analysis by reversed-phase HPLC under conditions analysis 2 above, to measure the amount of PPIX. The amount of PPIX in the reaction solution, which was added to the solution to (XIII)-convert (A1), was greater than the number PPIX in the reaction solution, to which solution was added (XIII)control (A1).

(4) the Conversion of compound (XVI) protein (A1) of the present invention

Except for the use of 12.5 ppm of the compound (XVI) instead of 5 ppm of the compound (XII), the reaction solutions were obtained and maintained is similar to the method described in example 41(2). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 200 μl of 50 mm potassium phosphate buffer (pH 7.0). The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 4(2), called "the solution to (XVII)-convert (A1)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 4(2), referred to as "solution (XVI)control (A1)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XVI), detected from the solution (XVI)control (A1), the concentration of the compound (XVI), detected from the solution to (XVI)transformation (A1)was lower. Then, from the solution to (XVI)transformation (A1) was detected peak, which is not detected from the solution (XVI)control (A1).

Twenty microliters (20 µl) 8 crtn the th dilution of the above solution to (XVI)transformation (A1) and 60 μl fractions plastids were mixed. In dark conditions was added 20 μl of substrate solution PPO and kept at 30°C for 1.5 hours. Further, instead of the above 20 ál 8-fold dilution of the solution to (XVI)transformation (A1) was prepared by the reaction solution, to which was added 20 μl of 8-fold dilution of the solution (XVI)control (A1), and added the substrate solution PPO and kept the same way. Analogously to example 41(2), after keeping prepared each of the reaction solutions was subjected to analysis by reversed-phase HPLC under conditions analysis 2 above, to measure the amount of PPIX. The amount of PPIX in the reaction solution, which was added to the solution to (XVI)transformation (A1), was greater than the amount of PPIX in the reaction solution, to which was added a solution of (XVI)control (A1).

(5) Conversion of the compound (XVII) protein (A1) of the present invention

Except for the use of 12.5 ppm of the compound (XVII) instead of 5 ppm of the compound (XII), the reaction solutions were obtained and maintained is similar to the method described in example 41(2). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 200 μl of 50 mm potassium phosphate buffer (pH 7.0). The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 2 μl of the supernatant fraction, obtained in example 4(2), called "the solution to (XVII)-convert (A1)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 4(2), referred to as "solution (XVII)control (A1)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XVII), detected from the solution (XVII)control (A1), the concentration of the compound (XVII), detected from the solution to (XVII)-convert (A1)was lower. Then, from the solution to (XVII)-convert (A1) was detected peak, which is not detected from the solution (XVII)control (A1).

Twenty microliters (20 µl) 32-fold dilution of the above solution to (XVII)-convert (A1) and 60 μl fractions plastids were mixed. In dark conditions was added 20 μl of substrate solution PPO and kept at 30°C for 1.5 hours. Further, instead of the above 20 μl of a 32-fold dilution of the solution to (XVII)-convert (A1) was prepared by the reaction solution, to which was added 20 μl of a 32-fold dilution solution (XVII)control (A1) and added the substrate solution PPO and kept the same way. Analogously to example 41(2), after keeping prepared each of the reaction solutions was subjected to analysis by reversed-phase HPLC under the conditions of analysis 2, Pref is given above, to measure the amount of PPIX. The amount of PPIX in the reaction solution, which was added to the solution to (XVII)-convert (A1), was greater than the amount of PPIX in the reaction solution, to which was added a solution of (XVII)control (A1).

(6) Conversion of the compound (VI) protein (A1) of the present invention

E. coli JM109/pKSN657F were cultured over night at 37°C in 3 ml of medium TV containing 50 μg/ml ampicillin. A milliliter (1 ml) of the obtained culture medium was transferred into 100 ml of medium TV containing 50 μg/ml of ampicillin, and cultured at 26°C. When the OD660 reached 0,5, was added 5-aminolevulinate acid to a final concentration of 500 μm, and the cultivation was continued. After 30 minutes, then IPTG was added to a final concentration of 1 mm and were additionally cultured for 20 hours.

Cells were removed from culture medium, washed with 0.1 M Tris-HCl-buffer (pH 7.5) and suspended in 10 ml of 0.1 M Tris-HCl buffer containing 1% glucose. The compound (VI) was added to the resulting cell suspension to a final concentration of 100 ppm and incubated with shaking at 30°C. At 0 hours and the next day after the start of shaking, 2 ml of this cell suspension was fractionally. Fifty microliters (50 ál) 2 N. HCl was added to each fraction and was extracted with 2 ml ethyl acetate. Received an ethyl acetate layers were analyzed with tomosunas provided reaction 1. Compared with the concentration of the compound (VI), detected from an ethyl acetate layer obtained from a cell suspension at 0 hours after the start of shaking, the concentration of the compound (VI), detected from an ethyl acetate layer obtained from a cell suspension the next day after the start of shaking, was lower. In addition, a peak is not detected from an ethyl acetate layer obtained from a cell suspension at 0 hours after the start of shaking, were detected from an ethyl acetate layer obtained from a cell suspension the next day after the start of shaking. Conducted mass spectrometry compounds contained in the specified peak. Weight compounds contained in the specified peak was 14 less than the mass of the compounds (VI).

(7) Conversion of the compound (VIII) of this protein (A1)

Except for using the compound (VIII) instead of compound (VI)was carried out according to the method described in example 41(6), the cultivation of E. coli JM109/pKSN657F, solution preparation of cell suspension, incubation with shaking of a solution of cell suspension to which was added the compound (VIII), reagent preparation from cell suspension solution and HPLC-analysis of these reagents. Compared with the concentration of the compound (VIII), detected from an ethyl acetate layer obtained from cleto is Noah suspension at 0 hours after the start of shaking, the concentration of compounds (VIII), detected from an ethyl acetate layer obtained from a cell suspension the next day after the start of shaking, was lower. In addition, 2 peak, which is not detected from an ethyl acetate layer obtained from a cell suspension at 0 hours after the start of shaking, were detected from an ethyl acetate layer obtained from a cell suspension the next day after the start of shaking. Conducted mass spectrometry compounds contained in said peaks. Weight compounds contained in one of these peaks was less than 14, and the weight compounds contained in the other peak was 28 less than the mass of the compound (VIII).

(8) Conversion of the compound (X) protein (A1) of the present invention

Except for using compound (X) instead of compound (VI)was carried out according to the method described in example 41(6), the cultivation of E. coli JM109/pKSN657F, solution preparation of cell suspension, incubation with shaking of a solution of cell suspension to which was added the compound (X), reagent preparation from cell suspension solution and HPLC-analysis of these reagents. Compared with the concentration of the compound (X), detected from an ethyl acetate layer obtained from a cell suspension at 0 hours after the start of shaking, the concentration is soedineniya (X), detected from an ethyl acetate layer obtained from a cell suspension the next day after the start of shaking, was lower. In addition, 2 peak, which is not detected from an ethyl acetate layer obtained from a cell suspension at 0 hours after the start of shaking, were detected from an ethyl acetate layer obtained from a cell suspension the next day after the start of shaking. Conducted mass spectrometry compounds contained in said peaks. Weight compounds contained in one of these peaks was less than 40, and the weight compounds contained in the other peak was 54 is less than the mass of compound (X).

(9) Conversion of the compound (XI) protein (A1) of the present invention

Except for using the compound (XI) instead of compound (VI)was carried out according to the method described in example 41(6), the cultivation of E. coli JM109/pKSN657F, solution preparation of cell suspension, incubation with shaking of a solution of cell suspension to which was added the compound (XI), reagent preparation from cell suspension solution and HPLC-analysis of these reagents. Compared with the concentration of the compound (XI), detected from an ethyl acetate layer obtained from a cell suspension at 0 hours after the start of shaking, the concentration of the compound (XI)program and is an ethyl acetate layer, obtained from cell suspension on the next day after the start of shaking, was lower. In addition, 2 peak, which is not detected from an ethyl acetate layer obtained from a cell suspension at 0 hours after the start of shaking, were detected from an ethyl acetate layer obtained from a cell suspension the next day after the start of shaking. Conducted mass spectrometry compounds contained in said peaks. Weight compounds contained in one of these peaks was less than 14, and the weight compounds contained in the other peak was 16 less than the mass of the compound (XI).

Example 42. The conversion of compounds protein (A11) this invention

(1) Conversion of the compound (X) protein (A11) this invention

E. coli JM109/pKSN849F and E. coli JM109/pKSN2 were cultured over night at 37°C in 3 ml of medium TV containing 50 μg/ml ampicillin. A milliliter (1 ml) of the obtained culture medium was transferred into 100 ml of medium TV containing 50 μg/ml of ampicillin, and cultured at 26°C. When the OD660 reached 0,5, was added 5-aminolevulinate acid to a final concentration of 500 μm, and the cultivation was continued. After 30 minutes, then IPTG was added to a final concentration of 1 mm and were additionally cultured for 18 hours.

Cells were removed from culture medium, washed with 0.1 M Tris-Cl buffer (pH 7.5) and suspended in 10 ml of 0.1 M Tris-HCl buffer, containing 1% glucose. The compound (X) was added to the resulting cell suspension to a final concentration of 25 ppm and incubated with shaking at 30°C. At 0 hours and 4 days after the start of shaking, 2 ml of this cell suspension was fractionally. Fifty microliters (50 ál) 2 N. HCl was added to each fraction and was extracted with 2 ml ethyl acetate. Received an ethyl acetate layers were analyzed by HPLC under the condition of reaction 1. Compared with the concentration of the compound (X), detected from an ethyl acetate layer obtained from cell suspension JM109/pKSN2, the concentration of the compound (X), detected from an ethyl acetate layer obtained from cell suspension JM109/pKSN849AF was lower. In addition, 3 peak, which is not detected from an ethyl acetate layer obtained from cell suspension JM109/pKSN2, were detected from an ethyl acetate layer obtained from cell suspension JM109/pKSN849AF. Of these three peaks, elution in HPLC 1 of the peaks coincided with the elution peak of the connection, which has a mass of 40 smaller than the compound (X), detektirovanie in example 41(8). Next, elution in HPLC peak coincided with the elution peak of the connection, which has mass, 54 less than the compound (X), detektirovanie in example 41(8).

After drying, respectively, 1 ml of an ethyl acetate layer obtained from the above-mentioned cell suspension JM109/pKSN2, and 1 ml of an ethyl acetate layer obtained from the above-mentioned cell suspension JM109/pKSN849AF, the residues were dissolved in 1 ml of dimethylsulfoxide (hereinafter, the solution obtained from an ethyl acetate layer obtained from JM109/pKSN849AF, called "the solution to (X)-transformation (A11)"; next, a solution obtained from an ethyl acetate layer obtained from a suspension of cells JM109/pKSN2, referred to as "solution (X)-control (A11)").

Mixing 20 ál of 128-fold dilution of the above solution for a (X)-transformation (A11) and 60 μl fractions plastids. In dark conditions was added 20 μl of substrate solution PPO and kept at 30°C for 1.5 hours. Further, instead of the above 20 µl 128-fold dilution of the solution for a (X)-transformation (A11) was prepared reaction solution, to which was added 20 μl of 128-fold dilution of the solution (X)-control (A11), and added the substrate solution PPO and kept the same way. Analogously to example 41(2), after keeping prepared each of the reaction solutions was subjected to analysis by reversed-phase HPLC under conditions analysis 2 above, to measure the amount of PPIX. The amount of PPIX in the reaction solution, to which solution was added to (X)-transformation (A11), was greater than the amount of PPIX in the reaction solution, to which was added the solution (X)-control (A11).

(2) Conversion of the compound (XII) protein (the 11) of the present invention

With the exception of using 20 μl of the supernatant fraction obtained in example 32(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were obtained and maintained is similar to the method described in example 41(2). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 100 μl of 50 mm potassium phosphate buffer (pH 7.0). The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 32(2), called "the solution to (XII)-conversion (A11)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 32(2), referred to as "solution (XII)control (A11)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A11), the concentration of the compound (XII)detected from the solution to (XII)-conversion (A11), was lower. Then, from the solution to (XII)-conversion (A11) was detected peak, which is not detected from the solution (XII)control (A11). Elution of the specified peak on HPLC coincided with the elution peak connections to the m this mass is 14 less than the mass of the compounds (XII)detected from the solution to (XII)-convert (A1)in example 41(2).

(3) Conversion of the compound (XIII) protein of this invention (A11)

With the exception of using 20 μl of the supernatant fraction obtained in example 32(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were obtained and maintained is similar to the method described in example 41(2). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 100 μl of dimethyl sulfoxide. The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 32(2), called "the solution to (XIII)-conversion (A11)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 32(2), referred to as "solution (XIII)control (A11)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XIII)detected from the solution (XIII)control (A11), the concentration of the compound (XIII)detected from the solution to (XIII)-conversion (A11), was lower. Then, from the solution to (XIII)-conversion (A11) b the l detected peak, which is not detected from the solution (XIII)control (A11). Elution of the specified peak on HPLC coincided with the elution peak of the connection in which this mass is 14 less than the mass of the compounds (XIII), detected from the solution to (XIII)-conversion (A11), example 41(3).

(4) the Conversion of compound (XVI) protein (A11) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 32(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were obtained and maintained is similar to the method described in example 41(4). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 200 μl of 50 mm potassium phosphate buffer (pH 7.0). The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 32(2), called "the solution to (XVI)transformation (A11)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 32(2), referred to as "solution (XVI)control (A11)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XVI), detektyw the constituent of the solution (XVI)control (A11), the concentration of compounds (XVI), detected from the solution to (XVI)transformation (A11), was lower. Then, from the solution to (XVI)transformation (A11) was detected peak, which is not detected from the solution (XVI)control (A11). Elution of the specified peak on HPLC coincided with the elution peak in example 41(4), which is detected from the solution to (XVI)transformation (A11) and not detected in solution (XVI)control (A11).

(5) Conversion of the compound (XVII) protein (A11) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 32(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were obtained and maintained is similar to the method described in example 41(5). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 200 μl of 50 mm potassium phosphate buffer (pH 7.0). The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 32(2), called "the solution to (XVII)-conversion (A11)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 32(2), referred to as a "rest the rum (XVII)control (A11)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XVII), detected from the solution (XVII)control (A11), the concentration of the compound (XVII), detected from the solution to (XVII)-conversion (A11), was lower. Then, from the solution to (XVII)-conversion (A11) was detected peak, which is not detected from the solution (XVII)control (A11). Elution of the specified peak on HPLC coincided with the elution peak in example 41(5), which is detected from the solution to (XVII)-convert (A1) and not detected in solution (XVII)control (A1).

Example 43. The conversion of compounds of proteins (A2), (A3), (A12), (A13), (A14) and (A15) of the present invention or the protein (a10) this invention

(1) Conversion of the compound (XII) protein (A2) of the present invention

With the exception of using 20 μl of the supernatant fraction obtained in example 7(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were obtained and maintained is similar to the method described in example 41(2). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 100 μl of 50 mm potassium phosphate buffer (pH 7.0). The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in PR the least 7(2), called "the solution to (XII)-convert (A2)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 7(2), referred to as "solution (XII)control (A2)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A2), the concentration of the compound (XII)detected from the solution to (XII)-convert (A2)was lower. Then, from the solution to (XII)-convert (A2) was detected peak, which is not detected from the solution (XII)control (A2). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XII)detected from the solution to (XII)-convert (A1) in example 41(2).

(2) Conversion of the compound (XII) protein (A3) of the present invention

With the exception of using 20 μl of the supernatant fraction obtained in example 12(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were obtained and maintained is similar to the method described in example 41(2). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 100 μl of 50 mm califofnia buffer (pH 7.0). The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 12(2), called "the solution to (XII)-conversion (A3)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 7(2), referred to as "solution (XII)control (A3)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A3), the concentration of the compound (XII)detected from the solution to (XII)-conversion (A3), was lower. Then, from the solution to (XII)-conversion (A3) was detected peak, which is not detected from the solution (XII)control (A3). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XII)detected from the solution to (XII)-convert (A1) in example 41(2).

(3) Conversion of the compound (XII) of this protein (a10)

With the exception of using 20 μl of the supernatant fraction obtained in example 10(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were obtained and maintained is similar to the method described in note the re 41(2). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 100 μl of 50 mm potassium phosphate buffer (pH 7.0). The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 10(2), called "the solution to (XII)-conversion (a10)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 12(3), referred to as "solution (XII)control (a10)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (a10), the concentration of the compound (XII)detected from the solution to (XII)-conversion (a10), was lower. Then, from the solution to (XII)-conversion (a10) was detected peak, which is not detected from the solution (XII)control (a10). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XII)detected from the solution to (XII)-convert (A1) in example 41(2).

(4) the Conversion of compound (XII) protein (A12) this invention

Except for the use of the Finance 20 μl of the supernatant fraction, obtained in example 34(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were obtained and maintained is similar to the method described in example 41(2). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 100 μl of 50 mm potassium phosphate buffer (pH 7.0). The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 34(2), called "the solution to (XII)-conversion (A12)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 34(2), referred to as "solution (XII)control (A12)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A12), the concentration of the compound (XII)detected from the solution to (XII)-conversion (A12), was lower. Then, from the solution to (XII)-conversion (A12) was detected peak, which is not detected from the solution (XII)control (A12). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds(XII), detected from the solution to (XII)-convert (A1) in example 41(2).

(5) Conversion of the compound (XII) protein (A13) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 36(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were obtained and maintained is similar to the method described in example 41(2). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 100 μl of 50 mm potassium phosphate buffer (pH 7.0). The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 7(2), called "the solution to (XII)-conversion (A13)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 36(2), referred to as "solution (XII)control (A13)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A13), the concentration of the compound (XII)detected from the solution to (XII)-conversion (A13), was lower. Then, from the solution to (XII)-conversion (A13) was detected peak, which is detected from the solution (XII)control (A13). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XII)detected from the solution to (XII)-convert (A1) in example 41(2).

(6) Conversion of the compound (XII) protein (A14) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 38(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were obtained and maintained is similar to the method described in example 41(2). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 100 μl of 50 mm potassium phosphate buffer (pH 7.0). The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 38(2), called "the solution to (XII)-conversion (A14)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 38(2), referred to as "solution (XII)control (A14)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A14), the concentration of soy is inane (XII), detected from the solution to (XII)-conversion (A14), was lower. Then, from the solution to (XII)-conversion (A14) was detected peak, which is not detected from the solution (XII)control (A14). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XII)detected from the solution to (XII)-convert (A1) in example 41(2).

(7) Conversion of compound (XII) protein (A15) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 40(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were obtained and maintained is similar to the method described in example 41(2). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 100 μl of 50 mm potassium phosphate buffer (pH 7.0). The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 40(2), called "the solution to (XII)-conversion (A15)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 40(2), referred to as a "rest the rum (XII)control (A15)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A15), the concentration of the compound (XII)detected from the solution to (XII)-conversion (A15), was lower. Then, from the solution to (XII)-conversion (A15) was detected peak, which is not detected from the solution (XII)control (A15). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XII)detected from the solution to (XII)-convert (A1) in example 41(2).

(8) Metabolism of compound (XIII) protein (A2) of the present invention

With the exception of using 20 μl of the supernatant fraction obtained in example 7(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were obtained and maintained is similar to the method described in example 41(3). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 100 μl of dimethyl sulfoxide. The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 7(2), called "the solution to (XIII)-convert (A2)"; then, the solution obtained from the reaction solution containing no com is onent And, component B, component C and the supernatant fraction obtained in example 7(2), referred to as "solution (XIII)control (A2)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XIII)detected from the solution (XIII)control (A2), the concentration of the compound (XIII)detected from the solution to (XIII)-convert (A2)was lower. Then, from the solution to (XIII)-convert (A2) was detected peak, which is not detected from the solution (XIII)control (A2). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XIII), detected from the solution to (XIII)-convert (A1) in example 41(3).

(9) Conversion of compound (XIII) protein (A3) of the present invention

With the exception of using 20 μl of the supernatant fraction obtained in example 12(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were obtained and maintained is similar to the method described in example 41(3). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 100 μl of dimethyl sulfoxide. The obtained solutions (the solution obtained from the reaction solution containing component a, component B, to mponent C and 20 μl of the supernatant fraction, obtained in example 12(2), called "the solution to (XIII)-conversion (A3)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 12(2), referred to as "solution (XIII)control (A3)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XIII)detected from the solution (XIII)control (A3), the concentration of the compound (XIII)detected from the solution to (XIII)-conversion (A3), was lower. Then, from the solution to (XIII)-conversion (A3) was detected peak, which is not detected from the solution (XIII)control (A3). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XIII), detected from the solution to (XIII)-convert (A1) in example 41(3).

(10) the Conversion of compounds (XIII) of this protein (a10)

With the exception of using 20 μl of the supernatant fraction obtained in example 10(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were obtained and maintained is similar to the method described in example 41(3). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved is in 100 μl of dimethyl sulfoxide. The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 10(2), called "the solution to (XIII)-conversion (a10)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 10(2), referred to as "solution (XIII)control (a10)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XIII)detected from the solution (XIII)control (a10), the concentration of the compound (XIII)detected from the solution to (XIII)-conversion (a10), was lower. Then, from the solution to (XIII)-conversion (a10) was detected peak, which is not detected from the solution (XIII)control (a10). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XIII), detected from the solution to (XIII)-convert (A1) in example 41(3).

(11) Conversion of compound (XIII) protein (A12) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 34(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were received and handled like JV is soba, described in example 41(3). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 100 μl of dimethyl sulfoxide. The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 34(2), called "the solution to (XIII)-conversion (A12)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 34(2), referred to as "solution (XIII)control (A12)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XIII)detected from the solution (XIII)control (A12), the concentration of the compound (XIII)detected from the solution to (XIII)-conversion (A12), was lower. Then, from the solution to (XIII)-conversion (A12) was detected peak, which is not detected from the solution (XIII)control (A12). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XIII), detected from the solution to (XIII)-convert (A1) in example 41(3).

(12) the Conversion of compound (XIII) protein (A13) this invention

For IP the connection using 20 μl of the supernatant fraction, obtained in example 36(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were obtained and maintained is similar to the method described in example 41(3). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 100 μl of dimethyl sulfoxide. The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 36(2), called "the solution to (XIII)-conversion (A13)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 36(2), referred to as "solution (XIII)control (A13)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XIII)detected from the solution (XIII)control (A13), the concentration of the compound (XIII)detected from the solution to (XIII)-conversion (A13), was lower. Then, from the solution to (XIII)-conversion (A13) was detected peak, which is not detected from the solution (XIII)control (A13). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XIII), detec the dummy from the solution to (XIII)-convert (A1) in example 41(3).

(13) Conversion of compound (XIII) protein (A14) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 38(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were obtained and maintained is similar to the method described in example 41(3). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 100 μl of dimethyl sulfoxide. The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 38(2), called "the solution to (XIII)-conversion (A14)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 38(2), referred to as "solution (XIII)control (A14)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XIII)detected from the solution (XIII)control (A14), the concentration of the compound (XIII)detected from the solution to (XIII)-conversion (A14), was lower. Then, from the solution to (XIII)-conversion (A14) was detected peak, which is not detected from the solution (XIII)control (A14). Elution of the specified picana HPLC coincided with the elution peak connections where weight is 14 less than the mass of the compounds (XIII), detected from the solution to (XIII)-convert (A1) in example 41(3).

(14) the Conversion of compound (XIII) protein (A15) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 40(2), instead of 20 μl of the supernatant fraction obtained in example 4(2), the reaction solutions were obtained and maintained is similar to the method described in example 41(3). Similarly to example 41(2), each of these reaction solutions after aging were extracted with ethyl acetate and the obtained residues were dissolved in 100 μl of dimethyl sulfoxide. The obtained solutions (the solution obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 40(2), called "the solution to (XIII)-conversion (A15)"; then, the solution obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 40(2), referred to as "solution (XIII)control (A15)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XIII)detected from the solution (XIII)control (A15), the concentration of the compound (XIII)detected from the solution to (XIII)-conversion (A15), was lower. Next, the solution to (XIII)-conversion (A15) was detected peak, which is not detected from the solution (XIII)control (A15). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XIII), detected from the solution to (XIII)-convert (A1) in example 41(3).

Example 44. Obtaining antibodies (A) of the present invention, capable of recognizing protein (A1) of the present invention (hereinafter referred to as antibody (A1) of the present invention")

(1) Obtaining an extract of E. coli expressing the protein (A1) of the present invention

In accordance with the method described in example 4(2), E. coli JM109/pKSN657F, which expresses the protein (A1) of the present invention, the pre-cultured overnight and then cultured in 1 l of medium TV containing 50 μg/ml ampicillin. After extraction and destruction of cells received fraction supernatant (extract of E. coli pKSN657F) from the solution obtained cell lysate.

(2) Purification of protein (A1) of the present invention

Protein (A1) of the present invention was purified in accordance with the method described in example 2(4) exposure to the supernatant fraction obtained in example 44(1) (extract of E. coli pKSN657F), alternately chromatography on a column Hiload HiLoad26/10 Q Sepharose HP and then on a column of SNT-1 type I Bio-Scale Ceramic Hydroxyapatite. Purified fractions were analyzed by electrophoresis on 10-20% of the LTO-page to confirm the CSOs, these fractions were only fractions of the protein (A1) of the present invention.

(3) Obtaining antibodies (A1) of the present invention

Protein (A1) of the present invention obtained in example 44(2), was dissolved in 0.05 M potassium phosphate buffer (pH 7.0) so that its concentration was equal to 1 mg/ml) was Added 40 μl of adjuvant system (RAS MPL (monophosphorylated A) + TDM (synthetic dikarenakan trehalose) + CWS (cell wall skeleton) (Sigma Company), already preincubating at 42-43°C and well stirred with 2 ml of the obtained solution. The resulting mixture was injected, respectively, new Zealand white rabbits (female, 14 weeks of age, with an average weight of 2.4 kg) in a dose of 1 ml per rabbit. Thus, were injected with 100 μl subcutaneously in 10 locations on the back. Approximately ¾ of the amount of the first injection was administered after 3 weeks and after 5 weeks. During this time, the antibody titer was measured by sampling blood from the ear vein of the rabbit. Because the antibody titer increased after the third injection, the blood was removed from the neck immunized rabbit 2 weeks after the third injection. The obtained blood was added into a test tube Separapit (Sekisui Chemical Company), incubated at 37°C for 2 hours and then centrifuged (3000 rpm, 20 minutes, at room temperature). Anticigarette (containing antibody (A1) of the present invention)) received the removing the supernatant.

Example 45. Detection of this protein antibody (A1) of the present invention and detecting expression of cells of a given protein

The immunoblot analysis was performed using antibodies (A1) of the present invention obtained in example 44, with each of the extracts of E. coli. Electrophoresis was performed in LTO-polyacrylamide (40 mA, 1 hour): extract of E. coli pKSN657F obtained in example 4(2) (containing about 0.5 pmol of protein (A1) of the present invention, containing approximately 0,78 mg protein); an extract of E. coli pKSN2 obtained in example 4(2) (containing about 0,78 mg protein); an extract of E. coli pKSN923F obtained in example 7(2) (containing about 2 pmol of the protein (A2) of the present invention); an extract of E. coli pKSN671F obtained in example 12(2) (containing about 2 pmol of the protein (A3) of the present invention); an extract of E. coli pKSN646F obtained in example 27(2) (containing about 2 pmol of the protein (A4) of the present invention); an extract of E. coli pKSN11796F obtained in example 10(2) (containing about 2 pmol of the protein (a10) of the present invention); an extract of E. coli pKSNSCA obtained in example 14(2) (containing about 2 pmol of the protein (A9) of the present invention); an extract of E. coli pKSN849F obtained in example 32(2) (containing about 2 pmol of the protein (A11) of the present invention); an extract of E. coli pKSN1618F obtained in example 34(2) (containing in listello 2 pmol of protein (A12) of the present invention); extract of E. coli pKSN474F obtained in example 36(2) (containing about 2 pmol of the protein (A13) of the present invention); an extract of E. coli pKSN1491F obtained in example 38(2) (containing about 2 pmol of the protein (A14) of the present invention) and an extract of E. coli pKSN1555F obtained in example 40(2) (containing about 2 pmol of the protein (A15) of the present invention). PVDF-membrane was placed on the gel. Proteins in the gel were transferred to PVDF-membrane using a blotting device BioRad at 4°C, 30 V for 2 hours under conditions of impregnation buffer transfer (25 mm Tris, 192 mm glycine, 10% methanol). After washing with a solution of TBS + tween-20 (50 mm Tris-HCl (pH 7.5), 200 mm NaCl, 0.05% tween-20) obtained PVDF-membrane was incubated for 30 minutes in a solution of TBS + tween 20 containing 3% BSA, and then used for the reaction with the above-described anticorodal diluted to 30,000 times, for 30 minutes in a solution of TBS + tween 20 containing 3% BSA. After this reaction, the PVDF-membrane was washed twice with a solution of TBS + tween-20. Then, the PVDF-membrane was used for reaction in a solution of TBS + tween 20 containing 3% BSA for 30 minutes at 3000-fold dilution of goat antisera against rabbit IgG labeled with alkaline phosphatase (Santa Cruz Biotechnology Company). After the reaction PVDF-membrane was washed twice with a solution of TBS + tween-20 and soaked in a solution of NBT-BCIP (Sigma Company). Were detected staining bands correspond to the her each of the proteins of this invention (A1), (A2), (A3), (A4), (A11), (A12), (A13), (A14) and (A15), and proteins (A9) and (a10) of the present invention. Not detected staining reagent strip with an extract of E. coli pKSN2 (containing approximately 0,78 mg protein)obtained in example 4(2).

Example 46. The acquisition and expression of DNA (A1) of the present invention, in which the use of codons was adapted for expression in soybean (hereinafter referred to as "DNA (A1)S of the present invention"))

(1) Obtaining DNA (A1)S of this invention

PCR was performed with DNA polymerase, Pyrobest (Takara Shuzo Company) in accordance with the attached guidance using a primer having the nucleotide sequence shown in SEQ ID NO:192, and a primer having the nucleotide sequence shown in SEQ ID NO:213. An aliquot of the obtained PCR product was used as template for PCR carried out in the same way using a primer having the nucleotide sequence shown in SEQ ID NO:191, and a primer having the nucleotide sequence shown in SEQ ID NO:212. Next, an aliquot of this PCR product was used as template for PCR carried out in the same way using a primer having the nucleotide sequence shown in SEQ ID NO:190, and a primer having the nucleotide sequence shown in SEQ ID NO:211. The resulting reaction solution was named actionem solution 1.

PCR was performed with DNA polymerase, Pyrobest (Takara Shuzo Company) in accordance with the attached guidance using a primer having the nucleotide sequence shown in SEQ ID NO:195, and a primer having the nucleotide sequence shown in SEQ ID NO:210. An aliquot of the obtained PCR product was used as template for PCR carried out in the same way using a primer having the nucleotide sequence shown in SEQ ID NO:194, and a primer having the nucleotide sequence shown in SEQ ID NO:209. Next, an aliquot of this PCR product was used as template for PCR carried out in the same way using a primer having the nucleotide sequence shown in SEQ ID NO:193, and a primer having the nucleotide sequence shown in SEQ ID NO:208. The resulting reaction solution was named the reaction solution 2.

PCR was performed with DNA polymerase, Pyrobest (Takara Shuzo Company) in accordance with the attached guidance using a primer having the nucleotide sequence shown in SEQ ID NO:198, and a primer having the nucleotide sequence shown in SEQ ID NO:207. An aliquot of the obtained PCR product was used as template for PCR carried out in the same way using a primer having a nucleotide serial is lnost, shown in SEQ ID NO:197, and a primer having the nucleotide sequence shown in SEQ ID NO:206. Next, an aliquot of this PCR product was used as template for PCR carried out in the same way using a primer having the nucleotide sequence shown in SEQ ID NO:196, and a primer having the nucleotide sequence shown in SEQ ID NO:205. The resulting reaction solution was named the reaction solution 3.

PCR was performed with DNA polymerase, Pyrobest (Takara Shuzo Company) in accordance with the attached guidance using a primer having the nucleotide sequence shown in SEQ ID NO:201, and a primer having the nucleotide sequence shown in SEQ ID NO:204. An aliquot of the obtained PCR product was used as template for PCR carried out in the same way using a primer having the nucleotide sequence shown in SEQ ID NO:200, and a primer having the nucleotide sequence shown in SEQ ID NO:203. Next, an aliquot of this PCR product was used as template for PCR carried out in the same way using a primer having the nucleotide sequence shown in SEQ ID NO:199, and a primer having the nucleotide sequence shown in SEQ ID NO:202. The resulting reaction solution was named reaction Rast is a PR 4.

The reaction solutions 1 to 4, thus obtained, was mixed. PCR was performed with DNA polymerase, Pyrobest (Takara Shuzo Company) in accordance with the attached manual, using as matrix aliquots of this mixture and using a primer having the nucleotide sequence shown in SEQ ID NO:190, and a primer having the nucleotide sequence shown in SEQ ID NO:202. The nucleotide sequence of the amplified DNA was confirmed. Received DNA having the sequence in which the nucleotide sequence 5'-cat-3' attached "to the left" from the 5'-end, and the nucleotide sequence 5'-aagctt-3' attached "to the right" from the 3'-end of the nucleotide sequence shown in SEQ ID NO:214.

The use of the codons of the DNA (A1) of the present invention having the nucleotide sequence shown in SEQ ID NO:6 (GC-content 70,58%)shown in table 22 and table 23. The use of codons soybean (GC-content 46,12%database usage codons, published Kazusa DNA Research Institute (http://www.kazusa.or.jp/codon)) shown in table 24 and table 25. The use of the codons of the DNA (A1) of the present invention having the nucleotide sequence shown in SEQ ID NO:214 (GC-content 51,59%)shown in table 26 and table 27.

Table 22
codon%codon%
TTT0,00TCT0,00
TTC3,18TCC1,71
TTA0,00TCA0,00
TTG1,22TCG2,20
CTT0,00CCT0,00
CTCto 3.67CCC4,16
HUNDRED0,00CCA0,00
CTG7,09CCG2,69
ATT0,24ACT0,24
ATS4,16 ACC2,69
ATA0,00ASA0,24
ATG2,69ACG1,96
GTT0,24GCT0,00
GTCto 3.67GCC7,58
GTA0,00GCA0,49
GTG3,18GCG3,42

Table 23
codon%codon%
TAT0,00TGT0,24
TAC1,47TGC0,98
TAATGA0,00
TAG0,24TGG0,98
CAT0,24CGT1,22
CAC2,20CGC4,40
CAA0,24CGA0,24
CAG2,93CGG4,16
AAT0,00AGT0,00
AAC1,22AGC0,49
AAA0,24AGA0,00
AAG0,98AGG0,00
GAT0,98GGT0,98
GAC7,82GGC3,42
GAA0,73GGA0,24
GAG5,38GGG1,22

Table 24
codon%codon%
TTT2,03TCT1,71
TTC2,09TCC1,21
TTA0,82TCA1,45
TTGof 2.21TCG0,44
CTT2,36CCT2,00
CTC1,66CCC1,01
HUNDRED0,82CCA2,05
CTG1,22CCG0,40
ATT2,61ACT1,78
ATS1,64ACC1,49
ATA1,27ASA1,51
ATG2,27ACG0,41
GTTto 2.67GCT2,81
GTC1,24GCC1,69
GTA0,73GCA2,27
GTG2,20GCG0,59

AGC
Table 25
codon%codon%
TAT1,61TGT0,72
TAC1,53TGC0,75
TAA0,11TGA0,09
TAG0,06TGG1,21
CAT1,33CGT0,72
CAC1,09CGC0,63
CAA2,04CGA0,38
CAG1,71CGG0,27
AAT2,10AGT1,21
AAC2,271,08
AAA2,63AGA1,42
AAG3,83AGG1,35
GAT3,29GGT2,17
GACto 2.06GGC1,38
GAA3,35GGA2,23
GAGof 3.46GGG1,29

Table 26
codon%codon%
TTT1,71TCT0,98
TTC1,47TCC0,73
TTA,98 TCA0,98
TTG2,93TCG0,24
CTT3,18CCT2,44
CTC2,20CCC1,22
HUNDRED0,98CCA2,69
CTG1,71CCG0,49
ATT2,20ACT1,71
ATS1,22ACC1,47
ATA0,98ASA1,47
ATG2,69ACG0,49
GTT2,93GCT4,16
GTC1,22GCC2,69
GTA0,73GCAto 3.67
GTG2,20GCG0,98

Table 27
codon%codon%
TAT0,73TGT0,73
TAC0,73TGC0,49
TAA0,00TGA0,00
TAG0,24TGG0,98
CAT1,47CGT1,47
CAC0,98CGC1,47
CAA1,71CGA0,73
CAG1,47CGG0,49
AAT0,73AGT0,73
AAC0,49AGC0,73
AAA0,49AGA2,93
AAG0,73AGG2,93
GAT5,38GGT1,71
GAC3,42GGC1,22
GAA2,69GGA1,96
GAG3,42GGG0,98

(2) Obtaining transformed E. coli with protein (A1)S this image is the shadow

DNA having the nucleotide sequence shown in SEQ ID NO:214, obtained in example 46(1), was digested with restrictase NdeI and HindIII. The resulting DNA and plasmid pKSN2, split NdeI and HindIII, ligated to obtain plasmid in which the DNA having the nucleotide sequence shown in SEQ ID NO:214, inserted between the NdeI site and the HindIII site pKSN2 (hereinafter called "pKSN657soy"). The indicated plasmid was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN657soy.

(3) protein Expression (A1) of the present invention in E. coli and the selection of the specified protein

Analogously to example 4(2), cultivated each of E. coli JM109/pKSN657soy obtained in example 46(2), and E. coli JM109/pKSN657 obtained in example 4(1). The cells were extracted. Received solutions of cell lysates. According to the method described in example 4(2), received a fraction of supernatants of these solutions cell lysates (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN657soy, called “extract of E. coli pKSN657soy”, and the supernatant fraction obtained from E. coli JM109/pKSN657, called “extract of E. coli pKSN657”). The number of P450 in the amount of protein contained in the extract of E. coli pKSN657soy, compared with the number of P450 in the amount of protein contained in the extract of E. coli pKSN657, and it was higher than the number of P450 in the amount of protein contained in the extract of E. coli pKSN657.

Example 47. Introduction DNA (A1)S this is subramania in plant

(1) Construction of chloroplast expression plasmids containing DNA (A1)S of the present invention, for direct introduction - part 1

A plasmid containing a chimeric DNA in which DNA (A1)S of this invention was added immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons, was designed as a plasmid for introduction of DNA (A1)S of the present invention in a plant method using guns particles.

First, a DNA containing the nucleotide sequence shown in SEQ ID NO:214, amplified using PCR. PCR was performed using as matrix pKSN657soy obtained in example 46(2), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:394, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:395. In PCR were using KOD-plus (Toyobo Company). PCR was performed after maintaining 94°C for 2 minutes as follows: 30 cycles, where each cycle consisted of maintaining 94°C for 30 seconds, then 50°C for 30 seconds and then 68°C for 60 seconds; and final maintenance 68°C for 30 seconds. Amplified DNA was isolated and purified using MagExtractor-PCR &Gel-Clen up (Toyobo Company) carrying out procedures in accordance with the attached manual. After cleavage of the purified DNA restrictase Eat and SacI, DNA containing the nucleotide sequence shown in SEQ ID NO:214, was isolated. After cleavage of plasmid pUCrSt657 obtained in example 16(2), restrictase Eat and SacI, was isolated DNA approximately 2,9 TPN having a nucleotide sequence derived from pUC19, and a sequence encoding a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack). The obtained DNA and the above-mentioned DNA containing the nucleotide sequence shown in SEQ ID NO:214, ligated with getting pUCrSt657soy (Fig. 48)containing a chimeric DNA in which DNA (A1)S of the present invention is attached immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons.

The obtained plasmid pUCrSt657soy were digested with restrictase BamHI and SacI for DNA containing the nucleotide sequence shown in SEQ ID NO:214. Specified DNA was built between the site restrictase BglII site of restrictase SacI plasmids pNdG6-ΔT obtained in example 16(2), to obtain plasmid pSUM-NdG6-rSt-657soy (Fig. 49), where the promoter CR16G6 attached after chimeric DNA in which DNA (A1)S of the present invention is attached immediately after the nucleotide sequence, the code is highlighted chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons.

Then, this plasmid was introduced into competent cells of E. coli DH5α (Takara Shuzo Company) and were selected by ampicillin-resistant cells. Further, the nucleotide sequence of the plasmid contained in the selected ampicillin-resistant strains, was determined using kit BigDye Terminator Cycle Sequencing Ready Reaction kit v3.0 (PE Applied Biosystems Company) and DNA sequencing machine 3100 (PE Applied Biosystems Company). In the result, it was confirmed that the plasmid pSUM-NdG6-rSt-657soy has the nucleotide sequence shown in SEQ ID NO:214.

(2) Construction of chloroplast expression plasmids containing DNA (A1)S of the present invention, for direct introduction - part 2

Designed plasmid for introduction of DNA (A1)S of the present invention in a plant method using guns particles. This plasmid contained a chimeric DNA in which DNA (A1)S of this invention was added immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without altering the reading frames in the codons. First, a DNA containing the nucleotide sequence shown in SEQ ID NO:214, amplified using PCR. PCR was performed using as matrix pKSN657soy obtained in example 46(2), and use the education as primers the oligonucleotide, having the nucleotide sequence shown in SEQ ID NO:395, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:396. In PCR were using KOD-plus (Toyobo Company). PCR was performed after maintaining 94°C for 2 minutes as follows: 25 cycles, where each cycle consisted of maintaining 94°C for 30 seconds, then 46°C for 30 seconds and then 68°C for 60 seconds; and final maintenance 68°C for 3 minutes. Amplified DNA was isolated and purified using MagExtractor-PCR &Gel Clean up (Toyobo Company) carrying out procedures in accordance with the attached manual. After cleavage of purified DNA with restriction enzyme SacI, DNA containing the nucleotide sequence shown in SEQ ID NO:214, was identified.

Plasmid pKFrSt12-657 obtained in example 16(3), were digested with restriction enzyme BspHI. Then this DNA was a small mistake and the 5'-end was dephosphorylated using a set of TaKaRa BKL Kit (Takara Shuzo Company) in accordance with the attached manual. Then, after DNA cleavage by the restriction enzyme SacI DNA derived from the plasmid pKFrSt12, were isolated. Specified DNA ligated with DNA, which was digested SacI and which contains the nucleotide sequence shown in SEQ ID NO:214, obtaining the plasmid pKFrSt12-657soy (Fig. 50)containing a chimeric DNA in which DNA (A1)S of this invention was added immediately after nucleotide after the outermost, encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without altering the reading frames in the codons.

The obtained plasmid pKFrSt12-657soy were digested with restrictase BamHI and SacI for DNA containing the nucleotide sequence shown in SEQ ID NO:214. Specified DNA was built between the site restrictase BglII site of restrictase SacI plasmids pNdG6-ΔT to obtain plasmid pSUM-NdG6-rSt12-657soy (Fig. 51), where the promoter CR16G6 attached after chimeric DNA in which the DNA is attached immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons.

Then, this plasmid was introduced into competent cells of E. coli DH5α (Takara Shuzo Company) and were selected by ampicillin-resistant cells. Further, the nucleotide sequence of the plasmid contained in the ampicillin-resistant strains, was determined using kit BigDye Terminator Cycle Sequencing Ready Reaction kit v3.0 (PE Applied Biosystems Company) and DNA sequencing machine 3100 (PE Applied Biosystems Company). In the result, it was confirmed that the plasmid pSUM-NdG6-rSt12-657soy has the nucleotide sequence shown in SEQ ID NO:214.

(3) the Introduction of DNA (A)S of the present invention in soybean

Globular embryos of soybean (cultivar: Fayette and Jack) received ACC is accordance with the method, described in example 17(1), except for the replacement source of vitamins MS source of vitamins environment B5 (O. L. Gamborg et al., Exp. Cell Res. (1986) 50 p 151).

Received globular embryo was transferred into a fresh environment for the cultivation of somatic embryos and cultured for 2-3 days. In accordance with the method described in example 17(2), plasmid pSUM-NdG6-rSt-657soy constructed in example 47(1), or plasmid pSUM-NdG6-rSt12-657soy constructed in example 47(2), was introduced in these globular embryos.

(4) the Selection of somatic embryo with hygromycin

Selection using hygromycin globular embryo after the introduction of a gene obtained in example 47(3), conducted in accordance with the method described in example 17(3), except for the replacement source of vitamins MS source of vitamins environment B5. However, after the second transplant used the environment to which was added 0.2 (wt./vol.)% Gellrich, or fluid, to which was added Gellrich, as the medium for selection of somatic embryo. In the case of a liquid medium, culturing was carried out at 90 careful rpm.

(5) the Selection of somatic embryo with compound (II)

Selection using the compound (II) globular embryo after the introduction of a gene obtained in example 47(3), conducted in accordance with the method described in example 17(4), and is the conclusion of a replacement source of vitamins MS source of vitamins environment B5.

(6) Regeneration of plants from somatic embryo, acclimate and cultivation of plants

In accordance with the method described in example 17(5), regeneration of plants was performed from globular embryos, selected in example 47(4) 47(5). However, the concentration of agar in the medium for development brought to 0.8 (wt./vol.)% or 1.0 (wt./vol.%. Further, the source of vitamins MS was replaced by a source of vitamins environment B5.

Plant with roots and Mature leaves subjected to acclimation and cultivation in accordance with the method described in example 17(6), and collect a lot of plants.

(7) Evaluation of resistance to the herbicide compound (II)

The degree of resistance against compounds (II) regenerated plants obtained in example 47(6), evaluated in accordance with the method described in example 17(4).

(8) Construction of chloroplast expression plasmids with DNA (A1)S of the present invention, for introduction using Agrobacterium

Designed plasmid for introduction of DNA (A1)S of the present invention into a plant by Agrobacterium. Plasmid pSUM-NdG6-rSt-657soy were digested with restriction enzyme NotI to obtain chimeric DNA in which DNA (A1)S of this invention was added immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons. Specified DNA was embedded in the site restrictase NotI above binary plasmid vector pBI121S obtained in example 18, to obtain the plasmid pBI-NdG6-rSt-657soy (Fig. 52). Next, the plasmid pSUM-NdG6-rSt12-657soy were digested with restriction enzyme NotI emitting chimeric DNA in which DNA (A1)S of this invention was added immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without altering the reading frames in the codons. This DNA was embedded in the site restrictase NotI above binary plasmid vector pBI121S with obtaining the plasmid pBI-NdG6-rSt12-657soy (Fig. 53).

(9) the Introduction of DNA (A1)S of this invention in tobacco

DNA (A1)S of the present invention was introduced into tobacco by means of Agrobacterium using pBI-NdG6-rSt-657soy and pBI-NdG6-rSt12-657soy obtained in example 47(8).

First, in accordance with the method described in example 19, each of the plasmid pBI-NdG6-rSt-657soy and pBI-NdG6-rSt12-657soy was introduced into Agrobacterium tumefaciens LBA4404 (Clontech Company). Allocated transgenic Agrobacterium bearing pBI-NdG6-rSt-657soy or pBI-NdG6-rSt12-657soy.

Then, with the exception of cultivation during the night transgenic Agrobacterium, bearing the above plasmid, at 30°C in liquid LB medium containing 25 mg/l kanamycin, MC is connected Agrobacterium used to introduce genes into the tobacco in accordance with the method, described in example 19. Received, respectively, transgenic tobacco plants, which included the area of the T-DNA pBI-NdG6-rSt-657soy or pBI-NdG6-rSt12-657soy.

(10) Evaluation of stability by using a piece of sheet transgenic tobacco with DNA (A1)S of this invention

Leaves were collected from 35 transgenic tobacco plants obtained in example 47(9). Each sheet was divided into pieces, and each piece had a width of 5-7 mm. Pieces of the sheet were dropped off on Wednesday with MS agar containing 0, of 0.05, 0.1 or 0.2 mg/l of compound (II), and cultured in the light at room temperature. On the 11th day of cultivation was observed herbicide damage to each of these pieces of paper. Then the pieces of the sheet were planted on the environment with MS agar containing 0, 0,01, 0,02, 0,05 or 0,1 mg/l of compound (XII), and cultured in the light at room temperature. On the 7th day of cultivation was observed herbicide damage to each of pieces of the sheet. As a control, 20 pieces of leaf tobacco, which have not performed a genetic introduction (hereinafter called "tobacco wild type"), were used at each concentration. Average rating (points) for each group was determined with the adoption of the evaluation 1 point for a piece of sheet, which is continuously growing, assessment, 0.5 points for a half-wilted piece of sheet, in which the observed chemical damage, and a score of 0 points for Coachella, which was white and had sevadal. Pieces of tobacco leaf, which was introduced DNA (A1)S of the present invention (the area of the T-DNA plasmid pBI-NdG6-rSt-657soy or pBI-NdG6-rSt12-657soy), gave a higher score than tobacco wild type with each of the compounds (II) and (XII).

Example 48. Obtaining DNA (A16) this invention

(1) preparation of chromosomal DNA of Streptomyces ornatus IFO 13069t

Chromosomal DNA of Streptomyces ornatus IFO 13069t was obtained according to the method described in example 31(1).

(2) Isolation of DNA having a partial nucleotide sequence of DNA (A16) this invention

PCR was performed using as a template the chromosomal DNA of Streptomyces ornatus IFO 13069t obtained in example 48(1), and using a pair of 14 primers in accordance with the method described in example 29. Similarly to example 31(2), amplified DNA was cloned into the cloning vector pCRII-TOPO (Invitrogen Company). Its nucleotide sequence was analyzed. The result was obtained nucleotide sequence consisting of nucleotides 343-1069 the nucleotide sequence shown in SEQ ID NO:225.

Next, the chromosomal DNA obtained in example 48(1), was digested with restriction enzyme PvuII. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), obtaining products PE the howling PCR, using this library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:265 and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:266, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1-501 the nucleotide sequence shown in SEQ ID NO:235.

Next, the chromosomal DNA obtained in example 48(1), was digested with restriction enzyme PvuII. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:267, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:268 and primer AR. Nucleotide posledovatel the activity of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1044-1454 the nucleotide sequence shown in SEQ ID NO:235.

(3) Analysis of DNA sequences (A16) this invention

The nucleotide sequence shown in SEQ ID NO:235, received by the connection of the nucleotide sequences provided DNA obtained in example 48(2). In the specified nucleotide sequence was attended by two open reading frames (ORF). Thus, it contained the nucleotide sequence (SEQ ID NO:225), consisting of 1251 nucleotides including the stop codon) and encoding a 416 amino acid residue (SEQ ID NO:215), and the nucleotide sequence (SEQ ID NO:225), consisting of 198 nucleotides including the stop codon) and encoding a 65 amino acid residues (SEQ ID NO:245). It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:215), encoded by the nucleotide sequence shown in SEQ ID NO:225, equal 46013 Yes. Further, it was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:245), encoded by the nucleotide sequence shown in SEQ ID NO:255, equal 6768 Yes.

Example 49. Expression of DNA (A16) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (A16) this invention

PCR carried out and using PCR-system GeneAmp High Fidelity (Applied Biosystems Japan Company) and using as the template the chromosomal DNA, derived from Streptomyces ornatus IFO 13069t in example 48(1). As primers used a couple of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:269, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:286. The reaction solution for PCR was brought up to 50 μl by adding each of the 2 primers, brought to 200 nm, 50 ng of the above chromosomal DNA, 5,0 ál dNTP mixture (a mixture of 2.0 mm each of the 4 types of dNTP; Clontech Company), 5,0 ál 10x buffer (containing MgCl2) and 0.5 μl of a mixture of enzymes GeneAmp HF and by adding distilled water. The PCR reaction conditions were as follows: after maintaining 97°C for 1 minute, repeating 10 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 60°C for 30 seconds, and then 72°C for 90 seconds; then 15 cycles, where each cycle consisted of maintaining 97°C for 15 seconds, then 60°C for 30 seconds, and then 72°C for 90 seconds (20 seconds added to maintain 72°C for each cycle); and then maintaining at 72°C for 7 minutes. Similarly to example 32(1), DNA was purified from the reaction solution for PCR and cloned into the cloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequence of the obtained plasmid DNA was analyzed using as primers the oligonucleotide having the nucleotide PEFC is the sequences, shown, respectively, in SEQ ID nos:57, 59, 267, 286 and 288. On the basis of the results obtained plasmid having the nucleotide sequence shown in SEQ ID NO:235, was named pCR452F. Similarly to example 32(1), pCR452F were digested with restrictase NdeI and HindIII. From the products of cleavage of the purified DNA size approximately 1.5 TPN Obtained DNA and plasmid pKSN2, split NdeI and HindIII, ligated to obtain plasmid containing the nucleotide sequence shown in SEQ ID NO:235, in which DNA encoding a protein (A16) of the present invention, inserted between the NdeI site and the HindIII site pKSN2 (hereinafter called “pKSN452F”). The indicated plasmid was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN452F.

(2) protein Expression (A16) of the present invention in E. coli and the selection of the specified protein

Like in example 4(2) cultivated each of E. coli JM109/pKSN452F and JM109/pKSN2. The cells were extracted. Prepared solutions of cell lysates. According to the method described in example 4(2)of these solutions cell lysates were obtained fractions supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN452F, called “extract of E. coli pKSN452F”, and the supernatant fraction obtained from E. coli JM109/pKSN2, called “extract of E. coli pKSN2”).

(3) the Discovery of an ability to convert compound (II) into the compound (III)

Similarly to example 32(3), the reaction solution and 30 μl prepared is kept for 10 minutes at 30°C. However, as a fraction of the supernatant used a fraction of the supernatant obtained in example 49(2) (extract of E. coli pKSN452F or extract of E. coli pKSN2). The reaction solutions after aging were extracted with ethyl acetate and extracted layers were analyzed using TLC. After developing TLC plates the presence of stains on it, corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). The spot corresponding to the compound (III), were detected from the reaction solution containing the extract of E. coli pKSN452F. In contrast, this spot is not detected from the reaction solution containing the extract of E. coli pKSN2.

Example 50. Obtaining DNA (A17) this invention

(1) preparation of chromosomal DNA of Streptomyces griseus ATCC 10137

Chromosomal DNA of Streptomyces griseus ATCC 10137 was obtained according to the method described in example 31(1).

(2) Isolation of DNA having a partial nucleotide sequence of DNA (A17) this invention

PCR was performed using as a template the chromosomal DNA of Streptomyces griseus ATCC 10137 obtained in example 50(1), and using a pair of 14 primers in accordance with the method described in example 29. Similarly to example 31(2), amplified DNA was cloned into the cloning vector pCRII-TOPO (Invitrogen Company). Its nucleotide sequence was analyzed. The result was the floor of the Jena nucleotide sequence, consisting of nucleotides 343-1069 the nucleotide sequence shown in SEQ ID NO:226.

Next, the chromosomal DNA obtained in example 50(1), was digested with restriction enzyme SmaI. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:270 and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:271, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1-361 the nucleotide sequence shown in SEQ ID NO:236.

Next, the chromosomal DNA obtained in example 50(1), was digested with restriction enzyme PvuII. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as a matrix and use what Lovanium of the oligonucleotide, having the nucleotide sequence shown in SEQ ID NO:272, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:273, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1035-1454 the nucleotide sequence shown in SEQ ID NO:236.

(3) Analysis of DNA sequences (A17) this invention

The nucleotide sequence shown in SEQ ID NO:236, received by the connection of the nucleotide sequences provided DNA obtained in example 50(2). In the specified nucleotide sequence was attended by two open reading frames (ORF). Thus, it contained the nucleotide sequence (SEQ ID NO:226), comprising 1251 nucleotides including the stop codon) and encoding a 416 amino acid residue (SEQ ID NO:216), and the nucleotide sequence (SEQ ID NO:226), consisting of 198 nucleotides including the stop codon) and encoding a 65 amino acid residues (SEQ ID NO:246). It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:216), encoded by the nucleotide sequence shown in SEQ ID N:226, equal 46082 Yes. Further, it was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:246), encoded by the nucleotide sequence shown in SEQ ID NO:256, equal 6768 Yes. The nucleotide sequence shown in SEQ ID NO:256, is 100% identical to the nucleotide sequence shown in SEQ ID NO:255. Amino acid sequence shown in SEQ ID NO:246, is 100% identical to the amino acid sequence shown in SEQ ID NO:245.

Example 51. Expression of DNA (A17) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (A17) this invention

PCR was performed similarly to example 32(1), except for use as matrix chromosomal DNA derived from Streptomyces griseus ATCC 10137 in example 50(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:274, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:275. Similarly to example 32(1), DNA was purified from the reaction solution for PCR and cloned into the cloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequence of the obtained plasmid DNA was analyzed using as primers the oligonucleotide having the nucleotide sequence shown corresponding to the o in SEQ ID NO:57, 59, 274, 276, and 277. On the basis of the results obtained plasmid having the nucleotide sequence shown in SEQ ID NO:236, was named pCR608F. Similarly to example 32(1), pCR608F were digested with restrictase NdeI and HindIII. From the products of cleavage of the purified DNA size approximately 1.5 TPN Obtained DNA and plasmid pKSN2, split NdeI and HindIII, ligated to obtain plasmid containing the nucleotide sequence shown in SEQ ID NO:236, in which DNA encoding a protein (A17) of the present invention, inserted between the NdeI site and the HindIII site pKSN2 (hereinafter called “pKSN608F”). The indicated plasmid was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN608F.

(2) the expression of the protein (A17) of the present invention in E. coli and the selection of the specified protein

Like in example 4(2) cultivated each of E. coli JM109/pKSN608F and JM109/pKSN2. The cells were extracted. Prepared solutions of cell lysates. According to the method described in example 4(2)of these solutions cell lysates were obtained fractions supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN608F, called “extract of E. coli pKSN608F”, and the supernatant fraction obtained from E. coli JM109/pKSN2, called “extract of E. coli pKSN2”).

(3) the Discovery of an ability to convert compound (II) into the compound (III)

Similarly to example 32(3), the reaction solution and 30 μl were prepared and kept for 10 mi the ut at 30°C. However, as a fraction of the supernatant used a fraction of the supernatant obtained in example 51(2) (extract of E. coli pKSN608F or extract of E. coli pKSN2). These reaction solutions after aging were extracted with ethyl acetate and extracted layers were analyzed using TLC. After developing TLC plates the presence of stains on it, corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). The spot corresponding to the compound (III), were detected from the reaction solution containing the extract of E. coli pKSN608F. In contrast, this spot is not detected from the reaction solution containing the extract of E. coli pKSN2.

Example 52. Obtaining DNA (A18) this invention

(1) preparation of chromosomal DNA of Streptomyces achromogenes IFO 12735

Chromosomal DNA of Streptomyces achromogenes IFO 12735 was obtained according to the method described in example 31(1).

(2) Isolation of DNA having a partial nucleotide sequence of DNA (A18) this invention

PCR was performed using as a template the chromosomal DNA of Streptomyces achromogenes IFO 12735 obtained in example 52(1), and using a pair of 17 primers in accordance with the method described in example 29. Similarly to example 31(2), amplified DNA was cloned into the cloning vector pCRII-TOPO (Invitrogen Company). Its nucleotide sequence was analyzed. In the result which was obtained nucleotide sequence, consisting of nucleotides 526-1048 the nucleotide sequence shown in SEQ ID NO:227.

Next, the chromosomal DNA obtained in example 52(1), was digested with restriction enzyme HincII. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:278, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:279, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1-361 the nucleotide sequence shown in SEQ ID NO:237.

Next, the chromosomal DNA obtained in example 52(1), was digested with restriction enzyme BalI. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as template and with the COI is the whether of the oligonucleotide, having the nucleotide sequence shown in SEQ ID NO:163, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:164, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 983-1449 the nucleotide sequence shown in SEQ ID NO:237.

(3) Analysis of DNA sequences (A18) this invention

The nucleotide sequence shown in SEQ ID NO:237, received by the connection of the nucleotide sequences provided DNA obtained in example 52(2). In the specified nucleotide sequence was attended by two open reading frames (ORF). Thus, it contained the nucleotide sequence (SEQ ID NO:227), consisting of 1230 nucleotides including the stop codon) and encoding a 409 amino acid residues (SEQ ID NO:217), and the nucleotide sequence (SEQ ID NO:257), consisting of 207 nucleotides including the stop codon) and encoding a 68 amino acid residues (SEQ ID NO:247). It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:217), encoded by the nucleotide sequence shown in SEQ ID O:227, equal 45099 Yes. Further, it was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:247), encoded by the nucleotide sequence shown in SEQ ID NO:257, equal 7193 Yes.

Example 53. Expression of DNA (A18) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (A18) this invention

PCR was performed similarly to example 49(1), except for use as matrix chromosomal DNA obtained from Streptomyces achromogenes IFO 12735 in example 52(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:183, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:280. Similarly to example 32(1), DNA was purified from the reaction solution for PCR and cloned into the cloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequence of the obtained plasmid DNA was analyzed using as primers the oligonucleotides having the nucleotide sequences shown, respectively, in SEQ ID NO:67, 68, 163, 279 and 281. On the basis of the results obtained, the plasmid having the nucleotide sequence shown in SEQ ID NO:237, was named pCR646F. Similarly to example 32(1), pCR646F were digested with restrictase NdeI and HindIII. From the products of cleavage of the purified DNA size priblisitelno,5 TPN The resulting DNA and plasmid pKSN2, split NdeI and HindIII, ligated to obtain plasmid containing the nucleotide sequence shown in SEQ ID NO:237, in which DNA encoding a protein (A18) of the present invention, inserted between the NdeI site and the HindIII site pKSN2 (hereinafter called “pKSN646F”). The indicated plasmid was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN646F.

(2) protein Expression (A18) of the present invention in E. coli and the selection of the specified protein

Like in example 4(2), cultivated each of E. coli JM109/pKSN646F and JM109/pKSN2. The cells were extracted. Prepared solutions of cell lysates. According to the method described in example 4(2)of these solutions cell lysates were obtained fractions supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN646F, called “extract of E. coli pKSN646F”, and the supernatant fraction obtained from E. coli JM109/pKSN2, called “extract of E. coli pKSN2”).

(3) the Discovery of an ability to convert compound (II) into the compound (III)

Similarly to example 32(3), the reaction solution and 30 μl were prepared and kept for 10 minutes at 30°C. However, as a fraction of the supernatant used a fraction of the supernatant obtained in example 53(2) (extract of E. coli pKSN646F or extract of E. coli pKSN2). These reaction solutions after aging were extracted with ethyl acetate and extracted layers were analyzed with the measures TLC. After developing TLC plates the presence of stains on it, corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). The spot corresponding to the compound (III), were detected from the reaction solution containing the extract of E. coli pKSN646F. In contrast, this spot is not detected from the reaction solution containing the extract of E. coli pKSN2.

Example 54. Obtaining DNA (A19) this invention

(1) preparation of chromosomal DNA of Streptomyces griseus IFO 13849T

Chromosomal DNA of Streptomyces griseus IFO 13849T was obtained according to the method described in example 31(1).

(2) Isolation of DNA having a partial nucleotide sequence of DNA (A19) this invention

PCR was performed using as a template the chromosomal DNA of Streptomyces griseus IFO 13849T obtained in example 54(1), and using a pair of 14 primers in accordance with the method described in example 29. Similarly to example 31(2), amplified DNA was cloned into the cloning vector pCRII-TOPO (Invitrogen Company). Its nucleotide sequence was analyzed. The result was obtained nucleotide sequence consisting of nucleotides 343-1069 the nucleotide sequence shown in SEQ ID NO:228.

Next, the chromosomal DNA obtained in example 54(1), was digested with restriction enzyme SmaI. Library genome walker was obtained using the obtained D Is in accordance with the method, described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:282, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:283, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1-358 of the nucleotide sequence shown in SEQ ID NO:238.

Next, the chromosomal DNA obtained in example 54(1), was digested with restriction enzyme HincII. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:284, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using oligonucleotide is a, having the nucleotide sequence shown in SEQ ID NO:285, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1005-1454 the nucleotide sequence shown in SEQ ID NO:238.

(3) Analysis of DNA sequences (A19) this invention

The nucleotide sequence shown in SEQ ID NO:238, received by the connection of the nucleotide sequences provided DNA obtained in example 54(2). In the specified nucleotide sequence was attended by two open reading frames (ORF). Thus, it contained the nucleotide sequence (SEQ ID NO:228), comprising 1251 nucleotides including the stop codon) and encoding a 416 amino acid residues (SEQ ID NO:218), and the nucleotide sequence (SEQ ID NO:258), consisting of 156 nucleotides including the stop codon) and encoding a 51 amino acid residue (SEQ ID NO:248). It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:218), encoded by the nucleotide sequence shown in SEQ ID NO:228, equal 45903 Yes. Further, it was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:248), encoded by the nucleotide sequence shown in SEQ ID NO:258, equal 5175 Yes.

Example 55. Exp is Essie DNA (A19) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (A19) this invention

PCR was performed similarly to example 49(1), except for use as matrix chromosomal DNA derived from Streptomyces griseus IFO 13849T in example 54(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:286, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:287. Similarly to example 32(1), DNA was purified from the reaction solution for PCR and cloned into the cloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequence of the obtained plasmid DNA was analyzed using as primers the oligonucleotides having the nucleotide sequences shown, respectively, in SEQ ID nos:57, 59, 284, 286 and 288. On the basis of the results obtained, the plasmid having the nucleotide sequence shown in SEQ ID NO:238, was named pCR1502F. Similarly to example 32(1), pCR1502F were digested with restrictase NdeI and HindIII. From the products of cleavage of the purified DNA size approximately 1.5 TPN Obtained DNA and plasmid pKSN2, split NdeI and HindIII, ligated to obtain plasmid containing the nucleotide sequence shown in SEQ ID NO:238, in which DNA encoding a protein (A19) of the present invention, inserted between the NdeI site and the HindIII site pKSN2 (hereinafter the so-called “pKSN1502F”). The indicated plasmid was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN1502F.

(2) protein Expression (A19) of the present invention in E. coli and the selection of the specified protein

Like in example 4(2) cultivated each of E. coli JM109/pKSN1502F and JM109/pKSN2. The cells were extracted. Prepared solutions of cell lysates. According to the method described in example 4(2)of these solutions cell lysates were obtained fractions supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN1502F, called “extract of E. coli pKSN1502F”, and the supernatant fraction obtained from E. coli JM109/pKSN2, called “extract of E. coli pKSN2”).

(3) the Discovery of an ability to convert compound (II) into the compound (III)

Similarly to example 32(3), the reaction solution and 30 μl were prepared and kept for 10 minutes at 30°C. However, as a fraction of the supernatant used a fraction of the supernatant obtained in example 55(2) (extract of E. coli pKSN1502F or extract of E. coli pKSN2). These reaction solutions after aging were extracted with ethyl acetate and extracted layers were analyzed using TLC. After developing TLC plates the presence of stains on it, corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). The spot corresponding to the compound (III), were detected from the reaction solution containing the extract of E. coli pKSN1502F. In protivopoloznostyu this spot is not detected from the reaction solution, containing extract of E. coli pKSN2.

Example 56. Obtaining DNA (A20) this invention

(1) preparation of chromosomal DNA of Streptomyces lanatus IFO 12787T

Chromosomal DNA of Streptomyces lanatus IFO 12787T was obtained according to the method described in example 31(1).

(2) Isolation of DNA having a partial nucleotide sequence of DNA (A20) this invention

PCR was performed using as a template the chromosomal DNA of Streptomyces lanatus IFO 12787T obtained in example 56(1), and using a pair of 14 primers in accordance with the method described in example 29. Similarly to example 31(2), amplified DNA was cloned into the cloning vector pCRII-TOPO (Invitrogen Company). Its nucleotide sequence was analyzed. The result was obtained nucleotide sequence consisting of nucleotides 304-1036 the nucleotide sequence shown in SEQ ID NO:229.

Next, the chromosomal DNA obtained in example 56(1), was digested with restriction enzyme PmacI. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:278, and primer AP1. Then PCR was performed in the services who were described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:289 and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1-318 the nucleotide sequence shown in SEQ ID NO:239.

Next, the chromosomal DNA obtained in example 56(1), was digested with restriction enzyme StuI. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:290, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:291, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 969-1461 the nucleotide sequence shown in SEQ ID NO:239.

(3) Analysis of DNA sequences (the 20) of the present invention

The nucleotide sequence shown in SEQ ID NO:239, received by the connection of the nucleotide sequences provided DNA obtained in example 56(2). In the specified nucleotide sequence was attended by two open reading frames (ORF). Thus, it contained the nucleotide sequence (SEQ ID NO:229), comprising 1218 nucleotides including the stop codon) and encoding a 405 amino acid residues (SEQ ID NO:219), and the nucleotide sequence (SEQ ID NO:259), consisting of 231 nucleotides (including the stop codon) and encoding a 76 amino acid residues (SEQ ID NO:249). It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:219), encoded by the nucleotide sequence shown in SEQ ID NO:229, equal 45071 Yes. Further, it was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:249), encoded by the nucleotide sequence shown in SEQ ID NO:259, equal 7816 Yes.

Example 57. Expression of DNA (A20) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (A20) this invention

PCR was performed similarly to example 49(1), except for use as matrix chromosomal DNA obtained from Streptomyces lanatus IFO 12787T in example 56(1), and using as primers the oligonucleotide, having the th nucleotide sequence, shown in SEQ ID NO:292, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:293. Similarly to example 32(1), DNA was purified from the reaction solution for PCR and cloned into the cloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequence of the obtained plasmid DNA was analyzed using as primers the oligonucleotides having the nucleotide sequences shown, respectively, in SEQ ID NO:67, 68, 188, 278 and 290. On the basis of the results obtained plasmid having the nucleotide sequence shown in SEQ ID NO:239, was named pCR1525F. Similarly to example 32(1), pCR1525F were digested with restrictase NdeI and HindIII. From the products of cleavage of the purified DNA size approximately 1.5 TPN Obtained DNA and plasmid pKSN2, split NdeI and HindIII, ligated to obtain plasmid containing the nucleotide sequence shown in SEQ ID NO:239, in which DNA encoding a protein (A20) of the present invention, inserted between the NdeI site and the HindIII site pKSN2 (hereinafter called “pKSN1525F”). The indicated plasmid was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN1525F.

(2) protein Expression (A20) of the present invention in E. coli and the selection of the specified protein

Like in example 4(2) cultivated each of E. coli JM109/pKSN1525F and JM109/pKSN2. The cells were extracted. Prepared solutions of cell lysates. By the way, describe the approach in example 4(2), from these solutions the cell lysates were obtained fractions supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN1525F, called “extract of E. coli pKSN1525F”, and the supernatant fraction obtained from E. coli JM109/pKSN2, called “extract of E. coli pKSN2”).

(3) the Discovery of an ability to convert compound (II) into the compound (III)

Similarly to example 32(3), the reaction solution and 30 μl were prepared and kept for 10 minutes at 30°C. However, as a fraction of the supernatant used a fraction of the supernatant obtained in example 57(2) (extract of E. coli pKSN1525F or extract of E. coli pKSN2). These reaction solutions after aging were extracted with ethyl acetate and extracted layers were analyzed using TLC. After developing TLC plates the presence of stains on it, corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). The spot corresponding to the compound (III), were detected from the reaction solution containing the extract of E. coli pKSN1525F. In contrast, this spot is not detected from the reaction solution containing the extract of E. coli pKSN2.

Example 58. Obtaining DNA (A21) this invention

(1) preparation of chromosomal DNA of Streptomyces misawanensis IFO 13855T

Chromosomal DNA of Streptomyces misawanensis IFO 13855T was obtained according to the method described in example 31(1).

(2) Isolation of DNA having a partial is nucleotide DNA sequence (A21) this invention

PCR was performed using as a template the chromosomal DNA of Streptomyces misawanensis IFO 13855T obtained in example 58(1), and using a pair of 14 primers in accordance with the method described in example 29. Similarly to example 31(2), amplified DNA was cloned into the cloning vector pCRII-TOPO (Invitrogen Company). Its nucleotide sequence was analyzed. The result was obtained nucleotide sequence consisting of nucleotides 328-1063 the nucleotide sequence shown in SEQ ID NO:230.

Next, the chromosomal DNA obtained in example 58(1), was digested with restriction enzyme SmaI. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:294, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:295, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Received a nucleotide sequence consisting of a nucleotide sequence that is the Chida 1-341 nucleotide sequence, shown in SEQ ID NO:240.

Next, the chromosomal DNA obtained in example 58(1), was digested with restriction enzyme HincII. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:296, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:297, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1017-1458 the nucleotide sequence shown in SEQ ID NO:240.

(3) Analysis of DNA sequences (A21) this invention

The nucleotide sequence shown in SEQ ID NO:240, received by the connection of the nucleotide sequences provided DNA obtained in example 58(2). In the specified nucleotide sequence was attended by two open reading frames (ORF). Thus, it contained the nucleotide sequence (SEQ ID NO:230), comprising as nucleotides including the stop codon) and encoding a 414 amino acid residues (SEQ ID NO:220), and the nucleotide sequence (SEQ ID NO:260), consisting of a nucleotide 201 (including the stop codon) and encoding a 66 amino acid residues (SEQ ID NO:250). It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:220), encoded by the nucleotide sequence shown in SEQ ID NO:230, equal 45806 Yes. Further, it was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:250), encoded by the nucleotide sequence shown in SEQ ID NO:260, equal 6712 Yes.

Example 59. Expression of DNA (A21) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (A21) this invention

PCR was performed similarly to example 32(1), except for use as matrix chromosomal DNA obtained from Streptomyces misawanensis IFO 13855T in example 58(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:298, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:299. Similarly to example 32(1), DNA was purified from the reaction solution for PCR and cloned into the cloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequence of the obtained plasmid DNA was analyzed using as primers the oligonucleotide having the nucleotide consistent is Telenesti, shown, respectively, in SEQ ID nos:57, 59, 296, 298 and 300. On the basis of the results obtained plasmid having the nucleotide sequence shown in SEQ ID NO:240, was named pCR1543F. Similarly to example 32(1), pCR1543F were digested with restrictase NdeI and HindIII. From the products of cleavage of the purified DNA size approximately 1.5 TPN Obtained DNA and plasmid pKSN2, split NdeI and HindIII, ligated to obtain plasmid containing the nucleotide sequence shown in SEQ ID NO:240, in which a DNA encoding a protein (A21) of the present invention, inserted between the NdeI site and the HindIII site pKSN2 (hereinafter called “pKSN1543F”). The indicated plasmid was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN1543F.

(2) the expression of the protein (A21) of the present invention in E. coli and the selection of the specified protein

Like in example 4(2) cultivated each of E. coli JM109/pKSN1543F and JM109/pKSN2. The cells were extracted. Prepared solutions of cell lysates. According to the method described in example 4(2)of these solutions cell lysates were obtained fractions supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN1543F, called “extract of E. coli pKSN1543F”, and the supernatant fraction obtained from E. coli JM109/pKSN2, called “extract of E. coli pKSN2”).

(3) the Discovery of an ability to convert compound (II) into the compound (III)

Similarly to example 32(3), the reaction solutions at 30 m the l was prepared and kept for 10 minutes at 30°C. However, as a fraction of the supernatant used a fraction of the supernatant obtained in example 59(2) (extract of E. coli pKSN1543F or extract of E. coli pKSN2). These reaction solutions after aging were extracted with ethyl acetate and extracted layers were analyzed using TLC. After developing TLC plates the presence of stains on it, corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). The spot corresponding to the compound (III), were detected from the reaction solution containing the extract of E. coli pKSN1543F. In contrast, this spot is not detected from the reaction solution containing the extract of E. coli pKSN2.

Example 60. Obtaining DNA (A22) this invention

(1) preparation of chromosomal DNA of Streptomyces pallidus IFO 13434T

Chromosomal DNA of Streptomyces pallidus IFO 13434T was obtained according to the method described in example 31(1).

(2) Isolation of DNA having a partial nucleotide sequence of DNA (A22) this invention

PCR was performed using as a template the chromosomal DNA of Streptomyces pallidus IFO 13434T obtained in example 60(1), and using 15 pairs of primers in accordance with the method described in example 29. Similarly to example 31(2), amplified DNA was cloned into the cloning vector pCRII-TOPO (Invitrogen Company). Its nucleotide sequence was analyzed. The result is the had been obtained nucleotide sequence, consisting of nucleotides 483-1048 the nucleotide sequence shown in SEQ ID NO:231.

Next, the chromosomal DNA obtained in example 60(1), was digested with restriction enzyme SmaI. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:301, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:302 and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 68-516 the nucleotide sequence shown in SEQ ID NO:241.

Next, the chromosomal DNA obtained in example 60(1), was digested with restriction enzyme HincII. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as template and with the COI is the whether of the oligonucleotide, having the nucleotide sequence shown in SEQ ID NO:302 and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:303 and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1-270 the nucleotide sequence shown in SEQ ID NO:241.

Next, the chromosomal DNA obtained in example 60(1), was digested with restriction enzyme HincII. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:304, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:305, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of h is of cleotides 982-1448 nucleotide sequence, shown in SEQ ID NO:241.

(3) Analysis of DNA sequences (A22) this invention

The nucleotide sequence shown in SEQ ID NO:241, received by the connection of the nucleotide sequences provided DNA obtained in example 60(2). In the specified nucleotide sequence was attended by two open reading frames (ORF). Thus, it contained the nucleotide sequence (SEQ ID NO:231), comprising 1230 nucleotides including the stop codon) and encoding a 409 amino acid residues (SEQ ID NO:221), and the nucleotide sequence (SEQ ID NO:261), consisting of 195 nucleotides including the stop codon) and 64 encoding amino acid residue (SEQ ID NO:251). It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:221), encoded by the nucleotide sequence shown in SEQ ID NO:231, equal 45050 Yes. Further, it was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:251), encoded by the nucleotide sequence shown in SEQ ID NO:261, equal 6914 Yes.

Example 61. Expression of DNA (A22) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (A22) this invention

PCR was performed similarly to example 32(1), except for use as matrix chromosomal DNA obtained from Streptomycespallidus IFO 13434T in example 60(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:306, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:307. Similarly to example 32(1), DNA was purified from the reaction solution for PCR and cloned into the cloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequence of the obtained plasmid DNA was analyzed using as primers the oligonucleotides having the nucleotide sequences shown, respectively, in SEQ ID NO:67, 68 and 308. On the basis of the results obtained plasmid having the nucleotide sequence shown in SEQ ID NO:241, was named pCR1558F. Similarly to example 32(1), pCR1558F were digested with restrictase NdeI and HindIII. From the products of cleavage of the purified DNA size approximately 1.5 TPN Obtained DNA and plasmid pKSN2, split NdeI and HindIII, ligated to obtain plasmid containing the nucleotide sequence shown in SEQ ID NO:241, in which DNA encoding a protein (A22) of the present invention, inserted between the NdeI site and the HindIII site pKSN2 (hereinafter called “pKSN1558F”). The indicated plasmid was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN1558F.

(2) the expression of the protein (A22) of the present invention in E. coli and the selection of the specified protein

Like in example 4(2) cultivated area is Yu from E. coli JM109/pKSN1558F and JM109/pKSN2. The cells were extracted. Prepared solutions of cell lysates. According to the method described in example 4(2)of these solutions cell lysates were obtained fractions supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN1558F, called “extract of E. coli pKSN1558F”, and the supernatant fraction obtained from E. coli JM109/pKSN2, called “extract of E. coli pKSN2”).

(3) the Discovery of an ability to convert compound (II) into the compound (III)

Similarly to example 32(3), the reaction solution and 30 μl were prepared and kept for 10 minutes at 30°C. However, as a fraction of the supernatant used a fraction of the supernatant obtained in example 61(2) (extract of E. coli pKSN1558F or extract of E. coli pKSN2). These reaction solutions after aging were extracted with ethyl acetate and extracted layers were analyzed using TLC. After developing TLC plates the presence of stains on it, corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). The spot corresponding to the compound (III), were detected from the reaction solution containing the extract of E. coli pKSN1558F. In contrast, this spot is not detected from the reaction solution containing the extract of E. coli pKSN2.

Example 62. Obtaining DNA (A23) this invention

(1) preparation of chromosomal DNA of Streptomyces roseorubens IFO 13682T

Chromoso the percent DNA of Streptomyces roseorubens IFO 13682T got in the way, described in example 31(1).

(2) Isolation of DNA having a partial nucleotide sequence of DNA (A23) this invention

PCR was performed using as a template the chromosomal DNA of Streptomyces roseorubens IFO 13682T obtained in example 62(1), and using a pair of 14 primers in accordance with the method described in example 29. Similarly to example 31(2), amplified DNA was cloned into the cloning vector pCRII-TOPO (Invitrogen Company). Its nucleotide sequence was analyzed. The result was obtained nucleotide sequence consisting of nucleotides 289-1015 the nucleotide sequence shown in SEQ ID NO:232.

Next, the chromosomal DNA obtained in example 62(1), was digested with restriction enzyme SmaI. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:309, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:310, and Primera. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1-354 the nucleotide sequence shown in SEQ ID NO:242.

Next, the chromosomal DNA obtained in example 62(1), was digested with restriction enzyme PvuII. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:311, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:312, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 966-1411 the nucleotide sequence shown in SEQ ID NO:242.

(3) Analysis of DNA sequences (A23) this invention

The nucleotide sequence shown in SEQ ID NO:242, received by the connection of the nucleotide sequences provided DNA obtained in example 62(2). In shows the nucleotide sequence was attended by two open reading frames (ORF). Thus, it contained the nucleotide sequence (SEQ ID NO:232), consisting of 1197 nucleotides including the stop codon) and encoding a 398 amino acid residues (SEQ ID NO:222), and the nucleotide sequence (SEQ ID NO:262), consisting of a nucleotide 201 (including the stop codon) and encoding a 66 amino acid residues (SEQ ID NO:252). It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:222), encoded by the nucleotide sequence shown in SEQ ID NO:232, equal 43624 Yes. Further, it was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:252), encoded by the nucleotide sequence shown in SEQ ID NO:262, equal 6797 Yes.

Example 63. Expression of DNA (A23) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (A23) this invention

PCR was performed similarly to example 49(1), except for use as matrix chromosomal DNA obtained from Streptomyces roseorubens IFO 13682T in example 62(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:313, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:314. Similarly to example 32(1), DNA was purified from the reaction solution for PCR and cloned into the cloning vector pCRII-TOPO (Invitroen Company). The nucleotide sequence of the obtained plasmid DNA was analyzed using as primers the oligonucleotides having the nucleotide sequences shown, respectively, in SEQ ID NO:67, 68, 309, 311 and 315. On the basis of the results obtained plasmid having the nucleotide sequence shown in SEQ ID NO:242, was named pCR1584F. Similarly to example 32(1), pCR1584F were digested with restrictase NdeI and HindIII. From the products of cleavage of the purified DNA size approximately 1.5 TPN Obtained DNA and plasmid pKSN2, split NdeI and HindIII, ligated to obtain plasmid containing the nucleotide sequence shown in SEQ ID NO:242, in which DNA encoding a protein (A23) of the present invention, inserted between the NdeI site and the HindIII site pKSN2 (hereinafter called “pKSN1584F”). The indicated plasmid was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN1584F.

(2) the expression of the protein (A23) of the present invention in E. coli and the selection of the specified protein

Like in example 4(2) cultivated each of E. coli JM109/pKSN1584F and JM109/pKSN2. The cells were extracted. Prepared solutions of cell lysates. According to the method described in example 4(2)of these solutions cell lysates were obtained fractions supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN1584F, called “extract of E. coli pKSN1584F”, and the supernatant fraction obtained from E. coli JM109/KSN2, called “extract of E. coli pKSN2”).

(3) the Discovery of an ability to convert compound (II) into the compound (III)

Similarly to example 32(3), the reaction solution and 30 μl were prepared and kept for 10 minutes at 30°C. However, as a fraction of the supernatant used a fraction of the supernatant obtained in example 63(2) (extract of E. coli pKSN1584F or extract of E. coli pKSN2). These reaction solutions after aging were extracted with ethyl acetate and extracted layers were analyzed using TLC. After developing TLC plates the presence of stains on it, corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). The spot corresponding to the compound (III), were detected from the reaction solution containing the extract of E. coli pKSN1584F. In contrast, this spot is not detected from the reaction solution containing the extract of E. coli pKSN2.

Example 64. Obtaining DNA (A24) this invention

(1) preparation of chromosomal DNA of Streptomyces rutgersensis IFO 15875T

Chromosomal DNA of Streptomyces rutgersensis IFO 15875T was obtained according to the method described in example 31(1).

(2) Isolation of DNA having a partial nucleotide sequence of DNA (A24) this invention

PCR was performed using as a template the chromosomal DNA of Streptomyces rutgersensis IFO 15875T obtained in example 64(1), and using pairs 14 p is amerov in accordance with the method, described in example 29. Similarly to example 31(2), amplified DNA was cloned into the cloning vector pCRII-TOPO (Invitrogen Company). Its nucleotide sequence was analyzed. The result was obtained nucleotide sequence consisting of nucleotides 322-1057 the nucleotide sequence shown in SEQ ID NO:233.

Next, the chromosomal DNA obtained in example 64(1), was digested with restriction enzyme SmaI. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:316, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:317, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1-384 the nucleotide sequence shown in SEQ ID NO:243.

Next, the chromosomal DNA obtained in example 64(1), was digested with restriction enzyme NaeI. Library genome walker received with use what Itanium obtained DNA in accordance with the method, described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:318, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:319, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 992-1466 the nucleotide sequence shown in SEQ ID NO:243.

(3) Analysis of DNA sequences (A24) this invention

The nucleotide sequence shown in SEQ ID NO:243, received by the connection of the nucleotide sequences provided DNA obtained in example 64(2). In the specified nucleotide sequence was attended by two open reading frames (ORF). Thus, it contained the nucleotide sequence (SEQ ID NO:233), consisting of 1245 nucleotides including the stop codon) and encoding a 414 amino acid residues (SEQ ID NO:223), and the nucleotide sequence (SEQ ID NO:263), consisting of 198 nucleotides including the stop codon) and 65 encoding aminokislotnykh residues (SEQ ID NO:253). It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:223), encoded by the nucleotide sequence shown in SEQ ID NO:233, equal 45830 Yes. Further, it was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:253), encoded by the nucleotide sequence shown in SEQ ID NO:263, equal 7034 Yes.

Example 65. Expression of DNA (A24) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (A24) this invention

PCR was performed similarly to example 49(1), except for use as matrix chromosomal DNA obtained from Streptomyces rutgersensis IFO 15875T in example 64(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:320, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:321. Similarly to example 32(1), DNA was purified from the reaction solution for PCR and cloned into the cloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequence of the obtained plasmid DNA sequenced using as primers the oligonucleotides having the nucleotide sequences shown, respectively, in SEQ ID NO:67, 68 and 322. On the basis of the results obtained plasmid having the nucleotide series is here, shown in SEQ ID NO:243, was named pCR1589F. Similarly to example 32(1), pCR1589F were digested with restrictase NdeI and HindIII. From the products of cleavage of the purified DNA size approximately 1.5 TPN Obtained DNA and plasmid pKSN2, split NdeI and HindIII, ligated to obtain plasmid containing the nucleotide sequence shown in SEQ ID NO:243, in which DNA encoding a protein (A24) of the present invention, inserted between the NdeI site and the HindIII site pKSN2 (hereinafter called “pKSN1589F”). The indicated plasmid was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN1589F.

(2) the expression of the protein (A24) of the present invention in E. coli and the selection of the specified protein

Like in example 4(2) cultivated each of E. coli JM109/pKSN1589F and JM109/pKSN2. The cells were extracted. Prepared solutions of cell lysates. According to the method described in example 4(2)of these solutions cell lysates were obtained fractions supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN1589F, called “extract of E. coli pKSN1589F”, and the supernatant fraction obtained from E. coli JM109/pKSN2, called “extract of E. coli pKSN2”).

(3) the Discovery of an ability to convert compound (II) into the compound (III)

Similarly to example 32(3), the reaction solution and 30 μl were prepared and kept for 10 minutes at 30°C. However, as a fraction of the supernatant used the supernatant fraction obtained is example 65(2) (extract of E. coli pKSN1589F or extract of E. coli pKSN2). These reaction solutions after aging were extracted with ethyl acetate and extracted layers were analyzed using TLC. After developing TLC plates the presence of stains on it, corresponding to compound (III)labeled14With, investigated (Rf value of 0.24 and 0.29 to). The spot corresponding to the compound (III), were detected from the reaction solution containing the extract of E. coli pKSN1589F. In contrast, this spot is not detected from the reaction solution containing the extract of E. coli pKSN2.

Example 66. Obtaining DNA (A25) this invention

(1) preparation of chromosomal DNA of Streptomyces steffisburgensis IFO 13446T

Chromosomal DNA of Streptomyces steffisburgensis IFO 13446T was obtained according to the method described in example 31(1).

(2) Isolation of DNA having a partial nucleotide sequence of DNA (A25) this invention

PCR was performed using as a template the chromosomal DNA of Streptomyces steffisburgensis IFO 13446T obtained in example 66(1), and using a pair of 14 primers in accordance with the method described in example 29. Similarly to example 31(2), amplified DNA was cloned into the cloning vector pCRII-TOPO (Invitrogen Company). Its nucleotide sequence was analyzed. The result was obtained nucleotide sequence consisting of nucleotides 289-1015 nucleotide placenta is successive, shown in SEQ ID NO:234.

Next, the chromosomal DNA obtained in example 66(1), was digested with restriction enzyme SmaI. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:323, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:324, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 1-303 the nucleotide sequence shown in SEQ ID NO:244.

Next, the chromosomal DNA obtained in example 66(1), was digested with restriction enzyme PmacI. Library genome walker was obtained using the obtained DNA in accordance with the method described in example 26(3). PCR was performed under the conditions described in example 26(3), with the receipt of the products of the first PCR, using the obtained library as the template and using an oligonucleotide having the nucleotide posledovatel the face, shown in SEQ ID NO:311, and primer AP1. Then PCR was performed under the conditions described in example 26(3), using the products of the first PCR as template and using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:325, and primer AR. The nucleotide sequence of the obtained DNA was analyzed. Got a nucleotide sequence consisting of nucleotides 966-1411 the nucleotide sequence shown in SEQ ID NO:244.

(3) Analysis of DNA sequences (A25) this invention

The nucleotide sequence shown in SEQ ID NO:244, received by the connection of the nucleotide sequences provided DNA obtained in example 66(2). In the specified nucleotide sequence was attended by two open reading frames (ORF). Thus, it contained the nucleotide sequence (SEQ ID NO:234), consisting of 1197 nucleotides including the stop codon) and encoding a 398 amino acid residues (SEQ ID NO:224), and the nucleotide sequence (SEQ ID NO:264), consisting of a nucleotide 201 (including the stop codon) and encoding a 66 amino acid residues (SEQ ID NO:254). It was estimated that the molecular weight of the protein consisting of the amino acid sequence (SEQ ID NO:224), encoded by the nucleotide sequence shown in SEQ ID NO:234, equal 44175 Yes. Further, it was estimated that MOLEKULYaRNAYa protein mass, consisting of the amino acid sequence (SEQ ID NO:254), encoded by the nucleotide sequence shown in SEQ ID NO:264, equal 6685 Yes.

Example 67. Expression of DNA (A25) of the present invention in E. coli

(1) Obtaining a transformed E. coli with DNA (A25) this invention

PCR was performed similarly to example 49(1), except for use as matrix chromosomal DNA obtained from Streptomyces steffisburgensis IFO 13446T in example 66(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:326, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:327. Similarly to example 32(1), DNA was purified from the reaction solution for PCR and cloned into the cloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequence of the obtained plasmid DNA sequenced using as primers the oligonucleotides having the nucleotide sequences shown, respectively, in SEQ ID NO:67, 68, 311, 315 and 323. On the basis of the results obtained plasmid having the nucleotide sequence shown in SEQ ID NO:244, was named pCR1609F. Similarly to example 32(1), pCR1609F were digested with restrictase NdeI and HindIII. From the products of cleavage of the purified DNA size approximately 1.5 TPN Obtained DNA and plasmid pKSN2, split NdeI and HindIII, the League is believed to obtain plasmid containing the nucleotide sequence shown in SEQ ID NO:244, in which DNA encoding a protein (A25) of the present invention, inserted between the NdeI site and the HindIII site pKSN2 (hereinafter called “pKSN1609F”). The indicated plasmid was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN1609F.

(2) the expression of the protein (A25) of the present invention in E. coli and the selection of the specified protein

Like in example 4(2) cultivated each of E. coli JM109/pKSN1609F and JM109/pKSN2. The cells were extracted. Prepared solutions of cell lysates. According to the method described in example 4(2)of these solutions cell lysates were obtained fractions supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN16093F, called “extract of E. coli pKSN1609F”, and the supernatant fraction obtained from E. coli JM109/pKSN2, called “extract of E. coli pKSN2”).

(3) the Discovery of an ability to convert compound (II) into the compound (III)

Similarly to example 32(3), the reaction solution and 30 μl were prepared and kept for 10 minutes at 30°C. However, as a fraction of the supernatant used a fraction of the supernatant obtained in example 67(2) (extract of E. coli pKSN1609F or extract of E. coli pKSN2). These reaction solutions after aging were extracted with ethyl acetate and extracted layers were analyzed using TLC. After developing TLC plates the presence of stains on it, the corresponding connection of the structure (III), labeled14With, investigated (Rf value of 0.24 and 0.29 to). The spot corresponding to the compound (III), were detected from the reaction solution containing the extract of E. coli pKSN1609F. In contrast, this spot is not detected from the reaction solution containing the extract of E. coli pKSN2.

Example 68. The conversion of compounds protein (A16), (A17), (A18), (A19), (A20), (A21), (A22), (A23), (A24) and (A25) this invention

(1) Conversion of the compound (XII) protein (A16) this invention

Received 100 μl of reaction solution of 50 mm potassium phosphate buffer (pH 7.0)containing 12,5 ppm of the compound (XII), 3 mm β-NADPH (hereinafter referred to as "component a") (Oriental Yeast Company), 1 mg/ml ferredoxin obtained from spinach (hereinafter referred to as "component B") (Sigma Company), and 0.15 u/ml paradoxicality (hereinafter referred to as "component C") (Sigma Company) and 20 μl of the supernatant fraction obtained in example 49(2). The reaction solution was kept at 30°C for 10 minutes. In addition, prepared and handled in the same way, 100 μl of reaction solution of 50 mm potassium phosphate buffer (pH 7.0) without adding at least one component used in the above reaction solution, selected from the component A, component B, component C and the supernatant fraction obtained in example 49(2). Added five microliters (5 ál) 2 N. HCl and 100 MK is ethyl acetate in each of these reaction solutions after the specified curing and stirred. The supernatant, centrifuged at 8000 g, was filtered using a filtration device UltraFree MC 0.22 μm (Millipore Company). Forty microliters (40 μl) of the liquid filtrate (the liquid filtrate obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 49(2), called "the solution to (XII)-conversion (A16)"; the liquid filtrate obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 49(2), referred to as "solution (XII)control (A16)") were analyzed using HPLC provided analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A16), the concentration of the compound (XII)detected from the solution to (XII)-conversion (A16), was lower. Then, from the solution to (XII)-conversion (A16) was detected peak, which is not detected from the solution (XII)control (A16). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XII), detektirovanie from the solution to (XII)-convert (A1) in example 41(2).

(2) Conversion of the compound (XII) protein (A17) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 51(2), instead of 20 μl of the supernatant fraction obtained in example 49(2), the reaction solution was obtained and was kept similar to the method described in example 68(1). Similarly to example 68(1)preparing a reaction solution after aging. Forty microliters (40 μl) obtained liquid filtrate (the liquid filtrate obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 51(2), called "the solution to (XII)-conversion (A17)"; the liquid filtrate obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 51(2), referred to as "solution (XII)control (A17)") analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A17), the concentration of the compound (XII)detected from the solution to (XII)-conversion (A17), was lower. Then, from the solution to (XII)-conversion (A17) was detected peak, which is not detected from the solution (XII)control (A17). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XII), detektirovanie from the solution to (XII)-convert (A1) in example 41(2).

(3) Pre the education of the compound (XII) protein (A18) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 53(2), instead of 20 μl of the supernatant fraction obtained in example 49(2), the reaction solution was obtained and was kept similar to the method described in example 68(1). Similarly to example 68(1), was prepared by the reaction solutions after aging. Forty microliters (40 μl) obtained liquid filtrate (the liquid filtrate obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 53(2), called "the solution to (XII)-conversion (A18)"; the liquid filtrate obtained from the reaction solution containing no component a, component B, component C and 20 μl of the supernatant fraction obtained in example 53(2), referred to as "solution (XII)control (A18)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A18), the concentration of the compound (XII)detected from the solution to (XII)-conversion (A18), was lower. Then, from the solution to (XII)-conversion (A18) was detected peak, which is not detected from the solution (XII)control (A18). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the weight specified with the organisations (XII), detektirovanie from the solution to (XII)-convert (A1) in example 41(2).

(4) the Conversion of compound (XII) protein (A19) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 55(2), instead of 20 μl of the supernatant fraction obtained in example 49(2), the reaction solution was obtained and was kept similar to the method described in example 68(1). Similarly to example 68(1), was prepared by the reaction solutions after aging. Forty microliters (40 μl) obtained liquid filtrate (the liquid filtrate obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 55(2), called "the solution to (XII)-conversion (A19)"; the liquid filtrate obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 55(2), referred to as "solution (XII)control (A19)") analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A19), the concentration of the compound (XII)detected from the solution to (XII)-conversion (A19), was lower. Then, from the solution to (XII)-conversion (A19) was detected peak, which is not detected from the solution (XII)control (A19). Time e is of the specified peak on HPLC coincided with the elution peak connections where weight is 14 less than the mass of the compounds (XII), detektirovanie from the solution to (XII)-convert (A1) in example 41(2).

(5) Conversion of the compound (XII) protein (A20) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 57(2), instead of 20 μl of the supernatant fraction obtained in example 49(2), the reaction solution was obtained and was kept similar to the method described in example 68(1). Similarly to example 68(1), was prepared by the reaction solutions after aging. Forty microliters (40 μl) obtained liquid filtrate (the liquid filtrate obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 57(2), called "the solution to (XII)-conversion (A20)"; the liquid filtrate obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 57(2), referred to as "solution (XII)control (A20)") analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A20), the concentration of the compound (XII)detected from the solution to (XII)-conversion (A20), was lower. Then, from the solution to (XII)-conversion (A20) was the detective who Rowan peak, which is not detected from the solution (XII)control (A20). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XII), detektirovanie from the solution to (XII)-convert (A1) in example 41(2).

(6) Conversion of the compound (XII) protein (A21) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 59(2), instead of 20 μl of the supernatant fraction obtained in example 49(2), the reaction solution was obtained and was kept similar to the method described in example 68(1). Similarly to example 68(1), was prepared by the reaction solutions after aging. Forty microliters (40 μl) obtained liquid filtrate (the liquid filtrate obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 59(2), called "the solution to (XII)-conversion (A21)"; the liquid filtrate obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 59(2), referred to as "solution (XII)control (A21)") analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A21), the concentration of the is program (XII), detected from the solution to (XII)-conversion (A21), was lower. Then, from the solution to (XII)-conversion (A21) was detected peak, which is not detected from the solution (XII)control (A21). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XII), detektirovanie from the solution to (XII)-convert (A1) in example 41(2).

(7) Conversion of compound (XII) protein (A22) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 61(2), instead of 20 μl of the supernatant fraction obtained in example 49(2), the reaction solution was obtained and was kept similar to the method described in example 68(1). Similarly to example 68(1), was prepared by the reaction solutions after aging. Forty microliters (40 μl) obtained liquid filtrate (the liquid filtrate obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 61(2), called "the solution to (XII)-conversion (A22)"; the liquid filtrate obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 61(2), referred to as "solution (XII)control (A22)") analyzed using HPLC with at asanam above condition analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A22), the concentration of the compound (XII)detected from the solution to (XII)-conversion (A22)was lower. Then, from the solution to (XII)-conversion (A22) was detected peak, which is not detected from the solution (XII)control (A22). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XII), detektirovanie from the solution to (XII)-convert (A1) in example 41(2).

(8) Conversion of compound (XII) protein (A23) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 63(2), instead of 20 μl of the supernatant fraction obtained in example 49(2), the reaction solution was obtained and was kept similar to the method described in example 68(1). Similarly to example 68(1), was prepared by the reaction solutions after aging. Forty microliters (40 μl) obtained liquid filtrate (the liquid filtrate obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 63(2), called "the solution to (XII)-conversion (A23)"; the liquid filtrate obtained from the reaction solution containing no component a, component B, component C and FR is the Ktsia supernatant, obtained in example 63(2), referred to as "solution (XII)control (A23)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A23), the concentration of the compound (XII)detected from the solution to (XII)-conversion (A23), was lower. Then, from the solution to (XII)-conversion (A23) was detected peak, which is not detected from the solution (XII)control (A23). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XII), detektirovanie from the solution to (XII)-convert (A1) in example 41(2).

(9) Conversion of compound (XII) protein (A24) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 65(2), instead of 20 μl of the supernatant fraction obtained in example 49(2), the reaction solution was obtained and was kept similar to the method described in example 68(1). Similarly to example 68(1), was prepared by the reaction solutions after aging. Forty microliters (40 μl) obtained liquid filtrate (the liquid filtrate obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 65(2), called "the solution to (XII)-transformations the Finance (A24)"; the liquid filtrate obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 65(2), referred to as "solution (XII)control (A24)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A24), the concentration of the compound (XII)detected from the solution to (XII)-conversion (A24), was lower. Then, from the solution to (XII)-conversion (A24) was detected peak, which is not detected from the solution (XII)control (A24). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XII), detektirovanie from the solution to (XII)-convert (A1) in example 41(2).

(10) Conversion of compound (XII) protein (A25) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 67(2), instead of 20 μl of the supernatant fraction obtained in example 49(2), the reaction solution was obtained and was kept similar to the method described in example 68(1). Similarly to example 68(1), was prepared by the reaction solutions after aging. Forty microliters (40 μl) obtained liquid filtrate (the liquid filtrate obtained from the reaction solution, from which containing a series of component a, component B, component C and 20 μl of the supernatant fraction obtained in example 67(2), called "the solution to (XII)-conversion (A25)"; the liquid filtrate obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 67(2), referred to as "solution (XII)control (A25)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XII)detected from the solution (XII)control (A25), the concentration of the compound (XII)detected from the solution to (XII)-conversion (A25), was lower. Then, from the solution to (XII)-conversion (A25) was detected peak, which is not detected from the solution (XII)control (A25). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XII), detektirovanie from the solution to (XII)-convert (A1) in example 41(2).

(11) Conversion of compound (XIII) protein (A17) this invention

Except for the use of 12.5 ppm of the compound (XIII) instead of 12.5 ppm of the compound (XII), the reaction solution was prepared and maintained in accordance with the method described in example 68(2). Similarly to example 68(1), was prepared for each of the reaction solutions after aging. Forty microliters (40 μl) obtained the CSOs liquid filtrate (the liquid filtrate, obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 51(2), called "the solution to (XIII)-conversion (A17)"; the liquid filtrate obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 51(2), referred to as "solution (XIII)control (A17)") was analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XIII)detected from the solution (XIII)control (A17), the concentration of the compound (XIII)detected from the solution to (XIII)-conversion (A17), was lower. Then, from the solution to (XIII)-conversion (A17) was detected peak, which is not detected from the solution (XIII)control (A17). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XIII), detektirovanie from the solution to (XIII)-convert (A1) in example 41(3).

(12) the Conversion of compound (XIII) protein (A18) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 53(2), instead of 20 μl of the supernatant fraction obtained in example 51(2), the reaction solution was received and handled like the method described in the example is 68(11). Similarly to example 68(1), was prepared for each of the reaction solutions after aging. Forty microliters (40 μl) obtained liquid filtrate (the liquid filtrate obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 53(2), called "the solution to (XIII)-conversion (A18)"; the liquid filtrate obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 53(2), referred to as "solution (XIII)control (A18)") analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XIII)detected from the solution (XIII)control (A18), the concentration of the compound (XIII)detected from the solution to (XIII)-conversion (A18), was lower. Then, from the solution to (XIII)-conversion (A18) was detected peak, which is not detected from the solution (XIII)control (A18). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XIII), detektirovanie from the solution to (XIII)-convert (A1) in example 41(3).

(13) Conversion of compound (XIII) protein (A19) this invention

With the exception of using 20 μl of the supernatant fraction, the floor is obtained in example 55(2), instead of 20 μl of the supernatant fraction obtained in example 51(2), the reaction solution was obtained and was kept similar to the method described in example 68(11). Similarly to example 68(1), was prepared for each of the reaction solutions after aging. Forty microliters (40 μl) obtained liquid filtrate (the liquid filtrate obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 55(2), called "the solution to (XIII)-conversion (A19)"; the liquid filtrate obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 55(2), referred to as "solution (XIII)control (A19)") analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XIII)detected from the solution (XIII)control (A19), the concentration of the compound (XIII)detected from the solution to (XIII)-conversion (A19), was lower. Then, from the solution to (XIII)-conversion (A19) was detected peak, which is not detected from the solution (XIII)control (A19). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XIII), detektirovanie from the solution to (XIII)-convert (A1) in the use of the s 41(3).

(14) the Conversion of compound (XIII) protein (A20) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 57(2), instead of 20 μl of the supernatant fraction obtained in example 51(2), the reaction solution was obtained and was kept similar to the method described in example 68(11). Similarly to example 68(1), was prepared for each of the reaction solutions after aging. Forty microliters (40 μl) obtained liquid filtrate (the liquid filtrate obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 57(2), called "the solution to (XIII)-conversion (A20)"; the liquid filtrate obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 57(2), referred to as "solution (XIII)control (A20)") analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XIII)detected from the solution (XIII)control (A20), the concentration of the compound (XIII)detected from the solution to (XIII)-conversion (A20), was lower. Then, from the solution to (XIII)-conversion (A20) was detected peak, which is not detected from the solution (XIII)control (A20). Elution of the specified peak on HPLC coincided with the time to ale the AI peak connections where weight is 14 less than the mass of the compounds (XIII), detektirovanie from the solution to (XIII)-convert (A1) in example 41(3).

(15) the Conversion of compound (XIII) protein (A21) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 59(2), instead of 20 μl of the supernatant fraction obtained in example 51(2), the reaction solution was obtained and was kept similar to the method described in example 68(11). Similarly to example 68(1), was prepared for each of the reaction solutions after aging. Forty microliters (40 μl) obtained liquid filtrate (the liquid filtrate obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 59(2), called "the solution to (XIII)-conversion (A21)"; the liquid filtrate obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 59(2), referred to as "solution (XIII)control (A21)") analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XIII)detected from the solution (XIII)control (A21), the concentration of the compound (XIII)detected from the solution to (XIII)-conversion (A21), was lower. Then, from the solution to (XIII)is converted to the I (A21) was detected peak, which is not detected from the solution (XIII)control (A21). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XIII), detektirovanie from the solution to (XIII)-convert (A1) in example 41(3).

(16) the Conversion of compound (XIII) protein (A23) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 63(2), instead of 20 μl of the supernatant fraction obtained in example 51(2), the reaction solution was obtained and was kept similar to the method described in example 68(11). Similarly to example 68(1), was prepared for each of the reaction solutions after aging. Forty microliters (40 μl) obtained liquid filtrate (the liquid filtrate obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 63(2), called "the solution to (XIII)-conversion (A23)"; the liquid filtrate obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 63(2), referred to as "solution (XIII)control (A23)") analyzed by HPLC under the above condition analysis 1. Compared with the concentration of the compound (XIII)detected from the solution (XIII)control (A23), end of entrace compounds (XIII), detected from the solution to (XIII)-conversion (A23), was lower. Then, from the solution to (XIII)-conversion (A23) was detected peak, which is not detected from the solution (XIII)control (A23). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XIII), detektirovanie from the solution to (XIII)-convert (A1) in example 41(3).

(17) the Conversion of compound (XIII) protein (A25) this invention

With the exception of using 20 μl of the supernatant fraction obtained in example 67(2), instead of 20 μl of the supernatant fraction obtained in example 51(2), the reaction solution was obtained and was kept similar to the method described in example 68(11). Similarly to example 68(1), was prepared for each of the reaction solutions after aging. Forty microliters (40 μl) obtained liquid filtrate (the liquid filtrate obtained from the reaction solution containing component a, component B, component C and 20 μl of the supernatant fraction obtained in example 67(2), called "the solution to (XIII)-conversion (A25)"; the liquid filtrate obtained from the reaction solution containing no component a, component B, component C and the supernatant fraction obtained in example 67(2), referred to as "solution (XIII)control (A25)") analyzed with OSU HPLC under the above condition analysis 1. Compared with the concentration of the compound (XIII)detected from the solution (XIII)control (A25), the concentration of the compound (XIII)detected from the solution to (XIII)-conversion (A25), was lower. Then, from the solution to (XIII)-conversion (A25) was detected peak, which is not detected from the solution (XIII)control (A25). Elution of the specified peak on HPLC coincided with the elution peak of the connection, in which the mass is 14 less than the mass of the compounds (XIII), detektirovanie from the solution to (XIII)-convert (A1) in example 41(3).

Example 69. Hybridization in which DNA (A1), (A2), (A3) or (A4) of this invention was probe

(1) Receiving probe

PCR was carried out according to the method described in example 30(1). However, as the matrix used 10 ng of chromosomal DNA of Streptomyces achromogenes IFO 12735, obtained in example 26(1), instead of above 50 ng of chromosomal DNA of Streptomyces phaeochromogenes IFO 12898, obtained in example 3(1). As primers used oligonucleotide having the nucleotide sequence shown in SEQ ID NO:328, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:329. DNA amplified using the PCR was extracted with obtaining a probe having the nucleotide sequence shown in SEQ ID NO:109, labeled digoxigenin (hereinafter called the so called "DIG-labeled probe (A4)").

(2) obtaining the solution of plasmid

PCR was performed using a mixture of genomic polymerase Advantage-GC (Clontech Company) and using as the template the chromosomal DNA of Streptomyces nogalator IFO 13445 obtained in example 31(1). As primers used oligonucleotide having the nucleotide sequence shown in SEQ ID NO:330, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:331. The reaction solution for PCR was brought up to 50 μl by adding each of the 2 primers, brought to 200 nm, 10 ng of chromosomal DNA of 4.0 μl dNTP mixture (a mixture of 2.5 mm each of the 4 types of dNTP; Clontech Company), 10,0 ál 5GC buffer with 2.2 μl of 25 mm Mg(OAc)2, 10,0 ál of 5 M GC-melt and 1.0 μl of a mixture of genomic polymerase Advantage-GC (Clontech Company) and distilled water. The PCR conditions were as follows: after the maintenance of 94°C for 1 minute, repeat 7 cycles, where each cycle consisted of maintaining 94°C for 10 seconds, and then 72°C for 3 minutes; repeat 36 cycles, where each cycle consisted of maintaining 94°C for 10 seconds and then 67°C for 3 minutes; and then maintaining the 67°C for 7 minutes. DNA was purified from the reaction solution for PCR using the cleaning kit QIAquick PCR (Qiagen Company) in accordance with the attached to this set by the automatic disconnection. The resulting DNA ligated to the cloning vector TA pCR2.1 (Invitrogen Company) in accordance with the PR, the proposed guidance and introduced into E. coli TOP10F' (Invitrogen Company). Plasmid DNA was obtained from the received transformant E. coli using the kit QIAprep Spin Miniprep (Qiagen Company) to obtain the solution of plasmid containing DNA (A11) this invention.

Similarly, PCR was performed using as a template the chromosomal DNA of Streptomyces tsusimaensis IFO 13782 obtained in example 33(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:332, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:333. DNA obtained using this PCR, ligated with the vector, as described above. Then was used to transform E. coli. From the received transformant E. coli was obtained plasmid to obtain a solution of a plasmid containing DNA (A12) this invention.

Similarly, PCR was performed using as a template the chromosomal DNA of Streptomyces thermocoerulesces IFO 14273t obtained in example 35(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:331, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:334. DNA obtained using this PCR, ligated with the vector, as described above. Then was used to transform E. coli. From the received transformant E. coli was obtained plasmid to obtain the Astor plasmids, containing DNA (A13) this invention.

Similarly, PCR was performed using as a template the chromosomal DNA of Streptomyces glomerulochromogenes IFO 13673t obtained in example 37(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:330, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:331. DNA obtained using this PCR, ligated with the vector, as described above. Then was used to transform E. coli. From the received transformant E. coli was obtained plasmid to obtain a solution of a plasmid containing DNA (A14) this invention.

Similarly, PCR was performed using as a template the chromosomal DNA of Streptomyces olivochromogenes IFO 12444, obtained in example 39(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:330, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:331. DNA obtained using this PCR, ligated with the vector, as described above. Then was used to transform E. coli. From the received transformant E. coli was obtained plasmid to obtain a solution of a plasmid containing DNA (A15) this invention.

Similarly, PCR was performed using as a template the chromosomal DNA Streptoyces ornatus IFO 13069t, obtained in example 48(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:335, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:336. DNA obtained using this PCR, ligated with the vector, as described above. Then was used to transform E. coli. From the received transformant E. coli was obtained plasmid to obtain a solution of a plasmid containing DNA (A16) this invention.

Similarly, PCR was performed using as a template the chromosomal DNA of Streptomyces griseus ATCC 10137 obtained in example 50(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:335, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:336. DNA obtained using this PCR, ligated with the vector, as described above. Then was used to transform E. coli. From the received transformant E. coli was obtained plasmid to obtain a solution of a plasmid containing DNA (A17) this invention.

Similarly, PCR was performed using as a template the chromosomal DNA of Streptomyces achromogenes IFO 12735 obtained in example 52(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ IDNO:330, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:331. DNA obtained using this PCR, ligated with the vector, as described above. Then was used to transform E. coli. From the received transformant E. coli was obtained plasmid to obtain a solution of a plasmid containing DNA (A18) this invention.

Similarly, PCR was performed using as a template the chromosomal DNA of Streptomyces griseus IFO 13849T obtained in example 54(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:333, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:335. DNA obtained using this PCR, ligated with the vector, as described above. Then was used to transform E. coli. From the received transformant E. coli was obtained plasmid to obtain a solution of a plasmid containing DNA (A19) this invention.

Similarly, PCR was performed using as a template the chromosomal DNA of Streptomyces lanatus IFO 12787T obtained in example 56(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:331, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:337. DNA obtained using this PCR, ligated with the vector an is logically described above. Then was used to transform E. coli. From the received transformant E. coli was obtained plasmid to obtain a solution of a plasmid containing DNA (A20) this invention.

Similarly, PCR was performed using as a template the chromosomal DNA of Streptomyces misawanensis IFO T obtained in example 58(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:331, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:338. DNA obtained using this PCR, ligated with the vector, as described above. Then was used to transform E. coli. From the received transformant E. coli was obtained plasmid to obtain a solution of a plasmid containing DNA (A21) of the present invention.

Similarly, PCR was performed using as a template the chromosomal DNA of Streptomyces roseorubens IFO 13682T obtained in example 62(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:331, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:339. DNA obtained using this PCR, ligated with the vector, as described above. Then was used to transform E. coli. From the received transformant E. coli was obtained plasmid to obtain a solution of a plasmid containing DNA (A23) this is about inventions.

Similarly, PCR was performed using as a template the chromosomal DNA of Streptomyces steffisburgensis IFO 13446T obtained in example 66(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:331, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:339. DNA obtained using this PCR, ligated with the vector, as described above. Then was used to transform E. coli. From the received transformant E. coli was obtained plasmid to obtain a solution of a plasmid containing DNA (A25) this invention.

Further, similarly, PCR was performed using as a template the chromosomal DNA of Streptomyces pallidus IFO 13434T obtained in example 60(1), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:340, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:341. DNA obtained using this PCR, ligated with the vector, as described above. Then was used to transform E. coli. From the received transformant E. coli was obtained plasmid to obtain a solution of a plasmid containing DNA (A22) this invention.

Similarly, PCR was performed using as a template the chromosomal DNA of Streptomyces rutgersensis IFO 15875T obtained in example 64(1),and using as primers the oligonucleotide, having the nucleotide sequence shown in SEQ ID NO:342, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:343. DNA obtained using this PCR, ligated with the vector, as described above. Then was used to transform E. coli. From the received transformant E. coli was obtained plasmid to obtain a solution of a plasmid containing DNA (A24) this invention.

(2) Dot-blot-hybridization

Approximately 100 ng and 10 ng of each plasmid obtained in example 69(2), struck by blotting on a nylon membrane Hybond N+ (Amersham Biosciences Company). These plasmids were: plasmid DNA containing DNA (A11) of the present invention, plasmid DNA containing DNA (A12) of the present invention, plasmid DNA containing DNA (A13) of the present invention, plasmid DNA containing DNA (A14) of the present invention, plasmid DNA containing DNA (A15) of the present invention, plasmid DNA containing DNA (A16) of the present invention, plasmid DNA containing DNA (A17) of the present invention, plasmid DNA containing DNA (A18) of the present invention, plasmid DNA containing DNA (A19) the present invention, plasmid DNA containing DNA (A20) of the present invention, plasmid DNA containing DNA (A21) of the present invention, plasmid DNA containing DNA (A23) of the present invention, plasmid DNA containing DNA (A25) and this is gaining. Ultraviolet light was directed on the obtained membrane using transilluminator within 5 minutes.

Hybridization and detection was performed in accordance with the method described in example 30(2). The probes obtained in example 30(1), kept at 100°C for 5 minutes and then cooled on ice. As the probes used DNA having the nucleotide sequence shown in SEQ ID NO:6, labeled digoxigenin (hereinafter called “DIG-labeled probe (A1)”), DNA having the nucleotide sequence shown in SEQ ID NO:7, labeled digoxigenin (hereinafter called “DIG-labeled probe (A2)”), DNA having the nucleotide sequence shown in SEQ ID NO:8, labeled digoxigenin (hereinafter called “DIG-labeled probe (A3)”), or DIG-labeled probe (A4)obtained in example 69(1). When using any of DIG-labeled probes (A1), (A2), (A3) or (A4) for hybridization signal was detected for each of these reagents at 10 ng and 100 ng of each of the above plasmid DNA.

Further, similarly, approximately 10 ng and 100 ng of each plasmid DNA containing DNA (A22) of this invention obtained in example 69(2), and plasmid DNA containing DNA (A24) of the present invention, inflicted by blotting on a nylon membrane Hybond N+ (Amersham Biosciences Company). Hybridization and detection was performed in the accordance with example 30(2).

Example 70. Obtaining DNA (A23) of the present invention, in which the use of codons was adapted for expression in soybean (hereinafter referred to as "DNA (A23)S of the present invention"))

(1) Obtaining DNA (A23)S of this invention

PCR was performed with DNA polymerase, Pyrobest (Takara Shuzo Company) in accordance with the attached manual, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:346, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:367. An aliquot of the obtained PCR product was used as template for PCR carried out in the same way, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:345, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:366. Next, an aliquot of this PCR product was used as template for PCR carried out in the same way, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:344, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:365. The resulting reaction solution was named the reaction solution 1.

Next, PCR was performed with DNA polymerase, Pyrobest (Takara Shuzo Company) in accordance with primegenerator, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:349, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:364. An aliquot of the obtained PCR product was used as template for PCR carried out in the same way, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:348, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:363. Next, an aliquot of this PCR product was used as template for PCR carried out in the same way, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:347, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:362. The resulting reaction solution was named the reaction solution 2.

Next, PCR was performed with DNA polymerase, Pyrobest (Takara Shuzo Company) in accordance with the attached manual, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:352, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:361. An aliquot of the obtained PCR product was used as template for PCR carried out in the same way IP is the use as primers the oligonucleotide, having the nucleotide sequence shown in SEQ ID NO:351, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:360. Next, an aliquot of this PCR product was used as template for PCR carried out in the same way, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:350, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:359. The resulting reaction solution was named the reaction solution 3.

Next, PCR was performed with DNA polymerase, Pyrobest (Takara Shuzo Company) in accordance with the attached manual, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:355, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:358. An aliquot of the obtained PCR product was used as template for PCR carried out in the same way, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:354, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:357. Next, an aliquot of this PCR product was used as template for PCR carried out in the same way, using as primers the oligonucleotide, have its nucleotide sequence, shown in SEQ ID NO:353, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:356. The resulting reaction solution was named the reaction solution 4.

The reaction solutions 1 to 4, thus obtained, was mixed. PCR was performed with DNA polymerase, Pyrobest (Takara Shuzo Company) in accordance with the attached manual, using as matrix aliquots of this mixture and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:344, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:356. The nucleotide sequence of this amplified DNA was confirmed. Was obtained DNA having the sequence in which the nucleotide sequence 5'-cat-3' attached to the left from the 5'-end and the nucleotide sequence 5'-aagctt-3' attached to the right of the 3'end of the nucleotide sequence shown in SEQ ID NO:368.

The use of the codons of the DNA (A23) of the present invention having the nucleotide sequence shown in SEQ ID NO:232 (GC-content 73,10%), shown in table 28 and table 29. The use of codons soybean (GC-content 46,12%) shown in table 24 and table 25. The use of the codons of the DNA (A23)S of the present invention having the nucleotide sequence shown in SEQ ID NO:368 (GC-content 52,38%)shown in table 30 and table 31.

Table 28
codon%codon%
TTT0,00TCT0,00
TTC4,01TCC1,50
TTA0,00TCA0,00
TTG0,00TCG0,50
CTT0,00CCT0,00
CTC4.26 deathsCCC5,76
HUNDRED0,00CCA0,00
CTGto 7.77CCGof 2.26
ATT0,00ACT 0,00
ATS4,51ACC3,76
ATA0,00ASA0,00
ATGof 2.26ACGwas 2.76
GTT0,00GCT0,25
GTC3,51GCC9,27
GTA0,00GCA0,75
GTGof 2.51GCG1,75

Table 29
codon%codon%
TAT0,00TGT0,00
TAC1,00 TGC0,75
TAA0,25TGA0,00
TAG0,00TGG0,75
CAT0,00CGT0,50
CACof 2.26CGC6,02
CAA0,50CGA0,25
CAGof 2.51CGG3,01
AAT0,00AGT0,00
AAC1,00AGC1,25
AAA0,25AGA0,00
AAG0,50AGG0,50
GAT 0,00GGT0,98
GAC7,27GGC6,27
GAA1,25GGA0,25
GAG5,26GGG1,00

Table 30
codon%codon%
TTT2,01TCT0,75
TTC2,01TCC0,50
TTA1,00TCA0,75
TTG3,01TCG0,25
CTT3,26CCT3,01
CTC of 2.26CCC1,50
HUNDRED1,00CCA3,01
CTG1,50CCG0,50
ATTof 2.26ACTof 2.26
ATS1,25ACC1,75
ATA1,00ASA2,01
ATGof 2.26ACG0,50
GTTof 2.26GCT4,51
GTC1,00GCCwas 2.76
GTA0,75GCA3,76
GTG2,01GCG 1,00

Table 31
codon%codon%
TAT0,50TGT0,25
TAC0,50TGC0,50
TAA0,25TGA0,00
TAG0,00TGG0,75
CAT1,25CGT1,50
CAC1,00CGC1,25
CAA1,75CGA0,75
CAG1,25CGG0,50
AAT0,50AGT 0,50
AAC0,50AGC0,50
AAA0,25AGA3,26
AAG0,50AGG3,01
GAT4,51GGTof 2.26
GACwas 2.76GGC1,50
GAA3,26GGAof 2.26
GAG3,26GGG1,50

(2) Obtaining transformed E. coli with protein (A23)S of this invention

DNA having the nucleotide sequence shown in SEQ ID NO:368, obtained in example 70(1), was digested with restrictase NdeI and HindIII. The resulting DNA and plasmid pKSN2, split NdeI and HindIII, ligated to obtain plasmid in which the DNA having the nucleotide sequence shown in SEQ ID NO:368, inserted between the NdeI site by iaitam HindIII pKSN2 (hereinafter called "pKSN1584soy"). The indicated plasmid was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN1584soy.

(3) the expression of the protein (A23) of the present invention in E. coli and the selection of the specified protein

Analogously to example 4(2), cultivated each of E. coli JM109/pKSN1584soy obtained in example 70(2), and E. coli JM109/pKSN1584F obtained in example 63(1). The cells were extracted. Received solutions of cell lysates. According to the method described in example 4(2), received a fraction of supernatants of these solutions cell lysates (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN1584soy, called “extract of E. coli pKSN1584soy”, and the supernatant fraction obtained from E. coli JM109/pKSN1584F, called “extract of E. coli pKSN1584F”). The number of P450 in the amount of protein contained in the extract of E. coli pKSN1584soy, compared with the number of P450 in the amount of protein contained in the extract of E. coli pKSN1584F, and it was higher than the number of P450 in the amount of protein contained in the extract of E. coli pKSN1584F.

Example 71. The acquisition and expression of DNA (A25) of the present invention, in which the use of codons was adapted for expression in soybean (hereinafter referred to as "DNA (A25)S of the present invention")

(1) Obtaining DNA (A25)S of this invention

PCR was performed with DNA polymerase, Pyrobest (Takara Shuzo Company) in accordance with the attached manual, using as primers the oligonucleotide, and housego nucleotide sequence, shown in SEQ ID NO:371, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:392. An aliquot of the obtained PCR product was used as template for PCR carried out in the same way, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:370, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:391. Next, an aliquot of this PCR product was used as template for PCR carried out in the same way, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:369, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:390. The resulting reaction solution was named the reaction solution 1.

Next, PCR was performed with DNA polymerase, Pyrobest (Takara Shuzo Company) in accordance with the attached manual, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:374, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:389. An aliquot of the obtained PCR product was used as template for PCR carried out in the same way, using as primers the oligonucleotide having the nucleotide sequence until annoy in SEQ ID NO:373, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:383. Next, an aliquot of this PCR product was used as template for PCR carried out in the same way, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:372, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:387. The resulting reaction solution was named the reaction solution 2.

Next, PCR was performed with DNA polymerase, Pyrobest (Takara Shuzo Company) in accordance with the attached manual, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:377, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:386. An aliquot of the obtained PCR product was used as template for PCR carried out in the same way, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:376, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:385. Next, an aliquot of this PCR product was used as template for PCR carried out in the same way, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:375, and ol is gonucleotide, having the nucleotide sequence shown in SEQ ID NO:384. The resulting reaction solution was named the reaction solution 3.

Next, PCR was performed with DNA polymerase, Pyrobest (Takara Shuzo Company) in accordance with the attached manual, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:380, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:383. An aliquot of the obtained PCR product was used as template for PCR carried out in the same way, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:379, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:382. Next, an aliquot of this PCR product was used as template for PCR carried out in the same way, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:378, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:381. The resulting reaction solution was named the reaction solution 4.

The reaction solutions 1 to 4, thus obtained, was mixed. PCR was performed with DNA polymerase, Pyrobest (Takara Shuzo Company) in accordance with the attached manual, using ka is este matrix aliquots of this mixture and using as primers the oligonucleotide, having the nucleotide sequence shown in SEQ ID NO:369, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:381. The nucleotide sequence of this amplified DNA was confirmed. Was obtained DNA having the sequence in which the nucleotide sequence 5'-cat-3' attached to the left from the 5'-end and the nucleotide sequence 5'-aagctt-3' attached to the right of the 3'end of the nucleotide sequence shown in SEQ ID NO:393.

The use of the codons of the DNA (A25) this invention having the nucleotide sequence shown in SEQ ID NO:234 (GC-content 71,93%)shown in table 32 and table 33. The use of codons soybean (GC-content 46,12%) shown in table 24 and table 25. The use of the codons of the DNA (A25)S of the present invention having the nucleotide sequence shown in SEQ ID NO:393 (GC-content 52,05%), shown in table 34 and table 35.

TCC
Table 32
codon%codon%
TTT0,00TCT0,00
TTC3,761,25
TTA0,00TCA0,25
TTG0,00TCG0,75
CTT0,00CCT0,25
CTC4,01CCC4,01
HUNDRED0,00CCA0,25
CTG9,52CCGwas 2.76
ATT0,00ACT0,25
ATS4.26 deathsACC4,01
ATA0,25ASA0,00
ATGof 2.26ACG1,75
GTT 0,00GCT0,00
GTC3,01GCCcharged 8.52
GTA0,00GCA0,50
GTGof 2.51GCG3,01

Table 33
codon%codon%
TAT0,00TGT0,25
TAC1,25TGC0,50
TAA0,25TGA0,00
TAG0,00TGG1,00
CAT0,25CGT0,75
CAC of 2.26CGC5,51
CAA0,00CGA1,25
CAG3,01CGG3,26
AAT0,00AGT0,00
AAC1,00AGC1,00
AAA0,25AGA0,25
AAG1,00AGG0,00
GAT0,00GGT0,25
GAC7,52GGC4,76
GAA1,00GGA0,25
GAG4,76GGG

Table 34
codon%codon%
TTT1,75TCT1,25
TTC2,01TCC0,50
TTA1,25TCA0,50
TTG3,26TCG0,00
CTT3,51CCTwas 2.76
CTCof 2.51CCC1,25
HUNDRED1,25CCAwas 2.76
CTG1,75CCG0,50
ATTof 2.26ACT 2,01
ATS1,25ACC1,75
ATA1,00ASA1,75
ATGof 2.26ACG0,50
GTTof 2.26GCT4,51
GTC1,00GCCwas 2.76
GTA0,50GCA3,76
GTG1,75GCG1,00

Table 35
codon%codon%
TAT0,50TGT0,25
TAC0,7 TGC0,50
TAA0,25TGA0,00
TAG0,00TGG1,00
CAT1,25CGT1,75
CAC1,25CGC1,50
CAA1,50CGA0,75
GAG1,50CGG0,75
AAT0,50AGT0,50
AAC0,50AGC0,50
AAA0,50AGA3,26
AAG0,75AGG3,01
GAT4,76GGT2,01
GACwas 2.76GGC1,25
GAAwas 2.76GGA2,01
GAG3,01GGG1,25

(2) Obtaining transformed E. coli with protein (A25)S of this invention

DNA having the nucleotide sequence shown in SEQ ID NO:393, obtained in example 71(1), was digested with restrictase NdeI and HindIII. The resulting DNA and plasmid pKSN2, split NdeI and HindIII, ligated to obtain plasmid in which the DNA having the nucleotide sequence shown in SEQ ID NO:393, inserted between the NdeI site and the HindIII site pKSN2 (hereinafter called "pKSN1609soy"). The indicated plasmid was introduced into E. coli JM109. The obtained transformant E. coli was named JM109/pKSN1609soy.

(3) the expression of the protein (A25) of the present invention in E. coli and the selection of the specified protein

Analogously to example 4(2), cultivated each of E. coli JM109/pKSN1609soy obtained in example 71(2), and E. coli JM109/pKSN1609F obtained in example 67(1). The cells were extracted. Received solutions of cell lysates. By the way, opican the mu in example 4(2), received a fraction of supernatants of these solutions cell lysates (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN1609soy, called “extract of E. coli pKSN1609soy”, and the supernatant fraction obtained from E. coli JM109/pKSN1609F, called “extract of E. coli pKSN1609F”). The number of P450 in the amount of protein contained in the extract of E. coli pKSN1609soy, compared with the number of P450 in the amount of protein contained in the extract of E. coli pKSN1609F, and it was higher than the number of P450 in the amount of protein contained in the extract of E. coli pKSN1609F.

Example 72. Obtaining antibodies (A) of the present invention, capable of recognizing protein (A25) of the present invention (hereinafter referred to as antibody (A25) of this invention")

(1) Obtaining an extract of E. coli expressing the protein (A25) this invention

In accordance with the method described in example 4(2), E. coli JM109/pKSN1609soy obtained in example 71(2), pre-cultured overnight. The obtained culture medium was inoculable in 1 l medium TV containing 50 μg/ml of ampicillin, and cultured at 26aboutC. Then was added 5-aminolevulinate acid to a final concentration of 500 μm was added IPTG to a final concentration of 1 mm and additionally cultivated. Cells were removed from culture medium, washed with 0.05 M Tris-HCl-buffer (pH 7.5) and then suspended in 100 ml of the specified buffer, soteriades is 1 mm PMSF. The obtained culture medium cells were subjected to 3 times the processing on the ultrasonic disintegrator (Sonifier (Branson Sonic Power Company)) for 10 minutes each time in terms of radiated power 5, load cycle 30%, to obtain solutions of cell lysates. After centrifugation of the solutions of cell lysates (9000 g, 10 min) supernatant were removed and centrifuged (200000 g, 70 minutes) to obtain fractions of supernatant (hereinafter the supernatant fraction obtained from E. coli JM109/pKSN1609soy, called “extract of E. coli pKSN1609soy”).

(2) Purification of protein (A25) this invention

The supernatant fraction obtained in example 72(1) (extract of E. coli pKSN1609soy) was injected into the column Hiload HiLoad16/10 Q Sepharose HP (Amersham Bioscience Company). Then, after passing 40 ml of 20 mm bestreplibng buffer (pH 7.0) through this column, 20 mm listresponse buffer proceeded with a linear gradient of NaCl (NaCl gradient was 0,00125 M/min, the concentration range of NaCl was from 0 M to 0,375 M, the flow rate was 3 ml/min) for the fractional extraction of 10 ml fractions, eluruumiks when NaCl concentration 0,088-0,100 M

The extracted fractions were subjected to purification by column PD-10 (Amersham Bioscience Company) and was suirable 20 mm mistresspopular buffer (pH 7.0) to extract the fractions containing protein. Then these fractions were introduced in MonoQ HR 10/10 (Amersham Bioscience Company). Sixteen milliliters (16 ml) 20 mm bestreplibng buffer passed is through the column. Then 20 mm listresponse buffer proceeded with a linear gradient of NaCl (NaCl gradient was 0,001042 M/min, the concentration range of NaCl was from 0 to 0.25 M, the flow rate was 4 ml/min) for fractional extraction 8 ml fractions, eluruumiks when NaCl concentration from up to 0.060 0,069 M

The extracted fractions were diluted 2.5 times with 20 mm mistresspopular buffer (pH 7.0) and injected into the column MonoQ HR 5/5 (Amersham Bioscience Company). Then, after passing 2 ml of 20 mm bestreplibng buffer (pH 7.0) through a column of 20 mm listresponse buffer proceeded with a linear gradient of NaCl (NaCl gradient was 0,008333 M/min, the concentration range of NaCl was from 0 M to 0.25 M, the flow rate was 1 ml/min) for the fractional extraction of 0.5 ml fractions, eluruumiks when NaCl concentration 0,073-0,077 M

Fractions, refined, therefore, were analyzed by electrophoresis in LTO-SDS page using “PAG mini Daiichi 10/20” (Daiichi Pure Chemicals Co., Ltd.) to confirm that these fractions were factions that contain mostly protein (A25) this invention.

(3) Obtaining antibodies (A25) this invention

Obtaining antibodies of the present invention was carried out according to the method described in example 44(3). However, instead of using protein (A1) of the present invention used protein (A25) of this invention obtained in example 72(2), obtaining antisera containing an antibody (A25) this izaberete the Oia.

Example 73. Detection of the protein of the present invention antibody (A25) this invention

The immunoblot analysis was performed using antibodies (A25) of this invention obtained in example 72(3), with each of the extracts of E. coli. Electrophoresis was performed in LTO-polyacrylamide (40 mA, 1 hour): extract of E. coli pKSN452F obtained in example 49(2) (containing about 2 pmol of the protein (A16) of the present invention); an extract of E. coli pKSN608F obtained in example 51(2) (containing about 2 pmol of the protein (A17) of the present invention); an extract of E. coli pKSN646F obtained in example 53(2) (containing about 2 pmol of the protein (A18) of the present invention); an extract of E. coli pKSN1502F obtained in example 55(2) (containing about 2 pmol of the protein (A19) of the present invention); an extract of E. coli pKSN1525F obtained in example 57(2) (containing about 2 pmol of the protein (A20) of the present invention); an extract of E. coli pKSN1543BF obtained in example 59(2) (containing about 2 pmol of the protein (A21) of the present invention); an extract of E. coli pKSN1558F obtained in example 61(2) (containing about 2 pmol of the protein (A22) of the present invention); an extract of E. coli pKSN1584F obtained in example 63(2) (containing about 2 pmol of the protein (A23) of the present invention); an extract of E. coli pKSN1589F obtained in example 65(2) (containing about 2 pmol of the protein (A24) this image is etenia); extract of E. coli pKSN1609F obtained in example 67(2) (containing about 0.5 pmol of protein (A25) of the present invention), an extract of E. coli pKSN1584soy obtained in example 70(3) (containing about 2 pmol of the protein (A23) of the present invention), an extract of E. coli pKSN1609soy obtained in example 71(3) (containing approximately 0.5 pmol of protein (A25) of the present invention) and an extract of E. coli pKSN2 obtained in example 67(2) (containing about 0.8 mg of protein). Proteins specified in the gel were transferred to PVDF-membrane in accordance with the method described in example 45. PDVF membrane obtained in example 45 (hereinafter called "PDVF-membrane (A)"), and the PDVF membrane obtained by the above method (hereinafter referred to as "PDVF-membrane (In)")were subjected to reaction with anticorodal obtained in example 72(3), in accordance with the method described in example 45. After that carried out the reaction with the second antibody, washing and dyeing in accordance with the method described in example 45. Staining of the bands corresponding to proteins of this invention (A1), (A2), (A3), (A4), (A11), (A12), (A13), (A14) and (A15), and proteins (A9) and (a10) this invention were detected on PDVF-membrane (A). Staining of the bands corresponding to proteins of this invention (A16), (A17), (A18), (A19), (A20), (A21), (A22), (A23), (A24) and (A25), were detected on PDVF-membrane (). Not detected staining reagent strip with ek the tract E. coli pKSN2 obtained in example 4(2) (containing about 0,78 mg protein), PVDF-membrane (a) and reagent extract of E. coli pKSN2 obtained in example 67(2) (containing 0.8 mg of protein), PVDF-membrane (B).

Example 74. Introduction DNA (A23)S of the present invention in a plant

(1) Construction of chloroplast expression plasmids containing DNA (A23)S of the present invention, for direct introduction - part 1

A plasmid containing a chimeric DNA in which DNA (A23)S of this invention was added immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons, was designed as a plasmid for introduction of DNA (A23)S of the present invention in a plant method using guns particles.

First, a DNA containing the nucleotide sequence shown in SEQ ID NO:398, amplified using PCR. PCR was performed using as matrix pKSN1584soy obtained in example 70(2), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:397, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:398. In PCR were using KOD-plus (Toyobo Company). PCR was performed after maintenance once 94°C for 2 minutes; the conduct is of 20 cycles, where each cycle consisted of maintaining 94°C for 30 seconds, then 53°C for 30 seconds and then 68°C for 90 seconds; and final maintenance at 68°C for 3 minutes. Amplified DNA was isolated and purified using MagExtractor-PCR &Gel Clean up (Toyobo Company) carrying out procedures in accordance with the attached manual. The processing of the resulting DNA using a set of TaKaRa BKL (Takara Shuzo Company) in accordance with the attached guidance, DNA was a small mistake and the 5'-end was fosforilirovanii. DNA containing the nucleotide sequence shown in SEQ ID NO:368, were isolated. After cleavage of plasmid pUC19 (Takara Shuzo Company) by the restriction enzyme SmaI 5'-end dephosphorylated alkaline phosphatase calf intestine (Takara Shuzo). Plasmid was obtained by legirovaniem received dephosphorylating DNA and DNA containing the nucleotide sequence shown in SEQ ID NO:368. After splitting the resulting plasmid by restrictase Eat and SacI were isolated DNA containing the nucleotide sequence shown in SEQ ID NO:368. After cleavage of plasmid pUCrSt657 obtained in example 16(2), restrictase EcoT22I and SacI were isolated DNA approximately 2,9 TPN having a nucleotide sequence derived from pUC19, and a sequence encoding a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack). The obtained DNA and the above-mentioned DNA, terzidou nucleotide sequence, shown in SEQ ID NO:368, ligated with getting pUCrSt1584soy (Fig. 54)containing a chimeric DNA in which DNA (A23)S of the present invention is attached immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons.

The obtained plasmid pUCrSt1584soy were digested with restrictase BamHI and SacI for DNA containing the nucleotide sequence shown in SEQ ID NO:368. Specified DNA was embedded between the restriction site BglII and restriction site SacI plasmids pNdG6-ΔT obtained in example 16(2), to obtain plasmid pSUM-NdG6-rSt-1584soy (Fig. 55), where the promoter CR16G6 attached right from the chimeric DNA in which the DNA is attached immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons.

Then, this plasmid was introduced into competent cells of E. coli DH5α (Takara Shuzo Company) and were selected by ampicillin-resistant cells. Further, the nucleotide sequence of the plasmid contained in these ampicillin-resistant strains, was determined using kit BigDye Terminator Cycle Sequencing Ready Reaction kit v3.0 (PE Applied Biosystems Company) and DNA sequencing machine 3100 (PE Applied Biosystems Company). In the result, it was confirmed that the plasmid pSUM-NdG6-rSt-1584soy have the t nucleotide sequence, shown in SEQ ID NO:368.

(2) Construction of chloroplast expression plasmids containing DNA (A23)S of the present invention, for direct introduction - part 2

Designed plasmid for introduction of DNA (A23)S of the present invention in a plant method using guns particles. This plasmid contained a chimeric DNA in which DNA (A23)S of this invention was added immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without altering the reading frames in the codons. First, a DNA containing the nucleotide sequence shown in SEQ ID NO:368, amplified using PCR. PCR was performed using as matrix pKSN1584soy obtained in example 70, using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:399, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:398. In PCR were using KOD-plus (Toyobo Company). PCR was performed after maintaining 94°C once for 2 minutes as follows: holding of 25 cycles, where each cycle consisted of maintaining 94°C for 30 seconds, then 46°C for 30 seconds and then 68°C for 90 seconds; and final maintenance 68°C for 3 mi the ut. Amplified DNA was isolated and purified using MagExtractor-PCR &Gel Clean up (Toyobo Company) carrying out procedures in accordance with the attached manual. The processing of the resulting DNA using a set of TaKaRa BKL (Takara Shuzo Company) in accordance with the attached guidance, DNA was a small mistake and the 5'-end was fosforilirovanii. DNA containing the nucleotide sequence shown in SEQ ID NO:368, were isolated. After cleavage of plasmid pKF19 ΔBs, obtained in example 15(3), the restriction enzyme SmaI 5'-end dephosphorylated alkaline phosphatase calf intestine (Takara Shuzo). Plasmid was obtained by legirovaniem received dephosphorylating DNA and DNA containing the nucleotide sequence shown in SEQ ID NO:368. After splitting the resulting plasmid by restrictase BspHI and SacI were isolated DNA containing the nucleotide sequence shown in SEQ ID NO:368. Then plasmid pKFrSt12-657 obtained in example 16(3), were digested with restrictase BspHI and SacI for DNA derived from the plasmid pKFrSt12. Specified DNA ligated with DNA, which was split restrictase SacI and BspHI and which contains the nucleotide sequence shown in SEQ ID NO:368, obtaining the plasmid pKFrSt12-1584soy (Fig. 56)containing a chimeric DNA in which DNA (A23)S of the present invention is attached immediately after the nucleotide sequence that encodes a chloroplast Tr is nsity peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without changing the reading frames in the codons.

The obtained plasmid pKFrSt12-1584soy were digested with restrictase BamHI and SacI for DNA containing the nucleotide sequence shown in SEQ ID NO:368. Specified DNA was built between the site restrictase BglII site of restrictase SacI plasmids pNdG6-ΔT to obtain plasmid pSUM-NdG6-rSt12-1584soy (Fig. 57), where the promoter CR16G6 attached right from the chimeric DNA in which the DNA is attached immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons.

Then, this plasmid was introduced into competent cells of E. coli DH5α (Takara Shuzo Company) and were selected by ampicillin-resistant cells. Further, the nucleotide sequence of the plasmid contained in the ampicillin-resistant strains, was determined using kit BigDye Terminator Cycle Sequencing Ready Reaction kit v3.0 (PE Applied Biosystems Company) and DNA sequencing machine 3100 (PE Applied Biosystems Company). In the result, it was confirmed that the plasmid pSUM-NdG6-rSt12-1584soy has the nucleotide sequence shown in SEQ ID NO:368.

(3) the Introduction of DNA (A23)S of this invention in soybean

Globular embryos of soybean (cultivar: Fayette and Jack) were received in accordance with the method described in example 47(3).

Received globular embryo was transferred into a fresh environment for vyrshinokamianka embryos and cultured for 2-3 days. In accordance with the method described in example 17(2), plasmid pSUM-NdG6-rSt-1584soy obtained in example 74(1), or plasmid pSUM-NdG6-rSt12-1584soy obtained in example 74(2), was introduced in these globular embryos.

(4) the Selection of somatic embryo with hygromycin

Selection using hygromycin globular embryo after the introduction of a gene obtained in example 74(3), conducted in accordance with the method described in example 47(4).

(5) the Selection of somatic embryo with compound (II)

Selection using the compound (II) globular embryo after the introduction of a gene obtained in example 74(3), conducted in accordance with the method described in example 47(5).

(6) Regeneration of plants from somatic embryo, acclimate and cultivation of plants

In accordance with the method described in example 47(6), the regeneration of plants was performed from globular embryos, selected in example 74(4) or 74(5).

Plant with roots and Mature leaves subjected to acclimation and cultivation in accordance with the method described in example 17(6), and collect a lot of plants.

(7) Evaluation of resistance to the herbicide compound (II)

The degree of resistance against compounds (II) regenerated plants obtained in example 74(6), evaluated in accordance with the method described in example 17(4).

(8) Design is the formation of chloroplast expression plasmids with DNA (A23)S of the present invention, for introduction using Agrobacterium

Designed plasmid for introduction of DNA (A23)S of the present invention into a plant by Agrobacterium. Plasmid pSUM-NdG6-rSt-1584soy were digested with restrictase HindIII and EcoRI to highlight the chimeric DNA in which DNA (A23)S of this invention was added immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons. Specified DNA was embedded between the restriction site HindIII and the restriction site EcoRI above binary plasmid vector pBI121S obtained in example 18, to obtain the plasmid pBI-NdG6-rSt-1584soy (Fig. 58). Next, the plasmid pSUM-NdG6-rSt12-1584soy were digested with restriction enzyme NotI emitting chimeric DNA in which DNA (A23)S of this invention was added immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without altering the reading frames in the codons. This DNA was embedded between the restriction site HindIII and the restriction site EcoRI above binary plasmid vector pBI121S with obtaining the plasmid pBI-NdG6-rSt12-1584soy (Fig. 59).

(9) the Introduction of DNA (A23)S of this invention in tobacco

DNA (A23)S of the present invention was introduced into tobacco by a method using Agrobacterium, using the plasmid pBI-NdG6-rSt-1584soy and pBI-NdG6-rSt12-1584soy obtained in example 74(8).

First, in accordance with the method described in example 19, each of the plasmid pBI-NdG6-rSt-1584soy and pBI-NdG6-rSt12-1584soy was introduced into Agrobacterium tumefaciens LBA4404 (Clontech Company). Allocated each transgenic Agrobacterium bearing pBI-NdG6-rSt-1584soy or pBI-NdG6-rSt12-1584soy.

Then these Agrobacterium carrying these plasmids were used to introduce genes into the tobacco in accordance with the method described in example 47(9), with, respectively, transgenic tobacco plants, which included the area of the T-DNA pBI-NdG6-rSt-1584soy or pBI-NdG6-rSt12-1584soy.

(10) Evaluation of stability by using a piece of sheet transgenic tobacco with DNA (A23)S of this invention

Leaves were collected from 35 transgenic tobacco plants obtained in example 74(9). Each sheet was divided into pieces, and each piece had a width of 5-7 mm. Pieces of the sheet were dropped off on Wednesday with MS agar containing the compound (II) or the compound (XII), and cultured in the light at room temperature. After several days of cultivation was observed herbicide damage to each of these pieces of paper. As control was used pieces of tobacco leaves of the wild type. Resistance of transgenic tobacco plants was determined by evaluation b is Allaah pieces of leaves, which continuously grow, pieces of leaves, which have chemical damage, pieces of leaves, which was white and had savagely.

Example 75. Introduction DNA (A25)S of the present invention in a plant

(1) Construction of chloroplast expression plasmids containing DNA (A25)S of the present invention, for direct introduction - part 1

A plasmid containing a chimeric DNA in which DNA (A25)S of this invention was added immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons, was designed as a plasmid for introduction of DNA (A25)S of the present invention in a plant method using guns particles.

First, a DNA containing the nucleotide sequence shown in SEQ ID NO:393, amplified using PCR. PCR was performed using as matrix pKSN1609soy obtained in example 71(2), and using as primers the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:400, and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:401. In PCR were using KOD-plus (Toyobo Company). PCR was performed after maintaining at 94°C for 2 minutes as follows: holding 20 cycles, where each CEC is included maintaining 94°C for 30 seconds, then, 53°C for 30 seconds and then 68°C for 90 seconds; and final maintenance at 68°C for 3 minutes. Amplified DNA was isolated and purified using MagExtractor-PCR &Gel Clean up (Toyobo Company) carrying out procedures in accordance with the attached manual. The processing of the resulting DNA using a set of TaKaRa BKL (Takara Shuzo Company) in accordance with the attached guidance, DNA was a small mistake and the 5'-end was fosforilirovanii. DNA containing the nucleotide sequence shown in SEQ ID NO:393, were isolated. After cleavage of plasmid pUC19 (Takara Shuzo Company) by the restriction enzyme SmaI 5'-end dephosphorylated alkaline phosphatase calf intestine (Takara Shuzo). Plasmid was obtained by legirovaniem received dephosphorylating DNA and DNA containing the nucleotide sequence shown in SEQ ID NO:393. After splitting the resulting plasmid by restrictase Eat and SacI were isolated DNA containing the nucleotide sequence shown in SEQ ID NO:393. After cleavage of plasmid pUCrSt657 obtained in example 16(2), restrictase EcoT22I and SacI were isolated DNA approximately 2,9 TPN having a nucleotide sequence derived from pUC19, and a sequence encoding a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack). The obtained DNA and the above-mentioned DNA containing the nucleotide sequence shown in SE ID NO:393, ligated with getting pUCrSt1609soy (Fig. 60)containing a chimeric DNA in which DNA (A25)S of the present invention is attached immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons.

The obtained plasmid pUCrSt1609soy were digested with restrictase BamHI and SacI for DNA containing the nucleotide sequence shown in SEQ ID NO:393. Specified DNA was embedded between the restriction site BglII and restriction site SacI plasmids pNdG6-ΔT to obtain plasmid pSUM-NdG6-rSt-1609soy (Fig. 61), where the promoter CR16G6 attached right from the chimeric DNA in which the DNA is attached immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons.

Then, this plasmid was introduced into competent cells of E. coli DH5α (Takara Shuzo Company) and were selected by ampicillin-resistant cells. Further, the nucleotide sequence of the plasmid contained in these ampicillin-resistant strains, was determined using kit BigDye Terminator Cycle Sequencing Ready Reaction kit v3.0 (PE Applied Biosystems Company) and DNA sequencing machine 3100 (PE Applied Biosystems Company). In the result, it was confirmed that the plasmid pSUM-NdG6-rSt-1609soy has the nucleotide sequence, pokazanno is in SEQ ID NO:393.

(2) Construction of chloroplast expression plasmids containing DNA (A25)S of the present invention, for direct introduction - part 2

Designed plasmid for introduction of DNA (A25)S of the present invention in a plant method using guns particles. This plasmid contained a chimeric DNA in which DNA (A25)S of this invention was added immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without altering the reading frames in the codons. First used plasmid pUCrSt1609soy obtained in example 75(1), embedded in its EcoT22I restriction site linker EcoT22I-12-AA-EcoT22I (Fig. 62)obtained by annealing of the oligonucleotide having the nucleotide sequence shown in SEQ ID NO:402, and the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO:403. Received plasmid pUCrSt12-1609soy (Fig. 63)containing a chimeric DNA in which DNA (A25)S of the present invention is attached immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without altering the reading frames in the codons.

The obtained plasmid pUCrSt12-1609soy were digested restr chasami BamHI and SacI for DNA extraction, containing the nucleotide sequence shown in SEQ ID NO:393. Specified DNA was built between the site restrictase BglII site of restrictase SacI plasmids pNdG6-ΔT obtained in example 16(2), to obtain plasmid pSUM-NdG6-rSt12-1609soy (Fig. 64), where the promoter CR16G6 attached right from the chimeric DNA in which the DNA is attached immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons.

Then, this plasmid was introduced into competent cells of E. coli DH5α (Takara Shuzo Company) and were selected by ampicillin-resistant cells. Further, the nucleotide sequence of the plasmid contained in the ampicillin-resistant strains, was determined using kit BigDye Terminator Cycle Sequencing Ready Reaction kit v3.0 (PE Applied Biosystems Company) and DNA sequencing machine 3100 (PE Applied Biosystems Company). In the result, it was confirmed that the plasmid pSUM-NdG6-rSt12-1609soy has the nucleotide sequence shown in SEQ ID NO:393.

(3) the Introduction of DNA (A25)S of this invention in soybean

Globular embryos of soybean (cultivar: Fayette and Jack) were received in accordance with the method described in example 47(3).

Received globular embryo was transferred into a fresh environment for the cultivation of somatic embryos and cultured for 2-3 days. In accordance with the method described in the ore 17(2), plasmid pSUM-NdG6-rSt-1609soy obtained in example 75(1), or plasmid pSUM-NdG6-rSt12-1609soy obtained in example 75(2), was introduced in these globular embryos.

(4) the Selection of somatic embryo with hygromycin

Selection using hygromycin globular embryo after the introduction of a gene obtained in example 75(3), conducted in accordance with the method described in example 47(4).

(5) the Selection of somatic embryo with compound (II)

Selection using the compound (II) globular embryo after the introduction of a gene obtained in example 75(3), conducted in accordance with the method described in example 47(5).

(6) Regeneration of plants from somatic embryo, acclimate and cultivation of plants

In accordance with the method described in example 47(6), the regeneration of plants was performed from globular embryos, selected in example 75(4) or 75(5).

Plant with roots and Mature leaves subjected to acclimation and cultivation in accordance with the method described in example 17(6), and collect a lot of plants.

(7) Evaluation of resistance to the herbicide compound (II)

The degree of resistance against compounds (II) regenerated plants obtained in example 75(6), evaluated in accordance with the method described in example 17(4).

(8) Construction of chloroplast expression plasmids, it is the MT DNA (A25)S of this invention, for the introduction using Agrobacterium

Designed plasmid for introduction of DNA (A25)S of the present invention into a plant by Agrobacterium. Plasmid pSUM-NdG6-rSt-1609soy were digested with restrictase HindIII and EcoRI to highlight the chimeric DNA in which DNA (A25)S of this invention was added immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack), without changing the reading frames in the codons. Specified DNA was embedded between the restriction site HindIII and the restriction site EcoRI above binary plasmid vector pBI121S obtained in example 18, to obtain the plasmid pBI-NdG6-rSt-1609soy (Fig. 65). Next, the plasmid pSUM-NdG6-rSt12-1609soy were digested with restriction enzyme NotI emitting chimeric DNA in which DNA (A25)S of this invention was added immediately after the nucleotide sequence that encodes a chloroplast transit peptide of the small subunit of RuBPC of the soybean cultivar Jack) and the coding then 12 amino acids of the Mature protein, without altering the reading frames in the codons. This DNA was embedded between the restriction site HindIII and the restriction site EcoRI above binary plasmid vector pBI121S with obtaining the plasmid pBI-NdG6-rSt12-1609soy (Fig. 66).

(9) the Introduction of DNA (A25)S of this invention in tobacco

DNA (A25)S of this invention centuries the Dili in tobacco method using Agrobacterium, using the plasmid pBI-NdG6-rSt-1609soy and pBI-NdG6-rSt12-1609soy obtained in example 75(8).

First, in accordance with the method described in example 19, each of the plasmid pBI-NdG6-rSt-1609soy and pBI-NdG6-rSt12-1609soy was introduced into Agrobacterium tumefaciens LBA4404 (Clontech Company). Allocated each transgenic Agrobacterium bearing pBI-NdG6-rSt-1609soy or pBI-NdG6-rSt12-1609soy.

Then these Agrobacterium carrying these plasmids were used to introduce genes into the tobacco in accordance with the method described in example 47(9), with, respectively, transgenic tobacco plants, which included the area of the T-DNA pBI-NdG6-rSt-1609soy or pBI-NdG6-rSt12-1609soy.

(10) Evaluation of stability by using a piece of sheet transgenic tobacco with DNA (A25)S of this invention

Leaves were collected from transgenic tobacco plants obtained in example 75(9). Such sheets are used to assess the resistance of transgenic tobacco against the compound (II) or compound (XII) in accordance with the method of example 74(10).

INDUSTRIAL APPLICABILITY

By means of this invention can be provided with a protein capable of metabolizing the PPO-inhibiting herbicide connection and make the connection in the connection of the lower herbicide activity; DNA encoding such protein; and the plant resistant to the herbicide compound, expressing such a protein.

The NAMES GIVEN WHAT'S IN the LIST of SEQUENCES

SEQ ID NO:35

Designed oligonucleotide primer for PCR

SEQ ID NO:36

Designed oligonucleotide primer for PCR

SEQ ID NO:37

Designed oligonucleotide primer for PCR

SEQ ID NO:38

Designed oligonucleotide primer for PCR

SEQ ID NO:39

Designed oligonucleotide primer for PCR

SEQ ID NO:40

Designed oligonucleotide primer for PCR

SEQ ID NO:41

Designed oligonucleotide primer for PCR

SEQ ID NO:42

Designed oligonucleotide primer for PCR

SEQ ID NO:43

Designed oligonucleotide primer for PCR

SEQ ID NO:44

Designed oligonucleotide primer for PCR

SEQ ID NO:45

Designed oligonucleotide primer for PCR

SEQ ID NO:46

Designed oligonucleotide primer for PCR

SEQ ID NO:47

Designed oligonucleotide primer for PCR

SEQ ID NO:48

Designed oligonucleotide primer for PCR

SEQ ID NO:49

Designed oligonucleotide primer for PCR

SEQ ID NO:50

Designed oligonucleotide primer for PCR

SEQ ID NO:51

Designed oligonucleotide primer for PCR

SEQ ID NO:52

Designed oligonucleotide primer for PCR

SEQ ID NO:53

Designed oligonucleotide the th primer for PCR

SEQ ID NO:54

Designed oligonucleotide primer for PCR

SEQ ID NO:55

Designed oligonucleotide primer for PCR

SEQ ID NO:56

Designed oligonucleotide primer for PCR

SEQ ID NO:57

Designed oligonucleotide primer for PCR

SEQ ID NO:58

Designed oligonucleotide primer for PCR

SEQ ID NO:59

Designed oligonucleotide primer for PCR

SEQ ID NO:60

Designed oligonucleotide primer for PCR

SEQ ID NO:61

Designed oligonucleotide primer for PCR

SEQ ID NO:62

Designed oligonucleotide primer for PCR

SEQ ID NO:63

Designed oligonucleotide primer for PCR

SEQ ID NO:64

Designed oligonucleotide primer for PCR

SEQ ID NO:65

Designed oligonucleotide primer for PCR

SEQ ID NO:66

Designed oligonucleotide primer for PCR

SEQ ID NO:67

Designed oligonucleotide primer for PCR

SEQ ID NO:68

Designed oligonucleotide primer for PCR

SEQ ID NO:70

Designed oligonucleotide primer for PCR

SEQ ID NO:71

Designed oligonucleotide primer for PCR

SEQ ID NO:72

Designed oligonucleotide primer for PCR

SEQ ID NO:73

Designed oligonucleotide is Reimer for PCR

SEQ ID NO:74

Designed oligonucleotide primer for PCR

SEQ ID NO:75

Designed oligonucleotide primer for PCR

SEQ ID NO:76

Designed oligonucleotide primer for PCR

SEQ ID NO:77

Designed oligonucleotide primer for PCR

SEQ ID NO:79

Designed oligonucleotide primer for PCR

SEQ ID NO:80

Designed oligonucleotide primer for PCR

SEQ ID NO:81

Designed oligonucleotide primer for PCR

SEQ ID NO:82

Designed oligonucleotide primer for PCR

SEQ ID NO:83

Designed oligonucleotide primer for PCR

SEQ ID NO:86

Designed oligonucleotide primer for PCR

SEQ ID NO:87

Designed oligonucleotide primer for PCR

SEQ ID NO:89

Designed oligonucleotide linker to construct expressing vector

SEQ ID NO:90

Designed oligonucleotide linker to construct expressing vector

SEQ ID NO:91

Designed oligonucleotide linker to construct expressing vector

SEQ ID NO:92

Designed oligonucleotide linker to construct expressing vector

SEQ ID NO:93

Designed oligonucleotide primer for PCR

SEQ ID NO:94

Designed oligo is nucleotidyl primer for PCR

SEQ ID NO:95

Designed oligonucleotide primer for PCR

SEQ ID NO:96

Designed oligonucleotide primer for PCR

SEQ ID NO:97

Designed oligonucleotide primer for PCR

SEQ ID NO:98

Designed oligonucleotide linker to construct expressing vector

SEQ ID NO:99

Designed oligonucleotide linker to construct expressing vector

SEQ ID NO:100

Designed oligonucleotide primer for PCR

SEQ ID NO:101

Designed oligonucleotide primer for PCR

SEQ ID NO:102

Designed oligonucleotide primer for PCR

SEQ ID NO:103

Designed oligonucleotide primer for PCR

SEQ ID NO:104

Designed oligonucleotide primer for PCR

SEQ ID NO:105

Designed oligonucleotide primer for PCR

SEQ ID NO:106

Designed oligonucleotide primer for PCR

SEQ ID NO:107

Designed oligonucleotide primer for PCR

SEQ ID NO:114

Designed oligonucleotide primer for PCR

SEQ ID NO:115

Designed oligonucleotide primer for PCR

SEQ ID NO:116

Designed oligonucleotide primer for PCR

SEQ ID NO:117

Designed oligonucleotide primer for PCR

SEQ ID NO:118

Designed oligo is nucleotidyl primer for PCR

SEQ ID NO:119

Designed oligonucleotide primer for PCR

SEQ ID NO:120

Designed oligonucleotide primer for PCR

SEQ ID NO:121

Designed oligonucleotide primer for PCR

SEQ ID NO:122

Designed oligonucleotide primer for PCR

SEQ ID NO:123

Designed oligonucleotide primer for PCR

SEQ ID NO:124

Designed oligonucleotide primer for PCR

SEQ ID NO:125

Designed oligonucleotide primer for PCR

SEQ ID NO:126

Designed oligonucleotide primer for PCR

SEQ ID NO:127

Designed oligonucleotide primer for PCR

SEQ ID NO:128

Designed oligonucleotide primer for PCR

SEQ ID NO:129

Designed oligonucleotide primer for PCR

SEQ ID NO:130

Designed oligonucleotide primer for PCR

SEQ ID NO:131

Designed oligonucleotide primer for PCR

SEQ ID NO:132

Designed oligonucleotide primer for PCR

SEQ ID NO:133

Designed oligonucleotide primer for PCR

SEQ ID NO:134

Designed oligonucleotide linker to construct expressing vector

SEQ ID NO:135

Designed oligonucleotide linker to construct expressing vector

SEQ ID NO:161

Constructed ol gonucleotide primer for PCR

SEQ ID NO:162

Designed oligonucleotide primer for PCR

SEQ ID NO:163

Designed oligonucleotide primer for PCR

SEQ ID NO:164

Designed oligonucleotide primer for PCR

SEQ ID NO:165

Designed oligonucleotide primer for PCR

SEQ ID NO:166

Designed oligonucleotide primer for PCR

SEQ ID NO:167

Designed oligonucleotide primer for PCR

SEQ ID NO:168

Designed oligonucleotide primer for PCR

SEQ ID NO:169

Designed oligonucleotide primer for PCR

SEQ ID NO:170

Designed oligonucleotide primer for PCR

SEQ ID NO:171

Designed oligonucleotide primer for PCR

SEQ ID NO:172

Designed oligonucleotide primer for PCR

SEQ ID NO:173

Designed oligonucleotide primer for PCR

SEQ ID NO:174

Designed oligonucleotide primer for PCR

SEQ ID NO:175

Designed oligonucleotide primer for PCR

SEQ ID NO:176

Designed oligonucleotide primer for PCR

SEQ ID NO:177

Designed oligonucleotide primer for PCR

SEQ ID NO:178

Designed oligonucleotide primer for PCR

SEQ ID NO:179

Designed oligonucleotide primer for PCR

SEQ ID NO:180

Designed oligonu etigny primer for PCR

SEQ ID NO:181

Designed oligonucleotide primer for PCR

SEQ ID NO:182

Designed oligonucleotide primer for PCR

SEQ ID NO:183

Designed oligonucleotide primer for PCR

SEQ ID NO:184

Designed oligonucleotide primer for PCR

SEQ ID NO:185

Designed oligonucleotide primer for PCR

SEQ ID NO:186

Designed oligonucleotide primer for DNA sequencing

SEQ ID NO:187

Designed oligonucleotide primer for DNA sequencing

SEQ ID NO:188

Designed oligonucleotide primer for DNA sequencing

SEQ ID NO:189

Designed oligonucleotide primer for DNA sequencing

SEQ ID NO:190

Designed oligonucleotide primer for PCR

SEQ ID NO:191

Designed oligonucleotide primer for PCR

SEQ ID NO:192

Designed oligonucleotide primer for PCR

SEQ ID NO:193

Designed oligonucleotide primer for PCR

SEQ ID NO:194

Designed oligonucleotide primer for PCR

SEQ ID NO:195

Designed oligonucleotide primer for PCR

SEQ ID NO:196

Designed oligonucleotide primer for PCR

SEQ ID NO:197

Designed oligonucleotide primer for PCR

SEQ ID NO:198

Designed oligonucleotide is first primer for PCR

SEQ ID NO:199

Designed oligonucleotide primer for PCR

SEQ ID NO:200

Designed oligonucleotide primer for PCR

SEQ ID NO:201

Designed oligonucleotide primer for PCR

SEQ ID NO:202

Designed oligonucleotide primer for PCR

SEQ ID NO:203

Designed oligonucleotide primer for PCR

SEQ ID NO:204

Designed oligonucleotide primer for PCR

SEQ ID NO:205

Designed oligonucleotide primer for PCR

SEQ ID NO:206

Designed oligonucleotide primer for PCR

SEQ ID NO:207

Designed oligonucleotide primer for PCR

SEQ ID NO:208

Designed oligonucleotide primer for PCR

SEQ ID NO:209

Designed oligonucleotide primer for PCR

SEQ ID NO:210

Designed oligonucleotide primer for PCR

SEQ ID NO:211

Designed oligonucleotide primer for PCR

SEQ ID NO:212

Designed oligonucleotide primer for PCR

SEQ ID NO:213

Designed oligonucleotide primer for PCR

SEQ ID NO:214

Designed polynucleotide encoding the amino acid sequence of SEQ ID NO:1

SEQ ID NO:265

Designed oligonucleotide primer for PCR

SEQ ID NO:266

Designed oligonucleotide primer for PCR

SEQ ID NO:67

Designed oligonucleotide primer for PCR

SEQ ID NO:268

Designed oligonucleotide primer for PCR

SEQ ID NO:269

Designed oligonucleotide primer for PCR

SEQ ID NO:270

Designed oligonucleotide primer for PCR

SEQ ID NO:271

Designed oligonucleotide primer for PCR

SEQ ID NO:272

Designed oligonucleotide primer for PCR

SEQ ID NO:273

Designed oligonucleotide primer for PCR

SEQ ID NO:274

Designed oligonucleotide primer for PCR

SEQ ID NO:275

Designed oligonucleotide primer for PCR

SEQ ID NO:276

Designed oligonucleotide primer for DNA sequencing

SEQ ID NO:277

Designed oligonucleotide primer for DNA sequencing

SEQ ID NO:278

Designed oligonucleotide primer for PCR

SEQ ID NO:279

Designed oligonucleotide primer for PCR

SEQ ID NO:280

Designed oligonucleotide primer for PCR

SEQ ID NO:281

Designed oligonucleotide primer for DNA sequencing

SEQ ID NO:282

Designed oligonucleotide primer for PCR

SEQ ID NO:283

Designed oligonucleotide primer for PCR

SEQ ID NO:284

Designed oligonucleotide primer for PCR

SEQ ID N:285

Designed oligonucleotide primer for PCR

SEQ ID NO:286

Designed oligonucleotide primer for PCR

SEQ ID NO:287

Designed oligonucleotide primer for PCR

SEQ ID NO:288

Designed oligonucleotide primer for DNA sequencing

SEQ ID NO:289

Designed oligonucleotide primer for PCR

SEQ ID NO:290

Designed oligonucleotide primer for PCR

SEQ ID NO:291

Designed oligonucleotide primer for PCR

SEQ ID NO:292

Designed oligonucleotide primer for PCR

SEQ ID NO:293

Designed oligonucleotide primer for PCR

SEQ ID NO:294

Designed oligonucleotide primer for PCR

SEQ ID NO:295

Designed oligonucleotide primer for PCR

SEQ ID NO:296

Designed oligonucleotide primer for PCR

SEQ ID NO:297

Designed oligonucleotide primer for PCR

SEQ ID NO:298

Designed oligonucleotide primer for PCR

SEQ ID NO:299

Designed oligonucleotide primer for PCR

SEQ ID NO:300

Designed oligonucleotide primer for DNA sequencing

SEQ ID NO:301

Designed oligonucleotide primer for PCR

SEQ ID NO:302

Designed oligonucleotide primer for PCR

SEQ ID NO:303

The designed the new oligonucleotide primer for PCR

SEQ ID NO:304

Designed oligonucleotide primer for PCR

SEQ ID NO:305

Designed oligonucleotide primer for PCR

SEQ ID NO:306

Designed oligonucleotide primer for PCR

SEQ ID NO:307

Designed oligonucleotide primer for PCR

SEQ ID NO:308

Designed oligonucleotide primer for DNA sequencing

SEQ ID NO:309

Designed oligonucleotide primer for PCR

SEQ ID NO:310

Designed oligonucleotide primer for PCR

SEQ ID NO:311

Designed oligonucleotide primer for PCR

SEQ ID NO:312

Designed oligonucleotide primer for PCR

SEQ ID NO:313

Designed oligonucleotide primer for PCR

SEQ ID NO:314

Designed oligonucleotide primer for PCR

SEQ ID NO:315

Designed oligonucleotide primer for DNA sequencing

SEQ ID NO:316

Designed oligonucleotide primer for PCR

SEQ ID NO:317

Designed oligonucleotide primer for PCR

SEQ ID NO:318

Designed oligonucleotide primer for PCR

SEQ ID NO:319

Designed oligonucleotide primer for PCR

SEQ ID NO:320

Designed oligonucleotide primer for PCR

SEQ ID NO:321

Designed oligonucleotide primer for PCR

SEQ ID NO:322

Designed oligonucleotide primer for DNA sequencing

SEQ ID NO:323

Designed oligonucleotide primer for PCR

SEQ ID NO:324

Designed oligonucleotide primer for PCR

SEQ ID NO:325

Designed oligonucleotide primer for PCR

SEQ ID NO:326

Designed oligonucleotide primer for PCR

SEQ ID NO:327

Designed oligonucleotide primer for PCR

SEQ ID NO:328

Designed oligonucleotide primer for PCR

SEQ ID NO:329

Designed oligonucleotide primer for PCR

SEQ ID NO:330

Designed oligonucleotide primer for PCR

SEQ ID NO:331

Designed oligonucleotide primer for PCR

SEQ ID NO:332

Designed oligonucleotide primer for PCR

SEQ ID NO:333

Designed oligonucleotide primer for PCR

SEQ ID NO:334

Designed oligonucleotide primer for PCR

SEQ ID NO:335

Designed oligonucleotide primer for PCR

SEQ ID NO:336

Designed oligonucleotide primer for PCR

SEQ ID NO:337

Designed oligonucleotide primer for PCR

SEQ ID NO:338

Designed oligonucleotide primer for PCR

SEQ ID NO:339

Designed oligonucleotide primer for PCR

SEQ ID NO:340

Designed oligo is nucleotidyl primer for PCR

SEQ ID NO:341

Designed oligonucleotide primer for PCR

SEQ ID NO:342

Designed oligonucleotide primer for PCR

SEQ ID NO:343

Designed oligonucleotide primer for PCR

SEQ ID NO:344

Designed oligonucleotide primer for PCR

SEQ ID NO:345

Designed oligonucleotide primer for PCR

SEQ ID NO:346

Designed oligonucleotide primer for PCR

SEQ ID NO:347

Designed oligonucleotide primer for PCR

SEQ ID NO:348

Designed oligonucleotide primer for PCR

SEQ ID NO:349

Designed oligonucleotide primer for PCR

SEQ ID NO:350

Designed oligonucleotide primer for PCR

SEQ ID NO:351

Designed oligonucleotide primer for PCR

SEQ ID NO:352

Designed oligonucleotide primer for PCR

SEQ ID NO:353

Designed oligonucleotide primer for PCR

SEQ ID NO:354

Designed oligonucleotide primer for PCR

SEQ ID NO:355

Designed oligonucleotide primer for PCR

SEQ ID NO:356

Designed oligonucleotide primer for PCR

SEQ ID NO:357

Designed oligonucleotide primer for PCR

SEQ ID NO:358

Designed oligonucleotide primer for PCR

SEQ ID NO:359

Designed oligonu etigny primer for PCR

SEQ ID NO:360

Designed oligonucleotide primer for PCR

SEQ ID NO:361

Designed oligonucleotide primer for PCR

SEQ ID NO:362

Designed oligonucleotide primer for PCR

SEQ ID NO:363

Designed oligonucleotide primer for PCR

SEQ ID NO:364

Designed oligonucleotide primer for PCR

SEQ ID NO:365

Designed oligonucleotide primer for PCR

SEQ ID NO:366

Designed oligonucleotide primer for PCR

SEQ ID NO:367

Designed oligonucleotide primer for PCR

SEQ ID NO:368

Designed polynucleotide encoding the amino acid sequence of SEQ ID NO:222

SEQ ID NO:369

Designed oligonucleotide primer for PCR

SEQ ID NO:370

Designed oligonucleotide primer for PCR

SEQ ID NO:371

Designed oligonucleotide primer for PCR

SEQ ID NO:372

Designed oligonucleotide primer for PCR

SEQ ID NO:373

Designed oligonucleotide primer for PCR

SEQ ID NO:374

Designed oligonucleotide primer for PCR

SEQ ID NO:375

Designed oligonucleotide primer for PCR

SEQ ID NO:376

Designed oligonucleotide primer for PCR

SEQ ID NO:377

Designed oligonucleotide primer for PCR

SEQ ID N:378

Designed oligonucleotide primer for PCR

SEQ ID NO:379

Designed oligonucleotide primer for PCR

SEQ ID NO:380

Designed oligonucleotide primer for PCR

SEQ ID NO:381

Designed oligonucleotide primer for PCR

SEQ ID NO:382

Designed oligonucleotide primer for PCR

SEQ ID NO:383

Designed oligonucleotide primer for PCR

SEQ ID NO:384

Designed oligonucleotide primer for PCR

SEQ ID NO:385

Designed oligonucleotide primer for PCR

SEQ ID NO:386

Designed oligonucleotide primer for PCR

SEQ ID NO:387

Designed oligonucleotide primer for PCR

SEQ ID NO:388

Designed oligonucleotide primer for PCR

SEQ ID NO:389

Designed oligonucleotide primer for PCR

SEQ ID NO:390

Designed oligonucleotide primer for PCR

SEQ ID NO:391

Designed oligonucleotide primer for PCR

SEQ ID NO:392

Designed oligonucleotide primer for PCR

SEQ ID NO:393

Designed polynucleotide encoding the amino acid sequence of SEQ ID NO:224

SEQ ID NO:394

Designed oligonucleotide primer for PCR

SEQ ID NO:395

Designed oligonucleotide primer for PCR

SEQ ID NO:396

The scone is trueromance oligonucleotide primer for PCR

SEQ ID NO:397

Designed oligonucleotide primer for PCR

SEQ ID NO:398

Designed oligonucleotide primer for PCR

SEQ ID NO:399

Designed oligonucleotide primer for PCR

SEQ ID NO:400

Designed oligonucleotide primer for PCR

SEQ ID NO:401

Designed oligonucleotide primer for PCR

SEQ ID NO:402

Designed oligonucleotide linker to construct expressing vector

SEQ ID NO:403

Designed oligonucleotide linker to construct expressing vector

1. DNA encoding a protein capable of converting in the presence of a system of electron transport containing electron donor, a compound of formula (II)

in the compound of formula (III)

where this protein is selected from the group consisting of
(A11) a protein containing the amino acid sequence of SEQ ID NO:159;
(A12) a protein containing the amino acid sequence of SEQ ID NO:160;
(A13) a protein containing the amino acid sequence of SEQ ID NO:136;
(A14) a protein containing the amino acid sequence of SEQ ID NO:137;
(A15) a protein containing the amino acid sequence of SEQ ID NO:138;
(A16) a protein containing the amino acid sequence of SEQ ID NO:215;
(A17) protein containing linakis is now the sequence of SEQ ID NO:216;
(A18) a protein containing the amino acid sequence of SEQ ID NO:217;
(A19) a protein containing the amino acid sequence of SEQ ID NO:218;
(A20) a protein containing the amino acid sequence of SEQ ID NO:219;
(A21) a protein containing the amino acid sequence of SEQ ID NO:220;
(A22) a protein containing the amino acid sequence of SEQ ID NO:221;
(A23) a protein containing the amino acid sequence of SEQ ID NO:222;
(A24) a protein containing the amino acid sequence of SEQ ID NO:223;
(A25) a protein containing the amino acid sequence of SEQ ID NO:224;
(A26) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence that is at least 80% identical to any amino acid sequence of SEQ ID NO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221 or SEQ ID NO:223, or amino acid sequence that is at least 90% identical to any amino acid sequence of SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:222, or SEQ ID NO:224; and
(A27) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence, to diraimo nucleotide sequence, which at least 90% identical to any nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223 or SEQ ID NO:224.

2. The DNA according to claim 1, containing a nucleotide sequence selected from the group consisting of
(A5) the nucleotide sequence of SEQ ID NO:139;
(A6) the nucleotide sequence of SEQ ID NO:140;
(A7) the nucleotide sequence of SEQ ID NO:141;
(A8) the nucleotide sequence of SEQ ID NO:142;
(A9) the nucleotide sequence of SEQ ID NO:143;
(a10) the nucleotide sequence of SEQ ID NO:225;
(a11) the nucleotide sequence of SEQ ID NO:226;
(A12) the nucleotide sequence of SEQ ID NO:227;
(A13) the nucleotide sequence of SEQ ID NO:228;
(A14) the nucleotide sequence of SEQ ID NO:229;
(A15) the nucleotide sequence of SEQ ID NO:230;
(A16) the nucleotide sequence of SEQ ID NO:231;
(A17) the nucleotide sequence of SEQ ID NO:232;
(A18) the nucleotide sequence of SEQ ID NO:233;
(A19) the nucleotide sequence of SEQ ID NO:234; and (A21) a nucleotide sequence that encodes the amino acid sequence of the protein capable of transforming in the presence of a system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (II), moreover, the specified nucleotide sequence at least 90% identical to any of the nucleotide sequence of SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, or SEQ ID NO:234.

3. The DNA according to claim 1, containing the nucleotide sequence encoding the amino acid sequence of the specified protein, where the frequency of use of codons in the specified nucleotide sequence is in the range of plus or minus 4% of the frequency of use of codons in the genes for the type of host cells in which the introduced DNA and GC contents of the specified nucleotide sequence is at least 40% and maximum 60%.

4. The DNA according to claim 3, containing the nucleotide sequence of SEQ ID NO:368.

5. The DNA according to claim 3, containing the nucleotide sequence of SEQ ID NO:393.

6. DNA in which a DNA having a nucleotide sequence encoding a transit signal sequence of intracellular organelles attached "to the left" from the DNA according to claim 1 in reading frame.

7. DNA in which the DNA according to claim 1 and a promoter functional in the cell-master, functionally linked.

8. The cloning vector containing the DNA according to claim 1.

9. The expression vector containing the DNA according to claim 1.

10. The way to obtain a cloning vector or expression, including the state introduced the I DNA according to claim 1 into a vector, replicated in the cell host.

11. A host cell in which the introduced DNA according to claim 1 and which expresses the specified DNA.

12. A host cell according to claim 11, which is a microorganism or a plant cell.

13. The method of producing transformant, which expresses the DNA according to claim 1, comprising a stage of introduction into the cell of the host DNA according to claim 1.

14. A method of obtaining a protein capable of converting the compound of formula (II) in the compound of formula (III), and the method includes the stage of culturing transformant on p.12 and allocation obtained the specified protein.

15. The use of DNA according to claim 1 for obtaining a protein capable of converting the compound of formula (II) in the compound of formula (III).

16. The method of imparting to a plant resistance to a herbicide, comprising the stage of introducing into a plant cell a DNA according to claim 1 and expression of DNA in the plant cell.

17. Polynucleotide, which is a primer or probe, and polynucleotide has a partial nucleotide sequence of at least 20 nucleotides of the DNA according to claim 1 or a nucleotide sequence complementary to the specified partial nucleotide sequence.

18. The method of detecting DNA that encodes a protein capable of converting the compound of formula (II) in the compound of formula (III), including the stage of discovery of DNA, which hybridizes probe when g is britishly using as a probe the DNA according to claim 1 or polynucleotide by 17.

19. The method of detecting DNA that encodes a protein capable of converting the compound of formula (II) in the compound of formula (III), including the stage of detection of DNA amplified in polymerase chain reaction with polynucleotide at 17 as a primer.

20. The method according to claim 19, where at least one of the primers selected from the group consisting of polynucleotide containing any nucleotide sequence of SEQ ID NO:124 and SEQ ID NO:128, and polynucleotide containing the nucleotide sequence of SEQ ID NO:129.

21. A method of obtaining a DNA that encodes a protein capable of converting the compound of formula (II) in the compound of formula (III), including the extraction of DNA, detektirovanii way p or 19.

22. Method of screening cells with DNA encoding a protein capable of converting the compound of formula (II) in the compound of formula (III), including the stage of detection of the indicated DNA from a test cell by the method according to p or 19.

23. Protein is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III), while this protein is selected from the group consisting of
(A11) a protein containing the amino acid sequence of SEQ ID NO:159;
(A12) a protein containing the amino acid sequence of SEQ ID NO:160;
(A13) a protein containing the amino acid sequence of SQ ID NO:136;
(A14) a protein containing the amino acid sequence of SEQ ID NO:137;
(A15) a protein containing the amino acid sequence of SEQ ID NO:138;
(A16) a protein containing the amino acid sequence of SEQ ID NO:215;
(A17) a protein containing the amino acid sequence of SEQ ID NO:216;
(A18) a protein containing the amino acid sequence of SEQ ID NO:217;
(A19) a protein containing the amino acid sequence of SEQ ID NO:218;
(A20) a protein containing the amino acid sequence of SEQ ID NO:219;
(A21) a protein containing the amino acid sequence of SEQ ID NO:220;
(A22) a protein containing the amino acid sequence of SEQ ID NO:221;
(A23) a protein containing the amino acid sequence of SEQ ID NO:222;
(A24) a protein containing the amino acid sequence of SEQ ID NO:223;
(A25) a protein containing the amino acid sequence of SEQ ID NO:224;
(A26) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing an amino acid sequence that is at least 80% identical to any amino acid sequence of SEQ ID NO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221 or SEQ ID NO:223, or amino acid sequence that is at least 90% identical to any amino acid pic is egovernance SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:222, or SEQ ID NO:224; and
(A27) protein, is able to transform into the presence system of electron transport containing electron donor, a compound of formula (II) in the compound of formula (III) and containing the amino acid sequence encoded by a nucleotide sequence which is at least 90% identical to any nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223 or SEQ ID NO:224.

24. The antibody recognizes a protein according to item 23, where the specified antibody obtained using the protein according to item 23.

25. The detection of protein in item 23, including
(1) the stage of contacting the test compound with the antibody that recognizes this protein, and
(2) the stage of detection of the complex specified protein and the indicated antibodies arising as a result of this contact.

26. Set for analysis or detection, containing antibody at point 24 and the instructions for use.



 

Same patents:

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2 tbl

FIELD: veterinary.

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1 tbl, 3 ex

FIELD: medicine.

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FIELD: medicine.

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1 tbl, 8 ex

FIELD: medicine.

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1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention can be applied for assessment of adverse effect of herpesvirus infection in third trimester of pregnancy on apoptosis development in symplast nuclei of placental stems. Assessment is performed on the basis of herpes infection severity level and respective antibody titre of pregnant women.

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1 dwg

FIELD: chemistry.

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8 ex, 1 tbl, 1 dwg

FIELD: medicine.

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38 cl, 8 dwg

FIELD: medicine.

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3 tbl, 3 ex

FIELD: medicine.

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7 ex, 6 tbl

FIELD: medicine.

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6 cl, 12 dwg, 8 ex

FIELD: agriculture.

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36 cl, 3 tbl, 4 ex

FIELD: medicine.

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1 tbl, 2 ex

FIELD: medicine.

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2 ex, 2 tbl

FIELD: medicine.

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2 cl, 1 dwg, 6 tbl, 5 ex

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21 cl, 13 dwg

FIELD: medicine.

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1 tbl, 3 ex

FIELD: medicine.

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2 dwg, 2 tbl, 4 ex

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3 cl, 5 dwg, 3 tbl

FIELD: medicine.

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2 ex, 3 tbl

FIELD: medicine.

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6 cl, 12 dwg, 8 ex

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