Method for production of l-amino acid

FIELD: biotechnology, in particular method for production of L-amino acid except L-glutamic acid.

SUBSTANCE: claimed method includes cultivation of bacteria Methylophilus, which is capable to grow utilizing methanol as a main carbon source and to produce L-amino acid; and collection L-amino acid from culture. For example, bacteria Methylophilus with increased activity of dihydrodipicolinate synthase and aspartokinase is used. Said activity is increased by introducing DNA encoding dihydrodipicolinate synthase which is not inhibited with L-lysine by negative back coupling, and DNA encoding aspartokinase which is not inhibited with L-lysine by negative back coupling, into cells.

EFFECT: increased yield of L-amino acids.

10 cl, 7 dwg, 6 tbl, 7 ex

 

The technical FIELD

This invention relates to methods in the field of microbiological industry. In particular, this invention relates to a method for production of L-amino acids by fermentation and by the microorganism used in this way.

BACKGROUND of the INVENTION

Amino acids such as L-lysine, L-glutamic acid, L-threonine, L-leucine, L-isoleucine, L-valine and L-phenylalanine, get industrially by fermentation using microorganisms that belong to the genera Brevibacterium, Corynebacterium, Bacillus, Escherichia, Streptomyces, Pseudomonas, Arthrobacter, Serratia, Penicillium, Candida and the like. In order to increase productivity, as these microorganisms used strains isolated in nature, or artificial mutants. Have been stated in various ways to strengthen the activities of the enzymes of biosynthesis of L-glutamic acid using recombinant DNA technology to enhance the ability to produce L-glutamic acid.

Productivity in obtaining L-amino acids was significantly increased through breeding of micro-organisms such as microorganisms, above, and improvement of production methods. However, to meet future needs increase in the future, you still have to develop ways to more effectively what about the production of L-amino acids.

As methods for producing amino acids by fermentation of methanol, which is a raw material for fermentation, available in large quantities at low cost, are traditionally known methods using microorganisms that belong to the genus Achromobacter or Pseudomonas (application for Japan patent (Kokoku) No. 45-25273/1970), Protaminobacter (published patent application Japan (Kokai) No. 49-125590/1974), Protaminobacter or Methanomonas (published patent application Japan (Kokai) No. 50-25790/1975), Microcyclus (published patent application Japan (Kokai) No. 52-18886/1977), Methylobacillus (published patent application Japan (Kokai) No. 4-91793/1992), Bacillus (published patent application Japan (Kokai) No.3-505284/1991) and so on.

However, to date, has not been known a method of obtaining L-amino acid using a bacterium Methylophilus. Although the methods described in EP 0035831 AND, ER 0037273 and EP 0066994 AND were known as the ways of transformation of Methylophilus bacteria using recombinant DNA, the use of recombinant DNA technology to improve the production of amino acids by Methylophilus bacteria was not known.

DISCLOSURE of the INVENTION

The purpose according to the invention is the provision of a new bacteria producing L-amino acid, and method of producing L-amino acids using bacteria producing L-amino acid.

As a result, popito the authors of the invention, dedicated to the achievement of the above objectives, it was found that the bacterium Methylophilus suitable for the production of L-amino acids. In addition, although traditionally it is considered that it is difficult to get auxotrophic mutants of Methylophilus bacteria (FEMS Microbiology Rev.39, 235-258 (1986) and Antonie van Leeuwenhoek 53, 47-53 (1987)), the authors of this invention have succeeded in obtaining auxotrophic mutants of these bacteria. Thus, this invention has been accomplished.

That is, this invention provides the following.

(1) the Methylophilus Bacterium having an ability to produce L-amino acid.

(2) the Methylophilus Bacterium according to p.(1), where L-amino acid is L-lysine, L-valine, L-leucine, L-isoleucine or L-threonine.

(3) the Methylophilus Bacterium according to p.(1), which has a resistance analogue of L-amino acids or auxotrophies on L-amino acid.

(4) the Methylophilus Bacterium according to p.(1), which increased the activity of enzymes of the biosynthesis of L-amino acids.

(5) the Methylophilus Bacterium according to p.(1), which increased the activity dihydrodipicolinate and activity aspartokinase, and when this bacterium has the ability to produce L-lysine.

(6) the Methylophilus Bacterium according to p.(1), which increased the activity dihydrodipicolinate, and when this bacterium has the ability to produce L-lysine.

(7) the Methylophilus Bacterium according to p.(1), which increased the activity of aspartokinase, and when this is m the bacterium has the ability to produce L-lysine.

(8) the Methylophilus Bacterium according to any one of paragraphs. (5) to (7), in which the activity or activity of one, two or three enzymes selected from the dehydrogenase of tolualdehyde aspartic acid, dihydrodipicolinate and diaminomaleonitrile, reinforced/strengthened.

(9) the Methylophilus Bacterium according to p.(5), whose activity dihydrodipicolinate and activity aspartokinase improved by transformation by introducing into the cells a DNA that encodes dihydrodipicolinate, which is not subject to inhibition by L-lysine on the basis of feedback, and the DNA that encodes aspartokinase, which is not subject to inhibition by L-lysine on the basis of feedback.

(10) the Bacterium under item(1), where activity aspartokinase, homoerythromycin, homoserine and trionychinae raised and the bacterium has the ability to produce L-threonine.

(11) the Bacterium according to any one of paragraphs. (1) through (10), where the bacterium Methylophilus is Methylophilus methylotrophus.

(12) the Method of obtaining L-amino acids, which comprises cultivating the bacterium Methylophilus, which is described in any of the above paragraphs. (1) through (11)in a medium to produce and accumulate the L-amino acid in the culture, and collecting the L-amino acid from the culture.

(13) the Method according to p.(12), wherein the medium contains methanol as a main carbon source.

(14) the Method according to the teachings of the bacterial cells of the bacterium Methylophilus with a high content of L-amino acids, which comprises cultivating the bacterium Methylophilus described in any of the above items (1) through (11)in a medium to produce and accumulate the L-amino acid in bacterial cells of this bacterium.

(15) the Method of obtaining bacterial cells of the bacterium Methylophilus at 14, where L-amino acid is L-lysine, L-valine, L-leucine, L-isoleucine or L-threonine.

(16) a DNA that encodes a protein described in the following paragraphs (a) And (b):

(A) a protein which has the amino acid sequence of SEQ ID NO: 6, or

(B) a protein which has the amino acid sequence of SEQ ID NO: 6, including replacement, deletions, insertions, join, or inversion of one or several amino acids, and has the activity of aspartokinase.

(17) DNA under item(16), which is a DNA described in the following paragraphs (a) or (b):

(a) DNA which has a nucleotide sequence comprising the nucleotide sequence from nucleotide number 510 to nucleotide number 1736 SEQ ID NO: 5; or

(b) DNA capable of gibridizatsiya with the sample having the nucleotide sequence from nucleotide number 510 to nucleotide number 1736 SEQ ID NO: 5, or its part, in hard conditions, and encodes a protein having the activity of aspartokinase.

(18) a DNA that encodes a protein described in the following paragraph shall nkth (C) or (D):

(C) a protein which has the amino acid sequence of SEQ ID NO: 8, or

(D) a protein which has the amino acid sequence of SEQ ID NO: 8, including replacement, deletions, insertions, join, or inversion of one or several amino acids, and has a dehydrogenase activity of tolualdehyde aspartic acid.

(19) DNA under item(18), which is a DNA described in the following paragraphs (C) or (d):

(c) DNA which has a nucleotide sequence comprising the nucleotide sequence from nucleotide number 98 to nucleotide room 1207 SEQ ID NO: 7; or

(d) DNA, which hybridizes with the sample having the nucleotide sequence from nucleotide number 98 to nucleotide room 1207 SEQ ID NO: 7 or its part, in hard conditions, and encodes a protein having a dehydrogenase activity of tolualdehyde aspartic acid.

(20) a DNA that encodes a protein described in the following paragraphs (E) or (F):

(E) a protein which has the amino acid sequence of SEQ ID NO: 10, or

(F) a protein which has the amino acid sequence of SEQ ID NO: 10, including replacement, deletions, insertions, join, or inversion of one or several amino acids, and has activity dihydrodipicolinate.

(21) DNA under item(20), which is a DNA, characterized in follow what their paragraphs (e) or (f):

(e) DNA which has a nucleotide sequence comprising the nucleotide sequence from nucleotide number 1268 to nucleotide number 2155 SEQ ID NO: 9; or

(f) DNA that hybridizes with the sample having the nucleotide sequence from nucleotide number 1268 to nucleotide number 2155 SEQ ID NO: 9 or its part, in hard conditions, and encodes a protein having the activity dihydrodipicolinate.

(22) a DNA that encodes a protein described in the following paragraphs (G) or (H):

(G) a protein which has the amino acid sequence of SEQ ID NO: 12, or

(H) a protein which has the amino acid sequence of SEQ ID NO: 12, including replacement, deletions, insertions, join, or inversion of one or several amino acids, and has activity dihydrodipicolinate.

(23) DNA under item(22), which is a DNA described in the following paragraphs (g) or (h):

(g) a DNA which has a nucleotide sequence comprising the nucleotide sequence from nucleotide number 2080 to nucleotide number 2883 SEQ ID NO: 11; or

(h) DNA, which hybridizes with the sample having the nucleotide sequence from nucleotide number 2080 to nucleotide number 2883 SEQ ID NO: 11 or its part, in hard conditions, and encodes a protein having the activity dihydrodipicolinate.

(24)DNA which encodes a protein described in the following paragraphs (I) or (J):

(I) a protein which has the amino acid sequence of SEQ ID NO: 14, or

(J) a protein which has the amino acid sequence of SEQ ID NO: 14, including replacement, deletions, insertions, join, or inversion of one or several amino acids, and has the activity of diaminomaleonitrile.

(25) DNA under item(24), which is a DNA described in the following paragraphs (i) or (j):

(i) a DNA which has a nucleotide sequence comprising the nucleotide sequence from nucleotide number 751 to nucleotide number 1995 SEQ ID NO: 13; or

(j) DNA, which hybridizes with the sample having the nucleotide sequence from nucleotide number 751 to nucleotide number 1995 SEQ ID NO: 13 or its part, in hard conditions, and encodes a protein having the activity of diaminomaleonitrile.

In this description “the ability to produce L-amino acid” refers to the ability to accumulate a significant amount of L-amino acids in the environment, or to the increase in the content of amino acids in bacterial cells, in the case where the microorganism according to the invention is cultivated in the environment.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 shows how to obtain the plasmid RSF24P with mutant dapA. “dapA* 24” refers to Tantau dapA, which encodes a mutant enzyme DDPS, in which the histidine residue 118 is replaced by a tyrosine residue.

Figure 2 shows the method of obtaining the plasmid RSFD80 with mutant dapA and mutant lysC. “lysC* 80” refers to a mutant lysC, which encodes a mutant AKIII, where the threonine residue 352 replaced by an isoleucine residue.

Figure 3 shows aspartokinase activity of transformed E. coli strains containing the gene ask.

Figure 4 shows the activity of the dehydrogenase of tolualdehyde aspartic acid transformed E. coli strains containing the asd gene.

Figure 5 shows dihydrodipicolinate activity of transformed E. coli strains containing dapA gene.

Figure 6 shows dihydrodipicolinate activity of transformed E. coli strain containing dapB gene.

7 shows diaminotrinitrobenzene activity of transformed E. coli strains containing the gene lysA.

The BEST WAY of carrying out the INVENTION

<1> the Microorganism of the present invention.

The microorganism according to the invention is a bacterium belonging to the genus Methylophilus and having the ability to produce L-amino acid. The Methylophilus bacterium according to the invention includes, for example, a strain of Methylophilus methylotrophus AS1 (NCIMB10515) and so on. The strain of Methylophilus methylotrophus AS1 (NCIMB10515) available for purchase from mi is selected collections of industrial and marine bacteria (National Collections of Industrial and Marine Bacteria) (address: NCIMB Lts., Torry Research Station, 135, Abbey Road, Aberdeen AB9 8DG, United Kingdom).

L-amino acids obtained according to the invention include L-lysine, L-glutamic acid, L-threonine, L-valine, L-leucine, L-isoleucine, L-tryptophan, L-phenylalanine, L-tyrosine, and so forth. You can get one or more types of amino acids.

The Methylophilus bacteria with the ability to produce L-amino acids, can be obtained by imparting the ability to produce L-amino acid bacteria strains wild-type Methylophilus. To give the ability to produce L-amino acids, can be used in ways that are traditionally taken in the selection of coryneform bacteria, bacteria, Escherichia and others like them, such as methods of obtaining auxotrophic mutant strains, strains resistant analogues of L-amino acids, or mutant strains in the control of metabolic processes, and methods for producing recombinant strains in which increased activity of enzymes of the biosynthesis of L-amino acids (see "Amino Acid Fermentation", the Japan Scientific Societies Press [Gakkai Shuppan Center], 1stEdition, published may 30, 1986, pp.77-100). When breeding bacteria produce amino acids, such characteristics as auxotrophy, the resistance analogue of L-amino acids and mutation of metabolic control, you can give the bacteria alone or in combination of two or more live in the VA characteristics. The activity of enzymes of the biosynthesis of L-amino acids can be amplified for each individual or combinations of one or more enzymes. In addition, making such characteristics as auxotrophy, the resistance analogue of L-amino acids and mutation of metabolic control, can be combined with increased activity of enzymes of the biosynthesis of L-amino acids.

For example, bacteria producing L-lysine obtained in breeding as mutants, demonstrating auxotrophy of L-homoserine or L-threonine and L-methionine (application for Japan patent (Kokoku) No. 48-28078/1973 and 56-6499/1981), mutants, demonstrating auxotrophy on Inositol or acetic acid (published patent application Japan (Kokai) No. 55-9784/1980 and 56-8692/1981), or mutants that are resistant to oxylysine, litigitimate, S-(2-amino-ethyl)cysteine, γ -methyllysine, α -chloropropane, DL-α -aminoε -caprolactam, α -aminophenylacetate, the analogue of aspartic acid, a sulfa medicine, chinaedu or N-eurolatin.

Additionally, bacteria producing L-glutamic acid, can be obtained by breeding as mutants, demonstrating auxotrophy on oleic acid or the like. Bacteria producing L-threonine can be obtained by selection as the resistant mutants α -aminoβ -hydroxyvalerenic acid. The tank is ' series, producing L-homoserine, can be obtained by breeding as mutants, demonstrating auxotrophy of L-threonine, or mutants resistant analogues of L-phenylalanine. Bacteria producing L-phenylalanine, can be obtained by breeding as mutants, demonstrating auxotrophy of L-tyrosine. Bacteria producing L-isoleucine, can be obtained by breeding as mutants, demonstrating auxotrophy on L-Latino. Bacteria producing L-Proline, can be obtained by breeding as mutants, demonstrating auxotrophy of L-isoleucine.

Moreover, as indicated in the examples below, the strains that produce one or more types of branched chain amino acids (L-valine, L-leucine and L-isoleucine), can be obtained as strains, demonstrating auxotrophy on Kazarinova acid.

To obtain mutants of Methylophilus bacteria, the inventors first studied the details of the optimal conditions for mutagenesis is used as the index of the frequency of occurrence of strains resistant to streptomycin. In the result, the maximum frequency of emergence of resistant to streptomycin strains was obtained in the case when the degree of survival after mutagenesis was approximately 0.5%, and the authors have reached the goal in getting the auxotrophic strains under these conditions. The authors also succeeded in obtaining the auxotrophic stammo is, to get which was considered difficult by a significant increase in scale screening of mutants compared to the previously held for E. coli and so forth.

As described above, because it was found that the mutants can be obtained by mutagenesis of Methylophilus bacteria in appropriate circumstances, it has become possible to easily obtain the desired mutants, choosing such suitable conditions under which the degree of survival after mutagenesis should correspond to about 0.5%, depending on the method of mutagenesis.

Methods mutagenesis to obtain mutants of Methylophilus bacteria include UV radiation and mutagenic treatment means used in conventional mutagenic treatments, such as N-methyl-N’-nitro-N-nitrosoguanidine (NTG) and nitrous acid. The Methylophilus bacteria with the ability to produce L-amino acids can also be obtained by selection of mutants of the bacterium Methylophilus natural origin.

Mutants resistant analogues of L-amino acids, can be obtained, for example, inocula mutant Methylophilus bacteria on agar medium containing analogue of L-amino acids in various concentrations, and selecting strains that form colonies.

Auxotrophic mutants can be obtained, enabling the Methylophilus bacteria to form colonies on agar medium containing the target pittel the substance (for example, L-amino acid), obtaining replicas of the colonies on agar medium not containing the specified nutrients, and selection of strains that cannot grow on agar medium not containing nutrients.

The ways of imparting or enhancing the ability to produce L-amino acids by increasing the activity of enzymes of the biosynthesis of L-amino acids will be shown in the examples below.

[L-lysine]

The ability to produce L-lysine can be given, for example, increasing activity dihydrodipicolinate and/or activity of aspartokinase.

Activity dihydrodipicolinate and/or activity of aspartokinase Methylophilus bacteria can be increased by ligating a fragment of the gene encoding dihydrodipicolinate, and/or fragment of the gene encoding aspartokinase, with a vector that functions in Methylophilus bacteria, preferably with vector multicopying type to create recombinant DNA, and the introduction of it in the bacteria-host Methylophilus to transform the host. By increasing the number of copies of the gene encoding dihydrodipicolinate, and/or the gene encoding aspartokinase, in cells transformed strain activity or enzyme activity will increase. Next dihydrodipicolinate, aspartokinase and aspartokinase III also indicated reductions in the DDPS, AK and AKIII, respectively.

As the microorganism, providing a gene that encodes DDPS, and the gene that encodes AK, can be used with any micro-organisms, provided that they have the genes responsible for the expression of activity DDPS activity and AK in microorganisms belonging to the genus Methylophilus. Such microorganisms may be strains of wild-type or received from mutant strains. In particular, examples of such microorganisms include strain K-12 E. coli (Escherichia coli)strain AS1 Methylophilus methylotrophus (NCIMB10515) and so on. Since the obtained nucleotide sequences of both genes of the gene encoding DDPS (dapA, Richaud, F. et al., J. Bacteriol., 297, (1986)), and the gene encoding AKIII (lysC, Cassan, M., Parsot, C., Cohen, G.N. and Patte, J.C., J. Biol. Chem., 261, 1052 (1986)), derived from the bacterium Escherichia, these genes can be obtained by PCR using primers synthesized based on the nucleotide sequences of these genes, and chromosomal DNA of this organism as E. coli K-12 or similar, as a matrix. As specific examples will be explained dapA and lysC, obtained from E. coli. However, the genes used in this invention is not limited to the above genes.

Preferably, DDPS and AK used according to the invention did not undergo inhibition of L-lysine on the basis of feedback. Know the IDT, that wild-type DDPS derived from E. coli, is undergoing inhibition of L-lysine on the basis of feedback, and that wild-type AKIII derived from E. coli, is undergoing suppression and inhibition on the basis of feedback L-lysine. Therefore, dapA and lysC entered in Methylophilus bacteria, preferably encode DDPS and AKIII having a mutation that desensibilisation to inhibition of L-lysine on the basis of feedback. Further DDPS that has a mutation that desensibilisation to inhibition of L-lysine on the basis of feedback, also referred to as “mutant DDPS, and DNA encoding a mutant DDPS, also referred to as “mutant dapA”. AKIII derived from E. coli, which has a mutation that desensibilisation to inhibition of L-lysine on the basis of feedback, also referred to as “mutant AKIII, and DNA encoding a mutant AKIII, also referred to as “mutant lysC”.

According to this invention does not necessarily need to DDPS and AK were mutant. It is known, for example, that DDPS derived from bacteria Corynebacterium, is not subject to inhibition by L-lysine on the basis of feedback.

The nucleotide sequence of wild-type dapA derived from E. coli, is shown in SEQ ID NO: 1. Amino acid sequence of wild-type DDPS encoded by the specified nucleotide sequence is illustrated in SEQ ID NO: 2. Nucleotide lysC wild-type, derived from E. coli, is illustrated by SEQ ID NO: 3. Amino acid sequence of ATIII wild-type encoded by the specified nucleotide sequence is illustrated in SEQ ID NO: 4.

DNA encoding a mutant DDPS, which is not subject to inhibition by the feedback principle L-lysine, includes DNA encoding DDPS, which has the amino acid sequence described in SEQ ID NO: 2, where the histidine residue 118 is replaced by a tyrosine residue. DNA encoding a mutant AKIII, which is not subject to inhibition by the feedback principle L-lysine, includes DNA encoding AKIII, which has the amino acid sequence described in SEQ ID NO: 4, where the threonine residue 352 replaced by an isoleucine residue.

The plasmid used for cloning of the gene can be any plasmid, provided that it can be replicated in a microorganism, such as bacteria Escherichia and the like, and in particular including pBR322, pTWV228, pMW119, pUC19, and so on.

A vector that functions in Methylophilus bacteria, is, for example, a plasmid, which can autonomously replicate in Methylophilus bacteria. In particular, we can mention RSF1010, which is a vector of a wide range of hosts, and its derivatives, for example, pAYC32 (Chistorerdov, A.Y., Tsygankov, Y.D. Plasmid, 16, 161-167, (1986)), pMFY42 (Gene, 44, 53, (1990)), pRP301, pTB70 (Nature, 287, 396, (1980)), and so on.

What is advised to prepare a recombinant DNA by legirovaniem dapA and lysC with vector, which operates in Methylophilus bacteria, the vector is digested with the restriction enzyme, which corresponds to the end of the DNA fragment containing dapA and lysC. Ligation is usually performed using a ligase such as DNA ligase T4. dapA and lysC can be individually included in a separate vectors or vector.

As the plasmid containing the mutant dapA encoding mutant DDPS, and mutant lysC, encoding a mutant AKIII, was known plasmid RSFD80 with a wide range of hosts (WO 95/16042). The strain JM109 E. coli, transformed with the indicated plasmid, named AJ12396 and deposited in National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (postal code 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on October 28, 1993, and received inventory number FERM P-13936, and transferred to international Deposit under the terms of the Budapest agreement November 1, 1994, and received inventory number FERM BP-4859. RSFD80 can be obtained from strain AJ12396 known method.

Mutant dapA available in RSFD80 has the nucleotide sequence of wild-type dapA SEQ ID NO: 1, including replacement With the nucleotide room 623 on T. Therefore, the encoded mutant DDPS has the amino acid sequence of SEQ ID NO: 2, including the replacement of the histidine residue 118 to the tyrosine residue. Mutant lysC, available in RSFD80 has the nucleotide sequence of lysC wild tee is as SEQ ID NO: 3, including replacement With the nucleotide room 1638 on T. Therefore, the encoded mutant AKIII has the amino acid sequence of SEQ ID NO: 4, including the replacement of a threonine residue 352 on the balance of isoleucine.

To introduce the recombinant DNA obtained as described above, in Methylophilus bacteria, you can use any method provided that it provides sufficient transformation efficiency. For example, you can use electroporation (Canadian Journal of Microbiology, 43, 197 (1997)).

The DDPS activity and/or activity of AK can be further enhanced in the presence of multiple copies of dapA and/or lysC in the chromosomal DNA of Methylophilus bacteria. In order to introduce multiple copies of dapA and/or lysC in the chromosomal DNA of the bacterium Methylophilus, carry out homologous recombination using a target sequence that is present in the chromosomal DNA of the bacterium Methylophilus many copies. As a sequence present in the chromosomal DNA in multiple copies, you can use repetitive DNA inverted repeats present at the end of transpositive element, and the like. Alternatively, as claimed in the published patent application Japan (Kokai) No. 2-109985/1990, multiple copies of dapA and/or lysC can be introduced into the chromosomal DNA inline in the transposon to move. In both ways in rez is ltate increased number of copies of dapA and/or lysC in the transformed strains the DDPS activity and the activity of AK will be raised.

In addition to the above gene amplification, the DDPS activity and/or activity of AK can be increased by replacing a sequence controlling the expression, such as promoters of dapA and/or lysC, stronger promoters (published patent application Japan (Kokai) No. 1-215280/1989). As strong promoters are known, for example, the lac promoter, trp promoter, trc promoter, tac promoter, the promoter PRand the promoter PLphage lambda, the tet promoter, the promoter of Amae, the spac promoter, and so forth. Substitution of these promoters enhances expression of dapA and/or lysC, and thus the DDPS activity and the activity of AK increases. Amplification of sequences controlling the expression can be combined with the increasing number of copies of dapA and/or lysC.

To prepare a recombinant DNA by legirovaniem gene fragment and vector, the vector is digested with the restriction enzyme corresponding to the end of the gene fragment. Ligation is usually performed using a ligase such as DNA ligase T4. As methods of digestion, ligation and other methods for DNA preparation of chromosomal DNA, PCR, obtaining plasmid DNA, transformation, design of oligonucleotides used as primers, and so forth, you can use the standard methods, well known to specialists in this field the tee. These methods are described in Sambrook, J., Fritsch, E.F., and Maniatis, T., "Molecular Cloning: A Laboratory Manual, 2ndEdition", Cold Spring Harbor Laboratory Press, (1989), and so on.

In addition to improving the DDPS activity and/or activity of AK can also be increased activity of another enzyme involved in the biosynthesis of L-lysine. Such enzymes include enzymes route of metabolism of diaminopimelate, such as dihydrodipicolinate, diaminomaleonitrile, diaminophenylmethane (WO 96/40934 for all of the above enzymes), fosfoenolpiruvatcarboksilaza (published patent application Japan (Kokai) No. 60-87788/1985), aspartate aminotransferase (published patent application Japan (Kokoku) No.6-102028/1994), diaminopimelate, dehydrogenase of tolualdehyde aspartic acid, and so forth, or the enzymes of the metabolic pathways of aminoadipate, such as homogentisate, and so on. Preferably increases the activity of at least one enzyme of the dehydrogenase of tolualdehyde aspartic acid, dihydrodipicolinate and diaminomaleonitrile.

Aspartokinase, dehydrogenase of tolualdehyde aspartic acid, dihydrodipicolinate, dihydrodipicolinate and diaminomaleonitrile derived from Methylophilus methylotrophus, will be described next.

In addition, the microorganisms according to the invention which may be reduced enzyme activity, that catalyses the reaction of formation of another compound other than L-lysine by branching the biosynthesis pathway of L-lysine, or they may be deficient in this enzyme. The enzyme that catalyzes the reaction of education is not L-lysine, and other compounds through the branches of the biosynthesis pathway of L-lysine, includes gomoserinlaktonazy (see WO 95/23864).

Similarly to the above-mentioned methods of increasing the activity of an enzyme involved in the biosynthesis of L-lysine, can be used for other amino acids, as described below.

[L-glutamic acid]

The ability to produce L-glutamic acid can be given Methylophilus bacteria, for example, by introducing a DNA that encodes any of the enzymes, including glutaraldehydes (published patent application Japan (Kokai) 61-268185/1986), glutamylcysteine, glutamates, izotsitratdegidrogenazy (published patent application Japan (Kokai) 62-166890/1987 and 63-214189/1988), genitalwarts (published patent application Japan (Kokai) 62-294086/1987), citratenazi (published patent application Japan (Kokai) 62-201585/1987 and 63-119688/1988), fosfoenolpiruvatcarboksilaza (published application the Japan patent (Kokai) 60-87788/1985 and 62-55089/1987), piruvatdegidrogenzu, piruwatkinaza, phosphoenolpyruvate, enolase, phosphoglyceromutase, phosphoglycerate, glyceraldehyde-3-ostat-dehydrogenase, triosephosphate, fruktozodifosfata, phosphofructokinase (published patent application Japan (Kokai) 63-102692/1988), glucosephosphate, glutamylcyclotransferase (WO 99/07853) and so on.

In addition, the microorganisms according to the invention can be reduced activity of the enzyme that catalyzes the reaction of formation of another compound other than L-glutamic acid by branching the biosynthesis pathway of L-glutamic acid, or they may be deficient in this enzyme. The enzyme that catalyzes the reaction of education is not L-glutamic acid, and other compounds through the branches of the biosynthesis pathway of L-glutamic acid, includes α -ketoglutarates (α KGDH), isocitrate, hospitalitytourism, azetidines, the synthase of ecotoxicology, acetolactate, formatterlistener, lactate dehydrogenase, glutamatdekarboksilazy, 1-pyrrolin-dehydrogenase and so on.

[L-threonine]

The ability to produce L-threonine can be given, or to increase, for example, increasing activity aspartokinase, homoerythromycin, homoserine and trionychinae. Activity of these enzymes can be increased, for example, by transformation of Methylophilus bacteria using recombinant plasmids containing the operon Treo the ina (published patent application Japan (Kokai) No.55-131397/1980, 59-31691/1984 and 56-15696/1981 and published a patent application in Japanese (Kohyo) No.3-501682/1991).

The capacity of production is also possible to impart or improve by amplification or the introduction of threonine operon containing the gene encoding aspartokinase, which is desensitized to inhibition by L-threonine on the basis of feedback (published patent application Japan (Kokoku) No.1-29559/1989), the gene encoding gomoserinlaktonazy (published patent application Japan (Kokai) No.60-012995/1985), or the gene encoding homoserine and gomoserinlaktonazy (published patent application Japan (Kokai) No.61-195695/1986).

In addition, the ability to produce L-threonine can be improved by introducing a DNA that encodes a mutant fosfoenolpiruvatcarboksilaza with mutation desensitization to the inhibition of aspartic acid on the basis of feedback.

[L-valine]

The ability to produce L-valine can be given, for example, by introducing into the bacterium Methylophilus gene for biosynthesis of L-valine, the mechanism of regulation which largely desensitized. It is also possible to introduce a mutation that substantially desensibilisation mechanism of gene regulation of the biosynthesis of L-valine, which is a microorganism belonging to the genus Methylophilus.

Examples of the gene for biosynthesis of L-valine include, for example, the ilvGMEDA operon of E. coli. Training samyaza, encoded by ilvA gene, catalyzes the deamination reaction, converting L-threonine into 2-kalimalang acid, which is determinative of the speed stage of the biosynthesis of L-isoleucine. Therefore, in order to achieve efficient amplification reactions for the synthesis of L-valine, preferably operon, which expresses not the activity trainingseminar. Examples of the ilvGMEDA operon, which expresses not so trainingseminar activity, include the ilvGMEDA operon, which ilvA introduced mutation elimination activity trainingseminar, or ilvA broken, and the operon in which ilvA deleterows.

As the ilvGMEDA operon is undergoing control the expression of the operon (weakening) of L-valine and/or L-isoleucine and/or L-leucine, the area required for the weakening of, preferably remove or subjected to mutation to eliminate sensitivity to suppression of expression of L-valine.

The ilvGMEDA operon, which expresses not trainingseminar activity and which is devoid of sensitivity to attenuation, as described above, can be obtained by subjecting the ilvGMEDA operon wild-type mutagenic processing or modifying it using technology recombination of genes (see WO 96/06926).

[L-leucine]

The ability to produce L-leucine can be given, or to increase, for example, by introducing into the microorganism belonging to the genus Methylphilus, the gene for biosynthesis of L-leucine, the mechanism of regulation which largely desensitized, in addition to the above characteristics, necessary for the production of L-valine. It is also possible to introduce a mutation, in which the mechanism of gene regulation of the biosynthesis of L-leucine in the microorganism belonging to the genus Methylophilus, will be largely eliminated. Examples of the specified gene include, for example, leuA gene that makes the enzyme, the inhibition of which L-leucine largely eliminated.

[L-isoleucine]

The ability to produce L-isoleucine can be given, for example, the introduction of the thrABC operon containing the thrA gene, encoding aspartokinase I/gomoserinlaktonazy I, obtained from E. coli, which largely desensitized to inhibition by L-threonine, and ilvGMEDA operon which contains a gene ilvA encoding trainingseminar, which largely desensitized to inhibition by L-isoleucine, and the area which is required for attenuation removed (published patent application Japan (Kokai) No.8-47397/1996).

[Other amino]

The biosynthesis of L-tryptophan, L-phenylalanine, L-tyrosine, L-threonine and L-isoleucine can be enhanced, increasing the ability of Methylophilus bacteria to produce phosphoenolpyruvate (WO 97/08333).

The ability to produce L-phenylalanine and L-tyrosine stand the shawls by amplification or introduction desensitized gene charismatise-preventdisease (CM-PDT) (published patent application Japan (Kokai) No. 5-236947/1993 and 62-130693/1987) and desensitized gene 3-deoxy-D-arabinoheptulosonate-7-phosphadites (DS) (published patent application Japan (Kokai) No.5-236947/1993 and 61-124375/1986).

The ability to produce L-tryptophan is increased by amplification or the introduction of a tryptophan operon containing a gene encoding desensitized anthranilate (published patent application Japan (Kokai) No.57-71397/1982, 62-244382/1987 and U.S. patent No.4371614).

In this description, the expression “the activity of the enzyme increased” usually refers to the fact that the intracellular enzyme activity higher than the activity of the wild-type strain, and the case when the strain in which the activity of the enzyme increased, is obtained by modification with the use of technology recombination of genes or similar manner, with the intracellular enzyme activity higher than the activity in strain before modification. The phrase “activity of the enzyme reduced” usually refers to the fact that the intracellular activity of the enzyme is lower than the activity of the wild-type strain, and the case when the strain in which the activity of the enzyme is reduced, is obtained by modification with the use of technology recombination of genes or similar way, while the intracellular activity of the enzyme is lower than the activity in strain before modification.

L-amino acids can be obtained, the stump is irua Methylophilus bacteria, with the ability to produce L-amino acids, obtained as described above in a medium to produce and accumulate the L-amino acids in the culture, and collecting the L-amino acid from the culture.

Bacterial cells Methylophilus bacteria with a high content of L-amino acids compared with strains of bacteria Methyiophilus wild-type can be obtained by cultivation of Methylophilus bacteria with the ability to produce L-amino acid in a medium to produce and accumulate the L-amino acids in bacterial cells bacteria.

The microorganisms used in this invention can be grown by methods commonly used for culturing microorganisms having the ability to metabolize methanol. Medium used according to the invention may be natural or synthetic environment, provided that it contains a carbon source, nitrogen source, inorganic ions and other organic micro-components if necessary.

Using methanol as a main carbon source, L-amino acids can be obtained at low cost. In the case where as the main carbon source use methanol, it is usually added to the medium in an amount of from 0.001 to 30%. As a source of nitrogen use ammonium sulfate or similar connection, adding it to the environment. Besides what about these compounds usually add a small amount of micro-components, such as potassium phosphate, sodium phosphate, magnesium sulfate, ferrous sulfate and manganese sulfate.

Culture usually contain in aerobic conditions, obtained, for example, by shaking or stirring for aeration at pH from 5 to 9 and a temperature of 20 to 45° and is usually completed within 24 to 120 hours.

Collecting the L-amino acid from the culture usually can be done by combining known methods such as methods using ion exchange resins, precipitation and others.

In addition, cells of Methylophilus bacteria can be separated from the environment by the standard methods of separation of the cells of microorganisms.

<2> Gene according to this invention

DNA according to the invention is a gene that encodes one of the enzymes, aspartokinase (henceforth also referred to as “AK”), dehydrogenase tolualdehyde aspartic acid (henceforth also referred to as “ASD”), dihydrodipicolinate (henceforth also referred to as “DDPS”), dihydrodipicolinate (henceforth also referred to as “DDPR) and diaminomaleonitrile (henceforth also referred to as “DPDC”), derived from Methylophilus methylotrophus.

DNA according to the invention can be obtained, for example, by transformation of the mutant microorganism strain, deficient in AK, ASD, DDPS, DDPR or DPDC, ispolzovaniya genes Methylophilus methylotrophus and clone selection, whose auxotrophy offset.

Library of genes Methylophilus methylotrophus, for example, be obtained as follows. First get the total chromosomal DNA from a strain of Methylophilus methylotrophus wild-type, for example, strain AS1 Methylophilus methylotrophus (NCIMB10515), by the method of Saito et al. (Saito, H. and Miura, K., Biochem. Biophys. Acta 72, 619-629, (1963)) or similar method, and partially digested with a suitable restriction enzyme, such as Sau3AI or Alul, to obtain a mixture of different fragments. Controlling the degree of digestion by regulating the duration of the digestion reaction, and so on, you can use a wide range of enzymes.

Then digested fragments of chromosomal DNA are ligated with a vector DNA autonomously can replicate in Escherichia coli cells to obtain recombinant DNA. In particular, the restriction enzyme that gives the same end of the nucleotide sequence as the sequence obtained using the restriction enzyme used in the digestion of chromosomal DNA, give the opportunity to act on a vector DNA to fully digest and split vector. The mixture is then fragments of the chromosomal DNA and digested and cleaved vector DNA mix, and allow ligase, preferably DNA-ligase T4, to act on the mixture, to obtain a recombinant DNA.

The solution bib is iteki genes can be obtained, transforming Escherichia coli, for example E. coli strain JM109 or similar, using the obtained recombinant DNA, and the preparation of recombinant DNA from the culture fluid of transformant. The above transformation can be done by way D.M. Morrison (Methods in Enzymology 68, 326 (1979)), a method of treating recipient cells with calcium chloride to increase the permeability of DNA (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)), and so on. In the referenced below examples used electroporation.

As examples of the above vector can be named pUC19, pUC18, pUC118, pUC119, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, pMW218, pSTV28, pSTV29 and so on. You can also use phage vectors. Because, for example, pUC118 and pUC119 contain the gene of resistance to ampicillin, and pSTV28 and pSTV29 contain the gene of resistance to chloramphenicol, only transformants that carry the vector or recombinant DNA, can be grown using medium containing ampicillin or chloramphenicol.

As a method of cultivation of the transformants and collection of recombinant DNA from bacterial cells can be called alkaline method with SDS and the like.

Mutant bacterial strain, deficient in AK, ASD, DDPS, DDPR or DPDC, transform, using a mortar libraries of genes Methylophilus methylotrophus, obtained as described above, and selected clones, in which the aucc is tropia offset.

Examples of mutant bacterial strain deficient in AK, include E. coli GT3, scarce three kinds of genes encoding AK (thrA, metLM, lysC). Examples of mutant bacterial strain deficient in ASD include Hfr3000 U482 E. coli (strain CGSC 5081). Examples of mutant bacterial strain deficient in DDPS include AT E. coli (strain CGSC 4547). Examples of mutant bacterial strain deficient in DDPR include AT E. coli (strain CGSC 4549). Examples of mutant bacterial strain deficient in DPDC include AT E. coli (strain CGSC 4505). These mutant strains can be obtained from the Center of cultures E. coli Genetic Stock Center (the Yale University, Department of Biology, Osborn Memorial Labs., P.O. box 6666, New Haven 06511-7444, Connecticut, U.S.).

Although all the above-mentioned mutant strains unable to grow on minimal medium M9, transformed strains that contain the gene encoding AK, ASD, DDPS, DDPR or DPDC, can grow on minimal medium M9, as in transformed function these genes. Therefore, selecting the transformed strains that can grow on minimal medium, and receiving recombinant DNA from these strains, it is possible to obtain DNA fragments containing a gene that encodes each enzyme. AT E. coli (strain CGSC 4549) shows a very low rate of growth even in an environment such as the environment L, in the case when diaminopimelic acid is e added to the environment. However, normal growth can be observed for the transformed strains of these bacteria that contain the gene encoding DDPR Methylophilus methylotrophus, due to the operation of the gene. Therefore, the transformed strain that contains the gene encoding DDPR also can be obtained by selection of the transformed strain, normally growing on the environment L.

Highlighting the integrated DNA fragment obtained from recombinant DNA and determine its nucleotide sequence, it is possible to determine the amino acid sequence of each enzyme and the nucleotide sequence encoding its gene.

The gene encoding AK according to the invention (henceforth also referred to as “ask”), encodes AK, which has the amino acid sequence of SEQ ID NO: 6, specified in the list of sequences. As a specific example of a gene ask you to specify a DNA having a nucleotide sequence consisting of nucleotide SEQ ID NO: 5. Gene ask according to this invention can have the sequence in which the codon corresponding to each of the amino acids are replaced by equivalent codon, provided that it encodes the same amino acid sequence as the amino acid sequence of SEQ ID NO: 6.

The gene that encodes ASD according to the invention (henceforth also referred to as "asd"), encodes ASD, to ora has the amino acid sequence of SEQ ID NO: 8, specified in the list of sequences. As a concrete example of the asd gene, you can specify DNA, which contains the nucleotide sequence consisting of nucleotide numbers of nucleotides 98-1207 in SEQ ID NO: 7. Gene asd according to the invention can have the sequence in which the codon corresponding to each of the amino acids are replaced by equivalent codon, provided that it encodes the same amino acid sequence as the amino acid sequence of SEQ ID NO: 8.

The gene that encodes DDPS according to the invention (henceforth also referred to as "dapA"), encodes DDPS, which has the amino acid sequence of SEQ ID NO: 10, specified in the list of sequences. As a specific example of a gene dapA you can specify a DNA which has a nucleotide sequence consisting of nucleotide numbers of nucleotides 1268-2155 in SEQ ID NO: 9. Gene dapA according to the invention can have the sequence in which the codon corresponding to each of the amino acids are replaced by equivalent codon, provided that it encodes the same amino acid sequence as the amino acid sequence of SEQ ID NO: 10.

The gene that encodes DDPR according to the invention (henceforth also referred to as "dapB"), encodes DDPR, which has the amino acid sequence of SEQ ID NO: 12, provided in the list is posledovatelnostei. As a specific example of a gene dapB you can specify a DNA which has a nucleotide sequence consisting of nucleotide numbers of nucleotides 2080-2883 in SEQ ID NO: 11. Gene dapB according to the invention can have the sequence in which the codon corresponding to each of the amino acids are replaced by equivalent codon, provided that it encodes the same amino acid sequence as the amino acid sequence of SEQ ID NO: 12.

The gene that encodes DPDC according to the invention (henceforth also referred to as "lysA"), encodes DPDC, which has the amino acid sequence of SEQ ID NO: 14, indicated in the list of sequences. As a specific example of a gene lysA, you can specify DNA, which contains the nucleotide sequence consisting of nucleotide numbers of nucleotides 751-1995 in SEQ ID NO: 13. Gene lysA according to the invention can have the sequence in which the codon corresponding to each of the amino acids are replaced by equivalent codon, provided that it encodes the same amino acid sequence as the amino acid sequence of SEQ ID NO: 14.

The gene of any of the enzyme according to the invention may have an amino acid sequence corresponding to any of amino acid sequence SEQ ID NO: 6, 8, 10, 12 or 14, including replacement, deletions, insertions, to recognize the giving or inversion of one or several amino acids, and can encode a protein having the activity of AK, ASD, DDPS, DDPR or DPDC. Used herein, the expression “one or more” preferably means a number from 1 to 10, more preferably a number from 1 to 5, more preferably a number from 1 to 2.

The DNA that encodes essentially the same protein, as AK, ASD, DDPS, DDPR or DPDC as those described above, can be obtained by modifying each nucleotide sequence to amino acid sequence contained a substitution, deletions, insertions, accession or inversion of amino acid residue or residues at a specific site, for example, by site-specific mutagenesis. Such a modified DNA, which is described above, can also be obtained by traditional mutagenic effects. Examples of mutagenic effects include processing in vitro DNA coding AK, ASD, DDPS, DDPR or DPDC, hydroxylamine or similar means, the processing of the microorganism, such as bacteria Escherichia containing the gene encoding AK, ASD, DDPS, DDPR or DPDC, UV radiation or mutagenic agents used in conventional mutagenic treatments, such as N-methyl-N’-nitro-N-nitrosoguanidine (NTG) and nitrous acid.

The above substitution, deletion, insertion, joining or inversion of nucleotides comprises a mutation of natural origin (Muta is t or a variant), such as mutations observed depending on differences between species or strains of microorganisms, containing AK, ASD, DDPS, DDPR or DPDC and so on.

The DNA that encodes essentially the same protein that AK, ASD, DDPS, DDPR or DPDC, you can get by creating the opportunity for the expression of DNA having such a mutation as described above in an appropriate cell, and evaluating the activity AK, ASD, DDPS, DDPR or DPDC product expression. The DNA that encodes essentially the same protein that AK, ASD, DDPS, DDPR or DPDC, can also be obtained by separation of the DNA coding for AK, ASD, DDPS, DDPR or DPDC, which have a mutation, or from cells containing each of the DNA, the DNA can gibridizatsiya with a sample containing a nucleotide sequence that comprises the nucleotide sequence of nucleotide numbers 510-1736 SEQ ID NO: 5, a nucleotide sequence comprising the nucleotide sequence of nucleotide numbers 98-1207 SEQ ID NO: 7, the nucleotide sequence comprising the nucleotide sequence of nucleotide numbers 1268-2155 SEQ ID NO: 9, the nucleotide sequence comprising the nucleotide sequence of nucleotide numbers 2080-2883 SEQ ID NO: 11, or the nucleotide sequence comprising the nucleotide sequence of nucleotide numbers 751-1995 SEQ ID O: 13, or part of these nucleotide sequences, in hard conditions, and encodes a protein having activity of AK, ASD, DDPS, DDPR or DPDC. In this description to have a nucleotide sequence or part of it means to have a nucleotide sequence or part of it, or she complementary nucleotide sequence.

Used herein, the term “stringent conditions” means conditions which allow for formation of a so-called specific hybrid and do not allow to form a non-specific hybrid. These conditions may vary depending on the nucleotide sequence and length of the sample. However this may be, for example, the condition that ensures hybridization highly homologous DNA, such as DNA having a homology of 40% or higher, but does not allow the hybridization of the DNA with lower homology than specified above, or condition that provides hybridization under conditions of washing for traditional southern hybridization at a temperature of 60° and salt concentrations corresponding to 1 × SSC and 0.1% SDS, preferably 0.1 to × SSC and 0.1% SDS.

As a sample you can also use a partial sequence of each gene. This sample can be obtained by PCR (polymerase chain reaction) using oligonucleotides derived from the nucleotide at which sledovatelnot each gene, as primers, and a DNA fragment containing each gene, as a matrix. In the case when the samples using a DNA fragment having a length of about 300 BP, conditions of leaching under hybridization can be, for example, 50°, 2 × SSC and 0.1% SDS.

Genes that hybridize under these conditions, listed above, also include genes with a stop codon in their sequence, and genes encoding the enzyme, not with more of their activity due to mutation of the active site. However, such genes can be easily excluded by ligating genes in commercially available vector expressing the activity, and measuring the activity of AK, ASD, DDPS, DDPR or DPDC.

Because the nucleotide sequences of genes that encode AK, ASD, DDPS, DDPR or DPDC derived from Methylophilus methylotrophus discovered by this invention, the DNA sequences that encode AK, ASD, DDPS, DDPR or DPDC, can be obtained from a library of genes Methylophilus methylotrophus by hybridization using oligonucleotide samples obtained on the basis of sequence data. In addition, DNA sequences that encode these enzymes can also be obtained by amplification from chromosomal DNA Methylophilus methylotrophus by PCR using oligonucleotide primers derived on the Nove of the above nucleotide sequences.

The above-mentioned genes can be properly used to enhance the ability of Methylophilus bacteria to produce L-lysine.

EXAMPLES

The invention will be further described specifically with reference to the following examples.

The used reagents were obtained from Wako Pure Chemicals or Nakarai Tesque, unless otherwise stated. The composition used in each example of the environments shown below. the pH was brought by using NaOH or Hcl for all environments.

Environment (L)

Lactotropes (DIFCO) 10 g/l

Yeast extract (DIFCO) 5 g/l

NaCl 5 g/l

[Steam sterilization at 120° C for 20 minutes]

(Agar medium L)

Environment L

Baktagir (DIFCO) 15 g/l

[steam sterilization at 120°C for 20 minutes]

(Wednesday SOC)

Lactotropes (DIFCO) 20 g/l

Yeast extract (DIFCO) 5 g/l

10 mm NaCl

2.5 mm KCl

10 mm MgSO4

10 mm MgCl2

20 mm glucose

[Components, with the exception of a solution of magnesium and glucose, were sterilized by steam (120° C, 20 minutes), and then thereto was added 2 M mother solution of magnesium (1 M MgSO4, 1 M MgCl2) and 2 M glucose solution, the solutions which have been previously passed through a filter with a pore diameter of 0.22 μm, and the mixture was passed again through a 0.22 μm filter.]

(Wednesday M)

To2NRA41.2 g/l

KH2PO40,62 g/l

NaCl 0.1 g/l

(NH4)2SO40.5 g/l

MgSO4· 7H2O 0.2 g/l

CaCl2·6N2About 0.05 g/l

Fl3·6H2O 1.0 mg/l

H3IN310 ág/l

CuSO4·5H2O 5 µg/l

MnSO4·5H2O 10 ág/l

ZnSO4·7H2O 70 µg/l

NaMoO4·2H2O 10 ág/l

CoCl2·6H2O 5 µg/l

Methanol 1% (vol./vol.), pH 7.0

[Components, with the exception of methanol, sterilized with steam at 121° C for 15 minutes. After sufficient cooling of the components were added methanol.]

(The composition of the medium 121 for production)

Methanol 2%

Phosphate potassium 0,12%

Potassium phosphate 0,062%

The uranyl chloride calcium 0,005%

The heptahydrate of magnesium sulfate 0,02%

Sodium chloride 0,01%

The uranyl chloride iron 1.0 mg/l

Ammonium sulfate 0,3%

The pentahydrate of copper sulfate 5 mg/l

Pentahydrate sulfate manganese 10 ág/l

The dihydrate of sodium molybdate 10 ág/l

Boric acid 10 ág/l

Heptahydrate zinc sulfate 70 µg/l

The uranyl chloride cobalt 5 µg/l

Calcium carbonate (Kanto Called) 3%

(pH 7.0)

(Agar medium N)

Wednesday M

Baktagir (DIFCO) 15 g/l

[Components, with the exception of methanol, sterilized with steam at 121° C for 15 minutes. After sufficient cooling of the components were added methanol.]

(Minimum with the food M9)

Na2HPO4·N2About 16 g/l

KH2PO43 g/l

NaCl 0.5 g/l

NH4Cl 1 g/l

MgSO4·7H2O 246,48 mg/l

glucose 2 g/l

pH 7.0

[MgSO4and glucose was sterilized separately (120° C, 20 minutes) was added. If necessary, was added the appropriate amount of amino acids and vitamins.]

(Minimum agar medium M9)

Minimal medium M9

Baktagir (DIFCO) 15 g/l

Example 1

Creating bacteria producing L-lysine (1)

(1) the Introduction of mutant lysC and mutant dapA in a Methylophilus bacterium.

Mutant lysC and mutant dapA was introduced into the Methylophilus bacterium, using the containing known plasmid RSFD80 (see WO 95/16042). RSFD80 is a plasmid pVIC40 (international patent application WO 90/04636 published patent application Japanese (Kohyo) No.3-501682/1991)obtained from the vector plasmid pAYC32 with a wide range of hosts (Chistorerdov, A.Y., Tsygankov, Y.D., Plasmid, 16, 161-167, (1986)), which is a derivative of RSF1010, in which the mutant dapA and mutant lysC, obtained from E. coli, localized in this order downstream of the promoter (tetP) resistance gene for tetracycline plasmid pVIC40, so that the direction of transcription of the genes relative to normal tetP. Mutant dapA encode mutant DDPS, in which the histidine residue 118 replaced by a tyrosine residue. Mutant lysC encode mutant AKIII, what toroi residue threonine 352 has been replaced by isoleucine residue.

RSFD80 was designed as follows. Mutant dapA plasmid pdapAS24 ligated with pVIC40 in position downstream of the promoter of the gene of resistance to tetracycline, to get RSF24P, as shown in figure 1. Then got plasmid RSFD80, which had a mutant dapA and mutant lysC, RSF24P and pLLC*80 containing mutant lysC, as shown in figure 2. That is, while pVIC40 contains the threonine operon, the specified threonine operon in RSFD80 replaced by a DNA fragment containing the mutant dapA, and the DNA fragment containing mutant lysC.

The strain E. coli JM109 transformed with the plasmid RSFD80, named AJ12396 and deposited in National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (postal code 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on October 28, 1993, and received inventory number FERM P-13936, and transferred to international Deposit under the terms of the Budapest agreement November 1, 1994, and received inventory number FERM BP-4859.

Strain AJ1239 E. coli was cultured in 30 ml of LB medium containing 20 mg/l streptomycin, at 30° within 12 hours, and plasmid RSFD80 was purified from the obtained cells using the system for purification of DNA Wizard® Plus Midipreps (sold Promega).

Plasmid RSFD80, obtained as described above was introduced into a strain AS1 Methylophilus methylotrophus (NCIMB10515) by electroporation (Canadian Journal of Microbiology, 43, 197 (1997)). As controleren DNA encoding threonine operon, delegated from the plasmid pVIC40 used to obtain the plasmid RSFD80, to obtain the plasmid pRS containing only vector area (see published patent application Japanese (Kohyo) No.3-501682/1991), and plasmid pRS was introduced into a strain AS1 in the same way, which is used for RSFD80.

(2) the Activity AKIII Methylophilus bacteria containing mutant lysC and mutant dapA, obtained from E. coli.

Received cell-free extracts of strain AS1 Methylophilus methylotrophus containing plasmid RSFD80 (also hereinafter referred to as “AS1/RSFD80”), and strain AS1 Methylophilus methylotrophus containing plasmid pRS (also hereinafter referred to as “AS1/pRS”), and measured the activity AK. Cell-free extracts (crude solutions of enzymes) was prepared as follows. Each of the strains AS1/RSFD80 and AS1/pRS was inoculable on Wednesday for producing 121 of the above composition containing 20 mg/ml of streptomycin, and cultured at 37° C for 34 hours with shaking, and then remove calcium carbonate and collected cells.

Bacterial cells obtained as described above were washed with 0.2% KCl in the conditions of 0° C, suspended in 20 mm potassium phosphate buffer (pH 7)containing 10 mm MgSO4, 0.8 M (NH4)2SO4and 0.03 M β -mercaptoethanol, and was destroyed by ultrasound (0° C, 200 W, 10 minutes). The suspension is treated with ultrasound cells centrifuging the Ali at 33000 rpm for 30 minutes under conditions of 0° With, and separated adosados. To nadeshiko was added ammonium sulfate to 80% saturation, and the mixture was left at 0° 1 hour, and centrifuged. The precipitate was dissolved in 20 mm potassium phosphate buffer (pH 7)containing 10 mm gSO4, 0.8 M (NH4)2SO4and 0.03 M β -mercaptoethanol.

Measurement of the activity AK was performed by the method of Stadtman (Stadtman, E.R., Cohen, G.N., LeBras, G., and Robichon-Szulmajster, H., J.Biol. Chem., 236, 2033 (1961)). Namely, the reaction solution of the following composition were incubated at 30° C for 45 minutes, and the manifestation of staining caused by a solution of Fl3(2,8 N Hcl: 0.4 ml, 12% THU: 0.4 ml, 5% Fl3·6N2O/a 0.1 N Hcl: 0.7 ml). The reaction solution was centrifuged, and measured the optical density nadeshiko at 540 nm. The activity represented in units of number of hydroxamic acids obtained in 1 minute (1 unit = 1 μmol/min). The molar extinction coefficient is assumed to be 600. As a blank control was used the reaction solution containing no potassium aspartate. After we measured the enzymatic activity, to the reaction solution of enzyme was added L-lysine in various concentrations, to assess the degree of inhibition of L-lysine. The results are shown in table 1.

(The composition of the reaction solution)

The reaction mixture*10.3 ml

A solution of hydroxylamine*2 0.2 ml

0.1 M potassium aspartate (pH 7.0) 0.2 ml

A solution of 0.1 ml enzyme

Water (the rest) total volume of 1 ml

* 1: 1 M Tris-Hcl (pH 8.1): 9 ml, 0.3 M MgSO4: 0.5 ml and 0.2 M ATP (pH 7.0): 5 ml

* 2: 8 M solution of hydroxylamine, neutralized CON immediately before use

Table 1
StrainActivity AK (specific activity*1)The specific activity in the presence of 5 mm L-lysineThe degree of desensitization of inhibition*2(%)
AS1/pBSto 7.939,07114
AS1/RSFD80made 13.3615,33115
* 1: nmol/min/mg protein

* 2: the Degree of preservation activity in the presence of 5 mm L-lysine.

As shown in table 1, the activity of AK was increased approximately 1.7-fold with the introduction of the plasmid RSFD80. In addition, it was confirmed that the AK derived from E. coli, which encodes a plasmid RSFD80 was completely desensitized to inhibition by L-lysine. In addition, it was found that AK, which in the initial state was preserved in strain AS1, not inhibited one L-lysine. The authors according to the invention have found that AK, obtained from strain AS1, and who was hyperovals 100% in the case when the reaction solution was attended by L-lysine and L-threonine to 2 mm each (mutual inhibition).

(3) Production of L-lysine by Methylophilus bacterium containing mutant lysC and mutant dapA, obtained from E. coli.

Then strain AS1/RSFD80 and strain AS1/pRS was inoculable on Wednesday for producing 121 containing 20 mg/l of streptomycin, and cultured at 37° C for 34 hours with shaking. After culturing bacterial cells and the calcium carbonate was removed by centrifugation, and nadeshiko culture was measured concentration of L-lysine in the amino acid analyzer (JASCO Corporation [Nihon Bunko], high performance liquid chromatography). The results are shown in table 2.

Table 2
StrainProduced amount of the hydrochloride of L-lysine (g/l)
AS1/pRS0
AS1/RSFD800,3

Example 2

Creating bacteria producing L-lysine (2)

(1) Introduction to the promoter region of tac in the vector with a wide range of hosts.

In order to obtain large quantities of the enzyme involved in the biosynthesis of L-lysine (Lys) from Methylophilus methylotrophus for gene expression of the target enzyme used the tac promoter. This promoter is frequently used in E. coli.

The promotor region tac amplification by PCR using as template DNA RCC-3 (Pharmacia), and as primers in DNA fragments having the nucleotide sequence of SEQ ID NO: 15 and 16, and thermoresistant DNA polymerase. PCR was performed using the cycle at 94° C for 20 seconds, at 60° C for 30 seconds, and 72° C for 60 seconds, which was repeated 30 times. Then amplificatory the DNA fragment was collected and treated with restriction enzymes EcoRI and > PST. On the other hand, the vector pRS with a wide range of hosts (see published patent application Japanese (Kohyo) No.3-501682/1991) was also digested with the same enzymes, and the above-mentioned DNA fragment, which contained the promoter region tac, was introduced between the ends, obtained by digestion with restriction enzymes, to construct pRS-tac.

(2) Obtaining the plasmid pRS-dapA24 expressing the dapA gene (gene dihydrodipicolinate), and the plasmid pRS-lysC80 expressing the gene lysC (gene aspartokinase).

Mutant gene (dapA* 24)encoding dihydrodipicolinate, enzymatic activity, which was partially desensitized towards the inhibition of amino acid Lys on the basis of feedback, was inserted in the plasmid pRS-tac, which was obtained by a method described above in (1).

First received the district dapA gene* 24 amplification by PCR, using as template DNA RSFD80 (see example 1), and the DNA fragments imeyushayasyaya sequence SEQ ID NO: 17 and 18, as primers. PCR was performed using the cycle at 94° C for 20 seconds, at 60° C for 30 seconds, and 72° C for 90 seconds, which was repeated 30 times. Then the fragment was treated with restriction enzymes Sse8387l and XbaI to obtain a fragment of the gene dapA* 24 having corresponding split ends. On the other hand, the vector pRS-tac also worked Sse8387I and partially digested XbaI in the same manner as described above. This digested plasmid ligated to the above fragment of the gene dapA* 24 using T4 ligase to obtain pRS-dapA24.

Similarly gene (ls* 80), encoding aspartokinase, enzymatic activity, which was partially desensitized towards the inhibition of amino acid Lys on the basis of feedback received by PCR, using as template DNA RSFD80, and the DNA fragments having the nucleotide sequence of SEQ ID NO: 19 and 20, as primers. PCR was performed using the cycle at 94° C for 20 seconds, at 60° C for 30 seconds, and 72° C for 90 seconds, which was repeated 30 times. Then, the obtained DNA fragment was treated with restriction enzymes Sse8387l and Sapl. On the other hand, the vector pRS-tac also worked Sse8387l and Sapl. This digested plasmid ligated to the above fragment of the gene lysC* 80, use the I ligase T4, to get the pRS-lysC80.

(3) the introduction of the pRS-dapA24 or pRS-lysC80 in Methylophilus methylotrophus and evaluation culture.

Each of the plasmid pRS-dapA24 and pRS-lysC80 obtained as described above was introduced into a strain AS1 Methylophilus methylotrophus (NCIMB10515) by electroporation to obtain respectively AS1/pRS-dapA24 and AS1/pRS-lys80. Each strain was inoculable on Wednesday for producing 121 containing 20 mg/l of streptomycin, and cultured at 37° C for 48 hours with shaking. As a control strain in a similar manner also cultivated strain AS1 carrying pRS. After completion of culturing cells and the calcium carbonate was removed by centrifugation, and nadeshiko culture was measured concentration of L-lysine in the amino acid analyzer (JASCO Corporation [Ninon Bunko], high performance liquid chromatography). The results are shown in table 3.

Table 3
StrainProduced amount of the hydrochloride of L-lysine (g/l)
AS1/pRS<0,01
AS1/pRS-lysC800,06
ASl/pRS-dapA240,13

Example 3

Creating bacteria producing L-lysine (3)

Strain AS1 Methylophilus methylotrophus (NCIMB10515) was inoculable on Wednesday M and cultivated at 37° C for 15 hours. The obtained bacterial cells on relatively NTG in a standard way (the concentration of NTG: 100 mg/l, 37° C, 5 minutes), and put on agar medium M containing 7 g/l S-(2-amino-ethyl)cysteine (AEC) and 3 g/l of L-threonine. Cells were cultured at 37° With from 2 to 8 days and collected formed the colony, getting resistant AES strains.

The above-resistant AES strains were inoculable on Wednesday for producing 121 and cultivated at 37°C for 38 hours in aerobic conditions. After culturing cells and the calcium carbonate was removed from the medium by centrifugation and nadeshiko culture was measured concentration of L-lysine in the amino acid analyzer (JASCO Corporation [Nihon Bunko], high performance liquid chromatography). Selected strain expressing enhanced ability to produce L-lysine in comparison with the parent strain, and called strain AR-166 Methylophilus methylotrophus. The amount of L-lysine produced by the parent strain (strain AS1) and strain AR-166, shown in table 4.

Table 4
StrainProduced amount of the hydrochloride of L-lysine (g/l)
AS15,8
AR-16680

Strain AR-166 Methylophilus methylotrophus got their own room AJ13608, and he deposited in National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (octavii index 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on 10 June 1999 and received inventory number FERM P-17416, and transferred to international Deposit under the terms of the Budapest agreement on March 31, 2000, and received inventory number FERM BP-7112.

Example 4

Creating bacteria producing L-threonine.

(1) the Introduction of plasmids with treningowy the operon in the bacterium Methylophilus.

Plasmid pVIC40 (international patent application WO 90/04636 published patent application Japanese (Kohyo) No.3-501682/1992)containing the threonine operon derived from E. coli was introduced into a strain AS1 Methylophilus methylotrophus (NCIMB10515) by electroporation (Canadian Journal of Microbiology, 43, 197 (1997))to obtain the strain AS1/pVIC40. As a control received pRS (published patent application Japanese (Kohyo) No.3-501682/1991)with vector only area, by delegation from the plasmid pVIC40 region DNA encoding the threonine operon, and this plasmid was introduced into a strain AS1 in the same way, which is used for pVIC40, getting strain AS1/pRS.

(2) Production of L-threonine by Methylophilus bacterium containing the threonine operon derived from E. coli.

Each of the strains AS1/pVIC40 and AS1/pRS was inoculable on Wednesday for producing 121 containing 20 mg/l streptomycin, 1 g/l L-valine and 1 g/l L-leucine, and cultivated at 37° C for 50 hours with shaking. After culturing cells and the calcium carbonate was removed what centrifugational, and nadeshiko culture was measured concentration of L-threonine in the amino acid analyzer (JASCO Corporation [Ninon Bunko], high performance liquid chromatography). The results are shown in table 5.

Table 5
StrainProduced amount of L-threonine (mg/l)
AS1/pRS15
AS1/pVIC4030

Example 5

Creating bacteria, producing the branched chain amino acids.

Strain AS1 Methylophilus methylotrophus (NCIMB10515) was inoculable on Wednesday M and cultivated at 37° C for 15 hours. The obtained bacterial cells were treated with NTG in the standard way (the concentration of NTG: 100 mg/l, 37° C, 5 minutes), and put on agar medium M containing 0.5% Kazarinova acids (DIFCO). Cells were cultured at 37° With from 2 to 8 days and allowed to form colonies. Formed colonies were collected and inoculable in agar medium M and agar medium M containing 0.5% Kazarinova acid. Selected strains exhibiting better growth in the latter environment, compared with the first environment, as the strains auxotrophic for Kazarinova acid. Thus, from 500 treated NTG strains received 9 strains auxotrophic for Kazarinova acid, maintaining the receiving residual level of expression. Of these auxotrophic for Kazarinova acid strains received one strain that accumulated in the environment more L-valine, L-leucine and L-isoleucine compared to its parent strain. The specified strain was named strain S Methylophilus methylotrophus.

Strain S Methylophilus methylotrophus got their own room AJ13609, and he deposited in National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (postal code 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on 10 June 1999 and received inventory number FERM P-17417, and transferred to international Deposit under the terms of the Budapest agreement on March 31, 2000, and received inventory number FERM BP-7113.

The parent strain (strain AS1) and strain S was inoculable on Wednesday for producing 121 and cultivated at 37° C for 34 hours in aerobic conditions. After culturing cells and the calcium carbonate was removed from the medium by centrifugation and nadeshiko culture was measured concentrations of L-valine, L-leucine and L-isoleucine at amino acid analyzer (JASCO Corporation [Ninon Bunko], high performance liquid chromatography). The results are shown in table 6.

Table 6
StrainL-valine (mg/l)L-leucine (mg/l)L-isoleucine (mg/l)
7,55,02,7
S330166249

Example 6

Obtaining a library of chromosomal DNA Methylophilus methylotrophus strain AS1.

(1) preparation of chromosomal DNA Methylophilus methylotrophus strain AS1.

One platinum loop Methylophilus methylotrophus strain AS1 (NCIMB10515) were sown in 5 ml of medium M in a test tube and cultivated at 37° C overnight with shaking. The obtained culture broth was inoculable in 50 ml of medium M in the flask Sakaguchi volume of 500 ml at a rate of 1% and cultured at 37° during the night, shaking. Then the cells were collected by centrifugation and suspended in 50 ml of TEN (solution containing 50 mm Tris-Hcl (pH 8.0), 10 mm EDTA and 20 mm NaCl (pH 8.0)). The cells were collected by centrifugation and again suspended in 5 ml of TEN containing 5 mg/ml lysozyme and 10 µg/ml RNase A. the Suspension was kept at 37° C for 30 minutes, and then thereto was added proteinase K and sodium dodecyl sulfate to a final concentration of 10 μg/ml and 0.5% (wt./about.) respectively.

The suspension is kept at 70° C for 2 hours and then added an equal amount of saturated solution of phenol (phenol saturated with 10 mm Tris-Hcl (pH 8.0)and stirred. The suspension was centrifuged, and the collected adosados. Added an equal amount of solution pheno is a/chloroform (phenol:chloroform:isoamyl alcohol = 25:24:1) and stirred, and the mixture was centrifuged. Adosados collected and to it was added an equal quantity of a solution of chloroform (chloroform:isoamyl alcohol = 24:1)to repeat the same procedure extraction. To nadeshiko was added 1/10 volume of 3 M sodium acetate (pH of 4.8) and 2.5-fold volume of ethanol to precipitate chromosomal DNA. Sediments were collected by centrifugation, washed with 70% ethanol, dried under reduced pressure and dissolved in an appropriate amount of THE solution (10 mm Tris-HCl, 1 mm EDTA (pH 8.0)).

(2) obtaining a library of genes.

Portions of the chromosomal DNA obtained as described above in (1)with a volume of 50 μl (1 μg/μl), 20 μl of buffer B (500 mm Tris-HCl, 100 mm MgCl2, 10 mm dithiothreitol, 1000 mm NaCl (pH 7.5)) and 8 units of the restriction enzyme Sau3AI (Takara Shuzo) was allowed to react at 37° C for 10 minutes in a total volume of 200 μl, and then to stop the reaction, was added 200 μl of phenol/chloroform and stirred. The reaction mixture was centrifuged and collected the top layer and separated by 0.8% agarose gel. DNA corresponding to from 2 to 5 thousand pairs of nucleotides (hereinafter abbreviated label “TPN”), collected using a system of rapid extraction from the gel Concert™ (set to collect DNA, GIBCO BRL Co.). Thus, received 50 μl of the DNA solution fractionated size.

On the other hand 2.5 ág plasmid pUC118 (Takara Shuzo), 2 m the l buffer (200 mm Tris-HCl, 100 mm MgCl2, 10 mm dithiothreitol, 1000 mm KCl (pH 8.5)and 10 units of restriction enzyme BamHI (Takara Shuzo) was allowed to react at 37° C for 2 hours in a total volume of 20 μl, was then added 20 units of alkaline phosphatase of the small intestine of the calf (Takara Shuzo) and stirred, the mixture was allowed to react for another 30 minutes. The reaction mixture was mixed with an equal amount of phenol/chloroform, and the mixture was centrifuged. Collected adosados and to it was added an equal quantity of a solution of chloroform, to repeat a similar procedure for the extraction. To nadeshiko was added 1/10 volume of 3 M sodium acetate (pH of 4.8) and 2.5-fold volume of ethanol to precipitate the DNA. DNA was collected by centrifugation, washed with 70% ethanol, dried under reduced pressure and dissolved in the appropriate amount of solution.

The product of digestion of chromosomal DNA Sau3AI, obtained as described above, and the product of digestion of pUC118 the restriction enzyme BamHI ligated using the set for ligating, version 2 (Takara Shuzo). To the reaction mixture were added 1/10 volume of 3 M sodium acetate (pH of 4.8) and 2.5-fold volume of ethanol to precipitate the DNA. DNA was collected by centrifugation, washed with 70% ethanol, dried under reduced pressure and dissolved in THE solution (solution And ligase).

In the same manner as in the above-described procedure, ligated fragments, the floor is built in a partial digestion of chromosomal DNA with the restriction enzyme AluI (Takara Shuzo), and SmaI digestion of the plasmid pSTV29 (Takara Shuzo) (solution for ligase).

One platinum loop of E. coli JM109 was inoculable in 5 ml of medium L in a test tube and cultivated at 37° during the night, shaking. The obtained culture broth was inoculable in 50 ml of medium L flask Sakaguchi volume of 500 ml quantity of 1%, were cultured at 37° before OD660culture reached values from 0.5 to 0.6, and cooled on ice for 15 minutes. Then the cells were collected by centrifugation at 4° C. the Cells are suspended in 50 ml of ice-cold water and centrifuged to wash the cells. This operation was repeated once again, and the cells suspended in 50 ml of ice-cold 10% solution of glycerol, and centrifuged to wash the cells. Cells suspended in 10% glycerin solution of the same volume as the volume of the cells, and divided into aliquots of 50 ál. To cells in a volume of 50 μl was added to 1 μl of the solution And ligase or solution for ligase obtained as described above. Then the mixture was placed in a special cell (with a gap of 0.1 cm, pre-cooled on ice) for a device for electroporation BioRad.

The settings of the instrument were equal to 1.8 kV and 25 μf, and the system settings of the control pulses is 200 Ohms. The cuvette was inserted into the device and applied to her impulses. Immediately after exposure pulses to it was added 1 ml Le is Jana environment SOC, and the mixture was transferred into a sterile test tube and cultivated at 37°C for 1 hour, shaking. The broth from each cell culture were applied to the agar medium L containing antibiotic (100 μg/ml of ampicillin in the case of using the solution And ligase or 20 µg/ml chloramphenicol in the case of using the solution for ligase), and incubated at 37° With during the night. Colonies that appeared on each agar medium were scraped, inoculable in 50 ml of medium L containing the appropriate antibiotic in the flask Sakaguchi 500 ml and cultured at 37° C for 2 hours, shaking. Plasmid DNA was extracted from each broth culture by the alkaline method with SDS to get the solution And the libraries of genes and the solution In the libraries of genes, respectively.

Example 7

Cloning of the gene for biosynthesis of lysine Methylophilus methylotrophus strain AS1.

(1) Cloning of the gene encoding aspartokinase (AK).

Cells of E. coli GT3, scarce three genes coding for AK (thrA, metLM and lysC), transformed solution In the libraries of genes through the same procedure of electroporation, as described above. To a solution for the transformation was added Wednesday SOC containing 20 μg/ml diaminopimelic acid and cultured at 37° C, shaking. Then the culture broth was spread upon the environment L containing 20 μg/ml diaminopimelic the howling acid and 20 μg/ml chloramphenicol, to obtain the emerging colony. With the obtained Cup as the main matrix of the received replica on agar medium M9 containing 20 μg/ml of chloramphenicol, and the replica incubated at 37° With 2 to 3 days. The owner could not grow on minimal medium M9, which did not contain diaminopimelic acid, as it had no activity AK. On the contrary, it was expected that the transformed strain that contains the gene encoding the AK derived from Methylophilus methylotrophus was able to grow in minimal medium M9 due to the operation of the gene.

Two transformant approximately 3000 transformants formed colonies on M9 medium. Of these colonies that appeared on the environment M9, were extracted and analyzed plasmids. The result was confirmed by the presence of the plasmid built-in fragment. Plasmids were named S-1 and pMMASK-2, respectively. Using these plasmids, again transformed E. coli GT3. The obtained transformants were able to grow on minimal medium M9. Then the transformant, which contained each of these plasmids were cultured overnight in medium L containing 20 μg/ml of chloramphenicol, and the cells were collected by centrifugation of the broth culture. Cell-free extracts were obtained, treating the cells with ultrasound, and measured the activity of the AK method Miyajima et al. (Journal of Biochemistry (Tokyo), vol.63, 139-148 (1968)) (3: pMMASK-1, pMMASK-2). In addition to t the th, the GT3 strain carrying the vector pSTV29, similarly cultured in medium L containing 20 μg/ml diaminopimelic acid and 20 μg/ml of chloramphenicol, and measured the activity of AK (3: Vector). In the observed increased activity AK in two clones containing embedded fragments, compared with transformants carrying only the vector. Therefore, it was confirmed that the gene that has been cloned into pSTV29, represented gene AK derived from Methylophilus methylotrophus. Specified gene was identified ask.

The sequence of DNA nucleotides ask gene was determined by the method using dideoxynucleotides. It was found that S-1 and pMMASK-2 contained a common fragment. The nucleotide sequence of the DNA fragment containing the gene ask derived from Methylophilus methylotrophus shown in SEQ ID NO: 5. Amino acid sequence that can encode the nucleotide sequence shown in SEQ ID NO: 5 and 6.

(2) Cloning of the gene encoding the dehydrogenase tolualdehyde aspartic acid (ASD).

Cells of E. coli Hfr3000 U482 (strain CGSC 5081), deficient in asd gene, transformed by electroporation using the solution In the libraries of genes, in the same way as described above. To a solution for the transformation was added Wednesday SOC containing 20 μg/ml diaminopimelic acid, and the mixture was cultured at 37° C, shaking. CL the TCI was collected by centrifugation. Cells were washed, suspending them in the environment L and centrifuger suspension. The same washing operation was again repeated once, and the cells suspended in the medium of L. Then the suspension was distributed on agar medium L containing 20 μg/ml of chloramphenicol, and incubated overnight at 37° C. the Owner showed very low growth environment L that does not contain diaminopimelic acid, as it was deficient in asd gene. On the contrary, it was expected that normal growth would be observed for the transformed strain that contains the gene encoding the ASD derived from Methylophilus methylotrophus, even in the environment of L due to the operation of the gene. In addition, the host cells E. coli was unable to grow in minimal medium M9, but it was expected that the transformed strain that contains the gene encoding the ASD derived from Methylophilus methylotrophus, will be able to grow on minimal medium M9 due to the operation of the gene. Therefore, colonies of transformants, which are normally grown in the medium L, gathered, were sown by the bar and were cultured on agar medium M9. In the observed growth. Thus, confirmed that the gene encoding the ASD, as expected, functioned in these transformants.

Of the three transformed strains that appeared on the M9 medium, and plasmids were extracted and confirmed the presence of the plasmid built-in fragment. Plasma the s were named pMMASD-1, pMMASD-2 and pMMASD-3, respectively. When E. coli Hfr3000 U482 again transformed using the plasmids, each transformant was grown on minimal medium M9. Then each transformant was cultured overnight in medium L containing 20 μg/ml of chloramphenicol, and the cells were collected by centrifugation of the broth culture. Cells were treated with ultrasound, receiving the crude solution of enzymes, and measured the activity of ASD method Boy et al. (Journal of Bacteriology, vol.112 (1), 84-92 (1972)) (4: pMMASD-1, pMMASD-2, pMMASD-3). In addition, as a control experiment, the host cells carrying the vector, similarly cultured in medium L containing 20 μg/ml diaminopimelic acid and 20 μg/ml of chloramphenicol, and measured the activity of ASD (figure 4: Vector). As a result of enzymatic activity was not detected in transformants carrying only the vector, while the activity of ASD could be defined in three clones containing the built-in fragment. Therefore, it was confirmed that the obtained gene was a gene that encodes ASD derived from Methylophilus methylotrophus (marked asd).

The nucleotide sequence of the DNA asd gene was determined by the method using dideoxynucleotides. It was found that all three of the resulting clone contains common code fragment. The nucleotide sequence of the DNA fragment containing the asd gene, derived from Methylophilus methyotrophus, shown in SEQ ID NO: 7. Amino acid sequence that can encode the nucleotide sequence shown in SEQ ID NO: 7 and 8.

(3) Cloning of the gene encoding dihydrodipicolinate (DDPS).

Cells of E. coli AT997 (strain CGSC 4547), scarce dapA gene, transformed by the same procedure of electroporation using a mortar And libraries of genes. To a solution for the transformation was added Wednesday SOC containing 20 μg/ml diaminopimelic acid, and the mixture was cultured at 37° C, shaking. Then the broth culture were distributed on the environment L containing 20 μg/ml diaminopimelic acid and 100 μg/ml ampicillin to obtain the emerging colony. With the obtained Cup as the main matrix of the received replica on minimal agar medium M9 containing 100 μg/ml ampicillin, and the replica incubated at 37° With 2 to 3 days. The owner could not grow on minimal medium M9, which did not contain diaminopimelic acid, as was deficient gene dapA. On the contrary, it was expected that the transformed strain that contains the gene encoding DDPS derived from Methylophilus methylotrophus was able to grow in minimal medium M9 due to the operation of the gene.

From the colonies of the two strains appeared on the environment M9, were extracted and analyzed plasmids. The result confirmed the presence of the plasmid built the first fragment. Plasmids were named pMMDAPA-1 and pMMDAPA-2, respectively. When E. coli AT again transformed using the plasmids, each transformant was grown on minimal medium M9. Then each transformant containing each plasmid was cultured overnight in medium L containing 100 μg/ml ampicillin, and the cells were collected by centrifugation of the broth culture. Cells were treated with ultrasound, receiving cell extract, and measured the activity of DDPS method Yugari et al. (Journal of Biological Chemistry, vol.240, and p.4710 (1965)) (5: pMMDAPA-1, pMMDAPA-2). In addition, as a control experiment, the host cells carrying the vector, similarly cultured in medium L containing 20 μg/ml diaminopimelic acid and 100 μg/ml ampicillin, and measured the activity of DDPS (figure 5: Vector). As a result of enzymatic activity was not detected in transformants carrying only the vector, while the DDPS activity can be determined in each of the transformants carrying plasmids that contain a built-in fragment. Therefore, it was confirmed that the obtained gene was a gene encoding DDPS derived from Methylophilus methylotrophus (indicated by the dapA).

The nucleotide sequence of the DNA dapA gene was determined by the method using dideoxynucleotides. It was found that the two built-in fragment contained a common fragment. Nucleotide on sledovatelnot DNA fragment, containing dapA gene derived from Methylophilus methylotrophus shown in SEQ ID NO: 9. Amino acid sequence that can encode the nucleotide sequence shown in SEQ ID NO: 9 and 10.

(4) Cloning of the gene encoding dihydrodipicolinate (DDPR).

Cells of E. coli AT999 (strain CGSC 4549), deficient dapB gene, transformed by the same procedure of electroporation, as described above, using a mortar And libraries of genes. To a solution for the transformation was added Wednesday SOC containing 20 μg/ml diaminopimelic acid, and the mixture was cultured at 37° C, shaking. Then the cells were collected by centrifugation. Cells were washed, suspending them in the environment L and centrifuger suspension. The same washing operation was again repeated once, and the cells suspended in the medium of L. Then the suspension was distributed on agar medium L containing 100 μg/ml ampicillin, and incubated overnight at 37° C. the Owner showed very low growth environment L that does not contain diaminopimelic acid, as it was deficient in the gene dapB. On the contrary, it was expected that normal growth can be observed for the transformed strain that contains the gene encoding DDPR derived from Methylophilus methylotrophus, even in the environment of L, due to the operation of the gene. In addition, the host cells E. coli was unable to grow in minimal is the ed M9, but it was expected that the transformed strain that contains the gene encoding DDPR derived from Methylophilus methylotrophus, can grow on minimal medium M9 due to the operation of the gene.

Therefore, a colony of transformant, which normally has grown in the medium L, was scraped and cultured on agar medium M9. In this case, also saw growth in the medium M9. Thus, confirmed that the gene encoding DDPR, functioned in the transformed strain. Of the colonies that appeared on the environment M9, were extracted plasmid and confirmed the presence of plasmid built-in fragment. When E. coli AT999 again transformed using a plasmid (pMMDAPB), the transformant was grown on minimal medium M9. Then, the transformant containing the plasmid were cultured overnight in medium L, and the cells were collected by centrifugation of the broth culture. Cells were treated with ultrasound, receiving cell extract, and measured the activity of the DDPR method Tamir et al. (Journal of Biological Chemistry, vol.249, p.3034 (1974)) (6: pMMDAPB). In addition, as a control experiment, the host cells carrying the vector, similarly cultured in medium L containing 20 μg/ml diaminopimelic acid and 100 μg/ml ampicillin, and measured the activity of the DDPR (6: Vector). As a result of enzymatic activity was not detected in transformants carrying only the vector, while the AK is Yunosti DDPR was possible to determine transformant, carrying pMMDAPB. Therefore, it was confirmed that the obtained gene was a gene that encodes a DDPR derived from Methylophilus methylotrophus (indicated by the dapB).

The nucleotide sequence of the DNA dapB gene was determined by the method using dideoxynucleotides. The nucleotide sequence of the DNA fragment containing dapB gene, derived from Methylophilus methylotrophus shown in SEQ ID NO: 11. Amino acid sequence that can encode the nucleotide sequence shown in SEQ ID NO: 11 and 12.

(5) Cloning of the gene encoding diaminopimelate (DPDC).

Cells of E. coli AT2453 (strain CGSC 4505), scarce lysA gene, transformed by the same procedure of electroporation, as described above, using a mortar And libraries of genes. To a solution for the transformation was added Wednesday SOC, and the mixture was cultured at 37° C, shaking. The cells were collected by centrifugation. Cells were washed, suspending them in 5 ml of sterile water and centrifuger suspension. The same washing operation was again repeated once, and the cells suspended in 500 µl of sterile water. Then the suspension was distributed on agar minimal medium M9 containing 20 μg/ml of chloramphenicol, and incubated at 37° With 2 to 3 days. The owner could not grow on minimal medium M9 containing lysine, as he was deficient in the gene lysA. Opposite the harbour is in, it was expected that the transformed strain that contains the gene encoding DPDC derived from Methylophilus methylotrophus, can grow in minimal medium M9 due to the operation of the gene.

So of the three transformed strains that appeared on the environment M9, were extracted and analyzed plasmids. The result confirmed the presence of the plasmid built-in fragment. Plasmids were named pMMLYSA-1, pMMLYSA-2 and pMMLYSA-3, respectively. When E. coli AT2453 again transformed using each of the above plasmids, each transformant was grown on minimal medium M9. Then each transformant containing each of the plasmids were cultured overnight in medium L containing 20 μg/ml of chloramphenicol, and the cells were collected by centrifugation of the broth culture. Cells were treated with ultrasound, receiving cell extract, and measured the activity DPDC method Cremer et al. (Journal of General Microbiology, vol.134, 3221-3229 (1988)) (7: pMMLYSA-1, pMMLYSA-2, pMMLYSA-3). In addition, as a control experiment, the host cells carrying the vector, similarly cultured in medium L containing 20 μg/ml of chloramphenicol, and measured the activity DPDC (7: Vector). As a result of enzymatic activity was not detected in transformants carrying only the vector, while the activity DPDC could be defined in three clones with a built-in fragment. So it was under the approved, the resulting gene was a gene that encodes a DPDC derived from Methylophilus methylotrophus (indicated by lysA).

The nucleotide sequence of the DNA lysA gene was determined by the method using dideoxynucleotides. Discovered that all three of the built-in fragment contained a total DNA fragment. The nucleotide sequence of the DNA fragment containing the gene lysA derived from Methylophilus methylotrophus shown in SEQ ID NO: 13. Amino acid sequence that can encode the nucleotide sequence shown in SEQ ID NO: 13 and 14.

Industrial application

According to this invention provided the Methylophilus bacterium having an ability to produce L-amino acid, a way of producing L-amino acid using a bacterium Methylophilus, and bacterial cells Methylophilus with a high content of L-amino acids. The method according to the invention have the opportunity to get L-amino acid using methanol as feedstock. In addition, this invention provides new genes for enzymes of the biosynthesis of L-lysine derived from Methylophilus bacteria.

1. The method of obtaining L-amino acids except L-glutamic acid, which involves the cultivation of Methylophilus bacteria with the ability to produce L-amino acid in a medium to produce and accumulate the L-amino acids in the culture, and collecting the L-amino acid from the culture, and the Methylophilus bacterium has a high enzymatic activity of the biosynthesis of L-amino acids.

2. The method according to claim 1, characterized in that the medium to cool the cultivated contains methanol as a main carbon source.

3. The method according to claim 1, wherein the L-amino acid is L-lysine, L-valine, L-leucine, L-isoleucine or L-threonine.

4. The method according to claim 1, characterized in that the Methylophilus bacterium has increased dihydrodipicolinate activity and aspartokinase activity and has the ability to produce L-lysine.

5. The method according to claim 1, characterized in that the Methylophilus bacterium has increased dihydrodipicolinate activity and has the ability to produce L-lysine.

6. The method according to claim 1, characterized in that the Methylophilus bacterium has increased aspartokinase activity and has the ability to produce L-lysine.

7. The method according to claim 1, characterized in that the bacterium Methylophilus has elevated activities of aspartokinase, homoerythromycin, homoserine and trionychinae and has the ability to produce L-threonine.

8. The method according to claim 1, characterized in that the bacterium Methylophilus is Methylophilus methylotrophus.

9. The method according to claim 4, characterized in that the bacterium Methylophilus activity dihydrodipicolinate and activity aspartokinase improved by transformation by introducing into the cells a DNA that encodes dihydrodipicolinate, which is not subject to inhibition under the action of L-lysine on the principle of negative feedback, and the NC, coding aspartokinase, which is not subject to inhibition under the action of L-lysine on the principle of negative feedback.

10. The method according to claims 4 to 6, characterized in that the Methylophilus bacterium has a high activity of one, two or three enzymes selected from the dehydrogenase of tolualdehyde aspartic acid, dihydrodipicolinate and diaminomaleonitrile.



 

Same patents:

FIELD: biotechnology, amino acids.

SUBSTANCE: method for preparing taurine involves milling a biological raw, its treatment and isolation of taurine. Waste from meat-processing industry is used as a raw and treatment is carried out with sodium hydroxide solution. Prepared solution is treated successively with protosubtilin, homogenized mass of cattle pancreas or liver. Isolation of taurine from hydrolyzate is carried out by sorption on anion-exchange resin AB-17-2 followed by precipitation of product with 10-fold hydromodulus of ethyl alcohol and the following purification. Method provides increasing yield of taurine in using inedible raw.

EFFECT: improved preparing method, increased yield.

3 ex

FIELD: biotechnology, L-amino acids.

SUBSTANCE: invention relates to a method for preparing such L-amino acids as L-threonine, L-isoleucine, L-valine, L-tryptophan and L-homoserine that involves fermentation with using corynebacteria wherein a nucleotide sequence encoding glutamic acid dehydrogenase is subjected for superexpression. Invention provides elevating yield of indicated amino acids.

EFFECT: improved preparing method.

9 cl, 5 tbl, 5 ex

The invention relates to biotechnology and is a way for L-amino acid using a microorganism belonging to the genus Escherichia or coryneform bacteria

The invention relates to a microbiological method of obtaining aminoacids collection aspartate and/or glutamate on PP 1-17 claims, genes of providerbased on PP 18-23 claims, gene structures by p. 24 claims, vectors for p. 25 claims, transformed cells in PP 26-31 claims, as well as to their application on PP 32-37 claims

The invention relates to biotechnology
The invention relates to the microbiological industry

The invention relates to biotechnology and relates to a method of constructing strains coryneform bacteria with enhanced productivity amino acids, and a method of producing amino acids by fermentation using engineered strains of bacteria coryneform

FIELD: biotechnology, in particular prephenate dehydrotase-chorismatmutase and DNA fragment encoding the same.

SUBSTANCE: prephenate dehydrotase-chorismatmutase is isolated from Methylophilus methylotropus and may contain replacements, deletions, inserts, or incorporations of one or more amino acids. Said enzyme plays an important role in L-phenylalanine biosynthesis. Method of present invention makes it possible to improve L-phenylalanine production due to increased activity of enzymes involving in L-phenylalanine biosynthesis pathway.

EFFECT: improved L-phenylalanine production.

2 cl, 2 dwg, 1 tbl

The invention relates to the field of biotechnology and concerns get a new polyketide-synthase, required for biosynthesis epothilones a and b

The invention relates to biotechnology, in particular genetic engineering

The invention relates to a microbiological method of obtaining aminoacids collection aspartate and/or glutamate on PP 1-17 claims, genes of providerbased on PP 18-23 claims, gene structures by p. 24 claims, vectors for p. 25 claims, transformed cells in PP 26-31 claims, as well as to their application on PP 32-37 claims

The invention relates to biotechnology, in particular the obtaining of cephalosporins using genetically modified organisms-owners

The invention relates to biotechnology, in particular to 3-deoxy-D-arabinoheptulosonate-7-phosphadites and fragment DNA, this enzyme codereuse

FIELD: biotechnology, in particular prephenate dehydrotase-chorismatmutase and DNA fragment encoding the same.

SUBSTANCE: prephenate dehydrotase-chorismatmutase is isolated from Methylophilus methylotropus and may contain replacements, deletions, inserts, or incorporations of one or more amino acids. Said enzyme plays an important role in L-phenylalanine biosynthesis. Method of present invention makes it possible to improve L-phenylalanine production due to increased activity of enzymes involving in L-phenylalanine biosynthesis pathway.

EFFECT: improved L-phenylalanine production.

2 cl, 2 dwg, 1 tbl

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