Polynucleotide coding acyltransferase that responds for modification of platenolid in position 3 (versions), polypeptide that represents acyltransferase, which is responsible for modification of platenolid in position 3 (versions), bacterial expression vector (versions), bacterial expression system, bacterial host cell, method for production of polypeptide, strain streptomyces ambofaciens, application of polynucleotide (versions), cell of bacterium streptomyces ambofaciens, host cell streptomyces ambofaciens and method of polypeptide production

FIELD: biotechnologies.

SUBSTANCE: invention is related to the field of biotechnology, specifically, to separation and identification of new genes of spiramycins biosynthesis track and to new polypeptides, which participate in this biosynthesis, and may be used to produce acyltransferase, which is responsible for modification of platenolid in position 3. Polynucleotide coding acyltransferase, which is responsible for modification of platenolid in position 3, cells of bacterium Streptomyces type are transformed, and strain-producer of end polypeptide is made.

EFFECT: increased extent of production and purity of produced spiramycin.

26 cl, 41 dwg, 44 tbl, 31 ex

 

The present invention relates to the isolation and identification of new genes of the biosynthesis pathway of spiramycin and new polypeptides involved in the biosynthesis. It also relates to the use of these genes to increase production and purity of spiramycin.

The invention relates also to the use of these genes for the design of mutants that can lead to the synthesis of new antibiotics or derivatives of spiramycin. The invention relates also to molecules produced by expression of these genes, and finally to a pharmacologically active composition comprising a molecule produced by expression of such genes.

Streptomyces are gram-positive filamentosa bacteria of the soil. They play an important role in the decomposition and mineralization of organic materials due to the large variety of digestive enzymes that they secrete. They are phenomena unique morphological differentsirovat in prokaryotes, accompanied by metabolic differentiation, characterized by the production of secondary metabolites, with a huge diversity of chemical structures and biological activities. Among these metabolites are spiramycine produced in their native state by the bacterium Streptomyces ambofaciens.

<> Spiramycin is an antibiotic of the family of macrolides, suitable for use both in veterinary and in human medicine. Macrolides are characterized by lactoovo cycle, to which are grafted one or more sugars. Streptomyces ambofaciens in the native state produces spiramycin I, II and III (see figure 1), however, the antibiotic activity of spiramycin I distinctly higher than that of spiramycin II and III (Liu and others, 1999). The molecule spiramycin I formed Laktionova the macrocycle called platanoides, and three sugars: forosamine, mycaminose and microsol (see figure 1). Antibiotic activity of spiramycin occurs due to inhibition of protein synthesis in prokaryotes due to the mechanism leading to the binding of the antibiotic from the bacterial ribosome.

Some compounds that are members of the family of macrolides and have also lackenby cycle, can be used in the field of antibiotics. So, the product FK506 has immunosuppressive effects and opens prospects for therapeutic use in organ transplantation, rheumatoid arthritis and, in General, in the case of pathologies associated with autoimmune reactions. Other macrolides, as avermectin, possess insecticidal and anthelmintic activities.

Numerous biosynthesis pathway related to antibiotics belonging to different to the Assam, and other secondary metabolites (according to the survey, K. Chater, 1990), to date, have been studied in the case of Streptomyces. However, knowledge of the pathways of biosynthesis of spiramycine currently is only very partial.

Biosynthesis spiramycin is a complex process involving multiple steps and mediated numerous enzymes (Omura and others, a; Omura and others, 1979b). Spiramycine belong to a large class of polyketides, which combines complex molecules, especially abundant in soil microorganisms. These molecules are United not by analogy patterns, and some similarity at the level of the stages of the path of their biosynthesis. In fact, are polyketides are produced by a number of complex reactions, but in the way of their biosynthesis together have a number of reactions catalyzed by the enzyme or enzymes called polyketide structures” (PKS). In the case of Streptomyces ambofaciens biosynthesis lactoovo macrocycle of spiramycin (platinoid) flows through a series of eight modules encoded by five different genes PKS (Kuhstoss, S., 1996; U.S. patent 5945320). Spiramycine formed from this lactoovo cycle. However, different stages, and the enzymes involved in the synthesis of sugars, as well as linking them with Laktionova cycle and modification of this cycle to get spiramycine to date remain unknown.

In U.S. patent 5514544 about anywayse cloning sequence, called srmR, in Streptomyces ambofaciens. This patent suggests the hypothesis that the srmR gene encodes a protein regulator of transcription of the genes involved in the biosynthesis of macrolides.

In 1987, Richardson and employees (Richardson and others, 1987) showed that resistance to spiramycin S. ambofaciens is attached by at least three genes, and these last were called srmA, srmB and srmC. In U.S. patent 4886757 in particular describes the characteristics of the DNA fragment of S. ambofaciens, containing the gene for srmC. However, the sequence of this gene was not represented. In 1990, Richardson and employees (Richardson and others, 1990) proposed the hypothesis of the existence of the three genes of the biosynthesis of spiramycin close to srmB. In U.S. patent 5098837 reported the cloning of five genes potentially involved in the biosynthesis of spiramycin. These genes were named srmD, srmE, srmF, srmG and srmH.

One of the great difficulties in obtaining such compounds as spiramycin, is that for relatively small amounts of product required very large amounts during fermentation. Therefore, it is desirable to have the possibility to increase the efficiency of obtaining such molecules in order to reduce production costs.

The path of the biosynthesis of spiramycin is a complex process and it is desirable to identify and eliminate parasitic reactions that can occur in BP is me in this process. The aim of this manipulation is to obtain a more pure antibiotic and/or improved performance. On this occasion, Streptomyces ambofaciens in the native state produces spiramycin I, II and III (see figure 1), however, the antibiotic activity of spiramycin I distinctly higher than that of spiramycin II and III (Liu and others, 1999). Therefore, it is desirable to be able to have strains producing only spiramycin I.

Because of the commercial interest to macrolide antibiotics, intensive research is being conducted in obtaining new derivatives, in particular, analogues of spiramycin with preferred properties. It is desirable to be able to generate sufficient mediators of the pathway of biosynthesis of spiramycin or derivative spiramycine, in particular, with the aim of obtaining hybrid antibiotics originating from spiramycine.

General description of the invention

The present invention is based on the cloning of genes, the product of which is involved in the biosynthesis of spiramycin. The invention relates primarily to new genes of the biosynthesis pathway of spiramycin and new polypeptides involved in the biosynthesis.

Were cloned genes of the biosynthesis pathway and associated coding sequences and determined the DNA sequence of each. The cloned caderoussechemin further designated as orf1*c, orf2*c, orf3*c, orf4*c, orf5*, orf6*, orf7*c, orf8*, orf9*, orf10*, orf1, orf2, orf3, orf4, orf5, orf6, orf7, orf8, orf9c, orf10, orf11c, orf12, orf13c, orf14, orf15c, orf16, orf17, orf18, orf19, orf20, orf21c, orf22c, orf23c, orf24c, orf25c, orf26, orf27, orf28c, orf29, orf30c, orf31, orf32c, orf33 and orf34c. The function of the proteins encoded by these sequences in the biosynthesis pathway of spiramycine disclosed in the following discussion, which is illustrated figure 4, 5, 6, and 8.

1) the First object of the invention relates to polynucleotide, codereuse polypeptide involved in the biosynthesis of spiramycine, characterized in that the sequence of the above polynucleotide represents:

(a) one of the sequences SEQ ID№ 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 28, 30, 34, 36, 40, 43, 45, 47, 49, 53, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 107, 109, 111, 113, 115, 118, 120, 141, 143, 145, 147 and 149,

(b) one of the sequences generated variants of the sequences (a);

(C) one of the sequences derived from the sequences (a) and (b) in accordance with the degeneracy of the genetic code.

2) the Object of the present invention is also polynucleotide, hybridization in hybridization conditions of high stringency with at least one polynucleotide according to the above item 1).

3) the Invention relates also to polynucleotide with at least 70%, 80%, 85%, 90%, 95% or 98% identity to nucleotides with polynucleotides, including at least 10, 12, 5, 18, 20-25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850 or 1900 consecutive nucleotides of polynucleotide according to the above item 1).

4) the Invention also relates to polynucleotide according to the above item 2) or 3), characterized in that it is isolated from bacteria of the genus Streptomyces.

5) the Invention relates also to polynucleotide according to the above item 2), 3 or 4, characterized in that it encodes a protein involved in the biosynthesis of the macrolide.

6) the Invention also relates to polynucleotide according to the above item 2), 3) or (4), characterized in that it encodes a protein having activity similar to the protein encoded by polynucleotides, with whom he hybridized or has identity.

7) the Invention also relates to the polypeptide resulting from the expression of polynucleotide in claim 1 above), 2), 3), 4), 5) or 6).

8) another aspect of the invention relates to a polypeptide involved in the biosynthesis of spiramycine, characterized in that the above sequence of the polypeptide is:

(a) one of the sequences SEQ ID№ 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 27, 29, 31, 32, 33, 35, 37, 38, 39, 41, 42, 44, 46, 48, 50, 51, 52, 54, 55, 56, 57, 58, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 108, 110, 112, 114, 116, 117, 119, 121, 142, 144, 146, 148 and 150,

(b) ar is the sequence, such as specified in paragraph(a), except that the entire length of the above sequence one or more amino acids replaced built or subject to deletion without affecting the functional properties;

(C) one of the sequences generated variants of the sequences (a) and (b).

9) Another aspect of the invention relates to a polypeptide having at least 70%, 80%, 85%, 90%, 95% or 98% identity to amino acids from a polypeptide comprising at least 10, 15, 20, 30-40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620 or 640 consecutive amino acids of the polypeptide according to item 8 above).

10) Another aspect of the invention relates to a polypeptide according to the above item 9), characterized in that it is isolated from bacteria of the genus Streptomyces.

11) Another aspect of the invention relates to a polypeptide according to the above item 9) or 10), characterized in that it encodes a protein involved in the biosynthesis of the macrolide.

12) another aspect of the invention relates to a polypeptide according to the above item 9), 10) or 11), characterized in that it has activity similar to that of the polypeptide with which it has an identity.

13) Another aspect of the invention relates to recombinant DNA, characterized in that it includes at least one Poliny eated according to any one of above items 1), 2), 3), 4), 5) or 6).

14) Another aspect of the invention relates to a recombinant DNA according to the above item 13, characterized in that the recombinant DNA is integrated into the vector.

15) the Next aspect of the invention relates to a recombinant DNA according to the above 14, characterized in that the above vector is chosen among the bacteriophages, plasmids, phagemid integrating vectors, fosmid, cosmid, Shuttle vectors, YOU or RACES.

16) Another aspect of the invention relates to a recombinant DNA according to the above item 15, characterized in that it is chosen among pOS49.1, pOS49.11, pOSC49.12, pOS49.14, pOS49.16, pOS49.28, pOS44.1, pOS44.2, pOS44.4, pSPM5, pSPM7, pOS49.67, pOS49.88, pOS49.106, pOS49.120, pOS49.107, pOS49.32, pOS49.43, pOS49.44, pOS49.50, pOS49.99, pSPM17, pSPM21, pSPM502, pSPM504, pSPM507, pSPM508, pSPM509, pSPM1, pBXL1111, pBXL1112, pBXL1113, pSPM520, pSPM521, pSPM522, pSPM523, pSPM524, pSPM525, pSPM527, pSPM528, pSPM34, pSPM35, pSPM36, pSPM37, pSPM38, pSPM39, pSPM40, pSPM41, pSPM42, pSPM43, pSPM44, pSPM45, pSPM47, pSPM48, pSPM50, pSPM51, pSPM52, pSPM53, pSPM55, pSPM56, pSPM58, pSPM72, pSPM73, pSPM515, pSPM519, pSPM74, pSPM75, pSPM79, pSPM83, pSPM107, pSPM543 and pSPM106.

17) another aspect of the invention relates to expressing vector, characterized in that it comprises at least one nucleotide sequence encoding a polypeptide according visheprislannogo 7), 8), 9), 10), 11) or 12).

18) the Invention also relates to an expression system that includes the appropriate expressing vector and a cell of the host, allowing the expression of one Il is several polypeptides according to the above item 7), 8), 9), 10), 11) or 12.

19) the Invention also relates to an expression system according to the above p, characterized in that it is chosen from prokaryotic expression systems or expressing eukaryotic systems.

20) the Invention also relates to an expression system according to claim 19 above), characterized in that it is chosen from expression systems, E. coli, baculovirus expression systems that allow for the expression in insect cells, expressioni of systems that allow for expression in yeast cells, expression systems that allow for the expression in mammalian cells.

21) the Invention also relates to a cell-master in which you have entered at least one polynucleotide and/or at least one recombinant DNA and/or at least one expressing the vector according to any one of above items 1), 2), 3), 4), 5), 6), 13), 14), 15), 16) or 17.

22) the Invention also relates to a method for producing the polypeptide according to the above item 7), 8), 9), 10), 11) or 12), characterized in that the above method includes the following stages:

a) insertion of at least one nucleic acid encoding the above polypeptide into an appropriate vector;

b) culturing, in the appropriate culture medium, host cells, preventernational or transfectional vector from step a);

(C) recovery of conditioned culture medium or cell extract;

d) isolation and purification of the above culture medium or else from the cell extract obtained(s) at the stage (C), the above polypeptide;

e) in the desired case, okharakterizovanie produced recombinant polypeptide.

23) Another aspect of the invention relates to a microorganism that are blocked at the stage of the biosynthesis pathway of at least one of the macrolide.

24) the Next aspect of the invention relates to a microorganism according to the above item 23), characterized in that it is produced by inactivation of the function of at least one protein involved in the biosynthesis of this or these macrolides in the microorganism, which is the producer of this or these macrolides.

25) Another aspect of the invention relates to a microorganism according to the above paragraph 24), characterized in that the inactivation of this protein or of these proteins is carried out by mutagenesis in the gene or genes encoding the above or the above-mentioned proteins, or by the expression of one or more antisense RNA complementary to the RNA or messenger RNA coding for the above or the above-mentioned proteins.

26) Another aspect of the invention relates to a microorganism according to the above A.25, characterized in that the inak is evalu this or these proteins carry out due to mutagenesis by irradiation, by mutagenic chemical agent, by site-directed mutagenesis, or by replacement of the gene.

27) another aspect of the invention relates to a microorganism according to the above A.25) or 26), characterized in that the mutagenesis or mutagenesis carried out in vitro or in situ by suppression, substitution, deletion and/or insertions of one or more bases in the case of the gene or genes considered or by gene inactivation.

28) Another aspect of the invention relates to a microorganism according to the above item 23), 24), 25), 26) or 27), characterized in that the above-mentioned microorganism is a bacterium of the genus Streptomyces.

29) Another aspect of the invention relates to a microorganism according to the above item 23), 24), 25), 26), 27) or 28), wherein the macrolide is spiramycin.

30) Another aspect of the invention relates to a microorganism according to the above item 23), 24), 25), 26), 27), 28) or 29, characterized in that the above-mentioned microorganism is a strain of S. ambofaciens.

31) Another aspect of the invention relates to a microorganism according to the above item 23), 24), 25), 26), 27), 28), 29) or 30), wherein the mutagenesis is carried out at least in a gene comprising a sequence according to any one of above items 1), 2), 3), 4), 5) or 6).

32) another aspect of the invention relates to a microorganism on visiprise the military A.25), 26), 27), 28), 29), 30) or 31, wherein the mutagenesis is carried out in one or more genes comprising one of the sequences corresponding to one or more of the sequences SEQ ID№ 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 28, 30, 34, 36, 40, 43, 45, 47, 49, 53, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 107, 109, 111, 113, 115, 118, 120, 141, 143, 145, 147 and 149.

33) Another aspect of the invention relates to a microorganism according to the above A.25), 26), 27), 28), 29), 30), 31) or 32, wherein the mutation is in the gene inactivation of a gene comprising the sequence corresponding to posledovatelnosti SEQ ID No. 13.

34) Another aspect of the invention refers to a strain of Streptomyces ambofaciens, characterized in that it is a strain selected from one of the strains deposited in the National collection of microorganism cultures (CNCM) on 10 July 2002 under registration number I-2909, 2911, 2912, 2913, 2914, 2915, 2916 or I-2917.

35) the Next aspect of the invention relates to a method for production of neurotransmitter biosynthesis of the macrolide, characterized in that it comprises the following stages:

a) culturing, in the appropriate culture medium, a microorganism according to any one of the above PP), 24), 25), 26), 27), 28), 29), 30), 31), 32), 33) or 34);

b) recovery of conditioned culture medium or cell extract;

C) isolation and purification of the above culture media is or else from the cell extract, the obtained(s) on stage b), the above mediator biosynthesis.

36) Another aspect of the invention relates to a method for obtaining molecules, derived from the macrolide, characterized in that the mediator biosynthesis according to the method according to the above p) and modify thus produced the mediator.

37) another aspect of the invention relates to a method of obtaining the above p), characterized in that the above mediator modifies the chemical, biochemical, enzymatic and/or microbial method.

38) Another aspect of the invention relates to a method of obtaining the above p) or 37), characterized in that the above-mentioned microorganism is administered one or more genes encoding proteins that can modify the mediator that serves for them as a substrate.

39) the Next aspect of the invention relates to a method of obtaining the above p), 37 or 38), wherein the macrolide is spiramycin.

40) Another aspect of the invention relates to a method of obtaining the above p), 37), 38 or 39), characterized in that the microorganism is a strain of S. ambofaciences.

41) another aspect of the invention relates to a microorganism which produces spiramycin I, but not producing spiramycin II and III.

42) the Other is the aspect of the invention relates to a microorganism according to paragraph 41 above), characterized in that it includes a set of genes required in the biosynthesis of spiramycin I, however, the gene comprising the sequence SEQ ID No. 13 or one of its variants, or one of the sequences originating from them in accordance with the degeneracy of the genetic code and encodes a polypeptide corresponding to the sequence SEQ ID No. 14 or one of its variants, is not expressed or inactivated.

43) another aspect of the invention relates to a microorganism according to the above § 42), characterized in that the above inactivation is carried out by mutagenesis in the gene encoding the above protein, or by expression of antisense RNA, complementary to the messenger RNA that encodes the above-mentioned protein.

44) Another aspect of the invention relates to a microorganism according to the above § 42), characterized in that the above mutagenesis is carried out in the promoter of this gene in the coding sequence or non-coding sequence with the purpose of inactivation of the encoded protein or prevent its expression or its broadcast.

45) another aspect of the invention relates to a microorganism according to the above item 43 or 44), wherein the mutagenesis is carried out by irradiation, by mutagenic chemical agent, directed by mutah is illegal or by replacement of the gene.

46) Another aspect of the invention relates to a microorganism according to the above item 43), 44 or 45), wherein the mutagenesis is carried out in vitro or in situ by suppression, substitution, deletion and/or insertions of one or more bases in the case of the gene or by gene inactivation.

47) another aspect of the invention relates to a microorganism according to the above paragraph 41 or 42), characterized in that the above-mentioned microorganism produced by the expression of genes of the biosynthesis pathway of spiramycin, and they do not include a gene containing a sequence corresponding to SEQ ID No. 13, or one of its variants, or one of the sequences originating from them in accordance with the degeneracy of the genetic code and encodes a polypeptide corresponding to the sequence SEQ ID No. 14 or one of its variants.

48) Another aspect of the invention relates to a microorganism according to paragraph 41 above), 42), 43), 44), 45), 46) (47), characterized in that the above-mentioned microorganism is a bacterium of the genus Streptomyces.

49) the Next aspect of the invention relates to a microorganism according to paragraph 41 above), 42), 43), 44), 45) 46), 47) and 48), characterized in that the above-mentioned microorganism derived from the original strain, producing spiramycin I, II and III.

50) Another aspect of the invention relates to a microorganism on visar is maintained paragraph 41), 42), 43), 44), 45), 46), 47), 48) or 49), characterized in that it is produced by mutagenesis in the gene comprising the sequence corresponding to SEQ ID No. 13, or one of its variants, or one of the sequences originating from them in accordance with the degeneracy of the genetic code, and encoding a polypeptide corresponding to the sequence SEQ ID No. 14 or one of its variants that have the same function.

51) another aspect of the invention relates to a microorganism according to paragraph 41 above), 42), 43), 44), 45), 46), 47), 48), 49) or 50), characterized in that the above-mentioned microorganism is obtained from a strain of S. ambofaciens, producing spiramycin I, II and III, in which gene inactivation of a gene comprising the sequence corresponding to the sequence SEQ ID No. 13, or one of the sequences originating from it in accordance with the degeneracy of the genetic code.

52) Another aspect of the invention refers to a strain of S. ambofaciens, characterized in that it is about the strain deposited at the National collection of microorganism cultures (CNCM) on 10 July 2002 under registration number I-2910.

53) another aspect of the invention relates to a method for producing spiramycin I, characterized in that it comprises the following stages:

a) culturing, in the appropriate culture medium, a microorganism p is any of the above PP), 42), 43), 44), 45), 46), 47), 48), 49), 50), 51) or 52);

b) recovery of conditioned culture medium or cell extract;

C) isolation and purification of the above culture medium or else from the cell extract obtained(s) on stage b), spiramycin I.

54) Another aspect of the invention relates to the application of polynucleotide according to any one of above items 1), 2), 3), 4), 5) or 6) to increase production of macrolides organism.

55) the Next aspect of the invention relates to a mutant microorganism producing the macrolide, characterized in that it has a genetic modification in at least one gene comprising a sequence according to any one of above items 1), 2), 3), 4), 5) or 6), and/or that he overproduce at least one gene comprising a sequence according to any one of above items 1), 2), 3), 4), 5) or 6).

56) Another aspect of the invention relates to a mutant microorganism according to the above § 55), wherein the genetic modification consists in the suppression, substitution, deletion and/or insertions of one or more bases in the case of the gene or genes considered with the aim of ekspressirovali one or more proteins having the highest activity, or ekspressirovali at a higher level or protein of the century

57) another aspect of the invention relates to a mutant microorganism according to the above § 55), characterized in that overproductive reach of the gene by increasing the number of copies of this gene and/or by introducing a more active promoter than the original promoter.

58) Another aspect of the invention relates to a mutant microorganism according to the above § 55 or 57), characterized in that overproductive reach of the gene by transformation of the microorganism, which is the producer of the macrolide, by using recombinant DNA according to the above item 13, 14 or 17, allowing overproduction this gene.

59) another aspect of the invention relates to a mutant microorganism according to the above § 55), 56), 57 or 58), wherein the genetic modification is carried out in one or more genes comprising one of the sequences corresponding to one or more of the sequences SEQ ID№ 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 28, 30, 34, 36, 40, 43, 45, 47, 49, 53, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 107, 109, 111, 113, 115, 118, 120, 141, 143, 145, 147 and 149, or one of its variants, or one of the sequences derived from them in accordance with the degeneracy of the genetic code.

60) Another aspect of the invention relates to a mutant microorganism according to enter the lists § 55), 56), 57), 58 or 59), wherein the microorganism overproduce one or more genes comprising one of the sequences corresponding to one or more of the sequences SEQ ID№ 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 28, 30, 34, 36, 40, 43, 45, 47, 49, 53, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 107, 109, 111, 113, 115, 118, 120, 141, 143, 145, 147 and 149, or one of its variants, or one of the sequences derived from them in accordance with the degeneracy of the genetic code.

61) Another aspect of the invention relates to a mutant microorganism according to the above § 55), 56), 57), 58), 59) or 60), characterized in that the above-mentioned microorganism is one of the bacteria of the genus Streptomyces.

62) the Next aspect of the invention relates to a mutant microorganism according to the above § 55), 56), 57), 58), 59), 60) or 61), wherein the macrolide is spiramycin.

63) Another aspect of the invention relates to a mutant microorganism according to the above § 55), 56), 57), 58), 59), 60), 61) or 62), characterized in that the above-mentioned microorganism is one of the strains of S. ambofaciens.

64) another aspect of the invention relates to a method for production of macrolides, characterized in that it comprises the following stages:

a) culturing, in the appropriate culture medium, a microorganism according to any one of the above PP), 56), 57), 58), 59), 60), 61), 62), 63) �if 64);

b) recovery of conditioned culture medium or cell extract;

C) isolation and purification of the above culture medium or else from the cell extract obtained(s) on stage b), the above produced the macrolide or above produced the macrolides.

65) Another aspect of the invention relates to the use of sequence and/or recombinant DNA and/or vector according to any one of above items 1), 2), 3), 4), 5), 6), 7), 8), 9), 10), 11), 12), 13), 14), 15), 16) or 17) for hybrid antibiotics.

66) the Next aspect of the invention concerns the use of at least one polynucleotide and/or at least one recombinant DNA and/or at least one expressing vector and/or at least one polypeptide and/or at least one host cell on one of the aforementioned items 1), 2), 3), 4), 5), 6), 7), 8), 9), 10), 11), 12), 13), 14), 15), 16), 17) or 21) to implement one or more bioconversion.

67) Another aspect of the invention relates to polynucleotide, characterized in that it comes polynucleotide, complementary to one of polynucleotides in claim 1 above), 2), 3), 4), 5) or 6).

68) another aspect of the invention relates to a microorganism, which is the producer of at least one spiramycin, characterized in that it is curprod ciruit:

- gene, which can be obtained by amplification by polymerase chain reaction (PCR) using a pair of primers having the following sequences: 5' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID No. 138) and 5' GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID No. 139) and as a matrix of Comedy pSPM36 or total DNA of Streptomyces ambofaciens;

or a gene derived from it in accordance with the degeneracy of the genetic code.

69) Another aspect of the invention relates to a microorganism on p or 90, wherein we are talking about bacteria of the genus Streptomyces.

70) the Next aspect of the invention relates to a microorganism on p, 69 or 90, wherein we are talking about bacteria species Streptomyces ambofaciens.

71) Another aspect of the invention relates to a microorganism on p, 69, 70 or 90, wherein overproductive above reach of the gene by transformation of the above mentioned microorganism expressing vector.

72) another aspect of the invention refers to a strain of Streptomyces ambofaciens, characterized in that it is about the strain OSC2/pSPM75(1) or the strain OSC2/pSPM75(2)deposited in the National collection of microorganism cultures (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex 15, France, October 6, 2003 under registration number I-3101.

73) Another aspect of the invention relates to recombinant DNA, characterized in that it includes:

- palynol acid, which can be obtained by amplification by polymerase chain reaction using a pair of primers having the following sequences: 5' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID No. 138) and 5' GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID No. 139) and as a matrix of Comedy pSPM36 or total DNA of Streptomyces ambofaciens;

or a fragment comprising at least 10, 12, 15, 18, 20-25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1460, 1470, 1480, 1490 or 1500 consecutive nucleotides of this polynucleotide.

74) the Next aspect of the invention relates to recombinant DNA in p or 91, wherein we are talking about vector.

75) Another aspect of the invention relates to recombinant DNA in p, 74 or 91, characterized in that it is about expressing vector.

76) another aspect of the invention relates to a cell-master in which you have entered at least one recombinant DNA according to any one of Pb, 74, 75 or 91.

77) Another aspect of the invention relates to a method for production of the polypeptide, characterized in that the above method includes the following stages:

a) transforming the host cell with at least one expressing the vector according to item 75;

b) culturing, in the appropriate culture medium, the above-mentioned host cell;

(C) regenerating air is ulturally medium or cell extract;

d) isolation and purification of the above culture medium or else from the cell extract obtained(s) at the stage (C), the above polypeptide;

e) in the desired case, okharakterizovanie produced recombinant polypeptide.

78) another aspect of the invention relates to a microorganism according to § 51, wherein the gene inactivation is carried out by the full-sized deletions of the gene or part of a gene comprising a sequence that corresponds to SEQ ID No. 13, or one of the sequences originating from it in accordance with the degeneracy of the genetic code.

79) Another aspect of the invention relates to a microorganism according to any one of Pb, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 78, wherein he also overproduced:

- gene, which can be obtained by amplification by polymerase chain reaction using a pair of primers having the following sequences: 5' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID No. 138) and 5' GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID No. 139) and as a matrix of Comedy pSPM36 or total DNA of Streptomyces ambofsciens;

or a gene derived from it in accordance with the degeneracy of the genetic code.

80) the Next aspect of the invention relates to expressing vector, characterized in that polynucleotide corresponding to the sequence SEQ ID No. 47, or polynuclear is Ted, what comes from it in accordance with the degeneracy of the genetic code, is under the control of a promoter that allows expression of the protein encoded by the specified polynucleotide in Streptomyces ambofaciens.

81) Another aspect of the invention relates to expressing the vector according to item 80, characterized in that it is a plasmid pSPM524 or pSPM525.

82) the Next aspect of the invention refers to a strain of Streptomyces ambofaciens, transformed by the vector according to item 80 or 81.

83. Another aspect of the invention relates to a microorganism according to any one of Pb, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 78, 79 or 92, wherein he also overproduce the gene corresponding to the coding sequence of SEQ ID 47, or the coding sequence derived from it in accordance with the degeneracy of the genetic code.

84. Another aspect of the invention relates to a microorganism on p, characterized in that it is about the strain SPM502pSPM525 deposited in the National collection of microorganism cultures (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex 15, France, on February 26, 2003 under registration number I-2977.

85) Another aspect of the invention relates to a method for producing spiramycin (spiramycin), characterized in that it comprises the following stages:

a) culturing, in the appropriate culture medium, mi is reorganiza on any of PP, 69, 70, 71, 72, 78, 79, 82, 83, 84, 90 or 92;

b) recovery of conditioned culture medium or cell extract;

C) isolation and purification of the above culture medium or else from the cell extract obtained(s) on stage b), spiramycin.

86) the Next aspect of the invention relates to the polypeptide, characterized in that its sequence includes the sequence SEQ ID No. 112, or the sequence SEQ ID No. 142.

87) Another aspect of the invention relates to the polypeptide, characterized in that its sequence corresponds to the sequence, we Express the coding sequence:

- gene, which can be obtained by amplification by polymerase chain reaction (R) when using a pair of primers having the following sequences: 5' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID No. 138) and 5' GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID No. 139) and as a matrix of Comedy pSPM36 or total DNA of Streptomyces ambofaciens,

or gene originating from him in accordance with the degeneracy of the genetic code.

88) the Next aspect of the invention relates to expressing vector allowing expression of the polypeptide in p, 87 or 93 in Streptomyces ambofaciens.

89) Another aspect of the invention relates to expressing the vector in p, characterized in that it comes plasmid pSPM75.

90) the Next aspect is subramania refers to the microorganism by p, characterized in that genome, which can be obtained by amplification by polymerase chain reaction, is the gene corresponding to the coding sequence SEQ ID No. 141, or a gene derived from it in accordance with the degeneracy of the genetic code.

91) Another aspect of the invention relates to recombinant DNA in p, characterized in that polynucleotides, which can be obtained by amplification by polymerase chain reaction is polynucleotide corresponding to the sequence SEQ ID No. 141.

92) another aspect of the invention relates to a microorganism on p, wherein the genome, which can be obtained by amplification by polymerase chain reaction, is the gene corresponding to the coding sequence SEQ ID No. 141, or a gene derived from it in accordance with the degeneracy of the genetic code.

93) Another aspect of the invention relates to the polypeptide, characterized in that its sequence is SEQ ID No. 142.

Common definitions

The term “isolated” in the sense of the present invention means a biological material (nucleic acid or protein)that is removed from its original environment (the environment in which it is localized in a natural way.

For example, polynucleotide, the presence of the plans in their native state in the plant or animal, is not selected. The same polynucleotide, separated adjacent nucleic acids, inside which it is naturally integrated into the genome of a plant or animal, regarded as “selected”.

This polynucleotide can be embedded in a vector and/or such polynucleotide may be included in the composition and nevertheless is selected due to the fact that the vector or composition does not form its native environment.

The term “purified” does not require that the material was in an absolutely pure form, excluding the presence of other compounds. Rather, we are talking about the relative definition.

Polynucleotide is in a clean state after treatment of the source material or natural material at least one order of magnitude, preferably 2 or 3 and particularly preferably 4 or 5 orders of magnitude.

According to the present invention, the term ORF (Open Reading Frame”, i.e. open reading frame) is used to denote, in particular, the coding sequence of a gene.

According to the present description, the expression “nucleotide sequence” can be used to denote indifferently of polynucleotide or nucleic acid. The expression “nucleotide sequence” includes the actual genetic material and, therefore, does not t aetsa limited information on its sequence.

The terms “nucleic acid”, “polynucleotide”, “oligonucleotide” or “nucleotide sequence” include RNA sequences, DNA, cDNA, or even a hybrid sequence RNA/DNA from more than one nucleotide, indifferent as the ordinary chain or double chain.

The term “nucleotide” means a natural nucleotides (A, T, G, C), and modified nucleotides that include at least one modification such as (1) a purine analog, (2) a pyrimidine analog, or (3) similar sugar, and measurements of such modified nucleotides are described, for example in PCT application no WO-95/04064.

According to the present invention, the first polynucleotide considered as “complementary” to a second polynucleotide, while each base of the first polynucleotide paired with the complementary base of the second polynucleotide, orientation which is the reverse. Complementary bases are a and T (or a and U), or C and G.

The term “genes of the biosynthesis pathway of spiramycine also includes genes regulators and genes giving resistance to microorganisms-producers.

By “fragment” of the standard nucleic acid according to the invention understand the nucleotide sequence of reduced length relative standartniy nucleic acid and comprising, in the General part, the nucleotide sequence that is identical to the standard nucleic acid.

A “fragment” of a nucleic acid according to the invention, in the desirable case may be embedded in polynucleotide greater magnitude, for which it is constitutive.

Such fragments comprise or alternatively, consist of polynucleotide length, reaching to 8, 10, 12, 15, 18, 20-25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850 or 1900 consecutive nucleotides of the nucleic acid according to the invention.

By “fragment” of a polypeptide according to the invention understand the polypeptide amino acid sequence which is shorter than that of a standard polypeptide, and which includes, in whole, part, in common with these standard polypeptides identical to the amino acid sequence.

Such fragments in the desired case, can be incorporated into a polypeptide of greater magnitude which they are a part.

Fragments of a polypeptide according to the invention may have a length, component 10, 15, 20, 30-40, 50, 60, 70, 80, 90,100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620 or 640 amino acids.

Under hybridization conditions of high stringency” according to the present invention understand the conditions of hybridization, unfavorable for hybridization of the strands of homologous nucleic acids. The hybridization conditions of high stringency can be described, for example, as conditions of hybridization in buffer described by Church and Gilbert (Church &Gilbert, 1984)at a temperature of from 55°C to 65°C, and, preferably, the hybridization temperature is 55°C, even more preferably, the hybridization temperature is 60°C and highly preferably, the hybridization temperature is 65°C, followed by one or more washes carried out in the buffer 2X SSC (buffer 1X SSC corresponds to a water solution of 0.15 M NaCl, 15 mm sodium citrate)at a temperature of from 55°C to 65°C, and, preferably, this temperature is 55°C, even more preferably, this temperature is 60°C and highly preferably, this temperature is 65°C, followed by one or more washes carried out in the buffer 0.5x SSC at a temperature of from 55°C to 65°C, and, preferably, this temperature is 55°C, even more preferably, this temperature is 60°C and, in highly preferably, this temperature is 65°C.

Needless to say that the above conditions of hybridization can be adapted depending on the length of nucleic acid hybridization which seek to implement, or in the alarm type marking according to the methods known to the person skilled in the art. Appropriate hybridization conditions can be adapted, for example, according to F. Ausubel and others (2002).

By “variant” nucleic acid according to the invention understand nucleic acid which differs by one or more bases compared with standard polynucleotides. A variant nucleic acid may be of natural origin, such as a naturally occurring allelic variant, or it may also be a native option, we get, for example, methods of mutagenesis.

In General, the differences between the standard nucleic acid or variant nucleic acid is limited so that the nucleotide sequence of the standard nucleic acid and the variant nucleic acids are very close and in many areas are identical. Modification of nucleotides located in a variant nucleic acid may be silent, which means that they do not alter the amino acid sequence encoded by the above variant nucleic acids.

However, changes in nucleotide in the variant nucleic acid can also occur as a result of substitutions, insertions, deletions in the polypeptide encoded by the variant nucleic acid in comparison with peptides, Cody who has been created a standard nucleic acid. In addition, modification of nucleotides in the coding regions may produce conservative or non-conservative substitutions in the amino acid sequence.

Preferably, the variants of nucleic acids according to the invention encode polypeptides that are substantially retain the same function or biological activity as the polypeptide corresponding to standard nucleic acid, or else the ability to be recognized by antibodies directed against polypeptides encoded by the original nucleic acid.

Some variants of nucleic acids encode and mutated forms of the polypeptides, a systematic study of which will allow to make a conclusion about the relationship of structure to activity of the corresponding proteins.

By “variant” polypeptide according to the invention understand, in principle, polypeptide, amino acid sequence which contains one or more substitutions, insertions or deletions of at least one amino acid residue compared to the amino acid sequence of standard polypeptide, having in mind that replacement of amino acids can be indifferent, conservative or nonconservative.

Preferably, polypetide variants according to the invention substantially retain the same function or biologicheskuyu activity as a standard polypeptide, or else the ability to be recognized by antibodies directed against the original polypeptide.

Under polypeptide “having activity similar to the” standard polypeptide according to the invention understand polypeptide having similar biological activity, but not necessarily identical standard polypeptide, such as measured in an appropriate biological test to determine the biological activity of the standard polypeptide.

Under “hybrid antibiotic” according to the invention understand the connection generated by artificial construct biosynthesis pathway using recombinant DNA technology.

Detailed description of the invention

The object of the present invention in particular are new genes of the biosynthesis pathway of spiramycin and new polypeptides involved in the biosynthesis, such as shown in the below detailed description.

Genes of the biosynthesis pathway were cloned and determined the DNA sequence of these genes. Obtained sequences were analyzed by the FramePlot program (J. Ishikawa & Hotta K., 1999). Among the open reading frames identified open reading frames with a typical codons Streptomyces. This analysis showed that this area includes 44 open reading frames (RFs), flanked on both sides by five genes encoding the enzyme “policyinstitute” (PKS) and with typical codons Streptomyces. With both sides of these five genes encoding PKS identified, respectively, 10 and 34 ORFs above and below (bottom and top are defined by the orientation of the 5 genes, all of which have the same semantic orientation) (see figure 3 and 37). Thus, 34 open reading frames of this type, covering an area of about 41,7 so-called (see SEQ ID No. 1, representing the first area length 31 so-called, containing 25 ORFs, and SEQ ID No. 140, an area with a length of about 12,1 so-called, so-called 1,4 which overlap the previous sequence (SEQ ID No. 1) and about 10.7 called which correspond to a contiguous sequence, and this last part of the length of about 10.7 called, contains 9 additional open reading frames (including the open reading frame (ORF) partial sequence), see also 3 and 37 below), have been identified above 5 genes encoding PKS, and 10 open reading frames, covering an area of about 11,1 so-called (SEQ ID No. 2 and figure 3), were identified below PKS genes. 10 Genes located below 5 PKS genes, thus, were named orf1*c, orf2*c, orf3*c, orf4*c, orf5*, orf6*, orf7*c, orf8*, orf9*, orf10* (SEQ ID№ 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21). The letter “C”added to the name of the gene means, for the appropriate reading frame, to yuusha sequence is in the reverse orientation (coding thread, therefore, a thread complementary to the sequence presented as SEQ ID No. 2, these genes). Using the same nomenclature, 34 reading frames above PKS genes were named orf1, orf2, orf3, orf4, orf5, orf6, orf7, orf8, orf9, orf10, orf11, orf12, orf13, orf14, orf15, orf16, orf17, orf18, orf19, orf20, orf21, orf22, orf23, orf24, orf25, orf26, orf27, orf28, orf29, orf30, orf31, orf32, orf33 and orf34 (SEQ ID№ 23, 25, 28, 30, 34, 36, 40, 43, 45, 47, 49, 53, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 107, 109, 111, 113, 115, 118, 120, 141, 143, 145, 147 and 149) (see figure 3 and 37).

Protein sequence resulting from these open reading frames were compared with those available in the various databases with a variety of programs: BLAST (Altschul and others, 1990; Altschul and others, 1997), CD-search, COGs (Cluster ontologica groups) (these three programs are available, in particular from NCBI (national center for biotechnology information) (Bethesda, Maryland, USA), FASTA (Pearson W. R. &Lipman, D. J., 1988) and (W. R. Pearson, 1990)), BEATY (K. C. Worley and others, 1995) (these two programs are available, in particular from the resource center INFOBIOGEN, Evry, France). These comparisons allowed us to formulate hypotheses concerning the function produced by these genes products and identify those that may participate in the biosynthesis of spiramycin.

Genes located below the genes encoding PKS

Schematic illustration of the structure of the region is presented in figure 3. Thus, as will be shown below, from 10 GE is s, identified below, genes encoding PKS, 9 are involved in the biosynthesis or resistance to spiramycin. We are talking about 9 the following genes: orf1*c, orf2*c, orf3*c, orf4*c, orf5*, orf6*, orf7*c, orf8* and orf9*.

In the following table 1 provides links to a sequence of DNA and amino acids 10 genes identified below 5 PKS genes.

Table 1
GeneThe position in the sequence SEQ ID No. 2The DNA sequencePolypeptide sequence
orf1*c10882-10172SEQ ID no 3SEQ ID no 4
orf2*c10052-8781SEQ ID no 5SEQ ID no 6
orf3*c8741-7476SEQ ID no 7SEQ ID no 8
orf4*c7459-6100SEQ ID no 9SEQ ID no 10
orf5*5302-5976SEQ ID no 11 SEQ ID no 12
orf6*4061-5305SEQ ID no 13SEQ ID no 14
orf7*c3665-2817SEQ ID no 15SEQ ID no 16
orf8*1925-2755SEQ ID no 17SEQ ID no 18
orf9*1007-1888SEQ ID no 19SEQ ID no 20
orf10*710-937SEQ ID no 21SEQ ID no 22

The letter “C”added to the name of the gene means that the coding sequence is in the reverse orientation (coding thread, therefore, is a thread which is complementary to the sequence presented as SEQ ID No. 2, these genes).

To determine the function of the identified polypeptides were conducted three types of experiments: comparison of the identified sequences with sequences with known functions; the experiments on the inactivation of genes leading to the construction of mutant strains; and analyses in relation to the production of these mutant PCs is the Mmax of spiramycin and mediators biosynthesis spiramycine.

Protein sequence resulting from these open reading frames, all are first compared with those available in the various databases with a variety of programs: BLAST (Altschul and others, 1990; Altschul and others, 1997), CD-search, COGs (Cluster ontologica groups), FASTA (Pearson W. R. &Lipman, D. J., 1988) and (W. R. Pearson, 1990)), BEATY (K. C. Worley and others, 1995). These comparisons allowed us to formulate hypotheses concerning the function produced by these genes products and identify those that can participate in the biosynthesis of spiramycin. Table 2 presents the proteins with greater affinity with the products produced by the 10 genes located below 5 PKS genes.

Table 2
Gene productProtein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
orf1*cTylMI(orf3*) (S. fradiae)CAA57473287N-methyltransferase
orf2*cGene product dnrQ (Streptomyces peucetius)AAD15266 153Unknown
orf3*cTylMII(orf2*) (S. fradiae)CAA57472448Glycosyl-transferase
orf4*cCrotonyl-CoA reductase (S. coelicolor)NP_630556772Crotonyl-CoA reductase
orf5*MdmC (S. mycarofaciens)B42719355O-methyltransferase
orf6*3-O-acyltransferase (S. mycarofaciens)Q00718494Alltrans-ferase
orf7*cMdmA (S. mycarofaciens)A60725380Protein involved in resistance to midecamycin
orf8*ABC-carrier (S. griseus)CAC22119191ABC-vector
orf9*ABC-carrier (S. griseus) CAC22118269ABC-vector
orf10*The estimated small-preserved hypothetical proteinNP_627432109Unknown
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990)

Experiments on inactivation of genes was carried out to confirm these results. Used methods are the implementation of the replacement gene. Interrupted gene target was replaced by a copy of this gene, interrupted by the cluster, which imparts resistance to antibiotics (such as, for example, apramycin, geneticin and hygromycin). Used the cluster is flanked on both sides by codons broadcast in all open reading frames and active terminators of transcription in Streptomyces. The insertion of the cluster in the gene target may be accompanied or not by a deletion in this gene target. The size of the regions flanking the cluster, up from several hundred to several thousand base pairs. For inactivation of genes can be used the second type of clusters: clusters, called “excesively clusters”. These clusters region is give the advantage what can be excitatory in the case of Streptomyces due to the phenomenon of recombination specific website after it has been integrated into the genome of S. ambofaciens. The goal is to inactivation of some genes in strains of Streptomyces, leaving in the end the strain of markers in breeding or large DNA sequences not belonging to the strain. After excision in the genome of strain has only a short sequence of the thirty base pairs (referred to as “cicatricial” site) (see figure 10). The implementation of this system is, first, the replacement copy of the wild-type gene target (due to two phenomena homologous recombination, see Fig.9) design, in which excesively cluster embedded in this gene target. The insertion of this cluster is accompanied by a deletion in the gene target (see Fig.9). Secondly, provoke excision excisional cluster from the genome of strain. Excesively cluster operates the system sitespecifically recombination and has the advantage that it allows to obtain mutants of Streptomyces, not containing, ultimately, gene resistance. Also overcome possible polar effects on the expression of genes below the inactivated gene or inactivated genes (see figure 10). Thus constructed the strains were tested for production of their spiramycine.

Inactivation of genes orf1*c, orf2*c, of3*c and orf4*c was not performed, as the experiments concerning the comparison of sequences allowed us to determine that these genes have a relatively high similarity with genes involved in the biosynthesis of relatively similar antibiotic. So, gene orf1* encodes a protein having an identity of 66% (defined according to the program BLAST) protein encoded by the gene tylMI, which encodes N-methyltransferase involved in the biosynthesis of tylosin and catalyzes the N-methylation during production of mycaminose in the case of Streptomyces fradiae (Gandecha, A. R. and others, 1997; the access number in the gene Bank: SAA; score from BLAST: 287). This similarity with a protein involved in the biosynthesis pathway of another, relatively close antibiotic and particularly in the biosynthesis of mycaminose, suggesting that the gene orf1*c encodes N-methyltransferase, responsible for N-methylation during biosynthesis of forosamine or mycaminose (see figure 5 and 6). This hypothesis is supported by the fact that the protein encoded by orf1 gene*c, has a great similarity with other proteins of similar function in other organisms (see table 3).

Table 3
Protein with significant similarityThe access number in the gene BankScore from BLAST* Correlated function
Methyltransferase (S. antibioticus)CAA05643277Methyltransferase
N,N-dimethylarsenate (S. venezuelae)AAC68678268N,N-dimethylarsenate
Probable N-methylase snogX (S. nogalater)T46679243N-methyltransferase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990)

Gene orf2*c encodes a protein having a relatively high similarity (35% identity) with the protein encoded by the gene tylMIII coding for NDP-hexose-3,4-isomerase involved in the biosynthesis of tylosin in the case Streptomycts fradiae (Gandecha, A. R. and others, 1997; the access number in the gene Bank: SAA; score from BLAST: 130). This similarity with a protein involved in the biosynthesis pathway for another close antibiotic and, in particular, in the biosynthesis of mycaminose, suggesting that orf2 gene*c encodes NDP-hexose-3,4-isomerase responsible for the isomerization during the biosynthesis of one of the sugars of spiramycin, in certain cases, mycaminose (see figure 5 and 6).

Orf3 gene*encodes FR is in, having a relatively high similarity (59% identity) with the protein encoded by the gene tylMII that encodes glycosyltransferase involved in the biosynthesis of tylosin in the case of Streptomyces fradiae (Gandecha, A. R. and others, 1997; the access number in the gene Bank: SAA; score from BLAST: 448). This similarity with a protein involved in the biosynthesis pathway for another close antibiotic, suggesting that orf3 gene*c encodes glycosyltransferase. This hypothesis is supported by the fact that the protein encoded by orf3 gene*c, has a great similarity with other proteins of similar function in other organisms (see table 4).

Table 4
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
Glycosyltransferase (S. venezuelae)AAC68677426Glycosyltransferase
Glycosyltransferase (S. antibioticus)CAA05642425Glycosyltransferase
Glycosyltransferase (Saccharopolyspora erythraea) CAA74710395Glycosyltransferase
Glycosyltransferase (S. antibioticus)CAA05641394Glycosyltransferase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990)

Orf4 gene*encodes a protein that has great similarity with several crotonyl-COA-reductase. In particular, the protein encoded by orf4 gene*c, has considerable similarity with crotonyl-COA-reductase in the case of Streptomyces coelicolor (Redenbach, M. and others, 1996; the access number in the gene Bank: NP_630556; score from BLAST: 772). This similarity with a protein involved in the biosynthesis pathway for another close antibiotic, highly suggesting that orf4 gene*c encodes also crotonyl-COA-reductase. This hypothesis is supported by the fact that the protein encoded by orf4 gene*c, has a great similarity with other proteins of similar function in other organisms (see table 5).

Table 5
Protein with significant similarityThe access number in the gene BankScore from BLAST Correlated function
TRANS-2-enoyl-CoA reductase (EC1.3.1.38) (S. collinus)S72400764TRANS-2-enoyl-CoA reductase
Crotonyl-CoA-reductase (S. fradiae)CAA57474757Crotonyl-CoA-reductase
Crotonyl-CoA-reductase (S. cinnamonensis)AAD53915747Crotonyl-CoA-reductase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990)

Orf6 gene* has a great similarity with the gene mdmB available in the case of Streptomyces mycarofaciens (Hara and Hutchisnson, 1992; access number in the gene Bank: A; score from BLAST: 489), which is the producer of macrolide antibiotic. In the case of this producer gene is involved in the acylation lactoovo cycle. Orf6 gene*, therefore, encodes an acyltransferase. This hypothesis is supported by the fact that the protein encoded by orf6 gene*has a great similarity with other proteins of similar function in other organisms (see table 6).

Table 6
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
AcyA (Streptomyces thermotolerans)J4001450Macrolide-3-O-acyltransferase
Midecamycin-4"-O-propionyl transferase (S. mycarofaciens)BAA09815234Midecamycin-4"-O-propionylcarnitine
Mycarose-O-acyltransferase (Micromonospora megalomicea subsp. Nigra)AAG13909189Mycarose-O-acyltransferase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990)

Inactivation of the gene orf6* was carried out by deletions/insertions open reading frame and it is shown that the resulting strain no longer produces spiramycin II and spiramycin III, and produces only spiramycin I (see figure 1). This confirms that the orf6 gene* really involved in the synthesis of spiramycin II and spiramycin III. The enzyme encoded by this gene, t is aetsa responsible for the formation of spiramycin II and III by fixing the acetyl or butyilkoy group on carbon 3. Strains not expressing more protein encoded by orf6 gene*, are of special interest because they do not produce more spiramycin II and III, and produce only spiramycin I. As indicated above, the antibiotic activity of spiramycin I distinctly higher than that of spiramycin II and III (Liu and others, 1999).

Orf5 gene* encodes a protein with a relatively high similarity with several O-methyltransferases. In particular, the protein encoded by orf5*, has significant similarity with O-methyltransferase (EC 2.1.1.-) MdmC Streptomyces mycarofaciens (Hara & Hutchinson, 1992; access number in the gene Bank: V; score from BLAST: 355). This similarity with a protein involved in the biosynthesis pathway of another antibiotic, highly suggesting that orf5 gene also encodes the O-methyltransferase. Orf5 gene* participates in the formation of the precursor, included in lackenby cycle. In fact, according to the comparisons of the sequences produced by the orf5 gene* the product is also relatively close FkbG, which is responsible for methylation of hydroxymethyl-ASR (according to Wu and others, 2000; Hoffmeister and others, 2000; the access number in the gene Bank: F86386; score from BLAST: 247) (see Fig). This hypothesis is supported by the fact that the protein encoded by orf5 gene*, has a great similarity with other proteins of similar function in other organisms (see table 7).

Table 7
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
Probable O-methyltransferase (EC 2.1.1.-) safC (Myxococcus xanthus)T18553223O-methyltransferase
4-O-methyltransferase (EC 2.1.1.-)-(Streptomyces sp.)JC4004222O-methyltransferase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990)

Due to the polar effect insertions excisional cluster in the gene orf6* determined that the orf5 gene* is essential in the biosynthesis pathway of spiramycin. In fact, the insertion excisional cluster in the coding part of the gene orf6* causes complete cessation of the production of spiramycin. However, in the case when a built-in cluster excision (and, therefore, when one gene orf6* inactivated, see examples 14 and 15), detect the production of spiramycin I. It shows that the orf5 gene* is significant in the way biosint is for spiramycine, since its inactivation causes complete cessation of the production of spiramycin.

Orf5 gene* encodes a protein having a relatively high similarity with several O-methyltransferases. Orf5 gene* is O-methyltransferase involved either directly in the synthesis platinoid, or in the synthesis of methylated predecessor (methoxymethyl, see Fig)included in platinoid with PKS. To confirm this hypothesis, experiments with analysis of the CL/SM (liquid chromatography/mass spectrometry and NMR was carried out using strain S.ambofaciens genotype orf6*::att1Ωhyg+. In this strain orf5 gene* is not expressed due to a polar effect of the insertions in the gene orf6*, cluster, which contains the transcription terminators (see example 27). It is shown that this strain produces a molecule, the UV spectrum which is similar to that of spiramycin I, however, the mass spectrum shows the molecular ion 829. The difference of the mass 14 relative to the weight spiramycin can be explained by the absence of bromide of oxygen at carbon No. 4 lactoovo cycle (the structure of this compound is presented on Fig). The results obtained using NMR compatible with this hypothesis. The presence of a compound with the molecular ion 829 allows you to recognize the validity of the hypothesis about the role of the orf5* in the biosynthesis of spiramycin. In addition, the product line is adequate to spiramycin without a methyl group, has a very low microbiological activity (lower by a factor of 10) compared to the unmodified spiramycin, when the test is carried out using a microorganism Micrococcus luteus.

Orf7 gene*encodes a protein having a relatively large similarity with the protein encoded by the genome of mdmA Streptomyces mycarofaciens, the latter encodes a protein involved in resistance to midecamycin in the case of this organism producer (Hara and others, 1990; access number in the gene Bank: A; score from BLAST: 380). This similarity with a protein involved in the biosynthesis pathway of another antibiotic, highly suggesting that orf7 gene*also encodes a protein involved in resistance to spiramycin. In particular, the enzyme encoded by orf7 gene*, has methyltransferase activity and is involved in resistance to spiramycin in the case of Streptomyces ambofaciens. It is shown that this gene gives resistance type MLS 1, the resistance of which is known that it occurs as a result of monomethylamine in position A 23S ribosomal RNA (Pernodet and others, 1996). This hypothesis is supported by the fact that the protein encoded by orf7 gene*, has a great similarity with other proteins of similar function in other organisms (see table 8).

Table 8
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
Determinant of resistance to macrolide-lincosamide-streptogramin In (S. fradiae)JC531923823S rRNA methyltransferase
23S ribosomal RNA methyltransferase ErmML (Micrococcus luteus)AAL6882711923S rRNA methyltransferase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990)

Orf8 gene* encodes a protein having a relatively large similarity with protein type vector ABC in Streptomyces griseus (Campelo, 2002; the access number in the gene Bank: SAS; score from BLAST: 191). This similarity with a protein of type vector ABC in the highest degree allows you to believe that orf8 gene* encodes the protein of type vector ABC, which could be involved in resistance to spiramycin. This hypothesis is supported by the fact that the protein encoded by the genome of orf8*, has a great similarity with other proteins of similar functions is the case of other organisms (see table 9).

Table 9
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
AcrW (Streptomyces galilaeus)BAB7206094The vector ABC
Transmembrane protein resistance to daunorubicin (Streptomyces peucetius)P3201189Resistance to daunorubicin
probable ABC-Transporter of the transmembrane component (Streptomyces coelicolor)NP_62650689The vector ABC
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990)

Orf9 gene* encodes a protein having a relatively large similarity with protein type vector ABC in the case of Streptomyces griseus (Campelo, 2002; the access number in the gene Bank: SAS; score from BLAST: 269). This similarity with a protein of type vector ABC in the highest degree allows palaghat is, that orf9 gene* encodes the protein of type vector ABC, which could be involved in resistance to spiramycin. This hypothesis is supported by the fact that the protein encoded by orf9 gene*, has a great similarity with other proteins of similar function in other organisms (see table 10).

Table 10
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
Probable transport protein type ABC, ATP-binding component (S. coelicolor)NP_626505231The vector ABC
The estimated carrier ABC, ATP-binding component (Streptomyces Coelicolor)NP_627624228The vector ABC
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990)

Orf10 gene* encodes a protein having a relatively high similarity to proteins of unknown function. However, genes such orf10*, about which the detected among several groups of genes, involved in the biosynthesis of antibiotics. So, gene, close to orf10*found in the case of S.coelicolor (Redenbach and others, 1996; the access number in the gene Bank: NP_627432; score from BLAST: 109). Closest gene (CouY) also found in the case S.rishiriensis (Wang and others, 2000; the access number in the gene Bank: G29779; score from BLAST: 97).

Genes located above the genes encoding PKS

In the DNA sequence above genes encoding PKS (bottom and top are defined by the orientation of the 5 genes, all of which have the same semantic orientation) (see figure 3)identified 34 open reading frames (see above). Thus, 34 open reading frames (ORFs) of this type, covering an area of about 41,7 so-called (see SEQ ID No. 1, representing the first area length 31 so-called, containing 25 ORFs, and SEQ ID No. 140, an area with a length of about 12,1 so-called, so-called 1,4 which overlap the previous sequence (SEQ ID No. 1) and about 10.7 called which correspond to a contiguous sequence), and this last part of the length of about 10.7 called, contains 9 additional reading frames (including reading frame (ORF) partial sequence), see also figure 3 and 37 below), have been identified above 5 genes encoding PKS. Schematic illustration of the organization of the region is presented in figure 3 and 37. 34 Identified gene were named: orf1, orf2, orf3, orf4, orf5, orf6, orf7, orf8, orf9, orf10, orf11, orf12, orf13, or14, orf15, orf16, orf17, orf18, orf19, orf20, orf21, orf22, orf23, orf24, orf25, orf26, orf27, orf28, orf29, orf30, orf31, orf32, orf33 and orf34.

The following table 11 presents denote the sequence of DNA and amino acids 34 genes identified above 5 PKS genes.

SEQ ID no 65
Table 11
Gene1The position in the sequence SEQ ID No. 1The DNA sequencePolypeptide(s) the sequence(ti)2
orf1658-1869SEQ ID no 23SEQ ID no 24
orf21866-2405SEQ ID no 25SEQ ID No. 26 and 27
orf32402-3568SEQ ID no 28SEQ ID no 29
orf43565-4473SEQ ID no 30SEQ ID No. 31, 32 and 33
orf54457-5494SEQ ID no 34SEQ ID no 35
orf6 5491-6294SEQ ID no 36SEQ ID No. 37, 38 and 39
orf76296-7705SEQ ID no 40SEQ ID No. 41 and 42
orf88011-9258SEQ ID no 43SEQ ID no 44
orf9c10081-9362SEQ ID no 45SEQ ID no 46
orf1010656-12623SEQ ID no 47SEQ ID no 48
orf11c14482-12734SEQ ID no 49SEQ ID No. 50, 51 and 52
orf1214601-16031SEQ ID no 53SEQ ID№ 54, 55, 56, 57, 58 and 59
orf13c17489-16092SEQ ID no 60SEQ ID no 61
orf1417809-18852SEQ ID no 62SEQ ID no 63
orf15c20001-18961SEQ ID no 64
orf1620314-21552SEQ ID no 66SEQ ID no 67
orf1721609-22879SEQ ID no 68SEQ ID no 69
orf1822997-24175SEQ ID no 70SEQ ID no 71
orf1924177-25169SEQ ID no 72SEQ ID no 73
orf2025166-26173SEQ ID no 74SEQ ID no 75
orf21c27448-26216SEQ ID no 76SEQ ID no 77
orf22c28560-27445SEQ ID no 78SEQ ID no 79
orf23c29770-28649SEQ ID no 80SEQ ID no 81
orf24c30074-29763SEQ ID no 82SEQ ID no 83
orf25c 30937-30071SEQ ID no 84SEQ ID no 85
Gene1The position in the sequence SEQ ID no 140The DNA sequencePolypeptide(s) the sequence(ti)2
orf261647-2864SEQ ID no 107SEQ ID no 108
orf272914-3534SEQ ID no 109SEQ ID no 110
orf28c4967-3804SEQ ID no 141SEQ ID no 142
orf295656-6663SEQ ID no 113SEQ ID no 114
orf30c7723-6686
7534-6686
SEQ ID no 115
SEQ ID no 143
SEQ ID No. 116 and 117
SEQ ID no 144
orf317754-8728SEQ ID no 118SEQ ID no 119
orf32c10488-8977SEQ ID no 145SE ID no 146
orf3310562-10837SEQ ID no 147SEQ ID no 148
orf34c12134-10899SEQ ID no 149SEQ ID no 150
1: The letter “C”added to the name of the gene means that the coding sequence is in the reverse orientation (coding thread, therefore, is a thread which is complementary to the sequence presented as SEQ ID No. 1 or SEQ ID No. 140 for these genes)
2: When one reading frames (ORFS) identified several protein sequences corresponding to the proteins originate from several possible sites begins broadcasting

To determine the function of the polypeptides identified according to the above table 11, was performed three types of experiments: a comparison of the identity of the identified sequences with sequences of known function, experiments on inactivation of genes and analysis of the production of spiramycin these mutant strains.

Protein sequence resulting from these open reading frames, were first compared with those available in the various databases, thanks to the various programs: BLAST (Altschul and others, 1990; Altschul and others, 1997), CD-search, COGs (Cluster ontologica groups), FASTA (Pearson W. R. &Lipman, D. J., 1988) and (W. R. Pearson, 1990)), BEATY (K. C. Worley and others, 1995) (see above). These comparisons allowed us to formulate hypotheses concerning the function produced by these genes products and identify those that may participate in the biosynthesis of spiramycin. Table 12 presents the proteins with greater affinity with 34 genes upstream 5 PKS genes.

Acyl-CoA-dehydrogenase
Table 12
GeneProtein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
orf1Cytochrome P450 tylI (Streptomyces fradiae)S49051530Cytochrome P450
orf2ORF15x4 (Listonella anguillarum)AAB81630113Unknown
orf3Such aminotransferase protein (Streptomyces antibioticus)AAF59939431 Aminotransferase
orf4α-D-glucose-1-phosphatidylserine (Streptomyces venezuelae)AAC68682404α-D-glucose-1-phosphati-meditrans-ferase
orf5AprE (Streptomyces tenebrarius)AAG18457476dTDP-glucose-4,6-dehydratase
orf6Thioesterase (Streptomyces avermitilis)BAB69315234Thioesterase
orf7TylCVI (Streptomyces fradiae)AAF29379461DNTP-hexose-2,3-dehydratase
orf8Probable aminotransferase (Saccharopolyspora spinosa)AAG23279465Aminotransferase
orf9cSrmX (Streptomyces ambofaciens)S25204445Methyltransferase
orf10SrmR (Streptomyces ambofaciens) S252031074Protein regulation
orf11cSrmB (Streptomyces ambofaciens)S25202955Resistance to spiramycin
orf12UrdQ (Streptomyces fradiae)AAF72550634NDP-hexose-3,4-dehydratase
orf13cSC4H2.17 (Streptomyces coelicolor)T35116619Unknown
orf14The putative reductase (Streptomyces coelicolor)CAB90862147Reductase
orf15cProbable 3-ketoreductase (Streptomyces antibioticus)T511022853-ketoreductase
orf16Hypothetical NDP-hexose-3,4-isomerase (Streptomyces fradiae)CAA57471209NDP-hexose-3,4-isomerase
orf17 Glycosyltransferase (Streptomyces venezuelae)AAC68677400Glycosyl-transferase
orf18Glycosyltransferase (Streptomyces rishiriensis)AAG29785185Glycosyl-transferase
orf19NDP-hexose-4-ketoreductase TylCIV (Streptomyces fradiae)AAD41822266NDP-hexose-4-ketoreductase
orf20EryBII (Saccharopolyspora erythraea)AAB84068491Aldoketoreductase
orf21cTylCIII (Streptomyces fradiae)AAD41823669NDP-hexose-3-C-methyltransferase
orf22cFkbH (Streptomyces hygroscopicus)AAF86387463Involved in the biosynthesis methoxymethyl
orf23cFkbI (Streptomyces hygroscopicus)AAF86388387
orf24cFkbJ (Streptomyces hygroscopicus)AAF8638987Involved in the biosynthesis methoxymethyl
orf25cFkbK (Streptomyces hygroscopicus)AAF86390268Acyl-CoA-dehydrogenase
orf26TylCV (Streptomyces fradiae)AAD41824471Mikrosil-transferase
orf27TylCVII (Streptomyces fradiae)AAD41825243NDP-hexose-3,5- (or 5-) epimerase
orf28cAcyB2 (Streptomyces thermotolerans)JC2032451Protein-controller
orf29Béta-mannanase (Sorangium cellulosum)AAK19890139Glycosyl-hydrolase
orf30cSharedocuments-parepimeria (Corynebacterium glutamicum) NP_60059089Sahariano-cleotide-phosphatability
orf31Oxidoreductase (Streptomyces coelicolor)NP_631148261Oxidoreductase
orf32cProtein regulator of the GntR family (Streptomyces avermitilis)NP_824604282Protein-controller
orf33Hypothetical protein (Xanthomonas campestris)NP_63556454Unknown
orf34cArabinofuranoside (Streptomyces coelicolor)NP_630049654Arabinfo-ranoside
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990)

Experiments on inactivation of genes was carried out to confirm these results. Used methods are the implementation of the replacement gene. Interrupted gene target was replaced by a copy of this gene, interrupted by the cluster, giving the cut is stadtnest to the antibiotic ( for example, apramycin or hygromycin). Used the cluster is flanked on both sides by codons broadcast in all reading frames and active transcription terminators in the case of Streptomyces. The insertion of the cluster in the gene target may be accompanied or not by a deletion in this gene target. The size of the regions flanking the cluster, up from several hundred to several thousand base pairs. For inactivation of genes can be used the second type of clusters: clusters, called “excesively clusters” (see above). Thus constructed the strains were tested for production of their spiramycine.

Gene orf1 encodes a protein with a relatively high similarity with several cytochromes P450. In particular, the protein encoded by orf1 gene, has considerable similarity with the protein encoded by the gene tylI involved in the biosynthesis of tylosin in Streptomyces fradiae (Merson-Davies L. A. and others, 1994; the access number in the gene Bank: S49051; score from BLAST: 530). This similarity with a protein involved in the biosynthesis pathway for another close antibiotic, highly suggesting that the gene orf1 encodes the cytochrome P450. This hypothesis is supported by the fact that the protein encoded by orf1 gene, has a great similarity with other proteins of similar function in other organisms (see table 13).

Table 13
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
Estimated cytochrome P450 YJIB (Bacillus subtilis)O34374248Cytochrome P450
Cytochrome-P450 113A1 (Saccharopolyspora erythraea)P48635237Cytochrome P450
The homologue of cytochrome-P-450 hydroxylase (Streptomyces caelestis)AAC46023208Cytochrome-P-450 hydroxylase
Cytochrome-P450 of monooxygenase (Streptomyces venezuelae)AAC64105206Cytochrome-P450 of monooxygenase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990)

Gene orf2 encodes a protein with a relatively high similarity to dTDP-6-deoxy-3,4-cathecolamines Aneurinibacillus thermoaerophilus (Pfoestl A. and others, 2003; the access number in the gene Bank: AAA score from BLAST: 118). This similarity in high suggesting that the gene orf2 encodes the isomerase responsible for the isomerization reaction required in the biosynthesis of one of the sugars present in the molecule spiramycin, and this sugar can be mycarose (see figure 5). Was carried out by inactivation of the gene orf2. It is shown that the resulting strain no longer produces spiramycin. This confirms that the orf2 gene is indeed involved in the biosynthesis of spiramycin.

Orf3 gene encodes a protein with a relatively high similarity with several aminotransferases. In particular, the protein encoded by orf3 gene, has considerable similarity with aminotransferase Streptomyces antibioticus (Draeger G. and others, 1999; the access number in the gene Bank: F59939; score from BLAST: 431). This similarity with a protein involved in the biosynthesis pathway for another close antibiotic, highly suggesting that orf3 gene encodes 3-aminotransferase responsible for the transamination reaction required in the biosynthesis of one of the amino sugars of spiramycin (see figure 5). This hypothesis is supported by the fact that the protein encoded by orf3 gene, has a great similarity with other proteins of similar function in other organisms (see table 15).

Table 15
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
Aminotransferase (Streptomyces antibioticus)T51111429Aminotransferase
Transaminase levels (Streptomyces venezuelae)AAC68680419Transaminase levels
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990).

Carried inactivation orf3 gene. Thus, it is shown that the resulting strain does not produce more spiramycine. This confirms that the orf3 gene is indeed involved in the biosynthesis of spiramycin. The enzyme encoded by this gene, therefore, is in fact responsible for the stage of bioconversion, essential in the biosynthesis of spiramycin. The production of spiramycin can be complemented by expression of the protein TylB S.fradiae (see example 23). This suggests that the orf3 gene encodes 3-aminotransferase responsible for the transamination reaction required in the biosynthesis of mycaminose (see figure 5). As mycaminose PR is dstanley the first sugar, fixed on platanoides expect that the strain interrupted in orf3 (OS49.67), accumulates platinoid.

Examined mediators of the biosynthesis of strain, interrupted in the orf3 gene (see example 20). These experiments allowed us to demonstrate that this strain produces two forms platinoid: platinoid and platinoid In, installed the structure of these two molecules presented on Fig. This strain also produces platinoid And + mycarose and platinoid In + mycarose (see example 20 and Fig). These compounds include sugar, but do not contain mycaminose. In addition, if they are compared with spiramycin (see figure 1), these compounds include mycarose instead mycaminose. These results are consistent with the participation produced by the orf3 gene product in the biosynthesis of mycaminose and its role as a 3-aminotransferase responsible for the transamination reaction required in the biosynthesis of mycaminose (see figure 5). You may notice that the specificity of glycosylation does not seem to be absolute, because detect molecules with microsol, fixed in the position usually occupied by mycaminose (see Fig).

Orf4 gene encodes a protein with a relatively high similarity with several NDP-glucosaminidase. In particular, the protein encoded by orf4 gene, has considerable similarity with a-D-glucose-1-phosphatidylinositol Streptomyces venezuelae (Y. Xue and others, 1998; DOS the UPA gene Bank: AAC68682; score from BLAST: 404). This similarity with a protein involved in the biosynthesis pathway for another close antibiotic, highly suggesting that orf4 gene also encodes NDP-glucosaminidase responsible for the synthesis of NDP-glucose needed in the biosynthesis of three atypical sugars that are built into the molecule spiramycin (see figure 4, 5 and 6). This hypothesis is supported by the fact that the protein encoded by orf4 gene, has a great similarity with other proteins of similar function in other organisms (see table 16).

Table 16
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
Glucose-1-phosphatidylserine (Streptomyces avermitilis)BAA84594402Glucose-1-phosphatidyl-
transferase
AclY (Streptomyces galilaeus)BAB72036400dTDP-1-glucosaminidase
The estimated glucose-1-phosphatidylserine (Saccharopolyspora spinosa)AAK83289 399Glucose-1-phosphatidyl-
transferase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990)

Orf5 gene encodes a protein with a relatively high similarity with several glucosegalactose. In particular, the protein encoded by orf5, has significant similarity to dTDP-glucose-4,6-dehydratase Streptomyces tenebrarius (Li T. B. and others, 2001; the access number in the gene Bank: AAG18457; score from BLAST: 476). This similarity with a protein involved in the biosynthesis pathway for another close antibiotic, highly suggesting that orf5 gene also encodes NDP-glucosegalactose required in the biosynthesis of three atypical sugars that are built into the molecule spiramycin (see figure 4, 5 and 6). This hypothesis is supported by the fact that the protein encoded by orf5 gene, has a great similarity with other proteins of similar function in other organisms (see table 17).

Table 17
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
DTDP-Lycosa-4,6-dehydratase (Saccharopolyspora spinosa) AAK83290464DTDP-glucose-4,6-dehydro-
pelvis
Timeinvestment-4,6-dehydratase (Saccharopolyspora erythraea)AAA68211445Timeinfo-polycose-4,6-dehydratase
DTDP-glucose-4,6-dehydratase (EC 4.2.1.46)-(Streptomyces fradiae)S49054443DTDP-glucose-4,6-dehydratase
TDP-glucose-4,6-dehydratase (Streptomyces venezuelae)AAC68681421TDP-glucose-4,6-dehydro-
pelvis
SgcA (Streptomyces globisporus)AAF13998418dNDP-glucose-4,6-dehydro-
pelvis
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990)

Orf6 gene encodes a protein with a relatively high similarity with several thioesterase. In particular, the protein encoded by orf6, has considerable similarity with thioesterase Streptomyces avermitilis (Omura S. and others, 2001; the access number in the gene Bank: BAB69315; score from BLAST: 234). This similarity with FR is given, involved in the biosynthesis pathway for another close antibiotic, highly suggesting that orf6 gene encodes also thioesterase. This hypothesis is supported by the fact that the protein encoded by orf6 gene, has a great similarity with other proteins of similar function in other organisms (see table 18).

Table 18
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
RifR (Amycolatopsis mediterranei)AAG52991216Thioesterase
Thioesterase (Streptomyces fradiae)S49055215Thioesterase
Thioesterase (Streptomyces avermitilis)BAB69188213Thioesterase
Thioesterase II (EC 3.1.2.-)-(Streptomyces venezuelae)T17413203Thioesterase
Protein PimI (Streptomycesnatalensis) CAC20922201Thioesterase
Thioesterase (Streptomyces griseus)CAC22116200Thioesterase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990)

Orf7 gene encodes a protein with a relatively high similarity with several exozodiacal. In particular, the protein encoded by orf7, has considerable similarity with dNTP-hexose-2,3-dehydratase (encoded by genome TylCVI) Streptomyces fradiae, involved in biosynthesis of tylosin (Merson-Davies L. A. and others, 1994; the access number in the gene Bank: AAF29379; score from BLAST: 461). This similarity with a protein involved in the biosynthesis pathway for another close antibiotic, highly suggesting that orf7 gene encodes also hexose-2,3-dehydratase required in the biosynthesis of two atypical sugars that are built into the molecule spiramycin (see figure 4 and 6). This hypothesis is supported by the fact that the protein encoded by orf7 gene, has a great similarity with other proteins of similar function in other organisms (see table 19).

Table 19
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
Rif18 (Amycolatopsis mediterranei)AAG52988459Existieras
SimB3 (Streptomyces antibioticus)AAK06810444dNDP-4-keto-6-detectplugin-2,3-dehydratase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990)

Orf8 gene encodes a protein with a relatively high similarity with several aminotransferases. In particular, the protein encoded by orf8, has considerable similarity with aminotransferase, possibly involved in the biosynthesis of forosamine in the case of Saccharopolyspora spinosa (C. Waldron and others, 2001; the access number in the gene Bank: AAG23279; score from BLAST: 465). This similarity with a protein involved in the biosynthesis pathway for another close antibiotic, highly suggesting that orf8 gene encodes 4-aminotransferase responsible for the transamination reaction required in the biosynthesis of forosamine (see Fig.6). This hypothesis connecting aleesa the fact, the protein encoded by the genome of orf8, has a great similarity with other proteins of similar function in other organisms (see table 20).

Table 20
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
The estimated protein biosynthesis of amino sugar (Bordetella bronchiseptica)CAA07666213Protein involved in the biosynthesis of amino sugar
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990)

Was carried out by inactivation of the gene orf8. Thus, it is shown that the resulting strain does not produce more spiramycine. This confirms that orf8 gene is indeed involved in the biosynthesis of spiramycin. The enzyme encoded by this gene, therefore, is in fact responsible for the stage of bioconversion, essential in the biosynthesis of spiramycin. In favor of the recognition of valid hypotheses about the role of the produced orf8 gene product in the bio is intese of forosamine, the fact that an inactivated mutant orf8 gene produces vorotilin therefore, this mutant is blocked at the stage of vorotilin and does not produce reaspiration (see Fig.7 and example 25). These results are consistent with the participation produced by the orf8 gene product in the biosynthesis of forosamine (see Fig.6).

Orf9 gene has already been identified in the case of Streptomyces ambofaciens was named srmX according Geistlich and others (Geistlich M. and others, 1992). The similarity of the protein encoded by this gene, with several methyltransferases involved in the biosynthesis pathway of other relatives antibiotics, highly suggesting that orf9 gene encodes a methyltransferase that is responsible for the methylation reaction required in the biosynthesis of mycaminose or forosamine (see figure 5 and 6). This hypothesis is supported by the fact that the protein encoded by orf9 gene, has a great similarity with other proteins of similar function in other organisms (see table 21).

Table 21
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
N,N-dimethylarsenate (Streptomyces venezuelae) AAC68678240N,N-dimethylarsenate
Methyltransferase (Streptomyces antibioticus)CAA05643232Methyltransferase
Estimated aminomethylated (Streptomyces nogalater)AAF01819219Aminomethylated
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others, 1990)

Orf10 gene has already been identified in the case of Streptomyces ambofaciens and named srmR according Geistlich and others (Geistlich M. and others, 1992). The protein encoded by this gene is involved in regulation of the biosynthesis pathway of spiramycin in the case of Streptomyces ambofaciens. Was carried out by inactivation of the gene orf10. Thus, it is shown that the resulting strain does not produce more spiramycine. This confirms that orf10 gene is indeed involved in the biosynthesis of spiramycin. The protein encoded by this gene, therefore, is in fact essential in the biosynthesis of spiramycin.

On the other hand, was determined the site of translation initiation orf10 and it is shown that overproduction this gene causes an increase in the production of spiramycin. The site of initiation) is tion corresponds ATG, located above ATG proposed Geistlich and others (Geistlich M. and others, 1992). In addition, it is shown that the 5'-end is essential for the function orf10, since shortened to 5' mRNA is inactive (see example 17). To achieve the desired effect in relation to the production of spiramycin therefore, it is important to place overproduction orf10, not wary of ekspressirovali shortened to 5' mRNA orf10 gene.

Gene orf11 was already identified in the case of Streptomyces ambofaciens and named srmB according Geistlich and others (Geistlich M. and others, 1992) and Schoener and others (Schoner B. and others, 1992). The protein encoded by this gene is involved in resistance to spiramycin in the case of Streptomyces ambofaciens and is a vector of type ABC.

Orf12 gene encodes a protein with a relatively high similarity with several exozodiacal. In particular, the protein encoded orf12, has considerable similarity with NDP-hexose-3,4-dehydratase encoded by the genome UrdQ Streptomyces fradiae and involved in biosynthesis organizing (Hoffmeister, D. and others, 2000; the access number in the gene Bank: AAF72550; score from BLAST: 634). This similarity with a protein involved in the biosynthesis pathway for another close antibiotic, highly suggesting that orf12 gene encodes 3,4-dehydratase responsible for the dehydration reaction required in the biosynthesis of forosamine (see Fig.6). This hypothesis supporting aetsa the fact, the protein encoded by the genome of orf12, has a great similarity with other proteins of similar function in other organisms (see table 22).

Table 22
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
AknP (Streptomyces galilaeus)AAF734526253-dehydratase
Homolog NDP-hexose-3,4-dehydratase (Streptomyces cyanogenus)AAD13547624NDP-hexose-3,4-dehydro-pelvis
RdmI (Streptomyces purpurascens)AAL24451608Hexose-C-3 - dehydratase
Likely CDP-4-keto-6-deoxyglucose-3-dehydratase (E1) (Streptomyces violaceoruber)T46528602CDP-4-keto-6-deoxyglucose-AOR-3-dehydratase
Probable NDP-hexose-3,4-dehydratase (Saccharopolyspora spinosa)AAG23278 582NDP-hexose-3,4-dehydratase
dNTP-existieras (Amycolatopsis mediterranei)AAC01730576dNTP-existieras
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others,1990)

Was carried out by inactivation of the gene orf12. It is shown that the resulting strain does not produce more spiramycine. This confirms that orf12 gene is indeed involved in the biosynthesis of spiramycin. The enzyme encoded by this gene, therefore, is in fact responsible for the stage of bioconversion, essential in the biosynthesis of spiramycin. In favor of the recognition of valid hypotheses about the role of Orf12 in the biosynthesis of forosamine, the fact that an inactivated mutant orf12 gene does not produce more forosamine. However, he produces a small number of vorotilin. Therefore, this mutant is blocked at the stage of vorotilin and does not produce reaspiration (see Fig.7 and example 26). This mutant, in addition, produces the connection patterns presented on Fig. This is the last connection includes two sugar, mycaminose and mycarose, but does not include forosamine. In addition, if the comparison is about the structure of spiramycin (see 1), this connection comprises a sugar mycarose instead of the expected forosamine. These results are consistent with the participation produced orf12 gene product in the biosynthesis of forosamine (see Fig.6). You may notice that the specificity of glycosylation is not absolute, because detect molecules in which mycarose fixed in the position usually occupied by forosamine (see Fig).

Gene orf13c encodes a protein with a relatively high similarity to proteins of unknown function in the case of Streptomyces coelicolor. This protein was named SC4H2.17 (access number in the gene Bank: T; score from BLAST: 619). The protein encoded by the gene orf13c, also has a great similarity with other proteins in other organisms (see table 23).

Table 23
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
Protein hflX (Mycobacterium leprae)S72938473Unknown
Possible ATP/GTP-binding protein (Mycobacterium leprae)NP_301739 470Protein retention ATP/GTP
GTP-binding protein (Mycobacterium tuberculosis CDC1551)AAK47114468Fixing GTP protein
ATP/GTP-binding protein (Streptomyces fradiae)T44592388Fixing ATP/GTP protein
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others,1990)

No precise function has not been identified in proteins that are close such that the encoded gene orf13c. Was made inactivation of the gene orf13c to study the function of this gene in the biosynthesis pathway of spiramycin in the case of Streptomyces ambofaciens. It is shown that the resulting strain produces spiramycin. This indicates that the gene orf13c is not essential in the biosynthesis of spiramycin and that it is not essential for the survival of bacteria. The enzyme encoded by this gene, therefore, is not responsible for the stage of bioconversion, essential in the biosynthesis of spiramycin.

Orf14 gene encodes a protein with a relatively large similarity with the proposed reductase (Redenbach, M. and others, 1996; Bentley and others, 2002; the number of access Bank g is new: SAW; score from BLAST: 147).

Was carried out by inactivation of the gene orf14. It is shown that the resulting strain does not produce more spiramycine. This confirms that orf14 gene is indeed involved in the biosynthesis of spiramycin. The enzyme encoded by this gene, therefore, is in fact responsible for the stage of bioconversion, essential in the biosynthesis of spiramycin. Investigated mediators biosynthesis in the case of strain, interrupted in the orf14 gene (see example 20). These experiments allowed us to demonstrate that this strain produces platinoid but does not produce platinoid (see Fig).

Gene orf15c encodes a protein with a relatively high similarity with several ketoreductase. In particular, the protein encoded orf15c, has significant similarity to 3-ketoreductase in the case of Streptomyces antibioticus (access number in the gene Bank: T72550; score from BLAST: 285). This similarity in high suggesting that the gene orf15c encodes 3-ketoreductase responsible for the reduction needed in the biosynthesis of forosamine (see Fig.6). This hypothesis is supported by the fact that the protein encoded by the gene orf15, has a great similarity with other proteins of similar function in other organisms (see table 24).

Table 24
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
The oxidoreductase homolog (Streptomyces cyanogenus)AAD13550272The oxidoreductase
D-Aliso-4-ketoreductase (Streptomyces argillaceus)CAB96550265D-Aliso-4-ketoreductase
AknQ (Streptomyces galilaeus)AAF73453263The alleged-may 3-ketoreductase
Probable NDP-hexose-3-ketoreductase (Saccharopolyspora spinosa)AAG23275253NDP-hexose-3-Ketorolac
pelvis
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others,1990)

Gene orf16 encodes a protein with a relatively high similarity with several isomerase. In particular, the protein encoded orf16, has considerable similarity with NDP-hexose-3,4-isomerase in the case of Streptomyces fradiae (Gandecha and others, 1997; number available is in the gene Bank: SAA; score from BLAST: 209). This similarity in high suggesting that the gene orf16 encodes a protein involved in the biosynthesis of one of the sugars of spiramycin (see figure 5 and 6). This hypothesis is supported by the fact that the protein encoded by the genome of orf16, has a great similarity with other proteins of similar function in other organisms (see table 25).

Table 25
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
Estimated tautomerase (Streptomyces venezuelae)AAC68676145Tautomerase
TDP-4-keto-6-deoxyhexose-3,4-isomerase (Micromonospora megalomicea subsp. nigra)AAG13907112TDP-4-keto-6-deoxyhexose-3,4-isomerase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschuel and others,1990)

Orf17 gene encodes a protein with a relatively high similarity with several glycosylation what verasamy. In particular, the protein encoded by orf17, has considerable similarity with glycosyltransferases Streptomyces venezuelae (Y. Xue and others, 1998; the access number in the gene Bank: AAS; score from BLAST: 400). The similarity of the protein encoded by the genome of orf17, with several glycosyltransferases involved in the biosynthesis pathway of other relatives antibiotics, highly suggesting that this gene also encodes glycosyltransferase. This hypothesis is supported by the fact that the protein encoded by the genome of orf17, has a great similarity with other proteins of similar function in other organisms (see table 26).

Table 26
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
Glycosyltransferase (Streptomyces fradiae)CAA57472399Glycosyltransferase
Glycosyltransferase (Streptomyces antibioticus)CAA05642378Glycosyltransferase
Glycosyltransferase (Streptomyces antibioticus CAA05641360Glycosyltransferase
Glycosyltransferase (Saccharopolyspora erythraea)CAA74710344Glycosyltransferase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschul and others,1990)

Gene orf18 encodes a protein with a relatively high similarity with several glycosyltransferases. In particular, the protein encoded orf18, has considerable similarity with glycosyltransferases Streptomyces rishiriensis (Wang and others, 1998; the access number in the gene Bank: G29785; score from BLAST: 185). The similarity of the protein encoded by the genome of orf18, with several glycosyltransferases involved in the biosynthesis pathway of other relatives antibiotics, highly suggesting that this gene also encodes glycosyltransferase. This hypothesis is supported by the fact that the protein encoded by the genome of orf18, has a great similarity with other proteins of similar function in other organisms (see table 27).

Table 27
Protein with significant similarity The access number in the gene BankScore from BLAST*Correlated function
NovM (Streptomyces spheroides)AAF67506184Glycosyltransferase
Likely glycosyltransferase (Streptomyces violaceoruber)T46519169Glycosyltransferase
The glycosyltransferase gene homologue (Streptomyces cyanogenus)AAD13553167Glycosyltransferase
The glycosyltransferase gene homologue (Streptomyces cyanogenus)AAD13555163Glycosyltransferase
dNTP-glycosyltransferase (Amycolatopsis mediterranei)AAC01731160Glycosyltransferase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschul and others,1990)

Gene orf19 encodes a protein with a relatively high similarity with several ketoreductase. In particular, the protein encoded orf19, the region is AET significant similarity with NDP-hexose-4-ketoreductase (TylCIV) Streptomyces fradiae (Bate and others, 2000; the access number in the gene Bank: D41822; score from BLAST: 266). The similarity of the protein encoded by the genome of orf19, this ketoreductases involved in the biosynthesis pathway similar antibiotic, highly suggesting that this gene encodes a 4-ketoreductase responsible for the reduction needed in the biosynthesis of mycarose (see figure 4). This hypothesis is supported by the fact that the protein encoded by the genome of orf19, has a great similarity with other proteins of similar function in other organisms (see table 28).

Table 28
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
NDP-4-keto-6-deoxyhexose-4-ketoreductase (Streptomyces venezuelae)AAL14256251NDP-4-keto-6-deoxyhexose-4-ketoreductase
EryBIV (Saccharopolyspora erythraea)AAB84071249Oxidoreductase-
TDP-4-keto-6 - deoxyhexose-4 - ketoreductase (Micromonospora megalomicea subsp. Nigra) AAG13916218TDP-4-keto-6-deoxyhexose-4-ketoreductase
dTDP-4-keto-6-deoxy-L-hexose-4-reductase (Streptomyces avermitilis)BAA84595212dTDP-4-keto-6-deoxy-L-hexose-4-
reductase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschul and others,1990)

Gene orf20 encodes a protein with a relatively high similarity with several hexatriacontane. In particular, the protein encoded by orf20, has considerable similarity with the gene EryBII Saccharopolyspora erythraea, which encodes a dTDP-4-keto-L-6-deoxyhexose-2,3-reductase Summers, R.G., and others, 1997; the access number in the gene Bank: AAV; score from BLAST: 491). The similarity of the protein encoded by the genome of orf20, with several hexatriacontane involved in the biosynthesis pathway of other relatives antibiotics, highly suggesting that this gene encodes 2,3-reductase responsible for the reduction needed in the biosynthesis of mycarose (see figure 4). This hypothesis is supported by the fact that the protein encoded by the genome of orf20, has a great similarity with other proteins of similar function in other organisms (see table 29).

Table 29
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
TylCII (Streptomyces fradiae)AAD41821464NDP-hexose-2,3-torridal-pelvis
TDP-4-keto-6-deoxyhexose-2,3-reductase (Micromonospora megalomicea subsp. Nigra)AAG13914446TDP-4-keto-6-detoxikacni-zo-2,3-reductase
dTDP-4-keto-6-deoxy-L-hexose-2,3-reductase (Streptomyces avermitilis)BAA84599377dTDP-4-keto-6-deoxy-L-hexose-2,3-reductase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschul and others,1990)

Gene orf21c encodes a protein with a relatively high similarity with several hexosaminidase. In particular, the protein encoded orf21, has considerable similarity with the gene TylCIII Streptomyces fradiae, which encodes NDP-hexose-3-C-methyltransferase (N. Bate and others, 2000; the number of access Bykhanov: D41823; score from BLAST: 669). The similarity of the protein encoded by the gene orf21c, with several hexosaminidase involved in the biosynthesis pathway of other relatives antibiotics, highly suggesting that this gene encodes hexosaminidase responsible for the methylation reaction required in the biosynthesis of mycarose (see figure 4). This hypothesis is supported by the fact that the protein encoded by the gene orf21, has a great similarity with other proteins of similar function in other organisms (see table 30).

Table 30
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
EryH (Saccharopolyspora erythraea)228448592Gene biosynthesis of erythromycin
S-adenocarcinoma methyltransferase (Coxiella burnetii)AAK71270358Methyltransferase
NovU (Streptomyces spheroides)AAF67514184 C-methyltransferase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschul and others,1990)

Gene orf22c encodes a protein with a relatively large similarity with the protein encoded by the gene fkbH Streptomyces hygroscopicus var. ascomyceticus, which encodes an enzyme involved in the biosynthesis methoxymethyl (K. Wu and others, 2000; the access number in the gene Bank: F86387; score from BLAST: 463). The similarity of the protein encoded by the gene orf22c, this protein is involved in the biosynthesis pathway of another close of the macrolide, highly suggesting that this gene also encodes an enzyme involved in the biosynthesis methoxymethyl in the case of Streptomyces ambofaciens (see Fig).

Gene orf23c encodes a protein with a relatively large similarity with the protein encoded by the genome of fkbI Streptomyces hygroscopicus var. ascomyceticus, which encodes an acyl-COA-dehydrogenase, involved in the biosynthesis methoxymethyl (K. Wu and others, 2000; the access number in the gene Bank: F86388; score from BLAST: 387). The similarity of the protein encoded by the gene orf23c, with multiple acyl-COA-dehydrogenase, involved in the biosynthesis pathway of other relatives antibiotics, highly suggesting that this gene encodes an acyl-COA-dehydrogenase, involved in the biosynthesis methoxymethyl (see Fig). This hypothesis is supported by the fact that the protein, the encoded gene orf23c, has a great similarity with other proteins of similar function in other organisms (see table 31).

Table 31
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
Acyl-CoA-dehydrogenase (Polyangium cellulosum)AAK19892171Acyl-CoA-dehydrogenase
Probable acyl-CoA dehydrogenase (Streptomyces coelicolor)T36802160Acyl-CoA-dehydrogenase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschul and others,1990)

Gene orf24c encodes a protein with a relatively large similarity with the protein encoded by the gene fkbJ Streptomyces hygroscopicus var. ascomyceticus, which encodes a protein binding acyl group (protein-acyl carrier group (ACP))involved in the biosynthesis methoxymethyl (K. Wu and others, 2000; the access number in the gene Bank: F86389; score from BLAST: 87). The similarity of the protein, dirremove genome orf24c, this protein is involved in the biosynthesis pathway of another close of the macrolide, highly suggesting that this gene encodes a protein involved in the biosynthesis methoxymethyl in the case of Streptomyces ambofaciens (see Fig).

Gene orf25c encodes a protein with a relatively large similarity with the protein encoded by the gene fkbK Streptomyces hygroscopicus var. ascomyceticus, which encodes an acyl-COA-dehydrogenase, involved in the biosynthesis methoxymethyl (K. Wu and others, 2000; the access number in the gene Bank: F86390; score from BLAST: 268). The similarity of the protein encoded by the gene orf25c, with multiple acyl-COA-dehydrogenase, involved in the biosynthesis pathway of other relatives antibiotics, highly suggesting that this gene encodes an acyl-COA-dehydrogenase, involved in the biosynthesis methoxymethyl (see Fig). This hypothesis is supported by the fact that the protein encoded by the gene orf25c, has a great similarity with other proteins of similar function in other organisms (see table 32).

Table 32
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
Veroia the Fes 3-hydroxybutyryl-CoA-dehydrogenase ( Bacillus subtilis)P458561773-hydroxybutyl-RIL-CoA-dehydrogenase
Protein 3-hydroxybutyryl-CoA-dehydrogenase (Bacillus thuringiensis serovar kurstaki)AAL322701743-hydroxybutyl-RIL-CoA-dehydrogenase
3-hydroxybutyryl-CoA-dehydrogenase (Deinococcus radiodurans)NP_2947921673-hydroxybutyl-RIL-CoA-dehydrogenase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschul and others,1990)

Orf26 gene encodes a protein with an identity of 65% (determined by the BLAST program) with the protein encoded by the genome of tylCV, which encodes microtitration involved in the biosynthesis of tylosin in the case of Streptomyces fradiae (N. Bate and others, 2000; the access number in the gene Bank: D41824; score from BLAST: 471). More specifically, TylCV is glycosyltransferase linking molecule mycarose during the synthesis of tylosin. This similarity with a protein involved in the biosynthesis pathway of another, relatively close antibiotic and particularly in the transfer mycarose, suggesting that orf26 gene encodes Glick is intransferable. This hypothesis is supported by the fact that the protein encoded by the genome of orf26, has a great similarity with other proteins of similar function in other organisms (see table 33).

Table 33
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
Glycosyltransferase (Streptomyces avermitilis)BAA84592218Glycosyltransferase
CalG4 (Micromonospora echinospora)AAM70365217Glycosyltransferase
CalG2 (Micromonospora echinospora)AAM70348197Glycosyltransferase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschul and others,1990)

Gene orf27 encodes a protein having an identity of 70% (determined by the BLAST program) with the protein encoded by the gene tylCVII that encodes NDP-hexose-3,5-(or 5-)epimers is, involved in the biosynthesis of tylosin in the case of Streptomyces fradiae (N. Bate and others, 2000; the access number in the gene Bank: D41825; score from BLAST: 243). More specifically, TylCVII is hexose-3,5-(or 5-)epimerase involved in the biosynthesis of mycarose. This similarity with a protein involved in the biosynthesis pathway of another, relatively close antibiotic and particularly in the biosynthesis of mycarose, suggesting that the gene orf27 encodes epimerase. This hypothesis is supported by the fact that the protein encoded by the gene orf27, has a great similarity with other proteins of similar function in other organisms (see table 34). A close analysis of the sequences obtained by BLAST program, highly suggesting that the gene orf27 encodes 5-epimerase responsible for the epimerization reaction required in the biosynthesis of mycarose (see figure 4).

Table 34
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
LanZ1 (Streptomyces cyanogenus)AAD13558172NDP-hexose-3,5-epimerase
Epi (Saccharopolyspora spinosa)AAK83288169TDP-4-keto-6-deoxyglucose-3,5-
epimerase
dNTP-hexose-3,5-epimerase (Amycolatopsis mediterranei)AAC01732166dNTP-hexose-3,5-epimerase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschul and others,1990)

The sequence orf28c, first, defined partly because the sequence of the segment with a length of approximately 450 base pairs was determined only after re-sequencing (this area is marked by the symbol “N” in the partial sequence SEQ ID No. 106). Partial sequence of this open reading frame (SEQ ID no. 111) however, was used for analysis using a variety of informational programs, as explained above. Thus, it is determined that the gene orf28c encodes a protein having an identity of 64% in a certain sequence (SEQ ID No. 112, which is a partial sequence of a protein Orf28c) (defined by the BLAST program) with the protein encoded by the gene acyB2, which encodes a protein regulator that is involved in the biosynthesis of carbomycin in the case of Streptomyces thermotolerans (Arisawa A. and others, 1993 the access number in the gene Bank: JC2032; score from BLAST: 329). This similarity with a protein involved in the biosynthesis pathway of relatively close antibiotic, suggesting that the gene orf28c encodes a protein regulator that is involved in the biosynthesis of spiramycin. This hypothesis is supported by the fact that the protein encoded by the gene orf28c, also has a great similarity with protein TylR, which is a protein regulator involved in the biosynthesis of tylosin in the case of Streptomyces fradiae (N. Bate and others, 1999; the access number in the gene Bank: AAF29380; score from BLAST: 167).

Gene orf28c was amplified by oligonucleotides that are located on either side of an indefinite sequence and subcloned in expressing vector. Thus, it is shown that overproduction gene orf28c significantly increases the production of spiramycin strain OSC2 (see example 24). This shows that overproduction gene orf28c leads to increased production of spiramycin and confirms its role as regulator of the biosynthesis pathway of spiramycin.

Partial sequence orf28c, therefore, was added and was found to be missing region with a length of about 450 base pairs (see SEQ ID No. 140 and SEQ ID No. 141). The full sequence of this open reading frame (SEQ ID No. 141) was used for analysis using a variety of informational programs, as explained above. Thisway, determined that the gene orf28c encodes a protein having an identity of 69% in a certain sequence (SEQ ID No. 142, which represents the complete sequence of the protein Orf28c) (defined by the BLAST program) with the protein encoded by the gene acyB2, which encodes a protein regulator that is involved in the biosynthesis of carbomycin in the case of Streptomyces thermotolerans (Arisawa A. and others, 1993; the access number in the gene Bank: JC2032; score from BLAST: 451). This similarity with a protein involved in the regulation of biosynthesis relatively close antibiotic, suggesting that the gene orf28c encodes a protein regulator that is involved in the biosynthesis of spiramycin. This hypothesis is supported by the fact that the protein encoded by the gene orf28c, also has a great similarity with protein TylR, which is a protein regulator involved in the regulation of biosynthesis of tylosin in the case of Streptomyces fradiae (N. Bate and others, 1999; the access number in the gene Bank: AAF29380; score from BLAST: 224). The results overproductive this gene (see example 24) confirm its role as regulator of the biosynthesis pathway of spiramycin.

Was made inactivation of the gene orf28c. Thus, it is shown that the resulting strain does not produce more spiramycine. This confirms that the gene orf28c really involved in the biosynthesis of spiramycin and is essential in the biosynthesis spiramycine the results together with the results overproducing this gene (see example 24) are consistent with the role of activator essential in the biosynthesis of spiramycine Orf28c.

Orf29 gene encodes a protein with an identity of 31% (determined by the BLAST program) with probable glycosylglycerols localized in the group of genes involved in the biosynthesis sarafina And (antifungal agent class of polyketides) in the case of Sorangium cellulosum (J. Ligon and others, 2002; the access number in the gene Bank: AAC; score from BLAST: 139). This similarity with a protein involved in the biosynthesis pathway relatively similar molecules, suggesting that orf29 gene encodes a protein having the activity of glucosylceramide. This hypothesis is supported by the fact that the protein encoded by the genome of orf29, has a great similarity with other proteins of similar function in other organisms (see table). Sequence analysis of the protein encoded orf29, the program CD-search (see above) also allows us to suppose that orf29 gene encodes glycosylglycerols.

Table 35
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
ManA (Caldicellulosiruptor saccharolyticus)AAC4432 136Beta-1,4-mannanase
ManA (Dictyoglomus thermophilum)AAB82454129Beta-mannanase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschul and others,1990)

Analysis of protein sequence, the next of orf29, the program SignalP (http://www.cbs.dtu.dk/services/SignalP/) (Nielsen H. and others, 1997) shows that this protein has a C-terminal signal sequence with a predicted cleavage site between positions 30 and 31 (QSA/QA). You can predict that this protein is extracellular. He can, as well as glycosylglycerols to play a role in the reactivation of inactivated spiramycine by glycosylation using glycosyltransferases GimA and/or GimB (Gourmelen and others, 1998).

Gene orf30c encodes a protein having an identity of 31% (determined by the BLAST program) sharedrecipesdotorg Corynebacterium glutamicum (access number in the gene Bank: NP_600590; score from BLAST: 89). This similarity allows us to suppose that the gene orf30c encodes epimerase. This hypothesis is supported by the fact that the sequence analysis program CD-search (see above) suggesting that the gene orf30c encodes epimerase.

Gene orf30c has two POS of the different initiation codon (see SEQ ID No. 115), which gives two possible protein length, respectively, 345 282 amino acids and amino acid (SEQ ID No. 116 and SEQ ID No. 117). However, the use of codons is typical of Streptomyces only from the second ATG, to the same protein sequence, the following sequence between the first ATG and the second ATG, not comparable by alignment with identified similar sequences, whereas most short protein sequence (from the second ATG: SEQ ID no 144) is well comparable by aligning with the beginning of these proteins. Therefore, we can conclude that the second ATG is a good initiation codon and the sequence of this open reading frame (orf), therefore, is presented as SEQ ID No. 143, which, once expressed, corresponds to a protein corresponding to the sequence SEQ ID No. 144.

Gene orf31 encodes a protein having an identity of 52% (determined by the BLAST program) oxidoreductase in Streptomyces coelicolor (access number in the gene Bank: NP_631148; score from BLAST: 261). This similarity allows us to suppose that the gene orf31 encodes the reductase. This hypothesis is supported by the fact that the sequence analysis program CD-search (see above) also allows you to believe that gene orf31 encodes the reductase. This hypothesis is also supported by the fact the f is that the protein encoded by the genome of orf31, has a great similarity with other proteins of similar function in other organisms (see table 36).

Table 36
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
Putative oxidoreductase (Streptomyces griseus)BAB79295173Oxidoreductase
MocA (Xanthomonas axonopodis)NP_640644171Oxidoreductase
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschul and others,1990)

Was made the inactivation of the orf31. Thus, it is shown that the resulting strain does not produce more spiramycine. This confirms that the gene orf31 really involved in the biosynthesis of spiramycin. The enzyme encoded by this gene, therefore, is in fact responsible for the stage of bioconversion, essential in the biosynthesis of ceramic is new.

The sequence orf32c was first identified in part (see example 19), because the coding sequence of the 5' was defined then. Partial sequence of this open reading frame (SEQ ID No. 120) however was used for analysis using a variety of informational programs, as explained above. Thus, it is determined that the gene orf32c encodes a protein having an identity of 47% in a certain sequence (SEQ ID No. 121, which is a partial sequence of a protein Orf32c) (determined by the BLAST program) protein regulator of the GntR family in the case of Streptomyces coelicolor (access number in the gene Bank: NP_625576; score from BLAST: 229). This similarity allows us to suppose that the gene orf32c encodes a transcription regulator GntR family. This hypothesis is supported by the fact that the protein encoded by the gene orf32c, has a great similarity with other proteins of similar function in other organisms.

Partial sequence orf32c was then added and was determined missing region (see SEQ ID No. 140 and SEQ ID no 145). The full sequence of this open reading frame encodes a protein having an identity of 44% (determined by the BLAST program) protein regulator of the GntR family in case of Streptomyces avermitilis (access number in the gene Bank: NP_824604; score from BLAST: 282). It is the similarities which allows you to put, what gene orf32c encodes a transcription regulator GntR family. This hypothesis is supported by the fact that the protein encoded by the gene orf32c, has a great similarity with other proteins of similar function in other organisms (see table 37).

Table 37
Protein with significant similarityThe access number in the gene BankScore from BLAST*Correlated function
Protein regulator of the GntR family (Streptomyces avermitilis)NP_828241270Protein regulator of the GntR family
Protein regulator of the GntR family of Streptomyces coelicolor)NP_625576266Protein regulator of the GntR family
SC5G8.04 (Streptomyces coelicolor)NP_628991258Protein regulator of the GntR family
The regulator of transcription (Streptomyces venezuelae)AAF01064224The regulator transcription
Protein regulator of the GntR family (Streptomyces avermitilis)NP_827432239Protein regulator of the GntR family
*: the greater the similarity of the sequences associated with the higher score on BLAST (Altschul and others,1990)

Was carried out by inactivation of the gene orf32c to study the function of this gene in the biosynthesis pathway of spiramycin in the case of Streptomyces ambofaciens. It is shown that the resulting strain produces spiramycin. This indicates that the gene orf32c is not essential in the biosynthesis of spiramycin and that it is not essential for the survival of bacteria.

Gene orf33 encodes a protein having an identity of 49% (determined by the BLAST program) hypothetical protein Xanthomnas campestris (access number in the gene Bank: NP_635564; score from BLAST: 54).

The sequence orf34c is partial. In fact, comparisons made between the product of this open reading frame and data banks suggests that the C-terminal part of this protein is not present in the product of the following nucleotide sequences, and, therefore, that this open reading frame is longer and extends outside the sequenced region. Partial sequence of this ORF, however, was using the and for analysis using a variety of informational programs, as specified above. Thus, it is determined that the gene orf34c encodes a protein having an identity of 91% in a certain sequence (SEQ ID No. 150, which is a partial sequence of a protein Orf34c) (determined by the BLAST program) arabinofuranoside in the case of Streptomyces coelicolor (Bentley and others, 2002; the access number in the gene Bank: NP_630049; score from BLAST: 654). In the case of S. coelicolor, the gene encoding this arabinofuranoside, apparently, is not involved in the biosynthesis of the secondary metabolite. This gene in S. ambofaciens, therefore, probably not involved in the biosynthesis of spiramycin.

The object of the present invention are also polynucleotide, hybridization in hybridization conditions of high stringency with at least one polynucleotide corresponding to the sequence SEQ ID№ 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 28, 30, 34, 36, 40, 43, 45, 47, 49, 53, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 107, 109, 111, 113, 115, 118, 120, 141, 143, 145, 147 and 149 or one of its variants, or one of the sequences originating from them in accordance with the degeneracy of the genetic code. Preferably, these polynucleotide was isolated from bacteria of the genus Streptomyces, more preferably, these polynucleotides encode proteins involved in the biosynthesis of the macrolide, and even more preferably, these polynucleotides encode a protein having the activity of these Pro is Ainu, encoded by polynucleotide with which they hybridize. The hybridization conditions of high stringency can be defined as hybridization conditions, blagopriyatstvuyut hybridization of homologous strands of nucleic acids. The hybridization conditions of high stringency can be, for example, the conditions described as conditions of hybridization in buffer described by Church and Gilbert (Church &Gilbert, 1984)at a temperature of from 55°C to 65°C, and, preferably, the hybridization temperature is 55°C, even more preferably, the hybridization temperature is 60°C and highly preferably, the hybridization temperature is 65°C, followed by one or more washes carried out in the buffer 2X SSC at a temperature of from 55°C to 65°C and, preferably, this temperature is 55°C, even more preferably, this temperature is 60°C and highly preferably, this temperature is 65°C, followed by one or more washes carried out in the buffer 0.5x SSC at a temperature of from 55°C to 65°C, and, preferably, this temperature is 55°C, even more preferably, this temperature is 60°C and highly preferably, this temperature is 65°C. the Above conditions are hybridization can be adapted depending on the length of nuclein the howling acid, hybridization investigate, or the type of the selected marking, according to methods known to the person skilled in the art. Suitable hybridization conditions, for example, can be adapted according to F. Ausubel and others, 2002.

The invention relates also to polynucleotide having at least 70%, more preferably 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, most preferably 98% identity to nucleotides, with polynucleotides, including at least 10, 12, 15, 18, 20-25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850 or 1900 consecutive nucleotides of polynucleotide selected from the group consisting of nucleotide sequences SEQ ID№ 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 28, 30, 34, 36, 40, 43, 45, 47, 49, 53, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 107, 109, 111, 113, 115, 118, 120, 141, 143, 145, 147 and 149 or one of its variants, or one of the sequences originating from them in accordance with the degeneracy of the genetic code, or polynucleotide with complementary sequence. Preferably, these polynucleotides isolated from bacteria of the genus Streptomyces, more preferably, these polynucleotides encode proteins involved in the biosynthesis of the macrolide, and even more preferably, the th polynucleotide encode proteins, with activities like proteins encoded by polynucleotide that they possess identity. Highly preferably, polynucleotide according to the invention are selected from the group consisting of nucleotide sequences SEQ ID№ 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 28, 30, 34, 36, 40, 43, 45, 47, 49, 53, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 107, 109, 111, 113, 115, 118, 120, 141, 143, 145, 147 and 149 or polynucleotide with complementary sequence.

The best comparison by alignment of sequences for comparison may be computer implemented method using known algorithms, for example, such a set of FASTA programs (W.R. Pearson &D.J. Lipman, 1988; Pearson W.R., 1990), available, particularly from the resource center INFOBIOGEN, Evry, France. As a clarification, the percent sequence identity may be determined using a software package LFASTA (Chao, K.-M. and others, 1992) or LALIGN (Huang and X. W. Miller, 1991). Program LFASTA and LALIGN form part of the FASTA software package. LALIGN aimed at optimal local comparison by alignment: this program is more accurate, but slower than LFASTA.

Another aspect of the invention relates to the polypeptide resulting from the expression of the nucleotide sequence, such as the above. Preferably, the polypeptides according to the invention possess at m is re 70%, more preferably 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, most preferably 98% identity on the amino acid, polypeptide, comprising at least 10, 15, 20, 30-40, 50, 60, 70, 80, 90,100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620 or 640 consecutive amino acids of a polypeptide selected among SEQ ID№ 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 27, 29, 31, 32, 33, 35, 37, 38, 39, 41, 42, 44, 46, 48, 50, 51, 52, 54, 55, 56, 57, 58, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 108, 110, 112, 114, 116, 117, 119, 121, 142, 144, 146, 148 and 150 or one of these sequences, except for the fact that, in General, for the above sequence one or more amino acids replaced built or subject to deletion without modifying the functional properties, or one of the variants of these sequences. Preferably, the polypeptides according to the invention expressed in the native state by a bacterium of the genus Streptomyces, more preferably, these polypeptides involved in the biosynthesis of the macrolide and, more preferably, these polypeptides have activity similar to that of the polypeptide with which it has identity. Preferably, the polypeptide according to the invention are selected from the group consisting of polypeptide sequences of SEQ ID№ 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 27, 29, 31, 32, 33, 35, 37, 38,39, 41, 42, 44, 46, 48, 50, 51, 52, 54, 55, 56, 57, 58, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 108, 110, 112, 114, 116, 117, 119, 121, 142, 144, 146, 148 and 150 or one of these sequences, except for the fact that, in General, throughout the length of the above sequence one or more amino acids replaced built or subject to deletion without modifying the functional properties, or one of the variants of these sequences. Highly preferably, the polypeptide according to the invention are selected from the group consisting of polypeptide sequences of SEQ ID№ 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 27, 29, 31, 32, 33, 35, 37, 38, 39, 41, 42, 44, 46, 48, 50, 51, 52, 54, 55, 56, 57, 58, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 108, 110, 112, 114, 116, 117, 119, 121, 142, 144, 146, 148 and 150.

The best comparison by alignment of sequences for comparison may be computer implemented method using known algorithms, for example, such a set of FASTA programs (W.R. Pearson &D.J. Lipman, 1988; Pearson W.R., 1990), available, particularly from the resource center INFOBIOGEN, Evry, France. As a clarification, the percent sequence identity may be determined using a software package LFASTA (Chao, K.-M. and others, 1992) or LALIGN (Huang and X. W. Miller, 1991), using the parameters on the defect, such as defined by the resource center INFOBIOGEN, Evry, France. Program LFASTA and LALIGN form part of the FASTA software package. LALIGN aimed at optimal local SOP is based by alignment: this program is more accurate however, it is also slower than LFASTA.

Another aspect of the invention relates to a recombinant DNA comprising at least one polynucleotide, such as described above. Preferably, this recombinant DNA is a vector. Even more preferably, the vector is chosen from bacteriophages, plasmids, phagemid integrating vectors, fosmid, cosmid, Shuttle vectors, YOU (bacterial artificial chromosome) or RACES (P1-derived artificial chromosome). As a clarification, as bacteriophages can be called phage lambda and M13 phage. As plasmids can be called plasmids that can replicate in E. coli, for example, pBR322 and its derivatives, pUC18 and its derivatives, pUC19 and its derivatives, pGB2 and its derivatives (G. Churchward and others, 1984), pACYC177 (access number in the gene Bank: H) and its derivatives, pACYC184 (access number in the gene Bank: H) and its derivatives. May also be called plasmids that can replicate in Streptomyces, such as pIJ101 and its derivatives, pSG5 and its derivatives, SLP1 and its derivatives, SCP2* and its derivatives (Kieser and others, 2000). As phagemid can be called as explanations, pBluescript II and its derivatives (produced, in particular, by the company Stratagene (LaJolla, CA, USA)), pGEM-T and its derivative (manufactured by a company Promega (Madison, Wisconsin, USA)), λZAPII and its derivatives (produced, in particular, by the company Stratagene (LaJolla, CA, USA)). As and tigerwomah vectors can be called, as a clarification, integrating vectors in the case of Streptomyces, such as those occurring from SLP1 (Kieser and others, 2000), those originating from pSAM2 (Kieser and others, 2000), vectors, systems integration phage PhiC31 (Kieser and others, 2000) (e.g., pSET152 (Bierman and others, 1992)) or VWB (Van Mellaert L. and others, 1998), as well as vectors, using the system integration IS117 (Kieser and others, 2000). As fosmid can be called as explanations fosmid pFOS1 (manufactured by New England Biolabs Inc., Beverly, Massachussetts, USA) and its derivatives. As cosmid can be called as explanations, kosmidou SuperCos and its derivatives (produced, in particular, by the company Stratagene (LaJolla, CA, USA)), kosmidou pWED15 (Wahl and others, 1987) and its derivatives. As Shuttle vectors can be called as explanations Shuttle plasmids E. coli/Streptomyces, such as pIJ903 and its derivatives, a number of plasmids pUWL, pCAO106, pWHM3, pOJ446 and their derivatives (Kieser and others, 2000), Shuttle YOU E. coli/Streptomyces, such as those described in the International patent application WO-01/40497. As YOU (bacterial artificial chromosome) can be called, as the explanations YOU pBeloBAC11 (access number in the gene Bank: U51113). As RACES (P1-derived artificial chromosome) can be called, as explanations, vector pCYPAC6 (access number in the gene Bank: AF133437). Highly preferably, the vector according to the invention, chosen among pOS49.1, pOS4.11, pOSC49.12, pOS49.14, pOS49.16, pOS49.28, pOS44.1, pOS44.2, pOS44.4, pSPM5, pSPM7, pOS49.67, pOS49.88, pOS49.106, pOS49.120, pOS49.107, pOS49.32, pOS49.43, pOS49.44, pOS49.50, pOS49.99, pSPM17, pSPM21, pSPM502, pSPM504, pSPM507, pSPM508, pSPM509, pSPM1, pBXL1111, pBXL1112, pBXL1113, pSPM520, pSPM521, pSPM522, pSPM523, pSPM524, pSPM525, pSPM527, pSPM528, pSPM34, pSPM35, pSPM36, pSPM37, pSPM38, pSPM39, pSPM40, pSPM41, pSPM42, pSPM43, pSPM44, pSPM45, pSPM47, pSPM48, pSPM50, pSPM51, pSPM52, pSPM53, pSPM55, pSPM56, pSPM58, pSPM72, pSPM73, pSPM515, pSPM519, pSPM74, pSPM75, pSPM79, pSPM83, pSPM107, pSPM543 and pSPM106.

Another aspect of the invention relates to an expression system that includes the appropriate expressing vector and a cell of the host, allowing the expression of one or more polypeptides, such as described above, in a biological system. Expression vectors according to the invention include the nucleotide sequence encoding one or more polypeptides, such as described above, and can be designed for expression of different polypeptides according to the invention in different cells-the masters, well-known specialists in this field. As an example, can be called a prokaryotic expression system, such as gene-expression system in the case of E. coli, eukaryotic expression systems such as baculovirus expression system, which allows for expression in insect cells, expression system, which allows for expression in yeast cells, expression systems, p is permitted to carry out the expression in mammalian cells, in particular, in human cells. Expression vectors used in such systems, well known to experts in the field; in regard to prokaryotic cells, can be called as an explanation, expression vectors in the case of E. coli, for example, family pet, manufactured by the company Stratagene (LaJolla, CA, USA), the vectors of the family GATEWAY, manufactured by Invitrogen (Carlsbad, CA, USA), the vectors of the family pBAD manufactured by Invitrogen (Carlsbad, CA, USA), the vectors of the family pMAL manufactured by New England Biolabs Inc. (Beverly, Massachussetts, USA), expression vectors, the inductive Ramezay specified in the publication Wilms B. and others, 2001, and their derivatives, may also be called expression vectors in Streptomyces, such as, for example, vectors pIJ4123, pIJ6021, pPM927, pANT849, pANT850, pANT851, pANT1200, pANT1201, pANT1202, and their derivatives (Kaiser and others, 2000). In regard to yeast cells, can be called as a clarification, the vector pESC manufactured by the company Stratagene (LaJolla, CA, USA). In regards baculovirus expression system, which allows for expression in insect cells, can be called as explanations, vector Basrak manufactured by BD Biosciences Clontech (Palo Alto, CA, USA). In regard to mammalian cells, can be called, as an example, vectors comprising the promoter of the early genes of the virus CMV (cytomegalovirus) (e.g. the R, the vector pCMV and its derivatives are produced by the company Stratagene (LaJolla, CA, USA), or the promoter predannih genes (the early promoter of SV40) vacuolating monkey virus SV40 (e.g., vector pSG5, manufactured by the company Stratagene (LaJolla, CA, USA).

The invention also relates to a method for production of the polypeptide, such as described above, with the above method includes the following stages:

a) insertion of a nucleic acid encoding the above polypeptide into an appropriate vector;

b) culturing, in the appropriate culture medium, host cells previously transformed or transfectional using the vector from step a);

(C) recovery of conditioned culture medium or cell extract, for example, by ultrasonic treatment or by osmotic shock;

d) isolation and purification of the above culture medium or else from the cell extract obtained(CSOs) at the stage (C), the above polypeptide;

e) in the desired case, okharakterizovanie produced recombinant polypeptide.

The recombinant polypeptide according to the invention can be purified by passing through a corresponding series of chromatographic columns according to methods known to experts in this field and are described, for example, F. Ausubel and Dr. who. (2002). As an example we can mention the way with his-tag label (“Tag-Histidine), which consists in adding short polyhistidine sequence to the polypeptide produced, the latter can then be purified on a Nickel column. The polypeptide according to the invention can also be obtained by means of synthesis in vitro. As explanations of such methods, the polypeptide according to the invention can be obtained by using a system known as “fast broadcast (RTS), which are produced, in particular, the company Roche Diagnostics France S.A., Meylan, France.

Another aspect of the invention relates to cells-owners that have at least one polynucleotide and/or at least one recombinant DNA and/or at least one expressing vector according to the invention.

Another aspect of the invention relates to microorganisms, blocked at one stage of the biosynthesis pathway of at least one of the macrolide. Of interest, on the one hand, the study of the function of mutated proteins and, on the other hand, the implementation of microorganisms, producing mediators biosynthesis. These mediators can be modified, if necessary, after separation, either by the addition of special components in the environment for production, either by introducing in this way the mutated microo the organisms of other genes, coding for proteins that are able to modify the mediator serving as substrate. Thus, these mediators can be modified chemical, biochemical, enzymatic and/or microbial method. The microorganisms blocked at one stage of the pathway of biosynthesis of macrolides, can be obtained by inactivation of the function of one or more proteins involved in the biosynthesis of this or these macrolides, microorganisms are producers of this or these macrolides. Accordingly inaktivirovannaja protein or inaktivirovannye proteins, thus, it is possible to obtain the microorganisms blocked at different stages of the biosynthesis pathway of this or these macrolides. Inactivation of this or these proteins can be carried out by any method known to specialists in this field, for example, by mutagenesis in the gene or genes encoding the above or the above-mentioned proteins, or by the expression of one or more antisense RNA complementary to the RNA or messenger RNA encoding the above or the above-mentioned proteins. Mutagenesis can be performed, for example, by irradiation, by mutagenic chemical agent, by site-directed mutagenesis by gene replacement or any other means known to experts in the data is the second region. Conditions suitable for such mutagenesis can be adapted, for example, according to the instructions found in the works Kieser T. and others (2000) and Ausubel and others (2002). Mutagenesis can be performed in vitro or in situ by suppression, substitution, deletion and/or insertions of one or more bases in the case of the gene or by gene inactivation. This mutagenesis can be carried out in a gene comprising a sequence such as described above. Preferably, the microorganisms blocked in one stage of the pathway of biosynthesis of macrolides, are bacteria of the genus Streptomyces. More preferably, the inactivation of the function of one or more proteins involved in the biosynthesis of this macrolide or consider macrolides, carried out by mutagenesis. Even more preferably, considering the macrolide is spiramycin and microorganisms, by mutagenesis or mutagenesis are strains of S. ambofaciens. More preferably, the mutagenesis is carried out in one or more genes comprising one of the sequences corresponding to one or more of the sequences SEQ ID№ 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 28, 30, 34, 36, 40, 43, 45, 47, 49, 53, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 107, 109, 111, 113, 115, 118, 120, 141, 143, 145, 147 and 149.

Preferably, the mutagenesis or mutagenesis carried out by the gene is aktivacii. Highly preferably, the mutation is in the gene inactivation of a gene comprising the sequence corresponding to the sequence SEQ ID No. 13.

As examples of such microorganisms can be named the following microorganisms: OS49.16 (orf3:; Ωhyg, see example 2); OS49.67 (deletion of the entire orf3, see example 6); OS49.107 (orf8:; Ωhyg, see example 7); OS49.50 (orf10:; Ωhyg, see example 8); SPM21 (orf2::att3Ωaac-see example 10); SPM22 (deletion of the entire orf2::att3, see example 10); SPM501 (orf6*::att1Ωhyg+, see example 14); SPM502 (deletion of the entire orf6*::att1, see example 14); SPM507 (orf2::att3Ωaac-see example 11); SPM508 (orf3c::att3Ωaac-see example 12); SPM509 (orf14::att3Ωaac-see example 13); SPM107 (orf28c::att3aac+, see example 29); SPM21 (orf31::att3aac+, see example 30); SPM543 (orf31::att3aac+, see example 30); SPM106 (orf32c::att3aac+, see example 31).

Another aspect of the invention relates to a method for production of neurotransmitter biosynthesis of the macrolide using microorganisms blocked at one stage of the pathway of biosynthesis of macrolides, such as described above. The method consists in the cultivation, the appropriate culture medium, a microorganism, blocked at one stage of the pathway of biosynthesis of macrolides, such as described above, the recovery of the conditioned culture medium or cell extract, for example, by treatment with ultrasound or osmotic shock, the separation and purification of the above culture medium or else from the cell extract, is received(CSOs) at the previous stage, the above mediator biosynthesis. Conditions of cultivation of such microorganisms can be determined according to methods well known to specialists in this field. Cultural environment, for example, may be the environment MP5 or the environment SL11 for Streptomyces and especially for Streptomyces ambofaciens (Pernodet and others, 1993). The specialist may apply, in particular, to work Kieser and others (2000) in regard to the cultivation of Streptomyces. Produced mediators can be recovered by any known specialists of ways. The specialist may apply, for example, by the methods specified in U.S. patent 3000785, and in particular to methods of extrabuy of spiramycine described in this patent.

Another object of the invention relates to a method for obtaining molecules, derived from the macrolide, when using microorganisms blocked at one stage of the pathway of biosynthesis of the macrolide, such as described above. The method consists of obtaining the mediator biosynthesis by the above method and in the modification of the thus obtained mediator, if necessary, after separation from the culture medium. Conditions of cultivation of such microorganisms can be determined by well-known specialists in this field. Cultural environment, for example, may be the environment MP5 or the environment SL11 for Streptomyces and especially for Streptomyces ambofacien (Pernodet and others, 1993). The specialist may apply, in particular, to work Kieser and others (2000) in regard to the cultivation of Streptomyces. Produced mediators can be modified, if necessary, after separation, either through supplements corresponding components in the environment for production, either by introduction into the organisms of other genes encoding proteins that are able to modify the mediator serving as substrate. These mediators, thus, can be modified by chemical, biochemical, enzymatic and/or microbial method. More preferably, considering the macrolide is spiramycin and microorganisms, by mutagenesis or mutagenesis are strains of S. ambofaciens.

The invention also relates to a microorganism which produces spiramycin I, but not producing spiramycin II and III. This organism includes a set of genes required in the biosynthesis of spiramycin I, however, do not provide spiramycin II and III as a gene comprising a sequence that corresponds to SEQ ID No. 13, or one of its variants, or one derived from these sequences in accordance with the degeneracy of the genetic code and encodes a polypeptide corresponding to the sequence SEQ ID No. 14 or one of its alternatives, is not expressed and the and inactivated. Inactivation of this protein can be carried out by any method known to specialists in this field, for example, by mutagenesis in the gene encoding the above protein, or by expression of antisense RNA, complementary to the messenger RNA that encodes the above-mentioned protein having in mind that, if the expression of orf5* changes due to this manipulation, you need to make another modification, in which case correctly expressed orf5 gene*. Mutagenesis can be carried out in the coding sequence or non-coding sequence to inactivate the encoded protein or to prevent its expression or its translation. Mutagenesis can be carried out by site-directed mutagenesis by gene replacement, or any other method known to the expert. Suitable for mutagenesis conditions, for example, can be adapted according to the instructions found in the works Kieser T. and others (2000) and Ausubel and others (2002). Mutagenesis can be performed in vitro or in situ, by suppression, substitution, deletion and/or insertions of one or more bases in the case of the gene or by gene inactivation. The microorganism can also be obtained by the expression of genes of the biosynthesis pathway of spiramycin, so that they do not include a gene comprising the sequence SEQ ID No. 13 or one of the e variants, or one derived from these sequences in accordance with the degeneracy of the genetic code, and encoding a polypeptide corresponding to the sequence SEQ ID No. 14 or one of its variants. Preferably, the microorganism is a bacterium of the genus Strepromyces. More preferably, the microorganism that produces spiramycin I, but not producing spiramycin II and III, obtained from the original microorganism producing spiramycin I, II and III. Even more preferably, the microorganism is obtained by mutagenesis in the gene comprising the sequence corresponding to SEQ ID No. 13, or one of its variants, or one of the sequences derived from them in accordance with the degeneracy of the genetic code, and encoding a polypeptide corresponding to the sequence SEQ ID No. 14 or one of its variants, having the same function. Even more preferably, the mutagenesis is carried out by gene inactivation. The microorganism is preferably derived from a strain of S. ambociens producing spiramycin I, II and III, in which gene inactivation of a gene comprising a sequence that corresponds to SEQ ID No. 13, or one of the sequences originating from it in accordance with the degeneracy of the genetic code. Highly preferably, the gene inactivation is carried out by the full-sized deletions of the gene or part of a gene comprising a sequence that corresponds to SEQ ID No. 13, or one of spoletolocalita, stemming from this in accordance with the degeneracy of the genetic code. As an example, the strain SPM502 (orf6*::att1, see example 14) can be specified as a microorganism that produces spiramycin I, but not producing spiramycin II and III.

The invention also relates to a microorganism which produces spiramycin I, but not producing spiramycin II and III, such as described above, characterized in that it is, in addition, overproductive :

- gene, which can be obtained by amplification by polymerase chain reaction using a pair of primers having the following sequences:

5' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID N° 138) and

5' GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID N° 139),

and as the matrix of Comedy pSPM36 or total DNA of Streptomyces ambofaciens, more preferably, it is about the gene corresponding to the coding sequence SEQ ID No. 141,

or a gene derived from them in accordance with the degeneracy of the genetic code.

An example of a sequence of such a gene is represented as a sequence SEQ ID No. 111 (DNA), however, this sequence is partial because it does not include the part 3' of the corresponding coding sequence. The translation of this part of the protein coding sequences represented in the sequence SEQ ID No. 112. The person skilled in the art can easily her to olnet, in particular, using the indication shown in example 24. Then was defined sequence that is not defined in the sequence SEQ ID No. 111, and the full sequence of this open reading frame (orf28) presented in the form of sequence SEQ ID No. 141. The translation of this protein coding sequences represented in the sequence SEQ ID No. 142. In example 24 describes the method for cloning the gene orf28c and get expressing vector that allows the expression of orf28c. This example also shows that overproduction gene orf28c in the strain OSC2 leads to increased production by this strain spiramycine. Overproduction gene orf28c can be achieved by increasing the number of copies of this gene and/or by introducing a more active promoter than the original promoter. Preferably, overproduction the above gene is achieved by introducing into the microorganism construction of recombinant DNA, allowing overproduction this gene. Preferably, this construction of recombinant DNA increases the number of copies of the above gene and allows you to reach overproductive the above gene. In this construction of recombinant DNA sequence encoding a gene can be under the control of a more active promoter than the similar promoter. As an example, can be called an active promoter ptrc available in the case of Streptomyces ambofaciens (Amann E. andetc., 1988), and the promoter ermE*. So, put a copy or copies of the gene orf28c preferably under the control of the promoter ermE*, as is the case in the design pSPM75 (see example 24).

The invention also relates to a microorganism which produces spiramycin I, but not producing spiramycin II and III, such as described above, characterized in that it overproduced, in addition, the gene corresponding to the coding sequence SEQ ID No. 47 or the coding sequence derived from it in accordance with the degeneracy of the genetic code. This microorganism preferably distinguished by the fact that we are talking about the strain SPM502 pSPM525 deposited in the National collection of microorganism cultures (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex 15, France, on February 26, 2003 under registration number I-2977.

The invention also relates to a method for producing spiramycin I, and the method consists in the cultivation, the appropriate culture medium, a microorganism that produces spiramycin I, but not producing spiramycin II and III, such as described above, the recovery of the conditioned culture medium or cell extract, the separation and purification of the above culture medium eliese from the cell extract, the obtained(s) at the previous stage, spiramycin I. conditions for the cultivation of such a microorganism can be determined according to methods well known to specialists in this field. The culture medium may be, for example, the environment MP5 or the environment SL11 for Streptomyces and especially for Streptomyces ambofaciens (Pernodet and others, 1993). Specialist, in particular, may apply to work Kieser and others (2000) in regard to the cultivation of Streptomyces. Produced spiramycin I can be recovered by any means known specialist. The specialist may apply, for example, by the methods specified in U.S. patent 3000785, and, in particular, to methods of extraction of spiramycine described in this patent.

Another object of the invention relates to the use of nucleotide sequences according to the invention to increase the production of macrolides by the microorganism. Thus, the invention relates to mutant, which is the producer of the macrolide to a microorganism, characterized in that it has a genetic modification in at least one gene comprising a sequence such as defined above, and/or that it overproduce at least one gene comprising a sequence such as defined above. Genetic modification may consist in suppression, substitution, deletion and/or insertions of one or more OS is Avani in the case of the gene or genes considered with the aim of ekspressirovali protein or proteins, having the highest activity, or ekspressirovali at a higher level of this or these proteins. Overproduction of the gene can be achieved by increasing the number of copies of this gene and/or the introduction of a more active promoter than the original promoter. As examples, an active promoter ptrc in the case of Streptomyces ambofaciens (E. Amann and others, 1988), and the promoter ermE* (Bibb and others, 1985; Bibb and others, 1994). So, overproduction of the gene can be achieved by introducing in this microorganism, which is the producer of the macrolide, construction of recombinant DNA according to the invention, allowing to be carried out to overproduction this gene. In fact, some stages of the biosynthesis of macrolides are limiting and, if Express one or more active protein or if a greater level of expression of the original protein or the source of proteins involved in these limiting stages, it is possible to increase the production of the corresponding macrolide or related macrolides. Increasing the production of tylosin, for example, in the case of Streptomyces fradiae was achieved by duplication of the gene encoding the rate-limiting the methyltransferase turning macrotin in tylosin (Baltz R., 1997). Achieving the expression of a more active protein can be carried out, in particular, by mutagenesis, and on this subject specialist may apply, for example, F. Ausubel and others (2002). Preferably, these mutant microorganisms, improved in relation to producing their macrolides are bacteria of the genus Streptomyces. More preferably, considering the macrolide is spiramycin and microorganisms that perform the mutagenesis or mutagenesis are strains of S. ambofaciens. More preferably, the genetic modification is carried out in one or more genes comprising one of the sequences corresponding to one or more of the sequences SEQ ID№ 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 28, 30, 34, 36, 40, 43, 45, 47, 49, 53, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 107, 109, 111, 113, 115, 118, 120, 141, 143, 145, 147 and 149 or one of its variants, or one of the sequences originating from them in accordance with the degeneracy of the genetic code. Preferably, the microorganisms overproducing one or more genes comprising one of the sequences corresponding to one or more of the sequences SEQ ID№ 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 28, 30, 34, 36, 40, 43, 45, 47, 49, 53, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 107, 109, 111, 113, 115, 118, 120, 141, 143, 145, 147 and 149 or one of its variants, or one of the sequences originating from them in accordance with the degeneracy of the genetic code. Preferably, the micro is organism overproduced gene comprising the sequence corresponding to the sequence SEQ ID No. 111 or 141, or one of its variants, or one of the sequences derived from them in accordance with the degeneracy of the genetic code. Presented as SEQ ID No. 111 sequence is partial, however, the specialist can easily Supplement it, in particular, by using the instructions given in example 24. An indefinite sequence in SEQ ID No. 111 was then determined and the full sequence of this open reading frame (orf28c) are presented as SEQ ID No. 141. The translation of this protein coding sequence presented as SEQ ID No. 142. Thus, in example 24 describes the method for cloning the gene orf28c and get expressing vector that allows expressio orf28c. This example also shows that overproduction gene orf28c in the strain OSC2 leads to increased production of spiramycin in this strain. Overproduction gene orf28c can be achieved by increasing the number of copies of this gene and/or by introducing a more active promoter than the original promoter. Preferably, overproduction gene orf28c is achieved by introducing into the microorganism construction of recombinant DNA, allowing overproduction this gene. Preferably, this design is I recombinant DNA increases the number of copies of the gene orf28c and allows you to reach overproductive gene orf28c. In this construction of recombinant DNA sequence encoding a gene orf28c, can be controlled by a more active promoter than the original promoter. As an example, can be called an active promoter ptrc available in the case of Streptomyces ambofaciens (Amann E. andetc., 1988), and the promoter ermE*. So, put a copy or copies of the gene orf28c preferably under the control of the promoter ermE*, as is the case in the design pSPM75 (see example 24).

Another aspect of the invention relates to a method for production of macrolides using mikroorganizmov described in the previous paragraph. This method consists in the cultivation, the appropriate culture medium, a microorganism, specified in the previous paragraph, the recovery of the conditioned culture medium or cell extract, the separation and purification of the above culture medium or else from the cell extract obtained(s) at the previous stage, the macrolide produced or produced macrolides. Conditions of cultivation of such microorganisms can be determined according to methods well known to specialists in this field. Cultural environment, for example, may be the environment MP5 or the environment SL11 for Streptomyces and especially for Streptomyces ambofaciens (Pernodet and others, 1993). The specialist may apply, in particular, to work Kieser and others (2000) that it is for the cultivation of Streptomyces. Produced macrolide or macrolides produced can be recovered by any means known to specialists in this field. The specialist may apply, for example, by the methods specified in U.S. patent 3000785, and in particular to methods of extraction of spiramycine described in this patent. Preferably, the microorganisms used in this method are bacteria of the genus Streptomyces. More preferably, considering the macrolide is spiramycin and mutant microorganisms, improved in relation to producing their spermicides are strains of S. ambofaciens.

Another aspect of the invention relates to the use of sequences and/or vector according to the invention for hybrid antibiotics. In fact, polynucleotide according to the invention can be used for obtaining microorganisms expressing one or more mutant proteins, enabling them to make a modification in substrate specificity or even which can be expressed in numerous organisms that are producers of antibiotics, with the purpose of generating hybrid antibiotics. So, polynucleotide according to the invention, by gene transfer between microorganisms-producers can afford to get a hybrid antibiotics, possessing interesting with FA is mikologicheskoe perspective properties (Hopwood and others, A; Hopwood and others, 1985b; Hutchinson and others, 1989). The principle on which genetic engineering can lead to the production of hybrid antibiotics was first proposed Hopwood (Hopwood, 1981). So, he suggested that the enzymes involved in the biosynthesis of antibiotics, often acceptat structurally related substrates, but other than their native substrate. As a rule, allow (Hopwood,1981; Hutchinson, 1988; Robinson, 1988)that the enzymes encoded by the genes of the biosynthesis pathway of antibiotics, have less strict specificity to the substrate than the enzymes of primary metabolism. It has been shown that a large number negativnyh substrates transformed producing the antibiotic by microorganisms, mutants or purified enzymes of the biosynthesis pathway of these antibiotics (Hutchinson, 1988). Using this instruction, the person skilled in the art can construct microorganisms, expression of one or more mutant proteins, allows to modify the specificity of the substrate, with the purpose of generating hybrid atibiotics.

The invention also relates to the use of at least one polynucleotide and/or at least one recombinant DNA, and/or at least one expressing vector, and/or at least one polypeptide and/or at least one host cell according to the invention, for the implementation of the od is Oh or several bioconversion. Thus, the invention enables the development of bacterial or fungal strains, which are under the control of appropriate expression signals are expressed one or more proteins according to the invention. These strains can then be used to implement the bioconversion or bioconversion. These bioconversion can be implemented using either whole cells or with cell extracts from these cells. These bioconversion can allow for the transformation of molecules in a derived form of the enzyme biosynthesis pathway. For example, Carreras and others describe the application of a strain of Saccharopolyspora erythraea and strain Streptomyces coelicolor for producing new derivatives eritromicina (Carreras and others, 2002). Walczar and others describe the application of monooxygenase P450 Streptomyces for the bioconversion of desacetylrifabutin (analog anthracycline) in new anthracyclines (Walzcar and others, 2001). Olonao and others describe the application of a modified strain of Streptomyces lividans for the bioconversion of rhodomycinone-Epsilon in sodomizing D (Olonao and others,1999). The expert can apply this principle in the case of any mediator biosynthesis.

The invention relates to recombinant DNA, characterized in that it includes:

- polynucleotide, which can be obtained by amplification by polymerase chain reaction using a pair of primers, and ausich the following sequence: 5' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID No. 138) and 5' GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID No. 139), and as the matrix of Comedy pSPM36 or total DNA of Streptomyces ambofaciens, more preferably it is about polynucleotide corresponding to the sequence SEQ ID No. 141;

or a fragment of at least 10, 12, 15, 18, 20-25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1460, 1470, 1480, 1490 or 1500 consecutive nucleotides of these polynucleotides.

Preferably, this recombinant DNA is a vector. Even more preferably, the vector is chosen from bacteriophages, plasmids, phagemid integrating vectors, fosmid, cosmid, Shuttle vectors, YOU (bacterial iskusstvennaja chromosome) or RACES (P1-derived artificial chromosome). As a clarification, may be called as bacteriophages phage lambda and M13 phage. As plasmids can be called plasmids that can replicate in E. coli, for example, pBR322 and its derivatives, pUC18 and its derivatives, pUC19 and its derivatives, GB2 and its derivatives (G. Churchward and others, 1984), pACYC177 (access number in the gene Bank: H) and its derivatives, pACYC184 (access number in the gene Bank: H) and its derivatives. May also be called plasmids that can replicate in Streptomyces, such as pIJ101 and its derivatives, pSG5 and its derivatives, SLP1 and its derivatives, SCP2* and its derivatives (Kieser and others, 2000). As phagemid can be called as explanations, pBluescript II and its derivatives (issued in cha is in the surrounding area, the company Stratagene (LaJolla, CA, USA)), pGEM-T and its derivative (manufactured by a company Promega (Madison, Wisconsin, USA)), λZAPII and its derivatives (produced, in particular, by the company Stratagene (LaJolla, CA, USA)). As an integrating vectors can be called as explanations integrating vectors in the case of Streptomyces, such as those occurring from SLP1 (Kieser and others, 2000), those originating from pSAM2 (Kieser and others, 2000), vectors, systems integration phage PhiC31 (Kieser and others, 2000) (e.g., pSET152 (Bierman and others, 1992)) or VWB (Van Mellaert L. and others, 1998), as well as vectors, using the system integration IS117 (Kieser and others, 2000). As fosmid can be called as explanations fosmid pFOS1 (manufactured by New England Biolabs Inc., Beverly, Massachussetts, USA) and its derivatives. As cosmid can be called as explanations, kosmidou SuperCos and its derivatives (produced, in particular, by the company Stratagene (LaJolla, CA, USA)), kosmidou pWED15 (Wahl and others, 1987) and its derivatives. As Shuttle vectors can be called as explanations Shuttle plasmids E. coli/Streptomyces, such as pIJ903 and its derivatives, a number of plasmid pUWL, pCAO106, pWHM3, pOJ446 and their derivatives (Kieser and others, 2000), Shuttle YOU E. coli/Streptomyces, such as those described in the International patent application WO-01/40497. As YOU (bacterial artificial chromosome) can be called, as the explanations YOU pBeloBAC11 (access number in the ANK genes: U51113). As RACES (P1-derived artificial chromosome) can be called, as explanations, vector pCYPAC6 (access number in the gene Bank: AF133437). More preferably, this recombinant DNA is expressing vector. Expression vectors used in expressing different systems, well known to experts in the field; in regard to prokaryotic cells, can be called as an explanation, expression vectors for E. coli, for example, family pet, manufactured by the company Stratagene (LaJolla, CA, USA), the vectors of the family GATEWAY, manufactured by Invitrogen (Carlsbad, CA, USA), the vectors of the family pBAD manufactured by Invitrogen (Carlsbad, CA, USA), the vectors of the family pMAL manufactured by New England Biolabs Inc. (Beverly, Massachussetts, USA), expression vectors, the inductive Ramezay specified in the publication Wilms B. and others, 2001, and their derivatives, may also be called the expression vectors in the case of Streptomyces, such as, for example, vectors pIJ4123, pIJ6021, pPM927, pANT849, pANT850, pANT851, pANT1200, pANT1201, pANT1202, and their derivatives (Kaiser and others, 2000). In regard to yeast cells, can be called as a clarification, the vector pESC manufactured by the company Stratagene (LaJolla, CA, USA). In regards baculovirus expression system, which allows for expression in insect cells, can be called as explanations vector Wasr the K6, manufactured by BD Biosciences Clontech (Palo Alto, CA, USA). In regard to mammalian cells, can be called, as an example, vectors comprising the promoter of the early genes of the virus CMV (cytomegalovirus) (for example, the vector pCMV and its derivatives are produced by the company Stratagene (LaJolla, CA, USA), or the promoter predannih genes (the early promoter of SV40) simian vacuolating virus SV40 (e.g., vector pSG5, manufactured by the company Stratagene (LaJolla, CA, USA). Another aspect of the invention relates to cells of the host, which are introduced at one recombinant DNA described in this paragraph.

Another aspect of the invention relates to a method for production of the polypeptide, characterized in that the above method includes the following stages:

a) transforming the host cell with at least one expressing vector, such as described in the paragraph above;

b) culturing, in the appropriate culture medium, the above-mentioned host cell;

(C) recovery of conditioned culture medium or cell extract;

d) isolation and purification of the above culture medium or else from the cell extract obtained(s) at the stage (C), the above polypeptide;

e) in the desired case, okharakterizovanie obtained recombinant poly is eptide.

Produced thus recombinant polypeptide can be purified by passing through a corresponding series of chromatographic columns according to methods known to experts in this field and are described, for example, F. Ausubel and others (2002). As an example we can mention the way with his-tag label (“Tag-Histidine), which consists in adding short polyhistidine sequence to the polypeptide produced, the latter can then be purified on a Nickel column. This polypeptide can also be obtained by means of synthesis in vitro. As explanations of such methods, the polypeptide may be obtained using a system known as “fast broadcast (RTS), which are produced, in particular, the company Roche Diagnostics France S.A., Meylan, France.

Another aspect of the invention relates to a microorganism that produces at least one spiramycin, characterized in that it overproducers :

- gene, which can be obtained by amplification by polymerase chain reaction (PCR) using a pair of primers having the following sequences: 5' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID No. 138) and 5' GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID No. 139), and as a matrix of Comedy pSPM36 or total DNA of Streptomyces ambofaciens, more preferably, it is about the gene corresponding to the coding sequence SEQ ID No. 141,

or a gene derived from it in accordance with the degeneracy of the genetic code.

An example of a sequence of such a gene is represented as a sequence SEQ ID No. 111 (DNA), however, this sequence is partial because it does not include the part 3' of the corresponding coding sequence. The translation of this part of the protein coding sequences represented in the sequence SEQ ID No. 112. The person skilled in the art can easily add, using, in particular, the indication shown in example 24. This example also shows how to clone a gene orf28c and get expressing vector that allows the expression of orf28c. This example also shows that overproduction gene orf28c in the strain OSC2 leads to increased production by this strain spiramycine. Preferably, the microorganism, overproducers

- gene, which can be obtained by amplification by polymerase chain reaction (PCR) using a pair of primers having the following sequences: 5' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID No. 138) and 5' GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID No. 139), and as a matrix of Comedy pSPM36 or total DNA of Streptomyces ambofaciens, more preferably, it is about the gene corresponding to the coding sequence SEQ ID No. 141,

or a gene derived from Negev accordance with the degeneracy of the genetic code,

is a bacterium of the genus Streptomyces, more preferably, it is about bacteria species Streptomyces ambofaciens. Preferably, overproduction the above gene is achieved by transforming the above-mentioned microorganism with expressing vector, highly preferably, the microorganism strain is the strain OSC2/pSPM75(1) or strain OSC2/pSPM(2)deposited in the National collection of microorganism cultures (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex 15, France, October 6, 2003 under registration number I-3101.

Another aspect of the invention relates to a method for producing spiramycin (spiramycin) using microorganisms, described in the previous paragraph. This method consists in the cultivation, the appropriate culture medium, a microorganism, specified in the previous paragraph, the recovery of the conditioned culture medium or cell extract, the separation and purification of the above culture medium or else from the cell extract obtained(s) at the previous stage, produced spiramycin or produced of spiramycin. Conditions of cultivation of such microorganisms can be determined according to methods well known to specialists in this field. The culture medium may be, for example, the environment MP5 or the environment SL11 on what I Streptomyces and especially for Streptomyces ambofaciens (Pernodet and others, 1993). Specialist, in particular, may apply to work Kieser and others (2000) in regard to the cultivation of Streptomyces. Produced spiramycin or produced spiramycine can be recovered by any means known specialista in this area. The specialist may apply, for example, by the methods specified in U.S. patent 3000785, and, in particular, to methods of extraction of spiramycine described in this patent.

Preferably, the microorganisms used in this method are bacteria of the genus Streptomyces. More preferably, the microorganisms are strains S.amboficiens.

Another aspect of the invention relates to expressing vector, characterized in that polynucleotide corresponding to the sequence SEQ ID No. 47, or polynucleotide occurring from him in accordance with the degeneracy of the genetic code, is under the control of a promoter that allows expression of the protein encoded by the said nucleotide in the case of Streptomyces ambofaciens. Examples expressing vectors used in the case of Streptomyces, are given above. This expressing vector preferably distinguished by the fact that we are talking about plasmids pSPM524 or pSPM525.

Another aspect of the invention refers to a strain of Streptomyces ambofaciens, transformed with a vector defined in the previous paragraph.

Another TSA is CT of the invention relates to the polypeptide, characterized in that its sequence includes the sequence SEQ ID No. 112. The invention also relates to the polypeptide, characterized in that its sequence corresponds to the sequence, we Express the coding sequence:

- gene, which can be obtained by amplification by polymerase chain reaction (PCR) using a pair of primers having the following sequences: 5' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID No. 138) and 5' GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID No. 139), and as a matrix of Comedy pSPM36 or total DNA of Streptomyces ambofaciens, more preferably, it is about the gene corresponding to the coding sequence SEQ ID No. 141,

or gene originating from him in accordance with the degeneracy of the genetic code.

Preferably, these polypeptides are expressed in the native state by a bacterium of the genus Streptomyces, more preferably, these polypeptides involved in the biosynthesis of spiramycin.

Another aspect of the invention relates to expressing vector that allows the expression of a polypeptide such as defined in the previous paragraph, in Streptomyces ambofaciens. The examples used expressing vectors in the case of Streptomyces given above. Consider expressing the vector is preferably a plasmid pSPM75.

List of figures

1: Chemical structure is ur spiramycin I, II and III.

2: Comedy used for sequencing the field.

3: The organization of groups of genes involved in the biosynthesis pathway of spiramycin.

4: The expected path of the biosynthesis mycarose.

5: The expected path of the biosynthesis mycaminose.

6: The expected path of the biosynthesis of forosamine.

Fig.7: The preferred order of introduction of sugars in the molecule spiramycin and mediators.

Fig: The expected path of the biosynthesis methoxymethyl if S.ambofaciens. This way it is expected by analogy with the biosynthesis methoxymethyl in the case of Streptomyces hygroscopicus var. ascomyceticus (K. Wu and others, 2000).

Fig.9: Stage, leading to inactivation of the gene:

A) cloning of the target genes in the vector that can replicate in E. coli but not in Streptomyces;

C) insertion of a cluster of resistance gene target (by cloning or recombination between identical short sequences);

(C) the introduction of plasmids in Streptomyces ambofaciens (by transformation or conjugation with E. coli and the selection of clones with integrated cluster, then the screening of clones that had lost part of a vector for gene replacement;

D) region of chromosome mutant strain in which the gene target inactivated by gene replacement.

Figure 10: Optional stage, following the inactivation of the gene according to the method, opican the mu according to Fig.9, which can be carried out, if the interrupt of the target genes was used excesively cluster:

E) introducing a mutant strain plasmids pOSV508 carrying genes xis and int pSAM2, which produced products allow for efficient excision by specific recombination sites between sequences attL and attR, flanking the cluster.

F) obtaining clones, lost excesively cluster and sensitive to the antibiotic to which the cluster gave resistance;

G) after growth and sporulation on solid medium without antibiotic, with high frequency lost the plasmid pOSV508. Thus it is possible to obtain clones that are sensitive to thiostrepton, in which gene target contains a deletion in the open reading frame. The sequence subjected to the deletion of the target genes can be controlled by amplification by R and sequencing of the PCR product.

11: Amplification excisional cluster to use for the experiment by homologous recombination. Technology homologous recombination at the expense of short homologous sequences described Chaveroche and others (2000). 39 or 40 Deoxynucleotides located at the 5' end of these oligonucleotides that include the sequence corresponding to the sequence of inactivating the gene, and 20 deoxynucleotides, u is routed towards the end of the 3', correspond to the sequence of one of the ends excisional cluster.

Fig: Getting the design for the inactivation of the target genes according to the method described Chaveroche and others (2000).

Fig: Map of the plasmid pWHM3. Strepto ori: replication to begin Streptomyces.

Fig: Map of the plasmid pOSV508.

Fig: An example of the structure excisional cluster. He formed a cluster Ωhyg (Blondelet-Rouault and others, 1997), flanked by sites attR and attL (Raynal and others, 1998), recombination between which allows excision of the cluster by gene expression xis and int.

Fig: Map of the plasmid pBXL1111.

Fig: Map of the plasmid pBXL1112.

Fig: Microbiological test for the production of spiramycin based on the sensitivity of the strain of Micrococcus luteus to spiramycin. Used a strain of Micrococcus luteus is a strain that is inherently sensitive to spiramycin, but resistant to conhecido. Various test strains of Streptomyces were cultured in Erlenmeyer flasks 500 ml, containing 70 ml of medium MP5, inoculation with an initial concentration of 2.5·106spores/ml and grown at a temperature of 27°C With orbital stirring at a speed of 250 Rev/min sampling fermentation of the wort was carried out after 48, 72 and 96 hours of cultivation and centrifugally. For the test used breeding ten times those with whom pertanto in sterile culture medium. The indicator strain Micrococcus luteus resistant to conhecido, but sensitive to spiramycin (Gourmelen and others, 1998), were cultured in a square Cup size 12 cm x 12 cm Discs of Whatman paper AA impregnated with 70 μl of ten-fold dilution of each supernatant was placed on the surface of the Cup. Discs impregnated with a solution spiramycine different concentrations (2-4-8 μg/ml of culture medium MP5), was used as the reference number. The cups were incubated at 37°C for 24-48 hours. If the disk contains spiramycin, it diffuses into the agar and inhibited growth of the indicator strain Micrococcus luteus. This inhibition causes the formation of “halo” around the disk, and this halo reflects the area where the strain Micrococcus luteus is not growing. The presence of this halo is, therefore, a sign of the presence of spiramycin can determine, produces or does not produce spiramycin consider the strain S.ambofaciens. Comparison with the diameters of inhibition was obtained in the case of the reference range, allows to obtain an indication of the number of spiramycin produced by this strain.

Fig: Chromatogram HPLC of the supernatant filtered from the culture medium of the strain OSC2.

Fig: Chromatogram HPLC of the supernatant filtered from the culture medium of strain SPM501.

Fig: Chromatogram HPLC su is inatant, filtered from the culture medium of strain SPM502.

Fig: Chromatogram HPLC of the supernatant filtered from the culture medium of strain SPM507.

Fig: Chromatogram HPLC of the supernatant filtered from the culture medium of strain SPM508.

Fig: Chromatogram HPLC of the supernatant filtered from the culture medium of strain SPM509.

Fig: Comparison by alignment of protein Orf3 (SEQ ID No. 29) c protein TylB (SEQ ID No. 87) S. fradiae carried out according to the program FASTA.

Fig: Comparison by alignment of protein MdmA S. mycarofaciens (SEQ ID No. 88) c protein SrmD (SEQ ID No. 16), carried out according to the program FASTA.

Fig: Example sequence of residual sites after excision excisional cluster. Bold specified minimum site att26, such as certain Raynal and others, 1998. The sequence of open reading frame 1 (att1) of 33 nucleotides presented in the sequence SEQ ID No. 104, the sequence of open reading frame 2 (att2) of 34 nucleotides presented in the sequence SEQ ID No. 105, the sequence of open reading frame 3 (att3) of 35 nucleotides presented in the sequence SEQ ID No. 95.

Fig: Schematic diagram of the orf10 gene, localization of the used PCR primers and design, obtained with each pair p is Amirov.

Fig: Design of a cluster of races-oritT.

FigMap of Comedy pWED2.

Fig: Schematic representation of groups of genes involved in the biosynthesis pathway of spiramycin and localization of the three probes used for selection of cosmid from the Bank genomic DNA of the strain OSC2 Streptomyces ambofaciens in the case of E. coli (see example 19).

Fig: Localization inserts cosmid allocated from the Bank genomic DNA of the strain OSC2 Streptomyces ambofaciens in the case of E. coli (see example 19). The new Bank komenich DNA.

Fig: Subclavian fragment > PST - > PST Comedy pSPM36 (insert plasmids pSPM58), subclavian fragment StuI-StuI of Comedy pSPM36 (insert plasmids pSPM72), subclavian fragment EcoRI-StuI (insert plasmids pSPM73).

Fig: Localization of open reading frames identified in box > PST - > PST plasmids pSPM58 and inserting EcoRI-StuI plasmids pSPM73.

Fig: Superposition of HPLC chromatograms of the supernatant filtered from the culture medium of strain OS49.67 received at 238 nm and 280 nm (top)and UV spectra of molecules, buervenich in the case of 33.4 minutes and 44.8 minutes (below).

Fig: The molecular structure of molecules platinoid and platinoid Century

Fig: The organization of groups of genes involved in the biosynthesis pathway of spiramycin.

Fig: Molecular structure of neurotransmitter biosynthesis, produced by strain SPM507.

Fig: Structure of m is Diadora biosynthesis, produced by a strain of S. ambofaciens genotype orf6*::att1Ωhyg+, derived from strain, overproducing spiramycine. The insertion of the cluster att1Ωhyg+ orf6* causes polar effect, preventing the expression of orf5*.

Fig: The molecular structure of molecules platinoid And + mycarose and platinoid In + mycarose produced by strain OS49.67.

Fig: Subclavian fragment > PST - > PST Comedy pSPM36 (insert plasmids (pSPM79)), localization of open reading frames identified in box > PST - > PST plasmids SPM79 and localization sequence SEQ ID No. 140.

The present invention is illustrated using the following examples, which need to be considered as explanatory and not limiting the scope of protection of the invention.

Briefly, the genes of the biosynthesis pathway of spiramycin was isolated from genomic DNA Bank of Streptomyces ambofaciens. This Bank was obtained by partial restriction of the genomic DNA of Streptomyces ambofaciens with restriction enzyme BamHI. Large DNA fragments, the average length of 35-45 called, cloned in cosmides pWED1 (Gourmelen and others, 1998), derived from Comedy pWED15 (Wahl and others, 1987). Comedy was introduced in E. coli due rahovym particles. Thus the Bank was subjected to hybridization with a probe corresponding to the sequence SEQ ID No. 86), the relevant part of the gene tylB S. fradiae (Merson-Davies & Cundliffe, 1994; the access number in the gene Bank: U08223). After hybridization in persons is nasty selected kosmidou 4 hybridizers with probe areas. This kosmidou called pOS49.1, then restrictively using SacI and a fragment of 3,3 called, contains the area of hybridizers probe cloned into the vector pUC19 and sequenced. Identified four open reading frames and one of them encodes a protein (SEQ ID No. 29), with large sequence similarities with protein TylB S. fradiae (SEQ ID No. 87) (cm. Fig). This gene was named orf3 (SEQ ID No. 28) and inactivated in S. ambjfaciens. It is shown that inactivated in the orf3 gene clones do not produce more spiramycine. This indicates the participation of the orf3 gene or genes located below in the biosynthesis of spiramycin.

Based on this confirmation sequenced a much larger area of Comedy pOS49.1 with both sides investigated previously SacI fragment. So, from Comedy pOS49.1 received sequence region that includes seven full open reading frames and the other two incomplete open reading frames located on both sides of these seven complete open reading frames. Through searching of the databases shows that one of the incomplete open reading frames corresponds to the locus srmG (region encoding the enzyme called as “policyinstitute” (PKS)). The corresponding genes were cloned according to S. Burgett and others in 1996 (U.S. patent 5945320). In addition, the databases were not identified what s other open reading frames: seven full open reading frames, named orf1, orf2, orf3, orf4, orf5, orf6, orf7 (SEQ ID№ 23, 25, 28, 30, 34, 36, 40), and the beginning of the eighth open reading frame, named orf8 (sequence SEQ ID No. 43).

Secondly, and with the aim of cloning other genes involved in the biosynthesis of spiramycine, in the same area were allocated to the other Comedy, including fragments of the genome of S. ambofaciens. This was carried out by a new series of hybridisable in the colonies, using three probe. The first probe corresponds to a DNA fragment with a length of 3.7 so called, containing orf1, orf2 and the beginning of orf3, subcloned from pOS49.1. The second probe corresponds to a fragment of length 2 so-called DNA containing part of the prf7 and part of orf8 and subcloned from pOS49.1. Was also used for the third probe. This last corresponds to a DNA fragment with a length of 1.8 so-called, aderasa gene srmD. Gene srmD is a gene isolated from S. ambofaciens capable of imparting resistance to spiramycin. In fact, according to previous works, carried out the cloning of several determinants of resistance of S. ambofaciens, giving resistance to spiramycin, in the strain of S. griseofuscus (strain, sensitive to spiramycin) (Pernodet and others, 1993; Pernodet and others, 1999). To highlight resistance genes was implemented Comedy Bank genomic DNA of a strain of S. ambofaciens in cosmides pKC505 (M.A. Richardson and others, 1987). This pool of cosmid was introduced into S. griseofuscus, inherently sensitive to spiramycin. Therefore clicks the zoom were obtained five cosmid, able to give resistance to apramycin and spiramycin strain S. griseofuscus. Among these 5 cosmid, one cosmid called pOS44.1 is inserted gene srmD, which encodes a protein with some similarity to the protein encoded by the genome of mdmA Streptomyces mycarofaciens and involved in resistance to midecamycin in this body-producer (Hara and others, 1990; access number in the gene Bank; A) (Fig). The third probe is used to locate genes of the biosynthesis of spiramycin, is a insert a length of approximately 1.8 so called, containing the gene srmD.

These three probes were used for hybridization Bank genomic DNA described above, and allowed to choose two of Comedy (pSPM7 and pSPM5), able to contain the longest insert and not having the common areas. Cosmid pSPM5 hybridizes with the first and second probe, but not hybridized with the third probe, whereas cosmid pSPM7 hybridized only with the third probe. These two Comedy fully sequenced by the method of “a shot from a shot gun. The sequence of the inserts of these two cosmid pSPM7 and pSPM5 can be connected, as, though not overlap, each of the inserts includes well-known at one of its ends the sequence. In fact, each of these inserts includes a fragment of the sequence of one of the genes encoding the enzyme called “policyinstitute” (PKS). These 5 genes b is whether the cloned S. Burgett and others in 1996 (U.S. patent 5945320) (see figure 2). Thus, it was determined one sequence of genomic DNA of S. ambafaciens. The sequence of 30943 nucleotides beginning, a 5'EcoRI site located in the first PKS gene, and goes up to the BamHI site at 3', presented in the form of sequence SEQ ID No. 1. This sequence corresponds to the region above the PKS genes (see figure 2 and 3). The second area of 11171 nucleotides, starting from site > PST 5' and goes up to the BstEII site in the 3', located in the fifth PKS gene, represented as sequences of SEQ ID No. 2. This area is the area below the PKS genes (and the bottom and top are defined by the orientation of the 5 PKS genes, all of which have the same semantic orientation) (see figure 2 and 3).

Thirdly, and with the aim of cloning other genes involved in the biosynthesis of spiramycine, were allocated to the other Comedy, including fragments of the genome of S. ambofaciens, in the same area.

Example 1

Construction Bank genomic DNA of strain ADS Streptomyces ambofaciens in E. coli

1.1. Extraction of genomic DNA from strain ADS Streptomyces ambofaciens

Strain ADS Streptomyces ambofaciens (available, in particular from the American type culture collection (ATSS) (Manassas, Virginie, USA) under the number 23877)) were cultured in YEME medium (yeast extract-malt extract) (Kieser, T. and others, 2000) and the genomic DNA of this strain was AKST who was agarawala and purified according to standard methods lysis and precipitation (Kieser T. and others, 2000).

1.2. Construction Bank genomic DNA

Genomic DNA of strain ADS Streptomyces ambofaciens, such as highlighted above, was partially restrictively the restriction enzyme BamHI to obtain DNA fragments ranging in length from approximately 35 so-called to 45 called These fragments were cloned in cosmides pWEDI (Gourmelen and others, 1998), pre-restrictional with BamHI. Cosmid pWED1 is formed from Comedy WED15 (Wahl and others, 1987) due to the deletion of the fragment of the HpaI-HpaI length of 4.1 so called, containing the active module expression in mammals (Gouemelan and others, 1998). The resulting ligation mixture was then uncapsizable in vitro into particles of lambda phage using system Packagene®Lambda DNA packaging system”manufactured by Promega according to the manufacturer's recommendations. The obtained phage particles were used to infect strain SURE®E.coli produced by the company Stratagene (LaJolla, CA, USA). The selection of clones was carried out on LB medium + ampicillin (50 μg/ml), because cosmides pWED1 give resistance to ampicillin.

Example 2

Isolation and characterization of genes involved in the biosynthesis of spiramycin in Streptomyces ambofaciens

2.1. Hybridization in the colony of clones of E.coli genomic Bank of Streptomyces ambofaciens ADS

Approximately 2000 clones of E. coli obtained above Bank were transferred to a filter for hybridization of colonies. Used for hybridization AOR the house is the NaeI fragment-NaeI DNA (SEQ ID No. 86), comprising part of the gene tylB Streptomyces fraidae. This fragment corresponds to nucleotides 2663-3702 DNA fragment described Merson-Davies L.A. and Cundliffe, E. (Merson-Davies L.A. & Cundliffe, E., 1994; the access number in the gene Bank: U08223), in which the coding sequence of the gene tylB corresponds to nucleotides 2677-3843.

The NaeI fragment-NaeI-DNA carrying a part of the gene tylB Streptomyces fraidae (SEQ ID No. 86), marked with32P according to the method of “arbitrary premirovany” (kit, manufactured by Roche) and used as probe for hybridization 2000 clones of the Bank, transferred to a filter. Used membrane is a nylon membrane Hybond N, manufactured by Amersham (Amersham Biosciences, Orsay, France), and hybridization was carried out at a temperature of 55°C in the buffer described by Church and Gilbert (Church &Gilbert, 1984). One leaching was carried out in 2X SSC at a temperature of 55°C for 15 minutes and two consecutive washing was then carried out in 0.5x SSC at a temperature of 55°With a duration of 15 minutes each. Under these conditions, hybridization and washing, 4 clone from 2000 hybridized had a strong hybridization signal. These 4 clones were cultured in LB medium + ampicillin (50 μg/ml) and corresponding 4 Comedy were extracted by standard alkaline lysis (Sambrook and others, 1989). Then confirmed that hybridization had taken place in the DNA fragment, present in box of these four KOs is ID. For Comedy was restrictively independently by several enzymes (BamHI, PST and SacI). Food restrictions were separated on agarose gel, transferred to Nylon membrane and was hybridisable using NaeI fragment-NaeI DNA comprising part of the gene tylB Streptomyces fraidae (see above), in the same conditions as above. Valid four Comedy and preferably was chosen as one of these cosmid and named pOS49.1.

2.2. Confirmation of participation identified region and sequencing of the insert of Comedy pOS49.1

Was subclinically a few fragments of the insertion of Comedy pOS49.1 and determined their sequences. Kosmidou pOS49.1 was restrictively enzyme SacI and by southern blotting under these conditions it is shown that a fragment of 3,3 contains the so-called region, hybridizers with probe tylB. This fragment of 3,3-called allocated by electroelution from 0.8%agarose gel, and then cloned into the vector pUC19 (access number in the gene Bank: M77789) and sequenced. The thus obtained phasmida was named pOS49.11. Four open reading frames with a typical use of the codons of Streptomyces were identified in this fragment (two full and two shortened) using FramePlot (J. Ishikawa & Hotta K., 1999). Comparison of sequences in the FASTA program (see W.R. Pearson &D.J. Lipman, 1988; and Ptarson W.R., 1990), available in particular from centresource INFOBIOGEN, Evry, France)revealed that the protein, following from one of these 4 open reading frames, has extensive sequence similarity with protein TylB (SEQ ID No. 87; the access number in the gene Bank: U08223) S. fradiae (see Fig). This protein was named Prf3 (SEQ ID No. 29).

To test whether the corresponding gene (orf3 gene (SEQ ID No. 28)) in the biosynthesis of spiramycin in the case of S. ambofaciens, this gene was interrupted by a cluster Ωhyg (Blondelet-Rouault M.-H. and others, 1997; the access number in the gene Bank: H). This plasmid pOS49.11 was restrictively enzyme XhoI and the fragment containing four open reading frames (two full and two shortened that orf3 is whole), was subclinical level XhoI site of the vector SK +manufactured by the company Stratagene (LaJolla, CA, USA). The thus obtained plasmid was named pOS49.12. The purpose of inactivation of orf3, internal PmlI fragment-BstEII orf3 was replaced by the cluster Ωhyg by cloning the free end of this latter plasmid. This plasmid pOS49.12 was restrictively enzymes PmlI and BstEII, the only site which is located in the coding sequence of orf3 gene. The ends of the fragment corresponding to the vector, did the free ends by treatment with enzyme maple (large fragment of DNA polymerase I). The cluster Ωhyg was obtained by restriction enzyme BamHI plasmid pHP45 Ωhyg (Blondelet-Rouault and others, 1997; the number of shortcuts is PA in the gene Bank: H). The fragment corresponding to the cluster Ωhyg, recuperable on the agarose gel and it did free ends by treatment with enzyme maple. Thus obtained the free ends of the two fragments (cluster Ωhyg and plasmid pOS49.12) ligated and the ligation product was used for transformation of E.coli bacteria. The thus obtained plasmid was named pOS40.14 and it contains the orf3 gene, interrupted by a cluster Ωhyg.

Insert plasmids pOS49.14, in the form of a fragment XhoI-XhoI, the ends of which were made non-sticky by processing enzyme maple, cloned at the level of the EcoRV site of plasmid pOJ260 (plasmid pOJ260 is conjugative plasmid that can replicate in E. coli but is unable to replicate in S. ambofaciens (M. Bierman and others, 1992). This plasmid gives resistance to apramycin strains of E. coli and Streptomyces). The resulting plasmid (insert plasmids pOS49.14, cloned in the plasmid pOJ260) was named pOS49.16. This last was transferred into strain ATS S. ambofaciens due to conjugation with conjugative strain E. coli S17-1 as described Mazodier and others (Mazodier and others, 1989). The strain E. coli S17-1 is derived from the strain E. coli 294 (Simon and others, 1983; Simon and others, 1986). Received transconjugate clones with token resistance to hygromycin on the cluster Ωhyg, and lost token resistance to apramycin located in the vector pOJ260. For this is about clones selected for resistance to hygromycin. Resistant to hygromycin clones were then perseval, respectively, on Wednesday with hygromycin (antibiotic) and on Wednesday with apramycin (antibiotic) (see Fig.9). Clones resistant to hygromycin (HygR) and sensitive to apramycin (ApraS)are in principle those in which there was a double phenomenon of recombination, and which therefore have the orf3 gene, interrupted by a cluster Ωhyg. Replacement original copy of prf3 on the interrupted copy confirmed by two consecutive hybridisable. So, the full DNA of the obtained clones was restrictively with the help of various enzymes, separated on agarose gel, transferred to membrane and hybridisable with a probe corresponding to the cluster Ωhyg (see above), to confirm the presence of the cluster in genomic DNA of the obtained clones. A second hybridization was carried out using as a probe insert XhoI-XhoI plasmid pOS49.11, which contains four open reading frames (two full and two shortened that orf3 is whole). Confirmation of the genotype can also be carried out by any method known to the expert, and, in particular, by PCR, using the appropriate oligonucleotides and sequencing of the PCR product. Was chosen one of the clones orf3:: Ωhyg thus obtained and the genotype of which has been confirmed, and named OS49.16.

The production of spiramycin thus receiving the essential clone OS49.16 were tested using test production, described below (see example 15). Thus, it was shown that this strain does not produce more spiramycin, which confirms the participation of orf3 and/or genes located below, as, for example, orf4, in the biosynthesis of spiramycin.

First, according to the received confirmation sequenced a much larger area of Comedy pOS49.1 with both sides investigated above SacI fragment. Thus, on the basis of Comedy pOS49.1 was obtained sequence region that includes seven full open reading frames and the other two incomplete open reading frames located on both sides of these seven complete open reading frames. Through searching of the databases shows that one partial open reading frames corresponds to the locus srmG (region encoding the enzyme called “policyinstitute” (PKS)). The corresponding genes have been cloned S. Burgett and others in 1996 (U.S. patent 5945320). In addition, the databases were not detected by other open reading frames: seven full open reading frames, named orf1, orf2, orf3, orf4, orf5, orf6, orf7 (SEQ ID№ 23, 25, 28, 30, 34, 36, 40), and the beginning of the eighth open reading frame, named orf8 (the full sequence of this open reading frame is represented as SEQ ID No. 43).

Example 3

Isolation and characterization of other genes involved in the biosynthesis of Speer is Mizinov in Streptomyces ambofaciens

Secondly, and with the aim of cloning other genes involved in the biosynthesis of spiramycine, in the same area were allocated to the other Comedy, including fragments of the genome of S. ambofaciens. This was carried out by a new series of hybridisable in the colonies, using three probe:

The first used the probe corresponds to a DNA fragment BamHI- > PST a length of 3.7 so called, containing a fragment of the PKS gene (genes, corresponding PKS were cloned S. Burgett and others in 1996 (U.S. patent 5945320)), orf1, orf2 and the beginning of orf3, subcloned, based on pOS49.1, and coming from the BamHI site located at 1300 base pairs above the EcoRI site, which determines the position 1 of the sequence SEQ ID No. 1, up to the customers > PST in 2472 (SEQ ID No. 1). This fragment BamHI- > PST was subclinically based on pOS49.1, the plasmid PBC SK+, which allowed to obtain the plasmid pOS49.28.

The second probe corresponds to a DNA fragment > PST -BamHI length of about 2 so called, containing a fragment of orf7 and a fragment of orf8, subcloned, based on pOS49.1, and going from site > PST in 6693 SEQ ID No. 1, up to the BamHI site located in position 8714 SEQ ID No. 1. This fragment > PST -BamHI was subclinically based on pOS49.1, the plasmid pBC SK+, which allowed to obtain the plasmid pOS49.76.

- Also was used for the third probe. This last corresponds to a DNA fragment with a length of 1.8 so-called EcoRI-HindIII containing gene srmD. Gene srmD is a gene isolated from S. ambofaciens capable of imparting resistance to spiramycin. In fact, according to previous works, carried out the cloning of several determinants of resistance of S. ambofaciens, giving resistance to spiramycin, in the strain of S. griseofuscus (strain, sensitive to spiramycin) (Pernodet and others, 1993; Pernodet and others, 1999). To highlight resistance genes was implemented Comedy Bank genomic DNA of a strain of S. ambofaciens ATS in cosmides pKC505 (M.A. Richardson and others, 1987). For this, genomic DNA of strain S.ambjfaciens ATS was partially restrictively with Sau3AI to obtain fragments ranging in length from about 30 so-called 40 so-called So restrictively genomic DNA (3 μg) ligated with 1 μg pKC505, pre restrictional enzyme BamHI (Pernodet and others, 1999). The mixture obtained by ligating, then uncapsizable in vitro into phage particles. The obtained phage particles were used to infect E.coli strain HB101 (available, in particular from the American type culture collection (ATSS) (Manassas, Virginie, USA) under No. 33694). Were received about 20,000 clones of E. coli resistant to apramycin, in the view of the pool and were proektirovanii of Comedy these clones. This pool of cosmid was introduced by transformation of protoplasts of the strain DSM 10191 S. griseofuscus (Cox K.L. & Baltz R.H., 1984), are inherently sensitive to spiramycin (R.N. Rao and others, 1987; this strain is deposited, in particular,in the German collection of microorganisms and cell cultures GmbH (DSMZ) (Braunschweig, Germany under the number DSM 10191). Transformants selected on medium containing apramycin. 1300 Clones growing on a medium containing apramycin, transferred to medium containing 5 μg/ml spiramycin. Several clones resistant to apramycin, was also grown on a medium containing spiramycin, and Comedy these colonies were extracted and used to transform E. coli S. griseofuscus (Pernodet and others, 1999). Thus, we have obtained five cosmid capable of imparting resistance to apramycin in the case of E. coli and artistintent to apramycin and spiramycin in the case of S. griseofuscus. Among these 5 cosmid it was determined that one cosmid called pOS44.1 in his box contains a gene (SEQ ID No. 15), which encodes a protein (SEQ ID No. 16), which has some similarity with the protein encoded by the genome of mdmA Streptomyces mycofaciens (SEQ ID No. 88); this gene was named srmD (see comparison by alignment presented on Fig, implemented under the program FASTA (cm. Pearson W.R. & D.J. Lipman, 1988; Pearson W.R., 1990); available, in particular from the resource center INFOBIOGEN, Evry, France).

To highlight the determinants of resistance contained in the plasmid pOS44.1, it was partially restrictively using the restriction enzyme Sau3AI to obtain fragments ranging in size from approximately 1.5 to the so-called 3 called, these fragments ligated in the vector pIJ486, linearized with the enzyme BamHI (Ward and others, 1986). Plasmid behold what was aktsioniroval on its ability to confer resistance to spiramycin strain DSM 10191 S. griseofuscus (R.N. Rao and others, 1987), are inherently sensitive to spiramycin (see above). A pool of plasmids corresponding to the Sau3AI fragment pOS44.1, legirovannykh in the vector pIJ486 (see above), was introduced by transformation of protoplasts of the strain DSM 10191 and transformants selected by their resistance to thiostrepton (occurring due to the tsr gene, located in pIJ486). Clones growing on a medium containing thiostrepton, transferred to medium containing spiramycin. Several clones resistant to thiostrepton, also cultivated on a medium containing spiramycin, and plasmids were extracted from these clones. Plasmid, giving resistance and containing insert a length of about 1.8 so-called, were selected and it was named pOS44.2. This insert length 1,8 called can be easily extracted with HindIII and EcoRI site located in the vector with the other hand insert. This insert a length of 1.8 so-called HindIII-EcoRI sequenced and gene resistance, which it includes, has been named the srmD. This fragment, containing the gene smrD, thus, can be easily subcloned into the vector pUC19 (access number in the gene Bank: M), restrictionon with EcoRI-HindIII, and the resulting plasmid was named pOS44.4. Insert a length of 1.8 so-called HindIII-EcoRI containing gene srmD, this plasmid was used as a probe to localize genes of the biosynthesis of spiramycin (see above).

A sample of the strain Escherichia Coli DH5α, containing plasmid pOS44.4, was deposited in the National collection of microorganism cultures (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex 15, France, July 10, 2002 under registration number I-2918.

Approximately 2000 clones Bank obtained above (see example 1), transferred to a filter for hybridization of colonies according to the classical methods (Sambrook and others, 1989).

Three probe described above was marked with32P according to the method of “arbitrary premirovany” (kit, manufactured by Roche) and used for hybridization 2000 clones of the Bank, transferred to a filter. Hybridization was performed at 65°C in the buffer described by Church and Gilbert (Church &Gilbert,1984). The leaching was carried out in 2X SSC at 65° for 15 minutes and then carried out two successive washing in 0.5x SSC at 65°With a duration of 15 minutes each. Under these conditions, hybridization and washing of 16 clones from 2000 hybridized had a strong hybridization signal with at least one of the probes. However, none of cosmid not hybridized with three probes. 16 Cosmid were extracted and restrictively using the restriction enzyme BamHI. Comparing with each other the restriction profiles of these different cosmid resulted in the selection of two cosmid able to contain the longest insert the field and not having the common areas. Thus, ibrani two Comedy, one of them was named pSPM5 and the other pSPM7. Cosmid pSPM5 hybridized with probes prf1 - orf4 and probe orf8, but not hybridized with probe srmD. Cosmid pSPM7 hybridized only with probe srmD and not hybridized with the other two probes.

These two Comedy were completely sequenced by the method of sequencing a shot from a shot gun. The sequence of the inserts of these two cosmid pSPM7 and pSPM5 can be combined, as, though not overlap, each of the inserts includes a known sequence at one of its ends. In fact, each of these inserts includes a fragment of the sequence of one of the genes encoding the enzyme called “policyinstitute” (PKS). These 5 genes were cloned S. Burgett and others in 1996 (U.S. patent 5945320) (see figure 2). Thus, it was determined one sequence of genomic DNA of S. ambofaciens. The sequence of 30943 nucleotides beginning, a 5'EcoRI site located in the first PKS gene, and going up to the BamHI site at 3', presented in the form of sequence SEQ ID No. 1. This sequence corresponds to the region above the PKS genes (see figure 2 and 3). The second area of 11171 nucleotides, starting from site > PST 5' and going up to the NcoI site at the 3', located in the fifth PKS gene, represented as sequences of SEQ ID No. 2. This second area is an area below the PKS genes (bottom and ver is determined by the orientation of the 5 PKS genes, all of which have the same semantic orientation) (see figure 2 and 3).

Example 4

Analysis of nucleotide sequences, determination of open reading frames and characterization of genes involved in the biosynthesis of spiramycin

Obtained sequences were analyzed using FramePlot program (J. Ishikawa & Hotta K.,1999). This analysis allows us to identify, among the open reading frames open reading frames representing typical usage codons Streptomyces. This analysis allowed us to determine that this area includes 35 open reading frames flanked on both sides by five genes encoding the enzyme “policyinstitute” (PKS). Respectively, 10 and 25 open reading frames were indetification below and above these genes (bottom and top are defined by the orientation of the 5 PKS genes, all of which are in the same semantic orientation) (see figure 3). Thus, 25 open reading frames of this type, covering an area of approximately 31 so-called (SEQ ID No. 1 and 3), have been identified above 5 genes encoding PKS, and 10 open reading frames, covering an area of approximately 11,1 so-called (SEQ ID No. 2 and figure 3), were identified below PKS genes. Genes upper area are related to:orf1, orf2, orf3, orf4, orf5, orf6, orf7, orf8, orf9c, orf10, orf11c, orf12, orf13c, orf14, orf15c, orf16, orf17, orf18, orf19, orf20, orf21c, orf22c, orf23c, orf24c et orf25c(SEQ ID№ 23, 25, 28, 30, 34, 36 40, 43, 45, 47, 49, 53, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82 and 84). Genes bottom area are related to:orf1*c, orf2*c, orf3*c, orf4*c, orf5*, orf6*, orf7*c, orf8*, orf9*, orf10*(SEQ ID№ 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21).

The letter “C”added to the end of the gene name for the given open reading frame means that the coding sequence is in the reverse orientation (coding thread, therefore, is a thread which is complementary to the sequence presented as SEQ ID No. 1 or SEQ ID No. 2 for these genes) (see figure 3).

Protein sequence resulting from these open reading frames were compared with those available in the various databases, using different programs: BLAST (Altschul and others, 1990; Altschul and others, 1997), CD-search, COGs (Cluster ontologica groups) (these three programs are available, in particular, from the National information center for biotechnology (NCBI) (Bethesda, Maryland, USA), FASTA (Pearson W. R. &Lipman, D. J., 1988) and (W. R. Pearson, 1990)), BEATY (K. C. Worley and others, 1995) (these two programs are available, in particular from the resource center INFOBIOGEN, Evry, France). These comparisons allowed us to formulate hypotheses concerning the function produced by these genes products and identify those that may be involved in the biosynthesis of spiramycin.

Example 5

Inactivation of the gene: concept design interrupted strain of Streptomyces ambofaciens

Used methods are done is no replacement gene. Interrupted gene target was replaced by a copy of this gene, interrupted by the cluster, which imparts resistance to antibiotics (such as, for example, apramycin or hygromycin), as illustrated in Fig. 9. Used the cluster is flanked on both sides by codons broadcast in all reading frames and active transcription terminators Streptomyces.

The insertion of the cluster in the gene target may be accompanied or not by a deletion in this gene target. The size of the flanking regions of a cluster can range from a few hundred to several thousand base pairs.

Structure required for inactivation of the gene by using cluster was obtained in E. coli, the standard body to produce structures of recombinant DNA. The interrupted gene was obtained plasmid that can replicate in E. coli but not able to replicate in Streptomyces.

Constructs were then subcloned into vectors for the possibility of transformation and inactivation of the desired gene in S. ambofaciens. For this we have used two plasmids:

- pOJ260 (M. Bierman and others, 1992) (see example 2), which gives resistance to apramycin in the case of E. coli and Streptomyces and which was used when gene target was interrupted by the cluster, giving resistance to hygromycin;

- pOSK1205 (472 base pairs). This plasmid is derived from plasmids RVC-CMV (manufactured by a company Stratagene (LaJolla, CA is ifornia, USA)), in which the AvrII fragment containing the sequence coding for resistance to neomycin/kanamycin, replaced by a sequence that encodes resistance to hygromycin, fully preserving the promoter P SV40. This plasmid pHP45-Ωhyg (Blondolet-Rouault and others, 1997) was restrictively enzymes NotI and PflmI and fragment, giving resistance to hygromycin, subclinical level AvrII site of the vector pBK-CMV after the ends have been made free ends by processing enzyme maple. In pOSK1205, the cluster, which gives resistance to hygromycin precedes the pSV40 promoter. This plasmid gives resistance to hygromycin in the case of E. coli and Streptomyces and was used when gene target was interrupted by the cluster, giving resistance to apramycin.

The clusters were embedded in the gene target or by cloning using restriction sites present in the gene of the target, either by recombination between short identical sequences, as described, for example, Chaveroche and others (Chaveroche MCI others, 2000).

A plasmid carrying the gene interrupted by the cluster can then be introduced into Streptomyces ambofaciens, for example, by conjugation between E. coli and Streptomyces (Mazodier, P. and others, 1989). This method is used in the case where the base vector is the vector of pOJ260. Can be used the second way: the way of transformation of protoplasts in the Le denaturation due to the alkaline processing DNA (T. Kieser and others, 2000) to increase the frequency of recombination as described, for example, Oh and Chater (Oh & Chater, 1997). This method was used in the case where the base vector is pOJ260 or pOSK1205 (see below). Transformants are then selected using the antibiotic corresponding to the cluster that is located in a gene target (see Fig.9, an antibiotic). Thus, selectionyou mixture of clones, among which was the integration at the expense of one or two recombination phenomena. Secondly, seek out clones sensitive to the antibiotic, in which the resistance gene is located in the vector (unclustered recombination) (see Fig.9, an antibiotic). You can also select clones that, in General, were two phenomena recombination, leading to the replacement of the original gene copy aborted by the cluster. These stages are schematically presented in Fig.9.

For interruption of target genes can be used multiple clusters. You can use, for example, the cluster Ωhyg, which gives resistance to hygromycin (Blondelet-Rouault and others, 1997; the access number in the gene Bank: H).

Example 6

Construction of a strain of Streptomyces ambofaciens, interrupted open reading frame orf3 gene

Orf3 gene was disrupted using cluster Ωhyg (see example 2.2.) it is shown that the strain orf3::Ωhyg not produce more spiramycin, which confirms the participation of one or ascolichen cloned region in the biosynthesis of spiramycin (see example 2.2). Given their orientation, then atranscript open reading frames 1-7 (see figure 3) and the observed phenotype (which is not a producer of spiramycine) may occur due to inactivation of one or more genes subjected atranscript with orf3. To confirm the participation of orf3 in the biosynthesis of spiramycine implemented a new gene inactivation orf3, and this last was carried out in the open reading frame. This was subjected to the deletion of the internal DraIII fragment length orf3 504 base pairs. The DNA fragment derived from pOS49.1 coming from the EcoRI site located in position 1 (SEQ ID No. 1) until the SacI site located in position 5274 (SEQ ID No. 1), and which includes a deletion between the two DraIII sites in the provisions of 2563 and 3067 (504 deleted nucleotides), cloned in the plasmid pOJ260 (M. Bierman and others, 1992). The thus obtained plasmid was named pOS49.67.

Insert pOS49.67, therefore, formed by the DNA fragment of S. ambofaciens containing genes orf1, orf2, orf3 with a deletion in the frame orf4 and part of orf5. Vector, which was subcloned this insert is pOJ260; plasmid pOS49.67, therefore, gives resistance to apramycin and was introduced by transformation of protoplasts of the strain OS49.16 (see example 2). Were obtained strain OS49.16 with resistance to hygromycin, hygR transformants and apraR. After two passages of such clones is selectively environment were detected clones sensitive apramycin and hygromycin (apraS and hygS). In some of these clones, in fact, expect that the phenomenon of recombination between homologous sequences leads to the substitution of copies of orf3, interrupted by a cluster Ωhyg (contained in the genome of strain OS49.16), a copy of orf3 with a deletion in the frame read in the vector. The clones obtained as a result of this recombination, as expected clones apraS and hygS after elimination sequences of the vector. The genotype of the resulting strains can be confirmed by hybridization or by PCR and sequencing of the PCR product (to ensure that one copy of orf3 with a deletion in the reading frame present in the genome of the obtained clones). There were thus obtained clones with only a copy of orf3 with a deletion in the frame is read, and the genotype was confirmed. In particular, he was selected as a clone having the desired characteristics, and named as OS49.67.

The sample strain OS49.67 was deposited in the National collection of microorganism cultures (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex 15, France, July 10, 2002 under registration number I-2916.

Example 7

Construction of a strain of Streptomyces ambofaciens, interrupted in the orf8 gene

To implement the inactivation of the gene orf8 received a construction in which the cluster Ωhyg was introduced in sequence, encoding orf8. Tothis first designed plasmid pOS49.88. Plasmid pOS49.88 produced from the plasmid pUC19 (access number in the gene Bank: M) due to the insertion of the fragment length is 3.7 so-called (>PST fragment-EcoRI obtained from Comedy pSPM5), containing the end of orf7, orf8 and the beginning of orf9, cloned sites > PST -pUC19 EcoRI. The cluster Ωyg (in the form of a BamHI fragment with the free ends of the through-processing enzyme maple) was cloned at the level of a single SalI site pOS49.88 located in orf8, after the ends have been made free ends by treatment with enzyme maple.

After cloning with obtaining loose ends were obtained two types of plasmids according to the direction of insertion of the cluster: pOS49.106 where hyg genes and orf8 are in the same orientation, and pOS49.120 where hyg genes and orf8 are in opposite orientations. Insert plasmids pOS49.106 then was subcloned into the plasmid pOJ260 to get pOS49.107. This plasmid pOS49.106 was restrictively using enzyme Asp7181 and the ends did the free ends by treatment with enzyme maple, this product restriction re restrictively using enzyme > PST and the fragment containing the orf8 gene, which is embedded in the cluster Ωhyg, cloned in the vector pOJ260 (see above). For this vector pOJ260 was restrictively enzymes EcoRI and > PST and used for ligation. This manipulation allows, therefore, to achieve directional ligation, since each of the two is Rahmanov has a free end on one side and > PST on the other hand. The obtained plasmid was named pOS49.107.

Plasmid pOS49.107 was introduced into the strain ADS S.ambofaciens by transformation of protoplasts (Kieser, T. and others, 2000). After transformation of protoplasts clones selected for resistance to hygromycin. Resistant to hygromycin clones were then perseval, respectively, on Wednesday with hygromycin (antibiotic) and the environment apramycin (antibiotic) (see Fig.9). Clones resistant to hygromycin (HygR) and sensitive to apramycin (ApraS), in principle, are such, which carried out the DCO and which have orf8 gene, interrupted by a cluster Ωhyg. Replacement original copy of the orf8 on a copy, interrupted by a cluster Ωhyg, was confirmed by southern blotting. Thus, the full DNA of the obtained clones was restrictively with several enzymes, separated on agarose gel, transferred to membrane and hybridisable with a probe corresponding to the cluster Ωhyg, to confirm the presence of the cluster in genomic DNA of the obtained clones. A second hybridization was carried out using as a probe insert > PST -EcoRI containing the end of orf7, orf8 and the beginning of orf9, the size of about 3.7 called plasmids pOS49.88. Confirmation of the genotype can be carried out by any method known to the expert, and, in particular, by PCR, using the appropriate oligonucleotides and sekhonyane product R.

Bilateral clone orf8:: Ωhyg and named OS49.107. The sample strain OS49.107 was deposited in the National collection of microorganism cultures (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex 15, France, July 10, 2002 under registration number I-2917.

Example 8

Construction of a strain of Streptomyces ambofaciens, interrupted in the orf10 gene

The DNA fragment with a length of 1.5 so called, internal to orf10 gene, was obtained by PCR using as template genomic DNA of S. ambofaciens and the following primers:

SRMR1: 5' CTGCCAGTCCTCTCCCAGCAGTACG 3' (SEQ ID No. 89)

SRMR2: 5' TGAAGCTGGACGTCTCCTACGTCGG 3' (SEQ ID NO. 90).

This DNA fragment obtained by PCR, cloned in the vector pCR2.1 manufactured by Invitrogen (carlsbad, CA, USA). The thus obtained plasmid was named pOS49.32. The cluster Ωhyg (in the form of a BamHI fragment, see above) was cloned at a BstEII site, internal to the fragment of the gene orf10, after all did the free ends through the processing enzyme maple. After cloning with obtaining loose ends, received two types of plasmids according to the direction of insertion of the cluster: pOS49.43 where hyg genes and orf10 are in the same orientation, and pOS49.44 where hyg genes and orf10 are in opposite orientations. Insert plasmids pOS49.43 transferred (in the form of Asp718I fragment-XbaI, the ends of which were released by treatment with enzyme maple) in the EcoRI site of plasmid pOJ260, what is solilo to obtain plasmid pOS49.50. Plasmid pOS49.50 containing a fragment of the orf10 gene, interrupted by a cluster Ωhyg, was introduced into strain ADS Streptomyces ambofaciens. After the transformation, the clones selected for resistance to hygromycin. Resistentie to hygromycin clones were then perseval, respectively, on Wednesday with hygromycin (antibiotic) and on Wednesday with apramycin (antibiotic) (see Fig.9). Clones resistant to hygromycin (HygR) and sensitive to apramycin (ApraS), in principle, are such, which carried out the DCO and which have orf10 gene, interrupted by a cluster Ωhyg. Thus clones were obtained, which had a token resistance to hygromycin located in the cluster, and lost token resistance to apramycin located in the vector pOJ260. Replacement original copy of the orf10 on a copy, interrupted by a cluster orf10::Ωhyg, was confirmed by southern blotting. Thus, the total genomic DNA of the obtained clones was restrictively with several enzymes, separated on agarose gel, transferred to membrane and hybridisable with a probe corresponding to the cluster Ωhyg, to confirm the presence of the cluster in genomic DNA of the obtained clones. A second hybridization was carried out using as a probe of the PCR product with a length of 1.5 so called, internal to orf10 gene (see above).

In particular, he was selected as a clone, which is idemia characteristics (orf10::Ωhyg), and named OS49.50. Actually, by two hybridisable it was confirmed that the cluster Ωhyg is in the genome of this clone and that in fact reach the expected profile restriction in the case of replacement, due to the dual effects of recombination, the source of the gene on a copy, interrupted by a cluster Ωhyg in the genome of this clone. Confirmation of the genotype can also be carried out in any known specialist way, and, in particular, by PCR, using the appropriate oligonucleotides and sequencing of the PCR product.

Example 9

Inactivation of genes: principle design of a strain of Streptomyces ambofaciens, interrupted by the method of “excesively clusters (see figures 9 and 10)

For inactivation of genes can be used the second type of clusters: clusters, called “excesively clusters”. These clusters have the advantage that can be excision from Streptomyces by sitespecifically recombination, after having been introduced into the genome of S. ambofaciens. The goal is to inactivation of some genes in Streptomyces strains without leaving in the final strain markers breeding or large sections of DNA that are not related to the strain. After excision there is only a short sequence length of about thirty base pairs (referred to as “cicatricial” site) in the genome of strain (see figure 10).

The implementation of this system sostoi is, first, in substitution of the original copies of the target genes (due to two phenomena homologous recombination, see Fig.9) design, in which excesively cluster embedded in this gene target. The insertion of this cluster is accompanied by a deletion in the gene target (see Fig.9). Secondly, provoked excision excisional cluster from the genome of strain. Excesively cluster operates the system sitespecifically recombination and has the advantage that it allows to obtain mutants of Streptomyces, not bearing in the end of the gene of resistance. Also get rid of possible polar effects on the expression of genes located below the inactivated gene or inactivated genes (see figure 10).

Use excesively clusters described and applied in the case of many organisms, including in the case of mammalian cells, yeast cells and in E. coli (Bayley and others, 1992; Brunelli and Pall, 1993; Camilli and others, 1994; Dale and Ow, 1991; Russel and others, 1992; Lakso and others, 1992). These excesively clusters, all use sitespecifically the recombinases Cre affecting the lox sites. This recombination system from bacteriophage P1.

For the design of the system type “excesively cluster in Streptomyces, took part of the system sitespecifically recombination, described for a mobile genetic element pSAM2 of Streptomyces ambofaciens (Boccard and others, 1989 a and b). the boost system is first, in constructing a recombinant vector comprising an interrupted gene that is built excesively cluster. The insertion in the gene target excisional cluster is accompanied by a deletion in the gene target. It can be implemented by cloning using restriction sites present in the gene of the target, or by recombination between short identical sequences, as described, for example, Chaveroche and others (Chaveroche M.K. and others, 2000). Excesively cluster can be constructed using, for example, cluster Ωhyg (Blondelet-Rouault and others, 1997). This cluster is flanked by sequences attR and attL, which usually flank the integrated copy of pSAM2 (see Fig). The sequence of attR and attL, all contain sites that are necessary for sitespecifically recombination, allowing the excision pSAM2 or any DNA fragment located between these two areas (Sezonov and others, 1997; Raynal and others, 1998). The design of this cluster, of course, not limited to the use of cluster Ωhyg, but other clusters resistantanti can serve as a basis for the design of this cluster (e.g. cluster Ωaac or Ωvph (Blondelet-Rouault and others, 1997).

After receiving this construction the strain Streptomyces transformed with recombinant plasmids. Transformants are then selected with the antibiotic, suitable for the cluster, located in a gene target (see Fig.9, the antibiotic; it is, for example, about breeding with hygromycin if excesively the cluster comes from the cluster Ωhyg). Thus, selectionyou mixture of clones, among which was the integration through the one or by two recombination phenomena. Secondly, seek out clones sensitive to the antibiotic, in which case the gene of resistance is the vector (unclustered recombination) (see Fig.9, an antibiotic). You can also selekcionirovat clones, which was, in principle, two phenomena recombination, leading to the replacement of the original gene copy aborted by the cluster. These stages are schematically presented in figure 9; the genotype of the thus obtained clones was confirmed by southern-blotting and selected strains with desirable characteristics (replace the original gene copy aborted excisional cluster).

Secondly, the strain selected above, transformed with plasmids allowing the expression of genes xis and int, which are both necessary for sitespecifically recombination between sites attR and attL. The result of this recombination is the removal excisional cluster from the genome of strain due to the phenomenon of recombination (see figure 10) (Raynal and others, 1998). Of interest is the choice of the vector carrying the genes xis and int, relatively unstable the vectors in Streptomyces (e.g., derived Streptomyces vector pWHM3 (Vara and others, 1989)), it allows to obtain a strain that has lost this last vector after several cycles of sporulation in the absence of selection pressure.

For excision of the cluster, for example, to use a plasmid pOSV508 (see Fig), which is introduced by transformation of protoplasts in a strain of S. ambofaciens, containing the gene, interrupted excisional cluster. Plasmid pOSV508 derived from plasmid pWHM3 (Vara J. and others, 1989) (see Fig), which added genes xis and int pSAM2 (Boccard F. and others, 1989 b), under the control of the promoter ptrc (Amann, E. and others, 1988). Genes xis and int, under the control of the ptrc promoter were subcloned in the plasmid pWHM3, on the basis of plasmids pOSint3 (Raynal and others, 1998) (see Fig). Introduction in the mutant strain plasmids pOSV508 carrying genes xis and int pSAM2 allows efficient excision by sitespecifically recombination excisional cluster between sites attL and attR, flanking the cluster (A. Raynal and others, 1998) (figure 10). Among the transformants, selected for their resistance to thiostrepton caused tsr gene, which is pOSV508, choose those that have become sensitive to the antibiotic, the presence of which in the cluster gives resistance (see figure 10). Excision is an effective and discovered that more than 90% of transformation are of this type. After one or more cycles of growth and is porously on solid medium, devoid of thiostrepton get clones that have lost the plasmid pOSV508. These clones are caught by their sensitivity to thiostrepton. The sequence of the target genes subjected to deletions, can be confirmed using PCR and sequencing of the PCR product.

Finally, the resulting strain at the level of the inactivated gene (for example, internal deletion) includes “scar” site att, corresponding to the minimal attB site (Raynal and others, 1998), which is derived from the recombination between sites attR and attL. This minimal attB site, which continues to exist, similar to that found naturally in strains of Streptomyces ambofaciens, Streptomyces pristinaespiralis and Streptomyces lividans (Sezonov and others, 1997).

Gene, I want to inactivate may be cotranscriptional with other genes, located below. To avoid that inactivation of one of the genes has a polar effect on the expression of genes that are lower in the operon, it is important to achieve deletions in the reading frame after the excision of the cluster. System excesively clusters, such as described above, allows you to meet this requirement. In fact, the specialist can easily construct three different excesively cluster, leaving after the excision sequence of 33, 34 or 35 nucleotides, respectively, without stop codon, which Bini was reading frame. Knowing the sequence of the target genes and the size of the deletions associated with insertion of the cluster, you can choose between these three excesively clusters so that excision resulted in deletions in the reading frame. 33, 34 or 35 of the added nucleotides 26 correspond meemalee attB sequence (see Fig).

In the case of this application, we used two excesively cluster. These two clusters are the following: att1Ωhyg+ (Seq ID No. 91) and att3Ωaac- (SEQ ID No. 92); these clusters after excision leave, respectively, 33 and 35 nucleotides. They include, respectively, the cluster Ωhyg or cluster Ωaac, and the signs + and - correspond to the orientation of the cluster of resistance. These two clusters were constructed and cloned in the EcoRV site level vector pBC SK+HindIII site which was previously eliminated. The resulting plasmids were named patt1Ωhyg+ and patt3Ωaac-, respectively. Excesively clusters can be easily eliminated by restriction analysis of the plasmid with EcoRV.

Example 10

Construction of a strain of Streptomyces ambofaciens, interrupted in the gene orf2

Inactivation of the gene orf2 implemented by the method using excesively clusters (see above). Used the original strain is Streptomyces ambofaciens OSC2, which is derived from the strain ADS. However, the strain OSC2 differs from strain ADS the fact that the n lost mobile genetic element pSAM2 (Boccard and others, 1989 a and b). This mobile element can be lost spontaneously in the process of protoplastic (effect of lysozyme for the restriction of bacterial wall and fragmentation of mycelium (Kieser and others, 2000)) and regeneration of protoplasts of strain ADS. For breeding clones that had lost the element pSAM2, was carried out by screening based on the repression of the gene pra using a repressor of transcription KorSA (Sezonov and others,1995; G. Sezonov and others, 2000). For this purpose, a DNA fragment containing the promoter of the gene pra above gene aph (giving resistance to kanamycin and devoid of its own promoter)was cloned in an unstable vector pWHM3Hyg, the latter is derived from the plasmid pWHM3 (Vara and others, 1989), in which the tsr gene was replaced with the hyg gene (giving resistance to hygromycin). The thus obtained plasmid was named pOSV510. Strain ATS transformed, after protoplastic, using plasmids pOSV510. The promoter Pra is a promoter that is repressed by the repressor KorSA, and the gene encoding this last, is a mobile element pSAM2 (Sezonov, G., and others, 2000). After transformation with plasmids pOSV510 transformed bacteria are selected by their resistance to kanamycin (called aph gene, located in pOSV510). Clones that had lost the integrative element pSAM2, lost repressor KorSA and promoter Pra, therefore, is not more depressed and allows the expression of a gene of resistance to kanamycin aph. Selection by kanamycin after transformation with plasmids pOSV510, therefore, allows you to select clones that have lost the integrative element pSAM2 (and, therefore, KorSA) and containing plasmid pOSV510. Because plasmid pOSV510 is unstable after several cycles of sporulation without antibiotic, the selected clones were perseval on medium with kanamycin, on Wednesday with hygromycin and on the medium without the antibiotic. The clones sensitive to kanamycin and to hygromycin, lost pOSV510. Loss element pSAM2 was confirmed by hybridization and PCR. He was selected one clone having the desired characteristics, and named OSC2.

A sample of the strain OSC2 was deposited in the National collection of microorganism cultures (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex 15, France, July 10, 2002 under registration number I-2908.

Inactivation of the gene orf2 was carried out according to the method with excesively clusters (see above and figure 10). To do this, insert a length of 4.5 so-called, the sequence of which comes from the EcoRI site located in position 1, up to the BamHI site located in position 4521 (SEQ ID No. 1)was subclinical level sites EcoRI and BamHI plasmid pUC19 (access number in the gene Bank: M), based on Comedy pSPM5. The thus obtained plasmid was named pOS49.99.

This plasmid was introduced into E.coli strain KS272, which already contained the plasmid pKOBEG (Chaveroche, etc, 2000) (see Fig).

In parallel, excesively cluster att3Ωaac- (SEQ ID No. 92, see above) amplified by PCR using as template a plasmid pOSK1102 (plasmid pOSK1102 is a plasmid, derived from vector pGP704Not (Chaveroche and others, 2000; Miller V.L. & J.J. Mekalanos, 1988), in which the cluster att3Ωaac was cloned as an EcoRV fragment in a single EcoRV site pGP704Not) and using the following primers:

ORF2A

5' CCCGCGCGGCAGCCTCTCCGTGATCGAGTCCGGCGTGACCATCGCGCGCGCTTCGTTCGG-3' (SEQ ID No. 93);

ORF2B

5' GCTCCGTGCGTCATGCAGGAAGGTGTCGTAGTCGCGGTAGATCTGCCTCTTCGTCCCGAA-3' (SEQ ID NO. 94).

40 Deoxynucleotides located at the 5' end of these oligonucleotides that include the sequence corresponding to the sequence in the gene target (in this case orf2), and 20 deoxynucleotides located closer to the 3' (shown in bold above)correspond to a sequence of one end excisional cluster att3Ωaac- (see 11).

The thus obtained PCR product was used to transform E.coli strain containing the plasmid pKOBEG and pOS49.99, as described by Chaveroche and others (2000) (see Fig). Thus, the bacteria were transformirovaniya by electroporation and selected for their resistance to apramycin. The plasmids obtained clones were extracted and restrictively with several restriction enzymes to confirm that the profile reached the hotel is AI match the expected profile, if there was the insertion of the cluster (attt3Ωaac-) gene target (orf2), that is, if it really happened homologous recombination between the ends of the PCR product and of the target genes (Chaveroche and others, 2000). Design confirmation can also be made by any method known to the expert, and, in Casinocity by PCR using the appropriate oligonucleotides and sequencing of the PCR product. He was selected clone, the plasmid which has the expected profile, and the corresponding plasmid was named pSPM17. This plasmid is derived from pOS49.99 in which orf2 broken by apromising cluster (see Fig).

The insertion of the cluster is accompanied by a deletion in orf2, between nucleotides 211 and 492 coding part of orf2.

Plasmid pSPM17 was restrictively by the enzyme EcoRI, and then the ends were made free ends by treatment with enzyme maple, this product restriction then restrictively using enzyme XbaI and the insert containing the lost orf2 gene, cloned in the vector pOSK1205 (see above). For this vector pOSK1205 was restrictively enzyme BamHI, and then the ends were made free ends by treatment with enzyme maple, this product since restrictively enzyme XbaI and used for ligation with the insert obtained from pSPM17, as described above. This manipulation therefore allows to achieve directional leagues is the way, because each of the two fragments has a free end with one hand and XbaI on the other hand. The thus obtained plasmid was named pSPM21, she carries the gene for resistance to hygromycin (vector and insert, in which the lost orf2 gene is replaced by a cluster att3Ωaac-.

Vector pSPM21 was introduced into a strain of Streptomyces ambofaciens OSC2 (see above) by transformation of protoplasts (Kieser, T. and others, 2000). After the transformation, the clones selected for resistance to apramycin. Resistant to apramycin clones were then perseval, respectively, on Wednesday with apramycin (antibiotic) and on Wednesday with hygromycin (antibiotic) (see Fig.9). Clones resistant to apramycin (ApraR) and sensitive to hygromycin (HygS), are, in principle, these where made DCO and which have orf2 gene, interrupted by a cluster att3Ωaac-. These clones selected and confirmed the replacement of the original copy of the orf2 copy, interrupted by the cluster. The presence of a cluster attΩaac was confirmed by PCR in the colony. Was also carried out hybridization. This full DNA of the obtained clones was restrictively with several enzymes, separated on agarose gel, transferred to membrane and hybridisable with probe length 3 so-called corresponding to the fragment EcoRI-BamHI insert of the plasmid pOS49.99 (see above). Confirmation of the genotype can also be assests is but any way well-known specialist, and, in particular, by PCR using the appropriate oligonucleotides and sequencing of the PCR product.

He was selected clone with the expected characteristics, and named SPM21. By PCR and hybridization can be confirmed that the cluster att3Ωaac - is really in the genome of this clone and that really made a profile restriction, expected in the case of replacement, due to the dual effects of recombination, the original gene copy aborted cluster att3Ωaac-in the genome of this clone. This clone therefore has the genotype: orf2:: att3Ωaac-.

The sample strain SPM21 was deposited in the National collection of microorganism cultures (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex 15, France, July 10, 2002 under registration number I-2914.

Strain SPM21 transformed with a vector POSV508 due to transformation of protoplasts with the aim of provoking the excision of the cluster (see Fig). Plasmid pOSV508 derived from plasmid pWHM3 (Vara J. and others, 1989) (see Fig), which added genes xis and int pSAM2 (Boccard F. and others, 1989 b), under the control of the promoter ptrc (Amann, E. and others, 1988) (see Fig). Introduction to strain SPM21 plasmids pOSV508 carrying genes xis and int pSAM2 allows efficient excision by sitespecifically recombination excisional cluster between sites attL and attR, flanking the cluster (A. Raynal and others, 1998) (f is g). Among the transformants, selected for their resistance to thiostrepton caused by the tsr gene, which is located in pOSV508, choose two that have become sensitive to apramycin, gene resistance which is in the cluster att3Ωaac-; a consequence of the excision, in fact, is the loss of this gene resistance (see figure 10). Plasmid pOSV508 is unstable and after two consecutive passages on medium without antibiotic selected clones subcultured on a medium with thiostrepton and the environment without thiostrepton. The clones sensitive to thiostrepton, lost pOSV508. That excision of the cluster really leads to deletions in the reading frame orf2 gene was confirmed using PCR and sequencing of the PCR product; in fact, after the excision of the cluster remains characteristic “scar” sequence att3 (which is similar to the attB site, formed after recombination sites attL and attR):

5' ATCGCGCGCGCTTCGTTCGGGACGAAGAGGTAGAT 3' (SEQ ID NO. 95).

Thus obtained and having the desired genotype (orf2::att3) strain was named SPM22.

The sample strain SPM22 was deposited in the National collection of microorganism cultures (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex 15, France, July 10, 2002 under registration number I-2915.

Example 11

Construction of a strain of Streptomyces ambofaciens, interrupted in orf12 gene

For inactivation of orf12, orf13c and rf14 used the same original plasmid (pSPM504) for introduction into the different positions of the cluster type “excisional cluster. This plasmid has an insertion length of 15.1 called, which corresponds to the region from orf7 to orf17. To construct this plasmid, the fragment BglII length of 15.1 called, derived from the restriction of Comedy pSPM7 (see above), cloned in the plasmid pMBL18 (Nakano and others, 1995), restrictional with BamHI. Since the ends of BamHI and BglII are compatible, after ligating receive plasmid pSPM502. In General, the insert pSPM502 then subclinically (fragment HindIII/NheI) plasmid pOSK1205 (restrictional using HindIII/NheI), which allowed to obtain the plasmid pSPM504.

This plasmid was introduced into E.coli strain KS272, which already contained the plasmid pKOBEG (Chaveroche and others, 2000) (see Fig).

In parallel excesively cluster att3Ωaac - amplified by PCR using as template a plasmid pOSK1102 (plasmid pOSK1102 is a plasmid, derived from vector pGP704Not (Chaveroche and others, 2000; Miller V.L. & J.J. Mekalanos, 1988), which was cloned cluster att3Ωaac as EcoRV fragment in a single EcoRV site pGP704Not); primers used are the following:

EDR8:5' CGGGATGATCGCTTGTCCGGCGGCCGGATGCCTAGCCTCATCGCGCGCGCTTCGTTCGG3' (SEQ ID No. 96);

EDR9:5' CCCGATCCAGAACGTCTGGTCGGTGATCAGGTCGCTGTTCATCTGCCTCTTCGTCCCGAA3' (SEQ ID NO. 97).

40 (only 39 in EDR8) Deoxynucleotides located at the 5' end of these oligonucleotides that include the sequence corresponding to the sequence in the gene target (in this case, orf12), and 20 deoxynucleotide is s, located mostly in the 3' (shown in bold above)correspond to a sequence of one end excisional cluster att3Ωaac- (see 11).

The thus obtained PCR product was used to transform E.coli strain KS272 containing plasmids pKOBEG and pSPM504 (see above), as described Chaveroche and others (Chaveroche and others, 2000) (see Fig on the principle of plasmid pOS49.99 must be replaced by the plasmid pSPM504 and the resulting plasmid is not more pSPM17, and is a plasmid pSPM507). Thus, the bacteria were transformed by electroporation using this PCR product and the clones were selected by their resistance to apramycin. The plasmids obtained clones were extracted and restrictively with several restriction enzymes to confirm that the obtained profile restriction corresponds to the expected profile, if there was the insertion of the cluster (att3Ωaac-) gene target (orf12), that is, if it really happened homologous recombination between the ends of the PCR product and of the target genes (Chaveroche and others, 2000). Design confirmation can also be made by any method known to the expert, and, in particular, by PCR using the appropriate oligonucleotides and sequencing of the PCR product. Clone, plasmid which has the expected profile was selected and soo the appropriate plasmid was named pSPM507. This plasmid is derived from pSPM504 in which orf12 gene interrupted by a cluster att3Ωaac- (see Fig). The insertion of the cluster is accompanied by a deletion in the gene orf12, interruption begins at the level of the thirtieth codon orf12. After the cluster remain 46 last codons of the gene orf12.

Vector pSPM507 was introduced into a strain of Streptomyces ambofaciens OSC2 (see above) by transformation of protoplasts (Kieser, T. and others, 2000). After the transformation, the clones selected for resistance to apramycin. Resistant to apramycin clones were then perseval, respectively, on Wednesday with apramycin (antibiotic) and on Wednesday with hygromycin (antibiotic) (see Fig.9). Clones resistant to apramycin (ApraR) and sensitive to hygromycin (HygS), in principle, represent that took place DCO and which have orf12 gene, interrupted by a cluster att3Ωaac-. These clones were preferably selected and replace the original copy of the orf12 gene copy aborted by the cluster was confirmed by hybridization. So, the full DNA of the obtained clones was restrictively with several enzymes, separated on agarose gel, transferred to membrane and hybridisable with a probe corresponding to the cluster att3Ωaac-to confirm the presence of the cluster in genomic DNA of the obtained clones. A second hybridization was carried out using as a probe a DNA fragment obtained by CR and the corresponding very large part of the coding sequence of the gene orf12.

Confirmation of the genotype can also be susestudio by any method known to the expert, and, in particular, by PCR, using the appropriate oligonucleotides and sequencing of the PCR product.

Preferably been selected clone with the expected characteristics (orf12:: att3Ωaac-), and named SPM507. In fact, by two hybridisable it was confirmed that the cluster att3Ωaac - is really in the genome of this clone and that in fact reach the expected profile restriction in the case of replacement, due to a double recombination, the original gene copy aborted cluster att3Ωaac-in the genome of this clone. This clone therefore has the genotype orf12:: att3Ωaac and was named SPM507. Unnecessary is the implementation of the excision of the cluster to study the effect of inactivation of orf12 from the point of view of the orientation of the genes (see figure 3). In fact, the fact that orf13c is oriented in the opposite direction orf12 gene, indicates that these genes are not cotranscriptional. Use excisional cluster, instead, allows to be able at any time to get rid of marker selection, in particular, by transformation with plasmids pOSV508.

The sample strain SPM507 was deposited in the National collection of microorganism cultures (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex 15, France, July 10, 2002 under registration NR the rum I-2911.

Example 12

Construction of a strain of Streptomyces ambofaciens, interrupted in the gene orf13c

Excesively cluster att3Ωaac - amplified by PCR using as template a plasmid pOSK1102 (see above), using the following primers:

EDR3: 5' ACCGGGGCGGTCCTCCCCTCCGGGGCGTCACGGCCGCGGAATCTGCCTCTTCGTCCCGAA 3'(SEQ ID No. 98);

EDR4: 5' CACGCAGCGAGCCGACGCACTGATGGACGACACGATGGCCATCGCGCGCGCTTCGTTCGG 3'(SEQ ID NO. 99).

40 Deoxynucleotides located at the 5' end of these oligonucleotides that include the sequence corresponding to the sequence in the gene target (in this case orf13c), and 20 deoxynucleotides located most 3' (shown in bold above)correspond to a sequence of one end excisional cluster att3Ωaac- (see 11).

The thus obtained PCR product was used to transform E.coli strain KS272 containing plasmids pKOBEG and pSPM504 (see above), as described Chaveroche and others (Chaveroche and others, 2000) (see Fig on the principle of plasmid pOS49.99 must be replaced by the plasmid pSPM504 and the resulting plasmid is not more pSPM17, and is a plasmid pSPM508). Thus, the bacteria were transformed by electroporation using the PCR product and the clones were selected by their resistance to apramycin. The plasmids obtained clones were extracted and restrictively using multiple enzymes with a purpose on the show, the obtained profile restriction corresponds to the expected profile, if there was the insertion of the cluster (att3Ωaac-) gene target (orf13c), that is, if it really happened homologous recombination between the ends of the PCR product and of the target genes (Chaveroche and others, 2000). Design confirmation can also be made by any method known to the expert, and, in particular, by PCR using the appropriate oligonucleotides and sequencing of the PCR product. Clone, plasmid which has the expected profile was selected and the corresponding plasmid was named pSPM508. This plasmid is derived from pSPM504, in which the gene orf13c interrupted apramycin cluster (see Fig). The insertion of the cluster is accompanied by a deletion in the gene orf13c, interruption begins at the level of the sixth codon orf13c. After the cluster remain in 3 of the last codon of the gene orf13c.

Vector pSPM508 was introduced into a strain of Streptomyces ambofaciens OSC2 (see above) by transformation of protoplasts (Kieser, T. and others, 2000). After the transformation, the clones selected for resistance to apramycin. Resistant to apramycin clones were then perseval, respectively, on Wednesday with apramycin (antibiotic) and on Wednesday with hygromycin (antibiotic) (see Fig.9). Clones resistant to apramycin (ApraR) and sensitive to hygromycin (HygS), in principle, represent that took place DCO and to the which possess genome orf13c, interrupted by the cluster att3Ωaac-. These clones were preferably selected and replace the original copy of the gene orf13c copy, interrupted by the cluster was confirmed by hybridization. So, the full DNA of the obtained clones was restrictively with several enzymes, separated on agarose gel, transferred to membrane and hybridisable with a probe corresponding to the cluster att3Ωaac-to confirm the presence of the cluster in genomic DNA of the obtained clones. A second hybridization was carried out using as a probe PCR product corresponding to sequence serving a hundred base pairs above and below the coding sequence orf13c. Confirmation of the genotype can also be susestudio by any method known to the expert, and, in particular, by PCR, using the appropriate oligonucleotides and sequencing of the PCR product.

Preferably been selected clone with the expected characteristics (orf13c:: att3Ωaac-), and named SPM508. In fact, by two hybridisable it was confirmed that the cluster att3Ωaac - is really in the genome of this clone and that in fact reach the expected profile restriction in the case of replacement, due to the dual effects of recombination, the original gene copy aborted cluster att3Ωaac-in the genome of this clone. This clone therefore has the genotype orf13c:: att3Ωaac and was called the Academy of Sciences SPM508. Unnecessary is the implementation of the excision of the cluster to study the effect of inactivation orf13c from the point of view of the orientation of the genes (see figure 3); the fact that orf14 is oriented in the direction opposite gene orf13c shows that these genes are not cotranscriptional. Use excisional cluster, instead, allows to be able at any time to get rid of marker selection.

The sample strain SPM508 was deposited in the National collection of microorganism cultures (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex 15, France, July 10, 2002 under registration number I-2912.

Example 13

Construction of a strain of Streptomyces ambofaciens, interrupted in the orf14 gene

Excesively cluster att3Ωaac - amplified by PCR using as template a plasmid pOSK1102 (see above), using the following primers:

EDR5: 5'GGGCGTGAAGCGGGCGAGTGTGGATGTCATGCGAGTACTCATCGCGCGCGCTTCGTTCGG3' (SEQ ID No. 100);

EDR6: 5'CGGGAAACGGCGTCGCACTCCTCGGGGGCCGCGTCAGCCCATCTGCCTCTTCGTCCCGAA3' (SEQ ID NO. 101).

40 Deoxynucleotides located at the 5' end of these oligonucleotides that include the sequence corresponding to the sequence in the gene target (in this case, orf14), and 20 deoxynucleotides located most 3' (shown in bold above)correspond to a sequence of one end excisional cluster att3Ωaac- (see 11).

Thus the m resulting PCR product was used to transform E.coli strain KS272, containing plasmids pKOBEG and pSPM504 (see above), as described Chaveroche and others (Chaveroche and others, 2000) (see Fig on the principle of plasmid pOS49.99 must be replaced by the plasmid pSPM504 and the resulting plasmid is not more pSPM17, and is a plasmid pSPM509). Thus, the bacteria were rasformirovan by electroporation using the PCR product and the clones were selected by their resistance to apramycin. The plasmids obtained clones were extracted and restrictively with several restriction enzymes to confirm that the obtained profile restriction corresponds to the expected profile, if there was the insertion of the cluster (att3Ωaac-) gene target (orf14), that is, if it really happened homologous recombination between the ends of the PCR product and of the target genes (Chaveroche and others, 2000). Design confirmation can also be made by any method known to the expert, and, in particular, by PCR using the appropriate oligonucleotides and sequencing of the PCR product. Clone, plasmid which has the expected profile was selected and the corresponding plasmid was named pSPM509. This plasmid is derived from pSPM504 in which orf14 gene interrupted apramycin cluster (see Fig). The insertion of the cluster is accompanied by a deletion in the gene orf14, interruption begins at the level of the fourth codon orf14. After the cluster is the last codon of the gene orf14.

Vector pSPM509 was introduced into a strain of Streptomyces ambofaciens OSC2 (see above) by transformation of protoplasts (Kieser, T. and others, 2000). After the transformation, the clones selected for resistance to apramycin. Resistant to apramycin clones were then perseval, respectively, on Wednesday with apramycin (antibiotic) and on Wednesday with hygromycin (antibiotic) (see Fig.9). Clones resistant to apramycin (ApraR) and sensitive to hygromycin (HygS), in principle, represent that took place DCO and which have orf14 gene, interrupted by a cluster att3Ωaac-. These clones were preferably selected and replace the original copy of the orf14 gene copy aborted by the cluster was confirmed by hybridization. So, the full DNA of the obtained clones was restrictively with several enzymes, separated on agarose gel, transferred to membrane and hybridisable with a probe corresponding to the cluster att3Ωaac-to confirm the presence of the cluster in genomic DNA of the obtained clones. A second hybridization was carried out using as a probe PCR product corresponding to sequence serving a hundred base pairs above and below the coding sequence orf14. Confirmation of the genotype can also be susestudio by any method known to the expert, and, in particular, by PCR, using the appropriate olig the nucleotides, and sequencing of the PCR product.

Preferably been selected clone with the expected characteristics (orf14:: att3Ωaac-), and named SPM509. In fact, by two hybridisable it was confirmed that the cluster att3Ωaac - is really in the genome of this clone and that in fact reach the expected profile restriction in the case of replacement, due to the dual effects of recombination, the original gene copy aborted cluster att3Ωaac-in the genome of this clone. This clone therefore has the genotype orf14:: att3Ωaac and was named SPM509. Unnecessary is the implementation of the excision of the cluster to study the effect of inactivation of orf14 from the point of view of the orientation of the genes (see figure 3); the fact that orf15c is oriented in the opposite direction orf14 gene, indicates that these genes are not cotranscriptional. Use excisional cluster , instead, allows to be able at any time to get rid of marker selection.

The sample strain SPM509 was deposited in the National collection of microorganism cultures (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex 15, France, July 10, 2002 under registration number I-2913.

Example 14

Construction of a strain of Streptomyces ambofaciens, interrupted in the gene orf6*

Inactivation of the gene orf6* was performed using the method with excesively clusters (see above and figure 10). For this kosmidou pSPM7 COI is litovali as template for amplification of a fragment of the gene orf6* using the following oligonucleotides:

C9583: 5'CTGCAGGTGCTCCAGCGCGTCGATCT 3' (oligo sens) (SEQ ID No. 102);

C9584: 5'CTGCAGACGGAGGCGGACCTGCGGCT 3' (oligo antisens) (SEQ ID No. 103).

20 Deoxynucleotides at the end 3' of these oligonucleotides correspond to a sequence located in the coding part of the gene orf6* (SEQ ID No. 13), and 6 deoxynucleotides are more than just 5'correspond to a sequence of customers > PST, allowing subsequently to facilitate cloning. Amplificatory DNA fragment had a size of approximately 1,11 called This PCR product was cloned in the vector pGEM-T Easy (produced by the company Promega (Madison, Wisconcin, USA), which allowed to obtain the plasmid, named pBXL1111 (see Fig).

Then in the coding sequence of a gene orf6* introduced excesively cluster attΩhyg+. This plasmid pBXL1111 was restrictively using enzymes SmaI and Asp718I and product were treated with restriction enzyme maple. This manipulation allows to implement an internal deletion of 120 base pairs in the coding sequence of the gene orf6* (see Fig). Moreover, with both sides of the site restrictions remain, respectively, 511 base pairs and 485 base pairs of sequence of orf6*, which allow for homologous recombination to inactivate the gene orf6*. Excesively cluster attΩhyg+ was obtained from the plasmid patt1Ωhyg+ (see above) by the restriction of this plasmid with EcoRV. This last ZAT what was subcloned into the vector pBXL1111, previously obtained as described above (restriction with SmaI and Asp718I, then processing by the enzyme maple). The obtained plasmid was named pBXL1112 (see Fig). In this design the orf6 gene* includes a deletion of 120 base pairs and terminated cluster attΩhyg+.

Plasmid pBXL1112 then restrictively using enzyme > PST (site flanking the cluster, as is the oligonucleotides PCR) and insert > PST a length of 3.7 called, comprising part of the orf6*, interrupted by a cluster attΩhyg+, then cloned at the site level > PST plasmid pOJ260 (see above). The thus obtained plasmid was named pBXL1113.

Vector pBXL1113 was introduced into a strain of Streptomyces ambofaciens OSC2 (see above) by transformation of protoplasts (Kieser, T. and others, 2000). After the transformation, the clones selected for resistance to hygromycin. Resistant to hygromycin clones were then perseval, respectively, on Wednesday with hygromycin (antibiotic) and on Wednesday with apramycin (antibiotic) (see Fig.9). Clones resistant to hygromycin (HygR) and sensitive to apramycin (ApraS), in principle, represent that took place DCO and which have orf6 gene*, interrupted by a cluster att1Ωhyg+. These clones were preferably selected and replace the original copy of the orf6 gene* copy aborted cluster was confirmed by southern blotting. So, the full DNA obtained is s clones were restrictively with several enzymes, separated on the agarose gel, transferred to membrane and hybridisable with a probe corresponding to the hyg gene (obtained by PCR to confirm the presence of the cluster in genomic DNA of the obtained clones. A second hybridization was carried out using as a probe insert > PST - > PST, containing the gene orf6* size of about 1.1 so-called and obtained from a plasmid pBXL1111 (see above and Fig). Confirmation of the genotype can also be susestudio by any method known to the expert, and, in particular, by PCR, using the appropriate oligonucleotides and sequencing of the PCR product.

Preferably been selected clone with the expected characteristics (orf6*:: att3Ωhyg+), and named SPM501. In fact, by two hybridisable it was confirmed that the cluster att3Ωhyg+ is really in the genome of this clone and that in fact reach the expected profile restriction in the case of replacement, due to the dual effects of recombination, the original gene copy aborted cluster att3Ωhyg+, in the genome of this clone. This clone therefore has the genotype orf6*::att3Ωhyg+ and was named SPM501.

The sample strain SPM501 was deposited in the National collection of microorganism cultures (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex 15, France, July 10, 2002 under registration number I-2909.

Strain SPM501 transformed with a vector POSV508 due to transformation FR the layers with the aim of provoking the excision of the cluster (see Fig). Plasmid pOSV508 derived from plasmid pWHM3 (Vara J. and others, 1989) (see Fig), which added genes xis and int pSAM2 (Boccard F., and others, 1989b), under the control of the promoter ptrc (Amann, E. and others, 1988) (see Fig). Introduction to strain SPM501 plasmids pOSV508 carrying genes xis and int pSAM2 allows efficient excision by sitespecifically recombination excisional cluster between sites attL and attR, flanking the cluster (A. Raynal and others, 1998) (figure 10). Among the transformants, selected for their resistance to thiostrepton caused by the tsr gene, which is located in pOSV508, choose two that have become sensitive to hygromycin, gene resistance which is in the cluster att3Ωhyg+; a consequence of the excision, in fact, is the loss of this gene resistance (see figure 10). Plasmid pOSV508 is unstable and after two consecutive passages on medium without antibiotic selected clones were perseval on Wednesday with thiostrepton and the environment without thiostrepton. The clones sensitive to thiostrepton, lost pOSV508. That excision of the cluster actually took place in the reading frame orf6 gene*, confirmed by PCR and sequencing of the PCR product. The interrupt starts with a 158-th codon, 40 codons have undergone deletions (120 base pairs) and after excision of the cluster remains characteristic “scar” sequence att1 from 33 base pairs: 5' ATCGCGCCTTCGTTCGGGACGAAGAGGTAGAT 3' (SEQ ID No. 104).

Thus obtained and having the desired genotype (orf6*::att1) strain was named SPM502.

The sample strain SPM502 was deposited in the National collection of microorganism cultures (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex 15, France, July 10, 2002 under registration number I-2910.

Example 15

Analysis of strains of Streptomyces ambofaciens, interrupted genes orf2, orf3, orf8, orf10, orf12, orf13c, orf14, orf6*

To test the production of spiramycin obtained by different strains developed microbiological test based on the sensitivity of the strain Micrococcus luteus (see Gourmelon A. and others, 1998). Used a strain of Micrococcus luteus is a strain derived from strain DSM1790 inherently sensitive to spiramycin (this strain, in particular, is available from the German collection of microorganisms and cell cultures GmbH, DSMZ) (Braunschweig, Germany) under the number DSM1790); used in the present test, the strain is different from the strain DSM1790 the fact that it is resistant to conhecido. This strain is a spontaneous mutant, obtained by selection on media containing increasing doses of kogiidena. This strain was selected because of Streptomyces ambofaciens simultaneously produces spiramycin and congalidis. For the purpose of determining the production of spiramycin get different strains using microbiological test osnovanog is on the sensitivity of the strain Micrococcus luteus, you must have a strain that is resistant to conhecido.

Various test strains of Streptomyces were cultured in Erlenmeyer flasks with baffles with a capacity of 500 ml, containing 70 ml of medium MP5 (Pernodet and others, 1993). The Erlenmeyer flask with baffles was inoculable when the initial concentration of 2.5·106spores/ml of different strains of S. ambofaciens and cultivated at a temperature of 27°C With orbital agitation of 250 rpm Selections 2 ml of the suspensions was carried out after 48, 72 and 96 hours of cultivation and centrifuged. Various supernatant then frozen at -20°C. Ten-fold dilution of these supernatants in sterile culture medium used for the test (see Fig).

Typical strain Micrococcus luteus resistant to conhecido, but sensitive to spiramycin, were cultured in 2TY medium (Sambrook and others, 1989)containing congalidis in an amount of 5 μg/ml, for 48 hours at 37°C. was Determined by optical density (DO) culture and this culture was diluted in such a way as to set the optical density equal to 4. 0.4 ml of This preculture was dissolved in 40 ml of medium DAM5 (Difco Antibiotic Medium 5, manufactured by Difco), previously brought to a temperature of about 45°C. the medium was then poured into a square Cup size 12 cm x 12 cm and left to stand to raise the temperature to room.

PR is using once chilled and utverzhdenii environment discs of Whatman paper AA (see Gourmelen A. and others, 1998) with a diameter of 12 mm was impregnated with 70 μl of ten-fold dilution of each supernatant was placed on the surface of the Cup. Discs impregnated with a solution spiramycine different concentrations (2-4-8 µg/ml in culture medium MP5), was used as the reference number. The Cup was kept at 4°C for 2 hours to give the opportunity to be the diffusion of antibiotics in agar, then incubated at 37°C for 24-48 hours.

If the disk contains spiramycin, it diffuses into the agar and inhibited the growth of the characteristic strain of Micrococcus luteus. This inhibition causes a “halo” around the disk, and this halo reflects the area where the strain Micrococcus luteus is not growing. The presence of this halo, therefore, is an indication of the presence of spiramycin and allows to determine whether a strain of S. ambofaciens corresponding to a given disk, producing or neproducyruth spiramycin. Comparison with the diameters of inhibition was obtained for the reference number, allows to obtain an indication of the number of spiramycin produced by this strain. Different strains described in the previous examples, were used in this test to test the production of their spiramycin. The results obtained are presented in table 38.

Table 38
StrainAn inactivated geneThe example, which describes the strainPhenotype: the producer (+) or reproduced (-) spiramycin
ATCC23877No1(+)
OS49.16orf3::the Ωhyg2(-)
OS49.67orf3 deletion in the box6(-)
OS49.107orf8::the Ωhyg7(-)
OS49.50orf10:: Ωhyg8(-)
OSC2No10(+)
SPM21orf2::att3Ωaac-10(-)
SPM22orf2::att3 deletion in the box10 (-)
SPM501orf6*::att1Ωhyg+14(-)
SPM502orf6*::att1 deletion in the box14(+)
SPM507orf12::att3Ωaac-11(-)
SPM508orf13c::att3Ωaac-12(+)
SPM509orf14::att3Ωaac-13(-)

These results allow to draw some conclusions in regard to the functions of the various genes involved in the biosynthesis of spiramycin. So, orf3 gene is essential in the biosynthesis of spiramycin. In fact, inactivation in the frame read in this gene leads to strain OS49.67 (see example 6), not producing more spiramycin. Inactivation in the frame Schetyna allows to reject the hypothesis about the possible impact of the embedded cluster on the expression of genes located below orf3.

Similarly, genes orf8 and orf10 encode proteins essential in the biosynthesis of spiramycin, since the strains OS49.107 and OS49.50 have reproducethe f is notion. In addition, in these two latter strains really happening inactivation of the corresponding gene, which is responsible for this nepreodolenny phenotype, because, from the point of view of orientation different reading frames (see figure 3), introduced the design may not have a polar effect.

The study of strains with excisional cluster, also allows to make some conclusions regarding the function of the interrupted gene. Strain SPM507 has the genotype orf12::att3Ωaac-. Unnecessary is the implementation of the excision of the cluster to study the effect of inactivation of orf12 from the point of view of the orientation of the genes (see figure 3). The fact that orf13c is oriented in the opposite direction orf12 gene, indicates that these genes are not cotranscriptional. Use excisional cluster, instead, allows to be able at any time to remove the marker selection. The phenotype of strain SPM507 is not a producer, therefore, we can conclude that orf12 gene is a gene essential in the biosynthesis of spiramycin in the case of S. ambofaciens.

Strain SPM508 has the genotype orf13c::att3Ωaac-. Unnecessary is the implementation of the excision of the cluster to study the effect of inactivation orf13c from the point of view of the orientation of the genes (see figure 3). The fact that orf14 is oriented in the direction opposite gene orf13c, shows that these GE is s are not cotranscriptional. Use excisional cluster, instead, allows to be able at any time to remove the marker selection. The phenotype of strain SPM508 is the producer, therefore, we can conclude that the gene orf13c genome is not essential in the biosynthesis of spiramycin in the case of S. ambofaciens.

Strain SPM509 has the genotype orf14::att3Ωaac-. Unnecessary is the implementation of the excision of the cluster to study the effect of inactivation of orf14 from the point of view of the orientation of the genes (see figure 3). The fact that orf15c is oriented in the opposite direction orf14 gene, indicates that these genes are not cotranscriptional. Use excisional cluster, instead, allows to be able at any time to remove the marker selection. The phenotype of strain SPM509 is not a producer, therefore, we can conclude that orf14 gene is a gene essential in the biosynthesis of spiramycin in the case of S. ambofaciens.

Strain SPM21 has the genotype orf2::att3Ωaac-. The phenotype of this strain is not a producer of spiramycin. However, the orientation of the genes orf1 - orf8 (see figure 3) suggesting that these genes are cotranscriptional. Thus, the observed phenotype may occur due to polar effects of the cluster embedded in orf2, on the expression of genes further downstream in the operon. Strain SPM22 has the genotype orf2::att3 and was obtained after excision of the frame please take the of the embedded cluster. As a result of excision of the cluster remains the only characteristic “scar” sequence (see example 10). In the case of strain SPM22, also with non-producer phenotype, we can conclude that the orf2 gene is a gene essential in the biosynthesis of spiramycin in S. ambofaciens. In this case, see only the effect caused by inactivation of orf2.

Strain SPM501 has the genotype orf6*::att3Ωhyg+. The phenotype of this strain is not a producer of spiramycin. However, as orf5 genes* and orf6* (see figure 3) have the same orientation, the observed phenotype may occur due to polar effects of the built-in orf6* cluster on the expression of orf5*. The design of these genes suggesting that they may be cotranscriptional. To answer this question, the strain SPM502 received after the excision of the reading frames of the embedded cluster. In this strain only see the effect of inactivation of orf6*. This strain has the genotype orf6*::att1 (see example 14). As a result of excision of the cluster in the frame read-only characteristic “scar” sequence (see example 14). Strain SPM502 has the phenotype-producer (this strain, however, produces only spiramycin I (see example 16)). Therefore, we can conclude that the orf5 gene* is a gene essential in the biosynthesis of spiramycin in S. ambofaciens, as its indirect inactive the Oia in strain SPM501 leads to the phenotype, non-producer. On the contrary, orf6 gene* is not a gene that is essential in the biosynthesis of spiramycin I in S. ambofaciens (on the contrary, it is essential in the production of spiramycin II and III (see example 16)).

Example 16

Quantitative determination producing spiramycin I, II and III in the resulting mutant strains

Various test strains were cultured every 7 Erlenmeyer flasks with baffles with a capacity of 500 ml, containing 70 ml of medium MP5 (Pernodet and others, 1993). The Erlenmeyer flask was inoculable using a 2.5·106spores/ml of different strains of S. ambofaciens and cultivated at temerature 27°C With orbital agitation of 250 rpm for 72 hours. Culture, corresponding to one and the same clone, combined, if necessary, filtered through a folded filter and centrifuged for 15 minutes at a speed of 7000 rpm./minutes Different supernatant then kept at a temperature of -30°C.

The determination was carried out by high performance liquid chromatography (HPLC) for the selection of ion pairs. Analysis by HPLC culture medium allows you to accurately determine the concentration of the three forms of spiramycin. Used column (Macherey-Nagel) completed phase Nucleosil (silica with grafted aktalnye groups). The grading is 5 microns and the pore size has a value of 100 Å. The internal diameter is of alanki equal to 4.6 mm and its length is 25 cm Mobile phase buffer is a mixture of N3RHO4(pH=2,2) and acetonitrile in the ratio of 70:30 (by volume)containing 6.25 g/l of sodium perchlorate (NaClO4). The analysis was carried out in isocratic mode with a flow rate fixed at 1 ml/min the Column was thermoregulatory at a temperature of 23°C. Detection was provided by UV spectrometry at a wavelength of 238 nm. The sample was cooled to a temperature of +10° and quantitative determination was carried out based on the peak areas (internal calibration). Under these conditions, the retention times of spiramycin I, II and III are, respectively, about 17, 21 and 30 minutes, as this was confirmed when using the sales model, including three forms of spiramycin.

The strain OSC2 has producenti phenotype of spiramycin. This parent strain used to obtain strains with excisional cluster (see example 15). This strain is, therefore, used as a positive control for the production of three forms of spiramycin. This strain really produces three forms of spiramycine, as was confirmed by HPLC (see Fig).

The study strains with excisional cluster, allows to draw a number of conclusions regarding the function of the interrupted gene. Strain SPM507 has the genotype orf2::att3Ωaac-. The phenotype of strain SPM507 Pawleys producer (see example 15)therefore, we can conclude that orf12 gene is a gene essential in the biosynthesis of spiramycin in the case of S. ambofaciens. This strain does not produce more spiramycin, as confirmed by HPLC (see Fig). This result confirms the significant orf12 gene in the biosynthesis of spiramycin.

Strain SPM508 has the genotype orf3::att3Ωaac-. The phenotype of strain SPM508 is the producer of spiramycin (see example 15)therefore, we can conclude that the gene orf13c genome is not essential in the biosynthesis of spiramycin in the case of S. ambofaciens. This strain produces spiramycin I, II and III, as confirmed by HPLC (see Fig). This result confirms that the gene orf13c genome is not essential in the biosynthesis of spiramycin I, II and III in the case of S. ambofaciens.

Strain SPM509 has the genotype orf4::att3Ωaac-. The phenotype of strain SPM509 is not a producer, therefore, we can conclude that orf14 gene is a gene essential in the biosynthesis of spiramycin in the case of S. ambofaciens. This strain does not produce more spiramycine, as confirmed by HPLC (see Fig). This result confirms the materiality orf14 gene in the biosynthesis of spiramycin.

Strain SPM501 has the genotype orf6*::att3Ωhyg+. The phenotype of this strain is not a producer of spiramycin. This strain does not produce more spiramycine, as confirmed by HPLC (see Fig). However, as orf5 genes* and orf6* (see figure 3) have the same orientation, the observed phenotype may be caused due to polar effects of the cluster embedded in orf6*, on the expression of orf5* in the operon. This leads us to conclude that these genes are cotranscriptional. To answer this question, the strain SPM502 was obtained by excision of the embedded cluster, leading to deletions in the reading frame orf6 gene* and restores the expression of orf5*. This strain has the genotype orf6*::att1 (see examples 14 and 15). As a result of excision of the cluster in the frame read only “scar” sequence att (see example 14). Strain SPM502 has producenti genotype spiramycin. However, as detected by HPLC, this strain produces only spiramycin I and does not produce spiramycin II and III (see Fig). Therefore, from these results we can conclude that the orf5 gene is a gene essential in the biosynthesis of spiramycin in the case of S. ambofaciens, because of its indirect inactivation in strain SPM501 leads to a phenotype that is not the producer of spiramycin (see Fig). On the contrary, orf6 gene* genome is not essential in the biosynthesis of spiramycin I in the case of S. ambofaciens, because inactivation of this gene leads to a phenotype, which is the producer of spiramycin I (see Fig). However, orf6* is essential in the production of spiramycin II and III (see example 16). Orf6 gene*therefore, indeed encodes an acyltransferase, from estwennu for modification platinoid in position 3 (see figure 1).

Example 17

Determination of the site of translation initiation orf10 and increase production of spiramycin

17.1. Construction of plasmids pSPM523, pSPM524 and pSPM525

Gene prf10 was identified in Streptomyces ambofaciens and named srmR according Geistlich and others (Geistlich, M. and others, 1992). Was made inactivation orf10 gene (see example 8). It has been shown that the resulting strain does not produce more spiramycin (see example 15). This confirms that orf10 gene is indeed involved in the biosynthesis of spiramycin. The protein encoded by this gene, therefore, is in fact essential in the biosynthesis of spiramycin. However, sequence analysis shows that for broadcast orf10 (see Fig) can be used two ATG codon located in the same reading frame. One of the two possible codons (above all) begins with the provisions 10656 sequence of representations in the form of SEQ ID No. 1, while the other possible codon, below, begins with the provisions 10809 (see SEQ ID No. 1). To test the possible impact of overproduction srmR on the production of spiramycin it is important to determine, firstly, the site of translation initiation.

To determine the site of translation initiation were implemented in three designs, including three forms of orf10. These latter were obtained by PCR with ISOE is lovanii oligonucleotides, including any restriction site HindIII, or the restriction site BamHI.

The first pair of primers used for amplification, the following oligonucleotides:

EDR39 : 5'CCCAAGCTTGAGAAGGGAGCGGACATTCATGGCCCGCGCCGAACGC3' (SEQ ID No. 122) (websiteHindIII in bold);

EDR42 : 5'CGGGATCCGGCTGACCATGGGAGACGGGCGCATCGCCGAGTTCAGC3' (SEQ ID No. 123) (websiteBamHI highlighted in bold).

Primer pair EDR39-EDR42 allows the amplification of the fragment of orf10, including ATG, located mostly in the 3' (position 10809 in the sequence presented as SEQ IDF No. 1) (see Fig). The resulting fragment has a size of about 2 and the so-called later to be called “short orf10”, it does not contain a promoter orf10. This fragment of length 2 so-called cloned in the vector pGEM-T easy to obtain plasmids pSPM520.

The second pair of primers used for amplification, the following oligonucleotides:

EDR40 :

5'CCCAAGCTTGAGAAGGGAGCGGACATTCAATGCTTTGGTAAAGCAC3' (SEQ ID No. 124) (websiteHindIII in bold);

EDR42 : 5'CGGGATCCGGCTGACCATGGGAGACGGGCGCATCGCCGAGTTCAGC3' (SEQ ID No. 123) (websiteBamHI highlighted in bold).

Primer pair EDR40-EDR42 allows the amplification of the fragment of orf10, including ATG, located mostly in the 5' position 10656 in the sequence presented as SEQ IDF No. 1) (see Fig). This fragment with a length of 2.2 so-called, referred to subsequently as “long orf0”, cloned in the vector pGEM-T easy to obtain plasmids pSPM521.

The third pair of primers used for amplification, the following oligonucleotides:

EDR41 :

5'-CCCAAGCTTTCAAGGAACGACGGGGTGGTCAGTCAAGT-3' (SEQ ID N° 125) (websiteHindIII in bold);

EDR42 :

5'CGGGATCCGGCTGACCATGGGAGACGGGCGCATCGCCGAGTTCAGC-3' (SEQ ID N° 123) (websiteBamHI highlighted in bold).

Primer pair EDR41-EDR42 allows the amplification of the fragment of orf10 with two ATG, as well as with its own promoter (see Fig). The fragment length of 2.8 so-called, referred to subsequently as “orf10 pro”, cloned in the vector pGEM-T easy to obtain plasmids pSPM522.

Slice orf10 pro” was obtained when using as the template the chromosomal DNA of the strain OSC2. Slices short orf10 and long orf10”, in regard to them, was received when using as a template DNA fragment “orf10 pro”pre-cleared.

Insert HindIII-BamHI plasmid pSPM520, pSPM521 and pSPM522 then was subcloned into the vector pUWL201 (a plasmid derived from plasmid pUWL199 (Wehmeier U.F., 1995), which was embedded fragment Cloned-BamHI site of the ermE promoter (see Bibb and others, 1985, especially figure 2), which has a mutation that increases the “strength” of the promoter (promoter ermE*) (Bibb and others, 1994); (see Doumith and others, 2000)), pre restrictionon enzymes HindIII-BamHI. Thus, we have obtained three plasmids: pSPM523 (occurring about the pUWL201 form short orf10 as inserts) pSPM524 (derived from pUWL201 form long orf10” as an insert) and pSPM525 (derived from pUWL201 form orf10 pro”) (Fig).

17.2. Transformation of strain OS49.50 c constructs pSPM523, pSPM524 and pSPM525

Strain OS49.50 (strain, interrupted in the orf10 gene, see example 8) independently transformed by transformation of protoplasts (Kieser, T. and others, 2000) of each of the plasmids pSPM523, pSPM524 and pSPM525. Also implemented the negative control, transforming the strain OS49.50 using plasmids pUWL201 without insert. After transformation of protoplasts clones selected for resistance to thiostrepton. Transformation of clones with each of the plasmids was confirmed by extraction of these plasmids. Thus, we have obtained four new strain: strain OSC49.50 pUWL201, derived from transformation with the plasmid pUWL201 without insert; strain OSC49.50 pSPM523, derived from transformation with the plasmid pSPM523; strain OSC49.50 pSPM524, derived from transformation with the plasmid pSPM524; and, finally, the strain OSC49.50 pSPM525, derived from transformation with the plasmid pSPM525.

The production of spiramycin each of these four strains were tested using HPLC. Different tested strains of S. ambofaciens were cultured in Erlenmeyer flasks with baffles with a capacity of 500 ml, containing 70 ml of medium MP5 (Pernodet and others, 1993). When the strain contains a plasmid pUWL201 or the bottom of its derivatives, added 5 μg/ml of thiostrepton. The Erlenmeyer flask with baffles was inoculable when the initial concentration of 2.5·106spores/ml of different strains of S. ambofaciens and the culture is incubated at a temperature of 27°C With orbital agitation of 250 rpm for 96 hours. The cells were then separated from the medium by centrifugation and the supernatant was analyzed by HPLC (see example 16) to determine the amount of biogas produced spiramycin. With the help of a reference sample and by opredeleniya the peak areas it was possible to determine the number of each spiramycine produced by these strains. The results of this analysis are presented in table 39, the data are expressed in mg per liter of supernatant. The results represent the total production of spiramycin (obtained by summing the production of spiramycin I, II and III).

Table 39
The production of spiramycin strains originating from pOS49.50 (results expressed in mg/l)
StrainThe production of spiramycin
OS49.50 pUWL2010
OS49.50 pSPM5230
OS49.50 pSPM524 93
OS49.50 pSPM525149

As the results, shown in table 39, the negative control (strain OS49.50 transformed by the plasmid pUWL201) do not provide spiramycin. When the strain OS4.50 introduced plasmid pSPM523 (which contains the form of “short orf10”), no production of spiramycin not see. Instead, the presence of plasmids pSPM524 (which contains the form of “long orf10) and plasmids pSPM525 (which contains the form “orf10 pro”) restores the production of spiramycin in the strain-host OS49.50. Thus, only fragments of orf10, containing situated entirely above ATG, allow you to restore the synthesis of spiramycin.

To confirm these results, plasmid pSPM521 (plasmid pGEM-T easy containing the form of “long orf10”) was restrictively using the restriction enzyme XhoI (this enzyme possesses a single site in this plasmid, localized between the two ATG (see Fig)). The ends of the XhoI then released by treatment with enzyme maple. The plasmid is then “closed” on itself through action of T4 DNA ligase to obtain the plasmid pSPM527. If ATG located entirely above (position 10656 in the sequence presented as SEQ ID No. 1), is actually used as a site of translation initiation, that of the development will cause an open reading frame at the level of the XhoI site and have the consequence of producing the protein, not possessing activating activity. On the contrary, if the initiation of translation occurs at the level of the ATG located completely below (position 10809 in the sequence presented as SEQ ID No. 1), this treatment will have little or no impact on the expression of Orf10 (taking into account the localization of the site of transcription initiation) and will have no impact on the produced protein.

The insertion of a BamHI-HimdIII pSPM527 then was subcloned into the vector pUWL201 to obtain plasmids pSPM528. This plasmid was integrated into strain OS49.50 and in particular selected clone having the desired plasmid. The production of spiramycin the resulting strain was then determined by HPLC (see example 16 above). In contrast to what was observed in the case of plasmids pSPM524 (see table 39), the presence of plasmids pSPM528 in strain OS49.50 does not restore the production of spiramycin. This confirms that the beginning of a broadcast orf10 gene ATG is located completely below (ATG 1, see Fig).

17.3. Increased production of spiramycin strain OSC2 S. ambofaciens

To test the impact of overproduction orf10 gene on the production of spiramycin, plasmids pSPM523, pSPM524 pSPM525 and pSPM528 was introduced into the strain OSC2. For this purpose, the protoplasts of the strain OSC2 transformed (Kieser, T. and others, 2000), independently, with each of plasmi the pSPM523, pSPM524 pSPM525 and pSPM528. Has also been implemented negative control by transformation of the strain OSC2 using plasmids pUWL201 without insert. After transformation of protoplasts clones selected for resistance to thiostrepton. Thus, we have obtained five new strains:the strain OSC2 pUWL201, derived from transformation with the plasmid pUWL201 without insert; the strain OSC2 pSPM523, derived from transformation with the plasmid pSPM523; strain OSC2 pSPM524, derived from transformation with the plasmid pSPM524; strain OSC2 pSPM525, derived from transformation with the plasmid pSPM525; and, finally, the strain OSC2 pSPM528, derived from transformation with the plasmid pSPM528. Then by HPLC analyzed the production of spiramycin these strains (in the same way as in the example 17.2). Also for comparison was also carried out analysis of the production of spiramycin strain OSC2. The results of this analysis are presented in table 40; data are expressed in mg per liter of supernatant. The results represent the total production of spiramycin (obtained by summing the production of spiramycin I, II and III).

Table 40
The production of spiramycin strains originating from OSC2 (results expressed in mg/l)
StrainThe production of spiramycin
OSC269
OSC2 pUWL201103
OSC2 pSPM52319
OSC2 pSPM524135
OSC2 pSPM525278
OSC2 pSPM52868

Thus, it was observed that the presence of plasmids pSPM524 or pSPM525 significantly increases the production of spiramycin strain OSC2. It really shows that overproduction Orf10 has a positive effect on the production of spiramycin. Plasmid pSPM528, by contrast, has no effect on the production of spiramycin.

Plasmids pSPM525 and pUWL201 were similarly introduced into the strain SPM502 (see example 14). Thus, we have obtained two new shitama: strain SPM502pUWL201, derived from transformation with the plasmid pUWL201 no insert, and the strain PSM502pSPM525, derived from transformation with the plasmid pSPM525.

The sample strain SPM502 pSPM525 (this strain contains the plasmid pSPM525, see above) was deposited in the National collection of microorganism cultures (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex15, France, 26 February 2003 under registration number I-2977.

The production of spiramycin strains SPM502pUWL201 and SPM502pSPM525 analyzed by HPLC (the same way as in the example 17.2). Also for comparison in parallel was the analysis of the production of spiramycin strain SPM502. The results of these analyses are presented in table 42; data are expressed in mg per liter of supernatant. The results correspond to the production of spiramycin I. In fact, none of these strains do not produce spiramycin II and III.

Table 41
The production of spiramycin I strains originating from SPM502 (results expressed in mg/l)
StrainSpiramycin I
SPM50247
SPM502 pUWL20172
SPM502 pSPM525130

Thus, we discovered that overproduction orf10 gene in strain SPM502 significantly increases the production of spiramycin I.

Example 18

Construction Bank genomic DNA of the strain OSC2 Streptomyces ambofaciens in E. coli in cosmides pWED2

18.1. The design of Comedy pWED2

Since the goal is to facilitate inactivation of genes in Streptomyces designed kosmidou, bearing sequence oriT plasmids RK2 (which allows its introduction by conjugation in Streptomyces on the basis of an appropriate strain of E. coli) and also containing the gene for resistance to the antibiotic, giving Streptomyces detectable phenotype. This cosmid containing large insert genomic DNA of Streptomyces ambofaciens, can be used in experiments on the inactivation of genes.

To construct this vector, the pac cluster-oriT (EcoRV fragment) was built in kosmidou pWEDl (Gourmelen and others, 1998), derived from Comedy pWED15 (Wahl and others, 1987), at the level of single HpaI site. The cluster pac-oriT was obtained by PCR. For this RAS gene amplified by PCR from the plasmid pVF 10,4 (Vara and others, 1985; Lacalle and others, 1989), using, as the first primer, primer (sequence 5'-CCAGTAGATATCCCGCCAACCCGGAGCTGCAC-3' (SEQ ID No. 126), where the EcoRV restriction site is underlined and in bold sequence of 20 nucleotides corresponds to the region above the promoter of the gene RAS) and, as the second primer, primer (sequence 5'-GAAAAGATCCGTCATGGGGTCGTGCGCTCCTT-3' (SEQ ID No. 127), which includes at its 5' end of the sequence of 12 nucleotides, corresponding to the beginning of the oriT sequence (double underlined), and a sequence of 20 nucleotides (in bold)corresponding to the end of the RAS gene (see Fig, the first PCR).

What kasambagan oriT, his amplified by PCR from the plasmid pPM803 (Mazodier, P. and others, 1989), using, as the first primer, primer (sequence 5'-CACGACCCCATGACGGATCTTTTCCGCTGCAT-3' (SEQ ID No. 128), which includes at its 5' end of the sequence of 12 nucleotides corresponding to the sequence below the coding sequence of the gene of races (in bold), and a sequence of 20 nucleotides corresponding to the beginning of the oriT sequence, and, as the second primer, primer D (5'sequence-GAGCCGGATATCATCGGTCTTGCCTTGCTCGT-3' (SEQ ID No. 129)), which includes the EcoRV restriction site (underlined with a single line) and a sequence of 20 nucleotides corresponding to the end of the oriT sequence (underlined by a double line) (see Fig, the second PCR).

The amplification product obtained with primers a and b, and that obtained with primers C and D, at one of its ends have a common sequence of 24 nucleotides. Was carried out by a third PCR, mixing both amplification product obtained above, and using as primers the primers a and D (see Fig, the third PCR). It is possible to obtain an amplification product corresponding to the combination of races+oriT. This fragment of RAS oriT then cloned in the vector pGEM-T Easy (produced by the company Promega (Madison, Wisconsin, USA), which allowed to obtain the plasmid pGEM-T-pac-oriT. The insert of this plasmid is then cloned in cosmides pWED1. This plasmid pGEM-T-pac-oriT was restrictively using enzyme EcoRV and EcoRV insert that contains a set of races+oriT, was built in kosmidou pWED1 previously subjected to restriction analysis with the enzyme HpaI. Thus obtained cosmid was named pWED2 (see Fig).

This cosmid facilitates the inactivation of genes in Streptomyces. In fact, it contains the oriT sequence (which allows her to enter by conjugation in Streptomyces on the basis of an appropriate strain of E. coli), but also contains the gene for resistance to the antibiotic, giving Streptomyces detectable phenotype. This cosmid containing large insert genomic DNA of Streptomyces ambofaciens, can be used in experiments on inactivation of genes.

So, cosmid originating from pWED2 containing gene target may be, for example, introduced into the strain E. coli KA272 containing plasmid pKOBEG (see Chaveroche and others, 2000), and the cluster can be introduced into the gene target according to the technology described Chaveroche and others (2000). Obtained in this way, cosmid (cosmid, in which gene target inactivated) can then be introduced into a strain of E. coli, such as strain S17.1, or any other strain, allowing the transfer by conjugation of plasmids containing the oriT sequence, in Streptomyces.

After conjugation between E. coli and Streptomyces, clones Streptomyces, in which the original copy of the target genes should be replaced prerana the copy, can be obtained as described in example 2.

Gene resistance, expressed in Streptomyces, which is available in this new cosmides, is a RAS gene of Streptomyces alboniger (Vara J. and others, 1985; Lacalle and others, 1989), which encodes puromycin-N-acetyltransferase and which imparts resistance to puromycin. During experiments on the inactivation of a gene are looking for the clones, which was double the phenomenon of recombination. If one of these clones to remove kosmidou pWED2, consequently, they will again become sensitive to puromycin.

18.2. Construction Bank genomic DNA of the strain OSC2 Streptomyces ambofaciens in E. coli in cosmides pWED2

Genomic DNA of the strain OSC2 Streptomyces ambofaciens was partially restrictively using the restriction enzyme BamHI to obtain DNA fragments ranging in size from approximately 35 so-called to 45 called These fragments are then cloned in cosmides pWED2, and the latter has been previously subjected to restriction analysis using BamHI, and then was treated with alkaline phosphatase. The mixture after ligating then uncapsizable in vitro into particles of phage lambda using system Packagene®Lambda DNA packaging system”manufactured by Promega (Madison, Wisconsin, USA)following the manufacturer's recommendations. The obtained phage particles were used to infect strain SURE®E.coli produced by the company Stratagene (LaJolla, CA, USA). The selection of clones was carried out with the food LB + ampicillin (50 μg/ml), cosmid pWED2 gives resistance to ampicillin.

Example 19

The selection of cosmid new Bank, covering the area of the biosynthesis pathway of spiramycin. Subclavian and sequencing of these fragments of cosmid

19.1. Hybridization in the colonies genomic Bank of Streptomyces ambofaciens OSC2

Allocated Comedy new Bank Streptomyces ambofaciens OSC2 (see example 18), covering the area from orf1* to orf10* or part or all of the area from orf1 to orf25c or large area above orf25c. This was carried out by sequential hybridization in colonies of E. coli, obtained according to example 18, using the following 3 probe (see Fig):

The first used the probe corresponds to a DNA fragment with a length of about 0.8 called, amplificatoare by PCR using as template Comedy pSPM5 and the following primers:

ORF23c : 5'-ACGTGCGCGGTGAGTTCGCCGTTGC-3' (SEQ ID No. 130), and

ORF25c : 5'-CTGAACGACGCCATCGCGGTGGTGC-3' (SEQ ID No. 131).

The thus obtained PCR product contains a fragment of the beginning orf23c, fully orf24c and end orf25c (see Fig, probe I).

The second used the probe corresponds to a DNA fragment with a length of approximately 0.7 called, amplificatoare by PCR using as template the complete DNA of a strain of S. ambofaciens ATSS and the following primers:

ORF1*c : 5'-GACCACCTCGAACCGTCCGGCGTCA-3' (SEQ ID No. 132), and

ORF2*c : 5'-GGCCCGGTCCAGCGTGCCGAAGC-3' (SEQ ID no : 133).

The thus obtained PCR product contains f is agment the end of orf1*c and the beginning of orf2*c (see Fig probe II).

The third used the probe corresponds to the fragment EcoRI-BamHI length of about 3 so called, containing orf1, orf2 and orf3 and obtained by the restriction analysis of the plasmids pOS49.99 (see Fig, probe III).

Approximately 2000 clones Bank, obtained according to example 18.2, transferred to a filter for hybridization of colonies according to the classical methods (Sambrook and others, 1989).

The first probe (see Fig, probe I) were labeled with32P according to the method of “arbitrary premirovany” (kit, manufactured by Roche) and used for hybridization 2000 clones of the Bank, after the transfer to the filter. Hybridization was performed at 65°C in the buffer described by Church and Gilbert (Church &Gilbert, 1984). Was carried out by two washing in 2X SSC with 0.1% sodium dodecyl sulfate at 65°, the first one within 10 minutes and the second for 20 minutes, and the third rinsing for 30 minutes then carried out at 0,2X SSC with 0.1% sodium dodecyl sulfate at 65°C. under these conditions, hybridization and washing with 20 clones from 2000 hybridized had a strong hybridization signal with the first probe. These 20 clones were cultured in LB medium + ampicillin (50 μg/ml) and 20 of the respective cosmid were extracted by alkaline lysis (Sambrook and others, 1989), then they were subjected to restriction analysis using the restriction enzyme BamHI. Food restrictions were then separated on agarose gel, transferred to Nylon membrane, gibri who was seravalli with the first probe (see above: the PCR product ORF23c-ORF25c, probe I) in the same conditions as above. Twelve cosmid had a BamHI fragment, highly hybridizers used with the probe. These 12 cosmid were named as pSPM34, pSPM35, pSPM36, pSPM37, pSPM38, pSPM39, pSPM40, pSPM41, pSPM42, pSPM43, pSPM44 and pSPM45. Profiles, after restriction with BamHI, these 12 cosmid were compared between themselves and the profile of Comedy pSPM5. In addition, experiments on the amplification by PCR using different primers corresponding to different genes, have been identified in the region of orf1 - orf25c allowed to set the insertion of some of these cosmid one relative to the other and also to determine the localization of these inserts in the known region of orf1-orf25c (see Fig). In particular was chosen cosmid pSPM36, as it is able to contain a large area above orf25c (see Fig and 32).

Secondly, and using the same conditions as those described above 2000 clones Bank Streptomyces ambofaciens OSC2 were hybridisable with a second probe corresponding to the PCR product ORF1*c-ORF2*c (see Fig probe II). This hybridization allowed us to identify Comedy, the insert which is located in the region of orf1*c - orf10*c. In the conditions of hybridization of 16 clones from 2000 hybridized had a strong hybridization signal with this second probe. These 16 clones were cultured in LB medium+ampicillin (50 μg/ml) and 16 prior of cosmid extra is Aravali by alkaline lysis (Sambrook and others, 1989), and then they were restrictively using the restriction enzyme BamHI. Profiles restriction after restriction with BamHI) of these 16 cosmid were compared between themselves and the profile of Comedy pSPM7. Experiments on the amplification by PCR using primers ORF1*c and ORF2*c allowed to choose Comedy, which really consisted of genes orf1*c and orf2*c and profiles which had common areas, but also different areas. In addition, other experiments on the amplification by the PCR using different primers corresponding to different genes, have been identified in the region of orf1*c - orf10*c, allowed to establish the insertion of these cosmid one relative to the other and to determine the localization of these inserts in the known region of orf1*c - orf10*c (see Fig). Selected features of the two Comedy were named pSPM47 and pSPM48 (see Fig).

Using the same conditions as those described above 2000 clones Bank Streptomyces ambofaciens OSC2 also hybridisable with the third probe corresponding to the DNA fragment EcoRI-BamHI plasmid pOS49.99 (see Fig, probe III). This hybridization allowed us to identify Comedy containing region from orf1 up to orf3 and can contain either a large part of PKS genes, or a large part of the genes orf1 - orf25c biosynthesis pathway of spiramycin. Under these conditions, hybridization of 35 clones from 2000 hybridized had a strong hybridization signal with Tr is Tim probe. These 35 clones were cultured in LB medium+ampicillin (50 μg/ml) and 35 of the respective cosmid were extracted by alkaline lysis (Sambrook and others, 1989), then restrictively using the restriction enzyme BamHI. Profiles, after restriction with BamHI, these 35 cosmid were compared between themselves and the profile of Comedy pSPM5. In addition, experiments on the amplification by PCR using primers corresponding to different genes, have been identified in the region of orf1-orf25c allowed to confirm that Comedy really include inserts originating from the region of orf1-orf25c, and install insert some in relation to others and in relation to the already known areas of orf1-orf25c (see Fig). In particular were selected five cosmid, they were called pSPM50, pSPM51, pSPM53, pSPM55 and pSPM56 (see Fig).

19.2. Subclavian and sequencing of one part of the insertion of Comedy pSPM36

The probe length is about 0.8 called, obtained by PCR with primers ORF23c and ORF25c (see above and Fig, probe I), was also used in experiments by southern-blotting full DNA of S. ambofaciens OSC2, restrictional enzyme > PST. The hybridization conditions described above, this probe has a single > PST fragment length of approximately 6 called when hybridized with full DNA of S. ambofaciens OSC2, restrictional using enzyme > PST. There is one website > PST level orf23c (see SEQ ID No. 80), but no other SAI is > PST and not until the end (BamHI site) known sequence (see SEQ ID NO. 1). This fragment > PST -BamHI has a size of about 1.4 so-called > PST Fragment length 6 so-called, hybridized with the full DNA of S. ambofaciens, restrictional using enzyme > PST, therefore, contains an area with a length of about 4.6 so called, situated above orf25c. This area can contain other genes, which produced the products involved in the biosynthesis pathway of spiramycin. By restriction analysis confirmed that cosmid pSPM36 actually contains the > PST fragment length 6 called This fragment was isolated from pSPM36 to determine the sequence above orf25c. For this kosmidou pSPM36 was restrictively using restriction enzyme > PST. Fragment > PST - > PST length of about 6 so-called allocated by electroelution from 0.8%agarose gel and then cloned in the vector pBK-CMV (4512 base pairs) (produced by the company Stratagene (La Jolla, CA, USA)). The thus obtained plasmid was named pSPM58 (see Fig) and was determined the sequence of the insert. The sequence of this insert presents in the form of sequence SEQ ID No. 134. However, the entire sequence has not been determined and there is a gap length of about 450 nucleotides, uncertain part of the sequence was identified as a sequence of “N” in the appropriate sequence.

19.3. A new analysis of the nucleotide sequences determined is a group of open reading frames and characterization of genes involved in the biosynthesis of spiramycin

The sequence of the insert received Comedy pSPM58 was analyzed using FramePlot program (J. Ishikawa & Hotta K., 1999). This allowed us to identify additional open reading frames open reading frames, which are typical codons Streptomyces. This analysis allowed us to determine that this box includes 4 new open reading frames above orf25c (see Fig). These genes were named orf26 (SEQ ID No. 107), orf27 (SEQ ID no. 109), orf28c (SEQ ID No. 111; the sequence of this open reading frame has not been completely defined, because there is a gap length of about 450 nucleotides during sequencing of the insert pSPM58, and these 450 nucleotides appear in the form of a number “N” in the sequence SEQ ID No. 111) and orf29 (this last sequence open reading frame is incomplete in this box). The letter “C”added to the name of the gene for this open reading frame means that the coding sequence has the opposite orientation (see Fig).

Protein sequence resulting from these open reading frames were compared with those found in different databases, using various programs: BLAST (Altschul and others, 1990; Altschul and others, 1997), CD-search (these tdve programs available, in particular from NCBI (national center for information on bi is technology) (Bethesda, Maryland, USA), FASTA (Pearson W. R. &Lipman, D. J., 1988) and (W. R. Pearson, 1990)) (this program is available, in particular from the resource center INFOBIOGEN, Evry, France). These comparisons allowed us to formulate hypotheses concerning the function produced by these genes products and identify those that may participate in the biosynthesis of spiramycin.

19.4. Subclavian and sequencing other side of the insert of Comedy pSPM36

The probe length is about 0.8 called, obtained by PCR with primers ORF23c and ORF25c (see above and Fig, probe I), was also used in experiments by southern-blotting full DNA of the strain OSC2, restrictional enzyme StuI. The hybridization conditions described above for this probe, this probe has a unique StuI fragment length of about 10 so-called, when hybridized with the full DNA of the strain OSC2, restrictional using enzyme StuI. Given the presence of a single StuI site in orf23c (see SEQ ID No. 80) and location of this site relative to the site > PST, the fragment length of 10 so-called includes the totality vysheskazannogo > PST fragment (insert pSPM58) and allows you to have access to have not studied the field length of about 4 so-called (see Fig). By restriction analysis confirmed that cosmid pSPM36 really contains this StuI fragment length of 10 so-called This fragment was isolated from Comedy pSPM36 to determine the sequence of the end of orf29 and other genes located above orf29. For this kosmidou pSPM36 was restrictively using the restriction enzyme StuI. Fragment StuI-StuI length of about 10 so-called allocated by electroelution from 0.8%agarose gel and then cloned in the vector pBK-CMV (4512 base pairs) (produced by the company Stratagene (La Jolla, CA, USA)). The thus obtained plasmid was named pSPM72 (see Fig). This last then restrictively with EcoRI (EcoRI site in box pSPM58) and HindIII (the HindIII site in the multiple cloning site of the vector, immediately after the StuI site of the end of the insert) (see Fig). The thus obtained DNA fragment EcoRI-HindIII corresponds to the fragment of the insert of plasmid pSPM72 (see Fig) and was cloned in the vector pBC-SK+ (manufactured by a company Stratagene (La Jolla, California, USA), pre-restrictionon using EcoRI and HindIII. The thus obtained plasmid was named pSPM73 and was determined the sequence of the insert. The sequence of this insert presents in the form of sequence SEQ ID No. 135.

The set of sequences of the inserts pSPM58 and pSPM73 presented in the form of sequence SEQ ID No. 106. This sequence starts from site > PST level orf23c (see SEQ ID No. 80) and goes up to the StuI site at the level of orf32c (see Fig). The sequence orf28c (SEQ ID No. 111) is not complete (see example 19.3), an area of approximately 450 nucleotides not sequenced, these 450 nucleotides appear is in the form of a number “N” in the sequence SEQ ID No. 106.

19.5. Analysis of new nucleotide sequences, determination of open reading frames and characterization of genes involved in the biosynthesis of spiramycin

A partial sequence of the insert received Comedy pSPM73 was analyzed using FramePlot program (J. Ishikawa & Hotta K., 1999). This allowed us to identify additional open reading frames open reading frames, which are typical codons Streptomyces. This analysis allowed us to determine that this box includes 4 new open reading frames: a partial and three complete (see Fig). Incomplete open reading frame corresponds to part of the 3' coding sequence orf29, which allowed us to complement the sequence of this gene due to partial sequences of the same open reading frame obtained during the sequencing of the inserts of plasmids pSPM58 (see examples 19.2 and 19.3), the combination of these two sequences thus allows to obtain the full sequence of orf29. Thus, 4 gene were named orf29 (SEQ ID No. 113), orf30c (SEQ ID No. 115), orf31 (SEQ ID No. 118) and orf32c (SEQ ID No. 120). The letter “C”added to the name of the gene for this open reading frame means that the coding sequence has the opposite orientation (see Fig).

Protein sequence resulting from these open amoxicilina (SEQ ID No. 114 to orf29, SEQ ID No. 116 and 117 for orf30c, SEQ ID No. 119 for orf31 and SEQ ID No. 121 for orf32c), were compared with those found in different databases, using various programs: BLAST (Altschul and others, 1990; Altschul and others, 1997), CD-search (these two programs are available, in particular from NCBI (national center for biotechnology information) (Bethesda, Maryland, USA), FASTA (Pearson W. R. &Lipman, D. J., 1988) and (W. R. Pearson, 1990)) (this program is available, in particular from the resource center INFOBIOGEN, Evry, France). These comparisons allowed us to formulate hypotheses concerning the function produced by these genes products and identify those that may participate in the biosynthesis of spiramycin.

19.6. Subclavian and sequencing of the third part of the insertion of Comedy pSPM36

Probe DNA fragment with a length of 0.8 so-called), corresponding to internal sequence orf32c, was obtained by PCR using as template the complete DNA of a strain of Streptomyces ambofaciens and the following primers:

KF36:5'- TTGCCGTAGCCGAGGACCAGCG-3' (SEQ ID No. 151), and

KF37:5'- CACATGGCCCTGGAGGACCCTG-3' (SEQ ID NO 152).

The thus obtained PCR product has internal consistency orf32c. This probe was used in experiments by southern-blot of chromosomal DNA of the strain OSC2 and DNA of Comedy pSPM36, restrictively using enzyme > PST. Using the same hybridization conditions as those described above (see example 19.1), this probe has two Pst fragment length of about 3.4 the so-called and 2.5 so-called, when hybridized with the full DNA of the strain OSC2 and DNA of Comedy pSPM36, restrictionenzyme using enzyme > PST. Given the availability of the site > PST used in the probe, it is possible to explain these results. The first DNA fragment which has a length of about 3.4 so-called, is a fragment, the sequence of which is already fully known. The sequence of fragment length 2.5 so-called known only partially, in the region of 700 base pairs. This fragment was isolated from Comedy pSPM36 to determine the sequence of end orf32c and other genes above this last. For this kosmidou pSPM36 was restrictively using restriction enzyme > PST. Fragment > PST - > PST a length of about 2.5 so-called was isolated by purification from a 0.8%agarose gel and then cloned in the vector pBK-CMV (4518 base pairs) (produced by the company Stratagene (La Jolla, CA, USA)). The thus obtained plasmid was named pSPM79 (see Fig) and was determined the sequence of the insert.

The sequence orf28c (SEQ ID No. 111) is incomplete (see example 19.3). In fact, the region length of about 450 nucleotides has not been determined, these 450 nucleotides appear in the form of a number “N” in the sequence SEQ ID No. 106. The entire sequence as a whole the missing region was identified by re-sequencing of this region. The sequence of the inserts pSPM58 and pSPM73, followed the Sabbath.) was fully defined. Complete sequence of the coding portion orf28c presented in the form of sequence SEQ ID No. 141 and protein, following from this sequence, as the sequence SEQ ID No. 142. The sequence of the insert of plasmid pSPM79 presented in the form of sequence SEQ ID No. 161.

The set of sequences of the inserts pSPM58, pSPM73 and pSPM79 presented in the form of sequence SEQ ID No. 140 (see Fig). This sequence starts from site > PST level orf23c (see SEQ ID No. 80) and goes all the way to customers > PST level orf34c (see Fig).

19.7. Analysis of new nucleotide sequences, determination of open reading frames and characterization of genes that may be involved in the biosynthesis of spiramycin

The sequence of the insert obtained plasmids pSPM79 was analyzed using FramePlot program (J. Ishikawa & Hotta K., 1999). This allowed us to identify additional open reading frames open reading frames, which are typical codons Streptomyces. This analysis allowed us to determine that this box contains 3 open reading frames: two incomplete (orf32c and orf34c) and one full (orf33) (see Fig).

The first incomplete open reading frame corresponds to a portion of 5' coding sequence orf32c. This allowed us to complement the sequence of this gene due to partial placenta is successive to the same open reading frame, obtained during the sequencing of the inserts of plasmids pSPM73 (see examples 19.4 and 19.5), the combination of these two sequences thus allows to obtain the full sequence orf32c (SEQ ID no. 145). The letter “C”added to the name of the gene for this open reading frame shows that the coding sequence is in the reverse orientation (see Fig). The complete open reading frame (ORF) was named orf33 (SEQ ID No. 147). The third ORF was named orf34c (SEQ ID No. 149). The letter “C”added to the name of the gene for this open reading frame shows that the coding sequence is in the reverse orientation (see Fig). Comparisons made between the product of this ORF and data banks suggests that the C-terminal part of this protein is not present in the product of the following nucleotide sequences, and, therefore, that this ORF is longer and extends outside the sequenced region.

Protein sequence resulting from these open reading frames were compared with those found in different databases, using various programs: BLAST (Altschul and others, 1990; Altschul and others, 1997), CD-search (these two programs are available, in particular from NCBI (national center for biotechnology information) (Bethesda, Maryland, USA), FASTA (Pearson W. R. &Lipman, D. J., 1988) and (Pearson W. R., 190)) (this program is available, in particular, the resource center INFOBIOGEN, Evry, France). These comparisons allowed us to formulate hypotheses concerning the function produced by these genes products and identify those that may participate in the biosynthesis of spiramycin.

Example 20

Analysis of the production of mediators of the biosynthesis of spiramycin

20.1. Sample preparation

Various test strains were cultured every 7 Erlenmeyer flasks with baffles with a capacity of 500 ml, containing 70 ml of medium MP5 (Pernodet and others, 1993). The Erlenmeyer flask was inoculable using a 2.5·106spores/ml of different strains of S. ambofaciens and cultivated at temerature 27°C With orbital agitation of 250 rpm for 96 hours. Culture, corresponding to one and the same clone, combined, if necessary, filtered through a folded filter and centrifuged for 15 minutes at a speed of 7000 rpm./minutes

The pH value of the supernatant was then brought to 9 with sodium hydroxide solution and the supernatant was extracted using methylisobutylketone (MIBK). The organic phase (MIBK) was then recuperable and evaporated. The dry extract was then treated with 1 ml of acetonitrile, and then diluted in the ratio 1:10 (100 µl to 1 ml with water) before use for analysis by liquid chromatography in combination with mass-SP is chromaturia (CL/SM).

20.2. Analysis of the samples by CL/SM

The samples were analyzed by CL/SM order to determine the mass of various products synthesized by the tested strains.

Used column for high performance liquid chromatography (HPLC) is the column Kromasil C8 150* with an inner diameter of 4.6 mm; grain size distribution is 5 microns and a pore size of 100 E.

Mobile phase is a gradient formed by a mixture of acetonitrile and 0.05%aqueous solution triperoxonane acid, the flow rate is fixed at 1 ml/min column Temperature was maintained at 30°C.

UV-detection at the outlet of the column was carried out at two different wavelength: 238 nm and 280 nm.

Mass spectrometer, coupled with a chromatographic column, is a Simple device Quadripole manufactured by Agilent, with a cone voltage of 30 volts and 70 volts.

20.3. Analysis of the biosynthesis of mediators produced by strain OS49.67

Strain OS49.67 in which orf3 gene inactivated by deletions in the frame read, does not produce spiramycin (see examples 6 and 15).

The sample prepared by the method described above (see paragraph 20.1) and analyzed using CL/SM as described above (see section 20.2).

More specifically, the analysis by chromatography was performed using a gradient solvent as a mobile phase: 20% acetonitrile in time is I T=0 to 5 minutes, then a linear increase up to 30% until T=35 minutes, followed by a period of stability until T=50 minutes.

In these conditions, particularly watched two products: absorption at 238 nm (retention time of 33.4 minutes) and absorbing at 280 nm (retention time 44.8 minutes) (see Fig). On Fig presents a superposition of HPLC chromatograms obtained at 238 nm and 280 nm (top)and UV spectra of molecules, buervenich when the 33.4 minutes and 44.8 minutes (below).

Conditions analysis using associated with HPLC mass spectrometry were as follows: the search was carried out by scanning, covering a range of masses ranging from 100 Da to 1000 Da. Increase electromedical was 1 volt. As for the source electronic spraying, pressure spraying gas was 35 pounds/inch2consumption of dry gas was 12.0 l/min-1the temperature of the dry gas was 350°C, capillary voltage was increased to ± 3000 volts. These experiments allowed us to determine the mass of two separate products. These masses are, respectively, 370 g/mol for a product lirovannomu first ([M-H2O]+=353, the main product) and 368 g/mol for the second product ([M-H2O]+=351, the main product).

To define the structure of the above products were isolated and purified under the following conditions: moveable the second phase is a mixture of 0.05%aqueous solution triperoxonane acid and acetonitrile in the ratio of 70:30 (V/V). Chromatography was carried out in isocratic mode with flow rate, fixed at 1 ml/min under these conditions, the retention times of 2 products are, respectively, 8 minutes and 13.3 minutes. In addition, in this case, prigotovlennyi sample (see paragraph 20.1) was diluted in water prior to injection of 10 µl.

2 Product was recuperable at the outlet of the chromatographic column and allocated in the following conditions: the Oasis HLB cartridge, 1 cm3, 30 mg (Waters)was kondicionirovanie sequentially with 1 ml of acetonitrile, and then 1 ml of a mixture of water and acetonitrile (20 rpm./about 80.) and 1 ml of a mixture of water and acetonitrile in the ratio of 80:20. Then entered the sample and the cartridge then washed with 1 ml of a mixture of water and acetonitrile (95:5), 1 ml of a mixture of water and deuterated acetonitrile (95:5), then suirable with 600 μl of a mixture of water and deuterated acetonitrile in the ratio of 40:60. The recovered solution is then directly analyzed using NMR.

The NMR spectra obtained for these two compounds are the following:

first suirvey product: platinoid And (range 9646V)

Range1N CD3CN (chemical shifts in ppm): from 0.90 (3H, t, J=6 Hz), of 0.93 (3H, d, J=5 Hz), of 1.27 (3H, d, J=5 Hz), between of 1.27 and 1.40 (3H, m)and 1.51 (1H, m), of 1.95 (1H, m), 2,12 (1H, m), 2,30 (1H, d, J=12 Hz), 2,50 (1H, d, J=11 Hz), 2,58 (1H, DD, J=9 and 12 Hz), 2,96 (1H, d, J=7 Hz), 3.43 points (3H, s), 3,70 (1H, d, J=9 Hz), 3,86 (1H, d, J=7 Hz), 4,10 (1H, m)5,08 (1H, m), to 5.58 (1H, dt, J=3 and 12 Hz), 5,70 (1H, DD, J=8 and 12 Hz), equal to 6.05 (1H, DD, J=9 and 12 Hz), 6,24 (1H, DD, J=9 and 12 Hz).

second suirvey product: platinoid (range 9647V)

Range1N CD3CN (chemical shifts in ppm): 0,81 (3H, t, J=6 Hz), 0,89 (1H, m)of 1.17 (3H, d, J=5 Hz), of 1.30 (4H, m)of 1.47 (2H, m)to 1.61 (1H, t, J=10 Hz), measuring 2.20 (1H, m), of 2.38 (1H, d, J=13 Hz), 2,52 (1H, m), 2,58 (1H, m), 2,68 (1H, DD, J=8 and 13 Hz), 3,10 (1H, d, J=7 Hz), 3,50 (3H, s), 3,61 (1H, d, J=8 Hz), 3,82 (1H, d, J=7 Hz), 5,09 (1H, m), of 6.20 (1H, m), and 6.25 (1H, DD, J=9 and 12 Hz), to 6.58 (1H, d, J=12 Hz), 7,19 (1H, DD, J=9 and 12 Hz).

These experiments, therefore, allowed to determine the structure of these two compounds. First elyuirovaniya product is platinoid and second - platinoid In; set the structure of these two molecules presented on Fig.

Also, using the method CL/SM, associated with NMR, under conditions slightly different from those described above, but the establishment of which is well known to the specialist, it is determined that the strain OS49.67 produces, besides platanoides a and b, derived from these two compounds. We are talking about connections platinoid And + mycarose and platinoid In + mycarose (the structure of these two compounds presented on Fig). The results of the analysis of the supernatant of strain OS49/67 presented in table 42.

Table 42
The results of the analysis of the CL/SM supernatant of strain OS49.67
Identity[ ] (mg/l)Mass ionsThe maximum absorption
Platinoid A
Exact mass=370
Molecular formula=C20H34O6
16,1[M+Na]+
393,0
[M+K]+
408,9
[M-H2O+H]+
353,0
[2M+Na]+
763,2
231 nm
Platinoid B
Exact mass=368
Molecular formula=C20H32O6
1,4[M-H2O+H]+
351,0
[M+Na]+
391,0
[M+K]+
406,9
[2M+Na]+
759,1
283 nm
Platinoid A + 'mycarose'
Exact mass=514
Molecular formula=C27H46O9
4,27[M+Na]+
537,0
[M+K]+
553,0
230 nm
Platinoid B + 'mycarose'
Exact mass=512
Molecular formula=C27H44O9
Not defined[M+Na]+
535,0
[M+K]+550,9
[PlatB-H2O+H]+350,9
284 nm

20.4. Analysis of mediators of biosynthesis, produciruemihkh SPM509

Strain SPM509 in which orf14 gene inactivated (orf14::attΩaac-), does not produce spiramycin (see examples 13, 15 and 16). The sample was obtained by the method described above (see paragraph 20.1) and analyzed by CL/SM, as described above (see paragraphs 20.2 and 20.3). Analysis of the biosynthesis of mediators present in the culture supernatant of strain SPM509, cultured in the medium MP5, showed that this strain produces only form In platinoid (“platinoid”, see Fig), but not the form A (“platinoid And see Fig).

Example 21

Interrupt orf14 gene in strain, interrupted in the orf3 gene (OS49.67)

Produced orf14 gene product is essential in the biosynthesis of spiramycin (see examples 13, 15 and 16: the strain SPM509 in which this gene is interrupted, does not produce more spiramycine). Analysis of the biosynthesis of mediators present in the culture supernatant of strain SPM509, cultured in the medium MP5, showed that this strain produces only form In platinoid, but not the form (see example 20). One hypothesis that may explain this observation is that the product orf14 is involved in the conversion form platinoid in the form And by enzymatic stage of recovery. To test this hypothesis, orf14 gene was inactivated with the formation of a mutant, not producing more spiramycine, but producing forms a and b platinoid. This is the case of strain OS49.67 (see section shall emery 6 and 20), in which orf3 gene inactivated by deletions reading frames (Δorf3). For inactivation of orf14 gene in this strain was introduced plasmid pSPM509 by transformation of protoplasts of strain OS49.67 (Rieser, T. and others, 2000). Inactivation orf14 gene has already been described in the case of the strain OSC2 (see example 13) and in the same way it was made to inactivate orf14 gene in strain OS49.67. After transformation with plasmids pSPM509 clones selected for resistance to apramycin. Resistant to apramycin clones were then perseval, respectively, on Wednesday with apramycin and the environment hygromycin. Clones resistant to apramycin (ApraR) and sensitive to hygromycin (HygS)are in principle those in which took place DCO and orf14 gene is replaced by a copy of orf14, interrupted by a cluster att3Ωaac-. In particular, these clones were selected and replace the original copy of the orf14 copy, interrupted by the cluster was confirmed by hybridization. So, the full DNA of the obtained clones was restrictively with several enzymes, separated on agarose gel, transferred to membrane and hybridisable with a probe corresponding to the cluster att3Ωaac-to confirm that the replacement gene actually took place. Confirmation of the genotype may also be implemented in any manner known to the expert, and, in particular, by PCR using appropriate is ejstvujuschij oligonucleotides and sequencing of the PCR product.

In particular was selected clone with the expected characteristics (Δorf3, orf14:: att3Ωaac-), and named SPM510. In fact, thanks to two hybridization it was confirmed that the cluster att3Ωaac - is really in the genome of this clone and that really gets the expected profile restriction in the case of replacement, due to double recombination, the original orf14 gene copy aborted cluster att3Ωaac-in the genome of this clone.

Example 22

Functional complementation interrupt orf14 gene

22.1. Construction of plasmids pSPM519

Orf14 gene amplified by PCR using the following pairs of oligonucleotides: EDR31 : 5' CCCAAGCTTCTGCGCCCGCGGGCGTGAA 3' (SEQ ID No. 136) and EDR37 : 5' GCTCTAGAACCGTGTAGCCGCGCCCCGG 3' (SEQ ID No. 137) and, as matrix, the chromosomal DNA of the strain OSC2. Oligonucleotides EDR31 and EDR37 contain, respectively, the restriction site HindIII and XbaI (sequence in bold). The thus obtained fragment length 1.2 so-called cloned in the vector pGEM-T easy (produced by the company Promega (Madison, Wisconcin, USA) to obtain plasmids pSPN515. This plasmid was then restrictively using the enzymes HindIII and XbaI. Obtained the insertion of a HindIII/XbaI length 1.2 so-called cloned in the vector pUWL201 (see example 17.1), preliminary restrictionon using the same enzymes. The thus obtained plasmid was named pSPM519.

22.1. Transformers the situation strains SPM509 and SPM510 using plasmids pSPM519

Plasmid pSPM519 introduced into strains SPM509 (see example 13) and SPM510 (see example 17) by transformation of protoplasts (Kieser, T. and others, 2000). After the transformation, the clones selected for resistance to thiostrepton. The clones were then perseval on containing thiostrepton environment.

Strain SPM509 is a strain that is not a producer of spiramycin (see examples 13, 15 and 16 and Fig). The production of spiramycin strain SPM509 transformed vector pSPM519 (strain called SPM509pSPM519), was analyzed by culturing this strain on the environment MP5 in the presence of thiostrepton. Cultural supernatant then analyzed by HPLC (see examples 16 and 17). The results of this analysis are presented in table 43, the data are expressed in mg per liter of supernatant. The results represent the total production of spiramycin (obtained by summing the production of spiramycin I, II and III). Found that the presence of the vector pSPM519 in strain SPM509 restores the production of spiramycin (see table 43).

Table 43
The production of spiramycin strain SPM509 transformed with a vector pSPM519 (results expressed in mg/l supernatant)
Strain The production of spiramycin
SPM509 pSPM51958

Strain SPM510 transformed with plasmids pSPM519, was named SPM510pSPM519.

Example 23

Functional complementation of the interrupted gene, orf3 gene tylB S. fradiae

23.1. Construction of plasmids pOS49.52

Plasmid pOS49.52 corresponds to the plasmid that allows expression of the protein TylB in S. ambofaciens. For the design, the coding sequence of a gene tylB S. fradiae (Merson-Davies & Cundliffe, 1994; the access number in the gene Bank: U08223 (region sequence), SFU08223 (DNA sequence) and AAA (protein sequence)) have been built into the plasmid RX (Bierman and others, 1992; Kieser and others, 2000; E. coli strain containing this plasmid available from ARS (NRRL) (culture Collection services research in agriculture) (Peoria, Illinois, USA), under the number B-14790). In addition, this coding sequence was under the control of the promoter ermE* (see above, in particular, the example 17.1; Bibb and others, 1985; Bibb and others, 1994).

23.1. Transformation of strain OS49.67 using plasmids pOS49.52

Strain OS49.67 in which orf3 gene inactivated by deletions in the frame read, does not produce spiramycin (see examples 6 and 15). Plasmid pOS49.52 was introduced into a strain OS49.67 by transformation of protoplasts (Kieser, T. and others, 2000). After the transformation, the clones seleccionara the Ali for their resistance to apramycin. The clones were then perseval on Wednesday, containing apramycin. In particular, he was selected one clone and named OS49.67 pOS49.52.

As was shown above, the strain OS49.67 not produces spiramycin (see examples 6 and 15). Produzione of spiramycine strain OS49.67 transformed vector pOS49.52, was analyzed by the method described in example 15. Thus, it was shown that this strain has the phenotype, which is the producer of spiramycin. Thus, the protein TylB allows functional complementation interrupt orf3 gene.

Example 24

Increased production of spiramycin by overproductive gene orf28c

24.1. Construction of plasmids pSPM75

Gene orf28c amplified by PCR, using a pair of nucleotides, including the restriction site HindIII or the restriction site BamHI. These primers have the following sequences:

KF30: 5'AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID No. 138) with the restriction site HindIII (in bold);

KF31: 5'GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID No. 139) with the restriction site BamHI (in bold).

Primers KF30 and KF31 contain, respectively, the restriction site HindIII and BamHI (sequence in bold). Primer pair KF30 and KF31 allows you to amplify the DNA fragment size of approximately 1.5 so called, containing the gene orf28c, using as matrix kosmidou pSPM36 (see the above). The thus obtained fragment with a length of 1.5 so-called cloned in the vector pGEM-T easy (produced by the company Promega (Madison, Wisconcin, USA) to obtain plasmids pSPM74. Plasmid pSPM74 then restrictively the enzymes HindIII and BamHI, and the resulting inserting the HindIII/BamHI length of approximately 1.5 so-called was subcloned into the vector pUWL201 (see example 17.1), pre-restrictionon using the same enzymes. The thus obtained plasmid was named pSPM75, it contains the totality of the coding sequence orf28c under the control of the promoter ermE*.

24.2. Transformation of the strain OSC2 using plasmids pSPM75

Plasmid pSPM75 introduced into strains OSC2 by transformation of protoplasts (Kieser, T. and others, 2000). After transformation of protoplasts clones selected for resistance to thiostrepton. The clones were then perseval on the medium containing thiostrepton, and transformation using plasmids was confirmed by extraction of plasmids. In particular, two were selected clone and named OSC2/pSPM75(1) and OSC2/pSPM75(2).

A sample of the strain OSC2/pSPM75(2) was deposited in the National collection of microorganism cultures (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex 15, France, October 6, 2003 under registration number I-3101.

To test the impact of overproduction gene orf289c on the production of spiramycin producing spiramycine clones OSC2/pSPM75(1 and OSC2/pSPM75(2) tested according to the method, described in example 15. Analysis of the production of spiramycin strain OSC2 also carried out in parallel for comparison. Thus, it was found that the presence of plasmids pSPM75 significantly increases the production of spiramycin strain OSC2. This shows that overproduction orf28c leads to increased production of spiramycin and confirms its role as a regulator.

The production of spiramycin clones OSC2/pSPM75(1) and OSC2/pSPM75(2) were also analyzed by HPLC (the same way as described in example 17.2). The analysis with regard to the production of spiramycin strain OSC2 also carried out in parallel for comparison. The results of this analysis are presented in table 44, the data are expressed in mg per liter of supernatant. The results represent the total production of spiramycin (obtained by summing the production of spiramycin I, II and III).

120
Table 44
The production of spiramycin strains derived from strain OSC2 transformed using pSPM75 (results expressed in mg/l)
StrainSpiramycine
OSC250
OSC2/pSPM75(1)
OSC2/pSPM75(2)155

Thus, found that the presence of plasmids pSPM75 significantly increases the production of spiramycin strain OSC2. It really shows that overproduction orf28c has a positive effect on the production of spiramycin.

Example 25

Analysis of the production of mediators of the biosynthesis of spiramycine strain, inaktivirovannye in orf8 gene

Strain OS49.107 in which orf8 gene inactivated by insertion of cluster Ωhyg, does not produce spiramycin (see examples 7 and 15). Orf8 gene encodes a protein with a relatively high similarity with several aminotransferases, and highly suggesting that orf8 gene encodes 4-amino-transferase, responsible for the transamination reaction required in the biosynthesis of forosamine (see Fig.6). Therefore, it is expected that the biosynthesis of spiramycin is blocked at the stage of vorotilin (see Fig.7). Strain OS49.107 that is not the producer of spiramycin, therefore, should be to produce vorotilin.

The sample supernatant of strain OS49.107 prepared according to the method described above (see example 16, without extraction with MIBK), and analyzed by CL/SM, as described above (see paragraphs 20.2 and 20.3). According to the method SIM was selected weight 558-related molecules is momu ion vorotilin, and it detected several peaks. The presence of the connection weight 558 is consistent with the hypothesis about the role of orf8 in the synthesis of forosamine.

Example 26

Analysis of the production of mediators of the biosynthesis of spiramycine strain, inaktivirovannye in orf12 gene

Strain SPM507 in which orf12 gene inactivated, does not produce spiramycin (see examples 11 and 15). Orf12 gene encodes 3,4-dehydratase responsible for the dehydration reaction necessary in the biosynthesis of forosamine (see Fig.6). Therefore, it is expected that the biosynthesis of spiramycin is blocked at the stage of vorotilin (see Fig.7). Strain SPM507 that is not the producer of spiramycin, therefore, should be to produce vorotilin.

The sample supernatant of strain SPM507 prepared according to the method described above (see example 16, without extraction with MIBK), and analyzed by CL/SM, as described above (see paragraphs 20.2 and 20.3). Under these conditions, the retention time of vorotilin is approximately 12.9 minutes. According to SIM, was selected weight 558 related to the molecular ion [M+H]+vorotilin, and was detected as one peak. The presence of the connection weight 558 allows you to recognize the validity of the hypothesis about the role of the produced orf12 gene product in the biosynthesis of spiramycin.

However, vorotilin is in a relatively small number, and in these conditions, in particular b is l found the product, absorption at 238 nm (retention time of 17.1 min). Analysis by LC/SM allowed us to determine the mass of this compound, which is 744,3 g/mol ([M+H]+=744,3; the main product).

To define the structure of the above products were isolated and purified under the conditions described above (see paragraph 20.1). The organic phase (MIBK) was then recuperable and evaporated. The dry extract was treated with water and extracted with heptane. The aqueous solution was then extragonadal by locking on an Oasis HLB cartridge, 1 g (Waters SAS, St-Quentin en Yvelines, France). The connection recuperable by elution with a mixture of water/acetonitrile in the ratio of 30:70. This solution was then injectively (100 μl) in the analytical column and fractions were collected in the Oasis HLB cartridge, 1 cm3, 30 mg (Waters). Before using Oasis HLB cartridges, 1 cm3, 30 mg (Waters)was kondicionirovanie successively with acetonitrile, and then a mixture of water/acetonitrile (20 rpm./about 80.) and a mixture of water/acetonitrile in the ratio of 80:20.

The Oasis HLB cartridge, 1 cm3, 30 mg (Waters), then washed sequentially with 1 ml of a mixture of water/acetonitrile (95:5), 1 ml of a mixture water/deuterated acetonitrile (95:5), then suirable with 600 μl of a mixture of water/deuterated acetonitrile (40:60). The recovered solution is then directly analyzed by NMR.

An NMR spectrum obtained for this connection is the following (an NMR spectrum 19312V):

When ECTR 1N CD3CN/D2O (chemical shifts in ppm): to 0.92 (3H, d, J=6 Hz), 1,10 (1H, m)to 1.14 (3H, s)of 1.17 (3H, d, J=6 Hz), 1,22 (3H, d, J=6 Hz), 1,25 (3H, d, J=6 Hz), 1,40 (1H, m)1,75 (1H, DD, J=12 and 2 Hz), is 1.81 (1H, m), 1,90 (1H, d, J=12 Hz), was 2.05 (1H, m), 2,12 (3H, s)to 2.15 (1H, m), 2,35 (2H, m), of 2.45 (6H, greatly.), of 2.53 (1H, m)of 2.64 (1H, DD, J=12 and 9 Hz), 2,80 (1H, DD, J=9, 16 Hz), 2,95 (1H, d, J=8 Hz), 3,23 (2H, m)to 3.34 (1H, d, J=7 Hz), of 3.45 (3H, s), 3,49 (1H, m), 3,93 (1H, DD, J=7 and 3 Hz), 4,08 (1H, m), 4,37 (1H, d, J=6 Hz), 4,88 (1H, m), of 5.05 (2H, m), the 5.65 (2H, m), between 6.08 (1H, DD, J=8 and 12 Hz), 6,40 (1H, DD, J=12 and 9 Hz), 9,60(1H, s).

These experiments, therefore, allowed to determine the structure of this compound will be presented on Fig.

Example 27

Analysis of the production of mediators of the biosynthesis of spiramycine strain, inaktivirovannye in orf5 gene*

Strain SPM501 has the genotype orf6*::attΩhyg+. Due to the polar effect of the insertion of the cluster attΩhyg+ orf6 gene* determined that the orf5 gene* required in the biosynthesis pathway of spiramycin. Actually, the insertion excisional cluster in the coding part of the gene orf6* causes complete cessation of the production of spiramycin due to polar effects on the expression of orf5 gene*. However, once the embedded cluster was excision (and, therefore, when one orf6 gene was inactivated, see examples 14 and 15), reduced production of spiramycin I. It shows that the orf5 gene* required in the biosynthesis of spiramycine because its inactivation causes a full prekrseni the production of spiramycin.

Orf5 gene* encodes a protein with a relatively high similarity with several O-methyltransferases. Therefore, orf5 gene* will be an O-methyltransferase involved in the biosynthesis platinoid. To confirm this hypothesis was conducted experiments on analysis using CL/SM and NMR when using a strain of S. ambofaciens genotype orf6*::attΩhyg+, obtained from strain, overproducing spiramycine.

A sample of the supernatant of this strain prepared by the method described above (see example 16, without extraction with MIBK) and analyzed by CL/SM, as described above (see paragraphs 20.2 and 20.3). However, the used column is column X-Terra (Waters SAS, St-Quentin en Yvelines, France) and cone voltage spectrometer installed at 380 volts to achieve fragmentation of the analyzed compounds. In these conditions was discovered product retention time of approximately 13.1 minutes. The mass spectrum of this compound is similar to that of spiramycin I, but the molecular ion has a mass 829. The difference between the mass 14 relative to the weight spiramycin can be explained by the absence of a methyl group to the oxygen, which contains carbon in position 4 lactoovo cycle (the structure of this compound is presented on Fig). The connection is a lot 829 allows you to confirm the hypothesis about the role of the orf5* in the biosynthesis of ceramic is new.

By microbiological test, carried out using a sensitive strain of M. luteus (see example 15 and Fig), it was shown that the mediator molecule (spiramycin without a methyl group, whose structure is presented on Fig)produced by strain orf6*::Ωatthyg+, is much less active (10 times)than the original spiramycin with a methyl group in position 4.

Example 28

Design of new “excesively clusters”

Were constructed new excesively clusters. These clusters are very similar to excesively clusters, already described in example 9. The main difference between the old and new clusters is the absence in these last sequences corresponding to the ends of interposing Ω, sequences that contain a transcription terminator, derived from phage T4.

In clusters without terminator gene, which imparts resistance to the antibiotic, flanked by sequences attR and attL, allowing the excision. Gene resistance is gene AAC(3)IV, which encodes acetyltransferase, giving resistance to apramycin. This gene is located in the cluster Ω (access number in the gene Bank: H; Blondelet-Rouault M.H. and others, 1997) and was amplified by PCR using, as a matrix, plasmids pOSK1102 (see above) and, as primers, Oleg the nucleotide KF42 and KF43, containing, each, the restriction site HindIII (bold) (AAGCTTin 5'.

KF42: 5'-AAGCTTGTACGGCCCACAGAATGATGTCAC-3' (SEQ ID No. 153), and

KF43: 5'-AAGCTTCGACTACCTTGGTGATCTCGCCTT-3' (SEQ ID NO. 154).

The resulting PCR product of approximately 1 the so-called cloned in the vector E. coli pGEMT Easy to obtain plasmid pSPM83.

Vector pSPM83 was restrictively the restriction enzyme HindIII. Fragment HindIII-HindIII insert was isolated by purification from a 0.8%agarose gel, and then cloned into the HindIII site located between the sequences attL and attR different plasmids carrying different possible excesively clusters (see example 9 and Fig), to replace the HindIII fragment corresponding to Ωacc, HindIII fragment corresponding to one gene AAS. It is possible to obtain clusters att1aac, att2aac and att3aac (according to the desired reading frame, see example 9). Depending on the orientation of the gene AAS compared to sequences attL and attR distinguish att1aac+, att1aac-, att2aac+, att2aac-, att3aac+ and att3aac- (according to the same conditions as those selected in example 9).

Example 29

Construction of a strain of S. ambofaciens, interrupted in the gene orf28c

Inactivation of the gene orf28c carried out according to the method of using excesively clusters. Excesively cluster att3aac+ (see example 28) amplified by PCR using as template a plasmid pSPM101 (plasmid pAPM101 is a plasmid, increasing asuu from vector pGP704Not (Chaveroche and others, 2000; Miller V.L. & J.J. Mekalanos, 1988), in which the cluster aat3taac+ was cloned in EcoRV fragment in a single EcoRV site pGP704Not) and using the following primers:

KF32:

5' CAACCGCTTGAGCTGCTCCATCAACTGCTGGGCCGAGGTATCGCGCGCGCTTCGTTCGGGACGAA3' (SEQ ID No. 155), and

KF33:

5' TGGGTCCCGCCGCGCGGCACGACTTCGACTCGCTCGTCTATCTGCCTCTTCGTCCCGAAGCAACT3' (SEQ ID No. 156)

39 Nucleotides located at the 5' end of these oligonucleotides that include the sequence corresponding to the sequence in the gene orf28c, and 26 nucleotides, located entirely in 3' (shown above in bold and underlined)correspond to a sequence of one end excisional cluster att3aac+.

The thus obtained PCR product was used for transformation hyperaccumulator E. coli strain DY330 (Yu and others, 2000) (this strain contains genes exo, bet and gam phage lambda integrated into its chromosome, these genes are expressed at a temperature of 42°C; this strain was used instead of E. coli strain KS272 (Chaveroche and others, 2000)), containing kosmidou pSPM36. Thus, bacteria transformed by electroporation using this PCR product and the clones selected for resistance to apramycin. Comedy obtained clones were extragonadal and restrictively using the restriction enzyme BamHI to confirm that the obtained profile restriction corresponds to the expected profile, if there was the insertion of class the EPA (att3aac+) gene orf28c, that is, if there really has been a homologous recombination between the ends of the PCR product and of the target genes. Design confirmation may also be implemented in any manner known to the expert, and, in particular, by PCR using the appropriate oligonucleotides and sequencing of the PCR product. He was selected clone, cosmid which has the expected profile, and the corresponding cosmid was named pSPM107. This cosmid is derived pSPM36 in which prf28c interrupted by cluster att3aac+. The insertion of the cluster is accompanied by a deletion in the gene orf28c, interruption begins at the 28-th codon orf28c. After the cluster remain 137 last codons orf28c.

Kosmidou pSPM107, first introduced in the strain E. coli DH5α, and then, in a strain of Streptomyces ambofaciens OSC2 by transformation of protoplasts. After the transformation, the clones selected for resistance to apramycin. Resistant to apramycin clones were then perseval, respectively, on Wednesday with apramycin (antibiotic) and on Wednesday with puromycin (antibiotic) (see Fig.9). Clones resistant to apramycin (ApraR) and sensitive to puromycin (PuroS)are in principle those in which took place DCO and which have a genome orf28c, interrupted by a cluster att3acc+. These clones are preferably selected and replace the original copy of the orf28c copy aborted cluster, was proof is and by hybridization. So, the full DNA of the obtained clones was restrictively with several enzymes, separated on agarose gel, transferred to membrane and hybridisable with a probe corresponding to the cluster att3aac+, to confirm the presence of the cluster in the expected locus in the genomic DNA of the obtained clones. Confirmation of the genotype can also be carried out by any method known to the expert, and, in particular, by PCR using the appropriate oligonucleotides and sequencing of the PCR product.

In particular was selected clone with the expected characteristics (orf28c::att3aac+), and named SPM107. This clone therefore has the genotype orf28c::att3aac+ and was named SPM107. You do not need to perform excision of the cluster to explore the impact of inactivation orf28c, given the orientation of the genes (see figure 3). Actually, the fact that orf29 is oriented in the opposite direction orf28c shows that these genes are not cotranscriptional. Use excisional cluster, instead, allows to be able at any time to remove the marker selection, in particular, by transformation with plasmids pOSV508.

For the definition of “quenching” effects of gene orf28c on the production of spiramycin, producing spiramycine strain SPM107 was tested by the method described in example 15. Thus, it is shown that this strain about the fully phenotype, not which is the producer of spiramycin. This shows that the gene orf28c is a gene essential in the biosynthesis of spiramycin in S. ambofaciens.

Example 30

Construction of a strain of S. ambofaciens, interrupted in the gene orf31

Inactivation of the gene orf31 carried out according to the method of using excesively clusters. Excesively cluster att3aac+ amplified by PCR using, as a matrix, plasmids pSPM101 and oligonucleotides EDR71 and EDR72:

5' CGTCATCGACGTGCGGGGAAGACAGAGGTGATACCGATGATCGCGCGCGCTTCGTTCGGGACGAA3' (SEQ ID No. 157);

EDR72:

5' GCCAGCACCTCGTCCAGCTGCTCGACGGAACTCACCCCCATCTGCCTCTTCGTCCCGAAGCAACT3' (SEQ ID No. 158)

39 Nucleotides located at the 5' end of these oligonucleotides that include the sequence corresponding to the sequence in the gene orf31, and 26 nucleotides, located entirely in 3' (shown above in bold and underlined)correspond to a sequence of one end excisional cluster att3aac+.

The thus obtained PCR product was used to transform E. coli strain KS272 containing plasmid pKOBEG and kosmidou pSPM36 as described Chaveroche and others (Chaveroche and others, 2000) (see Fig on the principle of plasmid pOS49.99 should be replaced by kosmidou pSPM36 and the resulting plasmid is no longer a plasmid pSPM17, and is a plasmid pSPM543). Thus, bacteria transformed by electroporation using this paragraph is oduct PCR and clones selected for resistance to apramycin. Comedy obtained clones were extracted and restrictively with several restriction enzymes to confirm that the obtained profile restriction corresponds to the expected profile, if there was the insertion of the cluster (att3aac+) gene orf31, that is, if there really has been a homologous recombination between the ends of the PCR product and of the target genes. Design confirmation may also be implemented in any manner known to the expert, and, in particular, by PCR using the appropriate oligonucleotides and sequencing of the PCR product. He was selected clone, cosmid which has the expected profile, and the corresponding cosmid was named pSPM543. This cosmid is derived pSPM36 in which prf31 interrupted by cluster att3aac+ (see Fig). The insertion of the cluster is accompanied by a deletion in the gene orf31, interruption begins at the level of the thirty-sixth codon orf31. After the cluster remain 33 last codon orf31.

Kosmidou pSPM543 was introduced into a strain of Streptomyces ambofaciens OSC2 (see above) by transformation of protoplasts (Kieser, T. and others, 2000). After the transformation, the clones selected for resistance to apramycin. Resistant to apramycin clones were then perseval, respectively, on Wednesday with apramycin (antibiotic) and on Wednesday with puromycin (antibiotic) (see Fig.9). Clones resistant to apramycin (ApraR) and coustical the data to puromycin (PuroS), represent, in principle, that took place DCO and which have a genome orf31, interrupted by a cluster att3acc+. These clones are preferably selected and replace the original copy of the orf31 copy, interrupted by the cluster was confirmed by hybridization. So, the full DNA of the obtained clones was restrictively with several enzymes, separated on agarose gel, transferred to membrane and hybridisable with a probe corresponding to the cluster att3aac+, to confirm the presence of the cluster in the expected locus in the genomic DNA of the obtained clones. A second hybridization was carried out using as a probe a DNA fragment obtained by PCR and containing a very large area of the coding sequence of the gene orf31.

Confirmation of the genotype can also be carried out by any method known to the expert, and, in particular, by PCR using the appropriate oligonucleotides and sequencing of the PCR product.

Preferably been selected clone with the expected characteristics (orf31::att3aac+), and named SPM543. In fact, thanks to two hybridization it was confirmed that the cluster att3aac+ is really in the genome of this clone and that really gets the expected profile restriction in the case of replacement, due to double recombination, the original gene copy aborted cluster tt3aac+ in the genome of this clone. This clone therefore has the genotype orf31::att3aac+ and was named SPM543. You do not need to perform excision of the cluster to explore the impact of inactivation of orf31, given the orientation of the genes (see figure 3). In fact, the fact that orf32c is oriented in the opposite direction orf31, shows that these genes are not cotranscriptional. Use excisional cluster, instead, allows to be able at any time to remove the marker selection, in particular, by transformation with plasmids pOSV508.

For the definition of “quenching” effects of gene orf31 on the production of spiramycin, producing spiramycine strain SPM543 was tested by the method described in example 15. Thus, it is shown that this strain has a phenotype that is not a producer of spiramycin. This shows that the gene orf31 is a gene essential in the biosynthesis of spiramycin in S. ambofaciens.

Example 31

Construction of a strain of S. ambofaciens, interrupted in the gene orf32c

Inactivation of the gene orf32c carried out according to the method of using excesively clusters. Excesively cluster att3aac+ amplified by PCR using as template a plasmid pSPM101 and using the following primers:

KF52:

5' GATCCGCCAGCCTCACGTCACGCCGCGCCGCCTCCCTGACATCGCGCGCGCTTCGTTCGGGACGAA3' (SEQ ID No. 159)

KF53:

5' GAGGCGGACGTCGGTACGCGGTGGGAGCCGGAGTTCGACAACTGCCTCTTCGTCCCGAAGCAACT 3' (SEQ ID NO. 160).

40 Nucleotides located at the 5' end of these oligonucleotides that include the sequence corresponding to the sequence in the gene orf32c, and 26 nucleotides, located entirely in 3' (shown above in bold and underlined)correspond to a sequence of one end excisional cluster att3aac+.

The thus obtained PCR product was used for transformation hyperaccumulator E. coli strain DY330 (Yu and others, 2000), containing kosmidou pSPM36. Thus, bacteria transformed by electroporation using this PCR product and the clones selected for resistance to apramycin. Comedy obtained clones were extragonadal and restrictively using the restriction enzyme BamHI to confirm that the obtained profile restriction corresponds to the expected profile, if there was the insertion of the cluster (att3aac+) gene orf32c, that is, if there really has been a homologous recombination between the ends of the PCR product and of the target genes. Design confirmation may also be implemented in any manner known to the expert, and, in particular, by PCR using the appropriate oligonucleotides and sequencing of the PCR product. He was selected clone, cosmid which has the expected profile, and the corresponding cosmid was named pSPM106. This cosmid going is it from pSPM36, in which prf32c interrupted by cluster att3aac+. The insertion of the cluster is accompanied by a deletion in the gene orf32c, interruption begins at the level of the 112-th codon orf31c. After the cluster remain 91 last codons orf32c.

Kosmidou pSPM106, first introduced in the strain E. coli DH5α, and then, in a strain of Streptomyces ambofaciens OSC2 by transformation. After the transformation, the clones selected for resistance to apramycin. Resistant to apramycin clones were then perseval, respectively, on Wednesday with apramycin (antibiotic) and on Wednesday with puromycin (antibiotic) (see Fig.9). Clones resistant to apramycin (ApraR) and sensitive to puromycin (PuroS)are in principle those in which took place DCO and which have a genome orf32c, interrupted by a cluster att3acc+. These clones are preferably selected and replace the original copy of the orf32c copy, interrupted by the cluster was confirmed by hybridization. So, the full DNA of the obtained clones was restrictively with several enzymes, separated on agarose gel, transferred to membrane and hybridisable with a probe corresponding to the cluster att3aac+, to confirm the presence of the cluster in genomic DNA of the obtained clones. Confirmation of the genotype can also be carried out by any method known to the expert, and, in particular, by PCR using the appropriate oligonucleotides and Sequeira the Oia of the PCR product.

In particular was selected clone with the expected characteristics (orf32c::att3aac+). This clone therefore has the genotype orf32c::att3aac+ and was named SPM106. You do not need to perform excision of the cluster to explore the impact of inactivation orf32c, given the orientation of the genes (see figure 3). Actually, the fact that orf33 is oriented in the opposite direction orf32c shows that these genes are not cotranscriptional. Use excisional cluster, instead, allows to be able at any time to remove the marker selection, in particular, by transformation with plasmids pOSV508.

For the definition of “quenching” effects of gene orf32c on the production of spiramycin, producing spiramycine strain SPM106 was tested by the method described in example 15. Thus, it is shown that this strain has a phenotype that is not a producer of spiramycin. This shows that the gene orf32c is a gene essential in the biosynthesis of spiramycin in S. ambofaciens.

The list of structures described in this application

List of abbreviations: Am: ampicillin; Hyg: hygromycin; Sp: spiramycin; Ts: thiostrepton; Cm: chloramphenicol; Kn: kanamycin; Apra: apramycin.

Name designsMarker selection Main featuresLink
pWE15Am(Wahl, et al., 1987)
pWED1AmpWE15, which is subjected to the deletion of the fragment of HpaI-HpaI length of 4.1 so-called(Gourmelen et al., 1998)
pOJ260ApraConjugium not replicated Streptomyces(Bierman et al., 1992)
pHP45 ΩhygHygThe cluster Ωhyg in pHP45.(Blondelet-Rouault et al., 1997)
pKC505ApraCosmid(Richardson MA et al., 1987)
pIJ486TsReplicated mnogokupolnaya plasmid of Streptomyces(Ward et al., 1986)
pOSint3Amptrc-xis-int dans pTrc99A(Raynal et al., 1998)
pWHM3Am, TsReplicative Shuttle vector for E. coli/Streptomyces(Vra et al., 1989)
pKOBEGCm(Chaveroche et al., 2000)
pGP704NotAm(Chaveroche et al., 2000)
pMBL18Am(Nakano et al., 1995)
pGEM-T EasyAmVector E. coli for cloning PCR productsMezei et al., 1994
pOS49.1AmpWED1 with inserting the BamHI site levelExample 2
pOS49.11AmThe SacI fragment pOS49.1 in pUC19.Example 2
pOSC49.12ChThe XhoI fragment pOS49.11 in pBC SK+Example 2
pOS49.14Cm , HygpOSC49.12 with orf3 gene, interrupted by a cluster ΩhygExample 2
pOS49.16Apra, HygInsert pOS49.14 in pOJ260 Example 2
pOS49.28CmFragment BamHI- > PST length 3,7kb so-called pOS49.1 in pBC SK+Example 3
pOS44.1Apra, SppKC505 containing an insert that gives resistance to spiramycin in S. griseofuscus(Pernodet et al., 1999)
pOS44.2Ts, SpThe Sau3AI fragment length 1,8 called pOS44.1 in pIJ486Example 3
pOS44.4.AmInsert pOS44.2 in pUC19Example 3
pSPM5AmpWED1 with insert DNA of S. ambofaciens level BamHI siteExample 3
pSPM7AmpWED1 with insert DNA of S. ambofaciens level BamHI siteExample 3
pOSK1205HygpBK-CMV, where hyg replaced by a neoExample 5
pOS49.67ApraThe fragment EcoRI-SacI de pOS49.1, including internal reality is, s 504 of nucleotides, in pOJ260Example 6
pOS49.88AmThe fragment length is 3.7 so-called > PST -EcoRI de pOS49.1 in pUC19Example 7
pOS49.106AmpO49.88 with hyg in orf8 (hyg and orf8 in the same orientation)Example 7
pOS49.120AmpOS49.88 with hyg in orf8 (hyg et orf8 in opposite orientations)Example 7
pOS49.107Apra, HygInsert pOS49.106 in pOJ260Example 7
pOS49.32Am, KnThe fragment length of 1.5 so-called internal to orf10 in pCR2.1-TOPOExample 8
pOS49.43Am, KnpOS49.32 c hyg in orf10 (hyg and orf10 in the same orientation)Example 8
pOS49.44Am, KnpOS49.32 c hyg in orf10 (hyg and orf10 in opposite orientations)Example 8
pOS49.50Apra, Hyg Insert pOS49.43 dans pOJ260Example 8
pWHM3HygAm, HygpWHM3, where tsr is replaced by hygExample 10
pOSV508Am, Tsptrc-xis-int pWHM3Example 9
patt1Ωhyg+Cm, HygThe cluster att1Ωhyg+ pBC SK+HindIII site where deletedExample 9
patt3Ωaac-Cm, GnThe cluster att3Ωaac in pBC SK+HindIII site where deletedExample 9
pOSV510Am, Hygpro pra-Amh dans pWHM3HygExample 10
pOS49.99AmThe fragment EcoRI-BamHI length of 4.5 so-called pSPM5 in pUC19Example 10
pOSK1102Am, AprapGP704Not containing the cluster att3Ωaac-Example 10
pSPM17Am, AprapOS49.99 where orf2 interrupted by cluster att3Ωaac-Example 10
pSPM21Hyg, AprapOSK1205 containing inserting EcoRI-XbaI pSPM17 (where orf2 interrupted by cluster att3Ωaac-)Example 10
pSPM502AmThe BglII fragment length 15,1 so-called pSPM7 in pMBL18Example 11
pSPM504HygInsert pSPM502 in pOSK1205Example 11
pSPM507Hyg, AprapSPM504 where orf12 interrupted by cluster att3Ωaac-Example 11
pSPM508Hyg, AprapSPM504 where orf13c interrupted by cluster att3Ωaac-Example 12
pSPM509Hyg, AprapSPM504 where orf14 interrupted by cluster att3Ωaac-Example 13
pBXL1111AmFragment length 1,11 so called, containing orf6*, amplificatory by PCR, based on pSPM7, in the vector pGEM-T EasyExample 14
pBXL1112Am, HygpBXL111, where the cluster att1Ωhyg+ entered after the deletion of 120 base pairs in the coding sequence of the gene orf6*Example 14
pBXL1113Apra, HygInsert > PST a length of 3.7 so-called de pBXL1112 in pOJ260Example 14
pSPM520AmThe PCR fragment, amplificatory oligonucleotide EDR39-EDR42 in pGEM-T EasyExample 17
pSPM521AmThe PCR fragment, amplificatory oligonucleotide EDR40-EDR42 in pGEM-T EasyExample 17
pSPM522AmThe PCR fragment, amplificatory oligonucleotide EDR41-EDR42 in pGEM-T EasyExample 17
pUWL201Am , Ts(Doumith et al., 2000)
pSPM523Am, TsFragment HindIII-BamHI insert of the plasmid pSPM520 in the vector pUWL201Example 17
pSPM524Am, TsFragment HindIII-BamHI insert of the plasmid pSPM521 in which the sector pUWL201 Example 17
pSPM525Am, TsFragment HindIII-BamHI insert of the plasmid pSPM522 in the vector pUWL201Example 17
pSPM527AmpSPM521 with a gap reading frames at the site level XhoIExample 17
pSPM528Am , TsFragment HindIII-BamHI insert of the plasmid pSPM527 in the vector pUWL201Example 17
pVF 10.4(Vara et al., 1985; Lacalle et al., 1989)
pPM803Ts(Mazodier,P. et al., 1989)
pGEM-T-pac-oriTAmThe cluster pac-oriT (amplificatory by PCR on the basis of pVF 10.4 and pPM803) in pGEM-T EasyExample 18
pWED2AmThe cluster pac-oriT obtained from pGEM-T-pac-oriT, built-in pWED1Example 18
pSPM34AmpWED2 what about inserting the BamHI site level Example 19
pSPM35AmpWED2 with inserting the BamHI site levelExample 19
pSPM36AmpWED2 with inserting the BamHI site levelExample 19
pSPM37AmpWED2 with inserting the BamHI site levelExample 19
pSPM38AmpWED2 with inserting the BamHI site levelExample 19
pSPM39AmpWED2 with inserting the BamHI site levelExample 19
pSPM40AmpWED2 with inserting the BamHI site levelExample 19
pSPM41AmpWED2 with inserting the BamHI site levelExample 19
pSPM42AmpWED2 with inserting the BamHI site levelExample 19
pSPM43AmpWED2 with inserting the BamHI site levelExample 19
pSPM44AmpWED2 with inserting the BamHI site levelExample 19
pSPM45AmpWED2 with inserting the BamHI site levelExample 19
pSPM47AmpWED2 with inserting the BamHI site levelExample 19
pSPM48AmpWED2 with inserting the BamHI site levelExample 19
pSPM50AmpWED2 with inserting the BamHI site levelExample 19
pSPM51AmpWED2 with inserting the BamHI site levelExample 19
pSPM52AmpWED2 with inserting the BamHI site levelExample 19
pSPM53Am pWED2 with inserting the BamHI site levelExample 19
pSPM55AmpWED2 with inserting the BamHI site levelExample 19
pSPM56AmpWED2 with inserting the BamHI site levelExample 19
pSPM58KnFragment > PST - > PST length of about 6 so-called insert pSPM36 in pBK-CMVExample 19
pSPM72KnFragment StuI-StuI length of about 10 so-called insert pSPM36 cloned into pBK-CMVExample 19
pSPM73CmThe fragment EcoRI-HindIII insert pSPM72 in pBC-SK+Example 19
pSPM515AmThe PCR fragment, amplificatory EDR31-EDR37 in pGEM-T easyExample 22
pSPM519Am, TsInserting a HindIII/XbaI pSPM515 in pUWL201Example 22
pOS49.52Apra The sequence encoding tylB under the control of the promoter ermE* plasmid pKC1218Example 23
pSPM74AmThe PCR fragment, amplificatory KF30-KF31 in pGEM-T easyExample 24
pSPM75Am, TsInserting a HindIII/BamHI pSPM74 in pUWL201Example 24
pSPM79KnFragment > PST - > PST a length of about 2.5 so-called insert pSPM36 in pBK-CMVExample 19
pSPM83AmThe PCR fragment, amplificatory KF42-KF43 in pGEM-T easyExample 28
pSPM107Am, AprapSPM36 where orf28c interrupted by cluster att3aac+Example 29
pSPM543Am, AprapSPM36 where orf31 interrupted by cluster att3aac+Example 30
pSPM106Am, AprapSPM36 where orf32c interrupted by cluster att3aac+Example 31

Pick biologists the definition of material

The following organisms were deposited on 10 July 2002 in the National collection of microorganism cultures (CNCM), 25 rue du Docteur Roux, 75724 Paris, Cedex 15, France, under the provisions of the Budapest Treaty.

The strain OSC2 under the registration number I-2908.

A strain SPM501 under the registration number I-2909.

A strain SPM502 under the registration number I-2910.

A strain SPM507 under the registration number I-2911.

A strain SPM508 under the registration number I-2912.

A strain SPM509 under the registration number I-2913.

A strain SPM21 under the registration number I-2914.

A strain SPM22 under the registration number I-2915.

A strain OS49.67 under the registration number I-2916.

A strain OS49.107 under the registration number I-2917.

A strain of Escherichia coli DH5α containing plasmid pOS44.4, under the registration number I-2918.

The following organisms were deposited on 26 February 2003 in the National collection of microorganism cultures (CNCM), 25 rue du Docteur Roux, 75724 Paris, Cedex 15, France, under the provisions of the Budapest Treaty.

A strain SPM502pSPM525 under the registration number I-2977.

The following organisms were deposited in the National collection of microorganism cultures (CNCM), 25 rue du Docteur Roux, 75724 Paris, Cedex 15, France, 6 October 2003, in accordance with the provisions of the Budapest Treaty.

The strain OSC2/pSPM75(2) under the registration number I-3101.

All cited publications and patents is turned off in this application by reference.

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1. Polynucleotide encoding an acyltransferase responsible for
modification platinoid in position 3, and the sequence of the above polynucleotide is:
(a) one of the sequences SEQ ID No. 111 or 141;
(b) one of the sequences derived sequences (a) in accordance with the degeneracy of the genetic the definition of the code.

2. Polynucleotide encoding an acyltransferase responsible for the modification platinoid in position 3, hybridization under conditions of high stringency with at least one polynucleotide according to claim 1, moreover, high rigidity imply the implementation of hybridization at a temperature of from 55 to 65°C, followed by one or more washes carried out in the buffer 2X SSC at a temperature of from 55 to 65°C, then with one or more leaching carried out in the buffer 0.5x SSC at a temperature of from 55 to 65°C.

3. Polynucleotide having at least 70, 80, 85, 90, 95, or 98% identity to nucleotides with polynucleotide according to claim 1 and encodes an acyltransferase responsible for the modification platinoid in position 3.

4. Polynucleotide according to claim 2 or 3, characterized in that it is isolated from bacteria of the genus Streptomyces.

5. Polynucleotide according to claim 2, 3 or 4, characterized in that it encodes a protein involved in the biosynthesis of the macrolide.

6. Polynucleotide according to claim 2, 3, 4 or 5, characterized in that it encodes a protein having activity similar to the protein encoded by polynucleotides, with whom he hybridized or has identity.

7. The polypeptide resulting from the expression of polynucleotide according to claim 1 and represents the acyltransferase responsible for the modification platinoid in position 3.

8. The polypeptide representing a acyltransferase, responsible for the modification platinoid in position 3, and the above sequence of the polypeptide is:
(a) one of the sequences SEQ ID No. 112 or 142;
(b) one of the sequences, such as those described in (a), except that the entire length of the above sequence one or more amino acids replaced built or subject to deletion without affecting the functional properties.

9. The polypeptide representing the acyltransferase responsible for the modification platinoid in position 3 and with at least 70, 80, 85, 90, 95, or 98% identity to amino acids from the polypeptide of claim 8.

10. The polypeptide according to claim 9, characterized in that it is isolated from bacteria of the genus Streptomyces.

11. The polypeptide of claim 10, characterized in that it is involved in the biosynthesis of the macrolide.

12. The polypeptide according to claim 9, characterized in that it has activity similar to the activity of the polypeptide with which it has an identity.

13. Bacterial expression vector, comprising at least one nucleotide sequence encoding a polypeptide according to claim 7 or 8.

14. Bacterial expression system comprising expressing a vector according to item 13 and the cage bacteria of the genus Streptomyces, allowing the expression of one or more polypeptides according to claim 7 or 8.

15. Expression C is theme for 14 characterized in that it is chosen from prokaryotic expression systems.

16. Bacterial cell host, which is a bacterial cell of the genus Streptomyces, for expression of the polypeptide according to claim 7 or 8, in which you have entered at least one polynucleotide and/or at least one expressing the vector of claim 1.

17. The method of producing the polypeptide according to claim 7 or 8, comprising the following stages:
a) insertion of polynucleotide according to claim 1 in expressing vector;
b) culturing, in a suitable for the expression of culture medium, the cells of bacteria of the genus Streptomyces, previously transformed or transtitional vector from step a);
c) recovery of conditioned culture medium or cell extract;
d) isolation and purification of the above culture medium or cell extract obtained(s) at the stage (C), the above polypeptide.

18. A strain of Streptomyces ambofaciens, superexpression spiramycine representing the strain deposited at the National collection of microorganism cultures (CNCM) of the Institut Pasteur, 25 Rue du Docter Roux 75724 Paris Cedex 15, France, October 6, 2003 under registration number I-3101.

19. The use of polynucleotide according to claim 1 for improving the production of macrolides in the cells of bacteria of the genus Streptomyces.

20. The application of a sequence according to claim 1 in rekombinantnogo or vector for producing spiramycin I, II and III.

21. Polynucleotide encoding an acyltransferase responsible for the modification platinoid in position 3, representing polynucleotide, complementary polynucleotide according to claim 1.

22. The cell of the bacterium Streptomyces ambofaciens, which is the producer of at least one spiramycin, superexpression:
the gene, which can be obtained by amplification by polymerase chain reaction (PCR) using a pair of primers having the following sequence:
5' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID No. 138) and
5' GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID No. 139),
and as the matrix of Comedy pSPM36 or total DNA of Streptomyces ambofaciens;
or gene, its derived in accordance with the degeneracy of the genetic code.

23. The cell according to item 22, wherein superexpression above reach of the gene by transformation of the above mentioned microorganism expressing vector.

24. Bacterial expressing vector, including:
polynucleotide, which can be obtained by amplification by polymerase chain reaction using a pair of primers having the following sequence:
5' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID No. 138) and
5' GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID No. 139),
and as the matrix of Comedy S36 or total DNA of Streptomyces ambofaciens;
or a fragment comprising the polynucleotide sequence according to claim 1.

25. CL is TKA-host Streptomyces ambofaciens, superexpression spiramycine, which introduced the vector at point 24.

26. The method of producing the polypeptide according to claim 7, comprising the following stages:
a) transforming cells of bacteria of the genus Streptomyces with at least one expressing vector at point 24;
b) culturing on appropriate culture medium above the cells of bacteria of the genus Streptomyces;
c) recovery of conditioned culture medium or cell extract;
d) isolation and purification of the above culture medium or cell extract obtained(s) at the stage (C), the above polypeptide.



 

Same patents:

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention proposes a method for purifying macrolide compounds. Invention describes a method for isolation of macrolide in a purified state that involves the following steps: (a) treatment of an unpurified macrolide or crude macrolide with a solvent not mixing with water; (b) not obligatory concentrating the mixture; (c) treatment with gaseous ammonia to precipitate impurities; (d) separation of impurities; (e) not obligatory concentration of macrolide-containing phase; (f) carrying out chromatography on silica gel column in not obligatory reversed-phase, or with preliminary treatment with silver and elution of macrolide; (g) preparing macrolide in the essential purified form; (h) not obligatory repeat of steps (f) and (g) for preparing macrolide in the essential purified form. Macrolide is represented preferably as tacrolimus, immunomycin or syrolimus. Proposed method provides preparing macrolides of high purity degree by simplified technology.

EFFECT: improved isolating and purifying method.

8 cl, 2 ex

FIELD: biotechnology, in particular antibiotic production and new erythromycin producer.

SUBSTANCE: claimed strain is obtained by mutant selection from S.erythraea strain. Said strain is characterized with high specific rate of antibiotic formation (120±10 mg of erythromycin/g of dry biomass/h). Level of erythromycin formation after seven days of culturing in discontinuous process is 8250 mug/ml. Strain of present invention makes it possible to increase by 10 % level of erythromycin formation in contrast to starting strain and to decrease by 15-20 % formed biomass amount. Maximum synthesis specific rate is by 30 % higher in contrast to starting strain.

EFFECT: strain for accelerated synthesis of antibiotic erythromycin.

1 tbl, 1 ex

FIELD: food-processing industry, in particular, compositions providing immediate antimycotic action and methods of applying such compositions.

SUBSTANCE: food composition comprises food material prone to decaying owing to growth of mold, fungi or yeast thereon, and natamicine material designed for people and made in the form of particles with average diameter of 10 micron or less, or with average particles surface area to weight ratio of at least 2 m2/g, said particles being dispersed onto surface or into perishable food product at concentrations of from 1 part of natamicine per million to 5 parts of natamicine per million. Method involves dispersing natamicine material so as to provide natamicine concentration of from 1 part of natamicine per million to 5 parts of natamicine per million, or dispersing bulk natamicine material including particles with average diameter of particles of 10 micron or less or with average particle surface area to weight ratio of at least 2 m2/g onto surface or inside perishable food product.

EFFECT: increased antimycotic effect owing to use of natamicine material without deteriorating quality of perishable food product.

27 cl, 2 dwg, 6 tbl, 4 ex

The invention relates to biotechnology, in particular, to the production of erythromycin
The invention relates to biotechnology

The invention relates to the field of biotechnology and related to tylosin is a macrolide antibiotic of broad-spectrum, used in agriculture

FIELD: agriculture.

SUBSTANCE: genetic modification of wheat in the form of mutation in gene SBEIIa with reduction of level of its activity.

EFFECT: grain with high content of amylose in its starch; wheat with reduced level of SBEIIb-activity with grain with non-wrinkled phenotype, with relatively high content of amylose.

62 cl, 28 dwg, 12 tbl, 16 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, specifically to a method of producing a version of glycolipid acyltransferase enzyme, which involves: (a) selection of an initial enzyme which is glycolipid acyltransferase, where the enzyme contains amino acid motif GDSX, where X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T, N, M or S; (b) modification of one or more amino acids to obtain a glycolipid acyltransferase version; (c) testing the glycolipid acyltransferase version on transferase activity, and optionally hydrolytic activity with respect to a galactolipid substrate and, optionally, a phospholipid substrate and/or, optionally, triglyceride substrate; (d) selection of the version of the enzyme with high activity with respect to galactolipids compared to the initial enzyme; and, optionally, (e) obtaining a large amount of the version of the enzyme. The invention also relates to versions of the lipidacyltransferase enzyme, where the enzyme contains amino acid motif GDSX, where X is one or more of the following amino acid residues L, A, V, T, F, Y, H, Q, T, N, M or S, and where, compared to the initial sequence, the version of the enzyme contains one or more amino acid modifications.

EFFECT: obtaining versions of an enzyme with high transferase activity compared to the initial enzyme.

36 cl, 61 dwg, 14 tbl, 10 ex

FIELD: biotechnologies.

SUBSTANCE: invention is related to biotechnology and may be used for site-specific hydrolysis of DNA, which contains C5-methylcytosin bases. Strain Paracoccus carotinifaciens 3K is extracted from soil, which provides for production of site-specific endonuclease, which recognises and splits both chains of nucleotide sequence of DNA containing four C5-methylcytosin bases in site of recognition 5'-WCGNNNNNNNCGW-3', with production of single-nucleotide 3'-protruding ends.

EFFECT: invention makes it possible to produce new site-specific endonuclease Pcsl, which may be used for detection and analysis of methylated DNA.

2 dwg, 3 ex

Mashing method // 2376347

FIELD: food industry.

SUBSTANCE: invention is represented by ferment composition containing GH10 xylanase in quantity at least 33 wt % of common ferment protein weight and GH12 endoglucanase in quantity at least 14 wt % of ferment protein common weight. This composition is used for mash preparartion.

EFFECT: mash with improved filtering ability.

20 cl, 22 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: method of producing cyclopropyl-condensed inhibitors of dipeptidyl peptidase IV involves using BOC-protected amine with structural formula (3) , obtained through reductive amination of acid with formula (1) by treating the said acid with ammonium formate, nicotinamide adenine dinucleotide, dithiothreitol and partially purified concentrate of phenyl alanine dehydrogenase and formate dehydrogenase (PDH/FDH) enzymes and without separation - by treating the obtained amine of formula (2) with ditertbutyl dicarbonate, obtaining BOC-protected amine.

EFFECT: cutting on costs.

13 cl, 7 ex

FIELD: chemistry.

SUBSTANCE: method of producing cyclopropyl-condensed inhibitors of dipeptidyl peptidase IV involves using BOC-protected amine with structural formula (3) , obtained through reductive amination of acid with formula (1) by treating the said acid with ammonium formate, nicotinamide adenine dinucleotide, dithiothreitol and partially purified concentrate of phenyl alanine dehydrogenase and formate dehydrogenase (PDH/FDH) enzymes and without separation - by treating the obtained amine of formula (2) with ditertbutyl dicarbonate, obtaining BOC-protected amine.

EFFECT: cutting on costs.

13 cl, 7 ex

FIELD: biotechnology.

SUBSTANCE: thrombin derivative is described that includes chain A and chain B where chain B has an aminoacid sequence whose aminoacids of the series active center in position 205 and histidine in position 43 in the aminoacid sequence of chain B of thrombin are substituted and where: the specified thrombin derivative decomposes thrombin substrate to the extent of 10% or less when interacting with thrombin substrate in 50 mM Tris-HCl (pH 7.4) containing 0.1 M NaCl at the temperature of 37°C for 3 hours and the specified thrombin derivative retains the ability of getting combined with C-terminal hirudin peptide immobilised in gel.

EFFECT: pharmaceutical formula is disclosed that contains the thrombin derivative described.

54 cl, 48 dwg, 2 tbl, 31 ex

FIELD: medicine.

SUBSTANCE: vitamin K dependent protein is made by separating a cultivated eukaryotic cell that contains an expressing vector that contains a nucleic acid molecule coding vitamin K dependent protein and associated sequences regulating expression. The associated sequences contain the first promoter and the nucleic acid molecule coding gamma-glutamylcarboxylase, and the second promoter. The first promoter represents a pre-early promoter of human cytomegalovirus (hCMV), and the second promoter is a pre-early promoter SV40. Herewith the expressing relation of vitamin K dependent protein and gamma-glutamylcarboxylase is 10:1 to 250:1.

EFFECT: invention allows for making gamma-carboxylated vitamin K dependent protein in production quantities.

29 cl, 5 dwg, 6 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used for producing esters of such compounds as carbohydrates, proteins, protein subunits and hydroxyacids. The method involves mixing an acyl group donor, acyl group acceptor and water with formation of a medium with high water content, containing 5 to 98% water. The acyl group donor used is a lipid substrate, chosen from phospolipid, lysophospholipid, triacylglyceride, diglyceride, glycolipid or lysoglycolipid, and the acyl group acceptor used is carbohydrate, protein, protein subunitor hydroxyacid. The mixture is brought into contact with lipidacyltransferase, which catalyses alcoholysis and/or re-esterification and is an enzyme which has acyltransferase activity, containing a GDSX fragment of amino acid sequence, where X stands for one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T, N, M or S.

EFFECT: invention allows for obtaining one or more carbohydrate esters, protein esters, esters of protein subunits or hydroxyacid esters using lipidacyltransferase.

18 cl, 51 dwg, 10 tbl, 13 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used for producing esters of such compounds as carbohydrates, proteins, protein subunits and hydroxyacids. The method involves mixing an acyl group donor, acyl group acceptor and water with formation of a medium with high water content, containing 5 to 98% water. The acyl group donor used is a lipid substrate, chosen from phospolipid, lysophospholipid, triacylglyceride, diglyceride, glycolipid or lysoglycolipid, and the acyl group acceptor used is carbohydrate, protein, protein subunitor hydroxyacid. The mixture is brought into contact with lipidacyltransferase, which catalyses alcoholysis and/or re-esterification and is an enzyme which has acyltransferase activity, containing a GDSX fragment of amino acid sequence, where X stands for one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T, N, M or S.

EFFECT: invention allows for obtaining one or more carbohydrate esters, protein esters, esters of protein subunits or hydroxyacid esters using lipidacyltransferase.

18 cl, 51 dwg, 10 tbl, 13 ex

FIELD: biotechnologies.

SUBSTANCE: invention is related to the field of biotechnology and immunology. Separated and cleaned DNA is presented, which codes receptor CTLA-4 (CD 152) of cat. The following is also suggested - diagnostic oligonucleotide, cloning vector, vaccine, methods of induction, strengthening and suppression of immune response in cat.

EFFECT: creation of model cat for research of retroviral infection.

24 cl, 10 dwg, 6 tbl, 8 ex

FIELD: pharmacology.

SUBSTANCE: present invention refers to immunology and biotechnology. There are antibody-antagonist to CD40 with their variable areas derived from an antibody produced of hybridoma 4D11 (FERM BP-7758). The constant areas of antibodies are derived from human IgG4 with mutations S228P and L235E. There are described related coding polynucleotides and the based expression vector. There is disclosed host-cell containing said vector. There is described method for preparing monoclonal antibody and application thereof in the pharmaceutical composition.

EFFECT: application of the invention provides reduced ADCC and CDC activity that can find application in therapy of autoimmune diseases and graft rejection.

10 cl, 26 dwg, 2 tbl, 22 ex

FIELD: pharmacology.

SUBSTANCE: invention concerns immunology and biotechnology. There is offered human monoclonal antibody specific to TNF-alpha containing light and heavy chain with appropriate CDR3 sites. There are described versions thereof including those based on heavy and light chains and coded by human genes VH3-33 and A30VK1 or VH3-53 and L2VK3 respectively. There are disclosed: the method for estimating the TNF-alpha content in the patient's sample with using specified antibodies, and application of antibodies for preparing a medical product. There are described: compositions for diagnostics and treatment of the conditions associated with TNF-alpha activity on the basis of antibodies. There is disclosed coding nucleic acid, a cell for making said antibodies and the method for making said antibodies.

EFFECT: application of the invention ensured high-affinity neutralizing monoclonal antibodies with improved Kd and IC50 in comparison with Infliximab, Adalimumab or Etanercept that can find application in medicine for treatment and diagnostics of the diseases associated with TNF-alpha hyperactivity.

35 cl, 13 dwg, 36 tbl, 14 ex

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