WO2004033689A2

WO2004033689A2 - Polypeptides involved in spiramycin biosynthesis, nucleotide sequences encoding said polypeptides and uses thereof

Info

Publication number: WO2004033689A2
Application number: PCT/FR2003/002962
Authority: WO
Inventors: Marie-Hélène BLONDELET-ROUAULT; Hélène DOMINGUEZ; Emmanuelle Darbon-Rongere; Claude Gerbaud; Anne Gondran; Fatma Karray; Patricia Lacroix; Nathalie Oestreicher-Mermet- Bouvier; Jean-Luc Pernodet; Karine Tuphile
Original assignee: Aventis Pharma S.A.; Cnrs
Priority date: 2002-10-08
Filing date: 2003-10-08
Publication date: 2004-04-22
Also published as: KR101110175B1; IL167854A; JP5042497B2; NO335995B1; CA2501445C; KR20050084843A; WO2004033689A3; EP1551975A2; AU2003300479B2; MXPA05002852A; NO20052160L; JP2006501863A; BRPI0314543B1; CA2501445A1; AU2003300479A1; BRPI0314543B8; BR0314543A

Abstract

The invention concerns isolation and identification of novel genes of spiramycin biosynthesis process and novel polypeptides involved in said biosynthesis. The invention also concerns a method for producing sand polypeptides. The invention further concerns the use of said genes for increasing the rate of production and purity in the resulting spiramycin. The invention concerns in particular a micro-organism producing spiramycin I but not producing spiramycin II and III and the use of such a micro-organism. The invention also concerns the use of genes in spiramycin biosynthesis process for constructing mutants capable of leading to synthesis of novel antibiotics or derivative forms of spiramycin. Finally the invention concerns molecules produced through expression of said genes and phamacologically active compositions of one molecule produced through expression of such genes.

Description

POLYPEPTIDES INVOLVED IN THE BIOSYNTHESIS OF SPIRAMYCINS. NUCLEOTIDIOUE SEQUENCES ENCODING THESE POLYPEPTIDES AND THEIR APPLICATIONS

The present invention relates to the isolation and identification of new genes from the biosynthetic pathway for spiramycins and to new polypeptides involved in this biosynthesis. It also relates to the use of these genes in order to increase the production rates and the purity of the spiramycin produced.

The invention also relates to the use of these genes for the construction of mutants which may lead to the synthesis of new antibiotics or to forms derived from spiramycins. The invention also relates to the molecules produced by the expression of these genes and finally to pharmacologically active compositions of a molecule produced by the expression of such genes.

Streptomyces are Gram-positive filamentous soil bacteria. They play an important role in the decomposition and mineralization of organic materials thanks to the great diversity of degradative enzymes that they secrete. They present unique morphological differentiation phenomena in prokaryotes accompanied by metabolic differentiation characterized by the production of secondary metabolites with an extraordinary diversity of chemical structures and biological activities. Among these metabolites are spiramycins produced naturally by the bacteria Streptomyces ambofaciens ,.

Spiramycin is an antibiotic from the macrolide family, useful in both veterinary and human medicine. Macrolides are characterized by the presence of a lactone cycle on which one or more sugars are grafted. Streptomyces ambofaciens produces in its natural state spiramycin I, II and III (cf. Figure 1), however the antibiotic activity of spiramycin I is clearly superior to that of spiramycins II and III (Liu et al, 1999). The spiramycin I molecule consists of a lactonic macro-cycle, called platenolide and three sugars: forosamine, mycaminose and mycarose (see Figure 1). The antibiotic activity of spiramycins is due to an inhibition of protein synthesis in prokaryotes by a mechanism involving the binding of the antibiotic to the bacterial ribosome.

Certain compounds which are members of the macrolide family and which also have a lactone cycle have given rise to uses outside the field of antibiotics.

Thus the product FK506 has immunosuppressive effects and offers prospects for therapeutic application in the field of organ transplantation, rheumatoid arthritis and more generally in pathologies linked to autoimmune reactions. Other macrolides like avermectin have insecticidal and anti-helminthic activities.

Numerous biosynthetic pathways, concerning antibiotics belonging to various classes as well as other secondary metabolites (for a review, Chater K. 1990), have to date already been studied in Streptomycetes. However, the ;; _; ; knowledge of the biosynthetic pathways of spiramycins is only very partial to date.

The biosynthesis of spiramycins is a complex process with many stages and involving many enzymes (Omura et al, 1979a, Omura et al, 1979b). Spiramycins belong to the large class of polyketides which groups together complex molecules which are particularly abundant in soil microorganisms. These molecules are grouped together not by analogy of structure but by a certain similarity at the level of stages of their path of biosynthesis. Indeed, polyketides are produced by a complex series of reactions but have in common that they have in their biosynthetic pathway a series of reactions catalyzed by one or more enzymes called “polyketides synthases” (PKS). In Streptomyces ambofaciens, the biosynthesis of the lactonic macro-cycle of spiramycins (platenolide) is carried out by a series of eight modules coded by five different PKS genes (Kuhstoss S., 1996, US patent 5,945,320). Spiramycins are obtained at from this lactone cycle. However, the various stages and enzymes involved in the synthesis of sugars, as well as their binding to the lactone cycle and the modification of this cycle to obtain spiramycins are still unknown to this day.

US Patent 5,514,544 describes the cloning of a sequence called srmR in Streptomyces ambofaciens. It is hypothesized in this patent that the srmR gene codes for a protein which regulates the transcription of genes involved in the biosynthesis of macrolides.

In 1987, Richardson et al. (Richardson et al., 1987) showed that the resistance to spiramycin in S. ambofaciens is conferred by at least three genes, the latter were called srmA, srmB and srmC. US Patent 4,886,757 more particularly describes the characterization of a DNA fragment of S. ambofaciens containing the srmC gene. However, the sequence of this gene has not been disclosed. In 1990, Richardson et al. (Richardson et al, 1990) hypothesized the existence of three genes for the biosynthesis of spiramycin close to srmB. US Patent 5,098,837 reports the cloning of five genes potentially involved in the biosynthesis of spiramycin. These genes have been named srmD, srmE, srmF, srmG and srmH.

One of the major difficulties in the production of compounds such as spiramycins resides in the fact that very large volumes of fermentation are necessary for the production of a relatively small quantity of product. It is therefore desirable to be able to increase the production efficiency of such molecules in order to reduce the production costs.

The biosynthetic pathway for spiramycins is a complex process and it would be desirable to identify and suppress unwanted reactions that may exist during this process. The purpose of such manipulation is to obtain a purer antibiotic and / or an improvement in productivity. In this regard, Streptomyces ambofaciens produces in its natural state spiramycin I, II and III (cf. Figure 1), however the antibiotic activity of spiramycin I is clearly superior to that of spiramycin II and III (Liu et al, 1999). It would therefore be desirable to have available strains producing only spiramycin I.

Due to the commercial interest of macrolide antibiotics, the obtaining of new derivatives, in particular analogues of spiramycins having advantageous properties is intensively sought. It would be desirable to be able to generate in sufficient quantity the biosynthesis intermediates of the biosynthesis pathway for spiramycins or derivatives of spiramycins, in particular for the purpose of manufacturing hybrid antibiotics derived from spiramycins.

General description of the invention

The present invention results from the cloning of genes whose product is involved in the biosynthesis of spiramycins. The invention relates first of all to new genes of the biosynthetic pathway for spiramycins and to new polypeptides involved in this biosynthesis.

The biosynthetic pathway genes and associated coding sequences were cloned and the DNA sequence of each was determined. The cloned coding sequences will hereinafter be designated orfI * c, orβ * c, orβ * c, orf4 * c, orβ *, orfό *, orβ * c, orβ *, orβ *, orflO *, orfl, orβ, orβ, orβ, orβ, orfό, orβ, orβ, orβc, orflO, orfllc, orf! 2, orfl 3c, orfI4, orfl 5c, orfl 6, orf! 7, orf! 8, orfl 9, orβO, orβ le, orβ2c, orβSc , orβ4c, orβ5c, orβό, orβ7, orβδc, orβ9, orβOc, orβl, orβ2c, orβ 3 and orβ4c. The function of the proteins encoded by these sequences in the biosynthetic pathway for spiramycins is developed in the discussion below which is illustrated by FIGS. 4, 5, 6 and 8.

1) A first subject of the invention relates to a polynucleotide encoding a polypeptide involved in the biosynthesis of spiramycins, characterized in that the sequence of said polynucleotide is: (a) one of the sequences SEQ ID N ° 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 constituted by the variants of the sequences (a), (c) one of the sequences derived from the sequences (a) and (b) due to the degeneration of the genetic code.

2) The present invention also relates to a polynucleotide which hybridizes under hybridization conditions of high stringency to at least one of the polynucleotides according to paragraph 1) above.

3) The invention also relates to a polynucleotide having at least 70%,

80%, 85%, 90%, 95% or 98% of nucleotide identity with a polynucleotide comprising at least 10, 12, 15, 18, 20 to 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 a polynucleotide according to paragraph 1) above.

4) The invention also relates to a polynucleotide according to paragraph 2) or 3) above, characterized in that it is isolated from a bacterium of the genus Streptomyces.

5) The invention also relates to a polynucleotide according to paragraph 2), 3) or 4) above, characterized in that it codes for a protein involved in the biosynthesis of a macrolide.

6) The invention also relates to a polynucleotide according to paragraph 2), 3) or 4) above characterized in that it codes for a protein having an activity similar to the protein encoded by the polynucleotide with which it hybridizes or it presents identity.

7) The invention also relates to a polypeptide resulting from the expression of a polynucleotide according to paragraph 1), 2), 3), 4), 5) or 6) above. 8) Another aspect of the invention relates to a polypeptide involved in the biosynthesis of spiramycins, characterized in that the sequence of said polypeptide is:

(a) one of the sequences SEQ ID N ° 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) one of the sequences as defined in (a) except that throughout said sequence one or more amino acids have been substituted, inserted or deleted without affecting their functional properties, (c) one of the sequences constituted by the variants of the sequences (a) and (b).

9) Another subject of the invention relates to a polypeptide having at least 70%, 80%, 85%, 90%, 95% or 98% of amino acid identity with a polypeptide comprising at least 10, 15, 20, 30 to 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 according to paragraph 8) above.

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

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

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

13) Another aspect of the invention relates to a recombinant DNA characterized in that it comprises at least one polynucleotide according to one of paragraphs 1), 2), 3), 4), 5) or 6) above . 14) Another aspect of the invention relates to a recombinant DNA according to paragraph 13) above, characterized in that the said recombinant DNA is included in a vector.

15) Another aspect of the invention relates to a recombinant DNA according to paragraph 14) above, characterized in that said vector is chosen from bacteriophages, plasmids, phagemids, integrative vectors, fosmids, cosmids, shuttle vectors, BAC or PAC.

16) Another aspect of the invention relates to a recombinant DNA according to paragraph 15) above, characterized in that it is chosen from 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, pSPMl, pBXLllll, pBXL1112, pBXL1113, pSPM520, pSPM521, pSPM522 pM522 pM522 pSPM35, pSPM36, pSPM37, pSPM38, pSPM39, pSPM40,; pSPM41, pSPM42, pSPM43, pSPM44, pSPM45, pSPM47, pSPM48, pSPM50, pSPM51, pSPM52, pSPM53, pSPM55, pSPM56, pSPM58, pSPM72, pSPM73, pSPM515, pSPM5 pSPM5 pMM pMM pMM pMM 17) Another aspect of the invention relates to an expression vector characterized in that it comprises at least one nucleic acid sequence coding for a polypeptide according to paragraph 7), 8), 9), 10), 11) or 12) above.

18) The invention also relates to an expression system comprising an appropriate expression vector and a host cell allowing the expression of one or more polypeptides according to paragraph 7), 8), 9), 10), 11) or 12) above.

19) The invention also relates to an expression system according to paragraph 18 above, characterized in that it is chosen from prokaryotic expression systems or eukaryotic expression systems. 20) The invention also relates to an expression system according to paragraph 19) above, characterized in that it is chosen from expression systems in the E. coli bacterium, baculovirus expression systems allowing expression in insect cells, expression systems for expression in yeast cells, expression systems for expression in mammalian cells.

21) The invention also relates to a host cell into which has been introduced at least one polynucleotide and / or at least one recombinant DNA and / or at least one expression vector according to one of the paragraphs 1), 2) , 3), 4), 5), 6), 13), 14), 15), 16) or 17) above.

22) The invention also relates to a method for producing a polypeptide according to paragraph 7), 8), 9), 10), 11) or 12) above characterized in that said method comprises the following steps : a) inserting at least one nucleic acid encoding said polypeptide into an appropriate vector; b) cultivating, in an appropriate culture medium, a host cell previously transformed or transfected with the vector of step a); c) recovering the conditioned culture medium or a cell extract; d) separating and purifying from said culture medium or also from the cell extract obtained in step c), said polypeptide; e) where appropriate, characterize the recombinant polypeptide produced.

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

24) Another aspect of the invention relates to a microorganism according to paragraph 23) above, characterized in that it is obtained by inactivating the function of at least one protein involved in the biosynthesis of this or these macrolides in a microorganism producing this or these macrolide (s). 25) Another aspect of the invention relates to a microorganism according to paragraph 24) above, characterized in that the inactivation of this or these proteins is carried out by mutagenesis in the gene or genes encoding said protein or proteins or by expression one or more antisense RNAs complementary to the RNA or messenger RNAs encoding said protein (s). "

26) Another aspect of the invention relates to a microorganism according to paragraph 25) above, characterized in that the inactivation of this or these proteins is carried out by mutagenesis by irradiation, by the action of chemical mutagenic agent, by mutagenesis directed or by gene replacement.

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

28) Another aspect of the invention relates to a microorganism according to paragraph 23), 24), 25), 26) or 27) above characterized in that said?; microorganism is a bacteria of the genus Streptomyces.

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

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

31) Another aspect of the invention relates to a microorganism according to paragraph 23), 24), 25), 26), 27), 28), 29) or 30) above characterized in that the mutagenesis is carried out in at least one gene comprising a sequence according to one of paragraphs 1), 2), 3), 4), 5) or 6) above. 32) Another aspect of the invention relates to a microorganism according to paragraph 25), 26), 27), 28), 29), 30) or 31) above characterized in that 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 No. 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 paragraph 25), 26), 27), 28), 29), 30), 31) or 32) above characterized in that the mutagenesis consists of l gene inactivation of a gene comprising a sequence corresponding to the sequence SEQ ID No. 13.

34) Another aspect of the invention relates to a strain of Streptomyces ambofaciens characterized in that it is a strain chosen from one of the strains deposited with the National Collection of Cultures of Microorganisms (CNCM) on 10 July 2002 under registration number 1-2909, 1-2911, 1-2912, 1- 2913, 1-2914, 1-2915, 1-2916 or 1-2917.

35) Another aspect of the invention relates to a process for the preparation of a macrolide biosynthesis intermediate, characterized in that it comprises the following steps: a) cultivating, in an appropriate culture medium, a microorganism according to one paragraphs 23), 24), 25), 26), 27), 28), 29), 30), 31), 32), 33) or 34) above, b) recover the conditioned culture medium or a cell extract, c) separate and purify from said culture medium or again from the cell extract obtained in step b), said biosynthesis intermediate.

36) Another aspect of the invention relates to a process for the preparation of a molecule derived from a macrolide, characterized in that a biosynthesis intermediate is prepared according to the process of paragraph 35) above and that one modifies the intermediary thus produced. 37) Another aspect of the invention relates to a preparation process according to paragraph 36) above, characterized in that said intermediate is modified by chemical, biochemical, enzymatic and / or microbiological means.

38) Another aspect of the invention relates to a preparation process according to paragraph 36) or 37) above, characterized in that one or more genes encoding proteins capable of modifying the intermediate are introduced into said microorganism. using it as a substrate.

39) Another aspect of the invention relates to a preparation process according to paragraph 36), 37) or 38) above, characterized in that the macrolide is spiramycin.

40) Another aspect of the invention relates to a preparation process according to paragraph 36), 37), 38) or 39) above characterized in that the microorganism used is a strain of S. ambofaciens.

41) Another aspect of the invention relates to a microorganism producing spiramycin I but not producing spiramycin II and m

42) Another aspect of the invention relates to a microorganism according to paragraph 41) above, characterized in that it comprises all of the genes necessary for the biosynthesis of spiramycin I but that the gene comprising the sequence SEQ ID N ° 13 or one of its variants or one of the sequences derived therefrom due to the degeneracy of the genetic code and encoding a polypeptide of sequence SEQ ID No 14 or one of its variants is not expressed or has been rendered inactive.

43) Another aspect of the invention relates to a microorganism according to paragraph 42) above, characterized in that said inactivation is carried out by mutagenesis in the gene encoding said protein or by the expression of a complementary antisense RNA messenger RNA encoding said protein. 44) Another aspect of the invention relates to a microorganism according to paragraph 43) above, characterized in that said mutagenesis is carried out in the promoter of this gene, in the coding sequence or in a non-coding sequence so as to render inactivate the encoded protein or prevent its expression or translation.

45) Another aspect of the invention relates to a microorganism according to claim 43) or 44) above characterized in that the mutagenesis is carried out by irradiation, by the action of a chemical mutagenic agent, by directed mutagenesis or by gene replacement .

46) Another aspect of the invention relates to a microorganism according to paragraph 43), 44) or 45) above, characterized in that the mutagenesis is carried out in vitro or in situ, by suppression, substitution, deletion and / or addition one or more bases in the gene under consideration or by gene inactivation.

47) Another aspect of the invention relates to a microorganism according to _; paragraph 41) or 42) above, characterized in that said microorganism is obtained; by expressing the genes of the spiramycin biosynthesis pathway without these comprising the gene comprising the sequence corresponding to SEQ ID No. 13 or one of its variants or one of the sequences derived from these in due to the degeneracy of the genetic code and encoding a polypeptide of sequence SEQ ID No. 14 or one of its variants.

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

49) Another aspect of the invention relates to a microorganism according to paragraph 41), 42), 43), 44), 45), 46), 47) or 48) above characterized in that said microorganism is obtained at from a starting strain producing spiramycins I, II and III. 50) Another aspect of the invention relates to a microorganism according to paragraph 41), 42), 43), 44), 45), 46), 47), 48) or 49) above, characterized in that it is obtained by mutagenesis in a gene comprising the sequence corresponding to SEQ ID No. 13 or one of its variants or one of the sequences derived therefrom due to the degeneration of the genetic code and coding a polypeptide of sequence SEQ ID No. 14 or one of its variants having the same function.

51) Another aspect of the invention relates to a microorganism according to paragraph 41), 42), 43), 44), 45), 46), 47), 48), 49) or 50) above characterized in that that said microorganism is obtained from a strain of S. ambofaciens producing spiramycins I, II and III, in which a gene inactivation of the gene comprising the sequence corresponding to SEQ ID No. 13 or one of the sequences derived is carried out of it due to the degeneration of the genetic code.

52) Another aspect of the invention relates to a strain of S. ambofaciens characterized in that it is the strain deposited with the National Collection of Cultures of Microorganisms (CNCM) on July 10, 2002 under the number, d 1-2910.

53) Another aspect of the invention relates to a process for the production of spiramycin I, characterized in that it comprises the following steps:

(a) cultivate, in an appropriate culture medium, a microorganism according to one of paragraphs 41), 42), 43), 44), 45), 46), 47), 48), 49), 50), 51) or 52) above,

(b) recovering the conditioned culture medium or a cell extract,

(c) separate and purify from said culture medium or also from the cell extract obtained in step b), spiramycin I.

54) Another aspect of the invention relates to the use of a polynucleotide according to one of paragraphs 1), 2), 3), 4), 5) or 6) above to improve the production of macrolides d 'a microorganism. 55) Another aspect of the invention relates to a mutant macrolide-producing microorganism characterized in that it has a genetic modification in at least one gene comprising a sequence according to one of paragraphs 1), 2), 3), 4 ), 5) or 6) above and / or that it overexpresses at least one gene comprising a sequence according to one of paragraphs 1), 2), 3), 4), 5) or 6) above .

56) Another aspect of the invention relates to a mutant microorganism according to paragraph 55) above, characterized in that the genetic modification consists of a deletion, a substitution, a deletion and / or an addition of one or more bases in the gene or genes considered for the purpose of expressing one or more proteins having a higher activity or of expressing a higher level of this or these proteins.

57) Another aspect of the invention relates to a mutant microorganism according to paragraph 55) above, characterized in that the overexpression of the gene considered is obtained by increasing the number of copies of this gene and / or by placing a more active promoter than the wild promoter.

58) Another aspect of the invention relates to a mutant microorganism according to paragraph 55) or 57) above, characterized in that the overexpression of the gene considered is obtained by transforming a macrolide-producing microorganism by a construction of recombinant DNA according to paragraph 13, 14 or 17 above, allowing the overexpression of this gene.

59) Another aspect of the invention relates to a mutant microorganism according to paragraph 55), 56), 57) or 58) above characterized in that 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 N ° 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 therefrom due to the degeneracy of the genetic code. 60) Another aspect of the invention relates to a mutant microorganism according to paragraph 55), 56), 57), 58) or 59) above characterized the microorganism overexpresses one or more genes comprising one of the sequences corresponding to one or more sequences SEQ ID N ° 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 therefrom due to the degeneracy of the genetic code.

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

62) Another aspect of the invention relates to a mutant microorganism according to paragraph 55), 56), 57), 58), 59), 60) or 61) above characterized in that the macrolide is spiramycin.

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

64) Another aspect of the invention relates to a process for producing macrolides, characterized in that it comprises the following steps:

(a) cultivate, in an appropriate culture medium, a microorganism according to one of paragraphs 55), 56), 57), 58), 59), 60), 61), 62), 63) or 64) ci -above,

(b) recovering the conditioned culture medium or a cell extract,

(c) separating and purifying from said culture medium or also from the cell extract obtained in step b), the said macrolide (s) produced.

65) Another aspect of the invention relates to the use of a sequence and / or of a recombinant DNA and / or of a vector according to one of paragraphs 1), 2), 3), 4), 5), 6), 7),

8), 9), 10), 11), 12), 13), 14), 15), 16) or 17) above for the preparation of hybrid antibiotics. 66) Another aspect of the invention relates to the use of at least one polynucleotide and / or at least one recombinant DNA and / or at least one expression vector and / or at least one polypeptide and / or at least one host cell according to one of paragraphs 1), 2), 3), 4), 5), 6), 7), 8), 9), 10), 11), 12), 13), 14), 15), 16), 17) or 21) above to carry out one or more bioconversions.

_**

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

68) Another aspect of the invention relates to a microorganism producing at least one spiramycin, characterized in that it overexpresses:

- a gene capable of being obtained by polymerase chain reaction (PCR) using the pair of primers with the following sequence: 5 'AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID No 138) and 5 'GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID N ° 139) and as a matrix the cosmid pSPM36 or the total DNA of Streptomyces ambofaciens,

- or a gene derived therefrom due to the degeneracy of the genetic code.

69) Another aspect of the invention relates to a microorganism according to paragraph 68 or 90 characterized in that it is a bacterium of the genus Streptomyces.

70) Another aspect of the invention relates to a microorganism according to paragraph 68, 69 or 90 characterized in that it is a bacterium of the species Streptomyces ambofaciens.

71) Another aspect of the invention relates to a microorganism according to paragraph 68, 69, 70 or 90 characterized in that the overexpression of said gene is obtained by transformation of said microorganism by an expression vector. 72) Another aspect of the invention relates to a strain of Streptomyces ambofaciens characterized in that it is the strain OSC2 / pSPM75 (l) or the strain OSC2 / pSPM75 (2) deposited with the National Collection of Cultures de Microorganismes (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, October 6, 2003 under registration number 1-3101.

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

- a polynucleotide capable of being obtained by polymerase chain reaction using the pair of primers with the following sequence: 5 'AAGCTTGTGTGCCCGGTGTACCTGGGGAGCC' '(SEQ ID No. 138) and 5' GGATCCCGCGACGGACACGACCGCCGCGCA 3 '(SEQ ID No. 139) and as a matrix the cosmid pSPM36 or the total DNA of Streptomyces ambofaciens,

- or a fragment of at least 10, 12, 15, 18, 20 to 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 nucleotides of this polynucleotide.

74) Another aspect of the invention relates to a recombinant DNA according to paragraph 73 or 91, characterized in that it is a vector.

75) Another aspect of the invention relates to a recombinant DNA according to paragraph 73, 74 or 91 characterized in that it is an expression vector.

76) Another aspect of the invention relates to a host cell into which at least one recombinant DNA has been introduced according to one of paragraphs 73, 74, 75 or 91.

77) Another aspect of the invention relates to a method for producing a polypeptide characterized in that said method comprises the following steps: a) transforming a host cell with at least one expression vector according to paragraph 75; b) cultivating, in an appropriate culture medium, said host cell; c) recovering the conditioned culture medium or a cell extract; d) separating and purifying from said culture medium or also from the cell extract obtained in step c), said polypeptide; e) where appropriate, characterize the recombinant polypeptide produced.

78) Another aspect of the invention relates to a microorganism 'according to paragraph 51 characterized in that the gene inactivation is carried out by deletion in phase of the gene or of part of the gene comprising the sequence corresponding to SEQ ID N ° 13 or one of the sequences derived therefrom due to the degeneracy of the genetic code.

79) Another aspect of the invention relates to a microorganism according to one of paragraphs 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 78 characterized in that it overexpresses in addition :

- a gene capable of being obtained by polymerase chain reaction using the pair of primers with the following sequence: 5 ′

AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3 '(SEQ ID N ° 138) and 5' 'GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID N ° 139) and as a matrix the cosmid pSPM36 or the total DNA of Streptomyces ambofaciens,

- or a gene derived from them due to the degeneracy of the genetic code.

80) Another aspect of the invention relates to an expression vector characterized in that the polynucleotide of sequence SEQ ID No. 47 or a polynucleotide derived therefrom due to the degeneration of the genetic code is placed under the control of 'a promoter allowing the expression of the protein coded by the said polynucleotide in Streptomyces ambofaciens.

81) Another aspect of the invention relates to an expression vector according to paragraph 80, characterized in that it is the plasmid pSPM524 or pSPM525. 82) Another aspect of the invention relates to a strain of Streptomyces ambofaciens transformed by a vector according to paragraph 80 or 81.

83) Another aspect of the invention relates to a microorganism according to one of paragraphs 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 78, 79 or 92 characterized in that it also overexpresses the coding sequence gene SEQ ID No. 47 or a coding sequence derived therefrom due to the degeneracy of the genetic code.

84) Another aspect of the invention relates to a microorganism according to paragraph 83 characterized in that it is the strain SPM502 pSPM525 deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, February 26, 2003 under registration number 1-2977.

85) Another aspect of the invention relates to a process for the production of spiramycin (s), characterized in that it comprises the following stages: (a) cultivating, in an appropriate culture medium, a microorganism according to ύή of paragraphs 68, 69, 70, 71, 72, 78, 79, 82, 83, 84, 90 or 92,

(b) recovering the conditioned culture medium or a cell extract,

(c) separating and purifying from said culture medium or also from the cell extract obtained in step b), the spiramycins.

86) Another aspect of the invention relates to a polypeptide characterized in that its sequence comprises the sequence SEQ ID No. 112 or the sequence SEQ ID No. 142.

87) Another aspect of the invention relates to a polypeptide characterized in that its sequence corresponds to the translated sequence of the coding sequence:

- a gene capable of being obtained by polymerase chain reaction (PCR) using the pair of primers with the following sequence: 5 '

AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3 '(SEQ ID N ° 138) and 5' GGATCCCGCGACGGACACGACCGCCGCGCA 3 '(SEQ ID No. 139) and as a matrix the cosmid pSPM36 or the total DNA of Streptomyces ambofaciens,

- or a gene derived therefrom due to the degeneracy of the genetic code.

88) Another aspect of the invention relates to an expression vector allowing the expression of a polypeptide according to paragraph 86, 87 or 93 in Streptomyces. ambofaciens.

89) Another aspect of the invention relates to an expression vector according to paragraph 88, characterized in that it is the plasmid pSPM75.

90) Another aspect of the invention relates to a microorganism according to paragraph 68 characterized in that the gene capable of being obtained by polymerase chain reaction is the gene with coding sequence SEQ ID No. 141 or a gene derived from that due to the degeneracy of the genetic code.

91) Another aspect of the invention relates to a recombinant DNA according to paragraph 73, characterized in that the polynucleotide capable of being obtained by chain amplification by polymerase is a polynucleotide of sequence SEQ ID No. 141.

92) Another aspect of the invention relates to a microorganism according to paragraph 79, characterized in that the gene capable of being obtained by amplification in a polymerase chain is the gene with coding sequence SEQ ID No 141 or a gene derived from that due to the degeneracy of the genetic code.

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

The term "isolated" in the sense of the present invention designates a biological material (nucleic acid or protein) which has been removed from its original environment (the environment in which it is naturally located).

For example, a polynucleotide naturally occurring in a plant or animal is not isolated. The same polynucleotide separated from adjacent nucleic acids within which it is naturally inserted into the genome of the plant or animal is considered to be "isolated".

Such a polynucleotide may be included in a vector and / or such a polynucleotide may be included in a composition and nevertheless remain in an isolated state since the vector or the composition does not constitute its natural environment.

The term "purified" does not require that the material be present in a form of absolute purity, exclusive of the presence of other compounds. Rather, it is a relative definition.

A polynucleotide is in the "purified" state after purification of the starting material or of the natural material of at least one order of magnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.

For the purposes of the present invention the term ORF ("Open Reading Fra e", that is to say open reading frame) has been used to designate in particular the coding sequence of a gene.

For the purposes of the present description, the expression "nucleotide sequence" can be used to denote either a polynucleotide or a nucleic acid. The term "nucleotide sequence" encompasses the genetic material itself and is therefore not limited to information regarding its sequence. The terms "nucleic acid", "polynucleotide", "oligonucleotide" or "nucleotide sequence" include RNA, DNA, cDNA or even RNA / DNA hybrid sequences of more than one nucleotide, in single chain form or in duplex form.

The term "nucleotide" designates both natural nucleotides (A, T, G, C) as well as modified nucleotides which comprise at least one modification such as (1) an analogue of a purine, (2) an analogue of 'a pyrimidine, or (3) a similar sugar, examples of such modified nucleotides being described for example in PCT application No. WO 95/04064.

For the purposes of the present invention, a first polynucleotide is considered to be "complementary" to a second polynucleotide when each base of the first polynucleotide is paired with the base complementary to the second polynucleotide whose orientation is reversed. The complementary bases are A and T (or A and U), or C and G.

The term 'genes of the spiramycin biosynthetic pathway' also includes regulatory genes and genes conferring resistance to producer microorganisms.

The term “fragment” of a reference nucleic acid according to the invention will be understood to mean a nucleotide sequence of reduced length compared to the reference nucleic acid and comprising, on the common part, a nucleotide sequence identical to the acid. reference nucleic acid.

Such a "fragment" of nucleic acid according to the invention may, where appropriate, be included in a larger polynucleotide of which it is constitutive.

Such fragments include or alternatively consist of polynucleotides of length ranging from 8, 10, 12, 15, 18, 20 to 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 a nucleic acid according to the invention.

By "fragment" of a polypeptide according to the invention, the expression a polypeptide whose amino acid sequence is shorter than that of the reference polypeptide and which comprises over the entire common _^ portion with these reference polypeptides, a sequence in identical amino acids.

Such fragments may, if appropriate, be included within a larger polypeptide of which they are part.

Such fragments of a polypeptide according to the invention can have a length of 10, 15, 20, 30 to 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.

By “high stringency hybridization conditions” within the meaning of the present invention, hybridization conditions are understood to disadvantage the hybridization of strands of non-homologous nucleic acids. High stringency hybridization conditions can for example be described as hybridization conditions in the buffer described by Church & Gilbert (Church & Gilbert, 1984) at a temperature between 55 ° C and 65 ° C, preferably the annealing temperature is 55 ° C, more preferably the annealing temperature is 60 ° C and most preferably the annealing temperature is 65 ° C, followed by one or more washes carried out in 2X SSC buffer (the IX SSC buffer corresponds to an 0.15M NaCl aqueous solution, 15 mM sodium citrate) at a temperature between 55 ° C and 65 ° C, preferably this temperature is 55 ° C, even more preferably this temperature is 60 ° C and very preferably this temperature is 65 ° C, followed by one or more washings in 0.5X SSC buffer at a temperature between 55 ° C and 65 ° C, preferably this e temperature is 55 ° C, more preferably this temperature is 60 ° C and most preferably this temperature is 65 ° C. It goes without saying that the hybridization conditions described above can be adapted as a function of the length of the nucleic acid for which hybridization is sought or of the type of labeling chosen, according to techniques known to those skilled in the art. . The suitable hybridization conditions can, for example, be adapted according to the work by F. Ausubel et al (2002).

By "variant" of a nucleic acid according to the invention, is meant a nucleic acid which differs by one or more bases with respect to the reference polynucleotide. A variant nucleic acid may be of natural origin, such as an allelic variant found naturally, or may also be an unnatural variant obtained for example by mutagenesis techniques.

In general, the differences between the reference nucleic acid and the variant nucleic acid are reduced so that the nucleotide sequences of the reference nucleic acid and of the variant nucleic acid are very close and, in many regions , identical. The nucleotide modifications present in a variant nucleic acid can be silent, which means that they do not alter the amino acid sequences encoded by said variant nucleic acid.

However, changes of nucleotides in a variant nucleic acid can also result from substitutions, additions, deletions in the polypeptide encoded by the variant nucleic acid with respect to the peptides encoded by the reference nucleic acid. In addition, modifications of nucleotides in the coding regions can produce substitutions, conservative or non-conservative in the amino acid sequence.

Preferably, the variant nucleic acids according to the invention encode polypeptides which retain substantially the same biological function or activity as the polypeptide of the reference nucleic acid or also the ability to be recognized by antibodies directed against the polypeptides encoded by l initial nucleic acid. Certain variant nucleic acids will thus encode mutated forms of the polypeptides whose systematic study will make it possible to deduce structure activity relationships from the proteins in question.

By "variant" of a polypeptide according to the invention, is mainly meant a polypeptide whose amino acid sequence contains one or more substitutions, additions or deletions of at least one amino acid residue, relative to the sequence amino acids of the reference polypeptide, it being understood that the amino acid substitutions can be either conservative or non-conservative.

Preferably, the variant polypeptides according to the invention retain substantially the same biological function or activity as the reference polypeptide or the ability to be recognized by antibodies directed against the initial polypeptides.

By polypeptide having "an activity similar" to a reference polypeptide within the meaning of the invention means a polypeptide having a biological activity close, but not necessarily identical, to that of the reference polypeptide as measured in a biological test suitable for measuring the biological activity of the reference polypeptide.

By "hybrid antibiotic" within the meaning of the invention means a compound, generated by the construction of an artificial biosynthesis pathway using recombinant DNA technology.

Detailed description of the invention

The present invention more particularly relates to new genes of the biosynthetic pathway for spiramycins and to new polypeptides involved in this biosynthesis as presented in the detailed description below.

The biosynthetic pathway genes have been cloned and the DNA sequence of these genes has been determined. The sequences obtained were analyzed using the FramePlot program (Ishikawa J & Hotta K. 1999). We have identified, among the open reading phases, the open reading phases presenting a use of the codons typical of Streptomyces. This analysis showed that this region comprises 44 ORFs, located on either side of five genes coding for the enzyme “polyketide synthase” (PKS), and having a codon use typical of Streptomyces. On either side of these five genes encoding PKS, respectively 10 and 34 ORFs were identified downstream and upstream (downstream and upstream being defined by the orientation of the 5 PKS genes all oriented _; in the same direction) (see Figures 3 and 37). Thus, the 34 open reading phases of this ,; type, occupying a region of approximately 41.7 kb (cf. SEQ ID N ° 1 presenting a first region of 31 kb containing 25 ORFs and SEQ ID N ° 140 presenting a region of approximately 12.1 kb including 1, 4 kb overlap the previous sequence (SEQ ID N ° 1) and approximately 10.7 kb correspond to the rest of the sequence, this last part of approximately 10.7 kb containing 9 additional ORFs (including one ORF of partial sequence), see also Figure 3 and 37 and below), have been identified upstream of the 5 genes encoding PKS and 10 occupying a region of approximately 11.1 kb (SEQ ID No. 2 and Figure 3), have been identified downstream of the PKS genes. The 10 genes located downstream of the 5 PKS genes were thus named orfl * c, orβ * c, orβ * c, orβ * c, orβ * orfό * orβ * c, orβ *, orβ *, orflO * (SEQ ID N ° 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21). The “c” added in the name of the gene signifying for the ORF in question that the coding sequence is in the reverse orientation (the coding strand is therefore the complementary strand of the sequence given in SEQ ID No. 2 for these genes) . Using the same nomenclature, the 34 ORFs upstream of the PKS genes were named orfl, orβ, orβ, orβ, orβ, orfό, orβ, orβ, orβc, orflO, orfl le, orfl2, orfl 3c, orfJ4, orfl 5c, orfl 6, orfl 7, orflδ, orfl 9, orβO, orβlc, orβ2c, orβ3c, orβ4c, orβ5c, orβό, orβ7, orβδc, orβ9, orβOc, orβl, orβ 2c, orβ 3 and orβ4c (SEQ ID N ° 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) (cf. figures 3 and 37).

The protein sequences deduced from these open reading phases were compared with those present in different databases using different programs: BLAST (Altschul et al, 1990) (Altschul et al, 1997), CD-search, COGs (Cluster of Orthologous Groups) (these three programs are accessible in particular from the National Center for Biotechnology Information (NCBI) (Bethesda, Maryland, USA)), FASTA ((Pearson W. R & DJ Lipman, 1988) and (Pearson WR, 1990), BEAUTY (Worley KC et al, 1995)) (these two programs are accessible in particular from the INFOBIOGEN resource center, Evry, France). These comparisons made it possible to formulate hypotheses on the function of the products of these genes and to identify those likely to be involved in the biosynthesis of spiramycins. Genes located downstream of the genes encoding PKS

A schematic representation of the organization of the region is presented in FIG. 3. As will be demonstrated below, of the 10 genes identified downstream of the genes encoding PKS 9 seem to be involved in biosynthesis or resistance to spiramycins. These are the following 9 genes: orfl * c, orβ * c, orβ * c, orβ * c, orβ *, orfό *, orβ * c, orβ * and orβ *.

In the following table 1 are presented the references to the DNA and amino acid sequence of the 10 genes identified downstream of the 5 PKS genes.

Table 1

The “c” added in the name of the gene indicates that the coding sequence is in the reverse orientation (the coding strand is therefore the complementary strand of the sequence given in SEQ ED No. 2 for these genes).

construction of mutant strains and analyzes of the production of spiramycins and biosynthesis intermediates of spiramycins by these mutant strains.

The protein sequences deduced from these open reading phases were first compared with those present in different databases using different programs: BLAST (Altschul et al, 1990) (Altschul et al, 1997), CD-search, COGs (Cluster of Orthologous Groups), FASTA ((Pearson W. R & DJ Lipman, 1988) and (Pearson WR, 1990), BEAUTY (Worley KC et al, 1995)). These comparisons made it possible to formulate hypotheses on the function of the products of these genes and to identify those likely to be involved in the biosynthesis of spiramycin. Table 2 shows the proteins having a strong similarity with the 10 gene products located downstream of the 5 PKS genes. Table 2

* greater sequence similarity is associated with a higher BLAST score (Altschul et al, 1990).

Gene inactivation experiments were carried out to confirm these results. The methods used consist in performing a gene replacement. The target gene to be interrupted is replaced by a copy of this interrupted gene by a cassette conferring resistance to an antibiotic (for example apramycin, geneticin or hygromycin). The cassettes used are bordered on either side by translation termination codons in all the reading phases and by transcription terminators active at Streptomyces. The insertion of the cassette into the target gene may or may not be accompanied by a deletion in this target gene. The size of the regions flanking the cassette can range from a few hundred to several thousand base pairs. A second type of cassette can be used for the inactivation of genes: cassettes known as “excisable cassettes”. These cassettes have the advantage of being able to be excised in Streptomyces by a site-specific recombination event after having been introduced into the genome of S. ambofaciens. The aim is to inactivate certain X genes in Streptomyces strains without leaving selection markers or large DNA sequences not belonging to the strain in the final strain. After excision there remains only a short sequence of around thirty base pairs (called the “scar” site) in the genome of the strain (cf. FIG. 10). The implementation of this system consists, firstly, in replacing the wild copy of the target gene (thanks to two homologous recombination events, cf. FIG. 9) by a construction in which an excisable cassette has been inserted into this uncomfortable ,; target. The insertion of this cassette is accompanied by a deletion in the target gene (cf. - •>^'• Figure 9). In a second step, the excision of the excisable cassette of the genome of the strain is caused. The excisable cassette functions thanks to a site-specific recombination system and has the advantage of making it possible to obtain mutants of Streptomyces which ultimately do not carry a resistance gene. It also frees itself from possible polar effects on the expression of genes located downstream of the inactivated gene (s) (cf. FIG. 10). The strains thus constructed were tested for their production of spiramycins. The inactivation of the orfl * c, orβ * c, orβ * c and orf4 * c genes was not carried out because the sequence comparison experiments made it possible to determine that these genes had a relatively significant similarity with genes involved in the biosynthesis of a relatively close antibiotic. Thus, the orfl * c gene codes for a protein with an identity of 66% (determined thanks to the BLAST program) with the protein coded by the tylMI gene which codes for an N-methyltransferase involved in the Tylosin biosynthesis and catalyzes 3 N-methylation during the production of mycaminosis in Streptomyces fradiae (Gandecha, AR et al, 1997; GenBank access number: CAA57473; BLAST score: 287). This similarity with a protein involved in the biosynthesis pathway of another relatively close antibiotic and more particularly in the biosynthesis of mycaminosis suggests that the orfl * c gene codes for an N-methyltransferase responsible for N-methylation during the biosynthesis of forosamine or mycaminosis (see Figures 5 and 6). This hypothesis is supported by the fact that the protein encoded by the orfl * c gene has a strong similarity with other proteins of similar function in other organisms (see Table 3).

Table 3

The orβ * c gene encodes a protein with relatively strong similarity (35% identity) with a protein encoded by the tylMIII gene encoding an NDP hexose 3,4 isomerase involved in the biosynthesis of tylosin in Streptomyces fradiae (Gandecha, AR , et al, 1997; GenBank access number: CAA57471; BMAST score: 130). This similarity with a protein involved in the biosynthesis pathway of another close antibiotic and more particularly in the biosynthesis of mycaminosis strongly suggests that the orβ2c gene codes for an NDP hexose 3,4 isomerase responsible isomerization during the biosynthesis of one of the sugars of spiramycin, possibly mycaminose (cf. FIGS. 5 and 6).

The orβ * c gene codes for a protein having relatively strong similarity (59% identity) with a protein coded by the tylMII gene coding for a glycosyltransferase involved in the biosynthesis of tylosin in Streptomyqes fradiae (Gandecha, AR et al, 1997; GenBank Access Number: CAA57472; BLAST Score: 448). This similarity to a protein involved in the biosynthesis pathway of another nearby antibiotic strongly suggests that the orβ3c gene codes for a glycosyltransferase. This hypothesis is supported by the fact that protein encoded by the orβ * c gene has a strong similarity with other proteins of similar function in other organisms (see Table 4).

Table 4

The orβ * c gene encodes a protein with relatively strong similarity to several crotonyl-CoA reductases. In particular, the protein encoded by orβ * c has significant similarity with a crotonyl CoA reductase from Streptomyces coelicolor (Redenbach, M et al, 1996; GenBank access number: NP_630556; Score BLAST: 772). This similarity with a protein involved in the biosynthetic pathway of another close antibiotic strongly suggests that the orβ * c gene also codes for a crotonyl-CoA reductase. This hypothesis is supported by the fact that the protein encoded by the orβ * c gene has a strong similarity with other proteins of similar function in other organisms (cf. table 5).

Table 5

The orfό * gene has some similarity with the mdmB gene present in Streptomyces mycarofaciens (Hara and Hutchisnson, 1992; GenBank access number: A42719; BLAST score: 489) producer of macrolide antibiotic. In this producer, the gene is involved in the acylation of the lactone cycle. The orfό * gene would therefore encode an acyltransferase. This hypothesis is supported by the fact that the protein encoded by the orfό * gene has a strong similarity with other proteins of similar function in other organisms (see Table 6). Table 6

Inactivation of the orfό * gene was carried out by deletion / insertion in phase and it was shown that the resulting strain no longer produced spiramycin II and III but only spiramycin I (see FIG. 1). This confirms that the orβ * gene is indeed involved in the synthesis of spiramycin II and III. The enzyme coded by this gene is responsible for the formation of spiramycin II and III by fixing an acetyl or butyryl group on carbon 3. The strains no longer expressing the protein coded by the orfό * gene are particularly interesting since they no longer produce spiramycin II and III but only spiramycin I. As stated above, the antibiotic activity of spiramycin I is clearly superior to that of spiramycin II and III (Un et al, 1999) .

The orβ * gene encodes a protein with relatively strong similarity to several O-methyltransferases. In particular, the protein encoded by orβ * has significant similarity with an O-methyltransferase (EC 2.1.1.-) MdmC of Streptomyces mycarofaciens (Hara & Hutchinson, 1992; GenBank access number: B42719; BLAST score: 355). This similarity with a protein involved in the biosynthetic pathway of another antibiotic strongly suggests that the orβ * gene also encodes an O-methyltransferase. The orβ * gene is implicated in the formation of precursors incorporated into the lactone cycle. Indeed, according to the sequence comparisons, the product of the orβ * gene is also relatively close to FkbG which is responsible for the methylation of hydroxymalonyl-ACP according to (Wu et al, 2000; Hoffrneister et al, 2000 ; GenBank access number: AAF86386; BLAST score: 247) (see Figure 8). This hypothesis is supported by the fact that the protein encoded ^'? by the orβ * gene has a strong similarity with other proteins of similar function in other organisms (see Table 7).

Table 7

Thanks to the polar effect of the insertion of a non-excised cassette into the orfό * gene, it could be determined that the orf 5 * gene is essential for the biosynthesis pathway of spiramycins. In fact, the insertion of the excisable cassette into the coding part of the orfό * gene leads to a total halt in the production of spiramycins. However, once the inserted cassette has been excised (and therefore when only the orfό * gene is inactivated, cf. examples 14 and 15), there is a production of spiramycin I. This shows that the 5 * orf gene is essential to the biosynthetic pathway of spiramycins since its inactivation results in a total halt in the production of spiramycins.

The orβ * gene encodes a protein with relatively strong similarity to several O-methyltransferases. The orβ * gene is an O-methyl transferase involved either directly in the synthesis of platenolide or in the synthesis of a precursor _^ methylated (methoxymalonyl, see Figure 8) incorporated into the platenolide by the PKS. To verify this hypothesis, CL / SM and NMR analysis experiments were carried out on a strain of S. ambofaciens of genotype: orfό * :: àttlΩhyg +. In this strain, the orβ * gene is not expressed because of the polar effect of the insertion, in the orfό * gene, of the cassette which contains transcription terminators (cf. example 27). This strain has been shown to produce a molecule with a similar spectrum UN

the hypothesis of the role of orβ * in the biosynthesis of spiramycins. In addition, the product corresponding to spiramycin without the methyl group has a very low microbological activity (lower by a factor of 10) compared to unmodified spiramycin, when tested on the microorganism Micrococcus luteus.

The orβ * c gene codes for a protein with relatively strong similarity to a protein encoded by the mdmA gene from Streptomyces mycarofaciens, the latter encoding a protein involved in resistance to midecamycin in this producer organism (Hara et al, 1990; Issue d '' GenBank access: A60725; BLAST score: 380). This similarity with a protein involved in the biosynthetic pathway of another antibiotic strongly suggests that the orβ * c gene also codes for a protein involved in resistance to spiramycin. More particularly, the enzyme coded by the orβ * c gene has a methyltransferase activity and is involved in resistance to spiramycin in Streptomyces ambofaciens. This gene has been shown to confer resistance of the MLS I type, resistance which is known to be due to the mono-methylation in position A2058 of the ribosomal RNA 23 S (Pernodet et al, 1996). This hypothesis is supported by the fact that the protein encoded by the orβ * c gene has a strong similarity with other proteins of similar function in other organisms (cf. table 8).

Table 8

* greater sequence similarity is associated with a BLAST plus score. ;;. "high (Altschul et al, 1990).

The orβ * gene encodes a protein with relatively strong similarity to an ABC transporter type protein in Streptomyces griseus (Campelo, 2002, GenBank access number: CAC22119; BLAST score: 191). This similarity with an ABC transporter type protein strongly suggests that the orβ * gene also codes for an ABC transporter type protein that may be involved in resistance to spiramycin. This hypothesis is supported by the fact that the protein encoded by the orβ * gene has a strong similarity with other proteins of similar function in other organisms (cf. table 9). Table 9

* greater sequence similarity is associated with a higher BLAST score (Altschul et a, 1990).

The or / 9 * gene encodes a protein with relatively strong similarity to uiιé, protein of the ABC transporter type in Streptomyces griseus (Campelo, 2002, Number;; GenBank access: CAC22118; BLAST score: 269). This similarity with an ABC transporter type protein strongly suggests that the orβ * gene also codes for an ABC transporter type protein that may be involved in resistance to spiramycin. This hypothesis is supported by the fact that the protein encoded by the orβ * gene has a strong similarity with other proteins of similar function in other organisms (cf. table 10).

Table 10

The orflO * gene encodes a protein with relatively strong similarity to a protein of unknown function. However, genes similar to orflO * are found in the middle of several groups of genes involved in the biosynthesis of antibiotics. Thus, a gene close to orflO * is found in S. coelicolor (Redenbach et al, 1996, GenBank access number: NP_627432, BLAST score: 109). A close gene (CouY) is also found in S. rishiriensis (Wang et al, 2000, GenBank access number: AAG29779, BLAST 97 score).

Genes located upstream of the genes encoding PKS; ..- ^' ,

In the DNA sequence located upstream of the genes encoding the PKS (downstream and; ': the upstream being defined by the orientation of the 5 PKS genes all oriented in the same direction) (cf. FIG. 3), 34 ORFs have been identified (see above). Thus, the 34 open reading phases of this type, occupying a region of approximately 41.7 kb (cf. SEQ ID N ° 1 having a first region of 31 kb containing 25 ORFs and SEQ ID N ° 140 having a region d '' approximately 12.1 kb of which 1.4 kb overlap the previous sequence (SEQ ID No. 1) and approximately 10.7 kb correspond to the rest of the sequence), this last part of approximately 10.7 kb containing 9 ORFs additional (including a partial sequence ORF), cf. also Figures 3 and 37 and below). A schematic representation of the organization of the region is presented in FIGS. 3 and 37. The 34 genes identified have been named: orfl, orβ, orβ, orβ, orβ, orfό, orβ, orβ, orβc, orflO, orfl le, orfl 2, orfl 3c, orfl4, orfl 5c, orfl 6, orfl 7, orfl 8, orfl 9, orβO, orβ le, orβ2c, orβ3c, orβ4c, orβSc, orβό, orβ 7, orβ8c, orβ9, orβOc, orβl, orβ2c, orβ 3 and orβ4c. In Table 11 below are presented the references to the DNA and amino acid sequence of the 34 genes identified upstream of the 5 PKS genes.

Table 11

41

The “c” added in the name of the gene indicates that the coding sequence is in the reverse orientation (the coding strand is therefore the complementary strand of the sequence given in SEQ ID No. 1 or SEQ ID No. 140 for these genes) .

When several protein sequences are indicated for a single orf, the corresponding proteins come from several possible sites for starting translation. In order to determine the function of the polypeptides identified in Table 11 above, three types of experiments were carried out: comparison of the identity of the identified sequences with sequences of known functions, gene inactivation experiments and analyzes of the spiramycin production of these mutant strains.

The protein sequences deduced from these open reading phases were first compared with those present in different databases using different programs: BLAST (Altschul et al, 1990) (Altschul et al, 1997), CD-search, COGs (Cluster of Orthologous Groups), FASTA ((Pearson W. R & DJ Lipman, 1988) and (Pearson WR, 1990), BEAUTY (Worley KC et al, 1995)), (see above). These comparisons made it possible to formulate hypotheses on the function of the products of these genes and to identify those likely to be involved in the biosynthesis of spiramycin. Table 12 shows the proteins having a strong similarity with the 34 genes located upstream of the 5 PKS genes. Table 12

* greater sequence similarity is associated with a higher BLAST score (Altschul et al, 1990). 45

Gene inactivation experiments were carried out to confirm these results. The methods used consist in performing a gene replacement. The target gene to be interrupted is replaced by a copy of this interrupted gene by a cassette conferring resistance to an antibiotic (for example apramycin or hygromycin). The cassettes used are bordered on either side by translation termination codons in all the reading phases and by transcription terminators active at Streptomyces. Insertion of the cassette into ^'the target gene can occur with or without a deletion in the target gene. The size of the regions flanking the cassette can range from a few hundred to several thousand base pairs. A second type of cassette can be used for the inactivation of genes: cassettes known as “excisable cassettes” (cf. above). The strains thus constructed were tested for their production of spiramycins.

The orfl gene encodes a protein with relatively strong similarity to

protein involved in the biosynthetic pathway of another close antibiotic strongly suggests that the orfl gene also encodes a cytochrome P450. This hypothesis is supported by the fact that the protein encoded by the orfl gene has a strong similarity with other proteins of similar function in other organisms (cf. table 13).

Table 13

* greater sequence similarity is associated with a higher BLAST score (Altschul et al., 1990).

The orβ gene encodes a protein with relatively strong similarity to a dTDP-6-deoxy-3,4-keto-hexulose isomerase from Aneuήnïbacillus thermoaerophilus (Pfoestl, A. Et al, 2003, GenBank access number: AAO06351; BLAST score : 118). This similarity strongly suggests that the orβ gene codes for an isomerase responsible for the isomerization reaction necessary for the biosynthesis of one of the sugars present in the spiramycin molecule, this sugar possibly being mycarose (cf. FIG. 5). The orβ gene was inactivated. It could be shown that the resulting strain no longer produced spiramycins. This confirms that the orβ gene is indeed involved in the biosynthesis of spiramycins.

The orβ gene encodes a protein with relatively strong similarity to several aminotransferases. In particular, the protein encoded by orβ has significant similarity with an aminotransferase from Streptomyces antibioticus involved in the biosynthesis of oleandomycin (Draeger, G., Et al, 1999; GenBank access number: AAF59939; BLAST score: 431). This similarity with a protein involved in the biosynthesis pathway of another close antibiotic strongly suggests that the orβ gene codes for a 3 amino transferase responsible for the transamination reaction necessary for the biosynthesis of one of the amino sugars of spiramycins (cf. figure 5). This hypothesis is supported by the fact that the protein encoded by the orβ gene 004/033

47

has a strong similarity with other proteins of similar function in other organisms (see Table 15).

Table 15

* greater sequence similarity is associated with a higher BLAST score (Altschul et al, 1990). ^"

The orβ gene was inactivated. It could thus be shown that the strain ^' ;; resulting no longer produces spiramycins. This confirms that the orβ gene is biéή; i | f involved in the biosynthesis of spiramycins. The enzyme encoded by this gene is therefore well responsible for a bioconversion step essential to the biosynthesis of spiramycins. The production of spiramycins can be complemented by the expression of the TylB protein of S. fradiae (cf. example 23). This demonstrates that the orβ gene codes for a 3 amino transferase responsible for the transamination reaction necessary for the biosynthesis of mycaminosis (cf. FIG. 5). As mycaminose is the first sugar to be fixed on platenolide, it is expected that the strain interrupted in orβ (OS49.67) accumulates platenolide.

Intermediaries of biosynthesis of the interrupted strain in the orβ gene were studied (cf. example 20). These experiments made it possible to demonstrate that this strain produces two forms of platenolide: platenolide A and platenolide B, the structure deduced from these two molecules is presented in FIG. 36. This strain also produces platenolide A + mycarose and platenolide B + mycarosis (see example 20 and figure 40). These compounds contain a sugar but do not contain mycaminosis. In addition, if we compare them to spiramycin (cf. Figure 1), these compounds contain mycarose instead of mycaminose. These results are in agreement with the implication of the orβ gene product in the biosynthesis of mycaminosis and its role as 3 amino transferase responsible for the transamination reaction necessary for the biosynthesis of mycaminosis (cf. FIG. 5). We can notice that the specificity of glycosylation does not seem absolute since we find molecules with mycarose fixed at the position noramally occupied by mycaminose (cf. figure 40).

The orβ gene encodes a protein with relatively strong similarity to several NDP-glucose synthetases. In particular, the protein encoded by otβ has significant similarity with an alpha-D-glucose-1-phosphate thymidylyltransferase from Streptomyces venezuelae (Xue Y and α /., 1998; GenBank access number: AAC68682; BLAST score 404). This similarity with a protein involved in the pathway of ^'- biosynthesis of another antibiotic close strongly suggests that the gene encodes a orβ ^ NDP-glucose syntéthase responsible for the synthesis of NDP-glucose necessary to lai;, biosynthesis three atypical sugars incorporated into the spiramycin molecule (see FIGS. 4, 5 and 6). This hypothesis is supported by the fact that the protein encoded by the orβ gene has a strong similarity with other proteins of similar function in other organisms (cf. table 16).

Table 16

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The orβ gene encodes a protein with relatively strong similarity to several glucose dehydratases. In particular, the protein encoded by orβ has significant similarity with a dTDP-glucose 4,6-dehydratase from Streptomyces tenebrarius (Li, T.B. Et al, 2001; GenBank access number: AAG18457, BLAST score: 476). This similarity with a protein involved in the biosynthetic pathway of another close antibiotic strongly suggests that the orβ gene codes for an NDP glucose dehydratase necessary for the biosynthesis of the three atypical sugars incorporated; , in the spiramycin molecule (see figures 4, 5 and 6). This hypothesis is supported by the fact that the protein encoded by the orβ gene has a strong similarity with other proteins of similar function in other organisms (cf. table 17).

Table 17

50

The orfό gene encodes a protein with relatively strong similarity to several thioesterases. In particular, the protein encoded by orfό has significant similarity with a thioesterase from Streptomyces avermitilis (Omura, S. Et al., 2001; .. GenBank access number: BAB69315; BLAST score: 234). This similarity to an X protein involved in the biosynthetic pathway of another close antibiotic strongly suggests that the orβ gene also codes for uαe ^ tMpestàa ^ é This hypothesis is supported ff by the fact that the protein encoded by the orfό gene strong similarity with other proteins of similar function in other organisms (see Table 18).

Table 18

The orβ gene encodes a protein with relatively strong similarity to several hexose dehydratases. In particular, the protein encoded by orβ has significant similarity to a dNTP hexose 2,3-dehydratase (encoded by the TylCNI gene) from Streptomyces fradiae involved in the biosynthesis of tylosin (Merson-Davies, LA et a, 1994; Issue number '' GenBank access: AAF29379; BLAST score: 461). This similarity with a protein involved in the biosynthesis pathway of another close antibiotic strongly suggests that the orβ gene also encodes a hexose 2-3 dehydratase necessary for the biosynthesis of two atypical sugars incorporated into the spiramycin molecule (see Figure 4 and 6). This hypothesis is supported by the fact that the protein encoded by the orβ gene has a strong similarity with other proteins of similar function in other organisms (cf. table 19).

Table 19

* greater sequence similarity is associated with a higher BLAST score (Altschul et al, 1990). 004/033

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The orβ gene encodes a protein with relatively strong similarity to several aminotransferases. In particular, the protein encoded by orβ has significant similarity with an aminotransferase probably involved in the biosynthesis of forosamine in Saccharopolyspora spinosa (Waldron, C. Et al, 2001; GenBank access number: AAG23279; BLAST score: 465). This similarity with a protein involved in the biosynthesis pathway of another close antibiotic, strongly suggests that the orβ gene codes for a 4 amino transferase responsible for the transamination reaction necessary for the biosynthesis of forosamine (see Figure 6). This hypothesis is supported by the fact that the protein encoded by the orβ gene has a strong similarity with other proteins of similar function in other organisms (cf. table 20).

Table 20

The orβ gene was inactivated. It has thus been possible to show that the resulting strain no longer produces spiramycins. This confirms that the orβ gene is indeed involved in the biosynthesis of spiramycins. The enzyme encoded by this gene is therefore well responsible for a bioconversion step essential to the biosynthesis of spiramycins. The validation of the hypothesis of the role played by the product of the orβ gene in the biosynthesis of forosamine is provided by the fact that an incativated mutant for the orβ gene produces forocidine, this mutant is therefore blocked at the forocidine stage. and does not produce neo-spiramycin (cf. FIG. 7 and example 25). These results are in 00

53

agreement with an implication of the orβ gene product in the biosynthesis of forosamine (cf. Figure 6).

The orβc gene has already been identified in Streptomyces ambofaciens and has been designated srmXpwc Geistlich et al (Geistlich, M., Et al, 1992). The similarity of the protein encoded by this gene with several methyltransferases involved in the biosynthesis pathway of other close antibiotics strongly suggests that the or9c gene codes for a methyltransferase responsible for the methylation reaction necessary for the biosynthesis of mycaminosis or forosamine (cf. . Figures 5 and 6). This hypothesis is supported by the fact that the protein encoded by the orβc gene has a strong similarity with other proteins of similar function in other organisms (cf. table 21).

Table 21

The orflO gene has already been identified in Streptomyces ambofaciens and has been designated .srmR by Geistlich et al. (Geistlich, M., Et al, 1992). The protein encoded by this gene is involved in the regulation of the biosynthetic pathway for spiramycins in Streptomyces ambofaciens. Inactivation of the orflO gene has been performed. It has thus been possible to show that the resulting strain no longer produces spiramycins. This confirms that the orflO gene is well involved in the biosynthesis of spiramycins. The protein encoded by this gene is therefore very essential for the biosynthesis of spiramycins.

On the other hand ; the starting point for the translation of orflO has been determined and it has been shown that overexpression of this gene leads to an improvement in the production of spiramycins. The translation start site corresponds to an ATG located upstream of the ATG proposed by Geistlich et al. (Geistlich, M., Et al, 1992). It has also been shown that this 5 ′ end is essential to the function of OrflO since a messenger truncated in 5 ′ is inactive (cf. example 17). To obtain the desired effect on the production of spiramycins, it is therefore essential that the overexpression of orflO is carried out while taking care not to express a messenger truncated in 5 ′ of orflO.

The orfl le gene has already been identified in Streptomyces ambofaciens and has been designated srmB by Geistlich et al (Geistlich, M. Et al, 1992) and Schoner et al (Schoner B et a, 1992). The protein encoded by this gene is involved in resistance to spiramycin in Streptomyces ambofaciens and is an ABC-type transporter.

The or72 gene encodes a protein with relatively strong similarity to several hexose dehydratases. In particular, the protein coded by orfl 2 has an important similarity with an NDP -hexose 3,4-dehydratase coded by the UrdQ gene of Streptomyces fradiae and involved in the biosynthesis of urdamycin (Hoffineister, D. Et al, 2000; Number GenBank access: AAF72550; BLAST score: 634). This similarity with a protein involved in the biosynthesis pathway of another close antibiotic strongly suggests that the orfl 2 gene codes for a 3,4 dehydratase responsible for the dehydration reaction necessary for the biosynthesis of forosamine (see Figure 6). This hypothesis is supported by the fact that the protein encoded by the orfl 2 gene has a strong similarity with other proteins of similar function in other organisms (cf. table 22). 004/033689

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Table 22

The or / 12 gene was inactivated. It could be shown that the resulting strain no longer produced spiramycins. This confirms that the orfl 2 gene is indeed involved in the biosynthesis of spiramycin. The enzyme encoded by this gene is therefore well responsible for a bioconversion step essential to the biosynthesis of spiramycin. The validation of the hypothesis of the role played by Orfl 2 in the biosynthesis of forosamine is provided by the fact that an incativated mutant for the orfl 2 gene no longer produces forosamine. However, it produces a small amount of forocidin. This mutant is therefore blocked at the forocidin stage and does not produce neo-spiramycin (cf. FIG. 7 and example 26). This mutant also produces a compound of structure presented in FIG. 38. The latter compound comprises two sugars, mycaminose and mycarose but does not contain forosamine. In addition, if we compare it to the structure of spiramycin (cf. FIG. 1), this compound contains the sugar mycarose in the expected place of the forosamine. These results are in agreement with the implication of the or / 72 'gene product in the biosynthesis of forosamine (cf. FIG. 6). It can be noted that the specificity of glycosylation is not absolute since we observe molecules in which the mycarose is fixed at the position normally occupied by forosamine (see Figure 38).

The orfl 3c gene encodes a protein with relatively strong similarity to a protein of unknown function in Streptomyces coelicolor. This protein was named SC4H2.17 (GeneBank access number: T35116; BLAST score: 619). The protein encoded by the orfl 3c gene also has a strong similarity with other proteins from other organisms (see Table 23).

Table 23

5 * greater sequence similarity is associated with a higher BLAST score (Altschul et al, 1990). No specific function has been assigned to proteins close to that encoded by orfl 3c. The orfl 3c gene was inactivated in order to study the function of this gene in the biosynthesis pathway for spiramycins in Streptomyces ambofaciens. It has been shown that the resulting strain produces spiramycins. This indicates that the orfl 3c gene is not essential for the biosynthesis of spiramycins and that it is not essential for the survival of the bacteria. The enzyme encoded by this gene is therefore not responsible for a bioconversion step essential to the biosynthesis of spiramycins.

The orfl 4 gene encodes a protein with relatively strong similarity to a putative reductase (Redenbach, M., And α /., 1996; Bentley et a, 2002; GenBank access number: CAB90862; BLAST score: 147).

Inactivation of the orfl.4 gene has been performed. It has thus been possible to show that the resulting strain no longer produces spiramycins. This confirms that the orfl4 gene is indeed involved in the biosynthesis of spiramycins. The enzyme encoded by this gene is therefore well responsible for a bioconversion step essential to the biosynthesis of spiramycins. Intermediate biosynthesis of the interrupted strain in ιë% orfl4 have been studied (cf. example 20). These experiments made it possible to demonstrate that this strain produces platenolide A but does not produce platenolide B (cf. FIG. 36).

The orfl5c gene encodes a protein with relatively strong similarity to several ketoreductases. In particular, the protein encoded by orfl 5c has significant similarity to a 3-ketoreductase in Streptomyces antibioticus (GenBak access number: T51102, BLAST score: 285). This similarity strongly suggests that the orfl 5c gene codes for a 3 keto-reductase responsible for the reduction reaction necessary for the biosynthesis of forosamine (cf. FIG. 6). This hypothesis is supported by the fact that the protein encoded by the orfl 5c gene has a strong similarity with other proteins of similar function in other organisms (cf. table 24). 58

Table 24

The orflό gene codes for a protein with relatively strong similarity to several isomerases. In particular, the protein encoded by orflό has significant similarity to an NDP hexose 3,4 isomerase in Streptomyces fradiae (Gandecha et al., 1997; GenBak access number: CAA57471, BLAST score: 209). This similarity strongly suggests that the orflό gene codes for a protein involved in the biosynthesis of one of the spiramycin sugars (see Figures 5 and 6). This hypothesis is supported by the fact that the protein encoded by the orflό gene has a strong similarity with other proteins of similar function in other organisms (see Table 25). Table 25

The orfl 7 gene encodes a protein with relatively strong similarity to several glycosyl transferases. In particular, the protein encoded by orfl 7 has significant similarity with a glycosyl transferase from Streptomyces venezuelae (Xue, Y. I and α /., 1998; Access number ^{^} GenBank: AAC68677; BLAST score: 400). ^' The similarity of the protein encoded by the orfl 7 gene with several glycosyl transferases involved in the biosynthesis pathway of other close antibiotics strongly suggests that this gene also encodes a glycosyl transferase. This hypothesis is supported by the fact that the protein encoded by the orfl 7 gene has a strong similarity with other proteins of similar function in other organisms (cf. table 26).

Table 26

The orfl 8 gene encodes a protein with relatively strong similarity to several glycosyl transferases. In particular, the protein encoded by orfl 8 has significant similarity with a glycosyl transferase from Streptomyces rishiriensis (Wang et al, 20Q0; GenBank access number: AAG29785; BLAST score: 185). The similarity of the protein coded by the orfl8 gene with several glycosyl transferases involved in the detection pathway with antibiotics: relatives strongly suggests that this gene also codes for a glycosyl transferase. This hypothesis is supported by the fact that the protein encoded by the or / 18 gene has a strong similarity with other proteins of similar function in other organisms (cf. table 27).

Table 27

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The or / 79 gene encodes a protein with relatively strong similarity to. several ketoreductases. Notably, the protein encoded by orfl 9 has a similarity> important with NDP-hexose-4-ketoreductase (TylCIV) Streptomyces _fradiae:; (Bâte et α /., 2000; GenBank access number: AAD41822; BLAST score: 266). The . :;, similarity of the protein coded by the or / 19 gene with this ketpreductase involved in the biosynthetic pathway of a close antibiotic strongly suggests that this gene ^" coded ^ also a 4-keto-reductase responsible for the necessary reduction reaction to the biosynthesis of mycarosis (see Figure 4). This hypothesis is supported by the fact that the protein encoded by the orfl 9 gene has a strong similarity with other proteins of similar function in other organisms (see Table 28 ).

Table 28

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The orβO gene encodes a protein with relatively strong similarity to several hexose reductases. In particular, the protein encoded by orβO has significant similarity with the EryBII gene of Saccharopolyspora erythraea which codes for a dTDP-4-keto-L-6-deoxy-hexose 2,3-reductase (Summers, RG, et al., 991) , GenBank Access Number: AAB84068; BLAST score: 491). The similarity of the protein encoded !: by the or / 20 gene with several hexose reductases involved in the biosynthesis pathway of other close antibiotics strongly suggests that this gene codes for a 2-3 reductase responsible for the reduction reaction necessary for biosynthesis mycarosis (see Figure 4). This hypothesis is supported by the fact that the protein encoded by the orβO gene has a strong similarity with other proteins of similar function in other organisms (cf. table 29).

Table 29

63

The orβlc gene codes for a protein with relatively strong similarity to several hexose methyltransferases. In particular, the protein encoded by orβlc has significant similarity with the TylCIII gene from Streptomyces fradiae which codes for a

methyltransferase responsible for the methylation reaction necessary for the biosynthesis of mycarosis (see Figure 4). This hypothesis is supported by the fact that the protein encoded by the orβlc gene has a strong similarity with other proteins of similar function in other organisms (cf. table 30).

Table 30

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The orβ2c gene codes for a protein with relatively strong similarity to the protein encoded by the fkbH gene from Streptomyces hygroscopicus var. ascomyceticus which codes for an enzyme involved in the biosynthesis of methoxymalonyl (Wu, K. and α /., 2000; GenBank access number: AAF86387; BLAST score: 463). The similarity of the protein encoded by the orβ2c gene with this protein involved in the biosynthetic pathway of another close macrolide strongly suggests that this gene also codes for an enzyme involved in the biosynthesis of methoxymalonyl in Streptomyces ambofaciens (see Figure 8).

The or / 23c gene encodes a protein with relatively strong similarity to the,

'' Protein encoded by the fkbl gene of Streptomyces hygroscopicus var. ascomyceticus which codes for an acyl CoA dehydrogenase involved in the biosynthesis of methoxymalonyl (Wu, K. et al, 2000; GenBank access number: AAF86388; BLAST score 387). The similarity of the protein coded by the orβ 3c gene with several acyl CoA dehydrogenases involved in the biosynthesis pathway of other close antibiotics strongly suggests that this gene codes for an acyl CoA dehydrogenase involved in the biosynthesis of methoxymalonyl (cf. FIG. 8). This hypothesis is supported by the fact that the protein encoded by the orβ 3c gene has a strong similarity with other proteins of similar function in other organisms (cf. table 31). 004/033

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Table 31

The orβ4c gene codes for a protein with relatively strong similarity to the protein encoded by the fkbJ gene from Streptomyces hygroscopicus var. ascomyceticus which, ^' - :, would code the binding protein of the acyl group (Acyl Carrier Protein (ACP)) ^' ,, y- involved in the biosynthesis of methoxymalonyl (Wu, K., et al, 2000; GenBank access number : ÀAJF86389; BLAST score: 87). The siώilitude of the protein ^' cόà ξli. by the or / 24c gene with this protein involved in the biosynthesis pathway of another close macrolide strongly suggests that this gene codes for a protein involved in the biosynthesis of methoxymalonyl in Streptomyces ambofaciens (see Figure 8).

The orβ5c gene codes for a protein with relatively strong similarity to the protein encoded by the βbK gene from Streptomyces hygroscopicus var. ascomyceticus which codes for an acyl CoA dehydrogenase involved in the biosynthesis of methoxymalonyl (Wu, K., et al, 2000; GenBank access number: AAF86390; BLAST score: 268). The similarity of the protein encoded by the orβ5c gene with several acyl CoA dehydrogenases involved in the biosynthesis pathway of other close antibiotics strongly suggests that this gene codes for an acyl CoA dehydrogenase involved in the biosynthesis of methoxymalonyl (cf. FIG. 8). This hypothesis is supported by the fact that the protein encoded by the orβ5c gene has a strong similarity with other proteins of similar function in other organisms (cf. table 32). 66

Table 32

The orβό gene codes for a protein with an identity of 65% (determined thanks to the BLAST program) with the protein coded by the tylCV gene which codes for a mycarosyl transferase involved in the tylosin biosynthesis in Streptomyces fradiae (Bate, N. Et al, 2000 ; GenBank access number: AAD41824, BLAST score: 471). More particularly, TylCV is a glycosyl transferase which binds the mycarose molecule during the synthesis of tylosin. This similarity with a protein involved in the biosynthetic pathway of another relatively close antibiotic and more particularly in the transfer of mycarosis, suggests that the orβό gene codes for a glycosyl transferase. This hypothesis is supported by the fact that the protein encoded by the orβό gene has a strong similarity with other proteins of similar function in other organisms (cf. table 33). 67

Table 33

The orβ 7 gene codes for a protein with an identity of 70% (determined thanks to the BLAST program) with the protein coded by the tylCVII gene which codes for an NDP- 3,5- (or 5-) epimerase hexose involved in the biosynthesis of tylosin at;. Streptomyces fradiae (Bate, N. And, ύf., 2000; GenBank access number: AAD41825j BLAST score: 243) ^ More specifically, TylCVII is a 3,5- (or 5-i epimerase hexose involved the biosynthesis of mycarosis. This similarity to a protein involved in the biosynthesis pathway of another relatively close antibiotic and more particularly in the biosynthesis of mycarosis, suggests that the orβ7 gene codes for an epimerase. This hypothesis is supported by the fact that the protein encoded by the orβ 7 gene exhibits strong similarity with other proteins of similar function in other organisms (see Table 34) .An analysis of close sequences obtained thanks to the BLAST program strongly suggests that the orβ7 gene codes for a 5 epimerase responsible for the epimerization reaction necessary for the biosynthesis of mycarosis (see Figure 4). 68

Table 34

The sequence of ^"Orβ8c has initially been partially determined, i: since the sequence of a region of about 450 base pairs was determined after resequencing (this region is symbolized by" N "In the incomplete sequence SEQ ID No. 106). The partial sequence of this ORF (SEQ ID No. 111) was nevertheless used for analysis with the various computer programs as explained above. It was thus possible to determine that the orβ8c gene codes for a protein with an identity of 64% over the determined sequence (SEQ ID No. 112, which is the partial sequence of the Orf28c protein) (determined thanks to the program

BLAST) with the protein coded by the acyB2 gene which codes for a regulatory protein involved in the biosynthesis of carbomycin in Streptomyces thermotolerans

(Arisawa, A., Et al, 1993; GenBank access number: JC2032, BLAST score: 329). This similarity with a protein involved in the biosynthetic pathway of a relatively close antibiotic suggests that the orβ8c gene codes for a regulatory protein involved in the biosynthesis of spiramycins. This hypothesis is supported by the fact that the protein encoded by the orβ8c gene also has a high similarity with the protein TylR which is a regulatory protein involved in the biosynthesis of tylosin in Streptomyces fradiae (Bate, N. et al, 1999; GenBank access number: AAF29380, BLAST score: 167).

The orβ8c gene could be amplified using oligonucleotides located on either side of the undetermined sequence and subcloned in an expression vector. It has thus been possible to demonstrate that the overexpression of the orβ8c gene significantly increases the production of spiramycins of the OSC2 strain (cf. example 24). This demonstrates that the overexpression of orβ8c leads to an increase in the production of spiramycins \ [and confirms its role as regulator of the biosynthetic pathway for spiramycins.

The partial sequence of orfl8c was subsequently completed and the missing region of approximately 450 base pairs was determined (cf. SEQ ID No. 140 and SEQ ID No. 141). The complete sequence of this ORF (SEQ ID No. 141) was used for analysis with the various computer programs as explained above. It has thus been possible to determine that the orf28c gene codes for a protein having an identity of 69% on the; ^;

15 determined sequence (SEQ ID NO: 142, which is the complete protein sequence

: -u Or £ 28c) (determined thanks to the BLÂST program) with the protein coded by the gee ^' acyB2 which codes a regulatory protein involved in the biosynthesis of carbomycin in Streptomyces thermotolerans (Arisawa, A., et al, 1993; Number d '' GenBank access: JC2032, BLAST score: 451). This similarity to a protein involved

20 in the regulation of the biosynthesis of a relatively close antibiotic suggests that the orβ8c gene codes for a regulatory protein involved in the biosynthesis of spiramycins. This hypothesis is supported by the fact that the protein encoded by the orβ8c gene also has a strong similarity with the protein TylR which is a regulatory protein involved in the regulation of tylosin biosynthesis in Streptomyces

25 fradiae (Bate, N. Et al, 1999; GenBank access number: AAF29380, BLAST score: 224). The results of the overexpression of this gene (cf. example 24) confirm its role as regulator of the biosynthetic pathway for spiramycins. 004/033689

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The orβ8c gene was inactivated. It has thus been possible to show that the resulting strain no longer produces spiramycins. This confirms that the orβ8c gene is indeed involved in the biosynthesis of spiramycins and is essential for the biosynthesis of spiramycins. These results associated with the results of the overexpression of this gene (cf. example 24) are in agreement with a role of activator essential to the biosynthesis of the spiramycins of Orf28c.

The orβ9 gene encodes a protein with an identity of 31% (determined thanks to the BLAST program) with a probable glycosyl hydrolase located in the group, of genes involved in the biosynthesis of soraphen A (an antifungal of the polyketide class) in Sorangium cellulosum (Ligon, J., Et al, 2002; GenBank access number: AAK19890, BLAST score: 139). This similarity with a protein involved in the biosynthetic pathway of a relatively close molecule suggests that the or / 29 gene codes for a protein with glycosyl hydrolase activity. This assumption; ; is supported by the fact that the protein encoded by the orβ9 gene has a strong X- similarity with other proteins of similar function in other organisms (cf. table 35). ,; , An analysis of the sequence of the protein encoded by orβ9 using the CD search program (see above) also suggests that the or / 29 gene codes for a glycosyl hydrolase! ^' ' - •

Table 35

Analysis of the protein sequence deduced from Vorβ9 by the SignalP program (http://www.cbs.dtu.dk/services/SignalP/) (Nielsen, H., Et a, 1997) shows that this 004/033689

71

protein has a C-terminal signal sequence with a predicted cleavage site between positions 30 and 31 (QSA / QA). We can predict that this protein is extra-cellular. It could, as a glycosyl hydrolase, have a role in the reactivation of spiramycin inactivated by glycosylation by the glycosyl transferases GimA and / or GimB (Gourmelen et al, 1998).

The orβOc gene encodes a protein with an identity of 31% (determined thanks to the BLAST program) with a sugar epimerase-nucleoside-diphosphate of Corynebacterium glutamicum (GenBank access number: NP 600590, BLAST score: 89). This similarity suggests that the orβOc gene codes for an epimerase. This hypothesis is supported by the fact that an analysis of the sequences using the CD-search program (cf. above) also suggests that the orβOc gene codes for an epimerase.

VorβOc presents two possible initiation codons (cf. SEQ ID No 115) which give two possible proteins of 345 and 282 amino acids respectively (SEQ ID No 116 and 117). However, the use of codons is typical of Streptomyces only at ^' J / ^' from the second ATG moreover, the protein sequence deduced from the sequence between | first ATG and the second born do not lie down with the identified close sequences, ^' l, || whereas the shortest protein sequence (from the 2 ^nd ATG: SEQ ID No. 144) aligns well with the start of these proteins. We can therefore deduce that the second ATG is the correct initiation codon and that the sequence of this orf is therefore that presented in SEQ ID No. 143 which once translated corresponds to the protein of sequence SEQ ID No. 144.

The orβ1 gene codes for a protein with an identity of 52% (determined thanks to the BLAST program) with an oxidoreductase in Streptomyces coelicolor (GenBank access number: NP_631148, BLAST score: 261). This similarity suggests that the orβ1 gene codes for a reductase. This hypothesis is supported by the fact that an analysis of the sequences using the CD-search program (cf. above) also suggests that the orβ1 gene codes for a reductase. This assumption is also supported by the fact that the protein encoded by the orβ1 gene has a strong similarity with other proteins of similar function in other organisms (see Table 36).

Table 36

* greater sequence similarity is associated with a higher BLAST score 5 (Altschul et al, 1990).

The orβ1 gene was inactivated. It has thus been possible to show that the resulting strain no longer produces spiramycins. This confirms that the orβ1 gene is well involved in the biosynthesis of spiramycins. The enzyme encoded by this gene is therefore g responsible for a bioconvergence step essential for the biosynthesis of? .

10 spiramycins.

The sequence d ^, orβ2c was first of all determined in part (cf. example 19), since the coding sequence in 5 ′ was only determined in a second step. The partial sequence of this orf (SEQ ID N ° 120) was nevertheless used for the analysis with the various computer programs as explained above. He has

15 thus it could be determined that the orβ2c gene codes for a protein having an identity of 47% on the determined sequence (SEQ ID No. 121, which is the partial sequence of the Orf32c protein) (determined thanks to the BLAST program) with a regulatory protein of the GntR family in Streptomyces coelicolor (GenBank access number: NP_625576, BLAST score: 229). This similarity suggests that the orβ2c gene codes for a

20 transcriptional regulator of the GntR family. This hypothesis is supported by the fact that the 73

protein encoded by the orβ2c gene has a strong similarity with other proteins of similar function in other organisms.

The partial LVorβ2c sequence was subsequently completed and the missing region was determined (cf. SEQ ID No. 140 and SEQ ID No. 145). The complete sequence of this orf encodes a protein with an identity of 44% (determined thanks to the program

BLAST) with a regulatory protein of the GntR family in Streptomyces avermitilis (GenBank access number: NP_824604, BLAST score: 282). This similarity suggests that the orβ2c gene codes for a transcriptional regulator of the GntR family. This hypothesis is supported by the fact that the protein encoded by the orβ2c gene has a strong similarity with other proteins of similar function in other organisms (cf. table 37).

Table 37

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The orβ2c gene was inactivated in order to study the function of this gene in the biosynthesis pathway for spiramycins in Streptomyces ambofaciens. It has been shown that the resulting strain produces spiramycins. This indicates that the orβ2c gene is not essential for the biosynthesis of spiramycins and that it is not essential for the survival of the bacteria.

The orβ 3 gene codes for a protein with an identity of 49% (determined through the BLAST program X) with a hypothetical protein from Xanthomonas campestris (GenBank access number: NP_635564, BLAST score: 54).

Of the ^sequence, orβ4c is partial. Indeed, the comparisons made between the product of this orf and the databases suggest that the C-terminal part of this protein is not in the product deduced from the nucleotide sequence and therefore that} this orf would be longer and would continue outside the sequenced region. The sequence <. ^' ii; f partial of this ORF was nevertheless used for the analysis with the different H computer programs as explained above. It was thus possible to determine that the ^' "-V <orβ 4c gene codes for a protein having an identity of 91% on the determined sequence (SEQ ID No. 150, which is the partial sequence of the Orf34c protein) (determined thanks to BLAST program) with an arabinofuranosidase from Streptomyces coelicolor (Bentley et al, 2002; GenBank access number: NP_630049, BLAST score: 654) In S. coelicolor the gene coding for this arabinofuranosidase does not seem to be involved in the metabolite biosynthesis In S. ambofaciens, this gene is therefore probably not involved in the biosynthesis of spiramycin.

The present invention also relates to polynucleotides which hybridize under hybridization conditions of high stringency to at least one of the polynucleotides of sequence SEQ ID No. 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 therefrom due to the degeneracy of the genetic code. Preferably, these polynucleotides are isolated from a bacterium of the genus Streptomyces, more preferentially, these polynucleotides encode proteins involved in the biosynthesis of a macrolide and even more preferentially, these polynucleotides encode a protein having an activity similar to the encoded protein by the polynucleotides with which they hybridize. The high stringency hybridization conditions can be defined as hybridization conditions which disadvantage the hybridization of strands of non-homologous nucleic acids. High stringency hybridization conditions can for example be described as: ..; hybridization conditions in the buffer described by Church & Gilbert (Church & Gilbert, 1984) at a temperature between 55 ° C and 65 ° C, preferably the annealing temperature is 55 ° C, even more preferred temperature

0.5X SSC at a temperature between 55 ° C and 65 ° C, preferably this temperature is 55 ° C, even more preferably this temperature is 60 ° C and most preferably this temperature is 65 ° C. The hybridization conditions described above can be adapted according to the length of the nucleic acid whose hybridization is sought or the type of labeling chosen, according to techniques known to those skilled in the art. The suitable hybridization conditions can for example be adapted according to the work by F. Ausubel et al, 2002.

The invention also relates to a polynucleotide having at least 70%, more preferably 80%, more preferably 85%, even more preferably 90%, even more preferably 95% and most preferably 98 % identity in nucleotides with a polynucleotide comprising at least 10, 12, 15, 18, 20 to 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 d a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID N ° 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 therefrom due to the degeneracy of the genetic code, or a polynucleotide of complementary sequence. Preferably, these so-called polynucleotides are isolated from a bacterium of the genus Streptomyces, more preferentially, these polynucleotides encode proteins involved in the biosynthesis of a macrolide and even more preferentially, these polynucleotides encode proteins having activities similar to the proteins encoded by the polynucleotides with which they present identity. Most preferably, a polynucleotide according to the invention is chosen from the group consisting of nucleotide sequences SEQ ID No. 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 w | polynucleotide of sequence mpl * enmémentt. ^' -ïfi ^' %

The optimal alignment of the sequences for the comparison can be achieved by computer using known algorithms, for example those of the FASTA package (Pearson W. R & DJ Lipman, 1988) and (Pearson WR, 1990), accessible notably with the INFOBIOGEN resource center, Evry, France. As an illustration, the percentage of sequence identity can be determined using the LFASTA software (Chao K.-M. et al., 1992) or LALIGN (Huang X. and Miller W., 1991). The LFASTA and LALIGN programs are part of the FASTA package. LALIGN returns the optimal local alignments: this program is more rigorous, but also slower than LFASTA.

Another aspect of the invention relates to a polypeptide resulting from the expression of a nucleic acid sequence as defined above. Preferably, the polypeptides according to the invention have at least 70%, more preferably 80%, of more preferably 85%, even more preferably 90%, even more preferably 95% and most preferably 98% of amino acid identity with a polypeptide comprising at least 10, 15, 20, 30 to 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 'chosen from SEQ ID N ° 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 that throughout said sequence one or H; / ;, several amino acids have been substituted, inserted or deleted without affecting the functional properties or one of the variants of these sequences. Preferably, the polypeptides according to the invention are expressed in the natural state by a bacterium of the genus Streptomyces, more preferably, these polypeptides are involved in the biosynthesis of a macrolide and even more preferably, these polypeptides have a

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 l one of these sequences except that throughout said sequence one or more amino acids have been substituted, inserted or deleted without affecting the functional properties or one of the variants of these sequences. Most preferably, a polypeptide according to the invention is chosen from the group consisting of polypeptide sequences SEQ ID No. 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 optimal alignment of the sequences for the comparison can be carried out by computer using known algorithms, for example those of the package FASTA (Pearson W. R & DJ Lipman, 1988) and (Pearson WR, 1990), available in particular from the INFOBIOGEN resource center, Evry, France. By way of illustration, the percentage of sequence identity can be determined using the LFASTA software (Chao K.-M. et a, 1992) or LALIGN (Huang X. and Miller W., 1991), in using the default settings as defined by the INFOBIOGEN resource center, Evry, France. The LFASTA and LALIGN programs are part of the FASTA package. LALIGN returns the optimal local alignments: this program is more rigorous, but also slower than LFASTA.

Another aspect of the invention relates to a recombinant DNA comprising at least one polynucleotide as described above. Preferably; this recombinant DNA is a vector. Even more preferably, the vector is chosen from bacteriophages, plasmids, phagemids, integrative vectors, fosmids,, cosmids, shuttle vectors, BAC (Bacterial Artificial Chromosome) or PAC (Pl-derived Artificial Chromosome) . By way of illustration, mention may be made, like bacteriophages, of phage lambda and phage M13. As plasmids, mention may be made of; plasmids replicating in E. coli for example pBR322 and its derivatives, pUC18 and its; ij, derivatives, pUC19 and its derivatives, pGB2 and its derivatives (Churchward G. et al, 1984), ρACYC177 (GenBank access number: X06402 ) and its derivatives, ρACYC184 (GenBank access number: X06403) and its derivatives. Mention may also be made of the plasmids replicating in Streptomyces such as for example pIJ101 and its derivatives, pSG5 and its derivatives, SLP1 and its derivatives, SCP2 * and its derivatives (Kieser et al. 2000). As phagemids there may be mentioned by way of illustration pBluescript II and its derivatives (marketed in particular by the company Stratagene (LaJolla, California, USA)), pGEM-T and its derivatives (marketed by the company Promega (Madison, Wisconcin, USA)), λZAPII and its derivatives (sold in particular by the company Stratagene (LaJolla, California, USA)). As integrative vectors there may be mentioned by way of illustration, the integrative vectors in Streptomyces such as for example those derived from SLP1 (Kieser et al, 2000), those derived from pSAM2 (Kieser et al, 2000), the vectors using the integration systems phages PhiC31 (Kieser et al, 2000) (for example pSET152 (Bierman et al, 1992)) or VWB (Van Mellaert L, et al. 1998), as well as the vectors using the IS117 integration system (Kieser et al. 2000). As fosmids, the fosmid pFOS1 (sold by the company New England Bioloabs Inc., Beverly, Massachussetts, USA) and its derivatives may be mentioned by way of illustration. As cosmids, there may be mentioned by way of illustration the cosmid SuperCos and its derivatives (sold in particular by

5 company Stratagene (LaJolla, California, USA)), the cosmid pWED 15 (Wahl et al, 1987) and its derivatives. As shuttle vectors, mention may be made, by way of illustration, of the E. coli I Streptomyces shuttle plasmids, such as, for example, pIJ903 and its derivatives, the series of plasmids pUWL, pC AO 106, ρWHM3, pO 446 and their derivatives (Kieser et al. 2000). , the BAC shuttle E. coli I Streptomyces such as for example those described in the application; ^•

10 of patent WO 01/40497. As BAC (Bacterial Artificial Chromosome) we can cite by way of illustration the BAC pBeloBACl l (GenBank access number: U51113). As PAC (Pl-derived Artificial Chromosome), we can cite as an illustration the vector pCYPACό (GenBank access number: AF133437). Very preferably, an i vector according to the invention is chosen from ρOS49.1, pOS49.11, ρOSC49.12, ρOS49.14, _: ;

15 pOS49.16, pOS49.28, pOS44.1, pOS44.2, pOS44.4, pSPM5, pSPM7, pOS49.67, H

• m pOS49.88, pOS49.106, pOS49.120, pOS49.107, pOS49.32, pOS49.43, pOS49.44j |

} -) .. pOS49.50, PO $ 49.99, ^pSPM17,. SPM21; # 3PM502, pSPM504, pSPM507, pSPM50ffj§ pSPM509, pSPMl, pBXLll ll, pBXL1112, pBXL1113, pSPM520, pSPM52Ï, pSPM522, pSPM523, pSPM524, pSPM525, pSPM527, pSPM527

20 pSPM36, pSPM37, pSPM38, pSPM39, pSPM40, pSPM41, pSPM42, pSPM43, pSPM44, pSPM45, pSPM47, pSPM48, pSPM50, pSPM51, pSPM52, pSPM53, pSPM55, pSPM56, pSPM5 pMM pMM pMM , pSPM79, pSPM83, pSPM107, pSPM543 and pSPM106.

Another aspect of the invention relates to an expression system comprising a suitable expression vector and a host cell allowing the expression of one or more polypeptides as described above in a biological system. The expression vectors according to the invention comprise a nucleic acid sequence encoding one or more polypeptides as described above and can be intended for the expression of the different polypeptides according to the invention in various host cells well known in the art. the skilled person. By way of example, mention may be made of the systems of prokaryotic expression such as expression systems in E. coli, eukaryotic expression systems, such as the baculovirus expression system allowing expression in insect cells, expression systems allowing expression in yeast cells, expression systems allowing expression in mammalian cells, in particular human cells. The expression vectors which can be used in such systems are well known to those skilled in the art, as regards prokaryotic cells, there may be mentioned by way of illustration the expression vectors in E. coli for example, of the pΕT family . marketed by the company Stratagene (LaJolla, California, USA), the vectors of the i ;; GATEWAY family marketed by Invitrogen (Carlsbad, California, USA), pBAD family vectors marketed by Invitrogen (Carlsbad, California, USA) vectors, pMAL family vectors marketed by New England Bioloabs Inc. ( Beverly, Massachussetts, USA), the rhamnose-inducible expression vectors cited in the publication Wilms B et al, 2001 and their derivatives, there may also be mentioned the expression vectors in Streptomyces;!; such as for example the vectors ρIJ4123, pIJ6021, pPM927, ρANT849, pANT 850. - X pANT 851, ρANT1200, pANT1201, pANT1202 and their derivatives (Kieser et al, 2000). As regards yeast cells, the vector pESC sold by the company Stratagene (LaJolla, California, USA) may be cited by way of illustration. As regards the baculovirus expression system allowing expression in insect cells, the vector BacPAK6 sold by the company BD Biosciences Clontech, (Palo Alto, California, USA) may be mentioned by way of illustration. As regards mammalian cells, mention may be made, by way of illustration, of vectors comprising the promoter of the early genes of the CMV virus (Cytomegalovirus) (for example the vector pCMV and its derivatives marketed by the company Stratagene (LaJolla, California, USA) , or the promoter of the immediate genes (S V40 early promoter) of the vacuolating simian virus SV40 (for example the vector pSG5 marketed by the company Stratagene (LaJolla, California, USA).

The invention also relates to a method for producing a polypeptide as described above, said method comprising the following steps: 004/033689

81

a) inserting a nucleic acid encoding said polypeptide into an appropriate vector

b) cultivating, in an appropriate culture medium, a host cell previously transformed or transfected with the vector of step a);

c) recovering the conditioned culture medium or a cell extract, for example by sonication or by osmotic shock;

d) separating and purifying from said culture medium or also from the cell extract obtained in step c), said polypeptide;

e) where appropriate, characterize the recombinant polypeptide produced.

A recombinant polypeptide according to the invention can be purified by passage through an appropriate series of chromatography columns, according to the methods known to those skilled in the art and described for example in FΛusubel et al (2002). By way of illustration, mention may be made of the “Tag-Histidine” technique, which consists in adding a short poly-histidine sequence to the polypeptide to be produced, the latter then being able to be; purified on a nickel column. A polypeptide according to the invention can also be prepared by in vitro synthesis techniques. By way of illustration of such techniques, a polypeptide according to the invention may be prepared using the "rapid translation System (RTS)" system, sold in particular by the company Roche Diagnostics France S.A, Meylan, France.

Another aspect of the invention relates to host cells into which at least one polynucleotide and / or at least one recombinant DNA and / or at least one expression vector according to the invention has been introduced.

Another aspect of the invention relates to microorganisms blocked in a stage of the biosynthetic pathway of at least one macrolide. The interest resides on the one hand in the study of the function of the mutated proteins and on the other hand in the realization of microorganisms producing intermediates of biosynthesis. These intermediaries may be modified, possibly after separation, either by adding particular components to the production media, or by introducing into the microorganisms thus mutated other genes coding for proteins capable of modifying the intermediate by using it as a substrate. These intermediates can thus be modified chemically, biochemically, enzymatically and / or microbiologically. The microorganisms blocked in a stage of the macrolide biosynthesis pathway can be obtained by inactivating the function of one or more proteins involved in the biosynthesis of this or these macrolides in microorganisms producing this or these macrolide. According to the protein or inactivated, so we can _get:, microorganisms blocked in the various stages of the biosynthetic pathway of this or these macrolide. The inactivation of this protein or these proteins can be carried out by any method known to those skilled in the art, for example by mutagenesis in the gene or genes encoding said protein (s) or by the expression of one or more anti RNA -sense complementary to the RNA or messenger RNAs encoding said protein or proteins. Mutagenesis can, for example, be carried out by irradiation, by the action of a mutagenic chemical agent, by site-directed mutagenesis, by gene replacement,]; ,: or any other known method ^{* 'I} like in the art. The suitable conditions ^for such a mutagenesis can, for example, be adapted according to the teaching contained in the works of Kieser, T et al (2000) and Ausubel et al., (2002). Mutagenesis can be carried out in vitro or in situ, by deletion, substitution, deletion and / or addition of one or more bases in the gene considered or by gene inactivation. This mutagenesis can be carried out in a gene comprising a sequence as described above. Preferably, the microorganisms blocked in a stage of the macrolide biosynthesis pathway are bacteria of the genus Streptomyces. More preferably, the inactivation of the function of one or more proteins involved in the biosynthesis of the macrolide (s) in question is carried out by mutagenesis. Even more preferably, the macrolide in question is spiramycin and the microorganisms in which the mutagenesis are carried out 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 N ° 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. From preferably, the mutagenesis (s) are carried out by gene inactivation. Most preferably, mutagenesis consists in the gene inactivation of a gene comprising a sequence corresponding to the sequence SEQ BD No. 13.

By way of illustration, the following microorganisms can be cited as an example of such microorganisms: OS49.16 (orβ :: Ωhyg, cf. example 2), OS49.67 (orβdeletion in phase, cf. example 6), OS49. 107 (orβ :: Ωhyg, see example 7), OS49.50 (orfl 0 :: Ωhyg, see example 8), SPM21 (orβ :: att3Ωaac-, see example 10), SPM22 (orβ :: att3 deletion in phase, see example 10), SPM501 (orfό * :: attlΩhyg +, see example 14), SPM502 (orfό * :: attl deletion in phase, see example 14), SPM507. (orfl2 :: att3Ωaac-, see example 11), SPM508 (orfl3c :: att3Ωaac-, see example 12), SPM509 (orfl4 :: att3Ωaac-, see example 13), SPM107 (orβ8c :: att3aac +, see example; 29), SPM543 (orβl :: att3aac +, see example 30), SPM106 (orβ2c :: att3aac +, see example 31). . èà

Another aspect of the invention relates to a process for the preparation of a macrolide biosynthesis intermediate using microorganisms blocked in a step of the macrolide biosynthesis pathway, as described above. The method consists in cultivating, in an appropriate culture medium, a microorganism blocked in a step of the macrolide biosynthesis pathway, as described above, recovering the conditioned culture medium or a cell extract, for example by sonication or by osmotic shock, separate and purify from said culture medium or from the cell extract obtained in the previous step, said biosynthesis intermediate. The culture conditions for such microorganisms can be determined according to techniques well known to those skilled in the art. The culture medium may for example be MP5 medium or SL11 medium for Streptomyces and in particular for Streptomyces ambofaciens (Pernodet et al, 1993). Those skilled in the art may in particular refer to the work by Kieser et al. (2000) regarding culture Streptomyces. The product intermediate can be recovered by any techniques known to those skilled in the art. Those skilled in the art may refer, for example, to the techniques taught in US patent US 3,000,785 and more particularly to the methods of extraction of spiramycins described in this patent.

Another object of the invention relates to a process for the preparation of a molecule derived from a macrolide using the microorganisms blocked in a stage of the pathway for biosynthesis of this macrolide, as described above. The method consists in obtaining a biosynthesis intermediate according to the above method and in modifying the intermediate thus produced, possibly after separation from the culture medium. The culture conditions for such microorganisms can be determined according to techniques well known to those skilled in the art. The culture medium could for example be MP5 medium or SL11 medium for Streptomyces and in particular for Streptomyces ambofaciens (Pernodet et al, 1993). Those skilled in the art may in particular refer to the work by Kieser et al. 2000 with regard to the culture of Streptomyces. The intermediates produced can be modified, optionally after separation, either by adding appropriate components to the production media, or by introduction into microorganisms of other genes coding for proteins capable of modifying the intermediary by using them as a substrate. These intermediates can thus be modified chemically, biochemically, enzymatically and / or microbiologically. More preferably, the macrolide in question is spiramycin and the microorganisms in which the mutagenesis are carried out are strains of S. ambofaciens.

The invention also relates to a microorganism producing spiramycin I but not producing spiramycin II and III. This microorganism comprises all of the genes necessary for the biosynthesis of spiramycin I but does not produce spiramycin II and III because the gene comprising the sequence corresponding to SEQ ID No. 13 or one of its variants or one of the sequences derived therefrom due to the degeneracy of the genetic code and encoding a polypeptide of sequence SEQ ID No. 14 or one of its variants is not expressed or has been rendered inactive. The inactivation of this protein can be carried out by any known method of those skilled in the art, for example by mutagenesis in the gene encoding said protein or by the expression of an antisense RNA complementary to the messenger RNA encoding said protein, it being understood that if the expression of orβ * is affected by this manipulation, it will be necessary to make another modification so that the orβ * gene is correctly expressed. Mutagenesis can be carried out in the coding sequence or in a non-coding sequence so as to render the eodinated protein inactive or to prevent its expression or translation. Mutagenesis can be carried out by site-directed mutagenesis, by gene replacement or any other method known to those skilled in the art. The suitable conditions for such a mutagenesis can, for example, be adapted according to the teaching contained in the works of Kieser, T et al (2000) and Ausubel et al, 2002. The mutagenesis can be carried out in vitro or in situ, by suppression , substitution, deletion and / or addition of one or more bases in the gene under consideration or by gene inactivation. The microorganism can also be obtained by expressing the genes of the spiramycin biosynthetic pathway, without these comprising the gene comprising the sequence SEQ ID No. 13 or one of its variants or one of the sequences derived therefrom. of these due to the degeneration of the X genetic code and coding a .x poxly-pe .pt • id • e. of. s -éque .nc ^• e.- ^• = SE -Q, LD. NOT . °. - 14 or. one of s xésW ^' variants. Preferably, the microorganism is a bacterium of the genus Streptomyces. More preferably, the microorganism producing spiramycin I but not producing spiramycin II and III is obtained from a starting microorganism producing spiramycins I, II and III. Even more preferably, the microorganism is obtained by mutagenesis in a gene comprising the sequence corresponding to SEQ ID No. 13 or one of its variants or one of the sequences derived therefrom due to the degeneracy of the genetic code and encoding a polypeptide of sequence SEQ ID No. 14 or one of its variants having the same function. Even more preferably, this mutagenesis is carried out by gene inactivation. Preferably, the microorganism is obtained from a strain of S. ambofaciens producing spiramycins I, II and III in which a gene inactivation is carried out of the gene comprising the sequence corresponding to SEQ ID No. 13 or one of the sequences derived from it due to the degeneracy of the genetic code. Of most preferably, gene inactivation is carried out by deletion in phase of the gene or part of the gene comprising the sequence corresponding to SEQ ID No. 13 or one of the sequences derived therefrom due to the degeneration of the genetic code. By way of illustration, the strain SPM502 (orfό * :: attl, cf. example 14) can be cited as a microorganism producing spiramycin I but not producing spiramycin II and III.

The invention also relates to a microorganism producing spiramycin I but not producing spiramycin II and III as described above, characterized in that it additionally overexpresses: - a gene capable of being obtained by polymerase chain reaction using the pair of primers with the following sequence: 5 'AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID No 138) and 5 'GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID No 139) or as the cosmid pSPM36 or l Total DNA of Streptomyces ambofaciens, more preferably it is the gene of coding sequence SEQ ID No. 141,.:

- or a gene derived from them due to the degeneracy of the genetic code. ; -ffë

An example of the sequence of such a gene is given in SEQ ID No. 111 (DNA), however this sequence is partial since it does not comprise the 3 ′ part of the corresponding coding sequence. The translation into protein of this part of the coding sequence is given in SEQ ID No. 112. Those skilled in the art can easily complete it in particular by using the teaching presented in Example 24. The undetermined sequence in SEQ ID No. 111 was determined in a second step and the complete sequence of this orf (orβ 8c) is given in SEQ ID No. 141. The protein translation of this coding sequence is given in SEQ ID No. 142. It is presented in Example 24 a method for cloning the orβ8c gene and for manufacturing an expression vector allowing the expression of orβ8c. It is also shown in this example that the overexpression of the orβ8c gene in the OSC2 strain leads to an improvement in the production of spiramycins of this strain. The overexpression of the orβ8c gene can be obtained by increasing the number of copies of this gene and / or by placing a promoter more active than the wild-type promoter. Preferably, the overexpression of said gene is obtained by bringing into the microorganism a construction of recombinant DNA, allowing the overexpression of this gene. Preferably, this construction of recombinant DNA increases the number of copies of said gene and makes it possible to obtain an overexpression of said gene. In this recombinant DNA construct, the coding sequence of the gene can be placed under the control of a promoter which is more active than the wild type promoter. By way of illustration, mention may be made of the ptrc promoter active in Streptomyces ambofaciens (Amann, E. et al, 1988) as well as the ermE * promoter. Thus, preferably, the copy or copies of the orβ8c gene supplied are placed under the control of the ermE * promoter as is the case in the construction pSPM75 (cf. example 24).

The invention also relates to a microorganism producing \; spiramycin I but not producing spiramycin II and III as described above, characterized in that it additionally overexpresses the gene of coding sequence SEQ ID No. 47 or of a coding sequence derived therefrom because of the degeneracy of the genetic code. Preferably, this microorganism is characterized in that it is added to the strain SPM502 pSPM525 deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, le February 26, 2003 under registration number 1-2977.

The invention also relates to a method for producing spiramycin I, the method consists in cultivating in a suitable culture medium a microorganism producing spiramycin I but not producing spiramycin II and III as described above, recover the conditioned culture medium or a cell extract, separate and purify from said culture medium or also from the cell extract obtained in the preceding step spiramycin I. The conditions for culturing such a microorganism may be determined according to techniques well known to those skilled in the art. The culture medium may for example be MP5 medium or SL11 medium for Streptomyces and in particular for Streptomyces ambofaciens (Pernodet et al, 1993). A person skilled in the art may in particular refer to the work by Kieser et al. 2000 with regard to the culture of Streptomyces. The spiramycin I produced can be recovered by any techniques known to those skilled in the art. Those skilled in the art may refer, for example, to the techniques taught in US patent US 3,000,785 and more particularly to the methods of extraction of spiramycins described in this patent.

Another aspect of the invention relates to the use of a nucleotide sequence according to the invention for improving the production of macrolides by a microorganism. Thus, the invention relates to a mutant macrolide-producing microorganism characterized in that it has a genetic modification in at least one gèήè] ^' - ^' comprising a sequence as defined above and / or that it overexpresses at least one gene comprising a sequence as defined above. The genetic modification can consist in a suppression, a substitution, a deletion and / or an addition of one or more bases in the genes considered in order to express one or more ^' -. ^: proteins with higher activity or express a higher level of this or ^'' -i these proteins. Overexpression of the gene under consideration can be obtained by increasing the g; number of copies of this gene and / or by placing a promoter more active than the prompter

• '- -. ^"' .î'H'i-Wiu' ^* wild. Mention may be made, by way of illustration, of the ptrc promoter active in Streptomyces' ambofaciens (Amann, E. et al, 1988) as well as the ermE * promoter (Bibb et al , 1985),

(Bibb et al, 1994). Thus, the overexpression of the gene considered can be obtained by bringing into a microorganism producing the macrolide considered a construction of recombinant DNA according to the invention, allowing the overexpression of this gene. Indeed, certain stages of macrolide biosynthesis are limiting and if one or more more active proteins or a higher level of expression of the wild protein (s) involved in these limiting stages are expressed, the production of the or the macrolides concerned. An increase in the rate of production of tylosin has for example been obtained in Streptomyces fradiae by duplication of the gene encoding a limiting methyl transferase converting macrocin into tylosin (Baltz R, 1997). Obtaining the expression of a more active protein can be obtained in particular by mutagenesis, a person skilled in the art may for example refer to this subject in the work by F. Ausubel et al (2002). Preferably, these mutant microorganisms improved for their production in macrolides are bacteria of the genus Streptomyces. More preferably, the macrolide in question is spiramycin and the microorganisms in which the mutagenesis are carried out 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 No. 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 due to the degeneracy of the genetic code. Preferably, the microorganism overexpresses one or more genes comprising one of the sequences corresponding to one or more of the sequences SEQ ID No. 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 therefrom due to the. degeneration of the genetic code. Preferably, the microorganism overexpresses the gene comprising a sequence corresponding to SEQ ID No. 111 or 141, or one of its% variants or one of the sequences derived therefrom due to the degeneration μj | $ dd genetic code. The sequence given in SEQ ID N ° 111 is partial, however the person skilled in the art can easily complete it in particular by using the teaching presented in example 24. The undetermined sequence in SEQ ID N ° 111 was determined in a second time and the complete sequence of this orf (orβ8c) is given in SEQ ID No. 141. The protein translation of this coding sequence is given in SEQ ID No. 142. Example 24 is thus presented a method for cloning the orβ8c gene and to manufacture an expression vector allowing the expression of orβ8c. It is also shown in this example that the overexpression of the orβ8c gene in the OSC2 strain leads to an improvement in the production of spiramycins of this strain. The overexpression of the orβ8c gene can be obtained by increasing the number of copies of this gene and / or by placing a promoter more active than the wild-type promoter. Preferably, the overexpression of the orβ8c gene is obtained by bringing a recombinant DNA construct into the microorganism, allowing the overexpression of this gene. Preferably, this construction of recombinant DNA increases the number of copies of the orβ 8c gene and makes it possible to obtain an overexpression of the orβ 8c gene. In this recombinant DNA construct, the coding sequence for orβ 8c can be placed under the control of a promoter more active than the wild-type promoter. By way of illustration, mention may be made of the ptrc promoter active in Streptomyces ambofaciens (Amann, E. et al, 1988) as well as the ermE * promoter. Thus, preferably, the copy or copies of the orβ8c gene supplied are placed under the control of the ermE * promoter as is the case in the construction pSPM75 (cf. example 24).

Another aspect of the invention relates to a process for producing macrolides using the microorganisms described in the preceding paragraph. This process consists in cultivating in a suitable culture medium a microorganism defined in the preceding paragraph, recovering the conditioned culture medium or a cell extract, separating and purifying from said culture medium or from the extract • '. cell obtained in the previous step the macrolide (s) produced. The conditions of X ^'culture of such microorganisms may be determined using techniques well known to ^¬ _t in the art. The culture medium may for example be miliefrj I MP5 or SL11 medium for Streptomyces and in particular for Streptomyces ^' X ambofaciens (Pernodet et al., 1993). Those skilled in the art may in particular refer to the work by Kieser et al. 2000 with regard to the culture of Streptomyces. The macrolide (s) produced can be recovered by any technique known to a person skilled in the art. Those skilled in the art may refer, for example, to the techniques taught in US patent US 3,000,785 and more particularly to the methods of extraction of spiramycins described in this patent. Preferably, the microorganisms used in such a process are bacteria of the genus Streptomyces. More preferably, the macrolide in question is spiramycin and the mutant microorganisms improved for their production in spiramycins are strains of S. ambofaciens.

Another aspect of the invention relates to the use of a sequence and / or of a vector according to the invention for the preparation of hybrid antibiotics. Indeed, the 004/033689

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polynucleotides according to the invention can be used to obtain microorganisms expressing one or more mutant proteins giving rise to a modification in the specificity of the substrate or also be expressed in multiple microorganisms producing antibiotics with the aim of generating hybrid antibiotics. Thus, the polynucleotides according to the invention can make it possible, by transfer of genes between producing organisms, to manufacture hybrid antibiotics having pharmacologically advantageous properties (Hopwood et al, 1985a, Hopwood et al, 1985b, Hutchinson and α /., 1989). The principle by which genetic engineering can lead to the production of hybrid antibiotics was first proposed by 'Hopwood (Hopwood 1981). It has thus been proposed that the enzymes participating in the biosynthesis of antibiotics often accept structurally linked substrates, but different from their natural substrate. It is generally accepted (Hopwood 1981, Hutchinson 1988, Robinson 1988) that the enzymes coded by the genes of the antibiotic biosynthetic pathway have a less specific substrate specificity than the enzymes

(Hutchinson 1988). Using this teaching, a person skilled in the art will be able to construct microorganisms expressing one or more mutant proteins giving rise to a modification in the specificity of the substrate in order to generate hybrid antibiotics.

The invention also relates to the use of at least one polynucleotide and / or at least one recombinant DNA and / or at least one expression vector and / or at least one polypeptide and / or at least one host cell according to the invention. invention for carrying out one or more bioconversions. Thus, the invention makes it possible to construct bacterial or fungal strains in which one or more proteins according to the invention are expressed, under the control of appropriate expression signals. Such strains can then be used to carry out one or more bioconversions. These bioconversions can be done either using whole cells, or using acellular extracts of said cells. These bioconversions can transform a molecule into a derived form, with an enzyme of a biosynthetic pathway. For example, Carreras et al. describes the use of a strain of Saccharopolyspora erythraea and Streptomyces coelicolor for the production of new derivatives of erithromycins (Carreras et al, 2002). Walczar et al. describes the use of a P450 monooxygenase from Streptomyces for the bioconversion of deacetyladriamycin (an anthracycline analog) into new anthracyclines (Walczar et al., 2001). Olonao et al. describes the use of a modified Streptomyces lividans strain for the bioconversion of rhodomycinone-epsilon to rhodomycin D (Olonao et al, 1999). Those skilled in the art can apply this principle to any biosynthesis intermediary.

The invention also relates to a recombinant DNA characterized in that it comprises:

- A polynucleotide capable of being obtained by polymerase chain reaction using the pair of primers with the following sequence: 5 'AAGCTTGTGTGCCCGGTGTACCTGGGGAGCC' '(SEQ ID No 138) and 5' GGATCCCGCGACGGACACGACCGCCGCGCA 3 '(SEQ ID No 139) and as a matrix the cosmid pSPM36 or the total DNA of Streptomyces ambofaciens, more preferably it is a polynucleotide of sequence SEQ ID No. 141,

- or a fragment of at least 10, 12, 15, 18, 20 to 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 nucleotides consecutive of these polynucleotides. Preferably; this recombinant DNA is a vector. Even more preferably, the vector is chosen from bacteriophages, plasmids, phagemids, integrative vectors, fosmids, cosmids, shuttle vectors, BAC (Bacterial Artificial Chromosome) or PAC (Pl-derived Artificial Chromosome). By way of illustration, phage lambda and phage Ml 3 may be mentioned as bacteriophages. As plasmids, mention may be made of plasmids replicating in E. coli, for example pBR322 and its derivatives, pUC18 and its derivatives, pUC19 and its derivatives, ρGB2 and its derivatives (Churchward G. et al, 1984), pACYC177 (access number GenBank: X06402) and its derivatives, ρACYC184 (GenBank access number: X06403) and its derivatives. Mention may also be made of the plasmids replicating in Streptomyces such as for example pIJ101 and its derivatives, pSG5 and its derivatives, SLP1 and its derivatives, SCP2 * and its derivatives (Kieser et al. 2000). As phagemids there may be mentioned by way of illustration pBluescript II and its derivatives (marketed in particular by the company Stratagene (LaJ © lla, California, USA)), pGEM-T and its derivatives (marketed by the company Promega (Madison, Wisconcin, USA) ), λZAPII and its derivatives (sold in particular by the company Stratagene (LaJolla, California, USA)). As integrative vectors, there may be mentioned by way of illustration, integrative vectors; ^''' .} _> in Streptomyces such as for example those derived from SLP1 (Kieser et al, 2000), those derived from pSAM2 (Kieser et al, 2000), the vectors using the phage integration systems PhiC31 (Kieser et al , 2000) (for example pSET152 (Bierman et al, 1992)) or VWB (Van Mellaert L, et al 1998), as well as the vectors using the integration system of IS117 (Kieser et al. 2000). As fosmids, the fosmid pFOS1 (sold by the company New England Bioloabs Inc., Beverly, Massachussetts, USA) and its derivatives may be mentioned by way of illustration. ^' : ^" - ^' β As cosmids, we can cite by way of illustration the cosmid SuperCos and its derivatives ι •: •; ^ k (sold in particular by the company Stratagene (LaJolla, California, USA)), the cosmid pWED15 (Wahl et al , 1987) and its derivatives Mention may be made, as shuttle vectors, of E. coli I Streptomyces shuttle plasmids, for example pIJ903 and its derivatives, the series of plasmids pUWL, pCAO106, pWHM3, pOJ446 and their derivatives (Kieser et al. 2000), the E. coli I Streptomyces shuttle BACs, such as, for example, those described in patent application WO 01/40497. As a BAC (Bacterial Artificial Chromosome), we can cite by way of illustration the BAC pBeloBACU (GenBank access number: U51113 As PAC (Pl-derived Artificial Chromosome), the vector pCYPAC6 (GenBank access number: AF 133437) may be cited by way of illustration. More preferably, this recombinant DNA is an expression vector. in dif Different expression systems are well known to those skilled in the art. As regards prokaryotic cells, the expression vectors in E. coli can be cited by way of illustration. example, of the pET family sold by the company Stratagene (LaJolla, California, USA), the vectors of the GATEWAY family sold by the company Invitrogen (Carlsbad, California, USA), the vectors of the pBAD family sold by the company Invitrogen ( Carlsbad, California, USA), the vectors of the pMAL family sold by the company New England Bioloabs Inc. (Beverly, Massachussetts, USA) _/ the rhamnose-inducible expression vectors cited in the publication Wilms B et al, 2001 and their derivatives, one can also cite the expression vectors in Streptomyces such as for example the vectors pIJ4123, pIJ6021, pPM927, pANT849, pANT 850, pANT 851, pANT1200, pANT1201, pANT1202 and their derivatives (Kieser et al, 2000). As regards yeast cells, the pESC vector sold by the company Stratagene (LaJolla, California, USA) may be cited by way of illustration. As regards the baculovirus expression system allowing expression in insect cells, the vector BacPAK6 sold by the company BD Biosciences Clontech, (Palo Alto, California, USA) may be mentioned by way of illustration. As regards mammalian cells, mention may be made, by way of illustration, of vectors comprising the promoter of the early genes of the CMV virus (Cytomegalovirus) (for example the vector pCMV and; = ^• its derivatives marketed by the company Stratagene (LaJolla, California , USA), or the promoter of the immediate genes (SV40 early promoter) of the vacuolating simian virus SV40 (for example the vector pSG5 marketed by the company Stratagene (LaJolla, California, USA). Another aspect of the invention relates to host cells. into which at least one recombinant DNA described in this paragraph has been introduced.

Another aspect of the invention relates to a method for producing a polypeptide characterized in that said method comprises the following steps: a) transforming a host cell with at least one expression vector as described in the paragraph above ; b) cultivating, in an appropriate culture medium, said host cell; c) recovering the conditioned culture medium or a cell extract; d) separating and purifying from said culture medium or also from the cell extract obtained in step c), said polypeptide; 004/033689

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e) where appropriate, characterize the recombinant polypeptide produced.

The recombinant polypeptide thus produced can be purified by passage through an appropriate series of chromatography columns, according to the methods known to those skilled in the art and described for example in F. Ausubel et al (2002). By way of illustration, mention may be made of the “Tag-Histidine” technique, which consists in adding a short poly-histidine sequence to the polypeptide to be produced, the latter being then able to be purified on a nickel column. This polypeptide can also be prepared by in vitro synthesis techniques. As an illustration of such techniques, the polypeptide may be prepared using the "rapid translation System (RTS)" system, sold in particular by the company Roche Diagnostics France S.A, Meylan, France.

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

a gene capable of being obtained by polymerase chain reaction (PCR) using the pair of primers with the following sequence: 5 ''. AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3 '(SEQ ID N ° 138) GGATCCCGCGACGOACACGACCGCCGCGCA 3' (SEQ ID N ° 139) and

matrix the cosmid pSPM36 or the total DNA of Streptomyces ambofaciens, more preferably it is the gene with coding sequence SEQ ID No. 141, - or a gene derived from these due to the degeneracy of the genetic code. An example of the sequence of such a gene is given in SEQ ID No. 111 (DNA), however this sequence is partial since it does not comprise the 3 ′ part of the corresponding coding sequence. The translation into protein of this part of the coding sequence is given in SEQ ID No. 112. A person skilled in the art can easily complete it in particular by using the teaching presented in example 24. A method is thus presented in this example to clone the orβ8c gene and to manufacture an expression vector allowing the expression of orβ 8c. It is also shown in this example that the overexpression of the orβ8c gene in the OSC2 strain leads to an improvement in the production of spiramycins of this strain. Preferably, the overexpressing microorganism - a gene capable of being obtained by polymerase chain reaction (PCR) using the pair of primers with the following sequence: 5 'AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID N ° 138) and 5 'GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID N ° 139) and as a matrix the cosmid pSPM36 or the total DNA of Streptomyces ambofaciens, more preferably it is the gene of coding sequence SEQ ID N ° 141,

- or a gene derived therefrom due to the degeneration of the genetic code is a bacterium of the genus Streptomyces, even more preferably, it is a bacterium of the species Streptomyces ambofaciens. Preferably, the overexpression of said gene is obtained by transformation of said microorganism with an expression vector, quite preferably, the microorganism strain is the strain OSC2 / pSPM75 (1) or of the strain OSC2 / pSPM75 (2 ) deposited with the Collection

spiramycin (s) using the microorganisms described in the previous paragraph. This'?;?: Method consists in cultivating in a suitable culture medium a microorganism defined in the preceding paragraph, recovering the conditioned culture medium or a cell extract, separating and purifying from said culture medium or from l cell extract obtained in the previous step the spiramycin (s) produced. The culture conditions for such microorganisms can be determined according to techniques well known to those skilled in the art. The culture medium may for example be MP5 medium or SL11 medium for Streptomyces and in particular for Streptomyces ambofaciens (Pernodet et al., 1993). Those skilled in the art may in particular refer to the work by Kieser et al. 2000 with regard to the culture of Streptomyces. The spiramycin (s) produced can be recovered by any techniques known to those skilled in the art. Those skilled in the art may refer, for example, to the techniques taught in US patent US 3,000,785 and more particularly to the methods of extraction of spiramycins described in this patent. Preferably, the microorganisms used in such a process are bacteria of the genus Streptomyces. More preferably, the microorganisms are strains of S. ambofaciens.

Another aspect of the invention relates to an expression vector characterized in that the polynucleotide of sequence SEQ ID No. 47 or a polynucleotide derived therefrom due to the degeneration of the genetic code is placed under the control of a promoter allowing the expression of the protein coded by the said polynucleotide in Streptomyces ambofaciens. Examples of expression vectors usable in Streptomyces have been given above. Preferably, such an expression vector is characterized in that it is the plasmid pSPM524 or pSPM525.

Another aspect of the invention relates to a strain of Streptomyces ambofaciens transformed by a vector defined in the preceding paragraph.

Another aspect of the invention relates to a polypeptide characterized in that, its sequence comprises the sequence SEQ ID No. 112. The invention is also>: 'relating to a polypeptide characterized in that its sequence corresponds to the translated sequence of the coding sequence:

- a gene capable of being obtained by polymerase chain reaction (PCR) using the pair of primers with the following sequence: 5 'AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID No. 138) and 5 'GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID No. 139) and as a matrix the cosmid pSPM36 or the total DNA of Streptomyces ambofaciens, more preferably it is the gene of coding sequence SEQ ID No. 141,

-or a gene derived therefrom due to the degeneracy of the genetic code.

Preferably, these polypeptides are expressed in the natural state by a bacterium of the genus Streptomyces, more preferably, these polypeptides are involved in the biosynthesis of spiramycins. Another aspect of the invention relates to an expression vector allowing the expression of a polypeptide as defined in the preceding paragraph in

Ambofacial streptomyces. Examples of expression vectors usable in

Streptomyces have been given above. Preferably, the expression vector in question is the plasmid pSPM75.

LIST OF FIGURES

Figure 1: Chemical structure of spiramycins I, II and III. ; .

Figure 2: Cosmids used for the sequencing of the region.

Figure 3: Organization of a group of genes involved in the biosynthetic pathway for spiramycins.

Figure 4: Proposed biosynthetic pathway for mycarosis.

Figure 5: Proposed biosynthetic pathway for mycaminosis.

Figure 6: Proposed biosynthetic pathway for forosamine.

Figure 7: Preferential order of addition of sugars in the spiramycin molecule and • intermediates. , -i - f

Figure 8: Proposed biosynthetic pathway for methoxymalonyl in S. ambofaciens. Cettig, route is proposed by analogy with the biosynthesis of methoxymalonyl in

Streptomyces hygroscopicus var. ascomyceticus (Wu, K. and β /., 2000).

Figure 9: Steps leading to gene inactivation: A) Cloning of the target gene into a vector replicating in E. coli but not in Streptomyces;

B) Insertion of the resistance cassette into the target gene (by cloning or recombination between short identical sequences);

C) Introduction of the plasmid into Streptomyces ambofaciens (by transformation or conjugation with E. coli) and selection of the clones having integrated the cassette then screening of clones having lost the vector part to have a gene replacement;

D) Region of the chromosome of the mutant strain in which the target gene is inactivated by gene replacement. Figure 10: Optional steps following the inactivation of a gene according to the method described in Figure 9, which can be carried out if an excisable cassette has been used to interrupt the target gene:

E) Introduction into the mutant strain of the plasmid pOSV508 carrying the xis and int genes of pSAM2, the products of which will allow efficient excision by site-specific recombination between the attL and attR sequences bordering the cassette;

F) Obtaining clones having lost the excisable cassette and sensitive to the antibiotic to which the cassette conferred resistance; G) After growth and sporulation on solid medium without antibiotic, the plasmid pOSV508 is lost at high frequency. One can thus obtain clones sensitive to thiostrepton in which the target gene contains a phase deletion. The sequence of the deleted target gene can be checked by PCR amplification and sequencing of the PCR product.

Figure 11: Amplification of the excisable cassette in order to use it for a homologous recombination experiment. The homologous recombination technique, thanks to short homologous sequences, has been described by (Chaveroche et al, 2000). The 39 or 40 deoxy-nucleotides located at the 5 ′ end of these oligonucleotides have a sequence corresponding to a sequence of the gene to be inactivated and the 20 deoxy-nucleotides located most in 3 ′ correspond to the sequence of one of the ends of the excisable cassette.

Figure 12: Obtaining a construct for the inactivation of a target gene using the technique described by (Chaveroche et al, 2000).

Figure 13: Map of plasmid pWHM3. Strepto on: origin of Streptomyces replication.

Figure 14: Map of plasmid pOSV508.

Figure 15: Example of the structure of an excisable cassette. This consists of the Ωhyg cassette (Blondelet-Rouault et al, 1997) bordered by the attR and attL sites (Raynal and al, 1998), between which a recombination event will allow excision of the cassette, thanks to the expression of the xis and int genes. Figure 16: Map of plasmid pBXLl 111. Figure 17: Map of plasmid pBXLl 112. Figure 18: Microbiological test for production of spiramycin, based on the sensitivity of a strain of Micrococcus luteus to spiramycin. The strain of Micrococcus luteus used is a strain naturally sensitive to spiramycin but resistant to congocidine. The different strains of Streptomyces to be tested were cultured in 500 ml Erlenmeyer flasks containing 70 ml of MP5 medium, inoculated at an initial concentration of 2.5 × 10 ⁶ spores / ml and pushed to 27 ° C. with orbital shaking of 250 rpm. Samples of fermentation musts were taken after 48, 72 and 96 hours of culture and centrifuged. A tenth dilution of these supernatants in sterile culture medium is used for the test. The indicator strain of Micrococcus luteus resistant to congocidine but sensitive to spiramycin (Gourmelen et al, 1998) was grown in a 12x12 cm square dish. Whatman: AA paper discs were soaked with 70 μl of the 1/10 dilution of each supernatant and placed X on the surface of the dish. Discs soaked in a solution of spiramycin of y different concentrations (2-4-8 μg / ml in MP5 culture medium) are used as standard range. The dishes are incubated at 37 ° C for 24 to 48 hours. If the disc contains spiramycin, it diffuses into the agar and inhibits the growth of the indicator strain of Micrococcus luteus. This inhibition creates a “halo” around the disc, this halo reflecting the area where the strain of Micrococcus luteus did not grow. The presence of this halo is therefore an indication of the presence of spiramycin and makes it possible to determine whether the strain of S. ambofaciens in question is producing or not producing spiramycin. A comparison with the inhibition diameters obtained for the standard range makes it possible to obtain an indication of the quantity of spiramycin produced by this strain.

Figure 19: HPLC chromatogram of the filtered supernatant of the culture medium of the OSC2 strain. Figure 20: HPLC chromatogram of the filtered supernatant from the culture medium of the strain SPM501.

Figure 21: HPLC chromatogram of the filtered supernatant of the culture medium of the strain SPM502. Figure 22: HPLC chromatogram of the filtered supernatant from the culture medium of the strain SPM507.

Figure 23: HPLC chromatogram of the filtered supernatant from the culture medium of the strain SPM508.

Figure 24: HPLC chromatogram of the filtered supernatant from the culture medium of the strain SPM509.

Figure 25: Alignment of the Orf3 protein (SEQ ID No. 29) with the TylB protein (SEQ

ID No. 87) of S. fradiae produced thanks to the FASTA program.

Figure 26: Alignment of the MdmA protein of S. mycarofaciens (SEQ ID N ° 88) with the SrmD protein (SEQ ID N ° 16) performed thanks to the FASTA program. Figure 27: Example of sequences of residual sites after excision of the excisable cassette. In bold is indicated the minimum att26 site as defined in Raynal et al.,

1998. The sequence of phase 1 (att1) of 33 nucleotides is presented in SEQ ID No. '

104, the sequence of phase 2 (att2) of 34 nucleotides is presented in SEQ ID No. 105, the sequence of phase 3 (att3) of 35 nucleotides is presented in SEQ ID No. 95. Figure 28: Schematic representation of the orflO gene, localization of PCR primers used and construction obtained with each pair of primers.

Figure 29: Construction of the pac-oritT cassette.

Figure 30: Map of the cosmid pWED2.

Figure 31: Schematic representation of the group of genes involved in the spiramycin biosynthesis pathway and the location of the three probes used to isolate the cosmids from the genomic DNA library of the OSC2 strain of Streptomyces ambofaciens in E. coli (cf. Figure 19: Location of the inserts of the cosmids isolated from the genomic DNA library of the OSC2 strain of Streptomyces ambofaciens in E. coli (cf. example 19). New cosmid DNA library. Figure 33: Under cloning of the Pstl-Pstl fragment of the cosmid pSPM36 (insert of the plasmid (pSPM58), under cloning of the Stul-Stul fragment of the cosmid pSPM36 (insert of the plasmid pSPM72) and under cloning of a EcoRl-Stul fragment (insert of the plasmid pSPM73) Figure 34: Location of the open reading phases identified in the Pstl-Pstl insert of the plasmid pSPM58 and in the EcoRl-Stul insert of the plasmid pSPM73. 'Figure 35: Overlay of the chromatograms HPLC of the filtered supernatant of the culture medium of the strain OS49.67 produced at 238 and 280nm (top) and UV spectra of the molecules eluted at 33.4 minutes and 44.8 minutes (bottom) Figure 36: Molecular structure of platenolide molecules A and platenolide B.

Figure 37: Organization of the group of genes involved in the biosynthetic pathway for spiramycins.

Figure 38: Molecular structure of a biosynthesis intermediate produced by the strain SPM507. Figure 39: Structure of a biosynthesis intermediate produced by a strain of S. ambofaciens of genotype orfό * :: attlΩhyg + obtained from a strain ^ s overproducing spiramycins. The insertion of the attlΩhyg + cassette into orfό * exerts a polar effect preventing the expression of orβ *. Figure 40: Molecular structure of the platenolide A + mycarose and platenolide B + mycarose molecules produced by the strain OS49.67

Figure 41: Under cloning of a Pstl-Pstl fragment of the cosmid pSPM36 (insert of the plasmid (pSPM79)), localization of the open reading phases identified in the Pstl-Pstl insert of the plasmid ρSPM79 and localization of the sequence SEQ ID No. 140.

The present invention is illustrated by means of the following examples, which should be considered as illustrative and not limiting.

In summary, the genes for the spiramycin biosynthetic pathway were isolated from a genomic DNA library of Streptomyces ambofaciens. This library was obtained by partial digestion of the genomic DNA of Streptomyces ambofaciens, with the restriction enzyme Bamlil. Large DNA fragments, from 35 to 45 kb on average, have been cloned into the cosmid pWED1 (Gourmelen et al, 1998) derived from the cosmid pWED15 (Wahl et al, 1987). These cosmids were introduced into E. coli using phage particles. The library thus obtained was hybridized with a probe (of sequence SΕQ ID No. 86) corresponding to a part of the tylB gene of S. fradiae (Merson-Davies & Cundliffe, 1994, GenBank access number: U08223). After hybridization, a cosmid out of the 4 hybridizing with the probe was more particularly selected. This cosmid named pOS49.1 was then digested with Sac1 and a 3.3 kb fragment containing the region hybridizing with the probe was cloned into the vector pUC19 and sequenced. Four open reading phases have been identified and one of them encodes a protein (SΕQ ID N ° 29) having strong sequence similarities with the ff TylB protein of S. fradiae (SΕQ ÏD N ° 87) ( see Figure 25). This gene was named orfi (SΕQ ID N ° 28) and was inactivated in S. ambofaciens. It has been shown that clones inactivated in the orβ gene no longer produce spiramycins. This shows the implication of the orβ gene or of genes located downstream in the biosynthesis of spiramycins.

Once this confirmation was obtained, a larger region of the cosmid pOS49.1 was sequenced, on either side of the previously studied fragment S cl. Thus, from the cosmid pOS49.1, the sequence of a region comprising seven whole open reading phases and two incomplete others, located on either side of these seven complete open phases, could be obtained. Thanks to a search in the databases, it could be shown that one of the open phases of incomplete reading corresponded to the locus srmG (region coding for an enzyme called “polyketide synthase” (PKS)). The corresponding genes were cloned by Burgett S. et al. in 1996 (US American patent 5945320). In addition, the other open reading phases, the seven whole ORFs named: orfl, orβ, orβ, orβ, orβ, orfό, orβ (SEQ ID N ° 23, 25, 28, 30, 34, 36, 40) and the beginning of the eighth ORF named orβ (sequence SEQ ID No. 43) were not found in the databases.

In a second step and in order to clone other genes involved in the biosynthesis of spiramycins, other cosmids comprising fragments of the genome of S. ambofaciens in this same region were isolated. For this, a new series of hybridizations on colonies was carried out using three probes. The first probe corresponds to a DNA fragment of 3.7 kb containing orfl, orβ and the start of orβ, subcloned from pOS49.1. The second probe corresponds to a 2 kb fragment of DNA containing a portion of orβ and a portion of orβ and subcloned from pOS49.1. A third probe was also used. The latter corresponds to a 1.8 kb DNA fragment containing the srmD gene. The srmD gene is a gene isolated from S. ambofaciens ^' capable of conferring resistance to spiramycin. Indeed, previous work had enabled the cloning of several determinants of resistance of S. ambofaciens, "conferring resistance to spiramycin to a strain of S. griseofuscus (strain sensitive to spiramycin) (Pernodet et al., 1993 ) (Pernodet et al, 1999). For the isolation of resistance genes, a cosmid library of the genomic DNA of the S. ambofaciens strain had been produced in the cosmid pKC505 (Richardson MA et al., 1987). This cosmid pool had been introduced in S. griseofuscus, which is naturally sensitive to spiramycin. Five cosmids capable of conferring resistance to apramycin and spiramycin in S. griseofuscus were thus obtained. Among these 5 cosmids, a cosmid named pOS44.1 has in its insert the srmD gene which codes for a protein having a certain similarity with the protein coded by the mdmA gene of Streptomyces mycarofaciens and implicated in the resistance to midecamycin in this producing organism ( Hara et al, 1990, Genbank access number: A60725) (Figure 26). The third probe used to locate the biosynthesis genes for spiramycin was an insert of approximately 1.8kb comprising the srmD gene. These three probes were used to hybridize the genomic DNA library described above and made it possible to choose two cosmids (pSPM7 and pSPM5) capable of containing the longest inserts and having no common bands. The cosmid pSMP5 hybridized with the first and the second probe, but did not hybridize with the third probe, while the cosmid pSPM7 hybridized with the third probe only. These two cosmids were completely sequenced by the technique of blind sequencing ("shotgun sequencing"). The sequences of the inserts of these two cosmids: pSPM7 and pSPM5 could be assembled because although not overlapping, each of the inserts included a known sequence at one of its ends. Indeed, J. each of these inserts contained a fragment of the sequence of one of the genes encoding an enzyme called “polyketide synthase” (PKS). These 5 genes were cloned by Burgett S. et al. in 1996 (US patent US 5,945,320) (see Figure 2). Thus, a unique genomic DNA sequence of S. ambofaciens could be determined. A sequence of 30943 nucleotides thus 5'EcόRl a site located in the first PKS gene _and:; going to a Bamϋl site in 3 'is presented in SEQ E) No. 1. This sequence J; corresponds to the upstream region of the PKS genes (cf. FIGS. 2 and 3). A second.;!;! region of 11171 nucleotides, starting from a Pstl site in 5 'and going to a Bs site site in 3' located in the fifth PKS gene is presented in SEQ ID No. 2. This region is the downstream region of PKS genes (downstream and upstream being defined by the orientation of the 5 PKS genes all oriented in the same direction) (cf. Figures 2 and 3).

Thirdly and in order to clone other genes involved in the biosynthesis of spiramycins, other cosmids comprising fragments of the genome of S. ambofaciens in this same region were isolated (cf. examples 18 and 19). EXAMPLE 1 Construction of a Genomic DNA Library of the Streptomyces ambofaciens strain ATCC23877 in E. coli

1.1. Extraction of genomic DNA from the strain ATCC23877 of Streptomyces ambofaciens.

5 'The strain ATCC23877 of Streptomyces ambofaciens (accessible in particular from the American Type Culture Collection (ATCC) (Manassas, Virginia, USA), under the number 23877) was cultivated in YEME medium (Yeast Extract-Malt Extract) (( Kieser, T, et al., 2000) and the genomic DNA of this strain was extracted and purified according to standard lysis and precipitation techniques (Kieser T. et al, 2000).

10 1.2. Construction of the genomic DNA library

The genomic DNA of the strain ATCC23877 from Streptomyces ambofaciens tel.! •: ,, as isolated above was partially digested with the restriction enzyme BamHl-der so as to obtain DNA fragments of size between approximately 35 and 45 lώ X

S i ^: These fragments were cloned into the cosmid pWEDl (Gourmelen et a, 1998) digested || ξ;

'„ ^' . '• ^• .- ^{' •} -. '^:'' - • • ^• •. • ^' . ^'"' -, ^"'':': - ^r -. ^{; ''} - ^'' -v ^'''""". ^: - ^^

15 beforehand by BamHI. The cosmid pWED1 is derived from the cosmid pWE15 (Wahl, ^" eiψk al, 1987) by deletion of the 4.1 kb Hpal-Hpal fragment containing the expression module active in mammals (Gourmelen et al, 1998). was then packaged in vitro in lambda phage particles thanks to the “Packagene® Lambda DNA packaging system” system marketed by the company

20 Promega following the manufacturer's recommendations. The phage particles obtained were used to infect the SURE® strain of E. coli marketed by the company Stratagene (LaJolla, California, USA). The selection of the clones was carried out on LB + ampicillin medium (50 μg / ml) since the cosmid pWΕDl confers resistance to ampicillin.

25 EXAMPLE 2 Isolation and Characterization of Genes Involved in the Biosynthesis of Spiramycins in Streptomyces Ambofaciens

2.1 Colony hybridization of E. clones. coli from the genomic bank of Streptomyces ambofaciens ATCC23877

About 2000 clones of E. coli from the library obtained above, were transferred to a filter for hybridization on colonies. The probe used for hybridization is a Nael-Nael fragment of DNA (SΕQ ID No. 86) comprising a part of the tylB gene from Streptomyces fradiae. This fragment corresponds to nucleotides 2663 to 3702 of the DNA fragment described by Merson-Davies, L.A. & Cundliffe Ε. (Merson-Davies, L.A. & Cundliffe Ε., 1994, GenBank access number: U08223), in which the coding sequence for the tylB gene corresponds to nucleotides 2677 to 3843.

The Nael-Nael fragment of DNA carrying a part of the tylB gene of Streptomyces fradiae (SΕQ ID No. 86) was labeled with ³² P by the technique of "random priming" (Kit sold by the company Roche) and used as probe for the hybridization of 2000 bank clones, transferred to a filter. The membranes used are ^" ',; Hybond N nylon membranes sold by the company Amersham (Amersham Biosciences, Orsay, France) and the hybridization was carried out at 55 ° C. in the buffer described by Church & Gilbert (Church & Gilbert, A washing was carried out in 2X SSC at 55 ° C for 15 minutes and two successive washings in 0.5X SSC were then carried out at 55 ° C, for a duration of 15 minutes each. and washing, 4 clones among the 2000 hybrids presented a strong hybridization signal. These 4 clones were cultured in LB + ampicillin medium (50 μg / ml) and the 4 corresponding cosmids were extracted by standard alkaline lysis (Sambrook et al , 1989). It was then verified that the hybridization was indeed due to a DNA fragment present in the insert of these four cosmids. For this, the cosmids were digested independently by several enzymes (R mHI, Psil and Digestio products ns were separated on agarose gel, transferred to a nylon membrane and hybridized with the Nael-Nael DNA fragment comprising a part of the tylB gene from Streptomyces fradiae (cf. above) in the same conditions as above. The four cosmids were able to be validated and one of these cosmids was more particularly selected and was named pOS49.1.

2.2 Verification of the involvement of the identified region and sequencing of the insert of the cosmid pOS49.1

Several fragments of the insert of the cosmid pOS49.1 have been subcloned and their sequences have been determined. The cosmid pOS49.1 was digested with the enzyme S cl and it was shown by Southern blotting, under the conditions described above, that a 3.3 kb fragment contains the region hybridizing with the tylB probe. This 3.3 _: kb fragment was isolated by electroelution from a 0.8% agarose gel and then cloned into the vector pUC19 (GenBank access number M77789) and sequence. The plasmid thus obtained was named pOS49.11. Four open reading phases with typical Streptomyces codon usage were identified in this fragment (two complete and two truncated), thanks to the FramePlot program (Ishikawa J & Hotta K. 1999). Sequence comparisons thanks to the FASTA program (cf. (Pearson W. R _J. & DJ Lipman, 1988) and (Pearson WR, 1990), accessible in particular from the hundredie of INFOBIOGEN resources, Evry, France) made it possible to show that protein X deduced from one of these 4 open reading phases has strong sequence similarities with the TylB protein (SEQ ID No. 87; GenBank access number: U08223) of S. fradiae (cf. FIG. 25 ). This protein was named Orf3 (SEQ ID No. 29).

In order to test whether the corresponding gene (the orβ gene (SEQ ID No. 28)) was involved in the biosynthesis of spiramycins in S. ambofaciens, this gene was interrupted by an Ωhyg cassette (Blondelet-Rouault MH. Et al ., 991, GenBank access number: X99315). For this, the plasmid pOS49.11 was digested with the Xhol enzyme and the fragment containing the four open reading phases (two complete and two truncated, including orβ in its entirety) was subcloned at the Xhol site of the vector. pBC SK + marketed by the company Stratagene (LaJolla, Califormie, USA). The plasmid thus obtained was named pOS49.12. In order to inactivate orβ, a PmR-BstEll fragment internal to orβ was replaced by the Ωhyg cassette by blunt-end cloning in this last plasmid. For this, the plasmid pOS49.12 was digested with the enzymes Pmll and BstEll, the unique site of which is found in the coding sequence of the orβ gene. The ends of the fragment corresponding to the vector were blunt-ended by treatment with the Klenow enzyme (large fragment of DNA polymerase I). The Ωhyg cassette was obtained by digestion with the enzyme BamHI of the plasmid pHP45 Ωhyg (Blondelet-Rouault et al, 1997, GenBank access number: X99315). The fragment corresponding to the Ωhyg cassette was recovered on agarose gel and its ends were blunt-ended by treatment with the Klenow enzyme. The two blunt-end fragments thus obtained (the Ωhyg cassette and the plasmid pOS49.12) were ligated and the ligation product was used for the transformation of E. coli bacteria. The plasmid thus obtained was named pOS49.14 and contains the orβ gene interrupted by the Ωhyg cassette.

The insert of the plasmid pOS49.14, in the form of an Xhόl-Xhol fragment, the ends of which were made non-cohesive by treatment with the Klenow enzyme, was cloned at the EcoRV site of the plasmid pOJ260 (the plasmid pOJ260 is a conjugative plasmid capable of replicating in E. coli but incapable of replicating> in S. ambofaciens (Bierman M et al, 1992). This plasmid confers resistance to apramycin in E. coli and Streptomyces). The plasmid obtained (insert of the plasmid pOS49.14 cloned into the plasmid pOJ260) was named pOS49.16. The latter was transferred into the strain ATCC23877 of S. ambofaciens by conjugation using the conjugative strain E. coli S17-1, as described by Mazodier et al. (Mazodier et al, 1989). The E. coli S17-1 strain is derived from the E. coli 294 strain (Simon et al, 1983) (Simon, et al, 1986). Transconjugating clones having the marker for resistance to hygromycin carried by the Ωhyg cassette and having lost the marker for resistance to apramycin carried by the vector pOJ260 could be obtained. For this, after conjugation, the clones are selected for their resistance to hygromycin. The clones resistant to hygromycin are then subcultured respectively on medium with hygromycin (antibiotic B) and on medium with apramycin (antibiotic A) (cf. FIG. 9). The clones resistant to hygromycin (HygR) and sensitive to apramycin (ApraS) are in principle those where a double recombination event has occurred and which therefore have the orβ gene interrupted by the Ωhyg cassette. The replacement of the wild-type copy of orβ by the interrupted copy was verified by two successive hybridizations. Thus, the total DNA of the clones obtained was digested with different enzymes, separated on agarose gel, transferred to the membrane and hybridized with a probe corresponding to the Ωhyg cassette (see above)) to verify the presence of the cassette in the genomic DNA of the clones obtained. A second hybridization was carried out using as a probe the Xhol-Xhόl insert of the plasmid pOS49.11 containing the four open reading phases (two complete and two truncated, including orβ in full). Verification of the genotype can also be carried out by any method known to those skilled in the art and in particular by PCR using the appropriate oligonucleotides and sequencing of the PCR product. One of the orβ :: Ωhyg clones thus obtained and whose genotype has been verified, was chosen and was called OS49.16.

The spiramycin production of the clone OS49.16 thus obtained was tested using the production test described below (cf. example 15). It has thus been possible to demonstrate that this strain no longer produces spiramycin, confirming the involvement of orβ and / or genes located downstream such as for example orβ in the biosynthesis of spiramycins. ^'! X

Once this confirmation was obtained, a larger region of the cosmid pOS49.1 was sequenced on either side of the previously studied Sαcl fragment. Thus, from the cosmid pOS49.1, the sequence of a region comprising seven whole open reading phases and two incomplete others, located on either side of these seven complete open phases, could be obtained. Thanks to a search in the databases, it could be shown that one of the open phases of incomplete reading corresponded to the locus srmG (region coding for an enzyme called “polyketide synthase” (PKS)). The corresponding genes were cloned by Burgett S. et al. in 1996 (US patent US 5,945,320). In addition, the other open reading phases: the seven whole ORFs named: orfl, orβ, orβ, orβ, orβ, orfό, orβ (SEQ ID N ° 23, 25, 28, 30, 34, 36 and 40) and the beginning of the eighth ORF named orβ (the entire sequence of this orf is given in SEQ ID N ° 43) were not found in the databases. EXAMPLE 3 Isolation and Characterization of Other Genes Involved in the Biosynthesis of Spiramycins in Streptomyces Ambofaciens

Secondly and in order to clone other genes involved in the biosynthesis of spiramycins, other cosmids comprising fragments of the genome of S. ambofaciens in this same region were isolated. For this, a new series of colony hybridizations was carried out using three probes:

- The first probe used corresponds to a 3.7 kb BamHl-Pstl DNA fragment containing a fragment of the PKS gene (The genes corresponding to PKS were cloned by Burgett S. et al. In 1996 (US patent US 5,945,320)), orfl, orβ and the start of orβ, subcloned from pOS49.1 and going from a BamHI site located 1300 base pairs upstream of the EcoRI site defining position 1 of SΕQ ID N ° 1, up to 'to the site

- The second probe used corresponds to a Pstl-BamHI DNA fragment of approximately JS 2kb containing a gold / 7 and orβ fragment, subcloned from pOS49.1 and going from a Pstl site located in position 6693 of SΕQ ID N ° 1 to the BamHI site located in position 8714 of SΕQ ID N ° 1. This Pstl-BamHl fragment was cloned from pOS49.1 in the pBC SK + plamside, which made it possible to '' obtain the plasmid pOS49.76.

- A third probe was also used. The latter corresponds to a DNA fragment of 1.8 kb EcoRl-Hindïl containing the srmD gene. The srmD gene is a gene isolated from S. ambofaciens capable of conferring resistance to spiramycin. Indeed, previous work had allowed the cloning of several determinants of resistance of S. ambofaciens, conferring resistance to spiramycin to a strain of S. griseofuscus (strain sensitive to spiramycin) (Pernodet et al, 1993) (Pernodet and al, 1999). To isolate resistance genes, a cosmid library of the genomic DNA of the S. ambofaciens strain ATCC23877 was produced in the cosmid pKC505. (Richardson MA et al, 1987). For this, the genomic DNA of the S. ambofaciens strain ATCC23877 had been partially digested with Sau3Al so as to obtain fragments of size between approximately 30 and 40 kb. The genomic DNA thus digested (3 μg) had been ligated with 1 μg of pKC505 previously digested with the enzyme BamHI (Pernodet et al, 1999). The ligation mixture was then packaged in vitro into phage particles. The phage particles obtained were used to infect the strain of E. coli HB101 (accessible in particular from the American Type Culture Collection (ATCC) (Manassas, Virginia, USA), under the number 33694). About 20,000 clones of E. apramycin resistant coli had been pooled and the cosmids of these: • clones had been extracted. This cosmid pool was introduced by transformation of protoplasts into the DSM 10191 strain of S. griseofuscus (Cox KL & Baltz RH. 1984), naturally sensitive to spiramycin (Rao RN et al, 1987, this strain is available in particular from the German Collection of Microorganisms and Cell Cultures _.: (Deutsche Sammlung von Mikro-organismen und Zellkulturen GmbH, DSMZ),; (Braunschweig, Germany), under number DSM 10191). Transformants were ^';: selected on a medium containing apramycin. 1300 of the clones growing on ,, i medium containing apramycin had been transferred to medium containing 5 μg ml of: - spiramycin. Several clones resistant to apramycin had also grown on: medium containing spiramycin and the cosmids of these colonies had been extracted and used to transform E. coli and S. griseofuscus (Pernodet et al, 1999). Five cosmids capable of conferring apramycin resistance to E. coli and co-resistance to apramycin and spiramycin in S. griseofuscus were thus obtained. Among these 5 cosmids, it was determined that a cosmid named pOS44.1 has in its insert a gene (SΕQ ID N ° 15) which codes for a protein (SΕQ ID N ° 16) having a certain similarity with a protein coded by the mdmA gene from Streptomyces mycarofaciens (SΕQ ID No. 88), this gene was named srmD (cf. alignment presented in FIG. 26 carried out thanks to the FASTA program (cf. (Pearson W. R & DJ Lipman, 1988) and ( Pearson WR, 1990), accessible in particular from the INFOBIOGEN resource center, Evry, France). To isolate the resistance determinant contained in the plasmid pOS44.1, it was partially digested with the restriction enzyme Sau3Al so as to obtain fragments of size of approximately 1.5 to 3 kb, these fragments were ligated into the vector pIJ486 linearized by the enzyme BamHI (Ward et al, 1986). A plasmid was selected for its ability to confer resistance to spiramycin in the DSM 10191 strain of S. griseofuscus (Rao RN et a, 1987), which is naturally sensitive to spiramycin (cf. above). For this, the pool of plasmids corresponding to the Sau3Al fragment of ρOS44.1 ligated in the vector pIJ486 (cf. above), was introduced by transformation of protoplasts in the strain DSM 10191 and the transformants were selected for their resistance to thiostrepton (due to the ter gene carried by pIJ486). Clones growing on medium containing thiostrepton were transferred to medium containing spiramycin. Several clones resistant to thiostrepton also grew on medium containing spiramycin and the plasmids of these colonies were extracted. A plasmid conferring resistance and containing an insert of approximately 1.8 kb was selected and named pOS44.2. This 1.8kb insert can be taken out easily thanks to a Hindïïl site. and an EcoKL site present in the vector on either side of the insert. This 1.8kb insert. , ^: HindlU-EcoRÎ was sequenced and the Resistance gene it contains was named srmDr This fragment containing the srmD gene could thus be easily subcloned into the vector pUC19 (GenBank access number M77789) opened by EcoRl -HindlU, the plasmid obtained was named pOS44.4. The 1.8kb Hindlll-EcoRl insert, containing the srmD gene, of this plasmid was used as a probe to locate the biosynthesis genes of spiramycin (see below).

A sample of an Escherichia Coli DH5α strain containing the plasmid pOS44.4 was deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, July 10, 2002 under registration number 1-2918.

About 2000 clones from the library, obtained above (cf. example 1), were transferred to a filter for hybridization on colonies according to conventional techniques (Sambrook et al, 1989). The three probes described above were labeled with ³² P by the technique of "random priming" (Kit marketed by Roche) and used for the hybridization of the 2000 clones of the bank, transferred to a filter. Hybridization was carried out at 65 ° C in the buffer described by Church & Gilbert (Church & Gilbert, 1984). A washing was carried out in 2X SSC at 65 ° C for 15 minutes and two successive washes were then carried out in 0.5X SSC at 65 ° C, lasting 15 minutes each. Under these hybridization and washing conditions, 16 clones among the 2000 hybrids exhibited a strong hybridization signal with at least one of the probes. However, no cosmid hybridized with the three probes. The 16 cosmids were extracted and digested with the restriction enzyme BamHI. The comparison of the restriction profiles of these different cosmids with one another has led to the choice of two cosmids capable of containing the longest inserts in the region and having no common bands. Thus two cosmids, one named pSPM5 and the other pSPM7, were chosen. The cosmid pSPM5

blind ("shotgun sequencing"). The sequences of the inserts of these two cosmids: pSPM7 and pSPM5 could be assembled because although not overlapping, each of the inserts included a known sequence at one of its ends. Each of these inserts contained a fragment of the sequence of one of the genes encoding an enzyme called "polyketide synthase" (PKS). These 5 genes were cloned by Burgett S. et al in 1996 (American patent US 5,945,320) (cf. FIG. 2). Thus, a unique genomic DNA sequence of S. ambofaciens could be determined. A sequence of 30,943 nucleotides starting at 5 'from an EeoRI site located in the first PKS gene and going to a BamHI site at 3' is presented in SΕQ ID No. 1. This sequence corresponds to the upstream region of the genes of PKS (see Figures 2 and 3). A second region of 11171 nucleotides, starting from a Pstl site in 5 'and going to a Ncol site in 3' located in the fifth gene of PKS is presented in SΕQ ID Ν ° 2. This second region is the downstream region of the PKS genes (downstream and upstream being defined by the orientation of the 5 PKS genes all oriented in the same direction) (cf. FIGS. 2 and 3).

EXAMPLE 4 Analysis of the nucleotide sequences, determination of the open reading phases and characterization of the genes involved in the biosynthesis of spiramycins.

The sequences obtained were analyzed using the FramePlot program (Ishikawa J & Hotta K. 1999). This made it possible to identify, among the open reading phases, the open reading phases exhibiting a use of codons typical of Streptomyces. This analysis made it possible to determine that this region comprises 35 ORFs located on either side of five genes coding for the enzyme “polyketide synthase” (PKS). Respectively 10 and 25 ORFs were identified downstream and upstream of these genes (downstream and upstream being defined by the orientation of the 5 PKS genes all oriented in the same direction) (cf. FIG. 3). Thus, the 25 open reading phases of this type, occupying a region of approximately 31 kb (SEQ ID No. 1 and FIG. 3) were identified upstream of the 5 genes encoding PKS and 10 occupying a region of approximately 11 , 1 kb (SEQ ID - _r -

^* iX N ° 2 and Figure 3), have been identified downstream of the PKS genes. The genes of the region (C i upstream were named orfl, orβ, orβ, orβ, orβ, orfό, orβ, orβ, orβc, orflO, orfl le, orfl 2, orfl 3c, orfl4, orfl 5c, orflό, orf 17 , orfl 8, orfl 9, orβO, orβlc, orβ2c, orβ3c, orβ4c and orβ5c (SEQ ID N ° 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) The genes of the downstream region were named orfl * c, orβ * c, orβ * c, orβ * c, orβ *, orfό *, orβ * c, orβ *, orβ *, orflO * (SEQ ID N ° 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21). The "c" added in the name of the gene signifying for the ORF in question that the coding sequence is in the reverse orientation (the coding strand is therefore the complementary strand of the sequence given in SEQ ID No. 1 or SEQ ID No. 2 for these genes) ( see Figure 3).

The protein sequences deduced from these open reading phases were compared with those present in different databases using different programs: BLAST (Altschul et al, 1990) (Altschul et al, 1997), CD-search, COGs (Cluster of Orthologous Groups) (these three programs are accessible in particular from the National Center for Biotechnology Information (NCBI) (Bethesda, Maryland, USA)), FASTA ((Pearson W. R & DJ Lipman, 1988) and (Pearson WR, 1990), BEAUTY (Worley KC et al, 1995)) , (these two programs are accessible in particular from the INFOBIOGEN resource center, Evry, France). These comparisons made it possible to formulate hypotheses on the function of the products of these genes and to identify those likely to be involved in the biosynthesis of spiramycin.

EXAMPLE 5 Gene Inactivation: Principle of Construction of an Interrupted Streptomyces ambofaciens Strain The methods used consist in performing a gene replacement. The target gene to be interrupted is replaced by a copy of this interrupted gene by a cassette conferring resistance to an antibiotic (for example apramycin or hygromycin), as illustrated in FIG. 9. The cassettes used are bordered on the side and others by translation termination codons in all the reading phases and by transcription terminators active in Streptomyces.

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

The constructs necessary for the inactivation of the gene by the cassette were obtained from E. coli, the reference organism for obtaining recombinant DNA constructs. The interrupted gene was obtained in a plasmid that replicates in E. coli but cannot replicate in Streptomyces.

The constructs were then subcloned into vectors to allow transformation and inactivation of the desired gene in S. ambofaciens. For this, two plasmids were used:

- pOJ260 (Bierman M. et al, 1992) (cf. example 2) which confers resistance to apramycin in E. coli and Streptomyces and which was used when the target gene was interrupted by a cassette conferring resistance to hygromycin. - pOSK1205 (4726pb). This plasmid is derived from the plasmid pBK-CMN (marketed by the company Stratagene (LaJolla, California, USA)) in which an Avril fragment containing the sequence coding for resistance to Νeomycin / Kanamycin has been replaced by a sequence coding for resistance to hygromycin, while retaining the promoter P SN40. For this, the plasmid \ iHP45-Ωhyg (Blondelet-

Rouault et a, 1997) was digested with the enzymes Nσtl and Pflml and the fragment conferring resistance to hygromycin was subcloned at the site _^ 4vrII of the vector pBK-CMV after all the ends had been rendered whole frank by treatment with Klenow's enzyme. In pOSK1205, the cassette which confers resistance to hygromycin is preceded by the promoter pSV40. This plasmid confers resistance to hygromycin in E. coli and Streptomyces and was used when the target gene was interrupted by a cassette conferring resistance to apramycin.

The plasmid carrying the gene interrupted by the cassette can then be introduced into Streptomyces ambofaciens, for example by conjugation between E. coli and Streptomyces (Mazodier P, et a, 1989). This technique was used in the case where the base vector is the vector pOJ260. A second technique can be used: the technique of transforming protoplasts after denaturation by alkaline treatment of ADΝ (Kieser, T et al, 2000), to increase the frequency of recombination as described for example by Oh & Chater (Oh & Chater, 1997). This technique has been used in the case where the basic vector is pOJ260 or pOSK1205 (see below). The transformants are then selected with the antibiotic corresponding to the cassette present in the target gene (cf. FIG. 9, antibiotic B). A mixture of clones is thus selected, among which there has been integration by one or by two recombination events. Secondly, we are looking for clones sensitive to the antibiotic for which the resistance gene is present in the vector (outside the recombination cassette) (cf. FIG. 9, antibiotic A). It is thus possible to select the clones for which there have in principle been two recombination events leading to the replacement of the wild-type gene by the copy interrupted by the cassette. These steps are shown diagrammatically in Figure 9.

Several cassettes can be used for the interruption of the target genes. One can for example use the Ωhyg cassette which confers resistance to hygromycin (Blondelet-Rouault et al, 1997, GenBank access number: X99315).

EXAMPLE 6 Construction of a Streptomyces ambofaciens strain interrupted in phase in the orβ gene

The orf 3 gene had been interrupted by the Ωhyg cassette (cf. example 2.2) and it could be demonstrated that an orβ :: Ωhyg strain no longer produced spiramycin, confirming the involvement of one or more genes from the region cloned in the biosynthesis of spiramycins (cf. example 2.2). In view of their orientation, a co-transcription of ORFs .; _n 1 to 7 is probable (cf. FIG. 3) and the phenotype observed (non-producer of spiramycins) may be due to the inactivation of one or more of the genes co-transcribed with orβ. To confirm the involvement of orβ in the biosynthesis of spiramycins, a new inactivation of the orβ gene was carried out, the latter being carried out in phase. For this, an internal Dralïl fragment with orβ of 504 base pairs was deleted. A DNA fragment obtained from pOS49.1, going from the EcoRI site located in position 1 (SΕQ ID N ° 1) to the SacI site located in position 5274 (SΕQ ID N ° 1) and which contains a deletion between the two Dralïl sites at positions 2563 and 3067 (504 nucleotides removed) was cloned into the plasmid pOJ260 (Bierman M. et al., 1992). The plasmid thus obtained was named pOS49.67.

The insert of pOS49.67 is therefore constituted by a DNA fragment of S. ambofaciens containing the orfl, orβ, orβ genes with the deletion in phase, orβ and part of orβ. The vector into which this insert has been subcloned is pOJ260, the plasmid pOS49.67 therefore confers resistance to apramycin and was introduced by transformation of protoplasts into the strain OS49.16 (cf. example 2). The strain OS49.16 being resistant to hygromycin, transformants hygR and apraR were obtained. After two passages of such clones on non-selective medium, the clones sensitive to apramycin and to hygromycin (apraS and hygS) were sought. In some of these clones, it is expected that a recombination event between homologous sequences will lead to the replacement of the copy of orβ interrupted by the Ωhyg cassette (contained in the genome of the strain OS49.16) by the copy of orβ with the phase deletion present on the vector. The clones resulting from this recombination are expected as apraS and hygS after the elimination of the vector sequences. The genotype of the strains thus obtained can be verified by hybridization or by PCR and sequencing of the PCR product (to verify that only a copy of orβ deleted in phase is present in the genome of the clones obtained). Clones having only the copy of orβ with a phase deletion were thus obtained and their genotype was verified. A clone of the _(desired characteristics was more particularly selected and was named.

Nationale de Cultures de Microorganismes (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, July 10, 2002 under registration number 1-2916.

EXAMPLE 7 Construction of a Streptomyces ambofaciens strain interrupted in the orβ gene

To achieve inactivation of the orβ gene, a construct in which the Ωhyg cassette was introduced into the coding sequence for orβ was obtained. For this, the plasmid pOS49.88 was firstly constructed. The plasmid pOS49.88 is derived from the plasmid pUC19 (GenBank access number M77789) by insertion of a 3.7 kb fragment (fragment Λtl-EeoRI obtained from the cosmid pSPM5), containing the end of orβ, orβ and the start of orβ, cloned into the Pstl-EcoRl sites of pUC19. The Ωhyg cassette (in the form of a BamHI fragment, made blunt by treatment with the Klenow enzyme) was cloned at the unique Sali site of pOS49.88 located in orβ after all the ends were made blunt by treatment with the Klenow enzyme.

Cloning being blunt end, two types of plasmids were obtained according to the ^' direction of insertion of the cassette: pOS49.106 in which the hyg and orβ genes are in the same orientation and pOS49.120 in which the hyg and orβ genes are in opposite directions. The insert of plasmid pOS49.106 was then subcloned into plasmid pOJ260 to give pOS49.107. For this, the plasmid pOS49.106 was digested with the enzyme AspllSl and the ends were made blunt-ended by treatment with the Klenow enzyme, this digestion product was redigested by the enzyme Pstl and the fragment containing the orβ gene into which the Ωhyg cassette has been inserted, has been cloned j- into the vector pOJ260 (cf. above). For this, the vector pOJ260 was digested with; EcoRV and Pstl enzymes and used for ligation. This manipulation therefore makes it possible to obtain an oriented ligation since each of the two fragments is a blunt end on ^one side and P ^ tl on the other. The plasmid obtained was named pOS49.107.

The plasmid ρOS49.107 was introduced into the strain ATCC23877 of S. ambofaciens by transformation of protoplasts (Kieser, T et al, 2000). After transformation of the protoplasts, the clones are selected for their resistance to hygromycin. The clones resistant to hygromycin are then subcultured respectively on medium with hygromycin (antibiotic B) and on medium with apramycin (antibiotic A) (cf. FIG. 9). The clones resistant to hygromycin (HygR) and sensitive to apramycin (ApraS) are in principle those where a double crossing over event has occurred and which have the orβ gene interrupted by the Ωhyg cassette. The replacement of the wild-type copy of orβ by the copy interrupted by the Ωhyg cassette was verified by Southern Blot. Thus, the total DNA of the clones obtained was digested with several enzymes, separated on agarose gel, transferred to the membrane and hybridized with a probe corresponding to the Ωhyg cassette to verify the presence of the cassette in the genomic DNA of the clones obtained. A second hybridization was carried out using as probe the Pstl-EcoRl insert containing the end of orβ, orβ and the start of orβ with a size of approximately 3.7 kb of the plasmid pOS49.88. Verification of the genotype can be carried out by any method known to a person skilled in the art and in particular by PCR using the appropriate oligonucleotides and sequencing of the PCR product.

A clone orβv.Ωhyg was chosen and named OS49.107. A sample of the strain OS49.107 was deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, on July 10, 2002 under registration number 1 -2917.

EXAMPLE 8 Construction of a Streptomyces ambofaciens strain interrupted in the orflO gene

using as template, the genomic DNA of S. ambofaciens and the following primers

SRMRl: 5 'CTGCCAGTCCTCTCCCAGCAGTACG 3' (SEQ ID NO "89)

SRMR2: 5 'TGAAGCTGGACGTCTCCTACGTCGG 3' (SEQ ID H "90)

This DNA fragment from the PCR was cloned into the vector pCR2.1 sold by the company Invitrogen (Carlsbad, California, USA). The plasmid thus obtained was named pOS49.32. The Ωhyg cassette (in the form of a BamHI fragment, cf. above) was cloned at the unique RstEII site internal to the fragment of the orf 10 gene, after all of the ends had been made blunt by treatment with Klenow enzyme. The cloning being blunt, two types of plasmids were obtained according to the direction of insertion of the cassette: pOS49.43 in which the genes hyg and orf 10 are in the same orientation and pOS49.44 in which the genes hyg and orflO are in opposite directions. The insert of the plasmid pOS49.43 was transferred (in the form of an Aspl1Sl-Xbal fragment, the ends of which were made blunt-ended by treatment with the Klenow enzyme) in the EcoRV site of the plasmid pOJ260 which made it possible to '' obtain the plasmid pOS49.50. The plasmid pOS49.50 containing the fragment of the orflO gene interrupted by the Ωhyg cassette was introduced into the strain ATCC23877 of Streptomyces ambofaciens. After transformation, the clones are selected for their resistance to hygromycin. The clones resistant to hygromycin are then subcultured respectively on medium with hygromycin (antibiotic B) and on medium with apramycin (antibiotic A) (cf. FIG. 9). The clones resistant to hygromycin (HygR) and sensitive to apramycin (ApraS) are in principle those where a double crossing over event has occurred and which have the orflO gene interrupted by the Ωhyg cassette. Clones which possessed the marker of resistance to hygromycin carried by the cassette and which had lost the marker of resistance to apramycin carried by the vector pOJ260 were thus obtained. The event of replacement of the wild copy of orflO by the interrupted copy orflO :: Ωhyg was verified by Southern Blot. Thus, the total genomic DNA of the clones obtained was digested with several enzymes, separated on agarose gel, transferred to the membrane and hybridized with a probe corresponding to the Ωhyg cassette to verify the presence of the cassette in the genomic DNA. clones obtained. A second hybridization was carried out using as a probe the 1.5 kb PCR product internal to the orf 10 gene (cf. above).

A clone with the expected characteristics (orflOv.Ωhyg) was more particularly selected and named OS49.50. It has indeed been possible to verify thanks to the two hybridizations that the Ωhyg cassette was indeed present in the genome of this clone and that the expected digestion profile is indeed obtained in the case of a replacement, following a double recombination event, of the wild-type gene by the copy interrupted by the Ωhyg cassette in the genome of this clone. Verification of the genotype can also be carried out by any method known to those skilled in the art and in particular by PCR using the appropriate oligonucleotides and sequencing of the PCR product. EXAMPLE 9 Gene Inactivation: Principle of Building a Strain of Streptomyces ambofaciens Interrupted Using the “Excisable Cassettes” Technique (cf. FIGS. 9 and 10)

"

A second type of cassette can be used for the inactivation of genes: cassettes known as “excisable cassettes”. These cassettes have the advantage of being able to be excised in Streptomyces by a site-specific recombination event after having been introduced into the genome of S. ambofaciens. The aim is to inactivate certain genes in Streptomyces strains without leaving selection markers or large DNA sequences not belonging to the strain in the final strain. After excision there remains only a short sequence of about thirty base pairs (called the “scar” site) in the genome of the strain (cf. FIG. 10).

The implementation of this system consists, firstly, in replacing the wild copy of the target gene (thanks to two homologous recombinaisόh events), cf. FIG. 9) by a construct in which an excisable cassette has been inserted into this target gene. The insertion of this cassette is accompanied by a deletion in the target gene (cf. FIG. 9). In a second step, the excision of the excisable cassette of the genome of the strain is caused. The excisable cassette functions thanks to a site-specific recombination system and has the advantage of making it possible to obtain mutants of Streptomyces which ultimately do not carry a resistance gene. It also frees itself from possible polar effects on the expression of genes located downstream of the inactivated gene (s) (cf. FIG. 10).

The use of excisable cassettes has been described and used in many organisms including mammalian, yeast cells and in E. coli (Bayley et a, 1992; Brunelli and Pall, 1993; Camilli et al, 1994; Dale and Ow , 1991; Russell et al, 1992; Lakso et al,

1992). These excisable cassettes all use the Cre site specific recombinase acting on the lox sites. This recombination system comes from the bacteriophage PI. For the construction of a “excisable cassette” type system in Streptomyces, use was made of the site-specific recombination system described for the mobile genetic element pSAM2 from Streptomyces ambofaciens (Boccard et al, 1989a and b). The system implemented consists first of all in constructing a recombinant vector comprising the gene to be interrupted in which an excisable cassette is inserted. The insertion into the target gene of the excisable cassette is accompanied by a deletion in the target gene. It can be carried out by cloning using restriction sites present in the target gene or by recombination between short identical sequences as described for example by Chaveroche et al (Chaveroche MK et a, 2000). The excisable cassette can be constructed using for example the Ωhyg cassette (Blondelet Rouault et al, 1997). This cassette was bordered by αttR and attL sequences which normally flank the integrated copy of pSAM2 (cf. FIG. 15). The attL and attR sequences contain all the sites necessary for site- recombination. specific allowing excision of pSAM2 or any DNA fragment located between these two regions (Sezonov et al, 1997, Raynal et /. 1998). The construction of such. cassette is obviously not limited to the use of an Ωhyg cassette, but others.;! ;; Resistance cassettes can be used as a basis for the construction of this cassette (for example; the Ωaac or Ωvph cassette (Blondelet Rouault et al, 1997)).

After obtaining this construction, the Streptomyces strain is transformed with the recombinant plasmid. The transformants are then selected with the antibiotic corresponding to the cassette present in the target gene (cf. FIG. 9, antibiotic B, this is for example a selection by hygromycin if the excisable cassette is derived from the Ωhyg cassette ). A mixture of clones is thus selected, among which there has been integration by a single or by two recombination events. Secondly, we are looking for clones sensitive to the antibiotic for which the resistance gene is present in the vector (outside the recombination cassette)

(cf. figure 9, antibiotic A). It is thus possible to select the clones for which there have in principle been two recombination events leading to the replacement of the wild-type gene by the copy interrupted by the cassette. These steps are shown diagrammatically in FIG. 9, the genotype of the clones thus obtained is verified by Southern blot and a strain with the desired characteristics (replacement of the wild-type gene with the copy interrupted by the excisable cassette) is selected.

In a second step, the strain selected above is transformed by a plasmid allowing the expression of the xis and int genes which are both necessary for site-specific recombination between the attR and attL sites. This recombination results in the departure of the excisable cassette of the genome of the strain, thanks to a recombination event (cf. FIG. 10) (Raynal et a, 1998). It is interesting to choose the vector carrying the xis and int genes from the relatively unstable vectors in; _: Streptomyces (e.g. Streptomyces derivative of the vector pWHM3 (Vara et ^al, 1989)), this allows to obtain a strain having lost the latter vector after sporulation few cycles in the absence of selection pressure.

To excise the cassette, one can for example use the plasmid pOSV508 (cf. figure; 14) which is introduced by transformation of protoplasts into the strain of S. ambofaciens i containing a gene interrupted by the excisable cassette. The plasmid pOSV508 is derived from the plasmid pWHM3 (Vara J et al, l9S9) (cf. FIG. 13) in which i have been added i to 1 § xis and int genes of pSAM2 (Boccard F. et al., 1989b) placed under the control of the -} ptrc promoter (Amann, E. et al, 1988). The xis and int genes placed under the control of the ptrc promoter were subcloned into the plasmid pWHM3 from the plasmid pOSint3 (Raynal et al, 1998) (cf. FIG. 14). The introduction into the mutant strain of the plasmid pOSV508 carrying the xis and int genes of pSAM2 will allow efficient excision by site-specific recombination of the excisable cassette between the attL and attR sites flanking this cassette (Raynal A. et al, 1998 ) (Figure 10). Among the transformants, selected for their resistance to thiostrepton due to the tsr gene carried by pOSV508, those chosen have become sensitive to the antibiotic to which the presence of the cassette confers resistance (cf. FIG. 10). Excision is effective and it has been observed that more than 90% of the transformants are of this type. After one or more growth and sporulation cycles on a solid medium devoid of thiostrepton, clones are obtained which have lost the plasmid pOSV508. These clones are identifiable by their sensitivity at thiostrepton. The sequence of the deleted target gene can be verified by PCR and sequencing of the PCR product.

Finally, the resulting strain carries, at the level of the inactivated gene (internal deletion for example) a “scar” att site corresponding to the minimal attB site (Raynal et al, 1998), resulting from the recombination between the αttR and attL sites. This minimal attB site which remains is similar to that naturally present in the strains of Streptomyces ambofaciens, Streptomyces pristinaespiralis and Streptomyces lividans (Sezonov et al, 1997).

The gene that we want to inactivate can be cotranscribed with other genes located downstream. To avoid the inactivation of one of the genes having a polar effect on the expression of genes downstream in the operon, it is important to obtain a deletion in phase after excision of the cassette. The excisable cassette system as described above makes it possible to meet such a requirement. Those skilled in the art can, in fact, easily construct three separate excisable cassettes, these leaving after excision a sequence of 33, 34 or 35 nucleptides respectively, without stop codon whatever the '*] reading phase. Knowing the target gene sequence and the size of the deletion; associated with the insertion of the cassette, it is possible to choose between these three excisable cassettes so that the excision leads to a deletion in phase. Of the 33, 34 or 35 nucleotides added, 26 correspond to the minimal attB sequence (cf. FIG. 27). In the case of the present application, two excisable cassettes were used. These two cassettes are as follows: attlΩhyg + (SEQ ID No. 91) and att3Ωaac- (SEQ ID No. 92), these cassettes leave 33 and 35 nucleotides respectively after excision. They respectively include the Ωhyg cassette or the Ωaac cassette, the + and - signs corresponding to the orientation of the resistance cassette. These two cassettes were constructed and cloned at the EcoRV site of the pBC SK + vector, the HindIII site of which was previously deleted. The plasmids obtained were named pattlΩhyg + and patt3Ωaac- respectively. The excisable cassettes can be easily removed by digestion of the plasmid with EcoRV. EXAMPLE 10 Construction of a Streptomyces ambofaciens strain interrupted in the orβ gene

The orβ gene was inactivated using the excisable cassette technique (see above). The starting strain used is the Streptomyces ambofaciens OSC2 strain which is derived from the ATCC23877 strain. However, the OSC2 strain differs from the ATCC23877 strain in that it has lost the mobile genetic element pSAM2 (Boccard et al, 1989a and b). This mobile element can be lost spontaneously during protoplastization (action of lysozyme to digest the wall; bacterial and fragment the mycelium (Kieser et al, 2000)) and the regeneration of protoplasts of the ATCC23877 strain. To select the clones having lost the pSAM2 element, a screen was set up, based on the repression of the pra gene by the transcription repressor KorSA (Sezonov et al, 1995) (Sezonov G. et al, 2000). ^< > For this, a DNA fragment containing the promoter of the pra gene placed upstream of the. ^' ; _. aph gene (conferring resistance to kanamycin and devoid of its own promoter)} .- _. - • was cloned into the unstable vector ρWHM3Hyg, the latter deriving from the plasmid; - ;;

. 'ÀiiΛ r pWHM3 (Vara et al., 1989) in which the tsr gene has been replaced by the ^' $$ ^κ $ gene (conferring resistance to hygromycin). The plasmid thus obtained was named pOSV510 ^s. The strain ATCC23877 was transformed after protoplastization with the plasmid pOSV510. The Pra promoter is a promoter repressed by the KorSA repressor, the gene encoding the latter being located within the mobile element pSAM2 (Sezonov G. et al, 2000). After transformation with the plasmid pOSV510, the transformed bacteria are selected for their resistance to kanamycin (due to the aph gene carried by pOSV510). The clones having lost the integrative element pS AM2 lost the repressor KorSA and the promoter Pra is therefore no longer repressed and allows the expression of the kanamycin aph resistance gene. Selection by kanamycin after transformation with the plasmid pOSV510 therefore makes it possible to select the clones having lost the integrative element pSAM2 (and therefore KorSA) and having the plasmid pOSV510. The plasmid pOSV510 being unstable, after a few cycles of sporulation without antibiotic, isolated clones are subcultured on medium with kanamycin, on medium with hygromycin and on medium without antibiotic. Clones sensitive to kanamycin and hygromycin lost pOSV510. The loss of the pSAM2 element was verified by hybridization and PCR. A clone with the desired characteristics was selected and was named OSC2.

A sample of the OSC2 strain was deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, on July 10, 2002 under the registration number I-2908 .

The orβ gene was inactivated using the excisable cassette technique (see above and Figure 10). For this, a 4.5 kb insert whose sequence starts from the EcoRI site located in position 1 to the BamHI site located in position 4521 (SΕQ ID No. 1) was subcloned at the EcoRI and BamHI sites of plasmid pUCl _. 9 (GenBank access number M77789) from the cosmid pSPM5. The plasmid thus obtained was named pOS49.99.

This plasmid was introduced into the E. coli KS272 strain which already contained the. plasmid pKOBΕG (Chaveroche et al, 2000) (cf. FIG. 12). ' ^?

In parallel, the excisable cassette att3Ωaac- (SEQ ID No. 92, cf. above) was amplified by PCR using as plasmid the plasmid pOSK1102 (The plasmid pOSK1102 is a plasmid derived from the vector ρGP704Not (Chaveroche et al, 2000) (Miller VL & Mekalanos JJ, 1988) in which the att3Ωaac- cassette was cloned as an EcoRV fragment in the unique EcoRV site of pGP704Not) and using the following primers:

ORF2A

5 'CCCGCGCGGCAGCCTCTCCGTGATCGAGTCCGGCGTGACCATCGCGCGCGCTTCGTTCGG-3' (SΕQ ID N ° 93) ORF2B

5 'GCTCCGTGCGTCATGCAGGAAGGTGTCGTAGTCGCGGTAGATCTGCCTCTTCGTCCCGAA-3' (SΕQ ID N ° 94) The 40 deoxy-nucleotides located at the 5 'end of these oligonucleotides have a sequence corresponding to a sequence in the target gene (orβ in this case) and the 20 deoxy-nucleotides located most in 3' (shown in bold ci above) correspond to the sequence of one of the ends of the att3Ωaac- excisable cassette (see Figure 11).

The PCR product thus obtained was used to transform the E. cpli strain containing the plasmids pKOBΕG and pOS49.99 as described (Chaveroche et al, 2000) (cf. FIG. 12). Thus, the bacteria were transformed by electroporation and selected for their resistance to apramycin. The plasmids of the clones obtained were extracted and digested with several restriction enzymes, in order to verify that the digestion profile obtained corresponds to the profile expected if there has been insertion of the cassette (att3Ωaac-) into the target gene ( orβ), i.e. if there was indeed homologous recombination between the ends of the PCR product and the target gene (Chaveroche et al,

plasmid derives from pOS49.99 in which orβ is interrupted by the apramycin cassette (cf. FIG. 12). The insertion of the cassette is accompanied by a deletion in orβ, between nucleotides 211 and 492 of the coding part of orβ.

The plasmid pSPM17 was digested with the EcoRI enzyme then the ends were made blunt by treatment with the Klenow enzyme, this digestion product was then digested with the Xbal enzyme and the insert containing the deleted orβ gene was cloned into the vector pOSK1205 (cf. above). For this, the vector pOSK1205 was digested with the enzyme BamHI then the ends were made blunt end by treatment with the Klenow enzyme, this product was then digested with the enzyme Xbal, and used for the ligation with l insert obtained from pSPM17 as above. This manipulation therefore makes it possible to obtain an oriented ligation since each of the two fragments is a blunt end on one side and Xbal on the other. The plasmid thus obtained was named pSPM21, it carries a hygromycin resistance gene (vector part) and an insert in which the deleted orβ gene is replaced by the att3Ωaac- cassette.

The vector pSPM21 was introduced into the Streptomyces ambofaciens OSC2 strain (see above) by transformation of protoplasts (Kieser, T et al, 2000). After transformation, the clones are selected for their resistance to apramycin. The clones resistant to apramycin are then subcultured respectively on milia with apramycin (antibiotic B) and on medium with hygromycin (antibiotic A) (cf. FIG. 9). The clones resistant to apramycin (ApraR) and sensitive to hygromycin (HygS) are in principle those where a double crossing over event has occurred and which have the orβ gene interrupted by the att3Ωaac- cassette. These clones were selected and the replacement of the wild-type copy of orβ by the copy interrupted by the cassette was verified. The presence of the att3Ωaac- cassette was verified by colony PCR. Hybridization was also carried out. For this, the total DNA of the clones obtained was digested with several enzymes, separated on an agarose gel, transferred to a membrane and hybridized with a 3 kb probe corresponding to an EcoRI-BamHI fragment from the insert of the plasmid pOS49. .99 (see above). Verification of the genotype; can also be carried out by any method known to those skilled in the art and ^' ' in particular by PCR using the appropriate oligonucleotides and sequencing of the PCR product.

A clone with the expected characteristics has been selected and named

SPM21. It was possible to verify by PCR and hybridization that the att3Ωaac- cassette was indeed present in the genome of this clone and that the expected digestion profile is obtained in the case of replacement, at the following a double recombination event, of the wild-type gene by the copy interrupted by the att3Ωaac- cassette in the genome of this clone. This clone therefore has the genotype: orβ :: att3Ωaac-.

A sample of the strain SPM21 was deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, on July 10, 2002 under the registration number I-2914 . The strain SPM21 was transformed by the vector pOSV508 by transformation of protoplasts to cause excision of the cassette (cf. FIG. 14). The plasmid pOSV508 is derived from the plasmid pWHM3 (Vara J and α., 1989) (cf. FIG. 13) in which the xis and int genes of pSAM2 (Boccard F. et al, 1989b) have been added, placed under the control of ptrc promoter (Amann, E. et al, 1988) (cf. FIG. 14). The introduction into the strain SPM21 of the plasmid pOSV508 carrying the xis and int genes of pSAM2 allows efficient excision by site-specific recombination of the excisable cassette between the attL and attR sites flanking this cassette (Raynal A. et a, 1998) (figure 10). Among the transformants selected for their resistance to thiostrepton due to the tsr gene carried by pOSV508, we choose those which have become sensitive to apramycin, the resistance gene of which is carried by the att3Ωaac- cassette, excision in fact leads to the loss of this gene resistance (see Figure 10). The plasmid pOSV508 is unstable and after two successive passages on medium without antibiotic, isolated clones are subcultured on; medium with thiostrepton and on medium without thiostrepton. Clones sensitive to; ^' = :. thiostrepton have lost pOSV508. It has been verified that the excision of the cassette is successful ^'• • ^; , to a deletion in phase in the orβ gene by PCR and sequencing of the PCR product, ^ i the excision of the cassette in fact left a characteristic “scarring” sequence att3% (which is similar to the original attB site after recombination between the attL and attR sites):

5 'ATCGCGCGCGCTTCGTTCGGGACGAAGAGGTAGAT 3' (SEQ ID N ° 95).

The strain thus obtained and having the desired genotype (orβ :: att3) was named SPM22.

A sample of the strain SPM22 was deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, on July 10, 2002 under the registration number I- 2915 . 004/033689

132

EXAMPLE 11 Construction of a Streptomyces ambofaciens strain interrupted in the orfl2 gene

For the inactivation of orfl2, orfl3c and orfl4, the same starting plasmid (pSPM504) was used to introduce a cassette of the “excisable cassette” type at different positions. This plasmid has a 15.1 kb insert that corresponds to the region to orβ orfl 7. To construct this plasmid a ^'BglII fragment of 15.1 kb derived from the digestion of the cosmid pSPM7 (see above) was clone in the plasmid pMBL18 (Nakano et al, 1995) digested with BamHI. The BamHI and BglTL ends being compatible, the plasmid pSPM502 is obtained after ligation. The entire insert of pSPM502 was then subcloned (in the form of a HindUl / Nhel fragment) in the plasmid pOSK1205 (digested with HindlUJNhei) which made it possible to obtain the plasmid pSPM504.

This plasmid was introduced into the E. coli KS272 strain which already contained the plasmid pKOBEG (Chaveroche and αl, 2000) (cf. FIG. 12).

In parallel, the excisable cassette αtt3Ωααc- was amplified by PCR using as plasmid the plasmid pOSKl 102 (the plasmid pOSKl 102 is a plasmid derived from the vector ρGP704Not (Chaveroche and αl, 2000) (Miller VL & Mekalanos JJ, 1988) in which the αtt3Ωααc- cassette has been cloned as an EcoRV fragment in the unique EcoRV site of pGP704Not), the primers used are the following:

ΕDR8: 5 'CGGGATGATCGCTTGTCCGGCGGCCGGATGCCTAGCCTCATCGCGCGCGCTTCGTTCGG 3' (SΕQ ID N ° 96)

ΕDR9: 5 'CCCGATCCAGAACGTCTGGTCGGTGATCAGGTCGCTGTTCATCTGCCTCTTCGTCCCGAA 3' (SΕQ ID N ° 97)

The 40 (only 39 for ΕDR8) deoxy-nucleotides located at the 5 'end of these oligonucleotides have a sequence corresponding to a sequence in the gene 004/033689

133

target (orfl 2 in this case) and the 20 deoxy-nucleotides located most in 3 '(shown in bold above) correspond to the sequence of one end of the excisable cassette att3Ωaac- (see Figure 11) .

The PCR product thus obtained was used to transform the E. coli KS272 strain containing the plasmids pKOBΕG and pSPM504 (cf. above), as described by Chaveroche et al. (Chaveroche ^* et al, 2000) (cf. FIG. 12 for the principle, the plasmid pOS49.99 must be replaced by the plasmid pSPM504 and the plasmid obtained is no longer pSPM17 but pSPM507). Thus, the bacteria were transformed by electroporation with this PCR product and the clones were selected for their resistance to apramycin. The plasmids of the clones obtained were extracted and digested with several restriction enzymes, in order to verify that the digestion profile obtained corresponds to the expected profile if there has been insertion of the cassette (att3Ωaac ~) into the target gene ( orfl 2), ie if there has indeed been homologous recombination between the ends of the PCR product and the target gene (Chaveroche et al, 2000). The verification of the construction can also be carried out by any method known to a person skilled in the art and in particular by PCR using the appropriate oligonucleotides and sequenced? ; (] of the PCR product. A clone whose plasmid has the expected profile was selected and the corresponding plasmid was named pSPM507 This plasmid is derived from pSPM504 in which orfl 2 is interrupted by the att3Ωaac- cassette (cf. FIG. 12 The insertion of the cassette is accompanied by a deletion in the orfl 2 gene, the interruption begins at the thirtieth codon of orf 12. The last 46 codons of orfl2 remain after the cassette.

The vector pSPM507 was introduced into the Streptomyces ambofaciens OSC2 strain (see above) by transformation of protoplasts (Kieser, T et al, 2000). After transformation, the clones are selected for their resistance to apramycin. The clones resistant to apramycin are then subcultured respectively on medium with apramycin (antibiotic B) and on medium with hy romycin (antibiotic A) (cf. FIG. 9). The clones resistant to apramycin (ApraR) and sensitive to hygromycin (HygS) are in principle those where a double crossing over event has occurred and which have the orf 12 gene interrupted by the att3Ωaac- cassette. These clones were more particularly selected and the replacement of the wild-type copy of orfl 2 by the copy interrupted by the cassette was verified by hybridization. Thus, the total DNA of the clones obtained was digested by several enzymes, separated on agarose gel, transferred to the membrane and hybridized with a probe corresponding to the att3Ωaac- cassette to verify the presence of the cassette in the genomic DNA. clones obtained. A second hybridization was carried out using as a probe a DNA fragment obtained by PCR and corresponding to a very large part of the coding sequence of the gold 72 gene.

Verification of the genotype can also be carried out by any method known to those skilled in the art and in particular by PCR using the appropriate oligonucleotides and sequencing of the PCR product.

A clone with the expected characteristics (orfl2 :: att3Ωaac-) was more particularly selected and named SPM507. It has indeed been possible to verify thanks to the two hybridizations that the att3Ωaac- cassette was indeed present in the genome of this 'clone and that the digestion profile expected is indeed obtained in the case of a' replacement, following of a double recombination event, of the wild-type gene by. '> η the copy interrupted by the att3Ωaac- cassette in the genome of this clone. This clone therefore has the genotype: orfl2 :: att3Ωaac- and was named SPM507. There is no need to excise the cassette to study the effect of inactivation of orfl2, given the orientation of the genes (see Figure 3). Indeed, the fact that orfl3c is oriented in the opposite direction to orfl 2 shows that these genes are not co-transcribed. The use of an excisable cassette, on the other hand, makes it possible to get rid of the selection marker at any time, in particular by transformation with the plasmid pOSV508. A sample of the strain SPM507 was deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, on July 10, 2002 under the registration number 1-2911 . EXAMPLE 12 Construction of a Streptomyces ambofaciens strain interrupted in the orfl3c gene

The att3Ωaac- excisable cassette was amplified by PCR using the plasmid pOSKl 102 (see above) as matrix using the following primers:

EDR3: 5 'ACCGGGGCGGTCCTCCCCTCCGGGGCGTCACGGCCGCGGAATCTGCCTCTTCGTCCCGAA 3' (SEQ ID N ° 98)

EDR4: 5 'CACGCAGCGAGCCGACGCACTGATGGACGACACGATGGCCATCGCGCGCGCTTCGTTCGG 3' (SEQ ID N ° 99)

10

The 40 deoxy-nucleotides located at the 5 ′ end of these oligonucleotides comprise a sequence corresponding to a sequence in the target gene (orfl 3 c in this case) and the 20 deoxy-nucleotides located most at 3 ′ (shown in bold above) 'correspond to the sequence of one of the ends of the att3Ωaac- excisable cassette (cf. =. 15 figure 11). NOT

: Y The PCR product thus obtained was used to transform the E. cόliX strain

KS272 containing the plasmids pKOBEG and pSPM504 (cf. above), as described by '• Chaveroche et al. (Chaveroche et al, 2000) (cf. Figure 12 for the principle, the plasmid pOS49.99 must be replaced by the plasmid pSPM504 and the plasmid obtained is no longer

20 pSPM17 but pSPM508). Thus, the bacteria were transformed by electroporation with the PCR product and the clones were selected for their resistance to apramycin. The plasmids of the clones obtained were extracted and digested with several restriction enzymes, in order to verify that the digestion profile obtained corresponds to the profile expected if there has been insertion of the cassette (att3Ωaac-) into the gene

25 target (orfl 3c), ie if there has indeed been homologous recombination between the ends of the PCR product and the target gene (Chaveroche et al, 2000). The verification of the construction can also be carried out by any method known to a person skilled in the art and in particular by PCR using the appropriate oligonucleotides and sequencing of the PCR product. A clone whose plasmid has the expected profile has been selected

30 and the corresponding plasmid was named pSPM508. This plasmid is derived from pSPM504 in which orfl 3c is interrupted by the apramycin cassette (cf. FIG. 12). The insertion of the cassette is accompanied by a deletion in the orfl3c gene, the interruption begins at the sixth codon of orfl3c. After the cassette, the last 3 codons of orfl3c remain.

The vector pSPM508 was introduced into the Streptomyces ambofaciens strain

OSC2 (see above) by transformation of protoplasts (Kieser, T et al, 2000). After transformation, the clones are selected for their resistance to apramycin. The clones resistant to apramycin are then subcultured respectively on medium with _; apramycin (antibiotic B) and on medium with hygromycin (antibiotic A) (cf. FIG. 9). The clones resistant to apramycin (ApraR) and sensitive to hygromycin (HygS) are in principle those where a double crossing over event has occurred and which have the orfl 3c gene interrupted by the att3Ωaac- cassette. These clones were more particularly selected and the replacement of the wild-type copy of orfl3c by the copy interrupted by the cassette was verified by hybridization. Thus, the total DNA of the clones obtained was digested with several enzymes, separated on agarose gel, transferred. on membrane and hybrid with a probe corresponding to the att3Ωaac- pό if cassette | verify the presence of the cassette in the genomic DNA of the clones obtained. A ^: second hybridization was carried out using as a probe a PCR product corresponding to a sequence extending over a hundred base pairs upstream and downstream of the coding sequence of orfl 3c. Verification of the genotype can also be carried out by any method known to those skilled in the art and in particular by PCR using the appropriate oligonucleotides and sequencing of the PCR product.

A clone exhibiting the expected characteristics (orfl 3c:: att3Ωaac-) was more particularly selected and named SPM508. It has indeed been possible to verify thanks to the two hybridizations that the att3Ωaac- cassette was indeed present in the genome of this clone and that the digestion profile expected is indeed obtained in the case of a replacement, following a double recombination event, of the wild-type gene by the copy interrupted by the att3Ωaac- cassette in the genome of this clone. This clone therefore has the genotype: orfl3c :: att3Ωaac- and was named SPM508. There is no point in excise the cassette to study the effect of inactivation of orfl3c, given the orientation of the genes (see Figure 3), the fact that orfl 4 is oriented in the opposite direction to orfl3c shows that these genes are not co-transcribed. The use of an excisable cassette, on the other hand, makes it possible to get rid of the selection marker at any time. A sample of the strain SPM508 was deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, on July 10, 2002 under the registration number 1-2912 .

EXAMPLE 13 Construction of a Streptomyces ambofaciens strain interrupted in the orfl4 gene

EDR5: 5'GGGCGTGAAGCGGGCGAGTGTGGATGTCaTGCGAGTACTCATCGCGCGCGCTTCGTTCGO 3 '(SEQ ID N ° 100)

EDR6: 5 'CGGGAAACGGCGTCGCACTCCTCGGGGGCCGCGTCAGCCCATCTGCCTCTTCGTCCCGAA 3' (SEQ ID N ° 101)

The 40 deoxy-nucleotides located at the 5 'end of these oligonucleotides have a sequence corresponding to a sequence in the target gene (orfl4 in this case) and the 20 deoxy-nucleotides located most at 3' (shown in bold here). above) correspond to the sequence of one of the ends of the att3Ωaac- excisable cassette (see Figure 11).

The PCR product thus obtained was used to transform the E. coli KS272 strain containing the plasmids pKOBEG and pSPM504 (cf. above), as described by Chaveroche et al. (Chaveroche et al, 2000) (cf. FIG. 12 for the principle, the plasmid pOS49.99 must be replaced by the plasmid pSPM504 and the plasmid obtained is no longer pSPM17 but pSPM509). Thus, the bacteria were transformed by electroporation with the PCR product and the clones were selected for their resistance to apramycin. The plasmids of the clones obtained were extracted and digested with several restriction enzymes, in order to verify that the digestion profile obtained corresponds to the expected profile if there has been insertion of the cassette (attSΩaac-) into the target gene ( orfl 4), ie if there has indeed been homologous recombination between the ends of the PCR product and the target gene (Chaveroche et al, 2000). The verification of the construction can also be carried out by any method known to a person skilled in the art and in particular by PCR using the appropriate oligonucleotides and sequencing of the PCR product. A clone whose plasmid has the expected profile has been selected

The vector pSPM509 was introduced into the Streptomyces ambofaciens OSC2 strain (see above) by transformation of protoplasts (Kieser, T et al, 2000). After transformation, the clones are selected for their resistance to apramycin. The clones resistant to apramycin are then subcultured respectively on medium with apramycin (antibiotic B) and on medium with hygromycin (antibiotic A) (cf. FIG. 9). The clones resistant to apramycin (ApraR) and sensitive to hygromycin (HygS) are in principle those where a double crossing over event has occurred and which have the orfl 4 gene interrupted by the att3Ωaac- cassette. These clones were more particularly selected and the replacement of the wild-type copy of orfl4 by the copy interrupted by the cassette was verified by hybridization. Thus, the total DNA of the clones obtained was digested by several enzymes, separated on agarose gel, transferred to the membrane and hybridized with a probe corresponding to the att3Ωaac- cassette to verify the presence of the cassette in the genomic DNA. clones obtained. A second hybridization was carried out using a PCR product as probe corresponding to a sequence extending over a hundred base pairs upstream and downstream of the coding sequence of orf 14. Verification of the genotype can also be carried out by any method known to those skilled in the art and in particular by PCR using appropriate oligonucleotides and sequencing of the PCR product.

A clone having the expected characteristics (orfl4 :: att3Ωaac-) was more particularly selected and named SPM509. It has indeed been possible to verify thanks to the two hybridizations that the att3Ωaac- cassette was indeed present in the genome of this clone and that the digestion profile expected is indeed obtained in the case of a replacement, following a double recombination event, of the wild-type gene by the copy interrupted by the att3Ωaac- cassette in the genome of this clone. This clone therefore has the genotype: orfl4 :: att3Ωaac- and was named SPM509. There is no need to excise the cassette to study the effect of inactivation of orfl.4, given the orientation of the genes (see Figure 3), the fact that orflSc is oriented opposite to or / 14 shows that these genes are not co-transcribed. The use of an excisable cassette, on the other hand, makes it possible to get rid of the selection marker at any time. A sample of the strain SPM509 has been deposited with; National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, July 10, 2002 under registration number 1-2913.

EXAMPLE 14 Construction of a Streptomyces ambofaciens strain interrupted in the orfό * gene

The orfό * gene was inactivated using the excisable cassette technique (see above and Figure 10). For this, the cosmid pSPM7 was used as a matrix to amplify a fragment of the orfό * gene using the following oligonucleotides:

C9583: 5 'CTGCAGGTGCTCCAGCGCGTCGATCT 3' (oligo sense) (SEQ ID N ° 102) C9584: 5 'CTGCAGACGGAGGCGGACCTGCGGCT 3' (oligo antisense) (SEQ ID N ° 103)

The 20 deoxy-nucleotides located at the 3 ′ end of these oligonucleotides correspond to a sequence located in the coding part of the orfό * gene (SEQ ID No. 13) and the 6 deoxy-nucleotides located most at 5 ′ correspond to the site sequence

Pstl to facilitate cloning thereafter. The amplified DNA fragment is approximately 1.11 kb in size. This PCR product is cloned into the vector pGEM-T Easy (marketed by the company Promega (Madison, Wisconcin, USA)) which made it possible to obtain the plasmid named pBXL111 (cf. FIG. 16).

The attlΩhyg + excisable cassette was then introduced into the coding sequence of the orfό * gene. For this, the plasmid pBXL1 ll 1 was digested with the restriction enzymes SmaI and Aspl1S1 and the digestion product was treated with the enzyme Klenow. This manipulation makes it possible to carry out an internal deletion of 120 bp in the coding sequence of the orfό * gene (cf. FIG. 15). In addition, on either side of the restriction sites, there remain respectively 511 bp and 485 bp of the sequence of orfό * which will allow homologous recombination for the inactivation of the orfό * gene. The attlΩhyg + excisable cassette was prepared from the plasmid pattlΩhyg + (cf. above) by digestion of this plasmid with EcoRV. The latter was then subcloned into the vector pBXLl l l l previously prepared as described above (Smal and AspllSl digestion then treatment with the Klenow enzyme). The plasmid obtained was named pBXL112 (cf. FIG. 17). In this construction, the orfό * gene has a 120bp deletion and is interrupted by the attlΩhyg + cassette.

The plasmid pBXL1112 was then digested with the enzyme Pstl (site bordering the cassette since it is present in the oligonucleotides PCR) and the Pstl insert of 3.7 kb comprising a part of orfό * interrupted by the attlΩhyg + cassette was then cloned at the level Pstl site of the plasmid pOJ260 (cf. above). The plasmid thus obtained was named pBXLl 113. The vector pBXL1113 was introduced into the Streptomyces ambofaciens OSC2 strain (see above) by transformation of protoplasts (Kieser, T et al, 2000). After transformation, the clones are selected for their resistance to hygromycin. The clones resistant to hygromycin are then subcultured respectively on medium with hygromycin (antibiotic B) and on medium with apramycin (antibiotic A) (cf. FIG. 9). The clones resistant to hygromycin (HygR) and sensitive to apramycin (ApraS) are in principle those where a double crossing over event has occurred and which have the orfό * gene interrupted by the attlΩhyg + cassette. These clones were more particularly selected and the replacement of the wild copy of orfό * by the copy interrupted by the cassette was verified by the technique of Southern blot. Thus, the total DNA of the clones obtained was digested with several enzymes, separated on agarose gel, transferred to a membrane and hybridized with a probe corresponding to the hyg gene (obtained by PCR) to verify the presence of the cassette in the Genomic DNA of the clones obtained. A second hybridization was carried out using as probe the Pstl-Pstl insert containing the orfό * gene, of a size of about 1.1 kb and obtained from r of the plasmid pBXLllll (cf. above and fig re 16). Verification of the μμ genotype can also be carried out by any method known to those skilled in the art and in particular ^' - \ by PCR using the appropriate oligonucleotides and sequencing of the PCR product.

A clone with the expected characteristics (orfό * :: attlΩhyg +) was more particularly selected and named SPM501. It has indeed been possible to verify thanks to the two hybridizations that the attlΩhyg + cassette was indeed present in the genome of this clone and that the expected digestion profile is indeed obtained in the case of a replacement, following a double recombination event, of the wild-type gene by the copy interrupted by the attlΩhyg + cassette in the genome of this clone. This clone therefore has the genotype: orfό * :: attlΩhyg + and was named SPM501. A sample of the strain SPM501 was deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, on July 10, 2002 under the registration number 1-2909 . The strain SPM501 was transformed by the vector pOSV508 by transformation of protoplasts to cause excision of the cassette (cf. FIG. 14). The plasmid pOSV508 is derived from the plasmid pWHM3 (Vara J et a /., 1989) (cf. FIG. 13) in which the xis and int genes of pSAM2 (Boccard F. et al, 1989b) placed under the control of the ptrc promoter ( Amann, E. et al, 1988) were added (see Figure 14). The introduction into the strain SPM501 of the plasmid pOSV508 carrying xis and int genes of pSAM2 allows efficient excision by site-specific recombination of the excisable cassette between the attL and αttR sites flanking this cassette (Raynal A. et al, 1998) (figure 10). Among the transformants, selected for their resistance to thiostrepton due to the tsr gene carried by pOSV508, we choose those that have become sensitive to hygromycin, the resistance gene of which is carried by the attlΩhyg + cassette, excision in fact leads to the loss of this gene resistance (see Figure 10). The plasmid pOSV508 is unstable and after two successive passages on medium without antibiotic isolated clones are subcultured on medium with thiostrepton and on medium without thiostrepton. Clones sensitive to thiostrepton lost pOSV508. It was verified that the excision of the cassette did take place in phase in the orfό * gene by PCR and sequencing of the PCR product. The interruption; H begins at 158 codon, 40 cPdons are deleted (120pb) and the excision of the cassette ¹ ^; leaves a “scar” attl sequence characteristic of 33 bp: 5 ′ ATCGCGCGCTTCGTTCGGGACGAAGAGGTAGAT 3 ′ (SEQ ID No. 104).

The strain thus obtained and having the desired genotype (orfό * :: attl) was named SPM502.

A sample of the strain SPM502 was deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, on July 10, 2002 under the registration number 1-2910 . EXAMPLE 15 Analysis of Streptomyces ambofaciens strains interrupted in the orβ, orβ, orβ, orflO, orfl2, orfl3c, orfl4, orfό * genes

To test the production of spiramycin of the various strains obtained, a microbiological test based on the sensitivity of a strain of Micrococcus luteus was developed (cf. (Gourmelen A. et al., L99S)). The strain of Micrococcus luteus used is a strain derived from the strain DSM 1790 naturally sensitive to spiramycin (this strain is available in particular from the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikro-organismen und Zellkulturen GmbH, DSMZ), (Braunschweig, Germany), under the number DSM 1790), the strain used in the present test differs from the strain DSM1790 in that it is resistant to congocidine. This strain is a spontaneous mutant obtained by selection on medium containing increasing doses of congocidine. Such a strain was selected because Streptomyces ambofaciens produces both spiramycin and y congocidine. The aim is to measure the production of spiramycin from the various strains _: f; ; - ^' ; obtained thanks to a microbiological test based on the sensitivity of a strain of Micrococcus luteus, it is necessary to have a strain resistant to ^! ; ïà; 'îf congocidine.

The different strains of Streptomyces to be tested were cultured in 500 ml baffled erlenmeyers (baffled erlenmeyers) containing 70 ml of MP5 medium (Pernodet et al, 1993). The baffled Erlenmeyer flasks were inoculated at an initial concentration of 2.5 × 10 ⁶ spores / ml of the different strains of S. ambofaciens and grown at 27 ° C. with orbital shaking at 250 rpm. Samples of 2 ml of suspensions were taken after 48, 72 and 96 hours of culture and centrifuged. The different supernatants were then frozen at -20 ° C. A dilution to a tenth of these supernatants in sterile culture medium is used for the test (cf. FIG. 18).

The indicator strain of Micrococcus luteus resistant to congocidin but sensitive to spiramycin was cultured in 2TY medium (Sambrook et al, 1989) containing congocidin at 5 μg / ml for 48 h at 37 ° C. The optical density (OD) of the culture is measured and this culture is diluted so as to adjust the optical density to 4. 0.4 ml of this preculture is diluted in 40 ml of DAM5 medium (Difco Antibiotic Medium 5, marketed by the company Difco), beforehand brought to a temperature of about 45 ° C. This medium is then poured into a 12 × 12 cm square box and left to stand at room temperature.

"

Once the medium had cooled and solidified, Whatman AA paper discs (cf. Gourmelen A. et al, 1998) 12 mm in diameter were soaked with 70 μl of the dilution to one tenth of each supernatant and deposited on the surface of the box. Discs soaked in a solution of spiramycin of different concentrations (2-4-8 μg / ml in MP5 culture medium) are used as standard range. The dishes are left at 4 ° C for 2 h so as to allow the diffusion of antibiotics in the agar and are then incubated at 37 ° C for 24 to 48 h.

If the disc contains spiramycin, it diffuses into the agar and inhibits the growth of the indicator strain of Micrococcus luteus. This inhibition creates a "halo" around the disc, this halo reflecting the area where the strain of Micrococcus luteus did not grow. The presence of this halo is therefore an indication of the presence of spiramycin and makes it possible to determine whether the strain of S. ambofaciens corresponding to the disc in question is producing or not producing spiramycin. A comparison with the inhibition diameters obtained for the standard range makes it possible to obtain an indication of the quantity of spiramycin produced by this strain.

The different strains described in the previous examples were used in this test to detect their production of spiramycin. The results obtained are collated in Table 38. Table 38

These results allow a number of conclusions to be drawn with regard to the function of the different genes involved in the biosynthesis of spiramycin. Thus the orβ gene is essential for the biosynthesis of spiramycin. Indeed, inactivation in phase in this gene leads to a strain (OS49.67, (cf. example 6)) no longer producing spiramycin. Inactivation in phase makes it possible to reject the hypothesis of a possible influence of the cassette introduced on the expression of the genes located downstream of orβ.

Likewise, the orβ and orf 10 genes encode proteins essential for the biosynthesis of spiramycin since the strains OS49.107 and OS49.50 have a non-producing phenotype. In addition, in these last two strains, it is the inactivation of the corresponding gene which is responsible for this non-producing phenotype, since in view of the orientation of the different orfs (cf. FIG. 3), the construction introduced does not may have a polar effect.

The study of strains having an excisable cassette also makes it possible to draw a certain number of conclusions with regard to the function of the interrupted genes. The SPM507 strain has the genotype: orfl2 :: att3Ωaac-. There is no need to excise the cassette to study the effect of inactivation of orf! 2, in view of! the orientation of the genes (cf. FIG. 3). The fact that orf! 3c is oriented in the opposite direction to orfl2 shows that these genes are not co-transcribed. The use of an excisable cassette, on the other hand, makes it possible to get rid of the selection marker at any time. The phenotype of the strain SPM507 is non-producing, it can therefore be concluded that the orfl2 gene is a gene essential for the biosynthesis of spiramycin in S. ambofaciens.

The SPM508 strain has the genotype: orfl3c :: att3Ωaac-. There is no need to excise the cassette to study the effect of inactivation of orfl 3c, given the orientation of the genes (see Figure 3). The fact that orf! 4 is oriented in the opposite direction to orf! 3c shows that these genes are not co-transcribed. The use of an excisable cassette, on the other hand, makes it possible to get rid of the selection marker at any time. The phenotype of the strain SPM508 is productive, we can therefore conclude that the orfl3c gene is not a gene essential for the biosynthesis of spiramycin in S. ambofaciens.

The SPM509 strain has the genotype: orfl4 :: att3Ωaac-. There is no need to excise the cassette to study the effect of inactivation of orfl.4, given the orientation of the genes (see Figure 3), the fact that orflSc is oriented opposite to orfl4 shows that these genes are not co-transcribed. The use of an excisable cassette, on the other hand, makes it possible to get rid of the selection marker at any time. The phenotype of the strain SPM509 is non-producer, it can therefore be concluded that the orfl 4 gene is a gene essential for the biosynthesis of spiramycin in S. ambofaciens.

The strain SPM21 has the genotype: orβ :: att3Ωaac-, The phenotype of this strain is not a producer of spiramycins. However, the orientation of the orfl to orβ genes (cf. FIG. 3) suggests that these genes are cotranscribed. Also, the phenotype observed may be due to a polar effect of the cassette introduced into orβ on the expression ;; of genes located downstream in the operation. The strain SPM22 has the genotype orβ :: att3, -. and was obtained after excision in phase of the inserted cassette. The excision of the cassette leaves only a characteristic “scar” sequence (cf. example 10). The strain SPM22 also being of non-producing phenotype, it can be concluded that the orβ gene is a gene essential for the biosynthesis of spiramycin in S. ambofaciens. We observe here only the effect due to the inactivation of orβ.

The strain SPM501 has the genotype: orfό * :: attlΩhyg +. The phenotype of this strain is not a producer of spiramycins. However, the orβ * and orfό * genes (cf. FIG. 3) having the same orientation, the phenotype observed may be due to a polar effect of the cassette introduced into orfό * on the expression of orβ *. The arrangement of these genes suggests that they can be cotranscribed. To answer this question, the strain SPM502 was obtained after excision in phase of the cassette introduced. In this strain, we only observe the effect of inactivation of orfό *. This strain has the orfό * :: attl genotype (see example 14). The excision of the cassette leaves only a “scar” sequence in phase (cf. example 14). The strain SPM502 has a producer phenotype (this strain only produces spiramycin I, however (cf. *. Example 16)). It can therefore be concluded that the orβ * gene is a gene essential for the biosynthesis of spiramycin in S. ambofaciens, since its indirect inactivation in the strain SPM501 leads to a non-producing phenotype. On the other hand ; the orfό * gene is not a gene essential for the biosynthesis of spiramycin I in S. ambofaciens (on the other hand, it is essential for the production of spiramycin II and Ht (cf. example 16)). ^!

EXAMPLE 16. Determination of the production of spiramycins I, II and III in the mutant strains obtained

The different strains to be tested were each cultured in 7 erlensmeyers; j, g baffled 500 ml containing 70 ml of MP5 medium (Pernodet et al, 1993). The plants were inoculated with 2.5 × 10 ⁶ spores / ml of the different strains of S. ambofaciens and allowed to grow at 27 ° C. with orbital stirring at 250 rpm for 72 hours. The cultures corresponding to the same clone are combined, optionally filtered on a pleated filter, and centrifuged for 15 min at 7000 rpm. The different supernatants were then stored at -30 ° C.

The assays were carried out by High Performance Liquid Chromatography

(HPLC) by ion pairing. HPLC analysis of the culture medium makes it possible to precisely determine the concentration of the three forms of spiramycin. The column used (Macherey-Nagel) is filled with a Nucleosil phase of octyl grafted silica. The particle size is 5 μm and the size of the pores 100. The internal diameter of the column is 4.6mm and its length 25cm. The mobile phase is a mixture of H ₃ PO ₄ buffer (pH2.2) and 70/30 acetonitrile (v / v) containing 6.25 g / L of NaGO ₄ perchlorate. The analysis is carried out in an isocratic regime with a flow rate set at 1 ml / min. The column is thermoregulated at 23 ° C. Detection is ensured by UV spectrophotometry at 238nm. The sample is refrigerated at + 10 ° C and the quantification is determined from the peak area (by external calibration). Under these conditions, the retention times of spiramycin I, II and III are approximately 17 respectively; 21 and 30 minutes, as it could be verified using a commercial sample comprising the three forms of spiramycin.

The OSC2 strain has a spiramycin-producing phenotype. It is the parental strain used to obtain the strains having an excisable cassette

(cf. example 15). This strain was therefore used as a positive control for the production of the three forms of spiramycin. This strain produces well the three forms of spiramycins as it was verified by HPLC (cf. FIG. 19).

The study of strains having an excisable cassette makes it possible to draw a certain number of conclusions with regard to the function of the interrupted genes. Strain ; SPM507 has the genotype: orf! 2 :: att3Ωaac-. The phenotype of the strain SPM507 is non-producer (cf. example 15), we can therefore conclude that the orfl 2 gene is μn ^' ; fi gene essential for the biosynthesis of spiramycin in S. ambofaciens. This strain no longer produces spiramycins as has been verified by HPLC (cf. FIG. 22). This result confirms the essential character of the orfl 2 gene in the biosynthesis of spiramycin.

The SPM508 strain has the genotype: orfl3c :: att3Ωaac-. The phenotype of the strain SPM508 is a producer of spiramycin (cf. example 15), it can therefore be concluded that the orfl3c gene is not a gene essential for the biosynthesis of spiramycin in S. ambofaciens. This strain produces spiramycin I, II and III as it was verified by HPLC (cf. FIG. 23). This result confirms that the orfl 3c gene is not a gene essential for the biosynthesis of spiramycins I, II and III in S. ambofaciens.

The SPM509 strain has the genotype: orfl4 :: att3Ωaac-. The phenotype of the strain SPM509 is non-producer, we can therefore conclude that the orfl 4 gene is a gene essential for the biosynthesis of spiramycin in S. ambofaciens. This strain no longer produces spiramycins as has been verified by HPLC (cf. FIG. 24). This result confirms the essentiality of the orfl 4 gene in the biosynthesis of spiramycin.

The strain SPM501 has the genotype: orfό * :: att! Ωhyg +. The phenotype of this strain is not a producer of spiramycins. This strain does not produce p spiramycins as it has been verified by HPLC (cf. FIG. 20). However, the ^' orβ * and orfό * genes (see Figure 3) having the same orientation, the observed phenotype may be due to a polar effect of the cassette introduced in orfό * on the expression of orβ * in the operon. This suggests that these genes are cotranscribed. To answer this question, the strain SPM502 was obtained by excision of the cassette introduced, producing a deletion in phase in the gold 6 * gene and restoring the expression of orβ *. This strain has the orfό * :: attl genotype (cf. examples 14 and 15). The excision of the cassette leaves only a “scar” att sequence in phase (cf. example 14). The SPM502 strain has a spiramycin-producing phenotype. However, as has been proven by HPLC, this spuche produces only spiramycin I and does not produce 1 of spiramycin II and El (cf. FIG. 21). It can therefore be concluded from these results that the 'orβ * gene is a gene essential for the biosynthesis of spiramycin in S. ambofaciens, since its indirect inactivation in the strain SPM501 leads to a phenotype which does not produce spiramycin (cf. FIG. 20) . On the other hand ; the orfό * gene is not a gene essential for the biosynthesis of spiramycin I in S. ambofaciens, since the inactivation of this gene leads to a phenotype producing spiramycin I (cf. FIG. 21). However, orfό * is essential for the production of spiramycin II and III (cf. example 16)). The orfό * gene therefore encodes an acyl transferase responsible for the modification of platenolide at position 3 (cf. FIG. 1). EXAMPLE 17 Determination of the starting point of the translation of orf 10 and improvement of the production of spiramycins

17.1 Construction of the plasmids pSPM523, pSPM524 and pSPM525:

The or / 70 gene was identified in Streptomyces ambofaciens and was designated srmR by Geistlich et al (Geistlich, M., et al, 992% The inactivation of the orflO gene was carried out (cf. Example 8). . thus be shown that the resulting strain no longer produces spiramycin (cf. example 15) this confirms that the orflO gene is indeed involved in the biosynthesis of spiramycin the protein encoded by this ^gene;. is therefore essential for the biosynthesis of Spiramycins, however, the analysis of the sequence shows that two ATG codons located in the same reading phase can be used for the translation of orflO (cf. Figure 28) .One of the two possible codons (the codon most upstream) starts at position 10656 in the sequence

In order to determine the translation start site, three constructions comprising three forms of orflO were carried out. The latter were obtained by PCR using oligonucleotides comprising either an H dlII restriction site or a BamHI restriction site.

The first pair used for the amplification corresponds to the following oligonucleotides:

EDR39:

5 'CCCAAGCTTGAGAAGGGAGCGGACATTCATGGCCCGCGCCGAACGC3' (SEQ ID No. 122) (the Hindlll site is in bold)

EDR42:

5'CGGGATCCGGCTGACCATGGGAGACGGGCGCATCGCCGAGTTCAGC3 '

(SEQ ID N ° 123) (the BamHI site is in bold) The pair of primers EDR39-EDR42 allows the amplification of a gold fragment 70 comprising the ATG located most in 3 '(position 10809 in the sequence given in

SEQ ID N ° 1) (see Figure 28). The fragment obtained is approximately 2 kb in size and will hereinafter be called "gold / 70 short", it does not contain the orflO promoter. This 2kb fragment was cloned into the vector pGEM-T easy to give rise to the plasmid pSPM520.

The second pair used for the amplification corresponds to the following oligonucleotides:

EDR40: 5 'CCCAAGCTTGAGAAGGGAGCGGACATTCAATGCTTTGGTAAAGCAC3'

(SEQ ID N ° 124) (the Hindlll site is in bold)

EDR42:

5'CGGGATCCGGCTGACCATGGGAGACGGGCGCATCGCCGAGTTCAGC3 '

(SEQ ID No. 123) (the BamHI site is shown in bold) The pair of primers EDR40-EDR42 allows the amplification of an orflO fragment comprising the ATG located most in 5 '(position 10656 in the sequence given in

SEQ ID N ° 1) (see Figure 28). This 2.2 kb fragment, subsequently called "long orflO", was cloned into the vector pGEM-T easy to give rise to the plasmid pSPM521, this plasmid does not contain the gold / 70 promoter. The third pair used for the amplification corresponds to the following oligonucleotides:

EDR41:

5 '-CCCAAGCTTTCAAGGAACGACGGGGTGGTCAGTCAAGT-3'

(SEQ ID N ° 125) (the Hindlll site is in bold) EDR42:

5'CGGGATCCGGCTGACCATGGGAGACGGGCGCATCGCCGAGTTCAGC3 '

(SEQ ID N ° 123) (the R mHI site is in bold)

The pair of primers EDR41-EDR42 allows the amplification of the or / 70 gene with the two ATGs, as well as its own promoter (cf. FIG. 28). This 2.8 kb fragment hereinafter called "orf 10 pro" was cloned into the vector pGEM-T easy to give rise to the plasmid pSPM522.

The "or 70 pro" fragment was obtained using the chromosomal DNA of the OSC2 strain as a template. The "gold / 70 short" and "gold / 70 long" fragments were obtained using the DNA from the previously purified "gold 70 pro" fragment as a template.

The HindlYL-BamHI inserts of the plasmids pSPM520, pSPM521 and pSPM522 were then subcloned into the vector ρUWL201 (plasmid derived from the plasmid pUWL199 (Wehmeier UF, 1995) in which the fragment Kpnl-BamHI of the region of the ermE promoter (cf Bibb et al., 1985, in particular FIG. 2) carrying a mutation increasing the strength of the promoter (ermΕ * promoter) (Bibb et al, 1994) was introduced (cf. Doumith et al, 2000)) previously digested by enzymes H dlII-iîαmHI. Thus, three plasmids were obtained: pSPM523 (derived from pUWL201 with the form "or / 70 short" as an insert), pSPM524 (derived from pUWL201 with the form "gold / 70 long" as an insert) and pSPM525 (derived ρUWL201 with the form "or / 70 pro") (Figure 28).

17.2 Transformation of the strain OS49.50 by the constructions pSPM523, pSPM524 and pSPM525:

The strain OS49.50 (strain interrupted in the or / 70 gene, cf. example 8) was transformed independently by transformation of protoplasts (Kieser, T et al, 2000) by each of the plasmids pSPM523, pSPM524 and pSPM525. A negative control was also produced by transforming the strain OS49.50 with the plasmid pUWL201 without insert. After transformation of the protoplasts, the clones are selected for their resistance to thiostrepton. The transformation of the clones by each of the plamides is verified by extraction of these plasmids. Four new strains were thus obtained: the OSC49.50 pUWL201 strain, resulting from the transformation by the plasmid pUWL201 without insert, the OSC49.50 pSPM523 strain, resulting from the transformation by the pSPM523 plasmid, the OSC49.50 pSPM524 strain, of transformation by the plasmid pSPM524 and finally the strain OS49.50 ρSPM525, resulting from the transformation by the plasmid ρSPM525.

The production of spiramycins of each of these four strains was tested by HPLC. For this, the various strains of Streptomyces to be tested were cultivated in 500 ml baffled erlenmeyers (baffled erlenmeyers) containing 70 ml of MP5 medium (Pernodet and α /., ^* 1993). When the strain contains the plasmid pUWL201 or one of its derivatives, 5 μg / ml of thiostrepton is added. The baffled Erlenmeyer flasks were inoculated at an initial concentration of 2.5 × 10 10 spores / ml of the different strains of S. ambofaciens and the cultures were incubated at 27 ° C. with orbital shaking of 250 μm for 96 hours. The cells were then separated from the medium by centrifugation and the supernatant was analyzed by HPLC (cf. example 16) to determine the amount of spiramycin produced. Thanks to a standard sample and measurement of the air in the peaks, it was possible to determine the quantity of each of the spiramycins 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 ^" correspond to the total production of spiramycins (obtained by adding the production of spiramycin I, II and lu).

Table 39. Production in spiramycins of strains derived from OS49.50, (results expressed in mg / 1).

As shown in the results given in Table 39, the negative control (strain OS49.50 transformed by the plasmid pUWL201) does not produce spiramycin. When the plasmid pSPM523 (which contains the form "gold / short 70") is introduced ^to the strain OS49.50 years, no spiramycin production is observed. In contrast, the presence of the plasmid pSPM524 (which contains the form "or / 70 long") and of the plasmid pSPM525 (which contains the form "gold / 70 pro") restores the production of spiramycin in the host strain OS49.50. Thus, only the gold / 70 fragments containing the most upstream ATG restore the synthesis of spiramycin.

In order to confirm these results, the plasmid pSPM521 (plasmid

10 pGEM-T easy containing the form "gold / 70 long") was digested with the enzyme,. XhoI restriction (this enzyme has a unique site in this plasmid, located between ^• 'both ATG (see Figure 28)). The Xhol ends were then made blunt end; jg

; ; by treatment with the Klenow enzyme. The plasmid was then closed on itself ;; _: ^ by action of T4 DNA ligase to give rise to the plasmid pSPM527. If 15 the most upstream ATG (position 10656 in the sequence given in SEQ ID No. 1) is indeed used as the translation initiation site, this processing will cause a reading frame shift at the site level. ^ ol and will have the effect of producing a protein not exhibiting activating activity. On the other hand, if the initiation of translation takes place at the level of the ATG further downstream (position 10809 in the sequence given in SEQ ID No. 1), this processing should have little or no effect on expression. OrflO (taking into account the location of the transcription start point) and no effect on the protein produced.

The BamHI-HindIII insert of pSPM527 was then subcloned into the vector pUWL201 to give rise to the plasmid pSPM528. This plasmid was introduced

25 in strain OS49.50 and a clone having the desired plasmid was more particularly selected. Spiramycin production of the strain The resultant was then tested by HPLC (see example 16 and above). Contrary to what had been observed with the plasmid pSPM524 (cf. table 39), the presence of the plasmid pSPM528 in the strain OS49.50 does not restore the production of spiramycin. This confirms that the start of translation of the orflO gene is the ATG located furthest downstream (ATG 1 cf. FIG. 28).

"

17.3 Improvement in the production of spiramycins of the OSC2 strain of S. ambofaciens:

In order to test the effect of overexpression of the or / 70 gene on the production of spiramycins, the plasmids pSPM523, pSPM524, pSPM525 and pSPM528 were

10 introduced into the OSC2 strain. For this, protoplasts of the OSC2 strain were transformed (Kieser, T et al, 2000) independently by each of the plasmids pSPM523, pSPM524, pSPM525 and ρSPM528. A negative control was also produced by transforming the OSC2 strain with the plasmid pUWL201 without insert. After transformation of the protoplasts, the clones are selected for their resistance to thiostrepton. Thus five new strains were obtained: the OSC2 strain pUWL201, resulting from the transformation by the plasmid pUWL201 without insert, the

OSC2 strain pSPM523, resulting from the transformation by the plasmid pSPM523, the OSC2 strain pSPM524, resulting from the transformation by the plasmid pSPM524, the OSC2 strain pSPM525, resulting from the transformation by the plasmid pSPM525 and

Finally, the OSC2 strain pSPM528, resulting from the transformation with the plasmid pSPM528. The production of spiramycins of these strains was then analyzed by HPLC (in the same way as in Example 17.2). Analysis of the spiramycin production of the OSC2 strain was also carried out in parallel for comparison. The results of this analysis are presented in Table 40, the

25 data are expressed in mg per liter of supernatant. The results correspond to the total production of spiramycins (obtained by adding the production of spiramycin I, II and III). Table 40. Production in spiramycins of strains derived from OSC2, (results expressed in mg / 1).

Thus, it is observed that the presence of the plasmid pSPM524 or of the plasmid pSPM525 significantly increases the production in spiramycins of the strain

OSC2. This clearly demonstrates that the overexpression of OrflO has a positive effect on the production of spiramycins. On the other hand, the plasmid pSPM528 has no effect on the production of spiramycins.

The plasmids pSPM525 and pUWL201 were similarly introduced into the strain SPM502 (cf. example 14). Thus two new strains were obtained: the strain SPM502 ρUWL201, resulting from the transformation by the plasmid pUWL201 without insert, and the strain SPM502 pSPM525, resulting from the transformation by the plasmid pSPM525.

A sample of the strain SPM502 pSPM525 (this strain contains the plasmid pSPM525, cf. above) was deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, February 26, 2003 under registration number 1-2977.

The production in spiramycins of the strains SPM502 ρUWL201 and SPM502 pSPM525 was analyzed by HPLC (in the same way as in Example 17.2). Analysis of the spiramycin production of the strain SPM502 was also carried out in parallel for comparison. The results of this analysis are presented in Table 41, the data are expressed in mg per liter of supernatant. The results correspond to the production of spiramycin I. Indeed, none of these strains produces spiramycin π and m.

Table 41. Production in spiramycin I of strains derived from SPM502, ^' (results ts i expressed in mg / 1). IIThere

Thus, it could be observed that the overexpression of the orflO gene in the strain SPM502 significantly increases the production of spiramycin I. EXAMPLE 18 Construction of a genomic DNA library of the OSC2 strain of Streptomyces ambofaciens in E. coli in the cosmid pWED2.

18.1 Construction of the cosmid pWED2:

In order to facilitate the inactivation of genes in Streptomyces, a cosmid carrying the oriT sequence of the plasmid RK2 (which allows its introduction by conjugation into Streptomyces from an appropriate strain of E. coli) and also carrying a gene resistance to an antibiotic conferring a phenotype identifiable in Streptomyces, was built. Such a cosmid containing large inserts of genomic DNA from Streptomyces ambofaciens can be used in gene inactivation experiments.

To construct this vector, a pac-oriT cassette (EcoRV fragment) was introduced into the cosmid pWΕDl (Gourmelen et al, 1998), derived from the cosmid pWΕD15 (Wahl et a, 1987), at the unique Hpal site. The pac-oriT cassette was obtained by PCR. For this, the pac gene was amplified by PCR from the plasmid pVF 10.4 (Vara et al., 1985; Lacalle et al, 1989) using as 4 '(first primer, primer A (of sequence 5'- '

CCAGTAGATATCCCGCCAACCCGGAGCTGCAC-3 '(SΕQ ID No. 126), the EcoRV restriction site has been underlined and the bold sequence of 20 nucleotides corresponds to a region upstream of the pac gene promoter) and as second primer, primer B ( 5'- sequence

GAAAAGATCCGTCATGGGGTCGTGCGCTCCTT-3 '(SΕQ ID N ° 127), which has at its 5' end a sequence of 12 nucleotides corresponding to the start of the oriT sequence (double underlined) and a sequence of 20 nucleotides (in bold) corresponding to the end of the pac gene (see Figure 29, ^1st PCR). The oriT gene was amplified by PCR from the plasmid pPM803

(Mazodier, P. Et al, 1989) using as first primer, primer C (of sequence 5'-CACGACCCCATGACGGATCTTTTCCGCTGCAT-3 '(SΕQ ID No. 128)), which has at its 5' end a sequence of 12 nucleotides corresponding to a sequence downstream of the coding sequence for the pac gene (in bold) and a sequence of 20 nucleotides corresponding to the start of the oriT sequence) and as second primer, primer D (of sequence 5'-

GAGCCGGATATCATCGGTCTTGCCTTGCTCGT-3 '(SEQ ID No 129)) which includes the restriction site EcoRV (single underlined) and a sequence of 20 nucleotides corresponding to the end of the oriT sequence (double underlined) (cf. Figure 29, ₂ nd _{pcR )}

The amplification product obtained with primers A and B and that obtained with primers C and D have at one of their ends a common sequence of 24 nucleotides. A third PCR was performed by mixing the two amplification products obtained above and using, as primers, the primers A and D (see Figure 29, 3 ^-th PCR). This made it possible to obtain an amplification product corresponding to the pac + oriT assembly. This pac-oriT fragment was then cloned into the vector pGΕM-T Easy (sold by the company Promega (Madison, Wisconcin, USA)), which made it possible to obtain the plasmid pGEM-T-> c-oπ. The insert of this "plasmid was then cloned into the cosmid pWEDl. For this, the plasmid; GEM-pac-oriT was digested with the enzyme EcoRV and the insert EcoRV containing: ^ the set pac + oriT was inserted into the cosmid pWΕDl opened beforehand by the enzyme Hpal. The cosmid thus obtained was named pWΕD2 (cf. FIG. 30).

This cosmid facilitates the inactivation of genes in Streptomyces. Indeed, it carries the oriT sequence (which allows its introduction by conjugation into Streptomyces from an appropriate strain of E. coli) but also a gene for resistance to an antibiotic conferring a phenotype identifiable in Streptomyces. Such a cosmid containing large inserts of genomic DNA from Streptomyces ambofaciens can be used in gene inactivation experiments.

Thus a cosmid derived from pWED2 containing the target gene could for example be introduced into the strain of E. coli KS272 containing the plasmid pKOBEG (cf. Chaveroche et al 2000) and a cassette will be introduced into the target gene according to the technique described by Chaveroche et al. 2000. The cosmid obtained by this technique (cosmid in which the target gene is inactivated), can then be introduced into an E. coli strain such as strain S 17.1 or any other strain making it possible to transfer, by conjugation, plasmids containing the oriT sequence to Streptomyces.

After conjugation between E. coli and Streptomyces, clones of Streptomyces in which the wild copy of the target gene has been replaced by the interrupted copy can be obtained as described in Example 2.

The resistance gene expressed in Streptomyces present on this new cosmid is the pac gene of Streptomyces alboniger (Vara, J et al, 1985; Lacalle et al, 1989) which codes for puromycin N-acetyl transferase and which confers resistance to puromycin . During gene inactivation experiments, we will look for clones in which a double recombination event has taken place. These clones will have eliminated the cosmid pWΕD2 and will therefore have become sensitive to puromycin again.

18.2 Construction of a genomic DNA library of the OSC2 strain of Streptomyces ambofaciens in E. coli in the cosmid pWΕD2,

The genomic DNA of the OSC2 strain of Streptomyces ambofaciens was _f

^{,, f} partially digested with the restriction enzyme BamHI so as to obtain DNA fragments of size between approximately 35 and 45 kb. These fragments were then cloned into the cosmid pWΕD2, the latter having been previously digested with

BamHI, then treated with alkaline phosphatase. The ligation mixture was then packaged in vitro into lambda phage particles using the “Packagene® Lambda DNA packaging System” system marketed by the company Promega (Madison, Wisconcin, USA) according to the manufacturer's recommendations. The phage particles obtained were used to infect the SURE® strain of E. coli marketed by the company Stratagene (LaJolla, California, USA). The selection of the clones was carried out on LB + ampicillin medium (50 μg / ml), the cosmid pWΕD2 conferring resistance to ampicillin. EXAMPLE 19 Isolation of Cosmids from the New Library Covering the Region of the Spiramycin Biosynthesis Pathway Under cloning and sequencing of fragments of these cosmids.

19.1 Hybridization on colonies of the genomic library of Streptomyces ambofaciens OSC2:

Cosmids from the new library of Streptomyces ambofaciens OSC2 (cf. example 18) covering orfl * to or / 70 * or part or all of orfl to orβ 5c, or a region further upstream of V orβ 5c were isolated . For this, successive hybridizations on E. coli. coli obtained according to example 18 were carried out using the following 3 probes (cf. FIG. 31):

- The first probe used corresponds to a DNA fragment of about 0.8 kb amplified by PCR using as matrix the cosmid pSPM5 and the following primers:

ORF23c: 5'-ACGTGCGCGGTGAGTTCGÇCGTTGC-3 '(SΕQ ID N ° 130) and> OR 25c: 5'-CTGAACGACGCCATCGCGGTGGTGC-3' (SEQ ID N ⁰ 131). j:

The PCR product thus obtained contains a fragment of the start of Yorf23c, the entire orβ4c and the end of the orβ 5c (cf. FIG. 31, probe I).

- The second probe used corresponds to a DNA fragment of approximately 0.7 kb amplified by PCR using as matrix the total DNA of the strain S. ambofaciens ATCC23877 and the following primers:

ORFl * c: 5 * -GACCACCTCGAACCGTCCGGCGTCA-3 '(SEQ ID N ° 132) and ORF2 * c: 5'-GGCCCGGTCCAGCGTGCCGAAGC-3' (SEQ ID N ° 133). The PCR product thus obtained contains a fragment of the end of orfl * c and the beginning of 1 orβ * c (cf. FIG. 31, probe II).

- The third probe used corresponds to an EcoRl-BamHI fragment of approximately 3kb containing the orfl, orβ and orβ and obtained by digestion of the plasmid pOS49.99 (cf. FIG. 31, probe III). About 2000 clones of the library obtained in Example 18.2 were transferred to a filter for hybridization on colonies according to conventional techniques (Sambrook et al, 1989).

The first probe (cf. FIG. 31, probe I) was marked with ³² P by the “random priming” technique (Kit sold by the company Roche) and used for hybridization on 2000 clones of the bank, after transfer to filtered. Hybridization was carried out at 65 ° C in the buffer described by Church & Gilbert (Church & Gilbert, 1984). Two washes were carried out in 2x SSC, 0.1% SDS at 65 ° C, the first for 10 minutes and the second for 20 minutes and a third wash lasting 30 minutes was then carried out in 0.2X SSC, 0.1% SDS at 65 ° C. Under these hybridization and washing conditions, 20 clones among the 2000 hybrids exhibited a strong hybridization signal with the first probe. These 20 clones were cultured in LB + ampicillin medium (50 μg / ml) and the 20 corresponding cosmids were extracted by alkaline lysis (Sambrook et al, 1989) and then they were digested with the restriction enzyme] 'BamHI. The digestion products were then separated on an agarose gel, transferred i \ r ₍ \ Φ, to a nylon membrane and hybridized by the first probe (cf. above: the product of ^{* j} PCR ORF23c-ORF25c, probe I) under the same conditions as above. Twelve cosmids had a BamHI fragment strongly hybridizing with the probe used. These 12 cosmids were named pSPM34, pSPM35, ρSPM36, pSPM37, ρSPM38, pSPM39, pSPM40, pSPM41, pSPM42, pSPM43, pSPM44 and pSPM45 The profiles, after digestion with BamHI, of these 12 cosmids were compared with each other and with that of cosmid pSPM5, plus PCR amplification experiments using different primers corresponding to different genes already identified in the region orfl- orβ5c made it possible to position the insert of some of these cosmids relative to each other and also to determine the location of these inserts in the already known orfl- orβ5c region (see Figure 32). pSPM36 was more particularly chosen because it was likely to contain a large region upstream of V orβ 5c (cf. Figures 31 and 32). In a second step, and using the same conditions as those described above, the 2000 clones of the Streptomyces ambofaciens OSC2 library were hybridized with the second probe corresponding to the PCR product: ORFl * c- ORF2 * c (cf. Figure 31, probe II). This hybridization made it possible to isolate cosmids whose insert is located in the region of orfl * c to orfl0 * c. Under the hybridization conditions used, 16 clones among the 2000 hybrids exhibited a strong hybridization signal with this second probe. These 16 clones were cultured in LB + ampicillin medium (50 μg / ml) and the 16 corresponding cosmids were extracted by alkaline lysis (Sambrook et al, 1989) then they were digested with the restriction enzyme BamHI. The digestion profiles (after digestion with BamHI) of these 16 cosmids were compared with each other and with that of the cosmid pSPM7. PCR amplification experiments with the primers ORFl * c and ORF2 * c made it possible to choose two cosmids which well contained the orfl * c and orβ * c genes and whose profiles had common bands but also different bands. In addition, other PCR amplification experiments in • ' ^• , using different primers corresponding to different genes already identified in the ^" -l region orfl * c to brfl0 * c have made it possible to position the insert of these cosmids. one pa i; relative to the other and also to determine the location of these inserts in the region? orfl * c to orfl0 * c already known (cf. Figure 32). The two more particularly selected cosmids were named pSPM47 and ρSPM48 (cf Figure 32).

Using the same conditions as those described above, the 2000 clones of the Streptomyces ambofaciens OSC2 library were also hybridized with the third probe corresponding to the DNA fragment EcoR1-BamHI of the plasmid pOS49.99 (cf. FIG. 31 probe III). This hybridization made it possible to isolate the cosmids containing the region of orfl up to Vorβ and capable of containing either a large part of the PKS genes, or a large part of the orfl to orβ 5c genes of the spiramycin biosynthesis pathway. . Under these hybridization conditions, 35 clones among the 2000 hybrids exhibited a strong hybridization signal with the third probe. These 35 clones were cultured in LB + ampicillin medium (50 μg / ml) and the 35 corresponding cosmids were extracted by alkaline lysis (Sambrook et al, 1989) then digested with the restriction enzyme BamHI. The profiles, after digestion with BamHI, of these 35 cosmids were compared with each other and with that of the cosmid pSPM5. Furthermore, PCR amplification experiments using different primers corresponding to different genes already identified in the orfl to orβ 5c region made it possible to verify that these cosmids indeed contained inserts originating from the orfl to orβ5c region and to position the inserts of these cosmids relative to each other and to the already known orfl to orβ 5c region (see Figure 32). Five cosmids were more particularly selected, they were named pSPM50, pSPM51, pSPM53, pSPM55 and pSPM56 (cf. Figure 32).

19.2 Under cloning and sequencing of part of the insert of the cosmid pSPM36.

The probe of around 0.8 kb obtained by PCR with the primers ORF23c and ORF25c (cf. above and FIG. 31, probe T) was also used in Southern Blot experiments on the total DNA of S. ambofaciens OSC2 digested with the enzyme Pstl. Under the conditions of hybridization described above, this probe reveals a single PstI fragment of approximately 6 kb when hybridized on the total DNA of S. ambofaciens OSC2 digested with the enzyme Pst1. There is a Pstl site at Yorβ3c (cf. SEQ ID No. 80) but no other Pstl site up to the end (BamHI site) of the known sequence (cf. SEQ ID No. 1). This Pstl-BamHI fragment has a size of approximately 1.4 kb. The 6 kb Pstl fragment hybridized on the total DNA of S. ambofaciens digested with the Pstl enzyme therefore contains a region of approximately 4.6 kb located upstream of orβ 5c. This region is likely to contain other genes whose products are involved in the biosynthesis pathway of spiramycin. It was verified by digestion that the cosmid pSPM36 did indeed contain this 6 kb PstI fragment. This fragment was isolated from pSPM36, in order to determine the sequence further upstream of orβ5c. For this, the cosmid pSPM36 was digested with the restriction enzyme Pstl. The Pstl-Pstl fragment with a size of approximately 6 kb was isolated by electroelution from a 0.8% agarose gel and then cloned into the vector pBK-CMV (4512bp) (sold by the company Stratagene (La Jolla, California, USA)). The plasmid thus obtained was named pSPM58 (cf. FIG. 33) and the sequence of its insert was determined. The sequence of this insert is presented in SEQ ID No. 134. However, the entire sequence has not been determined and there is a hole of approximately 450 nucleotides, the part of the sequence not determined has been noted by a succession of "N" in the corresponding sequence.

19.3 Analysis of new nucleotide sequences, determination of the open reading phases and characterization of the genes involved in the biosynthesis of spiramycins.

The sequence of the insert of the cosmid ρSPM58 obtained was analyzed using the FramePlot program (Ishikawa J & Hotta K. 1999). This made it possible to identify, among the open reading phases, the open reading phases presenting a use of

10 codons typical of Streptomyces. This analysis made it possible to determine that this insert includes 4 new ORFs upstream of Yorβ5c (cf. FIG. 34). These genes were named orβό (SEQ ID N ° 107), orβ7 (SEQ ID N ° 109), orβ8c (SEQ ID N ° 111, the sequence of this orf has not been determined completely since a hole of approximately 450 nucleotide subsite during sequencing of the insert of pSPM58, these 450 nucleotides

, 15 appear in the form of a sequence of "N" daμ $ the sequence SEQ ID No. 111) and or / 29 (there i sequence of this last orf was incomplete in this insert). The "c" added in the;! ^'Name of sense gene for the ORF in question that the coding sequence is in reverse orientation (Figure 34).

The protein sequences deduced from these open reading phases were compared with those present in different databases using different programs: BLAST (Altschul et al, 1990) (Altschul et al, 1997), CD-search, (these two programs are accessible in particular from the National Center for Biotechnology Information (NCBI) (Bethesda, Maryland, USA)), FASTA ((Pearson W. R & DJ Lipman, 1988) and (Pearson WR, 1990) (this program is accessible 25 in particular from the INFOBIOGEN resource center, Evry, France) These comparisons made it possible to formulate hypotheses on the function of the products of these genes and to identify those likely to be involved in the biosynthesis of spiramycins. 19.4 Under cloning and sequencing of another part of the insert of the cosmid pSPM36.

The approximately 0.8 kb probe obtained by PCR with the primers ORF23c and ORF25c (cf. above and FIG. 31, probe I) was also used in Southern Blot experiments on the total DNA of the OSC2 strain. digested with the enzyme Stul. Under the hybridization conditions described above for this probe, this probe reveals a single Stul fragment of approximately 10 kb when hybridized on the total DNA of the OSC2 strain digested by the enzyme StwI. Given the presence of a Stul site in Yorβ3c (cf. SEQ ID No. 80) and the location of this site with respect to the Pstl site, this 10 kb fragment includes the entire Pstl fragment previously studied (insert of pSPM58) and provides access to a region not yet studied of around 4 kb. (see figure 33). It was verified by digestion that the cosmid pSPM36 did indeed contain this 10 kb Stul fragment. This fragment was isolated from the cosmid pSPM36, in order to determine the sequence of the end of ^, orβ9 and other genes further upstream of orβ 9. For this, the cosmid pSPM36 was digested with the enzyme of Stul restriction The Stul-Stul fragment, about 10 kb in size, was isolated by electroelution from an 0.8% agarose gel, then cloned into the vector pBK-CMV (4512bp) (sold by the Stratagene company (La Jolla, California, USA)). The plasmid thus obtained was named pSPM72 (cf. FIG. 33). The latter was then digested with EcoRI (EcoRI site in the insert of pSPM58) and HmdlII (Hindlll site in the multiple cloning site of the vector, immediately after the Stul site of the end of the insert) (cf. FIG. 33). The EcoRI-H dlII DNA fragment thus obtained, corresponds to a fragment of the insert of the plasmid pSPM72 (cf. FIG. 33) and was subcloned into the vector pBC-SK + (marketed by the company Stratagene (La Jolla , California, USA)) digested beforehand with EcoRI and HmdlII. The plasmid thus obtained was named pSPM73 and the sequence of its insert was determined. The sequence of this insert is presented in SΕQ ID N ° 135. An assembly of the sequences of the inserts of pSPM58 and pSPM73 is presented in SΕQ ID N ° 106. This sequence starts from the Pstl site at Yorβ3c (cf. SΕQ ID N ° 80) and goes to the Stul site at Yorβ2c (see Figure 34). The sequence of Yorβ8c (SΕQ ID N ° 111), not being complete (cf. example 19.3), a region of approximately 450 nucleotides is not sequenced, these 450 nucleotides appear in the form of a series of "N" in the sequence SEQ ID No. 106).

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

The partial sequence of the insert of the cosmid pSPM73 obtained was analyzed using the FramePlot program (Ishikawa J & Hotta K. 1999). This made it possible to identify, among the open reading phases, the open reading phases exhibiting a codon use typical of Streptomyces. This analysis made it possible to determine that this insert includes 4 ORFs, one incomplete and three complete (cf. Figure 34). The incomplete ORF corresponds to the 3 ′ part of the orβ9 coding sequence, which made it possible to complete the sequence of this gene thanks to the partial sequence of this same orf obtained during; sequencing of the insert of the plasmid pSPM58 (cf. example 19.2 and 19.3), all of the v 15 two sequences thus made it possible to obtain the complete sequence of or / 29. The 4 genes were -j ;; l ^{• * ""} thus were n orhinies orf2 (SEQ BD N ° 113), ωβOc (SEQ ID N ⁰ 115), orβl (SEQ © # ^; S |? 118) and orβ 2c (SEQ ID No. 120). The “c” added in the name of the gene signifying for the ORF in question that the coding sequence is in the reverse orientation (cf. FIG. 34).

The protein sequences deduced from these open reading phases (SEQ ID N ° 20 114 for orβ9, SEQ ID N ° 116 and 117 for orβOc, SEQ ID N ° 119 for orβl and SEQ ID N ° 121 for orβ 2c) were compared with those present in different databases thanks to different programs: BLAST (Altschul et al, 1990) (Altschul et al, 1997), CD-search, (these two programs are accessible in particular from the National Center for Biotechnology Information (NCBI) (Bethesda, Maryland, USA)), 25 FASTA ((Pearson W. R & DJ Lipman, 1988) and (Pearson WR, 1990) (this program is available in particular from the INFOBIOGEN resource center, Evry, France). allowed to formulate hypotheses on the function of the products of these genes and to identify those likely to be involved in the biosynthesis of spiramycins.

19.6 Under cloning and sequencing of a third part of the insert of the cosmid pSPM36. A probe (DNA fragment of 0.8 kb) corresponding to an internal sequence to orβ2c was obtained by PCR using as matrix the total DNA of the strain of Streptomyces ambofaciens and the following primers

KF36: 5'- TTGCCGTAGCCGAGGACCAGCG-3 '(SEQ IDN ° 151) and KF37: 5'- CACATGGCCCTGGAGGACCCTG-3' (SEQIDN ° 152).

The PCR product thus obtained represents an internal sequence of Vorβ2c. This probe was used in Southern blot experiments on the chromosomal DNA of the OSC2 strain and on the DNA of the cosmid pSPM36 digested with the Pst1 enzyme. In., Using the same hybridization conditions as those described above (cf. example X \ 19.1), this probe reveals two PstI fragments of approximately 3.4kb and 2.5kb when hybridized on the total DNA of the OSC2 strain and on the DNA of the cosmid pSPM36 digested by the enzyme Pstl. Given the presence of a Pstl site in the probe used, these results can be explained. The first DNA fragment which has a size of approximately 3.4 kb is a fragment whose sequence is already known in full. The sequence of the 2.5 kb fragment is only partially known, over a region of 700 bp. This fragment was isolated from the cosmid pSPM36 in order to determine the sequence of the end of Y orβ 2c and other genes upstream of the latter. For this, the cosmid pSPM36 was digested with the restriction enzyme Pstl. The Pstl-Pstl fragment with a size of approximately 2.5 kb was isolated by purification from a 0.8% agarose gel and then cloned into the vector pBK-CMV (4518bp) (marketed by the company Stratagene (La Jolla, California, USA)). The plasmid thus obtained was named pSPM79 (cf. FIG. 41) and the sequence of its insert was determined. The sequence of Yorβ8c (SEQ ID No. 111) was not complete (cf. example 19.3). Indeed, a region of approximately 450 nucleotides could not be determined, these 450 nucleotides appear in the form of a sequence of "N" in the sequence SEQ ID No. 106. The sequence of the entire region missing was determined by resequencing this region. The sequence of the inserts of pSPM58 and pSPM73 was therefore determined in full. The complete sequence of the coding part of Yorβ8c is presented in SEQ ID No. 141 and the protein deduced from this sequence in SEQ ID No. 142. The sequence of the insert of the plasmid pSPM79 is presented in SEQ ED No. 161.

An assembly of the sequences of the inserts of pSPM58, pSPM73 and pSPM79 is presented in SEQ ID No. 140 (cf. FIG. 41). This sequence starts from the Pstl site at Yorβ3c (cf. SEQ ID No. 80) and goes to the Pstl site at Yorβ4c (cf. FIG. 41).

19.7 Analysis of new nucleotide sequences. determination of open reading phases and characterization of genes that may be involved in the _r , biosynthesis of spiramycins. ? -;

The sequence of the insert of the plasmid pSPM79 obtained was analyzed using the FramePlot program (Ishikawa J & Hotta K. 1999). This made it possible to identify, among the open reading phases, the open reading phases exhibiting a use of codons typical of Streptomyces. This analysis made it possible to determine that this insert includes 3 ORFs, two incomplete (orβ2c and orβ4c) and one complete (orβ3) (cf. Figure 41).

The first incomplete ORF corresponds to the 5 'part of the coding sequence for orβ2c. This made it possible to complete the sequence of this gene thanks to the partial sequence of this same orf obtained during the sequencing of the insert of the plasmid pSPM73 (cf. example 19.4 and 19.5), all of the two sequences thus allowing obtain the complete sequence of orβ2c. (SEQ ID No. 145). The "c" added in the name of the gene means for the ORF in question that the coding sequence is in the reverse orientation (cf. FIG. 41). The complete orf was named or / 33 (SEQ ID N ° 147). The third ORF was named orβ4c (SEQ ID No. 149). The “c” added in the name of the gene means for the ORF in question that the coding sequence is in the reverse orientation (cf. FIG. 5 37). The comparisons made between the product of this orf and the databases suggest that the C-terminal part of this protein is not in the product deduced from the nucleotide sequence and therefore that this orf would be longer and would continue outside the sequenced region.

The protein sequences deduced from these open reading phases were

10 compared with those present in different databases thanks to different programs: BLAST (Altschul et al., 1990) (Altschul et al., 1997), CD-search, (these two programs are accessible in particular from the National Center for

Biotechnology Information (NCBI) (Bethesda, Maryland, USA)), FASTA ((Pearson W.;!

R & DJ Lipman, 1988) and (Pearson WR, 1990) (this program is available ^" ; ^' /;

15

~ lx in particular from the INFOBIOGEN resource center, Evry, France). These weeks ^' Hi ι.τ comparisons made it possible to formulate hypotheses on the function of the products of these i' genes and to identify those likely to be involved in the biosynthesis of spiramycins.

EXAMPLE 20 Analysis of the production of spiramycin biosynthesis intermediates:

20.1 Preparation of samples:

The different strains to be tested were each cultivated in 7 500 ml baffled Erlenmeyer flasks containing 70 ml of MP5 medium (Pernodet et al., 1993). The erlens

25 were inoculated with 2.5 × 10 ⁶ spores / ml of the different strains of S. ambofaciens and grown at 27 ° C. with orbital stirring at 250 m for 96 hours. The cultures corresponding to the same clone are collected, optionally filtered on a pleated filter, and centrifuged 15 min at 7000 rpm.

The pH of the must is then adjusted to 9 with sodium hydroxide and the supernatant is extracted with methyl iso-butyryl ketone (MIBK). The organic phase (MIBK) is then recovered and evaporated. The dry extract is then taken up in 1 ml of acetonitrile, then diluted to 1/10 (100 μl qs 1 ml with water) before being used for the analyzes in liquid chromatography coupled with mass spectrometry (CL / SM ).

20.2 Analysis of samples in CL / SM:

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

The high performance liquid chromatography column used is a Kromasil C8 150 * 4.6mmm, 5mmm, 100A column.

The mobile phase is a gradient consisting of a mixture of acetonitrile and a solution;, ^' ; 0.05% trifluorpacetic acid aqueous, the flow rate is set at 1 ml min. The temperature of the colored oven is raised to 30 ° C ^ ^ '^' v _; : -

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

The mass spectrometer coupled to the chromatography column is a Simple Quadripole device sold by the company Agilent, with cone voltages at 30 and 70V.

20.3 Analysis of the biosynthesis intermediates produced by the strain OS49.67:

The strain OS49.67 in which the orβ gene is inactivated by a phase deletion does not produce spiramycins (cf. examples 6 and 15). A sample was prepared according to the method described above (cf. paragraph 20.1) and was analyzed by CL / SM as described above (cf. paragraph 20.2). More particularly, the analysis by chromatography was carried out using a solvent gradient with the mobile phase: 20% acetonitrile of time T = 0 to 5 min then linear rise to 30% at T = 35 minutes, followed by a plateau up to 'at T = 50 minutes.

Under these conditions, two products were more particularly observed: a

5 absorbent at 238 nm (retention time of 33.4 min) and an absorbent at 280 nm (retention time of 44.8 min) (see Figure 35). Figure 35 shows the supeφosition of HPLC chromatograms produced at 238 and 280nm (top) as well as the UV spectra of the molecules eluted at 33.4 minutes and 44.8 minutes (bottom).

The conditions for analysis in coupled mass spectrometry were as follows

10: the scan is done in scan mode, covering a mass range between 100 and 1000 Da. The gain of the electro-multiplier was IV. Regarding the electrospray source, the nebulizing gas pressure was 35 psig, the drying gas flow rate was 12.0 min ^"1 , the drying gas temperature was 350 ° C, the capillary voltage was

J- ï * •

In order to obtain the structure, the products mentioned above were isolated and purified under the following conditions: the mobile phase is a mixture of a solution

20 aqueous 0.05% trifluoroacetic acid and 70/30 acetonitrile (v / v). Chromatography is carried out under isocratic conditions with a fixed flow rate of 1 ml / min. Under these conditions, the retention times of the 2 products are 8 and 13.3 minutes respectively. In addition in this case, the prepared sample (see paragraph 20.1) is not diluted in water before injecting 10 μl.

25 The 2 products are recovered at the outlet of the chromatographic column and isolated under the following conditions: an Oasis HLB 1 cartridge 30 mg (Waters) is conditioned sequentially with 1 ml of acetonitrile, then 1 ml of water / acetonitrile (20v / 80v) and 1ml of water / acetonitrile 80/20. The sample is then introduced and the cartridge washed successively with 1 ml of water / acetonitrile (95/5), 1 ml water / deuterated acetonitrile (95/5), then elute with 600 μl of water / deuterated acetonitrile 40/60. The recovered solution is then directly analyzed by NMR.

The NMR spectra obtained for these two compounds are as follows: - First product eluted: Platenolide A: (Spectrum 9646V)

IH spectrum in CD3CN - (chemical shifts in ppm): 0.90 (3H, t, J = 6Hz), 0.93 (3H, d, J = 5Hz), 1.27 (3H-, d, J = 5Hz), between 1.27 and 1.40 (3H, m), 1.51 (IH, m), 1.95 (IH, m), 2.12 (IH, m), 2.30 (IH, d, J = 12Hz), 2.50 (IH, d, J = 11Hz), 2.58 (IH, dd, J = 9 and 12 Hz), 2.96 (IH, d, J = 7Hz), 3 , 43 (3H, s), 3.70 (IH, d, J = 9Hz), 3.86 (IH, d, J = 7Hz), Λ, 4.10 (IH, m), 5.08 (IH , m), 5.58 (IH, dt, J = 3 and 12Hz), 5.70 (IH, dd, J = 8 and 12 Hz), 6.05 (IH, dd, J = 9 and 12 Hz) , 6.24 (1H, dd, J = 9 and 12Hz).

-Second product elected: Platenolide B: (Specter 9647V)

IH spectrum in CD3CN (chemical shifts in ppm): 0.81 (3H, t, J = 6Hz), 0.89 d (IH, m), 1.17 (3H, d, J = 5Hz), 1.30 (4H, m), 1.47 (2H, m), 1.61 (1H, t, J = 10Hz), 2.20 ^' f _;

(IH, m), 2.38 (IH, d, J = 13Hz), 2.52 (IH, m), 2.58 (IH, m), 2.68 (IH, dd, J = 8 and 13Hzj || 3.10 (IH, d, J = 7Hz), 3.50 (3H, s), 3.6 \ (IH, d, J = 8flz), 3.82 (IH, d, J = 7Hz) , 5.09 (1 pm), 6.20 (1H, m), 6.25 (1H, dd, J = 9 and 12 Hz), 6.58 (1H, d, J = 12 Hz), 7 , 19 (1H, dd, J = 9 and 12Hz).

These experiments thus made it possible to determine the structure of these two compounds. The first product elected is platenolide A and the second is platenolide B, the structure deduced from these two meolecules is presented in FIG. 36.

It could also be determined using the CL / SM technique associated with the

NMR, under conditions slightly different from those described above but whose development is well known to those skilled in the art, that the strain OS49.67 produces, in addition to platenolide A and B, a derivative of these two compounds. These are platenolide A + mycaorse and platenolide B + mycarose (the structure of these two compounds is shown in Figure 40). The results of the analysis of the must of the strain OS49.67 are presented in Table 42.

Table 42. The results of the CL / SM analysis of the must of the strain OS49.67

20.4 Analysis of the biosynthesis intermediates produced by the strain SPM509:

The strain SPM509 in which the orfl4 gene is inactivated (orfl4 :: att3Ωaac-) does not produce spiramycins (cf. examples 13, 15 and 16). A sample was prepared according to the method described above (see paragraph 20.1) and was analyzed by CL / SM as described above (see paragraph 20.2 and 20.3). Analysis of the biosynthesis intermediates present in the culture supernatant of the strain SPM509 cultivated in MP5 medium showed that this strain produced only form B of platenolide (“platenolide B”, cf. FIG. 36) but not form A ("Platenolide A", see Figure 36).

EXAMPLE 21: Interruption of the orfl 4 gene in a strain interrupted in the orβ gene (OS49.67)

The product 4 orfl gene is essential to spiramycin biosynthesis (see X Example 13, 15 and 16: the SPM509 strain in which this gene is disrupted does ^ _r 'r X _i ¹ produces more spiramycin.). Analysis of the biosynthetic intermediates present at.

^"•"' the culture supernatant of the strain SPM509 cultivated in MP5 medium showed that X this strain produced only form B of platenolide but not form A (cf. example 20). One of the hypotheses that can explain this observation is that the product of orfl 4 is involved in the conversion of form B of platenolide to form A by an enzymatic reduction step. To test this hypothesis, the orf! 4 gene was inactivated in a mutant no longer producing spiramycin, but producing forms A and B of platenolide. This is the case of the strain OS49.67 (cf. example 6 and 20) in which the orβ gene is inactivated by a deletion in phase (Aorβ). To inactivate the orfl 4 gene in this strain, the plasmid pSPM509 was introduced by transformation of protoplasts of the strain OS49.67 (Kieser, T et al, 2000). The inactivation of the orf! 4 gene has already been described in the case of the OSC2 strain (cf. example 13) and the same procedure was followed for the inactivation of the orf! 4 gene in the OS49.67 strain. . After transformation with the plasmid pSPM509, the clones are selected for their resistance to apramycin. Clones resistant to apramycin are then subcultured respectively on medium with apramycin and on medium with hygromycin. The clones resistant to apramycin (ApraR) and sensitive to hygromycin (HygS) are in principle those where a double crossing over event has occurred and in which the orfl4 gene has been replaced by a copy of orfl4 interrupted by the att3Ωaac- cassette. These clones were more particularly selected and the replacement of the wild copy of orfl 4 by the copy interrupted by the cassette was verified by hybridization? Thus, the total DNA of the clones obtained was digested with several enzymes, separated on an agarose gel, transferred to a membrane and hybridized with a probe corresponding to the att3Ωaac- cassette to verify that the gene replacement had indeed taken place. Verification of the genotype can also be carried out by any method known to those skilled in the art and in particular by PCR using the appropriate oligonucleotides and sequencing of the PCR product.

A clone having the expected characteristics (Aorβ, orfl4 :: att3Ωaac-) was more particularly selected and named SPM510. It was indeed possible to verify thanks to the two hybridizations that the att3Ωaac- cassette was indeed present in the genome of ^• [, this clone and that the expected digestion profile is obtained in the case of a replacement i, following a double recombination event, of the wild-type gene orfl 4 by the copy interrupted by the att3Ωaac- cassette in the genome of this clone.

EXAMPLE 22 Functional complementation of the interruption of the orfl4 gene

22.1 Construction of the plasmid pSPM519:

The orf! 4 gene was amplified by PCR using the following pair 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 OSC2 strain. The oligonucleotides EDR31 and EDR37 carry the Hindlll and Xbal restriction site respectively (sequence in bold). The 1.2 kb fragment thus obtained was cloned into the vector pGEM-T easy (marketed by the company Promega (Madison, Wisconcin, USA)) to give rise to the plasmid pSPM515. This plasmid was then digested with the restriction enzymes HindIII and Xbal. The 1.2 kb Hindlll / Xbal insert obtained was cloned into the vector pUWL201 (cf. example 17.1) previously digested with the same enzymes. The plasmid thus obtained was named pSPM519.

22.1 Transformation of strains SPM5 ^* 09 and SPM510 with the plasmid PSPM519:

The plasmid pSPM519 was introduced into the strains SPM509 (cf. example 13) and SPM510 (cf. example 17) by transformation of protoplasts (Kieser, T et al, 2000). After transformation, the clones are selected for their resistance to thiostrepton.

culture supernatants were then analyzed by HPLC (cf. example 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 correspond to the total production of spiramycins (obtained by adding the production of spiramycin I, II and III). It was observed that the presence of the vector pSPM519 in the strain SPM509 restores the production of spiramycins (cf. table 43).

Table 43. Production in spiramycins of the strain SPM509 transformed by the vector pSPM519, (results expressed in mg / 1 of supernatant).

The strain SPM510 transformed by the plasmid pSPM519 was named SPM510 pSPM509.

EXAMPLE 23 Functional complementation of the interruption of the orβ gene by the tylB gene of S. fradiae

23.1 Construction of the plasmid pOS49.52:

The plasmid pOS49.52 corresponds to a plasmid allowing the expression of the TylB protein in S. ambofaciens. To construct it, the coding sequence of the tylB "r gene of S. fradiae (Merson-Davies & Cundliffe, 1994, GenBank access number: U08223 (region sequence), SFU08223 (DNA sequence) and AAA21342 (protein sequence) ) was introduced into the plasmid pKC1218 (Bierman et al, 1992, Kieser et al, 2000, an E. coli strain containing this plasmid is accessible in particular from the ARS (NRRL) Agricultural Rese rch Service Culture Collection) (Peoria , Illinois, X USA), under number B-14790), and this coding sequence has also been placed under

_: _:? x £ control of the promoter ermE * (see above, npt exemplehment example 17.1, Bibb et al., T9 j $ || i R Biibbhb e ett a all., Ï 1G9-C9v4iY) ^• . - ^{':' •} ' ^• - ^•'•> I

23.1 Transformation of the OS49.67 strain by the plasmid pOS49.52:

The strain OS49.67 in which the orβ gene is inactivated by a phase deletion does not produce spiramycins (cf. examples 6 and 15). The plasmid pOS49.52 was introduced into the strain OS49.67 by transformation of protoplasts (Kieser, T et a, 2000). After transformation, the clones are selected for their resistance to apramycin. The clones are then subcultured on a medium containing apramycin. A clone was more particularly selected and was named OS49.67 pOS49.52.

As it has been demonstrated above, the strain OS49.67 does not produce spiramycins (cf. examples 6 and 15). Spiramycin production of the strain

OS49.67 transformed by the vector pOS49.52 was analyzed by the technique described in Example 15. It was thus possible to demonstrate that this strain has a phenotype producer of spiramycins. Thus the TylB protein allows the functional complementation of the interruption of the orβ gene.

EXAMPLE 24 Improvement of the production of spiramycins by overexpression of the orβ8c gene

24.1 Construction of the plasmid pSPM75:

The orβ 8c gene was amplified by PCR using a pair of oligonucleotides comprising an H αTII restriction site or a BamHI restriction site. These primers have the following sequence: KF30: 5 'AAGCTTGTGTGCCCGGTGTACCTGGGGAGCC 3' (SEQ ID No. 138) with an HmdlII restriction site (which is shown in bold)

as a matrix the cosmid pSPM36 (cf. above). The 1.5 kb fragment thus obtained was cloned into the vector pGEM-T easy (sold by the company Promega (Madison, Wisconcin, USA)) to give rise to the plasmid pSPM74. The plasmid pSPM74 was then digested with the restriction enzymes Hindlll and BamHI and the Hindlll / BamHI insert of approximately 1.5 kb obtained was cloned into the vector pUWL201 (cf. example 17.1) previously digested with the same enzymes . The plasmid thus obtained was named pSPM75, it contains the entire coding sequence of orβ8c placed under the control of the ermE * promoter.

24.2 Transformation of the OSC2 strain by the plasmid pSPM75:

The plasmid pSPM75 was introduced into the OSC2 strains by transformation of protoplasts (Kieser, T et al, 2000). After transformation of the protoplasts, the clones are selected for their resistance to thiostrepton. The clones are then subcultured on a medium containing thiostrepton and the transformation by the plasmid is verified by extraction of plasmids. Two clones were more particularly selected and named OSC2 / pSPM75 (1) and OSC2 / ρSPM75 (2).

A sample of the strain OSC2 / pSPM75 (2) was deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, on October 6, 2003 under the number d 'record 1-3101.

In order to test the effect of the overexpression of the orβ8c gene on the production of - spiramycins, the production in spiramycins of the clones OSC2 / pSPM75 (1) and OSC2 / pSPM75 (2) was tested by the technique described in Example 15 Analysis of the spiramycin production of the OSC2 strain was also carried out in parallel

OSC2 / pSPM75 (2) was also analyzed by HPLC (in the same way as in Example 17.2). Analysis of the spiramycin production of the OSC2 strain was 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 correspond to the total production of spiramycins (obtained by adding the production of spiramycin I, II and III).

Table 44. Production in spiramycins of strains derived from OSC2 transformed by pSPM75, (results expressed in mg / 1).

Thus, it is observed that the presence of the plasmid pSPM75 significantly increases the production of spiramycins of the OSC2 strain. This clearly demonstrates that the overexpression of gold / 28c has a positive effect on the production of spiramycins.

EXAMPLE 25 Analysis of the production of biosynthesis intermediates for spiramycins of an inactivated strain in the orβ gene:

The strain OS49.107, in which the orβ gene is inactivated by insertion of the; ;

It is therefore expected that the biosynthesis of spiramycins will be blocked at the forocidin stage (cf. FIG. 7). The strain OS49.107 which is not a producer of spiramycin should therefore produce forocidin.

A sample of supernatant of the strain OS49.107 was prepared according to the method described above (cf. example 16, without extraction with MIBK) and was analyzed by CL / SM as described above (cf. paragraph 20.2 and 20.3). In SIM mode, mass 558 relating to the molecular ion of forocidin was selected and several peaks were detected. The presence of compounds of mass 558 is compatible with the hypothesis of the role of orf8 in the synthesis of forosamine. EXAMPLE 26 Analysis of the production of biosynthesis intermediates for spiramycins of an inactivated strain in the or / 72 gene:

The strain SPM507, in which the or / 72 gene is inactivated, does not produce spiramycins (cf. examples 11 and 15). The orfl 2 gene would encode a 3,4 dehydratase responsible for the dehydration reaction necessary for the biosynthesis of forosamine (see Figure 6). It is therefore expected that the biosynthesis of spiramycins will be blocked at the forocidin stage (cf. FIG. 7). The SPM507 strain which is not a producer of spiramycin should therefore produce forocidin.

A sample of supernatant of the strain SPM507 was prepared according to the method described above (cf. example 16, without extraction with MIBK) and was analyzed by CL / SM as described above (cf. paragraph 20.2 and 20.3 ). Under these conditions, the time of

con tions, a product absorbing at 238 nm was more particularly observed (retention time of 17.1 min). LC / MS analysis made it possible to determine the mass of this compound which is 744.3 g / mole ([M + H] ⁺ = 744.3 majority product). In order to obtain the structure, the product mentioned above was isolated and purified under the conditions described above (cf. paragraph 20.1)). The organic phase (MIBK) is then recovered and evaporated. The dry extract is taken up with water and extracted with heptane. The aqueous solution is then extracted by fixing on a 1 g Oasis HLB cartridge (Waters SAS, St-Quentin en-Y vélines, France). The compound is recovered by elution with a water / acetonitrile mixture 30/70. This solution is then injected (100 μL) onto the analytical column and the fractions recovered on the Oasis HLB 1 cartridge 30 mg (Waters). Before use, the Oasis HLB 1 ce 30mg cartridges (Waters) are packaged sequentially with racetonitrile, then a water / acetonitrile mixture (20v / 80v) and a water / acetonitrile 80/20 mixture. The Oasis HLB 1 cartridge 30 mg (Waters) is then washed successively with 1 ml of acetonitrile water (95/5), 1 ml of water / deuterated acetonitrile (95/5), then eluted with 600 μl of deuterated acetonitrile water 40 / 60. The recovered solution is then directly analyzed by NMR.

5 The NMR spectrum obtained for this compound is the following (NMR spectrum 19312 V):

_**

1H spectrum. In CD3CN / D2O (chemical shifts in ppm): 0.92 (3H, d, J = 6Hz), 1.10 (IH, m), 1.14 (3H, s), 1.17 (3H , d, J = 6Hz), 1.22 (3H, d, J = 6Hz), 1.25 (3H, d, J = 6Hz), 1.40 (IH, m), 1.75 (IH, dd , J = 12 and 2Hz), 1.81 (IH, m), 1.90 (IH, d, J = 12Hz), 2.05 (IH, m), 2.12 (3H, s), 2, 15 (IH, m), 2.35 (2H, m), 2.45 (6H, broad s), 2.53 (IH, m), 10 2.64 (IH, dd, J = 12 and 9Hz) , 2.80 (IH, dd, J = 9 and 16Hz), 2.95 (IH, d, J = 8Hz), 3.23 (2H, m), 3.34 (IH, d, J = 7Hz) , 3.45 (3H, s), 3.49 (1H, m), 3.93 (1H, dd, J = 7 and 3Hz), 4.08 (1H, m), 4.37 (1H, d , J = 6Hz), 4.88 (IH, m), 5.05 (2H, m), 5.65 (2H, m), 6.08 (IH, dd, J = 8 and 12Hz), 6, 40 (1H, dd, J = 12 and 9Hz), 9.60 (1H, s).

These experiments have determined the structure of this compound, _that: H ;. this is shown in figure 38. ^' i lfl

EXAMPLE 27 Analysis of the Production of Biosynthesis Intermediaries of Spiramycins of an Inactivated Strain in the orβ * Gene:

The strain SPM501 has the genotype orβ * :: attlΩhyg +. Thanks to the polar effect 20 of the insertion of the attlΩhyg + cassette into the orfό * gene, it could be determined that the orf 5 * gene is essential for the biosynthesis pathway of spiramycins. In fact, the insertion of the excisable cassette into the coding part of the orfό * gene leads to a total halt in the production of spiramycins by polar effect on the expression of the orβ * gene. However, once the inserted cassette has been excised (and therefore when only the orfό * gene is inactivated, cf. examples 14 and 15), a production of spiramycin I is restored. This demonstrates that the orf 5 * gene is essential for the biosynthesis of spiramycins since its inactivation leads to a total halt in the production of spiramycins. The orβ * gene encodes a protein with relatively strong similarity to several O-methyltransferases. The orβ * gene is said to be an O-methyl transferase involved in the biosynthesis of platenolide. To verify this hypothesis, CL / SM and NMR analysis experiments were carried out on a strain of S. ambofaciens of genotype orfό * :: attlΩhyg + obtained from a strain suφroducing spiramycins.

A sample of the supernatant of this strain was prepared according to the method described above (cf. example 16, without extraction with MIBK) and was analyzed by CL / SM as described above (cf. paragraph 20.2 and 20.3) . However, the column used is X an X-Terra column (Waters SAS, St-Quentin en-Yvelines, France), and the cone voltage of the spectrometer is adjusted to 380V to obtain the fragmentation of the compound analyzed. Under these conditions, a product whose retention time is approximately 13.1

By a microbioogic test carried out on a sensitive strain of M. luteus (cf. example 15 and figure 18) it has been demonstrated that the intermediate molecule (spiramycin minus methyl group whose structure is presented in figure 39) produced by the strain orfό * :: Ωatthyg + is much less active (by a factor of 10) than the original spiramycin with the methyl group in position 4.

EXAMPLE 28 Construction of new “excisable cassettes”:

New excisable cassettes have been built. These cassettes are very similar to the excisable cassettes already described in example 9. The main difference between the old and the new cassettes is the absence in the latter. sequences corresponding to the ends of the Ω inteφoson, sequences which contain a transcription terminator originating from phage T4.

In cassettes without terminators, the gene which confers resistance to an antibiotic is flanked by the sequences αttR and attL allowing excision. The resistance gene is the aac (3) IV gene which encodes an acetyltransferase which confers resistance to apramycin. This gene is present in the Ωaac cassette (GenBank access number: X99313, Blondelet-Rouault, MH et al, 1997) and was amplified by PCR using the plasmid pOSK1102 (see above) and as primers oligonucleotides KF42 and KF43 each containing the HindIII restriction site (in bold) (AAGCTT) in 5 ′.

KF42: 5'-AAGCTTGTACGGCCCACAGAATGATGTCAC-3 '(SEQ DD N ° 153) and

KF43: 5'-AAGCTTCGACTACCTTGGTGATCTCGCCTT-3 '(SEQ ID No. 154). The PCR product obtained of approximately 1 kb was cloned into the E. coli vector pGEMT> Easy giving rise to the plasmid pSPM83.

The vector pSPM83 was digested with the restriction enzyme HindlU. The fragment 'HiruiΩl-Hinά π. of the insert was isolated by purification from a 0.8% agarose gel ^and then cloned into the HindIII site located between the ttL and ttR sequences of the different plasmids carrying the different possible excisable cassettes (cf. example 9 and FIG. 27) so as to replace the HindIII fragment corresponding to Ωacc with the HindIII fragment corresponding to the aac gene alone. This made it possible to obtain the attlaac, att2aac and att3aac cassettes (depending on the desired phase, cf. example 9). Depending on the orientation of the aac gene with respect to the αttL and cttR sequences, a distinction is made between attlaac +, attlaac-, att2aac +, att2aac-, att3aac + and att3aac- (according to the same conventions as those adopted in Example 9). EXAMPLE 29 Construction of a strain of S. ambofaciens interrupted in the orβ8c gene:

The orβ8c gene was inactivated using the cassette technique

5 excisable. The att3aac + excisable cassette (cf. example 28) was amplified by PCR using as plasmid the plasmid pSPMIOl (The plasmid pSPMIOl is a plasmid derived from the vector ρGP704Not (Chaveroche et al, 2000) (Miller VL & Mekalanos JJ,

1988) in which the att3aac + cassette was cloned as an EcoRV fragment into the unique EcoRV site of pGP704Not) and using the following primers:

10 KF32:

5 'CAACCGCTTGAGCTGCTCCATCAACTGCTGGGCCGAGGTATCGCGCGCG

CTTCGTTCGGGACGAA 3 '(SEQ ID N ° 155)

sequence corresponding to a sequence in the orβ8c gene and the 26 nucleotides located most in 3 '(shown in bold and underlined above) correspond to the sequence of one of the

20 ends of the att3aac + excisable cassette

The PCR product thus obtained was used to transform the hyper-recombinant E. coli strain DY330 (Yu et al, 2000) (this strain contains the exo, bet and gam genes of phage lambda integrated into its chromosome, these genes are expressed at 42 ° C, it was used in place of the E. coli KS272 strain (Chaveroche et al, 2000)) containing the

25 cosmid pSPM36. Thus, the bacteria were transformed by electroporation with this PCR product and the clones were selected for their resistance to apramycin. The cosmids of the clones obtained were extracted and digested with the restriction enzyme BamHI, in order to verify that the digestion profile obtained corresponded to the expected profile if there was insertion of the cassette (att3aac +) in the orβ8c gene , i.e. if it

30 there was indeed homologous recombination between the ends of the PCR product and the gene target. The verification of the construction can also be carried out by any method known to those skilled in the art and in particular by PCR using the appropriate oligonucleotides and sequencing of the PCR product. A clone whose cosmid has the expected profile was selected and the corresponding cosmid was named pSPM107. This cosmid is a derivative of pSPM36 in which orβ 8c is interrupted by the att3aac + cassette. The insertion of the cassette is accompanied by a deletion in the orβ 8c gene, the interruption begins at the 28th codon of orf28c II remains after the cassette the last 137 codons of orf28c.

The cosmid pSPM107 was first introduced into the E. coli strain

DH5α then in the Streptomyces ambofaciens OSC2 strain by transformation of protoplasts. After transformation, the clones are selected for their resistance to apramycin. The clones resistant to apramycin are then subcultured respectively on medium with apramycin (antibiotic B) and on medium with puromycin (antibiotic _; A) (cf. FIG. 9). The clones resistant to apramycin (ApraR) and sensitive to puromycin (PuroS) are in principle those with a double oyer crossing event; f occurred and which have the genius σr2Sc interrupted by the att3aac + cassette. _, These clones were more particularly selected and the replacement of the wild-type copy of orβ8c by the copy interrupted by the cassette was verified by hybridization. Thus, the total DNA of the clones obtained was digested with several enzymes, separated on agarose gel, transferred to the membrane and hybridized with a probe corresponding to the att3aac + cassette to verify the presence of the cassette at the expected locus in the Genomic DNA of the clones obtained. Verification of the genotype can also be carried out by any method known to a person skilled in the art and in particular by PCR using the appropriate oligonucleotides and sequencing of the PCR product.

A clone exhibiting the expected characteristics (orβ8c :: att3aac +) was more particularly selected and named SPM107. This clone therefore has the genotype: orβ8c :: att3aac + and was named SPM107. There is no need to excise the cassette for the study of the effect of inactivation of orβ 8c, in view of the orientation of the genes (cf. FIG. 3). Indeed, the fact that orβ9 is oriented in the opposite direction to orβ8c shows that these genes are not co-transcribed. The use of an excisable cassette, on the other hand, makes it possible to get rid of the selection marker at any time, in particular by transformation with the plasmid pOSV508.

In order to test the effect of the extinction of the orβ8c gene on the production of

5 spiramycins, the production in spiramycins of the strain SPM107 was tested by the technique described in example 15. It could thus be demonstrated that this strain has a phenotype which does not produce spiramycins. This demonstrates that the orβ 8c gene is a gene essential for the biosynthesis of spiramycin in S. ambofaciens.

EXAMPLE 30 Construction of a strain of S. ambofaciens interrupted in the orβ1 gene:

GCTTCGTTCGGGACGAA 3 '(SEQ ID N ° 157)

20 EDR72:

5 'GCCAGCACCTCGTCCAGCTGCTCGACGGAACTCACCCCCATCTGCCTCT TCGTCCCGAAGCAACT 3' (SEQ ID N ° 158)

The 39 nucleotides located at the 5 ′ end of these oligonucleotides have a sequence corresponding to a sequence in the orβ1 gene and the 26 nucleotides located most in 3 ′ (shown in bold and underlined above) correspond to the sequence d 'one end of the att3aac + excisable cassette.

The PCR product thus obtained was used to transform the E. coli strain

KS272 containing the plasmid pKOBEG and the cosmid pSPM3, as described by

30 Chaveroche et al. (Chaveroche et al., 2000) (cf. Figure 12 for the principle, the plasmid pOS49.99 must be replaced by the cosmid pSPM36 and the plasmid obtained is no longer pSPM17 but pSPM543). Thus, the bacteria were transformed by electroporation with this PCR product and the clones were selected for their resistance to apramycin. The cosmids of the clones obtained were extracted and digested with several restriction enzymes, in order to verify that the digestion profile obtained corresponds to the expected profile if there has been insertion of the cassette (att3aac +) into the orβ1 gene, that is to say if there has indeed been homologous recombination between the ends of the PCR product and the target gene. The verification of the construction can also be carried out by any method known to those skilled in the art and in particular by PCR in; using the appropriate oligonucleotides and sequencing the PCR product. A clone whose cosmid has the expected profile was selected and the corresponding cosmid was named pSPM543. This cosmid is a derivative of pSPM36 in which orβ1 is interrupted by the att3aac + cassette (cf. FIG. 12). Inserting the cassette

transformation, the clones are selected for their resistance to apramycin. The clones resistant to apramycin are then subcultured respectively on medium with apramycin (antibiotic B) and on medium with puromycin (antibiotic A) (cf. FIG. 9). The clones resistant to apramycin (ApraR) and sensitive to puromycin (PuroS) are in principle those where a double crossing over event has occurred and which have the orβ1 gene interrupted by the att3aac + cassette. These clones were more particularly selected and the replacement of the wild-type copy of orβ1 by the copy interrupted by the cassette was verified by hybridization. Thus, the total DNA of the clones obtained was digested with several enzymes, separated on agarose gel, transferred to the membrane and hybridized with a probe corresponding to the att3aac + cassette to verify the presence of the cassette at the expected locus in the Genomic DNA of the clones obtained. A second hybridization was carried out using as probe a DNA fragment obtained by PCR and corresponding to a very large part of the coding sequence for the orβ1 gene.

Verification of the genotype can also be carried out by any method known to a person skilled in the art and in particular by PCR using the appropriate oligonucleotides and sequencing of the PCR product.

A clone exhibiting the expected characteristics (orβl :: att3aac +) was more particularly selected and named SPM543. It has indeed been possible to verify thanks to the two hybridizations that the att3aac + cassette was indeed present in the genome of this clone and that the expected digestion profile is indeed obtained in the case of a replacement, following a double recombination event, of the wild-type gene by the copy interrupted by the att3aac + cassette in the genome of this clone. This clone therefore has the genotype: orβl :: att3aac + and was named SPM543. There is no need to excise the cassette for the study of the effect of the inactivation of orβl, in view of the orientation of the genes (cf. FIG. 3). Indeed, the fact that orβ2c is oriented in the opposite direction to orβl shows that these genes are not co-transcribed. The use of an excisable cassette on the other hand allows the possibility of getting rid of the selection marker at any time, in particular by transformation with the plasmid pOSV508.

In order to test the effect of the extinction of the orβ1 gene on the production of spiramycins, the production of spiramycins of the strain SPM543 was tested by the technique described in Example 15. It was thus possible to demonstrate that this strain has a non-spiramycin-producing phenotype. This demonstrates that the orβ1 gene is a gene essential for the biosynthesis of spiramycin in S. ambofaciens.

EXAMPLE 31 Construction of a strain of S. ambofaciens interrupted in the orβ2c gene:

The orf32c gene was inactivated using the excisable cassette technique. The att3aac + excisable cassette was amplified by PCR using the plasmid pSPMIOl as a template and using the following primers: KF52:

5 'GATCCGCCAGCCTCACGTCACGCCGCGCCGCCTCCCTGACATCGCGCGC GCTTCGTTCGGGACGAA 3' (SEQ ID N ° 159). and KF53: 5 'GAGGCGGACGTCGGTACGCGGTGGGAGCCGGAGTTCGACAATCTGCCTC TTCGTCCCGAAGCAACT 3' (SEQ ID No. 160).

The 40 nucleotides located at the 5 'end of these oligonucleotides have a sequence corresponding to a sequence in the orβ2c gene and the 26 nucleotides located most in 3' (shown in bold and underlined above) correspond to the sequence of one end of the att3aac + excisable cassette.

The PCR product thus obtained was used to transform the hyper-recombinant E. coli strain DY330 (Yu et al, 2000) containing the cosmid pSPM36. Thus, the bacteria were transformed by electroporation with this PCR product and the clones were selected for their resistance to apramycin. The cosmids of the clones obtained were extracted and digested with the restriction enzyme BamHI, in order to verify that the digestion profile obtained corresponds to the profile expected if there has been insertion of the cassette (att3aac +) into the orβ2c gene , ie if there was indeed homologous recombination between the ends of the PCR product and the target gene. The verification of the construction can also be carried out by any method known to those skilled in the art and in particular by PCR using the appropriate oligonucleotides and sequencing of the PCR product. A clone whose cosmid has the expected profile was selected and the corresponding cosmid was named pSPMIOό. This cosmid is a derivative of pSPM36 in which orβ 2c is interrupted by the att3aac + cassette. The insertion of the cassette is accompanied by a deletion in the orβ2c gene, the interruption begins at the 112th codon of orβ 2c. After the cassette, the last 91 codons of orβ2c remain.

The cosmid pSPM106 was first introduced into the E. coli DH5α strain and then into the Streptomyces ambofaciens OSC2 strain by transformation. After transformation, the clones are selected for their resistance to apramycin. The clones resistant to apramycin are then subcultured respectively on medium with apramycin (antibiotic B) and on medium with puromycin (antibiotic A) (cf. FIG. 9). The clones resistant to apramycin (ApraR) and sensitive to puromycin (PuroS) are in principle those where a double crossing over event has occurred and which have the orβ2c gene interrupted by the att3aac + cassette. These clones were more particularly selected and the replacement of the wild-type copy of orβ2c by the copy interrupted by the cassette was verified by hybridization. Thus, the total DNA of the clones obtained was digested with several enzymes, separated on agarose gel, transferred ^_Y. ^' : on membrane and hybrid with a probe corresponding to the att3aac + cassette to verify the presence of the cassette in the genomic DNA of the clones obtained. Verification of the genotype can also be carried out by any method known to those skilled in the art and in particular by PCR using the appropriate oligonucleotides: and sequencing of the PCR product. ^" -Xi :.

' ^• ": - X {-

*. -. ^* , '*'_# : A clone with the expected characteristics (orβ2c :: att3aac +) was more _, particularly selected. This clone therefore has the genotype: orβ2c :: att3aac + ^' and has been named SPM106. There is no need to excise the cassette for the study of the effect of the inactivation of orβ2c, in view of the orientation of the genes (cf. FIG. 3). Indeed, the fact that or / 33 is oriented in the opposite direction to orβ2c shows that these genes are not co-transcribed. The use of an excisable cassette, on the other hand, makes it possible to get rid of the selection marker at any time, in particular by transformation with the plasmid pOSV508.

In order to test the effect of the extinction of the orβ2c gene on the production of spiramycins, the production of spiramycins of the strain SPM106 was tested by the technique described in Example 15. It was thus possible to demonstrate that this strain has a spiramycin-producing phenotype. This demonstrates that the orβ2c gene is not a gene essential for the biosynthesis of spiramycin in S. ambofaciens. List of constructions described in this request

List of abbreviations: Am: Ampicillin; Hyg: Hygromycin; Sp: Spiramycin; Ts: Thiostrepton; Cm: Chloramphenicol. Kn: Kanamycin, Apra: apramycin.

'' I-0'k

ïX 'y;^' ïj

x

Storage of biological material

The following organizations were deposited on July 10, 2002 with the National Collection of Cultures of Microorganisms (CNCM), 25 rue du Docteur Roux, 75724 Paris Cedex 15, France, according to the provisions of the Budapest Treaty.

5 - OSC2 strain under registration number 1-2908.

- SPM501 strain under registration number 1-2909.

- SPM502 strain under registration number 1-2910.

- SPM507 strain under registration number 1-2911.

- Strain SPM508 under registration number 1-2912.

10 - Strain SPM509 under the registration number 1-2913.

- Strain OS49.67 under the registration number 1-2916.

- Strain OS49.107 under registration number 1-2917.

15 - Escherichia coli DH5α strain containing the plasmid pOS44.4 under the registration number 1-2918.

The following organisms were deposited on February 26, 2003 with the National Collection of Cultures of Microorganisms (CNCM), 25 rue du Docteur Roux, 75724 Paris Cedex 15, France, according to the provisions of the Budapest Treaty.

20 - strain SPM502 pSPM525 under registration number 1-2977. The following organisms were deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, on October 6, 2003 according to the provisions of the Budapest Treaty.

- Strain OSC2 / pSPM75 (2) under the registration number 1-3101.

All cited publications and patents are incorporated into this application by reference.

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Claims

1. Polynucleotide encoding a polypeptide involved in the biosynthesis of spiramycins, characterized in that the sequence of said polynucleotide is: (a) one of the sequences SEQ ID No. 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 constituted by the variants of the sequences (a),

(c) one of the sequences derived from the sequences (a) and (b) due to the degeneracy of the genetic code.

2. Polynucleotide hybridizing under high stringency hybridization conditions to at least one of the polynucleotides according to claim 1.

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 a polynucleotide according to claim 1.

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

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

6. Polynucleotide according to claim 2, 3, 4 or 5 characterized in that it encodes a protein having an activity similar to the protein encoded by the polynucleotide with which it hybridizes or it has identity.

7. Polypeptide resulting from the expression of a polynucleotide according to claim 1, 2, 3, 4, 5 or 6.

8. Polypeptide involved in the biosynthesis of spiramycins, characterized in that the sequence of said polypeptide is:

(b) one of the sequences as defined in (a) except that throughout said sequence one or more amino acids have been substituted, inserted or deleted; without affecting its functional properties,

(c) one of the sequences constituted by the variants of the sequences (a) and (b).

9. Polypeptide having at least 70%, 80%, 85%, 90%, 95% or _; 98% ^ J of amino acid identity with a polypeptide comprising at least 10, 15, 20, 30 to X ' ^: -' 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 according to claim 8.

10. Polypeptide according to claim 9 characterized in that it is isolated from a bacterium of the genus Streptomyces.

11. A polypeptide according to claim 9 or 10 characterized in that it codes for a protein involved in the biosynthesis of a macrolide.

12. Polypeptide according to claim 9, 10 or 11 characterized in that it has an activity similar to that of the polypeptide with which it shares identity.

13. Recombinant DNA characterized in that it comprises at least one polynucleotide according to one of claims 1, 2, 3, 4, 5 or 6.

14. Recombinant DNA according to claim 13 characterized in that said recombinant DNA is included in a vector.

15. Recombinant DNA according to claim 14 characterized in that said vector is chosen from bacteriophages, plasmids, phagemids, integrative vectors, fosmids, cosmids, shuttle vectors, BACs or PACs.

16. Recombinant DNA according to claim 15 characterized in that it is chosen from 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,

pSPM524, pSPM525, pSPM527, pSPM528, pSPM34, pSPM35, pSPM36, pSPM37, pSPM38, pSPM39, pSPM40, pSPM41, pSPM42, pSPM43, pSPM44, pSPM45, pSPM47, pSPM48 pSPM55, pSPM55, pSPM55 pSPM72, pSPM73, pSPM515, pSPM519, pSPM74, pSPM75, pSPM79, pSPM83, pSPM107, pSPM543 and pSPM106.

17. Expression vector characterized in that it comprises at least one nucleic acid sequence encoding a polypeptide according to claim 7, 8, 9, 10,

11 or 12.

18. An expression system comprising an appropriate expression vector and a host cell allowing the expression of one or more polypeptides according to claim 7, 8, 9, 10, 11 or 12.

19. Expression system according to claim 18 characterized in that it is chosen from prokaryotic expression systems or eukaryotic expression systems.

', 20. An expression system according to claim 19 characterized in that it is chosen from expression systems in the E. coli bacteria, baculovirus expression systems allowing expression in insect cells, expression systems for expression in yeast cells, expression systems for expression in mammalian cells.

21. Host cell into which has been introduced at least one polynucleotide and / or at least one recombinant DNA and / or at least one expression vector according to one of claims 1, 2, 3, 4, 5, 6, 13 , 14, 15, 16 or 17.

22. A method of producing a polypeptide according to claim 7, 8, 9, 10, ⁽ r 11 or 12 characterized in that said method comprises the following steps: '[, a) inserting at least one nucleic acid encoding said polypeptide in an appropriate vector; b) cultivating, in an appropriate culture medium, a host cell previously transformed or transfected with the vector of step a); c) recovering the conditioned culture medium or a cell extract; d) separating and purifying from said culture medium or also from the cell extract obtained in step c), said polypeptide; e) where appropriate, characterize the recombinant polypeptide produced.

23. Microorganism blocked in a stage of the biosynthetic pathway of at least one macrolide.

24. Microorganism according to claim 23 characterized in that it is obtained by inactivating the function of at least one protein involved in the biosynthesis of this or these macrolides in a microorganism producing this or these macrolide.

25. Microorganism according to claim 24 characterized in that the inactivation of this or these proteins is carried out by mutagenesis in the or them. genes encoding said protein (s) or by expression of one or more antisense RNAs complementary to RNA or messenger RNAs encoding said protein (s).

26. Microorganism according to claim 25 characterized in that the inactivation of this or these proteins is carried out by mutagenesis by irradiation, by action of chemical mutagenic agent, by directed mutagenesis or by gene replacement.

27. Microorganism according to claim 25 or 26 characterized in that the pu ^' ^, ^' 1 mutagenesis are carried out in vitro or in situ, by deletion, substitutiόi deletion and / or addition of one or more bases in the gene or genes considered or by gene inactivation.

28. Microorganism according to claim 23, 24, 25, 26 or 27 characterized in that said microorganism is a bacterium of the genus Streptomyces.

29. Microorganism according to claim 23, 24, 25, 26, 27 or 28 characterized in that the macrolide is spiramycin.

30. Microorganism according to claim 23, 24, 25, 26, 27, 28 or 29 characterized in that said microorganism is a strain of S. ambofaciens.

31. Microorganism according to claim 23, 24, 25, 26, 27, 28, 29 or 30 characterized in that the mutagenesis is carried out in at least one gene comprising a sequence according to one of claims 1, 2, 3, 4 , 5 or 6.

32. Microorganism according to claim 25, 26, 27, 28, 29, 30 or 31 characterized in that. that 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 N ° 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. Microorganism according to claim 25, 26, 27, 28, 29, 30, 31 or 32 characterized in that the mutagenesis consists in the genetic inactivation of a gene comprising a sequence corresponding to the sequence SEQ ID No. 13.

34. Soμche of Streptomyces ambofaciens characterized in that it is a strain selected from: one of the strains deposited with the National Collection of Cultures of Microorganisms (CNCM) on July 10, 2002 under the number d 1-2909, 1-2911, 1-2912, 1-2913, 1-2914, 1-2915, 1-2916 or 1-2917 - strain SPM510.

35. Process for the preparation of a macrolide biosynthesis intermediate, characterized in that it comprises the following steps: a) cultivating, in an appropriate culture medium, a microorganism according to one of claims 23, 24, 25, 26 , 27, 28, 29, 30, 31, 32, 33 or 34, b) recovering the conditioned culture medium or a cell extract, c) separating and purifying from said culture medium or from the cell extract obtained in step b), said biosynthesis intermediate.

36. Process for the preparation of a molecule derived from a macrolide, characterized in that a biosynthesis intermediate is prepared according to the method of claim 35 and that the intermediate thus produced is modified.

5 37. A method of preparation according to claim 36 characterized in that one modifies said intermediate by chemical, biochemical, enzymatic and / or microbiological.

38. Preparation process according to claim 36 or 37 characterized in -y ^"10 which is introduced into said microorganism or more genes encoding proteins capable of modifying the intermediate by using it as substrate.

41. Microorganism producing spiramycin I but not producing spiramycin II and III.

42. Microorganism according to claim 41 characterized in that it comprises all of the genes necessary for the biosynthesis of spiramycin I but that the gene comprising the sequence SEQ ID No. 13 or one of its variants or one sequences

25 derived therefrom due to the degeneracy of the genetic code and encoding a polypeptide of sequence SEQ ID NO: 14 or one of its variants is not expressed or has been rendered inactive.

43. Microorganism according to claim 42 characterized in that said inactivation is carried out by mutagenesis in the gene encoding said protein or by the expression of an antisense RNA complementary to the messenger RNA encoding said protein.

44. Microorganism according to claim 43 characterized in that said mutagenesis is carried out in the promoter of this gene, in the coding sequence or in a non-coding sequence. So as to make the coded protein inactive or to prevent its expression or its translation.

45. Microorganism according to claim 43 or 44 characterized in that the mutagenesis is carried out by irradiation, by action of chemical mutagenic agent, by directed mutagenesis or by gene replacement.

46. Microorganism according to claim 43, 44 or 45 characterized in that the mutagenesis and / or gene addition,

47. Microorganism according to claim 41 or 42 characterized in that said microorganism is obtained by expressing the genes of the biosynthetic pathway for spiramycin without these comprising the gene comprising the sequence corresponding to SEQ ID No. 13 or 1 one of its variants or one of the sequences derived therefrom due to the degeneration of the genetic code and encoding a polypeptide of sequence SEQ ID No. 14 or one of its variants.

48. Microorganism according to claim 41, 42, 43, 44, 45, 46 or 47 characterized in that said microorganism is a bacterium of the genus Streptomyces.

49. Microorganism according to claim 41, 42, 43, 44, 45, 46, 47 or 48 characterized in that said microorganism is obtained from a starting strain producing spiramycins I, II and III.

50. Microorganism according to claim 41, 42, 43, 44, 45, 46, 47, 48 or 49 characterized in that it is obtained by mutagenesis in a gene comprising the sequence corresponding to SEQ ID No. 13 or one of its variants or one of

5 sequences derived therefrom due to the degeneracy of the genetic code and encoding a polypeptide of sequence SEQ ID No. 14 or one of its variants having the same function.

51. Microorganism according to claim 41, 42, 43, 44, 45, 46, 47, 48, 49 or 10 50 characterized in that said microorganism is obtained from a strain of S. ambofaciens producing spiramycins I, II and III, in which a gene inactivation is carried out of the gene comprising the sequence corresponding to SEQ ID No. 13 or one of the sequences derived therefrom due to the degeneracy of the genetic code.

15

52. Strain of S. ambofaciens characterized in that it is the strain & fîâ *

,. "'.''. • - _Λ l: deposited with the National Collection of Microorganism Qiltures (CNCJM); Ï?? On July 10, 2002 under the registration number 1-2910.

53. Process for the production of spiramycin I, characterized in that it comprises the following stages:

(a) cultivating, in an appropriate culture medium, a microorganism according to one of claims 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52,

(b) recovering the conditioned culture medium or a cell extract,

54. Use of a polynucleotide according to one of claims 1, 2, 3, 4, 5 or 6 to improve the production of macrolides of a microorganism. 30 4/033689

224

55. Macrolide-producing mutant microorganism characterized in that it has a genetic modification in at least one gene comprising a sequence according to one of claims 1, 2, 3, 4, 5 or 6 and / or that it overexpresses at least one gene comprising a sequence according to one of claims 1, 2, 3, 4, 5 or 6.

56. Mutant microorganism according to claim 55 characterized in that the genetic modification consists in a deletion, a substitution, a deletion and / or an addition of one or more bases in the gene or genes considered for the purpose of expressing a or proteins having a higher activity or expressing a higher level of this or these proteins.

57. Mutant microorganism according to claim 55 characterized in that the

microorganism producing macrolide by a recombinant DNA construct according to claim 13, 14 or 17, allowing the overexpression of this gene.

59. Mutant microorganism according to claim 55, 56, 57 or 58 characterized in that 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 No. 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 due to the degeneracy of the genetic code.

60. Mutant microorganism according to claim 55, 56, 57, 58 or 59 characterized in that the microorganism overexpresses one or more genes comprising one of the sequences corresponding to one or more of the sequences SEQ ID N ° 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 therefrom due to the degeneracy of the genetic code.

61. Mutant microorganism according to claim 55, 56, 57, 58, 59 or 60 characterized in that said microorganism is a bacteria of the genus Streptomyces.

62. Mutant microorganism according to claim 55, 56, 57, 58, 59, 60 or 61 characterized in that the macrolide is spiramycin.

63. Mutant microorganism according to claim 55, 56, 57, 58, 59, 60, 61 or 62 characterized in that said microorganism is a strain of S. ambofaciens.

64. Process for the production of macrolides, characterized in that it comprises the following steps: g; ^' - v ^' ; 't ff

(a) cultivating, in an appropriate culture medium, a microorganism according to one of claims 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64, (b) recovering the conditioned culture medium or a cell extract,

65. Use of a sequence and / or of a recombinant DNA and / or of a vector according to one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 for the preparation of hybrid antibiotics.

66. Use of at least one polynucleotide and / or at least one recombinant DNA and / or at least one expression vector and / or at least one polypeptide and / or at least one host cell according to one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 13, 14, 15, 16, 17 or 21 for producing one or more bioconversions.

67. Polynucleotide characterized in that it is a polynucleotide complementary to one of the polynucleotides according to claim 1, 2, 3, 4, 5, or 6.

68. Microorganism producing at least one spiramycin, characterized in that it overexpresses:

a gene capable of being obtained by polymerase chain reaction (PCR) using the pair of primers with the following sequence: 5 'AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID No. 138) and 5 '

GGATCCCGCGACGGACACGACCGCCGCGCA 3 '(SEQ ID No. 139) and as a matrix the cosmid pSPM36 or the total DNA of Streptomyces ambofaciens,

-or a gene derived therefrom due to the degeneracy of the genetic code.

69. Microorganism according to claim, 68 or 90 characterized in that qμ'i ï is a bacterium of the genus StrepiotήycësX '

70. Microorganism according to claim 68, 69 or 90 characterized in that it is a bacterium of the species Streptomyces ambofaciens.

11. Microorganism according to claim 68, 69, 70 or 90 characterized in that the overexpression of said gene is obtained by transformation of said microorganism with an expression vector.

72. Strain of Streptomyces ambofaciens characterized in that it is the strain OSC2 / pSPM75 (l) or the strain OSC2 / pSPM75 (2) deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Docteur Roux 75724 Paris Cedex 15, France, October 6, 2003 under registration number 1-3101.

73. Recombinant DNA characterized in that it comprises: a polynucleotide capable of being obtained by polymerase chain reaction using the pair of primers with the following sequence: 5 ′

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

GGATCCCGCGACGGACACGACCGCCGCGCA 3 '(SEQ ID No. 139) and as a matrix the cosmid pSPM36 or the total DNA of Streptomyces ambofaciens, or, a fragment of at least 10, 12, 15, 18, 20 to 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. Recombinant DNA according to claim 73 or 91 characterized in that it is a vector.

75. Recombinant DNA according to claim 73, 74 or 91 characterized in that it is an expression vector.

76. Host cell into which at least one recombinant DNA has been introduced according to one of claims 73, 74, 75 or 91.

77. A method of producing a polypeptide characterized in that said method comprises the following steps: a) transforming a host cell with at least one expression vector according to claim 75; b) cultivating, in an appropriate culture medium, said host cell; c) recovering the conditioned culture medium or a cell extract; d) separating and purifying from said culture medium or also from the cell extract obtained in step c), said polypeptide; e) where appropriate, characterize the recombinant polypeptide produced.

78. Microorganism according to claim 51 characterized in that the gene inactivation is carried out by deletion in phase of the gene or of a part of the gene comprising the sequence corresponding to SEQ ID No. 13 or one of the sequences derived therefrom due to the degeneracy of the genetic code.

79. Microorganism according to one of claims 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 78 characterized in that it also overexpresses: - a gene capable of be obtained by polymerase chain reaction using the pair of primers with the following sequence: 5 'AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID No 138) and 5 'GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID No 139) and as matrix the cosmid pSPM36 or total DNA of Streptomyces ambofaciens, or a gene derived therefrom due to the degeneracy of the genetic code.

80. Expression vector characterized in that the polynucleotide of sequence SEQ ID No. 47 or a polynucleotide derived therefrom due to the degeneration of the genetic code is placed under the control of a promoter allowing the expression of the protein encoded by said polynucleotide in Streptomyces ambofaciens.

81. Expression vector according to claim 80, characterized in that it is the plasmid pSPM524 or pSPM525.

82. A strain of Streptomyces ambofaciens transformed by a vector according to claim 80 or 81.

83. Microorganism according to one of claims 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 78, 79 or 92 characterized in that it additionally overexpresses the gene for coding sequence SEQ ID No. 47 or a coding sequence derived therefrom due to the degeneracy of the genetic code.

84. Microorganism according to claim 83 characterized in that it is the strain SPM502 pSPM525 deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Docteur Roux 4/033689

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75724 Paris Cedex 15, France, on February 26, 2003 under registration number 1-2977.

85. A process for the production of spiramycin (s), characterized in that it comprises the following stages: (a) cultivating, in an appropriate culture medium, a microorganism according to one of claims 68, 69, 70, 71, D2, 78, 79, 82, 83, 84, 90 or 92,

(b) recovering the conditioned culture medium or a cell extract,

(c) separate and purify from said culture medium or from. the cell extract obtained in step b), the spiramycins.

86. Polypeptide characterized in that its sequence comprises the sequence SEQ

ID No. 112 or the sequence SEQ ID No. 142.

87. Polypeptide characterized eμ that its sequence corresponds to the sequence. translated from the coding sequence:; : ^? ) ^

- a gene capable of being obtained by chain amplification by; : !! 'polymerase (PCR) using, l coupled to primers of following sequence ^' : •; .5 '; | ff§ff AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3' (SEQ ID N ° 138) and 5 'GGATCCCGCGACGGACACGACCGCCGCGCA 3' (SEQ ID N ° 139) and as a matrix the cosmid pSPM36 or the total DNA of Streptomyces ambofaciens,

-or a gene derived therefrom due to the degeneracy of the genetic code.

88. Expression vector allowing the expression of a polypeptide according to claim 86, 87 or 93 in Streptomyces ambofaciens.

89. Expression vector according to claim 88, characterized in that it is the plasmid pSPM75.

90) Microorganism according to claim 68 characterized in that the gene capable of being obtained by polymerase chain reaction is the gene for 4/033689

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coding sequence SEQ ID No. 141 or a gene derived therefrom due to the degeneracy of the genetic code.

91) recombinant DNA according to claim 73 characterized in that the polynucleotide capable of being obtained by amplification in a polymerase chain is a polynucleotide of sequence SEQ ID No. 141.

92) Microorganism according to claim 79 characterized in that the gene capable of being obtained by polymerase chain reaction is the gene with coding sequence SEQ ID No. 141 or a gene derived therefrom due to the: degeneration of the genetic code.

93) Polypeptide characterized in that its sequence is SEQ ID No. 142.