EP1551975A2 - Polypeptide, die an der biosynthese von spiramycinen beteiligt sind, nukleinsäuren, die diese polypeptide kodieren und ihre verwendungen - Google Patents

Polypeptide, die an der biosynthese von spiramycinen beteiligt sind, nukleinsäuren, die diese polypeptide kodieren und ihre verwendungen

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Publication number
EP1551975A2
EP1551975A2 EP03807883A EP03807883A EP1551975A2 EP 1551975 A2 EP1551975 A2 EP 1551975A2 EP 03807883 A EP03807883 A EP 03807883A EP 03807883 A EP03807883 A EP 03807883A EP 1551975 A2 EP1551975 A2 EP 1551975A2
Authority
EP
European Patent Office
Prior art keywords
gene
sequence
microorganism
seq
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03807883A
Other languages
English (en)
French (fr)
Inventor
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
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Aventis Pharma SA
Original Assignee
Centre National de la Recherche Scientifique CNRS
Aventis Pharma SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from FR0212489A external-priority patent/FR2845394A1/fr
Priority claimed from FR0302439A external-priority patent/FR2851773A1/fr
Application filed by Centre National de la Recherche Scientifique CNRS, Aventis Pharma SA filed Critical Centre National de la Recherche Scientifique CNRS
Priority to EP07019306.5A priority Critical patent/EP1905833B1/de
Priority to DK07019306.5T priority patent/DK1905833T3/en
Publication of EP1551975A2 publication Critical patent/EP1551975A2/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Definitions

  • 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.
  • 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.
  • 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.
  • 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).
  • PPS polyketides synthases
  • 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.
  • 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.
  • 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, or ⁇ * c, or ⁇ * c, orf4 * c, or ⁇ *, orf ⁇ *, or ⁇ * c, or ⁇ *, or ⁇ *, orflO *, orfl, or ⁇ , or ⁇ , or ⁇ , or ⁇ , orf ⁇ , or ⁇ , or ⁇ c, orflO, orfllc, orf! 2, orfl 3c, orfI4, orfl 5c, orfl 6, orf! 7, orf!
  • 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.
  • the invention also relates to a polynucleotide having at least 70%
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Another aspect of the invention relates to a microorganism blocked in a stage of the biosynthesis pathway of at least one macrolide.
  • 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).
  • 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).
  • 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.
  • 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.
  • microorganism is a bacteria of the genus Streptomyces.
  • 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.
  • 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.
  • 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.
  • 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.
  • CNCM National Collection of Cultures of Microorganisms
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Another aspect of the invention relates to a microorganism producing spiramycin I but not producing spiramycin II and m
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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.
  • CNCM National Collection of Cultures of Microorganisms
  • Another aspect of the invention relates to a process for the production of spiramycin I, characterized in that it comprises the following steps:
  • step b separate and purify from said culture medium or also from the cell extract obtained in step b), spiramycin I.
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Another aspect of the invention relates to a process for producing macrolides, characterized in that it comprises the following steps:
  • step b separating and purifying from said culture medium or also from the cell extract obtained in step b), the said macrolide (s) produced.
  • 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),
  • 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.
  • 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.
  • Another aspect of the invention relates to a microorganism producing at least one spiramycin, characterized in that it overexpresses:
  • telomere 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,
  • 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.
  • 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.
  • 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.
  • 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 Dondel Roux 75724 Paris Cedex 15, France, October 6, 2003 under registration number 1-3101.
  • CNCM National Collection of Cultures de Microorganismes
  • Another aspect of the invention relates to a recombinant DNA characterized in that it comprises:
  • 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,
  • Another aspect of the invention relates to a recombinant DNA according to paragraph 73 or 91, characterized in that it is a vector.
  • 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.
  • 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.
  • 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.
  • 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 :
  • 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.
  • Another aspect of the invention relates to an expression vector according to paragraph 80, 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 according to paragraph 80 or 81.
  • 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.
  • 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 Dondel Roux 75724 Paris Cedex 15, France, February 26, 2003 under registration number 1-2977.
  • CNCM National Collection of Cultures of Microorganisms
  • 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 ⁇ of paragraphs 68, 69, 70, 71, 72, 78, 79, 82, 83, 84, 90 or 92,
  • step b separating and purifying from said culture medium or also from the cell extract obtained in step b), the spiramycins.
  • 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.
  • Another aspect of the invention relates to a polypeptide characterized in that its sequence corresponds to the translated sequence of the coding sequence:
  • 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.
  • Another aspect of the invention relates to an expression vector according to paragraph 88, characterized in that it is the plasmid pSPM75.
  • 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.
  • 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.
  • 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.
  • Another aspect of the invention relates to a polypeptide characterized in that its sequence is SEQ ID No. 142.
  • 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).
  • 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”.
  • 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.
  • ORF Open Reading Fra e
  • nucleotide sequence can be used to denote either a polynucleotide or a nucleic acid.
  • nucleotide sequence encompasses the genetic material itself and is therefore not limited to information regarding its sequence.
  • nucleic acid include RNA, DNA, cDNA or even RNA / DNA hybrid sequences of more than one nucleotide, in single chain form or in duplex form.
  • 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.
  • 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.
  • 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,
  • nucleic acid 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.
  • 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.
  • 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
  • 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).
  • variant of a nucleic acid 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.
  • 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.
  • 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.
  • modifications of nucleotides in the coding regions can produce substitutions, conservative or non-conservative in the amino acid sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • hybrid antibiotic within the meaning of the invention means a compound, generated by the construction of an artificial biosynthesis pathway using recombinant DNA technology.
  • 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).
  • PPS polyketide synthase
  • 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, or ⁇ * c, or ⁇ * c, or ⁇ * c, or ⁇ * orf ⁇ * or ⁇ * c, or ⁇ *, or ⁇ *, 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) .
  • the 34 ORFs upstream of the PKS genes were named orfl, or ⁇ , or ⁇ , or ⁇ , or ⁇ , orf ⁇ , or ⁇ , or ⁇ , or ⁇ c, orflO, orfl le, orfl2, orfl 3c, orfJ4, orfl 5c, orfl 6, orfl 7, orfl ⁇ , orfl 9, or ⁇ O, or ⁇ lc, or ⁇ 2c, or ⁇ 3c, or ⁇ 4c, or ⁇ 5c, or ⁇ , or ⁇ 7, or ⁇ c, or ⁇ 9, or ⁇ Oc, or ⁇ l, or ⁇ 2c, or ⁇ 3 and or ⁇ 4c (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
  • FIG. 3 A schematic representation of the organization of the region is presented in FIG. 3.
  • 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, or ⁇ * c, or ⁇ * c, or ⁇ * c, or ⁇ *, orf ⁇ *, or ⁇ * c, or ⁇ * and or ⁇ *.
  • 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).
  • mutant strains constructed of mutant strains and analyzes of the production of spiramycins and biosynthesis intermediates of spiramycins by these mutant strains.
  • 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”.
  • 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).
  • 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 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).
  • the or ⁇ * 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).
  • the or ⁇ * 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 or ⁇ 3c gene codes for a glycosyltransferase.
  • This hypothesis is supported by the fact that protein encoded by the or ⁇ * c gene has a strong similarity with other proteins of similar function in other organisms (see Table 4).
  • the or ⁇ * c gene encodes a protein with relatively strong similarity to several crotonyl-CoA reductases.
  • the protein encoded by or ⁇ * 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 or ⁇ * c gene also codes for a crotonyl-CoA reductase.
  • This hypothesis is supported by the fact that the protein encoded by the or ⁇ * c gene has a strong similarity with other proteins of similar function in other organisms (cf. table 5).
  • the orf ⁇ * 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 orf ⁇ * gene would therefore encode an acyltransferase. This hypothesis is supported by the fact that the protein encoded by the orf ⁇ * gene has a strong similarity with other proteins of similar function in other organisms (see Table 6). Table 6
  • the or ⁇ * gene encodes a protein with relatively strong similarity to several O-methyltransferases.
  • the protein encoded by or ⁇ * 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 or ⁇ * gene also encodes an O-methyltransferase.
  • the or ⁇ * gene is implicated in the formation of precursors incorporated into the lactone cycle.
  • the product of the or ⁇ * 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).
  • 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)
  • the or ⁇ * gene encodes a protein with relatively strong similarity to several O-methyltransferases.
  • the or ⁇ * 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.
  • CL / SM and NMR analysis experiments were carried out on a strain of S. ambofaciens of genotype: orf ⁇ * :: àttl ⁇ hyg +.
  • the or ⁇ * gene is not expressed because of the polar effect of the insertion, in the orf ⁇ * 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 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 or ⁇ * 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 or ⁇ * c gene also codes for a protein involved in resistance to spiramycin.
  • the enzyme coded by the or ⁇ * 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 or ⁇ * c gene has a strong similarity with other proteins of similar function in other organisms (cf. table 8).
  • the or ⁇ * 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 or ⁇ * 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 or ⁇ * gene has a strong similarity with other proteins of similar function in other organisms (cf. table 9). Table 9
  • the or / 9 * gene encodes a protein with relatively strong similarity to ui ⁇ é, 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 or ⁇ * 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 or ⁇ * gene has a strong similarity with other proteins of similar function in other organisms (cf. table 10).
  • the orflO * gene encodes a protein with relatively strong similarity to a protein of unknown function.
  • genes similar to orflO * are found in the middle of several groups of genes involved in the biosynthesis of antibiotics.
  • 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).
  • the 34 genes identified have been named: orfl, or ⁇ , or ⁇ , or ⁇ , or ⁇ , orf ⁇ , or ⁇ , or ⁇ c, orflO, orfl le, orfl 2, orfl 3c, orfl4, orfl 5c, orfl 6, orfl 7, orfl 8, orfl 9, or ⁇ O, or ⁇ le, or ⁇ 2c, or ⁇ 3c, or ⁇ 4c, or ⁇ Sc, or ⁇ , or ⁇ 7, or ⁇ 8c, or ⁇ 9, or ⁇ Oc, or ⁇ l, or ⁇ 2c, or ⁇ 3 and or ⁇ 4c.
  • 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.
  • 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) .
  • 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
  • the or ⁇ gene encodes a protein with relatively strong similarity to a dTDP-6-deoxy-3,4-keto-hexulose isomerase from Aneu ⁇ n ⁇ bacillus thermoaerophilus (Pfoestl, A. Et al, 2003, GenBank access number: AAO06351; BLAST score : 118).
  • This similarity strongly suggests that the or ⁇ 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 or ⁇ gene was inactivated. It could be shown that the resulting strain no longer produced spiramycins. This confirms that the or ⁇ gene is indeed involved in the biosynthesis of spiramycins.
  • the or ⁇ gene encodes a protein with relatively strong similarity to several aminotransferases.
  • the protein encoded by or ⁇ 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 or ⁇ 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 or ⁇ gene 004/033
  • the or ⁇ gene was inactivated. It could thus be shown that the strain ' ;; resulting no longer produces spiramycins. This confirms that the or ⁇ gene is bié ⁇ ; i
  • 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 or ⁇ 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 or ⁇ (OS49.67) accumulates platenolide.
  • the or ⁇ gene encodes a protein with relatively strong similarity to several NDP-glucose synthetases.
  • the protein encoded by ot ⁇ has significant similarity with an alpha-D-glucose-1-phosphate thymidylyltransferase from Streptomyces venezuelae (Xue Y and ⁇ /., 1998; GenBank access number: AAC68682; BLAST score 404).
  • the or ⁇ gene encodes a protein with relatively strong similarity to several glucose dehydratases.
  • the protein encoded by or ⁇ 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 or ⁇ 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 or ⁇ gene has a strong similarity with other proteins of similar function in other organisms (cf. table 17).
  • the orf ⁇ gene encodes a protein with relatively strong similarity to several thioesterases.
  • the protein encoded by orf ⁇ 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 or ⁇ gene also codes for u ⁇ e ⁇ tMpestàa ⁇ é
  • This hypothesis is supported ff by the fact that the protein encoded by the orf ⁇ gene strong similarity with other proteins of similar function in other organisms (see Table 18).
  • the or ⁇ gene encodes a protein with relatively strong similarity to several hexose dehydratases.
  • the protein encoded by or ⁇ 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).
  • the or ⁇ gene encodes a protein with relatively strong similarity to several aminotransferases.
  • the protein encoded by or ⁇ 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 or ⁇ 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 or ⁇ gene has a strong similarity with other proteins of similar function in other organisms (cf. table 20).
  • the or ⁇ gene was inactivated. It has thus been possible to show that the resulting strain no longer produces spiramycins. This confirms that the or ⁇ 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 or ⁇ gene in the biosynthesis of forosamine is provided by the fact that an inactivted mutant for the or ⁇ gene produces forocidine, this mutant is therefore blocked at the forocidine stage. and does not produce neo-spiramycin (cf. FIG. 7 and example 25).
  • the or ⁇ c gene has already been identified in Streptomyces ambofaciens and has been designated srmXpwc Geistlich et al (Geistlich, M., Et al, 1992).
  • This hypothesis is supported by the fact that the protein encoded by the or ⁇ c gene has a strong similarity with other proteins of similar function in other organisms (cf. 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.
  • 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.
  • 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).
  • 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 inactivted 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.
  • this compound contains the sugar mycarose in the expected place of the forosamine.
  • 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).
  • the orfl 4 gene encodes a protein with relatively strong similarity to a putative reductase (Redenbach, M., And ⁇ /., 1996; Bentley et a, 2002; GenBank access number: CAB90862; BLAST score: 147).
  • the orfl5c gene encodes a protein with relatively strong similarity to several ketoreductases.
  • 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).
  • the orfl ⁇ gene codes for a protein with relatively strong similarity to several isomerases.
  • the protein encoded by orfl ⁇ 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 orfl ⁇ 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 orfl ⁇ 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.
  • the protein encoded by orfl 7 has significant similarity with a glycosyl transferase from Streptomyces venezuelae (Xue, Y. I and ⁇ /., 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).
  • the orfl 8 gene encodes a protein with relatively strong similarity to several glycosyl transferases.
  • 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 or / 79 gene encodes a protein with relatively strong similarity to. several ketoreductases.
  • the protein encoded by orfl 9 has a similarity> important with NDP-hexose-4-ketoreductase (TylCIV) Streptomyces fradiae:; (Bâte et ⁇ /., 2000; GenBank access number: AAD41822; BLAST score: 266).
  • TylCIV NDP-hexose-4-ketoreductase
  • the or ⁇ O gene encodes a protein with relatively strong similarity to several hexose reductases.
  • the protein encoded by or ⁇ O 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 or ⁇ lc gene codes for a protein with relatively strong similarity to several hexose methyltransferases.
  • the protein encoded by or ⁇ lc has significant similarity with the TylCIII gene from Streptomyces fradiae which codes for a
  • the or ⁇ 2c 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 ⁇ /., 2000; GenBank access number: AAF86387; BLAST score: 463).
  • the similarity of the protein encoded by the or ⁇ 2c 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
  • the or ⁇ 4c 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 : ⁇ AJF86389; BLAST score: 87).
  • ACP Adiyl Carrier Protein
  • the or ⁇ 5c gene codes for a protein with relatively strong similarity to the protein encoded by the ⁇ bK 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 or ⁇ 5c 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 or ⁇ 5c gene has a strong similarity with other proteins of similar function in other organisms (cf. table 32).
  • the or ⁇ 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 or ⁇ gene codes for a glycosyl transferase. This hypothesis is supported by the fact that the protein encoded by the or ⁇ gene has a strong similarity with other proteins of similar function in other organisms (cf. table 33). 67
  • the or ⁇ 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, ⁇ f., 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.
  • the or ⁇ 8c 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 or ⁇ 8c gene significantly increases the production of spiramycins of the OSC2 strain (cf. example 24). This demonstrates that the overexpression of or ⁇ 8c leads to an increase in the production of spiramycins ⁇ [and confirms its role as regulator of the biosynthetic pathway for spiramycins.
  • the or ⁇ 9 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).
  • soraphen A an antifungal of the polyketide class
  • Sorangium cellulosum Ligon, J., Et al, 2002
  • GenBank access number: AAK19890 BLAST score: 139
  • the or ⁇ Oc 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 or ⁇ Oc 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 or ⁇ Oc gene codes for an epimerase.
  • Vor ⁇ Oc 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).
  • the use of codons is typical of Streptomyces only at ' J / ' from the second ATG moreover, the protein sequence deduced from the sequence between
  • 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 or ⁇ 1 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 or ⁇ 1 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 or ⁇ 1 gene codes for a reductase. This assumption is also supported by the fact that the protein encoded by the or ⁇ 1 gene has a strong similarity with other proteins of similar function in other organisms (see Table 36).
  • the or ⁇ 1 gene was inactivated. It has thus been possible to show that the resulting strain no longer produces spiramycins. This confirms that the or ⁇ 1 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.
  • sequence d or ⁇ 2c 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
  • the or ⁇ 2c 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).
  • SEQ ID No. 121 which is the partial sequence of the Orf32c protein
  • BLAST program a regulatory protein of the GntR family in Streptomyces coelicolor
  • protein encoded by the or ⁇ 2c gene has a strong similarity with other proteins of similar function in other organisms.
  • the partial LVor ⁇ 2c 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
  • the or ⁇ 2c 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 or ⁇ 2c gene is not essential for the biosynthesis of spiramycins and that it is not essential for the survival of the bacteria.
  • the or ⁇ 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).
  • 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,
  • 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,
  • 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.
  • 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,, ' ?
  • 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.
  • 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.
  • 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
  • a polypeptide according to the invention is chosen from the group consisting of polypeptide sequences SEQ ID No.
  • 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.
  • 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.
  • a recombinant DNA comprising at least one polynucleotide as described above.
  • this recombinant DNA is a vector.
  • the vector is chosen from bacteriophages, plasmids, phagemids, integrative vectors, fosmids,, cosmids, shuttle vectors, BAC (Bacterial Artificial Chromosome) or PAC (Pl-derived Artificial Chromosome) .
  • BAC Bacterial Artificial Chromosome
  • PAC Pl-derived Artificial Chromosome
  • 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)), ⁇ ZAPII and its derivatives (sold in particular by the company Stratagene (LaJolla, California, USA)).
  • 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).
  • 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.
  • cosmids there may be mentioned by way of illustration the cosmid SuperCos and its derivatives (sold in particular by
  • 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, ⁇ WHM3, 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; •
  • BAC Bacterial Artificial Chromosome
  • PAC Pl-derived Artificial Chromosome
  • an i vector according to the invention is chosen from ⁇ OS49.1, pOS49.11, ⁇ OSC49.12, ⁇ OS49.14, : ;
  • 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.
  • 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 p ⁇ T 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.
  • the vector BacPAK6 sold by the company BD Biosciences Clontech, (Palo Alto, California, USA) may be mentioned by way of illustration.
  • 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).
  • CMV virus Cytomegalovirus
  • S V40 early promoter the immediate genes of the vacuolating simian virus SV40
  • the invention also relates to a method for producing a polypeptide as described above, said method comprising the following steps: 004/033689
  • step c) separating and purifying from said culture medium or also from the cell extract obtained in step c), said polypeptide;
  • 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 F ⁇ usubel et al (2002).
  • 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.
  • RTS rapid translation System
  • 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.
  • 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.
  • 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.
  • the mutagenesis (s) are carried out by gene inactivation.
  • mutagenesis consists in the gene inactivation of a gene comprising a sequence corresponding to the sequence SEQ BD No. 13.
  • microorganisms can be cited as an example of such microorganisms: OS49.16 (or ⁇ :: ⁇ hyg, cf. example 2), OS49.67 (or ⁇ deletion in phase, cf. example 6), OS49.
  • 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.
  • 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.
  • 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 or ⁇ * is affected by this manipulation, it will be necessary to make another modification so that the or ⁇ * 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.
  • 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.
  • 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.
  • 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.
  • strain SPM502 (orf ⁇ * :: 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,.:
  • SEQ ID No. 111 DNA
  • SEQ ID No. 112 The translation into protein of this part of the coding sequence is given in SEQ ID No. 112.
  • the undetermined sequence in SEQ ID No. 111 was determined in a second step and the complete sequence of this orf (or ⁇ 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 or ⁇ 8c gene and for manufacturing an expression vector allowing the expression of or ⁇ 8c.
  • the overexpression of the or ⁇ 8c gene in the OSC2 strain leads to an improvement in the production of spiramycins of this strain.
  • the overexpression of the or ⁇ 8c 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.
  • the overexpression of said gene is obtained by bringing into the microorganism a construction of recombinant DNA, allowing the overexpression of this gene.
  • this construction of recombinant DNA increases the number of copies of said gene and makes it possible to obtain an overexpression of said gene.
  • the coding sequence of the gene can be placed under the control of a promoter which is more active than the wild type promoter.
  • a promoter which is more active than the wild type promoter.
  • the copy or copies of the or ⁇ 8c 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.
  • 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 Dondel 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).
  • 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.
  • the invention relates to a mutant macrolide-producing microorganism characterized in that it has a genetic modification in at least one gè ⁇ è] ' - ' 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
  • 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.
  • 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).
  • mutagenesis a person skilled in the art may for example refer to this subject in the work by F. Ausubel et al (2002).
  • these mutant microorganisms improved for their production in macrolides are bacteria of the genus Streptomyces.
  • the macrolide in question is spiramycin and the microorganisms in which the mutagenesis are carried out are strains of S. ambofaciens.
  • 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.
  • the microorganism overexpresses one or more genes comprising one of the sequences corresponding to one or more of the sequences SEQ ID No.
  • the microorganism overexpresses the gene comprising a sequence corresponding to SEQ ID No.
  • SEQ ID N ° 111 or 141, or one of its% variants or one of the sequences derived therefrom due to the degeneration ⁇ j
  • 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 (or ⁇ 8c) 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 or ⁇ 8c gene and to manufacture an expression vector allowing the expression of or ⁇ 8c.
  • the overexpression of the or ⁇ 8c gene in the OSC2 strain leads to an improvement in the production of spiramycins of this strain.
  • the overexpression of the or ⁇ 8c 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.
  • the overexpression of the or ⁇ 8c gene is obtained by bringing a recombinant DNA construct into the microorganism, allowing the overexpression of this gene.
  • this construction of recombinant DNA increases the number of copies of the or ⁇ 8c gene and makes it possible to obtain an overexpression of the or ⁇ 8c gene.
  • the coding sequence for or ⁇ 8c can be placed under the control of a promoter more active than the wild-type promoter.
  • a promoter more active than the wild-type promoter.
  • the copy or copies of the or ⁇ 8c 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).
  • 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.
  • the microorganisms used in such a process are bacteria of the genus Streptomyces.
  • 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
  • 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.
  • 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 ⁇ /., 1989).
  • the principle by which genetic engineering can lead to the production of hybrid antibiotics was first proposed by 'Hopwood (Hopwood 1981).
  • 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.
  • 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.
  • 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.
  • 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.
  • this recombinant DNA is a vector.
  • the vector is chosen from bacteriophages, plasmids, phagemids, integrative vectors, fosmids, cosmids, shuttle vectors, BAC (Bacterial Artificial Chromosome) or PAC (Pl-derived Artificial Chromosome).
  • phage lambda and phage Ml 3 may be mentioned as bacteriophages.
  • 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, ⁇ GB2 and its derivatives (Churchward G.
  • pACYC177 access number GenBank: X06402
  • ⁇ ACYC184 GenBank access number: X06403
  • 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) ), ⁇ ZAPII and its derivatives (sold in particular by the company Stratagene (LaJolla, California, USA)).
  • 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).
  • 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, we can cite by way of illustration the cosmid SuperCos and its derivatives ⁇ •: •; ⁇ 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.
  • BAC Bacterial Artificial Chromosome
  • BAC pBeloBACU GenBank access number: U51113 As PAC (Pl-derived Artificial Chromosome)
  • the vector pCYPAC6 GenBank access number: AF 133437
  • this recombinant DNA is an expression vector.
  • 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.
  • the vector BacPAK6 sold by the company BD Biosciences Clontech, (Palo Alto, California, USA) may be mentioned by way of illustration.
  • 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
  • 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).
  • 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.
  • RTS rapid translation System
  • Another aspect of the invention relates to a microorganism producing at least one spiramycin, characterized in that it overexpresses:
  • SEQ ID No. 141 An example of the sequence of such a gene is given in SEQ ID No.
  • 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,
  • 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
  • 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).
  • 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.
  • 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.
  • 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:
  • telomere 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,
  • 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
  • the expression vector in question is the plasmid pSPM75.
  • 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.
  • FIG. 4 Proposed biosynthetic pathway for mycarosis.
  • FIG. 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 ⁇ /., 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;
  • 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.
  • FIG. 14 Map of plasmid pOSV508.
  • Figure 15 Example of the structure of an excisable cassette. This consists of the ⁇ hyg 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 was grown in a 12x12 cm square dish. Whatman: AA paper discs were soaked with 70 ⁇ l 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 ⁇ g / 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. 29) with the TylB protein (SEQ ID No. 29)
  • 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.,
  • phase 1 (att1) of 33 nucleotides is presented in SEQ ID No. '
  • 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 orf ⁇ * :: attl ⁇ hyg + obtained from a strain ⁇ s overproducing spiramycins. The insertion of the attl ⁇ hyg + cassette into orf ⁇ * exerts a polar effect preventing the expression of or ⁇ *.
  • 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 ⁇ SPM79 and localization of the sequence SEQ ID No. 140.
  • 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 S ⁇ Q ID No.
  • This gene was named orfi (S ⁇ Q ID N ° 28) and was inactivated in S. ambofaciens. It has been shown that clones inactivated in the or ⁇ gene no longer produce spiramycins. This shows the implication of the or ⁇ gene or of genes located downstream in the biosynthesis of spiramycins.
  • 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.
  • 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.
  • the cosmid pWED1 is derived from the cosmid pWE15 (Wahl, " ei ⁇ k 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
  • 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 ⁇ g / ml) since the cosmid pW ⁇ Dl confers resistance to ampicillin.
  • the probe used for hybridization is a Nael-Nael fragment of DNA (S ⁇ Q 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 ⁇ . (Merson-Davies, L.A. & Cundliffe ⁇ ., 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 (S ⁇ Q 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.
  • 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.
  • 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.
  • the corresponding gene (the or ⁇ gene (SEQ ID No. 28)) was involved in the biosynthesis of spiramycins in S. ambofaciens
  • this gene was interrupted by an ⁇ hyg cassette (Blondelet-Rouault MH. Et al ., 991, GenBank access number: X99315).
  • 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 or ⁇ 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.
  • a PmR-BstEll fragment internal to or ⁇ was replaced by the ⁇ hyg cassette by blunt-end cloning in this last plasmid.
  • 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 or ⁇ 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 ⁇ hyg cassette was obtained by digestion with the enzyme BamHI of the plasmid pHP45 ⁇ hyg (Blondelet-Rouault et al, 1997, GenBank access number: X99315).
  • the fragment corresponding to the ⁇ hyg 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 ⁇ hyg 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 or ⁇ gene interrupted by the ⁇ hyg cassette.
  • 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 ⁇ hyg cassette and having lost the marker for resistance to apramycin carried by the vector pOJ260 could be obtained.
  • 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 or ⁇ gene interrupted by the ⁇ hyg cassette.
  • the replacement of the wild-type copy of or ⁇ by the interrupted copy was verified by two successive hybridizations.
  • 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 ⁇ hyg 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-Xh ⁇ l insert of the plasmid pOS49.11 containing the four open reading phases (two complete and two truncated, including or ⁇ 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.
  • 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, or ⁇ and the start of or ⁇ , subcloned from pOS49.1 and going from a BamHI site located 1300 base pairs upstream of the EcoRI site defining position 1 of S ⁇ Q 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 or ⁇ fragment, subcloned from pOS49.1 and going from a Pstl site located in position 6693 of S ⁇ Q ID N ° 1 to the BamHI site located in position 8714 of S ⁇ Q 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.
  • 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).
  • 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 ⁇ g) had been ligated with 1 ⁇ g 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.
  • griseofuscus (Rao RN et a, 1987), which is naturally sensitive to spiramycin (cf. above).
  • the pool of plasmids corresponding to the Sau3Al fragment of ⁇ OS44.1 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 Hind ⁇ l 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 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 S ⁇ Q 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 S ⁇ Q ID ⁇ ° 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 genes of the region (C i upstream were named orfl, or ⁇ , or ⁇ , or ⁇ , or ⁇ , orf ⁇ , or ⁇ , or ⁇ , or ⁇ c, orflO, orfl le, orfl 2, orfl 3c, orfl4, orfl 5c, orfl ⁇ , orf 17 , orfl 8, orfl 9, or ⁇ O, or ⁇ lc, or ⁇ 2c, or ⁇ 3c, or ⁇ 4c and or ⁇ 5c (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, or ⁇ * c, or ⁇ * c, or ⁇ * c, or ⁇ *, orf ⁇ *, or ⁇ * c, or ⁇ *, or ⁇ * c, or ⁇ *, or ⁇ * c, or ⁇ *
  • 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.
  • an antibiotic for example apramycin or hygromycin
  • the insertion of the cassette into the targeted gene ei ⁇ 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.
  • constructs were then subcloned into vectors to allow transformation and inactivation of the desired gene in S. ambofaciens.
  • two plasmids were used:
  • plasmid ⁇ iHP45- ⁇ hyg (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 ⁇ eomycin / Kanamycin has been replaced by a sequence coding for resistance to hygromycin, while retaining the promoter P SN40.
  • the plasmid ⁇ iHP45- ⁇ hyg (Blondelet-
  • Rouault et a, 1997) was digested with the enzymes N ⁇ tl 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.
  • 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 AD ⁇ (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).
  • antibiotic B the antibiotic corresponding to the cassette present in the target gene
  • a mixture of clones is thus selected, among which there has been integration by one or by two recombination events.
  • clones sensitive to the antibiotic for which the resistance gene is present in the vector (outside the recombination cassette) cf. FIG. 9, antibiotic A
  • cassettes can be used for the interruption of the target genes.
  • the orf 3 gene had been interrupted by the ⁇ hyg cassette (cf. example 2.2) and it could be demonstrated that an or ⁇ :: ⁇ hyg 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).
  • 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 or ⁇ .
  • the insert of pOS49.67 is therefore constituted by a DNA fragment of S. ambofaciens containing the orfl, or ⁇ , or ⁇ genes with the deletion in phase, or ⁇ and part of or ⁇ .
  • 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.
  • clones sensitive to apramycin and to hygromycin were sought.
  • apraS and hygS apraS and hygS
  • apraS and hygS hygromycin
  • 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 or ⁇ deleted in phase is present in the genome of the clones obtained). Clones having only the copy of or ⁇ 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.
  • 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 ⁇ tl-EeoRI obtained from the cosmid pSPM5), containing the end of or ⁇ , or ⁇ and the start of or ⁇ , cloned into the Pstl-EcoRl sites of pUC19.
  • the ⁇ hyg 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 or ⁇ after all the ends were made blunt by treatment with the Klenow enzyme.
  • Plasmids 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 or ⁇ genes are in the same orientation and pOS49.120 in which the hyg and or ⁇ genes are in opposite directions.
  • the insert of plasmid pOS49.106 was then subcloned into plasmid pOJ260 to give pOS49.107.
  • 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 or ⁇ gene into which the ⁇ hyg cassette has been inserted, has been cloned j- into the vector pOJ260 (cf. above).
  • 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 ⁇ OS49.107 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 or ⁇ gene interrupted by the ⁇ hyg cassette.
  • the replacement of the wild-type copy of or ⁇ by the copy interrupted by the ⁇ hyg cassette was verified by Southern Blot.
  • 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 ⁇ hyg 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 or ⁇ , or ⁇ and the start of or ⁇ 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.
  • 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 Dondel Roux 75724 Paris Cedex 15, France, on July 10, 2002 under registration number 1 -2917.
  • CNCM National Collection of Cultures of Microorganisms
  • 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 ⁇ hyg 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.
  • plasmid pOS49.43 in which the genes hyg and orf 10 are in the same orientation
  • 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 ⁇ hyg 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 ⁇ hyg 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 :: ⁇ hyg was verified by Southern Blot.
  • 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 ⁇ hyg cassette to verify the presence of the cassette in the genomic DNA.
  • 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. ⁇ hyg) was more particularly selected and named OS49.50. It has indeed been possible to verify thanks to the two hybridizations that the ⁇ hyg 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 ⁇ hyg 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)
  • 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 recombinais ⁇ h 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 excisable cassette can be constructed using for example the ⁇ hyg cassette (Blondelet Rouault et al, 1997). This cassette was bordered by ⁇ ttR 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.
  • 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 ⁇ hyg cassette ).
  • antibiotic B this is for example a selection by hygromycin if the excisable cassette is derived from the ⁇ hyg cassette ).
  • a mixture of clones is thus selected, among which there has been integration by a single or by two recombination events.
  • 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).
  • a recombination event (cf. FIG. 10) (Raynal et a, 1998).
  • 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.
  • 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).
  • 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.
  • 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 ⁇ ttR 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.
  • 26 correspond to the minimal attB sequence (cf. FIG. 27).
  • two excisable cassettes were used. These two cassettes are as follows: attl ⁇ hyg + (SEQ ID No. 91) and att3 ⁇ aac- (SEQ ID No. 92), these cassettes leave 33 and 35 nucleotides respectively after excision. They respectively include the ⁇ hyg cassette or the ⁇ aac 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 pattl ⁇ hyg + and patt3 ⁇ aac- 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 or ⁇ gene
  • the or ⁇ 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.
  • 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.
  • 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).
  • the transformed bacteria After transformation with the plasmid pOSV510, the transformed bacteria are selected for their resistance to kanamycin (due to the aph gene carried by pOSV510).
  • 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.
  • the or ⁇ 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 (S ⁇ Q 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 pKOB ⁇ G (Chaveroche et al, 2000) (cf. FIG. 12). ' ?
  • the excisable cassette att3 ⁇ aac- (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 ⁇ GP704Not (Chaveroche et al, 2000) (Miller VL & Mekalanos JJ, 1988) in which the att3 ⁇ aac- cassette was cloned as an EcoRV fragment in the unique EcoRV site of pGP704Not) and using the following primers:
  • the 40 deoxy-nucleotides located at the 5 'end of these oligonucleotides have a sequence corresponding to a sequence in the target gene (or ⁇ 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 att3 ⁇ aac- excisable cassette (see Figure 11).
  • the PCR product thus obtained was used to transform the E. cpli strain containing the plasmids pKOB ⁇ G and pOS49.99 as described (Chaveroche et al, 2000) (cf. FIG. 12).
  • 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 (att3 ⁇ aac-) into the target gene ( or ⁇ ), 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 or ⁇ is interrupted by the apramycin cassette (cf. FIG. 12).
  • the insertion of the cassette is accompanied by a deletion in or ⁇ , between nucleotides 211 and 492 of the coding part of or ⁇ .
  • 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 or ⁇ gene was cloned into the vector pOSK1205 (cf. above).
  • 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 or ⁇ gene is replaced by the att3 ⁇ aac- 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 or ⁇ gene interrupted by the att3 ⁇ aac- cassette.
  • the strain SPM21 was deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Dondel 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 ⁇ ., 1989) (cf. FIG. 13) in which the xis and int genes of pSAM2 (Boccard F.
  • the strain thus obtained and having the desired genotype was named SPM22.
  • plasmid pSPM504 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 or ⁇ orfl 7.
  • 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.
  • pSPM502 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 ⁇ l, 2000) (cf. FIG. 12).
  • the excisable cassette ⁇ tt3 ⁇ c- was amplified by PCR using as plasmid the plasmid pOSKl 102 (the plasmid pOSKl 102 is a plasmid derived from the vector ⁇ GP704Not (Chaveroche and ⁇ l, 2000) (Miller VL & Mekalanos JJ, 1988) in which the ⁇ tt3 ⁇ c- cassette has been cloned as an EcoRV fragment in the unique EcoRV site of pGP704Not), the primers used are the following:
  • ⁇ DR8 5 'CGGGATGATCGCTTGTCCGGCGGCCGGATGCCTAGCCTCATCGCGCGCGCTTCGTTCGG 3' (S ⁇ Q ID N ° 96)
  • ⁇ DR9 5 'CCCGATCCAGAACGTCTGGTCGGTGATCAGGTCGCTGTTCATCTGCCTCTTCGTCCCGAA 3' (S ⁇ Q ID N ° 97)
  • the 40 (only 39 for ⁇ DR8) deoxy-nucleotides located at the 5 'end of these oligonucleotides have a sequence corresponding to a sequence in the gene 004/033689
  • the PCR product thus obtained was used to transform the E. coli KS272 strain containing the plasmids pKOB ⁇ G 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).
  • 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 (att3 ⁇ aac ⁇ ) 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.
  • pSPM507 This plasmid is derived from pSPM504 in which orfl 2 is interrupted by the att3 ⁇ aac- 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 att3 ⁇ aac- cassette.
  • 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 :: att3 ⁇ aac-) was more particularly selected and named SPM507. It has indeed been possible to verify thanks to the two hybridizations that the att3 ⁇ aac- 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. '> ⁇ the copy interrupted by the att3 ⁇ aac- cassette in the genome of this clone. This clone therefore has the genotype: orfl2 :: att3 ⁇ aac- and was named SPM507.
  • the att3 ⁇ aac- 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)
  • 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
  • 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 (att3 ⁇ aac-) into the gene
  • 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 att3 ⁇ aac- cassette.
  • a clone exhibiting the expected characteristics was more particularly selected and named SPM508. It has indeed been possible to verify thanks to the two hybridizations that the att3 ⁇ aac- 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 att3 ⁇ aac- cassette in the genome of this clone. This clone therefore has the genotype: orfl3c :: att3 ⁇ aac- and was named SPM508.
  • the att3 ⁇ aac- excisable cassette was amplified by PCR using the plasmid pOSKl 102 (see above) as matrix using the following primers:
  • 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 att3 ⁇ aac- 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).
  • 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 (attS ⁇ aac-) 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 att3 ⁇ aac- cassette.
  • 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.
  • 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 att3 ⁇ aac- cassette to verify the presence of the cassette in the genomic DNA.
  • 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.
  • EXAMPLE 14 Construction of a Streptomyces ambofaciens strain interrupted in the orf ⁇ * gene
  • the orf ⁇ * gene was inactivated using the excisable cassette technique (see above and Figure 10).
  • the cosmid pSPM7 was used as a matrix to amplify a fragment of the orf ⁇ * 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 orf ⁇ * gene (SEQ ID No. 13) and the 6 deoxy-nucleotides located most at 5 ′ correspond to the site sequence
  • 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 attl ⁇ hyg + excisable cassette was then introduced into the coding sequence of the orf ⁇ * gene.
  • 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 orf ⁇ * gene (cf. FIG. 15).
  • the attl ⁇ hyg + excisable cassette was prepared from the plasmid pattl ⁇ hyg + (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).
  • the orf ⁇ * gene has a 120bp deletion and is interrupted by the attl ⁇ hyg + 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 orf ⁇ * interrupted by the attl ⁇ hyg + 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).
  • 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 orf ⁇ * gene interrupted by the attl ⁇ hyg + cassette. These clones were more particularly selected and the replacement of the wild copy of orf ⁇ * by the copy interrupted by the cassette was verified by the technique of Southern blot.
  • 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 orf ⁇ * 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 ⁇ 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 (orf ⁇ * :: attl ⁇ hyg +) was more particularly selected and named SPM501. It has indeed been possible to verify thanks to the two hybridizations that the attl ⁇ hyg + 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 attl ⁇ hyg + cassette in the genome of this clone. This clone therefore has the genotype: orf ⁇ * :: attl ⁇ hyg + and was named SPM501.
  • the strain SPM501 was deposited with the National Collection of Cultures of Microorganisms (CNCM) Institut Pasteur, 25, rue du Dondel 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.
  • 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 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 ! ; ⁇ à; '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 ⁇ g / 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.
  • Whatman AA paper discs (cf. Gourmelen A. et al, 1998) 12 mm in diameter were soaked with 70 ⁇ l 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 ⁇ g / 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.
  • 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 or ⁇ 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.
  • 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 :: att3 ⁇ aac-. 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 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 :: att3 ⁇ aac-. 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 :: att3 ⁇ aac-. 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 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: or ⁇ :: att3 ⁇ aac-,
  • the phenotype of this strain is not a producer of spiramycins.
  • the orientation of the orfl to or ⁇ genes suggests that these genes are cotranscribed.
  • the phenotype observed may be due to a polar effect of the cassette introduced into or ⁇ on the expression ;; of genes located downstream in the operation.
  • the strain SPM22 has the genotype or ⁇ :: 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).
  • strain SPM22 also being of non-producing phenotype, it can be concluded that the or ⁇ gene is a gene essential for the biosynthesis of spiramycin in S. ambofaciens. We observe here only the effect due to the inactivation of or ⁇ .
  • the strain SPM501 has the genotype: orf ⁇ * :: attl ⁇ hyg +.
  • the phenotype of this strain is not a producer of spiramycins.
  • the or ⁇ * and orf ⁇ * genes (cf. FIG. 3) having the same orientation, the phenotype observed may be due to a polar effect of the cassette introduced into orf ⁇ * on the expression of or ⁇ *.
  • the arrangement of these genes suggests that they can be cotranscribed.
  • the strain SPM502 was obtained after excision in phase of the cassette introduced. In this strain, we only observe the effect of inactivation of orf ⁇ *.
  • This strain has the orf ⁇ * :: 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)).
  • the or ⁇ * 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.
  • the orf ⁇ * 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)). !
  • 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 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 ⁇ m 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
  • 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 :: att3 ⁇ aac-.
  • the phenotype of the strain SPM507 is non-producer (cf. example 15), we can therefore conclude that the orfl 2 gene is ⁇ n ' ; 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 :: att3 ⁇ aac-.
  • 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 :: att3 ⁇ aac-.
  • 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: orf ⁇ * :: att! ⁇ hyg +.
  • 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).
  • the ' or ⁇ * and orf ⁇ * genes (see Figure 3) having the same orientation, the observed phenotype may be due to a polar effect of the cassette introduced in orf ⁇ * on the expression of or ⁇ * in the operon. This suggests that these genes are cotranscribed.
  • 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 or ⁇ *.
  • This strain has the orf ⁇ * :: 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 'or ⁇ * 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.
  • the orf ⁇ * 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, orf ⁇ * is essential for the production of spiramycin II and III (cf. example 16)). The orf ⁇ * 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
  • 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
  • the first pair used for the amplification corresponds to the following oligonucleotides:
  • 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'
  • 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 ⁇ UWL201 (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 (erm ⁇ * promoter) (Bibb et al, 1994) was introduced (cf. Doumith et al, 2000)) previously digested by enzymes H dlII-iî ⁇ mHI.
  • 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
  • pSPM525 (derived ⁇ UWL201 with the form “or / 70 pro”)
  • 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.
  • 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.
  • the negative control (strain OS49.50 transformed by the plasmid pUWL201) does not produce spiramycin.
  • the plasmid pSPM523 (which contains the form "gold / short 70") is introduced to the strain OS49.50 years, no spiramycin production is observed.
  • 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.
  • the gold / 70 fragments containing the most upstream ATG restore the synthesis of spiramycin.
  • the plasmids pSPM525 and pUWL201 were similarly introduced into the strain SPM502 (cf. example 14). Thus two new strains were obtained: the strain SPM502 ⁇ UWL201, resulting from the transformation by the plasmid pUWL201 without insert, and the strain SPM502 pSPM525, resulting from the transformation by the plasmid pSPM525.
  • EXAMPLE 18 Construction of a genomic DNA library of the OSC2 strain of Streptomyces ambofaciens in E. coli in the cosmid pWED2.
  • 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.
  • pac-oriT cassette (EcoRV fragment) was introduced into the cosmid pW ⁇ Dl (Gourmelen et al, 1998), derived from the cosmid pW ⁇ D15 (Wahl et a, 1987), at the unique Hpal site.
  • the pac-oriT cassette was obtained by PCR.
  • 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'- '
  • primer C (of sequence 5'-CACGACCCCATGACGGATCTTTTCCGCTGCAT-3 '(S ⁇ Q 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'-
  • 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 pG ⁇ M-T Easy (sold by the company Promega (Madison, Wisconcin, USA)), which made it possible to obtain the plasmid pGEM-T-> c-o ⁇ .
  • 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.
  • 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 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.
  • 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 .
  • Vara J et al, 1985; Lacalle et al, 1989
  • puromycin N-acetyl transferase which confers resistance to puromycin .
  • the genomic DNA of the OSC2 strain of Streptomyces ambofaciens was f
  • Cosmids from the new library of Streptomyces ambofaciens OSC2 (cf. example 18) covering orfl * to or / 70 * or part or all of orfl to or ⁇ 5c, or a region further upstream of V or ⁇ 5c were isolated .
  • 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'-ACGTGCGCGGTGAGTTCG ⁇ CGTTGC-3 '(S ⁇ Q 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 or ⁇ 4c and the end of the or ⁇ 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 or ⁇ * c (cf. FIG. 31, probe II).
  • the third probe used corresponds to an EcoRl-BamHI fragment of approximately 3kb containing the orfl, or ⁇ and or ⁇ 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.
  • pSPM36 was more particularly chosen because it was likely to contain a large region upstream of V or ⁇ 5c (cf. Figures 31 and 32).
  • 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.
  • 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 Vor ⁇ and capable of containing either a large part of the PKS genes, or a large part of the orfl to or ⁇ 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.
  • the probe of around 0.8 kb obtained by PCR with the primers ORF23c and ORF25c 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 Yor ⁇ 3c (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 or ⁇ 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 or ⁇ 5c. 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.
  • the approximately 0.8 kb probe obtained by PCR with the primers ORF23c and ORF25c 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 Yor ⁇ 3c (cf. SEQ ID No.
  • 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 , or ⁇ 9 and other genes further upstream of or ⁇ 9.
  • 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 S ⁇ Q ID N ° 135.
  • An assembly of the sequences of the inserts of pSPM58 and pSPM73 is presented in S ⁇ Q ID N ° 106. This sequence starts from the Pstl site at Yor ⁇ 3c (cf.
  • 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 or ⁇ 9 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.
  • a probe (DNA fragment of 0.8 kb) corresponding to an internal sequence to or ⁇ 2c 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 Vor ⁇ 2c.
  • 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.
  • this probe 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 or ⁇ 2c and other genes upstream of the latter.
  • 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 Yor ⁇ 8c 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 Yor ⁇ 8c 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.
  • SEQ ID No. 140 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 Yor ⁇ 3c (cf. SEQ ID No. 80) and goes to the Pstl site at Yor ⁇ 4c (cf. FIG. 41).
  • 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 (or ⁇ 2c and or ⁇ 4c) and one complete (or ⁇ 3) (cf. Figure 41).
  • the first incomplete ORF corresponds to the 5 'part of the coding sequence for or ⁇ 2c. 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 or ⁇ 2c. (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 or ⁇ 4c (SEQ ID No. 149).
  • 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 were each cultivated in 7 500 ml baffled Erlenmeyer flasks containing 70 ml of MP5 medium (Pernodet et al., 1993). The erlens
  • the pH of the must is then adjusted to 9 with sodium hydroxide and the supernatant is extracted with methyl iso-butyryl ketone (MIBK).
  • MIBK methyl iso-butyryl ketone
  • 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 ⁇ l qs 1 ml with water) before being used for the analyzes in liquid chromatography coupled with mass spectrometry (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.
  • the strain OS49.67 in which the or ⁇ gene is inactivated by a phase deletion does not produce spiramycins (cf. examples 6 and 15).
  • Figure 35 shows the supe ⁇ osition 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 scan is done in scan mode, covering a mass range between 100 and 1000 Da.
  • the gain of the electro-multiplier was IV.
  • 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
  • the mobile phase is a mixture of a solution
  • 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 ⁇ l of water / deuterated acetonitrile 40/60. The recovered solution is then directly analyzed by NMR.
  • Platenolide B (Specter 9647V)
  • the strain SPM509 in which the orfl4 gene is inactivated 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 or ⁇ 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 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. .
  • 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 att3 ⁇ aac- 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?
  • 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 att3 ⁇ aac- 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 (Aor ⁇ , orfl4 :: att3 ⁇ aac-) was more particularly selected and named SPM510. It was indeed possible to verify thanks to the two hybridizations that the att3 ⁇ aac- 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 att3 ⁇ aac- cassette in the genome of this clone.
  • 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.
  • 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.
  • the strain SPM510 transformed by the plasmid pSPM519 was named SPM510 pSPM509.
  • the plasmid pOS49.52 corresponds to a plasmid allowing the expression of the TylB protein in S. ambofaciens.
  • 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
  • the strain OS49.67 in which the or ⁇ 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.
  • the strain OS49.67 does not produce spiramycins (cf. examples 6 and 15). Spiramycin production of the strain
  • the or ⁇ 8c gene was amplified by PCR using a pair of oligonucleotides comprising an H ⁇ TII 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)
  • 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 or ⁇ 8c placed under the control of the ermE * promoter.
  • 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 / ⁇ SPM75 (2).
  • 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).
  • strain OS49.107 in which the or ⁇ gene is inactivated by insertion of the; ;
  • 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
  • the compound is recovered by elution with a water / acetonitrile mixture 30/70. This solution is then injected (100 ⁇ L) 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 ⁇ l of deuterated acetonitrile water 40 / 60.
  • the recovered solution is then directly analyzed by NMR.
  • the strain SPM501 has the genotype or ⁇ * :: attl ⁇ hyg +. Thanks to the polar effect 20 of the insertion of the attl ⁇ hyg + cassette into the orf ⁇ * 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 orf ⁇ * gene leads to a total halt in the production of spiramycins by polar effect on the expression of the or ⁇ * gene. However, once the inserted cassette has been excised (and therefore when only the orf ⁇ * gene is inactivated, cf. examples 14 and 15), a production of spiramycin I is restored.
  • 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 or ⁇ * gene encodes a protein with relatively strong similarity to several O-methyltransferases.
  • the or ⁇ * gene is said to be an O-methyl transferase involved in the biosynthesis of platenolide.
  • CL / SM and NMR analysis experiments were carried out on a strain of S. ambofaciens of genotype orf ⁇ * :: attl ⁇ hyg + obtained from a strain su ⁇ roducing 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) .
  • 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
  • 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 ⁇ inte ⁇ oson, sequences which contain a transcription terminator originating from phage T4.
  • the gene which confers resistance to an antibiotic is flanked by the sequences ⁇ ttR 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 ⁇ aac 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)
  • 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 'Hirui ⁇ l-Hin ⁇ ⁇ . 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 ⁇ acc 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).
  • the or ⁇ 8c gene was inactivated using the cassette technique
  • 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 ⁇ GP704Not (Chaveroche et al, 2000) (Miller VL & Mekalanos JJ,
  • sequence corresponding to a sequence in the or ⁇ 8c gene and the 26 nucleotides located most in 3 '(shown in bold and underlined above) correspond to the sequence of one of the
  • 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
  • cosmid pSPM36 25 cosmid pSPM36.
  • 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 or ⁇ 8c gene , i.e. if it
  • the cosmid pSPM107 was first introduced into the E. coli strain
  • DH5 ⁇ 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 ⁇ r2Sc interrupted by the att3aac + cassette.
  • a clone exhibiting the expected characteristics (or ⁇ 8c :: att3aac +) was more particularly selected and named SPM107.
  • This clone therefore has the genotype: or ⁇ 8c :: att3aac + and was named SPM107.
  • the use of an excisable cassette makes it possible to get rid of the selection marker at any time, in particular by transformation with the plasmid pOSV508.
  • EXAMPLE 30 Construction of a strain of S. ambofaciens interrupted in the or ⁇ 1 gene:
  • the 39 nucleotides located at the 5 ′ end of these oligonucleotides have a sequence corresponding to a sequence in the or ⁇ 1 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
  • 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 or ⁇ 1 gene interrupted by the att3aac + cassette. These clones were more particularly selected and the replacement of the wild-type copy of or ⁇ 1 by the copy interrupted by the cassette was verified by hybridization.

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EP03807883A 2002-10-08 2003-10-08 Polypeptide, die an der biosynthese von spiramycinen beteiligt sind, nukleinsäuren, die diese polypeptide kodieren und ihre verwendungen Withdrawn EP1551975A2 (de)

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EP07019306.5A EP1905833B1 (de) 2002-10-08 2003-10-08 Polypeptide, die an der Biosynthese von Spiramycinen beteiligt sind, Nukleotidsequenzen, die diese Polypetide kodieren, und ihre Anwendungen
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FR0212489A FR2845394A1 (fr) 2002-10-08 2002-10-08 Polypeptides impliques dans la biosynthese des spiramycines, sequences nucleotidiques codant ces polypeptides et leurs applications
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FR0302439 2003-02-27
FR0302439A FR2851773A1 (fr) 2003-02-27 2003-02-27 Polypeptides impliques dans la biosynthese des spiramycines, sequences nucleotidiques codant ces polypeptides et leurs applications
US49349003P 2003-08-07 2003-08-07
US493490P 2003-08-07
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