Classifications

• • 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
  • 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

Definitions

• the present invention describes genes involved in the biosynthesis and transfer of 6-deoxyhexoses in Saccharopolyspora erythraea and their use in the production of erythromycin analogs by genetic manipulation.
• Erythromycin A is a clinically important macrolide antibiotic produced by the Gram-positive bacteria Sac. erythraea.
• the erythromycin biosynthesis genes are organized into an ery gene cluster which also includes the erythromycin self-resistance gene ermE.
• the ery cluster contains the three major genes eryAI, eryAII and eryAIII (locus eryA) encoding three polypeptides making up polyketide synthetase (called PKS) flanked by two regions comprising the genes involved in the later stages of conversion of the lactone nucleus (6-deoxyerythronolide B) in erythromycin A.
• eryAI eryAII
• eryAIII locus eryAIII
• PKS polyketide synthetase
• the biosynthesis of 6-deoxyhexoses includes all the enzymatic reactions leading from glucose-1-phosphate to the final activated sugar dTDP-L-mycarose or dTDP-D- desosamine.
• the dTDP-L-mycarose or the dTDP-D-desosamine thus produced are then used as substrates for the transfer of the two deoxyhexoses onto the lactone nucleus.
• the formation of erythromycin requires the attachment of the fungus via hydroxyl in position C-3 of the lactone nucleus and the attachment of desosamine via hydroxyl in position C-5.
• the set of eryB genes involved in the biosynthesis or transfer of mycarosis and the set of eryC genes involved in the biosynthesis or transfer of desosamine have not yet been clearly identified.
• the 56 kb ery cluster includes 21 open reading frames (ORFs), the numbering of which was established by Haydock et al. (1991) and Donadio et al. (1993).
• the eryA locus includes ORFs 10, 11 and 12.
• the eryF gene (ORF4) responsible for C6 hydroxylation has been identified (Weber et al., 1991) and the eryK gene (ORF20) responsible for hydroxylation. in C12 (Stassi et al., 1993).
• the eryG gene (ORF6) responsible for the O-methylation of mycarosis in cladinose (position 3 "OH) has been identified (Weber et al., 1989). Erythromycin A is thus formed via erythromycin B or erythromycin C from erythromycin D according to the proposed scheme ( Figure 1).
• the present invention relates to the functional characterization of ten Sac genes.
• erythraea involved in the biosynthesis or attachment of mycarosis and desosamine (eryBII, eryCIII and eryCII located downstream of the locus eryA and eryBIV, eryBV, eryCVI, eryBVI, eryCIV, eryCV and eryBVII located upstream) in the production of erythromycin analogs and a process for their preparation.
• the present invention therefore relates to an isolated single or double-stranded DNA sequence, represented in FIG. 2 (direct and complementary sequence of SEQ ID No. 1) corresponding to the eryG-eryAIII region of the gene cluster for biosynthesis of erythromycin and particularly relates to a DNA sequence above comprising: the sequence eryBII corresponding to 1ORF7 (sequence complementary to SEQ ID No. 1 from nucleotide 48 to nucleotide 1046) and coding for a dTDP-4-keto -L-6-deoxyhexose 2,3-reductase,
• sequence eryCIII corresponding to 1ORF8 sequence complementary to SEQ ID No. 1 from nucleotide 1046 to nucleotide 2308, and coding for a deosaminyltransferase and
• sequence eryCII corresponding to 1ORF9 sequence complementary to SEQ ID No. 1 from nucleotide 2322 to nucleotide 3404
• sequence complementary to SEQ ID No. 1 from nucleotide 2322 to nucleotide 3404 sequence complementary to SEQ ID No. 1 from nucleotide 2322 to nucleotide 3404
• the above DNA sequence shown in Figure 2 is a genomic DNA sequence which can be obtained for example by subcloning restriction fragments of a Sac genomic DNA fragment. erythraea, according to operating conditions, a detailed description of which is given below.
• the invention more particularly relates to an isolated DNA sequence represented in FIG. 2 chosen from the sequence eryBII corresponding to 1ORF7 (sequence complementary to SEQ ID No. 1 from nucleotide 48 to nucleotide 1046), the sequence eryCIII corresponding to 1ORF8 (sequence complementary to SEQ ID No. 1 from nucleotide 1046 to nucleotide 2308) or the sequence eryCII corresponding to 1ORF9 (sequence complementary to SEQ ID No. 1 from nucleotide 2322 to nucleotide 3404) and the sequences which hybridize and / or show homologies significant with this sequence or fragments thereof and having the same function.
• the eryBII sequence corresponding to 1ORF7 codes for a polypeptide having 333 amino acids (sequence of SEQ ID No. 2)
• the eryCIII sequence corresponding to 1ORF8 codes for a polypeptide having 421 amino acids (sequence of SEQ ID No. 5)
• the sequence eryCII corresponding to 1ORF9 codes for a polypeptide having 361 amino acids (sequence of SEQ ID No. 3).
• sequences which hybridize and having the same function we include the DNA sequences which hybridize with one of the above DNA sequences under standard conditions of high or medium stringency described by Sambrook et al.
• the high stringency conditions include for example hybridization at 65 ° C for 18 hours in a solution 5 x SSPE, 10 x Denhardt, 100 ⁇ g / ml DNAss, 1% SDS followed by 2 washes for 20 minutes with a solution
• the conditions of medium stringency include, for example, a final wash for 20 minutes in a 0.2 x SSC, 0.1% SDS solution at 65 ° C.
• sequences which have significant homologies and having the same function we include the sequences having a nucleotide sequence identity of at least 60% with one of the DNA sequences above and which code for a protein having the same enzymatic function .
• the invention also relates to a polypeptide encoded by one of the DNA sequences above and especially to a polypeptide corresponding to an ORF represented in FIG. 2, chosen from 1ORF7 (having the sequence of SEQ ID N ° 2), 1ORF8 (having the sequence of SEQ ID No. 5) or 1ORF9 (having the sequence of SEQ ID No. 3) and analogs of this polypeptide.
• an ORF represented in FIG. 2 chosen from 1ORF7 (having the sequence of SEQ ID N ° 2), 1ORF8 (having the sequence of SEQ ID No. 5) or 1ORF9 (having the sequence of SEQ ID No. 3) and analogs of this polypeptide.
• analogues we include peptides having an amino acid sequence modified by substitution, deletion or addition of one or more amino acids provided that these products retain the same enzymatic function.
• the modified sequences can for example be prepared using the site-directed mutagenesis technique known to those skilled in the art.
• a more specific subject of the invention is the polypeptide corresponding to ORF 8 represented in FIG. 2 (having the sequence of SEQ ID No. 5) and having a deosaminyltransferase activity, called EryCIII.
• the invention describes a recombinant Sac EryCIII protein. erythraea obtained by expression in a host cell according to known methods of genetic engineering and cell culture.
• a subject of the invention is also the thymidine 5 '- (tri-hydrogen diphosphate), P' - [3, 4, 6-tridesoxy-3- (dimethylamino) - D-.xylo.-hexopyranosyl] ester (dTDP-D -desosamine) and the addition salts with bases, an example of preparation of which is described later in the experimental part.
• the subject of the invention is also an isolated DNA sequence represented in FIG. 3 (sequence of SEQ ID No. 6) corresponding to the eryAI-eryK region of the gene cluster for the biosynthesis of erythromycin and particularly relates to a DNA sequence above comprising: - the eryBIV sequence corresponding to 1ORF13 (sequence of SEQ ID No. 6 from nucleotide 242 to nucleotide 1207) and coding for a dTDP-4-keto-L-6-deoxyhexose 4-reductase ,
• sequence eryBV corresponding to 1ORF14 sequence of SEQ ID No. 6 from nucleotide 1210 to nucleotide 2454
• sequence of SEQ ID No. 6 from nucleotide 1210 to nucleotide 2454
• coding for a mycarosyltransferase
• sequence eryCVI corresponding to 1ORF15 sequence of SEQ ID No. 6 from nucleotide 2510 to nucleotide 3220
• sequence of SEQ ID No. 6 from nucleotide 2510 to nucleotide 3220
• sequence eryBVI corresponding to 1ORF16 sequence of SEQ ID No. 6 from nucleotide 3308 to nucleotide 4837
• sequence of SEQ ID No. 6 sequence of SEQ ID No. 6 from nucleotide 3308 to nucleotide 4837
• sequence eryBVII corresponding to 1ORF19 sequence of SEQ ID No. 6 from nucleotide 7578 to nucleotide 8156
• sequence of SEQ ID No. 6 sequence of SEQ ID No. 6 from nucleotide 7578 to nucleotide 8156
• coding for a dTDP-4-keto-D-6-deoxyhexose 3.5 epimerase sequence of SEQ ID No. 6 from nucleotide 7578 to nucleotide 8156
• the above DNA sequence shown in Figure 3 is a genomic DNA sequence which can be obtained, for example, by subcloning restriction fragments of cosmids containing a Sac genomic DNA library. erythraea, according to operating conditions, a detailed description of which is given below.
• the subject of the invention is more particularly an isolated DNA sequence represented in FIG. 3 chosen from the sequence eryBIV corresponding to 1ORF13 (sequence of SEQ ID No. 6 from nucleotide 242 to nucleotide 1207), the sequence eryBV corresponding to 1ORF14 ( sequence of SEQ ID No 6 from nucleotide 1210 to nucleotide 2454), the sequence eryCVI corresponding to 1ORF15 (sequence of SEQ ID No 6 from nucleotide 2510 to nucleotide 3220), the sequence eryBVI corresponding to 1ORF16 (sequence of SEQ ID No 6 from nucleotide 3308 to nucleotide 4837), the eryCIV sequence corresponding to 1ORF17 (sequence of SEQ ID No.
• the subject of the invention is very particularly the isolated DNA sequence eryBV represented in FIG. 3 corresponding to 1ORF14 (sequence of SEQ ID No. 6 from nucleotide 1210 to nucleotide 2454) and coding for a mycarosyltransferase.
• the eryBIV sequence corresponding to 1ORF13 codes for a polypeptide having 322 amino acids (SEQ ID No. 7)
• the eryBV sequence corresponding to 1ORF14 codes for a polypeptide having 415 amino acids (SEQ ID No. 8)
• the corresponding eryCVI sequence to 1ORF15 code for a polypeptide having 237 amino acids (SEQ ID No. 9)
• the sequence eryBVI corresponding to 1ORF16 code for a polypeptide having 510 amino acids
• the sequence eryCIV corresponding to 1ORF17 code for a polypeptide having 401 amino acids SEQ ID No.
• the eryCV sequence corresponding to 1ORF18 codes for a polypeptide having 489 amino acids (SEQ ID No. 11) and the eryBVII sequence corresponding to 1ORF19 codes for a polypeptide having 193 amino acids (SEQ ID N "12).
• the demonstration of the respective enzymatic activities indicated above was carried out by introduction of an internal deletion to the corresponding gene as illustrated below in the experimental part.
• the DNA sequences which hybridize as well as the DNA sequences which exhibit significant homologies and having the same function have the same meaning as that indicated above.
• the invention also relates to a polypeptide encoded by one of the DNA sequences above and especially to a polypeptide corresponding to an ORF represented in FIG.
• a more specific subject of the invention is the polypeptide corresponding to 1ORF14 represented in FIG. 3 (having the sequence of SEQ ID No. 8) and having a mycarosyltransferase activity, called EryBV.
• polypeptides of the invention can be obtained by known methods, for example by chemical synthesis or by the methodology of recombinant DNA by expression in a prokaryotic or eukaryotic host cell.
• Another subject of the invention relates to the use of at least one of the DNA sequences chosen from sequences eryBII (sequence complementary to SEQ ID N ° 1 from nucleotide 48 to nucleotide 1046), eryCIII (sequence complementary to SEQ ID N ° 1 from nucleotide 1046 to nucleotide 2308) or eryCII (sequence complementary to SEQ ID N ° 1 to nucleotide 2322 at nucleotide 3404) shown in FIG.
• eryBIV sequence of SEQ ID No 6 from nucleotide 242 to nucleotide 1207), eryBV (sequence of SEQ ID No 6 from nucleotide 1210 to nucleotide 2454), eryCVI (sequence of SEQ ID No. 6 from nucleotide 2510 to nucleotide 3220), eryBVI (sequence of SEQ ID No. 6 from nucleotide 3308 to nucleotide 4837), eryCIV (sequence of SEQ ID No. 6 from nucleotide 4837 to nucleotide 6039), eryCV (sequence of SEQ ID No.
• hybrid secondary metabolites is meant either erythromycin analogs, that is to say erythromycin derivatives having one or more modifications relating to the sugar part and having antibiotic activity, or precursors of erythromycin such as 6-deoxyerythronolide B or erythronolide B to which are attached one or more modified or unmodified sugar residues and not having antibiotic activity.
• the modified sugar residue can be, for example, 4-keto-L-mycarose.
• the synthesis of hybrid secondary metabolites in Sac. erythraea by using DNA sequences eryB or eryC of the invention can be carried out for example, by the inactivation of one or more genes eryB or eryC above and the introduction of one or more exogenous genes or their derivatives obtained for example by mutagenesis, having nucleotide sequences coding for enzymes having the same function in strains producing other acrolides, for example tylosin, picromycin or methymycin.
• exogenous genes can be carried out by integration of a DNA sequence obtained according to the methodology of "DNA shuffling" (Stemmer, 1994) or by the construction of a chimeric DNA sequence, by example from an eryB or eryC sequence of the invention involved in the transfer of a sugar residue, for example the eryCIII or eryBV sequence, and of homologous genes isolated from macrolide producing strains, for example Streptomyces fradiae, Streptomyces olivaceus, Streptomyces venezuelae or Streptomyces antibioticus.
• the invention also relates to the use of at least one of the DNA sequences chosen from the sequences eryBII (sequence complementary to SEQ ID No. 1 from nucleotide 48 to nucleotide 1046), eryCIII (sequence complementary to SEQ ID N ° 1 from nucleotide 1046 to nucleotide 2308) or eryCII (sequence complementary to SEQ ID No 1 from nucleotide 2322 to nucleotide 3404) shown in FIG.
• eryBIV (sequence from SEQ ID No 6 from nucleotide 242 to nucleotide 1207), eryBV (sequence of SEQ ID No 6 from nucleotide 1210 to nucleotide 2454), eryCVI (sequence of SEQ ID No 6 of nucleotide 2510 to nucleotide 3220), eryBVI (sequence of SEQ ID No 6 of nucleotide 3308 to nucleotide 4837) , eryCIV (sequence of SEQ ID No 6 from nucleotide 4837 to nucleotide 6039), eryCV (sequence of SEQ ID No 6 from nucleotide 6080 to nucleotide 7546) or eryBVII (sequence of SEQ ID No 6 from nucleotide 7578 to nucleotide 8156 ) shown in Figure 3 or a fragment of this sequence, co same hybridization probes.
• the eryB or eryC DNA sequences of the invention can be used to constitute hybridization probes of at least 19 nucleotides, making it possible to isolate homologous genes in macrolide producing strains using the conventional hybridization methods of nucleic acids immobilized on filters or of amplification by PCR, according to the conditions described by Sambrook et al. (1989).
• the invention relates more particularly to the above use, in which the homologous genes are the genes for the biosynthesis of oleandomycin in S. antibioticus.
• the invention describes, by way of example, the use of the sequence of the eryCIII gene as a hybridization probe for isolating homologous genes in a strain producing oleandomycin.
• the eryCIII probe used made it possible to isolate the oleGl and oleG2 genes coding for glycosyltransferases in S. antibioticus involved in the transfer of desosamine and oleandrosis to the lactone nucleus.
• the functional characterization of the oleGl and oleG2 genes made it possible to define the organization of the right part of the cluster of genes for the biosynthesis of oleandomycin in S. antibioticus.
• the subject of the invention is therefore an isolated DNA sequence represented in FIG. 22 (sequence of SEQ ID No. 15) corresponding to a region of the gene cluster for the biosynthesis of oleandomycin comprising:
• the DNA sequence above shown in FIG. 22 is a genomic DNA sequence which can be obtained, for example, from a cosmid covering the right part of the gene cluster.
• sequence of SEQ ID No. 15 is a genomic DNA sequence which can be obtained, for example, from a cosmid covering the right part of the gene cluster.
• the subject of the invention is more particularly a isolated DNA sequence represented in FIG. 22 chosen from the sequence corresponding to the ORF oleG1 (sequence of SEQ ID No. 15 from nucleotide 1437 to nucleotide 2714 coding for glycosyltransferase activity and the sequence corresponding to the ORF oleG2 (sequence of SEQ ID No. 15 from nucleotide 2722 to nucleotide 3999) coding for glycosyltransferase activity.
• a very particular subject of the invention is a DNA sequence isolated above corresponding to the ORF oleG1 (sequence of SEQ ID No. 15 from nucleotide 1437 to nucleotide 2714) coding for a deosaminyltransferase activity, as well as a sequence of DNA isolated above corresponding to ORF o! EG2 (sequence of SEQ ID No. 15 from nucleotide 2722 to nucleotide 3999) coding for an oleandrosyl transferase activity.
• sequence corresponding to the ORF oleG1 codes for a polypeptide having 426 amino acids (sequence of SEQ ID No. 17) and the sequence corresponding to the ORF oleG2 codes for a polypeptide having 426 amino acids (sequence of SEQ ID No. 18 ).
• the demonstration of the respective enzymatic activities indicated above was carried out by alteration of the corresponding gene as illustrated later in the experimental part.
• the subject of the invention is also the polypeptide coded by the DNA sequence corresponding to the oleG1 ORF and having a deosaminyltransferase activity (sequence of SEQ ID No. 17) and the polypeptide coded by the DNA sequence corresponding to the ORF o! EG2 and having oleandrosyltransferase activity (sequence of SEQ ID No. 18).
• the invention also relates to a process for the preparation of hybrid secondary metabolites in Sac. erythraea in which:
• - a DNA sequence is isolated containing at least one eryB sequence or an eryC sequence from the bio- gene cluster synthesis of erythromycin shown in FIG. 2 (sequence complementary to SEQ ID No. 1) or in FIG. 3 (sequence of SEQ ID No. 6),
• the modified strain is cultivated under conditions allowing the formation of the hybrid secondary metabolite and - the hybrid secondary metabolite is isolated.
• the modification of the DNA sequence can be carried out for example by an addition and / or by deletion of DNA sequences of at least one nucleotide, in an eryB or eryC sequence of the invention which codes for one corresponding enzymes indicated above.
• the integration of the altered sequence into the host strain can be carried out for example by the methodology of homologous recombination which can be carried out according to the scheme shown in FIG. 4 and leads to the generation of chromosomal mutants of Sac strains. erythraea which is then cultivated according to the general known methods of cell culture.
• the invention particularly relates to the above method in which the DNA sequence codes for one of the enzymes chosen from a
• the invention more particularly relates to the above method in which the alteration of the sequence results in the inactivation of at least one of the enzymes indicated above.
• the inactivation of at least one of the enzymes is demonstrated, on the one hand by the absence of production of erythromycin, on the other hand by the accumulation of precursors of erythromycin such as 6-deoxyerythronolide B, erythronolide B or 3- ⁇ -mycarosyl erythronolide B and / or the accumulation of hybrid secondary metabolites as defined above in the culture supernatants of the corresponding modified strains.
• the invention relates more particularly to the above method in which the inactivated enzyme is a dTDP-4-keto-L-6-deoxyhexose 4-reductase or in which the inactivated enzyme is a dTDP-D-6-deoxyhexose 3 , 4-dehydratase or in which the inactivated enzyme is mycarosyltransferase or in which the inactivated enzyme is dTDP-4-keto-L-6-deoxyhexose 2, 3-reductase.
• the invention also relates to the above method in which the isolated hybrid secondary metabolite is an erythromycin analog chosen from 4 "-keto-erythromycin, 4'-hydroxy-erythromycin or 3" -C-desmethyl-2 ", 3" -ene- erythromycin or in which the isolated hybrid secondary metabolite is desosa inyl erythronolide B.
• the invention also relates to a Sac strain. modified erythraea in which at least one of the enzymes chosen from a - dTDP-4-keto-L-6-deoxyhexose 2, 3-reductase,
• the invention particularly relates to the Sac strain. modified BII92 erythraea in which a dTDP-4-keto-L-6-deoxyhexose 2, 3-reductase is inactivated and producing 3 "-C desmethyl-2", 3 "-ene-erythromycin C, the Sac strain. erythraea modified BIV87 in which a dTDP-4-keto-L-6-deoxyhexose 4-reductase is inactivated and producing 4 "- keto-erythromycin, the Sac strain.
• modified CIV89 erythraea in which a dTDP-D-6-deoxyhexose 3, 4-dehydratase is inactivated and producing 4'-hydroxyerythromycin D as well as the Sac strain.
• modified erythraea BV88 in which a mycarosyltransferase is inactivated and producing desoaminyl erythronolide B. Detailed constructions of the above strains are given later in the experimental part.
• the invention also relates to a process for the preparation of oleandomycin precursors in S. antibioticus in which
• an alteration is created of the gene sequence chosen from the DNA sequence corresponding to the oleG1 ORF (sequence of SEQ ID No. 15 from nucleotide 1437 to nucleotide 2714) and the DNA sequence corresponding to the oleG2 ORF (sequence of SEQ ID No. 15 from nucleotide 2722 to nucleotide 3999) in the chromosome of a host strain and obtains a modified strain,
• the modified strain is cultivated under conditions allowing the accumulation of the oleandomycin precursors and
• the alteration of the DNA sequence can be carried out for example by interruption of the target gene in the strain S. antibioticus, for example by integration of a plasmid by the methodology of homologous recombination and leads to the generation of chromosomal mutants of the wild strain.
• the invention particularly relates to a process above in which the alteration is created in the DNA sequence corresponding to the ORF oleGl (sequence of SEQ ID No. 15 from nucleotide 1437 to nucleotide 2714) and from which it results at least the elimination of the deoaminyltransferase activity and the accumulation of the precursor of oleandomycin 8, 8a-deoxyoleandolide.
• the Sac strain. erythraea used for carrying out the invention is a spontaneous phenotypic variant called "red variant" (Hessler et al., 1997) of the wild strain Sac. erythraea NRRL 2338, the growth of which is carried out routinely either on solid R2T2 medium (R2T medium described by Weber et al., 1985 without peptone), R2T20 (Yamamoto et al., 1986) or Ml-102 on agar (Kaneda et al. , 1962), or in TSB (Oxoid) liquid medium at 30 ° C.
• R2T2 medium described by Weber et al., 1985 without peptone
• R2T20 Yamamoto et al., 1986
• Ml-102 on agar
• TSB Oxoid
• the strain E. coli XLl-blue is used for routine cloning.
• the JM10O strain is used for cloning where restriction sites such as Bell are used.
• the DH5 ⁇ .MCR strain is used for the preparation of plasmids intended to be introduced to Sac. erythraea for optimal transformation.
• the plasmids Litmus28, pUCl ⁇ and pUC19 were used routinely for subcloning.
• the vector pIJ702 (Katz et al., 1983) was obtained from the John Inocc Institute.
• the vector pIJ486 (Ward et al., 1986) was obtained from C.J. Thompson (University of Basel, Switzerland).
• the phagmid pTZl ⁇ R was obtained from Pharmacia Biotech.
• the coli-streptomyces shuttle vector pUWL218 (Wehmeier, 1995) used for chromosomal integration in Sac. erythraea was obtained from W. Piepersberg (University of Wuppertal, Germany).
• DNA ligation System kit (Amersham) was used to perform the ligations and the Plasmid Midi kit (Quiagen) or RPM kit (BiolOl Inc.) to purify the plasmid DNA.
• the preparation of the bacteriophage ⁇ DNA was carried out according to Ausubel et al. (1995) and the isolation of chromosomal DNA from Sac. erythraea according to Hopwood et al. (1985).
• the transformation of S. lividans and the isolation of the plasmids were carried out according to Hopwood et al. (1985).
• Erythronolide B and 3- ⁇ -mycarosyl erythronolide B were purified from culture extracts of the eryCI mutant (clone WHB2221 described by Dhillon et al., 1989) by chromatography on aminopropyl gel (LichroprepNH2 25-40 ⁇ , Merck) with an elution gradient by successive butyl chloride / methylene chloride mixtures (100: 0, 80:20, 50:50 and 20:80) followed by a linear elution gradient with the chloride mixture butyl / methanol ranging from 99: 1 to 90:10.
• the solution is sterilized by autoclaving for 30 minutes at 120 ° C.
• the following sterile solutions are added: 20 ml of 50% glucose; 25 ml of 1M Tris-HCl, pH 7.0; 5 ml of 0.5% KH 2 P0 4 ; 2.5 ml of NaOH IN; 50 ml of 1M CaCl 2 ; 50 ml of MgCl 2 , 6H 2 0 1M and 2 ml of "trace element" solution (Hopwood et al., 1985).
• aqueous solution glucose 5 g; commercial brown sugar 10 g; tryptone 5 g; yeast extract 2.5 g; Versene 36 mg; running water 1000 ml; final pH adjusted to 7.0 to 7.2 with KOH.
• the solution is sterilized by autoclaving for 30 minutes at 120 ° C.
• the solution is sterilized by autoclaving for 30 minutes at 120 ° C.
• the following sterile solutions are added: 10 ml of 0.5% KH 2 P0 4 ; 20 ml of 1M CaCl 2 ; 15 ml of 20% L-proline; 20 ml of 50% glucose and 1 ml of 2 lOmM CuCl.
• the solution is sterilized by autoclaving for 30 minutes at 120 ° C.
• the following sterile solutions are added: 5 ml of CaCl 2 and 20 ml of 5.3% TES.
• FIG. 1 shows the biosynthetic pathway of
• FIG. 2 shows the nucleotide sequence
• FIG. 3 shows the nucleotide sequence
• sequence of SEQ ID No. 6 of the eryAI-eryK region of the erythromycin biosynthesis gene cluster comprising the
• Figure 4 shows the gene substitution scheme by homologous recombination.
• FIG. 5A represents the organization of the left part of the cluster of genes for the biosynthesis of erythromycin in Sac. erythraea whose ORFs 1 to 9 are indicated by arrows as well as a restriction map of the plasmids pK62, pBCKl, pKB22, pBK44, pBIISB, pEco2 and pK23, generated from the XSE5.5 genomic clone.
• restriction enzymes B, BajnHI; Bc, Bell; Bg, BgrlII; E, EcoRI; K, Kpnl; M, MluI; P, PstI; S, Sacl; Sa, Sali.
• FIG. 5B represents the organization of the right part of the cluster of genes for the biosynthesis of erythromycin in Sac. erythraea whose ORFs 13 to 21 are indicated by arrows as well as a restriction map of the plasmids pBK6-12, pCN9, pNC028, pNB49, pNC062, pPSP4, pNC062X and pBABl ⁇ .
• restriction enzymes B, BamKI; Ba, Bail; Bc, Bell; C, Clal; E, EcoRI; K, Kpnl; N, Ncol; Ns, Nsil; P, PstI; Pv, PvuII; S, Sacl ; Se, Seal; Sh, SphI; Sp, Spel; X, Xjal; Xh, Xhol).
• FIG. 6A represents the construction diagram of the plasmid pBH ⁇ .
• Figure 6B shows a restriction map of plasmid pUWL218.
• FIG. 6C represents a restriction map of the plasmid pBIl ⁇ .
• FIG. 7A represents the construction diagram of the plasmid pdel88.
• FIG. 7B represents the construction diagram of the plasmid pdel88A.
• FIG. 7C represents the construction diagram of the plasmid pOBB.
• FIG. 7D represents the construction diagram and a restriction map of the plasmid pCIU ⁇ .
• FIG. 8A represents the construction diagram of the plasmid pCIl ⁇ .
• Figure 8B shows a restriction map of plasmid pORTl.
• FIG. 8C represents a restriction map of the plasmid pCH ⁇ .
• FIG. 9A represents the construction diagram of the plasmid pBIV ⁇ .
• FIG. 9B represents a restriction map of the plasmid pBIV ⁇ .
• FIG. 10A represents the construction diagram of the plasmid pBV ⁇ .
• FIG. 10B represents a restriction map of the plasmid pBV ⁇ .
• FIG. 11A represents the construction diagram of the plasmid pPSTI.
• Figure 11B shows a restriction map of the plasmid pPSTI.
• FIG. 12A represents the construction diagram of the plasmid pXhol.
• Figure 12B shows a restriction map of the plasmid pXhol.
• FIG. 13A represents the construction diagram of the plasmid pCIV ⁇ .
• FIG. 13B represents a restriction map of the plasmid pCIV ⁇ .
• FIG. 14A represents the construction diagram of the plasmid pCV ⁇ .
• FIG. 14B represents a restriction map of the plasmid pCV ⁇ .
• FIG. 15 represents the analysis by Southern blot of the mutant strains BII92, CIII68, CII62, BIV87, BV88, CIV89 and CV90, compared to the wild strain "red variant" noted Wt.
• the restriction enzyme used is indicated below each blot and the size of the bands detected in front of each blot is estimated relative to the molecular weight markers ⁇ -HindlII and ⁇ -BstEII (not detectable by auto-radiography ).
• FIG. 16 represents the analysis by PCR of the mutant strains BII91, CIII68, CII62, BIV87, BV88, CIV89 and CV90, compared with the wild strain "red variant" denoted Wt and with the plasmids pBH ⁇ , pCIU ⁇ , pCH ⁇ , pBIV ⁇ , pBV ⁇ , PCIV ⁇ and pCV ⁇ used respectively to obtain the mutant by homologous recombination.
• the band sizes detected by staining with ethydium bromide are estimated relative to the molecular weight markers X174-HaeIII or ⁇ -BstEII.
• 17 represents the TLC analysis of the metabolites produced by the mutant strains BII92, CIII68, CII62, BIV87, BV88, CIV89 and CV90, compared to the standard products erythromycin A (Er A), erythronolide B and 3- ⁇ -mycarosyl erythronolide B (SEM).
• Er A erythromycin A
• SEM 3- ⁇ -mycarosyl erythronolide B
• FIG. 18 represents the analysis by SDS-PAGE of the purification of the protein EryCIII successively after extraction with urea 7M (line 2), Q Sepharose chromatography (line 3), Superdex chromatography (line 4), Q source chromatography ( lane 6) with standard molecular weight markers (lanes 1 and 5);
• FIG. 19 represents the analysis by CMM of the test of biological activity of the protein EryCIII, by incubation with d-TDP-D-desosamine (lane 2) or with d-TDP-D-desosamine and 3- ⁇ -mycarosyl erythronolide B (SEM) (line 3) compared to SEM control (line 1) and erythromycin A control (line 4).
• SEM 3- ⁇ -mycarosyl erythronolide B
• FIG. 20 represents the location of the six cosmids (cosAB35, cosAB76, COSAB87, COSAB67, cosAB63 and cosABôl) covering the entire cluster of genes for oleanomomycin biosynthesis.
• the BaraHI restriction fragments (denoted B) hybridizing with the probes denoted str M, D, E and the BamHI fragments (3.5 kb and 2.7 kb) hybridizing with the eryCIII probe are shown.
• FIG. 21 represents the organization of the right part of the cluster of genes for the biosynthesis of oleandromycin in S. antibioticus whose different ORFs (noted olePl, oleGl, oleG2, oleM, oleY, oleP and oleB) are indicated by arrows as well as a restriction map of plasmid pC035-S and the insert of plasmid pC03 generated from pC035 -S.
• the double arrow indicates the insert corresponding to the sequence of FIG. 22 (abbreviations of restriction enzymes: B, BamHI; Bg, Bgl I; K, Kpn; S, Sacl; Sh, SphI; the star indicates that it is not a single site).
• FIG. 22 represents the nucleotide sequence (sequence of SEQ ID No. 15) of the region covering the oleP1, oleG1, oleG2, oleM and oleY genes of oleanomomycin biosynthesis and their deduced protein sequences.
• EXAMPLE 1 Cloning and sequencing of the eryG-eryAIII region of the gene cluster for the biosynthesis of erythromycin. A fragment of genomic DNA from Sac.
• erythraea NRRL 2338 having> 20 kb downstream of the ermE gene covering in particular ORFs 3 to 9 and corresponding to the clone XSE5.5 as well as the nucleotide sequence of a 4.5 kb fragment corresponding to the region of the ery cluster between 3 , 7 kb and 8.0 kb from the 3 'end of the erm ⁇ gene and comprising ORFs 3, 4, 5 and 6 were described by Haydock et al. (1991).
• the plasmids pK62 and pK66 were directly constructed by subcloning the 5.8 kb Kpnl fragment into pUC19, the plasmid pK66 corresponding to the same Kpnl fragment subcloned with an inverted orientation of the insert relative to the vector.
• the plasmid pKB22 containing an insert of 2.9 kb was then derived from the plasmid pK66 by excision of the BamHI-Bg II fragment (2.9 kb) covering 1ORF8 as well as part of ORFs 7 and 9 by digestion with the enzymes of BamHI and Bgl II restriction.
• the plasmid pKB44 containing a 2.9 kb insert was obtained from the plasmid pK62 by excision of the BajnHI-BglII fragment (2.9 kb) covering the eryG gene corresponding to ORFs 5 and 6 and the gene eryF corresponding to 1 '0RF4.
• Plasmid pBIISB was derived from plasmid pBK44 by subcloning into pUC19 the 600 bp SalI fragment obtained from plasmid pBK44 digested with the restriction enzyme Sali (FIG. 5A).
• the plasmid pEco2 was directly constructed by subcloning the BcoRI fragment (2.2 kb) in pUC19.
• the subclones pKB22, pBK44, pBIISB and pEco2 thus obtained were then sequenced.
• the analysis was carried out on plasmid DNA samples, previously purified on a column of Quiagen 100 (Quiagen), on the sequencer automatic ABI prism 377.
• nucleotide sequences obtained made it possible to establish the nucleotide sequence of 3412 bp of FIG. 2 (direct and complementary sequence of SEQ ID No. 1) in which three ORFs (7, 8 and 9) were identified respectively from nucleotide 8957 to nucleotide 7959, from nucleotide 10219 to nucleotide 8957 and from nucleotide 11315 to nucleotide 10233 (numbered in FIG.
• EXAMPLE 2 Construction of the plasmid pBH ⁇ .
• the 598 bp fragment Bc I-BamHI was deleted in the plasmid pK62 obtained in Example 1 by digestion with the enzymes Bell and BamHI.
• the resulting plasmid pBCKl was then digested with the restriction enzymes M uI and Bg / III so as to delete a fragment having 853 bp inside 1ORF7 from nucleotide 8011 to nucleotide 8863 of the sequence of FIG. 2. After filling ends using the Klenow fragment of DNA polymerase I, the plasmid containing the deletion, was religated and transformed into E. coli XLl-blue.
• the plasmid pBH ⁇ was then transferred to the strain
• E. coli DH ⁇ MRC then used to transform Sac. erythraea.
• EXAMPLE 3 Construction of a Sac strain. erythraea ery BII ⁇ (BII92).
• the protoplast preparation was carried out according to the method described by Weber and Losick (1988), using PEG 3350 (Sigma) instead of PEG 1000 and a modified P buffer. (called PT) containing MgCl 2 , 6H 2 0 28 M and without P0 4 H 2 K instead of the buffers P, L or T described, according to the following operating conditions:
• Sac cells (at least 10 8 spores).
• erythraea "red variant” (a sample of which was deposited at the National Collection of Cultures of Microorganisms (CNCM) INSTITUT PASTEUR, 25, Rue du Dondel Roux 75724 PARIS CEDEX 15 FRANCE, July 16, 1997 under the number 1-1902) were grown in 50 ml of TBS medium for 3 to 5 days at 30 ° C, then washed in 10.3% sucrose.
• the cells were resuspended in 50 ml of PT buffer containing 2 to 5 mg / ml of lysozyme (Sigma), then incubated at 30 ° C for 1 to 2 hours by disaggregating the mycelium clusters every 15 minutes until conversion of at least 50% of the mycelium into protoplasts.
• the protoplasts were washed with 50 ml of PT buffer, resuspended in 12.5 to 25 ml of the same buffer, frozen slowly and then stored at -80 ° C in 200 l aliquots.
• plasmid DNA pBH ⁇ prepared in Example 2 from the strain E. coli DH5 ⁇ MRC were dissolved in 5 to 10 ⁇ l of TE buffer (Tris HC1 10 mM pH 7.5, EDTA 1 mM) then deposited on the wall of the inclined tube to which was then added 0.5 ml of a solution of PEG 3350 in the PT buffer prepared extemporaneously from a 50% aqueous solution which is diluted to half in the 2 x PT buffer.
• TE buffer Tris HC1 10 mM pH 7.5, EDTA 1 mM
• the selection of the integrants corresponding to the first recombination event was carried out by replicating the sporulated dishes using velvet or by spreading a suspension of the spores on R2T2 dishes containing thiostrepton then incubation at 32 ° C. , which allows the growth of clones of potential integrants.
• protoplasts were prepared from the cells as indicated above, so as to drive out the plasmid. The protoplasts were then spread on R2T2 dishes so as to obtain individualized colonies whose sensitivity to thiostrepton was determined by replication on R2T2 dishes containing thiostrepton.
• the phenotype of the thiostrepton-sensitive colonies is of the wild type or of the mutated type carrying the deletion.
• the selection of the mutants having the phenotype ery ⁇ was carried out by antibiogram on the strain B. pumilu ⁇ ATCC 14884 sensitive to erythromycin.
• the B. pumilus strain was used as an indicator strain to evaluate the production of erythromycin in bioassays by antibiogram. The colonies were spread with a platinum loop on R2T2 dishes, then incubated for 3 to 4 days at 30 ° C.
• Agar zones where the mutant has grown to confluence have then taken with a cookie cutter and then placed on A-Merck boxes covered with a 4 ml overlay of 0.5 x A Merck (Antibiotic agar No. 1 Merck) inoculated with a suspension of B. pumilus spores , then incubated overnight at 37 ° C.
• a Merck Antibiotic agar No. 1 Merck
• PCR analysis a 100 ⁇ l sample of a 3-day culture in TSB medium was centrifuged. The pellet obtained was resuspended in 10 ⁇ l of TSB medium, then used for amplification in the genA p PCR System 9600 device (Perkin El er Cetus). After heating of the sample for 3 min at 94 ° C, the following amplification conditions were used: 94 ° C, 1 min; 55 ° C, 1 min; 72 ° C, 3 min; 30 cycles; Ampli Taq polymerase (Perkin Elmer) in the presence of 10% dimethylsulfoxide (v / v) followed by an elongation of 3 min at 72 ° C.
• genA p PCR System 9600 device Perkin El er Cetus
• the amplification was carried out using the oligonucleotide B2S above and the oligonucleotide having the following sequence B2-R GCCGCTCGGCACGGTGAACTTCA (SEQ ID No. 31) corresponding to the sequence of the complementary strand of the DNA region located in the position 8873 at position 8892 of the sequence of FIG. 2 to which three nucleotides have been added at the 5 ′ end and making it possible to frame, by PCR amplification, the region carrying the internal deletion at 1 ORF7.
• the recombinant strain thus obtained designated BII92, was then cultured to identify the metabolites produced by the strain.
• EXAMPLE 4 Fermentation of the BII92 strain and identification of the secondary metabolites produced.
• Extracts of culture broth of the strain were analyzed by thin layer chromatography (cmc) with erythromycin A, erythronolide B and 3- ⁇ -mycarosyl erythronolide B as standards.
• PPE medium Solulys L-Corn steep liquor (Ro
• the culture supernatant was then extracted at pH 9-10 with ethyl acetate.
• the organic phases were dried over SO 4 Mg, brought to dryness under reduced pressure and then analyzed by ccm on silica gel 60 F254 (Merck) [dichloroethane / methanol (90:10, v / v) or isopropyl ether / methanol / 25% NH 4 OH (75: 35: 2, v / v)].
• the analysis was carried out by ccm on grafted silica gel plates of NH 2 F254 type (Merk) [butyl chloride / methanol (90:10, v / v)].
• the chemical development of the plates was carried out by spraying with a solution of p-anisaldehyde-sulfuric acid 98% -ethanol (1: 1: 9, v / v), followed by heating for a few minutes at 80 ° C.
• the potential antibiotic activities were analyzed by direct bioautography of the ccm plates on agar seeded with B. pumilus ATCC 14884.
• the results obtained by chemical revelation show that the strain BII92 preferentially accumulates erythronolide B as expected from a mutant eryB.
• RP-HPLC reverse-phase high performance liquid chromatography
• the RP-HPLC was carried out on a column (250 x 4.6 mm) of Kro asil C18 5 ⁇ using the acetonitrile / methanol / 0.065 M ammonium acetate pH 6.7 mixture as the mobile phase (350: 150: 500, v / v) on a Waters chromatograph equipped with a Finningan TSQ 7000 mass spectrometer.
• Ml gives a parent peak at m / z 704 and fragmentation products at m / z 576 and m / z 158.
• deaminerythronolide A indicates that the difference in m / z of 30 compared to erythromycin A (m / z 734) or 16 compared to erythromycin C (m / z 576) z 720) is carried by the sugar residue neutral.
• the proposed structure for Ml is 3 "-C desmethyl- 2", 3 "-ene-erythromycin C.
• - M2 gives a parent peak at m / z 706 and fragmentation products at m / z 576 and m / z 158.
• the presence of deaminerythronolide A indicates that the difference in m / z of 28 compared to 1 erythromycin A (m / z 734) or 14 compared to 1 erythromycin C (m / z 720) is carried by the neutral sugar residue.
• the proposed structure for M2 is 3 "-C ethyl ethyl erythromycin C.
• - M3 gives a parent peak at m / z 690 and fragmentation products at m / z 560 and m / z 158.
• deaminyl erythronolide B (m / z 560) indicates that the difference in m / z of 28 compared to erythromycin B (m / z 718) or of 14 compared to erythromycin D (m / z 704) is carried by the sugar residue neutral.
• the proposed structure for M3 is 3 "-C desmethyl- erythromycin D.
• M4 gives a parent peak at m / z 720 and fragmentation products at m / z 576 and m / z 158.
• the profile is identical to that of erythromycin C (m / z 720) with the presence of desosaminylerythronolide A (m / z 576) and the loss of the amino sugar residue (m / z 158), but the metabolite M4 does not have the same retention time in RP-HPLC as erythromycin.
• the proposed structure for M4 is 3 "-C desmethyl- erythromycin A.
• SM-SM minor metabolite Ml having an unsaturated neutral sugar (3" -C desmethyl-2 ", 3" -ene-erythromycin C) indicates that the eryBII gene codes for dTDP-4-keto-L-6-deoxyhexose 2,3-reductase in the biosynthetic pathway for dTDP-mycarose.
• the BII92 strain was deposited at the National Collection of Cultures of Microorganisms (CNCM) INSTITUT PASTEUR, 25, Rue du Dondel Roux 75724 PARIS CEDEX 15 FRANCE, on July 16, 1997 under the number 1-1903.
• a Sali deletion of 663 bp was introduced into 1ORF8 from nucleotide 9384 to nucleotide 10046 of the sequence of FIG. 2 by subcloning in the plasmid pUC19 the two SalI fragments (a: 794 bp and b: 631 bp shown in FIG. 5A) isolated from the plasmid pBK44 obtained in Example 1 to generate the plasmid pdel88 (FIG. 7A). The presence of the 663 bp deletion was confirmed by sequencing. The plasmid pdel88 was then subjected to two additional subcloning so as to widen the chromosomal regions which can be used for homologous recombination on both sides of the deletion site.
• the SacI fragment (450 bp) of the plasmid pdel88 was first replaced by the SacI fragment (1.1 kb) of the plasmid pEco2 obtained in Example 1 to generate the plasmid pdel88A (FIG. 7B). Then the EcoRI fragment (1.5 kb) carrying the deletion in 1ORF8 was isolated from the plasmid pdel ⁇ A and used to replace the BcoRI fragment (1.66 kb) carrying the intact ORF in the plasmid pOBB.
• the plasmid pOBB represented in FIG.
• a strain in which the eryCIII gene carries an internal deletion such as that introduced into the plasmid pCIU ⁇ obtained in Example 5 was prepared by transformation of the sac protoplasts. erythraea with the plasmid pCIU ⁇ .
• the protoplast preparation, the integration process and the selection of the mutants having the ery ⁇ phenotype were carried out as in Example 3.
• the presence of the expected deletion in the chromosome (663 bp deletion from nucleotide 9384 to nucleotide 10046 of the sequence of FIG. 2) was confirmed by genomic analysis by Southern blot as well as by PCR amplification according to the conditions described in Example 3.
• the PCR amplification was carried out using the oligonucleotide C3-S above and the oligonucleotide having the following sequence
• the recombinant strain thus obtained designated CIII68, was then cultured to identify the metabolites produced by the strain.
• EXAMPLE 7 Fermentation of the CIII68 strain and identification of the secondary metabolites produced.
• the ccm results show that the CII68 strain preferentially accumulates 3- ⁇ -mycarosyl erythronolide B as well as small amounts of erythronolide B as expected from an eryC mutant.
• the eryCIII sequence has strong homology with other putative glycosyltransferases such as DauH (43% identity at the protein level) and DnrS (47% identity) involved in the biosynthesis of daunorubicin in S. peucetiu ⁇ (Otten et al ., 1995) and in Streptomyces sp C5 (Dickens et al., 1996) as well as TylM2 (50% identity) involved in the transfer of mycaminosis to tylactone in the pathway of tylosin biosynthesis in S. fradiae (Gandecha et al., 1997).
• DauH 33% identity at the protein level
• DnrS 47% identity
• TylM2 50% identity
• Plasmid pK23 (FIG. 5A) was obtained by subcloning into pUC19 the 10 kb Kpn fragment isolated from the DNA of clone XSE5.5 digested with the restriction enzyme Kpn1.
• the shuttle vector pORT1 shown in FIG. 8B, was obtained by subcloning the PstI fragment of 4kb isolated by digestion of the plasmid pIJ486 with the restriction enzyme PstI including the thiostrepton resistance gene and the replicon Streptomyces, in the PstI site of pUC19.
• erythraea eryCIl ⁇ (CII62).
• a strain in which the eryCII gene carries an internal deletion such as that introduced into the plasmid pCH ⁇ obtained in Example 8 was prepared by transformation of the sac protoplasts. erythraea with the plasmid pCIl ⁇ . The preparation of the protoplasts, the integration process and the selection of the mutants having the ery ⁇ phenotype were carried out as in Example 3.
• PCR amplification analysis detected a band of approximately 760 bp in the wild strain and a band of approximately 460 bp in the mutant CII62 in an identical manner to the signal obtained with pCIl ⁇ .
• the results shown in FIG. 16 confirm that the deletion of approximately 400 bp detected by the Southern analysis is identical to that carried by the plasmid pCH ⁇ (304 bp).
• the recombinant strain thus obtained designated CII62, was then cultured to identify the metabolites produced by the strain.
• EXAMPLE 10 Fermentation of the CII62 strain and identification of the secondary metabolites produced.
• the ccm results show that the CII62 strain preferentially accumulates 3- ⁇ -mycarosyl erythronolide B as well as small amounts of erythronolide B as expected from an eryC mutant.
• the eryCII sequence has a strong homology with genes involved in the biosynthesis pathways of daunosamine (DnrQ, 38% identity at protein level, Otten et al., 1995) and mycaminose (protein encoded by ORFl *, 40% identity at the protein level, Gandecha et al., 1997) which also need to transfer a keto group in position 3 from an adjacent carbon.
• EXAMPLE 11 Cloning and Sequencing of the eryAI-eryK Region of the Gene Cluster for Erythromycin Biosynthesis.
• Cosmids containing the eryAI-eryK region of the ery gene cluster such as the cosmid Cos6B, were isolated by screening of a Sac genomic DNA library.
• erythraea in the cosmid vector pWE15 (Stratagene) using as probe a DNA fragment of 13.2 kb comprising the entire eryAI gene and corresponding to the region of DNA between the Wcol site located at position 44382 of the sequence in Figure 3 and the Wcol site located at position 392 of the sequence X62569 (Bevitt et al., 1992).
• the probe was prepared as follows: First, the 13.2 kb Ncol fragment was isolated from the plasmid pBK25 described by Bevitt et al., 1992 and subcloned into the Smal site of pUC18 after filling the Ncol ends with the Klenow fragment. From the plasmid pNC012 thus generated, the 13.2 kb fragment was isolated by digestion with the restriction enzyme Ncol.
• the cosmid cos6B thus obtained was digested with the restriction enzyme Ncol and the resulting fragments of 2.8 kb and 6.1 kb were cloned into the Ncol site of the vector Litmus28 generating the plasmids pNC028 and pNC062 respectively shown in the figure. 5B.
• Plasmid pNC028 was sequenced by generation of subclones using exonuclease III according to the protocol of the supplier of the Erase-a-Base Kit (Promega) by digesting with the restriction enzymes Sac / Xba and Nsil / BamBI respectively for the reverse direction. The sequence was completed using as primers the synthetic oligonucleotides having the following sequences 644 GATCACGCTCTTCGAGCGGCAG (SEQ ID N ° 36)
• templates were generated by sonication of the DNA according to Bankier et al. (1987) using pUC18 as a vector.
• the sequence was completed using as primers the synthetic oligonucleotides having the following sequences:
• the Ncol junctions were sequenced using as DNA the cosmid cos6B DNA obtained above, the regions covering the Ncol sites were sequenced using the primers having the sequences 644 and 645 indicated above.
• plasmid pBK6-12 shown in FIG. 5B was first generated by subcloning into phagmide pTZ18R the 4.5 kb Kpn1 fragment isolated from the plasmid pBK25 described by Bevitt et al., 1992.
• the subclone pCN9 was then generated by subcloning the 0.9 kb Clal-Ncol fragment isolated from the plasmid pBK6-12 in the Smal site of pUC19, after filling the ends with using the Klenow fragment.
• the plasmid pCN9 thus obtained (FIG. 5B) was sequenced. Arrays were generated by DNA sonication according to Bankier et al. (1987) using pUC18 as a vector. The sequence was completed using as an primer the oligonucleotide having the following sequence: 675 CGACGAGGTCGTGCATCAG (SEQ ID No. 44).
• DNA sequencing is carried out by Sanger's method (1977) using an automated sequencer on double stranded DNA templates with the Applied Biosystem 373 A sequencer.
• the assembly of the sequence data was carried out with the software SAP (Staden, 19 ⁇ 4).
• the sequences were analyzed using GCG software (Devereux, 1984).
• nucleotide sequences obtained made it possible to establish the nucleotide sequence of ⁇ 160 bp of FIG. 3 (sequence of SEQ ID No. 6) in which seven ORFs (13-19) were identified respectively from nucleotide 43641 to nucleotide 44806, from nucleotide 44809 at nucleotide 46053, from nucleotide 46109 to nucleotide 46819, from nucleotide 46907 to nucleotide 48436, from nucleotide 48436 to nucleotide 4963 ⁇ , from nucleotide 49679 to nucleotide 51145 and from nucleotide 51177 to nucleotide 51755 (numbered in Figure 3 from the site BamHI located at the 5 'end of the erraE gene) (respectively sequence of SEQ ID No.
• the plasmid pNC062 comprising the coding sequence for ORFs 17, l ⁇ and 19 as well as for part of 1ORF16 under the number 1-1900.
• EXAMPLE 12 Construction of the plasmid pBIV ⁇ .
• LORF13 being translationally coupled to 1ORF14 located downstream, a phase deletion had to be introduced.
• the plasmid pPSP4 (FIG. 5B) was first constructed by subcloning the PvuII-Spel fragment (2.7 kb) isolated from the plasmid pBK6-12 obtained in Example 11 and the Spel-PstI fragment (1 , 6 kb) isolated from the plasmid pNC02 ⁇ obtained in Example 11 in the vector pUC19 previously digested using the restriction enzymes SmaI and PstI.
• the plasmid pl9BIV ⁇ was generated by deleting the BclI-Ncol fragment of 510 bp internal to 1ORF13 and by replacing it with 45 bp coming from a synthetic adapter of 54 bp.
• This adapter was generated by pairing the 2 complementary oligonucleotides having the following sequences SEQ A
• the two oligonucleotides were brought to a final concentration of ⁇ ⁇ M in the 50 mM NaCl hybridization buffer, Tris, 20 mM HCl pH 7.4, MgCl 2 , 6H 2 0 2 mM, heated for 5 min at 100 ° C then slowly cooled to room temperature. After digestion with the restriction enzymes Ncol and Bell, a ligation was carried out in the plasmid pPSP4 from which the BclI-Ncol fragment of 510 bp had previously been eliminated.
• a strain in which the eryBIV gene carries an internal deletion such as that introduced into the plasmid pBIV ⁇ obtained in Example 12 was prepared by transformation of the sac protoplasts. erythraea with the plasmid pBIV ⁇ . The protoplast preparation, the integration process and the selection of the mutants having the ery ⁇ phenotype were carried out as in Example 3.
• the PCR amplification was carried out using the oligonucleotide having the following sequence B4-S CAATATAGGAAGGATCAAGAGGTTGAC (SEQ ID No. 48) corresponding to the DNA region located from position 43652 to position 43678 of the sequence of the figure 3 and the oligonucleotide B4-R having the sequence indicated above, making it possible to frame by PCR amplification the region carrying the internal deletion at 1ORF13.
• Analysis by PCR amplification made it possible to detect a band of approximately 1 kb in the wild-type strain and a band of approximately 500 bp in the mutant BIV87 in an identical manner to the signal obtained with the plasmid pBIV ⁇ 7 (FIG. 16).
• the recombinant strain thus obtained designated BIV67, was then cultured to identify the metabolites produced by the strain.
• EXAMPLE 14 Fermentation of the BIV87 strain and identification of the secondary metabolites produced.
• the culture of the BIV87 strain and the ccm analyzes were carried out according to the conditions indicated in Example 4.
• the ccm results show that the BIV87 strain preferentially accumulates erythronolide B as expected from a mutant eryB.
• Mass spectrum results indicate that modified forms of erythromycin A, B, C and D have been produced. One major metabolite and 3 minor metabolites were detected.
• the major metabolite M5 gives a parent peak at m / z 702 with dehydration and fragmentation products at m / z 684, m / z 560 and m / z 158 and corresponds to the elimination of 2 hydrogen atoms in l erythromycin D (m / z 704, m / z 686).
• the presence of desosaminyl erythronolide B fragment m / z at 560 indicates that the difference in mass is carried by the neutral sugar.
• the proposed structure for this metabolite is 4 "-keto erythromycin D.
• the minor metabolites also give a profile with a difference of 2 in the m / z values respectively:
• the proposed structures are respectively 4 "-keto erythromycin C for M6, 4" -keto erythromycin A for M7 and 4 "-keto erythromycin B for M8.
• a 726 bp deletion was generated in 1ORF14 of nucleotide 44963 to nucleotide 45688 of the sequence of FIG. 3 by ligation of the Bcll-Kpnl fragment (1.1 kb) isolated from the plasmid pBK6-12, obtained in Example 11, to the Kpnl-BamHI fragment (1.1 kb) isolated from the plasmid pNC028, obtained in Example 11, in the plasmid pUWL218 previously digested with the restriction enzyme BamHI.
• the integration plasmid pBV ⁇ thus obtained (FIG. 10B) was then transferred to the E. coli strain DH ⁇ MRC, then used to transform Sac. erythraea.
• EXAMPLE 16 Construction of a Sac strain. erythraea eryBV A (BV88).
• a strain in which the eryBV gene carries an internal deletion such as that introduced into the plasmid pBV ⁇ obtained in Example 15 was prepared by transformation of the sac protoplasts. erythraea with the plasmid pBV ⁇ . The protoplast preparation, the integration process and the selection of the mutants having the ery ⁇ phenotype were carried out as in Example 3.
• B5-R TCCGGAGGTGTGCTGTCGGACGGACTTGTCGGTCGGAAA (SEQ ID No. 49) corresponding to the complementary strand of the DNA region located from position 46060 to position 46098 of the sequence of FIG. 3, a band of 2.7 kb from the strain wild and a 2.0 kb band from the mutant BV88 were detected.
• the results shown in FIG. 15 indicate in the mutant the presence of a deletion of approximately 700 bp in this region of the chromosome.
• the PCR amplification was carried out using the oligonucleotide having the following sequence: B5-S AGGAGCACTAGTGCGGGTACTGCTGACGTCCTT (SEQ ID No.
• the recombinant strain thus obtained designated BV88, was then cultured to identify the metabolites produced by the strain.
• the culture of the BV88 strain and the ccm analyzes were carried out according to the conditions indicated in Example 4.
• the ccm results show that the BV88 strain preferentially accumulates erythronolide B as expected from an eryB mutant.
• EXAMPLE 18 Construction of a plasmid pCVl ⁇ (pPSTI).
• An integration plasmid called pPSTI and carrying a deletion in the eryCVI gene coding for 1ORF15, was constructed according to the diagram of FIG. 11A as follows:
• the plasmid pNB49 was generated by treatment with exonuclease III of the plasmid pNC028 obtained in Example 11 previously digested with the restriction enzymes Nsil and BamHI.
• the plasmid pNB49 (FIG. 5B) containing the nucleotides 44382 to 46562 of the sequence of FIG. 3, was then digested using the restriction enzyme PstI then treated with the Mung Bean nuclease (NE Biolabs) as described by Sambrook et al. (1989). After religion and transformation in E. coli XLl-Blue, ampicillin resistant colonies were selected by restriction analysis with the enzyme PstI.
• the loss of the PstI site was confirmed by sequencing a clone using the reverse M13 primer and the deletion of nucleotide 46364 from the sequence of FIG. 3 was observed creating a phase change in 1ORF15 in the plasmid pNB49 ⁇ Pst thus generated .
• the plasmid pIJ702 digested with the restriction enzyme Bg II was then ligated to the BglII site of the plasmid pNB49 ⁇ Pst generating the plasmid pPSTI.
• the orientation of pIJ702 in pPSTI was confirmed by the presence of a DNA fragment having 0.9 kb after digestion with the restriction enzyme Sphl.
• the integration plasmid pPSTI (FIG. 11B) thus obtained was transferred to the strain E. coli DH5 ⁇ MRC, then used to transform Sac. erythraea.
• EXAMPLE 19 Construction of a Sac strain. erythraea eryCVlA (PstlO).
• a strain in which the eryCVI gene carries an internal deletion such as that introduced into the plasmid pPSTI obtained in Example 18 was prepared by transformation of the sac protoplasts. erythraea with the plasmid pPSTI.
• B. subtilis sensitive to erythromycin instead of a B. pumilus strain as an indicator strain.
• B. subtilis ATCC 6633 was used to assess production of erythromycin in biological tests on agar dishes in Ml-102 medium inoculated with the mutant to be analyzed and incubated for 3 days at 30 ° C. Agar areas covered with bacteria were then taken with a cookie cutter and then placed on 2 x TY dishes covered with an overlay of 5 ml of agar in TY medium containing 200 ⁇ l of a B. subtilis culture. ATCC 6633, then incubated overnight at 37 ° C.
• the oligonucleotide 14-1 was designed so as to introduce a BamHI site and an NdeI site upstream of the sequence corresponding to the DNA region located from position 44811 to position 44833 of the sequence of FIG. 3.
• the oligonucleotide 14-2 was designed to introduce a BglII site downstream of the sequence corresponding to the complementary strand of the DNA region located from position 46027 to position 46053 of the sequence of FIG. 3. Chromosomal DNA previously digested with restriction enzymes Clal and PstI showed the expected bands of 4 kb and 7 kb from the integrant while the wild strain presented the expected 3 kb band.
• the recombinant strain thus obtained designated Pst10, was then cultured to identify the metabolites produced.
• EXAMPLE 20 Fermentation of the Pst10 strain and identification of the secondary metabolites produced.
• the Pst10 strain was cultivated in the sucrose-succinate medium described by Caffrey et al. (1992) for 3 days at 30 ° C. The culture supernatant was then extracted at pH 9 with ethyl acetate. The organic phases obtained were dried over SO 4 Mg 2 and then brought to dryness under reduced pressure. The residue was dissolved in acetonitrile-water (1: 1, v / v), then was analyzed by mass spectrometry on a BioQ (Micromass, Manchester, UK) or Finningan LCQ (Finningag, CA) spectrometer.
• BioQ Micromass, Manchester, UK
• Finningan LCQ Finningan LCQ
• erythomycin A (m / z 734 and m / z 716) was not observed but the presence of erythronolide B (MK +: m / z 441 and MNa +: m / z 425) as well as 3- ⁇ -mycarosyl Erythronolide B (MK +: m / z 585 and MNa +: m / z 569) demonstrated characterizes the Pst10 strain as an eryC mutant.
• the eryCVI sequence has strong homology with other methyltransferases such as SnoX involved in the biosynthesis of nogalamycin in S.
• EXAMPLE 21 Construction of a plasmid pBVl ⁇ (pXhol).
• FIG. 12A An integration plasmid, called pXhol and carrying a deletion in the eryBVI gene coding for 1ORF16, was constructed according to the diagram of FIG. 12A as follows:
• the Ncol-Xhol fragment (3.1 kb) of the plasmid pNC062 obtained in Example 11 and containing the nucleotides 47142 to 50254 of the sequence of FIG. 3 was subcloned in the Ncol and Xhol sites of the plasmid Litmus 28.
• the plasmid pNC062X (FIG. 5B) thus generated was digested with the restriction enzyme PstI and then treated with DNA polymerase T4 (Boehringer Mannheim). After religion and transformation in E. coli XLl-Blue, the loss of the PstI site at nucleotide 47397 of the sequence of FIG.
• a strain in which the eryBVI gene carries an internal deletion such as that introduced into the plasmid pXhol obtained in example 21 was prepared by transformation of the sac protoplasts. erythraea with the plasmid pXhol.
• the recombinant strain thus obtained and designated Xho91 was then cultured to identify the metabolites produced.
• the culture of the Xho91 strain and the analysis of the culture supernatant by mass spectrometry were carried out according to the conditions described in Example 20.
• erythomycin A (m / z 734 and m / z 716) was not observed but the presence of a majority amount of erythronolide B (MK + : m / z 441; MNa + : m / z 425; MH 2 0 H + : m / z 385) as well as the presence of desosaminyl erythronolide B (m / z 560) demonstrated characterize the strain Pst10 as a mutant eryB.
• the mass spectrometry results were confirmed by high resolution mass spectrometry on a Brucker FT-ICR spectrometer (Brucker, FRG).
• the eryBVI sequence has a strong homology with DnmT involved in the biosynthesis of daunorubicin in S. peucetius (accession number EMBL U77891) (43.9% identity at the protein level).
• EXAMPLE 24 construction of the plasmid pCIV ⁇ .
• the plasmid pNC062 obtained in Example 11 was digested with using the restriction enzymes Bail and BcII so as to eliminate a fragment having 949 bp inside 1ORF17 from nucleotide 48650 to nucleotide 49598 of the sequence of FIG. 3. After filling the ends with the Klenow fragment of DNA polymerase I, the plasmid was religated and transformed into E. coli XLl-blue. From the plasmid pBCB17 thus generated, the 2.68 kb fragment carrying the deletion was isolated by digestion using the enzymes Xbal and SphI, then subcloned into the corresponding sites of the plasmid pUWL218.
• EXAMPLE 25 Construction of a Sac strain. erythraea eryCIV A (CIV89).
• the PCR amplification was carried out using the oligonucleotide having the following sequence:
• the recombinant strain thus obtained designated CIV89, was then cultured to identify the metabolites produced by the strain.
• the CIV89 strain was deposited at the National Collection of Cultures of Microorganisms (CNCM) INSTITUT PASTEUR, 25, Rue du Dondel Roux 75724 PARIS CEDEX 15 FRANCE, on July 16, 1997 under the number 1-1905.
• EXAMPLE 27 Construction of the plasmid pCV ⁇ .
• FIG. 14A An integration plasmid, called pCV ⁇ and carrying a deletion in the eryCV gene coding for 1ORF18, was constructed according to the diagram of FIG. 14A as follows:
• a strain in which the eryCV gene carries an internal deletion such as that introduced into the plasmid pCV ⁇ obtained in Example 27 was prepared by transformation of the sac protoplasts. erythraea with the plasmid pCV ⁇ . The protoplast preparation, the integration process and the selection of the mutants having the ery ⁇ phenotype were carried out as in Example 3.
• the PCR amplification was carried out using the oligonucleotide having the following sequence: C5-S TTCGCTCCCCGATGAACACAACTCGTA (SEQ ID No. 56) corresponding to the DNA region located at position 49668 at position 49694 of the sequence of the FIG. 3 and the oligonucleotide C5-R having the sequence indicated above, making it possible to frame by PCR amplification the region carrying the internal deletion at 1ORF18.
• Analysis by PCR amplification made it possible to detect a band of approximately 1.6 kb in the wild-type strain and a band of approximately 500 bp in the mutant CV90 in an identical manner to the signal obtained with the plasmid pCV ⁇ .
• the results shown in FIG. 16 confirm that the deletion of approximately 1.1 kb detected by the Southern analysis is identical to that carried by the plasmid PCV ⁇ (1044 bp).
• the recombinant strain thus obtained designated CV90, was then cultured to identify the metabolites produced by the strain.
• EXAMPLE 29 Fermentation of the CV90 strain and identification of the secondary metabolites produced.
• the culture of the CV90 strain and the analyzes by ccm were carried out according to the conditions indicated in Example 4.
• the results of ccm show that the CV90 strain preferentially accumulates 3- ⁇ -mycarosyl erythronolide B as well as Erythronolide B as expected from an eryC mutant.
• sequence of residues 38-50 Val Thr Gly Ala Gly Asp Gly Asp Ala Asp Val Gin Ala (SEQ ID No 61) of the protein coded by eryCV (sequence of SEQ ID No 11) is close to the sequence NAD + binding consensus described by Wierenga et al., 1985 and by Scrutton et al., 1990.
• EXAMPLE 30 Overexpression of the eryCIII gene product in E. coli.
• the heterologous expression of the Sac eryCIII gene product. erythraea corresponding to 1ORF8 described in Example 1 and coding for the deosaminyltransferase activity identified in Example 7 was carried out using E. coli as the host strain. The protein thus produced in the form of inclusion bodies was then purified and its enzymatic activity determined in vitro. 1) Expression of the EryCIII protein in E. coli
• Expression was achieved using the pET11a vector (Stratagene) for cloning and expression of recombinant proteins in E. coli under the control of the promoter of RNA polymerase of bacteriophage T7.
• the eryCIII gene was amplified from the plasmid pK62 described in Example 1 in the manner next :
• the PCR amplification was carried out using the Native Pfu polymerase (Stratagene) and as primers the oligonucleotide A homologous to the strand coding for the eryCIII gene having the sequence
• a GAAGGAGATATACATATGCGCGTCGTCTTCTCCTC (SEQ ID N ° 57) making it possible to introduce an NdeI site upstream of the ATG initiating eryCIII and the oligonucleotide B homologous to the complementary strand of the eryCIII gene having the sequence B CGGGATCCTCATCGTGGTTCTCTCCTTCCTGC) to introduce a BamHI site downstream of the stop codon of the eryCIII gene.
• the amplified DNA was then digested with the restriction enzymes Ndel and BamHI, then the 1.2 kb Ndel-BamHI fragment obtained containing the entire eryCIII gene was ligated into the expression vector pETlla (Stratagene) which contains the ampicillin resistance ⁇ -lactamase gene, the origin of ColEl replication and the promoter of the T7 RNA polymerase gene located upstream of the Ndel cloning site, previously digested with the restriction enzymes Ndel and BamHI. After ligation and transformation in E. coli XLl-blue, the plasmid pCEIII thus obtained was confirmed by restriction card and sequencing.
• pETlla Stratagene
• IPTG isopropyl- -D- thiogalactopyranoside
• the overexpression of a protein having an apparent molecular weight of approximately 46 Kd corresponding to the expected PM for the EryCIII protein was observed compared to the total proteins of a control strain transformed by the plasmid pET11a.
• the induction of the EryCIII protein was monitored by SDS-PAGE (polyacrylamide gradient: 10 to 20%) and with Comassie blue staining after lysis on an aliquot in the 1% SDS buffer, at 100 ° C. for 5 min, either directly on the broth harvested, or on the bacterial pellet after a first lysis by sonication in a phosphate buffer.
• the pellet thus washed was then suspended in 2.5 volumes of a 7M urea solution in 50 mM tris buffer pH 7.5 (buffer A) so as to dissolve the EryCIII protein. After centrifugation under the same conditions, the collected supernatant obtained contains 2.1 g of total proteins determined by the Bradford method using 5 ⁇ a commercial kit (Pierce).
• the extract in 7M urea was then loaded at the speed of 0.5 meters / h and at 4-8 ° C. on a 180 ml (5 cm ⁇ 9 cm) column of Q sepharose (Pharmacia) previously balanced. with buffer A above and with detection at 280 nm.
• the EryCIII protein was then eluted with buffer A containing 0.3M NaCl.
• the combined fractions, containing the EryCIII protein demonstrated by SDS-PAGE (polyacrylamide gradient: 10 to 20%) revealed by staining with Comassie blue and 835 mg of total proteins, were then loaded onto a column of 5, 5 liters (10 cm x 70 cm) of Superdex 200 Prep grade (Pharmacia) previously balanced with buffer A above.
• FIG. 1 ⁇ shows the evolution of the purity of the EryCIII protein followed by SDS-PAGE (polyacrylamide gradient: 10 to 15%) for a deposit of 500 ng of total proteins and a revelation with silver nitrate successively after extraction with 7M urea (line 2), Q sepharose chromatography (line 3), chromatography
• the EryCIII protein was then renatured by diluting the homogeneous eluate with a solution of buffer A containing 10 mM DTT to obtain a final protein concentration of 0.1 mg / ml.
• the diluted solution was then dialyzed against 50 mM Tris buffer; 0.15 M NaCl; 0.3% n-octyl- ⁇ -D-glucopyranosyl (NOG); DTT 10 mM, pH ⁇ , 3 then concentrated to 4 mg / ml by ultrafiltration on a PLGC04310 membrane (Millipore) having a cutoff threshold of 10,000.
• the purified EryCIII protein was then stored frozen at -20 ° C in 500 ⁇ l aliquots. 3) Characterization of the EryCIII protein
• Electrophoresis by SDS-PAGE polyacrylamide gradient: 10 to 15%
• Phast System device Phast System device
• revelation with silver nitrate shows a purity greater than 99% for a deposit of 2000 ng.
• N-terminal amino acid sequence The N-terminal sequence was determined by microsequencing on a Model A492 protein microsequencer coupled to an HPLC PTH-amino acid analyzer (Applied Biosystems). No secondary sequence was detected for the first 10 residues which is in agreement with the amino acid sequence described in FIG. 2 (sequence of SEQ ID No. 5).
• the desosaminyl transferase activity of the EryCIII protein was determined in vitro by demonstrating the formation of erythromycin D from dTDP-D-desosamine, the preparation of which is described below and from 3- ⁇ -mycarosyl erythronolide B (SEM) whose preparation is described above in General Materials and Methods.
• the reaction medium contains 150 nmol of dTDP-D-desosamine, 137.4 nmol of SEM and 1 mg of EryCIII protein using the following operating conditions:
• the stoppered tube is placed for 5 h in a bath thermostatically controlled at 30 ° C., then the pH is adjusted to 9-10 with 32% NaOH and then the reaction mixture is extracted 3 times with 5 ml of acetate. ethyl.
• the extract obtained, brought to dryness under reduced pressure, then taken up in 100 ⁇ l of methylene chloride is then analyzed by ccm under the conditions indicated in Example 4 using as eluent the methylene chloride / methanol mixture (90:10 , v / v).
• the sequence of the Sac eryCIII gene. erythraea corresponding to the ORF ⁇ described in example 1 coding for the deosaminyltransferase activity was used to prepare a hybridization probe and made it possible to isolate homologous genes in the strain S. oleandomycin-producing ATCC 11691 antibioticus by Southern hybridization.
• the entire eryCIII gene was amplified by PCR from 6 ng of the plasmid pK62 obtained in Example 1 by following the operating conditions described in Example 3 using the native polymerase pfu (Stratagene) and as primers.
• oligonucleotide having the following sequence: eryCIII-1 CGGGTACCATGCGCGTCGTCTTCTCCTCCATG (SEQ ID No.
• oligonucleotide eryCIII2 having the following sequence: eryCIII-2 CGGGTACCTCATCGTGGTTCTCTCCTTCC (SEQ ID No. 60) comprising a KpnI site in its 5 'region and whose part 3' corresponds to the region of DNA located from position 6954 to position 8974 of the sequence of Figure 2.
• the approximately 1.2 kb band obtained by amplification was then digested with the restriction enzyme Kpnl and cloned into the plasmid pUC19 previously digested with the enzyme Kpnl.
• the plasmid pCIIIPCRl thus obtained was then used to re-isolate the 1.2 kb Kpnl fragment corresponding to the entire eryCIII gene shown in FIG. 2.
• the fragment thus isolated was then labeled with 32 P by the "random priming" technique "described by Sambrook et al., 1989 and used as an eryCIII probe to analyze by Southern hybridization cosmid clones obtained from a genomic DNA library of S. antibioticus ATCC 11691 and prepared as follows (FIG. 20):
• a series of six cosmids (cosAB35, cosAB76, cosAB87, cosAB67, cosAB63 and cosAB61) overlapping and covering approximately 100 kb of the region corresponding to the olandomycin biosynthesis gene cluster was isolated using the method described by Swan et al., 1994 using as probes the 2 kb Smal fragment internal to the third subunit of the polyketide synthase of Sac. erythraea in the erythromycin biosynthesis gene cluster (Cortes et al., 1990) followed by a walk on the chromosome.
• cosmid cosAB35 Swan et al., 1994
• Subcloning and subsequent sequencing show that these two fragments are separated by a 0.6 kb BamHI fragment not detected by hybridization.
• the clone pC035-S thus obtained was used to generate single-stranded templates by subcloning of different DNA fragments in the bacteriophages M13mpl ⁇ and MP13mpl9 (New England Biolabs), then the nucleotide sequence of these fragments was determined according to the method of Sanger et al. (1977) using a modified T7 polymerase (Sequenase version 2.0; US Biochemicals) in the presence of ⁇ [ 35 S] dCTP (Amersham) and 7-deaza-dGTP, according to the supplier's recommendations in order to limit compression problems of bands.
• the conventional primers supplied with the Sequenase kit as well as the internal synthetic primers (17 mer) were used.
• the assembly of the sequence data was carried out using the Fragment Assembly program (Genetic Computer Group, University of Wisconsin) and the identification of the open reading phases using the CODONPREFERENCE program (Devereux et al., 1984).
• the nucleotide sequences obtained made it possible to establish the nucleotide sequence of 6093 bp represented in FIG. 22 (sequence of SEQ ID No. 15), comprised between the SphI and Kpnl sites shown in FIG.
• the oleGl gene codes for a polypeptide having 426 amino acids (sequence of SEQ ID No. 17). However, the presence 0 of a CGC codon coding for an arginine very conserved in this class of glycosyltransferase in Streptomycetes located immediately upstream of the GTG codon, would indicate that the initiating codon could be the CTG codon at position 1431 of the sequence SEQ ID N ° 17.
• the o! EG2 gene codes for a polypeptide having 426 amino acids (sequence of SEQ ID NO: 18).
• a strain in which the oleG1 gene is interrupted was prepared by integration of a plasmid pC03 into the homologous regions of the chromosomal DNA of the strain of S. antibioticus ATCC 11891 producing oleandomycin.
• the 0.6 kb BamHI fragment internal to the oleGl gene obtained by digestion of the pC035-S plamide prepared above, with the restriction enzyme BamHI (FIG. 21), was subcloned into the BamHI site of plasmid pOJ260 NRRL B-14785.
• the plasmid pC03 thus generated was then transferred into the strain E. coli TG1 rec01504:: Tn5 (Kolodner et al., 1965), then used to transform the protoplasts of S. antibioticus.
• the selection of transformants was carried out by resistance to apramycin (Apramycin for injection, Rhône Mérieux).
• the protoplasts were prepared from the strain S. antibioticus ATCC ll ⁇ 91 following the conditions described by Hopwood et al., 1985.
• the transformation was carried out using 50 ⁇ l of aliquot of protoplasts, 5 ⁇ g of plasmid DNA pC03 and replacing the thiostrepton with apramycin at the final concentration of 25 ⁇ g / ml.
• the selection of integrants carried out by resistance to apramycin made it possible to isolate a clone called A35G1.
• the expected alteration in the corresponding region of the chromosome of S. antibioticus was confirmed by genomic analysis by Southern blot.
• the chromosomal DNA isolated and then digested with the restriction enzyme PstI from the strain S. antibioticus wild or from the mutant A35G1 was analyzed by Southern using, as hybridization probe, the 0.6 kb BamHI fragment indicated above. above.
• the replacement of the 4.7 kb PstI fragment thus detected in the wild-type strain by two PstI fragments of 2.4 and 6.5 kb in the mutant A35G1 confirms the integration of the plasmid pC03 into the chromosome of the strain A35G1 at the level of the oleGl ORF.
• the recombinant strain A35G1 thus obtained was then cultivated to identify the precursors produced by the strain.
• the strain A35G1 was cultured for 72 h in 50 ml Erlenmeyer flask in EP2 medium from a 48 h preculture in PPE medium under the conditions described in Example 4.
• the culture was diluted to 1/100 in medium containing 50% (w / v) of glycerol, then the cell suspension obtained was kept. at -20 ° C before use.
• the biological test was then carried out by introducing 150 ⁇ l of the thawed cell suspension into 100 ml of TSB medium containing 1% agar and maintained at 55 ° C. The mixture was then poured into petri dishes. After cooling, oxford cylinders containing 50 to 200 ⁇ l of ethyl acetate extracts were placed on the agar dishes, kept for 2 h at 4 ° C., then incubated overnight at 37 ° C.
• the ccm analysis shows that the strain A35G1 does not produce oleandomycin but preferentially accumulates a purple product having greater mobility than erythronolide B and close to 6-deoxyerythronolide B and which can be expected from the aglycone part 8.8a-deoxy-oleandolide.
• c) Analysis by RP-HPLC chromatography coupled to Mass spectrometry was carried out according to the conditions described in Example 4. Two major metabolites, called M9 and MIO, were detected (elution at 6.12 min and 17.23 min respectively). The two products give a parent peak m / z 373 and similar fragmentation profiles which may be in agreement with the structure [8, 8a-deoxyoleandolide] H + . However, only the retention time of the metabolite MIO is in agreement with the proposed structure whereas the metabolite M9 could correspond to an isomer structure or to the open lactone nucleus.
• a mixture containing 18.6 g of the product from stage B and 50 cm 3 of DMF is brought to 50 ° C. and 6.62 g of hydrazine acetate NH 2 NH 2 , ACOH are added.
• the reaction mixture is stirred and poured onto a saturated solution of sodium hydrogen carbonate.
• the aqueous phase is extracted with ethyl acetate.
• the organic phases are combined, dried, filtered and concentrated. Distillation is carried out under reduced pressure to remove the DMF by azeotrophic entrainment with toluene.
• 11.28 g of product are obtained which is chromatographed on silica eluting with an ethyl acetate-triethylamine mixture (90-10).
• 6.5 g of sought product is obtained which is used as it is in the following stage.
• STAGE D 3, 4, 6-trideoxy-3- (dimethylamino) -D-xylo-hexopyra-nose, 2-acetate bis (phenylmethyl) phosphate 5.7 cm 3 of a solution are added at -70 ° C of n-butyl lithium in hexane in a solution containing 1.738 g of the product of the previous stage and 40 cm 3 of THF. 10 g of extemporaneously prepared dibenzylphosphochloride are added at -70-75 ° C. (J. Chem. Soc. 1958, p. 1957),
• STAGE E 3, 4, 6-trideoxy-3- (dimethylamino) -D-xylohexopyranose, l- (dihydrogen phosphate), N, N-diethylethanamine Is placed under stirring and under a stream of hydrogen for 30 minutes at at room temperature, a mixture containing 1.070 g of the product of the preceding stage, 20 cm 3 of ethyl acetate, 10 cm 3 of methanol, 0.622 cm 3 of triethylamine and 200 mg of palladium on carbon. It is filtered, washed with methanol and ethyl acetate and the filtrate is concentrated. 1 g of an oil is obtained to which 10 cm 3 of methanol are added.
• STAGE G Thymidine 5'- (trihydrogen diphosphate), P '- [3, 4, 6- trideoxy-3- (dimethylamino) -D-xylo-hexopyranosy1] ester, N, N- diethylethanamine 228 mg of the product is mixed preparation 1, 6 cm J of pyridine and 544 mg of thymidine 5 '-monophosphate morpholi- date-4-morpholine-NN'-dicyclohexylcarboxamidine.
• the pyridine is removed under reduced pressure on the rotor-vapor while maintaining the temperature at 30 ° C. or below. 6 cm 3 of pyridine are added, which is removed under reduced pressure. The operation is repeated 2 times.
• Hopwood DA, Kieser T, Wright HM an Bibb MJ Journal of General Microbiology (1983), 129, 2257-2269.
• SEQ ID NO: 15 / gene ⁇ "olePl” / function ⁇ "glycosylation of 8, 8a-desoxyoleandolide” / gene ⁇ "oleGl”
• transl_except (pos: 1437 .. 1439, aa: Met) / function ⁇ "glycosylation of 8,8a-desoxyoleandolide” / gene ⁇ "oleG2" / gene ⁇ "oleY”

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