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EP0626806A1 - Procede permettant d'effectuer la biosynthese de polycetides specifiques - Google Patents

Procede permettant d'effectuer la biosynthese de polycetides specifiques

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Publication number
EP0626806A1
EP0626806A1 EP92905082A EP92905082A EP0626806A1 EP 0626806 A1 EP0626806 A1 EP 0626806A1 EP 92905082 A EP92905082 A EP 92905082A EP 92905082 A EP92905082 A EP 92905082A EP 0626806 A1 EP0626806 A1 EP 0626806A1
Authority
EP
European Patent Office
Prior art keywords
polyketide
dna sequence
enzymatic activities
plasmid
dna
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Withdrawn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
EP92905082A
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German (de)
English (en)
Inventor
Leonard Katz
Stefano Donadio
James B. Mcalpine
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.)
Abbott Laboratories
Original Assignee
Abbott Laboratories
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
Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Priority claimed from CA002100791A external-priority patent/CA2100791C/fr
Priority claimed from PCT/US1992/000427 external-priority patent/WO1993013663A1/fr
Publication of EP0626806A1 publication Critical patent/EP0626806A1/fr
Withdrawn legal-status Critical Current

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Definitions

  • the present invention relates to a method for directing the biosynthesis of specific polyketide analogs by genetic manipulation.
  • polyketide biosynthetic genes are manipulated to produce precise, novel polyketides of predicted structure.
  • Polyketides are a large class of natural products that includes many important antibiotics and immunosuppressants such as erythromycins, tetracyclines, and rapamycins. Their synthesis proceeds by an ordered condensation of acyl esters to generate carbon chains of varying length and
  • the present invention provides a method to produce novel structures from designing and introducing specified changes in the DNA governing the synthesis of the polyketide.
  • the biosynthesis of specific polyketide analogs is accomplished by genetic manipulation of a polyketide-producing microorganism comprising tiie steps of:
  • the present method is most useful when the segment of the chromosome modified is involved in an enzymatic activity associated with polyketide biosynthesis.
  • the present invention is especially useful in manipulating polyketide biosynthetic genes from Streptomyces, an organism which provides over one-half of the clinically useful antibiotics.
  • PT propionyltransferase
  • ACP acyl carrier protein
  • KS ⁇ -ketoacyl ACP synthase
  • RmT (2R) methylmalonyl
  • FIG. 2. illustrates the nucleotide sequence of eryA
  • Standard one letter codes for the amino acids appear beneath their respective nucleic acid codons.
  • the standard one letter codes for the amino acid sequences are as follows:
  • FIG. 3. is a schematic representation of Type I, Type II and Type UI changes in eryA and structures of corresponding novel polyketides produced.
  • ⁇ 69 (Type I) and ⁇ 33 (Type II) represent in-frame deletions of tiie base pairs in the DNA segments corresponding to the KR of module 2 and the ⁇ -ketoacyl ACP synthase of module 2, respectively. Insertion of a complete copy of module 4 within module 1 is also shown. Production of ll-epifluoro-15-norerythromycin in strain that carries ⁇ 33 occurs when substrate analog (2S,3S,4S ⁇ S)2,4-dimethyl-3-fluoro-5-hydroxyhexanoic aad-ethyl thioester is fed.
  • FIG. 4 illustrates the restriction site coordinates of cosmid pRl 5' to the sequence of eryA (Fig 2).
  • polyketide refers to a large and diverse class of natural products, including antibiotics, pigments, and immunosuppressants. Antibiotics include, but are not limited to anthracyclines, tetracyclines, polyethers, ansamycins, macrolides of different types (polyenes and avermectins as well as classical macrolides such as eiythromy ⁇ ns).
  • polyketide-producing microorganism as used herein includes any Actinomycetales which can produced a polyketide. Examples of Actinomycetes that produce polyketides include but are not limited to
  • polyketide synthase refers to the complex of enzymatic activities responsible for the biosynthesis of .*- 5 polyketides which include but are not limited to ⁇ -ketoreductase,
  • extender refers to a coenzyme A thioester of a dicarboxylate which is incorporated into a polyketide by a 1 0 polyketide synthase.
  • starter refers to a coenzyme A thioester of a carboxylic acid which is used by the polyketide synthase as the first building block of the polyketide.
  • eryA refers to the genes involved in the
  • condensation refers to the addition of an extender unit out to the nascent polyketide chain and requires the action of ⁇ -ketoacyl ACP synthase, acyltransferase, and acyl carrier protein.
  • module refers to the genetic element encoding one condensation step, as defined above, and one ⁇ -carbonyl processing step, as defined herein.
  • Type I change refers to changes in DNA sequence which will result in the production of polyketide rings of length identical to that of 6-deoxyerythronolide A, but with altered functional groups at specific ring positions.
  • Type ⁇ mutants are erythromycin non-producing (Ery) mutants.
  • the structure of the resulting macrolides will depend on the substrate employed. 3 5
  • Type in change refers to alterations which will result in the biosynthesis of macrolide rings of length reduced (deletion) or increased (insertion) by two carbon units, or macrolide rings altered in specific portions of the chain (replacement).
  • the present invention entails a general procedure for producing novel polyketide structures in vivo by selectively altering the genetic information of the organism that naturally produces a related polyketide.
  • a set of examples described herein are a series of novel polyketides that make use of the genetic information for tiie biosynthesis of the polyketide portion of the macrolide antibiotic erythromycin.
  • the organization of the segment of the Saccharapoly$pora erythrae chromosome, designated eryA, and the corresponding polypeptides which it encodes that determine the biosynthesis of the polyketide segment of erythromycin, are shown in FIG. 1.
  • eryA is organized in modules, as shown, and that each module takes care of one condensation step, through the action of the ⁇ -ketoacyl ACP synthase specified within, wherein an extender unit, methylmalonyl CoA, is added first to the starter unit, propionyl CoA, and then to the successively growing acyl chain.
  • the precise succession of elongation steps is dictated by the genetic order of the six modules: module 1 determines the first condensation; module 2, the second; module 3/ the third, and so on until the sixth condensation step has occurred.
  • the processing of the growing chain after each condensation is also determined by the information within each module.
  • ⁇ - ketoreduction of the ⁇ -carbonyl takes place after each step except for step 3, as determined by the presence of a functional ⁇ -ketoreductase in all modules except module 3, whereas dehydration and enoylreduction only take place after the fourth extender unit is added to the growing acyl chain, as determined by the presence of dehydratase and enoylreductase in module 4.
  • the choice of the correct enantiomer (2R or 2S) of methylmalonyl-CoA as the extender unit employed at each condensation is specified by the acyltransferase function determined by each module (FIG.1C).
  • novel polyketide molecules of desired structure are produced by the introduction of specific genetic alterations of the eryA sequence into the Sac. erythraea chromosome.
  • the complete nucleotide sequence of the eryA segment of the Sac. erythraea chromosome and the sequence of the corresponding polypeptides are shown in FIG.2.
  • Type I 5 changes will result in the production of polyketide rings of length identical to that of 6-deoxyerythronolide A, but with altered functional groups at specific ring positions. Strains carrying type II alterations will result in the production of macrolide rings only when fed exogenously with substrate analogs, e.g.thioesters of appropriate acyl compounds of 1 0 various length. Thus Type II mutants are erythromycin non-producing (Ery") mutants. The structure of the resulting macrolides will depend on the substrate employed. Type III changes will result in the biosynthesis of macrolide rings of length reduced (deletion) or increased (insertion) by two carbon units, or macrolide rings altered in specific portions of the
  • Type I, Type II and Type UI alterations in eryA and the corresponding novel polyketides produced in hosts that carry such alterations is shown in FIG. 3.
  • Step 1 requires standard recombinant DNA manipulations employing E. coli as the host.
  • Step 2 requires one or more plasmids out of the several E. coli-
  • Sac. erythraea non-replicating vector The plasmid carrying the altered - allele is then introduced into the host strain by transformation of protoplasts employing selection for a plasmid marker. Since the plasmid does not replicate, regenerated cells that carry the marker have undergone 3 5 a single homologous recombination between one of the two segments flanking the mutation on the plasmid and its homologous counterpart in the chromosome. Some of the colonies that have subsequently lost the marker will have undergone a second recombination between the other plasmid borne adjacent DNA segment and its homologous chromosomal counterpart resulting in the retention of the mutation in the chromosome, replacing the normal allele with t e mutant one.
  • the second method to introduce an altered allele into the chromosome employs gene conversion, described in Examples 37 and 43.
  • an Ery' Sac. erythraea strain carrying a deletion of a specified region of the eryA segment of the chromosome is used as a host.
  • Sac. erythraea multicopy plasmid that carries a selectable marker is cloned the wild type counterpart (segment 1) of the eryA segment mutant in the host.
  • the desired homologous or heterologous DNA segment to be introduced (segment 2) is cloned within the portion of segment 1 which is deleted in the mutant strain.
  • the resulting plasmid is then introduced into the host employing selection for the marker.
  • the transformants will be a population that have integrated segments 1 and 2 from the plasmid by the process of gene conversion which can be verified by examination of the DNA among t e colonies that have recovered the ability to produce erythromycin.
  • Types I, II and HI alterations to the eryA DNA sequence and the resultant novel polyketides produced are described in the examples described herein.
  • Examples 1 through 8, 9 through 12 and 13 through 16 describe the construction and effect of three Type I mutants.
  • Examples 17 through 22 and 23 through 27 describe the construction of two Type ⁇ mutants and the effects of feeding two different synthetic substrates to the mutant strains.
  • Examples 28 through 38 and 39 through 44 outline the steps in constructing Type HI changes and their respective effects on the structure of the novel polyketides produced.
  • a plasmid that contains a substantial deletion of the segment of the gene corresponding to the b-ketoreductase of module 5 is created, the altered gene is inserted into the Sac.
  • Example 8 the new strain is fermented and the novel polyketide 5-oxo-5,6-dideoxy-3 ⁇ -mycarosyl erythronolide B that results from the introduction of the mutant allele is isolated.
  • Examples 9 through 11 a mutation is introduced into the ⁇ -ketoreductase of module 2 and the mutated allele is then used to replace the wild type allele in the chromosome.
  • Example 12 the strain carrying the altered allele is fermented and the novel compound 11-oxo-ll-deoxyerythromycin A is isolated.
  • Examples 13 through 16 a mutation is introduced into the dehydratase of module 4 and the mutated allele is then used to replace the wild type allele in the chromosome. The strain carrying this altered allele is then fermented and the novel products 7- hydroxyerythromycin A and 6-deoxy-7-hydroxyerythromycin A are isolated.
  • Examples 17 through 21 a mutation is made in the DNA corresponding to the ⁇ -ketoacyl- ACP synthase of module 1 and introduced into the chromosome to replace the wild type allele. This mutation has the effect of arresting the synthesis of the polyketide chain and results in the Ery" phenotype.
  • the synthetic substrate (2S,3R,4S,5S)3,5-dihydroxy-2,4- dimethylhexanoic acid-ethyl ester is then made and fed to the mutant resulting in the production of the novel compound (14S,15S)14(1- hydroxyethyDerythromycin.
  • a mutation is created in the ⁇ -ketoacyl- ACP synthase of module 2 and introduced into the chromosome to replace the wild type allele.
  • Example 25 and 26 the synthetic substrate (2S,3S,4S,5S)2,4-dimethyl-3- fluoro-5-hydroxyhexanoic acid-ethyl thioester is made and fed to the module 2 ⁇ -ketoacyl- ACP synthase mutant and the resulting novel compound ll-epifluoro-15-norerythromycin is isolated.
  • Examples 27 through 38 a copy of the DNA sequence corresponding to module 4 is introduced into the deleted segment of the ⁇ -ketoacyl- ACP synthase of module 1 resulting in the production of the novel compound 14(1- propyDerythromycin.
  • Examples 40 through 44 a copy of the DNA sequence corresponding to module 5 is introduced into the deleted segment of the ⁇ -ketoacyl ACP synthase of module 1 resulting in the production of the novel compound 14[l(l-hydroxypropyl)]erythromycin.
  • Restriction endonucleases T4 DNA ligase, nick-translation kit, competent E. coli DH5 ⁇ cells , X-gal, IPTG, and plasmids pUC19 and pUC12 are purchased from Bethesda Research Laboratories (BRL), Gaithersburg, MD. [ ⁇ -32p]dCTP and Hybond N are from Amersham Corp., Chicago, IL. Seakem LE agarose and Seaplaque low gelling temperature agarose are from FMC Bioproducts, Rockland, ME. E. coli K12 strains carrying the E. coli-Sac. shuttle plasmids pWHM3 or pWHM4 (Vara et al.,T. Bacteriol..
  • Staphylococcus aureus Th R (thiostrepton resistant) is obtained by plating 10 8 cells of S. aureus on agar medium containing 10 mg/ml thiostrepton and picking a survivor after 48 hr growth at 37°C. Thiostrepton is obtained from Squibb-Bristol Myers, New Brunswick, NJ. All other chemical and reagents are from standard commercial sources unless specified otherwise.
  • Standard conditions (Maniatis et al.. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982) are employed for restriction endonuclease digestion, agarose gel-electrophoresis, nick translation of DNA to make 32p-labeled probes, DNA ligation, and transformation of E, coli employing selection for ampi ⁇ llin resistance (Ap R ) on LB agar plates. Plasmid DNA is isolated from minipreps of E. coli transformants by the boiling method (Maniatis et al., 1982, supra). DNA fragments are recovered from low melting agarose gels using the metiiod of Langridge et al., 1980.
  • Total DNA from Sac, erythraea strains is prepared according to described procedures (Hopwood et al.. Genetic Manipulation of Streptomyces, A Laboratory Manual, John Lines Foundation, Norwich, U.K., 1985). DNA is transferred from agarose gels onto Hybond N following the manufacturer's instructions.
  • Amplification of DNA fragments is performed by the polymerase chain reaction (PCR) employing a Coy thermocycler. Reactions contain 100 pmol c «.ch primer, 1 ⁇ g of template
  • Thermus aquaticus DNA polymerase in a 100 ml volume of PCR buffer [50 mM KC1, 10 mM TrisHCl (pH 8.0) 2 mM 5 MgCl2, 0.01% gelatin) containing 200 mM of the 4 dNTPs.
  • the above reagents are from Perkin Elmer Cetus, Norwalk, CT.
  • the reaction mixture is overlaid with a drop of paraffin oil and subjected to 30-50 cycles. Each cycle consists of one 94 °C, one 55 °C and one 72 °C period, each of the duration of 3 min.
  • the progress of the amplification is monitored by 1 0 agarose gel-electrophoresis.
  • the PCR primers described in the examples below are derived from the nucleotide sequence of eryA of FIG. 2.
  • Protoplasts of Sac. erythraea strains are prepared and transformed
  • Th ⁇ thiostrepton-sensitive colonies arise at a frequency of 10"2 (Donadio et al., 1990). The retention of the mutant allele is established by Southern hybridization of a few ThS colonies.
  • Th-R colonies obtained by transformation of an eryA strain with pWHM4 derivatives are screened for antibiotic production by the agar-plug assay employing Staphylococcus aureus as Th R organism as described (Tuan et al.-Gene, 90: 21 (1990)).
  • Sac erythraea cells are inoculated into 100 ml SCM medium (1.5% soluble starch, 2.0% Soytone [Difco], 0.15% Yeast Extract [Difco], 0.01% CaCl2) and allowed to grow at 32°C for 3 to 6 days. The entire culture is then inoculated into 10 liters of fresh SCM medium. The fermenter is operated for a period of 7 days at 32°C maintaining constant aeration and pH at 7.0. After fermentation is complete, the cells are removed by centrifugation at 4°C and the fermentation beer is kept in the cold until further use.
  • the present invention will now be illustrated, but is not intended to be limited, by the following examples:
  • plasmid pABX9 prepared as described in Example 1, were transformed into E. coli K12 DH5 ⁇ and a few of the resulting white ApR colonies that appeared on the LB-agar plates containing X-gal and ampicillin were analyzed for their plasmid content.
  • One colony was found to carry pABX9, as verified by the observation of fragments of 3.93, 3.39, 2.01, 1.56, 0.87, and 0.48 kb in size upon agarose gel electrophoresis after Smal digestion of the plasmid.
  • Plasmid pABX9 isolated from E. coli K12 DH5 ⁇ /pABX9, was digested with Ncol and then treated with T4 DNA ligase. The resulting mixture contained the desired plasmid pABX9DN.
  • Example 4 Construction of E. coli K12 DH5a/pABX9DN
  • plasmid pABX9DN prepared as described $ 5 in Example 3, were transformed into E. coli K12 DH5 ⁇ and a few of the ft resulting white ApR colonies that appeared on the LB-agar plates ⁇ containing X-gal and ampicillin were analyzed for their plasmid content.
  • Colonies carrying pABX9DN exhibited a single Ncol fragment of 11.5 kb visible by agarose gel electrophoresis, confirming that the 813 bp Ncol - 1 0 Ncol fragment from pABX9 has been deleted in pABX9DN.
  • Plasmid pABX95DN was digested with EcoRI and Hindm and ligated to pWHM3 digested with the same two enzymes. The resulting mixture contained the desired plasmid pABX95DN.
  • Colonies carrying pABX95DN exhibited fragments of 8.8 and 7.2 kb visible in agarose gels after EcoRI and Hindlll digestion.
  • Example 8 Isolation, purification and properties of 5-oxo-5,6-dideoxy-3-a-mycarosyl erythronolide B from Sac, erythraea AKR5
  • a 10-liter fermentation of Sac. erythrea AKR5 carrying the eryAKR5 allele in a Biolafitte fermentor using SNC Media was inoculated with 100 ml of a 3 day old seed.
  • the p ⁇ 2 was initially 80 ppm and the temperature was maintained at 32°C.
  • the pH was controlled to 7.0 ⁇ 0.2 by addition of propionic acid or potassium hydroxide as needed.
  • the whole broth was extracted three times with 4-liter portions of ethylacetate. The combined extracts were concentrated under reduced pressure and the residue was chromatographed on a column (50 x
  • the 1.3 kb DNA segment comprised between coordinates 8.63-9.93 (fragment 1) is amplified by PCR employing two oligodeoxynucleotides, la
  • plasmid pALeryAKR2 prepared as described in Example 9, are transformed into E. coli K12 DHS ⁇ and a few of the resulting white ApR colonies that appear on the LB-agar plates containing X-gal and ampicillin are analyzed for t eir plasmid content.
  • pALeryA2KR2 The identity of plasmid pALeryA2KR2, 9.8 kb in size, and carrying a 2.6 kb EcoRI-SphI insert with an internal PstI site, is verified by Sail digestion (fragments at 2.91, 2.21, 1.61, 1.42, 1.08, 0.29, 0.12 and 0.10 kb are released, visible by agarose gel electrophoresis).
  • pALeryAKR2 contains an in-frame deletion of 102 base pairs of the corresponding segment of the wild type eryA chromosomal DNA. The cloned segment in pALeryAKR2 is designated the eryAKR2 allele.
  • K12 DH5 ⁇ /pALeryAKR2 is transformed into Sac. erythraea protoplasts and stable Th R colonies are isolated. Serial dilutions of one of these colonies are screened for loss of the antibiotic resistance marker, and six ThS colonies are analyzed for their genotype by Southern hybridization. Total DNA from the six ThS colonies and from untransformed Sac. erythraea NRRL2338 is digested with PstI and witii Sail and is then examined by Southern hybridization using the 2.6 kb EcoRI-SphI insert from pALeryAKR2 as probe.
  • NRRL2338 contains a 39 kb PstI hybridizing band
  • colonies in which the mutation in KR2 has been introduced exhibit two bands of approximately equal intensity, one at 27 kb and the other at 12 kb.
  • the Sail digest with bands at 1.04, 0.75, 0.29, 0.12 and 0.10 kb common to NRRL2338 and AKR2, but with the 1.16 kb band in NRRL2338 replaced by the 1.06 kb band in AKR2, confirms that tiie only change introduced into strain AKR2 is the deletion of tiie 102 bp segment from KR2, resulting in a strain carrying the eryAKR2 allele.
  • Example 12 Isolation and purification of 11-deoxy-ll-oxoerythromycin A
  • the fermentation beer of strain AKR2, cooled to 4°C is adjusted to
  • 11-oxo-ll-deoxyerythromycin A 1 5 desired 11-oxo-ll-deoxyerythromycin A were combined, concentrated, digested in methylene chloride, washed well with water and concentrated on rotary evaporator under reduced pressure to yield 11-deoxy-ll- oxoerythromycin A as an off-white solid froth. Its identity is confirmed by comparison with antibiotic L53-18A. 11-Deoxy-ll-oxoerythromycin A is
  • Primers 3a (GCGCGAGCTCGACGACCAGGGCGGCATGGT) and 3b (GGTGGCATGCTGCGACCACTGCGCGTCGGC) are used to PCR-amplify the 1.05 kb eryA segment of the Sac, erythraea chromosome between sequence coordinates 18.47-20.07 (fragment 3), and primers 4a 3 5 (AGCTGCATGCTCTGGACTGGGGACGGCTAG) and 4b
  • fragment 4 (CGCGGGATCCCAGCTCCCACGCCGATACCG) are used to amplify the 1.35 kb segment between sequence coordinates 20.58-21.96 (fragment 4) as described in Example 1. Fragment 3 and 4, after digestion wit SstI + SphI and with SphI + BamHI, respectively, are ligated to SstI -, BamHI-digested pWHM3. The resulting ligation mixture contains the desired plasmid pALeryADH4.
  • pALeryADH4 Approximately 10 ng of pALeryADH4, prepared as described in Example 13, are transformed transformed into E. coli K12 DH5 ⁇ and a few of the resulting white Ap colonies that appear on the LB-agar plates containing X-gal and ampicillin are analyzed for their plasmid content. The identity of plasmid pALeryADH4, 9.6 kb in size, is verified by SphI + EcoRI digestion (fragments at 7.2, 1.35 and 1.05 kb are released). pALeryADH4 carries a 498 base pair in-frame deletion of the corresponding segment of the wild type eryA DNA. The cloned segment in p ALeryADH4 is designated the ervADH4 allele.
  • plasmid pALeryADH4 isolated from E. coli K12 DH5 ⁇ /pALeryADH4, is used for transformation into Sac. erythraea protoplasts and stable Th R colonies are isolated. Serial dilutions of one of these colonies are screened for loss of the antibiotic resistance marker, and six Th.S colonies are analyzed for their genotype by Southern hybridization. Total DNA from the six Th ⁇ colonies and from untransformed Sac. erythraea NRRL2338 is digested with SphI and with SstI and examined by Southern hybridization using tiie 2.4 kb Sstl-BamHI insert from pALeryADH4 as probe.
  • the fermentation beer of strain ADH4 is cooled to 4°C and the pH is adjusted to 5.0.
  • the mixture is extracted once with an equal volume of methylene chloride.
  • the pH of the aqueous layer is readjusted to 9.0 and two further methylene chloride extracts are carried out. These two extracts are combined, washed with water and concentrated to a residue.
  • This is digested in 10 ml of the upper phase of a (3:7:5, v/v/v) mixture of hexane, ethylacetate, aqueous phosphate buffer (0.05 M, pH 7.5) and chromatographed on an Ito Coil Planet Centrifuge in the same system.
  • fragment 5 fragment 5 and the 1.5 kb segment between sequence coordinates 2.88- 4.37 (fragment 6) are PCR-amplified using primers 5a (TGCAGAATTCGCTGGCCGCGCTCTGGCGCT) and 5b (GAGAGCTGCAGCATGAGCCGCTGCTGCGGG), and 6a (CATGCTGCAGGACTTCAGCCGGATGAACTC) and 6b
  • plasmid pALeryAKSl Approximately 10 ng of pALeryAKSl, prepared as described in Example 17, are transformed into E. coli K12 DH5 ⁇ _. and a few of the resulting white ApR colonies that appear on the LB-agar plates containing X-gal and ampicillin are analyzed for their plasmid content.
  • the identity of plasmid pALeryAKSl, 10.1 kb in size, is verified by digestion with PstI + Hin llT (fragments of 8.6 and 1.5 kb are observed by agarose gel electrophoresis) and with Sail (fragments of 2.93, 2.21, 1.42, 1.37, 0.86, 0.54, 0.27, 0.14, 0.13, and 0.10 kb are observed).
  • pALeryAKSl carries an in-frame deletion of 282 base pairs of the corresponding wild type eryA DNA-
  • the cloned insert in plasmid pALeryAKSl is designated the eryAK
  • plasmid pALeryAKSl isolated from E. coli K12 DH5 ⁇ /pALeryAKSl, is used for transformation into Sac. erythraea protoplasts and stable T ⁇ colonies are isolated. Serial dilutions of one of these colonies are screened for loss of the antibiotic resistance marker, and six ThS colonies are analyzed for their genotype by Southern hybridization. Total DNA from the six T .S colonies and from unfransformed Sac. erythraea NRRL2338 is digested with PstI and with
  • a convenient source of this compound in chiral purity is the antibiotic oleandomycin.
  • Oleandomy ⁇ n (5 g) is dissolved in an aprotic solvent such as toluene and treated with diazabicyclo[5.4.0]undecene-5 (1 g) and heated for one hour.
  • the resulting solution is poured into iced water, agitated well and the organic layer is drawn off and concentrated to a residue.
  • the residue is digested in methylene chloride and treated exhaustively with a solution of ozone.
  • the resulting ozonide is oxidatively decomposed with dilute hydrogen peroxide in sufficient aqueous ethanol to yield a monophasic mixture. This is further diluted with water and made 0.1 N with sodium hydroxide.
  • the mixture is warmed for one hour at 70°C and then cooled before being acidified to pH 2.5 with dilute sulfuric acid.
  • the mixture is then exhaustively extracted with methylene chloride.
  • the combined extracts are concentrated to an oily residue and the desired lactone is recovered by chromatography on silica gel eluted with a gradient of toluene- isopropanol.
  • the ⁇ -lactone is converted to the butyl thioester before feeding to Sac. erythrea AKS1 by refluxing with n-butylthiol in the presence of a catalytic amount of triethylamine.
  • the fermentation broth of AKS1 is cooled to 4°C and adjusted to pH 4.0 and extracted once with methylene chloride.
  • the aqueous layer is readjusted to pH 9.0 and extracted twice with methylene chloride and the combined extracts are concentrated to a solid residue.
  • This is digested in methanol and chromatographed over a column of Sephadex LH-20 in methanol. Fractions are tested for bioactivity against a sensitive organism, such as Staphylococcus aureus Th- , and active fractions are combined.
  • the combined fractions are concentrated and the residue is digested in 10 ml of the upper phase of a solvent system consisting of n-heptane, benzene, acetone, isopropanol, 0.05 M, pH 7.0 aqueous phosphate buffer (5:10:3:2:5, v/v/v/v/v), and chromatographed on an Ito Coil Planet Centrifuge in the same system. Active fractions are combined, concentrated and partitioned between methylene chloride and dilute ammonium hydroxide (pH 9.0). The methylene chloride layer is separated and concentrated to yield the desired product as a white foam.
  • Primers 7a CGCCCGAATTCGAGGCGCTGGGCGCCCGGAC
  • 7b CCACCTGCAGCGCGGGACCTTCCAGCCCC
  • primers 8a GTGGGTCGCTGCAGACGGTGACTGCGG
  • 8b GGTCAAGCTTCGTCGGCGAGCAGCTTCTC
  • fragment 7 fragment 7
  • fragment 8 fragment 8
  • the two fragments are ligated to pWHM3 cut with EcoRI + Hindlll.
  • the resulting mixture contains the desired plasmid pALeryAKS2.
  • Plasmid pALeryAKS2 carries an in ⁇ frame deletion of 60 base pairs of the corresponding wild type eryA DNA.
  • K12 DH5 ⁇ /pALeryAKS2 is used for transformation into Sac, erythraea protoplasts and stable Th-R colonies are isolated. Serial dilutions of one of these colonies are screened for loss of the antibiotic resistance marker, and six ThS colonies are analyzed for their genotype by Southern hybridization. Total DNA from the six ThS colonies and from unfransformed Sac, erythraea NRRL2338 is digested with PstI and with Sst-Q and examined in Southern hybridization employing the 2.9 kb EcoRI- Hind ⁇ insert from pALeryAKS2 as probe.
  • Colonies in which the wild type allele has been replaced by the mutated copy show two PstI bands, one at 34.5 and the other at 4.4 kb, whereas the wild type strain exhibits a single band at 39 kb.
  • the Sst ⁇ pattern with the 0.78 kb band from NRRL2338 being replaced in AKS2 by a 0.72 kb band, confirms that the 60 bp created in plasmid pALeryAKS2 has been transferred into strain AKS2.
  • Strains that carry the eryAKS2 allele are designated Sac, erythraea AKS2.
  • the lactone is then converted to the n-butyl thiolester by refluxing in n-butyl thiol with a catalytic amount of triethylamine. Solvent is removed and the residue is digested in DMSO before feeding to fermentations of Sac, erythraea AKS2.
  • the fermentation broth of strain AKS2 is cooled to 4°C and adjusted to pH 4.0 and extracted once with ethylacetate.
  • the aqueous layer is adjusted to pH 9.0 and extracted twice with methylene chloride and the combined extracts are concentrated to a white solid.
  • This is chromatographed over a column of Sephadex LH-20 in a mixture of heptane, chloroform, ethanol (10:10:1, v/v/v) and fractions containing the desired product are combined and concentrated to a solid residue.
  • Primers 9a GCGCCGAATTCTCGAGACGGCGTGGGAGGCA
  • 9b TGCGGTACCAGTAGGAGGCGTCCATCGCG
  • fragment 9 After digestion with EcoRI + Kpnl, fragment 9 is ligated to pUC19 cut with the same two enzymes The resulting mixture contains the desired plasmid pALeryAM4.1.
  • Example 28 Construction of E. coli K12 DH5a/pALeryAM4.1
  • plasmid pALeryAM4.1 Approximately 10 ng of pALeryAM4.1, prepared as described in Example 27, are transformed into E. coli K12 DH5a_. and a few of the resulting white ApR colonies that appear on the LB-agar plates containing X-gal and ampicillin are analyzed for their plasmid content. The identity of plasmid pALeryAM4.1, 4.7 kb in size, is verified by digestion with Sail (fragments of 2.8, 0.85, 0.53, 0.27 and 0.22 kb are observed by agarose gel electrophoresis).
  • plasmid pALeryAM4.2 Approximately 10 ng of pALeryAM4.2, prepared as described in Example 29, are transformed into E. coli K12 DH5a ⁇ and a few of the resulting white ApR colonies that appear on the LB-agar plates containing X-gal and ampicillin are analyzed for their plasmid content.
  • Example 31 Construction of plasmid pALeryAMl
  • the 2.9 kb Smal(4)-Smal(20) fragment from cosmid clone pRl is ligated to pUC12 cut with Smal.
  • the resulting mixture contains plasmid pALeryAMl.
  • plasmid pALeryAMl Approximately 10 ng of pALeryAMl, prepared as described in Example 31, are transformed into E. coli K12 DH5 ⁇ r and a few of the resulting white Ap R colonies that appear on the LB-agar plates containing X-gal and ampicillin are analyzed for their plasmid content-.
  • Plasmid pALeryAMl is cut with Xhol to completion, partially with SphI, and the resulting 5.25 kb band, isolated from an agarose gel, is ligated to the 6.65 kb insert released from pALeryAM4.2 by Xhol + SphI digestion The resulting mixture contains the desired plasmid pALeryAM4.3.
  • Plasmid pALeryAM4.3 carries the entire eryA module 4 inserted into the KS region of module 1. The cloned insert in pALeryAM4.3 is degnated the eryAM412 allele.
  • Plasmid pALeryAM4.3 is cut with EcoRI + Malawi!, and the resulting 9.2 kb band, recovered from an agarose gel, is ligated to pWHM4 cut with the same two enzymes. The resulting mixture contains the desired plasmid pALeryAM4.4.
  • Example 35 are transformed into E. coli K12 DH5 ⁇ x and a few of the resulting white Ap R colonies that appear on the LB-agar plates containing X-gal and ampicillin are analyzed for their plasmid content.
  • the identity of plasmid pALeryAM4.1, 16.5 kb in size, is verified by EcoRI + Hindi ⁇ digestion, with fragments of 9.2 and 7.3 kb released.
  • Plasmid pALeryAM4.4 carries the eryAM412 allele on the Sac, erythraea multicopy vector pWHM4.
  • plasmid pALeryAM4.4 isolated from E. coli K12 DH5 ⁇ /pALeryAM4.4, is used for transformation into Sac, erythraea strain AKSl protoplasts. A few hundred transformants are screened for antibiotic production by the agar-plug assay, and one of the colonies found to produce antimicrobial activity is cured of pALeryAM4.4 by protoplast formation and regeneration as described in General Methods.
  • the SphI band at 0.8 kb in strain AKSl is seen to be replaced by a 7.5 kb band in strain AM412, whereas the other two bands at 2.4 kb and 5.2 kb are unaffected, h the Xhol digest, the AKSl band at 2.9 kb is replaced by a 9.6 kb band in AM412, with the other band at 5.2 kb conserved in both strains.
  • strain AKSl exhibits one band at 25.5 kb and one at 17.9 kb in the SphI and Xhol digests, respectively, whereas, in addition to these bands, strain AM412 shows one SphI band at 7.5 kb and one Xhol band at 9.6 kb. L this way, it is established that the eryAKSl allele has been converted into tiie eryAM412 allele in strain AM412.
  • the fermentation is adjusted to pH 9.5 and extracted twice witii equal volumes of methylene chloride.
  • the combined extracts are washed once with water and concentrated to an oily residue.
  • This is partitioned in a heptane methanol water (5:5:1, v/v/v) system and the lower layer is washed once with heptane and then concentrated to a semisolid residue.
  • This is digested in methanol and chromatographed over a column of Sephadex LH-20 in methanol. Fractions are tested for bioactivity in an agar diffusion assay on plates seeded with ihe macrolide- sensitive strain Staphylococcus aureus Th R .
  • fragment 11 The 4.7 kb eryA fragment between sequence coordinates 23.65-28.36 (fragment 11) is PCR-amplified employing primers 11a (ATGCTCGAGATCTCGTGGGAGGCGCTGGA) and lib (AGAACTCGGTGAGCATGCCCGGGCCCGCCA). Fragment 11, after digestion with Xhol + SphI, is ligated to the 5.25 kb fragment resulting from complete Xhol and partial SphI digestion of pALeryAMl, as in Example 33. The resulting mixture contains the desired plasmid pALeryAM5.1.
  • Example 40 Construction of E. coli K12 DH5 ⁇ /pALeryAM5.1
  • Example 39 are transformed into E. coli K12 DH5 ⁇ _; and a few of the resulting white A R colonies that appear on the LB-agar plates containing X-gal and ampicillin are analyzed for their plasmid content.
  • the identity of plasmid pALeryAM5.1, 9.95 kb in size, is verified by SphI + Xhol digestion, with fragments of 5.25 and 4.7 kb released, and by Smal digestion where fragments of 3.39, 2.68 and 1.94 (doublet) kb are observed.
  • Plasmid pALeryAM5.1 carries the entire eryA module 5 inserted into the ⁇ -ketoacyl ACP synthase region of modulel.
  • the cloned insert in plasmid pALeryAM5.1 is designated the eryA512 allele.
  • Plasmid pALeryAM5.1 is cut with EcoRI + HindHI and the resulting 6.3 kb fragment, recovered from an agarose gel, is ligated to pWHM4 cut with the same two enzymes. The resulting mixture contains the desired plasmid pALeryAM5.2.
  • Plasmid pALeryAM5.2 contains the eryAM512 allele on the Sac, erythraea multicopy vector pWHM4.
  • K12 DH5 ⁇ /pALeryAM5.2 is used for transformation into Sac, erythraea strain AKSl protoplasts.
  • a few hundred transformants are screened for antibiotic production by the agar-plug assay, and one of the colonies found to produce antimicrobial activity is cured of pALeryAM5.2 by protoplast formation and regeneration as described in General Methods.
  • Total DNA from six antibiotic-producing, Th ⁇ colonies (strain AM512)and from strain AKSl is digested with SphI and with Xhol and the resulting Southern blot is hybridized first to the 2.9 kb insert from pALeryAMl, and then to the 0.8 kb NcoI(119)-NcoI(123) fragment from plasmid pALeryAM5.1.
  • the SphI band at 0.8 kb in sfrain AKSl is replaced by a 5.5 kb band in strain AM512, whereas the other two bands at 2.4 kb and 5.2 kb are unaffected.
  • the AKSl band at 2.9 kb is replaced by a 7.6 kb band in AM512, with the other band at 5.2 kb conserved in both strains.
  • strain AKSl exhibits one band at 25.5 kb and one at 17.9 kb in the SphI and Xhol digests, respectively, whereas,-in addition to these bands, strain AM512 shows one SphI band at 5.5 kb and one Xhol band at 7.6 kb. In this way, it is established that tiie eryAKSl allele has been converted into the eryAM512 allele in strain AM512.
  • the combined ethylacetate extracts are washed with water, dried and partitioned in a heptane, methanol, water (5:5:1, v/v/v) system.
  • the lower (methanolic phase) is washed with an equal volume of heptane and is concentrated to a residue.
  • Examples of combinations of two Type I alterations leading to useful compounds include but are not limited to: mutants of the the ⁇ -ketoreductase of module 2 (KR2) and the ⁇ -ketoreductase of module 4 (KR4) leading to the formation of 7,ll-dioxo-7,ll-dideoxyerythromycin A; mutants of KR2 and the ⁇ -ketoreductase of module 6 (KR6) leading to the formation of 3,11- dioxo-3,ll-dideoxy-5-desosaminylerythronolide A; mutants of KR2 and the dehydratase of module 4 (DH4) leading to the synthesis of 7-hydroxy- 11-oxo-ll-deoxyerythromycin A; mutants of KR2 and the enoylreductase of module 4 (ER4) leading to the synthesis of ⁇ -6,7-anhydro-ll-oxo-ll- deoxyerythromycin A; mutants of KR4 and KR
  • Examples of combinations of three Type I alterations leading to the synthesis of novel polyketides include but are not limited to: mutants of KR2, KR4 and KR6 leading to the synthesis of 3,7,1 l-trioxo-3,7,ll-trideoxy-5- desosaminylerythronolide A; mutants of KR2, KR6 and DH4 leading to the synthesis of 3,ll-dioxo-3,ll-dideoxy-5-desosaminyl-7- hydroxyerythronolide A; mutants of KR2, KR6 and ER4 leading to the synthesis of 3,ll-dioxo-3,ll-dideoxy-5-desosaminyl-D-6,7- anhydroerythronolide A. All combinations of two or three Type I mutants, the Sac, erythraea strains that carry said combinations and the corresponding polyketides produced from said strains, therefore, are included within the scope of the present invention.
  • Type II mutants specified herein have been constructed in the ⁇ -ketoacyl ACP synthase of module 1 (KS1) and the ⁇ — ketoacyl ACP synthase of module 2 (KS2), other Type II mutants can be constructed in other domains to result in the synthesis of novel polyketide structures upon feeding with appropriate substrate analogs.
  • Other Type II mutants include but are not limited to: inactivation of the either of the acyltransferases or acyl carrier proteins of module 1, or the acyltransferase or acyl carrier protein of module 2, the ⁇ -ketoacyl ACP synthase, acyltransferase or acyl carrier protein of module 3, module 4 or module 5.
  • compounds other than (2S,3R,4S,5S)3,5- dihydroxy-2,4-dimethylhexanoic acid-ethyl thioester and (2S,3S,4S,5S)2,4- dimethyl-3-fluoro-5-hydroxyhexanoic acid-ethyl thioester specified herein can be synthesized and fed to strains AKSl or AKS2 specified herein or other strains that carry other Type IE mutations to result in the creation of novel polyketides that are within the scope of the present invention.
  • Type HI alterations are specified herein, it is apparent to those skilled in the art that many other examples of Type HI changes are possible.
  • Strains of Sac. erythraea carrying changes of this type offer the very high potential for the production of novel polyketides of specified structure, since they do not require synthetic substrates as do Type H mutants and they are not limited to the formation of derivatives of erythromycin, as in the case of Type I mutants, hi the embodiments of
  • Type HI mutants specified herein we have illustrated how a second copy of a complete module can be introduced at a desired position by gene conversion to result in tiie synthesis of 14-(l-propyl)erythromycin A or 14- [l(l-hydroxypropyl])erythromycin A.
  • These alterations make use of the high conservation and simultaneous lack of specificity of the ⁇ -ketoacyl ACP synthases of modules 1 and 2, thereby making possible the construction of hybrid ⁇ -ketoacyl ACP synthase functions consisting of portions of proteins derived from different modules.
  • any segment of eryA by ligation of two non-contiguous PCR-generated fragments and to subsequently construct strains, therefore, devoid of any or all portions of any module.
  • Such strains deleted of a full module can be employed for reinfroduction of either the same or a different module at a different location. It is possible, therefore, to determine the novel structures desired and then create a series of Sac. erythraea strains containing the corresponding arrangements of eryA modules that would produce said novel structures that are included within the scope of the present invention.
  • Additional examples of novel compounds produced from the construction of Type III alterations include but are not limited to 11-deoxyerythromycin, resulting from the insertion of the eryA segment encoding DH4 and ER4 in module 2.
  • two or more modules can be excised and introduced into various sites of the Sac. erythraea chromosome to produce novel polyketides of predicted structure such as the introduction of the eryA segment encoding DH4 and ER4 in both module 1 and module 2 to result in the production of 14(R)[1- hydroxypropyl]ll-deoxyerythromycin A. All combinations, therefore, of Type HI alterations and the strains of Sac. erythraea that carry said alterations as well as the polyketides produced from said strains are included within the scope of the present invention.
  • Type I, Type ⁇ and Type HI alterations and insert such alterations into Sac. erythraea to produce novel polyketides.
  • Examples of such combinations include but are not limited to the following.
  • Type I alteration such as an alteration in DH4
  • Type -CH alteration such as found in Sac. erythraea strain AM412
  • a copy of the DNA segment of module 4 is introduced in module 1
  • Sac. erythraea strain so constructed produces the compound 7-hydroxy-14-propylerythromycin A.
  • All combinations of two or more alterations of Type I, Type II and Type HI alterations, the Sac. erythraea strains that carry such alterations, and the polyketides produced from such strains are included within the scope of the present invention.
  • erythraea strains AM412 and AM512 in Examples 29 and 35, respectively, does not rely on homologous recombination between the incoming eryA module and the host chromosome. Rather, gene conversion of the host allele with the eryA allele residing on the multicopy plasmid requires DNA sequences homologous to the host allele flanking the incoming module. Thus, any module carrying the desired specificities, either from homologous or heterologous sources, can be employed in gene conversion of the host allele, provided that is flanked by segments of homology.
  • Exogenous modules constitute the source of specificities for starter and extender units other than those employed by Sac. erythraea for erythromycin biosynthesis, making it thereby possible to employ, for example, malonylCoA or (2R)- or (2S)ethylmalonylCoA, etc. as extender units, and acetyl CoA, butyryl CoA, etc. as the starter unit.
  • the result will be the formation of erythromycin analogs containing the desired functional groups and side chains with the desired stereochemistry.
  • erythraea strain carrying a heterologous module inserted into eryA requires: (i) cloning of the genes from any other Actinomyces producing a polyketide with desired structural features; (ii) mapping of the modular organization of the cloned genes by low stringency hybridization and restriction analysis; (iii) locating the module carrying the desired specificities by partial sequencing; (iv) precise excision of the desired genetic element and cloning into a vector suitable for gene conversion; (v) construction and transformation of a Sac. erythraea strain suitable for gene conversion and screening for the novel compound. Any module, or portion thereof, can thus be precisely excised from the genome of a polyketide-producing microorganism and introduced into suitable Sac.
  • erythraea strains to create a novel polyketide of predicted structure.
  • replacement of the acyltransferase segments of modules 1, 2, 3, 4, 5,or 6 in eryA with the acyltransferase segment specific for malonyl CoA such as can be found in the polyketide synthase genes for the synthesis of pikromycin in Streptomyces venezuelae, to result in t e synthesis of 12- norerythromycin A, 10-norerythromycin A, 8-norerythromycin A, 6- norerythromycin A, 4-norerythromycin A and 2-norerythromycin A, respectively, that are included within the scope of the present invention.
  • acyltransferase segments of modules 1, 2, 3, 4, 5,or 6 in eryA with an acyltransferase specific for (2R)-ethylmalonyl CoA, such as can be found in the polyketide synthase genes for the synthesis of spiramycin in Streptomyces ambofasciens, will result in the formation of 12-homoerythromycin A, 10-homoerythromycin A, 8- epihomoerythromycin A, 6-epihomoerythromycin A, 4- epihomoerythromycin A and 2-homoerythromycin A, respectively, all of which are included within the scope of the present invention.
  • acyltransferase segments carrying desired specificities for the starter or extender unit into eryA DNA that results in the synthesis of novel compounds are included within the scope of the present invention.
  • the erythromycin analogs produced by tiie method of this invention are structurally similar to known antibacterial and prokinetic agents.
  • Suitable hosts are any other polyketide-producing Actinomyces where DNA can be precisely inserted into the chromosome.
  • the choice of a convenient host is based solely on the relatedness of the novel polyketide to a natural counterpart so as to minimize the number of module rearrangements required for its biosynthesis. Therefore, Type I, Type II and Type HI alterations can be constructed in other Actinomyces employing either endogenous or exogenous modules to produce novel polyketides employing strategies analogous to those described herein for Sac. erythraea.
  • Type I, Type E or Type HI mutations or various combinations thereof constructed in other actinomycetes according to the principles described herein, and the respective polyketides produced from such strains are included within the scope of the present invention.
  • polyketides that can be altered by creating Type I, Type H or Type HI changes in the producing microorganisms include, but are not limited to macrolide antibiotics such as erythromycin, tylosin, spiramycin, etc.; ansamacrolides such as rifamycins, maytansines, etc.; polyketide antibiotics such as tetracycline; polyethers such as monesin, salinomycin, etc.; polyenes such as candicidin, amphothericins; immunosuppressants such as FK506, ascomycin, rapamycin, etc. and other complex polyketides such as avermectin.
  • erythraea such as strain AKR2
  • strain AKR2 for example, would be expected to produce the corresponding members of the ll-oxo-ll-deoxyerythromycin family, including ll-oxo-ll-deoxyerythromycin A, ll-oxo-ll-deoxyerythromycin
  • strain AM412 would be expected to produce not only 14(1- propyl)erythromycin A but also the other members of the 14(1- propyl)erythromycin family including 14(l-propyl)erythromycin B, 14(1- propyDerythromycin C and 14(l-propyl)erythromycin D. Similarly, all other modified strains of Sac.
  • erythraea shuttle vectors other vectors can be employed wherein all or part of pWHM3 or pWHM4 is replaced by other DNA segments that function in a similar manner, such as replacing the pUC19 component of pWHM3 and pWHM4 with pBR322, available from BRL, employing different segments of the pIJlOl or pJVl replicons in pWHM3 and pWHM4, respectively, or employing selectable markers other than thiostrepton- and ampicillin-resistance.
  • pWHM3 and pWHM4 DNA segments that function in a similar manner, such as replacing the pUC19 component of pWHM3 and pWHM4 with pBR322, available from BRL, employing different segments of the pIJlOl or pJVl replicons in pWHM3 and pWHM4, respectively, or employing selectable markers other than thiostrepton- and ampicillin-resistance.
  • the segments of the eryA locus subcloned into pWHM3 for generating strains AKSl, AKS2, etc. specified herein can readily be substituted for other segments of different length encoding the same fimctions, either produced by PCR-amplification of genomic DNA or of an isolated clone, or by isolating suitable restriction fragments from Sac. erythraea.
  • Sac. erythraea strains with mutant alleles other than the ⁇ - ketoacyl ACP synthase portions of eryA can be employed as hosts for gene conversion; Type HI mutants can be constructed by double reciprocal crossover as exemplified for Type I and Type II mutants rather than by the gene conversion method described herein. Additional modifications include changes in the restriction sites used for cloning or in the general methodologies described above. All such changes are included in the scope of the invention. It will also occur to those skilled in the art that different methods are available to ferment Sac. erythraea, to extract the novel polyketides specified herein, and to synthesize substrate analogs, and that all such methods are also included within the scope of the present invention. It will be apparent that many modifications and variations of the invention as set forth herein are possible without departing from the spirit and scope thereof, and that, accordingly, such limitations are imposed only as indicated by the appended claims.

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Abstract

Procédé de production de nouvelles structures de polycétides dans lequel on détermine et on introduit des modifications prédéterminées dans l'ADN commandant la synthèse du polycétide. On effectue la biosynthèse d'analogues de polycétides spécifiques en modifiant un microorganisme producteur de polycétide par manipulation génétique en isolant une séquence d'ADN contenant un gène biosynthétique de polycétide, en identifiant les activités enzymatiques associées au sein de la séquence d'ADN, en introduisant une ou plusieurs modifications prédéterminées dans la séquence d'ADN qui code une des activités enzymatiques produisant une séquence d'ADN modifiée, en introduisant cette séquence d'ADN modifiée dans le microorganisme producteur de polycétide à la place de la séquence d'origine, en cultivant le microorganisme modifié dans des conditions adaptées à la formation de l'analogue du polycétide spécifique, puis en isolant l'analogue du polycétide spécifique de cette culture. Ce procédé est plus particulièrement utile lorsque le segment du chromosome modifié est impliqué dans une activité enzymatique associée à la biosynthèse du polycétide, plus spécifiquement pour manipuler des gènes de polycétide synthase provenant du genre Saccharapolyspora ou du genre Streptomyces.
EP92905082A 1992-01-17 1992-01-17 Procede permettant d'effectuer la biosynthese de polycetides specifiques Withdrawn EP0626806A1 (fr)

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CA002100791A CA2100791C (fr) 1991-01-17 1992-01-17 Methode pour diriger la biosynthese de polycetides specifiques
PCT/US1992/000427 WO1993013663A1 (fr) 1992-01-17 1992-01-17 Procede permettant d'effectuer la biosynthese de polycetides specifiques

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