+

WO1993013663A1 - Procede permettant d'effectuer la biosynthese de polycetides specifiques - Google Patents

Procede permettant d'effectuer la biosynthese de polycetides specifiques Download PDF

Info

Publication number
WO1993013663A1
WO1993013663A1 PCT/US1992/000427 US9200427W WO9313663A1 WO 1993013663 A1 WO1993013663 A1 WO 1993013663A1 US 9200427 W US9200427 W US 9200427W WO 9313663 A1 WO9313663 A1 WO 9313663A1
Authority
WO
WIPO (PCT)
Prior art keywords
polyketide
dna sequence
enzymatic activities
plasmid
dna
Prior art date
Application number
PCT/US1992/000427
Other languages
English (en)
Inventor
Leonard Katz
Stefano Donadio
James B. Mcalpine
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 to CA002100791A priority Critical patent/CA2100791C/fr
Priority to EP92905082A priority patent/EP0626806A1/fr
Priority to PCT/US1992/000427 priority patent/WO1993013663A1/fr
Priority to AU12450/92A priority patent/AU665526B2/en
Priority claimed from CA002100791A external-priority patent/CA2100791C/fr
Publication of WO1993013663A1 publication Critical patent/WO1993013663A1/fr

Links

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 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.

Landscapes

  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Saccharide Compounds (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

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.
PCT/US1992/000427 1991-01-17 1992-01-17 Procede permettant d'effectuer la biosynthese de polycetides specifiques WO1993013663A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002100791A CA2100791C (fr) 1991-01-17 1992-01-17 Methode pour diriger la biosynthese de polycetides specifiques
EP92905082A EP0626806A1 (fr) 1992-01-17 1992-01-17 Procede permettant d'effectuer 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
AU12450/92A AU665526B2 (en) 1991-01-17 1992-01-17 Method of directing biosynthesis of specific polyketides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
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

Publications (1)

Publication Number Publication Date
WO1993013663A1 true WO1993013663A1 (fr) 1993-07-22

Family

ID=25676384

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/000427 WO1993013663A1 (fr) 1991-01-17 1992-01-17 Procede permettant d'effectuer la biosynthese de polycetides specifiques

Country Status (1)

Country Link
WO (1) WO1993013663A1 (fr)

Cited By (78)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0725778A1 (fr) * 1993-09-20 1996-08-14 The Leland Stanford Junior University Production de nouveaux polycetides par recombinaison
WO1997013845A3 (fr) * 1995-10-13 1997-06-26 Harvard College Transferases de phosphopantetheinyles et leurs utilisations
EP0791656A2 (fr) * 1996-02-22 1997-08-27 Eli Lilly And Company Gène de la platenolide-synthase
WO1997023630A3 (fr) * 1995-12-21 1997-09-25 Abbott Lab Genes de biosynthese de sucres associes a des polyketides
US5672491A (en) * 1993-09-20 1997-09-30 The Leland Stanford Junior University Recombinant production of novel polyketides
WO1998001571A3 (fr) * 1996-07-05 1998-02-19 Biotica Tech Ltd Erythromycines et leur procede de preparation
EP0836649A1 (fr) * 1995-07-06 1998-04-22 The Leland Stanford Junior University Synthese de polycetides, exempte de cellule
US5866549A (en) * 1996-09-04 1999-02-02 Abbott Laboratories 6-O-substituted ketolides having antibacterial activity
WO1998051695A3 (fr) * 1997-05-16 1999-02-04 Abbott Lab Nouveaux derives de polyketides et procedes de recombinaison pour produire ces derives
US5876991A (en) * 1996-02-22 1999-03-02 Eli Lilly And Company Polyketide synthase genes
WO1999036546A1 (fr) * 1998-01-14 1999-07-22 Glaxo Group Limited Polycetides et leur synthese
WO1999037815A1 (fr) * 1998-01-22 1999-07-29 Akzo Nobel N.V. ANALYSE FONDEE SUR UNE TRANSCRIPTION ISOTHERME ET DESTINEE A LA DETECTION ET A LA QUANTIFICATION DE CHIMIOKINES RANTES, MIP-1α ET MIP1-$g(b)
WO1999046387A1 (fr) * 1998-03-09 1999-09-16 Dow Agrosciences Llc Genes biosynthetiques de production d'insecticides a base de spinosyne
US5962290A (en) * 1993-09-20 1999-10-05 The Leland Stanford Junior University Recombinant production of novel polyketides
US6028181A (en) * 1996-09-04 2000-02-22 Abbott Laboratories 6-0-Substituted antibacterial erythromycin ketolides and methods of making
WO2000000620A3 (fr) * 1998-06-26 2000-04-13 Univ Minnesota Adn codant pour la methymycine et la pikromycine
US6054435A (en) * 1999-03-19 2000-04-25 Abbott Laboratories 6-O-substituted macrolides having antibacterial activity
US6066721A (en) * 1995-07-06 2000-05-23 Stanford University Method to produce novel polyketides
WO2000031247A2 (fr) 1998-11-20 2000-06-02 Kosan Biosciences, Inc. Matieres et procedes recombinants destines a la production d'epothilone et de derives d'epothilone
US6090601A (en) * 1998-01-23 2000-07-18 Kosan Bioscience Sorangium polyketide synthase
US6117659A (en) * 1997-04-30 2000-09-12 Kosan Biosciences, Inc. Recombinant narbonolide polyketide synthase
US6150513A (en) * 1998-09-16 2000-11-21 Kosan Biosciences, Inc. Polyketide synthase enzymes and recombinant DNA constructs therefor
WO2000022139A3 (fr) * 1998-10-09 2001-01-18 Biotechnolog Forschung Gmbh Sequences d'adn destinees a la synthese enzymatique de composes a base de polyketides ou d'heteropolyketides
US6177262B1 (en) 1998-09-22 2001-01-23 Kosan Biosciences, Inc. Recombinant host cells for the production of polyketides
US6187757B1 (en) 1995-06-07 2001-02-13 Ariad Pharmaceuticals, Inc. Regulation of biological events using novel compounds
US6271255B1 (en) 1996-07-05 2001-08-07 Biotica Technology Limited Erythromycins and process for their preparation
US6280999B1 (en) 1998-01-23 2001-08-28 Kosan Bioscience Sorangium polyketide synthases and encoding DNA therefor
US6403775B1 (en) 1998-10-28 2002-06-11 Kosan Biosciences, Inc. Erythronolide compounds
US6495348B1 (en) 1993-10-07 2002-12-17 Regents Of The University Of Minnesota Mitomycin biosynthetic gene cluster
US6500960B1 (en) 1995-07-06 2002-12-31 Stanford University (Board Of Trustees Of The Leland Stanford Junior University) Method to produce novel polyketides
US6503741B1 (en) 1998-05-28 2003-01-07 Kosan Biosciences, Inc. Polyketide synthase genes from Streptomyces venezuelae
US6503737B1 (en) 1998-10-02 2003-01-07 Kosan Biosciences, Inc. Isolated nucleic acids relating to the fkbA gene within the FK-520 polyketide synthase gene cluster
US6558942B1 (en) 1994-05-06 2003-05-06 The Leland Stanford Junior University Combinatorial polyketide libraries produced using a modular PKS gene cluster as scaffold
US6569867B2 (en) 1999-10-01 2003-05-27 Kosan Biosciences, Inc. Polyketide derivatives
US6600029B1 (en) 1995-12-19 2003-07-29 Regents Of The University Of Minnesota Metabolic engineering of polyhydroxyalkanoate monomer synthases
WO2003070908A2 (fr) 2002-02-19 2003-08-28 Dow Agrosciences Llc Nouvelles polyketide synthases produisant des spinosynes
US6753173B1 (en) 1999-02-09 2004-06-22 Board Of Trustees Of The Leland Stanford Junior University Methods to mediate polyketide synthase module effectiveness
US6794366B2 (en) 1999-04-16 2004-09-21 Kosan Biosciences, Inc. Macrolide antiinfective agents
US6825171B2 (en) 1998-01-02 2004-11-30 Pfizer, Inc. Erythromycin derivatives
US6861513B2 (en) 2000-01-12 2005-03-01 Schering Corporation Everninomicin biosynthetic genes
US6902913B2 (en) 1997-04-30 2005-06-07 Kosan Biosciences, Inc. Recombinant narbonolide polyketide synthase
US6960453B1 (en) 1996-07-05 2005-11-01 Biotica Technology Limited Hybrid polyketide synthases combining heterologous loading and extender modules
US6984635B1 (en) 1998-02-13 2006-01-10 Board Of Trustees Of The Leland Stanford Jr. University Dimerizing agents, their production and use
WO2006009276A1 (fr) * 2004-07-20 2006-01-26 Eisai R & D Management Co., Ltd. Adn codant pour un polypeptide participant à la biosynthèse d'un pladienolide
US7001748B2 (en) 1999-02-09 2006-02-21 The Board Of Trustees Of The Leland Stanford Junior University Methods of making polyketides using hybrid polyketide synthases
EP1602727A3 (fr) * 1996-07-05 2006-03-15 Biotica Technology Limited Nouvelles érythromycines et méthodes de fabrication
US7015203B2 (en) 1998-01-02 2006-03-21 Pfizer Inc. Macrolides
WO2004053065A3 (fr) * 2002-12-06 2006-03-30 Kosan Biosciences Inc Polynucleotides codant la synthase de polycetide disorazole
WO2004029220A3 (fr) * 2002-09-26 2006-04-06 Kosan Biosciences Inc Genes artificiels
US7067526B1 (en) 1999-08-24 2006-06-27 Ariad Gene Therapeutics, Inc. 28-epirapalogs
US7196192B2 (en) 1999-08-24 2007-03-27 Ariad Gene Therapeutics, Inc. 28-epirapalogs
US7229814B2 (en) 2004-05-05 2007-06-12 Regents Of The University Of Minnesota Nucleic acids and polypeptides involved in the production of cryptophycin
EP1967520A2 (fr) 2002-07-16 2008-09-10 Biotica Technology Limited Production de polykétines et autres produits naturels
US7427493B2 (en) 2002-06-28 2008-09-23 Kosan Biosciences Incorporated Recombinant genes for polyketide modifying enzymes
EP1978098A2 (fr) 1999-12-10 2008-10-08 Invitrogen Corporation Utilisation de sites à recombinaison multiples avec une spécificité unique de clonage recombinatoire
US7482137B2 (en) 2000-04-13 2009-01-27 Biotica Technology Limited Hybrid glycosylated products and their production and use
US7566558B2 (en) 2006-07-28 2009-07-28 Regents Of The University Of Michigan Nucleic acids and polypeptides involved in the production of cryptophycin
US7595156B2 (en) 2003-10-23 2009-09-29 Korea Advanced Institute Of Science And Technology Genes for synthesis of FR-008 polyketides
WO2009147984A1 (fr) 2008-06-04 2009-12-10 エーザイ・アール・アンド・ディー・マネジメント株式会社 Adn codant un polypeptide utilisé dans la biosynthèse de l’herboxidiène
EP2181704A2 (fr) 2002-12-30 2010-05-05 Angiotech International Ag Liberation de medicaments a partir d'une compostion polymere a gelification rapide
US7741456B2 (en) 1993-09-20 2010-06-22 The Board Of Trustees Of The Leland Stanford Junior University Polyketides and antibiotics produced by combinatorial techniques
WO2010106366A1 (fr) 2009-03-17 2010-09-23 Biotica Technology Limited Analogues de fk506 et pk520 et leurs utilisations pharmaceutiques
EP2261222A2 (fr) 2003-11-28 2010-12-15 Biotica Technology Limited Erythromycines et leur procede de preparation
WO2011021036A1 (fr) 2009-08-20 2011-02-24 Biotica Technology Limited Analogues de polycétides et leurs procédés de production
US7932083B2 (en) 2003-11-27 2011-04-26 Mercian Corporation DNA participating in hydroxylation of macrolide compound
EP2407473A2 (fr) 2002-02-01 2012-01-18 ARIAD Pharmaceuticals, Inc Procédé de préparation de composés contenant du phosphore
WO2012103516A1 (fr) 2011-01-28 2012-08-02 Amyris, Inc. Criblage de micro-colonies encapsulées dans du gel
WO2012158466A1 (fr) 2011-05-13 2012-11-22 Amyris, Inc. Procédés et compositions pour détecter la production microbienne de composés immiscibles avec l'eau
CN103408571A (zh) * 2013-08-23 2013-11-27 成都樵枫科技发展有限公司 利福布丁的晶型i及其制备方法和用途
WO2014025941A1 (fr) 2012-08-07 2014-02-13 Jiang Hanxiao Méthodes de stabilisation de la production de composés dérivés de l'acétyl-coenzyme a
US8759031B2 (en) 2007-04-12 2014-06-24 Wisconsin Alumni Research Foundation Type I polyketide synthase extender units
WO2014144135A2 (fr) 2013-03-15 2014-09-18 Amyris, Inc. Utilisation de phosphocétolase et de phosphotransacétylase pour la production de composés dérivés d'acétyl-coenzyme a
US8921642B2 (en) 2008-01-11 2014-12-30 Massachusetts Eye And Ear Infirmary Conditional-stop dimerizable caspase transgenic animals
WO2015020649A1 (fr) 2013-08-07 2015-02-12 Amyris, Inc. Procédés pour stabiliser la production de composés dérivés de l'acétyl-coenzyme a
US9115359B2 (en) 2007-04-02 2015-08-25 Newsouth Innovations Pty Limited Methods for producing secondary metabolites
WO2016210350A1 (fr) 2015-06-25 2016-12-29 Amyris, Inc. Dégrons dépendant du maltose, promoteurs sensibles au maltose, constructions de stabilisation, et leur utilisation dans la production de composés non cataboliques
WO2018020272A1 (fr) 2016-07-29 2018-02-01 Isomerase Therapeutics Limited Nouveaux procédés
EP3663405A1 (fr) 2013-06-11 2020-06-10 Takara Bio USA, Inc. Microvésicules enrichies en protéines et leurs procédés de fabrication et d'utilisation

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4874748A (en) * 1986-03-24 1989-10-17 Abbott Laboratories Cloning vectors for streptomyces and use thereof in macrolide antibiotic production
US4935340A (en) * 1985-06-07 1990-06-19 Eli Lilly And Company Method of isolating antibiotic biosynthetic genes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4935340A (en) * 1985-06-07 1990-06-19 Eli Lilly And Company Method of isolating antibiotic biosynthetic genes
US4874748A (en) * 1986-03-24 1989-10-17 Abbott Laboratories Cloning vectors for streptomyces and use thereof in macrolide antibiotic production

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. of Bacteriology, Volume 164, No. 1, issued October 1985, J.M. WEBER et al., "Genetic Analysis of Erythromycin Production in Streptomyces erythreus", pages 425-433, see the entire document. *
J. of Bacteriology, Volume 172, No. 5, issued May 1990, J.M. WEBER et al., "Organization of a Cluster of Erythromycin Genes in Saccharomyces erythraea", pages 2372-2383, see the entire document. *

Cited By (151)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5843718A (en) * 1993-09-20 1998-12-01 The Leland Stanford Junior University Recombinant production of novel polyketides
US6399382B1 (en) 1993-09-20 2002-06-04 The Leland Stanford Junior University Recombinant production of novel polyketides
US7312048B2 (en) 1993-09-20 2007-12-25 The Board Of Trustees Of The Leland Stanford University Recombinant production of novel polyketides
US6215007B1 (en) 1993-09-20 2001-04-10 The Leland Stanford Junior Univ. Recombinant production of novel polyketides
US5672491A (en) * 1993-09-20 1997-09-30 The Leland Stanford Junior University Recombinant production of novel polyketides
EP0725778A1 (fr) * 1993-09-20 1996-08-14 The Leland Stanford Junior University Production de nouveaux polycetides par recombinaison
US6214573B1 (en) 1993-09-20 2001-04-10 The Leland Stanford Junior Unv. Recombinant production of novel polyketides
EP1118665A1 (fr) * 1993-09-20 2001-07-25 The Leland Stanford Junior University Production recombinante de polykétides aromatiques
US5830750A (en) * 1993-09-20 1998-11-03 The John Innes Institute Recombinant production of novel polyketides
US6077696A (en) * 1993-09-20 2000-06-20 The Johns Innes Institute Recombinant production of novel polyketides
US6022731A (en) * 1993-09-20 2000-02-08 The Leland Stanford Junior University Recombinant production of novel polyketides
US6461838B2 (en) 1993-09-20 2002-10-08 The Leland Stanford Junior University Recombinant production of novel polyketides
EP0725778A4 (fr) * 1993-09-20 1998-11-11 Univ Leland Stanford Junior Production de nouveaux polycetides par recombinaison
US6391594B1 (en) 1993-09-20 2002-05-21 Kosan Biosciences, Inc. Modified modular PKS with retained scaffold
US6969611B2 (en) 1993-09-20 2005-11-29 The Board Of Trustees Of The Leland Stanford University Methods to prepare cells comprising heterologous polyketide synthase expression systems
US5962290A (en) * 1993-09-20 1999-10-05 The Leland Stanford Junior University Recombinant production of novel polyketides
US7741456B2 (en) 1993-09-20 2010-06-22 The Board Of Trustees Of The Leland Stanford Junior University Polyketides and antibiotics produced by combinatorial techniques
EP1568774A3 (fr) * 1993-09-20 2005-11-30 The Leland Stanford Junior University Production recombinante de polykétides
US6495348B1 (en) 1993-10-07 2002-12-17 Regents Of The University Of Minnesota Mitomycin biosynthetic gene cluster
US6558942B1 (en) 1994-05-06 2003-05-06 The Leland Stanford Junior University Combinatorial polyketide libraries produced using a modular PKS gene cluster as scaffold
US6649595B2 (en) 1995-06-07 2003-11-18 Ariad Gene Therapeutics, Inc. Regulation of biological events using novel compounds
US6187757B1 (en) 1995-06-07 2001-02-13 Ariad Pharmaceuticals, Inc. Regulation of biological events using novel compounds
US6080555A (en) * 1995-07-06 2000-06-27 Stanford University Synthesis of polyketides from diketides
US6274560B1 (en) 1995-07-06 2001-08-14 Brown University Research Foundation Cell-free synthesis of polyketides
US6750040B1 (en) 1995-07-06 2004-06-15 Board Of Trustees Of The Leland Stanford Junior University Cell-free synthesis of polyketides
US6500960B1 (en) 1995-07-06 2002-12-31 Stanford University (Board Of Trustees Of The Leland Stanford Junior University) Method to produce novel polyketides
US6066721A (en) * 1995-07-06 2000-05-23 Stanford University Method to produce novel polyketides
US6531299B1 (en) 1995-07-06 2003-03-11 Stanford University Cell-free synthesis of polyketides
US6710189B2 (en) 1995-07-06 2004-03-23 Stanford University Method to produce novel polyketides
EP1493814A1 (fr) 1995-07-06 2005-01-05 The Leland Stanford Junior University Synthèse de polycetides, exempté de cellule
US6261816B1 (en) 1995-07-06 2001-07-17 Stanford University Method to produce novel polyketides
EP0836649A4 (fr) * 1995-07-06 2000-04-05 Univ Leland Stanford Junior Synthese de polycetides, exempte de cellule
EP0836649A1 (fr) * 1995-07-06 1998-04-22 The Leland Stanford Junior University Synthese de polycetides, exempte de cellule
US7101684B2 (en) 1995-07-06 2006-09-05 Stanford University Modified modular polyketide synthase
US7192735B2 (en) 1995-10-13 2007-03-20 President & Fellows Of Harvard College Phosphopantetheinyl transferases and uses thereof
US6579695B1 (en) 1995-10-13 2003-06-17 President And Fellows Of Harvard College Phosphopantetheinyl transferases and uses thereof
WO1997013845A3 (fr) * 1995-10-13 1997-06-26 Harvard College Transferases de phosphopantetheinyles et leurs utilisations
US6600029B1 (en) 1995-12-19 2003-07-29 Regents Of The University Of Minnesota Metabolic engineering of polyhydroxyalkanoate monomer synthases
WO1997023630A3 (fr) * 1995-12-21 1997-09-25 Abbott Lab Genes de biosynthese de sucres associes a des polyketides
US5998194A (en) * 1995-12-21 1999-12-07 Abbott Laboratories Polyketide-associated sugar biosynthesis genes
US5945320A (en) * 1996-02-22 1999-08-31 Eli Lilly And Company Platenolide synthase gene
EP0791655A3 (fr) * 1996-02-22 2000-10-18 Eli Lilly And Company Gènes de polyketide-synthase
EP0791656A2 (fr) * 1996-02-22 1997-08-27 Eli Lilly And Company Gène de la platenolide-synthase
US5876991A (en) * 1996-02-22 1999-03-02 Eli Lilly And Company Polyketide synthase genes
EP0791656A3 (fr) * 1996-02-22 2000-10-18 Eli Lilly And Company Gène de la platenolide-synthase
EP2182067A3 (fr) * 1996-07-05 2010-07-14 Biotica Technology Limited Polykétide synthases hybrides
EP1602727A3 (fr) * 1996-07-05 2006-03-15 Biotica Technology Limited Nouvelles érythromycines et méthodes de fabrication
GB2331518B (en) * 1996-07-05 2001-03-14 Biotica Tech Ltd Erythromycins and process for their preparation
WO1998001571A3 (fr) * 1996-07-05 1998-02-19 Biotica Tech Ltd Erythromycines et leur procede de preparation
US6271255B1 (en) 1996-07-05 2001-08-07 Biotica Technology Limited Erythromycins and process for their preparation
WO1998001546A3 (fr) * 1996-07-05 1998-04-09 Biotica Tech Ltd Polyketides et leur synthese
US6960453B1 (en) 1996-07-05 2005-11-01 Biotica Technology Limited Hybrid polyketide synthases combining heterologous loading and extender modules
GB2331518A (en) * 1996-07-05 1999-05-26 Biotica Tech Ltd Erythromycins and process for their preparation
AP1029A (en) * 1996-07-05 2001-12-11 Biotica Tech Limited Erythromycins and process for their preparation.
US7807418B2 (en) 1996-07-05 2010-10-05 Biotica Technology Limited Method for producing hybrid polyketide synthases
EP2182067A2 (fr) * 1996-07-05 2010-05-05 Biotica Technology Limited Polykétide synthases hybrides
US5866549A (en) * 1996-09-04 1999-02-02 Abbott Laboratories 6-O-substituted ketolides having antibacterial activity
USRE39591E1 (en) * 1996-09-04 2007-04-24 Abbott Laboratories 6-O-substituted ketolides having antibacterial activity
US6028181A (en) * 1996-09-04 2000-02-22 Abbott Laboratories 6-0-Substituted antibacterial erythromycin ketolides and methods of making
US6075133A (en) * 1996-09-04 2000-06-13 Abbott Laboratories 6-O-substituted antibacterial erythromycin ketolides and methods of making
US6147197A (en) * 1996-09-04 2000-11-14 Or; Yat Sun 6-O-substituted erythromycin ketolides having antibacterial activity
US6509455B1 (en) 1997-04-30 2003-01-21 Kosan Biosciences, Inc. Recombinant narbonolide polyketide synthase
US6117659A (en) * 1997-04-30 2000-09-12 Kosan Biosciences, Inc. Recombinant narbonolide polyketide synthase
US6902913B2 (en) 1997-04-30 2005-06-07 Kosan Biosciences, Inc. Recombinant narbonolide polyketide synthase
WO1998051695A3 (fr) * 1997-05-16 1999-02-04 Abbott Lab Nouveaux derives de polyketides et procedes de recombinaison pour produire ces derives
EP1642901A1 (fr) 1998-01-02 2006-04-05 Pfizer Products Inc. Nouveaux derives d'Erythromycine
US6825171B2 (en) 1998-01-02 2004-11-30 Pfizer, Inc. Erythromycin derivatives
US7015203B2 (en) 1998-01-02 2006-03-21 Pfizer Inc. Macrolides
WO1999036546A1 (fr) * 1998-01-14 1999-07-22 Glaxo Group Limited Polycetides et leur synthese
US6218154B1 (en) 1998-01-22 2001-04-17 Akzo Nobel N.V. Isothermal transcription based assay for the detection and quantification of chemokines rantes, MIP-1α and MIP-1β
WO1999037815A1 (fr) * 1998-01-22 1999-07-29 Akzo Nobel N.V. ANALYSE FONDEE SUR UNE TRANSCRIPTION ISOTHERME ET DESTINEE A LA DETECTION ET A LA QUANTIFICATION DE CHIMIOKINES RANTES, MIP-1α ET MIP1-$g(b)
US6121023A (en) * 1998-01-22 2000-09-19 Akzo Nobel N.V. Isothermal transcription based assay for the detection and quantification of the chemokine rantes
US6280999B1 (en) 1998-01-23 2001-08-28 Kosan Bioscience Sorangium polyketide synthases and encoding DNA therefor
US6090601A (en) * 1998-01-23 2000-07-18 Kosan Bioscience Sorangium polyketide synthase
US6984635B1 (en) 1998-02-13 2006-01-10 Board Of Trustees Of The Leland Stanford Jr. University Dimerizing agents, their production and use
US6143526A (en) * 1998-03-09 2000-11-07 Baltz; Richard H. Biosynthetic genes for spinosyn insecticide production
WO1999046387A1 (fr) * 1998-03-09 1999-09-16 Dow Agrosciences Llc Genes biosynthetiques de production d'insecticides a base de spinosyne
US6274350B1 (en) 1998-03-09 2001-08-14 Dow Agrosciences Llc Biosynthetic genes for spinosyn insecticide production
US7015001B2 (en) 1998-03-09 2006-03-21 Dow Agrosciences Llc DNA encoding SPNF of the spinosyn cluster
US6521406B1 (en) 1998-03-09 2003-02-18 Dow Agrosciences Llc SpnG, a gene for spinosyn insecticide biosynthesis
US6503741B1 (en) 1998-05-28 2003-01-07 Kosan Biosciences, Inc. Polyketide synthase genes from Streptomyces venezuelae
US6265202B1 (en) 1998-06-26 2001-07-24 Regents Of The University Of Minnesota DNA encoding methymycin and pikromycin
WO2000000620A3 (fr) * 1998-06-26 2000-04-13 Univ Minnesota Adn codant pour la methymycine et la pikromycine
US6150513A (en) * 1998-09-16 2000-11-21 Kosan Biosciences, Inc. Polyketide synthase enzymes and recombinant DNA constructs therefor
US6177262B1 (en) 1998-09-22 2001-01-23 Kosan Biosciences, Inc. Recombinant host cells for the production of polyketides
US6759536B2 (en) 1998-10-02 2004-07-06 Kosan Biosciences, Inc. Polynucleotides encoding the fkbA gene of the FK-520 polyketide synthase gene cluster
US6503737B1 (en) 1998-10-02 2003-01-07 Kosan Biosciences, Inc. Isolated nucleic acids relating to the fkbA gene within the FK-520 polyketide synthase gene cluster
US7714118B2 (en) 1998-10-02 2010-05-11 Kosan Biosciences Incorporated Polynucleotides encoding the fkbB gene of the FK-520 polyketide synthase gene cluster
US6660862B2 (en) 1998-10-02 2003-12-09 Kosan Biosciences, Inc. Polyketide synthase enzymes and recombinant DNA constructs therefor
USRE39762E1 (en) * 1998-10-02 2007-08-07 Kosan Biosciences, Inc. Polyketide synthase enzymes and recombinant DNA constructs therefor
WO2000022139A3 (fr) * 1998-10-09 2001-01-18 Biotechnolog Forschung Gmbh Sequences d'adn destinees a la synthese enzymatique de composes a base de polyketides ou d'heteropolyketides
US6403775B1 (en) 1998-10-28 2002-06-11 Kosan Biosciences, Inc. Erythronolide compounds
WO2000031247A2 (fr) 1998-11-20 2000-06-02 Kosan Biosciences, Inc. Matieres et procedes recombinants destines a la production d'epothilone et de derives d'epothilone
US7001748B2 (en) 1999-02-09 2006-02-21 The Board Of Trustees Of The Leland Stanford Junior University Methods of making polyketides using hybrid polyketide synthases
US6753173B1 (en) 1999-02-09 2004-06-22 Board Of Trustees Of The Leland Stanford Junior University Methods to mediate polyketide synthase module effectiveness
US6054435A (en) * 1999-03-19 2000-04-25 Abbott Laboratories 6-O-substituted macrolides having antibacterial activity
US6794366B2 (en) 1999-04-16 2004-09-21 Kosan Biosciences, Inc. Macrolide antiinfective agents
US7196192B2 (en) 1999-08-24 2007-03-27 Ariad Gene Therapeutics, Inc. 28-epirapalogs
US7067526B1 (en) 1999-08-24 2006-06-27 Ariad Gene Therapeutics, Inc. 28-epirapalogs
US6624302B2 (en) 1999-10-01 2003-09-23 Kosan Biosciences, Inc. Polyketide derivatives
US6569867B2 (en) 1999-10-01 2003-05-27 Kosan Biosciences, Inc. Polyketide derivatives
EP1978098A2 (fr) 1999-12-10 2008-10-08 Invitrogen Corporation Utilisation de sites à recombinaison multiples avec une spécificité unique de clonage recombinatoire
EP2210948A2 (fr) 1999-12-10 2010-07-28 Life Technologies Corporation Utilisation de sites à recombinaison multiples avec une spécificité unique de clonage recombinatoire
US7947480B2 (en) 2000-01-12 2011-05-24 Schering Corporation Everninomicin biosynthetic genes
US7229813B2 (en) 2000-01-12 2007-06-12 Schering Corporation Everninomicin biosynthetic proteins
US7790411B2 (en) 2000-01-12 2010-09-07 Schering Corporation Everninomicin biosynthetic genes
US6861513B2 (en) 2000-01-12 2005-03-01 Schering Corporation Everninomicin biosynthetic genes
US7482137B2 (en) 2000-04-13 2009-01-27 Biotica Technology Limited Hybrid glycosylated products and their production and use
EP2407473A2 (fr) 2002-02-01 2012-01-18 ARIAD Pharmaceuticals, Inc Procédé de préparation de composés contenant du phosphore
WO2003070908A2 (fr) 2002-02-19 2003-08-28 Dow Agrosciences Llc Nouvelles polyketide synthases produisant des spinosynes
US7427493B2 (en) 2002-06-28 2008-09-23 Kosan Biosciences Incorporated Recombinant genes for polyketide modifying enzymes
EP2277898A2 (fr) 2002-07-16 2011-01-26 Biotica Technology Limited Analogues de Rapamycine
EP1967520A2 (fr) 2002-07-16 2008-09-10 Biotica Technology Limited Production de polykétines et autres produits naturels
JP2006517090A (ja) * 2002-09-26 2006-07-20 コーサン バイオサイエンシーズ， インコーポレイテッド 合成遺伝子
WO2004029220A3 (fr) * 2002-09-26 2006-04-06 Kosan Biosciences Inc Genes artificiels
US7364877B2 (en) 2002-12-06 2008-04-29 Kosan Biosciences, Inc. Polynucleotides encoding disorazole polyketide synthase polypeptides
WO2004053065A3 (fr) * 2002-12-06 2006-03-30 Kosan Biosciences Inc Polynucleotides codant la synthase de polycetide disorazole
EP2181704A2 (fr) 2002-12-30 2010-05-05 Angiotech International Ag Liberation de medicaments a partir d'une compostion polymere a gelification rapide
US7595156B2 (en) 2003-10-23 2009-09-29 Korea Advanced Institute Of Science And Technology Genes for synthesis of FR-008 polyketides
US7932083B2 (en) 2003-11-27 2011-04-26 Mercian Corporation DNA participating in hydroxylation of macrolide compound
EP2261222A2 (fr) 2003-11-28 2010-12-15 Biotica Technology Limited Erythromycines et leur procede de preparation
US7662599B2 (en) 2004-05-05 2010-02-16 Regents Of The University Of Minnesota Nucleic acids and polypeptides involved in the production of cryptophycin
US7229814B2 (en) 2004-05-05 2007-06-12 Regents Of The University Of Minnesota Nucleic acids and polypeptides involved in the production of cryptophycin
JPWO2006009276A1 (ja) * 2004-07-20 2008-05-01 エーザイ・アール・アンド・ディー・マネジメント株式会社 プラジエノライドの生合成に関与するポリペプチドをコードするｄｎａ
JP4599357B2 (ja) * 2004-07-20 2010-12-15 エーザイ・アール・アンド・ディー・マネジメント株式会社 プラジエノライドの生合成に関与するポリペプチドをコードするｄｎａ
WO2006009276A1 (fr) * 2004-07-20 2006-01-26 Eisai R & D Management Co., Ltd. Adn codant pour un polypeptide participant à la biosynthèse d'un pladienolide
US8008049B2 (en) 2004-07-20 2011-08-30 Eisai R&D Management Co., Ltd. DNA coding for polypeptide participating in biosynthesis of pladienolide
US8313936B2 (en) 2006-07-28 2012-11-20 Regents Of The University Of Michigan Nucleic acids and polypeptides involved in the production of cryptophycin
US7566558B2 (en) 2006-07-28 2009-07-28 Regents Of The University Of Michigan Nucleic acids and polypeptides involved in the production of cryptophycin
US9115359B2 (en) 2007-04-02 2015-08-25 Newsouth Innovations Pty Limited Methods for producing secondary metabolites
US8759031B2 (en) 2007-04-12 2014-06-24 Wisconsin Alumni Research Foundation Type I polyketide synthase extender units
US8921642B2 (en) 2008-01-11 2014-12-30 Massachusetts Eye And Ear Infirmary Conditional-stop dimerizable caspase transgenic animals
US8512995B2 (en) 2008-06-04 2013-08-20 Eisai R&D Management Co., Ltd. DNA encoding polypeptide involved in biosynthesis of herboxidiene
WO2009147984A1 (fr) 2008-06-04 2009-12-10 エーザイ・アール・アンド・ディー・マネジメント株式会社 Adn codant un polypeptide utilisé dans la biosynthèse de l’herboxidiène
EP2546345A1 (fr) 2008-06-04 2013-01-16 Eisai R&D Management Co., Ltd. ADN codant un polypeptide intervenant dans la biosynthèse d'herboxidiène
WO2010106366A1 (fr) 2009-03-17 2010-09-23 Biotica Technology Limited Analogues de fk506 et pk520 et leurs utilisations pharmaceutiques
WO2011021036A1 (fr) 2009-08-20 2011-02-24 Biotica Technology Limited Analogues de polycétides et leurs procédés de production
WO2012103516A1 (fr) 2011-01-28 2012-08-02 Amyris, Inc. Criblage de micro-colonies encapsulées dans du gel
WO2012158466A1 (fr) 2011-05-13 2012-11-22 Amyris, Inc. Procédés et compositions pour détecter la production microbienne de composés immiscibles avec l'eau
WO2014025941A1 (fr) 2012-08-07 2014-02-13 Jiang Hanxiao Méthodes de stabilisation de la production de composés dérivés de l'acétyl-coenzyme a
WO2014144135A2 (fr) 2013-03-15 2014-09-18 Amyris, Inc. Utilisation de phosphocétolase et de phosphotransacétylase pour la production de composés dérivés d'acétyl-coenzyme a
EP4491726A2 (fr) 2013-06-11 2025-01-15 Takara Bio USA, Inc. Microvésicules enrichies en protéines et leurs procédés de fabrication et d'utilisation
EP3663405A1 (fr) 2013-06-11 2020-06-10 Takara Bio USA, Inc. Microvésicules enrichies en protéines et leurs procédés de fabrication et d'utilisation
EP3663392A1 (fr) 2013-08-07 2020-06-10 Amyris, Inc. Procédés pour stabiliser la production de composés dérivés de l'acétyl-coenzyme a
WO2015020649A1 (fr) 2013-08-07 2015-02-12 Amyris, Inc. Procédés pour stabiliser la production de composés dérivés de l'acétyl-coenzyme a
CN103408571A (zh) * 2013-08-23 2013-11-27 成都樵枫科技发展有限公司 利福布丁的晶型i及其制备方法和用途
CN103408571B (zh) * 2013-08-23 2015-11-18 成都樵枫科技发展有限公司 利福布丁的晶型i及其制备方法和用途
WO2016210343A1 (fr) 2015-06-25 2016-12-29 Amyris, Inc. Dégrons dépendant du maltose, promoteurs sensibles au maltose, constructions de stabilisation, et leur utilisation dans la production de composés non cataboliques
WO2016210350A1 (fr) 2015-06-25 2016-12-29 Amyris, Inc. Dégrons dépendant du maltose, promoteurs sensibles au maltose, constructions de stabilisation, et leur utilisation dans la production de composés non cataboliques
WO2018020272A1 (fr) 2016-07-29 2018-02-01 Isomerase Therapeutics Limited Nouveaux procédés
EP3960857A1 (fr) 2016-07-29 2022-03-02 Isomerase Therapeutics Limited Nouveaux procédés

Similar Documents

Publication Publication Date Title
US5824513A (en) Recombinant DNA method for producing erythromycin analogs
WO1993013663A1 (fr) Procede permettant d'effectuer la biosynthese de polycetides specifiques
Ruan et al. Acyltransferase domain substitutions in erythromycin polyketide synthase yield novel erythromycin derivatives
US6200813B1 (en) Polyketide derivatives and recombinant methods for making same
US10047363B2 (en) NRPS-PKS gene cluster and its manipulation and utility
CA2322449C (fr) Genes biosynthetiques de production d'insecticides a base de spinosyne
CA2197160C (fr) Gene codant la platenolide synthase
AU731654B2 (en) Polyketides and their synthesis
WO1997023630A2 (fr) Genes de biosynthese de sucres associes a des polyketides
JP2005529579A (ja) ポリケチドの生合成のための過剰産生宿主
EP1414969B1 (fr) Genes biosynthetiques destines a la production d'un insecticide de butenyle-spinosyne
AU665526B2 (en) Method of directing biosynthesis of specific polyketides
EP0626806A1 (fr) Procede permettant d'effectuer la biosynthese de polycetides specifiques
US7807418B2 (en) Method for producing hybrid polyketide synthases
WO2001068867A1 (fr) Polycetides et leur synthese
US7595156B2 (en) Genes for synthesis of FR-008 polyketides
He Molecular analysis of the aureothin biosynthesis gene cluster from streptomyces thioluteus HKI-227: new insights into polyketide assemply
CA2241139A1 (fr) Genes de biosynthese de sucres associes a des polyketides
JPH07177892A (ja) マクロライド抗生物質の３位アシル化酵素をコードする遺伝子
AU2002305118A1 (en) Biosynthetic genes for butenyl-spinosyn insecticide production
JP2009213479A (ja) ポリケチド類及びそれらの合成

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 1992905082

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2100791

Country of ref document: CA

AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP KR

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU MC NL SE

WWP Wipo information: published in national office

Ref document number: 1992905082

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1992905082

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载