WO2003068955A2 - Streptomyces avermitilis gene directing the ratio of b2:b1 avermectins - Google Patents

Streptomyces avermitilis gene directing the ratio of b2:b1 avermectins Download PDF

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WO2003068955A2
WO2003068955A2 PCT/IB2003/000348 IB0300348W WO03068955A2 WO 2003068955 A2 WO2003068955 A2 WO 2003068955A2 IB 0300348 W IB0300348 W IB 0300348W WO 03068955 A2 WO03068955 A2 WO 03068955A2
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Prior art keywords
amino acid
avec
avermitilis
cyclohexyl
combination
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English (en)
French (fr)
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WO2003068955A3 (en
Inventor
Kim Jonelle Stutzman-Engwall
Claes Eric Daniel Gustafsson
Anke Krebber
Sun Ai Raillard
Jeremy Stephen Minshull
Seran Kim
Yan Chen
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Pfizer Products Inc
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Pfizer Products Inc
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Priority to CN038037416A priority Critical patent/CN1630712B/zh
Priority to KR1020047012437A priority patent/KR100718378B1/ko
Priority to JP2003568070A priority patent/JP2005517406A/ja
Priority to SI200331553T priority patent/SI1476539T1/sl
Priority to DE60326824T priority patent/DE60326824D1/de
Priority to AU2003245052A priority patent/AU2003245052B2/en
Priority to MEP-2008-730A priority patent/ME00472B/me
Priority to IL16286603A priority patent/IL162866A0/xx
Priority to EP03739390A priority patent/EP1476539B1/en
Priority to YUP-700/04A priority patent/RS50813B/sr
Priority to DK03739390T priority patent/DK1476539T3/da
Application filed by Pfizer Products Inc filed Critical Pfizer Products Inc
Priority to BRPI0307620A priority patent/BRPI0307620B8/pt
Priority to CA2475214A priority patent/CA2475214C/en
Priority to HK05106917.2A priority patent/HK1074644B/xx
Priority to MXPA04007777A priority patent/MXPA04007777A/es
Publication of WO2003068955A2 publication Critical patent/WO2003068955A2/en
Publication of WO2003068955A3 publication Critical patent/WO2003068955A3/en
Priority to IL162866A priority patent/IL162866A/en
Anticipated expiration legal-status Critical
Priority to IL192371A priority patent/IL192371A/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
    • 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
    • C12P19/623—Avermectin; Milbemycin; Ivermectin; C-076

Definitions

  • the present invention is directed to compositions and methods for the efficient production of avermectins such as "doramectin", which are primarily useful in the field of animal health. More particularly, the present invention relates to polynucleotide molecules comprising nucleotide sequences encoding an AveC gene product, which can be used to modulate the ratio of class 2:1 avermectins produced by fermentation cultures of Streptomyces avermitilis. The present invention further relates to vectors, transformed host cells, and novel mutant strains of S. avermitilis in which the aveC gene has been mutated so as to modulate the ratio of class 2:1 avermectins produced.
  • Streptomyces species produce a wide variety of secondary metabolites, including the avermectins, which comprise a series of eight related sixteen-membered macrocyclic lactones having potent anthelmintic and insecticidal activity.
  • the eight distinct but closely related compounds are referred to as A1a, A1b, A2a, A2b, B1a, B1b, B2a and B2b.
  • the "a” series of compounds refers to the natural avermectin where the substituent at the C25 position is (S)- sec-butyl, and the "b” series refers to those compounds where the substituent at the C25 position is isopropyl.
  • the designations "A” and “B” refer to avermectins where the substituent at the C5 position is methoxy and hydroxy, respectively.
  • the numeral “1” refers to avermectins where a double bond is present at the C22.23 position
  • the numeral “2” refers to avermectins having a hydrogen at the C22 position and a hydroxy at the C23 position.
  • the B1 type of avermectin such as doramectin, is recognized as having the most effective antiparasitic and pesticidal activity, and is therefore the most commercially desirable avermectin.
  • avermectins and their production by aerobic fermentation of strains of S. avermitilis are described in United States Patent Nos. 4,310,519 and 4,429,042.
  • the biosynthesis of natural avermectins is believed to be initiated endogenously from the CoA thioester analogs of isobutyric acid and S-(+)-2-methyl butyric acid.
  • Fermentation of such mutants in the presence of exogenously supplied fatty acids results in production of only the four avermectins corresponding to the fatty acid employed.
  • S. avermitilis ATCC 53567
  • S-(+)-2-methylbutyric acid results in production of the natural avermectins A1 a, A2a, B1a and B2a
  • isobutyric acid results in production of the natural avermectins A1b, A2b, B1 b, and B2b
  • supplementing fermentations with cyclopentanecarboxylic acid results in the production of the four novel cyclopentylavermectins A1 , A2, B1 , and B2.
  • novel avermectins are produced. By screening over 800 potential precursors, more than 60 other novel avermectins have been identified. (See, e.g., Dutton ef al., 1991 , J. Antibiot. 44:357-365; and Banks et al., 1994, Roy. Soc. Chem. 147:16-26). In addition, mutants of S. avermitilis deficient in 5-O-methyltransferase activity produce essentially only the B analog avermectins. Consequently, S.
  • avermitilis mutants lacking both branched-chain 2-oxo acid dehydrogenase and 5-O-methyltransferase activity produce only the B avermectins corresponding to the fatty acid employed to supplement the fermentation.
  • supplementing such double mutants with S-(+)-2- methylbutyric acid results in production of only the natural avermectins B1a and B2a
  • supplementing with isobutyric acid or cyclopentanecarboxylic acid results in production of the natural avermectins B1b and B2b or the novel cyclopentyl B1 and B2 avermectins, respectively.
  • portions of a DNA library from an organism capable of producing a particular metabolite are introduced into a non- producing mutant and transformants screened for production of the metabolite.
  • hybridization of a library using probes derived from other Streptomyces species has been used to identify and clone genes in biosynthetic pathways.
  • Genes involved in avermectin biosynthesis like the genes required for biosynthesis of other Streptomyces secondary metabolites (e.g., PKS), are found clustered on the chromosome.
  • a number of ave genes have been successfully cloned using vectors to complement S. avermitilis mutants blocked in avermectin biosynthesis.
  • ivermectin a potent anthelmintic compound
  • avermectin B2a such a single component producer of avermectin B2a is considered particularly useful for commercial production of ivermectin.
  • U.S. Patent No. 6,248,579 to Stutzman-Engwall et al., issued June 19, 2001 describes certain mutations to the aveC gene of Streptomyces avermitilis leading to a reduction in the ratio of cyclohexyl B2:cyclohexyl B1 ratio to about 0.75:1.
  • the present invention provides a polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as the Streptomyces avermitilis aveC allele, the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604) or the nucleotide sequence of the aveC ORF of S. avermitilis as presented in FIGURE 1 (SEQ ID NO:1 ), or a degenerate variant thereof, but which nucleotide sequence further comprises mutations encoding a combination of amino acid substitutions at amino acid residues corresponding to the amino acid positions of SEQ ID NO:2, such that cells of S.
  • avermitilis strain ATCC 53692 in which the wild-type aveC allele has been inactivated and that express the polynucleotide molecule comprising the mutated nucleotide sequence are capable of producing a class 2:1 ratio of avermectins that is reduced compared to the ratio produced by cells of S.
  • the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.30:1 or less. In a more preferred embodiment, the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.25:1 or less. In a more preferred embodiment, the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.20:1 or less.
  • the combination of amino acid substitutions comprises a combination selected from the group consisting of: (a) D48E, A61T, A89T, S138T, A139T, G179S, A198G, P289L;
  • the present invention further provides a polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as the Streptomyces avermitilis aveC allele, the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604) or the nucleotide sequence of the aveC ORF of S. avermitilis as presented in FIGURE 1 (SEQ ID NO:1), or a degenerate variant thereof, but which nucleotide sequence further comprises mutations encoding a combination of amino acid substitutions at amino acid residues corresponding to the amino acid positions of SEQ ID NO:2, such that cells of S.
  • avermitilis strain ATCC 53692 in which the wild-type aveC allele has been inactivated and that express a polynucleotide molecule comprising the mutated nucleotide sequence are capable of producing a class 2:1 ratio of avermectins that is reduced compared to the ratio produced by cells of S.
  • avermitilis strain ATCC 53692 that instead express only the wild-type aveC allele, wherein when the class 2:1 avermectins are cyclohexyl B2:cyclohexyl B1 avermectins, the ratio of class 2:1 avermectins is reduced to about 0.40:1 or less, and wherein the combination of amino acid substitutions comprises a combination selected from the group consisting of: (bf) D48E, S138T, A139T, G179S, E238D; and (bg) Y28C, Q38R, D48E, L136P, G179S, E238D.
  • the present invention further provides a recombinant vector comprising a polynucleotide molecule of the present invention.
  • the present invention further provides a host cell comprising a polynucleotide molecule or a recombinant vector of the present invention.
  • the host cell is a Streptomyces cell.
  • the host cell is a cell of Streptomyces avermitilis.
  • the present invention further provides a method for making a novel strain of Streptomyces avermitilis, comprising (i) mutating the aveC allele in a cell of a strain of S. avermitilis, which mutation results in a combination of amino acid substitutions in the AveC gene product, or (ii) introducing into a cell of a strain of S. avermitilis a mutated aveC allele or degenerate variant thereof encoding an AveC gene product ' comprising a combination of amino acid substitutions, wherein the combination of amino acid substitutions is selected from (a) through (be) listed above.
  • the present invention further provides a method for making a novel strain of Streptomyces avermitilis, comprising (i) mutating the aveC allele in a cell of a strain of S. avermitilis, which mutation results in a combination of amino acid substitutions in the AveC gene product, or (ii) introducing into a cell of a strain of S.
  • avermitilis a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions, wherein cells comprising the mutated aveC allele or degenerate variant are capable of producing cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of 0.35:1 or less.
  • the mutated aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (a) through (be) listed above.
  • the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.30:1 or less.
  • the mutated aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (f) through (be) listed above.
  • the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.25:1 or less.
  • the mutated aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (w) through (be) listed above.
  • the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.20:1 or less.
  • the mutated aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (ao) through (be) listed above.
  • the present invention further provides a method for making a novel strain of
  • Streptomyces avermitilis comprising (i) mutating the aveC allele in a cell of a strain of S. avermitilis, which mutation results in a combination of amino acid substitutions in the AveC gene product, or (ii) introducing into a cell of a strain of S. avermitilis a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions, wherein the combination of amino acid substitutions is selected from the group consisting of (bf) and (bg) listed above.
  • cells of S are selected from the group consisting of (bf) and (bg) listed above.
  • avermitilis comprising such a mutated aveC allele or degenerate variant are capable of producing cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of about 0.40:1 or less.
  • the present invention further provides a cell of a Streptomyces species that comprises a mutated S. avermitilis aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from (a) through (be) listed above.
  • the species of Streptomyces is S. avermitilis.
  • the present invention further provides a cell of Streptomyces avermitilis capable of producing cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of 0.35:1 or less.
  • the cell comprises a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (a) through (be) listed above.
  • the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.30:1 or less.
  • the cells comprise a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (f) through (be) listed above.
  • the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.25:1 or less.
  • the cells comprise a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (w) through (be) listed above.
  • the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.20:1 or less.
  • the cells comprise a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (ao) through (be) listed above.
  • the present invention further provides a cell of a Streptomyces species, comprising a mutated S. avermitilis aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (bf) and (bg) listed above.
  • the species of Streptomyces is S. avermitilis.
  • the cell is a cell of S. avermitilis capable of producing cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of about 0.40:1 or less.
  • the present invention further provides a process for producing avermectins, comprising culturing a strain of Streptomyces avermitilis cells of the present invention in culture media under conditions that permit or induce the production of avermectins therefrom, and recovering said avermectins from the culture.
  • the present invention further provides a composition of cyclohexyl B2:cyclohexyl B1 avermectins produced by cells of Streptomyces avermitilis, comprising the cyclohexyl B2:cyclohexyl B1 avermectins present in a culture medium in which the cells have been cultured, wherein the ratio of the cyclohexyl B2:cyclohexyl B1 avermectins present in the culture medium is 0.35:1 or less, preferably about 0.30:1 or less, more preferably about 0.25:1 or less, and more preferably about 0.20:1 or less.
  • the composition of cyclohexyl B2:cyclohexyl B1 avermectins is produced by cells of a strain of S. avermitilis that express a mutated aveC allele or degenerate variant thereof which encodes a gene product that results in the reduction of the class 2:1 ratio of cyclohexyl B2:cyclohexyl B1 avermectins produced by the cells compared to cells of the same strain of S. avermitilis that do not express the mutated aveC allele but instead express only the wild-type aveC allele.
  • the composition is cyclohexyl B2:cyclohexyl
  • the composition is produced by cells comprising a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (a) through (be) listed above.
  • the composition is cyclohexyl
  • B2 cyclohexyl B1 avermectins in a ratio of about 0.30:1 or less, the composition is produced by cells comprising a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (f) through (be) listed above.
  • the composition is cyclohexyl
  • composition B2 cyclohexyl B1 avermectins in a ratio of about 0.25:1 or less, the composition is produced by cells comprising a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (w) through (be) listed above.
  • the composition is cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of about 0.20:1 or less
  • the composition is produced by cells comprising a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (ao) through (be) listed above.
  • the present invention further provides a composition of cyclohexyl B2:cyclohexyl B1 avermectins produced by cells of Streptomyces avermitilis, comprising the cyclohexyl B2:cyclohexyl B1 avermectins present in a culture medium in which the cells have been cultured, wherein the ratio of the cyclohexyl B2:cyclohexyl B1 avermectins present in the culture medium is about 0.40:1 or less, and produced by cells comprising a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (bf) and (bg) listed above.
  • FIGURE 1 DNA sequence (SEQ ID NO:1 ) comprising the S. avermitilis aveC ORF, and deduced amino acid sequence (SEQ ID NO:2).
  • FIGURE 2 Plasmid vector pSE186 (ATCC 209604) comprising the entire ORF of the aveC gene of S. avermitilis.
  • FIGURE 3 Gene replacement vector pSE180 (ATCC 209605) comprising the ermE gene of Sacc. erythraea inserted into the aveC ORF of S. avermitilis.
  • FIGURE 4 BamHl restriction map of the avermectin polyketide synthase gene cluster from S. avermitilis with five overlapping cosmid clones identified (i.e., pSE65, pSE66, pSE67, pSE68, pSE69). The relationship of pSE118 and pSE119 is also indicated.
  • FIGURE 5 HPLC analysis of fermentation products produced by S. avermitilis strains. Peak quantitation was performed by comparison to standard quantities of cyclohexyl B1. Cyclohexyl B2 retention time was 7.4-7.7 min; cyclohexyl B1 retention time was 11.9-12.3 min.
  • FIG. 5A S. avermitilis strain SE180-11 with an inactivated aveC ORF.
  • FIG. 5B. S. avermitilis strain SE180-11 transformed with pSE186 (ATCC 209604).
  • FIG. 5C S. avermitilis strain SE180-11 transformed with pSE187.
  • FIG. 5D S. avermitilis strain SE180-11 transformed with pSE188.
  • FIGURE 6A-M Compiled list of combinations of amino acid substitutions encoded by mutations to the aveC allele as identified by a second round of "gene shuffling", and their effects on the ratio of cyclohexyl B2:cyclohexyl B1 production.
  • the upper box lists the amino acid substitutions
  • the lower box lists the nucleotide base changes resulting in those amino acid substitutions. Nucleotide base changes in parentheses are silent changes, i.e., they do not result in changes to the amino acid sequence.
  • the present invention relates to the identification and characterization of polynucleotide molecules having nucleotide sequences that encode the AveC gene product from Streptomyces avermitilis, the construction of novel strains of S. avermitilis that can be used to screen mutated AveC gene products for their effect on avermectin production, and the discovery that certain mutated AveC gene products can reduce the ratio of B2:B1 avermectins produced by S. avermitilis.
  • the invention is described in the sections below for a polynucleotide molecule having either a nucleotide sequence that is the same as the S.
  • avermitilis AveC gene product-encoding sequence of plasmid pSE186 ATCC 209604
  • the nucleotide sequence of the ORF of FIGURE 1 SEQ ID NO:1
  • the principles set forth in the present invention can be analogously applied to other polynucleotide molecules, including aveC homolog genes from other Streptomyces species including, e.g., S. hygroscopicus and S. griseochromogenes, among others.
  • the present invention provides an isolated polynucleotide molecule comprising the complete aveC ORF of S. avermitilis or a substantial portion thereof, which isolated polynucleotide molecule lacks the next complete ORF that is located downstream from the aveC ORF in situ in the S. avermitilis chromosome.
  • the isolated polynucleotide molecule of the present invention preferably comprises a nucleotide sequence that is the same as the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or that is the same as the nucleotide sequence of the ORF of FIGURE 1 (SEQ ID NO:1) or substantial portion thereof.
  • a "substantial portion" of an isolated polynucleotide molecule comprising a nucleotide sequence encoding the S. avermitilis AveC gene product means an isolated polynucleotide molecule comprising at least about 70% of the complete aveC ORF sequence shown in FIGURE 1 (SEQ ID NO:1), and that encodes a functionally equivalent AveC gene product.
  • a "functionally equivalent” AveC gene product is defined as a gene product that, when expressed in S. avermitilis strain ATCC 53692 in which the native aveC allele has been inactivated, results in the production of substantially the same ratio and amount of avermectins as produced by S. avermitilis strain ATCC 53692 which instead expresses only the wild-type, functional aveC allele native to S. avermitilis strain ATCC 53692.
  • the isolated polynucleotide molecule of the present invention can further comprise nucleotide sequences that naturally flank the aveC gene in situ in S. avermitilis, such as those flanking nucleotide sequences shown in FIGURE 1 (SEQ ID NO:1 ).
  • the present invention further provides an isolated polynucleotide molecule comprising the nucleotide sequence of SEQ ID NO:1 or a degenerate variant thereof, as based on the known degeneracy of the genetic code.
  • polynucleotide molecule polynucleotide sequence
  • coding sequence "open-reading frame,” and “ORF” are intended to refer to both DNA and RNA molecules, which can either be single-stranded or double-stranded, and that can be transcribed and translated (DNA), or translated (RNA), into an AveC gene product, or into a polypeptide that is homologous to an AveC gene product in an appropriate host cell expression system when placed under the control of appropriate regulatory elements.
  • a coding sequence can include but is not limited to prokaryotic sequences, cDNA sequences, genomic DNA sequences, and chemically synthesized DNA and RNA sequences.
  • the nucleotide sequence shown in FIGURE 1 (SEQ ID NO:1 ) comprises four different GTG codons at bp positions 42, 174, 177 and 180.
  • FIGURE 1 The nucleotide sequence shown in FIGURE 1 (SEQ ID NO:1 ) comprises four different GTG codons at bp positions 42, 174, 177 and 180.
  • multiple deletions of the 5' region of the aveC ORF (FIGURE 1 ; SEQ ID NO:1) were constructed to help define which of these codons could function in the aveC ORF as start sites for protein expression. Deletion of the first GTG site at bp 42 did not eliminate AveC activity. Additional deletion of all of the GTG codons at bp positions 174, 177 and 180 together eliminated AveC activity, indicating that this region is necessary for protein expression.
  • the present invention thus encompasses variable length aveC ORFs.
  • the present invention further provides a polynucleotide molecule having a nucleotide sequence that is homologous to the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or to the nucleotide sequence of the ORF presented in FIGURE 1 (SEQ ID NO:1) or substantial portion thereof.
  • the term "homologous" when used to refer to a polynucleotide molecule that is homologous to an S. avermitilis AveC gene product-encoding sequence means a polynucleotide molecule having a nucleotide sequence: (a) that encodes the same AveC gene product as the S.
  • avermitilis AveC gene product- encoding sequence of plasmid pSE186 ATCC 209604, or that encodes the same AveC gene product as the nucleotide sequence of the aveC ORF presented in FIGURE 1 (SEQ ID NO:1), but that includes one or more silent changes to the nucleotide sequence according to the degeneracy of the genetic code (i.e., a degenerate variant); or (b) that hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence encoded by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604) or that encodes the amino acid sequence shown in FIGURE 1 (SEQ ID NO:2) under moderately stringent conditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHP0 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C,
  • AveC gene product encodes a functionally equivalent AveC gene product as defined above.
  • the homologous polynucleotide molecule hybridizes to the complement of the AveC gene product-encoding nucleotide sequence of plasmid pSE186 (ATCC 209604) or to the complement of the nucleotide sequence of the aveC ORF presented in FIGURE 1 (SEQ ID NO:1) or substantial portion thereof under highly stringent conditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHP0 4 , 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1xSSC/0.1% SDS at 68°C (Ausubel et al., 1989, above), and encodes a functionally equivalent AveC gene product as defined above.
  • an AveC gene product .and potential functional equivalents thereof can be determined through HPLC analysis of fermentation products, as described in the examples below.
  • Polynucleotide molecules having nucleotide sequences that encode functional equivalents of the S. avermitilis AveC gene product may include naturally occurring aveC genes present in other strains of S. avermitilis, aveC homolog genes present in other species of Streptomyces, and mutated aveC alleles, whether naturally occurring or engineered.
  • the present invention further provides a polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide having an amino acid sequence that is homologous to the amino acid sequence encoded by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or the amino acid sequence of FIGURE 1 (SEQ ID NO:2) or substantial portion thereof.
  • a "substantial portion" of the amino acid sequence of FIGURE 1 (SEQ ID NO:2) means a polypeptide comprising at least about 70% of the amino acid sequence shown in FIGURE 1 (SEQ ID NO:2), and that constitutes a functionally equivalent AveC gene product, as defined above.
  • the term "homologous” refers to a polypeptide which otherwise has the amino acid sequence of FIGURE 1 (SEQ ID NO:2), but in which one or more amino acid residues has been conservatively substituted with a different amino acid residue, wherein said amino acid sequence has at least about 70%, more preferably at least about 80%, and most preferably at least about 90% amino acid sequence identity to the polypeptide encoded by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604) or the amino acid sequence of FIGURE 1 (SEQ ID NO:2) as determined by any standard amino acid sequence identity algorithm, such as the BLASTP algorithm (GENBANK, NCBI), and where such conservative substitution results in a functionally equivalent gene product, as defined above.
  • Conservative amino acid substitutions are well known in the art. Rules for making such substitutions include those described by Dayhof, M.D., 1978, Nat. Biomed. Res. Found., Washington, D.C., Vol. 5, Sup. 3, among others. More specifically, conservative amino acid substitutions are those that generally take place within a family of amino acids that are related in the acidity or polarity.
  • One or more replacements within any particular group e.g., of a leucine with an isoleucine or valine, or of an aspartate with a glutamate, or of a threonine with a serine, or of any other amino acid residue with a structurally related amino acid residue, e.g., an amino acid residue with similar acidity or polarity, or with similarity in some combination thereof, will generally have an insignificant effect on the function of the polypeptide.
  • Polynucleotide clones encoding AveC gene products or AveC homolog gene products can be identified using any method known in the art, including but not limited to the methods set forth in Section 7, below. Genomic DNA libraries can be screened for aveC and aveC homolog coding sequences using techniques such as the methods set forth in Benton and Davis, 1977, Science 196:180, for bacteriophage libraries, and in Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci.
  • oligonucleotide probes can be synthesized that correspond to nucleotide sequences deduced from partial or complete amino acid sequences of the purified AveC homolog gene product.
  • the present invention further provides recombinant cloning vectors and expression vectors which are useful in cloning or expressing polynucleotide molecules of the present invention comprising, e.g., the aveC ORF of S. avermitilis or any aveC homolog ORFs.
  • the present invention provides plasmid pSE186 (ATCC 209604), which comprises the complete ORF of the aveC gene of S. avermitilis. All of the following description regarding the aveC ORF from S.
  • avermitilis or a polynucleotide molecule comprising the aveC ORF from S. avermitilis or portion thereof, or an S. avermitilis AveC gene product, also refers to mutated aveC alleles as described below, unless indicated explicitly or by context.
  • Recombinant vectors of the present invention are preferably constructed so that the coding sequence for the polynucleotide molecule of the invention is in operative association with one or more regulatory elements necessary for transcription and translation of the coding sequence to produce a polypeptide.
  • regulatory element includes but is not limited to nucleotide sequences that encode inducible and non-inducible promoters, enhancers, operators and other elements known in the art that serve to drive and/or regulate expression of polynucleotide coding sequences.
  • the coding sequence is in "operative association" with one or more regulatory elements where the regulatory elements effectively regulate and allow for the transcription of the coding sequence or the translation of its mRNA, or both.
  • Typical plasmid vectors that can be engineered to contain a polynucleotide molecule of the present invention include pCR-Blunt, pCR2.1 (Invitrogen), pGEM3Zf (Promega), and the shuttle vector pWHM3 (Vara et al., 1989, J. Bact. 171 :5872-5881), among many others.
  • Methods are well-known in the art for constructing recombinant vectors containing particular coding sequences in operative association with appropriate regulatory elements, and these can be used to practice the present invention. These methods include in vitro recombinant techniques, synthetic techniques, and in vivo genetic recombination. See, e.g., the techniques described in Maniatis et al., 1989, above; Ausubel et al., 1989, above; Sambrook et al., 1989, above; Innis et al., 1995, above; and Erlich, 1992, above.
  • the regulatory elements of these vectors can vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements can be used.
  • transcriptional regulatory regions or promoters for bacteria include the ⁇ -gal promoter, the T7 promoter, the TAC promoter, ⁇ left and right promoters, trp and lac promoters, trp-lac fusion promoters and, more specifically for Streptomyces, the promoters ermE, melC, and tipA, etc.
  • an expression vector can be generated that contains the aveC ORF or mutated ORF thereof cloned adjacent to a strong constitutive promoter, such as the ermE promoter from Saccharopolyspora erythraea.
  • a strong constitutive promoter such as the ermE promoter from Saccharopolyspora erythraea.
  • a vector comprising the ermE promoter was transformed into S. avermitilis, and subsequent HPLC analysis of fermentation products indicated an increased titer of avermectins produced compared to production by the same strain which instead expresses only the wild-type aveC allele.
  • Fusion protein expression vectors can be used to express an AveC gene product- fusion protein.
  • the purified fusion protein can be used to raise antisera against the AveC gene product, to study the biochemical properties of the AveC gene product, to engineer AveC fusion proteins with different biochemical activities, or to aid in the identification or purification of the expressed AveC gene product.
  • Possible fusion protein expression vectors include but are not limited to vectors incorporating sequences that encode ⁇ -galactosidase and trpE fusions, maltose-binding protein fusions, glutathione-S-transferase fusions and polyhistidine fusions (carrier regions).
  • an AveC gene product or a portion thereof can be fused to an AveC homolog gene product, or portion thereof, derived from another species or strain of Streptomyces, such as, e.g., S. hygroscopicus or S. griseochromogenes.
  • Such hybrid vectors can be transformed into S. avermitilis cells and tested to determine their effect, e.g., on the ratio of class 2:1 avermectin produced.
  • AveC fusion proteins can be engineered to comprise a region useful for purification.
  • AveC-maltose-binding protein fusions can be purified using amylose resin; AveC-glutathione-S-transferase fusion proteins can be purified using glutathione-agarose beads; and AveC-polyhistidine fusions can be purified using divalent nickel resin.
  • antibodies against a carrier protein or peptide can be used for affinity chromatography purification of the fusion protein.
  • a nucleotide sequence coding for the target epitope of a monoclonal antibody can be engineered into the expression vector in operative association with the regulatory elements and situated so that the expressed epitope is fused to the AveC polypeptide.
  • a nucleotide sequence coding for the FLAGTM epitope tag (International Biotechnologies Inc.), which is a hydrophilic marker peptide, can be inserted by standard techniques into the expression vector at a point corresponding, e.g., to the carboxyl terminus of the AveC polypeptide.
  • the expressed AveC polypeptide- FLAGTM epitope fusion product can then be detected and affinity-purified using commercially available anti-FLAGTM antibodies.
  • the expression vector encoding the AveC fusion protein can also be engineered to contain polylinker sequences that encode specific protease cleavage sites so that the expressed AveC polypeptide can be released from the carrier region or fusion partner by treatment with a specific protease.
  • the fusion protein vector can include DNA sequences encoding thrombin or factor Xa cleavage sites, among others.
  • a signal sequence upstream from, and in reading frame with, the aveC ORF can be engineered into the expression vector by known methods to direct the trafficking and secretion of the expressed gene product.
  • signal sequences include those from ⁇ -factor, immunoglobulins, outer membrane proteins, penicillinase, and T-cell receptors, among others.
  • the vector can be engineered to further comprise a coding sequence for a reporter gene product or other selectable marker.
  • a coding sequence is preferably in operative association with the regulatory element coding sequences, as described above.
  • Reporter genes that are useful in the invention are well-known in the art and include those encoding green fluorescent protein, luciferase, xylE, and tyrosinase, among others.
  • Nucleotide sequences encoding selectable markers are well known in the art, and include those that encode gene products conferring resistance to antibiotics or anti- metabolites, or that supply an auxotrophic requirement. Examples of such sequences include those that encode resistance to erythromycin, thiostrepton or kanamycin, among many others.
  • the present invention further provides transformed host cells comprising a polynucleotide molecule or recombinant vector of the invention, and novel strains or cell lines derived therefrom.
  • Host cells useful in the practice of the invention are preferably Streptomyces cells, although other prokaryotic cells or eukaryotic cells can also be used.
  • Such transformed host cells typically include but are not limited to microorganisms, such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA vectors, or yeast transformed with recombinant vectors, among others.
  • the polynucleotide molecules of the present invention are intended to function in
  • Streptomyces cells can also be transformed into other bacterial or eukaryotic cells, e.g., for cloning or expression purposes.
  • a strain of E. coli can typically be used, such as, e.g., the DH5 ⁇ strain, available from the American Type Culture Collection (ATCC), Rockville, MD, USA (Accession No. 31343), and from commercial sources (Stratagene).
  • Preferred eukaryotic host cells include yeast cells, although mammalian cells or insect cells can also be utilized effectively.
  • the recombinant expression vector of the invention is preferably introduced, e.g., transformed or transfected, into one or more host cells of a substantially homogeneous culture of cells.
  • the expression vector is generally introduced into host cells in accordance with known techniques, such as, e.g., by protoplast transformation, calcium phosphate precipitation, calcium chloride treatment, microinjection, electroporation, transfection by contact with a recombined virus, liposome-mediated transfection, DEAE-dextran transfection, transduction, conjugation, or microprojectile bombardment. Selection of transformants can be conducted by standard procedures, such as by selecting for cells expressing a selectable marker, e.g., antibiotic resistance, associated with the recombinant vector, as described above.
  • a selectable marker e.g., antibiotic resistance
  • the integration and maintenance of the aveC coding sequence either in the host cell chromosome or episomally can be confirmed by standard techniques, e.g., by Southern hybridization analysis, restriction enzyme analysis, PCR analysis, including reverse transcriptase PCR (rt-PCR), or by immunological assay to detect the expected gene product.
  • standard techniques e.g., by Southern hybridization analysis, restriction enzyme analysis, PCR analysis, including reverse transcriptase PCR (rt-PCR), or by immunological assay to detect the expected gene product.
  • Host cells containing and/or expressing the recombinant aveC coding sequence can be identified by any of at least four general approaches which are well-known in the art, including: (i) DNA-DNA, DNA-RNA, or RNA-antisense RNA hybridization; (ii) detecting the presence of "marker" gene functions; (iii) assessing the level of transcription as measured by the expression of aveC-specific mRNA transcripts in the host cell; and (iv) detecting the presence of mature polypeptide product as measured, e.g., by immunoassay or by the presence of AveC biological activity (e.g., the production of specific ratios and amounts of avermectins indicative of AveC activity in, e.g., S. avermitilis host cells).
  • AveC biological activity e.g., the production of specific ratios and amounts of avermectins indicative of AveC activity in, e.g., S. avermitilis host
  • the transformed host cell is clonally propagated, and the resulting cells can be grown under conditions conducive to the maximum production of the native or mutated
  • AveC gene product Such conditions typically include growing cells to high density.
  • the expression vector comprises an inducible promoter
  • appropriate induction conditions such as, e.g., temperature shift, exhaustion of nutrients, addition of gratuitous inducers (e.g., analogs of carbohydrates, such as isopropyl- ⁇ -D-thiogalactopyranoside (IPTG)), accumulation of excess metabolic by-products, or the like, are employed as needed to induce expression.
  • gratuitous inducers e.g., analogs of carbohydrates, such as isopropyl- ⁇ -D-thiogalactopyranoside (IPTG)
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the cells are harvested and lysed, and the product isolated and purified from the lysate under extraction conditions known in the art to minimize protein degradation such as, e.g., at 4°C, or in the presence of protease inhibitors, or both.
  • the expressed AveC gene product is secreted from the host cells, the exhausted nutrient medium can simply be collected and the product isolated therefrom.
  • the expressed AveC gene product can be isolated or substantially purified from cell lysates or culture medium, as appropriate, using standard methods, including but not limited to any combination of the following methods: ammonium sulfate precipitation, size fractionation, ion exchange chromatography, HPLC, density centrifugation, and affinity chromatography. Where the expressed AveC gene product exhibits biological activity, increasing purity of the preparation can be monitored at each step of the purification procedure by use of an appropriate assay. Whether or not the expressed AveC gene product exhibits biological activity, it can be detected as based, e.g., on size, or reactivity with an antibody otherwise specific for AveC, or by the presence of a fusion tag.
  • an AveC gene product is “substantially purified” where the product constitutes more than about 20 wt% of the protein in a particular preparation. Also, as used herein, an AveC gene product is “isolated” where the product constitutes at least about 80 wt% of the protein in a particular preparation.
  • the present invention thus provides a recombinantly-expressed isolated or substantially purified S. avermitilis AveC gene product comprising the amino acid sequence encoded by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or the amino acid sequence of FIGURE 1 (SEQ ID NO:2) or a substantial portion thereof, and mutated versions and degenerate variants thereof.
  • the present invention further provides a method for producing an AveC gene product, comprising culturing a host cell transformed with a recombinant expression vector, said vector comprising a polynucleotide molecule having a nucleotide sequence encoding the AveC gene product, which polynucleotide molecule is in operative association with one or more regulatory elements that control expression of the polynucleotide molecule in the host cell, under conditions conducive to the production of the recombinant AveC gene product, and recovering the AveC gene product from the cell culture.
  • the recombinantly expressed S. avermitilis AveC gene product is useful for a variety of purposes, including for screening compounds that alter AveC gene product function and thereby modulate avermectin biosynthesis, and for raising antibodies directed against the AveC gene product.
  • an AveC gene product of sufficient purity can be characterized by standard methods, including by SDS-PAGE, size exclusion chromatography, amino acid sequence analysis, biological activity in producing appropriate products in the avermectin biosynthetic pathway, etc.
  • the amino acid sequence of the AveC gene product can be determined using standard peptide sequencing techniques.
  • the AveC gene product can be further characterized using hydrophilicity analysis (see, e.g., Hopp and Woods, 1981 , Proc. Natl. Acad. Sci. USA 78:3824), or analogous software algorithms, to identify hydrophobic and hydrophilic regions of the AveC gene product. Structural analysis can be carried out to identify regions of the AveC gene product that assume specific secondary structures. Biophysical methods such as X-ray crystallography (Engstrom, 1974, Biochem. Exp. Biol. 11: 7-13), computer modelling (Fletterick and Zoller (eds), 1986, in: Current Communications in Molecular Biology.
  • a primary objective of the present invention is to identify novel mutations in the aveC allele of S. avermitilis that result in a change, and most preferably a reduction, in the ratio of B2:B1 avermectins.
  • the present invention thus provides polynucleotide molecules useful to produce novel strains of S.
  • avermitilis cells that exhibit a detectable change in avermectin production compared to cells of the same strain but which instead express only the wild-type aveC allele.
  • polynucleotide molecules are useful to produce novel strains of S. avermitilis cells that produce avermectins in a reduced class 2:1 ratio compared to cells of the same strain which instead express only the wild-type aveC allele.
  • the cells of such strains can also comprise additional mutations to produce an increased amount of avermectins compared to cells of the same strain that instead express only a single wild-type aveC allele.
  • Mutations to the aveC allele or coding sequence include any mutations that introduce one or more amino acid substitutions, deletions and/or additions into the AveC gene product, or that result in truncation of the AveC gene product, or any combination thereof, and that produce the desired result.
  • Such mutated aveC allele sequences are intended to include any degenerate variants thereof.
  • the present invention provides a polynucleotide molecule comprising the nucleotide sequence of the aveC allele or a degenerate variant thereof, or the AveC gene product-encoding sequence of plasmid pSEl86 (ATCC 209604) or a degenerate variant thereof, or the nucleotide sequence of the aveC ORF of S. avermitilis as present in FIGURE 1 (SEQ ID N0:1 ) or a degenerate variant thereof, but that further comprises mutations that encode a combination of amino acid substitutions at selected positions in the AveC gene product.
  • such substitutions occur at one or more amino acid positions of the AveC gene product corresponding to amino acid positions 25, 28, 35, 36, 38, 40, 41 , 48, 55, 61 , 78, 84, 89, 90, 99, 107, 108, 111 , 120, 123, 136, 138, 139, 141 , 154, 159, 163, 179, 192, 196, 198, 200, 202, 220, 228, 229, 230, 231 , 234, 238, 239, 250, 252, 266, 275, 278, 289 or 298 of SEQ ID NO:2.
  • Preferred combinations of amino acid positions to be substituted comprise one or more of amino acid residues D48, A61, A89, L136, S138, A139, R163, G179, V196, A198, E238 and P289.
  • Specifically preferred combinations of amino acid substitutions comprise substitutions at both D48 and G179, and more specifically D48E and G179S.
  • Specific examples of combinations of amino acid substitutions that result in a reduction in cyclohexylB2;cyclohexyl B1 ratios are listed in FIGURE 6A-J.
  • the present invention thus provides a polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as the Streptomyces avermitilis aveC allele, the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604) or the nucleotide sequence of the aveC ORF of S. avermitilis as presented in FIGURE 1 (SEQ ID NO:1), or a degenerate variant thereof, but which nucleotide sequence further comprises mutations encoding a combination of amino acid substitutions at amino acid residues corresponding to the amino acid positions of SEQ ID NO:2, such that cells of S.
  • avermitilis strain ATCC 53692 in which the wild-type aveC allele has been inactivated and that express the polynucleotide molecule comprising the mutated nucleotide sequence are capable of producing a class 2:1 ratio of avermectins that is reduced compared to the ratio produced by cells of S.
  • the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.30:1 or less. In a more preferred embodiment, the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.25:1 or less. In a more preferred embodiment, the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.20:1 or less.
  • the combination of amino acid substitutions comprises the combination of group (a): D48E, A61T, A89T, S138T, A139T, G179S, A198G, P289L.
  • group (a): D48E, A61T, A89T, S138T, A139T, G179S, A198G, P289L A non-limiting example of a plasmid encoding these amino acid substitutions is pSE538 (see FIGURE 6).
  • the combination of amino acid substitutions comprises the combination of group (b): G40S, D48E, L136P, G179S, E238D.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE559.
  • the combination of amino acid substitutions comprises the combination of group (c): D48E, L136P, R163Q, G179S.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE567.
  • the combination of amino acid substitutions comprises the combination of group (d): D48E, L136P, R163Q, G179S, E238D.
  • Non-limiting examples of plasmids encoding these amino acid substitutions are pSE570 and pSE572.
  • the combination of amino acid substitutions comprises the combination of group (e): D48E, L136P, R163Q, G179S, A200G, E238D.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE571.
  • the combination of amino acid substitutions comprises the combination of group (f): D48E, L136P, G179S, E238D.
  • Non-limiting examples of plasmids encoding these amino acid substitutions are pSE501 and pSE546.
  • the combination of amino acid substitutions comprises the combination of group (g): D48E, A61T, L136P, G179S, E238D.
  • group (g): D48E, A61T, L136P, G179S, E238D A non-limiting example of a plasmid encoding these amino acid substitutions is pSE510.
  • the combination of amino acid substitutions comprises the combination of group (h): D48E, A61T, L136P, G179S.
  • group (h): D48E, A61T, L136P, G179S A non-limiting example of a plasmid encoding these amino acid substitutions is pSE512.
  • the combination of amino acid substitutions comprises the combination of group (i): D48E, A89T, S138T, A139T, G179S.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE519.
  • the combination of amino acid substitutions comprises the combination of group (j): D48E, A61T, L136P, G179S, A198G, P202S, E238D, P289L.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE526.
  • the combination of amino acid substitutions comprises the combination of group (k): D48E, A61T, L136P, S138T, A139F, G179S, E238D, P289L.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE528.
  • the combination of amino acid substitutions comprises the combination of group (I): D48E, L136P, G179S, A198G, E238D, P289L.
  • a non- limiting example of a plasmid encoding these amino acid substitutions is pSE530.
  • the combination of amino acid substitutions comprises the combination of group (m): D48E, A61T, S138T, A139F, G179S, A198G, P289L.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE531.
  • the combination of amino acid substitutions comprises the combination of group (n): D48E, L84P, G111V, S138T, A139T, G179S, A198G, P289L.
  • group (n): D48E, L84P, G111V, S138T, A139T, G179S, A198G, P289L is pSE534.
  • the combination of amino acid substitutions comprises the combination of group (o): Y28C, D48E, A61T, A89T, S138T, A139T, G179S, E238D.
  • group (o): Y28C, D48E, A61T, A89T, S138T, A139T, G179S, E238D is pSE535.
  • the combination of amino acid substitutions comprises the combination of group (p): D48E, A61T, A107T, S108G, L136P, G179S, S192A, E238D, P289L.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE542.
  • the combination of amino acid substitutions comprises the combination of group (q): D48E, L136P, G179S, R250W.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE545.
  • the combination of amino acid substitutions comprises the combination of group (r): D48E, A89T, S138T, A139T, R163Q, G179S.
  • a non- limiting example of a plasmid encoding these amino acid substitutions is pSE548.
  • the combination of amino acid substitutions comprises the combination of group (s): D48E, L136P, G179S, A198G, P289L.
  • group (s): D48E, L136P, G179S, A198G, P289L is pSE552.
  • the combination of amino acid substitutions comprises the combination of group (t): D48E, F78L, A89T, L136P, G179S.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE557.
  • the combination of amino acid substitutions comprises the combination of group (u): D48E, A89T, S138T, A139T, G179S, E238D, F278L.
  • group (u): D48E, A89T, S138T, A139T, G179S, E238D, F278L Non-limiting examples of plasmids encoding these amino acid substitutions are pSE564 and pSE565.
  • the combination of amino acid substitutions comprises the combination of group (v): D48E, A89T, L136P, R163Q, G179S.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE568.
  • the combination of amino acid substitutions comprises the combination of group (w): D48E, A61T, A89T, G111V, S138T, A139F, G179S, E238D, P289L .
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE543.
  • the combination of amino acid substitutions comprises the combination of group (x): D25G, D48E, A89T, L136P, S138T, A139T, V141A, I159T, R163Q, G179S.
  • group (x) D25G, D48E, A89T, L136P, S138T, A139T, V141A, I159T, R163Q, G179S.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE504.
  • the combination of amino acid substitutions comprises the combination of group (y): D48E, A89T, S90G, L136P, R163Q, G179S, E238D.
  • group (y): D48E, A89T, S90G, L136P, R163Q, G179S, E238D A non-limiting example of a plasmid encoding these amino acid substitutions is pSE508.
  • the combination of amino acid substitutions comprises the combination of group (z): D48E, A61T, A89T, G111V, S138T, A139T, G179S, E238D, P289L.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE511.
  • the combination of amino acid substitutions comprises the combination of group (aa): D48E, A89T, S138T, A139T, G179S.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE520.
  • the combination of amino acid substitutions comprises the combination of group (ab): D48E, L136P, R163Q, G179S, S231 L.
  • group (ab) D48E, L136P, R163Q, G179S, S231 L.
  • a non- limiting example of a plasmid encoding these amino acid substitutions is pSE523.
  • the combination of amino acid substitutions comprises the combination of group (ac): D48E, L136P, S138T, A139F, G179S, V196A, E238D.
  • group (ac) D48E, L136P, S138T, A139F, G179S, V196A, E238D.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE527.
  • the combination of amino acid substitutions comprises the combination of group (ad): D48E, A61T, A89T, F99S, S138T, A139T, G179S, E238D.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE539.
  • the combination of amino acid substitutions comprises the combination of group (ae): G35S, D48E, A89T, S138T, A139T, G179S, P289L.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE540.
  • the combination of amino acid substitutions comprises the combination of group (af): D48E, A61T, A89T, S138T, A139T, G179S, V196A, E238D.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE547.
  • the combination of amino acid substitutions comprises the combination of group (ag): D48E, A89T, G111V, S138T, A139T, G179S, A198G, E238D.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE550.
  • the combination of amino acid substitutions comprises the combination of group (ah): S41G, D48E, A89T, L136P, G179S.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE558.
  • the combination of amino acid substitutions comprises the combination of group (ai): D48E, A89T, L136P, R163Q, G179S, P252S.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE563.
  • the combination of amino acid substitutions comprises the combination of group (aj): D48E, A89T, L136P, G179S, F234S.
  • group (aj): D48E, A89T, L136P, G179S, F234S is pSE566.
  • the combination of amino acid substitutions comprises the combination of group (ak): D48E, A89T, L136P, R163Q, G179S, E238D.
  • Non- limiting examples of plasmids encoding these amino acid substitutions are pSE573 and pSE578.
  • the combination of amino acid substitutions comprises the combination of group (al): Q36R, D48E, A89T, L136P, G179S, E238D.
  • group (al) Q36R, D48E, A89T, L136P, G179S, E238D.
  • a non- limiting example of a plasmid encoding these amino acid substitutions is pSE574.
  • the combination of amino acid substitutions comprises the combination of group (am): D48E, A89T, L136P, R163Q, G179S.
  • Non-limiting examples of plasmids encoding these amino acid substitutions are pSE575 and pSE576.
  • the combination of amino acid substitutions comprises the combination of group (an): D48E, A89T, S138T, G179S.
  • group (an) D48E, A89T, S138T, G179S.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE577.
  • the combination of amino acid substitutions comprises the combination of group (ao): D48E, A89T, L136P, G179S, E238D.
  • Non-limiting examples of plasmids encoding these amino acid substitutions are pSE502 and pSE524.
  • the combination of amino acid substitutions comprises the combination of group (ap): D48E, A89T, L136P, K154E, G179S, E238D.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE503.
  • the combination of amino acid substitutions comprises the combination of group (aq): D48E, A89T, S138T, A139T, K154R, G179S, V196A, P289L.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE505.
  • the combination of amino acid substitutions comprises the combination of group (ar): D48E, A89T, S138T, A139F, G179S, V196A, E238D.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE506.
  • the combination of amino acid substitutions comprises the combination of group (as): D48E, A61T, A89T, L136P, G179S, V196A, A198G, P289L.
  • group (as): D48E, A61T, A89T, L136P, G179S, V196A, A198G, P289L is pSE507.
  • the combination of amino acid substitutions comprises the combination of group (at): D48E, A61T, S138T, A139F, G179S, G196A, E238D, P289L.
  • group (at): D48E, A61T, S138T, A139F, G179S, G196A, E238D, P289L is pSE509.
  • the combination of amino acid substitutions comprises the combination of group (au): D48E, A89T, L136P, G179S.
  • Non-limiting examples of plasmids encoding these amino acid substitutions are pSE514 and pSE525.
  • the combination of amino acid substitutions comprises the combination of group (av): D48E, A89T, V120A, L136P, G179S.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE515.
  • the combination of amino acid substitutions comprises the combination of group (aw): D48E, A61T, A89T, S138T, A139F.G179S, V196A, A198G, E238D.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE517.
  • the combination of amino acid substitutions comprises the combination of group (ax): D48E, A61T, A89T, G111V, S138T, A139F, G179S, V196A, E238D.
  • group (ax) D48E, A61T, A89T, G111V, S138T, A139F, G179S, V196A, E238D.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE518.
  • the combination of amino acid substitutions comprises the combination of group (ay): D48E, A61T, A89T, S138T, A139T, G179S, V196A, E238D, P289L.
  • Non-limiting examples of plasmids encoding these amino acid substitutions are pSE529 and pSE554.
  • the combination of amino acid substitutions comprises the combination of group (az): D48E, A61T, A89T, L136P, S138T, A139F, G179S, A198G, E238D.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE532.
  • the combination of amino acid substitutions comprises the combination of group (ba) D48E, A89T, S138T, A139F, G179S, A198G, V220A.
  • a non-limiting example of a plasmid encoding these amino acid substitutions is pSE536.
  • the combination of amino acid substitutions comprises the combination of group (bb): D48E, A61T, A89T, S138T, A139T, G179S, V196A, E238D, R239H, P289L.
  • group (bb) D48E, A61T, A89T, S138T, A139T, G179S, V196A, E238D, R239H, P289L.
  • the combination of amino acid substitutions comprises the combination of group (be): D48E, A61T, A89T, L136P, G179S, P289L.
  • a non- limiting example of a plasmid encoding these amino acid substitutions is pSE541.
  • the combination of amino acid substitutions comprises the combination of group (bd): D48E, A89T, S138T, A139T, G179S, V196A, E238D, P289L.
  • Non-limiting examples of plasmids encoding these amino acid substitutions are pSE549 and pSE553.
  • the combination of amino acid substitutions comprises the combination of group (be): D48E, A61T, A89T, S138T, A139F, G179S, V196A, E238D.
  • group (be): D48E, A61T, A89T, S138T, A139F, G179S, V196A, E238D A non-limiting example of a plasmid encoding these amino acid substitutions is pSE551.
  • the present invention further provides a polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as the Streptomyces avermitilis aveC allele, the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604) or the nucleotide sequence of the aveC ORF of S. avermitilis as presented in FIGURE 1 (SEQ ID NO:1), or a degenerate variant thereof, but which nucleotide sequence further comprises mutations encoding a combination of amino acid substitutions at amino acid residues corresponding to the amino acid positions of SEQ ID NO:2, such that cells of S.
  • avermitilis strain ATCC 53692 in which the wild-type aveC allele has been inactivated and that express a polynucleotide molecule comprising the mutated nucleotide sequence are capable of producing a class 2:1 ratio of avermectins that is reduced compared to the ratio produced by cells of S.
  • avermitilis strain ATCC 53692 that instead express only the wild-type aveC allele, wherein when the class 2:1 avermectins are cyclohexyl B2:cyclohexyl B1 avermectins, the ratio of class 2:1 avermectins is reduced to about 0.40:1 or less, and wherein the combination of amino acid substitutions comprises a combination selected from the group consisting of:
  • Non-limiting examples of a plasmid encoding the amino acid substitutions of group (bf) are pSE556 and pSE569.
  • a non-limiting example of a plasmid encoding the amino acid substitutions of group (bg) is pSE561.
  • the present invention contemplates that any of the aforementioned amino acid substitutions can be accomplished by any modification to the nucleotide sequence of the aveC allele or a degenerate variant thereof that results in such substitutions. For example, it is possible to effect most of the amino acid substitutions described herein by changing a native codon sequence or a degenerate variant thereof to any one of several alternative codons that encode the same amino acid substitution.
  • the phrase "the combination of amino acid substitutions comprises the combination of group ... ", and the like, means that the amino acid substitutions in the AveC gene product according to the present invention include at least those substitutions that are specifically recited, and may include other amino acid substitutions, or amino acid deletions, or amino acid additions, or some combination thereof, wherein the expression of the resulting AveC gene product in the S. avermitilis cell yields a desirable reduction in the ratio of B2:B1 avermectins.
  • Mutations to the aveC allele or degenerate variant thereof can be carried out by any of a variety of known methods, including by use of error-prone PCR, or by cassette mutagenesis.
  • oligonucleotide-directed mutagenesis can be employed to alter the sequence of the aveC allele or ORF in a defined way such as, e.g., to introduce one or more restriction sites, or a termination codon, into specific regions within the aveC allele or ORF.
  • Methods such as those described in U.S. Patent 5,605,793, U.S. Patent 5,830,721 and U.S.
  • Patent 5,837,458 which involve random fragmentation, repeated cycles of mutagenesis, and nucleotide shuffling, can also be used to generate large libraries of polynucleotides having nucleotide sequences encoding aveC mutations.
  • Targeted mutations can be useful, particularly where they serve to alter one or more conserved amino acid residues in the AveC gene product.
  • a comparison of the deduced amino acid sequence of the AveC gene product of S. avermitilis (SEQ ID NO:2) with AveC homolog gene products from S. griseochromogenes (SEQ ID NO:5) and S. hygroscopicus (SEQ ID NO:4), as described in U.S. Patent No. 6,248,579 indicates sites of significant conservation of amino acid residues between these species.
  • Targeted mutagenesis that leads to a change in one or more of these conserved amino acid residues may be effective in producing novel mutant strains that exhibit desirable alterations in avermectin production.
  • Random mutagenesis can also be useful, and can be carried out by exposing cells of S. avermitilis to ultraviolet radiation or x-rays, or to chemical mutagens such as N-methyl-N'- nitrosoguanidine, ethyl methane sulfonate, nitrous acid or nitrogen mustards. See, e.g., Ausubel, 1989, above, for a review of mutagenesis techniques. Once mutated polynucleotide molecules are generated, they are screened to determine whether they can modulate avermectin biosynthesis in S. avermitilis.
  • a polynucleotide molecule having a mutated nucleotide sequence is tested by complementing a strain of S. avermitilis in which the aveC gene has been inactivated to give an aveC negative (aveC) background.
  • the mutated polynucleotide molecule is spliced into an expression plasmid in operative association with one or more regulatory elements, which plasmid also preferably comprises one or more drug resistance genes to allow for selection of transformed cells.
  • This vector is then transformed into aveC host cells using known techniques, and transformed cells are selected and cultured in appropriate fermentation media under conditions that permit or induce avermectin production, for example, by including appropriate starter subunits in the medium, and culturing under optimal conditions for avermectin production as known in the art. Fermentation products are then analyzed by HPLC to determine the ability of the mutated polynucleotide molecule to complement the host cell.
  • plasmid vectors bearing mutated polynucleotide molecules capable of reducing the B2:B1 ratio of avermectins including pSE188, pSE199, pSE231 , pSE239, and pSE290 through pSE297, are exemplified in Section 8.3, below. Other examples of such plasmid vectors are recited in FIGURE 6.
  • Any of the aforementioned methods of the present invention can be carried out using fermentation culture media preferably supplemented with cyclohexane carboxylic acid, although other appropriate fatty acid precursors, such as any one of the fatty acid precursors listed in TABLE 1 , or methylthiolactic acid, can also used.
  • a mutated polynucleotide molecule that modulates avermectin production in a desirable direction the location of the mutation in the nucleotide sequence can be determined.
  • a polynucleotide molecule having a nucleotide sequence encoding a mutated AveC gene product can be isolated by PCR and subjected to DNA sequence analysis using known methods.
  • the mutation(s) responsible for the alteration in avermectin production can be determined. For example, S.
  • avermitilis AveC gene products comprising either single amino acid substitutions at any of residues 55 (S55F), 138 (S138T), 139 (A139T), or 230 (G230D), or double substitutions at positions 138 (S138T) and 139 (A139T or A139F), yielded changes in AveC gene product function such that the ratio of class 2:1 avermectins produced was altered (see Section 8, below), wherein the recited amino acid positions correspond to those presented in FIGURE 1 (SEQ ID N0:2).
  • the present invention provides fifty-nine (59) additional combinations of mutations that are shown to reduce the cyclohexyl B2:cyclohexyl B1 ratio of avermectins, and these are presented in FIGURE 6 and recited in the appended claims.
  • the aforementioned designations such as A139T, indicate the original amino acid residue by single letter designation, which in this example is alanine (A), at the indicated position, which in this example is position 139 (referring to SEQ ID N0:2) of the polypeptide, followed by the amino acid residue which replaces the original amino acid residue, which in this example is threonine (T).
  • amino acid residue encoded by an aveC allele in the S. avermitilis chromosome, or in a vector or isolated polynucleotide molecule of the present invention is referred to as "corresponding to" a particular amino acid residue of SEQ ID NO:2, or where an amino acid substitution is referred to as occurring at a particular position "corresponding to” that of a specific numbered amino acid residue of SEQ ID NO:2, this is intended to refer to the amino acid residue at the same relative location in the AveC gene product, which the skilled artisan can quickly determine by reference to the amino acid sequence presented herein as SEQ ID NO:2.
  • a polynucleotide molecule of the present invention may be "isolated", which means either that it is: (i) purified to the extent that it is substantially free of other polynucleotide molecules having different nucleotide sequences, or (ii) present in an environment in which it would not naturally occur, e.g., where an aveC allele from S. avermitilis, or a mutated version thereof, is present in a cell other than a cell of S.
  • avermitilis or (iii) present in a form in which it would not naturally occur, e.g., as a shorter piece of DNA, such as a restriction fragment digested out of a bacterial chromosome, comprising predominantly the aveC coding region or a mutated version thereof, with or without any associated regulatory sequences thereof, or as subsequently integrated into a heterologous piece of DNA, such as the chromosome of a bacterial cell (other than a cell of S. avermitilis) or the DNA of a vector such as a plasmid or phage, or integrated into the S. avermitilis chromosome at a locus other than that of the native aveC allele.
  • a shorter piece of DNA such as a restriction fragment digested out of a bacterial chromosome, comprising predominantly the aveC coding region or a mutated version thereof, with or without any associated regulatory sequences thereof, or as subsequently integrated into a hetero
  • the present invention further provides a recombinant vector comprising a polynucleotide molecule of the present invention.
  • a recombinant vector can be used to target any of the polynucleotide molecules comprising mutated nucleotide sequences of the present invention to the site of the aveC allele of the S. avermitilis chromosome to either insert into or replace the aveC ORF or a portion thereof, e.g., by homologous recombination.
  • a polynucleotide molecule comprising a mutated nucleotide sequence of the present invention provided herewith can also function to modulate avermectin biosynthesis when inserted into the S. avermitilis chromosome at a site other than at the aveC allele, or when maintained episomally in S. avermitilis cells.
  • the present invention further provides vectors comprising a polynucleotide molecule comprising a mutated nucleotide sequence of the present invention, which vectors can be used to insert the polynucleotide molecule at a site in the S.
  • the vector is a gene replacement vector that can be used to insert a mutated aveC allele or degenerate variant thereof according to the present invention into cells of a strain of S. avermitilis, thereby generating novel strains of S. avermitilis, the cells of which can produce avermectins in a reduced class 2:1 ratio compared to cells of the same strain which instead express only the wild-type aveC allele.
  • Such gene replacement vectors can be constructed using mutated polynucleotide molecules present in expression vectors provided herewith, such as those expression vectors exemplified in Section 8 below.
  • the present invention further provides vectors that can be used to insert a mutated aveC allele or degenerate variant thereof into cells of a strain of S. avermitilis to generate novel strains of cells that produce altered amounts of avermectins compared to cells of the same strain which instead express only the wild-type aveC allele.
  • the amount of avermectins produced by the cells is increased.
  • such a vector comprises a strong promoter as known in the art, such as, e.g., the strong constitutive ermE promoter from Saccharopolyspora erythraea, that is situated upstream from, and in operative association with, the aveC ORF.
  • a strong promoter as known in the art, such as, e.g., the strong constitutive ermE promoter from Saccharopolyspora erythraea, that is situated upstream from, and in operative association with, the aveC ORF.
  • Such vectors can be constructed using the mutated aveC allele of plasmid pSE189, and according to methods described in U.S. Patent No. 6,248,579,
  • the present invention provides gene replacement vectors that are useful to inactivate the aveC gene in a wild-type strain of S. avermitilis.
  • such gene replacement vectors can be constructed using the mutated polynucleotide molecule present in plasmid pSE180 (ATCC 209605), which is exemplified in Section 8.1 , below (FIGURE 3).
  • the present invention further provides gene replacement vectors that comprise a polynucleotide molecule comprising or consisting of nucleotide sequences that naturally flank the aveC gene in situ in the S.
  • avermitilis chromosome including, e.g., those flanking nucleotide sequences shown in FIGURE 1 (SEQ ID NO:1), which vectors can be used to delete the S. avermitilis aveC ORF.
  • the present invention further provides a host cell comprising a polynucleotide molecule or recombinant vector of the present invention.
  • the host cell can be any prokaryotic or eukaryotic cell capable of use as a host for the polynucleotide molecule or recombinant vector.
  • the host ceil is a bacterial cell.
  • the host cell is a Streptomyces cell.
  • the host cell is a cell of Streptomyces avermitilis.
  • the present invention further provides a method for making a novel strain of Streptomyces avermitilis, comprising (i) mutating the aveC allele in a cell of a strain of S. avermitilis, which mutation results in a combination of amino acid substitutions in the AveC gene product, or (ii) introducing into a cell of a strain of S. avermitilis a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions, wherein the combination of amino acid substitutions is selected from (a) through (be) listed above.
  • the present invention further provides a method for making a novel strain of S.
  • avermitilis comprising (i) mutating the aveC allele in a cell of a strain of S. avermitilis, which mutation results in a combination of amino acid substitutions in the AveC gene product, or (ii) introducing into a cell of a strain of S. avermitilis a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions, wherein cells comprising the mutated aveC allele or degenerate variant are capable of producing cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of 0.35:1 or less.
  • the mutated aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (a) through (be) listed above.
  • the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.30:1 or less.
  • the mutated aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (f) through (be) listed above.
  • the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.25:1 or less.
  • the mutated aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (w) through (be) listed above.
  • the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.20:1 or less.
  • the mutated aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (ao) through (be) listed above.
  • the present invention further provides a method for making a novel strain of Streptomyces avermitilis, comprising (i) mutating the aveC allele in a cell of a strain of S. avermitilis, which mutation results in a combination of amino acid substitutions in the AveC gene product, or (ii) introducing into a cell of a strain of S. avermitilis a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions, wherein the combination of amino acid substitutions is selected from the group consisting of (bf) and (bg).
  • cells of S comprising (i) mutating the aveC allele in a cell of a strain of S. avermitilis, which mutation results in a combination of amino acid substitutions in the AveC gene product, or (ii) introducing into a cell of a strain of S. avermitilis a mutated a
  • avermitilis comprising such a mutated aveC allele or degenerate variant are capable of producing cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of about 0.40:1 or less.
  • the present invention further provides a cell of a Streptomyces species that comprises a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from (a) through (be) listed above.
  • the species of Streptomyces is S. avermitilis.
  • the present invention further provides a cell of S. avermitilis capable of producing cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of 0.35:1 or less.
  • the cell comprises a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (a) through (be) listed above.
  • the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.30:1 or less.
  • the cell comprises a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (f) through (be) listed above.
  • the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.25:1 or less.
  • the cell comprises a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of
  • the ratio of cyclohexyl B2:cyclohexyl B1 avermectins is about 0.20:1 or less.
  • the cell comprises a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of
  • the present invention further provides a cell of a Streptomyces species, comprising a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (bf) and (bg) listed above.
  • the species of Streptomyces is S. avermitilis.
  • the cell is a cell of S. avermitilis capable of producing cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of about 0.40:1 or less.
  • any of the recited mutations can be present in cells of the present invention on an extrachromosomal element such as a plasmid, it is preferred that such mutations are present in an aveC coding sequence integrated into the S. avermitilis chromosome, and preferably, though not necessarily, at the site of the native aveC allele.
  • the present invention further provides a process for producing avermectins, comprising culturing the S. avermitilis cells of the present invention in culture media under conditions that permit or induce the production of avermectins therefrom, and recovering said avermectins from the culture.
  • the cells used in the process produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of 0.35:1 or less, more preferably in a ratio of about 0.30:1 or less, more preferably in a ratio of about 0.25:1 or less, and more preferably in a ratio of about 0.20:1 or less.
  • cells producing cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of 0.35:1 or less comprise a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (a) through (be) listed above.
  • cells producing cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of about 0.30:1 or less comprise a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (f) through (be) listed above.
  • B1 avermectins in a ratio of about 0.25:1 or less comprise a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (w) through (be) listed above.
  • cells producing cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of about 0.20:1 or less comprise a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (ao) through (be) listed above.
  • the cells produce cyclohexyl B2:cyclohexyI B1 avermectins in a ratio of about 0.40:1 or less and comprise a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (bf) and (bg) listed above.
  • the process of the invention provides increased efficiency in the production of commercially valuable avermectins such as doramectin.
  • the present invention further provides a composition of cyclohexyl B2:cyclohexyl B1 avermectins produced by cells of Streptomyces avermitilis, comprising the cyclohexyl B2:cyclohexyl B1 avermectins present in a culture medium in which the cells have been cultured, wherein the ratio of the cyclohexyl B2:cyclohexyl B1 avermectins present in the culture medium is 0.35:1 or less, preferably about 0.30:1 or less, more preferably about 0.25:1 or less, and more preferably about 0.20:1 or less.
  • the composition of cyclohexyl B2:cyclohexyl B1 avermectins is produced by cells of a strain of S. avermitilis that express a mutated aveC allele or degenerate variant thereof which encodes a gene product that results in the reduction in the ratio of cyclohexyl B2:cyclohexyl B1 avermectins produced by the cells compared to cells of the same strain of S. avermitilis that do not express the mutated aveC allele but instead express only the wild-type aveC allele.
  • the composition is cyclohexyl B2:cyclohexyl
  • the composition is produced by cells comprising a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (a) through (be) listed above.
  • the composition is cyclohexyl
  • composition B2 cyclohexyl B1 avermectins in a ratio of about 0.30:1 or less, the composition is produced by cells comprising a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (f) through (be) listed above.
  • the composition is cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of about 0.25:1 or less
  • the composition is produced by cells comprising a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (w) through (be) listed above.
  • the composition is cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of about 0.20:1 or less
  • the composition is produced by cells comprising a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (ao) through (be) listed above.
  • the present invention further provides a composition of cyclohexyl B2:cyclohexyl B1 avermectins produced by cells of Streptomyces avermitilis, comprising the cyclohexyl B2:cyclohexyl B1 avermectins present in a culture medium in which the cells have been cultured, wherein the ratio of the cyclohexyl B2:cyclohexyl B1 avermectins present in the culture medium is about 0.40:1 or less, and which is produced by cells comprising a mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (bf) and (bg) listed above.
  • the novel avermectin composition is present in a culture medium in which the cells have been cultured, e.g., in partially or totally exhausted fermentation culture fluid
  • the avermectin composition may alternatively be partially or substantially purified from the culture fluid by known biochemical techniques of purification, such as by ammonium sulfate precipitation, dialysis, size fractionation, ion exchange chromatography, HPLC, etc.
  • cells of the present invention can further comprise modifications to increase the production level of avermectins.
  • such cells can be prepared by (i) mutating the aveC allele in a cell of S. avermitilis, or (ii) introducing a mutated aveC allele or degenerate variant thereof into cells of a strain of S.
  • avermitilis wherein the expression of the mutated allele results in an increase in the amount of avermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allele compared to cells of the same strain that instead express only a single wild-type aveC allele, and selecting transformed cells that produce avermectins in an increased amount compared to the amount of avermectins produced by cells of the strain that instead express only the single wild-type aveC allele.
  • the aveC allele can be modified so that it comprises a strong promoter, such as the strong constitutive ermE promoter from Saccharopolyspora erythraea, inserted upstream from and in operative association with the aveC ORF.
  • a strong promoter such as the strong constitutive ermE promoter from Saccharopolyspora erythraea
  • one or more mutations can be introduced into the aveR1 and/or aveR2 genes of S. avermitilis, thereby increasing the level of avermectin production as described in U.S. Patent No. 6,197,591 to Stutzman-Engwall et al., issued March 6, 2001.
  • Avermectins are highly active antiparasitic agents having particular utility as anthelmintics, ectoparasiticides, insecticides and acaricides.
  • Avermectin compounds produced according to the methods of the present invention are useful for any of these purposes.
  • avermectin compounds produced according to the present invention are useful to treat various diseases or conditions in humans, particularly where those diseases or conditions are caused by parasitic infections, as known in the art. See, e.g., Ikeda and Omura, 1997, Chem. Rev. 97(7):2591-2609.
  • avermectin compounds produced according to the present invention are effective in treating a variety of diseases or conditions caused by endoparasites, such as parasitic nematodes, which can infect humans, domestic animals, swine, sheep, poultry, horses or cattle.
  • avermectin compounds produced according to the present invention are effective against nematodes that infect humans, as well as those that infect various species of animals.
  • nematodes include gastrointestinal parasites such as Ancylostoma, Necator, Ascaris, Strongyloides, Trichinella, Capillaria, Trichuris, Enterobius, Dirofilaria, and parasites that are found in the blood or other tissues ' or organs, such as filarial worms and the extract intestinal states of Strongyloides and Trichinella.
  • avermectin compounds produced according to the present invention are also useful in treating ectoparasitic infections including, e.g., arthropod infestations of mammals and birds, caused by ticks, mites, lice, fleas, blowflies, biting insects, or migrating dipterous larvae that can affect cattle and horses, among others.
  • the avermectin compounds produced according to the present invention are also useful as insecticides against household pests such as, e.g., the cockroach, clothes moth, carpet beetle and the housefly among others, as well as insect pests of stored grain and of agricultural plants, which pests include spider mites, aphids, caterpillars, and orthopterans such as locusts, among others.
  • Animals that can be treated with the avermectin compounds produced according to the present invention include sheep, cattle, horses, deer, goats, swine, birds including poultry, and dogs and cats.
  • an avermectin compound produced according to the present invention is administered in a formulation appropriate to the specific intended use, the particular species of host animal being treated, and the parasite or insect involved.
  • an avermectin compound produced according to the present invention can be administered orally in the form of a capsule, bolus, tablet or liquid drench or, alternatively, can be administered as a pour-on, or by injection, or as an implant.
  • Such formulations are prepared in a conventional manner in accordance with standard veterinary practice.
  • capsules, boluses or tablets can be prepared by mixing the active ingredient with a suitable finely divided diluent or carrier additionally containing a disintegrating agent and/or binder such as starch, lactose, talc, magnesium stearate, etc.
  • a drench formulation can be prepared by dispersing the active ingredient in an aqueous solution together with a dispersing or wetting agent, etc.
  • Injectable formulations can be prepared in the form of a sterile solution, which can contain other substances such as, e.g., sufficient salts and/or glucose to make the solution isotonic with blood.
  • Such formulations will vary with regard to the weight of active compound depending on the patient, or species of host animal to be treated, the severity and type of infection, and the body weight of the host.
  • a dose of active compound of from about 0.001 to 10 mg per kg of patient or animal body weight given as a single dose or in divided doses for a period of from 1 to 5 days will be satisfactory.
  • higher or lower dosage ranges are indicated, as determined, e.g., by a physician or veterinarian, as based on clinical symptoms.
  • an avermectin compound produced according to the present invention can be administered in combination with animal feedstuff, and for this purpose a concentrated feed additive or premix can be prepared for mixing with the normal animal feed.
  • an avermectin compound produced according to the present invention can be applied as a spray, dust, emulsion and the like in accordance with standard agricultural practice.
  • Streptomyces avermitilis ATCC 53692 was stored at -70°C as a whole broth prepared in seed medium consisting of: Starch (Nadex, Laing National) - 20g; Pharmamedia (Trader's
  • the column used was a Beckman Ultrasphere C-18, 5 ⁇ m, 4.6 mm x 25 cm column maintained at 40°C. Twenty-five ⁇ l of the above methanol solution was injected onto the column. Elution was performed with a linear gradient of methanol-water from 80:20 to 95:5 over 40 min at 0.85/ml min. Two standard concentrations of cyclohexyl B1 were used to calibrate the detector response, and the area under the curves for B2 and B1 avermectins was measured. 6.2. Results
  • the mycelia grown in the above medium were used to inoculate 10 ml of TSB medium (Difco Tryptic Soy Broth - 30 g, in 1 liter dH 2 0, autoclaved at 121 °C for 25 min) in a 25 mm x 150 mm tube which was maintained with shaking (300 rpm) at 28°C for 48-72 hrs. 7.1.2. Chromosomal DNA Isolation From Streptomyces Aliquots (0.25 ml or 0.5 ml) of mycelia grown as described above were placed in 1.5 ml microcentrifuge tubes and the cells concentrated by centrifugation at 12,000 x g for 60 sec.
  • the supernatant was discarded and the cells were resuspended in 0.25 ml TSE buffer (20 ml 1.5 M sucrose, 2.5 ml 1 M Tris-HCI, pH 8.0, 2.5 ml 1 M EDTA, pH 8.0, and 75 ml dH 2 0) containing 2 mg/ml Iysozyme.
  • TSE buffer 20 ml 1.5 M sucrose, 2.5 ml 1 M Tris-HCI, pH 8.0, 2.5 ml 1 M EDTA, pH 8.0, and 75 ml dH 2 0
  • the samples were incubated at 37 C C for 20 min with shaking, loaded into an AutoGen 540TM automated nucleic acid isolation instrument (Integrated Separation Systems, Natick, MA), and genomic DNA isolated using Cycle 159 (equipment software) according to manufacturer's instructions.
  • the lysate was incubated at 65°C for 10 min, cooled to rm temp, split into two 1.5 ml Eppendorf tubes, and extracted 1x with 0.5 ml phenol/chloroform (50% phenol previously equilibrated with 0.5 M Tris, pH 8.0; 50% chloroform). The aqueous phase was removed and extracted 2 to 5x with chloroform :isoamyl alcohol (24:1).
  • the DNA was precipitated by adding 1/10 volume 3M sodium acetate, pH 4.8, incubating the mixture on ice for 10 min, centrifuging the mixture at 15,000 rpm at 5°C for 10 min, and removing the supernatant to a clean tube to which 1 volume of isopropanol was added.
  • 1.5 ml of mycelia were placed in 1.5 ml microcentrifuge tubes and the cells concentrated by centrifugation at 12,000 x g for 60 sec. The supernatant was discarded, the cells were resuspended in 1.0 ml 10.3% sucrose and concentrated by centrifugation at 12,000 x g for 60 sec, and the supernatant discarded. The cells were resuspended in 0.5 ml TSE buffer containing 2 mg/ml Iysozyme, and incubated at 37°C for 15-30 min.
  • E. coli Plasmid DNA Isolation From E. coli
  • 5 ml Luria-Bertani (LB) medium Bacto-yeast extract - 5 g, and NaCI - 10 g in 1 liter dH 2 0, pH 7.0, autoclaved at 121°C for 25 min, and supplemented with 100 ⁇ g/ml ampicillin).
  • the culture was incubated overnight, and a 1 ml aliquot placed in a 1.5 ml microcentrifuge tube.
  • the culture samples were loaded into the AutoGen 540TM automated nucleic acid isolation instrument and plasmid DNA was isolated using Cycle 3 (equipment software) according to manufacturer's instructions.
  • YEME medium contains per liter: Difco Yeast Extract - 3 g; Difco Bacto-peptone - 5 g; Difco Malt Extract - 3 g; Sucrose - 300 g.
  • the mycelia were grown at 30°C for 48-72 hrs and harvested by centrifugation in a 50 ml centrifuge tube (Falcon) at 3,000 rpm for 20 min. The supernatant was discarded and the mycelia were resuspended in P buffer, which contains: sucrose - 205 g; K 2 S0 4 - 0.25 g; MgCI 2 ' 6H 2 0 - 2.02 g; H 2 0 - 600 ml; K 2 P0 4 (0.5%) - 10 ml; trace element solution * - 20 ml; CaCI 2 • 2H 2 0 (3.68%) - 100 ml; and MES buffer (1.0 M, pH 6.5) - 10 ml.
  • P buffer which contains: sucrose - 205 g; K 2 S0 4 - 0.25 g; MgCI 2 ' 6H 2 0 - 2.02 g; H 2 0 - 600 ml; K 2 P0 4 (
  • Trace element solution contains per liter: ZnCI 2 - 40 mg; FeCI 3 ' 6H 2 0 - 200 mg; CuCI 2 • 2H 2 0 - 10 mg; MnCI 2 ' 4H 2 0 - 10 mg; Na 2 B 4 0 7 ' 10H 2 0 - 10 mg; (NH 4 ) 6 Mo 7 0 24 ' 4H 2 0 - 10 mg).
  • the pH was adjusted to 6.5, final volume was adjusted to 1 liter, and the medium was filtered hot through a 0.45 micron filter.
  • the mycelia were pelleted at 3,000 rpm for 20 min, the supernatant was discarded, and the mycelia were resuspended in 20 ml P buffer containing 2 mg/ml Iysozyme.
  • the mycelia were incubated at 35°C for 15 min with shaking, and checked microscopically to determine extent of protoplast formation. When protoplast formation was complete, the protoplasts were centrifuged at 8,000 rpm for 10 min. The supernatant was removed and the protoplasts were resuspended in 10 ml P buffer.
  • the protoplasts were centrifuged at 8,000 rpm for 10 min, the supernatant was removed, the protoplasts were resuspended in 2 ml P buffer, and approximately 1 x 10 9 protoplasts were distributed to 2.0 ml cryogenic vials (Nalgene).
  • T buffer base contains: PEG-1000 (Sigma) - 25'g; sucrose - 2.5 g; H 2 0 - 83 ml. The pH was adjusted to 8.8 with 1 N NaOH (filter sterilized), and the T buffer base was filter-sterilized and stored at 4°C.
  • T buffer base 8.3 ml
  • K 2 P0 4 (4 mM) - 1.0 ml
  • TES (1 M, pH 8) - 0.5 ml.
  • Each component of the working T buffer was individually filter-sterilized.
  • the protoplasts were then plated on RM 14 media, which contains: sucrose - 205 g; K 2 S0 4 - 0.25 g; MgCI 2 ' 6H 2 0 - 10.12 g; glucose - 10 g; Difco Casamino Acids - 0.1 g; Difco Yeast Extract - 5 g; Difco Oatmeal Agar - 3 g; Difco Bacto Agar - 22 g; dH 2 0 - 800 ml.
  • the solution was autoclaved at 121°C for 25 min.
  • the protoplasts were incubated in 95% humidity at 30°C for 20-24 hrs.
  • 1 ml of overlay buffer containing 125 ⁇ g per ml thiostrepton was spread evenly over the RM14 regeneration plates.
  • Overlay buffer contains per 100 ml: sucrose - 10.3 g; trace element solution (same as above) - 0.2 ml; and MES (1 M, pH 6.5) - 1 ml.
  • the protoplasts were incubated in 95% humidity at 30°C for 7-14 days until thiostrepton resistant (Thio r ) colonies were visible.
  • Preform medium contains: soluble starch (either thin boiled starch or KOSO, Japan Corn Starch Co., Nagoya) - 20 g/L; Pharmamedia - 15 g/L; Ardamine pH - 5 g/L (Champlain Ind., Clifton, NJ); CaC0 3 - 2 g/L; 2x bcfa ("bcfa" refers to branched chain fatty acids) containing a final concentration in the medium of 50 ppm 2-(+/-)-methyl butyric acid, 60 ppm isobutyric acid, and 20 ppm isovaleric acid. The pH was adjusted to 7.2, and the medium was autoclaved at 121 °C for 25 min.
  • soluble starch either thin boiled starch or KOSO, Japan Corn Starch Co., Nagoya
  • Pharmamedia 15 g/L
  • Ardamine pH - 5 g/L Ardamine pH - 5 g/L (Champlain Ind.,
  • the tube was shaken at a 17° angle at 215 rpm at 29°C for 3 days.
  • a 2-ml aliquot of the seed culture was used to inoculate a 300 ml Erlenmeyer flask containing 25 ml of production medium which contains: starch (either thin boiled starch or KOSO) - 160 g/L; Nutrisoy (Archer Daniels Midland, Decatur, IL) - 10 g/L; Ardamine pH - 10 g/L; K 2 HP0 4 - 2 g/L; MgS0 4 .4H 2 0 - 2 g/L; FeS0 4 .7H 2 0 - 0.02 g/L; MnCI 2 - 0.002 g/L; ZnS0 4 .7H 2 0 - 0.002 g/L; CaC0 3 - 14 g/L; 2x bcfa (as above); and cyclohexane carboxy
  • a cosmid library of S. avermitilis (ATCC 31272, SC-2) chromosomal DNA was prepared and hybridized with a ketosynthase (KS) probe made from a fragment of the Saccharopolyspora erythraea polyketide synthase (PKS) gene.
  • KS ketosynthase
  • PKS polyketide synthase
  • Cosmid clones containing ketosynthase-hybridizing regions were identified by hybridization to a 2.7 Kb ⁇ /cfel/Eco47lll fragment from pEX26 (kindly supplied by Dr. P. Leadlay, Cambridge, UK). Approximately 5 ng of pEX26 were digested using A/del and Eco47lll. The reaction mixture was loaded on a 0.8% SeaPlaque GTG agarose gel (FMC BioProducts, Rockland, ME). The 2.7 Kb ⁇ /del/Eco47lll fragment was excised from the gel after electrophoresis and the DNA recovered from the gel using GELaseTM from Epicentre Technologies using the Fast Protocol.
  • the 2.7 Kb ⁇ /del/Eco47lll fragment was labeled with [ ⁇ - 32 P]dCTP (deoxycytidine 5'-triphosphate, tetra (triethylammonium) salt, [alpha- 32 P]-) (NEN-Dupont, Boston, MA) using the BRL Nick Translation System (BRL Life Technologies, Inc., Gaithersburg, MD) following the supplier's instructions.
  • BRL Nick Translation System BRL Life Technologies, Inc., Gaithersburg, MD
  • a typical reaction was performed in 0.05 ml volume. After addition of 5 ⁇ l Stop buffer, the labeled DNA was separated from unincorporated nucleotides using a G-25 Sephadex Quick SpinTM Column (Boehringer Mannheim) following supplier's instructions.
  • avermitilis genomic SamHI restriction map of the five cosmids (i.e., pSE65, pSE66, pSE67, pSE68, pSE69) was constructed by analysis of overlapping cosmids and hybridizations (FIGURE 4).
  • pSE66 5 ⁇ g was digested with Sacl and BamHl. The reaction mixture was loaded on a 0.8%
  • Protoplasts of S. avermitilis strain 1100-SC38 were prepared and transformed with pSE119 as described in Section 7.1.5 above.
  • Strain 1100-SC38 is a mutant that produces significantly more of the avermectin cyclohexyl-B2 form compared to avermectin cyclohexyI-B1 form when supplemented with cyclohexane carboxylic acid (B2:B1 of about 30:1).
  • pSE119 used to transform S. avermitilis protoplasts was isolated from either E. coli strain GM2163 (obtained from Dr. B. J. Bachmann, Curator, E. coli Genetic Stock Center, Yale University), E.
  • AveC mutant the insert DNA was sequenced. Approximately 10 ⁇ g of pSE119 were isolated using a plasmid DNA isolation kit (Qiagen, Valencia, CA) following manufacturer's instructions, and sequenced using an ABI 373A Automated DNA Sequencer (Perkin Elmer,
  • FIGURE 1 SEQ ID NO:1 .
  • a new plasmid designated as pSE118, was constructed as follows. Approximately 5 ⁇ g of pSE66 was digested with Sphl and BamHl. The reaction mixture was loaded on a 0.8% SeaPlaque GTG agarose gel (FMC BioProducts), a 2.8 Kb Sphl/BamHi fragment was excised from the gel after electrophoresis, and the DNA was recovered from the gel using GELaseTM (Epicentre Technologies) using the Fast Protocol. Approximately 5 ⁇ g of the shuttle vector pWHM3 was digested with Sphl and SamHI.
  • Protoplasts of S. avermitilis strain 1100-SC38 were transformed with pSE118 as above. Thiostrepton resistant transformants of strain 1100-SC38 were isolated and analyzed by HPLC analysis of fermentation products. Transformants of S. avermitilis strain 1100-SC38 containing pSE118 were not altered in the ratios of avermectin cyclohexyl-B2: avermectin cyclohexyl-B1 compared to strain 1100-SC38 (TABLE 2).
  • a -1.2 Kb fragment containing the aveC ORF was isolated from S. avermitilis chromosomal DNA by PCR amplification using primers designed on the basis of the aveC nucleotide sequence obtained above.
  • the PCR primers were supplied by Genosys
  • the PCR reaction was carried out with Deep VentTM polymerase (New England Biolabs) in buffer provided by the manufacturer, and in the presence of 300 ⁇ M dNTP, 10% glycerol, 200 pmol of each primer, 0.1 ⁇ g template, and 2.5 units enzyme in a final volume of
  • extension step The subsequent 24 cycles had a similar thermal profile except that the denaturation step was shortened to 45 sec and the annealing step was shortened to 1 min.
  • the PCR product was electrophoresed in a 1 % agarose gel and a single DNA band of -1.2 Kb was detected.
  • This DNA was purified from the gel, and ligated with 25 ng of linearized, blunt pCR-Blunt vector (Invitrogen) in a 1 :10 molar vector-to-insert ratio following manufacturer's instructions.
  • the ligation mixture was used to transform One ShotTM Competent E. coli cells (Invitrogen) following manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the -1.2 Kb insert was confirmed by restriction analysis. This plasmid was designated as pSE179.
  • the insert DNA from pSE179 was isolated by digestion with BamYMIXbal, separated by electrophoresis, purified from the gel, and ligated with shuttle vector pWHM3, which had also been digested with BamH Xbal, in a total DNA concentration of 1 ⁇ g in a 1 :5 molar vector-to-insert ratio.
  • the ligation mixture was used to transform competent E. coli DH5 ⁇ cells according to manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the -1.2 Kb insert was confirmed by restriction analysis.
  • This plasmid which was designated as pSE186 (FIGURE 2, ATCC 209604), was transformed into E. coli DM1 , and plasmid DNA was isolated from ampicillin resistant transformants.
  • This example describes the construction of several different S. avermitilis AveC mutants using the compositions and methods described above.
  • a general description of techniques for introducing mutations into a gene in Streptomyces is described by Kieser and Hopwood, 1991 , Meth. Enzym. 204:430-458.
  • a more detailed description is provided by Anzai et al., 1988, J. Antibiot. XLI(2):226-233, and by Stutzman-Engwall et al., 1992, J. Bacteriol.
  • AveC mutants containing inactivated aveC genes were constructed using several methods, as detailed below.
  • pSE118 (described in Section 7.1.9, above) was digested with Sph ⁇ and BamHl, the digest electrophoresed, and the -2.8 Kb Sph ⁇ IBamH ⁇ insert purified from the gel.
  • pSE119 was digested with Pstl and EcoRl, the digest electrophoresed, and the -1.5 Kb PsttlEcoRl insert purified from the gel.
  • Shuttle vector pWHM3 was digested with BamHl and EcoRl.
  • pSE27 was digested with Pstl and Sphl, the digest electrophoresed, and the -1.7 Kb Pstl/Sphl insert purified from the gel.
  • All four fragments (i.e., -2.8 Kb, ⁇ 1.5Kb, ⁇ 7.2Kb, -1.7 Kb) were ligated together in a 4-way ligation.
  • the ligation mixture was transformed into competent E. coli DH5 ⁇ cells following manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid was designated as pSE180 (FIGURE 3; ATCC 209605).
  • pSE180 was transformed into S. lividans TK64 and transformed colonies identified by resistance to thiostrepton and erythromycin.
  • pSE180 was isolated from S. lividans and used to transform S. avermitilis protoplasts.
  • Total chromosomal DNA was isolated from strain SE180-11, digested with restriction enzymes BamHl, Hindlll, Pstl, or Sphl, resolved by electrophoresis on a 0.8% agarose gel, transferred to nylon membranes, and hybridized to the ermE probe. These analyses showed that chromosomal integration of the ermE resistance gene, and concomitant deletion of the 640 bp Pstl/Sphl fragment had occurred by a double crossover event. HPLC analysis of fermentation products of strain SE180-11 showed that normal avermectins were no longer produced (FIGURE 5A) .
  • the 1.7 Kb ermE gene was removed from the chromosome of S. avermitilis strain SE180-11 , leaving a 640 bp Pstl/Sphl deletion in the aveC gene.
  • a gene replacement plasmid was constructed as follows: pSE180 was partially digested with Xbal and an -11.4 Kb fragment purified from the gel. The ⁇ 11.4 Kb band lacks the 1.7 Kb ermE resistance gene. The DNA was then ligated and transformed into E. coli DH5 cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis.
  • This plasmid which was designated as pSE184, was transformed into E. coli DM1 , and plasmid DNA isolated from ampicillin resistant transformants.
  • This plasmid was used to transform protoplasts of S. avermitilis strain SE180-11. Protoplasts were prepared from thiostrepton resistant transformants of strain SE180-11 and were plated as single colonies on RM14. After the protoplasts had regenerated, single colonies were screened for the absence of both erythromycin resistance and thiostrepton resistance, indicating chromosomal integration of the inactivated aveC gene and loss of the free replicon containing the ermE gene.
  • SE184-1-13 Fermentation analysis of SE184-1-13 showed that normal avermectins were not produced and that SE184-1-13 had the same fermentation profile as SE180-11.
  • a frameshift was introduced into the chromosomal aveC gene by adding two G's after the C at nt position 471 using PCR, thereby creating a BspE1 site.
  • the presence of the engineered 6spE1 site was useful in detecting the gene replacement event.
  • the PCR primers were designed to introduce a frameshift mutation into the aveC gene, and were supplied by Genosys Biotechnologies, Inc.
  • the rightward primer was: 5'-GGTTCCGGATGCCGTTCTCG-3' (SEQ ID NO:8) and the leftward primer was: 5'-AACTCCGGTCGACTCCCCTTC-3' (SEQ ID NO:9).
  • the PCR conditions were as described in Section 7.1.10 above.
  • the 666 bp PCR product was digested with Sphl to give two fragments of 278 bp and 388 bp, respectively.
  • the 388 bp fragment was purified from the gel.
  • the gene replacement plasmid was constructed as follows: shuttle vector pWHM3 was digested with EcoRl and BamHl. pSE119 was digested with SamHI and Sphl, the digest electrophoresed, and a -840 bp fragment was purified from the gel. pSE119 was digested with EcoRl and Xmnl, the digest was resolved by electrophoresis, and a -1.7 Kb fragment was purified from the gel. All four fragments (i.e., -7.2 Kb, -840 bp, -1.7 Kb, and 388 bp) were ligated together in a 4-way ligation. The ligation mixture was transformed into competent E. coli DH5 cells.
  • Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis.
  • This plasmid which was designated as pSE185, was transformed into E. coli DM1 and plasmid DNA isolated from ampicillin resistant transformants.
  • This plasmid was used to transform protoplasts of S. avermitilis strain 1100-SC38. Thiostrepton resistant transformants of strain 1100-SC38 were isolated and analyzed by HPLC analysis of fermentation products.
  • pSE185 did not significantly alter the B2:B1 avermectin ratios when transformed into S. avermitilis strain 1100-SC38 (TABLE 2).
  • pSE185 was used to transform protoplasts of S.
  • avermitilis to generate a frameshift mutation in the chromosomal aveC gene.
  • Protoplasts were prepared from thiostrepton resistant transformants and plated as single colonies on RM14. After the protoplasts had regenerated, single colonies were screened for the absence of thiostrepton resistance. Chromosomal DNA from thiostrepton sensitive colonies was isolated and screened by PCR for the presence of the frameshift mutation integrated into the chromosome. The PCR primers were designed based on the aveC nucleotide sequence, and were supplied by Genosys Biotechnologies, Inc. (Texas).
  • the rightward PCR primer was: 5'- GCAAGGATACGGGGACTAC-3' (SEQ ID NO:10) and the leftward PCR primer was: 5'- GAACCGACCGCCTGATAC-3' (SEQ ID NO:11 ), and the PCR conditions were as described in Section 7.1.10 above.
  • the PCR product obtained was 543 bp and, when digested with BspE1 , three fragments of 368 bp, 96 bp, and 79 bp were observed, indicating chromosomal integration of the inactivated aveC gene and loss of the free replicon.
  • mutations in the aveC gene that change both: (i) nt position 970 from G to A, which changes the amino acid at position 266 from a glycine (G) to an aspartate (D), and (ii) nt position 996 from T to C, which changes the amino acid at position 275 from tyrosine (Y) to histidine (H), were produced.
  • An S. avermitilis strain with these mutations did not produce normal avermectins and had the same fermentation profile as strains SE180- 11 , SE184-1-13, and SE185-5a.
  • pSE186 which contains a wild-type copy of the aveC gene, was transformed into E. coli DM1 , and plasmid DNA was isolated from ampicillin resistant transformants. This pSE186 DNA was used to transform protoplasts of S. avermitilis strain SE180-11. Thiostrepton resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and Thio r Erm r transformants were analyzed by HPLC analysis of fermentation products. The presence of the functional aveC gene in trans was able to restore normal avermectin production to strain SE180-11 (FIGURE
  • S. avermitilis strain SE180-11 containing an inactive aveC gene was complemented by transformation with a plasmid containing a functional aveC gene
  • Strain SE180-11 was also utilized as a host strain to characterize other mutations in the aveC gene, as described below. Chromosomal DNA was isolated from strain 1100-SC38, and used as a template for
  • the 1.2 Kb ORF was isolated by PCR amplification using primers designed on the basis of the aveC nucleotide sequence.
  • the rightward primer was SEQ ID NO:6 and the leftward primer was SEQ ID NO:7 (see Section 7.1.10, above).
  • the PCR and subcloning conditions were as described in Section 7.1.10.
  • DNA sequence analysis of the 1.2 Kb ORF shows a mutation in the aveC gene that changes nt position 337 from C to T, which changes the amino acid at position 55 from serine (S) to phenylalanine (F).
  • the aveC gene containing the S55F mutation was subcloned into pWHM3 to produce a plasmid which was designated as pSE187, and which was used to transform protoplasts of S. avermitilis strain SE180-11. Thiostrepton resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and Thio r Erm r transformants were analyzed by HPLC analysis of fermentation products.
  • a mutation in the aveC gene was identified that changes nt position 588 from G to A, which changes the amino acid at position 139 from alanine (A) to threonine (T).
  • the aveC gene containing the A139T mutation was subcloned into pWHM3 to produce a plasmid which was designated pSE188, and which was used to transform protoplasts of S. avermitilis strain SE180-11.
  • Thiostrepton resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and Thio r Erm r transformants were analyzed by HPLC analysis of fermentation products.
  • pSE186 was digested with EcoRl and cloned into pGEM3Zf (Promega) which had been digested with EcoRl.
  • This plasmid which was designated as pSE186a, was digested with Apal and Kpnl, the DNA fragments separated on an agarose gel, and two fragments of -3.8 Kb and -0.4 Kb were purified from the gel.
  • the -1.2 Kb insert DNA from pSE186 was used as a PCR template to introduce a single base change at nt position 585.
  • the PCR primers were designed to introduce a mutation at nt position 585, and were supplied by Genosys Biotechnologies, Inc. (Texas).
  • the rightward PCR primer was: 5'- GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCCCTGGCGACG-3' (SEQ ID NO:12); and the leftward PCR primer was: 5'-GGAACCGACCGCCTGATACA-3' (SEQ ID NO:13).
  • the PCR reaction was carried out using an Advantage GC genomic PCR kit (Clonetech Laboratories, Palo Alto, CA) in buffer provided by the manufacturer in the presence of 200 ⁇ M dNTPs, 200 pmol of each primer, 50 ng template DNA, 1.0 M GC-Melt and 1 unit KlenTaq Polymerase Mix in a final volume of 50 ⁇ l.
  • the thermal profile of the first cycle was 94°C for 1 min; followed by 25 cycles of 94°C for 30 sec and 68°C for 2 min; and 1 cycle at 68°C for 3 min.
  • a PCR product of 295 bp was digested with Apal and Kpnl to release a 254 bp fragment, which was resolved by electrophoresis and purified from the gel. All three fragments (-3.8 Kb, -0.4 Kb and 254 bp) were ligated together in a 3-way ligation.
  • the ligation mixture was transformed into competent E. coli DH5 ⁇ cells. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid was designated as pSE198.
  • pSE198 was digested with EcoRl, cloned into pWHM3, which had been digested with EcoRl, and transformed into E. coli DH5 cells.
  • Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis.
  • This plasmid DNA was transformed into E. coli DM1 , plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis.
  • This plasmid which was designated as pSE199, was used to transform protoplasts of S. avermitilis strain SE180-11. Thiostrepton resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and Thio r Erm r transformants were analyzed by HPLC analysis of fermentation products.
  • the PCR primers were designed to introduce mutations at nt positions 585 and 588, and were supplied by Genosys Biotechnologies, Inc. (Texas).
  • the rightward PCR primer was: 5'- GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCGCTGGCGACGACC-3' (SEQ ID NO:14); and the leftward PCR primer was: 5'-GGAACATCACGGCATTCACC-3' (SEQ ID NO:15).
  • the PCR reaction was performed using the conditions described immediately above in this Section.
  • a PCR product of 449 bp was digested with Apal and Kpnl to release a 254 bp fragment, which was resolved by electrophoresis and purified from the gel.
  • pSE186a was digested with Apal and Kpnl, the DNA fragments separated on an agarose gel, and two fragments of -3.8 Kb and -0.4 Kb were purified from the gel. All three fragments (-3.8 Kb, -0.4 Kb and 254 bp) were ligated together in a 3-way ligation, and the ligation mixture was transformed into competent E. coli DH5 ⁇ cells. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis.
  • This plasmid was designated as pSE230.
  • pSE230 was digested with EcoRl, cloned into pWHM3, which had been digested with EcoRl, and transformed into E. coli DH5 ⁇ cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. This plasmid DNA was transformed into E. coli DM1 , plasmid DNA isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid, which was designated as pSE231 , was used to transform protoplasts of S. avermitilis strain SE180-11.
  • Thiostrepton resistant transformants of SE180-11 were isolated, the presence of erythromycin resistance was determined, and Thio r Erm r transformants were analyzed by fermentation.
  • Another mutation was constructed to further reduce the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1. Because the S138T/A139T mutations altered the B2:B1 ratios in the more favorable B1 direction, a mutation was constructed to introduce a threonine at amino acid position 138 and a phenylalanine at amino acid position 139.
  • the -1.2 Kb insert DNA from pSE186 was used as a PCR template.
  • the PCR primers were designed to introduce mutations at nt positions 585 (changing a T to A), 588 (changing a G to T), and 589 (changing a C to T), and were supplied by Genosys Biotechnologies, Inc. (Texas).
  • the rightward PCR primer was: 5'-GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCGC TGGCGACGTTC-3' (SEQ ID NO:16); and the leftward PCR primer was: 5'- GGAACATCACGGCATTCACC-3' (SEQ ID NO: 15).
  • the PCR reaction was carried out using an Advantage GC genomic PCR kit (Clonetech Laboratories, Palo Alto, CA) in buffer provided by the manufacturer in the presence of 200 ⁇ M dNTPs, 200 pmol of each primer, 50 ng template DNA, 1.1 mM Mg acetate, 1.0 M GC-Melt and 1 unit Tth DNA Polymerase in a final volume of 50 ⁇ l.
  • the thermal profile of the first cycle was 94°C for 1 min; followed by 25 cycles of 94°C for 30 sec and 68°C for 2 min; and 1 cycle at 68°C for 3 min.
  • a PCR product of 449 bp was digested with Apal and Kpnl to release a 254 bp fragment, which was resolved by electrophoresis and purified from the gel. All three fragments (-3.8 Kb, -0.4 Kb and 254 bp) were ligated together in a 3-way ligation.
  • the ligation mixture was transformed into competent E. coli DH5 cells. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid was designated as pSE238.
  • pSE238 was digested with EcoRl, cloned into pWHM3, which had been digested with EcoRl, and transformed into E. coli DH5 ⁇ cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. This plasmid DNA was transformed into E. coli DM1 , plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid, which was designated as pSE239, was used to transform protoplasts of S. avermitilis strain SE180-11.
  • Plasmid DNA was isolated from ampicillin resistant transformants, and used to transform protoplasts of S. avermitilis strain SE180-11. Thiostrepton resistant transformants of strain SE180-11 were isolated and screened for the production of avermectins with a cyclohexyl-B2:cyclohexyl-B1 ratio of 1 :1 or less.
  • the DNA sequence of plasmid DNA from SE180-11 transformants producing avermectins with a B2:B1 ratio of 1 :1 or less was determined.
  • pSE290 contains 4 nucleotide mutations at nt position 317 from T to A, at nt position
  • pSE291 contains 4 nucleotide mutations at nt position 272 from G to A, at nt position 585 from T to A, at nt position 588 from G to A, and at nt position 708 from G to A.
  • the nucleotide change at nt position 585 changes the amino acid at AA position 138 from S to T
  • the nucleotide change at nt position 588 changes the amino acid at AA position 139 from A to T
  • the nucleotide change at nt position 708 changes the amino acid at AA position 179 from G to S.
  • the B2:B1 ratio produced by cells carrying this plasmid was 0.57:1 (TABLE 4).
  • pSE292 contains the same four nucleotide mutations as pSE290.
  • the B2:B1 ratio produced by cells carrying this plasmid was 0.40:1 (TABLE 4).
  • pSE293 contains 6 nucleotide mutations at nt 24 from A to G, at nt position 286 from A to C, at nt position 497 from T to C, at nt position 554 from C to T, at nt position 580 from T to C, and at nt position 886 from A to T.
  • the nucleotide change at nt position 286 changes the amino acid at AA position 38 from Q to P
  • the nucleotide change at nt position 580 changes the amino acid at AA position 136 from L to P
  • the nucleotide change at nt position 886 changes the amino acid at AA position 238 from E to D.
  • pSE294 contains 6 nucleotide mutations at nt 469 from T to C, at nt position 585 from T to A, at nt position 588 from G to A, at nt position 708 from G to A, at nt position 833 from C to T, and at nt position 1184 from G to A. In addition, nts at positions 173, 174, and 175 are deleted.
  • the nucleotide change at nt position 469 changes the amino acid at AA position 99 from F to S
  • the nucleotide change at nt position 585 changes the amino acid at AA position 138 from S to T
  • the nucleotide change at nt position 588 changes the amino acid at AA position 139 from A to T
  • the nucleotide change at nt position 708 changes the amino acid from AA position 179 from G to S.
  • the B2:B1 ratio produced by cells carrying this plasmid was 0.53:1 (TABLE 4).
  • pSE295 contains 2 nucleotide mutations at nt 588 from G to A and at nt 856 from T to
  • pSE296 contains 5 nucleotide mutations at nt position 155 from T to C, at nt position 505 from G to T, at nt position 1039 from C to T, at nt position 1202 from C to T, and at nt position 1210 from T to C.
  • the nucleotide change at nt position 505 changes the amino acid at AA position 111 from G to V and the nucleotide change at nt position 1039 changes the amino acid at AA position 289 from P to L.
  • the B2:B1 ratio produced by cells carrying this plasmid was 0.73:1 (TABLE 4).
  • pSE297 contains 4 nucleotide mutations at nt position 377 from G to T, at nt position
  • the nucleotide change at nt position 588 changes the amino acid at AA position 139 from A to T
  • the nucleotide change at nt position 633 changes the amino acid at AA position 154 from K to E
  • the nucleotide change at nt position 1067 changes the amino acid at AA position 298 from Q to H.
  • the B2:B1 ratio produced by cells carrying this plasmid was 0.67:1 (TABLE 4).
  • the production plates were incubated at 30° under humidity for 12-14 days. Sporulation of the strains occurred after 5-8 days of incubation.
  • the production plates were made essentially as described in PCT International Publication WO 99/41389 by Pfizer Inc., with the exception of adding 1 % agarose to ensure a solid surface. Extraction and ESI-MS/MS screening
  • the MS/MS transition for B1 sodiated ion is from m/z 921 to m/z 777 and for B2 sodiated ion is from m/z 939 to m/z 795 in positive mode.
  • a Finnigan TSQ-7000, Micromass Quattro-LC mass spectrometer and a Leap Technology Twin-Pal autosampler were used for this high throughput screening. Integration of the separate B1 and B2 chromatograms for each well location identified the hits.

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DK03739390T DK1476539T3 (da) 2002-02-12 2003-01-31 Streptomyces avermitilis gen dirigerende forholdet af B2:B1 avermectiner
JP2003568070A JP2005517406A (ja) 2002-02-12 2003-01-31 Ｂ２：Ｂ１アベルメクチン比を支配するＳｔｒｅｐｔｏｍｙｃｅｓａｖｅｒｍｉｔｉｌｉｓ遺伝子
SI200331553T SI1476539T1 (sl) 2002-02-12 2003-01-31 Gen streptomyces avermitilis, ki uravnava razmerje B2:B1 avermektinov
DE60326824T DE60326824D1 (de) 2002-02-12 2003-01-31 Streptomyces avermitilis gen zur regulierung des verhältnisses der b2:b1 avermectine
AU2003245052A AU2003245052B2 (en) 2002-02-12 2003-01-31 Streptomyces avermitilis gene directing the ratio of B2:B1 avermectins
MEP-2008-730A ME00472B (me) 2002-02-12 2003-01-31 Streptomyces avermitilis gen koji određuje odnos b2:b1 avermektina
IL16286603A IL162866A0 (en) 2002-02-12 2003-01-31 Streptomyces a vermitilis gene directing the ratioof b2:b1 avermectins
EP03739390A EP1476539B1 (en) 2002-02-12 2003-01-31 Streptomyces avermitilis gene directing the ratio of b2:b1 avermectins
BRPI0307620A BRPI0307620B8 (pt) 2002-02-12 2003-01-31 moléculas de polinucleotídeo codificando variantes de avec, métodos para produzir uma cepa recombinante de streptomyces avermitilis, bem como as referidas células recombinantes e composição de avermectinas
CN038037416A CN1630712B (zh) 2002-02-12 2003-01-31 指导b2:b1除虫菌素比例的除虫链霉菌基因
KR1020047012437A KR100718378B1 (ko) 2002-02-12 2003-01-31 Ｂ2:ｂ1 아베르멕틴의 비를 제어하는 스트렙토마이세스아베르미틸리스 유전자
YUP-700/04A RS50813B (sr) 2002-02-12 2003-01-31 Streptomyces avermitilis gen koji određuje odnos b2:b1 avermektina
CA2475214A CA2475214C (en) 2002-02-12 2003-01-31 Streptomyces avermitilis gene directing the ratio of b2:b1 avermectins
HK05106917.2A HK1074644B (en) 2002-02-12 2003-01-31 Streptomyces avermitilis gene directing the ratio of b2:b1 avermectins
MXPA04007777A MXPA04007777A (es) 2002-02-12 2003-01-31 Gen de streptomyces avertmitilis que rige la relacion de avermectinas b2:b1.
IL162866A IL162866A (en) 2002-02-12 2004-07-05 A gene of Streptomyces avermitilis that directs the ratio of Avermectins B2: B1
IL192371A IL192371A (en) 2002-02-12 2008-06-22 POLYNUCLEOTIDE SEQUENCES TO A STREPTOMYCES AVERMITILIS aveC ALLELE PRODUCING A REDUCED CLASS 2:1 RATIO OF AVERMECTINS

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CN102241750A (zh) * 2010-05-14 2011-11-16 中国科学院微生物研究所 一种生产阿维菌素的基因工程方法及其专用菌株

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CN102093997B (zh) * 2010-12-01 2013-03-13 中国科学院上海有机化学研究所 一种改造除虫链霉菌产生多拉菌素的方法
CN109576196B (zh) * 2019-01-25 2022-01-04 北大方正集团有限公司 一种生产多拉菌素的发酵培养基及多拉菌素的生产方法

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EP2050815A1 (en) * 2002-02-12 2009-04-22 Pfizer Products Inc. Streptomyces Avermitilis gene directing the ratio of B2:B1 avermectins
US8008078B2 (en) 2002-02-12 2011-08-30 Pfizer Inc. Streptomyces avermitilis gene directing the ratio of B2:B1 avermectins
CN102241750A (zh) * 2010-05-14 2011-11-16 中国科学院微生物研究所 一种生产阿维菌素的基因工程方法及其专用菌株
CN102241750B (zh) * 2010-05-14 2014-05-28 中国科学院微生物研究所 一种生产阿维菌素的基因工程方法及其专用菌株

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