JP4011036B2 - Streptomyces avermitilis gene for B2: B1 avermectin ratio - Google Patents

Description

1. TECHNICAL FIELD The present invention relates to a composition and a method for producing avermectin (mainly in the field of animal health). More particularly, the present invention relates to a polynucleotide molecule comprising a nucleotide sequence encoding an AveC gene product, a composition, and a method for screening said polynucleotide molecule. The AveC gene product can be used to adjust the ratio of class 2: 1 avermectins produced by fermentation of Streptomyces avermitilis cultures. The present invention further relates to vectors, transformed host cells, and novel mutants of Streptomyces avermitilis in which the aveC gene has been mutated to adjust the ratio of class 2: 1 avermectins produced.

2. 2. Background of the Invention 2.1. Avermectin Streptomyces species produce a wide variety of secondary metabolites. The secondary metabolites include avermectin, which includes a group of eight related 16-membered macrocyclic lactones with effective anthelmintic and insecticidal activity. Eight different (but closely related) compounds are referred to as A1a, A1b, A2a, A2b, B1a, B1b, B2a, and B2b. A series of compounds with “a” means natural avermectin in which the substituent at the C25 position is an (S) -sec-butyl group, and a series of compounds with “b” has a substituent at the C25 position with an isopropyl group. Means a compound that is The designations “A” and “B” mean avermectins in which the substituent at the C5 position is a methoxy group and a hydroxy group, respectively. The number “1” means avermectin having a double bond at the C22 and 23 positions, and the number “2” means avermectin having a hydrogen atom at the C22 position and a hydroxy group at the C23 position. Among the related avermectins, the B1 type avermectin is the most commercially desirable avermectin since it has been found to have the most effective antiparasitic and pesticidal activity.

  Their production by aerobic fermentation of Avermectin and Streptomyces avermitilis strains is described in US Pat. Nos. 4,310,519 and 4,429,042. Natural avermectin biosynthesis is thought to be intrinsically initiated from CoA thioester analogs of isobutyric acid and S-(+)-2-methylbutyric acid.

  The effective production of avermectin analogs has been accomplished by combining both strain improvement by random mutagenesis and the use of exogenously supplied fatty acids. A Streptomyces avermitilis mutant deficient in branched-chain 2-oxoacid dehydrogenase (bkd-deficient mutant) can only produce avermectin when fermented under fatty acid supplementation. . Methods for screening and isolation of mutants lacking branched chain dehydrogenase activity (eg, Streptomyces avermitilis, ATCC 53567) are described in EP 276103. When the mutant is fermented in the presence of exogenously supplied fatty acids, only four types of avermectins corresponding to the fatty acids used are produced. Thus, when Streptomyces avermitilis (ATCC 53567) is fermented with S-(+)-2-methylbutyric acid supplementation, natural avermectins A1a, A2a, B1a, and B2a are produced. Moreover, when fermented under isobutyric acid supplementation, natural avermectins A1b, A2b, B1b, and B2b are produced. In addition, when fermented under cyclopentanecarboxylic acid supplementation, four novel cyclopentyl avermectins A1, A2, B1, and B2 are produced.

  If another fatty acid is supplemented, new avermectin is produced. By screening over 800 potential precursors, over 60 other novel avermectins have been identified (Dutton et al., 1991, J. Antibiot. 44: 357-365; and Banks et al., 1994, Roy. Soc. Chem. 147: 16-26). Moreover, mutants of Streptomyces avermitilis without 5-O-methyltransferase activity essentially produce only the B-like avermectin. As a result, the Streptomyces avermitilis mutant lacking both branched 2-oxo acid dehydrogenase and 5-O-methyl transferase has only B avermectin corresponding to the fatty acid used to supplement the fermentation. Generate. Thus, supplementing the double mutant with S-(+)-2-methylbutyric acid produces only natural avermectins B1a and B2a, but when supplementing with isobutyric acid or cyclopentanecarboxylic acid, Natural avermectins B1b and B2b or novel cyclopentyl B1 and B2 avermectins are produced, respectively. Supplementing the double mutant with cyclohexanecarboxylic acid is a preferred method for producing cyclohexyl avermectin B1 (doramectin), a commercially important new avermectin. The isolation and characterization of such double mutants [eg Streptomyces avermitilis (ATCC53692)] is described in EP 276103.

2.2. Genes associated with avermectin biosynthesis In many cases, genes associated with the production of secondary metabolites and genes encoding specific antibiotics have been found together on the chromosome. Examples thereof include polyketide synthase gene cluster (PKS) in Streptomyces (see Hopwood and Sherman, 1990, Ann. Rev. Genet. 24: 37-66). Thus, one strategy for cloning a gene in the biosynthetic pathway is to isolate a drug resistance gene and then test the adjacent region of the chromosome for another gene for the biosynthesis of that particular antibiotic. there were. Another strategy for cloning genes involved in the biosynthesis of important metabolites was mutant complementation. For example, a part of a DNA library derived from an organism capable of producing a specific metabolite is introduced into a mutant that does not produce it, and a transformant is screened for the production of the metabolite. Furthermore, hybridization of libraries with probes from other Streptomyces species has been used for gene identification and cloning in the biosynthetic pathway.

Genes related to the biosynthesis of avermectin (ave gene) are found as clusters on the chromosome like genes required for the biosynthesis of another Streptomyces secondary metabolite (eg, PKS). Many ave genes have been successfully cloned using vectors to complement Streptomyces avermitilis mutants in which avermectin biosynthesis is impeded. The cloning of the gene is described in US Pat. No. 5,252,474. Furthermore, Ikeda et al., 1995, J. MoI. Antibiot. 48: 532-534 found that the chromosomal region associated with the C22,23 dehydration process (aveC) was within the 4.82 Kb BamHI fragment of Streptomyces avermitilis and single component B2a Mutations in the aveC gene that cause producer production have been described. Since ivermectin, an effective anthelmintic compound, can be chemically generated from avermectin B2a, a single component producer of avermectin B2a is believed to be particularly useful for commercial production of ivermectin.
Identifying mutations in the aveC gene that minimize the complexity of avermectin production (eg, mutations that reduce the B2: B1 ratio of avermectin) simplify the production and purification of commercially important avermectins Will.

3. SUMMARY OF THE INVENTION The present invention relates to an isolated polynucleotide molecule comprising the complete aveC-ORF of Streptomyces avermitilis or a substantial part thereof, wherein the isolated polynucleotide molecule is a Streptomyces avermitilis. An isolated polynucleotide molecule is provided that lacks the next complete ORF located in the chromosome downstream of the aveC-ORF in vivo. The isolated polynucleotide molecule according to the present invention is preferably the same as the sequence encoding the Streptomyces avermitilis AveC gene product of plasmid pSE186 (ATCC 209604), or FIG. 1 (SEQ ID NO: 1 in the sequence listing) A nucleotide sequence that is the same as the nucleotide sequence of the aveC-ORF or a substantial part thereof.

Furthermore, the present invention relates to a sequence encoding the Streptomyces avermitilis AveC gene product of plasmid pSE186 (ATCC209604), or to the aveC-ORF shown in FIG. 1 (SEQ ID NO: 1 in the sequence listing) or a substantial part thereof. A polynucleotide molecule comprising a nucleotide sequence homologous to the nucleotide sequence of is provided.
Furthermore, the present invention relates to the amino acid sequence encoded by the sequence encoding the AveC gene product of plasmid pSE186 (ATCC209604), or to the amino acid sequence of FIG. 1 (SEQ ID NO: 2 of the sequence listing) or a substantial part thereof. Thus, a polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide having a homologous amino acid sequence is provided.

  The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding an AveC homologous gene product. In a preferred embodiment, the isolated polynucleotide molecule is an AveC homologous gene product derived from Streptomyces hygroscopicus (provided that the homologous gene product comprises the amino acid sequence of SEQ ID NO: 4 in the sequence listing or a substantial part thereof). A nucleotide sequence encoding In a preferred embodiment, the isolated polynucleotide molecule of the present invention encoding the Streptomyces hygroscopicus AveC homologous gene product comprises the nucleotide sequence of SEQ ID NO: 3 in the sequence listing or a substantial part thereof.

Furthermore, the present invention provides a polynucleotide molecule comprising a nucleotide sequence homologous to the nucleotide sequence of Streptomyces hygroscopicus of SEQ ID NO: 3 in the sequence listing. Furthermore, the present invention provides a polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide homologous to a Streptomyces hygroscopicus AveC homologous gene product having the amino acid sequence of SEQ ID NO: 4 in the Sequence Listing.
Furthermore, the present invention relates to a polynucleotide molecule having the nucleotide sequence of FIG. 1 (SEQ ID NO: 1 of the sequence listing) or SEQ ID NO: 3 of the sequence listing, or FIG. 1 (SEQ ID NO: 1 of the sequence listing) or sequence listing. An oligonucleotide is provided that hybridizes to a polynucleotide molecule having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3.

  Furthermore, the present invention provides recombinant cloning and expression vectors comprising a polynucleotide molecule comprising a Streptomyces avermitilis aveC-ORF or aveC homologous ORF. The vector is useful for cloning or expressing the polynucleotide of the present invention. In a non-limiting aspect, the present invention provides plasmid pSE186 (ATCC 209604), which comprises the complete ORF of the Streptomyces avermitilis aveC gene. Furthermore, the present invention provides transformed host cells comprising the polynucleotide molecules or recombinant vectors of the present invention, and novel strains or cell lines derived therefrom.

  Furthermore, the present invention provides substantially purified or isolated recombinantly expressed aveC gene product or AveC homologous gene product, or a substantial part thereof, and homologues thereof. Furthermore, the present invention provides a recombinant expression vector [wherein the recombinant expression vector comprises a polynucleotide molecule having a nucleotide sequence encoding an AveC gene product or an AveC homologous gene product, and the polynucleotide molecule and The regulatory element 1 or more that controls the expression of the polynucleotide molecule in is in an influential relationship] under conditions that promote the production of a recombinant AveC gene product or an AveC homologous gene product A method for producing a recombinant AveC gene product comprising culturing and recovering an AveC gene product or an AveC homologous gene product from the cell culture is provided.

  Furthermore, the present invention provides a cell of the Streptomyces avermitilis strain ATCC 53692 in which a wild-type aveC allele is inactivated and a polynucleotide molecule comprising a nucleotide sequence further comprising mutation 1 or more is expressed. Wherein said mutant nucleotide sequence is such that it produces a different ratio or amount of avermectin as compared to avermectin produced by cells of the Streptomyces avermitilis strain ATCC 53692 that instead expresses only the wild type aveC allele. Except for further inclusion, a sequence encoding the Streptomyces avermitilis AveC gene product of the plasmid pSE186 (ATCC209604), or a Streptomyces avermitilis a described in FIG. 1 (SEQ ID NO: 1 in the sequence listing) It contains the same nucleotide sequence as the nucleotide sequence of eC-ORF, provides polynucleotide molecules. According to the present invention, said polynucleotide molecule is a novel strain of Streptomyces avermitilis that exhibits a detectable change in the production of avermectin compared to the same strain that instead expresses only the wild type aveC allele. Can be used to generate. In a preferred embodiment, the polynucleotide molecule is a novel Streptomyces avermitilis that produces avermectin with a reduced class 2: 1 ratio compared to the same strain that instead expresses only the wild type aveC allele. Useful for generating strains. In a further preferred embodiment, said polynucleotide molecule produces a new strain of Streptomyces avermitilis that produces avermectin at an increased level compared to the same strain that instead expresses only the wild type aveC allele. Useful for. In a further preferred embodiment, the polynucleotide molecule is useful for generating a new strain of Streptomyces avermitilis in which the aveC gene is inactivated.

  Furthermore, the present invention provides a method for identifying a mutation in the AveC-ORF of Streptomyces avermitilis that can alter the production ratio and / or amount of avermectin. In a preferred embodiment, the present invention provides: (a) a natural aveC allele is inactivated and a polynucleotide molecule comprising a nucleotide sequence encoding a mutated AveC gene product is introduced and expressed. Determining the class 2: 1 ratio of avermectins produced by cells of the Streptomyces avermitilis strain; (b) the nucleotide sequence of the ORF of FIG. 1 (SEQ ID NO: 1 of the Sequence Listing) or a nucleotide sequence homologous thereto Determining the class 2: 1 ratio of avermectins produced by cells of the same Streptomyces avermitilis strain as in step (a) except that instead of expressing only the aveC allele having Avermectin produced by Streptomyces avermitilis cells in step (a) Comparing the class 2: 1 ratio to the class 2: 1 ratio of avermectins produced by Streptomyces avermitilis cells in step (b); produced by the Streptomyces avermitilis cells of step (a) Changing the class 2: 1 ratio of avermectins when the class 2: 1 ratio of avermectins differs from the class 2: 1 ratio of avermectins produced by Streptomyces avermitilis cells in step (b) A method for identifying aveC-ORF mutations capable of altering the class 2: 1 ratio of avermectins produced is provided.

  Further, in a preferred embodiment, the present invention provides: (a) the natural aveC allele is inactivated and contains a nucleotide sequence that encodes a mutated AveC gene product or encodes an AveC gene product. Determining the amount of avermectin produced by cells of a Streptomyces avermitilis strain that has been introduced and expressed with a polynucleotide molecule comprising a genetic construct comprising the nucleotide sequence to: (b) FIG. It is produced by the same Streptomyces avermitilis cell as in step (a) except that only the aveC allele having the nucleotide sequence of ORF of number 1) or a homologous nucleotide sequence thereof is expressed instead. Determining the amount of avermectin; and (c) the strike of step (a) Comparing the amount of avermectin produced by P. avermitilis cells with the amount of avermectin produced by Streptomyces avermitilis cells in step (b); Streptomyces avermitilis in step (a) An aveC-ORF or gene construct capable of changing the amount of avermectin when the amount of avermectin produced by the cell is different from the amount of avermectin produced by the Streptomyces avermitilis cell in step (b) A method for identifying mutations in a gene construct comprising aveC-ORF or aveC-ORF capable of altering the amount of avermectin produced is provided. In a preferred embodiment, the amount of avermectin produced is increased by mutation.

  Furthermore, the present invention provides a recombinant vector useful for the production of a new strain of Streptomyces avermitilis with altered avermectin production. For example, the present invention targets any polynucleotide molecule comprising the mutated nucleotide sequence of the present invention, targeting the AveC gene site of the Streptomyces avermitilis chromosome, by homologous recombination, and aveC-ORF or a part thereof Vectors that can be used to insert or substitute into are provided. However, according to the present invention, the polynucleotide molecule comprising the mutant nucleotide sequence of the present invention provided together with the present invention is inserted into a portion other than the aveC gene in the Streptomyces avermitilis chromosome, or When maintained episomally in Streptomyces avermitilis cells, it can also function to modulate avermectin biosynthesis. Accordingly, the present invention also provides a vector comprising a polynucleotide molecule comprising a mutated nucleotide sequence of the present invention. The vector can be used to insert a polynucleotide molecule at a position other than the aveC gene of the Streptomyces avermitilis chromosome or to maintain it episomally. In a preferred embodiment, the present invention relates to class 2 cells by inserting a mutant aveC allele into the Streptomyces avermitilis chromosome as compared to cells of the same strain that instead express only the wild type aveC allele. Provided are gene replacement vectors that can be used to produce new strains of cells that produce avermectins with a low: 1 ratio.

  Furthermore, the present invention provides a ratio and / or amount as compared to cells of the same strain of Streptomyces avermitilis that expresses a mutated aveC allele and that instead expresses only the wild type aveC allele. Provides a method for producing a new strain of Streptomyces avermitilis comprising cells that produce different avermectins. In a preferred embodiment, the present invention relates to a class 2: cell as compared to cells of the same strain of Streptomyces avermitilis that expresses a mutated aveC allele and expresses only the wild type aveC allele instead. A method for producing a new strain of Streptomyces avermitilis comprising cells that produce avermectins having different ratios, wherein the cells of Streptomyces avermitilis strain are mutated with an aveC allele (wherein said gene is Changes in the class 2: 1 ratio of avermectins produced by cells of the Streptomyces avermitilis strain expressing the mutated allele compared to cells of the same strain that instead express only the wild type aveC allele And a vector carrying the gene product to be transformed, and Selecting a transformed cell that produces an avermectin having a different class 2: 1 ratio compared to a class 2: 1 ratio produced by cells of a strain that instead expresses only the wild type aveC allele, The manufacturing method is provided. In a preferred embodiment, the class 2: 1 ratio of avermectins produced is reduced in the cells of said novel strain.

  In a more preferred embodiment, the present invention provides a method for producing a new strain of Streptomyces avermitilis comprising cells that produce different amounts of avermectins, wherein the cells of the Streptomyces avermitilis strain are mutated with a mutated aveC allele. A gene construct comprising a gene or aveC allele, provided that the amount of avermectin produced by the cells of the Streptomyces avermitilis strain expressing the mutated aveC allele or gene construct as a result of its expression is the wild type aveC Avermectin produced by a cell carrying a strain carrying the wild-type aveC allele instead, and transformed with a vector carrying the same allele instead of the same strain of cells Different amounts of avermectin compared to Comprises selecting transformed cells that formed, to provide the manufacturing method. In a preferred embodiment, the amount of avermectin produced is increased in the cells of the novel strain.

  Furthermore, in a preferred embodiment, the present invention provides a method for producing a new strain of Streptomyces avermitilis comprising an inactivated aveC allele, wherein the cell expresses a wild-type aveC allele. A method for the production is provided, comprising transforming a cell of a Mytilis strain with a vector that inactivates an aveC allele and selecting a transformed cell in which the aveC allele is inactivated.

  Furthermore, the present invention provides a new strain of Streptomyces avermitilis comprising cells transformed with any polynucleotide molecule or vector comprising the mutated nucleotide sequence of the present invention. In a preferred embodiment, the present invention provides a novel strain of Streptomyces avermitilis comprising cells expressing a mutated aveC allele instead of or in addition to the wild type aveC allele, wherein said The new strain of cells produces avermectins with different class 2: 1 ratios compared to cells of the same strain that instead express only the wild type aveC allele. In a more preferred embodiment, the new strain of cells produces an avermectin with a reduced class 2: 1 ratio compared to cells of the same strain that instead express only the wild type aveC allele. The novel strain is useful for large-scale production of commercially desirable avermectins (eg, doramectin).

Further, in a preferred embodiment, the present invention provides a cell expressing a mutated aveC allele or a gene construct comprising an aveC allele instead of or in addition to the wild type aveC allele [wherein said gene is Of Streptomyces avermitilis, including the production of different amounts of avermectin (by the cell) as compared to the amount of avermectin produced by cells of the same strain that instead express only the wild type aveC allele] Offer new stocks. In a preferred embodiment, the novel cells produce increased amounts of avermectin.
In a more preferred embodiment, the present invention provides a novel strain of Streptomyces avermitilis comprising cells in which the aveC gene is inactivated. The strain determines the spectrum of avermectin (produced by the strain) that is different from the wild-type strain and either targeted or random mutagenesis of the aveC gene that affects avermectin production Useful with both complementation screening assays described in the literature.

  Furthermore, the present invention relates to a cell of a Streptomyces avermitilis strain, wherein said cell does not express a mutated aveC allele but instead expresses only a wild type aveC allele. Expressing a mutated aveC allele that encodes a gene product that alters the class 2: 1 ratio of avermectins produced by cells of the Streptomyces avermitilis strain that expresses the mutated aveC allele) Is produced in a culture medium under conditions that permit or induce the production of avermectin by the cells, and avermectin is recovered from the culture. In a preferred embodiment, the class 2: 1 ratio of avermectins produced by cells expressing the mutation is reduced. This manufacturing method provides increased efficiency in the production of commercially valuable avermectins (eg, doramectin).

  Furthermore, the present invention relates to a cell of a Streptomyces avermitilis strain, wherein said cell replaces only the wild type aveC allele without expressing a mutated aveC allele or a gene construct comprising the aveC allele. A mutated aveC allele that causes a change in the amount of avermectin produced by a cell of a Streptomyces avermitilis strain expressing a mutated aveC allele or gene construct compared to a cell of the same strain that expresses A method for producing avermectin comprising culturing a gene construct in a culture medium under conditions that permit or induce production of avermectin by the cell and recovering avermectin from the culture is provided . In a preferred embodiment, the amount of avermectin produced by cells expressing the mutation or gene construct is increased.

  Furthermore, the present invention relates to an avermectin produced by a Streptomyces avermitilis strain that expresses a mutated aveC allele of the present invention, wherein said avermectin is wild-type without expressing the mutated aveC allele. Produced in a reduced class 2: 1 ratio compared to the class 2: 1 ratio of avermectins produced by cells of the same strain of Streptomyces avermitilis that instead express only the type aveC allele) Novel compositions are provided. The novel avermectin compositions may be present as produced in fermentation broth, or may be recovered from them, and partially or substantially purified therefrom Can be things.

4). BRIEF DESCRIPTION OF THE FIGURES FIG. DNA sequence (SEQ ID NO: 1 in the sequence listing) containing Streptomyces avermitilis aveC-ORF and deduced amino acid sequence (SEQ ID NO: 2 in the sequence listing).
FIG. Plasmid vector pSE186 (ATCC 209604) containing the complete ORF of the aveC gene of Streptomyces avermitilis.
FIG. Saccharopolyspora inserted into the aveC-ORF of Streptomyces avermitilis
erythraea) gene replacement vector pSE186 (ATCC 209605) containing the ermE gene.
FIG. BamHI restriction map of the avermectin polyketide synthase gene cluster from Streptomyces avermitilis and five overlapping cosmid clones identified (ie, pSE65, pSE66, pSE67, pSE68, pSE69). The relationship between pSE118 and pSE119 is also shown.
FIG. HPLC analysis of fermentation products produced by Streptomyces avermitilis strains. Peak quantification was performed by comparison with a standard amount of cyclohexyl B1. The retention time of cyclohexyl B2 is 7.4 to 7.7 minutes; the retention time of cyclohexyl B1 is 11.9 to 12.3 minutes. FIG. 5A. Streptomyces avermitilis strain SE180-11 having an inactive aveC-ORF. FIG. 5B. Streptomyces avermitilis strain SE180-11 transformed with pSE186 (ATCC 209604). FIG. 5C. Streptomyces avermitilis strain SE180-11 transformed with pSE187. FIG. 5D. Streptomyces avermitilis strain SE180-11 transformed with pSE188.
FIG. A deduced amino acid sequence encoded by aveC-ORF (SEC ID NO: 2) of Streptomyces avermitilis and a deduced amino acid sequence encoded by aveC homologous partial ORF derived from Streptomyces griseochromogenes Comparison of (SEQ ID NO: 5 in the sequence listing) with the deduced amino acid sequence (SEQ ID NO: 4 in the sequence listing) encoded by the aveC homologous ORF derived from Streptomyces hygroscopicus. The bold valine residue is the putative start site for the protein. Conserved residues are shown in capital letters if all three sequences are homologous and in lower case if two of the three sequences are homologous. The amino acid sequence contains about 50% sequence identity.
FIG. A hybrid plasmid construct comprising a 564 bp BsaAI / KpnI fragment from the Streptomyces hygroscopicus aveC homologous gene inserted into the BsaAI / KpnI site in Streptomyces avermitilis aveC-ORF.

5). Detailed Description of the Invention The present invention identifies and characterizes polynucleotide molecules having a nucleotide sequence encoding an AveC gene product from Streptomyces avermitilis, mutated AveC gene products and their effects on avermectin production. Of a new strain of Streptomyces avermitilis that can be used to screen for and a mutated AveC gene product reduces the ratio of B1: B2 avermectins produced by Streptomyces avermitilis Concerning the discovery that you can. For example, for a polynucleotide molecule having the same nucleotide sequence as the sequence encoding the Streptomyces avermitilis AveC gene product of plasmid pSE186 (ATCC 209604) or the nucleotide sequence of the ORF of FIG. 1 (SEQ ID NO: 1 of the Sequence Listing) The invention is described in a later section with respect to polynucleotide molecules having mutated nucleotide sequences derived therefrom. However, the principle described in the present invention is based on the fact that AveC from another polynucleotide molecule, such as another Streptomyces species, such as Streptomyces hygroscopicus and Streptomyces glyceochromogenes. The same applies to homologous genes.

5.1. Polynucleotide molecule encoding Streptomyces avermitilis AveC gene product The present invention is an isolated polynucleotide molecule comprising the complete aveC-ORF of Streptomyces avermitilis or a substantial part thereof, wherein said isolated polynucleotide An isolated polynucleotide molecule is provided wherein the molecule lacks the next complete ORF located downstream of the aveC-ORF in vivo on the Streptomyces avermitilis chromosome.

  Said isolated polynucleotide molecule of the invention is preferably the same as the sequence encoding the Streptomyces avermitilis AveC gene product of plasmid pSE186 (ATCC 209604) or alternatively as shown in FIG. A nucleotide sequence that is the same as the nucleotide sequence of the ORF of 1) or a substantial part thereof. In the present specification, the “substance part” of an isolated polynucleotide molecule comprising a polynucleotide sequence encoding the Streptomyces avermitilis AveC gene product refers to the complete aveC − shown in FIG. 1 (SEQ ID NO: 1 in the Sequence Listing). By an isolated polynucleotide molecule comprising at least about 70% of the ORF sequence and encoding a functionally equivalent AveC gene product. In this context, a “functionally equivalent” AveC gene product is expressed when expressed in the Streptomyces avermitilis ATCC53692 strain, in which the natural aveC allele is inactivated. Production of substantially the same ratio and amount as the avermectin produced by the Streptomyces avermitilis ATCC53692 strain that instead expresses only the wild type (natural functional aveC allele in the Streptomyces avermitilis ATCC53692 strain) Is defined as the gene product that causes

  In addition to the aveC-ORF nucleotide sequence, the isolated polynucleotide molecule of the present invention may further comprise a nucleotide sequence naturally adjacent to the aveC gene in Streptomyces avermitilis [eg, FIG. The adjacent nucleotide sequence shown in SEQ ID NO: 1) in the column table] can be included.

  As used herein, the terms “polynucleotide molecule”, “polynucleotide sequence”, “coding sequence”, “open reading frame”, and “ORF” can be either single stranded or double stranded. It is intended to mean DNA and RNA molecules, and when placed under the control of appropriate regulatory elements, in an appropriate host cell expression system, into the AveC gene product, or As shown, it can be transcribed and translated (DNA) or translated (RNA) into an AveC homologous gene product or into an AveC gene product or a polypeptide homologous to an AveC homologous gene product. 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 FIG. 1 (SEQ ID NO: 1 in the sequence listing) contains 4 different GTG codons at bp positions 42, 174, 177 and 180. As described in Section 9 below, by constructing a complex deletion in the 5 ′ region of the aveC-ORF (FIG. 1; SEQ ID NO: 1 of the Sequence Listing), which of these codons is aveC It was defined whether it can function as a start site for protein expression in the ORF. Deletion of the first GTG position in bp42 did not abolish AveC activity. Further deletion along with all GTG codons at bp positions 174, 177 and 180 abolish AveC activity, indicating that this region is required for protein expression. Accordingly, the present invention relates to various lengths of aveC-ORFs that initiate translation at any GTG site located at bp 174, 177, or 180 bp shown in FIG. 1 (SEQ ID NO: 1 of the Sequence Listing), and corresponding to each Including polypeptides.

Furthermore, the present invention relates to a sequence encoding the Streptomyces avermitilis AveC gene product of plasmid pSE186 (ATCC209604), or to the aveC-ORF shown in FIG. 1 (SEQ ID NO: 1 in the sequence listing) or a substantial part thereof. A polynucleotide molecule having a nucleotide sequence homologous to the nucleotide sequence of The term “homologous” when used in reference to a polynucleotide molecule that is homologous to a sequence encoding a Streptomyces avermitilis AveC gene product means a polynucleotide molecule having the following nucleotide sequence:
(A) encoding the same AveC gene product as that encoding the Streptomyces avermitilis AveC gene product of plasmid pSE186 (ATCC209604), or the aveC-ORF shown in FIG. 1 (SEQ ID NO: 1 in the sequence listing) A nucleotide sequence encoding the same AveC gene product as the nucleotide sequence, but comprising one or more silent changes to the nucleotide sequence due to the degeneracy of the genetic code; or (b) by a sequence encoding the AveC gene product of plasmid pSE186 (ATCC209604) A moderate stringency for the complement of a polynucleotide molecule that encodes the encoded amino acid sequence or that has the nucleotide sequence that encodes the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2) Hybridizing [ie such conditions, 0.5M-NaHPO _4, 7% sodium dodecyl sulfate (SDS), in 1 mM-EDTA, at 65 ° C., hybridization to filter-bound DNA, and, 0.2 × SSC / Wash in 0.1% SDS at 42 ° C.] [Ausubel et al. (Eds.), 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. , And John Wiley & Sons, Inc. , New York, p2.10.3] and a nucleotide sequence encoding a functionally equivalent AveC gene product as defined above.
In a preferred embodiment, the homologous polynucleotide molecule is the complement of the nucleotide sequence encoding the AveC gene product of plasmid pSE186 (ATCC 209604), or the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1 of the Sequence Listing) or an entity thereof. Hybridize to the complement of the part under highly stringent conditions [ie, 0.5 M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 ° C. to filter-bound DNA Hybridize and wash at 68 ° C. in 0.1 × SSC / 0.1% SDS] (Ausubel et al., 1989, supra) and encode the functionally equivalent AveC gene product defined above. To do.

  The activity of the AveC gene product and its potential functional equivalents can be determined by HPLC analysis of the fermentation product, as described in the Examples below. A polynucleotide molecule having a nucleotide sequence that encodes a functional equivalent of the Streptomyces avermitilis AveC gene product may be used in another strain of Streptomyces avermitilis regardless of whether it appears in nature or is designed. Included are naturally occurring AveC genes that are present, AveC homologous genes that are present in other species of Streptomyces, and mutated aveC alleles.

  Furthermore, the present invention relates to the amino acid sequence encoded by the sequence encoding the AveC gene product of plasmid pSE186 (ATCC209604), or to the amino acid sequence of FIG. 1 (SEQ ID NO: 2 in the sequence listing) or a substantial part thereof. A polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide having a homologous amino acid sequence. In the present specification, the “substance” of the amino acid sequence of FIG. 1 (SEQ ID NO: 2 of the sequence listing) includes at least about 70% of the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2 of the sequence listing), and It means a polypeptide constituting the functionally equivalent AveC gene product defined above.

  As used herein, the term “homology” when referring to an amino acid sequence that is homologous to the amino acid sequence of the AveC gene product from Streptomyces avermitilis is encoded by the sequence encoding the AveC gene product of plasmid pSE186 (ATCC209604). Or having the amino acid sequence of FIG. 1 (SEQ ID NO: 2 in the sequence listing), wherein one or more amino acid residues therein are conservatively substituted with another amino acid residue. (The conservative substitution described above results in a functionally equivalent gene product as defined above). Conservative amino acid substitutions are well known to those skilled in the art. The rules for carrying out the above substitution are described in “Dayhof, MD, 1978, Nat. Biomed. Res. Found., Washington, DC, Vol. 5, Sup. Things can be mentioned. More specifically, conservative amino acid substitutions are those that commonly occur within related amino acid families of side chain acidity, polarity, or bulkiness. Genetically encoded amino acids are generally classified into four types: (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine; (3) nonpolar = alanine, Valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polarity = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. In addition, phenylalanine, tryptophan, and tyrosine are collectively classified as aromatic amino acids. One or more substitutions within any particular group [eg, substitution of leucine with isoleucine or valine, substitution of aspartic acid with glutamic acid, substitution of threonine with serine, or structurally related amino acids Substitution of any other amino acid residue by a residue (eg, an amino acid residue having similar acidity, polarity, bulkiness, or similarity in some combination thereof) is generally Will have a trivial effect on function.

  The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding an AveC homologous gene product. In the present specification, the “AveC homologous gene product” refers to the amino acid sequence encoded by the sequence encoding the AveC gene product of the plasmid pSE186 (ATCC 209604), or the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2 in the sequence listing). It is defined as a gene product having at least about 50% amino acid sequence identity to the AveC gene product of Streptomyces avermitilis comprising. In a non-limiting embodiment, the AveC homologous gene product is derived from Streptomyces hygroscopicus (described in European application 0298423; deposited FERM BP-1901) and comprises the amino acid sequence of SEQ ID NO: 4 in the sequence listing or a substantial part thereof. A “substance” of the amino acid sequence of SEQ ID NO: 4 in the sequence listing means a polypeptide comprising at least about 70% of the amino acid sequence of SEQ ID NO: 4 in the sequence listing and constituting a functionally equivalent AveC homolog product. A “functional equivalent” AveC homologous gene product is expressed in the wild-type (Streptomyces hygroscopicus strain FERM BP− when expressed in the Streptomyces hygroscopicus strain FERM BP-1901 in which the natural aveC homologous allele is inactivated. 1901 is defined as a gene product that causes the production of substantially the same ratio and amount as milbemycin produced by Streptomyces hygroscopicus strain FERM BP-1901, which instead expresses only the natural functional aveC homologous allele in 1901. In a non-limiting embodiment, an isolated polynucleotide molecule of the invention that encodes a Streptomyces hygroscopicus AveC homologous gene product comprises the nucleotide sequence of SEQ ID NO: 3 of the Sequence Listing, or a substantial part thereof. In this connection, an “entity part” of an isolated nucleotide molecule comprising the nucleotide sequence of SEQ ID NO: 3 of the sequence listing comprises at least 70% of the nucleotide sequence of SEQ ID NO: 3 of the sequence listing, and the functional group defined immediately above It refers to an isolated polynucleotide molecule that encodes an equivalent AveC homologous gene product.

Furthermore, the present invention provides a polynucleotide molecule containing a nucleotide sequence homologous to the nucleotide sequence of Streptomyces hygroscopicus of SEQ ID NO: 3 in the Sequence Listing. The term “homologous” when used to mean a polynucleotide molecule comprising a nucleotide sequence homologous to the sequence encoding the Streptomyces hygroscopicus AveC homologous gene product of SEQ ID NO: 3 in the sequence listing, when Means a polynucleotide molecule having:
(A) a nucleotide sequence that encodes the same gene product as the nucleotide sequence of SEQ ID NO: 3 in the sequence listing or a substantial part thereof, but comprising one or more silent changes to the nucleotide sequence due to degeneracy of the gene code; or (b) Hybridizes under moderately stringent conditions to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 4 in the sequence listing [ie 0.5M-NaHPO ₄ , 7% sodium dodecyl sulfate (SDS) Hybridize to filter-bound DNA in 1 mM EDTA at 65 ° C. and wash at 42 ° C. in 0.2 × SSC / 0.1% SDS] (see Ausubel et al. Supra). In addition, a nucleotide encoding the functionally equivalent AveC homologous gene product defined above. Reochido array.
In a preferred embodiment, the homologous polynucleotide molecule hybridizes under highly stringent conditions to the nucleotide sequence encoding the AveC homologous gene product of SEQ ID NO: 3 in the sequence listing or a substantial part thereof [ie 0 Hybridize to filter-bound DNA at 65 ° C. in 5M-NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA and 68 ° C. in 0.1 × SSC / 0.1% SDS (Ausubel et al., 1989, supra) and encodes a functionally equivalent AveC homologous gene product as defined above.

  The present invention further provides a polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide homologous to the Streptomyces hygroscopicus AveC homologous gene product. In the present specification, the term “homology” when referring to a polypeptide homologous to the AveC homologous gene product of SEQ ID NO: 4 in the sequence listing derived from Streptomyces hygroscopicus has the amino acid sequence of SEQ ID NO: 4 in the sequence listing. A polypeptide in which one or more amino acid residues therein are conservatively substituted with another amino acid residue (the above-mentioned conservative substitution results in the functionally equivalent homologous gene product defined above) Means.

  Furthermore, the present invention relates to a polynucleotide molecule having the nucleotide sequence of FIG. 1 (SEQ ID NO: 1 of the sequence listing) or SEQ ID NO: 3 of the sequence listing, or FIG. An oligonucleotide that hybridizes to a polynucleotide molecule having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 is provided. The oligonucleotide is at least about 10 nucleotides in length, preferably from about 15 to about 30 nucleotides in length, and hybridizes with one of the polynucleotide molecules under highly stringent conditions (ie, 6 × In SSC / 0.5% sodium pyrophosphate at ˜37 ° C. for ˜14 base oligo, ˜48 ° C. for ˜17 base oligo, ˜55 ° C. for ˜20 base oligo, and Wash at ˜60 ° C. for ˜23 base oligo). In a preferred embodiment, the oligonucleotide is complementary to a portion of one of the polynucleotide molecules. These oligonucleotides are useful for a variety of purposes, for example, to encode or act on antisense molecules useful for gene regulation, or primers in amplification of polynucleotide molecules encoding aveC- or aveC homologues. is there.

  Additional aveC homologous genes can be identified in another species or strain of Streptomyces by using the polynucleotide molecules or oligonucleotides described herein in connection with known techniques. For example, an oligonucleotide molecule comprising a part of the nucleotide sequence of Streptomyces avermitilis in FIG. 1 (SEQ ID NO: 1) or a part of the nucleotide sequence of Streptomyces hygroscopicus of SEQ ID NO: 3 in the sequence listing Can be detectably labeled and used to screen genomic libraries composed of DNA from organisms of interest. The stringency of hybridization conditions is selected based on the relationship between the reference organism (in this example, Streptomyces avermitilis or Streptomyces hygroscopicus) and the organism of interest. The requirements for various stringency conditions are well known to those of skill in the art, and the conditions will change predictably according to the particular organism that provides the library and label sequence. The oligonucleotide is preferably at least about 15 nucleotides in length, and examples include those described in the examples below. Amplification of homologous genes can be performed using these and other oligonucleotides and applying standard techniques [eg, polymerase chain reaction (PCR)], but other amplifications known in the art. Techniques (eg, ligase chain reaction) can also be used.

  Clones identified as containing aveC homologous nucleotide sequences can be tested for the ability to encode a functional AveC homologous gene product. For this purpose, the clones are sequenced to confirm proper reading frames and start and stop signals. Alternatively or additionally, the cloned DNA sequence can be inserted into an appropriate expression vector (ie, a vector containing elements necessary for transcription and translation of the inserted protein coding sequence). Any of a variety of host / vector systems can be used as follows, including but not limited to bacterial systems such as plasmids, bacteriophages, or cosmid expression vectors. Next, a suitable host cell transformed with the vector containing a sequence encoding a potential aveC homolog is analyzed by HPLC analysis of the fermentation product, eg, as described in Section 7 below. Can be used to analyze for AveC-type activity.

The generation and manipulation of the polynucleotide molecules disclosed herein are within the skill of the artisan, for example, Maniatis et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, Cold Spring Harbor, Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates & Wiley Interscience, NY; Sambrook et al., 1989, Molecular.
Cloning: A Laboratory Manual, 2d ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Innis et al. (Eds), 1995, PCR Strategies, Academic Press, Inc. , San Diego; and Errich (ed), 1992, PCR Technology, Oxford University Press, New.
Can be performed according to the recombinant techniques described in York, all of which are incorporated herein by reference. Polynucleotide clones encoding the AveC gene product or AveC homologous gene product can be identified using any method known to those of skill in the art (eg, but not limited to, the method described in Section 7 below). it can. Genomic DNA libraries are described in, for example, Benton and Davis, 1977, Science, 196: 180 for bacteriophage libraries and Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. USA, 72: 3961-3965, can be screened for sequences encoding aveC and aveC homologues. Polynucleotide molecules having a nucleotide sequence known to contain aveC-ORF [eg, present in plasmid pSE186 (ATCC209604) or plasmid pSE119 (described in Section 7 below)] are probes in their screening experiments. Can be used as Alternatively, an oligonucleotide probe corresponding to a nucleotide sequence deduced from a part or all of the amino acid sequence of the purified AveC homologous gene product can be synthesized.

5.2. Recombination system 5.2.1. Cloning and Expression Vectors Further, the present invention provides recombinant cloning useful for cloning or expression of a polynucleotide molecule of the present invention (eg, including the aveC-ORF of Streptomyces avermitilis or any aveC homologue ORF). Supply vectors and expression vectors. In a non-limiting aspect, the present invention provides plasmid pSE186 (ATCC) containing the complete ORF of the aveC gene of Streptomyces avermitilis.
209604).
All of the following descriptions relating to aveC-ORF from Streptomyces avermitilis, polynucleotide molecules comprising aveC-ORF from Streptomyces avermitilis or parts thereof, or Streptomyces avermitilis AveC gene product Means aveC homologue and AveC homologous gene product unless otherwise specified.

  A variety of different vectors such as phage, high copy number plasmids, low copy number plasmids, and E. coli. E. coli-streptomyces shuttle vectors and the like have been developed for specific use in Streptomyces, and any of them can be used in the practice of the present invention. In addition, many drug resistance genes have been cloned from Streptomyces, and some of these genes have been incorporated into vectors as selectable markers. Examples of current vectors for use in Streptomyces exist elsewhere (Hutchinson, 1980, Applied Biochem. Biotech. 16: 169-190).

  The recombinant vector of the invention (especially the expression vector) is preferably a regulatory element 1 wherein the coding sequence for the polynucleotide molecule of the invention is necessary for the transcription and translation of the coding sequence to produce a polypeptide. It is configured to exist effectively in association with more than one. As used herein, the term “regulatory element” includes, but is not limited to, nucleotide sequences encoding inducible and non-inducible promoters, enhancers, operators, and other elements. The other elements are other elements known to those skilled in the art used to induce and / or regulate the expression of a sequence encoding a polynucleotide. Also, as used herein, the coding sequence is “effectively associated” with one or more regulatory elements, wherein the regulatory element is responsible for transcription of the coding sequence or translation of its mRNA, or both. Effectively adjust and allow.

  Typical plasmid vectors that can be designed to contain the polynucleotide molecules of the present invention include, among many others, pCR-Blunt, pCR2.1 (Invitrogen), pGEM3Zf (Promega), and shuttle vectors pWHM3 (Vara et al., 1989, J. Bact. 171: 5872-5881).

  Methods of constructing recombinant vectors containing specific coding sequences effectively associated with appropriate regulatory elements are well known to those of skill in the art and can be used in the practice of the present invention. These methods include, for example, in vitro recombination techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Maniatis et al., 1989 (supra); Ausubel et al., 1989 (supra); Sambrook et al., 1989 (supra); and Errich, 1992 (supra).

The regulatory elements of these vectors can vary in strength and specificity. Depending on the host / vector system used, any of a number of suitable transcription and translation elements can be used. Non-limiting examples of transcriptional regulatory regions or promoters for bacteria can include β-gal promoter, T7 promoter, TAC promoter, λ left and right promoter, trp and lac promoter, trp-lac fusion promoter, More specifically, the promoters ermE, melC, tipA and the like can be mentioned for the Mrs. In a particular embodiment, described below in Section 11, Saccharopolyspora (Saccharopolyspora)
An expression vector containing the AveC-ORF cloned adjacent to the strong constitutive ermE promoter from erythraea) was generated. The vector was transformed into Streptomyces avermitilis, followed by HPLC analysis of the fermentation product, which was instead produced compared to avermectin produced by the same strain expressing the wild type aveC allele. It became clear that the amount of avermectin increased.

  Fusion protein expression vectors can be used to express the AveC gene product-fusion protein. The purified fusion protein can be used to generate antisera against the AveC gene product, to study the biochemical properties of the AveC gene product, to construct AveC fusion proteins with various biochemical activities, or Can be used to help identify or purify the expressed AveC gene product. Possible fusion protein expression vectors include, but are not limited to, β-galactosidase and trpE fusions, maltose binding protein fusions, glutathione-S-transferase fusions, and polyhistidine fusions (carrier regions). Includes vectors incorporating the coding sequence. In another aspect, the AveC gene product or part thereof is obtained from another species or strain of Streptomyces [eg, AveC homologous gene product or one of them from Streptomyces hygroscopicus or Streptomyces glyceochromenes]. Can be fused with the part. A particularly preferred embodiment is described in section 12 below and is described in FIG. The chimeric plasmid was constructed to include the 564 bp region of the Streptomyces avermitilis aveC-ORF homologous 564 bp region instead of the Streptomyces hygroscopicus aveC homologous ORF. The hybrid vectors can be transformed into Streptomyces avermitilis cells and tested for their effects (eg, effects on the class 2: 1 ratio of avermectins produced).

AveC fusion proteins can be constructed to contain regions useful for purification. For example, the AveC-maltose-binding protein fusion can be purified using amylose resin; the AveC-glutathione-S-transferase fusion protein can be purified using glutathione-agarose beads; and AveC -The polyhistidine fusion can be purified using a divalent nickel resin. Alternatively, antibodies against carrier proteins or peptides can be used for affinity chromatography purification of the fusion protein. For example, a nucleotide sequence encoding a target epitope of a monoclonal antibody can be constructed in an expression vector that is effectively associated with a regulatory element and positioned such that the expressed epitope is fused to an AveC polypeptide. For example, the nucleotide sequence encoding the FLAG ^™ epitope tag (International Biotechnology, Inc.), a hydrophilic marker peptide, is inserted into the expression vector at the corresponding position (eg, the carboxyl terminus of the AveC polypeptide) by standard techniques. be able to. The expressed AveC polypeptide-FLAG ^™ epitope fusion product can then be detected and affinity purified using a commercially available anti-FLAG ^™ antibody.

In addition, an expression vector encoding an AveC fusion protein can be constructed to include a polylinker sequence encoding a specific protease cleavage site, and as a result, the expressed AveC polypeptide can be supported by treatment with a specific protease. Or it can be released from the fusion partner. For example, a fusion protein vector can include a DNA sequence that specifically encodes a thrombin or factor Xa cleavage site.
The signal sequence present upstream of the aveC-ORF (to be in reading frame with the aveC-ORF) is expressed in an expression vector to direct the transfer (trafficking) and secretion of the expressed gene product by known methods. Can be built in. Non-limiting examples of signal sequences can include, among others, those derived from α-factor, immunoglobulins, outer membrane proteins, penicillinases, and T-cell receptors.

  To aid in selection of host cells transformed or transfected with the cloning or expression vectors of the invention, the vectors can be further constructed to include coding sequences for reporter gene products or other selectable markers. . Such a coding sequence is preferably present in effective association with the sequence encoding the regulatory element. Reporter genes useful in the present invention are well known in the art and can include, for example, those that specifically encode green fluorescent protein, luciferase, xylE, and tyrosinase. Nucleotide sequences encoding selectable markers are well known in the art and include those that encode gene products that confer resistance to antibiotics or anti-metabolites or that supply auxotrophic requirements. Examples of such sequences include, among others, those encoding resistance to erythromycin, thiostrepton, or kanamycin.

5.2.2. In addition, the present invention provides transformed host cells containing the polynucleotide molecules or recombinant vectors of the present invention, and novel strains or cell lines derived therefrom. Host cells useful in the practice of the present invention are preferably Streptomyces cells, although other prokaryotic or eukaryotic cells can be used. Typically, the transformed host cell is not limited to, in particular, a bacterium transformed with a microorganism, such as a recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA vector, Alternatively, yeast transformed with a recombinant vector is included.

  The polynucleotide molecules of the present invention are intended to function in Streptomyces cells, but can also be transformed into other bacteria or eukaryotic cells, eg, for cloning or expression purposes. Typically, E.I. E. coli strains, such as the DH5α strain [available from the American Type Culture Collection (ATCC), Rockville, MD, USA (Accession Number 31343) or Stratagene], can be used. Preferred eukaryotic host cells include yeast cells, but mammalian cells or insect cells can also be used effectively.

  The recombinant expression vectors of the invention preferably transform or transfect one or more host cells of a substantially homogeneous cell culture. Expression vectors are generally obtained using known techniques (eg, protoplast transformation, calcium phosphate precipitation, calcium chloride treatment, microinjection, electroporation, transfection by contact with recombinant viruses, liposome-mediated transfection, DEAE-dextran transfection. Introduction, transduction, conjugation, or microprojectile bombardment). Selection of transformants can be performed by standard procedures, eg, selection for cells that express a selectable marker (eg, antibiotic resistance associated with the recombinant vector as described above).

  If the expression vector has been introduced into the host cell, integration and maintenance of the aveC coding sequence in the host cell chromosome or in the episome can be performed using standard techniques such as Southern hybridization analysis, restriction enzyme analysis, PCR analysis [reversal]. Including transcriptase PCR (rt-PCR)] or by immunological assays that detect the predicted gene product. Host cells containing and / or expressing a recombinant aveC coding sequence can be identified by any of at least four general approaches well known in the art: (i) DNA-DNA, DNA-RNA, or RNA-antisense RNA hybridization; (ii) detection of the presence of “marker” gene function; (iii) assessment of the level of transcription as measured by expression of an aveC-specific mRNA transcript in the host cell; iv) mature poly, as measured, for example, by immunoassay or by the presence of AveC biological activity (eg, a specific rate and amount of avermectin production that revelates AveC activity in, eg, Streptomyces avermitilis host cells) Detection of the presence of peptide product.

5.2.3. Expression and Characterization of Recombinant AveC Gene Product If the aveC coding sequence has been stably introduced into a suitable host cell, the transformed cell host is clonally propagated and the resulting cell is It can be grown under conditions that aid in maximal production of the aveC gene product. Such conditions typically include the growth of cells to a high density. If the expression vector contains an inducible promoter, suitable induction conditions such as temperature shift, nutrient depletion, extra inducers [eg, carbohydrate analogs such as isopropyl-β-D-thiogalactopyranoside ( Addition of IPTG)] or accumulation of excess metabolic byproducts is used when expression needs to be induced.

  If the expressed AveC gene product is retained in the host cell, the cells are harvested and lysed, and from the lysate, extraction conditions known in the art to minimize proteolysis (eg, 4 ° C. And / or in the presence of protease inhibitors, or both). If the expressed AveC gene product is secreted from the host cell, the spent nutrient medium can simply be collected and the product isolated therefrom.

  The expressed AveC gene product is optionally obtained by standard methods such as, but not limited to, the following methods (ie ammonium sulfate precipitation, size fractionation, ion exchange chromatography, HPLC, density centrifugation, and affinity Any combination of chromatography) can be used to isolate or substantially purify from cell lysates or culture media. If the expressed AveC gene product exhibits biological activity, the increased purity of the preparation can be observed at each step of the purification procedure by using an appropriate assay. Whether the expressed AveC gene product exhibits or does not exhibit biological activity, for example, by size or otherwise based on reactivity with antibodies specific for AveC, or a fusion tag Can be detected by the presence of.

Therefore, the present invention provides a recombinant expression comprising the amino acid sequence encoded by the sequence encoding the AveC gene product of plasmid pSE186 (ATCC209604), or the amino acid sequence of FIG. 1 (SEQ ID NO: 2 of the sequence listing) or a substantial part thereof. Streptomyces avermitilis AveC gene product and homologues thereof are provided.
Furthermore, the present invention provides a recombinantly expressed Streptomyces hygroscopicus AveC homologous gene product containing the amino acid sequence of SEQ ID NO: 4 in the Sequence Listing or a substantial part thereof, and homologues thereof.

  Furthermore, the present invention provides a recombinant expression vector [wherein the vector comprises a polynucleotide molecule having a nucleotide sequence encoding an AveC gene product, wherein the polynucleotide molecule controls the expression of the polynucleotide molecule in a host cell. Culturing the host cell transformed with at least one regulatory element under conditions that promote the production of a recombinant AveC gene product and recovering the AveC gene product from the cell culture. A method for producing an AveC gene product is provided.

  The recombinantly expressed Streptomyces avermitilis AVEc gene product is used for various purposes (for example, to screen for compounds that alter the function of AveC gene production and thereby modulate avermectin biosynthesis, and antibodies to the AveC gene product. This is useful for the purpose of

Once a sufficiently pure AveC gene product is obtained, it can be converted to biological activity in standard methods (eg, SDS-PAGE, size exclusion chromatography, amino acid sequence analysis, production of appropriate products in the avermectin biosynthetic pathway). Etc.). For example, the amino acid sequence of the AveC gene product can be determined using standard peptide sequencing techniques. In addition, to identify the hydrophobic and hydrophilic regions of the AveC gene product, the AveC gene product was analyzed using a hydrophilicity analysis (see, eg, Hopp and Woods, 1981, Proc.
Natl. Acad. Sci. USA 78: 3824), or similar software algorithms. Structural analysis can be performed to identify regions of the AveC gene product that assume a particular secondary structure. To map and study the site of interaction between the AveC gene product and its substrate, biophysical methods such as X-ray crystallography (Engstrom, 1974, Biochem. Exp. Biol. 11: 7-13) Computer modeling (Fleterick and Zoller (eds), 1986, Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), and nuclear magnetic resonance (NMR) can be used. The information obtained from these studies is used to select new sites for mutations in the AveC ORF to help develop new strains of Streptomyces avermitilis with more desirable avermectin production characteristics can do.

5.3. Construction and Use of AveC Mutants The present invention further comprises mutation 1 or more (so that a polynucleotide molecule comprising a mutated nucleotide sequence in which the wild type aveC allele has been inactivated) Expressing Streptomyces avermitilis strain ATCC53692 cells avermectins in different proportions and amounts than those produced by Streptomyces avermitilis strain ATCC53692 cells that instead express only the wild type aveC allele. A sequence encoding the AveC gene product of Streptomyces avermitilis in plasmid pSE186 (ATCC209604), or the Streptomyces avermitilis sequence shown in FIG. 1 (SEQ ID NO: 1 in the sequence listing) aveC A polynucleotide molecule comprising a nucleotide sequence that is identical to the nucleotide sequence of the ORF is provided.

  According to the present invention, S. cerevisiae exhibits a detectable change in avermectin production compared to the same strain that instead expresses only the wild type aveC allele. The polynucleotide molecule can be used to produce a new strain of avermectin. In a preferred embodiment, the polynucleotide molecule produces an avermectin in a reduced class 2: 1 ratio compared to the same strain that instead expresses only the wild type aveC allele. It is useful for producing a new strain of avermectin. In a further preferred embodiment, said polynucleotide molecule produces an avermectin at an increased level compared to the same strain that instead expresses only the wild type aveC allele. It is useful for producing a new strain of avermectin. In a further preferred embodiment, the polynucleotide molecule is useful for producing a new strain of Streptomyces avermitilis in which the aveC gene is inactivated.

  Mutations to the aveC coding sequence cause amino acid deletions, additions, or substitutions in the aveC gene product, or cause truncation of the aveC gene product (or any combination thereof), and Any mutation that yields the desired result is included. For example, the present invention includes the sequence encoding the AveC gene product of plasmid pSE186 (ATCC209604), or the nucleotide sequence of the AveC-ORF of Streptomyces avermitilis as shown in FIG. 1 (SEQ ID NO: 1 of the Sequence Listing), Further provided is a polynucleotide molecule comprising one or more mutations encoding that an amino acid residue is replaced by another amino acid residue at a selected position in the AveC gene product. In some non-limiting aspects exemplified below, the substitution can be performed at amino acid positions 55, 138, 139, or 230, or some combination thereof.

  Mutations to the aveC coding sequence can be performed by various known methods (eg, by using error prone PCR or cassette mutagenesis). For example, oligonucleotide directed mutagenesis can be performed in a defined manner (eg, by introducing restriction sites or stop codons 1 or more into specific regions in the aveC-ORF sequence) and aveC-ORF sequences. Can be used to change. For example, the method described in US Pat. No. 5,605,793 (including random fragmentation, repeated cycles of mutagenesis, and nucleotide shuffling) is also used to form a large library of polynucleotides having nucleotide sequences encoding aveC mutations. be able to.

  Targeted mutations can be useful, especially when they serve to change one or more conserved amino acid residues in the AveC gene product. For example, as shown in FIG. 6, the deduced amino acid sequence of the AveC gene product, Streptomyces avermitilis (SEQ ID NO: 2 in the sequence listing), Streptomyces griseochromogenes (SEQ ID NO: 5 in the sequence listing), and Comparison with the AveC homologous gene product from Streptomyces hygroscopicus (SEQ ID NO: 4 in the sequence listing) reveals important conserved positions of amino acid residues between the species. Targeted mutagenesis that results in changes in one or more of these conserved amino acid residues can be particularly effective in producing novel mutants that exhibit the desired changes in avermectin production.

  Random mutagenesis can also be useful, and UV or X-ray, or chemical mutagens (eg, N-methyl-N′-nitrosoguanidine, ethyl methanesulfonate, nitrous acid, or nitrogen) Can be performed by exposing cells of Streptomyces avermitilis to mustard). For example, see Ausubel, 1989 (supra) for reference on mutagenesis techniques.

  Once mutated polynucleotide molecules are generated, they are screened to determine if they can modulate avermectin biosynthesis in Streptomyces avermitilis. In a preferred embodiment, a polynucleotide molecule having a mutated nucleotide sequence is complemented in a Streptomyces avermitilis strain that exhibits an aveC negative background due to inactivation of the aveC gene (aveC negative background). ) To test. In a non-limiting method, the mutated polynucleotide molecule is an expression plasmid that is effectively associated with one or more regulatory elements (this plasmid may also contain one or more drug resistance genes capable of selecting transformed cells). (Preferably). This vector is then transformed into aveC host cells using known techniques, and the transformed cells are selected and cultured in an appropriate fermentation medium under conditions that permit or induce avermectin production. . The fermentation product is then analyzed by HPLC to determine the ability of the mutated polynucleotide molecule to complement the host cell. Several vectors (eg, pSE188, pSE199, and pSE231) carrying mutated polynucleotide molecules capable of reducing the B2: B1 ratio of avermectin are illustrated in Section 8.3 below.

  The present invention provides a method for identifying mutations in the aveC-ORF of Streptomyces avermitilis that can alter the ratio and / or amount of avermectins produced. In a preferred embodiment, the present invention provides: (a) a natural aveC allele is inactivated and a polynucleotide molecule comprising a nucleotide sequence encoding a mutated AveC gene product is introduced and expressed. Determining the class 2: 1 ratio of avermectins produced by cells of the Streptomyces avermitilis strain; (b) the nucleotide sequence of the ORF of FIG. 1 (SEQ ID NO: 1 of the Sequence Listing) or a nucleotide sequence homologous thereto Determining the class 2: 1 ratio of avermectins produced by cells of the same Streptomyces avermitilis strain as in step (a) except that instead of expressing only the aveC allele having Avermectin produced by Streptomyces avermitilis cells in step (a) Comparing the class 2: 1 ratio to the class 2: 1 ratio of avermectins produced by Streptomyces avermitilis cells in step (b); produced by the Streptomyces avermitilis cells of step (a) Changing the class 2: 1 ratio of avermectins when the class 2: 1 ratio of avermectins differs from the class 2: 1 ratio of avermectins produced by Streptomyces avermitilis cells in step (b) A method for identifying aveC-ORF mutations capable of altering the class 2: 1 ratio of avermectins produced is provided. In a preferred embodiment, the mutation reduces the class 2: 1 ratio of avermectins.

  In a further preferred embodiment, the present invention provides that (a) the natural aveC allele is inactivated and comprises a nucleotide sequence encoding a mutated AveC gene product or encodes an AveC gene product Determining the amount of avermectin produced by cells of a Streptomyces avermitilis strain that has been introduced and expressed with a polynucleotide molecule comprising a gene construct comprising a nucleotide sequence; (b) FIG. 1 (SEQ ID NO: Avermectin produced by cells of the same Streptomyces avermitilis strain as in step (a) except that only the aveC allele having the nucleotide sequence of ORF of 1) or a homologous nucleotide sequence thereof is expressed instead And (c) the strain of step (a) Comparing the amount of avermectin produced by Tomisces avermitilis cells with the amount of avermectin produced by Streptomyces avermitilis cells in step (b); Streptomyces avermitilis in step (a) An aveC-ORF or gene construct capable of changing the amount of avermectin when the amount of avermectin produced by the cell is different from the amount of avermectin produced by the Streptomyces avermitilis cell in step (b) A method for identifying mutations in a gene construct comprising an aveC-ORF or an aveC-ORF capable of altering the amount of avermectin produced is provided.

  All of the above methods for identifying mutations are performed by using a fermentation culture medium (preferably a fermentation culture medium supplemented with cyclohexanecarboxylic acid), but another suitable fatty acid precursor (eg, a table). 1 can also be used.

  Once a mutated polynucleotide molecule that modulates the desired orientation of avermectin production has been identified, the location of the mutation in the nucleotide sequence can be determined. For example, 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. By comparing the DNA sequence of the mutated aveC allele with the DNA sequence of the wild type aveC allele, the mutational reactivity for changes in avermectin production can be determined. In a specific but non-limiting aspect of the invention, a single amino acid substitution at any residue of 55 (S55F), 138 (S138T), 139 (A139T), or 230 (G230G), or 138 ( The Streptomyces avermitilis AveC gene product containing either a double substitution at positions S138T) and 139 (A139T) produced a change in the function of the AveC gene product. For example, the ratio of class 2: 1 avermectins produced has changed (see section 8 below). Thus, a polynucleotide molecule having a nucleotide sequence encoding a mutated Streptomyces avermitilis AveC gene product comprising an amino acid substitution at one or more of 55, 138, 139, or 230 amino acid residues or any combination thereof is Are included in the present invention.

  Furthermore, the present invention provides a composition for producing a novel strain of Streptomyces avermitilis, the cell containing a mutant aveC allele that results in altered avermectin production. For example, the present invention relates to an aveC-ORF by homologous recombination or one of them by directing all polynucleotide molecules comprising the mutant nucleotide sequence of the present invention to the site of the AveC gene of the Streptomyces avermitilis chromosome. Recombinant vectors that can be used to insert or replace parts are provided. However, according to the present invention, the polynucleotide molecule comprising the mutant nucleotide sequence of the present invention provided therewith is inserted into a site other than the aveC gene in the Streptomyces avermitilis chromosome, or It can also function to modulate avermectin biosynthesis when maintained episomally in Mrs. avermitilis cells. Accordingly, the present invention also provides a vector comprising a polynucleotide molecule comprising a mutated nucleotide sequence of the present invention. The vector can be used to insert a polynucleotide molecule at a site other than the aveC gene in the Streptomyces avermitilis chromosome or to maintain it episomally.

  In a preferred embodiment, the present invention comprises inserting a mutated aveC allele into a cell of a strain of Streptomyces avermitilis to form a novel strain of Streptomyces avermitilis (the cell is a wild type aveC allele). Provides a gene replacement vector that can be used to generate avermectins in different class 2: 1 ratios as compared to cells of the same strain that instead express only. In a preferred embodiment, the class 2: 1 ratio of avermectins produced by the cells is reduced. The gene replacement vector is a mutated poly present in the expression vectors provided therewith, such as pSE188, pSE199, and pSE231 (these expression vectors are exemplified in section 8.3 below). It can be constructed using nucleotide molecules.

  In a further preferred embodiment, the present invention provides for the production of a new strain that produces a different amount of avermectin compared to cells of the same strain that instead express only the wild type aveC allele. Provided are vectors that can be used to insert mutated aveC alleles into cells of a strain. In a preferred embodiment, the amount of avermectin produced by the cell is increased. In a specific but non-limiting embodiment, the vector further comprises a strong promoter known to those skilled in the art, such as a strong constitutive ermE promoter from Saccharopolyspora erythraea (this is , Located upstream of the aveC-ORF and effectively associated with the aveC-ORF). The vector can be plasmid pSE189 (described in Example 11 below) or can be constructed using the mutated aveC allele of plasmid pSE189.

  In a further preferred embodiment, the present invention provides a gene replacement vector useful for inactivating the aveC gene in a wild type strain of Streptomyces avermitilis. In a non-limiting embodiment, the gene replacement vector is a mutated polynucleotide molecule present in plasmid pSE180 (ATCC 209605) [this is exemplified in section 8.1 below (FIG. 3)]. Can be built using. Furthermore, the present invention comprises, in the Streptomyces avermitilis chromosome, a nucleotide sequence naturally adjacent to the AveC gene in vivo [for example, the adjacent nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1 in the sequence listing)], Alternatively, a gene replacement vector comprising a polynucleotide molecule comprising it is provided. This vector can be used to delete Streptomyces avermitilis aveC-ORF.

  Furthermore, the present invention relates to avermectins in different ratios and / or amounts as compared to the same strain of Streptomyces avermitilis that expresses a mutated aveC allele and that instead expresses only the wild type aveC allele A method for producing a new strain of Streptomyces avermitilis comprising cells that produce In a preferred embodiment, the present invention relates to a class 2: cell as compared to cells of the same strain of Streptomyces avermitilis that expresses a mutated aveC allele and expresses only the wild type aveC allele instead. A method for producing a novel strain of Streptomyces avermitilis comprising cells that produce avermectins having different ratios, wherein the cells of Streptomyces avermitilis strain are mutated with an aveC allele (wherein the gene is Compare the class 2: 1 ratio of avermectins produced by cells of the Streptomyces avermitilis strain expressing the mutated aveC allele compared to cells of the same strain that instead express only the wild type aveC allele. Transformation with a vector carrying the gene product to be altered) Selecting a transformed cell that produces an avermectin with a different class 2: 1 ratio compared to a class 2: 1 ratio produced by cells of a strain that instead expresses only the wild type aveC allele. Including the manufacturing method. In a preferred embodiment, the altered class 2: 1 ratio of avermectin is reduced.

In a further preferred embodiment, the present invention is a method for producing a novel strain of Streptomyces avermitilis comprising cells that produce different amounts of avermectin,
A cell of a Streptomyces avermitilis strain is transformed into a gene construct containing a mutated aveC allele or aveC allele (provided that the expression results in a Streptomyces avermitis avermitilis strain expressing the mutated aveC allele or gene construct). The amount of avermectin produced by cells of the Tirith strain is transformed with the vector carrying the wild type aveC The method is provided comprising selecting transformed cells that produce a different amount of avermectin as compared to the amount of avermectin produced by cells of a strain that instead expresses only the allele. In a preferred embodiment, the amount of avermectin produced in the transformed cell is increased.

  In a further preferred embodiment, the present invention transforms cells of a strain of Streptomyces avermitilis expressing a wild type aveC allele with a vector that inactivates the aveC allele, and the aveC allele is inactive A method for producing a novel strain of Streptomyces avermitilis, which cell contains an inactivated aveC allele, comprising selecting transformed cells that have been activated. In a preferred but non-limiting embodiment, cells of the Streptomyces avermitilis strain carry an aveC allele that has been inactivated by mutation or by replacement of a portion of the aveC allele by a heterologous gene sequence. Transform with a gene replacement vector and select for transformed cells (the cell's natural aveC allele has been replaced with an inactivated aveC allele). Inactivation of the aveC allele can be determined by HPLC analysis of fermentation products (see below). In a specific but non-limiting embodiment described below in Section 8.1, the aveC allele is inserted by inserting the ermE gene from Saccharopolyspora erythraea into the aveC-ORF. Inactivate genes.

  Furthermore, the present invention provides a novel strain of Streptomyces avermitilis comprising cells transformed with any of the polynucleotide molecules or vectors of the present invention. In a preferred embodiment, the present invention provides a novel strain of Streptomyces avermitilis comprising a cell expressing a mutated aveC allele instead of or in addition to the wild type aveC allele (herein The cells of the new strain produce avermectins in a different class 2: 1 ratio compared to the class 2: 1 ratio of avermectins produced by cells of the same strain that instead express only the wild type aveC allele Provide). In a preferred embodiment, the different class 2: 1 ratio produced by the new cells is reduced. The new strains are useful for large-scale production of commercially desirable avermectins (eg, doramectin).

  The primary purpose of the screening assays described herein is to change (especially reduce) the aveC class 2: 1 ratio of avermectins produced by expression in Streptomyces avermitilis cells. The goal is to identify mutated alleles of the gene. In a preferred embodiment, the B2: B1 avermectin produced by cells of a novel strain of Streptomyces avermitilis of the present invention expressing a mutated aveC allele that reduces the class 2: 1 ratio of avermectins produced The ratio is less than 1.6: 1 to about 0: 1; in a more preferred embodiment, the ratio is between about 1: 1 to about 0: 1; The ratio is from about 0.84: 1 to about 0: 1. In specific embodiments described below, the novel cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectins in a ratio of less than 1.6: 1. In another later specific embodiment, the novel cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectins in a ratio of about 0.94: 1. In yet another specific embodiment described below, the novel cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectins in a ratio of about 0.88: 1. In yet another specific embodiment described below, the novel cells of the present invention produce cyclohexyl 2: cyclohexyl B1 avermectins in a ratio of about 0.84: 1.

  In a further preferred embodiment, the present invention provides a genetic construct comprising a mutated aveC allele or aveC allele instead of, or in addition to, the wild type aveC allele (and thereby the wild type aveC allele). A new strain of Streptomyces avermitilis comprising cells expressing different amounts of said new strain of cells producing avermectin) compared to cells of the same strain that instead express only the allele provide. In a preferred embodiment, the novel strain produces an increased amount of avermectin. In a non-limiting embodiment, the genetic construct is further derived from a Saccharospora erythraea, which is a strong promoter (eg, upstream of the aveC-ORF and is effectively associated with the aveC-ORF). A strong constitutive ermE promoter).

  In a further preferred embodiment, the present invention provides a novel strain of Streptomyces avermitilis containing cells in which the aveC gene has been inactivated. The strain determines the spectrum of avermectin (generated by the strain) that is different from the wild type strain and either targeted or random mutagenesis of the aveC gene that affects avermectin production. Useful for both the complementation screening assays described in. In specific embodiments described below, Streptomyces avermitilis host cells were genetically designed to contain an inactivated aveC gene. For example, using the gene replacement plasmid pSE180 (ATCC 209605) (FIG. 3) constructed to inactivate the Streptomyces avermitilis aveC gene by inserting an ermE resistance gene into the aveC coding region, strain SE180- 11 (described in the examples below) was produced.

  Furthermore, the present invention provides Streptomyces avermitilis AveC gene product encoded by any of the polynucleotide molecules of the present invention, recombinantly expressed and mutated, and a method for producing the same.

  In addition, the present invention provides a cell of a Streptomyces avermitilis strain, wherein said cell expresses a mutated aveC allele as compared to a cell of the same strain that instead expresses only the wild type aveC allele. Expresses a mutated aveC allele that encodes a gene product that alters the class 2: 1 ratio of avermectins produced by cells of a Streptomyces avermitilis strain that permits or allows production of avermectins by the cells A method for producing avermectin comprising culturing in a culture medium under inducing conditions and recovering avermectin from the culture is provided. In a preferred embodiment, the class 2: 1 ratio of avermectins produced in culture by cells expressing the mutated aveC allele is reduced. This manufacturing method provides increased efficiency in the production of commercially valuable avermectins (eg, doramectin).

  Furthermore, the present invention relates to a cell of a Streptomyces avermitilis strain, wherein said cell replaces only the wild type aveC allele without expressing a mutated aveC allele or a gene construct comprising the aveC allele. A mutated aveC allele that causes a change in the amount of avermectin produced by a cell of a Streptomyces avermitilis strain expressing a mutated aveC allele or gene construct compared to a cell of the same strain that expresses A method for producing avermectin comprising culturing a gene construct in a culture medium under conditions that permit or induce production of avermectin by the cell and recovering avermectin from the culture is provided . In a preferred embodiment, the amount of avermectin produced in culture by cells expressing a mutated aveC allele or gene construct is increased.

  Furthermore, the present invention relates to a Streptomyces avermitilis strain, wherein said strain is a Streptomyces that expresses a mutated aveC allele as compared to cells of the same strain that instead express only the wild type aveC allele. A novel composition of avermectins produced by expressing a mutated aveC allele encoding a gene product that reduces the class 2: 1 ratio of avermectins produced by cells of the M. avermitilis strain, comprising: The avermectin in the novel composition is reduced compared to the class 2: 1 ratio of avermectin produced by cells of the same strain of Streptomyces avermitilis that instead express only the wild type aveC allele Provided is the composition produced in a class 2: 1 ratio. The novel avermectin composition can be present as produced in the spent fermentation broth or can be collected therefrom. The novel avermectin composition can be partially or substantially purified from the culture medium by known biochemical purification techniques (eg, ammonium sulfate precipitation, dialysis, size fractionation, ion exchange chromatography, HPLC, etc.). .

5.4. Use of Avermectin Avermectin is a highly active antiparasitic agent with particular utility as an anthelmintic, ectoparasiticide, insecticide, and acaricide. And the avermectin compounds produced by the method of the present invention are useful for all of their purposes. For example, avermectins produced by the present invention are useful for treating various diseases or conditions in humans (particularly where the disease or condition is caused by a parasitic infection known in the art). . See, for example, Ikeda and Omura, 1997, Chem. Rev. 97 (7): 2591-1609. More specifically, avermectin compounds prepared according to the present invention are caused by endoparasites (eg, parasitic nematodes) that can infect humans, livestock, pigs, sheep, poultry, horses, or cattle. It is effective in the treatment of various diseases and conditions.

  More specifically, avermectin compounds prepared in accordance with the present invention are effective against nematodes that infect humans and nematodes that infect various animal species. The nematodes include gastrointestinal parasites [eg, Ancylostomoma, Necator, Ascaris, Strongyloides, Trichinella, Capillaria Trichuri, Tricris (Enterobius, Dirofilaria)], and parasites found in blood or other tissues or organs (eg, the filamentous worms, and the extracted intestinal state of Stromboloides and Trichinella).

  Also, avermectin compounds prepared in accordance with the present invention may have ectoparasite infections [eg, ticks, ticks, lice, fleas, blowfly, stings insects, or cattle and horses that can affect cattle and horses. It is also useful in the treatment of mammalian and avian arthropod invasion caused by sexual dipterous larvae.

  Also, avermectin compounds prepared according to the present invention are useful for indoor pests such as cockroaches, moths, carpet beetles, and house flies, as well as insects that damage stored cereals and crops such as spider mites, aphids, It is also useful as an insecticide against caterpillars and straight worms (for example, grasshoppers in particular).

  Animals that can be treated with avermectin compounds prepared in accordance with the present invention include sheep, cows, horses, deer, goats, pigs, birds (including poultry), and dogs and cats.

  The avermectin compounds prepared according to the present invention are administered in a formulation suitable for the particular use intended, the individual species of host animal to be treated, and the associated parasite or insect. For use as a parasiticide, the avermectin compounds prepared according to the invention can be administered orally in the form of capsules, boluses, tablets or liquid drinks, or It can also be administered as a pour-on, by injection or as an implant. The formulations are prepared in the usual manner according to standard veterinary techniques. Thus, by mixing the active ingredient with a finely divided suitable diluent or carrier, and further including a disintegrant and / or binder (eg starch, lactose, talc, magnesium stearate, etc.) Capsules, boluses, or tablets can be prepared. Drinkable formulations can be prepared by dispersing the active ingredient in an aqueous solution with a dispersing or wetting agent or the like. Injectable preparations can be prepared in the form of sterile solutions that can contain other substances (eg, enough salts and / or glucose) to make the solution isotonic with blood.

  The formulation will vary with respect to the weight of active compound depending on the patient, or the type of host animal to be treated, the severity and type of infection, and the body weight of the host. In general, for oral administration, a dose of about 0.001 to 10 mg of active compound per kg patient or animal body weight as a single dose or divided dose for 1 to 5 days would be satisfactory. . However, there are cases where higher or lower dosage ranges are indicated based on clinical symptoms, for example, as determined by a physician or veterinarian.

  Alternatively, the avermectin compound prepared according to the present invention can be administered in combination with animal feed and for this purpose a concentrated feed additive or premix for mixing with normal animal feed Can be prepared.

  For use as pesticides and for the treatment of agricultural pests, the avermectin compounds prepared according to the invention can be applied as sprays, fine powders, emulsions, etc., according to standard agricultural techniques.

6). Example: Fermentation of Streptomyces avermitilis and B2: B1 avermectin analysis Strains lacking both branched 2-oxoacid dehydrogenase activity and 5-O-methyltransferase activity are not supplemented with fatty acids in the fermentation medium In some cases, it does not produce avermectin. This example demonstrates that a wide range of B2: B1 ratios of avermectin can be obtained when biosynthesis is initiated in the presence of various fatty acids in such mutants.

6.1. Materials and Methods Streptomyces avermitilis ATCC53692 was added to Starch [Nadex, Laing National] 20 g; 15 g; Ardamine pH [Yeast Products Inc.] 5 g; stored at −70 ° C. as a complete broth prepared in a seed medium consisting of 1 g of calcium carbonate. The final volume was adjusted to 1 liter with tap water, the pH was adjusted to 7.2, and the medium was autoclaved at 121 ° C. for 25 minutes.

  2 mL of the thawed suspension of the preparation was used to inoculate a flask containing 50 mL of the same medium. After incubation at 28 ° C. for 48 hours on a rotary shaker at 180 rpm, 2 mL of broth was added to 80 g of starch; 7 g of calcium carbonate; 5 g of Pharmamedia; 1 g of dipotassium hydrogen phosphate; 1 g of magnesium sulfate; It was used to inoculate a flask containing 50 mL of production medium consisting of 0.01 g of iron heptahydrate; 0.001 g of zinc sulfate; 0.001 g of manganese sulfate. The final volume was adjusted to 1 liter with fresh water, the pH was adjusted to 7.2, and the medium was autoclaved at 121 ° C. for 25 minutes.

  Various carboxylic acid substrates (see Table 1) were dissolved in methanol and added to the fermentation broth 24 hours after inoculation so that the final concentration was 0.2 g / L. After incubating the fermentation broth at 28 ° C. for 14 days, the broth was centrifuged (2,500 rpm for 2 minutes), and the supernatant was discarded. The mycelium pellet was extracted with acetone (15 mL) followed by dichloromethane (30 mL), the organic phase was separated, filtered and then evaporated to dryness. The residue was taken up in methanol (1 mL) and analyzed at 240 nm by HPLC using a Hewlett-Packard 1090A liquid chromatograph equipped with a scanning diode-array detector set. The column used was a Beckman Ultrasphere C-18, 5 μm, 4.6 mm × 25 cm column maintained at 40 ° C. 25 μL of the methanol solution was injected into the column. Elution was carried out at a ratio of 80:20 to 95: 5 methanol-water linear gradient at 0.85 / mL min for 40 minutes. The detector response was calibrated with two standard concentrations of cyclohexyl B1 and the area under the curve for B2 and B1 avermectins was measured.

6.2. Results The HPLC retention times observed for B2 and B1 avermectin, and the 2: 1 ratio are shown in Table 1.

  The data shown in Table 1 proves that the ratio of B2: B1 avermectin production is very wide, which depends on the nature of the fatty acid side chain starter unit supplied. It shows that there is a considerable difference in the results of dehydration conversion from compounds to class 1 compounds. This indicates that the change in B2: B1 ratio resulting from alterations to the AveC protein can be specific for a particular substrate. Therefore, screening for mutants that exhibit a change in the B2: B1 ratio obtained with a particular substrate needs to be performed in the presence of that substrate. In the following examples, cyclohexanecarboxylic acid is used as the screening substrate. However, this substrate is merely used to illustrate possibilities and is not intended to limit the applicability of the present invention.

7). Example: Isolation of the aveC gene This example describes the isolation and characterization of a region of the Streptomyces avermitilis chromosome encoding the aveC gene product. As shown below, the aveC gene was identified as being capable of altering the ratio of cyclohexyl-B2 to cyclohexyl-B1 (B2: B1) avermectins produced.

7.1. Materials and Methods 7.1.1. Growth of Streptomyces for DNA isolation To perform the growth of Streptomyces, the following procedure was followed. 1. A single colony of Streptomyces avermitilis ATCC 31272 (single colony isolate # 2) was added to Difco Yeast Extract 5 g; Difco Bact-peptone 5 g; Dextrose 5 g; MOPS 5 g; isolated on 1/2 strength YPD-6 containing 15 g Difco Bacto agar. The final volume was adjusted to 1 liter with dH ₂ O, the pH was adjusted to 7.0, and the medium was autoclaved at 121 ° C. for 25 minutes.

Mycelium grown in the above medium was TSB medium [Difco Tryptic Soy Broth 30 g in 1 liter dH ₂ O, autoclaved at 121 ° C. for 25 minutes] in a 25 mm × 150 mm test tube 10 mL And inoculated at 28 ° C. for 48-72 hours with shaking (300 rpm).

7.1.2. Isolation of chromosomal DNA from Streptomyces Place an aliquot (0.25 mL or 0.5 mL) of the mycelium grown as described above into a 1.5 mL microcentrifuge tube and centrifuge for 60 seconds at 12,000 x g. Was concentrated. The supernatant is discarded, and the cells are washed with TSE buffer containing 2 mg / mL lysozyme [1.5 M sucrose 20 mL, 1 M Tris-HCl (pH 8.0) 2.5 mL, 1 M EDTA (pH 8.0) 2.5 mL, and dH ₂ O 75 mL] resuspended in 0.25 mL. The sample was incubated for 20 minutes at 37 ° C. with shaking, and the AutoGen 540 ^™ automated nucleic acid isolator [Integrated Separation System, Natick, Mass.] And genomic DNA was isolated using Cycle 159 (instrument software) according to the manufacturer's instructions.

  Instead, 5 mL of mycelium was placed in a 17 mm × 100 mm test tube, and the cells were concentrated by centrifugation at 3,000 rpm for 5 minutes, and the supernatant was removed. The cells were resuspended in 1 mL of TSE buffer, concentrated by centrifugation at 3,000 rpm for 5 minutes, and the supernatant was removed. Cells were resuspended in 1 mL TSE buffer containing 2 mg / mL lysozyme and incubated at 37 ° C. for 30-60 minutes with shaking. After incubation, 0.5 mL of 10% sodium dodecyl sulfate (SDS) was added and the cells were incubated at 37 ° C. until lysis was complete. The lysate is incubated at 65 ° C. for 10 minutes, cooled to room temperature, placed in two 1.5 mL Eppendorf tubes and pre-equilibrated with phenol / chloroform [0.5 M Tris, pH 8.0]. 50% phenol; 50% chloroform] was extracted once (1 ×) with 0.5 mL. The aqueous phase was removed and extracted 2-5 times (2-5x) with chloroform: isoamyl alcohol (24: 1). The DNA is precipitated by adding 1/10 volume of 3M sodium acetate (pH 4.8), the mixture is incubated on ice for 10 minutes, and the mixture is centrifuged at 15,000 rpm for 10 minutes at 5 ° C. The supernatant was removed to a clean test tube and 1 volume of isopropanol was added thereto. The supernatant and isopropanol mixture is then incubated on ice for 20 minutes, centrifuged at 15,000 rpm for 20 minutes at 5 ° C., the supernatant removed, and the DNA pellet once with 70% ethanol ( 1x) Washed. After drying the pellet, the DNA was resuspended in TE buffer [10 mM Tris, 1 mM EDTA (pH 8.0)].

7.1.3. Plasmid DNA isolation from Streptomyces Mycelium aliquots (1.0 mL) were placed in 1.5 mL microcentrifuge tubes and the cells were concentrated by centrifugation at 12,000 × g for 60 seconds. The supernatant was discarded and the cells were resuspended in 1.0 mL of 10.3% sucrose and concentrated by centrifugation at 12,000 × g for 60 seconds, and the supernatant was discarded. The cells were then resuspended in TSE buffer containing 2 mg / mL lysozyme, incubated for 20 minutes at 37 ° C. with shaking, and subjected to an Autogen 540 ^™ automated nucleic acid isolator. Plasmid DNA was isolated using cycle 106 (equipment software) according to the manufacturer's instructions.

  Instead, 1.5 mL of mycelium was placed in a 1.5 mL microcentrifuge tube, and the cells were concentrated by centrifugation at 12,000 × g for 60 seconds. The supernatant was discarded and the cells were resuspended in 1.0 mL of 10.3% sucrose and concentrated by centrifugation at 12,000 × g for 60 seconds, and the supernatant was discarded. Cells were resuspended in 0.5 mL TSE buffer containing 2 mg / mL lysozyme and incubated at 37 ° C. for 15-30 minutes. After incubation, 0.25 mL of alkaline SDS (0.3 N NaOH, 2% SDS) was added and the cells were incubated at 55 ° C. for 15-30 minutes or until the solution was clear. Sodium acetate (0.1 mL, 3M, pH 4.8) was added to the DNA solution and then incubated on ice for 10 minutes. This DNA sample was centrifuged at 14,000 rpm for 10 minutes at 5 ° C. The supernatant was taken out into a clean test tube, and 0.2 mL of phenol: chloroform (50% phenol: 50% chloroform) was added thereto and mixed gently. This DNA solution was centrifuged at 14,000 rpm for 10 minutes at 5 ° C., and the upper layer was taken out into a clean Eppendorf tube. Isopropanol (0.75 mL) was added and the solution was mixed gently then incubated at room temperature for 20 minutes. The DNA solution was centrifuged at 14,000 rpm for 15 minutes at 5 ° C., the supernatant was removed, the DNA pellet was washed with 70% ethanol, dried and resuspended in TE buffer.

7.1.4. Plasmid DNA Isolation transformed single E.coli colony from E. coli (E. coli), Luria - Bertani (Luria-Bertani) (LB) medium [dH ₂ in O1 l, Bacto - tryptone (Bacto-Tryptone) 10g Bacto-Yeast extract (5 g) and NaCl 10 g (pH 7.0), autoclaved at 121 ° C. for 25 minutes, and supplemented with 100 μg / mL ampicillin]. The culture was incubated overnight and a 1 mL aliquot was placed in a 1.5 mL microcentrifuge tube. This cultured sample was run on an Autogen 540 ^™ automatic nucleic acid isolation device and plasmid DNA was isolated using cycle 3 (device software) according to the manufacturer's instructions.

7.1.5. Preparation and transformation of Streptomyces avermitilis protoplasts A single colony of Streptomyces avermitilis was isolated on 1/2 strength YPD-6. The mycelium was inoculated into 10 mL of TSB medium in a 25 mm × 150 mm test tube and then incubated at 28 ° C. for 48 hours with shaking (300 rpm). 1 mL of mycelium was inoculated into 50 mL of YEME medium. YEME medium per liter: 3 g Difco East Extract; 5 g Difco Bacto-Peptone; Difco Malt Extract (Difco)
(Mal Extract) 3g; Sucrose 300g. After autoclaving at 121 ° C. for 25 minutes, the following ingredients were added: 2.5 mL of 2.5 M MgCl ₂ .6H ₂ O (separately autoclaved at 121 ° C. for 25 minutes); and glycine (20%) (filter sterilized) Finished) 25 mL.

The mycelium was grown at 30 ° C. for 48-72 hours and collected in a 50 mL centrifuge tube [Falcon] by centrifugation at 3,000 rpm for 20 minutes. The supernatant was discarded and the mycelium was resuspended in P buffer. P buffer is sucrose 205 g; K ₂ SO ₄ 0.25 g; MgCl ₂ .6H ₂ O 2.02 g; H ₂ O 600 mL; K ₂ PO ₄ (0.5%) 10 mL; trace element solution 20 mL; CaCl ₂ .2H ₂ O (3.68%) 100mL; and MES buffer (1.0 M, pH 6.5) containing a 10 mL (trace elements solution per _{liter, ZnCl 2 40mg; FeCl 3 ·} 6H 2 O200mg; CuCl 2 · 2H ₂ O 10 mg; MnCl ₂ .4H ₂ O 10 mg; Na ₂ B ₄ O ₇ .10H ₂ O ₁₀ mg; (NH ₄ ) 6Mo ₇ O ₂₄ · 4H ₂ O ₁₀ mg). The pH was adjusted to 6.5, the final volume was adjusted to 1 liter, and the medium was hot filtered through a 0.45 micron filter.

The mycelium was pelleted at 3,000 rpm for 20 minutes, the supernatant was discarded, and the mycelium was resuspended in 20 mL of P buffer containing 2 mg / mL lysozyme. The mycelium was incubated at 35 ° C. for 15 minutes with shaking, and the degree of protoplast formation was measured by a microscope and examined. When protoplast formation was complete, the protoplasts were subjected to a 10 minute centrifugation at 8,000 rpm. The supernatant was removed and the protoplasts were resuspended in 10 mL P buffer. The protoplasts are centrifuged at 8,000 rpm for 10 minutes, the supernatant is removed, the protoplasts are resuspended in 2 mL of P buffer, and approximately 1 × 10 ⁹ protoplasts are added to a 2.0 mL cryogenic vial [ Distributed to Nalgene.

A vial containing 1 × 10 ⁹ protoplasts was centrifuged at 8,000 rpm for 10 minutes, the supernatant was removed, and the protoplasts were resuspended in 0.1 mL P buffer. 2-5 μg of transforming DNA was added to the protoplasts, followed immediately by 0.5 mL working T buffer. T buffer base, PEG-1000 [Sigma (Sigma)] 25g; contains H ₂ O83mL; sucrose 2.5 g. The pH was adjusted to 8.8 using 1N NaOH (filter sterilized) and the T buffer base was filter sterilized and stored at 4 ° C. Working T buffer prepared on the day of use was T buffer base 8.3 mL; K ₂ PO ₄ (4 mM) 1.0 mL; CaCl ₂ · 2H ₂ O (5 M) 0.2 mL; and TES (1 M, pH 8) 0. 5 mL. Each component of working T buffer was individually filter sterilized.

Within 20 seconds of adding T buffer to the protoplasts, 1.0 mL of P buffer was also added, and the protoplasts were centrifuged at 8,000 rpm for 10 minutes. The supernatant was discarded and the protoplasts were resuspended in 0.1 mL P buffer. The protoplasts were then plated on RM14 medium. This RM14 medium consists of sucrose 205 g; K ₂ SO ₄ 0.25 g; MgCl ₂ .6H ₂ O 10.12 g; glucose 10 g; Difco Casaamino Acids 0.1 g; 5 g; 3 g Difco Oatmeal Agar; 22 g Difco Bacto Agar; 800 mL dH ₂ O. This solution was autoclaved at 121 ° C. for 25 minutes. Following autoclaving, the following sterile stocks were added: K ₂ PO ₄ (0.5%) 10 mL; CaCl ₂ · 2H ₂ O (5M) 5 mL; L-proline (20%) 15 mL; MES buffer (1.0 M PH 6.5) 10 mL; trace element solution (same as above) 2 mL; cycloheximide stock (25 mg / mL) 40 mL; and 1 N NaOH 2 mL. 25 mL of RM14 medium was aliquoted per plate and the plates were allowed to dry for 24 hours prior to use.

Protoplasts were incubated at 95% humidity and 30 ° C. for 20-24 hours. To select thiostrepton resistant transformants, 1 mL of overlay buffer containing 125 μg / mL thiostrepton was spread evenly on the RM14 regeneration plate. The overlay buffer contains 10.3 g sucrose; 0.2 mL trace element solution (as above); and 1 mL MES (1M, pH 6.5) per 100 mL. The protoplasts were incubated for 7-14 days at 30 ° C. and 95% humidity until thiostrepton resistant (Thio ^r ) colonies were visible.

7.1.6. Transformation of Streptomyces lividans protoplasts Streptomyces lividans TK64 [supplied by John Inns Institute, Norwich, UK (UK)] in some cases Used for transformation. Methods and constructions for the growth, protoplast formation, and transformation of Streptomyces lividans are described in Hopwood et al., 1985, Genetic Manipulation of Streptomyces, A Laboratory Manual, John Innes Foundation, Norwich, UK. Performed as described in the book. Plasmid DNA was isolated from Streptomyces lividans transformants as described in section 7.1.3 above.

7.1.7. Fermentation analysis of Streptomyces avermitilis strains Mycelium of Streptomyces avermitilis grown for 4-7 days on 1/2 strength YPD-6, 1 × 6 inch test containing 8 mL preform medium and two 5 mm glass beads The tube was inoculated. The preform medium is soluble starch [thin cooked starch or KOSO; Japan Corn Starch Co., Nagoya] 20 g / L; Pharmamedia 15 g / L; Aldamine ( Ardamine) pH 5 g / L [Champlan India. (Camplain Ind.), Clifton, NJ (NJ)]; CaCO ₃ 2 g / L; final concentration 50 ppm 2-(+/−)-methylbutyric acid, 60 ppm isobutyric acid, and 20 ppm in the medium 2xbcfa containing “isovaleric acid” (“bcfa” refers to a branched chain fatty acid). The pH was adjusted to 7.2 and the medium was autoclaved at 121 ° C. for 25 minutes.

The test tube was shaken for 3 days at 29 ° C. at 215 rpm with an angle of 17 °. A 2 mL aliquot of this seed culture was inoculated into a 300 mL Erlenmeyer flask containing 25 mL of production medium. This production medium consists of starch [weakly boiled starch or coso] 160 g / L; Nutrisoy [Archer Daniels Midland, Decatur, Illinois (IL)] 10 g / L; Aldamine pH 10 g / L; K ₂ HPO ₄ 2 g / L; MgSO ₄ · 4H ₂ O ₂ g / L; FeSO ₄ · 7H ₂ O 0.02 g / L; MnCl ₂ 0.002 g / L; ZnSO ₄ · 7H ₂ O 0.002 g / L CaCO ₃ 14 g / L; 2 × bcfa (same as above); and cyclohexanecarboxylic acid (CHC) (prepared as a 20% solution at pH 7.0) 800 ppm. The pH was adjusted to 6.9 and the medium was autoclaved at 121 ° C. for 25 minutes.

  After inoculation, the flask was incubated at 29 ° C. for 12 days with shaking at 200 rpm. After incubation, 2 mL of sample was removed from the flask, diluted with 8 mL of methanol, mixed, and the mixture was centrifuged at 1,250 × g for 10 minutes to pellet the debris. The supernatant was then analyzed by HPLC using a Beckman Ultrasphere ODS column (25 cm x 4.6 mm diameter) with a flow rate of 0.75 mL / min and detection by 240 nm absorption. The mobile phase was 86 / 8.9 / 5.1 methanol / water / acetonitrile.

7.1.8. Isolation of Streptomyces avermitilis PKS gene A cosmid library of Streptomyces avermitilis (ATCC31272, SC-2) chromosomal DNA was prepared and prepared from a fragment of the Saccharopolyspora erythraea polyketide synthase (PKS) gene Hybridized with a ketosynthase (KS) probe. A detailed description of the preparation of a cosmid library can be found in Sambrook et al., 1989, supra. A detailed description of the preparation of the Streptomyces chromosomal DNA library is given in Hopwood et al., 1985, supra. A cosmid clone containing a ketosynthase-hybridization region is described in [Doctor P. et al. Identified by hybridization to a 2.7 Kb NdeI / Eco47III fragment derived from pEX26, kindly supplied by Dr. P. Leadlay, Cambridge, UK. About 5 ng of pEX26 was digested with NdeI and Eco47III. The reaction mixture was run on a 0.8% SeaPlaque GTG agarose gel (FMC BioProducts, Rockland, ME). After electrophoresis, a 2.7 Kb NdeI / Eco47III fragment was excised from the gel and the gel was gelled using GELase ^™ from Epicenter Technologies using Fast Protocol. Recovered from. This 2.7 Kb NdeI / Eco47III fragment was added to the BRL Translation System (BRL Life Technologies, Inc., Gaithersburg, MD). [Α- ³² P] dCTP [deoxycytidine 5′-triphosphate, tetra (triethylammonium) salt, [α- ³² P]-] [NEN-Dupont] according to the manufacturer's instructions , Boston, Massachusetts]. A typical reaction was performed in a 0.05 mL volume. After addition of 5 μL of stop buffer, labeled with unincorporated nucleotides using G-25 Sephadex Quick Spin ^™ column [Boehringer Mannheim] according to manufacturer's instructions DNA was isolated.

About 1,800 cosmid clones were screened by colony hybridization. Ten clones were identified that hybridize strongly to the Saccharopolyspora erythraea KS probe. Escherichia coli colonies containing cosmid DNA are grown in LB liquid medium and cosmid DNA from each culture using Cycle 3 (device software) according to the manufacturer's instructions on an Autogen 540 ^™ automatic nucleic acid isolator. Was isolated. Restriction endonuclease mapping and Southern blot hybridization analysis showed that 5 clones contained partially overlapping chromosomal regions. A genomic BamHI restriction map of Streptomyces avermitilis for 5 cosmids (ie, pSE65, pSE66, pSE67, pSE68, pSE69) was generated by analysis of partially overlapping cosmids and hybridization (FIG. 4).

7.1.9. Identification of DNA that regulates the avermectin B2: B1 ratio and identification of the aveC-ORF Using the following method, subcloned fragments obtained from the pSE66 cosmid clone were used to regulate the B2: B1 ratio of avermectin in the AveC mutant Tested for ability to do. pSE66 (5 μg) was digested with SacI and BamHI. The reaction mixture was run on a 0.8% Seaplak ^™ GTG agarose gel (FMC Bioproducts), and after electrophoresis, a 2.9 Kb SacI / BamHI fragment was excised from the gel and GELase (1) using a fast protocol. DNA was recovered from the gel using GELase ^™ ) (Epicenter Technologies). About 5 μg of shuttle vector pWHM3 [Vara et al., 1989, Jay. Bacteriol. (J. Bacteriol.) 171: 5872-5881] was digested with SacI and BamHI. Approximately 0.5 μg of the 2.9 Kb insert and 0.5 μg of digested pWHM3 were mixed together and 1 unit of ligase [New England Biolabs, Inc., Beverly, Mass. State] and a total volume of 20 μL and incubated overnight at 15 ° C. according to the manufacturer's instructions. After incubation, 5 μL of the ligation mixture was incubated at 70 ° C. for 10 minutes, cooled to room temperature, and used to transform competent E. coli DH5α cells (BRL) according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants and confirmed by restriction analysis for the presence of a 2.9 Kb SacI / BamHI insert. This plasmid was designated as pSE119.

  Protoplasts of Streptomyces avermitilis strain 1100-SC38 (Pfizer in-house strain) were prepared and transformed with pSE119 described in Section 7.1.5 above. Strain 1100-SC38 is a mutant that produces a significantly higher amount of avermectin cyclohexyl-B2 form (approximately 30: 1 B2: B1) when supplemented with cyclohexanecarboxylic acid compared to avermectin cyclohexyl-B1 form. PSE119 used to transform the protoplast of Streptomyces avermitilis is Escherichia coli strain GM2163 [Doctor Bee. Jay. Dr. BJ Bachmann, Curator, e. E. coli Genetic Stock Center, Yale University, E. coli strain DM1 (BRL), or Streptomyces lividans strain TK64. A thiostrepton resistant transformant of strain 1100-SC38 was isolated and analyzed by HPLC analysis of the fermentation product. Transformants of Streptomyces avermitilis strain 1100-SC38 containing pSE119 produced avermectin cyclohexyl-B2: cyclohexyl-B1 with a change ratio of approximately 3.7: 1 (Table 2).

  After confirming that pSE119 can regulate the ratio of avermectin B2: B1 in the AveC mutant, the insert DNA was sequenced. Approximately 10 μg of pSE119 is isolated using a plasmid DNA isolation kit [Qiagen, Valencia, Calif. (CA)] according to the manufacturer's instructions, and the ABI373A automated DNA sequencer (Automated DNA Sequencer). The sequence was determined using [Perkin Elmer, Foster City, CA]. Sequence data was assembled and edited using Genetic Computer Group programs [GCG, Madison, WI (WI)]. The DNA sequence and aveC-ORF are shown in FIG. 1 (SEQ ID NO: 1 in the sequence listing).

A new plasmid designated pSE118 was constructed as follows. About 5 μg of pSE66 was digested with SphI and BamHI. The reaction mixture was run on a 0.8% sieve plaque GTG agarose gel (FMC Bioproducts), and the 2.8 Kb SphI / BamHI fragment was excised from the gel after electrophoresis and GELase ^™ (GELase ^™ ) using the fast protocol ( DNA was recovered from the gel using Epicenter Technologies. About 5 μg of shuttle vector pWHM3 was digested with SphI and BamHI. About 0.5 μg of 2.8 Kb insert and 0.5 μg of digested pWHM3 are mixed together to make 1 unit of ligase (New England Biolabs), total volume 20 μL, manufacturer's instructions And incubated overnight at 15 ° C. After incubation, 5 μL of the ligation mixture was incubated at 70 ° C. for 10 minutes, cooled to room temperature, and used to transform competent E. coli DH5α cells according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants and confirmed by restriction analysis for the presence of a 2.8 Kb SphI / BamHI insert. This plasmid was designated as pSE118. The insert DNA in pSE118 and pSE119 is partially overlapped by approximately 838 nucleotides (FIG. 4).

  A protoplast of Streptomyces avermitilis strain 1100-SC38 was transformed with the above-mentioned pSE118. A thiostrepton resistant transformant of strain 1100-SC38 was isolated and analyzed by HPLC analysis of the fermentation product. Transformants of Streptomyces avermitilis strain 1100-SC38 containing pSE118 had no change in the ratio of avermectin cyclohexyl-B2: avermectin cyclohexyl-B1 compared to strain 1100-SC38 (Table 2).

7.1.10. PCR amplification of the aveC gene from Streptomyces avermitilis chromosomal DNA by PCR amplification of the ~ 1.2 Kb fragment containing the aveC-ORF using primers designed based on the previously obtained aveC nucleotide sequence Isolated from Streptomyces avermitilis chromosomal DNA. PCR primers were supplied by Genosys Biotechnologies, Inc., Texas. The right-facing primer is 5′-TCACGAAACCGGACACAC-3 ′ (SEQ ID NO: 6 in the sequence listing), and the left-facing primer is 5′-CATGATCCGCTGAACCGAG-3 ′ (SEQ ID NO: 7 in the sequence listing). there were. The PCR reaction was performed using Deep Vent ^™ polymerase (New England Biolabs) in buffer supplied by the manufacturer, 300 μM dNTP, 10% glycerol, 200 bimol of each primer, 0.1 μg of template. , And 2.5 units of enzyme in a final volume of 100μ using a Perkin-Elmer Cetus thermal cycler. The thermal history of the first cycle was 95 ° C. for 5 minutes (denaturation step), 60 ° C. for 2 minutes (annealing step), and 72 ° C. for 2 minutes (extension step). The subsequent 24 cycles had a similar thermal history except that the denaturation step was shortened to 45 seconds and the annealing step was shortened to 1 minute.

The PCR product was electrophoresed in a 1% agarose gel and a single DNA band of ˜1.2 Kb was detected. The DNA was purified from the gel and 25 ng of linearized blunt pCR-Blunt vector [Invitrogen] and 1:10 vector to insert according to manufacturer's instructions. Were linked at a molar ratio of This ligation mixture was used to transform One Shot ^™ Competent E. coli cells (Invitrogen) according to the 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 BamHI / XbaI, separated by electrophoresis, purified from gel, and similarly digested with BamHI / XbaI and at a total DNA concentration of 1 μg. Ligated at a 1: 5 vector to insert molar ratio. This ligation mixture was used to transform competent E. coli DH5α cells according to the 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, designated pSE186 (FIG. 2, ATCC 209604), was transformed into E. coli DM1 and plasmid DNA was isolated from ampicillin resistant transformants.

7.2. Results The 2.9 Kb SacI / BamHI fragment from pSE119 was confirmed to significantly change the ratio of B2: B1 avermectin production when transformed into Streptomyces avermitilis strain 1100-SC38. Streptomyces avermitilis strain 1100-SC38 typically has a B2: B1 ratio of about 30: 1, but when transformed with a vector containing a 2.9 Kb SacI / BamHI fragment, B2: B1 avermectin Ratio decreased to about 3.7: 1. Post-fermentation analysis of the transformant culture confirmed the presence of transforming DNA.

  The sequence of the 2.9 Kb pSE119 fragment was determined and the .about.0.9 Kb ORF was identified (FIG. 1) (SEQ ID NO: 1 in the sequence listing). This includes a PstI / SphI fragment that has been previously mutated elsewhere to produce only the B2 product [Ikeda et al., 1995, supra]. Comparison of this ORF or its corresponding putative polypeptide against a known database (GenEMBL, SWISS-PROT) did not show any strong homology with known DNA or protein sequences.

  Table 2 shows the fermentation analysis of Streptomyces avermitilis strain 1100-SC38 transformed with various plasmids.

8). Example: Construction of Streptomyces avermitilis AveC mutants This example describes the construction of several different Streptomyces avermitilis AveC mutants using the compositions and methods described above. A general description of techniques for introducing mutations into Streptomyces genes is given by Kieser and Hopwood, 1991, Meth. Enzym. 204: 430-458. A more detailed description can be found in Anzai et al. Antibiot. , XLI (2): 226-233 and Stutzman-Engwall et al., 1992, J. MoI. Bacteriol. 174 (1): 144-154. These references are hereby incorporated by reference in their entirety.

8.1. Inactivation of Streptomyces avermitilis aveC gene AveC mutants containing inactivated aveC gene were constructed using several methods, as described below.

  In the first method, the 640 bp SphI / PstI fragment endogenous to the aveC gene in pSE119 (the plasmid described in Section 7.1.9 above) was replaced with the ermE gene (for erythromycin resistance) from Saccharopolyspora erythraea. It was. The ermE gene was supplied by pIJ4026 [John Inns Institute, Norwich, UK by restriction enzyme digestion with BglII and EcoRI followed by electrophoresis; Bibb et al., 1985, Gene, 41: 357-. See also 368] and purified from the gel. The ˜1.7 Kb fragment was ligated into pGEM7Zf [Promega] digested with BamHI and EcoRI and the ligation mixture was transformed into competent E. coli DH5α cells according to the manufacturer's instructions. Converted. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of a ~ 1.7 Kb insert was confirmed by restriction analysis. This plasmid was designated as pSE27.

  pSE118 (described in Section 7.1.9 above) was digested with SphI and BamHI, the digestion product was electrophoresed, and the ˜2.8 Kb SphI / BamHI insert was purified from the gel. pSE119 was digested with PstI and EcoRI, the digestion product was electrophoresed, and the ˜1.5 Kb PstI / EcoRI insert was purified from the gel. The shuttle vector pWHM3 was digested with BamHI and EcoRI. pSE27 was digested with PstI and SphI, the digest was electrophoresed, and the ˜1.7 Kb PstI / SphI insert was purified from the gel. All four fragments (ie ˜2.8 Kb, ˜1.5 Kb, ˜7.2 Kb, ˜1.7 Kb) were ligated together by four ligation methods. This ligation mixture was transformed into competent E. coli DH5α cells according to the 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 (FIG. 3; ATCC 209605).

pSE180 was transformed into Streptomyces lividans strain TK64 and transformed colonies were identified by resistance to thiostrepton and erythromycin. pSE180 was isolated from Streptomyces lividans and used to transform Streptomyces avermitilis protoplasts. Four thiostrepton resistant Streptomyces avermitilis transformants were identified and their protoplasts were prepared and plated under non-selective conditions on RM14 medium. After the protoplasts were regenerated, single colonies were screened for the presence of erythromycin resistance and the absence of thiostrepton resistance, indicating chromosomal integration of the inactivated aveC gene and loss of free replicon. One Erm ^r Thio ^s transformant was identified and designated strain SE180-11. The entire chromosomal DNA is isolated from strain SE180-11, digested with restriction enzymes BamHI, HindIII, PstI, or SphI and separated by electrophoresis on a 0.8% agarose gel, resulting in a nylon membrane. Transfer and hybridize to ermE probe. These analyzes indicated that chromosomal integration of the ermE resistance gene and concomitant deletion of the 640 bp PstI / SphI fragment was caused by double crossover events. HPLC analysis of the fermentation product of strain SE180-11 showed that normal avermectin was no longer produced (FIG. 5A).

In the second method of inactivating the aveC gene, a 1.7 Kb ermE gene was removed from the chromosome of Streptomyces avermitilis strain SE180-11 leaving a 640 bp PstI / SphI deletion in the aveC gene. A gene replacement plasmid was constructed as follows. That is, pSE180 was partially digested with XbaI and the ˜11.4 Kb fragment was purified from the gel. This ˜11.4 Kb band lacks the 1.7 Kb ermE resistance gene. This DNA was then ligated and transformed into E. coli DH5α cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis. The plasmid designated pSE184 was transformed into E. coli DM1, and the plasmid DNA was isolated from ampicillin resistant transformants. This plasmid was used to transform Streptomyces avermitilis strain SE180-11 protoplasts. Protoplasts were prepared from thiostrepton resistant transformants of strain SE180-11 and plated as single colonies on RM14 medium. After regenerating the protoplasts, single colonies were screened for the absence of both erythromycin resistance and thiostrepton resistance, indicating chromosomal integration of the inactivated aveC gene and a free replicon containing the ermE gene. One Erm ^s Thio ^s transformant was identified and designated SE184-1-13. Fermentation analysis of SE184-1-13 indicated that normal avermectin was not produced and that SE1844-1-13 had the same fermentation characteristics as SE180-11.

  In the third method of inactivating the aveC gene, a frame shift was introduced into the chromosomal aveC gene by using PCR to add two Gs after C at nt position 471 to create a BspE1 site. . The presence of the created BspE1 site was effective in detecting gene replacement events. PCR primers were designed to introduce frameshift mutations into the aveC gene, which was supplied by Genosys Biotechnologies. The right-facing primer was 5'-GGTTCCGGATGCCGTTCCT-3 '(SEQ ID NO: 8 in the sequence listing), and the left facing primer was 5'-AACTCCGGTCCGACTCCCCCTTC-3' (SEQ ID NO: 9 in the sequence listing). PCR conditions were as described in Section 7.1.10. The 666 bp PCR product was digested with SphI, resulting in two fragments of 278 bp and 388 bp, respectively. A 388 bp fragment was purified from the gel.

  A gene replacement plasmid was constructed as follows. That is, the shuttle vector pWHM3 was digested with EcoRI and BamHI. pSE119 was digested with BamHI and SphI, the digestion product was electrophoresed, and the ˜840 bp fragment was purified from the gel. pSE119 was digested with EcoRI and XmnI, the digestion products were separated by electrophoresis, and the ˜1.7 Kb fragment was purified from the gel. All four fragments (ie ˜7.2 Kb, ˜840 bp, ˜1.7 Kb, and 388 bp) were ligated together in four ways. This ligation mixture was transformed into competent E. coli DH5α 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. The plasmid designated pSE185 was transformed into E. coli DM1, and plasmid DNA was isolated from ampicillin resistant transformants. This plasmid was used to transform Streptomyces avermitilis strain 1100-SC38 protoplasts. A thiostrepton resistant transformant of strain 1100-SC38 was isolated and analyzed by HPLC analysis of the fermentation product. pSE185 did not significantly change the B2: B1 avermectin ratio when transformed into Streptomyces avermitilis strain 1100-SC38 (Table 2).

  pSE185 was used to transform Streptomyces avermitilis protoplasts, causing a frameshift mutation in the chromosomal aveC gene. Protothrust was prepared from thiostrepton resistant transformants and plated as a single colony on RM14. After regenerating protoplasts, single colonies were screened for the absence of thiostrepton resistance. Chromosomal DNA was isolated from thiostrepton sensitive colonies and screened by PCR for the presence of frameshift mutations integrated into the chromosome. This PCR primer was designed based on the aveC nucleotide sequence and was supplied by Genosys Biotechnology (Texas). The right-pointing PCR primer is 5'-GCAAGGATACGGGGAACTAC-3 '(SEQ ID NO: 10 in the sequence listing), the left-pointing primer is 5'-GAACCGACCGCCTGATAC-3' (SEQ ID NO: 11 in the sequence listing), and the PCR conditions are As described in Section 7.1.10. The resulting PCR product 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 deletion of the free replicon.

Fermentation analysis of Streptomyces avermitilis mutants containing a frameshift mutation in the aveC gene indicates that normal avermectin is no longer produced and that these mutants are identified as strains SE180-11 and SE1844-1-13. It was shown to have the same fermentation HPLC characteristics. One Thio ^s mutant was identified and designated strain SE185-5a.

  In addition, a mutation in the aveC gene was made that changed nt position 520 from G to A, resulting in a change in the codon encoding tryptophan (W) at position 116 to a stop codon. The Streptomyces avermitilis strain with this mutation did not produce normal avermectin and had the same fermentation characteristics as strains SE180-11, SE184-1-13, and SE185-5a.

  Further, (i) changing the amino acid from glycine (G) to aspartic acid (D) at position 256, changing from G to A at nt position 970, and (ii) changing the amino acid from tyrosine (Y) to histidine at position 275 A mutation in the aveC gene was made that both changed to (H), changing both T to C at nt position 996. The Streptomyces avermitilis strain with these mutations (G256D / Y275H) does not produce normal avermectin and has the same fermentation characteristics as strains SE180-11, SE1844-1-13, and SE185-5a. Was.

Streptomyces avermitilis aveC-inactivating mutant strains SE180-11, SE184-1-13, SE185-5a, and other strains described herein may affect the effects of other mutations in the aveC gene. Provide screening tools for evaluation. pSE186 containing a wild type copy of the aveC gene was transformed into E. coli DM1, and plasmid DNA was isolated from ampicillin resistant transformants. The pSE186 DNA was used to transform Streptomyces avermitilis strain SE180-11 protoplasts. Transformants thiostrepton resistant strains SE180-11 were isolated, the presence of erythromycin resistance was determined, and analyzed by HPLC analysis of fermentation products to Thio ^r Erm ^r transformants. The presence of a functional aveC gene [in trans] allowed strain SE180-11 to restore normal avermectin production (FIG. 5B).

8.2. Analysis of mutations in the aveC gene that alter the ratio of class B2: B1 As described above, the Streptomyces avermitilis strain SE180-11 containing an inactive aveC gene is a plasmid containing a functional aveC gene (pSE186) Was complemented by transformation with. Strain SE180-11 was also used 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 PCR amplification of the aveC gene. The 1.2 Kb ORF was isolated by PCR amplification using primers designed on the basis of the aveC nucleotide sequence. The right-facing primer was SEQ ID NO: 6 in the Sequence Listing, and the left-facing primer was SEQ ID NO: 7 in the Sequence Listing (see Section 7.1.10 above). 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, changing the amino acid at position 55 from serine (S) to phenylalanine (F). Yes. The aveC gene containing the S55F mutation was subcloned into pWHM3 to form a plasmid designated pSE187, which was used to transform the protoplast of Streptomyces avermitilis strain SE180-11. Transformants thiostrepton resistant strains SE180-11 were isolated, the presence of erythromycin resistance was determined, and analyzed by HPLC analysis of fermentation products to Thio ^r Erm ^r transformants. The presence of the aveC gene encoding a change at amino acid residue 55 (S55F) allowed strain SE180-11 to restore normal avermectin production (FIG. 5C). However, the ratio of cyclohexyl B2: cyclohexyl B1 is about 26: 1 compared to strain SE180-11 transformed with pSE186 having a B2: B1 ratio of about 1.6: 1 ( Table 3), indicating that a single mutation (S55F) regulates the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1.

  Another mutation in the aveC gene that changes nt position 862 from G to A was identified, which changes the amino acid at position 230 from glycine (G) to aspartic acid (D). A Streptomyces avermitilis strain carrying this mutation (G230D) produces avermectins with a B2: B1 ratio of about 30: 1.

8.3. Mutations that reduce the B2: B1 ratio Several mutations that reduce the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1 were constructed as follows.

A mutation in the aveC gene that changes nt position 588 from G to A was identified, which changes the amino acid at position 139 from alanine (A) to threonine (T). The aveC gene containing this A139T mutation was subcloned into pWHM3 to form a plasmid designated pSE188, which was used to transform the protoplast of Streptomyces avermitilis strain SE180-11. Transformants thiostrepton resistant strains SE180-11 were isolated, the presence of erythromycin resistance was determined, and analyzed by HPLC analysis of fermentation products to Thio ^r Erm ^r transformants. Strain SE180-11 was able to restore avermectin production due to the presence of a mutant aveC gene encoding a change at amino acid residue 139 (A139T) (FIG. 5D). However, the B2: B1 ratio is about 0.94: 1, indicating that this mutation reduces the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1. This result was unexpected. This is because the results described in the publication, as well as the results of the mutations described above, only showed inactivation of the aveC gene or increased production of B2 type relative to B1 type of avermectin.

  Since the A139T mutation changed the B2: B1 ratio in the more favorable B1 direction, a mutation was constructed that encoded threonine instead of serine at amino acid position 138. Thus, pSE186 was digested with EcoRI and cloned into pGEM3Zf (Promega) that had been digested with EcoRI. The plasmid designated pSE186a was digested with ApaI and KpnI, the DNA fragments were separated on an agarose gel, and two fragments of ˜3.8 Kb and ˜0.4 Kb were purified from the gel. A single base change was introduced at nt position 585 using the ~ 1.2 Kb insert DNA from pSE186 as a PCR template. This PCR primer was designed to introduce a mutation at nt position 585 and was supplied by Genosis Biotechnologies (Texas). The right-pointing PCR primer was 5'-GGGGGGGGGCCCGGGGTGCGGAGGCGGAAATGCCCCCTGCGCAGCG-3 '(SEQ ID NO: 12 in the sequence listing), and the PCR primer facing left was 5'-GGAACCGACCGCCTGATACA-3' (SEQ ID NO: 13 in the sequence listing). The PCR reaction was performed using the Advantage GC genomic PCR kit (Clonetech Laboratories, Palo Alto, Calif.) In the buffer supplied by the manufacturer, Performed in a final volume of 50 μL in the presence of 200 μM dNTPs, 200 pmoles of each primer, 50 ng template DNA, 1.0 M GC-melt (GC-Melt) and 1 unit of Clentack Polymerase Mix (KlenTaq Polymerase Mix) did. The thermal history of the first cycle was 94 ° C. for 1 minute, followed by 25 cycles of 94 ° C. for 30 seconds and 68 ° C. for 2 minutes, and 1 cycle of 68 ° C. for 3 minutes. The 295 bp PCR product was digested with ApaI and KpnI to release a 254 bp fragment, which was separated by electrophoresis and purified from the gel. All three fragments (-3.8 Kb, -0.4 Kb and 254 bp) were ligated together in three ways. This ligation mixture was transformed into competent E. coli DH5α 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 EcoRI, cloned into pWHM3 that had been digested with EcoRI, and transformed into E. coli DH5α 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. A plasmid designated pSE199 was used to transform a protoplast of Streptomyces avermitilis strain SE180-11. Transformants thiostrepton resistant strains SE180-11 were isolated, the presence of erythromycin resistance was determined, and the Thio ^r Erm ^r transformants were analyzed by HPLC analysis of fermentation products. Strain SE180-11 was able to restore normal avermectin production due to the presence of a mutated aveC gene encoding a change at amino acid residue 138 (S138T). However, the B2: B1 ratio is about 0.88: 1, indicating that this mutation reduces the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1 ( Table 3). This B2: B1 ratio is even lower than the 0.94: 1 ratio observed for the A139T mutation formed by transformation of strain SE180-11 with pSE188 as described above.

  Another mutation was constructed to introduce threonine at both amino acid positions 138 and 139. A ~ 1.2 Kb insert DNA from pSE186 was used as a PCR template. PCR primers were designed to introduce mutations at nt positions 585 and 588 and were supplied by Genosis Biotechnology (Texas). The right-pointing PCR primer was 5'-GGGGGGGGGCCCGGGGTGCGGAGGCGGAAATGCCGCTGGCGACGACCACC-3 '(SEQ ID NO: 14 in the sequence listing), and the PCR primer facing left was 5'-GGAACATACGGCATTCACC-3' (SEQ ID NO: 15 in the sequence listing). PCR reactions were performed using the conditions described immediately above in this section. The 449 bp PCR product was digested with ApaI and KpnI to release a 254 bp fragment that was separated by electrophoresis and purified from the gel. pSE186a was digested with ApaI and KpnI, the DNA fragments were 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 three ways and the ligation mixture was transformed into competent E. coli DH5α 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 EcoRI, cloned into pWHM3 that had been digested with EcoRI, and transformed into E. coli DH5α 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 was designated as pSE231, and this plasmid was used to transform a protoplast of Streptomyces avermitilis strain SE180-11. Transformants of thiostrepton resistance SE180-11 were isolated, the presence of erythromycin resistance was determined, and the Thio ^r Erm ^r transformants were analyzed by fermentation. The presence of doubly mutated aveC gene encoding S138T / A139T allowed strain SE180-11 to restore normal avermectin production. However, the ratio of B2: B1 is about 0.84: 1, indicating that this mutation is less than the reduction caused by transformation of strain SE180-11 with pSE188 or pSE199 as described above. Furthermore, it shows that the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1 is reduced (Table 3).

  These results are the first to show specific mutations in the aveC gene that result in increased production of the more commercially desirable class 1 versus class 2 avermectins.

9. Example: Construction of 5 'deletion mutants As explained in section 5.1 above, the nucleotide sequence of Streptomyces avermitilis (SEQ ID NO: 1 in the sequence listing) shown in FIG. It contains four different GTG codons at bp positions 42, 174, 177 and 180. This section describes the organization of multiple deletions in the 5 ′ region of the aveC-ORF (FIG. 1; SEQ ID NO: 1 in the sequence listing), which of these codons begins in the aveC-ORF for protein expression It helps to define whether it could function as a position.

  Fragments of the aveC gene that were variously deleted at the 5 'end were isolated from Streptomyces avermitilis chromosomal DNA by PCR amplification. PCR primers were designed based on the aveC DNA sequence and were supplied by Genosys Biotechnologies. The primer facing right is 5′-AACCCATCCGAGCCCTC-3 ′ (SEQ ID NO: 16 in the sequence listing) (D1F1); 5′-TCGGCCTGCCCAACGAAC-3 ′ (SEQ ID NO: 17 in the sequence listing) (D1F2); (SEQ ID NO: 18 of the Sequence Listing) (D1F3); and 5′-TGCAGGCGTACGTGTCAGC-3 ′ (SEQ ID NO: 19 of the Sequence Listing) (D2F2); The left-facing primers were 5′-CATGATCCGCTGAACCGA-3 ′ (SEQ ID NO: 20 in the sequence listing); 5′-CATGATCCGCTGAACCGAGGA-3 ′ (SEQ ID NO: 21 in the sequence listing); and 5′-AGGAGTGTGGTGGCGTCTTGGA-3 ′ (sequence listing SEQ ID NO: 22). This PCR reaction was performed as described in section 8.3 above.

The PCR products were separated by electrophoresis in a 1% agarose gel and a single DNA band of ˜1.0 Kb or ˜1.1 Kb was detected. The PCR product was purified from the gel and ligated with 25 ng of linearized pCR2.1 vector (Invitrogen) at a 1:10 molar vector to insert ratio according to the manufacturer's instructions. This ligation mixture was used to transform One Shot ^™ Competent E. coli cells (Invitrogen) according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the insert was confirmed by restriction analysis and DNA sequence analysis. These plasmids were pSE190 (obtained using primer D1F1), pSE191 (obtained using primer D1F2), pSE192 (obtained using primer D1F3), and pSE193 (obtained using primer D2F2). Displayed).

These insert DNAs were each digested with BamHI / XbaI, separated by electrophoresis, purified from gel and separately digested with BamHI / XbaI and the 1: 5 vector to insert moles. The DNA was ligated at a total DNA concentration of 1 μg. This ligation mixture was used to transform competent E. coli DH5α cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the insert was confirmed by restriction analysis. These plasmids are designated as pSE194 (D1F1), pSE195 (D1F2), pSE196 (D1F3) and pSE197 (D2F2), each of which is separately transformed into E. coli strain DM1, and the plasmid DNA is transformed into ampicillin resistant transformants. And the presence of the correct insert was confirmed by restriction analysis. This DNA was used to transform Streptomyces avermitilis strain SE180-11 protoplasts. Transformants thiostrepton resistant strains SE180-11 were isolated, the presence of erythromycin resistance was determined, and the Thio ^r Erm ^r transformants were analyzed by HPLC analysis of fermentation products, which GTG sites aveC expression Identified what is needed. This result indicates that pSE194, pSE195, and pSE196, which each lack a GTG site at position 42 but all contain 3 GTG sites at positions 174, 177, and 180, are transformed into SE180-11. Since it was able to restore normal avermectin production when converted, it indicates that the GTG codon at position 42 can be removed without affecting aveC expression. Normal avermectin production was not restored when strain SE180-11 was transformed with pSE197 lacking all four GTG sites (Table 4).

10. Example: Cloning of aveC homologues from S. hygroscopicus and S. griseochromogenes The present invention produces other avermectins or milbemycin. It makes it possible to identify and clone aveC homologous genes from species. For example, a cosmid library of Streptomyces hygroscopicus (FERM BP-1901) was hybridized with the 1.2 Kb aveC probe derived from Streptomyces avermitilis. Several strongly cosmid clones were identified. Chromosomal DNA was isolated from these cosmids and a 4.9 Kb KpnI fragment hybridized with the aveC probe was identified. The sequence of this DNA was determined, and an ORF (SEQ ID NO: 3 in the sequence listing) having significant homology with the aveC-ORF of Streptomyces avermitilis was identified. The amino acid sequence deduced from the aveC homologous ORF of Streptomyces hygroscopicus is shown in FIG.

  Further, a cosmid library of Streptomyces glyceochromogenes genomic DNA was hybridized with the 1.2 Kb aveC probe derived from Streptomyces avermitilis described above. Several strongly cosmid clones were identified. Chromosomal DNA was isolated from these cosmids and a 5.4 Kb PstI fragment hybridized with the aveC probe was identified. The DNA was sequenced and a partial ORF of the aveC homolog with significant homology to the aveC-ORF of Streptomyces avermitilis was identified. The deduced partial amino acid sequence (SEQ ID NO: 5 in the sequence listing) is shown in FIG.

  DNA and amino acid sequence analysis of aveC homologues from Streptomyces hygroscopicus and Streptomyces griseochromogenes shows that these regions are both relative to each other and to the aveC-ORF and AveC gene products of Streptomyces avermitilis Share significant homology (˜50% sequence identity at the amino acid level) (FIG. 6).

11. Example: Construction of a plasmid using the aveC gene after the ermE promoter A 1.2 Kb aveC-ORF from pSE186 was shuttled with a 300 bp ermE promoter inserted as a KpnI / BamHI fragment in the KpnI / BamHI site of pWHM3. Subcloned into the vector pWHM3, pSE34 [Ward et al., 1986, Mol. Gen. Genet. , 203: 468-478]. pSE186 was digested with BamHI and HindIII, the digestion products were separated by electrophoresis, and the 1.2 Kb fragment was separated from the agarose gel and ligated with pSE34 that had been digested with BamHI and HindIII. This ligation mixture was transformed into competent E. coli DH5α cells according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of a 1.2 Kb insert was confirmed by restriction analysis. This plasmid was designated pSE189, transformed into E. coli DM1, and plasmid DNA was isolated from ampicillin resistant transformants. Protoplasts of Streptomyces avermitilis strain 1100-SC38 were transformed with pSE189. A thiostrepton resistant transformant of strain 1100-SC38 was isolated and analyzed by HPLC analysis of the fermentation product.

  A transformant of Streptomyces avermitilis strain 1100-SC38 containing pSE189 is produced in strain 1100-SC38 (approximately 34: 1) in the ratio of avermectin cyclohexyl-B2: avermectin cyclohexyl-B1 produced (approximately 3: 1). ) And the total production of avermectin increased about 2.4-fold compared to the strain 1100-SC38 transformed with pSE119 (Table 5).

  pSE189 was similarly transformed into protoplasts of wild-type Streptomyces avermitilis strains. Thiostrepton resistant transformants were isolated and analyzed by HPLC analysis of the fermentation product. The total amount of avermectin produced by wild-type Streptomyces avermitilis transformed with pSE189 increased approximately 2.2-fold compared to wild-type Streptomyces avermitilis transformed with pSE119 (Table 5).

12 Example: A chimeric plasmid containing sequences derived from both the aveC-ORF of Streptomyces avermitilis and the aveC homologue of Streptomyces hygroscopicus The 564 bp homology portion of the aveC-ORF of Streptomyces avermitilis A hybrid plasmid designated as pSE350 (FIG. 7), which was replaced with the 564 bp portion of the aveC homologue, was constructed as follows. pSE350 was constructed using a BsaAI restriction site conserved in both sequences (aveC position 225) and a KpnI restriction site (aveC position 810) present in the aveC gene of Streptomyces avermitilis. The KpnI site was prepared by using the PCR conditions described in Section 7.1.10 above, the primer 5′-CTTCAGGTGTACGTGTTTCG-3 ′ (SEQ ID NO: 23 in the sequence listing) and the primer 5′-GAACTGGTACCATGGCCC-3 ′ (designated to the left) It was introduced into DNA of Streptomyces hygroscopicus by PCR using SEQ ID NO: 24 (supplied by Genosys Biotechnology). The PCR product was digested with BsaAI and KpnI, the fragments were separated by electrophoresis in a 1% agarose gel, and the 564 bp BsaAI / KpnI fragment was separated from the gel. pSE179 (described in Section 7.1.10 above) was digested with KpnI and HindIII, the fragments were separated by electrophoresis in a 1% agarose gel, and the ˜4.5 Kb fragment was separated from the gel. pSE179 was digested with HindIII and BsaAI, the fragments were separated by electrophoresis in a 1% agarose gel, and the ˜0.2 Kb BsaAI / HindIII fragment was separated from the gel. A ~ 4.5 Kb HindIII / KpnI fragment, a ~ 0.2 Kb BsaAI / HindIII fragment and a 564 bp BsaAI / KpnI fragment from Streptomyces hygroscopicus were ligated together in three ways and the ligation mixture was competed. Transformation into E. coli DH5α cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis using KpnI and AvaI. This plasmid was digested with HindIII and XbaI to release a 1.2 Kb insert, which was then ligated with pWHM3 that had been digested with HindIII and XbaI. This ligation mixture was transformed into competent E. coli DH5α cells, plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis using HindIII and AvaI. This plasmid DNA was transformed into E. coli DM1, the 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 was designated as pSE350 and was used to transform a protoplast of Streptomyces avermitilis strain SE180-11. Transformants thiostrepton resistant strains SE180-11 were isolated, the presence of erythromycin resistance was determined, and the Thio ^r Erm ^r transformants were analyzed by HPLC analysis of fermentation products. The results show that transformants containing the Streptomyces avermitilis / Streptomyces hygroscopicus hybrid plasmid have an average B2: B1 ratio of about 109: 1.

Deposit of Biological Materials The following biological materials are available from the American Type Culture Collection, US, 20852, Rockville, Maryland 12301, Parklawn Drive. Collection) (ATCC) was deposited on January 29, 1998 and was given the following deposit number.

Plasmid Accession Number Plasmid pSE180 209605
Plasmid pSE186 209604

All patents, patent applications and publications mentioned above are hereby incorporated by reference in their entirety.
The present invention is not intended to limit the scope of the invention by the specific embodiments described herein, which are provided as a sole explanation of the individual aspects of the invention. Yes, functionally equivalent methods and elements are within the scope of the invention. Indeed, it will be apparent to those skilled in the art from the description herein and accompanying drawings that there are various aspects of the present invention in addition to the aspects shown and described herein. Such embodiments are intended to be encompassed within the scope of the claims.

DNA sequence (SEQ ID NO: 1 in the sequence listing) containing Streptomyces avermitilis aveC-ORF and deduced amino acid sequence (SEQ ID NO: 2 in the sequence listing). Plasmid vector pSE186 (ATCC 209604) containing the complete ORF of the aveC gene of Streptomyces avermitilis. A gene replacement vector pSE186 (ATCC 209605) containing the ermE gene of Saccharopolyspora erythraea inserted into the aveC-ORF of Streptomyces avermitilis. BamHI restriction map of the avermectin polyketide synthase gene cluster from Streptomyces avermitilis and five overlapping cosmid clones identified (ie, pSE65, pSE66, pSE67, pSE68, pSE69). The relationship between pSE118 and pSE119 is also shown. HPLC analysis of fermentation products produced by Streptomyces avermitilis strains. Peak quantification was performed by comparison with a standard amount of cyclohexyl B1. The retention time of cyclohexyl B2 is 7.4 to 7.7 minutes; the retention time of cyclohexyl B1 is 11.9 to 12.3 minutes. Streptomyces avermitilis strain SE180-11 having an inactive aveC-ORF. HPLC analysis of fermentation products produced by Streptomyces avermitilis strains. Peak quantification was performed by comparison with a standard amount of cyclohexyl B1. The retention time of cyclohexyl B2 is 7.4 to 7.7 minutes; the retention time of cyclohexyl B1 is 11.9 to 12.3 minutes. Streptomyces avermitilis strain SE180-11 transformed with pSE186 (ATCC 209604). HPLC analysis of fermentation products produced by Streptomyces avermitilis strains. Peak quantification was performed by comparison with a standard amount of cyclohexyl B1. The retention time of cyclohexyl B2 is 7.4 to 7.7 minutes; the retention time of cyclohexyl B1 is 11.9 to 12.3 minutes. Streptomyces avermitilis strain SE180-11 transformed with pSE187. HPLC analysis of fermentation products produced by Streptomyces avermitilis strains. Peak quantification was performed by comparison with a standard amount of cyclohexyl B1. The retention time of cyclohexyl B2 is 7.4 to 7.7 minutes; the retention time of cyclohexyl B1 is 11.9 to 12.3 minutes. Streptomyces avermitilis strain SE180-11 transformed with pSE188. A deduced amino acid sequence encoded by the aveC-ORF (SEC ID NO: 2) of Streptomyces avermitilis and a deduced amino acid sequence encoded by the aveC homologous partial ORF derived from Streptomyces griseochromogenes Comparison of (SEQ ID NO: 5 in the Sequence Listing) with the deduced amino acid sequence (SEQ ID NO: 4 in the Sequence Listing) encoded by the aveC homologous ORF derived from Streptomyces hygroscopicus. The bold valine residue is the putative start site for the protein. Conserved residues are shown in capital letters if all three sequences are homologous and in lower case if two of the three sequences are homologous. The amino acid sequence contains about 50% sequence identity. A hybrid plasmid construct comprising a 564 bp BsaAl / Kpnl fragment from the Streptomyces hygroscopicus aveC homologous gene inserted into the BsaAl / Kpnl site in Streptomyces avermitilis aveC-ORF.

Claims (9)

An isolated polynucleotide molecule comprising the base sequence of any one of the following (a) to (d):
(A) Hybridization under high stringency conditions with the base sequence of the complete aveC open reading frame (ORF) of Streptomyces hygroscopicus consisting of the base sequence of SEQ ID NO: 3 in the sequence listing (b) (a) And the aveC gene encoded by the nucleotide sequence of the nucleotide sequence (d) (a) encoding the polypeptide of the aveC gene product encoded by the nucleotide sequence of the nucleotide sequence (c) (a) encoding the polypeptide having aveC activity A nucleotide sequence encoding a polypeptide in which one or several amino acids have been deleted, substituted or added in the product polypeptide, and encoding a polypeptide having aveC activity A probe or amplification for detecting a polynucleotide according to claim 1, which is an oligonucleotide molecule that hybridizes under highly stringent conditions to the following polynucleotide (a) or (b): Oligonucleotide molecule that functions as a primer for
(A) a polynucleotide comprising the base sequence of SEQ ID NO: 3 in the sequence listing (b) a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 3 of the sequence listing The oligonucleotide molecule according to claim 2, which is complementary to the following (a) or (b).
(A) a polynucleotide comprising the base sequence of SEQ ID NO: 3 in the sequence listing (b) a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 3 of the sequence listing A recombinant vector comprising the polynucleotide molecule of claim 1. The recombinant vector according to claim 4, further comprising a base sequence encoding a selectable marker. A host cell comprising the recombinant vector according to claim 4. The host cell according to claim 6, which is Streptomyces hygroscopicus. The polypeptide of any of the following (a) to (c).
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 in the sequence listing (b) consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the polypeptide of (a), and A polypeptide encoded by a nucleotide sequence that hybridizes under high stringency conditions with a nucleotide sequence encoding the polypeptide (c) (a) or (b) having aveC activity, and aveC Polypeptide having activity A method for producing a recombinant AveC gene product, comprising culturing host cells transformed with a recombinant expression vector under conditions that promote the production of a recombinant AveC gene product, and recovering the AveC gene product from the cell culture. A production method, wherein the recombinant expression vector is the recombinant vector according to claim 4.

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