OA11449A

OA11449A - Streptomyces avermitilis gene directing the ratio of B2:B1 avermectins.

Info

Publication number: OA11449A
Application number: OA1200000217A
Authority: OA
Inventors: Kim Jonelle Stutzman-Engwall; Yoshihiro Katoh; Hamish Alastair Irvin Mcarthur
Original assignee: Pfizer Prod Inc
Priority date: 1998-02-13
Filing date: 2000-08-04
Publication date: 2003-12-03
Also published as: HUP0100784A2; CA2521289A1; TW585910B; KR20010052166A; DZ2720A1; MA24760A1; NZ505676A; CO5070698A1; EP1053335A1; EA200000758A1; IS5567A; PL342468A1; NZ518657A; KR20040053390A; NO20004044D0; AU752343B2; PA8467601A1; WO1999041389A1; ZA991138B; AU752343C

Abstract

The present invention relates to polynucleotide molecules comprising nucleotide sequences encoding an aveC gene product, which polynucleotide molecules can be used to alter the ratio or amount of class 2:1 avermectins produced in fermentation cultures of S. avermitilis. The present invention further relates to vectors, host cells, and mutant strains of S. avermitilis in which the aveC gene has been inactivated, or mutated so as to change the ratio or amount of class 2:1 avermectins produced.

Description

011442

STREPTOMYCES AVERMITIUS GENE

DIRECTING THE RATIO OF B2.B1 AVERMECTINS

1. FIELD OF THE INVENTION

The présent invention is directed to compositions and methods for producingavermectins, and is primarily in the field of animal health. More particularly, the présentinvention relates to polynucleotide molécules comprising nucléotide sequences encoding anAveC gene product, which can be used to modulate the ratio of class 2:1 avermectinsproduced by fermentation of cultures of Streptomyces avermitilis, and to compositions andmethods for screening for such polynucleotide molécules. The présent invention furtherrelates to vectors, transformed host cells, and novel mutant strains of S. avermitilis in whichthe avec gene has been mutated so as to modulate the ratio of class 2:1 avermectinsproduced.

2. BACKGROUND OF THE INVENTION 2.1. Avermectins

Streptomyces species produce a wide variety of secondary métabolites, including theavermectins which comprise a sériés of eight related sixteen-membered macrocyclic lactoneshaving potent anthelmintic and insecticidal activity. The eight distinct but closely relatedcompounds are referred to as A1a, A1b, A2a, A2b, B1a, 81b, B2a and B2b. The "a" sériés ofcompounds refers te the natural avermectin where the substituent at the C25 position is (S)-sec-butyl, and the "b" sériés refers to those compounds where the substituent at the C25position is isopropyl. The désignations "A" and "B" refer to avermectins where the substituentat the C5 position is methoxy and hydroxy, respectively. The numéral "1" refers toavermectins where a double bond is présent at the C22.23 position, and the numéral "2"refers to avermectins having a hydrogen at the C22 position and a hydroxy at the C23position. Among the related avermectins, the B1 type of avermectin is recognized as havingthe most effective antiparasitic and pesticidal activity, and is therefore the most commerciallydésirable avermectin.

The avermectins and their production by aérobic fermentation of strains of S.avermitilis are described in United States Patents 4,310,519 and 4,429,042. The biosynthesisof natural avermectins is believed to be initiated endogenously from the CoA thioester analogsof isobutyric acid and S-(+)-2-methyl butyric acid. A combination of both strain improvement through random mutagenesis and the useof exogenously supplied fatty acids has led to the efficient production of avermectin analogs.Mutants of S. avermitilis that are déficient in branched-chain 2-oxo acid dehydrogenase (bkddéficient mutants) can only produce avermectins when fermentations are supplemented with 011449 fatty acids. Screening and isolation of mutants déficient in branched-chain dehydrogenaseactivity (e.g., S. avermitilis, ATCC 53567) are described in European Patent (EP) 276103Fermentation of such mutants in the presence of exogenously supplied fatty acids results inproduction of only the four avermectins corresponding to the fatty acid employed. Thus, 5 supplementing fermentations of S. avermitilis (ATCC 53567) with S-(+)-2-methylbutyric acidresults in production of the natural avermectins A1a, A2a, B1a and B2a; supplementingfermentations with isobutyric acid results in production of the natural avermectins A1b, A2b,B1b, and B2b; and supplementing fermentations with cyclopentanecarboxylic acid results inthe production of the four novel cyclopentylavermectins A1, A2, B1, and B2. 10 If supplemented with other fatty acids, novel avermectins are produced By screening over 800 potential precursors, more than 60 other novel avermectins hâve been identified(See, e.g., Dutton et al., 1991, J. Antibiot. 44:357-365; and Banks ef al., 1994, Roy. Soc.Chem. 147:16-26). In addition, mutants of S. avermitilis déficient in 5-O-methyltransferaseactivity produce essentially only the B analog avermectins. Consequently, S. avermitilis 15 mutants lacking both branched-chain 2-oxo acid dehydrogenase and 5-O-methyltransferaseactivity produce only the B avermectins corresponding to the fatty acid employed tosupplément the fermentation. Thus, supplementing such double mutants with S-(+)-2-methylbutyric acid results in production of only the natural avermectins B1a and B2a, whilesupplementing with isobutyric acid or cyclopentanecarboxylic acid results in production of the 20 natural avermectins B1b and B2b or the novel cyclopentyl B1 and B2 avermectins,respectively. Supplémentation of the double mutant strain with cyclohexane carboxylic acid isa preferred method for producing the commercially important novel avermectin,cyclohexylavermectin B1 (doramectin). The isolation and characteristics of such doublemutants, e.g., S. avermitilis (ATCC 53692), is described in EP 276103. 25 2.2. Genes Involved In Avermectin Biosynthesis

In many cases, genes involved in production of secondary métabolites and genes encoding a particular antibiotic are found clustered together on the chromosome. Such is thecase, e.g., with the Streptomyces polyketide synthase gene cluster (PKS) (see Hopwood andSherman, 1990, Ann. Rev. Genet. 24:37-66). Thus, one strategy for cloning genes in a 30 biosynthetic pathway has been to isolate a drug résistance gene and then test adjacentrégions of the chromosome for other genes related to the biosynthesis of that particularantibiotic. Another strategy for cloning genes involved in the biosynthesis of importantmétabolites has been complémentation of mutants. For example, portions of a DNA libraryfrom an organism capable of producing a particular métabolite are introduced into a non- 2 Οι 1449 producing mutant and transformants screened for production of the métabolite. Additionally,hybridization of a library using probes derived from other Streptomyces species has beenused to identify and clone genes in biosynthetic pathways.

Genes involved in avermectin biosynthesis (ave genes), like the genes required forbiosynthesis of other Streptomyces secondary métabolites (e.g., PKS), are found clustered onthe chromosome. A number of ave genes hâve been successfully cloned using vectors tocomplément S. avermitilis mutants blocked in avermectin biosynthesis. The cloning of suchgenes is described in U.S. Patent 5,252,474. In addition, Ikeda et al., 1995, J. Antibiot.48:532-534, describes the localization of a chromosomal région involving the C22.23déhydration step (aveC) to a 4.82 Kb SamHI fragment of S. avermitilis, as well as mutations inthe aveC gene that resuit in the production of a single component B2a producer. Sinceivermectin, a potent anthelmintic compound, can be produced chemically from avermectinB2a, such a single component producer of avermectin B2a is considered particularly usefulfor commercial production of ivermectin.

Identification of mutations in the aveC gene that minimize the complexity ofavermectin production, such as, e.g., mutations that decrease the B2.B1 ratio of avermectins,would simplify production and purification of commercially important avermectins.

3. SUMMARY OF THE INVENTION

The présent invention provides an isolated polynucleotide molécule comprising thecomplété aveC ORF of S. avermitilis or a substantial portion thereof, which isolatedpolynucleotide molécule lacks the next complété ORF that is located downstream from theaveC ORF in situ in the S. avermitilis chromosome. The isolated polynucleotide molécule ofthe présent invention preferably comprises a nucléotide sequence that is the same as the S.avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or thatis the same as the nucléotide sequence of the aveC ORF of FIGURE 1 (SEQ ID NO:1) orsubstantial portion thereof.

The présent invention further provides a polynucleotide molécule having a nucléotidesequence that is homologous to the S. avermitilis AveC gene product-encoding sequence ofplasmid pSE186 (ATCC 209604), or to the nucléotide sequence of the aveC ORF presentedin FIGURE 1 (SEQ ID NO:1) or substantial portion thereof.

The présent invention further provides a polynucleotide molécule comprising anucléotide sequence that encodes a polypeptide having an amino acid sequence that ishomologous to the amino acid sequence encoded by the AveC gene product-encoding 3 sequence of plasmid pSE186 (ATCC 209604), or the amino acid sequence of FIGURE 1(SEQ ID NO:2) or substantial portion thereof.

The présent invention further provides an isolated polynucleotide molécule comprisinga nucléotide sequence encoding an AveC homolog gene product. In a preferred embodiment,

5 the isolated polynucleotide molécule comprises a nucléotide sequence encoding the AveChomolog gene product from S. hygroscopicus, which homolog gene product comprises theamino acid sequence of SEQ ID NO:4 or a substantial portion thereof. In a preferredembodiment, the isolated polynucleotide molécule of the présent invention that encodes the S.hygroscopicus AveC homolog gene product comprises the nucléotide sequence of SEQ ID 10 NO:3 or a substantial portion thereof.

The présent invention further provides a polynucleotide molécule comprising a nucieotide sequence that is homologous to the S. hygroscopicus nucléotide sequence of SEQID NO:3. The présent invention further provides a polynucleotide molécule comprising anucieotide sequence that encodes a polypeptide that is homologous to the S. hygroscopicus 15 AveC homolog gene product having the amino acid sequence of SEQ ID NO:4.

The présent invention further provides oligonucleotides that hybridize to a polynucleotide molécule having the nucieotide sequence of FIGURE 1 (SEQ ID NO:1) or SEQID NO.3, or to a polynucleotide molécule having a nucieotide sequence which is thecomplément of the nucieotide sequence of FIGURE 1 (SEQ ID NO.1) or SEQ ID NO:3. 20 The présent invention further provides recombinant cloning vectors and expression vectors, that are useful in cloning or expressing a polynucleotide of the présent invention,including polynucleotide molécules comprising the aveC ORF of S. avermitilis or an aveChomolog ORF. In a non-limiting embodiment, the présent invention provides plasmid pSE186(ATCC 209604), which comprises the entire ORF of the aveC gene of S. avermitilis. The 25 présent invention further provides transformed host cells comprising a polynucleotidemolécule or recombinant vector of the invention, and novel strains or cell lines derivedtherefrom.

The présent invention further provides a recombinantly expressed AveC gene productor AveC homolog gene product, or a substantial portion thereof, that has been substantially 30 purified or isolated, as well as homologs thereof. The présent invention further provides amethod for producing a recombinant AveC gene product, comprising culturing a host celltransformed with a recombinant expression vector, said recombinant expression vectorcomprising a polynucleotide molécule having a nucieotide sequence encoding an AveC geneproduct or AveC homolog gene product, which polynucleotide molécule is in operative 4 011449 association with one or more reguiatory éléments that control expression of the polynucleotidemolécule in the host cell, under conditions conducive to the production of the recombinantAveC gene product or AveC homolog gene product, and recovering the AveC gene product orAveC homolog gene product from the cell culture. 5 The présent invention further provides a polynucleotide molécule comprising a nucléotide sequence that is otherwise the same as the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604) or the nucléotide sequence of theaveC ORF of S. avermitilis as presented in FIGURE 1 (SEQ ID NO:1), but that furthercomprises one or more mutations, so that cells of S. avermitilis strain ATCC 53692 in which 10 the wild-type aveC allele has been inactivated and that express the polynucleotide moléculecomprising the mutated nucléotide sequence produce a different ratio or amount ofavermectins than are produced by cells of S. avermitilis strain ATCC 53692 that insteadexpress only the wild-type aveC allele. According to the présent invention, suchpolynucleotide molécules can be used to produce novel strains of S. avermitilis that exhibit a 15 détectable change in avermectin production compared to the same strain that insteadexpresses only the wild-type aveC allele. In a preferred embodiment, such polynucleotidemolécules are useful to produce novel strains of S. avermitilis that produce avermectins in areduced class 2:1 ratio compared to that from the same strain that instead expresses only thewild-type aveC allele. In a further preferred embodiment, such polynucleotide molécules are 20 useful to produce novel strains of S. avermitilis that produce increased levels of avermectinscompared to the same strain that instead expresses only the wild-type aveC allele. In afurther preferred embodiment, such polynucleotide molécules are useful to produce novelstrains of S. avermitilis in which the aveC gene has been inactivated.

The présent invention provides methods for identifying mutations of the aveC ORF of 25 S. avermitilis capable of altering the ratio and/or amount of avermectins produced. In apreferred embodiment, the présent invention provides a method for identifying mutations ofthe aveC ORF capable of altering the class 2:1 ratio of avermectins produced, comprising: (a)determining the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis inwhich the native aveC allele has been inactivated, and into which a polynucleotide molécule 30 comprising a nucléotide sequence encoding a mutated AveC gene product has beenintroduced and is being expressed; (b) determining the class 2:1 ratio of avermectinsproduced by cells of the same strain of S. avermitilis as in step (a) but which instead expressonly an aveC allele having the nucléotide sequence of the ORF of FIGURE 1 (SEQ ID NO:1)or a nucléotide sequence that is homologous thereto; and (c) comparing the class 2:1 ratio of 5 υ ι i *-t *·· avermectins produced by the S. avermitilis cells of step (a) to the class 2:1 ratio ofavermectins produced by the S. avermitilis cells of step (b); such that if the class 2:1 ratio ofavermectins produced by the S. avermitilis cells of step (a) is different from the class 2:1 ratioof avermectins produced by the S. avermitilis cells of step (b), then a mutation of the aveCORF capable of altering the class 2:1 ratio of avermectins has been identified. In a preferredembodiment, the class 2:1 ratio of avermectins is reduced by the mutation.

In a further preferred embodiment, the présent invention provides a method foridentifying mutations of the aveC ORF or genetic constructs comprising the aveC ORFcapable of altering the amount of avermectins produced, comprising: (a) determining theamount of avermectins produced by cells of a strain of S. avermitilis in which the native aveCallele has been inactivated, and into which a polynucleotide molécule comprising a nucléotidesequence encoding a mutated AveC gene product or comprising a genetic constructcomprising a nucléotide sequence encoding an AveC gene product has been introduced andis being expressed; (b) determining the amount of avermectins produced by cells of the samestrain of S. avermitilis as in step (a) but which instead express only an aveC allele having thenucléotide sequence of the ORF of FIGURE 1 (SEQ ID NO:1) or a nucléotide sequence thatis homologous thereto; and (c) comparing the amount of avermectins produced by the S.avermitilis cells of step (a) to the amount of avermectins produced by the S. avermitilis cells ofstep (b); such that if the amount of avermectins produced by the S. avermitilis cells of step (a)is different from the amount of avermectins produced by the S. avermitilis cells of step (b),then a mutation of the aveC ORF or a genetic construct capable of altering the amount ofavermectins has been identified. In a preferred embodiment, the amount of avermectinsproduced is increased by the mutation.

The présent invention further provides recombinant vectors that are useful for makingnovel strains of S. avermitilis having altered avermectin production. For example, the présentinvention provides vectors that can be used to target any of the polynucleotide moléculescomprising the mutated nucléotide sequences of the présent invention to the site of the aveCgene of the S. avermitilis chromosome to either insert into or replace the aveC ORF or aportion thereof by homologous recombination. According to the présent invention, however, apolynucleotide molécule comprising a mutated nucléotide sequence of the présent inventionprovided herewith can also function to modulate avermectin biosynthesis when inserted intothe S. avermitilis chromosome at a site other than at the aveC gene, or when maintainedepisomally in S. avermitilis cells. Thus, the présent invention also provides vectorscomprising a polynucleotide molécule comprising a mutated nucléotide sequence of the 011449 présent invention, which vectors can be used to insert the polynucleotide molécule at a site inthe S. avermitilis chromosome other than at the aveC gene, or to be maintained episomally.In a preferred embodiment, the présent invention provides gene replacement vectors that canbe used to insert a mutated aveC allele into the S. avermitilis chromosome to generate novel 5 strains of cells that produce avermectins in a reduced class 2.Ί ratio compared to the cells ofthe same strain which instead express only the wild-type aveC allele.

The présent invention further provides methods for making novel strains of S.avermitilis comprising cells that express a mutated aveC allele and that produce altered ratiosand/or amounts of avermectins compared to cells of the same strain of S. avermitilis that 10 instead express only the wild-type aveC allele. In a preferred embodiment, the présentinvention provides a method for making novel strains of S. avermitilis comprising cells thatexpress a mutated aveC allele and that produce an altered class 2:1 ratio of avermectinscompared to cells of the same strain of S. avermitilis that instead express only a wild-typeaveC allele, comprising transforming cells of a strain of S. avermitilis with a vector that carries

15 a mutated aveC allele that encodes a gene product that alters the class 2:1 ratio ofavermectins produced by cells of a strain of S. avermitilis expressing the mutated allelecompared to cells of the same strain that instead express only the wild-type aveC allele, andselecting transformed cells that produce avermectins in an altered class 2:1 ratio compared tothe class 2:1 ratio produced by cells of the strain that instead express the wild-type aveC 20 allele. In a preferred embodiment, the class 2:1 ratio of avermectins produced is reduced incells of the novel strain.

In a further preferred embodiment, the présent invention provides a method formaking novel strains of S. avermitilis comprising cells that produce altered amounts ofavermectin, comprising transforming cells of a strain of S. avermitilis with a vector that carries 25 a mutated aveC allele or a genetic construct comprising the aveC allele, the expression ofwhich results in an altered amount of avermectins produced by cells of a strain of S.avermitilis expressing the mutated aveC allele or genetic construct as compared to cells of thesame strain that instead express only the wild-type aveC allele, and selecting transformedcells that produce avermectins in an altered amount compared to the amount of avermectins 30 produced by cells of the strain that instead express only the wild-type aveC allele. In apreferred embodiment, the amount of avermectins produced is increased in cells of the novelstrain.

In a further preferred embodiment, the présent invention provides a method for making novel strains of S. avermitilis, the cells of which comprise an inactivated aveC allele. 7 comprising transforming cells of a strain of S. avermitilis that express a wild-type aveC allelewith a vector that inactivâtes the aveC allele, and selecting transformed cells in which theaveC allele has been inactivated.

The présent invention further provides novel strains of S. avermitilis comprising cells 5 that hâve been transformed with any of the polynucleotide molécules or vectors comprising amutated nucléotide sequence of the présent invention. In a preferred embodiment, theprésent invention provides novel strains of S. avermitilis comprising cells which express amutated aveC allele in place of, or in addition to, the wild-type aveC allele, wherein the cells ofthe novel strain produce avermectins in an altered class 2:1 ratio compared to cells of the 10 same strain that instead express only the wild-type aveC allele. In a more preferredembodiment, the cells of the novel strain produce avermectins in a reduced class 2.Ί ratiocompared to cells of the same strain that instead express only the wild-type aveC allele. Suchnovel strains are useful in the large-scale production of commercially désirable avermectinssuch as doramectin. 15 In a further preferred embodiment, the présent invention provides novel strains of S. avermitilis comprising cells which express a mutated aveC allele, or a genetic constructcomprising the aveC allele, in place of, or in addition to, the wild-type aveC allele, whichrésulte in the production by the cells of an altered amount of avermectins compared to theamount of avermectins produced by cells of the same strain that instead express only the 20 wild-type aveC allele. In a prefemed embodiment, the novel cells produce an increasedamount of avermectins.

In a further preferred embodiment, the présent invention provides novel strains of S.avermitilis comprising cells in which the aveC gene has been inactivated. Such strains areuseful both for the different spectrum of avermectins that they produce compared to the wild- 25 type strain, and in complémentation screening assays as described herein, to déterminewhether targeted or random mutagenesis of the aveC gene affects avermectin production.

The présent invention further provides a process for producing avermectins,comprising culturing cells of a strain of S. avermitilis, which cells express a mutated aveCallele that encodes a gene product that alters the class 2:1 ratio of avermectins produced by 30 cells of a strain of S. avermitilis expressing the mutated aveC allele compared to cells of thesame strain which do not express the mutated aveC allele but instead express only the wild-type aveC allele, in culture media under conditions that permit or induce the production ofavermectins therefrom, and recovering said avermectins from the culture. In a preferredembodiment, the class 2:1 ratio of avermectins produced by cells expressing the mutation is G1 1 4 4 9 reduced. This process provides increased efficiency in the production of commerciallyvaluable avermectins such as doramectin.

The présent invention further provides a process for producing avermectins,comprising culturing cells of a strain of S. avermitilis, which cells express a mutated aveCalleie or a genetic construct comprising an aveC allele that results in the production of analtered amount of avermectins produced by cells of a strain of S. avermitilis expressing themutated aveC allele or genetic construct compared to cells of the same strain which do notexpress the mutated aveC allele or genetic construct but instead express only the wild-typeaveC allele, in culture media under conditions that permit or induce the production ofavermectins therefrom, and recovering said avermectins from the culture. In a preferredembodiment, the amount of avermectins produced by cells expressing the mutation or geneticconstruct is increased.

The présent invention further provides a novel composition of avermectins producedby a strain of S. avermitilis expressing a mutated aveC allele of the présent invention, whereinthe avermectins are produced in a reduced class 2:1 ratio as compared to the class 2.Ί ratioof avermectins produced by cells of the same strain of S. avermitilis that do not express themutated aveC allele but instead express only the wild-type avec allele. The novel avermectincomposition can be présent as produced in fermentation culture fluid, or can be harvestedtherefrom, and can be partially or substantially purified therefrom.

4. BRIEF DESCRIPTION OF THE FIGURES FIGURE 1. DNA sequence (SEQ ID NO:1) comprising the S. avermitilis aveC ORF,and deduced amino acid sequence (SEQ ID NO:2). FIGURE 2. Plasmid vector pSE186 (ATCC 209604) comprising the entire ORF of theaveC gene of S. avermitilis. FIGURE 3. Gene replacement vector pSE180 (ATCC 209605) comprising the ermEgene of Sacc. erythraea inserted into the aveC ORF of S. avermitilis. FIGURE 4. SamHI restriction map of the avermectin polyketide synthase genecluster from S. avermitilis with five overlapping cosmid clones identifîed (i.e., pSE65, pSE66,pSE67, pSE68, pSE69). The relationship of pSE118 and pSE119 is also indicated. FIGURE 5. HPLC analysis of fermentation products produced by S. avermitilisstrains. Peak quantitation was performed by comparison to standard quantities of cyclohexylB1. Cyclohexyl B2 rétention time was 7Α-Ί1 min; cyclohexyl B1 rétention time was 11.9-12.3min. FIG. 5A. S. avermitilis strain SE180.-11 with an inactivated aveC ORF. FIG. 5B. S.avermitilis strain SE180-11 transformed with pSE186 (ATCC 209604). FIG. 5C. S. avermitilis 9 011449 strain SE18O-11 transformed with pSE187. FIG 5D. S. avermitilis strain SE180-11transformed with pSE188. FIGURE 6. Comparison of deduced amino acid sequences encoded by the aveCORF of S. avermitilis (SEQ ID NO:2), an aveC homolog partial ORF from S. 5 gnseochromogenes (SEQ ID NO.5), and the aveC homolog ORF from S. hygroscopicus(SEQ ID NO:4). The valine residue in bold is the putative start site for the protein. Conservedresidues are shown in capital letters for homology in ail three sequences and in lower caseletters for homology in 2 of the 3 sequences. The amino acid sequences containapproximately 50% sequence identity. 10 FIGURE 7. Hybrid plasmid construct containing a 564 bp BsaMIKpnl fragment from the S. hygroscopicus aveC homolog gene inserted into the BsaAl/Kpnl site in the S.avermitilis aveC ORF.

5. DETAILED DESCRIPTION OF THE INVENTION

The présent invention relates to the identification and characterization of 15 polynucleotide molécules having nucléotide sequences that encode the AveC gene productfrorr Streptomyces avermitilis, the construction of novel strains of S. avermitilis that can beused to screen mutated AveC gene products for their effect on avermectin production, and thediscovery that certain mutated AveC gene products can reduce the ratio of B2.B1 avermectinsproduced by S. avermitilis. By way of example, the invention is described in the sections 20 below for a polynucleotide molécule having either a nucléotide sequence that is the same asthe S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604),or the nucléotide sequence of the ORF of FIGURE 1 (SEQ ID NO:1), and for polynucleotidesmolécules having mutated nucléotide sequences derived therefrom. However, the principlesset forth in the présent invention can be analogously applied to other polynucleotide 25 molécules, including aveC homolog genes from other Streptomyces species including, e.g., S. hygroscopicus and S. griseochromogenes, among others. 5.1. Polynucleotide Molécules EncodingThe S. avermitilis AveC Gene Product

The présent invention provides an isolated polynucleotide molécule comprising the 30 complété aveC ORF of S. avermitilis or a substantial portion thereof, which isolatedpolynucleotide molécule lacks the next complété ORF that is located downstream from theaveC ORF in situ in the S. avermitilis chromosome.

The isolated polynucleotide molécule of the présent invention preferably comprises anucléotide sequence that is the same as the S. avermitilis AveC gene product-encoding 10 sequence of plasmid pSE186 (ATCC 209604), or that is the same as the nucléotide sequenceof the ORF of FIGURE 1 (SEQ ID NO:1) or substantial portion thereof. As used herein, a“substantial portion" of an isolated polynucleotide molécule comprising a nucléotide sequenceencoding the S. avermiiilis AveC gene product means an isolated polynucleotide moléculecomprising at least about 70% of the complété aveC ORF sequence shown in FIGURE 1(SEQ ID NO:1), and that encodes a functionally équivalent AveC gene product. In this regard,a “functionally équivalent' AveC gene product is defined as a gene product that, whenexpressed in S. avermitilis strain ATCC 53692 in which the native aveC allele has beeninactivated, results in the production of substantially the same ratio and amount ofavermectins as produced by S. avermitilis strain ATCC 53692 which instead expresses onlythe wild-type, functional aveC allele native to S. avermitilis strain ATCC 53692.

In addition to the nucléotide sequence of the aveC ORF, the isolated polynucleotidemolécule of the présent invention can further comprise nucléotide sequences that naturallyflank the aveC gene in situ in S. avermitilis, such as those flanking nucléotide sequencesshown in FIGURE 1 (SEQ ID NO:1).

As used herein, the terms “polynucleotide molécule,” “polynucleotide sequence,”“coding sequence,” “open-reading frame," and "ORF'' are intended to refer to both DNA andRNA molécules, which can either be single-stranded or double-stranded, and that can betranscribed and translated (DNA), or translated (RNA), into an AveC gene product or, asdescribed below, into an AveC homolog gene product, or into a polypeptide that ishomologous to an AveC gene product or AveC homolog gene product in an appropriate hostcell expression System when placed under the control of appropriate regulatory éléments. Acoding sequence can include but is not limited to prokaryotic sequences, cDNA sequences,genomic DNA sequences, and chemically synthesized DNA and RNA sequences.

The nucléotide sequence shown in FIGURE 1 (SEQ ID NO:1) comprises four differentGTG codons at bp positions 42, 174, 177 and 180. As described in Section 9 below, multipledélétions of the 5' région of the aveC ORF (FIGURE 1; SEQ ID NO:1) were constructed tohelp define which of these codons could function in the aveC ORF as start sites for proteinexpression. Délétion of the first GTG site at bp 42 did not eliminate AveC activity. Additionaldélétion of ail of the GTG codons at bp positions 174, 177 and 180 together eliminated AveCactivity, indicating that this région is necessary for protein expression. The présent inventionthus encompasses variable length aveC ORFs that initiate translation at any of the GTG siteslocated at bp 174, 177 or 180 bp, as presented in FIGURE 1 (SEQ ID NO:1), andcorresponding polypeptides for each. 11

The présent invention further provides a polynucleotide molécule having a nucléotidesequence that is homologous to the S. avermitiiis AveC gene product-encoding sequence ofplasmid pSE186 (ATCC 209604), or to the nucléotide sequence of the aveC ORF presentedin FIGURE 1 (SEQ ID NO:1) or substantiel portion thereof. The term "homologous” whenused to refer to a polynucleotide molécule that is homologous to an S. avermitiiis AveC geneproduct-encoding sequence means a polynucleotide molécule having a nucléotide sequence:(a) that encodes the same AveC gene product as the S. avermitiiis fweC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or that encodes the same AveC geneproduct as the nucléotide sequence of the aveC ORF presented in FIGURE 1 (SEQ ID NO:1 ),but that includes one or more silent changes to the nucléotide sequence according to thedegeneracy of the genetic code; or (b) that hybridizes to the complément of a polynucleotidemolécule having a nucléotide sequence that encodes the amino acid sequence encoded bythe AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or thatencodes the amino acid sequence shown in FIGURE 1 (SEQ ID NO:2), under moderatelystringent conditions, i.e., hybridization to fiiter-bound DNA in 0.5 M NaHPO4, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.2xSSC/0.1% SDS at 42°C (seeAusubel ef al. (eds,), 1989, Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3), and encodes afunctionally équivalent AveC gene product as defined above. In a preferred embodiment, thehomologous polynucleotide molécule hybridizes to the complément of the AveC gene product-encoding nucléotide sequence of plasmid pSE186 (ATCC 209604), or to the complément ofthe nucléotide sequence shown in FIGURE 1 (SEQ ID NO:1) or substantial portion thereof,under highly stringent conditions, i.e., hybridization to fiiter-bound DNA in 0.5 M NaHPO4, 7%sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.1xSSC/0.1% SDS at68°C (Ausubel étal., 1989, above), and encodes a functionally équivalent AveC gene productas defined above.

The activity of an AveC gene product and potential functional équivalents thereof canbe determined through HPLC analysis of fermentation products, as described in the examplesbelow. Polynucleotide molécules having nucléotide sequences that encode functionaléquivalents of the S. avermitiiis AveC gene product include naturally occurring aveC genesprésent in other strains of S. avermitiiis, aveC homolog genes présent in other species ofStreptomyces, and mutated aveC alleles, whether naturally occurring or engineered.

The présent invention further provides a polynucleotide molécule comprising anucléotide sequence that encodes a polypeptide having an amino acid sequence that is 12 611449 homologous to the amino acid sequence encoded by the AveC gene product-encodingsequence of plasmid pSE186 (ATCC 209604), or the amino acid sequence of FIGURE 1(SEQ ID NO:2) or substantial portion thereof. As used herein, a "substantial portion'' of theamino acid sequence of FIGURE 1 (SEQ ID NO:2) means a polypeptide comprising at least 5 about 70% of the amino acid sequence shown in FIGURE 1 (SEQ ID NO:2), and thatconstitutes a functionally équivalent AveC gene product, as defined above.

As used herein to refer to amino acid sequences that are homologous to the aminoacid sequence of an AveC gene product front S. avermitilis, the term “homologous" refers to apolypeptide encoded by the AveC gene product-encoding sequence of plasmid pSE186 10 (ATCC 209604), or having the amino acid sequence of FIGURE 1 (SEQ ID NO:2) but in whichone or more amino acid residues has been conservatively substituted with a different aminoacid residue, where such conservative substitution results in a functionally équivalent geneproduct, as defined above. Conservative amino acid substitutions are well-known in the art.Rules for making such substitutions include those described by Dayhof, M.D., 1978, Nat. 15 Biomed. Res. Found., Washington, D.C., Vol. 5, Sup. 3, among others. More specifically,conservative amino acid substitutions are those that generally take place within a family ofamino acids that are related in the acidity, polarity, or bulkiness of their side chains.Genetically encoded amino acids are generally divided into four groups: (1) acidic =aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) non-polar = alanine, valine, 20 leucine, isoleucine, proline, phenylalanine, méthionine, tryptophan; and (4) uncharged polar =glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine,tryptophan and tyrosine are also jointly classified as aromatic amino acids. One or morereplacements within any particular group, e.g., of a leucine with an isoleucine or valine, or ofan aspartate with a glutamate, or of a threonine with a serine, or of any other amino acid 25 residue with a structurally related amino acid residue, e.g., an amino acid residue with similaracidity, polarity, bulkiness, or with similarity in some combination thereof, will generally hâvean insignificant effect on the fonction of the polypeptide.

The présent invention further provides an isolated polynucleotide molécule comprisinga nucléotide sequence encoding an AveC homolog gene product. As used herein, an “AveC

30 homolog gene product” is defined as a gene product having at least about 50% amino acidsequence identity to an AveC gene product of S. avermitilis comprising the amino acidsequence encoded by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC209604), or the amino acid sequence shown in FIGURE 1 (SEQ ID NO.2). In a non-limitingembodiment the AveC homolog gene product is from S. hygroscopicus, (described in EP 13 U11449 application 0298423; deposit FERM BP-1901) and comprises the amino acid sequence ofSEQ ID NO:4, or a substantial portion thereof. A “substantial portion" of the amino acidsequence of SEQ ID NO:4 means a polypeptide comprising at least about 70% of the aminoacid sequence of SEQ ID NO:4, and that constitutes a functionally équivalent AveC homologgene product. A "functionally équivalent" AveC homolog gene product is defined as a geneproduct that, when expressed in S. hygroscopicus strain FERM BP-1901 in which the nativeavec homolog allele has been inactivated, results in the production of substantially the sameratio and amount of milbemycins as produced by S. hygroscopicus strain FERM BP-1901expressing instead only the wild-type, functional avec homolog allele native to S,hygroscopicus strain FERM BP-1901. In a non-limiting embodiment, the isolatedpolynucleotide molécule of the présent invention that encodes the S. hygroscopicus AveChomolog gene product comprises the nucléotide sequence of SEQ ID NO;3 or a substantialportion thereof. In this regard, a “substantial portion” of the isolated polynucleotide moléculecomprising the nucléotide sequence of SEQ ID NO;3 means an isolated polynucleotidemolécule comprising at least 70% of the nucléotide sequence of SEQ ID NO.3, and thatencodes a functionally équivalent AveC homolog gene product, as defined immediatelyabove.

The présent invention further provides a polynucleotide molécule comprising anucieotide sequence that is homologous to the S. hygroscopicus nucléotide sequence of SEQID NO:3. The term "homologous” when used to refer to a polynucleotide molécule comprisinga nucieotide sequence that is homologous to the S. hygroscopicus AveC homolog geneproduct-encoding sequence of SEQ ID NO:3 means a polynucleotide molécule having anucieotide sequence; (a) that encodes the same gene product as the nucieotide sequence ofSEQ ID NO:3, or a substantial portion thereof, but that includes one or more silent changes tothe nucieotide sequence according to the degeneracy of the genetic code; or (b) thathybridizes to the complément of a polynucleotide molécule having a nucieotide sequence thatencodes the amino acid sequence of SEQ ID NO:4, under moderately stringent conditions,i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1mM EDTA at 65°C, and washing in 0.2xSSC/0.1% SDS at 42°C (see Ausubel ef ai. above),and encodes a functionally équivalent AveC homolog gene product as defined above. In apreferred embodiment, the homologous polynucleotide molécule hybridizes to thecomplément of the AveC homolog gene product-encoding nucieotide sequence of SEQ IDNO;3 or substantial portion thereof, under highly stringent conditions, i.e., hybridization tofilter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, 14 C11449 and washing in 0.1xSSC/0.1% SDS at 68°C (Ausubel et al., 1989, above), and encodes afunctionally équivalent AveC homolog gene product as defined above.

The présent invention further provides a polynucleotide molécule comprising anucléotide sequence that encodes a polypeptide that is homologous to the S. hygroscopicusAveC homolog gene product. As used herein to refer to polypeptides that are homologous tothe AveC homolog gene product of SEQ ID NO:4 from S. hygroscopicus, the term“homologous" means a polypeptide having the amino acid sequence of SEQ ID NO:4, but inwhich one or more amino acid residues has been conservatively substituted with a differentamino acid residue, where such conservative substitution results in a functionally équivalentAveC homolog gene product, as defined above.

The présent invention further provides oligonucleotides that hybridize to apolynucleotide molécule having the nucléotide sequence of FIGURE 1 (SEQ ID NO:1) or SEQID NO:3, or to a polynucleotide molécule having a nucléotide sequence which is thecomplément of the nucléotide sequence of FIGURE 1 (SEQ ID NO:1) or SEQ ID NO:3. Sucholigonucleotides are at least about 10 nucléotides in length, and preferably from about 15 toabout 30 nucléotides in length, and hybridize to one of the aforementioned polynucleotidemolécules under highly stringent conditions, i.e., washing in 6xSSC/0.5% sodiumpyrophosphate at -37°C for -14-base oligos, at ~48°C for ~17-base oligos, at ~55°C for '20-base oligos, and at ~60°C for ~23-base oligos. In a preferred embodiment, theoligonucleotides are complementary to a portion of one of the aforementioned polynucleotidemolécules. These oligonucleotides are useful for a variety of purposes including to encode oract as antisense molécules useful in gene régulation, or as primers in amplification of aveC-or aveC homolog-encoding polynucleotide molécules.

Additional aveC homolog genes can be identified in other species or strains ofStreptomyces by using the polynucleotide molécules or oligonucleotides disclosed herein inconjunction with known techniques. For example, an oligonucleotide molécule comprising aportion of the S. avermitilis nucléotide sequence of FIGURE 1 (SEQ ID NO:1) or a portion ofthe S. hygroscopicus nucléotide sequence of SEQ ID NO.3 can be detectably labeled andused to screen a genomic library constructed from DNA derived from the organism of interest.The stringency of the hybridization conditions is selected based on the relationship of theréférencé organism, in this example S. avermitilis or S. hygroscopicus, to the organism ofinterest. Requirements for different stringency conditions are well known to those of skill inthe art, and such conditions will vary predictably depending on the spécifie organisme fromwhich the library and the labeled sequences are derived. Such oligonucleotides are 15 CI 1442 preferably at least about 15 nucléotides in length and include, e.g., those described in theexamples below. Amplification of homolog genes can be carried out using these and otheroligonucleotides by applying standard techniques such as the polymerase Chain reaction(PCR), although other amplification techniques known in the art, e.g., the ligase Chain 5 reaction, can also be used.

Clones identified as containing aveC homolog nucléotide sequences can be tested fortheir ability to encode a functional AveC homolog gene product. For this purpose, the clonescan be subjected to sequence analysis in order to identify a suitable reading frame, as well asinitiation and termination signais, Altematively or additionally, the cloned DNA sequence can 10 be inserted into an appropriate expression vector, i.e., a vector that contains the necessaryéléments for the transcription and translation of the inserted protein-coding sequence. Any ofa variety of host/vector Systems can be used as described below, including but not limited tobacterial Systems such as plasmid, bactériophage, or cosmid expression vectors. Appropriatehost cells transformed with such vectors comprising potential aveC homolog coding 15 sequences can then be analyzed for AveC-type activity using methods such as HPLCanalysis of fermentation products, as described, e.g., in Section 7, below.

Production and manipulation of the polynucleotide molécules disclosed herein arewithin the skill in the art and can be carried out according to recombinant techniquesdescribed, e.g., in Maniatis, et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring 20 Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel, et al., 1989, Current Protocols InMolecular Biology, Greene 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, Academie Press, Inc., SanDiego; and Erlich (ed), 1992, PCR Technology, Oxford University Press, New York, ail of 25 which are incorporated herein by référencé. Polynucleotide clones encoding AveC geneproducts or AveC homolog gene products can be identified using any method known in theart, including but not limited to the methods set forth in Section 7, below. Genomic DNAlibraries can be screened for aveC and aveC homolog coding sequences using techniquessuch as the methods set forth in Benton and Davis, 1977, Science 196:180, for bactériophage 30 libraries, and in Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. USA, 72:3961-3965, forplasmid libraries. Polynucleotide molécules having nucléotide sequences known to includethe aveC ORF, as présent, e.g., in plasmid pSE186 (ATCC 209604), or in plasmid pSE119(described in Section 7, below), can be used as probes in these screening experiments.Altematively, oligonucleotide probes can be synthesized that correspond to nucléotide 16 011449 sequences deduced from partial or complété amino acid sequences of the purified AveChomolog gene product. 5.2. Recombinant Systems5.2.1. Cloning And Expression Vectors 5 The présent invention further provides recombinant cloning vectors and expression vectors which are useful in cloning or expressing polynucleotide molécules of the présentinvention comprising, e.g., the aveC ORF of S. avermitilis or any aveC homolog ORFs. In anon-limiting embodiment, the présent invention provides plasmid pSE186 (ATCC 209604),which comprises the complété ORF of the aveC gene of S. avermitilis. 10 Ail of the following description regarding the aveC ORF from S. avermitilis, or a polynucleotide molécule comprising the aveC ORF from S. avermitilis or portion thereof, or anS. avermitilis AveC gene product, also refers to aveC homologs and AveC homolog geneProducts, unless indicated explicitly or by context. A variety of different vectors hâve been developed for spécifie use in Streptomyces, 15 including phage, high copy number plasmids, low copy number plasmids, and E.coli-Streptomyces shuttle vectors, among others, and any of these can be used to practice theprésent invention. A number of drug résistance genes hâve also been cloned fromStreptomyces, and several of these genes hâve been incorporated into vectors as selectablemarkers. Examples of current vectors for use in Streptomyces are presented, among other 20 places, in Hutchinson, 1980, Applied Biochem. Biotech. 16:169-190.

Recombinant vectors of the présent invention, particularly expression vectors, are preferably constructed so that the coding sequence for the polynucleotide molécule of theinvention is in operative association with one or more regulatory éléments necessary fortranscription and translation of the coding sequence to produce a polypeptide. As used 25 herein, the term “regulatory element’’ includes but is not limited to nucléotide sequences thatencode inducible and non-inducible promoters, enhancers, operators and other élémentsknown in the art that serve to drive and/or regulate expression of polynucleotide codingsequences. Also, as used herein, the coding sequence is in “operative association" with oneor more regulatory éléments where the regulatory éléments effectively regulate and allow for 30 the transcription of the coding sequence or the translation of its mRNA, or both.

Typical plasmid vectors that can be engineered to contain a polynucleotide molécule of the présent invention include pCR-Blunt, pCR2.1 (Invitrogen), pGEM3Zf (Promega), andthe shuttle vector pWHM3 (Vara et al., 1989, J. Bact. 171:5872-5881), among many others. 17 011449

Methods are well-known in the art for constructing recombinant vectors containingparticular coding sequences in operative association with appropriate regulatory éléments,and these can be used to practice the présent invention. These methods include in vitrorecombinant techniques, synthetic techniques, and in vivo genetic recombination. See, e.g., 5 the techniques described in Maniatis et al., 1989, above; Ausubel et al., 1989, above;Sambrook et al., 1989, above; Innis et al., 1995, above; and Erlich, 1992, above.

The regulatory éléments of these vectors can vary in their strength and specificities.Depending on the host/vector System utilized, any of a number of suitable transcription andtranslation éléments can be used. Non-limiting examples of transcriptional regulatory régions 10 or promoters for bacteria include the β-gal promoter, the T7 promoter, the TAC promoter, λleft and right promoters, trp and lac promoters, trp-lac fusion promoters and, more specificallyfor Streptomyces, the promoters ermE, melC, and tipA, etc. In a spécifie embodimentdescribed in Section 11 below, an expression vector was generated that contained the aveCORF cloned adjacent to the strong constitutive ermE promoter from Saccharopolyspora 15 erythraea. The vector was transformed into S. avermitilis, and subséquent HPLC analysis offermentation products indicated an increased titer of avermectins produced compared toproduction by the same strain but which instead expresses the wild-type aveC allele.

Fusion protein expression vectors can be used to express an AveC gene product-fusion protein. The purified fusion protein can be used to raise antisera against the AveC 20 gene product, to study the biochemical properties of the AveC gene product, to engineerAveC fusion proteins with different biochemical activities, or to aid in the identification orpurification of the expressed AveC gene product. Possible fusion protein expression vectorsinclude but are not limited to vectors incorporating sequences that encode β-galactosidaseand trpE fusions, maltose-binding protein fusions, glutathione-S-transferase fusions and 25 polyhistidine fusions (carrier régions). In an alternative embodiment, an AveC gene productor a portion thereof can be fused to an AveC homolog gene product, or portion thereof,derived from another species or strain of Streptomyces, such as, e.g., S. hygroscopicus or S.griseochromogenes. In a particular embodiment described in Section 12, below, and depictedin FIGURE 7, a chimeric plasmid was constructed that contains a 564 bp région of the S. 30 hygroscopicus aveC homolog ORF replacing a homologous 564 bp région of the S. avermitilisaveC ORF. Such hybrid vectors can be transformed into S. avermitilis cells and tested todétermine their effect, e.g., on the ratio of class 2:1 avermectin produced.

AveC fusion proteins can be engineered to comprise a région useful for purification.For example, AveC-maltose-binding protein fusions can be purified using amylose resin; 18 011449

AveC-glutathione-S-transferase fusion proteins can be purified using glutathione-agarosebeads; and AveC-polyhistidine fusions can be purified using divalent nickel resin.Altematively, antibodies against a carrier protein or peptide can be used for affinitychromatography purification of the fusion protein. For example, a nucléotide sequence codingfor the target epitope of a monoclonal antibody can be engineered into the expression vectorin operative association with the regulatory éléments and situated so that the expressedepitope is fused to the AveC polypeptide. For example, a nucléotide sequence coding for theFLAG™ epitope tag (International Biotechnologies Inc.), which is a hydrophilic markerpeptide, can be inserted by standard techniques into the expression vector at a pointcorresponding, e.g., to the carboxyl terminus of the AveC polypeptide. The expressed Avecpolypeptide-FLAG™ epitope fusion product can then be detected and affinity-purified usingcommercially available anti-FLAG™ antibodies.

The expression vector encoding the AveC fusion protein can also be engineered tocontain polylinker sequences that encode spécifie protease cleavage sites so that theexpressed AveC polypeptide can be released from the carrier région or fusion partner bytreatment with a spécifie protease. For example, the fusion protein vector can include DNAsequences encoding thrombin or factor Xa cleavage sites, among others. A signal sequence upstream from, and in reading frame with, the aveC ORF can beengineered into the expression vector by known methods to direct the trafficking and sécrétionof the expressed gene product. Non-limiting examples of signal sequences include thosefrom α-factor, immunoglobulins, outer membrane proteins, penicillinase, and T-cell receptors,among others.

To aid in the sélection of host cells transformed or transfected with cloning orexpression vectors of the présent invention, the vector can be engineered to further comprisea coding sequence for a reporter gene product or other selectable marker. Such a codingsequence is preferably in operative association with the regulatory element codingsequences, as described above. Reporter genes that are useful in the invention are well-known in the art and include those encoding green fluorescent protein, luciferase, xylE, andtyrosinase, among others. Nucléotide sequences encoding selectable markers are well-known in the art, and include those that encode gene products conferring résistance toantibiotics or anti-metabolites, or that supply an auxotrophic requirement. Examples of suchsequences include those that encode résistance to erythromycin, thiostrepton or kanamycin,among many others. 19 C11449 5.2.2. Transformation Of Host Celis

The présent invention further provides transformed host cells comprising apolynucleotide molécule or recombinant vector of the invention, and novel strains or cell linesderived therefrom. Host cells useful in the practice of the invention are preferably 5 Streptomyces cells, although other prokaryotic cells or eukaryotic cells can also be used.Such transformed host cells typically include but are not limited to microorganisms, such asbacteria transformed with recombinant bactériophage DNA, plasmid DNA or cosmid DNAvectors, or yeast transformed with recombinant vectors, among others.

The polynucleotide molécules of the présent invention are intended to function in 10 Streptomyces cells, but can also be transformed into other bacteria! or eukaryotic cells, e.g.,for cloning or expression purposes. A strain of E. coli can typically be used, such as, e.g.,the DH5a strain, available from the American Type Culture Collection (ATCC), Rockville, MD,USA (Accession No. 31343), and from commercial sources (Stratagene). Preferredeukaryotic host cells include yeast cells, although mammalian cells or insect cells can also be 15 utilized effectively.

The recombinant expression vector of the invention is preferably transformed ortransfected into one or more host cells of a substantially homogeneous culture of cells. Theexpression vector is generally introduced into host cells in accordance with known techniques,such as, e.g., by protoplast transformation, calcium phosphate précipitation, calcium chloride 20 treatment, microinjection, electroporation, transfection by contact with a recombined virus,liposome-mediated transfection, DEAE-dextran transfection, transduction, conjugation, ormicroprojectile bombardment. Sélection of transformants can be conducted by standardprocedures, such as by selecting for cells expressing a selectable marker, e.g., antibioticrésistance, associated with the recombinant vector, as described above. 25 Once the expression vector is introduced into the host cell, the intégration and maintenance of the aveC coding sequence either in the host cell chromosome or episomallycan be confirmed by standard techniques, e.g., by Southern hybridization analysis, restrictionenzyme analysis, PCR analysis, including reverse transcriptase PCR (rt-PCR), or byimmunological assay to detect the expected gene product. Host cells containing and/or 30 expressing the recombinant aveC coding sequence can be identified by any of at least fourgeneral approaches which are well-known in the art, including: (i) DNA-DNA, DNA-RNA, orRNA-antisense RNA hybridization; (ii) detecting the presence of “marker” gene fonctions; (iii)assessing the level of transcription as measured by the expression of aveC-specific mRNAtranscripts in the host cell; and (iv) detecting the presence of mature polypeptide product as 20 0114/19 measured, e.g., by immunoassay or by the presence of AveC biological activity (e.g., theproduction of spécifie ratios and amounts of avermectins indicative of AveC activity in, e.g., S.avermitilis host cells). 5.2.3. Expression And Characterization

Of A Recombinant AveC Gene Product

Once the aveC coding sequence has been stably introduced into an appropriate hostcell, the transformed host cell is clonally propagated, and the resulting cells can be grownunder conditions conducive to the maximum production of the AveC gene product. Suchconditions typically include growing cells to high density. Where the expression vectorcomprises an inducible promoter, appropriate induction conditions such as, e.g., températureshift, exhaustion of nutrients, addition of gratuitous inducers (e.g., analogs of carbohydrates,such as isopropyl-p-D-thiogalactopyranoside (IPTG)), accumulation of excess metabolic by-products, or the like, are employed as needed to induce expression.

Where the expressed AveC gene product is retained inside the host cells, the cellsare harvested and lysed, and the product isolated and purified from the lysate underextraction conditions known in the art to minimize protein dégradation such as, e.g., at 4°C, orin the presence of protease inhibitors, or both. Where the expressed AveC gene product issecreted from the host cells, the exhausted nutrient medium can simply be collected and theproduct isolated therefrom.

The expressed AveC gene product can be isolated or substantially purified from celllysâtes or culture medium, as appropriate, using standard methods, including but not limitedto any combination of the foliowing methods: ammonium sulfate précipitation, sizefractionation, ion exchange chromatography, HPLC, density centrifugation, and affinitychromatography. Where the expressed AveC gene product exhibits biological activity,increasing purity of the préparation can be monitored at each step of the purificationprocedure by use of an appropriate assay. Whether or not the expressed AveC gene productexhibits biological activity, it can be detected as based, e.g., on size, or reactivity with anantibody otherwise spécifie for AveC, or by the presence of a fusion tag.

The présent invention thus provides a recombinantly-expressed S. avermitilis AveCgene product comprising the amino acid sequence encoded by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or the amino acid sequence ofFIGURE 1 (SEQ ID NO:2) or a substantial portion thereof, and homologs thereof. 21 U I I s· y

The présent invention further provides a recombinantiy-expressed S. hygroscopicusAveC homolog gene product comprising the amino acid sequence of SEQ ID NO:4 or asubstantiel portion thereof, and homologs thereof.

The présent invention further provides a method for producing an AveC gene product, 5 comprising culturing a host cell transformed with a recombinant expression vector, said vectorcomprising a polynucleotide molécule having a nucléotide sequence encoding an AveC geneproduct, which polynucleotide molécule is in operative association with one or more regulatoryéléments that control expression of the polynucleotide molécule in the host cell, underconditions conducive to the production of the recombinant AveC gene product, and recovering 10 the AveC gene product from the cell culture.

The recombinantly expressed S. avermitilis AveC gene product is useful for a varietyof purposes, including for screening compounds that alter AveC gene product fonction andthereby modulate avermectin biosynthesis, and for raising antibodies directed against theAveC gene product. 15 Once an AveC gene product of sufficient purity has been obtained, it can be

characterized by standard methods, including by SDS-PAGE, size exclusion chromatography,amino acid sequence analysis, biological activity in producing appropriate products in theavermectin biosynthetic pathway, etc. For example, the amino acid sequence of the AveCgene product can be determined using standard peptide sequencing techniques. The AveC 20 gene product can be further characterized using hydrophilicity analysis (see, e.g., Hopp andWoods, 1981, Proc. Natl. Acad. Sci. USA 78:3824), or analogous software algorithme, toidentify hydrophobie and hydrophilic régions of the AveC gene product. Structural analysiscan be carried out to identify régions of the AveC gene product that assume spécifiesecondary structures. Biophysical methods such as X-ray crystallography (Engstrom, 1974, 25 Biochem. Exp. Biol. 11: 7-13), computer modelling (Fletterick and Zoller (eds), 1986, in:Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold SpringHarbor, NY), and nuclear magnetic résonance (NMR) can be used to map and study sites ofinteraction between the AveC gene product and its substrate. Information obtained fromthese studies can be used to select new sites for mutation in the avec ORF to help develop 30 new strains of S. avermitilis having more désirable avermectin production characteristics . 5.3. Construction And Use Of AveC Mutants

The présent invention provides a polynucleotide molécule comprising a nucléotidesequence that is otherwise the same as the S. avermitilis AveC gene product-encodingsequence of plasmid pSE186 (ATCC 209604) or the nucléotide sequence of the aveC ORF of 22 011449 S. avermitilis as presented in FIGURE 1 (SEQ ID NO:1), but that further comprises one ormore mutations, so that cells of S. avermitilis strain ATCC 53692 in which the wild-type aveCallele has been inactivated and that express the polynucleotide molécule comprising themutated nucléotide sequence produce a different ratio or amount of avermectins than areproduced by cells of S. avermitilis strain ATCC 53692 that instead express only the wild-typeaveC allele.

According to the présent invention, such polynucleotide molécules can be used toproduce novel strains of S. avermitilis that exhibit a détectable change in avermectinproduction compared to the same strain which instead expresses only the wild-type aveCallele. In a preferred embodiment, such polynucleotide molécules are useful to produce novelstrains of S. avermitilis that produce avermectins in a reduced class 2:1 ratio compared to thesame strain which instead expresses only the wild-type aveC allele. In a further preferredembodiment, such polynucleotide molécules are useful to produce novel strains of S.avermitifis that produce increased levels of avermectins compared to the same strain whichinstead expresses only the wild-type aveC allele. In a further preferred embodiment, suchpolynucleotide molécules are useful to produce novel strains of S. avermitilis in which theaveC gene has been inactivated.

Mutations to the aveC coding sequence include any mutations that introduce aminoacid délétions, additions, or substitutions into the AveC gene product, or that resuit intruncation of the AveC gene product, or any combination thereof, and that produce the desiredresuit. For example, the présent invention provides polynucleotide molécules comprising theAveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or the nucléotidesequence of the aveC ORF of S. avermitilis as presented in FIGURE 1 (SEQ ID NO:1), butthat further comprise one or more mutations that encode the substitution of an amino acidresidue with a different amino acid residue at selected positions in the AveC gene product. Inseveral non-limiting embodiments, which are exemplified below, such substitutions can becarried out at any of amino acid positions 55,138, 139, or 230, or some combination thereof.

Mutations to the aveC coding sequence are carried out by any of a variety of knownmethods, including by use of error-prone PCR, or by cassette mutagenesis. For example,oligonucleotide-directed mutagenesis can be employed to aiter the aveC ORF sequence in adefined way such as, e.g., to introduce one or more restriction sites, or a termination codon,into spécifie régions within the aveC ORF sequence. Methods such as those described inU.S. Patent 5,605,793, which involve random fragmentation, repeated cycles of mutagenesis, 23 011449 and nucléotide shuffling, can also be used to generate large libraries of polynucleotideshaving nucléotide sequences encoding aveC mutations.

Targeted mutations can be useful, particularly where they serve to alter one or moreconserved amino acid residues in the AveC gene product. For example, a comparison of 5 deduced amino acid sequences of AveC gene products and AveC homolog gene productsfrom S. avermitilis (SEQ ID NO;2), S. griseochromogenes (SEQ ID NO:5), and S.hygroscopicus (SEQ ID NO:4), as presented in FIGURE 6, indicates sites of significantconservation of amino acid residues between these species. Targeted mutagenesis thatleads to a change in one or more of these conserved amino acid residues can be particularly 10 effective in producing novel mutant strains that exhibit désirable alterations in avermectinproduction.

Random mutagenesis can also be useful, and can be carried out by exposing cells ofS. avermitilis to ultraviolet radiation or x-rays, or to Chemical mutagens such as N-methyl-N'-nitrosoguanidine, ethyl methane sulfonate, nitrous acid or nitrogen mustards. See, e.g., 15 Ausubel, 1989, above, for a review of mutagenesis techniques.

Once mutated polynucleotide molécules are generated, they are screened todétermine whether they can modulate avermectin biosynthesis in S. avermitilis. In a preferredembodiment, a polynucleotide molécule having a mutated nucléotide sequence is tested bycomplementing a strain of S. avermitilis in which the aveC gene has been inactivated to give 20 an aveC négative (aveC') background. In a non-limiting method, the mutated polynucleotidemolécule is spliced into an expression plasmid in operative association with one or moreregulatory éléments, which plasmid also preferably comprises one or more drug résistancegenes to allow for sélection of transformed cells. This vector is then transformed into aveC'host cells using known techniques, and transformed cells are selected and cultured in 25 appropriate fermentation media under conditions that permit or induce avermectin production.Fermentation products are then analyzed by HPLC to détermine the ability of the mutatedpolynucleotide molécule to complément the host cell. Several vectors bearing mutatedpolynucleotide molécules capable of reducing the B2.B1 ratio of avermectins, includingpSE188, pSE199, and pSE231, are exemplified in Section 8.3, below. 30 The présent invention provides methods for identifying mutations of the aveC ORF of S. avermitilis capable of altering the ratio and/or amount of avermectins produced. In apreferred embodiment, the présent invention provides a method for identifying mutations ofthe aveC ORF capable of altering the class 2:1 ratio of avermectins produced, comprising: (a)determining the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis in 24 011449 which the native aveC allele has been inactivated, and into which a pofynucleotide moléculecomprising a nucléotide sequence encoding a mutated AveC gene product has beenintroduced and is being expressed; (b) determining the class 2:1 ratio of avermectinsproduced by cells of the same strain of S. avermitiiis as in step (a) but which instead expressonly an aveC allele having the nucléotide sequence of the ORF of FIGURE 1 (SEQ ID NO:1)or a nucléotide sequence that is homologous thereto; and (c) comparing the class 2:1 ratio ofavermectins produced by the S. avermitiiis cells of step (a) to the class 2:1 ratio ofavermectins produced by the S. avermitiiis cells of step (b); such that if the class 2:1 ratio ofavermectins produced by the S. avermitiiis cells of step (a) is different from the class 2:1 ratioof avermectins produced by the S. avermitiiis cells of step (b), then a mutation of the aveCORF capable of altering the class 2:1 ratio of avermectins has been identified. In a preferredembodiment, the class 2:1 ratio of avermectins is reduced by the mutation.

In a further preferred embodiment, the présent invention provides a method foridentifying mutations of the aveC ORF or genetic constructs comprising the avec ORFcapable of altering the amount of avermectins produced, comprising: (a) determining theamount of avermectins produced by cells of a strain of S. avermitiiis in which the native aveCallele has been inactivated, and into which a polynucleotide molécule comprising a nucléotidesequence encoding a mutated AveC gene product or comprising a genetic constructcomprising a nucléotide sequence encoding an AveC gene product has been introduced andis being expressed; (b) determining the amount of avermectins produced by cells of the samestrain of S. avermitiiis as in step (a) but which instead express only an aveC allele having thenucléotide sequence of the ORF of FIGURE 1 (SEQ ID NO:1) or a nucléotide sequence thatis homologous thereto; and (c) comparing the amount of avermectins produced by the S.avermitiiis cells of step (a) to the amount of avermectins produced by the S. avermitiiis cells ofstep (b); such that if the amount of avermectins produced by the S. avermitiiis cells of step (a)is different from the amount of avermectins produced by the S. avermitiiis cells of step (b),then a mutation of the aveC ORF or a genetic construct capable of altering the amount ofavermectins has been identified. In a preferred embodiment, the amount of avermectinsproduced is increased by the mutation.

Any of the aforementioned methods for identifying mutations are carried out by usingfermentation culture media preferably supplemented with cyclohexane carboxylic acid,although other appropriate fatty acid precursors, such as any one of the fatty acid precursorslisted in TABLE 1, can also used. 25 011449

Once a mutated polynucleotide molécule that modulâtes avermectin production in adésirable direction has been identified, the location of the mutation in the nucléotide sequencecan be determined. For example, a polynucleotide molécule having a nucléotide sequenceencoding a mutated AveC gene product can be isolated by PCR and subjected to DNA 5 sequence analysis using known methods. By comparing the DNA sequence of the mutatedaveC allele to that of the wild-type aveC allele, the mutation(s) responsible for the alteration inavermectin production can be determined. In spécifie, though non-limiting, embodiments ofthe présent invention, S. avermitilis AveC gene products comprising either single amino acidsubstitutions at any of residues 55 (S55F), 138 (S138T), 139 (A139T), or 230 (G230D), or a 10 double substitution at positions 138 (S138T) and 139 (A139T), yielded changes in AveC geneproduct function such that the ratio of class 2:1 avermectins produced was altered (seeSection 8, below). Accordingly, polynucleotide molécules having nucléotide sequences thatencode mutated S. avermitilis AveC gene products comprising amino acid substitutions at oneor more of amino acid residues 55, 138, 139 or 230, or any combination thereof, are 15 encompassed by the présent invention.

The présent invention further provides compositions for making novel strains of S. avermitilis, the cells of which contain a mutated aveC allele that results in the alteration ofavermectin production. For example, the présent invention provides recombinant vectors thatcan be used to target any of the polynucleotide molécules comprising mutated nucléotide 20 sequences of the présent invention to the site of the aveC gene of the S. avermitilischromosome to either insert into or replace the aveC ORF or a portion thereof by homologousrecombination. According to the présent invention, however, a polynucleotide moléculecomprising a mutated nucléotide sequence of the présent invention provided herewith canalso function to modulate avermectin biosynthesis when inserted into the S. avermitilis 25 chromosome at a site other than at the aveC gene, or when maintained episomally in S.avermitilis cells. Thus, the présent invention also provides vectors comprising apolynucleotide molécule comprising a mutated nucléotide sequence of the présent invention,which vectors can be used to insert the polynucleotide molécule at a site in the S. avermitilischromosome other than at the aveC gene, or to be maintained episomally. 30 In a preferred embodiment, the présent invention provides gene replacement vectors that can be used to insert a mutated aveC allele into cells of a strain of S. avermitilis, therebygenerating novel strains of S. avermitilis, the cells of which produce avermectins in an alteredclass 2:1 ratio compared to cells of the same strain which instead express only the wild-typeaveC allele. In a preferred embodiment, the class 2:1 ratio of avermectins produced by the 26 011449 cells is reduced. Such gene replacement vectors can be constructed using mutatedpolynucleotide molécules présent in expression vectors provided herewith such as pSE188,pSE199, and pSE231, which expression vectors are exemplified in Section 8.3 below.

In a further preferred embodiment, the présent invention provides vectors that can beused to insert a mutated aveC allele into cells of a strain of S. avermitilis to generate novelstrains of cells that produce altered amounts of avermectins compared to cells of the samestrain which instead express only the wild-type aveC allele. In a preferred embodiment, theamount of avermectins produced by the cells is increased. In a spécifie, though non-limiting,embodiment, such a vector further comprises a strong promoter as known in the art, such as,e.g., the strong constitutive ermE promoter from Saccharopolyspora erythraea, that is situatedupstream from, and in operative association with, the aveC ORF. Such a vector can beplasmid pSE189, described in Example 11 below, or can be constructed by using the mutatedaveC allele of plasmid pSE189.

In a further preferred embodiment, the présent invention provides gene replacementvectors that are useful to inactivate the aveC gene in a wild-type strain of S. avermitilis. In anon-limiting embodiment, such gene replacement vectors can be constructed using themutated polynucleotide molécule présent in plasmid pSE180 (ATCC 209605), which isexemplified in Section 8.1, below (FIGURE 3). The présent invention further provides genereplacement vectors that comprise a polynucleotide molécule comprising or consisting ofnucléotide sequences that naturally flank the aveC gene in situ in the S. avermitilischromosome, including, e.g., those flanking nucléotide sequences shown in FIGURE 1 (SEQID NO:1), which vectors can be used to delete the S. avermitilis aveC ORF.

The présent invention further provides methods for making novel strains of S.avermitilis comprising cells that express a mutated aveC allele and that produce an alteredratio and/or amount of avermectins compared to cells of the same strain of S. avermitilis thatinstead express only the wild-type aveC allele. In a prefemed embodiment, the présentinvention provides a method for making novel strains of S. avermitilis comprising cells thatexpress a mutated aveC allele and that produce an altered class 2:1 ratio of avermectinscompared to cells of the same strain of S. avermitilis that instead express only a wild-typeaveC allele, comprising transforming cells of a strain of S. avermitilis with a vector that carriesa mutated aveC allele that encodes a gene product that alters the class 2:1 ratio ofavermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allelecompared to cells of the same strain that instead express only the wild-type aveC allele, andselecting transformed cells that produce avermectins in an altered class 2:1 ratio compared to 27 011449 C11 4 / 9 the class 2:1 ratio produced by cells of the strain that instead express only the wild-type aveCallele. In a preferred embodiment, the altered class 2:1 ratio of avermectins is reduced.

In a further preferred embodiment, the présent invention provides a method formaking novel strains of S. avermitilis comprising cells that produce altered amounts ofavermectin, comprising transforming cells of a strain of S. avermitilis with a vector that carriesa mutated aveC allele or a genetic construct comprising the aveC allele, the expression ofwhich results in an alteration in the amount of avermectins produced by cells of a strain of S.avermitilis expressing the mutated aveC allele or genetic construct as compared to cells of thesame strain that instead express only the wild-type aveC allele, and selecting transformedcells that produce avermectins in an altered amount compared to the amount of avermectinsproduced by cells of the strain that instead express only the wild-type aveC allele. In apreferred embodiment, the amount of avermectins produced in the transformed cells isincreased.

In a further preferred embodiment, the présent invention provides a method formaking novel strains of S. avermitilis, the cells of which comprise an inactivated aveC allele,comprising transforming cells of a strain of S. avermitilis that express a wild-type aveC allelewith a vector that inactivâtes the aveC allele, and selecting transformed cells in which theaveC allele has been inactivated. In a preferred, though non-limiting, embodiment, cells of astrain of S. avermitilis are transformed with a gene replacement vector that carries an aveCallele that has been inactivated by mutation or by replacement of a portion of the aveC allelewith a heterologous gene sequence, and transformed cells in which the native aveC allele ofthe cells has been replaced with the inactivated aveC allele are selected. Inactivation of theaveC allele can be determined by HPLC analysis of fermentation products, as describedbelow. In a spécifie, though non-limiting, embodiment described in Section 8.1 below, theaveC allele is inactivated by insertion of the ermE gene from Saccharopolyspora erythraeainto the aveC ORF.

The présent invention further provides novel strains of S. avermitilis comprising cellsthat hâve been transformed with any of the polynucleotide molécules or vectors of the présentinvention. In a preferred embodiment, the présent invention provides novel strains of S.avermitilis comprising cells which express a mutated avec allele in place of, or in addition to,the wild-type aveC allele, wherein the cells of the novel strain produce avermectins in analtered class 2:1 ratio compared to the class 2:1 ratio of avermectins produced by cells of thesame strain that instead express only the wild-type aveC allele. In a preferred embodiment,the altered class 2:1 ratio produced by the novel cells is reduced. Such novel strains are 28 011449 useful in the large-scale production of commercially désirable avermectins such asdoramectin.

It is a primary objective of the screening assays described herein to identify mutatedalleles of the aveC gene the expression of which, in S. avermitilis cells, alters and, moreparticularly, reduces the ratio of class 2:1 avermectins produced. In a preferred embodiment,the ratio of B2:B1 avermectins produced by cells of a novel S. avermitilis strain of the présentinvention expressing a mutated aveC allele which reduces the ratio of class 2:1 avermectinsproduced is between less than 1,6:1 to about 0:1; in a more preferred embodiment, the ratio isbetween about 1:1 to about 0:1; and in the most preferred embodiment, the ratio is betweenabout 0.84:1 to about 0:1. In a spécifie embodiment described below, novel cells of theprésent invention produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of less than1.6:1. In a different spécifie embodiment described below, novel cells of the présentinvention produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of about 0.94:1. In afurther different spécifie embodiment described below, novel cells of the présent inventionproduce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of about 0.88:1. In a furtherdifferent spécifie embodiment described below, novel cells of the présent invention producecyclohexyl 2:cyclohexyl B1 avermectins in a ratio of about 0.84:1.

In a further preferred embodiment, the présent invention provides novel strains of S.avermitilis comprising cells which express a mutated aveC allele, or a genetic constructcomprising an aveC allele, in place of, or in addition to, the wild-type aveC allele, wherein thecells of the novel strain produce an altered amount of avermectins compared to cells of thesame strain that instead express only the wild-type aveC allele. In a preferred embodiment,the novel strain produces an increased amount of avermectins. In a non-limiting embodiment,the genetic construct further comprises a strong promoter, such as the strong constitutiveermE promoter from Saccharopolyspora erythraea, upstream from and in operativeassociation with the aveC ORF.

In a further preferred embodiment, the présent invention provides novel strains of S.avermitilis comprising cells in which the aveC gene has been inactivated. Such strains areuseful both for the different spectrum of avermectins that they produce compared to the wild-type strain, and in complémentation screening assays as described herein, to déterminewhether targeted or random mutagenesis of the aveC gene affects avermectin production. Ina spécifie embodiment described below, S. avermitilis host cells were genetically engineeredto contain an inactivated aveC gene. For example, strain SE180-11, described in theexamples below, was generated using the gene replacement plasmid pSE18O (ATCC 29 011449 209605) (FIGURE 3), which was constructed to inactivate the S. avermitilis aveC gene byinsertion of the ermE résistance gene into the aveC coding région.

The présent invention further provides recombinantly expressed, mutated S.avermitilis AveC gene products encoded by any of the aforementioned polynucleotide 5 molécules of the invention, and methods of preparing the same.

The présent invention further provides a process for producing avermectins, comprising culturing cells of a strain of S. avermitilis, which cells express a mutated aveCallele that encodes a gene product that alters the class 2:1 ratio of avermectins produced bycells of a strain of S. avermitilis expressing the mutated aveC allele compared to cells of the 10 same strain that instead express only the wild-type aveC allele, in culture media underconditions that permit or induce the production of avermectins therefrom, and recovering saidavermectins from the culture. In a preferred embodiment, the class 2:1 ratio of avermectinsproduced in the culture by cells expressing the mutated aveC allele is reduced. This processprovides increased efficiency in the production of commercially valuable avermectins such as 15 doramectin.

The présent invention further provides a process for producing avermectins,comprising culturing cells of a strain of S. avermitilis, which cells express a mutated aveCallele or a genetic construct comprising an aveC allele that results in the production of analtered amount of avermectins produced by cells of a strain of S. avermitilis expressing the 20 mutated aveC allele or genetic construct compared to cells of the same strain which do notexpress the mutated aveC allele or genetic construct but instead express only the wild-typeaveC allele, in culture media under conditions that permit or induce the production ofavermectins therefrom, and recovering said avermectins from the culture. In a preferredembodiment, the amount of avermectins produced in culture by cells expressing the mutated 25 aveC allele or genetic construct is increased.

The présent invention further provides a novel composition of avermectins produced by a strain of S. avermitilis expressing a mutated aveC allele that encodes a gene product thatreduces the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilisexpressing the mutated aveC allele compared to cells of the same strain that instead express 30 only the wild-type aveC allele, wherein the avermectins in the novel composition are producedin a reduced class 2:1 ratio as compared to the class 2:1 ratio of avermectins produced bycells of the same strain of S. avermitilis that instead express only the wild-type aveC allele.The novel avermectin composition can be présent as produced in exhausted fermentationculture fluid, or can be harvested therefrom. The novel avermectin composition can be 30 011449 partially or substantially purified from the culture fluid by known biochemical techniques ofpurification, such as by ammonium sulfate précipitation, dialysis, size fractionation, ionexchange chromatography. HPLC, efc. 5.4. Uses Of Avermectins

Avermectins are highly active antiparasitic agents having particular utility asanthelmintics, ectoparasiticides, insecticides and acaricides. Avermectin compoundsproduced according to the methods of the présent invention are useful for any of thesepurposes. For example, avermectin compounds produced according to the présent inventionare useful to treat various diseases or conditions in humans, particularly where thosediseases or conditions are caused by parasitic infections, as known in the art. See, e.g.,Ikeda and Omura, 1997, Chem. Rev. 97(7):2591-2609. More particularly, avermectincompounds produced according to the présent invention are effective in treating a variety ofdiseases or conditions caused by endoparasites, such as parasitic nematodes, which caninfect humans, domestic animais, swine, sheep, poultry, horses or cattle.

More specifically, avermectin compounds produced according to the présent inventionare effective against nematodes that infect humans, as well as those that infect variousspecies of animais. Such nematodes include gastrointestinal parasites such as Ancylostoma,Necator, Ascaris, Strongyloides, Trichinella, Capillaria, Trichuris, Enterobius, Dirofilaria, andparasites that are found in the blood or other tissues or organs, such as filariat worms and theextract intestinal States of Strongyloides and Trichinella.

The avermectin compounds produced according to the présent invention are alsouseful in treating ectoparasitic infections including, e.g., arthropod infestations of mammalsand birds, caused by ticks, mites, lice, fleas, blowflies, biting insects, or migrating dipterouslarvae that can affect cattle and horses, among others.

The avermectin compounds produced according to the présent invention are alsouseful as insecticides against household pests such as, e.g., the cockroach, clothes moth,carpet beetle and the housefly among others, as well as insect pests of stored grain and ofagricultural plants, which pests include spider mites, aphids, caterpillars, and orthopteranssuch as locusts, among others.

Animais that can be treated with the avermectin compounds produced according tothe présent invention include sheep, cattle, horses, deer, goats, swine, birds including poultry,and dogs and cats.

An avermectin compound produced according to the présent invention isadministered in a formulation appropriate to the spécifie intended use, the particular species 31 011449 of host animal being treated, and the parasite or insect involved. For use as a parasiticide, anavermectin compound produced according to the présent invention can be administered orallyin the form of a capsule, bolus, tablet or liquid drench or, alternatively, can be administered asa pour-on, or by injection, or as an implant. Such formulations are prepared in a conventional 5 manner in accordance with standard veterinary practice. Thus, capsules, boluses or tabletscan be prepared by mixing the active ingrédient with a suitable fînely divided diluent or carrieradditionally containing a disintegrating agent and/or binder such as starch, lactose, talc,magnésium stéarate, etc. A drench formulation can be prepared by dispersing the activeingrédient in an aqueous solution together with a dispersing or wetting agent, etc. Injectable 10 formulations can be prepared in the form of a stérile solution which can contain othersubstances such as, e.g., sufficient salts and/or glucose to make the solution isotonie withblood.

Such formulations will vary with regard to the weight of active compound dependingon the patient, or species of host animal to be treated, the severity and type of infection, and 15 the body weight of the host. Generally, for oral administration a dose of active compound offrom about 0.001 to 10 mg per kg of patient or animal body weight given as a single dose or individed doses for a period of from 1 to 5 days will be satisfactory. However, there can beinstances where higher or lower dosage ranges are indicated, as determined, e.g., by aphysician or veterinarian, as based on clinical symptoms. 20 As an alternative, an avermectin compound produced according to the présent invention can be administered in combination with animal feedstuff, and for this purpose aconcentrated feed additive or premix can be prepared for mixing with the normal animal feed.

For use as an insecticide, and for treating agricultural pests, an avermectin compoundproduced according to the présent invention can be applied as a spray, dust, émulsion and 25 the like in accordance with standard agricultural practice.

6. EXAMPLE: FERMENTATION OF STREPTOMYCESAVERMITIUS AND B2:B1 AVERMECTIN ANALYSIS

Strains lacking both branched-chain 2-oxo acid dehydrogenase and 5-O- methyltransferase activities produce no avermectins if the fermentation medium is not 30 supplemented with fatty acids. This example demonstrates that in such mutants a wide rangeof B2:B1 ratios of avermectins can be obtained when biosynthesis is initiated in the presenceof different fatty acids. 32 011449 6.1. Materials And Methods

Streptomyces avermitilis ATCC 53692 was stored at -70°C as a whole broth preparedin seed medium consisting of: Starch (Nadex, Laing National) - 20g; Pharmamedia (Trader'sProtein, Memphis, TN) -15 g; Ardamine pH (Yeast Products Inc.) - 5 g; calcium carbonate -1g. Final volume was adjusted to 1 liter with tap water, pH was adjusted to 7.2, and themedium was autoclaved at 121 °C for 25 mm.

Two ml of a thawed suspension of the above préparation was used to inoculate aflask containing 50 ml of the same medium. After 48 hrs incubation at 28°C on a rotaryshaker at 180 rpm, 2 ml of the broth was used to inoculate a flask containing 50 ml of aproduction medium consisting of: Starch - 80 g; calcium carbonate - 7 g; Pharmamedia - 5 g;dipotassium hydrogen phosphate -1 g; magnésium sulfate -1 g; glutamic acid - 0.6 g; ferroussulfate heptahydrate - 0.01 g; zinc sulfate - 0.001 g; manganous sulfate - 0.001 g. Finalvolume was adjusted to 1 liter with tap water, pH was adjusted to 7.2, and the medium wasautoclaved at 121 °C for 25 min.

Various carboxylic acid substrates (see TABLE 1) were dissolved in methanol andadded to the fermentation broth 24 hrs after inoculation to give a final concentration of 0.2g/liter. The fermentation broth was incubated for 14 days at 28°C, then the broth wascentrifuged (2,500 rpm for 2 min) and the supernatant discarded. The mycelial pellet wasextracted with acetone (15 ml), then with dichloromethane (30 ml), and the organic phaseseparated, filtered, then evaporated to dryness. The residue was taken up in methanol (1 ml)and analyzed by HPLC with a Hewlett-Packard 1090A liquid cbromatograph equipped with ascanning diode-array detector set at 240 nm. The column used was a Beckman UltrasphereC-18, 5 pm, 4.6 mm x 25 cm column maintained at 40°C. Twenty-five μΙ of the abovemethanol solution was injected onto the column. Elution was performed with a linear gradientof methanol-water from 80:20 to 95:5 over 40 min at 0.85/ml min. Two standardconcentrations of cyclohexyl B1 were used to calibrate the detector response, and the areaunder the curves for B2 and B1 avermectins was measured. 6.2. Results

The HPLC rétention times observed for the B2 and B1 avermectins, and the 2:1ratios, are shown in TABLE 1. 33 TABLE 1 01 1449 HPLC Rétention Time (min) Ratio Substrate B2 B1 B2:B1 4-Tetrahydropyran carboxylic acid 8.1 14.5 0.25 Isobutyric acid 10.8 18.9 0.5 3-Furoic acid 7.6 14.6 0.62 S-(+)-2-methylbutyric acid 12.8 21.6 1.0 Cyclohexanecarboxylic acid 16.9 26.0 1.6 3-Thiophenecarboxylic acid 8.8 16.0 1.8 Cyclopentanecarboxylic acid 14.2 23.0 2.0 3-Trifluoromethylbutyric acid 10.9 18.8 3.9 2-Methylpentanoic acid 14.5 24.9 4.2 Cycloheptanecarboxylic acid 18.6 29.0 15.0

The data presented in TABLE 1 demonstrates an extremely wide range of B2:B1avermectin product ratios, indicating a considérable différence in the results of dehydrative 5 conversion of class 2 compounds to class 1 compounds, depending on the nature of the fattyacid side Chain starter unit supplied. This indicates that changes in B2:B1 ratios resultingfrom alterations to the AveC protein may be spécifie to particular substrates. Consequently,screening for mutants exhibiting changes in the B2:B1 ratio obtained with a particularsubstrate needs to be done in the presence of that substrate. The subséquent examples 10 described below use cyclohexanecarboxylic acid as the screening substrate. However, thissubstrate is used merely to exemplify the potential, and is not intended to limit theapplicability, of the présent invention. 7. EXAMPLE: ISOLATION OF THE aveC GENEThis example describes the isolation and characterization of a région of the 15 Streptomyces avermitilis chromosome that encodes the AveC gene product. Asdemonstrated below, the aveC gene was identified as capable of modifying the ratio ofcyclohexyl-B2 to cyclohexyl-B1 (B2:B1) avermectins produced. 34 011449 7.1. Materials And Methods 7.1.1. Growth Of Streptomyces For DNA Isolation

The following method was followed for growing Streptomyces. Single colonies of S.avermitilis ATCC 31272 (single colony isolate #2) were isolated on 1/2 strength YPD-6 5 containing: Difco Yeast Extract - 5 g; Difco Bacto-peptone - 5 g; dextrose - 2.5 g; MOPS - 5 g;Difco Bacto agar -15 g. Final volume was adjusted to 1 liter with dH20, pH was adjusted to7.0, and the medium was autoclaved at 121°C for 25 min.

The mycelia grown in the above medium were used to inoculate 10 ml of TSBmedium (Difco Tryptic Soy Broth - 30 g, in 1 liter dK2O, autoclaved at 121°C for 25 min) in a 10 25 mm x 150 mm tube which was maintained with shaking (300 rpm) at 28°C for 48-72 hrs. 7.1.2. Chromosomal DNA Isolation Froro Streptomyces

Aliquots (0.25 ml or 0.5 ml) of mycelia grown as described above were placed in 1.5ml microcentrifuge tubes and the cells concentrated by centrifugation at 12,000 x g for 60 sec.The supematant was discarded and the cells were resuspended in 0.25 ml TSE buffer (20 ml 15 1.5 M sucrose, 2.5 ml 1 M Tris-HCI, pH 8.0, 2.5 ml 1 M EDTA, pH 8.0, and 75 ml dH2O) containing 2 mg/ml lysozyme. The samples were incubated at 37°C for 20 min with shaking,loaded into an AutoGen 540™ automated nucleic acid isolation instrument (IntegratedSéparation Systems, Natick, MA), and genomic DNA isolated using Cycle 159 (equipmentsoftware) according to manufacturées instructions. 20 Altematively, 5 ml of mycelia were placed in a 17 mm x 100 mm tube, the cells concentrated by centrifugation at 3,000 rpm for 5 min, and the supernatant removed. Cellswere resuspended in 1 ml TSE buffer, concentrated by centrifugation at 3,000 rpm for 5 min,and the supernatant removed. Cells were resuspended in 1 ml TSE buffer containing 2 mg/mllysozyme, and incubated at 37°C with shaking for 30-60 min. After incubation, 0.5 ml 10% 25 sodium dodecyl sulfate (SDS) was added and the cells incubated at 37°C until lysis wascomplété. The lysate was incubated at 65°C for 10 min, cooled to rm temp, split into two 1.5ml Eppendorf tubes, and extracted 1x with 0.5 ml phenol/chloroform (50% phénol previouslyequilibrated with 0.5 M Tris, pH 8.0; 50% chloroform). The aqueous phase was removed andextracted 2 to 5x with chloroform:isoamyl alcohol (24:1). The DNA was precipitated by adding 30 1/10 volume 3M sodium acetate, pH 4.8, incubating the mixture on ice for 10 min, centrifuging the mixture at 15,000 rpm at 5°C for 10 min, and removing the supernatant to a clean tube towhich 1 volume of isopropanol was added. The supernatant plus isopropanol mixture wasthen incubated on ice for 20 min, centrifuged at 15,000 rpm for 20 min at 5°C, the supematant 35 removed, and the DNA pellet washed 1x with 70% éthanol. After the pellet was dry, the DNAwas resuspended in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). 7.1.3. Plasmid DNA Isolation From StreptomycesAn aliquot (1.0 ml) of mycelia was placed in 1.5 ml microcentrifuge tubes and the cells

5 concentrated by centrifugation at 12,000 x g for 60 sec. The supernatant was discarded, thecells were resuspended in 1.0 ml 10.3% sucrose and concentrated by centrifugation at 12,000x g for 60 sec, and the supernatant discarded. The cells were then resuspended in 0.25 mlTSE buffer containing 2 mg/ml lysozyme, and incubated at 37eC for 20 min with shaking andloaded into the AutoGen 540™ automated nucleic acid isolation instrument. Plasmid DNA 10 was isolated using Cycle 106 (equipment software) according to manufacturées instructions.

Alternatively, 1.5 ml of mycelia were placed in 1.5 ml microcentrifuge tubes and the cells concentrated by centrifugation at 12,000 x g for 60 sec The supernatant was discarded,the cells were resuspended in 1.0 ml 10.3% sucrose and concentrated by centrifugation at12,000 x g for 60 sec, and the supernatant discarded. The cells were resuspended in 0.5 ml 15 TSE buffer containing 2 mg/ml lysozyme, and incubated at 37°C for 15-30 min. Afterincubation, 0.25 ml alkaline SDS (0.3N NaOH, 2% SDS) was added and the cells incubated at55°C for 15-30 min or until the solution was clear. Sodium acetate (0.1 ml, 3M, pH 4.8) wasadded to the DNA solution, which was then incubated on ice for 10 min. The DNA sampleswere centrifuged at 14,000 rpm for 10 min at 5°C. The supernatant was removed to a clean 20 tube, and 0.2 ml phenokchloroform (50% phenol:50% chloroform) was added and gentlymixed. The DNA solution was centrifuged at 14,000 rpm for 10 min at 5°C and the upperlayer removed to a clean Eppendorf tube. Isopropanol (0.75 ml) was added, and the solutionwas gently mixed and then incubated at rm temp for 20 min. The DNA solution wascentrifuged at 14,000 rpm for 15 min at 5°C, the supernatant removed, and the DNA pellet 25 was washed with 70% éthanol, dried, and resuspended in TE buffer. 7.1.4. Plasmid DNA Isolation From E. coli A single transformed E. coli colony was inoculated into 5 ml Luria-Bertani (LB)medium (Bacto-Tryptone -10 g, Bacto-yeast extract - 5 g, and NaCI -10 g in 1 liter dH2O, pH7.0, autoclaved at 121°C for 25 min, and supplemented with 100 pg/ml ampicillin). The 30 culture was incubated overnight, and a 1 ml aliquot placed in a 1.5 ml microcentrifuge tube.

The culture samples were loaded into the AutoGen 540™ automated nucleic acid isolation instrument and plasmid DNA was isolated using Cycle 3 (equipment software) according to manufacturer^ instructions. 36 011449 7.1.5. Préparation And TransformationOf S. avermitilis Protoplasts

Single colonies of S. avermitilis were isolated on 1/2 strength YPD-6. The myceliawere used to inoculate 10 ml of TSB medium in a 25 mm x 150 mm tube, which was then 5 incubated with shaking (300 rpm) at 28°C for 48 hrs. One ml of mycelia was used to inoculate50 ml YEME medium. YEME medium contains per liter. Difco Yeast Extract - 3 g; DifcoBacto-peptone - 5 g; Difco Malt Extract - 3 g; Sucrose - 300 g. After autoclaving at 121°C for25 min, the following were added: 2.5 M MgCI2 6H2O (separately autoclaved at 121°C for 25min) - 2 ml; and glycine (20%) (filter-sterilized)- 25 ml. 10 The mycelia were grown at 30°C for 4.8-72 hrs and harvested by centrifugation in a 50 ml centrifuge tube (Falcon) at 3,000 rpm for 20 min. The supernatant was discarded and themycelia were resuspended in P buffer, which contains: sucrose - 205 g; K2SO4 - 0.25 g;MgCI2 6H20 - 2.02 g; H2O - 600 ml; K2PO4 (0.5%) - 10 ml; trace element solution - 20 ml;CaCI2 2H20 (3.68%) - 100 ml; and MES buffer (1.0 M, pH 6.5) - 10 ml. (‘Trace element 15 solution contains per liter; ZnCI2 - 40 mg; PeCI3 ' 6H20 - 200 mg; CuCI2 2H20 - 10 mg;MnCI2' 4H20 -10 mg; Na2B4O7 10H20 -10 mg; (NH4)6 Mo7O24 ' 4H20 -10 mg). The pH wasadjusted to 6.5, final volume was adjusted to 1 liter, and the medium was filtered hot through a0.45 micron filter.

The mycelia were pelleted at 3,000 rpm for 20 min, the supernatant was discarded, 20 and the mycelia were resuspended in 20 ml P buffer containing 2 mg/ml lysozyme. Themycelia were incubated at 35°C for 15 min with shaking, and checked microscopically todétermine extent of protoplast formation, When protoplast formation was complété, theprotoplasts were centrifuged at 8,000 rpm for 10 min. The supernatant was removed and theprotoplasts were resuspended in 10 ml P buffer. The protoplasts were centrifuged at 8,000 25 rpm for 10 min, the supernatant was removed, the protoplasts were resuspended in 2 ml Pbuffer, and approximately 1 x 109 protoplasts were distributed to 2.0 ml cryogénie vials(Nalgene). A vial containing 1 x 109 protoplasts was centrifuged at 8,000 rpm for 10 min, thesupernatant was removed, and the protoplasts were resuspended in 0.1 ml P buffer. Two to 5 30 pg of transforming DNA were added to the protoplasts, immediately followed by the addition of0.5 ml working T buffer. T buffer base contains: PEG-1000 (Sigma) - 25 g; sucrose - 2.5 g;H2O - 83 ml. The pH was adjusted to 8.8 with 1 N NaOH (filter sterilized), and the T bufferbase was filter-sterilizéd and stored at 4°C. Working T buffer, made the same day used, was 37 T buffer base - 8.3 ml; K2PO4 (4 mM) -1.0 ml; CaCI2 2H20 (5 M) - 0.2 ml; and TES (1 M, pH8) - 0.5 ml. Each component of the working T buffer was individually filter-sterilized.

Within 20 sec of adding T buffer to the protoplasts, 1.0 ml P buffer was also addedand the protoplasts were centrifuged at 8,000 rpm for 10 min. The supernatant was discarded 5 and the protoplasts were resuspended in 0.1 ml P buffer. The protoplasts were then plated onRM14 media, which contains: sucrose - 205 g; K2SO4 - 0.25 g; MgCI2 6H20 - 10.12 g;glucose -10 g; Difco Casamino Acids - 0.1 g; Difco Yeast Extract - 5 g; Difco Oatmeal Agar- 3 g; Difco Bacto Agar - 22 g; dH2O - 800 ml. The solution was autoclaved at 121°C for 25min. After autoclaving, stérile stocks of the following were added. K2PO4 (0.5%) - 10 ml; 10 CaCI2'2H20 (5 M) - 5 ml; L-proline (20%) -15 ml; MES buffer (1.0 M, pH 6.5) -10 ml; traceelement solution (same as above) - 2 ml; cycloheximide stock (25 mg/ml) - 40 ml; and 1NNaOH - 2 ml. Twenty-five ml of RM14 medium were aliquoted per plate, and plates dried for24 hr before use.

The protoplasts were incubated in 95% humidity at 30’C for 20-24 hrs. To select 15 thiostrepton résistant transformants, 1 ml of overlay buffer containing 125 μ g per mlthiostrepton was spread evenly over the RM14 régénération plates. Overlay buffer containsper 100 ml: sucrose -10.3 g; trace element solution (same as above) - 0.2 ml; and MES (1 M,pH 6.5) -1 ml. The protoplasts were incubated in 95% humidity at 30°C for 7-14 days untilthiostrepton résistant (Thior) colonies were visible. 20 7.1.6. Transformation Of Streptomyces lividans Protoplasts S. lividans TK64 (provided by the John Innés Institute, Norwich, U.K) was used for transformations in some cases. Methods and compositions for growing, protopiasting, andtransforming S. lividans are described in Hopwood et al., 1985, Genetic Manipulation ofStreptomyces, A Laboratory Manual, John Innés Foundation, Norwich, U.K., and performed 25 as described therein. Plasmid DNA was isolated from S. lividans transformants as describedin Section 7.1.3, above. 7.1.7. Fermentation Analysis Of S. avermitilis StrainsS. avermitilis mycelia grown on 1/2 strength YPD-6 for 4-7 days were inoculated into 1x6 inch tubes containing 8 ml of preform medium and two 5 mm glass beads. Preform 30 medium contains: soluble starch (either thin boiled starch or KOSO, Japan Corn Starch Co.,Nagoya) - 20 g/L; Pharmamedia -15 g/L; Ardamine pH - 5 g/L (Champlain Ind., Clifton, NJ);CaCO3 - 2 g/L; 2x bcfa ("bcfa" refers to branched Chain fatty acids) containing a finalconcentration in the medium of 50 ppm 2-(+/-)-methyl butyric acid, 60 ppm isobutyric acid, 38 011449 and;20 ppm isovaleric acid. The pH was adjusted to 7.2, and the medium was autoclaved at121°C for 25 min.

The tube was shaken at a 17° angle at 215 rpm at 29°C for 3 days. A 2-ml aliquot ofthe seed culture was used to inoculate a 300 ml Erlenmeyer flask containing 25 ml ofproduction medium which contains: starch (either thin boiled starch or KOSO) - 160 g/L;Nutrisoy (Archer Daniels Midland, Decatur, IL) -10 g/L; Ardamine pH -10 g/L; K2HPO4 - 2 g/L;MgSO4.4H2O - 2 g/L; FeSO4.7H2O - 0.02 g/L; MnCI2 - 0.002 g/L; ZnSO4.7H2O - 0.002 g/L;CaCO3 -14 g/L; 2x bcfa (as above); and cyclohexane carboxylic acid (CHC) (made up as a20% solution at pH 7.0) - 800 ppm. The pH was adjusted to 6.9, and the medium wasautoclaved at 121°C for 25 min.

After inoculation, the flask was incubated at 29°C for 12 days with shaking at 200rpm. After incubation, a 2 ml sample was withdrawn from the flask, diluted with 8 ml ofmethanol, mixed, and the mixture centrifuged at 1,250 x g for 10 min to pellet débris. Thesupernatant was then assayed by HPLC using a Beckman Ultrasphere ODS column (25 cm x 4.6 mm ID) with a flow rate of 0.75 ml/min and détection by absorbance at 240 nm. Themobile phase was 86/8.9/5.1 methanol/water/ acetonitrile. 7.1.8. Isolation Of S, avermitilis PKS Genes A cosmid library of S. avermitilis (ATCC 31272, SC-2) chromosomal DNA wasprepared and hybridized with a ketosynthase (KS) probe made from a fragment of theSaccharopolyspora erythraea polyketide synthase (PKS) gene. A detailed description of thepréparation of cosmid libraries can be found in Sambrook et al., 1989, above. A detaileddescription of the préparation of Streptomyces chromosomal DNA libraries is presented inHopwood et al., 1985, above. Cosmid clones containing ketosynthase-hybridizing régionswere identified by hybridization to a 2.7 Kb Ndel/Eco47lli fragment from pEX26 (kindlysupplied by Dr. P. Leadlay, Cambridge, UK). Approximately 5 ng of pEX26 were digestedusing Nde\ and Eco47lll. The reaction mixture was loaded on a 0.8% SeaPlaque GTGagarose gel (FMC BioProducts, Rockland, ME). The 2.7 Kb Ndel/Eco47lll fragment wasexcised from the gel after electrophoresis and the DNA recovered from the gel usingGELase™ from Epicentre Technologies using the Fast Protocol. The 2.7 Kb NdellEco47tt\fragment was labeled with fa-32P]dCTP (deoxycytidine 5’-triphosphate, tetra(triethylammonium) sait, [alpha-32P]-) (NEN-Dupont, Boston, MA) using the BRL NickTranslation System (BRL Life Technologies, Inc., Gaithersburg, MD) following the supplier’sinstructions. A typical réaction was performed in 0.05 ml volume. After addition of 5 μΙ Stop 39 buffer, the labeled DNA was separated from unincorporated nucléotides using a G-25Sephadex Quick Spin™ Column (Boehringer Mannheim) following supplier's instructions.

Approximately 1,800 cosmid clones were screened by colony hybridization. Tenclones were identified that hybridized strongly to the Sacc. erythraea KS probe. E. coli

5 colonies containing cosmid DNA were grown in LB liquid medium and cosmid DNA wasisolated from each culture in the AutoGen 540™ automated nucleic acid isolation instrumentusing Cycle 3 (equipment software) according to manufactureras instructions. Restrictionendonuclease mapping and Southern blot hybridization analyses revealed that five of theclones contained overlapping chromosomal régions. An S. avermitilis genomic SamHI 10 restriction map of the five cosmids (/.e, pSE65, pSE66, pSE67, pSE68, pSE69) was constructed by analysis of overlapping cosmids and hybridizations (FIGURE 4).

7.1.9. Identification Of DNA That ModulâtesAvermectin B2:B1 Ratios AndIdentification Of An aveC ORF 15 The following methods were used to test subcloned fragments derived from the pSE66 cosmid clone for their ability to modulate avermectin B2:B1 ratios in AveC mutants.pSE66 (5 pg) was digested with Sacl and SamHI, The reaction mixture was loaded on a0.8% SeaPlaque™ GTG agarose gel (FMC BioProducts), a 2.9 Kb Sacl/SamHI fragment wasexcised from the gel after electrophoresis, and the DNA was recovered from the gel using 20 GELase™ (Epicentre Technologies) using the Fast Protocol. Approximately 5 pg of theshuttle vector pWHM3 (Vara et al., 1989, J. Bacteriol. 171:5872-5881) was digested with Sacland SamHI. About 0.5 pg of the 2.9 Kb insert and 0.5 pg of digested pWHM3 were mixedtogether and incubated ovemight with 1 unit of ligase (New England Biolabs, Inc, Beverly,MA) at 15°C, in a total volume of 20 pl, according to supplier’s instructions. After incubation, 5 25 pl of the ligation mixture was incubated at 70°C for 10 min, cooled to mn temp, and used totransform competent E. coli DH5a cells (BRL) according to manufacturées instructions.Plasmid DNA was isolated from ampicillin résistant transformants and the presence of the 2.9Kb Sacl/SamHI insert was confirmed by restriction analysis. This plasmid was designated aspSE119. 30 Protoplasts of S. avermitilis strain 1100-SC38 (Pfizer in-house strain) were prepared and transformed with pSE119 as described in Section 7.1.5 above. Strain 1100-SC38 is amutant that produces significantly more of the avermectin cyclohexyl-B2 form compared toavermectin cyclohexyl-B1 form when supplemented with cyclohexane carboxylic acid (B2:B1of about 30:1). pSE119 used to transform S. avermitilis protoplasts was isolated from either 40 011449 E. coli strain GM2163 (obtained from Dr. B. J. Bachmann, Curator, E. coli Genetic StockCenter, Yale University), E. coli strain DM1 (BRL), or S. lividans strain TK64. Thiostreptonrésistant transformants of strain 1100-SC38 were isolated and analyzed by HPLC analysis offermentation products. Transformants of S. avermitilis strain 1100-SC38 containing pSE119 5 produced an altered ratio of avermectin cyclohexyl-B2:cyclohexyl-B1 of about 3.7:1 (TABLE2).

Having established that pSE119 was able to modulate avermectin B2:B1 ratios in anAveC mutant, the insert DNA was sequenced. Approximately 10 pg of pSE119 were isolatedusing a plasmid DNA isolation kit (Qiagen, Valencia, CA) following manufacturées 10 instructions, and sequenced using an ABI 373A Automated DNA Sequencer (Perkin Elmer,Foster City, CA). Sequence data was assembled and edited using Genetic Computer Groupprograms (GCG, Madison, Wl). The DNA sequence and the aveC ORF are presented inFIGURE 1 (SEQ ID NO:1). A new plasmid, designated as pSE118, was constructed as follows. Approximately 5 15 pg of pSE66 was digested with Sph\ and BamHI. The reaction mixture was loaded on a 0.8%SeaPlaque GTG agarose gel (FMC BioProducts), a 2.8 Kb Sphl/SamHI fragment was excisedfrom the gel after electrophoresis, and the DNA was recovered from the gel using GELase™(Epicentre Technologies) using the Fast Protocol. Approximately 5 pg of the shuttle vectorpWHM3 was digested with Sph\ and BamHI. About 0.5 pg of the 2.8 Kb insert and 0.5 pg of 20 digested pWHM3 were mixed together and incubated overnight with 1 unit of ligase (NewEngland Biolabs) at 15°C in a total volume of 20 pl according to supplier's instructions. Afterincubation, 5 pl of the ligation mixture was incubated at 70°C for 10 min, cooled to rm temp,and used to transform competent E. coli DH5oc cells according to manufacturées instructions.Plasmid DNA was isolated from ampicillin résistant transformants, and the presence of the 2.8 25 Kb Spbl/BamHI insert was confirmed by restriction analysis. This plasmid was designated aspSE118. The insert DNA in pSE118 and pSE119 overlap by approximately 838 nucléotides(FIGURE 4).

Protoplasts of S. avermitilis strain 1100-SC38 were transformed with pSE118 asabove. Thiostrepton résistant transformants of strain 1100-SC38 were isolated and analyzed 30 by HPLC analysis of fermentation products. Transformants of S. avermitilis strain 1100-SC38containing pSE118 were not altered in the ratios of avermectin cyclohexyl-B2: avermectincyclohexyl-B1 compared to strain 1100-SC38 (TABLE 2). 41 011449

7.1.10. PCR Amplification Of The aveC GeneFrom S. avermitilis Chromosomal DNA

A -1.2 Kb fragment containing the aveC ORF was isolated from S. avermitilischromosomal DNA by PCR amplification using primers designed on the basis of the aveC 5 nucléotide sequence obtained above. The PCR primers were supplied by GenosysBiotechnologies, Inc. (Texas). The rightward primer was: 5’-TCACGAAACCGGACACAC-3’(SEQ ID NO:6); and the leftward primer was: 5 - CATGATCGCTGAACCGAG-3' (SEQ IDNO:7). The PCR reaction was carried out with Deep Vent™ polymerase (New EnglandBiolabs) in buffer provided by the manufacturer, and in the presence of 300 μΜ dNTP, 10% 10 glycerol. 200 pmol of each primer, 0.1 pg template, and 2.5 units enzyme in a final volume of100 μΙ, using a Perkin-Elmer Cetus thermal cycler. The thermal profile of the first cycle was95°C for 5 min (dénaturation step), 60°C for 2 min (annealing step), and 72°C for 2 min(extension step). The subséquent 24 cycles had a similar thermal profile except that thedénaturation step was shortened to 45 sec and the annealing step was shortened to 1 min. 15 The PCR productwas electrophoresed in a 1% agarose gel and a single DNA band of -1.2 Kb was detected. This DNA was purified from the gel, and ligated with 25 ng oflinearized, blunt pCR-Blunt vector (Invitrogen) in a 1.10 molar vector-to-insert ratio followingmanufacturées instructions. The ligation mixture was used to transform One Shot™Competent E. coli cells (Invitrogen) following manufacturées instructions. Plasmid DNA was 20 isolated from ampicillin résistant transformants, and the presence of the -1.2 Kb insert wasconfirmed by restriction analysis. This plasmid was designated as pSE179.

The insert DNA from pSE179 was isolated by digestion with BamHI/Xbal, separatedby electrophoresis, purified from the gel, and ligated with shuttle vector pWHM3, which hadalso been digested with BamHI/Xbal, in a total DNA concentration of 1 pg in a 1:5 molar 25 vector-to-insert ratio. The ligation mixture was used to transform competent E. cofi DH5acells according to manufacturées instructions. Plasmid DNA was isolated from ampicillinrésistant transformants and the presence of the -1.2 Kb insert was confirmed by restrictionanalysis. This plasmid, which was designated as pSE186 (FIGURE 2, ATCC 209604), wastransformed into E. coli DM1, and plasmid DNA was isolated from ampicillin résistant 30 transformants. 7.2. Results A 2.9 Kb Sacl/fîamHI fragment from pSE119 was identified that, when transformedinto S. avermitilis strain 1100-SC38, significantly altered the ratio of B2:B1 avermectinproduction. S. avermitilis strain 1100-SC38 normally has a B2:B1 ratio of about 30:1, but 42 011449 when transformée! with a vector comprising the 2.9 Kb Sacl/fîamHI fragment, the ratio ofB2:B1 avermectin decreased to about 3,7:1. Post-fermentation analysis of transformantcultures verified the presence of the transforming DNA.

The 2.9 Kb pSEH9 fragment was sequenced and a -0.9 Kb ORF was identified5 (FIGURE 1) (SEQ ID NO:1), which encompasses a Psti/SpM fragment that had previouslybeen mutated elsewhere to produce B2 products only (Ikeda ef al., 1995, above). Acomparison of this ORF, or its corresponding deduced polypeptide, against known databases(GenEMBL, SWISS-PROT) did not show any strong homology with known DNA or protein sequences. 10 TABLE 2 présents the fermentation analysis of S. avermitilis strain 1100-SC38 transformed with various plasmids. TABLE 2 S. avermitilis strain (transforming piasmid) No. TransformantsTested Avg. B2:B1 Ratio 1100-SC38 (none) 9 30.66 1100-SC38 (pWHM3) 21 31.3 1100-SC38 (pSE119) 12 3.7 1100-SC38 (pSE118) 12 30.4 1100-SC38 (pSE185) 14 27.9

8. EXAMPLE: CONSTRUCTION OFS. AVERMITILIS AveC MUTANTS

This example describes the construction of several different S. avermitilis AveCmutants using the compositions and methods described above. A general description of 20 techniques for introducing mutations into a gene in Streptomyces is described by Kieser andHopwood, 1991, Meth. Enzym. 204:430-458. A more detailed description is provided byAnzai ef al., 1988, J. Antibiot. XLI(2):226-233, and by Stutzman-Engwall ef al., 1992, J.Bacteriol. 174(1):144-154. These référencés are incorporated herein by référencé in theirentirety. 25 8.1. Inactivation Of The S. avermitilis aveC Gene

AveC mutants containing inactivated aveC genes were constructed using several methods, as detailed below. 43 011449

In the first method, a 640 bp SphllPstl fragment internai to the aveC gene in pSE119(plasmid described in Section 7.1.9, above) was replaced with the ermE gene (forerythromycin résistance) from Sacc. erythraea. The ermE gene was isolated from plJ4026(provided by the John Innés Institute, Norwich, U.K.; see also Bibb et al., 1985, Gene 41:357- 5 368) by restriction enzyme digestion with fig/ll and EcoRI, followed by electrophoresis, and was purified from the gel. This -1.7 Kb fragment was ligated into pGEM7Zf (Promega) whichhad been digested with fîamHI and EcoRI, and the ligation mixture transformed intocompetent E. coli DH5a cells following manufacturer^ instructions. Plasmid DNA wasisolated from ampicillin résistant transformants, and the presence of the -1.7 Kb insert was 10 confirmed by restriction analysis. This plasmid was designated as pSE27. pSE118 (described in Section 7.1.9, above) was digested with Sphï and EamHI, the digest electrophoresed, and the -2.8 Kb SphllBamH] insert purified from the gel. pSE119was digested with Psîl and EcoRI, the digest electrophoresed, and the -1.5 Kb Pst\IEcoR\insert purified from the gel. Shuttle vector pWHM3 was digested with BamHI and EcoRI. 15 pSE27 was digested with Psîl and Sph\, the digest electrophoresed, and the -1.7 KbPst\ISph\ insert purified from the gel. Ail four fragments (/.e., -2.8 Kb, -1.5Kb, -7.2Kb, -1.7Kb) were ligated together in a 4-way ligation. The ligation mixture was transformed intocompetent E. coli DH5a cells following manufacturées instructions. Plasmid DNA wasisolated from ampicillin résistant transformants, and the presence of the correct insert was 20 confirmed by restriction analysis. This plasmid was designated as pSE180 (FIGURE 3; ATCC209605). pSE180 was transformed into S. lividans TK64 and transformed colonies identified byrésistance to thiostrepton and erythromycin. pSE180 was isolated from S. lividans and usedto transform S. avermitilis protoplasts. Four thiostrepton résistant S. avermitilis transformants 25 were identified, and protoplasts were prepared and plated under non-selective conditions onRM14 media. After the protoplasts had regenerated, single colonies were screened for thepresence of erythromycin résistance and the absence of thiostrepton résistance, indicatingchromosomal intégration of the inactivated aveC gene and loss of the free replicon. One ErmrThio’ transformant was identified and designated as strain SE180-11. Total chromosomal 30 DNA was isolated from strain SE180-11, digested with restriction enzymes SamHI, HindW,Pstl, or Sphl, resolved by electrophoresis on a 0.8% agarose gel, transferred to nylonmembranes, and hybridized to the ermE probe. These analyses showed that chromosomalintégration of the ermE résistance gene, and concomitant délétion of the 640 bp PstllSphl 44 011449 fragment had occurred by a double crossover event. HPLC analysis of fermentation productsof strain SE180-11 showed that normal avermectins were no longer produced (FIGURE 5A).

In a second method for inactivating the aveC gene, the 1.7 Kb ermE gene wasremoved from the chromosome of S. avermitilis strain SE180-11, leaving a 640 bp PstllSphldélétion in the aveC gene. A gene replacement plasmid was constructed as follows: pSE180was partially digested with Xbal and an -11.4 Kb fragment purified from the gel. The -11.4Kb band lacks the 1.7 Kb ermE résistance gene. The DNA was then ligated and transformedinto E. coli DH5a cells. Plasmid DNA was isolated from ampicillin résistant transformants andthe presence of the correct insert was confirmed by restriction analysis. This plasmid, whichwas designated as pSE184, was transformed into E. coli DM1, and plasmid DNA isolatedfrom ampicillin résistant transformants. This plasmid was used to transform protoplasts of S.avermitilis strain SE180-11. Protoplasts were prepared from thiostrepton résistanttransformants of strain SE180-11 and were plated as single colonies on RM14. After theprotoplasts had regenerated, single colonies were screened for the absence of botherythromycin résistance and thiostrepton résistance, indicating chromosomal intégration of theinactivated aveC gene and loss of the free replicon containing the ermE gene. One ErmsThios transformant was identified and designated as SE184-1-13. Fermentation analysis ofSE184-1-13 showed that normal avermectins were not produced and that SE184-1-13 had thesame fermentation profile as SE180-11.

In a third method for inactivating the aveC gene, a frameshift was introduced into thechromosomal aveC gene by adding two G’s after the C at nt position 471 using PCR, therebycreating a SspE1 site. The presence of the engineered BspEî site was useful in detecting thegene replacement event. The PCR primers were designed to introduce a frameshift mutationinto the aveC gene, and were supplied by Genosys Biotechnologies, Inc. The rightwardprimer was: 5’-GGTTCCGGATGCCGTTCTCG-3’ (SEQ ID NO:8) and the leftward primerwas: 5’-AACTCCGGTCGACTCCCCTTC-3' (SEQ ID NO:9). The PCR conditions were asdescribed in Section 7.1.10 above. The 666 bp PCR product was digested with Spril to givetwo fragments of 278 bp and 388 bp, respectively. The 388 bp fragment was purified from thegel.

The gene replacement plasmid was constructed as follows: shuttle vector pWHM3was digested with EcoRI and ÔamHI. pSE119 was digested with BarnHl and Sphl, the digestelectrophoresed, and a -840 bp fragment was purified from the get. pSE119 was digestedwith EcoRI and Xmnl, the digest was resolved by electrophoresis, and a -1.7 Kb fragmentwas purified from the gel. Ail four fragments (/.e., -7.2 Kb, -840 bp, -1.7 Kb, and 388 bp) 45

U I were ligated together in a 4-way ligation. The ligation mixture was transformed intocompetent E. coli DH5a cells. Plasmid DNA was isolated from ampicillin résistanttransformants and the presence of the correct insert was confirmed by restriction analysis andDNA sequence analysis. This plasmid, which was designated as pSE185, was transformed 5 into E. coli DM1 and plasmid DNA isolated from ampicillin résistant transformants. Thisplasmid was used to transform protoplasts of S. avermitiiis strain 1100-SC38. Thiostreptonrésistant transformants of strain 1100-SC38 were isolated and analyzed by HPLC analysis offermentation products. pSE185 did not significantly alter the B2:B1 avermectin ratios whentransformed into S. avermitiiis strain 1100-SC38 (TABLE 2). 10 pSE185 was used to transform protoplasts of S. avermitiiis to generate a frameshift

mutation in the chromosomal aveC gene. Protoplasts were prepared from thiostreptonrésistant transformants and plated as single colonies on RM14. After the protoplasts hadregenerated, single colonies were screened for the absence of thiostrepton résistance.Chromosomal DNA from thiostrepton sensitive colonies was isolated and screened by PCR 15 for the presence of the frameshift mutation integrated into the chromosome. The PCR primerswere designed based on the aveC nucléotide sequence, and were supplied by GenosysBiotechnologies, Inc. (Texas). The rightward PCR primer was: 5'- GCAAGGATACGGGGACTAC-3' (SEQ ID NO: 10) and the leftward PCR primer was: 5'-GAACCGACCGCCTGATAC-3’ (SEQ ID NO:11), and the PCR conditions were as described 20 in Section 7.1.10 above. The PCR product obtained was 543 bp and, when digested withBspE1, three fragments of 368 bp, 96 bp, and 79 bp were observed, indicating chromosomalintégration of the inactivated aveC gene and loss of the free replicon.

Fermentation analysis of S. avermitiiis mutants containing the frameshift mutation inthe aveC gene showed that normal avermectins were no longer produced, and that these 25 mutants had the same fermentation HPLC profile as strains SE180-11 and SE184-1-13. OneThio* transformant was identified and designated as strain SE185-5a.

Additionally, a mutation in the aveC gene that changes nt position 520 from G to A,which results in changing the codon encoding a tryptophan (W) at position 116 to atermination codon, was produced. An S. avermitiiis strain with this mutation did not produce 30 normal avermectins and had the same fermentation profile as strains SE180-11, SE184-1-13,and SE185-5a.

Additionally, mutations in the aveC gene that change both: (i) nt position 970 from Gto A, which changes the amino acid at position 256 from a glycine (G) to an aspartate (D), and(ii) nt position 996 from T to C, which changes the amino acid at position 275 from tyrosine (Y) 46 011449 to histidine (H), were produced. An S. avermitilis strain with these mutations (G256D/Y275H)did not produce normal avermectins and had the same fermentation profile as strains SE180-11, SE184-1-13, and SE185-5a.

The S. avermitilis aveC inactivation mutant strains SE180-11, SE184-1-13, SE185-5a, and others provided herewith, provide screening tools to assess the impact of othermutations in the aveC gene. pSE186, which contains a wild-type copy of the aveC gene, wastransformed into E. coli DM1, and plasmid DNA was isolated from ampicillin résistanttransformants. This pSE186 DNA was used to transform protoplasts of S. avermitilis strainSE180-11. Thiostrepton résistant transformants of strain SE180-11 were isolated, thepresence of erythromycin résistance was determined, and Thior ErnY transformants wereanalyzed by HPLC analysis of fermentation products. The presence of the functional aveCgene in trans was able to restore normal avermectin production to strain SE180-11 (FIGURE5B). 8.2. Analysis Of Mutations In The aveCGene That Alter Class B2:B1 Ratios

As described above, S. avermitilis strain SE180-11 containing an inactive aveC genewas complemented by transformation with a plasmid containing a functional aveC gene(pSE186). Strain SE180-11 was also utilized as a host strain to characterize other mutationsin the aveC gene, as described below.

Chromosomal DNA was isolated from strain 1100-SC38, and used as a template forPCR amplification of the aveC gene. The 1.2 Kb ORF was isolated by PCR amplificationusing primers designed on the basis of the aveC nucléotide sequence. The rightward primerwas SEQ ID NO.6 and the leftward primer was SEQ ID NO:7 (see Section 7.1.10, above).The PCR and subcloning conditions were as described in Section 7.1.10. DNA sequenceanalysis of the 1.2 Kb ORF shows a mutation in the aveC gene that changes nt position 337from C to T, which changes the amino acid at position 55 from serine (S) to phenylalanine (F).The aveC gene containing the S55F mutation was subcloned into pWHM3 to produce aplasmid which was designated as pSE187, and which was used to transform protoplasts of S.avermitilis strain SE180-11. Thiostrepton résistant transformants of strain SE180-11 wereisolated, the presence of erythromycin résistance was determined, and Thior Ermrtransformants were analyzed by HPLC analysis of fermentation products. The presence ofthe aveC gene encoding a change at amino acid residue 55 (S55F) was able to restorenormal avermectin production to strain SE180-11 (Fig. 5C); however, the cyclohexylB2:cyclohexyl B1 ratio was about 26:1, as compared to strain SE180-11 transformed with 47 011449 pSE186, which had a ratio of B2:B1 of about 1.6:1 (TABLE 3), indicating that the singlemutation (S55F) modulâtes the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1.

Another mutation in the aveC gene was identified that changes nt position 862 from Gto A, which changes the amino acid at position 230 from glycine (G) to aspartate (D). An S. 5 avermitilis strain having this mutation (G230D) produces avermectins at a B2:B1 ratio of about30:1. 8.3. Mutations That Reduce The B2:B1 Ratio

Several mutations were constructed that reduce the amount of cyclohexyl-B2produced relative to cyclohexyl-B1, as follows. 10 A mutation in the aveC gene was identified that changes nt position 588 from G to A, which changes the amino acid at position 139 from alanine (A) to threonine (T). The aveCgene containing the A139T mutation was subcloned into pWHM3 to produce a plasmid whichwas designated pSE188, and which was used to transform protoplasts of S. avermitilis strainSE180-11. Thiostrepton résistant transformants of strain SE180-11 were isolated, the 15 presence of erythromycin résistance was determined, and Thior Errrf transformants wereanalyzed by HPLC analysis of fermentation products. The presence of the mutated aveCgene encoding a change at amino acid residue 139 (A139T) was able to restore avermectinproduction to strain SE180-11 (FIGURE 5D); however, the B2:B1 ratio was about 0.94:1,indicating that this mutation reduces the amount of cyclohexyl-B2 produced relative to 20 cyclohexyl-B1. This resuit was unexpected because published results, as well as the resultsof mutations described above, hâve only demonstrated either inactivation of the aveC gene orincreased production of the B2 form of avermectin relative to the B1 form (TABLE 3).

Because the A139T mutation altered the B2:B1 ratios in the more favorable B1direction, a mutation was constructed that encoded a threonine instead of a serine at amino 25 acid position 138. Thus, pSE186 was digested with EcoRI and cloned into pGEM3Zf(Promega) which had been digested with EcoRI. This plasmid, which was designated aspSE186a, was digested with Apal and Kpn], the DNA fragments separated on an agarose gel,and two fragments of -3.8 Kb and -0.4 Kb were purified from the gel. The -1.2 Kb insertDNA from pSE186 was used as a PCR template to introduce a single base change at nt 30 position 585. The PCR primers were designed to introduce a mutation at nt position 585, andwere supplied by Genosys Biotechnologies, Inc. (Texas). The rightward PCR primer was: 5’-GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCCCTGGCGACG-3' (SEQ ID NO: 12);and the leftward PCR primer was: 5'-GGAACCGACCGCCTGATACA-3’ (SEQ ID NO: 13).The PCR reaction was carried out using an Advantage GC genomic PCR kit (Clonetech 48 011449

Laboratories, Palo Alto, CA) in buffer provided by the manufacturer in the presence of 200 μΜdNTPs, 200 pmol of each primer, 50 ng template DNA, 1.0 M GC-Melt and 1 unit KlenTaqPolymerase Mix in a final volume of 50 μΙ. The thermal profile of the first cycle was 94°C for 1min; followed by 25 cycles of 94°C for 30 sec and 68°C for 2 min; and 1 cycle at 68°C for 3min. A PCR product of 295 bp was digested with Apa\ and Kpnl to release a 254 bp fragmentwhich was resolved by electrophoresis and purified from the gel. Ail three~fragments (~3.8Kb, ~0.4 Kb and 254 bp) were ligated together in a 3-way ligation. The ligation mixture wastransformed into competent E. coli DH5a cells. Plasmid DNA was isolated from ampicillinrésistant transformants, and the presence of the correct insert was confirmed by restrictionanalysis. This plasmid was designated as pSE 198. pSE198 was digested with EcoRI, cloned into pWHM3 which had been digested withEcoRI, and transformed into E. coli DH5a cells. Plasmid DNA was isolated from ampicillinrésistant transformants and the presence of the correct insert was confirmed by restrictionanalysis and DNA sequence analysis. This plasmid DNA was transformed into £. coli DM1,plasmid DNA was isolated from ampicillin résistant transformants, and the presence of thecorrect insert was confirmed by restriction analysis. This plasmid, which was designated aspSE199, was used to transform protoplasts of S. avermitilis strain SE180-11. Thiostreptonrésistant transformants of strain SE180-11 were isolated, the presence of erythromycinrésistance was determined, and Thior Ermr transformants were analyzed by HPLC analysis offermentation products. The presence of the mutated aveC gene encoding a change at aminoacid residue 138 (S138T) was able to restore normal avermectin production to strain SE180-11; however, the B2;B1 ratio was 0.88:1 indicating that this mutation reduces the amount ofcyclohexyl-B2 produced relative to cyclohexyl-B1 (TABLE 3). This B2:B1 ratio is even lowerthan the 0.94:1 ratio observed with the A139T mutation produced by transformation of strainSE180-11 with pSE188, as described above.

Another mutation was constructed to introduce a threonine at both amino acidpositions 138 and 139. The -1.2 Kb insert DNA from pSE186 was used as a PCR template.The PCR primers were designed to introduce mutations at nt positions 585 and 588, and weresupplied by Genosys Biotechnologies, Inc. (Texas). The rightward PCR primer was: 5'-GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCGCTGGCGACGACC-3’ (SEQ IDNO: 14); and the leftward PCR primer was: 5’-GGAACATCACGGCATTCACC-3’ (SEQ IDNO: 15). The PCR reaction was performed using the conditions described immédiately abovein this Section. A PCR product of 449 bp was digested with Apa\ and Kpn\ to release a 254bp fragment, which was resolved by electrophoresis and purified from the gel. pSE186a was 49 011449 digested with Apal and Kpnl, the DNA fragments separated on an agarose gel, and twofragments of -3.8 Kb and ~0.4 Kb were purified from the gel. Ail three fragments (-3.8 Kb,-0.4 Kb and 254 bp) were ligated together in a 3-way ligation, and the ligation mixture wastransformed into competent E. co/ι DH5a cells. Plasmid DNA was isolated from ampicillin 5 résistant transformants, and the presence of the correct insert was confirmed by restrictionanalysis. This plasmid was designated as pSE230. pSE230 was digested with EcoRI, cloned into pWHM3 which had been digested withEcoRI, and transformed into £. coli DH5a cells. Plasmid DNA was isolated from ampicillinrésistant transformants and the presence of the correct insert was confirmed by restriction 10 analysis and DNA sequence analysis. This plasmid DNA was transformed into E. coli DM1,plasmid DNA isolated from ampicillin résistant transformants, and the presence of the correctinsert was confirmed by restriction analysis. This plasmid, which was designated as pSE231,was used to transform protoplasts of S. avermitilis strain SE180-11. Thiostrepton résistanttransformants of SE180-11 were isolated, the presence of erythromycin résistance was 15 determined, and Thior Ermr transformants were analyzed by fermentation. The presence ofthe double mutated aveC gene, encoding S138T/A139T, was able to restore normalavermectin production to strain SE180-11; however, the B2.B1 ratio was 0.84:1 showing thatthis mutation further reduces the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1(TABLE 3), over the réductions provided by transformation of strain SE180-11 with pSEl88 or 20 pSEl99, as described above. TABLE 3 S. avermitilis strain(transforming plasmid) No. transformants tested Relative B2 Conc. RelativeB1 Conc. Avg. B2:B1 Ratio SE180-11 (none) 30 0 0 0 SE180-11 (pWHM3) 30 0 0 0 SE180-11 (pSE186) 26 222 140 1.59 SE180-11 (pSE187) 12 283 11 26.3 SE180-11 (pSE188) 24 193 206 0.94 SE180-11 (pSE199) 18 155 171 0.88 SE180-11 (pSE231) 6 259 309 0.84 50 011449

These results are the first to demonstrate spécifie mutations in the aveC gene thatresuit in the production of increased levels of the more commercially désirable class 1avermectins relative to class 2 avermectins.

9. EXAMPLE: CONSTRUCTION OF 5’ DELETION MUTANTS

As explained in Section 5.1, above, the S. avermitilis nucléotide sequence shown inFIGURE 1 (SEQ ID ΝΟ.Ί) comprises four different GTG codons at bp positions 42, 174, 177and 180 which are potential start sites. This section describes the construction of multipledélétions of the 5' région of the aveC ORF (FIGURE 1; SEQ ID ΝΟ.Ί) to help define which ofthese codons could function as start sites in the aveC ORF for protein expression.

Fragments of the aveC gene variously deleted at the 5’ end were isolated from S.avermitilis chromosomal DNA by PCR amplification. The PCR primers were designed basedon the aveC DNA sequence, and were supplied by Genosys Biotechnologies, Inc. Therightward primers were 5'-AACCCATCCGAGCCGCTC-3' (SEQ ID NO:16) (D1F1); 5’-TCGGCCTGCCAACGAAC-3’ (SEQ ID NO:17) (D1F2); 5’-CCAACGAACGTGTAGTAG-3’(SEQ ID NO: 18) (D1F3); and 5’-TGCAGGCGTACGTGTTCAGC-3' (SEQ ID NO: 19) (D2F2).The leftward primers were 5'-CATGATCGCTGAACCGA-3’ (SEQ ID NO:20); 5-CATGATCGCTGAACCGAGGA-3’ (SEQ ID NO:21); and 5'-AGGAGTGTGGTGCGTCTGGA-3' (SEQ.ID NO:22). The PCR reaction was carried out as described in Section 8.3, above.

The PCR products were separated by electrophoresis in a 1% agarose gel and singleDNA bands of either -1.0 Kb or -1.1 Kb were detected. The PCR products were purified fromthe gel and ligated with 25 ng of linearized pCR2.1 vector (Invitrogen) in a 1:10 molar vector-to-insert ratio following the manufacturer’s instructions. The ligation mixtures were used totransform One Shot™ Competent E. coli cells (Invitrogen) following manufacturer’sinstructions. Plasmid DNA was isolated from ampicillin résistant transformants and thepresence of the insert was confirmed by restriction analysis and DNA sequence analysis.These plasmids were designated as pSE190 (obtained with primer D1F1), pSE191 (obtainedwith primer D1F2), pSE192 (obtained with primer D1F3), and pSE193 (obtained with primerD2F2).

The insert DNAs were each digested with BamHI/Xbal, separated by electrophoresis,purified from the gel, and separately ligated with shuttle vector pWHM3, which had beendigested with BamHI/Xbal, in a total DNA concentration of 1 pg in a 1:5 molar vector-to-insertratio. The ligation mixtures were used to transform competent E. coli DH 5a cells. PlasmidDNA was isolated from ampicillin résistant transformants and the presence of the insert wasconfirmed by restriction analysis. These plasmids, which were designated as pSE194 51 (D1F1). pSE195 (D1F2), pSE196 (D1F3), and pSE197 (D2F2), were each separatelytransformed into E. coli strain DM1, plasmid DNA isolated from ampicillin résistanttransformants, and the presence of the correct insert confirmed by restriction analysis. ThisDNA was used to transform protoplasts of S. avermitilis strain SE180-11. Thiostrepton 5 résistant transformants of strain SE180-11 were isolated, the presence of erythromycinrésistance was determined, and Thior Ermf transformants were analyzed by HPLC analysis offermentation products to détermine which GTG sites were necessary for avec expression.The results indicate that the GTG codon at position 42 can be eliminated without affectingaveC expression, since pSE194, pSE195, and pSE196, each of which lack the GTG site at 10 position 42, but which ail contain the three GTG sites at positions 174, 177, and 180, wereeach able to restore normal avermectin production when transformed into SE180-11. Nomalavermectin production was not restored when strain SE180-11 was transformed with pSE197,which lacks ail four of the GTG sites (TABLE 4). 15 TABLE 4 S. avermitilis strain(transforming plasmid) No. transformantstested RelativeB2 Conc. RelativeB1 Conc. Avg. B2.B1 Ratio SE180-11 (none) 6 0 0 0 SE180-11 (pWHM3) 6 0 0 0 SE180-11 (pSE186) 6 241 152 1.58 SE180-11 (pSE194) 6 35 15 2.43 SE180-11 (pSE195) 6 74 38 1.97 SE180-11 (pSE196) 6 328 208 1.58 SE180-11 (pSE197) 12 0 0 0 52

10. EXAMPLE: CLONING OF aveC HOMOLOGS FROM20 S. HYGROSCOPICUS AND S. GRISEOCHROMOGENES

The présent invention allows aveC homolog genes from other avermectin- or milbemycin-producing species of Streptomyces to be identified and cloned. For example, a cosmid library of S. hygroscopicus (FERM BP-1901) genomic DNA was hybridized with the 1.2 Kb aveC probe from S. avermitilis described above. Several cosmid clones were 011449 identified that hybridized strongly. Chromosomal DNA was isolated from these cosmids, anda 4.9 Kb Kpn\ fragment was identified that hybridized with the aveC probe. This DNA wassequenced and an ORF (SEQ ID NO:3) was identified having significant homology to theaveC ORF of S. avermitilis. An amino acid sequence (SEQ ID NO:4) deduced from the S.hygroscopicus aveC homolog ORF is presented in FIGURE 6.

In addition, a cosmid library of S. griseochromogenes genomic DNA was hybridizedwith the 1.2 Kb aveC probe from S. avermitilis described above. Several cosmid clones wereidentified that hybridized strongly. Chromosomal DNA was isolated from these cosmids, anda 5.4 Kb Psfl fragment was identified that hybridized with the aveC probe. This DNA wassequenced and an aveC homolog partial ORF was identified having significant homology tothe aveC ORF of S. avermitilis. A deduced partial amino acid sequence (SEQ ID NO:5) ispresented in FIGURE 6. DNA and amino acid sequence analysis of the aveC homologs from S. hygroscopicus and S. griseochromogenes indicates that these régions share significant homology (~50%

sequence identity at the amino acid level) both to each other and to the S. avermitilis aveC ORF and AveC gene product (FIGURE 6).

11. EXAMPLE: CONSTRUCTION OF A PLASMID WITHTHE aveC GENE BEHIND THE ermE PROMOTER

The 1.2 Kb aveC ORF from pSE186 was subcloned in pSE34, which is the shuttlevector pWHM3 having the 300 bp ermE promoter inserted as a Kpnl/fiamHI fragment in theKpnl/SamHI site of pWHM3 (see Ward ef al., 1986, Mol. Gen. Genet. 203:468-478). pSE186was digested with SamHI and HindM, the digest resolved by electrophoresis, and the 1.2 Kbfragment was isolated from the agarose gel and ligated with pSE34 which had been digestedwith SamHI and H/ndlll. The ligation mixture was transformed into competent E. coli DH5acells according to manufacturées instructions. Plasmid DNA was isolated from ampicillinrésistant transformants, and the presence of the 1.2 Kb insert was confirmed by restrictionanalysis. This plasmid, which was designated as pSE189, was transformed into E. coli DM1,and plasmid DNA isolated from ampicillin résistant transformants. Protoplasts of S. avermitilisstrain 1100-SC38 were transformed with pSE189. Thiostrepton résistant transformants ofstrain 1100-SC38 were isolated and analyzed by HPLC analysis of fermentation products. S. avermitilis strain 1100-SC38 transformants containing pSE189 were altered in theratios of avermectin cyclohexyl-B2:avermectin cyclohexyl-B1 produced (about 3:1) comparedto strain 1100-SC38 (about 34:1), and total avermectin production was increasedapproximately 2.4-fold compared to strain 1100-SC38 transformed with pSE119 (TABLE 5). 53 011449 pSE189 was also transformed into protoplasts of a wild-type S. avermitilis strain.

Thiostrepton résistant transformants were isolated and analyzed by HPLC analysis of fermentation products. Total avermectins produced by S. avermitilis wild-type transformed with pSE189 were increased approximately 2.2-fold compared to wild-type S. avermitilis 5 transformed with pSE119 (TABLE 5). TABLE 5 S. avermitilisstrain (transforming plasmid) No. Trans-formantsTested Relative [B2] Relative [B1] Relative TotalAvermectins Avg. B2:B1 Ratio 1100-SC38 6 155 4.8 176 33.9 1100-SC38 (pSE119) 9 239 50.3 357 4.7 1100-SC38 (PSE189) 16 546 166 849 3.3 wild type 6 59 42 113 1.41 wild type(pSE119) 6 248 151 481 1.64 wild type(pSE189) 5 545 345 1,071 1.58

12. EXAMPLE: CHIMERIC PLASMID CONTAININGSEQUENCES FROM BOTH S. AVERMITILIS aveCORF AND $. HYGROSCOPICUS aveC HOMOLOG A hybrid plasmid designated as pSE350 was constructed that contains a 564 bp15 portion of the S. hygroscopicus aveC homolog replacing a 564 bp homologous portion of theS. avermitilis aveC ORF (FIGURE 7), as follows. pSE350 was constructed using a fîsaAlrestriction site that is conserved in both sequences (aveC position 225), and a Kpn\ restrictionsite that is présent in the S. avermitilis aveC gene (aveC position 810). The Kpnl site wasintroduced into the S. hygroscopicus DNA by PCR using the rightward primer 5’- 20 CTTCAGGTGTACGTGTTCG-3’ (SEQ ID NO:23) and the leftward primer 5’-GAACTGGTACCAGTGCCC-3' (SEQ ID NO:24) (supplied by Genosys Biotechnologies)using PCR conditions described in Section 7.1.10, above. The PCR product was digestedwith BsaAl and Kpnl, the fragments were separated by electrophoresis in a 1% agarose gel,and the 564 bp BsaAl/Kpnl fragment was isolated from the gel. pSE179 (described in Section 54 011449 7.1.10, above) was digested with Kpn\ and H/'ndlII, the fragments separated byelectrophoresis in a 1% agarose gel, and a fragment of -4.5 Kb was isolated from the gel.pSE179 was digested with W/ndlII and SsaAl, the fragments separated by electrophoresis in a1% agarose gel, and a -0.2 Kb SsaAl/HzndlII fragment isolated from the gel. The -4.5 KbΗίηύ\\\ΙΚρη\ fragment, the -0.2 Kb SsaAl/H/'ndlII fragment, and the 564 bp BsaMIKpnlfragment from S. hygroscopicus were ligated together in a 3-way ligation and the ligationmixture transformed into competent E. coli DH5a cells. Plasmid DNA was isolated fromampicillin résistant transformants and the presence of the correct insert was confirmed byrestriction analysis using Kpn\ and Aval. This plasmid was digested with H/ndlII and Xbal torelease the 1.2 Kb insert, which was then ligated with pWHM3 which had been digested withH/'ndlll and Xbal. The ligation mixture was transformed into competent E. coli DH5a cells,plasmid DNA was isolated from ampicillin résistant transformants, and the presence of thecorrect insert was confirmed by restriction analysis using /-Y/ndlII and Aval. This plasmid DNAwas transformed into E. coli DM1, plasmid DNA was isolated from ampicillin résistanttransformants, and the presence of the comect insert was confirmed by restriction analysisand DNA sequence analysis. This plasmid was designated as pSE350 and used to transformprotoplasts of S. avermitilis strain SE180-11. Thiostrepton résistant transformants of strainSE180-11 were isolated, the presence of erythromycin résistance was determined and ThiorErmr transformants were analyzed by HPLC analysis of fermentation products. Results showthat transformants containing the S. avermitilis/S. hygroscopicus hybrid plasmid hâve anaverage B2:B1 ratio of about 109:1 (TABLE 6). TABLE 6 S. avermitilis strain(transforming plasmid) No. transformantstested RelativeB2 Conc. RelativeB1 Conc. Avg. B2:B1 Ratio SE180-11 (none) 8 0 0 0 SE180-11 (pWHM3) 8 0 0 0 SE 180-11 (pSE350) 16 233 2 109 55

DEPOSIT OF BIQLOGICAL MATERIALS

The following biotogical material was deposited with the American Type CultureCollection (ATCC) at 12301 Parklawn Drive, Rockville, MD, 20852, USA, on January 29,1998, and was assigned the following accession numbers:

Plasmid plasmid pSE180plasmid pSE186

Accession No.209605209604 10 Ail patents, patent applications, and publications cited above are incorporated herein by référencé in their entirety.

The présent invention is not to be limited in scope by the spécifie embodimentsdescribed herein, which are intended as single illustrations of individual aspects of theinvention, and functionally équivalent methods and components are within the scope of the 15 invention. Indeed, various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art from the foregoingdescription and accompanying drawings. Such modifications are intended to fall within thescope of the appended daims. 56

Claims

011449 WHATIS CLAIMEO IS:

1. An isolated polynucleotide molécule comprising the complété avecopen-reading frame (ORF) of Streptomyces avermitilis or a substantial portionthereof, which isolated polynucleotide molécule lacks the next complété ORF that is 5 located downstream from the aveC ORF in situ In the Streptomyces avermitilischromosome, and which ORF has the nucléotide sequence of FIGURE 1 (SEQ IDNO;1).
2. The isolated polynucleotide molécule of claim 1, further comprisingnucléotide sequences that naturally flank the avec gene in situ in S avermititis.
3. An Isolated polynucleotide molécule, comprising a nucléotide sequence that is homologous to the Streptomyces avermrtilis AveC gene product-encoding nudeotide sequence of the avsC ORF presented in FIGURE 1 {SEQ IDNO;1) or substantial portion thereof.
4. An isolated polynucleotide molécule comprising the nudeotide 15 sequence of SEQ ID NO:3 or a substantial portion thereof, or a nudeotide sequence that is homologous to the nudeotide sequence of SEQ ID NO:3.
5. Ah oligonucleotide molécule that hybridizes to a polynucleotidemolécule having the nudeotide sequence of FIGURE 1 (SEQ 10 NQ:1) or SEQ IDNO:3, or to a polynudeotide molécule having a nudeotide sequence that is the 20 complément of the nudeotide sequence of FIGURE 1 (StQ ID ΝΟ.Ί) or SEQ IDNO:3, under highly stringent conditions.
6. The oligonudeotide molécule of daim 5, which is complementary tothe nudeotide sequence of FIGURE 1 (SEQ ID NO:1) or SEQ ID NO:3, or to apolynucleotide motecuîe having a nudeotide sequence that is the complément of the 25 nudeotide sequence of FIGURE 1 (SEQ ID N0:1) or SEQ ID NO;3.
7. A recombinant vector comprising a polynucleotide moléculecomprising a nudeotide sequence selected from lhe group consisting of thenucléotide sequence of the aveC ORF of FIGURE 1 (SEQ ID NO:1) or substantialportion thereof; and a nudeotide sequence that is homologous to the S. avermititis 30 AveC gene product-encoding ORF of FIGURE 1 (SEQ ID NO:1) or substantial portionthereof. 57 5 10 15 20 25 30 011449
8. The recombinant vector of claim 7, further comprising a nucieotidesequence encoding one or more reguiatory éléments, which reguiatory eiament-encoding nucléotide ssquence is in operative association with the nucléotidessquence of the polynucleotide molécule of the vector of ctaim 7.
9. The recombinant vector of claim 8, further comprising a nucléotidesequence encoding a selectable marker.
10. The recombinant vector of claim 9, which is plasmid pSE1S6 (ATCC209604).
11. A host call comprising the recombinant vector of claim 9.
12. The host cetl of claim 11. which is Strepiomyces avermitiiis.
13. A recombinant vector comprising a polynucleotide moléculecomprising a nucléotide sequence selected from the group consisting of thenucléotide sequence of SEQ ID NO:3 or a substantiel portion thereof; a nucléotidesequence thaï is homologous to the nucléotide sequence of SEQ ID NO:3; and anucléotide sequence that encodes a polypeptide having the amino acid sequence ofSEQ ID N0:4.
14. The recombinant vector of claim 13, Further comprising a nucléotidesequence encoding one or more reguiatory éléments, which reguiatory element-encoding nucléotide sequence is in operative association with the nucléotidesequence of the polynucleotide molécule of the vector of claim 13.
15. The recombinant vector of claim 14, further comprising a nucléotidesequence encoding a selectable marker.
16. A host cetl comprising the recombinant vector of daim 15.
17. The host oeil of claim 16, which is Straptomyces hygrOscopteus.
18. An isolated S. avôrmmïts AveC gene product having the amino acidsequence encoded by the S. avermftifis AveC gens product-encoding nudeotidesequence of plasmid pSE186 (ATCC 209604), or the amino acid sequence of SEQID NO:2 or substantiat portion thereof, or a homologous polypeptide thereof. 19 An isolated S. hygroscopicus AveC homolog gene product having theamino acid sequence of SEQ ID NO;4. 58 011449
20. A method for producing a recombinant AveC gene product, comprisingculturing a host cell transformed with a recombinant expression vector, saidrecombinant expression vector comprising a polynudeotide molécule having anucleolide sequence encoding the amino acid sequence of SEQ ID NO:2 or 5 substantial portion thereof, whïch polynudeotide molécule is in operative associationwïth one or more regulatory éléments that control expression of the polynudeotidemolécule in the host cell, under conditions conducivs lo the production of therecombinant AveC gene product, and recovering the AveC gene product from the cellculture. 10 21. A method for produdng a recombinant AveC homolog gene product, comprising culturing a host cell transformed with a recombinant expression vector,said recombinant expression vector comprising a polynudeotide molécule having anucléotide sequence encoding the amino add sequence of SEQ ID NO:4 orsubstantial portion thereof, which potynucleotide molécule is in operative association 15 with one or more regulatory éléments thaï control expression of the potynucleotidemolécule in the host cell, under conditions conducive to the production of therecombinant AveC homolog gene product, and recovering the AveC homolog geneproduct from the celi culture.
22. A polynudeotide molécule comprising a nucléotide sequence that is 20 otherwise the same as the S. avermitilis AveC gene product-encoding sequence ofplasmid pSE186 (ATCC 209604) or the nucléotide sequence of thesveC ORF of S.avermitills as presented in FIGURE 1 (SEQ ID NQ;1) or a degenerate variant thereof,but which nucléotide sequence further comprises one or more mutations, so that oellsof S, avermitilis strain ATCC 53692 in whïch the wild-type sveC allele has been 25 inactivated and that express the polynudeotide molécule comprising the mutatednucléotide sequence produce a reduced cydohexyl B2:cyclohexyl B1 ratio ofavermectins when fermented in the présence of cyclohexanecarboxylic acid than isproduced by celis of S. avermitilis strain ATCC 53692 that instead express only thewild-type aveC allele.
23. The polynudeotide molécule of daim 22, wherein the reduced ratio of cydohexyl B2:cyclohexyl 31 is less than 1.6:1. 53
24. The polynucleotide molécule of claim 23, wherein the reduced ratio ofcyclohexyl B2:cyclohexyl B1 is about 0.94:1.
25. The polynucleotide molécule of claim 24, wherein the mutation to theS. avermitilis aveC ORF of FIGURE 1 (SEQ ID NQ:1) encodes an amino acid 5 substitution at residue 139 of the AveC gene product from alanine to threonine.
26. The polynucleotide molécule of claim 23, wherein the reduced ratio ofcyclohexyl B2:cyclohexyl B1 is about 0.88:1.
27. The polynucleotide molécule of claim 26, wherein the mutation to theS. avermitilis aveC ORF of FIGURE 1 (SEQ ID NO:1) encodes an amino acid 1 o substitution at residue 138 of the AveC gene product from serine to threonine.
28. The polynucleotide molécule of claim 23. wherein the reduced ratio ofcyclohexyl S2:cydohexyl B1 is about 0.84:1.
29. The polynucleotide molscuie of claim 28, wherein the mutation to theS. avermitilis aveC ORF of FIGURE 1 (SEQ ID NO:1) encodes s first amino acid 15 substitution at résidus 138 of the AveC gene product from serine to threonine, and asecond amino acid substitution at residue 139 of the AveC gene product from alanineto threonine.
30. A potynucieotide molécule comprising a strong promoter in operativeassociation with the S. avermitilis aveC ORF of FIGURE 1 (SEQ ID NO:1).
31. The polynucleotide molacuie of claim 30, wherein the strong promoter is the srm£ promoter from Saccharopolyspora erythraea.
32. A polynucleotide molécule comprising a nucléotide sequenceencoding the AveC gene product-encoding ORF of FIGURE 1 (SEQ ID NO:1) thathas been inactivated by insertion into said nucléotide sequence of a heterologous 25 nucléotide sequence.
33. A polynucleotide molécule comprising an aveC allele that has beeninactivated by deleting a 640 bp Psil/Spftl fragment from the aveC ORF of FIGURE 1(SEQ ID NO:1).
34. A polynucleotide molécule comprising an aveC allele that has been30 inactivated by introducing a frameshifl mutation into the avec ORF of FIGURE 1 (SEQ ID NO:1). 60 011449
35. The polynucleotide molécule of claim 34, in which lhe frameshiftmutation was introduced by adding two G nucléotides after the C nucléotide at ntposition 471 of the aveC ORF of FIGURE 1 (SEQ ID NO:1).
36. A polynucleotide molécule comprising an aveC allele thai has been5 inaciivated by introducing a termination codon at the nucîeotide position that encodes amino acid 116 of the S. avermitilis AveC gene product encoded by the aveC ORF ofFIGURE 1 (SEQ ID NO:1).
37. A polynucleotide molécule comprising an aveC allele lhat has beeninactivated by introducing a first mutation at amino acid position 256 of the AveC 10 gene product that changes a glycine to an aspartate, and a second mutation atposition 275 of the AveC gene product that changes a tyrosine to a histidine.
38. A gene replacement vector comprising a polynucleotide moléculecomprising nucîeotide sequences that naturally flank the aveC ORF in situ in the S.avermitilis chromosome. 15 39. A recombinant vector comprising the polynucleotide moiecule of any of daims 22 to 37.
40. A recombinant vector comprising the polynucleotide moiecule of claim32, which vector is pSE180 (ATCC 209605).
41. A host Streptomyces cell comprising the recombinant vector of daim 20 39.
42. A method for identifying mutations of the aveC ORF capable of reducingthe class 2:1 ratio of avermeciins prcduced, comprising: (a) determining the class2:1 ratio of avermectins produced by cells of a strain of S. avermitilis in which thenative aveC allele has been inactivated, and into which a polynucleotide moiecule 25 comprising a nucîeotide sequence encoding a mutated AveC gene product has beenintroduced and is being expressed; (b) determining the class 2:1 ratio of avermectinsproduced by cells of the same strain of S. avermitilis as in step (a) but which insteadexpress only an aveC allele having the nudeotide sequence of the ORF of FIGURE 1{SEQ ID NO:1) or a nucîeotide sequence that is homologcus thereto; and (c) 30 comparing the dass 2:1 ratio of avermectins produced by the S. avermiïiïis cells ofstep (a) to the dass 2:1 ratio of avermectins produced by the S. avermiïiïis cells of 61 0Ί1449 step (b); such that if the dass 2:1 ratio cf svermectins prcduced by the$. evemitiliscells of step (a) is lower than the class 2:1 ratio of avermectins produced by the S.avermiïifis cells of step (b), then a mutation of theaveC ORF capable of reducing thedass 2:1 ratio of avermectins bas been identified. 5 43. A method for makïng novel strains of S. avermiïilis comprising cells that express a mutated avec alîele and that produces a reduced class 2:1 ratio ofavermectins compared to cells of the same strain of S. avenwtilis that insieadexpress only a wild'type avec allele, comprising transforming cells of a strain of S.avermiïilis wrth a vector that carnes a mutated aveC aliele that encodes a gene 10 product that reduces the class 2:1 ratio of avermectins produced by cells of a strain ofS. svermitilis exprèssing the mutated aveC allais compared to cells of the samestrain that instead express only the wild-type aveC aliele, and selecling transformedcells that produce avermectins ir. a reduced ciass 2:1 ratio compared to the class 2:1ratio produced by cells of the strain that instead express the wild-type aveC allele. 15 44. A method for making novel strains of S. avermiti/is, the cells of which comprise an inactivated aveC allele, comprising transforming ceils of a strain of-S.avermiïilis with a vector thai inactivâtes the aveC allele, and setecting transformedcells in which the aveC allele bas been inactivated.
45. The method of daim 44, wherein the vector is pSE180 (ATCC 20 209605).
46. A strain of S. avermitilis comprising cells expressing a mutated aveCallele which results in the production by the cells of avermectins in a reduced dass2:1 ratio compared to cells of the same strain that instead express only the wild-typeaveC allele.
47. The strain of toiaim 46, wherein the cells produce cyclohexyl B2:cyclohexyl 31 avermectins in a ratio of iess than 1.6:1.
48. The strain of daim 46, wherein the cells produce cyclohexylB2;cyclohexyl B1 avermectins in a ratio of about 0.94:1.
49. The strain of daim 46, wherein the cells produce cyciûhexyl30 B2:cyclohexyl B1 avermectins in a ratio of about 0.88:1. 62 011449
50. The strain cf ciaim 46, wherein the cells produce cyclohexylB2;cyclohexyl 31 avermectins in s ratio of about 0.84:1,
51. A strain of S. avermiïilis comprising cells in which the aveC gene hasbeen inactivated. 5 52. A process for producing avermectins, comprising culturing cells of s strain of S. avermitiiïs. which cells express s mutated aveC allele that encodes agene product that reduces the class 2:1 ratio of avermectins produced by cells of astrain of S. avermitiiïs expressing the mutated aveC allele compared to cells of thesame strain which do not express the mutated aveC allele but instead express only 10 the wild-type aveC allele, in culture media under conditions that permit or induce theproduction of avermectins therefrom, and recovering said avermectins from theculture.
53. A composition of avermectins produced by cells of a strain of S.avermitiiïs, which cells express a mutated aveC allele that encodes a gene product 15 that reduces the class 2:1 ratio of avermectins produced by cells of a strain ofS.avermitiiïs expressing the mutated aveC allele compared to cells of the same strainwhich do not express lhe mutated aveC allele but instead express only the wild-typeaveC allele, wherein the avermectins are produced in s reduced class 2:1 ratio ascompared to the class 2:1 ratio of avermectins produced by cells of the same strain of 20 S. avermiïilis that do not express the mutated aveC allele but instead express onlythe wild-type avsC aliele. 53 SEQUENCE LISTING <110> Pfizer Products Inc. (Ali Non-U.S. Applications) <120> STREPTOMYCES AVERMITILIS GENE DIRECTING THE PATIO OFB2:3i AVERMECTINS <130> PC9916A < 14 0> < 141> <150> 60/074,636 <151> 1998-02-13 <160> 24 10 < 170> <210><211><212><213> Patentln Ver. 2.0 - beta 1 1229 DNA Streptomyces avermitilis 15 <220> <221> <222> < 4 00> CDS ( 174 ). . (1085) 1 tcacgaaacc ggacacacca cacacacgaa ggtgagacag cgtgaaccca tccgagccgc 60 tcggcctgcc caacgaacgt gtagtagaca cccgaccgtc cgatgccacg ctctcacccg 120 aggccggcct gaacaggtca ggagcgctgc cccgtgaact gctgtcgttg ccg gtg 176 Val 1 gtg Val gtg val tgg gcc ggg Gly gtc Val ggc ctg ctg ttt ctg Leu gcc Ala ctg Leu cag Gin 15 gcg Ala tac Tyr 224 Trp Ala 5 Gly Leu Leu 10 Phe gtg ttc agc cgc tgg gcg gcc gac ggt ggc tac cgg ctg atc gag acg 272 Val Phe Ser Arg Trp Ala Ala Asp Gly Gly Tyr Arg Leu Ile Glu Thr 20 25 30 gcg ggc cag ggt cag ggc ggc agc aag gat acg ggg act acc gat gtg 320 Ai a Gly Gin Gly Gin Gly Gly Ser Lys Asp Thr Gly Thr Thr Asp Val 35 40 45 gtc tat ccc gtg att tcc gtc gtc tgc atc acc gcc gcg gcg gcg tgg 368 30 64 416 Val 50 Tyr Pro Val lie Ser 55 Val Val Cys Ile Thr 60 Ala Ala Ala Ala Trp 65 CtC ttc cgg agg tgc cgt gtc gaa ega cgg ctg ctg ttc gac gcc ctt Leu Phe Arg Arg Cys Arg Val Glu Arg Arg Leu Leu Phe Asp Ala Leu 70 75 80 etc etc etc ggg ctg ctg ttc gcg age tgg cag age ccg etc atg aac Leu Phe Leu Gly Leu Leu Phe Ala Ser Trp Gin Ser Pro Leu Met Asn 85 90 95 tgg ttc cat tcc gtt etc gtc tcc aac gcg agt gtg tgg ggc gcg gtg Trp Phe His Ser Val Leu Val Ser Asn Ala Ser Val Trp Gly A.la Val 100 105 110 ggt tcc tgg ggt ccg tat gtg ccc ggc tgg cag ggg gcg ggc ccg ggt Gly Ser Trp Gly Pro Tyr Val Pro Gly Trp Gin Gly Ala Gly Pro Gly 115 120 125 gcg gag gcg gaa atg ccg ctg gcg teg gcc tcc gtc tgc atg teg gct Al a Glu Ala Glu Met Pro Leu Ala Ser Ala Ser Val Cys Met Ser Ala 130 135 140 145 ctg atc gtc acc gtg ctg tgc age aag gea ctg ggg tgg atc aag gcc Leu Ile Val Thr Val Leu Cys Ser Lys Ala Leu Gly Trp Ile Lys Ala 150 155 160 cgc cgg ccg gea tgg cgg acc tgg cgg ctg gtc ctg gcc gtg ttc ttc Arg Arg Pro Ala Trp Arg Thr Trp Arg Leu Val Leu Ala Val Phe Phe 165 170 175 a:c ggc atc gtg etc ggt ctg tcc gag ccg ctg ccg tcc gcc tcc ggg lie Gly Ile Val Leu Gly Leu Ser Glu Pro Leu Pro Ser Ala Ser Gly 180 185 190 atc age gta tgg gcc aga gcg ctg ccc gag gtg acc ttg tgg agt ggc Ile Ser Val Trp Ala Arg Ala Leu Pro Glu Val Thr Leu Trp Ser Gly 195 200 205 gag tgg tac cag ttc ccc gtg tat cag gcg gtc ggt tcc ggc ctg gtc Glu Trp Tyr Gin Phe Pro Val Tyr Gin Ala Val Gly Ser Gly Leu Val 210 215 220 225 tgc tgc atg ctg ggc teg ctg cgc ttc ttc cgc gac gaa cgc gat gag Cys Cys Met Leu Gly Ser Leu Arg Phe Phe Arg Asp Glu Arg Asp Glu 230 235 240 tcç tgg gtg gaa cgg gga gcc tgg cgg ttg ccg caa cgg gea gcg aac Ser Trp Val Glu Arg Gly Ala Trp Arg Leu Pro Gin Arg Ala Ala Asn 245 250 255 464 512 560 608 656 704 800 848 896 752 65 944 tgg Trp Qcg Al a cgt Arg 260 ttc Phe etc Leu gcc Al a gtg Val gtc Val 265 ggt Gly ggg Gly gtg Val aat Asn gcc Ala 270 gtg Val atg Met ttc PhiS 992 etc tac acc tgt ttc est atc etc ctg tcc etc gtc ggt gga cag ccg 1040 Leu Tyr Thr Cys Phe His lie Leu Leu Ser Leu Val Gly Gly Gin Prc n cz. ι 0 280 285 ccc gac caa ctg ccg gac tcc ttc caa gcg ccg gcc gct tac tga 1085 Pro Asp Gin Leu Pro Asp Ser Phe Gin Ala Pro Ala Ala Tyr 290 295 300 gttcagggca ggtcggagga gacggagaag gggaggcgac cggagttccg gtcacctccc 1145 ctttgtgcat gggtggacgg ggatcacgct cccatggcgg cgggctcctc cagacgcacc 1205 acactcctcg gttcagcgat catg 1229 <210 2 <211> 303 <212> PRT <213> Streptomyces avermitilis <400> 2 Ala Leu Gin 15 Ala Val 1 Val Val Trp Ala Gly 5 Val Gly Leu Leu Phe10 Leu Tyr Val Phe Ser Arg Trp Ala Ala Asp Gly Gly Tyr Arg Leu Ile Glu 20 25 30 Thr Ala Gly Gin Gly Gin Gly Gly Ser Lys Asp Thr Gly Thr Thr Asp 35 40 45 Val Val Tyr Pro Val Ile Ser Val Val Cys Ile Thr Ala Ala Ala Ala 50 55 60 Trp Leu Phe Arg Arg Cys Arg Val Glu Arg Arg Leu Leu Phe Asp Ala 65 70 75 80 Leu Leu Phe Leu Gly Leu Leu Phe Ala Ser Trp Gin Ser Pro Leu Met 85 90 95 Asn Trp Phe His Ser Val Leu Val Ser Asn Ala Ser Val Trp Gly Ala 100 105 110 Val Gly Ser Trp Gly Pro Tyr Val Pro Gly Trp Gin Gly Ala Gly Pro 115 120 125 66 01 14 49 Gly Ala 130 Glu Ala Glu Met Pro 135 Leu Ala Ser Ala Ser 140 Val Cys Met Ser Ala Leu Ile Val Thr Val Leu Cys Ser Lys Ala Leu Gly Trp Ile Lys 145 150 155 160 Ala Arg Arg Pro Ala Trp Arg Thr Trp Arg Leu Val Leu Ala Val Phe 165 170 175 Phe lie Gly Ile Val Leu Gly Leu Ser Glu Pro Leu Pro Ser Ala Ser 180 185 190 Gly Ile Ser Val Trp Ala Arg Ala Leu Pro Glu Val Thr Leu Trp Ser 195 200 205 Gly Glu Trp Tyr Gin Phe Pro Val Tyr Gin Ala Val Gly Ser Gly Leu 210 215 220 Val Cys Cys Met Leu Gly Ser Leu Arg Phe Phe Arg Asp Glu Arg Asp 225 230 235 240 Glu Ser Trp Val Glu Arg Gly Ala Trp Arg Leu Pro Gin Arg Ala Ala 245 250 255 Asn Trp Ala Arg Phe Leu Ala Val Val Gly Gly Val Asn Ala Val Met 260 265 270 Phe Leu Tyr Thr Cys Phe His Ile Leu Leu Ser Leu Val Gly Gly Gin 275 280 285 Pro Pro Asp Gin Leu Pro Asp Ser Phe Gin Ala Pro Ala Ala Tyr 290 295 300 <210> 3 <211> 1150 <212> DNA <213> Streptomyces hygroscopicus <220> <221> CDS <222> (58)..(990) <400> 3 gtcgacgaag accggccgga ggccgtcggc cgggccgata ccgtacgcgg cctgcgg gtg ttc acc ctt ccc gta aca ctg tgg gcg tgt gtc ggc gcg ctg gtg Val Phe Thr Leu Pro Val Thr Leu Trp Ala Cys Val Gly Ala Leu Val 1 5 10 15 57 105 67 U114 49

68 011449 210 215 220 gct Al a 225 tcg Ser gcg Ala etc Leu ttc Phe ggc Gly 230 gcc Ala tet Ser ttg Leu ggg Gly gcc Ala 235 gcg Ala cgc Arg cac His ttt Phe cgc Arg 240 777 aac cgg cgc ggc gaa aeg tgt ctg gag tcc ggg gcg gcc etc cta ccg 825 Asn Arg Arg Gly Glu Thr Cys Leu Glu Ser Gly Ala Al a Leu Leu Pro 245 250 255 gag ggc ccg agg cca tgg gtc cgg ctg ctg gcg gtg gtg ggc ggg gcc 873 Glu Gly Pro Arg Pro Trp Val Arg Leu Leu Ala Val val Gly Gly Ala 260 265 270 aac atc agc atc gcc etc tac acc ggc gea cac ggc gea cac atc ctg 921 Asn Ile Ser Ile Ala Leu Tyr Thr Gly Ala His Gly Ala His Ile Leu 275 280 285 ttc tcg ctg atg gac ggc gct ccc ccg gac cgg etc ccc gaa ttc ttc 969 Phe Ser Leu Met Asp Gly Ala Pro Pro Asp Arg Leu Pro Glu Phe Phe 290 295 300 cgt ccg gcg gcc ggc tac tga gaccgccggc accacccacg tacccgatgt 1020 Arg Pro Ala Ala Gly Tyr 305 310 gcgcgatgtg cctgatgcgc ctgatgtacc cggggtgtca tcggctcacc tgtggcgcct 1080 catgcggtga gcgctccgcc tcgtccttgt tccggctcct gggctccacg accatacgga 1140 gcggccgggg 1150 20 <21Ο> 4 <211> 310 <212> PRT <213> Streptomyces hygroscopicus <400> 4 Val Phe Thr Leu Pro Val Thr Leu Trp Ala Cys Val Gly Ala Leu Val 1 5 10 15 Leu Gly Leu Gin Val Tyr Val Phe Ala Ala Trp Leu Ala Asp Ser Gly 20 25 30 Tyr Arg Ile Glu Lys Ala Ser Pro Ala Arg Gly Gly Gly Asp Ser Glu 35 40 45 Arg Ile Ala Asp Val Leu Ile Pro Leu Leu Ser Val Val Gly Ala Val 50 55 60 30 69 011449 Val Leu Ala Val Cys65 Thr Phe Asp Ala Ser35 Ser Pro Leu Met Asn100 $ Val Prie Gly Ala Val115 Gly Ala Gly Ala His130 Ile Cys Met Thr Ala145 Gly Leu Ala Ala Ala165 10 Ala Leu Gly Phe Leu180 Val Ser Phe Ala Gly195 Thr Ile Trp Ser Gly210 Ala Ser Ala Leu Phe225 15 Asn Arg Arg Gly Glu245 Glu Gly Pro Arg Pro260 Asn île Ser Ile Ala275 20 Phe Ser Leu Met Asp290 A.rg Pro Ala Ala Gly305 Leu Tyr Arg Arg Cys Arg70 75 Leu Phe Ile Gly Leu Leu 90 Trp Ile Asn Pro Val Leu105 Ala Ser Trp Gly Pro Tyr120 Gin Glu Ala Glu Leu Pro135 Met Met Ala Ala Val Ala150 155 Arg Trp Pro Arg Leu Gly170 Leu Val Val Leu Leu Asp185 Val Ser Val Trp Thr Arg200 His Trp Tyr Gin Phe Pro215 Gly Ala Ser Leu Gly Ala230 235 Thr Cys Leu Glu Ser Gly250 Trp Val Arg Leu Leu Ala265 Leu Tyr Thr Gly Ala His280 Gly Ala Pro Pro Asp Arg 295 Tyr 310 Ala Arg Arg Arg Leu80 Ser Ala Ser Trp Gin95 Ala Ser Asn Val Asn110 Val Pro Gly Trp Gin125 Leu Ala Thr Leu Ser140 Cys Gly Lys Gly Met160 Pro Leu Arg Leu Ile175 Ile Ala Glu Pro Leu190 Ala Val Pro Glu Leu205 Leu Tyr Gin Met Val220 Ala Arg His Phe A.rg240 Ala Ala Leu Leu Pro255 Val Val Gly Gly Ala270 Gly Ala His Ile Leu285 Leu Pro Glu Phe Phe300 70 <210> 5 <211> 215 <212> PRT <213> Streptomyces griseochromoaenes 011449 5 <400> 5 Al a Leu Gly Ala Val 10 Phe Leu Val Leu Gin 15 Val Val 1 Ile Gly Trp Ala 5 Tyr Val Phe Ala Arg Trp Thr Ala Asp Gly Gly Tyr His Leu Ala Asp 20 25 30 Val Ser Gly Pro Asp Gly Arg Glu Pro Gly His Arg Arg Ile Ile Asp 35 40 45 10 Val Leu Leu Pro Ala Leu ser Met Ala Gly Val Val Gly Leu Ala Phe 50 55 60 Trp Leu Val Arg Arg Trp Arg Ala Glu Arg Arg Leu Ser Phe Asp Ala 65 70 75 80 Leu Leu Phe Thr Gly Val Leu Phe Ala Gly Trp Leu Ser Pro Leu Met 85 90 95 Asn Trp Phe His Pro Val Leu Met Ala Asn Thr His Val Trp Gly Ala 100 105 110 15 Val Gly Ser Trp Gly Pro Tyr Val Pro Gly Trp Arg Gly Leu Pro Pro 115 120 125 Gly Lys Glu Ala Glu Leu Pro Leu Val Thr Phe Ser Leu Gly Ser Thr 130 135 140 Val Leu Leu Gly Val Leu Gly Cys Cys Gin Val Met Ser Arg Val Arg 145 150 155 160 Glu Arg Trp Pro Gly Val Arg Pro Trp Gin Leu Val Gly Leu Ala Phe 165 170 175 20 Leu Thr Ala Val Ala Phe Asp Leu Ser Glu Pro Phe Ile Ser Phe Ala 180 185 190 Gly Val Ser Val Trp Ala Arg Ala Leu Pro Thr Val Thr Leu Trp Arg 195 200 205 Gly Ala Trp Tyr Arg Ala Arg 210 215 25 <210> 6 71 <211> 18 <212> DNA <213> Streptomyces avermitilis <400> 6 tcacgaaacc ggacacac 18 011449 <210> 7<211> 18<212> DNA <213> Streptomyces avermitilis yQ <4QQ> 7 catgatcgct gaaccgag 18 <210> 8<211> 20<212> DNA <213> Streptomyces avermitilis <400> 8 ggttccggat gccgttctcg 20 <210> S<211> 21<212> DNA <213> Streptomyces avermitilis <400> 9 aactccggtc gactcccctt c 21 <210> 10<211> 19<212> DNA <213> Streptomyces avermitilis <400> 10 gcaaggatac ggggactac <210> 11<211> 18<212> DNA <213> Streptomyces avermitilis <400> 11 72 18 5 gcccctggcg acg gaaccgaccg cctgatac 011449 10 15 20 25 <210 <211> <212> <213> iZ 43 DNA Streptomyces avermitilis <400> 12 gggggcgggc ccgggtgcgg aggcggaaat <210> 13 <211> 20 <212> DNA <213> Streptomyces avermitilis <400> 13 ggaaccgacc gcctgataca <210> 14 <211> 46 <212> DNA <213> Streptomyces avermitilis <400 14 gggggcgggc ccgggtgcgg aggcggaaat <210> 15 <211> 20 <212> DNA <213> Streptomyces avermitilis <400> 15 ggaacatcac gacattcacc <210> 16 <211> 18 <212> DNA <213> Streptomyces avermitilis gccgctggcg acgacc 20 46 20 <400 16 aacccatccg agccgctc <210> 17<211> 17 18 30 73 011449 <212> DNA <213> Streptomyces avermitilis <400> 17 tcggcctgcc aacgaac 27 <210> 18 <211> 18 <212> DNA <213> Streptomyces avermitilis <400> 18 ccaacgaacg tgtagtag 18 <210> 19 <211> 20 <212> DNA <213> Streptomyces avermitilis <400> 19 tgcaggcgta cgtgttcagc 20 <210> 20 <211> 17 <212> DNA <213> Streptomyces avermitilis <400> 20 catgatcgct gaaccga 17 25 <210> 21 <211> 20 <212> DNA <213> Streptomyces avermitilis <400> 21 catgatcgct gaaccgagga 20 <210> 22 <211> 20 <212> DNA <213> Streptomyces avermitilis <400> 22 aggagtgtgg tgcgtctgga 20 74 01 1449 <210> 23 <211> 19 <212> DNA <213> Streptomyces avermitilis <400> 23 crtcaggtgt acgtgttcg <210> 24 <211> 18 <212> DNA <213> Streptomyces avermitilis <400> 24 gaactggtac cagtgccc 75