AU752343B2 - Streptomyces avermitilis gene directing the ratio of B2:B1 avermectins - Google Patents

Description

WO 99/41389 PCT/IB99/00130 STREPTOMYCES A VERMITILIS GENE DIRECTING THE RATIO OF B2:B1 AVERMECTINS 1. FIELD OF THE INVENTION The present invention is directed to compositions and methods for producing avermectins, and is primarily in the field of animal health. More particularly, the present invention relates to polynucleotide molecules comprising nucleotide sequences encoding an AveC gene product, which can be used to modulate the ratio of class 2:1 avermectins produced by fermentation of cultures of Streptomyces avermitilis, and to compositions and methods for screening for such polynucleotide molecules. The present invention further relates to vectors, transformed host cells, and novel mutant strains of S. avermitilis in which the aveC gene has been mutated so as to modulate the ratio of class 2:1 avermectins produced.

2. BACKGROUND OF THE INVENTION 2.1. Avermectins Streptomyces species produce a wide variety of secondary metabolites, including the avermectins which comprise a series of eight related sixteen-membered macrocyclic lactones having potent anthelmintic and insecticidal activity. The eight distinct but closely related compounds are referred to as Ala, Alb, A2a, A2b, Bla, B1b, B2a and B2b. The series of compounds refers to the natural avermectin where the substituent at the C25 position is sec-butyl, and the series refers to those compounds where the substituent at the position is isopropyl. The designations and refer to avermectins where the substituent at the C5 position is methoxy and hydroxy, respectively. The numeral refers to avermectins where a double bond is present at the C22,23 position, and the numeral "2" refers to avermectins having a hydrogen at the C22 position and a hydroxy at the C23 position. Among the related avermectins, the B1 type of avermectin is recognized as having the most effective antiparasitic and pesticidal activity, and is therefore the most commercially desirable avermectin.

The avermectins and their production by aerobic fermentation of strains of S.

avermitilis are described in United States Patents 4,310,519 and 4,429,042. The biosynthesis of natural avermectins is believed to be initiated endogenously from the CoA thioester analogs of isobutyric acid and S-(+)-2-methyl butyric acid.

A combination of both strain improvement through random mutagenesis and the use of exogenously supplied fatty acids has led to the efficient production of avermectin analogs.

Mutants of S. avermitilis that are deficient in branched-chain 2-oxo acid dehydrogenase (bkd deficient mutants) can only produce avermectins when fermentations are supplemented with WO 99/41389 PCT/IB99/00130 fatty acids. Screening and isolation of mutants deficient in branched-chain dehydrogenase activity S. avermitilis, ATCC 53567) are described in European Patent (EP) 276103.

Fermentation of such mutants in the presence of exogenously supplied fatty acids results in production of only the four avermectins corresponding to the fatty acid employed. Thus, supplementing fermentations of S. avermitilis (ATCC 53567) with S-(+)-2-methylbutyric acid results in production of the natural avermectins Ala, A2a, Bla and B2a; supplementing fermentations with isobutyric acid results in production of the natural avermectins Alb, A2b, B1b, and B2b; and supplementing fermentations with cyclopentanecarboxylic acid results in the production of the four novel cyclopentylavermectins A1, A2, B1, and B2.

If supplemented with other fatty acids, novel avermectins are produced. By screening over 800 potential precursors, more than 60 other novel avermectins have been identified.

(See, Dutton et al., 1991, J. Antibiot. 44:357-365; and Banks et al., 1994, Roy. Soc.

Chem. 147:16-26). In addition, mutants of S. avermitilis deficient in activity produce essentially only the B analog avermectins. Consequently, S. avermitilis mutants lacking both branched-chain 2-oxo acid dehydrogenase and activity produce only the B avermectins corresponding to the fatty acid employed to supplement the fermentation. Thus, supplementing such double mutants with methylbutyric acid results in production of only the natural avermectins Bla and B2a, while supplementing with isobutyric acid or cyclopentanecarboxylic acid results in production of the natural avermectins Bib and B2b or the novel cyclopentyl B1 and B2 avermectins, respectively. Supplementation of the double mutant strain with cyclohexane carboxylic acid is a preferred method for producing the commercially important novel avermectin, cyclohexylavermectin B1 (doramectin). The isolation and characteristics of such double mutants, S. avermitilis (ATCC 53692), is described in EP 276103.

2.2. Genes Involved In Avermectin Biosynthesis In many cases, genes involved in production of secondary metabolites and genes encoding a particular antibiotic are found clustered together on the chromosome. Such is the case, with the Streptomyces polyketide synthase gene cluster (PKS) (see Hopwood and Sherman, 1990, Ann. Rev. Genet. 24:37-66). Thus, one strategy for cloning genes in a biosynthetic pathway has been to isolate a drug resistance gene and then test adjacent regions of the chromosome for other genes related to the biosynthesis of that particular antibiotic. Another strategy for cloning genes involved in the biosynthesis of important metabolites has been complementation of mutants. For example, portions of a DNA library from an organism capable of producing a particular metabolite are introduced into a nonproducing mutant and transformants screened for production of the metabolite.

Additionally, hybridization of a library using probes derived from other Streptomyces species has been used to identify and clone genes in biosynthetic pathways.

Genes involved in avermectin biosynthesis (ave genes), like the genes required for biosynthesis of other Streptomyces secondary metabolites PKS), are found clustered on the chromosome. A number of ave genes have been successfully cloned using vectors to complement S. avermitilis mutants blocked in avermectin biosynthesis. The cloning of such genes 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 region involving the C22,23 dehydration step (aveC) to a 4.82 Kb BamH1 fragment of S. avermitilis, as well as mutations in the aveC gene that result in the production of a single component B2a producer. Since ivermectin, a potent anthelmintic compound, can be produced chemically from avermectin B2a, such a single component producer of avermectin B2a is considered particularly useful for commercial production of ivermectin.

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

3. Summary of the Invention 20 The present invention provides in a first aspect an isolated polynucleotide molecule comprising the complete aveC ORF of S. avermitilis, which isolated polynucleotide molecule lacks the next complete ORF that is located downstream from the aveC ORF in situ in the S. avermitilis chromosome and which ORF comprises the nucleotide sequence of FIGURE 1 (SEQ ID NO:1). The isolated polynucleotide molecule of the present invention preferably comprises a nucleotide sequence that is the same as the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or that is the same as the nucleotide sequence of the aveC ORF of FIGURE 1 (SEQ ID NO:1) or substantial portion thereof.

The present invention further provides in a second aspect an isolated polynucleotide molecule comprising a nucleotide sequence that is homologous to the S. avermitilis AveC gene product-encoding nucleotide sequence of the aveC ORF, which ORF comprises the nucleotide sequence presented in FIGURE 1 (SEQ ID NO:1).

A third aspect of the invention provides an oligonucleotide molecule that hybridizes So a polynucleotide molecule having the nucleotide sequence of FIGURE 1 (SEQ ID [I:\DayLib\L1BA]41429spec.doc:gcc 4 NO:1), or to a polynucleotide molecule having a nucleotide sequence that is the complement of the nucleotide sequence of FIGURE 1 (SEQ ID NO:1), under highly stringent conditions.

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

The present invention further provides oligonucleotides that hybridize to a o0 polynucleotide molecule having the nucleotide sequence of FIGURE 1 (SEQ ID NO: 1) or to a polynucleotide molecule having a nucleotide sequence which is the complement of the nucleotide sequence of FIGURE 1 (SEQ ID NO:1).

A fourth aspect of the present invention provides a recombinant vector comprising a polynucleotide molecule comprising a nucleotide sequence encoding a protein comprising 15 the amino acid sequence of SEQ ID NO:2.

A fifth aspect of the present invention provides a host cell comprising the recombinant vector of the fourth aspect of the invention.

A sixth aspect of the present invention provides an isolated S. avermitilis AveC gene product comprising the amino acid sequence encoded by the S. avermitilis AveC 20 gene product-encoding nucleotide sequence of plasmid pSE186 (ATCC 209604) or the amino acid sequence of SEQ ID NO:2.

.The present invention further provides recombinant cloning vectors and expression vectors, that are useful in cloning or expressing a polynucleotide of the present invention, including polynucleotide molecules comprising the aveC ORF of S. avermitilis or an aveC homolog ORF. In a non-limiting embodiment, the present invention provides plasmid pSE186 (ATCC 209604), which comprises the entire ORF of the aveC gene of S. avermitilis. The present invention further provides transformed host cells comprising a polynucleotide molecule or recombinant vector of the invention, and novel strains or cell lines derived therefrom.

The present invention further provides a recombinantly expressed AveC gene product or AveC homolog gene product, or a substantial portion thereof, that has been substantially purified or isolated, as well as homologs thereof. The present invention further provides in a seventh aspect a method for producing a recombinant AveC gene product, comprising culturing a host cell transformed with a recombinant expression r 351- vector- said recombinant expression vector comprising a polynucleotide molecule [I:\DayLib\LBA]41 429spec.doc:gcc comprising a nucleotide sequence encoding an amino acid sequence comprising the amino acid sequence of SEQ ID NO:2, which polynucleotide molecule is in operative association with one or more regulatory elements that control expression of the polynucleotide molecule in the host cell, under conditions conducive to the production of the recombinant AveC gene product, and recovering the AveC gene product from the cell culture.

The present invention further provides in an eighth aspect a polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604) or the to nucleotide sequence of the aveC ORF of S. avermitilis as presented in FIGURE 1 (SEQ ID NO:1 or a degenerative variant thereof, but which nucleotide sequence further comprises one or more mutations, such that cells of S. avermitilis strain ATCC 53692 in which the wild-type aveC allele has been inactivated and that express the polynucleotide molecule comprising the mutated nucleotide sequence produce a reduced cyclohexyl 15 B2:cyclohexyl B1 ratio of avermectins when fermented in the presence of cyclohexanecarboxylic acid than is produced by cells of S. avermitilis strain ATCC 53692 that instead express only the wild-type aveC allele. According to the present invention, such polynucleotide molecules can be used to produce novel strains of S. avermitilis that iexhibit a detectable change in avermectin production compared to the same strain that 20 instead expresses only the wild-type aveC allele. In a preferred embodiment, such polynucleotide molecules are useful to produce novel strains of S. avermitilis that produce avermectins in a reduced class 2:1 ratio compared to that from the same strain that instead expresses only the wild-type aveC allele. In a further preferred embodiment, such o. polynucleotide molecules are useful to produce novel strains of S. avermitilis that produce increased levels of avermectins compared to the same strain that instead expresses only the wild-type aveC allele. In a further preferred embodiment, such polynucleotide molecules are useful to produce novel strains of S. avermitilis in which the aveC gene has been inactivated.

In a ninth aspect of the present invention there is provided a polynucleotide molecule comprising a strong promoter in operative association with the S. avermitilis aveC ORF of FIGURE 1 (SEQ ID NO:1).

In a tenth aspect of the present invention there is provided a polynucleotide molecule comprising a nucleotide sequence encoding the AveC gene product-encoding ^IA^ ORF of FIGURE 1 (SEQ ID NQ:1) that has been inactivated by insertion into said ucleotide sequence of a heterologous nucleotide sequence.

[I:\DayLib\LIBA]41429spec.doc:gcc In an eleventh aspect of the present invention there is provided a polynucleotide molecule comprising an aveC allele that has been inactivated by deleting a 640 bp Pstl/Sphl fragment from the aveC ORF of FIGURE 1 (SEQ ID NO:1).

In a twelfth aspect of the present invention there is provided a polynucleotide molecule comprising an aveC allele that has been inactivated by introducing a frameshift mutation into the aveC ORF of FIGURE 1 (SEQ ID NO:1).

In a thirteenth aspect of the present invention there is provided a polynucleotide molecule comprising an aveC allele that has been inactivated by introducing a termination codon at the nucleotide position that encodes amino acid 116 of the S. avermitilis AveC gene product encoded by the aveC ORF of FIGURE 1 (SEQ ID NO:1).

In a fourteenth aspect of the present invention there is provided a polynucleotide molecule comprising an aveC allele that has been inactivated by introducing a first mutation at amino acid position 256 of the S. avermitilis AveC gene product that changes a glycine to an aspartate, and a second mutation at position 275 of the S. avermitilis AveC gene product that changes a tyrosine to a histidine.

In a fifteenth aspect of the present invention there is provided a gene replacement vector comprising a polynucleotide molecule comprising nucleotide sequences that naturally flank the aveC ORF in situ in the S. avermitilis chromosome.

The present invention provides in a sixteenth aspect a method for identifying 20 mutations of the aveC ORF capable of producing the class 2:1 ratio of avermectins produced, comprising: determining the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis in which the native aveC allele has been inactivated, and into which a polynucleotide molecule comprising a nucleotide sequence encoding a mutated AveC gene product has been introduced and is being expressed; determining 25 the class 2:1 ratio of avermectins produced by cells of the same strain of S. avermitilis as in step but which instead express only an aveC allele having the nucleotide sequence of the ORF of FIGURE 1 (SEQ ID NO:1) or a nucleotide sequence that is homologous thereto; and comparing the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step to the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step such that if the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step is lower than the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step then a mutation of the aveC ORF capable of reducing the class 2:1 ratio of avermectins has been identified. In a preferred i e% odiment, the class 2:1 ratio of avermectins is reduced by the mutation.

[I:\DayLib\LIBA]41429spec.doc:gcc In a further preferred embodiment, the present invention provides a method for identifying mutations of the aveC ORF or genetic constructs comprising the aveC ORF capable of altering the amount of avermectins produced, comprising: determining the amount of avermectins produced by cells of a strain of S. avermitilis in which the native aveC allele has been inactivated, and into which a polynucleotide molecule comprising a nucleotide sequence encoding a mutated AveC gene product or comprising a genetic construct comprising a nucleotide sequence encoding an AveC gene product has been introduced and is being expressed; determining the amount of avermectins produced by cells of the same strain of S. avermitilis as in step but which instead express only an to aveC allele having the nucleotide sequence of the ORF of FIGURE 1 (SEQ ID NO:1) or a nucleotide sequence that is homologous thereto; and comparing the amount of avermectins produced by the S. avermitilis cells of step to the amount of avermectins *produced by the S. avermitilis cells of step such that if the amount of avermectins produced by the S. avermitilis cells of step is different from the amount of avermectins 15 produced by the S. avermitilis cells of step then a mutation of the aveC ORF or a genetic construct capable of altering the amount of avermectins has been identified. In a preferred embodiment, the amount of avermectins produced is increased by the mutation.

The present invention further provides recombinant vectors that are useful for making novel strains of S. avermitilis having altered avermectin production. For example, 20 the present invention provides vectors that can be used to target any of the polynucleotide S* molecules comprising the mutated nucleotide sequences of the present invention to the site of the aveC gene of the S. avermitilis chromosome to either insert into or replace the aveC ORF or a portion thereof by homologous recombination. According to the present S...invention, however, a polynucleotide molecule comprising a mutated nucleotide sequence of the present invention provided herewith can also function to modulate avermectin biosynthesis when inserted into the S. avermitilis chromosome at a site other than at the aveC gene, or when maintained episomally in S. avermitilis cells. Thus, the present invention also provides vectors comprising a polynucleotide molecule comprising a mutated nucleotide sequence of the present invention, which vectors can be used to insert the polynucleotide molecule at a site in the S. avermitilis chromosome other than at the aveC gene, or to be maintained episomally. In a preferred embodiment, the present invention provides gene replacement vectors that can be used to insert a mutated aveC allele into the S. avermitilis chromosome to generate novel strains of cells that produce avermectins in a reduced class 2:1 ratio compared to the cells of the same strain which S 35 n.stead.express only the wild-type aveC allele.

[I:\DayLib\LIBA]41429spc.dc:gcc The present invention further provides methods for making novel strains of S.

avermitilis comprising cells that express a mutated aveC allele and that produce altered ratios and/or amounts of avermectins compared to cells of the same strain of S. avermitilis that instead express only the wild-type aveC allele. In a seventeenth aspect, the present invention provides a method for making novel strains of S. avermitilis comprising cells that express a mutated aveC allele and that produce a reduced class 2:1 ratio of avermectins compared to cells of the same strain of S. avermitilis that instead express only a wild-type aveC allele, comprising obtaining cells of a strain of S. avermitilis, mutating the aveC allele in a cell of step or introducing into a cell of step a 0o mutated aveC allele or degenerate variant thereof which mutated aveC allele or degenerative variant thereof encodes a gene product that alters the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis expressing the mutated allele compared to cells of the same strain that instead express only the wild-type aveC allele, and selecting transformed cells from step that produce avermectins in an altered class S 15 2:1 ratio compared to the class 2:1 ratio produced by cells of the strain that instead express the wild-type aveC allele. In a preferred embodiment, the class 2:1 ratio of avermectins produced is reduced in cells of the novel strain.

In a further preferred embodiment, the present invention provides a method for making novel strains of S. avermitilis comprising cells that produce altered amounts of avermectin, comprising transforming cells of a strain of S. avermitilis with a vector that carries a mutated aveC allele or a genetic construct comprising the aveC allele, the expression of which 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 the same strain that instead express only the wild-type aveC allele, and selecting transformed cells that produce avermectins in an altered amount compared to the amount of avermectins produced by cells of the strain that instead express only the wild-type aveC allele. In a preferred embodiment, the amount of avermectins produced is increased in cells of the novel strain.

In an eighteenth aspect, the present invention provides a method for making novel strains of S. avermitilis, the cells of which comprise an inactivated aveC allele, comprising introducing into cells of a strain of S. avermitilis a vector that inactivates the aveC allele, and selecting those cells in which the aveC allele has been inactivated.

The present invention further provides novel strains of S. avermitilis comprising

S

1 <ls that have been transformed with any of the polynucleotide molecules or vectors c ~prising a mutated nucleotide sequence of the present invention. In a preferred [I:\DayLib\LIBA]41429spec.doc:gcc embodiment, the present 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 an altered class 2:1 ratio compared to cells of the same strain that instead express only the wild-type aveC allele. In a nineteenth aspect of the invention, there is provided a strain of S. avermitilis comprising cells expressing a mutated aveC allele which results in the production by the cells of avermectins in a reduced class 2:1 ratio compared to cells of the same strain that instead express only the wild-type aveC allele. Such novel strains are useful in the largescale production of commercially desirable avermectins such as doramectin.

In a further preferred embodiment, the present invention provides novel strains of S.

avermitilis comprising cells which express a mutated aveC allele, or a genetic construct comprising the aveC allele, in place of, or in addition to, the wild-type aveC allele, which results in the production by the cells of an altered amount of avermectins compared to the amount of avermectins produced by cells of the same strain that instead express only the 15 wild-type aveC allele. In a preferred embodiment, the novel cells produce an increased amount of avermectins.

In a further preferred embodiment, the present invention provides novel strains of S.

avermitilis comprising cells in which the aveC gene has been inactivated. Such strains are useful both for the different spectrum of avermectins that they produce compared to the S 20 wild-type strain, and in complementation screening assays as described herein, to determine whether targeted or random mutagenesis of the aveC gene affects avermectin production.

The present invention further provides in a twentieth aspect a process for producing avermectins, comprising culturing cells of a strain of S. avermitilis, which cells express a mutated aveC allele that encodes a gene product that alters the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allele compared to cells of the same 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 of avermectins therefrom, and recovering said fI:\DayLib\LIBA]41 429spec.doc:gcc 9a avermectins from the culture. In a preferred embodiment, the class 2:1 ratio of avermectins produced by cells expressing the mutation is reduced. This process provides increased efficiency in the production of commercially valuable avermectins such as doramectin.

The present invention further provides a process for producing avermectins, comprising culturing cells of a strain of S. avermitilis, which cells express a mutated aveC allele or a genetic construct comprising an aveC allele that results in the production of an altered amount of avermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allele or genetic construct compared to cells of the same strain which do not express the mutated aveC allele or genetic construct but instead express only the wildtype aveC allele, in culture media under conditions that permit or induce the production of avermectins therefrom, and recovering said avermectins from the culture. In a preferred embodiment, the amount of avermectins produced by cells expressing the mutation or genetic construct is increased.

5 The present invention further provides a composition of cyclohexyl B2:cyclohexyl B1 avermectins present in exhausted fermentation medium in a ratio of less than 1.6:1, S* produced by cells of a strain of S. avermitilis that express a mutated aveC allele that encodes a gene product that reduces the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allele compared to cells of the same strain which do not express the mutated aveC allele but instead express only the o Swild-type aveC allele. The novel composition can be present as produced in fermentation culture fluid or can be harvested therefrom 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 the aveC gene of S. avermitilis.

FIGURE 3. Gene replacement vector pSE180 (ATCC 209605) comprising the ermE gene of Sacc. erythraea inserted into the aveC ORF of S. avermitilis.

FIGURE 4. BamH1 restriction map of the avermectin polyketide synthase gene cluster from S. avermitilis with five overlapping cosmid clones identified pSE66, pSE67, pSE68, pSE69). The relationship ofpSE118 and pSE119 is also indicated.

[I:\DayLib\LIBA]41429spec.doc:gcc 9b FIGURE 5. HPLC analysis of fermentation products produced by S. avermitilis strains. Peak quantitation was performed by comparison to standard quantities of cyclohexyl B1. Cyclohexyl B2 retention time was 7.4-7.7 min; cyclohexyl B1 retention time was 11.9-12.3 min. FIG. 5A. S. avermitilis strain SE180-11 with an inactivated aveC s ORF. FIG. 5B. S. avermitilis strain SE180-11 transformed with pSE186 (ATCC 209604).

FIG. 5C. S. avermitilis a. a *a* [I:\DayLib\LIBA]41429spec.doc:gcc WO 99/41389 PCT/IB99/00130 strain SE180-11 transformed with pSE187. FIG. 5D. S. avermitilis strain SE180-11 transformed with pSE188.

FIGURE 6. Comparison of deduced amino acid sequences encoded by the aveC ORF of S. avermitilis (SEQ ID NO:2), an aveC homolog partial ORF from S.

griseochromogenes (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. Conserved residues are shown in capital letters for homology in all three sequences and in lower case letters for homology in 2 of the 3 sequences. The amino acid sequences contain approximately 50% sequence identity.

FIGURE 7. Hybrid plasmid construct containing a 564 bp BsaAIIKpnl fragment from the S. hygroscopicus aveC homolog gene inserted into the BsaAI/Kpnl site in the S.

avermitilis aveC ORF.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the identification and characterization of polynucleotide molecules having nucleotide sequences that encode the AveC gene product from Streptomyces avermitilis, the construction of novel strains of S. avermitilis that can be used to screen mutated AveC gene products for their effect on avermectin production, and the discovery that certain mutated AveC gene products can reduce the ratio of B2:B1 avermectins produced by S. avermitilis. By way of example, the invention is described in the sections below for a polynucleotide molecule having either a nucleotide sequence that is the same as the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or the nucleotide sequence of the ORF of FIGURE 1 (SEQ ID NO:1), and for polynucleotides molecules having mutated nucleotide sequences derived therefrom. However, the principles set forth in the present invention can be analogously applied to other polynucleotide molecules, including aveC homolog genes from other Streptomyces species including, S.

hygroscopicus and S. griseochromogenes, among others.

5.1. Polynucleotide Molecules Encoding The S. avermitilis AveC Gene Product The present invention provides an isolated polynucleotide molecule comprising the complete aveC ORF of S. avermitilis or a substantial portion thereof, which isolated polynucleotide molecule lacks the next complete ORF that is located downstream from the aveC ORF in situ in the S. avermitilis chromosome.

The isolated polynucleotide molecule of the present invention preferably comprises a nucleotide sequence that is the same as the S. avermitilis AveC gene product-encoding WO 99/41389 PCT/IB99/00130 sequence of plasmid pSE186 (ATCC 209604), or that is the same as the nucleotide sequence of the ORF of FIGURE 1 (SEQ ID NO:1) or substantial portion thereof. As used herein, a "substantial portion" of an isolated polynucleotide molecule comprising a nucleotide sequence encoding the S. avermitilis AveC gene product means an isolated polynucleotide molecule comprising at least about 70% of the complete aveC ORF sequence shown in FIGURE 1 (SEQ ID NO:1), and that encodes a functionally equivalent AveC gene product. In this regard, a "functionally equivalent" AveC gene product is defined as a gene product that, when expressed in S. avermitilis strain ATCC 53692 in which the native aveC allele has been inactivated, results in the production of substantially the same ratio and amount of avermectins as produced by S. avermitilis strain ATCC 53692 which instead expresses only the wild-type, functional aveC allele native to S. avermitilis strain ATCC 53692.

In addition to the nucleotide sequence of the aveC ORF, the isolated polynucleotide molecule of the present invention can further comprise nucleotide sequences that naturally flank the aveC gene in situ in S. avermitilis, such as those flanking nucleotide sequences shown in FIGURE 1 (SEQ ID NO:1).

As used herein, the terms "polynucleotide molecule," "polynucleotide sequence," "coding sequence," "open-reading frame," and "ORF" are intended to refer to both DNA and RNA molecules, which can either be single-stranded or double-stranded, and that can be transcribed and translated (DNA), or translated (RNA), into an AveC gene product or, as described below, into an AveC homolog gene product, or into a polypeptide that is homologous to an AveC gene product or AveC homolog gene product in an appropriate host cell expression system when placed under the control of appropriate regulatory elements. A coding sequence can include but is not limited to prokaryotic sequences, cDNA sequences, genomic DNA sequences, and chemically synthesized DNA and RNA sequences.

The nucleotide sequence shown in FIGURE 1 (SEQ ID NO:1) comprises four different GTG codons at bp positions 42, 174, 177 and 180. As described in Section 9 below, multiple deletions of the 5' region of the aveC ORF (FIGURE 1; SEQ ID NO:1) were constructed to help define which of these codons could function in the aveC ORF as start sites for protein expression. Deletion of the first GTG site at bp 42 did not eliminate AveC activity. Additional deletion of all of the GTG codons at bp positions 174, 177 and 180 together eliminated AveC activity, indicating that this region is necessary for protein expression. The present invention thus encompasses variable length aveC ORFs that initiate translation at any of the GTG sites located at bp 174, 177 or 180 bp, as presented in FIGURE 1 (SEQ ID NO:1), and corresponding polypeptides for each.

WO 99/41389 PCT/IB99/00130 The present invention further provides a polynucleotide molecule having a nucleotide sequence that is homologous to the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or to the nucleotide sequence of the aveC ORF presented in FIGURE 1 (SEQ ID NO:1) or substantial portion thereof. The term "homologous" when used to refer to a polynucleotide molecule that is homologous to an S. avermitilis AveC gene product-encoding sequence means a polynucleotide molecule having a nucleotide sequence: that encodes the same AveC gene product as the S. avermitilis AveC gene productencoding sequence of plasmid pSE186 (ATCC 209604), or that encodes the same AveC gene product as the nucleotide sequence of the aveC ORF presented in FIGURE 1 (SEQ ID NO:1), but that includes one or more silent changes to the nucleotide sequence according to the degeneracy of the genetic code; or that hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence encoded by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or that encodes the amino acid sequence shown in FIGURE 1 (SEQ ID NO:2), under moderately stringent conditions, hybridization to filter-bound DNA in 0.5 M NaHPO 4 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 0 C, and washing in 0.2xSSC/0.1% SDS at 42 0 C (see Ausubel et al 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley Sons, Inc., New York, at p. 2.10.3), and encodes a functionally equivalent AveC gene product as defined above. In a preferred embodiment, the homologous polynucleotide molecule hybridizes to the complement of the AveC gene productencoding nucleotide sequence of plasmid pSE186 (ATCC 209604), or to the complement of the nucleotide sequence shown in FIGURE 1 (SEQ ID NO:1) or substantial portion thereof, under highly stringent conditions, hybridization to filter-bound DNA in 0.5 M NaHPO 4 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 0 C, and washing in 0.1xSSC/0.1% SDS at 68 0 C (Ausubel et al., 1989, above), and encodes a functionally equivalent AveC gene product as defined above.

The activity of an AveC gene product and potential functional equivalents thereof can be determined through HPLC analysis of fermentation products, as described in the examples below. Polynucleotide molecules having nucleotide sequences that encode functional equivalents of the S. avermitilis AveC gene product include naturally occurring aveC genes present in other strains of S. avermitilis, aveC homolog genes present in other species of Streptomyces, and mutated aveC alleles, whether naturally occurring or engineered.

The present invention further provides a polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide having an amino acid sequence that is WO 99/41389 PCT/IB99/00130 homologous to the amino acid sequence encoded by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or the amino acid sequence of FIGURE 1 (SEQ ID NO:2) or substantial portion thereof. As used herein, a "substantial portion" of the amino acid sequence of FIGURE 1 (SEQ ID NO:2) means a polypeptide comprising at least about 70% of the amino acid sequence shown in FIGURE 1 (SEQ ID NO:2), and that constitutes a functionally equivalent AveC gene product, as defined above.

As used herein to refer to amino acid sequences that are homologous to the amino acid sequence of an AveC gene product from S. avermitilis, the term "homologous" refers to a polypeptide encoded by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or having the amino acid sequence of FIGURE 1 (SEQ ID NO:2) but in which one or more amino acid residues has been conservatively substituted with a different amino acid residue, where such conservative substitution results in a functionally equivalent gene product, as defined above. Conservative amino acid substitutions are well-known in the art.

Rules for making such substitutions include those described by Dayhof, 1978, Nat.

Biomed. Res. Found., Washington, Vol. 5, Sup. 3, among others. More specifically, conservative amino acid substitutions are those that generally take place within a family of amino acids that are related in the acidity, polarity, or bulkiness of their side chains.

Genetically encoded amino acids are generally divided into four groups: acidic aspartate, glutamate; basic lysine, arginine, histidine; non-polar alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and uncharged polar glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are also jointly classified as aromatic amino acids. One or more replacements within any particular group, of a leucine with an isoleucine or valine, or of an aspartate with a glutamate, or of a threonine with a serine, or of any other amino acid residue with a structurally related amino acid residue, an amino acid residue with similar acidity, polarity, bulkiness, or with similarity in some combination thereof, will generally have an insignificant effect on the function of the polypeptide.

The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding an AveC homolog gene product. As used herein, an "AveC homolog gene product" is defined as a gene product having at least about 50% amino acid sequence identity to an AveC gene product of S. avermitilis comprising the amino acid sequence encoded by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or the amino acid sequence shown in FIGURE 1 (SEQ ID NO:2). In a non-limiting embodiment the AveC homolog gene product is from S. hygroscopicus, (described in EP WO 99/41389 PCT/IB99/00130 application 0298423; deposit FERM BP-1901) and comprises the amino acid sequence of SEQ ID NO:4, or a substantial portion thereof. A "substantial portion" of the amino acid sequence of SEQ ID NO:4 means a polypeptide comprising at least about 70% of the amino acid sequence of SEQ ID NO:4, and that constitutes a functionally equivalent AveC homolog gene product. A "functionally equivalent" AveC homolog gene product is defined as a gene product that, when expressed in S. hygroscopicus strain FERM BP-1901 in which the native aveC homolog allele has been inactivated, results in the production of substantially the same ratio and amount of milbemycins as produced by S. hygroscopicus strain FERM BP-1901 expressing instead only the wild-type, functional aveC homolog allele native to S.

hygroscopicus strain FERM BP-1901. In a non-limiting embodiment, the isolated polynucleotide molecule of the present invention that encodes the S. hygroscopicus AveC homolog gene product comprises the nucleotide sequence of SEQ ID NO:3 or a substantial portion thereof. In this regard, a "substantial portion" of the isolated polynucleotide molecule comprising the nucleotide sequence of SEQ ID NO:3 means an isolated polynucleotide molecule comprising at least 70% of the nucleotide sequence of SEQ ID NO:3, and that encodes a functionally equivalent AveC homolog gene product, as defined immediately above.

The present invention further provides a polynucleotide molecule comprising a nucleotide sequence that is homologous to the S. hygroscopicus nucleotide sequence of SEQ ID NO:3. The term "homologous" when used to refer to a polynucleotide molecule comprising a nucleotide sequence that is homologous to the S. hygroscopicus AveC homolog gene product-encoding sequence of SEQ ID NO:3 means a polynucleotide molecule having a nucleotide sequence: that encodes the same gene product as the nucleotide sequence of SEQ ID NO:3, or a substantial portion thereof, but that includes one or more silent changes to the nucleotide sequence according to the degeneracy of the genetic code; or that hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:4, under moderately stringent conditions, hybridization to filter-bound DNA in 0.5 M NaHPO 4 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 0 C, and washing in 0.2xSSC/0.1% SDS at 420C (see Ausubel et al. above), and encodes a functionally equivalent AveC homolog gene product as defined above. In a preferred embodiment, the homologous polynucleotide molecule hybridizes to the complement of the AveC homolog gene product-encoding nucleotide sequence of SEQ ID NO:3 or substantial portion thereof, under highly stringent conditions, hybridization to filter-bound DNA in 0.5 M NaHPO 4 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 0

C,

WO 99/41389 PCT/IB99/00130 and washing in 0.1xSSC/0.1% SDS at 68°C (Ausubel et al., 1989, above), and encodes a functionally equivalent AveC homolog gene product as defined above.

The present invention further provides a polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the S. hygroscopicus AveC homolog gene product. As used herein to refer to polypeptides that are homologous to the 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 in which one or more amino acid residues has been conservatively substituted with a different amino acid residue, where such conservative substitution results in a functionally equivalent AveC homolog gene product, as defined above.

The present invention further provides oligonucleotides that hybridize to a polynucleotide molecule having the nucleotide sequence of FIGURE 1 (SEQ ID NO:1) or SEQ ID NO:3, or to a polynucleotide molecule having a nucleotide sequence which is the complement of the nucleotide sequence of FIGURE 1 (SEQ ID NO:1) or SEQ ID NO:3. Such oligonucleotides are at least about 10 nucleotides in length, and preferably from about 15 to about 30 nucleotides in length, and hybridize to one of the aforementioned polynucleotide molecules under highly stringent conditions, washing in 6xSSC/0.5% sodium pyrophosphate at ~37 0 C for -14-base oligos, at ~48 0 C for -17-base oligos, at -55 0 C for base oligos, and at ~60 0 C for ~23-base oligos. In a preferred embodiment, the oligonucleotides are complementary to a portion of one of the aforementioned polynucleotide molecules. These oligonucleotides are useful for a variety of purposes including to encode or act as antisense molecules useful in gene regulation, or as primers in amplification of aveCor aveC homolog-encoding polynucleotide molecules.

Additional aveC homolog genes can be identified in other species or strains of Streptomyces by using the polynucleotide molecules or oligonucleotides disclosed herein in conjunction with known techniques. For example, an oligonucleotide molecule comprising a portion of the S. avermitilis nucleotide sequence of FIGURE 1 (SEQ ID NO:1) or a portion of the S. hygroscopicus nucleotide sequence of SEQ ID NO:3 can be detectably labeled and used 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 the reference organism, in this example S. avermitilis or S. hygroscopicus, to the organism of interest. Requirements for different stringency conditions are well known to those of skill in the art, and such conditions will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. Such oligonucleotides are WO 99/41389 PCT/IB99/00130 preferably at least about 15 nucleotides in length and include, those described in the examples below. Amplification of homolog genes can be carried out using these and other oligonucleotides by applying standard techniques such as the polymerase chain reaction (PCR), although other amplification techniques known in the art, the ligase chain reaction, can also be used.

Clones identified as containing aveC homolog nucleotide sequences can be tested for their ability to encode a functional AveC homolog gene product. For this purpose, the clones can be subjected to sequence analysis in order to identify a suitable reading frame, as well as initiation and termination signals. Alternatively or additionally, the cloned DNA sequence can be inserted into an appropriate expression vector, ie., a vector that contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Any of a variety of host/vector systems can be used as described below, including but not limited to bacterial systems such as plasmid, bacteriophage, or cosmid expression vectors. Appropriate host cells transformed with such vectors comprising potential aveC homolog coding sequences can then be analyzed for AveC-type activity using methods such as HPLC analysis of fermentation products, as described, in Section 7, below.

Production and manipulation of the polynucleotide molecules disclosed herein are within the skill in the art and can be carried out according to recombinant techniques described, in Maniatis, et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel, et al., 1989, Current Protocols In Molecular 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, Academic Press, Inc., San Diego; and Erlich 1992, PCR Technology, Oxford University Press, New York, all of which are incorporated herein by reference. Polynucleotide clones encoding AveC gene products or AveC homolog gene products can be identified using any method known in the art, including but not limited to the methods set forth in Section 7, below. Genomic DNA libraries can be screened for aveC and aveC homolog coding sequences using techniques such as the methods set forth in Benton and Davis, 1977, Science 196:180, for bacteriophage libraries, and in Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. USA, 72:3961-3965, for plasmid libraries. Polynucleotide molecules having nucleotide sequences known to include the aveC ORF, as present, in plasmid pSE186 (ATCC 209604), or in plasmid pSE119 (described in Section 7, below), can be used as probes in these screening experiments.

Alternatively, oligonucleotide probes can be synthesized that correspond to nucleotide WO 99/41389 PCT/IB99/00130 sequences deduced from partial or complete amino acid sequences of the purified AveC homolog gene product.

5.2. Recombinant Systems 5.2.1. Cloning And Expression Vectors The present invention further provides recombinant cloning vectors and expression vectors which are useful in cloning or expressing polynucleotide molecules of the present invention comprising, the aveC ORF of S. avermitilis or any aveC homolog ORFs. In a non-limiting embodiment, the present invention provides plasmid pSE186 (ATCC 209604), which comprises the complete ORF of the aveC gene of S. avermitilis.

All of the following description regarding the aveC ORF from S. avermitilis, or a polynucleotide molecule comprising the aveC ORF from S. avermitilis or portion thereof, or an S. avermitilis AveC gene product, also refers to aveC homologs and AveC homolog gene products, unless indicated explicitly or by context.

A variety of different vectors have been developed for specific use in Streptomyces, 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 the present invention. A number of drug resistance genes have also been cloned from Streptomyces, and several of these genes have been incorporated into vectors as selectable markers. Examples of current vectors for use in Streptomyces are presented, among other places, in Hutchinson, 1980, Applied Biochem. Biotech. 16:169-190.

Recombinant vectors of the present invention, particularly expression vectors, are preferably constructed so that the coding sequence for the polynucleotide molecule of the invention is in operative association with one or more regulatory elements necessary for transcription and translation of the coding sequence to produce a polypeptide. As used herein, the term "regulatory element" includes but is not limited to nucleotide sequences that encode inducible and non-inducible promoters, enhancers, operators and other elements known in the art that serve to drive and/or regulate expression of polynucleotide coding sequences. Also, as used herein, the coding sequence is in "operative association" with one or more regulatory elements where the regulatory elements effectively regulate and allow for the transcription of the coding sequence or the translation of its mRNA, or both.

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

WO 99/41389 PCT/IB99/00130 Methods are well-known in the art for constructing recombinant vectors containing particular coding sequences in operative association with appropriate regulatory elements, and these can be used to practice the present invention. These methods include in vitro recombinant techniques, synthetic techniques, and in vivo genetic recombination. See, e.g., the techniques described in Maniatis et al., 1989, above; Ausubel et al., 1989, above; Sambrook et al., 1989, above; Innis et al., 1995, above; and Erlich, 1992, above.

The regulatory elements of these vectors can vary in their strength and specificities.

Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements can be used. Non-limiting examples of transcriptional regulatory regions or promoters for bacteria include the P-gal promoter, the T7 promoter, the TAC promoter, X left and right promoters, trp and lac promoters, trp-lac fusion promoters and, more specifically for Streptomyces, the promoters ermE, melC, and tipA, etc. In a specific embodiment described in Section 11 below, an expression vector was generated that contained the aveC ORF cloned adjacent to the strong constitutive ermE promoter from Saccharopolyspora erythraea. The vector was transformed into S. avermitilis, and subsequent HPLC analysis of fermentation products indicated an increased titer of avermectins produced compared to production by the same strain but which instead expresses the wild-type aveC allele.

Fusion protein expression vectors can be used to express an AveC gene productfusion protein. The purified fusion protein can be used to raise antisera against the AveC gene product, to study the biochemical properties of the AveC gene product, to engineer AveC fusion proteins with different biochemical activities, or to aid in the identification or purification of the expressed AveC gene product. Possible fusion protein expression vectors include but are not limited to vectors incorporating sequences that encode p-galactosidase and trpE fusions, maltose-binding protein fusions, glutathione-S-transferase fusions and polyhistidine fusions (carrier regions). In an alternative embodiment, an AveC gene product or a portion thereof can be fused to an AveC homolog gene product, or portion thereof, derived from another species or strain of Streptomyces, such as, S. hygroscopicus or S.

griseochromogenes. In a particular embodiment described in Section 12, below, and depicted in FIGURE 7, a chimeric plasmid was constructed that contains a 564 bp region of the S.

hygroscopicus aveC homolog ORF replacing a homologous 564 bp region of the S. avermitilis aveC ORF. Such hybrid vectors can be transformed into S. avermitilis cells and tested to determine their effect, on the ratio of class 2:1 avermectin produced.

AveC fusion proteins can be engineered to comprise a region useful for purification.

For example, AveC-maltose-binding protein fusions can be purified using amylose resin; WO 99/41389 PCT/IB99/00130 AveC-glutathione-S-transferase fusion proteins can be purified using glutathione-agarose beads; and AveC-polyhistidine fusions can be purified using divalent nickel resin.

Alternatively, antibodies against a carrier protein or peptide can be used for affinity chromatography purification of the fusion protein. For example, a nucleotide sequence coding for the target epitope of a monoclonal antibody can be engineered into the expression vector in operative association with the regulatory elements and situated so that the expressed epitope is fused to the AveC polypeptide. For example, a nucleotide sequence coding for the

FLAG

T

epitope tag (International Biotechnologies Inc.), which is a hydrophilic marker peptide, can be inserted by standard techniques into the expression vector at a point corresponding, to the carboxyl terminus of the AveC polypeptide. The expressed AveC polypeptide-FLAG T M epitope fusion product can then be detected and affinity-purified using commercially available anti-FLAGTM antibodies.

The expression vector encoding the AveC fusion protein can also be engineered to contain polylinker sequences that encode specific protease cleavage sites so that the expressed AveC polypeptide can be released from the carrier region or fusion partner by treatment with a specific protease. For example, the fusion protein vector can include DNA sequences encoding thrombin or factor Xa cleavage sites, among others.

A signal sequence upstream from, and in reading frame with, the aveC ORF can be engineered into the expression vector by known methods to direct the trafficking and secretion of the expressed gene product. Non-limiting examples of signal sequences include those from a-factor, immunoglobulins, outer membrane proteins, penicillinase, and T-cell receptors, among others.

To aid in the selection of host cells transformed or transfected with cloning or expression vectors of the present invention, the vector can be engineered to further comprise a coding sequence for a reporter gene product or other selectable marker. Such a coding sequence is preferably in operative association with the regulatory element coding sequences, as described above. Reporter genes that are useful in the invention are wellknown in the art and include those encoding green fluorescent protein, luciferase, xylE, and tyrosinase, among others. Nucleotide sequences encoding selectable markers are wellknown in the art, and include those that encode gene products conferring resistance to antibiotics or anti-metabolites, or that supply an auxotrophic requirement. Examples of such sequences include those that encode resistance to erythromycin, thiostrepton or kanamycin, among many others.

WO 99/41389 PCT/IB99/00130 5.2.2. Transformation Of Host Cells The present invention further provides transformed host cells comprising a polynucleotide molecule or recombinant vector of the invention, and novel strains or cell lines derived therefrom. Host cells useful in the practice of the invention are preferably Streptomyces cells, although other prokaryotic cells or eukaryotic cells can also be used.

Such transformed host cells typically include but are not limited to microorganisms, such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA vectors, or yeast transformed with recombinant vectors, among others.

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

The recombinant expression vector of the invention is preferably transformed or transfected into one or more host cells of a substantially homogeneous culture of cells. The expression vector is generally introduced into host cells in accordance with known techniques, such as, by protoplast transformation, calcium phosphate precipitation, calcium chloride treatment, microinjection, electroporation, transfection by contact with a recombined virus, liposome-mediated transfection, DEAE-dextran transfection, transduction, conjugation, or microprojectile bombardment. Selection of transformants can be conducted by standard procedures, such as by selecting for cells expressing a selectable marker, antibiotic resistance, associated with the recombinant vector, as described above.

Once the expression vector is introduced into the host cell, the integration and maintenance of the aveC coding sequence either in the host cell chromosome or episomally can be confirmed by standard techniques, by Southern hybridization analysis, restriction enzyme analysis, PCR analysis, including reverse transcriptase PCR (rt-PCR), or by immunological assay to detect the expected gene product. Host cells containing and/or expressing the recombinant aveC coding sequence can be identified by any of at least four general approaches which are well-known in the art, including: DNA-DNA, DNA-RNA, or RNA-antisense RNA hybridization; (ii) detecting the presence of "marker" gene functions; (iii) assessing the level of transcription as measured by the expression of aveC-specific mRNA transcripts in the host cell; and (iv) detecting the presence of mature polypeptide product as WO 99/41389 PCT/IB99/00130 measured, by immunoassay or by the presence of AveC biological activity the production of specific ratios and amounts of avermectins indicative of AveC activity in, 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 host cell, the transformed host cell is clonally propagated, and the resulting cells can be grown under conditions conducive to the maximum production of the AveC gene product. Such conditions typically include growing cells to high density. Where the expression vector comprises an inducible promoter, appropriate induction conditions such as, temperature shift, exhaustion of nutrients, addition of gratuitous inducers analogs of carbohydrates, such as isopropyl-P-D-thiogalactopyranoside (IPTG)), accumulation of excess metabolic byproducts, or the like, are employed as needed to induce expression.

Where the expressed AveC gene product is retained inside the host cells, the cells are harvested and lysed, and the product isolated and purified from the lysate under extraction conditions known in the art to minimize protein degradation such as, at 4 0 C, or in the presence of protease inhibitors, or both. Where the expressed AveC gene product is secreted from the host cells, the exhausted nutrient medium can simply be collected and the product isolated therefrom.

The expressed AveC gene product can be isolated or substantially purified from cell lysates or culture medium, as appropriate, using standard methods, including but not limited to any combination of the following methods: ammonium sulfate precipitation, size fractionation, ion exchange chromatography, HPLC, density centrifugation, and affinity chromatography. Where the expressed AveC gene product exhibits biological activity, increasing purity of the preparation can be monitored at each step of the purification procedure by use of an appropriate assay. Whether or not the expressed AveC gene product exhibits biological activity, it can be detected as based, on size, or reactivity with an antibody otherwise specific for AveC, or by the presence of a fusion tag.

The present invention thus provides a recombinantly-expressed S. avermitilis AveC gene product comprising the amino acid sequence encoded by the AveC gene productencoding sequence of plasmid pSE186 (ATCC 209604), or the amino acid sequence of FIGURE 1 (SEQ ID NO:2) or a substantial portion thereof, and homologs thereof.

WO 99/41389 PCT/IB99/00130 The present invention further provides a recombinantly-expressed S. hygroscopicus AveC homolog gene product comprising the amino acid sequence of SEQ ID NO:4 or a substantial portion thereof, and homologs thereof.

The present invention further provides a method for producing an AveC gene product, comprising culturing a host cell transformed with a recombinant expression vector, said vector comprising a polynucleotide molecule having a nucleotide sequence encoding an AveC gene product, which polynucleotide molecule is in operative association with one or more regulatory elements that control expression of the polynucleotide molecule in the host cell, under conditions conducive to the production of the recombinant AveC gene product, and recovering the AveC gene product from the cell culture.

The recombinantly expressed S. avermitilis AveC gene product is useful for a variety of purposes, including for screening compounds that alter AveC gene product function and thereby modulate avermectin biosynthesis, and for raising antibodies directed against the AveC gene product.

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 the avermectin biosynthetic pathway, etc. For example, the amino acid sequence of the AveC gene product can be determined using standard peptide sequencing techniques. The AveC gene product can be further characterized using hydrophilicity analysis (see, Hopp and Woods, 1981, Proc. Natl. Acad. Sci. USA 78:3824), or analogous software algorithms, to identify hydrophobic and hydrophilic regions of the AveC gene product. Structural analysis can be carried out to identify regions of the AveC gene product that assume specific secondary structures. Biophysical methods such as X-ray crystallography (Engstrom, 1974, Biochem. Exp. Biol. 11: 7-13), computer modelling (Fletterick and Zoller (eds), 1986, in: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), and nuclear magnetic resonance (NMR) can be used to map and study sites of interaction between the AveC gene product and its substrate. Information obtained from these studies can be used to select new sites for mutation in the aveC ORF to help develop new strains of S. avermitilis having more desirable avermectin production characteristics 5.3. Construction And Use Of AveC Mutants The present invention provides a polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604) or the nucleotide sequence of the aveC ORF of WO 99/41389 PCT/IB99/00130 S. avermitilis as presented in FIGURE 1 (SEQ ID NO:1), but that further comprises one or more mutations, so that cells of S. avermitilis strain ATCC 53692 in which the wild-type aveC allele has been inactivated and that express the polynucleotide molecule comprising the mutated nucleotide sequence produce a different ratio or amount of avermectins than are produced by cells of S. avermitilis strain ATCC 53692 that instead express only the wild-type aveC allele.

According to the present invention, such polynucleotide molecules can be used to produce novel strains of S. avermitilis that exhibit a detectable change in avermectin production compared to the same strain which instead expresses only the wild-type aveC allele. In a preferred embodiment, such polynucleotide molecules are useful to produce novel strains of S. avermitilis that produce avermectins in a reduced class 2:1 ratio compared to the same strain which instead expresses only the wild-type aveC allele. In a further preferred embodiment, such polynucleotide molecules are useful to produce novel strains of S.

avermitilis that produce increased levels of avermectins compared to the same strain which instead expresses only the wild-type aveC allele. In a further preferred embodiment, such polynucleotide molecules are useful to produce novel strains of S. avermitilis in which the aveC gene has been inactivated.

Mutations to the aveC coding sequence include any mutations that introduce amino acid deletions, additions, or substitutions into the AveC gene product, or that result in truncation of the AveC gene product, or any combination thereof, and that produce the desired result. For example, the present invention provides polynucleotide molecules comprising the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or the nucleotide sequence of the aveC ORF of S. avermitilis as presented in FIGURE 1 (SEQ ID NO:1), but that further comprise one or more mutations that encode the substitution of an amino acid residue with a different amino acid residue at selected positions in the AveC gene product. In several non-limiting embodiments, which are exemplified below, such substitutions can be carried 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 known methods, including by use of error-prone PCR, or by cassette mutagenesis. For example, oligonucleotide-directed mutagenesis can be employed to alter the aveC ORF sequence in a defined way such as, to introduce one or more restriction sites, or a termination codon, into specific regions within the aveC ORF sequence. Methods such as those described in U.S. Patent 5,605,793, which involve random fragmentation, repeated cycles of mutagenesis, WO 99/41389 PCT/IB99/00130 and nucleotide shuffling, can also be used to generate large libraries of polynucleotides having nucleotide sequences encoding aveC mutations.

Targeted mutations can be useful, particularly where they serve to alter one or more conserved amino acid residues in the AveC gene product. For example, a comparison of deduced amino acid sequences of AveC gene products and AveC homolog gene products from 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 significant conservation of amino acid residues between these species. Targeted mutagenesis that leads to a change in one or more of these conserved amino acid residues can be particularly effective in producing novel mutant strains that exhibit desirable alterations in avermectin production.

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

Once mutated polynucleotide molecules are generated, they are screened to determine whether they can modulate avermectin biosynthesis in S. avermitilis. In a preferred embodiment, a polynucleotide molecule having a mutated nucleotide sequence is tested by complementing a strain of S. avermitilis in which the aveC gene has been inactivated to give an aveC negative (aveC) background. In a non-limiting method, the mutated polynucleotide molecule is spliced into an expression plasmid in operative association with one or more regulatory elements, which plasmid also preferably comprises one or more drug resistance genes to allow for selection of transformed cells. This vector is then transformed into aveC host cells using known techniques, and transformed cells are selected and cultured in appropriate fermentation media under conditions that permit or induce avermectin production.

Fermentation products are then analyzed by HPLC to determine the ability of the mutated polynucleotide molecule to complement the host cell. Several vectors bearing mutated polynucleotide molecules capable of reducing the B2:B1 ratio of avermectins, including pSE188, pSE199, and pSE231, are exemplified in Section 8.3, below.

The present 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 a preferred embodiment, the present invention provides a method for identifying mutations of the 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 WO 99/41389 PCT/IB99/00130 which the native aveC allele has been inactivated, and into which a polynucleotide molecule comprising a nucleotide sequence encoding a mutated AveC gene product has been introduced and is being expressed; determining the class 2:1 ratio of avermectins produced by cells of the same strain of S. avermitilis as in step but which instead express only an aveC allele having the nucleotide sequence of the ORF of FIGURE 1 (SEQ ID NO:1) or a nucleotide sequence that is homologous thereto; and comparing the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step to the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step such that if the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step is different from the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step then a mutation of the aveC ORF capable of altering the class 2:1 ratio of avermectins has been identified. In a preferred embodiment, the class 2:1 ratio of avermectins is reduced by the mutation.

In a further preferred embodiment, the present invention provides a method for identifying mutations of the aveC ORF or genetic constructs comprising the aveC ORF capable of altering the amount of avermectins produced, comprising: determining the amount of avermectins produced by cells of a strain of S. avermitilis in which the native aveC allele has been inactivated, and into which a polynucleotide molecule comprising a nucleotide sequence encoding a mutated AveC gene product or comprising a genetic construct comprising a nucleotide sequence encoding an AveC gene product has been introduced and is being expressed; determining the amount of avermectins produced by cells of the same strain of S. avermitilis as in step but which instead express only an aveC allele having the nucleotide sequence of the ORF of FIGURE 1 (SEQ ID NO:1) or a nucleotide sequence that is homologous thereto; and comparing the amount of avermectins produced by the S.

avermitilis cells of step to the amount of avermectins produced by the S. avermitilis cells of step 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 then a mutation of the aveC ORF or a genetic construct capable of altering the amount of avermectins has been identified. In a preferred embodiment, the amount of avermectins produced is increased by the mutation.

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

WO 99/41389 PCT/IB99/00130 Once a mutated polynucleotide molecule that modulates avermectin production in a desirable direction has been identified, the location of the mutation in the nucleotide sequence can be determined. For example, a polynucleotide molecule having a nucleotide sequence encoding a mutated AveC gene product can be isolated by PCR and subjected to DNA sequence analysis using known methods. By comparing the DNA sequence of the mutated aveC allele to that of the wild-type aveC allele, the mutation(s) responsible for the alteration in avermectin production can be determined. In specific, though non-limiting, embodiments of the present invention, S. avermitilis AveC gene products comprising either single amino acid substitutions at any of residues 55 (S55F), 138 (S138T), 139 (A139T), or 230 (G230D), or a double substitution at positions 138 (S138T) and 139 (A139T), yielded changes in AveC gene product function such that the ratio of class 2:1 avermectins produced was altered (see Section 8, below). Accordingly, polynucleotide molecules having nucleotide sequences that encode mutated S. avermitilis AveC gene products comprising amino acid substitutions at one or more of amino acid residues 55, 138, 139 or 230, or any combination thereof, are encompassed by the present invention.

The present 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 of avermectin production. For example, the present invention provides recombinant vectors that can be used to target any of the polynucleotide molecules comprising mutated nucleotide sequences of the present invention to the site of the aveC gene of the S. avermitilis chromosome to either insert into or replace the aveC ORF or a portion thereof by homologous recombination. According to the present invention, however, a polynucleotide molecule comprising a mutated nucleotide sequence of the present invention provided herewith can also function to modulate avermectin biosynthesis when inserted into the S. avermitilis chromosome at a site other than at the aveC gene, or when maintained episomally in S.

avermitilis cells. Thus, the present invention also provides vectors comprising a polynucleotide molecule comprising a mutated nucleotide sequence of the present invention, which vectors can be used to insert the polynucleotide molecule at a site in the S. avermitilis chromosome other than at the aveC gene, or to be maintained episomally.

In a preferred embodiment, the present invention provides gene replacement vectors that can be used to insert a mutated aveC allele into cells of a strain of S. avermitilis, thereby generating novel strains of S. avermitilis, the cells of which produce avermectins in an altered class 2:1 ratio compared to cells of the same strain which instead express only the wild-type aveC allele. In a preferred embodiment, the class 2:1 ratio of avermectins produced by the WO 99/41389 PCT/IB99/00130 cells is reduced. Such gene replacement vectors can be constructed using mutated polynucleotide molecules present 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 present invention provides vectors that can be used to insert a mutated aveC allele into cells of a strain of S. avermitilis to generate novel strains of cells that produce altered amounts of avermectins compared to cells of the same strain which instead express only the wild-type aveC allele. In a preferred embodiment, the amount of avermectins produced by the cells is increased. In a specific, though non-limiting, embodiment, such a vector further comprises a strong promoter as known in the art, such as, the strong constitutive ermE promoter from Saccharopolyspora erythraea, that is situated upstream from, and in operative association with, the aveC ORF. Such a vector can be plasmid pSE189, described in Example 11 below, or can be constructed by using the mutated aveC allele of plasmid pSE189.

In a further preferred embodiment, the present invention provides gene replacement vectors that are useful to inactivate the aveC gene in a wild-type strain of S. avermitilis. In a non-limiting embodiment, such gene replacement vectors can be constructed using the mutated polynucleotide molecule present in plasmid pSE180 (ATCC 209605), which is exemplified in Section 8.1, below (FIGURE The present invention further provides gene replacement vectors that comprise a polynucleotide molecule comprising or consisting of nucleotide sequences that naturally flank the aveC gene in situ in the S. avermitilis chromosome, including, those flanking nucleotide sequences shown in FIGURE 1 (SEQ ID NO:1), which vectors can be used to delete the S. avermitilis aveC ORF.

The present invention further provides methods for making novel strains of S.

avermitilis comprising cells that express a mutated aveC allele and that produce an altered ratio and/or amount of avermectins compared to cells of the same strain of S. avermitilis that instead express only the wild-type aveC allele. In a preferred embodiment, the present invention provides a method for making novel strains of S. avermitilis comprising cells that express a mutated aveC allele and that produce an altered class 2:1 ratio of avermectins compared to cells of the same strain of S. avermitilis that instead express only a wild-type aveC allele, comprising transforming cells of a strain of S. avermitilis with a vector that carries a mutated aveC allele that encodes a gene product that alters the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allele compared to cells of the same strain that instead express only the wild-type aveC allele, and selecting transformed cells that produce avermectins in an altered class 2:1 ratio compared to WO 99/41389 PCT/IB99/00130 the class 2:1 ratio produced by cells of the strain that instead express only the wild-type aveC allele. In a preferred embodiment, the altered class 2:1 ratio of avermectins is reduced.

In a further preferred embodiment, the present invention provides a method for making novel strains of S. avermitilis comprising cells that produce altered amounts of avermectin, comprising transforming cells of a strain of S. avermitilis with a vector that carries a mutated aveC allele or a genetic construct comprising the aveC allele, the expression of which 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 the same strain that instead express only the wild-type aveC allele, and selecting transformed cells that produce avermectins in an altered amount compared to the amount of avermectins produced by cells of the strain that instead express only the wild-type aveC allele. In a preferred embodiment, the amount of avermectins produced in the transformed cells is increased.

In a further preferred embodiment, the present invention provides a method for making 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 allele with a vector that inactivates the aveC allele, and selecting transformed cells in which the aveC allele has been inactivated. In a preferred, though non-limiting, embodiment, cells of a strain of S. avermitilis are transformed with a gene replacement vector that carries an aveC allele that has been inactivated by mutation or by replacement of a portion of the aveC allele with a heterologous gene sequence, and transformed cells in which the native aveC allele of the cells has been replaced with the inactivated aveC allele are selected. Inactivation of the aveC allele can be determined by HPLC analysis of fermentation products, as described below. In a specific, though non-limiting, embodiment described in Section 8.1 below, the aveC allele is inactivated by insertion of the ermE gene from Saccharopolyspora erythraea into the aveC ORF.

The present invention further provides novel strains of S. avermitilis comprising cells that have been transformed with any of the polynucleotide molecules or vectors of the present invention. In a preferred embodiment, the present 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 an altered class 2:1 ratio compared to the class 2:1 ratio of avermectins produced by cells of the same strain that instead express only the wild-type aveC allele. In a preferred embodiment, the altered class 2:1 ratio produced by the novel cells is reduced. Such novel strains are WO 99/41389 PCT/IB99/00130 useful in the large-scale production of commercially desirable avermectins such as doramectin.

It is a primary objective of the screening assays described herein to identify mutated alleles of the aveC gene the expression of which, in S. avermitilis cells, alters and, more particularly, 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 present invention expressing a mutated aveC allele which reduces the ratio of class 2:1 avermectins produced is between less than 1.6:1 to about 0:1; in a more preferred embodiment, the ratio is between about 1:1 to about 0:1; and in the most preferred embodiment, the ratio is between about 0.84:1 to about 0:1. In a specific embodiment described below, novel cells of the present invention produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of less than 1.6:1. In a different specific embodiment described below, novel cells of the present invention produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of about 0.94:1. In a further different specific embodiment described below, novel cells of the present invention produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of about 0.88:1. In a further different specific embodiment described below, novel cells of the present invention produce cyclohexyl 2:cyclohexyl B1 avermectins in a ratio of about 0.84:1.

In a further preferred embodiment, the present invention provides novel strains of S.

avermitilis comprising cells which express a mutated aveC allele, or a genetic construct comprising an aveC allele, in place of, or in addition to, the wild-type aveC allele, wherein the cells of the novel strain produce an altered amount of avermectins compared to cells of the same 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 constitutive ermE promoter from Saccharopolyspora erythraea, upstream from and in operative association with the aveC ORF.

In a further preferred embodiment, the present invention provides novel strains of S.

avermitilis comprising cells in which the aveC gene has been inactivated. Such strains are useful both for the different spectrum of avermectins that they produce compared to the wildtype strain, and in complementation screening assays as described herein, to determine whether targeted or random mutagenesis of the aveC gene affects avermectin production. In a specific embodiment described below, S. avermitilis host cells were genetically engineered to contain an inactivated aveC gene. For example, strain SE180-11, described in the examples below, was generated using the gene replacement plasmid pSE180 (ATCC WO 99/41389 PCT/IB99/00130 209605) (FIGURE which was constructed to inactivate the S. avermitilis aveC gene by insertion of the ermE resistance gene into the aveC coding region.

The present invention further provides recombinantly expressed, mutated S.

avermitilis AveC gene products encoded by any of the aforementioned polynucleotide molecules of the invention, and methods of preparing the same.

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

The present invention further provides a process for producing avermectins, comprising culturing cells of a strain of S. avermitilis, which cells express a mutated aveC allele or a genetic construct comprising an aveC allele that results in the production of an altered amount of avermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allele or genetic construct compared to cells of the same strain which do not express the mutated aveC allele or genetic construct but instead express only the wild-type aveC allele, in culture media under conditions that permit or induce the production of avermectins therefrom, and recovering said avermectins from the culture. In a preferred embodiment, the amount of avermectins produced in culture by cells expressing the mutated aveC allele or genetic construct is increased.

The present 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 that reduces the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allele compared to cells of the same strain that instead express only the wild-type aveC allele, wherein the avermectins in the novel composition are produced in a reduced class 2:1 ratio as compared to the class 2:1 ratio of avermectins produced by cells of the same strain of S. avermitilis that instead express only the wild-type aveC allele.

The novel avermectin composition can be present as produced in exhausted fermentation culture fluid, or can be harvested therefrom. The novel avermectin composition can be WO 99/41389 PCT/IB99/00130 partially or substantially purified from the culture fluid by known biochemical techniques of purification, such as by ammonium sulfate precipitation, dialysis, size fractionation, ion exchange chromatography, HPLC, etc.

5.4. Uses Of Avermectins Avermectins are highly active antiparasitic agents having particular utility as anthelmintics, ectoparasiticides, insecticides and acaricides. Avermectin compounds produced according to the methods of the present invention are useful for any of these purposes. For example, avermectin compounds produced according to the present invention are useful to treat various diseases or conditions in humans, particularly where those diseases or conditions are caused by parasitic infections, as known in the art. See, e.g., Ikeda and Omura, 1997, Chem. Rev. 97(7):2591-2609. More particularly, avermectin compounds produced according to the present invention are effective in treating a variety of diseases or conditions caused by endoparasites, such as parasitic nematodes, which can infect humans, domestic animals, swine, sheep, poultry, horses or cattle.

More specifically, avermectin compounds produced according to the present invention are effective against nematodes that infect humans, as well as those that infect various species of animals. Such nematodes include gastrointestinal parasites such as Ancylostoma, Necator, Ascaris, Strongyloides, Trichinella, Capillaria, Trichuris, Enterobius, Dirofilaria, and parasites that are found in the blood or other tissues or organs, such as filarial worms and the extract intestinal states of Strongyloides and Trichinella.

The avermectin compounds produced according to the present invention are also useful in treating ectoparasitic infections including, arthropod infestations of mammals and birds, caused by ticks, mites, lice, fleas, blowflies, biting insects, or migrating dipterous larvae that can affect cattle and horses, among others.

The avermectin compounds produced according to the present invention are also useful as insecticides against household pests such as, the cockroach, clothes moth, carpet beetle and the housefly among others, as well as insect pests of stored grain and of agricultural plants, which pests include spider mites, aphids, caterpillars, and orthopterans such as locusts, among others.

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

An avermectin compound produced according to the present invention is administered in a formulation appropriate to the specific intended use, the particular species WO 99/41389 PCT/IB99/00130 of host animal being treated, and the parasite or insect involved. For use as a parasiticide, an avermectin compound produced according to the present invention can be administered orally in the form of a capsule, bolus, tablet or liquid drench or, altemrnatively, can be administered as a pour-on, or by injection, or as an implant. Such formulations are prepared in a conventional manner in accordance with standard veterinary practice. Thus, capsules, boluses or tablets can be prepared by mixing the active ingredient with a suitable finely divided diluent or carrier additionally containing a disintegrating agent and/or binder such as starch, lactose, talc, magnesium stearate, etc. A drench formulation can be prepared by dispersing the active ingredient in an aqueous solution together with a dispersing or wetting agent, etc. Injectable formulations can be prepared in the form of a sterile solution which can contain other substances such as, sufficient salts and/or glucose to make the solution isotonic with blood.

Such formulations will vary with regard to the weight of active compound depending on the patient, or species of host animal to be treated, the severity and type of infection, and the body weight of the host. Generally, for oral administration a dose of active compound of from about 0.001 to 10 mg per kg of patient or animal body weight given as a single dose or in divided doses for a period of from 1 to 5 days will be satisfactory. However, there can be instances where higher or lower dosage ranges are indicated, as determined, by a physician or veterinarian, as based on clinical symptoms.

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

For use as an insecticide, and for treating agricultural pests, an avermectin compound produced according to the present invention can be applied as a spray, dust, emulsion and the like in accordance with standard agricultural practice.

6. EXAMPLE: FERMENTATION OF STREPTOMYCES A VERMITILIS AND B2:B1 AVERMECTIN

ANALYSIS

Strains lacking both branched-chain 2-oxo acid dehydrogenase and methyltransferase activities produce no avermectins if the fermentation medium is not supplemented with fatty acids. This example demonstrates that in such mutants a wide range of B2:B1 ratios of avermectins can be obtained when biosynthesis is initiated in the presence of different fatty acids.

WO 99/41389 PCT/IB99/00130 6.1. Materials And Methods Streptomyces avermitilis ATCC 53692 was stored at -70 0 C as a whole broth prepared in seed medium consisting of: Starch (Nadex, Laing National) 20g; Pharmamedia (Trader's Protein, Memphis, TN) 15 g; Ardamine pH (Yeast Products Inc.) 5 g; calcium carbonate 1 g. Final volume was adjusted to 1 liter with tap water, pH was adjusted to 7.2, and the medium was autoclaved at 121°C for 25 min.

Two ml of a thawed suspension of the above preparation was used to inoculate a flask containing 50 ml of the same medium. After 48 hrs incubation at 280C on a rotary shaker at 180 rpm, 2 ml of the broth was used to inoculate a flask containing 50 ml of a production medium consisting of: Starch 80 g; calcium carbonate 7 g; Pharmamedia 5 g; dipotassium hydrogen phosphate 1 g; magnesium sulfate 1 g; glutamic acid 0.6 g; ferrous sulfate heptahydrate 0.01 g; zinc sulfate 0.001 g; manganous sulfate 0.001 g. Final volume was adjusted to 1 liter with tap water, pH was adjusted to 7.2, and the medium was autoclaved at 121°C for 25 min.

Various carboxylic acid substrates (see TABLE 1) were dissolved in methanol and added to the fermentation broth 24 hrs after inoculation to give a final concentration of 0.2 g/liter. The fermentation broth was incubated for 14 days at 280C, then the broth was centrifuged (2,500 rpm for 2 min) and the supernatant discarded. The mycelial pellet was extracted with acetone (15 ml), then with dichloromethane (30 ml), and the organic phase separated, filtered, then evaporated to dryness. The residue was taken up in methanol (1 ml) and analyzed by HPLC with a Hewlett-Packard 1090A liquid chromatograph equipped with a scanning diode-array detector set at 240 nm. The column used was a Beckman Ultrasphere C-18, 5 pm, 4.6 mm x 25 cm column maintained at 400C. Twenty-five pI of the above methanol solution was injected onto the column. Elution was performed with a linear gradient of methanol-water from 80:20 to 95:5 over 40 min at 0.85/ml min. Two standard concentrations of cyclohexyl B1 were used to calibrate the detector response, and the area under the curves for B2 and B1 avermectins was measured.

6.2. Results The HPLC retention times observed for the B2 and B1 avermectins, and the 2:1 ratios, are shown in TABLE 1.

WO 99/41389 PCT/IB99/00130 TABLE 1 HPLC Retention Ratio Time (min) Substrate B2 B1 B2:B1 4-Tetrahydropyran carboxylic acid 8.1 14.5 0.25 Isobutyric acid 10.8 18.9 3-Furoic acid 7.6 14.6 0.62 S-(+)-2-methylbutyric acid 12.8 21.6 Cyclohexanecarboxylic acid 16.9 26.0 1.6 3-Thiophenecarboxylic acid 8.8 16.0 1.8 Cyclopentanecarboxylic acid 14.2 23.0 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:B1 avermectin product ratios, indicating a considerable difference in the results of dehydrative conversion of class 2 compounds to class 1 compounds, depending on the nature of the fatty acid side chain starter unit supplied. This indicates that changes in B2:B1 ratios resulting from alterations to the AveC protein may be specific to particular substrates. Consequently, screening for mutants exhibiting changes in the B2:B1 ratio obtained with a particular substrate needs to be done in the presence of that substrate. The subsequent examples described below use cyclohexanecarboxylic acid as the screening substrate. However, this substrate is used merely to exemplify the potential, and is not intended to limit the applicability, of the present invention.

7. EXAMPLE: ISOLATION OF THE aveC GENE This example describes the isolation and characterization of a region of the Streptomyces avermitilis chromosome that encodes the AveC gene product. As demonstrated below, the aveC gene was identified as capable of modifying the ratio of cyclohexyl-B2 to cyclohexyl-B1 (B2:B1) avermectins produced.

WO 99/41389 PCT/IB99/00130 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 were isolated on 1/2 strength YPD-6 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 dH 2 0, pH was adjusted to 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 TSB medium (Difco Tryptic Soy Broth 30 g, in 1 liter dH20, autoclaved at 121 0 C for 25 min) in a 25 mm x 150 mm tube which was maintained with shaking (300 rpm) at 28 0 C for 48-72 hrs.

7.1.2. Chromosomal DNA Isolation From Streptomyces Aliquots (0.25 ml or 0.5 ml) of mycelia grown as described above were placed in ml microcentrifuge tubes and the cells concentrated by centrifugation at 12,000 x g for 60 sec.

The supernatant was discarded and the cells were resuspended in 0.25 ml TSE buffer (20 ml 1.5 M sucrose, 2.5 ml 1 M Tris-HCI, pH 8.0, 2.5 ml 1 M EDTA, pH 8.0, and 75 ml dH 2

O)

containing 2 mg/ml lysozyme. The samples were incubated at 37 0 C for 20 min with shaking, loaded into an AutoGen 5 40 TM automated nucleic acid isolation instrument (Integrated Separation Systems, Natick, MA), and genomic DNA isolated using Cycle 159 (equipment software) according to manufacturer's instructions.

Alternatively, 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. Cells were 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/ml lysozyme, and incubated at 37 0 C with shaking for 30-60 min. After incubation, 0.5 ml sodium dodecyl sulfate (SDS) was added and the cells incubated at 37 0 C until lysis was complete. The lysate was incubated at 65 0 C for 10 min, cooled to rm temp, split into two ml Eppendorf tubes, and extracted 1x with 0.5 ml phenol/chloroform (50% phenol previously equilibrated with 0.5 M Tris, pH 8.0; 50% chloroform). The aqueous phase was removed and extracted 2 to 5x with chloroform:isoamyl alcohol The DNA was precipitated by adding 1/10 volume 3M sodium acetate, pH 4.8, incubating the mixture on ice for 10 min, centrifuging the mixture at 15,000 rpm at 5°C for 10 min, and removing the supernatant to a clean tube to which 1 volume of isopropanol was added. The supernatant plus isopropanol mixture was then incubated on ice for 20 min, centrifuged at 15,000 rpm for 20 min at 50C, the supernatant WO 99/41389 PCT/IB99/00130 removed, and the DNA pellet washed 1x with 70% ethanol. After the pellet was dry, the DNA was resuspended in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.1.3. Plasmid DNA Isolation From Streptomyces An aliquot (1.0 ml) of mycelia was placed in 1.5 ml microcentrifuge tubes and the cells concentrated by centrifugation at 12,000 x g for 60 sec. The supernatant was discarded, the cells were resuspended in 1.0 ml 10.3% sucrose and concentrated by centrifugation at 12,000 x g for 60 sec, and the supernatant discarded. The cells were then resuspended in 0.25 ml TSE buffer containing 2 mg/ml lysozyme, and incubated at 37 0 C for 20 min with shaking and loaded into the AutoGen 540TM automated nucleic acid isolation instrument. Plasmid DNA was isolated using Cycle 106 (equipment software) according to manufacturer's 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 at 12,000 x g for 60 sec, and the supernatant discarded. The cells were resuspended in 0.5 ml TSE buffer containing 2 mg/ml lysozyme, and incubated at 37 0 C for 15-30 min. After incubation, 0.25 ml alkaline SDS (0.3N NaOH, 2% SDS) was added and the cells incubated at 0 C for 15-30 min or until the solution was clear. Sodium acetate (0.1 ml, 3M, pH 4.8) was added to the DNA solution, which was then incubated on ice for 10 min. The DNA samples were centrifuged at 14,000 rpm for 10 min at 5°C. The supernatant was removed to a clean tube, and 0.2 ml phenol:chloroform (50% phenol:50% chloroform) was added and gently mixed. The DNA solution was centrifuged at 14,000 rpm for 10 min at 5 0 C and the upper layer removed to a clean Eppendorf tube. Isopropanol (0.75 ml) was added, and the solution was gently mixed and then incubated at rm temp for 20 min. The DNA solution was centrifuged at 14,000 rpm for 15 min at 5 0 C, the supernatant removed, and the DNA pellet was washed with 70% ethanol, 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 dHzO, pH autoclaved at 1210C for 25 min, and supplemented with 100 Ig/ml ampicillin). The culture was incubated overnight, and a 1 ml aliquot placed in a 1.5 ml microcentrifuge tube.

The culture samples were loaded into the AutoGen 540TM automated nucleic acid isolation instrument and plasmid DNA was isolated using Cycle 3 (equipment software) according to manufacturer's instructions.

WO 99/41389 PCT/IB99/00130 7.1.5. Preparation And Transformation Of S. avermitilis Protoplasts Single colonies of S. avermitilis were isolated on 1/2 strength YPD-6. The mycelia were used to inoculate 10 ml of TSB medium in a 25 mm x 150 mm tube, which was then incubated with shaking (300 rpm) at 280C for 48 hrs. One ml of mycelia was used to inoculate ml YEME medium. YEME medium contains per liter: Difco Yeast Extract 3 g; Difco Bacto-peptone 5 g; Difco Malt Extract 3 g; Sucrose 300 g. After autoclaving at 121 0 C for min, the following were added: 2.5 M MgCI, 6H 2 0 (separately autoclaved at 121°C for min) 2 ml; and glycine (filter-sterilized)- 25 ml.

The mycelia were grown at 300C for 48-72 hrs and harvested by centrifugation in a ml centrifuge tube (Falcon) at 3,000 rpm for 20 min. The supernatant was discarded and the mycelia were resuspended in P buffer, which contains: sucrose 205 g; K 2

SO

4 0.25 g; MgC2I 6H 2 0 2.02 g; H 2 0 600 ml; K 2

PO

4 10 ml; trace element solution 20 ml; CaCI 2 2H 2 0 100 ml; and MES buffer (1.0 M, pH 6.5) 10 ml. (*Trace element solution contains per liter: ZnCI 2 40 mg; FeCI 3 6H 2 0 200 mg; CuCI 2 2H 2 0 10 mg; MnCI 2 4H 2 0 10 mg; Na 2

B

4 07 10H 2 0 10 mg; (NH 4 6 Mo 7

O

24 4H0 10 mg). The pH was adjusted to 6.5, final volume was adjusted to 1 liter, and the medium was filtered hot through a 0.45 micron filter.

The mycelia were pelleted at 3,000 rpm for 20 min, the supernatant was discarded, and the mycelia were resuspended in 20 ml P buffer containing 2 mg/ml lysozyme. The mycelia were incubated at 35°C for 15 min with shaking, and checked microscopically to determine extent of protoplast formation. When protoplast formation was complete, the protoplasts were centrifuged at 8,000 rpm for 10 min. The supernatant was removed and the protoplasts were resuspended in 10 ml P buffer. The protoplasts were centrifuged at 8,000 rpm for 10 min, the supernatant was removed, the protoplasts were resuspended in 2 ml P buffer, and approximately 1 x 109 protoplasts were distributed to 2.0 ml cryogenic vials (Nalgene).

A vial containing 1 x 109 protoplasts was centrifuged at 8,000 rpm for 10 min, the supernatant was removed, and the protoplasts were resuspended in 0.1 ml P buffer. Two to pg of transforming DNA were added to the protoplasts, immediately followed by the addition of ml working T buffer. T buffer base contains: PEG-1000 (Sigma) 25 g; sucrose 2.5 g;

H

2 0 83 ml. The pH was adjusted to 8.8 with 1 N NaOH (filter sterilized), and the T buffer base was filter-sterilized and stored at 4°C. Working T buffer, made the same day used, was WO 99/41389 PCT/IB99/00130 T buffer base 8.3 ml; K 2

PO

4 (4 mM) 1.0 ml; CaCI 2 2H 2 0 (5 M) 0.2 ml; and TES (1 M, pH 8) 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 added and the protoplasts were centrifuged at 8,000 rpm for 10 min. The supernatant was discarded and the protoplasts were resuspended in 0.1 ml P buffer. The protoplasts were then plated on RM14 media, which contains: sucrose 205 g; K 2 SO4 0.25 g; MgCI 2 6H 2 0 10.12 g; glucose 10 g; Difco Casamino Acids 0.1 g; Difco Yeast Extract 5 g; Difco Oatmeal Agar 3 g; Difco Bacto Agar 22 g; dH20 800 ml. The solution was autoclaved at 121°C for min. After autoclaving, sterile stocks of the following were added: K 2

PO

4 10 ml; CaCI 2 2H 2 0 (5 M) 5 ml; L-proline 15 ml; MES buffer (1.0 M, pH 6.5) 10 ml; trace element solution (same as above) 2 ml; cycloheximide stock (25 mg/ml) 40 ml; and 1N NaOH 2 ml. Twenty-five ml of RM14 medium were aliquoted per plate, and plates dried for 24 hr before use.

The protoplasts were incubated in 95% humidity at 30 0 C for 20-24 hrs. To select thiostrepton resistant transformants, 1 ml of overlay buffer containing 125 pg per ml thiostrepton was spread evenly over the RM14 regeneration plates. Overlay buffer contains per 100 ml: sucrose 10.3 g; trace element solution (same as above) 0.2 and MES (1 M, pH 6.5) 1 ml. The protoplasts were incubated in 95% humidity at 300C for 7-14 days until thiostrepton resistant (Thio') colonies were visible.

7.1.6. Transformation Of Streptomyces lividans Protoplasts S. lividans TK64 (provided by the John Innes Institute, Norwich, U.K) was used for transformations in some cases. Methods and compositions for growing, protoplasting, and transforming S. lividans are described in Hopwood et al., 1985, Genetic Manipulation of Streptomyces, A Laboratory Manual, John Innes Foundation, Norwich, and performed as described therein. Plasmid DNA was isolated from S. lividans transformants as described in Section 7.1.3, above.

7.1.7. Fermentation Analysis Of S. avermitilis Strains S. avermitilis mycelia grown on 1/2 strength YPD-6 for 4-7 days were inoculated into 1 x 6 inch tubes containing 8 ml of preform medium and two 5 mm glass beads. Preform medium contains: soluble starch (either thin boiled starch or KOSO, Japan Corn Starch Co., Nagoya) 20 g/L; Pharmamedia 15 g/L; Ardamine pH 5 g/L (Champlain Ind., Clifton, NJ); CaCO 3 2 g/L; 2x bcfa ("bcfa" refers to branched chain fatty acids) containing a final concentration in the medium of 50 ppm 2-(+/-)-methyl butyric acid, 60 ppm isobutyric acid, WO 99/41389 PCT/IB99/00130 and 20 ppm isovaleric acid. The pH was adjusted to 7.2, and the medium was autoclaved at 121°C for 25 min.

The tube was shaken at a 170 angle at 215 rpm at 29 0 C for 3 days. A 2-ml aliquot of the seed culture was used to inoculate a 300 ml Erlenmeyer flask containing 25 ml of production medium which contains: starch (either thin boiled starch or KOSO) 160 g/L; Nutrisoy (Archer Daniels Midland, Decatur, IL) 10 g/L; Ardamine pH 10 g/L; K 2

HPO

4 2 g/L; MgSO 4 .4H 2 0 2 g/L; FeSO 4 .7H 2 0 0.02 g/L; MnCI, 0.002 g/L; ZnSO 4 .7H 2 0 0.002 g/L; CaCO3 14 g/L; 2x bcfa (as above); and cyclohexane carboxylic acid (CHC) (made up as a solution at pH 7.0) 800 ppm. The pH was adjusted to 6.9, and the medium was autoclaved at 121 0 C for 25 min.

After inoculation, the flask was incubated at 29 0 C for 12 days with shaking at 200 rpm. After incubation, a 2 ml sample was withdrawn from the flask, diluted with 8 ml of methanol, mixed, and the mixture centrifuged at 1,250 x g for 10 min to pellet debris. The supernatant 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 detection by absorbance at 240 nm. The mobile 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 was prepared and hybridized with a ketosynthase (KS) probe made from a fragment of the Saccharopolyspora erythraea polyketide synthase (PKS) gene. A detailed description of the preparation of cosmid libraries can be found in Sambrook et al., 1989, above. A detailed description of the preparation of Streptomyces chromosomal DNA libraries is presented in Hopwood et al, 1985, above. Cosmid clones containing ketosynthase-hybridizing regions were identified by hybridization to a 2.7 Kb Ndel/Eco47ll1 fragment from pEX26 (kindly supplied by Dr. P. Leadlay, Cambridge, UK). Approximately 5 ng of pEX26 were digested using Ndel and Eco47111. The reaction mixture was loaded on a 0.8% SeaPlaque

GTG

agarose gel (FMC BioProducts, Rockland, ME). The 2.7 Kb NdellEco47ll fragment was excised from the gel after electrophoresis and the DNA recovered from the gel using GELaseT from Epicentre Technologies using the.yast Protocol. The 2.7 Kb Ndel/Eco47ll fragment was labeled with [a- 32 P]dCTP (deoxycytidine 5'-triphosphate, tetra (triethylammonium) salt, [alpha- 3 2 (NEN-Dupont, Boston, MA) using the BRL Nick Translation System (BRL Life Technologies, Inc., Gaithersburg, MD) following the supplier's instructions. A typical reaction was performed in 0.05 ml volume. After addition of 5 pl Stop WO 99/41389 PCT/IB99/00130 buffer, the labeled DNA was separated from unincorporated nucleotides using a Sephadex Quick SpinTM Column (Boehringer Mannheim) following supplier's instructions.

Approximately 1,800 cosmid clones were screened by colony hybridization. Ten clones were identified that hybridized strongly to the Sacc. erythraea KS probe. E. coli colonies containing cosmid DNA were grown in LB liquid medium and cosmid DNA was isolated from each culture in the AutoGen 540TM automated nucleic acid isolation instrument using Cycle 3 (equipment software) according to manufacturer's instructions. Restriction endonuclease mapping and Southern blot hybridization analyses revealed that five of the clones contained overlapping chromosomal regions. An S. avermitilis genomic BamHI restriction map of the five cosmids pSE65, pSE66, pSE67, pSE68, pSE69) was constructed by analysis of overlapping cosmids and hybridizations (FIGURE 4).

7.1.9. Identification Of DNA That Modulates Avermectin B2:B1 Ratios And Identification Of An aveC ORF 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 Sad and BamHI. The reaction mixture was loaded on a 0.8% SeaPlaqueTM GTG agarose gel (FMC BioProducts), a 2.9 Kb Sacl/BamHI fragment was excised from the gel after electrophoresis, and the DNA was recovered from the gel using GELaseTM (Epicentre Technologies) using the Fast Protocol. Approximately 5 pIg of the shuttle vector pWHM3 (Vara et al., 1989, J. Bacteriol. 171:5872-5881) was digested with Sad and BamHI. About 0.5 pg of the 2.9 Kb insert and 0.5 pg of digested pWHM3 were mixed together and incubated overnight with 1 unit of ligase (New England Biolabs, Inc., Beverly, MA) at 15 0 C, in a total volume of 20 pl, according to supplier's instructions. After incubation, pl of the ligation mixture was incubated at 70 0 C for 10 min, cooled to rm temp, and used to transform competent E. coli DH5a cells (BRL) according to manufacturer's instructions.

Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the 2.9 Kb Sacl/BamHI insert was confirmed by restriction analysis. This plasmid was designated as pSE119.

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 a mutant that produces significantly more of the avermectin cyclohexyl-B2 form compared to avermectin cyclohexyl-B1 form when supplemented with cyclohexane carboxylic acid (B2:B1 of about 30:1). pSE119 used to transform S. avermitilis protoplasts was isolated from either WO 99/41389 PCT/IB99/00130 E. coli strain GM2163 (obtained from Dr. B. J. Bachmann, Curator, E. coli Genetic Stock Center, Yale University), E. coli strain DM1 (BRL), or S. lividans strain TK64. Thiostrepton resistant transformants of strain 1100-SC38 were isolated and analyzed by HPLC analysis of fermentation products. Transformants of S. avermitilis strain 1100-SC38 containing pSE119 produced an altered ratio of avermectin cyclohexyl-B2:cyclohexyl-B1 of about 3.7:1 (TABLE 2).

Having established that pSE119 was able to modulate avermectin B2:B1 ratios in an AveC mutant, the insert DNA was sequenced. Approximately 10 pg of pSE119 were isolated using a plasmid DNA isolation kit (Qiagen, Valencia, CA) following manufacturer's instructions, and sequenced using an ABI 373A Automated DNA Sequencer (Perkin Elmer, Foster City, CA). Sequence data was assembled and edited using Genetic Computer Group programs (GCG, Madison, WI). The DNA sequence and the aveC ORF are presented in FIGURE 1 (SEQ ID NO:1).

A new plasmid, designated as pSE118, was constructed as follows. Approximately pg of pSE66 was digested with Sphl and BamHI. The reaction mixture was loaded on a 0.8% SeaPlaque GTG agarose gel (FMC BioProducts), a 2.8 Kb Sphl/BamHI fragment was excised from the gel after electrophoresis, and the DNA was recovered from the gel using GELaseTM (Epicentre Technologies) using the Fast Protocol. Approximately 5 pg of the shuttle vector pWHM3 was digested with Sphl and BamHI. About 0.5 pg of the 2.8 Kb insert and 0.5 pg of digested pWHM3 were mixed together and incubated overnight with 1 unit of ligase (New England Biolabs) at 15°C in a total volume of 20 pl according to supplier's instructions. After incubation, 5 pl of the ligation mixture was incubated at 70 0 C for 10 min, cooled to rm temp, and used to transform competent E. coli DH5a cells according to manufacturer's instructions.

Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the 2.8 Kb Sphl/BamHI insert was confirmed by restriction analysis. This plasmid was designated as pSE118. The insert DNA in pSE118 and pSE119 overlap by approximately 838 nucleotides (FIGURE 4).

Protoplasts of S. avermitilis strain 1100-SC38 were transformed with pSE118 as above. Thiostrepton resistant transformants of strain 1100-SC38 were isolated and analyzed by HPLC analysis of fermentation products. Transformants of S. avermitilis strain 1100-SC38 containing pSE118 were not altered in the ratios of avermectin cyclohexyl-B2: avermectin cyclohexyl-B1 compared to strain 1100-SC38 (TABLE 2).

WO 99/41389 PCT/IB99/00130 7.1.10. PCR Amplification Of The aveC Gene From S. avermitilis Chromosomal DNA A -1.2 Kb fragment containing the aveC ORF was isolated from S. avermitilis chromosomal DNA by PCR amplification using primers designed on the basis of the aveC nucleotide sequence obtained above. The PCR primers were supplied by Genosys Biotechnologies, Inc. (Texas). The rightward primer was: 5'-TCACGAAACCGGACACAC-3' (SEQ ID NO:6); and the leftward primer was: CATGATCGCTGAACCGAG-3' (SEQ ID NO:7). The PCR reaction was carried out with Deep Vent T M polymerase (New England Biolabs) in buffer provided by the manufacturer, and in the presence of 300 pM dNTP, glycerol, 200 pmol of each primer, 0.1 pg template, and 2.5 units enzyme in a final volume of 100 gl, using a Perkin-Elmer Cetus thermal cycler. The thermal profile of the first cycle was 0 C for 5 min (denaturation step), 60°C for 2 min (annealing step), and 720C for 2 min (extension step). The subsequent 24 cycles had a similar thermal profile except that the denaturation step was shortened to 45 sec and the annealing step was shortened to 1 min.

The PCR product was electrophoresed in a 1% agarose gel and a single DNA band of -1.2 Kb was detected. This DNA was purified from the gel, and ligated with 25 ng of linearized, blunt pCR-Blunt vector (Invitrogen) in a 1:10 molar vector-to-insert ratio following manufacturer's instructions. The ligation mixture was used to transform One ShotTM Competent E. coli cells (Invitrogen) following manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the -1.2 Kb insert was confirmed by restriction analysis. This plasmid was designated as pSE179.

The insert DNA from pSE179 was isolated by digestion with BamHI/Xbal, separated by electrophoresis, purified from the gel, and ligated with shuttle vector pWHM3, which had also been digested with BamHI/Xbal, in a total DNA concentration of 1 pg in a 1:5 molar vector-to-insert ratio. The ligation mixture was used to transform competent E. coli cells according to manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the -1.2 Kb insert was confirmed by restriction analysis. This plasmid, which was designated as pSE186 (FIGURE 2, ATCC 209604), was transformed into E coli DM1, and plasmid DNA was isolated from ampicillin resistant transformants.

7.2. Results A 2.9 Kb Sacl/BamHI fragment from pSE119 was identified that, when transformed into S. avermitilis strain 1100-SC38, significantly altered the ratio of B2:B1 avermectin production. S. avermitilis strain 1100-SC38 normally has a B2:B1 ratio of about 30:1, but WO 99/41389 PCT/IB99/00130 when transformed with a vector comprising the 2.9 Kb Sacl/BamHI fragment, the ratio of B2:B1 avermectin decreased to about 3.7:1. Post-fermentation analysis of transformant cultures verified the presence of the transforming DNA.

The 2.9 Kb pSE119 fragment was sequenced and a -0.9 Kb ORF was identified (FIGURE 1) (SEQ ID NO:1), which encompasses a PstllSphl fragment that had previously been mutated elsewhere to produce B2 products only (Ikeda et 1995, above). A comparison 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.

TABLE 2 presents the fermentation analysis of S. avermitilis strain 1100-SC38 transformed with various plasmids.

TABLE 2 S. avermitilis strain No. Transformants Avg.

(transforming plasmid) Tested 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

OF

S. AVERMITILIS AveC MUTANTS This example describes the construction of several different S. avermitilis AveC mutants using the compositions and methods described above. A general description of techniques for introducing mutations into a gene in Streptomyces is described by Kieser and Hopwood, 1991, Meth. Enzym. 204:430-458. A more detailed description is provided by Anzai et al., 1988, J. Antibiot. XLI(2):226-233, and by Stutzman-Engwall et al., 1992, J.

Bacteriol. 174(1):144-154. These references are incorporated herein by reference in their entirety.

8.1. Inactivation Of The S. avermitilis aveC Gene AveC mutants containing inactivated aveC genes were constructed using several methods, as detailed below.

WO 99/41389 PCT/IB99/00130 In the first method, a 640 bp SphllPstl fragment internal to the aveC gene in pSE119 (plasmid described in Section 7.1.9, above) was replaced with the ermE gene (for erythromycin resistance) from Sacc. erythraea. The ermE gene was isolated from plJ4026 (provided by the John Innes Institute, Norwich, see also Bibb et aL, 1985, Gene 41:357- 368) by restriction enzyme digestion with Bgll and EcoRI, followed by electrophoresis, and was purified from the gel. This -1.7 Kb fragment was ligated into pGEM7Zf (Promega) which had been digested with BamHI and EcoRI, and the ligation mixture transformed into competent E. coli DH5a cells following manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the -1.7 Kb insert was confirmed by restriction analysis. This plasmid was designated as pSE27.

pSE118 (described in Section 7.1.9, above) was digested with Sphl and BamHI, the digest electrophoresed, and the -2.8 Kb SphllBamHI insert purified from the gel. pSE119 was digested with Pstl and EcoRI, the digest electrophoresed, and the -1.5 Kb Pstl/EcoRI insert purified from the gel. Shuttle vector pWHM3 was digested with BamHI and EcoRI.

pSE27 was digested with Pstl and Sphl, the digest electrophoresed, and the -1.7 Kb PstllSphl insert purified from the gel. All four fragments -2.8 Kb, -1.5Kb, -7.2Kb, -1.7 Kb) were ligated together in a 4-way ligation. The ligation mixture was transformed into competent E. coli DH5a cells following manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid was designated as pSE180 (FIGURE 3; ATCC 209605).

pSE180 was transformed into S. lividans TK64 and transformed colonies identified by resistance to thiostrepton and erythromycin. pSE180 was isolated from S. lividans and used to transform S. avermitilis protoplasts. Four thiostrepton resistant S. avermitilis transformants were identified, and protoplasts were prepared and plated under non-selective conditions on RM14 media. After the protoplasts had regenerated, single colonies were screened for the presence of erythromycin resistance and the absence of thiostrepton resistance, indicating chromosomal integration of the inactivated aveC gene and loss of the free replicon. One Ermr Thios transformant was identified and designated as strain SE180-11. Total chromosomal DNA was isolated from strain SE180-11, digested with restriction enzymes BamHI, Hindlll, Pstl, or Sphl, resolved by electrophoresis on a 0.8% agarose gel, transferred to nylon membranes, and hybridized to the ermE probe. These analyses showed that chromosomal integration of the ermE resistance gene, and concomitant deletion of the 640 bp Pstl/Sphl WO 99/41389 PCT/IB99/00130 fragment had occurred by a double crossover event. HPLC analysis of fermentation products of strain SE180-11 showed that normal avermectins were no longer produced (FIGURE In a second method for inactivating the aveC gene, the 1.7 Kb ermE gene was removed from the chromosome of S. avermitilis strain SE180-11, leaving a 640 bp PstllSphl deletion in the aveC gene. A gene replacement plasmid was constructed as follows: pSE180 was partially digested with Xbal and an -11.4 Kb fragment purified from the gel. The ~11.4 Kb band lacks the 1.7 Kb ermE resistance gene. The DNA was then ligated and transformed into E. coli DH5c cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis. This plasmid, which was designated as pSE184, was transformed into E. coli DM1, and plasmid DNA isolated from ampicillin resistant transformants. This plasmid was used to transform protoplasts of S.

avermitilis strain SE180-11. Protoplasts were prepared from thiostrepton resistant transformants of strain SE180-11 and were plated as single colonies on RM14. After the protoplasts had regenerated, single colonies were screened for the absence of both erythromycin resistance and thiostrepton resistance, indicating chromosomal integration of the inactivated aveC gene and loss of the free replicon containing the ermE gene. One Erms Thios transformant was identified and designated as SE184-1-13. Fermentation analysis of SE184-1-13 showed that normal avermectins were not produced and that SE184-1-13 had the same fermentation profile as SE180-11.

In a third method for inactivating the aveC gene, a frameshift was introduced into the chromosomal aveC gene by adding two G's after the C at nt position 471 using PCR, thereby creating a BspE1 site. The presence of the engineered BspE1 site was useful in detecting the gene replacement event. The PCR primers were designed to introduce a frameshift mutation into the aveC gene, and were supplied by Genosys Biotechnologies, Inc. The rightward primer was: 5'-GGTTCCGGATGCCGTTCTCG-3' (SEQ ID NO:8) and the leftward primer was: 5'-AACTCCGGTCGACTCCCCTTC-3' (SEQ ID NO:9). The PCR conditions were as described in Section 7.1.10 above. The 666 bp PCR product was digested with Sphl to give two fragments of 278 bp and 388 bp, respectively. The 388 bp fragment was purified from the gel.

The gene replacement plasmid was constructed as follows: shuttle vector pWHM3 was digested with EcoRI and BamHI. pSE119 was digested with BamHI and Sphl, the digest electrophoresed, and a -840 bp fragment was purified from the gel. pSE119 was digested with EcoRI and Xmnl, the digest was resolved by electrophoresis, and a -1.7 Kb fragment was purified from the gel. All four fragments -7.2 Kb, -840 bp, -1.7 Kb, and 388 bp) WO 99/41389 PCT/IB99/00130 were ligated together in a 4-way ligation. The ligation mixture was transformed into competent E coli DH5a cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. This plasmid, which was designated as pSE185, was transformed into E coli DM1 and plasmid DNA isolated from ampicillin resistant transformants. This plasmid was used to transform protoplasts of S. avermitilis strain 1100-SC38. Thiostrepton resistant transformants of strain 1100-SC38 were isolated and analyzed by HPLC analysis of fermentation products. pSE185 did not significantly alter the B2:B1 avermectin ratios when transformed into S. avermitilis strain 1100-SC38 (TABLE 2).

pSE185 was used to transform protoplasts of S. avermitilis to generate a frameshift mutation in the chromosomal aveC gene. Protoplasts were prepared from thiostrepton resistant transformants and plated as single colonies on RM14. After the protoplasts had regenerated, single colonies were screened for the absence of thiostrepton resistance.

Chromosomal DNA from thiostrepton sensitive colonies was isolated and screened by PCR for the presence of the frameshift mutation integrated into the chromosome. The PCR primers were designed based on the aveC nucleotide sequence, and were supplied by Genosys Biotechnologies, Inc. (Texas). The rightward PCR primer was: GCAAGGATACGGGGACTAC-3' (SEQ ID NO:10) and the leftward PCR primer was: GAACCGACCGCCTGATAC-3' (SEQ ID NO: and the PCR conditions were as described in Section 7.1.10 above. The PCR product obtained was 543 bp and, when digested with BspE1, three fragments of 368 bp, 96 bp, and 79 bp were observed, indicating chromosomal integration of the inactivated aveC gene and loss of the free replicon.

Fermentation analysis of S. avermitilis mutants containing the frameshift mutation in the aveC gene showed that normal avermectins were no longer produced, and that these mutants had the same fermentation HPLC profile as strains SE180-11 and SE184-1-13. One Thios 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 at position 116 to a termination codon, was produced. An S. avermitilis strain with this mutation did not produce 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: nt position 970 from G to A, which changes the amino acid at position 256 from a glycine to an aspartate and (ii) nt position 996 from T to C, which changes the amino acid at position 275 from tyrosine (Y) WO 99/41389 PCT/IB99/00130 to histidine 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 other mutations in the aveC gene. pSE186, which contains a wild-type copy of the aveC gene, was transformed into E. coli DM1, and plasmid DNA was isolated from ampicillin resistant transformants. This pSE186 DNA was used to transform protoplasts of S. avermitilis strain SE180-11. Thiostrepton resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and Thio' Erm' transformants were analyzed by HPLC analysis of fermentation products. The presence of the functional aveC gene in trans was able to restore normal avermectin production to strain SE180-11 (FIGURE 8.2. Analysis Of Mutations In The aveC Gene That Alter Class B2:B1 Ratios As described above, S. avermitilis strain SE180-11 containing an inactive aveC gene was 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 mutations in the aveC gene, as described below.

Chromosomal DNA was isolated from strain 1100-SC38, and used as a template for PCR amplification of the aveC gene. The 1.2 Kb ORF was isolated by PCR amplification using primers designed on the basis of the aveC nucleotide sequence. The rightward primer was SEQ ID NO:6 and the leftward primer was SEQ ID NO:7 (see Section 7.1.10, above).

The PCR and subcloning conditions were as described in Section 7.1.10. DNA sequence analysis of the 1.2 Kb ORF shows a mutation in the aveC gene that changes nt position 337 from C to T, which changes the amino acid at position 55 from serine to phenylalanine The aveC gene containing the S55F mutation was subcloned into pWHM3 to produce a plasmid which was designated as pSE187, and which was used to transform protoplasts of S.

avermitilis strain SE180-11. Thiostrepton resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and Thio' Erm' transformants were analyzed by HPLC analysis of fermentation products. The presence of the aveC gene encoding a change at amino acid residue 55 (S55F) was able to restore normal avermectin production to strain SE180-11 (Fig. 5C); however, the cyclohexyl B2:cyclohexyl B1 ratio was about 26:1, as compared to strain SE180-11 transformed with WO 99/41389 PCT/IB99/00130 pSE186, which had a ratio of B2:B1 of about 1.6:1 (TABLE indicating that the single mutation (S55F) modulates the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1.

Another mutation in the aveC gene was identified that changes nt position 862 from G to A, which changes the amino acid at position 230 from glycine to aspartate An S.

avermitilis strain having this mutation (G230D) produces avermectins at a B2:B1 ratio of about 30:1.

8.3. Mutations That Reduce The B2:B1 Ratio Several mutations were constructed that reduce the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1, as follows.

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 to threonine The aveC gene containing the A139T mutation was subcloned into pWHM3 to produce a plasmid which was designated pSE188, and which was used to transform protoplasts of S. avermitilis strain SE180-11. Thiostrepton resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and Thior Ermr transformants were analyzed by HPLC analysis of fermentation products. The presence of the mutated aveC gene encoding a change at amino acid residue 139 (A139T) was able to restore avermectin production 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 cyclohexyl-B1. This result was unexpected because published results, as well as the results of mutations described above, have only demonstrated either inactivation of the aveC gene or increased 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 B1 direction, a mutation was constructed that encoded a threonine instead of a serine at amino 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 as pSE186a, was digested with Apal and Kpnl, the DNA fragments separated on an agarose gel, and two fragments of -3.8 Kb and -0.4 Kb were purified from the gel. The -1.2 Kb insert DNA from pSE186 was used as a PCR template to introduce a single base change at nt position 585. The PCR primers were designed to introduce a mutation at nt position 585, and were supplied by Genosys Biotechnologies, Inc. (Texas). The rightward PCR primer was: 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 WO 99/41389 PCT/IB99/00130 Laboratories, Palo Alto, CA) in buffer provided by the manufacturer in the presence of 200 pM dNTPs, 200 pmol of each primer, 50 ng template DNA, 1.0 M GC-Melt and 1 unit KlenTaq Polymerase Mix in a final volume of 50 pl. The thermal profile of the first cycle was 940C for 1 min; followed by 25 cycles of 94°C for 30 sec and 68°C for 2 min; and 1 cycle at 68 0 C for 3 min. A PCR product of 295 bp was digested with Apal and Kpnl to release a 254 bp fragment which was resolved by electrophoresis and purified from the gel. All three fragments (-3.8 Kb, -0.4 Kb and 254 bp) were ligated together in a 3-way ligation. The ligation mixture was transformed into competent E. coli DH5a cells. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid was designated as pSE198.

pSE198 was digested with EcoRI, cloned into pWHM3 which had been digested with EcoRI, and transformed into E coli DH5a cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. This plasmid DNA was transformed into E. coli DM1, plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid, which was designated as pSE199, was used to transform protoplasts of S. avermitilis strain SE180-11. Thiostrepton resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and Thior Ermr transformants were analyzed by HPLC analysis of fermentation products. The presence of the mutated aveC gene encoding a change at amino acid 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 of cyclohexyl-B2 produced relative to cyclohexyl-B1 (TABLE This B2:B1 ratio is even lower than the 0.94:1 ratio observed with the A139T mutation produced by transformation of strain SE180-11 with pSE188, as described above.

Another mutation was constructed to introduce a threonine at both amino acid positions 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 were supplied by Genosys Biotechnologies, Inc. (Texas). The rightward PCR primer was: GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCGCTGGCGACGACC-3' (SEQ ID NO:14); and the leftward PCR primer was: 5'-GGAACATCACGGCATTCACC-3' (SEQ ID The PCR reaction was performed using the conditions described immediately above in this Section. A PCR product of 449 bp was digested with Apal and Kpnl to release a 254 bp fragment, which was resolved by electrophoresis and purified from the gel. pSE186a was 49 WO 99/41389 PCT/IB99/00130 digested with Apal and Kpnl, the DNA fragments separated on an agarose gel, and two fragments of -3.8 Kb and ~0.4 Kb were purified from the gel. All three fragments Kb, -0.4 Kb and 254 bp) were ligated together in a 3-way ligation, and the ligation mixture was transformed into competent E. coli DH5 cells. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid was designated as pSE230.

pSE230 was digested with EcoRI, cloned into pWHM3 which had been digested with EcoRI, and transformed into E. coli DH5a cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. This plasmid DNA was transformed into E. coli DM1, plasmid DNA isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid, which was designated as pSE231, was used to transform protoplasts of S. avermitilis strain SE180-11. Thiostrepton resistant transformants of SE180-11 were isolated, the presence of erythromycin resistance was determined, and Thior Ermr transformants were analyzed by fermentation. The presence of the double mutated aveC gene, encoding S138T/A139T, was able to restore normal avermectin production to strain SE180-11; however, the B2:B1 ratio was 0.84:1 showing that this mutation further reduces the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1 (TABLE over the reductions provided by transformation of strain SE180-11 with pSE188 or pSE199, as described above.

TABLE 3 S. avermitilis strain No. Relative Relative Avg.

(transforming plasmid) transformants B2 Conc. B1 Conc. B2:B1 tested 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 WO 99/41389 PCT/IB99/00130 These results are the first to demonstrate specific mutations in the aveC gene that result in the production of increased levels of the more commercially desirable class 1 avermectins relative to class 2 avermectins.

9. EXAMPLE: CONSTRUCTION OF 5' DELETION MUTANTS As explained in Section 5.1, above, the S. avermitilis nucleotide sequence shown in FIGURE 1 (SEQ ID NO:1) comprises four different GTG codons at bp positions 42, 174, 177 and 180 which are potential start sites. This section describes the construction of multiple deletions of the 5' region of the aveC ORF (FIGURE 1; SEQ ID NO:1) to help define which of these 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 based on the aveC DNA sequence, and were supplied by Genosys Biotechnologies, Inc. The rightward primers were 5'-AACCCATCCGAGCCGCTC-3' (SEQ ID NO:16) (D1F1); 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); CGCTGAACCGAGGA-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 single DNA bands of either -1.0 Kb or ~1.1 Kb were detected. The PCR products were purified from the gel and ligated with 25 ng of linearized pCR2.1 vector (Invitrogen) in a 1:10 molar vectorto-insert ratio following the manufacturer's instructions. The ligation mixtures were used to transform One Shot T Competent E. coli cells (Invitrogen) following manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the insert was confirmed by restriction analysis and DNA sequence analysis.

These plasmids were designated as pSE190 (obtained with primer D1F1), pSE191 (obtained with primer D1F2), pSE192 (obtained with primer D1F3), and pSE193 (obtained with primer D2F2).

The insert DNAs were each digested with BamHIIXbal, separated by electrophoresis, purified from the gel, and separately ligated with shuttle vector pWHM3, which had been digested with BamHIIXbal, in a total DNA concentration of 1 pg in a 1:5 molar vector-to-insert ratio. The ligation mixtures were used to transform competent E. coli DH5a cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the insert was confirmed by restriction analysis. These plasmids, which were designated as pSE194 WO 99/41389 PCT/IB99/00130 (D1F1), pSE195 (D1F2), pSE196 (D1F3), and pSE197 (D2F2), were each separately transformed into E. coli strain DM1, plasmid DNA isolated from ampicillin resistant transformants, and the presence of the correct insert confirmed by restriction analysis. This DNA was used to transform protoplasts of S. avermitilis strain SE180-11. Thiostrepton resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and Thio' Erm' transformants were analyzed by HPLC analysis of fermentation products to determine which GTG sites were necessary for aveC expression.

The results indicate that the GTG codon at position 42 can be eliminated without affecting aveC expression, since pSE194, pSE195, and pSE196, each of which lack the GTG site at position 42, but which all contain the three GTG sites at positions 174, 177, and 180, were each able to restore normal avermectin production when transformed into SE180-11. Nomal avermectin production was not restored when strain SE180-11 was transformed with pSE197, which lacks all four of the GTG sites (TABLE 4).

TABLE 4 S. avermitilis strain No. transformants Relative Relative Avg.

(transforming plasmid) tested B2 Conc. B1 Conc. 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 EXAMPLE: CLONING OF aveC HOMOLOGS FROM S. HYGROSCOPICUS AND S. GRISEOCHROMOGENES The present 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 WO 99/41389 PCT/IB99/00130 identified that hybridized strongly. Chromosomal DNA was isolated from these cosmids, and a 4.9 Kb Kpnl fragment was identified that hybridized with the aveC probe. This DNA was sequenced and an ORF (SEQ ID NO:3) was identified having significant homology to the aveC 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 hybridized with the 1.2 Kb aveC probe from S. avermitilis described above. Several cosmid clones were identified that hybridized strongly. Chromosomal DNA was isolated from these cosmids, and a 5.4 Kb Pstl fragment was identified that hybridized with the aveC probe. This DNA was sequenced and an aveC homolog partial ORF was identified having significant homology to the aveC ORF of S. avermitilis. A deduced partial amino acid sequence (SEQ ID NO:5) is presented in FIGURE 6.

DNA and amino acid sequence analysis of the aveC homologs from S. hygroscopicus and S. griseochromogenes indicates that these regions share significant homology 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 WITH THE aveC GENE BEHIND THE ermE PROMOTER The 1.2 Kb aveC ORF from pSE186 was subcloned in pSE34, which is the shuttle vector pWHM3 having the 300 bp ermE promoter inserted as a Kpnl/BamHI fragment in the Kpnl/BamHI site of pWHM3 (see Ward et al., 1986, Mol. Gen. Genet. 203:468-478). pSE186 was digested with BamHI and HindIll, the digest resolved by electrophoresis, and the 1.2 Kb fragment was isolated from the agarose gel and ligated with pSE34 which had been digested with BamHI and Hindlll. The ligation mixture was transformed into competent E. coli cells according to manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the 1.2 Kb insert was confirmed by restriction analysis. This plasmid, which was designated as pSE189, was transformed into E. coli DM1, and plasmid DNA isolated from ampicillin resistant transformants. Protoplasts of S. avermitilis strain 1100-SC38 were transformed with pSE189. Thiostrepton resistant transformants of strain 1100-SC38 were isolated and analyzed by HPLC analysis of fermentation products.

S. avermitilis strain 1100-SC38 transformants containing pSE189 were altered in the ratios of avermectin cyclohexyl-B2:avermectin cyclohexyl-Bi produced (about 3:1) compared to strain 1100-SC38 (about 34:1), and total avermectin production was increased approximately 2.4-fold compared to strain 1100-SC38 transformed with pSE119 (TABLE WO 99/41389 PCT/IB99/00130 pSE189 was also transformed into protoplasts of a wild-type S. avermitilis strain.

Thiostrepton resistant 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 transformed with pSE119 (TABLE TABLE S. avermitilis strain No. Trans- Relative Relative Relative Total Avg.

(transforming formants [B2] Avermectins B2:B1 plasmid) Tested [B1] Ratio 1100-SC38 6 155 4.8 176 33.9 1100-SC38 9 239 50.3 357 4.7 (pSE119) 1100-SC38 16 546 166 849 3.3 (pSE189) wild type 6 59 42 113 1.41 wild type 6 248 151 481 1.64 (pSE119) wild type 5 545 345 1,071 1.58 (pSE189) 12. EXAMPLE: CHIMERIC PLASMID CONTAINING SEQUENCES FROM BOTH S. A VERMITILIS aveC ORF AND S. HYGROSCOPICUS aveC HOMOLOG A hybrid plasmid designated as pSE350 was constructed that contains a 564 bp portion of the S. hygroscopicus aveC homolog replacing a 564 bp homologous portion of the S. avermitilis aveC ORF (FIGURE as follows. pSE350 was constructed using a BsaAl restriction site that is conserved in both sequences (aveC position 225), and a Kpnl restriction site that is present in the S. avermitilis aveC gene (aveC position 810). The KpnI site was introduced into the S. hygroscopicus DNA by PCR using the rightward primer CTTCAGGTGTACGTGTTCG-3' (SEQ ID NO:23) and the leftward primer GAACTGGTACCAGTGCCC-3' (SEQ ID NO:24) (supplied by Genosys Biotechnologies) using PCR conditions described in Section 7.1.10, above. The PCR product was digested with BsaAl and Kpnl, the fragments were separated by electrophoresis in a 1% agarose gel, and the 564 bp BsaAIIKpnl fragment was isolated from the gel. pSE179 (described in Section WO 99/41389 PCT/IB99/00130 7.1.10, above) was digested with Kpnl and Hindlll, the fragments separated by electrophoresis in a 1% agarose gel, and a fragment of -4.5 Kb was isolated from the gel.

pSE179 was digested with Hindlll and BsaAI, the fragments separated by electrophoresis in a 1% agarose gel, and a -0.2 Kb BsaAI/Hindlll fragment isolated from the gel. The -4.5 Kb HindllllKpnl fragment, the -0.2 Kb BsaAlIHindlll fragment, and the 564 bp BsaAIIKpnl fragment from S. hygroscopicus were ligated together in a 3-way ligation and the ligation mixture transformed into competent E. coli DH5a cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis using Kpnl and Aval. This plasmid was digested with Hindlll and Xbal to release the 1.2 Kb insert, which was then ligated with pWHM3 which had been digested with Hindlll and Xbal. The ligation mixture was transformed into competent E. coli DH5a cells, plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis using Hindlll and Aval. This plasmid DNA was transformed into E. coli DM1, plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. This plasmid was designated as pSE350 and used to transform protoplasts of S. avermitilis strain SE180-11. Thiostrepton resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined and Thio' Ermr transformants were analyzed by HPLC analysis of fermentation products. Results show that transformants containing the S. avermitilis/S. hygroscopicus hybrid plasmid have an average B2:B1 ratio of about 109:1 (TABLE 6).

TABLE 6 S. avermitilis strain No. transformants Relative Relative Avg.

(transforming plasmid) tested B2 Conc. B1 Conc. B2:B1 Ratio SE180-11 (none) 8 0 0 0 SE180-11 (pWHM3) 8 0 0 0 SE180-11 (pSE350) 16 233 2 109 WO 99/41389 PCT/IB99/00130 DEPOSIT OF BIOLOGICAL MATERIALS The following biological material was deposited with the American Type Culture Collection (ATCC) at 12301 Parklawn Drive, Rockville, MD, 20852, USA, on January 29, 1998, and was assigned the following accession numbers: Plasmid Accession No.

plasmid pSE180 209605 plasmid pSE186 209604 All patents, patent applications, and publications cited above are incorporated herein by reference in their entirety.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

EDITORIAL NOTE 18878/99 SEQUENCE LISTING PAGES 1 TO 12 FOLLOW PAGE 56 OF THE

DESCRIPTION.

WO 99/41389 PCT/1B99/00130 SEQUENCE LISTING <110> Pfizer Products Inc. (All Non-U.S. Applications) <120> STREPTOMYCES AVERMITILIS GENE DIRECTING THE RATIO OF B2:B1 AVERMECTINS <130> PC9916A <140> <141> <150> 60/074,636 <151> 1998-02-13 <160> 24 <170> Patentln Ver. 2.0 beta <210> 1 <211> 1229 <212> DNA <213> Streptomyces avermitilis <220> <221> CDS <222> (174)..(1085) <400> 1 tcacgaaacc ggacacacca cacacacgaa ggtgagacag cgtgaaccca tccgagccgc tcggcctgcc caacgaacgt gtagtagaca cccgaccgtc cgatgccacg ctctcacccg 120 aggccggcct gaacaggtca ggagcgctgc cccgtgaact gctgtcgttg ccg gtg 176 Vai 1 gtg gtg tgg gcc ggg gtc ggc ctg ctg ttt ctg gcc ctg cag gcg tac 224 Val Val Trp Ala Giy Val Gly Leu Leu Phe Leu Ala Leu Gin Ala Tyr 10 gtg ttc agc cgc tgg gcg gcc gac ggt ggc tac cgg ctg atc gag acg 272 Vai Phe Ser Arg Trp Ala Ala Asp Gly Gly Tyr Arg Leu Ile Giu Thr 25 gcg ggc cag ggt cag ggc ggc agc aag gat acg ggg act acc gat gtg 320 Ala Gly Gin Gly Gin Giy Gly Ser Lys Asp Thr Gly Thr Thr Asp Val 40 gtc tat ccc gtg att tcc gtc gtc tgc atc acc gcc gcg gcg gcg tgg 368 1 WO 99/41389 WO 9941389PCT/IB99/00130 Val1 Tyr Pro Val Ile Se r 55 Val Val Cys Ile Thr Ala Ala Ala Ala 60 T rp ctc ttc cgg agg Leu Phe Arg Arg tgc Cys cgt gtc gaa cga Arg Val Glu Arg cgg Arg 75 ctg ctg ttc gac Leu Leu Phe Asp gcc ott Ala Leu ctc ttc ctc Leu Phe Leu tgg ttc cat Trp Phe His 100 ggg Gly ctg ctg ttc gcg Leu Leu Phe Ala agc Ser 90 tgg cag agc ccg Trp Gin Ser Pro ctc atg aac Leu Met Asn ggc gcg gtg Gly Ala Val 464 512 tcc gtt ctc gtc Ser Val Leu Val tcc Ser 105 aac gcg agt gtg Asn Ala Ser Val tgg T rp 110 ggt tcc Gly Ser 115 tgg ggt ccg tat Trp Gly Pro Tyr gtg Val1 120 ccc ggc tgg cag Pro Gly Trp Gin ggg Gl y 125 gcg ggc ccg ggt Ala Gly Pro Gly gcg Ala 130 gag gcg gaa atg Glu Ala Glu Met ccg Pro 135 ctg gcg tcg gc Leu Ala Ser Ala tcc Ser 140 gto tgo atg tcg Val Cys Met Ser gct Ala 145 560 608 656 704 ctg atc gtc acc Leu Ile Val Thr gtg Val 150 ctg tgc agc aag Leu Gys Ser Lys gca Ala 155 ctg ggg tgg atc Leu Gly Trp Ile aag gc Lys Ala 160 ttc ttc Phe Phe cgc cgg ccg Arg Arg Pro atc ggc atc Ile Gly Ile 180 gca Ala 165 tgg cgg acc tgg Trp Arg Thr Trp cgg Arg 170 ctg gtc ctg gcc Leu Val Leu Ala gtg Val1 175 gtg ctc ggt ctg Vai Leu Gly Leu tcc Ser 185 gag ccg ctg ccg Glu Pro Leu Pro tcc Ser 190 gcc tcc ggg Ala Ser Gly atc agc Ile Ser 195 gta tgg gcc aga Val Trp Ala Arg gcg Al a 200 ctg ccc gag gtg Leu Pro Glu Val acc Thr 205 ttg tgg agt ggc Leu Trp Ser Gly gag Giu 210 tgg tac cag ttc Trp Tyr Gin Phe ccc Pro 215 gtg tat cag gcg Vai Tyr Gin Ala gtc Val1 220 ggt tcc ggc ctg Gly Ser Gly Leu gtc Val1 225 800 848 896 tgc tgc atg ctg Cys Cys Met Leu ggc Gly 230 tcg ctg cgc ttc Ser Leu Arg Phe ttc Phe 235 cgc gac gaa cgc Arg Asp Giu Arg gat gag Asp Glu 240 tog tgg gtg Ser Trp Val gaa Giu 245 cgg gga gcc tgg Arg Gly Ala Trp cgg Arg 250 ttg cog caa cgg Leu Pro Gin Arg gca gcg aac Ala Ala Asn 255 944 WO 99/41389 WO 9941389PCT/1B99/00130 tgg gcg cgt ttc ctc gcc gtg gtc 99t ggg gtg aat gcc gtg atg ttc Trp Ala Arg Phe Leu Ala Vai Val Gly Gly Vai Asn Ala Vai Met Phe 260 265 270 ctc tac acc tgt ttc cat atc ctc ctg tcc ctc gtc ggt gga cag ccg Leu Tyr Thr Cys Phe His Ile Leu Leu Ser Leu Val Gly Giy Gin Pro 275 280 285 ccc gac caa ctg ccg gac tcc ttc caa gcg ccg gcc gct tac tga Pro Asp Gin Leu Pro Asp Ser Phe Gin Ala Pro Ala Ala Tyr 290 295 300 gttcagggca ggtcggagga gacggagaag gggaggcgac cggagttccg gtcacctccc ctttgtgcat gggtggacgg ggatcacgct cccatggcgg cgggctcctc cagacgcacc acactcctcg gttcagcgat catg <210> 2 <211> 303 <212> PRT <213> Streptornyces avermitilis <400> 2 992 1040 1085 1145 1205 1229 Val1 1 Tyr Thr Val1 Trp Leu Asn Val Val Val Val Phe Ala Gly Val Tyr Leu Phe Leu Phe Trp Phe Gly Ser 115 Trp Ala 5 Ser Arg Gin Gly Pro Val Arg Arg Leu Giy His Ser 100 Trp Gly Giy Val Trp Ala Gin Gly Ile Ser 55 Cys Arg 70 Leu Leu Val Leu Pro Tyr Gly Ala Gly 40 Val1 Val1 Phe Val1 Val 120 Leu Leu Phe 10 Asp Giy Gly 25 Ser Lys Asp Vai Cys Ile Giu Arg Arg 75 Ala Ser Trp, 90 Ser Asn Ala 105 Pro Gly Trp Leu Ala Leu Gin Tyr Arg Leu Ile Thr Giy Thr Thr Thr Ala Ala Ala Leu Leu ?he Asp Gin Ser Pro Leu Ser Val Trp Gly 110 Gin Giy Ala Gly 125 Ala Giu Asp Ala Ala Met Aila Pro WO 99/41389 Gly Ala Glu Ala 130 Giu Met Pro Leu Ala Ser Ala Ser Val Cys Met Ala 145 Ala Phe Gly Gly Val1 225 Giu Asn Phe Leu Arg Ile Ile Glu 210 Cys Ser Trp Le u Ile Arg Gly Ser 195 T rp Cys Trp Ala Tyr 275 Val1 Pro Ile 180 Val Tyr Met Val1 Arg 260 Thr Thr Ala 165 Val1 Trp Gin Leu Glu 245 Phe Cys Val1 150 Trp Leu Al a Phe Gly 230 Arg Leu Phe Leu Arg Gly Arg Pro 215 Se r Gly Al a His Cys Thr Leu Al a 200 Val1 Leu Ala Vai I le 280 Ser Lys Trp Arg 170 Ser Glu 185 Leu Pro Tyr Gin Arg Phe Trp Arg 250 Val Gly 265 Leu Leu Ala 155 Leu Pro Giu Ala Phe 235 Leu Gly Se r Leu Val1 Leu Val Val1 220 Arg Pro Val Leu Gi y Leu Pro Thr 205 Gly Asp Gin As n Val 285 Trp Ile Ala Vai 175 Ser Ala 190 Leu Trp Ser Gly Giu Arq Arg Ala 255 Ala Val 270 Gly Gly PCTIIB99/00130 Ser Lys 160 Phe Ser Ser Leu Asp 240 Ala Met Gin Pro Pro Asp Gin Leu Pro Asp Ser Phe Gln 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 gtcgacgaaq accggccgga ggccgtcggc cgggccgata ccgtacgcgg cctgcgg gtg ttc aco ctt ccc gta aca ctg tgg gcg. tgt gtc ggc gcg cig gtg Val Phe Thr Leu Pro Val Thr Leu Trp Ala Cys Vai Gly Ala Leu Val 1 5 10 57 105 WO 99/41389 WO 9941389PCT/1B99/00130 ctg gga ctt Leu Gly Leu cag Gin gtg tac gtg ttc gcc Val Tyr Val Phe gcc tgg ctc gcc Ala Trp Leu Ala Al a 25 gac agc ggc Asp Ser Gly gac tcg gag Asp Ser Glu 153 tac cgc Tyr Arg atc Ile gag aag gcg tcc Giu Lys Ala Ser ccg Pro 40 gcc agg ggc ggt Ala Arg Gly Gly ggg Gi y cgg atc Arg Ile gcc gat gtg ctg Ala Asp Val Leu atc Ile 55 ccg ctg ctg tcc Pro Leu Leu Ser gtg Val1 gtg gga gcg gtg Val Gly Ala Val 249 gtc Val1 ctc gca gtg tgt Leu Ala Val Cys ctg Le u 70 tac cgg agg tgt Tyr Arg Arg Cys cgg gcc agg agg Arg Ala Arg Arg 75 ctg tcg gcc agt Leu Ser Ala Ser cgg Arg ctg Leu 297 345 acg ttc gac gcg Thr Phe Asp Ala tcg Ser ctc ttc atc ggg Leu Phe Ile Gly ctg Leu 90 tgg cag Trp Gin agt ccc ttg Ser Pro Leu gtg ttc gga Val Phe Gly 115 atg Met 100 aac tgg atc aat Asn Trp Ile Asn ccg Pro 105 gtg ctc gcg tca Val Leu Ala Ser aac gtc aat Asn Val Asn 110 ggt tgg cag Gly Trp Gin 393 441 gcg gtg gcc tcg Ala Val Ala Ser tgg Trp 120 ggg ccg tat gtg Gly Pro Tyr Val ccc Pro 125 ggg gcg Gly Ala 130 ggg gcg cac cag Gly Ala His Gin gag Giu 135 gcc gag q~tg ccg Ala Giu ZIe u Pro ctg Leu 140 gcg acc ctg agc Ala Thr Leu Ser atc Ile 145 tgt atg acg gcc Cys Met Thr Ala atg Met 150 atg goc gcc gtg Met Ala Ala Val gcc Al a 155 tgc ggc aag ggc Cys Gly Lys Gly atg Met 160 atc Ile 489 537 585 ggt ctt gcc gcc Gly Leu Ala Ala gcc Ala 165 cgg tgg ccg cgg Arg Trp Pro Arg ctg Leu 170 ggg ccg ctc cgg Gly Pro Leu Arg ctg Leu 175 gcg ctc ggc Ala Leu Gly gtg toc ttc Val Ser Phe 195 ttt Phe 180 ctg ctc gtc gtg Leu Leu Val Val ct C Leu 185 ctc gac atc gcc Leu Asp Ile Ala gag ccg ctg Giu Pro Leu 190 ccc gag ctg Pro Glu Leu gcg ggc gtc tc Ala Gly Val Ser gtg Val 200 tgg acg cgg gca Trp Thr Arg Ala gtg Val 205 acc atc tgg agt ggg cac tgg tat cag ttc ccg ctg tat cag atg gtg Thr Ile Trp Ser Gly His Trp Tyr Gin Phe Pro Leu Tyr Gin Met Val WO 99/41389 WO 9941389PCTIIB99/001 210 215 220 gct Al a 225 tog gcg ctc ttc Ser Ala Leu Phe ggc Gly 230 gcc tct ttg ggg Ala Ser Leu Gly gc Ala 235 gcg cgc cac ttt Ala Arg His Phe ogo Arg 240 777 825 aac cgg cgc ggc Asn Arg Arg Gly gaa Glu 245 acg tgt ctg gag Thr Cys Leu Glu toc Ser 250 ggg gog gco otc Gly Ala Ala Leu cta cog Leu Pro 255 gag ggc cog Glu Gly Pro aac atc ago Asn Ile Ser 275 agg Arg 260 oca tgg gtc cgg Pro Trp Val Arg ctg Leu 265 ctg gcg gtg gtg Leu Ala Val Val ggc ggg gcc Gly Gly Ala 270 cac ato ctg His Ile Leu 873 921 ato gcc ctc tao Ile Ala Leu Tyr acc Thr 280 ggc gca cac ggc Gly Ala His Gly gca Ala 285 ttc tcg Phe Ser 290 otg atg gac ggc Leu Met Asp Gly got Ala 295 000 oog gao ogg Pro Pro Asp Arg oto Leu 300 000 gaa tto tto Pro Glu Phe Phe 969 ogt Arg 305 oog gog goo ggo Pro Ala Ala Gly tao Tyr 310 tga gaoogooggo aooaoooacg taooogatgt 1020 gcgogatgtg ootgatgogo otgatgtaoo oggggtgtoa toggotoaoo tgtggogoot. 1080 oatgoggtga gogotoogoc togtoottgt tooggotoot gggctooaog aooataogga 1140 gcggoogggg 1150 <210> 4 <211> 310 <212> PRT <213> Streptomyoes hygrosoopious <400> 4 Val Phe Thr Leu Pro Val Thr Leu Trp Ala 1 5 10 Cys Val Gly Ala Leu Val Leu Gly Leu Gin Val Tyr Val Phe Ala 25 Tyr Arg Ile Glu Lys Ala Ser Pro Ala 40 Ala Trp Leu Ala Asp Ser Gly Asp Ser Glu Arg Gly Giy Gl y Arg Ile Ala Asp Val Leu Ile Pro Leu Leu Ser Val 55 Val Giy Ala Val WO 99/41389 WO 9941389PCT/1B99/00130 Val1 Thr Ser Val Gi y Ile 145 Gly Al a Val Thr Ala 225 Asn Glu Asn Phe Arg 305 Leu Phe Pro Phe Ala 130 Cys Leu Leu Ser Ile 210 Ser Arg Gly Ile Ser 290 Pro Ala Asp Leu Gly 115 Gly Met Ala Gly Phe 195 T rp Ala Arg Pro Ser 275 Leu Ala Val Cys Leu Tyr Arg Arg Cys Arg Ala Arq Arg Arg Leu 70 75 Al a Met 100 Ala Ala Thr Al a Phe 180 Al a Ser Leu Gl y Arg 260 Ile Met Al a Ser Asri Val1 His Ala Ala 165 Leu Gly Gly Phe Glu 245 Pro Ala Asp Gly Leu Trp Al a Gin Met 150 Arg Leu Val1 His Gly 230 Thr Trp Leu Gly Tyr 310 Phe Ile Ser Giu 135 Met Trp Val Ser Trp 215 Al a Cys Val Tyr Ala 295 Ile Asn Trp 120 Al a Al a Pro Val1 Val 200 Tyr Ser Leu Arg Thr 280 Pro Gly Pro 105 Gly Glu Ala Arg Leu 185 Trp Gin Le u Glu Leu 265 Gl y Pro Leu 90 Val Pro Leu Val Leu 170 Leu Thr Phe Gi y Ser 250 Leu Ala Asp Leu Ser Leu Ala Tyr Val Pro Leu 140 Ala Cys 155 Gly Pro Asp Ile Arg Ala Pro Leu 220 Ala Ala 235 Gly Ala Ala Val His Gly Arg Leu 300 Ala Ser Pro 125 Al a Gly Leu Al a Val 205 Tyr Arg Ala Val1 Ala 285 Pro Ser Asn 110 Gly Thr Lys Arg Giu 190 Pro Gin His Leu Gi y 270 His Giu Trp Val1 Trp Leu Gly Leu 175 Pro Glu Met Phe Leu 255 Gly Ile Phe Gin Asn Gin Ser Met 160 Ile Leu Leu Val Arg 240 Pro Ala Leu Phe WO 99/41389 <210> <211> 215 <212> PRT <213> Streptomyces griseochromogenes PCTIIB99/00130 <400> Val Ile Tyr Val Val Ser Val Leu Trp Leu Leu Leu Asn Trp Val Gly Gly Lys 130 Val Leu 145 Glu Arg Leu Thr Gly Val Gly Ala 210 <210> 6 Gly Phe Gly Leu Val Phe Phe Ser 115 Glu Leu T rp Ala Ser 195 T rp T rp Ala Pro Pro Arg Thr His 100 Trp Al a Gly Pro Val1 180 Val1 Tyr Ala Arg Asp Ala Arg Gly Pro Gly Giu Val Giy 165 Al a Trp Arg Al a Trp Gi y Leu T rp 70 Val Val Pro Leu Le u 150 Val Phe Ala Ala Leu Thr Arg Ser 55 Arg Leu Leu Tyr Pro 135 Gly Arg Asp Arg Arg 215 Gly Ala Glu 40 Met Ala Phe Met Val1 120 Leu Cys Pro Leu Ala 200 Ala Asp 25 Pro Ala Glu Al a Ala 105 Pro Val Cys T rp Ser 185 Leu Val1 10 Gly Gly Gl y Arg Gly 90 Asn Gly Thr Gin Gin 170 Gi u Pro Phe Gly His Val Arg 75 Trp Thr Trp Phe Val 155 Leu Pro Thr Le u Tyr Arg Val1 Leu Leu His Arg Ser 140 Met Val Phe Val1 Val1 His Arg Gly Ser Ser Val Gly 125 Leu Ser Gly Ile Thr 205 Le u Leu Ile Le u Phe Pro Trp 110 Leu Gly Arg Le u Ser 190 Leu Gln Ala Ile Al a Asp Leu Gly Pro Ser Val1 Ala 175 Phe T rp Val Asp Asp Phe Ala Met Ala Pro Thr Arg 160 Phe Ala Arg WO 99/41389 WO 9941389PCTIIB99/001 <211> 18 <212> DNA <213> Streptomyces averinitilis <400> 6 tcacgaaacc ggacacac 18 <210> 7 <211> 18 <212> DNA <213> Streptamyces avermitilis <400> 7 catgatcgct gaaccgag 18 <210> 8 <211> <212> DNA <213> Streptomyces avermitilis <400> 8 ggttccggat gccgttctcg <210> 9 <211> 21 <212> DNA <213> Streptomyces averinitilis <400> 9 aactccggtc gactcccctt c 21 <210> <211> 19 <212> DNA <213> Streptornyces avermitilis <400> qcaaqgatac ggggactac 19 <210> 11 <211> 18 <212> DNA <213> Streptomyces avermitilis <400> 11 WO 99/41389 WO 9941389PCTIIB99/00130 gaaccgaccg cctgatac 18 <210> 12 <211> 43 <212> DNA <213> Streptomyces avermitilis <400> 12 gggggcgggc ccgggtgcgg aggcggaaat gcccctggcg acg 43 <210> 13 <211> <212> DNA <213> Streptomyces avermitilis <400> 13 ggaaccgacc gcctqataca <210> 14 <211> 46 <212> DNA <213> Streptomyces avermitilis <400> 14 gggggcgggc ccgggtgcgg aggcggaaat gccgctggcg acgacc 46 <210> <211> <212> DNA <213> Streptomyces avermitilis <400> ggaacatcac ggcattcacc <210> 16 <211> 18 <212> DNA <213> Streptomyces avermitilis <400> 16 aacccatccg agccgctc 18 <210> 17 <211> 17 WO 99/41389 <212> DNA <213> Streptomyces avermitilis <400> 17 tcggcctgcc aacgaac <210> 18 <211> 18 <212> DNA <213> Streptomyces avermitilis <400> 18 ccaacgaacg tgtagtag <210> 19 <211> <212> DNA <213> Streptomyces avermitilis <400> 19 tgcaggcgta cgtgttcagc <210> <211> 17 <212> DNA <213> Streptomyces avermitilis <400> catgatcgct gaaccga <210> 21 <211> <212> DNA <213> Streptomyces avermitilis <400> 21 catgatcgct gaaccgagga <210> 22 <211> <212> DNA <213> Streptomyces avermitilis <400> 22 aqgagtgtgg tgcgtctgga PCT/1B99/00130 17 18 WO 99/41389 PT19/03 PCT/IB99/00130 <210> 23 <211> 19 <212> DNA <213> Streptomyces avermitilis <400> 23 V cttcaggtgt acgtgttcg 19 r (210> 24 (211> 18 <212> DNA <213> Streptomyces avermitilis <400> 24 gaactggtac cagtgccc 18

Claims (62)

1. An isolated polynucleotide molecule comprising the complete aveC open- reading frame (ORF) of Streptomyces avermitilis, which isolated polynucleotide molecule lacks the next complete ORF that is located downstream from the aveC ORF in situ in the Streptomyces avermitilis chromosome, and which ORF comprises the nucleotide sequence of FIGURE 1 (SEQ ID NO:1).

2. An isolated polynucleotide molecule, comprising a nucleotide sequence that is homologous to the Streptomyces avermitilis AveC gene product-encoding nucleotide sequence of the aveC ORF, which ORF comprises the nucleotide sequence presented in FIGURE 1 (SEQ ID NO:1).

3. An oligonucleotide molecule that hybridizes to a polynucleotide molecule having the nucleotide sequence of FIGURE 1 (SEQ ID NO:1), or to a polynucleotide molecule having a nucleotide sequence that is the complement of the nucleotide sequence ,of FIGURE 1 (SEQ ID NO:1), under highly stringent conditions. S 15 4. A recombinant vector comprising a polynucleotide molecule comprising a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID NO:2.

5. The recombinant vector of claim 4, wherein the polynucleotide molecule comprises the nucleotide sequence of the aveC ORF of FIGURE 1 (SEQ ID NO: 1).

6. The recombinant vector of claim 4, wherein the polynucleotide molecule is in operative association with a nucleotide sequence encoding one or more regulatory .elements.

7. The recombinant vector of claim 6, further comprising a nucleotide sequence encoding a selectable marker.

8. The recombinant vector of claim 7, which is plasmid pSE186 (ATCC 209604).

9. A recombinant vector comprising a polynucleotide molecule comprising a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID NO:2, substantially as hereinbefore described with reference to any one of the Examples.

10. A host cell comprising the recombinant vector of any one of claims 4 to 9.

11. The host cell of claim 10, which is Streptomyces avermitilis.

12. An isolated S. avermitilis AveC gene product comprising the amino acid sequence encoded by the S. avermitilis AveC gene product-encoding nucleotide sequence i/ T of plasmid pSE186 (ATCC 209604) or the amino acid sequence of SEQ ID NO:2. [I:\DayLib\LIBA]41429spec.doc:gcc 58

13. An isolated S. avermitilis AveC gene product comprising the amino acid sequence encoded by the S. avermitilis AveC gene product-encoding nucleotide sequence of plasmid pSE186 (ATCC 209604) or the amino acid sequence of SEQ ID NO:2, substantially as hereinbefore described with reference to any one of Examples 7-9.

14. A method for producing a recombinant AveC gene product, comprising culturing a host cell transformed with a recombinant expression vector, said recombinant expression vector comprising a polynucleotide molecule comprising a nucleotide sequence encoding an amino acid sequence comprising the amino acid sequence of SEQ ID NO:2, which polynucleotide molecule is in operative association with one or more 0o regulatory elements that control expression of the polynucleotide molecule in the host cell, under conditions conducive to the production of the recombinant AveC gene product, and recovering the AveC gene product from the cell culture. o*

15. A method for producing a recombinant AveC gene product, comprising 0*5 culturing a host cell transformed with a recombinant expression vector, said recombinant expression vector comprising a polynucleotide molecule comprising a nucleotide sequence encoding an amino acid sequence comprising the amino acid sequence of SEQ ID NO:2, which polynucleotide molecule is in operative association with one or more regulatory elements that control expression of the polynucleotide molecule in the host cell, under conditions conducive to the production of the recombinant AveC gene product, and recovering the AveC gene product from the cell culture, substantially as hereinbefore described with reference to any one of Examples 7-9.

16. A recombinant AveC gene product produced according to the method of claim 14 or claim

17. A polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604) or the nucleotide sequence of the aveC ORF of S. avermitilis as presented in FIGURE 1 (SEQ ID NO:1) or a degenerate variant thereof, but which nucleotide sequence further comprises one or more mutations, such that cells of S. avermitilis strain ATCC 53692 in which the wild-type aveC allele has been inactivated and that express the polynucleotide molecule comprising the mutated nucleotide sequence produce a reduced cyclohexyl B2:cyclohexyl B1 ratio of avermectins when fermented in the presence of cyclohexanecarboxylic acid than is produced by cells of S. avermitilis strain ATCC 53692 that instead express only the wild-type aveC allele.

18. The polynucleotide molecule of claim 17, wherein the reduced ratio of yclohexyl B2:cyclohexyl B1 is less than 1.6:1. \4 _o [I:\DayLib\LIBA]41 429spc.doc:gcc 59

19. The polynucleotide molecule of claim 18, wherein the reduced ratio of cyclohexyl B2:cyclohexyl B1 is about 0.94:1. The polynucleotide molecule of claim 19, wherein the mutation to the S. avermitilis aveC ORF of FIGURE 1 (SEQ ID NO:1) encodes an amino acid substitution at residue 139 of the AveC gene product from alanine to threonine.

21. The polynucleotide molecule of claim 18, wherein the reduced ratio of cyclohexyl B2:cyclohexyl B1 is about 0.88:1.

22. The polynucleotide molecule of claim 21, wherein the mutation to the S. avermitilis aveC ORF of FIGURE 1 (SEQ ID NO:1) encodes an amino acid 0o substitution at residue 138 of the AveC gene product from serine to threonine.

23. The polynucleotide molecule of claim 18, wherein the reduced ratio of cyclohexyl B2:cyclohexyl B1 is about 0.84:1.

24. The polynucleotide molecule of claim 23, wherein the mutation to the S. avermitilis aveC ORF of FIGURE 1 (SEQ ID NO:1) encodes a first amino acid 15 substitution at residue 138 of the AveC gene product from serine to threonine, and a second amino acid substitution at residue 139 of the AveC gene product from alanine to threonine.

25. A polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 S 20 (ATCC 209604) or the nucleotide sequence of the aveC ORF of S. avermitilis as presented in FIGURE 1 (SEQ ID NO:1) or a degenerate variant thereof, but which nucleotide sequence further comprises one or more mutations, such that cells of S. avermitilis strain ATCC 53692 in which the wild-type aveC allele has been inactivated and that express the polynucleotide molecule comprising the mutated nucleotide sequence produce a reduced cyclohexyl B2:cyclohexyl Bl ratio of avermectins when fermented in the presence of cyclohexanecarboxylic acid than is produced by cells of S. avermitilis strain ATCC 53692 that instead express only the wild-type aveC allele, substantially as hereinbefore described with reference to "Example 8: Construction of S. Avermitilis AveC Mutants" and "Example 9: Construction of 5' Deletion Mutants".

26. A polynucleotide molecule comprising a strong promoter in operative association with the S. avermitilis aveC ORF of FIGURE 1 (SEQ ID NO:1).

27. The polynucleotide molecule of claim 26, wherein the strong promoter is the ermE promoter from Saccharopolyspora erythraea. [I:\DayLib\LIBA]41429spec.doc:gcc

28. A polynucleotide molecule comprising a strong promoter in operative association with the S. avermitilis aveC ORF of FIGURE 1 (SEQ ID NO:1), substantially as hereinbefore described with reference to Example 11.

29. A polynucleotide molecule comprising a nucleotide sequence encoding the AveC gene product-encoding ORF of FIGURE 1 (SEQ ID NQ:1) that has been inactivated by insertion into said nucleotide sequence of a heterologous nucleotide sequence. A polynucleotide molecule comprising a nucleotide sequence encoding the AveC gene product-encoding ORF of FIGURE 1 (SEQ ID NQ:1) that has been inactivated by insertion into said nucleotide sequence of a heterologous nucleotide sequence, substantially as hereinbefore described with reference to any one of Examples 7-9.

31. A polynucleotide molecule comprising an aveC allele that has been inactivated by deleting a 640 bp Pstl/Sph fragment from the aveC ORF of FIGURE 1 15 (SEQ ID NO:1).

32. A polynucleotide molecule comprising an aveC allele that has been inactivated by deleting a 640 bp PstVlSphl fragment from the aveC ORF of FIGURE 1 (SEQ ID NO:1), substantially as hereinbefore described with reference to Example 8.

33. A polynucleotide molecule comprising an aveC allele that has been inactivated by introducing a frameshift mutation into the aveC ORF of FIGURE 1 (SEQ ID NO:1).

34. The polynucleotide molecule of claim 33, in which the frameshift mutation was introduced by adding two G nucleotides after the C nucleotide at nt position 471 of the aveC ORF of FIGURE 1 (SEQ ID NO:1).

35. A polynucleotide molecule comprising an aveC allele that has been inactivated by introducing a frameshift mutation into the aveC ORF of FIGURE 1 (SEQ ID NO:1), substantially as hereinbefore described with reference to Example 8.

36. A polynucleotide molecule comprising an aveC allele that has been inactivated by introducing a termination codon at the nucleotide position that encodes amino acid 116 of the S. avermitilis AveC gene product encoded by the aveC ORF of FIGURE 1 (SEQ ID NO:1).

37. A polynucleotide molecule comprising an aveC allele that has been Sinactivated by introducing a termination codon at the nucleotide position that encodes Iamino acid 116 of the S. avermitilis AveC gene product encoded by the aveC ORF of [I:\DayLib\LIBA]41 429spec.doc:gcc 61 FIGURE 1 (SEQ ID NO:1), substantially as hereinbefore described with reference to Example 8.

38. A polynucleotide molecule comprising an aveC allele that has been inactivated by introducing a first mutation at amino acid position 256 of the S. avermitilis AveC gene product that changes a glycine to an aspartate, and a second mutation at position 275 of the S. avermitilis AveC gene product that changes a tyrosine to a histidine.

39. A polynucleotide molecule comprising an aveC allele that has been inactivated by introducing a first mutation at amino acid position 256 of the AveC gene product that changes a glycine to an aspartate, and a second mutation at position 275 of o0 the AveC gene product that changes a tyrosine to a histidine, substantially as hereinbefore described with reference to Example 8. A gene replacement vector comprising a polynucleotide molecule comprising nucleotide sequences that naturally flank the aveC ORF in situ in the S. avermitilis chromosome.

41. A gene replacement vector comprising a polynucleotide molecule comprising nucleotide sequences that naturally flank the aveC ORF in situ in the S. avermitilis chromosome, substantially as hereinbefore described with reference to any one of Examples 7-9.

42. A recombinant vector comprising the polynucleotide molecule of any one of 20 claims 17 to 39.

43. A recombinant vector comprising the polynucleotide molecule of claim 29 or claim 30, which vector is pSE180 (ATCC 209605).

44. A recombinant vector comprising a polynucleotide molecule comprising a nucleotide sequence encoding the AveC gene product-encoding ORF of FIGURE 1 (SEQ ID NQ:1) that has been inactivated by insertion into said nucleotide sequence of a heterologous nucleotide sequence, substantially as hereinbefore described with reference to any one of Examples 7-9.

45. A host Streptomyces cell comprising the recombinant vector of any one of claims 42 to 44.

46. A method for identifying mutations of the aveC ORF capable of reducing the class 2:1 ratio of avermectins produced, comprising: determining the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis in which the native aveC allele has been inactivated, and into which a polynucleotide molecule comprising a nucleotide Osequence encoding a mutated AveC gene product has been introduced and is being e pressed; determining the class 2:1 ratio of avermectins produced by cells of the [I:\DayLib\LIBA]41429spec.doc:gcc 62 same strain of S. avermitilis as in step but which instead express only an aveC allele having the nucleotide sequence of the ORF of FIGURE 1 (SEQ ID NO; 1) or a nucleotide sequence that is homologous thereto; and comparing the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step to the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step such that if the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step is lower than the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step then a mutation of the aveC ORF capable of reducing the class 2:1 ratio of avermectins has been identified.

47. A method for identifying mutations of the aveC ORF capable of reducing the to class 2:1 ratio of avermectins produced, comprising: determining the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis in which the native aveC allele has been inactivated, and into which a polynucleotide molecule comprising a nucleotide sequence encoding a mutated AveC gene product has been introduced and is being expressed; determining the class 2:1 ratio of avermectins produced by cells of the Is same strain of S. avermitilis as in step but which instead express only an aveC allele having the nucleotide sequence of the ORF of FIGURE 1 (SEQ ID NO;1) or a nucleotide sequence that is homologous thereto; and comparing the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step to the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step such that if the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step is lower than the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step then a mutation of the aveC ORF capable of reducing the class 2:1 ratio of avermectins has been identified, substantially as hereinbefore described with reference to Example 8.

48. A method for making a novel strain of S. avermitilis comprising cells that express a mutated aveC allele and that produce a reduced class 2:1 ratio of avermectins compared to cells of the same strain of S. avermitilis that instead express only a wild-type aveC allele, comprising: obtaining cells of strain of S. avermitilis; mutating the aveC allele in a cell of step or introducing into a cell of step a mutated aveC allele or degenerate variant thereof, which mutated aveC allele or degenerate variant thereof encodes a gene product that reduces the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allele compared to cells of the same strain that instead express only the wild-type aveC allele, and selecting cells from tep that produce avermectins in a reduced class 2:1 ratio compared to the class 2:1 S//t tao produced by cells of the strain that instead express the wild-type aveC allele. [I:\DayLib\LIBA]41 429spec.doc:gcc 63

49. A method for making a novel strain of S. avermitilis comprising cells that express a mutated aveC allele and that produce a reduced class 2:1 ratio of avermectins compared to cells of the same strain of S. avermitilis that instead express only a wild-type aveC allele, comprising: obtaining cells of strain of S. avermitilis; mutating the aveC allele in a cell of step or introducing into a cell of step a mutated aveC allele or degenerate variant thereof, which mutated aveC allele or degenerate variant thereof encodes a gene product that reduces the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allele compared to cells of the same strain that instead express only the wild-type aveC allele, and selecting cells from step that produce avermectins in a reduced class 2:1 ratio compared to the class 2:1 ratio produced by cells of the strain that instead express the wild-type aveC allele, S. substantially as hereinbefore described with reference to Example 8 or Example 9. .50. A method for making novel strains of S. avermitilis, the cells of which comprise an inactivated aveC allele, comprising introducing into cells of a strain of 15 S. avermitilis a vector that inactivates the aveC allele, and selecting those cells in which the aveC allele has been inactivated. 4

51. The method of claim 50, wherein the vector is pSE180 (ATCC 209605).

52. A method for making novel strains of S. avermitilis, the cells of which comprise an inactivated aveC allele, comprising introducing into cells of a strain of 9. 20 S. avermitilis a vector that inactivates the aveC allele, and selecting those cells in which the aveC allele has been inactivated, substantially as hereinbefore described with reference to Example 8.

53. A novel strain of S. avermitilis when produced according to the method of any one of claims 48 to 52.

54. A strain of S. avermitilis comprising cells expressing a mutated aveC allele which results in the production by the cells of avermectins in a reduced class 2:1 ratio compared to cells of the same strain that instead express only the wild-type aveC allele. The strain of claim 54, wherein the cells produce cyclohexyl B2:cyclohexyl Bl avermectins in a ratio of less than 1.6:1.

56. The strain of claim 54, wherein the cells produce cyclohexyl B2:cyclohexyl B avermectins in a ratio of about 0.94:1.

57. The strain of claim 54, wherein the cells produce cyclohexyl B2:cyclohexyl Bl avermectins in a ratio of about 0.88:1. ^i 58. The strain of claim 54, wherein the cells produce cyclohexyl B2:cyclohexyl s3 B avermectins in a ratio of about 0.84:1. [1:\DayLib\LIBA]41429spec.doc:gcc 64

59. A strain of S. avermitilis comprising cells expressing a mutated aveC allele which results in the production by the cells of avermectins in a reduced class 2:1 ratio compared to cells of the same strain that instead express only the wild-type aveC allele, substantially as hereinbefore described with reference to any one of Examples 7-9.

60. A strain of S. avermitilis comprising cells in which the aveC gene has been inactivated.

61. A strain of S. avermitilis comprising cells in which the aveC gene has been inactivated, substantially as hereinbefore described with reference to Example 8.

62. A process for producing avermectins, comprising culturing cells of a strain of S. avermitilis, which cells express a mutated aveC allele that encodes a gene product that reduces the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allele compared to cells of the same 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 of avermectins S 15 therefrom, and recovering said avermectins from the culture.

63. A process for producing avermectins, comprising culturing cells of a strain of S. avermitilis, which cells express a mutated aveC allele that encodes a gene product that reduces the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allele compared to cells of the same strain which do not 20 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 of avermectins therefrom, and recovering said avermectins from the culture, substantially as hereinbefore described with reference to any one of Examples 7-9.

64. Avermectins produced by the process of claim 62 or claim 63.

65. A composition of cyclohexyl B2:cyclohexyl B1 avermectins present in exhausted fermentation medium in a ratio of less than 1.6:1, produced by cells of a strain of S. avermitilis that express a mutated aveC allele that encodes a gene product that reduces the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allele compared to cells of the same strain which do not express the mutated aveC allele but instead express only the wild-type aveC allele.

66. The composition of claim 65, wherein the ratio of cyclohexyl B2:cyclohexyl Bl avermectins is about 0.94:1.

67. The composition of claim 65, wherein the ratio of cyclohexyl B2:cyclohexyl 1 avermectins is about 0.88:1. (I:\DayLib\LIBA]41 429spec.doc:gcc

68. The composition of claim 65, wherein the ratio of cyclohexyl B2:cyclohexyl Bl avermectins is about 0.84:1.

69. A composition of cyclohexyl B2:cyclohexyl B1 avermectins present in exhausted fermentation medium in a ratio of less than 1.6:1, produced by cells of a strain of S. avermitilis that express a mutated aveC allele that encodes a gene product that reduces the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allele compared to cells of the same strain which do not express the mutated aveC allele but instead express only the wild-type aveC allele, substantially as hereinbefore described with reference to any one of Examples 7 to 9. Dated 7 January, 2002 Pfizer Products Inc. Patent Attorneys for the Applicant/Nominated Person sSPRUSON FERGUSON S• 0 *F [I:\DayLib\LIBA]41 4 29spec.doc:gcc