BG64911B1 - Streptomyces avermitilis gene directing the ratio of b2:b1 avermectins - Google Patents

Abstract

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

Description

The present invention relates to compositions and methods for the production of avermectins, and in particular to the field of animal health. In particular, the present invention relates to polynucleotide molecules comprising nucleotide sequences encoding an aveC gene product that can be used to modulate the ratio of class 2: 1 avermectins produced by the fermentation process of cultures of Streptomyces avermitilis, and to compositions and methods for screening such polynucleotide molecules. The present invention also relates to vectors, transformed host cells, and novel S. avermitilis mutant strains in which the aveC gene has mutated to modulate the ratio of the produced 2: 1 avermectins.

BACKGROUND OF THE INVENTION

Avermectins

Streptomyces species produce a wide variety of secondary metabolites, including avermectins, which includes a series of eight coupled 16-membered macrocyclic lactones possessing strong anthelmintic and insecticidal activity. The eight separate but closely related compounds are denoted as Ala, Alb, A2a, A2b, Bia, Bib, B2aB2b. The compounds of series "a" refer to natural avermectin, where the substituent at the C25 position is (S) -sec-6ynui, and the "b" series refers to those compounds where the substituent at the C25 position is isopropyl. The terms "A" and "B" refer to avermectins in which the substituent at the C25 position is methoxy and hydroxy, respectively. The number "1" corresponds to avermectins having a double bond at position C22,23, and the number "2" refers to avermectins having hydrogen at position C22 and a hydroxyl group at position C23. Among the associated avermectins, the B1 type avermectin is widely known to have the most effective antiparasitic and pesticidal action, and is therefore the most commercially available avermectin.

Avermectins and their production by aerobic fermentation of S. avermitilis strains are described in US 4,310,519 and US 4,429,042. It is claimed that the biosynthesis of natural avermectins is initiated endogenously by the CoA thioester analogues of isobutyric acid and S - (+) - 2-methyl butyric acid.

A combination of the two, improvement of the strain by random mutagenesis and use of exogenously delivered fatty acids led to efficient production of avermectin analogues. S. avermitilis mutants lacking branched-chain 2-oxo acid dehydrogenase (mutants with bkd deficiency) can only produce avermectins when fatty acids are added to the fermentation process. The screening and isolation of branched-chain 2-oxo acid dehydrogenase deficient mutants (e.g., S. avermitilis ATCC 53567) are described in EP 276103. Fermentation of such mutants in the presence of exogenously delivered fatty acids results in the production of only four avermectin corresponding to the fatty acids used. Therefore, feeding the fermentation of S. avermitilis (ATCC 53567) with S (+) - 2-methylbutyric acid results in the production of the natural avermectins Ala, A2a, B1a and B2a; the addition of isobutyric acid to the fermentation results in the production of natural avermectins Alb, A2b, B1a and B2b; and the addition of cyclopentanecarboxylic acid to the fermentations results in the production of four new cyclopentyl avermectins A1, A2, B1 and B2.

New avermectins are produced when other fatty acids are added. By screening over 80 potential precursors, over 60 other new avermectins have been identified (see, for example, Dutton et al., 1991, J. Antibiot. 44: 357-36 /; and Banks et al., 1994, Roy. Soc. Chem. 147 : 16-26). In addition, S. avermitilis mutants with deficient 5-O-methyltransferase activity mainly produce only the avermectins analogue. Therefore, S. avermitilis mutants, which lack both, 2-oxo acid dehydrogenase activity and 5-O-methyltransferase activity, only produce B avermectins corresponding to the fatty acid used to supplement the fermentation. Thus, feeding these double mutants with S - (+) 2-methyl butyric acid results in the production of only natural avermectins B1 a and B2a, while the addition of isobutyric acid or cyclopentanecarboxylic acid leads to the production of natural avermectins B1b and B2b or of the new cyclopentyl B1 and B2 avermectins, respectively. The addition to the double mutant strain of cyclohexane carboxylic acid is the preferred method of producing the commercially important new avermectin, cyclohexylvermectin Bl (doramectin). The isolation and characterization of such double mutants, for example S. avermitilis (ATCC 53692), is described in EP 276103.

Genes involved in avermectin biosynthesis

In many cases, genes involved in the production of secondary metabolites and genes encoding a particular antibiotic have been found clustered together on the chromosome. This is the case for, for example, the polyketide synthetase gene (PKS) of Streptomyces (see Hopwood and Sherman, 1990, Ann. Rev. Genet. 24: 37-66). Thus, a strategy for cloning genes by the biosynthetic pathway was to isolate a drug resistance gene and then test the adjacent chromosome regions for other genes related to the biosynthesis of this particular antibiotic. Another strategy for cloning genes involved in biosynthesis of important metabolites has been complementation of mutants. For example, portions of a DNA library of an organism capable of producing a particular metabolite are introduced into a non-producing mutant and transformants are screened for metabolite production. According to this, library hybridization using samples derived from other Streptomyces species has been used to identify and clone genes in biosynthetic pathways.

The genes involved in the biosynthesis of avermectin (ave genes), similar to the genes required for the biosynthesis of other secondary metabolites of Streptomyces (eg, PKS) were found packed on the chromosome. A large number of genes have been successfully cloned using vectors to complement S. avermitilis mutants blocked in avermectin biosynthesis. The cloning of such genes is described in US 5,252,474. In addition, Ikeda et al., 1995, J. Antibiot. 48: 582-534, describes the localization of a chromosomal region including C22,23 dehydration steps (aveC) to a 4.82 kb BamHI fragment of S. avermitilis, as well as mutations in the aveC gene that lead to the production of a single ingredient of the B2a producer. Because ivermectin, a strong anthelmintic compound, can be chemically produced by avermectin B2a, such a one-component producer of avermectin B2a is considered extremely useful for the commercial production of ivermectin.

Identifying mutations in the aveC gene that simplify the complexity of avermectin production, such as mutations that reduce the B2: B1 ratio of avermectins, would facilitate the production and purification of commercially available avermectins.

SUMMARY OF THE INVENTION

The present invention provides an isolated polynucleotide molecule comprising the complete aveC ORF of S. avermitilis or a substantial portion thereof, the isolated polynucleotide molecule lacking the next complete ORF that is downstream of the aveC ORF in situ in the chromosome of S. avermitilis. The isolated polynucleotide molecule of the present invention preferably includes a nucleotide sequence that is the same as aveC or is the same as the nucleotide sequence of the aveC ORF of Figure 1 (SEQ ID NO: 1) or a substantial portion thereof.

The present invention also provides a polynucleotide molecule having a nucleotide sequence that is homologous to the product-coding sequence of the S. avermitilis aveC gene of plasmid pSE 186 (ATCC 209604), or to the aveC ORF nucleotide sequence shown in Figure 1 (SEQ (ID) NO: 1) or a substantial part thereof.

The present invention also 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 of the product-coding sequence of the aveC gene of S. avermitilis of plasmid CCSE 186 or plasmid BCSE 186 the sequence of Figure 1 (SEQ ID NO: 2) or a substantial portion thereof.

The present invention also provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding an aveC homolog gene product. In a preferred embodiment of the present invention, the isolated polynucleotide molecule comprises a nucleotide sequence encoding a gene product of an aveC homolog of S. hygroscopicus, which homologous gene product includes the amino acid sequence of (SEQ ID NO: 4) or a substantial portion thereof. In a preferred embodiment, the isolated polynucleotide molecule of the present invention that encodes the product of the homologous aveC gene of S. hygroscopicus includes the nucleotide sequence of SEQ ID NO: 3 or a substantial portion thereof.

The present invention also provides a polynucleotide molecule comprising a nucleotide sequence that is homologous to the nucleotide sequence of S. hygroscopicus SEQ ID NO: 3. The present invention also provides a polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the gene product of the aveC gene of the S. hygroscopicus homologue having the amino acid sequence of SEQ ID NO: 4.

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 that is complementary to a nucleotide sequence of Figure 1 (SEQ ID NO: 1) or SEQ ID NO: 3.

The present invention also provides recombinant cloning vectors and expression vectors for use in the cloning or expression of the polynucleotide of the present invention, including polynucleotide molecules comprising the aveC ORF of S. avermitilis or the aveC homolog ORF. In a non-limiting embodiment, the present invention provides plasmid pSE186 (ATCC 209604) that includes the complete ORF of the aveC gene of S. avermitilis. The present invention also provides transformed host cells comprising a polynucleotide molecule or recombinant vector of the invention, new strains or cell lines derived therefrom.

The present invention also provides a recombinantly expressed aveC gene product or gene product of an aveC homolog, or a substantial portion thereof, which is substantially purified or isolated, and homologues thereof. The present invention also provides a method of 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 having a nucleotide sequence coding for a nucleotide sequence with one or more regulatory elements that control the expression of the polynucleotide molecule in the host cell at words resulting in the production of the recombinant aveC gene product or aveC homolog gene product and the cell culture of an aveC gene product or aveC homolog gene product.

The present invention also provides a polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as the coding sequence of the aveC gene product of S. avermitilis on plasmid pSE (ATCC 209604) or as the nucleotide sequence of aveC ORF of S. avermitilis, as represented by S. avermitilis Figure 1 (SEQ ID NO: 1), but which further comprises one or more mutations such that S. avermitilis cells strain ATCC 53692 in which the wild-type aveC allele is inactivated and which expresses a polynucleotide molecule comprising the mutated nucleotide after it produces a different ratio or amount of avermectins than that produced by S. avermitilis cells, strain ATCC 53692, which instead expresses only the wild-type aveC allele. According to the present invention, such polynucleotide molecules can be used to produce novel S. avermitilis strains that exhibit a significant change in the production of avermectin, compared to the same strain that instead expresses only the wild-type aveC allele. In a preferred embodiment, such polynucleotide molecules are used to produce strains of S. avermitilis that produce avermectins at a reduced ratio of class 2: 1 avermectins, compared to those of the same strain, which instead express only the aveC allele of wild type. In another preferred embodiment, such polynucleotide molecules are used to produce new strains of S. avermitilis that produce increased levels of avermectins, compared to the same strain that instead expresses only wild-type aveC appeals. In another preferred embodiment, such polynucleotide molecules are used to produce new strains of S. avermitilis in which the aveC gene has been inactivated.

The present invention provides methods for identifying aveC ORF mutations of S. avermitilis capable of altering the ratio and / or amount of avermectin produced. In a preferred embodiment, the present invention provides a method for identifying aveC ORF mutations capable of altering the class 2: 1 ratio of produced avermectins, including: (a) determining the class 2: 1 ratio of avermectins produced by cells from a strain of S. avermitilis in which the native aveC allele is inactivated and in which a polynucleotide molecule comprising a nucleotide sequence encoding a mutated aveC gene product is introduced and expressed; (B) determining the class 2: 1 ratio of produced avermectins from cells of the same S. avermitilis strain as in step (a), but which instead express only the aveC allele having the ORF nucleotide sequence of Figure 1 (SEQ ID NO: 1) or a nucleotide sequence homologous thereto; and (c) comparing the class 2: 1 ratio of avermectins produced by S. avermitilis cells of step (a) with the class 2: 1 ratio of avermectins produced by S. avermitilis cells of step (b); so if the class 2: 1 ratio of avermectins produced by S. avermitilis cells from step (a) is different from the class 2: 1 ratio of avermectins produced by S. avermitilis cells from step (b), then a mutation of aveC ORF capable of altering the class 2: 1 ratio of avermectins. In a preferred embodiment of the invention, the class 2: 1 ratio of avermectins is reduced by the mutation.

In another preferred embodiment, the present invention provides a method for identifying mutations of aveC ORFs or genetic structures including aveC ORFs capable of altering the amount of avermectins produced, which includes: (a) determining the amount of avermectins produced by cells from a strain of S. avermitilis, in which the native aveC allele is inactivated and in which a polynucleotide molecule comprising a nucleotide sequence encoding a mutated aveC gene product or incorporating genetic structures is introduced and expressed, contains a nucleotide sequence comprising an aveC gene product; (B) determining the amount of avermectins produced from cells of the same S. avermitilis strain as in step (a) but which instead express only the aveC allele having the nucleotide sequence of the ORF of Figure 1 (SEQ ID NO: 1) or nucleotide a sequence that is homologous to it; and (c) comparing the amount of avermectins produced by S. avermitilis cells from step (a) with the amount of avermectins produced by S. avermitilis cells from step (b); so that the amount of avermectins produced by S. avermitilis cells from step (a) is different from the amount of avermectins produced by S. avermitilis cells from step (b), then a mutation of aveC ORF or genetic structure capable of altering the amount of avermectins. In a preferred embodiment of the invention, the amount of avermectins produced is increased by mutation.

The present invention further provides recombinant vectors that are used to produce new S. avermitilis strains having altered avermectin production. For example, the present invention provides vectors that can be used to target any of the polynucleotide molecules, including the mutated nucleotide sequences of the present invention, to the aveC gene site of the S. avermitilis chromosome or to insert or replace aveC ORF or homologous recombination portion thereof. According to the present invention, however, a polynucleotide molecule comprising the mutated nucleotide sequence of the present invention provided herein may also function to modulate avermectin biosynthesis when inserted into the S. avermitilis chromosome at a site other than that of 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 into a site of the S. avermitilis chromosome other than the aveC gene site, or to keep it episomal. 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 produce a new strain of cells that produce class 2 reduced avermectins. : 1, compared to cells from the same strain that instead express only the wild-type aveC allele.

The present invention also provides methods for producing new strains of S. avermitilis, including cells that express the mutated aveC allele, and which produce altered ratios and / or amounts of avermectins, compared to cells from the same S. avermitilis strain, which instead only express the wild-type aveC allele. In a preferred embodiment, the present invention provides a method for producing new strains of S. avermitilis, including cells that express a mutated aveC allele, and which produce a modified class 2: 1 ratio of avermectins compared to the same strain of S. avermitilis , which instead expresses only a wild-type aveC allele involving transformation of S. avermitilis strain cells with a vector-carrying mutated aveC allele that encodes a gene product that changes the class 2: 1 ratio of avermectins produced by S. avermitilis, co it expresses the mutated allele, compared to cells of the same strain that instead express only the wild-type aveC allele, and selection of transformed cells that produce avermectins at an altered class 2: 1 ratio of avermectins compared to the class 2 ratio : 1 produced by cells from a strain that instead expresses aveC wild-type allele. In a preferred embodiment of the invention, the ratio of class 2: 1 produced avermectins is reduced in cells of the new strain.

In another preferred embodiment, the present invention provides a method for producing new strains of S. avermitilis, including cells that produce altered amounts of avermectin, including transforming cells from a strain of S. avermitilis with a vector that carries a mutated aveC allele or genetic structure , comprising the aveC allele, the expression of which results in an altered amount of avermectins produced by cells from a strain of S. avermitilis that express the mutated aveC allele or genetic structure, compared to cells from the same strain that instead of ca express only an aveC allele from the wild type, 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 an aveC allele from the wild type. In a preferred embodiment of the invention, the amount of avermectins produced is increased in cells from the new strain.

In another preferred embodiment, the present invention provides a method for producing new strains of S. avermitilis, the cells of which include an inactivated aveC allele involving the transformation of cells of a strain of S. avermitilis that express the wild-type aveC allele with a vector , which inactivates the aveC allele, and selects the transformed cells in which the aveC allele has been inactivated.

The present invention also provides novel strains of S. avermitilis comprising cells that have been transformed with any of the polynucleotide molecules or vectors comprising the mutated nucleotide sequence of the present invention. In a preferred embodiment, the present invention provides strains of S. avermitilis comprising cells that express a mutated aveC allele instead of, or in addition to, a wild-type aveC allele, with the cells of the new strain producing avermectins at a modified class 2 ratio : 1 compared to cells from the same strain that instead express only the wild-type aveC allele. In a more preferred embodiment of the invention, cells from the new strain produce avermectin in a reduced class 2: 1 ratio compared to cells from the same strain, which instead express only the wild-type aveC allele. Such new strains are useful in the large-scale production of commercially available avermectins and doramectin.

In another preferred embodiment, the present invention provides novel strains of S. avermitilis, including cells that express a mutated aveC allele or genetic structure including the aveC allele, instead of or in addition to a wild-type aveC allele that results in the production of cells with altered avermectins, compared to the amount of avermectins produced by cells of the same strain, which instead express only the wild-type aveC allele. In a preferred embodiment, the new cells produce an increased amount of avermectins.

In another preferred embodiment, the present invention provides novel strains of S. avermitilis, including cells in which the aveC gene has been inactivated. Such strains are useful for both the different avermectin spectra they produce, compared with the wild-type strain, and for the complementary screening assay described herein to determine whether targeted or incidental mutagenesis of the aveC gene affects the production of the aveC gene. avermectin.

The present invention further provides a method of producing avermectins comprising culturing cells from 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 S. avermitilis expressing the mutated aveC allele compared to cells of the same strain that do not express the mutated aveC allele but instead express only the wild-type aveC allele in a culture medium under conditions that allow or induce the production of avermectins by not oh, and getting avermectins from the culture. In a preferred embodiment of the invention, the ratio of class 2: 1 avermectins produced by cells expressing the mutation is reduced. The method provides increased efficacy in the production of commercially available avermectins, such as doramectin.

The present invention further provides a method for producing avermectins, which involves culturing cells from a strain of S. avermitilis, which cells express a mutated aveC allele or a genetic structure including the aveC allele, which results in the production of a changed amount of avermectins produced by cells of S. avermitilis strain expressing the mutated aveC allele or genetic structure, compared with cells of the same strain that do not express the mutated aveC allele or genetic structure, but instead express only the wild-type aveC allele in culture. under conditions that permit or induce the production of avermectins from them and the production of said avermectins from the culture. In a preferred embodiment, the amount of avermectins produced by cells expressing the mutation or genetic structure is increased.

The present invention further provides a new composition of avermectins, reduced by a strain of S. avermitilis expressing the mutated aveC allele of the present invention, wherein the avermectins are produced at a reduced ratio of class 2: 1, in comparison with the ratio of class 2: 1 of avermectins produced by cells of the same strain of S. avermitilis, which does not express the mutated aveC allele but instead expresses only the wild-type aveC allele. The new composition of avermectins may be present in the form produced in the fermentation culture medium or may be derived from it and may be partially or completely purified from the medium.

Short description of the figures

Figure 1. DNA sequence (SEQ ID NO: 1) comprising the aveC ORF of S. avermitilis and the deduced amino acid sequence (SEQ ID NO: 2).

Figure 2. Plasmid vector pSEl 86 (ATCC 209604) comprising the complete ORF of the aveC gene of S. avermitilis.

Figure 3. Vector pSE180 (ATCC 209605) for replacement of a gene comprising the Sacc eppE gene. erythraea in the aveC ORF of S. avermitilis.

Figure 4. BamHI restriction map of the avermectin polyketide synthetase gene cluster of S. avermitilis with five identified overlapping cosmid clones (i.e., pSE65, pSE66, pSE67, pSE68, pSE69. The relationship HapSE118HpSE119 is also indicated.

Figure 5. HPLC analysis of fermentation products produced from S. avermitilis strains. Peak amounts are obtained by comparing standard amounts of cyclohexyl B1. The retention time of cyclohexyl B2 is 7.4-7.7 min; the retention time of cyclohexyl B1 is 11.9 - 12.3 min.

Figure 5A. S. avermitilis strain SE180-11 with aveC ORF inactivated.

Figure 5B. S. avermitilis strain SE180-11 transformed with pSE186 (ATCC 209604).

Figure 5C. S. avermitilis strain pSEl80-11 transformed with pSE187.

Figure 5D. S. avermitilis strain SE180-11 transformed with pSEI88.

Figure 6. Comparison of derived amino acid sequences encoding the aveC ORF of S. avermitilis (SEQ ID NO: 2), the aveC homolog partial partial ORF of S. hygroscopicus (SEQ ID NO: 4). The bold valine residue is the putative protein start site. Reserved residues are shown in uppercase letters for homology in all three sequences, and in the lower margins are letters for homology in two of the three sequences. The amino acid sequences have approximately 50% sequence identity.

Figure 7. Hybrid plasmid structure containing 564 bp BsaAI / Kpnl site in the aveC ORF of S. avermitilis.

Detailed description of the invention

The present invention relates to the identification and characterization of polynucleotide molecules having nucleotide sequences encoding the AveC gene product of Streptomyces avermitilis, constructing new strains of S. avermitilis that can be used to screen for mutated AveC gene products avermectins, and to the discovery that some mutated AveC gene products may reduce the B2: B1 ratio of avermectins produced by S. avermitilis. As an example, the invention is described in the sections below for a polynucleotide molecule having a nucleotide sequence that is the same as the AveC product-encoding sequence of plasmid pSEl 86 (ATCC 209604} of S. avermitilis, or the nucleotide sequence of ORF of Figure 1 (SEQ ID NO: 1) and polynucleotide molecules having mutated nucleotide sequences derived therefrom, However, the principles of the present invention can be similarly applied to other polynucleotide molecules, including the aveC homologous genes of Streptomyces species, including, for example, S. hygroscopicus and S. griseochromogenes, among others.

Polynucleotide molecules encoding the AveC gene product of S. avermitilis

The present invention provides isolated polynucleotide molecules comprising the complete aveC ORF of S. avermitilis or a substantial portion thereof, the isolated polynucleotide molecule missing the next complete ORF, which is located downstream of the aveC ORF in situ in the S. avermitilis chromosome.

The isolated polynucleotide molecule of the present invention preferably includes a nucleotide sequence that is the same as the AveC gene product coding sequence of S. avermitilis of plasmid pSE 186 (ATCC 209604) or which is the same as the nucleotide sequence of ORF of Figure 1 (SEQ ID NO : 1) or a substantial part thereof. As used herein, "substantial hour" of an isolated polynucleotide molecule comprising a nucleotide sequence encoding the AveC gene product of S. avermitilis means an isolated polynucleotide molecule comprising at least approximately 70% of the complete ORF aveC sequence shown in Figure 1 ( SEQ ID NO: 1), which encodes a functionally equivalent AveC gene product. In this ratio, 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, produces the same substantially ratio and amount of avermectins, such 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 sequences of the aveC ORF, the isolated nucleotide molecule of the present invention may also include nucleotide sequences that naturally limit the aveC gene in situ in S. avermitilis, such borderline nucleotide sequences shown in Figure 1 (SEQ ID NO: 1) .

As used herein, "polynucleotide molecule", "polynucleotide sequence", "coding sequence", "open reading frame", and "ORF" refer to both DNA and RNA molecules, which may be either single stranded or double stranded , and which can be transcribed and translated (DNA) or translated (RNA) into an AveC gene product or, as described below, into an AveC gene product homologue, or into a polypeptide homologous to an AveC gene product or a homologous AveC gene product in a suitable host cell when o put under the control of appropriate regulatory elements. The coding sequence may 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) includes four different GTG codons at the bp positions 42, 174, 177 and 180. As described in section 9 below, numerous deletions are constructed in the 5 'region of the aveC ORF Figure 1 (SEQ ID NO: 1) to aid in determining which of these codons could act in the aveC ORF as starting sites for protein expression. The divisions in the first GTG site at Lp 42 did not remove the aveC activity. Further division of all GTG codons together at the bp positions 174,177 and 180 eliminates AveC activity, indicating that this region is required for protein expression. The present invention thus encompasses different lengths of the aveC ORF that initially initiate translation of any of the GTG sites localized at bp 174,177 and 180 bp, as represented in Figure 1 (SEQ ID NO: 1) and the corresponding polypeptides for each.

The present invention further provides a polynucleotide molecule having a nucleotide sequence homologous to the AveC gene product-coding sequence of plasmid pSE186 (ATCC 209604) or to the aveC ORF nucleotide sequence represented in Figure 1 (SEQ ID NO: 1) or SEQ ID NO thereof part. When the term "homologous" is used to refer to a polynucleotide molecule that is homologous to the AveC gene product encoding sequence of S. avermitilis, then it means a polynucleotide molecule having a nucleotide sequence: (a) which encodes the same AveC gene product as AveC the gene product coding sequence of S. avermitilis HapSE186 (ATCC 209604), or which encodes the same AveC gene product as the nucleotide sequence of the AveC ORF presented in Figure 1 (SEQ ID NO: 1) but which shows one or more cilient changes in nucleotide th sequence according to degeneracy of the genetic code; or (b) which hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence encoded by the AveC gene product-coding sequence of plasmid pSE186 (ATCC 209604), or which encodes the amino acid sequence N1 sequence of the N-amino acid sequence : 2), under medium-stringent conditions, i.e., hybridization to coupled-to-filter DNA in 0.5 M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 ° C, and washing in 0.2 x SSC / 0.1% SDS at 42 ° C (see Ausubel et al., (Eds.), 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing As sociates, 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 of the invention, the homologous polynucleotide molecule hybridizes to the complement of the AveC gene product encoding the nucleotide sequence of plasmid pSE186 (ATCC 209604), or the complement of the nucleotide sequence shown in Figure 1 (SEC ID NO: 1) or substantial under very stringent conditions, i.e., hybridization to DNA-coupled DNA in 0.5 M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 ° C, and washing at 0, 1 x SSC / 0.1% SDS at 68 ° C (see Ausubel et al., 1989, supra) and encodes a functionally equivalent AveC g accordingly product as defined above.

The activity of the AveC product and its potential functional equivalents can be determined by HPLC analysis of the fermentation products, as described in the examples below. Polynucleotide molecules having nucleotide sequences that encode functional equivalents of the AveC gene product of S. avermitilis include naturally occurring aveC genes present in other strains of S. avermitilis, aveC homologous genes present in other strains and Streptomites, and alleles, both naturally occurring in nature and genetically engineered.

The present invention further provides a polynucleotide molecule having a nucleotide sequence that encodes a polypeptide having an amino acid sequence homologous to the amino acid sequence encoded by the AveC gene product encoding the nucleotide sequence of plasmid pSE186 or the plasmidTSE186 sequence ID NO: 2} or a substantial portion thereof As used herein, the “substantial portion” of the amino acid sequence of Figure 1 (SEQ ID NO: 2) means a polype a bird comprising at least 70% of the amino acid sequence of Figure 1 (SEQ ID NO: 2), and which constitutes a functionally equivalent AveC gene product as defined above.

As used herein, to refer to amino acid sequences homologous to the amino acid sequence of the AveC gene product of S. avermitilis, the term "homologous" refers to a polypeptide encoded by the AveC gene encoding the nucleotide sequence of plasmid pSEl 86 (pSE1604) (pSEl 86) or: having the amino acid sequence of Figure 1 (SEQ ID NO: 2), but in which one or more amino acid residues have been conservatively substituted by different amino acid residues when such conservative substitutions result in an uniquely equivalent gene product as defined above. Conservative amino acid substitutions are well known in the art. Rules for making such substitutions include those described by Dayhof, M.D., 1978, Nat. Biomed. Really. Found., Washington, D.C., Vol. 5, Sup. 3, among others. In particular, conservative amino acid substitutions are those that usually consist within a family of amino acids that are linked by the acidity, polarity, or volume of their side chains. Genetically encoded amino acids are usually divided into four groups: (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) polar uncharged - glycine, asparagine, glutamag, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are also classified as aromatic amino acids. One or more substitutions within a particular group, for example of leucine with isoleucine or valine, or of aspartate with glutamate, or of threonine with serine, or of any other amino acid residue with a structurally related amino acid residue, for example an amino acid residue with a similar activity, polarity, bulk, or similarity to some combinations thereof, would have a negligible effect on the function of the polypeptide.

The present invention also provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a homologous AveC gene product. As used herein, a "homologous AveC gene product" is defined as a gene product having at least 50% amino acid sequence identity with an AveC gene product of S. avermitilis comprising the amino acid sequence encoded by the AveC gene product encoding the nucleotide sequence of plasmid pSEl86 (ATCC 209604), or from the amino acid sequence shown in Figure 1 (SEQ ID NO: 2). In a non-limiting embodiment, the homologous AveC gene product is from S. hygroscopicus (described in EP application 0298423; deposit FFRM BP-1901) and includes the amino acid sequence of SEQ ID NO: 4, or a substantial portion thereof. An "essential honor" of the amino acid sequence of SEQ ID NO: 4 means a polypeptide comprising at least 70% of the amino acid sequence of SEQ ID NO: 4 and which constitutes a functionally equivalent homolog of the AveC gene product. The "functionally equivalent" homolog of an AveC gene product is defined as a gene product that, when expressed in S. hygroscopicus strain FFRM BP-1901, in which the homolog of the native AveC allele is inactivated, results in the production of essentially the same ratio and an amount of milbemycin as produced by S. hygroscopicus strain FERN BP-1901, which instead expresses the only wild-type, functional homolog of the AveC allele native to S. hygroscopicus strain FERM BP-1901. In a non-limiting embodiment of the invention, the isolated polynucleotide molecule of the present invention that encodes the S. hygroscopicus homolog of an AveC gene product includes the nucleotide sequence of SEQ ID NO: 3 or a substantial portion thereof. In this sense, an "essential portion" of an isolated polynucleotide molecule comprising the nucleotide sequence of SEQ ID NO: 3 means an isolated polynucleotide molecule comprising at least 70% of the isolated polynucleotide molecule of SEQ ID NO: 3 and which encodes a functionally equivalent AveC homologue gene product as defined immediately above.

The present invention further provides a polynucleotide molecule comprising a nucleotide sequence that is homologous to the nucleotide sequence of S. hygroscopicus of SEQ ID NO: 3. The term "homologous" when used for a polynucleotide molecule having a nucleotide sequence that is homologous to a homologous sequence AveC homologous gene product-coding sequence of S. hygroscopicus of SEQ ID NO: 3 means a polynucleotide molecule having a nucleotide sequence: (a) which encodes the same gene product as the nucleotide sequence the identity of SEQ ID NO: 3 or a substantial portion thereof, but including one or more saylent changes in the nucleotide sequence, in accordance with the cleavage of the genetic code; (b) which hybridizes to the complement of the polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 4 under medium stringent conditions, i. hybridization to DNA-coupled filter in 0.5 M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 ° C, and washing in 2 x SSC / 0.1% SDS at 42 ° C ( see Ausubel et al., supra), and encodes a functionally equivalent homolog of an AveC gene product as defined above. In a preferred embodiment of the present invention, the homologous polynucleotide molecule hybridizes to the complement of the homologous AveC gene product encoding the nucleotide sequence of SEQ ID NO: 3 or a substantial portion thereof, under very stringent conditions, i.e., hybridization to a bound -c-filter DNA in 0.5 M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 ° C, and washing in 2 x 0.1 SSC / 0.1% SDS at 68 ° C ( see Ausubel et al., 1989, supra) and encodes a functionally equivalent AveC homologous gene product, as defined above.

The present invention also provides a polynucleotide molecule having a nucleotide sequence that encodes a polypeptide that is homologous to the AveC homologous gene product of S. hygroscopicus. As used herein, to refer to polypeptides that are homologous to the AveC homologous gene product of S. hygroscopicus SEQ ID NO: 4, 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 have been conservatively substituted and different amino acid residues, such conserved shifts leading to a functionally equivalent AveC homologous 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 to SEQ ID NO: 3 or to a polynucleotide molecule having a nucleotide sequence that is complementary to a nucleotide sequence Figure 1 (SEQID NO: 1) or SEQ ID NO: 3. Such oligonucleotides are at least 10 nucleotides in length, and preferably 15 to 30 nucleotides in length, and hybridize to one of the aforementioned polynucleotide molecules at very stringent levels. conditions, i.e., washing in 6 x SSC / 0.5% sodium pyrophosphate at ~ 37 ° C for ~ 14-base oligo, at ~ 48 ° C for ~ 17-base oligo, at ~ 55 ° C for 20 -base oligo, and ~ 60 ° C for 23-base oligo. In one embodiment of the invention, the oligonucleotides are complementary to part of one of the aforementioned molecules. These oligonucleotides are used for a variety of purposes, including to encode or act as antisense molecules useful for gene regulation or as primers for amplifying aveC or AveC homologous gene-producing polynucleotide molecules.

Additional AveC homologues can be identified in other Streptomyces species or strains using the polynucleotide molecules or oligonucleotides described herein, in conjunction with other known techniques. For example, an oligonucleotide molecule comprising part of the nucleotide sequence of S. avermitilis of Figure 1 (SEQ ID NO: 1) or part of the nucleotide sequence of S. hygroscopicus of (SEQ ID NO: 3) can be labeled in such a way that was determined and used to screen a genomic library constructed from DNA derived from the organism of interest. The severity of the hybridization conditions is chosen on the basis of the relationship of the comparative organism, in this example S. avermitilis or S. hygroscopicus, to the organism of interest. Requirements for varying severity of hybridization conditions are well known to those skilled in the art and these conditions will vary in a predictable manner depending on the specific organisms from which the library and sequences are derived. Such oligonucleotides are preferably of at least about 15 nucleotides in length and include, for example, those described in the examples below. Amplification of homologous genes can be carried out using these as well as other oligonucleotides by the use of standard techniques such as polymerase chain reaction (PCR), although other amplification techniques known in the art can also be used. technique, for example, a ligase chain reaction.

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

The production and handling of the polynucleotide molecules described herein is encompassed by the prior art and can be carried out according to the recombinant techniques described, e.g., 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 Intrscience, 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 Erich (ed.), 1992, PCR Technology, Oxford University Press, New York, which are incorporated herein by reference. Polynucleotide clones encoding aveC gene products or homologs of aveC gene products can be identified using any known state of the art, including, but not limited to, the methods set forth in section 7 below. A genomic DNA library can be screened for aveC and aveC homolog coding sequences using techniques such as the method described 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 comprising nucleotide sequences known to include aveC ORFs, such as the present ones, for example, in plasmid pSE186 (ATCC 209604) or in plasmid pSEl 19 (described in the examples below) can be used as probes in these screening experiments. Alternatively, oligonucleotide probes corresponding to nucleotide sequences derived from partial or complete amino acid sequences of the purified homologous aveC gene product can be synthesized.

Recombinant systems

Cloning and expression vectors

The present invention further provides recombinant cloning vectors and expression vectors that are used to clone or express the polynucleotide molecules of the present invention, for example, the aveC ORF of S. avermitilis or any homologous aveC ORF. In a non-limiting embodiment, the present invention provides plasmid pSEl86 (ATCC 209604) which contains the complete ORF of the aveC gene of S. avermitilis.

All the following description relating to the aveC ORF of S. avermitilis or a polynucleotide molecule comprising the aveC ORF of S. avermitilis or part thereof, or the aveC gene product of S. avermitilis, also relates to aveC homologs and aveC gene products, as long as it is not explained or understood in context.

A wide variety of different vectors have been developed for specific use in Streptomyces, including, among others, phages, large copy number plasmids, and an E. coli-Streptomyces vector shuttle, and any of these can be used to practically implement the present invention. A number of drug resistance genes have also been cloned by Streptomyces, with some of these genes being included in vectors as selection markers. Examples of commonly used vectors in Streptomyces are provided, including Hutchinson, 1980, Applied Biochem. Biotech. 16: 169-190.

The recombinant vectors of the present invention, in particular expression vectors, are preferably constructed such that the coding sequence for the polynucleotide molecule of the invention is operatively linked to one or more regulatory elements necessary for transcription and translation of the coding sequence for the production of polypeptides . As used herein, the term "regulatory element" includes, but is not limited to, nucleotide sequences encoding inducers and non-inducible promoters, enhancers, operators, and other elements known in the art for conducting and / or regulation of expression of polynucleotide encoding sequences. Also, as used herein, the coding sequence is "operatively linked" to one or more regulatory elements when the regulatory elements effectively regulate and allow the transcription of the coding sequence or the translation of its mRNA, or both.

Typical plasmid vectors that can be obtained by genetic engineering methods to comprise a polynucleotide molecule of the present invention include pCR-Blunt, pCR2.1 (Invitrogen), pGEM3Zf (Promega), and the vector shuttle pWHM3 (Vara et al., 1989, J. Bact. 171 & 5872-5881) among many others.

Methods for the design of recombinant vectors containing certain coding sequences operatively linked to suitable regulatory elements are well known in the art, and these methods can be used for the practical implementation of the invention. These methods include in vitro recombinant techniques, synthetic techniques and in vitro genetic recombination. See, for example, the technical description in Maniatis et al., 1989, supra; Ausubel et al., 1989, supra; Sambrook et al., 1989, no-rope; Innis et al., 1995, supra; and Erich, 1992, no-rope.

The regulatory elements of these vectors can be varied in their length and specificity. According to the host / vector system used, any of a number of suitable translation and transcription elements may be used. Non-limiting examples of transcriptional regulatory regions or bacterial promoters include beta-gal promoter, T7 promoter, TAC promoter, left and right lambda promoters, trp and lac promoters, trp-lac fusion promoters, and in particular for Streptomyces, ermE promoters, and type A and others. In one embodiment of the invention described below in Example 11, an expression vector is generated that contains the cloned aveC ORF bordering the full constitutive ermE promoter for Saccharopolyspora erythraea. The vector was transformed into S. avermitilis and subsequent HPLC analysis of the fermentation product showed an increased titer of produced avermectins compared to the production of the same strain but which instead expressed a wild-type aveC allele.

Fusion protein expression vectors can be used to express fusion proteins of aveC gene products. The purified fusion protein can be used to produce antisera against an aveC gene product, to study the biochemical properties of the aveC gene product, to design aveC fusion proteins with different biochemical activities, or to help identify or purify an expressed aveC gene product. Possible expression vectors for fusion proteins include, but are not limited to, vectors containing sequences encoding beta-gal actosidase and trpE fusion, maltose-binding protein fusion, tutathione-8-transferase fusion, and polyhistidine fusion (transportable region). In another embodiment of the invention, the aveC gene product or portion thereof may be fused to an aveC homologous gene product, or a portion thereof may be fused to an aveC homologous gene product or part thereof derived from other Streptomyces species or strains , such as S. hygroscopicus or S. griseochromogenes. In one particular embodiment of the invention described in Example 12 below and shown in Figure 7, a chimeric plasmid containing a 5 64 bp region of S. hygroscopicus homologous aveC ORF is constructed, replacing a homologous 564 aveC ORF region on S. avermitilis. Such hybrid vectors can be transformed into S. avermitilis cells and tested to determine their effect, for example, on the class 2: 1 ratio of avermectins produced.

AveC fusion proteins can be designed to include an area useful for purification. For example, the aveC-maltose-binding fusion protein can be purified using an amylose resin; the α-pgutathione-8-transferase fusion proteins can be purified using glutathione-agarose beads; and the aveC-polyhistidine fusion can be purified using a divalent nickel resin. Alternatively, antibodies against a carrier protein or peptide can be used for purification by affinity chromatography of the fusion protein. For example, a nucleotide sequence encoding for the epitope of a monoclonal antibody target (target) can be engineered into the expression vector for operative binding to regulatory elements and arranged so that the expressed epitope is fused to the aveC polypeptide. For example, a nucleotide sequence encoding the FLAG ™ 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, for example, the carboxyl terminus of the aveC polypeptide. The expressed aveC polypeptide-FLAG ™ epitope fusion product can then be detected and affinity purified using commercially available anti-FLAGO antibodies.

Also, the expression vector encoding the aveC fusion protein can be designed to contain a polylinker sequence that encodes specific proteinase cleavage sites so that the expression aveC polypeptide can be released from the region of the carrier or fusion partner by treatment with specific protease. The fusion protein vector, for example, may include, among others, a DNA sequence encoding thrombin or factor Xa cleavage sites.

The signal sequence in the upstream direction στ, and in the reading frame with, the aveC ORF can be engineered into the expression vector by known methods for directing the movement and secretion of the expressed gene product. Non-limiting examples of signal sequences include those of alpha-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 vectors or expression vectors, the present invention may be designed to also include a coding sequence for a product of a repeater gene or other selection markers. Such a coding sequence is preferably operatively linked to the coding sequence of the regulatory element, as described below. Reporter genes useful in the invention are well known in the art and include those encoding green fluorescent protein, luciferase, xy1E and tyrosinase, among others. The nucleotide sequences encoding selection markers are well known in the art and include those that encode gene products that confer resistance to antibiotics or antimetabolites, or that provide an autotrophic need. Examples of such sequences include those that code for resistance to erythromycin, thiostrepton, or kanamycin, among many others.

Transformation of host cells

The present invention also provides transformed host cells comprising polynucleotide molecules or recombinant vectors according to the invention, and novel strains or cell lines derived therefrom. Host cells useful for the practical implementation of the invention are preferably Streptomyces cells, although other prokaryotic and eukaryotic cells may be used. Such transformed host cells typically include, but are not limited to, microorganisms, such as bacteria transformed with recombinant DNA from bacteriophages, plasmid DNA or DNA from cosmid vectors, or yeast transformed with recombinant vectors, among others.

The polypeptide molecules of the present invention are intended to act in Streptomyces cells, but may also be transformed into other bacterial or eukaryotic cells, for example, for cloning or expression purposes. Typically, an E. coli strain may be used, such as the OH5alpha strain provided by the American Type Culture Collection (ATCC), Rockville, MD, USA (accession no. 31343), and from commercially available sources (Stratagene). Preferred eukaryotic host cells include yeast cells, although mammalian and insect cells can also be used efficiently.

The recombinant expression vector of the invention is preferably transformed into one or more host cells into a substantially homogeneous cell culture. The expression vector is typically introduced into the host cell according to known techniques, such as protoplast transformation, calcium phosphate precipitation, calcium chloride treatment, microinjection, transfection by contact with recombinant virus, transfection mediatedE, liposomal mediated transfection, conjugation or bombardment is micro-projectiles. Transformant selection can be performed by standard procedures, such as selection for cells expressing selection markers, for example for antibiotic resistance associated with recombinant vectors, as described below.

Once the expression vector is introduced into the host cell, the integration and maintenance of the aveC coding sequence or into the chromosome of the host cell or episomally can be confirmed by standard techniques, for example, by Southern hybridization analysis, restriction enzyme analysis, PCR analysis, including Reverse transcriptase PCR (rt-PCR), or by immunological assays, to detect the expected gene product. Host cells containing and / or expressing recombinant aveC coding sequences can be identified by any of at least four common approaches well known in the art, including: (i) DNADNA, DNA-RNA, or RNA- antisense RNA hybridization; (n) detecting the presence of marker gene functions; (iii) examining the level of transcription as measured by the expression of aveC-specific RNA transcripts in the host cell; and (iv) detecting the presence of a mature polypeptide product, as measured by immunoassay or by the presence of aveC biological activity (e.g., producing a specific ratio and amounts of avermectins indicative of aveC activity; for example, in S. avermitilis host cells).

Expression and characterization of a recombinant aveC gene product

Once the aveC coding sequence has been stably introduced into the appropriate host cell, the transformed host cell is propagated clonally and the resulting cells can grow under conditions leading to maximum production of the aveC gene product. Such conditions typically include the development of cells to high density. When the expression vector includes an inducible promoter, suitable induction conditions, such as temperature displacement, depletion of nutrients, addition of unnecessary inducers (e.g., carbohydrate analogues such as isopropyl-beta-O-thiogalactopyranoside (IPTG)), accumulation of excess by-products or the like are used as needed to induce expression.

When the expressed aveC gene product is retained inside the host cell, the cells are harvested and lysed, and the product is isolated and purified from the lysis under extraction conditions known in the art to reduce protein degradation, such as , at 4 ° C or in the presence of protease inhibitors, or both. When the expressed aveC gene product is secreted by the host cells, the depleted culture medium can simply be collected and the product isolated from it.

The expressed aveC gene product can be isolated or substantially purified from cell lysates or from the culture medium, as appropriate, using standard methods, including but not limited to, any combination of the following methods: ammonium precipitation sulfate, size fractionation, ion exchange chromatography, HPLC, density centrifugation and affinity chromatography. When the expressed aveC gene product exhibits biological activity, the increased purity of the preparation can be observed at each stage of the purification process using appropriate studies. Whether or not the expressed aveC gene product exhibits biological activity, this can be determined on the basis of, for example, the size or reactivity with an antibody, otherwise specific to aveC, or by the presence of a fused tag.

Therefore, the present invention provides a recombinantly expressed aveC gene product of Streptomyces, comprising the amino acid sequence encoded by the aveC gene product coding sequence of plasmid pSE186 (ATCC 209604), or the amino acid sequence of Figure 1 (SEQ ID NO: 2 or part thereof) his homologues.

The present invention further provides a recombinant-expressed homolog of an aveC gene product comprising the amino acid sequence of SEQ ID NO: 4 or a substantial portion thereof, and homologs thereof.

The present invention also provides a method of producing an aveC gene product comprising culturing host cells with a transformed expression vector, said vector comprising a polynucleotide molecule having a nucleotide sequence encoding an aveC gene product whose polynucleotide molecule is operatively linked regulatory elements that control expression of the polynucleotide molecule in the host cell under conditions leading to the production of the recombinant aveC gene roducts and to obtain the aveC gene product from the cell culture.

The recombinantly expressed aveC gene product of S. avermitilis is used for many purposes, including the screening of compounds that alter the functions of the aveC gene product and therefore modulate avermectin biosynthesis, and to produce antibodies directed against the aveC gene product.

Once an aveC gene product of sufficient purity has been obtained, it can then be characterized by standard methods, including SDS-PAGE, gel (sizeexclusion) chromatography, amino acid sequence analysis, biological activity in producing suitable products in the biosynthetic avermectic pathway. and others. For example, the amino acid sequence of the aveC gene product can be determined using standard peptide sequencing techniques. the aveC gene product can also be characterized using a hydrophilicity assay (see, e.g., Hopp and Woods, 1981, Proc. Natl. Acad. Sci. USA 78: 3824), or a similar software algorithm, to determine hydrophobic and hydrophilic regions of the aveC gene product. Structural analysis can be performed to identify the aveC gene product that permits specific secondary structures. Biophysical methods such as crystallographic X-ray structural analysis (Engstrom 1974, Biochem. Exp. Biol. 11: 7-13), computer modeling (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 substrates. The information obtained from these assays can be used to select new mutations sites in the aveC ORF to aid in the development of new S. avermitilis strains having more desirable avermectin production characteristics.

Construction and use of an aveC mutant

The present invention provides a polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as the coding sequence for the aveC gene product of S. avermitilis of plasmid pSEl86 (ATCC 209604) or the nucleotide sequence of the aveC ORF of S. avermitilis, as represented by FIG. SEQ ID NO: 1) but which also includes one or more mutations such that S. avermitilis cells strain ATCC 53692, in which a wild-type aveC allele has been activated, and which expresses a polynucleotide molecule comprising the mutated nucleotide sequence, etc producing the different ratio or amount of avermectins than are produced by cells of S. avermitilis strain ATCC 53692 that instead express only an aveC allele from the wild type.

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

Mutations of the aveC coding sequence include any mutations that introduce amino acid deletions, additions, or substitutions into the aveC gene product, or that lead to the replacement of the aveC gene product, or any combinations thereof, and that lead to the desired result . For example, the present invention provides polynucleotide molecules comprising the sequence encoding the aveC gene product of plasmid pSE 186 (ATCC 209604), or the nucleotide sequence of the aveC ORF of S. avermitilis, as presented in Figure 1 (SEQ ID NO: 1), but which in addition, it includes one or more mutations that encode the substitution of an amino acid residue with a different amino acid residue at the selected position in the aveC gene product. In several non-limiting embodiments of the invention, which are exemplified below, such substitutions may be made at any of the amino acid positions 55, 138, 139 or 230, or a combination thereof.

Mutations of the aveC coding sequences are carried out by any method known in the art, including by using error-prone PCR or by cassette mutagenesis. For example, oligonucleotide-directed mutagenesis can be used to alter the aveC ORF sequence in a particular way, such as inserting one or more restriction sites, or an end codon (terminal codon) into a specific region in the aveC ORF sequence. Methods such as those described in US 5,605,793, which include random fragmentation, repetitive cycles of mutagenesis, and nucleotide displacement, can also be used to generate large libraries of polynucleotides having nucleotide sequences encoding aveC mutations.

Targeted mutations can be used especially when they serve to alter one or more amino acid residues in the aveC gene product. For example, a comparison of the deduced amino acid sequences of the aveC gene product and the homolog of the aveC gene product of S. avermitilis (SEQ ID NO: 2), S. griseochromogenes (SEQ ID NO: 5), and S. hygroscopicus (SEQ ID NO: 4 ), as shown in Figure 6, means sites of significant conservation of amino acid residues between these species. Targeted mutagenesis leading to alteration of one or more of these conservative amino acid residues can be extremely effective in producing new mutants and strains that exhibit changes in avermectin production

Random mutagenesis can also be useful and can be done by exposing S. avermitilis cells to ultraviolet radiation or X-rays, or to chemical mutagenesis such as methyl-N-nitrosoguanidine, ethyl methane sulfonate, nitric acids or nitrogen mustards . See, for example, Ausubel, 1989 above, for an overview of mutagenesis techniques.

Once mutated polynucleotide molecules are generated, they are screened to determine whether they can modulate the biosynthesis of avermectin 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) base. In a non-limiting method, the mutated polynucleotide molecule is attached to an expression plasmid in operative association with one or more regulatory elements, which plasmid also preferably includes one or more drug resistance genes to enable selection of the transformed cells. This vector is then transformed into an aveC host cell using known techniques, and the transformed cells are selected and cultured in a suitable fermentation medium under conditions that allow or induce the production of avermectin. The fermentation products are then analyzed by HPLC to determine the ability of the mutated polynucleotide molecules to complement the host cell. A number of cells carrying mutated polynucleotide molecules capable of reducing the ratio of B2: B1 avermectins including pSE: 188, pSE199 npSE231 are indicated by the examples in Example 8 below.

The present invention provides methods for identifying S. avermitilis aveC ORF mutations capable of altering the ratio and / or amount of avermectins produced. In one preferred embodiment, the present invention provides a method for identifying aveC ORF mutations capable of altering the class 2: 1 ratio of avermectins produced, including: (a) determining the ratio of class 2: 1 avermectins produced by cells from a strain of S. avermitilis, in which the native aveC allele has been inactivated, and into which a polynucleotide molecule has been introduced comprising a nucleotide sequence encoding a mutated aveC gene product and expressed; (b) determining the ratio of class 2: 1 avermectins produced by cells of the same S. avermitilis strain as in step (a), but instead expressing only aveC appeal having the nucleotide sequence of the ORF of Figure 1 (SEQ ID NO : 1) or a nucleotide sequence homologous thereto; and (c) comparing the class 2: 1 ratio of avermectins produced by S. avermitilis cells from step (b) such that if the class 2: 1 ratio of avermectins produced by S. avermitilis cells from step (a) is different from the class 2: 1 ratio of avermectins produced by S. avermitilis cells from step (b), then a mutation of aveC ORF capable of altering the class 2: 1 ratio of produced avermectins is identified. In a preferred embodiment of the invention, the class 2: 1 ratio of avermectins is reduced by the mutation.

In another preferred embodiment, the present invention provides a method for identifying mutations of aveC ORFs or genetic structures including aveC ORFs capable of altering the amount of avermectins produced, including: (a) determining the amount of avermectins produced by cells from strain S avermitilis, in which the native aveC allele has been inactivated, and in which a polynucleotide molecule comprising a nucleotide sequence encoding a mutated aveC gene product or containing a genetic structure comprising a nucleotide sequence is introduced a sequence encoding the aveC gene product and expressed; (b) determining the amount of avermectins produced by cells of the same strain of S. avermitilis as in step (a) but which instead expresses only the aveC allele having the nucleotide sequence of the ORF of Figure 1 (SEQ ID NO: 1) or a nucleotide sequence homologous thereto; and (c) comparing the amount of avermectins produced by S. avermitilis cells of step (a) with the avermectins produced by S. avermitilis cells of step (b); so if the amount of avermectins produced by S. avermitilis cells from step (a) is different from the amount of avermectins produced by S. avermitilis cells from step (b), then a mutation of the aveC ORF or genetic structure capable of identifying changes the amount of avermectins. In a preferred embodiment of the invention, the amount of avermectins produced is increased by the mutation.

Any of the above methods for determining mutations is carried out using a fermentation culture medium, preferably with the addition of cyclohexane carboxylic acid, although other suitable precursors of fatty acids, such as any precursor to fatty acids, may also be used. fatty acids listed in Table 1.

Once the polynucleotide molecules that modulate the production of avermectin in the desired direction have 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 sequencing analysis using known methods. By comparing the DNA sequence of the mutated aveC allele with that of the wild-type aveC allele, the mutations (s) responsible for altering the production of avermectin can be determined. In certain, non-limiting embodiments of the present invention, the aveC genomic products of S. avermitilis, including or single amino acid substitutions at any residue 55 (S55F), 138 (S138T), 139 (A139T) or 230 (G230D) ), or double substitution at positions 138 (S138T) and 139 (A 139T), bring about a change in the function of the aveC gene product, such that the class 2: 1 ratio of avermectins produced changes (see: Example 8, below). Accordingly, polynucleotide molecules having nucleotide sequences that encode S. avermitilis mutated aveC genius products, including amino acid substitutions at one or more amino acid residues 55, 138, 139 or 230, or any combination thereof, are encompassed by this invention.

The present invention also provides compositions for the preparation of new strains of

S. avermitilis, the cells of which contain a mutated aveC allele that results in a change in the production of avermectin. For example, the present invention provides recombinant vectors that can be used to direct any of the polynucleotide molecules, including mutated nucleotide sequences of the present invention, to the site of the aveC gene of the S. avermitilis chromosome or to insert or replace the aveC ORF or part thereof, by homologous recombination. According to the present invention, however, a polynucleotide molecule comprising the mutated nucleotide sequence of the present invention provided herein may also act to modulate avermectin biosynthesis when inserted into the chromosome of S. avermitilis at a site other than the aveC gene. or when maintained episomally in S. avermitilis cells. Therefore, the present invention also provides vectors containing a polynucleotide molecule comprising a mutated nucleotide sequence of the present invention, which vector may be used to insert the polynucleotide sequence into a site in the chromosome of S. avermitilis other than the aveC gene, or is 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 S. avermitilis strain, whose cells produce avermectins at a modified 2: 1 ratio of avermectins. produced by cells. Such gene replacement vectors can be constructed using the mutated polynucleotide molecules present in the expression vectors provided herein, KaropSE188, pSE199 and BBE23, which expression vectors are explained in Example 8 below.

In another 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 new strains of cells that produce an altered avermectin ratio in compared to cells from the same strain that instead express a single wild-type aveC allele. In one preferred embodiment, the amount of avermectins produced by the cells is increasing. In a typical, non-limiting embodiment, such vectors further include a strong promoter known in the art, such as a strong constitutive ermE promoter from Saccharopolyspora erythraea, upstream, and operatively associated with the aveC ORF. Such a vector can be the plasmid pSE189 described in Example 11 below, or it can be constructed using a mutated aveC allele from plasmid pSE 189.

In another preferred embodiment, the present invention provides gene replacement vectors that are used to inactivate the aveC gene in wild-type S. avermitilis strain. In a non-limiting embodiment of the invention, such a gene replacement vector can be constructed using a mutated polynucleotide molecule present in plasmid pSE 180 (ATCC 20605), which is explained in Example 8 below (Figure 3) . The present invention further provides a vector comprising a polynucleotide molecule comprising or consisting of nucleotide sequences that naturally border the in situ gene of the aveC gene in the S. avermitilis chromosome, including, for example, those nucleotide sequencing shown in Figure 1 (SEQ ID NO: 1) that can be used to excrete S. avermitilis aveC ORF.

The present invention also provides methods for producing new strains of S. avermitilis, including cells that express a mutated aveC allele and produce a modified ratio and / or amount of avermectins, compared to cells from the same S. avermitilis strain that instead express only aveC wild-type allele. In a preferred embodiment, the invention provides a method for producing new strains of S. avermitilis, which include cells that express the mutated aveC allele, and which produce a modified class 2: 1 ratio of avermectins compared to cells of the same strain S avermitilis, which instead express only the wild-type aveC allele, involving transformation of S. avermitilis strain cells with a vector carrying a mutated aveC appeal that encodes a gene product that alters the 2: 1 ratio of avermectins produced by cells from a strain of S. ave rmitilis expressing the mutated aveC allele, compared to cells from the same S. avermitilis strain, which instead express only wild-type aveC alleles and selection of transformed cells that produce avermectins at a modified 2: 1 ratio, relative to the ratio of class 2: 1 produced by strain cells that instead express wild-type aveC alleles. In one preferred embodiment of the invention, the altered class 2: 1 ratio of avermectins is reduced.

In another preferred embodiment, the present invention provides a method for producing new strains of S. avermitilis, including cells that produce altered amounts of avermectin, including transforming cells from a strain of S. avermitilis with a vector carrying a mutated aveC allele or genetic structure, containing an aveC allele, the expression of which leads to a change in the amount of avermectins produced by cells of S. avermitilis strain expressing the mutated aveC allele or genetic structure, compared to cells from the same S. avermitilis strain which instead of ca express only an aveC allele from the wild type and selecting transformed cells in an altered amount compared to the amount of avermectins produced by cells of the strain that instead express only an aveC allele from the wild type. In a preferred embodiment, the amount of avermectins produced in the transformed cells is increasing.

In another preferred embodiment, the present invention provides a method for producing new strains of S. avermitilis, the cells of which include an inactivated aveC allele, comprising transforming cells from a strain of S. avermitilis that express the aveC allele with a vector that inactivates the aveC allele, and selection of transformed cells in which the aveC allele has been inactivated. In a preferred non-limiting embodiment, cells of S. avermitilis strain are transformed with a vector replacing gene that carries the aveC allele that has been inactivated by mutation or by replacing part of the aveC allele with a heterologous gene sequence, and selection of transformed cells in which the native aveC allele of the cells was replaced by the inactivated aveC allele. Inactivation of the aveC allele can be determined by HPLC analysis of the fermentation products as described below. In one specific non-limiting embodiment of the invention described in Example 8 below, the aveC allele is inactivated by inserting the eppE gene from Saccharopolyspora erythraea into the aveC ORF.

The present invention further provides a new strain of S. avermitilis comprising cells that have been transformed with any of the polynucleotide molecules or vectors of the present invention. In one preferred embodiment, the present invention provides a new strain of S. avermitilis comprising cells that express a mutated aveC allele instead of or in addition to a wild-type aveC allele, wherein said cells of the new strain produce avermectins at a changed ratio of class 2: 1 avermectins produced by cells of the same strain, which instead express only the wild-type aveC allele. In one preferred embodiment, the altered class 2: 1 ratio produced by the new cells is reduced. Such new strains are used in the production of commercially available avermectins, such as doramectin, on a large scale.

A first objective of the screening study described herein is to identify mutated alleles of the aveC gene, whose expression in S. avermitilis alters, and in particular reduces, the production ratio of class 2: 1 avermectins. In one preferred embodiment, the B2: B 1 ratio of avermectins produced by cells of the new S. avermitilis strain of the present invention expressing a mutated aveC allele that reduces the class 2: 1 ratio of produced avermectins between less than 1.6: 1 to ca. 0: 1, in a more preferred embodiment, the ratio is between about 0.84: 1 and about 0: 1. In the specific embodiment described below, the new cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectins at a ratio below 1.6: 1. In another specific preferred variant described below, the new cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectins in a ratio of approximately 0.94: 1. In another specific preferred embodiment described below, the new cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectins in a ratio of approximately 0.88: 1. In another specific preferred embodiment described below, the new cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectins in a ratio of approximately 0.84: 1.

In another specific preferred embodiment, the present invention provides a novel strain of S. avermitilis comprising cells expressing a mutated avcC allele, or a genetic structure containing an aveC allele at the site of, or in addition to, a wild-type aveC allele, wherein the cells of the new strain produce altered avermectins compared to cells of the same strain, which instead express only the wild-type aveC allele. In one preferred embodiment, the new strain produces an increased amount of avermectins. In a non-limiting embodiment, the genetic structure includes a strong promoter, such as the strong constitutive promoter of Saccharopolyspora erythraea, upstream and operatively linked to the aveC ORF.

In another embodiment, the present invention provides novel strains of S. avermitilis, including cells in which the aveC gene has been inactivated. Such strains are useful both for the different spectra of avermectins they produce in comparison with wild-type strains and for complementary screening assays as described herein to determine whether targeted or incidental mutagenesis of the aveC gene affects production of avermectins. In one specific embodiment described below, host cells of S. avermitilis are genetically engineered to contain an inactivated aveC gene. For example, strain SE180-11 described in the exemplary description is generated by replacing plasmid pSE180 (ATCC 209605) (Figure 3), which is designed to inactivate the aveC gene of S. avermitilis by inserting the ermE resistance gene into the aveC encoding area.

The present invention further provides recombinantly expressed, mutated S. avermitilis gene products encoded by any of the above polynucleotide molecules of the invention, and methods for preparing them.

The invention also provides a method for the preparation of avermectins comprising culturing cells from a strain of S. avermitilis, which cells express a mutated aveC allele encoding a gene product that changes the ratio of class 2: 1 avermectins produced by cells to a strain of S. avermitilis expressing the mutated aveC allele, in comparison with cells of the same strain, which instead express only the wild-type aveC allele in a culture medium, under conditions that allow or induce the production of avermectins from them, and the production of said cultured avermectins s. 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 method provides increased efficiency in the production of commercially important avermectins, such as doramectin.

The present invention also provides a method for the production of avermectins comprising culturing cells from a strain of S. avermitilis, which cells express a mutated aveC allele or a genetic structure comprising the aveC allele, which results in the production of a changed amount of avermectins produced by cells of a strain of S. avermitilis that expresses a mutated aveC allele or genetic structure, compared to the same strain that does not express the mutated aveC allele or genetic structure, but instead expresses only a wild-type aveC allele in the culture lines under conditions that permit or induce avermectin production thereof, and recovering said avermectins from the culture medium. In one preferred embodiment, the amount of avermekgins produced in the culture by cells expressing the mutated aveC allele or genetic structure is increased.

The present invention also provides a novel composition of avermectins produced by a strain of S. avermitilis expressing a matured aveC allele that encodes a gene product that reduces the ratio of class 2: 1 avermectins produced by cells of a strain of S. avermitilis expressing mutated aveC alleles. , compared to cells of the same strain that instead express only the wild-type aveC allele, whereby the avermekgins in the new composition are produced at a reduced class 2: 1 ratio of avermectins produced by cells of the same S. avermitilis strain, which instead but expresses only an aveC allele from the wild type. The new composition of avermectins may be presented as produced in the culture medium by depletion fermentation. The new avermectins composition may be partially or substantially purified from the culture fluid by known biochemical purification techniques such as ammonium sulfate precipitation, dialysis, size fractionation, ion exchange chromatography, HPLC and others.

Use of avermectins

Avermectins are highly active anti-parasitic agents having exceptional applications such as anthelmintic agents, ectoparasiticidal agents, insecticidal agents and acaricidal agents. Avermectin compounds produced according to the methods of the present invention are administered for any of these purposes. For example, avermectin compounds produced according to the methods of the present invention are used to treat a variety of human diseases or conditions, in particular when those diseases or conditions are caused by infection by parasites, as is known in the art. See, e.g., Ikeda and Omura, 1997, Chem. Rev. 97 (7): 2591-2609. In particular, the avermectin compounds produced according to the present invention are effective in treating diseases and conditions caused by ectoparasites, such as parasitic nematodes, which can infect humans, domestic animals, pigs, sheep, chickens, horses or cattle.

In particular, the avermectin compounds produced according to the present invention are useful against nematodes that infect humans as well as those that infect different species of animals. Such nematodes include gastrointestinal parasites, such as Ancylostoma, Necator, Ascaris, Strodyloides, Trichinella, Capillaria, Trichuris, Enterobius, Dirofilaria, and parasites that are found in blood or other tissues and organs, such as filamentous worms and extracts of intestinal cochlea and intestinal extract and Trichinella.

Avermectin compounds produced according to the present invention are also used to treat ectoparasite infections, including, for example, infecting mammals and birds with arthropods caused by ticks, worms, lice, fleas, butcher's flies, biting insects, or migrating larvae of bivalve insects affecting livestock and horses, among others.

The avermectin compounds produced according to the present invention are also used as insecticides against domestic pests, such as cockroaches, moths, carpet beetles and house flies among others, as well as insect pests for stored cereals and for crops that pest include worms, aphids, caterpillars, and right winged insects, such as locust, among others.

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

The avermectin compound produced according to the present invention is administered in a suitable form for specific use, the specific host animal species being treated and parasites or insects included. When used as a parasiticide, an avermectin compound produced according to the present invention can be administered orally in the form of capsules, bolus tablets, tablets or penetrating liquids, or alternatively, may be administered by injection or as an implant. Such dosage forms are formulated in conventional manner according to standard veterinary practice. Therefore, capsules, tablets or bolus tablets may be prepared by mixing the active ingredients with a finely dispersed solvent or carrier further containing a disintegrant and / or a binder such as starch, lactose, talc, magnesium stearate and the like. Doses may be prepared by dispersing the active ingredient in aqueous solution together with a dispersing or wetting agent, and the like. Injectable forms can be prepared in the form of sterile solutions that may contain other substances, such as enough salts and / or glucose to make the solution isotonic with blood.

Such dosage forms will vary according to the weight of the active ingredient, depending on the patient or the type of host being treated, the severity and type of infection, and the body weight of the host. A dose of the active ingredient of approximately 0.001 to 10 mg per kg of body weight of the patient or animal administered as a single dose or as divided doses over a period of 1 to 5 days would generally be satisfactory for oral administration. However, there may be cases where higher or lower doses are prescribed, as determined, for example, by the doctor or veterinarian, based on the clinical symptoms.

Alternatively, the avermectin compound produced according to the present invention can be administered in combination with animal feed mixtures, and for this purpose concentrated nutritional supplements can be prepared to be mixed with ordinary animal feed.

For use as an insecticide, and for the treatment of agricultural pests, the avermectin compound produced according to the present invention can be administered as a spray, powder, emulsion and the like. similar, according to standard agricultural practice.

Examples of carrying out the invention

Fermentation of Streptomyces avermitilis and analysis of B2: B1 avermectins

Strains lacking branched-chain 2-oxo acid dehydrogenase and 5-O-methyltransferase activity do not produce avermectins unless the fermentation medium is supplemented with fatty acids. These examples show that a wide variety of B2 ratios can be obtained in such mutants. B1 avermectins when biosynthesis is initiated in the presence of various fatty acids.

Materials and methods

Streptomyces avermitilis ATCC 53692 is stored at -70 ° C as a complete broth prepared in starch medium consisting of starch (Nadex, Laing National - 20 g: Pharmamedia (Trader's Protein, Memphis, TN) 15 g; Ardamine pH (Yeast Products Inc.) - 5 g: calcium carbonate - 1 g. The final volume is brought to 1 l with tap water, the pH is adjusted to 7.2 and the medium is autoclaved at 121 ° C for 25 min.

ml of the brown suspension of the above preparation are used to inoculate a flask containing 50 ml of the same medium. After 48 h incubation at 28 ° C on a rotary shaker at 180 rpm, 2 ml of broth was used to inoculate a flask containing 50 ml of starch-80 starch production medium; calcium carbonate - 7 g: Pharmamedia - 5 g: acidic potassium phosphate -1 g: magnesium sulfate -1 g; glutamic acid - 0.6 g; ferrous sulfate tetrahydrate - 0.01 g; zinc sulfate - 0.001 g; magnesium sulfate - 0.001 g. The final volume was adjusted to 11 with tap water, the 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 medium 24 h after inoculation to obtain a final concentration of 0.2 g / l. The fermentation medium was incubated for 14 days at 28 ° C, after which the medium was centrifuged (2,500 rpm for 2 min) and the supernatant removed. The micellar pellet (solid precipitate) was extracted with acetone (15 ml), then with dichloromethane (30 ml) and the organic phase separated, filtered and then evaporated to dryness. The residue was taken up in methanol (1 ml) and analyzed by HPLC with a Hewlett-Packard 1090A liquid chromatography apparatus equipped with a scanning diode detector adjusted to 240 nm. The column used is a Beckman Ultasphere C-18, 5 pm, 4.6 mm x 25 cm column maintained at 40 ° C. 25 μΐ of the above methanol solution was injected onto the column. The elution was performed with a linear gradient of methanol-water from 80:20 to 95: 5 for 40 min at a rate of 0.85 ml / min. Two standard concentrations of cyclohexyl B1 are used to calibrate the response of the detector, and the areas under the curve for B2 avermectins are measured.

Results

Table 1 shows the retention time for HPLC observed for B2 and B1 avermectins and the 2: 1 ratio __ TABLE 1

Time of at HPLC retention (min) Ratio Substrate B2 B1 B2: B1 4-T Etrahydropyran carboxylic acid 8.1 14.5 0.25 Isobutyric acid 10.8 18.9 0.5 3- Furancarboxylic acid 7,6 14.6 0.62 8 - (+) - 2-methylbutyrate acid 12,8 21.6 1.0 Cyclohexanecarboxylic acid 16.9 26,0 1.6 3-T Iofenecarboxylic acid 8.8 16.0 1.8 Cyclopentanecarboxylic acid 14.2 23,0 ^2.0 3-T Rifluoromethylbutyric 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 show extremely wide ranges of the ratio of B 2: B1 avermectin products, indicating a significant difference in the results of the dehydrated conversion of class 2 compounds into class 1 compounds, depending on the type of chain 40 of the starting fatty acid supplied. This indicates that changes in the B2: B1 ratio obtained from the variation of the AveC protein may be specific to certain substrates. Therefore, screening for mutants that exhibit a change in the B2: B1 ratio obtained with a particular substrate should be performed in the presence of that substrate. In the following examples described below, cyclohexanecarboxylic acid is used as the screening substrate. However, this substrate is primarily used as an example to explain the potential and is not intended to limit the application of the present invention.

Example 7. Isolation of the aveC gene

This example describes the isolation and characterization of a chromosome region of Streptomyces avermitilis that encodes the aveC gene product. As shown below, the aveC gene is identified as being able to modify the ratio of cyclohexyl-B2 to cyclohexyl-B2 (B2: B2) produced avermectins.

Materials and methods

Cultivation of Streptomyces for DNA Isolation

The following methods are followed by culturing Streptomyces. Single colonies of S. avermitilis ATCC 31272 (isolate # 2 from single colonies) were isolated on 1/2 strength YPD-6 containing: Difco Yeast Extract - 5 g; Difco Bactopeptone - 5 g; dextrose - 2.5 g; MOPS - 5 g; Difco Bacto arap - 15 g. The final volume was adjusted to 1 L with dH ₂ O, the pH was adjusted to 7.0 and the medium was autoclaved at 121 ° C for 25 min.

The micelles sprouted in the above medium were used to inoculate 10 ml TSB medium (Difco Tryptic Soya Broth - 30 g, in 1 1 dH ₂ O, autoclave at 121 ° C for 25 min) in 25 mm x 150 mm tubes which maintained at shaking (300 rpm) at 28 ° C for 48-72 h.

Isolation of chromosomal DNA from Streptomyces

Aliquots (0.25 ml or 0.5 ml) of the mycelium cultured as indicated above were placed in 1.5 ml centrifuge tubes and the cells were concentrated by centrifugation at 12,000 x g for 60 s. The supernatant was removed and the cells resuspended in 0.25 ml TSE buffer (20 ml 1.5M sucrose, 2.5 ml 1 M TrisHC1, pH 8.0 2.5 ml 1 M EDTA, pH 8.0 and 75 ml dH ₂ O) containing 2 mg / ml lysozyme. Samples are incubated at 37 ° C for 20 min with shaking, placed in an AutoGen 540 ™ Integrated Separation Systems (Natick, MA) and genomic DNA isolated using Cycle 159 (specialized software). according to the manufacturer's instructions.

Alternatively, 5 ml of mycelium was placed in 17 mm x 100 mm tubes, the cells were concentrated by centrifugation at 3000 rpm for 5 min and the supernatant removed. The cells were resuspended in 1 ml TSE buffer, concentrated by centrifugation at 3000 rpm for 5 min, and the supernatant removed. The cells were resuspended in 1 ml TSE buffer containing 2 mg / ml lysozyme and concentrated at 37 ° C with shaking for 3060 min. After incubation, 0.5 ml of 10% sodium dodecyl sulfate (SDS) was added and the cells were incubated at 37 ° C until the lysis process was complete. The lysate was incubated at 65 ° C for 10 min, cooled to room temperature, separated into two eppendorf tubes and extracted with 0.5 ml phenol / chloroform (50% phenol pre-equilibrated with 0.5 M Tris, pH 8 , 0; 50% chloroform). The aqueous phase was removed and extracted 2 to 5 times with chloroform: isoamyl alcohol (24: 1). The DNA was precipitated by adding 1/10 volumes of 3 M sodium acetate, pH 4.8, incubating the mixture on ice for 10 min, centrifuging at 15,000 rpm at 5 ° C for 10 min and moving the precipitate into a clean tube, to which is added 1 ml of isopropanol. The supernatant plus the isopropanol mixture was then incubated on ice for 20 min, centrifuged at 15,000 rpm for 20 min at 5 ° C, the supernatant removed and the solid DNA precipitate washed with 1 x 70% ethanol. After drying the precipitate, the DNA was resuspended in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).

Plasmid DNA isolation from Streptomyces

Aliquots (1.0 ml) of mycelium cultured as indicated above were placed in 1.5 ml microcentrifuge tubes and the cells were concentrated by centrifugation at 12,000 x g for 60 s. The supernatant was removed and the cells resuspended in 1.0 ml of 10.3% sucrose and concentrated by centrifugation at 12,000 x g for 60 s and the supernatant removed. The cells were resuspended in TSE buffer containing 2 mg / ml lysozyme and incubated at 37 ° C for 20 min with shaking, placed in an AutoGen540 ™ nucleic acid isolation device. Plasmid DNA was isolated using Cycle 106 (specialized software) according to the manufacturer's instructions.

Alternatively, 1.5 ml of the micelles were placed in 1.5 ml microcentrifuge tubes, the cells were concentrated by centrifugation at 12,000 x g for 60 s and the supernatant removed. The cells were resuspended in 1.0 ml in 10.3% sucrose and concentrated by centrifugation at 12,000 x g for 60 s and the supernatant removed. The cells were resuspended in TSE buffer containing 2 mg / lysozyme and concentrated at 3 7 ° C for 15-3 0 min. After incubation, 0.25 ml of alkaline SDS (0.3N NaOH, 2% SDS) was added and the cells were incubated at 5 5 ° C for 15-30 min until the solution clarified. Sodium acetate (0.1 ml, 3M pH 4.8) was added to the DNA solution, then the solution was incubated on ice for min. DNA samples were centrifuged at 14,000 rpm for 10 min at 5 ° C. The supernatant was transferred to a clean tube and 0.2 ml of phenol: chloroform (50% phenol: 50% chloroform) was added and carefully mixed. The DNA was centrifuged at 14,000 rpm for 10 min at 5 ° C and the top layer was transferred to a clean Eppendorf tube. Isopropanol was added and the solution was gently stirred, then incubated for 20 min at room temperature. The DNA solution was centrifuged at 14,000 rpm for 15 min at 5 ° C, the supernatant was transferred and the DNA pellet was washed with 70% ethanol, dried and resuspended in TE buffer.

Plasmid DNA isolation from E. coli

A single transformed colony of E. coli was inoculated into 5 ml of Luria-Bertani (LB) medium (Bacto-Tryptone - 10 g, Bacto-yeast extract - 5 g, and NaCl - 10 g in 1 1 dH ₂ O, pH 7 , 0, autoclave at 121 ° C for 25 min and add 100 pg / ml ampicillin). The culture was incubated overnight, and an aliquot of 1 ml was placed in a 1.5 ml microcentrifuge tube. The culture samples were placed in an AutoGen 540 ™ automatic nucleic acid isolation device and the plasmid DNA was isolated using Cycle 3 (specialized software) according to the manufacturer's instructions.

Preparation and transformation of protoplasts of S. avermitilis

Single colonies of S. avermitilis were isolated on 1/2 power YPD-6. The micelles were used to inoculate 10 ml of TSB medium in 25 mm x 150 mm tubes, which were then incubated with shaking (300 rpm) at 28 ° C for 48 h. 12 ml of mycelium was used to inoculate 50 ml of 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 ° C for 25 min, the following was added: 2.5 M MgCl ₂ 6H ₂ O (separately autoclaved at 121 ° C for 25 min) - 2 ml; and glycine (20%) (filter sterilized) - 25 ml.

The micelles were cultured at 30 ° C for 4872 h and collected by centrifugation in a 50 ml centrifuge tube (Falcon) at 3,000 rpm for 20 min. The supernatant was removed and the micelles were resuspended in P buffer containing: sucrose - 205 g; K ₂ SO ₄ 0.25 g; MgCl ₂ 6H ₂ O - 2.02 g; H ₂ O - 600 ml; ΐςΡΟ ₄ (0.5%) -10 ml; trace element solution - 20 ml; CaCl ₂ 2H ₂ O (3.68%) -100 ml; and MES buffer (1.0 M, pH 6.5) - 10 ml. (* the trace element solution contains for 1: ZnCl ₂ - 40 mg; FeCl ₃ 6H ₂ O - 200 mg; CuCl ₂ 2H ₂ O -10 mg; MnCl ₂ 4H ₂ O -10 mg; Na ^ O, 10H ₂ O - 10 mg; (NH ₄ ) ₆ Mo ₇ 0 ₂₄ 4H ₂ O -10 mg). The pH was adjusted to 6.5 and the final volume to 11 and the medium filtered warm through a 0.45 micron filter.

The micelles were precipitated in a solid precipitate at 3,000 rpm for 20 min, the supernatant removed and the micelles resuspended in 20 ml P buffer containing 2 mg / ml lysozyme. The micelles were incubated at 35 ° C for 15 min with shaking and examined microscopically to determine the size of protoplast formation. At the completion of protoplast formation, the protoplasts were centrifuged at 8,000 rpm for 10 min. The supernatant was removed and the protoplasts resuspended in 10 ml P buffer. The protoplasts were centrifuged at 8.00 rpm for 10 min, the supernatant removed, the protoplasts resuspended in 2 ml P buffer, and approximately 1x10 'protoplasts distributed in 2.0 ml cryogenic ampoules (Nalgene).

An ampoule containing 1x10 ⁹ protoplasts was centrifuged at 8,000 rpm for 10 min, the supernatant removed and the protoplasts resuspended in 0.1 ml P buffer. The protoplasts were added to 5 g of transforming DNA, immediately followed by the addition of 0.5 ml of working T buffer. The basic T buffer contains: PEG - 1000 (Sigma) - 25 g; sucrose 2.5 g; H ₂ O - 83 ml. The pH was adjusted to 8.8 with 1 N NaOH (sterilized by filtration) and the basic T buffer was sterilized by filtration and stored at 4 ° C. The working T buffer that is being prepared during the day in which it is to be used is the basic T buffer - 8.3 ml; K ₂ PO ₄ (4 mM) - 1.0 ml; CaCl ₂ 2H ₂ O (5M) - 0.2 ml; and TES (1 M, pH 8) - 0.5 ml. Each component of the T buffer is self-sterilized by filtration.

s after the addition of T buffer to the protoplasts, P buffer is also added and the protoplasts are centrifuged at 8,000 rpm for 10 min. The supernatant was removed and the protoplasts resuspended in 0.1 ml P buffer. The protoplasts were then seeded onto RM 14 medium containing: sucrose - 205 g;

K ₂ SO ₄ - 0.25 g; MgCl ₂ 6H ₂ O - 10.12 g; glucose 10 g; Difco Casamino Acids - 0.1 g; Difco Yeast Extract - 5 g; Difco Oatmeal Agar - 3 g; Difco Bacto Agar -22 g; dH ₂ O - 800 ml. The solution was autoclaved at 121 ° C for 25 min. After autoclaving, a sterile stock of the following was added: ΐςΡΟ ₄ (0.5%) -10 ml; CaCl ₂ 2H ₂ O (5 M) - 5 ml; MES buffer (1.0 M, pH 6.5) - 10 ml; trace element solution (same as above) - 2 ml; maternal cycloheximide (25 mg / ml) - 40 ml; and IN NaOH - 2 ml. 25 ml of RM14 medium were aliquoted into dishes and the dishes were dried 24 hours before use.

The protoplasts were incubated at 95% humidity at 30 ° C for 20-24 h. To select thiostrepton resistant transformants over RM14, regenerated dishes were sprayed with 1 ml of a layering buffer containing 125 g per ml of thiostrepton. The layering buffer contains for 100 ml: sucrose - 10.3 g; Trace element solution (same as above) - 0.2 ml; and MES (1 M, pH 6.5) - 1 ml. Protoplasts were incubated at 95% humidity at 30 ° C for 7-14 days (until thiostrepton-resistant colonies (Thio ') became visible).

Transformation of protoplasts by Streptomyces lividans

In some cases, S. lividans TK64 (supplied by John Innes Institute, Norwich, UK) is used for transformation. Methods and compositions for the cultivation, protoplast preparation and transformation of S. lividans have been described by Hopwood et al., 1985, Genetic Manipulation of Streptomyces, A Laboratory Manual ;, John Innes Foundation, Norwickm UK, and conducted as described herein . Plasmid DNA was isolated from S. lividans transformants as described in section 7.1.3 above.

Fermentation analysis of S. lividans strains

S. lividans micelles cultured on YPD-6 1/2 power for 4-7 days were inoculated into 1x6 inch tubes containing 8 ml pre-media and 5 mm glass beads. Preliminary medium contains: soluble starch (or lightly cooked starch or KOSO, Japan Com Starch Co., Nagoys) - 20 g / l; Pharmamedia 15 g / l; Ardamine pH - 5 g / l (Champlain Ind., Clifton, NJ); CaCO ₃ - 2 g / l; 2 x bcfa ("bcfa" refers to branched chain fatty acids) with a final concentration in the middle of 50 ppm 2 - (+/-) - methyl butyric acid, 60 ppm isobutyric acid, and 20 ppm isovaleric acid. The pH was adjusted to 7.2 and the medium was autoclaved at 121 ° C for 25 min.

The tube was shaken at an angle of 17 ° at 215 gr at 29 ° C for 3 days. 2 ml aliquots of the seed medium are used to inoculate 300 ml Erlenmayer flasks containing 25 ml of production medium containing: starch (or lightly cooked starch or KOSO) - 160 g / l; Nutrisoya (Archer Daniels Midland, Decatm, IL) - 10 g / l; Ardamine pH -10 g / l; K ₂ HPO ₄ - 2 g / l; MgSO ₄ .4H ₂ O) - 2 g / l; FeSO ₄ .7H ₂ O - 0.02 g / l; MnCl ₂ - 0.002 g / l; ZnSO ₄ .7H ₂ O - 0.002 g / l; CaCO ₃ - 14 g / l; 2 x bcfa (as above); and cyclohexane carboxylic acid (CHC) (prepared as a 20% solution with pH 7.0) - 800 ppm. The pH was adjusted to 6.9 and the medium was autoclaved at 121 ° C for 25 min.

After inoculation, the flask was incubated at 29 ° C for 12 days, shaking at 200 g. After incubation, a sample of 2 ml is taken from the flask, diluted with 8 ml of methanol, mixed and the mixture is centrifuged at 1,250 x g for 10 min to precipitate the cell debris. The supernatant was then assayed by HPLC using a Beckman Ultrashere ODS column (25 cm × 4.6 mm ID) at a flow rate of 0.75 ml / mm and determined; by absorption at 240 nm. The mobile phase is 865 / 8,9 / 5,1 metaiol / water / acetonitrile.

Isolation of PKS genes from S. avermitilis

A cosmid library of chromosomal DNA from S. avermitilis (ATCC 31272, SC-2) was prepared and hybridized with a ketosynthetase (LS) sample made from a fragment of the polyketide synthase (PKS) gene of Saccharopolyspora erythraea. A detailed description of the preparation of cosmid libraries can be found in Sambrook et al., 1989, supra. A detailed description of the preparation of chromosomal DNA libraries by Streptomyces is presented in Hopewood et al., 1985, supra. Cosmid clones containing ketosynthetase synthesizing regions were identified by hybridization to a 2.7 Kb Ndel / Eco47 III fragment of pEX26 (kindly provided by Dr. R. Leadlay, Cambridge, UK). Approximately 5 ng of pEX26 was digested using Ndel and Eco47 III. The reaction mixture was loaded onto 0.8% SeaPlaque GTG agarose gel (FMC bioProducts, Rocjland, ME). The 2.7 Kb Ndel / Eco47III fragment was excised from the gel after electrophoresis and the gel was obtained DNA using GELase ™ by Epicentre Technologies using Fast Protocol. The 2.7 Kb NdeI / Eco47III fragment was labeled with [alpha- ³² PDOSTP (deoxycytidine 5'-triphosphate, tetra (triethylammonium) salt, [alpha- ³² P] -) (NEN-Dupont, Boston, MA) using BRL Nick Trandlation System (BRL Life Technologies, Inc. Gaithersburg, MD) following the manufacturer's instructions. A typical reaction was carried out in 0.005 ml volume. After the addition of 5 μΐ stop buffer, the labeled DNA was separated from the unplugged nucleotides using G-25 Sephadex Quick Spin ™ Colonm * Boehring Manheim) following the manufacturer's instructions.

Approximately 1,800 cosmid clones were screened by colony hybridization. The clones are then identified as highly hybridizing to Sacc. erythraea KS sample. E. coli colonies were cultured containing cosmid DNA in LB liquid medium and cosmidia DNA was isolated from each culture in an AutoGen 540 ™ nucleic acid automated isolation device using Cycle 3 (specialized software) according to the manufacturer's recommendations. Mapping with restriction endonucleases and Southern blot hybridization analysis showed that five of the clones contained overlapping chromosomal regions. An S. avermitilis genomic BamHI restriction map of the cosmids (i.e., pSE65, pSE66, pSE67, pSE68, pSE69) was constructed by overlapping cosmid analysis and hybridization (Figure 4).

Identification of DNA modulating the avermectin B2: B1 ratio and identification of the aveC ORF

The following methods are used to test subcloned fragments derived from the pSE66 cosmid clone for their ability to modulate the avermectin B2: B1 ratio in AveC mutants. pSE66 (5 μβ) was digested with SacI and BamHI. The reaction mixture was loaded onto 0.8% SeaPlaque ™ GTG agarose gel (FMC BioProducts), a 2.9 kb SacI / BamHI fragment was excised from the gel after electrophoresis, and DNA was isolated from the gel using GELase ™ (Epicentre Technologies) using on Fast

Protocol. Approximately 5 μβ of the shuttle vector pWHM3 (Vara et al., 1989, J. Bacteriol. 171: 5872-5881) was digested with SacI and BamHI. About 0.5 pg of 2.9 kb inserts and 0.5 μβ of digested pWHM3 were mixed together and incubated overnight with 1 unit of ligase (New Efland Biolabs, Inc., Beverly, MA) at 15 ° C in total volume of 20 μΐ as recommended by the manufacturer. After incubation, 5 μΐ of the ligation mixture was incubated at 70 ° C for 10 min, cooled to room temperature and used to transform competent E. coli DH alpha cells (BRL) as recommended by the manufacturer. Plasmid DNA was isolated from ampicillin-resistant transformants and the presence of 2.9 kb Saci / BamHI insert was confirmed by restriction analysis. The plasmid was designated pSE119.

Protoplasts of S. avermitilis strain 1100-SC3 8 (Pfizer in-house strain) were prepared and transformed with pSEl 19 as described in Example 7 above. Strain 1100-SC38 is a mutant that produces significantly greater amounts of the avermectin cyclohexyl-B2 form than the cyclohexyl-B1 form when cyclohexane carboxylic acid (B1: B2 of approximately 30: 1) is added. pSE119, which is used to transform protoplasts from S. avermitilis, is isolated from either 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 TK64. Thiostrepton-resistant transformants from strain 1100-SC38 were isolated by HPLC analysis of the fermentation process products. Transformants of S. avermitilis strain 1100-SC38 containing pSE119 produced an altered ratio of cyclohexyl-B2: cyclohexyl-B 1 of approximately 3.7: 1 (Table 2).

Once pSE1 19 was found to be able to modulate the avermectin B2: B1 ratio in one aveC mutant, the inserted DNA was sequenced. Approximately 10 μβ pSEl 19 was isolated using a plasmid DNA isolation kit (Qiagen, Valencia, CA), following the manufacturer's recommendations, and sequenced using an ABI373A automated DNA sequencer (Perkin Elmer, Foater City, CA). Sequence data are aggregated and published using Computer Group programs (GCG, Madisson, WI). The DNA sequence and the aveC ORF are shown in Figure 1 (SEQ ID NO: 1).

A new plasmid, designated pSEl 18, was constructed as follows. Approximately 5 pg of pSE66 was digested with SphI and BamHI. The reaction mixture was loaded onto 0.8% SeaPlaque ™ GTG agarose gel (FMC BioProducts), a 2.8 kb SphI / BamHI fragment was excised from the gel after electrophoresis, and DNA was extracted from the gel using GELase ™ (Epicentre Technologies) using on Fast Protocol. Approximately 0.5 pg of the shuttle vector pWHM3 was digested with SphI and BamHI. Approximately 0.5 pg of 2.8 kb inserts and 0.5 pg of ground pWHM3 were mixed together and incubated overnight with 1 unit of ligase (New England Biokabs) at 15 ° C in a total volume of 20 pl, as recommended of the manufacturer. After incubation, 5 µl of the ligation mixture was incubated at 70 ° C for 10 min, cooled to room temperature and used to transform competent cells of E. coli OH5alpha, as recommended by the manufacturer. Plasmid DNA was isolated from ampicillin-resistant transformants and the presence of 2.8 kb Sphl / BamHI insert was confirmed by restriction analysis. The plasmid was designated as pSE1 18. The inserted DNA in pSE1 18 and pSE1 19 overlapped with approximately 838 nucleotides (Figure 4).

Protoplasts of S. avermitilis strain 1100SC38 were transformed with pSEl 18 as above. Thiostrepton resistant transformants of strain 110-SC38 were isolated and analyzed by HPLC analysis of fermentation products. Transformants of S. avermitilis strain 1100-SC38 containing pSRl 18 had no change in the ratio of avermectin cyclohexyl-B2: avermectin cyclohexyl-B1 compared to strain 1100-SC38 (Table 2).

PCR amplification of aveC gene from S. avermitilis chromosomal DNA A ~ 1.2 kb fragment containing 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. PCR primers are supplied by Genosys Biotechnologies, Inc. (Texas). Right-standing primer:

5'-TCACGAAACCGGACACAC-3 '(SEQ ID NO: 6);

and the left-handed primer is:

5-CATGATCGCTGAACCGAG-3 '(SEQ ID NO: 7).

The PCR reaction was performed with Deep Vent ™ polymerase (New England Biolabs) in buffer supplied by the manufacturer and in the presence of 300 pm dNTP, 10% glycerol, 200 pmol of each primer, 0.1 pg template and 2.5 units of enzyme in a final volume of 100 pl using a PerkinElmer Cetus thermal cycler. The thermal profile of the first cycle is 95 ° C for 5 min (denaturation step), 60 ° C for 2 min (hybridization step) and 72 ° C for 2 min (elongation step). The next 24 cycles have a similar thermal profile except that the denaturation step is reduced to 45 s and the hybridization step is reduced to 1 min.

The PCR product was electrophoresed on a 1% agarose gel and the single stranded DNA band was determined to be ~ 1.2 kb. This DNA was gel purified and ligated with a 25 ng linear pCR-Blunt vector with blunt edges (Invitrogen) in a 1:10 vector-to-insert molar ratio as recommended by the manufacturer. The ligation mixture was used to transform One Shot ™ Competent E. coli cells (Invitrogen) according to the manufacturer's recommendations. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of 1,21.2 Kb insert was confirmed by restriction analysis. The plasmid is designated PSE179.

The DNA insert of pSEl 79 was isolated by digestion with BamHI / Xbal, separated by electrophoresis, gel purified and ligated with a vector shuttle pWHM3, which was also digested with BamHI / Xbal, at a total DNA concentration of 1 pg in molar vector-insert ratio 1: 5. The ligation mixture was used to transform competent E. coli DH5alpha cells as recommended by the manufacturer. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of 1,21.2 Kb insert was confirmed by restriction analysis. The plasmid; referred to as pSE186 (Figure 2, ATCC 209604), was transformed into E. coli DM1, and plasmid DNA was isolated from ampicillin-resistant transformants.

The result

The 2.9 Kb SacI / BamHI fragment of pSEl 19 was determined such that when transformed into S. avermitilis strain 1100-SC38, it significantly altered the B2 .B 1 ratio of avermectin produced. 5 S. avermitilis strain 11OO-SC3 8 typically has a B2: B1 ratio of about 30: 1, but when transformed with a vector containing a 2.9 Kb SacI / BamHI fragment, the B2: B1 ratio of avermectin decreases to about 3, 7: 1. Post-fermentation analysis of transformant cultures confirms the presence of transforming DNA.

The 2.9 Kb pSEl 19 fragment was sequenced and identified ~ 0.9 KB ORF (Figure 1) (SEQ ID NO: 1), which included a Pstl / SphI fragment that had previously been mutated to produce only B2 products (Ikeda et al., 1995, supra). Comparison of this ORF, or its corresponding derived polypeptide, against a known database (GenEMBL, SWISS-PROT) does not show any strong homology with known DNA or protein sequences.

Table 2 presents the fermentation analysis of S. avermitilis strain 1100-SC3 8 transformed with different plasmids.

Table 2

S. avermitilis strain No.Tested On average (transforming transformants ratio plasmid) on B2: B1 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

Example 8. 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 the Streptomyces gene is described by Kieser and Hopwood, 1991, Meth. Enzym. 204: 430458. A more detailed description is provided by Anzai et al., 1988, J. Antibiot. XLI (2): 226-233, and from Stutzman-Engwall et al., 1992, J. Bacteriol. 174 (1): 144-154. These references are incorporated herein by reference.

Inactivation of the aveC gene of S. avermitilis

AveC mutants containing the aveC genes were constructed using the various methods described below.

In the first method, the 640 bp Sphl / PstI fragment fragment internal to the aveC gene in pSEl 19 (plasmid described in Example 7 above) was replaced with the exSe gene (erythromycin resistance) of Sacc. erytraea. The ermE gene was isolated from plJ4026 (provided by John Innes Institute, Norwick, UK; see also Bibb etal., 1985, Gene 41: 357-368) by restriction enzyme Bg / II and EcoRI followed by electrophoresis and purified from the gel. This ~ 1.7 K fragment was ligated into pGEM7Zf (Promega), which was digested with BamHI and EcoRI, and the ligation mixture was transformed into E. coli OH5alpha cells according to the manufacturer's recommendations. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of 1,71.7 Kb insert was confirmed by restriction analysis. Plasmid is referred to as pSE27.

pSEl 18 (described in Example 7 above) was digested with SphI and BamHI, electrophoresed, and ~ 2.8 kb Sphl / BamHI insert was gel purified. pSE1 19 was digested with PstI and EcoRI, electrophoresed, and 1,51.5 kb of PstI / EcoRI insert was gel purified. The vector shuttle pWHM3 was ground with BamHI and EcoRI. pSE27 was digested with PstI and SphI, electrophoresed, and и1.7 kb Pstl / SphI insert was gel purified. All four fragments (i.e., ~ 2.8 kb, ~ 1.5 kb, ~ 7.2 kb, ~ 1.7 kb) are ligated together in four-way ligation. The ligation mixture was transformed into competent E. coli OH5alpha cells following the manufacturer's recommendations. Plasmid DNA was selected from ampicillin-resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid was designated pSE180 (Figure 3; ATCC 209605).

pSEl80 was transformed into S. lividans TK64, and the transformed colonies were recognized by thiostrepton resistance and erythromycin resistance. pSE180 is isolated from S. lividans and used to transform protoplasts from S. avermitilis. Four thiostrepton-resistant S. avermitilis transformants were identified and protoplasts were prepared by seeding under non-selective conditions on RM14 medium. After regeneration of the protoplasts, single colonies are screened for erythromycin resistance and lack of thiostrepton resistance, which means chromosomal integration of the inactivated aveC gene and loss of free replicon. One Erm 'Thio ^s trans formant is identified and designated SE18 ΟΙ 1. Total chromosomal DNA is isolated from strain SE 180-11, digested with restriction enzymes BamHI, HindIII, PstI or SphI, separated by electrophoresis at 0.8 % agarose gel, transformed onto nylon membranes and hybridized to an ermE sample. This analysis shows that the chromosomal integration of the ermE resistance gene and the simultaneous deletion of the 640 bp Pst1 / SphI fragment is achieved by a double crossover event. HPLC analysis of fermentation products from strain SE 180-11 showed that normal avermectins were no longer produced (Figure 5A).

In a second method of inactivating the aveC gene, the 1.7 kb ermE gene is removed from the chromosome of S. avermitilis strain SE180-11, leaving a 640 bp Pstl / SphI deletion in the aveC gene. A plasmid was designed to replace the gene as follows: pSEl80 was partially digested with Xbal and a ~ 11.4 kb fragment of the gel was purified. The -11.4 kb band lacks the 1.7 kb ermE resistance gene. The DNA was then ligated and transformed into E. coli OH5alpha cells. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. The plasmid referred to as pSE184 was transformed into E. coli DM1 cells and the plasmid DNA was isolated from ampicillin resistant transformants. This plasmid was used to transform protoplasts of S. avermitilis strain SE180-11. The protoplasts are prepared from transformants of strain SE180-11 resistant to thiostrepton and sown as single colonies on RM14 medium. After protoplast regeneration, single colonies are screened for the absence of both erythromycin resistance as well as thiostrepton resistance, indicating chromosomal integration of the aveC gene and loss of the free replicon containing the ermE gene. An Erm ^s Thio ^s transformant is identified and designated SE184-1-13. The fermentation analysis of SE184-1-13 has the same fermentation profile as SE 18 0-11.

In a third method of inactivating the aveC gene, a frame is introduced into the chromosomal aveC gene by adding two G after C at nucleotide position 471 using PCR, thereby creating a BspE1 site. The presence of the designed BspEl site is used to detect gene replacement events. PCR primers are designed to introduce a frame mutation into the aveC gene, and are supplied by Genosys Biotechnologies, Inc. The right-standing primer is:

5'-GGTTCCGGATGCCGTTCTCG-3 '(SEQ ID NO: 8), and the left primer is:

5'-AACTCCGGTCGACTCCCCTTC-3 '(SEQ ID NO: 9). The PCR conditions are described in Example 7 above. The 666 bp PCR product was digested with SphI 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: vector shuttle pWHM3 was digested with EcoRI and BamHI. pSE 119 was digested with BamHI and

SphI, electrophoresed, and a ~ 840 bp fragment was purified from bodies. pSE1 19 was digested with EcoRI and XmnI, separated by electrophoresis, and a ~ 1.7 kb fragment was purified from the gel. All four fragments (i.e., ~ 7.2 kb, ~ 840 kb, ~ 1.7 kb, and 388 bp) were ligated together by four-way ligation. The ligation mixture was transformed into competent E. coli OH5alpha cells. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the correct insert was confirmed by restriction analysis and DNA sequencing analysis. This plasmid was designated pSE 185, transformed into E. coli DM 1, and isolated plasmid DNA from ampicillin resistant transformants. This plasmid was used to transform protoplasts of S. avermitilis strain 1100-SC38. Transformants of strain 1100-SC38 resistant to thiostrepton were isolated and analyzed by HPLC analysis of fermentation products. pSE 185 did not significantly alter the B2: B 1 ratio of avermectin when transformed into S. avermitilis strain 1100-SC38 (Table 2).

pSEl 85 is used to transform protoplasts of S. avermitilis to achieve a frame mutation in the chromosomal aveC gene. Protoplasts are prepared from thiostrepton-resistant transformants and sown as single colonies on RM14 medium. After regeneration of the protoplasts, single columns are screened for the absence of thiostrepton resistance. Chromosomal DNA was isolated from thiostrepton-sensitive colonies and screened by PCR for the presence of a frame mutation integrated into the chromosome. PCR primers were constructed on the basis of an aveC nucleotide sequence and provided by Genosys Biotechnologies, Inc. (Texas). The right-handed PCR primer is:

5'-GCAAGGATACGGGGACTAC-3 '(SEQ ID NO: 10), and the left PCR primer is:

5'-GAACCGACCGCCTGATAC-3 '(SEQ ID NO: 11) as PCR conditions are the same as in Example Ί above. The resulting PCR product was 543 bp and, when digested with BspE 1, three fragments of 368 bp, 96 bp and 79 bp were observed, which means chromosomal integration of the inactivated aveC gene and loss of the free replicon.

Fermentation analysis of S. avermitilis mutants including the frame mutation in the aveC gene shows that normal avermectins are no longer produced, and that these mutants have the same fermentation HPLC profile as strains SE180-11 and SE184-1-13. One Thio ^s trans formant is identified and designated as strain SE185-5a.

Additionally, a mutation in the aveC gene is obtained that changes nucleotide position 520 from G to A, resulting in a change in the codon encoding tryptophan (W) at position 116 in a terminal codon. One strain of S. avermitilis with this mutation does not produce normal avermectins and has the same fermentation profile as strains SE180-11, SE184-1-13 and SE185-5a.

In addition, mutations in the aveC gene are obtained that alter both: (i) nucleotide position 970 from G to A, resulting in a change in the amino acid at position 256 from glycine (G) to aspartate (D), and (ii) nucleotide position 996 of T to C, which changes the amino acid at position 275 from tyrosine (Y) to histidine (H). One strain of S. avermitilis with these mutations (G256D / Y275H) does not produce normal avermectins and has the same fermentation profile as strains SE180-11, SE184-1-13 and SE185-5a.

Inactivating an aveC mutant of S. avermitilis strains SE180-11l, SE184-l-13HSE185-5aH others presented herein provides a screening tool for determining the effects of other mutations in the aveC gene. pSE186 containing a wild-type copy of the aveC gene was transformed into E. coli DM 1, and plasmid DNA was isolated from ampicillin-resistant transformants. This pSE186 DNA was used to transform protoplasts of S. avermitilis strain SE18011. Thiostrepton-resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and Tio ^r Erm ^r transformants were analyzed by HPLC analysis of fermentation products. The presence of the functional aveC gene in trans makes it possible to restore strain SE 180-11 to produce normal avermectin (Figure 5B).

Analysis of mutations in the aveC gene that alter the class B2: B 1 ratio

As described above, S. avermitilis strain SE180-11 containing an inactivated aveC gene was complemented with a plasmid containing a functional aveC gene (pSE186). Strain SE180-11 is also used as a host strain to harbor other mutations in the aveC gene as described above.

Chromosomal DNA from strain 1100-SC38 was isolated 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 an aveC nucleotide sequence. The right-hand primer is SEQ ID NO: 6, and the left-hand primer is SEQ ID NO: 7 (see example 7 above). The PCR and subcloning conditions are described in Example 7. DNA sequencing analysis of 1.2 kb ORF shows a mutation in the aveC gene that changes nucleotide position 337 from C to T, which changes the amino acid at position 55 of the (S) phenylalanine series (F). the aveC gene containing the S55F mutation was subcloned into pWHM3 to produce a plasmid, designated pSEl 87, which was used to transform S. avermitilis protoplasts strain SE18O-11. Thiostrepton-resistant transformants were isolated, the presence of erythromycin resistance was determined and Tio ^r Erm ^r was analyzed by HPLC analysis of the fermentation products. The presence of an aveC gene encoding a change in amino acid residue 55 (S55F) made it possible to restore the normal production of avermekgin in strain SE180-11 (Figure 5C); however, the ratio of cyclohexyl B2: cyclohexyl B1 is approximately 26: 1, compared with strain SE18O-11 transformed with pSE186, which has a B2: B1 ratio of approximately 1.6: 1 (Table 3), which means that the single mutation (S55F) modulates the amount of cyclohexyl B2 produced relative to cyclohexyl B2.

Another mutation in the aveC gene is identified that changes nucleotide position 862 from G to A, which changes the amino acid at position 230 from glycine (G) to aspartate (D). One strain of S. avermitilis possessing this mutation (G230D) produces avermectins at a B2: B1 ratio of approximately 30: 1.

Mutations that reduce the B2: B1 ratio

Some mutations have been constructed that reduce the amount of cyclohexyl B2 produced relative to cyclohexyl B2 as follows.

A mutation in the aveC gene is identified which alters nucleotide position 5 88 of G to A, which changes the amino acid at position 139 from alanine (A) to threonine (T). The AveC gene containing the A139T mutation was subcloned into pWHM3 to produce a plasmid, designated pSE 188, which was used to transform S. avermitilis protoplasts strain SE180-11. Thiostrepton-resistant transformants were isolated, the presence of erythromycin resistance was determined, and Thio ^r Erm ^r transformants were analyzed by HPLC analysis of fermentation products. The presence of a mutated aveC gene encoding a change in amino acid residue 139 (A139T) is able to restore strain SE180-11 (Figure 5D) to produce avermectin; however, the B2: B 1 ratio is approximately 0.94: 1, which means that this mutation reduces the amount of cyclohexyl B2 produced by the cyclohexyl B1 ratio. The result is unexpected, as the published results, as well as the mutation results described above, only show either inactivation of the aveC gene or increased production of the B2 form of avermectin by the ratio of the B1 form (Table 3).

As the A139T mutation alters the ratio of B2: B1 in a more favorable direction to B1, a mutation is constructed that encodes threonine instead of series at amino acid position 138. In this case, pSE186 is digested with EcoRI and cloned into pGEEM3Zf (Promega). was digested with EcoRI. This plasmid, designated pSE 18 6 A, was digested with Apal and ΚρηΙ, the DNA fragments were separated on an agarose gel, and two fragments of -3.8 kb and ~ 0.4 kb were purified from the gel. The ~ 1.2 kb inserted DNA from pSE186 was used as a PCR template to introduce a single base change at nucleotide position 585. The PCR primers were designed to introduce a mutation at nucleotide position 585 and were supplied by Genosys Biotechnologies, Inc. (Texas). The right-handed PCR primer is:

5'-GGGGGCGGGCCCGGGTGCGGAGG CGGAAATGCCCCTGGC-GACG -3 '(SEQ ID NO: 12); and the left PCR primer is:

5-GGAACCGACCGCCTGATACA-3 '(SEQ ID NO: 13). The PCR reaction was performed using an Adventage GC genomic PCR kit (Clontech Laboratories, Palo Alto, CA) in buffer provided by the manufacturer in the presence of

200 μΜ dMTPs, 200 pmol from each primer, 50 ng DNA template, 1.0 M GC-Melt, and 1 unit of KlcnTaq Polymerase Mix in a final volume of 50 μΐ. The thermal profile of the first cycle is 94 ° C for 1 min; followed by 25 cycles at 94 ° C for 30 s and 68 ° C for 2 min; and one cycle at 68 ° C for 3 min. The 295 bp PCR product was ground with Apal and ΚρηΙ to release a 294 bp fragment, which was separated by electrophoresis and gel purified. All three fragments (~ 3.8 kb, ~ 0.4 KB and 254 bp) were ligated together in three-way ligation. The ligation mixture was transformed into competent E. coli OH5alpha cells. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. The plasmid was designated pSE198.

pSE198 was digested with EcoRI, cloned into pWHM3, which was digested with EcoRI and transformed into E. coli OH5alpha cells. Plasmid DNA is isolated from ampicillin-resistant transformants and the presence of the correct insert is confirmed by restriction and DNA sequence analysis. This plasmid DNA was transformed into E. coli DM 1, plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. The plasmid referred to as pSE199 was used to transform protoplasts of S. avermitilis strain SE 180-11. Thiostrepton-resistant strain SE 180-11 transformants were isolated, erythromycin resistance was determined, and Thio ^r Erm ^r transformants were analyzed by HPLC analysis of the fermentation products. The presence of a mutated aveC gene encoding a change in amino acid residue 138 (S138T) allows the restoration of strain AE 180-11 to restore normal production of avermectin; however, the B2: B1 ratio was 0.88: 1, indicating that this mutation reduced the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1 (Table 3). This B2: B1 ratio is even lower than the 0.94: 1 ratio observed with the A139T mutation produced by transformation of strain SE180-11 with pSE188 as described above.

Another mutation was constructed to introduce threonine at two amino acid positions 138 and 139. A 1.2 kb DNA insert from pSE 186 was used as a PCR template. PCR primers are designed to introduce mutations at nucleotide positions 585 and 588 and are supplied by Genosy's Biotechnologies, Inc. (Texas). The right-handed PCR primer is:

5-GGGGGCGGGCCCGGGTGCGGAGG CGGAAATGCCGCTGG-CGACGACC - 3 '(SEQ ID NO: 14); and the left PCR primer is:

5'-GGAACATCACGGCATTCACC-3 '(SEQ ID NO: 15). The PCR reaction was performed under the conditions described immediately above in this section. The PCR product of 449 bp was digested with Apal and ΚρηΙ to release a 294 bp fragment, which was separated by electrophoresis and purified from the gel. pSEl86a was digested with Apal and ΚρηΙ, the DNA fragments were separated on an agarose gel, and two fragments of ~ 3.8 kb and ~ 0.4 kb were purified from the gel. All three fragments (~ 3.8 kb, ~ 0.4 kb and 254 bp) were ligated together in three-way ligation and the ligation mixture was transformed into competent E. coli OH5alpha cells. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid is referred to as pSE230.

PSE230 was digested with EcoRI, cloned into pWHM3, which was digested with EcoRI and transformed into E. coli OH5alpha cells.

Plasmid DNA is isolated from ampicillin-resistant transformants and the presence of the correct insert is confirmed by restriction 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. The plasmid referred to as pSE281 was used to transform protoplasts of S. avermitilis strain SE 180-11. Thiostrepton-resistant SE 180-11 transformants were isolated, the presence of erythromycin resistance was determined, and Thio ^r Erm ^r transformants were analyzed by fermentation. The presence of a double mutated aveC gene encoding S138T / A139T makes it possible to restore to strain SE18011 the normal production of avermectin; however, the B2: B1 ratio is 0.84: 1, which35 indicates that this mutation further reduces the amount of cyclohexyl-B2 produced relative to cyclohexyl-Bl (Table 3) over the production provided by transformation of strain SE180 -11 with pSE188 or pSEI99 as described above.

Table 3

strain of S. avermitilis No.Tecr- Relationships- Relationships- On average (transforming baths body body correlation- plasmid) trans- conc. conc. will of formants on B2 on B1 B2: B1 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 (pSE189) 18 155 171 0.88 SE180-11 (pSE231) 6 259 309 0.84

These results are the first to show specific mutations in the aveC gene that lead to the production of increased levels of commercially preferred class 1 avermectins over class 2 avermectins.

Example 9. Construction of 5 'deletion mutants

As explained above, the nucleotide sequence of S. avermitilis shown in Figure 1 (SEQ ID NO: 1) includes four different GTG codons at the 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 facilitate the definition of which of these codons could function as sites of origin in the aveC ORF for protein expression .

PCR amplification isolated fragments from the aveC gene from chromosonal DNA of S. avermitilis, differently delegated at the 5 'end. PCR primers were constructed based on the aveC DNA sequence and supplied by Genosys Biotechnologies, Inc. Right-standing primers are:

5'-AACCCATCCGAGCCGCTC-3 '(SEQ ID NO: 16) (D1F1);

5'-TCGGCCTGCCAACGAAC-3 '(SEQ ID NO: 17) (D1F2);

5'-CCAACGAACGTGTAGTAG-3 '(SEQ ID NO: 18) (D1F3); and

5'-TGCAGGCGTACGTGTTCAGC-3 '(SEQ ID NO: 19) (D2F2).

Left-handed primers are:

5′-CATGATCGCTGAACCGA-3 ′ (SEQ ID NO: 20);

5'-CATGATCGCTGAACCGAGGA-3 '(SEQ ID NO: 21); and

5'-AGGAGTGTGGTGCGTCTGGA-3 '(SEQ ID NO: 22).

The PCR reaction was performed as described in Example 8 above.

The PCR products were separated by electrophoresis on a 1% agarose gel and single DNA bands were detected either at ~ 1.0 kb or ~ 1.1 kb. PCR products were gel purified and ligated with 25 ng linearized pCR2.1 vector (Invitrogen) at a 1:10 molar ratio, following the manufacturer's recommendations. The ligation mixture was used to transform One Shot ™ competent E. coli cells (Invitrogen) according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin-resistant transformants and the presence of the insert was confirmed by restriction and DNA sequence analysis. Plasmids are referred to as pSE190 (obtained with primer D1F1), pSE191 (obtained with primer 10 D1F2), pSE192 (obtained with primer D1F3), and pSE193 (obtained with primer D2F2).

Each inserted DNA was digested with BamH / Xbal, separated by electrophoresis, purified by gel and separately ligated with vector 15 shuttle pWHM3, which was digested with BamHI / Xbal, at a total DNA concentration of 1 pg at a molar ratio of 1: 5 of Insert Vector. Ligation mixtures are used to transform competent E. coli OH5alpha cells. Plasmid DNA was isolated from ampicillin-resistant transformants and the presence of the insert was confirmed by restriction analysis. These plasmids, designated as pSE194 (D1F1), pSE195 (D1F2), pSE196 (D1F3) 25 and pDE197 (D2F2), were transformed separately into E. coli strain DM1. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This DNA is used to transform protoplasts of S. avermitilis strain SE180-11. Thiostrepton-resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and Thio ^r Erm ^r transformants were analyzed by HPLC analysis of fermentation products to determine which GTG sites were required for aveC expression. The results show that the GTG codon at position 42 can be eliminated without affecting aveC expression, while pSE194, pSE195, and pSE196, each of which lacks the GTG site at position 42, but which all contain the three GTG sites at positions 174, 177 and 180, all sp20 are capable of restoring the normal production of avermectin when transformed into SE180-11. Normal production of avermectin did not recover when strain SE180-11 was transformed with pSE197, which lacked all four of the GTG sites (Table 4).

Table 4

strain of S. avermitilis No.Tecr- Relationships- Relationships- On average (transforming baths tel at tel at correlation- plasmid) trans- conc. conc. will of formants on B2 on B1 B2: B1 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 (pSE297) 12 0 0 0

Example 10. Cloning of aveC homologues from S. higroscopicus and S. griseochromogenes

The present invention enables the identification and cloning of aveC homologous genes from other types of Streptomyces producing avermectin or milbemycin. For example, genomic DNA from a S. 5 0 hygroscopicus cosmid library (FERM BP-1901) was hybridized with a 1.2 kb aveC sample of S. avermitilis described above. Several cosmid clones that strongly hybridize are identified. Chromosonal DNA was isolated from these cosmids and a 4.9 kb ΚρηΙ fragment was identified that hybridized to the aveC sample. This DNA is sequenced and an ORF (SEQ ID NO: 3) is identified that has significant homology with the aveC ORF of

S. avermitilis. Figure 6 shows an amino acid sequence (SEQ ID NO: 4) derived from an aveC homologous ORF of S. hygroscopicus.

In addition, genomic DNA from a S. griseochromogenes cosmid library hybridized with a 1.2 kb aveC sample of S. avermitilis described above. Several cosmid clones that strongly hybridize are identified. Chromosomal DNA was isolated from these cosmids and a 5.4 kb PstI fragment was identified that hybridized to the aveC sample. This DNA is sequenced and an ORF partial aveC homolog is identified that has significant homology to the S. avermitilis aveC ORF. Figure 6 shows the deduced partial amino acid sequence (SEQ ID NO: 5).

DNA and amino acid sequence analysis of the aveC homologs of S. hygroscopicus and S. griseochromogenes show that these regions share significant homology (~ 50% amino acid sequence identity) to each other and to the aveC ORF of S. avermitilis and to the aveC gene product (Figure 6).

Example 11. Construction of a plasmid with the aveC gene after the eppE promoter

The 1.2 kb aveC ORF of pSE186 is subcloned into pSE34, which is the vector shuttle pWHM3 that has a 300 bp eppE promoter inserted as a KpnI / BamHI fragment into the KpnI / BamHI site of pWHM3 (see Ward et al., 1986, Mol. Gen. Gen. 203: 468-478). pSE186 was digested with BamHI and HindIII, separated by electrophoresis, and a 1.2 kb fragment of agarose gel was isolated and ligated with pSE34, which was digested with BamHI and HindIII. The ligation mixture was transformed into competent E. coli OH5alpha cells according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of 1.2 kb inserts was confirmed by restriction analysis. The plasmid referred to as pSE 189 was transformed into E. coli DM 1 and plasmid DNA was isolated from ampicillin resistant transformants. S. avermitilis protoplasts of strain 1100-SC38 were transformed with pSEl 89. Thiostrepton resistant transformants isolated from strain 1100SE38 were analyzed by HPLC analysis of fermentation products.

S. avermitilis strain 1100-SC38 transformants containing pSE 189 undergo a change in the ratio of avermectin cyclohexyl-B2 produced: avermectin cyclohexyl-B1 (approximately 3: 1) compared to strain 1100SC38 (approximately 34: 1), and total avermectin production increased approximately 2.4-fold compared to strain 1100-SC38 transformed with pSEl 19 (Table 5).

pSE189 was also transformed into protoplasts of S. avermitilis wild-type strain. Thiostrepton-resistant transformants were isolated and analyzed by HPLC analysis of fermentation products. The total amount of avermectins produced by S. avermitilis wild-type transformed with pSE189 increased approximately 2.2-fold compared to S. avermitilis wild-type transformed with pSE 119 (Table 5).

Table 5

S. avermitilis strain (transforming plasmid) No. Transformed Transformers Relative [B2] Relative [B1] Relative general Avermectins Average B2: B1 ratio 1100-SC38 6 155 4,8 176 33,9 1100-SC38 (pSE119) 6 239 50.3 357 4.7 SE180-11 (pSE189) 16 546 166 849 S.S. wild type 6 59 42 113 1.41 wild type (PSE119) 6 248 151 481 1.64 wild type (PSE189) 5 545 345 1,071 th most common 1.58

Example 12. Chimeric plasmid containing sequences of aveC ORF of S. avermitilis and aveC homolog of S. hygroscopicus

A hybrid plasmid designated pSE350 was constructed that contained a 564 bp aveC homologous region of S. hygroscopicus replacing the 564 bp homologous region of aveC ORF of S. avermitilis (Figure 7) as follows. pSE 350 was constructed using the BsaAI restriction site, which is conserved at both 35 sequences (aveC position 225), and the ΚρηΙ restriction site present in the aveC gene of S. avermitilis (aveC position 810). The ΙρηΙ site was introduced into S. hygroscopicus DNA by PCR using a right-handed primer 5′- 40 CTTCAGGTGTACGTGTTCG-3 ′ (SEQ ID NO: 23) and a left-handed primer 5′-GAACTGGTACCAGTGC CC-3 ′ (SEQ ID NO: 24 ) (supplied by Genosys Biotechnologies) using the PCR conditions described in Example 7 above. The PCR product was ground with BsaAI and ΚρηΙ, the fragments were separated by electrophoresis into a 1% agarose gel, and 564 bp BsaAI / ΚρηΙ fragment isolated from the gel. pSE179 (described in Example 7 above) was digested with ΚρηΙ and HindIII, the fragments were separated by 5 O electrophoresis in a 1% agarose gel and a ~ 4.5 kb fragment was isolated from the gel. pSE179 was digested with HindIII and BsaAI, the fragments were separated by electrophoresis into a 1% agarose gel and 30 ~ 0.2 kb BsaAI / HindIII fragment was isolated from the gel. The ~ 4.5 kb Hindlll / Kpnl, ~ 0.2 kb BsaAI / Hindlll and 564 bp BsaAI / ΚρηΙ fragments of S. hygroscopicus were ligated together into three-way ligation and the ligation mixture was transformed into competent E. coll DH5 alpha cells. Plasmid DNA was isolated from ampicillin-resistant transformants and the presence of the correct insert was confirmed by restriction analysis using ΚρηΙ and Aval. This plasmid was digested with HindIII and Xbal to release 1.2 kb of insert, which was then ligated with pWHM3, which was digested with HindIII and Xbal. The ligation mixture was transformed into competent E. coli OH5alpha cells, plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis using HindIII and Aval. This plasmid DNA was transformed into E. coli DM 1, plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the correct insert was confirmed by restriction and DNA sequence analysis. This plasmid is designated pSE35O and is used to transform protoplasts of S. avermitilis strain SE18O-11. Thiostrepton resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and Thio ^r Epp ^r transformants were analyzed by HPLC analysis of the fermentation products. The results show that S. avermitilis / S. hygroscopicus hybrid plasmids have an average B2: B1 ratio of approximately 109: 1 (Table 6).

Table 6

strain of S. avermitilis No. Test- Relationships- Relationships- On average (transforming baths tel at tel at correlation- plasmid) trans- conc. conc. sion of formants on B2 on B1 B2: B1 SE180-11 (none) 8 0 0 0 SE180-11 (pWHM3) 8 0 0 0 SE180-11 (pSE350) 16 233 2 109

Deposition of biological materials

The following biological material was deposited on 25 wounds at the American Type Culture Collection (ATCC) at 12301 Parklawn Drive, Rockville, MD, 20852, USA on 29/01/1998 and received the following entries

Incoming number

209605

209604 current numbers:

Plasmid

Plasmid pSE 180

Plasmid pSEl 86

All patents, patent applications and cited publications are incorporated herein by reference. 3 5

The scope of the present invention is not limited to the specific embodiments described herein, which are presented solely to illustrate individual aspects of the invention. In fact, from the foregoing descriptions and the accompanying figures, many other various modifications will be apparent to those skilled in the art, in addition to those shown and described herein. Such modifications are considered to fall within the scope of the appended claims.

Claims (53)

Claims 1. An isolated polynucleotide molecule, 5 0 comprising the complete aveC open reading frame (ORF) from Streptomyces avermitilis, or a substantial portion thereof, to which an isolated polynucleotide molecule lacks the next complete ORF downstream of the aveC ORF in situ the chromosome of Streptomyces avermitilis, and whose ORF has the nucleotide sequence of Figure 1 (SEQ ID NO: 1). An isolated polynucleotide molecule according to claim 1, characterized in that it further includes nucleotide sequences that typically limit the aveC gene in situ in S. avermitilis. An isolated polynucleotide molecule having a nucleotide sequence that is homologous to an aveC product encoding a nucleotide sequence from Streptomyces avermitilis of the aveC ORF shown in Figure 1 (SEQ ID NO: 1) or a substantial portion thereof. An isolated polynucleotide molecule comprising the nucleotide sequence of SEQ ID NO: 3 or a substantial portion thereof, or a nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO: 3. An oligo nucleotide molecule that hybridizes to the polynucleotide molecule of Figure 1 (SEQ ID NO: 1) or to SEQ ID NO: 3, or a polynucleotide molecule having a nucleotide sequence complementary to the nucleotide sequence of Figure 1 (SEQ ID NO: 1) ) or SEQ ID NO: 3, under very stringent conditions. An oligonucleotide molecule according to claim 5, characterized in that it is complementary to the nucleotide sequence of Figure 1 (SEQ ID NO: 1) or SEQ ID NO: 3, or to a polynucleotide molecule having a nucleotide sequence that is complementary to the nucleotide the sequence of Figure 1 (SEQ ID NO: 1) or SEQ ID NO: 3. A recombinant vector comprising a polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of the aveC ORF of Figure 1 (SEQ ID NO: 1) or a substantial portion thereof; and a nucleotide sequence homologous to the S. avermitilis-producing ORF aveC gene of Figure 1 (SEQ ID NO: 1) or a substantial portion thereof. The recombinant vector of claim 7, further comprising a nucleotide sequence encoding one or more regulatory elements, which regulatory element encoding the nucleotide sequence is operatively linked to the nucleotide sequence of the polynucleotide molecule of the vector of claim 7. The recombinant vector of claim 8, further comprising a nucleotide sequence encoding a selection marker. The recombinant vector of claim 9, which is plasmid pSE186 (ATCC 209604). A host cell comprising the recombinant vector of claim 9. A host cell according to claim 11, characterized in that it is Streptomyces avermitilis. A recombinant vector comprising a polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 3 or a substantial portion thereof; a nucleotide sequence homologous to a nucleotide sequence of SEQ ID NO: 3; and a nucleotide sequence that encodes a poly peptide having the amino acid sequence of SEQ ID NO: 4. A recombinant vector according to claim 13, further comprising a nucleotide sequence encoding one or more regulatory elements, which nucleotide sequence encoding regulatory elements is operatively linked to the nucleotide sequence of the polynucleotide molecule of the vector of claim 13 . 15. The recombinant vector of claim 14, further comprising a nucleotide sequence encoding a selection marker. A host cell comprising the recombinant vector of claim 15. A host cell according to claim 16, characterized in that it is Streptomyces hygroscopicus. 18. An isolated aveC gene product of S. avermitilis having an amino acid sequence encoded by S. avermitilis aveC gene producing the nucleotide sequence of plasmid pSEl86 (ATCC 209604), or the amino acid sequence of SEQ ID NO: 2 or 2 polypeptide. 19. An isolated homologous aveC gene product of S. hygroscopicus having the amino acid sequence of SEQ ID NO: 4. 20. A method of producing a recombinant aveC gene product, characterized in that host cells transformed with the recombinant expression vector are recited, said recombinant expression vector comprising a polynucleotide molecule having the nucleotide sequenceQ of the nucleotide sequence : 2 or a substantial portion thereof, which is a polynucleotide molecule operatively linked to one or more regulatory elements that control the expression of the polynucleotide molecule and 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. 21. A method for producing a recombinant aveC homologous gene product, characterized in that the cells are cultured by a state expression vector, said recombinant expression vector comprising a polynucleotide molecule having a nucleotide sequence encoding the amino acid sequence N or the amino acid sequence NQQ a portion of which a polynucleotide molecule is operatively linked to one or more regulatory elements that control the expression of the polynucleotide molecule in the host cell, when catching leading to the production of the recombinant Aues homologous gene product, and obtaining the AveC homologous gene product from cell culture. 22. A polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as the coding product sequence of the aveC gene of S. avermitilis from plasmid pSE 186 (ATCC 209604) or the nucleotide sequence of aveC ORF of S. avermitilis, as shown in Figure 1 (Figure 1) SEQ ID NO: 1) or a variant thereof of the genetic code, but which nucleotide sequence further includes one or more mutations such that S. avermitilis cells strain ATCC 53692 in which the wild-type aveC appeal has been inactivated and which express a polynucleotide molecule comprising the mutated nucleotide sequence produce a reduced ratio of cyclohexyl B2: cyclohexyl B1 avermectins, when fermented in the presence of cyclohexanecarboxylic acid than produced cells of S. avermitilis strain ATCC 53692 that instead express only an aveC allele from the wild type. A polynucleotide molecule according to claim 22, characterized in that the reduced ratio of cyclohexyl B2: cyclohexyl B1 is less than 1.6: 1. A polynucleotide molecule according to claim 23, characterized in that the reduced ratio of cyclohexyl B2: cyclohexyl B1 is less than 0.94: 1. A polynucleotide molecule according to claim 24, wherein the mutation of the aveC ORF of S. avermitilis of Figure 1 (SEQ ID NO: 1) encodes a substitution of an amino acid residue 139 of the aveC gene product from alanine to threonine. A polynucleotide molecule according to claim 23, characterized in that the reduced ratio of cyclohexyl B2: cyclohexyl B1 is approximately 0.88: 1. A polynucleotide molecule according to claim 26, wherein the mutation of the aveC ORF of S. avermitilis of Figure 1 (SEQ ID NO: 1) encodes a substitution of an amino acid residue 138 of the aveC gene product from serine to threonine. A polynucleotide molecule according to claim 23, characterized in that the reduced ratio of cyclohexyl B2: cyclohexyl B1 is approximately 0.84: 1. A polynucleotide molecule according to claim 28, characterized in that the mutation of the aveC ORF of S. avermitilis of Figure 1 (SEQ ID NO: 1) encodes a first substitution of an amino acid residue 138 of the aveC gene product from serine to threonine and one second substitution of the amino acid residue at position 139 of the aveC gene product from alanine to threonine. 30. A polynucleotide molecule comprising a strong promoter operatively linked to S. avermitilis aveC ORF of Figure 1 (SEQ ID NO: 1). A polynucleotide molecule according to claim 30, wherein the strong promoter is an eppE promoter of Saccharopolyspora erythraea. 32. A polynucleotide molecule comprising a nucleotide sequence encoding the ORF of the product encoding the aveC gene of Figure 1 (SEQ ID NO: 1), which has been inactivated by inserting into said nucleotide sequence a heterologous nucleotide sequence. 33. A polynucleotide molecule comprising an aveC allele that has been inactivated by delegation of a 640 bp Pstl / SphI fragment from the aveC ORF of Figure 1 (SEQ ID NO: 1). 34. A polynucleotide molecule comprising an aveC allele that has been inactivated by introducing a frame mutation into the aveC ORF of Figure 1 (SEQ ID NO: 1). A polynucleotide molecule according to claim 34, characterized in that the frame mutation is introduced by the addition of two G nucleotides after the C nucleotide at nucleotide position 471 on the aveC ORF of Figure 1 (SEQ ID NO: 1). 36. A polynucleotide molecule comprising an aveC allele that has been inactivated by inserting a terminal codon at a 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 inactivated by introducing a first mutation at amino acid position 256 of the aveC gene product that alters glycine to aspartate, and a second mutation at position 275 of the aveC gene product that alters tyrosine of histidine. 38. A gene replacement vector comprising a polynucleotide molecule comprising nucleotide sequences that limit the aveC ORF in situ in the chromosome of S. avermitilis. A recombinant vector comprising the polynucleotide molecule of any one of claims 22 to 3 7. A recombinant vector comprising a polynucleotide molecule according to claim 32, characterized in that it is pSEl80 (ATCC209605). 41. A Streptomyces host cell comprising the recombinant vector of claim 39. 42. A method for identifying an aveC ORF mutation capable of reducing the production of a 2: 1 ratio of avermekgins, characterized in that it includes: (a) determining the ratio of class 2: 1 avermectins produced by cells of a strain of S. avermitilis in which the native aveC allele is inactivated, and into which a polynucleotide molecule comprising a nucleotide sequence encoding a mutated aveC gene is introduced and expressed; b) determining the ratio of class 2: 1 avermectins produced by cells of the same strain of S. avermitilis as in step a) but which instead express only the aveC allele having the nucleotide sequence of the ORF of Figure 1 (SEQ ID NO: 1) or a homologous nucleotide sequence thereof; and c) comparing the class 2: 1 ratio of avermectins produced by S. avermitilis cells from step a) to the class 2: 1 ratio of avermectins produced by S. avermitilis cells from step b); so that if the ratio of class 2: 1 avermectins produced by S. avermitilis cells from step a) is lower than the ratio of class 2: 1 avermectins produced by S. avermitilis cells from step b), then a mutation of the aveC ORF capable of reducing the class 2: 1 ratio of avermectins is identified 43. A method for producing new strains of S. avermitilis, including cells that express the mutated aveC allele and that produce a reduced class 2: 1 ratio of avermectins, compared to cells of the same S. avermitilis strain that instead express only aveC wild-type allele, characterized in that cells of S. avermitilis strain are transformed with a vector that carries a mutated aveC allele encoding a gene product that reduces the class 2: 1 ratio of avermekgins produced by cells of strain S avermitilis expressing a mutated aveC allele compared to cells of the same strain, which instead express only the wild-type aveC allele, and select transformed cells producing avermekgins at a reduced class 2: 1 ratio, compared to the class 2: 1 ratio produced by the cells of the strain, which instead express the wild-type aveC allele. 44. A method of producing new strains of S. avermitilis whose cells contain an inactivated aveC allele, characterized in that cells of S. avermitilis are transformed with a vector that inactivates the aveC allele and selected transformed cells into which the aveC allele was inactivated. The method of claim 44, wherein the vector is pSE180 (ATCC 209605). 46. A strain of S. avermitilis comprising cells expressing a mutated aveC allele that results in the production of avermectin cells in a reduced 2: 1 ratio, compared to cells of the same strain that instead express only the aveC allele from wild type. The strain of claim 46, wherein the cells produce cyclohexyl B2: cyclohexyl B1 avermectins at a ratio of less than 1.6: 1. 48. The strain of claim 46, wherein the cells produce cyclohexyl B2: cyclohexyl B1 avermectins in a ratio of approximately 0.94: 1. 49. The strain of claim 46, wherein the cells produce cyclohexyl B2: cyclohexyl B1 avermectins in a ratio of approximately 0.88: 1. 5 50. The strain of claim 46, wherein the cells produce cyclohexyl B2: cyclohexyl B1 avermectins in a ratio of approximately 0.84: 1. 51. A strain of S. avermitilis comprising cells in which the aveC gene has been inactivated. 52. Avermectin production method, characterized in that cultured cells of a strain of S. avermitilis that express a mutated aveC allele that encodes a gene product 15 reducing the class 2: 1 ratio of avermectins produced by strain cells from S. avermitilis expressing the mutated aveC allele, compared to cells from the same strain that do not express the mutated aveC allele and instead 20 express only wild-type aveC alleles in culture, under conditions that allow or induce the production of avermectins and said avermectins were semi avat culture. 53. Composition of avermectins 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 , 10 compared to cells of the same strain that do not express the mutated aveC allele but instead express only the wild-type aveC allele, which is produced at a reduced 2: 1 ratio compared to the ratio of class 2: 1 avermectins produced by cells of the same strain m from S. avermitilis, which do not express the mutated aveC allele but instead express only the wild-type aveC allele.