HRP20000539A2 - Streptomyces avermitilis gene directing the ratio of b2:b1 avermectins - Google Patents

Description

The field of technology

The described invention is directed to mixtures and processes for the production of avermectin and primarily belongs to the field of animal health care. More specifically, the described invention relates to polynucleotide molecules comprising nucleotide sequences encoding the AveC gene product, which can be used to modulate the class 2:1 ratio of avermectins produced by fermentation of cultures of Streptomyces avermitilis, and to mixtures and methods for testing such polynucleotides. molecule. The described invention further relates to vectors, transformed host cells and new mutant S. avermitilis species in which the AveC gene is mutated so that it modulates the class 2:1 avermectin ratio.

State of the art

1 Avermectins

Streptomyces species produce a large variety of secondary metabolites that include avermectins, which comprise a series of eight similar 16-membered macrocyclic lactones with potent anthelmintic and insecticidal activity. These eight different but very similar compounds are designated as A1a, A1b, A2a, A2b, B1a, B1b, B2a and B2b. Series "a" of these compounds refer to natural avermectin where the substituent at the C25 position is (S)-sec-butyl, and series "b" refer to those compounds where the substituent at the C25 position is isopropyl. The designations "A" and "B" refer to avermectins where the substituent at the C5 position is methoxy and hydroxy, respectively. The designation "1" refers to avermectins in which a double bond is present at the C22, 23 position, and the designation "2" refers to avermectins that have a hydrogen at the C22 position and a hydroxy at the C23 position. Among similar avermectins, the B1 type of avermectin is known to have the most effective antiparasitic and pesticidal activity and is therefore the most commercially preferred avermectin.

Avermectins and their production by aerobic fermentation of S. avermitilis species are described, inter alia, in US patents no. 4,310,519 and 4,429,042. The biosynthesis of natural avermectins is believed to start endogenously from the CoA thioester analogues of isobutyric acid and S-(+)-2-methyl butyric acid.

Combining the two effects, the improvement of the species through undirected mutagenesis and the use of exogenous fatty acid supply leads to the efficient production of avermectin analogues. S. avermitilis mutants deficient in branched chain 2-oxo acid dehydrogenase (bkd deficient mutants) can only produce avermectins when fermentations are supplied with fatty acids. Testing and isolation of mutants deficient in branched chain dehydrogenase activity (eg, S. avermitilis, ATCC 53567) is described in European Patent (EP) No. 276103. Fermentation of such mutants in the presence of exogenously supplied fatty acids leads to the production of only four avermectins corresponding to the fatty acid used. Thus, supplying fermentation of S. avermitilis (ATCC 53567) with S-(+)-2-methylbutyric acid will lead to the production of natural avermectins A1a, A2a, B1a and B2a; supplying fermentations with isobutyric acid will lead to the production of natural avermectins A1b, A2b, B1b and B2b; and supply fermentations with cyclopentanecarboxylic acid will lead to the production of four new cyclopentyl avermectins A1, A2, B1 and B2.

If supplied with other fatty acids, new avermectins will be produced. Through screening of over 800 potential precursors, more than 60 other novel avermectins have been identified (see, for example, Dutton et al.: "J. Antibiot.", 44: 357-365, (1991); and Banks et al.: " Roy. Soc. Chem.", 147:16-26, (1994)). In addition to this, S. avermitilis mutants deficient in 5-O-methyltransferase activity produce mostly only the B analog avermectins. Therefore, S. avermitilis mutants lacking branched-chain 2-oxo acid hydrogenase and 5-O-methyltransferase activity produce only B avermectins corresponding to the fatty acid used to supply the fermentation. Thus, supplying such double mutants with S-(+)-2-methylbutyric acid leads to the production of only natural avermectins B1a and B2a, while supplying with isobutyric acid or with cyclopentanecarboxylic acid leads to the production of natural avermectins B1b and B2b, or new cyclopentyl B1 and B2 avermectin, in the same order. Supplying the double mutant species with cyclohexane carboxylic acid is the preferred procedure for the production of a commercially important new avermectin, cyclohexyl avermectin B1 (doramectin). The isolation and characterization of such double mutants, for example, S. avermitilis (ATCC 53692) is described in European Patent (EP) no. 276103.

2 Genes involved in avermectin biosynthesis

In many cases, genes involved in the production of secondary metabolites and genes encoding a specific antibiotic are clustered together on the chromosome. Such is the case, for example, with the Stretomyces polyketide synthesis gene cluster (PKS) (see Hopwood and Sherman: "Ann. Rev. Genet.", 24:37-66, (1990)). Thus, one strategy for cloning genes in the biosynthetic pathway has been to isolate a drug-resistant gene and then test adjacent regions of the chromosome for other similar genes for the biosynthesis of a particular antibiotic. Another strategy for cloning genes involved in the biosynthesis of important metabolites was the complementation of mutants. For example, portions of a DNA library from an organism capable of producing a particular metabolite are introduced into a non-producing mutant and the transformants are tested for metabolite production. Additionally, hybridization libraries using probes derived from other Streptomyces species have been used to identify and clone genes in biosynthetic pathways.

Genes involved in the biosynthesis of avermectin (Ave genes), similar to genes required for the biosynthesis of other Streptomyces secondary metabolites (for example, PKS) were found clustered on the chromosome. A number of Ave genes have been successfully cloned using vectors to complement S. avermitilis mutants that are blocked in avermectin synthesis. Cloning of such genes is described in US Pat. No. 5,252,474. In addition to this, Ikeda et al.: "J. Antibiot." 48:532-534, (1995), describe the localization of the chromosomal region encompassing the C22.23 degree of dehydration (AveC) in the 4.82 Kb BamHI fragment of S. avermitilis, as well as mutations in the AveC gene that lead to the production of a single-component B2a producer. As ivermectin, a potent anthelmintic compound, can be produced chemically from avermectin B2a, such a one-component producer of avermectin B2a was considered particularly applicable for the commercial production of ivermectin.

Identification of mutations in the AveC gene that minimize the complexity of avermectin production, such as, for example, mutations that lower the B2:B1 ratio of avermectin should simplify the production and purification of commercially relevant avermectins.

Brief description of the invention

The described invention provides an isolated polynucleotide molecule comprising the complete AveC ORF of S. avermitilis or an essential part thereof, where the isolated polynucleotide molecule does not have a subsequent ORF located downstream of AveC in situ in the S. avermitilis chromosome. The isolated polynucleotide molecule of the described invention preferably comprises a nucleotide sequence, which is the same as the sequence of the plasmid pSE 186 (ATCC 209604) encoding the S. avermitilis AveC gene product, or which is the same as the nucleotide sequence of the AveC ORF in Figure 1 (SEQ ID NO:1) or its essential part.

The described invention further provides a polynucleotide molecule having a nucleotide sequence that is homologous to the sequence of plasmid pSE 186 (ATCC 209604) encoding the S. avermitilis AveC gene product or to the nucleotide sequence of the AveC ORF present in Figure 1 (SEQ ID NO:1) or its essential part.

The described invention further provides a polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence that is homologous to the amino acid sequence encoded by the sequence of plasmid pSE 186 (ATCC 209604) encoding the AveC gene product or the amino acid sequence in Figure 1 ( SEQ ID NO:2) or its essential part.

The described invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding an AveC homologous gene product. In a preferred embodiment, the isolated polynucleotide molecule comprises a nucleotide sequence encoding an AveC homologous genetic product from S. hygroscopicus, where the homologous genetic product comprises the amino acid sequence of SEQ ID NO: 4 or an essential part thereof. In a preferred embodiment, the isolated polynucleotide molecule of the described invention is one that encodes a S. hygroscopicus AveC homologous genetic product comprising the nucleotide sequence of SEQ ID NO: 3 or a substantial portion thereof.

The described invention further provides a polynucleotide molecule comprising a nucleotide sequence that is homologous to the S. hygroscopicus nucleotide sequence of SEQ ID NO: 3. The described invention further provides a polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the S. hygroscopicus AveC homologous genetic product that has the amino acid sequence of SEQ ID NO: 4.

The described invention further provides oligonucleotides that hybridize to a polynucleotide molecule having a nucleotide sequence in Figure 1 (SEQ ID NO: 1) or SEQ ID NO: 3 or a polynucleotide molecule having a nucleotide sequence that is the complement of the nucleotide sequence in Figure 1 (SEQ ID NO: 1 ) or SEQ ID NO: 3.

The described invention further provides recombinant cloning vectors and expression vectors that are applicable in cloning or expressing polynucleotides of the described invention, including polynucleotide molecules comprising the AveC ORF of S. avermitilis or the AveC homologous ORF. In a non-limiting embodiment, the described invention provides the plasmid pSE 186 (ATCC 209604) which comprises the entire ORF of the AveC gene of S. avermitilis. The described invention further provides transformed host cells comprising a polynucleotide molecule or recombinant vector of the invention and novel species or cell lines derived therefrom.

The described invention further provides a recombinantly expressed AveC gene product or an AveC homologous gene product or an essential part thereof, which is substantially purified or isolated as well as its homologues. The described invention further provides a method for the production of a recombinant genetic product comprising culturing a host cell transformed with a recombinant expression vector, wherein said recombinant expression vector comprises a polynucleotide molecule having a nucleotide sequence encoding an AveC gene product or an AveC homologous gene product, where the polynucleotide molecule is in operative association with one or more regulatory elements that control the expression of the polynucleotide molecule in the host cell, under conditions that lead to the production of a recombinant AveC gene product or AveC homologous gene product, and isolation of the AveC gene product or AveC homologous gene product from cell culture.

The described invention further provides a polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as the sequence of plasmid pSE 186 (ATCC 209604) encoding the S. avermitilis gene product or nucleotide sequence of the AveC ORF as shown in Figure 1 (SEQ ID NO: 1), but which further comprises one or more mutations, such that S. avermitilis ATCC 53692 cells in which the wild-type avaC allele is inactivated and which express a polynucleotide molecule comprising the mutated nucleotide sequence produce a different ratio or amount of avermectin than that produced by cells of S. avermitilis species ATCC 53692 which, in addition, express only the AveC allele of the original type. According to the described invention, such polynucleotide molecules can be used to produce new strains of S. avermitilis that show a detectable change in avermectin production compared to the same strains that instead express only the AveC allele of the original type. In a preferred embodiment, such polynucleotide molecules are applicable to the production of new species of S. avermitilis that produce avermectins in a reduced class 2:1 ratio compared to that of the same species that instead express only the wild-type AveC allele. In a further preferred embodiment, such polynucleotide molecules are applicable to the production of new strains of S. avermitilis which produce increased levels of avermectin compared to the same strain which in addition expresses only the AveC allele of the original type. In a further preferred embodiment, such polynucleotide molecules are applicable for the production of new strains of S. avermitilis in which the AveC gene is deactivated.

The disclosed invention provides methods for identifying mutations in the AveC ORF of S. avermitilis that may alter the ratio and/or amount of avermectin produced. In a preferred embodiment, the described invention provides a method for identifying AveC ORF mutations that can alter the class 2:1 ratio of produced avermectins, comprising: (a) determining the class 2:1 ratio of avemerctins produced by S. avermitilis species where the natural AveC allele is deactivated and where a polynucleotide molecule comprising a nucleotide sequence encoding a mutated AveC gene product is introduced to be expressed; (b) determining the class 2:1 ratio of avermectins that are produced using cells of the same S. avermitilis species as in step (a) but which in addition express only the AveC allele having the nucleotide sequence of the ORF of Figure 1 (SEQ ID NO: 1) or a sequence homologous to it; and (c) comparing the class 2:1 ratio of avermectins produced by S. avermitilis cells in step (a) to the class 2:1 ratio of avermectins produced by S. avermitilis cells in step (b), such that if the class 2:1 ratio of avermectins produced by S. avermitilis cells in step (a) is different from the class 2:1 ratio of avermectins produced by S. avermitilis cells in step (b), then the AveC ORF mutation that can change the class A 2:1 ratio of avermectin is identified. In a preferred embodiment, the class 2:1 ratio of avermectin is reduced by mutation.

In a further preferred embodiment, the described invention provides a method for identifying mutations of the AveC ORF of S. avermitilis or genetic constructs comprising the AveC ORF that can alter the amount of avermectin produced which comprises: (a) determining the amount of avemerctin produced by a S. avermitilis species where the natural AveC allele is inactivated and where a polynucleotide molecule comprising a nucleotide sequence encoding a mutated AveC gene product or comprising a genetic construct comprising a nucleotide sequence encoding an AveC gene product is introduced to be expressed; (b) determining the amount of avermectin produced by cells of the same S. avermitilis species as in step (a) but which in addition express only the AveC allele having the nucleotide sequence ORF of Figure 1 (SEQ ID NO: 1) or a nucleotide sequence that is homologous to it; and (c) comparing the amount of avermectin produced by S. avermitilis cells in step (a) to the amount of avermectin produced by S. avermitilis cells in step (b), such that if the amount of avermectin produced by S. avermitilis cells in step (a) is different from the amount of avermectin produced by S. avermitilis cells in step (b), then a mutation in the AveC ORF or a genetic construct that can alter the amount of avermectin is identified. In a preferred embodiment, the amount of avermectin produced is increased by mutation.

The described invention further provides recombinant vectors that are applicable for obtaining new species of S. avermitilis that have altered avermectin production. For example, the described invention provides vectors that can be used to obtain some polynucleotide molecules comprising the mutated nucleotide sequences of the described invention at the AveC gene site of the S. avermitilis chromosome for insertion into or replacement of the AveC ORF or part thereof by homologous recombination. According to the described invention, even more, the polynucleotide molecule comprising the mutated nucleotide sequence of the described invention provided herein may also function to modulate avermectin biosynthesis when inserted into the S. avermitilis chromosome at a site other than the AveC gene site, or when retained episomally in S. avermitilis cells. Thus, the described invention also provides vectors comprising a polynucleotide molecule comprising a mutated nucleotide sequence of the described invention, where the vectors may be used to insert the polynucleotide molecule into a site in the S. avermitilis chromosome other than the site of the AveC gene, or be maintained episomally. In a preferred embodiment, the described invention provides gene exchange vectors that can be used to insert a mutated AveC allele into the S. avermitilis chromosome to generate new types of cells that produce avermections at a reduced class 2:1 ratio compared to cells of the same species that instead express only AveC allele of the original type.

The described invention further provides methods for obtaining new species of S. avermitilis comprising cells that express a mutated AveC allele and that produce altered ratios and/or amounts of avermectin compared to cells of the same species of S. avermitilis that, in addition, express only the AveC allele of the original type. In a preferred embodiment, the described invention provides a method for obtaining new species of S. avermitilis comprising cells expressing a mutated AveC allele and producing an altered class 2:1 ratio of avermectin compared to cells of the same species of S. avermitilis which in addition express only the AveC allele of the original type, which involves transforming S. avermitilis cells with a vector carrying a mutated AveC allele that encodes a gene product that alters the 2:1 class ratio of avermectins produced by S. avermitilis cells expressing the mutated allele relative to cells of the same species adjacent to that express only the wild-type AveC allele, and selection of transformed cells that produce avermectins in an altered 2:1 class ratio compared to the 2:1 class ratio produced by cells of the species that additionally express only the wild-type AveC allele. In a preferred embodiment, the class 2:1 ratio of avermectins produced is reduced in cells of the new species.

In a further preferred embodiment, the described invention provides a method for obtaining new S. avermitilis species comprising cells that produce altered amounts of avermectin, comprising transforming S. avermitilis cells with a vector carrying a mutated AveC allele or a genetic construct comprising an AveC allele, the expression of which leads to an altered amount of avermectin produced by S. avermitilis cells that express the mutated allele or genetic construct compared to cells of the same species that in addition express only the AveC allele of the original type, and the selection of transformed cells that produce avermectins in an altered amount compared to by the amount of avermectin produced by cells of a species that in addition express only the AveC allele of the original type. In a preferred embodiment, the amount of avermectin produced is increased in the cells of the new species.

In a further preferred embodiment, the described invention provides a method for obtaining new species of S. avermitilis, cells that comprise a deactivated AveC allele, which comprises transforming S. avermitilis species that express the AveC allele of the original type with a vector that deactivates the AveC allele and selection of transformed cells where the AveC allele is deactivated.

The described invention further includes new species of S. avermilitis comprising cells transformed with one of the polynucleotide molecules or vectors comprising the mutated nucleotide sequence of the described invention. In a preferred embodiment, the described invention provides new species of S. avermitilis comprising cells expressing a mutated AveC allele in place of, or in addition to, the wild-type AveC allele, where the cells of the new species produce avermectins in an altered class 2:1 ratio compared to cells of the same species which, in addition, express only the AveC allele of the original type. In an even more preferred embodiment, the cells of the new type produce avermectins in a reduced class 2:1 ratio compared to cells of the same type which in addition express only the AveC allele of the original type. Such new species are applicable in the production of a large number of commercially desirable avermectins such as doramectin.

In a further preferred embodiment, the described invention provides new strains of S. avermitilis comprising cells expressing a mutated AveC allele or a genetic construct comprising an AveC allele in place of, or additionally, an AveC allele of the original type, which leads to the production by the cells of an altered amount of avermectin in relation to on the amount of avermectin that was produced by cells of the same species that, in addition, express only the AveC allele of the original type. In a preferred embodiment, the new cells produce an increased amount of avermectin.

In a further preferred embodiment, the described invention provides new species of S. avermitilis comprising cells where the AveC gene has been deactivated. Such strains are applicable both to the different spectrum of avermectin they produce compared to the original type strain and to complement the testing assays described herein to determine whether targeted or non-targeted mutagenesis of the AveC gene affects avermectin production.

The described invention further provides a process for the production of avermectins, which comprises culturing cells of the S. avermitilis species where the cells express a mutated AveC allele encoding a gene product that alters the 2:1 class ratio of avermectins produced by S. avermitilis species expressing the mutated AveC allele in comparison with cells of the same species that do not express the mutated AveC allele, but in addition express only the AveC allele of the original type, in the culture medium under conditions that allow or induce the production of this avermectin, and the extraction of said avermectin from the culture. In a preferred embodiment, the class 2:1 ratio of avermectins obtained by cells expressing the mutation is reduced. This process ensures increased efficiency in the production of commercially valuable avermectins, such as doramectin.

The described invention further provides a process for the production of avermectin, which comprises culturing cells of the S. avermitilis species where the cells express a mutated AveC allele or a genetic construct comprising the AveC allele leading to the production of an altered amount of avermectin produced by the S. avermitilis species expressing the mutated AveC allele or genetic construct in comparison with cells of the same species that do not express the mutated AveC allele or genetic construct but which in addition express only the AveC allele of the original type, in the culture medium under conditions that allow or induce the production of avermectin, and the isolation of said avermectin from the culture. In a preferred embodiment, the amount of avermectin produced by cells expressing the mutation or genetic construct is increased.

The described invention further provides a new mixture of avermectins that are produced using the species S. avermitilis expressing the mutated AveC allele of the described invention, where the avermectins are obtained in a reduced 2:1 class ratio compared to the 2:1 class ratio of avermectins that are obtained using the same species S avermitilis which does not express the mutated AveC allele but in addition expresses only the AveC allele of the original type. The novel avermectin preparation may be present as obtained in the fermentation culture fluid, or may be recovered from this, and may be partially or substantially purified therefrom.

Short description of the pictures

Figure 1. DNA sequence (SEQ ID NO: 1) encompassing the S. avermitilis AveC ORF, and deduced acid sequence (SEQ ID NO: 2).

Figure 2 Plasmid vector pSE 186 (ATCC 209604) which includes the entire ORF of the AveC gene of S. avermitilis.

Figure 3 Gene exchange vector pSE 180 (ATCC 209605) containing the ermE gene of Sacc erythraea inserted into the AveC ORF of S. avermitilis.

Figure 4 BamHI restriction map of the avermectin polyketide synthesis gene cluster from S. avermitilis with five overlapping cosmid clones identified (eg, pSE 65, pSE 66, pSE 67, pSE 68, pSE 69). The relationship of pSE 118 and pSE 119 is also indicated.

Figure 5 HPLC analysis of fermentation products obtained by S. avermitilis species. Peak quantification was performed by comparison with standard amounts of cyclohexyl B1. Cyclohexyl B2 retention time was from 7.4 to 7.7 minutes; cyclohexyl B1 retention time was from 11.9 to 12.3 minutes.

Figure 5A S. avermitilis strain SE 180-11 with inactivated AveC ORF.

Figure 5B S. avermitilis strain SE 180-11 transformed with pSE 186 (ATCC 209604).

Figure 5C S. avermitilis strain SE 180-11 transformed with pSE 187.

Figure 5D S. avermitilis strain SE 180-11 transformed with pSE 188.

Figure 6 Comparison of the deduced amino acid sequences encoded by the AveC ORF of S. avermitilis (SEQ ID NO: 2), the AveC homolog of the partial ORF from S. griseochromogenes (SEQ ID NO: 5) and the AveC homolog of the partial ORF from S. hygroscopicus (SEQ ID NO: 4). The valine residue in bold is the supposed starting site for the protein. Conserved residues are shown in capital letters for homology in all three sequences and in lowercase letters for homology in 2 of the three sequences. Amino acid sequences contain approximately 50% sequence identity.

Figure 7 Hybrid plasmid construct containing a 564 bp BsaAl/Kpnl fragment from the S. hygroscopicus AveC homologous gene inserted into the BsaAl/Kpnl site of the S. avermitilis AveC ORF.

Detailed description of the invention

The described invention relates to the identification and characterization of polynucleotide molecules having nucleotide sequences encoding the AveC gene product from Streptomyces avermitilis, the construction of new strains of S. avermitilis that can be used to test mutated AveC gene products for their effect on avermectin production, and the discovery that some mutated AveC gene products can reduce the B2:B1 ratio of avermectins produced by S. avermitilis. By way of example, the invention is described in the sections below for a polynucleotide molecule comprising either a nucleotide sequence that is the same as the nucleotide sequence of plasmid pSE 186 (ATCC 209604) encoding the S. avertimilis AveC gene product or the ORF sequence of Figure 1 (SEQ ID NO : 1) and for polynucleotide molecules that have mutated nucleotide sequences derived from these. However, the principles provided in the described invention may be analogously applied to other polynucleotide molecules including AveC homologous genes from other Streptomyces species including, for example, S. hygroscopicus and S. griseochromogenes, among others.

1 Polynucleotide molecules

S. avermitilis AveC gene product

The described invention provides an isolated polynucleotide molecule comprising the complete AveC ORF of S. avermitilis or an essential part thereof, where the isolated polynucleotide molecule does not contain the following complete ORF that is located downstream of the AveC ORF in situ in the S. avermitilis chromosome.

The isolated polynucleotide molecule of the described invention preferably comprises a nucleotide sequence which is the same as the sequence of the plasmid pSE 186 (ATCC 209604) which encodes the S. avermitilis AveC gene product or which is like the nucleotide sequence of the ORF of Figure 1 (SEQ ID NO: 1) or an essential part thereof . As used herein, a "substantial portion" of an isolated polynucleotide molecule comprising the nucleotide sequence encoding the S. avermitilis AveC gene product means an isolated polynucleotide molecule comprising at least about 70% of the complete AveC ORF sequence shown in Figure 1 (SEQ ID NO: 1 ) and which encodes a functionally equivalent AveC gene product. In this regard, a "functionally equivalent" AveC gene product is defined as a gene product that, when expressed in S. avermitilis ATCC 53692 where the native AveC allele has been inactivated, leads to the production of substantially the same ratio and amount of avermectin as produced by S. avermitilis strain ATCC 53692 which in addition expresses only the functional AveC allele of the original type in S. avermitilis ATCC 53692.

In addition to the nucleotide sequence of the AveC ORF, the isolated polynucleotide molecule of the described invention may further comprise a nucleotide sequence that naturally flanks the AveC gene in situ in S. avermitilis, such as those flanking the nucleotide sequences shown in Figure 1 (SEQ ID NO: 1).

As used herein, the terms "polynucleotide molecule", "polynucleotide sequence", coding sequence", "open reading frame (ORF)" and the like are intended to refer to both DNA and RNA molecules, which may be either single-stranded or double-stranded and which can be transcribed into a translated (DNA) or translated (RNA) AveC gene product or, as described hereinbelow, an AveC homologous gene product, or a polypeptide that is homologous to an AveC gene product or an AveC homolog by a genetic product in an appropriate host cell expression system when placed 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) comprises four different GTG codons at bp positions 42, 174 and 180. As described in Chapter 9 below, multiple deletions of the 5' region of the AveC ORF (Figure 1 ; SEQ ID NO: 1) were performed to help define which of these codons may function in the AveC ORF as an initiation site for protein expression. Deletion of the first GTG site at bp 42 does not eliminate AveC activity. Additional deletion of all GTG codons at bp positions 174, 177, and 180 collectively eliminated AveC activity, demonstrating that this region is required for protein expression. The described invention thus includes variable length AveC ORFs that initiate translation at one of the GTG sites located at bp 174, 177 or 180 bp, as shown in Figure 1 (SEQ ID NO:1) and corresponding polypeptides for each.

The described invention further encompasses a polynucleotide molecule having a nucleotide sequence that is homologous to the sequence of plasmid pSE 186 (ATCC 209604) encoding the S. avermitilis AveC gene product or to the nucleotide sequence of the AveC ORF shown in Figure 1 (SEQ ID NO: 1) or with its essential part. The term "homologous" when used to refer to a polynucleotide molecule that is homologous to the sequence encoding the S. avermitilis AveC gene product means a polynucleotide molecule having the nucleotide sequence: (a) that encodes the same AveC gene product as the sequence of plasmid pSE 186 (ATCC 209604) which encodes the S. avermitilis AveC gene product or which encodes the same AveC gene product as the AveC ORF nucleotide sequence shown in Figure 1 (SEQ ID NO: 1), but which includes one or more invisible changes in the nucleotide sequence according to the degeneracy of the genetic code; or (b) that hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes an amino acid sequence that is encoded by the sequence of plasmid pSE 186 (ATCC 209604) that encodes the AveC gene product or that encodes an amino acid sequence that is shown in Figure 1 (SEQ ID NO: 2) under moderately binding conditions, i.e. hybridization in filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 °C and washing in 0.2 × SSC/ 0.1% SDS at 42°C (see Ausubel et al.: "Current Protocols in Molecular Biology", Vol. I, p. 2.10.3, (1989), published by Green Publishing Associates Inc. and John Wiley & Sons Inc. , New York,) and encodes a functionally equivalent gene product as defined herein. In a preferred embodiment, the homologous polynucleotide molecule hybridizes to the complement sequence of the plasmid pSE 186 (209604) encoding the Avec gene product or to the complement of the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1) or a substantial portion thereof, under moderately stringent conditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 °C and washing in 0.1 × SSC/0.1% SDS at 68 °C ( see Ausubel et al., supra) and encodes a functionally equivalent AveC gene product as defined hereinbefore.

The activity of the AveC gene product and its potential functional equivalents can be determined by HPLC analysis of the fermentation product, as described in the examples herein below. Polynucleotide molecules having nucleotide sequences encoding functional equivalents of the S. avermitilis AveC gene product include naturally occurring AveC genes present in other S. avermitilis species, AveC homologous genes present in other Streptomyces species, and mutated AveC alleles, whether naturally occurring or engineered. .

The described invention further encompasses a polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide having an amino acid sequence that is homologous to the amino acid sequence that is encoded by the sequence of plasmid pSE 186 (ATCC 209604) that encodes the AveC gene product or the amino sequence in Figure 1 (SEQ ID NO: 2) or with its essential part. As used herein, a "substantial portion" of the amino acid sequence of Figure 1 (SEQ ID NO: 2) means a polypeptide comprising at least about 70% of the amino acid sequence shown in Figure 1 (SEQ ID NO: 2) and which functionally equivalent AveC gene product, as defined hereinafter.

As used herein to refer to amino acid sequences that are homologous to the amino acid sequence of the AveC gene product from S. avermitilis, the term "homologous" refers to a polypeptide that is encoded by the sequence of plasmid pSE 186 (ATCC 209604) encoding the AveC gene product or having the amino acid sequence of Figure 1 (SEQ ID NO: 2) but in which one or more amino acid residues are conservatively substituted with a different amino acid residue, where such conservative substitution results in a functionally equivalent genetic product, as defined hereinabove. Conservative amino acid substitutions are well known in the art. Rules for making such substitutions include those described by M.D. Dayhof in "Nat. Biomed. Res. Found.", Volume 5 Supplement 3, (1978), Washington D.C., among others. More specifically, conservative amino acid substitutions are those that typically occur in a family of amino acids that are similar in the acidity, polarity, or bulkiness of their side chains. Genetically encoded amino acids are usually divided into two groups: (a) acidic = aspartate, glutamate; (2) bases = lysine, arginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylalanine, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are thus classified together as aromatic amino acids. One or more substitutions in a particular group, for example leucine with isoleucine or valine, or aspartate with glutamate or threonine with serine or another amino acid residue with a structurally similar amino acid residue, for example an amino acid residue with similar acidity, polarity, or bulkiness in its same combination, it will have an insignificant effect on the function of the polypeptide.

The described invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding an AveC homologous gene product. As used herein, an "AveC homologous gene product" is defined as a gene product having at least about 50% amino acid sequence identity to the S. avermitilis AveC gene product comprising the amino acid sequence encoded by plasmid pSE 186 (ATCC 209604) encoding AveC gene product or amino acid sequence shown in Figure 1 (SEQ ID NO: 2). In a non-limiting embodiment, the AveC homologous genetic product is from S. Hygroscopicus, (described in EP Patent Application No. 0298423; FERM Deposit BP-1901) and comprises the amino acid sequence of SEQ ID NO: 4, or a substantial portion thereof. "Essential portion" of the amino acid sequence of SEQ ID NO: 4 means a polypeptide comprising at least about 70% of the amino acid sequence of SEQ ID NO: 4, and which constitutes a functionally equivalent AveC homologous gene product. A "functionally equivalent" AveC homologous gene product is defined as a gene product that, when expressed in the S. hygroscopicus strain FERM BP-1901 in which the native AveC homologous allele has been inactivated, leads to the production of substantially the same ratio and amount of milbemycin as those produced using S. hygroscopicus strain FERM BP-1901, which expresses in addition only a functional aceC homologous allele of the original type naturally present in S. hygroscopicus strain FERM BP-1901. In a non-limiting embodiment, the isolated polynucleotide molecule of the described invention encoding the S. hygroscopicus AveC homologous genetic product comprises the nucleotide sequence of SEQ ID NO: 3 or a substantial portion thereof. In this regard, the "substantial 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 nucleotide sequence of SEQ ID NO: 3 and which encodes a functionally equivalent AveC homologous gene product, as defined right here ahead.

The described invention further provides a polynucleotide molecule comprising a nucleotide sequence that is homologous to the S. hygroscopicus nucleotide sequence of SEQ ID NO: 3. The term "homologous" when used to refer to a polynucleotide molecule comprising a nucleotide sequence that is homologous to the sequence of SEQ ID NO: 3 which encodes S. hygroscopicus AveC homologous genetic product means a polynucleotide molecule having a nucleotide sequence: (a) which encodes the same genetic product as the nucleotide sequence of SEQ ID NO: 3 or an essential part thereof, but which includes one or more undetectable changes in the nucleotide sequence according to degeneration of the genetic code; or (b) that hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4, under moderate binding conditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate ( SDS), 1 mM EDTA at 65 °C and washing in 0.2 × SSC/0.1% SDS at 42 °C (see Ausubel et al., supra), and encodes a functionally equivalent AveC homologous gene product as defined hereinbefore . In a preferred embodiment, the homologous polynucleotide molecule hybridizes to the complement of the nucleotide sequence SEQ ID NO: 3 encoding the AveC homologous gene product or its essential part, under conditions of high binding, i.e. hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 °C and washing in 0.1 × SSC/0.1% SDS at 68 °C (see Ausubel et al., forward), and encodes a functionally equivalent AveC homologous gene product as defined hereinafter.

The described invention further provides a polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide that is homologous to the S. hygroscopicus AveC homologous gene product. As used herein to refer to polypeptides that are homologous to the AveC homologous gene product of SEQ ID NO: 4 from S. hygroscopicus, the term "homologous" means a polypeptide having the amino acid sequence of SEQ ID NO: 4, but in which one or more amino acid residues conservatively substituted with a different amino acid residue, where such conservative substitution results in a functionally equivalent AveC homologous gene product, as defined hereinabove.

The described invention further provides oligonucleotides that hybridize to a polynucleotide molecule that has the nucleotide sequence from Figure 1 (SEQ ID NO: 1) or SEQ ID NO: 3 or to a polynucleotide molecule that has a nucleotide sequence that is the complement of the nucleotide sequence from Figure 1 (SEQ ID NO : 1) or SEQ ID NO: 3. Such oligonucleotides are at least about 10 nucleotides in length, and preferably about 15 to about 3 nucleotides in length, and hybridize to one of the aforementioned polynucleotide molecules under high-binding conditions, i.e. washing in 6×SSC /0.5% sodium phosphate at approximately 37 °C for approximately 14-base oligos, at approximately 48 °C for approximately 17-base oligos, at approximately 55 °C for approximately 20-base oligos, and at approximately 60 °C for approximately 23-base oligos. In a preferred embodiment, the oligonucleotides are complementary to a portion of one of the aforementioned polynucleotide molecules. These oligonucleotides are useful for a variety of purposes including encoding or acting as antisense molecules useful in genetic regulation, or as primers in the amplification of AveC- or AveC homolog-encoding polynucleotide molecules.

Additionally AveC homologous genes can be identified in other Streptomyces species or genera using the polynucleotide molecules or oligonucleotides described herein in conjunction with known techniques. For example, an oligonucleotide molecule comprising a portion of the S. avermitilis nucleotide sequence of Figure 1 (SEQ ID NO: 1) or a portion of the S. hygroscopicus nucleotide sequence of SEQ ID NO: 3 can be detectably labeled and used to test a genomic library formed from DNA derived from the organism of interest. The binding conditions for hybridization are chosen based on the relationship of the reference organism, in this example S. avermitilis or S. hygroscopicus, to the organism of interest. Regimes for different binding conditions are well known to those skilled in the art, and such conditions will vary largely depending on the specific organisms from which the library and labeled sequences are derived. Such oligonucleotides are at least about 15 nucleotides in length and include, for example, those described in the examples hereinbelow. Amplification of homologous genes can be performed using these and other oligonucleotides using standard techniques, such as polymerase chain reaction (PCR), although other amplification techniques are known in the art, such as, for example, ligase chain reaction can also be used.

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

The production and manipulation of the polynucleotide molecules described herein are within the skill of those skilled in the art, and may be performed according to recombinant techniques described, for example, in Maniatis et al.: "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al.: "Current Protocols in Molecular Biology", (1989), Greene Publishing Associates & Wiley Interscience, NY; Sambrook et al.: "Molecular Cloning: A Laboratory Manual", 2nd ed. (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Innis et al.: "PCR Strategies", (1995), published by Academic Press Inc., San Diego; and Erlich: "PCR Technology", Oxford University Press, New York, which is incorporated herein by reference. Polynucleotide clones encoding AveC gene products or AveC homologous gene products can be identified using procedures known in the art, including, but not limited to, the procedures provided in Chapter 7, herein below. Genomic DNA libraries can be screened for AveC and AveC homologous coding sequences using techniques such as the procedures given in Benton and Davis: "Science", 196:180, (1977), for bacteriophage libraries; and in Grunstein and Hognes: "Proc. Natl. Acad. Sci. USA", 72: 3961-3965, (1975), for plasmid libraries. Polynucleotide molecules having nucleotide sequences known to include the AveC ORF as present, for example, in plasmid pSE 186 (ATCC 209604) or in plasmid pSE 119 (which is described in Chapter 7, herein below) can be used as probes in these experiments. for testing. Alternatively, oligonucleotide probes can be synthesized to match nucleotide sequences deduced from partial or complete amino acid sequences of a purified AveC homologous gene product.

2. Recombinant systems

Cloning and expression vectors

The described invention further provides recombinant cloning vectors and expression vectors, which are applicable in the cloning or expression of polynucleotide molecules of the described invention comprising, for example, either the AveC ORF of S. avermitilis or some AveC homologous ORFs. In a non-limiting embodiment, the described invention provides plasmid pSE 186 (ATCC 209604), which comprises the complete ORFs of the AveC gene of S. avermitilis.

The entire description that follows with respect to an AveC ORF from S. avermitilis or a polynucleotide molecule comprising an AveC ORF from S. avermitilis or a portion thereof or a S. avermitilis AveC gene product also refers to AveC homologs and AveC homologous gene products, unless otherwise stated. indicated explicitly or in context.

Variants of various vectors have been developed for specific use in Streptomyces, including phage, high copy number plasmids, low copy number plasmids, and E. coli-Streptomyces travel vectors among others, and any of these can be used in the practice of the described invention. A number of drug resistance genes have also been cloned from Streptomyces, and most of these genes have been incorporated into vectors as selectable markers. Examples of currently available vectors for use in Streptomyces are provided, inter alia, in Huntchinson: "Applied Biochem. Biotech.", 16:169-190, (1980).

Recombinant vectors of the described invention, especially expression vectors, are preferably formed so that the coding sequence for the polynucleotide molecule of the described invention is in operative association with one or more regulatory elements that are required for transcription and translation of the coding sequence for polypeptide production. As used herein, the term "regulatory element" includes, but is not limited to, nucleotide sequences encoding inducible and non-inducible promoters, enhancers, operators, and other elements known in the art that serve to direct and/or regulate the expression of a polynucleotide that exerts sequence coding. Also, as used herein, a coding sequence is in "operable association" with one or more regulatory elements that effectively regulate and permit transcription of the coding sequence or translation of its mRNA, or both.

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

Procedures for generating recombinant vectors containing appropriate coding sequences in operative association with appropriate regulatory elements are well known in the art, and these may be used to practice the described invention. These methods include in vitro recombinant techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Maniatis et al., supra; Ausubel et al., forward; Sambrook et al., forward; Innis et al., forward; and Erlich, ahead.

The regulatory elements of these vectors can vary in strength and specificity. Depending on the host/vector system used, any number of suitable transcriptional and translational elements may be used. Non-limiting examples of transcriptional regulatory regions or promoters for bacteria include the β-gal promoter, T7 promoter, TAC promoter, λ left and right promoters, trp and lac promoters, trp-lac fusion promoters and more specifically for Streptomyces, the ermE, melC and tipA promoters, etc. In a specific embodiment described in Chapter 11 below, the expression vector is constructed to contain the AveC ORF cloned with the strong constitutive ermE promoter from Saccharopolyspora erythraea. The vector was transformed into S. avermitilis and after that HPLC analysis of the fermentation products showed an increased titer of avermectins that were produced compared to the production by the same species but which in addition expresses the AveC allele of the original type.

Fusion protein expression vectors can be used to express an AveC gene protein fusion product. The purified fusion protein can be used to raise antisera against the AveC gene product, to study the biochemical properties of the AveC gene product, to form AveC fusion proteins with different biochemical activities, or to aid in the identification or purification of the expressed AveC gene product. Possible fusion protein expression vectors include, but are not limited to, vectors incorporating sequences encoding β-galactosidase and trpE fusions, maltose binding protein fusions, glutathione-S-transferase fusions, and polyhistidine fusions (carrier regions). In an alternative embodiment, the AveC gene product or part thereof may be fused to an AveC homologous gene product or part thereof, derived from other species or genera of Streptomyces such as, for example, S. hygroscopicus or S. griseochromogenes. In a particular embodiment described in Chapter 12 below and shown in Figure 7, a chimeric plasmid is constructed to contain a 564 bp region of the S. hygroscopicus AveC homologous ORF replacing the 564 bp region of the S. avermitilis AveC ORF. Such hybrid vectors can be transformed into S. avermitilis cells and tested to determine their effect, for example, on the 2:1 class ratio of avermectins produced.

AveC fusion proteins can be designed to encompass a region that is applicable for purification. For example, AveC-maltose-binding protein fusions can be purified using amylose resin; AveC-glutathione-S-transferase fusion proteins can be purified using glutathione-agarose beads; and AveC-polyhistidine fusions can be purified using divalent nickel resin. Alternatively, antibodies against the carrier protein or peptide can be used to purify the fusion protein by affinity chromatography. For example, a nucleotide sequence encoding a target epitope of a monoclonal antibody can be incorporated into an expression vector in operative association with regulatory elements and positioned such that the expressed epitope is fused to an AveC polypeptide. For example, a nucleotide sequence encoding a FLAGTM epitope tag (International Biotechnologies Inc.), which is a hydrophilic peptide marker, can be inserted using standard techniques into an expression vector at a point corresponding to, for example, the carboxyl terminus of the AveC polypeptide. The expressed AveC polypeptide-FLAGTM epitope fusion product can then be detected and affinity-purified using commercially available anti-FLAGTM antibodies.

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

A signal sequence above, and in reading frame with, the AveC ORF can be incorporated into an expression vector using known methods to direct the movement and secretion of the expressed gene product. Non-limiting examples of signal sequences include those from α-factor, immunoglobulins, outer membrane proteins, penicillinase, and T-cell receptors, among others.

To aid in the selection of host cells transformed or transfected with the cloning or expression vectors of the described invention, the vector may be designed to further comprise a coding sequence for a reporter gene product or other selectable marker. Such a coding sequence is preferably in operative association with a regulatory element coding sequence, as described above. Reporter genes that can be used in the invention are well known in the art and include those encoding green fluorescent protein, luciferase, xyIE, and tyrosinase, among others. Nucleotide sequences encoding selectable markers are well known in the art and include those encoding gene products that have resistance to antibiotics or antimetabolites, or those that fulfill an auxotrophic requirement. Examples of such sequences include those encoding resistance to erythromycin, thiostrepton, or kanamycin, among others.

Transformation of the host cell

The disclosed invention further provides transformed host cells comprising the polynucleotide molecule or recombinant vector of the invention, and novel species or cell lines derived therefrom. Host cells applicable in the practical application of the invention are preferably Streptomyces cells, although other prokaryotic cells or eukaryotic cells may also be used. Such transformed host cells typically include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA vectors, or yeast transformed with recombinant vectors, among others.

The polynucleotide molecules of the described invention are intended to function in Streptomyces cells, but may also be transformed into other bacterial or eukaryotic cells, for example, for cloning or expression purposes. An E. coli strain may be commonly used, such as, for example, the DH5α strain available from the American Type Culture Collection (ATCC), Rockville, MD, USA (Accession No. 31343) and from commercial sources (Stratagene). Preferred eukaryotic host cells include yeast cells, although mammalian or insect cells may also be effectively used.

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

Once an expression vector is introduced into a host cell, the integration and maintenance of the AveC coding sequence, either in the host cell chromosome or episomally, can be confirmed using standard techniques, for example, by Southern hybridization analysis, restriction enzyme analysis, PCR analysis including reverse transcriptase PCR (rt-PCR), or using an immunoassay to detect the expected genetic product. Host cells containing and/or expressing a recombinant AveC coding sequence can be identified using one of four general approaches well known in the art and include: (i) DNA-DNA, DNA-RNA, or RNA-antisense RNA hybridization; (ii) detecting the presence of "marker" genetic functions; (iii) examining the level of transcription as measured by the expression of AveC-specific mRNA transcripts in the host cell; and (iv) detecting the presence of the mature polypeptide product as measured, for example, by immunoassay or by the presence of AveC biological activity (eg, production of specific ratios and amounts of avermectin indicative of AveC activity in, for example, S. aveermitilis host cells).

Expression and characterization of the recombinant AveC gene product

Once the AveC coding sequence is stably introduced into the appropriate host cell, the transformed host cell is clonally multiplied and the resulting cells are grown under conditions that lead to maximal production of the AveC gene product. Such conditions usually involve high cell growth. When the expression vector comprises an inducible promoter, appropriate induction conditions, such as, for example, temperature change, nutrient consumption, addition of optional inducers (for example, carbohydrate analogs, such as isopropyl-β-D-thiogalactopyranoside (IPTG) ), accumulation of excess metabolic by-products, or the like, are used as necessary to induce expression.

When the expressed AveC gene product is retained within the host cells, the cells are harvested and lysed and the product is isolated and purified from the lysate under extraction conditions known in the art to minimize protein degradation such as, for example, at 45°C or in presence of protease inhibitors or both. When the expressed AveC gene product is secreted from host cells, the spent nutrient 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 culture medium, as appropriate, using standard procedures including, but not limited to, some combination of the following methods: ammonium sulfate precipitation, size fractionation, ion-exchange chromatography, HPLC, density centrifugation and affinity chromatography. When the expressed AveC gene product shows biological activity, the increase in the purity of the preparation can be monitored at each stage of the purification procedure using an appropriate assay. Whether or not the expressed AveC gene product exhibits biological activity, this may be detected on the basis of, for example, size or reactivity with an antibody otherwise specific for AveC, or by the presence of a fusion tag.

The described invention thus provides a recombinantly-expressed S. avermitilis AveC genetic product comprising the amino acid sequence of plasmid pSE 186 (ATCC 209604) which encodes the AveC genetic product or the amino acid sequence from Figure 1 (SEQ ID NO: 2) or its essential part and its homologues.

The described invention thus provides a recombinantly expressed S. hygroscopicus AveC homologous genetic product comprising the amino acid sequence of SEQ ID NO: 4 or its essential part and its homologs.

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

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

Once an AveC gene product of sufficient purity is obtained, it can be characterized using standard methods including SDS-PAGE, size exclusion chromatography, amino acid sequence analysis, biological activity in the production of the corresponding products in the avermectin biosynthetic pathway, etc. For example, amino the acid sequence of the AveC gene product can be determined using standard peptide sequencing techniques. The AveC gene product can be further characterized using hydrophilic analysis (see, for example, Hopp and Woods: "Proc. Natl. Acad.Sci. USA", 78:3824, (1981)) or analogous software algorithms, to identify hydrophobic and hydrophilic regions of the AveC genetic product. Structural analysis can be performed to identify regions of the AveC gene product for presumed specific secondary structures. Biophysical methods, such as X-ray crystallography (Engstrom: "Biochem. Exp. Biol.", 11:7-13, (1974)), computer modeling (Fletterick and Zoller: "Current Communications in Molecular Biology", (1986), published by Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and nuclear magnetic resonance (NMR) can be used to map and study the site of interaction between the AveC gene product and its substrate. The information obtained from these studies can be used to select new mutation sites in the AveC ORF to help develop new strains of S. avermitilis that have even more desirable avermectin production characteristics.

3 Construction and use of AveC mutants

The described invention provides a polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as the sequence of plasmid pSE 186 (ATCC 209604) encoding the S. avermitilis AveC gene product or the nucleotide sequence of the AveC ORF of S. avermitilis as shown in Figure 1 (SEQ ID NO: 1 ), but which further comprises one or more mutations, so that cells of S. avermitilis species ATCC 53692 in which the AveC allele of the original type is inactivated and which express a polynucleotide molecule comprising the mutated nucleotide sequence produce a different ratio or amount of avermectin compared to those which produced using cells of S. avermitilis strain ATCC 53692 which, in addition, express only the AveC allele of the original type.

According to the described invention, such polynucleotide molecules can be used to produce new strains of S. avermitilis that show a detectable change in avermectin production compared to the same strain that in addition expresses only the AveC allele of the original type. In a preferred embodiment, such polynucleotide molecules are applicable for the production of new species of S. avermitilis that produce avermectins in a reduced 2:1 class ratio compared to the same species that in addition expresses only the allele of the original type. In a further preferred embodiment, such polynucleotide molecules are applicable to the production of new strains of S. avermitilis which produce increased levels of avermectin compared to the same strain which in addition expresses only the AveC allele of the original type. In a further preferred embodiment, such polynucleotide molecules are applicable for the production of new strains of S. avermitilis in which the AveC gene is deactivated.

Mutations in the AveC coding sequence include those mutations which introduce amino acid deletions, additions or substitutions, into the AveC gene product, or which lead to truncation of the AveC gene product, or some combination thereof, and which produce the desired result. For example, the described invention provides polynucleotide molecules comprising the sequence of plasmid pSE 186 (ATCC 209604) encoding the AveC gene product or the nucleotide sequence of the AveC ORF of S. avermitilis as shown in Figure 1 (SEQ ID NO: 1), but further comprising a or more mutations that encode the substitution of an amino acid residue with a different amino acid residue at a selected position in the AveC gene product. In several non-limiting embodiments provided by way of example below, such substitutions may be made at any of amino acid positions 55, 138, 139 or 230, or some combination thereof.

Mutations in the AveC coding sequence are made using any of a number of known methods including the use of missense-prone PCR or cassette mutagenesis. For example, oligonucleotide-directed mutagenesis can be used to alter the AveC ORF sequence in a defined manner such as, for example, introducing one or more restriction sites, or a terminal codon into specific regions of the AveC ORF sequence. Procedures such as those described in US Pat. No. 5,605,793 involving non-directed fragmentation, repeat mutagenesis cycles, and nucleotide shuffling, can also be used to generate large libraries of polynucleotides having nucleotide sequences encoding AveC mutations.

Targeted mutations are applicable, particularly where these serve to alter one or more conserved amino acid residues in the AveC gene product. For example, comparing the deduced amino acid sequences of AveC gene products and AveC homologous gene products from S. avermitilis (SEQ ID NO: 2), S. gnseochromogenes (SEQ ID NO: 5) and S. hygroscopicus (SEQ ID NO: 4) as is shown in Figure 6, indicates sites of significant conservation of amino acid residues between these species. Targeted mutagenesis that leads to a change in one or more of these conserved amino acid residues can be particularly effective in producing new mutant species that exhibit desirable changes in avermectin production.

Non-directed mutagenesis may also be applicable and may be performed by exposing S. avermitilis cells to ultraviolet radiation or X-rays or chemical mutagens such as N-methyl-N'-nitrosoguanidine, ethyl methane sulfonate, nitric acid or nitrogen mustards. See, for example, Ausubel, forward for a review of techniques for mutagenesis.

Once mutated polynucleotide molecules are formed and tested to determine whether they modulate avermectin biosynthesis in S. avermitilis. In a preferred embodiment, the polynucleotide molecule having the mutated nucleotide sequence is tested using a complementing strain of S. avermitilis in which the AveC gene has been inactivated to produce an AveC negative (AveC-) base. In a non-limiting method, the mutated polynucleotide molecule is inserted into an expression plasmid in operative association with one or more regulatory elements, wherein the plasmid also preferably comprises one or more drug resistance genes to ensure selection of transformed cells. The vector is then transformed into AveC host cells using known techniques, and the transformed cells are selected and cultured in an appropriate fermentation medium under conditions that allow induction of avermectin production. The fermentation products are then analyzed by HPLC to determine the ability of the mutated polynucleotide molecule to complement the host cell. More vectors carrying mutated polynucleotide molecules capable of reducing the B2:B1 ratio of avermectin, including pSE 188, pSE 199 and pSE 231, are exemplified in Section 8.3, herein below.

The disclosed invention further provides methods for identifying mutations of the AveC ORF of S. avermitilis that may alter the ratio and/or amount of avermectins produced. In a preferred embodiment, the described invention provides a method for identifying AveC ORF mutations that can alter the 2:1 class ratio of produced avermectins, comprising: (a) determining the 2:1 class ratio of avermectins produced by S. avermitilis cells in which native AveC the allele is deactivated and in which a polynucleotide molecule comprising the nucleotide sequence encoding the mutated AveC gene product is introduced and expressed; (b) determination of the class 2:1 ratio of avermectins which are produced using cells of the same S. avermitilis species as in step (a) but which in addition express only the AveC allele having the nucleotide sequence of the ORF of Figure 1 (SEQ ID NO: 1) or a nucleotide sequence that is homologous to this; and (c) comparing the 2:1 class ratio of avermectins produced by S. avermitilis cells of stage (a) to the 2:1 class ratio of avermectins produced by S. avermitilis cells of stage (b), such that if the class ratio 2:1 of avermectins produced by S. avermitilis cells of stage (a) is different from the 2:1 class ratio of avermectins produced by S. avermitilis cells of stage (b), then an AveC ORF mutation that can change the class ratio of avermectins can be identified. In a preferred embodiment, the class 2:1 ratio of avermectins produced is lowered by mutation.

In a further preferred embodiment, the described invention provides a method for identifying mutations of the AveC ORF or genetic constructs comprising the AveC ORF that can alter the amount of avermectin produced, comprising: (a) determining the amount of avermectin produced by S. avermitilis cells in which the natural AveC allele is inactivated and in which a polynucleotide molecule comprising a nucleotide sequence encoding a mutated AveC gene product or comprising a genetic construct comprising a nucleotide sequence encoding an AveC gene product is introduced and expressed; (b) determining the amount of avermectin produced by cells of the same S. avermitilis species as in step (a) but which in addition express only the AveC allele having the nucleotide sequence of the ORF of Figure 1 (SEQ ID NO: 1) or a nucleotide sequence that is homologous to this; and (c) comparing the amount of avermectin produced by the S. avermitilis cells of stage (a) versus the amount of avermectin produced by the S. avermitilis cells of stage (b); so that if the amount of avermectin produced by S. avermitilis cells of stage (a) is different from the amount of avermectin produced by S. avermitilis cells of stage (b), then a mutation of the AveC ORF or a genetic construct that can alter the amount of avermectin can be identified . In a preferred embodiment, the amount of avermectin is increased by mutation.

Some of the aforementioned methods for identifying mutations are carried out by using fermentation culture medium preferably supplemented with cyclohexane carboxylic acid, although other suitable fatty acid precursors, such as the fatty acid precursors given in Table 1, may also be used.

Once the mutated polynucleotide molecule that modulates avermectin production in the desired direction has been identified, the location of the mutation in the nucleotide sequence can be determined. For example, a polynucleotide molecule having a nucleotide sequence encoding a mutated AveC gene product can be isolated by PCR and subjected to DNA sequence analysis using known methods. By comparing the DNA sequence of the mutated AveC allele with that of the wild-type AveC allele, the mutation(s) responsible for the change in avermectin production can be determined. In specific, although non-limiting embodiments of the described invention, the S. avermitilis AveC genetic product comprises either a single amino acid substitution at one of residues 55 (S55F), 138 (S138T), 139 (A139T) or 230 (G230D), or a double substitution at positions 138 (S138T) and 139 (A139T) confer changes in the production function of the AveC gene so that the 2:1 class ratio of produced avermectins is altered (see Chapter 8, below). Therefore, polynucleotide molecules having nucleotide sequences encoding S. avermitilis AveC gene products comprising amino acid substitutions at one or more amino acid residues 55, 138, 139 or 230 or some combination thereof are encompassed by the present invention.

The described invention further provides mixtures for obtaining new species of S. avermitilis, whose cells contain a mutated AveC allele that leads to a change in the production of avermectin. For example, the described invention provides recombinant vectors that can be used to target some of the polynucleotide molecules comprising the mutated nucleotide sequences of the described invention to the AveC gene site of the S. avermitilis chromosome to insert or replace the AveC ORF or part thereof by homologous recombination. According to the described invention, even more, the polynucleotide molecule comprising the mutated sequence of the described invention may also function to modulate avermectin biosynthesis when inserted into the S. avermitilis chromosome at a site other than the AveC gene, or when retained episomally in S. avermitilis cells. . Thus, the described invention also provides vectors comprising a polynucleotide molecule comprising a mutated nucleotide sequence of the described invention, where the vector may be used to insert the polynucleotide molecule into a site in the S. avermitilis chromosome that is not on the AveC gene or may be episomally retained.

In a preferred embodiment, the described invention provides gene replacement vectors that can be used to introduce a mutated AveC allele into S. avermitilis cells, thereby forming new S. avermitilis species whose cells produce avermectins in an altered class 2:1 ratio compared to cells the same species which in addition express only the AveC allele of the original type. In a preferred embodiment, the class 2:1 ratio of avermectin produced by the cells is reduced. Such gene replacement vectors can be formed using the mutated polynucleotide molecules present in the expression vectors provided herein, such as PSE 188, pSE 199 and pSE 231, where the expression vectors are exemplified in section 8.3 below.

In a further preferred embodiment, the described invention provides vectors that can be used to introduce a mutated AveC allele into cells of the S. avermitilis species in order to form new types of cells that produce altered amounts of avermectin compared to cells of the same species that, in addition, express only the AveC allele of the original type. In a preferred embodiment, the amount of avermectin produced by the cells is increased. In a specific but non-limiting embodiment, such a vector further comprises a strong promoter known in the art, such as, for example, the strong constitutive ermeE promoter from Saccharopolyspora erythraea, located upstream of, and in operative association with, the AveC ORF. Such a vector may be plasmid pSE 189, which is described in Example 11 hereinbelow, or may be formed using a mutated AveC allele of plasmid pSE 189.

In a further preferred embodiment, the described invention provides gene replacement vectors that are useful in deactivating the AveC gene in the original type species S. avermitilis. In a non-limiting embodiment, such gene replacement vectors can be formed using the mutated polynucleotide molecule present in plasmid pSE 180 (ATCC 209605), exemplified in section 8.1 below (Figure 3). The described invention further encompasses gene replacement vectors comprising a polynucleotide molecule comprising or consisting of a nucleotide sequence naturally flanking the AveC gene in situ in the S. avermitilis chromosome including, for example, those flanking nucleotide sequences shown in Figure 1 (SEQ ID NO: 1) where vectors can be used to delete the S. avermitilis AveC ORF.

The described invention further provides methods for obtaining new species of S. avermitilis that express a mutated AveC allele and that produce an altered ratio and/or amount of avermectin compared to cells of the same species of S. avermitilis that, in addition, express only the AveC allele of the original type. In a preferred embodiment, the described invention provides a method for obtaining new species of S. avermitilis that include cells that express a mutated AveC allele and that produce an altered 2:1 class ratio of avermectin in relation to the same species of S. avermitilis that, in addition, only expresses the AveC allele of the original type , which involves transforming S. avermitilis cells with a vector carrying a mutated AveC allele that encodes a gene product that alters the 2:1 class ratio of avermectins produced by S. avermitilis cells expressing the mutated AveC allele relative to cells of the same species that that express only the wild-type AveC allele and selecting transformed cells that produce avermectins in an altered 2:1 class ratio compared to the 2:1 class ratio produced by cells that additionally express only the wild-type AveC allele. In a preferred embodiment, the altered class 2:1 ratio of avermectin is reduced.

In a further preferred embodiment, the described invention provides a method for obtaining new S. avermitilis species comprising cells that produce altered amounts of avermectin, comprising transforming S. avermitilis cells with a vector carrying a mutated AveC allele or a genetic construct comprising an AveC allele, the expression of which leads to a change in the amount of avermectins produced by S. avermitilis cells that express a mutated AveC allele or a genetic construct compared to cells of the same species that, in addition, express only the AveC allele of the original type and the selection of transformed cells that produce avermectins in an altered amount in relation to on the amount of avermectin produced by cells that, in addition, express only the AveC allele of the original type. In a preferred embodiment, the amount of avermectin produced in the transformed cells is increased.

In a further preferred embodiment, the described invention provides a method for obtaining new species of S. avermitilis, whose cells comprise a deactivated AveC allele, which comprises transforming cells of the species S. avermitilis that express the AveC allele of the original type with a vector that deactivates the AveC allele, and selecting the transformed cells in in which the AveC allele is deactivated. In a preferred, non-limiting embodiment, S. avermitilis cells are transformed with a gene replacement vector carrying an AveC allele that has been inactivated by mutation or by replacing a portion of the AveC allele with a heterologous genetic sequence, and transformed cells in which the native AveC allele of the cell is replaced with the inactivated AveC allele are selected. Deactivation of the AveC allele can be determined by HPLC analysis of the fermentation products, as described hereinbelow. In a specific, say non-limiting embodiment described in section 8.1 below, the AveC allele is inactivated by inserting the ermE gene from Saccharopolyspora erythraea into the AveC ORF.

The described invention further provides new species of S. avermitilis comprising cells transformed with one of the polynucleotide molecules or vectors of the described invention. In a preferred embodiment, the described invention provides new species of S. avermitilis comprising cells that express a mutated AveC allele in place of, or in addition to, an untreated type of AveC allele, where the cells of the new species produce avermectins in an altered class 2:1 ratio compared to class 2: 1 ratio of avermectins that are produced using cells of the same species that, in addition, express only the AveC allele of the original type. In a preferred embodiment, the altered class 2:1 ratio produced by the new cells is reduced. Such new species are applicable in the large-scale production of commercially desirable avermectins, such as doramectin.

The primary object of the experimental tests described here is to identify mutated alleles of the AveC gene whose expression in S. avermitilis cells alters, and more precisely, reduces the class 2:1 ratio of produced avermectins. In a preferred embodiment, the ratio of B2:B1 avermectins produced by a novel S. avermitilis strain of the described invention expressing a mutated AveC allele that lowers the class 2:1 ratio of avermectins produced is between 1.6:1 to about 0: 1, in an even more preferred embodiment, the ratio is between about 1:1 to about 0:1, and in the most preferred embodiment, the ratio is between about 0.84:1 to about 0:1. In a specific embodiment described below, the novel cells of the described invention produce cyclohexyl B2: cyclohexyl B1 avermectins in a ratio of less than 1.6:1. In various specific embodiments described hereinbelow, the novel cells of the described invention produce cyclohexyl B2:cyclohexyl B1 avermectins at a ratio of about 0.94:1. In a further different specific embodiment described hereinbelow, the novel cells of the described invention produce cyclohexyl B2:cyclohexyl B1 avermectins at a ratio of about 0.88:1. In a further different specific embodiment described hereinbelow, the novel cells of the described invention produce cyclohexyl B2:cyclohexyl B1 avermectins at a ratio of about 0.84:1.

In a further preferred embodiment, the described invention provides new species of S. avermitilis comprising cells expressing a mutated AveC allele or a genetic construct comprising an AveC allele, instead of or in addition to the AveC allele of the original type, wherein the cells of the new species produce an altered amount of avermectin compared to cells the same species which in addition express only the AveC allele of the original type. In a preferred embodiment, the new species produces an increased amount of avermectin. In a non-limiting embodiment, the genetic construct further comprises a strong promoter, such as a strong constitutive promoter from Saccharopolypora erythraea, upstream of and in operative association with the AveC ORF.

In a further preferred embodiment, the described invention provides new species of S. avermitilis comprising cells in which the AveC gene has been deactivated. Such species are applicable both to the different spectrum of avermectins that are produced in relation to the original type species, and to the complementation of experimental tests as described herein, to determine whether targeted or non-targeted mutagenesis of the AveC gene affects avermectin production. In a specific embodiment described below, S. avermitilis host cells are genetically engineered to contain an inactivated AveC gene. For example, strain SE 180-11, which is described in the examples below, was made using the gene exchange plasmid pSE 180 (ATCC 209605) (Figure 3) which was made to inactivate the S. avermitilis AveC gene by inserting the ermE resistance gene into AveC coding region.

The described invention further provides recombinantly expressed, S. avermitilis AveC genetic products that are encoded by one of the aforementioned polynucleotide molecules of the invention and methods for obtaining the same.

The described invention further provides a process for the production of avermectin, which comprises culturing cells of the species S. avermitilis, which express a mutated AveC allele that encodes a genetic product that alters the class 2:1 ratio of avermectins that are produced by a strain of S. avermitilis that expresses a mutated AveC allele in the ratio to cells of the same species that, in addition, express only the AveC allele of the original type, in the culture medium under conditions that allow or induce the production of this avermectin, and the isolation of said avermectin from the culture. In a preferred embodiment, the class 2:1 ratio of avermectins produced in culture by cells expressing the mutated AveC allele is reduced. This process ensures increased efficiency in the production of commercially valuable avermectins, such as doramectin.

The described invention further provides a process for the production of avermectin, which comprises culturing cells of the S. avermitilis species, which express a mutated AveC allele or a genetic construct that leads to the production of an altered amount of avermectins that are produced by a S. avermitilis species that expresses a mutated AveC allele or a genetic construct in in relation to cells of the same species which, in addition, express only the AveC allele of the original type, in the culture medium under conditions that allow or induce the production of this avermectin, and the extraction of said avermectin from the culture. In a preferred embodiment, the amount of avermectin produced in culture by cells expressing the mutated AveC allele or genetic construct is increased.

The described invention further provides a novel mixture of avermectins produced by S. avermitilis expressing a mutated AveC allele encoding a gene product that reduces the class 2:1 ratio of avermectins produced by S. avermitilis expressing a mutated AveC allele relative to cells of the same species that in addition express only the AveC allele of the original type, where the avermectins in the new mixture are produced in a reduced class 2:1 ratio compared to the class 2:1 ratio of avermectins produced by cells of the same species S. avermitilis that in addition express only AveC allele of the original type. The new avermectin mixture may be present as produced in the spent fermentation culture fluid or may be collected from it. The novel avermectin mixture may be partially or substantially purified from the culture fluid using known biochemical purification techniques, such as ammonium sulfate precipitation, dialysis, size fractionation, ion exchange chromatography, HPLC, and so on.

4. Applications of avermectin

Avermectins are very active antiparasitic agents that are particularly useful as anthelmintics, ectoparasiticides, insecticides and acaricides. Avermectin compounds which are produced according to the processes of the described invention are applicable for any of these purposes. For example, the avermectin compounds obtained according to the described invention are applicable for the treatment of various diseases and conditions in humans, especially those diseases or conditions caused by parasitic infections, as known in the art. See, for example, "Ikeda and Omura: Chem. Rev.", 97(7):2591-2609, (1997). More specifically, the avermectin compounds obtained by the described invention are effective in treating various diseases or conditions caused by endoparasites, such as parasitic nematodes, which can infect humans, domestic animals, pigs, sheep, cattle, horses or cattle.

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

The avermectin compounds obtained according to the present invention are also applicable in the treatment of ectoparasitic infections including, for example, arthropod infestations in mammals and birds caused by ticks, mites, fleas, buzzing flies, biting insects or migrating dipteran larvae that can act on cattle and horses, among others.

The avermectin compounds obtained according to the invention are also useful as insecticides against household pests such as, for example, cockroaches, fabric-biting moths, carpet-biting beetles and houseflies among others, as well as insect pests that lodge in cereals and agricultural plants which include spider mites, aphids, caterpillars and orthopterans, such as grasshoppers, among others.

Animals that can be treated with the avermectin compounds of the described invention include sheep, cattle, horses, deer, goats, pigs, birds including animals, dogs and cats.

The avermectin compound obtained according to the described invention is applied in a formulation that corresponds specifically to the intended application, the specific species of the animal host being treated and the parasite or insecticide in question. For use as a parasiticide, the avermectin compound of the described invention may be administered orally in the form of a capsule, bolus, tablet or liquid drink or, alternatively, may be administered by pouring on, or by injection, or in the form of implants. Such formulations are obtained in the usual way in accordance with standard veterinary practice. Thus, capsules, boluses or tablets can be obtained by mixing the active ingredient with a suitable finely ground diluent or carrier that additionally contains a disintegrating agent and/or binder, such as starch, lactose, talc, magnesium stearate, etc. The beverage formulation can be obtained by dispersing the active ingredient in an aqueous solution together with a dispersing or wetting agent, etc. Injectable formulations can be obtained in the form of a sterile solution that may contain other substances, such as, for example, sufficient salt and/or glucose to achieve isotonicity of the solution with blood.

Such formulations will vary with respect to the mass of the active compound depending on the patient or species of animal host being treated, the severity and type of infection and the host's body weight. Usually, for oral administration, a dose of the active compound of about 0.001 to 10 mg per kg of body weight of the patient or animal, given as a single dose or in the form of several smaller (divided) doses, and it will be sufficient to give it over a period from 1 to 5 days. However, there may be cases where higher or lower dosage levels are indicated, as determined, for example, by a physician or veterinarian and based on clinical symptoms.

As an alternative, the avermectin compound obtained according to the described invention can be applied in combination with animal feed and for this purpose a concentrated nutrient additive or premix can be obtained for mixing with normal animal feed.

For application in the form of insecticides, and for the treatment of agricultural pests, the compounds of the described invention can be applied in the form of sprays, powders, emulsions and the like, in accordance with standard agricultural practice.

EXAMPLE: Fermentation of streptomyces avermitilis species and analysis of B2:B1 avermectin

Species that do not contain both activities, i.e. 2-oxo branched chain acid hydrogenase and 5-O-methyltarnasferase, do not produce avermectins if the fermentation medium is not supplemented with fatty acids. This example shows that in such mutants a wide range of B2:B1 ratios of avermectin can be obtained when biosynthesis is initiated in the presence of different fatty acids.

1 Materials and procedures

The strain Streptomyces avermitilis ATCC 53692 which was stored at -70 °C as a whole broth which was obtained in a seeded medium consisting of starch (Nadex, Laing National) 20g; Pharmamedia (Trader's Protein, Memphis, TN) 15 g; Ardamine pH (Champlain Inc. Clifton, NJ) 5 g; CaCO3 1 g. The final volume is adjusted to 1 liter with plain water, the pH is adjusted to 7.2 and the medium is autoclaved at 121 °C for 25 minutes.

Two milliliters of the dissolved suspension of the above preparation is used to inoculate a bottle containing 50 ml of the same medium. After 48 hours of incubation at 28 °C on a rotating shaker at 180 revolutions per minute, 2 ml of broth is used to inoculate a bottle containing 50 ml of production medium consisting of: starch 80 g, calcium carbonate 7 g; Pharamamedia 5 g; dicalcium phosphate 1 g; magnesium sulfate 1 g; glutamic acid 0.6 g; ferrous sulfate heptahydrate 0.001 g; magnesium sulfate 0.001 g. The final volume was adjusted to 1 liter with plain water, the pH was adjusted to 7.2 and the medium was autoclaved at 121 °C for 25 minutes.

Various carboxylic acid substrates (see Table 1) were dissolved in methanol and added to the fermentation broth 24 hours after inoculation to obtain a final concentration of 0.2 g/liter. The fermentation broth was incubated for 14 days at 28 °C, then the broth was centrifuged (2,500 rpm for 2 minutes) and the supernatant was discarded. The mycelial pellet is extracted with acetone (15 ml), then with dichloromethane (30 ml) and the organic phase is separated, filtered, then evaporated to dryness. The residue was taken up in methanol (1 ml) and analyzed by HPLC with a Hewlett-Packard 1090A liquid chromatograph equipped with a scanning arrow diode detector set at 240 nm. A Beckman Ultrasphere C-18, 5 µm, 4.6 mm × 25 cm column maintained at 40 °C was used. Twenty-five µl of the above methanol solution is injected onto the column. Elution is performed with a linear gradient of methanol-water mixture from 80:20 to 95:5 over 40 minutes at 0.85 ml/minute. Two standard concentrations of cyclohexyl B1 were used to calibrate the detector response, and the area under the curves for B2 and B1 avermectins was measured.

2 Results

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

The data given in Table 1 show an extremely wide range of B2:B1 ratios of the produced avermectins, indicating a significant difference in the results of the dehydrative conversion of class 2 compounds to class 1 compounds, depending on the nature of the fatty acid side chain as the plasma unit supplied. This indicates that changes in the B2:B1 ratio resulting from changes in the AveC protein may be specific for given substrates. Therefore, testing for mutants showing changes in the B2:B1 ratio obtained with a particular substrate should be performed in the presence of the substrate. The examples that follow in the description below use cyclohexanecarboxylic acid as the test substrate. However, this substrate is used only as an exemplary potential and is not intended to limit the applicability of the described invention.

Table 1

[image]

EXAMPLE: Isolation of the AveC gene

This example describes the isolation and characterization of the region of the Streptomyces avermitilis chromosome encoding the AveC gene product. As shown below, the AveC gene has been identified as being able to modify the cyclohexyl-B2 to cyclohexyl-B1 (B2:B1) ratio of the produced avermectins.

1 Materials and procedures

Cultivation of Streptomyces species for DNA isolation

The following method was followed for the growth of Streptomyces. Single colonies of S. avermitilis ATCC 31272 (single colony isolate #2) were isolated on 1/2 strength YPD-6 medium containing: Difco Yeast Extract 5 g; Difco Bacto-peptone 5 g; dextrose 2.5 g; MOPS 5 g; Difco Bacto agar 15 g. The final volume was adjusted to 1 liter with dH2O, the pH was adjusted to 7.0 and the medium was autoclaved at 121 °C for 25 minutes.

The grown mycelia in the upper medium were used to inoculate 10 ml of TSB medium (Difco Tryptic Soy Broth 30 g in 1 liter of dH2O autoclaved at 121 °C for 25 min) in a 25 mm × 150 mm test tube maintained under shaking (300 rpm in minutes) at 28 °C for 48 to 72 hours.

Isolation of chromosal DNA from Streptomyces species

Aliquots (0.25 ml or 0.5 ml) of mycelia grown as described above were placed in 1.5 ml microcentrifuge tubes and cells were concentrated by centrifugation at 12,000 x g for 60 seconds. The supernatant was discarded and the cells were resuspended in 0.25 ml of TSE buffer (20 ml of 1.5 M sucrose, 2.5 ml of 1 M Tris-HCl, pH 8.0, 2.5 ml of 1 M EDTA, pH 8.0 and 75 ml dH2O) containing 2 mg/ml lysozyme. Samples were incubated at 37 °C for 20 min with shaking, introduced into an AutoGen 540TM instrument for automated nucleic acid extraction (Integrated Separation Systems, Natick, MA), and genomic DNA was isolated using Cycle 159 (equipment software) according to the manufacturer's instructions.

Alternatively, 5 ml of mycelia were placed in a 17 mm × 100 mm tube, the cells were concentrated by centrifugation at 3,000 rpm for 5 min and the supernatant was removed. Cells were resuspended in 1 ml of TSE buffer, concentrated by centrifugation at 3,000 rpm for 5 minutes and the supernatant was removed. Cells were resuspended in 1 ml of TSE buffer containing 2 mg/ml lysozyme and incubated at 37 °C with agitation for 30 to 60 minutes. After incubation, 0.5 ml of 10% sodium dodecyl sulfate (SDS) was added and cells were incubated at 37 °C until lysis was complete. The lysate was incubated at 65 °C for 10 min, cooled to room temperature, divided into two 1.5 Eppendorf tubes, and extracted at once with 0.5 ml of a phenol/chloroform mixture (50% phenol previously equilibrated with 0.5 M Tris, pH 8.0; 50% chloroform). The aqueous phase was removed and extracted 2 to 5 times with a mixture of chloroform:isoamyl alcohol (24:1). DNA was precipitated by adding 1/10 volume of 3M sodium acetate, pH 4.8, incubating the mixture on ice for 10 min, centrifuging the mixture at 15,000 rpm at 5 °C for 10 min, and removing the supernatant to clean the tube into which it was added. one volume of isopropanol. The supernatant plus the isopropanol mixture were then incubated on ice for 20 minutes, centrifuged at 15,000 rpm for 20 minutes at 5°C, the supernatant was removed and the DNA pellet was washed once with 70% ethanol. After drying the pellet, the DNA was resuspended in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).

Isolation of plasmid DNA from S. avermitilis species

An aliquot (1.0 ml) of the mycelia was placed in a 1.5 ml microcentrifuge tube and the cells were concentrated by centrifugation at 12,000 x g for 60 seconds. The supernatant was discarded and the cells were resuspended in 1.0 ml of 10.3% sucrose and concentrated by centrifugation at 12,000 x g for 60 seconds and the supernatant was discarded. Cells were then resuspended in 0.25 ml of TSE buffer containing 2 mg/ml of lysosomes, incubated at 37°C for 20 minutes with shaking, and introduced into an AutoGen 540TM automated nucleic acid extraction instrument. Plasmid DNA was isolated using Cycle 106 (equipment software) according to the manufacturer's instructions.

Alternatively, 1.5 ml of mycelia were placed in a 1.5 ml microcentrifuge tube and the cells were concentrated by centrifugation at 12,000 x g for 60 seconds. The supernatant was discarded, the cells were resuspended in 1 ml of 10.3% sucrose, concentrated by centrifugation at 12,000 x g for 60 seconds and the supernatant was discarded. Cells were resuspended in 0.5 ml TSE buffer containing 2 mg/ml lysosomes and incubated at 37 °C for 15 to 30 minutes. After incubation, 0.25 ml of alkaline SDS (0.3 N NaOH, 2% SDS) was added and the cells were incubated at 55°C for 15 to 30 minutes or until a clear solution was obtained. Sodium acetate (0.1 ml, 3M, pH 4.8) was then added to the DNA solution and this solution was incubated on ice for 10 minutes. DNA samples were centrifuged at 14,000 rpm for 10 minutes at 5 °C. The supernatant was removed to clean the tube and 0.2 ml of a phenol:chloroform mixture (50% phenol:50% chloroform) was added and mixed gently. The DNA solution was centrifuged at 14,000 rpm for 10 minutes at 5 °C and the upper layer was introduced into a clean Ependorf tube. Isopropanol (0.75 ml) was added and the solution was mixed gently and then incubated at room temperature for 20 minutes. The DNA solution was centrifuged at 14,000 rpm for 15 minutes at 5 °C, the supernatant was discarded and the DNA pellet was washed at once with 70% ethanol, dried and the DNA was resuspended in TE buffer.

Isolation of plasmid DNA from E. coli species

A single transformed E. coli colony 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 liter of dH2O, pH 7.0 which was autoclaved at 121 ° C for 25 minutes and supplemented with 100 µg/ml ampicillin). The culture was incubated overnight and a 1.0 ml aliquot was placed in a 1.5 ml microcentrifuge tube. Culture samples were batched into an AutoGen 540TM instrument for automatic isolation of nucleic acids and plasmids. DNA was isolated using Cycle 3 (equipment software) according to the manufacturer's instructions.

Obtaining and transformation of S. avermitilis protoplast

Individual colonies of S. avermitilis were isolated on 1/2 strength YPD-6 medium. Mycelia were used to inoculate 10 ml of TSB medium in a 25 mm × 150 mm tube which was then incubated with agitation (at 300 rpm) at 28 °C for 48 hours. One ml of mycelium was used to inoculate 50 ml of YEME medium. YEME medium contains in 1 liter: Difco yeast extract 3 g; Difco Baco-peptone 5 g; Difco malt extract 3 g and sucrose 300 g. After autoclaving at 121 °C for 25 minutes, the following was added: 2.5 M MgCl2 • H2O (which was separately autoclaved at 121 °C for 25 minutes) 2 ml; and glycine (20%) (filter-sterilized) 25 ml.

Mycelia were grown at 30°C for 48 to 72 hours and harvested by centrifugation in a 50 ml centrifuge tube (Falcon) at 3,000 rpm for 20 minutes. The supernatant was discarded and the mycelia were resuspended in P buffer containing: sucrose 205 g; K2SO4 0.25 g, MgCl2 • 6H2O 2.02 g; H2O 600 ml; K2PO4 (0.5%) 10 ml; solution of labeled element* 20 ml; CaCl2 • 2H2O (3.68%) 100 ml; MES buffer (1.0 M, pH 6.5) 10 ml. (*The solution of the marked element contains 1 liter each: ZnCl2 40 mg, FeCl3 • 6H2O 200 mg; CuCl2 • 2H2O 10 mg; MnCl2 • 4H20 10 mg; Na2B4O7 • 10H20 10 mg; (NH4)6Mo7O24 • 4H2O 10 mg). The pH was adjusted to 6.5, the final volume was adjusted to 1 liter and the medium was hot filtered through a 0.45 micron filter.

The mycelia were pelleted at 3,000 rpm for 20 min, the supernatant was removed and the mycelia were resuspended in 20 ml P buffer containing 2 mg/ml lysosomes. The mycelia were incubated at 35 °C for 15 minutes with mixing and microscopic examination to determine the progress of protoplast formation. When protoplast formation was complete, the protoplasts were centrifuged at 8,000 rpm for 10 minutes. The supernatant was discarded and the protoplasts were resuspended in 10 ml of P buffer. Protoplasts were centrifuged at 8,000 rpm for 10 min, the supernatant was discarded, the protoplasts were resuspended in 2 ml P buffer, and approximately 1 × 109 protoplasts were dispensed into a 2.0 ml cryogenic vial (Nalgene).

An ampoule containing 1 × 109 protoplasts was centrifuged for 10 min at 8,000 rpm, the supernatant was removed, and the protoplasts were resuspended in 0.1 ml of P buffer. Two to five µg of transformed DNA was added to the protoplasts, which was immediately followed by the addition of 0.5 ml of working T buffer. Basic T buffer contains: PEG 1,000 (Sigma) 25 g; sucrose 2.5 g; and H2O 83 ml. The pH was adjusted to 8.8 with 1N NaOH (filter-sterilized) and the basic T buffer was filter-sterilized and stored at 4 °C. Working buffer T, which was made on the same day as it was used, contains 8.3 ml of base buffer T; K2PO4 (4 mM) 1.0 ml; CaCl2 • 2H2O (5 M) 0.2 ml; and TES (1M, pH 8) 0.5 ml. Each component of the working T buffer is filter-sterilized and stored at 4 °C.

During the 20 second addition of buffer T to the protoplasts, 1.0 ml of buffer P was also added and the protoplasts were centrifuged at 8,000 rpm for 10 minutes. The supernatant was removed and the protoplasts were resuspended in 0.1 ml P buffer. Protoplasts were then placed on RM14 medium containing sucrose 205 g; K2SO4 0.25 g; MgCl2 • 6H2O -0.12 g; glucose 10 g; Difco Casamino acid 0.1 g; Difco yeast extract 5 g; Difco oat agar 3 g; Difco Bacto agar 22 g and H2O 800 ml. The solution was autoclaved at 121 °C for 25 minutes. After autoclaving, the following sterile batches were added: K2PO4 (0.5%) 10 ml; CaCl2 • 2H2O (5 M) 5 ml; L-proline (20%) 15 ml; MES buffer (1.0 M, pH 6.5) 10 ml; solution of labeled element (same as before) 2 ml; cycloheximide batch (25 mg/ml) 40 ml and 1N NaOH 2 ml. Twenty-five ml of RM14 medium was aliquoted per plate and the plates were dried for 24 hours before use.

Protoplasts were incubated in 95% humidity at 30 °C for 20 to 24 hours. To select for erythromycin-resistant transformants, 1 ml of overlay buffer plus 125 µg of erythromycin (to obtain a final concentration of 5 µg/ml of erythromycin) was plated nondirectionally over RM14 regeneration plates. Covering buffer contains per 100 ml: sucrose 10.3 g; labeled element solution (same as above) 0.2 ml and MES (1M, pH 6.5) 1 ml. Protoplasts were incubated in 95% humidity at 30°C for 7 to 14 days until erythromycin-resistant (Thior) colonies were observed.

Transformation of Streptomyces lividans protoplasts

S. lividans TK 64 (provided by the John Innes Institute, Norwich, U.K.) was used for transformations in some cases. Methods and mixtures for growth, protoplastization and transformation of S. lividans are described by Hapwood et al. in "Genetic Manipulation of Streptomyces, A. Laboratory Manual", (1985), John Innes Foundation, Norwich, U.K., and are carried out as there described. Plasmid DNA was isolated from S. lividans transformants as described in section 7.1.3, hereinabove.

Fermentation analysis of S. avermitilis species

S. avermitilis mycelia grown on 1/2 strength YPD-6 medium for 4 to 7 days were inoculated into 2.54 × 15.24 cm (1 × 6 inch) tubes containing 8 ml of medium and two glass beads of 5 mm. This medium contains: soluble starch (either thin boiled starch or KOSO, Japan Corn Starch Co., Nagoya) 20 g/l; Pharmamedia (Trader's Protein, Memphis TN) 15 g/l; Ardamine pH (Champlain Inc. Clifton, NJ) 5 g/l; CaCO3 2 g/l; 2 × bcfa ("bcfa" stands for branched chain fatty acids) giving a final concentration in the medium of 50 ppm 2-(+/-)-methyl butyric acid, 60 ppm isobutyric acid, and 20 ppm isovaleric acid. The pH was adjusted to 7.2 and the medium was autoclaved at 121 °C for 25 minutes.

The tube was shaken at an angle of 17 ° at 215 revolutions per minute for 3 days. An aliquot of 2 ml of the seeded culture was used to inoculate a 300 ml Erlenmeyer flask containing 25 ml of production medium containing: starch (either thin boiled starch or KOSO) 160 g/l; Nutrisoy (Archer Daniels Midland, Decatur, IL) 10 g/l; Ardamine pH 10 g/l; K2HPO4 2 g/l; MgSO4 • 4H2O 2 g/l; FeSO4 • 7H2O 0.02 g/l; MnCl2 0.002 g/l; ZnSO4 • 7H2O 0.002 g/l; CaCO3 14g/l; and 2 × bcfa (as above) 800 ppm. The pH was adjusted to 6.9 and the medium was autoclaved at 121 °C for 25 minutes.

After inoculation, the flask was incubated at 29 °C for 12 days with agitation at 200 rpm. After incubation, a 2 ml sample was taken from the bottle, diluted with 8 ml of methanol, mixed and the mixture was centrifuged at 1,250 × g for 10 min to obtain a debris pellet. The supernatant was then analyzed by high performance liquid chromatography (HPLC) using a Beckman Ultrasphere ODS column (25 cm × 4.6 mm ID) at a flow rate of 0.75 ml/min and detection by measuring absorbance at 240 nm. The mobile phase was a mixture of methanol/water/acetonitrile = 86/8.9/5.1.

Isolation of the S. avermitilis PKS gene

A cosmid library of S. avermitilis (ATCC 31272, SC-2) chromosomal DNA was obtained and hybridized with a ketosynthesis probe (KS) that was formed from a fragment of the Saccharopolyspora erytheraea polyketide synthesis gene (PKS). A detailed description of obtaining cosmid libraries can be found in Sambrook et al., supra. A detailed description of obtaining Streptomyces chromosomal DNA libraries is given in Hopwood et al., supra. Cosmid clones containing ketosynthesis-hybridizing regions were identified by hybridization to a 2.7 Kb Ndel/Eco47III fragment from pEX26 (obtained courtesy of Dr. P. Leadley, Cambridge, UK). About 5 µg of pEX26 was digested using restriction endonucleases Ndel and Eco47III. The reaction mixture was loaded onto a 0.8% SeaPlaqueTM GTG agarose gel (FMC BioProducts, Rockland, ME). The 2.7 Kb Ndel/Eco47III fragment was excised from the gel after electrophoresis and DNA was isolated from the gel using GELaseTM from Epicenter Technologies using a rapid protocol. The 2.7 Kb Ndel/Eco47III fragment was labeled with [α-32P]dCTP (deoxycytidine 5'-triphosphate, tetra(triethylammonium) salt, [α-32P]-) (NEN-Dupont, Boston, MA) using BRL Nick Translation System (BRL Life Technologies, Inc., Gaithersburg, MD) according to the supplier's instructions. Usually, the reaction is carried out in a volume of 0.05 ml. After adding 5 µl of buffer that stops the reaction (Stop buffer), labeled DNA is separated from unincorporated nucleotides using a G-25 Sephadex Quick SpinTM column (Boehringer Mannheim) according to the supplier's instructions.

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

Identification of DNA that modulates avermectin B2:B1 ratios and identification of the AveC ORF

The following methods were 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 µg) was digested with SacI and BamHI. The reaction mixture was loaded onto a 0.8% SeaPlaqueTM GTG agarose gel (FMC BioProducts), the 2.9 Kb Sac1/BamHI fragment was excised from the gel after electrophoresis, and DNA was extracted from the gel using GELaseTM (Epicentre Technologies) using a rapid protocol . About 5 µg of the walking vector pWHM3 (Vera et al.: "J. Bacteriol.", 171: 5872-5881, (1989)) was digested with SacI and BamHI. About 0.5 µg of the 2.9 Kb insert and 0.5 µg of digested pWHM3 were mixed together and incubated overnight with 1 unit of ligase (New England Biolabs, Inc., Beverly MA) at 15 °C in a total volume of 20 µl according to supplier's instructions. 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 E. coli DH5α cells (BRL) according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin-resistant transformants and the presence of an approximately 2.9 Kb Sac1/BamHI insert was confirmed by restriction analysis. This plasmid was designated pSE 119.

Protoplasts of S. avermitilis strain 1100-SC38 (Pfizer's proprietary strain) were obtained and transformed with pSE 119 as described in section 7.1.5 below. Strain 1100-SC38 is a mutant that produces significantly more avermectin cyclohexyl-B2 form than avermectin cyclohexyl-B1 form when supplied with cyclohexane carboxylic acid (B2:B1 of about 30:1). pSE 119 that was used to transform S. avermitilis was isolated from either E. coli strain GM 2163 (which was obtained from Dr. B.J. Bachmann, Curator, E. coli Genetic Stock Center, Yale University), E. coli strain DM1 (BRL ) or S. lividans species TK64. Thiostrepton resistant transformants of type 1100-SC38 were isolated and analyzed by HPLC analysis of fermentation products. Transformants of S. avermitilis strain 1100-SC83 containing pSE 119 produce an altered avermectin cyclohexyl-B2:cyclohexyl-B1 ratio of about 3.7:1 (Table 2).

As pSE 119 was shown to modulate B2:B1 ratios in the AveC mutant, the DNA insert was sequenced. Approximately 10 µg of pSE 119 was isolated using a plasmid DNA isolation kit (Qiagen, Valencia, CA) following the manufacturer's instructions and sequenced using an ABI 373A automated DNA sequencer (Perkin Elmer, Foster City, CA). Sequencing data were merged and edited using the Genetic Computer Group program (GCG, Madison, WI). The DNA sequence and AveC ORF are given in Figure 1 (SEQ ID NO: 1).

A new plasmid, designated pSE 118, was generated as follows. Approximately 5 µg of pSE 66 was digested with SphI and BamHI. The reaction mixture was loaded onto a 0.8% SeaPlaque agarose gel (FMC BioProducts), the 2.8 Kb Sphl/BamHI fragment was excised from the gel after electrophoresis and DNA was isolated from the gel using GELaseTM (Epicentre Technologies) using a rapid protocol. Approximately 5 µg of the walking vector pWHM3 is digested with SphI and BamHI. About 0.5 µg of the 2.8 Kb insert and 0.5 µg of digested pWHM3 were mixed together and incubated overnight with one unit of ligase (New England Biolabs) at 15 °C in a total volume of 20 µl according to the supplier's instructions. 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 E. coli DH5α cells according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the 2.8 Kb Sphl/BamHI insert was confirmed by restriction analysis. This plasmid is designated pSE 118. The insert DNA in pSE 118 and pSE 119 overlaps by approximately 838 nucleotides (Figure 4).

Protoplasts of S. avermitilis strain 1100-SC38 were transformed with pSE 118 as before. Thiostrepton resistant transformants of type 1100-SC38 were isolated and analyzed by HPLC analysis of fermentation products. Transformants of S. avermitilis strain 1100-SC38 containing pSE 118 did not change 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

An approximately 1.2 Kb fragment containing the AveC ORF was isolated from S. avermitilis chromosomal DNA by PCR amplification using primers derived from the AveC nucleotide sequence provided hereinabove. PCR primers were obtained from Genosys Biotechnologies, Inc. (Texas). The right upper primer was: 5'-TCACGAAACCGGACACAC-3' (SEQ ID NO: 6) and the left upper primer was 5'-CATGATCGCTGAACC GAG-3' (SEQ ID NO: 7). The PCR reaction was performed with Deep VentTM polymerase (New England Biolabs) in a buffer obtained from the manufacturer, and in the presence of 300 µM dNTP 10% glycerol, 200 pmol of each primer, 0.1 template and 2.5 units of enzyme in a final volume of 100 µl, using a Perkin-Elmer Cetus thermal cycler. The thermal profile of the first cycle was 95 °C for 5 min (denaturation step), 60 °C for 2 min (relaxation step) and 72 °C for 2 min (extension step). The next 24 cycles have a similar profile with the exception of the denaturation step which is shortened to 45 seconds and the relaxation step which is shortened to 1 minute.

The PCR product was electrophoresed in a 1% agarose gel and a unique DNA band of approximately 1.2 Kb was detected. This DNA was gel purified and ligated with 25 ng of linearized, pCR-Blunt vector (Invitrogen) at a 1:10 molar vector-to-insert ratio according to the manufacturer's instructions. The ligation mixture was used to transform One ShotTM components of E. coli cells (Invitrogen) according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of an approximately 1.2 Kb insert was confirmed by restriction analysis. This plasmid was designated pSE 179.

Insert DNA from pSE 179 was isolated by digestion with BamHI/XbaI, separated by electrophoresis, purified from the gel and ligated with the walking vector pWHM3, which was also digested with BamHI/XbaI, at a total DNA concentration of 1 µg in 1: 5 molar vector-to-insert ratio. The ligation mixture was used to transform competent E. coli DH5α cells according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin-resistant transformants and the presence of an approximately 1.2 Kb insert was confirmed by restriction analysis. This plasmid, designated pSE 186 (Figure 2, ATCC 209604), was transformed into E. coli DM1, and plasmid DNA was isolated from the ampicillin-resistant transformants.

2 Results

A 2.9 Kb Sacl/BamHI fragment from pSE 119 was identified as one that, when transformed into S. avermitilis strain 1100-SC38, significantly altered the B2:B1 ratio of avermectin production. S. avermitilis strain 1100-SC38 normally has a B2:B1 ratio of about 30:1, but when transformed with a vector containing the 2.9 Kb Sac1/BamHI fragment, the B2:B1 avermectin ratio drops to about 3.7:1. Post-fermentation analysis of transformant cultures confirmed the presence of transforming DNA.

A 2.9 Kb pSE 119 fragment was sequenced and an approximately 0.9 Kb ORF was identified (Figure 1) (SEQ ID NO: 1), encompassing a Pstl/Sphl fragment that had previously been mutated to produce only the B2 product (Ikeda et al. , forward). Comparison of this ORF or its corresponding deduced polypeptide with known databases (GeneEMBL, SWISS-PROT) did not show any strong homology with known DNA or protein sequences.

Table 2

[image]

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

EXAMPLE: Construction of S. avermitilis AveC mutants

This example describes the formation of several different S. avermitilis AveC mutants using the mixtures and procedures described hereinabove. A general description of techniques for introducing gene mutations into Streptomyces is described by Keiser and Hopwood: "Meth. Enzym.", 204:430-458, (1991). An even more detailed description was given by Anzai et al.: "J. Antibiot.", XLI(2):226-233, (1998); and Stutzman-Engwall et al.: "J. Bacterial.", 174(1):144-154, (1992). These publications are incorporated herein by reference in their entirety.

1. Deactycellostation of the S. avermitilis AveC gene

AveC mutants containing inactivated AveC genes were generated using multiple procedures, as detailed below.

In the first method, the 640 bp Sphl/Pstl internal fragment in the AveC gene in pSE 119 (the plasmid described in section 7.1.9, forward) was replaced with the ermE gene (for erythromycin resistance) from Sacc. erythraea. The ermE gene was isolated from pIJ4026 (which was obtained from the John Innes Institute, Norwich, U.K.; see also Bibb et al.: "Gene", 41:357-368, (1985)) by restriction enzyme digestion with Bg/ii and EcoRI , which was followed by electrophoresis and gel purification. This approximately 1.7 Kb fragment was ligated into pGEM7Zf (Promega) that had been digested with BamHI and EcoRI, and the ligation mixture was transformed into competent E. coli DH5α cells according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of an approximately 1.7 Kb insert was confirmed by restriction analysis. This plasmid is designated as pSE 27.

pSE 118 (which is described in section 7.1.9 above) was digested with Sphl and BamHI, the digest was electrophoresed, and the approximately 2.8 Kb Sphl/BamHI insert was gel purified. pSE 119 was digested with PstI and EcoRI, the digest was electrophoresed and the approximately 1.5 Kb PstI/EcoRI insert was gel purified. The travel vector pWHM3 was digested with BamHI and EcoRI. pSE 27 was digested with PstI and Sphl, the digest was electrophoresed and the approximately 1.7 Kb PstI/Sphl insert was gel purified. All four fragments (eg, approximately 2.8 Kb, approximately 1.5 Kb, approximately 7.2 Kb, approximately 1.7 Kb) were ligated together in a 4-strand ligation. The ligation mixture was transformed in competent E. coli DH5α cells according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid was designated pSE 180 (Figure 3, ATCC 209605).

pSE 180 was transformed into S. lividans TK64 and transformed colonies were identified by resistance to thiostrepton and erythromycin. pSE 180 was isolated from S. lividans and used to transform S. avermitilis protoplasts. 4 thiostrepton-resistant S. avermitilis transformants were identified, and protoplasts were obtained and plated under non-selective conditions on RM14 medium. After the protoplasts were regenerated, individual colonies were tested for the presence of erythromycin resistance and the absence of triostrepton resistance, which indicated integration of the deactivated AveC gene and loss of free replication. One Ermr Thios transformant was identified and designated strain SE 180-11. Total chromosomal DNA was isolated from strain SE 180-11, digested with restriction enzymes BamHI, HindIII, Pstl or Sphl, resolved using electrophoresis on 0.8% agarose gel, transferred to nylon membranes and hybridized to the ermeE probe. These analyzes showed that the chromosomal integration of the ermE resistance gene and the simultaneous deletion of the 640 bp Pstl/Sphl fragment occurs by a double crossover event. HPLC analysis of fermentation products of strain SE 180-11 showed that normal avermectins were no longer produced (Figure 5A).

In another method for inactivating the AveC gene, approximately 1.7 Kb of the ermE gene was removed from the chromosome of S. avermitilis strain SE 180-11, leaving a 640 bp Pstl/Sphl deletion in the AveC gene. The genetic modification plasmid was constructed as follows: pSE 180 was partially digested with XbaI and an approximately 11.4 Kb fragment was gel purified. The approximately 11.4 Kb band does not contain the 1.7 Kb ermE resistance gene. The DNA was then ligated and transformed into E. coli DH5α cells. Plasmid DNA was isolated from ampicillin-resistant transformants and the presence of the correct insert was confirmed by restriction analysis. This plasmid, designated pSE 184, was transformed into E. coli DM1, and plasmid DNA was isolated from ampicillin-resistant transformants. This plasmid was used to transform protoplasts of S. avermitilis strain SE 180-11. Protoplasts were obtained from thiostrepton-resistant transformants of SE 180-11 and were plated as single colonies on RM14. After the protoplasts were regenerated, individual colonies were tested for the absence of erythromycin resistance and thiostrepton resistance, indicating chromosomal integration of the deactivated AveC gene and loss of the free replicon containing the ermE gene. One Erms Tios transformant was identified and designated as SE 184-1-13. Fermentation analysis of SE 184-1-13 showed that normal avermectins were not produced and that SE 184-1-13 had the same fermentation profile as SE 180-11.

In a third method for inactivating the AveC gene, a frameshift is introduced into the chromosomal AveC gene by adding two Gs after the C at nt position 471 using PCR, thus forming a BspE1 site. The presence of a formed BspE1 site was useful in detecting gene replacement events. PCR primers were labeled to introduce a frameshift mutation in the AveC gene, and were obtained from Genosys Biotechnologies, Inc. The right primer was: 5'-GGTTCCGGATGCCGTTCTCG-3' (SEQ ID NO: 8) and the left primer was 5'-AACTCCGGTCGACTCCCCTTC-3' (SEQ ID NO: 9). PCR conditions were as described in section 7.1.10 above. The 666 bp PCR product was digested with Sphl to obtain two fragments of 278 bp and 388 bp, in the same order. A 388 bp fragment was purified from the gel.

The gene replacement plasmid was constructed as follows: the travel vector pWHM3 was digested with EcoRI and BamHI. pSE 119 was digested with BamHI and SphI, the digest was electrophoresed and an approximately 840 bp fragment was gel purified. pSE 119 was digested with EcoRI and XmnI, the digest resolved by electrophoresis and an approximately 1.7 Kb fragment was gel purified. All four fragments (eg, approximately 7.2, Kb, approximately 840, bp, approximately 1.7 Kb, and 380, bp) were ligated together in a four-step ligation. The ligation mixture was transformed in competent E. coli DH5α cells. Plasmid DNA was isolated from ampicillin-resistant transformants and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. This plasmid was designated pSE 185, was transformed into E. coli DM1 and plasmid DNA was isolated from ampicillin resistant transformants. This plasmid was used to transform protoplasts of S. avermitilis strain 1100-SC38. Thiostrepton-resistant transformants of type 1100-SC38 were isolated and analyzed by HPLC analysis of fermentation products. pSE 185 did not significantly alter B2:B1 avermectin ratios when transformed into S. avermitilis strain 1100-SC38 (Table 2).

pSE 185 was used to transform protoplasts of S. avermitilis to generate a frameshift mutation in the chromosomal AveC gene. Protoplasts were obtained from thiostrepton-resistant transformants and plated as single colonies on RM14. After the protoplasts were regenerated, individual colonies were tested for the absence of thiostrepton resistance. Chromosomal DNA from thiostrepton-susceptible colonies was isolated and tested by PCR for the presence of a frameshift mutation integrated into the chromosome. PCR primers were designed based on the AveC nucleotide sequence, and were obtained from Genosys Biotechnologies, Inc. (Texas). The right primer was: 5'-GCAAGGATACGGGGACTAC-3' (SEQ ID NO: 10) and the left primer was 5'-GAACCGACCGCCTGATAC-3' (SEQ ID NO: 11) and the PCR conditions were as described in chapter 7.1 .10 here forward. The resulting PCR product was 543 bp, and digested with BspE1, three fragments of 368 bp, 96 bp and 79 bp were observed, indicating chromosomal integration of the AveC gene and loss of the free replicon.

Fermentation analysis of S. avermitilis mutants containing a frameshift mutation in the AveC gene showed that normal avermectins were no longer produced and that these mutants had the same fermentation profile as SE 180-11 and SE 184-1-13. One Thios transformant was identified and designated strain SE 185-5a.

Additionally, a mutation in the AveC gene that changes nt position 520 from G to A, leading to a change of the codon encoding Tryptophan (W) at position 116 to a termination codon, was produced. The S. avermitilis strain with this mutation did not produce normal avermectins and had the same fermentation profile as strains SE 180-11, SE 184-1-13 and SE 185-5a.

Additionally, mutations in the AveC gene that change both: (i) nt position 970 from G to A, which changes the amino acid at position 256 from glycine (G) to aspartate (D); and (ii) nt position 996 T to C, which changes the amino acid at position 275 from tyrosine (Y) to histidine (H) were produced. S. avermitilis strains with these mutations (G256D/Y275H) do not produce normal avermectins and have the same fermentation profile as strains SE 180-11, SE 184-1-13 and SE 185-5a.

The S. avermitilis AveC inactivated mutant strains SE 180-11, SE 184-1-13, SE 185-5a, and other strains described herein provide a test tool for examining the impact of other mutations in the AveC gene. pSE 186, containing a copy of the wild-type AveC gene, was transformed into E. coli DM1 and plasmid DNA was isolated from ampicillin-resistant transformants. This pSE 186 DNA was used to transform protoplasts of S. avermitilis strain SE 180-11. Thiostrepton-resistant transformants of the SE 180-11 species were isolated, the presence of erythromycin resistance was determined and Thior Ermr transformants were analyzed using HPLC analysis of fermentation products. The presence of the functional AveC gene in trans could disrupt the normal avermectin production in SE 180-11 strain (Figure 5B).

2 Analysis of mutations in the AveC gene that change the class B2:B1 relationships

As described hereinabove, S. avermitilis strain SE 180-11 containing an inactivated AveC gene was complemented by transformation with a plasmid containing a functional AveC gene (pSE 186). Strain SE 180-11 was also used as a host strain to characterize other mutations in the AveC gene as described hereinbelow.

Chromosomal DNA was isolated from strain 1100-SC38 and was used as a template for PCR amplification of the AveC gene. The 1.2 Kb ORF was isolated by PCR amplification using primers based on the AveC nucleotide sequence. The right primer was SEQ ID NO: 6 and the left primer was SEQ ID NO: 7 (see section 7.1.10, hereinabove). PCR and subcloning conditions were as described in section 7.1.10. DNA sequence analysis of the 1.2 Kb ORF shows a mutation in the AveC gene that changes nt position 337 from C to T, which changes the amino acid at position 55 from serine (S) to phenylalanine (F). The AveC gene containing the S55F mutation was subcloned into pWHM3 to obtain a plasmid designated pSE 187, which was used to transform S. avermitilis SE 180-11 protoplasts. Thiostrepton-resistant SE 180-11 transformants were isolated, the presence of erythromycin resistance was determined and Thior Ermr transformants were analyzed by HPLC analysis of fermentation products. The presence of the AveC gene encoding a change of amino acid residue 55 (S55F) was able to disrupt normal avermectin production in strain SE180-11 (Fig. 5C); however, the cyclohexyl B2:cyclohexyl B1 ratio was about 26:1 compared to strain SE 180-11 transformed with pSE 186, which had a B2:B1 ratio of about 1.6:1 (Table 3) indicating that a single mutation (s55F) modulates the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1.

Another mutation in the AveC gene was identified to change nt position 862 from G to A, which changes the amino acid at position 230 from glycine (G) to aspartate (D). The S. avermitilis species that has this mutation (G230D) produces avermectins at a B2:B1 ratio of about 30:1.

3 Mutations that lower the B2:B1 ratio

Multiple mutations have been formed that reduce the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1, as follows.

A mutation in the AveC gene was identified to change nt position 588 from G to A, which changes the amino acid at position 139 from alanine (A) to threonine (T). The AveC gene containing the A139T mutation was subcloned into PWM3 to obtain a plasmid designated pSE 188, which was used to transform S. avermitilis SE 180-11 protoplasts. Thiostrepton-resistant SE 180-11 transformants were isolated, the presence of erythromycin resistance was determined and Thior Ermr transformants were analyzed by HPLC analysis of fermentation products. The presence of a mutated AveC gene encoding a change at amino acid residue 139 (A139T) was able to disrupt avermectin production in strain SE 180-11 (Figure 5D); even more, the B2:B1 ratio was about 0.94:1, indicating that this mutation reduces the amount of cyclohexyl-B2 that is produced relative to cyclohexyl-B1. This result is unexpected, because the published results, as well as the mutation results described above, only showed either inactivation of the AveC gene or increased production of the B2 form of avermectin compared to the B1 form (Table 3).

Because the A139T mutation changed the B2:B1 ratios toward the preferred B1, a mutation encoding a threonine instead of a serine at amino acid position 138 was formed. Thus, pSE 186 was digested with EcoRI and cloned into pGEM3Zf (Promega) which had been digested with EcoRI. This plasmid, designated pSE 186a, was digested with ApaI and KpnI, the DNA fragments were separated on an agarose gel and two fragments of approximately 3.8 Kb and approximately 0.4 Kb were purified from the gel. An approximately 1.2 Kb insert DNA from pSE 186 was used as a PCR template to introduce a single base pair at nt position 585. PCR primers were designed to introduce a mutation at position 585, and were supplied by Genosys Biotechnologies, Inc. (Texas). The right PCR primer was 5'-GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCCCTGGCGACG-3' (SEQ ID NO: 12) and the left PCR primer was 5'-GGAACCGACCGCCTGATACA-3' (SEQ ID NO: 13). The PCR reaction was performed using a modern GC genomic PCR kit (Clonetech Laboratories, Palo Alto, CA) in a buffer obtained from the manufacturer in the presence of 200 µM dNTPs, 200 pmol of each primer, 50 ng of template DNA, 1.0 M GC-solution and 1 unit of Klen Taq Polymerase Mixture in a final volume of 50 µl. The thermal profile of the first cycle was 94 °C for 1 minute; which was followed by 25 cycles at 94 °C for 30 seconds and 68 °C for 2 minutes; and 1 cycle at 68 °C for 3 minutes. The 295 bp PCR product was digested with ApaI and KpnI to release a 254 bp fragment that was resolved by electrophoresis and purified from the gel. All three fragments (approximately 3.8 Kb, approximately 0.4 Kb and 254 bp) were ligated together in a three-step ligation. The ligation mixture was transformed into competent E. coli DH5α cells. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid was designated pSE 198.

pSE 198 was digested with EcoRI, cloned into pWHM3 which had been digested with EcoRI and transformed into E. coli DH5α cells. Plasmid DNA was isolated from ampicillin-resistant transformants and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. This plasmid DNA was transformed into E. coli DM1, plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid, designated pSE 199, was used to transform S. avermitilis SE 180-11 protoplasts. Thiostrepton-resistant SE 180-11 transformants were isolated, the presence of erythromycin resistance was determined, and Thior Ermr transformants were analyzed by HPLC analysis of fermentation products. The presence of a mutated AveC gene encoding a change at amino acid residue 138 (S138T) was able to disrupt normal avermectin production in strain SE 180-11; even more, the B2:B1 ratio was 0.88:1 indicating that this mutation reduces the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1 (Table 3). This B2:B1 ratio is even lower than the 0.94:1 ratio observed with the A139T mutation produced by transformation of strain SE 180-11 with pSE 188, as described hereinabove.

Another mutation was made to introduce threonine at both amino acid positions 138 and 139. An approximately 1.2 Kb insert DNA from pSE 186 was used as a PCR template. PCR primers were designed to introduce mutations at nt positions 585 and 588 and were available from Genosys Biotechnologies, Inc. (Texas). The right-hand PCR primer was 5'-GGGGGCGGGCCCGGGTGCGGAGGCGAAA-TGCCGCTGGGCGACGACC-3' (SEQ ID NO: 14) and the left-hand PCR primer was 5'-GGAACATCACGGCATTCACC-3' (SEQID NO: 15). The PCR reaction was performed using the conditions described immediately earlier in this chapter. The 449 bp PCR product was digested with ApaI and KpnI to release a 254 bp fragment, which was resolved by electrophoresis and purified from the gel. pSE 186a was digested with ApaI and KpnI, the DNA fragments were separated on an agarose gel and two fragments of approximately 3.8 Kb and approximately 0.4 Kb were purified from the gel. All three fragments (approximately 3.8 Kb, approximately 0.4 Kb and 254 bp) were ligated together in a three-step ligation and the ligation mixture was transformed in competent E. coli DH5α cells. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid is designated as pSE 230.

Table 3

[image]

pSE 230 was digested with EcoRI, cloned into pWHM3 which had been digested with EcoRI and transformed into E. coli DH5α cells. Plasmid DNA was isolated from ampicillin-resistant transformants and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. This plasmid DNA was transformed into E. coli DM1, plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid, designated pSE 231, was used to transform S. avermitilis SE 180-11 protoplasts. Thiostrepton-resistant SE 180-11 transformants were isolated, the presence of erythromycin resistance was determined, and Thior Ermr transformants were analyzed by HPLC analysis of fermentation products. The presence of the double mutated AveC gene encoding S138T/A139T could disrupt the normal avermectin production in strain SE 180-11; even more, the B2:B1 ratio was 0.84:1 indicating that this mutation reduces the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1 (Table 3), relative to the reductions obtained using the SE180-11 strain transformation with pSE 188 or pSE 199, as described hereinabove.

These results are provided first to demonstrate specific mutations in the AveC gene that lead to the production of increased levels of the more commercially preferred class 1 avermectin relative to class 2 avermectin.

EXAMPLE: Construction of 5'-region deletion mutants

As discussed in section 5.1 above, the S. avermitilis nucleotide sequence shown in Figure 1 (SEQ ID NO: 1) comprises four different codons at bp positions 42, 174, 177 and 180 which are potential start sites. This chapter describes the construction of multiple deletions of the 5' region of the AveC ORF (Figure 1; SEQ ID NO: 1) to help define which of these codons may function as start sites in the AveC ORF for protein expression.

AveC gene fragments differently deleted at the 5' end were isolated from S. avermitilis chromosomal DNA by PCR amplification. PCR primers were labeled based on the AveC DNA sequence and were obtained from Genosys Biotechnologies, Inc. Right primers were 5'-AACCCATCCGAGCCGCTC-3' (SEQ ID NO: 16) (D1F1); 5'-TCGGCCTGCCAACGAAC-3' (SEQ ID NO: 17); 5'-CCAACGAACGTGTAGTAG-3' (SEQ ID NO: 18) (D1F3) and 5'-TGCAGGCGTACGTGTTCAGC-3' (SEQ ID NO: 19) (D2F2). Left primers were 5'-CATGATCGCTGAACCGA-3' (SEQ ID NO: 20); 5'-CATGATCGCTGAACCGAGGA-3' (SEQ ID NO: 21); and 5'-AGGAGTGTGGTGCGTCTGGA-3' (SEQ ID NO: 22). The PCR reaction was performed as described in section 8.3 above.

PCR products were separated by 1% agarose gel electrophoresis and unique DNA bands of approximately 1.0 Kb or approximately 1.1 Kb were detected. PCR products were gel purified and ligated with 25 ng of linearized pCR2.1 vector (Invitrogen) at a 1:10 molar vector-to-insert ratio according to the manufacturer's instructions. Ligation mixtures were used to transform One ShotTM competent E. coli cells (Invitrogen) according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin-resistant transformants and the presence of the insert was confirmed by restriction analysis and DNA sequence analysis. These plasmids are designated as pSE 190 (obtained with primer D1F1), pSE 191 (obtained with primer D1F2), pSE 192 (obtained with primer D1F3) and pSE 193 (obtained with primer D2F2).

The inserted DNAs were each digested with BamHI/Xbal, separated by electrophoresis, purified from the gel and specifically ligated with the traveling vector pWHM3, which had been digested with BamHI/Xbal, at a total DNA concentration of 1 µg in a 1:5 molar ratio vector- according to the concentration insert. The ligation mixture was used to transform competent E. coli DH5α cells. Plasmid DNA was isolated from ampicillin-resistant transformants and the presence of the insert was confirmed by restriction analysis. These plasmids, designated pSE 194 (D1F1), pSE (D1F2), pSE 196 (D1F3), and pSE 197 (D2F2), were each separately transformed into E. coli strain DM1, plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the correct insert was confirmed using restriction analysis. This DNA was used to transform protoplasts of S. avermitilis strain SE 180-11. Thiostrepton-resistant transformants of strain SE 180-11 were isolated, the presence of erythromycin resistance was determined and Thior Ermr transformants were analyzed by HPLC analysis of fermentation products to determine which GTG sites are required for AveC expression. The results show that the GTG codon at position 42 can be eliminated without AveC expression, as pSE 194, pSE 195, and pSE 196 each contain no GTG site at position 42, but all contain three GTG sites at positions 174, 177, and 180, and each can disrupt normal avermectin production when transformed into SE 180-11. Normal avermectin production was not impaired when strain SE 180-11 was transformed with pSE 197, which lacks all four GTG sites (Table 4).

Table 4

[image]

EXAMPLE: Cloning of AveC homologs from S. Hygroscopicus and S. Griseockromognes

The described invention ensures that AveC homologous genes from other avermectin- or milbemecin-producing Streptomyces species are identified and cloned. For example, a cosmid library of S. hygroscopicus (FERM BP-1901) genomic DNA was hybridized with the 1.2 Kb AveC probe from S. avermitilis described hereinabove. Multiple cosmid clones were identified that were highly hybridized. Chromosomal DNA was isolated from these junctions and a 4.9 Kb Kpnl fragment was identified which hybridized with the vaeC probe. This DNA was sequenced and an ORF (SEQ ID NO: 3) was identified which has significant homology to AveC of S. avermitilis. The amino acid sequence (SEQ ID NO: 4) deduced from the S. hygroscopicus AveC ORF is shown in Figure 6.

Additionally, a cosmid library of S. griseochromogenes genomic DNA was hybridized with the 1.2 Kb AveC probe from S. avermitilis described hereinabove. Multiple cosmid clones were identified that hybridized strongly. Chromosomal DNA was isolated from these cosmids, and a 5.4 Kb Pstl fragment was identified that hybridized with the AveC probe. This DNA was sequenced and an AveC homologous partial ORF was identified that has significant homology to the AveC ORF of S. avermitilis. The deduced partial amino acid sequence (SEQ ID NO: 5) is shown in Figure 6.

DNA and amino acid sequence analysis of AveC homologues from S. hygroscopicus and S. griseochromogenes indicate that these regions share significant homology (approximately 50% sequence identity at the amino acid level) in both the other S. avermitilis AveC ORF and the AveC gene product ( picture 6).

EXAMPLE: Construction of a plasmid with the AveC gene behind the ermE promoter

The 1.2 Kb AveC ORF from pSE 186 was subcloned into pSE 34, which is the travel vector pWHM3 having the 300 bp ermE promoter inserted as a Kpnl/BamHI fragment into the Kpnl/BamHI site of pWHM3 (see Ward et al.: "Mol. Gen. Genet .", 203:468-478, (1986)). pSE 186 was digested with BamHI and HindIII, the digest resolved by electrophoresis, and a 1.2 Kb fragment was isolated from an agarose gel and ligated to pSE 34 which had been digested with BamHI and HindIII. The ligation mixture was transformed into competent E. coli DH5α cells according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the 1.2 Kb insert was confirmed by restriction analysis. This plasmid, designated pSE 189, was transformed into E. coli DM1, and plasmid DNA was isolated from ampicillin-resistant transformants. Protoplasts of S. avermitilis type 1100-SC38 were transformed with pSE 189. Thiostrepton-resistant transformants of type 1100-SC38 were isolated and analyzed using HPLC analysis of fermentation products.

S. avermitilis strain 1100-SC38 transformants containing pSE 189 were altered in the ratios of produced avermectin cyclohexyl-B2:avermectin cyclohexyl-B1 (about 3:1) compared to strain 1100-SC 38 (about 34:1) and total avermectin production was increased approximately 2.4 times compared to strain 1100-SC38 transformed with pSE 119 (Table 5).

Table 5

[image]

pSE 189 was also transformed in S. avermitilis protoplasts of the original type species. Thiostrepton-resistant transformants were isolated and analyzed using HPLC analysis of fermentation products. Total avermectins produced by wild-type S. avermitilis transformed with pSE were increased approximately 2.2-fold compared to wild-type S. avermitilis transformed with pSE 119 (Table 5).

EXAMPLE: A chimeric plasmid containing sequences from both the S. avermitilis AveC ORF and the Hygroscopicus AveC homolog

A hybrid plasmid designated pSE 350 was constructed to contain a 564 bp portion of the S. hygroscopicus AveC homolog replacing the 564 bp homologous portion of the S. avermitilis AveC ORF (Figure 7) as follows. pSE 350 was constructed with the formation of a BsaAl restriction site that is conserved in both sequences (AveC position 225) and a KpnI restriction site that is present in the S. avermitilis AveC gene (AveC position 810). The Kpnl site was introduced into S. hygroscopicus DNA by PCR using the right primer 5'-CTTCAGGTGTACGTG TTCG-3' (SEQ ID NO: 23) and the left primer 5'-GAACTGGTACCAGTGCCC-3' (SEQ id NO: 24) (which obtained from Genosys Biotechnologies) using the PCR conditions described in section 7.1.0, hereinabove. The PCR product was digested with BsaAl and Kpnl, the fragments were separated by 1% agarose gel electrophoresis, and the 564 bp BsaAl/Kpnl fragment was isolated from the gel. pSE 179 (which is described in section 7.1.10 hereinabove) was digested with KpnI and HindIII, the fragments were separated by 1% agarose gel electrophoresis, and a fragment of approximately 4.5 Kb was isolated from the gel. pSE 179 was digested with HindIII and BsaAl, the fragments were separated by 1% agarose gel electrophoresis and an approximately 0.2 Kb BsaAl/HindIII fragment was isolated from the gel. An approximately 4.5 Kb HindIII/Kpnl fragment, an approximately 0.2 Kb BsaAl/HindIII fragment, and a 564 bp BsaAl/Kpnl fragment from S. hygroscopicus were ligated together in a three-step ligation and the ligation mixture was transformed into competent E. coli DH5α cells. . Plasmid DNA was isolated from ampicillin-resistant transformants and the presence of the correct insert was confirmed by restriction analysis using KpnI and Aval. This plasmid was digested with HindIII and XbaI to release a 1.2 Kb insert, which was then ligated to pWHM3 which had been digested with HindIII and XbaI. The ligation mixture was transformed into competent E. coli DH5α cells, plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the correct insert was confirmed by restriction analysis using HindIII and Aval. This plasmid DNA was transformed into E. coli DM1, plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. This plasmid was designated pSE 350 and was used to transform the protoplast of S. avermitilis strain SE 180-11. Thiostrepton-resistant SE 180-11 transformants were isolated, the presence of erythromycin resistance was determined and Thior ERMr transformants were analyzed by HPLC analysis of fermentation products. The results show that transformants containing the S. avermitilis/S.Hygroscopicus hybrid plasmid have an average B2:B1 ratio of about 109:1 (Table 6).

Table 6

[image]

Depositing biological materials

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

[image]

All patents, patent applications and publications cited hereinbefore are incorporated herein by reference in their entirety.

The described invention is not intended to be limited in its scope by its specific embodiments provided herein and which are provided only to illustrate individual aspects of the invention, and the functionality of equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will be apparent to one skilled in the art from the above description and accompanying drawings. Such modifications are also within the scope of the appended claims.

Claims (71)

1. An isolated polynucleotide molecule, characterized in that it comprises the complete AveC open reading arm (ORF) of Streptomyces avermitilis or an essential part thereof, where the isolated polynucleotide molecule does not contain the following complete ORF that is located below the AveC ORF in situ in the Streptomyces avermitilis chromosome and where the ORF has nucleotide sequence from Figure 1 (SEQ ID NO: 1). 2. An isolated polynucleotide molecule according to claim 1, characterized in that it further comprises nucleotide sequences that naturally flank the AveC gene in situ in S. avermitilis. 3. An isolated polynucleotide molecule, characterized in that it comprises a nucleotide sequence that is homologous to the nucleotide sequence of the AveC ORF encoding the Streptomyces avermitilis AveC gene product that is present in Figure 1 (SEQ ID NO: 1) or its essential part. 4. An isolated polynucleotide molecule, characterized by the fact that it comprises the nucleotide sequence of SEQ ID NO: 3 or its essential part, or a nucleotide sequence that is homologous to the nucleotide sequence of SEQ ID NO: 3. 5. An oligonucleotide molecule that hybridizes to a polynucleotide molecule, indicated by having the nucleotide sequence in Figure 1 (SEQ ID NO: 1) or SEQ ID NO: 3 or in a polynucleotide molecule having a nucleotide sequence that is the complement of the nucleotide sequence in Figure 1 (SEQ ID NO: 1) or SEQ ID NO: 3, under high binding conditions. 6. Oligonucleotide molecule according to claim 5, indicated by the fact that it is complementary to the nucleotide sequence in Figure 1 (SEQ ID NO: 1) or SEQ ID NO: 3 or to a polynucleotide molecule having a nucleotide sequence that is the complement of the nucleotide sequence in Figure 1 (SEQ ID NO: 1) or SEQ ID NO:3. 7. Recombinant vector comprising a polynucleotide molecule, characterized in that it comprises a nucleotide sequence selected from the group consisting of the AveC ORF nucleotide sequence from Figure 1 (SEQ ID NO: 1) or its essential part; and a nucleotide sequence that is homologous to the ORF encoding the S. avermitilis AveC gene product of Figure 1 (SEQ ID NO: 1) or an essential part thereof. 8. Recombinant vector according to claim 7, characterized in that it further comprises a nucleotide sequence encoding one or more regulatory elements, where the nucleotide sequence encoding the regulatory element is in operative association with the nucleotide sequence of the vector polynucleotide molecule according to claim 7. 9. Recombinant vector according to claim 8, characterized in that it further comprises a nucleotide sequence encoding a selectable marker. 10. Recombinant vector according to claim 9, characterized in that the plasmid is pSE 186 (ATCC 209604). 11. Host cell, characterized in that it comprises a recombinant vector according to claim 9. 12. The host cell according to claim 11, characterized in that it is Streptomyces avermitilis. 13. Recombinant vector comprising a polynucleotide molecule, characterized in that it comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence SEQ ID NO: 3 or its essential part; a nucleotide sequence that is homologous to the nucleotide sequence of SEQ ID NO: 3; and a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4. 14. The recombinant vector according to claim 13, characterized in that it further comprises a nucleotide sequence encoding one or more regulatory elements, where the nucleotide sequence encoding the regulatory element is in operative association with the nucleotide sequence of the vector polynucleotide molecule according to claim 13. 15. Recombinant vector according to claim 14, characterized in that it further comprises a nucleotide sequence encoding a selectable marker. 16. Host cell, characterized in that it comprises the recombinant vector according to claim 15. 17. The host cell according to claim 16, characterized in that it is Streptomyces avermitilis. 18. An isolated S. avermitilis AveC gene product, characterized in that it has an amino acid sequence that is encoded by the sequence of plasmid pSE 186 (ATCC 209604) that encodes an S. avermitilis AveC gene product or an amino acid sequence of SEQ ID NO: 2 or an essential part thereof or its homologous polypeptide. 19. An isolated S. hygroscopicus AveC homologous genetic product, characterized in that it has the amino acid sequence of SEQ ID NO: 4. 20. A method for the production of a recombinant AveC genetic product, characterized in that it comprises culturing a host cell that has been transformed with a recombinant expression vector, said recombinant expression vector comprising a polynucleotide molecule having a nucleotide sequence encoding the amino acid sequence SEQ ID NO: 2 or its essential part, where the polynucleotide molecule is in operative association with one or more regulatory elements that control the expression of the polynucleotide molecule in the host cell, under conditions that lead to the production of the recombinant genetic product, and isolating the AveC genetic product from the cell culture. 21. A method for the production of a recombinant AveC homologous genetic product, characterized in that it comprises culturing a host cell that has been transformed with a recombinant expression vector, said recombinant expression vector comprising a polynucleotide molecule having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4 or its essential part, where the polynucleotide molecule is in operative association with one or more regulatory elements that control the expression of the polynucleotide molecule in the host cell, under conditions that lead to the production of a recombinant AveC homologous gene product, and isolating the AveC homologous gene product from the cell culture. 22. A polynucleotide molecule, characterized in that it comprises a nucleotide sequence that is otherwise the same as the sequence of plasmid pSE 186 (ATCC 209604) encoding the S. avermitilis gene product or the nucleotide sequence of AveC ORF of S. avermitilis as shown in Figure 1 (SEQ ID NO: 1) or its degenerative variant, but where the nucleotide sequence further comprises one or more mutations, so that S. avermitilis ATCC 53692 cells in which the AveC allele of the original type is deactivated and which express a polynucleotide molecule comprising the mutated nucleotide sequence produce reduced cyclohexyl B2: cyclohexyl B1 ratio of avermectin when fermentation is carried out in the presence of cyclohexanecarboxylic acid than that produced by cells of S. avermitilis strain ATCC 53692 which in addition express only the AveC allele of the original type. 23. Polynucleotide molecule according to claim 22, characterized in that the reduced cyclohexyl B2:cyclohexyl B1 ratio is less than 1.6:1. 24. Polynucleotide molecule according to claim 23, characterized in that the reduced cyclohexyl B2:cyclohexyl B1 ratio is less than 0.94:1. 25. Polynucleotide molecule according to claim 24, characterized in that the mutation in the S. avermitilis AveC ORF from Figure 1 (SEQ ID NO: 1) encodes an amino acid substitution at residue 139 of the AveC genetic product from alanine to threonine. 26. Polynucleotide molecule according to claim 23, characterized in that the reduced cyclohexyl B2:cyclohexyl B1 ratio is less than 0.88:1. 27. Polynucleotide molecule according to claim 26, characterized in that the mutation in the S. avermitilis AveC ORF from Figure 1 (SEQ ID NO: 1) encodes an amino acid substitution at residue 138 of the AveC genetic product from serine to threonine. 28. Polynucleotide molecule according to claim 28, characterized in that the reduced cyclohexyl B2:cyclohexyl B1 ratio is less than 0.84:1. 29. The polynucleotide molecule according to claim 28, characterized in that the mutation in the S. avermitilis AveC ORF of Figure 1 (SEQ ID NO: 1) encodes an amino acid substitution at residue 138 of the AveC genetic product from serine to threonine, and another amino acid substitution at residue 139 AveC gene product from alanine to threonine. 30. A polynucleotide molecule characterized by comprising a strong promoter in operative association with the S. avermitilis AveC ORF from Figure 1 (SEQ ID NO: 1). 31. Polynucleotide molecule according to claim 30, characterized in that the strong promoter is the ermE promoter from Saccharopolyspora erythraea. 32. A polynucleotide molecule, indicated by the fact that it comprises the nucleotide sequence ORF encoding the AveC genetic product from Figure 1 (SEQ ID NO: 1) which is deactivated by inserting a heterologous nucleotide sequence into said nucleotide sequence. 33. A polynucleotide molecule, characterized in that it comprises an AveC allele that has been deactivated by deleting the 640 bp Pstl/Sphl fragment from the AveC ORF from Figure 1 (SEQ ID NO: 1). 34. A polynucleotide molecule, characterized in that it comprises an AveC allele that has been deactivated by introducing a frameshift mutation into the AveC ORF of Figure 1 (SEQ ID NO: 1). 35. The polynucleotide molecule according to claim 34, characterized in that the frameshift mutation is introduced by adding two G nucleotides after the C nucleotide at nt position 471 of the AveC ORF from Figure 1 (SEQ ID NO: 1). 36. A polynucleotide molecule, characterized in that it comprises an AveC allele that has been deactivated by introducing a terminal codon at the nucleotide position encoding amino acid 116 of the S. avermitilis genetic product encoded by the AveC ORF of Figure 1 (SEQ ID NO: 1). 37. A polynucleotide molecule characterized by the fact that it comprises an AveC allele that is deactivated by introducing a first mutation at amino acid position 256 of the AveC genetic product that changes glycine to aspartate and a second mutation at position 275 of the AveC genetic product that changes tyrosine to histidine. 38. A gene replacement vector comprising a polynucleotide molecule comprising nucleotide sequences naturally flanking the AveC ORF in situ in the S. avermitilis chromosome. 39. Recombinant vector, characterized in that it comprises a polynucleotide molecule according to any of claims 22 to 37. 40. Recombinant vector, characterized in that it comprises a polynucleotide molecule according to claim 32, where the vector is pSE 180 (ATCC 209605). 41. Streptomyces host cell, characterized in that it comprises the recombinant vector according to claim 39. 42. A method for identifying mutations of the AveC ORF of S. avermitilis that can reduce the class 2:1 ratio of produced avermectins, characterized by comprising: (a) determining the class 2:1 ratio of avemerctins that are produced by S. avermitilis cells where the natural AveC allele is inactivated and where a polynucleotide molecule comprising a nucleotide sequence encoding a mutated AveC gene product is introduced to be expressed; (b) determining the class 2:1 ratio of avermectins that are produced using cells of the same S. avermitilis species as in step (a) but which in addition express only the AveC allele having the nucleotide sequence of the ORF of Figure 1 (SEQ ID NO: 1) or a sequence that is homologous to it; and (c) comparing the class 2:1 ratio of avermectins produced by S. avermitilis cells in step (a) to the class 2:1 ratio of avermectins produced by S. avermitilis cells in step (b) such that if the class The 2:1 ratio of avermectins produced by S. avermitilis cells in step (a) is lower than the class 2:1 ratio of avermectins produced by S. avermitilis cells in step (b), then the AveC ORF mutation that can lower the class 2 :1 ratio of avermectin is identified. 43. Method for obtaining new species of S. avermitilis comprising cells that express a mutated AveC allele and that produce a reduced 2:1 class ratio of avermectin compared to cells of the same species of S. avermitilis that, in addition, express only the AveC allele of the original type, characterized in that involves transforming S. avermitilis cells with a vector carrying a mutated AveC allele that encodes a gene product that lowers the 2:1 class ratio of avermectins produced by S. avermitilis cells expressing the mutated AveC allele relative to cells of the same species that additionally express wild-type AveC allele alone, and selection of transformed cells that produce avermectins at a reduced 2:1 class ratio compared to the 2:1 class ratio produced by cells of the species that additionally express the wild-type AveC allele. 44. Method for obtaining new types of S. avermitilis cells that include a deactivated AveC allele, characterized in that it includes transforming S. avermitilis cells with a vector carrying a deactivated AveC allele and selection of transformed cells in which the AveC allele is deactivated. 45. The method according to claim 44, characterized in that the vector is pSE 180 (ATCC 209605). 46. The species S. avermilitis, characterized by the fact that it includes cells that express a mutated AveC allele that leads to the production by cells of avermectin in a reduced class 2:1 ratio compared to cells of the same species that, in addition, express only the AveC allele of the original type. 47. The species according to claim 46, characterized in that the cells produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio that is less than 1.6:1. 48. The species according to claim 46, characterized in that the cells produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of less than 0.94:1. 49. The species according to claim 46, characterized in that the cells produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of less than 0.88:1. 50. The species according to claim 46, characterized in that the cells produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of less than 0.84:1. 51. The species S. avermitilis, characterized by the fact that it includes cells in which the AveC gene is deactivated. 52. A method for the production of avermectin, characterized in that it comprises culturing cells of the S. avermitilis species where the cells express a mutated AveC allele that encodes a genetic product that lowers the class 2:1 ratio of avermectins produced by a S. avermitilis species that expresses a mutated AveC allele in comparison with cells of the same species that do not express the mutated AveC allele, but in addition express only the AveC allele of the original type, in the medium of the culture under conditions that allow or induce the production of avermectin from it, and the extraction of said avermectin from the culture. 53. An avermectin mixture, characterized in that it is produced by S. avermitilis cells, wherein the cells express a mutated AveC allele encoding a gene product that lowers the class 2:1 ratio of avermectins produced by S. avermitilis cells expressing a mutated AveC allele in compared to cells of the same species that do not express the mutated AveC allele but in addition express only the AveC allele of the original type, where avermectins are produced in a reduced class 2:1 ratio compared to the class 2:1 ratio of avermectins produced by cells of the same species S. avermitilis that do not express the mutated AveC allele but in addition express only the AveC allele of the original type. 54. The method according to claim 53, characterized in that the class 2:1 ratio of avermectins produced is reduced by mutation. 55. A method for obtaining new species of S. avermitilis comprising cells that produce altered amounts of avermectin, characterized in that it comprises transforming cells of the S. avermitilis species with a vector carrying a mutated AveC allele or a genetic construct comprising an AveC allele, expression leading to an altered amount of avermectins produced by S. avermitilis cells expressing a mutated AveC allele or a genetic construct compared to cells of the same species expressing only the AveC allele of the original type, and selection of transformed cells that produce avermectins in an altered amount compared to the amount of avermectins that were produced using cells of a species that in addition express only the AveC allele of the original type. 56. The method according to claim 55, characterized in that the amount of avermectin produced is increased by mutation. 57. Method for obtaining new species of S. avermitilis, cells that include a deactivated AveC allele, characterized in that it includes transforming cells of the species S. avermitilis with a vector that deactivates the AveC allele and selection of transformed cells where the AveC allele is deactivated. 58. The method according to claim 57, characterized in that the vector is pSE 180 (ATCC 209605). 59. The species S. avermilitis, characterized by the fact that it includes cells that express a mutated AveC allele that leads to the production by cells of avermectin in a changed class 2:1 ratio compared to cells of the same species that, in addition, express only the AveC allele of the original type. 60. The species according to claim 59, characterized in that the class 2:1 ratio of avermectin is reduced by mutation. 61. The species according to claim 60, characterized in that the cells produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio that is less than 1.6:1. 62. The species according to claim 61, characterized in that the cells produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of less than 0.94:1. 63. The species according to claim 61, characterized in that the cells produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of less than 0.88:1. 64. The species according to claim 61, characterized in that the cells produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of less than 0.84:1. 65. The species S. avermitilis, characterized by the fact that it includes cells that express a mutated AveC allele or a genetic construct that leads to the production by the cells of an altered amount of avermectin compared to the cells of the same species that, in addition, express only the AveC allele of the original type. 66. A species of S. avermitilis, characterized by the fact that it includes cells in which the AveC gene has been deactivated. 67. A method for the production of avermectin, characterized in that it comprises culturing cells of the S. avermitilis species where the cells express a mutated AveC allele that encodes a genetic product that changes the class 2:1 ratio of avermectins produced by a S. avermitilis species that expresses a mutated AveC allele in comparison with cells of the same species that do not express the mutated AveC allele, but in addition express only the AveC allele of the original type, in the medium of the culture under conditions that allow or induce the production of avermectin from it, and the extraction of said avermectin from the culture. 68. The method of claim 67, wherein the class 2:1 ratio is reduced by mutation. 69. A method for the production of avermectin, characterized in that it comprises culturing cells of the S. avermitilis species where the cells express a mutated AveC allele or a genetic construct comprising the AveC allele leading to the production of an altered amount of avermectins that are produced by a S. avermitilis species expressing a mutated AveC allele or genetic construct compared to cells of the same species that do not express the mutated AveC allele or genetic construct but in addition express only the AveC allele of the original type, in the medium of the culture under conditions that allow or induce the production of avermectin from this, and the extraction of said avermectin from the culture. 70. The method according to claim 69, characterized in that the amount of avermectin produced is increased by expression of a mutated AveC allele or genetic construct. 71. An avermectin mixture, characterized in that it is produced by S. avermitilis cells where the cells express a mutated AveC allele encoding a gene product that lowers the class 2:1 ratio of avermectins that are produced by S. avermitilis cells expressing a mutated AveC allele in the ratio to cells of the same species that do not express the mutated AveC allele but in addition express only the AveC allele of the original type, where avermectins are produced in a reduced 2:1 class ratio of avermectins produced by cells of the same species of S. avermitilis that do not express the mutated AveC allele but in addition of that they express only the AveC allele of the original type.