HK1168383B - Streptomyces avermitilis gene directing the ratio of b2:b1 avermectins - Google Patents

Description

Streptomyces avermitilis gene directing B2: B1 avermectin ratio

The application is a divisional application of an invention with the application date of 2003, 1 month and 31 days, Chinese application number of 03803741.6 and the invention name of streptomyces avermitilis gene for guiding the ratio of B2 to B1 avermectin.

1. Field of the invention

The present invention relates to compositions of avermectins (avermectins), such as doramectin, and methods for efficiently producing avermectins, primarily for use in the field of animal health. More particularly, the present invention relates to polynucleotide molecules comprising nucleotide sequences encoding an AveC gene product, which are useful for modulating the class 2:1 ratio of avermectins produced by fermentation of cultures of S.avermitilis (Streptomyces avermitilis). The invention also relates to vectors, transformed host cells, and novel mutants of S.avermitilis in which the aveC gene has been mutated to modulate the class 2:1 ratio of avermectins produced.

2. Background of the invention

2.1.Avermectin

Streptomyces produces a wide variety of secondary metabolites, including avermectins, which contain a series of eight related macrolides, 16 members, that produce potent anthelmintic and insecticidal activity. These eight distinct but closely related compounds are primarily referred to as: a1a, A1B, A2a, A2B, B1a, B1B, B2a, and B2B. The "a" series of compounds refers to natural avermectins, the substituent at position C25 being (S) -sec-butyl, and the "b" series of compounds refers to those compounds in which the substituent at position C25 is isopropyl. The designations "A" and "B" refer to avermectins in which the substituent at C5 is methoxy and hydroxy, respectively. The number "1" refers to avermectins with double bonds at the C22 and C23 positions; and the number "2" is avermectin having a hydrogen atom at the C22 position and a hydroxyl group at the C23 position. Among these related avermectins, the avermectins of type B1 (e.g., doramectin) are recognized as having the most potent antiparasitic and insecticidal activity and are therefore also the most commercially promising avermectins.

Avermectins and methods for their production by aerobic fermentation of strains of S.avermitilis are described in U.S. Pat. Nos. 4,310,519 and 4,429,042. It is believed that biosynthesis of natural avermectins can be endogenously initiated by thioester analogs of coenzyme A of isobutyric acid and S- (+) -2-methylbutyric acid.

Strain improvement by random mutagenesis in combination with exogenously supplied fatty acids can lead to efficient production of avermectin analogs. A Streptomyces avermitilis mutant deficient in branched-chain 2-oxoacid dehydrogenase (bkd-deficient mutant) produces avermectins only when supplemented with fatty acids during fermentation. Screening and isolation of mutants deficient in branched-chain dehydrogenase activity (e.g., S.avermitilis, ATCC 53567) is described in European Patent (EP) 276103. Fermentation of these mutants in the presence of exogenously supplied fatty acids results in the production of only four avermectins corresponding to the fatty acids used. Thus, supplementation of S- (+) -2-methylbutyric acid with S.avermitilis (ATCC 53567) fermentation resulted in the production of natural avermectins A1a, A2a, B1a and B2 a; supplementation with isobutyric acid resulted in the production of natural avermectins A1B, A2B, B1B and B2B; supplementation of the fermentation with cyclopentanecarboxylic acid, in turn, resulted in the production of four novel cyclopentylacetins A1, A2, B1 and B2.

Supplemental fermentation with other fatty acids can produce novel avermectins. Through the screening of over 800 potential precursors, over 60 other novel avermectins have been identified (see, e.g., Dutton et al, 1991, J.Antibiot., 44: 357-365; and Bank et al, 1994, Roy.Soc. chem.147: 16-26). Furthermore, mutants of S.avermitilis deficient in 5-O-methyltransferase activity produce essentially only B-avermectins, and thus mutants of S.avermitilis lacking both branched-chain 2-oxoacid dehydrogenase and 5-O-methyltransferase activity produce only B-avermectins, corresponding to supplementation of fatty acids used in fermentation. Thus, supplementation of such double mutants with S- (+) -2-methylbutyric acid resulted in the production of only natural avermectins B1a and B2a, whereas supplementation with isobutyric acid or cyclopentanecarboxylic acid resulted in the production of natural avermectins B1B and B2B or novel cyclopentyl B1 and B2 avermectins, respectively. Supplementation of the double mutant strain with cyclohexane carboxylic acid is the preferred method for producing the commercially important novel avermectin cyclohexyl avermectin B1 (doramectin). The isolation and characterization of such double mutants, such as S.avermitilis (ATCC 53692), is described in EP 276103.

2.2.Genes involved in avermectin biosynthesis

In many cases, the locations on the chromosome of genes involved in the production of secondary metabolites and genes encoding a particular antibiotic are often clustered together. For example, the Streptomyces polyketide synthase gene cluster (PKS) (see Hopwood and Sherman, 1990, Ann. Rev. Genet., 24: 37-66). Thus, one strategy for cloning genes in the biosynthetic pathway is to isolate drug resistance genes and then detect other genes in adjacent regions on the chromosome that are associated with the biosynthesis of that particular antibiotic. Another strategy for cloning genes involved in the biosynthesis of important metabolites is mutant complementation. For example, a portion of a DNA library from an organism that produces a particular metabolite is introduced into a mutant that does not produce the metabolite, and transformants that produce the metabolite are screened. In addition, library hybridization methods using probes from other streptomyces have been used to identify and clone genes in biosynthetic pathways.

Genes involved in avermectin biosynthesis (ave genes), like those essential for biosynthesis of other secondary metabolites of Streptomyces (e.g., PKS), are clustered on the chromosome. Vectors have been used to successfully clone a number of ave genes to complement S.avermitilis mutants with hindered avermectin biosynthesis. The cloning of these genes has been described in U.S. Pat. No. 5,252,474. Furthermore, Ikeda et al, 1995, J.Antibiot.48: 532-534 described the mapping of the chromosomal region (aveC) involved in the dehydration step of C22, C23 to the 4.82Kb BamHI fragment of S.avermitilis and the mutation of the aveC gene to produce a single component B2a producer. Since ivermectin (a potential anthelmintic compound) can be chemically synthesized from avermectin B2a, a single component producer of avermectin B2a is believed to be particularly useful for commercial production of ivermectin.

U.S. Pat. No. 6,248,579 to Stutzman-Engwall et al, 6/19, 2001, describes mutations in the aveC gene of S.avermitilis that result in a reduction in the ratio of cyclohexyl B2 to cyclohexyl B1 to about 0.75: 1.

PCT application WO01/12821, published on 22.2.2001, by Pezizer Products Inc., describes additional mutations in the aveC gene of S.avermitilis that result in a reduction of the ratio of cyclohexyl B2 to cyclohexyl B1 to about 0.40: 1.

Identification of mutations or combinations of mutations in the aveC gene that result in further reduction of the complexity of avermectin production, e.g., mutations that further reduce the ratio of avermectin B2: B1, can simplify production and purification of commercially important avermectins.

3. Brief description of the invention

The present invention provides a polynucleotide molecule comprising a nucleotide sequence that is identical to an aveC allele of S.avermitilis, a nucleotide sequence that is identical to a sequence on plasmid pSE186(ATCC209604) that encodes the AveC gene product of S.avermitilis, or a nucleotide sequence that is identical to the aveC ORF from S.avermitilis as shown in FIG. 1(SEQ ID NO: 1), or a degenerate variant thereof, but which further comprises a mutation that encodes a combination of amino acid substitutions that occur in a nucleotide sequence that corresponds to the amino acid sequence of SEQ ID NO:2, such that those cells of streptomyces avermitilis ATCC53692 that have inactivated the wild-type aveC allele and that express a polynucleotide molecule comprising the mutant nucleotide sequence, produce a reduced ratio of class 2:1 avermectins relative to cells of streptomyces avermitilis ATCC53692 that express only the wild-type aveC allele, wherein the ratio of class 2:1 avermectins is 0.35: 1 or less when the class 2:1 avermectins are cyclohexyl B2: cyclohexyl B1 avermectins. In a preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.30: 1 or less. In a more preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.25: 1 or less. In a more preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.20: 1 or less.

In one embodiment, the combination of amino acid substitutions comprises a combination selected from the group consisting of:

(a)D48E，A61T，A89T，S138T，A139T，G179S，A198G，P289L；

(b)G40S，D48E，L136P，G179S，E238D；

(c)D48E，L136P，R163Q，G179S；

(d)D48E，L136P，R163Q，G179S，E238D；

(e)D48E，L136P，R163Q，G179S，A200G，E238D；

(f)D48E，L136P，G179S，E238D；

(g)D48E，A61T，L136P，G179S，E238D；

(h)D48E，A61T，L136P，G179S；

(i)D48E，A89T，S138T，A139T，G179S；

(j)D48E，A61T，L136P，G179S，A198G，P202S，E238D，P289L；

(k)D48E，A61T，L136P，S138T，A139F，G179S，E238D，P289L；

(l)D48E，L136P，G179S，A198G，E238D，P289L；

(m)D48E，A61T，S138T，A139F，G179S，A198G，P289L；

(n)D48E，L84P，G111V，S138T，A139T，G179S，A198G，P289L；

(o)Y28C，D48E，A61T，A89T，S138T，A139T，G179S，E238D；

(p)D48E，A61T，A107T，S108G，L136P，G179S，S192A，E238D，P289L；

(q)D48E，L136P，G179S，R250W；

(r)D48E，A89T，S138T，A139T，R163Q，G179S；

(s)D48E，L136P，G179S，A198G，P289L；

(t)D48E，F78L，A89T，L136P，G179S；

(u)D48E，A89T，S138T，A139T，G179S，E238D，F278L；

(v)D48E，A89T，L136P，R163Q，G179S；

(w)D48E，A61T，A89T，G111V，S138T，A139F，G179S，E238D，P289L；

(x)D25G，D48E，A89T，L136P，S138T，A139T，V141A，I159T，R163Q，G179S；

(y)D48E，A89T，S90G，L136P，R163Q，G179S，E238D；

(z)D48E，A61T，A89T，G111V，S138T，A139T，G179S，E238D，P289L；

(aa)D48E，A89T，S138T，A139T，G179S；

(ab)D48E，L136P，R163Q，G179S，S231L；

(ac)D48E，L136P，S138T，A139F，G179S，V196A，E238D；

(ad)D48E，A61T，A89T，F99S，S138T，A139T，G179S，E238D；

(ae)G35S，D48E，A89T，S138T，A139T，G179S，P289L；

(af)D48E，A61T，A89T，S138T，A139T，G179S，V196A，E238D；

(ag)D48E，A89T，G111V，S138T，A139T，G179S，A198G，E238D；

(ah)S41G，D48E，A89T，L136P，G179S；

(ai)D48E，A89T，L136P，R163Q，G179S，P252S；

(aj)D48E，A89T，L136P，G179S，F234S；

(ak)D48E，A89T，L136P，R163Q，G179S，E238D；

(al)Q36R，D48E，A89T，L136P，G179S，E238D；

(am)D48E，A89T，L136P，R163Q，G179S；

(an)D48E，A89T，S138T，G179S；

(ao)D48E，A89T，L136P，G179S，E238D；

(ap)D48E，A89T，L136P，K154E，G179S，E238D；

(aq)D48E，A89T，S138T，A139T，K154R，G179S，V196A，P289L；

(ar)D48E，A89T，S138T，A139F，G179S，V196A，E238D；

(as)D48E，A61T，A89T，L136P，G179S，V196A，A198G，P289L；

(at)D48E，A61T，S138T，A139F，G179S，G196A，E238D，P289L；

(au)D48E，A89T，L136P，G179S；

(av)D48E，A89T，V120A，L136P，G179S；

(aw)D48E，A61T，A89T，S138T，A139F，G179S，V196A，A198G，E238D；

(ax)D48E，A61T，A89T，G111V，S138T，A139F，G179S，V196A，E238D；

(ay)D48E，A61T，A89T，S138T，A139T，G179S，V196A，E238D，P289L；

(az)D48E，A61T，A89T，L136P，S138T，A139F，G179S，A198G，E238D；

(ba)D48E，A89T，S138T，A139F，G179S，A198G，V220A；

(bb)D48E，A61T，A89T，S138T，A139T，G179S，V196A，E238D，R239H，P289L；

(bc)D48E，A61T，A89T，L136P，G179S，P289L；

(bd) D48E, a89T, S138T, a139T, G179S, V196A, E238D, P289L; and

(be)D48E，A61T，A89T，S138T，A139F，G179S，V196A，E238D。

the present invention also provides a polynucleotide molecule comprising a nucleotide sequence that is identical to the aveC allele of S.avermitilis, a nucleotide sequence that is identical to the sequence encoding the AveC gene product of S.avermitilis on plasmid pSE186(ATCC209604), or a nucleotide sequence that is identical to the aveC ORF of S.avermitilis as shown in FIG. 1(SEQ ID NO: 1), or a degenerate variant thereof, but which further comprises a mutation encoding a combination of amino acid substitutions that occur in a nucleotide sequence that corresponds to the amino acid sequence of SEQ ID NO:2, and expressing a polynucleotide molecule comprising a mutant nucleotide sequence, results in a reduced ratio of class 2:1 avermectins relative to a cell of streptomyces avermitilis ATCC53692 that expresses only the wild-type aveC allele, wherein the ratio of class 2:1 avermectins is about 0.40: 1 or less when the class 2:1 avermectins are cyclohexyl B2: cyclohexyl B1 avermectins, and wherein the combination of amino acid substitutions comprises a combination selected from the group consisting of:

(bf) D48E, S138T, a139T, G179S, E238D; and

(bg)Y28C，Q38R，D48E，L136P，G179S，E238D。

the invention also provides recombinant vectors comprising the polynucleotide molecules of the invention.

The invention also provides host cells containing the polynucleotide molecules or recombinant vectors of the invention. In a preferred embodiment, the host cell is a Streptomyces cell. In a more preferred embodiment, the host cell is a Streptomyces avermitilis cell.

The present invention also provides a method for making a novel strain of S.avermitilis, comprising (i) mutating an aveC allele of a cell of a strain of S.avermitilis, the mutation resulting in a combination of amino acid substitutions in the AveC gene product, or (ii) introducing into a cell of a strain of S.avermitilis a mutated aveC allele or a degenerate variant thereof, said mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions, wherein said combination of amino acid substitutions is selected from (a) - (be) listed above.

The present invention also provides a method for making a novel strain of S.avermitilis, comprising (i) mutating an aveC allele of a cell of a strain of S.avermitilis, the mutation resulting in a combination of amino acid substitutions in the AveC gene product, or (ii) introducing into a cell of a strain of S.avermitilis a mutant aveC allele or a degenerate variant thereof, said mutant aveC allele or degenerate variant thereof encoding an AveC gene product comprising the combination of amino acid substitutions, wherein a cell comprising the mutant aveC allele or degenerate variant thereof is capable of producing a ratio of cyclohexyl B2: cyclohexyl B1 avermectins of 0.35: 1 or less. In one non-limiting embodiment, the mutant aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from those set forth in (a) - (be) above.

In a preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.30: 1 or less. In one non-limiting embodiment, the mutant aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from those set forth in (f) - (be) above.

In a more preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.25: 1 or less. In one non-limiting embodiment, the mutant aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (w) - (be) above.

In a more preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.20: 1 or less. In one non-limiting embodiment, the mutant aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (ao) - (be) above.

The present invention also provides a method for making a novel strain of S.avermitilis, comprising (i) mutating an aveC allele of a cell of a strain of S.avermitilis, the mutation resulting in a combination of amino acid substitutions in the AveC gene product, or (ii) introducing into a cell of a strain of S.avermitilis a mutated aveC allele or a degenerate variant thereof, said mutated aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions, wherein said combination of amino acid substitutions is selected from the group set forth in (bf) and (bg) above. In a preferred embodiment, S.avermitilis cells comprising such a mutant aveC allele or degenerate variant thereof produce a ratio of cyclohexyl B2 to cyclohexyl B1 avermectins of about 0.40: 1 or less.

The present invention also provides a Streptomyces cell comprising a mutant S.avermitilis aveC allele or a degenerate variant thereof, which mutant aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from those set forth in (a) - (be) above. In a preferred embodiment, the streptomyces is streptomyces avermitilis.

The invention also provides Streptomyces avermitilis cells that produce a ratio of cyclohexyl B2 to cyclohexyl B1 avermectins of 0.35: 1 or less. In one non-limiting embodiment, the cell comprises a mutant aveC allele or degenerate variant thereof, which encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of those listed in (a) - (be) above.

In a preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.30: 1 or less. In one non-limiting embodiment, the cell comprises a mutant aveC allele or degenerate variant thereof, which encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of those listed in (f) - (be) above.

In a more preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.25: 1 or less. In one non-limiting embodiment, the cell comprises a mutant aveC allele or degenerate variant thereof, which encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (w) - (be) above.

In a more preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.20: 1 or less. In one non-limiting embodiment, the cell comprises a mutant aveC allele or degenerate variant thereof, which encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (ao) - (be) above.

The present invention also provides a Streptomyces cell comprising a mutant S.avermitilis aveC allele or degenerate variant thereof, which mutant aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of those listed in (bf) and (bg) above. In a preferred embodiment, the streptomyces is streptomyces avermitilis. In a more preferred embodiment, the cell is a Streptomyces avermitilis cell that produces a ratio of cyclohexyl B2: cyclohexyl B1 avermectins of about 0.40: 1 or less.

The invention also provides a method of producing avermectins comprising culturing cells of the S.avermitilis strain of the invention in a culture medium under conditions that permit or induce production of avermectins, and recovering the avermectins from the culture.

The present invention also provides a composition of cyclohexyl B2: cyclohexyl B1 avermectins produced by cells of S.avermitilis, comprising cyclohexyl B2: cyclohexyl B1 avermectins in a medium in which the cells have been cultured, wherein the ratio of cyclohexyl B2: cyclohexyl B1 avermectins in the medium is 0.35: 1 or less, preferably about 0.30: 1 or less, more preferably about 0.25: 1 or less, more preferably about 0.20: 1 or less. In a particular embodiment, the combination of cyclohexyl B2: cyclohexyl B1 avermectins is produced by a cell of a strain of S.avermitilis that expresses a mutant aveC allele or degenerate variant thereof, wherein the gene product encoded by the mutant aveC allele or degenerate variant thereof results in a class 2:1 ratio of cyclohexyl B2: cyclohexyl B1 avermectins produced by the cell that is reduced relative to a cell of the same strain of S.avermitilis that does not express the mutant aveC allele but instead expresses only the wild-type aveC allele.

In a preferred embodiment, when the composition is a 0.35: 1 or lower ratio of cyclohexyl B2: cyclohexyl B1 avermectin, the composition is produced by a cell comprising a mutant aveC allele or degenerate variant thereof, which mutant aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (a) - (be) above.

In a more preferred embodiment, the composition is produced by a cell comprising a mutant aveC allele, or a degenerate variant thereof, that encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (f) - (be) above, when the composition is cyclohexyl B2: cyclohexyl B1 avermectin, at a ratio of about 0.30: 1 or less.

In a more preferred embodiment, the composition is produced by a cell comprising a mutant aveC allele, or a degenerate variant thereof, that encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (w) - (be) above, when the composition is cyclohexyl B2: cyclohexyl B1 avermectin, at a ratio of about 0.25: 1 or less.

In a more preferred embodiment, the composition is produced by a cell comprising a mutant aveC allele, or a degenerate variant thereof, that encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (ao) - (be) above, when the composition is cyclohexyl B2: cyclohexyl B1 avermectin, at a ratio of about 0.20: 1 or less.

The present invention also provides compositions of cyclohexyl B2: cyclohexyl B1 avermectins produced by a Streptomyces avermitilis cell, comprising cyclohexyl B2: cyclohexyl B1 avermectins in a medium in which the cell has been cultured, wherein the ratio of cyclohexyl B2: cyclohexyl B1 avermectins in the medium is about 0.40: 1 or less, and compositions of cyclohexyl B2: cyclohexyl B1 avermectins produced by a cell comprising a mutant aveC allele or degenerate variant thereof that encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (bf) and (bg) above.

The invention also relates to the following.

1. A polynucleotide molecule comprising a nucleotide sequence that is identical to the nucleotide sequence of the aveC allele of streptomyces avermitilis, or the aveC gene product-encoding sequence of the aveC ORF of streptomyces avermitilis as shown in figure 1(SEQ ID NO: 1), or a degenerate variant thereof, but which further comprises a mutation that encodes a nucleotide sequence corresponding to SEQ ID NO:2, said mutation(s) resulting in production of avermectins by those cells of the strain of streptomyces avermitilis ATCC53692 in which the wild-type aveC allele has been inactivated but which express a polynucleotide molecule comprising said mutant nucleotide sequence and which produce avermectins having a reduced ratio of class 2:1 avermectins relative to the ratio produced by those cells of the strain of streptomyces avermitilis ATCC53692 which express only the wild-type aveC allele, wherein when said class 2:1 avermectins are cyclohexyl B2: cyclohexyl B1 avermectins, the ratio of said class 2:1 avermectins is 0.35: 1 or less.

2. The polynucleotide molecule of item 1, wherein the ratio of cyclohexyl B2: cyclohexyl B1 avermectin is 0.20: 1 or less.

3. The polynucleotide molecule of item 1, wherein said combination of amino acid substitutions comprises a combination of amino acid substitutions selected from the group consisting of those listed in (a) - (be) above.

4. A polynucleotide molecule comprising a nucleotide sequence that is identical to the nucleotide sequence of the S.avermitilis aveC gene product-encoding sequence of S.avermitilis on plasmid pSE186(ATCC209604), or the aveC ORF of S.avermitilis as shown in FIG. 1(SEQ ID NO: 1), or a degenerate variant thereof, but which further comprises a mutation that encodes a nucleotide sequence corresponding to SEQ ID NO:2, said mutation results in production of avermectins by those cells of the strain of streptomyces avermitilis ATCC53692 that have had the wild-type aveC allele inactivated but which are capable of expressing a polynucleotide molecule comprising said mutant nucleotide sequence and produce avermectins having a reduced ratio of class 2:1 avermectins relative to the ratio produced by those cells of the strain of streptomyces avermitilis ATCC53692 that express only the wild-type aveC allele, wherein when said class 2:1 avermectins are cyclohexyl B2: cyclohexyl B1 avermectins, the ratio of said class 2:1 avermectins is reduced to about 0.40: 1 or less, and wherein said combination of amino acid substitutions comprises a combination of amino acid substitutions selected from the group consisting of the amino acid substitutions set forth in (bf) and (bg) above.

5. A host cell comprising the polynucleotide molecule of item 1 or 4.

6. A method of producing a streptomyces avermitilis strain, comprising: (i) mutating an aveC allele in a cell of a Streptomyces avermitilis strain, the mutation resulting in a combination of amino acid substitutions in the AveC gene product, or (ii) introducing into a cell of a Streptomyces avermitilis strain a mutated aveC allele or a degenerate variant thereof, the mutated aveC allele or the degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions, the combination of amino acid substitutions in the AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of the amino acid substitutions set forth in (a) - (be) above.

7. A method of producing a streptomyces avermitilis strain, comprising: (i) mutating an aveC allele in a cell of a strain of S.avermitilis, the mutation resulting in a combination of amino acid substitutions in the AveC gene product, or (ii) introducing into a cell of a strain of S.avermitilis a mutated aveC allele or a degenerate variant thereof, the mutated aveC allele or the degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions, wherein the cell comprising the mutated aveC allele or the degenerate variant thereof is capable of producing cyclohexyl B2: cyclohexyl B1 avermectin at a ratio of 0.35: 1 or less.

8. The method of item 7, wherein said combination of amino acid substitutions comprises a combination of amino acid substitutions selected from the group consisting of (a) - (be) above.

9. The process of item 7, wherein the ratio of cyclohexyl B2: cyclohexyl B1 avermectin is about 0.20: 1 or less.

10. A method of producing a streptomyces avermitilis strain, comprising: (i) mutating an aveC allele in a cell of a Streptomyces avermitilis strain, the mutation resulting in a combination of amino acid substitutions in the AveC gene product, or (ii) introducing into a cell of a Streptomyces avermitilis strain a mutated aveC allele or a degenerate variant thereof, the mutated aveC allele or the degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions, and wherein the combination of amino acid substitutions comprises a combination of amino acid substitutions selected from the group consisting of (bf) and (bg) listed above.

11. A Streptomyces cell comprising a mutant S.avermitilis aveC allele or degenerate variant thereof, said mutant aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (a) - (be) above.

12. The cell of item 11, which is a streptomyces avermitilis cell.

13. A Streptomyces cell comprising a mutant S.avermitilis aveC allele or degenerate variant thereof, said mutant aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of those set forth in (bf) and (bg) above.

14. A composition of cyclohexyl B2: cyclohexyl B1 avermectins produced by cells of S.avermitilis, said composition comprising cyclohexyl B2: cyclohexyl B1 avermectins in a medium in which the cells are cultured, and the ratio of cyclohexyl B2: cyclohexyl B1 avermectins in the medium being 0.35: 1 or less.

4. Description of the drawings

FIG. 1. DNA sequence (SEQ ID NO: 1) containing the aveC ORF of S.avermitilis and its putative amino acid sequence (SEQ ID NO: 2).

FIG. 2. plasmid vector pSE186(ATCC209604) containing the complete ORF of the aveC gene of S.avermitilis.

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

FIG. 4. BamHI restriction map of avermectin polyketide synthase gene cluster from S.avermitilis, and five overlapping cosmid clones identified (i.e., pSE65, pSE66, pSE67, pSE68, PSE 69). The relationship of pSE118 to pSE119 is also indicated.

FIGS. 5A-5D HPLC analysis of fermentation products of S.avermitilis strains. Peak quantification was performed by comparison with standard amounts of cyclohexyl B1. The retention time of cyclohexyl B2 was 7.4 to 7.7 minutes and the retention time of cyclohexyl B1 was 11.9 to 12.3 minutes. FIG. 5A. Streptomyces avermitilis SE180-11 strain carrying an inactivated aveC ORF. FIG. 5B, S.avermitilis strain SE180-11 transformed with pSEI86(ATCC 209604). FIG. 5C, S.avermitilis SE180-11 strain transformed with pSE 187. FIG. 5D S.avermitilis SE180-11 strain transformed with pSE 188.

FIGS. 6A-6M. A list is compiled showing the combinations of amino acid substitutions encoded by the aveC allelic mutations identified in the second round of "Gene shuffling" and their effect on the ratio of cyclohexyl B2: cyclohexyl B1 production. For each plasmid, in the column entitled "mutations," the amino acid substitutions are listed in the upper panel and the nucleotide base changes that result in these amino acid substitutions are listed in the lower panel. Nucleotide base changes in parentheses are silent changes, i.e., they do not change the amino acid sequence.

5. Detailed description of the invention

The present invention relates to the identification and characterization of polynucleotide molecules having nucleotide sequences encoding S.avermitilis AveC gene products, the construction of novel strains of S.avermitilis useful for screening for the effect of mutated AveC gene products on avermectin production, and the discovery that some mutated AveC gene products can reduce the ratio of avermectins B2: B1 produced by S.avermitilis. For example, the present invention describes hereinafter a polynucleotide molecule having the same nucleotide sequence as the S.avermitilis AveC gene product-encoding sequence of plasmid pSE186(ATCC209604), or the nucleotide sequence of the ORF of FIG. 1(SEQ ID NO: 1), and polynucleotide molecules having mutant nucleotide sequences derived from those sequences, and degenerate variants thereof. The principles of the invention may be similarly applied to other polynucleotide molecules, including aveC homologous genes from other Streptomyces species, such as S.hygroscopicus and S.griseochromogenes.

5.1.Polynucleotide molecules encoding S.avermitilis AveC gene products

The present invention provides an isolated polynucleotide molecule comprising the entire aveC ORF of S.avermitilis, or a substantial portion thereof, that lacks the next entire ORF that is downstream from the location of the aveC ORF in the S.avermitilis chromosome.

The isolated polynucleotide molecule of the present invention preferably comprises a nucleotide sequence that is identical to the S.avermitilis AveC gene product-encoding sequence of plasmid pSE186(ATCC209604), or identical to the nucleotide sequence of the ORF of FIG. 1(SEQ ID NO: 1), or substantial portion thereof. As used herein, a "substantial portion" of an isolated polynucleotide molecule comprising a nucleotide sequence that encodes an AveC gene product of S.avermitilis refers to an isolated polynucleotide molecule that comprises at least about 70% of the complete aveC ORF sequence shown in FIG. 1(SEQ ID NO: 1) and that encodes a functionally equivalent AveC gene product. Herein, a "functionally equivalent" AveC gene product is defined as a gene product that, when expressed in S.avermitilis strain ATCC53692 in which the native aveC allele is inactivated, results in the production of substantially the same ratio and amount of avermectins as compared to S.avermitilis strain ATCC53692 which only expresses the native wild-type functional aveC allele of S.avermitilis strain ATCC 53692.

In addition to the nucleotide sequence of the aveC ORF, the isolated polynucleotide molecules of the invention may further comprise nucleotide sequences naturally flanking the aveC gene in situ in S.avermitilis, such as the flanking nucleotide sequences shown in FIG. 1(SEQ ID NO: 1).

The invention also provides an isolated polynucleotide molecule comprising, based on the known degeneracy of the genetic code, SEQ ID NO:1 or a degenerate variant thereof.

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

The nucleotide sequence shown in FIG. 1(SEQ ID NO: 1) contains four different GTG codons at bp positions 42, 174, 177 and 180. As shown in U.S. Pat. No. 6,248,579, multiple deletions of the 5' region of the aveC ORF (FIG. 1; SEQ ID NO: 1) can be constructed to help determine which of these codons can serve as initiation sites for protein expression in the aveC ORF. Deletion of the first GTG site at bp 42 did not abolish AveC activity. Further deletion of all GTG codons at bp positions 174, 177 and 180 abolished AveC activity, indicating that this region is essential for protein expression. The present invention thus encompasses aveC ORFs having different lengths.

The present invention also provides a polynucleotide molecule comprising a nucleotide sequence that is homologous to the S.avermitilis aveC gene product-encoding sequence of plasmid pSE186(ATCC209604), or to the nucleotide sequence of the aveC ORF shown in FIG. 1(SEQ ID NO: 1), or substantial portion thereof. The term "homologous" when used in reference to a polynucleotide molecule that is homologous to the S.avermitilis AveC gene product-encoding sequence, refers to a polynucleotide molecule having the nucleotide sequence: (a) the AveC gene product encoding sequence of S.avermitilis on plasmid pSE186(ATCC209604) encodes the same AveC gene product, or the aveC gene product encoding the same AveC ORF as shown in FIG. 1(SEQ ID NO: 1), but contains one or more silent changes in the nucleotide sequence based on the degeneracy of the genetic code (i.e., degeneracy)Variants); or (b) hybridizes under moderately stringent conditions, i.e., 0.5M NaHPO at 65 ℃, to the complement of a polynucleotide molecule comprising a nucleotide sequence encoding the amino acid sequence encoded by the AveC gene product coding sequence on plasmid pSE186(ATCC209604), or encoding the amino acid sequence depicted in FIG. 1(SEQ ID NO: 2), and encodes a functionally equivalent AveC gene product as described above₄7% Sodium Dodecyl Sulfate (SDS), hybridization with DNA bound to the filter in 1mM EDTA, and washing at 42 ℃ in 0.2 XSSC/0.1% SDS (see, e.g., Ausubel et al (ed.), 1989, Current Protocols in molecular Biology, Vol.1, Green Publishing Associates, Inc., and John Wiley&Sons, inc., New York, p.2.10.3). In a preferred embodiment, the homologous polynucleotide molecule hybridizes to the complement of the nucleotide sequence encoding the AveC gene product in plasmid pSE186(ATCC209604) or to the complement of the nucleotide sequence shown in FIG. 1(SEQ ID NO: 1), or a substantial portion thereof, under high stringency conditions, i.e., 0.5M NaHPO at 65 ℃, and encodes a functionally equivalent AveC gene product as described above₄Hybridization with DNA bound to the filter in 7% SDS, 1mM EDTA, and washing at 68 ℃ in 0.1 XSSC/0.1% SDS (Ausubel et al, 1989, supra).

The activity of the AveC gene product, and its potential functional equivalents, can be determined by HPLC analysis of the fermentation product, see examples below. Polynucleotide molecules comprising nucleotide sequences encoding functional equivalents of the aveC gene product of S.avermitilis, including the aveC gene naturally occurring in other strains of S.avermitilis, aveC homologous genes occurring in other species of S.avermitilis, and mutant aveC alleles, whether naturally or artificially synthesized.

The present invention also 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 AveC gene product-encoding sequence of plasmid pSE186(ATCC209604), or to the amino acid sequence depicted in FIG. 1(SEQ ID NO: 2), or substantial portion thereof. As used herein, a "substantial portion" of the amino acid sequence depicted in FIG. 1(SEQ ID NO: 2) refers to a polypeptide that includes at least about 70% of the amino acid sequence depicted in FIG. 1(SEQ ID NO: 2) and constitutes a functionally equivalent AveC gene product as described above.

As used herein to describe an amino acid sequence that is homologous to an amino acid sequence of an AveC gene product of S.avermitilis, the term "homologous" refers to a polypeptide comprising the amino acid sequence shown in FIG. 1(SEQ ID NO: 2) wherein one or more amino acid residues have been conservatively substituted by a different amino acid residue, wherein said amino acid sequence has at least about 70%, more preferably at least about 80%, and most preferably at least about 90% amino acid sequence identity as determined by any standard amino acid sequence identity algorithm, such as the BLASTP algorithm (GENBANK, NCBI), as compared to the polypeptide encoded by the AveC gene product coding sequence on plasmid pSE186(ATCC209604), or to the amino acid sequence shown in FIG. 1(SEQ ID NO: 2), wherein said conservative amino acid substitution results in a functionally equivalent gene product, as defined above. Conservative amino acid substitutions are well known in the art. Rules for making such substitutions are found in Dayhof, m.d., 1978, nat. biomed. res. foundation, Washington, d.c., vol.5, sup.3, etc. More specifically, conservative amino acid substitutions are those that typically occur in a family of amino acids with associated acidity or polarity. The amino acids encoded by a gene are generally divided into four groups: (1) acid-aspartic acid, glutamic acid; (2) basic ═ lysine, arginine, histidine; (3) non-polar ═ alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar ═ glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine can also be classified as aromatic amino acids. One or more substitutions in any particular group will generally have no significant effect on the function of the polypeptide, e.g., a leucine substituted with an isoleucine or valine, or an aspartate substituted with a glutamate, or a threonine substituted with a serine, or any other amino acid residue substituted with a structurally related amino acid residue (e.g., with a similar acidity or polarity, or with some combination of similar amino acid residues).

The production and manipulation of the polynucleotide molecules disclosed herein is well within the skill of the art and may be performed according to recombinant techniques, e.g., Maniatis et al, 1989,Molecular Cloning， A Laboratory Manualcold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; ausubel et al, 1989,Current Protocols In Molecular Biology，GreenePublishing Associates &wiley Interscience, NY; the contents of Sambrook et al, 1989,Molecular Cloning：A Laboratory Manual2d ed., Cold Spring Harbor Laboratory pressure Cold Spring Harbor, NY; innis et al (eds.), 1995,PCR Strategiesacademic pressinc, San Diego; the results of the work by Erlich (eds.), 1992,PCR Technologyoxford University Press, New York (incorporated herein by reference). Polynucleotide clones encoding an AveC gene product or an AveC homologous gene product can be identified by any method known in the art, including, but not limited to, the methods listed in section 7, below. By using a method such as Benton and Davis, 1977, Science, 196: 180 and Grunstein and Hogness, 1975, proc.natl.acad.sci.usa, 72: 3961-3965, the method for screening plasmid libraries can screen aveC and aveC homolog coding sequences from genomic DNA libraries. Polynucleotide molecules having nucleotide sequences known to include the aveC ORF, such as those present in plasmid pSE186(ATCC209604), or plasmid pSE119 (see section 7 below), can be used as probes in these screening experiments. Alternatively, the corresponding oligonucleotide probes can be synthesized based on the deduced nucleotide sequence of part or all of the amino acid sequence of the purified AveC homologous gene product.

5.2.Recombination system

5.2.1.Cloning and expression vectors

The present invention also provides recombinant cloning and expression vectors that can be used to clone or express the polynucleotide molecules of the present invention that comprise, for example, the aveC ORF of S.avermitilis or any of the aveC homolog ORFs. In a non-limiting embodiment, the present invention provides plasmid pSE186(ATCC209604), which comprises the complete ORF of the aveC gene of S.avermitilis.

All of the following descriptions of the S.avermitilis aveC ORF, or polynucleotide molecules comprising the S.avermitilis aveC ORF or portions thereof, or S.avermitilis AveC gene product, also refer to mutated aveC alleles as described below, unless explicitly indicated or clear from the context.

A variety of different vectors have been developed for use specifically in Streptomyces, including phage, high copy number plasmids, low copy number plasmids, and E.coli-Streptomyces shuttle plasmids, among others, any of which can be used to practice the present invention. In addition, a number of drug resistance genes have been cloned from Streptomyces, some of which are incorporated into vectors as selectable markers. Examples of the use of these vectors in streptomyces are found, for example, in Hutchinson, 1980, Applied biochem. 169 to 190.

The recombinant vectors of the invention, particularly expression vectors, are preferably constructed such that the coding sequence of the polynucleotide molecule of the invention is operably linked to one or more regulatory elements necessary for the transcription or translation of the coding sequence in order to produce the polypeptide. The term "regulatory element" as used herein includes, but is not limited to, nucleotide sequences encoding inducible and non-inducible promoters, enhancers, operators, and other elements known in the art for driving and/or regulating expression of a polynucleotide coding sequence. In addition, as used herein, a coding sequence is "operably linked" to one or more regulatory elements, wherein the regulatory elements are effective to regulate and allow transcription of the coding sequence and/or translation of its mRNA.

Exemplary plasmid vectors which have been engineered to contain the polynucleotide molecules of the present invention include pCR-Blunt, pCR2.1(Invitrogen), pGEM3Zf (Promega), and the shuttle vector pWHM3(Vara et al, 1989, J.Bact., 171: 5872-5881), among others.

Methods for constructing recombinant vectors containing specific coding sequences and operably linked to the appropriate regulatory elements are well known in the art and may be used to practice the present invention. These methods include in vitro recombinant techniques, synthetic techniques, and in vivo gene recombination. See Maniatis et al, 1989, supra; ausubel et al, 1989, supra; sambrook et al, 1989, supra; innis et al, 1995, supra; and techniques described in Erlich, 1992, supra.

The regulatory elements of these vectors may differ in strength and specificity. Any of a variety of suitable transcription and translation elements may be used depending on the host/vector system used. Non-limiting examples of transcriptional regulatory regions or promoters for bacteria include the β -gal promoter, the T7 promoter, the TAC promoter, the left and right lambda promoters, the trp and lac promoters, the trp-lac fusion promoter, and in particular for Streptomyces, the promoters are ermE, melC, tipA, and the like. In one embodiment, an expression vector is prepared containing the aveC ORF or mutated ORF thereof that has been cloned adjacent to a strong constitutive promoter, such as the ermE of Saccharopolyspora erythraea. Transformation of a vector containing the ermE promoter into S.avermitilis, as described in U.S. Pat. No. 6,248,579, followed by HPLC analysis of the fermentation product, showed that the titer of avermectins produced was increased relative to the production of the same strain that expressed only the wild-type aveC allele.

The fusion protein expression vector can be used to express an AveC gene product-fusion protein. The purified fusion proteins can be used to generate antisera against an AveC gene product, to study the biochemical properties of an AveC gene product, to engineer AveC fusion proteins with different biochemical activities, or to aid in the identification or purification of an expressed AveC gene product. Possible fusion protein expression vectors include, but are not limited to, vectors incorporating a sequence encoding beta-galactosidase and trpE fusions, maltose binding protein fusions, glutathione-S-transferase fusions, and polyhistidine fusions (carrier regions). In another embodiment, the AveC gene product, or a portion thereof, can be fused to an AveC homologous gene product, or portion thereof, derived from another species or strain of Streptomyces (e.g., S.hygroscopicus or S.griseochromogenes). Such hybrid vectors are transformed into S.avermitilis cells and the effect, e.g., on the class 2:1 ratio of avermectins produced, is examined.

AveC fusion proteins can be engineered to contain regions useful for purification. For example, an AveC-maltose binding protein fusion can be purified using amylose resin; the AveC-glutathione-S-transferase fusion protein can be purified using glutathione-agarose beads; the AveC-polyhistidine fusion can be purified using a divalent nickel resin. Alternatively, affinity purification of the fusion protein may be performed with antibodies against the carrier protein or peptide. For example, a nucleotide sequence encoding a target epitope of a monoclonal antibody can be inserted into an expression vector at an appropriate location, operably linked to regulatory elements, and the expressed epitope fused to an AveC polypeptide. For example, the encoded FLAG may be encoded by standard techniques^TMThe nucleotide sequence of the epitope tag (international biotechnologies Inc.) which is a hydrophilic tag peptide is inserted into an expression vector such that its insertion position corresponds to, for example, the carboxyl terminus of the AveC polypeptide. Expressed AveC polypeptide-FLAG^TMThe epitope fusion product can then be used with a commercially available anti-FLAG^TMAntibody for detection and affinity purification.

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

The signal sequence, located upstream of and in frame with the aveC ORF, can be inserted into an expression vector using known methods in order to direct the trafficking and secretion of the expressed gene product. Non-limiting examples of signal sequences include those derived from alpha factor, immunoglobulins, outer membrane proteins, penicillinase, and T cell receptors, among others.

To aid in the selection of host cells transformed or transfected with a cloning or expression vector of the invention, the vector may be engineered to further include sequences encoding a reporter gene product or other selectable marker. Preferably, the coding sequence is operably linked to a regulatory element coding sequence as described above. Reporter genes useful in the present invention are known in the art and include those encoding green fluorescent protein, luciferase, xylE, tyrosinase, and the like. Nucleotide sequences encoding selectable markers are known in the art, including those encoding gene products that confer antibiotic resistance or antimetabolite resistance, or those that provide auxotrophic requirements. Examples of such sequences include those encoding resistance to erythromycin, thiostrepton, kanamycin, or the like.

5.2.2Transformation of host cells

The invention further provides transformed host cells containing the polynucleotide molecules or recombinant vectors of the invention, or novel strains or cell lines derived therefrom. The host cells useful in the practice of the present invention are preferably Streptomyces cells, but other prokaryotic or eukaryotic cells may be used. Such transformed host cells generally include, but are not limited to, microorganisms such as bacteria transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA vectors, or yeast transformed with recombinant vectors, and the like.

The polynucleotide molecules of the invention are intended to function in Streptomyces cells, but may also be transformed into other bacterial or eukaryotic cells for purposes such as cloning or expression. Coli strains, such as the DH5 alpha strain, which is available from American Type Culture Collection (ATCC), Rockville, MD, USA (accession number 31343), or commercially available (Stratagene), are generally used. Preferred eukaryotic host cells include yeast cells, and mammalian cells or insect cells can also be effectively utilized.

The recombinant expression vectors of the invention are preferably introduced (e.g., transformed or transfected) into one or more host cells that are substantially uniformly cultured. Expression vectors are typically introduced into host cells using known techniques such as protoplast transformation, calcium phosphate precipitation, calcium chloride treatment, microinjection, electroporation, transfection by contact with recombinant viruses, liposome-mediated transfection, DEAE-dextran transfection, transduction, conjugation, or microprojectile bombardment. Transformants can be selected by standard methods, for example, by selecting cells that express a selectable marker associated with the recombinant vector, such as antibiotic resistance, as described above.

The fact that the aveC coding sequence has been integrated and maintained in the host chromosome or in episomal form once the expression vector is introduced into the host cell can be confirmed by standard techniques such as Southern hybridization analysis, restriction enzyme analysis, PCR (including reverse transcriptase PCR (rt-PCR)) analysis, or by detecting the desired gene product by immunological assays. A host cell containing and/or expressing a recombinant aveC coding sequence can be identified by any of at least four conventional methods known in the art, including: (i) DNA-DNA, DNA-RNA, or RNA-antisense RNA hybridization; (ii) detecting the presence of a marker gene function; (iii) (iii) assessing the level of transcription by measuring the expression of an aveC-specific mRNA transcript in the host cell, and (iv) determining the presence of a mature polypeptide product by immunoassay or the presence of AveC biological activity (e.g., production of a particular ratio and amount of avermectin indicates the presence of AveC activity in, for example, a S.avermitilis host cell).

5.2.3.Expression and identification of recombinant AveC Gene products

Once the native or mutated aveC coding sequence has been stably introduced into an appropriate host cell, the transformed host cell can be clonally propagated, and the resulting cells can be cultured under conditions conducive to the maximum production of the native or mutated AveC gene product. The conditions typically include culturing the cells to high density. When the expression vector contains an inducible promoter, appropriate induction conditions, e.g., temperature change, nutrient depletion, addition of a obligatory inducer (e.g., an analog of a carbohydrate such as isopropyl- β -D-thiogalactopyranoside (IPTG)), accumulation of excess metabolic byproducts, etc., can be employed as needed to induce expression.

While the expressed AveC gene product remains inside the host cell, 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, e.g., at 4 ℃ and/or in the presence of a protease inhibitor. When the expressed AveC gene product is secreted from the host cell, it is only necessary to collect the depleted nutrient medium and isolate the product therefrom.

The expressed AveC gene product can be isolated or substantially purified from the corresponding cell lysate or culture medium using standard methods, including, but not limited to, any combination of the following methods: ammonium sulfate precipitation, size fractionation, ion exchange chromatography, HPLC, density centrifugation and affinity chromatography. Where the expressed AveC gene product exhibits biological activity, the increasing purity of the preparation can be monitored at each step of the purification step using appropriate assays. Whether or not the expressed AveC gene product exhibits biological activity, it can be detected by its size or reactivity with an AveC-specific antibody, or in the presence of a fusion tag. As used herein, an AveC gene product is "substantially purified" when it comprises more than about 20% by weight of the protein in a particular formulation. Also, as used herein, an AveC gene product is "isolated" when it comprises at least about 80% by weight of the protein in a particular formulation.

The present invention thus provides an isolated or substantially purified recombinant S.avermitilis AveC gene product comprising an amino acid sequence that can be encoded by the AveC gene product-encoding sequence of plasmid pSE186(ATCC209604), or the amino acid sequence depicted in FIG. 1(SEQ ID NO: 2), or a substantial portion thereof, as well as mutants and degenerate variants of said gene product.

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

The recombinantly expressed S.avermitilis AveC gene product can be used for a variety of purposes, including screening for compounds that alter AveC gene product function and thereby modulate avermectin biosynthesis, and producing antibodies against AveC gene products.

Once the AveC gene product is obtained in sufficiently high purity, it can be identified by standard methods, such as SDS-PAGE, size exclusion chromatography, amino acid sequence analysis, biological activity in the avermectin biosynthetic pathway to produce an appropriate product, and the like. For example, the amino acid sequence of the AveC gene product can be determined using standard peptide sequencing techniques. Hydrophilic and hydrophobic regions of the AveC gene product can be identified using hydrophilicity assays (see, e.g., Hopp and Woods, 1981, Proc. Natl. Acad. Sci. USA 78: 3824) or similar software algorithms, thereby further identifying the AveC gene product. Structural analysis can be performed to identify regions of the AveC gene product that have particular secondary structures, for example, using biophysical methods such as X-ray crystallography (Engstrom, 1974, biochem. exp. biol. 11: 7-13), computer modeling (Fletterick and Zoller (eds.), 1986,Communications in Molecular Biologycold Spring Harbor Laboratory, Cold Spring Harbor, NY), and Nuclear Magnetic Resonance (NMR) to map and study the sites 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 with more appropriate avermectin production characteristics.

5.3.Construction and application of aveC mutant

It is a primary object of the present invention to identify novel mutations in the aveC allele of S.avermitilis that result in an altered (most preferably reduced) ratio of B2: B1 avermectins. The present invention thus provides a polynucleotide molecule useful for the production of cells of a novel strain of S.avermitilis that exhibit a detectable change in avermectin production as compared to cells of the same strain that express only the wild-type aveC allele. In a preferred embodiment, the polynucleotide molecules can be used to produce cells of a novel strain of S.avermitilis that produce avermectins in a lower class 2:1 ratio than cells of the same strain that express only the wild-type aveC allele. The cells of the strain may also contain additional mutations to produce greater amounts of avermectins than cells of the same strain that express only a single wild-type aveC allele.

Mutations in the aveC allele or coding sequence include any mutation that introduces one or more amino acid substitutions, deletions, and/or additions to the AveC gene product, or that results in truncation of the AveC gene product, or any combination thereof, and produces the desired result. The mutated aveC allele sequence also includes any degenerate variant thereof. For example, the present invention provides a polynucleotide molecule comprising the nucleotide sequence of the aveC allele, or a degenerate variant thereof, or comprising the AveC gene product-encoding sequence on plasmid pSE186(ATCC209604), or a degenerate variant thereof, or the nucleotide sequence of the aveC ORF of S.avermitilis as shown in FIG. 1(SEQ ID NO: 1), or a degenerate variant thereof, but which further comprises mutations encoding combinations of amino acid substitutions at selected positions in the AveC gene product. In one non-limiting embodiment, such substitutions occur in the AveC gene product corresponding to SEQ id no:2, at one or more amino acid positions 25, 28, 35, 36, 38, 40, 41, 48, 55, 61, 78, 84, 89, 90, 99, 107, 108, 111, 120, 123, 136, 138, 139, 141, 154, 159, 163, 179, 192, 196, 198, 200, 202, 220, 228, 229, 230, 231, 234, 238, 239, 250, 252, 266, 275, 278, 289, or 298. Preferably, the combination of amino acid positions to be substituted comprises one or more of D48, a61, a89, L136, S138, a139, R163, G179, V196, a198, E238 and P289. Particularly preferred combinations of amino acid substitutions comprise substitutions at D48 and G179, more preferably D48E and G179S. Specific examples of combinations of amino acid substitutions that result in a decreased ratio of cyclohexyl B2 to cyclohexyl B1 are shown in FIGS. 6A-J.

The present invention therefore provides a polynucleotide molecule comprising a nucleotide sequence that is identical to the aveC allele of S.avermitilis, a nucleotide sequence that is identical to the sequence encoding the AveC gene product of S.avermitilis on plasmid pSE186(ATCC209604), or a nucleotide sequence that is identical to the aveC ORF of S.avermitilis as shown in FIG. 1(SEQ ID NO: 1), or a degenerate variant thereof, but which further comprises a mutation encoding a combination of amino acid substitutions that occur in a nucleotide sequence that corresponds to the amino acid sequence of SEQ ID NO:2, such that the ratio of class 2:1 avermectins produced by those cells of streptomyces avermitilis ATCC53692 in which the wild-type aveC allele has been inactivated at the amino acid residue(s) of amino acid position(s) of s avermitilis ATCC53692 and which express a polynucleotide molecule comprising the mutant nucleotide sequence described above is reduced relative to cells of streptomyces avermitilis ATCC53692 which express only the wild-type aveC allele, wherein the ratio of class 2:1 avermectins is 0.35: 1 or less when class 2 avermectins is cyclohexyl B2: cyclohexyl B1 avermectins. In a preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.30: 1 or less. In a more preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.25: 1 or less. In a preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.20: 1 or less.

In a specific embodiment, the combination of amino acid substitutions comprises a combination of group (a): D48E, a61T, a89T, S138T, a139T, G179S, a198G, P289L. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE538 (see FIG. 6).

In another specific embodiment, the combination of amino acid substitutions comprises a combination of group (b): G40S, D48E, L136P, G179S, E238D. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 559.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of group (c): D48E, L136P, R163Q, G179S. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 567.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of group (d): D48E, L136P, R163Q, G179S, E238D. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 572.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of group (e): D48E, L136P, R163Q, G179S, a200G, E238D. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 571.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of group (f): D48E, L136P, G179S, E238D. Non-limiting examples of plasmids encoding these amino acid substitutions are pSE501 and pSE 546.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (g): D48E, a61T, L136P, G179S, E238D. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 510.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (h): D48E, a61T, L136P, G179S. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 512.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of group (i): D48E, a89T, S138T, a139T, G179S. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 519.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of group (j): D48E, a61T, L136P, G179S, a198G, P202S, E238D, P289L. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 526.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of group (k): D48E, a61T, L136P, S138T, a139F, G179S, E238D, P289L. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 528.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of group (i): D48E, L136P, G179S, a198G, E238D, P289L. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 530.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (m): D48E, a61T, S138T, a139F, G179S, a198G, P289L. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 531.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (n): D48E, L84P, G111V, S138T, a139T, G179S, a198G, P289L. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 534.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of group (o): Y28C, D48E, a61T, a89T, S138T, a139T, G179S, E238D. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 535.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (p): D48E, a61T, a107T, S108G, L136P, G179S, S192A, E238D, P289L. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 542.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (q): D48E, L136P, G179S, R250W. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 545.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of sets (r): D48E, a89T, S138T, a139T, R163Q, G179S. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 548.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups(s): D48E, L136P, G179S, a198G, P289L. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 552.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (t): D48E, F78L, a89T, L136P, G179S. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 557.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of group (u): D48E, a89T, S138T, a139T, G179S, E238D, F278L. Non-limiting examples of plasmids encoding these amino acid substitutions are pSE564 and pSE 565.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (v): D48E, a89T, L136P, R163Q, G179S. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 568.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (w): D48E, a61T, a89T, G111V, S138T, a139F, G179S, E238D, P289L. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 543.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (x): D25G, D48E, a89T, L136P, S138T, a139T, V141A, I159T, R163Q, G179S. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 504.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (y): D48E, a89T, S90G, L136P, R163Q, G179S, E238D. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 508.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of group (z): D48E, a61T, a89T, G111V, S138T, a139T, G179S, E238D, P289L. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 511.

In another specific embodiment, said combination of amino acid substitutions comprises the combination of group (aa): D48E, a89T, S138T, a139T, G179S. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 520.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (ab): D48E, L136P, R163Q, G179S, S231L. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 523.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (ac): D48E, L136P, S138T, a139F, G179S, V196A, E238D. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 527.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (ad): D48E, a61T, a89T, F99S, S138T, a139T, G179S, E238D. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 539.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (ae): G35S, D48E, a89T, S138T, a139T, G179S, P289L. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 540.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (af): D48E, a61T, a89T, S138T, a139T, G179S, V196A, E238D. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 547.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (ag): D48E, a89T, G111V, S138T, a139T, G179S, a198G, E238D. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 550.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (ah): S41G, D48E, a89T, L136P, G179S. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 558.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (ai): D48E, a89T, L136P, R163Q, G179S, P252S. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 563.

In another specific embodiment, said combination of amino acid substitutions comprises a combination of groups (aj): D48E, a89T, L136P, G179S, F234S. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 566.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (ak): D48E, a89T, L136P, R163Q, G179S, E238D. Non-limiting examples of plasmids encoding these amino acid substitutions are pSE573 and pSE 578.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (al): Q36R, D48E, a89T, L136P, G179S, E238D. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 574.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (am): D48E, a89T, L136P, R163Q, G179S. Non-limiting examples of plasmids encoding these amino acid substitutions are pSE575 and pSE 576.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of sets (an): D48E, a89T, S138T, G179S. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 577.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (ao): D48E, a89T, L136P, G179S, E238D. Non-limiting examples of plasmids encoding these amino acid substitutions are pSE502 and pSE 524.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of sets (ap): D48E, a89T, L136P, K154E, G179S, E238D. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 503.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (aq): D48E, a89T, S138T, a139T, K154R, G179S, V196A, P289L. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 505.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of sets (ar): D48E, a89T, S138T, a139F, G179S, V196A, E238D. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 506.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of sets (as): D48E, a61T, a89T, L136P, G179S, V196A, a198G, P289L. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 507.

In another specific embodiment, said combination of amino acid substitutions comprises the combination of group (at): D48E, a61T, S138T, a139F, G179S, G196A, E238D, P289L. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 509.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (au): D48E, a89T, L136P, G179S. Non-limiting examples of plasmids encoding these amino acid substitutions are pSE514 and pSE 525.

In another specific embodiment, said combination of amino acid substitutions comprises a combination of groups (av): D48E, a89T, V120A, L136P, G179S. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 515.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (aw): D48E, a61T, a89T, S138T, a139F, G179S, V196A, a198G, E238D. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 517.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of sets (ax): D48E, a61T, a89T, G111V, S138T, a139F, G179S, V196A, E238D. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 518.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (ay): D48E, a61T, a89T, S138T, a139T, G179S, V196A, E238D, P289L. Non-limiting examples of plasmids encoding these amino acid substitutions are pSE529 and pSE 554.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (az): D48E, a61T, a89T, L136P, S138T, a139F, G179S, a198G, E238D. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 532.

In another specific embodiment, said combination of amino acid substitutions comprises a combination of groups (ba): D48E, a89T, S138T, a139F, G179S, a198G, V220A. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 536.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of group (bb): D48E, a61T, a89T, S138T, a139T, G179S, V196A, E238D, R239H, P289L. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 537.

In another specific embodiment, said combination of amino acid substitutions comprises a combination of the groups (bc): D48E, a61T, a89T, L136P, G179S, P289L. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 541.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of groups (bd): D48E, a89T, S138T, a139T, G179S, V196A, E238D, P289L. Non-limiting examples of plasmids encoding these amino acid substitutions are pSE549 and pSE 553.

In another specific embodiment, the combination of amino acid substitutions comprises a combination of sets (be): D48E, a61T, a89T, S138T, a139F, G179S, V196A, E238D. A non-limiting example of a plasmid encoding these amino acid substitutions is pSE 551.

The present invention also provides a polynucleotide molecule comprising a nucleotide sequence that is identical to the aveC allele of S.avermitilis, a nucleotide sequence that is identical to the sequence encoding the AveC gene product of S.avermitilis on plasmid pSE186(ATCC209604), or a nucleotide sequence that is identical to the aveC ORF of S.avermitilis as shown in FIG. 1(SEQ ID NO: 1), or a degenerate variant thereof, but which further comprises a mutation encoding a combination of amino acid substitutions that occur in a nucleotide sequence that corresponds to the amino acid sequence of SEQ ID NO:2, resulting in a reduced ratio of class 2:1 avermectins produced by those cells of streptomyces avermitilis ATCC53692 that have inactivated the wild-type aveC allele and that express a polynucleotide molecule comprising the mutant nucleotide sequence as described above, relative to cells of streptomyces avermitilis ATCC53692 that express only the wild-type aveC allele, wherein the ratio of class 2:1 avermectins is about 0.40: 1 or less when the class 2:1 avermectins are cyclohexyl B2: cyclohexyl B1 avermectins, and wherein the combination of amino acid substitutions comprises a combination selected from the group consisting of:

(bf) D48E, S138T, a139T, G179S, E238D; and

(bg)Y28C，Q38R，D48E，L136P，G179S，E238D。

non-limiting examples of plasmids encoding the amino acid substitutions of group (bf) are pSE556 and pSE 569. One non-limiting example of a plasmid encoding the (bg) set of amino acid substitutions is pSE 561.

The present invention contemplates that any of the above amino acid substitutions can be made by any modification of the nucleotide sequence of the aveC allele or a degenerate variant thereof that results in such substitution. For example, by changing the natural codon sequence or its degenerate variants to encode the same amino acid substitution in any of several alternative codons, it is possible to achieve the majority of amino acid substitutions described herein. The various possible sequences encoding the amino acid substitutions described above can be readily and clearly determined by those skilled in the art based on the degeneracy of the genetic code as described and known herein. In one non-limiting embodiment of each of the specific combinations listed above, the amino acid substitutions are effected by non-silent nucleotide changes as shown in FIG. 6.

As used herein, "amino acid substitution combinations including the following combination …" and the like, means that amino acid substitutions in the AveC gene product of the invention include at least those specifically listed, and may include other amino acid substitutions, or amino acid deletions, or amino acid additions, or some combination thereof, wherein expression of the resulting AveC gene product in S.avermitilis cells results in a reduction in the ratio of B2: B1 avermectins, as expected.

Mutation of the aveC allele or degenerate variant thereof can be carried out by any of a variety of known methods, including the use of error-prone PCR, or by cassette mutagenesis. For example, oligonucleotide-directed mutagenesis may be used to alter the sequence of the aveC allele or ORF in a defined manner, such as by introducing one or more restriction sites or a stop codon at a particular region within the aveC allele or ORF. Methods involving random fragmentation, iterative rounds of mutagenesis, and nucleotide shuffling can also be used to generate large libraries of polynucleotides having nucleotide sequences encoding aveC mutations, as described in U.S. Pat. Nos. 5,605,793, 5,830,721, 5,837,458.

Targeted mutations are effective, particularly when they alter one or more conserved amino acid residues in the AveC gene product. For example, comparison of the putative amino acid sequence of the AveC gene product of S.avermitilis (SEQ ID NO: 2) with the putative amino acid sequences of the AveC homologous gene products from S.griseochromogenes (SEQ ID NO: 5) and S.hygroscopicus (SEQ ID NO: 4) (U.S. Pat. No. 6,248,579) reveals a significant conserved amino acid residue position between these species. Targeted mutagenesis resulting in changes to one or more of the conserved amino acid residues is highly effective for generating new mutants that can exhibit the desired altered avermectin production.

Random mutagenesis is also useful, and may be performed by exposing cells of S.avermitilis to ultraviolet or X-ray radiation, or to chemical mutagens such as N-methyl-N' -nitrosoguanidine, ethylmethane sulfonate, nitrous acid or nitrogen mustard, among others. For a review of mutagenesis techniques, see Ausubel, 1989, supra.

Once the mutated polynucleotide molecules are generated, they can be screened to determine whether they can modulate avermectin biosynthesis in S.avermitilis. In a preferred embodiment, polynucleotide molecules having a mutated nucleotide sequence are detected by complementing (complementing) strains of S.avermitilis that have an aveC gene inactivated to give an aveC-negative (aveC-) background. In one non-limiting method, the mutated polynucleotide molecule is spliced into an expression plasmid, operably linked to one or more regulatory elements, which preferably further comprises one or more drug resistance genes, to allow for selection of transformed cells. The vector is transformed into an aveC-host cell using known techniques, the transformed cells are selected and cultured in a suitable fermentation medium under conditions that permit or induce the production of avermectins, e.g., including a suitable starter subunit in the culture medium, and under conditions known in the art to be most suitable for avermectin production. The fermentation product is then analyzed by HPLC to determine the ability of the mutated polynucleotide molecule to complement the host cell. Several vectors carrying mutant polynucleotide molecules that reduce the avermectin B2: B1 ratio, including pSE188, pSE199, pSE231, pSE239, and pSE290 through pSE297, are illustrated in section 8.3 below. Other examples of such plasmid vectors are shown in FIG. 6.

Any of the above processes of the invention may be carried out using a fermentation medium, preferably supplemented with cyclohexanecarboxylic acid, but other suitable fatty acid precursors may be used, for example, any of the fatty acid precursors listed in Table 1, or methylthiolactic acid (methylthiolactic acid).

Once a mutated polynucleotide molecule that modulates avermectin production in a desired direction is identified, the location of the mutation in the nucleotide sequence can be determined. For example, a polynucleotide molecule having a nucleotide sequence encoding a mutant AveC gene product can be isolated by PCR and subjected to DNA sequence analysis using known methods. By comparing the DNA sequences of the mutant and wild-type aveC alleles, mutations responsible for altering avermectin production can be identified. For example, a S.avermitilis AveC gene product contains a single amino acid substitution at any of position 55 (S55F), position 138 (S138T), position 139 (A139T), or position 230 (G230D), or a double substitution at position 138 (S138T) and position 139 (A139T or A139F), which corresponds to the amino acid positions shown in FIG. 1(SEQ ID NO: 2), resulting in an altered function of the AveC gene product and an altered class 2:1 ratio of avermectins produced (see section 8 below). In addition, the following 7 combinations of mutations were shown to be effective in reducing class 2:1 ratios of avermectins, respectively: (1) D48E/A89T; (2) S138T/A139T/G179S; (3) Q38P/L136P/E238D; (4) F99S/S138T/A139T/G179S; (5) A139T/M228T; (6) G111V/P289L; (7) A139T/K154E/Q298H. The present invention includes an additional 59 combinations of mutations shown to reduce the ratio of avermectin cyclohexyl B2 to cyclohexyl B1, which are shown in FIG. 6 and recited in the claims.

Here, the above expression (for example, A139T) means that the starting amino acid residue indicated by one letter (alanine A in this case) is substituted at the specified position (position 139 in the polypeptide of SEQ ID NO:2 in this case) with an amino acid residue (threonine T in this case) replacing the starting amino acid residue.

Herein, when an amino acid residue encoded by the aveC allele in the S.avermitilis chromosome, or in a vector or isolated polynucleotide molecule of the invention, is described as "corresponding to" the amino acid sequence of SEQ ID NO:2, or when an amino acid substitution is described as occurring at a position "corresponding to" SEQ ID NO:2, when referring to the amino acid residue at the same position of the AveC gene product, those skilled in the art can refer to SEQ ID NO:2 quickly determines the residue.

Herein, when a particular mutation in the aveC allele that encodes a particular mutation is described as "corresponding to" SEQ ID NO:1, or when the nucleotide position in the aveC allele is described as "corresponding to" the nucleotide position in SEQ ID NO:1, when referring to the nucleotide at the same relevant position in an aveC nucleotide sequence or degenerate variant thereof, the skilled person will refer to the nucleotide sequence shown in SEQ ID NO:1 can quickly determine the nucleotide.

The term "about" as used herein to describe the ratio of avermectin cyclohexyl B2 to cyclohexyl B1 means that the specified value is plus or minus 10% of the specified value.

The polynucleotide molecules of the invention may be "isolated", meaning that: (i) it has been purified and is therefore substantially free of other polynucleotide molecules having different nucleotide sequences; or (ii) it is in an environment that is not native, e.g., the aveC allele or mutated form thereof from S.avermitilis is in another cell that is not a cell of S.avermitilis; or (iii) it may take a form other than its native form, e.g., a shorter DNA fragment, such as a restriction fragment obtained by digesting a bacterial chromosome, which may or may not contain any of its helper regulatory sequences, which predominantly contains the aveC coding region or mutated form thereof, or which is subsequently integrated into a heterologous DNA segment, such as the chromosome of a bacterial cell (other than a S.avermitilis cell), or the DNA of a vector such as a plasmid or phage, or into a location on the S.avermitilis chromosome where the aveC allele is not naturally located.

The invention also provides recombinant vectors comprising the polynucleotide molecules of the invention. The recombinant vectors can be used to target any polynucleotide molecule comprising a mutant nucleotide sequence of the present invention to the aveC allelic site on the S.avermitilis chromosome for insertion or substitution of the aveC ORF, or portions thereof, by, for example, homologous recombination. However, in accordance with the present invention, the polynucleotide molecules provided herein comprising the mutant nucleotide sequences of the present invention may also function to modulate avermectin biosynthesis when inserted into a site other than the aveC allele on the S.avermitilis chromosome or when maintained as episome in S.avermitilis cells. Thus, the present invention also provides vectors comprising a polynucleotide molecule comprising a mutated nucleotide sequence of the present invention, which vectors are useful for inserting the polynucleotide molecule into the S.avermitilis chromosome at a position other than the aveC gene locus, or maintained episomally.

In a non-limiting embodiment, the vector is a gene replacement vector that can be used to insert a mutated aveC allele of the invention, or a degenerate variant thereof, into cells of a strain of S.avermitilis that produces a reduced ratio of class 2:1 avermectins as compared to cells of the same strain that express only the wild-type aveC allele, thereby producing a novel strain of S.avermitilis. Such gene replacement vectors can be constructed using the mutant polynucleotide molecules in expression vectors provided herein (e.g., those exemplified in section 8 below).

The present invention also provides a vector that can be used to insert a mutated aveC allele or a degenerate variant thereof into cells of a Streptomyces avermitilis strain, such that cells of the novel strain are produced that have an altered production of avermectins as compared to cells of the same strain that express only the wild-type aveC allele. In a preferred embodiment, the novel cell produces an increased amount of avermectins. In a specific non-limiting embodiment, the vector comprises a strong promoter known in the art, e.g., a strong constitutive ermE promoter from Saccharopolyspora erythraea (Saccharopolyspora erythraea), such that it is located upstream of and operably linked to the aveC ORF. The vector may be constructed using the mutated aveC allele in plasmid pSE189, following the procedure described in U.S. Pat. No. 6,248,579.

The present invention provides gene replacement vectors that can be used to inactivate the aveC gene in wild-type S.avermitilis strains. In a non-limiting embodiment, the gene replacement vector can be constructed using the mutated polynucleotide molecule present in plasmid pSE180(ATCC 209605), described in section 8.1 below (FIG. 3). The present invention also provides gene replacement vectors comprising or consisting of a nucleotide sequence of the aveC gene naturally flanking the S.avermitilis chromosome in situ, including, for example, those flanking nucleotide sequences as shown in FIG. 1(SEQ ID NO: 1), which vectors are useful for deleting the aveC ORF of S.avermitilis.

The invention also provides a host cell comprising a polynucleotide molecule or recombinant vector of the invention. The host cell may be any prokaryotic or eukaryotic cell capable of hosting the polynucleotide molecule or recombinant vector. In a preferred embodiment, the host cell is a bacterial cell. In a more preferred embodiment, the host cell is a cell of the genus Streptomyces. In a more preferred embodiment, the host cell is a cell of S.avermitilis.

The present invention also provides methods for producing novel strains of S.avermitilis, comprising (i) mutating the aveC allele in cells of a S.avermitilis strain resulting in the occurrence of a combination of amino acid substitutions in the AveC gene product, or (ii) introducing into cells of a S.avermitilis strain a mutated aveC allele or a degenerate variant thereof, which gene or variant encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (a) - (be) above.

The present invention also provides methods for producing novel strains of S.avermitilis, comprising (i) mutating the aveC allele in cells of a S.avermitilis strain, resulting in the occurrence of a combination of amino acid substitutions in the AveC gene product, or (ii) introducing into cells of a S.avermitilis strain a mutated aveC allele or a degenerate variant thereof, which gene or variant encodes an AveC gene product comprising a combination of amino acid substitutions, wherein the cells containing the mutated aveC allele or the degenerate variant thereof are capable of producing cyclohexyl B2: cyclohexyl B1 avermectins at a ratio of 0.35: 1 or less. In one non-limiting embodiment, the mutated aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from those listed in (a) - (be) above.

In a preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.30: 1 or less. In one non-limiting embodiment, the mutated aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (f) - (be) set forth above.

In a more preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.25: 1 or less. In one non-limiting embodiment, the mutated aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (w) - (be) listed above.

In a more preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.20: 1 or less. In one non-limiting embodiment, the mutated aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (ao) - (be) listed above.

The present invention also provides methods for producing novel strains of S.avermitilis, comprising (i) mutating the aveC allele in cells of a S.avermitilis strain resulting in a combination of amino acid substitutions in the AveC gene product, or (ii) introducing into cells of a S.avermitilis strain a mutated aveC allele or a degenerate variant thereof, which gene or variant encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (bf) and (bg). In a preferred embodiment, cells containing the mutated aveC allele or degenerate variant thereof are capable of producing cyclohexyl B2: cyclohexyl B1 avermectins in a ratio of about 0.40: 1 or less.

After mutating the aveC allele according to the above procedure, or introducing a mutated aveC allele or a degenerate variant thereof, a new strain of S.avermitilis can be produced.

The invention also provides a Streptomyces cell comprising a mutated aveC allele or a degenerate variant thereof, which gene or variant encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of those listed in (a) - (be) above. In a preferred embodiment, the species of Streptomyces is Streptomyces avermitilis.

The invention also provides Streptomyces avermitilis cells that produce cyclohexyl B2: cyclohexyl B1 avermectins at a ratio of 0.35: 1 or less. In one non-limiting embodiment, the cell comprises a mutated aveC allele or a degenerate variant thereof, which encodes an aveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (a) - (be) above.

In a preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.30: 1 or less. In one non-limiting embodiment, the cell comprises a mutant aveC allele or degenerate variant thereof, which encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (f) - (be) above.

In a more preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.25: 1 or less. In one non-limiting embodiment, the cell comprises a mutant aveC allele or degenerate variant thereof, which encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (w) - (be) set forth above.

In a more preferred embodiment, the ratio of cyclohexyl B2 to cyclohexyl B1 avermectin is about 0.20: 1 or less. In one non-limiting embodiment, the cell comprises a mutant aveC allele or degenerate variant thereof, which encodes an aveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (ao) - (be) set forth above.

The present invention also provides a cell of a streptomyces species comprising a mutant aveC allele or a degenerate variant thereof, encoding an aveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (bf) and (bg) listed above. In a preferred embodiment, the Streptomyces species is S.avermitilis. In a more preferred embodiment, the cells are S.avermitilis cells that produce cyclohexyl B2: cyclohexyl B1 avermectins at a ratio of about 0.40: 1 or less.

Any of the mutations described above may be present on an extrachromosomal element (e.g., a plasmid) in a cell of the invention, but preferably the mutation is present in the aveC coding sequence integrated into the S.avermitilis chromosome, preferably, but not necessarily, at the position of the native aveC allele.

Cells of the novel strains can be used for the large-scale production of commercially valuable avermectins such as doramectin.

The invention also provides a method of producing avermectins comprising culturing cells of the Streptomyces avermitilis of the invention in a culture medium under conditions that permit or induce production of avermectins by the cells, and recovering the avermectins from the culture. In a preferred embodiment, the cells used in the method produce cyclohexyl B2: cyclohexyl B1 avermectin at a ratio of 0.35: 1 or less, more preferably at a ratio of about 0.30: 1 or less, more preferably at a ratio of about 0.25: 1 or less, more preferably at a ratio of about 0.20: 1 or less.

In a preferred embodiment, a cell that produces cyclohexyl B2: cyclohexyl B1 avermectin at a ratio of 0.35: 1 or less, comprises a mutant aveC allele or degenerate variant thereof, which mutant aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (a) - (be) above.

In another preferred embodiment, a cell that produces a cyclohexyl B2: cyclohexyl B1 avermectin, at a ratio of about 0.30: 1 or less, comprises a mutant aveC allele or degenerate variant thereof, which mutant aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (f) - (be) above.

In another preferred embodiment, a cell that produces a cyclohexyl B2: cyclohexyl B1 avermectin, at a ratio of about 0.25: 1 or less, comprises a mutant aveC allele or degenerate variant thereof, which mutant aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (w) - (be) above.

In another preferred embodiment, a cell that produces a cyclohexyl B2: cyclohexyl B1 avermectin, at a ratio of about 0.20: 1 or less, comprises a mutant aveC allele or degenerate variant thereof, which mutant aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (ao) - (be) above.

In another preferred embodiment, the cell produces cyclohexyl B2: cyclohexyl B1 avermectin, at a ratio of about 0.40: 1 or less, and comprises a mutant aveC allele or degenerate variant thereof, which encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of those listed under (bf) and (bg) above.

The methods of the invention provide for the enhanced efficacy of the production of commercially valuable avermectins, such as doramectin.

The present invention also provides a composition of cyclohexyl B2: cyclohexyl B1 avermectins produced by cells of S.avermitilis comprising cyclohexyl B2: cyclohexyl B1 avermectins in a medium in which the cells are cultured, wherein the ratio of cyclohexyl B2: cyclohexyl B1 avermectins in the medium is 0.35: 1 or less, preferably about 0.30: 1 or less, more preferably about 0.25: 1 or less, preferably about 0.20: 1 or less. In a specific embodiment, the cyclohexyl B2: cyclohexyl B1 avermectin composition is produced by a cell of a strain of S.avermitilis that expresses a mutant aveC allele or degenerate variant thereof, which mutant aveC allele or degenerate variant thereof encodes a gene product that results in the cell producing a reduced ratio of cyclohexyl B2: cyclohexyl B1 avermectins as compared to a cell of the same strain of S.avermitilis that does not express the mutant aveC allele but instead expresses only the wild-type aveC allele.

In a preferred embodiment, when the composition is a 0.35: 1 or lower ratio of cyclohexyl B2: cyclohexyl B1 avermectin, the composition is produced by a cell comprising a mutant aveC allele or degenerate variant thereof, which encodes an aveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (a) - (be) above.

In another preferred embodiment, when the composition is a cyclohexyl B2: cyclohexyl B1 avermectin, in a ratio of about 0.30: 1 or less, the composition is produced by a cell comprising a mutant aveC allele or degenerate variant thereof, which mutant aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (f) - (be) above.

In another preferred embodiment, when the composition is a cyclohexyl B2: cyclohexyl B1 avermectin, in a ratio of about 0.25: 1 or less, the composition is produced by a cell comprising a mutant aveC allele or degenerate variant thereof, which mutant aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (w) - (be) above.

In another preferred embodiment, when the composition is a cyclohexyl B2: cyclohexyl B1 avermectin, in a ratio of about 0.20: 1 or less, the composition is produced by a cell comprising a mutant aveC allele or degenerate variant thereof, which mutant aveC allele or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (ao) - (be) above.

The present invention also provides a composition of cyclohexyl B2: cyclohexyl B1 avermectins produced by a streptomyces avermitilis cell comprising cyclohexyl B2: cyclohexyl B1 avermectins in a medium that cultures the cell, wherein the ratio of cyclohexyl B2: cyclohexyl B1 avermectins in the medium is about 0.40: 1 or less, and is produced by a cell comprising a mutant aveC allele or a degenerate variant thereof, wherein the mutant aveC allele or degenerate variant thereof encodes an aveC gene product comprising a combination of amino acid substitutions selected from the group consisting of (bf) and (bg) above.

Preferably, the novel avermectin composition is present in the culture medium of the cultured cells, e.g., in a partially or fully depleted fermentation broth, and the avermectin composition may alternatively be partially or substantially purified from the culture broth using known biochemical purification techniques, such as ammonium sulfate precipitation, dialysis, size fractionation, ion exchange chromatography, HPLC, and the like.

In addition to producing novel strains of S.avermitilis as described above, such that they comprise cells that produce a reduced ratio of cyclohexyl B2 to cyclohexyl B1, the present invention also relates to the introduction of other mutations into S.avermitilis cells in order to further improve avermectin production characteristics. In a non-limiting embodiment, the cells of the invention further comprise modifications to increase the level of avermectin production. In one embodiment, the cell may be prepared as follows: (i) mutating an aveC allele in a cell of S.avermitilis, or (ii) introducing a mutated aveC allele or a degenerate variant thereof into a cell of a strain of S.avermitilis, wherein expression of the mutated allele results in an increased production of avermectins by a cell of the strain of S.avermitilis that expresses the mutated aveC allele as compared to a cell of the same strain that expresses only a single wild-type aveC allele, and then selecting transformed cells that have increased production of avermectins as compared to a cell of a strain that expresses only a single wild-type aveC allele. For example, the aveC allele can be modified to include a strong promoter, such as the strong constitutive ermE promoter of Saccharopolyspora erythraea, upstream of and operably linked to the aveC ORF. In another embodiment, one or more mutations may be introduced into the aveR1 and/or aveR2 gene of S.avermitilis to increase avermectin production, as described in U.S. Pat. No. 3/6/2001 to Stutzman-Engwall 6,197,5591.

5.4.Use of avermectins

Avermectins are highly effective antiparasitic agents which can be used in particular as anthelmintics, ectoparasiticides, insecticides and acaricides. Avermectin compounds produced by the methods of the invention are useful for these purposes. For example, avermectin compounds produced according to the methods of the present invention are useful in the treatment of various diseases or conditions in humans, particularly those diseases known in the art to be caused by parasitic infections. See Ikeda and Omura, 1997, chem.rev.97 (7): 2591-2609. More specifically, the avermectin compounds produced by the methods of the present invention are effective in treating a variety of diseases caused by endoparasites, such as parasitic nematodes that infect humans, domestic animals, pigs, sheep, poultry, horses or cattle.

More specifically, avermectin compounds produced by the methods of the present invention are effective against nematodes that infect humans as well as nematodes that infect a variety of animals. Such nematodes include gastrointestinal parasites such as hookworms (Ancylostoma), nematodes (Necator), roundworms (Ascaris), strongylidae (Strongyloides), Trichinella (Trichinella), Trichinella (Capillaria), Trichuris (Trichuris), pinworms (Enterobius), heartworms (Dirofilaria), and parasites found in blood vessels or other tissues or organs such as filarial worms and intestinal extracts of roundworm and Trichinella.

The avermectin compounds produced by the methods of the present invention are also useful in the treatment of ectoparasitic infestations, including arthropod infestations of mammals or birds, such as infestations caused by ticks (ticks), mites, lice, fleas, green head flies, biting insects (biting insects), or migratory dipteran larvae, among others.

The avermectin compounds produced by the process of the present invention are also useful as insecticides against indoor pests (household pest) such as cockroaches, clothiantha, carpet beetles, and house flies, as well as against pests of stored grain and crops, including red spiders (spider mite), aphids, caterpillars, and orthopteran larvae such as locusts (locusts), among others.

Animals which can be treated with avermectin compounds produced by the methods of the present invention include sheep, cattle, horses, deer, goats, pigs, birds including poultry, dogs and cats.

The avermectin compounds produced by the methods of the present invention may be administered in a dosage form that is appropriate for the particular application, the particular species of host animal being treated, the parasite or insect involved, and the like. When used as parasiticides, the avermectin compounds produced by the methods of the present invention may be administered orally in the form of capsules, pills, tablets, or drenches, or in pour-on, injection, or implant form. Such formulations may be prepared in conventional manner according to standard veterinary practice. Thus, capsules, pills or tablets may be prepared by mixing the active ingredient with a suitable diluent or carrier for precise dispensing which additionally contains disintegrating agents and/or binding agents such as starch, lactose, talc, magnesium stearate and the like. The drench is prepared by dispersing the active ingredient in an aqueous solution together with a dispersant or wetting agent. Injectables can be formulated as sterile solutions, which can contain other substances, such as sufficient salts and/or glucose to render the solution isotonic with respect to blood.

The weight of active compound in these formulations may vary depending on the patient, or the type of host animal being treated, the severity and type of infection, the weight of the host, and the like. Generally, one dose for oral administration is about 0.001 to 10mg of active compound per kg of body weight of the patient or animal, administered in a single dose or divided over 1 to 5 days. But higher or lower dosages may sometimes be determined by a physician or veterinarian based on clinical symptoms.

Alternatively, the avermectin compounds produced by the methods of the present invention may also be administered with animal feed, for which purpose concentrated food additives or premixes may be prepared for mixing with conventional animal feed.

When used as pesticides, and for treating agricultural pests, the avermectin compounds produced by the methods of the present invention may also be used in sprays, dusts, emulsions and the like in accordance with standard agricultural practice.

6.Example (b): fermentation of Streptomyces avermitilis And disinsectizationAnalysis of rhzomorphs B2: B1

Strains lacking both branched-chain 2-oxoacid dehydrogenase and 5-O-methyltransferase activity will not produce avermectins if the fermentation medium is not supplemented with fatty acids. This example demonstrates that such mutants can achieve a wide range of avermectin B2: B1 ratios when biosynthesized in the presence of different fatty acids.

6.1.Materials and methods

Streptomyces avermitilis ATCC53692 was stored in a whole broth inoculation medium at-70 ℃ consisting of: starch (Nadex, lying National) -20 g; pharmamedia (Trader's protein, Memphis, TN) -15 g; ardamine pH (Yeast Products Inc.) -5 g; 1g of calcium carbonate. The final volume was adjusted to 1 liter with tap water, the pH was adjusted to 7.2 and the medium was autoclaved at 121 ℃ for 25 minutes.

2ml of the thawed suspension of the above preparation were inoculated into a flask containing 50ml of the same medium. After 48 hours of incubation at 28 ℃ on a rotary shaker at 180rpm, 2ml of broth was inoculated into a flask containing 50ml of production medium containing: 80g of starch; 7g of calcium carbonate; pharmamedia-5 g; 1g of dipotassium phosphate; magnesium sulfate-1 g; glutamic acid-0.6 g; ferrous sulfate heptahydrate-0.01 g; zinc sulfate-0.001 g; 0.001g of magnesium sulfite. The final volume was adjusted to 1 liter with tap water, the pH was adjusted to 7.2 and the medium was autoclaved at 121 ℃ for 25 minutes.

The different carboxylic acid substrates (see Table 1) were dissolved in methanol and added to the fermentation broth 24 hours after inoculation to a final concentration of 0.2 g/l. The fermentation broth was incubated at 28 ℃ for 14 days, followed by centrifugation (2,500rpm, 2 minutes) to remove the supernatant. The hyphal precipitate was extracted with acetone (15ml) and then dichloromethane (30ml), the organic phase separated, filtered and then evaporated to dryness. The residue was taken up in 1ml of methanol and analyzed by HPLC at 240nm using Hewlett-Packard 1090A liquid chromatography equipped with a scanning diode array detector using a Beckman Ultrasphere C-18, 5 μm, 4.6mmx25cm column maintained at 40 ℃. Mu.l of the above methanol solution was injected into the column. Elution was carried out with a linear gradient of methanol-water from 80: 20 to 95: 5 at a flow rate of 0.85/ml min for 40 minutes. The detector response was calibrated with two standard concentrations of cyclohexyl B1 and the area under the curve for avermectins B2 and B1 was measured.

6.2. Results

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

TABLE 1

The data presented in Table 1 demonstrate a relatively broad range of B2: B1 avermectin production ratios, which indicates that depending on the nature of the fatty acid side chain starter units provided, the dehydrating conversion results for compounds of class 2 versus compounds of class 1 are very different. This indicates that the change in the ratio of B2: B1 due to the change in AveC protein is specific for a particular substrate only. Thus, screening for mutants with altered ratios of B2 to B1 obtained with a particular substrate requires the presence of that substrate. The following examples describe the use of cyclohexane carboxylic acid as a screening substrate. However, this substrate is merely illustrative of the potential utility of the invention and is not intended to be limiting.

7.Example (b): isolation of the aveC Gene

This example describes the isolation and identification of a region of the S.avermitilis chromosome encoding an AveC gene product, which, as demonstrated below, was identified to alter the ratio of cyclohexyl B2 to cyclohexyl B1 avermectins produced (B2: B1).

7.1.Materials and methods

7.1.1.Growing Streptomyces species for isolation of DNA

The following procedure was used to culture Streptomyces. Streptomyces avermitilis ATCC 31272 strain single colony (Single colony isolate #)2) YPD-6 was isolated at 1/2 strength, and YPD-6 contained: difco yeast extract-5 g; difco bactopeptone-5 g; 2.5g of glucose; MOPS-5 g; difco bacterial agar-15 g, dH for final volume₂O was adjusted to 1 liter, pH was adjusted to 7.0, and the medium was autoclaved at 121 ℃ for 25 minutes.

The mycelia grown in the above medium were inoculated into 10ml of TSB medium (Difco Tryptic Soy Broth-30g in 1 liter dH) in a 25mmX150mm test tube₂O, autoclaved at 121 ℃ for 25 minutes), and cultured at 28 ℃ for 48 to 72 hours with shaking at 300 rpm.

7.1.2.Isolation of chromosomal DNA from Streptomyces

An aliquot (0.25ml or 0.5ml) of the mycelium grown as described above was placed in a 1.5ml microcentrifuge tube and the cells were concentrated by centrifugation at 12,000Xg for 60 seconds. The supernatant was removed and the cells resuspended in 0.25ml of TSE buffer (20ml of 1.5M sucrose, 2.5ml of 1M Tris-HCl, pH 8.0, 2.5ml of 1M EDTA, pH 8.0, and 75ml dH₂O), the buffer contained 2mg/ml lysozyme. Samples were incubated at 37 ℃ for 20 minutes with shaking and loaded into AutoGen540^TMAn automatic nucleic acid isolation system (Integrated isolation Systems, Natick, MA) used Cycle 159 (Instrument software) to isolate genomic DNA according to the manual.

Alternatively, 5ml of the mycelium was placed in a test tube of 17mmx100mm, centrifuged at 3,000rpm for 5 minutes to concentrate the cells, and the supernatant was removed. The cells were resuspended in 1ml of TSE buffer, centrifuged at 3,000rpm for 5 minutes to concentrate the cells, and the supernatant was removed. The cells were resuspended in 1ml of TSE buffer containing 2mg/ml lysozyme and incubated with shaking at 37 ℃ for 30-60 minutes. After incubation, 0.5ml of 10% Sodium Dodecyl Sulfate (SDS) was added and the cells were incubated at 37 ℃ until lysis was complete. The lysate was incubated at 60 ℃ for 10 minutes, cooled to room temperature, split into two 1.5ml Eppendorf tubes and extracted 1 time with 0.5ml phenol/chloroform (50% phenol, equilibrated beforehand with 0.5M Tris, pH 8.0; 50% chloroform). The aqueous phase was removed and extracted 2 to 5 times with chloroform/isoamyl alcohol (24: 1). The DNA was settled by adding 1/10 volumes of 3M sodium acetate, pH 4.8, the mixture was warmed in ice for 10 minutes, the mixture was centrifuged at 15,000rpm at 5 ℃ for 10 minutes, the supernatant was transferred to a clean tube, and 1-fold volume of isopropanol was added thereto. The supernatant was then incubated with isopropanol in ice for 20 minutes, the mixture was centrifuged at 15,000rpm at 5 ℃ for 20 minutes, the supernatant was removed, and the DNA pellet was washed 1 time with 70% ethanol. After the pellet was dried, the DNA was resuspended in TE buffer (10mM Tris, 1mM EDTA, pH 8.0).

7.1.3.Isolation of plasmid DNA from Streptomyces

An aliquot of mycelium (1.0ml) was placed in a 1.5ml microcentrifuge tube and centrifuged at 12,000Xg for 60 seconds to concentrate the cells. The supernatant was removed, the cells were resuspended in 1.0ml of 10.3% sucrose, centrifuged at 12,000Xg for 60 seconds, and the supernatant was removed. The cells were then resuspended in 0.25ml TSE buffer containing 2mg/ml lysozyme, incubated at 37 ℃ for 20 minutes with shaking, and then loaded onto AutoGen540^TMIn an automated nucleic acid isolation apparatus. Plasmid DNA was isolated using Cycle 106 (instrument software) according to the operating manual.

Alternatively, 1.5ml of mycelium was placed in a 1.5ml microcentrifuge tube and centrifuged at 12,000Xg for 60 seconds to concentrate the cells. The supernatant was removed, the cells were resuspended in 1.0ml of 10.3% sucrose, centrifuged at 12,000Xg for 60 seconds, and the supernatant was removed. The cells were resuspended in 0.5ml of TSE buffer containing 2mg/ml lysozyme and incubated for 15-30 minutes at 37 ℃. After incubation, 0.25ml of alkaline SDS (0.3N NaOH, 2% SDS) was added and incubated at 55 ℃ for 15-30 minutes or until the solution became clear. Sodium acetate (0.1ml, 3M, pH 4.8) was added to the DNA solution, which was incubated in ice for 10 minutes. The DNA sample was centrifuged at 14,000rpm at 5 ℃ for 10 minutes. The supernatant was transferred to a clean tube, to which 0.2ml of phenol/chloroform (50% phenol: 50% chloroform) was added and gently mixed. The DNA solution was centrifuged at 14,000rpm for 10 minutes at 5 ℃ and the supernatant was transferred to a clean Eppendorf tube. 0.75ml of isopropanol was added, the solution was gently mixed and then incubated at room temperature for 20 minutes. The DNA solution was centrifuged at 14,000rpm at 5 ℃ for 15 minutes, the supernatant was removed, and the DNA pellet was washed with 70% ethanol, dried, and resuspended in TE buffer.

7.1.4.Isolation of plasmid DNA from E.coli

Individual E.coli transformants were plated on 5ml Luria-Bertani (LB) medium (bactopeptone-10 g, bactopeptone yeast extract-5 g, and NaCl-10g at 1 liter dH₂O, pH7.0, autoclaved at 121 ℃ for 25 minutes, and incubated with 100. mu.g/ml ampicillin). Cultures were grown overnight, 1 μm aliquots were placed in 1.5ml microcentrifuge tubes, and culture samples were loaded into AutoGen540^TMIn the automatic nucleic acid separator, according to the operation manual, using Cycle 3 (instrument software) separation of plasmid DNA.

7.1.5.Preparation and transformation of S.avermitilis protoplast

A single colony of S.avermitilis was isolated in 1/2 strength YPD-6. The mycelia were inoculated into 10ml of TSB medium in a 25mmX150mm test tube and cultured with shaking at 300rpm at 28 ℃ for 48 hours. 1ml of mycelium was inoculated into 50ml of YEME medium. YEME medium contained per liter: difco yeast extract-3 g; difco bactopeptone-5 g; difco Malt Extract-3 g; sucrose-300 g. After autoclaving at 121 ℃ for 25 minutes, the following were added: 2.5M MgCl₂·6H₂O (autoclaved 25 min at 121 ℃ C. alone) -2 ml; and glycine (20%) (filter-sterilized) -25 ml.

The mycelia were incubated at 30 ℃ for 48-72 hours and then harvested by centrifugation in a 50ml centrifuge tube (Falcon) at 3,000rpm for 20 minutes. The supernatant was removed and the mycelium was resuspended in P buffer, which contained: sucrose-205 g; k₂SO₄-0.25g；MgCl₂6H₂O-2.02g；H₂O-600ml；K₂PO₄(0.5%) -10 ml; trace element solution-20 ml; CaCl₂2H₂O (3.68%) -100 ml; and MES buffer (1.0M, pH 6.5) -10ml (Trace element solution containing ZnCl per liter)₂-40mg；FeCl₃·6H₂O-200mg；CuCl₂·2H₂O-10mg；MnCl₂·4H₂O-10mg；Na₂B₄O₇·10H₂O-10mg；(NH₄)₆Mo₇O₂₄·4H₂O-10 mg). The pH was adjusted to 6.5, the final volume was adjusted to 1 liter and the medium was filtered hot through a 0.45 micron filter.

The mycelia were centrifuged at 3,000rpm for 20 minutes to obtain a pellet, the supernatant was removed, and the mycelia were resuspended in 20ml of P buffer containing 2mg/ml of lysozyme. The mycelium was incubated at 35 ℃ for 15 minutes with shaking and the degree of protoplast formation was determined by microscopic examination. When the formation of protoplasts was complete, it was centrifuged at 8,000rpm for 10 minutes. The supernatant was removed and the protoplasts were resuspended in 10ml of P buffer. Protoplasts were centrifuged at 8,000rpm for 10 minutes, the supernatant removed, resuspended in 2ml of P buffer, and approximately 1X109 protoplasts were dispensed into 2.0ml cryovials (Nalgene).

Vials containing 1X109 protoplasts were centrifuged at 8,000rpm for 10 minutes, the supernatant removed, and the protoplasts resuspended in 0.1ml of P buffer. 2-5. mu.g of the transformed DNA was added to the protoplasts followed by 0.5ml of T working solution. The T buffer matrix contains: PEG-1000(Sigma) -25 g; 2.5g of cane sugar; h₂O-83 ml. The pH was adjusted to 8.8 with 1N NaOH (filter-sterilized), the T-buffer substrate was filter-sterilized and stored at 4 ℃. The working solution (prepared on the day of use) was T buffer base-8.3 ml; k₂PO₄(4mM)-1.0ml；CaCl₂·2H₂O (5M) -0.2 ml; and TES (1M, pH 8) -0.5 ml. And each component of the working solution T is respectively subjected to filtration sterilization.

The T buffer was added to the protoplasts over 20 seconds, 1.0ml of P buffer was also added, and then the protoplasts were centrifuged at 8,000rpm for 10 minutes. After removal of the supernatant the protoplasts were resuspended in 0.1mlP buffer. Protoplasts were then plated onto RM14 medium containing: sucrose-205 g; k₂SO₄-0.25g；MgCl₂.6H₂O-10.12 g; 10g of glucose; difco casein amino acid-0.1 g; difco yeast extract-5 g; difco oat agar-3 g; agar-22 g for Difco bacteria; dH₂O-800 ml. The solution was at 121 deg.CAutoclaved for 25 minutes. The following sterile stock solutions were then added: k₂PO₄(0.5％)-10ml；CaCl₂.2H₂O (5M) -5 ml; l-proline (20%) -15 ml; MES buffer (1.0M, pH 6.5) -10 ml; trace elements (supra) -2 ml; cycloheximide stock solution (25mg/ml) -40 ml; and 2ml of 1N NaOH. 25ml of RM14 medium were aliquoted on each plate and the plates were dried for 24 hours prior to use.

The protoplast is kept at the humidity of 95 percent and the temperature of 30 ℃ for 20 to 24 hours. To select thiostrepton resistant transformants, 1ml of the upper buffer matrix containing 125. mu.g/ml thiostrepton was evenly spread on RM14 regeneration plates. Every 100ml of the upper buffer substrate contains: 10.3g of cane sugar; trace element solution (same as above) -0.2 ml; and MES (1M, pH 6.5) -1 ml. Protoplasts were incubated at 95% humidity and 30 ℃ for 7-14 days until thiostrepton resistance (Thio) was observed^r) And (5) bacterial colonies.

7.1.6.Transformation of Streptomyces lividans protoplasts

Transformation was carried out in some cases with Streptomyces lividans TK64 (supplied by John Innes institute, Norwich, U.K). Methods and compositions for growth, protoplasting, and transformation of Streptomyces lividans are described and performed in accordance with Hopwood et al, 1985, Genetic management of Streptomyces, organism Manual, John Innes Foundation, Norwich, U.K. Plasmid DNA was isolated from the S.lividans transformants as described in 7.1.3 above.

7.1.7.Fermentation analysis of Streptomyces avermitilis strains

After culturing mycelia of S.avermitilis on YPD-6 of 1/2 strength for 4-7 days, they were inoculated into 1X6 inch tubes containing 8ml of a pre-formed medium containing: soluble Starch (as soluble Starch or KOSO, Japan Com Starch Co., Nagoya) -20 g/L; pharmamedia-15 g/L; ardamine pH-5g/L (Champlain Ind., Clifton, NJ); CaCO₃-2 g/L; 2x bcfa ("bcfa" refers to branched fatty acids) which is present in the culture medium to a final concentration of 50ppm of 2- (+/-) -methyl butyric acid, 60ppm of isobutyric acid, and 20ppm of isovaleric acid. The pH was adjusted to 7.2 and the medium was autoclaved at 121 ℃ for 25 minutes.

The tubes were incubated at 29 ℃ for 3 days at 17 ℃ with shaking. A 2ml aliquot of the seed culture was inoculated into a 300ml Erlenmeyer flask containing 25ml of growth medium containing: starch (soluble starch or KOSO) -160 g/L; nutrisoy (Archer Daniels Midland, Decatur, IL)10 g/L; the pH value of the Ardamine is-10 g/L; k₂HPO₄-2g/L；MgSO₄.4H₂O-2g/L；FeSO₄.7H₂O-0.02g/L；MnCl₂-0.002g/L；ZnSO₄.7H₂O-0.002g/L；CaCO₃14 g/L2 x bcfa (supra); and cyclohexane carboxylic acid (CHC) (made as a 20% solution at pH 7.0) -800 ppm. The pH was adjusted to 6.9 and the medium was autoclaved at 121 ℃ for 25 minutes.

After inoculation, the flasks were incubated at 29 ℃ for 12 days with shaking at 200 rpm. After incubation, 2ml of sample was taken from the flask, diluted with 8ml of methanol, mixed, and the mixture was centrifuged at 1,250Xg for 10 minutes to precipitate debris. The supernatant was analyzed by HPLC using a Beckman Ultrasphere ODS column (25 cm. times.4.6 mm ID) at a flow rate of 0.75ml/min, and the absorbance at 240nm was measured. The mobile phase was 86/8.9/5.1 methanol/water/acetonitrile.

7.1.8.Isolation of the PKS Gene of Streptomyces avermitilis

A cosmid library of chromosomal DNA from S.avermitilis (ATCC 31272, SC-2) was prepared and hybridized with a Ketone Synthase (KS) probe made from a fragment of the Glycopolyspora erythraea polyketide synthase (PKS) gene. Cosmid libraries were prepared as described in Sambrook et al, 1989, supra. Streptomyces chromosomal DNA libraries are prepared as described in Hopwood et al, 1985, supra. Cosmid colonies with the ketosynthase-hybridizing region were identified by hybridization with the 2.7kb ndei/Eco47III fragment of pEX26 (supplied by dr. About 5ng of pEX26 was digested with Ndel and Eco47 III. The reaction mixture was loaded onto 0.8% SeaPlaque GTG agarose gel (FMC BioProducts, Rockland, ME). After electrophoresis, 2.7Kb of Ndel/Eco47III fragment was cut out of the gelGELase from FastProtocol^TM(Epicentre Technologies) DNA was recovered from the gel. 2.7Kb of the Ndel/Eco47III fragment [ alpha-³²P]dCTP (deoxycytidine 5' -triphosphate, tetra (triethylamine) salt, [ alpha-³²P]- (NEN-Dupont, Boston, MA) marker. A typical reaction is carried out in a volume of 0.05 ml. After addition of 5. mu.l of stop buffer, G-25Sephadex Quick Spin was used^TMColumn (Boehringer Mannheim) separates the labeled DNA molecules from the nucleic acid molecules which have not been incorporated according to the instructions.

About 1,800 cosmid colonies were screened by colony hybridization. 10 colonies that hybridized strongly with the saccharopolyspora erythraea KS probe were identified. Coli colonies containing cosmid DNA were cultured in LB liquid medium and cultured in AutoGen540 using Cycle 3 (Instrument software)^TMCosmid DNA from each culture was isolated in an automated nucleic acid separator according to the protocol. Restriction endonuclease mapping and Southern blot analysis revealed that five colonies contained overlapping chromosomal regions. The BamH1 cleavage maps of the genome of five cosmids (i.e., pSE65, pSE66, pSE67, pSE68, pSE69) were constructed by analysis of overlapping cosmids and hybridization (FIG. 4).

7.1.9.Identification of DNA capable of regulating avermectin B2: B1 ratio And identification of the aveC ORF

The following protocol was used to test the ability of subcloned fragments from the pSE66 cosmid clone to modulate the ratio of avermectins B2: B1 in AveC mutants. pSE66 (5. mu.g) was digested with Sacl and BamHI. The reaction mixture was loaded to 0.8% Seaplaque^TMGTG agarose gel (FMC BioProducts), after electrophoresis, 2.9Kb of Sacl/BamHl fragment was excised from the gel and used as GELase of Fast Protocol^TM(Epicentre Technologies) DNA was recovered from the gel. About 5. mu.g of the shuttle vector pWHM3(Vara et al, 1989, J.Bacteriol.171: 5872-5881) was digested with Sacl and BamHI. Approximately 0.5. mu.g of the 2.9Kb insert was mixed with 0.5. mu.g of digested pWHM3 and ligated with 1 unit of ligase according to the manufacturer's instructions (New England Biolabs, Inc.,beverly, MA) was incubated overnight at 15 ℃ in a total volume of 20. mu.l. After incubation, 5. mu.l of the ligation mixture was incubated at 70 ℃ for 10 minutes, cooled to room temperature and used to transform competent E.coli DH 5. alpha. cells (BRL) according to the operating manual. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the 2.9Kb Sacl/BamHI insert could be confirmed by restriction analysis. This plasmid was designated pSE 119.

Protoplasts of S.avermitilis 1100-SC38 strain (the internal (in-house) strain of Pfizer) were prepared and transformed with pSE119 as described in section 7.1.5 above. The 1100-SC38 strain is a mutant that produces significantly more of the avermectin cyclohexyl-B2 form when supplemented with cyclohexanecarboxylic acid relative to the avermectin cyclohexyl-B1 form (the ratio of B2: B1 is approximately 30: 1). pSE119 used to transform S.avermitilis protoplasts was isolated from E.coli GM2163 strain (obtained from Dr. B.J.Bachmann, Curator, E.coli Genetic Stock Center, Yale University), E.coli DM1 strain (BRL), or S.lividans TK64 strain. Thiostrepton-resistant transformants of the 1100-SC38 strain were isolated and analyzed by HPLC analysis of the fermentation product. Transformants of the S.avermitilis strain 1100-SC38 containing pSE119 produced the avermectin cyclohexyl B2: cyclohexyl-B1 in an altered ratio of approximately 3.7: 1 (Table 2).

After determining that pSE119 can modulate the ratio of avermectin B2: B1 in AveC mutants, the inserted DNA sequence was determined. Approximately 10. mu.g of pSE119 was isolated using a plasmid DNA isolation kit (Qiagen, Valencia, Calif.) according to the operating manual and then sequenced using an ABI 373A automated DNA sequencer (Perkin Elmer, Foster City, Calif.). Sequencing data were assembled and compiled using a genetic computer set program (GCG, Madison, Wis.). The DNA sequence and the aveC ORF are shown in FIG. 1(SEQ ID NO: 1).

A new plasmid, designated pSE118, was constructed as follows. Approximately 5. mu.g of pSE66 was digested with SphI and BamHI. The reaction mixture was applied to 0.8% SeaPlaque GTG agarose gel (FMCBioproducts), and after electrophoresis, 2.8Kb of SphI/BamHI fragment was excised from the gel, using GELase from FastProtocol^TM(Epicentre Technologies) DNA was recovered from the gel. About 5. mu.g of shuttle vector pWHM3 was digested with SphI and BamHI. Mu.g of the 2.8Kb insert were mixed with 0.5. mu.g of digested pWHM3 and incubated overnight with 1 unit of ligase (New England Biolabs, Inc., Beverly, Mass.) at 15 ℃ in a total volume of 20. mu.l according to the manufacturer's instructions. After incubation, 5. mu.l of the ligation mixture were incubated at 70 ℃ for 10 minutes, cooled to room temperature and used to transform the competent E.coli DH 5. alpha. cells according to the operating manual. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the 2.8Kb SphI/BamHI insert was confirmed by restriction analysis. This plasmid was designated pSE 118. The DNA inserts in pSE118 and pSE119 overlap by about 838 nucleotides (FIG. 4).

Protoplasts of S.avermitilis strain 1100-SC38 were transformed with pSE118 as described above. Thiostrepton-resistant transformants of the 1100-SC38 strain were isolated and analyzed by HPLC analysis of the fermentation product. The ratio of avermectin cyclohexyl B2 to cyclohexyl-B1 was unchanged for the pSE 118-containing transformants of S.avermitilis strain 1100-SC38 relative to strain 1100-SC38 (Table 2).

7.1.10.PCR amplification of aveC Gene on chromosomal DNA of Streptomyces avermitilis

A1.2 Kb fragment containing the aveC ORF was isolated from S.avermitilis chromosomal DNA by PCR amplification using primers designed based on the aveC nucleotide sequence obtained above. PCR primers were provided by Genosys Biotechnologies, inc. (Texas). The right primer is: 5' -TCACGAAACCGGACACAC-3(SEQ ID NO: 6); the left primer is: 5'-CATGATCGCTGAACCGAG-3' (SEQ ID NO: 7). In a buffer provided by the manufacturer, Deep Vent was used in a final volume of 100. mu.l in the presence of 300. mu.M dNTP, 10% glycerol, 200pmol of each primer, 0.1. mu.g template and 2.5 units enzyme^TMPolymerase (New England-Biolabs) performed the PCR reaction in a Perkin-Elmer Cetus thermal cycler. The first cycle was 95 ℃ for 5 minutes (denaturation step), 60 ℃ for 2 minutes (annealing step), and 72 ℃ for 2 minutes (extension step). The subsequent 24 cycles of temperature change were similar, but the time of the denaturation step was shortened to 45 seconds and annealedThe time is shortened to 1 minute.

After electrophoresis of the PCR products in a 1% agarose gel, a single DNA band of about 1.2Kb was detected. The DNA was purified from the gel and ligated with 25ng of linearized Blunt-ended PCR-Blunt vector (Invitrogen) at a vector: insert ratio of 1: 10 molar according to the operating manual. The ligation mixture was used to transform One Shot according to the operating manual^TMColi competent cells (Invitrogen). Plasmid DNA was isolated from ampicillin resistant transformants and the presence of this-1.2 Kb insert was confirmed by restriction analysis. This plasmid was designated pSE 179.

The DNA insert from pSE179 was isolated by BamHI/XbaI digestion, electrophoretically separated, purified from the gel and ligated with the BamHI/XbaI digested shuttle vector pWHM3 at a total DNA concentration of 1. mu.g and a vector to insert molar ratio of 1: 5. Coli DH 5. alpha. competent cells were transformed with the ligation mixture according to the manual. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the-1.2 Kb insert was confirmed by restriction analysis. This plasmid, designated pSE186 (FIG. 2, ATCC209604), was transformed into E.coli DM1 and plasmid DNA was isolated from ampicillin resistant transformants.

7.2.Results

A2.9 Kb SacI/BamHI fragment from pSE119 was identified which, when transformed into S.avermitilis 1100-SC38 strain, significantly altered the B2: B1 avermectin production ratio. In general, the B2: B1 ratio of S.avermitilis 1100-SC38 strain was approximately 30: 1, but when transformed with a vector containing 2.9KbSacI/BamHI cleavage fragments, the ratio of avermectins B2: B1 was reduced to approximately 3.7: 1. Post-fermentation analysis of the transformant culture confirmed the presence of the transforming DNA.

Sequencing the 2.9Kb pSE119 fragment identified an ORF of about 0.9Kb (FIG. 1) (SEQ ID NO: 1) comprising a PstI/SphI fragment which has been mutated elsewhere to produce only the B2 product (Ikeda et al, 1995, supra). This ORF, or its corresponding deduced polypeptide, does not show any homology to known DNA or protein sequences, in comparison with the known database (GenEMBL, SWISS-PROT).

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

TABLE 2

Streptomyces avermitilis strain (transformation plasmid)	Numbering of the transformants tested	Average B2: B1 ratio
			1100-SC38 (none)	9	30.66
1100-SC38(pWHM3)	21	31.3
			1100-SC38(pSE119)	12	3.7
1100-SC38(pSE118)	12	30.4
			1100-SC38(pSE185)	14	27.9

8. Example (b):construction of S.avermitilis aveC mutant Strain

This example describes the construction of several different S.avermitilis aveC mutants using the compositions and methods described above. Techniques for introducing mutations into streptomyces genes are described in Kieser and Hopwood, 1991, meth.enzym.204: 430-458. See, Anzai et al, 1988, j.anibiot XLI (2): 226-233 and Stutzman-Engwall et al, 1992, J.Bacteriol.174 (1): 144-154. These references are incorporated herein in their entirety.

8.1.Inactivation of the AveC Gene of S.avermitilis

AveC mutants containing an inactivated AveC gene can be constructed by several methods as described below.

In the first approach, the 640bp SphI/PstI fragment inside the aveC gene on pSE119 (plasmid 7.1.9 above) was replaced with the ermE gene of Saccharopolyspora erythraea (conferring erythromycin resistance). The ermE Gene was isolated from pIJ4026 (from John Innes Institute, Norwich, U.K.; see also Bibb et al, 1985, Gene 41: 357 368) by digestion with BglII and EcoRI, followed by electrophoresis and purification from the gel. This fragment of about 1.7Kb was ligated into pGEM7Zf (Promega) digested with BamHI and EcoRI and the ligation mixture was transformed into competent cells of E.coli DH5 α according to the operating manual. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the approximately 1.7Kb insert was confirmed by restriction analysis. This plasmid was designated pSE 27.

pSE118 (described above in section 7.1.9) was digested with SphI and BamHI, the digests were electrophoresed, and approximately 2.8Kb of SphI/BamHI inserts were purified from the gel. pSE119 was digested with PstI and EcoRI, followed by electrophoresis, and the PstI/EcoRI insert of about 1.5Kb was purified from the gel. The shuttle vector pWHM3 was digested with BamHI and EcoRI. pSE27 was digested with PstI and SphI. The PstI/SphI insert, about 1.7Kb, was then purified from the gel by electrophoresis. All four fragments (i.e., about 2.8Kb, about 1.5Kb, about 7.2Kb, about 1.7Kb) were ligated together by a 4-step (4-way) ligation reaction. The ligation mixture was transformed into competent cells of E.coli DH5 alpha according to the operating manual. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis. This plasmid was designated pSE180 (FIG. 3; ATCC 209605).

pSE180 was transformed into S.lividans TK64 cells and transformed colonies were identified by resistance to thiostrepton and erythromycin. pSE180 was isolated from S.lividans and used to transform protoplasts of S.avermitilis. Four S.thiostrepton-resistant S.avermitilis transformants were identified, protoplasts were prepared and plated under non-selective conditions into RM14 medium. Protoplasts were regenerated and single colonies were then screened for erythromycin resistance and thiostrepton-free resistance, indicating that the inactivated aveC gene had integrated on the chromosome but the episomal replicon had been lost. Identification of an Erm^rThio^sThe transformant was named as SE180-11 strain. Total chromosomal DNA was isolated from SE180-11 strain, digested with restriction enzymes BamHI, HindIII, PstI or SphI, electrophoresed in 0.8% agarose gel, transferred to a nylon membrane, and hybridized with ermE probe. These analyses showed that the ermE resistance gene was integrated into the chromosome by a double crossover event and that the 640bp PstI/SphI fragment was deleted at the same time. HPLC analysis of the fermentation product of strain SE180-11 showed that normal avermectins were no longer produced (FIG. 5A).

In a second method for inactivating the aveC gene, 1.7Kb of the ermE gene was removed from the chromosome of S.avermitilis strain SE180-11, resulting in a deletion of 640bp PstI/SphI in the aveC gene. The gene replacement plasmid was constructed as follows: pSE180 was partially digested with XbaI and the fragment of about 11.4Kb was purified from the gel. This band of about 11.4Kb lacks the 1.7Kb ermE resistance gene. This DNA was ligated and transformed into E.coli DH 5. alpha. cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert could be confirmed by restriction analysis. This plasmid (designated pSE184) was transformed into E.coli DM1, and the ampicillin-resistant transformant was isolated as a plasmidAnd (3) granular DNA. Protoplasts of S.avermitilis strain SE180-11 were transformed with this plasmid. Protoplasts were prepared from thiostrepton-resistant transformants of strain SE180-11 and plated as single colonies on RM 14. Protoplasts were regenerated and single colonies were then screened for no erythromycin resistance and thiostrepton resistance, indicating that the inactivated aveC gene had integrated into the chromosome but the episomal replicon containing the ermE gene had been deleted. Identification of an Erm^sThio^sThe transformant was designated SE 184-1-13. Fermentation analysis of SE184-1-13 showed that normal avermectins were no longer produced and that SE184-1-13 had the same fermentation characteristics as SE 180-11.

In a third method for inactivating the aveC gene, two Gs were added by PCR after nucleotide C at position 471, resulting in the introduction of a frameshift mutation in the aveC gene of the chromosome, thereby creating a Bspel site. The presence of the engineered EspEl site can be used to detect gene substitution events. PCR primers were designed to introduce frameshift mutations in the aveC gene, and were provided by Genosys Biotechnologies. The right primer is: 5'-GGTTCCGGATGCCGTTCTCG-3' (SEQ ID NO: 8), the left primer is: 5'-AACTCCGGTCGACTCCCCTTC-3' (SEQ ID NO: 9). PCR conditions were as described above in section 7.1.10. A666 bp PCR product was digested with Sphl to give 278bp and 388bp fragments, respectively. The 388bp fragment was purified from the gel.

The gene replacement plasmid was constructed as follows: the shuttle vector pWHM3 was digested with EcoRI and BamHI. pSE119 was digested with BamHI and SphI, followed by electrophoresis to isolate an approximately 840bp fragment from the gel. pSE119 was digested with EcoRI and XmmI, electrophoretically separated, and a fragment of about 1.7Kb was purified from the gel. The four fragments (i.e.: 7.2Kb,. about.840 bp,. about.1.7 Kb and 388bp) were ligated together by a 4-step ligation reaction. The ligation mixture was transformed into competent cells of E.coli DH5 alpha. 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 (designated pSE185) was transformed into E.coli DM1 cells, and plasmid DNA was isolated from the ampicillin-resistant transformant. Protoplasts of the S.avermitilis strain 1100-SC38 were transformed with the plasmid. Thiostrepton-resistant transformants of the 1100-SC38 strain were isolated and analyzed by HPLC analysis of the fermentation products. When pSE185 was transformed into S.avermitilis strain 1100-SC38, the ratio of avermectins B2: B1 was not significantly altered (Table 2).

Transformation of S.avermitilis protoplasts with pSE185 resulted in a frameshift mutation in the aveC gene of the chromosome. Protoplasts were prepared from thiostrepton-resistant transformants and plated as single colonies on RM14 medium. Protoplasts were regenerated and single colonies were screened for thiostrepton-free resistance. Chromosomal DNA from thiostrepton-sensitive colonies was isolated and screened by PCR for the presence of frameshift mutations integrated into the chromosome. PCR primers were designed based on the aveC nucleotide sequence and were supplied by Genosys Biotechnologies (Texas). The right-hand PCR primers were: 5'-GCAAGGATACGGGGACTAC-3' (SEQ ID NO: 10), left PCR primers: 5'-GAACCGACCGCCTGATAC-3' (SEQ ID NO: 11), PCR conditions were as described above in section 7.1.10. The PCR product obtained was 543bp, and when digested with Bspel, three fragments of 368bp, 96bp, and 79bp were detected, which indicated chromosomal integration of the inactivated aveC gene and deletion of the episomal 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 HPLC profile of fermentation products as SE180-11 and SE 184-1-13. Identification of a Thio^SThe transformant was designated SE185-5a.

In addition, a mutation was made in the aveC gene which changed nucleotide 520 from G to A, resulting in a codon encoding tryptophan (W) at position 116 being changed to a stop codon. The S.avermitilis strain carrying this mutation does not produce normal avermectins and has the same fermentation characteristics as the SE180-11, SE184-1-13, and SE185-5a strains.

Furthermore, mutations were generated in the aveC gene which resulted in: (i) changing nucleotide 970 from G to A to change amino acid 256 from glycine (G) to aspartic acid (D), (ii) changing nucleotide 996 from T to C to change amino acid 275 from tyrosine (Y) to histidine (H). The S.avermitilis strain with these mutations (G256D/Y275H) did not produce normal avermectins but had the same fermentation characteristics as the SE180-11, SE184-1-13, and SE185-5a strains.

The S.avermitilis aveC-inactivated mutants SE180-11, SE184-1-13, SE185-5a, and other strains described herein, provide screening tools for assessing the effects of other mutations in the aveC gene. pSE186 containing the wild-type aveC gene was transformed into E.coli DM1 cells and plasmid DNA was isolated from ampicillin-resistant transformants. The pSE186 DNA was used to transform S.avermitilis SE180-11 strain. Thiostrepton-resistant transformants of strain SE180-11 were isolated, assayed for the presence of erythromycin resistance, and analyzed for Thio by HPLC analysis of the fermentation products^rErm^rA transformant. The presence of an in trans (in trans) of a functional aveC gene restored normal production of avermectins to levels of SE180-11 strain (FIG. 5B).

8.2Analysis of mutations in the AveC Gene that alter the B2: B1 ratio

As described above, S.avermitilis SE180-11 strain containing an inactivated aveC gene was supplemented by transformation with a plasmid containing a functional aveC gene (pSE 186). The SE180-11 strain can also be used as a host strain to identify additional mutations in the aveC gene, as described below.

Chromosomal DNA was isolated from the 1100-SC38 strain and used as a template for PCR amplification of the aveC gene. The 1.2Kb ORF was isolated by PCR amplification using primers designed according to the aveC nucleotide sequence. The right primer is SEQ ID NO: 6, the left primer is SEQ ID NO: 7 (see section 7.1.10). PCR and subcloning conditions were as described in section 7.1.10. DNA sequence analysis of the 1.2Kb ORF shows a mutation of the aveC gene from C to T at nucleotide 337 and a change from serine (S) to phenylalanine (F) at amino acid 55. The aveC gene containing the S55F mutation was subcloned into pWHM3 to generate a plasmid, designated pSE187, for transformation of protoplasts of S.avermitilis strain SE 180-11. Isolating thiostrepton-resistant transformants of strain SE180-11 to determine the presence of erythromycin resistance,analysis of Thio by HPLC analysis of fermentation products^rErm^rA transformant. The presence of the aveC gene encoding the amino acid change at position 55 (S55F) restored normal avermectin production to levels of SE180-11 strain (FIG. 5C); however, the ratio of cyclohexyl B2: cyclohexyl B2 was about 26: 1, whereas the B2: B1 ratio of SE180-11 strain transformed with pSE186 was about 1.6: 1 (Table 3), indicating that this single mutation (S55F) modulated the amount of cyclohexyl B2 relative to cyclohexyl B1.

Another mutation in the aveC gene was identified which changed nucleotide 862 from G to A, and thus changed the amino acid at position 230 from glycine (G) to aspartic acid (D). The S.avermitilis strain containing this mutation (G230D) produced avermectins B2: B1 in a ratio of about 30: 1.

8.3.Mutations that decrease the ratio of B2 to B1

Several mutations that reduce the amount of cyclohexyl-B2 relative to cyclohexyl-B1 can be constructed as follows.

A mutation in the aveC gene was identified which changed nucleotide 588 from G to A, and amino acid 139 from alanine (A) to threonine (T). The aveC gene containing the A139T mutation was subcloned into pWHM3 to generate a plasmid (designated pSE188) which was used to transform protoplasts of S.avermitilis strain SE 180-11. Thiostrepton-resistant transformants of strain SE180-11 were isolated, assayed for the presence of erythromycin-resistant strains, and analyzed for Thio by HPLC analysis of the fermentation products^rErm^rA transformant. The presence of the mutant aveC gene encoding the amino acid 139 alteration (A139T) restored avermectin production to levels of SE180-11 strain (FIG. 5D); however, the ratio of B2: B1 was about 0.94: 1, indicating that this mutation reduced the amount of cyclohexyl B2 relative to cyclohexyl B1. This result is unexpected because the published results, as well as the above mutation results, demonstrate only the inactivation of the aveC gene or increased production of avermectins type B2 relative to type B1 (see Table 3).

Since the A139T mutation changed the ratio of B2 to B1 in favor of B1, a mutation was constructed which changed the amino acid at position 138 from serine to threonine. For this purpose, pSE186 was digested with EcoRI and cloned into pGEM3Zf (Promega) which had been digested with EcoRI. The plasmid named psE186a was digested with ApaI and KpnI, the DNA fragment was separated from the agarose gel, and the two fragments-3.8 Kb and-0.4 Kb were purified from the gel. A single base change at nucleotide 585 was induced using the 1.2Kb DNA insert of pSE186 as a template for PCR. The PCR primers were designed to introduce a mutation at nucleotide 585, and were provided by Genosys Biotechnologies Inc. (Texas). The right-hand PCR primers were: 5'-GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCCCTGGCGACG-3' (SEQ ID NO: 12); the left PCR primers were: 5'-GGAACCGACCGCCTGATACA-3' (SEQ ID NO: 13). PCR reactions were carried out in a final volume of 50. mu.l in the presence of 200. mu.M dNTPs, 200pmol of each primer, 50ng template DNA, 1.0M GC-Melt and 1 unit of KlenaQ polymerase mixture in a buffer provided by the manufacturer using the Advantage GC genomic PCR kit (Clonetech Laboratories, Palo Alto, Calif.). The first cycle was 94 ℃ for 1 minute; followed by 25 cycles at 94 ℃ for 30 seconds and 68 ℃ for 2 minutes; then one cycle was performed at 68 ℃ for 3 minutes. The 295bp PCR product was digested with ApaI and KpnI to release a 254bp fragment, which was resolved by electrophoresis and purified from the gel. All three fragments (. about.3.8 Kb,. about.0.4 Kb and 254bp) were ligated together using a 3-step ligation reaction. The ligation mixture was transformed into intact E.coli DH5 alpha 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.

pSE198 was digested with EcoRI, cloned into pWHM3 which had been digested with EcoRI and transformed into E.coli DH 5. alpha. cells. Plasmid DNA was isolated from the ampicillin-resistant transformant, and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. The plasmid DNA was transformed into E.coli DM1, the plasmid DNA was isolated from the ampicillin-resistant transformant, and the presence of the correct insert was confirmed by restriction analysis. This plasmid, designated pSE199, was used to transform protoplasts of the S.avermitilis SE180-11 strain. Isolation of S.thiostrepto strain of SE180-11 StrainPeptide-resistant transformants, determination of the Presence of erythromycin resistance, analysis of Thio by analysis of the fermentation products by HPLC method^rErm^rAnd (4) transforming the strain. The presence of the mutant aveC gene encoding the amino acid change at position 138 (S138T) restored normal avermectin production to levels of strain SE 180-11; however, the ratio of B2: B1 was 0.88: 1, indicating that this mutation reduced the amount of cyclohexyl-B2 relative to cyclohexyl-B1 (see Table-3). The ratio of B2: B1 was even lower than 0.94: 1 (this is the ratio observed with the A139T mutation resulting from transformation of the SE180-11 strain with pSE188, as described above).

Another mutation was constructed to introduce a threonine at both amino acid positions 138 and 139. An approximately 1.2Kb DNA insert of pSE186 was used as a template for PCR. PCR primers were designed to induce mutations at nucleotide positions 585 and 588, and were provided by Genosys Biotechnologies Inc. (Texas). The right-hand PCR primers were: 5'-GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCGCTGGCGACGACC-3' (SEQ ID NO: 14); the left PCR primers were: 5'-GGAACATCACGGCATTCACC-3' (SEQ ID NO: 15). PCR reactions were performed using the conditions described above in this section. The 449bp PCR product was digested with Apal and Kpnl to release a 254bp fragment, which was resolved by electrophoresis and purified from the gel. pSE186a was digested with ApaI and KpnI, the DNA fragment was separated by agarose gel electrophoresis, and both fragments of 3.8Kb and 0.4Kb were purified from the gel. All three fragments (. about.3.8 Kb,. about.0.4 Kb and 254bp) were ligated in a 3-step ligation and the ligation mixture was transformed into competent cells of E.coli DH5 α. 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 230.

pSE230 was digested with EcoRI, cloned into the plasmid pWHM3 which had been digested with EcoRI, and transformed into E.coli DH 5. alpha. cells. Plasmid DNA was isolated from ampicillin-resistant transformants and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. The plasmid 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. Using this designation pSThe plasmid of E231 was transformed into protoplasts of the S.avermitilis strain SE 180-11. Isolation of Thioflavin-resistant transformants of SE180-11, determination of the Presence of erythromycin resistance and analysis of Thio by fermentation^rErm^rA transformant. The presence of the double mutant aveC gene (encoding S138T/A139T) restored normal avermectin production to levels of strain SE 180-11; however, the ratio of B2: B1 was 0.84: 1, showing that this mutation further reduced the production of cyclohexyl-B2 relative to cyclohexyl B1, relative to the reduction provided by the SE180-11 strain transformed with pSE188 or pSE199 (see Table 3).

Another mutation was constructed to further reduce the production of cyclohexyl B2 relative to cyclohexyl B1. Since the mutation S138T/A139T changed the ratio of B2 to B1 in favor of B1, a mutation was constructed that introduced a threonine at amino acid 138 and a phenylalanine at amino acid 139. An approximately 1.2kb DNA insert from pSE186 was used as a template for PCR. PCR primers designed to introduce a mutation at nucleotide 585 (to change T to A), nucleotide 588 (to change G to T), and nucleotide 589 (to change C to T) were provided by Genosys Biotechnologies, Inc. (Texas). The forward PCR primers were: 5'-GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCGCTGGCGACGTTC-3' (SEQ ID NO: 16); the reverse PCR primers are: 5'-GGAACATCACGGCATTCACC-3' (SEQ ID NO: 15). PCR reactions were carried out using the Advantage GC genomic PCR kit (Clonetech Laboratories, Palo Alto, Calif.) in a buffer provided by the manufacturer with 200. mu.M dNTPs, 200pmol of each primer, 50ng template DNA, 101mM magnesium acetate, 1.0M GC-Melt and 1 unit of Tth DNA polymerase in a final volume of 50. mu.l. The thermal cycling process was carried out for the first cycle at 94 ℃ for 1 minute; then 25 cycles of 94 ℃ for 30 seconds and 68 ℃ for 2 minutes; one cycle was carried out at 68 ℃ for 3 minutes. The 449bp PCR product was digested with ApaI and KpnI, releasing a 254bp fragment which was isolated by electrophoresis and purified on a gel. All three fragments (about 3.8Kb, about 0.4Kb and 254bp) were ligated together by a three-step ligation reaction. The ligation was mixed without transformation into competent E.coli DH5 alpha cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis. This plasmid was designated as pSE 238.

pSE238 was digested with EcoRI, cloned into the plasmid pWHM3 which had been digested with EcoRI, and transformed into E.coli DH 5. alpha. cells. Plasmid DNA was isolated from ampicillin-resistant transformants and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. The plasmid 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 pSE239, was used to transform protoplasts of S.avermitilis strain SE 180-11. Isolation of thiostrepton-resistant transformants of the SE180-11 Strain, determination of the Presence of erythromycin resistance and analysis of the thios by HPLC analysis of the fermentation products^rErm^rA transformant. The presence of the double mutant aveC gene (encoding S138T/A139F) restored normal avermectin production to levels of strain SE 180-11; however, the ratio of B2: B1 was 0.75: 1, indicating that this mutation further reduced the production of cyclohexyl-B2 relative to cyclohexyl-B1, relative to the reduction provided by the SE180-11 strain transformed with pSE188, pSE199 or pSE231, as described above (see Table 3).

TABLE 3

Additional mutations can be constructed using DNA shuffling (shuffling) techniques to further reduce the yield of cyclohexyl B2 relative to cyclohexyl B1, such as Stemmer, 1994, Nature 370: 389-391; stemmer, 1994, proc.natl.acad.sci.usa 91: 10747-10751; see also us patents 5605793, 5811238, 5830721 and 5837458 for details.

The DNA shuffling plasmid containing the mutant aveC gene was transformed into competent dam dcm E.coli cells. Plasmid DNA was isolated from ampicillin resistant transformants and used to transform protoplasts of the S.avermitilis strain SE 180-11. Thioflavin-resistant transformants of strain SE180-11 were isolated, and those producing avermectins having a ratio of cyclohexyl B2: cyclohexyl B1 of 1: 1 or less were selected therefrom. The DNA sequence of plasmid DNA in those transformants which produce avermectins but which have a B2: B1 ratio of 1: 1 or less was determined.

8 transformants were identified that produced reduced amounts of cyclohexyl B2 relative to cyclohexyl B1. The minimum B2: B1 ratio in these transformants was 0.40: 1 (Table 4). Plasmid DNA from each of these 8 transformants was isolated and the DNA sequence determined to identify mutations in the aveC gene. The mutations are as follows.

pSE290 contains 4 nucleotide mutations, i.e., nucleotide 317 changed from T to A, nucleotide 353 changed from C to A, nucleotide 438 changed from G to A, and nucleotide 1155 changed from T to A. The change at nucleotide 317 changes the amino acid at position 48 from D to E and the change at nucleotide 438 changes the amino acid at position 89 from A to T. Cells harboring the plasmid produced a ratio of B2: B1 of 0.42: 1 (Table 4).

pSE291 contains 4 nucleotide mutations, i.e., nucleotide 272 changed from G to A, nucleotide 585 changed from T to A, nucleotide 588 changed from G to A, and nucleotide 708 changed from G to A. The change in nucleotide 585 results in the change from S to T at the amino acid 138, the change in nucleotide 588 results in the change from A to T at the amino acid 139, and the change in nucleotide 708 results in the change from G to S at the amino acid 179. Cells harboring this plasmid produced a ratio of B2: B1 of 0.57: 1 (Table 4).

pSE292 contained the same 4 nucleotide mutations as pSE 290. Cells harboring the plasmid produced a ratio of B2: B1 of 0.40: 1 (Table 4).

pSE293 contains 6 nucleotide mutations, i.e., nucleotide 24 changed from A to G, nucleotide 286 changed from A to C, nucleotide 497 changed from T to C, nucleotide 554 changed from C to T, nucleotide 580 changed from T to C, and nucleotide 886 changed from A to T. The change at nucleotide 286 changes the amino acid at position 38 from Q to P, the change at nucleotide 580 from L to P, and the change at nucleotide 886 from E to D. Cells harboring the plasmid produced a ratio of B2: B1 of 0.68: 1 (Table 4).

pSE294 contains 6 nucleotide mutations, i.e., nucleotide 469 was changed from T to C, nucleotide 585 was changed from T to A, nucleotide 588 was changed from G to A, nucleotide 708 was changed from G to A, nucleotide 833 was changed from C to T, and nucleotide 1184 was changed from G to A. In addition, nucleotides 173, 174, and 175 have been deleted. The change of the nucleotide 469 th results in the change of the amino acid at position 99 from F to S, the change of the nucleotide 585 th results in the change of the amino acid at position 138 from S to T, the change of the nucleotide 588 results in the change of the amino acid at position 139 from A to T, and the change of the nucleotide 708 results in the change of the amino acid at position 179 from G to S. Cells harboring the plasmid produced a ratio of B2: B1 of 0.53: 1 (Table 4).

pSE295 contains 2 nucleotide mutations, i.e., nucleotide 588 changed from G to A and nucleotide 856 changed from T to C. The change at nucleotide 588 changes the amino acid at 139 from A to T and the change at nucleotide 856 changes the amino acid at 228 from M to T. Cells harboring the plasmid produced a ratio of B2: B1 of 0.80: 1 (Table 4).

pSE296 contains 5 nucleotide mutations, nucleotide 155 changed from T to C, nucleotide 505 changed from G to T, nucleotide 1039 changed from C to T, nucleotide 1202 changed from C to T, and nucleotide 1210 changed from T to C. The change at nucleotide 505 changes the amino acid at position 111 from G to V and the change at nucleotide 1039 changes the amino acid at position 289 from P to L. Cells harboring the plasmid produced a ratio of B2: B1 of 0.73: 1 (Table 4).

pSE297 contains 4 nucleotide mutations, i.e., the nucleotide at position 377 was changed from G to T, the nucleotide at position 588 was changed from G to A, the nucleotide at position 633 was changed from A to G, and the nucleotide at position 1067 was changed from A to T. The change at nucleotide 588 changes the amino acid at 139 from A to T, the change at nucleotide 633 changes the amino acid at 154 from K to E, and the change at nucleotide 1067 changes the amino acid at 298 from Q to H. Cells harboring the plasmid produced a ratio of B2: B1 of 0.67: 1 (Table 4).

TABLE 4

Another round of DNA shuffling was performed as follows in order to reduce the production of cyclohexyl-B2 avermectin relative to cyclohexyl-B1 avermectin.

Semi-synthetic shuffling

The best clones were shuffled using the method described in WO97/20078 to Maxygen Inc. Individual oligonucleotides encoding beneficial substitutions optimally corresponding to a reduced B2: B1 ratio were added in a shuffling reaction in a 5 molar excess relative to the aveC template strand. Each nucleotide mismatch of the oligonucleotide is flanked by 20 nucleotides with perfect identity (perfect identity) in order to ensure correct insertion during the shuffling reaction. Oligonucleotides were purchased from Operon technologies (Alameda, Calif.).

HTP growth of Streptomyces avermitilis

Individual colonies were picked from the transformation plates and inoculated into 200. mu.l of R5 medium (Kieser, T et al, "Practical Streptomyces Genetics", 2000, Norwich, U.K., John Innes Foundation) in 96-well deep-well plates and cultured with shaking at 30 ℃. Glass beads were dispensed in each well to disperse the mycelium and aerate the culture. During this period, smooth and dense growth of the culture was achieved. After 4-5 days, 20. mu.l of the inoculum culture was dispensed into production plates and the remaining volume was frozen at-80 ℃ as master plates after addition of glycerol to a final concentration of 20%. The production plate was incubated at 30 ℃ for 12-14 days in a humid environment. After 5-8 days of incubation, these strains started to sporulate. Production plates were prepared essentially as described in WO 99/41389 of Pfizer Inc, Pfizer, except that 1% agarose was added to ensure a solid surface.

Extraction and ESI-MS/MS screening

A total of 1ml of ethyl acetate was added to each well and incubated for 20 minutes at room temperature with shaking. Approximately 750. mu.l of the ethyl acetate phase was recovered, transferred to a 96-well plate and evaporated overnight. The pellet was resuspended in 100. mu.l methanol 1mM NaCl, and 5. mu.l of the solution was taken up by an autosampler in 96-well plate format and injected into the mass spectrometer for direct analysis of the mobile injection phase without liquid chromatography or other separation. Two rounds of MS/MS transitions (transitions) were monitored for two different channels by ionizing the compound by electrospray ionization (ionization). The MS/MS transition for the B1 sodium ion (conditioning ion) was from m/z 921 to m/z777, and for the B2 sodium ion was from m/z 939 to m/z 795. The high throughput screening was performed using a Finnigan TSQ-7000, Micromass Quattro-LC mass spectrometer and a Leap Technology Twin-Pal autosampler. Integration of the respective maps of B1 and B2 at each well position identified a hot spot (hit).

Finally, 57 novel combinations of amino acid substitutions were identified that produced a ratio of cyclohexyl B2: cyclohexyl B1 avermectin of 0.35: 1 or less (FIG. 6). Wherein some of the novel mutations result in a ratio of about 0.30: 1 or less of cyclohexyl B2: cyclohexyl B1 avermectin; some can produce a ratio of cyclohexyl B2: cyclohexyl B1 avermectins of about 0.25: 1 or less, and some can produce a ratio of cyclohexyl B2: cyclohexyl B1 avermectins of about 0.20: 1 or less. 2 novel mutations were identified that produced a ratio of 0.37 or 0.38 for cyclohexyl B2 to cyclohexyl B1 avermectin.

Preservation of biological materials

On 29.1.1998, the following biological materials were deposited at the American Type Culture Collection (ATCC), 12301Parklawn Drive, Rockville, MD, 20852, USA under the accession numbers:

all patents, patent applications, and publications cited above are hereby incorporated by reference in their entirety.

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

Claims (8)

1. A polynucleotide molecule comprising a mutation in the nucleotide sequence of the aveC ORF of S.avermitilis as set forth in SEQ ID No. 1, or a degenerate variant thereof, wherein said mutation encodes a combination of amino acid substitutions corresponding to the amino acid residue at amino acid position 2 of SEQ ID No. 2, and wherein said mutation results in production of avermectins by those cells of the S.avermitilis ATCC53692 strain in which the wild-type aveC allele has been inactivated and which express the polynucleotide molecule comprising said mutant nucleotide sequence, and wherein the ratio of class 2:1 avermectins produced is reduced relative to the ratio produced by those cells of the S.avermitilis ATCC53692 strain which express only the wild-type aveC allele, wherein when said class 2:1 avermectin is cyclohexyl B2: cyclohexyl B1 avermectin, the ratio of said class 2:1 avermectin is 0.2:1 or less, and wherein said combination of amino acid substitutions comprises substitutions at both D48 and G179, and wherein the combination of amino acid substitutions is a combination selected from the group consisting of:

(ao)D48E,A89T,L136P,G179S,E238D;

(ap)D48E,A89T,L136P,K154E,G179S,E238D;

(aq)D48E,A89T,S138T,A139T,K154R,G179S,V196A,P289L;

(ar)D48E,A89T,S138T,A139F,G179S,V196A,E238D;

(as)D48E,A61T，A89T,L136P,G179S,V196A,A198G,P289L;

(at)D48E,A61T,S138T,A139F,G179S,G196A,E238D,P289L;

(au)D48E,A89T,L136P,G179S;

(av)D48E,A89T,V120A,L136P,G179S;

(aw)D48E,A61T,A89T,S138T,A139F,G179S,V196A,A198G,E238D;

(ax)D48E,A61T,A89T,G111V,S138T,A139F,G179S,V196A,E238D;

(ay)D48E,A61T,A89T,S138T,A139T,G179S,V196A,E238D,P289L;

(az)D48E,A61T,A89T,L136P,S138T,A139F,G179S,A198G,E238D;

(ba)D48E,A89T,S138T，A139F,G179S,A198G,V220A;

(bb)D48E,A61T,A89T,S138T,A139T,G179S,V196A,E238D,R239H,P289L;

(bc)D48E,A61T,A89T,L136P,G179S,P289L;

(bd) D48E, A89T, S138T, A139T, G179S, V196A, E238D, P289L, and

(be)D48E,A61T,A89T,S138T,A139F,G179S,V196A,E238D。

2. a recombinant vector comprising the polynucleotide molecule of claim 1.

3. A host cell comprising the recombinant vector of claim 2, which is a streptomyces cell.

4. The host cell of claim 3, which is a Streptomyces avermitilis cell.

5. A method of producing a streptomyces avermitilis strain, comprising: (i) mutating the nucleotide sequence of the aveC ORF shown in SEQ ID NO:1 in cells of a Streptomyces avermitilis strain, wherein the mutation encodes an amino acid substitution pattern corresponding to the amino acid residue at amino acid position SEQ ID NO:2, or (ii) introducing into cells of a Streptomyces avermitilis strain a polynucleotide molecule or degenerate variant thereof comprising a mutation in the nucleotide sequence of the aveC ORF shown in SEQ ID NO:1, wherein the polynucleotide molecule or degenerate variant thereof encodes an AveC gene product comprising an amino acid substitution pattern corresponding to the amino acid residue at amino acid position SEQ ID NO:2, wherein cells comprising the polynucleotide molecule or degenerate variant thereof are capable of producing cyclohexyl B2: cyclohexyl B1 avermectins at a ratio of 0.2:1 or less, and wherein the amino acid substitution pattern comprises substitutions at both D48 and G179, wherein the combination of amino acid substitutions in the AveC gene product is a combination selected from the group consisting of:

(ao)D48E,A89T,L136P,G179S,E238D;

(ap)D48E,A89T,L136P,K154E,G179S,E238D;

(aq)D48E,A89T,S138T,A139T,K154R,G179S,V196A,P289L;

(ar)D48E,A89T,S138T,A139F,G179S,V196A,E238D;

(as)D48E,A61T,A89T,L136P,G179S,V196A,A198G,P289L;

(at)D48E,A61T,S138T,A139F,G179S,G196A,E238D,P289L;

(au)D48E,A89T,L136P,G179S;

(av)D48E,A89T,V120A,L136P,G179S;

(aw)D48E,A61T,A89T,S138T,A139F,G179S,V196A,A198G,E238D;

(ax)D48E,A61T,A89T,G111V,S138T,A139F,G179S,V196A,E238D;

(ay)D48E,A61T,A89T,S138T,A139T,G179S,V196A,E238D,P289L;

(az)D48E,A61T,A89T,L136P,S138T,A139F,G179S,A198G,E238D;

(ba)D48E,A89T,S138T,A139F,G179S,A198G,V220A;

(bb)D48E,A61T,A89T,S138T,A139T，G179S,V196A,E238D,R239H,P289L;

(bc)D48E,A61T,A89T,L136P,G179S,P289L;

(bd) D48E, A89T, S138T, A139T, G179S, V196A, E238D, P289L, and

(be)D48E,A61T,A89T,S138T,A139F,G179S,V196A,E238D。

6. a Streptomyces cell comprising a polynucleotide molecule comprising a mutation in the nucleotide sequence of the aveC ORF of S.avermitilis as set forth in SEQ ID No. 1, or a degenerate variant thereof, wherein said polynucleotide or degenerate variant thereof encodes an AveC gene product comprising a combination of amino acid substitutions corresponding to the amino acid residue at amino acid position 2 of SEQ ID NO:

(ao)D48E,A89T,L136P,G179S,E238D;

(ap)D48E,A89T,L136P,K154E,G179S,E238D;

(aq)D48E,A89T,S138T,A139T,K154R,G179S,V196A,P289L;

(ar)D48E,A89T,S138T,A139F,G179S,V196A,E238D;

(as)D48E,A61T,A89T,L136P,G179S,V196A,A198G,P289L;

(at)D48E,A61T,S138T,A139F,G179S,G196A,E238D,P289L;

(au)D48E,A89T,L136P,G179S;

(av)D48E,A89T,V120A,L136P,G179S;

(aw)D48E,A61T,A89T,S138T,A139F,G179S,V196A,A198G,E238D;

(ax)D48E,A61T,A89T,G111V,S138T,A139F,G179S,V196A,E238D;

(ay)D48E,A61T,A89T,S138T,A139T,G179S,V196A,E238D,P289L;

(az)D48E,A61T,A89T,L136P,S138T,A139F,G179S,A198G,E238D;

(ba)D48E,A89T,S138T,A139F,G179S,A198G,V220A;

(bb)D48E,A61T,A89T,S138T,A139T,G179S,V196A,E238D,R239H,P289L;

(bc)D48E,A61T,A89T,L136P,G179S,P289L;

(bd) D48E, A89T, S138T, A139T, G179S, V196A, E238D, P289L, and

(be)D48E,A61T,A89T,S138T，A139F,G179S,V196A,E238D，

wherein a cell comprising the mutated aveC allele or degenerate variant thereof is capable of producing cyclohexyl B2: cyclohexyl B1 avermectin at a ratio of 0.2:1 or less.

7. The cell of claim 6, which is a Streptomyces avermitilis cell.

8. A method for producing avermectins, comprising culturing cells of the streptomyces avermitilis strain of claim 7 in a culture medium under conditions that allow or induce the production of avermectins, and recovering the avermectins from the culture.