IP Library › Granted Patent US 8,008,078

Granted Patent B2

US 8,008,078 · App. 12/107,949 · Granted Aug 30, 2011

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Patent No.: US 8,008,078
App. No.: 12/107,949
Granted: Aug 30, 2011
Kind: B2

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Abstract

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

Claims (299)

1. An isolated Streptomyces avermitilis cell which produce a cyclohexyl B2:cyclohexyl B1 avermectin ratio of about 0.35:1 or less, comprising a nucleotide sequence that is otherwise the same as the S.avermitilis aveC allele, the S. avermititis AveC gene product-encoding sequence of plasmid pSE 186 (ATCC 209604) or the nucleotide sequence of the aveC ORF of S.avermitilis of SEQ ID NO:1, or a degenerate variant thereof, which nucleotide encodes a functionally equivalent aveC gene product and which nucleotide sequence further comprises mutations encoding one or more amino acid substitutions in the amino acid sequence of SEQ. ID. NO:2 wherein said amino acid substitution is selected from the group consisting of:

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

(b) G40S, D48E, L136P, G179S, E238D;

(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;

(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, G 179S;

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

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

(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, F2345;

(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, V196A, 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;

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

(da) S41G, D48E, A61T, R71L, A89T, L136M, S138T, A139T, T149S, G179S, V196A, E238D, F278L, P289L;

(db) S41G, D48E, A61T, R71L, A89T, L136M, S138T, A139T, T149S, F176C, G179S, V196A, E238D, P289L;

(de) D48E, R71L, A89T, L136P, T149S, F176C, G179S, E238D, I280V;

(dd) D48E, A61T, R71L, W110L, T149S, G179S, V196A, L206M, E238D, V271A, I280V;

(de) D48E, A61T, R71L, A89T, L136M, S138T, A139T, T149S, G179S, V196A, E238D, H279Q, P289L;

(df) D48E, A61T, R71L, A89T, L136M, S138T, A139T, T149S, G179S, V196A, E238D, G287E, P289Q;

(dg) D48E, A61T, R71L, A89T, L136M, S138T, A139T, T149S, G179S, V196A, E238D, P289L;

(dh) D48E, A61T, R71L, A89T, A139T, T149S, F176C, G179S, V196A, E238D, V285G, P289L;

(di) Q38R, D48E, A61T, R71L, L87V, A89T, L136M, S138T, A139T, T149S, G179S, V196A, E238D, P289L;

(dj) D48E, A61T, L87V, A89T, W110L, S138T, A139T, T149S, G179S, V196A, E238D, P289L;

(dk) D48E, A61T, R71L, A89T, L136M, S138T, A139T, T149S, G179S, V196A, E238D, P289L;

(dl) D48E, A89T, L136P, K154E, G179S, S231L, E238D;

(ea) D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, I280V;

(eb) D48E, R71L, A89T, L136P, T149S, F176C, G179S, E238D, I280V;

(ec) Q36P, D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, I280V;

(ed) D48E, A61T, R71L, A89T, A139T, T149S, F176C, G179S, V196A, E238D, I280V;

(ee) V2M, D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, I280V;

(ef) D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, I280V, A302T;

(eg) D48E, R71L, A89T, L136P, T149S, F176C, G179S, E238D, P289L;

(eh) D48E, R71L, A89T, L136P, T149S, F176C, G179S, E238D, A302T;

(ei) D48E, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, I280V;

(ej) D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D;

(ek) V2M, D48E, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, I280V;

(el) D48E, A61T, R71L, A89T, L136P, T149S, R162H, F176C, G179S, V196A, E238D, I280V;

(em) D48E, R71L, A89T, V120A, L136P, T149S, K154E, G179S, S231L, E238D;

(en) D48E, R71L, A89T, V120A, L136P, T149S, F176C, G179S, S231L, E238D, I280V;

(eo) D48E, A61T, R71L, L87V, A89T, S90N, A139T, T149S, F176C, G179S, V196A, E238D, V285G, P289L;

(ep) D48E, A61T, R71L, L87V, A89T, A139T, T149S, F176C, G179S, V196A, E238D, V285G, P289L;

(eq) D48E, R71L, A89T, L136P, K154E, G179S, S231L, E238D;

(er) D48E, R71L, A89T, V120A, L136P, K154E, F176C, G179S, S231L, E238D; and

(es) D48E, R71L, A89T, V120A, L136P, T149S, K154E, F176C, G179S, S231L, E238D.

2. The isolated S.avermitilis cell of claim 1 wherein said amino acid substitution is selected from the group consisting of:

(g) D48E, A61T, L136P, G179S, E238D;

(h) D48E, A61T, L136P, 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, R163D, 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,I 159T, R163Q, G179S;

(y) D48E, A89T, 590G, L136P, R163D, G179S, E238D;

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

(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, V196A, 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;

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

(da) S41G, D48E, A61T, R71L, A89T, L136M, S138T, A139T, T149S, G179S, V196A, E238D, F278L, P289L;

(db) S41G, D48E, A61T, R71L, A89T, L136M, S138T, A139T, T149S, F176C, G179S, V196A, E238D, P289L;

(dc) D48E, R71L, A89T, L136P, T149S, F176C, 0179S, E238D, I280V;

(dd) D48E, A61T, R71L, W110L,T149S, G179S, V196A, L206M, E238D, V271A, I280V;

(de) D48E, A61T, R71L, A89T, L136M, S138T, A139T, T149S, G179S, V196A, E238D, H279Q, P289L;

(df) D48E, A61T, R71L, A89T, L136M, S138T, A139T, T149S, G179S, V196A, E238D, G287E, P289Q;

(dg) D48E, A61T, R71L, A89T, L136M, S138T, A139T, T149S, G179S, V196A, E238D, P289L;

(dh) D48E, A61T, R71L, A89T, A139T, T149S, F176C, G179S, V196A, E238D, V285G, P289L;

(di) Q38R, D48E, A61T, R71L, L87V, A89T, L136M, S138T, A139T, T149S, G179S, V196A, E238D, P289L;

(dj) D48E, A61T, L87V, A89T, W110L, S138T, A139T, T149S, G179S, V196A, E238D, P289L;

(dk) D48E, A61T, R71L, A89T, L136M, S138T, A139T, T149S, G179S, V196A, E238D, P289L;

(dl) D48E, A89T, L136P, K154E, G179S, S231L, E238D;

(ea) D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, I280V;

(eb) D48E, R71L, A89T, L136P, T149S, F176C, G179S, E238D, I280V;

(ec) Q36P, D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, I280V;

(ed) D48E, A61T, R71L, A89T, A139T, T149S, F176C, G179S, V196A, E238D, I280V;

(ee) V2M, D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, I280V;

(ef) D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, I280V, A302T;

(eg) D48E, R71L, A89T, L136P, T149S, F176C, G179S, E238D, P289T,

(eh) D48E, R71L, A89T, L136P, T149S, F176C, G179S, E238D, A302T;

(el) D48E, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, I280V;

(ej) D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D;

(ek) V2M, D48E, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, I280V,

(el) D48E, A61T, R71L, A89T, L136P, T149S, R162H, F176C, G179S, V196A, E238D, I280V;

(em) D48E, R71L, A89T, V120A, L136P, T149S, K154E, G179S, S231L, E238D;

(en) D48E, R71L, A89T, V120A, L136P, T149S, F176C, G179S, S231L, E238D, I280V;

(eo) D48E, A61T, R71L, L87V, A89T, S90N, A139T, T149S, F176C, G179S, V196A, E238D, V285G, P289L;

(ep) D48E, A61T, R71L, L87V, A89T, A139T, T149S, F176C, G179S, V196A, E238D, V285G, P289L;

(eq) D48E, R71L, A89T, L136P, K154E, G179S, S231L, E238D;

(er) D48E, R71L, A89T, V120A, L136P, K154E, F176C, G179S, S231L, E238D; and

(es) D48E, R71L, A89T, V120A, L136P, T149S, K154E, F176C, G179S, S231L, E238D.

3. The isolated S.avermitilis cell of claim 1 wherein said amino acid substitution is selected from the group consisting of:

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

(x) D25G, D48E, A89T, L136P, S138T, A139T, V141A,I 159T, R163Q, G179S;

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

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

(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, V196A, 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;

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