US 8,008,078 B2Granted

Streptomyces avermitilis gene directing the ratio of B2:B1 avermectins

AssigneesPfizer Inc., Pfizer Products Inc.

InventorsKim J. Stutzman-Engwall, Anke Krebber, Claes Gustafsson, Jeremy S. Minshull, Sun Ai Raillard, Seran Kim, Yan Chen

Life of this patent

Estimating the term…

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.

Claims5

Dependent claims

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;
(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;
(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.

84 more elements

Dependencies are inferred from the claim text.

Facts

Application: 12/107,949Filed 23 Apr 2008 · division
Priority: 12 Feb 2002
Published: US 2009/0017508 A115 Jan 2009
Granted: 30 Aug 2011
Term ends: Not estimatedThe source records don’t support an estimate.

Classification7 codes

C12N 9/88
Enzymes; Proenzymes; Compositions thereof ; Processes for preparing, activating, inhibiting, separating or purifying enzymes: lyases
A61P 33/00
Antiparasitic agents
C12P 17/181
Containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin: heterocyclic…
C12P 19/623
The hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin: avermectin;…

Family

Application60/356,222Us provisional application · filed 12 Feb 2002
Application10/361,376Parent of this division · filed 10 Feb 2003
US 2009/0017508 A1Pre-grant publication · published 15 Jan 2009
US 7,388,085 B2Parent of this division
US 2003/0166165 A1Published application
US 8,008,078 B2This patent

From the US filing history. Foreign family members aren’t listed.

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Abstract

(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;
(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.

84 more elements

Facts

Application

12/107,949Filed 23 Apr 2008 · division

Priority

12 Feb 2002

Published

US 2009/0017508 A115 Jan 2009

Granted

30 Aug 2011

Term ends

Not estimatedThe source records don’t support an estimate.

Classification7 codes

C12N 9/88
Enzymes; Proenzymes; Compositions thereof ; Processes for preparing, activating, inhibiting, separating or purifying enzymes: lyases
A61P 33/00
Antiparasitic agents
C12P 17/181
Containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin: heterocyclic…
C12P 19/623
The hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin: avermectin;…

Family

Application60/356,222Us provisional application · filed 12 Feb 2002

Application10/361,376Parent of this division · filed 10 Feb 2003

US 2009/0017508 A1Pre-grant publication · published 15 Jan 2009

US 7,388,085 B2Parent of this division

US 2003/0166165 A1Published application

US 8,008,078 B2This patent

From the US filing history. Foreign family members aren’t listed.