Streptomyces avermitilis gene directing the ratio of B2:B1 avermectins
AssigneePfizer 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.
Claims21
A polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as the Streptomyces avermitilis aveC allele, the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604) or the nucleotide sequence of the aveC ORF of S. avermitilis as presented in FIG. 1 (SEQ ID NO:1), or a degenerate variant thereof, but which nucleotide sequence further comprises mutations encoding a combination of amino acid substitutions at amino acid residues corresponding to the amino acid positions of SEQ ID NO:2, such that cells of S. avermitilis strain ATCC 53692 in which the wild-type aveC allele has been inactivated and that express the polynucleotide molecule comprising the mutated nucleotide sequence are capable of producing a class 2:1 ratio of avermectins that is reduced compared to the ratio produced by cells of S. avermitilis strain ATCC 53692 that instead express only the wild-type aveC allele, wherein when the class 2:1 avermectins are cyclohexyl B2:cyclohexyl B1 avermectins the ratio of class 2:1 avermectins is 0.35:1 or less.
A polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as the Streptomyces avermitilis aveC allele, the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604) or the nucleotide sequence of the aveC ORF of S. avermitilis as presented in FIG. 1 (SEQ ID NO:1), or a degenerate variant thereof, but which nucleotide sequence further comprises mutations encoding a combination of amino acid substitutions at amino acid residues corresponding to the amino acid positions of SEQ ID NO:2, such that cells of S. avermitilis strain ATCC 53692 in which the wild-type aveC allele has been inactivated and that express a polynucleotide molecule comprising the mutated nucleotide sequence are capable of producing a class 2:1 ratio of avermectins that is reduced compared to the ratio produced by cells of S. avermitilis strain ATCC 53692 that instead express only the wild-type aveC allele, wherein when the class 2:1 avermectins are cyclohexyl B2:cyclohexyl B1 avermectins, the ratio of class 2:1 avermectins is reduced to about 1.17:1 or less, and wherein the combination of amino acid substitutions comprises a combination selected from the group consisting of:
- (bf) D48E, S138T, A139T, G179S, E238D;
- (bg) Y28C, Q38R, D48E, L136P, G179S, E238D;
- (ca) V3L, L136M;
- (cb) G26A, D48Y, R75W, S93N;
- (cc) R71L;
- (cd) T471, W110L, A139T;
- (ce) V1041, S138T, V2201, F2341;
- (cf) G45R, A64V, R69K;
- (cg) S90N;
- (ch) G26D, W 110L, R233H;
- (ci) Q36R, V1041, P128S, C152W, T276A;
- (ck) C142Y, A302T;
- (cl) V2M, V56D;
- (cm) S41G, L87V, A139T, L206M, G209R, 1280V;
- (cn) A62V, A139D;
- (co) F176C;
- (cp) T149S;
- (cq) A64T, C142Y;
- (cr) A130V, C142Y, L224M, E238V, L293M;
- and
- (cs) A16T, K154M, L206F
A method for making a strain of Streptomyces avermitilis, comprising (i) mutating the aveC allele in a cell of a strain of S. avermitilis, which mutation results 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 degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions, wherein the combination of amino acid substitutions in the AveC gene product 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, G11V, S138T, A139F, G179S, E238D, P289L;
- (x) D25G, D48E, A89T, L136P, S138T, A139T, V411A, 1159T, 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;
- (be) D48E, A61T, A89T, S138T, A139F, G179S, V196A, E238D;
- (ca) V3L, L136M;
- (cb) G26A, D48Y, R75W, S93N;
- (cc) R71L;
- (cd) T471, W110L, A139T;
- (ce) V1041,S138T, V220I,F234I;
- (cf) G45R, A64V, R69K;
- (cg) S90N;
- (ch) G26D, W110L, R233H;
- (ci) Q36R, V1041, P128S, C152W, T276A;
- (ck) C142Y, A302T;
- (cl) V2M, V56D;
- (cm) S41G, L87V, A139T, L206M, G209R, 1280V;
- (cn) A62V, A139D;
- (co) F176C;
- (cp) T149S;
- (cq) A64T, C142Y;
- (cr) A130V, C142Y, L224M, E238V, L293M;
- (cs) A16T, K154M, L206F;
- (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, G179S, E238D, 1280V;
- (dd) D48E, A61T, R71L, W110L, T149S, G179S, V196A, L206M, E238D, V271A, 1280V;
- (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, 1280V;
- (eb) D48E, R71L, A89T, L136P, T149S, F176C, G179S, E238D, 1280V;
- (ec) Q36P, D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, 1280V;
- (ed) D48E, A61T, R71L, A89T, A139T, T149S, F176C, G179S, V196A, E238D, 1280V;
- (ee) V2M, D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, 1280V;
- (ef) D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, 1280V, 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, 1280V;
- (ej) D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D;
- (ek) V2M, D48E, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, 1280V;
- (el) D48E, A61T, R71L, A89T, L136P, T149S, R162H, F176C, G179S, V196A, E238D, 1280V;
- (em) D48E, R71L, A89T, V120A, L136P, T149S, K154E, G179S, S231L, E238D;
- (en) D48E, R71L, A89T, V120A, L136P, T149S, F176C, G179S, S231L, E238D, 1280V;
- (eo) D48E, A61T, R71L, L87V, A89T, A139T, T149S, F176C, G179S, V196A, E238D, V285G, P289L;
- (ep) D48E, A61T, R71L, L87V, A89T, S90N, 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.
A method for making a strain of Streptomyces avermitilis, comprising (i) mutating the aveC allele in a cell of a strain of S. avermitilis, which mutation results 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 degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions, wherein cells comprising the mutated aveC allele or degenerate variant are capable of producing cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of 0.35:1 or less.
A method for making a novel strain of Streptomyces avermitilis, comprising (i) mutating the aveC allele in a cell of a strain of S. avermitilis, which mutation results 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 degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions, wherein the combination of amino acid substitutions is selected from the group consisting of:
- (bf) D48E, S138T, A139T, G179S, E238D;
- and
- (bg) Y28C, Q38R, D48E, L136P, G179S, E238D;
- (ca) V3L, L136M;
- (cb) G26A, D48Y, R75W, S93N;
- (cc) R71L;
- (cd) T471, W110L, A139T;
- (ce) V1041, S138T, V2201, F2341;
- (cf) G45R, A64V, R69K;
- (cg) S90N;
- (ch) G26D, W110L, R233H;
- (ci) Q36R, V1041, P128S, C152W, T276A;
- (ck) C142Y, A302T;
- (cl) V2M, V56D;
- (cm) S41 G, L87V, A139T, L206M, G209R, 1280V;
- (cn) A62V, A139D;
- (co) F176C;
- (cp) T149S;
- (cq) A64T, C142Y;
- (cr) A130V, C142Y, L224M, E238V;
- and
- (cs) A16T, K154M, L206F;
A cell of a Streptomyces species that comprises a mutated S. avermitilis 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) 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, 1159T, 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;
- (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, G179S, E238D, 1280V;
- (dd) D48E, A61T, R71L, W110L, T149S, G179S, V196A, L206M, E238D, V271A, 1280V;
- (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, 1280V;
- (eb) D48E, R71L, A89T, L136P, T149S, F176C, G179S, E238D, 1280V;
- (ec) Q36P, D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, 1280V;
- (ed) D48E, A61T, R71L, A89T, A139T, T149S, F176C, G179S, V196A, E238D, 1280V;
- (ee) V2M, D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, 1280V;
- (ef) D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, 1280V, 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, 1280V;
- (ej) D48E, A61T, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D;
- (ek) V2M, D48E, R71L, A89T, L136P, T149S, F176C, G179S, V196A, E238D, 1280V;
- (el) D48E, A61T, R71L, A89T, L136P, T149S, R162H, F176C, G179S, V196A, E238D, 1280V;
- (em) D48E, R71L, A89T, V120A, L136P, T149S, K154E, G179S, S231L, E238D;
- (en) D48E, R71L, A89T, V120A, L136P, T149S, F176C, G179S, S231L, E238D, 1280V;
- (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.
A cell of a Streptomyces species, comprising a mutated S. avermitilis aveC allele or degenerate variant thereof encoding an AveC gene product comprising a combination of amino acid substitutions selected from the group consisting of
- (bf) D48E, S138T, A139T, G179S, E238D;
- and
- (bg) Y28C, Q38R, D48E, L136P, G179S, E238D;
- (ca) V3L, L136M;
- (cb) G26A, D48Y, R75W, S93N;
- (cc) R71L;
- (cd) T471, W110L, A139T;
- (ce) V1041, S138T, V2201, F2341;
- (cf) G45R, A64V, R69K;
- (cg) S90N;
- (ch) G26D, W 110L, R233H;
- (ci) Q36R, V1041, P128S, C152W, T276A;
- (ck) C142Y, A302T;
- (cl) V2M, V56D;
- (cm) S41G, L87V, A139T, L206M, G209R, 1280V;
- (cn) A62V, A139D;
- (co) F176C;
- (cp) T149S;
- (cq) A64T, C142Y;
- (cr) A130V, C142Y, L224M, E238V, L293M;
- (cs) A16T, K154M, L206F;
A composition of cyclohexyl B2:cyclohexyl B1 avermectins produced by cells of Streptomyces avermitilis, comprising the cyclohexyl B2:cyclohexyl B1 avermectins present in a culture medium in which the cells have been cultured, wherein the ratio of the cyclohexyl B2:cyclohexyl B1 avermectins present in the culture medium is 0.35:1 or less.
Dependencies are inferred from the claim text.