Polynucleotides and methods of directing the ratio of B2:B1 avermectins in
View Patent ↗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.
1. A purified polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide, wherein the amino acid sequence of the polypeptide is at least 90% identical to the amino acid sequence of SEQ ID NO:2, said polypeptide comprising substitutions at positions corresponding to amino acid residues selected from the group consisting of:
(a) D48E/A89T;
(b) S138T/A139T/G179S;
(c) Q38P/L136P/E238D;
(d) F99S/S138T/A139T/G179S;
(e) A139T/M228T;
(f) G111V/P289L; and
(g) A139T/K154E/Q298H
of SEQ ID NO:2; and
said polypeptide has the activity of altering the ratio of class 2:1 avermectins to a ratio of about 0.94:1 to about 0.4:1 when the polypeptide is present in Streptomyces avermitilis strain ATCC 53692.
2. The polynucleotide molecule of claim 1 , wherein the ratio of class 2:1 avermectins is about 0.68:1.
3. The polynucleotide molecule of claim 1 , wherein the ratio of class 2:1 avermectins is about 0.53:1.
4. The polynucleotide molecule of claim 1 , wherein the ratio of class 2:1 avermectins is about 0.42:1.
5. The polynucleotide molecule of claim 1 , wherein the ratio of class 2:1 avermectins is about 0.40:1.
6. The polynucleotide molecule of claim 1 , wherein the mutations in the polynucleotide encoding D48E/A89T comprise a base change from T to A at a nucleotide position corresponding to a nucleotide 317 of SEQ ID NO:1 and a base change from G to A at a nucleotide position corresponding to nucleotide 438 of SEQ ID NO:1.
7. The polynucleotide molecule of claim 6 , further comprising a base change from C to A at a nucleotide position corresponding to nucleotide 353 of SEQ ID NO:1, and a base change from T to A at a nucleotide position corresponding to nucleotide 1155 of SEQ ID NO:1.
8. The polynucleotide molecule of claim 1 , wherein the mutations in the polynucleotide encoding S138T/A139T/G179S comprise a base change from T to A at a nucleotide position corresponding to nucleotide 585 of SEQ ID NO:1, a base change from C to A at a nucleotide position corresponding to nucleotide 588 of SEQ ID NO:1, and a base change from G to A at a nucleotide position corresponding to nucleotide 708 of SEQ ID NO:1.
9. The polynucleotide molecule of claim 8 , further comprising a base change from C to A at a nucleotide position corresponding to nucleotide 272 of SEQ ID NO:1.
10. The polynucleotide molecule of claim 1 , wherein the mutations in the polynucleotide encoding Q38P/L136P/E238D comprise a base change from A to C at a nucleotide position corresponding to nucleotide 286 of SEQ ID NO:1, a base change from T to C at a nucleotide position corresponding to nucleotide 580 of SEQ ID NO:1, and a base change from A to T at a nucleotide position corresponding to nucleotide 886 of SEQ ID NO:1.
11. The polynucleotide molecule of claim 10 , further comprising a base change from A to G at a nucleotide position corresponding to nucleotide 24 of SEQ ID NO:1, a base change from T to C at a nucleotide position corresponding to nucleotide 497 of SEQ ID NO:1, and a base change from C to T at a nucleotide position corresponding to nucleotide 554 of SEQ ID NO:1.
12. The polynucleotide molecule of claim 1 , wherein the mutations in the polynucleotide encoding F99S/S138T/A139T/G179S comprise a 3 base pair deletion at nucleotide positions corresponding to nucleotides 173, 174 and 175 of SEQ ID NO:1, a base change from T to C at a nucleotide position corresponding to nucleotide 469 of SEQ ID NO:1, a base change from T to A at a nucleotide position corresponding to nucleotide 585 of SEQ ID NO:1, a base change from G to A at a nucleotide position corresponding to nucleotide 588 of SEQ ID NO:1, and a base change from G to A at a nucleotide position corresponding to nucleotide 708 of SEQ ID NO:1.
13. The polynucleotide molecule of claim 12 , further comprising a base change from C to T at a nucleotide position corresponding to nucleotide 833 of SEQ ID NO:1, and a base change from G to A at a nucleotide position corresponding to nucleotide 1184 of SEQ ID NO:1.
14. The polynucleotide molecule of claim 1 , wherein the mutations in the polynucleotide encoding A139T/M228T comprise a base change from G to A at a nucleotide position corresponding to nucleotide 588 of SEQ ID NO:1, and a base change from T to C at a nucleotide position corresponding to nucleotide 856 of SEQ ID NO:1.
15. The polynucleotide molecule of claim 1 , wherein the mutations in the polynucleotide encoding G111V/P289L comprise a base change from C to T at a nucleotide position corresponding to nucleotide 505 of SEQ ID NO:1, and a base change from C to T at a nucleotide position corresponding to nucleotide 1039 of SEQ ID NO:1.
16. The polynucleotide molecule of claim 15 , further comprising a base change from T to C at a nucleotide position corresponding to nucleotide 155 of SEQ ID NO:1, a base change from C to T at a nucleotide position corresponding to nucleotide 1202 of SEQ ID NO:1, and a base change from T to C at a nucleotide position corresponding to nucleotide 1210 of SEQ ID NO:1.
17. The polynucleotide molecule of claim 1 , wherein the mutations in the polynucleotide encoding A139T/K154E/Q298H comprise a base change from G to A at a nucleotide position corresponding to nucleotide 588 of SEQ ID NO:1, a base change from A to G at a nucleotide position corresponding to nucleotide 633 of SEQ ID NO:1, and a base change from A to T at a nucleotide position corresponding to nucleotide 1067 of SEQ ID NO:1.
18. The polynucleotide molecule of claim 17 , further comprising a base change from C to T at nucleotide position corresponding to nucleotide 377 of SEQ ID NO:1.
19. A purified recombinant vector comprising the polynucleotide molecule of claim 1 .
20. A purified host cell comprising the purified recombinant vector of claim 19 .
21. The purified host cell of claim 20 , which is a Streptomyces cell.