Patent EA-003905-B1 (EA)
Title: Streptomyces avermitilis gene directing the ratio of b2:b1 avermectins
Inventor: STUTZMAN-ENGWALL KIM JONELLE
IPC: C12Q1/68
Filed: 1999-01-25
Abstract: 1. An isolated polynucleotide molecule comprising the complete aveC open-reading frame (ORF) of Streptomyces avermitilis or a substantial portion thereof, comprising at least about 70% of the complete aveC encoding functionally equevalent AveC gene product, which isolated polynucleotide molecule lacks the next complete ORF that is located downstream from the aveC ORF in situ in the Streptomyces avermitilis chromosome, and which ORF has the nucleotide sequence of FIGURE 1 (SEQ ID NO:1). 2. The isolated polynucleotide molecule of claim 1, further comprising nucleotide sequences that naturally flank the aveC gene in situ in S avermitilis. 3. An isolated polynucleotide molecule, comprising a nucleotide sequence that is homologous to the Streptomyces avarmitilis AveC gene product-encoding nucleotide sequence of the aveC ORF presented in FIGURE 1 (SEQ ID NO:1) or substantial portion thereof, comprising at least about 70% of the complete aveC which encodes functionally equevalent AveC gene product. 4. An isolated polynucleotide molecule comprising the nucleotide sequence of SEQ ID ID:3 or a substantial portion thereof, or a nucleotide sequence that is homologous to the nucleotide sequence of SEQ ID NO:3. 5. An oligonucleotide molecule that hybridizes to a polynucleotide molecule having the nucleotide sequence of FIGURE 1 (SEQ ID NO:1) or to a polynucleotide molecule having a nucleotide sequence that is the complement of the nucleotide sequence of FIGURE 1 (SEQ ID NO:1),under highly stringent conditions. 6. An oligonucleotide molecule that hybridizes to a polynucleotide molecule having the nucleotide sequence of SEQ ID NO:3, or to a polynucleotide molecule having a nucleotide sequence that is the complement of the nucleotide sequence of SEQ ID NO:3, under highly stringent conditions. 7. The oligonucleotide molecule of claim 5, which is complementary to the nucleotide sequence of FIGURE 1 (SEQ ID NO:1) or to a polynucleotide molecule having a nucleotide sequence that is the complement of the nucleotide sequence of FIGURE 1 (SEQ ID NO:1) 8. The oligonucleotide molecule of claim 5, which is complementary to the nucleotide sequence of SEQ ID NO:3, or to a polynucleotide molecule having a nucleotide sequence that is the complement of the nucleotide sequence of SEQ ID NO:3. 9. A recombinant vector comprising a polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of the aveC ORF of FIGURE 1 (SEQ ID NO:1) or substantial portion thereof; and a nucleotide sequence that is homologous to the S. avermitilis AveC gene product-encoding ORF of FIGURE 1 (SEQ ID NO: 1) or substantial portion thereof. 10. The recombinant vector of claim 9, further comprising a nucleotide sequence encoding one or more regulatory elements, which regulatory element encoding nucleotide sequence is in operative association with the nucleotide sequence of the polynucleotide molecule of the vector of claim 9. 11. The recombinant vector of claim 10, further comprising a nucleotide sequence encoding a selectable marker. 12. The recombinant vector of claim 11, which is plasmid pSE186 (ATCG 209604). 13. A host cell comprising the recombinant vector of claim 11. 14. The host cell of claim 13, which is Stratomyces avermitilis. 15. A recombinant vector comprising polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of the nucleotide Sequence of SEQ ID NO:3 or a substantial portion thereof; a nucleotide sequence that is homologous to the nucleotide sequence of SEQ ID NO:3; and a nucleotide sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO:4. 16. The recombinant vector of claim 15, further comprising a nucleotide sequence encoding one or more regulatory elements, which regulatory element encoding nucleotide sequence is in operative association with the nucleotide sequence of the polynucleotide molecule of the vector of claim 15. 17. The recombinant vector of claim 16, further comprising a nucleotide sequence encoding a selectable marker. 18. A host cell comprising the recombinant vector of claim 17. 19. The host cell of claim 18, which is Streptomyces hygroscopicus. 20. An isolated S. avermitilis AveC gene product having the amino acid sequence encoded by the S. avermitilis AveC gene product-encoding nucleotide sequence of plasmid pSE186 (ATCC 209604), or the amino acid sequence of SEQ ID NO:2 or substantial portion thereof, comprising at least about 70% of the complete aveC which is a functionally equevalent AveC gene product, in which optionally one or more amino acid residue is conservatively replaced with another amino acid residue, wherein such a replacement brings to a functionally equevalent gene product. 21. An isolated S. hygroscopicus AveC homolog gene product having the amino acid sequence of SEQ ID NO:4. 22. A method for producing a recombinant AveC gene product, comprising culturing a host cell transformed with a recombinant expression vector according to any one of claims 9 to 11 under conditions conducive to the production of the recombinant AveC gene product and recovering the AveC gene product from the cell culture. 23. A method for producing a recombinant AveC gene product, comprising culturing a host cell transformed with a recombinant expression vector according to any one of claims 15 to 17 under conditions conducive to the production of the recombinant AveC gene product and recovering the AveC gene product from the cell culture. 24. A polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as 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 FIGURE 1 (SEQ IS NO:1) or a degenerate variant thereof, but which nucleotide sequence further comprises one or more mutations, so 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 produce a reduced cyclohexyl B2:cyclohexyl B1 ratio of avermectins when fermented in the presence of cyclohexanecarboxylic acid than is produced by cells of S. avermitilis strain ATCC 53692 that instead express only the wild-type aveC allele. 25. The polynucleotide molecule of claim 24, wherein the reduced ratio of cyclohexyl B2:cyclohexyl B1 is less than 1.6:1. 26. The polynucleotide molecule of claim 25, wherein the reduced ratio of cyclohexyl B2:cyclohexyl B1 is about 0.94:1. 27. The polynucleotide molecule of claim 26, wherein the mutation to the S. avermitilis aveC ORF of FIGURE 1 (SEQ ID NO:1) encodes an amino acid substitution at residue 139 of the AveC gene product from alanine to threonine. 28. The polynucleotide molecule of claim 25, wherein the reduced ratio of cyclohexyl B2:cyclohexyl B1 is about 0.88:1. 29. The polynucleotide molecule of claim 28, wherein the mutation to the S. avermitilis aveC ORF of FIGURE 1 (SEQ ID NO:1) encodes an amino acid substitution at residue 138 of the AveC gene product from serine to threonine. 30. The polynucleotide molecule of claim 25, wherein the reduced ratio of cyclohexyl B2:cyclohexyl B1 is about 0.84:1. 31. The polynucleotide molecule of claim 30, wherein the mutation to the S. avermitilis aveC ORF of FIGURE 1 (SEQ ID NO:1) encodes a first amino acid substitution at residua 138 of the AveC gene product from serine to threonine, and a second amino and substitution at residue 139 of the AveC gene product from alanine to threonine. 32. A polynucleotide molecule comprising a strong promoter in operative association with the S. avermitilis aveC ORF of FIGURE 1 (SEQ ID NO:1). 33. The polynucleotide molecule of claim 32, wherein the strong promoter is the ermE promoter from Saccharopolyspora erythraea. 34. A polynucleotide molecule comprising a nucleotide sequence encoding the AveC gene product-encoding ORF of FIGURE 1 (SEQ ID NO:1) that has been inactivated by insertion into said nucleotide sequence of a heterologous nucleotide sequence. 35. A polynucleotide molecule comprising an aveC allele that has been inactivated by deleting a 640 by Psf/Sph1 fragment from the aveC ORF of FIGURE 1 (SEQ ID NO: 1). 36. A polynucleotide molecule comprising an aveC allele that has been inactivated by introducing a frameshift mutation into the aveC ORF of FIGURE 1 (SEQ ID NO:1). 37. The polynucleotide molecule of claim 36, in which the frameshift mutation was introduced by adding two G nucleotides after the C nucleotide at position 471 of the aveC ORF of FIGURE 1 (SEQ ID NO:1). 38. A polynucleotide molecule comprising an aveC allele that has been inactivated by introducing a termination codon at the nucleotide position that encodes amino acid 116 of the S. avermitilis AveC gene product encoded by the aveC ORF of FIGURE 1 (SEQ ID NO:1). 39. A polynucleotide molecule comprising an aveC allele that has been inactivated by introducing a first mutation at amino acid position 256 of the AveC gene product that changes a glycine to an aspartate, and a second mutation at position 275 of the AveC gene product that changes a tyrosine to a histidine. 40. A recombinant vector comprising the polynucleotide molecule of any of claims 24 to 39. 41. A recombinant vector comprising the polynucleotide molecule of claim 34, which vector is pSE180 (ATCC 209605). 42. A host Streptomyces cell comprising the recombinant vector of claim 40. 43. A strain of S. avermitilis comprising cells expressing a mutated aveC allele which results in the production by the cells of avermectins in a reduced class 2:1 ratio compared to cells of the same strain that instead express only the wild-type aveC allele. 44. The strain of claim 43, wherein the cells produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of less than 1.6:1. 45. The strain of claim 43, wherein the cells produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio