Patent EA-004569-B1 (EA)
Title: Streptomyces avermitilis gene directing the ratio of b2:b1 avermectins
Inventor: STUTZMAN-ENGWALL KIM JONELLE
IPC: C12N15/76
Filed: 2000-07-19
Abstract: 1. A polynucleotide molecule comprising a nucleotide sequence of the S. avermitilis aveC allele or a plasmide sequence pSE186 (ATCC 209604), the AveC gene product-encoding or the nucleotide sequence of the aveC ORF of S. avermitilis as presented in FIGURE 1 (SEQ ID NO: 1) or a degenerate variant thereof, but which nucleotide sequence further comprises at least a first mutation encoding an amino acid substitution from serine to threonine at an amino acid residue of the AveC gene product corresponding to amino acid position 138 in SEQ ID NO: 2 and a second mutation encoding an amino acid substitution from alanine to phenylalanine at an amino acid residue of the AveC gene product corresponding to amino acid position 139 in 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 produce a class 2: 1 ratio of avermectins that is reduced compared to the class 2: 1 ratio of avermectins produced by cells of S. avermitilis strain ATCC 53692 that instead express only the wild-type aveC allele. 2. The polynucleotide molecule of claim 1, wherein the class 2: 1 avermectins are cyclohexyl B2: cyclohexyl B1 avermectins. 3. The polynucleotide molecule of claim 7, wherein the reduced ratio of cyclohexyl B2: cyclohexyl B1 is about 0.75: 1 or less. 4. A recombinant vector comprising the polynucleotide molecule of claim 1. 5. A host Streptomyces cell comprising the polynucleotide molecule of claim 1 or the recombinant vector of claim 4. 6. A method for making a novel strain of S. avermitilis comprising cells that express a mutated aveC allele and that produce a reduced class 2: 1 ratio of avermectins compared to cells of the same strain of S. avermitilis that instead express only the wild-type aveC allele, comprising: (a) transforming cells of a strain of S. avermitilis with a polynucleotide molecule that carries a mutated aveC allele or degenerate variant thereof that encodes a gene product having at least a first amino acid substitution from serine to threonine at an amino acid residue corresponding to amino acid position 138 in SEQ ID NO: 2 and a second mutation encoding an amino acid substitution from alanine to phenylalanine at an amino acid residue corresponding to amino acid position 139 in SEQ ID NO: 2, wherein the expression of the resulting gene product results in a reduction of the class 2: 1 ratio of avermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allele or degenerate variant thereof compared to cells of the same strain that instead express only the wild-type aveC allele, and selecting transformed cells that produce avermectins in a reduced class 2: 1 ratio compared to the class 2: 1 ratio produced by cells of the strain that instead express only the wild-type aveC allele; or (b) transforming cells of a strain of S. avermitilis with one or more polynucleotide moleculesmoleculescapable of introducing one or more mutations into the aveC allele so that such transformed cells encode an AveC gene product having at least a first amino acid substitution from serine to threonine at an amino acid residue corresponding to amino acid position 138 in SEQ ID NO: 2 and a second mutation encoding an amino acid substitution from alanine to phenylalanine at an amino acid residue corresponding to amino acid position 139 in SEQ ID NO: 2, wherein the expression of the gene product results in a reduction of the class 2: 1 ratio of avermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allele compared to cells of the same strain that instead express only the wild-type aveC allele, and selecting transformed cells that produce avermectins in a reduced class 2: 1 ratio compared to the class 2: 1 ratio produced by cells of the strain that instead express only the wild-type aveC allele. 7. The method of claim 6, wherein the class 2: 1 avermectins are cyclohexyl B2: cyclohexyl B1 avermectins. 8. The method of claim 7, wherein the reduced ratio of cyclohexyl B2: cyclohexyl B1 is about 0.75: 1 or less. 9. A Streptomyces avermitilis cell comprising a mutated aveC allele comprising a first mutation encoding an amino acid substitution from serine to threonine at an amino acid residue corresponding to amino acid position 138 in SEQ ID NO: 2 and a second mutation encoding an amino acid substitution from alanine tophenylalanine at an amino acid residue corresponding to amino acid position 139 in SEQ ID NO: 2. 10. The cell of claim 9, which produces a reduced class 2: 1 ratio of avermectins compared to a cell of the same strain of Streptomyces avermitilis that instead comprises only the wild-type aveC allele. 11. The cell of claim 10, wherein the class 2: 1 avermectins are cyclohexyl B2: cyclohexyl B1 avermectins. 12. The cell of claim 11, wherein the reduced ratio of cyclohexyl B2: cyclohexyl B1 is about 0.75: 1 or less. 13. A process for producing avermectins, comprising culturing cells of claim 9 in culture media under conditions that permit or induce the production of avermectins therefrom, and recovering said avermectins from the culture. 14. A composition of cyclohexyl B2:cyclohexyl B1 avermectins produced by cells of a strain of Streptomyces avermitilis that express a mutated aveC allele which encodes a gene product that results in the reduction of the class 2: 1 ratio of cyclohexyl B2: cyclohexyl B1 avermectins, wherein the reduced ratio of cyclohexyl B2: cyclohexyl B1 is about 0.75: 1 or less.