Cloning genes from Streptomyces cyaneogriseus subsp. Noncyanogenus for biosynthesis of antibiotics and methods of use

A method for the preparation of a 23-oxo-LL-F28249 compound, which comprises the following steps of:

• (a) mutating a module 3 ketoreductase domain of a polyketide synthase gene cluster to render said ketoreductase domain nonfunctional wherein the polyketide synthase gene cluster is responsible for the biosynthesis of an LL-F28249 compound and wherein said module 3 ketoreductase domain either has the amino acid sequence from position 3287 through position 3466, inclusive, of SEQ ID NO:6 or has a nucleotide sequence region that hybridizes at about 42° C. in a solution containing 6×SSC and 50% formamide with a nucleic acid molecule having the nucleic acid sequence from position 29,723 to position 30,262, inclusive, of SEQ ID NO:1
• (b) transforming or transfecting an isolated prokaryotic or eukaryotic host cell with the mutated nucleic acid molecule;
• (c) growing the transformed or transfected host cell under suitable nutrient conditions in a manner allowing expression of the 23-oxo-LL-F28249 compound;
• and
• (d) recovering the 23-oxo-LL-F28249 compound.

A method for the preparation of a 23-(O-methyloxime)-LL-F28249 compound, which comprises the following steps of:

• (a) mutating a module 3 ketoreductase domain of a polyketide synthase gene cluster to render said ketoreductase domain nonfunctional wherein the polyketide synthase gene cluster is responsible for the biosynthesis of an LL-F28249 compound and wherein said module 3 ketoreductase domain either has the amino acid sequence from position 3287 through position 3466, inclusive, of SEQ ID NO:6 or has a nucleotide sequence region that hybridizes at about 42° C. in a solution containing 6×SSC and 50% formamide with a nucleic acid molecule having the nucleic acid sequence from position 29,723 to position 30,262, inclusive, of SEQ ID NO:1
• (b) transforming or transfecting an antibiotic-producing wild-type or mutant Streptomyces strain with the mutated nucleic acid molecule to replace the original module 3 ketoreductase domain with the mutated module 3 ketoreductase domain;
• (c) culturing the transformed or transfected Streptomyces strain in a suitable nutrient medium in a manner allowing production of a 23-oxo-LL-F28249 compound;
• (d) recovering the 23-oxo-LL-F28249 compound;
• (e) converting the 23-oxo-LL-F28249 compound to the 23-(O-methyloxime)-LL-F28249 compound under suitable reaction conditions;
• and
• (f) isolating the 23-(O-methyloxime)-LL-F28249 compound.

A method for the preparation of a 23-(O-methyloxime)-LL-F28249 compound, which comprises the following steps of:

• (a) mutating a module 3 ketoreductase domain of a polyketide synthase gene cluster to render said ketoreductase domain nonfunctional wherein the polyketide synthase gene cluster is responsible for the biosynthesis of an LL-F28249 compound and wherein said module 3 ketoreductase domain either has the amino acid sequence from position 3287 through position 3466, inclusive, of SEQ ID NO:6 or has a nucleotide sequence region that hybridizes at about 42° C. in a solution containing 6×SSC and 50% formamide with a nucleic acid molecule having the nucleic acid sequence from position 29,723 to position 30,262, inclusive, of SEQ ID NO:1
• (b) transforming or transfecting an isolated prokaryotic or eukaryotic host cell with the mutated nucleic acid molecule;
• (c) growing the transformed or transfected host cell under suitable nutrient conditions in a manner allowing expression of a 23-oxo-LL-F28249 compound;
• (d) recovering the 23-oxo-LL-F28249 compound;
• (e) converting the 23-oxo-LL-F28249 compound to the 23-(O-methyloxime)-LL-F28249 compound under suitable reaction conditions;
• and
• (f) isolating the 23-(O-methyloxime)-LL-F28249 compound.