Recombinant plasmidstransformed mutants blocked in tylosin biosynthesis and complementedtylF (the structural gene for MacOMeTase) and tyl mutations of eightother classes.
fradiae and to optimize mutagenesis procedures to maximize the probability of isolating mutants with improved tylosin production or with other specific phenotypes; (b) to develop efficient recombination systems to reassort mutaÂ tions that affect antibiotic yields in additive or synergistic ways; (c) to define the pathway for tylosin biosynthesis and to determine the rate-limiting steps; (d) to define the structural organization and regulation of tylosin biosynthetic genes; and (e) to develop gene cloning methodologies to improve the levels or catalytic properties of enzymes involved in rate-limiting steps by gene amplification, promoter replacement, or site-directed mutagenesis.
Macrocin-O-methyltransferase (MacOMeTase) catalyzes the final enzymatic step in the biosynthesis of tylosin in Streptomyces fradiae. A 44-base mixed oligonucleotide probe containing only guanosine and cytidine in the third position of degenerate codons was synthesized based on the amino acid sequence of the amino terminus of MacOMeTase. Plaque blot hybridization to a bacteriophage lambda library and colony blot hybridization to a cosmid library of S. fradiae DNA identified recombinants that contained overlapping fragments of chromosomal DNA. The nucleotide sequence of the cloned DNA verified that the DNA contained the coding sequence for MacOMeTase. Recombinant plasmids transformed mutants blocked in tylosin biosynthesis and complemented tylF (the structural gene for MacOMeTase) and tyl mutations of eight other classes.
Tylosin, a macrolide antibiotic, was co-produced with four structurally similar antibiotics in fermentation cultures of Streptomyces fradiae. Macrocin, desmycosin, lactenocin, and relomycin were found to be components of a common pathway that functions in tylosin biosynthesis. Data obtained by the addition of the purified 14C-labeled antibiotics to cultures of S. fradiae revealed that macrocin and desmycosin were direct precursors of tylosin, whereas lactenocin was an immediate precursor of both macrocin and desmycosin. Incubation of these cultures with [14C]tylosin resulted in an equivalent distribution of radioactive label between relomycin and an unidentified component. The kinetics of incorporation of label into the two species were similar, suggesting that both were derived directly from tylosin. A system that supported that methylation of macrocin to tylosin by cell-free extracts of S. fradiae was developed. A proposed scheme defining the terminal stages of tylosin biosynthesis is presented.