The use of recombinant DNA techniques to study tylosin biosynthesis and resistance in Streptomyces fradiae

A substantial amount of information on the biosynthesis of tylosin has been obtained over the past ten years. Physiological studies and experiments with tylosin-blocked (tyl) mutants have suggested the probable pathway by which tylactone is converted to tylosin. The development of recombinant DNA methodology for streptomycetes in general, and for Streptomyces fradiae in particular, has allowed us to apply gene cloning techniques in further studies of tylosin biosynthesis in S. fradiae. The macrocin O-methyltransferase (MOMT), which catalyzes the last step in tylosin biosynthesis, was purified, and the sequence of the 35 amino acids at its amino-terminus was determined. A synthetic 44 base oligonucleotide probe was constructed on the basis of the amino acid sequence. The probe was used to identify sequences containing the MOMT structural gene in bacteriophage and cosmid libraries of S. fradiae DNA. Complementation of tyl mutants with the cloned DNA sequences identified nine tyl biosynthetic genes (tylC, D, E, F, H, J, K, L, and M) in a 42 kb stretch of DNA. Genes complementing four mutant classes, tylA, B, G, and I were not found. A tylosin-resistance gene, tlrB, was located just left of the tyl gene cluster. Tylosin-sensitive mutants of S. fradiae, which were isolated from regenerated protoplasts and which have pleiotropic deficiencies in tylosin biosynthesis, contained deletions which included at least some of the identified tyl loci and one or both of two tylosin-resistance genes, tlrB and tlrC. Possible schemes for the functional organization of the tyl region of the S. fradiae genome are discussed.