Impact of thioesterase activity on tylosin biosynthesis in Streptomyces fradiae

Background: The polyketide lactone tylactone is produced in Streptomyces fr adiae by the TylG complex of five multifunctional proteins. As with other t ype I polyketide synthases, the enzyme catalysing the final elongation step (TylGV) possesses an integral thioesterase domain that is believed to be r esponsible for chain termination and ring closure to form tylactone, which is then glycosylated to yield tylosin. In common with other macrolide produ cers, S, fradiae also possesses an additional thioesterase gene (orf5) loca ted within the cluster of antibiotic biosynthetic genes. The function of th e Orf5 protein is addressed here, Results: Disruption of orf5 reduced antibiotic accumulation in S. fradiae b y at feast 85%. Under such circumstances, the strain accumulated desmycosin (demycarosyl-tylosin) due to a downstream polar effect on the expression o f orf6, which encodes a mycarose biosynthetic enzyme. High levels of desmyc osin production were restored in the disrupted strain by complementation wi th intact orf5, or with the corresponding thioesterase gene, nbmB, from S. narbonensis, but not with DNA encoding the integral thioesterase domain of TylGV. Conclusions: Polyketide metabolism in S. fradiae is strongly dependent on t he thioesterase activity encoded by orf5 (tylO). It is proposed that the Ty lG complex might operate with a significant error frequency and be prone to blockage with aberrant polyketides. A putative editing activity associated with TylO might be essential to unblock the polyketide synthase complex an d thereby promote antibiotic accumulation.