Identification and characterization of wblA-dependent tmcT regulation during tautomycetin biosynthesis in Streptomyces sp. CK4412.

Tautomycetin (TMC) is an unusual linear polyketide compound esterified with a cyclic anhydride. It exhibits novel activated T cell-specific immunosuppressant as well as anti-cancer activities. Previously, we isolated and characterized the entire TMC biosynthetic gene cluster from Streptomyces sp. CK4412, including a TMC pathway-specific gene, tmcN, the over-expression of which led to a significant increase in TMC productivity. In addition, we also reported that WblA acts as a global down-regulator of antibiotic biosynthesis through pathway-specific regulation in Streptomyces species. Here, we confirm that TmcT acts as another TMC pathway-specific regulator within the TMC biosynthetic cluster. Specifically, tmcT deletion resulted in the complete loss of TMC production, whereas complementation with a tmcT-carrying integrative plasmid significantly restored TMC biosynthesis. We also identified a 0.39kb wblA ortholog (named wblA(tmc)) from Streptomyces sp. CK4412 via genomic DNA library screening that showed 96% amino acid identity compared to a previously-known S. coelicolor wblA. Targeted gene disruption of wblA(tmc) in Streptomyces sp. CK4412 exhibited approximately 3-fold higher TMC productivity than that in the wild-type strain. Moreover, transcription analyses of the TMC biosynthetic and regulatory genes revealed that the expression of tmcT was strongly down-regulated by wblA(tmc). These results imply that the TMC biosynthetic regulation network is controlled by two pathway-specific positive regulator, WblA(tmc)-dependent TmcT as well as WblA(tmc)-independent TmcN in Streptomyces sp. CK4412.