Functional Characterization of Novel Tautomycetin Analogues through tmcG or tmcR Gene Disruption in Streptomyces CK4412

초록

Tautomycetin (TMC), which is produced by Streptomyces sp. CK4412, is a novel activated T cell-specific immunosuppressivea and anti-cancer, anti-fungal compound with an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. According to recent studies, TMC blocks T cell receptor-mediated Src homology-2 domain phosphatase(SHP2), which is a positive transducer of growth facter and cytokine signalingb. Previously, we isolated and characterized the entire TMC biosynthetic gene cluster from Streptomyces sp.CK4412c. Within the TMC biosynthetic gene cluster, we identified two putative genes, tmcG and tmcR encoding TMC post-PKS tailoring enzymes through in silico sequence comparison. TmcG and tmcR were proposed as a TMC 3'-hydroxyase, and 5-oxygenase, respectively. Here we disrupted and complemented each gene to increase our understanding of its biosynthetic function. We found that these mutant strains produced regio-specifically modified TMC analogues, whose structures were confirmed through HPLC, MS, and NMR analyses. We further purified each modified compound to determine its biological activity. Remarkably, 5-deoxidized TMC in tmcR mutant strain maintains significant level of protein phosphatase inhibition and anti-fungal activities, even though it failed to show the existing anti-cancer activity. The more detailed results will be further discussed.