Functional Characterization Of Several Genes In The Biosynthesis Of Tautomycetin

Tautomycetin (TMC) is a polyketide that was first isolated from the Streptomyces griseochromogenes (S. griseochromogenes). TMC is a dialkylmaleic anhydride antibiotic with an unique chemical structure of an ester bond linkage between a dialkylmaleic anhydride (DA) moiety and a linear polyketide chain. Recently it was discovered that TMC is a specific protein phosphatase I inhibitor (PP1), with antifungi, antitumor and immunosuppressive activities. The unique chemical structure and important activities of TMC draw strong attention to the biosynthesis mechanism of TMC, which is not only critical for development of novel TMC analogs through combinatorial biosynthesis, semi-synthesis or heterologous expression, but also will contribute to a better understanding of the chemistry biology of polyketide synthase (PKS).The biosynthesis gene cluster of TMC has been cloned and fully sequenced. By analyzing the sequence of the gene cluster of TMC, it was determined that the polyketide backbone of TMC is synthesized by type I PKS. However, the mechanisms underlying the biosynthesis of DA, post-PKS tailoring and the formation of ester bond linkage between DA and the linear polyketide moiety have not been verified.In this thesis, an intergeneric genetic transfer system for S. griseochromogenes was first established and optimized, and we demonstrated that Ca2+and especially glycin, could increase the efficiency of exconjugates, which may be very helpful for genetic manipulation of Streptomyces.To elucidate the biosynthesis of dialkylmaleic anhydride, the two candidate genes ttnK and ttnO, which encode a carboxyl-esterase (TtnK) and a citryl-CoA lyase (TtnO) respectively, in the TMC biosynthesis gene cluster have been deleted and complemented. The results indicated that TtnO was involved in the biosynthesis of DA, whereas the mutant△ttnK accumulated dialkylmaleic anhydride, indicating a critical role for TtnK in the enzyme-dependent events related to incorporation of DA with the polyketide moiety.To determine the post-PKS modification mechanism, ttnF, ttnC, ttnD and ttnl genes were inactivated. These genes putatively encode a L-carnitine dehydratase, a flavoprotein decarboxylase, an UbiD family decarboxylase and a P450oxidase enzymes, respectively. The mutant△ttnF accumulatetd TMC-F1as a dominant metabolite. Compared to TMC, TMC-F1lacks the C5-ketone and the terminal alkene of TMC, but with a3-β-hydroxy-propionyloxyl group. The result indicated that the TtnF is directly responsible for the dehydration of the polyketide, the first post-PKS tailoring step. The inactivation of ttnC, resulted in the production of TMC at the wild-type levels, whereas the inactivation of ttnD resulted in the accumulation of TMC-D1as a dominant metabolite. Compared to TMC-F1which has a3-β-hydroxypropanoic acid moiety, TMC-D1had an acrylic acid moiety attached to C3.The results identified TtnF and TtnD as the enzymes to form the conjugated double bonds to install the carboxyl-diene, and TtnD as the decarboxylase for the post-PKS modification steps in TMC biosynthesis, whereas the function of TtnC remains unknown. The disruption of ttnl led to the accumulation of5-des-keto-tautomycetin, revealing that TtnI was responsible for the oxidation at C5as the last step of TMC biosynthesis.Notably, biotransformation experiments in mutants△ttnO and△ttnl suggested that the C5-hydroxyl in TMC analogs was transformed to C5-ketone via Oxidation by TtnI. Thus, there is a considerable degree of promiscuity of some of the post-PKS modification enzymes in the biosynthesis of TMC in S. griseochromogenes.In conclusion, this thesis provided novel insights into the mechanism of the biosynthesis of TMC and the basis for developing new analogs of TMC-based small molecule drug candidates by combinatorial biosynthesis.