Heterologous Expression Of Tautomycetin Biosynthetic Gene TtnN And Investigation Into Its Function

Tautomycetin(TMC) is a tumor proliferation resistant, anti-fungi, and immunouppressive polyketide-derived compound, acquired from fermentation of Streptomyces griseochromogene. This unique protein phosphatase 1-specific inhibitor contains a polyketide chain coupling with a rare dialkymaleic anhydride moiety(DAMA) through esterification. Over years the question that in which phase of polyketide chain biosynthesis is DAMA esterificated and coupled with it has been dramatically attractive and investigation into this enigma necessitates insights into esterification correlated enzymes and their gene maneuver.Through preliminary Blast work a gene named ttnK was identified as a single esterase encoding gene in TMC biosynthetic gene cluster. Meanwhile, a function-unknown gene ttnN close to ttnK attracted our interests and according to biochemical patterns of esterification in vivo, ttnN may cooperate with ttnK in esterification and play an important role in reaction initiating, substrate selecting or reaction equilibrium determining.In order to contribute to investigating phase of TMC esterification, ttnN, in this study, was firstly cloned and linked with expression vectors pET-32a(+) and pQE-9 and two E.coli strains expressing soluble ttnN were attained with their induction conditions optimized. To further study its function, by virtue of REDIRECT technology, through PCR amplification, electrotransformation and homologous recombination a gene-targeting vector was constructed and introduced into wild Streptomyces griseochromogenes by conjugational transfer, then gene ttnN was knocked out through replacement and recombinant strain SG3(â–³ttnN) was obtained. Fermentation experiments and HPLC assay showed that SG3(â–³ttnN) failed to produce TMC, thus it was proved that ttnN was directly correlated with TMC biosynthesis. Further by function comparison and referring to esterification mechanisms, a hypothetic theory was advanced that ttnN was involved in substrate activation by catalyzing energy-rich bond forming. All this work enriches insights into TMC esterification significantly and builds solid foundation for construction of in vitro esterification systems, which will facilitate study of TMC esterification phase and show potentials of solving it effectively at last.