Biosynthetic Studies Of Safracin And Naphthyridinomycin That Belong To The Tetrahydroisoquinoline Alkaloids Family

Three topics are discussed in this dissertation, including (1) identifing the upstream boundary of the biosynthetic gene cluster of naphthyridinomycin by using PCR-targeting method.(2) confirming that NptM2was involved in the biosynthesis of naphthyridinomycin by in vivo gene knockout and homologous complementation.(3) biochemical characterization of two methyltransferase, SfcG and SfcF, involved in the biosynthesis of safracin.Tetrahydroisoquinoline family alkaloids are a class of novel antibiotics with special structures and high biological activity. Especially Ecteinascidin743(ET-743), isolated from marine Ecteinascidia turbinate, is an alkaloid with a core skeleton similar to that of safracin (SAC) and naphthyridinomycin, possessing the high antitumor activity as fifty times as that of taxol (LC50<1pM, ID500.2-0.6nM in vitro). ET-743(Yondelis) was approved by the EMEA in September2007for the treatment of advanced soft tissue sarcoma, and is in Phase III clinical trials for the treatment of ovarian cancer (with J&J in the US) and other ongoing Phase II trials include paediatric sarcomas, breast and prostate cancers. As a marine natural product, the scarcity of ET-743may limit the practical value of preparing it as a low cost drug.To elucidate the functions of some hypothetic genes in naphthyridinomycin biosynthesis, double exchanged mutants of these genes are constructed by using PCR-targeting method. And through this method the upstream boundary of biosynthetic gene cluster of naphthyridinomycin was identified. In the biosynthetic studies of SAC-B, Velasco et al had proved the biosynthetic pathway of the3h5mOmTyr which is a biosynthetic precursor of SAC-B through heterologous expression. In1986, Steven J. Gould et al confirmed that both13C-labeled3m-Tyr and5m-Dopa could be incorporated into the biosynthesis of naphthyridinomycin, while Dopa couldn’t be incorporated, in labeled precursor feeding experiments. These data suggested that a common biosynthetic precursor maybe involved in both the biosynthetic pathway of naphthyridinomycin and safracin, which maybe3h5mOmTyr and had a similar biosynthetic pathway as confirmed in the biosynthesis of safracin. And in the biosynthetic gene cluster of naphthyridinomycin the homologous genes that resposible for the biosynthesis of3h5mOmTyr as listed in the gene clusters of SFM-A and SAC-B were also found. And one of them is NptM2, which is a SAM depedent methyltransferase. To confirm the putative function of NptM2in the biosynthesis of the precursor of naphthyridinomycin, the gene knock-out and homologous complementation experiments with NptM2in the producing strain of naphthyridinomycin were done. And the results confirmed that NptM2is involved in the biosynthesis of naphthyridinomycin.What’s more, the two methylation steps in the biosynthesis of safracin through the expression and in vitro biochemical characterization of two methyltransferases were elucidated, SfcF and SfcG. And the kinetic parameters of SfcF, the carbon-methyltransferase were also detected.Through our studies described above, the appropriate mechanism for the biosynthesis of the target molecule more clear and precise can be clarified, so as to provide a theoretical basis and practical guidance for the metabolic engineering of tetrahydroisoquinoline family alkaloids.