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Isolation, Structure Identification of Novel Tautomycetin Analogues, and Preliminary Study on Biosynthesis Mechanism

Author: LiuBo
Tutor: TangLi
School: Dalian University of Technology
Course: Biochemical Engineering
Keywords: Tautomycetin Isolation Purification Structure Identification Biosynthesis
CLC: R284
Type: Master's thesis
Year: 2011
Downloads: 38
Quote: 2
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Abstract


Natural products have provided considerable value to the pharmaceutical industry over the past half century, and continue to provide significant value in the discovery of novel chemical structures and bioactive lead molecules for clinical development. Polyketides mostly produced by microorganisms are a remarkable class of compounds, which exhibit a staggering range of functional and structural diversity, and boast a wealth of medicinally important activities. Besides, their unque biosynthesis mechanism makes them valuable in combinatorial biosynthesis.Tautomycetin(TMC), which was isolated from Streptomyces griseochromogennes in 1989, is a highly specific inhibitor of protein phosphatase I (PP1) and possesses anti-fungal and immunosuppressive activities. Thus, TMC is not only an interesting drug lead in antitumor drugs and immunosuppressive drugs development, but also a powerful biochemical tool in elucidation the roles of PP1 in various biological pathways. In addition, the unique structure of TMC featuring a rare dialkymalic anhydride moiety (DAM) and a polyketide chain draws attention to its biosynthetic chemistry. The tau biosynthetic gene cluster for TMC has been cloned and sequenced, and on the basis of functional assignments for each gene in the tau cluster from sequence analysis and feeding experiments, a model for TMC biosynthesis has been proposed.In this thesis, novel TMC analogues were isolated from the recombinant mutant strain STQ1306 and STQ1010, and their biosynthsis mechanism was studied by biotransformation experiment.Using RP-C18 column chromatography and semi-preparative HPLC,3 compounds (TMC-Ds, compounds 7-9) from strain STQ1306 and 5 compounds (TMC-Ks, compounds 10-14) from strain STQ1010 were obtained, and their structures were confirmed by UV, MS, 1D-NMR and 2D-NMR.On the basis of TMC-Ks (compounds 10-14), we proposed that there is non-specific oxidase involving the hydroxylation of C-5 and C-7 beyond the cloned gene cluster of TMC. And we proposed that the acyltransferase(AT) domains of module 8 in polyketide synthase TauB has relaxed substrate specificity, so it could accept either ethylmalonyl CoA, or malonyl CoA and methylmalonyl CoA as extender unit. The components of medium on the production of TMC-Ds by strain STQ1306 were studied, and it was found that CaCO3 affects production of TMC-Ds by affecting pH.Based on the results of the biotransformation experiments, we proposed that the decarboxylase TauD and P450 oxidase TauK have relaxed substrate specificity, and they could catalyze a variety of compounds. We proposed that TMC-D2 could be decarboxylated by TauD to produce TMC, TMC-D3 and TMC-D4 could be oxidated by TauK and decarboxylated by TauD to produce TMC. Besides, TMC-D3 and TMC-D4 also may only be decarboxylated by TauD to produce TMC-K6 and TMC-K5 respectively. Therefore, in addition to the main pathway of the biosynthesis of TMC, there are a number of shunt ways, which is also one of the reasons that there are more intermediate analogues of TMC.On the basis of the results in this thesis, the biosynthsis mechanism of TMC and its analogues was further studied, on the other side various compounds were provided for its structure activity relationships study.

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