Isolation And Elucidation Of Natural Products And Study Of Enzymatic Reaction Guided By Biosynthesis

Microbial genome sequencing is an important part of microbial genomics study. Through bioinformatics analysis of biosynthetic gene clusters combined with the knowledge about natural product biosynthetic pathways which were well understood, it has found that the number of compounds combined from the majority of Microorganism far more than the current number of compounds have been isolated and identified through fermentation. Based on the study of Biosynthesis mechanism of compounds produced by wide-type Bacterial,we firstly predict structure of new compounds may produced by mutants;Then, by analyzing the relative molecular weight and the UV characteristic absorption of each component in fermentation broth of mutants useful new compounds were detected;Finally, new compounds were isolated, purified and elucidation. The efficiency of discover new compounds can be greatly imporved and the structure information of these compounds are great significant to evaluate biosynthetic mechinery and biosynthetic pathway of its Family.Three parts are discussed in this paper, including1) Isolation and elucidation of analogues of Yatakemycin;2)Isolation and structure identification of biosynthesis intermediates involved in Kosinostatin;3)The synthesis of nicotinic acid labeled by isotopes through biotransformation in vitro.Yatakemycin is a kind of natural product isolated from Streptomyces sp.TP-A0365by Igarashi in2003with high antifungal and antitumor activity,and it is a new member of the DNA alkylation agent family including CC-1065, Duocarmycin A, and Duocarmycin SA. Structurally, it is made of pyrroloindoles and indole connected by two amide bonds. The most notable structural feature of this molecule is the cyclopropane that controls its reaction regioselectivity. The mutants of Streptomyces sp.TP-A0365were fermented and the contents of fermentation broth were analyzed by HPLC and LC-MS.When the analysis results were compared with the wide-type, we found two new compounds in fermentation broth of mutant YT-Tyr.The new compounds were obtained through repeated column chromatography (CC) on silica gel, Sephadex LH-20and semipreparative high performance liquid chromatography (SP-HPLC).The structures of these compounds were elucidated by means of spectroscopic methods including IR, UV, ESI-MS, HR-ESI-MS/MS,1D-NMR (’H-NMR,13C-NMR and DEPT) and2D-NMR (’H-’H COSY, HMBC, HMQC and NOESY). The skeleton of these new compounds were similar to Yatakemycin, which belongs to pyrroloindole heterocyclic compounds.Kosinostatin, an anthracycline antitumor antibiotics isolated from a marine bacteria Micromonospora. sp. A1304by Japanese scientists in2002, shows higher antitumor activity (ICso^0.10μM) than the widely used anticacer drug Adriamycin, and its chemical structual is very special. In our study, The mutants of Micromonospora.sp.Al3O4were fermented and the biosynthesis intermediates in fermentation broth were isolated through repeated column chromatography (CC) on silica gel, Sephadex LH-20and semipreparative high performance liquid chromatography (SP-HPLC).The structures of these compounds were elucidated by means of spectroscopic methods.Nicotinic acid is not only one essential vitamin but also an direct precursor of important coenzymes NAD and NADP in all living systems; Nicotinic acid labed by isotopes play an important role in study the biosynthetic pathways of natural products and the metabolism of matters. L-aspartic acid can be transformed into Nicotinic acid at the catalysis of aspartate oxidase(NadB), nicotinic acid synthase (NadA), quinolinic acid phosphoribosyl transferase (NadC), nicotinic acid phosphoribosyl transferase(PncB) in Escherichia coli. In this study, we planed to get nicotinic acid labeled by isotopes from labeled aspartic acid by biocatalysis of enzymes from E.coli in vitro. The genes, which encoding enzymes required in the biosynthesis of nicotinic acid, were cloned using E. coli DNA as template and then ligated into vector to generate expression plasmids. After sequenced, the expression plasmids were transformed into E. coli BL21(DE3) to express and then target enzymes were purified. We have successfully detected the activity of quinolinic acid phosphoribosyl transferase (NadC), nicotinic acid phosphoribosyl transferase (PncB) and detection of other enzymes activity were in progress.