Molecular Analysis and Application of Biosynthetic Systems Featuring the Diversity of Biologically Active Secondary Metabolites

次生代谢物的主要特征是其化学结构和生物活性的多样性。对造成生物多样性的生物合成机制进行了分子解剖和应用研究。克隆了长约64 kbp的巨内酰胺聚酮苷维森司他汀生物合成基因簇，并进行了功能鉴定。最重要的发现是，获得聚酮起始物的负载域是最简单的pks之一。异种表达了最后一步功能的糖基转移酶VinC，并将其应用于制备具有维森胺氨基糖的各种疏水糖苷。在搜索PKS基因的过程中，发现了一个额外的PKS基因簇，并在预测orf的同源性搜索的基础上提出了其代谢物的部分化学结构。事实上，两个新的相关代谢物被分离出来，并且推导出的化学结构与预测的结构完全吻合，这清楚地表明了基于基因组的新代谢物搜索的巨大潜力。并对含2-脱氧链胺氨基糖苷类抗生素的生物合成体系进行了详细的研究。一些放线菌菌株的遗传分析表明，orf的排列与产丁丁香的环状芽孢杆菌有显著差异。通过使用基于机制的抑制剂（磷酸葡萄糖）和广泛的LC-MS分析共价修饰酶的蛋白水解酶，对2-脱氧-酰基-肌糖合成酶（BtrC）进行了活性位点定位，并阐明了Lys-141是整个反应的关键碱基。此外，BtrS被证实是生物合成2-脱氧链胺的双功能转氨酶，BtrM被认为是产生paromamine的糖合转移酶，BtrD被认为是一种新的dtdp -葡萄糖胺合酶。我们的方法采用通路开关、高氘标记和1H-NMR光谱精确地确定了链霉菌中二萜环化酶的机制。少

Major feature of secondary metabolites is found in their diversity in chemical structure and biological activity. Molecular dissection and application studies of the biosynthetic machinery that is responsible for the diversity were pursued. The gene cluster spanning ca. 64 kbp for the biosynthesis of macrolactam polyketide glycoside vicenistatin was cloned and functionally confirmed. The most significant finding was found that the loading domain acquiring the polyketide starter is among the simplest PKSs. The glycosyltransferase VinC functioning at the final step was heterologously expressed and was applied for the preparation of various hydrophobic glycosides having the vicenisamine aminosugar. During the search of PKS genes, an additional PKS gene cluster was found and a partial chemical structure of its metabolite was proposed on the bases of homology search of the predicted ORFs. Two new related-metabolites were in fact isolated and the deduced chemical structures were completely c … More ompatible to the predicted structure, that clearly demonstrated significant potential of the genome-based search for new metabolites. The biosynthetic systems of the 2-deoxystreptamine-containing aminoglycoside antibiotics were also studied in detail. Genetic analyses of some Actinomycetes strains showed significant difference in the arrangement of ORFs from that of butirosin-producing Bacillus circulans. Active-site mapping was carried out for 2-deoxy-scyllo-inosose synthase (BtrC) by the use of mechanism-based inhibitor (carbaglucose phosphate) and extensive LC-MS analysis of proteolytic digests of the covalently-modified enzyme, and Lys-141 was elucidated to be a crucial base in the over-all reaction. Further, BtrS was confirmed as the doubly-functional aminotransferase for the biosynthesis of 2-deoxystreptamine, BtrM was proposed to be a glycosyntransferase giving rise to paromamine, and BtrD appeared to be a novel dTDP-glucosamine synthase. The mechanism of a diterpene cyclase derived from a Streptomyces was precisely determined by our methodology featuring pathway switch, hyperdeuterium labeling and 1H-NMR spectroscopy. Less