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

具有生物活性次生代谢物多样性的生物合成系统的分子分析及应用

• 批准号: 13480185
• 负责人: KAKINUMA Katsumi
• 金额: $ 9.6万
• 依托单位: Tokyo Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13480185/
• 关键词: secondary metabolites; polyketides; polyketide synthases; antitumor antibiotics; aminoglycoside antibiotics; glycosyltransferase; mechanism-based inhibitor; 2-deoxystreptamine; マクロリド; 基質特異性; 糖質環化酵素; ビセニスタチン; ラクタム配糖体; イソプレノイド; 重水素化; カナマイシン /

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

次生代谢产物的主要特征是其化学结构和生物活性的多样性。进行了负责多样性的生物合成机制的分子解剖和应用研究。基因簇跨越ca。64 kbp的大环内酰胺聚酮糖苷维西他汀的生物合成的克隆和功能证实。最重要的发现是，获得聚酮起始物的加载域是最简单的PKS之一。在最后一步起作用的糖基转移酶VinC被异源表达，并被应用于制备具有vicenisamine氨基糖的各种疏水糖苷。在PKS基因的搜索过程中，发现了一个新的PKS基因簇，并根据预测的开放阅读框进行同源性搜索，提出了其代谢产物的部分化学结构。两个新的相关代谢产物实际上是分离和推导的化学结构完全符合 ...更多信息 与预测的结构相匹配，这清楚地表明了基于基因组的新代谢物搜索的巨大潜力。对含2-脱氧链霉胺的氨基糖苷类抗生素的生物合成体系进行了详细的研究。对部分放线菌的遗传分析表明，其ORF的排列方式与产布替罗星的环状芽孢杆菌有显著差异。通过对2-脱氧-青蟹肌醇合酶（BtrC）的酶活性位点进行了定位，并对该酶的蛋白水解酶进行了LC-MS分析，确定了Lys-141在整个反应中起关键作用。此外，BtrS被证实为生物合成2-脱氧链霉胺的双功能氨基转移酶，BtrM被认为是一种糖基转移酶，产生巴龙胺，BtrD似乎是一种新的dTDP-氨基葡萄糖合酶。利用途径转换、高氘标记和1H-NMR技术对一株链霉菌二萜环化酶的作用机制进行了研究。少

项目成果

Katsumi Kakinuma: "New Approach to Multiply Deuterated Isoprenoids Using Triply Engineered Escherichia coli and Its Potential for Mechanistic Enzymology."J.Am.Chem.Soc.. 123. 1238-1239 (2001)

Katsumi Kakinuma：“使用三重工程大肠杆菌繁殖氘代类异戊二烯的新方法及其机械酶学潜力。”J.Am.Chem.Soc.. 123. 1238-1239 (2001)

Hideyuki Tamegai: "Identification of L-Glutamine : 2-Deoxy-scyllo-inosose Aminotransferase Required for the Biosynthesis of Butirosin in Bacillus circulans"J.Antibiot.. 55. 707-714 (2002)

Hideyuki Tamegai：“L-谷氨酰胺的鉴定：环状芽孢杆菌中 Butirosin 生物合成所需的 2-脱氧青蟹肌糖转氨酶”J.Antibiot.. 55. 707-714 (2002)

為我井秀行: "ものが秘める可能性-アミノグリコシド抗生物質の生合成に学ぶ"化学と生物. 41. 763-771 (2003)

Hideyuki Tamegai：“隐藏的潜力 - 从氨基糖苷类抗生素的生物合成中学习”化学与生物学 41. 763-771 (2003)。

Hiroshi Nishida et al.: "Amino Acid Starter Unit in the Biosynthesis of Macrolactam Polyketide Antitumor Antibiotic Vicenistatin"Tetrahedron. 57. 8237-8242 (2001)

Hiroshi Nishida 等人：“大环内酰胺聚酮化合物抗肿瘤抗生素维西他汀生物合成中的氨基酸起始单元”四面体。

Hideynki Tamegal et al.: "First Identification of Streptomyces genes involved in the Biosynthesis of 2-Deoxystreptamine-containing Aminoglycoside Antibiotics. Genetic and Evolutionary Analysis of L-Glutamine :2-deoxy-scyllo-inosose Aminotransferase Genes"

Hideynki Tamegal 等人：“首次鉴定参与含 2-脱氧链霉胺氨基糖苷类抗生素生物合成的链霉菌基因。L-谷氨酰胺：2-脱氧青蟹肌糖氨基转移酶基因的遗传和进化分析”