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

在化学结构和生物学活性方面的多样性中发现了继发代谢产物的主要特征。追求了负责多样性的生物合成机制的分子解剖和应用研究。跨越大约的基因簇。 64 kbp用于克隆大花生酰胺聚酯糖苷乙二醇糖苷的生物合成并在功能上证实。最重要的发现发现，在最简单的PKS之一中，负载域是多酮启动器。最后一步的糖基转移酶VINC的功能是异源表达的，并用于制备具有Vicenisamine aminosugar的各种疏水性糖苷。在搜索PKS基因时，发现了一个额外的PKS基因簇，并在对预测的ORF的同源性搜索基础上提出了其代谢产物的部分化学结构。实际上，两种新的相关超代谢产物被分离出来，推导的化学结构完全与预测的结构相兼容，这清楚地表明了基于基因组的新代谢产物的显着潜力。含2-脱氧毒素的氨基糖苷抗生素的生物合成系统也详细研究了。对某些放线菌菌株的遗传分析表明，ORF的排列与产生键球蛋白的芽孢杆菌的排列显着差异。通过使用基于机制的抑制剂（磷酸碳葡萄糖），对2-脱氧 - 丝氨酸 - 肌瘤合酶（BTRC）进行了活跃位点映射，并对共价修饰的酶的蛋白水解消化物进行了广泛的LC-MS分析，并且是crys-141的质量反应，这是一个过度的反应。此外，BTR被证实为2-脱氧肽胺的生物合成的双功能氨基转移酶，认为BTRM被认为是一种糖合成酶会引起寄生虫，而BTRD似乎是一种新型的DTDP-glucosamine合成酶。源自链霉菌的二萜环化酶的机理由我们的方法学确定，该方法具有途径开关，高去核标记和1H-NMR光谱法。较少的

项目成果

Tadashi Eguchi et al.: "Structure Revision of FD-891, a 16-Membered Macrolide Antibiotic"J. Antibiot.. 57[2]. 156-157 (2004)

Tadashi Eguchi 等人：“16 元大环内酯类抗生素 FD-891 的结构修订”J.

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”

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)。

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)

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)