Functional Analysis of Biosynthetic Enzymes for Natural Products

天然产物生物合成酶的功能分析

• 批准号: 12045213
• 负责人: EBIZUKA Yutaka
• 金额: $ 11.26万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12045213/
• 关键词: biosynthetic enzyme; biosynthetic gene; functional expression; polyketide; polyketide synthase; filamentous fungi; triterpene; triterpene synthase; カルコン; スチルベン; オキシドスクワレン; サポニン; ポロケタイド; 生合成; 酵素; メラニン; 分生子; ソラナピロン; Aspergillno fumigatuo /

Diverse structures of natural products are elaborated by biosynthetic enzymes, and those enzymes which work at the most upstream of biosynthetic pathways are thus regarded as the primary origin of structural diversty exhibited by natural products.Our attentions have been focused on these biosynthetic enzymes for future engineering of rationally designed biosynthetic machineries for production of new chemicals. In this study, gene cloning and functional analysis of some of these enzymes have been attempted.Continuous attempts for cloning of new polyketide synthases from filamentous fungi yielded several new clones.Enzyme functions of these clones were analyzed by heterologous expression in fungi and/or yeast. Construction of chimeric enzymes and site-directed mutants of these clones identified a new functional domain for aromatic pokyketide production.Over thirty triterpene synthase cDNAs have been cloned from various triterpene producing plants by homology based PCR methods.Their enzyme functions have been identified by heterologous expression in yeast.Functional analysis of the eight out of thirteen Arabidopsis thaliana triterpene synthase clones was attempted.Only two clones are product specific synthases for known triterpenes, while the others are multifunctional clones.In this four-year project, functional analysis of biosynthetic enzymes for polyketides and triterpenoids was extensively conducted.The results obtained in this study clearly show that functional genomics approach would provide a new methodology for the search of new natural products.Furthermore, by employing recombinant technology of these biosynthetic enzymes, development of practical production system for useful chemicals and construction of artificial chemical library comprising of "un-natural natural products" with diverse structures wouldbe expected.

天然产物的多样性结构是由生物合成酶精心设计的，因此，那些在生物合成途径最上游起作用的酶被认为是天然产物所表现出的结构多样性的主要来源。我们的注意力一直集中在这些生物合成酶上，以用于未来合理设计生物合成机械以生产新化学品的工程。在本研究中，尝试了其中一些酶的基因克隆和功能分析。不断尝试从丝状真菌中克隆新的聚酮合酶，产生了几个新的克隆。通过在真菌和/或酵母中的异源表达来分析这些克隆的酶功能。这些克隆的嵌合酶和定点突变体的构建鉴定了用于芳香聚酮化合物生产的新功能结构域。通过基于同源性的PCR方法从各种三萜生产植物中克隆了超过30个三萜合酶cDNA。通过在酵母中的异源表达鉴定了它们的酶功能。对13种拟南芥中的8种进行了功能分析 尝试了三萜合酶克隆。只有两个克隆是已知三萜的产物特异性合酶，而其他克隆都是多功能克隆。在这个为期四年的项目中，广泛进行了聚酮化合物和三萜类生物合成酶的功能分析。本研究获得的结果清楚地表明，功能基因组学方法将为寻找新的天然产物提供新的方法。 利用这些生物合成酶的重组技术，有望开发有用化学品的实用生产系统，并构建由具有多种结构的“非天然天然产物”组成的人工化学库。

项目成果

Miho Izumikawa, Michio Murata, Kazuo Tachibana, Yutaka Ebizuka, Isao Fujii.: "Cloning of Modular Type I Polyketide Synthase Genes from Salinomycin Producing Strain of Streptomyces albus."Bioorg. Med. Chem.. 11. 3401-3405 (2003)

Miho Izumikawa、Michio Murata、Kazuo Tachibana、Yutaka Ebizuka、Isao Fujii.：“从产盐霉素的白色链霉菌菌株中克隆模块化 I 型聚酮合酶基因。”Bioorg。

I.Fujii: "Enzymatic Synthesis of 1,3,6,8-Tetrahydroxynaphthalene solely from Maloxyl Coenzyme A by a Fungal Iterative Type I polykatide synthase PKSI."Biochemistry. 39. 8853-8858 (2000)

I.Fujii：“通过真菌迭代 I 型聚肽合酶 PKSI 仅从丙二酰辅酶 A 酶促合成 1,3,6,8-四羟基萘。”生物化学。

Akira Watanabe, Yutaka Ebizuka.: "A Novel Hexaketide Naphthalene Synthesized by a Chimeric Polyketide Synthase Composed of Fungal Pentaketide and Heptaketide Synthases."Tetrahedron Lett.. 43. 369-374 (2002)

Akira Watanabe、Yutaka Ebizuka.：“由真菌五肽和七肽合成酶组成的嵌合聚酮化合物合成酶合成的新型六酮化合物萘。”Tetrahedron Lett.. 43. 369-374 (2002)

Hiroaki Hayashi, Pengu Huang, Keiichiro Inoue, Noboru Hiraoka, Yasumasa Ikeshiro, Kazufumi Yazaki, Shige Tanaka, Tetsuo Kushiro, Masaaki Shibuya, Yutaka Ebizuka.: "Molecular Cloning and Characterization of Isomultiflorenol Synthase, a New Triterpene Synth

Hiroaki Hayashi、Pengu Huang、Keiichiro Inoue、Noboru Hiraoka、Yasumasa Ikeshiro、Kazufumi Yazaki、Shige Tanaka、Tetsuro Kushiro、Masaaki Shibuya、Yutaka Ebizuka。：“异多花烯醇合成酶（一种新的三萜合成物）的分子克隆和表征

Yutaka Ebizuka, Yuji Katsube, Takehiko Tsutsumi, Tetsuo Kushiro, Masaaki Shibuya: "Functional Genomics Approach to the Study of Triterpene Biosynthesis."Pure Appl. Chem.. 75. 369-374 (2003)

Yutaka Ebizuka、Yuji Katsube、Takehiko Tsutsumi、Tetsuro Kushiro、Masaaki Shibuya：“三萜生物合成研究的功能基因组学方法”。