The Basic Research for the Production of New Triterpene Skeletons by Extensive Use of the Technique of Molecular Biology

广泛运用分子生物学技术生产新三萜骨架的基础研究

• 批准号: 12480171
• 负责人: SHIBUYA Masaaki
• 金额: $ 9.47万
• 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 (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12480171/
• 关键词: triterpene; biosynthesis; β-amyrin; α-amyrin; cucurbitadienol; isomultiflorenol; lupeol; イソマルチフレノール

Cyclization of oxidosqualene into tetra- and pentacyclic carbon skeleton of sterols and triterpenes, catalyzed by oxidosqualene cyclases (OSCs), is one of the most complex and fascinating reactions found in nature. OSCs generate multiple stereogenic centers in a single reaction, and are responsible for the diverse triterpene skeletons. In order to investigate the origin of structural diversity of triterpene skeletons, cDNA cloning of OSCs and analysis of their product specificity were carried out. From triterpene producing plants, over twenty-five OSC clones were obtained, and their enzyme function established by expression in yeast. They included cycloartenol, cucurbitadienol, lupeol, β-amyrin, isomultiflorenol and mixed amyrin synthases. Studies of chimeric proteins between β-amyrin synthase and lupeol synthase, and mutant proeins constructed by site directed mutagenesis identified the amino acid residues responsible for their product specificity. Trp259 of β-amyrin synthase (PNY) was identified to be the critical residue controlling β-amyrin formation. In further mutation studies, PNYY261H mutant produced dammara-18,21-dien-3β-o1 (as a 3:5 mixture of E/Z isomer at Δ^<18>) together with minor amount of dammara-18(28),21-dien-3β-o1. These triterpenes have not been reported from nature, and therefore, could be categorized as "unnatural" natural products. The results of this study opened up the possibility of generating new triterpene synthases with additional novel functions through point mutations.

氧化角鲨烯环化酶（oxidosqualene cyclases，OSC）催化氧化角鲨烯环化为甾醇和三萜的四环和五环碳骨架，是自然界中最复杂、最吸引人的反应之一。OSC在一个反应中产生多个立体中心，并负责不同的三萜骨架。为了探讨三萜骨架结构多样性的来源，进行了OSCs的cDNA克隆和产物特异性分析。从生产三萜的植物中，获得了超过25个OSC克隆，并通过在酵母中表达建立了它们的酶功能。它们包括环阿屯醇、葫芦二烯醇、羽扇豆醇、β-香树脂醇、异多花烯醇和混合香树脂醇脱氢酶。对β-香树脂素合酶和羽扇豆醇合酶之间的嵌合蛋白以及通过定点诱变构建的突变蛋白的研究鉴定了负责其产物特异性的氨基酸残基。β-香树脂素合成酶（β-amyrin synthase，PNY）的Trp 259是控制β-香树脂素合成的关键残基。在进一步的突变研究中，PNYY 261 H突变体产生达玛拉-18，21-二烯-3 β-α 1（作为在Δ^处的E/Z异构体的3：5混合物<18>）以及少量的达玛拉-18（28），21-二烯-3 β-α 1。这些三萜类化合物在自然界中未见报道，因此可以归类为“非天然”的天然产物。这项研究的结果开辟了通过点突变产生具有额外新功能的新三萜类糖苷酶的可能性。

项目成果

Hiroaki Hayashi, et al.: "Molecular Cloning and Characterization of Isomultiflorenol Synthase, a New Triterpene Synthase"Eur. J. Biochem.. 268. 6311-6317 (2001)

Hiroaki Hayashi 等人：“异多花烯醇合酶（一种新的三萜合酶）的分子克隆和表征”Eur。

合田幸広, 他: "遺伝子組換えパパイヤからの組み換え遺伝子の検知"食品衛生学雑誌. 42. 231-236 (2001)

Yukihiro Goda 等人：“转基因木瓜重组基因的检测”《食品卫生杂志》42. 231-236 (2001)。

Hiroaki Hayashi, Pengyu Huang, Kenichiro Inoue, Noboru Hiraoka, Yasumasa Ikeshiro, Kazufumi Yazaki, Shigeo Tanaka, Tetsuo Kushiro, Masaaki Shibuya, Yutaka Ebizuka: "Molecular Cloning and Characterization of Isomultiflorenol Synthase, a New Triterpene synt

Hiroaki Hayashi、Pengyu Huang、Kenichiro Inoue、Noboru Hiraoka、Yasumasa Ikeshiro、Kazufumi Yazaki、Shigeo Tanaka、Tetsuo Kushiro、Masaaki Shibuya、Yutaka Ebizuka：“异多花酚合成酶（一种新的三萜合成酶）的分子克隆和表征

渋谷 雅明: "高等植物におけるステロール及びトリテルペンの生合成研究"Natural Medicines. 55. 1-6 (2001)

涉谷正明：《高等植物甾醇和三萜生物合成的研究》《天然药物》55. 1-6 (2001)。

Hiroaki Hayashi, Pengyu Huang, Ara Kirakosyan, Kenichiro Inoue, Noboru Hiraoka, Yasumasa Ikeshiro, Tetsuo Kushiro, Masaaki Shibuya and Yutaka Ebizuka: "Cloning and Characterization of a cDNA Encoding β-Amyrin Synthase Involved in Glycyrrhizin and Soyasapo

Hiroaki Hayashi、Pengyu Huang、Ara Kirakosyan、Kenichiro Inoue、Noboru Hiraoka、Yasumasa Ikeshiro、Tetsuo Kushiro、Masaaki Shibuya 和 Yutaka Ebizuka：“编码参与甘草酸和 Soyasapo 的 β-香树脂素合成酶的 cDNA 的克隆和表征