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Engineering of Triterpene Biosynthesis to Develop a Production System for Useful Compounds

三萜生物合成工程开发有用化合物的生产系统

基本信息

批准号：
14380285
负责人：
SHIBUYA Masaaki
金额：
$ 10.18万
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：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

cDNA cloning of triterpene synthases was carried out from triterpene producing plants. As a result, a new clone was cloned from a root of dandelion (Taraxacum officinale) which was identified to be achilleol synthase by its expression in yeast. Amino acid sequence of achilleol synthase has a high homology to those of beta-amyrin synthases, and this clone has joined to the branch of beta-amyrin synthases in phylogenetic analysis. It is very interesting that this clongives a mono-cyclic products instead of penta-cyclic product such as beta-amyrin. This clone has a glycine residue at the position of two amino acid upstream from DCTAE motif which is found in all oxidosqualene cyclase although all of beta-amyrin synthasq as valine residue at this position. When this glycine residue in achilleol synthase was changed to valine residue by site-direct mutagenesis, product was changed to beta-amyrin, suggesting that achilleol synthase was evolved from beta-amyrin synthase. The approach of this study is considered to be a new approach for isolation of a new compound by functional genomics since achilleol had not been isolated from dandelion itself although it was isolated from the other composite plant.cDNA cloning of triterpene hydroxylase as also carried out from soybean (Glycine max) which produced soyasaponins. More than two hundreds kinds of P450 EST clone from soybean were registered in data base. Among them, the clones (6 clones) which cover full-length region and function was not identified were cloned from soybean seedlings and expressed in yeast. CYP93E1, one of tested clones, showed 24-hydroxylation activity against beta-amyrin.

从产生三萜的植物中进行三萜脱氢酶的cDNA克隆。结果，从蒲公英（Taraxacum officinale）的根中克隆了一个新的克隆，该克隆通过在酵母中的表达而被鉴定为achilleol synthase。该基因的氨基酸序列与β-香树脂醇脱氢酶的氨基酸序列具有很高的同源性，在系统发育分析中，该克隆属于β-香树脂醇脱氢酶的分支。非常有趣的是，该克隆产生单环产物而不是五环产物，如β-香树脂素。该克隆在DCTAE基序上游两个氨基酸的位置处具有甘氨酸残基，其存在于所有氧化角鲨烯环化酶中，尽管所有β-香树脂蛋白在该位置处均为缬氨酸残基。当通过定点突变将该甘氨酸残基变为缬氨酸残基时，产物变为β-香树脂素，表明该Achilleol合酶是由β-香树脂素合酶进化而来的。本研究的方法被认为是一个新的方法分离一个新的化合物的功能基因组学，因为它没有被分离出的阿齐叶醇本身，但它是从其他复合植物分离。三萜羟化酶的cDNA克隆也进行了从大豆（Glycine max），产生大豆皂甙。在数据库中登记了200多个大豆P450 EST克隆。其中，6个克隆覆盖了全长区域，功能尚未鉴定，是从大豆幼苗中克隆的，并在酵母中表达。CYP 93 E1是一个被测的克隆，对β-香树素具有24-羟基化活性。

项目成果

期刊论文数量（21）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Shui-Qin Wu, Naohiro Watanabe, Storu Mita, Yoshihiro Ueda, Masaaki Shibuya, Yutaka Ebizuka: "Two O-Methyltransferases Isolated from Flower Petals of Rosa chinensis var. spontanea Involved in Scent Biosynthesis"J.Bioscience and Bioengineering. 96. 119-128

Shui-Qin Wu、Naohiro Watanabe、Storu Mita、Yoshihiro Ueda、Masaaki Shibuya、Yutaka Ebizuka：“从玫瑰花瓣中分离出的两种 O-甲基转移酶参与气味生物合成”J.Bioscience and Bioengineering。

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Masaaki Shibuya: "Structural Diversity and Molecular Origin of Triterpene Saponins"YAKUGAKU ZASSHI. 123, suppl3. 158-161 (2003)

涩谷正明：“三萜皂苷的结构多样性和分子起源”药学杂志。

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Yutaka Ebizuka, Yuji Katsube, Takehiko, Tsutsumi, Testuo Kushiro, Masaaki Shibuya: "Functional Genomics Approach to the Study Triterpene Biosynthesis"Pure Appl.Chem.. 75. 361-366 (2003)

Yutaka Ebizuka、Yuji Katsube、Takehiko、Ttsutsumi、Testuo Kushiro、Masaaki Shibuya：“研究三萜生物合成的功能基因组学方法”Pure Appl.Chem.. 75. 361-366 (2003)

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Yuichi Sakano, Masaaki Shibuya, Atsuko Matsumoto, Yoko Takahashi, Hiroshi Tomoda, Satosi Omura, Yutaka Ebizuka: "Lanopylins A1, B1, A2 and B2, Novel Lanosterol Synthase Inhibitors from, treptomyces sp. K99-5041"J.Antibiotics. 56. 817-826 (2003)

Yuichi Sakano、Masaaki Shibuya、Atsuko Matsumoto、Yoko Takahashi、Hiroshi Tomoda、Satosi Omura、Yutaka Ebizuka：“Lanopylins A1、B1、A2 和 B2，来自链霉菌 K99-5041 的新型羊毛甾醇合酶抑制剂”J.Antibiotics。