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Studies on production and improvement of terpenoids using biosynthetic gene cluster in fungi and plants.

利用真菌和植物生物合成基因簇生产和改良萜类化合物的研究。

基本信息

批准号：
16208012
负责人：
SASSA Takeshi
金额：
$ 32.03万
依托单位：
Yamagata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16208012/
关键词：
terpenoid biosynthesis gene cluster cyclase P450 oxygenase

项目摘要

A cDNA encoding fusicoccadiene synthase (PaFS) was isolated from mycelia of fusicoccin-producing fungi. PaFS possesses two domain, terpene cyclase domain at N-termini and prenyltransferase domain at C-termini, and shows multifunctional catalyzation. We have clarified the detailed cyclization mechanism forming fusicoccane skeleton from GGDP, and have succeeded in synthesizing a fucicoccin derivative that has a differentiation inducing activity. Mechanism on construction of molecular skeleton of diterpene, phomaction was investigated by chemical methods. Heterologous expression of the genes responsible for biosynthesis of diterpene, aphidicolin using yeast was explored. A common biosynthetic intermediate of indole diterpenoide was synthesized as non-and deuterium-labeled form. The deuterium-labeled intermediate was converted into paxilline and emindole DA via epoxidation and cyclization in two different fungi in an intact manner. These results give first evidence that various indole dite … More rpenoide are biosynthesized from a common intermediate. We have cloned three diterpene cyclase genes and one tetraterpene cyclase gene from actinomycetes. Of the four cyclases, enzymatic properties of the two diterpene cyclases were investigated with recombinant enzymes. The functional analyses were performed in respect to Rv3377c and Rv3378c involved in Mycobacterium tuberculosis H37 strain. These genes encode diterpene synthases for producing halimane skeleton. Studies on squalene cyclase were also conducted to gain more insights into the catalytic mechanism. Successful site-specific incorporation of fluorophenylalanines into the catalytic sites demonstrated the importance of cation-pi interaction. In vitro translation system revealed the function of a diterpene cyclase gene from the moss Physcomoitrella patens. This system was also available for determination of production and localization of cytochrome P450 monooxygenase proteins. The function of P450 monooxygenases responsible for biosynthesis of plant hormones, brassinosteroid and gibberellin, was revealed by heterologous expression in Pichia pastoris. We identified 6 cDNAs encoding diterpene cyclases responsible for phytoalexin bisynthesis in rice. We indicated strong evidence for the presence on chromosome 4 of a gene cluster involved in momilactone biosynthesis. The presence of phytocassane biosynthesis gene cluster on chromosome 2 was also suggested. Less

从产梭孢菌素的真菌菌丝体中分离到一个编码梭孢二烯合酶（PaFS）的cDNA。PaFS具有两个结构域，N-末端的萜烯环化酶结构域和C-末端的异戊烯基转移酶结构域，并且显示多功能催化作用。我们已经阐明了由GGDP形成岩藻素骨架的详细环化机理，并且已经成功地合成了具有分化诱导活性的岩藻素衍生物。用化学方法研究了二萜类化合物的结构和作用机理。探索了利用酵母异源表达负责二萜、蚜虫菌素生物合成的基因。合成了吲哚二萜类化合物的一种常见的生物合成中间体--非标记和氘标记形式。氘标记的中间体在两种不同的真菌中以完整的方式通过环氧化和环化转化为帕西林和依吲哚DA。这些结果首次证明， ...更多信息萜类化合物是从一个共同的中间体生物合成的。我们从放线菌中克隆了三个二萜环化酶基因和一个四萜环化酶基因。在四种环化酶中，用重组酶研究了两种二萜环化酶的酶性质。对结核分枝杆菌H37株中涉及的Rv 3377 c和Rv 3378 c进行功能分析。这些基因编码用于产生卤烷骨架的二萜脱氢酶。对角鲨烯环化酶也进行了研究，以获得更多的了解催化机制。成功的位点特异性掺入到催化位点的氟苯基丙氨酸证明了阳离子π相互作用的重要性。通过体外翻译系统研究了小立碗藓二萜环化酶基因的功能。该系统也可用于测定细胞色素P450单加氧酶蛋白的产生和定位。通过在毕赤酵母中的异源表达，揭示了植物激素油菜素类固醇和赤霉素生物合成的P450单加氧酶的功能。我们鉴定了6个编码水稻植保素双合成的二萜环化酶的cDNA。我们指出了强有力的证据4号染色体上的一个基因簇参与momilactone的生物合成。在第2染色体上可能存在植物卡桑生物合成基因簇。少

项目成果

期刊论文数量（44）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Fusicoccins are biosynthesized by an unusual chimera diterpene synthase in fungi

DOI：
10.1073/pnas.0608426104
发表时间：
2007-02-27
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
影响因子：
11.1
作者：
Toyomasu, Tomonobu;Tsukahara, Mai;Sassa, Takeshi
通讯作者：
Sassa, Takeshi

Proposed mechanism for diterpene synthases in the formation of Phomactatriene and taxadiene

双萜合酶在形成磷三烯和紫杉二烯过程中提出的机制