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

从产褐霉素真菌的菌丝体中分离到一个编码镰刀菌烯合酶(PAFS)的基因。PAF具有N-末端的萜烯环化酶结构域和C-末端的异戊烯转移酶结构域两个结构域，具有多功能催化作用。我们阐明了GGDP形成甘蔗骨架的详细环化机理，并成功地合成了具有诱导分化活性的岩藻素衍生物。用化学方法研究了二萜分子骨架的构筑机理。探索了利用酵母菌异源表达与生物合成二萜、蜂王素有关的基因。合成了一种常见的吲哚二萜生物合成中间体。在两种不同的真菌中，通过环氧化和环合反应，将氚标记的中间体完整地转化为帕西林和吲哚DA。这些结果首次证明了不同的吲哚对…有抑制作用更多的环烯内酯是从一种常见的中间体中生物合成的。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。用重组酶研究了四种环化酶中两种二萜环化酶的酶学性质。对结核分枝杆菌H37株涉及的Rv3377c和Rv3378c进行了功能分析。这些基因编码二萜合成酶，以产生卤烷骨架。此外，还对角鲨烯环化酶进行了研究，以期对催化机理有更深入的了解。成功地将氟苯丙氨酸结合到催化中心，证明了阳离子-pi相互作用的重要性。体外翻译系统揭示了青霉二萜环化酶基因的功能。该体系也可用于细胞色素P450单加氧酶蛋白的生产和定位。P450单加氧酶在巴斯德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的生物合成功能。我们鉴定了6个编码水稻植保素双合成的二萜环化酶基因。我们提出了强有力的证据表明，在4号染色体上存在一个与吗丁内酯生物合成有关的基因簇。2号染色体上可能存在植物钙类生物合成基因簇。较少

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