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

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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