73. ATP再生系導入によるテルペノイドの効率的な酵素合成法(口頭発表,植物化学調節学会第46回大会)

概要

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Terpenoids are widely distributed in nature and contain hormones and many bioactive secondary metabolites. We have set up a system for enzymatic total synthesis of terpenoids. This system dependent on mevalonate pathway is used for a novel strategy aimed at functional analysis of terpene biosynthetic genes. During reaction process by the enzymatic synthesis system, consumption of ATP will be one of the rate limiting factors. ATP is used for phosphorylation reactions at the steps of mevalonate, phosphomevalonate and diphosphomevalonate. To improve this issue, the enzaymatic synthesis system was modified by introduction of ATP regeneration using a bacterial polyphosphate kinase (PPK). A PPK gene was cloned from Escherichia coli and produced the recombinant PPK protein in E. coli. ^<31>P-NMR analysis revealed that PPK catalyzed the phosphorylation of ADP to form ATP in the presence of polyphosphate and magnesium ion. For enzymatic synthesis of farnesyl diphosphate (FDP) from mevalonate, PPK was added into an enzyme cocktail containing mevalonate, recombinant enzymes involved in FDP production were confirmed by monitoring of ^<31>P-NMR measurement. Thus, ATP regeneration system could be introduced for improvement of enzymatic terpenoid synthesis.
植物化学調節学会の論文
2011-10-03