Functional Analysis of Biosynthetic Enzymes for Natural Products

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Diverse structures of natural products are elaborated by biosynthetic enzymes, and those enzymes which work at the most upstream of biosynthetic pathways are thus regarded as the primary origin of structural diversty exhibited by natural products.Our attentions have been focused on these biosynthetic enzymes for future engineering of rationally designed biosynthetic machineries for production of new chemicals. In this study, gene cloning and functional analysis of some of these enzymes have been attempted.Continuous attempts for cloning of new polyketide synthases from filamentous fungi yielded several new clones.Enzyme functions of these clones were analyzed by heterologous expression in fungi and/or yeast. Construction of chimeric enzymes and site-directed mutants of these clones identified a new functional domain for aromatic pokyketide production.Over thirty triterpene synthase cDNAs have been cloned from various triterpene producing plants by homology based PCR methods.Their enzyme functions have been identified by heterologous expression in yeast.Functional analysis of the eight out of thirteen Arabidopsis thaliana triterpene synthase clones was attempted.Only two clones are product specific synthases for known triterpenes, while the others are multifunctional clones.In this four-year project, functional analysis of biosynthetic enzymes for polyketides and triterpenoids was extensively conducted.The results obtained in this study clearly show that functional genomics approach would provide a new methodology for the search of new natural products.Furthermore, by employing recombinant technology of these biosynthetic enzymes, development of practical production system for useful chemicals and construction of artificial chemical library comprising of "un-natural natural products" with diverse structures wouldbe expected.