Functional Analysis of Biosynthetic Enzymes for Natural Products

天然产物的结构多样性是由生物合成酶来完成的，而那些位于生物合成途径最上游的酶被认为是天然产物结构多样性的主要来源，因此，我们的注意力集中在这些生物合成酶上，以便将来设计合理的生物合成机器来生产新的化学品。本研究对其中一些酶的基因克隆和功能分析进行了尝试，通过对丝状真菌中新的聚酮酶的克隆，获得了几个新的克隆，并通过在真菌和/或酵母中的异源表达分析了这些克隆的酶功能。嵌合酶和位点的构建这些克隆的定向突变体鉴定了一个用于芳香族pokyketide生产的新功能结构域。通过基于同源性的PCR方法，从各种三萜产生植物中克隆了三十多个三萜合酶cDNA。通过酵母异源表达鉴定了它们的酶功能。尝试对十三个拟南芥三萜合酶克隆中的八个进行功能分析。只有两个克隆是本研究对聚酮化合物和三萜类化合物的生物合成酶进行了广泛的功能分析，结果表明，功能基因组学方法为寻找新的天然产物提供了新的方法学途径，并利用重组技术对这些生物合成酶进行了研究，开发实用的有用化学品生产系统和构建由具有不同结构的“非天然天然产物”组成的人工化学品库是值得期待的。

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.