The Basic Research for the Production of New Triterpene Skeletons by Extensive Use of the Technique of Molecular Biology

氧化角鲨烯环化酶（OSCs）催化氧化角鲨烯环化成甾醇和三萜的四环和五环碳骨架，是自然界中最复杂和最迷人的反应之一。OSCs在单一反应中产生多个立体中心，并负责不同的三萜骨架。为了探讨三萜骨架结构多样性的来源，对OSCs进行了cDNA克隆和产物特异性分析。从产三萜植物中获得了25个以上的OSC克隆，并通过酵母表达确定了它们的酶功能。它们包括环artenol、葫芦二烯醇、luppeol、β-amyrin、异多氯肾上腺醇和混合amyrin合成酶。对β-amyrin合成酶和luppeol合成酶的嵌合蛋白以及位点定向诱变构建的突变蛋白进行了研究，确定了其产物特异性的氨基酸残基。β-amyrin synthase （PNY）的Trp259是控制β-amyrin形成的关键残基。在进一步的突变研究中，PNYY261H突变体产生了damara -18,21-dien-3β- 01（在Δ^<18 b>处E/Z异构体为3:5混合物）和少量的damara -18(28),21-dien-3β- 01。这些三萜尚未从自然界中发现，因此可归类为“非天然”天然产品。本研究的结果开辟了通过点突变产生具有新功能的新三萜合成酶的可能性。

Cyclization of oxidosqualene into tetra- and pentacyclic carbon skeleton of sterols and triterpenes, catalyzed by oxidosqualene cyclases (OSCs), is one of the most complex and fascinating reactions found in nature. OSCs generate multiple stereogenic centers in a single reaction, and are responsible for the diverse triterpene skeletons. In order to investigate the origin of structural diversity of triterpene skeletons, cDNA cloning of OSCs and analysis of their product specificity were carried out. From triterpene producing plants, over twenty-five OSC clones were obtained, and their enzyme function established by expression in yeast. They included cycloartenol, cucurbitadienol, lupeol, β-amyrin, isomultiflorenol and mixed amyrin synthases. Studies of chimeric proteins between β-amyrin synthase and lupeol synthase, and mutant proeins constructed by site directed mutagenesis identified the amino acid residues responsible for their product specificity. Trp259 of β-amyrin synthase (PNY) was identified to be the critical residue controlling β-amyrin formation. In further mutation studies, PNYY261H mutant produced dammara-18,21-dien-3β-o1 (as a 3:5 mixture of E/Z isomer at Δ^<18>) together with minor amount of dammara-18(28),21-dien-3β-o1. These triterpenes have not been reported from nature, and therefore, could be categorized as "unnatural" natural products. The results of this study opened up the possibility of generating new triterpene synthases with additional novel functions through point mutations.