Engineering of Triterpene Biosynthesis to Develop a Production System for Useful Compounds

从三萜类植物中克隆了三萜类合成酶的基因。因此，从蒲公英(Taraxacum Office Inale)的根中克隆了一个新的克隆，通过其在酵母中的表达鉴定为无花果醇合成酶。Achilleol合成酶的氨基酸序列与β-淀粉酶的氨基酸序列有很高的同源性，该克隆在系统发育分析中已加入β-淀粉酶的分支。非常有趣的是，这个克隆得到的是单环产物，而不是五环产物，如β-香菇蛋白。该克隆在DCTAE基序的上游两个氨基酸的位置有一个甘氨酸残基，该残基存在于所有氧化橙烯环化酶中，尽管所有的β-香兰素合成酶都是在这个位置上的Valine残基。当Achilleol合成酶中的甘氨酸残基通过定点突变转变为Valine残基时，产物变成了β-香兰素，这表明Achilleol合成酶是由β-香兰素合成酶进化而来的。本研究为从蒲公英中分离到一种新化合物提供了一种新的功能基因组学方法，因为它虽然是从蒲公英中分离出来的，但并不是从蒲公英中分离出来的。从产生大豆皂苷的大豆中克隆三萜羟基酶。在数据库中登记了200多种大豆P450 EST克隆。其中，从大豆幼苗中克隆并在酵母中表达的克隆(6个克隆)，其全长区域和功能未被鉴定。被测克隆之一的CYP93E1具有24-羟基化活性，可对抗β-香草素。

cDNA cloning of triterpene synthases was carried out from triterpene producing plants. As a result, a new clone was cloned from a root of dandelion (Taraxacum officinale) which was identified to be achilleol synthase by its expression in yeast. Amino acid sequence of achilleol synthase has a high homology to those of beta-amyrin synthases, and this clone has joined to the branch of beta-amyrin synthases in phylogenetic analysis. It is very interesting that this clongives a mono-cyclic products instead of penta-cyclic product such as beta-amyrin. This clone has a glycine residue at the position of two amino acid upstream from DCTAE motif which is found in all oxidosqualene cyclase although all of beta-amyrin synthasq as valine residue at this position. When this glycine residue in achilleol synthase was changed to valine residue by site-direct mutagenesis, product was changed to beta-amyrin, suggesting that achilleol synthase was evolved from beta-amyrin synthase. The approach of this study is considered to be a new approach for isolation of a new compound by functional genomics since achilleol had not been isolated from dandelion itself although it was isolated from the other composite plant.cDNA cloning of triterpene hydroxylase as also carried out from soybean (Glycine max) which produced soyasaponins. More than two hundreds kinds of P450 EST clone from soybean were registered in data base. Among them, the clones (6 clones) which cover full-length region and function was not identified were cloned from soybean seedlings and expressed in yeast. CYP93E1, one of tested clones, showed 24-hydroxylation activity against beta-amyrin.