Tansakul Pimpimon, Shibuya Masaaki, Kushiro Tetsuo, Ebizuka Yutaka
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
FEBS Lett. 2006 Oct 2;580(22):5143-9. doi: 10.1016/j.febslet.2006.08.044. Epub 2006 Sep 1.
Panax ginseng produces triterpene saponins called ginsenosides, which are classified into two groups by the skeleton of aglycones, namely dammarane type and oleanane type. Dammarane-type ginsenosides dominate over oleanane type not only in amount but also in structural varieties. However, their sapogenin structure is restricted to two aglycones, protopanaxadiol and protopanaxatriol. So far, the genes encoding oxidosqualene cyclase (OSC) responsible for formation of dammarane skeleton have not been cloned, although OSC yielding oleanane skeleton (beta-amyrin synthase) has been successfully cloned from this plant. In this study, cDNA cloning of OSC producing dammmarane triterpene was attempted from hairy root cultures of P. ginseng by homology based PCR method. A new OSC gene (named as PNA) obtained was expressed in a lanosterol synthase deficient (erg7) Saccharomyces cerevisiae strain GIL77. LC-MS and NMR analyses identified the accumulated product in the yeast transformant to be dammarenediol-II, demonstrating PNA to encode dammarenediol-II synthase.
人参产生称为人参皂苷的三萜皂苷，根据苷元骨架可分为两组，即达玛烷型和齐墩果烷型。达玛烷型人参皂苷不仅在数量上而且在结构种类上都比齐墩果烷型占优势。然而，它们的皂苷元结构仅限于两种苷元，即原人参二醇和原人参三醇。到目前为止，尽管已经成功地从该植物中克隆出产生齐墩果烷骨架的氧化鲨烯环化酶（OSC）（β-香树脂醇合酶），但负责达玛烷骨架形成的氧化鲨烯环化酶（OSC）的编码基因尚未被克隆。在本研究中，尝试通过基于同源性的PCR方法从人参发根培养物中克隆产生达玛烷三萜的OSC的cDNA。获得的一个新的OSC基因（命名为PNA）在羊毛甾醇合酶缺陷型（erg7）酿酒酵母菌株GIL77中表达。液相色谱-质谱联用（LC-MS）和核磁共振（NMR）分析确定酵母转化体中积累的产物为达玛烯二醇-II，证明PNA编码达玛烯二醇-II合酶。
