人参达玛烯二醇-Ⅱ合酶在酿酒酵母中的表达、定位及功能研究  

Expression, subcellular localization and characterization of dammarenediol-Ⅱ synthase of Panax ginseng in Saccharomyces cerevisiae

作　　者：梁会超 高丽丽 胡宗风 巩婷 陈晶晶 杨金玲 朱平 

机构地区：[1]中国医学科学院、北京协和医学院药物研究所,天然药物活性物质与功能国家重点实验室,国家卫生计生委天然药物生物合成重点实验室,北京100050

出　　处：《药学学报》2016年第6期998-1003,共6页Acta Pharmaceutica Sinica

基　　金：北京市自然科学基金资助项目(7122115);天然药物活性物质与功能国家重点实验室自主课题

摘　　要：为了研究人参达玛烯二醇-Ⅱ合酶(dammarenediol-Ⅱ synthase,DS)在酿酒酵母中的重组表达和定位,本研究克隆了人参中达玛烯二醇-Ⅱ合酶基因ds,并将其与绿色荧光蛋白基因gfp融合,构建了相应的重组表达质粒pESC-HIS-DS和pESC-HIS-DS-GFP,转化酿酒酵母INVSc1,获得了重组菌INVSc1-DS和INVSc1-DS-GFP。通过差速离心法制备重组菌微粒体,荧光显微镜下观察达玛烯二醇-Ⅱ合酶的表达及亚细胞定位,并通过酶促反应对该酶进行了功能鉴定,结果表明,DS为膜结合型蛋白,在体外能催化2,3-氧化鲨烯生成达玛烯二醇-Ⅱ。对重组菌发酵产物进行分析,结果表明,重组菌中产生了达玛烯二醇-Ⅱ,且通过将ds与gfp融合,重组菌中的达玛烯二醇-Ⅱ产量由7.53 mg·g^(-1)提高到12.24 mg·g^(-1),该结果为优化在酿酒酵母中构建的人参皂苷代谢途径提供了新思路。To study the expression and subcellular localization of recombinant dammarenediol-Ⅱ synthase（DS） in Saccharomyces cerevisiae, the dammarenediol-Ⅱ synthase gene ds was cloned from Panax ginseng, and the gene ds was fused with the gene of green fluorescent protein to obtain the fusion gene ds-gfp. The recombinant expression plasmids pESC-HIS-DS and pESC-HIS-DS-GFP were constructed and transformed into S. cerevisiae INVSc1 to obtain recombinant strains INVSc1-DS and INVSc1-DS-GFP. Microsomes of recombinant strains were prepared by differential centrifugation and observed by fluorescence microscope. The green fluorescence was only detected in INVSc1-DS-GFP microsomes, which indicated that DS was a membrane protein. It was also proved that dammarenediol-Ⅱ was produced from the cyclization of 2,3-oxidosqualene catalyzed by DS through in vitro enzymatic reaction. In addition, our results revealed that the fusion expression of ds with gfp significantly improved the production of dammarenediol-Ⅱ from 7.53 mg·g^（-1） to 12.24 mg·g^（-1）. This study provides a new strategy in the optimization of the pathway of ginsenosides biosynthesis in S.cerevisiae.

关 键 词：达玛烯二醇-Ⅱ合酶 绿色荧光蛋白 融合表达 酿酒酵母 

分 类 号：R931[医药卫生—生药学]