过表达HMGR催化结构域以优化酵母工程菌原人参二醇代谢途径的研究  

Optimization of the protopanoxadiol metabolic pathway by overexpression of the catalytic domain of HMGR in Saccharomyces cerevisiae

作　　者：梁会超 胡宗风[1] 梁兰[1] 巩婷[1] 陈晶晶[1] 杨金玲[1] 朱平[1] 

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：《药学研究》2016年第8期444-448,493,共6页Journal of Pharmaceutical Research

基　　金：北京市自然科学基金资助项目(No.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：目的过表达3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMGR)催化结构域提高酵母工程菌中原人参二醇的产量。方法分别克隆酿酒酵母中编码3-羟基-3-甲基戊二酸单酰辅酶A还原酶催化结构域的基因t HMG1、人参中达玛烯二醇-Ⅱ合酶基因ds和原人参二醇合酶基因cyp1、拟南芥中CYP450还原酶基因cyp-re,构建相应表达质粒,转化酿酒酵母INVSc1,并检测重组菌中原人参二醇产量。结果重组菌INVSc1-DS-GFP/CYP1/CYP-Re和INVSc1-DS-GFP/CYP1/CYP-Re/t HMG1中原人参二醇产量分别为3.04 mg·L-1和26.5 mg·L-1。结论导入基因t HMG1,使原人参二醇产量提高了8.7倍,该结果为优化酵母工程菌中人参皂苷代谢途径奠定了基础。Objective To improve the production of protopanoxadiol in engineered Saccharomyces cerevisiae by overexpressing the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme.Methods The truncated gene t HMG1 encoding the catalytic domain of HMGR was cloned from S. cerevisiae,the dammarenediol-Ⅱ synthase gene ds and the protopanoxadiol synthase gene cyp1 were cloned from Panax ginseng and the CYP450 reductase synthase gene cyp-re was cloned from Arabidopsis thaliana.The recombinant expression plasmids were constructed and then transformed into S. cerevisiae INVSc1. The production of protopanoxadiol in recombinant strains were detected.Results The production of protopanoxadiol in recombinant strains INVSc1-DS-GFP / CYP1 / CYP-Re and INVSc1-DS-GFP / CYP1 / CYP-Re / t HMG1 was 3. 04 mg·L-1and26.5 mg·L-1,respectively.Conclusion The overexpression of catalytic domain of HMGR significantly improved the production of protopanoxadiol by 8.7 times.This research laid the foundation to optimize the metabolic pathway of ginsenosides constructed in Saccharomyces cerevisiae.

关 键 词：原人参二醇 3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR) 酿酒酵母 代谢途径 

分 类 号：Q814[生物学—生物工程]