Expression, subcellular localization and characterization of dammarenediol-Ⅱ synthase of Panax ginseng in Saccharomyces cerevisiae
10.16438/j.0513-4870.2016-0276
- VernacularTitle: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
- Author:
Hui-chao LIANG
1
;
Li-li GAO
1
;
Zong-feng HU
1
;
Ting GONG
1
;
Jing-jing CHEN
1
;
Jin-ling YANG
1
;
Ping ZHU
1
Author Information
1. State Key Laboratory of Bioactive Substance and Function of Natural Medicines and Key Laboratory of Biosynthesis of Natural Products of National Health and Family Planning Commission, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
- Publication Type:ORIGINAL ARTICLES
- Keywords:
dammarenediol-Ⅱ synthase;
green fluorescent protein;
fusion expression;
Saccharomyces cerevisiae
- From:
Acta Pharmaceutica Sinica
2016;51(6):998-
- CountryChina
- Language:Chinese
-
Abstract:
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.
