药学学报, 2016, 51(6): 998-1003
引用本文:
梁会超, 高丽丽, 胡宗风, 巩婷, 陈晶晶, 杨金玲, 朱平. 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[J]. 药学学报, 2016, 51(6): 998-1003.
LIANG Hui-chao, GAO Li-li, HU Zong-feng, GONG Ting, CHEN Jing-jing, YANG Jin-ling, ZHU Ping. Expression, subcellular localization and characterization of dammarenediol-Ⅱ synthase of Panax ginseng in Saccharomyces cerevisiae[J]. Acta Pharmaceutica Sinica, 2016, 51(6): 998-1003.

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
梁会超, 高丽丽, 胡宗风, 巩婷, 陈晶晶, 杨金玲, 朱平
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
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。
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     
Expression, subcellular localization and characterization of dammarenediol-Ⅱ synthase of Panax ginseng in Saccharomyces cerevisiae
LIANG Hui-chao, GAO Li-li, HU Zong-feng, GONG Ting, CHEN Jing-jing, YANG Jin-ling, ZHU Ping
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
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.
Key words:    dammarenediol-Ⅱ synthase    green fluorescent protein    fusion expression    Saccharomyces cerevisiae   
收稿日期: 2016-03-25
DOI: 10.16438/j.0513-4870.2016-0276
基金项目: 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.
通讯作者: 杨金玲,Tel:86-10-63165199,Fax:86-10-63165197,E-mail:yangjl@imm.ac.cn;朱平,zhuping@imm.ac.cn
Email: yangjl@imm.ac.cn;zhuping@imm.ac.cn
相关功能
PDF(2531KB) Free
打印本文
0
作者相关文章
梁会超  在本刊中的所有文章
高丽丽  在本刊中的所有文章
胡宗风  在本刊中的所有文章
巩婷  在本刊中的所有文章
陈晶晶  在本刊中的所有文章
杨金玲  在本刊中的所有文章
朱平  在本刊中的所有文章

参考文献:
[1] Hebshi LD, Angres BM, Li XL, et al. Green Fluorescent Protein (GFP)[M]//Encyclopedia of Immunotoxicology. Berlin Heidelberg:Springer, 2016, 10:351-351.
[2] Wang H, Zhen W, Yang L, et al. Construction of Saccharomyces cervisiae expression vector with GFP as report gene[J]. Guangdong J Med (广东医学), 2002, 23:1239-1240.
[3] Tie L. Studies of cellular distribution of PS1 using PS1/GFP fusing protein[J]. Chin Biotechnol (中国生物工程杂志), 2006, 26:17-22.
[4] Soundrarajan N, Cho HS, Ahn B, et al. Green fluorescent protein as a scaffold for high efficiency production of functional bacteriotoxic proteins in Escherichia coli[J]. Sci Rep, 2016, 6:20661.
[5] Kushiro T, Ohno Y, Shibuya M, et al. In vitro conversion of 2,3-oxidosqualene into dammarenediol by Panax ginseng microsomes[J]. Biol Pharm Bull, 1997, 20:292-294.
[6] Kim OT, Lee JW, Bang KH, et al. Characterization of a dammarenediol synthase in Centella asiatica (L.) Urban[J]. Plant Physiol Biochem, 2009, 47:998-1002.
[7] Lee MH, Han JY, Kim HJ, et al. Dammarenediol-Ⅱ production confers TMV tolerance in transgenic tobacco expressing Panax ginseng dammarenediol-Ⅱ synthase[J]. Plant Cell Physiol, 2012, 53:173-182.
[8] Gietz RD, Schiestl RH, Willems AR, et al. Studies on the transformation of intact yeast cells by the LiAC/SS-DNA/PEG procedure[J]. Yeast, 1995, 11:355-360.
[9] Tansakul P, Shibuya M, Kushiro T, et al. Dammarenediol-Ⅱ synthase, the first dedicated enzyme for ginsenoside biosynthesis in Panax ginseng[J]. FEBS Lett, 2006, 580:5143-5149.
[10] Prasher DC, Eckenrode VK, Ward WW, et al. Primary structure of the Aequorea victoria green fluorescent protein[J]. Gene, 1992, 111:229-233.
[11] Popova E, Rentzsch B, Bader M, et al. Generation and characterization of a GFP transgenic rat line for embryological research[J]. Transgenic Res, 2008, 17:955-963.
[12] Cai XD, Liu WW. GFP expression as an indicator of somatic hybrids between transgenic Satsuma mandarin and Calamondin at embryoid stage[J]. Plant Cell Tiss Org, 2006, 87:245-253.
[13] Peng C, Fan X, Chen J. Function and subcellular localization of Gcn5, a histone acetyltransferase in Candida albicans[J]. Fungal Genet Biol, 2015, 81:132-141.
[14] Wang H. Expression of the fusion gene of Chlamydomonas reinhardtii actin and green fluorescent protein in yeast[J]. J Agric Biotechnol (农业生物技术学报), 2003, 11:347-350.
[15] Bensidoun P, Raymond P, Oeffinger M, et al. Imaging single mRNAs to study dynamics of mRNA export in the yeast Saccharomyces cerevisiae[J]. Methods, 2016, 98:104-114.
[16] Yong JL. Fusion tags technology and their applications[J]. Chin J Biotechnol (生物工程学报), 2006, 22:523-527.