文献类型：期刊文献

英文题名：Metabolic engineering of Pichia pastoris for the production of dammarenediol-II

作者：Liu, Xin-Bin[1];Liu, Min[1];Tao, Xin-Yi[1];Zhang, Zhong-Xi[1];Wang, Feng-Qing[1];Wei, Dong-Zhi[1]

机构：[1]E China Univ Sci & Technol, Newworld Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2015

卷号：216

起止页码：47

外文期刊名：JOURNAL OF BIOTECHNOLOGY

收录：;EI(收录号：20154301449191);WOS:【SCI-EXPANDED(收录号:WOS:000365769000006)】；

基金：This research was financially supported by the Fundamental Research Funds for the Central Universities of China, the National Special Fund for State Key Laboratory of Bioreactor Engineering (2060204), National Basic Research Program of China (973, Program No. 2012CB721103) and the National Major Science and Technology Projects of China (No. 2012ZX09304009).

语种：英文

外文关键词：Dammarenediol-II; Metabolic engineering; Pichia pastoris; Ginsenosides; 2,3-Oxidosqualene

摘要：Dammarenediol-II is the nucleus of dammarane-type ginsenosides, which are a group of active triterpenoids exhibiting various pharmacological activities. Based on the native triterpene synthetic pathway, a dammarenediol-II synthetic pathway was established in Pichia pastoris by introducing a dammarenediol-II synthase gene (PgDDS) from Panax ginseng, which is responsible for the cyclization of 2,3-oxidosqualene to dammarenediol-II in this study. To enhance productivity, a strategy of "increasing supply and reducing competitive consumption of 2,3-oxidosqualene" was used. To increase the supply of 2,3-oxidosqualene, we augmented expression of the ERG1 gene, which is responsible for 2,3-oxidosqualene synthesis. This significantly improved the yield of dammarenediol-II over 6.7-fold, from 0.030 mg/g dry cell weight (DCW) to 0.203 mg/g DCW. Subsequently, to reduce competition for 2,3-oxidosqualene from ergosterol biosynthesis without affecting the normal growth of P. pastoris, we targeted the ERG7gene, which is responsible for conversion of 2,3-oxidosqualene to lanosterol. This gene was downregulated by replacing its native promoter with a thiamine-repressible promoter, using a marker-recycling and gene-targeting Cre-lox71/66 system developed for P. pastoris herein. The yield of dammarenediol-II was further increased more than 3.6-fold, to 0.736 mg/g DCW. Furthermore, the direct addition of 0.5 g/L squalene into the culture medium further enhanced the yield of dammarenediol-II to 1.073 mg/g DCW, which was 37.5-fold higher than the yield from the strain with the PgDDS gene introduction only. The P. pastoris strains engineered in this study constitute a good platform for further production of ginsenosides in Pichia species. (C) 2015 Elsevier B.V. All rights reserved.