title: Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae.
abstract: Ginsenosides, the major bioactive components of Panax ginseng, are regarded as promising high-value pharmaceutical compounds. In ginseng, ginsenosides are produced from their precursor protopanaxadiol. Recently, an artificial biosynthetic pathway of protopanaxadiol was built in Saccharomyces cerevisiae by introducing a P. ginseng dammarenediol-II synthase, a P. ginseng cytochrome P450-type protopanaxadiol synthase (PPDS), and a Arabidopsis thaliana NADPH-cytochrome P450 reductase (ATR1). In this engineered yeast strain, however, the low metabolic flux through PPDS resulted in a low productivity of protopanaxadiol. Moreover, health of the yeast cells was significantly affected by reactive oxygen species released by the pool coupling between PPDS and ATR1. To overcome the obstacles in protopanaxadiol production, PPDS was modified through transmembrane domain truncation and self-sufficient PPDS-ATR1 fusion construction in this study. The fusion enzymes conferred approximately 4.5-fold increase in catalytic activity, and 71.1% increase in protopanaxadiol production compared with PPDS and ATR1 co-expression. Our in vivo experiment indicated that the engineered yeast carrying fusion protein effectively converted 96.8% of dammarenediol-II into protopanaxadiol. Protopanaxadiol production in a 5 L bioreactor in fed-batch fermentation reached 1436.6 mg/L. Our study not only improved protopanaxadiol production in yeast, but also provided a generic method to improve activities of plant cytochrome P450 monooxygenases. This method is promising to be applied to other P450 systems in yeast. Biotechnol. Bioeng. 2016;113: 1787-1795. © 2016 Wiley Periodicals, Inc.
authors.affiliation: School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, PR China.
authors.forename: Fanglong
authors.initials: F
authors.lastname: Zhao
authors.affiliation: School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, PR China.|Key Laboratory of System Bioengineering (Tianjin University), Ministry of Education, Tianjin, PR China.
authors.forename: Peng
authors.initials: P
authors.lastname: Bai
authors.affiliation: School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, PR China.
authors.forename: Ting
authors.initials: T
authors.lastname: Liu
authors.affiliation: School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, PR China.
authors.forename: Dashuai
authors.initials: D
authors.lastname: Li
authors.affiliation: School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, PR China.
authors.forename: Xiangmei
authors.initials: X
authors.lastname: Zhang
authors.affiliation: School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, PR China. wenyulu@tju.edu.cn.|Key Laboratory of System Bioengineering (Tianjin University), Ministry of Education, Tianjin, PR China. wenyulu@tju.edu.cn.|SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, PR China. wenyulu@tju.edu.cn.
authors.forename: Wenyu
authors.initials: W
authors.lastname: Lu
authors.affiliation: School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, PR China.|Key Laboratory of System Bioengineering (Tianjin University), Ministry of Education, Tianjin, PR China.|SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, PR China.
authors.forename: Yingjin
authors.initials: Y
authors.lastname: Yuan
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D060847:Metabolic Engineering / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012502:Sapogenins / Q000032:analysis / Q000378:metabolism*; D012503:Saponins / Q000032:analysis / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods*; D014315:Triterpenes / Q000032:analysis / Q000378:metabolism
chemical_list: D012502:Sapogenins; D012503:Saponins; D014315:Triterpenes; C106188:dammarenediol; D003577:Cytochrome P-450 Enzyme System; C062916:protopanaxadiol
keywords: Saccharomyces cerevisiae; dammarenediol-II; protopanaxadiol; protopanaxadiol synthase; self-sufficient P450s; synthetic biology