Metabolic engineering of Pichia pastoris for the production of dammarenediol-II

• Published in: Journal of biotechnology 2015-12, Vol.216, p.47-55
• Main Authors: Liu, Xin-Bin, Liu, Min, Tao, Xin-Yi, Zhang, Zhong-Xi, Wang, Feng-Qing, Wei, Dong-Zhi
• Format: Article
• Language: English
• Subjects: 2,3-Oxidosqualene; Biosynthetic Pathways; Consumption; Culture; Dammarenediol-II; Ergosterol - metabolism; Gene expression; Genes; Genes, Fungal; Genetic Vectors - metabolism; Ginsenosides; Metabolic engineering; Metabolic Engineering - methods; Nuclei; Panax ginseng; Pathways; Pichia - genetics; Pichia - growth & development; Pichia - metabolism; Pichia pastoris; Promoter Regions, Genetic - genetics; RNA Editing; Saponins - biosynthesis; Squalene - analogs & derivatives; Strain; Triterpenes
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2015.10.005

•Increasing supply and reducing competitive consumption of 2,3-oxidosqualene was used.•A marker-recycling and gene-targeting Cre-lox71/66 system was developed herein.•The yield of dammarenediol-II in P. pastoris was increased significantly.•This study constitutes a good platform for production of ginsenosides in Pichia. 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 0.030mg/g dry cell weight (DCW) to 0.203mg/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 0.736mg/g DCW. Furthermore, the direct addition of 0.5g/L squalene into the culture medium further enhanced the yield of dammarenediol-II to 1.073mg/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.