Metabolic engineering of Pichia pastoris for the production of dammarenediol-II.
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.
['Liu|Xin-Bin|XB|', 'Liu|Min|M|', 'Tao|Xin-Yi|XY|', 'Zhang|Zhong-Xi|ZX|', 'Wang|Feng-Qing|FQ|', 'Wei|Dong-Zhi|DZ|']
D053898:Biosynthetic Pathways D004875:Ergosterol D005800:Genes, Fungal D005822:Genetic Vectors D060847:Metabolic Engineering D010843:Pichia D011401:Promoter Regions, Genetic D017393:RNA Editing D012503:Saponins D013185:Squalene D014315:Triterpenes
Metabolic engineering Pichia pastoris Dammarenediol-II Ginsenosides 2,3-Oxidosqualene