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-oxidosqua... More
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.
