title: Metabolic engineering of Pichia pastoris for the production of dammarenediol-II.
abstract: 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.
authors.affiliation: State key Lab of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Xin-Bin
authors.initials: XB
authors.lastname: Liu
authors.affiliation: State key Lab of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.affiliation: State key Lab of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Xin-Yi
authors.initials: XY
authors.lastname: Tao
authors.affiliation: State key Lab of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Zhong-Xi
authors.initials: ZX
authors.lastname: Zhang
authors.affiliation: State key Lab of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China. Electronic address: fqwang@ecust.edu.cn.
authors.forename: Feng-Qing
authors.initials: FQ
authors.lastname: Wang
authors.affiliation: State key Lab of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China. Electronic address: dzhwei@ecust.edu.cn.
authors.forename: Dong-Zhi
authors.initials: DZ
authors.lastname: Wei
mesh_terms: D053898:Biosynthetic Pathways; D004875:Ergosterol / Q000378:metabolism; D005800:Genes, Fungal; D005822:Genetic Vectors / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D010843:Pichia / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D017393:RNA Editing; D012503:Saponins / Q000096:biosynthesis*; D013185:Squalene / Q000031:analogs & derivatives; D014315:Triterpenes
chemical_list: C002821:2,3-oxidosqualene; D012503:Saponins; D014315:Triterpenes; C106188:dammarenediol; D013185:Squalene; D004875:Ergosterol
keywords: 2,3-Oxidosqualene; Dammarenediol-II; Ginsenosides; Metabolic engineering; Pichia pastoris