Biosynthesis Research Of Dammarenediol-Ⅱin Pichia Pastoris

Ginseng is an economically important medicinal plant. The major bioactive ingredients of ginseng are ginsenosides, which are triterpene saponins. Obtain ginseng saponin by ginseng planting and ginseng cell culture is very difficult and costly. With the development of metabolic engineering and synthetic biology, building ginseng saponin biosynthesis pathway in the microorganism caused great attention. Pichia is very suitable for large-scale industrial production. In order to develop efficient microbial strains which can produce ginsenosides, this study explores building efficient biosynthesis pathway of precursor of ginsenosides in Pichia.Our study is based on original ergosterol synthesis pathways of Pichia, and introduced into a metabolic branch of ginseng saponin syntheses after 2.3-oxidosqualene. This thesis carried out the relevant work mainly around the 2,3-oxidosqualene conversion to key precursor of ginsenosides——Dammarenediol-II. Firstly, Dammarenediol-II synthase gene was introduced into Pichia by plasmid pPIC3.5K, resulting in high copy strain GSKD which had a low Dammarenediol-II production, approximately 0.03 mg/g DCW; For increasing the supply of 2,3-oxidosqualene, we obtained recombinant strain GSKED by co-expression of erg1 and PgDDS genes with Promoter AOX. The yield of Dammarenediol-II was 0.203 mg/g DCW which was about 6 times as large as the strain GSKD; Finally, in order to reduce the 2,3-oxidosqualene in metabolic pathways to original ergosterol metabolic flux, we developed the Cre/mutated lox mediated unmarked gene manipulation tool. By this tool, we replaced the promoter of erg1 to promoter PTHI11 which can be inhibited by thiamine, and finally got strain GSKED-PTHI11. When added thiamine in GSKED-PTHI11 fermentation medium, more 2,3-oxidosqualene were accumulated and converted into Dammarenediol-II and the production of the strain GSKED-PTHI11 was 0.736 mg/g DCW, about 3 times more than strain GSKED; In addition, this study improves the production of Dammarenediol-II to 1.073 mg/g DCW further by adding squalene in GSKED-PTHI11 fermentation culture. The work of this paper laid a good foundation for further developing the efficient recombinant Pichia strain which produces ginsenosides.