Construction Of Saccharomyces Cerevisiae Cell Factories For Production Of β-Carotene And Ole Anolic Acid

β-carotene has a wide range of application in food, pharmaceutical and cosmetic industries. For microbial production of β-carotene in Saccharomyces cerevisiae, the supply of geranylgeranyl diphosphate (GGPP) was firstly increased in S. cerevisiae BY4742to obtain strain BY4742-T2through overexpressing truncated3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR), which is the major rate-limiting enzyme in the mevalonate (MVA) pathway, and GGPP synthase (GGPS), which is a key enzyme in the diterpenoid synthetic pathway. The β-carotene synthetic genes of Pantoea agglomerans and Xanthophyllomyces dendrorhous were further integrated into strain BY4742-T2for comparing β-carotene production. Over-expression of tHMGR and GGPS genes led to26-fold increase of β-carotene production. In addition, genes from X. dendrorhous was more efficient than those from P. agglomerans for β-carotene production in S. cerevisiae. Strain BW02was obtained which produced1.56mg/g (dry cell weight) β-carotene.All oleanane-type triterpenoids derive from β-amyrin, and oleanolic acid is a representative which exhibits hepatoprotective effects and anti-oxidant and anti-cancer activities. S. cerevisiae was metabolically engineered in this work for production of β-amyrin and oleanolic acid. A starting strain BY-T1for triterpenoids production was firstly constructed through overexpressing a truncated3-hydroxy-3-methylglutaryl-CoA reductase, leading to15.7-fold increase of squalene production. β-amyrin synthase gene{bAS) were then introduced into strain BY-T1for β-amyrin production, while squalene synthase and squalene epoxidase genes were overexpressed at the same time for increasing precursor supply. Introducing Glycyrrhiza glabra bAS gene (GgbAS) resulted in strain BY-βA-G which produced107.0mg/L β-amyrin that was55-fold higher than that of Panax ginseng bAS gene (PgbAS). Finally, Medicago truncatula oleanolic acid synthase (MtOAS), Arabidopsis thaliana cytochrome P450reductase (AtCPRl) and an additional GgbAS genes were introduced into strain BY-βA-G for oleanolic acid production. The resulting strain BY-OA produced71.0mg/L oleanolic acid with a yield of6.1mg/g DCW. The yeast strains engineered in this work can serve as the basis for creating an alternative way for producing ginsenosides in place of extractions from plant sources.