Construction Of Saccharomyces Cerevisiae Cell Factories For Terpenoids Production

Terpenoids are widely used in medical and fragrance industries. In particular, many diterpenoids can be used for the treatment of cancer and cardiovascular disease. Direct extraction from plant sources or chemical synthesis of terpenoids is inherently difficult and has low yield. Metabolic engineering of microorganisms is an alternative and attractive route for production of these valuable compounds. This strategy is used in this study to engineer Saccharomyces cerevisiae for successful production of terpenoids and important precursors in their biosynthesis pathways, such as miltiradiene and lycopene. As a step toward microbial production of tanshinones, copalyl diphosphate (CPP) synthase, and normal CPP kaurene synthase-like genes, which convert the universal diterpenoid precursor geranylgeranyl diphosphate (GGPP) to miltiradiene (an important intermediate of the tanshinones synthetic pathway), was introduced into Saccharomyces cerevisiae, resulting in production of 4.2 mg/L miltiradiene. Improving supplies of isoprenoid precursors was then investigated for increasing miltiradiene production. Although over-expression of a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase (tHMGR) and a mutated global regulatory factor (upc2.1)gene did improve farnesyl diphosphate (FPP) supply, production of miltiradiene was not increased while large amounts of squalene (78 mg/L) were accumulated. In contrast, miltiradiene production increased to 8.8 mg/L by improving supply of GGPP through over-expression of a fusion gene of FPP synthase (ERG20) and endogenous GGPP synthase (BTS1) together with a heterologous GGPP synthase from Sulfolobus acidocaldarius (SaGGPS). Auxotrophic markers in the episomal plasmids were then replaced by antibiotic markers, so that engineered yeast strains could use rich medium to obtain better cell growth while keeping plasmid stabilities. Over-expressing ERG20-BTS1 and SaGGPS genes increased miltiradiene production from 5.4 to 28.2 mg/L. Combinatorial overexpression of tHMGR-upc2.1 and ERG20-BTS1-SaGGPS genes had a synergetic effects on miltiradiene production, increasing titer to 61.8 mg/L.In order to verify the role of combinatorial overexpression, engineered yeast strains for lycopene production were constructed. Combinatorial overexpression of tHMGR-upc2.1 and ERG20-BTS1-SaGGPS genes also had a synergetic effect on lycopene production, increasing titer from 0.69 mg/L to 8.49 mg/L.Finally, fed-batch fermentation was performed.488 mg/L miltiradiene,852 mg/L squalene and 21.17 mg/L lycopene were produced, respectively. The yeast strains engineered in this work provide a basis for creating an alternative way for production of terpenoids in place of extraction from plant sources.