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(+)-Valencene and (+)-nootkatone are high value-added sesquiterpenoids found in grapefruit. The synthesis of (+)-nootkatone by chemical oxidation from (+)-valencene cannot meet the increasing demand in natural aromatics markets. Development of a viable bioprocess using microorganisms is attractive. According to the yields of β-nootkatol and (+)-nootkatone by strains harboring different expression cassettes in the resting cell assay, premnaspirodiene oxygenase from Hyoscyamus muticus (HPO), cytochrome P450 reductase from Arabidopsis thaliana (AtCPR) and alcohol dehydrogenase (ADH1) from Saccharomyces cerevisiae were finally selected and overexpressed in CEN·PK2-1Ca, yielding β-nootkatol and (+)-nootkatone with 170.5 and 45.6 mg L -1 ethyl acetate, respectively. A combinational engineering strategy including promoter change, regulator ROX1 knockout, squalene pathway inhibition, and tHMGR overexpression was performed to achieve de novo (+)-valencene production. Subsequent culture investigations found that galactose as the induced carbon source and a lower temperature (25 °C) were beneficial to target accumulation. Also, replacing the inducible promoters ( GAL1 ) of HPO and AtCPR with constitutive promoters ( HXT7 and CYC1 ) dramatically increased the β-nootkatol accumulation from 108.2 to 327.8 mg L -1 ethyl acetate in resting-cell experiments using (+)-valencene as a substrate. Finally, the total terpenoid titer of the engineered strain of PK2-25 using glucose as a carbon source was improved to 157.8 mg L -1 cell culture, which was 56 times the initial value. We present a new candidate for production of (+)-valencene and its related sesquiterpenoids with attraction for industry.

Stepwise engineering of Saccharomyces cerevisiae to produce (+)-valencene and its related sesquiterpenes