Stepwise Engineering Of Saccharomyces Cerevisiae To Produce (+)-valencene And Its Related Sesquiterpenes

(+)-Valencene and(+)-nootkatone are sesquiterpenoids,which are a class of natural terpenoids containing 15 carbon atoms in the molecule.(+)-Valencene is an aroma component of citrus fruits and citrus flavors,and is also an important precursor compound for(+)-nootkatone.(+)-Nootkatone has a unique aromatic scent.(+)-Valencene and(+)-nootkatone are widely used in food,beverage,flavor and fragrance industries and have high market value.When the(+)-nootkatone is present in the form of a spray,it has a good insecticidal effect.Recent studies have shown that(+)-nootkatone has the effect of inhibiting cancer cell proliferation and anti-platelet aggregation.In recent years,the development of synthetic biology has made it extremely attractive to engineer microorganisms through metabolic engineering to produce high value-added compounds with complex structures.This study first introduced cytochrome P450 monooxygenase(CYP450)HPO derived from Hyoscyamus muticus,(+)-valencene oxidase CnVO derived from yellow cypress,and four mutants of P450 monooxygenase gene that were successfully constructed by overlap extension PCR.They were co-expressed with cytochrome reductase CYP450 derived from Arabidopsis thaliana(cytochrome P450 reductase,CPR)AtCPR in the host strain Saccharomyces cerevisiae CEN.PK2-1Ca.Through resting cell experiments,we found that the catalytic effect of HPO was the best,and the conversion rate of(+)-valencene reached 10.73%(mol/mol).The results showed that when HPO and AtCPR were co-expressed on the same vector,the stability was significantly improved.The yield of(+)-nootkatol and(+)-nootkatone was 108.0 mg/L,19.1 mg / L ethyl acetate,respectively.In order to further increase the yield of(+)-nootkatone,two alcohol dehydrogenases,ADH1 and ADH3,were amplified from the Saccharomyces cerevisiae genome.The two dehydrogenases were overexpressed in the host strain,and the overexpression of ADH1 significantly improved the yield of(+)-nootkatol and(+)-nootkatone.The conversion rate reached 49.1%.Therefore,this study initially successfully constructed an engineered strain of semi-synthetic(+)-nootkatone.In order to solve the problem of strong volatility of exogenous added substrate,this study further introduced the(+)-valencene synthase gene CnVS from the Callitropsis nootkatensis into the yeast,and the endogenous yield of(+)-valencene was 2.60 ± 0.23 mg/L.A combinational engineering strategy including promoter change,regulator ROX1 knockout,squalene pathway inhibition,and tHMGR overexpression was performed to achieve de novo(+)-valencene production.,and finally the yield of(+)-valencene reached 28.97 mg/L.Based on this,both HPO,AtCPR and ADH1 genes were expressed,and the yields of(+)-valencene and(+)-nootkatol were 20.6 and 24.5 mg/L,respectively.Subsequent culture investigations found that galactose as induced carbon source and lower temperature(25 °C)were beneficial to target accumulation,the total yield of the shake flask level reached 130.5 mg/ L.Finally,based on this culture condition,the best combination of engineered strain PK2-24 was selected,and the total terpenoid yield was 310.94 mg/L,which was about 111 times higher than that of the original strain of 2.79 mg/L.Unfortunately,no(+)-nootkatone was detected.We present a new candidate for(+)-valencene and its related sesquiterpenoids production with industry attractions.