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

Abstract

(+)-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.

Scheme 1. (+)-Nootkatone biosynthesis pathway in S. cerevisiae.

Fig. 1. Analysis and identification of the hydroxylation of (+)-valencene (sample 1) to β-nootkanol (sample 2) and (+)-nootkatone (sample 3) by different S. cerevisiae strains expressing HPO, CnVO or their mutants, respectively. (A) GC-FID chromatogram patterns of the products obtained by 150 OD₆₀₀ yeast cells incubated with 2 mM (+)-valencene at 30 °C for 18 h. Retention times of (+)-valencene, β-nootkanol and (+)-nootkatone were 14.639 min, 18.349 min and 19.540 min, respectively. Mass spectra of the products are compared with mass patterns of authentic standards (+)-valencene (B), β-nootkatol (C) and (+)-nootkatone (D) standard.

Fig. 2. (A) Resting cell assays of (+)-valencene hydroxylation to β-nootkanol and (+)-nootkatone using different cytochrome P450s under the control of galactose inducible promoter of P_GAL1 in YEplac181 in S. cerevisiae. Amino acid exchange of V482I/A484I (mHPO) in HPO was achieved through site-directed mutagenesis. No β-nootkanol or (+)-nootkatone was detected in strain PK2-C. The asterisk (****) indicates statistically significant differences in terpenoids formation (p < 0.0001, student's t-test). (B) Changes of (+)-valencene oxidation capabilities in yeast PK2-01 when cells incubated in glycerol stocks at during one month of storage. Recombinant yeast strains transformed freshly or from −80 °C freezer were cultivated on SD plates at 30 °C for two days. Then the recombinant single yeast colonies were inoculated into tubes containing 5 mL of SD medium supplemented with appropriate amino acids for auxotrophic selection. Cultivation of strains (OD₆₀₀ ≈ 1 to 2) was scaled up to 50 mL of total volume in 250 mL baffled culture flasks to a starter OD₆₀₀ of 0.1, which was prepared for subsequent resting cell assays. The total terpenoids was calculated as the sum of β-nootkanol and (+)-nootkatone in mg L⁻¹ ethyl acetate. All experiments were performed in triplicates.

Fig. 3. (A) Resting cell assays of (+)-valencene hydroxylation to β-nootkanol and (+)-nootkatone using different constructs in S. cerevisiae. Hosts (CEN·PK2-1Ca, BY4741 and BJ5464), constructed based on backbone (YEplac181 and YEp352) and alcohol dehydrogenase isozymes (ADH1 and ADH3) were compared. (B) Changes of (+)-valencene oxidation capabilities in yeast PK2-32 when cells incubated over a period of 8 months. The total terpenoids was calculated as the sum of β-nootkanol and (+)-nootkatone in mg L⁻¹ ethyl acetate. Data is representative of triplicate determinations and error bars indicate standard deviations.

Fig. 4. De novo production of (+)-valencene in different S. cerevisiae strains cultured in 50 mL flask cultures containing 10 mL containing 20% n-dodecane. The valencene synthase (CnVS) from C. nootkatensis was cloned under different promoters of TDH3 and TEF1. The platform was based on CEN·PK2-1Ca combining the suppression of erg9, knockout of the negative regulator rox1 and overexpression of truncated form of HMG1 gene (tHMGR). Error bars represent standard deviation from a triplicate analysis. The asterisk (****) indicates statistically significant differences in terpenoids formation (p < 0.0001, student's t-test).

Fig. 5. De novo production of (+)-valencene and β-nootkatol using PK2-24 in 50 mL bi-phasic conical flask cultures. One μL aliquots of n-dodecane phase for each sample were analyzed by GC-FID. (A) Carbon source in induction medium effects on the products accumulation. Yeasts were cultured in 20 g L⁻¹ glucose at 30 °C for 14–16 h for cell growth. Cells were collected by centrifugation and transferred into fresh induction medium containing galactose to induce HPO and AtCPR expression, SG (2% galactose), SGG (2% galactose and 10% glycerol) and SGR (2% galactose and 0.7 raffinose), respectively. (B) Time and temperature responses of product accumulation. 24, 48, 72 and 96 represent the cultivation time in hours. Mean values and standard deviations of biological triplicates are shown. The asterisk (***) indicates statistically significant differences in (+)-valencene formation (p < 0.001, student's t-test).

Fig. 6. (A) Resting cell assays of (+)-valencene hydroxylation to β-nootkanol and (+)-nootkatone using different constructs in S. cerevisiae CEN·PK2-1Ca. Promoters of HPO and AtCPR were noted at the bottom of the diagram. (B) Time responses of product accumulation. 24, 48, 72 and 96 represent the cultivation time in hours with PK2-25 harboring p181-V2H and p352-A1HC3. Mean values and standard deviations of biological triplicates are shown. The asterisk (***) indicates statistically significant differences in β-nootkanol formation (p < 0.001, student's t-test). The asterisk (**) indicates statistically significant differences in (+)-nootkatone formation (p < 0.05, student's t-test).