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.