4.10. Yeast Heterologous Expression

The full-length 2,3-oxidosqualene cyclase PgbAS1 gene was inserted into pESC-URA vector, and the full-length of PgCYP716 genes were inserted into the pESC-TRP vector. The engineered yeast strain Saccharomyces cerevisiae WAT11, which has been modified to incorporate the P450 reductase from A. thaliana, serves as an optimal heterologous host for the expression of P450 enzymes. This strain is equipped with the mevalonate (MVA) metabolic pathway, enabling it to synthesize the triterpenoid precursor 2,3-oxidosqualene [56]. To validate the function of the PgCYP716s, the pESC-PgbAS1-URA vector was used to transform WAT11, after which the cyclization of 2, 3-oxidosqualene was catalyzed to generate β-amyrin [57]. Further, the pESC-PgCYP716s-TRP vector was used to transform the WAT11 containing pESC-PgbAS1-URA as described previously [10]. pESC-PgbAS1-URA and pESC-TRP (the empty vector) were also co-transformed into WAT11 as the control. The transformed yeast was inoculated in 5 mL SD/-TRP-URA liquid medium and grown till the OD₆₀₀ reached 1.5. Further, the yeast cells were harvested by centrifugation at 1000 rpm for 1 min, washed two times using sterile water, resuspended in 100 mL SC/-TRP-URA liquid medium, which added 2 g galactose, and cultured for 3 days in the dark at 28 °C to induce metabolite biosynthesis in yeast. Further, we use a mixture of acetone and ethyl acetate (1:1 v/v) to extract yeast metabolites as described previously [56]. The liquid was evaporated using a low-temperature concentration method, and the remaining residue was dissolved in 1.5 mL methanol for the next step of analysis. The metabolites isolated from yeast culture were detected using the vanillin-perchloric acid method and high-performance liquid chromatography (HPLC) [58,59]