2.4 In vitro enzymatic reactions
The yeast strains CPR-CYP5139G1 and CPR-EV (Table S3) were cultivated for microsome isolation. Microsomal isolation was performed as previously described (Wang et al., 2018). The enzymatic assay was conducted in a total volume of 500 μL of 90 mM Tris-HCl (pH 7.5) containing 500 μg microsomal protein and 2 mM NADPH; and 100 µM of a substrate - 3-hydroxy-lanosta-8,24-dien-26-ol (HLDO), 3-hydroxy-lanosta-8,24-dien-26-al (HLDA), or HLDOA was added, respectively. After incubation at 30 °C for 4 h, the product was extracted by ethyl acetate. The ethyl acetate layer was collected, evaporated, and redissolved in methanol for LC-MS analysis.