The clarification of two hydroxylation steps in the conversion of cholesterol to OSW‐1 in Ornithogalum saundersiae

Summary OSW‐1 is a cholestane saponin uniquely produced in Ornithogalum saundersiae and exhibits a potential antitumor activity. The enzymes responsible for OSW‐1 biosynthesis in O. saundersiae , however, remain unclear. Herein, cholesterol was confirmed to be the precursor of OSW‐1 using stable‐isotope labeling method. Next, two cytochrome P450s, Os CYP90B94 and Os CYP90G11, and their partner cytochrome P450 reductase Os CPR2 were mined by comparable transcriptome analysis and demonstrated to be involved in OSW‐1 biosynthesis. Specifically, Os CYP90B94 catalyses cholesterol to 22 R ‐hydroxycholesterol, which is then converted to 16 β ‐hydroxy‐22‐oxo‐cholesterol, a precursor of OSW‐1 by the second P450 O sCYP90G11 via two consecutive oxidation reactions. Unlike their homologues, Os CYP90G11 and Os CYP90B94 displayed broader catalytic promiscuity. Os CYP90B94 recognizes both cholesterol and 7 β ‐hydroxycholesterol, while Os CYP90G11 can react with cholesterol, 22 R ‐hydroxycholesterol and 16 β ,22 R ‐dihydroxycholesterol, suggesting their involvement in more than one pathway besides OSW‐1 biosynthesis. This study lays a foundation for the complete characterization of OSW‐1 biosynthesis.