The clarification of two hydroxylation steps in the conversion of cholesterol to <scp>OSW</scp>‐1 in <i>Ornithogalum saundersiae</i>

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, OsCYP90B94 and OsCYP90G11, and their partner cytochrome P450 reductase OsCPR2 were mined by comparable transcriptome analysis and demonstrated to be involved in OSW-1 biosynthesis. Specifically, OsCYP90B94 catalyses cholesterol to 22R-hydroxycholesterol, which is then converted to 16β-hydroxy-22-oxo-cholesterol, a precursor of OSW-1 by the second P450 OsCYP90G11 via two consecutive oxidation reactions. Unlike their homologues, OsCYP90G11 and OsCYP90B94 displayed broader catalytic promiscuity. OsCYP90B94 recognizes both cholesterol and 7β-hydroxycholesterol, while OsCYP90G11 can react with cholesterol, 22R-hydroxycholesterol and 16β,22R-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.