Abstract

Kadsura heteroclita (Roxb.) Craib, commonly known as "Xuetong", is a traditional Tujia ethnomedicine with anti-rheumatoid arthritis (RA) activity, and schizanlactone E (Xuetongsu) is its major bioactive component whose biosynthetic pathway remains uncharacterized. As a cycloartane-type tetracyclic triterpenoid, Xuetongsu's biosynthesis is likely to involve multiple oxidation steps. Cytochrome P450 (CYP450) is a versatile monooxygenase encoded by a large and diverse gene superfamily and plays a critical role in various oxidation reactions in plants' secondary metabolism. In this study, 367 KhCYP450s were identified and systematically analyzed for their physicochemical properties, phylogenetic analysis, conserved motifs, gene structures, collinearity, and cis-acting elements. Weighted gene co-expression network analysis (WGCNA) revealed a turquoise module strongly associated with Xuetong root tissue, which had the highest Xuetongsu accumulation; 32 candidate KhCYP450s within this module were screened via correlation analysis between gene expression and xuetongsu content and partially validated by qRT-PCR. Five of these candidates showed significant homology with known triterpenoid biosynthetic genes via protein structure analyses. This study deepened our comprehension of the CYP450 superfamily in Xuetong and provided a valuable reference for further research on the biosynthesis of Xuetongsu.