Discovery of pseudobaptigenin synthase, completing the (-)-maackiain biosynthetic pathway

Pterocarpans are structurally complex defence compounds produced by legumes (Fabaceae). They are commonly associated with antimicrobial activity and thought to be synthesized de novo or accumulated in response to microbial pathogens. (-)-Maackiain is a lineage-specific pterocarpan detected in some legumes, including red clover ( Trifolium pratense ). The biosynthesis of (-)-maackiain involves a distinctive methylenedioxy bridge formation step, predicted to be catalyzed by a cytochrome P450. Specifically, this elusive P450 catalyzes the conversion of calycosin to pseudobaptigenin. We integrated metabolomic and transcriptomic datasets of red clover roots treated with the fungi, Fusarium oxysporum and Phoma medicaginis , to identify candidate P450 genes. Over 40 molecular features were characterized as (iso)flavonoid structures, including the highly abundant O -methylated isoflavones (formononetin and biochanin A), as well as their derivatives. Long infection with P. medicaginis resulted in significant increases in (-)-maackiain, trifolirhizin and other pterocarpans. Concurrently, fungal infections led to upregulation of core and specialized metabolism-related transcripts, including those encoding phenylpropanoid and (iso)flavonoid biosynthetic enzymes. Using weighted gene co-expression network analysis (WGCNA), variance-stabilized expression patterns, and enzyme-class phylogeny, we were able to curate five candidate cytochrome P450s for pseudobaptigenin synthase (PbS) activity, assayed in engineered yeast ( Saccharomyces cerevisiae ). One candidate P450 was capable of methylenedioxy bridge formation, converting calycosin to pseudobaptigenin and pratensein to 5-hydroxypseudobaptigenin. Therefore, it was renamed T. pratense pseudobaptigenin synthase (TpPbS/CYP76F319). The discovery of TpPbS facilitates the reconstruction of the complete (-)-maackiain biosynthetic pathway and the production of this pterocarpan chemistry at scale for health and agricultural applications.