Abstract
Berberine is an effective antimicrobial and antidiabetic alkaloid, primarily extracted from divergent botanical lineages, specifically Coptis (Ranunculales, early-diverging eudicot) and Phellodendron (Sapindales, core eudicot). In comparison with its known pathway in Coptis species, its biosynthesis in Phellodendron species remains elusive. Using chromosome-level genome assembly, coexpression matrix, and biochemical assays, we identified six key steps in berberine biosynthesis from Phellodendron amurense, including methylation, hydroxylation, and berberine bridge formation. Notably, we discovered a specific class of O-methyltransferases (NOMT) responsible for N-methylation. Structural analysis and mutagenesis of PaNOMT9 revealed its unique substrate-binding conformation. In addition, unlike the classical FAD-dependent berberine bridge formation in Ranunculales, Phellodendron uses a NAD(P)H-dependent monooxygenase (PaCYP71BG29) for berberine bridge formation, originating from the neofunctionalization of tryptamine 5-hydroxylase. Together, these findings reveal the convergence of berberine biosynthesis between Coptis and Phellodendron and signify the role of the convergent evolution in plant specialized metabolisms.