Convergent evolution of berberine biosynthesis.

Berberine is an effective antimicrobial and antidiabetic alkaloid, primarily extracted from divergent botanical lineages, specifically (Ranunculales, early-diverging eudicot) and (Sapindales, core eudicot). In comparison with its known pathway in species, its biosynthesis in species remains elusive. Using chromosome-level genome assembly, coexpression matrix, and biochemical assays, we identified six key steps in berberine biosynthesis from , including methylation, hydroxylation, and berberine bridge formation. Notably, we discovered a specific class of -methyltransferases (NOMT) responsible for -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, 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 and and signify the role of the convergent evolution in plant specialized metabolisms.