Gene duplication and clustering underlie the conservation and diversification of benzylisoquinoline alkaloid biosynthesis in plants

Benzylisoquinoline alkaloids (BIAs), comprising ~2500 compounds with pharmacological significance, are well-studied in Ranunculales but poorly understood in Magnoliids, an early-diverging angiosperm group. This study characterizes key enzymes in Houttuynia cordata-including 6-OMT, NMT, CYP80B, and 4'OMT-that form BIA backbones and uncovers a CYP80G-mediated phenol coupling reaction in isoboldine biosynthesis. Functional analysis reveals conservation of BIA backbone formation genes between Magnoliids and Ranunculales, with evidence of gene duplication and neofunctionalization in H. cordata. Genome-wide analysis identifies dynamic clustering of CYP80B with 4'OMT and 6-OMT genes across angiosperms, reflecting their interlinked biochemical roles in the formation of BIA backbones. These findings suggest that such gene clustering may evolved through biochemical coordination, offering insights into the evolutionary mechanisms behind plant gene cluster formation. The study provides a foundation for understanding BIA biosynthesis across flowering plants and supports synthetic biology strategies to produce high-value BIAs.