Gymnosperm-specific CYP90Js enable biflavonoid biosynthesis and microbial production of amentoflavone

Biflavonoids, a unique subclass of flavonoids with superior clinical activity compared to their monomeric counterparts, offer distinct therapeutic benefits by targeting multiple pathways in neurodegenerative disorders. However, the mechanism of flavonoid dimerization in plants remains enigmatic. Here, we identify CYP90J orthologs as the missing link in biflavonoid biosynthesis. We demonstrate that gymnosperm-specific CYP90Js catalyze intermolecular C-C bond formation in the biosynthesis of biaryl natural products. Together with the identified O-methyltransferases, CYP90Js are responsible for the production of ginkgo biflavonoids. Phylogenetic analysis reveals that the CYP90J subfamily evolved from CYP90E of lycophytes and is found exclusively in gymnosperms. Molecular dynamics simulations show that regioselective dimerization of apigenin to amentoflavone is driven by spatial constraints and π-π stacking interactions. QM/MM calculations support a heme-induced diradical coupling mechanism. Notably, the de novo reconstruction of amentoflavone was achieved in an engineered L-tyrosine E. coli strain, with a titer of 4.75 mg/L. Overall, the discovery of CYP90Js represents a crucial step toward understanding flavone dimerization, and the engineering of biflavonoids in microorganism provides a promising biotechnology platform for expanding therapeutic applications of biflavonoids.