Sarcandra glabra genome unveils functional conservation and divergence of O-methyltransferases catalyzing isofraxidin biosynthesis in angiosperms.

Isofraxidin, a characteristic coumarin extracted from the root of Sarcandra glabra, prevents human hepatoma cell invasion, while its biosynthesis remains elusive. Here, we construct a 4.23-Gb chromosome-level genome of S. glabra. We find that its intergenic and genic regions are profoundly longer than those of other angiosperms. Among the root-enriched O-methyltransferase candidates, SgOMT3 produces comparable amounts of isofraxidin and the byproduct fraxidin from fraxetin, while SgOMT5 only produces fraxidin. SgOMT3 and SgOMT5 are dispersed duplication genes, and site-directed mutagenesis reveals that Leu127 in SgOMT3 and Val292 in SgOMT5 are the key residues for isofraxidin and fraxidin biosynthesis, respectively. SgOMT3 orthologs from eudicot species preferentially produce more isofraxidin than fraxidin. The molecular docking and site-directed mutagenesis reveal that a Leu-to-Ile substitution at position 307 alters the regioselectivity of SgOMT3, shifting the conversion predominantly toward isofraxidin. These findings accelerate germplasm improvement of S. glabra and provide evidence for isofraxidin biosynthesis in chassis.