Functional characterization of two <i>O</i> ‑methyltransferases and a glycosyltransferase catalyzing the last two steps of phillyrin biosynthesis in <i>Forsythia suspensa</i>

Phillyrin, a unique antiviral lignan exclusive to Forsythia suspensa, shows potent anti-influenza activity. However, its broad pharmaceutical application is hindered by low and variable natural accumulation, underscoring the need to elucidate its biosynthetic pathway for sustainable production. In this study, we used an integrated metabolomic and transcriptomic approach to investigate tissue-specific phillyrin biosynthesis in F. suspensa. We identified two O-methyltransferases (FsE4MT01 and FsE4MT02) that catalyze the methylation of (+)-epipinoresinol to form phillygenin and a UDP-glycosyltransferase (FsP4'GT) that glucosylates phillygenin to yield phillyrin. Using recombinant proteins expressed in Escherichia coli, we validated their in vitro catalytic activities and determined their kinetic parameters. Molecular docking and site-directed mutagenesis of key residues further revealed the structural basis of their catalytic mechanisms. The in vivo functions of these enzymes were confirmed through heterologous expression in Nicotiana benthamiana and F. suspensa leaves. Importantly, by developing a transient leaf-expression system in F. suspensa, we demonstrated that overexpression of FsE4MTs and FsP4'GT substantially increased the production of phillygenin and phillyrin, respectively, whereas virus-induced gene silencing (VIGS) of these genes reduced the accumulation of the corresponding products. Notably, enhanced phillyrin production was also achieved by expressing FsE4MTs and FsP4'GT in the related species Forsythia × intermedia, highlighting the potential of these key enzymes for metabolic engineering. Our findings elucidate the terminal steps of phillyrin biosynthesis and provide a strategic foundation for engineering the sustainable production of this pharmaceutically valuable compound.