Functional Analysis of FsUGT74F1 in the Biosynthetic Pathway of Phillyrin from Forsythia suspensa

In this study, a UDP-glycosyltransferase gene from Forsythia suspensa was systematically identified and functionally characterized. Phylogenetic analysis showed that this gene clustered with members of the UGT74 subfamily, where it clustered closely with members of known function; accordingly, it was provisionally designated as FsUGT74F1. Prokaryotic expression experiments determined that soluble recombinant protein could be obtained under induction conditions of 0.1 mM IPTG at 16 °C for 8 h. In vitro enzyme activity assays suggested that FsUGT74F1 might catalyze the glycosylation of phillygenin using UDP-glucose under the tested conditions, yielding a product whose HPLC retention time was consistent with that of authentic phillyrin. Subcellular localization results indicated that FsUGT74F1 was predominantly associated with the plasma membrane. Transient overexpression of FsUGT74F1 in F. suspensa leaves resulted in upregulation of gene expression by approximately 247.6%, and the phillyrin content increased to 124.7% of that in the control group. Conversely, virus-induced gene silencing (VIGS) of FsUGT74F1 led to a reduction in transcript levels by approximately 35.6%, and the phillyrin content decreased to 72.5% of the control level. Molecular docking suggested that phillygenin could interact with FsUGT74F1, with Leu169 predicted to participate in substrate positioning near the C4′-OH group. Collectively, these results support the involvement of FsUGT74F1 in phillyrin biosynthesis and provide a theoretical basis for its application in metabolic engineering.