Identification and Characterization of a Putative Kaempferol Glucosyltransferase UGT78G3 in Medicago truncatula

UDP-glycosyltransferases (UGTs) represent a large multigene family that play a central role in glycosylating a highly diverse array of natural products, underscoring their critical importance in various biological processes. However, the functional roles of a substantial majority of UGTs remain to be elucidated. In the present study, we characterized the glycosyltransferase UGT78G3, a member of the UGT78 glycosyltransferase family in the model legume <i>Medicago truncatula</i>. Amino-acid sequence analysis revealed a conserved PSPG motif at the C-terminus of UGT78G3. Liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) analysis demonstrated that UGT78G3 catalyzes the formation of kaempferol 3-<i>O</i>-glucoside in vitro. However, neither <i>UGT78G3</i> overexpression nor CRISPR/Cas9-mediated mutagenesis resulted in significant changes to the endogenous levels of kaempferol 3-<i>O</i>-glucoside, indicating that UGT78G3 does not play a predominant role in the biosynthesis of kaempferol 3-<i>O</i>-glucoside in vivo. Our findings identify a putative glycosyltransferase in <i>M. truncatula</i> and provide a target for biocatalyst design aimed at synthesizing flavonoid glucosides.