Identification and functional analysis of glycosyltransferase catalyzing the synthesis of phlorizin and trilobatin in Lithocarpus polystachyus Rehd.

The leaves of Lithocarpus polystachyus Rehd. are rich in dihydrochalcone compounds phlorizin and trilobatin, which are substances of great medicinal importance and may serve as potential sources for apple-derived phlorizin and trilobatin substitutes. In this study, the uridine diphosphate-glycosyltransferase (UGT) gene family in the L. polystachyus transcriptome was identified and analyzed at different developmental stages, and 107 UGTs were obtained, containing 16 subfamilies. The comparative analysis confirmed that a more complete UGT gene family was obtained for L. polystachyus. Following the comparative analysis of UGT gene expression and metabolite content, four candidate phloretin 2'-O-glucosyltransferase (LpP2`GT) genes catalyzing phlorizin synthesis and three phloretin 4'-O-glucosyltransferase (LpP4`GT) genes catalyzing trilobatin synthesis were screened in the E and L subgroups of the L. polystachyus UGT gene family. The functions of LpP2`GT (Cluster-6439.143031, Cluster-6439.111627) and LpP4`GT (Cluster-6439.98883) were expressed and validated in vitro; enzyme kinetic analysis showed that the affinity and catalytic efficiency of Cluster-6439.143031 recombinant for phloretin were higher than Cluster-6439.98883. Molecular docking results suggest that this may have been caused by a difference in the efficiency of the LpUGT proteins in catalyzing sugar donor protonation, which affected the nucleophilic substitution reaction with uridine diphosphate-glucose (UDP-glucose), leading to differences in activity.