Functional Investigation Of Bibenzyl And Flavonoid Glycosyltransferases From Marchantia Polymorpha

Flavonoids and bibenzyls are two major classes of phenylpropanoid-derived secondary metabolites.Flavonoids are widespread in the plant kingdom,while bibenzyls are exclusively distributed in liverworts and some higher plants such as orchidaceae.Their glycosylation modifications greatly enrich the structure of natural products and have significant effects on the dissolution,storage and metabolism of compounds.Some bibenzyl glycosides have the activity of inhibiting tyrosinase.Flavonoid glycosides have antioxidant,anti-inflammatory and neuroprotective capabilities.Among them,flavonoid glucuronides have received extensive attention due to their medicinal potential and specific metabolism in humans.In plants,the glycosylation of natural products is usually catalyzed by the uridine diphosphate-dependent glycosyltransferases(UGTs),which forms various glycosides by catalyzing the connection between active sugar molecules and glycosyl receptors.Therefore,identification of glycosyltransferases that efficiently and specifically catalyze flavonoids and bibenzyls will provide candidate genes to produce these active compounds using synthetic biology.Liverworts are the first plant lineage that produce flavonoids and bibenzyls.These secondary metabolites have a wide range of biological activities in plants,such as antifungal,antioxidant and insect antifeedant.In the present investigation,the glycosyltransferases involved in the biosynthesis of flavonoids and bibenzyls were functionally characterized from M.polymorpha.1.Gene cloning and functional characterization of bibenzyl glycosyltransferase in M.polymorpha.Two UGTs with characteristic conserved PSPG(Plant Secondary Product Glycosyltransferase)motifs were screened from M.polymorpha genome and named as MpUGT737B1 and MpUGT741A1,respectively.On the phylogenetic tree,MpUGT737B1 and MpUGT741A1 were located in a separate cluster at the root of the 7-O-glycosyltranferase cluster.In vitro enzymatic activity revealed that MpUGTs functioned as bibenzyl glycosyltransferases and specifically accepted UDP-glucose as sugar donor.In addition to bibenzyls,MpUGT737B1 is also active against some structurally similar compounds such as phloretin and phenylpropanoids.Interestingly,MpUGT737B1 could act at the 4-O position of phloretin to produce the rare natural product phloretin-4-O-glucoside.MpUGT741A1 exhibited strict substrate selectivity and could completely convert lunularin into the corresponding glycoside.Feeding experiments in E.coil showed that MpUGT737B1 could convert dihydroresveratrol and phloretin into the corresponding glycosides in vivo,and both the medium and the substrate concentration had an effect on the product yield.Molecular modeling and docking of MpUGTs were carried out to analyze the difference of substrate binding pocket,and explain the possible mechanisms of the divergences in substrate selectivity of MpUGTs from a structural perspective.The gene expression of MpUGT737B1 and MpUGT741 A1 was increased after UV-B irradiation stress,indicating that they may play a role in plant stress resistance.Subcellular localization in tobacco showed that MpUGTs were located in cytoplasm and nucleus.2.Gene cloning and functional characterization of flavonoid glucuronosyltransferases in M.polymorpha.Two candidate UGATs were screened and cloned based on the sequence analysis of the M.polymorpha genome.Phylogenetic tree analysis indicated that they are clustered in the UGT88 family,which contains most of the UGATs.In vitro functional identification results showed that MpUGT742A1 and MpUGT736B1 are flavonoid glucose/glucuronic acid glycosyltransferases.When UDP-glucose was used as the substrate,MpUGT742A1 had a higher-affinity for flavanones and could catalyze the formation of flavanones 7-O-glucoside,while MpUGT736B1 had weak catalytic activity for several flavones.When UDP-glucuronic acid acts as a sugar donor for the catalytic reaction,MpUGT742A1 and MpUGT736B1 had universal conversion ability for flavonoids,catalyzing the production of monoglycosides and diglycosides including some pharmaceutically active compounds,such as scutellarin.The expression levels of MpUGATs genes in M.polymorpha increased significantly after UV-B stress,and they were localized in the nucleus and cytoplasm.This is also the first identified glucuronosyltransferase in liverworts.In this study,four glycosyltransferase genes were identified from M.polymorpha.MpUGT737B1 and MpUGT741A1 are bibenzyl glycosyltransferases,and MpUGT742A1 and MpUGT736B1 are flavonoid glucuronyltransferase with striking broad-spectrum catalytic ability.This is the first time that specific bibenzyl glycosyltransferases have been identified in plants and flavonoid glucuronosyltransferases have been characterized from liverworts.The present investigation will provide candidate genes with different substrate selectivity to synthesize a variety of active glycosides using synthetic biology.