Screening And Functional Identification Of UDP-glycosyltransferases Involved In Biosynthesis Of Astringent Phenolic Compounds

Catechins(flavan-3-ols)and flavonol 3-glycosides are the pivotal components that contribute to health care and pharmacological functions.These compouds are of high content among tea polyphenols.Moreover,they are believed to be the typical astringent taste compounds in tea infusions.In previous study work,the biosynthesis of galloylated catechins has been confirmed to involve two enzymes,and 1-O-galloyl-β-D-glucose(βG)is the immediate biosynthetic precursor.βG was biosynthesized by UDP-glucose:galloyl-1-O-β-D-glucosyltransferase(UGGT)with the substrates gallic acid(GA)and UDP-glucose.As for flavonol 3-glycosides,their biosyntheses depend on the catalysis of flavonol 3-glycosyltransferases(F3GT).Termed UGGT and F3 GT,they both belong to glycosyltransferase family 1 UGTs(GT1),and participate in the biosyntheses of tea astringent compounds.Thus,it makes great difference in breeding and quality regulation of tea plants to study on the UGT genes as well as their functions which involves biosynthesis of astringent metabolites.In this paper,candidate UGT genes were screened out from Camellia sinensis transcriptome database based on NCBI-blastp analysis or online Jpred prediction and cloned by full length race,then function prediction was made by phylogenetic analysis and homology modeling,finally candidate Camellia sinensis UGTs were expressed in E.coli novablue and functionally identified by assaying recombinant enzymes in vitro.The main research results are as follows:1.Blastp search against NCBI database for GT sequences in transcriptome sequencing data revealed CsUGT84A1 displayed more than 80% identities with three V.vinifera VvgGTs,thus CsUGT84A1 was screened out as the candidate UUGT gene for further function identification.In addition,Jpred,the online software was used to make function prediction.CsUGT78E1 and CsUGT78F1 were found the most close to the identified V.vinifera UDP-glucose: anthocyanidin 3-O-glucosyltransferase(accession number:P51094.2,PDB id: 2c9 z,VvGT1),and exhibited 55% and 53% identities,respectively.So CsUGT78E1 and Cs UGT78F1 were predicted as flavonoid related 3-O-glycosyltransferase genes,and screened as candidate F3 GT genes.2.CsUGT84A1、CsUGT78E1 and Cs UGT78F1 were cloned by full length race,and designated uniformly as CsUGT84A22 、 CsUGT78A14 and CsUGT78A15 by UGT Nomenclature Committee.Phylogenetic analysis and function prediction for the amino acid sequences of these three UGTs were performed by MEGA.As a result,CsUGT84A22 clustered with the 1-O-ester forming GTs,and was predicted as a UGGT gene,while CsUGT78A14 and CsUGT78A15 were grouped into flavonoid3-O-glycosyltransferase cluster,and predicted as flavonol related 3-O-glycosyltransferase genes.3.In order to further make function prediction for CsUGT78A14 and CsUGT78A15 proteins,homology models of CsUGT78A14 and CsUGT78A15 were constructed with the matching crystal structure 2c9z(VvGT1)by PyMoL Molecular Graphics System.Homology modeling for these two Cs UGTs revealed that they shared a highly conserved tertiary structure with VvGT1.Multiple sequences alignment by ClustalX indicated CsUGT78A14 and CsUGT78A15 possessed the most similar catalytic residues with VvGT1.However,It is noteworthy that the last residue of the conserved PSPG box with Q(Gln)in CsUGT78A14 and H(His)in CsUGT78A15.we thus predicted CsUGT78A14 and CsUGT78A15 may encode flavonoid 3-O-glucosyltransferase(F3GlcT)and flavonoid3-O-galactosyltransferase(F3Gal T),respectively,in tea.4.The recombinant plasmids of pMAL-c2x-CsUGT84A22,pMAL-c2x-CsUGT78A14 and pMAL-c2x-CsUGT78A15 were constructed and transformed into expression of host Novablue(DE3).The genes were induced by 0.1mmol/L IPTG with the temperature of 28℃,and the recombinant proteins are purified by affinity chromatography on an amylase resin.Enzymatic products were analyzed by HPLC and UPLC-MS/MS techniques.As supposed,r CsUGT84A22 could catalyze the glucose conjugation of gallic acid to form βG,Meanwhile,CsUGT78A14 and CsUGT78A15 were found to be responsible for the biosynthesis of flavonol3-O-glucosides and flavonol 3-O-galactosides,respectively.5.Kinetic assays indicated that gallic acid among hydroxybenzoic acids(C6-C1)and p-coumaric acid among hydroxycinnamic acids(C6-C3)were the preferred substrates for.UDP-glucose and quercetin were the proper sugar donor and acceptor substrates for rCsUGT78A14,while UDP-galactose and myricetin were the best sugar donor and sugar acceptor for rCsUGT78A15.To conclude the results above,CsUGT84A22、CsUGT78A14 and CsUGT78A15 are characterized as Camellia sinensis UDP-glycosyltransferases,directly or indirectly participating the biosynthesis of astringent metabolites in tea plant.