Cloning And Functional Identification Of A Novel Plant Glycosyltransferase Gene CtGT-I From Cistanche Deserticola

Cistanche.tubulosa(Schrenk)Wight(syn.Cistanche Hoffing.et Link,Orobanchaceae)is a precious traditional Chinese medicine,used for nourishing the kidney,benefiting essence-blood,increasing digestion and elimination,and so on.In recent years,the wild resource of C.tubulosa is decreasing fastly and nearly extincted because of adverse living condition and growing medicinal demands.Phenylethanoid glycosides(PhGs)are the main active ingredients in C.tubulosa with obvious structural diversity,cause different number(mono-glucosides,di-glucosides and tri-glucosides)and different types(such as glucosylation,rhamnosylation)of sugars moieties were substituted at different positions(for example,center nucleus is substituted by β-D-glucose,and it’s 3-OH is regio-selectively substitued by rhamnose.).In addition,lignanosides,iridoid glycosides and other kinds of glycosides are also rich in C.tubulosa.So,it can be infered that various and novel glycosyltransferases(GTs)must be existed in the genome of C.tubulosa.Cloning novel GTs genes in C.tubulosa would be take useful hints for exploring related genes which involved in the biosynthesis of phenylethanoid glycosides,on one hand,the obtained glycosyltransferases with novel catalysis activities could be also applied in the enzymatic synthesis of different types of natural or unnatural glycosylated products;on the other hand,building a "unnatural glycosides library",which provide a source for the discovery of pharmaceutically useful leading compounds.In this paper,five novel GTs(CtGT-G、CtGT-H,CtGT-I、CtGT-J、CtGT-K)genes were cloned from C.tubulosa.Among them,CtGT-I was successfully expressed in E.Coli.To identify the function of CtGT-I,enzymatic reactions in vitro combined with HPLC-HR-ESI-MS were performed.The data indicated that CtGT-I proteins exhibited a broad range of acceptor tolerance,accepting a vast arry of compounds including PhGs,flavonoids,2-(2-phenylethyl)chromones,anthraquinones,and coumarins.In addition to UDPG,CtGT-I can accept UDPAG as donors at the same time.Remarkably,CtGT-I could catalyze O-/S-/N-glycosylations,showing promiscuous catalytic behaviours.Acteoside and echinacoside were chosen as glycosyl receptors to perform the large-scale enzymatic reaction.Glycosylated productsto were separated and structully identified with HR-EST-MS and NMR analysis.Data showed that CtGT-I can catalyze glycosylation of acteoside and echinacoside at 3’-OH position of caffeoyl group to form novel glucosidic compounds.In addition,CtGT-I can catalyze glycosylations of three 2-(2-phenylethyl)chromones which are substitued by different types of hydroxyl and chlorine groups at 6-OH,7-OH or 8-OH positions on ring A to form three novel(2-(2-phenylethyl)chromones glucosides,which were rarely reported in natural.Besides,the configuration of the glycosylated products was β-glucose.Our subsquential investigations aim at revealing GTs genes involved in the biosynthetic pathways of the active ingredient PhGs in C.tubulosa.So,the syntheses of UDP-rhamnose donor(3-OH position of β-D-glucose is region-selectively substitued by rhamnose moiety in PhGs)and acyl donors(4-OH/6-OH of β-D-glucose are usually substitued by coumaroyl,caffeoyl and other acyl groups)were of great significance for follow-up research.In this study,rhamnose synthase RHM2 from Arabidopsis thaliana were applied to synthesize UDP-Rha with two-step enzyme reactions using UDP-Glc as substrate.The reported glycosyltransferase AtUGT78D1 from A.thaliana with known activity as flavonols:3-O-rhamnosyltransferase was used as the positive catalyst to verify the synthesized UDP-Rha product.Surprisingly,expanding enzymatic reactions and functional characterizations revealed the extensive activities of AtUGT78D1 cause it can not only catalyze glycosylation of flavonols at 3-OH position,other types of flavonoids as well as a vast arry of compounds including coumarin,anthraquinone,2-(2-phenylethyl)chromones can also be accepted to produce a series of corresponding rhamnosylated product.In addition,4-coumarate coenzyme A ligase(4CL)from A.thaliana was cloned and expressed to catalyze eight kinds of organic acids with CoA to synthesize acyl donors.In conclusion,in this study novel GTs gene CtGT-I were cloned from C.tubulosa,and functional characterization of CtGT-I exhibited a broad range of acceptor tolerance,such as a vast arry of compounds including PhGs,flavonoids and flavonoid glycosides,2-(2-phenylethyl)chromones,coumarin,anthraquinone and different types of natural products.Two novel PhGs with glucosylation at caffeoyl 3’-OH position and three 2-(2-phenylethyl)chromones glucosides were chemo-enzymatic synthesized.Besides,enzymatic synthesis methods of UDP-Rha and acyl-CoA donor were successfully established which laid solid foundation for revealing genes involved in the biosynthesis of phenylethanoid glycosides in subsequent studies.Cloning and functional characterization of CtGT-I suggested it could be used as a universal glucosyltransferase being applied in the enzymatic synthesis of different types of natural or unnatural glycosylated products,to provide a source for the discovery of pharmaceutically useful leading compounds,with both theoretical and practical significance.