The Discovery And Identification Of Ornithogalum Glycosyltransferases And Their Applications In Glycodiversification

Glycosylation of natural products is widespread in animals,plants and microorganisms.The glycosylation-modified small molecule compounds have better pharmacological activity,higher bioavailability and less toxicity.In organisms,glycosylation was accomplished by different functional glycosyltransferases.Through in-depth study of the functions and properties of glycosyltransferases,highlighting their advantages to achieve the production of natural glycosides by using biosynthesis techniques.This article explores the glycosyltransferase genes OcUGT1 and OsUGT1 which were cloned from Omithogalum,and finished functional identification and analysis of enzymatic characterization.We found that OcUGT1 not only use UDP-sugar to achieve the classical glycosylation reaction,but also can use low-cost glycosides in transglycosylation to achieve glycosylation without adding UDP or UDP-sugar.In addition,OcUGTl can catalyzes three kinds of reversible reactions,namely UDP-sugar synthesis reaction,sugar or aglycone exchange reaction and hydrolysis reaction.As an aurone glycosyltransferase,OsUGTl can specifically catalyzes the glycosylation reaction of sulfuretin and also has transglycosylation and hydrolysis activity.The finding of this result indicates that OcUGTl has both activitives of glycosyltransferase and glycosidase.This result broadens the application prospects of OcUGT1,and the verification of multifunction lays a solid foundation for the subsequent use of biosynthesis techniques for the large-scale preparation of small-molecule glycosides.1.Isolation and characterization of a multifunctional flavonoid glycosyltransferase OcUGTl from Ornithogalum caudatumGlycosyltransferases(GTs)are bidirectional biocatalysts catalyzing the glycosylation of diverse molecules.However,the extensive applications of GTs in glycosides formation are limited due to their requirements of expensive nucleotide diphosphate(NDP)-sugars or NDP as the substrates.Here,in an effort to characterize flexible GTs for glycodiversification of natural products,we isolated a cDNA,designated as OcUGT1 from Ornithogalum caudatum,which encoded a flavonoid GT that was able to catalyze the trans-glycosylation reactions,allowing the formation of glycosides without the additions of NDP-sugars or NDP.In addition,OcUGTl was observed to exhibit additional five types of functions,including classical glycosylation reaction and three reversible reactions namely NDP-sugar synthesis,sugars exchange and aglycons exchange reactions,as well as enzymatic hydrolysis reaction,suggesting OcUGTl displays both glycosyltransferase and glycosidase activities.2.Biosynthesis of 7,8-dihydroxyflavone glycosides via OcUGT1-catalyzed glycosylation and transglycosylationOcUGT1 was determined to be able to attack C-8 position of 7,8-dihydroxyflavone(7,8-DHF)via both glycosylation and transglycosylation reactions.OcUGT1-catalyzed glycosylation of 7,8-DHF resulted in the formation of two monoglycosides 7-O-β-D-glucosyl-8-hydroxyflavone,7-hydroxy-8-O-β-D-glucosylflavone,as well as one diglycoside 7,8-di-O-β-D-glucosylflavone.Under the action of OcUGT 1,inter-molecular trans-glycosylations from aryl β-glycosides to 7,8-DHF to form two monoglycosides were observable.3.OcUGTl-catalyzed glycosylation of testosterone with alternative donor substratesTestosterone 17-O-β-D-glucoside(T-17-G)is a precursor of anticancer compound 3’-acetylated testosterone 17-O-β-D-glucoside(3’-AT-G).The biosynthesis of T-17-G was achieved by glycosyltransferase(GT)-mediated glycosylation of testosterone with UDP-Glc.The low availability of expensive UDP-Glc was detrimental to the synthesis of T-17-G,therby limiting the druggability study of 3’-AT-G.Therefore,finding cheap alternatives of sugar donors is particularly urgent for T-17-G biosynthesis.Specifically,a flavonoid glycosyltransferase OcUGT1 from Ornithogalum caudatum was used as a biocatalyst to test the reactivities of various sugar donors with testosterone.Results indicated that OcUGT1 was able to catalyze the glycosylation of testosterone with UDP-Glc,thereby forming T-17-G and a diglucoside.Also,the generation of T-17-G was achieved by OcUGT1-assisted transglucosylation between testosterone with oNP-Glc or 4-nitrophenylβ-D-glucopyranoside(pNP-Glc).4.Discovery and identification of an aurone glycosyltransferase from Ornithogalum saundersiaeAurone glycosides display a variety of biological activities.However,the reports about GTs responsible for aurones glycosylation are limited.Here,the discovery and identification of an aurone glycosyltransferase is reported.Specifically,a cDNA,designated as OsUGTl,was isolated from the plant Ornithogalum saundersiae based on transcriptome mining.Conserved domain(CD)-search speculated OsUGT1 as a flavonoid GT.Phylogenetically,OsUGT1 did not cluster into the same group with other known flavonoid GTs,hinting that OsUGT1 encoded a new group of flavonoid GT capable of glycosylating aurones.In vitro enzymatic analyses showed that OsUGT1 was able to catalyze sulfuretin to form corresponding monoglycosides.OsUGT1 was observed to be a flavonoid GT,specific for flavonoid substrates.Moreover,OsUGT1 was demonstrated to display glycosidase activity,exerting intermolecular transglucosylation and hydrolysis actions.