Functional Characterization Of Key Enzymes In The Biosynthesis Of Flavonol Glycosides From Cudrania Tricuspidata

Flavonoids are a class of secondary metabolites that are widely distributed in plants.In plants,flavonoids have the functions of regulating auxin transport,male fertility,protection against UV-B radiation,participating in the coloration of flowers and fruits,attracting symbionts,and recruiting pollinating insects,as well as participating in plant responses to specific hormones.Flavonoids have a wide range of health benefits and various pharmacological effects.Flavonols and their glycoside derivatives have been shown to have pharmacological effects such as antioxidant,antitumor,cardiovascular protection and so on.Flavonoid glycosides are widely present in plants,the largest number of which is flavonol oxyglycosides.Glycosylation is usually the last step of natural product modification.Glycosylation can improve the water solubility of flavonoids,thereby improving their bioavailability.Moreover,the production of flavonoid glycosides using methods such as enzymatic catalysis of plant glycosyltransferase and engineering bacterial transformation is relatively economical and convenient,and has a great application prospect.The mulberry plant Cudrania tricuspidata contains a large number of flavonoids,however,the biosynthetic pathway of flavonoids in Cudrania tricuspidata has not yet been characterized.This study mainly focuses on the biosynthesis of flavonol glycosides.F3H(Flavanone 3-hydroxylase)catalyzes the formation of dihydroflavonols from flavanones.FLS(Flavanol synthesis)catalyzes the formation of flavonols from dihydroflavonols,and flavonols are glycosylated by UGT(UDP glycosyltransferase)to form corresponding glycosides.The purpose of this study is to identify the key enzymes F3H,FLS and UGT in the biosynthesis of flavonol glycosides in Cudrania tricuspidata and study their catalytic properties to provide candidate genes for the biosynthesis of flavonol glycosides.The following studies have been conducted:1.Gene cloning and functional characterization of F3H and FLS in Cudrania tricuspidataThe candidate genes of F3H and FLS were screened by searching for the keyword"flavonoid 3-O-glucosyltransferase" from the transcriptome database of Cudrania tricuspidata and bioinformatics analysis.The recombinant protein was heterologously expressed,and the purified protein was subjected to enzymatic activity verification in vitro.CtrF3H1 and CtrF3H2 had the activity of flavanone 3-hydroxylase(F3H),which catalyzed naringin to produce dihydrokaempferol.CtrFLS had the activity of flavonol synthase(FLS),which catalyzed dihydrokaempferol to produce kaempferol.The results of substrate selectivity experiments showed that CtrF3H1 and CtrF3H2 had a difference in substrate selectivity,and CtrF3H2 had a more extensive substrate specificity.CtrF3H2 had strong catalytic activity for the four flavanones of naringin,eriodictyol,hesperidin,liquiritigeninan,while CtrF3H1 had strong catalytic activity only for naringin and eriodictyol.Through amino acid site-directed mutagenesis,the enhancement of substrate specificity of CtrF3H1 had been achieved,and the key amino acid site that affects the substrate specificity of CtrF3H1 and CtrF3H2 had been identified.CtrF3H1 also had the function of F3H in Escherichia coli.The function of CtrF3H1 in plants was further studied,and the results showed that the content of flavonols such as kaempferol and quercetin and flavone such as chrysoeriol and apigenin in CtrF3H1 transgenic Arabidopsis thaliana was increased compared to tt6 plants,indicating that CtrF3H1 performed a dual function of FNS I/F3H in plants.2.Gene cloning and functional characterization of UGT in Cudrania tricuspidataThe candidate genes of UGT were screened by searching for the keyword"flavonoid 3-O-glucosyltransfer" from the transcriptome database of C.tricuspidata and bioinformatics analysis.The recombinant UGT protein was heterologously expressed,and enzymatic activity of purified protein in vitro had been studied by using UDP-glucose as sugar donor and flavonoids as substrate.The results showed that CtrUGT1 and CtrUGT2 had glycosylation activity against various flavonoids.CtrUGT1 catalyzed more flavonoids and had stronger activity than CtrUGT2,and CtrUGT1 had the activity to catalyze the formation of corresponding glucosides at positions 3,7,and 3’,respectively,while CtrUGT2 catalyzed glycosylation at positions 3 and 7,respectively.CtrUGT1 also had stronger catalytic activity and efficiency in Escherichia coli.CtrUGT1 can be used as a candidate enzyme for flavonoid glycoside biosynthesis.