Functional Identification And Biocatalysis Of Key Enzymes In Safflower Flavonol Glycoside Biosynthesis

Safflower,a traditional Chinese medicine with anti-tumor,anti-oxidation and anti-inflammatory effects,is the dried corolla of Carthamus tinctorius L.Flavonols and their glycosides are one of the main active components.To explore the biosynthesis pathway of flavonol glycosides,we studied the catalytic properties systematically towards the key enzymes in flavonol glycosides metabolism of C.tinctorius,including flavanone 3-hydroxylase(F3H),flavonol synthase(FLS)and glycosyltransferase(GT).Results are as follows..The flavanone 3-hydroxylase(CtF3H),flavonol synthase(CtFLS)and glycosyltransferase genes were successfully searched and cloned from safflower,and the exogenous expression of the genes were realized in E.coli.CtF3H could catalyze the C-3 hydroxylation of flavanones to form dihydroflavonols.The catalytic activity of flavanone 3-hydroxylase CtF3H was evaluated by several pairs of flavanone isomers.The results of in vitro enzymatic reaction showed that the recombinant enzyme CtF3H could specifically recognize S-flavanone substrate including(2S)-naringenin,(2S)-eriodictyol,(2S)-liquiritigenin,(2S)-hesperetin and(2S)-farrerol,then corresponding 3-hydroxylation products were efficiently generated with the conversion rate of over 70%.The 3-hydroxy configurations were all ?-configuration,indicating that CtF3H had strict position selectivity and stereoselectivity.The structure and configuration of enzymatic reaction products were determined by MS,NMR and optical rotation after amplifing five dihydroflavonol products.Enzymological properties showed that the optimum temperature and pH of CtF3H were 35? and 7.0 respectively,depending on the divalent metal ion Fe2+.Kinetic analysis of enzymatic reaction showed that the KM value of CtF3H to(2,S)-naringenin was 16.2 ?M under the optimal conditions.CtFLS could catalyze the oxidative dehydrogenation of dihydrokaempferol and dihydroquercetin to form corresponding products kaempferol and quercetin respectively.Enzymological properties showed that CtFLS exhibited high catalytic activity at 25? and pH 6.0,and depended on divalent metal ion Fe2+.Kinetic analysis of enzymatic reaction showed that the KM value of CtFLS pair(2R,3R)-dihydrokaempferol was 73.9?M under the optimal conditions.In vitro reaction results showed that recombinant glycosyltransferase UGT88B2 had high O-glycosylation activity for flavonoids of various structural types,and could catalyze some substrates to form multiple glycosylation products.For example,the enzyme could catalyze kaempferol to form not only 3 mono-O-glucosides,but also at least 2 bis-O-glucosides.UGT88B2 showed high efficiency and diverse catalytic performance,which was expected to become an efficient biocatalyst for enzymatic synthesis of various bioactive glycoside drugs.Enzymological properties showed that UGT88B2 had high catalytic activity at 45? and pH 8.0.Kinetic analysis of enzymatic reaction showed that the KM value of UGT88B2 to kaempferol was 39.1 ?M under the optimum conditions.Based on the above,a glucuronosyltransferase(UGT88D7)gene derived from Perilla frutescens and CtF3H,CtFLS were introduced into E.coli to construct a whole-cell catalytic system,which could efficiently convert naringenin into kaempferol-7-O-?-D-glucuronide with high PTP(IB)inhibitory activity.Adding 8 mg naringenin into the system,the yield of the product could reach 70%.In conclusion,three genes were cloned and expressed from safflower in this paper.Recombinant enzymes CtF3H,CtFLS and UGT88B2 had good catalytic characteristics and could be used as tool enzymes for enzymatic synthesis of flavonols and their glycosides.Besides,a whole-cell catalytic system for efficiently producing kaempferol-7-O-?-D-glucuronide from naringenin was constructed.These results provided experimental basis for the biosynthesis of active flavonols and their glycosides,and had theoretical value and practical application potential.