Study On Key Enzymes In The Biosynthesis Of Active Glycosides From Safflower

Safflower,which is the dried corolla of Carthamus tinctorius L.in Composite family,is a traditional Chinese herb.It has anti-inflammatory and analgesic effects,improves heart and brain ischemia,anti-cancer,anti-tumor and anti-oxidant effects.The active glycosides in safflower are mainly flavonoid oxygen glycosides and quinoid chalcone carbon glycosides.The key enzymes of biosynthesis about these glycosides mainly include glycosyltransferase and hydroxylase,especially C-glycosyltransferase responsible for the synthesis of quinoid chalcone carbon glycosides and oxidative monooxygenase,which have not been reported.In order to discover the key enzymes responsible for the synthesis of active glycoside compounds in safflower and clarify the biosynthetic pathway of active glycosides,this study explored the glycosyltransferase and cytochrome P450 monooxygenase in safflower and their catalytic properties were studied.Contents and results of this study are as follows.The O-glycosyltransferase gene CtGT3 was cloned from the fresh corolla of C.tinctorius and its functional expression was realized in E.coli.In vitro enzyme catalytic activity studies,CtGT3 could catalyzed the glycosylation of seven flavonoids.In addition,CtGT3 has a broad substrate spectrum,and could also catalyze the glycosylation of a variety of non-flavonoids or even non-aromatics compounds,and the conversion rate of some non-flavonoid substrates could reach 100%.CtGT3 can be used as a tool enzyme for the enzymatic synthesis of active natural/non-natural glycosides.C-glycosyltransferase AbCGT from Aloe vera L.was used to successfully introduce C-glycosyl groups into the A ring of naringenin chalcone to generate 3'-C-?-D-glucosylnaringenin,a key intermediate for the biosynthesis of hydroxysafflor yellow A.After optimizing the catalytic conditions,the conversion rate of AbCGT to naringenin chalcone could reach more than 50%.The product of the enzymatic reaction was identified by MS and NMR as 3'-C-?-D-glucosylnaringenin chalcone,which spontaneously cyclized to naringenin 6-C-?-D-glucoside and naringenin 8-C-?-D-glucoside.It not only realized the C-glycosylation reaction in the biosynthesis of hydroxysafflor yellow pigment,but also provided an intermediate for the study of subsequent key steps.In order to explore the hydroxylation process of active glycosides,the differential transcriptome analysis of the red flower,yellow flower and white flower varieties of C.tinctorius was completed,and 15 CYP450 monooxygenase genes were locked and cloned,which were expressed in the Saccharomyces cerevisiae WAT11 system.The experimental results showed that 15 candidate oxidases did not show catalytic activity.The expression system will be optimized later and more possible substrates will be tried.In addition,it is necessary to expand the screening range of candidate genes.In summary,this paper explored aglycon promiscuity of an O-glycotransferase CtGT3 in the active glycoside biosynthesis pathway of safflower,synthesized an important intermediate of hydroxysafflor yellow A with C-glycotransferase AbCGT from A.vera,and excavated the hydroxylase gene in the active glycoside biosynthesis pathway of safflower and identified its function.These results provide experimental data for further studies on the biosynthesis pathway of safflower active glycosides.