Identification Of Glycosyltransferases Related To Panax Notoginseng Saponins Biosynthesis

Panax notoginseng,a perennial herb of the genus Panax in the family Araliaceae,is one of the traditional precious medicinal materials in China,which is mainly produced in Yunnan and Guangxi provinces.It has the functions of dispelling blood stasis,stopping bleeding,reducing swelling and pain.The main active components in Panax notoginseng is Panax notoginseng saponin(PNS),which have a wide range of pharmacological effects,such as hemostasis and activating blood,anti-cancer,anti-tumor,and anti-oxidation activities.Most PNSs exit in free form or in the form of glycosides or esters combined with sugars.According to the number of rings in their structure,they were mainly divided into tetracyclic dammarane-type saponins and pentacyclic oleanane-type saponins.Dammarane-type saponins were divided into three categories due to the different hydroxyl modification of the skeleton:dammarenediol-type,protopanaxadiol-type and protopanaxatriol-type.Glycosylation modification of saponin is the most important modification process in the biosynthesis of PNSs,which is mainly catalyzed by UDP-glycosyltransferase and cellulose synthase like glycosyltransferase.Nowadays,with the rapid development of synthetic biology,the production of natural products by microbial cell engineering has become a trend.However,the synthetic pathway of PNSs is not clear,especially the synthesis pathway of pentacyclic oleanane-type saponins is still unknown,which makes it difficult to carry out extensive and in-depth research on notoginsenosides.Therefore,exploring and perfecting the biosynthetic pathway of PNSs is the focus of current research.In this paper,we treated Panax notoginseng callus at low temperature to analyze the changes in the expression levels of genes involved in the synthesis of PNSs to discover the glycosyl-modifying genes involved in the biosynthetic pathways of different types of PNSs.The following experimental results were obtained:(1)We performed RNA-seq on Panax notoginseng callus treated at 4℃ for 12 hours and untreated control cells.The analysis found that cold treatment changed the expression levels of genes involved in the synthesis pathway of PNSs,among which the expression level of genes(dammarenediol synthase and protopanaxadiol synthase etc.)related to the synthesis of dammarane-type tetracyclic saponins increased,while the expression level of genes(β-Amyrin synthase and oleanic acid synthase,etc.)related to the synthesis of oleanane-type pentacyclic saponins decreased;(2)We constructed a phylogenetic tree with all glycosyltransferases from Panax notoginseng and glycosyltransferases with known functions in other species involved in the synthesis of triterpene saponins.Combining with the differential expression patterns of glycosyltransferase genes after cold treatment,we screened and cloned 16 UDP-glycosyltransferases and 1 cellulose synthase-like that may be involved in the synthesis of saponin in Panax notoginseng.By constructing expression vectors and expressing heterologously,we obtained the proteins encoded by these genes(3)The functional analysis of the candidate glycosyltransferase and cellulose synthase was carried out with glycosyl universal receptor and saponin as substrates.Among these candidate glycosyltransferases,three UDP-glycosyltransferases involved in the synthesis of notoginseng tetracyclic triterpene saponins,namely PnUGT74A,have glucosyltransferase activity at position C3 and can convert ginsenoside CK into ginsenosides F2;PnUGT94C,with C3 lateral glucose glycosyltransfer activity,can convert ginsenoside Rh2 into ginsenoside Rg3,and ginsenoside F2 into ginsenoside Rd;PnUGT71A,with C20 glycosyltransfer activity,can convert protopanaxatriol(PPT)into ginsenoside F1,and protopanaxadiol(PPD)into ginsenoside CK;(4)We have also obtained two other glycosyltransferases involved in the synthesis of pentacyclic oleanane-type saponins in P.notoginseng,namely:PnUGT74B,which has the glucose glycosyl transfer activity at position C28 and can convert calenduloside E into chikusetsusaponin Ⅳa,and zingibroside R1 into ginsenoside Ro;PnCs1,a cellulose synthase-like,with C3 glucuronic acid glycosyl transfer activity,can convert oleanolic acid into calenduloside E.In this study,we discovered that low temperature can regulate the biosynthetic pathway of PNSs,and clarified the biosynthetic pathway of pentacyclic oleanane saponins in P.notoginseng for the first time,which provides a solid foundation for the discovery of the biosynthetic pathways of panax notoginseng saponins.