Analysis And Application Of The Biosynthesis Pathway Of Notoginsenosides

Panax notoginseng is a rare Chinese herbal medicine of panax.As the main active ingredient,Panax notoginseng saponins(PNS)have strong pharmacological activities in anti-tumor,anti-oxidation and anti-aging,and they are mainly used in clinical treatment of cardiovascular and cerebrovascular diseases.Recently,these active ingredients are heavily dependent on the resources of traditional Chinese medicine.The directed breeding methods of fermentation by synthetic biology technology or improving the effective components of medicinal plants are beneficial to relieve the pressure of current resources.However,biosynthetic pathways of Panax notoginseng saponins which is the fundamental prerequisites for this work have not been explored clearly,it is crucial to analyze this synthetic pathway in the next."Plug and Play" resolution of biosynthetic pathways is an efficient and reliable method.The characteristics of the notoginsenosides are composed of triterpenoids and glycosyl groups.Firstly,we integrated the Panax ginseng-derived Dammarenediol synthase(SynPgDDS),Protopanaxadiol synthase(SynPgPPDS),Protopanaxatriol synthase(SynPgPPTS)genes and the Arabidopsis thaliana-derived Cytochrome P450 reductase(AtCPR1)gene into yeast triterpene chassis strain BY-T3 and obtained the transformant BY-PPT.Secondly,the regulation of the expression of functional module PGMI-PGM2-UGP1 which consists of genes such as phosphoglucomutase 1(PGM1),a-phosphoglucomutase(PGM2),UDP-glucose pyrophosphorylase(UGP1)in Saccharomyces cerevisiae increase the supply of UDP-glucose by 8 times.Thirdly,the"plug and play" chassis strain BY-PPT-GM was established.On this basis,the UDP-glucosyltransferase gene Pg3-29,catalyzing the PPT to ginsenoside FI,was transferred into BY-PPT-GM to finish the test of UGTs "plug and play" identification platform.Thus,it established a foundation for functional testing of the Panax notoginseng-derived glycosyltransferase.In the process of analysis of the biosynthesis pathway of notoginsenosides,the transformant BY-T3-GM was constructed by the UDP-glucose supply module PGM1-PGM2-UGP1 transferred into the Saccharowyces cerevisiae chassis cell BY-T3.Integrated SynPgDDS,SynPgPPDS,SynPgPPTS and AtCPR1 genes into BY-T3-GM,obtained chassis cell BY-GM-PPT produces triterpene aglycone protopanaxadiol and protopanaxatriol,providing a basis for functional testing of the glycosyltransferase.For building candidate function module libraries,the 64 Panax notoginseng-derived glycosyltransferase genes extracted by bioinformatics analysis were numbered and flanked with the strong promoter pTEF1 and terminator CYC1t respectivelyOn that basis,we transferred the candidate functional modules into the chassis cell BY-GM-PPT.It was proved that Pnl-31 catalyze the C3-OH glycosylation of PPD,and Pn3-29 catalyze the C20-OH glycosylation of PPD and PPT.Pn1-31 gene was integrated into the chassis cell BY-PPT together with the UDP-glucose supply module to gain a second-generation chassis cell BY-Rh2 which produce ginsenoside Rh2.The functional module library was transferred into BY-Rh2 for the second round of testing,and it showed that Pn3-29 catalyze the C20-OH glycosylation of ginsenoside Rh2 to produce ginsenoside F2.Pn3-31 catalyze ginsenoside Rh2 to obtain ginsenoside Rg3 after adding another glucose to the C3-OH glycosylation product.These three genes and the UDP-glucose supply module were integrated into the chassis cell BY-PPT to obtain a third-generation chassis cell BY-Rd which can produce the ginsenoside Rd.The functional module library was transferred into BY-Rd for the third round of testing,and finally Pn3-32 catalyze ginsenoside Rd to produce ginsenoside Rb1.Finally,the biosynthetic network pathways for the synthesis of saponins such as Rh2,CK,F2,Rg3,Rd and Rb1 in Panax notoginseng were successfully analyzed.