Study On Identification Of Notoginsenoside Biosynthesis Genes And Function Of Glycosyltransferase From Panax Notoginseng

Panax notoginseng(Burk.)F.H.Chen(Sanqi)belongs to Panax genus and is only distributed in China.The dry radix and rhizome of P.notoginseng(also named Sanqi,or Notoginseng Radix et Rhizoma)is an important medicine in traumatology,which has the functions of removing blood stasis,prompting hemostasis,relieving detumescence and reducing pain.In addition,P.notoginseng is also used in the treatment of cardiovascular and cerebrovascular diseases.The increasingly consumption amount of P.notoginseng in clinic has promoted the researches on its breeding,cultivation,chemical composition and pharmacological mechanism.Protopanaxadiol(PPD)-type ginsenosides Rd and Rb1,and protopanaxatriol(PPT)-type ginsenosides Rg1,Re and notoginsenoside R1,are five of the main active components in roots of P.notoginseng.Although the biosynthetic pathway of ginsenosides has been partially uncovered,there are still many ginsenosides containing rare sugars(rhamnose,xylose,arabinose,etc.),such as ginsenosides Re,Rb3,Rc,and notoginsenoside Fc,and malonylated ginsenosides such as m Rb1,m Rb3,m Rc,m Re,and m Rg1,whose biosynthetic pathways are not completely resolved.Further elucidation of the biosynthetic pathways of saponins in P.notoginseng is of great significance for finding new regulatory targets for ginsenosides biosynthesis,improving the quality of P.notoginseng,and synthesizing specific saponins by biotechnology.In this paper,we first conducted transcriptomic and genomic studies on different samples of P.notoginseng to mine the function genes concerning ginsenosides biosynthesis.Then,we conducted in vitro identification of candidate UDP-dependent glycosyltransferases(UGTs)by gene cloning,heterologous protein expression and bioactivity screening.Furthermore,rare ginsenosides Rh2 and Rg3 were synthesized by a one-step enzymatic cascade reactions using PPD as substrate.The main contents are as follows:1.Transcriptome analysis and mining genes in saponin biosynthesis from P.notoginseng.By comparing transcriptomic data of different P.notoginseng samples with six main databases(GO,COG,KEGG,Swiss-Prot,NR,and Pfam)and with high quality genome data of P.notoginseng,52 function genes related to saponin skeleton synthesis,89 candidate UGTs in saponin glycosylation,10 malonyltransferases(Ma Ts)possibly involved in ginsenoside malonylation,and 29 genes related to sugar donor synthesis were identified.The synthetic pathway of malonylginsenosides was analyzed for the first time.2.Function study of UGTs catalyzing ginsenoside biosynthesis.Eight UGTs of five groups including UGT71 A,UGT74AE,UGT74 AH,UGT94Q,and UGT94 M subfamily,were identified by gene cloning,heterologous protein expression and bioactivity screening.Four novel UGTs,namely Pn UGT71A3,Pn UGT74AH3,Pn UGT74AH4,Pn UGT94M1,were included.Pn UGT71A3 has only about 70%sequence similarity with Pg UGT71A53 reported in literature.It catalyzes not only the C6 hydroxyl glycosylation of PPT and F1 to form Rh1 and Rg1,respectively,but also the C20 hydroxyl glycosylation of PPD-type Rg3 to generate Rd.Pn UGT74AH3 catalyzes the C3 hydroxyl glycosylation of both PPD-and PPT-type ginsenosides,synthesizing ginsenodide F2 from CK,Rh2 from PPD,Rh19 from PPT,and Ia from F1,respectively.Ginsenosides Rh19 and Ia are two rare saponins first identified from the leaves of P.ginseng.The characterization of Pn UGT74AH3 suggested that C3 hydroxyl glycosylation bypathway of minor PPT-type ginsenosides might be ubiquitous in Panax plants.Pn UGT74AH4 only glycosylates the C3 hydroxyl of PPD-type ginsenosides.Especially,Pn UGT94M1 is a UDP-β-L-rhamnose(UDP-Rha)dependent UGT,regioselectively catalyzing the C2’ hydroxyl of C6 glucosyl rhamnosylation of the PPTtype ginsenosides to generate ginsenosides Re from Rg1,and Rg2 from Rh1,respectively.However,it can’t use UDP-α-D-glucose or UDP-α-D-xylose as sugar donors.This study also finds that glycosyltransferases in UGT71 A subfamily mainly catalyze the glycosylation of C6 and C20 hydroxyl groups of PPD and/or PPT-type ginsenosides,UGT74 family glycosyltransferases mainly catalyze the glycosylation of C3 hydroxyl of PPD and/or PPT-type ginsenosides,while glycosyltransferases in UGT94 family mainly catalyze the further extension of sugar chain of PPD and/or PPTtype ginsenosides.These results may prompt further identification of new function UGTs in notoginsenoside biosynthesis.3.Biosynthesis of rare ginsenosides in cascade reactions.Rare ginsenoside Rg3 was synthesized from PPD by a one-step enzymatic cascade reaction for the first time.The molar conversion rate of Rg3 was 60-70%.First,six sucrose synthases(SUSs)from different sources were cloned and their activities were compared,and the SUS with highest activities was selected.The catalytic efficiency of Pn UGT74AE1,Pn UGT74AH3,Pn UGT74AH4,and Sc UGT51＿M7 for the conversion of PPD to Rh2 was compared,and Sc UGT51＿M7 with the best activities was selected.The effects of p H,UDP,sucrose and enzyme loading on the conversion were investigated.When the mass ratio of UDP to PPD was more than 2/5,the concentration of sucrose was more than 30 g/L,and the p H range was from 6.5 to 7.5,the conversion rate of Rg3 from PPD was the highest.When PPD concentration was 0.2 and 1 g/L,the molar conversion rate of Rg3 from PPD was about 70% and 60%,respectively.The crystallographic study of Pn UGT74AE1 was carried out in order to obtain more active mutants for the biosynthesis of ginsenoside Rh2 and Rg3.The single crystal of Pn UGT74AE1 was obtained and its structure was resolved.Then a round of mutation was conducted to study the key amino acid residues by determining the catalytic activities of mutants.In conclusion,the biosynthetic pathways of saponins in P.notoginseng was perfected by mining and function study of UGTs.One-step synthesis system for rare ginsenoside Rg3 using PPD as the starting substrate was successfully constructed by enzymatic cascade reactions.These reults provide new targets for the regulation of saponin biosynthesis in P.notoginseng,lay solid biological foundation for cultivating high quality medicins of Panax,and put forward new solutions for solving the problem of the shortage of rare ginsenosides and other rare natural glycosylated products.