Identification Of The Biosynthesis Pathway Of Oleanane-type Ginsenosides And Establishment Of The Cell Factory For Ginsenosides Production

Ginsenosides are a group of triterpenoids,which are the main active substances in Panax ginseng,with anti-tumor and anti-inflammation effects.Based on the structure difference,ginsenosides can be divided into dammarane-type tetracyclic triterpenoids and oleanane-type pentacyclic triterpenoids.The biosynthesis pathway of dammarane-type ginsenosides has been well illustrated,but the glycosylation processes of oleanane-type ginsenoside are still unclear.The oleanane-type ginsenosides were formed by hydroxylation and glycosylation at the specific sites of β-amyrin.However,the glycosyltransferases involved in the synthesis of oleanane-type ginsenosides were still unknown.In this study,by analyzing the correlation between ginsenosides contents and gene expression levels,we pinpoint several glycosyltransferases.Then,three glycosyltransferases in biosynthetic pathway of ginsenoside Ro were identified using in vitro enzymatic assays and Agrobacterium-mediated genetic transformation into ginseng callus.Moreover,five different oleanane-type ginsenosides were de novo synthesized in the Saccharomyces cerevisiae.The main points are listed as bellow:The highest accumulation of oleanane-type ginsenosides such as ginsenoside Ro and chikusetsusaponin IVa in the rhizomes and roots of ginseng was significantly correlated with the gene expression levels of PgAS and PgOAS,two key genes in the synthesis pathway of oleanolic acid.Eight glycosyltransferases were screened by phylogenetic analysis and correlation analysis with PgOAS.The function of candidate glycosyltransferases was identified by protein expression and in vitro enzyme assays,combined with transgenic function verification of ginseng callus.The results showed that the PgCSyGT1 was the key enzyme for transferring a glucuronosyl moiety to the free C3-OH of oleanolic acid to synthesize calenduloside E.The PgUGT8 catalyzed the glycosylation reaction of the C28 position of oleanane-type ginsenoside,which can convert oleanolic acid,calenduloside E and zingibroside R1 to oleanolic acid 28-O-glucopyranosyl ester,chikusetsusaponin IVa and ginsenoside Ro,respectively.PgUGT18 added a glucosyl group at the C2 position of the glucuronyl group of calenduloside E and chikusetsusaponin IVa to produce zingibroside R1 and ginsenoside Ro respectively.By constructing the UDPGA biosynthetic pathway and increasing the flux of the mevalonate pathway,introducing two key enzymes in oleanolic acid biosynthetic pathway,PgAS1 and PgOAS1,three novel glycosyltransferases PgCSyGT1,PgUGT8 and PgUGT18 into yeast cells,six oleanane-type ginsenosides such as calenduloside E,chikusetsusaponin IVa,zingibroside R1 and ginsenoside Ro,were successfully synthesized in yeast.PgUGT8 was identified to have the activity of glycosylation and deglycosylation at C28 position using in vitro enzymatic reaction assays.PgUGT8 can use UDPG as donor to transfer glycosyl at C28 position of zingibroside R1 to generate ginsenoside Ro,and at the same time,it can also deglycosyl at position C28 of ginsenoside Ro to generate zingibroside R1.The key amino acid sites in the active pocket of PgUGT8 were predicted by protein structural simulation.After the in vitro enzymatic assays of the site-directed mutants,it was found that G20,V243,N245 and S283 sites were the key amino acids that affected the glycosylation and deglycosylation activities of PgUGT8.The above results demonstrated that the three glycosyltransferases PgCSyGT1,PgUGT8 and PgUGT18 are involved in the glycosyl modification process in the biosynthetic pathway of ginsenoside Ro.PgUGT8 has the activity of glycosylation and deglycosylation and plays multiple roles in the biosynthetic pathway.In summary,we identified the biosynthesis pathway of oleanane-type ginsenosides,elucidated the glycosylation and deglycosylation mechanism of glycosyltransferases,and established a cell factory for ginsenosides production using yeast.This study not only provides insights into the ginsenoside glycosylation,but also provides the genetic elements for industrial produce oleanane-type ginsenosides in microbial cell factories.