Reconstruction and optimization of the biosynthetic pathway for the mass production of minor ginsenosides

Abstract

Ginseng is a medicinal herb that requires cultivation under shade conditions, typically for 6 yr, before harvesting. The principal components of ginseng are ginsenosides, glycosylated tetracyclic terpenes. Ginseno-sides are classified into two groups, protopanaxadiol (PPD) and protopanaxatriol (PPT), based on their hy-droxylation patterns, and further diverge to diverse ginsenosides through differential glycosylation. Three early enzymes, Dammarenediol-II synthase (PgDS) and two P450 enzymes, protopanaxadiol synthase (PgPPDS) and protopanaxatriol synthase (PgPPTS), have been reported, but glycosyltransferases that are necessary to synthesize specific ginsenosides have not yet been fully identified. To discover glycosyltransferases responsible for ginsenoside biosynthesis, we sequenced and assembled Panax ginseng transcriptome de novo and characterized four UDP-glycosyltransferases (PgUGTs): PgUGT74AE2, PgUGT94Q2, PgUGT71A27 and GpUGT23. PgUGT74AE2 transfers a glucose moiety from UDP-glucose (UDP-Glc) to the C3 hydroxyl groups of PPD and compound K to form Rh2 and F2, respectively, whereas PgUGT94Q2 transfers a glucose moiety from UDP-Glc to Rh2 and F2 to form Rg3 and Rd, respectively. PgUGT71A27 introduced glucose moiety to C20 hydroxyl group of PPD, Rh2, Rg3 and PPT and produced CK, F2, Rd and F1, respectively. GpUGT23 introduced glucose moiety to C20 position glucose of CK, F2, Rd and F1 and produced Gypenoside XLLV, gypenoside XVII, ginsenoside Rd and notoginsenoside U, respectively. Introduction of the combination of four UGTs into yeast together with PgDS and PgPPDS resulted in the de novo production of Rg3, F2, F1, Rd, CK and gypenoside XVII. Our results indicate that these four UGTs are key enzymes for the synthesis of ginsenosides and provide a method for producing specific ginsenosides through yeast fermentation