Two Ginseng UDP-Glycosyltransferases Synthesize Ginsenoside Rg3 and Rd

Abstract

Ginseng is a medicinal herb that requires cultivation under shade conditions, typically for 4-6 years, before harvesting. The principal components of ginseng are ginsenosides, glycosylated tetracyclic terpenes. Dammarene-type ginsenosides are classified into two groups, protopanaxadiol (PPD) and protopanaxatriol (PPT), based on their hydroxylation patterns, and further diverge to diverse ginsenosides through differential glycosylation. Three early enzymes, dammarenediol-II synthase (DS) and two P450 enzymes, protopanaxadiol synthase (PPDS) and protopanaxatriol synthase (PPTS), 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 the ginseng transcriptome de novo and characterized two UDP-glycosyltransferases (PgUGTs): PgUGT74AE2 and PgUGT94Q2. 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. Introduction of the two UGT genes into yeast together with PgDS and PgPPDS resulted in the de novo production of Rg3. Our results indicate that these two UGTs are key enzymes for the synthesis of ginsenosides and provide a method for producing specific ginsenosides through yeast fermentation.

Biosynthesis and biotechnological production of ginsenosides

“…Furthermore, the importance of DDS in synthesizing ginsenosides was shown by experiments using hairy root of transgenic plant suppressing other branch CAS; these roots showed increased DDS enzyme activity and contained 50-100% higher ginsenoside levels compared with control roots (Liang et al, 2009). Hetero-overexpression of DDS successfully produced dammarenediol-II in tobacco cells (Han et al, 2014;Lee et al, 2012) providing a new way to produce ginsenosides with other downstream genes in yeast cell system (Dai et al, 2014;Jung et al, 2014;Yan et al, 2014). DDS is conserved across the Panax species (Niu et al, 2014), and it contains 769 amino acids with six QW-motifs and the substrate binding DCTAE motif.…”

“…In ginseng plants, UGTs are assumed to play an important role in producing different ginsenosides by adding monosaccharides to triterpene aglycones mainly at C-3 and/or C-20 for PPD-type ginsenosides and at C-6 and/or C-20 for PPT-type ginsenosides. Even though several UGT enzymes have been identified in glycosylating triterpene aglycones (Jung et al, 2014;Khorolragchaa et al, 2014;Luo et al, 2011;Xiang et al, 2012;Yan et al, 2014;Yue and Zhong, 2005), the biochemical functions of UGTs in ginseng plants remain largely unknown. Two UGTs, UGRdGT from P. notoginseng and UGRh2GT from P. ginseng, were purified, and were shown to be responsible for the synthesis of ginsenoside Rb1 from Rd, and Rg3 from Rh2, respectively (He and Yue, 2010;Yue and Zhong, 2005).…”

“…Jung et al (2014) characterized that PgUGT74AE2 catalyzes the transfer of a glucose moiety from UDP-glucose to the C3 hydroxyl groups of PPD and compound K, yielding Rh2 and F2, respectively, PgUGT94Q2 can transfer a glucose moiety from UDP-glucose to Rh2 and F2 to generate Rg3 and Rd, respectively. Khorolragchaa et al (2014) showed 12 putative UGTs from ginseng plants, future biochemical characterization of the UGTs may reveal the mechanism controlling ginsenoside biosynthesis, and provide new approaches for modifying ginsenoside production.…”

“…Yan et al (2014) employed the expression of UGTPg1 together with the overexpression of truncated HMGR and UPC2.1, a semi-dominant mutant UPC2 which acts as a global transcription factor for the synthesis of sterols, and obtained a yeast strain with a yield of up to 1.4 mg/L for compound K. This milestone work provides an inexpensive way to manufacture compound K to meet potential clinical applications. Furthermore, Jung et al (2014) expressed two newly identified PgUGTs: PgUGT74AE2 and PgUGT94Q2, together with PgDDS and PgPPDS and produced ginsenoside Rg3 in yeast cells. These works provide a new method for large-scale production of specific ginsenosides via yeast fermentation.…”

De novo assembly and comparative analysis of root transcriptomes from different varieties of Panax ginseng C. A. Meyer grown in different environments

“…Among those four cytochrome p450s, one (comp50843_c0) was also identified by Wu et al [17] in North American ginseng as being potentially related to dammarane-type ginsenoside biosynthesis. For DS, nine cytochrome p450s and seven glycosyltransferases showed a high expression correlation pattern, and among them, one cytochrome p450 (comp44704_c0), which catalyzes the glycosylation of certain kinds of protopanaxadiol [40] showed strong homology (E-value=3×10 -23 ) with PgUGT74AE2 (GenBank: JX8985-29).…”