Biocatalytic synthesis of ginsenoside Rh2 using Arabidopsis thaliana glucosyltransferase-catalyzed coupled reactions.

• UGT73C5 and sucrose synthase from A. thaliana were expressed in E. coli. • UGT73C5 selectively transformed protopanaxadiol into ginsenoside Rh2. • UGT73C5–sucrose synthase coupled reaction was optimized for UDP-glucose recycle. • The highest yield of ginsenoside Rh2 was obtained by one-pot reaction. Ginsenoside Rh2, a rare protopanaxadiol (PPD)-type triterpene saponin isolated from Panax ginseng , exhibits notable anticancer and immune-system-enhancing activities. Glycosylation catalyzed by uridine diphosphate-dependent glucosyltransferase (UGT) is the final biosynthetic step of ginsenoside Rh2. In this study, UGT73C5 isolated from Arabidopsis thaliana was demonstrated to selectively transfer a glucosyl moiety to the C3 hydroxyl group of PPD to synthesize ginsenoside Rh2. UGT73C5 was coupled with sucrose synthase (SuSy) from A. thaliana to regenerate costly uridine diphosphate glucose (UDPG) from cheap sucrose and catalytic amounts of uridine diphosphate (UDP). The UGT73C5/SuSy ratio, temperature, pH, cofactor UDP, and PPD concentrations for UGT73C5–SuSy coupled reactions were optimized. Through the stepwise addition of PPD, the maximal ginsenoside Rh2 production was 3.2 mg mL−1, which was the highest yield reported to date. These promising results provided an efficient and cost-effective approach to semisynthesize the highly valuable ginsenoside Rh2. [ABSTRACT FROM AUTHOR]