Search Results (3)

Ginsenosides, the main active compounds in Panax species, are glycosides of protopanaxadiol (PPD) or protopanaxatriol (PPT). PPT-type ginsenosides have unique pharmacological activities on the central nervous system and cardiovascular system. As an unnatural ginsenoside, 3,12-Di-O-β-D-glucopyranosyl-dammar-24-ene-3β,6α,12β,20S-tetraol (3β,12β-Di-O-Glc-PPT) can be synthesized through enzymatic reactions but is limited by the expensive substrates and low catalytic efficiency. In the present study, we successfully produced 3β,12β-Di-O-Glc-PPT in Saccharomyces cerevisiae with a titer of 7.0 mg/L by expressing protopanaxatriol synthase (PPTS) from Panax ginseng and UGT109A1 from Bacillus subtilis in PPD-producing yeast. Then, we modified this engineered strain by replacing UGT109A1 with its mutant UGT109A1-K73A, overexpressing the cytochrome P450 reductase ATR2 from Arabidopsis thaliana and the key enzymes of UDP-glucose biosynthesis to increase the production of 3β,12β-Di-O-Glc-PPT, although these strategies did not show any positive effect on the yield of 3β,12β-Di-O-Glc-PPT. However, the unnatural ginsenoside 3β,12β-Di-O-Glc-PPT was produced in this study by constructing its biosynthetic pathway in yeast. To the best of our knowledge, this is the first report of producing 3β,12β-Di-O-Glc-PPT through yeast cell factories. Our work provides a viable route for the production of 3β,12β-Di-O-Glc-PPT, which lays a foundation for drug research and development. Full article

Ginsenoside Rg3 is a bioactive compound from Panax ginseng and exhibits diverse notable biological properties. Glycosylation catalyzed by uridine diphosphate-dependent glycosyltransferase (UGT) is the final biosynthetic step of ginsenoside Rg3 and determines its diverse pharmacological activities. In the present study, promiscuous UGT Bs-YjiC from Bacillus subtilis 168 was expressed in Escherichia coli and purified via one-step nickel chelate affinity chromatography. The in vitro glycosylation reaction demonstrated Bs-Yjic could selectively glycosylate the C12 hydroxyl group of ginsenoside Rg3 to synthesize an unnatural ginsenoside Rd12. Ginsenoside Rd12 was about 40-fold more water-soluble than that of ginsenoside Rg3 (90 μM). Furthermore, in vitro cytotoxicity of ginsenoside Rd12 against diverse cancer cells was much stronger than that of ginsenoside Rg3. Our studies report the UGT-catalyzed synthesis of unnatural ginsenoside Rd12 for the first time. Ginsenoside Rd12 with antiproliferative activity might be further exploited as a potential anticancer drug. Full article

Glycosylation, which is catalyzed by UDP-glycosyltransferases (UGTs), is an important biological modification for the structural and functional diversity of ginsenosides. In this study, the promiscuous UGT109A1 from Bacillus subtilis was used to synthesize unnatural ginsenosides from natural ginsenosides. UGT109A1 was heterologously expressed in Escherichia coli and then purified by Ni-NTA affinity chromatography. Ginsenosides Re, Rf, Rh1, and R1 were selected as the substrates to produce the corresponding derivatives by the recombinant UGT109A1. The results showed that UGT109A1 could transfer a glucosyl moiety to C3-OH of ginsenosides Re and R1, and C3-OH and C12-OH of ginsenosides Rf and Rh1, respectively, to produce unnatural ginsenosides 3,20-di-O-β-d-glucopyranosyl-6-O-[α-l-rhamnopyrano-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (1), 3,20-di-O-β-d-glucopyranosyl-6-O-[β-d-xylopyranosyl-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (6), 3-O-β-d-glucopyranosyl-6-O-[β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (3), 3,12-di-O-β-d-glucopyranosyl-6-O-[β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (2), 3,6-di-O-β-d-glucopyranosyl-dammar-24-ene-3β,6α,12β,20S-tetraol (5), and 3,6,12-tri-O-β-d-glucopyranosyl-dammar-24-ene-3β,6α,12β,20S-tetraol (4). Among the above products, 1, 2, 3, and 6 are new compounds. The maximal activity of UGT109A1 was achieved at the temperature of 40 °C, in the pH range of 8.0–10.0. The activity of UGT109A1 was considerably enhanced by Mg²⁺, Mn²⁺, and Ca²⁺, but was obviously reduced by Cu²⁺, Co²⁺, and Zn²⁺. The study demonstrated that UGT109A1 was effective in producing a series of unnatural ginsenosides through enzymatic reactions, which could pave a way to generate promising leads for new drug discovery. Full article