Efficient Production of Various Minor Ginsenosides from PPD- and PPT-type Major Ginsenosides Using a Single Recombinant BglFc Isolated from Flavobacterium chilense

Abstract

Background: Rare ginsenosides (F2, Rg3, Gyp-XVII, and C-K) are pharmaceutically active components of Panax ginseng, which are derived from the conversion of major ginsenosides through various transformation methods. To date, most studies have failed to identify a competent bacterial strain and recombinant enzyme for converting protopanaxadiol (PPD)- and protopanaxatriol (PPT)-type ginsenosides to target minor ginsenosides. Method: Our study identified and employed nine sets of clones from different glycoside hydrolase bacterial strains for major ginsenoside bioconversion. Among these nine clones, only BglFc was selected based on its strong biotransformation ability and capacity to generate complete minor ginsenosides. bglFc was cloned and expressed in Escherichia coli using the pGEX-4T-1 vector system, and the recombinant enzyme was used for efficiently producing minor ginsenosides. Results: Recombinant BglFc is 2,394 bp and 798 amino acid residues long, with a predicted molecular mass of 78.8 kDa. BglFc belongs to the glycoside hydrolase family 3, and demonstrates a promising ability to convert major ginsenosides into minor ones. The Km and Vmax values of pNPG were 0.81 ± 0.06 and 4.0 ± 0.2 mM·min−1·mg−1 of protein, respectively. Under optimal conditions (37°C, pH 7.0), the ginsenoside transformation pathways for BglFc were as follows: Rb1→Rd→Rg3(S)→Rh2(S); GypXVII→GypLXXV→C-K; GypLXXV→C-K; F2→C-K; Rb2→C-O→C-Y; Rb3→C-Mx1→C-Mx; Rc→C-Mcl→C-Mc; Re→Rg2(S); and Rgl→Rhl(S), respectively. Conclusion: These results suggest that recombinant BglFc demonstrates a strong transformation activity for both PPD- and PPT-type major ginsenosides. Therefore, we conclude that BglFc can be used for gram unit production of various minor ginsenosides. Significance and Impact of Study: Previously, researchers have used a combination of enzymes for the production of minor ginsenosides. However, in this study, we found a favorable enzyme that can be used alone for the production of a different type of minor ginsenoside using the proposed conversion pathway.