# Progress on the Studies of the Key Enzymes of Ginsnoside Biosynthesis
## 8 UDP-Glycosyltransferase
(2018) cloned and identified a UGT gene named Pq3-O-UGT2 from _P. quinquefolius_. In vitro enzymatic assay confirmed that Pq3-O-UGT2 catalyzed the glycosylation of Rh2 and F2 to produce Rg3 and Rd. High identity of 99.32% between Pq3-O-UGT2 and PgUGT94Q2 indicated a close evolutionary relationship between _P. ginseng_ and _P. quinquefolius_. Figure 2: Glycosylation of ginsenosides by UGTs from microorganisms. (**a**) 3-O-\(\beta\)-D-glucopyranosyl-6-O-\(\beta\)-D-glucopyranosyl-20(\(S\))-protonanxatriol; (**b**) (205)-3\(\beta\),\(\delta\),\(\alpha\),12\(\beta\),20-tetrahydroxydammar-24-ene-20-O-\(\beta\)-D-glucopyrananoyl-3-O-\(\beta\)-D-glucopyranoside (ginsenoside Ia); (**c**) 3-O-\(\beta\)-D-glucopyranosyl-dammar-24-ene-3\(\beta\),205-diol; (**d**) 3,20-Di-O-\(\beta\)-D-glucopyranosyl-dammar-24-ene-3\(\beta\),208-diol; (**e**) 3,12-Di-O-\(\beta\)-D-glucopyranosyl-dammar-24-ene-3\(\beta\),12\(\beta\),205-triol; (**f**) ginsenoside Rh2; (**g**) 3,12-Di-O-\(\beta\)-D-glucopyranosyldammar-24-ene-3\(\beta\),\(\delta\),\(\alpha\),12\(\beta\),205-tetraol. Microorganisms contain rich UGTs capable of using UDP-sugar as the sugar donor and small molecules as the sugar acceptors [48; 49]. In recent years, several UGTs which can transfer glucose moieties to ginseng or their aglycones have been identified from microorganisms. The YjiC1 gene cloned from _Bacillus subtilis_ was expressed in _E. coli_ and the recombinant enzyme converted ginseng Rh1 into an unnatural ginsengoside 3-O-\(\beta\)-d-glucopyranosyl-6-O-\(\beta\)-d-glucopyranosyl-20(\(S\))-protopanaxatriol, which is the first saponin possessing \(\beta\)-glucopyrananoyll at both C3 and C6 sites (Figure 2a) [50]. [PERSON] et al. [51] cloned an UDP-glycosyltransferase BSGT1 gene from _B. subtilis_. The recombinant BSGT1 was purified and characterized to enzymatically transform F1 into (205)-3\(\beta\),6\(\alpha\),12\(\beta\),20-tetrahydrodoxydammar-24-ene-20-O-\(\beta\)-d-glucopyranosyl-3-O-\(\beta\)-d-glucopyranoside (ginsenoside Ia), which possesses an additional glucose linked into the C3 position of substrate (Figure 2b). In our study, a new UDP-glycosyltransferase UGT109A1 was also identified from _B. subtilis_. This enzyme transferred a glucose moiety to C3-OH and C20-OH of DM, and C3-OH and C12-OH of PPD and PPT respectively to produce unnatural ginsengosides 3-O-\(\beta\)-d-glucopyranosyl-dammar-24-ene-3\(\beta\),205-diol (Figure 2c), 3,20-Di-O-\(\beta\)-d-glucopyranosyl-dammar-24-ene-3\(\beta\),205-diol (Figure 2d), 3,12-Di-O-\(\beta\)-d-glucopyranosyl-dammar-24-ene-3\(\beta\),12\(\beta\),205-triol (Figure 2e) and 3,12-Di-O-\(\beta\)-d-glucopyranosyldammmar-24-ene-3\(\beta\),6\(\alpha\),12\(\beta\),205-tetraol (Figure 2g). The metabolically engineered yeasts were then constructed to produce 3,12-Di-O-\(\beta\)-d-glucopyranosyl-dammar-24-ene-3\(\beta\),12\(\beta\),205-triol by introducing the gene encoding UGT109A1 into the PPD producing strain [52]. [PERSON] et al. [53] cloned a promiscuous glycosyltransferase UGT51 from _S. cerevisiae_. The semi-rationally designed UGT51 presented an 1800-fold enhanced catalytic efficiency for converting PPD to ginseng Rh2 in vitro and then demonstrated high-level production of Rh2 in _S. cerevisiae_ by synthetic biology technology (Figure 2f). Glycosylation is the most downstream step of ginseng biosynthesis. It is of great significance to enrich the diversity of ginseng by mining more UGTs from _Panax_ species or microorganisms in the future.