Five plant-derived uridine diphosphate glycosyltransferases (UGTs) that catalyzed the glucosylation of stevia glycosides (SGs) were uncovered as the result of sequence mining considering the catalytic residues and conserved motifs of the known UGTs. Thereinto, LbUGT from Lycium barbarum with high activity toward rubusoside has been enzymatically characterized. The recombinant LbUGT was demonstrated to catalyze the β-1,6-glucosylation at C19 of rubusoside, producing a monoglucosyl derivative 13-[(O-β-D-glucopyranosyl) oxy] ent-kaur-16-en-19-oic acid-[(6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester], which was then submitted to a β-1,2-glucosylation by LbUGT, resulting in a diglucosyl derivative 13-[(O-β-D-glucopyranosyl) oxy] entkaur-16-en-19-oic acid-[(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester].The di-glycosylated product of rubusoside showed an obvious increase in sweetness intensity (134 times sweeter than 5% sucrose) and almost eliminated the unpleasant bitter taste. This work will provide a reference for the taste improvement of SGs.
Sucrose synthase (SuSy, EC 2.4.1.13) is a unique glycosyltransferase
(GT) for developing cost-effective glycosylation processes. Up to now,
some SuSys derived from plants and bacteria have been used to recycle
uridine 5’-diphosphate glucose in the reactions catalyzed by Leloir GTs.
In this study, after sequence mining and experimental verification, a
SuSy from Micractinium conductrix (McSuSy), a single-cell green alga,
was identified. In the direction of sucrose cleavage, the optimum
temperature and pH of the recombinant McSuSy were 60 °C and pH 7.0. The
mutations of the predicted N-terminal phosphorylation site (S31D) and
the QN motif (K684T and N685D) significantly stimulated the activity of
McSuSy. When the mutant S31D/684T/685D of McSuSy, with the highest
activity, was applied by coupling the engineered yeast
glycosyltransferase UGT51 in a one-pot two-enzyme reaction, 8 mM
protopanaxadiol was transformed into 6.02 mM (3.75 g/L) ginsenoside Rh2
within 3 h at 37 °C. The yield was comparable to the control reaction of
AtSuSy1 from Arabidopsis thaliana. This work reveals the lower
eukaryotes as a promising resource for SuSys of industrial interest.
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