Screening UDP-Glycosyltransferases for Effectively Transforming Stevia Glycosides: Enzymatic Synthesis of Glucosylated Derivatives of Rubusoside

Abstract

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.

Herb Stevia rebaudiana’s functionalities, safety and applications: a review

“…The transglycosylation rate of stevioside could reach 85.6% in the end. More lately, it is also observed that efforts were made to screen different glycosyltransferase enzymes to ultimately increase sweetness intensity and reduce bitter taste (Pan et al , 2022).…”

Directed Evolution of the UDP-Glycosyltransferase UGTBL1 for Highly Regioselective and Efficient Biosynthesis of Natural Phenolic Glycosides

“…However, the diversity of glycosylation sites leads to variations in the biological activities and functions of glycoside products . When polyphenols are used as aglycone acceptors, chemical glycosylation requires cumbersome protection/deprotection steps to achieve high regioselectivity. , Therefore, the enzymatic glycosylation of polyphenols offers numerous advantages, including precise tuning of regioselectivity, adherence to green chemistry principles, and high efficiency. − UGTs from various species, including Arabidopsis thaliana, Vitis vinifera, and Streptomyces antibioticus, have been extensively characterized. − Indeed, most of the characterized UGTs, including UGT BL 1, exhibit poor regioselectivity and catalytic activity. Simultaneous improvements in the catalytic efficiency and regioselectivity of UGTs remain a challenge.…”