Three Glycosyltransferase Mutants in a One-Pot Multi-enzyme System with Enhanced Efficiency for Biosynthesis of Quercetin-3,4′-O-diglucoside

Abstract

Quercetin-3,4′-O-diglucoside (Q3,4′G), among the major dietary flavonoids, is superior to quercetin aglycone or quercetin monoglucoside in solubility. However, its low content in nature makes it hard to be prepared in large quantities by traditional extraction methods. In the present study, the F378S mutant of UGT78D2 (78D2_F378S) derived from Arabidopsis thaliana with improved regioselectivity and the V371A mutant of UGT73G1 (73G1_V371A) derived from Allium cepa were adopted to realize a two-step continuous glycosylation of quercetin to produce Q3,4′G. The mutation S31D was introduced to the sucrose synthase from Micractinium conductrix with enhanced activity, which was responsible for regenerating UDP-glucose by coupling with 78D2_F378S and 73G1_V371A. Using the aforementioned enzymes, prepared from the three-enzyme co-expression strain, 4.4 ± 0.03 g/L (7.0 ± 0.05 mM, yield 21.2%) Q3,4′G was produced from 10 g/L quercetin after reaction for 24 h at 45 °C.

Solvent Engineering for Nonpolar Substrate Glycosylation Catalyzed by the UDP-Glucose-Dependent Glycosyltransferase UGT71E5: Intensification of the Synthesis of 15-Hydroxy Cinmethylin β-d-Glucoside

“…Sugar nucleotide-dependent (Leloir) glycosyltransferases (GTs) catalyze the glycosylation of acceptor substrates that represent a broad diversity of chemical structures. – Certain classes of GT acceptor, including natural products and xenobiotics in particular, involve a highly nonpolar aglycone core. ,– Due to the specificity and flexibility in catalysis offered in useful combination, GTs are promising enzymes for the synthesis of the corresponding acceptor glycosides. ,,– The glycosides involve considerable interest for the diverse uses they have, ranging from chemical reference for studies of the biological metabolism , to functional ingredients in food ,,, and cosmetic applications. ,,, However, GTs are generally perceived as “difficult” enzymes for use in applied biocatalysis. Besides their requirement of a nucleotide-activated sugar donor for activity, they also exhibit comparably low robustness, , falling short of widely used “industrial workhorse” enzymes such as hydrolases or nicotinamide coenzyme-dependent dehydrogenases. , Low robustness of GTs may have been the reason that solvent engineering strategies have not been widely explored with these enzymes .…”

“…Besides their requirement of a nucleotide-activated sugar donor for activity, they also exhibit comparably low robustness, , falling short of widely used “industrial workhorse” enzymes such as hydrolases or nicotinamide coenzyme-dependent dehydrogenases. , Low robustness of GTs may have been the reason that solvent engineering strategies have not been widely explored with these enzymes . Nevertheless, glycosylation reactions with nonpolar acceptors are expected to benefit strongly from enhanced solubilization of the substrate into the aqueous phase containing the enzyme. ,– , …”

“… 17 – 19 Certain classes of GT acceptor, including natural products and xenobiotics in particular, involve a highly nonpolar aglycone core. 17 , 20 – 24 Due to the specificity and flexibility in catalysis offered in useful combination, GTs are promising enzymes for the synthesis of the corresponding acceptor glycosides. 17 , 22 , 25 – 27 The glycosides involve considerable interest for the diverse uses they have, ranging from chemical reference for studies of the biological metabolism 28 , 29 to functional ingredients in food 23 , 26 , 30 , 31 and cosmetic applications.…”

“… 36 Nevertheless, glycosylation reactions with nonpolar acceptors are expected to benefit strongly from enhanced solubilization of the substrate into the aqueous phase containing the enzyme. 17 , 22 – 24 , 27 …”

Identification of sucrose synthase from Micractinium conductrix to favor biocatalytic glycosylation

“…However, most of the receptors to be glycosylated have poor water solubility, so dimethyl sulfoxide (DMSO) is added as cosolvent to increase the solubility of the substrate ( Pei et al, 2019 ; Chu et al, 2021 ; Tao et al, 2023 ). The low stability and intolerance to organic cosolvents of plant SuSys have limited the further application of such SuSy-GT cascades ( Schmölzer et al, 2016 ).…”