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

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.

Bioconversion of Stevioside to Rebaudioside E Using Glycosyltransferase UGTSL2.

Rebaudioside E, one of the minor components of steviol glycosides, was first isolated and identified from Stevia rebaudiana in 1977. It is a high-intensity sweetener that tastes about 150-200 times sweeter than sucrose and is also a precursor for biosynthesis of rebaudioside D and rebaudioside M, the next-generation Stevia sweeteners. In this work, new unknown steviol glycosides were enzymatically synthesized from stevioside by coupling UDP-glucosyltransferase UGTSL2 from Solanum lycopersicum and sucrose synthase StSUS1 from Solanum tuberosum. Rebaudioside E was speculated to be the main product of glucosylation of the Glc(ß1→C-19) residue of stevioside along with the formation of a (ß1→2) linkage based on the analysis of the regioselectivity and stereoselectivity of UGTSL2, and verified afterwards by LC-MS/MS with standard. In a 20-ml bioconversion reaction of 20 g/l stevioside by UGTSL2 and StSUS1, 15.92 g/l rebaudioside E was produced for 24 h.

Improved soluble bacterial expression and properties of the recombinant flavonoid glucosyltransferase UGT73G1 from Allium cepa.

Glycosylation of quercetin using flavonol-specific glycosyltransferases offers an alternate method for isoquercitrin production. Obtaining sufficient quantities of bioactive enzymes is an important prerequisite for highly effective biocatalysis and biotransformation. In this study, a codon-optimized gene for the flavonoid glucosyltransferase UGT73G1 from Allium cepa was heterologously expressed in the preferred prokaryotic expression host Escherichia coli. By combining expression as a fusion protein with 6-histidine tags with coexpression with molecular chaperones, increased soluble expression of UGT73G1 was achieved in E. coli. Two-terminal 6-histidine tags contributed more to the expression than molecular chaperones, as demonstrated by comparison of specific activities in crude extracts obtained from the recombinant E. coli strains. Studies of the catalytic properties of purified UGT73G1 indicated that its activity was significantly promoted by Mn2+ and Mg2+, while it was strongly inhibited by Cu2+. These expression strategies enhanced the solubility and activity of the overexpressed protein and enabled characterization of this plant-derived glucosyltransferase expressed in a prokaryotic host.