Enhancement of Rebaudioside M Production by Structure-Guided Engineering of Glycosyltransferase UGT76G1

Abstract

Owing to zero-calorie and advanced organoleptic properties similar to sucrose, the plant-derived rebaudioside M (Reb M) has been considered as a next generation sweetener. However, a low content of Reb M in Stevia rebaudiana Bertoni and low enzymatic activity of UGT76G1, which is an uridine diphosphate glucose (UDPG)-dependent glycosyltransferase with the ability to glycosylate rebaudioside D (Reb D) to produce Reb M through the formation of β-1,3 glycosidic bond, restrict its commercial usage. To improve the catalytic activity of UGT76G1, a variant UGT76G1-T284S/M88L/L200A was obtained by structure-guided evolution, whose catalytic activity toward Reb D increased by 2.38 times compared with UGT76G1-T284S. This allowed us to prepare Reb M on a large-scale with a great yield of 90.50%. Moreover, molecular dynamics simulation illustrated that UGT76G1-T284S/M88L/L200A reduced distances from Reb D to catalytic residues and UDPG. Hence, we report an efficient method for the potential scale production of Reb M in this study.

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

“…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][23][24]27 In the current study, we focused on β-D-glucosylation of 15hydroxy cinmethylin (15HCM) from uridine 5′-diphosphate (UDP)-glucose by UGT71E5 of safflower plant (Carthamus tinctorius), as shown in Figure 1A. 37,38 Cinmethylin (Figure 1B) is an agricultural herbicide, and its detoxification in planta involves C15 hydroxylation and glycosylation.…”

“…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. ,– , …”

Plant glycosyltransferases for expanding bioactive glycoside diversity

“…56 In another study, a UGT76G1 T284S/M88L/ L200A mutant was found to have an increased catalytic activity towards Reb D and was able to convert Reb D to Reb M at up to 90.50% yield in fed-batch fermentation culture of E. coli. 60 Steviol, Reb A, D, and M showcase the importance of the number and identity of sugars in determining the properties of glycosides. The production of Reb A, D, and M shows that alterations to UGT expression level or specicity can serve as a tool in enhancing the yield of desired glycosides.…”

“…56 In another study, a UGT76G1 T284S/M88L/L200A mutant was found to have an increased catalytic activity towards Reb D and was able to convert Reb D to Reb M at up to 90.50% yield in fed-batch fermentation culture of E. coli . 60…”