Semirational Design\nof a UDP-Glycosyltransferase from Nicotiana tomentosiformis for Efficient Biosynthesis\nof Rebaudioside M2

Rebaudioside M2 (RebM2) is characterized\nas 13-[(2-<i>O</i>-β-d-glucopyranosyl-3-<i>O</i>-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy]\nent-kaur-16-en-19-oic\nacid-[(2-<i>O</i>-β-d-glucopyranosyl-6-<i>O</i>-β-d-glucopyranosyl-β-d-glucopyranosyl)\nester], an isomer of rebaudioside M with a 1 → 6 sugar linkage.\nThe product was found in the biotransformation of rebaudioside D (RebD)\ncatalyzed by a glycosyltransferase from Nicotiana tomentosiformis (<i>Nt</i>UGT). Herein, guided by consensus engineering\nand molecular dynamics simulations, a variant <i>Nt</i>UGT<sub>F72L/L123P/L157P</sub> with enhanced activity and thermostability\nwas obtained. It exhibits a strikingly reduced <i>K</i><sub>m</sub> (22.47 mM to 0.15 mM) toward RebD, and the catalytic efficiency\nwas over 5000-fold higher than that of the wildtype. When an Arabidopsis sucrose synthase <i>At</i>SuSy\nwas used for UDP-glucose recycling, <i>Nt</i>UGT<sub>F72L/L123P/L157P</sub> effectively converted 80 g/L RebD to 90.14 g/L RebM2. In a one-pot\nthree-enzyme reaction involving an engineered glycosyltransferase\nUGTSL2<sub>N358F</sub>, which catalyzed the conversion of RebA into\nRebD, 78.8 g/L of RebM2 (with a yield of 84.56%) was produced from\n70 g/L of RebA, avoiding the use of the naturally rare and poorly\nsoluble RebD as the starting material. This work will provide a promising\nbiocatalyst for RebM2 biosynthesis on a large scale and create an\nopportunity to accelerate the exploration of the biological activity\nof RebM2 and its potential as a candidate for superior SG sweeteners.