Semirational Design of a UDP-Glycosyltransferase from <i>Nicotiana tomentosiformis</i> for Efficient Biosynthesis of Rebaudioside M2

Rebaudioside M2 (RebM2) is characterized as 13-[(2-<i>O</i>-β-d-glucopyranosyl-3-<i>O</i>-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(2-<i>O</i>-β-d-glucopyranosyl-6-<i>O</i>-β-d-glucopyranosyl-β-d-glucopyranosyl) ester], an isomer of rebaudioside M with a 1 → 6 sugar linkage. The product was found in the biotransformation of rebaudioside D (RebD) catalyzed by a glycosyltransferase from <i>Nicotiana tomentosiformis</i> (<i>Nt</i>UGT). Herein, guided by consensus engineering and molecular dynamics simulations, a variant <i>Nt</i>UGT_{F72L/L123P/L157P} with enhanced activity and thermostability was obtained. It exhibits a strikingly reduced <i>K</i>_m (22.47 mM to 0.15 mM) toward RebD, and the catalytic efficiency was over 5000-fold higher than that of the wildtype. When an <i>Arabidopsis</i> sucrose synthase <i>At</i>SuSy was used for UDP-glucose recycling, <i>Nt</i>UGT_{F72L/L123P/L157P} effectively converted 80 g/L RebD to 90.14 g/L RebM2. In a one-pot three-enzyme reaction involving an engineered glycosyltransferase UGTSL2_N358F, which catalyzed the conversion of RebA into RebD, 78.8 g/L of RebM2 (with a yield of 84.56%) was produced from 70 g/L of RebA, avoiding the use of the naturally rare and poorly soluble RebD as the starting material. This work will provide a promising biocatalyst for RebM2 biosynthesis on a large scale and create an opportunity to accelerate the exploration of the biological activity of RebM2 and its potential as a candidate for superior SG sweeteners.