Rebaudioside M2 (RebM2) is characterized as 13-[(2--β-d-glucopyranosyl-3--β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(2--β-d-glucopyranosyl-6--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 (UGT). Herein, guided by consensus engineering and molecular dynamics simulations, a variant UGT with enhanced activity and thermostability was obtained. It exhibits a strikingly reduced (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 sucrose synthase SuSy was used for UDP-glucose recycling, UGT effectively converted 80 g/L RebD to 90.14 g/L RebM2. In a one-pot three-enzyme reaction involving an engineered glycosyltransferase UGTSL2, 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.

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http://dx.doi.org/10.1021/acs.jafc.4c09051DOI Listing

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Semirational Design of a UDP-Glycosyltransferase from for Efficient Biosynthesis of Rebaudioside M2.

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Rebaudioside M2 (RebM2) is characterized as 13-[(2--β-d-glucopyranosyl-3--β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(2--β-d-glucopyranosyl-6--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 (UGT). Herein, guided by consensus engineering and molecular dynamics simulations, a variant UGT with enhanced activity and thermostability was obtained.

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