Semirational engineering of glycosyltransferase UGTPg29 enables efficient biosynthesis of rebaudioside M and M2

Steviol glycosides (SGs) from Stevia rebaudiana are widely used as low-calorie sweeteners. Rebaudioside M (RebM), valued for its superior sensory characteristics, occurs at low abundance in S. rebaudiana, whereas its isomer rebaudioside M2 (RebM2), which features a distinctive β-1,6 glycosidic linkage at the C-19 position, has so far been obtained mainly through enzymatic conversion. Here, semi-rational engineering of the Panax ginseng glycosyltransferase UGTPg29 generated the triple mutant UGTPg29_{C41I/A140S/A342L} for rebaudioside D biosynthesis. This mutant exhibited a catalytic efficiency (k_cat/K_m) 16.5-fold that of the wild type toward rebaudioside A (RebA) and retained 85.18% of its activity after 3 h at 50 °C. When coupled with UGT76G4 or NtUGT_{F72L/L123P/L157P}, and Arabidopsis thaliana sucrose synthase, this enzyme drove one-pot cascades converting abundant RebA into 62.74 mM RebM and 41.01 mM RebM2, with molar yields of 75.8% and 66.1%. These results establish an efficient multi-enzyme strategy for the green production of high-value SGs.