Fig. 1: Schematic view of the enzyme reaction and design process.

a The glycosyl transfer pathway mediated by UGT76G1. The glucoses conjugated to steviol aglycon are abbreviated as G1, G2, respectively. The newly attached glucose moieties resulting from the conversion reaction are highlighted in yellow. During the conversion of ST to RebA, UGT76G1 transfers the glucose of UDPG to the first glucose (G1) of C13 in stevioside with a beta-1,3-glycosidic bond. If the glucose of UDPG is transferred to the glucose (G1) of C19 in rebaudioside A, rebaudioside I is produced, which is an unwanted byproduct. b A schematic representation of the design strategies to enhance the thermodynamic stability of UGT76G1 through computational design. c The mutation profile of the final 10 variants. The components of the N-terminal and C-terminal domains of UGT are shown in the first line. The binding regions for steviol aglycon and glucose moieties are represented as yellow rectangles, while the binding regions for UDPG are displayed as blue rectangles. Catalytic residues (H25, D104) are highlighted in red rectangles. The mutation map for each variant is aligned to the exact location on the domain map, with each mutation represented as a rectangle. The overlapping mutations found in more than 5 variants are labeled with green. Mutations in the substrate binding region are marked with orange. The remaining mutations elsewhere are colored by gray. The number of mutations for each variant is displayed as horizontal bars.