Enhanced Production of Rebaudioside D and Rebaudioside M through V155T Substitution in the Glycosyltransferase UGT91D2 from <i>Stevia rebaudiana</i>

Steviol glycosides (SGs) are noncaloric natural sweeteners found in the leaves of stevia (<i>Stevia rebaudiana</i>). These diterpene glycosides are biosynthesized by attaching varying numbers of monosaccharides, primarily glucose, to steviol aglycone. Rebaudioside (Reb) D and Reb M are highly glucosylated SGs that are valued for their superior sweetness and organoleptic properties, yet they are present in limited quantities in stevia leaves. This study aims to improve the substrate preference and catalytic efficiency of UDP-sugar-dependent glycosyltransferase UGT91D2 from stevia, which acts as a bottleneck in the biosynthesis of Reb D and Reb M. We modeled the structure of UGT91D2 and substituted two amino acid residues, Y134 and V155, which are located near the glycosyl acceptor and donor, respectively. Expression of the <i>UGT91D2</i>^<i>V155T</i> in budding yeast significantly enhanced the production of Reb D and Reb M. Furthermore, transient expression in <i>Nicotiana benthamiana</i> revealed that the V155T substitution improved the glucosylation activity of UGT91D2, suggesting that this substitution enhances UDP-glucose binding and reduces side reactions involving nonglucose donors. By coexpressing multiple stevia <i>UGT</i> genes in <i>N. benthamiana</i>, we successfully produced highly glucosylated SGs from steviol. Our results provide insights into the substrate specificity of UGT91D2 and contribute to the engineering of SG biosynthesis.