Highly Efficient Biosynthesis of Rebaudioside M8 through Structure-Guided Engineering of Glycosyltransferase UGT94E13

作者：Lifeng Yang, Mengliang Yang, Zhiwei Deng, Zhengshan Luo, Zhenbo Yuan, Yijian Rao, Yan Zhang · 发表于：Journal of Agricultural and Food Chemistry · 年份：2024 · DOI：10.1021/acs.jafc.4c03565 · 被引用次数：10 · 研究领域：Biochemical Analysis and Sensing Techniques、melanin and skin pigmentation、RNA and protein synthesis mechanisms

Given the low-calorie, high-sweetness characteristics of steviol glycosides (SGs), developing SGs with improved taste profiles is a key focus. Rebaudioside M8 (Reb M8), a novel non-natural SG derivative obtained through glycosylation at the C-13 position of rebaudioside D (Reb D) using glycosyltransferase UGT94E13, holds promise for further development due to its enhanced sweetness. However, the low catalytic activity of UGT94E13 hampers further research and commercialization. This study aimed to improve the enzymatic activity of UGT94E13 through semirational design, and a variant UGT94E13-F169G/I185G was obtained with the catalytic activity improved by 13.90 times. A cascade reaction involving UGT94E13-F169G/I185G and sucrose synthase At SuSy was established to recycle uridine diphosphate glucose, resulting in an efficient preparation of Reb M8 with a yield of 98%. Moreover, according to the analysis of the distances between the substrate Reb D and enzymes as well as between Reb D and the glucose donor through molecular dynamics simulations, it is found that the positive effect of shortening the distance on glycosylation reaction activity accounts for the improved catalytic activity of UGT94E13-F169G/I185G. Therefore, this study addresses the bottleneck in the efficient production of Reb M8 and provides a foundation for its widespread application in the food industry.