Improving the thermostability of glycosyltransferase UGT76G1 by computer-aided target analysis for highly efficient biosynthesis of rebaudioside M

Natural sweetener rebaudioside M (Reb M), with high sweetness, good taste profile and zero-calorie, has abundant market potential. Currently, it is mainly from the glycosylation of rebaudioside D (Reb D) by glycosyltransferase UGT76G1. However, its poor thermostability hampers the large-scale preparation of Reb M in industry. In this study, through computer-aided target mutagenesis and structure analysis, a four-point mutant M3 (I30M/K53A/R141P/G349P) of UGT76G1-T284S/M88L/L200A (M0) with increased melting temperature of 6.89 °C and 1.16-fold improvement of catalytic activity is obtained from the limited mutant library. Based on molecular dynamics simulations and molecular mechanism analysis, it reveals that the improved thermostability is attributed to tighter core packing, redistribution of surface electrostatic charges and higher loop rigidity. Importantly, this variant increases the yield of Reb M to 90.60% from 25 mM Reb D in a fed-batch reaction at 45 °C. This work not only provides the thermostable M3 for potential scale-up preparation of Reb M, but also develops robust combinatorial strategies to enhance the enzyme thermostability.