Improving the thermostability of glycosyltransferase YojK by targeting mutagenesis for highly efficient biosynthesis of rebaudioside D

Rebaudioside D (Reb D), as a healthy natural sweetener, has abundant market potential due to the good taste profile, high sweetness and zero calories. Glycosyltransferase YojK from Bacillus subtilis 168 has been identified with the ability to glycosylate Rebaudioside A (Reb A) to produce Reb D, but the poor thermostability still limits its industrial application. Herein, combined with folding energy calculation, structure analysis and consensus analysis, a combinational mutant YojKM1 (S158E/A218H/I241T/G327N/A369K) with a great increase of half-time at 50°C from 2.16 h to 58.64 h was obtained. Moreover, its catalytic activity was also improved with a 1.39-fold increase. Next, its crystal structure was solved and well demonstrated that the newly formed hydrogen bond interactions, salt bridges and the optimization of surface electrostatic charges enhance its thermostability. Last, a fed-batch reaction was established to prepare Reb D with a great yield of 87.70% at high concentration of Reb A at 50°C. Therefore, an efficient and thermostable YojKM1 was developed for the potential industrial production of Reb D.