Improving The Thermostability Of Glycosyltransferase YojK By Targeting Mutagenesis For Highly Efficient Biosynthesis Of Rebaudioside D

Rebaudioside D（Reb D）,as a natural sweetener derived from Stevia rebaudiana,has great market potential due to low calorie,high sweetness and good taste profile.However,due to the low content in stevia,the enzymatic preparation of Reb D with rebaudioside A（Reb A）as a substrate is considered a feasible solution.Glycosyltransferase YojK derived from Bacillus subtilis 168 was mined in previous study.The enzyme YojK can efficiently convert Reb A to Reb D,but the poor thermostability limits its industrial application.To enhance the application value of YojK,the mutantion library was constructed by rational design in this article.Next,the molecular mechanism of improved thermostability was analyzed based on the crystal structure of mutant and molecular dynamics simulation.The results well demonstrated that the newly formed hydrogen bond interactions,salt bridges and the optimization of surface electrostatic charges enhance its thermostability.The research content is as follows:（1）Obtaining the mutation library of YojK by rational design.First,the online serve PROSS and Fire Prot were used to predict mutations,the overlapped ones,a total of 44 mutants,were selected.Then,Deep DDG was employed for folding energy calculation to delete the false positives.In order to avoid the loss of enzymatic activity,structure analysis and multiple sequence analysis were performed to further narrow down the size of mutant library,and the remaining 9 mutations（D87I,V115I,S158E,D169K,L199N,A128H,L253F,T273I and A369K）were finally obtained.（2）Obtaining combinational mutant YojK_M1 with enhanced thermostability.The enzymatic activity and thermostability analysis of the remaining mutations showed that the mutants S158E,A218H and A369K all had better thermostability and enzyme activity.The mutant YojK_M1(YojK_M0-S158E/A218H/A369K)was obtained through combined mutations.The denaturation temperature and optimum temperature of YojK_M1 were increased by 3.36 and10°C,respectively,and the half-life values was 58.64 h at 50°C,which was 27.15 times higher than that of YojK_M0.At the same time,the enzymatic activity of the mutant YojK_M1 was also improved with a 1.39-fold increase.（3）Analyzing the molecular mechanism of enhanced thermostability of YojK_M1.To understand the molecular mechanism of improved thermostability,the protein crystallization conditions of the mutant YojK_M1 were screened and optimized,and the crystal structure of YojK_M1 with a resolution of 2.5（?）was obtained（PDB ID:8H5D）.Through the molecular dynamics simulation analysis,the region with higher rigidity than YojK_M0 in the structure of YojK_M1 was obtained.Further structural analysis found that new interactions were generated at the mutation sites or nearby regions.The results showed that the formation of new hydrogen bonds interactions and salt bridges,and the optimization of surface electrostatic charges improved the thermostability of YojK.（4）Highly efficient synthesis of Reb D enabled by YojK_M1.Finally,the mutant YojK_M1was used for long-term reaction to prepare Reb D.Reb A was efficiently converted into Reb D by YojK_M1 with a great yield of 87.70%from the high concentration,up to 25 m M.However,only a moderate yield of Reb D（64.00%）was realized by YojK_M0.