Rational Evolution Of Xylulokinase Based On Molecular Docking

Cellulosic ethanol research is of great concern in recent years, many researches have been done on improvement of xylose utilizing ability and co-utilization of glucose and xylose present in cellulose hydrolyzates. Phosphorylation of xylulose to xylulose 5-phosphate catalyzed by xylulokinase is the key step in xylose fermentation, through which xylulose enters the pentose phosphate pathway. Endogenous xylulokinase activity of Saccharomyces cerevisiae is too low to meet the needs of xylose metabolism.This study focused on structure-activity relationship of xylulokinase. Docking analysis of xylulokinase from different origin provided targets for rational evolution. In this study, comparison was done among engineered Saccharomyces cerevisiae with xylulokinase from different origin. According to the fermentation results, the strain with xylulokinase derived from Scheffersomyces stipitis represented the fastest rate of xylose utilization, and the ethanol yield achieved 0.38 g/g xylose. Xylulokinase derived from Candida tropicalis has the lowest enzyme activity, resulting in low ability of xylose utilization.Docking analysis between enzyme and ligand showed that the enzyme acitivity was associated with the binding energy with ATP. With the binding energy with ATP increased, the enzyme activity improved. But the ADP binding energy for XKS from Candida tropicalis was much higher than others, leading to the much lower enzyme activity. The docking results indicated the 502 th amino acid Lys played an important role in combination with ADP. Site-directed mutation of Lys502 improved the performance of the strains on xylose utilization obviously. Xylose consumption rate was 2.3 times of the original strain, and the ethanol yield was increased from 0.11 g/g xylose to 0.39 g/g xylose. Mutations in conservative amino acid didn’t result in desired mutants, suggesting that these sites play a crucial role in substrate binding and enzyme activities. These also confirmed the credibility of the results of molecular docking.Aiming at synchronous utilization of xylose and glucose, we made an attempt on co-utilization of glucose and xylose by deletion of RPE1. The resulting strain presented synchronous utilizing of the sugars, and xylose consumption is about one-tenth of glucose consumption.