Advanced Whole-cell Conversion for D-allulose Production Using an Engineered Corynebacterium glutamicum

D-allulose has received considerable attention as an alternative functional sugar for its zero caloric value with 70% relative sweetness compared to D-sucrose. Despite its strong potential as an alternative sweetener, recent industrial productions rely on a high-cost enzymatic method. Here, we advanced whole-cell conversion at high temperatures using Corynebacterium glutamicum expressing D-allulose 3-epimerase (DAE). By varying the reaction temperature from 25°C to 70°C, D-allulose conversion could reach the reaction equilibrium at high temperatures. The C. glutamicum showed superior reusability of cells at 60°C compared to Escherichia coli. We simplified the cell growth media and whole-cell conversion reaction solution. Clostridium hylemonae DAE (ChDAE) showed the highest thermostability and reusability among various DAE candidates. Finally, the ChDAE expression under the synthetic 2X-cT-T5 promoter could reduce the reaction time by 25%. Our result showed that 120 g/L of D-allulose can be produced from 400 g/L of D-fructose by reusable whole-cell conversion at 55°C in 1.5 h. This study can be highly applicable in industrial economic production.