Efficient biosynthesis of D-allulose in Bacillus subtilis through D-psicose 3-epimerase translation modification

The combined catalysis of glucose isomerase (GI) and D-psicose 3-epimerase (DPEase) provided a convenient route for the direct synthesis of D-allulose from d-glucose, whose cost is lower than d-fructose. In the present research, the weak activity of DPEase was the key rate-limiting step and resulted in the accumulation of d-fructose in engineered Bacillus subtilis. Then, the 5'-untranslated region (5'-UTR) structure of the mRNA translational initiation region was optimized for the precise control of DPEase expression. The manipulation of the 5'-UTR region promoted the accessibility to ribosome binding and the stability of mRNA, resulting in a maximum of 1.73- and 1.98-fold increase in DPEase activity and intracellular mRNA amount, respectively. Under the optimal catalytic conditions of 75 °C, pH 6.5, 110 g/L d-glucose, and 1 mmol/L Co²⁺, the reaction equilibrium time was reduced from 7.6 h to 6.1 h. We hope that our results could provide a facilitated strategy for large-scale production of D-allulose at low-cost.