Engineering of glycerol-derived aldolase pathway improves D-allulose synthesis in a glucose-glycerol dual-carbon...

D-allulose is a rare sugar with multiple physiological functions and industrial applications. Efficient microbial production of D-allulose requires appropriate carbon allocation between cell growth and product formation. In this study, an engineered Escherichia coli BL21(DE3) strain was constructed for D-allulose biosynthesis using a glucose-glycerol co-utilization strategy. First, we established a phosphorylation-driven D-allulose pathway, achieving a titer of 1.43 g/L of D-allulose. However, knocking out the pfkA gene impaired cell growth, and the biomass OD₆₀₀ value was only 1.54. To restore biomass accumulation, the glycerol kinase mutant GlpK^G913S and the transcriptional regulator Mlc were overexpressed to enhance glycerol utilization efficiency, resulting in a 51.9% increase in biomass, but the glycerol utilization rate was only 45.0%. To further utilize the remaining glycerol, an aldol reaction pathway was constructed by expressing the genes aldO (alditol oxidase), rhaD (L-rhamnulose-1-phosphate aldolase), and yqaB (fructose-1-phosphate), which increased the glycerol utilization rate to 62.5% and the D-allulose yield to 3.46 g/L. Under optimized carbon source ratio conditions, the engineered strain produced 3.92 g/L of D-allulose with a productivity of 0.054 g/L/h, and the cell biomass was successfully increased to an OD₆₀₀ of 10.4. These results demonstrate that using glycerol as a co-substrate can promote cell growth and enhance D-allulose synthesis in engineered E. coli, providing a basis for further metabolic optimization of rare sugar production.