Improvement of d-Allulose Synthesis from Sucrose through Dual PTS-Dependent Transport in Engineered Escherichia coli

d-Allulose, a rare sugar characterized by its high sweetness and low-calorie profile, is gaining attention in the sweetener market. This study introduces an innovative method for converting sucrose into d-allulose through microbial fermentation. An irreversible synthesis pathway was constructed by expressing the scrA, scrB, alsE, and a6PP genes in Escherichia coli JM109 (DE3), enhancing substrate utilization via dual PTS-dependent transport of sucrose and d-fructose. A fructose-1,6-bisphosphatase mutant (GlpX [K29A]) was used to facilitate the influx of fructose-1-phosphate into the synthesis pathway. The Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways were weakened by deleting the pfkA and rpiA genes. To further regulate carbon fluxes, a structurally stable antisense RNA (asRNA) was employed to inhibit FbaA expression. The fermentation medium was optimized using response surface methodology. Finally, the d-allulose titer reached 12.8 g/L, with a yield of 0.23 g/g on sucrose, achieved through fed-batch fermentation in a 5 L fermenter.