Sucrose　is　a　commonly　utilized　nutritive　sweetener　in　food　and　beverages　due　to　its　abundance　in　nature　and　low　production　costs.　However,　excessive　intake　of　sucrose　increases　the　risk　of　metabolic　disorders,　including　diabetes　and　obesity.　Therefore,　there　is　a　growing　demand　for　the　development　of　nonnutritive　sweeteners　with　almost　no　calories.　d-Allulose　is　an　ultra-low-calorie,　rare　six-carbon　monosaccharide　with　high　sweetness,　making　it　an　ideal　alternative　to　sucrose.　In　this　study,　we　developed　a　cell　factory　for　d-allulose　production　from　sucrose　using　Escherichia　coli　JM109　(DE3)　as　a　chassis　host.　The　genes　cscA,　cscB,　cscK,　alsE,　and　a6PP　were　co-expressed　for　the　construction　of　the　synthesis　pathway.　Then,　the　introduction　of　ptsG-F　and　knockout　of　ptsG,　fruA,　ptsI,　and　ptsH　to　reprogram　sugar　transport　pathways　resulted　in　an　improvement　in　substrate　utilization.　Next,　the　carbon　fluxes　of　the　Embden-Meyerhof-Parnas　and　the　pentose　phosphate　pathways　were　regulated　by　the　inactivation　of　pfkA　and　zwf,　achieving　an　increase　in　d-allulose　titer　and　yield　of　154.2%　and　161.1%,　respectively.　Finally,　scaled-up　fermentation　was　performed　in　a　5　L　fermenter.　The　titer　of　d-allulose　reached　11.15　g/L,　with　a　yield　of　0.208　g/g　on　sucrose.　©　2024　Wiley　Periodicals　LLC.