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.　©　2025　American　Chemical　Society