d-Allulose　is　a　rare　hexose　with　great　application　potential,　owing　to　its　moderate　sweetness,　low　energy,　and　unique　physiological　functions.　The　current　strategies　for　d-allulose　production,　whether　industrialized　or　under　development,　utilize　six-carbon　sugars　such　as　d-glucose　or　d-fructose　as　a　substrate　and　are　usually　based　on　the　principle　of　reversible　Izumoring　epimerization.　In　this　work,　we　designed　a　novel　route　that　coupled　the　pathways　of　methanol　reduction,　pentose　phosphate　(PP),　ribulose　monophosphate　(RuMP),　and　allulose　monophosphate　(AuMP)　for　Escherichia　coli　to　irreversibly　synthesize　d-allulose　from　d-xylose　and　methanol.　After　improving　the　expression　of　AlsE　by　SUMO　fusion　and　regulating　the　carbon　fluxes　by　knockout　of　FrmRAB,　RpiA,　PfkA,　and　PfkB,　the　titer　of　d-allulose　in　fed-batch　fermentation　reached　≈70.7　mM,　with　a　yield　of　≈0.471　mM/mM　on　d-xylose　or　≈0.512　mM/mM　on　methanol.　©　2022　American　Chemical　Society.　All　rights　reserved.