d-Allulose,　a　C-3　epimer　of　d-fructose,　has　great　market　potential　in　food,　healthcare,　and　medicine　due　to　its　excellent　biochemical　and　physiological　properties.　Microbial　fermentation　for　d-allulose　production　is　being　developed,　which　contributes　to　cost　savings　and　environmental　protection.　A　novel　metabolic　pathway　for　the　biosynthesis　of　d-allulose　from　a　d-xylose-methanol　mixture　has　shown　potential　for　industrial　application.　In　this　study,　an　artificial　antisense　RNA　(asRNA)　was　introduced　into　engineered　Escherichia　coli　to　diminish　the　flow　of　pentose　phosphate　(PP)　pathway,　while　the　UDP-glucose-4-epimerase　(GalE)　was　knocked　out　to　prevent　the　synthesis　of　byproducts.　As　a　result,　the　d-allulose　yield　on　d-xylose　was　increased　by　35.1%.　Then,　we　designed　a　d-xylose-sensitive　translation　control　system　to　regulate　the　expression　of　the　formaldehyde　detoxification　operon　(FrmRAB),　achieving　self-inductive　detoxification　by　cells.　Finally,　fed-batch　fermentation　was　carried　out　to　improve　the　productivity　of　the　cell　factory.　The　d-allulose　titer　reached　98.6　mM,　with　a　yield　of　0.615　mM/mM　on　d-xylose　and　a　productivity　of　0.969　mM/h.　©　2024　American　Chemical　Society.