d-Allose　has　great　potential　for　application　in　the　food　and　pharmaceutical　industries　owing　to　its　remarkable　physiological　properties.　Most　studies　on　d-allose　production　have　primarily　focused　on　enzyme　catalysis　using　the　Izumoring　strategy,　which　typically　requires　the　use　of　expensive　d-allulose　as　a　substrate.　Herein,　a　metabolically　engineered　strain　of　Escherichia　coli　was　developed　to　synthesize　d-allose　directly　from　inexpensive　d-glucose.　The　synthesis　pathway　was　systematically　optimized　through　a　modular　metabolic　engineering.　The　functionality　of　the　isomerases　involved　in　the　conversion　of　d-allulose　to　d-allose　was　confirmed　in　vivo,　while　the　byproduct　and　transporter　pathways　were　blocked　to　positively　pull　the　reversible　epimerization.　Gene　knockouts　were　employed　to　weaken　glycolytic　pathways,　redirecting　the　carbon　flux　toward　product　synthesis.　Additionally,　the　nonphosphorylated　transport　of　d-glucose　was　introduced　to　enhance　substrate　utilization.　In　fed-batch　fermentation,　the　engineered　strain　achieved　a　d-allose　titer　of　4.17　g/L,　with　a　yield　of　0.103　g/g　from　d-glucose.　Our　achievements　are　expected　to　advance　the　industrial　production　of　d-allose,　and　this　strategy　is　also　applicable　for　producing　other　rare　sugars.　©　2025　American　Chemical　Society.