D-Allose　is　a　potential　alternative　to　sucrose　in　the　food　industries　and　a　useful　additive　for　the　healthcare　products　in　the　future.　At　present,　the　methods　for　large-scale　production　of　D-allose　are　still　under　investigation,　most　of　which　are　based　on　in　vitro　enzyme-catalyzed　Izumoring　epimerization.　In　contrast,　fermentative　synthesis　of　D-allose　has　never　been　reported,　probably　due　to　the　absence　of　available　natural　microorganisms.　In　this　work,　we　co-expressed　D-galactose:　H+　symporter　(GalP),　D-glucose　isomerase　(DGI),　D-allulose　3-epimerase　(DAE),　and　ribose-5-phosphate　isomerase　(RPI)　in　Escherichia　coli,　thereby　constructing　an　in　vivo　Izumoring　pathway　for　yielding　D-allose　from　D-glucose.　The　carbon　fluxes　and　carbon　catabolite　repression　(CCR)　were　rationally　regulated　by　knockout　of　FruA,　PtsG,　Glk,　Mak,　PfkA,　and　PfkB　involved　in　the　pathways　capable　of　phosphorylating　D-fructose,　D-glucose,　and　fructose-6-phosphate.　Moreover,　the　native　D-allose　transporter　was　damaged　by　inactivation　of　AlsB,　thus　driving　the　reversible　Izumoring　reactions　towards　the　target　product.　Fermentation　was　performed　in　the　M9　medium　supplemented　with　glycerol　as　a　carbon　source　and　D-glucose　as　a　substrate.　The　results　show　that　the　engineered　E.　coli　cell　factory　was　able　to　produce　approximately　127.35 mg/L　of　D-allose　after　84 h.　Our　achievements　in　the　fermentative　production　of　D-allose　in　this　work　may　further　promote　the　green　manufacturing　of　rare　sugars.　Copyright　©　2022　Zheng,　Guo,　Zhang,　Liu,　Fan　and　Zheng.