D-Allulose　is　an　ultra-low　calorie　sweetener　with　broad　market　prospects.　As　an　alternative　to　Izumoring,　phosphorylation-dephosphorylation　is　a　promising　method　for　D-allulose　synthesis　due　to　its　high　conversion　of　substrate,　which　has　been　preliminarily　attempted　in　enzymatic　systems.　However,　in　vitro　phosphorylation-dephosphorylation　requires　polyphosphate　as　a　phosphate　donor　and　cannot　completely　deplete　the　substrate,　which　may　limit　its　application　in　industry.　Here,　we　designed　and　constructed　a　metabolic　pathway　in　Escherichia　coli　for　producing　D-allulose　from　D-fructose　via　in　vivo　phosphorylation-dephosphorylation.　PtsG-F　and　Mak　were　used　to　replace　the　fructose　phosphotransferase　systems　(PTS)　for　uptake　and　phosphorylation　of　D-fructose　to　fructose-6-phosphate,　which　was　then　converted　to　D-allulose　by　AlsE　and　A6PP.　The　D-allulose　titer　reached　0.35 g/L　and　the　yield　was　0.16 g/g.　Further　block　of　the　carbon　flux　into　the　Embden-Meyerhof-Parnas　(EMP)　pathway　and　introduction　of　an　ATP　regeneration　system　obviously　improved　fermentation　performance,　increasing　the　titer　and　yield　of　D-allulose　to　1.23 g/L　and　0.68 g/g,　respectively.　The　E.　coli　cell　factory　cultured　in　M9　medium　with　glycerol　as　a　carbon　source　achieved　a　D-allulose　titer　of　≈1.59 g/L　and　a　yield　of　≈0.72 g/g　on　D-fructose.　Copyright　©　2022　Guo,　Liu,　Zheng,　Zheng,　Zhang,　Zhao,　Zheng,　Fan　and　Lin.