D-Allulose　is　an　ultra-low-calorie　sweetener　with　broad　market　prospects　in　the　fields　of　food,　beverage,　health　care,　and　medicine.　The　fermentative　synthesis　of　D-allulose　is　still　under　development　and　considered　as　an　ideal　route　to　replace　enzymatic　approaches　for　large-scale　production　of　D-allulose　in　the　future.　Generally,　D-allulose　is　synthesized　from　D-fructose　through　Izumoring　epimerization.　This　biological　reaction　is　reversible,　and　a　high　temperature　is　beneficial　to　the　conversion　of　D-fructose.　Mild　cell　growth　conditions　seriously　limit　the　efficiency　of　producing　D-allulose　through　fermentation.　FryABC　permease　was　identified　to　be　responsible　for　the　transport　of　D-allulose　in　Escherichia　coli　by　comparative　transcriptomic　analysis.　A　cell　factory　was　then　developed　by　expression　of　ptsG-F,　dpe,　and　deletion　of　fryA,　fruA,　manXYZ,　mak,　and　galE.　The　results　show　that　the　newly　engineered　E.　coli　was　able　to　produce　32.33 ± 1.33 g L−1　of　D-allulose　through　a　unique　thermo-swing　fermentation　process,　with　a　yield　of　0.94 ± 0.01 g g−1　on　D-fructose.　©　2023　Wiley-VCH　GmbH.