D-allulose,　a　naturally　occurring　monosaccharide,　is　present　in　small　quantities　in　nature.　It　is　considered　a　valuable　low-calorie　sweetener　due　to　its　low　absorption　in　the　digestive　tract　and　zero　energy　for　growth.　Most　of　the　recent　efforts　to　produce　D-allulose　have　focused　on　in　vitro　enzyme　catalysis.　However,　microbial　fermentation　is　emerging　as　a　promising　alternative　that　offers　the　advantage　of　combining　enzyme　manufacturing　and　product　synthesis　within　a　single　bioreactor.　Here,　a　novel　approach　was　proposed　for　the　efficient　biosynthesis　of　D-allulose　from　glycerol　using　metabolically　engineered　Escherichia　coli.　FbaA,　Fbp,　AlsE,　and　A6PP　were　used　to　construct　the　D-allulose　synthesis　pathway.　Subsequently,　PfkA,　PfkB,　and　Pgi　were　disrupted　to　block　the　entry　of　the　intermediate　fructose-6-phosphate　(F6P)　into　the　Embden−Meyerhof−Parnas　(EMP)　and　pentose　phosphate　(PP)　pathways.　Additionally,　GalE　and　FryA　were　inactivated　to　reduce　D-allulose　consumption　by　the　cells.　Finally,　a　fed-batch　fermentation　process　was　implemented　to　optimize　the　performance　of　the　cell　factory.　As　a　result,　the　titer　of　D-allulose　reached　7.02　g/L　with　a　maximum　yield　of　0.287　g/g.　©　2024　Elsevier　B.V.