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D-Allulose is the C-3 epimer of D-fructose, and widely regarded as a promising substitute for sucrose. It’s an excellent low-calorie sweetener, with 70% sweetness of sucrose, 0.4 kcal/g dietary energy, and special physiological functions. It has been approved as GRAS by the U.S. Food and Drug Administration, and is allowed to be excluded from total and added sugar counts on the food labels. Therefore, D-allulose gradually attracts more public attention. Owing to scarcity in nature, the bioproduction of D-allulose by using ketose 3-epimerase (KEase) has become the research hotspot. Herein, we give a summary of the physicochemical properties, physiological function, applications, and the chemical and biochemical synthesis methods of D-allulose. In addition, the recent progress in the D-allulose bioproduction using KEases, and the possible solutions for existing challenges in the D-allulose industrial production are comprehensively discussed, focusing on the molecular modification, immobilization, food-grade expression, utilizing low-cost biomass as feedstock, overcoming thermodynamic limitation, as well as the downstream separation and purification. Finally, Prospects for further development are also proposed.

D-allulose, a versatile rare sugar: recent biotechnological advances and challenges