D-Psicose has been attracting attention in recent years because of its alimentary activities and is used as an ingredient in a range of foods and dietary supplements. To develop a one-step enzymatic process of D-psicose production, thermoactive D-glucose isomerase and the D-psicose 3-epimerase obtained from Bacillus sp. and Ruminococcus sp., respectively, were successfully co-expressed in Escherichia coli BL21 strain. The substrate of one-step enzymatic process was D-glucose. The co-expression system exhibited maximum activity at 65 degrees C and pH 7.0. Mg2+ could enhance the output of D-psicose by 232 fold to 1.6 g/L from 10 g/L of D-glucose. When using high-fructose corn syrup (HFCS) as substrate, 135 g/L D-psicose was produced under optimum conditions. The mass ratio of D-glucose, D-fructose, and D-psicose was almost 3.0:2.7:1.0, when the reaction reached equilibrium after an 8 h incubation time. This co-expression system approaching to produce D-psicose has potential application in food and beverage products, especially softdrinks. (C) 2014 Elsevier Inc. All rights reserved.