The high-demand rare sugars D-tagatose and D-allulose may be produced from lactose in a combined process, though this dual conversion has not yet been demonstrated. Four enzymes from thermophilic organisms were cloned and expressed in E. coli to synthesize D-tagatose and D-allulose from lactose: β-galactosidase (Geobacillus stearothermophilus), L-arabinose isomerase (Geobacillus stearothermophilus US100), D-glucose isomerase (Weizmannia coagulans) and D-allulose epimerase (Arthrobacter globiformis). A single-pot reaction mixture yielded 9.9 + 1.5 mg mL-1 D-allulose and 10.7 + 1.1 mg mL-1 D-tagatose after 24h at 70°C. Enzyme addition order did not have an impact on yields (p=0.3527). Higher lactose concentrations resulted in higher D-tagatose concentrations (p