Underutilized dairy streams are predominantly composed of lactose. The high-demand rare sugars D-tagatose and D-allulose may theoretically be produced from lactose via a four-enzyme pathway in a single reaction, though this conversion has not yet been demonstrated. With the demand for these rare sugars being comparable to the lactose supply, such a demonstration could set the stage for commercial scale-up. To produce D-tagatose and D-allulose from lactose, four enzymes from thermophilic organisms were cloned and expressed in E. coli: 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 5.2-10.8 mg mL-1 D-allulose and 9.5- 13.1 mg mL-1 D-tagatose after 24 hours at 70°C. Enzyme addition order did not have an impact on yields. Higher lactose concentrations resulted in higher D-allulose and D-tagatose concentrations, but specific yields for D-allulose were higher with lower lactose starting concentrations.