Enzyme mining and D-tagatose synthesis using a dual-enzyme redox system.

The global risk of diabetes and obesity has risen sharply in recent years. D-tagatose, a low-calorie sweetener with medicinal properties, has gained significant attention in the food industry. D-tagatose was produced from D-galactose using L-arabinose isomerase as a catalyst. However, due to the limitation of thermodynamic equilibrium, the conversion rate is relatively low, and the product separation and purification processes are bottlenecks. Recently, the production of D-tagatose via redox reactions has been demonstrated as feasible. However, due to the low enzyme activity of known redox enzymes, the conversion rates remain limited. In this study, high-activity xylose reductase from Meyerozyma guilliermondii (MgXR) and galactitol dehydrogenase from Rhizobium meliloti (RmGDH) were obtained by enzyme screening. The activity of MgXR was 42.2 ± 1.1 U mg, and that of RmGDH was 10.2 ± 0.8 U mg. These two enzymes were co-expressed in E. coli BL21(DE3) to convert D-galactose into galactitol and then into D-tagatose, showing a titer of 15.48 g/L. In addition, a NADPH regeneration system was used, which further increased the D-tagatose titer to 21.3 g/L. Finally, using lactose as substrate, the E. coli MgRm-trcf strain produced 34.7 g/L D-tagatose during a fed-batch fermentation with a productivity of 0.482 g/L·h, which is the highest among reported. In summary, our study demonstrated the high efficiency of D-tagatose synthesis using a redox dual-enzyme system within the E. coli cell factory.