Coproduction of 2-O-α-d-Glucosyl Glycerol and D-Allulose from Sucrose and Glycerol by Dual-Enzyme Cascade

The efficient and cost-effective bioconversion of low-cost substrates into high-value products is a major challenge in industrial biotechnology. In response to the increasing demand for moisturizing agents (e.g., 2-O-α-d-glucosyl glycerol) and low-calorie sweeteners (e.g., D-allulose), we developed a dual-enzyme one-pot cascade system for the coproduction of 2-O-α-d-glucosyl glycerol (2-αGG) and D-allulose from sucrose and glycerol. The system integrates sucrose phosphorylase (SPase) and D-allulose-3-epimerase (DAEase) and was further enhanced through strategies, including dual-enzyme coexpression, sequential reaction, and protein engineering. A semirational design approach guided by molecular modeling led to I66V mutation in DAEase, which improved both catalytic activity and thermal stability by reinforcing local structural rigidity. Under optimized conditions, the system achieved titers of 215 g/L for 2-αGG and 104.6 g/L for D-allulose, with conversion rates of 97.7% for sucrose, 42.3% for glycerol, and 60.0% for D-fructose. This work presents a robust and economical enzymatic platform for the sustainable production of high-value biobased compounds with potential applications in the food and cosmetic industries.