Microbial production of the low-caloric sweetener d-allulose from d-glucose by evolutionary engineering

d-Allulose is a low-caloric natural sweetener and a promising alternative to d-sucrose and high-fructose corn syrup. Its current production relies on immobilized enzymes operating at approximately 60°C. In this research article, we developed a whole-cell process that converts d-glucose to d-allulose at 30°C, eliminating the need for enzyme purification and immobilization. To overcome inefficient d-glucose conversion to d-fructose at this temperature, we subjected a bacterial selection strain, whose growth was coupled to d-glucose isomerase activity, to adaptive laboratory evolution. This yielded improved d-glucose isomerase variants and mutations that strongly enhanced sugar uptake. Their integration into a highly engineered chassis strain expressing d-allulose 3-epimerase, together with another adaptive evolution experiment, enabled d-allulose yields of up to 19%, exceeding those obtained with immobilized enzymes at high temperatures. Together, our findings establish a strategy to overcome the limitations of a cell-free enzymatic process using a microbial whole-cell catalyst, offering alternative solutions for the food industry.