Metabolic engineering of Corynebacterium glutamicum for the production of the low-caloric natural sweetener D-allulose via phosphorylated intermediates

D -Allulose is a natural, low-calorie sweetener providing ∼70% of the sweetness of D-sucrose but ≤10% of its caloric value, making it an attractive alternative to conventional sugars. Recently, a phosphorylation–dephosphorylation pathway for D -allulose production was established, involving the formation and irreversible dephosphorylation of D -allulose 6-phosphate. Although this pathway has been demonstrated in Escherichia coli , efficient production involved complex medium, complicating downstream processing. Here, we report D -allulose production in minimal medium by implementing the phosphorylation–dephosphorylation pathway in a Corynebacterium glutamicum strain unable to metabolize D -fructose. Growth- and production-based screenings identified fructokinase Mak EC and D -allulose 6-phosphate 3-epimerase AlsE EC from E. coli , together with D -allulose 6-phosphate phosphatase AlsP CT from Clostridium thermocellum , as the most effective enzyme combination for D -allulose formation. Further metabolic engineering of C. glutamicum including deletion of zwf ( D -glucose 6-phosphate dehydrogenase), overexpression of fbp ( D -fructose 1,6-bisphosphatase), and downregulation of pgm (phosphoglucomutase) partially redirected central carbon flux toward D -allulose synthesis, resulting in a 2.3-fold increase in production. The engineered strain produced ∼3.6 g L −1 D -allulose from a D -glucose– D -fructose mixture with a yield of 9.1%.