Metabolic engineering of Corynebacterium glutamicum for 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 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 MakEC and D-allulose 6-phosphate 3-epimerase AlsEEC from E. coli, together with D-allulose 6-phosphate phosphatase AlsPCT 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%.