A gene for 2,5-diketo-d-gluconate (25DKG) reductase, which encodes an enzyme composed of 277 amino acid residues catalyzing the reduction of 25DKG to 2-keto-l-gulonate (2KLG), was cloned from Corynebacterium sp. strain SHS752001 and expressed in Erwinia citreus SHS2003, a strain which oxidizes glucose to 25DKG. The recombinant microorganism converted glucose to 2KLG, a compound which can be readily converted to l-ascorbate (vitamin C). Improvements in the yield of 2KLG were obtained by changing fermentation conditions, using the p(l) promoter of bacteriophage lambda to express the reductase, and selecting a mutant of E. citreus which could use neither 25DKG nor 2KLG as a sole carbon source for growth. When a culture of the recombinant strain was fed with glucose to a total of 40 g/liter, 49.4% of the glucose was converted to 2KLG during a 72-h fermentation.