Cofactor engineering is a common strategy to improve amino acid production. 4-hydroxyisoleucine (4-HIL), a nonproteinogenic amino acid, exhibits unique insulinotropic and insulin-sensitizing activities, therefore has potential medical value in treating diabetes. In our previous study, t-isoleucine (Ile) dioxygenase gene ido was overexpressed in an Ile-producing Corynebacterium glutamicum strain, and 4-HIL was de novo synthesized from glucose. In this study, to increase the NADPH supply, the endogenous NAD(+) kinase gene ppnK and glucose-6-phosphate dehydrogenase gene zwf were co-expressed with ido. The resulting strain SL01 produced 81.12 +/- 5.96 mM 4-HIL, 62% higher than the ido-mere expressing strain SN02. However, the strain SL02 coexpressing exogenous NADH kinase gene POS5 with ido grew slowly and its 4-HIL production decreased by 12%, perhaps due to the lower 2-oxoglutarate (OG) level and slightly weaker membrane permeability. To increase OG availability for 4-HIL conversion, the serine/threonine protein kinase G gene pknG was deleted and replaced by ido gene in SL02. The growth of the resulting strain SL04 was restored and 4-HIL production was improved to 84.14 +/- 6.38 mM; meanwhile, the conversion ratio of Ile to 4-HIL reached up to 0.98 +/- 0.01 mol/mol. Therefore, sufficient NADPH supply and OG availability may be benefit to 4-HIL de novo biosynthesis in recombinant C. glutamicum.