Industrially important compounds such as carotenoids, polymers and chiral alcohols, among others, require reducing equivalents, e.g. NAD(P)H, for their biosynthesis. The use of whole-cell biocatalysis can reduce process cost by acting as catalyst and cofactor regenerator at the same time; however product yields might be limited by cofactor availability within the cell. Thus, our study focuses on the genetic manipulation of a whole-cell system by modifying metabolic pathways and enzymes to improve the overall production process. In the present work we genetically engineered an Escherichia coli strain to increase NADPH availability and thus improve product yield. Our approach involved the alteration of the glycolysis step where glyceraldehyde-3-phosphate (G3P) is oxidized to 3-phosphoglycerate (3-PG). This reaction is catalyzed by an endogenous NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by the gapA gene. The gapA gene was inactivated and the heterologous NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC, was overexpressed. As a result, the recombinant E. coli produced NADPH instead of NADH during the mentioned reaction. The increase in NADPH availability in our system was assessed using monooxygenase enzymes that require stoichiometric amounts of NADPH to convert linear or cyclic ketones into the corresponding esters and lactones. These enzymes catalyze chemo- and stereo- specific reactions leading to products with high optical purity which are extremely important for chemical and pharmaceutical applications.