Enzymatic synthesis of some industrially important compounds depends heavily on cofactor NADPH as the reducing agent. This is especially true in the synthesis of chiral compounds that are often used as pharmaceutical intermediates to generate the correct stereochemistry in bioactive products. The high cost and technical difficulty of cofactor regeneration often pose a challenge for such biocatalytic reactions. In this study, to increase NADPH bioavailability, the native NAD(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gapA gene in Escherichia coli was replaced with a NADP(+)-dependent gapB from Bacillus subtilis. To overcome the limitation of NADP(+) availability, E. coli NAD kinase, nadK was also coexpressed with gapB. The recombinant strains were then tested in three reporting systems: biosynthesis of lycopene, oxidation of cyclohexanone with cyclohexanone monooxygenase (CHMO), and an anaerobic system utilizing 2-haloacrylate reductase (CAA43). In all the reporting systems, replacing NAD(+)-dependent GapA activity with NADP(+)-dependent GapB activity increased the synthesis of NADPH-dependent compounds. The increase was more pronounced when NAD kinase was also overexpressed in the case of the one-step reaction catalyzed by CAA43 which approximately doubled the product yield. These results validate this novel approach to improve NADPH bioavailability in E. coli and suggest that the strategy can be applied in E. coli or other bacterium-based production of NADPH-dependent compounds.