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The nicotinamide cofactor regeneration is essential for various oxidoreductase reactions.1,2) Herein, feasibility of a novel cofactor regeneration system was demonstrated using lactate dehydrogenase as a coupling reaction model. This regeneration system employed a permeabilized E.coli cell, in which glpD and gldA genes were deleted and glycerol-3-phosphate dehydrogenase (G3PDH) was overexpressed. These genetic manipulations were designed to block outflow of glycerol co-substrate and to substitute the reverse reaction of the G3PDH for the glpD gene encoding enzyme reaction. This novel cofactor regeneration efficiency could be easily verified using a simple colorimetric assay designed for monitoring pyruvate concentration.3) The results showed the cofactor regeneration system constructed in this study, where NADH produced by G3PDH was consumed by lactate dehydrogenase and then NAD+ was regenerated to NADH by G3PDH, worked very well as predicted. In addition, a capacity of NADPH regeneration can be drawn from the results of G3PDH activity assay 4) and further improvement of the G3PDH efficiency will be possible without constructing other screening system. Therefore, cofactor regeneration system of this study may have some advantages over other approaches and will be applicable to various oxidoreductase reactions.