Screening to isolate Bacillus subtilis mutants with enhanced NADPH levels

As the first step toward understanding how NADPH levels are regulated in Bacillus subtilis, we sought to obtain mutant strains with enhanced NADPH levels. Our previous study demonstrated that in a strain of B. subtilis expressing bacterial luciferase derived from Photorhabdus luminescens, artificially enhancing NADPH levels enhanced luciferase luminescence in the colonies. In this study, from a library of ethyl methanesulfonate-treated mutants, those with enhanced luciferase luminescence in colonies were isolated, and five isolates were further selected by luminescence in microplate culture. Finally, we measured intracellular NADPH levels of them and found that all the five strains had significantly enhanced NADPH levels compared to the parental strain. In addition, four strains significantly increased total NADP(H) levels. These results demonstrate the effectiveness of our strategy as a methodology for obtaining mutant strains useful for elucidating the mechanisms for regulation of NADPH levels in B. subtilis.