Molecular characterization of the missing electron pathways for butanol synthesis in Clostridium acetobutylicum

Abstract Clostridium acetobutylicum is a promising biocatalyst for the production of n-butanol at high yield from renewable resources. Several metabolic strategies have already been developed to increase butanol yields, most often based on carbon pathway redirection. However, it was previously demonstrated that the activities of both ferredoxin-NADP+ reductase and ferredoxin-NAD+ reductase, whose encoding genes remained unknown until this study, were necessary to produce the NADPH and the extra NADH needed for butanol synthesis under solventogenic conditions. Here, we purified, identified and characterized the proteins responsible for both ferredoxin-NADP+ reductase and ferredoxin-NAD+ reductase activities and demonstrated the involvement of the identified enzymes in butanol synthesis through a reverse genetic approach. We further demonstrated the yield of butanol formation was limited by the level of expression of CAC_0764, the ferredoxin-NADP+ reductase encoding gene. The integration of these enzymes into metabolic engineering strategies introduces new opportunities for developing a homobutanologenic C. acetobutylicum strain.