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

Clostridium acetobutylicum is a promising biocatalyst for the renewable production of n-butanol. Several metabolic strategies have already been developed to increase butanol yields, most often based on carbon pathway redirection. However, it has previously demonstrated that the activities of both ferredoxin-NADP⁺ reductase and ferredoxin-NAD⁺ reductase, whose encoding genes remain unknown, are necessary to produce the NADPH and the extra NADH needed for butanol synthesis under solventogenic conditions. Here, we purify, identify and partially characterize the proteins responsible for both activities and demonstrate the involvement of the identified enzymes in butanol synthesis through a reverse genetic approach. We further demonstrate the yield of butanol formation is limited by the level of expression of CA_C0764, the ferredoxin-NADP⁺ reductase encoding gene and the bcd operon, encoding a ferredoxin-NAD⁺ reductase. The integration of these enzymes into metabolic engineering strategies introduces opportunities for developing a homobutanologenic C. acetobutylicum strain.