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.

丙酮丁醇梭菌是一种很有前景的生物催化剂，可用于正丁醇的可再生生产。已经开发了几种代谢策略来增加丁醇产量，最常见的是基于碳途径重定向。然而，先前已经证明铁氧还蛋白-NADP ⁺还原酶和铁氧还蛋白-NAD ^+的活性还原酶的编码基因仍然未知，它是产生 NADPH 和在致溶剂条件下合成丁醇所需的额外 NADH 所必需的。在这里，我们纯化、鉴定和部分表征负责这两种活性的蛋白质，并通过反向遗传方法证明所鉴定的酶参与丁醇合成。我们进一步证明丁醇形成的产量受到CA_C0764、铁氧还蛋白-NADP ⁺还原酶编码基因和bcd操纵子（编码铁氧还蛋白-NAD ⁺还原酶）的表达水平的限制。将这些酶整合到代谢工程策略中为开发产同型丁醇的机会带来了机遇C. acetobutylicum菌株。