Elucidating the contributions of multiple aldehyde/alcohol dehydrogenases to butanol and ethanol production in Clostridium acetobutylicum.

Ethanol and butanol biosynthesis in Clostridium acetobutylicum share common aldehyde/alcohol dehydrogenases. However, little is known about the relative contributions of these multiple dehydrogenases to ethanol and butanol production respectively. The contributions of six aldehyde/alcohol dehydrogenases of C. acetobutylicum on butanol and ethanol production were evaluated through inactivation of the corresponding genes respectively. For butanol production, the relative contributions from these enzymes were: AdhE1 > BdhB > BdhA ≈ YqhD > SMB_P058 > AdhE2. For ethanol production, the contributions were: AdhE1 > BdhB > YqhD > SMB_P058 > AdhE2 > BdhA. AdhE1 and BdhB are two essential enzymes for butanol and ethanol production. AdhE1 was relatively specific for butanol production over ethanol, while BdhB, YqhD, and SMB_P058 favor ethanol production over butanol. Butanol synthesis was increased in the adhE2 mutant, which had a higher butanol/ethanol ratio (8.15:1) compared with wild type strain (6.65:1). Both the SMB_P058 mutant and yqhD mutant produced less ethanol without loss of butanol formation, which led to higher butanol/ethanol ratio, 10.12:1 and 10.17:1, respectively. To engineer a more efficient butanol-producing strain, adhE1 could be overexpressed, furthermore, adhE2, SMB_P058, yqhD are promising gene inactivation targets. This work provides useful information guiding future strain improvement for butanol production.