Abstract
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.
Highlights
1,4-butanediol production in E. coli[4]
This phenomenon does occur in C. acetobutylicum engineered for butanol production; when E. coli are engineered for butanol biosynthesis via the classic clostridial fermentation pathway or reverse β-oxidation pathway[3,13], ethanol is produced as by-product
In C. acetobutylicum, several different aldehyde/alcohol dehydrogenases are involved in butanol and ethanol production, each with specific order of expression, expression level and substrate preference
Summary
1,4-butanediol production in E. coli[4]. Notably, in an attempt to obtain an engineered E. coli strain capable of producing a high-titer of 1,3-propanediol, the broad-spectrum hypothetical dehydrogenase (encoded by yqhD) from E. coli showed better performance than the specific 1,3-propanediol dehydrogenase (encoded by dhaT) from Klebsiella pneumoniae[5], indicating that exogenous dehydrogenases might not always be the optimal enzymes for target production. In a C. acetobutylicum solR gene knockout strain, overexpression of the bifunctional alcohol/aldehyde dehydrogenase gene adhE1 increased butanol production by 21%, this increased ethanol production by 62%11. When the adhE1D485G gene was overexpressed in a pta-buk double deficient C. acetobutylicum strain, butanol production increased by 160% compared with wild-type, ethanol production increased by 233%12. This phenomenon does occur in C. acetobutylicum engineered for butanol production; when E. coli are engineered for butanol biosynthesis via the classic clostridial fermentation pathway or reverse β-oxidation pathway[3,13], ethanol is produced as by-product. The six genes were individually disrupted, and their physiological functions and specific contributions to butanol and ethanol synthesis were investigated
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