Engineering Clostridium acetobutylicum for alcohol production.

While Clostridium acetobutylicum has been used for large-scale butanol production (ABE fermentation), its by-product acetone cannot be used as a biofuel. In this study, C. acetobutylicum was engineered for alcohol titers (butanol plus ethanol). The adc gene was inactivated to eliminate acetone production, and glutathione biosynthetic capability was introduced into C. acetobutylicum to improve the strain's robustness by expressing Escherichia coli's gshAB genes in the adc locus. Acetone production was reduced from 2.64±0.22 g/L to 0.15±0.08 g/L in the engineered strain 824adc::gsh, whereas butanol production was increased from 5.17±0.26 g/L to 8.27±0.27 g/L. To further improve the alcohol titers, the metabolic flux in the alcohol biosynthesis pathways was enhanced. Overlapping PCR was used to generate expression cassette EC, which expresses the hbd, thl, crt, and bcd genes, and the Sol operon was amplified to express the adhE and ctfAB genes. Butanol and alcohol production reached 14.86±0.26 g/L and 18.11±0.66 g/L, respectively, in 824adc::gsh Sol-EC. Furthermore, the butanol and alcohol yields were 0.336 g/g and 0.409 g/g, respectively, in 824adc::gsh Sol-EC. This study provided a combined strategy for enhancing alcohol production in C. acetobutylicum.