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Biobutanol is a sustainable green biofuel that can substitute for gasoline. Carbon flux has been redistributed in Clostridium tyrobutyricum via metabolic cell engineering to produce biobutanol. However, the lack of reducing power hampered the further improvement of butanol production. The objective of this study was to improve butanol production by rebalancing redox. Firstly, a metabolically-engineered mutant CTC-fdh-adhE2 was constructed by introducing heterologous formate dehydrogenase (fdh) and bifunctional aldehyde/alcohol dehydrogenase (adhE2) simultaneously into wild-type C. tyrobutyricum. The mutant evaluation indicated that the fdh-catalyzed NADH-producing pathway improved butanol titer by 2.15-fold in the serum bottle and 2.72-fold in the bioreactor. Secondly, the medium supplements that could shift metabolic flux to improve the production of butyrate or butanol were identified, including vanadate, acetamide, sodium formate, vitamin B12 and methyl viologen hydrate. Finally, the free-cell fermentation produced 12.34 g/L of butanol from glucose using the mutant CTC-fdh-adhE2, which was 3.88-fold higher than that produced by the control mutant CTC-adhE2. This study demonstrated that the redox engineering in C. tyrobutyricum could greatly increase butanol production.

Biobutanol is a sustainable green biofuel that can substitute for gasoline. Carbon flux has been redistributed in Clostridium tyrobutyricum via metabolic cell engineering to produce biobutanol. However, the lack of reducing power hampered the further improvement of butanol production. The objective of this study was to improve butanol production by rebalancing redox. Firstly, a metabolically-engineered mutant CTC- fdh - adhE2 was constructed by introducing heterologous formate dehydrogenase ( fdh ) and bifunctional aldehyde/alcohol dehydrogenase ( adhE2 ) simultaneously into wild-type C. tyrobutyricum . The mutant evaluation indicated that the fdh -catalyzed NADH-producing pathway improved butanol titer by 2.15-fold in the serum bottle and 2.72-fold in the bioreactor. Secondly, the medium supplements that could shift metabolic flux to improve the production of butyrate or butanol were identified, including vanadate, acetamide, sodium formate, vitamin B12 and methyl viologen hydrate. Finally, the free-cell fermentation produced 12.34 g/L of butanol from glucose using the mutant CTC- fdh - adhE2 , which was 3.88-fold higher than that produced by the control mutant CTC- adhE2 . This study demonstrated that the redox engineering in C. tyrobutyricum could greatly increase butanol production.