Redox-Driven Metabolic Shift in <i>Clostridium acetobutylicum</i> for Enhanced Butanol Production

Butanol is a valuable industrial chemical and a potential transportation biofuel with superior fuel properties compared to ethanol. However, its production through traditional acetone–butanol–ethanol fermentation using Clostridium species is limited by the low titer, productivity, and yield. In this study, we employed a redox-based strategy to enhance butanol production by adding external electron shuttles. Various electron shuttles (anthraquinone-2-sulfonate, anthraquinone-1,5-disulfonic acid, riboflavin, methyl viologen, neutral red, and benzyl viologen) were screened for their ability to modulate intracellular redox states. Optimal concentrations and addition times were determined. We found that adding benzyl viologen (5 mg/L) at 0 h resulted in the highest butanol titer of 15.2 ± 0.9 g/L and a yield of 0.29 g/g, representing a 1.4- and 1.5-fold increase in titer and yield, respectively, compared to the control. The results suggest that the inherent redox potential of the electron shuttles plays a key role in redirecting metabolic fluxes toward reduced end-products (butanol and ethanol) by altering electron transfer pathways. To explore the underlying mechanisms, GC–MS-based metabolomics was used to study global metabolic shifts following the addition of benzyl viologen. Specifically, the upregulation of amino acids such as tryptophan and aspartic acid may enhance NADH biosynthesis. Electron shuttles likely divert electron flow from ferredoxin to NADH regeneration, thereby increasing butanol production. Overall, this study offers mechanistic insights and a potential approach for using redox-active electron shuttles to improve butanol production through metabolic redirection.