(485a) Redox Rewiring in Engineered Clostridium Tyrobutyricum: Comparative Transcriptomic Insights into Enhanced n-Butanol Production in a Hydrogenase-Null Mutant

Optimizing butanol production in Clostridium tyrobutyricum through metabolic engineering requires detailed insights into redox balance and global metabolic adjustments. This study compares transcriptomic and fermentation profiles between engineered strains MΔcat1::adhE2 and its hydrogenase knockout derivative, MΔcat1::adhE2ΔhydA. Batch fermentations revealed that the HydA knockout significantly increased butanol and reduced acids production, achieving higher alcohol-to-acid (AL/AC) and C4/C2 product ratios. Transcriptomic analysis identified extensive metabolic reprogramming with over 200 significant differentially expressed genes (FDR < 0.05), notably the downregulation of hydrogenase-associated electron transfer pathways and glycolytic flux and upregulation of nitrogen metabolism and ATP synthesis pathways. Compared to MΔcat1::adhE2, MΔcat1::adhE2ΔhydA also showed stable metabolic performance with higher butanol production in repeated batch fermentations. These findings demonstrate the critical role of hydrogenase in electron distribution and redox homeostasis, highlighting the effectiveness of redox-focused genetic interventions for enhancing butanol production in C. tyrobutyricum.