Cac3319 Inactivation Sustains Solventogenesis Through Proteomic and Phosphoregulatory Remodeling in Clostridium acetobutylicum

ABSTRACT Clostridium acetobutylicum is a promising biofuel producer for acetone‐butanol‐ethanol (ABE) fermentation, yet sustained butanol production is constrained by the short duration of solventogenic activity. Although the orphan histidine kinase Cac3319 has been implicated in Spo0A phosphorylation, sporulation, and solvent production, metabolic and phosphorylation changes following cac3319 inactivation have not been systematically characterized. Here, we integrated fermentation kinetics, comparative proteomics, and Ti 4+ ‐IMAC phosphoproteomics to investigate the effects of cac3319 inactivation in C. acetobutylicum ATCC 55025. The cac3319 knockout prolonged late‐stage metabolic activity without impairing early growth, enabling continued glucose utilization and solvent accumulation after entry into solventogenesis. Final butanol and total ABE titers increased from 13.0 to 17.5 g L −1 and from 18.5 to 28.5 g L −1 , respectively. Proteomic analysis revealed increased protein abundance spanning central carbon metabolism, acid reassimilation, energy metabolism, and protein protection. Phosphoproteomic analysis further revealed phase‐dependent remodeling of detectable phosphorylation events, indicating that post‐translational regulation was reshaped during the acidogenic‐to‐solventogenic transition. These coordinated changes supported a metabolically active and stress‐tolerant state conducive to sustained solventogenesis. Collectively, these findings provide new insights into histidine kinase‐mediated regulation of clostridial solventogenesis and highlight cac3319 inactivation as a promising strategy for sustaining late‐stage metabolic activity and improving butanol production in ABE fermentation.