Clostridium tyrobutyricum stands out as a promising anaerobic bacterium for the biosynthesis of organic acids such as acetic and butyric acids from glucose derived biomass. However, its industrial application is limited by microbial aging, which leads to decreased cell viability, reduced fermentation performance, and diminished tolerance to environmental stresses such as solvents and biomass derived inhibitors. This age associated declines in productivity are particularly detrimental during the production of biofuels such as n-butanol. A key approach to enhance cellular robustness and prolong chronological lifespan (CLS) is microbial membrane engineering. The integrity and fluidity of the cell membrane are vital for withstanding chemical stresses, as the membrane acts as the first line of defense against toxic solvents. Modifying membrane composition, particularly by altering the ratio of saturated to unsaturated fatty acids, has been shown to affect membrane permeability, resistance to oxidative stress and overall microbial resilience. The goal of this study was to enhance the CLS and stress tolerance of C. tyrobutyricum by modulating intracellular levels of malonyl-CoA by targeted regulation of acetyl-CoA carboxylase carboxyltransferase subunit beta (AccC). AccC catalyzes the conversion of acetyl-CoA to malonyl-CoA, a critical and rate limiting step in fatty acid biosynthesis. Malonyl-CoA serves as the precursor for long chain fatty acid elongation, and thus, its intracellular abundance significantly impacts fatty acid chain length and membrane lipid composition. We hypothesized that downregulation of AccC would reduce malonyl-CoA levels, thereby limiting fatty acid chain elongation. This metabolic shift is expected to decrease the production of long chain unsaturated fatty acids while increasing the proportion of shorter chain saturated fatty acids. As a result, the membrane would become more rigid with improved integrity and decreased permeability under stress conditions such as butanol exposure. In this study, we used CRISPR/Cas genome editing tool to knock out AccC and successfully constructed C. tyrobutyricum ΔAccC mutant strain. The effects of the mutant strain were evaluated during the non-dividing (stationary) phase which is a key phase for assessing CLS. Compared to the control strain, the ΔAccC mutant strain with enhanced membrane rigidity exhibited enhanced tolerance to butanol and biomass hydrolysate inhibitors, suggesting improved membrane robustness and prolonged CLS. Fine-tuning central metabolic pathways, such as malonyl-CoA biosynthesis, thus offers a strategic route to enhance stress resilience and prolong chronological lifespan, resulting in improved butanol production and fermentation efficiency.
