Adaptive metabolic rewiring and ion homeostasis enhance oxidative stress resistance in Lacticaseibacillus casei.

Enhancing the oxidative stress tolerance of microorganisms is critical for maintaining their viability and functionality in industrial fermentation under aerobic conditions. The present study used adaptive laboratory evolution to enhance the oxidative stress resistance of Lacticaseibacillus casei, a widely used lactic acid bacterium (LAB) in fermentation. By applying serial propagations with batch cultures under high oxygen conditions, the evolved strain exhibited enhanced H2O2 tolerance, improved metal ion chelation, and increased biofilm formation. By combining whole genome sequencing and re-sequencing, these phenotypic changes were attributed to key mutations involved in cell structure and metal ion homeostasis. Moreover, transcriptomic and metabolomic analyses revealed that, although both wild type and evolved strains employed a conserved stringent response by repressing de novo purine synthesis, the evolved strain adopted additional mechanisms to achieve this. These included enhanced purine salvage to support nucleic acid synthesis, increased transmembrane transport to improve carbon utilization, and upregulated citrate metabolism to generate proton motive force and maintain intracellular redox balance. Furthermore, strengthened copper ion homeostasis helped mitigate Fenton reaction-induced oxidative damage. These findings provide new insights into molecular adaptation and suggest a practical strategy to improve probiotic robustness in fermented foods. IMPORTANCE: Enhancing the oxidative stress tolerance of microorganisms is critical for maintaining their viability and functionality in industrial fermentation under aerobic conditions. This study demonstrates a non-GMO approach using adaptive laboratory evolution to enhance the robustness of Lacticaseibacillus casei. By integrating multiple characterization methods and combining genomics, transcriptomics, and metabolomics, the results revealed that the evolved strain developed significantly improved hydrogen peroxide resistance through key phenotypic and molecular adaptations. These findings uncover adaptive strategies in lactic acid bacteria and provide practical insights for optimizing probiotic performance in food and health applications without genetic engineering.