Time-resolved transcriptomics reveals mechanisms of acute and sustained low-pH adaptation in Corynebacterium glutamicum

Corynebacterium glutamicum is a major industrial cell factory for amino acid production. Acidic byproduct accumulation can lower broth pH, disrupt cytoplasmic pH homeostasis, induce oxidative stress, and ultimately compromise productivity. As low-pH stress is dynamic and process-dependent, distinguishing responses to acute acid shock versus sustained acidification is important for improving strain robustness. Here, we investigated low-pH adaptation under two regimes: acute, unbuffered pH 4.0 shock and sustained pH 5.5 stress with pH control, by integrating viability assays, time-series RNA-seq, and genetic validation. Acute shock caused 93% viability loss within 2 h, followed by broth neutralisation and regrowth, whereas sustained stress led to a 4.1-log 10 decline over 18 h. Transcriptomics identified 905/847 differentially expressed genes at 1/4 h under acute shock and 643/608/1857 genes at 1/8/18 h under sustained stress. Shared responses included repression of central metabolism and induction of β-ketoadipate catabolism, potassium uptake, sodium/proton antiport, urease-mediated ammonium release, amino acid biosynthesis, and oxidative and membrane-stress defences. Acute shock showed rapid global reprogramming dominated by oxidative protection, chaperone induction, and ion-flux control, whereas sustained stress induced progressive metabolic rewiring, cell envelope reinforcement, and redox buffering. Functional validation using both overexpression and knockout mutants confirmed the contribution of ion transport, iron regulation, β-ketoadipate metabolism, urease, and respiratory modules to low-pH tolerance in a regime-dependent manner. This study provides the first time-resolved, regime-specific transcriptomic dissection of low-pH adaptation in C. glutamicum and identifies key modules for engineering acid-resilient strains.