Adaptive laboratory evolution of Actinobacillus succinogenes for enhanced acid tolerance and succinic acid production

The acid tolerance and succinic acid (SA) production in Actinobacillus succinogenes 130Z were improved using adaptive laboratory evolution (ALE). A three-phase continuous culture strategy was employed, gradually decreasing the pH from 6.3 to 5.4. During the ALE process, the medium composition and CO 2 supply were optimized to mitigate acid stress. To relieve acid stress and maintain optimal ionic strength, 0.1 M NaCl was supplied, and after maintaining a continuous CO 2 supply during ALE, it was optimized to 2 bar and 0.1 vvm. The evolved strain, BAT1, demonstrated superior performance in batch fermentation at pH 5.6, producing 12.8 g/L of SA within 48 hours, indicating a substantial increase compared to the wild-type strain (4.9 g/L). Whole-genome sequencing revealed critical mutations in transport systems (BCCT and NupC/NupG), membrane maintenance proteins (LrgB and acyltransferase), and metabolic regulators (DksA and NAD+ kinase). This study suggests that the efficacy of ALE in developing robust strains for low-pH fermentation and provides potential genetic targets for metabolic engineering of acid tolerance. • Actinobacillus succinogenes was evolved for high acid tolerance via three-phase ALE. • The evolved BAT1 strain produced 2.6-fold more succinic acid than WT at pH 5.6. • WGS revealed mutations in transport, membrane maintenance, and metabolic regulators. • Biofilm formation and membrane thickening were identified as key adaptive mechanisms.