Engineered Hypermutation Adapts <i>Escherichia coli</i> to Octanoic Acid Stress

Medium-chain fatty acids (MCFAs) are valuable biochemicals, yet their inherent toxicity limits microbial productivity. Here, we developed a tunable hypermutation system in <i>Escherichia coli</i> by modulating translesion synthesis (TLS) pathways to accelerate adaptive laboratory evolution (ALE) for enhanced octanoic acid (C8) tolerance. Overexpression of <i>dinB</i>, encoding error-prone DNA polymerase IV, under T7 and BAD promoters yielded mutation rates 28.4-fold and 397-fold higher than the wild-type strain. ALE using these hypermutator strains yielded a robust variant capable of tolerating 50 mM C8. Whole-genome resequencing and reverse validation identified mutations related to membrane integrity and oxidative stress responses. Phenotypic analysis showed improved membrane integrity, reduced hydrophobicity, and lower reactive oxygen species (ROS) levels in the evolved strain under C8 stress. This study presents a hypermutation-assisted ALE strategy for improving microbial stress tolerance.