Genome-wide analysis of the Escherichia coli Keio collection reveals genetic determinants associated with ferulic acid responses.

Ferulic acid (FA) is a widespread plant derived phenolic compound with diverse biological activities, including antimicrobial, antioxidant, and anti-inflammatory properties. In addition to its importance in food, cosmetic, and pharmaceutical applications, FA is abundant in lignocellulosic biomass, where it functions both as a microbial stress factor and as a metabolizable aromatic intermediate during bioconversion processes. Despite its broad biological and biotechnological relevance, the genetic basis underlying bacterial adaptation and tolerance to FA remains poorly understood. In this study, we performed a genome-wide screen using the Escherichia coli Keio knockout collection to identify genes associated with FA sensitivity and tolerance. The analysis revealed that bacterial responses to FA involve multiple interconnected cellular systems rather than a single resistance mechanism. Mutants defective in outer membrane lipid homeostasis (Mla system) and aromatic acid efflux functions (aaeA/aaeR, acrB) displayed pronounced sensitivity to FA, highlighting the importance of membrane integrity and transport systems during phenolic stress. Genes involved in Fe-S cluster biogenesis (sufS, sufA), hydrogenase maturation (hypD, hybF), molybdenum transport and cofactor metabolism (modB, modC, modE) and electron transport associated functions (ccmE) were also associated with altered FA responses. In addition, disruption of global regulatory genes (seqA, dksA, and rlmE) impaired tolerance, whereas deletion of mutL increased resistance. Functional enrichment and network analyses further linked FA responses to pathways associated with membrane homeostasis, redox-associated processes, transport systems, and metabolic adaptation. Overall, this study provides a genome wide genetic framework for understanding bacterial responses to FA and identifies candidate genes and cellular systems potentially involved in phenolic stress adaptation. These findings expand current knowledge of microbial adaptation to plant derived phenolic compounds and provide a basis for future studies investigating microbial stress physiology, phenolic tolerance mechanisms, and strategies to modulate bacterial sensitivity under phenolic stress conditions.