Dynamic Regulation of Efflux Pump Protein AcrA by Quorum Sensing System to Improve Free Fatty Acids Production in <i>Escherichia coli</i>

Microbial production of free fatty acids (FFAs) is an eco-friendly and promising approach. However, FFAs accumulation within microbial cells imposes stress and toxicity, impairing biosynthetic performance. These challenges can be alleviated by exporting FFAs through the efflux system AcrAB-TolC in Escherichia coli, but the expression of these membrane channels needs to be fine-tuned. In this study, we employed a quorum sensing (QS) system to dynamically regulate the expression of AcrAB-TolC, thereby enhancing FFAs efflux and production. Two regulatory patterns of gene expression were designed to respond to increasing cell density: EsaI/R-P_esaS drives an initial increase followed by a decrease pattern, and EsaI/R-P_esaR enables a slow linear increase pattern. The L19IR-P_R-acrA strain, in which acrA expression is auto-induced by P_esaR, exhibited a 142% increase in extracellular FFAs and an 11% increase in total FFAs titer compared to the L19IR strain, which lacks acrA regulation. Auto-induced dynamic regulation of acrA demonstrates significant improvements in both FFAs efflux and overall production, while maintaining cell growth and membrane stability. Our results highlight the potential of QS-mediated dynamic regulation of efflux pumps to enhance the synthesis of bioproducts toxic to microorganisms.