Dynamic Rewiring of Biofilm Formation and l -Threonine Production through an Autonomous Quorum-Sensing Regulatory Circuit in Escherichia coli

Microbial production of l -threonine is an essential component of the global amino acid industry. However, large-scale processes are still hindered by prolonged fermentation cycles and seed preparation for each batch. Biofilm-based immobilized fermentation represents a promising approach to address these challenges, yet metabolic bottlenecks in biofilm formation and product export remain unresolved. Here, motA (a key gene promoting biofilm formation) and rhtA (a key gene promoting threonine transport) were identified as optimal regulatory targets. We engineered a bidirectional EsaI/R quorum-sensing (QS) circuit in Escherichia coli to autonomously regulate early-stage biofilm development and late-stage threonine transport. It accelerated biofilm formation, maintained metabolic activity, and enhanced product efflux by alleviating intracellular feedback inhibition. The engineered strain THW39 achieved an l -threonine titer of 17.1 g/L with a productivity of 0.61 g/L/h and yield of 0.57 g/g in biofilm-based immobilized fermentation, without external inducers or manual intervention. Overall, this work demonstrated that QS-mediated bidirectional regulation provided a general strategy for coordinating complex metabolic and physiological processes and established a broadly applicable framework for dynamic regulation, thereby enabling continuous biomanufacturing in microbial cell factories.