Design of a Novel Diamine Biosensor to Guide the Engineering of Escherichia coli for Cadaverine Overproduction.

The development of in vivo biosensor systems represents a powerful strategy for engineering cell factories with desired production. Here, we reported a paradigm for the design, mechanistic elucidation, and optimization of the novel biosensor to detect cadaverine, an important polymer monomer. We identified a diamine-responsive promoter PpyrF via transcriptomic analysis for biosensor construction. Further investigation suggested that Ihfβ plays a key role in PpyrF activity regulation, while cadaverine and structurally related amines may activate PpyrF through modulation of Ihfβ-DNA interactions. Stepwise engineering was then performed to enhance biosensor performance, including mutagenesis of the promoter DNA sequence and the Ihfβ sequence, as well as RBS tuning, resulting in more than a 2.33-fold increase in fold induction. Guided by the biosensor, we implemented a multi-level engineering strategy that spanned tuning a specific exporter, mining optimized pathways from SCRaMbLE libraries, and genome-wide identification of beneficial gene targets. These iterative efforts improved cadaverine titer by 44% to a record 114.2 g/L in Escherichia coli, concurrently uncovering the critical roles of biofilm formation and carbon flux rewiring for overproduction. Our study establishes a biosensor-driven framework for optimizing complex pathways and uncovering novel targets in strain engineering.