Engineering AraC L9K for inducer-independent pBAD activation in Salmonella Gallinarum

The AraC-pBAD promoter system is widely used for tunable gene expression in bacteria, but its dependence on arabinose as an inducer limits industrial scalability and complicates therapeutic deployment. In this study, an inducer-independent AraC variant was generated in Salmonella enterica serovar Gallinarum by combining host genome modification with targeted mutagenesis. Deletion of the chromosomal araBAD operon eliminated endogenous arabinose metabolism, while site-directed mutagenesis of the AraC N-terminal domain identified variants with altered regulatory behavior. Among these, the L9K substitution conferred strong constitutive expression of green fluorescent protein in the absence of inducer, with activity levels comparable to or exceeding those of the fully induced wild-type system. Growth kinetics and electrophoresis analysis confirmed that high-level expression was achieved without metabolic burden. Structural modeling suggested that the L9K mutation disrupts arabinose binding and stabilizes an "active" conformation, thus bypassing the native repressor-to-activator switch. This study establishes a robust, inducer-free AraC-based expression system directly compatible with widely used pBAD vectors, offering practical advantages for microbial biomanufacturing, synthetic biology, and therapeutic biotechnology where external inducers are undesirable or impractical.