Engineering <i>Escherichia coli</i> for the Production of the PK/NRP Hybrid Antibiotic Salivabactin

Salivabactin, a newly discovered hybrid polyketide/nonribosomal peptide (PK/NRP) antibiotic with a unique scaffold, exhibits potent activity against Gram-positive pathogens such as <i>Streptococcus pyogenes</i>. Despite its promising bioactivity, the low yield of the native producer <i>Streptococcus salivarius</i> (SAL) limits further investigation into salivabactin's mode of action and pharmaceutical applications. Here, we report the first heterologous production of salivabactin in <i>Escherichia coli</i>. The biosynthetic genes were co-expressed in <i>E. coli</i>, followed by optimizations in gene expression and precursor feeding to improve biosynthetic efficiency. Additional metabolic engineering strategies, including the knockout of a 4-hydroxybenzoic acid (4-HBA) exporter to increase intracellular precursor availability and overexpression of a multidrug exporter, significantly improved the titer. The final engineered strain achieved a titer of 5.48 mg/L of salivabactin, representing a 22-fold increase in production compared to the native SAL strain. This work establishes <i>E. coli</i> as a scalable microbial platform for salivabactin production and demonstrates its potential as a versatile chassis for the biosynthesis of complex PK/NRP antibiotics.