Engineering Escherichia coli 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 Streptococcus pyogenes. Despite its promising bioactivity, the low yield of the native producer Streptococcus salivarius (SAL) limits further investigation into salivabactin's mode of action and pharmaceutical applications. Here, we report the first heterologous production of salivabactin in Escherichia coli. The biosynthetic genes were co-expressed in E. coli, 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 E. coli as a scalable microbial platform for salivabactin production and demonstrates its potential as a versatile chassis for the biosynthesis of complex PK/NRP antibiotics.