Abstract
<jats:p>Nonribosomal peptide synthetases (NRPSs) assemble structurally diverse bioactive natural products through selective communication between adenylation (A) and carrier protein (CP) domains. Although rational rewiring of these protein-protein interactions could enable customized NRPS design, this remains challenging due to the dynamic nature of protein interfaces. Here we show that structure-guided interface reprogramming enables productive non-cognate A-CP pairings across enterobactin, vibriobactin, pyochelin, and vicenistatin biosynthetic systems. Engineered interactions between the A domains EntE, VibE, or PchD and the non-cognate CPs VinL or EntB were validated by biochemical, kinetic, and structural analyses. Reconstituted pathways containing engineered VibE, EntB, and EntF restored enterobactin production and increased yield to 2.2-fold that of the native EntE-EntB-EntF system. Interface-engineered PchD also enhanced production of a non-native salicylic acid-norspermidine conjugate. An X-ray structure and molecular dynamics simulations of an engineered non-cognate A-CP complex reveal recognition principles for programmable NRPS interface design.</jats:p>