The study describes a high-throughput approach to engineer condensation (C) domains in nonribosomal peptide synthetases (NRPSs), which are responsible for peptide elongation. The researchers used yeast display to interact a functional 120-kDa NRPS module with an upstream module in solution, producing amide products tethered to the yeast surface. By screening a large C-domain library, they successfully reprogrammed a surfactin synthetase module to accept fatty acid donors, increasing catalytic efficiency for this noncanonical substrate by over 40-fold. This methodology has potential to facilitate the precise engineering of NRPS assembly lines, enhancing the production of bioactive natural product analogs. The study also highlights the importance of screening large combinatorial libraries over rational design to identify variants with improved substrate specificity.The study describes a high-throughput approach to engineer condensation (C) domains in nonribosomal peptide synthetases (NRPSs), which are responsible for peptide elongation. The researchers used yeast display to interact a functional 120-kDa NRPS module with an upstream module in solution, producing amide products tethered to the yeast surface. By screening a large C-domain library, they successfully reprogrammed a surfactin synthetase module to accept fatty acid donors, increasing catalytic efficiency for this noncanonical substrate by over 40-fold. This methodology has potential to facilitate the precise engineering of NRPS assembly lines, enhancing the production of bioactive natural product analogs. The study also highlights the importance of screening large combinatorial libraries over rational design to identify variants with improved substrate specificity.