This study presents a high-throughput method for engineering condensation (C) domains in nonribosomal peptide synthetases (NRPSs), which are essential for peptide elongation. The approach involves displaying a functional NRPS module on yeast, allowing it to interact with an upstream module in solution to produce amide products. By screening a large C-domain library, the researchers reprogrammed a surfactin synthetase module to accept fatty acid donors, significantly increasing catalytic efficiency for this noncanonical substrate. The method enables precise engineering of NRPS assembly lines, which are crucial for the sustainable production of bioactive natural products. NRPSs are multifunctional enzymes that biosynthesize nonribosomal peptides (NRPs), which are valuable sources of clinical therapeutics. These enzymes use dedicated modules to incorporate building blocks selectively and sequentially into the peptide scaffold. Each module contains three core domains: adenylation (A), thiolation (T), and condensation (C). The C domain is responsible for forming amide bonds between amino acids or peptides, and its selectivity can be modified to accept noncanonical substrates. The study highlights the challenges of engineering C-domain specificity, as current methods have low throughput and limited ability to identify rare mutations. The yeast display strategy described here allows for high-throughput screening of C-domain variants, enabling the identification of efficient catalysts. The researchers successfully reprogrammed the SrfA-C C domain to accept fatty acid substrates, demonstrating a significant increase in catalytic efficiency compared to the native amino acid substrate. The engineered C domain was then transplanted into the tyrocidine synthetase system, where it successfully facilitated the incorporation of fatty acid substrates into the peptide chain. Structural analysis of the engineered C domain revealed changes in the binding pocket that accommodate fatty acid substrates, enhancing the enzyme's activity. The study also shows that the yeast display system can be used to explore the molecular recognition determinants underlying substrate selection by C domains. Overall, this work demonstrates the potential of high-throughput yeast display for engineering NRPSs, enabling the precise modification of C-domain specificity to expand the range of substrates that can be incorporated into NRPs. This approach has significant implications for the sustainable production of bioactive natural products and the development of new antibiotics.This study presents a high-throughput method for engineering condensation (C) domains in nonribosomal peptide synthetases (NRPSs), which are essential for peptide elongation. The approach involves displaying a functional NRPS module on yeast, allowing it to interact with an upstream module in solution to produce amide products. By screening a large C-domain library, the researchers reprogrammed a surfactin synthetase module to accept fatty acid donors, significantly increasing catalytic efficiency for this noncanonical substrate. The method enables precise engineering of NRPS assembly lines, which are crucial for the sustainable production of bioactive natural products. NRPSs are multifunctional enzymes that biosynthesize nonribosomal peptides (NRPs), which are valuable sources of clinical therapeutics. These enzymes use dedicated modules to incorporate building blocks selectively and sequentially into the peptide scaffold. Each module contains three core domains: adenylation (A), thiolation (T), and condensation (C). The C domain is responsible for forming amide bonds between amino acids or peptides, and its selectivity can be modified to accept noncanonical substrates. The study highlights the challenges of engineering C-domain specificity, as current methods have low throughput and limited ability to identify rare mutations. The yeast display strategy described here allows for high-throughput screening of C-domain variants, enabling the identification of efficient catalysts. The researchers successfully reprogrammed the SrfA-C C domain to accept fatty acid substrates, demonstrating a significant increase in catalytic efficiency compared to the native amino acid substrate. The engineered C domain was then transplanted into the tyrocidine synthetase system, where it successfully facilitated the incorporation of fatty acid substrates into the peptide chain. Structural analysis of the engineered C domain revealed changes in the binding pocket that accommodate fatty acid substrates, enhancing the enzyme's activity. The study also shows that the yeast display system can be used to explore the molecular recognition determinants underlying substrate selection by C domains. Overall, this work demonstrates the potential of high-throughput yeast display for engineering NRPSs, enabling the precise modification of C-domain specificity to expand the range of substrates that can be incorporated into NRPs. This approach has significant implications for the sustainable production of bioactive natural products and the development of new antibiotics.