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Many clinically used drugs are derived from or inspired by bacterial natural products that often are produced through nonribosomal peptide synthetases (NRPSs), megasynthetases that activate and join individual amino acids in an assembly line fashion. In this work, we describe a detailed phylogenetic analysis of several bacterial NRPSs that led to the identification of yet undescribed recombination sites within the thiolation (T) domain that can be used for NRPS engineering. We then developed an evolution-inspired “eXchange Unit between T domains” (XUT) approach, which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments. Editor’s summary Many clinically used drugs are derived from natural microbial products that are assembled in a stepwise fashion by the condensation of amino acids or acyl groups. Using insights from evolutionary analysis, two independent groups now show that the cumbersome enzyme complexes that produce these molecules can be pieced together to create new products on demand—if one knows the right spot for joining the pieces. Working with nonribosomal peptide synthetases, Bozhüyük et al. developed an approach called XUT (“exchange unit between T domains”) and demonstrated the production of a proteasome inhibitor by an enzyme complex containing fragments of five separate systems. Mabesoone et al. worked with polyketide synthases, demonstrating facile deletion and insertion of conceptually similar exchange units, producing a large number of related polyketide products with diverse modifications. These approaches are an important step forward for rational engineering of large enzyme complexes for small-molecule drug discovery and production. —Michael A. Funk Bioinformatic analysis reveals sequence sites ideal for engineering in enzymes that produce non-ribosomal peptide natural products. INTRODUCTION Natural products (NPs) have played a pivotal role in drug discovery, contributing to 48% of new medicines developed between 1981 and 2019. Despite their significance, there are obstacles in translating NPs into clinical drugs owing to their structural complexity and limitations to derivatize or synthesize them. Genetic engineering or synthetic biology present promising avenues for the efficient and cost-effective discovery of tailored biological drugs. Bacterial NPs, especially those derived from nonribosomal peptide synthetases (NRPSs), have emerged as ideal targets for synthetic biology and have the potential to enhance the pharmacological properties of nonribosomal peptides (NRPs) in clinical development. However, rational engineering of NRPSs remains complex despite technological advancements. RATIONALE Recent advancements in bioinformatic and genetic engineering technologies as well as structural data have propelled synthetic biology strategies for manipulating megasynthetases, offering innovative solutions for the production of NRP analogs. Building on the growing understanding of NRPS evolution, this study emphasizes the importance of intragenomic recombination, speciation, horizontal gene transfer, and recombination as driving forces behind the diversification and functionalization within NRP families. We hypothesized that recognizing the central evolutionary mechanisms is essential for the redesign of assembly lines, with the aim of achieving greater structural diversity and increased production yields. By using phylogenetic hidden Markov models and principal component analysis–based machine learning approaches, our study tries to expand the understanding of intragenomic recombination for NRPS engineering, identifying regions with inconsistent evolutionary histories as potential synthetic breakpoints. RESULTS The analysis of NRPS evolution, combined with systematic experimental analysis and in silico methods, unveils a previously undocumented recombination site within NRPSs. Using fusion point screening, we identified evolution-inspired synthetic engineering sites and designed more than 50 artificial peptides by combining building blocks from unrelated natural NRPS systems. The developed engineering framework, named the evolution-inspired eXchange Unit between T domains (XUT), aligns with structural revelations, substantially converging with the recently proposed unified model for the evolution of NRPSs. Important to this work was the identification of an additional yet undescribed recombination site within NRPS’s thiolation domains, which allows the combination of NRPS building blocks that differ in taxonomy, biochemistry, and GC content. CONCLUSION This study applies insights into the evolution of NRPS as a foundation to improve engineering of these megasynthetases. The XUT approach broadens the synthetic biology toolkit and facilitates the creation of tailor-made bioactive peptides. This approach is versatile and complementary to previous engineering strategies and holds great potential for advancing synthetic biology and NP engineering for clinical drug discovery, development, and optimization. Illustration of the XUT engineering concept. Two different NRPSs are shown in red and blue producing different schematic cyclic peptides. A modified blue NRPS is illustrated with one module exchanged against a red NRPS module through the XUT approach, resulting in a rationally engineered peptide product. Structural data for illustrations were derived from PDB 6MFZ and PDB 2CB9.