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Abstract:
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. 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.
