High-Throughput Engineering of Nonribosomal Extension Modules

Nonribosomal peptides constitute an important class of natural products that display a wide range of bioactivities. They are biosynthesized by large assembly lines called nonribosomal peptide synthetases (NRPSs). Engineering NRPS modules represents an attractive strategy for generating customized synthetases for the production of peptide variants with improved properties. Here, we explored the yeast display of NRPS elongation and termination modules as a high-throughput screening platform for assaying adenylation domain activity and altering substrate specificity. Depending on the module, display of A-T bidomains or C-A-T tridomains, which also include an upstream condensation domain, proved to be most effective. Reprograming a tyrocidine synthetase elongation module to accept 4-propargyloxy-phenylalanine, a noncanonical amino acid that is not activated by the native protein, illustrates the utility of this approach for altering NRPS specificity at internal sites.