Directed Evolution of Piperazic Acid Incorporation by a Nonribosomal Peptide Synthetase

Engineering of biosynthetic enzymes is increasingly employed to synthesize structural analogues of antibiotics. Of special interest are nonribosomal peptide synthetases (NRPSs) responsible for the production of important antimicrobial peptides. Here, directed evolution of an adenylation domain of a Pro-specific NRPS module completely switched substrate specificity to the non-standard amino acid piperazic acid (Piz) bearing a labile N-N bond. This success was achieved by UPLC-MS/MS-based screening of small, rationally designed mutant libraries and can presumably be replicated with a larger number of substrates and NRPS modules. The evolved NRPS produces a Piz-derived gramicidin S analogue. Thus, we give new impetus to the too-early dismissed idea that widely accessible low-throughput methods can switch the specificity of NRPSs in a biosynthetically useful fashion.

Automated summary

Engineering of biosynthetic enzymes is used to synthesize antibiotics analogues. This study focuses on nonribosomal peptide synthetases (NRPSs) responsible for producing antimicrobial peptides. By directed evolution, the substrate specificity of an adenylation domain of a Pro-specific NRPS module was completely switched to a non-standard amino acid. This achievement was made through screening of mutant libraries and can be replicated with different substrates and NRPS modules. New possibilities for modifying NRPS specificity in a biosynthetically useful way are presented.