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...

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Bibliographic Details
Published in:Angewandte Chemie International Edition 2023-08, Vol.62 (35), p.e202304843-n/a
Main Authors: Stephan, Philipp, Langley, Chloe, Winkler, Daniela, Basquin, Jérôme, Caputi, Lorenzo, O'Connor, Sarah E., Kries, Hajo
Format: Article
Language:English
Subjects:
Adenylation
Amino acids
Antibiotics
Antimicrobial peptides
Directed Evolution
Evolution
Gramicidin
Gramicidin S
Modules
NRPS
Peptides
Piperazic Acid
Protein Engineering
Substrate specificity
Substrates
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Summary: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. Efficient directed evolution protocols for nonribosomal peptide synthetases are needed to adapt the structures of antibiotic peptides for the fight against antimicrobial resistance. Here, an easily reproducible directed evolution protocol was used to reprogram the synthetase for the antibiotic peptide gramicidin S. A few mutations were sufficient to incorporate the non‐standard building block piperazic acid instead of proline with perfect specificity.
ISSN:1433-7851
1521-3773
DOI:10.1002/anie.202304843