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bioRxiv · 10.1101/2023.04.03.535426

Directed evolution of piperazic acid incorporation by a nonribosomal peptide synthetase

Stephan, P.·Langley, C.·Winkler, D.·Basquin, J.·Caputi, L.·O'Connor, S. E.·Kries, H.
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Abstract

Engineering of biosynthetic enzymes is increasingly employed to synthesize structural analogues of antibiotics. Of special interest are non-ribosomal peptide synthetases (NRPSs) responsible for 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 LC-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 analog. 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.

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Stephan, P., Langley, C., Winkler, D., Basquin, J., Caputi, L., O'Connor, S. E., Kries, H.. 2023-04-03. Directed evolution of piperazic acid incorporation by a nonribosomal peptide synthetase. https://doi.org/10.1101/2023.04.03.535426

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