The rise of antibiotic resistance demands the acceleration of molecular diversification strategies to inspire new chemical entities for antibiotic medicines. We report here on the large-scale engineering of ribosomally synthesized and post-translationally modified antimicrobial peptides carrying the ring-forming amino acid lanthionine. New-to-nature variants featuring distinct properties were obtained by combinatorial shuffling of peptide modules derived from 12 natural antimicrobial lanthipeptides and processing by a promiscuous post-translational modification machinery. For experimental characterization, we developed the nanoFleming, a miniaturized and parallelized high-throughput inhibition assay. On the basis of a hit set of >100 molecules, we identified variants with improved activity against pathogenic bacteria and shifted activity profiles, and extrapolated design guidelines that will simplify the identification of peptide-based anti-infectives in the future.
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Data from next generation sequencing of the module libraries, from mass spectrometry and from MIC measurements were analyzed using scripts written in the programming language R. The code of the scripts is available from the corresponding authors upon reasonable request.
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We thank A. van de Vries (Department of Biosystems Science and Engineering (D-BSSE), ETH Zürich) for assistance in protocol development, R. Pellaux, A. Meyer (both FGen GmbH), T. Roberts (D-BSSE) and A.J. van Heel (Department of Molecular Genetics, University of Groningen) for their valuable suggestions during the whole project. We thank the Genomics Facility Basel (C. Beisel, K. Eschbach, E.V. Burcklen, I. Nissen-Naidanow and M. Kohler, D-BSSE) for their help with next generation sequencing and S. Posada-Céspedes (D-BSSE) for her help with sequence analysis. Furthermore, the authors would like to thank A. Femmer (D-BSSE) for her excellent technical assistance during peptide characterization and thank S. Smits for the pNZ-SV-SaNSR construct. Last, S.S., M.M.-L., D.P., R.W., O.P.K. and S.P. would like to acknowledge funding from the ESF EUROCORES project ‘SYNMOD’ (grant number FP-017) and the EU FP7 project ‘SYNPEPTIDE’ (grant number 613981) and J.D. funding from the Chinese Scholarship Council (CSC).
The authors declare no competing interests.
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Schmitt, S., Montalbán-López, M., Peterhoff, D. et al. Analysis of modular bioengineered antimicrobial lanthipeptides at nanoliter scale. Nat Chem Biol 15, 437–443 (2019). https://doi.org/10.1038/s41589-019-0250-5
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