Analysis of modular bioengineered antimicrobial lanthipeptides at nanoliter scale

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Abstract

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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Fig. 1: Modular assembly of antimicrobial lanthipeptides.
Fig. 2: Discovery platform for antimicrobial peptides.
Fig. 3: Characterization of screening hits.

Data availability

The raw data from next generation sequencing of the peptide-encoding DNA libraries in Supplementary Fig. 2 are available in the NCBI Sequence Read Archive (SRA), accession number PRJNA511380.

Code availability

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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Acknowledgements

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).

Author information

O.P.K. and S.P. conceived the study. D.P. and R.W. developed the DNA synthesis strategy. S.S., M.M.-L., D.P., R.W. and O.P.K. designed and prepared the combinatorial library. S.S. performed the library quality control and developed the nanoFleming assay, the idea for which had been conceived by M.H. O.P.K., M.M.-L. and S.S. designed and prepared the screening strains. S.S. did the screening, characterization and purification of the screening hits. S.S., M.M.-L. and J.D. determined the MICs of the peptides. R.W., M.H., O.P.K. and S.P. supervised the work. S.S., M.H., M.M.-L., O.P.K. and S.P. wrote the paper. All authors discussed the results and commented on the manuscript.

Correspondence to Oscar P. Kuipers or Sven Panke.

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Supplementary Figures 1–11 and Supplementary Tables 1–7

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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) doi:10.1038/s41589-019-0250-5

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