Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Analysis of modular bioengineered antimicrobial lanthipeptides at nanoliter scale

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1.

    Newman, D. J. & Cragg, G. M. Natural products as sources of new drugs from 1981 to 2014. J. Nat. Prod. 79, 629–661 (2016).

    CAS  Article  Google Scholar 

  2. 2.

    Zengler, K. et al. Cultivating the uncultured. Proc. Natl Acad. Sci. USA 99, 15681–15686 (2002).

    CAS  Article  Google Scholar 

  3. 3.

    Baltz, R. H. Marcel Faber Roundtable: is our antibiotic pipeline unproductive because of starvation, constipation or lack of inspiration? J. Ind. Microbiol. Biotechnol. 33, 507–513 (2006).

    CAS  Article  Google Scholar 

  4. 4.

    Nichols, D. et al. Use of iChip for high-throughput in situ cultivation of ‘uncultivable’ microbial species. Appl. Environ. Microbiol. 76, 2445–2450 (2010).

    CAS  Article  Google Scholar 

  5. 5.

    Ling, L. L. et al. A new antibiotic kills pathogens without detectable resistance. Nature 517, 455–459 (2015).

    CAS  Article  Google Scholar 

  6. 6.

    Fischbach, M. A. & Walsh, C. T. Antibiotics for emerging pathogens. Science 325, 1089–1093 (2009).

    CAS  Article  Google Scholar 

  7. 7.

    Walsh, C. T. Insights into the chemical logic and enzymatic machinery of NRPS assembly lines. Nat. Prod. Rep. 33, 127–135 (2016).

    CAS  Article  Google Scholar 

  8. 8.

    Menzella, H. G. et al. Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes. Nat. Biotechnol. 23, 1171–1176 (2005).

    CAS  Article  Google Scholar 

  9. 9.

    Winn, M., Fyans, J. K., Zhuo, Y. & Micklefield, J. Recent advances in engineering nonribosomal peptide assembly lines. Nat. Prod. Rep. 33, 317–347 (2016).

    CAS  Article  Google Scholar 

  10. 10.

    Weissman, K. J. Genetic engineering of modular PKSs: from combinatorial biosynthesis to synthetic biology. Nat. Prod. Rep. 33, 203–230 (2016).

    CAS  Article  Google Scholar 

  11. 11.

    Arnison, P. G. et al. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 30, 108–160 (2013).

    CAS  Article  Google Scholar 

  12. 12.

    van Heel, A. J., Montalban-Lopez, M. & Kuipers, O. P. Evaluating the feasibility of lantibiotics as an alternative therapy against bacterial infections in humans. Expert Opin. Drug Metab. Toxicol 7, 675–680 (2011).

    Article  Google Scholar 

  13. 13.

    Legala, O. E., Yassi, H., Pflugmacher, S. & Neubauer, P. Pharmacological and pharmacokinetic properties of lanthipeptides undergoing clinical studies. Biotechnol. Lett. 39, 473–482 (2017).

    CAS  Article  Google Scholar 

  14. 14.

    Li, B. et al. Catalytic promiscuity in the biosynthesis of cyclic peptide secondary metabolites in planktonic marine Cyanobacteria. Proc. Natl Acad. Sci. USA 107, 10430–10435 (2010).

    CAS  Article  Google Scholar 

  15. 15.

    Zhang, Q., Yang, X., Wang, H. & van der Donk, W. A. High divergence of the precursor peptides in combinatorial lanthipeptide biosynthesis. ACS Chem. Biol. 9, 2686–2694 (2014).

    CAS  Article  Google Scholar 

  16. 16.

    Oman, T. J. & van der Donk, W. A. Follow the leader: the use of leader peptides to guide natural product biosynthesis. Nat. Chem. Biol. 6, 9–18 (2010).

    CAS  Article  Google Scholar 

  17. 17.

    van Heel, A. J. et al. Discovery, production and modification of five novel lantibiotics using the promiscuous nisin modification machinery. ACS Synth. Biol. 5, 1146–1154 (2016).

    Article  Google Scholar 

  18. 18.

    Montalbán-López, M., van Heel, A. J. & Kuipers, O. P. Employing the promiscuity of lantibiotic biosynthetic machineries to produce novel antimicrobials. FEMS Microbiol. Rev. 41, 5–18 (2017).

    Article  Google Scholar 

  19. 19.

    Majchrzykiewicz, J. A. et al. Production of a class II two-component lantibiotic of Streptococcus pneumoniae using the class I nisin synthetic machinery and leader sequence. Antimicrob. Agents Chemother. 54, 1498–1505 (2010).

    CAS  Article  Google Scholar 

  20. 20.

    Dischinger, J., Basi Chipalu, S. & Bierbaum, G. Lantibiotics: promising candidates for future applications in health care. Int. J. Med. Microbiol. 304, 51–62 (2014).

    CAS  Article  Google Scholar 

  21. 21.

    Fleming, A. On the antibacterial action of cultures of a Penicillium, with special reference to their use in the isolation of B. influenzae. Br. J. Exp. Pathol. 10, 226–236 (1929).

    CAS  PubMed Central  Google Scholar 

  22. 22.

    Walser, M. et al. Novel method for high-throughput colony PCR screening in nanoliter-reactors. Nucleic Acids Res. 37, e57 (2009).

    Article  Google Scholar 

  23. 23.

    Walser, M., Leibundgut, R. M., Pellaux, R., Panke, S. & Held, M. Isolation of monoclonal microcarriers colonized by fluorescent E. coli. Cytom. Part A 73A, 788–798 (2008).

    Article  Google Scholar 

  24. 24.

    Meyer, A. et al. Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors. Nat. Chem. 7, 673–678 (2015).

    CAS  Article  Google Scholar 

  25. 25.

    Roberts, T. M. et al. Identification and characterisation of a pH-stable GFP. Sci. Rep. 6, 28166 (2016).

    CAS  Article  Google Scholar 

  26. 26.

    Zhou, L., van Heel, A. J. & Kuipers, O. P. The length of a lantibiotic hinge region has profound influence on antimicrobial activity and host specificity. Front. Microbiol 6, 11 (2015).

    PubMed  PubMed Central  Google Scholar 

  27. 27.

    Montalbán-López, M., Deng, J., van Heel, A. J. & Kuipers, O. P. Specificity and application of the lantibiotic protease NisP. Front. Microbiol 9, 160 (2018).

    Article  Google Scholar 

  28. 28.

    Rogers, A. Improved agar diffusion assay for nisin quantification. Food Biotechnol. 5, 161–168 (1991).

    CAS  Article  Google Scholar 

  29. 29.

    Khosa, S., Lagedroste, M. & Smits, S. H. J. Protein defense systems against the lantibiotic nisin: function of the immunity protein NisI and the resistance protein NSR. Front. Microbiol 7, 504 (2016).

    Article  Google Scholar 

  30. 30.

    Khosa, S. et al. Structural basis of lantibiotic recognition by the nisin resistance protein from Streptococcus agalactiae. Sci. Rep. 6, 18679 (2016).

    CAS  Article  Google Scholar 

  31. 31.

    Zhou, L., van Heel, A. J., Montalban-Lopez, M. & Kuipers, O. P. Potentiating the activity of nisin against Escherichia coli. Front. Cell Dev. Biol 4, 7 (2016).

    Article  Google Scholar 

  32. 32.

    Casini, A. et al. One-pot DNA construction for synthetic biology: the modular overlap-directed assembly with Linkers (MODAL) strategy. Nucleic Acids Res. 42, e7 (2014).

    CAS  Article  Google Scholar 

  33. 33.

    Kuipers, A. et al. NisT, the transporter of the lantibiotic nisin, can transport fully modified, dehydrated, and unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides. J. Biol. Chem. 279, 22176–22182 (2004).

    Google Scholar 

  34. 34.

    Bernard, P. & Couturier, M. Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes. J. Mol. Biol. 226, 735–745 (1992).

    CAS  Article  Google Scholar 

  35. 35.

    van der Vossen, J. M., van der Lelie, D. & Venema, G. Isolation and characterization of Streptococcus cremoris Wg2-specific promoters. Appl. Environ. Microbiol. 53, 2452–2457 (1987).

    PubMed  PubMed Central  Google Scholar 

  36. 36.

    Dominy, C. N. & Andrews, D. W. Site-directed mutagenesis by inverse PCR. Methods Mol. Biol 235, 209–223 (2003).

    CAS  PubMed  Google Scholar 

  37. 37.

    Zheng, L. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res. 32, e115–e115 (2004).

    Article  Google Scholar 

  38. 38.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    CAS  Article  Google Scholar 

  39. 39.

    Schindelin, J., Rueden, C. T., Hiner, M. C. & Eliceiri, K. W. The ImageJ ecosystem: an open platform for biomedical image analysis. Mol. Reprod. Dev. 82, 518–529 (2015).

    CAS  Article  Google Scholar 

  40. 40.

    Green, M. R. & Sambrook, J. Molecular Cloning: A Laboratory Manual 4th edn (Cold Spring Harbor Laboratory Press, 2012).

  41. 41.

    Anthis, N. J. & Clore, G. M. Sequence-specific determination of protein and peptide concentrations by absorbance at 205 nm. Protein Sci. 22, 851–858 (2013).

    CAS  Article  Google Scholar 

  42. 42.

    Pelillo, M. et al. Calculation of the molar absorptivity of polyphenols by using liquid chromatography with diode array detection: the case of carnosic acid. J. Chromatogr. A 1023, 225–229 (2004).

    CAS  Article  Google Scholar 

  43. 43.

    Wiegand, I., Hilpert, K. & Hancock, R. E. W. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc. 3, 163–175 (2008).

    CAS  Article  Google Scholar 

  44. 44.

    Gibb, S. & Strimmer, K. Maldiquant: a versatile R package for the analysis of mass spectrometry data. Bioinformatics 28, 2270–2271 (2012).

    CAS  Article  Google Scholar 

Download references

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

Affiliations

Authors

Contributions

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.

Corresponding authors

Correspondence to Oscar P. Kuipers or Sven Panke.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–11 and Supplementary Tables 1–7

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing