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Metagenomic domain substitution for the high-throughput modification of nonribosomal peptides

Nature Chemical Biology volume 20, pages 251–260 (2024)Cite this article

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Abstract

The modular nature of nonribosomal peptide biosynthesis has driven efforts to generate peptide analogs by substituting amino acid-specifying domains within nonribosomal peptide synthetase (NRPS) enzymes. Rational NRPS engineering has increasingly focused on finding evolutionarily favored recombination sites for domain substitution. Here we present an alternative evolution-inspired approach that involves large-scale diversification and screening. By amplifying amino acid-specifying domains en masse from soil metagenomic DNA, we substitute more than 1,000 unique domains into a pyoverdine NRPS. Initial fluorescence and mass spectrometry screens followed by sequencing reveal more than 100 functional domain substitutions, collectively yielding 16 distinct pyoverdines as major products. This metagenomic approach does not require the high success rates demanded by rational NRPS engineering but instead enables the exploration of large numbers of substitutions in parallel. This opens possibilities for the discovery and production of nonribosomal peptides with diverse biological activities.

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Fig. 1: Nonribosomal peptide synthesis and metagenomic domain substitution.
Fig. 2: Analysis of candidate recombination sites.
Fig. 3: Experimental testing of candidate recombination sites.
Fig. 4: Diversity of the metagenomic domain substitution libraries.
Fig. 5: Screening and diversity of functional substitutions.
Fig. 6: Pyoverdine yield for top hits.

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Data availability

The data that support the findings of this study are available within the main text and its Supplementary Information file. Mass spectrometry data are available from https://doi.org/10.25345/C5S46HG94. The PDB file 6P1J is available from https://doi.org/10.2210/pdb6P1J/pdb. Source data are provided with this paper. Data are also available from the corresponding authors upon request.

Code availability

Code generated during this study is available from https://doi.org/10.5281/zenodo.8361572.

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Acknowledgements

This work was supported by the Royal Society of New Zealand Marsden Fund (grant no. 18-VUW-082 to M.J.C. and G.L.C.), and the Health Research Council of New Zealand (contract 16/172 to D.F.A. and J.G.O.). S.R.M. was supported by a New Zealand Lottery Health Research Scholarship (contract LHR-2020-129624). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

  1. School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand

    Sarah R. Messenger, Edward M. R. McGuinniety, Luke J. Stevenson, Jeremy G. Owen, David F. Ackerley & Mark J. Calcott

  2. Maurice Wilkins Centre for Molecular Biodiscovery, Victoria University of Wellington, Wellington, New Zealand

    Sarah R. Messenger, Edward M. R. McGuinniety, Luke J. Stevenson, Jeremy G. Owen, David F. Ackerley & Mark J. Calcott

  3. Ferrier Research Institute, Victoria University of Wellington, Wellington, New Zealand

    Luke J. Stevenson

  4. Department of Chemistry, University of Warwick, Coventry, UK

    Gregory L. Challis

  5. Warwick Integrative Synthetic Biology Centre, University of Warwick, Coventry, UK

    Gregory L. Challis

  6. Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia

    Gregory L. Challis

  7. ARC Centre of Excellence for Innovations in Peptide and Protein Science, Monash University, Clayton, Victoria, Australia

    Gregory L. Challis

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Contributions

S.R.M., D.F.A. and M.J.C. conceived and designed the work. S.R.M., E.M.R.M. and M.J.C. performed the experiments. S.R.M. and M.J.C. performed the data analysis and prepared all figures. L.J.S., J.G.O. and M.J.C. created the SL and RX metagenomic libraries. M.J.C., G.L.C., D.F.A. and J.G.O. obtained funding for this study. S.R.M., D.F.A. and M.J.C. wrote the manuscript. S.R.M., G.L.C., J.G.O., D.F.A. and M.J.C. edited the manuscript, and all authors approved the final version.

Corresponding authors

Correspondence to David F. Ackerley or Mark J. Calcott.

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Nature Chemical Biology thanks Donatella de Pascale, Sergey Zotchev and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Figs. 1–11 and Tables 1–4.

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Supplementary Data 1

Raw data for Supplementary Fig. 8.

Supplementary Data 2

DNA sequences and oligonucleotides.

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Messenger, S.R., McGuinniety, E.M.R., Stevenson, L.J. et al. Metagenomic domain substitution for the high-throughput modification of nonribosomal peptides. Nat Chem Biol 20, 251–260 (2024). https://doi.org/10.1038/s41589-023-01485-1

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