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De novo design of ribosomally synthesized and post-translationally modified peptides

Nature Chemistry volume 17, pages 233–245 (2025)Cite this article

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Abstract

In nature, peptides are enzymatically modified to constrain their structure and introduce functional moieties. De novo peptide structures could be built by combining enzymes from different pathways, but determining the rules of their use is difficult. We present a biophysical model to combine enzymes sourced from bacterial ribosomally synthesized and post-translationally modified peptide (RiPP) gene clusters. Using a pipeline to evaluate more than 1,000 peptides, the model was parameterized under uniform conditions in Escherichia coli for enzymes from different classes (graspetide, spliceotide, pantocin, cyanobactin, glycocin, lasso peptide and lanthipeptide). Synthetic leader peptides with recognition sequences for up to three enzymes were designed to modify core sequences sharing no identity to natural RiPPs. Empirically, RiPPs with the desired modifications constituted 7–67% of the total peptides produced, and 6 of our 8 peptide designs were successfully modified. This work is an example of the design of enzyme-modified peptides and libraries, using a framework that can be expanded to include new enzymes and chemical moieties.

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Fig. 1: Determination of leader-dependent enzyme RSs and their positioning rules.
Fig. 2: Extraction of core sequence rules.
Fig. 3: Synthetic precursor peptides that are modified by multiple enzymes.
Fig. 4: Pathway design to produce target RiPPs.

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Data availability

Data associated with this Article can be found in the Supplementary Information. All strains and plasmids are available upon request.

Code availability

Code is available on GitHub at https://github.com/VoigtLab/ripp-design (ref. 96). Instructions for its use are provided in Supplementary Note 12.

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Acknowledgements

This research was funded by the US Defense Advanced Research Projects Agency’s Living Foundries programme award (HR0011-12-C-0067), the US Defense Advanced Research Project Agency’s 1KM programme award (HR0011-15-C-0084) and a research grant from Novartis Institutes for Biomedical Research (NIMBR), a research grant from DSM Research, and a research grant from the US Office of Naval Research (N00014-18-1-2632). Research was additionally sponsored by the Army Research Office and was accomplished under cooperative agreement no. W911NF-22-2-0246. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the Army Research Office or the US Government. The US Government is authorized to reproduce and distribute reprints for Government purposes notwithstanding any copyright notation herein. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: Emerson Glassey, Zhengan Zhang.

Authors and Affiliations

  1. Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Emerson Glassey, Zhengan Zhang, Andrew M. King, David L. Niquille & Christopher A. Voigt

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  1. Emerson Glassey
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Contributions

E.G. and C.A.V. conceived the study and designed the experiments. E.G. wrote the software. Z.Z., A.M.K., D.L.N. and E.G. performed MS/MS and analysed the data. Z.Z. performed NMR and analysed the data. E.G. and Z.Z. performed all other experiments and analysed the data. E.G., Z.Z. and C.A.V. wrote the manuscript.

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Correspondence to Christopher A. Voigt.

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Supplementary Information

Supplementary Notes 1–13, Figs. 1–45 and Tables 1–6.

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Supplementary Table 1

Source data for Figs. 1 and 2 and for Supplementary notes 3–11.

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Glassey, E., Zhang, Z., King, A.M. et al. De novo design of ribosomally synthesized and post-translationally modified peptides. Nat. Chem. 17, 233–245 (2025). https://doi.org/10.1038/s41557-024-01685-9

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