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Synthetic fermentation of bioactive non-ribosomal peptides without organisms, enzymes or reagents

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Abstract

Microbial fermentation can rapidly provide potent compounds that can be easily screened for biological activity, and the active components can be isolated. Its success in drug discovery has inspired extensive efforts to modulate and control the products. In this Article, we document a ‘synthetic fermentation’ of bioactive, unnatural peptides ‘grown’ from small building blocks in water using amide-forming ligations. No organisms, enzymes or reagents are needed. The sequences, structures and compositions of the products can be modulated by adjusting the building blocks and conditions. No specialized knowledge of organic chemistry or handling of toxic material is required to produce complex organic molecules. The ‘fermentations’ can be conducted in arrays and screened for biological activity without isolation or workup. As a proof-of-concept, about 6,000 unnatural peptides were produced from just 23 building blocks, from which a hepatitis C virus NS3/4A protease inhibitor with a half-maximum inhibitory concentration of 1.0 μM was identified and characterized.

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Figure 1: The concept of NRPS and synthetic fermentation both involve initiation, elongation and termination steps.
Figure 2: The chemistry of synthetic fermentation relies on the KAHA ligation, which forms peptide bonds at a moderate temperature in water without any coupling reagents or protecting groups.
Figure 3: Two preliminary cultures were synthesized and poorly performing moieties were eliminated.
Figure 4: A focused culture was constructed with selected building blocks and the most-active compounds were predicted from biased deconvolution cultures.
Figure 5: To confirm the exact active structure, the proposed compound was recultured, isolated and characterized.
Figure 6: Selected examples of initiators, elongation monomers and terminators known to be suitable for synthetic fermentations.

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Elizabeth L. Bell, William Finnigan, … Sabine L. Flitsch

Change history

  • 07 October 2014

    In the HTML version of this Article originally published, a production error led to the 'Competing financial interests' statement incorrectly reading that the authors had no competing financial interests. The statement should have read: "ETH Zürich has filed a patent application on technology related to the processes described in this article. Both authors are listed as inventors on the patent application." This is correct in the PDF and print versions, and has now been amended in the HTML version.

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Acknowledgements

This work was supported by Swiss National Science Foundation (200021_131957), ETH Zürich and the David and Lucille Packard Foundation (Fellowship to J.W.B.). We thank D. Hilvert and R. Obexer for helpful discussions, S. Yu, T. Gerfaud and F. Schuler for preliminary experiments, and G. Erős for selected compounds.

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J.W.B. designed the project and wrote the paper with contributions by Y-L.H.; Y-L.H. performed the experiments and wrote the Supplementary Information. Both authors contributed to discussions.

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Correspondence to Jeffrey W. Bode.

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ETH Zürich has filed a patent application on technology related to the processes described in this article. Both authors are listed as inventors on the patent application.

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Huang, YL., Bode, J. Synthetic fermentation of bioactive non-ribosomal peptides without organisms, enzymes or reagents. Nature Chem 6, 877–884 (2014). https://doi.org/10.1038/nchem.2048

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