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Nonribosomal biosynthesis of backbone-modified peptides


Biosynthetic modification of nonribosomal peptide backbones represents a potentially powerful strategy to modulate the structure and properties of an important class of therapeutics. Using a high-throughput assay for catalytic activity, we show here that an L-Phe-specific module of an archetypal nonribosomal peptide synthetase can be reprogrammed to accept and process the backbone-modified amino acid (S)-β-Phe with near-native specificity and efficiency. A co-crystal structure with a non-hydrolysable aminoacyl-AMP analogue reveals the origins of the 40,000-fold α/β-specificity switch, illuminating subtle but precise remodelling of the active site. When the engineered catalyst was paired with downstream module(s), (S)-β-Phe-containing peptides were produced at preparative scale in vitro (~1 mmol) and high titres in vivo (~100 mg l–1), highlighting the potential of biosynthetic pathway engineering for the construction of novel nonribosomal β-frameworks.

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Figure 1: Loading amino acids onto NRPS assembly lines.
Figure 2: Engineering the substrate specificity of TycAF for (S)-β-Phe.
Figure 3: Structural analysis of the engineered β-A domains.
Figure 4: Biosynthesis of dipeptide analogues.
Figure 5: Biosynthesis of a β-amino-acid-containing pentapeptide.

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The authors thank A. Schütz and K. Malgorzata from the ETH Zurich Flow Cytometry Core Facility, C. Stutz-Ducommun and B. Blattmann from the Protein Crystallization Core Facility at the University of Zurich, N. Trapp from the Small Molecule Crystallography Center ETH Zurich, L. Bertschi from the Mass Spectrometry Service of the Laboratory of Organic Chemistry at ETH Zurich and the staff at the Swiss Light Source (Paul Scherrer Institute) for technical support. The authors also thank P. Mittl for assistance with data acquisition for X-ray crystallography, H.-M. Fischer for assistance with radiochemical experiments and J. Piel, P. Kast, T. Edwardson, X. Garrabou, S. Mantri and S. Studer for discussions. This work was supported by the ETH Zurich. Fellowships from the ETH (to D.A.H.), the Daiichi Sankyo Foundation of Life Science (to T.M.), the Scholarship Fund of the Swiss Chemical Industry (to H.K.) and the Studienstiftung des deutschen Volkes (to H.K.) are acknowledged.

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All authors were involved in project design. D.L.N., D.A.H., T.M., D.F. and H.K. executed experiments. The manuscript was written by D.L.N., D.A.H. and D.H., and revised by all authors.

Corresponding author

Correspondence to Donald Hilvert.

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The authors declare no competing financial interests.

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Crystallographic data for compound 9. (CIF 1683 kb)

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Crystallographic data for compound 10. (CIF 2692 kb)

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Niquille, D., Hansen, D., Mori, T. et al. Nonribosomal biosynthesis of backbone-modified peptides. Nature Chem 10, 282–287 (2018).

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