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Nonribosomal biosynthesis of backbone-modified peptides

Abstract

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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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  PubMed  Google Scholar 

  2. 2

    Cane, D. E., Walsh, C. T. & Khosla, C. Harnessing the biosynthetic code: combinations, permutations, and mutations. Science 282, 63–68 (1998).

    CAS  PubMed  Google Scholar 

  3. 3

    Kim, E., Moore, B. S. & Yoon, Y. J. Reinvigorating natural product combinatorial biosynthesis with synthetic biology. Nat. Chem. Biol. 11, 649–659 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Schwarzer, D., Finking, R. & Marahiel, M. A. Nonribosomal peptides: from genes to products. Nat. Prod. Rep. 20, 275–287 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Felnagle, E. A. et al. Nonribosomal peptide synthetases involved in the production of medically relevant natural products. Mol. Pharm. 5, 191–211 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Süssmuth, R. D. & Mainz, A. Nonribosomal peptide synthesis—principles and prospects. Angew. Chem. Int. Ed. 56, 3770–3821 (2017).

    Google Scholar 

  7. 7

    Sieber, S. A. & Marahiel, M. A. Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics. Chem. Rev. 105, 715–738 (2005).

    CAS  PubMed  Google Scholar 

  8. 8

    Fischbach, M. A. & Walsh, C. T. Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: logic, machinery, and mechanisms. Chem. Rev. 106, 3468–3496 (2006).

    CAS  PubMed  Google Scholar 

  9. 9

    Hur, G. H., Vickery, C. R. & Burkart, M. D. Explorations of catalytic domains in non-ribosomal peptide synthetase enzymology. Nat. Prod. Rep. 29, 1074–1098 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    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  PubMed  Google Scholar 

  11. 11

    Eppelmann, K., Stachelhaus, T. & Marahiel, M. A. Exploitation of the selectivity-conferring code of nonribosomal peptide synthetases for the rational design of novel peptide antibiotics. Biochemistry 41, 9718–9726 (2002).

    CAS  PubMed  Google Scholar 

  12. 12

    Chen, C.-Y., Georgiev, I., Anderson, A. C. & Donald, B. R. Computational structure-based redesign of enzyme activity. Proc. Natl Acad. Sci. USA 106, 3764–3769 (2009).

    CAS  PubMed  Google Scholar 

  13. 13

    Evans, B. S., Chen, Y., Metcalf, W. W., Zhao, H. & Kelleher, N. L. Directed evolution of the nonribosomal peptide synthetase AdmK generates new andrimid derivatives in vivo. Chem. Biol. 18, 601–607 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Villiers, B. & Hollfelder, F. Directed evolution of a gatekeeper domain in nonribosomal peptide synthesis. Chem. Biol. 18, 1290–1299 (2011).

    CAS  PubMed  Google Scholar 

  15. 15

    Thirlway, J. et al. Introduction of a non-natural amino acid into a nonribosomal peptide antibiotic by modification of adenylation domain specificity. Angew. Chem. Int. Ed. 51, 7181–7184 (2012).

    CAS  Google Scholar 

  16. 16

    Zhang, K. et al. Engineering the substrate specificity of the DhbE adenylation domain by yeast cell surface display. Chem. Biol. 20, 92–101 (2013).

    CAS  PubMed  Google Scholar 

  17. 17

    Kries, H. et al. Reprogramming nonribosomal peptide synthetases for ‘clickable’ amino acids. Angew. Chem. Int. Ed. 53, 10105–10108 (2014).

    CAS  Google Scholar 

  18. 18

    Kaljunen, H. et al. Structural elucidation of the bispecificity of A domains as a basis for activating non-natural amino acids. Angew. Chem. Int. Ed. 54, 8833–8836 (2015).

    CAS  Google Scholar 

  19. 19

    Shrestha, S. K. & Garneau-Tsodikova, S. Expanding substrate promiscuity by engineering a novel adenylating-methylating NRPS bifunctional enzyme. ChemBioChem 17, 1328–1332 (2016).

    CAS  PubMed  Google Scholar 

  20. 20

    Kudo, F., Miyanaga, A. & Eguchi, T. Biosynthesis of natural products containing β-amino acids. Nat. Prod. Rep. 31, 1056–1073 (2014).

    CAS  PubMed  Google Scholar 

  21. 21

    Seebach, D. et al. New open-chain and cyclic tetrapeptides, consisting of α-, β2-, and β3-amino-acid residues, as somatostatin mimics—a survey. Helv. Chim. Acta 91, 1736–1786 (2008).

    CAS  Google Scholar 

  22. 22

    Cheloha, R. W., Maeda, A., Dean, T., Gardella, T. J. & Gellman, S. H. Backbone modification of a polypeptide drug alters duration of action in vivo. Nat. Biotechnol. 32, 653–655 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Werner, H. M. & Horne, W. S. Folding and function in α/β-peptides: targets and therapeutic applications. Curr. Opin. Chem. Biol. 28, 75–82 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Mootz, H. D. & Marahiel, M. A. The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains. J. Bacteriol. 179, 6843–6850 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Boder, E. T. & Wittrup, K. D. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15, 553–557 (1997).

    CAS  PubMed  Google Scholar 

  26. 26

    Tornøe, C. W., Christensen, C. & Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalysed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67, 3057–3064 (2002).

    PubMed  Google Scholar 

  27. 27

    Rostovtsev, V. V., Green, L. G., Fokin, V. V. & Sharpless, K. B. A stepwise Huisgen cycloaddition process: copper(I)-catalysed regioselective ‘ligation’ of azides and terminal alkynes. Angew. Chem. Int. Ed. 41, 2596–2599 (2002).

    CAS  Google Scholar 

  28. 28

    Conti, E., Stachelhaus, T., Marahiel, M. A. & Brick, P. Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S. EMBO J. 16, 4174–4183 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Miyanaga, A., Cieślak, J., Shinohara, Y., Kudo, F. & Eguchi, T. The crystal structure of the adenylation enzyme VinN reveals a unique β-amino acid recognition mechanism. J. Biol. Chem. 289, 31448–31457 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Miyanaga, A., Kudo, F. & Eguchi, T. Mechanisms of β-amino acid incorporation in polyketide macrolactam biosynthesis. Curr. Opin. Chem. Biol. 35, 58–64 (2016).

    CAS  PubMed  Google Scholar 

  31. 31

    Otten, L. G., Schaffer, M. L., Villiers, B. R. M., Stachelhaus, T. & Hollfelder, F. An optimized ATP/PPi-exchange assay in 96-well format for screening of adenylation domains for applications in combinatorial biosynthesis. Biotechnol. J. 2, 232–240 (2007).

    CAS  PubMed  Google Scholar 

  32. 32

    Stachelhaus, T., Mootz, H. D., Bergendahl, V. & Marahiel, M. A. Peptide bond formation in nonribosomal peptide biosynthesis. Catalytic role of the condensation domain. J. Biol. Chem. 273, 22773–22781 (1998).

    CAS  PubMed  Google Scholar 

  33. 33

    Tseng, C. C. et al. Characterization of the surfactin synthetase C-terminal thioesterase domain as a cyclic depsipeptide synthase. Biochemistry 41, 13350–13359 (2002).

    CAS  PubMed  Google Scholar 

  34. 34

    Krätzschmar, J., Krause, M. & Marahiel, M. A. Gramicidin S biosynthesis operon containing the structural genes grsA and grsB has an open reading frame encoding a protein homologous to fatty acid thioesterases. J. Bacteriol. 171, 5422–5429 (1989).

    PubMed  PubMed Central  Google Scholar 

  35. 35

    Hahn, M. & Stachelhaus, T. Harnessing the potential of communication-mediating domains for the biocombinatorial synthesis of nonribosomal peptides. Proc. Natl Acad. Sci. USA 103, 275–280 (2006).

    CAS  PubMed  Google Scholar 

  36. 36

    Hoyer, K. M., Mahlert, C. & Marahiel, M. A. The iterative gramicidin S thioesterase catalyses peptide ligation and cyclization. Chem. Biol. 14, 13–22 (2007).

    CAS  PubMed  Google Scholar 

  37. 37

    Gruenewald, S., Mootz, H. D., Stehmeier, P. & Stachelhaus, T. In vivo production of artificial nonribosomal peptide products in the heterologous host Escherichia coli. Appl. Environ. Microbiol. 70, 3282–3291 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Mootz, H. D. et al. Decreasing the ring size of a cyclic nonribosomal peptide antibiotic by in-frame module deletion in the biosynthetic genes. J. Am. Chem. Soc. 124, 10980–10981 (2002).

    CAS  PubMed  Google Scholar 

  39. 39

    Nguyen, K. T. et al. Combinatorial biosynthesis of novel antibiotics related to daptomycin. Proc. Natl Acad. Sci. USA 103, 17462–17467 (2006).

    CAS  PubMed  Google Scholar 

  40. 40

    Fischbach, M. A., Lai, J. R., Roche, E. D., Walsh, C. T. & Liu, D. R. Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes. Proc. Natl Acad. Sci. USA 104, 11951–11956 (2007).

    CAS  PubMed  Google Scholar 

  41. 41

    Butz, D. et al. Module extension of a non-ribosomal peptide synthetase of the glycopeptide antibiotic balhimycin produced by Amycolaptosis balhimycina. ChemBioChem 9, 1195–1200 (2008).

    CAS  PubMed  Google Scholar 

  42. 42

    Calcott, M. J., Owen, J. G., Lamont, I. L. & Ackerley, D. F. Biosynthesis of novel pyoverdines by domain substitution in a nonribosomal peptide synthetase of Pseudomonas aeruginosa. Appl. Environ. Microbiol. 80, 5723–5731 (2014).

    PubMed  PubMed Central  Google Scholar 

  43. 43

    Murphy, A. C. Metabolic engineering is key to a sustainable chemical industry. Nat. Prod. Rep. 28, 1406–1425 (2011).

    CAS  PubMed  Google Scholar 

  44. 44

    McKay, C. S. & Finn, M. G. Click chemistry in complex mixtures: bioorthogonal bioconjugation. Chem. Biol. 21, 1075–1101 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Jäckel, C. & Hilvert, D. Biocatalysts by evolution. Curr. Opin. Biotechnol. 21, 753–759 (2010).

    PubMed  Google Scholar 

  46. 46

    Röttig, M. et al. NRPSpredictor2—a web server for predicting NRPS adenylation domain specificity. Nucleic Acids Res. 39, W362–W367 (2011).

    PubMed  PubMed Central  Google Scholar 

  47. 47

    Jin, M., Fischbach, M. A. & Clardy, J. A biosynthetic gene cluster for the acetyl-CoA carboxylase inhibitor andrimid. J. Am. Chem. Soc. 128, 10660–10661 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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.

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Correspondence to Donald Hilvert.

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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). https://doi.org/10.1038/nchem.2891

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