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De novo design and engineering of non-ribosomal peptide synthetases

Nature Chemistry volume 10, pages 275–281 (2018)Cite this article

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Abstract

Peptides derived from non-ribosomal peptide synthetases (NRPSs) represent an important class of pharmaceutically relevant drugs. Methods to generate novel non-ribosomal peptides or to modify peptide natural products in an easy and predictable way are therefore of great interest. However, although the overall modular structure of NRPSs suggests the possibility of adjusting domain specificity and selectivity, only a few examples have been reported and these usually show a severe drop in production titre. Here we report a new strategy for the modification of NRPSs that uses defined exchange units (XUs) and not modules as functional units. XUs are fused at specific positions that connect the condensation and adenylation domains and respect the original specificity of the downstream module to enable the production of the desired peptides. We also present the use of internal condensation domains as an alternative to other peptide-chain-releasing domains for the production of cyclic peptides.

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Figure 1: Domain and module swaps in the ambactin-producing NRPS AmbS.
Figure 2: XU-concept workflow.
Figure 3: De novo design of XtpS and GxpS NRPSs for xenotetrapeptide and GameXPeptide production.
Figure 4: Production of novel peptides.
Figure 5: Internal C domains as alternative termination domains.
Figure 6: Production of novel peptides by applying internal C domains as termination domains.

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References

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Walsh, C. T. The chemical versatility of natural-product assembly lines. Acc. Chem. Res. 41, 4–10 (2008).

    Article  CAS  Google Scholar 

  4. Kopp, F. & Marahiel, M. A. Macrocyclization strategies in polyketide and nonribosomal peptide biosynthesis. Nat. Prod. Rep. 24, 735–715 (2007).

    Article  CAS  Google Scholar 

  5. Marahiel, M. A. A structural model for multimodular NRPS assembly lines. Nat. Prod. Rep. 33, 136–140 (2016).

    Article  CAS  Google Scholar 

  6. Cai, X. et al. Biosynthesis of the antibiotic nematophin and its elongated derivatives in entomopathogenic bacteria. Org. Lett. 19, 806–809 (2017).

    Article  CAS  Google Scholar 

  7. Cai, X. et al. Entomopathogenic bacteria use multiple mechanisms for bioactive peptide library design. Nat. Chem. 9, 379–386 (2017).

    Article  CAS  Google Scholar 

  8. Gao, X. et al. Cyclization of fungal nonribosomal peptides by a terminal condensation-like domain. Nat. Chem. Biol. 8, 823–830 (2012).

    Article  CAS  Google Scholar 

  9. Winn, M., Fyans, J. K., Zhuo, Y. & Micklefield, J. Recent advances in engineering nonribosomal peptide assembly lines. Nat. Prod. Rep. 33, 317–347 (2016).

    Article  CAS  Google Scholar 

  10. Weist, S. & Süssmuth, R. D. Mutational biosynthesis—a tool for the generation of structural diversity in the biosynthesis of antibiotics. Appl. Microbiol. Biotechnol. 68, 141–150 (2005).

    Article  CAS  Google Scholar 

  11. Calcott, M. J. & Ackerley, D. F. Genetic manipulation of non-ribosomal peptide synthetases to generate novel bioactive peptide products. Biotechnol. Lett. 36, 2407–2416 (2014).

    Article  CAS  Google Scholar 

  12. Stachelhaus, T., Mootz, H. D. & Marahiel, M. A. The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem. Biol. 6, 493–505 (1999).

    Article  CAS  Google Scholar 

  13. Challis, G. L., Ravel, J. & Townsend, C. A. Predictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains. Chem. Biol. 7, 211–224 (2000).

    Article  CAS  Google Scholar 

  14. Kries, H., Niquille, D. L. & Hilvert, D. A subdomain swap strategy for reengineering nonribosomal peptides. Chem. Biol. 22, 640–648 (2015).

    Article  CAS  Google Scholar 

  15. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Kries, H. Biosynthetic engineering of nonribosomal peptide synthetases. J. Pept. Sci. 22, 564–570 (2016).

    Article  CAS  Google Scholar 

  18. Samel, S. A., Schoenafinger, G., Knappe, T. A., Marahiel, M. A. & Essen, L.-O. Structural and functional insights into a peptide bond-forming bidomain from a nonribosomal peptide synthetase. Structure 15, 781–792 (2007).

    Article  CAS  Google Scholar 

  19. Chiocchini, C., Linne, U. & Stachelhaus, T. In vivo biocombinatorial synthesis of lipopeptides by COM domain-mediated reprogramming of the surfactin biosynthetic complex. Chem. Biol. 13, 899–908 (2006).

    Article  CAS  Google Scholar 

  20. Miller, B. R., Sundlov, J. A., Drake, E. J., Makin, T. A. & Gulick, A. M. Analysis of the linker region joining the adenylation and carrier protein domains of the modular nonribosomal peptide synthetases. Proteins 82, 2691–2702 (2014).

    Article  CAS  Google Scholar 

  21. Schimming, O., Fleischhacker, F., Nollmann, F. I. & Bode, H. B. Yeast homologous recombination cloning leading to the novel peptides ambactin and xenolindicin. ChemBioChem 15, 1290–1294 (2014).

    Article  CAS  Google Scholar 

  22. Tanovic, A., Samel, S. A., Essen, L.-O. & Marahiel, M. A. Crystal structure of the termination module of a nonribosomal peptide synthetase. Science 321, 659–663 (2008).

    Article  CAS  Google Scholar 

  23. Sundlov, J. A., Shi, C., Wilson, D. J., Aldrich, C. C. & Gulick, A. M. Structural and functional investigation of the intermolecular interaction between NRPS adenylation and carrier protein domains. Chem. Biol. 19, 188–198 (2012).

    Article  CAS  Google Scholar 

  24. Drake, E. J. et al. Structures of two distinct conformations of holo-non-ribosomal peptide synthetases. Nature 529, 235–238 (2016).

    Article  CAS  Google Scholar 

  25. Liu, Y., Zheng, T. & Bruner, S. D. Structural basis for phosphopantetheinyl carrier domain interactions in the terminal module of nonribosomal peptide synthetases. Chem. Biol. 18, 1482–1488 (2011).

    Article  CAS  Google Scholar 

  26. Gulick, A. M. Conformational dynamics in the acyl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and firefly luciferase. ACS Chem. Biol. 4, 811–827 (2009).

    Article  CAS  Google Scholar 

  27. Tan, X.-F. et al. Structure of the adenylation-peptidyl carrier protein didomain of the Microcystis aeruginosa microcystin synthetase McyG. Acta. Crystallogr. D 71, 873–881 (2015).

    Article  CAS  Google Scholar 

  28. Strieker, M., Tanovic, A. & Marahiel, M. A. Nonribosomal peptide synthetases: structures and dynamics. Curr. Opin. Struct. Biol. 20, 234–240 (2010).

    Article  CAS  Google Scholar 

  29. Duerfahrt, T., Doekel, S., Sonke, T., Quaedflieg, P. J. L. M. & Marahiel, M. A. Construction of hybrid peptide synthetases for the production of α-L-aspartyl-L-phenylalanine, a precursor for the high-intensity sweetener aspartame. Eur. J. Biochem. 270, 4555–4563 (2003).

    Article  CAS  Google Scholar 

  30. Bode, H. B. et al. Determination of the absolute configuration of peptide natural products by using stable isotope labeling and mass spectrometry. Chem. Eur. J. 18, 2342–2348 (2012).

    Article  CAS  Google Scholar 

  31. Nollmann, F. I. et al. Insect-specific production of new GameXPeptides in Photorhabdus luminescens TTO1; widespread natural products in entomopathogenic bacteria. ChemBioChem 16, 205–208 (2015).

    Article  CAS  Google Scholar 

  32. Bode, H. B. et al. Structure elucidation and activity of Kolossin A, the D-/L-pentadecapeptide product of a giant nonribosomal peptide synthetase. Angew. Chem. Int. Ed. 54, 10352–10355 (2015).

    Article  CAS  Google Scholar 

  33. Kegler, C. et al. Rapid determination of the amino acid configuration of xenotetrapeptide. ChemBioChem 15, 826–828 (2014).

    Article  CAS  Google Scholar 

  34. Fuchs, S. W. et al. Neutral loss fragmentation pattern based screening for arginine-rich natural products in Xenorhabdus and Photorhabdus. Anal. Chem. 84, 6948–6955 (2012).

    Article  CAS  Google Scholar 

  35. Haynes, S. W., Ames, B. D., Gao, X., Tang, Y. & Walsh, C. T. Unraveling terminal C-domain-mediated condensation in fungal biosynthesis of imidazoindolone metabolites. Biochemistry 50, 5668–5679 (2011).

    Article  CAS  Google Scholar 

  36. Flissi, A. et al. Norine, the knowledge-base dedicated to non-ribosomal peptides, is now open to crowdsourcing. Nucleic Acids Res. 44, D1113–D1118 (2016).

    Article  CAS  Google Scholar 

  37. Medema, M. H. et al. Minimum information about a biosynthetic gene cluster. Nat. Chem. Biol. 11, 625–631 (2015).

    Article  CAS  Google Scholar 

  38. 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).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by a European Research Council Starting Grant to H.B.B. (grant agreement no. 311477) and the LOEWE program of the state of Hesse as part of the MegaSyn research cluster. The initial phase of this project was funded by the BMBF project BioPep in collaboration with Merck (Darmstadt). The authors are grateful to C. Kegler for useful discussions.

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Authors and Affiliations

  1. Merck Stiftungsprofessur für Molekulare Biotechnologie, Fachbereich Biowissenschaften, Goethe Universität Frankfurt, Max-von-Laue-Strasse 9, Frankfurt am Main, 60438, Germany

    Kenan A. J. Bozhüyük, Florian Fleischhacker, Annabell Linck, Frank Wesche, Andreas Tietze & Helge B. Bode

  2. Performance Materials/Process Technologies, Merck KGaA, Frankfurter Strasse 250, Darmstadt, 64293, Germany

    Claus-Peter Niesert

  3. Buchmann Institute for Molecular Life Sciences (BMLS), Goethe Universität Frankfurt, Max-von-Laue-Strasse 15, Frankfurt am Main, 60438, Germany

    Helge B. Bode

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Contributions

K.A.J.B. and H.B.B. conceived and designed the experiments. K.A.J.B., F.F., A.L. and A.T. performed the experiments and analysed the data together with H.B.B. and C.-P.N. F.W. performed the chemical synthesis of all of the peptides. K.A.J.B. and H.B.B. wrote the paper. All of the authors discussed the results and commented on the manuscript.

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Correspondence to Helge B. Bode.

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Bozhüyük, K., Fleischhacker, F., Linck, A. et al. De novo design and engineering of non-ribosomal peptide synthetases. Nature Chem 10, 275–281 (2018). https://doi.org/10.1038/nchem.2890

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