De novo design and engineering of non-ribosomal peptide synthetases


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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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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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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The authors declare no competing financial interests.

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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).

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