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Modification and de novo design of non-ribosomal peptide synthetases using specific assembly points within condensation domains

Abstract

Non-ribosomal peptide synthetases (NRPSs) are giant enzyme machines that activate amino acids in an assembly line fashion. As NRPSs are not restricted to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would enable microbial production of a diverse range of peptides; however, the structural requirements for reprogramming NRPSs to facilitate the production of new peptides are not clear. Here we describe a new fusion point inside the condensation domains of NRPSs that results in the development of the exchange unit condensation domain (XUC) concept, which enables the efficient production of peptides, even containing non-natural amino acids, in yields up to 280 mg l−1. This allows the generation of more specific NRPSs, reducing the number of unwanted peptide derivatives, but also the generation of peptide libraries. The XUC might therefore be suitable for the future optimization of peptide production and the identification of bioactive peptide derivatives for pharmaceutical and other applications.

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The data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

The authors thank M. Lindner and C. Zizka for help with the construction of selected plasmids and C. Kegler for helpful discussions. This work was funded in part by the LOEWE programme of the state of Hesse as part of the MegaSyn and TBG research clusters. H.B.B. acknowledges the Deutsche Forschungsgemeinschaft for funding of the Impact II qTof mass spectrometer (INST 161/810-1).

Author information

K.A.J.B. and H.B.B. designed the experiments. K.A.J.B., A.L., A.T., J.K., S.N. and F.F. performed all molecular biology and biochemical experiments. F.W. synthesized all peptide standards that were used for the high-performance liquid chromatography–mass spectrometry-based quantification performed by A.L. and A.T. J.K., Y.-N.S. and P.G. isolated selected peptides and Y.-N.S. performed their NMR analysis. All authors analysed the results and K.A.J.B., A.L., A.T., J.K. and H.B.B. wrote the manuscript. All authors saw and approved the manuscript.

Competing interests

The authors declare no competing interests.

Correspondence to Helge B. Bode.

Supplementary Information

Supplementary Information

Supplementary Information includes methods, Supplementary Tables 1–7 (containing mass spectrometry data of the produced peptides, strains, plasmids and oligonucleotides used in this study) and Supplementary Figs. 1–42 (including NMR spectra of new peptides).

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Fig. 1: Modulation of C domain substrate specificity.
Fig. 2: Design of recombinant NRPS for peptide production.
Fig. 3: Elongation XUCs can be used as starting XUC.
Fig. 4: Creation of functionalized xenotetrapeptide derivatives.
Fig. 5: Targeted randomization of GxpS.