Programming Nonribosomal Peptide Synthetases with DNA templates Huiyun Peng, Hsin-Mei Huang, Philipp Stephan, Hajo Kries Hans Knöll Institute, Germany Nonribosomal peptide synthetases (NRPSs) are important enzyme machineries that have supplied the pharmaceutical industry with numerous bioactive natural products. 1 Their modular architecture facilitates production of new compounds through NRPS engineering. However, this strategy faces major challenges because the manipulation of gigantic NRPS genes is time‑consuming and error-prone. 2, 3 To enable fast, targeted rearrangement of NRPS modules, we have established DNA-templated nonribosomal peptide synthetases ( DT‑NRPSs ) by taking advantage of the sequence-specific DNA recognition of fused zinc-finger motifs. 4 DT- NRPSs have been engineered from the gramicidin S (GrS) assembly line in four steps: I) constructing standalone NRPS modules; II) fusing docking domains; III) attaching zinc‑fingers to each module; and IV) optimizing the system for peptide formation. 4 The production of GrS and peptide intermediates was monitored in each step. NRPS programming with DNA templates has been demonstrated by the enhanced production of the targeted peptides when different DNA templates were added. Lastly, we harnessed DT-NRPSs to produce several new peptides by adding alternative NRPS-zinc-finger bricks. In conclusion, we have successfully reconstituted the GrS assembly line in vitro in a DNA-templated format. The engineering potential of DT‑NRPSs to rationally produce new peptides has been demonstrated. In the future, DT‑NRPSs will be developed into an efficient tool for biocatalytic on-demand peptide synthesis by enhancing enzyme activity and enlarging the collection of suitable DT-NRPS modules.
References 1. Süssmuth, R. D.; Mainz, A., Nonribosomal peptide synthesis-principles and prospects. Angew. Chem. Int. Ed. 2017, 56 (14), 3770-3821. 2. Bozhüyük, K. A. J.; Fleischhacker, F.; Linck, A.; Wesche, F.; Tietze, A.; Niesert, C. P.; Bode, H. B., De novo design and engineering of non-ribosomal peptide synthetases. Nat. Chem. 2018, 10 (3), 275-281. 3. Bozhüyük, K. A. J.; Linck, A.; Tietze, A.; Kranz, J.; Wesche, F.; Nowak, S.; Fleischhacker, F.; Shi, Y. N.; Grun, P.; Bode, H. B., Modification and de novo design of non-ribosomal peptide synthetases using specific assembly points within condensation domains. Nat. Chem. 2019, 11 (7), 653-661. 4. Huang, H. M.; Stephan, P.; Kries, H., Engineering DNA-templated nonribosomal peptide synthesis. Cell Chem. Biol. 2021, 28 (2), 221-227.
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