Short talk:
Engineering DNA-templated nonribosomal peptide synthetases

Hajo Kries1

1Leibniz-HKI, Jena, Germany

Nonribosomal peptide synthetases (NRPSs) protect microorganisms against environmental threats by producing siderophores or antibiotics, for instance, and are predisposed for biosynthetic engineering because of their modular molecular structure. We have explored several strategies for the redesign of NRPS specificity. Notable examples are the incorporation of a clickable amino acid through targeted binding pocket mutagenesis [1] or specificity transfer through swapping of small protein fragments [2, 3]. Incorporation of clickable amino acids has further enabled a strategy for high-throughput sorting of mutagenized NRPSs displayed on yeast [4]. Here, we demonstrate the engineering of DNA-templated supramolecular complexes to facilitate NRPS reprogramming [5]. We have split the NRPS for the cyclic decapeptide gramicidin S into modules. Up to four modules were later reassembled on a DNA template using DNA binding domains with high specificity and affinity, and loosely binding intermodular docking domains. The complex nonribosomal machinery showed astonishing tolerance for structural variations when the DNA spacers between modules were altered in length. In the future, DNA programmable NRPSs might allow to write the sequences of natural product-like peptides into short DNA templates to speed up NRPS design.

[1] H. Kries et al.; Angew. Chem. Int. Ed. Engl. 2014, 53, 10105.
[2] H. Kries et al.; Chem. Biol. 2015, 22, 640.
[3] A. Stanisic et al.; Chem. Sci 2019, 10, 10395.
[4] D. L. Niquille et al.; Nat. Chem. 2018, 10, 282.
[5] H.-M. Huang et al.; Cell Chem. Biol. 2021, 28, 221-227.e7.

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