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Efficient nonenzymatic cyclization and domain shuffling drive pyrrolopyrazine diversity from truncated variants of a fungal NRPS.

  • Berry D School of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand.
  • Mace W Grasslands Research Centre, AgResearch Ltd., Palmerston North 4442, New Zealand.
  • Grage K School of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand.
  • Wesche F Fachbereich Biowissenschaften, Molekulare Biotechnologie, Goethe Universität Frankfurt, 60438 Frankfurt am Main, Germany.
  • Gore S Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, 07745 Jena, Germany.
  • Schardl CL Department of Plant Pathology, University of Kentucky, Lexington, KY 40506.
  • Young CA Noble Research Institute, LLC, Ardmore, OK 73401.
  • Dijkwel PP School of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand.
  • Leuchtmann A Institute of Integrative Biology, ETH Zürich, CH-8092 Zürich, Switzerland.
  • Bode HB Fachbereich Biowissenschaften, Molekulare Biotechnologie, Goethe Universität Frankfurt, 60438 Frankfurt am Main, Germany; h.bode@bio.uni-frankfurt.de d.b.scott@massey.ac.nz.
  • Scott B School of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand; h.bode@bio.uni-frankfurt.de d.b.scott@massey.ac.nz.
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  • 2019-12-06
Published in:
  • Proceedings of the National Academy of Sciences of the United States of America. - 2019
allelic neofunctionalization
diketopiperazine
nonribosomal peptide synthetase
pyrrolopyrazine
secondary metabolism
Cyclization
DNA Shuffling
Diketopiperazines
Epichloe
Fungal Proteins
Peptide Synthases
Pyrazines
Recombinant Proteins
English Nonribosomal peptide synthetases (NRPSs) generate the core peptide scaffolds of many natural products. These include small cyclic dipeptides such as the insect feeding deterrent peramine, which is a pyrrolopyrazine (PPZ) produced by grass-endophytic Epichloë fungi. Biosynthesis of peramine is catalyzed by the 2-module NRPS, PpzA-1, which has a C-terminal reductase (R) domain that is required for reductive release and cyclization of the NRPS-tethered dipeptidyl-thioester intermediate. However, some PpzA variants lack this R domain due to insertion of a transposable element into the 3' end of ppzA We demonstrate here that these truncated PpzA variants utilize nonenzymatic cyclization of the dipeptidyl thioester to a 2,5-diketopiperazine (DKP) to synthesize a range of novel PPZ products. Truncation of the R domain is sufficient to subfunctionalize PpzA-1 into a dedicated DKP synthetase, exemplified by the truncated variant, PpzA-2, which has also evolved altered substrate specificity and reduced N-methyltransferase activity relative to PpzA-1. Further allelic diversity has been generated by recombination-mediated domain shuffling between ppzA-1 and ppzA-2, resulting in the ppzA-3 and ppzA-4 alleles, each of which encodes synthesis of a unique PPZ metabolite. This research establishes that efficient NRPS-catalyzed DKP biosynthesis can occur in vivo through nonenzymatic dipeptidyl cyclization and presents a remarkably clean example of NRPS evolution through recombinant exchange of functionally divergent domains. This work highlights that allelic variants of a single NRPS can result in a surprising level of secondary metabolite diversity comparable to that observed for some gene clusters.
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  • English
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hybrid
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https://folia.unifr.ch/global/documents/55235
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