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

Daniel Berry, Wade Mace, Katrin Grage, Frank Wesche, Sagar Gore, Christopher L. Schardl, Carolyn A. Young, Paul P. Dijkwel, Adrian Leuchtmann, Helge B. Bode, Barry Scott

Research output: Contribution to journalArticlepeer-review

27 Scopus citations


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.

Original languageEnglish
Pages (from-to)25614-25623
Number of pages10
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number51
StatePublished - Dec 17 2019

Bibliographical note

Publisher Copyright:
© 2019 National Academy of Sciences. All rights reserved.


  • Allelic neofunctionalization
  • Diketopiperazine
  • Nonribosomal peptide synthetase
  • Pyrrolopyrazine
  • Secondary metabolism

ASJC Scopus subject areas

  • General


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