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.
|Number of pages||10|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Dec 17 2019|
Bibliographical noteFunding Information:
19. D. J. Winter et al., Repeat elements organise 3D genome structure and mediate transcription in the filamentous fungus Epichloë festucae. PLoS Genet. 14, e1007467 (2018). Materials and Methods A comprehensive description of the materials and methods used in this study is available in SI Appendix. Homologous recombination was used to generate E. festucae ΔppzA-2 strains. Constructs for ppzA expression in P. paxilli were assembled with all ppzA variants placed under transcriptional control of the same regulatory sequences from the native P. paxilli secondary metabolism gene paxM. These constructs were introduced into the P. paxilli genome via nontargeted integration, with RT-PCR used to select at least 3 independent transformants expressing each ppzA variant. Cultures for metabolite analysis were grown for 6 d under standardized conditions, with substrate feeding performed at 4 and 5 d postinoculation. 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Work in the H.B.B. laboratory was funded in part by the LOEWE Centre for Translational Biodiversity Genomics. D.B. was supported by a Massey University PhD scholarship from Massey University and funding from the New Zealand Tertiary Education Commission provided through the Bioprotection Research Center. B.S. was supported by an Alexander von Humboldt Research Award. We thank Shaun Bushman (US Department of Agriculture) and Devish Singh (Barenbrug) for providing access to field trials for sampling and Dr. Patrick Edwards (Massey University) for assistance in generating NMR data.
© 2019 National Academy of Sciences. All rights reserved.
- Allelic neofunctionalization
- Nonribosomal peptide synthetase
- Secondary metabolism
ASJC Scopus subject areas