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

doi:10.1073/pnas.1913080116

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

Plant Pathology

Research output: Contribution to journal › Article › peer-review

18 Scopus citations

Abstract

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 language	English
Pages (from-to)	25614-25623
Number of pages	10
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	116
Issue number	51
DOIs	https://doi.org/10.1073/pnas.1913080116
State	Published - Dec 17 2019

Bibliographical note

Funding 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. PPZ metabolites were extracted from lyophilized P. paxilli mycelia or Epichloë-infected plant material and were analyzed using hydrophilic-interaction chromatography-coupled positive electrospray ionization mass spectroscopy (LCMS). Data Availability. DNA sequence data generated during this study have been deposited in the GenBank database under accession numbers MN605951 to MN605962. Raw PPZ concentrations from all LCMS analyses, MSn spectra for all PPZ metabolites, and NMR spectra for 2a are available in SI Appendix. ACKNOWLEDGMENTS. 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. 20. M. A. Marahiel, T. Stachelhaus, H. D. Mootz, Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem. Rev. 97, 2651–2674 (1997). 21. T. Stachelhaus, H. D. Mootz, M. A. Marahiel, The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem. Biol. 6, 493–505 (1999). 22. K. D. Craven, J. D. Blankenship, A. Leuchtmann, K. Hignight, C. L. Schardl, Hybrid fungal endophytes symbiotic with the grass Lolium pratense. Sydowia 53, 44–73 (2001). 23. D. Berry et al., Orthologous peramine and pyrrolopyrazine-producing biosynthetic gene clusters in Metarhizium rileyi, Metarhizium majus and Cladonia grayi. Environ. Microbiol. 21, 928–939 (2019). 24. T. Stachelhaus, H. D. Mootz, V. Bergendahl, M. A. Marahiel, Peptide bond formation in nonribosomal peptide biosynthesis. Catalytic role of the condensation domain. J. Biol. Chem. 273, 22773–22781 (1998). 25. A. Leuchtmann, C. W. Bacon, C. L. Schardl, J. F. White, Jr, M. Tadych, Nomenclatural realignment of Neotyphodium species with genus Epicholë. Mycologia 106, 202–215 (2014). 26. A. Rokas, J. H. Wisecaver, A. L. Lind, The birth, evolution and death of metabolic gene clusters in fungi. Nat. Rev. Microbiol. 16, 731–744 (2018). 27. A. L. Lind et al., Drivers of genetic diversity in secondary metabolic gene clusters within a fungal species. PLoS Biol. 15, e2003583 (2017). 28. C. A. Young et al., Genetics, genomics and evolution of ergot alkaloid diversity. Toxins (Basel) 7, 1273–1302 (2015). 29. X. Wang, Y. Li, X. Zhang, D. Lai, L. Zhou, Structural diversity and biological activities of the cyclodipeptides from fungi. Molecules 22, 2026 (2017). 30. W.-B. Yin, A. Grundmann, J. Cheng, S.-M. Li, Acetylaszonalenin biosynthesis in Neosartorya fischeri. Identification of the biosynthetic gene cluster by genomic mining and functional proof of the genes by biochemical investigation. J. Biol. Chem. 284, 100–109 (2009). 31. K. Mundt, B. Wollinsky, H. L. Ruan, T. Zhu, S. M. Li, Identification of the verruculogen prenyltransferase FtmPT3 by a combination of chemical, bioinformatic and biochemical approaches. ChemBioChem 13, 2583–2592 (2012). 32. S. Maiya, A. Grundmann, S. M. Li, G. Turner, The fumitremorgin gene cluster of As-pergillus fumigatus: Identification of a gene encoding brevianamide F synthetase. ChemBioChem 7, 1062–1069 (2006). 33. C. García-Estrada et al., A single cluster of coregulated genes encodes the biosynthesis of the mycotoxins roquefortine C and meleagrin in Penicillium chrysogenum. Chem. Biol. 18, 1499–1512 (2011). 34. T. Correia, N. Grammel, I. Ortel, U. Keller, P. Tudzynski, Molecular cloning and analysis of the ergopeptine assembly system in the ergot fungus Claviceps purpurea. Chem. Biol. 10, 1281–1292 (2003). 35. C. J. Balibar, C. T. Walsh, GliP, a multimodular nonribosomal peptide synthetase in Aspergillus fumigatus, makes the diketopiperazine scaffold of gliotoxin. Biochemistry 45, 15029–15038 (2006). 36. D. E. Stewart, A. Sarkar, J. E. Wampler, Occurrence and role of cis peptide bonds in protein structures. J. Mol. Biol. 214, 253–260 (1990). 37. X. Gao et al., Cyclization of fungal nonribosomal peptides by a terminal condensation-like domain. Nat. Chem. Biol. 8, 823–830 (2012). 38. J. A. Baccile et al., Diketopiperazine formation in fungi requires dedicated cyclization and thiolation domains. Angew. Chem. Int. Ed. Engl. 58, 14589–14593 (2019). Downloaded at Elsevier Science London on December 20, 2019 39. A. W. Schultz et al., Biosynthesis and structures of cyclomarins and cyclomarazines, prenylated cyclic peptides of marine actinobacterial origin. J. Am. Chem. Soc. 130, 4507–4516 (2008). 40. S. G. Lee, F. Lipmann, Isolation of amino acid activating subunit–pantetheine protein complexes: Their role in chain elongation in tyrocidine synthesis. Proc. Natl. Acad. Sci. U.S.A. 74, 2343–2347 (1977). 41. S. Gruenewald, H. D. Mootz, P. Stehmeier, T. Stachelhaus, In vivo production of ar-tificial nonribosomal peptide products in the heterologous host Escherichia coli. Appl. Environ. Microbiol. 70, 3282–3291 (2004). 42. F. Brotzel, H. Mayr, Nucleophilicities of amino acids and peptides. Org. Biomol. Chem. 5, 3814–3820 (2007). 43. M. Crüsemann, C. Kohlhaas, J. Piel, Evolution-guided engineering of nonribosomal peptide synthetase adenylation domains. Chem. Sci. (Camb.) 4, 1041–1045 (2013). 44. D. P. Fewer et al., Recurrent adenylation domain replacement in the microcystin synthetase gene cluster. BMC Evol. Biol. 7, 183 (2007). 45. B. Mikalsen et al., Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in Microcystis strains. J. Bacteriol. 185, 2774–2785 (2003). 46. J. Klein, Origin of major histocompatibility complex polymorphism: The trans-species hypothesis. Hum. Immunol. 19, 155–162 (1987). 47. S. Sommer, The importance of immune gene variability (MHC) in evolutionary ecol-ogy and conservation. Front. Zool. 2, 16 (2005). 48. O. J. Ball et al., Importance of host plant species, Neotyphodium endophyte isolate, and alkaloids on feeding by Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae. J. Econ. Entomol. 99, 1462–1473 (2006). 49. O. J. P. Ball, C. O. Miles, R. A. Prestidge, Ergopeptine alkaloids and Neotyphodium lolii-mediated resistance in perennial ryegrass against adult Heteronychus arator (Coleoptera: Scarabaeidae). J. Econ. Entomol. 90, 1382–1391 (1997). 50. H. Izumida, N. Imamura, H. Sano, A novel chitinase inhibitor from a marine bacterium, Pseudomonas sp. J. Antibiot. (Tokyo) 49, 76–80 (1996). 51. H. Izumida, M. Nishijima, T. Takadera, A. M. Nomoto, H. Sano, The effect of chitinase inhibitors, cyclo(Arg-Pro) against cell separation of Saccharomyces cerevisiae and the morphological change of Candida albicans. J. Antibiot. (Tokyo) 49, 829–831 (1996). 52. V. N. Minin, K. S. Dorman, F. Fang, M. A. Suchard, Dual multiple change-point model leads to more accurate recombination detection. Bioinformatics 21, 3034–3042 (2005). BIOCHEMISTRY

Funding Information:
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.

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

Keywords

Allelic neofunctionalization
Diketopiperazine
Nonribosomal peptide synthetase
Pyrrolopyrazine
Secondary metabolism

ASJC Scopus subject areas

General

Access to Document

10.1073/pnas.1913080116

Cite this

Berry, D., Mace, W., Grage, K., Wesche, F., Gore, S., Schardl, C. L., Young, C. A., Dijkwel, P. P., Leuchtmann, A., Bode, H. B., & Scott, B. (2019). Efficient nonenzymatic cyclization and domain shuffling drive pyrrolopyrazine diversity from truncated variants of a fungal NRPS. Proceedings of the National Academy of Sciences of the United States of America, 116(51), 25614-25623. https://doi.org/10.1073/pnas.1913080116

@article{7467a00c55be41f29bfa2ea5832d25c6,

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

abstract = "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 Epichlo{\"e} 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.",

keywords = "Allelic neofunctionalization, Diketopiperazine, Nonribosomal peptide synthetase, Pyrrolopyrazine, Secondary metabolism",

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

note = "Funding Information: 19. D. J. Winter et al., Repeat elements organise 3D genome structure and mediate transcription in the filamentous fungus Epichlo{\"e} 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. PPZ metabolites were extracted from lyophilized P. paxilli mycelia or Epichlo{\"e}-infected plant material and were analyzed using hydrophilic-interaction chromatography-coupled positive electrospray ionization mass spectroscopy (LCMS). Data Availability. DNA sequence data generated during this study have been deposited in the GenBank database under accession numbers MN605951 to MN605962. Raw PPZ concentrations from all LCMS analyses, MSn spectra for all PPZ metabolites, and NMR spectra for 2a are available in SI Appendix. ACKNOWLEDGMENTS. 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. 20. M. A. Marahiel, T. Stachelhaus, H. D. Mootz, Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem. Rev. 97, 2651–2674 (1997). 21. T. Stachelhaus, H. D. Mootz, M. A. Marahiel, The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem. Biol. 6, 493–505 (1999). 22. K. D. Craven, J. D. Blankenship, A. Leuchtmann, K. Hignight, C. L. Schardl, Hybrid fungal endophytes symbiotic with the grass Lolium pratense. Sydowia 53, 44–73 (2001). 23. D. Berry et al., Orthologous peramine and pyrrolopyrazine-producing biosynthetic gene clusters in Metarhizium rileyi, Metarhizium majus and Cladonia grayi. Environ. Microbiol. 21, 928–939 (2019). 24. T. Stachelhaus, H. D. Mootz, V. Bergendahl, M. A. Marahiel, Peptide bond formation in nonribosomal peptide biosynthesis. Catalytic role of the condensation domain. J. Biol. Chem. 273, 22773–22781 (1998). 25. A. Leuchtmann, C. W. Bacon, C. L. Schardl, J. F. White, Jr, M. Tadych, Nomenclatural realignment of Neotyphodium species with genus Epichol{\"e}. Mycologia 106, 202–215 (2014). 26. A. Rokas, J. H. Wisecaver, A. L. Lind, The birth, evolution and death of metabolic gene clusters in fungi. Nat. Rev. Microbiol. 16, 731–744 (2018). 27. A. L. Lind et al., Drivers of genetic diversity in secondary metabolic gene clusters within a fungal species. PLoS Biol. 15, e2003583 (2017). 28. C. A. Young et al., Genetics, genomics and evolution of ergot alkaloid diversity. Toxins (Basel) 7, 1273–1302 (2015). 29. X. Wang, Y. Li, X. Zhang, D. Lai, L. Zhou, Structural diversity and biological activities of the cyclodipeptides from fungi. Molecules 22, 2026 (2017). 30. W.-B. Yin, A. Grundmann, J. Cheng, S.-M. Li, Acetylaszonalenin biosynthesis in Neosartorya fischeri. Identification of the biosynthetic gene cluster by genomic mining and functional proof of the genes by biochemical investigation. J. Biol. Chem. 284, 100–109 (2009). 31. K. Mundt, B. Wollinsky, H. L. Ruan, T. Zhu, S. M. Li, Identification of the verruculogen prenyltransferase FtmPT3 by a combination of chemical, bioinformatic and biochemical approaches. ChemBioChem 13, 2583–2592 (2012). 32. S. Maiya, A. Grundmann, S. M. Li, G. Turner, The fumitremorgin gene cluster of As-pergillus fumigatus: Identification of a gene encoding brevianamide F synthetase. ChemBioChem 7, 1062–1069 (2006). 33. C. Garc{\'i}a-Estrada et al., A single cluster of coregulated genes encodes the biosynthesis of the mycotoxins roquefortine C and meleagrin in Penicillium chrysogenum. Chem. Biol. 18, 1499–1512 (2011). 34. T. Correia, N. Grammel, I. Ortel, U. Keller, P. Tudzynski, Molecular cloning and analysis of the ergopeptine assembly system in the ergot fungus Claviceps purpurea. Chem. Biol. 10, 1281–1292 (2003). 35. C. J. Balibar, C. T. Walsh, GliP, a multimodular nonribosomal peptide synthetase in Aspergillus fumigatus, makes the diketopiperazine scaffold of gliotoxin. Biochemistry 45, 15029–15038 (2006). 36. D. E. Stewart, A. Sarkar, J. E. Wampler, Occurrence and role of cis peptide bonds in protein structures. J. Mol. Biol. 214, 253–260 (1990). 37. X. Gao et al., Cyclization of fungal nonribosomal peptides by a terminal condensation-like domain. Nat. Chem. Biol. 8, 823–830 (2012). 38. J. A. Baccile et al., Diketopiperazine formation in fungi requires dedicated cyclization and thiolation domains. Angew. Chem. Int. Ed. Engl. 58, 14589–14593 (2019). Downloaded at Elsevier Science London on December 20, 2019 39. A. W. Schultz et al., Biosynthesis and structures of cyclomarins and cyclomarazines, prenylated cyclic peptides of marine actinobacterial origin. J. Am. Chem. Soc. 130, 4507–4516 (2008). 40. S. G. Lee, F. Lipmann, Isolation of amino acid activating subunit–pantetheine protein complexes: Their role in chain elongation in tyrocidine synthesis. Proc. Natl. Acad. Sci. U.S.A. 74, 2343–2347 (1977). 41. S. Gruenewald, H. D. Mootz, P. Stehmeier, T. Stachelhaus, In vivo production of ar-tificial nonribosomal peptide products in the heterologous host Escherichia coli. Appl. Environ. Microbiol. 70, 3282–3291 (2004). 42. F. Brotzel, H. Mayr, Nucleophilicities of amino acids and peptides. Org. Biomol. Chem. 5, 3814–3820 (2007). 43. M. Cr{\"u}semann, C. Kohlhaas, J. Piel, Evolution-guided engineering of nonribosomal peptide synthetase adenylation domains. Chem. Sci. (Camb.) 4, 1041–1045 (2013). 44. D. P. Fewer et al., Recurrent adenylation domain replacement in the microcystin synthetase gene cluster. BMC Evol. Biol. 7, 183 (2007). 45. B. Mikalsen et al., Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in Microcystis strains. J. Bacteriol. 185, 2774–2785 (2003). 46. J. Klein, Origin of major histocompatibility complex polymorphism: The trans-species hypothesis. Hum. Immunol. 19, 155–162 (1987). 47. S. Sommer, The importance of immune gene variability (MHC) in evolutionary ecol-ogy and conservation. Front. Zool. 2, 16 (2005). 48. O. J. Ball et al., Importance of host plant species, Neotyphodium endophyte isolate, and alkaloids on feeding by Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae. J. Econ. Entomol. 99, 1462–1473 (2006). 49. O. J. P. Ball, C. O. Miles, R. A. Prestidge, Ergopeptine alkaloids and Neotyphodium lolii-mediated resistance in perennial ryegrass against adult Heteronychus arator (Coleoptera: Scarabaeidae). J. Econ. Entomol. 90, 1382–1391 (1997). 50. H. Izumida, N. Imamura, H. Sano, A novel chitinase inhibitor from a marine bacterium, Pseudomonas sp. J. Antibiot. (Tokyo) 49, 76–80 (1996). 51. H. Izumida, M. Nishijima, T. Takadera, A. M. Nomoto, H. Sano, The effect of chitinase inhibitors, cyclo(Arg-Pro) against cell separation of Saccharomyces cerevisiae and the morphological change of Candida albicans. J. Antibiot. (Tokyo) 49, 829–831 (1996). 52. V. N. Minin, K. S. Dorman, F. Fang, M. A. Suchard, Dual multiple change-point model leads to more accurate recombination detection. Bioinformatics 21, 3034–3042 (2005). BIOCHEMISTRY Funding Information: 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. Publisher Copyright: {\textcopyright} 2019 National Academy of Sciences. All rights reserved.",

year = "2019",

month = dec,

day = "17",

doi = "10.1073/pnas.1913080116",

language = "English",

volume = "116",

pages = "25614--25623",

number = "51",

}

Berry, D, Mace, W, Grage, K, Wesche, F, Gore, S, Schardl, CL, Young, CA, Dijkwel, PP, Leuchtmann, A, Bode, HB & Scott, B 2019, 'Efficient nonenzymatic cyclization and domain shuffling drive pyrrolopyrazine diversity from truncated variants of a fungal NRPS', Proceedings of the National Academy of Sciences of the United States of America, vol. 116, no. 51, pp. 25614-25623. https://doi.org/10.1073/pnas.1913080116

Efficient nonenzymatic cyclization and domain shuffling drive pyrrolopyrazine diversity from truncated variants of a fungal NRPS. / Berry, Daniel; Mace, Wade; Grage, Katrin et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 116, No. 51, 17.12.2019, p. 25614-25623.

Research output: Contribution to journal › Article › peer-review

TY - JOUR

T1 - Efficient nonenzymatic cyclization and domain shuffling drive pyrrolopyrazine diversity from truncated variants of a fungal NRPS

AU - Berry, Daniel

AU - Mace, Wade

AU - Grage, Katrin

AU - Wesche, Frank

AU - Gore, Sagar

AU - Schardl, Christopher L.

AU - Young, Carolyn A.

AU - Dijkwel, Paul P.

AU - Leuchtmann, Adrian

AU - Bode, Helge B.

AU - Scott, Barry

N1 - Funding 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. PPZ metabolites were extracted from lyophilized P. paxilli mycelia or Epichloë-infected plant material and were analyzed using hydrophilic-interaction chromatography-coupled positive electrospray ionization mass spectroscopy (LCMS). Data Availability. DNA sequence data generated during this study have been deposited in the GenBank database under accession numbers MN605951 to MN605962. Raw PPZ concentrations from all LCMS analyses, MSn spectra for all PPZ metabolites, and NMR spectra for 2a are available in SI Appendix. ACKNOWLEDGMENTS. 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. 20. M. A. Marahiel, T. Stachelhaus, H. D. Mootz, Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem. Rev. 97, 2651–2674 (1997). 21. T. Stachelhaus, H. D. Mootz, M. A. Marahiel, The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem. Biol. 6, 493–505 (1999). 22. K. D. Craven, J. D. Blankenship, A. Leuchtmann, K. Hignight, C. L. Schardl, Hybrid fungal endophytes symbiotic with the grass Lolium pratense. Sydowia 53, 44–73 (2001). 23. D. Berry et al., Orthologous peramine and pyrrolopyrazine-producing biosynthetic gene clusters in Metarhizium rileyi, Metarhizium majus and Cladonia grayi. Environ. Microbiol. 21, 928–939 (2019). 24. T. Stachelhaus, H. D. Mootz, V. Bergendahl, M. A. Marahiel, Peptide bond formation in nonribosomal peptide biosynthesis. Catalytic role of the condensation domain. J. Biol. Chem. 273, 22773–22781 (1998). 25. A. Leuchtmann, C. W. Bacon, C. L. Schardl, J. F. White, Jr, M. Tadych, Nomenclatural realignment of Neotyphodium species with genus Epicholë. Mycologia 106, 202–215 (2014). 26. A. Rokas, J. H. Wisecaver, A. L. Lind, The birth, evolution and death of metabolic gene clusters in fungi. Nat. Rev. Microbiol. 16, 731–744 (2018). 27. A. L. Lind et al., Drivers of genetic diversity in secondary metabolic gene clusters within a fungal species. PLoS Biol. 15, e2003583 (2017). 28. C. A. Young et al., Genetics, genomics and evolution of ergot alkaloid diversity. Toxins (Basel) 7, 1273–1302 (2015). 29. X. Wang, Y. Li, X. Zhang, D. Lai, L. Zhou, Structural diversity and biological activities of the cyclodipeptides from fungi. Molecules 22, 2026 (2017). 30. W.-B. Yin, A. Grundmann, J. Cheng, S.-M. 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BMC Evol. Biol. 7, 183 (2007). 45. B. Mikalsen et al., Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in Microcystis strains. J. Bacteriol. 185, 2774–2785 (2003). 46. J. Klein, Origin of major histocompatibility complex polymorphism: The trans-species hypothesis. Hum. Immunol. 19, 155–162 (1987). 47. S. Sommer, The importance of immune gene variability (MHC) in evolutionary ecol-ogy and conservation. Front. Zool. 2, 16 (2005). 48. O. J. Ball et al., Importance of host plant species, Neotyphodium endophyte isolate, and alkaloids on feeding by Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae. J. Econ. Entomol. 99, 1462–1473 (2006). 49. O. J. P. Ball, C. O. Miles, R. A. Prestidge, Ergopeptine alkaloids and Neotyphodium lolii-mediated resistance in perennial ryegrass against adult Heteronychus arator (Coleoptera: Scarabaeidae). J. Econ. Entomol. 90, 1382–1391 (1997). 50. H. Izumida, N. Imamura, H. Sano, A novel chitinase inhibitor from a marine bacterium, Pseudomonas sp. J. Antibiot. (Tokyo) 49, 76–80 (1996). 51. H. Izumida, M. Nishijima, T. Takadera, A. M. Nomoto, H. Sano, The effect of chitinase inhibitors, cyclo(Arg-Pro) against cell separation of Saccharomyces cerevisiae and the morphological change of Candida albicans. J. Antibiot. (Tokyo) 49, 829–831 (1996). 52. V. N. Minin, K. S. Dorman, F. Fang, M. A. Suchard, Dual multiple change-point model leads to more accurate recombination detection. Bioinformatics 21, 3034–3042 (2005). BIOCHEMISTRY Funding Information: 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. Publisher Copyright: © 2019 National Academy of Sciences. All rights reserved.

PY - 2019/12/17

Y1 - 2019/12/17

N2 - 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.

AB - 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.

KW - Allelic neofunctionalization

KW - Diketopiperazine

KW - Nonribosomal peptide synthetase

KW - Pyrrolopyrazine

KW - Secondary metabolism

UR - http://www.scopus.com/inward/record.url?scp=85076670383&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85076670383&partnerID=8YFLogxK

U2 - 10.1073/pnas.1913080116

DO - 10.1073/pnas.1913080116

M3 - Article

C2 - 31801877

AN - SCOPUS:85076670383

VL - 116

SP - 25614

EP - 25623

IS - 51

ER -

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

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