Inactivation of the indole-diterpene biosynthetic gene cluster of Claviceps paspali by Agrobacterium-mediated gene replacement

László Kozák, Zoltán Szilágyi, Barbara Vágó, Annamária Kakuk, László Tóth, Istvan Molnar, István Pócsi

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

The hypocrealean fungus Claviceps paspali is a parasite of wild grasses. This fungus is widely utilized in the pharmaceutical industry for the manufacture of ergot alkaloids, but also produces tremorgenic and neurotoxic indole-diterpene (IDT) secondary metabolites such as paspalitrems A and B. IDTs cause significant losses in agriculture and represent health hazards that threaten food security. Conversely, IDTs may also be utilized as lead compounds for pharmaceutical drug discovery. Current protoplast-mediated transformation protocols of C. paspali are inadequate as they suffer from inefficiencies in protoplast regeneration, a low frequency of DNA integration, and a low mitotic stability of the nascent transformants. We adapted and optimized Agrobacterium tumefaciens-mediated transformation (ATMT) for C. paspali and validated this method with the straightforward creation of a mutant strain of this fungus featuring a targeted replacement of key genes in the putative IDT biosynthetic gene cluster. Complete abrogation of IDT production in isolates of the mutant strain proved the predicted involvement of the target genes in the biosynthesis of IDTs. The mutant isolates continued to produce ergot alkaloids undisturbed, indicating that equivalent mutants generated in industrial ergot producers may have a better safety profile as they are devoid of IDT-type mycotoxins. Meanwhile, ATMT optimized for Claviceps spp. may open the door for the facile genetic engineering of these industrially and ecologically important organisms.

Original languageEnglish (US)
Pages (from-to)3255-3266
Number of pages12
JournalApplied Microbiology and Biotechnology
Volume102
Issue number7
DOIs
StatePublished - Apr 1 2018

Fingerprint

Claviceps
Agrobacterium
Diterpenes
Multigene Family
Ergot Alkaloids
Agrobacterium tumefaciens
Fungi
Protoplasts
Genes
Genetic Engineering
Food Supply
Mycotoxins
Drug Industry
Drug Discovery
Poaceae
Agriculture
Regeneration
Parasites
Safety
indole

Keywords

  • Agrobacterium tumefaciens
  • Claviceps paspali
  • Ergot
  • Indole-diterpene
  • Paspaline
  • Paspalitrem

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

Cite this

Inactivation of the indole-diterpene biosynthetic gene cluster of Claviceps paspali by Agrobacterium-mediated gene replacement. / Kozák, László; Szilágyi, Zoltán; Vágó, Barbara; Kakuk, Annamária; Tóth, László; Molnar, Istvan; Pócsi, István.

In: Applied Microbiology and Biotechnology, Vol. 102, No. 7, 01.04.2018, p. 3255-3266.

Research output: Contribution to journalArticle

Kozák, László ; Szilágyi, Zoltán ; Vágó, Barbara ; Kakuk, Annamária ; Tóth, László ; Molnar, Istvan ; Pócsi, István. / Inactivation of the indole-diterpene biosynthetic gene cluster of Claviceps paspali by Agrobacterium-mediated gene replacement. In: Applied Microbiology and Biotechnology. 2018 ; Vol. 102, No. 7. pp. 3255-3266.
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