Abstract
Claviceps purpurea is an important food contaminant and well known for the production of the toxic ergot alkaloids. Apart from that, little is known about its secondary metabolism and not all toxic substances going along with the food contamination with Claviceps are known yet. We explored the metabolite profile of a gene cluster in C. purpurea with a high homology to gene clusters, which are responsible for the formation of epipolythiodiketopiperazine (ETP) toxins in other fungi. By overexpressing the transcription factor, we were able to activate the cluster in the standard C. purpurea strain 20.1. Although all necessary genes for the formation of the characteristic disulfide bridge were expressed in the overexpression mutants, the fungus did not produce any ETPs. Isolation of pathway intermediates showed that the common biosynthetic pathway stops after the first steps. Our results demonstrate that hydroxylation of the diketopiperazine backbone is the critical step during the ETP biosynthesis. Due to a dysfunctional enzyme, the fungus is not able to produce toxic ETPs. Instead, the pathway end-products are new unusual metabolites with a unique nitrogen-sulfur bond. By heterologous expression of the Leptosphaeria maculans cytochrome P450 encoding gene sirC, we were able to identify the end-products of the ETP cluster in C. purpurea. The thioclapurines are so far unknown ETPs, which might contribute to the toxicity of other C. purpurea strains with a potentially intact ETP cluster.
Highlights
The biotrophic plant pathogen Claviceps purpurea infects a broad range of grasses including economically important cereal crop plants [1]
By heterologous expression of a L. maculans sirodesmin cluster gene in C. purpurea and detailed nuclear magnetic resonance (NMR) as well as high resolution mass spectrometric (HRMS) studies, we identified so far unknown ETPs as end-products of the C. purpurea ETP cluster
Bioinformatic analysis revealed the presence of a C. purpurea nonribosomal peptide synthetases (NRPSs)-encoding gene (CPUR_02680) with significant sequence similarity to the ETP-toxin producing NRPSs in A. fumigatus and L. maculans [5]
Summary
The biotrophic plant pathogen Claviceps purpurea infects a broad range of grasses including economically important cereal crop plants [1]. In the sclerotia, which are the overwintering structure of the fungus, C. purpurea produces the toxic ergot alkaloids. In the Middle Ages, the PLOS ONE | DOI:10.1371/journal.pone.0158945. The Epipolythiodiketopiperazine Gene Cluster in Claviceps purpurea consumption of rye products contaminated with C. purpurea sclerotia led to the so-called St. Anthony’s Fire epidemics and in the 20th century this risk was still present [2]. Biochemistry and genetics of the ergot alkaloids biosynthesis have been well studied in C. purpurea [3], but apart from that, little is known about other secondary metabolites contributing to the toxicity of the ergot sclerotia
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