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. 2016 Jul 8;11(7):e0158945.
doi: 10.1371/journal.pone.0158945. eCollection 2016.

The Epipolythiodiketopiperazine Gene Cluster in Claviceps Purpurea: Dysfunctional Cytochrome P450 Enzyme Prevents Formation of the Previously Unknown Clapurines

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The Epipolythiodiketopiperazine Gene Cluster in Claviceps Purpurea: Dysfunctional Cytochrome P450 Enzyme Prevents Formation of the Previously Unknown Clapurines

Julian Dopstadt et al. PLoS One. .
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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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Organization of the different ETP biosynthesis gene clusters and structure of gliotoxin and sirodesmin.
(A) Shown is the ETP gene cluster in C. purpurea in comparison to the gliotoxin and sirodesmin producing gene clusters from A. fumigatus and L. maculans. Orientation of the arrows indicates the direction of transcription. Genes in black are common ETP moiety genes present in all three clusters. For gene designations see Table 1. (B) Structure of gliotoxin and sirodesmin PL with the bolded characteristic diketopiperazine moiety with an internal disulfide bridge.
Fig 2
Fig 2. Gene expression and co-regulation of the ETP cluster genes.
(A) In planta gene expression of tcpG. Shown are three independent experiments in the C. purpurea wild-type strain in planta 10, 15 and 20 days post-infection (dpi) and in axenic culture. Expression levels were normalized against the housekeeping genes encoding β-tubulin, γ-actin, and glyceraldehyde-3-phosphate dehydrogenase. (B) Co-regulation of the ETP cluster genes. The wild-type as well as three independent OE::tcpZ mutants were grown for seven days in liquid Mantle media and northern blot analysis was performed as described in methods.
Fig 3
Fig 3
Comparison of the HPLC-MS metabolite profile (total ion chromatograms) between the wild type of Claviceps purpurea strain 20.1 (A), OE::tcpZ (B) and OE::tcpZ/OE::tcpN (C). (D) Extracted ion signals of compounds 1a/b, 2a/b and 3 of OE::tcpZ.
Fig 4
Fig 4. Newly identified secondary metabolites as products of an ETP gene cluster in Claviceps purpurea.
(A) Compounds 13 were produced by OE::tcpZ strain in planta and in axenic culture. (B) Selected 2D- NMR data for 2. Red arrows indicate the specific couplings of H-7a/b and the blue arrows those of S-CH3. (C) HRMSn fragmentation of 1a as [M+H]+ illustrating the rapid cleavage of the thiomethyl group and dimethylallyl group.
Fig 5
Fig 5. Identified structures of the OE::tcpZ/OE::tcpN strain.
(A) 4-8 occur in axenic culture. (B) HRMSn analysis of 4a as [M+H]+ clarifies the characteristic fragmentation and underlines the differences concerning the N-4 substitution. This characteristic fragmentation allows structure elucidation of other unknown intermediates based on HRMSn.
Fig 6
Fig 6. Phylogenetic analysis of TcpC and its homologs from other fungal ETP clusters.
Amino acid sequences were obtained from GenBank database (GliC EDP49542.1, GliF AAW03300.1, SirE AAS92549.1, SirB XP_003842422.1, SirC AAS92547.1, ataF XM_001212649.1 ataTC XM_001212652.1). Sequences were aligned with MUSCLE (v3.8.31) and the phylogenetic tree was constructed with the maximum likelihood method using Phylogeny.fr [83]. Branch support values are indicated.
Fig 7
Fig 7. The characteristic MS/MS spectra of the identified clapurines as end-products of the ETP gene cluster in C. purpurea.
(A) The clapurines are so far unknown ETPs and were characterized in the OE::tcpZ/OE::sirC strain. The HRMSn experiments show a neutral loss of the sulfur groups and the common cleavage of the dimethylallyl group for dithioclapurine (B), trithioclapurine (C) and tetrathioclapurine (D) as [M+H]+.
Fig 8
Fig 8. Proposed biochemical pathway for the formation of diketopiperazine metabolites 1-8 and the clapurines.
Compounds 1215 are hypothesized intermediates.
Fig 9
Fig 9. Tentative biochemical pathway for the diastereomers 1a/b and 2a/b.
Compounds 16 and 18 are hypothesized intermediates.

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Grant support

This research was funded by Deutsche Forschungsgemeinschaft grants 50/18 1 (to PT) and 730/11-1 (to HUH).
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