Biosynthetisches und toxisches Potential des Mutterkornpilzes: Wirkungsanalyse der Ergotalkaloide und "Genome Mining" zur Identifizierung weiterer potentieller toxischer Sekundärmetabolite
Lebensmittelchemie
Zusammenfassung der Projektergebnisse
Claviceps purpurea is a plant pathogenic fungus which is well known for the production of toxic ergot alkaloids. Besides this not much is known about the regulation of ergot alkaloid synthesis (EAs) and the production of other secondary metabolites (SM). In the first step expression levels of the key cluster genes were tested under various conditions. The results show that some secondary metabolite gene clusters are regulated differently in different C. purpurea strains, but sequence comparison of regulatory genes revealed no obvious explanation. Studies evaluating the influence of phosphate-dependent regulation, global regulators as well as histone modifications on the expression levels of EAS genes revealed that the regulation of the EAS gene cluster is rather complex and so far no clear schema could be established. Furthermore, genome mining was performed in order to identify so far uncharacterized SM gene clusters in C. purpurea. Two new secondary metabolite gene clusters, namely the genes around the nonribosomal peptide synthetase NRPS3 and polyketide synthase PKS4 were selected and characterized in detail. The nrps3 gene shows homology to key enzymes of clusters encoding epipolythiodiketopiperazine (ETP) toxins, characterized by a diketopiperazine ring with an internal disulfide-bridge. Unexpectedly, however, no molecules with the typical ETP structures could be detected. Instead, several intermediates with an unusual nitrogen-sulfur bond were identified. Further experiments showed that due to a dysfunctional cytochrome P450 oxidase (TcpC) the fungus is not able to produce these toxic metabolites. Only the overexpression of a cytochrome P450 oxidase from the sirodesmin ETP cluster from Leptosphaeria (sirC) revealed new compounds with the typical ETP structure which were termed clapurines. This clearly illustrates the key role of the cytochrome P450 monooxygenase on the biosynthesis of ETPs in C. purpurea. The cluster around PKS4 turned out to be the ergochrome gene cluster responsible for the typical pigment formation of C. purpurea. Overexpression of the cluster-specific transcription factor led to activation of the gene cluster and to the production of several known ergot pigments. Knock out of the cluster key enzyme, a nonreducing polyketide synthase, clearly showed that this cluster is responsible for the production of red anthraquinones as well as yellow ergochromes. Pigment production was induced by high phosphate and low sucrose concentrations. This is the first functional analysis of a secondary metabolite gene cluster in the ergot fungus besides that for the classical ergot alkaloids.
Projektbezogene Publikationen (Auswahl)
- (2013) Plant-Symbiotic Fungi as Chemical Engineers: Multi-Genome Analysis of the Clavicipitaceae Reveals Dynamics of Alkaloid Loci. PLOS Genetics 9:2 ,e1003323
Schardl C, Young C, Hesse U, Amyotte SG, Andreeva K, Calie PJ, Fleetwood DJ, Haws DC, Moore N, Oeser B, Panaccione DG , Schweri KK, Voisey CR, Farman ML, Jaromczyk JW, Roe BA, O’Sullivan DM, Scott B, Tudzynski P et al.
(Siehe online unter https://doi.org/10.1371/journal.pgen.1003323) - (2014) Ergot alkaloids. In: Biosynthesis and molecular genetics of fungal secondary metabolites (Martin J-F, Garcia Estrada C, Zeilinger S edts) pp 303-316. Springer New York
Tudzynski P, Neubauer L
(Siehe online unter https://doi.org/10.1007/978-1-4939-1191-2_14) - (2014) Ergot alkaloids: Biosynthetic pathways. Toxins 6: 3281-3295
Gerhards N, Neubauer L, Tudzynski P, Li SM
(Siehe online unter https://doi.org/10.3390/toxins6123281) - (2016) Identification and charcterization of the ergochrome gene cluster in the plant pathogenic fungus C. purpurea. Fungal Biology and Biotechnology 3:2
Neubauer L, Dopstadt J, Humpf H-U, Tudzynski P
(Siehe online unter https://doi.org/10.1186/s40694-016-0020-z) - (2016) The epipolythiodiketopiperazine gene cluster in Claviceps purpurea: dysfunctional Cytochrome P450 enzyme prevents formation of the previously unknown clapurines. PLoS One 11:7, e0158945
Dopstadt J, Neubauer L, Tudzynski P, Humpf H-U
(Siehe online unter https://doi.org/10.1371/journal.pone.0158945)
