Produktion und Identifikation phyto- und cytotoxischer Metabolite des opportunistisch humanpathogenen Pilzes Aspergillus terreus

This work aimed at the identification of specific signals and signaling pathways inducing secondary metabolite production in Aspergillus terreus. The question is addressed how secondary metabolites can support competitive fitness of the fungus in its natural habitats, the rhizosphere and decaying organic matter. Mining of the A. terreus genome revealed an orphan polyketide-nonribosomal-peptide synthetase gene cluster. Activation of the gene cluster by expression of a neighbouring transcriptional activator required a combined approach of systematic investigation of physiologically inducing conditions followed by an adapted promoter exchange. This led to the identification of the phytotoxic products (dihydro-)isoflavipucine. Phytotoxin formation relied on the presence of asparagine, was stimulated at alkaline pH, but strictly repressed in the presence of glucose. Therefore, global catabolite repression may overrule gene cluster specific transcription factors, which should be kept in mind when performing genome mining experiments. Aspergilli generally utilise a naphthopyrone synthase for conidial pigment production. This pigment prevents phagolysosome acidification and enhances escape from macrophages by germination. A. terreus lacks this conserved PKS. Therefore, phagolysosomes acidify and prevent fungal escape. However, A. terreus conidia persist in phagolysosomes and may use immune cells as vehicle for dissemination. The different origin of the conidial pigment was confirmed by recombinant expression of the Aspergillus nidulans wA gene in A. terreus, which resulted in naphthopyrone production and enhanced macrophage escape rates. The search for a naphthopyrone synthase-like PKS in A. terreus serendipitously led to the identification of the terrein gene cluster. In agreement to its expression during growth on fruits, phytotoxic activities such as induction of fruit surface lesions and inhibition of seed germination were observed. While nitrogen starvation is a major inducer of terrein production on fruits, methionine - a trigger of plant defence mechanisms – also activated the cluster. Both signals are mediated by the transcriptional activators AtfA and AreA. Unexpectedly, iron starvation also induced the cluster via the iron regulator HapX and coincides with the ability of terrein to reduce ferric to ferrous iron increasing the iron availability for A. terreus. All inducing signals mimic conditions in the rhizosphere and combined with its broad spectrum of biological activities, terrein provides an excellent example for adaptation of A. terreus to environmental competition in the soil. Finally, the exceptionally strong promoter activity of the terrein synthase gene led to the development of an inducible heterologous expression system that based on regulatory elements of the cluster. Expression of PKS genes and domain swapping experiments confirmed the suitability of the expression system especially for high-level metabolite production in heterologous hosts