Molekularbiologische und biochemische Untersuchungen zur Biosynthese von Mykotoxinen aus Ascomyceten

Fungi are rich sources of secondary metabolites. Among these secondary metabolites, the ergot alkaloids are both pharmaceutically useful and toxic. Ergot alkaloids are indole derivatives produced by fungi of the genera Claviceps, Aspergillus and Penicillium with Claviceps purpurea (C. purpurea) as the most important producer for medical use. C. purpurea produces ergotamine and ergocryptine as main ergot alkaloids. The clavine-type alkaloids, e.g. festuclavine, pyroclavine and fumigaclavines A, B and C are mainly produced by the fungi Aspergillus fumigatus (A. fumigatus) and Penicillium commune (P. commune). Chanoclavine-I-aldehyde is a branch point of ergot alkaloid biosynthesis for ergotamine in C. purpurea and fumigaclavines C and A, respectively in A. fumigatus and P. commune. Ergotamine is characterized by a peptide moiety which is absent in the fumigaclavines. Instead, the fumigaclavines contain additional substituents, e.g. alcohol or acetoxy group at C-9 and a reverse prenyl moiety at C-2. Furthermore, the C-8 stereochemistry of fumigaclavine C from A. fumigatus and fumigaclavine A from P. commune differs. In this study, the presence of genes for ergot alkaloid biosynthesis in ascomycetous fungi was investigated. For this purpose, 138 available genomes of 53 fungi families of the NCBI database were screened with the sequences of the seven orthologous genes from C. purpurea and A. fumigatus. 23 ascomycetous fungi belonging to the families Trichocomaceae, Clavicipitaceae and Arthrodermataceae were identified to contain putative ergot alkaloid gene clusters. The family Clavicipitaceae is assigned to the class of Sordariomycetes, whereas the families Trichocomaceae and Arthrodermataceae are members of the class of Eurotiomycetes. The two identified fungi of the genus Metarhizium belong to the family Clavicipitaceae. Until now, no ergot alkaloids have been isolated from fungi neither of the genus Metarhizium nor of fungi of the family Arthrodermataceae. To prove functions, the gene fgaOx1 from A. fumigatus and its orthologue easE from C. purpurea were cloned into expression vectors for Escherichia coli (E. coli). In case of fgaOx1 Saccharomyces cerevisiae (S. cerevisiae) was also tested as expression strain. Unfortunately, no overexpression was detectable under different conditions. Additional protein sequence analysis indicated that FgaOx1 contains two membrane domains. Furthermore, analysis of genes of ergot alkaloid biosynthesis in ascomycetous fungi revealed that the intron-exon structure for easE from C. purpurea is incorrect in the NCBI database. The investigations of key enzymes from C. purpurea, A. fumigatus and P. commune in this study provided new insights in the differentiation of ergot alkaloid biosynthesis. The expression of the genes easA and easG from C. purpurea and the purification and biochemical characterization of the recombinant proteins showed that EasG alone catalyses, via a non-enzymatic adduct with reduced glutathione, the conversion of chanoclavine-I aldehyde to agroclavine. The overproduced EasA seems inactive and may lose its function during evolution. A reaction mechanism was proposed for the conversion of chanoclavine-I aldehyde to agroclavine, whereas one isomerisation and one reduction step was necessary. It was demonstrated that EasG was responsible for the reduction step and a non-enzymatic adduct with reduced glutathione (GSH) for the isomerisation. NADPH was required as cofactor for the EasG reaction. The structure of agroclavine was unequivocally elucidated by NMR and MS analyses. By using a combination of FgaOx3 from A. fumigatus and EasG from C. purpurea it was demonstrated that both enzymes catalyse the reaction from chanoclavine-I aldehyde to festuclavine and pyroclavine. It had been shown previously, that FgaOx3 and FgaFS from A. fumigatus together produce only festuclavine. For detailed investigations on the conversion of chanoclavine-I aldehyde to festuclavine and pyroclavine the orthologues of fgaOx3 and fgaFS from P. commune NRRL2033, fgaOx3pc and fgaFSpc, were amplified and expressed. The combinations of all orthologues from C. purpurea, A. fumigatus and P. commune revealed that in A. fumigatus and P. commune, in contrast to the reaction mechanism in C. purpurea, two reduction steps are required for the conversion of chanoclavine-I aldehyde to festuclavine and pyroclavine. It was shown that FgaOx3 or FgaOx3pc catalysed the first reduction step and the imine reductases FgaFS, FgaFSpc or EasG the second. More importantly, the ratio of pyroclavine to festuclavine was controlled by the reduction of an imine intermediate catalysed by FgaFS, FgaFSpc or EasG, and not by the reduction step catalysed by the old yellow enzymes FgaOx3 or FgaOx3pc.