Chemische, molekularbiologische und biochemische Untersuchungen zur Biosynthese von Mykotoxinen aus Ascomyceten

To understand the biological diversity of natural products produced by different microorganism, it is very important to elucidate the biosynthesis of these products. In most cases, the biosynthetic genes are located as a cluster on the genome and can be identified by bioinformatic approaches. Based on the structure feature of natural products, it is possible to find the biosynthetic gene cluster in the genome sequence. After successful identification of a cluster in the genome sequence, it is possible to modify the original biosynthesis by chemoenzymatic synthesis or by genetic manipulations. For that purpose, FtmPT1 in the biosynthesis of verruculogen was overproduced in E. coli M15 cells and purified with Ni-NTA-Agarose. In addition, the four stereoisomers of cyclo-Trp-Pro and cyclo-Trp-Ala were synthesised by chemical synthesis. These and further six cyclic dipeptides were subsequently incubated with the overproduced prenyltransferase FtmPT1, resulting in the formation of regularly C2-prenylated derivatives. These chemoenzymatically produced diketopierazines were tested in cooperation with Prof. Kassack from Düsseldorf for their biological activities with their non-prenylated precursors as controls. The results showed that the cytotoxic effects of the prenylated diketopierazines were significant higher than those of the non-prenylated precursors. This seems that the prenyl moiety, rather than the prolin moiety is essential for the biological activity. In addition, all tested stereoisomers of cyclo-Trp-Pro and cyclo-Trp-Ala showed comparable cytotoxic activities towards the tested cell lines. So the stereochemistry at C-11 and C-14 and the corresponding substituents have no significant influence on the cytotoxicity. During the isolation of the C2-prenylated diketopiperazines, detailed analysis of the incubation mixtures of FtmPT1 revealed the presence of additional product peaks in the HPLC chromatograms. Seven regularly C3-prenylated hexahydropyrrolo[2,3-β] indoles and two regularly N1-prenylated diketopiperazines were isolated and identified by HR-ESI-MS and NMR analysis including HMBC, HSQC and NOESY experiments. L-tryptophan-containing cyclic dipeptides were converted to C3β-prenylated diketopiperazines and D-tryptophan containing cyclic dipeptides to C3α-prenylated diketopiperazines, so that in both cases a syn-cis configuration was generated. Based on the given crystal structure of FtmPT1, a putative reaction mechanism was proposed. The independent product formation was confirmed by time dependency assays and elucidation of KM-values. In addition to the chemoenzymatic production of tryprostatin B analoga the natural compound fumigaclavin A was isolated from Penicillium commune NRRL2033. The structure and stereochemistry of (8R,9S)-fumigaclavin A was confirmed by ESI-MS and NMR analysis including HMBC, HSQC and NOESY experiments. Isolation of fumitremorgin A from N. fischeri NRRL181 provided the evidence for its role as end product of the biosynthesis of fumitremorgins in this fungus. Furthermore nine natural products could be isolated from N. fischeri NRRL181 and identified via HR-ESI-MS and NMR analysis. First of all, the known products aszonalenin, acetylaszonalenin and the precursors of fumitremorgin A: 12,13-dihydroxyfumitremrogin C, fumitremorgin B and verruculogen. Four additional substances of the fumitremorgin-type with unknown biosynthetic pathways could be isolated from N. fischeri NRRL181. Via HR-ES-MS and NMR analysis, these compounds could be identified as verruculogen TR-2, spirotryprostatin A, 6-methoxyspirotryprostain B and compound 15. Spirotryprostatin A and 6-methoxyspirotryprostain B were first isolated from N. fischeri NRRL181 in this study and compound 15 has not been reported previously. The prenyltransferase gene ftmPT3 is not located in the known verruculogen-cluster at chromosome 8, but at a different chromosome. It was therefore speculated that the genes around ftmPT3 could be responsible for the biosynthesis of verruculogen TR-2, spirotryprostatin A, 6-methoxyspirotryprostain B or compound 15. Three genes were therefore amplified by PCR from gDNA or cDNA and were cloned into expression vectors (pQE60, pQE70, pHis8, pYES2/NT C). After heterologous gene expression in E. coli or S. cerevisiae and purification of the recombinant proteins, enzyme assays with different substrates and cofactors were carried out; unfortunately, no additional product peak could be detected in the HPLC chromatograms of the enzyme assays. Transformation of the prenyltransferase gene brePT into A. nidulans TN02A7 containing the NRPS gene ftmPS, was used as a strategy for in vivo production of deoxybrevianamid E. Unfortunately, no expected product could be detected with HPLC.