Molekularbiologische und biochemische Untersuchungen von nichtribosomalen Peptidsynthetasen und Prenyltransferasen in der Biosynthese von Sekundärmetaboliten aus Ascomyceten
Im Laufe der Evolution, haben Mikroorganismen die Fähigkeit entwickelt, eine Vielzahl an strukturell vielseitigen und komplexen Substanzen zu bilden. Unter bestimmten Umweltbedingungen werden Biosynthesewege von komplexen Metaboliten induziert, die wiederum oft eine toxische Wirkung gegenüber den ko...
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- In the course of evolution, microorganisms have developed the ability for production of a multiplicity of structural diverse and complex compounds. Under given environmental conditions, biosynthetic pathways are induced to synthesize complex metabolites that are often toxic toward their competing organisms. Especially production of secondary metabolites of filamentous fungi is one of the most important source of biological active substances, which can be used for the benefit of human health. Notably, the enzyme classes of polyketide synthases (PKS), nonribosomal peptide synthetases (NRPS) and NRPS/PKS-hybrids are responsible for the production of novel natural products that exhibit considerable therapeutic potential, e.g. antibiotics, antifungal and immunospuppressive drugs. These natural products can be further modified by other enzymes e.g. prenyltransferases, consequently leading to a huge structural diversity. For discovery and development of new drugs, understanding of reaction mechanism of enzymes in nature is mandatory and especially biochemical investigations become more important. In this thesis, functions of two unknown prenyltransferases (FtmPT3, CdpC2PT) of Neosartorya fischeri (N. fischeri) were biochemically identified and characterised. The gene ftmPT3, encodes a prenyltransferase, which catalyses in the presence of DMAPP the prenylation of verruculogen at OH-13 and therefore acts as the long-time missing O-prenyltransferase in the fumitremorgin A biosynthesis. Intensive sequence analyses of ftmPT3 and its adjacent neighbour genes revealed, in comparison to Aspergillus fumigatus (A. fumigatus), an additional locus with a size of 9.6 kb and seven putative genes. The protein CdpC2PT, shows sequence identities of 40 % with the C2-prenyltransferase BrePT from Aspergillus versicolor and 42 % with NotF from Aspergillus sp. on the amino acid level. Investigations of reaction mixtures of CdpC2PT with DMAPP, showed a broad substrate specifity towards cyclic dipetides with preference for (S)-benzodiazepindione and cyclo-L-Trp-L-Trp. NMR and MS analyses of the enzymatic products revealed unequivocally that CdpC2PT acts as a reverse C2-prenyltransferase and catalyses the mono- and diprenylation of cyclo-L-Trp-L-Trp. Literature search showed the existence of mono- and diprenylated derivatives of cyclo-L-Trp-L-Trp in Penicillium fellutanum (P. fellutanum), socalled fellutanines. Therefore, it can be speculated that CdpC2PT could be involved in the biosynthesis of fellutanines in N. fischeri. Sequence analyses of the neighbour genes revealed the existence of a putative amino oxidase EAW25547, which could be contribute to the biosynthesis of fellutanines. The coding gene NFIA_043660 was successfully amplified from genomic DNA (gDNA) of N. fischeri and subsequently cloned into both expression vectors pQE60 and pHIS8. After incubation under different expression conditions no protein overproduction could be shown. Furthermore, the gene NFIA_062330, encoding for the last unkonwon putative prenyltransferase of N. fischeri, was biochemically investigated. Sequence analyses revealed an orthologous gene ACLA_042210 in Aspergillus clavatus (A. clavatus), with a sequence identity of 62 % on the amino acid level. Both genes NFIA_062330 and ACLA_042210 were amplified from gDNA and cloned into the expression vector pQE60. Expression of the genes was carried out successfully in the optimized overexpression E. coli strain M15 [pREP4]. Unfortunately no activity could be detected after testing a number of different substrates. Another project focused on the function of two homologous NRPS genes Pc21g15480 of Penicillium chrysogenum (P. chrysogenum) and NFIA_074300 of N. fischeri. Both genes were amplified from gDNA and successfully cloned into the expression vector pJW24. After protoplastation and transformation of the wildtype Aspergillus nidulans (A. nidulans) TN02A7 with the expression constructs, integration of genes was proven by PCR screening. After extraction of Pc21g15480 transformants, cyclo-L-Trp-L-His was detected by HPLC and NMR analyses. Analysis of the NFIA_074300 transformants revealed, that one transformant produced an additional product peak in comparison to the wildtype. Surprisingly this substance was identified by NMR and MS analyses as pseurotin A, a product of a NRPS/PKS-hybrid and was not the expected substance. In addition, production of secondary metabolites by targeted co-expression of prenyltransferases genes with a NRPS gene was carried out in the dissertation. The genes NFIA_043650 and NFIA_074280, encoding for the prenyltransferases CdpC2PT and CdpC3PT, respectively, were primarily amplified from gDNA and afterwards cloned into the expression construct pCaW34. The expression constructs were transferred into transformant A. nidulans CaW03 (ftmPS). PCR screening of the transformants confirmed the ectopic integration of the prenyltransferase genes into the genome. HPLC analysis revealed clearly product accumulation in the resulted transformants and showed the successful strategy for production of biologically active substances by synthetic biology. Structure elucidation of the isolated products was carried out by NMR and MS analyses.