Molekularbiologische und biochemische Untersuchungenvon Prenyltransferasen und NRPS-Enzymen aus Ascomyceten

Die Produktion sekundärer Stoffwechselprodukte durch Mikroorganismen stellt ein beachtliches Reservoir an biologisch-aktiven Naturstoffen da, die auch pharmazeutische Bedeutung haben können. Darunter fallen Antibiotika, Zytostatika oder auch Immunsuppressiva. Diese sind im Gegensatz zu Metaboliten d...

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Bibliographic Details
Main Author: Wunsch, Carsten
Contributors: Li, Shu-Ming (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:German
Published: Philipps-Universität Marburg 2014
Pharmazeutische Biologie
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Table of Contents: The production of secondary metabolites by microorganisms represents a considerable reservoir of biologically active natural products, which can exhibit pharmaceutical importance like antibiotics, antitumor drugs or immunosuppressives. These are in contrast to metabolites of primary metabolism initially dispensable for growth and development, but at second glance essential for the management of numerous interactions of the individual with its environment. The filamentous fungi contribute to the diversity of natural products. The backbone structures of these substances are often based on the participation of nonribosomal peptide synthetases (NRPS), NRPS-like enzymes, Polyketide synthases (PKS), and hybrid enzymes of NRPS/PKS. Due to the influence of other tailoring enzymes like prenyltransferases the biodiversity and biological activity is significantly increased. For selective chemoenzymatic synthesis of new natural products a well-founded understanding of the reaction mechanisms, as well as the biochemical properties of these classes of enzymes are of crucial significance. With this work, five putative prenyltransferases from A. terreus should be characterized. For two representatives, CdpC7PT and EAU34068 this intension was successfully realized. CdpC7PT is responsible for the C7-prenylation of tryptophan-containing cyclic dipeptides and additionally functions as the first reported prenyltransferase of the DMATS superfamily which performs an O-prenylation in the presence of tyrosine-containing cyclic dipeptides. In the case of EAU34068 sequence identities of up to 42 % at the amino acid level were found between this enzyme and other characterized cyclic dipeptide prenyltransferases. This prompted us to test such compounds but without success. After extensive testing of different substrate classes, the flavanones eriodictyol and naringenin, the hydroxynaphthalenes 1-naphthol and 1,7-dihydroxynaphthalene, and the xanthone 1,3,6-trihydroxyxanthone could be confirmed as aromatic substrates for geranylation. EAU34068 exhibited a high regioselectivity of the prenylation for all of these substrates. Two additional putative prenyltransferase genes ATEG_02823 and ATEG_01730 are currently at the cloning vectors pCaW5 and pCaW17, respectively, and another putative prenyltransferase gene ATEG_06111 is already subcloned in the expression vector pCaW9 to obtain the desired recombinant protein (EAU33872). Another focus of this work was the characterization of the nonribosomal peptide synthetases EAA61517 and EAW25548, as well as the NRPS-like enzyme EAU36364. The corresponding genes AN9226, NFIA_043670 and ATEG_03090 from A. nidulans, N. fischeri and A. terreus, respectively, should be cloned in a suitable construct for expression in A. nidulans TN02A7. At least for AN9226 and for NFIA_043670, positive transformants were obtained. Screening for additional secondary metabolites were performed by comparing the transformants with the untransformed strain A. nidulans TN02A7. In particular, for AN9226 numerous strategies were carried out to gain an increased product formation, e.g. expression mediated by the constitutive gpdA promoter. However, until now no product formation could be identified, with one exception of the unexpected accumulation of the polyketide Sterigmatocystin. By combination of the NRPS FtmPS from N. fischeri, whose ortholog from A. fumigatus is known to produce cyclo-L-Trp-L-Pro, with different prenyltransferases of the DMATS superfamily in A. nidulans, the results of in vitro assays with recombinant proteins should be confirmed in vivo. Additionally, a suitable technique to produce desired prenylated derivatives by in vivo synthesis should be established. For this purpose the nonribosomal peptide synthetase gene NFIA_093690 was placed under the control of the constitutive gpdA promoter, trpC terminator and afterwards integrated in the appropriate vector pJW24 for transformation of A. nidulans TN02A7. After successful transformation of A. nidulans TN02A7 with this vector, an additional and distinctive product could be identified by analyzing secondary metabolite production in comparison to the untransformed strain. This product could be confirmed by structure elucidation as the favoured cyclo-L-Trp-L-Pro. By coexpression of the prenyltransferase genes in one of the transformants which already comprises the ftmPS gene prenylated derivatives should be produced. As representatives two C2 reverse prenyltransferases CdpC2PT and BrePT from N. fischeri and A. versicolor, as well as the reverse C3-prenyltransferases CdpC3PT and CdpNPT from N. fischeri and A. fumigatus were selected. The encoding genes have also been combined with the aforementioned regulatory elements and the pyroA selection marker for the transformation of the selected transformant A. nidulans CaW03 (ftmPS). By further work of Dr. Kathrin Mundt concerning the identification of additional products in comparision to the untransformed strain additional products were observed. After structure elucidation the products of CdpC2PT and BrePT were confirmed as reversely C2-prenylated cyclo-L-Trp-L-Pro. In the case of CdpNPT a syn-cis-configured reversely C3-prenylated derivative and for CdpC3PT both reversly C3- and C2-prenylated derivatives, as well as a regular prenylation at position N1 were obtained.