Molekularbiologische Untersuchungen von Nicht-ribosomalen Peptidsynthetase-ähnlichen Enzymen aus Ascomyceten

Zusammenfassung Mikroorganismen haben im Laufe ihrer Evolution zur Anpassung an ihren Lebensraum eine Vielfalt an strukturell diversen und zum Teil sehr komplexen Metaboliten entwickelt. Diese verschaffen ihnen Vorteile gegenüber anderen Bewohnern ihrer Umwelt. Die Nutzung dieser Substanzen führte...

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Bibliographic Details
Main Author: Hühner, Elisabeth
Contributors: Li, Shu-Ming (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2020
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Summary In the course of their evolution, microorganisms have developed a variety of structurally diverse and sometimes very complex metabolites to adapt to their habitat. These give them advantages over other inhabitants of their environment. The use of these substances led to the development of antibiotics, immunosuppressants and antitumor agents. The backbone structures of these substances are often synthesized by the enzyme classes of polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), NRPS / PKS hybrids and NRPS-like enzymes. The resulting structures can be further modified by other tailoring enzymes such as methyltransferases or prenyltransferases, which often increases the biological activity. A detailed knowledge of the biosynthesis and of the involved enzymes is a prerequisite for the fermentative, semisynthetic or chemoenzymatic production of these natural substances and their derivatives. Although the individual domains of NRPS and PKS have biochemically and structurally been investigated, understanding of the interaction of the domain and the mechanisms involved is still very poor. Since simple systems like the NRPS-like enzymes can be investigated more easily, the NRPS-like enzymes with an A-T-TE architecture from Aspergillus terreus (A. terreus) and another from Chaetomium globosum (C. globosum) will be characterized in the present work. First, the NRPS-like genes apvA, melA, atrAAt, pgnA from A. terreus with their own promoter- and terminator-sequence should be analysed by heterologous expression in Aspergillus nidulans. Although an integration for melA and atrAAt into the genome was observed, no product could be detected in the ethyl acetate extracts of the culture. For the expression of the NRPS-like genes in Saccharomyces cerevisiae (S. cerevisiae), cDNA or gDNA was used for the amplification of the genes. This made it possible to revise the exon-intron structures for apvA, melA, btyA and atrAAt. Furthermore, the domain architecture of the putative NRPS-like gene atrAAt, which according to the NCBI database only consisted of one A and one T domains, could be corrected by amplifying cDNA to an A-T-TE structure. The NRPS-like genes have a size of 2763-2874 bp and accordingly code for proteins of 920-958 aa. Expression of the seven NRPS-like genes in S. cerevisiae led to the five products aspuvinone E, butyrolactone IIa, atromentine, phenguignardic acid and didemethylasterrichinone D. The products were extracted and the structures were determined by means of mass spectrometry and NRM spectroscopy. For aspulvinone E and phenguignardic acid, a production of 13 and 15 mg/l and for butyrolactone IIa even up to 35 mg/l could be calculated on one liter of culture. In subsequent attempts to isolate the NRPS-like proteins, it turned out that affinity tags at the C-terminus strongly inhibit enzyme activity. In contrast, an N-terminal His6 tag had no negative influence on the activity. On the basis of the protein detection by Western blot, the optimal point in time for isolation was determined to be around 16 hours after the induction of protein expression by galactose. The recombinant proteins could be purified with a concentration of about 1 ml/l culture. However, no catalytic activity could be detected. To test an alternative expression system, the NRPS-like genes and the phosphpantethenyltransferase gene npgA were cloned into the vectors pQE60 and pET28a (+) for the expression in Escherichia. Due to time limits the gene expression and protein isolation could no longer be carried out. The A-T-TE domains of the NRPS-like enzymes theoretically form autonomous units that can be exchanged between the enzymes. In order to test this theory and in addition to generate unnatural products, the substrate-activating A domains were combined with the TE domains, which are responsible for the dimerisation. For this purpose, the A domains of Pgna, ApvA, AstA and AtqA for activation of phenyl-, 4-hydroxy or indole pyruvate, were recombined with the TE domain of AvpA, BtyA, PgnA, AtrAAt and AstA, respectively. The T domain was taken over from one of the original enzymes. A total of 34 constructs were prepared in this way which were used for expression in S. cerevisiae. 22 of the expected products could be detected. The structure was elucidated on the basis of the molecular ions and their fragmentation as well as NMR spectroscopy. In this way, indole butyrolactone and indole guignaric acid could be obtained, which had never before been described in the literature. It is also known that the products of the NRPS-like enzymes often only form the basic structure for secondary metabolites. Therefore, the genetic environment of the NRPS-like genes was investigated. 4 prenyltransferase genes were identified which were selected for co-expression with the NRPS-like ones in S. cerevisiae. Unfortunately, the prenylated product could not be detected in the baker's yeast transformants.