Molekularbiologische Untersuchungen zu nichtribosomalen Peptidsynthetasen, Polyketidsynthasen und Prenyltransferasen aus Ascomyceten
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Philipps-Universität Marburg
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The connection between humans and fungi has been documented for a long time. Human recognize that fungi have helpful compunds and uses, but they can also cause a threat for humans. In the last years the number of genomes of fungi from biological nischens and biodiverse hotspots has been increased. This leads to the discovery of new biosynthetic gene clusters, that could produce previously unknown secondary metabolites. These new secondary metabolites harbor the chance of being new pharmacologically active substances or strucures. The most important enzymes, that catalyse the main strucure of secondary metabolites, are polyketide synthases (PKS), nonribosomal peptidesynthetases (NRPS) and terpene synthases. These enzymes are able to create complex structures out of simple universally available small molecules. PKS and terpene synthetases use acyl-CoA as a substrate, while NRPS usally use amino acids as their substrates. After the synthesis of the main structure, it can be further modified. For the further modification a cytochrome P450 enzyme or a prenyltransferases can accept the main structures as their substrates.
In the first part of this work, the function of the NRPS Pc21g15480 from Penicillium chrysogenum Wisconsin 54-1255 and the NRPS NFIA_074300 from Neosartorya fischeri NRRL 181 should be investigated. Both NRPS are part of gene clusters that produce Roquefortin C.
In the gene cluster of Pc21g15480 further genes for the production of meleagrin are present. The first step of roquefortin C biosynthesis is the building of cyclo-L-Trp-L-His by a NRPS. During the expression of Pc21g15480 and NFIA_074300 performed by Dr. Kathrin Mundt the production of cyclo-L-Trp-L-His could be verified. Unexpectedly cyclo-L-Trp-L-Pro was also detected. This should be verified by the expression of the NRPS Pc21g15480 and NFIA_074300 in S. cerevisiae and A. nidulans in this thesis. First there was a need for new yeast strains that are adapted to the expression of NRPS in S. cerevisiae and the needs of the laboratory. They are also able to express or coexpress prenyltransferases. After the production of suitable yeast strains and expression constructs, both NRPSs were expressed in S. cerevisiae. Eventhough the conditions oft he expression were optimized, only a low intensity of the mass of both products could be observed. However, these results could not be reliably reproduced. So the NRPS Pc21g15480 was to be expressed in A. nidulans further. The NRPS Pc21g15480 was successfully integrated into the genome of A. nidulans LO8030. At first both products could be detected, but the results could not be reproduced. Even a new integration of the NRPS Pc21g15480 in A. nidulans LO8030 yielded no production of cyclo-L-Trp-L-His or cyclo-L-Trp-L-Pro.
In the second part the function of two cryptic biosynthetic gene clusters from P. crustosum PRB-2 should be identified. Both gene clusters contain a PKS with a KS-AT-DH-cMeT-ER-KR-ACP domain structure, they are annotated as Pcr5819_g and Pcr11425_g. First the PKS5819_g should be heterologously expressed in A. nidulans. After the cloning of an expression construct, it was integrated into the genome of A. nidulans LO8030. During the analysis of the A. nidulans LO8030 transformants, in whose genome Pcr5819_g was integrated, no product could be identified. Secondly, both gene clusters should be specificly activated by the integration oft he constitutive promotor gdpA in front of the hypothetical transcriptionfactors Pcr5819_g and Pcr11422_g. Until now constructs for the activation have been produced. Unfortunately the integration of these constructs in P. crustosum JZ02 has not been successful yet.
In the third part, the function of the prenyltransferase HK57_00371 from Aspergillus ustus 3.3904 should be identified. Due to a genomic analysis HK57_00371 could be responsible for the production of phenylahistin. To confirm this, a construct for the expression of HK57_00371 in E. coli was produced. Until now only a small amount of HK57_00371 could be isolated from E. coli culture. In an enzyme assay no activity against the proposed substrate cyclo-L-Phe-L-His could be observed.
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Created: 2025Issued: 2025-06-23Updated: 2025-06-23
Faculty
Fachbereich Pharmazie
Publisher
Philipps-Universität Marburg
Language
ger
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DoctoralThesis
DFG-subjects
natural productsNRPSbiosynthesisNaturstoffecyclic dipeptidesSekundärmetaboliteRoquefortin CPKSBiosynthesepolyketidesPT
DDC-Numbers
615
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Öqvist, Malin Kristin: Molekularbiologische Untersuchungen zu nichtribosomalen Peptidsynthetasen, Polyketidsynthasen und Prenyltransferasen aus Ascomyceten. : Philipps-Universität Marburg 2025-06-23. DOI: https://doi.org/10.17192/z2025.0240.