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PDF-Volltext Molekularbiologische und biochemische Untersuchungen zur Biosynthese von Ergotalkaloiden in Pilzen der Familien Trichocomaceae und Arthrodermataceae Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Pharmazie der Philipps-Universität Marburg vorgelegt von Christiane Wallwey aus Dortmund Marburg/Lahn, 2012 Vom Fachbereich Pharmazie der Philipps-Universität Marburg als Dissertation angenommen am 25. Mai 2012. Erstgutachter: Prof. Dr. Shu-Ming Li Zweitgutachter: Prof. Dr. Michael Keusgen Tag der mündlichen Prüfung: 29. Mai 2012 INHALTSVERZEICHNIS I Publikationen und Präsentationen ............................................................................. IV Abkürzungen ............................................................................................................. VI Zusammenfassung ...................................................................................................... 1 Summary ..................................................................................................................... 4 1 Einleitung .............................................................................................................. 5 1.1 Die Geschichte der Ergotalkaloide ................................................................ 5 1.2 Einteilung der Ergotalkaloide und ihre pharmazeutische Wirkung ................. 6 1.2.1 Alkaloide vom Clavin-Typ ............................................................................ 7 1.2.2 D-Lysergsäureamide: Ergoamide ................................................................ 9 1.2.3 D-Lysergsäurepeptide: Ergopeptine ............................................................ 9 1.2.4 Substanzen mit ergotalkaloidähnlichen Strukturen ................................... 11 1.3 Produzenten von Ergotalkaloiden ................................................................ 12 1.3.1 Produzenten von Alkaloiden des Clavin-Typs ........................................... 12 1.3.2 Produzenten von Ergoamiden und Ergopeptinen ...................................... 13 1.4 Gencluster für die Ergotalkaloidbiosynthese ............................................... 14 1.4.1 Identifizierung der Gencluster in den Produzenten ................................... 14 1.4.2 Der Zusammenhang zwischen den Informationen in den Genclustern und den Strukturmerkmalen der Ergotalkaloide ........................................ 16 1.5 Stand der Biosynthese von Ergotalkaloiden zu Beginn der Promotion ........ 18 1.5.1 Die Bildung des tetrazyklischen Ergolinringsystems ................................. 18 1.5.2 Die Biosynthese von Fumigaclavinen ....................................................... 21 2 Zielsetzung der vorliegenden Arbeit .................................................................... 22 3 Material und Methoden ........................................................................................ 24 3.1 Chemikalien, Säulenmaterialien und Enzyme ............................................. 24 3.1.1 Chemikalien .............................................................................................. 24 3.1.2 Materialien zur Chromatographie .............................................................. 24 3.1.3 Enzyme und Kits ....................................................................................... 25 3.2 Nährmedien und Puffer ............................................................................... 25 3.2.1 Nährmedien zur Kultivierung von E. coli ................................................... 25 3.2.2 Antibiotika .................................................................................................. 26 3.2.3 Puffer und Lösungen ................................................................................. 26 3.3 Plasmide, Primer, Bakterienstämme ............................................................ 29 3.3.1 Plasmide ................................................................................................... 29 3.3.2 Primer........................................................................................................ 31 3.3.3 Bakterienstämme ...................................................................................... 32 3.4 Methoden der Molekularbiologie ................................................................. 33 3.4.1 Polymerase-Kettenreaktion (PCR) ............................................................ 33 3.4.2 Agarosegel-Elektrophorese ....................................................................... 36 3.4.3 Isolierung von DNA-Fragmenten aus Agarosegelen ................................. 36 INHALTSVERZEICHNIS II 3.4.4 Restriktion von DNA .................................................................................. 36 3.4.5 Ligation von DNA-Fragmenten .................................................................. 37 3.4.6 CaCl2-vermittelte Transformation von E. coli ............................................. 37 3.4.7 Isolierung von Plasmiden aus E. coli (Alkalische Lyse) ............................. 38 3.5 Methoden der Biochemie ............................................................................. 39 3.5.1 Heterologe Expression von Genen in E. coli ............................................. 39 3.5.2 Reinigung von Hexahistidin-Fusionsproteinen mittels Nickel-Affinitäts- chromatographie ........................................................................................ 39 3.5.3 Denaturierende Polyacrylamidgelelektrophorese (SDS-PAGE) und Coomassie-Färbung ................................................................................. 40 3.5.4 Proteinkonzentrationsbestimmung nach Bradford ..................................... 40 3.5.5 Bestimmung des Molekulargewichtes von Proteinen mittels Größen- ausschlusschromatographie ..................................................................... 41 3.5.6 Enzymatische Reaktionsansätze .............................................................. 41 3.6 Isolierung von Sekundärstoffen aus Ascomyceten ...................................... 46 3.6.1 Extraktion der Kulturüberstände von Arthroderma benhamiae .................. 46 3.6.2 Isolierung von (8R,9S)-Fumigaclavin A ..................................................... 46 3.7 Instrumentelle Analytik niedermolekularer Substanzen ............................... 47 3.7.1 Hochleistungsflüssigkeitschromatographie (HPLC) .................................. 47 3.7.2 Dünnschichtchromatographie (DC) ........................................................... 48 3.7.3 Massenspektrometrie (MS) ....................................................................... 48 3.7.4 Kernresonanzspektroskopie (NMR) .......................................................... 49 4 Ergebnisse .......................................................................................................... 50 4.1 Untersuchungen zur Ergotalkaloidbiosynthese in Pilzen der Familie Trichocomaceae .......................................................................................... 50 4.1.1 Charakterisierung der Chanoclavin-I-Dehydrogenase FgaDH aus Aspergillus fumigatus ................................................................................ 50 4.1.2 Charakterisierung des Old Yellow Enzyme FgaOx3 aus Aspergillus fumigatus .................................................................................................. 59 4.1.3 Charakterisierung der Festuclavin-Synthase FgaFS aus Aspergillus fumigatus .................................................................................................. 69 4.2 Versuche zur Prenylierung verschiedener Ergotalkaloide durch FgaPT1 aus Aspergillus fumigatus ........................................................................... 78 4.2.1 Die Prenyltransferase FgaPT1 .................................................................. 78 4.2.2 Optimierung der Expressionsbedingungen von fgaPT1 ............................ 79 4.2.3 Aktivitätstest mit His6-FgaPT1 und neuen Substraten ............................... 81 4.2.4 Isolierung von (8R,9S)-Fumigaclavin A aus Extrakten von Penicillium commune NRRL 2033 ............................................................................... 83 INHALTSVERZEICHNIS III 4.3 Untersuchungen zur Ergotalkaloidbiosynthese in Pilzen der Familie Arthrodermataceae ...................................................................................... 87 4.3.1 Identifikation eines putativen Genclusters für die Biosynthese von Ergotalkaloiden in Pilzen der Familie Arthrodermataceae ......................... 87 4.3.2 Charakterisierung der Chanoclavin-I-Dehydrogenase ChaDH aus Arthroderma benhamiae ........................................................................... 93 4.3.3 Untersuchung der Gene in Nachbarschaft zu den fünf homologen Genen aus Arthroderma benhamiae ....................................................... 105 5 Diskussion ......................................................................................................... 116 5.1 Die Biosynthese von Ergotalkaloiden in Aspergillus fumigatus .................. 116 5.1.1 Die Chanoclavin-I-Dehydrogenase FgaDH ............................................. 116 5.1.2 Die durch FgaOx3 und FgaFS katalysierte Bildung von Festuclavin ....... 117 5.1.3 Fortschritte in der Aufklärung des Biosynthesewegs von (8S,9S)- Fumigaclavin C in Aspergillus fumigatus ................................................ 120 5.1.4 Die Substratspezifität der Prenyltransferase FgaPT1 ............................
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