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KONSTRUKTION VON ESCHERICHIA COLI PRODUKTIONSSTÄMMEN ZUR FERMENTATIVEN HERSTELLUNG VON SUCCINAT AUS GLYCERIN Stefan Söllner

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KONSTRUKTION VON ESCHERICHIA COLI PRODUKTIONSSTÄMMEN ZUR FERMENTATIVEN HERSTELLUNG VON SUCCINAT AUS GLYCERIN Stefan Söllner KONSTRUKTION VON ESCHERICHIA COLI PRODUKTIONSSTÄMMEN ZUR FERMENTATIVEN HERSTELLUNG VON SUCCINAT AUS GLYCERIN Von der Fakultät 4 (Energie-, Verfahrens- und Biotechnik) der Universität Stuttgart zur Erlangung der Würde eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte Abhandlung Vorgelegt von Stefan Söllner aus Schweinfurt Hauptberichter: Prof. Dr. rer. nat. Ralf Mattes Mitberichter: Prof. Dr.-Ing. Ralf Takors Tag der mündlichen Prüfung: 29.02.2012 Institut für Industrielle Genetik Universität Stuttgart 2012 Vielen Dank! … Herrn Prof. Dr. R. Mattes danke ich für die Überlassung des interessanten Themas, des Arbeitsplatzes, für gelegentliches Aufmuntern und für die Erstellung des Erstgutachtens dieser Arbeit. … Herrn Prof. Dr. R. Takors danke ich für die freundliche Übernahme des Zweitgutachtens und für spannende Diskussionen. … Herrn Dr. Josef Altenbuchner danke ich für die praktische Betreuung dieser Arbeit, für diverse Einladungen zu Grillfesten und ganz besonders für die intensive Durchsicht des Manuskriptes!!! … Herrn Dr. Martin Siemann-Herzberg danke ich für die motivierende, überschwängliche Begeisterung, die meine Ideen und Ergebnisse bei deren Besprechung jedesmal auslösten. … Herrn Prof. Dr. Reuss danke ich für die Initiierung des Projektes lange vor meiner Zeit. … Meinen Kollegen und Exkollegen danke ich für die gute Zusammenarbeit, das abwechslungsreiche Arbeitsklima sowie die vielen fachlichen und nichtfachlichen Gespräche, welche die Arbeit immer spannend gestalteten. Vor allem danke ich für das Verständnis für die von mir durchgeführten, absolut notwendigen, regelmäßigen Arbeitskontrollen. … Frau Dr. Anne Völker hat mir die Integration zu Beginn meines Aufenthaltes am IIG sehr erleichtert. Herzlichen Dank dafür! … Herrn Kambiz Morabbi Heravi danke ich recht herzlich für die Einladung ans National Institute of Genetic Engineering and Biotechnology in Teheran, Iran und für die internationale Freundschaft. … Ich danke Frau Gisela Wajant vielmals für die Keksgespräche mit Masir-Tee. … Ich danke Frau Gisela Kwiatkowski, meiner einzigen, dauerhaften Laborkollegin für problemlose Abläufe, Gespräche und viele kleine Geschenke. … Ich danke Frau Dr. Hildegard Watzlawick für Aufmunterungen, stetige Hilfsbereitschaft, viele konstruktive fachliche Ratschläge, den stetigen Austausch und die angenehme Büroatmosphäre. … Herrn Tobias Vallon danke ich für die Erstellung des ersten Prototyps der Abbildungen und zahlreiche Gespräche. … Frau Michaela Moses-Endewardt danke ich für pantomimische Aufheiterungen. … Frau Corinna Kempter danke ich ebenfalls für die gute Zusammenarbeit und Diskussionen. … Frau Maria Rahnert danke ich für die Zusammenarbeit während der letzten Jahre, für die Auswertung und Generierung der 13C-Rohdaten und für durchgeführte Fermentationen. … Herrn Dr. Oliver Vielhauer und insbesondere Frau Mira Lenfers-Lücker danke ich für die Hauptarbeit bei den durchgeführten HPLC-Analysen. Ich habe noch nie eine derart niedrige Fehlerquote und Reproduzierbarkeit von unabhängigen Messläufen erleben dürfen! … Nicht zuletzt danke ich meinen Eltern dafür, dass sie mir alle Freiheiten gelassen haben und mein Studium wohlwollend unterstützt haben. Inhaltsverzeichnis ABKÜRZUNGSVERZEICHNIS .......................................................................................................... 5 ZUSAMMENFASSUNG .................................................................................................................... 10 SUMMARY ......................................................................................................................................... 12 1 EINLEITUNG 1.1 Relevanz des Projektes .......................................................................................................................... 15 1.1.1 Glycerin als geeignetes Edukt für die Succinatproduktion ................................................................. 15 1.1.2 Produkt Succinat als bedeutende Plattformchemikalie...................................................................... 19 1.1.3 Auswahl von E. coli als Organismus für die Succinatproduktion ........................................................ 21 1.1.4 Stand der Forschung und Beispiele aus der Industrie ........................................................................ 24 1.2 Strategien zur Succinatproduktion ......................................................................................................... 27 1.2.1 Übersicht über den Zentralstoffwechsel auf Glukose und Glycerin ................................................... 27 1.2.2 Succinatproduktion durch PEP-Carboxylierung oder über den Glyoxylatzyklus................................. 41 1.2.3 Succinatproduktion durch Carboxylierung von Pyruvat ..................................................................... 44 1.2.4 Succinatproduktion ausschließlich durch Carboxylierung von PEP .................................................... 48 1.2.5 Export von Succinat aus der Zelle ....................................................................................................... 52 1.2.6 Übersicht über Energetik, Ausbeuten und Redoxbilanzen der einzelnen Routen .............................. 54 1.3 Ausblick auf das Endziel ......................................................................................................................... 59 2 MATERIALIEN UND METHODEN 2.1 Materialien ............................................................................................................................................ 61 2.1.1 Enzyme ............................................................................................................................................... 61 2.1.2 Chemikalien ........................................................................................................................................ 61 2.1.3 Verbrauchsmaterialien ....................................................................................................................... 62 2.1.4 Verwendete Kits ................................................................................................................................. 62 2.1.5 Geräte ................................................................................................................................................. 62 1 Inhaltsverzeichnis 2.1.6 Medien ............................................................................................................................................... 63 2.1.7 Antibiotika und andere Zusätze .......................................................................................................... 64 2.1.8 Puffer und Lösungen........................................................................................................................... 65 2.2 Bakterienstämme .................................................................................................................................. 66 2.3 Bakteriophagen ..................................................................................................................................... 68 2.4 Verwendete synthetische Oligodesoxynukleotide ................................................................................. 69 2.5 Plasmide ................................................................................................................................................ 72 2.6 Methoden .............................................................................................................................................. 74 2.6.1 Stammhaltung von E. coli ................................................................................................................... 74 2.6.2 Routinemethoden der Molekularbiologie .......................................................................................... 74 2.6.3 Biotransformationen im Kleinmaßstab............................................................................................... 83 2.6.4 Bestimmung der extrazellulären Metabolitkonzentrationen durch HPLC .......................................... 87 2.6.5 Gaschromatographie und Massenspektrometrie der exkretierten Metabolite ................................. 88 2.6.6 Wachstumskurven und Wachstumsraten ........................................................................................... 89 2.6.7 Gezielte Gendeletionen mit dem -Red-System ................................................................................ 91 2.6.8 Aerobe Selektion auf schnelleres Wachstum auf Glycerin durch serielle Transfers ........................... 98 3 ERGEBNISSE 3.1 Herstellung und Charakterisierung der Stämme ................................................................................... 101 3.1.1 Herstellung der Stämme ................................................................................................................... 101 3.1.2 Wachstumsverhalten von Stämmen ohne LipA ................................................................................ 104 3.1.3 Wachstumsverhalten von Stämmen ohne Pyruvatkinasen auf Glycerin .......................................... 105 3.1.4 Versuch der Beseitigung des Wachstumsdefekts auf Glycerin und Auftreten von Spontanmutationen.......................................................................................................................... 107 3.2 Aerobe Selektion der ΔpykA ΔpykF Stämme auf Glycerin und Charakterisierung der Mutanten .......... 109 3.3 Entwicklung der Biotransformation im Kleinmaßstab
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