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Exopolysaccharides by Paenibacilli from Genetic Strain

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Exopolysaccharides by Paenibacilli from Genetic Strain Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt Campus Straubing für Biotechnologie und Nachhaltigkeit Lehrstuhl für Chemie Biogener Rohstoffe Exopolysaccharides by Paenibacilli: from Genetic Strain Engineering to Industrial Application Marius Paul Rütering Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Prof. Dr. Cordt Zollfrank Prüfer der Dissertation: 1. Prof. Dr. Volker Sieber 2. Prof. Dr. Wolfgang Liebl 3. Prof. Dr. Jochen Schmid Die Dissertation wurde am 01.04.2019 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 16.12.2019 angenommen. Dissertation Marius Rütering Exopolysaccharides by Paenibacilli: from Genetic Strain Engineering to Industrial Application Danksagung An erster Stelle möchte ich meinem Doktorvater Prof. Dr. Volker Sieber danken. Du hast durch deine Laufbahn den Weg von Nordrhein-Westfalen nach Niederbayern gepflastert, welchem ich später folgen durfte. Stets hast du mir das richtige Maß an individueller Freiheit und wissenschaftlicher Unterstützung zukommen lassen und somit maßgeblich dazu beigetragen, dass ich die Zeit in Straubing immer in guter Erinnerung behalten werde. Ich bin dankbar, dass ich in deiner Arbeitsgruppe promovieren durfte. Mein Dank gilt auch Prof. Wolfgang Liebl für die Übernahme des Korreferats und Prof. Cordt Zollfrank für die Übernahme des Prüfungsvorsitzes. Prof. Dr. Jochen Schmid - dir gilt ein ganz besonderes und herzliches Dankeschön. Über die gesamte Zeit meiner Promotion warst du weit mehr, als nur mein direkter wissenschaftlicher Betreuer. Du hattest immer ein offenes Ohr für Fragestellungen jeder Art– auch nach meiner Zeit am Lehrstuhl. Von Auslandsaufenthalten über Nachtschichten am Fermenter bis hin zu angeregten Diskussionen bei einem Glas Bier – auf deine Unterstützung konnte ich immer zählen. Dafür danke ich dir von ganzem Herzen. Ein weiterer besonderer Dank gebührt Dr. Martin Schilling. Seit Beginn meiner Masterarbeit bist du ein wichtiger Mentor für mich. Du hast einen großen Teil dazu beigetragen, dass ich diese Sätze schreiben darf. Dr. Petra Allef danke ich für die initiale Projektidee und die essentielle Unterstützung dieser Kooperationsarbeit. Bei Prof. Mattheos Koffas und Dr. Brady Cress möchte ich mich für eine wissentschaftlich, wie auch persönlich sehr lehrreiche Zeit am Rensselaer Polytechnic Institute in Troy bedanken. Bei euch durfte ich die wahre Macht der Molekularbiolgie begreifen und erlernen. Des Weiteren danke ich Dr. Broder Rühmann für die professionelle Unterstützung bei analytischen Untersuchungen, Steven Koenig für die Einführung in die faszinierende Welt der Rheologie und Sumanth Ranganathan für das Korrekturlesen dieser Arbeit. Für das überaus angenehme Arbeitsklima und viele schöne Stunden möchte ich allen Kollegen danken, mit denen ich während meiner Zeit in Straubing zusammenarbeiten durfte. Ganz besonders möchte ich hier meine guten Freunde José Guillermo Ortiz Tena, Hendrik Hohagen, Daniel Bauer und Dominik Schwarz erwähnen. Meinen Eltern Hiltrud und Ulrich Rütering kann ich gar nicht genug danken für dieses wunderschöne Leben, das ihr mir geschenkt habt. Die Sicherheit, dass ihr mich jederzeit auffangen würdet verleiht mir grenzenlose Freiheit. Auch euren Partnern Klaus Wandjo und Elisabeth Reiter, meiner Schwester Jennifer, Burkhardt Ross und „den Dudes“ möchte ich dafür danken, dass ihr jeden Tag meines Lebens bereichert. Mögen wir noch viele Feste feiern. Danke auch an Elke, Torsten und Carolin Diekmann. Ihr habt mich aufgenommen wie einen Sohn und Bruder und ward immer mein Ruhepol in bewegten Zeiten. Das werde ich euch nie vergessen. Mein größter Dank geht an meine Ehefrau Sandra Rütering. Danke, dass du mir nach Bayern gefolgt bist, mich immer wieder auffängst und seit mehr als 12 Jahren tapfer und treu an meiner Seite stehst. Du bist das Glück meines Lebens. Table of Contents 7 Table of Contents 1 Introduction ....................................................................................................................... 13 1.1 Polysaccharides ....................................................................................................................... 13 1.2 Microbial Polysaccharides ..................................................................................................... 14 1.2.1 Structure of exopolysaccharides .................................................................................. 15 1.2.2 Rheology of polysaccharides ....................................................................................... 17 1.2.3 Biosynthesis of exopolysaccharides ............................................................................ 19 1.2.4 Characteristics and challenges of exopolysaccharide production ................................ 21 1.2.5 Application of exopolysaccharides .............................................................................. 23 1.2.6 Exopolysaccharide engineering ................................................................................... 25 1.3 Genome editing and CRISPR-Cas ........................................................................................ 26 1.3.1 Functionality of CRISPR-Cas...................................................................................... 27 1.3.2 CRISPR Editing ........................................................................................................... 28 1.4 Paenibacillus spp. .................................................................................................................... 29 1.4.1 Exopolysaccharides by Paenibacilli ............................................................................ 31 1.5 Scope of the work .................................................................................................................... 33 2 Material and Methods ....................................................................................................... 34 2.1 Material ............................................................................................................................. 34 2.1.1 Equipment .................................................................................................................... 34 2.1.2 Software and databases ................................................................................................ 35 2.1.3 Bacterial strains ........................................................................................................... 36 2.1.4 Chemicals and reagents ............................................................................................... 37 2.1.5 Kits, enzymes and special consumables ...................................................................... 38 2.1.6 Plasmids, primer and oligos ......................................................................................... 39 2.2 Media and Buffer .................................................................................................................... 40 2.2.1 Media preparation ........................................................................................................ 40 2.2.2 Cultivation media ........................................................................................................ 40 2.2.3 Trace element solution ................................................................................................. 41 2.2.4 Buffers for chemical competent E. coli cells ............................................................... 41 2.3 Microbiological methods ........................................................................................................ 41 2.3.1 Bacterial strain storage ................................................................................................ 41 2.3.2 Strain cultivation .......................................................................................................... 41 2.3.3 Antibiotic sensitivity tests............................................................................................ 41 2.3.4 Fermentations .............................................................................................................. 42 2.3.5 EPS extraction ............................................................................................................. 42 2.3.6 Chemically competent E. coli cells ............................................................................. 42 2.3.7 Conjugation ................................................................................................................. 43 2.4 Molecular biological methods ................................................................................................ 43 2.4.1 Agarose gel electrophoresis ......................................................................................... 43 2.4.2 Bacterial strain identification ....................................................................................... 43 2.4.3 Isolation of genomic DNA from bacterial strains ........................................................ 43 2.4.4 Genome editing in P. polymyxa ................................................................................... 44 2.4.5 pCasPP plasmid construction .....................................................................................
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