Genetic tools for high yield protein production with Bacillus megaterium Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Simon Stammen aus Kevelaer 1. Referent: Professor Dr. Dieter Jahn 2. Referent: Professor Dr. Petra Dersch eingereicht am: 01.09.2010 mündliche Prüfung (Disputation) am: 08.10.2010 Druckjahr 2010 Vorveröffentlichungen der Dissertation: Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen: Stammen, S. , Schuller, F., Dietrich, S., Gamer, M., Biedendieck R. and D. Jahn. 2010. Application of Escherichia coli phage K1E DNA-dependent RNA polymerase for in vitro RNA synthesis and in vivo protein production in Bacillus megaterium . Appl Microbiol Biotechnol 88 :529-539. Stammen, S. , Müller, B. K., Korneli, C., Biedendieck, R., Gamer, M., Franco-Lara, E. and D. Jahn. 2010. High yield intra- and extracellular protein production using Bacillus megaterium . Appl Environ Microbiol 76 :4037-4046. Bunk, B., Schulz, A., Stammen, S. , Münch, R., Warren, M. J., Rohde, M., Jahn, D. and R. Biedendieck. 2010. A short story about a big magic bug. Bioengineered Bugs 1:1-7. Gamer, M., Fröde, D., Biedendieck, R., Stammen, S. and D. Jahn. 2009. A T7 RNA polymerase-dependent gene expression system for Bacillus megaterium . Appl Microbiol Biotechnol 82 :1195-1203. Tagungs- und Industriebeiträge: Stammen, S. , Schuller, F., Dietrich, S., Gamer, M., Biedendieck, R. and D. Jahn. 2010. A novel DNA-dependent RNA polymerase of Escherichia coli phage K1E: production, characterization and application. (Poster) World Congress on Industrial Biotechnology & Bioprocessing, Washington D.C., DC, USA. Stammen, S. , Müller, B. K., Hellert, J., Busch, M., Biedendieck, R. and D. Jahn. 2010. Construction of a high performance plasmid for intra- and extracellular protein production in Bacillus megaterium . (Vortrag) 3. Gemeinsame Tagung der Vereinigung für allgemeine und angewandte Mikrobiologie (VAAM) und der deutschen Gesellschaft für Hygiene und Mikrobiologie (DGHM), Hannover, Deutschland. S. Stammen. 2010. Advances in heterologous protein production in Bacillus megaterium . (Vortrag) Technologietransfergespräche beim Kooperationspartner Codexis Inc., Redwood City, CA, USA. Stammen, S. , Gamer, M., Müller, B. K. and D. Jahn. 2009. A Plasmid borne system for high level protein production in Bacillus megaterium . (Poster) Symbiosis, the 14 th European Congress on Biotechnology, Barcelona, Spain. Stammen, S. , Gamer, M., Müller, B. K., Schwerk, J., Schlums, H. and D. Jahn. 2009. Improved plasmids for heterologous protein production in Bacillus megaterium . (Poster) Jahrestagung der Vereinigung für allgemeine und angewandte Mikrobiologie (VAAM), Bochum, Deutschland. Stammen, S. , Gamer, M., Müller, B. K., Schwerk, J., Schlums, H. and D. Jahn. 2008. Improved plasmids for heterologous protein production in Bacillus megaterium . (Poster) 6th Conference on Recombinant Protein Production, Alghero, Sardina, Italy. Stammen, S. , Gamer, M., Schwerk, J., Schlums, H. and D. Jahn. 2008. Construction of strong inducible promoters for Bacillus megaterium expression vectors. (Poster) Jahrestagung der Vereinigung für allgemeine und angewandte Mikrobiologie (VAAM), Frankfurt, Deutschland. Gamer, M., Stammen, S. , Biedendieck, R., Yang, Y., Fröde, D., Bendig, C. and D. Jahn. 2007. Bacillus megaterium - an alternative expression system. (Poster) Symbiosis, the 13 th European Congress on Biotechnology, Barcelona, Spain. In den Wissenschaften ist viel Gewisses, sobald man sich von den Ausnahmen nicht irremachen lässt und die Probleme zu ehren weiß. (Wilhelm Meisters Wanderjahre - Buch 2, J. W. von Goethe, 1749-1832) Contents Contents Contents……………………………………………...…………..….I Abbreviations………………………………..………………….....V I. Introduction.................................................................... 1 I.1 Biotechnology ...............................................................................................1 I.2 Recombinant protein production ...................................................................2 I.3 Bacillus megaterium ......................................................................................3 I.4 The xylose-inducible gene expression system for Bacillus megaterium .......5 I.5 Phage RNA polymerase-based gene expression systems ...........................6 I.6 Protein secretion in Bacilli.............................................................................8 I.7 Model proteins for protein production studies .............................................11 I.7.1 Green fluorescent protein (Gfp) ...........................................................11 I.7.2 Hydrolase of Thermobifida fusca (Tfh).................................................12 I.8 Aim of this work...........................................................................................13 II. Materials and Methods................................................. 15 II.1 Instruments, chemicals and kits ..................................................................15 II.1.1 Instruments ..........................................................................................15 II.1.2 Chemicals and reagents ......................................................................17 II.2 Bacterial strains and plasmids ....................................................................18 II.2.1 Plasmid construction............................................................................25 II.2.1.1 Vectors used for the optimization of the P xylA system ...................25 II.2.1.2 Plasmids constructed for the Ecoco system .................................27 II.2.1.3 Plasmids developed for the phage RNA polymerase systems .....27 II.3 Growth media and media additives.............................................................29 II.3.1 Growth media ......................................................................................29 II.3.2 Media additives....................................................................................30 II.4 Microbiological techniques..........................................................................31 II.4.1 Sterilization ..........................................................................................31 II.4.2 Plate cultures .......................................................................................31 II.4.3 Liquid cultures of Escherichia coli ........................................................31 II.4.4 Liquid cultures of Bacillus megaterium .................................................31 -I- Contents II.4.5 Determination of cell density................................................................32 II.4.6 Storage of bacteria ..............................................................................32 II.5 Molecular biology techniques......................................................................32 II.5.1 Preparation and transformation of chemically competent ....................... Escherichia coli ....................................................................................32 II.5.2 Preparation and transformation of Bacillus megaterium protoplasts....33 II.5.3 Preparation of plasmid DNA from Escherichia coli ..............................35 II.5.4 Quantification of nucleic acids .............................................................36 II.5.5 Electrophoretic separation of DNA ......................................................37 II.5.6 Amplification of DNA fragments by polymerase chain reaction (PCR) 37 II.5.7 Phosphorylation and hybridization of oligo-nucleotides .......................39 II.5.8 Enzymatic digestion of DNA ................................................................41 II.5.9 Ligation of DNA fragments...................................................................41 II.5.10 DNA sequencing..................................................................................42 II.6 Protein biochemical techniques ..................................................................42 II.6.1 Expression of recombinant genes in Bacillus megaterium ...................42 II.6.2 Analysis of basal gfp expression in Escherichia coli ............................43 II.6.3 Production of recombinant proteins in Escherichia coli .......................43 II.6.4 Preparation of intracellular protein fractions ........................................43 II.6.5 Preparation of extracellular protein fractions .......................................44 II.6.6 Electrophoretic separation of proteins (SDS-PAGE)............................45 II.6.7 Purification of His 6-tagged fusion proteins ...........................................46 II.6.8 Quantification of purified protein..........................................................47 II.6.9 Fluorescence measurements of green fluorescent protein..................47 II.6.10 Determination of hydrolase activity......................................................48 II.6.11 Phage RNA polymerase driven in vitro transcription ...........................49 II.7 Fluorescence microscopy ...........................................................................50 III. Results and Discussion ...............................................51 III.1 Directed optimization of the P xylA -based gene expression system ..............51