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Optimization of Lipase Production in Burkholderia Glumae

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Optimization of Lipase Production in Burkholderia Glumae Optimization of lipase production in Burkholderia glumae Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie an der Internationalen Graduiertenschule Biowissenschaften der Ruhr-Universität Bochum angefertigt am Institut für Molekulare Enzymtechnologie vorgelegt von Anke Beselin aus Frankfurt a. Main Bochum August 2005 Optimierung der Lipaseproduktion in Burkholderia glumae Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie an der Internationalen Graduiertenschule Biowissenschaften der Ruhr-Universität Bochum angefertigt am Institut für Molekulare Enzymtechnologie vorgelegt von Anke Beselin aus Frankfurt a. Main Bochum August 2005 Die vorliegende Arbeit wurde im Rahmen des Europäischen Graduiertenkollegs der Ruhr- Universität Bochum (EGC 795): Regulatory Circuits in Cellular Systems: Fundamentals and Biotechnological Applications angefertigt. Referent: Prof. Dr. K.-E. Jäger Korreferent: Prof. Dr. W. J. Quax Tag der mündlichen Prüfung: 28.10.2005 ___________________________________________________________________________ Danksagungen Herrn Prof. Dr. K.-E. Jäger danke ich für die Überlassung des interessanten und aktuellen Themas, für das rege Interesse am Fortschritt meiner Arbeit, die konstruktiven Diskussionen und die mir gebotene Möglichkeit, die experimentelle Arbeit frei und selbständig zu gestalten. I would like to thank Prof. Dr. W. J. Quax, Laboratory of Pharmaceutical Biology- Rijksuniversität Groningen (NL) for agreeing to co-supervise this project. Herrn Dr. M. Breuer und Herrn Prof. Dr. B. Hauer, BASF AG (Ludwigshafen), danke ich für die sehr gute Kooperation, die finanzielle Unterstützung im Rahmen des Projektes und die ausgesprochen freundliche, zeitweilige Aufnahme in die Arbeitsgruppe der Abteilung „Forschung Feinchemikalien & Biokatalyse“ in Ludwigshafen. Des Weiteren danke ich Herrn Prof. Dr. M. Rögner am PhD-Programm des Europäischen Graduiertenkollegs EGC 795 teilnehmen zu können, sowie allen Organisatoren (innen) für die Gestaltung zahlreicher interessanter Kurse und Tagungen. An dieser Stelle auch ein herzlicher Dank an alle Mitglieder des Kollegs für die interessanten Diskussionen und die nette Atmosphäre auf den Tagungen. Herrn Dr. F. Rosenau danke ich für die freundschaftliche Unterstützung bei der Planung und Durchführung dieser Promotionsarbeit, für die Hilfe bei der Lösung großer und kleiner wissenschaftlicher Probleme und für die aufmerksame Durchsicht des Manuskriptes. A special thanks goes to Dr. M. Tsoli (Sydney, Australia) for good advices during writing my thesis and the critical reading of the manuscript. Thanks for the funny times we had in Germany and for always being a very good friend. Den Mitarbeitern des Instituts für Molekulare Enzymtechnologie danke ich für das angenehme und freundschaftliche Arbeitsklima während meiner gesamten Promotionszeit. Ein besonderer Dank an alle, die dazu beigetragen haben, den „kleinen und großen Rückschlägen“ im Labor mit Heiterkeit und Optimismus entgegenzutreten. Für die Unterstützung bei der Durchführung einiger Experimente danke ich Frau K. Range, Frau I. Frindi-Wosch und Frau V. Svensson. Außerdem, danke ich Herrn Dr. T. Drepper für die zahlreichen, wissenschaftlichen Diskussionen und hilfreichen Anregungen beim Zusammenschreiben der Arbeit. Herrn ´Dr.` C. Leggewie danke ich ebenfalls für die aufmerksame Durchsicht des Manuskriptes. Last but not least, möchte ich mich ganz herzlich bei meiner Familie bedanken, für die stetige Unterstützung und den Rückhalt während meiner gesamten Studien- und Promotionszeit. Insbesondere geht ein ganz großer Dank an Ulrich, für seine liebevolle Unterstützung und für seinen unermüdlichen Optimismus, mit dem er mich in „schlechten Zeiten“ aufgemuntert und sich in „guten Zeiten“ mit mir gefreut hat. ___________________________________________________________________________ ___________________________________________________________________________ Veröffentlichungen im Rahmen der Promotion: Boukema, B., Beselin A., Rosenau, F., Jaeger, K.-E. and Tommassen, J. (2005) Improvement of lipase production by addition of inert compounds to Burkholderia glumae cultures. In Vorbereitung Beselin, A., Breuer, M., Hauer, B., Rosenau, F. and Jaeger, K.-E. (2005) Erhöhung der Expression und/oder Faltung und Sekretion der Lipase LipA in Burkholderia glumae durch Co-Expression einer putativen, cytoplasmatischen Protease. Patent-Nr. 56817, BASF AG (Ludwigshafen) Tagungsbeiträge: Beselin, A., Rosenau, F. and Jaeger, K.-E. (2003) Identification of bottlenecks in lipase production of Burkholderia glumae. Pseudomonas 2003, Québec, Canada (Posterbeitrag) Beselin, A., Rosenau, F. and Jaeger, K.-E. (2003) Overexpression of lipase in Burkholderia glumae. EGC-Symposium, Groningen, The Netherlands (Posterbeitrag) Beselin, A., Rosenau, F., Breuer, M., Hauer, B. and Jaeger, K.-E. (2005) Construction of a Burkholderia glumae strain suitable for lipase overexpression. Pseudomonas 2005, Marseille, France (Posterbeitrag) Beselin, A., Rosenau, F., Breuer M., Hauer B., Jaeger, K.-E. (2005) Optimization of lipase production in Burkholderia glumae. EGC-Symposium, Groningen, The Netherlands (Vortrag) ___________________________________________________________________________ Contents Contents Abbreviations and units List of Figures List of Tables 1. Introduction………………………………………………….…….. 1 1.1 Lipolytic enzymes: Lipases………………………………………………..……. 2 1.1.1 Structure and enzymatic reactions of lipases……………………………. 3 1.1.2 Biotechnological applications of lipases………………………………… 5 1.2 Regulation of gene expression, folding and secretion of lipases………………... 6 1.2.1 Regulation of lipase gene expression……………………………………. 6 1.2.2 Periplasmic folding of lipases…………………………………………… 8 1.2.3 Secretion of lipases……………………………………………………… 11 1.3 Burkholderia glumae - A lipase-producing plant pathogen…………………….. 13 1.4 Expression systems……………………………………………………………… 15 1.4.1 General aspects………………………………………………………….. 15 1.4.2 The T7 RNA polymerase-based expression system………...…………... 17 1.5 Aims of this study………………………………………………………………. 19 2. Materials……………………………………………………………. 21 2.1 Chemicals and enzymes…………………………………………………………. 21 2.2 Strains and Plasmids…………………………………………………………….. 21 2.3 Oligonucleotides……………………………………………………………….... 26 2.4 Culture media and plates………………………………………………….…….. 26 2.5 Buffers and solutions……………………………………………………………. 27 2.6 Molecular weight standards……………………………………………….…….. 28 2.7 Kits………………………………………………………………………………. 28 2.8 Laboratory instruments………………………………………………………….. 28 3. Methods…………………………………………………………….. 30 3.1 Bacterial strain and growth conditions………………………………………….. 30 3.1.1 Cultivation of E. coli strains…………………………………………….. 30 3.1.2 Cultivation of B. glumae strains…………………………………….…... 30 3.1.3 Table of applied antibiotics……………………………………………... 30 3.1.4 Storage of microorganisms………………………………………….…... 30 3.2 Isolation of nucleic acid…………………………………………………………. 30 3.2.1 Isolation of plasmid and cosmid DNA………………………………….. 30 3.2.2 Isolation of chromosomal DNA……………………………………….... 31 3.2.3 Isolation of RNA………………………………………………...…….... 31 3.3 Agarose gel electrophoresis…………………………………………………….. 31 3.4 In vitro recombination of DNA…………………………………………………. 31 3.5 Construction of a cosmid library……………………………………..………… 31 3.6 Polymerase chain reaction (PCR)………………………………………………. 31 3.7 Primer extension analysis………………………………………………………. 32 3.8 DNA sequencing………………………………………………………………... 32 3.9 Transformation of bacteria……………………………………………………… 33 3.9.1 Chemical transformation of E. coli……………………………………... 33 3.9.2 Electroporation of B. glumae……………………………………………. 33 3.9.3 Conjugational transfer of plasmids or cosmids into B. glumae…………. 33 3.10 Determination of protein concentration…………………………………………. 34 ________________________________________________________________________I Contents 3.11 TCA precipitation of proteins…………………………………………………… 34 3.12 Polyacrylamide gel electrophoresis…………………………..…………………. 34 3.12.1 SDS-polyacrylamide gel electrophoresis..............................................…. 34 3.12.2 Native polyacrylamide gel electrophoresis………………………...……. 34 3.13 Western blot……………………………………………………………………... 34 3.14 Protein chromatography…………………………………………………………. 34 3.15 Preparative gel filtration…………………………………………………………. 35 3.16 Enzyme activity assays…………………………………………………………... 35 3.17 Fluorescence measurements……………………………………………………... 35 3.18 Computational methods………………………………………………………….. 36 4. Results………………………………………………………………. 37 4.1 Development of a T7 RNA polymerase-based expression system in B. glumae……………………………………………………………… 37 4.1.1 Expression of plasmid-encoded T7 RNA polymerase in B. glumae…….. 37 4.1.2 Construction of a T7-expression strain of B. glumae……………………. 38 4.1.3 Construction of expression vectors for high-level production of lipase in B. glumae………………………………………….……………. 40 4.2 Identification of bottlenecks for an improved lipase production in B. glumae….. 42 4.2.1 Construction of two cosmid libraries of B. glumae PG1 and B. glumae LU8093………………………………………………………. 42 4.2.2 Screening of the cosmid library of B. glumae PG1 led to the identi- fication of 15 cosmids influencing lipase production in B. glumae.…….. 43 4.2.3 Homologous expression of subcloned genomic DNA of B. glumae PG1.………………………………………………………….. 44 4.2.4 Co-expression of a gene encoding a putative protease increases lipase production in B. glumae………………………………………….. 48 4.2.5 The putative protease does not affect foldase production in B. glumae…. 49 4.3 Characterization
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