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High Quality 13C Metabolic Flux Analysis Using GC-MS“ 10 APPLIED MICROBIOLOGY Volume 10 To reduce the demand for fossil resources, biotechnological processes are increasingly applied. Therefore, the biological hosts used for manufacturing of products of interest have to be optimized in order to achieve the highest possible yield. For this optimization, Andreas Schmitz which is usually carried out by means of metabolic engineering strategies, the intracel- lular processes need to be investigated in advance by methods such as 13C-Metabolic 13 Flux Analysis (MFA). High Quality C metabolic flux The present thesis focused on improving analytical techniques for the measurement of analysis using GC-MS 13C-labeling patterns of metabolites. Parameters influencing the data quality of existing analytical techniques based on gas chromatography-mass spectrometry (GC-MS) for the classic 13C-MFA, were investigated. Focusing on the analysis of proteinogenic amino acids, a detailed protocol for sample preparation and GC-MS analysis was established to enable scientists to easily produce high quality labeling data for 13C-MFA. Additionally, sample preparation and measurement methods for labeling determination of intracellular metabolites were implemented and further developed. The application of the developed methods facilitates an in-depth analysis of the glucose uptake in Pseu- domonas and the examination of cyclic Entner-Doudoroff-Pathway fluxes. Compared to classic 13C-MFA, the use of intracellular metabolites increased the information content and therefore additional fluxes could be resolved. Furthermore, the potential of GC-MS/MS analyses was investigated to increase the po- sitional 13C-label information of the MS data by introducing a second fragmentation 13 C metabolic flux analysis using GC-MS step. This positional information on C-isotopes can be exploited to resolve additional 13 metabolic fluxes. Leucine and lysine were found to be promising amino acids in terms of acetyl-CoA labeling determination when analyzed in tandem MS. Labeling experiments with Saccharomyces cerevisiae revealed differences in the labeling of cytosolic and mi- tochondrial acetyl-CoA, indicating that there is no significant exchange of these two compartmental pools. High Quality The methods presented in this study are highly sophisticated and are suited for determi- nation of the labeling of various metabolites. The information gained by the application of these methods is suitable for use in 13C-MFA and thus enables a deeper insight into the metabolic activity inside the cell. Andreas Schmitz „High Quality 13C metabolic flux analysis using GC-MS“ Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von M.Sc. Angewandte Chemie Andreas Schmitz aus Mechernich, Nordrhein Westfalen, Deutschland Berichter: Universitätsprofessor Dr.-Ing. Lars M. Blank Universitätsprofessor Dr. rer. nat. Marco Oldiges Tag der mündlichen Prüfung: 16.07.2018 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar. Bibliografische Information der Deutschen Nationalbibliothek Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über https://portal.dnb.de abrufbar. Andreas Schmitz: High Quality 13C metabolic flux analysis using GC-MS 1. Auflage, 2018 Gedruckt auf holz- und säurefreiem Papier, 100% chlorfrei gebleicht. Apprimus Verlag, Aachen, 2018 Wissenschaftsverlag des Instituts für Industriekommunikation und Fachmedien an der RWTH Aachen Steinbachstr. 25, 52074 Aachen Internet: www.apprimus-verlag.de, E-Mail: [email protected] Printed in Germany ISBN 978-3-86359-660-6 D 82 (Diss. RWTH Aachen University, 2018) EIDESSTATTLICHE ERKLÄRUNG Eidesstattliche Erklärung “Hiermit erkläre ich, Andreas Schmitz, an Eides statt, dass ich die vorliegende Dissertation selbstständig verfasst und keine anderen als die angegebenen Quellen und Hilfsmittel benutzt habe.“ I DANKSAGUNG Danksagung An dieser Stelle möchte ich mich gerne bei einigen Personen bedanken, ohne welche diese Arbeit nicht entstanden wäre. Zunächst möchte ich mich bei meinem Doktorvater Prof. Dr. Lars M. Blank für die Möglichkeit bedanken, dieses interessante Thema im Rahmen meiner Doktorarbeit am Institut für Angewandte Mikrobiologie bearbeiten zu dürfen. Danke auch für die guten Gespräche und Diskussionen. Ein großer Dank geht ebenfalls an Dr. Birgitta Ebert, welche mir als Gruppenleiterin bei der Bearbeitung der Doktorarbeit stets zur Seite stand. Viele anregende Diskussionen führten letztlich zur erfolgreichen Planung der durchzuführenden Experimente als Grundlage für die vorliegende Arbeit. Weiterhin bedanke ich mich recht herzlich bei Prof. Dr. Marco Oldiges, für die bereitwillige Übernahme des Zweitgutachtens. Danken möchte ich auch meinen Studenten Vincent Wiebach, Michael Osthege, Tobias Alter, Isabella Albert und Sarah Maurer, welche im Rahmen von Bachelor- und Masterarbeiten sowie Forschungspraktika zu dieser Arbeit beigetragen haben. Meinen Kolleginnen und Kollegen am Institut für Angewandte Mikrobiologie, Ahmed, Andrea, Annette, Arnoldine, Benedikt, Bernd, Birthe, Carola, Christoph H., Christoph L., Christoph T., Dario, Eda, Eik, Elena, Elke, Gisela, Hamed, Henrik, Jan, Jannis, Kalle, Kerstin, Lars K., Maike, Manja, Mathias, Martin, Monika, Nick, Rabea, Salome, Sandra H., Sandra S., Sebastian K., Sebastian Z., Suresh, Theresa, Thiemo, Till, Ulf, Ulrike, Wie und Wing-Jin möchte ich ebenfalls an dieser Stelle danken. Ihr habt die Zeit am Institut zu einer für mich unvergesslichen gemacht! Es war schön mit euch nicht nur auf der Arbeit, sondern auch abseits davon viele gemeinsame und spaßige Augenblicke verbracht zu haben. Besonders großer Dank gilt an dieser Stelle meiner gesamten Familie. Ohne die großartige Unterstützung meiner Eltern, Helga und Helmut Schmitz, wäre dies alles nicht möglich gewesen. Ihr wart immer für mich da und habt mir immer unter die Arme gegriffen, wo Ihr nur konntet. Danke dafür! Der größte Dank gilt meiner Freundin Hannah! Danke, dass Du an meiner Seite bist und danke für die vielen aufmunternden Worte sowie die Unterstützung, besonders während des Zusammenschreibens! Es war nicht immer einfach, aber Du hast mich ermutigt, die Arbeit zu vollenden! DANKE! III TABLE OF CONTENTS EIDESSTATTLICHE ERKLÄRUNG ................................................................................................... I DANKSAGUNG ........................................................................................................................... III SUMMARY ................................................................................................................................ VII ZUSAMMENFASSUNG ................................................................................................................. IX LIST OF ABBREVIATIONS ........................................................................................................... XI LIST OF FIGURES .................................................................................................................... XIII LIST OF TABLES ..................................................................................................................... XVII 1. GENERAL INTRODUCTION ................................................................................................ 1 1.1. General motivation of flux quantification in organisms ................................................. 2 1.2. 13C-metabolic flux analysis ............................................................................................ 3 1.3. NMR and GC-MS techniques for stable isotope distribution quantification ................. 5 1.3.1. Amino acids determination .................................................................................. 7 1.3.2. Intracellular metabolite determination ............................................................... 10 1.4. GC-MS/MS technique .................................................................................................. 13 1.4.1. Amino acid fragmentation .................................................................................. 13 1.5. Aims of the Thesis ........................................................................................................ 14 2. RESULTS AND DISCUSSION ............................................................................................. 17 2.1. Mass isotopomer distribution measurements for high quality 13C-metabolic flux analysis ......................................................................................................................... 19 2.1.1. Summary ............................................................................................................ 20 2.1.2. Introduction ........................................................................................................ 20 2.1.3. Materials and Methods ....................................................................................... 22 2.1.4. Results and Discussion ....................................................................................... 23 2.1.5. Conclusion .......................................................................................................... 34 2.2. GC-MS based determination of mass isotopomer distributions for 13C-based metabolic flux analysis ................................................................................................
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