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Organic Acid Production from Glycerol with Ustilaginaceae

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Organic Acid Production from Glycerol with Ustilaginaceae 5 APPLIED MICROBIOLOGY Volume 5 The production process of biodiesel results in a huge waste stream of crude glycerol, reducing the economic and ecological advantage. Thus, the overall aim of this thesis was to establish a produc- tion process for organic acids from glycerol with Ustilaginaceae. In a screening of 126 Ustilagina- Thiemo Zambanini ceae, two candidates for organic acid production were found - Ustilago trichophora for malic acid and U. vetiveriae for itaconic acid. Glycerol uptake and growth rate of both strains were improved by adaptive laboratory evolution. Selection of the best growing colony for each strain and medium and process optimization drastically improved the production. What can we do with smut? The itaconic acid titer of U. vetiveriae TZ1 was increased to 35 g L-1 (rate: 0.09 g L-1 h-1). Simultane- ously about 60 g L-1 malic acid were formed. First metabolic engineering approaches overexpressing the mitochondrial transporter Mtt1 and the regulator of the itaconic acid gene cluster Ria1, both Organic acid production from from U. maydis, increased the itaconic acid titer by 1.5- and 2.0-fold, respectively. Simultaneously, the malic acid titer was reduced by 96 % and 61 %, respectively. glycerol with Ustilaginaceae For U. trichophora TZ1 the malic acid titer was improved to 200 g L-1 produced within 264 h reaching a maximal production rate of 1.53 g L-1 h-1. Potential native target genes for metabolic engineering were identified after de novo genome sequencing. To enable the improvement of malic acid production with U. trichophora TZ1 by metabolic engineering, existing tools were investigated and adapted to be suitable for creation of overexpression mutants. Using these tools, overexpressi- on mutants for two different malate dehydrogenases (Mdh1 and Mdh2), pyruvate-carboxylase (Pyc) and two different malic acid transporters (Ssu1 and Ssu2) were generated. While overexpression of Pyc did not improve malic acid production, transformants overexpressing Mdh1, Mdh2, Ssu1 and Ssu2 showed an up to 38 % increased malic acid production rate and an up to 54 % increased yield in shake flasks compared to U. trichophora TZ1. In bioreactor cultivations with the mutant over- expressing Ssu2, an increased production rate could not be observed. Due to a drastically lowered optical density, however, this strain had a 29 % higher specific production rate. Additionally, the product yield was improved by 1.4-fold. These results clearly strengthen the applicability of Ustilaginaceae as production organisms. By valorization of crude glycerol, the overall biodiesel bio refinery concept is improved. What can we do with smut? Organic acid production from glycerol with Ustilaginaceae ISBN 978-3-86359-492-3 Thiemo Zambanini ³What can we do with smut? Organic acid production from glycerol with Ustilaginaceae´ 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. Biologie Thiemo Zambanini aus Aachen, NRW, Germany %HULFKWHU8QLY3URI'U,QJ/DUV0%ODQN 8QLY3URI'UUHUQDW-DQ6FKLUDZVNL 7DJGHUPQGOLFKHQ3UIXQJ 'LHVH'LVVHUWDWLRQLVWDXIGHQ,QWHUQHWVHLWHQGHU+RFKVFKXOELEOLRWKHNRQOLQH YHUIJEDU Bibliografische Information der Deutschen Nationalbibliothek Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über http://dnb.ddb.de abrufbar. Thiemo Zambanini: What can we do with smut? Organic acid production from glycerol with Ustilaginaceae 1. Auflage, 2017 Gedruckt auf holz- und säurefreiem Papier, 100% chlorfrei gebleicht. Apprimus Verlag, Aachen, 2017 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 978-3-86359-492-3 D 82 (Diss. RWTH Aachen University, 2016) Danksagung Zunächst möchte ich mich bei meinem Doktorvater Prof. Lars M. Blank bedanken. Seit meinem Masterstudium konnte ich immer auf anregende Diskussionen und leitende Ideen zählen. Vielen Dank für die Möglichkeit dieses überaus interessante Thema während meiner Promotion zu bearbeiten, wobei du mir immer die nötige Freiheit gelassen hast mich selbst und das Projekt zu entwickeln. Prof. Jan Schirawski möchte ich dafür danken, dass er das Zweitgutachten für diese Arbeit übernommen hat und während der gesamten Zeit meiner Promotion für mich ansprechbar war. Durch deine kritischen Fragen und Anregungen wurde ich dazu bewegt, Sachverhalte und Ideen neu zu überdenken und oftmals zu neuen, besseren Lösungsansätzen zu kommen. Prof. Jochen Büchs danke ich dafür, dass er sich als dritter Prüfer für die Verteidigung bereit erklärt hat. Selbstverständlich möchte ich mich auch recht herzlich bei meinem Betreuer Nick Wierckx bedanken. Du warst und bist ein wirklich hilfsbereiter und motivierender Betreuer, der es verstanden hat, mich mit genau dem richtigen Input zur rechten Zeit zu versorgen, mich zu fordern und zu fördern und mir den Freiraum zu lassen meine eigenen Ideen umzusetzen. Ich kann mit Sicherheit behaupten, dass du zu großen Teilen zum Erfolg dieser Arbeit beigetragen hast. Ich bin froh, dass ich unter deiner Betreuung meine Arbeit anfertigen durfte. Auch bedanken möchte ich mich bei GHQ ]ZHL IODPPHQGHQ 7¶V GLH HV leider niemals auf die Laborkittel geschafft haben. Vielen Dank an ³7HDP 7KLHPR´ mit Melanie Beudels, Alina Huth, Ronald Jäpel, Wiebke Kleineberg, Judith Krabbe, Christian Lieven, Dorothee Merker, Eda Sarikaya, Sarah Schleese, Lisa Marie Schmitz, Christiane Katharina Sonntag, Hamed Tehrani und Dominik Venc. Ohne eure ausdauernde Arbeit im Labor in der Woche und am Wochenende, am Tag und in der Nacht wäre sicherlich nicht diese Menge an Ergebnissen zusammengetragen worden und schon gar nicht in dieser Zeit. Somit verdanke ich einen großen Teil des Erfolges auf den nächsten Seiten euch. Danke an das gesamte iAMB. Miriam Agler-Rosenbaum, Nisha Agrawal, Theresia Desi Askitosari, Vanessa Baier, Hendrik Ballerstedt, Johanna, Becker, Gisela Beissel, Carola Berger, Lars Blank, Nilam Borah, Erick Bosire, Monika Brehm, Henrik Cordes, Eik Czarnotta, Tatiana de Campos Rodrigues, Arnoldine Carlier, Frank Drechsler, Deiziane Dutra, Birgitta Ebert, Ute Ewodo, Jan Förster, Elena Geiser, Andrea Germer, Hao Guo, Christoph Halbfeld, Birthe Halmschlag, Malte Heyer, Hamed Hosseinpour Tehrani, Kalle Hüser, Elke Kaußen, Kristina Kirchner, Thomas Kirchner, Sebastian Köbbing, Manja Kropp, Lars Küpfer, Jannis Küpper, Mathias Lehnen, Christoph Lenzen, Bernd Leuchtle, Wing-Jin Li, Ulf Liebal, Roberta Lovaglio, Bastian Molitor, Salome Nies, Maike Otto, Liesa Pötschke, Alana Poloni, Sandra Kathrin Przybilla, Eda Sarikaya, Jan Schirawski, Ivan Schlembach, Ulrike Schmitt, Andreas Schmitz, Simone Schmitz, Annette Schreer, Julia Schütte, Sandra Schulte, Dario Silva Neves, Suresh Sudarsan, Christoph Thiel, Till Tiso, Ronny Uhlig, Kerstin Walter, Nick Wierckx, Theresa Wollenberg, Benedikt Wynands, Wie Xie, Ahmed Zahoor ul Hassan, Yulei Zhao, Rabea Zauter, Martin Zimmermann, Sebastian Zobel. Danke an euch alle für eine wunderschöne Zeit am iAMB während der vielen letzten Jahre. Ihr habt die Zeit echt zu einem Erlebnis und das iAMB zu dem, was es ist, gemacht. Diese Erfahrungen möchte ich um keinen Preis missen. Ein besonderer Dank geht an meine beiden langjährigen Büro- und Labornachbarn, Benedikt und Christoph. Ohne euch hätte so mancher anstrengende Tag im Institut sicherlich nicht ansatzweise so unterhaltsam geendet oder auch bereits begonnen, obwohl einige Tage sicherlich früher geendet hätten. Außerdem möchte ich mich bei Dr. Martin Zimmermann für die außergewöhnliche Betreuung während des gesamten Studiums bedanken. Ohne dich wäre so mancher Student, mich eingeschlossen, in einigen Situationen echt verloren gewesen. Durch deinen Einsatz wurden neue Perspektiven eröffnet und verloren geglaubte Möglichkeiten zurückgewonnen. Bedanken möchte ich mich auch bei Jörg Büscher und Guido Meurer, die durch Input in unzähligen Telefonkonferenzen und Meetings zum überaus erfolgreichen Gelingen des Projektes beigetragen haben. Ein großes ÄDanke³ geht an meine Jungenz (und zwar nicht nur den ursprünglichen Kern, sondern alle, die über die Jahre hinzugekommen sind) dafür, dass ihr mein Arbeitspensum und die damit verbunden wenige Zeit akzeptiert. Auch wenn wir oftmals nur noch selten etwas unternehmen können, steht ihr mir wirklich in allen Situationen zur Seite und ich weiß, dass ich mich immer 100prozentig auf euch verlassen kann, wenn ich es brauche. Danke dafür. Ganz besonderer Dank geht an das Possum. Last but not least möchte ich mich bei meiner gesamten Familie bedanken. Danke, dass ihr immer an mich glaubt und mich unterstützt, auch wenn ihr mich und meine Entscheidungen sicherlich nicht immer verstehen könnt. Außerdem holt ihr mich oftmals auf den Boden der Tatsachen zurück ± oder versucht es zumindest. Auch wenn ich mir das meistens nicht eingestehe, brauche ich das ziemlich sicher von Zeit zu Zeit. Funding The project was partially funded by Biotechnology Research And Information Network AG (BRAIN AG) and by the German Federal Ministry of Education and Research (BMBF) as part of the Strategic Alliance ZeroCarbFP (grant no. FKZ 031A217F) Table of Contents SUMMARY ......................................................................................................................................
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