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Molecular Genetic Approaches to Decrease Mis-Incorporation of Non Molecular genetic approaches to decrease mis-incorporation of non-canonical branched chain amino acids into a recombinant protein in Escherichia coli Ángel Córcoles García Molecular genetic approaches to decrease mis- incorporation of non-canonical branched chain amino acids into a recombinant protein in Escherichia coli Ángel Córcoles García - Dissertation Abstract II Molecular genetic approaches to decrease mis-incorporation of non-canonical branched chain amino acids into a recombinant protein in Escherichia coli vorgelegt von M. Sc. Ángel Córcoles García ORCID: 0000-0001-9300-5780 von der Fakultät III-Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktor der Naturwissenschaften - Dr. rer. nat. - genehmigte Dissertation Promotionsausschuss: Vorsitzender: Prof. Dr. Juri Rappsilber, Institut für Biotechnologie, TU Berlin, Berlin Gutachter: Prof. Dr. Peter Neubauer, Institut für Biotechnologie, TU Berlin, Berlin Gutachter: Prof. Dr. Pau Ferrer, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), Spain Gutachter: Dr. Heinrich Decker, Sanofi-Aventis Deutschland GmbH, Frankfurt am Main Tag der wissenschaftlichen Aussprache: 11. Dezember 2019 Berlin 2020 Molecular genetic approaches to decrease mis-incorporation of non-canonical branched chain amino acids into a recombinant protein in Escherichia coli Ángel Córcoles García Abstract The incorporation of non-canonical branched chain amino acids (ncBCAA) such as norleucine, norvaline and β-methylnorleucine into recombinant proteins during E.coli production processes has become a crucial matter of contention in the pharmaceutical industry, since such mis-incorporation can lead to production of altered proteins, having non optimal characteristics. Hence, a need exists for novel strategies valuable for preventing the mis-incorporation of ncBCAA into recombinant proteins. This work presents the development of novel E. coli K-12 BW25113 strain mutants allowing exogenous tunable expression of target genes involved in the BCAA biosynthetic pathway. For that purpose, following single genes were knocked out by homologous recombination in order to eliminate endogenous genetic expression: thrA, ilvA, leuA, ilvIH, ilvBN, ilvGM and ilvC. Expression regulation of previously knocked-out genes was carried out by transforming arabinose-based tunable expression plasmids containing the native sequence of target genes into the generated E. coli KO mutants. The engineered E. coli hosts were screened in a mini-reactor system in fed-batch mode under both standard cultivation conditions and under conditions reproducing large-scale effects. During screening three different concentrations of L-arabinose were tested for each strain in order to trigger different expression levels of a target gene. Screening was performed by comparing the impurity profile of the recombinant mini-proinsulin expressed in each tested strain with the non- engineered E. coli host. After screening, potential E. coli mutants showing the most significant ncBCAA reduction were scaled-up to a 15L stirred tank bioreactor and analysed to confirm their behaviour. Screening experiments carried out in the mini-reactor system indicated that an up-regulation of ilvC, ilvIH and ilvGM and down-regulation of leuA and ilvBN trigger a reduction of norvaline and norleucine biosynthesis and mis-incorporation into mini-proinsulin. Results for ilvA and thrA suggest that the threonine pathway is not the one redirecting more metabolic flux to α-ketobutyrate. Moreover, it is noteworthy that norleucine was the most mis-incorporated ncBCAA and β- methylnorleucine levels did not significantly change under tested experimental conditions. Furthermore, a novel cultivation strategy consisting of pyruvate pulsing and oxygen limitation was demonstrated to successfully mimic large-scale effects since biosynthesis of ncBCAA and metabolites of the overflow metabolism were reported under those conditions. Among the tested genes, up- regulation of ilvIH and ilvGM showed the highest reduction of ncBCAA biosynthesis and mis- incorporation. Reproducibility of potential ilvIH and ilvGM-tunable E. coli strains was confirmed in the 15L reactor.These novel E. coli strains with a reduced ncBCAA mis-incorporation may have a crucial role in the pharmaceutical industry since product quality is pivotal for commercial approval of recombinant proteins that are to be used as human therapeutics. I Abstract Molecular genetic approaches to decrease mis-incorporation of non-canonical Ángel Córcoles García branched chain amino acids into a recombinant protein in Escherichia coli Zusammenfassung Der Fehleinbau von nichtkanonischen verzweigtkettigen Aminosäuren (ncBCAA) wie Norleucin, Norvalin und β-Methylnorleucin in rekombinante Proteine während E. coli - Proteinproduktionsprozessen ist in der pharmazeutischen Industrie zu einem entscheidenden Kriterium geworden, da ein solcher Fehleinbau zur Produktion veränderter rekombinanter Proteine mit suboptimalen Eigenschaften führen kann. Daher besteht ein Bedarf an neuen Strategien, um den Fehleinbau von ncBCAA in rekombinante Proteine zu verhindern. Diese Arbeit beinhaltet die Entwicklung von neuen E. coli K-12 BW25113-Mutantstämmen, in denen die Expression von Zielgenen, die am BCAA-Biosyntheseweg beteiligt sind, extern gesteuert werden kann. Zu diesem Zweck wurden folgende Gene mittels homologer Rekombination inaktiviert: thrA, ilvA, leuA, ilvIH, ilvBN, ilvGM und ilvC. Gleichzeitig wurden diese Mutanten durch Plasmide komplementiert, die das chromosomal inaktivierte Gen unter Kontrolle eines regulierbaren Arabinose-Promoters enthalten. Diese E. coli-Stämme wurden in einem Mini-Bioreaktorsystem im Fed-batch-Modus sowohl unter Standardkultivierungsbedingungen als auch im Scale-down-Simulator mit pulsbasierter Zufütterung untersucht. Während des Screenings wurden für jeden Stamm drei verschiedene Konzentrationen von L-Arabinose getestet, um unterschiedliche Expressionsniveaus eines Zielgens auszulösen. Für jeden Stamm wurde dann der Fehleinbau nichtkanonischer Aminosäuren in rekombinant exprimiertes Mini-Proinsulins quantifiziert und mit dem nicht- manipulierten E. coli-Kontrollstamm verglichen. Nach dem Screening wurden E. coli-Mutanten mit einem signifikant verminderten Fehleinbau zur Validierung der Ergebnisse im Laborreaktor unter Scale-down Bedingungen getestet. Bei dem im Mini-Reaktorsystem durchgeführten Screening zeigte eine Erhöhung der Expression von ilvC, ilvIH bzw. ilvGM sowie eine Verminderung der Expression von leuA bzw. ilvBN eine Reduktion der Norvalin- und Norleucin-Biosynthese und einen geringeren Fehleinbau in das Mini-Proinsulin. Die Änderung der Expression der Gene ilvA und thrA zeigten keinen signifikanten Einfluss. Dies bestätigt die Hypothese, dass die Synthese der nichtkanonischen Aminosäuren nicht über den Threoninweg, sondern durch die direkte Kettenverlängeurng von Pyruvat verursacht wird. Außerdem, ist es bemerkenswert, dass Norleucin die am häufigsten falsch eingebaute nichtkanonische Aminosäure war und sich die β-Methylnorleucin-Spiegel unter getesteten Versuchsbedingungen nicht signifikant änderten. Darüber hinaus konnte gezeigt werden, dass eine neuartige Kultivierungsstrategie, die auf Pyruvat-Pulsen bei gleichzeitiger Sauerstofflimitierung beruht, Scale-down-Effekte erfolgreich imitieren kann. Von den getesteten Zielgenen zeigte eine Expressionssteigerung von ilvIH bzw. ilvGM den signifikantesten Effekt bezüglich einer verminderten Synthese der nichtkanonischen Aminosären und dem Fehleinbau. Diese neuartigen E. coli-Stämme, die einen verminderten Fehleinbau nichtkanonischer Aminosäuren zur Folge haben, können eine entscheidende Rolle in der pharmazeutischen Industrie spielen, da die Produktqualität für die Zulassung rekombinanter Proteine als Humantherapeutika von entscheidender Bedeutung ist. Zusammenfassung II Molecular genetic approaches to decrease mis-incorporation of non-canonical branched chain amino acids into a recombinant protein in Escherichia coli Ángel Córcoles García List of Contents Abstract .................................................................................................................................................... I Zusammenfassung ................................................................................................................................... II List of Contents ....................................................................................................................................... III Acknowledgements .............................................................................................................................. VIII List of Abbreviations and Symbols .......................................................................................................... X Abbreviations ........................................................................................................................................ X Symbols .............................................................................................................................................. XIII 1. Introduction .................................................................................................................................. 1 2. Literature Review .......................................................................................................................... 3 2.1 E. coli as industrial biofactory ......................................................................................................
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