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Implementation of CO2 Fixation Pathways Into Methylorubrum Extorquens AM1

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Implementation of CO2 Fixation Pathways Into Methylorubrum Extorquens AM1 Implementation of CO2 fixation pathways into Methylorubrum extorquens AM1 Dissertation kumulativ zur Erlangung des Grades eines Doktor der Naturwissenschaften (Dr. rer.nat.) des Fachbereichs Biologie der Philipps-Universität Marburg Vorgelegt von Martina Carrillo Camacho Aus Quito, Ecuador Marburg, 2020 Originaldokument gespeichert auf dem Publikationsserver der Philipps-Universität Marburg http://archiv.ub.uni-marburg.de Dieses Werk bzw. Inhalt steht unter einer Creative Commons Namensnennung Keine kommerzielle Nutzung Keine Bearbeitung 3.0 Deutschland Lizenz (CC BY-NC-ND 3.0 DE). Die vollständige Lizenz finden Sie unter: https://creativecommons.org/licenses/by-nc-nd/3.0/de/ Die vorliegende Dissertation wurde von September 2015 bis April 2020 am Fachbereich Biologie unter Leitung von Prof. Dr. Tobias J. Erb angefertigt. Vom Fachbereich Biologie der Philipps-Universität Marburg (Hochschulkennziffer 1180) als Dissertation angenommen am 08.07.2020. Erstgutachter: Prof. Dr. Tobias J. Erb Zweitgutachterin: Prof. Dr. Anke Becker Weitere Mitglieder der Prüfungskommission: Prof. Dr. Lennart Randau Prof. Dr. Victor Sourjik Tag der Disputation: 17.07.2020. Erklärung Ich versichere, dass ich meine Dissertation mit dem Titel „Implementation of CO2 fixation pathways into Methylorubrum extorquens AM1“ selbstständig ohne unerlaubte Hilfe angefertigt und mich dabei keiner anderen als der von mir ausdrücklich bezeichneten Quellen und Hilfsmittel bedient habe. Diese Dissertation wurde in der jetzigen oder einer ähnlichen Form noch bei keiner anderen Hochschule eingereicht und hat noch keinen sonstigen Prüfungszwecken gedient. Marburg, den 23. April 2020 Martina Carrillo Camacho “So let us then try to climb the mountain, not by stepping on what is below us, but to pull us up at what is above us, for my part at the stars…” M. C. Escher A mathematician and artist. TABLE OF CONTENTS Part One: Initial Remarks ......................................................................................... 9 Summary ................................................................................................................ 11 Zusammenfassung .................................................................................................. 13 Publications ............................................................................................................ 15 Introduction ........................................................................................................... 17 Part Two: Results .................................................................................................... 37 Chapter I ................................................................................................................. 39 Design and control of extrachromosomal elements in Methylorubrum extorquens AM1‡ ............................................................................................................................... 39 Chapter II ................................................................................................................ 69 Initial engineering of the Calvin-Benson-Bassham (CBB) cycle in Methylorubrum extorquens AM1‡ ............................................................................................................ 69 Chapter III ............................................................................................................. 107 Engineering and evolving a CO2 fixing ribose metabolism in Methylorubrum extorquens AM1‡ ............................................................................................................................. 107 Chapter IV ............................................................................................................ 137 Exploring in vivo implementation of synthetic carbon fixation cycles‡ ....................... 137 Part Three: Final Remarks ..................................................................................... 177 Discussion ............................................................................................................. 179 Acknowledgements .............................................................................................. 191 Curriculum Vitae ................................................................................................... 192 9 PART ONE: INITIAL REMARKS 10 Part One: Initial Remarks 11 SUMMARY Growth is a prerequisite for life and metabolism plays a central role to achieve and sustain growth. Its function is to obtain energy needed for metabolic (inter)conversions as well as converting nutrients into cellular building blocks used to synthesize macromolecules and thereby the cell itself. The metabolic pathways present in an organism define and limit its growth capabilities. Nowadays, an organism’s core metabolic network might be altered with relative ease through genetic engineering. The modification of biological systems to expand their growth potential was at the core of this thesis with an emphasis on the Alphaproteobacterium Methylorubrum extorquens AM1. The following results were achieved: 1. Novel inducible promoters were designed for M. extorquens AM1 based on the LacI-lacO system that are tight before induction and exhibit good dynamic range. The expression level achieved with these promoters exceeds that of the standard PmxaF promoter making them the strongest promoters described to date for this organism. New extrachromosomal elements termed ‘mini-chromosomes’ based on repABC cassettes were also identified, which maintain a stable unit copy number and faithful inheritance even in the absence of selection. These new DNA vehicles are compatible with each other and with the broad host plasmid used for M. extorquens AM1 so far. A conditionally unstable replicon, Mex-CM4, showed the behavior needed to establish CRISPR-Cas and similar techniques for which transient expression and fast extinction of the replicon are prerequisites. 2. The Calvin-Benson-Bassham (CBB) cycle was introduced into M. extorquens AM1 for heterologous CO2 fixation and assimilation of methanol was disrupted to provide the cell with energy consistent with (chemo)organoautotrophic growth. The operation of a functional CBB cycle was confirmed with 13C-tracer analysis in the engineered strain. Furthermore, a positive growth phenotype and increased cell viability was found dependent on a functional RubisCO. 3. M. extorquens AM1 was rationally engineered and further evolved by serial transfer to improve growth on the novel substrate ribose. Mutations throughout the evolutionary landscape were identified by whole genome (re)sequencing and further characterized genetically and biochemically. The CBB cycle was introduced into ribose evolved clones for simultaneous CO2 fixation and acetate was provided for additional energy. These strains were evolved in semi-chemostat conditions to promote carbon fixation via the CBB cycle. 4. In vivo implementation of a synthetic CO2 fixation cycle, the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, was explored 12 Part One: Initial Remarks in M. extorquens AM1. Suitable candidate enzymes were identified and characterized biochemically for every step of the crotonyl-CoA regeneration module. These new candidates were combined to close the CETCH cycle and various M. extorquens AM1 strains were created to test growth-based selection schemes. 13 ZUSAMMENFASSUNG Wachstum ist eine Voraussetzung für das Leben und der Stoffwechsel spielt eine zentrale Rolle, um Wachstum zu erreichen und zu erhalten. Seine Funktion besteht in der Konservierung von Energie, die für die metabolischen Reaktionen benötigt wird, sowie in der Umwandlung von Nährstoffen in zelluläre Bausteine, die zur Synthese von Makromolekülen und damit der Zelle selbst dienen. Die in einem Organismus vorhandenen Stoffwechselwege definieren und begrenzen daher seine Wachstumsfähigkeit. Heutzutage kann das zentrale Stoffwechselnetzwerk eines Organismus relativ einfach mithilfe der Gentechnik verändert werden. Die Modifikation biologischer Systeme zur Erweiterung ihres Wachstumspotentials stand im Mittelpunkt dieser Arbeit, wobei der Fokus auf das Alphaproteobakterium Methylorubrum extorquens AM1 gelegt wurde. Die folgenden Ergebnisse wurden erzielt: 1. Es wurden neuartige induzierbare Promotoren für M. extorquens AM1 auf der Basis des LacI-lacO-Systems entwickelt, die vor der Induktion dicht sind und einen guten dynamischen Bereich aufweisen. Das mit diesen Promotoren erreichte Expressionsniveau übertrifft das des Standard-PmxaF-Promotors, was sie zu den stärksten Promotoren macht, die bisher für diesen Organismus beschrieben wurden. Weiterhin wurden neue extrachromosomale Elemente identifiziert, die als "Mini-Chromosomen" bezeichnet werden und auf repABC-Kassetten basieren, die auch ohne Selektion eine stabile Einheitskopienzahl und eine sichere Vererbung erhalten. Diese neuen DNA-Vehikel sind untereinander und mit dem Broad-Host-(Range-)Plasmid, das bisher für M. extorquens AM1 verwendet wurde, kompatibel. Ein bedingt instabiles Replikon, Mex-CM4, zeigte das Verhalten, das zur Etablierung von CRISPR-Cas und ähnlichen Techniken erforderlich und für die eine transiente Expression und eine schnelle Auslöschung des Replikons Voraussetzung ist. 2. Der Calvin-Benson-Bassham (CBB)-Zyklus wurde in M. extorquens AM1 zur heterologen CO2-Fixierung eingeführt. Dazu wurde die Kohlenstoffassimilierung aus Methanol unterbrochen, um die Zelle im Einklang mit dem (chemo)organotrophen Wachstum mit Energie zu versorgen. Der Betrieb
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