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Engineering of Cytochrome P450s CYP109E1 and CYP109A2 from Bacillus Megaterium DSM319 for The Engineering of Cytochrome P450s CYP109E1 and CYP109A2 from Bacillus megaterium DSM319 for the production of vitamin D3 metabolites Kumulative Dissertation zur Erlangen des Grades des Doktors der Naturwissenschaften der Naturwissenschaftlich-Technischen Fakultät der Universität des Saarlandes von Ammar Abdulmughni Saarbrücken 2018 Tag des Kolloquiums : 14.08.2018 Dekan : Prof. Dr. Guido Kickelbick Berichterstatter : Prof. Dr. Rita Bernhardt Prof. Dr. Gert-Wieland Kohring Vorsitz : Prof. Dr. Uli Müller Akad. Mitarbeiter: Dr. Ing. Michael Kohlstedt Abstract Active vitamin D3 metabolites play an essential role in the maintenance of calcium and phosphorus homeostasis. The conventional chemical synthesis of this metabolite is time- consuming, environmentally unfriendly and often results in low yield. Therefore, the biotechnological production of active vitamin D3 metabolites is of great importance to the pharmaceutical industry. Hereby, cytochrome P450 enzymes have the potential to achieve this goal. The present work reports on the optimization of a biotechnological process in Bacillus megaterium MS941 for the production of vitamin D3 metabolites. On that account, two cytochrome P450 enzymes were used as biocataylsts, namely CYP109E1 and CYP109A2 from the Gram-positive bacterium Bacillus megaterium DSM319. Both enzymes were subjected for functional and structural characterization in order to optimize their activity and/or regio-selectivity towards vitamin D3. In terms of hydroxylation activity, it has been shown that the conversion of vitamin D3 with CYP109E1 results in the formation of several derivatives, while CYP109A2 shows clearly a higher regio-selectivity towards 25- hydroxylation. The elucidation of the crystal structure of both enzymes provides detailed insights into the geometry of these enzymes. By means of molecular docking, site-directed mutagenesis was successfully performed, resulting in the creation of mutants with higher regio-selectivity compared to the wild type, in particular when using CYP109E1. The enhancement of the regio-selectivity of this P450 resulted in a higher yield of the valuable metabolite 25-hydroxyvitamin D3. A two-fold increase of the 25-hydroxyvitamin D3 yield was achieved after the substitution of I85 by tryptophan or alanine. The engineering of CYP109A2 resulted also in a slight increase in the 25-hydroxyvitamin D3 yield compared to the wild type. Summarizing, it has been shown that CYP109E1-I85W, CYP109A2-, - or CYP109-T103A-based whole-cell systems are a competitive methods for the production of 25-hydroxyvitamin D3 among the previously established biotechnological processes. I Zusammenfassung Aktive Vitamin-D3-Metaboliten spielen eine wesentliche Rolle bei der Aufrechterhaltung der Calcium- und Phosphor-Homöostase. Die herkömmliche chemische Synthese dieser Metaboliten ist zeitaufwendig, umweltschädlich und führt oft zu geringer Ausbeute. Daher ist die biotechnologische Herstellung von aktiven Vitamin-D3-Metaboliten für die pharmazeutische Industrie von großer Bedeutung. Der Einsatz von Cytochromen P450 gilt hierbei als vielversprechend. Die vorliegende Arbeit beschäftigt sich mit der Optimierung eines biotechnologischen Prozesses in Bacillus megaterium MS941 zur Produktion von Vitamin-D3-Metaboliten. Als Biokatalysatoren wurden zwei Cytochrome-P450-Enzyme verwendet, nämlich CYP109E1 und CYP109A2 aus dem Gram-positiven Bakterium Bacillus megaterium DSM319. Beide Enzyme wurden einer funktionellen und strukturellen Charakterisierung unterzogen, um ihre Aktivität und / oder Regio-Selektivität gegenüber Vitamin-D3 zu optimieren. Bezüglich der Hydroxylierungsaktivität wurde gezeigt, dass die Umwandlung von Vitamin-D3 mit CYP109E1 zur Bildung mehrerer Derivate führt, während CYP109A2 deutlich eine höhere Regio-Selektivität bezüglich einer 25-Hydroxylierung zeigt. Die Aufklärung der Kristallstruktur beider Enzyme liefert detaillierte Einblicke in die Geometrie dieser Enzyme. Mit Hilfe des molekularen Dockings wurde eine ortsgerichtete Mutagenese erfolgreich durchgeführt, wodurch im Vergleich zum Wildtyp, insbesondere von CYP109E1, Mutanten mit höherer Regio-Selektivität hergestellt wurden. Die Optimierung der Regio-Selektivität dieses P450 führte zu einer höheren Ausbeute des wertvollen Metaboliten 25- Hydroxyvitamin-D3. Die Produktion von 25-Hydroxyvitamin-D3 konnte durch die Substitution von I85 durch Tryptophan oder Alanin verdoppelt werden. Die Optimierung von CYP109A2 führte ebenfalls zu einem leichten Anstieg der 25-Hydroxyvitamin-D3- Ausbeute im Vergleich zum Wildtyp. Zusammenfassend wurde gezeigt, dass CYP109E1- I85W-, CYP109A2-, oder CYP109-T103A-basierte Ganzzellsysteme eine wettbewerbsfähige Methode zur Herstellung von 25-Hydroxyvitamin-D3 unter den bisher etablierten biotechnologischen Verfahren darstellen. II Danksagung Ich bedanke mich herzlich bei all den Menschen, die mich immer unterstützt und mir dadurch ermöglicht haben, diese Arbeit zu beenden. Mein besonderer Dank gilt meiner Doktormutter Frau Prof. Rita Bernhardt. Ich danke ihr für die Möglichkeit, dass ich meine Doktorarbeit in ihrem Arbeitskreis anfertigen konnte. Ich bedanke mich bei ihr auch für die großartige wissenschaftliche Betreuung und die wertvollen Anregungen und Diskussionen sowohl in beruflichen als auch persönlichen Angelegenheiten. Von ihr habe ich sehr viel gelernt. Prof. Dr. Gert-Wieland Kohring danke ich herzlich für die Übernahme des Korreferates. Ich bedanke mich bei Dr. Frank Hannemann für Unterstützung und die hervorragende wissenschaftliche Beratung. Ganz besonders bedanke ich mich bei Frau Dr. Ilona Jóźwik und Dr. Andy-Mark Thunnissen von der Reichuniversität Groningen für die ausgezeichnete und unkomplizierte Zusammenarbeit. Ich bedanke mich bei allen Mitgliedern der Arbeitsgruppe für die angenehme und hervorragende Zusammenarbeit. Mein Dank gilt besonderes Dr. Simone Brixius-Anderko, Lisa König, Benjamin Stenger, Natalia Putkaradze, Dr. Martin Litzenburger und Tanja Sagadin. Ich bedanke mich auch bei Birgit Heider-Lips und Antje Eiden-Plach für ihre stetige Hilfsbereitschaft und ihr Interesse, welche auch über die Arbeit hinausgehen. Im Speziellen bedanke ich mich bei Dr. Mohammed Milhim und Philip Hartz für die zahlreichen und nützlichen Tipps und sehr interessanten Diskussionen in allen möglichen fachlichen und nicht-fachlichen Gebieten. Für ihre Hilfe bei allen bürokratischen Angelegenheiten möchte ich mich auch bei Gabi Schon bedanken. Meinen Freunden Danae, Didi, Ghamdan, Jannis, Katrin, Lisa, Shadi, Sherif und seiner Frau Iva, Sheikh, Tarik und Verena danke ich für die Unterstützung. Ich bin sehr froh, dass es euch gibt. Ich danke besonders meiner Esther dafür, dass sie immer an mich glaubt und mir bei schwierigen Zeiten zur Seite steht. III Mein größter Dank gilt meiner großen Familie, insbesondere meinen Eltern, ohne deren bedingungslose Liebe, Geduld und Unterstützung all das nicht möglich wäre. An dieser Stelle möchte ich an einen wunderbaren Menschen erinnern, meinen besten Freund Musaab Al-Tuwaijary, dem ich immer mein Herz ausschütten konnte. Ich vermisse dich. IV Contents Abstract……..………………………………………………………………………………I Zusammenfassung…………………………………………………………………………II Danksagung………………………………………………………………………………..III Contents.…………………………………………………………………………………....V Scientific contributions…………………………………………………………………..VII 1. Introduction……………………………………………………………………………...1 1.1 Protein engineering…………………………………………………………….1 1.2 Vitamin D3……………………………………………………………………..2 1.3 Metabolism of vitamin D3…………………………………………………….. 4 1.4 Action of the hormone 1α,25-dihydroxyvitamin D3…………………………...6 1.5 Cytochrome P450 monooxygenases…………………………………………...6 1.6 Electron transfer in cytochrome P450 systems………………………………...9 1.7 Structure of P450 enzymes…………………………………………………….9 1.8 Cytochrome P450s for the production of active vitamin D3 metabolites……..13 1.8 Aim of this work……………………………………………………………....14 2. Scientific articles………………………………………………………………………..15 2.1 Abdulmughni et al (2017a)……………………………………………………15 2.2 Abdulmughni et al (2017b)…………………………………………………...26 2.3 Jóźwik et al (2016)….………………………………………………………...44 2.4 Putkaradze et al (2017)………………………………………………………..72 3. General discussion ……………………………………………………………………103 3.1 Bioconversion of vitamin D3 by CYP109E1 and CYP109A2………………104 3.2 Structural aspects of CYP109E1 and CYP109A2…………………………...108 3.3 Engineering of CYP109E1…………………………………………………..109 3.4 Engineering of CYP109A2…………………………………………………..112 V 4. Outlook……………………………………………………………………...................117 5. Appendix………………………………………………………………………………119 6. Abbreviations…………………………………………………………………………122 7. References……………………………………………………………………………..123 VI Scientific contributions The work is based on four original research papers reproduced in chapter 2 with permission of Journal of Biotechnology (2.1 Abdulmughni et al 2017a), FEBS Journal (2.2 Abdulmughni et al 2017b and 2.3 Jóźwik et al (2016)) and Appl Microbiol Biotechnol (2.4 Pukaradze et al (2017)). 2.1 Abdulmughni A, Jóźwik IK, Putkaradze N, Brill E, Zapp J, Thunnissen AW, Hannemann F, Bernhardt R. (2017a). Characterization of cytochrome P450 CYP109E1 from Bacillus megaterium as a novel vitamin D3 hydroxylase. J Biotechnol. 243, 38 –47. The author performed all in vitro and in vivo experiments with CYP109E1 and its mutants, designed and carried out the site-directed mutagenesis. Furthermore, the author participated in the product purification and to writing the manuscript. 2.2 Abdulmughni A, Jóźwik IK, Brill E, Hannemann F, Thunnissen AMWH, Bernhardt R (2017b) Biochemical and structural
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