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Analyse Funktioneller Gene Des Abbaues Tertiärer Etherstrukturen in Dem Bakterienstamm Aquincola Tertiaricarbonis L32: Anhand Von Knock-Out Mutanten

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Analyse Funktioneller Gene Des Abbaues Tertiärer Etherstrukturen in Dem Bakterienstamm Aquincola Tertiaricarbonis L32: Anhand Von Knock-Out Mutanten Analyse funktioneller Gene des Abbaues tertiärer Etherstrukturen in dem Bakterienstamm Aquincola tertiaricarbonis L32: anhand von knock-out Mutanten Von der Fakultät für Biowissenschaften, Pharmazie und Psychologie der Universität Leipzig genehmigte Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt von Diplom-Biologin Judith Christina Schuster geboren am 44. Mai 3;:3 in Dresden Dekan Professorin Dr. Andrea Robitzki Gutachter Professor Dr. Hauke Harms Professor Dr. Michael Schlömann Tag der Verteidigung: 4:. März 4236 Bibliographische Darstellung Judith Christina Schuster Analyse funktioneller Gene des Abbaues tertiärer Etherstrukturen in dem Bakterienstamm Aquincola tertiaricarbonis L32: anhand von knock-out Mutanten Fakultät für Biowissenschaften, Pharmazie und Psychologie Universität Leipzig Dissertation 343 Seiten, 3;9 Literaturangaben, 75 Abbildungen, 5 Tabellen Methyl-tertiär-butylether (MTBE) ist ein synthetischer Ether mit einem verzweigten Koh- lenstoffgerüst, der seit mehreren Jahrzehnten dem Benzin in großen Mengen als Oxygenat zugesetzt wird. Der β-Proteobakterienstamm Aquincola tertiaricarbonis L32: gehört zu den wenigen bisher beschriebenen Reinkulturen mit einem etablierten Metabolismus zur spezifischen und vollständigen, wachstumsgebundenen Mineralisation der xenobiotischen Oxygenat-Ether zu CO2. Der MTBE-Abbau erfolgt oxidativ über die zentralen Interme- diate tert-Butylalkohol (TBA) und 4-Hydroxyisobuttersäure (4-HIBA). In dieser Arbeit werden Mutationsstudien vorgestellt, mit deren Hilfe der bisher postulierte Stoffwechsel bestätigt und die Spezifität der untersuchten Enzyme in den jeweiligen Reak- tionsschritten bewiesen wurde. Parallel wird gezeigt, dass längerkettige tertiäre Substrate über einen abweichenden Abbauweg prozessiert werden. Der Etherabbau wird durch die in Kapitel 4 behandelte Cytochrom P-672-Monooxygenase EthB initiiert. Der weitere Abbau involviert die Alkoholmonooxygenase MdpJ, deren Sub- stratspektrum und Spezifität im Alkoholabbau in Kapitel 5 dargestellt werden. TBA wird durch MdpJ zu 4-Methyl-3,4-propandiol (MPD) hydroxyliert. Dieses Diol wird schließlich zu der Säure 4-HIBA oxidiert, deren weiterhin verzweigte Struktur erst durch die spezi- fische 4-Hydroxyisobutyryl-CoA-Mutase (HCM) entfernt wird. In Kapitel 6 schließt sich die Mutationsstudie der beiden Untereinheiten der HCM (HcmA und HcmB) an. Interessanterweise wird der Metabolit tert-Amylalkohol (TAA) des nur um eine CH4- Gruppe längeren tert-Amylmethylether (TAME) durch die Alkoholmonooxygenase anders prozessiert. MdpJ wirkt hier als Desaturase und leitet damit einen zum TBA-Abbau ab- weichenden metabolischen Weg über ungesättigte Alkohole, sogenannte Hemiterpene ein. Dieser neu entdeckte Weg wird in Kapitel 7 vorgestellt. Die flüchtigen Alkohole TBA bzw. TAA stellen nützliche Indikatoren des natürlichen, in- itialen Abbaus in der Umwelt dar und sind zudem kosteneffizient per Gaschromatographie (GC) analysierbar. Weiterer TBA-Abbau wird anschließend per high performance liquid chromatography (HPLC) detektiert. Der TAA-Abbau kann hingegen über die Hemiterpene direkt mit der primären GC-Analyse verfolgt werden. Das wird zukünftig noch relevanter, denn der Trend geht zu immer längeren tertiären Ethern als Benzinadditive, die eventuell auch eher desaturiert werden. ii Eidesstattliche Erklärung Ich, Judith Christina Schuster, habe diese Arbeit selbständig verfasst und jeden Bezug zu anderen Quellen entsprechend gekennzeichnet. Es wurden nur die in dieser Arbeit ausdrücklich benannten Quellen und Hilfsmittel verwendet. Die kumulative Arbeit enthält vier akzeptierte und bereits veröffentlichte Manuskripte, welche in Zusammenarbeit mit mehreren Co-Autoren entstanden sind. Deren Anteile wur- den jeweils benannt, gegengezeichnet und im Anhang angefügt. Leipzig, 33. Mai 4236 Judith Schuster iii Danksagung An dieser Stelle möchte ich den vielen Menschen meinen Dank aussprechen, die mich auf meinem Weg zur Promotion begleitet und in vielfältiger Art und Weise unterstützt haben. • Professor Dr. Hauke Harms danke ich für seine Unterstützung und die sehr gute Be- treuung meiner Arbeit im Department Umweltmikrobiologie am Helmholtzzentrum für Umweltforschung GmbH – UFZ. • Ihm und Professor Dr. Michael Schlömann von der TU Bergakademie Freiberg bin ich für die Begutachtung meiner Arbeit dankbar. • Dr. Roland Müller und Dr. Thore Rohwerder danke ich für die vielen anregenden Diskussionen zu Fragen, wie „Wie organisiert man ein Paper, wie forscht man effizient ohne viel Datenmüll, wie programmiert man HPLC, GC und Fermenter?“. • Ich danke Cornelia Dilßner, Monika Neytschev, Hans-Jürgen Große, Birgit Würz und Rita Remer für die Hilfe bei der Erarbeitung kleiner und Großer technischer und analytischer Details. • Meinen Kollegen Dr. Franziska Schäfer, Sabine Leibeling, Nadya Yaneva, Denise Przybylski, Torsten Paproth, Annelie Steinbach, Jessica Hübner, Theresa Weichler und Sebastian Eisen sowie meinen Praktikanten Sina Riemschneider, Laura Rose und Ünsal Erdem danke ich für die hervorragende Zusammenarbeit. Besonders meiner Kollegin und Freundin Dr. Franziska Schäfer gilt ein großer Dank für die zahlreichen gemeinsamen Experimente, Kaffees und Publikationen. • Ebenso danke ich allen weiteren Co-Autoren für ihren Anteil an den Manuskripten und damit an meiner kumulativen Arbeit. Meiner spanischen Kollegin Dr. Jessica Purswani danke ich zusätzlich für drei Winter-Monate voll Sonne im Büro und Labor und die Regen-Überschuhe, die mich seitdem das UFZ weitaus komfortabler erreichen ließen. • Mein größter Dank gilt meiner Familie und meinen Freunden, die mich in jeder Hinsicht unterstützt und in meinem Vorhaben bekräftigt haben. Meinem Freund und Helden Daniel Fichtner danke ich von Herzen für seinen Halt in vielerlei Hinsicht. Für Motivation, aber auch Ablenkung durch Überraschungspartys. Für meinen unkaputtbaren Laptop, die Hilfe bei der Transformation meiner Arbeit in LATEX, die anschließende Layout-Optimierung und Korrektur. Kornelia Ehrlich danke ich ebenfalls sehr für ihre Korrekturhilfe. Ich danke euch Allen! iv Abstract The switch to unleaded fuels in the 3;92s and the high air pollution in areas of high population density due to traffic particularly since the 3;;2s required the use of alternative fuel additives to achieve an improvement of the combustion. The utilization of oxygenated hydrocarbons as antiknock additives and so-called oxygenates provided a more complete and efficient combustion with simultaneously less harmful and polluting emissions. These include the synthetic ethers methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), tert-amyl methyl ether (TAME) and tert-amyl ethyl ether (TAEE). MTBE has a particular position as within some years it became the dominant oxygenate worldwide. Since then, over 322.222 leakages, most often in close proximity to gas stations, resulted in just as many oxygenate-contaminated sites of soil and groundwater within few years. The high water solubility of these ethers leads to an especially fast and extensive spread of the contamination plumes. Ether-contaminated groundwater has a turpentine-like taste that is noticed already in really low concentrations. Thus, such water can no longer serve as drinking water and requires a counter-measure. The chemical parameters of oxygenates decrease the efficiency of otherwise successfully applied techniques such as adsorption or aeration. In addition the ethers proved recalcitrant against microbial attack. The search for microorganisms that could degrade these synthetic oxygenates indeed resul- ted in the enrichment of many isolates. The majority of these isolates oxidize the ethers in a cometabolic manner either partially or completely to CO2. However, only few cultures are capable of independent growth on these oxygenates. These include the β-proteobacteria Methylibium petroleiphilum PM3 and Aquincola tertiaricarbonis L32:, of which the latter is of particular interest for the present work. Strain L32: is characterized by good growth on MTBE and is presently the only known isolate which is able to mineralize ETBE, TAME and TAEE at similar rates. This work examined the seemingly particularly well adapted oxygenate ether metabo- lism of strain L32:, that was formerly isolated from an aquifer highly contaminated with MTBE. Via diverse deletion studies key enzymes of the degradative metabolism and their genetic background were clearly identified. Hence, the results of this work contribute to verify so far just hypothesized metabolic steps by detailed enzymatic and genetic studies. Based on detected metabolites, first studies on MTBE biodegradation already postulated an oxidative pathway via TBA, 4-methyl-3,4-propane-diol (MPD) and 4-HIBA. In case of a monoxygenatic hydroxylation of the methoxy group of MTBE a hemiacetale results as reaction product, from which the tertiary alcohol TBA can be formed easily in subsequent reactions. By comparing wild type strain L32: with the spontaneous mutant strain L32, we were now able to clearly show that the cytochrome P-672 monoxygenase system EthABCD accounts solely for this MTBE-oxidizing activity. It is also the only enzyme catalyzing the corresponding hydroxylation of ETBE, TAME and TAEE. In strain L32: this enzyme complex is expressed constitutively. v vi TBA, which is also generated from hydroxylation of ETBE, is, as postulated and verified by this study, degraded by a different monoxygenase resulting in MPD. Via Tn7-mediated mutations this enzyme was confirmed as Rieske non-heme
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