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 spezifische 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 abweichenden 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, initialen 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 wurden 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 resulted 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 metabolism 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 mononuclear iron monooxygenase MdpJ. MPD is further altered to the corresponding branched acid 4-HIBA, presumably by two dehydrogenation reactions. For the degradation of 4-HIBA, diverse hypotheses exist on the basis of known enzymatic reactions. Another Tn7 mutation now gave evidence, that in the mentioned β-proteobacteria the novel mutase HcmAB linearizes 4-HIBA to 5-hydroxybutyric acid (5-HB) dependent on cobalamin and coenzyme A (CoA). Sequence comparison revealed, that strain L32: acquired all three key enzyme complexes, EthABCD, MdpJ and HcmAB via horizontal gene transfer (HGT). For TAME and TAEE a completely new degradation pathway was found. In strain L32:, the resulting degradation product tert-amyl alcohol (TAA) of these ethers is, like TBA, also specifically oxidized by MdpJ. In Tn7-deletion studies and metabolite analyzes, however, no hydroxylation could be detected. Instead, TAA is rather desaturated. Therefore within the metabolism no diols or acids analogue to MPD or 4-HIBA were formed. Instead, via MdpJ TAA is initially degraded to the unsaturated tertiary alcohol and hemiterpene 4-methyl-5-butene-4-ol. Prenol (5-methyl-4-buten-4-ol), prenal (5-methyl-4-buten-4-al) and 5-methyl crotonic acid were detected as additional metabolites. Hence, an isomerization of the branched acid by HcmAB is apparently irrelevant in TAA biodegradation. Accordingly it could be shown, that deletion mutants for HcmAB indeed could not grow on TBA, but are still able to grow on TAA, just as fast as the wild type, in fact. The tertiary alcohol 4-methyl-5-butene-4-ol is presumably transformed via another isomerase resulting in the primary alcohol prenol. Prenol is further oxidized by postulated dehydrogenases to 5-methyl crotonic acid. This would also be in correlation to the already observed degradation pathway of the monoterpene linalool in other bacteria. However, the responsible enzymes in strain L32: are not yet identified. Besides this principal gain of knowledge in the degradation of xenobiotic ether structures and the evolution of degradative microorganism, the now confirmed key enzymes EthB, MdpJ and HcmAB, respectively their coding genes can be used as specific markers to monitor natural degradation processes in in situ studies. On this basis, the presence of active microorganisms and additionally — derived from the confirmed single key enzymes — a potentially complete degradation can be concluded. In the long run, it might be possible to stimulate the natural microbiological activity, e.g. via bioaugmentation with degradation specialists. Furthermore, regarding the potential progress of remediation procedures, potentially li- miting steps can be distinguished via respective markers and narrowed down as possible cause of deficient degradation activity. However, such function-based monitoring requires specific verification. Therefore, subsequent studies have to analyze, if there is a sequence diversity among these three key enzymes. Previous sequence comparisons hypothesize that up to 82% accordance in the protein sequence in homologues of MdpJ and HcmA they can still be assumed to possess the same enzymatic function. This diversity has to be considered in the development of specific probes.

Dissertation Judith Schuster Kurzfassung

Die Einführung bleifreien Benzins in den 3;92er-Jahren und die hohe Emissionsbelastung von Ballungszentren durch den Straßenverkehr insbesondere seit den 3;;2er-Jahren erforderte den Einsatz alternativer Benzinadditive, um eine Verbesserung der Verbrennung zu erreichen. Die Nutzung sauerstoffhaltiger Kohlenwasserstoffe als Antiklopfmittel und als sogenannte Oxygenate bot sich an, da diese eine effizientere Verbrennung mit gleichzeitig niedrigeren gesundheits- und umweltschädigenden Emissionen fördern. Zu den Oxygenaten gehören die synthetischen Ether Methyl-tert-butylether (MTBE), Ethyl-tert-butylether (ETBE), tert-Amylmethylether (TAME) und tert-Amylethylether (TAEE). Eine herausragende Stellung nimmt MTBE ein. Innerhalb weniger Jahre wurde es zum hauptsächlich verwendeten Oxygenat weltweit. Seitdem führten jedoch über 322.222 Leckagen, zumeist in Tankstellennähe, innerhalb weniger Jahre zu ebenso zahlreichen Kontaminationen des Grundwassers mit Oxygenaten. Aufgrund der hohen Wasserlöslichkeit kommt es dabei zu einer besonders schnellen und großflächigen Ausbreitung der Ether. Derart belastetes Grundwasser weist schon bei geringsten Etherkonzentrationen einen als terpentinartig wahrgenommenen Geruch und Geschmack auf und kann daher nicht mehr als Trinkwasserzufuhr genutzt werden. Es bedarf einer Lösung dieses Problems. Die chemischen Parameter der Ether senken allerdings die Effizienz anderweitig erfolgreich genutzter technischer Sanierungsverfahren auf Basis von z. B. Adsorption oder Aerisierung. Auch gegenüber mikrobiellen Abbau erweisen sie sich als rekalzitrant. Die Suche nach oxygenatabbauenden Mikroorganismen führte zwar zur Anreicherung vie- ler Isolate, welche die Oxygenate cometabolisch partiell oder sogar komplett oxidieren, nur sehr wenige Kulturen sind aber zu autarkem Wachstum auf diesen Ethern fähig. Dazu gehören die β-Proteobacteria Methylibium petroleiphilum PM3 und der dieser Arbeit zugrunde liegende Aquincola tertiaricarbonis L32:. Der Stamm L32: zeichnet sich durch ein vergleichsweise gutes Wachstum auf MTBE aus und ist als bisher einzig bekanntes Isolat in der Lage, auch ETBE, TAME und TAEE ähnlich schnell zu mineralisieren. Die vorliegende Arbeit handelt von dem scheinbar besonders gut an den Oxygenatabbau adaptierten Stoffwechsel des ursprünglich aus MTBE-kontaminiertem Grundwasser ange- reicherten Stammes L32:. Durch verschiedene Deletionsstudien wurden Schlüsselenzyme des Abbaus und deren genetischer Hintergrund eindeutig identifiziert. Die Ergebnisse der genetischen, enzymatischen und physiologischen Studien des Wildtyps im Vergleich zu den erzeugten Deletionsstämmen tragen dazu bei, bisher nur postulierte Reaktionsschritte zu verifizieren. Schon seit den ersten Studien zum MTBE-Abbau wird anhand markanter Metabolite ein oxidativer Abbau via TBA, 4-Methyl-3,4-propandiol (MPD) und 4-Hydroxyisobuttersäure (4-HIBA) vermutet. Im Fall einer Hydroxylierung der Methoxygruppe von MTBE wird ein Hemiacetal als Reaktionsprodukt erzeugt, aus dem nachfolgend leicht der tertiäre Alkohol TBA entstehen kann. Durch den Vergleich des Wildtyps mit der Spontanmutante Stamm L32 konnte jetzt gezeigt werden, dass hierfür allein das Cytochrom-P672-Monooxygenasesystem EthABCD

vii viii verantwortlich ist. Dieses katalysiert auch exklusiv die entsprechende Hydroxylierung von ETBE, TAME und TAEE. In Stamm L32: wird das Enzym konstitutiv exprimiert. TBA, das auch aus der Hydroxylierung von ETBE resultiert, wird, wie postuliert und in dieser Arbeit verifiziert, durch eine weitere Monooxygenase zu MPD abgebaut. Durch eine Tn7-Transposon-vermittelte Mutation konnte verifiziert werden, dass es sich bei diesem Enzym um die Rieske-nicht-Häm-Monooxygenase MdpJ handelt. MPD wird im weiteren Verlauf voraussichtlich durch zwei Dehydrogenierungen zur korrespondierenden, verzweigten Säure 4-HIBA gewandelt. Zum 4-HIBA-Abbau gibt es, basierend auf bekannten Enzymreaktionen, diverse Hypo- thesen. Anhand einer weiteren Tn7-Mutation konnte jetzt bestätigt werden, dass in den genannten β-Proteobacteria die neuartige Mutase HcmAB wirksam ist, welche 4-HIBA abhängig von Cobalamin und Coenzym A (CoA) zu 5-Hydroxybuttersäure (5-HB) linearisiert. Sequenzvergleiche ergaben, dass Stamm L32: die Schlüsselenzyme des Etherabbaus, EthABCD, MdpJ und HcmAB, durch horizontalen Gentransfer erworben hat. Für TAME und TAEE wurde ein völlig neuer Abbauweg gefunden. In Stamm L32: wird der beim Abbau dieser Ether entstehende tert-Amylalkohol (TAA) wie TBA ebenfalls exklusiv durch MdpJ oxidiert. Durch die Tn7-Deletionsstudien und durch Analyse der Metabolite konnte allerdings keine Hydroxylierung nachgewiesen werden. TAA wird durch MdpJ vielmehr desaturiert. Somit entstehen im Abbauweg keine zu MPD und 4-HIBA analogen Diole und Säuren, sondern TAA wird zunächst durch MdpJ zu einem ungesättigten tertiären Alkohol, dem Hemiter- pen 4-Methyl-5-buten-4-ol, abgebaut. Prenol (5-Methyl-4-buten-4-ol), Prenal (5-Methyl-4-buten-4-al) und 5-Methylcrotonsäure wurden als weitere Metabolite des TAA-Stoffwechsels detektiert. Somit spielt eine Isome- risierung einer tertiär verzweigten Säure durch HcmAB im TAA-Abbauweg offensichtlich keine Rolle. Entsprechend konnte gezeigt werden, dass Deletionsmutanten für hcmAB zwar nicht mehr auf TBA, aber immer noch auf TAA wachsen können, und das genauso schnell, wie der Wildtyp. Der tertiäre Alkohol 4-Methyl-5-buten-4-ol wird wahrscheinlich durch eine andere Isome- rase zum primären Alkohol Prenol umgewandelt und dieser dann durch Dehydrogenasen zur Methylcrotonsäure oxidiert. Dies würde dem bereits in anderen Bakterien beobachteten Abbauweg des Monoterpens Linalool entsprechen. Die in Stamm L32: dafür verantwortlichen Enzyme wurden aber noch nicht identifiziert. Neben diesem grundsätzlichen Erkenntnisgewinn zum Abbau der xenobiotischen Ether- verbindungen und der Evolution degradativer Mikroorganismen, können die hier bestä- tigten Schlüssel-enzyme EthABCD, MdpJ und HcmAB bzw. deren codierende Gene als spezifische Marker zum Monitoring natürlicher Abbauprozesse für in-situ-Untersuchungen genutzt werden. Auf dieser Basis kann auf die Anwesenheit aktiver Mikroorganismen und zudem noch — abgeleitet aus der Präsenz der einzelnen Schlüsselenzyme — auf einen potenziell kompletten Abbau geschlossen werden. Darauf aufbauend kann die natürliche mikrobiologische Aktivität durch nachfolgende biotechnologische Maßnahmen stimuliert werden, zum Beispiel durch eine Bioaugmentation mit Abbauspezialisten. Des weiteren können mögliche limitierende Schritte hinsichtlich des potenziellen Verlaufs der Sanierungsmaßnahme über Präsenztiter der betreffenden Marker gezielter verfolgt und als etwaige Ursachen defizitärer Abbauleistungen eingegrenzt werden. Voraussetzung für dieses funktionsbasierte Monitoring ist allerdings der spezifische Nachweis. Somit sollte in nachfolgenden Studien analysiert werden, ob es bei den drei Schlüsselenzymen eine

Dissertation Judith Schuster ix

Sequenzdiversität gibt. Die bisherigen Sequenzvergleiche lassen zumindest vermuten, dass bis etwa 82% Übereinstimmung der Proteinsequenzen bei Homologen von MdpJ und HcmA noch mit der gleichen Enzymfunktion zu rechnen ist. Diese Diversität sollte bei der Entwicklung von speziﬁschen Sonden berücksichtigt werden.

Dissertation Judith Schuster Inhaltsverzeichnis

Bibliographische Darstellung...... ii Eidesstattliche Erklärung...... iii Danksagung...... iv Abstract...... v Kurzfassung...... vii Abkürzungsverzeichnis...... xi

3. Einleitung 3 3.3. Tertiäre Ether als Benzin-Oxygenate — Hintergrund und Umweltproblematik 3 3.4. Mikrobiologischer Abbau tertiärer Ether...... 6 3.5. Postulierter Abbauweg...... 9 3.6. Monitoring-Tools für biologischen Abbau...... 34 3.7. Ziel dieser Arbeit...... 35 3.8. Referenzen der Einleitung...... 36

4. Die initiale Etherspaltung des Stammes L32: 3; 4.3. Die Ethermonooxygenase EthB...... 3; 4.4. Supplemental Material...... 49

5. Die speziﬁsche Alkoholmonooxygenase MdpJ 53 5.3. Die Alkoholmonooxygenase MdpJ als Hydroxylase und Reduktase...... 53 5.4. Supplemental Material...... 59

6. Die 4-HIBA-Mutase HcmAB des Stammes L32: 67 6.3. Die 4-HIBA-Mutase HcmAB...... 67 6.4. Supplemental Material...... 78

7. Der TAA-Abbau des Stammes L32: 84 7.3. Der TAA-Abbau des Stammes L32: ...... 84 7.4. Supplemental Material...... 95

8. Diskussion :5 8.3. Nachweis der Schlüsselenzyme in Stamm L32: durch Mutation...... :5 8.4. Nutzen für den Nachweis natürlichen Abbaus...... :6 8.5. Der TAA-Metabolismus als neuartiger Abbauweg...... :8 8.6. Mikrobiologische Anpassung an Xenobiotika am Beispiel MTBE...... :; 8.7. Ausblick...... ;3 8.8. Referenzen der Diskussion...... ;7

Anhang ;: Curriculum Vitæ...... ;; Publikationsverzeichnis...... 322 Tagungsbeiträge...... 323 Nachweis über Anteile der Co-Autoren...... 324

x Abkürzungsverzeichnis

% (w/v) Gewichtsprozent, Massenanteil 4-HIBA 4-Hydroxyisobuttersäure 4-HIBAl 4-Hydroxyisobutyraldehyd 5-HB 5-Hydroxybuttersäure 5M5MP 5-Methoxy-5-methylheptan 6-NP 6(5’,7’-Dimethyl-5’-heptyl)-phenol, technisches Nonylphenol BLAST Basic Local Alignment Search Tool CAA Clean Air Act CoA Coenzym A CSIA compound-speciﬁc stable isotope analysis DCPK Dicyclopropylketon DIPE Diisopropylether ETBE Ethyl-tert-Butylether EthABCD Ether abbauender Enzymkomplex GAC Granular activated carbon GC Gaschromatographie HCM 4-Hydroxyisobutyryl-CoA-Mutase HcmA/B substratbindende bzw. cobalaminbindende Untereinheit der HCM HGT horizontaler Gentransfer HPLC high performance liquid chromatography IARC International Agency for Research on Cancer’s IS5 Insertions-Sequenz des Typs 5

Km Michaelis-Konstante kb Kilobasen kt/a Kilotonnen pro Jahr LiuBD Carboxylase des Leucin-Abbaus, Anteil des Enzymkomplexes LiuABCDE MdpJK TBA-Monooxygenase aus MTBE degradation protein J and K MdpP Acyl-CoA-Synthase der HCM, Annotation analog zu M. petroleiphilum PM3 mM Millimolar MPD 4-Methyl-3,4-propandiol, Methylhydroxypropanol

xi Inhaltsverzeichnis xii

MTBE Methyl-tert-Butylether MTHxE Methyl-tert-hexylether MTOcE Methyl-tert-octylether NA natural attenuation NCBI National Center for Biotechnology Information NDO Naphthalen-Dioxygenase PCR Polymerasekettenreaktion PMO Propanmonooxygenase ppb parts per billion ppbV parts per billion Volume ppm parts per million Prenal 5-Methyl-4-buten-4-al Prenol 5-Methyl-4-buten-4-ol (r)DNA (Ribosomale) Desoxyribonukleinsäure RFG reformulated gasoline (RT)-qPCR (Reverse Transkriptase) quantitative PCR SIP stable isotope probing t, Mt Tonnen, Megatonnen TAA tert-Amylalkohol TAEE tert-Amyl-Ethylether TAME tert-Amyl-Methylether TBA tert-Butylalkohol, tert-Butanol, 4-Methyl-4-Propanol THF Tetrahydrofuran Tn7 Typ-7-Transposon US-EPA United States Environmental Protection Agency

Vmax maximale Abbau-Rate von Nanomol Substrat pro Minute und Milligramm Biotrockenmasse

Ymax maximaler Biomasseertrag in Gramm pro Gramm Substrat

µmax Wachstumsrate pro Stunde kb Kilobasen

Dissertation Judith Schuster 3. Einleitung

3.3. Tertiäre Ether als Benzin-Oxygenate — Hintergrund und Umweltproblematik

xygenate sind sauerstoffhaltige Verbindungen, die dem Benzin als Additive zugesetzt werden, um eine vollständigere Verbrennungsleistung zu erzielen. Dadurch O werden weniger Stickstoffoxide, Kohlenmonoxid und unvollständig verbrannte Kohlenwasserstoffe emittiert. Auch die bodennahe Bildung von Ozon wird reduziert. Die bessere Verbrennungsleistung erhöht gleichzeitig die sogenannte Klopffestigkeit der Otto- Motoren, was den Motorenverschleiß reduziert [3]. Die zugesetzten Oxygenate sind meist Alkohole oder Ether. Die bekanntesten Vertreter beider Gruppen sind Ethanol und der synthetische, tertiäre Ether Methyl-tert-butylether. MTBE setzt sich aus einem tertiär (-tert-) verzweigten Butylrest und einer über eine Etherstruktur gebundenen Methylgruppe zusammen (siehe Abbildung 3.3). Daneben wird Ethyl-tert-butylether (ETBE) als Oxygenat verwendet, welches analog zum MTBE eine Ethylgruppe aufweist. Die beiden anderen eingesetzten tertiären Oxygenat-Ether sind tert- Amylmethylether (TAME) und tert-Amylethylether (TAEE), hier sind die Methyl- bzw. Ethylreste über eine Etherbrücke an einen tert-Amylrest gebunden.

O O O O

MTBE ETBE TAME TAEE

Abb. 3.3.: Schematisierte Strukturen der vier gebräuchlichen tertiären Oxygenat-Ether MTBE, ETBE, TAME und TAEE.

Andere sauerstoffhaltige Additive sind zum Beispiel Methanol und der tertiäre Alkohol tert-Butylalkohol (TBA) sowie Diisopropylether (DIPE), jedoch ist MTBE weltweit das bislang am verbreitetsten eingesetzte Oxygenat. Die Ursachen dafür sind vielfältig. Es kann ökonomisch sehr effizient säurekatalysiert aus Isobuten und Methanol synthetisiert werden. Zudem mischt es sich stabil und homogen mit Benzin und kann problemlos gemeinsam gelagert und transportiert werden [4]. Die Einführung von MTBE als Benzinzusatz begann in den USA mit dem 3;92 ins Leben gerufenen Clean Air Act (CAA), welcher die US-amerikanische Umweltschutzbehörde (US-EPA) dazu ermächtigte, verpflichtende nationale Qualitätsstandards zur Verbesse- rung der Luftqualität zu etablieren. Die Verwendung des giftigen Tetraethylbleis wurde schrittweise eingestellt und seit 3;9; endgültig durch alternative Antiklopfmittel abgelöst [5]. Zuerst waren dies Aromaten wie Toluol oder Benzol. Aufgrund der bekanntgeworde-

3 3. Einleitung 4 nen Kanzerogenität von Benzol wurde dieses dann durch methylsubstituierte Benzole wie Toluol, zunehmend jedoch durch MTBE fast vollständig abgelöst. Zuerst komplett „blei- frei“ war Japan (3;:8), es folgten die USA, Kanada und 4222 die Europäische Union [6, 7]. Anfangs wurde MTBE nur in geringen Mengen als Antiklopfmittel eingesetzt. Entschei- dend für dessen großen Erfolg war erst die Verwendung in deutlich höheren Anteilen als sauerstoffzuführendes Oxygenat. Bedingt durch die ständige Grenzwertüberschreitung ge- sundheitsgefährdender Partikel in vielen US-amerikanischen Großstädten, wurden 3;;2 per Nachtrag zum Clean Air Act stark gesteigerte, emissionssenkende Oxygenatanteile in Benzin vorgeschrieben. In Regionen mit zu hohen Belastungen an Kohlenmonoxid musste Benzin während der Wintermonate mindestens 4,9% (w/v) Sauerstoff enthalten (Oxyfuel), in Regionen mit zu hohem troposphärischem Ozongehalt musste reformuliertes Benzin (RFG) ganzjährig 4% (w/v) Sauerstoff aufweisen. Die Wahl des verwendeten Oxygenats blieb frei und fiel aufgrund der genannten Vorteile in mehr als :2% der Fälle auf MTBE. Um die geforderten Sauerstoffanteile der beiden Benzinarten (Oxyfuel und RFG) zu erhalten, mussten 37% bzw. 32% MTBE als Oxygenat beigemischt werden. Solchermaßen oxygenierte Benzine machten Ende der 3;;2er-Jahre schon ein Drittel des gesamten Benzinmarktes in den USA aus. Die gesteigerte Nachfrage machte MTBE 3;;: mit fast 34 Milliarden Litern (etwa ; Mt) zu einer der am häufigsten produzierten Chemikalien der USA [5]. Der Einsatz der Oxygenat-Ether verursacht jedoch nicht nur positive Effekte. Seit der Produktion in großem Maßstab ab den 3;;2er-Jahren kam es weltweit zu unzähligen Fällen von Havarien und Leckagen an Treibstofflagerstätten, Rohrfernleitungen oder den Raffinerien selbst. 3;;; wurden allein in den USA über 5:7.222 Schadensfälle durch aus- laufende unterirdische Benzinlagertanks bestätigt, von denen geschätzte 472.222 auch MTBE aufwiesen [8]. Durch die schlechte Adsorption an die Bodenmatrix und gleichzeitig sehr gute Wasserlös- lichkeit erreichen die Ether alsbald das Grundwasser und breiten sich dort in Fließrich- tung viel schneller und großflächiger aus, als beispielsweise aromatische Benzinadditive. Dadurch werden immer größere Anteile des Grundwassersystems kontaminiert. Dieses ist eine wichtige Quelle der Trinkwasserversorgung. Etherkontaminiertes Trinkwasser wird jedoch bereits in geringsten Spuren durch einen terpentinartig wahrgenommenen Geruch und Geschmack ungenießbar. Die niedrigsten Schwellenwerte wurden für MTBE und TAME mit 9 – 38 ppb, für ETBE sogar mit nur 3 – 4 ppb angegeben [9]. In etwa 3% der erwähnten Schadensfälle überstie- gen die MTBE-Konzentrationen 42 ppb [:]. Ein viel zitiertes drastisches Beispiel ist die kalifornische Stadt Santa Monica (USA), wo 3;;8 bis zu 832 ppb MTBE in zwei zentralen Brunnenfeldern die Hälfte des städtischen Trinkwassers unbrauchbar machten [5]. Derartig kontaminierte Grundwasserleiter werden meist von der Trinkwasserzufuhr abgeschnitten. Die teilweise gesetzlich regulierten Grenzwerte für Oxygenat-Ether im Trinkwasser orien- tieren sich an den organoleptischen Eigenschaften, aber auch an toxikologischen Beurtei- lungen. Durch die US-EPA wurde ein Richtwert von 42 – 62 ppb angegeben [:]. Diverse Staaten der USA haben gesetzlich regulierte Standards von 7 ppb (Kalifornien) bis 92 ppb (Massachusetts) eingeführt [;, 32]. Ausgehend von der Schadensbilanz in den USA wurden weltweit Kontaminationen des Grundwassers mit MTBE detektiert. Deren Anzahl ist in Europa, bedingt durch den ge- ringeren Etheranteil in Benzin, zwar sehr viel geringer als in den USA [7, 33], trotzdem sind

Dissertation Judith Schuster 3. Einleitung 5 mehrere größere Schadensfälle aufgetreten. In Deutschland wurden die höchsten MTBE- Konzentrationen am Standort einer ehemaligen Raffinerie in Leuna (Sachsen-Anhalt) mit bis zu 3:7 ppm sowie auf dem Gelände eines ehemals durch sowjetische Streitkräfte ge- nutzten Flugplatzes/Tanklagers in Münchenbernsdorf (Thüringen) mit bis zu 4 ppm gefunden [34]. Durch Leckagen verursachte, punktuell stark erhöhte Werte wurden häufig an Tankstellen beobachtet [7], so z. B. ein Fall in England mit einer Konzentration von 325 ppm MTBE sowie 339 ppb TAME. Laut selbiger Studie wurden in Dänemark an einer Tankstelle sogar 752 ppm MTBE detektiert. In Zürich (Schweiz) führte der Unfall eines MTBE-Transportzuges zum Eintrag von 7 t MTBE ins Grundwasser, welches auch Jahre später im Umfeld noch bis zu 972 ppb MTBE enthielt [7]. MTBE wird aufgrund seiner hohen Flüchtigkeit auch in die Atmosphäre eingetragen, wo es aufgrund radikalischer Abbau-Mechanismen allerdings eine bedeutend geringere Halb- wertszeit aufweist, als im Grundwasser. Abhängig von der Besiedelungsdichte, der eingesetzten Oxygenatmenge und den klimatischen Bedingungen ist die Konzentrationsvertei- lung sehr unterschiedlich [35, 36]. Konzentrationsspitzen gibt es erwartungsgemäß während des Betankungsprozesses, also in unmittelbarer Umgebung von Tankstellen. Aber auch die durchschnittlichen Luftwerte können in stark belasteten Ballungsräumen problematisch hoch sein. Ein drastisches Beispiel ist Mexico City mit 62 – 382 ppbV MTBE [35]. Eine Auswaschung aus der Atmosphäre durch Niederschlag bei Temperaturen unter 37°C, im Besonderen durch Schnee, führt zu einer weiteren Verbreitung von MTBE über den Wasserkreislauf und die Atmosphäre und macht dieses zu einem omnipräsenten Schadstoff [36]. Lange Zeit wurde über eine Gesundheitsgefährdung von MTBE für den Menschen diskutiert. Direkter Kontakt mit MTBE wurde mit Atemnot, Asthma, Kopfschmerzen, Schwin- delanfällen, Schlafstörungen und Hautausschlag in Verbindung gebracht [37-39]. Eine Kan- zerogenität, wie sie in Tierstudien mit Mäusen und Ratten erst durch die chronische Expo- sition toxischer Dosen über die Flüssigkeitszufuhr erzeugt wurde, wird für den Menschen derzeit nicht angenommen [39]. Das Gleiche gilt für die Gesundheitsbewertung von TBA. Beobachtete Tumorbildungen in Niere und Hoden männlicher Ratten sowie der Schilddrüse von Mäusen sind an den nagerspezifischen Stoffwechsel gebunden und gelten als nicht für den Menschen relevant [3:]. TBA wurde durch die International Agency for Research on Cancer’s (IARC) in Ka- tegorie 5 als „Nicht einstufbar als kanzerogen für den Menschen“ bewertet. Damit ist die derzeitige TBA-Einstufung sehr ähnlich der von MTBE. Jedoch wirkt Formaldehyd als Metabolit der MTBE-Oxidation genotoxisch und karzinogen [3;], wodurch „die Eliminati- on menschlicher Exposition zu MTBE weiterhin ein wichtiger Bedarf im Gesundheitswesen bleibt“ [42]. Aufgrund der genannten Trinkwasserproblematik sowie der kontrovers geführten Debatte um eine mögliche Gesundheitsgefährdung durch MTBE entschied die US-amerikanische Regierung im März 4222, MTBE als Benzinadditiv zum nächstmöglichen Zeitpunkt komplett vom Markt zu nehmen und durch Ethanol zu ersetzen [43]. Bis 422; war diese Umstellung bereits in 47 Staaten der USA erfolgt [44]. In Europa ist eine dazu gegenläufige Entwicklung zu verzeichnen. Vorerst ist keine Re- duzierung, oder gar ein Bann der Etheranteile geplant. Im Gegenteil wurden die Anteile sogar signifikant erhöht, seit per EU-Verordnung eine Absenkung der Aromatengehalte auf unter 57% festgelegt wurde [8]. Die Aufrechterhaltung der notwendigen Klopffestig- keit der neuen Benzine erforderte eine entsprechende Steigerung der Oxygenatanteile als alternative Additive auf einen Gehalt von bis zu 37%[8].

Dissertation Judith Schuster 3. Einleitung 6

Seitdem wurden die verabschiedeten Direktiven zu erneuerbaren Energien und Benzinqua- lität Haupttreiber bezüglich der Veränderungen künftiger Benzinzusammensetzungen [8, 45]. Durch beide Verordnungen wurden partiell aus nachwachsenden Rohstoffen produ- zierbare Oxygenate, wie ETBE mit der Ethoxygruppe aus biogenem Ethanol, gefördert. Die europäische Produktionskapazität für MTBE, ETBE und TAME stieg innerhalb von acht Jahren von etwa 6 Mt/a auf über 8 Mt/a im Jahr 4232 [46], wobei der Marktanteil von ETBE zu Lasten von MTBE von 37% auf über 82% anstieg. Die Produktionskapazität für TAME wurde 4232 mit 2,7 Mt/a angegeben [46]. Der aktuelle Diskurs zeigt den Spannungsbogen zwischen der derzeitigen Notwendigkeit des Einsatzes von Oxygenaten auf der einen und der Abwendung von Gefahren durch deren weitverbreiteten Einsatz auf der anderen Seite auf. Wie soll man also dieser Gefahr begegnen? In einem aktuellen Review diskutieren Hao et al., [47] die derzeit angewandten Verfahren zur Reinigung von MTBE aus entsprechend verunreinigtem Wasser aus Grundwasser und Kläranlagen. Diverse Adsorptionstechniken, chemische und mikrobiologische Abbaustra- tegien sind inzwischen bekannt. Die ungünstigen physiko-chemischen Eigenschaften des MTBE erschweren jedoch die technischen Reinigungsverfahren, wodurch diese finanziell anspruchsvoll werden und dennoch die Kontaminationen nicht immer vollständig beseiti- gen. Daher sind „neue, einfache und kostengünstige Alternativen, wie ex-situ Reaktoren, na- türliche Abbauprozesse (natural attenuation, NA) und Bioaugmentation zur erfolgreichen Anwendung in der Sanierung MTBE-kontaminierten Grundwas- sers erforderlich“ [5]. Die natural attenuation beinhaltet die Entfernung von Umweltkontaminanten sowohl durch chemische und physikalische Prozesse (Hydrolyse, Verflüchtigung, Verdünnung und Ad- sorption) als auch durch Bioremediation [48, 49]. Erstere NA-Prozesse verlagern aber das Problem eher, als es zu lösen. Favorisiert wird daher die Bioremediation, also letztendlich die vollständige mikrobiologische Mineralisierung des Schadstoffes.

3.4. Mikrobiologischer Abbau tertiärer Ether

Seit dem Bekanntwerden der ersten MTBE-Kontaminationen in Boden und Trinkwasser begann daher besonders die Erforschung des Abbaus durch natürlich vorkommende Mikro- organismen. Anfänglich wurde die Abbaubarkeit unter anaeroben und aeroben Bedingun- gen durch mikrobiologische Aktivitäten als problematisch eingeschätzt und MTBE und die anderen Etherverbindungen prinzipiell als rekalzitrant angesehen [4, 4:]. Diverse anaerobe Proben benzinbelasteten Grundwassers oder Flusssediments des Ohio Rivers (USA) zeigten unter methanogenen, sulfat- und nitratreduzierenden Bedingungen nur in Einzelfällen einen sehr langsamen MTBE-Umsatz, bestenfalls wurden in einem Beispiel 48 der eingesetzten 6: ppm MTBE nach 374 Tagen zu TBA umgesetzt [4:]. Demnach ist anaerober Abbau prinzipiell möglich, jedoch sind die Halbwertszeiten sehr lang. Unter Fe(III)- oder Sulfat-reduzierenden Konditionen wurden diese mit 34 Jahren als nur sehr langsam oder nicht existent angegeben [5]. Johnson et al., [4;] und Wilson et al., [52] schätzten dagegen die Halbwertszeit des MTBE-Abbaus in aerobem Grundwasser auf 4 bis maximal 5 Jahre.

Dissertation Judith Schuster 3. Einleitung 7

Die meisten bisher im Zusammenhang mit Oxygenatabbau isolierten Mikroorganismen sind aerob zu MTBE-Abbau in der Lage. Es lassen sich dabei anhand des Metabolismus zwei Gruppen unterscheiden: Die Mehrzahl der Stämme kann MTBE nur durch Cometa- bolismus abbauen, viele davon sogar nur partiell bis zu TBA. Nur sehr wenige Abbauer weisen hingegen einen eigenen Metabolismus zur vollständigen Mineralisierung und damit verbundenen, heterotrophen Vermehrung auf. Die Fähigkeit, die tertiären Ether abzubauen, ist dabei nicht an ein spezielles Phylum gebunden, sondern zeichnet sich durch eine große Diversität aus, angefangen bei Pilzen wie Graphium sp. und Fusarium solani über Gram-positive Bakterienstämme der Gattungen Rhodococcus und Mycobacterium bis zu Gram-negativen Proteobacteria der Gattungen Pseudomonas, Methylibium und Aquincola [53-59]. Ebenso reicht die Isolationsquelle von MTBE-kontaminierten Böden, Sedimenten und Grundwasser bis zu unbelasteten Böden, Torfboden und Klärschlamm. Ein Überblick über das Spektrum relevanter Stämme ist, gegliedert nach der Art des MTBE-Metabolismus, in Tabelle 3.3 gegeben. Diese ist unterteilt in partielle cometabolische Oxidation, cometabolische Mineralisation sowie heterotrophen Abbau. Die mit 2,3: nmol min−1 mg−1 Biomasse niedrigste Rate wurde für den partiellen cometabolischen MTBE-Abbau zu TBA des Pilzes Graphium sp. ATCC 7:622 ermittelt [53]. Die Gram-positiven Klärschlammisolate R. ruber IFP 4223, R. zopfii IFP 4227 und Gordonia sp. IFP 422; weisen ebenso insignifikante MTBE-Abbauraten auf. In diesen Stämmen wird eine spezifisch durch ETBE induzierbare Reaktion detektiert. Dieses wird sehr gut zu TBA umgesetzt und ermöglicht durch die Mineralisation der Ethoxygruppe sogar Wachs- tum. Die jeweils höchsten Abbauraten werden mit ETBE und ETBE-gewachsenen Zellen erreicht, aber auch die MTBE-Verwertung wird nach ETBE-Induktion deutlich gesteigert (Tabelle 3.3), [5:]. Cometabolische Mineralisation ist in Gegenwart diverser Alkane wie Propan als Kohlenstoff- und Energiequelle u. a. für Gram-positive Actinobacteria, wie Rhodococcus sp. BU5 [5;], Nocardia sp. ENV647 [62], die Mycobacterium austroafricanum Stämme ENV643 und JOB7 [62, 63] sowie Mycobacterium duvalii TA7 [64] beschrieben. Letzterer baut bei Wachstum auf Propan oder Glukose cometabolisch 67% von 44,9 mM MTBE in einer Woche ab. Die für Stamm TA7 angegebene Rate von 3.252 nmol min−1 mg−1 Biomasse entspricht dabei dem theoretisch möglichen Vmax und bezieht sich auf Abbau- versuche mit 2,3 bis 32 mM MTBE in Gegenwart von Glukose. Der ermittelte zugehörige Km-Wert von 8,: mM ist allerdings ziemlich hoch [64].

Dissertation Judith Schuster 3. Einleitung 8

Tab. 3.3.: Maximale MTBE-Abbauraten diverser Reinkulturen mit Primär- und Cometabolismus zum Abbau tertiärer Ether; abgewandelt und erweitert nach Deeb et al., 4222 [65], Schmidt et al., 4226 [66] und Nava et al., 4229 [67].

V MTBE Y µ max andere max max Refe- Kultur Isolationsquelle (nmol min−1mg−1 MTBE MTBE Ether renzen Biotrockenmasse) (g g−1) (h−1)

heterotrophe Mineralisation

Variovorax Anreicherung aus ETBE, 5,72 2,6; 2,234 [68] paradoxus CL-: Aktivschlamm TAME

MTBE- ETBE, 2,267 Aquincola 2,77 kontaminiertes 37,52 TAME, 2,282ETBE [69] tertiaricarbonis L32: 2,:3T AME Grundwasser TAEE 2,277T AME

Methylibium Bioﬁlter zum 33,72 TAME 2,3: [6:] petroleiphilum PM3 MTBE-Abbau

Kläranlage ohne Mycobacterium ETBE, MTBE- austroafricanum 32,22 TAME, 2,66 [6;] Belastungs- IFP 4234 TAEE nachweis

Hydrogenophaga Bioreaktor zum 65,22 n.a. 2,62 [72] ﬂava ENV957 MTBE-Abbau

cometabolische Mineralisation

Pseudomonas benzinhaltiger Bo- 3,;7 n.a. [73] aeruginosa den

Nocardia sp. Propananreicherung ETBE, 4,52 [62] ENV647 aus Torfboden TAME

Mycobacterium sp. Propananreicherung ETBE, 6,82 [62] ENV643 aus Torfboden TAME

Mycobacterium 4-Methylbutan- ETBE, austroafricanum 34,42 [63] Anreicherung TAME JOB7

n-/iso- Xanthobacter sp. 47,72 n.a. [64] Alkanoxidierer

Ethan- Mycobacterium oxidierendes 3.252,22* n.a. [64] duvalii TA7 Bodenisolat

cometabolisch partieller Abbau

Graphium sp. n-Butan/Propan- 2,3: n.a. [53] ATCC 7:622 oxidierende Kultur

Pseudomonas putida Pentan-Oxidierer 5,:9 TAME [74] GPo3 aus Boden

Pseudomonas n-Alkanoxidierer ETBE, 52,72 [75] mendocina KR3 aus Boden TAME

Rhodococcus ruber 2 (5,47E ) ETBE: ETBE, Aktivschlamm [5:] IFP 4223 5,73 (57,5E ) TAME

Rhodococcus zopﬁi 2 (2,:5E ) ETBE: ETBE, Aktivschlamm [5:] IFP 4227 6,68 (7:,7E ) TAME

2,7: (3,84E ) Gordonia sp. ETBE, Aktivschlamm ETBE: 4,2: [5:] IFP 422; TAME (49,2E )

Dissertation Judith Schuster 3. Einleitung 9

Zur Ermittlung der MTBE-Abbau-Rate in Tabelle 3.3 wurden proteinbasierte Werte hal- biert, da je 72% Proteingehalt in der Biomasse angenommen wurde. N.a. bedeutet nicht analysiert; die ∗-Rate entspricht dem laut Kinetik theoretisch möglichen Maximalwert; E verweist auf Induktion der Zellen mit ETBE (ebenso bei der Abbau-Rate von ETBE). Nur wenige Reinkulturen sind für ihre heterotrophe Mineralisation von Oxygenat-Ethern durch Primärmetabolismus bekannt. Eine der ersten diesbezüglich erfolgreichen Studien wies mit Isolaten der Gattungen Arthrobacter, Rhodococcus und Methylobacterium [76] einen langsamen und unvollständiger Abbau von 82 ppm der eingesetzten 422 ppm MTBE innerhalb von zwei Wochen nach3. Weitere autark auf den Ethern wachsende Isolate sind die Gram-positiven Mycobacterium austroafricanum Stämme IFP 4234 [6;] und IFP 4237 [77] sowie die β-Proteobacteria Methylibium petroleiphilum PM3 [6:], Hydrogenophaga flava ENV957 [72], Variovorax paradoxus CL-: [68] und der in dieser Arbeit untersuchte Stamm Aquincola tertiaricarbonis L32:. Deren Aktivitäten, Wachstumsraten und Biomasseerträge sind sehr unterschiedlich (siehe Tabelle 3.3). In den Stämmen ENV957, IFP 4234 und IFP 4237 wird das akkumulierende TBA erst in Abwesenheit von MTBE für Wachstum genutzt, was eine besondere Regulation des Stoffwechsels andeutet. Der Stamm A. tertiaricarbonis L32: wurde 4225 aus einem MTBE-kontaminiertem Grundwasserleiter am Standort der ehemaligen Leuna-Werke in Sachsen-Anhalt angerei- chert. Das Isolat gehört zu den β-Proteobacteria und ist relativ nah mit dem bereits länger bekannten MTBE-Abbauer M. petroleiphilum PM34 verwandt. Eine Besonderheit des Stammes L32: ist die Fähigkeit, alle tertiären Oxygenat-Ether als einzige Energie- und C-Quelle effizient (also ohne wesentliche Ausscheidung von Metabo- liten) für Wachstum nutzen zu können. Dabei werden, trotz der vergleichsweise niedrigen Abbaurate (37,52 nmol min−1 mg−1 Biomasse), hohe Wachstumsraten von etwa 2,27 h−1 erzielt [6;]. Die Ertragskoeffizienten der Biomassetrockensubstanz pro umgesetztem Sub- strat liegen zwischen 2,77 und 2,:3 g g−1, was 85% bis :2% der theoretisch ermittelten −1 möglichen Maximalwerte der Kohlenstoffkonversion Ymax theor MT BE 2,:8; g g und −1 Ymax theor ET BE 2,972 g g entspricht [78].

3.5. Postulierter Abbauweg

Der natürliche Abbau von MTBE in den phylogenetisch diversen Kulturen warf früh die Frage nach den metabolischen Routen und den beteiligten Enzymen auf. Schon in den ersten Studien zu mikrobiologischem MTBE-Abbau [53, 62, 79] wurde 14C-markiertes MTBE eingesetzt, um resultierende 14C-Stoﬀwechselintermediate analysieren zu können und dadurch mögliche metabolische Routen abzuleiten. In der Analytik des MTBE-Abbaus des Pilzes Graphium sp. durch Hardison et al., [53] wurden tert-Butylformiat (TBF) und TBA als initiale MTBE-Abbauprodukte detektiert. Die Abbaukinetik ließ erkennen, dass MTBE über TBF und durch dessen Hydrolyse zu TBA prozessiert wird. In der Studie von Steﬀan et al. (3;;9) wurde der cometabolische MTBE-Abbau der Propanoxidierer und Mycobacterium sp. ENV643 untersucht [62], die jeweils MTBE vollständig zu CO2 abbauen. Als erstes messbares Produkt akkumulierte TBA. Der weitere TBA-Abbau erzeugte MPD und 4-HIBA, welche 35% bzw. 8% der 14C-Markierung aufwiesen.

3 14 14 Die einwöchige Inkubation mit uniform C-markiertem MTBE resultierte in :% C-markiertem CO2, was eine Mineralisierung lediglich der Methylgruppe nahelegt. 4Das Isolat wurde in die Roseateles-Leptothrix-Ideonella-Gruppe der Familie Comamonadaceae der β- Proteobacteria eingeordnet.

Dissertation Judith Schuster 3. Einleitung :

Aufgrund solcher generierter Daten wurde der oxidative Abbauweg für MTBE über die Metabolite TBA und 4-HIBA schon früh postuliert und resultierte in der Etablierung eines oxidativen Abbauwegs von MTBE und ETBE, wie er in Abbildung 3.4 dargestellt ist. Erstes Produkt der Hydroxylierung von MTBE ist das Hemiacetal tert-Butoxymethanol, welches entweder durch spontane Dismutation zu Formaldehyd und TBA zerfällt, oder enzymgekoppelt (vermutlich über eine Alkoholdehydrogenase und eine Esterase) über TBF zu Formiat und TBA umgesetzt wird [;, 48, 53, 79, 7:, 7;]. ETBE wird analog zu tert-Butoxyethanol hydroxyliert. Die strukturverwandten Amylether TAME und TAEE resultieren in tert-Amoxymethanol bzw. -ethanol. Die Nebenprodukte Formaldehyd bzw. Formiat bei MTBE und TAME sowie Acetaldehyd bzw. Acetat bei ETBE und TAEE können, abhängig von der Enzymausstattung, komplett zu CO2 mineralisiert werden. Die verzweigten Alkohole TBA bzw. TAA sind entweder akkumulierendes Endprodukt oder werden durch zusätzliche Enzyme weiter metabolisiert. Im zweiten Fall erzeugt die Hydroxylierung von TBA das Diol MPD, welches über 4-Hydroxyisobutyraldehyd (4- HIBAl) zu 4-HIBA umgesetzt wird, wie es unter anderem für den hier behandelten Stamm L32: nachgewiesen wurde [69]. Die Intermediate MPD, 4-HIBAl und 4-HIBA treten quasi in allen MTBE-mineralisierenden Isolaten auf. Zum weiteren Abbau von 4-HIBA stehen theoretisch mehrere Mechanismen zur Verfügung. Bisher nachgewiesen ist nur die Isome- risierung der CoA-aktivierten Säure zu dem regulären Metaboliten 5-HB-CoA, welches normal zu CO2 abbaubar ist (Abbildung 3.4), [82]. Die strukturverwandten Ether TAME und TAEE werden analog dazu über tert- Amylformiat bzw. tert-Amylacetat zu tert-Amylalkohol (TAA) oxidiert [57, 62, 69, 83]. Der mikrobielle Abbau von TAA ist jedoch noch nicht beschrieben worden, die Datenlage hinsichtlich möglicher Intermediate ist nicht so oﬀensichtlich, wie im Fall von TBA. Hinwei- se zum möglichen Stoﬀwechsel liegen bisher nur aus Toxizitätsstudien mit 13C-markiertem TAME in Ratten und freiwilligen Testpersonen vor [38, 84, 85]. In diesen verläuft der TAME-Abbau oxidativ über TAA zu den drei Diolen 4-Methyl- 3,4-butandiol, 4-Methyl-4,6-butandiol und 4-Methyl-4,5-butandiol, welche weiter zu 4- Hydroxy-4-methylbuttersäure, Methylacetoin bzw. 5-Hydroxy-5-methylbuttersäure oxidiert werden können. Die drei Diole sind als hypothetische Produkte in Abbildung 3.4 mit dargestellt. Um das unterschiedlich ausgeprägte Abbaupotenzial der verschiedenen Isolate in einen Zu- sammenhang mit der jeweils zugrundeliegenden Enzymausstattung stellen zu können und so die Ursache der Restriktionen im jeweiligen Substratverwertungsspektrum zu erkennen [78, 86], wurden in einer Vielzahl von Studien meist durch Induktionsexperimente entsprechende Enzymfunktionen abgeleitet und postuliert. Der Enzymschritt zur Hydroxylierung der Ether und derjenige zum Umsatz der ebenso als rekalzitrant geltenden tertiären Al- kohole standen dabei im Mittelpunkt des Interesses (Schritte 3 und 6 in Abbildung 3.4). Die folgenden Ausführungen behandeln die diversen Stämme und Reaktionen der in Ab- bildung 3.4 gezeigten einzelnen Enzymschritte 3 – : am Beispiel des MTBE-Abbaus.

Dissertation Judith Schuster 3. Einleitung ;

O O O O

MTBE ETBE TAME TAEE 1

R Hemiacetale R

O [R = H / CH3 bei O Methyl-/Ethylrest] OH OH 2

tert.-Alkyl-Ester 3 CO2 Formaldehyd / Acetaldehyd Formiat / Acetat CO2

OH tert.-Alkohole OH

TBA TAA

4 4?

OH OH OH OH OH OH OH

2-Methylpropan-1,2-diol (MPD) 2-Methyl- 2-Methyl- 2-Methyl- OH butan-1,2-diol butan-2,3-diol butan-2,4-diol 5 + 6

O OH HO

2-Hydroxyisobuttersäure (2-HIBA) 7 + 8

O OH

CoAS 3-Hydroxybutyryl-CoA (3-HB-CoA)

2 Acetyl-CoA

CO2

Abb. 3.4.: Oxidative Abbauwege der tertiären Oxygenat-Ether MTBE, ETBE, TAME und TAEE. Die initiale Hydroxylierung (3) erzeugt Hemiacetale, welche spontan durch Dis- mutation oder enzymatisch Dehydrogenase- (4) und Esterase-vermittelt (5) zu TBA bzw. TAA umgesetzt werden. TBA wird durch eine weitere Hydroxylase (6) zu MPD oxidiert, welches durch Dehydrogenasen (7+8) über 4-HIBAl zu 4-HIBA oxidiert wird. Diese wird CoA-aktiviert (9) zu 5-HB-CoA linearisiert (:). Ein analoger TAA-Abbau durch (6) könnte bis zu drei verschiedene Diole erzeugen. Das Schema ist abgewandelt nach [65, 69].

Dissertation Judith Schuster 3. Einleitung 32

Die initiale Ether-Oxidation (Schritt 3)

Die Analyse des jeweils für die initiale Etherhydroxylierung verantwortlichen Enzyms lässt eine große Vielfalt an Monooxygenasen vermuten, vom Häm-haltigen Cytochrom P-672-Typ zu nicht-Häm short-chain Alkanmonooxygenasen des AlkB-Typs und nicht- Häm THF-Monooxygenasen [87]. Letztere wird in Pseudonocardia tetrahydrofuranoxydans K3 und Pseudonocardia sp. ENV69: nach Wachstum auf Tetrahydrofuran (THF) für den cometabolischen Abbau von MTBE verantwortlich gemacht [88, 89]. Eine short-chain Alkanmonooxygenase des AlkB-Typs wird z. B. in den Gram-positiven Stämmen Mycobacterium austroafricanum JOB7, IFP 4234 und IFP 4237 [56, 63, 77, 8:] im Zusammenhang mit Schritt 3 genannt, ist aber auch in Gram-negativen Stäm- men, wie Pseudomonas putida GPo3 [74] und Methylibium petroleiphilum PM3 [8;, 92] gefunden worden. In den cometabolisch MTBE-abbauenden Stämmen wird diese durch Wachstum auf Propan oder längerkettigen Alkanen (bis C;) induziert, wobei die Rate in Abhängigkeit der Substratlänge und -komplexität variiert [63, 73, 93]. In P. putida GPo3 wirkt Dicyclopropylketon (DCPK) als bester Induktor [74]. In dem heterotrophen MTBE-Verwerter PM3 wird MdpA als neuartiges AlkB5 durch MTBE induziert [92]. Eine durch Cytochrom P-672-Monooxygenasen katalysierte initiale Reaktion wurde hingegen in Nocardia sp. ENV642 und ENV643 [62], R. ruber IFP 4223, R. zopfii IFP 4227 und Mycobacterium sp. IFP 422; [8:] gefunden. In den propanoxidierenden Nocardia-Stämmen wurde vermutet, dass es sich um die Propanmonooxygenase (PMO) selbst handelt, welche bekanntlich ein breites Substratverwertungsspektrum aufweist [62]. Eine andere, neuartige Cytochrom P-672 Monooxygenase, codiert durch das ethRABCD-Gencluster, wurde in den Gram-positiven Stämmen R. ruber IFP 4223, R. zopfii IFP 4227 und Mycobacterium sp. IFP 422; besonders durch ETBE induziert und durch Southern Hybridisierung und PCR- Amplifikation nachgewiesen [94, 95]. Die ethABC-Gene codieren eine Ferredoxinreduktase, ein Cytochrom P-672 und ein Ferredoxin, wie es für bakterielle P-672-Systeme zum Abbau anderer Substrate typisch ist.

Die weitere Prozessierung der Hemiacetale (Schritte 4 und 5)

In manchen Isolaten wie dem Pilz Graphium sp. [53], den Mycobacterium-Stämmen IFP 4234, IFP 4237 und JOB7 [63, 6;, 77] sowie dem Stamm L32: [78], wird tert-Butyl- formiat (TBF) gebildet, was durch Dehydrogenierung des Hemiacetals durch eine (noch unbekannte) Dehydrogenase (Schritt 4) erklärt werden kann. TBF kann abiotisch hy- drolysiert oder durch eine (ebenso unbekannte) Esterase (Schritt 5) zu TBA und Formiat gespalten werden. Parallel zu beiden Schritten ist auch eine spontane (enzymunabhängige) Dismutation zu Formaldehyd und TBA möglich.

Die alkoholoxidierende Reaktion (Schritt 6)

Aufgrund von Induktions- und Inhibitionsstudien wird vermutet, dass in Mycobacterien diverse nicht-Häm-Monooxygenasen (auch des AlkB-Typs) TBA hydroxylieren können [56, 77, 96]. Teilweise werden diese auch für die Umsetzung der Ether verantwortlich gemacht. In H. ﬂava ENV957 wird MTBE konstitutiv abgebaut, während die abweichende

5MdpA weist eine zu allen AlkB-Typen abweichende, stammspeziﬁsche Aminosäure (Thr59) an der konservierten, substratgrößenbestimmenden Position auf (Position 77 des P. putida GPo3-AlkB), [92].

Dissertation Judith Schuster 3. Einleitung 33

TBA-Monooxygenase erst induziert werden muss [72]. Induktionsstudien mit TBA ga- ben in Stamm L32: Hinweise auf eine Monooxygenase des nicht-Häm-Rieske-Typs mit mononuklearem Eisen als zentralem Enzym des tertiären Alkoholabbaus [97]. Ein Se- quenzvergleich zur NCBI-Datenbank zeigte die bisherige Einzigartigkeit dieses Enzyms. Lediglich ein Treﬀer fast identischer Sequenz zum MTBE degradation protein J (MdpJ) des Stammes M. petroleiphilum PM3, welches ebenso durch Induktion gefundenen wurde [8;], führte zur Übernahme der Gen- und Proteinbezeichnung [97].

Die folgenden Reaktionen 7 und 8

Für den Stamm IFP 4234 wurden durch heterologe Expression einzelner Gene des mpd- Operons in Mycobacterium smegmatis mc4 die MPD- und 4-HIBAl-Dehydrogenasen MpdB und MpdC [8:] als Enzyme des TBA-Folgestoﬀwechsels charakterisiert. In allen MTBE- mineralisierenden Stämmen wird eine analoge Oxidation zu 4-HIBA durch zwei Dehy- drogenasen vermutet. Die beiden Enzyme MpdB und MpdC sind jedoch nicht im Ge- nom des Stammes PM3 nachweisbar. Bei diesem wird anhand der Induktionsresultate der Transkriptomstudie eine Beteiligung anderer Dehydrogenasen (MdpH und Mpe_A2583) postuliert [8;].

Der Abbau von 4-HIBA (Schritte 9 und :)

In Stamm L32: wurde durch Induktionsstudien mit TBA sowie die beobachtete strikte Abhängigkeit des 4-HIBA-Abbaus von Cobalamin eine neuartige Mutase postuliert [82], welche 4-HIBA nach CoA-Aktivierung zu dem natürlichen Metaboliten 5-HB umwandelt und in den zentralen Stoffwechsel einschleust. Die 4-HIBA-CoA-Mutase (HCM) weist eine substratbindende Untereinheit (HcmA) und eine cobalaminbindende Untereinheit (HcmB) auf. Die korrespondierenden Gene für diese neuartige Mutase wurden auch im Stamm PM3 gefunden [8;, 98], so dass der Mutase-Weg möglicherweise eine verbreitete metabolische Route im Oxygenat-Etherabbau darstellt. Dagegen konnten für die z. B. von Steffan et al., [62] diskutierten alternativen 4-HIBA-Abbauwege über Dehydration, Decarboxylierung und Hydroxylierung bisher keine konkreten Enzymreaktionen nachgewiesen werden. Inwieweit alle diese Enzyme spezifisch für die jeweiligen Reaktionen verantwortlich sind, wurde allerdings in den meisten Fällen bislang noch nicht eindeutig bewiesen. Die in den diversen Mikroorganismen zum Oxygenatabbau korrelierten Enzymaktivitäten basieren nach wie vor größtenteils lediglich auf Erkenntnissen aus diversen Induktions- und Inhibitionsstudien. Das gilt auch für den dieser Arbeit zugrunde liegenden Stamm L32:. Funktionelle Beweise der postulierten Schlüsselenzyme EthB, MdpJ und HcmAB fehlen bislang. Weiterführende Transkriptomanalysen oder heterologe Expression können die Aussagen der zugewiesenen Funktionen stärken, ein eindeutig sicherer Beweis ist jedoch letztendlich nur durch die Deletion (knock-out) der Enzymfunktion zu erzielen. Derzeit existieren eindeutige molekularbiologische Nachweise ihrer spezifischen Funktion im Oxygenatstoffwechsel nur für die Etherhydroxylasen der Stämme IFP 4223, GPo3 und PM3. In erstem Fall beweist die spontane und Transposase-vermittelte ethABCD-Deletion die Funktion von EthB als Etherhydroxylase [94]. Für den Stamm GPo3 gilt der mit dem Verlust des AlkB-codierenden OCT-Plasmides einhergehende ausbleibende MTBE-Abbau (resultierend in dem defizitären Stamm GPo34) als Funktionsbeweis [74]. Die AlkB-Typ Ethermonooxygenase MdpA des Stammes PM3 wurde durch gezielten knock-out nachgewiesen [92].

Dissertation Judith Schuster 3. Einleitung 34

Die Beteiligung aller anderen Enzyme bleibt spekulativ. So ist ebenfalls die Funktion der per PCR auch in Stamm L32: nachgewiesenen IFP4223-ähnlichen ethABCD-Sequenz [99] bisher noch nicht als in diesem Stamm spezifisch verantwortliche Etherhydroxylase verifiziert. Solche Funktionsbeweise dienen dann nicht nur der Aufklärung des Stoffwechsels allein. Sie ermöglichen auch die Verwendung als Biomarker in der in-situ-Analyse des Schadstoff- abbaus aus Umweltproben für einen Nachweis biologischer Aktivität.

3.6. Monitoring-Tools für biologischen Abbau

Die Analyse des Oxygenatverhaltens im Grundwasser sowie der Effizienz eventuell vorhandener natural attenuation-Prozesse ist besonders im Hinblick auf die mögliche, weitere Verbreitung in Wasserschutzgebiete (in Fließrichtung) wichtig, um das Gefährdungspo- tenzial abschätzen und gegebenenfalls rechtzeitig Schutzmaßnahmen einleiten zu können. Biologischer Abbau ist laut Hyman et al., [86] „typischerweise der bedeutendste Prozess zur Massereduktion bezüglich organischer Chemikalien im Grundwasser“, was schon ge- genüber einer Vielzahl von Verbindungen, inklusive der Oxygenate, als effektiv wirksam nachgewiesen wurde. Dabei stellt sich die Frage nach einem geeigneten Nachweis dieser Bioremediation, der möglichst sensitiv, zeit- und kosteneffizient ist, und im besten Fall sogar eine Quantifizie- rung des Abbaus zulässt. Der direkte Nachweis von Metaboliten (v. a. von TBF und TBA) in kontaminiertem Grundwasser verweist möglicherweise nur auf einen unvollständigen Abbau. Abbauraten lassen sich daraus nur sehr bedingt ableiten. Die Untersuchung von Mikrokosmen mit beispielsweise Aquiferproben benötigt dagegen viel Zeit, ermöglicht dann aber meist eine Quantifizierung des Abbaus. Allerdings sind diese unter Laborbedingungen bestimmten Abbauraten nicht unbedingt auf die in-situ-Bedingungen am kontaminierten Standort übertragbar. Am sinnvollsten erscheinen deshalb kultivierungsunabhängige in-situ-Techniken, die durch den Nachweis von Biomarkern z. B. auf DNA und RNA-Ebene durch PCR bzw. RT-PCR oder sogar durch Quantifizierung mittels qPCR eine Analyse des biologischen Abbaus ermöglichen. Ursprünglich standen dafür nur phylogenetische Tools auf Basis der 38S-rDNA-Sequenzen zur Verfügung. Aufgrund der beobachteten Diversität des mikrobiellen Oxygenatabbaus (Tabelle 3.3) lässt sich damit jedoch nicht feststellen, ob die detektierten Organismen auch die genetische Ausstattung zum Abbau aufweisen. So ist die Phylogenie meist nicht funktionskorreliert. Im Fall der in dieser Arbeit untersuchten Art A. tertiaricarbonis kann beispielsweise nur Stamm L32: die Oxygenat-Ether abbauen, während die Stämme L32 und CIP-I 4274 bei nahezu identischer 38S-rDNA dies nicht können [59]. Erst eindeutig verifizierte essentielle Schlüsselenzyme des biologischen Abbaus bieten sich als aussagekräftige Monitoring-Tools zur Analyse von Bioremediation an. Deren Identifi- kation ist stammspezifisch nur durch Deletion des betreffenden Gens eindeutig möglich, wie es für die Monooxygenasen EthB und MdpA der Stämme IFP 4223 und PM3 durch- geführt wurde. Diese dienen nun erfolgreich als spezifische Biomarker für das Monitoring aktiven in-situ-MTBE-Abbaus [9:]. Der Nachweis des initialen Enzyms reicht aber nicht aus, um Aussagen darüber machen zu können, ob der mikrobielle Abbau zu der gewünschten vollständigen Mineralisierung führt.

Dissertation Judith Schuster 3. Einleitung 35

So wird zwar MTBE und ETBE in den bisher bekannten Gram-positiven Stämmen durch EthB abgebaut, aber es akkumuliert stöchiometrisch TBA. Erst durch den zusätzlichen Nachweis von für den Folgestoffwechsel spezifischen Biomarkern lässt sich ein genaueres Bild des in-situ-Sanierungspotenzials zeichnen. Hierfür ist die eindeutige Identifikation weiterer, den TBA-Folgestoffwechsel betreffender spezifischer Gene bzw. Schlüsselenzyme notwendig [9;]. Ziele solcher zusätzlichen Funktionsbeweise sind besonders die postulierten Enzymschritte der TBA-Monooxygenase MdpJ und der großen Untereinheit der 4-HIBA-Mutase HcmA.

3.7. Ziel dieser Arbeit

In dieser Arbeit sollten die bisher anhand von Induktionsstudien und Sequenzvergleichen als spezifisch postulierten zentralen Enzymfunktionen im Oxygenatabbau des Stammes Aquincola tertiaricarbonis L32: eindeutig nachgewiesen werden. Dies sollte auf genetischer Ebene mittels Deletionsstudien und darauf aufbauend auf physiologischer Ebene durch Abbautests erfolgen. Es handelt sich im Einzelnen um 3. das für die initiale Reaktion verantwortliche Enzymsystem EthABCD, 4. die potenziell für die Metabolisierung der zentralen Metabolite TBA bzw. TAA in Rede stehende Monooxygenase MdpJK sowie 5. die für die Einschleusung in den zentralen Stoffwechsel durch Linearisierung der tertiären Säure 4-HIBA verantwortlich gemachte Mutase HcmAB. Zweites Ziel war die Aufklärung des bisher unbekannten mikrobiologischen Metabolismus von TAA. Dies sollte für den Stamm L32: ebenso auf Basis von Stoffwechselmutation und Abbautests erfolgen. Vorangegangene vorläufige Ergebnisse, wie das Auftreten ungesättigter Stoffwechselinter- mediate, hatten den Verdacht erweckt, dass hier ein ganz anderer Weg beschritten wird. Weil bekannt ist, dass Monooxygenasen auch Desaturierungsreaktionen katalysieren kön- nen, wurde dies ins Kalkül genommen und Untersuchungen auch in diese Richtung unter- nommen.

Dissertation Judith Schuster 3. Einleitung 36

3.8. Referenzen der Einleitung

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3:. Hard, G. C., R. H. Bruner, S. M. Cohen, J. M. Pletcher, and K. S. Regan. 4233. Renal histopathology in toxicity and carcinogenicity studies with tert-butyl alcohol administered in drinking water to F566 rats: A pathology working group review and re-evaluation. Regulatory Toxicology and Pharmacology 7;:652–658. 3;. International Agency for Research on Cancer (IARC). 4228. Formaldehyde, 4-butoxyethanol and 3-tert-butoxypropan-4-ol. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. ::. 42. Burns, K. M., and R. L. Melnick. 4234. MTBE: recent carcinogenicity studies. International Journal of Occupational and Environmental Health. 3::88–8:. 43. US-EPA. 4222. Clinton-Gore administration acts to eliminate MTBE, Boost Ethanol. U.S. EPA. Headquarters Press Release. 42.25.4222. 44. Weaver, J. W., L. R. Exum, and L. M. Prieto. 4232. Gasoline composition regulations affecting LUST sites. United States Environmental Protection Agency. Office of Research and Development, Washington DC. 42682. 45. European Commission. 422;. Directive 422;/52/EC of the European Parliament and of the Council of 45 April 422; amending Directive ;:/92/EC as regards the specification of petrol, diesel and gas-oil and introducing a mechanism to monitor and reduce greenhouse gas emissions and amending Council Directive 3;;;/54/EC as regards the specification of fuel used by inland waterway vessels and repealing Directive ;5/34. Official Journal of the European Union. 46. CONCAWE. 4234. Gasoline ether oxygenate occurrence in Europe, and a review of their fate and transport characteristics in the environment. 26/4234. 47. Hao, Q., X.-R. Xu, S. Li, J.-L. Liu, Y.-Y. Yu, and H.-B. Li. 4234. Degradation and Removal of Methyl tert-Butyl Ether. International Journal of Environment and Bioenergy. 3:;5–326. 48. Fiorenza, S., and H. S. Rifai. 4225. Review of MTBE Biodegradation and Bioremediation. Bioremediation Journal 9:3–57. 49. Scow, K. M., and K. A. Hicks. 4227. Natural attenuation and enhanced bioremediation of organic contaminants in groundwater. Current Opinion in Biotechnology. 38:468–475. 4:. Mormile, M. R., S. Liu, and J. M. Suflita. 3;;6. Anaerobic biodegradation of gasoline oxygenates: extrapolation of information to multiple sites and redox conditions. Environmental Sciences and Technology. 4::3949–3954. 4;. Johnson, R., J. Pankow, D. Bender, C. Price, and J. Zogorski. 4222. MTBE: To What Extent Will Past Releases Contaminate Community Supply Wells? Environmental Science and Technology. 56:432A–439A. 52. Wilson, J. T. 4225. Fate and transport of MTBE and other gasoline components. In MTBE remediation handbook (pp. 3; – 83). Springer US. 53. Hardison, L. K., S. S. Curry, L. M. Ciuffetti, and M. R. Hyman. 3;;9. Metabolism of Diethyl Ether and Cometabolism of Methyl tert-Butyl Ether by a Filamentous Fungus, a Graphium sp. Applied and Environmental Microbiology. 85:527;–5289. 54. Magana-Reyes, M., M. Morales, and S. Revah. 4227. Methyl tert-butyl ether and tert- butyl alcohol degradation by Fusarium solani. Biotechnology Letters. 49:39;9–3:23. 55. Fayolle, F., G. Hernandez, F. Le Roux, and J. P. Vandecasteele. 3;;:. Isolation of two aerobic bacterial strains that degrade efficiently ethyl t-butyl ether (ETBE). Biotechnology Letters. 42:4:5–4:8. 56. Lopes Ferreira, N., H. Mathis, D. Labbe, F. Monot, C. W. Greer, and F. Fayolle- Guichard. 4229. n-Alkane assimilation and tert-butyl alcohol (TBA) oxidation capacity in Mycobacterium austroafricanum strains. Applied Microbiology and Biotechnology. 97:;2;–;3;.

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57. Morales, M., V. Nava, E. Velasquez, E. Razo-Flores, and S. Revah. 422;. Mineralization of methyl tert-butyl ether and other gasoline oxygenates by Pseudomonads using short n-alkanes as growth source. Biodegradation. 42:493–4:2. 58. Nakatsu, C. H., K. R. Hristova, S. Hanada, X. Y. Meng, J. R. Hanson, K. M. Scow, and Y. Kamagata. 4228. Methylibium petroleiphilum gen. nov., sp. nov., a novel methyl tert-butyl ether-degrading methylotroph of the Betaproteobacteria. International Journal of Systematic and Evolutionary Microbiology. 78:;:5–;:;. 59. Lechner, U., D. Brodkorb, R. Geyer, G. Hause, C. Härtig, G. Auling, F. Fayolle-Guichard, P. Piveteau, R. H. Müller, and T. Rohwerder. 4229. Aquincola tertiaricarbonis gen. nov., sp. nov., a tertiary butyl moiety-degrading bacterium. International Journal of Systematic and Evolutionary Microbiology. 79:34;7–3525. 5:. Malandain, C., F. Fayolle-Guichard, and T. M. Vogel. 4232. Cytochromes P672-mediated degradation of fuel oxygenates by environmental isolates. FEMS Microbiology Ecology. 94:4:;–4;8. 5;. Haase, K., K. D. Wendlandt, A. Gräber, and U. Stottmeister. 4228. Cometabolic Degradation of MTBE Using Methane-, Propane- and Butane-Utilizing Enrichment Cultures and Rhodococcus sp. BU5. Engineering in Life Sciences. 8:72:–735. 62. Steﬀan, R. J., K. McClay, S. Vainberg, C. W. Condee, and D. Zhang. 3;;9. Biodegradation of the gasoline oxygenates methyl tert-butyl ether, ethyl tert-butyl ether, and tert-amyl methyl ether by propane-oxidizing bacteria. Applied and Environmental Microbiology. 85:6438–6444. 63. Smith, C. A., K. T. O’Reilly, and M. R. Hyman. 4225. Characterization of the initial reactions during the cometabolic oxidation of methyl tert-butyl ether by propane-grown Mycobacterium vaccae JOB7. Applied and Environmental Microbiology. 8;:9;8–:26. 64. Ohkubo, N., A. H. Hashimoto, K. Iwasaki, and O. Yagi. 422;. Biodegradation of Methyl tert-Butyl Ether by Mycobacterium spp. Journal of Environmental Biotechnology. ;:335–344. 65. Deeb, R. A., K. M. Scow, and L. Alvarez-Cohen. 4222. Aerobic MTBE biodegradation: an examination of past studies, current challenges and future research directions. Biodegradation. 33:393–3:8. 66. Schmidt, C. T., M. Schirmer, H. Weiss, and S. B. Haderlein. 4226. Microbial degradation of methyl tert-butyl ether and tert-butyl alcohol in the subsurface. Journal of Contaminant Hydrology. 92:395–425. 67. Nava, V., M. Morales, and S. Revah. 4229. Cometabolism of methyl-tert-butyl ether (MTBE) with alkanes. Reviews in Environmental Sciences and Biotecnology. 8:55;–574. 68. Zaitsev, G. M., J. S. Uotila, and M. M. Haggblom. 4229. Biodegradation of methyl tert-butyl ether by cold-adapted mixed and pure bacterial cultures. Applied Microbiology and Biotechnology. 96:32;4–3324. 69. Müller, R. H., T. Rohwerder, and H. Harms. 422:. Degradation of fuel oxygenates and their main intermediates by Aquincola tertiaricarbonis L32:. Microbiology. 376:3636–3643. 6:. Hanson, J. R., C. E. Ackerman, and K. M. Scow. 3;;;. Biodegradation of methyl tert-butyl ether by a bacterial pure culture. Applied and Environmental Microbiology. 87:69::– 69;4. 6;. Francois, A., H. Mathis, D. Godefroy, P. Piveteau, F. Fayolle, and F. Monot. 4224. Biodegradation of methyl tert-butyl ether and other fuel oxygenates by a new strain, Mycobacterium austroafricanum IFP 4234. Applied and Environmental Microbiology. 8::4976–4984. 72. Hatzinger, P. B., K. McClay, S. Vainberg, M. Tugusheva, C. W. Condee, and R. J. Steﬀan. 4223. Biodegradation of methyl tert-butyl ether by a pure bacterial culture. Applied and Environmental Microbiology. 89:7823–7829.

Dissertation Judith Schuster 3. Einleitung 39

73. Garnier, P. M., R. Auria, C. Augur, and S. Revah. 3;;;. Cometabolic biodegradation of methyl t-butyl ether by Pseudomonas aeruginosa grown on pentane. Applied Microbiology and Biotechnology. 73:6;:–725. 74. Smith, C. A., and M. R. Hyman. 4226. Oxidation of methyl tert-butyl ether by alkane hydroxylase in dicyclopropylketone-induced and n-octane-grown Pseudomonas putida GPo3. Applied and Environmental Microbiology. 92:6766–6772. 75. Smith, C. A., K. T. O’Reilly, and M. R. Hyman. 4225. Cometabolism of methyl tertiary butyl ether and gaseous n-alkanes by Pseudomonas mendocina KR-3 grown on C7 to C: n- alkanes. Applied and Environmental Microbiology. 8;:95:7–95;6. 76. Mo, K., C. O. Lora, A. E. Wanken, M. Javanmardian, X. Yang, and C. F. Kulpa. 3;;9. Biodegradation of methyl t-butyl ether by pure bacterial cultures. Applied Microbiology and Biotechnology. 69:8;–94. 77. Lopes Ferreira, N., H. Maciel, H. Mathis, F. Monot, F. Fayolle-Guichard, and C. W. Greer. 4228. Isolation and characterization of a new Mycobacterium austroafricanum strain, IFP 4237, growing on MTBE. Applied Microbiology and Biotechnology. 92:57:–587. 78. Müller, R. H., T. Rohwerder, and H. Harms. 4229. Carbon conversion eﬃciency and limits of productive bacterial degradation of methyl tert-butyl ether and related compounds. Applied and Environmental Microbiology. 95:39:5–39;3. 79. Salanitro, J. P., L. A. Diaz, M. P. Williams, and H. L. Wisniewski. 3;;6. Isolation of a Bacterial Culture That Degrades Methyl t-Butyl Ether. Applied and Environmental Microbiology. 82:47;5–47;8. 7:. Church, C. D., L. M. Isabelle, J. F. Pankow, D. L. Rose, and P. G. Tratnyek. 3;;9. Method for Determination of Methyl tert-Butyl Ether and Its Degradation Products in Water. Environmental Science and Technology. 53:5945–5948. 7;. Fortin, N. Y., M. Morales, Y. Nakagawa, D. D. Focht, and M. A. Deshusses. 4223. Methyl tert-butyl ether (MTBE) degradation by a microbial consortium. Environmental Microbiology. 5:629–638. 82. Rohwerder, T., U. Breuer, D. Benndorf, U. Lechner, and R. H. Müller. 4228. The alkyl tert-butyl ether intermediate 4-hydroxyisobutyrate is degraded via a novel cobalamin- dependent mutase pathway. Applied and Environmental Microbiology. 94:634:–6357. 83. Hernandez-Perez, G., F. Fayolle, and J. P. Vandecasteele. 4223. Biodegradation of ethyl t-butyl ether (ETBE), methyl t-butyl ether (MTBE) and t-amyl methyl ether (TAME) by Gordonia terrae. Applied Microbiology and Biotechnology. 77:339–343. 84. Amberg, A., U. Bernauer, D. Scheutzow, and W. Dekant. 3;;;. Biotransformation of [12C]- and [13C]-tert-Amyl Methyl Ether and tert-Amyl Alcohol. Chemical Research in Toxicology. 34:;7:–;86. 85. Sumner, S. C. J., B. Asgharian, T. A. Moore, H. D. Parkinson, C. M. Bobbitt, and T. R. Fennell. 4225. Characterization of metabolites and disposition of tertiary amyl methyl ether in male F566 rats following inhalation exposure. Journal of Applied Toxicology. 45:633–639. 86. Prince, R. C. 4222. Biodegradation of Methyl tertiary-Butyl Ether (MTBE) and Other Fuel Oxygenates. Critical Reviews in Microbiology. 48:385–39:. 87. Hyman, M. 4234. Biodegradation of gasoline ether oxygenates. Current Opinion in Biotechnology. 46:xx–yy. 88. Thiemer, B., J. R. Andreesen, and T. Schrader. 4225. Cloning and characterization of a gene cluster involved in tetrahydrofuran degradation in Pseudonocardia sp. strain K3. Archives of Microbiology. 39;:488–499.

Dissertation Judith Schuster 3. Einleitung 3:

89. Vainberg S., K. McClay, H. Masuda, D. Root, C. Condee, G. J. Zylstra, and R. J. Steffan. 4228. Biodegradation of ether pollutants by Pseudonocardia sp. ENV69:. Applied and Environmental Microbiology. 94:743:–7446. 8:. Lopes Ferreira, N., C. Malandain, and F. Fayolle-Guichard. 4228. Enzymes and genes involved in the aerobic biodegradation of methyl tert-butyl ether (MTBE). Applied Microbiology and Biotechnology. 94:474–484. 8;. Hristova, K. R., R. Schmidt, A. Y. Chakicherla, T. C. Legler, J. Wu, P. S. Chain, K. M. Scow, and S. R. Kane. 4229. Comparative transcriptome analysis of Methylibium petroleiphilum PM3 exposed to the fuel oxygenates methyl tert-butyl ether and ethanol. Applied and Environmental Microbiology. 95:9569–9579. 92. Schmidt, R., V. Battaglia, K. Scow, S. Kane, and K. R. Hristova. 422:. Involvement of a novel enzyme, MdpA, in methyl tert-butyl ether degradation in Methylibium petroleiphilum PM3. Applied and Environmental Microbiology. 96:8853–885:. 93. House, A. J., and M. R. Hyman. 4232. Effects of gasoline components on MTBE and TBA cometabolism by Mycobacterium austroafricanum JOB7. Biodegradation. 43:747–763. 94. Chauvaux, S., F. Chevalier, C. Le Dantec, F. Fayolle, I. Miras, F. Kunst, and P. Beguin. 4223. Cloning of a genetically unstable cytochrome P-672 gene cluster involved in degradation of the pollutant ethyl-tert-butyl ether by Rhodococcus ruber. Journal of Bacteriology. 3:5:8773–8779. 95. Beguin, P., S. Chauvaux, I. Miras, A. François, F. Fayolle, and F. Monot. 4225. Genes involved in the degradation of ether fuels by bacteria of the Mycobacterium/Rhodococcus group. Oil and Gas Science and Technology — Review IFP. 7::6:;–6;7. 96. Francois, A., L. Garnier, H. Mathis, F. Fayolle, and F. Monot. 4225. Roles of tert-butyl formate, tert-butyl alcohol and acetone in the regulation of methyl tert-butyl ether degradation by Mycobacterium austroafricanum IFP 4234. Applied Microbiology and Biotechnology. 84:478–484. 97. Schäfer, F., U. Breuer, D. Benndorf, M. von Bergen, H. Harms, and R. Müller. 4229. Growth of Aquincola tertiaricarbonis L32: on tert-Butyl Alcohol Leads to the Induction of a Phthalate Dioxygenase-related Protein and its Associated Oxidoreductase Subunit. Engineering in Life Sciences. 9:734-73;. 98. Kane, S. R., A. Y. Chakicherla, P. S. Chain, R. Schmidt, M. W. Shin, T. C. Legler, K. M. Scow, F. W. Larimer, S. M. Lucas, P. M. Richardson, and K. R. Hristova. 4229. Whole-genome analysis of the methyl tert-butyl ether-degrading beta-proteobacterium Methylibium petroleiphilum PM3. Journal of Bacteriology. 3:;:3;53–3;67. 99. Breuer, U., C. Bäjen, T. Rohwerder, R. H. Müller, H. Harms, and L. Bastiaens. 4229. 5rd European Conference on MTBE and Other Fuel Oxygenates, Antwerp, Belgium. 4229. 9:. Jechalke, S., M. Rosell, P. M. Martínez-Lavanchy, P. Pérez-Leiva, T. Rohwerder, C. Vogt, and H. H. Richnow. 4233. Linking low-level stable isotope fractionation to expression of the cytochrome P672 monooxygenase-encoding ethB gene for elucidation of methyl tert- butyl ether biodegradation in aerated treatment pond systems. Applied and Environmental Microbiology. 99:32:8–32;8. 9;. Aslett, D., J. Haas, and M. Hyman. 4233. Identification of tertiary butyl alcohol (TBA)- utilizing organisms in Bio-GAC reactors using 13C-DNA stable isotope probing. Biodegradation. 44:;83–;94.

Dissertation Judith Schuster 4. Die initiale Etherspaltung des Stammes L32:

hema dieses Kapitels ist das für die initiale Etheroxidation speziﬁsch verantwortliche Enzym aus A. tertiaricarbonis L32:. Dieses ist ein Cytochrom-P-672 T gesteuerter Komplex aus der Monooxygenase EthB, der Ferredoxinreduktase EthA, dem Ferredoxin EthC und dem unbekannten Protein EthD, welche durch das entsprechende Gencluster ethABCD codiert werden. In der Studie wird die Expression, Regulation und Evolution dieses Enzymkomplexes charakterisiert. Der Artikel wurde in der Fachzeitschrift Applied Environmental Microbiology der American Society for Microbiology veröﬀentlicht (Appl. Environ. Microbiol. April 4235 vol. 9; No. 9 4543-4549). Die Online-Version ist seit dem 47. Januar 4235 verfügbar (doi:32.334:/AEM.2556:-34). Es folgt das Manuskript sowie das zugehörige Supplement. Die Anteile der Co-Autoren an der Studie sind im Anhang angefügt.

3; 4. Die initiale Etherspaltung des Stammes L32: 42

Constitutive Expression of the Cytochrome P450 EthABCD Monooxygenase System Enables Degradation of Synthetic Dialkyl Ethers in Aquincola tertiaricarbonis L108

Judith Schuster,a Jessica Purswani,b Uta Breuer,c Clementina Pozo,b Hauke Harms,a Roland H. Müller,a Thore Rohwerdera Helmholtz Centre for Environmental Research-UFZ, Department of Environmental Microbiology, Leipzig, Germanya; Grupo de Microbiología Ambiental, Departamento de Microbiología e Instituto del Agua, Universidad de Granada, Granada, Spainb; Fachhochschule Nordhausen, Umwelt- und Recyclingtechnik, Nordhausen, Germanyc

In Rhodococcus ruber IFP 2001, Rhodococcus zopfii IFP 2005, and Gordonia sp. strain IFP 2009, the cytochrome P450 monooxygenase EthABCD catalyzes hydroxylation of methoxy and ethoxy residues in the fuel oxygenates methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME). The expression of the IS3-type transposase-flanked eth genes is ETBE dependent and controlled by the regulator EthR (C. Malandain et al., FEMS Microbiol. Ecol. 72:289–296, 2010). In contrast, we demonstrated by reverse transcription-quantitative PCR (RT-qPCR) that the betaproteobacterium Aquincola tertiaricarbonis L108, which possesses the ethABCD genes but lacks ethR, constitutively expresses the P450 system at high levels even when growing on nonether substrates, such as glucose. The mutant strain A. tertiaricarbonis L10, which is unable to degrade dialkyl ethers, resulted from a transposition event mediated by a rolling-circle IS91-type element flanking the eth gene cluster in the wild-type strain L108. The constitutive expression of Eth monooxygenase is likely initiated by the housekeeping sigma factor ␴70, as indicated by the presence in strain L108 of characteristic ؊10 and ؊35 binding sites upstream of ethA which are lacking in strain IFP 2001. This enables efficient degradation of diethyl ether, diisopropyl ether, MTBE, ETBE, TAME, and tert-amyl ethyl ether (TAEE) without any lag phase in strain L108. However, ethers with larger residues, n-hexyl methyl ether, tetrahydrofuran, and alkyl aryl ethers, were not attacked by the Eth system at significant rates in resting-cell experiments, indicating that the residue in the ether molecule which is not hydroxylated also contributes to the determination of substrate specificity.

ynthetic alkyl and aryl ethers are widely used in industry and sis is needed for assessing degradation activities at contaminated Sagriculture as organic solvents, detergents, and pesticides, sites (5). among other uses. The most prominent examples of important The best-studied fuel oxygenate monooxygenase for MTBE xenobiotic dialkyl ethers are the fuel oxygenates methyl tert-butyl and ETBE hydroxylation is the cytochrome P450 EthABCD sys- ether (MTBE), ethyl tert-butyl ether (ETBE), tert-amyl methyl tem found in several Gram-positive bacterial strains of the genera ether (TAME), and tert-amyl ethyl ether (TAEE), which since the Rhodococcus, Gordonia, and Mycobacterium (7, 8, 9). The corre- late 1970s have been added to gasoline for optimizing combustion sponding genes are located in the ethRABCD gene cluster encod- and thus reducing carbon monoxide emissions (1). The inten- ing an AraC/XylS-type positive transcriptional regulator (ethR), a tional and unintentional release of synthetic ethers both lead to ferredoxin reductase (ethA), a cytochrome P450 monooxygenase ubiquitous pollution of water resources. Due to the low reactivity (ethB), a ferredoxin (ethC), and a protein with an unknown func- of the ether bridge, all of these pollutants generally possess a high tion (ethD). This cytochrome P450 system was classified as the degree of resistance to abiotic and biotic attack and tend to persist first member of the CYP249A1 family (8, 9). In Rhodococcus ruber in the environment (2). In the case of MTBE, its detection at the IFP 2001 and other strains, expression of the eth genes is induced ppb level or even higher concentrations in innumerable drinking by ETBE but not by MTBE or TAME (9). In addition, the eth gene water sources (3) has already led to a ban of its use as a gasoline cluster can undergo homologous recombination using two identical IS3-type class II transposases located on either side of the additive in the United States (4). gene cluster (Fig. 1), orientated in the same direction and forming In principle, dialkyl and alkyl aryl ethers can be degraded in a hairpin and releasing the eth genes (7, 8). Surprisingly, a similar aerobic microorganisms by monooxygenase-catalyzed hydroxyla- Eth system is also present in the fuel oxygenate-degrading beta- tion to hemiacetals, which can spontaneously dismutate in aque- proteobacterium Aquincola tertiaricarbonis L108 (10, 11). How- ous solutions to the corresponding alcohols plus aldehydes/ke- ever, unlike the Gram-positive eth-bearing strains, which stoichi- tones (2). However, corresponding monooxygenases are not widespread for all synthetic ethers, as has been demonstrated for MTBE and the other aforementioned fuel oxygenate ethers. Only Received 30 October 2012 Accepted 23 January 2013 a couple of enzymes are currently known to attack these chemicals Published ahead of print 25 January 2013 at significant rates. Among them are different AlkB-type and cy- Address correspondence to Thore Rohwerder, [email protected]. tochrome P450 systems (5, 6). Interestingly, these monooxyge- Supplemental material for this article may be found at http://dx.doi.org/10.1128 nases differ not only in their substrate specificity (e.g., hydroxyla- /AEM.03348-12. tion of only methoxy or methoxy and ethoxy groups of tert-alkyl Copyright © 2013, American Society for Microbiology. All Rights Reserved. ethers) but also in their isotope fractionation, and thus a critical doi:10.1128/AEM.03348-12 evaluation of two-dimensional (D/H and 13C/12C) isotope analy-

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Dissertation Judith Schuster 4. Die initiale Etherspaltung des Stammes L32: 43

Schuster et al.

FIG 1 Comparison of the eth gene regions sequenced thus far in R. ruber IFP 2001 (20,138 bp, GenBank/EMBL/DDBJ database number AF333761) (A) and A. tertiaricarbonis L108 (6,909 bp, KC333876) (B), which encode fuel oxygenate ether monooxygenase Eth, consisting of ferredoxin reductase EthA, cytochrome P450 monooxygenase EthB, ferredoxin EthC, the unknown-function protein EthD, and other proteins. The box marks the DNA region showing 99.9% identical nucleotide sequences in the two strains. In contrast, strain IFP 2001 ethABCD genes are ﬂanked by two identical copies of a 5.5-kb IS3-type class II transposon, the ethR gene, and other open reading frames (4 to 6), whereas the four eth genes in strain L108 are ﬂanked by a 957-bp gene fragment (X) encoding a protein showing 99% sequence identity to the C-terminal part of a putative peptidyl-prolyl cis-trans isomerase from the MTBE-degrading strain Methylibium petroleiphilum PM1 (Mpe_A1955) and one copy of an 1.5-kb IS91-type Y2 transposon element.

ometrically accumulate the tert-alcohol metabolites during ether (dry weight) literϪ1 (17). The cell suspensions were incubated with 2.5 degradation, Aquincola tertiaricarbonis L108 possesses metabolic mM target ether substrate at 30°C in glass serum bottles sealed with butyl pathways for complete mineralization. tert-Butyl alcohol (TBA) rubber stoppers (19). For monitoring degradation, samples were taken as from MTBE and ETBE is degraded via hydroxylation to 2-methyl- described previously (19) by puncturing the rubber stoppers with syringes 1,2-propanediol by the monooxygenase MdpJK (12, 13) and un- equipped with disposable Luer Lock needles. Volatile ethers, alcohols, and dergoes further enzymatic oxidation to 2-hydroxyisobutyric acid, other metabolites were quantified by headspace gas chromatography (GC) using flame ionization detection (FID) employing an Optima Delta which is then activated by coenzyme A (CoA) and isomerized to 3 column (60 m by 0.32 mm by 0.35 ␮m; Macherey-Nagel, Düren, Ger- the common metabolite 3-hydroxybutyryl-CoA by the cobala- many) either isothermally at 50°C (MTBE, ETBE, diethyl ether, diisopro- min-dependent mutase HcmAB (14, 15). In the same strain, the pyl ether, ethanol, acetaldehyde, isopropanol, acetone, tetrahydrofuran, TAME and TAEE metabolite tert-amyl alcohol (TAA) is de- and TBA) or applying a temperature gradient from 100 to 180°C (TAME, saturated by MdpJK to the hemiterpene 2-methyl-3-buten-2- TAEE, n-hexyl methyl ether, and TAA) (19). In the case of aryl alkyl ethers ol, which is isomerized to prenol, oxidized to 3-methylcroto- (anisole, phenetole, and isopropoxybenzene), the column oven program nyl-CoA, and then degraded via the leucine pathway (16). was set at 120°C for 2 min and then increased to 250°C at 15°C per min Consequently, strain L108 is able to grow on all four fuel oxy- and, finally, kept at 250°C for 0.67 min. The shown data represent mean genate ethers (16, 17). values and standard deviations of results from at least four replicate ex- In this study, we characterized the cluster of eth genes from A. periments. Batch cultivation. Strain L108 was grown on fuel oxygenate ethers tertiaricarbonis L108 which is lost in the spontaneous deletion (either MTBE, ETBE, TAME or TAEE) in serum bottles at 30°C as de- mutant strain L10. The latter strain is unable to degrade any of the scribed above. Cultivation on glucose was done in a 2-liter BioStat B-DCU four fuel oxygenate ethers. Furthermore, we compared the regu- II bioreactor system (Sartorius Stedim Systems GmbH, Melsungen, Ger- lation of eth gene expression in A. tertiaricarbonis L108 with the many) at automatically controlled conditions (temperature at 30°C, pH at ETBE-dependent induction previously found in R. ruber IFP 7.0, oxygen concentration in culture at 20% of saturation). Cells of strain 2001, Rhodococcus zopfii IFP 2005, and Gordonia sp. strain IFP L108 pregrown on MTBE were harvested by centrifugation as described 2009 (9). In contrast to the latter strains, A. tertiaricarbonis L108, above, and washed twice with MSM to remove residual MTBE com- whose eth gene cluster is lacking the regulator gene ethR, shows pletely. These cells were immediately used as inoculum (20 mg biomass literϪ1) for cultivation on 2.5 g literϪ1 glucose in 1.5 liters MSM supple- constitutive expression of the eth genes when incubated on any Ϫ1 fuel oxygenate ether or glucose as a growth substrate. This lack of mented with vitamins, 500 mg N liter (as ammonium chloride), and 0.5 ml antifoam solution (Carl Roth GmbH, Germany). For PCR, sterile vol- regulation enables strain L108 to degrade MTBE, ETBE, TAME, umes of 1 ml at an OD700 of 0.5 from the growing cultures were harvested and TAEE, as well as diethyl and diisopropyl ether, at high rates. by centrifugation and stored at Ϫ80°C. Two replicates were taken at each sampling point, serving for DNA and RNA isolation, respectively. The MATERIALS AND METHODS fermentation experiments were performed in triplicate. Chemicals, bacterial strains, and culture conditions. For a list of the Sequencing of genomic DNA. For sequencing of the eth gene cluster suppliers and the purities of ethers and other chemicals used in this study, of strain A. tertiaricarbonis L108, highly pure genomic DNA from an see the supplemental material. A. tertiaricarbonis L108 (14, 18) was culti- MTBE-grown culture was extracted using the MasterPure DNA purifica- vated at 30°C in liquid mineral salt medium (MSM) supplemented with tion kit (Epicentre) and sequenced by Illumina’s HiSeq 2000 technology vitamins, as described previously (19), on MTBE or other fuel oxygenate (GATC Biotech, Konstanz, Germany). A 6,909-bp sequence, including ethers at concentrations of 0.3 g literϪ1. The spontaneous mutant strain A. the ethABCD genes, was obtained (Fig. 1). Sequence similarities were eval- tertiaricarbonis L10 (14) was grown in MSM on TBA at 0.5 g literϪ1 sup- uated using the BLAST alignment tool (20). Promoter prediction was plemented with vitamins. Growth was monitored by measuring optical done using the prokaryotic promoter prediction tool at http:

density at 700 nm (OD700). //bioinformatics.biol.rug.nl/websoftware/ppp/ (21). The IS91-type trans- Resting-cell experiments and chemical analyses. Precultures were posase was deﬁned by BLAST and the ISﬁnder software at http://www-is harvested for degradation experiments by centrifugation at 13,000 ϫ g at .biotoul.fr (22). 4°C for 10 min. After washing twice with nitrogen-free MSM, cells were RT-qPCR, qPCR, and PCR. For reverse transcription-quantitative immediately used as inoculum for resting-cell experiments adjusted to an PCR (RT-qPCR), RNA was isolated from the frozen cell pellets of ether-

OD700 value of 2.5, corresponding to a concentration of 1.35 g biomass and glucose-grown cells using the RNeasy mini kit (Qiagen) and tran-

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Dissertation Judith Schuster 4. Die initiale Etherspaltung des Stammes L32: 44

Eth Monooxygenase in Aquincola tertiaricarbonis L108

TABLE 1 Primer pairs used for standard PCRs and qPCRs Primer name Sequence (5= ¡ 3=) Target gene Length (bp) iso-for TCGGTCCGGCATGGGACCTGAT L108 isomerase upstream of ethA 1,164 iso-rev AGGCGCGGAAGGACCCTG ethAup-for TCGGTCCGGCATGGGACCTGAT eth promoter region 1,618 ethA-rev1 ATCGCGCAGCGACATCGCTGCCTGCA ethA-for CAGGGTCCTTCCGCGCCT ethA 1,654 ethA-rev TCAGCGAGCCGTGGCGACCT ethB-for ATGACACTGTCACTGGCCA ethB 1,203 ethB-rev TCACTTCGGGTAGATCCGCA ethC-for ACGGCATCCTCGCCGAGTGC ethC 321 ethC-rev TCAGAACGCGTCGGGGACCT ethD-for ATGTATCAGATCGTGGCCTG ethD 312 ethD-rev CTAGGTCCGGTCGACCTCAT tnp-for ATTCCTGCGGCTACG L108 IS91-type Y2 tnp 1,346 tnp-rev GACACCGTACACATCACA mpdJ-2rev_c CGTCGACGGCAGCCTGCTGG mpdJ 380 mpdJ-3for_c TGTTGTCATCGGTCGGGTGC hcmA-c2-for GACTTCTTCGAGGAGGTCGC hcmA 425 hcmA-RPr-2 GTGCCACCGCGCTTCTCG unibac27f AGAGTTTGATCTGGCTCAG 16S rRNA gene 511 univ519r GTATTACCGCGGCTGCTG tnpR-for TCGTCGCGTGGCAGAGCGCTACCGCGCG R. ruber IFP 2001 tnpR 401 tnpR-rev CGATCACCGCCCTGGCCGACGCGCCGCC tnpA-for a ATGAACACCGTGACGT R. ruber IFP 2001 tnpA 810 tnpA-revC CGCAGTCAACGTCTCCACGC

scribed into cDNA using the RevertAid ﬁrst-strand cDNA synthesis kit ethA was ampliﬁed by standard PCR. By EcoRI digestion and blunting

(Thermo Scientific) according to the manufacturers’ protocols. The with mung bean nuclease, the promoter region of about 500 bp (PL108eth) cDNA was directly used as the template for RT-qPCR. For DNA isolation, was obtained. With the T4-DNA ligation kit, this fragment was ligated each frozen cell pellet was thawed and then suspended in 30 ␮l sterile into HindIII-linearized, blunt-ended, and, with Antarctic phosphatase, distilled water. The samples were heated up for 1 min at 350 W in a dephosphorylated pCM130. Each step was done according to the respec- ϫ microwave and centrifuged at 4°C for 3 min at 16,000 g to get the DNA tive protocols with enzymes from NEB. The resulting pCM130::PL108eth in the supernatant. DNA content and purity were determined via a Nano- was transformed into chemically competent Escherichia coli DH5␣ ac- Drop ND-1000 UV-Vis spectrophotometer (Thermo Scientific) and ad- cording to the rubidium chloride (RbCl) method (24) and selected over- justed to 5 ng ␮lϪ1 with sterile distilled water for qPCR. For nonquanti- night at 37°C on LB agar with 20 ␮gmlϪ1 tetracycline. Grown colonies tative PCR testing for the IFP 2001 IS3-type tnp genes, DNA was isolated were sprayed with an aqueous solution of 100 mM catechol. Expression of from 1 ml bacterial culture. active catechol dioxygenase resulted in instant yellow-green staining of Standard PCR with the Taq PCR master mix kit (Qiagen) with 5 ng the colonies due to 2-hydroxymuconic semialdehyde formation. Equally DNA in 10 ␮l was performed under the following conditions: 3 min of processed religated pCM130 served as negative control. initial denaturation at 94°C, followed by 30 cycles of 30 s at 94°C, anneal- Nucleotide sequence accession number. The complete sequence of ing for 30 s at 60°C, and 1 min kbϪ1 elongation at 72°C, terminated by a the ethABCD genetic cluster of strain A. tertiaricarbonis L108 obtained in final elongation of 5 min at 72°C. For IS3-type tnp PCR, 10% dimethyl this study has been deposited in the GenBank/EMBL/DDBJ database un- sulfoxide (DMSO) (Sigma-Aldrich) was added to the reaction and DNA der the accession number KC333876. from strain IFP 2001 was used as positive control. For qPCR of the eth genes, the following protocol was applied: initial denaturation at 98°C for RESULTS 2 min, followed by 40 cycles of3sat98°C,5sat60°C,3sat82°C plus plate read followed by the melt curve (65°C to 95°C, increment 0.5°C for 5 s), Sequence of the ethABCD gene cluster in strain L108. In strains and final plate read. Based on this protocol, ethC resulted in the highest L108 and IFP 2001, the ethABCD gene sequence, including the qPCR efficiency and was chosen as the target for qPCR and RT-qPCR 103-bp noncoding region directly upstream from ethA,is experiments. As control genes, we chose mdpJ and hcmA, which encode 99.9% identical (Fig. 1), indicating a recent horizontal gene key enzymes of TBA metabolism in strain L108, i.e., for the TBA mono- transfer event. However, the other genes found in the neigh- oxygenase (12, 13), and the large subunit of the 2-hydroxyisobutyryl-CoA borhood close to the eth cluster in strain IFP 2001 (8), com- mutase (15), respectively, and the housekeeping 16S rRNA gene. The prising the regulator gene ethR, the open reading frames 4 to 6, ␮ Ϫ1 qPCR was processed in triplicates with 5 ng l DNA for each reaction and the identical copies of the IS3-type class II transposon and target gene by using the SsoFast EvaGreen super mix according to the element, are missing in strain L108. In contrast, in the latter manufacturer’s protocol on the CFX96 real-time system and analyzed by case the ethABCD genes are flanked by a single gene fragment using the CFX Manager software (Bio-Rad). All PCR primer pairs of target gene fragments used in this study are listed in Table 1. For RT-qPCR, encoding a protein showing 99% sequence identity to the C- 1 ␮l synthesized cDNA was used as the template. terminal part of a putative peptidyl-prolyl cis-trans isomerase Promoter analysis. The predicted L108 eth promoter was tagged to from the MTBE-degrading strain Methylibium petroleiphilum the promoterless catechol-dioxygenase gene xylE of the broad-host-range PM1 (Mpe_A1955) (25, 26) and a putative IS91-type rolling- promoter-probe vector pCM130 (23). A 1.6-kb fragment upstream from circle transposase most similar to a sequence found in the

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FIG 2 RT-qPCR results for the target gene ethC from RNA samples of strain L108 cells exponentially growing on fuel oxygenate ethers as indicated. Nor- ⌬⌬ malized expression ( quantiﬁcation cycle [Cq]) with standard deviations determined from results obtained with cDNA of MTBE cultures as the control and expression of 16S rRNA and mpdJ genes as the references. Also shown are MTBE degradation rates in nmol minϪ1 mg biomassϪ1 in resting cells derived from the respective ether cultures.

Burkholderiales bacterium JOSHI_001 (ZP_09752764, 73% amino acid identity). The absence of IS3-type elements in the genome of strain L108 was verified by PCR targeting of the IFP 2001 tnpA and tnpR genes. Spontaneous eth deletion mutant strain L10. As has been previously reported for MTBE and ETBE (14), the mutant strain L10 FIG 3 MTBE-to-glucose shift experiments. (A) MTBE degradation and TBA did not show any growth or degradation activities when incubated accumulation rates in nmol minϪ1 mg biomassϪ1 of strain L108 cells pre- with TAME or TAEE. On the other hand, strain L10 readily de- grown on MTBE (inoculum) and after two to five generations (G2 to G5) of graded TBA and TAA at the same rates as the wild-type strain L108 growth on glucose, as indicated. (B) RT-qPCR (cDNA) and qPCR (DNA) without accumulation of metabolites (data not shown). Standard results for the target genes ethC, mdpJ, and hcmA from DNA and RNA samples of strain L108 cells pregrown on MTBE (inoculum) and after two and five PCRs designed for detection of the ethABCD genes, the IS91 ele- ⌬⌬ generations (G2, G5) on glucose. Normalized expression levels ( Cq) were ment, and the peptidyl-prolyl cis-trans isomerase gene fragment in calculated by normalization with control samples (DNA and cDNA from in- strain L108 (Table 1) revealed that all these genes were undetect- oculum samples, respectively) and the 16S rRNA gene as reference. able with DNA from strain L10 (see Fig. S1 in the supplemental material), indicating that a larger DNA fragment was lost during the recombination process. Expression of ethC in strain L108 growing on glucose. To Expression of ethC in strain L108 growing on fuel oxygenate analyze expression of the EthABCD monooxygenase on a none- ethers. For quantifying expression by RT-qPCR, the target gene ther growth substrate, the ethC gene was quantified in glucose- ethC was chosen as a proxy for the complete eth gene cluster. grown cultures. After a lag phase of about 2 h, MTBE-pregrown Cultures of strain L108 exponentially growing on either MTBE, cells of strain L108 began to grow exponentially on glucose with a ETBE, TAME, or TAEE as the sole source of energy and carbon generation time of 2.95 h (Fig. 3). RNA samples from the MTBE were sampled to isolate RNA for RT-qPCR and to test MTBE preculture and from the glucose culture at generations two and degradation rates in resting-cell experiments (Fig. 2). In all cases, five were analyzed. As already shown above for the different ether similar expression levels of the tested target gene ethC were found, cultures (Fig. 2), expression of ethC was not affected by glucose as indicating that transcription of the eth genes is not particularly the growth substrate. Even after five generations, expression levels induced by ETBE in strain L108, i.e., unlike previously reported were similar to those obtained for the MTBE preculture (Fig. 3B). results for the eth-bearing Gram-positive strains (9), or by any In contrast, expression of the other MTBE degradation-related other fuel oxygenate ether tested. An MTBE degradation rate of genes mdpJ and hcmA was completely downregulated in glucose- about 12 nmol minϪ1 mg biomassϪ1 was obtained with resting growing cells, whereas these genes were well induced in the MTBE cells pregrown on either MTBE, ETBE, or TAME. Similar activi- preculture. Hence, this significant repression of TBA and 2-hy- ties have also been measured for MTBE and TAME degradation droxyisobutyric acid metabolism led to a nearly stoichiometric with strain L108 in a previous study (19). However, only about accumulation of TBA and a reduction of about 40% of Eth mono- 70% of the maximal rate was observed with TAEE-grown cells. oxygenase activity when MTBE degradation was tested with glu- Most likely, this is not caused by different amounts of the cose-grown cells (Fig. 3A), as has also been observed with TAEE- EthABCD monooxygenase but by lower induction of the down- grown cells (Fig. 2). As a control, qPCR with DNA samples proved stream pathways processing TBA and formaldehyde/formic acid that all three catabolic genes, ethC, mdpJ, and hcmA, were not from MTBE. This incomplete degradation could result in an im- subjected to deletion events during the growth of strain L108 on balance in reducing equivalents (e.g., NADH) essential for mono- glucose (Fig. 3B). oxygenase activity. Accordingly, TAEE-grown cells significantly Degradation of other synthetic ethers by strain L108. For accumulated TBA from MTBE (data not shown). testing degradation capabilities of strain L108, several dialkyl and

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FIG 4 Prediction of promoter regions upstream of the ethA gene. A sigma factor binding site that possessed the Ϫ10 and Ϫ35 regions characteristic for ␴70-initiated transcription (score Ϫ7.4, E value 2.2) was detected only in strain L108. For inspection, the 468- and 186-bp intergenic spacers upstream of ethA in strains L108 and IFP 2001, respectively, were analyzed using the prokaryotic promoter prediction tool at http://bioinformatics.biol.rug.nl/websoftware/ppp/. Highlighted in bold are the Ϫ35 and Ϫ10 regions, the ϩ1 transcription start, the ribosomal binding site (RBS), and the translation start of ethA. Boxes indicate identical nucleotide sequences in the two strains.

aryl alkyl ethers not used as fuel oxygenates were tested with cells the fuel oxygenates MTBE, ETBE, TAME, and TAEE, at high rates pregrown on MTBE. In these resting-cell experiments, diethyl and and without any lag phase. diisopropyl ether were readily degraded, although only at about 30 The high sequence similarity (Ͼ99%) of the ethABCD genes in and 15% of the degradation rate obtained with MTBE, respec- all bacterial strains tested thus far indicates that the cluster of eth tively (see Table S1 in the supplemental material). In both cases, genes has been recently transferred by horizontal gene transfer. It accumulation of volatile metabolites was revealed by GC measure- is likely that the flanking transposon elements were the cause for ments, i.e., ethanol from diethyl ether and acetone from diisopro- high mobility. In principle, during the recombination event the pyl ether. On the other hand, the dialkyl ether n-hexyl methyl excision of the eth genes results in a circular structure, which can ether, the cyclic alkyl ether tetrahydrofuran, and the aryl alkyl then integrate into other genomes (7, 8). Invading transposons are ethers anisole, phenetole, and isopropoxybenzene were not de- major agents of interspecies gene transfer (27). This is also true for graded at detectable rates by strain L108. Diethyl and diisopropyl genes related to xenobiotic degradation (28). Interestingly, the eth ether as well as all other ethers tested were not degraded by the gene transfer was not restricted to Gram-positive strains of the mutant strain L10 pregrown on TBA. family Corynebacterineae, but the gene cluster was also transferred Transcription initiation site upstream of ethA. Inspection of to A. tertiaricarbonis L108, belonging to the Betaproteobacteria. the eth gene cluster sequences of strains L108 and IFP 2001 re- However, different transposon systems, the IS3- and IS91-type vealed that a sigma factor binding site is present upstream of ethA elements (Fig. 1), have been observed to flank the monooxygenase in strain L108 (Fig. 4). Although in the two strains the 103-bp genes in the two phylogenetically distant groups, indicating a region directly upstream from the ethA translation start is identi- more complex history of the eth gene cluster transfer. cal, and thus they share a ribosomal binding site, more distant The detection of a gene fragment upstream of ethA in strain sequences are totally different, and only in strain L108 were the L108 which is nearly identical to a sequence found in the betapro- 70 characteristic binding sites at Ϫ10 and Ϫ35 for a ␴ -initiated teobacterium M. petroleiphilum PM1 (Mpe_A1955) suggests that transcription clearly detected (Fig. 4). Further inspection revealed PM1-related strains and other Gram-negative bacteria might also no promoter regions between ethR and ethA in strain IFP 2001 bear eth genes. Strain PM1, however, does not possess the Eth (data not shown). Integration of the predicted L108 eth promoter monooxygenase (29, 30). region in pCM130 directly upstream of promoterless xylE allowed Due to the transposon-mediated genetic instability, prolonged active expression of the catechol dioxygenase (see Fig. S2 in the cultivation on nonselective growth substrates resulted in the for- supplemental material), establishing this DNA region as a consti- mation of eth deletion mutants, e.g., from R. ruber IFP 2001 (7) tutive promoter site. and from A. tertiaricarbonis L108 (14). The inability of these mutants to degrade ethers shows that the Eth monooxygenase is re- DISCUSSION sponsible for the degradation of all four fuel oxygenates in the Comparison of the eth gene cluster encoding the cytochrome P450 wild-type strains. The MTBE-to-glucose shifts revealed, however, monooxygenase system EthABCD found in the fuel oxygenate- that at least in strain L108 the transposon-mediated recombina- degrading strains A. tertiaricarbonis L108 and R. ruber IFP 2001 tion event does not occur during exponential growth, as the ex- revealed that both gene regions are subjected to transposon-me- pression and gene copy number of ethC did not differ even after diated recombination, resulting in eth deletion mutants, e.g., A. five generations on the nonselective substrate from those of the tertiaricarbonis L10, that are unable to degrade dialkyl ethers. inoculum. In line with this, it has been demonstrated that trans- However, although the high similarity between the ethABCD se- posase activities are typically correlated with stress conditions, quences indicates a recent horizontal gene transfer event, the un- such as cold shock, subinhibitory concentrations of antibiotics, or derlying deletion mechanisms are different (IS3-type transposon entry into stationary phase, rather than with exponential growth element in strain IFP 2001 versus rolling-circle IS91 type in strain (31, 32, 33). L108). In addition, the absence of the regulator EthR, resulting in The Eth monooxygenase system seems to be widespread the constitutive expression of the Eth monooxygenase, enables A. among Gram-positive bacteria, as it has already been found in tertiaricarbonis L108 to degrade synthetic dialkyl ethers, including several strains of the genera Rhodococcus, Mycobacterium, and

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Gordonia (7, 34). However, all these bacteria do not possess the catalyzed degradation of diethyl and diisopropyl ether. In addi- tert-alcohol degradation pathways needed for complete mineral- tion, ETBE-induced cells of strain IFP 2001 were already shown to ization of the fuel oxygenate ethers. On the other hand, strain M. attack n-butyl ethyl ether, di-n-butyl ether, diisopropyl ether, and petroleiphilum PM1 can degrade TBA and TAA completely (25, various short-chain glycol ethers (39). However, synthetic ethers 26) but attacks the ethers by an AlkB-type monooxygenase system with larger residues, such as n-hexyl methyl ether and the three (35), which can hydroxylate only MTBE and TAME but not the alkyl aryl ethers tested in this study, anisole, phenetole, and iso- ethyl ethers (19, 29). Thus far, only the betaproteobacterium A. propoxybenzene, are not substrates for the Eth monooxygenase. tertiaricarbonis L108 is known to possess Eth monooxygenase and Neither was the cyclic ether tetrahydrofuran. In conclusion, al- the complete pathways for the mineralization of tert-alcohols. In- though hydroxylation of methoxy, ethoxy, isopropoxy, and n- terestingly, these degradation capacities combined here in a single butoxy groups has been demonstrated, the second, nonreacting strain have been demonstrated in a mixed culture of R. ruber IFP residue in the ether molecule may not exceed the size of tert-amyl 2001 and strain IFP 2003, another A. tertiaricarbonis strain lacking as the largest residue that tested positive with the Eth system. ether-hydroxylating activity but able to degrade TBA and TAA (18, 36). However, to grow on MTBE this consortium had to be ACKNOWLEDGMENTS amended with ethanol or isopropanol, obviously because the We thank C. Dilssner, M. Neytschev, and B. Würz (UFZ) for excellent strain IFP 2001 could not gain energy from the formaldehyde/ technical and analytical assistance. We are also indebted to K. Glaser and formic acid released from the ether or from TBA, which was ex- D. Türkowsky (UFZ) for the introduction to qPCR, T. Weichler (UFZ) for clusively degraded by the strain IFP 2003 (37). the promoter probe experiment, and T. Unger (UFZ) for providing the Previously, Malandain and coworkers (9) demonstrated that vector pCM130. We are grateful to EMBO (European Molecular Biology Organiza- the transcriptional regulator EthR was responsible for ETBE-in- tion) for the financial support of J.P. (ASTF, 262-09). duced expression of the Eth monooxygenase in the Gram-positive strains IFP 2001, IFP 2005, and IFP 2009. Without induction, REFERENCES these strains attacked ETBE at Ͼ10-fold reduced rates, and deg- 1. Barceló D. 2007. Fuel oxygenates. Springer-Verlag, Berlin, Heidelberg, radation of MTBE and TAME was almost insignificant. 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Nakatsu CH, Hristova K, Hanada S, Meng XY, Hanson J, Scow KM, rial fuel oxygenate degradation. Environ. Sci. Technol. 41:2036–2043. Kamagata Y. 2006. Methylibium petroleiphilum PM1T gen. nov., sp. nov., 39. Hernandez-Perez G, Fayolle F, Vandecasteele JP. 2001. Biodegradation a new methyl tert-butyl ether (MTBE) degrading methylotroph of the of ethyl tert-butyl ether (ETBE), methyl tert-butyl ether (MTBE) and tert- beta-Proteobacteria. Int. J. Syst. Evol. Microbiol. 56:983–989. amyl methyl ether (TAME) by Gordonia terrae. Appl. Microbiol. Biotech- 27. Wiedenbeck J, Cohan FM. 2011. Origins of bacterial diversity through nol. 55:117–121.

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Dissertation Judith Schuster 4. Die initiale Etherspaltung des Stammes L32: 49

Supplemental Material

Applied and Environmental Microbiology

Constitutive expression of cytochrome P450 EthABCD monooxygenase system

enables degradation of synthetic dialkyl ethers in Aquincola tertiaricarbonis L108

Judith Schuster1, Jessica Purswani2, Uta Breuer3, Clementina Pozo2, Hauke Harms1,

Roland H. Müller1, Thore Rohwerder1*

1 Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, Permoserstr. 15, 04318 Leipzig, Germany 2 Grupo de Microbiología Ambiental, Departamento de Microbiología e Instituto del Agua, Universidad de Granada, C/ Ramon y Cajal Nº4, 18071 Granada, Spain 3 Fachhochschule Nordhausen, Umwelt- und Recyclingtechnik, Weinberghof 4, 99734 Nordhausen, Germany

* Corresponding author: Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, Permoserstr. 15, 04318 Leipzig, Germany, Phone: +49 341 235 1317. Fax: +49 341 235 1351. E-mail: [email protected]

Dissertation Judith Schuster 4. Die initiale Etherspaltung des Stammes L32: 4:

1. Purity and supply sources of ethers and other chemicals

MTBE (≥99% pure), ETBE (>97% pure), TBA (≥99% pure), acetaldehyde (>99% pure), and TAA (>99% pure) were purchased from Merck Schuchardt (Hohenbrunn, Germany). 2-Propanol (>99.95% pure), diethyl ether (>99.8% pure), anisole (≥99% pure), ethanol (≥99.9% pure), and acetone (>99.5)% pure) were purchased from Carl Roth GmbH & Co KG (Karlsruhe, Germany). Diisopropyl ether (>99.9%) and TAME (97%) were purchased from Sigma-Aldrich (Taufkirchen, Germany). Phenetole (>98%) and n-hexyl methyl ether (98% pure) were from Alfa Aesar GmbH & Co KG (Karlsruhe, Germany). TAEE (98% pure) was purchased from ABCR (Karlsruhe, Germany) and isopropoxybenzene was from Fluorochem Ltd. (Hadfield, UK) at the highest purity available.

2. Detailed description of MSM culture medium

-1 The mineral salt medium (MSM) contained in mg L : NH4Cl, 760; KH2PO4, 680;

K2HPO4, 970; CaCl2 × 6 H2O, 27; MgSO4 × 7 H2O, 71.2; initial pH was 7.5. MSM also

-1 contained trace elements (in mg L ): FeSO4 × 7 H2O, 14.94; CuSO4 × 5 H2O, 0.785;

MnSO4 × 4 H2O, 0.81; ZnSO4 ×7 H2O, 0.44; Na2MoO4 × 2 H2O, 0.25; CoCl2 x 6 H2O, 0.040. A vitamin solution was added (in µg L-1): biotin, 20; folic acid, 20; pyridoxine- HCl, 100; thiamine-HCl, 50; riboflavin, 50; nicotinic acid, 50; DL-Ca-pantothenate, 50; p-amino-benzoic acid, 50; lipoic acid, 50, and cobalamin, 50. Nitrogen-free MSM was

prepared by omitting the nitrogen source NH4Cl.

Dissertation Judith Schuster 4. Die initiale Etherspaltung des Stammes L32: 4;

3. Standard PCRs with genomic DNA from strains L108 and L10

M1 Iso ethA ethB ethC ethD tnp mdpJ hcmA M2 Iso ethA ethB ethC ethD tnp mdpJ hcmA

primer dimers primers A L108 B L10

Figure S1. Standard PCRs for eth and related genes as well as mdpJ and hcmA (see Table 1). All target genes could be detected in wild-type strain L108, whereas PCRs were negative for the eth, transposase (tnp), and isomerase (iso) genes with DNA from the deletion mutant strain L10. However, mdpJ and hcmA were still detected in strain L10. The DNA markers were the NEB 1kb ladder (M1) and NEB 100 bp ladder (M2), respectively.

4. Promoter analysis

(A) (B)

Figure S2: Promoter probe cloning results. (A) Colonies of E. coli

DH5α(pCM130::PL108-eth) before catechol treatment and (B) after treatment with an aqueous solution of 100 mM catechol were yellow-green colored colonies show 2- hydroxymuconic semialdehyde formation from active catechol dioxygenase XylE. The control of E. coli DH5α(pCM130) with re-ligated promoter-less pCM130 did not show any semialdehyde formation from catechol (data not shown). Both strains were selected on LB agar containing 20 µg mL-1 tetracycline as selection marker.

Dissertation Judith Schuster 4. Die initiale Etherspaltung des Stammes L32: 52

5. Degradation of non-fuel oxygenate ethers

Table S1. Degradation rates for various synthetic ethers in resting-cell experiments with strain A. tertiaricarbonis L108 pregrown on MTBE. Rate Name Structure (nmol min-1 Metabolites mg biomass-1)

diethyl ether O 3.3 ± 0.2 ethanol

diisopropyl ether 1.9 ± 0.2 acetone O

n-hexyl methyl not not ether O detectable detectable

not not tetrahydrofuran O detectable detectable

not not anisole O detectable detectable

not not phenetole O detectable detectable

isopropoxy- O not not benzene detectable detectable

Dissertation Judith Schuster 5. Die speziﬁsche Alkoholmonooxygenase MdpJ

hema dieses Kapitels ist die zentrale Alkoholmonooxygenase MdpJ des Stammes L32:. In Abhängigkeit von der Substratstruktur werden verschiedene Reaktionsme- T chanismen aufgezeigt. Das Enzym wurde bisher hauptsächlich im Zusammenhang mit der Verwertung tertiärer Substrate charakterisiert. In diesem Kapitel wird durch die Reaktivität gegenüber sekundären Alkoholen ein größeres Substratverwertungsspektrum dargestellt. Die erzeugten mdpJ-Deletionsmutanten veriﬁzieren die Speziﬁtät des Enzyms in Bezug auf sekundäre und tertiäre Alkoholformen. Der Artikel wurde in gleichwertiger Erstautorschaft mit Franziska Schäfer in der Fach- zeitschrift Applied and Environmental Microbiology (Appl. Environ. Microbiol. September 4234 vol. 9: No. 39 84:2-84:6) der American Society for Microbiology publiziert. Die Online-Version ist seit dem 4;. Juni 4234 verfügbar (doi: 32.334:/AEM.23656-34). Es folgt der Artikel samt Supplemental Material. Die Anteile der Co-Autorenschaften erscheinen im Anhang.

53 5. Die speziﬁsche Alkoholmonooxygenase MdpJ 54

Synthesis of Short-Chain Diols and Unsaturated Alcohols from Secondary Alcohol Substrates by the Rieske Nonheme Mononuclear Iron Oxygenase MdpJ

Franziska Schäfer, Judith Schuster, Birgit Würz, Claus Härtig, Hauke Harms, Roland H. Müller, and Thore Rohwerder Helmholtz Centre for Environmental Research, Department of Environmental Microbiology, Leipzig, Germany

The Rieske nonheme mononuclear iron oxygenase MdpJ of the fuel oxygenate-degrading bacterial strain Aquincola tertiaricarbonis L108 has been described to attack short-chain tertiary alcohols via hydroxylation and desaturation reactions. Here, we demonstrate that also short-chain secondary alcohols can be transformed by MdpJ. Wild-type cells of strain L108 converted 2-propanol and 2-butanol to 1,2-propanediol and 3-buten-2-ol, respectively, whereas an mdpJ knockout mutant did not show such activity. In addition, wild-type cells converted 3-methyl-2-butanol and 3-pentanol to the corresponding desaturation products 3-methyl-3-buten-2-ol and 1-penten-3-ol, respectively. The enzymatic hydroxylation of 2-propanol resulted in an enantiomeric excess of about 70% for the (R)-enantiomer, indicating that this reaction was favored. Likewise, desaturation of (R)-2-butanol to 3-buten-2-ol was about 2.3-fold faster than conversion of the (S)-enantiomer. The biotechnological potential of MdpJ for the synthesis of enantiopure short-chain alcohols and diols as building block chemicals is discussed.

nzymes catalyzing the hydroxylation and desaturation of ali- The natural substrates of MdpJ-catalyzed hydroxylations and Ephatic tertiary alcohols seem to be rare and have not been well desaturations might be tertiary alcohols, e.g., the fuel oxygenate characterized thus far. In previous studies of the biodegradation of intermediates TBA and TAA. Accordingly, it has already been the fuel oxygenates methyl tert-butyl ether (MTBE) and tert-amyl shown that MdpJ also catalyzes the desaturation of the tertiary methyl ether (TAME), at least one tertiary alcohol-hydroxylating alcohol 3-methyl-3-pentanol to 3-methyl-1-penten-3-ol (35). Here, enzymatic reaction has been proposed for the transformation of we demonstrate that MdpJ can also attack the secondary alcohols the central ether metabolites tert-butyl and tert-amyl alcohol 2-propanol, 2-butanol, 3-methyl-2-butanol, and 3-pentanol. In 1,2- (TBA and TAA, respectively) (9, 38). Though this reaction can be propanediol formation from 2-propanol via hydroxylation and expected in several bacterial strains known to completely degrade 2-butanol desaturation to 3-buten-2-ol, a preference for the (R)- MTBE and TAME under aerobic conditions (11, 13, 21, 27, 37), enantiomer was found, underpinning the potential of MdpJ for the only in the fuel oxygenate-degrading bacterial strain Aquincola synthesis of enantiopure short-chain diols and unsaturated alcohols tertiaricarbonis L108 has the identity of the tertiary alcohol-attack- from secondary alcoholic precursors. ing enzyme recently been revealed by gene knockout experiments (35). In this bacterium, the oxygenase MdpJ catalyzes not only the MATERIALS AND METHODS hydroxylation of TBA to 2-methylpropane-1,2-diol (MPD) but ϩ also the desaturation of TAA to 2-methyl-3-buten-2-ol (35). Chemicals. (S)-( )-2-Phenylbutyryl chloride was synthesized as de- ϩ MdpJ and its corresponding reductase, MdpK, have been described by Hammarström and Hamberg (12) using (S)-( )-2-phenylbu- scribed for the ﬁrst time by Hristova and coworkers as MTBE- tyric acid (99% pure; Sigma-Aldrich Chemie GmbH, Steinheim, Ger- many) and thionyl chloride (99% pure; Merck Schuchardt, Hohenbrunn, induced proteins and subunits of the postulated TBA-hydroxylat- Germany). Suppliers and purities of other chemicals used in this study are ing enzyme in Methylibium petroleiphilum PM1 (14). MdpJK listed in the supplemental material. belongs to the family of Rieske nonheme iron aromatic ring-hy- Bacterial strains and growth media. Aquincola tertiaricarbonis L108, droxylating oxygenases (RHO), such as the well-characterized isolated from an MTBE-contaminated aquifer (Leuna, Germany) (21, phthalate and naphthalene dioxygenases (5, 20, 28, 36). These are 31), was cultivated in liquid mineral salt medium (MSM; see the supple- multicomponent enzymes which use reduced pyridine nucleotide mental material) containing MTBE at a concentration of 0.3 g literϪ1. The as the electron donor. The electrons are transported via a ﬂavin previously generated knockout mutant strain A. tertiaricarbonis L108 cofactor of the reductase subunit to a [2Fe-2S] iron-sulfur cluster (⌬mdpJ) K24 (35) was cultivated on 2-methylpropane-1,2-diol (MPD) at Ϫ Ϫ located on the same protein component as in MdpK (32)orona 0.5 g liter 1 in MSM supplemented with kanamycin (50 mg liter 1). separate ferredoxin subunit. The electrons are further passed to a [2Fe-2S] Rieske cluster and a mononuclear iron center of the oxygenase subunit (23, 40). The multifunctionality of MdpJ to cat- Received 4 May 2012 Accepted 25 June 2012 alyze hydroxylation as well as desaturation reactions (35) has like- Published ahead of print 29 June 2012 wise been found for other RHO enzymes, e.g., naphthalene Address correspondence to Thore Rohwerder, [email protected]. dioxygenase catalyzes mono- and dihydroxylations as well as de- F.S. and J.S. contributed equally to this work. saturations (22). Practically all known RHO enzymes, however, Supplemental material for this article may be found at http://aem.asm.org/. are described to exclusively attack aromatic compounds (19). Copyright © 2012, American Society for Microbiology. All Rights Reserved. Thus, the use of aliphatic substrates underlines the uniqueness of doi:10.1128/AEM.01434-12 MdpJ among the RHO family.

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MdpJ-Catalyzed Transformations of Secondary Alcohols

FIG 1 Degradation of 2-propanol and accumulation of metabolites in resting-cell experiments of A. tertiaricarbonis wild-type strain L108 (A) and mdpJ knockout strain L108 (⌬mdpJ) K24 (B). In the latter case, 1,2-propanediol was below the detection limit (10 ␮M) throughout the experiment. The sum of 2-propanol and metabolites represents the percentage of all analyzed 2-propanol-derived compounds (2-propanol, acetone, and 1,2-propanediol) relative to the initial substrate concentration.

Resting-cell experiments. Cultures were incubated at 30°C on rotary 2-butanol, 3-methyl-2-butanone, 3-pentanol) were quantified by head- shakers. Bacterial cells were pregrown on MPD (0.5 g literϪ1) and then space GC using flame ionization detection (FID) (33). Compounds were incubated on TBA (0.5 g literϪ1) overnight. Afterwards, cells were har- assigned according to retention times of pure GC standards. Additionally, vested by centrifugation at 13,000 ϫ g and 4°C for 10 min. After being ketonic metabolites of 2-butanol as well as metabolites of 3-methyl-2- washed twice with MSM, cells were immediately used, and biomass was butanol and 3-pentanol were identified by GC-MS analysis (see Fig. S3 to adjusted to values of 1.4 g (dry weight) per liter by dilution with MSM for S6 in the supplemental material). Quantitative analysis of 1,2-propane- the 2-butanol experiments and 2.2 g per liter when cells were incubated diol was performed using high-performance liquid chromatography with 2-propanol, 3-methyl-2-butanol, and 3-pentanol. During the exper- (HPLC) with refractive index detection (RID) as described elsewhere iments, bacteria were incubated at 30°C in 25 ml MSM in glass serum (24, 25). bottles, sealed gas tight with butyl rubber stoppers. Liquid and gas samples were taken as described before (33) by puncturing the butyl rubber stop- RESULTS pers with syringes equipped with 0.6- by 30-mm Luer Lock needles. For Whole-cell assay for analyzing MdpJ substrate specificity. For chiral analysis of 1,2-propanediol, the complete culture liquid was har- studying the substrate and catalysis specificity of MdpJ, a test sys- vested by centrifugation at 13,000 ϫ g and 4°C for 10 min in order to tem employing the isolated enzyme is recommended. However, obtain a clear supernatant. The shown data represent mean values and purification of the active enzyme from cells of strain L108 or after standard deviations of results from four replicate experiments. heterologous expression in Escherichia coli was not successful (see Chiral analysis of 1,2-propanediol. Methods of Powers et al. for chiral the supplemental material). Therefore, we developed a whole-cell analysis of short-chain diols and hydroxy carboxylic acids (30) and Jenske and Vetter for chiral analysis of hydroxy fatty acids (15) were modified for assay comparing the substrate usage and product formation of determining the ratios of different 1,2-propanediol enantiomers formed wild-type strain L108 with that of the mdpJ knockout strain L108 in bacterial cultures. Samples from resting-cell experiments (200 ml) were (⌬mdpJ) K24 (35). MPD-grown cells of both strains were incu- saturated with NaCl and extracted two times with 100 ml diethyl ether. bated in the presence of TBA for 10 to 16 h. This procedure was

The diethyl ether phase was dried with Na2SO4 and evaporated com- sufficient to induce MdpJ and MdpK in the wild-type strain (see pletely. Standards of the 1,2-propanediol racemate and (S)-enantiomer Fig. S1 in the supplemental material); however, the mutant strain were applied directly (3 ␮l). Pyridine (400 ␮l) and (S)-(ϩ)-2-phenylbu- K24 did not synthesize these enzymes due to the insertion muta- ␮ tyryl chloride (100 l) were added for derivatization. After2hofincuba- tion in the mdpJ gene. In line with this, subsequent resting-cell O (5 ml) and one spatula tip tion on a slow shaker at room temperature, H2 experiments revealed that only the wild-type cells were able to of K CO were added. Then, the solutions were extracted with MTBE (5 2 3 degrade TBA (see Fig. S2 in the supplemental material). However, ml) via shaking for1hatroom temperature. The MTBE phase was dried the formation of small amounts of the alkene isobutene from TBA with Na2SO4 and evaporated to 0.5 ml. Analysis was performed using gas chromatography (GC) (model 7890A chromatograph; Agilent) coupled was observed with both strains. This dehydration activity is a side to mass spectrometry (MS) (mass selective detector [MSD] model 5975C; reaction of MTBE metabolism in strains L108 and PM1 and has Agilent) on an RTX-5Sil MS column (30 m, 250 ␮m, 0.25 ␮m; Restek) previously been ascribed to MdpJ (33). The dehydration activity with an Integra-Guard column (5 m; Restek). GC conditions were as of the mutant strain clearly shows now that an enzyme other than follows: the carrier gas was helium, constant flow was nominally 0.8 ml MdpJ must be involved. Likely, the same dehydratase that has Ϫ min 1, the injector temperature was 230°C, the split injection ratio was recently been considered for the conversion of the tertiary al- 20:1, programmed oven temperatures were 70°C for 1 min, with an in- Ϫ1 Ϫ1 cohols TAA and 2-methyl-3-buten-2-ol to isoamylene and iso- crease of 1°C min to 76°C and then 6°C min to 350°C, after which the prene, respectively (35), forms isobutene from TBA. Neverthe- temperature was held for 1 min, and the MSD transfer line temperature less, knockout cells formed only less than 30% of the alkene was 250°C. MSD conditions were as follows: full-scan mode (m/z 40 to 600) was used, the ion source temperature was 230°C, and the quadrupole amount of wild-type cells, indicating a higher dehydration ac- temperature was 150°C. Compounds were identified by comparison with tivity during complete TBA metabolization. calibrated retention times and mass spectra of authentic standards. Hydroxylation of 2-propanol to 1,2-propanediol. During the Other analytics. Volatile compounds (TBA, isobutene, 2-propanol, whole-cell assay, wild-type cells of strain L108 formed 1,2-pro- acetone, 2-butanol, 3-buten-2-ol, 2-butanone, 3-buten-2-one, 3-methyl- panediol from the secondary alcohol 2-propanol (Fig. 1), by anal-

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Dissertation Judith Schuster 5. Die speziﬁsche Alkoholmonooxygenase MdpJ 56

Schäfer et al.

favored, resulting in an enantiomeric excess (EE) of about 70% (Fig. 2). During resting-cell experiments with the wild-type strain L108, substrate recovery of the conversion products 1,2-propanediol and acetone was below three-quarters, as these metabolites represented only less than 25 and 50% of the converted substrate, respectively. In contrast, knockout cells, which converted only one-third of the 2-propanol amount of wild-type cells, accumulated exclusively acetone, with close to 100% substrate recovery. This confirms that acetone is not degraded by strain L108 (21) and that 1,2-propanediol may be converted slowly to other metabolites not accumulating under the experimental conditions. By analogy to TBA and MPD metabolism, likely degradation products of 1,2-propanediol are lactaldehyde and lactic acid. Desaturation of 2-butanol, 3-methyl-2-butanol, and 3-pentanol. As 2-propanol turned out to be a substrate for MdpJ, the higher homologue 2-butanol was also tested. Surprisingly, hydroxylation products were not obtained with wild-type cells; only the desaturation product 3-buten-2-ol was formed from racemic FIG 2 GC-MS analysis of the (S)-2-phenylbutyryl derivatives of a racemic 2-butanol (Fig. 3). Both secondary alcohols, 2-butanol and 3-buten- (RS)-1,2-propanediol standard, a pure (S)-1,2-propanediol standard, and a 2-ol, were oxidized to their corresponding ketones, 2-butanone sample from a resting-cell experiment of A. tertiaricarbonis wild-type strain and 3-buten-2-one, respectively (Fig. 3 and see Fig. S3 and S4 in L108 incubated on 2-propanol. (A) Total ion chromatogram signals; (B) mass spectra of peaks occurring in the total ion chromatograms of the sample and the supplemental material). In line with the assumption that the racemic standard at 37.9 min, representing the (S)-2-phenylbutyryl deriv- MdpJ is responsible for 2-butanol desaturation, neither 3-buten- ative of (R)-1,2-propanediol. 2-ol nor 3-buten-2-one was formed by the knockout mutant strain. Likely due to the high dehydrogenase activity, resulting in a nearly complete substrate conversion to 2-butanone, only very ogy with the hydroxylation of TBA to MPD. In addition, the de- small amounts of 2-butanol of maximally 2% and 1% were recov- hydrogenation product acetone accumulated. In contrast, the ered as the desaturation product 3-buten-2-ol and its correspond- mdpJ knockout cells converted 2-propanol exclusively to acetone, ing ketone, 3-buten-2-one, respectively. In experiments with providing evidence that MdpJ catalyzes the hydroxylation of enantiopure substrates, conversion of (R)-2-butanol to 3-buten- 2-propanol in the wild-type strain L108. Acetone formation, on 2-ol was about 2.3 times faster than desaturation of the (S)-enan- the other hand, is probably caused by an unspecific secondary tiomer (Fig. 4). alcohol dehydrogenase expressed in both strains under the exper- In line with the observed transformation of 2-butanol to imental conditions. Formation of 1,2-propanediol was about 7 3-buten-2-ol, 3-methyl-2-butanol and 3-pentanol were also con- times slower than TBA conversion (1.5 and 10 nmol minϪ1 mgϪ1 verted to the corresponding desaturation products 3-methyl-3- [dry biomass], respectively), indicating that TBA is a much better buten-2-ol and 1-penten-3-ol, respectively, by wild-type cells of substrate for MdpJ. As 1,2-propanediol is asymmetric, the ste- strain L108 (see Fig. S5 and S6 in the supplemental material). In reospecificity of the hydroxylation reaction was investigated. After addition, both wild-type and knockout mutant cells oxidized the derivatization to the corresponding (S)-phenylbutyryl diester, the secondary alcoholic substrates to the corresponding ketones. In (S)- and (R)-enantiomers of 1,2-propanediol could be distin- the case of incubation with 3-methyl-2-butanol, the oxidation to guished by GC analysis. Both enantiomers were formed by MdpJ. the unsaturated ketone 3-methyl-3-buten-2-one by the wild-type However, the hydroxylation to (R)-1,2-propanediol was slightly cells was also observed (see Fig. S5 in the supplemental material).

FIG 3 Degradation of racemic 2-butanol and accumulation of metabolites in resting-cell experiments of A. tertiaricarbonis wild-type strain L108 (A) and mdpJ knockout strain L108 (⌬mdpJ) K24 (B). In the latter case, 3-buten-2-ol and 3-buten-2-one were below the detection limit (2 ␮M) throughout the experiment.

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Dissertation Judith Schuster 5. Die speziﬁsche Alkoholmonooxygenase MdpJ 57

MdpJ-Catalyzed Transformations of Secondary Alcohols

FIG 5 Favored substrate and corresponding product enantiomers of MdpJ- catalyzed hydroxylation of 2-propanol to 1,2-propanediol and desaturation of 2-butanol to 3-buten-2-ol. FIG 4 Formation of 3-buten-2-ol from 2-butanol by resting cells of A. tertiaricarbonis wild-type strain L108 after application of pure enantiomers as the substrate. unique. In the context of MTBE metabolism, a few other strains which should possess enzymes with a similar substrate spectrum have been described. Accordingly, the conversion of TBA to MPD Generally, desaturation activities were lower than with 2-butanol, has also been shown for the strains Mycobacterium vaccae JOB5 and prolonged incubation periods of up to 8 h were necessary to (37), Hydrogenophaga flava ENV 735 (13), Mycobacterium aus- accumulate sufficient amounts of desaturation products for troafricanum IFP 2012 (11), and Methylibium petroleiphilum PM1 GC-MS analysis. (27). However, only the MdpJ of strain L108 has been characterized for oxidation of TBA in MTBE metabolism. In addition, DISCUSSION strain PM1 possesses a nearly identical enzyme, showing 97% se- The Rieske nonheme mononuclear iron oxygenase MdpJ of quence identity to MdpJ of strain L108, which has been proposed Aquincola tertiaricarbonis L108 has previously been found to con- to be involved in tertiary alcohol metabolism (14). Moreover, vert the tertiary alcohols TBA, TAA, and 3-methyl-3-pentanol to MdpJ has been detected in environmental samples. The mdpJ gene the corresponding diols and unsaturated alcohols (35). By com- is present in MTBE-degrading enrichment cultures from a con- paring the product formations of wild-type and mdpJ knockout taminated groundwater treatment plant in Leuna, Germany (33), mutant cells, this study now demonstrates that short-chain sec- and the enzyme has been detected in oxygenate-degrading mixed ondary alcohols can also be attacked by MdpJ. cultures by 13C metagenomic and metaproteomic stable-isotope As indicated recently by Schuster and coworkers in the context probing (SIP) experiments (2, 4). These findings underline the of TAA and 3-methyl-3-pentanol metabolism (35), the mode of important role of MdpJ in bacterial MTBE degradation. MdpJ catalysis obviously depends strongly on the molecule struc- Short-chain chiral alcohols and diols are interesting building ture of the substrate, allowing either hydroxylation or desatura- blocks for the synthesis of pharmaceuticals and other active tion reactions. An overview of the products obtained from the compounds (3, 10, 18, 39). Enantiopure alcohols, for example,

thus-far-tested substrates (C3 to C5 secondary alcohols and C4 to are key intermediates in the side chain synthesis of serum cho- C6 tertiary alcohols) is given in Fig. S7 in the supplemental mate- lesterol-reducing drugs, i.e., 3-hydroxy-3-methylglutaryl-co- rial. The structures of 2-propanol and TBA do not contain an ethyl enzyme A (CoA) reductase inhibitors (29). In addition, 1,2-pro- group that can be desaturated. Accordingly, only hydroxylation to panediol is a chiral building block for the synthesis of antiviral diols has been observed. On the other hand, 2-butanol, 3-methyl- drugs like tenofovir or efavirenz (26, 17). Thus far, the most im- 2-butanol, 3-pentanol, TAA, and also 3-methyl-3-pentanol are portant enzyme-based method to synthesize enantiopure alcohols desaturated mainly by MdpJ, as they all possess at least one pair of is lipase-catalyzed kinetic resolution of alcoholic and diolic race- vicinal carbon atoms which can form a double bond. It can also be mates (6, 18, 34). In addition, enantioselective reduction of ke- speculated whether the unsaturated alcohols 3-buten-2-ol and tones (29) or carboxylic acids (6) with specific dehydrogenases can 2-methyl-3-buten-2-ol, considering their structural similarity be applied. Enantiopure 1,2-propanediol for commercial pur- with the MdpJ substrates 2-butanol and TAA, can be used by poses, on the other hand, is currently synthesized only via chem- MdpJ. As these compounds are already desaturated, it appears ical routes (1, 6). A more straightforward approach for the syn- likely that hydroxylation products would be formed (see Fig. S7 in thesis of enantiopure unsaturated alcohols and, particularly, diols the supplemental material). However, when testing both unsatu- may be the stereospecific desaturation and hydroxylation of the rated alcohols in the whole-cell assay, we were not able to detect corresponding saturated alcoholic compounds. However, a one- the formation of 1,2-diols, but HPLC analysis indicated their con- step synthesis employing MdpJ would require a high stereospeci- version to other products, likely unsaturated 2-hydroxy carboxy- ficity. In our preliminary experiments on MdpJ catalysis, only an lic acids (data not shown). Thus far, these compounds have not EE value of about 70% of that of (R)-1,2-propanediol and a 2.3- been unambiguously assigned by GC-MS measurements, and ad- fold preference for (R)-2-butanol as a desaturation substrate were ditional identification work is needed. achieved. This indicates that at least for the secondary alcohols The capacity of MdpJ for hydroxylation and desaturation of tested, the same orientation in the reaction center of the enzyme is small aliphatic alcohols as found in strain L108 seems to be quite favored (Fig. 5). On the basis of this finding, the poor stereospec-

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Dissertation Judith Schuster 5. Die speziﬁsche Alkoholmonooxygenase MdpJ 58

Schäfer et al.

ificity could be improved by site-directed mutagenesis or novel 2009. Efficient enantioselective synthesis of optically active diols by asym- approaches like directed enzyme evolution (7, 8). The stereospec- metric hydrogenation with modular chiral metal catalysts. Angew. Chem. ificity of the MdpJ-catalyzed desaturation of 3-methyl-2-butanol Int. Ed. Engl. 48:7556–7559. 18. Kourist R, Bornscheuer UT. 2011. Biocatalytic synthesis of optically and 3-pentanol to the hemiterpenic 3-methyl-3-buten-2-ol and to active tertiary alcohols. Appl. Microbiol. Biotechnol. 91:505–517. 1-penten-3-ol, respectively, has not yet been analyzed. Neverthe- 19. Kweon O, et al. 2008. A new classification system for bacterial Rieske less, a preference similar to that with (R)-2-butanol could be ex- non-heme iron aromatic ring-hydroxylating oxygenases. BMC Biochem. pected. Hence, an improved MdpJ enzyme showing higher-than- 9:11. normal stereospecificity could be employed for the synthesis of 20. Larkin MJ, Allen CCR, Kulakov LA, Lipscomb DA. 1999. Purification and characterization of a novel naphthalene dioxygenase from Rhodococ- chiral allylic alcohols and related compounds currently available cus sp. NCIMB12038. J. Bacteriol. 181:6200–6204. only through multistep chemical synthesis (3, 16). At the present 21. Lechner U, et al. 2007. Aquincola tertiaricarbonis gen. nov., sp. nov., a stage of our research, however, we exclude the possibility of ap- tertiary butyl moiety-degrading bacterium. Int. J. Syst. Evol. Microbiol. plying pure MdpJ, because preparation of the cell-free enzyme 57:1295–1303. always resulted in a complete loss of enzymatic activity. Alterna- 22. Lee K, Gibson DT. 1996. Toluene and ethylbenzene oxidation by purified tively, application in whole-cell systems is quite promising but naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. Appl. Environ. Microbiol. 62:3101–3106. requires either metabolic manipulation of the wild-type strain 23. Mason JR, Cammak R. 1992. The electron-transport proteins of hy- L108 in order to avoid the ketone-forming side reaction or the droxylating bacterial dioxygenases. Annu. Rev. Microbiol. 46:277–305. transformation of the mdpJK genes into a more appropriate host 24. Müller RH, Rohwerder T, Harms H. 2007. Carbon conversion efficiency strain lacking such side activities. and limits of productive bacterial degradation of methyl tert-butyl ether and related compounds. Appl. Environ. Microbiol. 73:1783–1791. ACKNOWLEDGMENT 25. Müller RH, Rohwerder T, Harms H. 2008. Degradation of fuel oxygenates and their main intermediates by Aquincola tertiaricarbonis L108. Mi- We are grateful to the Deutsche Bundesstiftung Umwelt (DBU) for finan- crobiology 154:1414–1421. cial support of F.S. (grant AZ 20008/994). 26. Murakami H. 2007. From racemates to single enantiomers—chiral synthetic drugs over the last 20 years. Top. Curr. Chem. 269:273–299. REFERENCES 27. Nakatsu CH, et al. 2006. Methylibium petroleiphilum PM1T gen. nov., sp. 1. Altaras NE, Cameron D. 1999. Metabolic engineering of 1,2-propanediol nov., a new methyl tert-butyl ether (MTBE) degrading methylotroph of pathway in Escherichia coli. Appl. Environ. Microbiol. 65:1180–1185. the beta-Proteobacteria. Int. J. Syst. Evol. Microbiol. 56:983–989. 2. Aslett D, Haas J, Hyman M. 2011. Identification of tertiary butyl alcohol 28. Nomura Y, Nakagawa M, Ogawa N, Harashima S, Oshima Y. 1992. (TBA)-utilizing organisms in BioGAC reactors using 13C-DNA stable iso- Genes in PHT plasmid encoding the initial degradation pathway of phtha- tope probing. Biodegradation 22:961–972. late in Pseudomonas putida. J. Ferment. Bioeng. 74:333–344. 3. Balmer E, Germain A, Jackson WP, Lygo B. 1993. Larger scale prepara- 29. Panke S, Held M, Wubbolts M. 2004. Trends and innovations in indus- tion of optically-active allylic alcohols. J. Chem. Soc. Perkin Trans. 1:399– trial biocatalysis for the production of fine chemicals. Curr. Opin. Bio- 400. technol. 15:272–279. 4. Bastida F, et al. 2010. Elucidating MTBE degradation in a mixed consor- 30. Powers L, et al. 1994. Assay of the enantiomers of 1,2-propanediol, 1,3- tium using a multidisciplinary approach. FEMS Microbiol. Ecol. 73:370– butanediol, 1,3-pentanediol and the corresponding hydroxyacids by gas 384. chromatography-mass spectrometry. Anal. Biochem. 221:323–328. 5. Batie C, La Haie JE, Ballou DP. 1987. Purification and characterization 31. Rohwerder T, Breuer U, Benndorf D, Lechner U, Müller RH. 2006. The of phthalate oxygenase and phthalate oxygenase reductase from Pseu- alkyl tert-butyl ether intermediate 2-hydroxyisobutyrate is degraded via a domonas cepacia. J. Biol. Chem. 262:1510–1518. novel cobalamin-dependent mutase pathway. Appl. Environ. Microbiol. 6. Breuer M, et al. 2004. Industrial methods for the production of optically 72:4128–4135. active intermediates. Angew. Chem. Int. Ed. Engl. 43:788–824. 32. Schäfer F, et al. 2007. Growth of Aquincola tertiaricarbonis L108 on 7. Chen R. 2001. Enzyme engineering: rational redesign versus directed tert-butyl alcohol leads to the induction of a phthalate dioxygenase-related evolution. Trends Biotechnol. 19:13–14. protein and its associated oxidoreductase subunit. Eng. Life Sci. 7:512– 8. Dalby PA. 2003. Optimising enzyme function by directed evolution. 519. Curr. Opin. Struct. Biol. 13:500–505. 33. Schäfer F, et al. 2011. Alkene formation from tertiary alkyl ether and 9. Fayolle F, Vandecasteele JP, Monot F. 2001. Microbial degradation and alcohol degradation by Aquincola tertiaricarbonis L108 and Methylibium fate in the environment of methyl tert-butyl ether and related fuel oxygen- spp. Appl. Environ. Microbiol. 77:5981–5987. ates. Appl. Microbiol. Biotechnol. 56:339–349. 34. Schmid A, Hollmann F, Byung Park J, Bühler B. 2002. The use of 10. FDA. 1992. FDA’s statement for the development of new stereoisomeric enzymes in the chemical industry in Europe. Curr. Opin. Biotechnol. 13: drugs. Chirality 4:338–340. 359–366. 11. François A, et al. 2002. Biodegradation of methyl tert-butyl ether and 35. Schuster J, et al. 2012. Bacterial degradation of tert-amyl alcohol proceeds other fuel oxygenates by a new strain, Mycobacterium austroafricanum IFP 2012. Appl. Environ. Microbiol. 68:2754–2762. via hemiterpene 2-methyl-3-buten-2-ol by employing the tertiary alcohol 12. Hammarström S, Hamberg M. 1973. Steric analysis of 3-, ␻4-, ␻3- and desaturase function of the Rieske nonheme mononuclear iron oxygenase ␻2-hydroxy acids and various alkanols by gas-liquid chromatography. MdpJ. J. Bacteriol. 194:972–981. Anal. Biochem. 52:169–179. 36. Simon MJ, et al. 1993. Sequences of genes encoding naphthalene dioxygenase 13. Hatzinger PB, et al. 2001. Biodegradation of methyl tert-butyl ether by a in Pseudomonas putida strains G7 and NCIB 98 16-4. Gene 127:31–37. pure bacterial culture. Appl. Environ. Microbiol. 63:5601–5607. 37. Smith CA, O’Reilly T, Hyman MR. 2003. Characterization of the initial 14. Hristova KR, et al. 2007. Comparative transcriptome analysis of Methyl- reactions during the cometabolic oxidation of methyl tert-butyl ether by ibium petroleiphilum PM1exposed to the fuel oxygenates methyl tert-butyl propane-grown Mycobacterium vaccae JOB5. Appl. Environ. Microbiol. ether and ethanol. Appl. Environ. Microbiol. 73:7347–7357. 69:796–804. 15. Jenske R, Vetter W. 2007. Highly selective gas-chromatography-electron- 38. Steffan RJ, McClay K, Vainberg S, Condee CW, Zhang D. 1997. Bio- capture negative-ion mass spectrometry method for the indirect enantio- degradation of the gasoline oxygenates methyl tert-butyl ether, ethyl tert- selective identification of 2- and 3-hydroxy fatty acids in food and biolog- butyl ether and tert-amyl methyl ether by propane-oxidizing bacteria. ical samples. J. Chromatogr. A 1146:225–231. Appl. Environ. Microbiol. 63:4216–4222. 16. Jones S, Valette D. 2009. Enantioselective synthesis of allylic alcohols via 39. Vanhessche KPM, Sharpless KB. 1997. Catalytic asymmetric synthesis of an oxazaborolidinium ion catalyzed Diels-Alder/Retro-Diels-Alder se- new halogenated chiral synthons. Chem. Eur. J. 3:517–522. quence. Org. Lett. 11:5358–5361. 40. Wackett LP. 2002. Mechanism and applications of Rieske non-heme iron 17. Kadyrov R, Koenigs RM, Brinkmann C, Voigtlaender D, Rueping M. dioxygenases. Enzyme Microbial Technol. 31:577–587.

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Dissertation Judith Schuster 5. Die speziﬁsche Alkoholmonooxygenase MdpJ 59

Supplemental Material

Applied and Environmental Microbiology 8 pages 5 Figures

Synthesis of short-chain diols and unsaturated alcohols from

secondary alcoholic substrates by the Rieske non-heme

mononuclear iron oxygenase MdpJ

Franziska Schäfer#, Judith Schuster#, Birgit Würz, Claus Härtig, Hauke

Harms, Roland H. Müller, and Thore Rohwerder*

Department of Environmental Microbiology, Helmholtz Centre for Environmental

Research - UFZ, Permoserstr. 15, 04318 Leipzig, Germany.

# F. S and J. S. contributed equally to this work.

* Corresponding author. Mailing address: Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, Permoserstr. 15, 04318 Leipzig, Germany, Phone: +49 341 235 1317. Fax: +49 341 235 1351. E-mail: [email protected].

Dissertation Judith Schuster 5. Die speziﬁsche Alkoholmonooxygenase MdpJ 5:

1. Purity and supply sources of tertiary alcohols and other chemicals

MTBE (≥ 99% pure) and TBA (≥ 99% pure) were purchased from Merck Schuchardt (Hohenbrunn, Germany). Isobutene (≥ 99% pure), 1,2-propanediol (highest purity available), (R)-(-)-2-butanol (99% pure), (S)-(+)-2-butanol (99% pure), and 2-butanone (99% pure) were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). 2-Propanol (>99.95% pure), diethylether (>99.8% pure) and acetone (>99.57% pure) were purchased from Carl

Roth GmbH & Co KG (Karlsruhe, Germany). 2-Butanol (>99.5% pure), sodium sulfate

(Na2SO4, >99% pure) and potassium carbonate (K2CO3, >99% pure) were purchased from Merck KGAa (Darmstadt, Germany). (S)-(+)-1,2-Propandiol (99% pure) and 3-buten-2-one (90% pure) were purchased from ABCR GmbH & Co KG (Karlsruhe, Germany). 3-Buten-2- ol (97% pure) was purchased from Alfa Aesar GmbH & Co KG (Karlsruhe, Germany). Pyridine (>99.5% pure) was purchased from Riedel-de Haën AG (Selze, Germany), and MPD at the highest purity available was from Taros Chemicals (Dortmund, Germany).

2. Detailed description of MSM culture medium

-1 The mineral salt medium (MSM) contained in mg L : NH4Cl, 760; KH2PO4, 680; K2HPO4,

970; CaCl2 6 H2O, 27; MgSO4 7 H2O, 71.2; initial pH was 7.5. MSM also contained trace -1 elements (in mg L ): FeSO4 7 H2O, 14.94; CuSO4 5 H2O, 0.785; MnSO4 4 H2O, 0.81;

ZnSO4 7 H2O, 0.44; Na2MoO4 2 H2O, 0.25; CoCl2 x 6 H2O, 0.040. A vitamin solution was added (in μg L-1): biotin, 20; folic acid, 20; pyridoxine-HCl, 100; thiamine-HCl, 50; riboflavin, 50; nicotinic acid, 50; DL-Ca-pantothenate, 50; p-amino-benzoic acid, 50; lipoic acid, 50, and cobalamin, 50.

Dissertation Judith Schuster 5. Die speziﬁsche Alkoholmonooxygenase MdpJ 5;

3. Induction of MdpJK in A. tertiaricarbonis L108 wild-type strain by incubation on TBA

MW 0h 1h 2h 3h 4h 5h 6h MW

170 130

100 70

55 MdpJ 40 MdpK 35

Figure S1. SDS-PAGE of samples from wild-type A. tertiaricarbonis L108 resting-cell experiments. Protein patterns of cells pregrown on MPD and then shifted to TBA are shown for samples taken after 0 to 6 hours of incubation as indicated. SDS-PAGE was performed according to protocols of Schägger et al. (1987) and Laemmli (1970). MdpJ (55 kDa) and MdpK (38.6 kDa) proteins were assigned according to Schäfer et al. (2007). (MW: kDa molecular weight marker, PageRuler Prestained Protein Ladder; Fermentas GmbH, St. Leon- Rot, Germany).

Dissertation Judith Schuster 5. Die speziﬁsche Alkoholmonooxygenase MdpJ 62

4. Degradation of TBA by A. tertiaricarbonis L108 wild-type strain and mdpJ knockout mutant K24 in resting-cell experiments

5 TBA 50 5 TBA 50 Isobutene Isobutene

4 40 4 40

3 30 3 30

TBA (mM) 2 20 TBA (mM) 2 20 Isobutene (μM) Isobutene (μM) 1 10 1 10

0 0 0 0 0123456 0123456 A Time (hours) B Time (hours)

Figure S2. Degradation of TBA in resting-cell experiments (1.4 g biomass dry weight per liter). (A) Complete degradation of TBA and isobutene formation by wild-type strain L108. (B) No significant degradation of TBA but isobutene formation by mdpJ knockout strain L108 (ΔmdpJ) K24. For a direct comparison with TBA conversion, isobutene values refer to concentrations in the liquid phase, although it was exclusively found in the gas phase of the close incubation bottles.

Dissertation Judith Schuster 5. Die speziﬁsche Alkoholmonooxygenase MdpJ 63

5. Identification of the 2-butanol metabolites 2-butanone and 3-buten-2-one

Peak 1 t3 Peak 1, 2.66 min t1 t0

Abundance

Peak 2 Peak 2, 2.76 min

Retention time (min)

Figure S3. GC-MS analysis of 2-butanol metabolites. Overlay of total ion chromatograms of samples from resting-cell experiments incubating A. tertiaricarbonis L108 wild-type cells with 2-butanol. Incubation times are indicated as t0 = 0, t1 = 1, and t3 = 3 hours. An increase of peaks 1 and 2 was observed which were identified as 2-butanone and 3-buten-2-one (see Figure S4), respectively. Left: overview showing complete areas of peaks 1 and 2; right: blow-up of peak 2. GC method: Analyses were carried out using a 7890A GC instrument with mass detector MSD 5975C (Agilent) and a DB-23 column (30 m, 0.25 mm, 0.25 μm, Agilent). Helium was used as carrier gas at 0.8 ml min-1. Temperature program: initial oven temperature 50°C for 2.8 min, then increase to 120°C at 30°C min-1.

Dissertation Judith Schuster 5. Die speziﬁsche Alkoholmonooxygenase MdpJ 64

t3, peak1 = 2-butanone

Abundance

m/z

t3, peak2 = 3-buten-2-one

Abundance

m/z

Figure S4. GC-MS analysis of 2-butanol metabolites. Mass spectra of peaks 1 and 2 of sample t3 (see Figure S3) and most probable matches by GC-MS spectral NIST data base (NIST 02).

Dissertation Judith Schuster 5. Die speziﬁsche Alkoholmonooxygenase MdpJ 65

6. Substrates and products of MdpJ-catalyzed reactions

OH OH

OH OH OH * OH

TBA MPD 2-propanol 1,2-propanediol

OH * OH OH OH * * R R

TAA (R = H) / 2-methyl-3-buten-2-ol / 2-butanol 3-buten-2-ol 3-methyl-3-pentanol 3-methyl-1-penten-3-ol

(R = CH3) (with chiral center)

OH OH ? ? OH OH * OH * * OH

2-methyl-3-buten-2-ol 2-methyl-3-buten- 3-buten-2-ol 3-buten-1,2-diol 1,2-diol

Figure S5. Substrates and corresponding products of MdpJ-catalyzed hydroxylation and desaturation of tertiary (left) and secondary (right) alcohols. Hydroxylation of unsaturated alcohols has not yet been demonstrated (indicated by “?”). Chiral centers of substrates and products are indicated by an asterisk.

Dissertation Judith Schuster 5. Die speziﬁsche Alkoholmonooxygenase MdpJ 66

References

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.

Schäfer, F., U. Breuer, D. Benndorf, M. von Bergen, H. Harms, R. H. Müller. (2007). Growth of Aquincola tertiaricarbonis L108 on tert.-butyl alcohol leads to the induction of a phthalate dioxygenase-related protein and its associated oxidoreductase subunit. Eng. Life Sci. 7: 512- 519.

Schägger, H., G. Jagow. (1987). Tricine-sodium dodecylsulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Analytical Biochem. 166: 368-379.

Dissertation Judith Schuster 6. Die 4-HIBA-Mutase HcmAB des Stammes L32:

achfolgend wird die Spezifität der neuartigen, cobalaminabhängigen 4-HIBA- Mutase in Bezug auf die Linearisierung der tertiären Säure 4-HIBA zu 5-HB N anhand von Deletionsstudien der beiden Untereinheiten HcmA und HcmB verifiziert. Das Enzym wird detailliert vorgestellt und biochemisch charakterisiert. Das Substrat- spektrum wird in Abhängigkeit zum HCM-spezifischen und hochkonservierten Isoleucin der Substratbindestelle an Aminosäure-Position ;2 der Wildtyp-HcmA im Vergleich zu diversen Substitutionsmutanten gezeigt. Die Publikation ist im Mai 4234 in The Journal of Biological Chemistry (J. Biol. Chem. 4234 4:9: 37724-37733.) veröffentlicht worden. Die Online-Version ist seit dem 42. März 4234 (doi: 32.3296/jbc.M333.5368;2) verfügbar. Es folgt der Artikel samt Supplemental Material. Die Belege der Co-Autorenschaften sind im Anhang angefügt.

67 6. Die 4-HIBA-Mutase HcmAB des Stammes L32: 68

Bacterial Acyl-CoA Mutase Specifically Catalyzes Coenzyme B12-dependent Isomerization of 2-Hydroxyisobutyryl-CoA and (S)-3-Hydroxybutyryl-CoA*□S Received for publication, October 17, 2011, and in revised form, March 19, 2012 Published, JBC Papers in Press, March 20, 2012, DOI 10.1074/jbc.M111.314690 Nadya Yaneva, Judith Schuster, Franziska Schäfer, Vera Lede, Denise Przybylski, Torsten Paproth, Hauke Harms, Roland H. Müller, and Thore Rohwerder1 From the Department of Environmental Microbiology, Helmholtz Centre for Environmental Research (UFZ), 04318 Leipzig, Germany

Background: Carbon skeleton rearrangements of acyl-CoA esters are catalyzed by coenzyme B12-dependent mutases. Results: A bacterial mutase specifically catalyzes the isomerization of 2-hydroxyisobutyryl- and (S)-3-hydroxybutyryl-CoA. Conclusion: Substrate affinity and enzyme activity depend strongly on the active site amino acid Ile90. Significance: This is the first characterization of an enzyme isomerizing hydroxylated short chain carboxylic acids.

Coenzyme B12-dependent acyl-CoA mutases are radical of the main metabolic pathways. However, it is one of the uri- enzymes catalyzing reversible carbon skeleton rearrangements nary organic acids found in humans with lactic acidosis (1), and in carboxylic acids. Here, we describe 2-hydroxyisobutyryl-CoA several metabolic sequences can be proposed leading to this mutase (HCM) found in the bacterium Aquincola tertiaricarbo- unusual short chain carboxylic acid (Fig. 1). A major natural nis as a novel member of the mutase family. HCM specifically source of 2-HIBA might be the plant cyanoglycoside linamarin catalyzes the interconversion of 2-hydroxyisobutyryl- and (S)-3- (2), as the nitrile corresponding to 2-HIBA is an intermediate of hydroxybutyryl-CoA. Like isobutyryl-CoA mutase, HCM con- linamarin biosynthesis and catabolism. In addition, 2-HIBA sists of a large substrate- and a small B12-binding subunit, HcmA could be produced during degradation of isobutane and and HcmB, respectively. However, it is thus far the only acyl- isobutene via oxidation of the corresponding alcoholic and CoA mutase showing substrate specificity for hydroxylated car- diolic metabolites. Besides, anthropogenic sources of 2-HIBA boxylic acids. Complete loss of 2-hydroxyisobutyric acid degra- exist, because it is a pharmaceutical intermediate and by-prod- dation capacity in hcmA and hcmB knock-out mutants uct of industrial processes, e.g. the production of poly(methyl established the central role of HCM in A. tertiaricarbonis for methacrylate) (PMMA) (3). It has also been identified as a degrading substrates bearing a tert-butyl moiety, such as the fuel metabolite in the bacterial degradation of the gasoline additive oxygenate methyl tert-butyl ether (MTBE) and its metabolites. methyl tert-butyl ether (MTBE) (4), which is used at large scale Sequence analysis revealed several HCM-like enzymes in other since the 1990s as a fuel oxygenate for reducing carbon mon- bacterial strains not related to MTBE degradation, indicating oxide emissions and now threatens drinking water resources that HCM may also be involved in other pathways. In all strains, due to its persistence in contaminated aquifers (5). Although hcmA and hcmB are associated with genes encoding for a puta- biodegradation of 2-HIBA and its methyl ester has already been tive acyl-CoA synthetase and a MeaB-like chaperone. Activity observed in one early study in 1984 when investigating the bac- and substrate specificity of wild-type enzyme and active site terial degradation of wastewater compounds of a PMMA plant mutants HcmA I90V, I90F, and I90Y clearly demonstrated that (6), convincing enzymatic steps for its conversion to common HCM belongs to a new subfamily of B -dependent acyl-CoA 12 metabolites have not been proposed for Ͼ20 years. mutases. Recently, we found a coenzyme B12-dependent rearrangement reaction in the bacterial degradation pathway of MTBE via 2-HIBA, likely catalyzed by a novel acyl-CoA mutase (7). In The tertiary carbon-bearing 2-hydroxyisobutyric acid this enzymatic step, the CoA ester of 2-HIBA is converted to (2-HIBA)2 is rarely found in Nature and is not an intermediate 3-hydroxybutyryl-CoA (Fig. 1). Thus, in a single reaction the branched-chain ␣-hydroxy carboxylic acid is rearranged into * This work was supported by the Helmholtz Centre for Environmental an easily metabolizable linear isomer, now possessing an oxi- Research within the Chemicals in the Environment program and by Evonik dizable secondary hydroxyl group at the ␤ position. Conse- Industries. □S This article contains supplemental Figs. S1–S7. quently, the proposed 2-hydroxyisobutyryl-CoA mutase The nucleotide sequence(s) reported in this paper has been submitted to the Gen- (HCM) would play a central role in MTBE metabolism enabling BankTM/EBI Data Bank with accession number(s) JQ708092. its complete mineralization. Similar rearrangements in carbox- 1 To whom correspondence should be addressed. Tel.: 493412351317; Fax: 493412351351; E-mail: [email protected]. ylic acids have already been observed with several acyl-CoA 2 The abbreviations used are: 2-HIBA, 2-hydroxyisobutyric acid; ECM, ethyl- mutases (8, 9, 10), namely methylmalonyl-CoA mutase (MCM), malonyl-CoA mutase; HCM, 2-hydroxyisobutyryl-CoA mutase; ICM, isobu- isobutyryl-CoA mutase (ICM) and ethylmalonyl-CoA mutase tyryl-CoA mutase; IcmF, isobutyryl-CoA mutase-fused; MCM, methylmalonyl-CoA mutase; MSM, mineral salt medium; MTBE, methyl tert-butyl ether; (ECM). Recently, a variant of ICM (IcmF) has been character- PMMA, poly(methyl methacrylate); TBA, tert-butyl alcohol. ized as a fusion of the ICM sequence with the G protein chap-

15502 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 19•MAY 4, 2012

Dissertation Judith Schuster 6. Die 4-HIBA-Mutase HcmAB des Stammes L32: 69

2-Hydroxyisobutyryl-CoA Mutase

FIGURE 1. Possible metabolic routes leading to 2-HIBA. Degradation of valine, linamarin, alkyl tert-butyl ethers, and branched C4 hydrocarbons could result in 2-HIBA formation. Further degradation proceeds via isomerization to the common metabolite 3-hydroxybutyric acid by the B12-dependent acyl-CoA mutase HCM discovered in the MTBE-degrading bacterial strain A. tertiaricarbonis L108 (7). erone MeaI (11), a paralog of the MCM-associated MeaB pro- EXPERIMENTAL PROCEDURES tein. However, HCM would be distinct from all other known Materials acyl-CoA mutases because it would catalyze the conversion of Tert-butyl alcohol (TBA) (Ն99%), 2-HIBA (Ͼ98%), tert-amyl hydroxylated carboxylic acids. alcohol (Ͼ99%), and anhydrides of butyric (98%) and isobutyric This study aimed at elucidating the biochemistry of the acyl- (98%) acids were purchased from Merck Schuchardt. 2-Meth- CoA mutase activity from the MTBE-degrading bacterium ylpropan-1,2-diol was from Taros Chemicals at the highest Aquincola tertiaricarbonis L108. It was found that HCM con- purity available. Coenzyme B (Ն97%), CoA (Ն93%), methyl- sists of a large acyl-CoA-binding and a small B -binding sub- 12 12 malonyl-CoA (Ն96%, racemic mixture), and succinyl-CoA unit, HcmA and HcmB, respectively. Destroying the corre- Ն sponding genes hcmA and hcmB by insertional mutation ( 94%) were purchased from Sigma. Enantiopure (R)- and (S)- 3-hydroxybutyryl-CoA were provided by Evonik Industries resulted in complete loss of 2-HIBA-degrading capability. The Ͼ wild-type mutase genes were cloned in Escherichia coli strains, (53–54% acyl-CoA enantiomer, 99.9 and 98.9% enantiomeric and enzyme activity of heterologously expressed subunits was excess, respectively; 40% NaCl; 6–7% N-hydroxysuccinimide). characterized. Catalysis by purified recombinant HCM was specific for 2-hydroxyisobutyryl- and (S)-3-hydroxybutyryl- Syntheses CoA. Sequence comparison with known acyl-CoA mutases Synthesis of Acyl-CoA Esters—Acyl-CoA esters not commer- identified a single active site amino acid residue present in cially available were synthesized according to two methods. HcmA likely being important for determining substrate speci- Isobutyryl-CoA and butyryl-CoA were prepared from their ficity. To understand the function of this residue, we analyzed anhydrides (12). 2-Hydroxyisobutyryl-CoA was synthesized activities of three mutant HcmA subunits having relevant from its free acid via thiophenyl ester by the method of Padma- amino acid substitutions. kumar et al. (13).

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Dissertation Judith Schuster 6. Die 4-HIBA-Mutase HcmAB des Stammes L32: 6:

2-Hydroxyisobutyryl-CoA Mutase

Identification of Synthesis Products—Identity of synthesized 0.4 and further incubation at 12 °C to an OD550 of 0.5. Then, acyl-CoA esters was verified by electrospray ionization-tandem cells were centrifuged and suspended in Tris buffer. mass spectrometry (ESI-MS/MS) using a 4000 QTRAP LC/MS/MS system consisting of a liquid chromatograph and a Purification of Recombinant Proteins high performance triple quadrupole/linear ion trap mass spec- For the purification of recombinant mutase subunits, crude trometer (Applied Biosystems). Acyl-CoA esters were detected extracts of induced E. coli cells were prepared by disruption directly by ESI-MS/MS. Isocratic elution was achieved using a using a mixer mill (MM 400, Retsch GmbH, Germany) with Ϫ1 mobile phase containing 17.5 mM ammonium acetate, 0.5 vol- glass beads (212–300 ␮m, Sigma) at 30 s for 30 min. The ume % acetic acid, and 30 volume % acetonitrile (14). Charac- recombinant HcmA and HcmB subunits were purified with the teristic mass spectra for the respective acyl-CoA compounds help of their His and Strep tags, respectively. All purification were obtained (supplemental Fig. S1). steps were performed at 12 °C. HcmA Subunit—Crude extracts of E. coli TOP10 Bacterial Strains and Growth Conditions pASG-IBA43::hcmA were loaded on a nickel-nitrilotriacetic Strain A. tertiaricarbonis L108 isolated from an MTBE-con- acid Superflow 10-ml column (IBA Goettingen). After washing taminated aquifer at Leuna, Germany (7, 15) was cultivated in with 20 column volumes of imidazole buffer (50 mM sodium liquid mineral salt medium (MSM) containing MTBE at con- phosphate, 300 mM NaCl, 20 mM imidazole, pH 8.0), HcmA was centrations of 0.3 g literϪ1 as described previously (16). For eluted with the same buffer containing 250 mM imidazole. Frac- growth and resting-cell studies, also tert-amyl alcohol, TBA, tions containing HcmA were concentrated via viva spin col- 2-methylpropan-1,2-diol, and 2-HIBA were supplied as sole umns (30 kDa; GE Healthcare) and diluted with conservation source of carbon and energy at 0.5 g literϪ1. E. coli TOP10 and buffer (50 mM potassium phosphate, 10% glycerol, pH 7.4). ArcticExpress (DE3) were grown in Luria-Bertani broth. HcmB Subunit—Crude extracts of E. coli ArcticExpress Growth was monitored by measuring optical density (OD) of (DE3) pPR-IBA1::hcmB were loaded on a Strep-Tactin Super- cultures at 700 or 550 nm as indicated. flow high capacity 10-ml column (IBA Goettingen). After washing with 20 column volumes of Tris buffer, HcmB was eluted Sequencing of Genomic DNA from A. tertiaricarbonis with elution buffer (Tris buffer containing 2.5 mM desthiobio- tin). Fractions containing HcmB were concentrated using viva For sequencing a larger fragment of the hcm gene region of spin columns (10 kDa) and diluted with conservation buffer. wild-type strain L108, genomic DNA was extracted using Mas- terPure DNA Purification kit (Epicenter) and sequenced by Expression and Purification of Site-specific HcmA Mutants Illumina HiSeq 2000 technology (GATC Biotech). The The hcmA mutant genes cloned into pASG-IBA43 were pur- obtained DNA sequences were analyzed for open reading chased from GeneCust Europe. Site-directed mutagenesis frames using Rast (Rapid Annotation using Subsystem Tech- resulted in HcmA I90Y, I90F, and I90V mutants (with the point nology). A 4.5 kb sequence including both hcm genes was mutations a268t plus t269a, a268t, and a268g, respectively). obtained. The vectors were transformed into E. coli TOP10. Expression and purification were performed as described for the wild-type Cloning and Heterologous Expression of hcmA and hcmB from recombinant HcmA. A. tertiaricarbonis in E. coli The gene encoding for the large subunit of HCM (hcmA) was Quantitative Enzymatic Measurements of Recombinant amplified from strain L108 genomic DNA by applying the for- Enzyme ward primer 5Ј-AATG ACC TGG CTT GAG CCG CAG A-3Ј HCM Activity—Enzyme activity was routinely measured in and reverse primer 5Ј-TCCC GAA GAC CGG GTC TCG 1–2 ml of 50 mM potassium phosphate buffer, pH 6.6, contain- Ј ␮ CGG-3 . PCR was accomplished with Pfu DNA polymerase ing 10% glycerol, 833 M coenzyme B12, and 10 mM MgCl2,at (Promega) for 30 cycles, including denaturation at 94 °C for 1 30 °C in the dark. This reaction mixture was incubated in 10-ml min, annealing at 57 °C for 1 min, and extension at 72 °C for 2 headspace glass vials sealed with rubber stoppers. As variation min. The PCR product was cloned into expression vector of HcmA and HcmB ratios (up to 5:1 and 1:5, respectively) did pASG-IBA43 (IBA Goettingen) and transformed into E. coli not result in increased activities, the recombinant subunits TOP10 according to the protocol of IBA Goettingen. Induction were added at equimolar ratios (3 ␮M) throughout the study. ␮ Ϫ1 was performed at OD550 of 0.5 with 200 g liter anhydrotet- After a 5-min preincubation in the presence of coenzyme B12, racycline for3hat30°C.Cells were centrifuged and suspended the reaction was started by adding acyl-CoA substrates. in Tris buffer (100 mM Tris, 150 mM NaCl, pH 8.0) for further Throughout the experiments, oxygen concentrations were analysis. The cloning vector pPR-IBA1::hcmB for the small sub- minimized by permanently flushing the incubation vials with unit of HCM was purchased from DNA2.0 and transformed nitrogen. For determination of pH optimum, pH values of the into 100 ␮l of electrocompetent E. coli ArcticExpress (DE3) phosphate buffer and a phosphate/acetate buffer (phosphate (Novagen) at 300 mV for 5 ms in chilled 0.1-cm cuvettes in a buffer plus 50 mM sodium acetate buffer) were adjusted to val- MicroPulser (Bio-Rad). Induction was performed with 0.5 mM ues between 5.0 and 7.8. The temperature optimum was deter- isopropyl 1-thio-␤-D-galactopyranoside for 20 h at 12 °C, after mined by incubating at temperatures between 20 and 55 °C. growth at 30 °C in Luria-Bertani medium containing 20 mg HPLC Analysis of Acyl-CoA Esters—Concentrations of acyl- Ϫ1 Ϫ1 liter gentamycin and 50 mg liter kanamycin to an OD550 of CoA ester substrates and products were quantified by ion-pair

15504 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 19•MAY 4, 2012

Dissertation Judith Schuster 6. Die 4-HIBA-Mutase HcmAB des Stammes L32: 6;

2-Hydroxyisobutyryl-CoA Mutase

FIGURE 2. HCM genetic structure. Comparison of the 4.5-kb hcm gene environment in A. tertiaricarbonis L108 with the closely related sequences of R. sphaeroides ATCC 17029 (NCBI locus tag Rsph17029_3654 to Rsph17029_3657) and Nocardioides sp. JS614 (Noca_2129 to Noca_2131). Genes of the HCM subunits hcmB and hcmA are drawn in black; the acyl-CoA synthetase gene is gray; and meaH, encoding for a MeaB-like chaperone, is white. The Ez-Tnp5ϽKan- 2ϾTnp inserts from the two knock-out mutants L108(⌬hcmB)K7 and L108(⌬hcmA)K5 are shown fasciated, whereas the disrupted genes stay black. The different gene length and overlapping intensity between meaH and hcmA are based on the real data from NCBI. An identical organization of the hcm operon was also detected in the genomes of strains M. petroleiphilum PM1, R. sphaeroides KD131, S. novella DSM 506, Marinobacter algicola DG893, Mesorhizobium alhagi CCNWXJ12-2 and X. autotrophicus Py2.

chromatography using an HPLC system (Shimadzu) with a Purification kit). Conditions were 4 min initial denaturation at Nucleosil 100–5 C18 column (250 mm ϫ 3 mm, 5 ␮m; Mach- 95 °C followed by 60 cycles of 30 s at 95 °C and 4 min 60 °C, erey-Nagel) and a mobile phase of 14.5 vol% acetonitrile, 10 mM finally cooled down to 8 °C. The products were cleaned with tetrabutylammonium hydrogen sulfate and 100 mM sodium Centri-Sep columns (Applied Biosystems) and sequenced using phosphate at pH 4.5 for the separation of 2-hydroxyisobutyryl-, an ABI PRISM 3100 Genetic Analyzer with the BigDye Termi- 3-hydroxybutyryl-, methylmalonyl-, and succinyl-CoA, with nator v1.1 Cycle sequencing kit (Applied Biosystems). The retention times of about 35, 24, 30, and 40 min, respectively. For resulting sequences were analyzed with BLAST (19) and separation of isobutyryl- and butyryl-CoA, acetonitrile content aligned via Sequencher 5.0 software (Gene Codes Corporation). of the eluent was 21.6 vol%, resulting in retention times of 35 and 40 min, respectively (17). Eluent flow was adjusted to 0.6 ml RESULTS minϪ1, and column oven temperature was 30 °C. Absorbance at Insertional Inactivation of hcmA and hcmB, the Putative 260 nm was used for quantifying acyl-CoA esters. In addition, Genes Encoding for HCM Large and Small Subunits, and Char- spectra from 190 to 280 nm were recorded for distinguishing acterization of Mutant Strains—Sequencing of genomic DNA peaks of CoA esters from unspecific signals (18). Detection of wild-type A. tertiaricarbonis L108 revealed that a 4.5-kb frag- limit at 260 nm was 0.5 ␮M CoA ester. For stopping enzymatic ment comprised both hcmA and hcmB separated by two genes catalysis, samples were mixed with an equal volume of stop encoding for a putative acyl-CoA synthetase and a MeaB-like G buffer (100 mM acetate, pH 3.5) and incubated at 60 °C for 5 min protein chaperone, respectively (Fig. 2). A highly similar prior to HPLC analysis. sequence (Ͼ97% identity) having the same gene organization has already been found in the genome of the MTBE-degrading Knock-out Mutant Construction and Strain Isolation strain Methylibium petroleiphilum PM1 (20), supporting the To establish the enzymatic function of HcmAB in 2-HIBA hypothesis that HCM is involved in degradation of the MTBE degradation, we generated knock-out mutants of strain A. ter- metabolite 2-HIBA (7). However, the organization of hcm-like tiaricarbonis L108 by electroporation of 1 ␮l of EZ-Tn5ϽKAN- genes interrupted by genes likely encoding for a acyl-CoA syn- 2ϾTnp transposome (Epicenter Biotechnologies) to 70 ␮lof thetase and MeaB-like chaperone is also present in other bac- electrocompetent L108 cells in chilled cuvettes at 1.8 kV for teria, such as Rhodobacter sphaeroides ATCC 17029 and about 5 ms (MicroPulser; Bio-Rad). Transformed cells were Nocardioides sp. JS614 (Fig. 2), thus far not related to fuel oxy- rescued in 5 ml of MSM amended with 10 mM fructose for6hat genate ether degradation. As has already been suggested on the 30 °C and 150 rpm. Dilutions were plated on MSM fructose basis of transcriptome analysis of MTBE-grown cells of strain agar containing 50 ␮gmlϪ1 kanamycin and incubated for 2 days PM1 (21), the acyl-CoA synthetase could be involved in CoA at 30 °C. As the transposome integrates randomly into the activation of 2-HIBA (Fig. 1). The G protein chaperone may DNA, all colonies obtained had to be analyzed for loss of their play a role in HCM assembly and stabilization, as has already capability to grow on MSM agar containing 0.5 g literϪ1 TBA. found for MeaB and MeaI associated with MCM and ICM/ In addition, copies of the colonies were maintained on MSM IcmF, respectively (11, 22). Therefore, we propose for the fructose agar for further analysis. Colonies with restricted or MeaB/MeaI paralog associated with HCM the name MeaH even lost TBA degradation potential were transferred on MSM (Fig. 2). 2-HIBA agar plates. The mutants that failed to grow were fur- For precisely studying the role of HCM in bacterial 2-HIBA ther analyzed. degradation, we tried to create hcmA and hcmB knock-out The exact integration site of the kanamycin cassette into the mutants of strain L108 by a site-specific mutagenesis approach genomic DNA was determined by direct DNA sequencing via homologous recombination. However, even after several using flanking KAN-2 primers (Epicenter Biotechnologies) of 1 trials corresponding knock-out mutants were not obtained. On mg literϪ1 high concentrated genomic DNA (MasterPure DNA the other hand, unspecific transposon-mediated mutagenesis

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Dissertation Judith Schuster 6. Die 4-HIBA-Mutase HcmAB des Stammes L32: 72

2-Hydroxyisobutyryl-CoA Mutase

FIGURE 3. Phenotypic characterization of hcm knock-out mutants. Nearly stoichiometric conversion of TBA (A) and 2-methylpropan-1,2-diol (B) to 2-HIBA by resting cells of the knock-out mutant strains L108(⌬hcmB)K7 and L108(⌬hcmA)K5 pre-grown on tert-amyl alcohol (mean values of experiments performed with both mutants) is shown. In the case of incubation with TBA, also small amounts of 2-methylpropan-1,2-diol accumulated.

and screening for loss of capability to grow on 2-HIBA resulted in two hcm mutants, L108(⌬hcmA)K5 and L108(⌬hcmB)K7, bearing stable insertions of a functionally active kanamycin resistance gene in the hcmA and hcmB genes, respectively (Fig. 2). In contrast to the wild-type L108 strain, both mutant strains were not able to grow on TBA and 2-methylpropan-1,2-diol, precursors of 2-HIBA in the tert-butyl alkyl ether degradation pathway (Fig. 1), establishing the postulated central role of HCM in the degradation of organic compounds possessing the tert-butyl moiety (7). Accordingly, TBA and 2-methylpropan- 1,2-diol were stoichiometrically converted to 2-HIBA by both knock-out mutants in resting-cell experiments (Fig. 3), indicating that only the 2-HIBA-metabolizing enzymatic activity was affected by the hcm knockouts. Contrarily, the mutants still grew well on tert-amyl alcohol, suggesting an alternative degra- FIGURE 4. HPLC assay for the quantification of enzymatic transforma- dation pathway for this C5 homolog of TBA (23). tion of 2-hydroxyisobutyryl-CoA. HPLC chromatograms of samples after 10 min of incubation show formation of 3-hydroxybutyryl-CoA only in the pres- Expression of hcm Genes in E. coli and Purification of HCM ence of reconstituted wild-type mutase subunits HcmA and HcmB. Subunits—For biochemical characterization, wild-type hcmA and hcmB as well as hcmA mutant genes were heterologously enzymatic conversion of 2-hydroxyisobutyryl-CoA was observed, expressed in E. coli. N-terminal His-tagged and C-terminal having a maximal activity at pH 6.6 (supplemental Fig. S4). The Strep-tagged proteins, respectively, were purified from crude temperature optimum of the mutase activity was in the meso- extracts by one-step affinity chromatography. Denaturing gel philic range at about 30 °C. At 20 °C, however, still 50% of the electrophoresis established the presence of the mutase subunits maximal activity was achieved, whereas a complete loss of in crude extracts of induced E. coli cells and successful purifi- catalysis was shown at 45 °C (supplemental Fig. S5), which cor- cation of the tagged proteins (supplemental Fig. S2). Yields of responds well to the temperature spectrum described for purified HcmA (HcmA Ile90 and HcmA mutants) and HcmB growth of wild-type strain L108 on TBA (24). Interestingly, as were about 100 and 80 mg literϪ1 of E. coli culture, respectively. demonstrated by analyzing the 3-hydroxybutyryl-CoA esters Characterization of Reconstituted Hcm Enzyme Activity—As produced, after derivatization to propyl esters and enantiose- has already been shown for the small and large ICM subunits lective separation on a chiral GC column (supplemental Fig. (9), neither recombinant HcmA nor HcmB alone was able to S6), 2-hydroxyisobutyryl-CoA was predominantly converted to catalyze an acyl-CoA mutase reaction. However, a combination (S)-3-hydroxybutyryl-CoA, and only minor amounts of about of both purified subunits resulted in an enzymatic rearrange- 20% of the (R)-enantiomer were obtained, indicating stereospe- ment of 2-hydroxyisobutyryl- into 3-hydroxybutyryl-CoA (Fig. cific catalysis. 4). Under the experimental conditions applied, a linear increase Kinetic Parameters of HCM Catalysis—Possible enzymatic in 3-hydroxybutyryl-CoA for about 10 min could be achieved, rearrangement of substrates other than 2-hydroxyisobutyryl- thus, enabling determination of turnover rates by tracing CoA was tested at optimal pH and temperature conditions changes in substrate and product concentrations with HPLC (Table 1 and Fig. 5). As expected, purified wild-type HCM sub- (supplemental Fig. S3). Within a pH range of 5–8, significant units were able to convert (S)-3-hydroxybutyryl-CoA at high

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Dissertation Judith Schuster 6. Die 4-HIBA-Mutase HcmAB des Stammes L32: 73

2-Hydroxyisobutyryl-CoA Mutase

TABLE 1 Kinetic parameters of reconstituted HCM subunits HcmA (wild-type Ile90 and mutant I90V) and wild-type HcmB incubated with various acyl-CoA substrates at pH 6.6 and 30 °C (Fig. 5). a Subunit and substrate Vmax Km kcat kcat/Km Vmax/Km Ϫ1 Ϫ1 Ϫ1 Ϫ1 Ϫ1 Ϫ1 Ϫ1 nmol min mg ␮M min mM min liters min mg HcmA Ile90 (S)-3-Hydroxybutyryl-CoA 140 Ϯ 5.6 (100)b 128 Ϯ 22 12 Ϯ 0.5 90 Ϯ 16 1,080 Ϯ 190 2-Hydroxyisobutyryl-CoA 36 Ϯ 2.8 (26) 104 Ϯ 25 3.0 Ϯ 0.2 29 Ϯ 7.3 350 Ϯ 87 (R)-3-Hydroxybutyryl-CoA 2.4 Ϯ 0.15 (2) 1,660 Ϯ 367 0.20 Ϯ 0.01 0.12 Ϯ 0.03 1.4 Ϯ 0.3 Butyryl-CoA 2.4 Ϯ 0.26 (2) 3,340 Ϯ 880 0.20 Ϯ 0.02 0.06 Ϯ 0.02 0.7 Ϯ 0.2 Isobutyryl-CoA 0.67 Ϯ 0.03 (0.5) 550 Ϯ 140 0.06 Ϯ 0.003 0.10 Ϯ 0.03 1.2 Ϯ 0.3 HcmA I90V (S)-3-Hydroxybutyryl-CoA 24 Ϯ 1.1 (17) 1,760 Ϯ 240 2.0 Ϯ 0.1 1.1 Ϯ 0.16 14 Ϯ 2.0 2-Hydroxyisobutyryl-CoA 15 Ϯ 1.1 (11) 1,840 Ϯ 370 1.2 Ϯ 0.1 0.7 Ϯ 0.14 8.1 Ϯ 1.7 a Activities obtained with the wild-type HcmAB for methylmalonyl- and succinyl-CoA and with the HcmA I90V mutant reconstituted with wild-type HcmB for all acyl-CoA esters tested, except for (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA, were below detection limit (Ͻ0.01 nmol minϪ1 mgϪ1). b For calculating relative activities, Vmax obtained with wild-type HcmAB for (S)-3-hydroxybutyryl-CoA was set to 100%. rates, demonstrating the reversibility of the rearrangement suggests that this enzyme represents a new mutase subfamily.

reaction. Compared with Vmax obtained with 2-hydroxyisobu- This is also supported by a recent study on different IcmF tyryl-CoA, rates with (S)-3-hydroxybutyryl-CoA were nearly enzymes demonstrating that 3-hydroxybutyryl-CoA is not con- four times higher, indicating that at least in vitro not the direc- verted by this type of mutase (25). BLAST analysis of L108 tion toward degradation of tert-butyl group molecules (Fig. 1) hcmA as query against the NCBI data base (March 2012) but the reverse reaction is favored. As reconstituted HcmAB resulted in only a few HcmA-like proteins as closest matches remained active for extended times, not only steady-state rates with Ͼ60% identity to the L108 sequence. N-terminal regions were measured but also equilibrium between 2-hydroxyisobu- of all matches were aligned with the corresponding sequences tyryl- and 3-hydroxybutyryl-CoA could be reached (Fig. 6). of representatives of the other mutase subfamilies ICM, IcmF, Accordingly, with the higher specific activities obtained for (S)- MCM, and ECM (supplemental Fig. S7). Particularly, compar- 3-hydroxybutyryl-CoA (Table 1), the equilibrium lies on side of ison of two sequence sections containing amino acid residues 2-hydroxyisobutyryl-CoA, resulting in an equilibrium constant known to be responsible for binding the acyl-CoA substrates (9)

Keq of approximately 1.5. In line with the finding that conver- revealed a single Ile residue that clearly distinguishes HcmA- sion of 2-hydroxyisobutyryl-CoA was stereospecific for the (S)- like sequences from other mutase proteins (Ile90 in the L108 isomer of 3-hydroxybutyryl-CoA (Ͼ80% enantiomeric excess), sequence, Fig. 7). In addition, HCM, ICM, and IcmF sequences only very low activity was observed with pure (R)-3-hydroxybu- possess a Gln at position 208 (numbering as in HcmA of strain

tyryl-CoA, resulting in approximately 2% of the Vmax value L108) corresponding to an Arg residue in MCM and ECM. obtained with the (S)-enantiomer. As expected, reconstituted HcmAB did not show detectable conversion of the MCM sub- DISCUSSION strates methylmalonyl- and succinyl-CoA. However, with the Role of HCM in 2-HIBA Catabolism—Comparison of the ICM substrates isobutyryl- and butyryl-CoA low rearrange- wild-type A. tertiaricarbonis L108 and the mutant strains ment activities were observed, corresponding to approximately L108(⌬hcmA)K5 and L108(⌬hcmB)K7 clearly shows that integ- 0.5 and 2% of the (S)-3-hydroxybutyryl-CoA conversion rate. rity of both HCM subunits is required for degradation of With the conservative HcmA I90V mutant, significant activi- 2-HIBA. In line with the assigned role in the degradation path- ties were only obtained with (S)-3-hydroxybutyryl- and 2-hy- way proposed for compounds bearing a tert-butyl group (7) droxyisobutyryl-CoA, showing Ͻ20% of the wild-type rates (Fig. 1), reconstituted HcmAB specifically catalyzes the revers- (Table 1 and Fig. 5C). The nonconservative substitutions I90Y ible rearrangement of 2-hydroxyisobutyryl- and 3-hydroxybu-

and I90F resulted in complete loss of enzyme activity. tyryl-CoA. Conversion of substrates of other B12-dependent With the wild-type HcmA, the Km values for 2-hydroxyisobu- acyl-CoA mutases, such as methylmalonyl- and succinyl-CoA, tyryl- and (S)-3-hydroxybutyryl CoA were both determined to be is negligible, thus preventing interference of HCM activity with ␮ in the 100 M range, whereas affinities to (R)-3-hydroxybutyryl- central carbon metabolism. However, Keq and kcat values deter- CoA and the ICM substrates were approximately 5–30 times mined for the purified recombinant enzyme reveal that not the lower (Table 1). Consequently, with these latter substrates the cat- catalysis of 2-hydroxyisobutyryl-CoA conversion but the

alytic efficiency (kcat/Km) was approximately 1000-fold lower than reverse reaction is favored, suggesting that HCM is not only with (S)-3-hydroxybutyryl-CoA. The substitution I90V resulted in employed for channeling tert-butyl moieties toward central

Km values close to 2 mM and a nearly 100-fold diminution of the metabolism. A role in synthesis of 2-HIBA and related mole- catalytic efficiency with (S)-3-hydroxybuytryl-CoA. cules, on the other hand, would be surprising, as these com-

HCM Activity Defines New Subfamily of B12-dependent Acyl- pounds are rarely found in Nature. Further investigations are CoA Mutases—HcmAB showed only significant activity with required for testing whether catalysis of 3-hydroxybutyryl-CoA 2-hydroxyisobutyryl- and (S)-3-hydroxybutyryl-CoA (Table 1). conversion is also favored under in vivo conditions. In this con- Likewise, narrow substrate specificity has also been demon- nection, interplay with other proteins has to be considered, e.g.

strated for the thus far known subfamilies of B12-dependent it has previously been reported that interaction of MCM with acyl-CoA mutases (8, 9, 10). Hence, the distinct specificity for the P-loop GTPase MeaB facilitates GTP-dependent assembly hydroxylated short chain acyl residues found for HCM catalysis of the holo-MCM and protects against the radical intermedi-

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Dissertation Judith Schuster 6. Die 4-HIBA-Mutase HcmAB des Stammes L32: 74

2-Hydroxyisobutyryl-CoA Mutase

FIGURE 5. Kinetic plots of acyl-CoA rearrangement activities catalyzed by reconstituted HcmA and HcmB. A and B, conversion of 2-hydroxyisobutyryl- and (S)-3-hydroxybutyryl-CoA (A) and (R)-3-hydroxybutyryl-, butyryl-, and isobutyryl-CoA by wild-type HcmAB (B). C, conversion of 2-hydroxyisobutyryl- and (S)-3-hydroxybutyryl-CoA by HcmA I90V mutant reconstituted with the wild-type HcmB. All enzyme activities were measured at pH 6.6 and 30 °C. By using nonlinear regression analysis (Graph Pad Prism 5.0 software), Km and Vmax values were revealed (Table 1), applying either the Michaelis-Menten equation (A and B) or an allosteric sigmoidal model (C).

natural substrates isobutyryl- and butyryl-CoA do not possess chirality, catalysis is also predominantly stereospecific with retention of configuration, as has been revealed by testing labeled substrates in vivo (28) and with partially purified enzyme (29). Now, due to the structural similarities between the 2-hydroxyisobutyryl and isobutyryl substrate residues, in HCM an ICM-like orientation of the two methyl groups of the branched-chain carboxylic acid could be expected (30) which would result in catalysis predominantly toward (R)-3-hydroxybutyryl-CoA (Fig. 8). However, considering the observed stereospecificity of HCM catalysis favoring the (S)-enantiomer of 3-hydroxybutyryl-CoA, it can be concluded that in HCM the hydroxyacyl residue of its substrates is mainly oriented in the same way as the carboxyacyl substrate moiety in MCM and ECM. In HCM, the polar but uncharged hydroxyl group would FIGURE 6. Equilibrium of HCM-catalyzed hydroxyacyl-CoA rearrange- specifically interact with the amido function of Gln208 (num- ments. Appearance of equilibrium between (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA conversions by wild-type HcmAB after prolonged incu- bering as in HcmA of strain L108), whereas in MCM/ECM the bation is shown. negatively charged carboxyl group is close to the guanidinium ion of Arg207/Arg131 (numbering as in Figs. 7 and 8). Interest- ates formed during mutase catalysis (22). Accordingly, a strong ingly, a second polar amino acid residue corresponding to the operonic association of meaB and mcm genes has been Tyr89/Tyr13 in MCM/ECM is missing in HCM. Instead, the observed (26). In case of HCM, a quite similar association is hydrophobic Ile90 likely interacts with the hydroxyl and methyl found (Fig. 2), indicating that MeaH also functions as chaper- groups of the HCM acyl substrates. However, the ICM-like ori- one with this mutase. Possibly, interaction with MeaH or other entation of the methyl groups of the 2-hydroxyisobutyryl resi- proteins could also modulate enzyme kinetics in favor of due is not completely excluded, but takes place in about 2% of 2-HIBA degradation. On the other hand, an efficient processing substrate binding events, as can be deduced from the transfor- of (S)-3-hydroxybutyryl-CoA by a suitable dehydrogenase, and mation rates of (R)-3-hydroxybutyryl-CoA. This deviation further conversion into acetyl-CoA (Fig. 1) might be sufficient from complete stereospecificity may also explain the low but to shift the equilibrium of the mutase catalysis toward the 3-hy- significant conversion of the ICM substrates isobutyryl- and droxy carboxylic acid. In line with this, growth rate of the wild- butyryl-CoA (Table 1), as it can be assumed that these sub- type L108 strain on 2-HIBA is in the same range as found with strates and (R)-3-hydroxybutyryl-CoA have a similar orienta- other efficient substrates, such as fructose and lactic acid (24). tion in the substrate binding site of HCM (Fig. 8). Both HCM Retention of Configuration during HCM Catalysis—As and ICM/IcmF share the uncharged Gln residue (Gln208 and already found with MCM and ECM, only converting (R)-meth- Gln198/Gln733 in Fig. 8), suggesting that this amino acid is not ylmalonyl- and (R)-ethylmalonyl-CoA (10, 27), respectively, determining substrate orientation and stereospecificity. How- HCM catalysis is stereospecific, favoring the rearrangement of ever, the ICM-like substrate orientation is likely discriminated (S)-3-hydroxybutyryl-CoA versus the (R)-enantiomer. This in HCM due to the replacement of the aromatic Phe80/Phe589 specificity of catalysis may also shed light on the orientation of postulated to be specifically interacting with the substrate in the hydroxyacyl substrate at the catalytic site of HCM. The ICM/IcmF (9, 11) with the smaller aliphatic Ile90 (Fig. 8). rearrangement by MCM and ECM proceeds strictly with reten- Accordingly, the conservative substitution I90V still possess tion of configuration (Fig. 8). In the case of ICM, although its activity toward (S)-3-hydroxybutyryl- and 2-hydroxyisobu-

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FIGURE 7. ClustalW2 multiple sequence alignment of acyl-CoA binding domains of B12-dependent mutases. Comparison of HCM from A. tertiaricarbonis L108 with orthologous sequences from M. petroleiphilum PM1 (Mpe_B0541), R. sphaeroides KD131 (RSKD131_3116), R. sphaeroides ATCC 17029 (Rsph17029_3657), S. novella DSM 506 (Snov_2770), X. autotrophicus Py2 (Xaut_5021), M. alhagi CCNWXJ12–2 (ZP_09295256), M. algicola DG893 (MDG893_09606), and Nocardioides sp. JS614 (Noca_2131) is shown. In addition, the paralogous domains of ICM from S. cinnamonensis A3823.5 (icm, AAC08713), IcmF from Geobacillus kaustophilus HTA426 (GK3391), MCM from Propionibacterium freudenreichii subsp. shermanii CIRM-BIA1 (YP_003687736), and ECM from R. sphaeroides ATCC 17029 (Rsph17029_2621) were aligned. Bold amino acids show conserved residues directly involved in substrate binding (9). Residues highlighted in gray background are known to interact specifically with the CoA moiety. White-typed amino acids on black highlight two residues thus far identified to interact specifically with the acyl group of the substrates. Asterisks indicate identical residues in all sequences. A more extended alignment of these mutase sequences is presented in supplemental Fig. S7.

archaeal MCM sequences (10). In addition, HCM and ICM

share the same subunit organization consisting of a small B12- binding and a large protein encoded by two distinct genes,

whereas in bacterial MCM and ECM the B12- and acyl-CoA binding domains are both located on the same subunit (8, 10). The high sequence and structural similarity between HCM and ICM may allow studying substrate binding and catalysis of specific mutant enzymes. Comparable single amino acid mutations in ICM and MCM resulted mainly in loss of enzymatic activity (31), possibly due to the larger sequence and structural deviations between these mutase subfamilies. However, the activities of the amino acid mutants analyzed in this study clearly show that even in HCM single active site residue substitutions are not sufficient to change substrate specificity, e.g. achieving ICM activity with an HcmA I90F mutant. Occurrence and Evolution of HCM—As already outlined, the HCM enzyme is, besides ICM, the second representative within

the B12-dependent acyl-CoA mutase subfamilies thus far identified to be organized as small and large subunits, binding the

coenzyme B12 and the acyl-CoA ester substrate, respectively. In addition, HCM activity is different, as this enzyme specifically catalyzes the rearrangement of hydroxylated carboxylic acids which has not been observed with other mutases. However, the origin of HCM is enigmatic. BLAST analysis revealed a quite heterogeneous group of bacteria, including phylogenetically distant proteobacteria and a Gram-positive strain, which all possess the four genes encoding for the HCM subunits plus a putative acyl-CoA synthetase and the chaperone MeaH orga- FIGURE 8. Orientation of substrates and retention of configuration in the nized in an operon-like structure (Figs. 2 and 7). The high B12-dependent rearrangement of the carbon skeleton of acyl-CoA sequence similarity and the identical gene grouping suggest an esters. The observed isomerization of the 3-hydroxybutyryl-CoA enantio- interchange of the complete hcm operon by horizontal gene mers catalyzed by HCM is compared with transformations catalyzed by MCM/ ECM and ICM/IcmF. Active site amino acid residues proposed to interact spe- transfer. Unlike strains A. tertiaricarbonis L108 and M. petro- cifically with acyl group of the substrates are indicated. Numbering of leiphilum PM1 (15, 32), the other bacterial strains with an hcm residues is as in Fig. 7. operon have not been associated with the degradation of fuel oxygenates MTBE and TBA. An alternative source for com- tyryl-CoA, whereas the nonconservative mutations I90F and pounds bearing the tert-butyl group is the industrial production I90Y completely lost rearrangement activity. of PMMA (3, 6), as the corresponding wastewater contains In contrast to the observed deviation in the predominant 2-HIBA and its methyl ester. Accordingly, it has already been substrate orientation at the catalytic sites, HCM is phylogeneti- demonstrated that strains R. sphaeroides ATCC 17029, Xan- cally close to bacterial ICM, together forming a cluster within thobacter autotrophicus Py2 and Nocardioides sp. JS614 are

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able to grow on 2-HIBA (33). However, worldwide production and characterization of the recombinant enzyme produced in Escherichia of PMMA at large scale started not earlier than in the late 1930s coli. J. Biol. Chem. 274, 31679–31685 (34), whereas the hcm operon bearing strain Starkeya novella 10. Erb, T. J., Rétey, J., Fuchs, G., and Alber, B. E. (2008) Ethylmalonyl-CoA mutase from Rhodobacter sphaeroides defines a new subclade of coen- DSM 506 has already been isolated from uncontaminated agri- zyme B12-dependent acyl-CoA mutases. J. Biol. Chem. 283, 32283–32293 cultural soil in 1934 (35), indicating that other drivers than this 11. Cracan, V., Padovani, D., and Banerjee, R. (2010) IcmF is a fusion between

kind of anthropogenic contamination for the evolution of HCM the radical B12 enzyme isobutyryl-CoA mutase and its G-protein chaperone. J. Biol. Chem. 285, 655–666 may exist. The other known B12-dependent acyl-CoA mutases are widely distributed among bacteria playing central roles in 12. Simon, E. J., and Shemin, D. (1953) The preparation of S-succinyl coen- primary and secondary carbon metabolism, e.g. in branched- zyme A. J. Am. Chem. Soc. 75, 2520 13. Padmakumar, R., Gantla, S., and Banerjee, R. (1993) A rapid method for chain amino acid catabolism (8) and in the recently discovered the synthesis of methylmalonyl-coenzyme A and other CoA-esters. Anal. ethylmalonyl-CoA pathway for acetate assimilation (10) as well Biochem. 214, 318–320 as in the synthesis of macrolide and polyether antibiotics (9). In 14. Dalluge, J. J., Gort, S., and Hobson, R. (2002) Separation and identification contrast, HCM would be the first mutase exclusively employed of organic acid-coenzyme A thioesters using liquid chromatography/elec- for the dissimilatory degradation of a single substrate, i.e. trospray ionization-mass spectrometry. Anal. Bioanal. Chem. 374, 835–840 2-HIBA. In this connection, several not yet identified mutases 15. Lechner, U., Brodkorb, D., Geyer, R., Hause, G., Härtig, C., Auling, G., have been postulated to be involved in bacterial degradation Fayolle-Guichard, F., Piveteau, P., Müller, R. H., and Rohwerder T. (2007) pathways for the mineralization of alkanes, ethylbenzene, and Aquincola tertiaricarbonis gen. nov., sp. nov., a tertiary butyl moiety-de- the quaternary carbon-bearing pivalic acid (36, 37). Hence, it grading bacterium. Int. J. Syst. Evol. Microbiol. 57, 1295–1303 could be speculated that all of these mutases employed in dis- 16. Schäfer, F., Muzica, L., Schuster, J., Treuter, N., Rosell, M., Harms, H., similatory pathways may have the same origin being adapted to Müller, R. H., and Rohwerder, T. (2011) Formation of alkenes via degradation of tert-alkyl ethers and alcohols by Aquincola tertiaricarbonis L108 their specific substrates by a moderate variation of the amino and Methylibium spp. Appl. Environ. Microbiol. 77, 5981–5987 acid residues in the substrate binding site. The previously 17. Valentin, H. E., and Steinbüchel, A. (1993) Expression of an ␣-galactosid- described close phylogenetic relationship of bacterial HCM and ase gene under control of the homologous inulinase promoter in ICM sequences with archaeal mutases (10) is also interesting, as Kluyveromyces marxianus. Appl. Microbiol. Biotechnol. 39, 309–317 it suggests an origin of HCM outside the domain Bacteria. 18. Hermans-Lokkerbol, A., van der Heijden, R., and Verpoorte, R. (1996) Isocratic high-performance liquid chromatography of coenzyme A esters involved in the metabolism of 3S-hydroxy-3-methylglutaryl coenzyme A. Acknowledgments—We thank C. Schumann (Helmholtz Centre for Detection of related enzyme activities in Catharanthus roseus plant cell Environmental Research, UFZ), M. Neytschev (UFZ) and S. Kluge cultures. J. Chromatogr. A 752, 123–130 (UFZ) for technical assistance and B. Würz (UFZ) for excellent ana- 19. Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, lytical advice. W., and Lipman, D. J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 REFERENCES 20. Kane, S. R., Chakicherla, A. Y., Chain, P. S., Schmidt, R., Shin, M. W., 1. Kumps, A., Duez, P., and Mardens, Y. (2002) Metabolic, nutritional, iat- Legler, T. C., Scow, K. M., Larimer, F. W., Lucas, S. M., Richardson, P. M., rogenic, and artifactual sources of urinary organic acids: a comprehensive and Hristova, K. R. (2007) Whole-genome analysis of the methyl tert-butyl table. Clin. Chem. 48, 708–717 ether-degrading ␤-proteobacterium Methylibium petroleiphilum PM1. J. 2. Forslund, K., Morant, M., Jørgensen, B., Olsen, C. E., Asamizu, E., Sato, S., Bacteriol. 189, 1931–1945 Tabata, S., and Bak, S. (2004) Biosynthesis of the nitrile glucosides rhodio- 21. Hristova, K. R., Schmidt, R., Chakicherla, A. Y., Legler, T. C., Wu, J., Chain, cyanoside A and D and the cyanogenic glucosides lotaustralin and lina- P. S., Scow, K. M., and Kane, S. R. (2007) Comparative transcriptome marin in Lotus japonicus. Plant Physiol. 135, 71–84 analysis of Methylibium petroleiphilum PM1 exposed to the fuel oxygen- 3. Chisholm, M. S. (2000) Artificial glass: the versatility of poly(methyl meth- ates methyl tert-butyl ether and ethanol. Appl. Environ. Microbiol. 73, acrylate) from its early exploitation to the new millenium. J. Chem. Edu. 7347–7357 77, 841–845 22. Padovani, D., and Banerjee, R. (2006) Assembly and protection of the 4. Steffan, R. J., McClay, K., Vainberg, S., Condee, C. W., and Zhang, D. radical enzyme, methylmalonyl-CoA mutase, by its chaperone. Biochem- (1997) Biodegradation of the gasoline oxygenates methyl tert-butyl ether, istry 45, 9300–9306 ethyl tert-butyl ether, and tert-amyl methyl ether by propane-oxidizing 23. Schuster, J., Schäfer, F., Hübler, N., Brandt, A., Rosell, M., Härtig, C., bacteria. Appl. Environ. Microbiol. 63, 4216–4222 Harms, H., Müller, R. H., and Rohwerder, T. (2012) Bacterial degradation 5. Moran, M. J., Zogorski, J. S., and Squillace, P. J. (2005) MTBE and gasoline of tert-amyl alcohol proceeds via hemiterpene 2-methyl-3-buten-2-ol by hydrocarbons in ground water of the United States. Ground Water 43, employing the tertiary alcohol desaturase function of the Rieske nonheme 615–627 mononuclear iron oxygenase MdpJ. J. Bacteriol. 194, 972–981 6. Holowach, L. P., Swift, G. W., Wolk, S. W., and Klawiter, L. (1994) in 24. Müller, R. H., Rohwerder, T., and Harms, H. (2008) Degradation of fuel Polymers from Agricultural Coproducts (Fishman, M. L., Friedman, R. B., oxygenates and their main intermediates by Aquincola tertiaricarbonis and Huang, S. J., eds) pp. 202–211, ASC, Washington, DC L108. Microbiology 154, 1414–1421

7. Rohwerder, T., Breuer, U., Benndorf, D., Lechner, U., and Müller, R. H. 25. Cracan, V., and Banerjee, R. (2012) Novel coenzyme B12-dependent inter- (2006) The alkyl tert-butyl ether intermediate 2-hydroxyisobutyrate is de- conversion of isovaleryl-CoA and pivalyl-CoA. J. Biol. Chem. 287, graded via a novel cobalamin-dependent mutase pathway. Appl. Environ. 3723–3732 Microbiol. 72, 4128–4135 26. Korotkova, N., and Lidstrom, M. E. (2004) MeaB is a component of the 8. Birch, A., Leiser, A., and Robinson, J. A. (1993) Cloning, sequencing, and methylmalonyl-CoA mutase complex required for protection of the en- expression of the gene encoding methylmalonyl-coenzyme A mutase zyme from inactivation. J. Biol. Chem. 279, 13652–13658 from Streptomyces cinnamonensis. J. Bacteriol. 175, 3511–3519 27. Sprecher, M., Clark, M. J., and Sprinson, D. B. (1966) The absolute con- 9. Ratnatilleke, A., Vrijbloed, J. W., and Robinson, J. A. (1999) Cloning and figuration of methylmalonyl coenzyme A and stereochemistry of the

sequencing of the coenzyme B12-binding domain of isobutyryl-CoA mu- methymalonyl coenzyme A mutase reaction. J. Biol. Chem. 241, 872–877 tase from Streptomyces cinnamonensis, reconstitution of mutase activity, 28. Reynolds, K. A., O’Hagan, D., Gani, D., and Robinson, J. A. (1988) Butyrate

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metabolism in Streptomyces: characterization of an intramolecular vicinal proteobacteria. Int. J. Syst. Evol. Microbiol. 56, 983–989 interchange rearrangement linking isobutyrate and butyrate in Strepto- 33. Rohwerder, T., Breuer, U., Benndorf, D., Lechner, U., and Müller, R. H. myces cinnamonensis. J. Chem. Soc. Perkin Trans. 1, 3195–3207 (2007) in Proceedings of the 3rd European Conference on MTBE and Other 29. Moore, B. S., Eisenberg, R., Weber. C., Bridges, A., Nanz, D., and Robinson Fuel Oxygenates (Bastiaens, L., ed) pp. 11–14, VITO, Mol

J. A. (1995) On the stereospecificity of the coenzyme B12-dependent 34. Bauer, W. (2002) in Ullmann’s Encyclopedia of Industrial Chemistry, Vol. isobutyryl-CoA mutase reaction. J. Am. Chem. Soc. 117, 11285–11291 21, pp. 585–597, Wiley-VCH, Weinheim 30. Rohwerder, T., and Müller, R. H. (2010) Biosynthesis of 2-hydroxyisobu- 35. Starkey, R. L. (1934) Cultivation of organisms concerned in the oxidation tyric acid (2-HIBA) from renewable carbon. Microbial Cell Fact. 9, 13 of thiosulfate. J. Bacteriol. 28, 365–386 31. Vlasie, M. D., and Banerjee, R. (2004) When a spectator turns killer: sui- 36. Rohwerder, T., and Müller, R. H. (2007) in Vitamin B: New Research (El- cidal electron transfer from cobalamin in methylmalonyl-CoA mutase. liot, C. M., ed) pp. 81–98, Nova Science Publisher, Hauppauge Biochemistry 43, 8410–8417 37. Probian, C., Wülfing, A., and Harder, J. (2003) Anaerobic mineralization 32. Nakatsu, C. H., Hristova, K., Hanada, S., Meng, X. Y., Hanson, J. R., Scow, of quaternary carbon atoms: isolation of denitrifying bacteria on pivalic K. M., and Kamagata, Y. (2006) Methylibium petroleiphilum gen. nov., sp. acid (2,2-dimethylpropionic acid). Appl. Environ. Microbiol. 69, nov., a novel methyl tert-butyl ether-degrading methylotroph of the Beta- 1866–1870

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Supplemental Material

Journal of Biological Chemistry

2-hydroxyisobutyryl-CoA NL scan

EPI scan

butyryl-/isobutyryl-CoA NL scan

EPI scan

Supplementary Fig. S1. Analysis of synthesized acyl-CoA esters by ESI-MS/MS. In the Neutral Loss Scan mode (NL), masses of ionized acyl-CoA esters were detected, whereas in the Enhanced Product Ion Scan mode (EPI), also masses of smaller fragments were obtained. Characteristic fragments of 2-hydroxyisobutyryl-CoA (853.5 g mol-1) and butyryl-/isobutyryl- CoA (837.5 g mol-1) are the corresponding acyl-pantethein moieties of 347.4 and 331.4 g mol-1, respectively, as indicated by arrows.

Dissertation Judith Schuster 6. Die 4-HIBA-Mutase HcmAB des Stammes L32: 79

0h 20h eluate 0h 3h eluate

70 60 HcmA 50

HcmB

115

Supplementary Fig. S2. SDS PAGE analysis of crude extracts and eluates after heterologous expression of wild type HcmA and HcmB and their purification by one-step affinity chromatography, respectively (12% SDS-PAGE). Sampling times starting at induction of expression in E. coli strains TOP10 (HcmA) and ArcticExpress (DE3) (HcmB). Lanes: 0h, 30 µg E. coli cell extract protein before induction; 20 and 3h, 30 µg E. coli cell extract protein after 20 and 3 hours of induction, respectively; eluate, 30 µg purified protein. The molecular masses (kDa) of standard proteins are indicated. SDS PAGE analysis of the expression and purification of the HcmA mutants I90Y, I90F and I90V gave similar results (not shown).

Dissertation Judith Schuster 6. Die 4-HIBA-Mutase HcmAB des Stammes L32: 7:

25 slope = 3.053 µmol L-1 min-1 r = 0.994 20

10 3-hydroxybutyryl-CoA (µM) 3-hydroxybutyryl-CoA 5

0 02468 Time (min)

Supplementary Fig. S3. HPLC assay for quantifying HCM activity. Reconstituted wild type HcmAB (1.4 µM) was incubated with 250 µM 2-hydroxyisobutyryl-CoA at pH 6.6 and 30°C. The shown increase in 3-hydroxybutyryl-CoA represents mean values and SD of four replicates.

45 45 40 40

) 35

) 35 -1 -1 30 30 mg mg -1 -1 25 25 20 20 15 15 (nmol min (nmol min 10 10 5 5 0 0 5.0 5.5 6.0 6.5 7.0 7.5 8.0 20 30 40 50 60 pH Temperature (°C) Supplementary Fig. S4. pH optimum Supplementary Fig. S5. Temperature of HCM activity (conversion of optimum of HCM activity (conversion 2-hydroxyisobutyryl-CoA) obtained of 2-hydroxyisobutyryl-CoA) obtained with reconstituted wild type HcmA with reconstituted wild type HcmA and HcmB subunits incubated at 30°C and HcmB subunits incubated at pH 6.6. in phosphate buffer (solid symbols) or phosphate/acetate buffer (open symbols).

Dissertation Judith Schuster 6. Die 4-HIBA-Mutase HcmAB des Stammes L32: 7;

40.000 µV 38.000 36.000 34.000 32.000 A: initial sample 30.000 28.000 2-hydroxyisobutyric 26.000 24.000 acid propyl ester 22.000 20.000

FID signal 18.000 16.000 14.000 12.000

10.000 8.000 RT [min] 6.000 18 18,5 19 19,5 20 20,5 21 21,5 22 22,5 23 23,5 24 24,5 25 25,5 26 26,5 27 27,5 28 28,5 29 29,5 30 Retention time (min)

40.000 µV 38.000 36.000 34.000 B: after two hours (S)-3- (R)-3- 32.000 hydroxybutyric hydroxybutyric 30.000 of incubation acid propyl ester acid propyl ester 28.000 26.000 24.000 22.000 20.000

FID signal 18.000 16.000 14.000 12.000 10.000 8.000 RT [min] 6.000 18 18,5 19 19,5 20 20,5 21 21,5 22 22,5 23 23,5 24 24,5 25 25,5 26 26,5 27 27,5 28 28,5 29 29,5 30 Retention time (min)

Supplementary Fig. S6. Analysis of stereospecificity of 2-hydroxyisobutyryl-CoA conversion to 3-hydroxybutyryl-CoA esters by reconstituted wild type HcmA and HcmB. The acyl-CoAs produced were determined as propyl esters by gas chromatography (GC) using a HP 6890 system from Agilent Technologies with a Chirasil-DEX CB column (25 m by 0.25 mm by 0.25 µm) and flame ionization detector (FID). The carrier gas was helium and the injector and detector temperature was 250°C. The following GC oven temperature program was applied. The initial temperature was 35°C increasing to finally 170°C at 3°C min-1 and holding this temperature for 5 min. Samples were dried under vacuum and then incubated with 1 mL propanolic HCl (3 M) at 110°C for 45 min. The propyl esters formed were dissolved in 150 µL toluene prior to injection (1 µL, split 1:100).

A: Initial assay sample, immediately after addition of 12 mM 2-hydroxyisobutyryl-CoA.

B: For generating a sufficient amount of 3-hydroxybutyryl-CoA esters, the enzyme was incubated two hours with 12 mM 2-hydroxyisobutyryl-CoA under optimal assay conditions.

Dissertation Judith Schuster 6. Die 4-HIBA-Mutase HcmAB des Stammes L32: 82

HcmA_L108 TYTAAD-IADTPLEDIG------LPGRYPFTRGPYPTMYRSRTWTMRQIAGFG 94 HcmA_PM1 TYTAAD-IADTPLEDIG------LPGRYPFTRGPYPTMYRSRTWTMRQIAGFG 94 HcmA_KD131 TYTAAD-IADTPLEDIG------LPGKYPFTRGPYPTMYRGRNWTMRQIAGFG 95 HcmA_17029 TYTAAD-IADTPLEDIG------LPGKYPFTRGPYPTMYRGRNWTMRQIAGFG 95 HcmA_Dsm506 TYTAAD-LADTPVEDIG------LPGRYPFTRGPYPTMYRSRTWTMRQIAGFG 100 HcmA_Py2 VYTAAD-AAATPIEDIG------LPGRYPFTRGPYPTMYRSRNWTMRQIAGFG 94 HcmA_CCNWXJ12-2 TYIAAD-IAGTPAEDIG------LPGRYPFTRGPYPTMYRGRNWTMRQIAGFG 95 HcmA_DG893 TYTPLD-VKNTPFEDIG------FPGQYPFTRGPYPTMYRGRNWTMRQIAGFG 97 HcmA_JS614 VYTPAD-LPED-WNDIG------LPGQFPFTRGPYPTMYRGRHWTMRQIAGFG 101 IcmF_HTA426 TLSGLD-IPKVVLPKFKDYGEILRWVYKENVPGSFPYTAGVFPFKRQG-EDPKRQFAGEG 593 Icm_A3823.5 VYGPRPGDTYDGFERIG------WPGEYPFTRGLYATGYRGRTWTIRQFAGFG 84 ECM_17029 ------MTQKDS------PWLFRTYAGHS 17 MCM_CIRM-BIA1 -LFNEDVYKDMDWLDTY------AGIPPFVHGPYATMYAFRPWTIRQYAGFS 93 * ** .

HcmA_L108 TGEDTNKRFKYLIAQGQTG-ISTDFDMPTLMGYDSDH-PMSDGEVGREGVAIDTLADMEA 152 HcmA_PM1 TGEDTNKRFKYLIAQGQTG-ISTDFDMPTLMGYDSDH-PMSDGEVGREGVAIDTLADMEA 152 HcmA_KD131 TGEDTNKRFKFLIEQGQTG-ISTDFDMPTLMGYDSDH-PMSDGEVGREGVAIDTLADMRA 153 HcmA_17029 TGEDTNKRFKFLIEQGQTG-ISTDFDMPTLMGYDSDH-PMSDGEVGREGVAIDTLADMRA 153 HcmA_Dsm506 TGEDTNKRFKYLIAQGQTG-ISTDFDMPTLMGYDSDH-PMSEGEVGREGVAIDTLADMEA 158 HcmA_Py2 TGEDTNKRFKYLIEQGQTG-ISTDFDMPTLMGYDSDH-PMSDGEVGREGVAIDTLADMEA 152 HcmA_CCNWXJ12-2 TGEDTNRRFKFLIEQGQTG-ISTDFDMPTLMGYDSDH-PMSDGEVGREGVAIDTLADMEA 153 HcmA_DG893 TARETNGRFKYLIAQGQTG-LSIDFDMPTLMGYDSSH-AMSQGEVGREGVAIDTLADMEE 155 HcmA_JS614 QAEETNKRFQYLINQGQTG-LSVDFDMPTLMGLDSDD-PMSLGEVGREGVAVDVLSDMEA 159 IcmF_HTA426 TPERTNRRFHYLCKEDKAKRLSTAFDSVTLYGEDPDYRPDIFGKVGESGVSVCTLDDMKK 653 Icm_A3823.5 NAEQTNERYKMILANGGGG-LSVAFDMPTLMGRDSDD-PRSLGEVGHCGVAIDSAADMEV 142 ECM_17029 TAKASNALYRTNLAKGQTG-LSVAFDLPTQTGYDSDD-ALARGEVGKVGVPICHLGDMRM 75 MCM_CIRM-BIA1 TAKESNAFYRRNLAAGQKG-LSVAFDLPTHRGYDSDN-PRVAGDVGMAGVAIDSIYDMRE 151 . :* :: . :* ** * * *.. . *.** **.: **.

HcmA_L108 LLADIDLEK--ISVSFTINPSAWILLAMYVALGEK------RGY 188 HcmA_PM1 LLADIDLEK--ISVSFTINPSAWILLAMYVALGEK------RGY 188 HcmA_KD131 LLDGIDLEK--ISVSLTINPTAWILLAMYIALCEE------RGY 189 HcmA_17029 LLDGIDLEK--ISVSLTINPTAWILLAMYIALCEE------RGY 189 HcmA_Dsm506 LFDGIDLEK--ISVSMTINPSAWILLAMYIVLAEK------RGY 194 HcmA_Py2 LFDGIDLEK--ISVSMTINPSAWILLAMYIVLAQK------RGY 188 HcmA_CCNWXJ12-2 LLADIDLEK--ISVSLTINPTAWILFAMYVALAEK------RGY 189 HcmA_DG893 LFDDIDLTK--ISVSMTINPSAWILYAMYIALAQK------RGY 191 HcmA_JS614 LFDGIDLEN--ISVSMTINPSAWILLAMYIAVAED------KGY 195 IcmF_HTA426 LYKGFDLCDPLTSVSMTINGPAPILLAMFMNTAIDQQVEKKEAELGRPLTPEEYEQVKEW 713 Icm_A3823.5 LFKDIPLGD--VTTSMTISGPAVPVFCMYLVAAER------QGV 178 ECM_17029 LFDQIPLEQ--MNTSMTINATAPWLLALYIAVAEE------QGA 111 MCM_CIRM-BIA1 LFAGIPLDQ--MSVSMTMNGAVLPILALYVVTAEE------QGV 187 * : * . ..*:*:. .. : .::: :

HcmA_L108 DLNKLSGTVQADILKEYMAQKEYIYPIAPSVRIVRDIITYSAKNLKR-YNPINISGYHIS 247 HcmA_PM1 DLNKLSGTVQADILKEYMAQKEYIYPIAPSVRIVRDIITYSAKNLKR-YNPINISGYHIS 247 HcmA_KD131 DLNKVSGTVQADILKEYMAQKEYIFPIAPSVRIVRDIISHSTRTMKR-YNPINISGYHIS 248 HcmA_17029 DLNKVSGTVQADILKEYMAQKEYIFPIAPSVRIVRDIISHSTRTMKR-YNPINISGYHIS 248 HcmA_Dsm506 DLNKLSGTIQADILKEYMAQKEYVFPIEPSVRIVRDCITYCARNMKR-YNPINISGYHIS 253 HcmA_Py2 DLDKLSGTVQADILKEYMAQKEYIYPIAPSVRIVRDCITYCAKNMKR-YNPINISGYHIS 247 HcmA_CCNWXJ12-2 DLNKLSGTVQADILKEFMAQKEYIFPIAPSVRIVRDLIAYSTRHMKR-YNPINISGYHIS 248 HcmA_DG893 DLNDLSGTIQNDILKEYIAQKEWIFPVRPSVRLVRDCIQYGSENMNR-YNPINISGYHIS 250 HcmA_JS614 DLNRLSGTIQNDILKEYVAQKEWIFPVRPSMRIVRDCIAYCAENMAR-YNPVNISGYHIS 254 IcmF_HTA426 TLQTVRGTVQADILKEDQGQNTCIFSTDFALKMMGDIQEYFIKHRVRNYYSVSISGYHIA 773 Icm_A3823.5 DPAVLNGTLQTDIFKEYIAQKEWLFQPEPHLRLIGDLMEHCARDIPA-YKPLSVSGYHIR 237 ECM_17029 DISKLQGTVQNDLMKEYLSRGTYICPPRPSLRMITDVAAYTRVHLPK-WNPMNVCSYHLQ 170 MCM_CIRM-BIA1 KPEQLAGTIQNDILKEFMVRNTYIYPPQPSMRIISEIFAYTSANMPK-WNSISISGYHMQ 246 : **:* *::** : : :::: : : : .:.:..**:

HcmA_L108 EAGSSPLQEAAFTLANLITYVNEV--TKTGMHVDEFAPRLAFFFVSQGDFFEEVAKFRAL 305 HcmA_PM1 EAGSSPLQEAAFTLANLITYVNEV--TETGMHVDEFAPRLAFFFVSQGDFFEEVAKFRAL 305 HcmA_KD131 EAGSSPLHEAAFTLANLIVYVEEV--LKTGVEVDDFAPRLAFFFVCQADFFEEIAKFRAL 306 HcmA_17029 EAGSSPLHEAAFTLANLIVYVEEV--LKTGVEVDDFAPRLAFFFVCQADFFEEIAKFRAL 306 HcmA_Dsm506 EAGSSPLHEAAFTLANLIVYVEEV--LKTGMQVDEFAPRLAFFFVCQADFFEEIAKFRAL 311 HcmA_Py2 EAGSSPVDEVAFTLANLIVYVEEV--LKTGMKVDDFAPRLAFFFVCQADFFEEIAKFRAV 305 HcmA_CCNWXJ12-2 EAGSSPLHEAAFALANLIVYVEEV--TKLGIDVDDFAPRLAFFFVSQADFFEEVAKFRAL 306 HcmA_DG893 EAGSTAVQEVAYTMATTMEYVRTA--IDAGVDVNDFGPRLSFFFVSQADFFEEIAKFRAA 308 HcmA_JS614 EAGANAVQEVAFTMAITRAYVSDV--IAAGVDVDTFAPRLSFFFVSQADFFEEAAKFRAV 312 IcmF_HTA426 EAGANPITQLAFTLANGFTYVEYY--LSRGMHIDDFAPNLSFFFSNGLDPEYSVIG-RVA 830 Icm_A3823.5 EAGATAAQELAYTLADGFGYVELG--LSRGLDVDVFAPGLSFFFDAHVDFFEEIAKFRAA 295 ECM_17029 EAGATPEQELAFALATGIAVLDDLRTKVPAEHFPAMVGRISFFVNAGIRFVTEMCKMRAF 230 MCM_CIRM-BIA1 EAGATADIEMAYTLADGVDYIRAG--ESVGLNVDQFAPRLSFFWGIGMNFFMEVAKLRAA 304 ***:.. : *:::* : . .. : ::** . *.

Dissertation Judith Schuster 6. Die 4-HIBA-Mutase HcmAB des Stammes L32: 83

HcmA_L108 RRCYAKIMKERFGARNPESMRLRFHCQTAAATLTKPQYMVNVVRTSLQALSAVLG---GA 362 HcmA_PM1 RRCYAKIMKERFGAKNPESMRLRFHCQTAAATLTKPQYMVNVVRTSLQALSAVLG---GA 362 HcmA_KD131 RRCYAKIMKERFGAKKPESMRLRFHCQTAAASLTKPQYMVNVMRTTTQALAAVLG---GA 363 HcmA_17029 RRCYAKIMKERFGAKKPESMRLRFHCQTAAASLTKPQYMVNVMRTTTQALAAVLG---GA 363 HcmA_Dsm506 RRCYAKIMKERFGAQNPESMRLRFHCQTAAASLTKPQYMVNVVRTAMQALAAALG---GT 368 HcmA_Py2 RRCYAKIMKERFGARNPESMRLRFHCQTAAASLTKPQFMVNVVRTTLQALAAVLG---GC 362 HcmA_CCNWXJ12-2 RRCYAKIMKERFGARQPESMRLRFHCQTAAASLTKPQYMVNVVRTAMQALSAVLG---GT 363 HcmA_DG893 RRVYAKIMREKFGATKPEASRLRFHAQTAAATLTKPQYTINPIRTALQALSAVLG---GA 365 HcmA_JS614 RRFYAKMMRDEFGAENEQSMRLRFHAQTAAATLTKPQPMNNIIRTTLQALSAILG---GA 369 IcmF_HTA426 RRIWAIVMREKYGANE-RSQKLKYHIQTSGRSLHAQEIDFNDIRTTLQALLAIYD---NC 886 Icm_A3823.5 RRIWARWLRDEYGAKTEKAQWLRFHTQTAGVSLTAQQPYNNVVRTAVEALAAVLG---GT 352 ECM_17029 VDLWDEICRDRYGIEEEKYRRFRYGVQVNSLGLTEQQPENNVYRILIEMLAVTLSKKARA 290 MCM_CIRM-BIA1 RMLWAKLVHQ-FGPKNPKSMSLRTHSQTSGWSLTAQDVYNNVVRTCIEAMAATQG---HT 360 : :: :* . :: *. . * : * * : : . .

HcmA_L108 QSLHTNGYDEAFAIPTEDAMKMALRTQQIIAEESGVADVIDPLGGSYYVEALTTEYEKKI 422 HcmA_PM1 QSLHTNGYDEAFAIPTEDAMKMALRTQQIIAEESGVADVIDPLGGSYYVEALTTEYEKKI 422 HcmA_KD131 QSLHTNGYDEAFAIPTEHAMQLALRTQQVIADETGVTQVVDPLGGSYFVESLTNDYEKKI 423 HcmA_17029 QSLHTNGYDEAFAIPTEHAMQLALRTQQVIADETGVTQVVDPLGGSYFVESLTNDYEKKI 423 HcmA_Dsm506 QSLHTNGYDEAFAIPTEDAMRMALRTQQVIAEETNVTQVVDPLGGSYYVESLTTEYEKRI 428 HcmA_Py2 QSLHTNGFDEAFAIPTEEAMRLALRTQQVIAEESNVTQVIDPVGGSYYVETLTTEYEKRI 422 HcmA_CCNWXJ12-2 QSLHTNGFDEAFAIPTEEAMQLALRTQQVIADETNVTQVVDPLGGSYYVEALTNEYEKRI 423 HcmA_DG893 QSLHTNGMDEAFAIPTEEAMRIALRTQQIIAYETNITQVVDPLGGSYYVENLTDEIEKEV 425 HcmA_JS614 QSLHTNGLDEAYTIPSETAMKIALRTQQVIAHETGVPSIVDPLGGSYYVEALTDEIETGI 429 IcmF_HTA426 NSLHTNAYDEAITTPTEESVRRAMAIQLIITKEFGLTKNENPLQGSFIIEELTDLVEEAV 946 Icm_A3823.5 NSLHTNALDETLALPSEQAAEIALRTQQVLMEETGVANVADPLGGSWYIEQLTDRIEADA 412 ECM_17029 RAVQLPAWNEALGLPRPWDQQWSLRMQQILAYESDLLEYEDLFDGNPAIERKVEALKDGA 350 MCM_CIRM-BIA1 QSLHTNSLDEAIALPTDFSARIARNTQLFLQQESGTTRVIDPWSGSAYVEELTWDLARKA 420 .::: . :*: * . : * .: * . : *. :* .

Supplementary Fig. S7. Extended ClustalW2 multiple sequence alignment of substrate binding domains of B12-dependent acyl-CoA mutases shown in Fig. 7. Comparison of HcmA from A. tertiaricarbonis L108 to orthologous sequences from M. petroleiphilum PM1 (Mpe_B0541), R. sphaeroides KD131 (RSKD131_3116), R. sphaeroides ATCC 17029 (Rsph17029_3657), S. novella DSM 506 (Snov_2770), X. autotrophicus Py2 (Xaut_5021), M. alhagi CCNWXJ12-2 (ZP_09295256), M. algicola DG893 (MDG893_09606) and Nocardioides sp. JS614 (Noca_2131). In addition, the paralogous domains of ICM from S. cinnamonensis A3823.5 (icm, AAC08713), IcmF from G. kaustophilus HTA426 (GK3391), MCM from P. freudenreichii subsp. shermanii CIRM-BIA1 (YP_003687736) and ECM from R. sphaeroides ATCC 17029 (Rsph17029_2621) were aligned. Bold amino acids show conserved residues directly involved in substrate binding. Residues highlighted on gray background are known to interact specifically with the CoA moiety. White-typed amino acids on black highlight the residues thus far identified to interact specifically with the acyl group of the substrates. Asterisks indicate identical residues in all sequences, ":" mark conserved substitutions and "." assign semi-conserved substitutions with similar shapes.

Dissertation Judith Schuster 7. Der TAA-Abbau des Stammes L32:

apitel 7 beinhaltet die Analyse des Reaktionsmechanismus der in dieser Arbeit schon in Kapitel 5 vorgestellten Alkoholmonooxygenase MdpJ in Bezug auf diverse K längerkettige tertiäre Alkohole. Interessanterweise werden diese durch MdpJ nicht oxidiert, sondern durch die Desaturase- funktion des Enzyms zu Hemiterpenen reduziert. Der weitere Hemiterpenabbau ist strikt biotinabhängig. Eine Cobalaminabhängigkeit wie im TBA-Metabolismus besteht jedoch nicht. Im TAA-Abbau entsteht 5-Methylcrotonat als Intermediat. Diese Tatsachen legen eine Kopplung des TAA-Abbaus an den etablierten Leucinabbau nahe. In diesem ist die biotinabhängige 5-Methylcrotonyl-CoA-Carboxylase LiuC für den Abbau der tertiären Aminosäure Leucin verantwortlich. Als Beispiel dient das Liu-Operon aus Thauera sp. MZ3T. Ein entsprechendes Gencluster für die analoge Enzymfunktion konnte auch in Aquincola tertiaricarbonis L32: nachgewiesen werden. Der Artikel erschien im März 4234 in der Fachzeitschrift Journal of Bacteriology der American Society for Microbiology (ASM, J. Bacteriol. March 4234 vol. 3;6 No. 7 ;94-;:3). Die Online-Version ist seit dem 44. Dezember 4233 verfügbar (doi: 32.334:/JB.285:6-33). Es folgt der Artikel sowie das Supplemental Material. Der Beleg über die Co-Autorschaften ist im Anhang angefügt.

84 7. Der TAA-Abbau des Stammes L32: 85

Bacterial Degradation of tert-Amyl Alcohol Proceeds via Hemiterpene 2-Methyl-3-Buten-2-ol by Employing the Tertiary Alcohol Desaturase Function of the Rieske Nonheme Mononuclear Iron Oxygenase MdpJ

Judith Schuster,a Franziska Schäfer,a Nora Hübler,a Anne Brandt,a Mònica Rosell,b Claus Härtig,a Hauke Harms,a Roland H. Müller,a and Thore Rohwerdera Departments of Environmental Microbiologya and Isotope Biogeochemistry,b Helmholtz Centre for Environmental Research-UFZ, Leipzig, Germany

Tertiary alcohols, such as tert-butyl alcohol (TBA) and tert-amyl alcohol (TAA) and higher homologues, are only slowly degraded microbially. The conversion of TBA seems to proceed via hydroxylation to 2-methylpropan-1,2-diol, which is further oxidized to 2-hydroxyisobutyric acid. By analogy, a branched pathway is expected for the degradation of TAA, as this molecule possesses several potential hydroxylation sites. In Aquincola tertiaricarbonis L108 and Methylibium petroleiphilum PM1, a likely candidate catalyst for hydroxylations is the putative tertiary alcohol monooxygenase MdpJ. However, by comparing metabolite accumulations in wild-type strains of L108 and PM1 and in two mdpJ knockout mutants of strain L108, we could clearly show that MdpJ is not hydroxylating TAA to diols but functions as a desaturase, resulting in the formation of the hemiterpene 2-methyl-3-buten-2-ol. The latter is further processed via the hemiterpenes prenol, prenal, and 3-methylcrotonic acid. Likewise, 3-methyl-3-pentanol is degraded via 3-methyl-1-penten-3-ol. Wild-type strain L108 and mdpJ knockout mutants formed isoamylene and isoprene from TAA and 2-methyl-3-buten-2-ol, respectively. It is likely that this dehydratase activity is catalyzed by a not-yet-characterized enzyme postulated for the isomerization of 2-methyl-3-buten-2-ol and prenol. The vitamin requirements of strain L108 growing on TAA and the occurrence of 3-methylcrotonic acid as a metabolite indicate that TAA and hemiterpene degradation are linked with the catabolic route of the amino acid leucine, including an involvement of the biotin-dependent 3-methylcrotonyl coenzyme A (3-methylcrotonyl-CoA) carboxylase LiuBD. Evolutionary aspects of favored desaturase versus hydroxylation pathways for TAA conversion and the possible role of MdpJ in the degradation of higher tertiary alcohols are discussed.

n nature, molecules bearing tertiary alcohol groups are not un- soluble and render water unfit for drinking even at concentrations Iusual and can even be central metabolites, such as citric acid and in the parts-per-billion range (44). These properties often impede mevalonic acid, which are processed by nearly all living beings. the removal of fuel oxygenate ethers from contaminated sites be- However, not much is known about the catabolism of simple ter- low threshold values, making fuel oxygenate ethers and also their tiary alcohols not possessing additional functional groups. The tertiary alcohol intermediates very problematic pollutants. homologous series starts with tert-butyl alcohol (TBA) (2-methyl- Although fuel oxygenate ethers are now banned in the United 2-propanol) as the smallest molecule, with only four carbon at- States (47) and may be phased out in other countries, they will oms, followed by tert-amyl alcohol (TAA) (2-methyl-2-butanol). persist at polluted sites for a long time, as they all turned out to be In the last years, some progress on elucidating the catabolism of quite recalcitrant to biodegradation (10, 36). The main reason for these compounds has been made when research focused on the this poor degradability might be the xenobiotic character of their environmental fate of fuel oxygenates, since tertiary alcohols have tert-butyl and tert-amyl moieties, as the catabolism of these highly been identified as intermediates in bacterial degradation pathways branched structures likely requires the evolution of novel enzymes of branched-chain dialkyl ethers (10, 27, 42). Since the 1980s, linking the ether-specific pathways with common metabolic methyl tert-butyl ether (MTBE) and tert-amyl methyl ether routes. In the case of the alkyl tert-butyl ethers MTBE and ETBE, (TAME) have been used as gasoline additives in Europe. Initially there is a general agreement that aerobic degradation proceeds via introduced as an octane booster at low concentrations, oxygen- several specific monooxygenase- and dehydrogenase-catalyzed ated gasoline is now amended with up to 15 vol% of these ethers to steps, resulting in the formation of TBA, 2-methylpropan-1,2- improve combustion efficiency and reduce carbon monoxide emissions. Currently, the corresponding ethyl ethers, ethyl tert- diol, and 2-hydroxyisobutyric acid (Fig. 1) (27, 40, 42). 2- butyl ether (ETBE) and tert-amyl ethyl ether (TAEE), have gained the market share due to their lower vapor pressures and higher boiling points (8). The use of ethyl tert-alkyl ethers is also pro- Received 17 October 2011 Accepted 13 December 2011 moted by legislation in some countries, as the ethyl moiety can be Published ahead of print 22 December 2011 easily derived from bioethanol (34), thus helping to reduce carbon Address correspondence to Thore Rohwerder, [email protected]. dioxide emissions from fossil resources. The extensive use of fuel J. Schuster and F. Schäfer contributed equally to this work. oxygenate ethers, however, has resulted in the contamination of Supplemental material for this article may be found at http://jb.asm.org/. numerous groundwater sites in the United States and Europe due Copyright © 2012, American Society for Microbiology. All Rights Reserved. to accidental spills and leaking storage tanks (14, 24, 29, 45). Com- doi:10.1128/JB.06384-11 pared to other gasoline compounds, the ethers are highly water

972 jb.asm.org 0021-9193/12/$12.00 Journal of Bacteriology p. 972–981

Dissertation Judith Schuster 7. Der TAA-Abbau des Stammes L32: 86

Hemiterpene Formation from tert-Amyl Alcohol

FIG 1 Proposed pathways for the bacterial degradation of tert-butyl alkyl (MTBE and ETBE) and tert-amyl alkyl (TAME and TAEE) ethers (27, 31). (A) Initially, the methyl/ethyl groups of the ethers are hydroxylated, resulting in the formation of instable hemiacetals (not shown). The latter can spontaneously dismutate to the corresponding tertiary alcohols (TBA and TAA) and formaldehyde/acetaldehyde. Alternatively, the hemiacetals are oxidized to esters, which are hydro- lyzed to the tertiary alcohols and formic/acetic acid. (B) The tertiary alcohols are hydroxylated to diols. In M. petroleiphilum PM1 and A. tertiaricarbonis L108, this step is likely catalyzed by the Rieske nonheme mononuclear iron oxygenase MdpJ (18, 39). (C) Dehydrogenation of primary and secondary alcohols of the diol intermediates to aldehyde and ketone groups. In the case of the aldehydes, further dehydrogenation to the corresponding carboxylic acids is possible. Asterisks indicate chiral carbon centers in metabolite molecules.

Hydroxyisobutyric acid is then converted into the common metabo- enzymes are responsible for the hydroxylation of TBA to lite 3-hydroxybutyric acid in a cobalamin-dependent mutase 2-methylpropan-1,2-diol (18, 39). In strain PM1, the oxygenase reaction (35). The biochemistry of tert-amyl alkyl ether catabolism, and reductase are encoded by the mdpJ and mdpK genes, respec- on the other hand, has not been elucidated in much detail. By analogy tively, which show about 97% identity to the corresponding se- with MTBE and ETBE, it can be proposed that TAME and TAEE are quences found in strain L108. Recently, the importance of MdpJ degraded via the TBA homologue TAA. However, in contrast to in TBA metabolism has also been demonstrated by 13C metabolo- 2-methylpropan-1,2-diol formation from TBA, the hydroxylation of mic and proteomic stable isotope probing (SIP) approaches inves- TAA would result in three possible diol products (Fig. 1), as has tigating oxygenate degradation in mixed cultures (3, 4). It is likely already been shown for TAME and TAA conversion in rats and that MdpJ is also involved in TAA degradation, as strains L108 and humans (2, 43). The 1,2- and 1,3-diols could be further oxidized PM1 could metabolize TAME and TAA (31, 38). However, the to 2-hydroxy-2-methyl- and 3-hydroxy-3-methylbutyric acids, hydroxylation of TAA by MdpJ or other bacterial tert-alcohol mo- respectively, while the dehydrogenation of the 2,3-diol would re- nooxygenases has not been investigated so far. sult in the formation of methylacetoin. Hence, it can be expected In order to identify the role of the putative monooxygenase that compared to MTBE and ETBE degradation, bacterial path- MdpJ in tert-amyl alkyl ether catabolism, we analyzed cultures of ways for TAME and TAEE are branched and likely involve a larger the bacteria A. tertiaricarbonis strain L108 and M. petroleiphilum number of metabolites. strain PM1 for TAME- and TAEE-related metabolites. Further- For the bacteria Aquincola tertiaricarbonis strain L108 and more, two mdpJ knockout mutants of A. tertiaricarbonis L108 Methylibium petroleiphilum strain PM1, it has been found that the were characterized. Surprisingly, it was shown that MdpJ is not expressions of a putative Rieske nonheme mononuclear iron hydroxylating the tert-amyl alkyl ether intermediate TAA and its monooxygenase and its corresponding reductase are upregulated higher homologue 3-methyl-3-pentanol but formed the corre- when cells are grown on MTBE and TBA, suggesting that these sponding desaturation products. Thus, MdpJ plays a central role

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Dissertation Judith Schuster 7. Der TAA-Abbau des Stammes L32: 87

Schuster et al.

in the TAME and TAEE degradation pathways by linking the con- the transposome integrates randomly into the DNA, all colonies obtained version of TAA with the routes for catabolizing the hemiterpene had to be analyzed for the loss of their capability to grow on MSM TBA 2-methyl-3-buten-2-ol and the amino acid leucine. agar (containing 0.5 g literϪ1 TBA). In addition, copies of the colonies were maintained on MSM fructose agar for further analysis. About 5,000 colonies were screened in this way. Colonies with restricted or even lost MATERIALS AND METHODS TBA degradation potential were transferred again onto MSM TBA agar Chemicals, bacterial strains, and growth medium. The purity and sup- plates. The mutants that failed to grow were analyzed further. ply sources of tertiary alcohols and other chemicals used in this study are The exact integration site of the transposome into the genomic DNA listed in the supplemental material. Aquincola tertiaricarbonis strain L108, was determined by direct sequencing with ﬂanking KAN-2 primers (Epi- previously isolated from an MTBE-contaminated aquifer in Leuna, Ger- centre) out of 1 ␮g ␮lϪ1 high-concentrated genomic DNA (Master Pure many (25, 35), was cultivated in liquid mineral salt medium (MSM) (see DNA puriﬁcation kit; Epicentre Biotechnologies) according to the follow- the supplemental material) containing MTBE at a concentration of 0.3 g ing protocol. Sequencing forward primer KAN-2 FP-1 (5=-ACCTACAA literϪ1. Methylibium petroleiphilum strain PM1 (32), obtained from the CAAAGCTCTCATCAACC-3=) and reverse primer KAN-2 RP-1 (5=-GC American Type Culture Collection (ATCC BAA-1232), was grown under AATGTAACATCAGAGATTTTGAG-3=) were used. Direct sequencing the same conditions. Nitrogen-free and cobalt-free MSM was prepared by conditions were 4 min at 95°C and 60 cycles of 30 s at 95°C and 4 min at omitting NH4Cl and CoCl2 ·6H2O, respectively. 60°C. The products were cleaned with Centri-Sep columns (Applied Bio- Growth and resting-cell experiments. Cultures were incubated at systems) and sequenced according to a method described previously by 30°C on rotary shakers. Bacterial cells used in experiments were pregrown Sanger et al. (37), using an ABI Prism 3100 genetic analyzer and the Big- on the respective substrates in closed glass bottles in up to 1 liter of culture Dye Terminator v1.1 cycle sequencing kit (Applied Biosystems). The re- medium and harvested by centrifugation at 13,000 ϫ g at 4°C for 10 min. sulting sequences were analyzed with BLAST (1) and aligned via Se- After washing twice with MSM or nitrogen-free MSM, cells were imme- quencher, version 5.0, sequence analysis software (Gene Codes diately used as an inoculum for growth or resting-cell experiments. For Corporation, Ann Arbor, MI). the latter experiments, the cell concentration was adjusted to values be- Nucleotide sequence accession numbers. Sequences of the mdpJK tween 1.4 and 2.2 g biomass (dry weight) per liter by dilution with MSM, gene environment and the liu operon obtained by the sequencing of whereas growth experiments were typically started with 30 to 60 mg bio- genomic DNA of A. tertiaricarbonis strain L108 have been deposited in the mass per liter. The data shown in this study represent the mean values and GenBank/EMBL/DDBJ database under accession numbers JQ062962 and standard deviations (SD) of data from at least three replicate experiments. JQ001939. Sampling and analytics. Liquid and gas samples were taken as previously described (38), by puncturing the butyl rubber stoppers of incuba- RESULTS tion bottles with syringes equipped with 0.6- by 30-mm Luer Lock needles. The biomass was monitored by measuring the optical density at 700 TAA formation in cultures grown on TAME and TAEE. When

nm (OD700), using a multiplication factor of 0.54 for calculating the dry grown on TAME, cells of M. petroleiphilum strain PM1 temporar- biomass in g per liter (31). Volatile compounds (MTBE, TAME, TAEE, ily accumulated TAA in significant amounts (see Fig. S1 in the TBA, TAA, isoamylene, isoprene, 2-methyl-3-buten-2-ol, prenol, prenal, supplemental material), proving that TAA is a central metabolite 3-methyl-3-pentanol, 3-methyl-1-penten-3-ol, and methylacetoin) were of the TAME degradation pathway. TAEE did not support the quantified by headspace gas chromatography (GC) using flame ionization growth of strain PM1. In contrast, A. tertiaricarbonis L108 metab- detection (FID) (38). Compounds in samples were identified according to olized TAME and also TAEE without an accumulation of TAA the retention times of pure GC standards. In addition, assignments were (see Fig. S1 and S2 in the supplemental material), indicating that verified by GC mass spectrometry analysis (see Fig. S3 to S7 in the supplemental material). Diols and carboxylic acids were quantified by using TAA is more efficiently degraded by this strain. high-performance liquid chromatography (HPLC) with refractive index mdpJ gene environment and knockout mutants. By the se- (RI) detection as described elsewhere previously (30, 31), applying an quencing of genomic DNA from strain L108, a 6.2-kb fragment eluent of 0.01 N sulfuric acid at 0.6 ml per min and a Nucleogel Ion 300 containing the mdpJ and mdpK genes was obtained. An alignment OA column (300 by 7.7 mm; Macherey-Nagel). In addition, carboxylic with the corresponding gene region of strain PM1 showed a very acid metabolites were identified as methyl esters by GC mass spectrometry high level of similarity (Fig. 2). Both the gene and intergenic se- (see the supplemental material). quences possess Ͼ97% identity. The only substantial difference Sequencing of wild-type strain L108 DNA. Genomic DNA of A. ter- that could be found were two transposase genes present in strain tiaricarbonis wild-type strain L108 was extracted by using the MasterPure PM1 and interrupting Mpe_B0551 and Mpe_B0548. At the cor- DNA purification kit (Epicentre) and sequenced by Illumina HiSeq 2000 responding position in strain L108, only a single gene encoding a technology (GATC Biotech, Konstanz, Germany). The obtained DNA sequences were analyzed for open reading frames by using Rast (Rapid hypothetical protein with 290 amino acids could be identified. Annotation Using Subsystem Technology) (http://rast.nmpdr.org/). For precisely studying the role of MdpJ in the bacterial degra- Knockout mutants. In order to prove the enzymatic function of MdpJ dation of tertiary alcohols, we tried to create mdpJ knockout mu- in tertiary alcohol degradation, we generated knockout mutants of A. tants of strain L108 by a site-specific mutagenesis approach. How- tertiaricarbonis strain L108. Site-directed mutagenesis by the homologous ever, even after several trials, corresponding knockout mutants recombination of the designed modified target gene mdpJ::tet (our un- were not obtained. Unspecific transposon mutagenesis, on the published data) out of diverse special knockout vectors failed. However, other hand, and screening for a loss of the capability to grow on mutants were successfully obtained by using the high-transposable but TBA resulted in two mdpJ knockout mutants, which possessed unspecific 1.2-kb small linear EZ-Tn5ϽKAN-2Ͼ Tnp transposome (Epi- ␮ insertions at different positions in the wild-type mdpJ gene (Fig. centre Biotechnologies). We transformed 1 l of the transposome into 70 2). In contrast to wild-type strain L108, both mutant strains were ␮l highly competent mid-exponential-phase bacterial cells by electroporation (MicroPulser; Bio-Rad) in 0.1-cm cuvettes at 600 ⍀ and 1.8 kV for not able to grow on the tertiary alcohols TBA and TAA, whereas about 5 ms. Transformed cells were rescued in 5 ml MSM amended with 2-methylpropan-1,2-diol, the putative product of TBA hydroxy- 10 mM fructose as a carbon source, incubated for6hat30°C and 150 rpm. lation by MdpJ catalysis (Fig. 1), could still be used as the sole Dilutions were then plated onto MSM fructose agar containing 50 ␮g source of energy and carbon. In addition, the postulated mlϪ1 kanamycin as a selection marker and incubated for 2 days at 30°C. As 2-methylpropan-1,2-diol intermediate 2-hydroxyisobutyric acid

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FIG 2 Comparison of the mdpJ gene regions in M. petroleiphilum PM1 and A. tertiaricarbonis L108. The location of transposome integration into mdpJ found in the two knockout mutants of strain L108 resulting in a complete loss of the capability to grow on TBA and TAA is indicated.

was metabolized by wild-type and mutant strains at the same dehydration of the tertiary alcohols TAA and 2-methyl-3-buten- rates, indicating that only the tertiary alcohol-attacking enzymatic 2-ol (see also the next section). Both dehydration products repre- step was affected by the mdpJ knockout. sented about 1.6% of TAA conversion (data not shown). In the Accumulation of TAA metabolites by resting cells. When first hours of the experiment, cumulative concentrations of the shifting TBA-grown cells of A. tertiaricarbonis wild-type strain substrate and all identified metabolites resulted in a nearly com- L108 to TAA and concomitantly inhibiting protein synthesis by plete recovery of the carbon initially added. Toward the end of the chloramphenicol, significant amounts of various metabolites ac- experiment, this value steadily decreased. However, even after the cumulated (Fig. 3). Surprisingly, not the expected diol com- complete degradation of TAA, still about 60% of the substrate was pounds and derived carboxylic acids (Fig. 1) but several hemiter- recovered as the metabolites listed in Fig. 3. This final analytical penes were detected. About 30% of the TAA was converted to the gap of about 40% may be attributed to common metabolites pro- primary alcohol prenol (3-methyl-2-buten-1-ol), while the unsat- duced from 3-methylcrotonic acid, such as acetoacetate and acetyl urated tertiary alcohol 2-methyl-3-buten-2-ol and the aldehyde coenzyme A (acetyl-CoA), which are readily processed via consti- prenal (3-methyl-2-butenal) corresponded to less than 5% of the tutive pathways. Overall, the observed accumulation of metabo- metabolized substrate. In addition, significant amounts of lites indicates that the degradation of TAA proceeds dominantly 3-methylcrotonic acid accumulated. However, in contrast to the via hemiterpenes and that the enzymes required for catabolizing previously found alkene formation by TAA-grown cells (38), iso- them are not well induced in TBA-grown cells of A. tertiaricarbo- amylene was not produced from TAA in significant amounts by nis L108. In contrast, TBA-grown cells not incubated in the pres- TBA-grown cells. Interestingly, not pure isoamylene but a mixture ence of the translation inhibitor chloramphenicol accumulated of this alkene and isoprene was formed, indicating the enzymatic 2-methyl-3-buten-2-ol, prenol, and the corresponding aldehyde

FIG 3 Accumulation of metabolites in resting-cell experiments with TBA-grown wild-type A. tertiaricarbonis L108 cells incubated on TAA in nitrogen-free MSM in the presence of chloramphenicol (2 mM). (A) Concentrations of TAA, the sum of quantiﬁed metabolites, and the percentages of all TAA-derived compounds compared to the initial TAA concentration. (B) Concentrations of the metabolites 2-methyl-3-buten-2-ol, prenol, prenal, and 3-methylcrotonic acid.

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FIG 4 Degradation of TAA and 2-methyl-3-buten-2-ol by resting cells of wild-type strain L108 (closed symbols) and the L108(⌬mdpJ) K2 knockout mutant strain (open symbols). (A) Isoamylene, as the sum of gamma- and beta-isomers (diamonds), was formed from TAA (squares). In all cases, mainly beta- isoamylene was emitted, representing 96 to 97% of the alkenes produced (data not shown). In addition, the accumulation of small amounts of 2-methyl-3- buten-2-ol (triangles) from TAA with the wild-type strain but not with the mutant was observed. (B) Isoprene (diamonds) was formed from 2-methyl-3-buten- 2-ol (triangles). For a direct comparison with alcohol conversion, values of dehydration products refer to concentrations in the liquid phase, although they were found exclusively in the gas phase of the closed incubation bottles.

only temporarily in the first2hoftheexperiment (data not eficial for a high level of dehydration activity. Accordingly, iso- shown). The concentration of metabolites then decreased steadily, prene was emitted from the metabolizable tertiary alcohol indicating the induction of enzymes involved in the TAA- and 2-methyl-3-buten-2-ol by mutant and wild-type strains at similar hemiterpene-specific degradation pathway. rates (Fig. 4B), representing less than 2% of the substrate turnover. Resting cells pregrown on TAA, on the other hand, did not Degradation of 3-methyl-3-pentanol. Wild-type strain L108 show a significant accumulation of metabolites. When these cells and the mdpJ knockout mutant strains were also tested for their were incubated with or without chloramphenicol on TAA, the capabilities to metabolize the tertiary alcohol 3-methyl-3- latter was rapidly degraded, and neither prenol nor prenal or pentanol, a C6 homologue of TBA and TAA. In analogy with 3-methylcrotonic acid was detectable (data not shown). Only 2-methyl-3-buten-2-ol accumulation from TAA, resting cells of small amounts of 2-methyl-3-buten-2-ol were produced, reach- the wild-type strain pregrown on the tertiary hemiterpene alcohol ing maximal concentrations of less than 0.1 mM, which repre- produced the unsaturated tertiary alcohol 3-methyl-1-buten-3-ol. sented about 2% of the TAA metabolized. Interestingly, resting However, compared to hemiterpene accumulation from TAA, the cells pregrown on 2-methyl-3-buten-2-ol gave similar results (Fig. formation of 3-methyl-1-buten-3-ol was more pronounced, tem- 4), suggesting that all enzymes necessary for efficient TAA degra- porarily amounting to up to more than 30% of the substrate con- dation were induced under these conditions. Like L108 wild-type version (Fig. 5). In line with the assumption that MdpJ is respon- and mutant strains, strain PM1 was able to grow on 2-methyl-3- sible for tertiary alcohol degradation, the mutant strains neither buten-2-ol as the sole source of carbon and energy (data not produced 3-methyl-1-buten-3-ol from 3-methyl-3-pentanol nor shown), indicating that this hemiterpene could be metabolized in converted the substrate at all (Fig. 5). The wild-type strain not case it was also occurring as a TAA metabolite in this strain. only metabolized the C tertiary alcohol in short-term experi- Dehydration of TAA and 2-methyl-3-buten-2-ol. As the mu- 6 tant strains of L108 were not able to grow on TAA but could still ments but also could grow on it as the sole source of carbon and use 2-methyl-3-buten-2-ol as a growth substrate, resting cells pre- energy (data not shown). However, with generation times of grown on this hemiterpene were used to test whether the mutant about 40 h, the growth of strain L108 on 3-methyl-3-pentanol was strains accumulated metabolites from TAA. As expected, and in significantly slower than that on TAA (31). contrast to the wild-type strain, with both mutant strains, a for- Vitamin dependence of tertiary alcohol degradation. The vi- mation of 2-methyl-3-buten-2-ol or other hemiterpene com- tamin requirements of strain L108 for the degradation of TAA, Ͼ pounds was not observed. However, as was described previously 3-methyl-3-pentanol, and TBA were studied. Biotin at 8ngli- Ϫ for cells of the wild-type strain grown on TAA (38) and now for ter 1 was essential for growth on TAA and 3-methyl-3-pentanol, TBA-grown cells (see the section above), the alkenes gamma- and whereas TBA was still used as a growth substrate without biotin beta-isoamylene were still formed from TAA by resting cells of (Fig. 6). Interestingly, cobalamin was not essential for growth on both mutant strains, as shown exemplarily for the L108(⌬mdpJ) TAA and 3-methyl-3-pentanol, whereas it was previously shown K2 strain in Fig. 4A, proving that this dehydration reaction is not to be required for TBA metabolism in strain L108 as a component catalyzed by MdpJ. Interestingly, the level of alkene formation of a cobalamin-dependent CoA-carbonyl mutase for the conver- from TAA by the wild-type strain was about 5 times higher, indi- sion of the TBA metabolite 2-hydroxyisobutyric acid (35). Con- cating that a complete metabolization of the alcohol may be ben- sequently, both TAA and 3-methyl-3-pentanol are obviously de-

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FIG 5 Accumulation of the desaturation product 3-methyl-1-penten-3-ol (closed triangles) by resting cells of wild-type A. tertiaricarbonis L108 from the tertiary alcohol substrate 3-methyl-3-pentanol (closed squares). Both L108(⌬mdpJ) K2 and L108(⌬mdpJ) K24 knockout mutant strains did not metabolize the tertiary alcohol (open squares). Accordingly, desaturation products were not detected (open triangles). Cells were pregrown on 2-methyl-3-buten-2-ol.

graded via a totally different route not involving the mutase FIG 6 Comparison of vitamin dependences of wild-type A. tertiaricarbonis reaction. L108 cells grown on TAA (A), 3-methyl-3-pentanol (B), and TBA (C) as the Leucine degradation pathway in strains PM1 and L108. The sole source of energy. In all cases, biomass increase and substrate turnover refer to incubation periods required for the complete consumption of the respective occurrence of 3-methylcrotonic acid in the course of TAA conver- tertiary alcohol substrates in the reference cultures supplemented with all vi- sion and the strict dependence of strain L108 on biotin for growth tamins of complete MSM. Incubation without vitamins was achieved by dilut- on this tertiary alcohol let us assume that 3-methylcrotonyl-CoA ing cultures pregrown in complete MSM with cobalt- and vitamin-free MSM, carboxylase is involved in the degradation pathway. Normally, resulting in a 2,500-fold dilution of cobalt and all vitamins. The same dilution was applied when only single vitamins, i.e., cobalamin or biotin, were omitted. this biotin-dependent enzyme is part of the leucine catabolism, catalyzing the carboxylation of 3-methycrotonyl-CoA produced from the branched-chain amino acid via 2-oxoisocaproic acid and isovaleryl-CoA (21). The genes encoding the relevant enzymes tial for TAA degradation. However, the latter compound is not LiuABCDE are organized in the liu (leucine-isovalerate-utilizing) converted to diols by hydroxylation reactions, but instead, the operon. Accordingly, a complete set of liu genes can be found on hemiterpene 2-methyl-3-buten-2-ol is formed. This implies that the chromosome of strain PM1. The sequencing of the corre- MdpJ shows desaturase activity when attacking tertiary alcohols, sponding DNA fragment in strain L108 gave a similar result (Fig. thereby causing the formation of a double bond by the removal of 7). Interestingly, a gene encoding a putative acyl-CoA synthetase is two hydrogen radicals from vicinal carbon atoms. A similar mul- present only in the liu operon found in strain L108, while two tifunctionality was found previously for the MdpJ-related Rieske extra genes, encoding a putative transmembrane protein nonheme mononuclear iron naphthalene dioxygenase from Pseu- (Mpe_A3357) and a hypothetical protein (Mpe_A3355), were domonas sp. strain NCIB 9816-4 (11). Besides showing mono- and found only in strain PM1. Another difference might be the regu- dihydroxylations of aromatic compounds, naphthalene dioxyge- lation of gene expression, as genes encoding proteins belonging to nase is also catalyzing double-bond formations (26). On the other the different regulator families TetR and AraC are found upstream hand, diiron center-containing desaturases not only are responsi- from the liu genes in strains PM1 and L108, respectively. ble for double-bond formation in fatty acids but also can catalyze hydroxylations (41). The speciﬁcity of catalysis depends mainly DISCUSSION on the substrate structure and its mobility at the catalytic site of While the metabolic sequences responsible for tert-butyl alkyl the enzyme (5, 49). TBA does not possess vicinal carbons allowing ether and TBA degradation in bacteria have been largely eluci- hydrogen removal and double-bond formation. Consequently, dated, practically nothing is known about the metabolism of tert- only hydroxylation can occur, resulting in the diolic reaction amyl alkyl ethers and TAA. The work presented here revealed that product 2-methylpropan-1,2-diol. In contrast, the structure of the putative Rieske nonheme mononuclear iron oxygenase MdpJ TAA would allow both hydroxylation and desaturation reactions. functions as a desaturase enabling the degradation of the TAME Interestingly, MdpJ obviously catalyzes mainly double-bond for- and TAEE metabolite TAA via hemiterpenes to intermediates of mation in this alcohol, as TAA was predominantly converted to the leucine catabolism. hemiterpenes by resting cells of strain L108 when pregrown on Comparison of the wild type and the two knockout mutants of TBA and incubated in the presence of a translation inhibitor. A. tertiaricarbonis L108 clearly demonstrated that MdpJ is essen- Sequence comparisons revealed that MdpJ is quite unique.

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FIG 7 liu operons found in M. petroleiphilum PM1 and A. tertiaricarbonis L108. For comparison, the corresponding region present in the genome of Thauera sp. strain MZ1T is also shown (Tmz1t_0747 to Tmz1t_0753). Genes not yet related to leucine and isovaleric acid catabolism are highlighted in black.

Thus far, all mdpJ genes and the corresponding proteins detected strains 6A and MCM1/1 were recently demonstrated to degrade in pure cultures and enrichments associated with MTBE or TBA MTBE (3a, 46). degradation were highly similar (3, 38). The amino acid sequences The initial attack of tertiary alcohols seems to be the result of of MdpJ found for strains PM1 and L108, for example, showed recent evolutionary processes referred to as the signiﬁcant release 97% identity. A BLAST search with the complete MdpJ sequence of fuel oxygenates into the environment in the last decades. How- of strain PM1 as a query against the NCBI nonredundant database ever, all other enzymatic steps downstream from MdpJ catalysis (November 2011) resulted in only a couple of MdpJ-like proteins required for TAA mineralization are related to metabolic se- as closest matches, with about 60% identity to the PM1 sequence. quences already established for a long time in nature for the deg- These predicted proteins all belong to strains of Bordetella parap- radation of nonxenobiotic compounds. As proposed previously ertussis, Bordetella bronchiseptica, and Achromobacter xylosoxi- by Malone and coworkers (28), the product of TAA desaturation, dans, which are known to cause respiratory diseases and other the unsaturated tertiary alcohol 2-methyl-3-buten-2-ol, is likely infections but have not often been associated with the degradation catabolized via the primary alcohol prenol and its corresponding of fuel oxygenate ethers or tertiary alcohols. Hence, the origin of aldehyde prenal (Fig. 8). The latter is then oxidized to MdpJ remains quite enigmatic. However, at least A. xylosoxidans 3-methylcrotonic acid. This metabolic sequence would require an

FIG 8 New proposal for a bacterial degradation pathway for the tertiary alcohols TAA and 3-methyl-3-pentanol involving an initial desaturation step catalyzed by the Rieske nonheme mononuclear iron oxygenase MdpJ and its reductase, MdpK. Underlined compounds indicate metabolites detected by GC mass spectrometry analysis. Broken lines indicate postulated but not-yet-characterized enzymatic reactions (28).

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allylic rearrangement previously shown for the interconversion of the presence of 2-methyl-3-buten-2-ol and other hemiterpenes in the monoterpenes linalool and geraniol (13). Recently, the en- the environment for millions of years, hemiterpene degradation is zyme responsible for the rearrangement in the bacterium Castel- likely widespread, and an efficient bacterial degradation pathway laniella defragrans strain 65Phen was identified (6). Besides could have evolved. Consequently, linking the conversion of the isomerization, this so-called linalool dehydratase-isomerase also truly xenobiotic TAA with an already well-established hemiter- catalyzes the dehydration of linalool to myrcene, very similar to pene catabolism by the desaturase activity of MdpJ is straightfor- the allylic rearrangement and dehydration of unsaturated tertiary ward, whereas the degradation of hydroxylation products would alcohols catalyzed by plant monoterpene cyclases (48). As the rel- require the invention of several novel enzymatic steps (Fig. 1). evant structures in both hemi- and monoterpenes are identical, it The product of prenal oxidation, 3-methylcrotonic acid, could could be that in all these cases, the same catalytic mechanism is be metabolized via the leucine degradation route. This would re- employed. As already described for monoterpene cyclases, a uni- quire activation to the corresponding CoA ester, putatively by an form cationic intermediate which is formed after the removal of AMP-forming acyl-CoA synthetase (Fig. 8). 3-Methylcrotonyl- the hydroxyl group could be postulated (see Fig. S8 in the supple- CoA is then carboxylated (LiuBD) to 3-methylglutaconyl-CoA. mental material). In the case of unsaturated alcohols, stabilization After the addition of water (LiuC), the resulting 3-hydroxy-3- by mesomerism can be observed. Either deprotonation or capture methylglutaryl-CoA is split (LiuE) to acetoacetate and acetyl- by water then resulted in elimination or isomerization, respec- CoA. In addition, the involvement of the biotin-dependent car- tively. With saturated alcohols, on the other hand, only the elim- boxylase LiuBD in the TAA degradation pathway is indicated by ination reaction is possible. This dehydratase function of the pos- the biotin dependence observed for strain L108. In line with a tulated 2-methyl-3-buten-2-ol isomerase would explain the rising demand for this vitamin when employing a biotin- alkene formation from TBA and TAA observed previously for dependent enzymatic step in a dissimilatory route, biotin- strains L108 and PM1 (38) and also the isoamylene and isoprene auxotrophic strain L108 cannot grow on TAA in MSM containing formation found in this study. However, conversion of saturated only about 8 ng literϪ1 biotin, while TBA is still used as a growth tertiary alcohols and hemiterpenes has not yet been tested with the substrate under these conditions. enzyme from strain 65Phen or with terpene cyclases. In addition, It is likely that MdpJ is also employed for tertiary alcohol deg- a BLAST search based on the complete genome sequence of strain radation in strain PM1 (18). However, it is quite surprising that PM1 clearly confirms that a sequence homologous to the linalool this strain shows a temporary accumulation of TAA when grown dehydratase-isomerase is not present. on TAME. The nearly identical mdpJ gene environments in strains The second step in hemiterpene catabolism (Fig. 8), the PM1 and L108, likely due to a recent horizontal gene transfer prenol-oxidizing activity, was described previously for NAD- event, imply similar enzymatic activities for TAA conversion. dependent benzyl alcohol dehydrogenase-like enzymes found in Consequently, differences in the degradation pathways more Pseudomonas putida strains MB-1 and mt-2 as well as in Acineto- likely exist downstream from the MdpJ activity. Accordingly, a bacter calcoaceticus NCIB 8250 (28). All these enzymes show rather comparison of the liu operons in strains PM1 and L108 revealed broad substrate specificities for both allylic and aromatic alcohols, low levels of similarity regarding the sequence, length, and num- indicating that a hemiterpene-specific dehydrogenase is not re- ber of genes, indicating only a distant phylogenetic relationship. quired for oxidizing prenol to prenal. Nevertheless, prenol dehy- The leucine degradation pathway belongs to the branched-chain drogenase activity was highly induced in strain MB-1 when cells amino acid catabolism present in many bacteria (21). In the case were grown on 2-methyl-3-buten-2-ol (28). Likewise, prenol did of betaproteobacteria, the activation of the liu genes is normally not accumulate when TAA-degrading cells of strain L108 were regulated by LiuR and/or LiuQ, belonging to the MerR and TetR pregrown on TAA or 2-methyl-3-buten-2-ol, indicating that ei- families of transcriptional regulators, respectively. As was re- ther TAA itself or the resulting hemiterpene alcohols can act as an ported previously (21), in strain PM1, the operon is under the inducer. The final oxidation to 3-methylcrotonic acid has not control of LiuQ and consists of the degradation genes liuABCDE. been characterized so far. However, at least prenal dehydrogenase In strain L108, on the other hand, two additional genes not present activity was detected in strain MB-1 (28). In summary, although in strain PM1 are found directly upstream from liuA, encoding a the metabolic sequence from 2-methyl-3-buten-2-ol via prenol transcription factor of the AraC family and a putative acyl-CoA and prenal to 3-methylcrotonic acid is consistent with the ob- synthetase (Fig. 7). A similar genetic organization is also present in served accumulation of metabolites, the identity of the enzymes the betaproteobacterium Thauera sp. strain MZ1T. Possibly, the involved in bacterial hemiterpene catabolism remains unclear. On AraC-like transcriptional factor is better suited to activate the the basis of our findings and considering the substantial bioinfor- genes of the liu operon when not leucine but only its catabolites matic data already available for strains L108 and PM1, it now are supplied as the sole carbon source. In addition, in a pathway seems to be worthwhile to elucidate the biochemistry of hemiter- running via the free 3-methylcrotonic acid (Fig. 8), a specific pene degradation in these fuel oxygenate-metabolizing bacteria. 3-methylcrotonyl-CoA synthetase is required, which is not neces- 2-Methyl-3-buten-2-ol is part of the complex mixture of vol- sary when the catabolic route starts from leucine. The putative atile organic compounds released by biota, mainly by plants (12), acyl-CoA synthetase encoded by the additional gene found in the and involved in the tropospheric organic aerosol formation influ- liu operon of strain L108 may play this role, likely resulting in a encing the world climate (7, 22). Moreover, it has been found that more efficient degradation of leucine catabolites. this hemiterpene can be the dominating nonmethane volatile or- Tertiary alcohols, such as TBA and TAA, are rarely found in ganic compound emitted, e.g., by a North American pine forest nature. However, anthropogenic sources do exist and tend to pol- (17, 23). Currently, P. putida MB-1 and strains L108 and PM1 are lute the environment. Besides fuel oxygenate ether degradation, the only bacteria known to be capable of using this hemiterpene as the hydroxylation of branched-chain alkanes, such as isobutane, the sole source of carbon and energy (28). However, considering 2-methylbutane, and higher homologues, at the tertiary carbon

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position by various monooxygenases may lead to tertiary alcohol tium using a multidisciplinary approach. FEMS Microbiol. Ecol. 73:370– formation (9, 19, 33). In this context, it can also be speculated 384. whether MdpJ or similar enzymes might be suitable for the de- 5. Behrouzian B, Buist PH. 2002. Fatty acid desaturation: variations on an oxidative theme. Curr. Opin. Chem. Biol. 6:577–582. saturation of larger aliphatic compounds bearing a tertiary alco- 6. Brodkorb D, Gottschall M, Marmulla R, Lüddeke F, Harder J. 2010. hol group, e.g., the C9 alcohols 2,3,5-trimethyl-2-hexanol, 3,6- Linalool dehydratase-isomerase, a bifunctional enzyme in the anaerobic dimethyl-3-heptanol, and 2-methyl-2-octanol, formed in the degradation of monoterpenes. J. Biol. Chem. 285:30436–30442. course of the bacterial degradation of nonylphenols, which are 7. Chan AW, et al. 2009. Photooxidation of 2-methyl-3-buten-2-ol (MBO) as a potential source of secondary organic aerosol. Environ. Sci. Technol. widely used as surfactants in cleaning products (15, 16). At least 43:4647–4652. the C6 alcohol 3-methyl-3-pentanol can be attacked by MdpJ and 8. Chauvaux S, et al. 2001. Cloning of a genetically unstable cytochrome is converted to the expected unsaturated tertiary alcohol. More- P-450 gene cluster involved in degradation of the pollutant ethyl tert-butyl over, strain L108 is also able to use it as the sole source of carbon ether by Rhodococcus ruber. J. Bacteriol. 183:6551–6557. and energy, suggesting that not only MdpJ but also the enzymes 9. Dubbels BL, Sayavedra-Soto L, Arp DJ. 2007. Butane monooxygenase of ‘Pseudomonas butanovora’: purification and biochemical characterization involved in hemiterpene and leucine catabolism are able to pro- of a terminal-alkane hydroxylating diiron monooxygenase. Microbiology cess molecules larger than C5. 153:1808–1816. Our finding that TAA and the higher homologue 3-methyl-3- 10. Fayolle F, Vandecasteele J-P, Monot F. 2001. Microbial degradation and pentanol are not hydroxylated to the corresponding diols but that fate in the environment of methyl tert-butyl ether and related fuel oxygenates. Appl. Microbiol. Biotechnol. 56:339–349. unsaturated alcohols are formed might be surprising at first sight. 11. Ferraro DJ, Gakhar L, Ramaswamy S. 2005. Rieske business: structure- However, by linking the desaturase reaction with hemiterpene function of Rieske non-heme oxygenases. Biochem. Biophys. Res. Com- and branched-chain amino acid catabolism, an efficient linear mun. 338:175–190. degradation pathway has evolved. The alternative hydroxylation 12. Fisher AJ, Baker BM, Greenberg JP, Fall R. 2000. Enzymatic synthesis of can lead to a significant number of metabolites, including stereo- methylbutenol from dimethylallyl diphosphate in needles of Pinus sabiniana. Arch. Biochem. Biophys. 383:128–134. isomers (Fig. 1). Consequently, a large number of enzymatic steps 13. Foss S, Harder J. 1997. Microbial transformation of a tertiary allylalcohol: would be required for the complete mineralization of the tertiary regioselective isomerization of linalool to geraniol without nerol forma- alcohols. This branching of the degradation pathway can be pre- tion. FEMS Microbiol. Lett. 149:71–75. vented only by employing highly specific enzymatic catalysts. 14. Fraile J, et al. 2002. Monitoring of the gasoline oxygenate MTBE and BTEX compounds in groundwater in Catalonia (Northeast Spain). Sci. However, especially at the beginning of pathway evolution, when World J. 2:1235–1242. only enzymes not well adapted to a new substrate can be recruited, 15. Gabriel FLP, et al. 2005. A novel metabolic pathway for degradation of it is unlikely that the catalysis of a highly selective monooxygenase 4-nonylphenol environmental contaminants by Sphingomonas xenophaga could be employed, resulting in only one hydroxylation product. Bayram. J. Biol. Chem. 280:15526–15533. In a theoretic study based on the Y concept, we have already 16. Gabriel FLP, Giger W, Guenther K, Kohler H-PE. 2005. Differential ATP degradation of nonylphenol isomers by Sphingomonas xenophaga Bayram. shown that fuel oxygenates, such as MTBE and ETBE, are formally Appl. Environ. Microbiol. 71:1123–1129. good growth substrates, allowing high theoretical biomass yields 17. Goldan PD, Kuster WC, Fehsenfeld FC, Montzka SA. 1993. The obser- (30). However, when including an extended Monod equation, it vation of a C5 alcohol in a North American pine forest. Geophys. Res. Lett. became obvious that low growth rates would result in mainte- 20:1039–1042. nance requirements too high for supporting productive degrada- 18. Hristova KR, et al. 2007. Comparative transcriptome analysis of Methyl- ibium petroleiphilum PM1 exposed to the fuel oxygenates methyl tert-butyl tion. In this connection, it is quite consistent that not a compli- ether and ethanol. Appl. Environ. Microbiol. 73:7347–7357. cated pathway with several branched metabolic sequences has 19. Imai T, et al. 1986. Microbial oxidation of hydrocarbons and related evolved for degrading TAME and TAEE metabolites but only a compounds by whole-cell suspensions of the methane-oxidizing bacte- superior linear route via hemiterpenes is employed in A. tertiari- rium H-2. Appl. Environ. Microbiol. 52:1403–1406. 20. Reference deleted. carbonis L108 and likely also in M. petroleiphilum PM1. 21. Kazakov AE, et al. 2009. Comparative genomics of regulation of fatty acid and branched-chain amino acid utilization in proteobacteria. J. Bacteriol. ACKNOWLEDGMENTS 191:52–64. 22. Kiendler-Scharr A, et al. 2009. New particle formation in forests inhibited This study was supported by the UFZ within the CITE program. We are by isoprene emissions. Nature 461:381–384. grateful to the DBU (Deutsche Bundesstiftung Umwelt) for financial sup- 23. Kim S, et al. 2010. Emissions and ambient distributions of biogenic port of F.S. (AZ: 20008/994). volatile organic compounds (BVOC) in a ponderosa pine ecosystem: in- We thank C. Schumann (UFZ) and M. Neytschev (UFZ) for technical terpretation of PTR-MS mass spectra. Atmos. Chem. Phys. 10:1759–1771. assistance and B. Würz (UFZ) for excellent analytical advice. 24. Kolb A, Püttmann W. 2006. Comparison of MTBE concentrations in groundwater of urban and nonurban areas in Germany. Water Res. 40: 3551–3558. REFERENCES 25. Lechner U, et al. 2007. Aquincola tertiaricarbonis gen. nov., sp. nov., a 1. Altschul SF, et al. 1997. Gapped BLAST and PSI-BLAST: a new genera- tertiary butyl moiety-degrading bacterium. Int. J. Syst. Evol. Microbiol. tion of protein database search programs. Nucleic Acids Res. 25:3389– 57:1295–1303. 3402. 26. Lee K, Gibson DT. 1996. Toluene and ethylbenzene oxidation by purified 2. Amberg A, Rosner E, Dekant W. 2000. Biotransformation and kinetics of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. excretion of tert-amyl-methyl ether in humans and rats after inhalation Appl. Environ. Microbiol. 62:3101–3106. exposure. Toxicol. Sci. 55:274–283. 27. Lopes Ferreira N, Malandain C, Fayolle-Guichard F. 2006. Enzymes and 3. Aslett D, Haas J, Hyman M. 2011. Identification of tertiary butyl alcohol genes involved in the aerobic biodegradation of methyl tert-butyl ether (TBA)-utilizing organisms in BioGAC reactors using 13C-DNA stable iso- (MTBE). Appl. Microbiol. Biotechnol. 72:252–262. tope probing. Biodegradation 22:961–972. 28. Malone VF, et al. 1999. Characterization of a Pseudomonas putida allylic 3a.Basbera MJ, Mateo E, Monkaityte R, Constanti M. 2011. Biodegrada- alcohol dehydrogenase induced by growth on 2-methyl-3-buten-2-ol. tion of methyl tert-butyl ether by newly identified soil microorganisms in Appl. Environ. Microbiol. 65:2622–2630. a simple mineral solution. World J. Microbiol. Biotechnol. 27:813–821. 29. Moran MJ, Zogorski JS, Squillace PJ. 2005. MTBE and gasoline hydro- 4. Bastida F, et al. 2010. Elucidating MTBE degradation in a mixed consor- carbons in ground water of the United States. Ground Water 43:615–627.

980 jb.asm.org Journal of Bacteriology

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Hemiterpene Formation from tert-Amyl Alcohol

30. Müller RH, Rohwerder T, Harms H. 2007. Carbon conversion efficiency Methylibium petroleiphilum PM1. Appl. Environ. Microbiol. 74:6631– and limits of productive bacterial degradation of methyl tert-butyl ether 6638. and related compounds. Appl. Environ. Microbiol. 73:1783–1791. 41. Shanklin J, Guy JE, Mishra G, Lindqvist Y. 2009. Desaturases: emerging 31. Müller RH, Rohwerder T, Harms H. 2008. Degradation of fuel oxygen- models for understanding functional diversification of diiron-containing ates and their main intermediates by Aquincola tertiaricarbonis L108. Mi- enzymes. J. Biol. Chem. 284:18559–18563. crobiology 154:1414–1421. 42. Steffan RJ, McClay K, Vainberg S, Condee CW, Zhang D. 1997. Bio- 32. Nakatsu CH, et al. 2006. Methylibium petroleiphilum PM1T gen. nov., sp. degradation of the gasoline oxygenates methyl tert-butyl ether, ethyl tert- nov., a new methyl tert-butyl ether (MTBE) degrading methylotroph of butyl ether, and tert-amyl methyl ether by propane-oxidizing bacteria. the beta-Proteobacteria. Int. J. Syst. Evol. Microbiol. 56:983–989. Appl. Environ. Microbiol. 63:4216–4222. 33. Patel RN, Hou CT, Laskin AI, Felix A. 1982. Microbial oxidation of 43. Sumner SCJ, et al. 2003. Characterization of metabolites and disposition hydrocarbons: properties of a soluble methane monooxygenase from a of tertiary amyl methyl ether in male F344 rats following inhalation expo- facultative methane-utilizing organism, Methylobacterium sp. strain CRL- sure. J. Appl. Toxicol. 23:411–417. 26. Appl. Environ. Microbiol. 44:1130–1137. 44. US Environmental Protection Agency. 1997. Drinking water advisory: 34. Poitrat E. 1999. The potential of liquid biofuels in France. Renew. Energy consumer acceptability advice and health effects analysis on methyl ter- 16:1084–1089. tiary butyl ether (MTBE). EPA-822-F-97-008. Office of Water, US Envi- 35. Rohwerder T, Breuer U, Benndorf D, Lechner U, Müller RH. 2006. The ronmental Protection Agency, Washington, DC. alkyl tert-butyl ether intermediate 2-hydroxyisobutyrate is degraded via a 45. van Wezel A, Puijker L, Vink C, Versteegh A, de Voogt P. 2009. Odour novel cobalamin-dependent mutase pathway. Appl. Environ. Microbiol. and flavour thresholds of gasoline additives (MTBE, ETBE and TAME) 72:4128–4135. and their occurrence in Dutch drinking water collection areas. Chemo- 36. Salanitro JP. 1995. Understanding the limitations of microbial metabo- sphere 76:672–676. lism of ethers used as fuel octane enhancers. Curr. Opin. Biotechnol. 46. Vosahlikova-Kolarova M, Krejcik Z, Cajthaml T, Demnerova K, Pazla- 6:337–340. rova J. 2008. Biodegradation of methyl tert-butyl ether using bacterial 37. Sanger F, Nicklen S, Coulson AR. 1977. DNA sequencing with chain- strains. Folia Microbiol. (Praha) 53:411–416. terminating inhibitors. Proc. Natl. Acad. Sci. U. S. A. 74:5463–5467. 47. Weaver JW, Exum LR, Prieto LM. 2010. Gasoline composition regula- 38. Schäfer F, et al. 2011. Alkene formation from tertiary alkyl ether and tions affecting LUST sites. EPA 600/R-10/001. Office of Research and De- alcohol degradation by Aquincola tertiaricarbonis L108 and Methylibium velopment, US Environmental Protection Agency, Washington, DC. spp. Appl. Environ. Microbiol. 77:5981–5987. 48. Wheeler CJ, Croteau R. 1986. Monoterpene cyclases: use of the noncycl- 39. Schäfer F, et al. 2007. Growth of Aquincola tertiaricarbonis L108 on izable substrate analog 6,7-dihydrogeranyl pyrophosphate to uncouple tert-butyl alcohol leads to the induction of a phthalate dioxygenase-related the isomerization step of the coupled isomerization-cyclization reaction. protein and its associated oxidoreductase subunit. Eng. Life Sci. 7:512– Arch. Biochem. Biophys. 246:733–742. 519. 49. Whittle EJ, Tremblay AE, Buist PH, Shanklin J. 2008. Revealing the 40. Schmidt R, Battaglia V, Scow K, Kane S, Hristova KR. 2008. Involve- catalytic potential of an acyl-ACP desaturase: tandem selective oxidation ment of a novel enzyme, MdpA, in methyl tert-butyl ether degradation in of saturated fatty acids. Proc. Natl. Acad. Sci. U. S. A. 105:14738–14743.

March 2012 Volume 194 Number 5 jb.asm.org 981

Dissertation Judith Schuster 7. Der TAA-Abbau des Stammes L32: 95

SUPPLEMENTAL MATERIAL

Journal of Bacteriology

Bacterial degradation of tert-amyl alcohol proceeds via hemiterpene 2-methyl-3-buten-2-ol by employing the tertiary alcohol desaturase function of the Rieske non-heme mononuclear iron oxygenase MdpJ

Judith Schuster1, Franziska Schäfer1, Nora Hübler1, Anne Brandt1, Mònica Rosell2, Claus Härtig1, Hauke Harms1, Roland H. Müller1 and Thore Rohwerder*1

1 Department of Environmental Microbiology and 2Isotope Biogeochemistry, Helmholtz Centre for Environmental Research - UFZ, Permoserstr. 15, 04318 Leipzig, Germany.

Dissertation Judith Schuster 7. Der TAA-Abbau des Stammes L32: 96

1. Purity and supply sources of tertiary alcohols and other chemicals

TAA (>99%) and isoprene (≥98%) were purchased from Merck Schuchardt (Hohenbrunn, Germany). Alkene GC standards of ≥99% purity (alpha, beta and gamma isomers of isoamylene), 3-methylcrotonic acid (≥95%), prenal (3-methyl-2- butenal, 97%), 3-methyl-3-pentanol (>98%), 3-methyl-1,3-butandiol (≥97%), 2- methyl-2,3-butandiol (racemic mixture), methylacetoin (3-hydroxy-3-methyl-2- butanone, 95%) and TAME (97%) were purchased from Sigma-Aldrich (Taufkirchen, Germany). TAEE (98%) and 2-hydroxy-2-methylbutyric acid (98%, racemic mixture) was from ABCR (Karlsruhe, Germany). 2-Methyl-3-buten-2-ol (>98%), prenol (3- methyl-2-buten-1-ol, >98%), 3-methyl-1-buten-3-ol (98%) and 3-hydroxy-3- methylbutyric acid (98%) were from Alfa Aesar (Karlsruhe, Germany).

2. Detailed description of MSM culture medium

-1 The mineral salt medium (MSM) contained in mg L : NH4Cl, 760; KH2PO4, 680;

K2HPO4, 970; CaCl2  6 H2O, 27; MgSO4  7 H2O, 71.2; initial pH was 7.5. MSM also -1 contained trace elements (in mg L ): FeSO4  7 H2O, 14.94; CuSO4  5 H2O, 0.785;

MnSO4  4 H2O, 0.81; ZnSO4 7 H2O, 0.44; Na2MoO4  2 H2O, 0.25; CoCl2 x 6 H2O, 0.040. A vitamin solution was added (in µg L-1): biotin, 20; folic acid, 20; pyridoxine- HCl, 100; thiamine-HCl, 50; riboflavin, 50; nicotinic acid, 50; DL-Ca-pantothenate, 50; p-amino-benzoic acid, 50; lipoic acid, 50, and cobalamin, 50.

Dissertation Judith Schuster 7. Der TAA-Abbau des Stammes L32: 97

3. Degradation of tert-amyl alkyl ethers by M. petroleiphilum PM1 and A. tertiaricarbonis L108

6 TAME 1.0 6 TAME 1.0 TAA TAA 5 Optical density 5 Optical density 0.8 0.8

4 4 0.6 0.6

3 3

0.4 0.4

TAME,TAA (mM) 2 2 TAME, TAA (mM) Optical density (700 nm) Optical density (700 nm) 0.2 0.2 1 1

0 0.0 0 0.0 0123 0123 A Time (days) B Time (days) Figure S1. Degradation of TAME. (A) Biomass increase and temporarily accumulation of TAA by M. petroleiphilum PM1. (B) Biomass increase by A. tertiaricarbonis L108. Accumulation of TAA was not detectable (detection limit 5 µM).

5 TAEE 0.8 5 TAEE 0.8 TAA TAA Optical density Optical density 4 4 0.6 0.6

3 3 0.4 0.4 2 2 TAEE,TAA (mM) TAEE,(mM) TAA Optical density (700 nm) 0.2 nm) (700 density Optical 0.2 1 1

0 0.0 0 0.0 02468101214 0123456 A Time (days) B Time (days)

Figure S2. Degradation of TAEE. (A) TAEE was not degraded by strain M. petroleiphilum PM1. (B) Biomass increase by A. tertiaricarbonis L108. Accumulation of TAA was not detectable (detection limit 5 µM).

Dissertation Judith Schuster 7. Der TAA-Abbau des Stammes L32: 98

4. Identification of hemiterpenes, 3-methyl-1-buten-3-ol and 3-methylcrotonic acid by GC-MS analysis

For confirming the identification of volatile metabolites, a 7890A gas chromatograph coupled to a 5975C mass spectrometer (Agilent Technologies, Waldbronn, Germany) was used. Gas samples were injected manually by a 1000-µL-Lock valve-gastight syringe (SGE, Darmstadt, Germany) into the GC injector held at 250°C and split ratio of 1:3. In all cases, the analytes were separated on a capillary column DB-MTBE (60 m length x 0.32 mm I.D. x 1.8 µm film thickness from Agilent Technologies, Waldbronn, Germany) using helium as carrier gas at a flow rate of 2 mL min-1. For identifying TAME metabolites the following temperature program was used: 70°C isotherm for 2 min, then 2°C/min heating up to 100°C and at 20°C/min up to 260°C; whereas for 3-methyl-3-pentanol experiments, the isotherm was held at 50ºC for 10 min and then the oven was heated up to 260ºC at 20°C/min. The mass spectrometer acquisition was performed in full-scan from 10 to 100 m/z. The different compounds were identified according to retention times obtained for pure standards as well as by comparison of the experimental spectra with those of the NIST database (NIST/EPA/NIH Mass Spectral Library, version 2.0, 2005), see Figure S4 and S7.

3-Methylcrotonic acid was identified as methyl ester. A cell-free 800-µL-sample was incubated with 400 µL methanol (containing 3 vol-% sulfuric acid) at 95°C for 2 hours. Then, the ester was extracted with 500 µL n-hexane. GC analysis was performed using gas chromatography (GC 7890A, Agilent) coupled to mass spectrometry (MSD 5975C, Agilent) on a DB35ms column (30 m; 0.25 mm; 0.25 µm, Agilent). GC conditions: carrier gas helium, constant flow nominal 1 mL/min, injector temperature 250°C, splitless injection (purge time 1 min), programmed oven temperature (1 min isothermal at 50°C, ramp 4 K/min to 330°C), transfer line temperature 300°C. MSD conditions: full scan mode (m/z 40-440), ion source temperature 230°C, quadrupol temperature 150°C, scan rate 3.58 scans/sec, threshold abundance 50. Analytes were identified by comparison of calibrated retention times of authentic standards and mass spectra data in the GC/MS NIST02 library, see Figure S5 B.

Dissertation Judith Schuster 7. Der TAA-Abbau des Stammes L32: 99

55000 initial sample 50000 45000 40000

35000 TAA, 8.35 min 30000 25000 20000 15000 10000

5000

Peak1, 7.35 min 105000 100000 after 6 hours of incubation Peak3, 14.5 min 95000

Abundance 90000 85000 80000 75000 70000 Peak2, 14.14 min 65000 60000 55000 50000 45000 40000 35000 TAA, 8.35 min 30000 25000 20000 15000 10000 5000

Time (min)

Figure S3. GC-MS analysis. Total ion chromatogram (TIC) signals of samples from resting-cell experiments incubating TBA pre-grown cells of A. tertiaricarbonis L108 on TAA in the presence of chloramphenicol (2 mM). During incubation, a significant increase of peaks1 to 3 was observed.

Dissertation Judith Schuster 7. Der TAA-Abbau des Stammes L32: 9:

71 100

43 50 peak1 59 39 41 53 67 27 14 16 18 25 29 31 42 44 48 51 55 58 60 62 65 69 72 75 81 83 85 91 0 10 20 30 40 50 60 70 80 90 100 (Text File) Sca n 5388 (7.354 min): T26_T8_VIP.D\ d a ta .ms

71 100 2-methyl-3-buten-2-ol 43 OH 50 41 59 27 31 39 29 53 58 15 18 26 32 42 45 51 55 60 67 69 72 0 10 20 30 40 50 60 70 80 86 90 100 (replib) 3-Buten-2-ol, 2-methyl-

71 100 67 peak2

39 53 68 41 50 43 28 40 86 42 51 18 38 44 55 65 69 84 15 26 31 57 59 63 72 77 81 87 0 10 20 30 40 50 60 70 80 90 100 (Text File) Sca n 10419 (14.139 min): T26_T8_VIP.D\ d a ta .ms

71 100

OH prenol 41 50 43 39 53 27 29 68 86 31 42 55 25 33 36 44 46 51 57 59 61 63 65 69 72 81 83 87 0 10 20 30 40 50 60 70 80 90 100 (replib) 2-Buten-1-ol, 3-methyl-

84 100 peak3

55 50 39 29 50 53 14 18 32 44 56 61 65 69 77 81 85 89 96 0 10 20 30 40 50 60 70 80 90 100 110 (Text File) Sca n 10688 (14.502 min): T26_T8_VIP.D\ d a ta .ms

84 100 55 O prenal 41 50 29 28 53 38 43 56 59 65 69 85 0 10 20 30 40 50 60 70 80 90 100 110 (mainlib) 2-Butenal, 3-methyl-

Figure S4. GC-MS analysis. Mass spectra of peaks1 to 3 (see Figure S3) and most probable matches by GC-MS spectral NIST data base (≥ 90% similarity).

Dissertation Judith Schuster 7. Der TAA-Abbau des Stammes L32: 9;

Abundance after 7 hours of incubation TIC: 11025_T489.D\ data.ms 4200000 4000000 3800000 3600000 peak1, 5.4 min 3400000 3200000 3000000 2800000 2600000 2400000 peak2 2200000 2000000 1800000

Abundance 1600000 1400000 1200000 1000000 800000 600000 400000 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 ATime--> Time (min)

83 100

50 55 114 peak1

39 29 43 59 71 99 153 191 0 20 40 60 80 100 120 140 160 180 200 (Text File) Sca n 1118 (5.401 min): 11025_T489.D\ d a ta .ms (-1197)

3-methylcrotonic acid 83 100 methyl ester

O 55 50 114 29 39 O

43 59 67 91 99 B 0 20 40 60 80 100 120 140 160 180 200 (mainlib) 2-Butenoic acid, 3-methyl-, methyl ester

Figure S5. GC-MS analysis after methyl ester derivatization. A: TIC signal of a sample from resting-cell experiments incubating TBA pre-grown cells of A. tertiaricarbonis L108 on TAA in the presence of chloramphenicol (2 mM). During incubation, a significant increase of peak1 was observed. Peak2 is the methoxylation product of isoprene formed from 2-methyl-3-buten-2-ol and prenol during derivatization by the sulfuric acid-containing methanol solution. B: Mass spectra of peak1 and most probable matches by GC-MS spectral NIST02-database (95% similarity).

Dissertation Judith Schuster 7. Der TAA-Abbau des Stammes L32: :2

360000 initial sample 340000 320000 300000 280000 3-methyl-3- 260000 240000 pentanol, 220000 13.98 min 200000 peak1, 180000 160000 13.24 min 140000 120000 100000 80000 60000 40000 20000

360000 340000 after 3 hours of incubation 320000 300000 280000 Abundance 260000 peaks between 10.5 and 3-methyl-3- peak1, 240000 12.5 min pentanol, 220000 13.24 min 200000 13.98 min 180000 160000 140000 120000 100000 80000 60000 40000 20000

Time (min)

Figure S6. GC-MS analysis. Total ion chromatogram (TIC) signals of samples from resting-cell experiments incubating 2-methyl-3-buten-2-ol pre-grown cells of A. tertiaricarbonis L108 on 3-methyl-3-pentanol in the presence of chloramphenicol (2 mM). After 3 hours of incubation, a significant increase of peak1 was observed. In addition, several peaks between 10.5 and 12.5 min occurred. The latter represent a not further analyzed complex mixture of dehydration products of 3-methyl-3-pentanol and 3-methyl-1-penten-3-ol (alkenes and dienes).

Dissertation Judith Schuster 7. Der TAA-Abbau des Stammes L32: :3

71 100

67 50 43 peak1 82 41 55 85 27 65 73 14 17 42 45 58 61 69 79 83 86 98 0 10 20 30 40 50 60 70 80 90 100 110 (Text File) Sca n 9753 (13.241 min): T30_T6_SLOPE.D\ d a ta .ms

71 100 3-methyl-1- OH 43 50 penten-3-ol 41 55 85 27 67 73 31 42 45 50 58 77 82 100 0 10 20 30 40 50 60 70 80 90 100 110 (mainlib) 1-Penten-3-ol, 3-methyl-

Figure S7. GC-MS analysis. Mass spectra of peak1 (see Figure S6) and most probable match by GC-MS spectral NIST data base (≥ 90% similarity).

Dissertation Judith Schuster 7. Der TAA-Abbau des Stammes L32: :4

5. Possible mechanism for the enzymatic isomerization and dehydration of tertiary alcohols

isomerization R OH - + OH

R -OH- R R + -H unsaturated tertiary alcohol OH R dehydration

OH isobutene

-OH- -H+ TBA

beta- and gamma- OH isoamylene

-OH- -H+ TAA

Figure S8. Possible mechanism for the isomerization and dehydration of tertiary alcohols by the postulated 2-methyl-3-buten-2-ol isomerase (R = aliphatic residue). After removal of the hydroxyl group by an unknown activation step, the cationic intermediate of unsaturated tertiary alcohols may be converted either to a

dehydration product or isomerized to a primary alcohol, e. g., with R = CH3 to isoprene and prenol, respectively. The saturated alcohols TBA and TAA can only be dehydrated to the corresponding alkenes, as has already been demonstrated in whole-cell experiments for the strains A. tertiaricarbonis L108 and M. petroleiphilum PM1 (Appl. Environ. Microbiol. 77:5981).

Dissertation Judith Schuster 8. Diskussion

8.3. Nachweis der Schlüsselenzyme in Stamm L32: durch Mutation

ittels Deletionsstudien wurden in dieser Arbeit eindeutige Nachweise der bisher postulierten reaktionsspezifischen Funktionen der drei Schlüsselenzyme des Oxy- M genatabbaues (3.) der Ethermonooxygenase EthABCD, (4.) der Alkoholmono- oxygenase MdpJK und (5.) der 4-HIBA-Mutase HcmAB von A. tertiaricarbonis L32: erbracht. Diese Enzymfunktionen wurden sowohl molekularbiologisch als auch physiologisch und biochemisch charakterisiert und als essentiell für die entsprechenden katalytischen Schritte verifiziert. 3. Die in Kapitel 4 vorgestellte Studie [3] belegt die spezifische Etherspaltung durch die ursprünglich nur für Gram-positive Stämme beschriebene Cytochrom-P-672- Monooxygenase EthB des Enzymkomplexes EthABCD [4, 5]. Die spontane Deletion des DNA-Fragments mit dem ethABCD-Gencluster erfolgt in Stamm L32: vermutlich stressinduziert durch die Aktivität der EthABCD- nachgeschalteten IS;3-Y4-Typ Transposase. Der erzeugte ethABCD-negative Stamm L32 hat damit lediglich die Fähigkeit der Etherspaltung verloren, die Folgeschritte im Oxygenat-Stoffwechsel bleiben davon unbeeinflusst erhalten. Im Gegensatz zu den Genen, welche die Funktion der Etherspaltung codieren, sind die Gene des Folgestoffwechsels stabil im L32:-Genom integriert. Deren Mutation sollte ursprünglich rekombinationsbasiert durch spezifische Vektoren erfolgen. Jedoch wurde kein vollständiger Austausch gegen die mutierten Gene erhalten. Daraufhin wurde Transposon-Mutagenese angewandt.

Die ungerichtete, stabile Insertion je eines 3,7 kb langen linearen Transposoms pro Zelle („Ez-Tn7Tnp“, Epicentre Biotechnologies) war erfolgreich. Über 7.222 kanamyzinresistente Klone wurden, im Vergleich zu Positivkontrollen nicht betroffener Stoffwechselsubstrate, auf ausbleibendes Wachstum auf den jeweils enzymspezifischen Substraten hin analysiert. Die Selektion führte zur erfolgreichen Detektion von Mutationen in den Zielgenen, was anhand der Transposon-spezifischen Primer nachgewiesen werden konnte. Durch die zufällige Integration in das Genom wurden sogar mehrere unterschiedlich mutierte Klone pro gewünschtem Zielgen erhalten (mdpJ, hcmA und hcmB). Es wurden je zwei Klone für die weitere Untersuchung ausgewählt. Die resultierenden Stämme L32:(∆mdpJ)K4 und K46,L32:(∆hcmA)K7 und K53 bzw. L32:(∆hcmB)K9 und K; weisen erwartungsgemäß jeweils den gleichen Phänotyp auf, wodurch sie für die beabsichtigte Interpretation gleichwertig nutzbar sind. Das gilt sogar für die Mutanten beider Mutase-Untereinheiten, die zwar für die Funktionalität des Enzyms essentiell sind, aber verschiedene Teilfunktionen (HcmA und HcmB für Substrat- bzw. Cobalamin-Bindung) ausüben.

:5 8. Diskussion :6

4. Die Veröffentlichungen der Kapitel 5 und 7 belegen anhand der Deletionstämme L32:(∆mdpJ)K4 und K46 die spezifische, essentielle Funktion der Alkoholmonooxy- genase MdpJ des Enzymkomplexes MdpJK, welche in der TBA-Induktions-Studie von Schäfer et al. (4229) postuliert wurde [6 – 8]. Gleichzeitig wurde MPD als das resultierende diolische Produkt dieser Oxidation physisch nachgewiesen (Kapitel 5). Für den Abbau von TAA wurde ebenfalls die spezifische Reaktion dieses Enzyms als essentiell erkannt, wobei MdpJK hier, im Gegensatz zu der Hydroxylierung von TBA, überraschenderweise als Desaturase wirkt (Kapitel 7). Die in beiden Kapiteln beschriebene Fähigkeit, noch weitere Alkohole mit tertiärer und auch sekundärer Struktur umzusetzen, zeigt das breite Substrat- und Reaktionsspektrum des Enzymkomplexes MdpJK. 5. Auch die reaktionsspezifische Funktion der neuartigen Mutase HcmAB in der Linearisierung der Kohlenstoffkette durch die Umlagerung der CoA-aktivierten Carboxylgruppe von 4-HIBA zu 5-HB wurde durch Mutation beider Untereinheiten in Kapitel 6 nachgewiesen [9]. Die Stämme L32:(∆hcmA) und L32:(∆hcmB) sind nicht mehr zum Abbau von 4-HIBA fähig und akkumulieren diese Säure in stöchiometrischen Quantitäten nach Umsatz von TBA bzw. MPD. Dagegen bleiben die enzymatischen Schritte vom Halbacetal zu TBA oder TAA, welche in der Abbildung 3.4 einleitend als Dehydrogenase und Esterase vorgestellt wurden, weiterhin unbekannt. Auch die Reaktionsschritte für die weitere Oxidation des Diols MPD über das Aldehyd zur Säure 4-HIBA bleiben vorerst hypothetisch, betreffende Enzyme konnten in den Deletionsstudien nicht selektiert werden.

8.4. Nutzen für den Nachweis natürlichen Abbaus

Neben einem Beitrag zum Verständnis des bakteriellen Oxygenat-Stoffwechsels liefern die aus den Deletionsstudien gewonnenen Erkenntnisse auch eine Grundlage, um Werkzeuge für den direkten Nachweis natürlichen Abbaus abzuleiten. Durch die Verifizierung der spezifischen Funktionen von EthB, MdpJ und HcmA im Abbau der weiterhin als rekalzitrant geltenden Oxygenat-Ether ist es jetzt möglich, deren Nachweis für das Monitoring von mikrobiologischen in-situ-Abbauaktivitäten an kontaminierten Standorten zu nutzen. Beispielsweise können auf Basis der DNA-Sequenzen abgeleitete Sonden einen sensitiven Nachweis der Gene in Umweltproben und damit eine detailliertere Prognose über das vorhandene natürliche Abbaupotenzial des untersuchten Standortes erbringen. Ebenso wären Aussagen über die in-situ aktiven Mikroorganismen bzw. Enzyme durch den Nachweis der Expression der relevanten Gene mittels RT-PCR und sogar eine Quantifizierung mittels RT-qPCR möglich. Bisher waren nur EthB aus Gram-positiven Stämmen und MdpA aus Stamm PM3 als Ethermonooxygenasen verifiziert [4, :]. Jetzt ist der endgültige Beweis der Funktion von EthB auch in einem Gram-negativen Stamm erbracht worden (4). Des weiteren wurden die spezifischen Funktionen der Alkoholmonooxygenase MdpJK und der 4-HIBA-Mutase HcmAB im TBA-Folgestoffwechsel bestätigt. Durch die Verwendung entsprechender Sonden könnten somit jetzt auch Organismen erfasst werden, die TBA bzw. 4-HIBA verstoffwechseln. Die Betrachtung auch dieser Schlüsselenzyme ermöglicht es, Aussagen über die Vollständigkeit der am Etherabbau beteiligten Stoffwechseltypen (siehe Tabelle 3.3 auf Seite 8) an kontaminierten Standorten zu treffen. Dies erlaubt realistischere Prognosen zum tatsächlichen Sanierungspotenzial und -verlauf.

Dissertation Judith Schuster 8. Diskussion :7

Die Identifizierung der am Etherabbau beteiligten Enzyme kann auch für die korrekte Beurteilung von compound-specific stable isotope analysis (CSIA) Resultaten von entschei- dender Bedeutung sein. Die CSIA ist eine angewandte Methode zur (prinzipiell enzym- unabhängigen) Analyse von Ausmaß und Mechanismus der Etheroxidation in Labor- und Umweltproben. Die CSIA klärt die C-H-Spaltungsmechanismen isotopmarkierter Ether anhand der Isotopen-Analyse der 13C/12C- und D/H-Fraktionierungen auf. Die Unterscheidung des Abbaus unter anoxischen oder abiotischen Bedingungen (wie Säure- hydrolyse) wird ebenfalls ermöglicht. Die CSIA-Resultate mit Reinkulturen aeroben MTBE-Abbaus zeigen jedoch hoch-variable Fraktionierungen, was auf die offensichtlich vorhandene Vielfalt der reaktionsspezifischen Enzyme zurückgeführt wird [;, 32]. Teilweise können die Ergebnisse nicht von Isotopen- mustern anaerober Spaltung (Stamm JOB7) oder abiotischer Säurehydrolyse (Stamm GPo3) unterschieden werden oder erzeugen fast keine detektierbare Fraktionierung (wie in den Stämmen IFP 4223 und L32:), wodurch der Abbau übersehen werden könnte [32]. Insbesondere diese sehr geringe Fraktionierung erschwert die Interpretation der CSIA- Daten, so dass oft durch die Analyse der Isotopenmuster allein keine Aussage über den Anteil des biologischen Abbaus an den NA-Prozessen getroffen werden kann. Es werden zusätzliche Informationen über die beteiligten Enzyme benötigt. Ein konkretes Beispiel erfolgreicher Kombination von CSIA mit dem Nachweis des funktionell verifizierten Biomarkers EthB ist die Studie zur Analyse mikrobiellen MTBE- Abbaus in Biofilmen eines aerated treatmend pond systems von Jechalke et al., [33]. Erst durch die Kombination mit Expressionsanalysen von ethB konnten die CSIA-Resul- tate eindeutig mit aerobem Metabolismus in Verbindung gebracht werden. Von dieser methodischen Kombination profitierte auch eine aktuelle Studie zum Monitoring eines mit 522 ppm ETBE belasteten Standortes durch Fayolle-Guichard et al., [34]. Die wiederum insignifikante Fraktionierung wurde per qPCR als eth-abhängiges Resultat bestätigt. Der eindeutige Funktionsnachweis von EthB in Stamm L32: bestätigt jetzt, dass die in den oben erwähnten Laborstudien beobachtete niedrige Fraktionierung tatsächlich auf die Monooxygenaseaktivität durch die Stämme IFP 4223 oder aber L32: zurückzuführen ist [3, 4]. Um jedoch verbindliche Aussagen über den vorherrschenden Stoffwechseltyp und damit zur Vollständigkeit des in-situ-Sanierungspotenzials ableiten zu können, ist die Identifikation weiterer, den TBA-Folgestoffwechsel betreffender spezifischer Gene/Schlüsselenzyme sowie deren Diversität notwendig [35]. Ziele solcher neuen Funktionsbeweise sind besonders die TBA-Monooxygenase MdpJ und die substratbindenden Untereinheit der 4-HIBA-Mutase HcmA. Im Gegensatz zu der Diversität der Ethermonooxygenasen, wie sie durch die CSIA- Resultate vermutet wurde [32, 36], scheint es für den Abbau der tertiären Alkohole TBA und TAA nur wenige Enzyme zu geben. Die Verifizierung von MdpJ als verantwortliche Alkoholmonooxygenase in Stamm L32: ist somit von besonderer Bedeutung. Nur für eine bestimmte AlkB-Typ-Monooxygenase in Mycobacterium austroafricanum IFP 4234 und 4237 wird eine ähnliche Funktion vorhergesagt [37]. Diese Funktion ist allerdings noch nicht durch entsprechende Deletionsstudien bestätigt. Zudem besitzen Mycobacterium-Stämme i.d.R. eine Vielzahl von AlkB- und weiteren Monooxygenasen, so dass z. B. durch Induktionsstudien bei diesen Stämmen bisher keine eindeutige enzymatische Zuordnung für den Stoffwechsel tertiärer Alkohole erfolgen konnte.

Dissertation Judith Schuster 8. Diskussion :8

Die mdpJ-Sequenz erscheint dagegen einzigartig, wie eine entsprechende BLAST- Recherche in der NCBI-Datenbank zeigte [6, 7]. Die Entwicklung einer Sonde für MdpJ erscheint dadurch gut möglich. Die DNA-Ebene erfasst jedoch nicht die gesamte funktionelle Diversität. Auf der Enzymebene sind die nächsten Verwandten zwar mit nur noch 82% Amino- säureidentität als Dioxygenasen der humanpathogenen Arten Bordetella bronchiseptica, Bordetella parapertussis und Achromobacter xylosoxidans annotiert, kürzlich wurde aber für einige Achromobacter-Stämme auch MTBE-Abbau nachgewiesen [38]. Möglicherweise erfolgt der Abbau des MTBE-Metaboliten TBA dort über ein MdpJ-Homolog. Es wäre also erst zu überprüfen, inwieweit die Sonde das gesamte tatsächliche Spektrum funktioneller Biodiversität bis 82% Aminosäureidentität abdecken soll und ob hoch-konservierte Bereiche innerhalb dieses Spektrums existieren und eine Ableitung einer Sonde überhaupt gewährleisten. Das könnte aber die Spezifität der Sonde für tertiäre Alkoholmonooxygenasen beeinträchtigen. Auch die Ableitung einer funktionellen Sonde für das hcmA-Gen ist nicht unproblematisch. Ein Datenbankvergleich des Proteins HcmA unter Berücksichtigung des in Kapitel 6 identifizierten Ile90-Substratbindemotivs erzeugt zehn Treffer mit erneut wenigstens 82% Aminosäureidentität bei Nocardioides sp. JS836, wobei der Stamm JS836 ebenfalls 4-HIBA abbaut [39]. Diese HCM-Sequenzen sollten beim Primer-Design auf jeden Fall berücksichtigt werden, um aus möglichst spezifisch konservierten Bereichen angepasste Primer abzuleiten, welche die gesamte Biodiversität der HCM-Aktivität abdecken. Die Problematik der Spezifität ist durch die über diverse Mutasen verbreiteten hochkonservierten Bereiche gegeben und durch das in Kapitel 6 gezeigte Alignment der zehn HCM-Sequenzen zu Isobutyryl- und Methylmalonyl-CoA-Mutasen verdeutlicht [9]. Auch die N-terminale Region des 4-HIBA-spezifischen Ile90 ist davon betroffen. Eine Ableitung von zu spezifischen DNA-Sonden aus den Referenzgenen z. B. der Stämme IFP 4223, PM3 und L32: würde also sicher nicht die gesamte funktionelle Biodiverstiät des Etherabbaus erfassen. Der Ausweg ist eventuell eine andere Herangehensweise. So berücksichtigt die stable isotope probing (SIP) Methode besser die Diversität, erfordert aber auch dazu eine möglichst genaue Kenntnis der untersuchten Funktion. In einer 13C-DNA-SIP-Studie eines GAC3-Bioreaktors zur Diversitätsanalyse von TBA-Oxidierern wurden u. a. die Gene mdpJ und hcmA anhand der 13C-Markierung detektiert [35]. Die davon abgeleiteten Proteine wiesen jeweils nur wenige Aminosäuresubstitutionen zu der PM3-Referenz Mpe_B2777 (MdpJ) bzw. Mpe_B2763 (HcmA) auf. MdpJ zeigte sich mit nur zwei Substitutionen als genetisch besonders konserviert, HcmA deutete mit dreizehn Aminosäuresubstitutionen zur PM3-Sequenz auf eine leicht höhere Diversität in-situ hin. Diese Methode ermöglicht also auch die Detektion von relevanten Enzymen trotz abweichender Sequenz. Allerdings ist bei SIP eine Inkubation mit markiertem Substrat nötig, während z. B. eine Grundwasserprobe mit qPCR direkt untersucht werden kann.

8.5. Der TAA-Metabolismus als neuartiger Abbauweg

Der neu entdeckte TAA-Metabolismus des Stammes L32: ist das überraschendste Ergebnis aus den Abbau-Studien der MdpJ-Deletionsmutanten L32:(∆mdpJ). Es konnte gezeigt werden, dass die Rieske non-heme iron type Oxygenase MdpJ in Abhängigkeit von der Substratstruktur für die unterschiedliche Prozessierung von TBA und TAA

3Granular activated carbon; Aktivkohle ist Adsorptionsmaterial für TBA und mikrobielle Besiedlungshilfe.

Dissertation Judith Schuster 8. Diskussion :9 verantwortlich ist. TBA verfügt als kleinstmöglicher tertiärer Alkohol über drei gleichwertige Methylgruppen, von denen eine hydroxyliert wird und somit das Diol MPD entsteht. Hingegen bietet das nur um eine C-Gruppe längere TAA mehrere Möglichkeiten für oxidative Vorgänge, sowohl betreffs Position als auch der Reaktionsart. Erfolgt eine Hydroxylierung, können die drei Diole 4-Methyl-3,4-butandiol, 4-Methyl-4,5-butandiol und 4-Methyl-4,6-butandiol gebildet werden, wie dies auch in Toxizitätsstudien mit TAME in Ratten nachgewiesen wurde [3:] und in Abbildung 3.4 der Einleitung auf Seite 8 dargestellt ist. Im Stamm L32: konnte aber keines dieser potenziellen Diol-Produkte nachgewiesen werden. Allerdings fand sich als Hauptprodukt des TAA-Umsatzes der ungesättigte tertiäre Alkohol 4-Methyl-5-buten-4-ol. Die Enzymreaktion, die TAA zu diesem Hemiterpen umsetzt, ist folglich eine Desaturierung und keine Hydroxylierung. Dass der durch MdpJ katalysierte Mechanismus beide Reaktionen ermöglicht, ist in Oxy- genasen nicht ungewöhnlich. Eine ähnliche Reaktion wurde für die entfernt verwandte Naphthalen-Dioxygenase (NDO) aus Pseudomonas sp. NCIB ;:38-6 beobachtet [3;]. In der Oxidation des Aromaten Ethylbenzen über (S)-3-Phenethylalkohol zu 4-Hydroxyaceto- phenon ist zusätzlich die Desaturaseaktivität der NDO aktiv und desaturiert Ethylbenzen zu Styren. Die Desaturasefunktion von MdpJ eröffnet für TAA eine vollkommen andere metabolische Route als für TBA. Aufgrund der gefundenen Metabolite (4-Methyl-5-buten-4-ol, Prenol und 5-Methylcrotonsäure) sowie der strikten Bindung an das Vitamin B7 (Biotin), scheint der TAA-Stoffwechsel an den etablierten Leucin-Abbau gekoppelt zu sein. Das wurde in Kapitel 7 inklusive der Genkassette für den Leucin-Metabolismus (liuABCDE) vorgestellt [8]. Der resultierende Abbau der Oxygenat-Ether des Stammes L32:, dem, vermittelt durch MdpJ, in Abhängigkeit von der Länge der tertiären Seitenkette nun zwei Stoffwechselwege zugrunde liegen, ist in Abbildung 8.3 dargestellt. Die im TAA-Abbau gefundenen Metabolite 4-Methyl-5-buten-4-ol, Prenol, Prenal, 5- Methylcrontonsäure und Isopren gehören zu den natürlichen Hemiterpen-Verbindungen, für welche diverse Stoffwechsel (besonders im Pflanzenreich) etabliert sind. So zählen 4-Methyl-5-buten-4-ol und Isopren zu stark emittierten Gasen, die zu signifikanten Biosphären-Atmosphären-Interaktionen beitragen. Die globale jährliche Isopren-Emission aus der Vegetation beträgt laut Modellierung über 822 Mt, was äquivalent zur Methan-Emission ist und 1/3 der atmosphärisch einge- tragenen Gesamt-Kohlenwasserstoffe entspricht [42]. Isopren wird hauptsächlich von einer Vielzahl diverser Pflanzen wie Moosen, Farnen, Nackt- und Bedecktsamern emittiert, daneben wird es sowohl durch Mikroorganismen und Algen produziert als auch z. B. über die Atemluft des Menschen abgegeben [42]. Ein großer Anteil an der Emission von 4-Methyl-5-buten-4-ol wird den Pinien (Pinus sabiniana) in Nordwest-Amerika zugeordnet, deren spezifische Methylbutenol-Synthase 4-Methyl-5-buten-4-ol und Isopren im Verhältnis ;;:3 erzeugt [43]. Neben der Isoprenbildung aus 4-Methyl-5-buten-4-ol ist die Bildung der Alkene Isobuten aus TBA und Isoamylen aus TAA ein besonderes Merkmal der oxygenatverwertenden Stämme L32: und PM3. Dies wurde ursprünglich als mögliche Nebenreaktion der MdpJ diskutiert [44], was aber die vorliegenden Untersuchungen jetzt widerlegen, indem Alkenbildung in den MdpJ-Mutanten weiterhin stattfindet. Es ist folglich von einem anderen, bisher unbekannten Enzym mit Dehydratasefunktion für tert. Alkohole auszugehen. In Kapitel 7 wurde dies als mögliche Nebenreaktion der für den Umsatz von 4-Methyl-5-buten-4-ol zu Prenol postulierten Isomerase diskutiert. Als Beispiel wurde die Linalool-Dehydratase-Isomerase von Castellania defragrans 87Phen

Dissertation Judith Schuster 8. Diskussion ::

O O O O

MTBE ETBE TAME TAEE

X Hydroxylase EthABCD R R O O Hemiacetale

OH [R = H / CH3 OH bei Methyl- / Ethylrest]

Dehydrogenase Dismutation tert.-Alkyl-Formiat / -Acetat Esterase Formaldehyd / Acetaldehyd Formiat / Acetat OH Dehydratase OH Dehydratase tert.-Alkohole

TBA TAA beta- gamma- Isobuten Isoamylen Isoamylen Hydroxylase Desaturase X MdpJ X MdpJ

OH OH OH Dehydratase

2-Methylpropan-1,2-diol (MPD) 2-Methyl-3-buten-2-ol Isopren

MPD- und Aldehyd- Isomerase Dehydrogenasen O OH OH HO 3-Methyl-2-buten-1-ol (Prenol)

2-Hydroxyisobuttersäure (2-HIBA) Prenol- und Prenal-Dehydrogenasen Acyl-CoA-Synthetase MdpP X OH und B12-abh. Mutase HcmAB O OH O 3-Methylcrotonsäure CoAS Acyl-CoA-Synthetase 3-Hydroxybutyryl-CoA (3-HB-CoA) Dehydrogenase und 3-Methylcrotonyl-CoA beta-Kethothiolase

2 Acetyl-CoA B7-abh. Carboxylase LiuBD

Leucin Stoffwechsel

CO2 CO2

Abb. 8.3.: Abbauwege der tertiären Oxygenat-Ether MTBE, ETBE, TAME und TAEE in Stamm L32:. Die initiale Oxidation durch EthABCD erzeugt Hemiacetale, welche spontan dismutieren oder enzymatisch zu TBA bzw. TAA umgesetzt werden. TBA wird durch MdpJ zum Diol MHP hydroxyliert und weiter zu 4-HIBA oxidiert, welches CoA-aktiviert (MdpP-vermittelt) und cobalaminabhäng durch HcmAB zu 5-HB-CoA umgewandelt wird. TAA wird durch MdpJ zu 4-Methyl-5-buten-4-ol reduziert, was durch eine postulierte Isomerase zu Prenol umgelagert, weiter zu 5-Methylcrotonsäure oxidiert und CoA-aktiviert und biotinabhängig über den Leucin-Metabolismus (LiuABCDE) abgebaut wird. Alle Intermediate, außer [ ] markierte, wurden GC-/HPLC-analytisch nachgewiesen. Gepunktete Pfeile verweisen auf postulierte Enzymfunktionen. × markiert die Deletionen von EthABCD, MdpJ, HcmA und HcmB.

Dissertation Judith Schuster 8. Diskussion :; angeführt, welche in analoger Weise wie im vorliegenden TAA-Stoffwechsel beschrieben, Linalool und Geraniol isomerisiert sowie über die Dehydratasefunktion aus Linalool auch Myrcen bildet [45]. Die Studie untersuchte jedoch nicht den Umsatz von 4-Methyl-5-buten- 4-ol durch Stamm 87Phen, was durch die vorliegenden Resultate nun eventuell interessant wird. Bei der postulierten 4-Methyl-5-buten-4-ol Isomerase-Dehydratase handelt es sich aber offenbar um ein anderes Enzym. Im Genom von Stamm PM3, für den der gleiche 4-Methyl-5-buten-4-ol Stoffwechsel wie für Stamm L32: gefunden wurde (Kapitel 7), ist jedoch kein Homolog einer Linalool-Dehydratase-Isomerase nachweisbar. Das lässt auf eine neuartige Isomerase schließen. Die Existenz dieser postulierten Isomerase aufzuklären, bleibt Aufgabe künftiger Untersuchungen. Auch dieses Enzym könnte dann ein Schlüsselenzym im Monitoring des natürlichen Abbaus werden. Unabhängig von der Aufklärung der zugrundeliegenden Enzymfunktion können die Alkene als Metabolite des bakteriellen Etherstoffwechsels für ein schnelleres und funktionsgebundenes Monitoring genutzt werden, da diese sehr flüchtigen Verbindungen per GC leicht, schnell und sensitiv messbar sind [44]. In Abhängigkeit der verwendeten GC-Methode kann sogar eine zeitgleiche Bestimmung von Ethern, tert.-Alkoholen und den Alkenen erfolgen. Grundsätzlich ergeben sich aus der Alkenbildung darüber hinaus neue Beurteilungen und Konsequenzen für die Sanierung oxygenatkontaminierten Grundwassers, da von diesen Verbindungen eine potenziell größere Gesundheitsgefährdung ausgeht, als von den Ethern selbst. In Tierversuchen mit Ratten und Mäusen wurde für Isopren, besonders durch die im Stoffwechsel gebildeten Monoepoxide (5,6-Epoxy-5-methyl-3-buten und 5,6-Epoxy-4-methyl-3-buten) und das Diepoxid 4-Methyl-3,4:5,6-diepoxybutan, karzinogene und erbgutverändernde Wirkungen festgestellt, wodurch Isopren selbst als gesundheitsschädlich angesehen wird [46, 47, 48]. Ebenso gelten die Epoxide von Isobuten und Isoamylen 4-Methyl-3,4-epoxypropan [49] bzw. 4-Methyl-4,5-epoxybutan [4:] als erbgutverändernd und toxisch.

8.6. Mikrobiologische Anpassung an Xenobiotika am Beispiel MTBE

Die sehr hohe Sequenzübereinstimmung der Schlüsselenzyme des Oxygenatabbaus von Stamm L32: auf Protein- und sogar DNA-Ebene zu homologen Enzymen in diversen Phyla (z. B. EthB in Rhodococcus ruber IFP 4223 oder MdpJ und HcmA in M. petroleiphilum PM3) lässt auf einen Erwerb dieser Eigenschaften durch horizontalen Gentransfer (HGT) schließen, z. B. über den Austausch eines (Mega-)Plasmids oder transposabler, mobiler Elemente. MTBE kann zumindest als Induktor der Verbreitung passender Enzyme als Antwort auf die Konfrontation der Umwelt mit den xenobiotischen Oxygenaten gelten. Ein in dieser Hinsicht interessantes Untersuchungsobjekt ist das in Kapitel 4 vorgestellte eth-Fragment des Stammes L32:. Dessen Ursprung ist höchstwahrscheinlich die durch ﬂankierende IS5-Elemente als mobil beschriebene ethRABCD-Genkassette des Gram-positiven Stammes IFP 4223 [4]. Besonders ähnlich ist das Enzym dem Thc-System von Rhodococcus sp. NI:8/43, welches die N-Dealkylierung von (S)-Ethyldipropylthiocarbamat ermöglicht, aber auch MTBE umsetzen kann [4;]. In Stamm L32: ist aber nur ein zu IFP 4223 nahezu identischer Abschnitt ethABCD

Dissertation Judith Schuster 8. Diskussion ;2 zu finden, die flankierende DNA-Umgebung ist dagegen komplett verschieden. Das Regulatorgen ethR und alle weiteren in IFP 4223 gefundenen DNA-Sequenzen fehlen. Abweichend von Stämmen mit der kompletten Genkassette ethRABCD erfolgt deshalb die Expression in Stamm L32: konstitutiv, offensichtlich initiiert durch den nur hier gefundenen σ70-Promotor. Vor diesem deutet eine Sequenz mit ;;% Identität zu einem Genfragment aus dem Chromosom des Stammes PM3 (Mpe_A3;77) eine Einbindung von M. petroleiphilum in den eth-Genaustausch an. In Stamm PM3 selbst ist zwar kein eth-Gencluster vorhanden, es erscheint aber möglich, dass andere Methylibium-Stämme auch mit der EthB-Monooxygenase ausgestattet sein könnten [33]. Abweichend von Stämmen mit der kompletten Genkassette ethRABCD erfolgt deshalb die Expression in Stamm L32: konstitutiv, offensichtlich initiiert durch den nur hier gefundenen σ70-Promotor. Vor diesem deutet eine Sequenz mit ;;-prozentiger Übereinstimmung zu einem Genfragment aus dem Chromosom des Stammes PM3 (Mpe_A3;77) eine Einbindung von M. petroleiphilum in den eth-Genaustausch an. In Stamm PM3 selbst ist zwar kein eth-Gencluster vorhanden, es erscheint aber möglich, dass andere Methylibium-Stämme auch mit der EthB-Monooxygenase ausgestattet sein könnten [33]. Die Schlüsselenzyme des Folgestoffwechsels MdpJK und HcmAB sind auf dem PM3-Megaplasmid codiert (Mpe_B2777/B2776 und Mpe_B275:/B2763)[52]. Auch diese sind von Transposasen umgeben, was einen Transfer dieser DNA-Fragmente in L32: ermöglicht haben könnte. Es kann somit spekuliert werden, dass sich ursprünglich in einem Methylibium-Stamm der gesamte Stoffwechsel in Form diverser Genkassetten auf dem Megaplasmid zusammengefunden hat, welches dann, vermittelt durch die Prinzipien des HGT, mobiler Elemente und deren Insertion, verbreitet werden konnte. Das zeigt, dass sich die Mikroorganismen durch Neukombination vorhandener Ressourcen an die xenobiotischen Oxygenate angepasst haben. Den optimalen Selektionsmarker stellt dabei das System dar, welches durch einen vollständigen Abbauweg sowie dessen passende Regulation bzw. Induzierbarkeit sogar autarkes Wachstum ermöglicht. Die Auswirkungen der erworbenen Fähigkeiten können im spezifischen Stoff- wechsel deutlich abweichen, wie die unterschiedlichen Oxidationsmechanismen verschiedener Alkoholformen durch MdpJK. Alkohole, welche wie TAA strukturell mehrere Reaktionsmöglichkeiten aufweisen, wie dessen höheres Homolog 5-Methyl-5- pentanol sowie die sekundären Alkohole 4-Butanol, 5-Methyl-4-butanol und 5-Pentanol, werden durch MdpJK ebenfalls zu ungesättigten Alkoholen desaturiert. Durch die Desaturasefunktion entsteht jeweils nur ein Reaktionsprodukt für den folgenden Stoffwechsel, während Hydroxylierung zu mehreren diolischen Produkten führen würde, wie sie beispielsweise im Fall von TAA im eukaryotischen System nachgewiesen wurden (siehe Abbildung 3.4 auf Seite ;). Eine gerichtete Metabolisierung dieser diversen Diole erscheint unwahrscheinlich und eine effektive Nutzung des heterotrophen Substrates in Bezug auf schnelles Wachstum eingeschränkt oder dieses gar vermeidend. Für den tierischen Organismus ergeben sich durch die Oxidation an verschiedenen Stellen keine Nachteile, weil nach Funktionalisierung als Diol die Ausscheidung über die Niere unspezifisch erfolgt. Hingegen bedeutet die Prozessierung zu den genannten drei verschiedenen Diolen für einen darauf autark wachsenden Mikroorganismus die Notwendigkeit zur Etablierung ganz unterschiedlicher Abbauwege. Das kann nicht zielführend sein, weil die auf diese Weise erzeugten Raten sicherlich immer kleiner sind als eine gerichtete Oxidation nur eines Produktes über einen Weg. Nur so wird eine Energiereproduktionsrate erzeugt, die mit Wahrscheinlichkeit größer ist, als der Bedarf für den Erhaltungsstoffwechsel (maintenance). Erst dann kann Wachstum und Vermehrung stattfinden. Die Desaturasefunktion der MdpJK löst dieses Problem und bietet den

Dissertation Judith Schuster 8. Diskussion ;3

Mikroorganismen den genannten Vorteil, jeweils nur ein Produkt zu erhalten, welches effizient über bestehende metabolische Routen abgebaut werden kann. Die bisher als L32:-spezifisch erscheinende Kombination aus σ70-gesteuerter Ethermono- oxygenase EthABCD, Alkoholmonooxygenase MdpJK und 4-HIBA-Mutase HcmAB ist offensichtlich für dessen optimale Verwertung der vier Oxygenate als einziger Energie- und Kohlenstoffquelle verantwortlich. Das Resultat ist ein breiteres Oxygenatverwertungs- spektrum im Vergleich zum Stamm PM3, der nur MTBE und TAME abbauen kann. Diese Enzymausstattung versetzt L32: in die Lage, die Oxygenate mit hohen Wachstumsraten produktiv zu verwerten, welche bei 52°C mit 2,267 h−1 für MTBE und sogar 2,28 h−1 für ETBE angegeben sind [53]. Für die Intermediate TAA, TBA und 4-HIBA liegen die Wachstumsraten mit 2,2: h−1 bzw. 2,3 h−1 und 2,39 h−1 sogar schon im Bereich üblicher heterotropher Substrate wie Succinat und Fruktose. Damit wird deutlich, dass auch die beteiligten Enzyme des Folgestoffwechsels so gut angepasst sind, dass die sonst ebenfalls als rekalzitrant geltenden tertiär verzweigten Folgesubstrate effizient metabolisiert werden und einen vollständigen Abbau der Ether ermöglichen.

8.7. Ausblick

Die in Stamm L32: gefundenen Schlüsselenzyme für den Abbau der xenobiotischen, verzweigten Oxygenate können durch ihre ungewöhnlichen Eigenschaften hinsichtlich der Substratspezifität und teilweise auch des Katalysemechanismus sogar von industriellem Interesse sein. Zwei dieser Enzyme wurden bislang auf ihre biotechnologische Anwendungsmöglichkeit untersucht. Das ist zum einen MdpJK mit Potenzial zur Synthese enantiomerspe- zifischer alkoholischer und diolischer building blocks. Diese sind wichtige Ausgangs- verbindungen für Wirkstoffe im pharmazeutischen (Profene) bzw. agrochemischen Bereich (Phenoxypropionat-Herbizide) [7, 54]. Ein weiteres Beispiel vielversprechender Anwendungsmöglichkeit ist die 4-HIBA-Mutase (HcmAB). Durch ihre Fähigkeit reversibel zu funktionieren und zudem das Gleichgewicht auf Seiten von 4-HIBA zu haben [9], können heterolog HcmAB exprimierende Mikroorganismen mit einen Stoffwechsel zur Bereitstellung (over production) von 5-HB daraus 4-HIBA produzieren (z. B. PHB-produzierende Bakterien wie Cupriavidus necator,[55]). Aus dieser Säure können dann wichtige Polymere wie Poly(methylmethacrylat) hergestellt werden. In Bezug auf beide Biokatalysatoren sind vorher jedoch weitergehende gentechnische Maßnahmen zur Optimierung der enzymatischen Stabilität sowie Spezifität und Ausbeute betreffender Produkte vorzunehmen, denn in beiden Fällen wurde bisher eine ungeeignete Stereospezifität4 beobachtet, wodurch eine industrielle Anwendung jeweils noch nicht relevant ist. Besonders problematisch ist die beobachtete enzymatische Instabilität von MdpJ. Diese verhindert die Extraktion eines funktionellen Proteins aus TBA-gewachsenem Wildtyp ebenso wie aus heterologer Expression, wodurch in vitro keine spezifische Enzymaktivität bestimmt werden konnte [7, 8, 54]. Die Kenntnisse zu Substrat- und Stereospezifität basieren auf Abbaustudien, welche anhand der Deletion L32:(∆mdpJ) als MdpJ-spezifisch erkannt wurden. Spezielle degradative Stämme, die zudem leistungsstabil auf günstigen Substraten vermehrt werden können, bieten sich als Biokatalysatoren für den Einsatz zur

4Durch MdpJ wird aus 4-Propanol 92%(R)-3,4-Propandiol erzeugt [7]; durch HcmAB wird 4-HIBA-CoA zu :2%(S)-5-HB-CoA gewandelt [9], industriell relevant wäre aber das (R)-Enantiomer.

Dissertation Judith Schuster 8. Diskussion ;4 naturnahen Sanierung mittels Bioaugmentation an. Diese Sanierungstechnik ist im Fall von Oxygenaten durchaus möglich und eine Alternative zu anderen, meist kostenintensiven Reinigungsverfahren. Beispiele liefern diesbezügliche Studien mit den verwandten Stämmen H. flava ENV957 [56] und M. petroleiphilum PM3 [57]. Aber auch A. tertiaricarbonis L32: ist aufgrund seines bestens adaptierten, vollständigen Etherstoffwechsels als Biokatalysator für Bioaugmentation (z. B. in Form von Biofilmen, Kiesfiltern oder Pflanzenkläranlagen) in der Reinigung belasteter Wässer nutzbar. Dazu sollte jedoch vorher das Problem der in Stamm L32: beobachteten Instabilität der Ethermonooxygenase EthABCD durch die Erzeugung bzw. Selektion eines leistungsstabilen Stammes gelöst werden. Die in dieser Arbeit für den Stamm L32: gefundenen Enzymmechanismen des TBA- und TAA-Stoffwechsels lassen sich möglicherweise auch auf die Abbaubarkeit anderer Chemikalien mit ähnlich komplex verzweigten Strukturen übertragen. So wird darüber diskutiert, zukünftig auch höhere Ether als Oxygenate einzusetzen, welche in Abbildung 8.4 dargestellt sind.

O O O O O

MTBE TAME 3M3MP MTHxE MTOcE

Abb. 8.4.: Struktureller Vergleich der Oxygenate MTBE und TAME mit den höherwertigen Ethern 5-Methoxy-5-methylheptan (5M5MP), Methyl-tert-hexylether (MTHxE) und Methyl-tert-octylether (MTOcE).

Für 5-Methoxy-5-methylheptan [58], Methyl-tert-hexylether (MthxE) und Methyl-tert- octylether (MTocE) [59, 5:] sind die jeweiligen Synthesen technisch einfach umsetzbar. Diese wirken zudem ebenso gut als Oxygenate wie MTBE. Dabei wird außerdem für diese höheren Ether ein geringeres Umweltgefährdungsrisiko angenommen, da sie aufgrund der niedrigeren Polarität eine geringere Mobilität im Grundwasser besitzen. Trotzdem könnte es auch bei diesen Verbindungen zu Schadensfällen kommen, für die dann ebenfalls der biologische Abbau als am besten geeignete Sanierungsmethode zum Einsatz kommen könnte. Eine Analyse des Abbaupotenzials dieser Verbindungen u. a. durch den in dieser Hinsicht vielversprechenden Stamm L32: ist also anzustreben. Ein positives Vorzeichen für einen diesbezüglichen Erfolg ist die Fähigkeit des Stammes L32:, das C6-TAA-Analog 5-Methyl-5-pentanol als einzige Energie- und Kohlenstoffquelle zu mineralisieren. Das zeigt, dass neben MdpJ auch der Hemiterpen- und Leucin-Stoffwechsel in der Lage ist, größere Substrate umzusetzen. Noch höhere tert.-Alkohole mit neun C-Atomen können im Stoffwechsel von Nonylphenolen entstehen. Technisches Nonylphenol ist eine Mischung aus verschiedenen Isomeren, bestehend aus dem Phenolring und einer unterschiedlich stark verzweigten Nonylseitenkette. Nonylphenole sind stark umweltgefährdende, ätzende und als Xenoöstrogen hormonell aktive Substanzen. Die linearen Formen gelten als leicht abbaubar, wohingegen verzweigte Formen wie das häufig eingesetzte 6(5’,7’-Dimethyl-5’-heptyl)-phenol (6-NP), rekalzitrante Verbindungen und so persistente Belastungen darstellen. Bisher sind nur Abbauer (Pseudomonas putida, Alcaligenes sp. und Sphingomonas sp.) bekannt [5;, 62, 63], welche zur initialen Spaltung der verzweigten Seitenkette vom Aromaten fähig sind.

Dissertation Judith Schuster 8. Diskussion ;5

In Stamm TTNP5 wurde eine Typ-II Ipso-Substituierung zur hydrolytischen Spal- tung der zwei verzweigten Nonylphenolisomere 6(4’,8’-Dimethyl-4’-heptyl)-phenol und 6(5’,8’-Dimethyl-5’-heptyl)-phenol beobachtet [64], welche je in Hydrochionon und einem tertiären Nonylol (C9-Alkohol) resultiert. Die Nonylole weisen im Vergleich zu TBA oder TAA eine längere Seitenkette auf und stellen in Stamm TTNP5 nicht abbaubare, akkumulierende Endprodukte dar [64]. Die Analogien in der chemischen Grundstruktur der beiden gebildeten Nonylole zu TBA bzw. TAA lassen eine im Moment nur hypothetische Übertragung des in dieser Arbeit gezeigten Prinzips zur Metabolisierung verschiedener tertiärer Alkohole (Abbildung 8.3) auf die höherwertigen Alkohole zu. Die benötigten Enzymschritte werden in der Abbildung 8.5 deshalb nur als MdpJ-, HcmAB- und LiuBD-ähnlich vorgestellt.

4(2′,6′-Dimethyl-2′-heptyl)-phenol 4(3′,6′-Dimethyl-3′-heptyl)-phenol

Ipso- Monooxygenase Ipso- Monooxygenase

OH Monooxygenase (MdpJ-ähnlich) HO

Dehydrogenasen & Desaturase CoA-Aktivierung (MdpJ-ähnlich)

O Isomerase OH (HCM-ähnlich) CoAS Isomerase & HO Dehydrogenasen

O SCoA O CoAS

HO Carboxylase (LiuBD-ähnlich)

Abb. 8.5.: Mögliche Abbaumechanismen zweier tert. Nonylphenolisomere. Die Typ-II Ipso-mono- oxygenatische Hydroxylierung durch den Stamm Sphingomonas sp. TTNP5 resultiert jeweils in Hydrochinon und einem tertiären Nonylol-Endprodukt. Deren strukturelle Ähnlichkeiten zu TBA (links) bzw. TAA (rechts) lassen Spekulationen über eine MdpJ-ähnliche Hydroxylierung bzw. Desaturierung zu. Das im ersten Fall (links) resultierende Diol könnte anschließend über Dehydrogenasen und eine HCM-ähnliche Isomerisierung weiter prozessiert werden. Das Desaturaseprodukt (rechts) erlaubt analog zu 4-Methyl-5-buten-4-ol nach der Isomerisierung und Dehydrogenierung eine LiuBD-ähnliche Carboxylierung der ungesättigten CoA-aktivierten Säure. Die kleinen Pfeile markieren jeweils die reagierende Gruppe.

Dissertation Judith Schuster 8. Diskussion ;6

Die Nonylseitenketten könnten demnach entweder zu einem Diol hydroxyliert oder zu einem ungesättigten Alkohol desaturiert werden, wie es in Abbildung 8.5 gezeigt ist. Das Diol würde dann, analog zu MPD, weiter zur korrespondierenden tertiären Säure prozessiert und durch eine HcmAB-ähnliche Mutase zu einem linearen Metaboliten gewandelt werden. Der im zweiten Fall gebildete tertiäre ungesättigte Alkohol würde durch ein zur 4-Methyl-5-buten-4-ol-Isomerase ähnliches Enzym zu einem linearen ungesättigten Alkohol umgelagert werden. Die nachfolgend erzeugte, korrespondierende Säure würde durch Carboxylierung durch ein LiuBD-ähnliches Enzym in den zentralen Stoffwechsel überführt werden. Auch in diesem Fall wäre eine mögliche Anwendung des Stammes L32: zu untersuchen, da dieser durch die besondere Enzymausstattung aus MdpJK, HcmAB und LiuABCD, deren Aktivität bisher für C4- bis C6-Strukturen nachgewiesen wurde [8], eine optimale Voraussetzung zum Abbau auch dieser höherwertigen Substrate bietet. Eventuell ist dazu jedoch eine Evolution zumindest des Enzyms MdpJ nötig, damit die Umwandlung zum Diol bzw. ungesättigten Alkohol eingeleitet werden kann. Das im besten Fall resultierende Hydrochinon stünde in einer entsprechend syntrophen Kultur einem Aromatenabbauer zur Verfügung. Die Hauptaussage dieses Ausblicks ist, dass der neu gefundene TAA-Abbauweg, ebenso wie der TBA-Weg, auf höhere Substrate übertragbar ist. Dennoch bleibt vorerst unklar, ob diese höheren Verbindungen überhaupt funktionelle Substrate für die Schlüsselenzyme des Stammes L32: darstellen und trotz ihrer sterisch komplexeren Seitenketten eine Reaktion erlauben. Ist dies nicht möglich, ist die Konsequenz eine Suche nach neuen Stämmen, deren Schlüsselenzyme an längere Seitenkennen angepasst sind. Der knock-out ist ein probates Mittel, um solche neuartigen Enzymfunktionen zu studieren, besonders wenn das betreffende Enzym in-vitro selbst zu instabil ist, wie es für MdpJ der Fall ist [7, 54]. Diese Technik kann eventuell zukünftig für die Identifikation relevanter Schlüsselenzyme für den Abbau solcher höheren Verbindungen in anderen Stämmen genutzt werden und so zur weiteren Aufklärung spezifischer Stoffwechsel führen.

Dissertation Judith Schuster 8. Diskussion ;7

8.8. Referenzen der Diskussion

3. Schuster, J., J. Purswani, U. Breuer, C. Pozo, H. Harms, R. H. Müller, and T. Rohwerder. 4235. Constitutive expression of cytochrome P672 EthABCD monooxygenase system enables degradation of synthetic dialkyl ethers in Aquincola tertiaricarbonis L32:. Applied and Environmental Microbiology. 9;:4543-4549. 4. Chauvaux, S., F. Chevalier, C. Le Dantec, F. Fayolle, I. Miras, F. Kunst, and P. Beguin. 4223. Cloning of a genetically unstable cytochrome P-672 gene cluster involved in degradation of the pollutant ethyl-tert-butyl ether by Rhodococcus ruber. Journal of Bacteriology. 3:5:8773-8779. 5. Beguin, P., S. Chauvaux, I. Miras, A. François, F. Fayolle, and F. Monot. 4225. Genes involved in the degradation of ether fuels by bacteria of the Mycobacterium/ Rhodococcus group. Oil and Gas Science and Technology — Review IFP. 7::6:;-6;7. 6. Schäfer, F., U. Breuer, D. Benndorf, M. Von Bergen, H. Harms, and R. H. Müller. 4229. Growth of Aquincola tertiaricarbonis L32: on tert-Butyl Alcohol Leads to the Induction of a Phthalate Dioxygenase-related Protein and its Associated Oxidoreductase Subunit. Engineering in Life Sciences. 9:734-73;. 7. Schäfer, F., J. Schuster, B. Würz, C. Härtig, H. Harms, R. H. Müller, and T. Rohwerder. 4234. Synthesis of Short-Chain Diols and Unsaturated Alcohols from Secondary Alcohol Substrates by the Rieske Nonheme Mononuclear Iron Oxygenase MdpJ. Applied and Environmental Microbiology. 9::84:2-84:6. 8. Schuster, J., F. Schäfer, N. Hübler, A. Brandt, M. Rosell, C. Härtig, H. Harms, R. H. Müller, and T. Rohwerder. 4234. Bacterial degradation of tert-amyl alcohol proceeds via hemiterpene 4-methyl-5-buten-4-ol by employing the tertiary alcohol desaturase function of the Rieske nonheme mononuclear iron oxygenase MdpJ. Journal of Bacteriology. 3;6:;94-;:3. 9. Yaneva, N., J. Schuster, F. Schäfer, V. Lede, D. Przybylski, T. Paproth, H. Harms, R. H. Müller, and T. Rohwerder. 4234. Bacterial Acyl-CoA Mutase Speciﬁcally Catalyzes Coenzyme B12-dependent Isomerization of 4-Hydroxyisobutyryl-CoA and (S)-5-Hydroxybutyryl-CoA. Journal of Biological Chemistry. 4:9:37724-37733. :. Schmidt, R., V. Battaglia, K. Scow, S. Kane, and K. R. Hristova. 422:. Involvement of a novel enzyme, MdpA, in methyl tert-butyl ether degradation in Methylibium petroleiphilum PM3. Applied and Environmental Microbiology. 96:8853-885:. ;. Hyman, M. 4234. Biodegradation of gasoline ether oxygenates. Current Opinion in Biotechnology. 32. Rosell, M., R. Gonzalez-Olmos, T. Rohwerder, K. Rusevova, A. Georgi, F.-D. Kopinke, and H. H. Richnow. 4234. Critical Evaluation of the 4D-CSIA Scheme for Distinguishing Fuel Oxygenate Degradation Reaction Mechanisms. Environmental Science and Technology. 68:6979-6988. 33. Jechalke, S., M. Rosell, P. M. Martínez-Lavanchy, P. Pérez-Leiva, T. Rohwerder, C. Vogt, and H. H. Richnow. 4233. Linking low-level stable isotope fractionation to expression of the cytochrome P672 monooxygenase-encoding ethB gene for elucidation of methyl tert- butyl ether biodegradation in aerated treatment pond systems. Applied and Environmental Microbiology. 99:32:8-32;8. 34. Fayolle-Guichard, F., J. Durand, M. Cheucle, M. Rosell, R. J. Michelland, J.-P. Tracol, F. Le Roux, G. Grundman, O. Atteia, H. H. Richnow, A. Dumestre, and Y. Benoit. 4234. Study of an aquifer contaminated by ethyl tert-butyl ether (ETBE): Site characterization and on-site bioremediation. Journal of Hazardous Materials. 423-424:458- 465.

Dissertation Judith Schuster 8. Diskussion ;8

35. Aslett, D., J. Haas, M. Hyman. 4233. Identification of tertiary butyl alcohol (TBA)-utilizing organisms in Bio-GAC reactors using 13C-DNA stable isotope probing. Biodegradation. 44:;83- ;94. 36. Rosell, M., D. Barceló, T. Rohwerder, U. Breuer, M. Gehre, and H. H. Richnow. 4229. Variations in 13C/12C and D/H enrichment factors of aerobic bacterial fuel oxygenate degradation. Environmental Science and Technology. 63:4258-4265. 37. Ferreira, N. L., H. Mathis, D. Labbé, F. Monot, C. W. Greer, and F. Fayolle- Guichard. 4229. n-Alkane assimilation and tert-butyl alcohol (TBA) oxidation capacity in Mycobacterium austroafricanum strains. Applied Microbiology and Biotechnology. 97:;2;-;3;. 38. Barberà, M. J., E. Mateo, R. Monkaityte, and M. Constantí. 4233. Biodegradation of methyl tert-butyl ether by newly identified soil microorganisms in a simple mineral solution. World Journal of Microbiology and Biotechnology. 49::35-:43. 39. Rohwerder T., U. Breuer, D. Benndorf, U. Lechner, and R. H. Müller. 4229. The MTBE and ETBE intermediate 4-HIBA is degraded via a cobalamin-dependent mutase pathway. Proceedings of the 5rd European conference on MTBE and other fuel oxygenates Mol. VITO Bastiaens. L:33-36. 3:. Amberg, A., E. Rosner, and W. Dekant. 4222. Biotransformation and kinetics of excretion of tert-amyl-methyl ether in humans and rats after inhalation exposure. Toxicological Sciences. 77:496-4:5. 3;. Lee K., and D. T. Gibson. 3;;8. Toluene and ethylbenzene oxidation by purified naphthalene dioxygenase from Pseudomonas sp. strain NCIB ;:38-6. Applied and Environmental Microbiology. 84:5323-5328. 42. Guenther, A., T. Karl, P. Harley, C. Wiedinmyer, P. Palmer, and C. Geron. 4228. Estimates of global terrestrial isoprene emissions using MEGAN (Model of Emissions of Gases and Aerosols from Nature). Atmospheric Chemistry and Physics. 8:53:3-5432. 43. Gray, D. W., S. R. Breneman, L. A. Topper, and T. D. Sharkey. 4233. Biochemical Characterization and Homology Modeling of Methylbutenol Synthase and Implications for Understanding Hemiterpene Synthase Evolution in Plants. Journal of Biological Chemistry. 4:8:427:4-427;2. 44. Schäfer, F., L. Muzica, J. Schuster, N. Treuter, M. Rosell, H. Harms, R. H. Müller, and T. Rohwerder. 4233. Formation of alkenes via degradation of tert-alkyl ethers and alcohols by Aquincola tertiaricarbonis L32: and Methylibium spp. Applied and Environmental Microbiology. 99:7;:3-7;:9. 45. Brodkorb D., M. Gottschall, R. Marmulla, F. Lüddeke, J. Harder. 4232. Linalool Dehydratase-Isomerase, a Bifunctional Enzyme in the Anaerobic Degradation of Monoterpenes. Journal of Biological Chemistry. 4:7:52658-52664. 46. Melnick, R. L., R. C. Sills, J. H. Roycroft, B. J. Chou, H. A. Ragan, and R. A. Miller. 3;;6. Isoprene, an endogenous hydrocarbon and industrial chemical, induces multiple organ neoplasia in rodents after 48 weeks of inhalation exposure. Cancer research. 76:7555- 755;. 47. Hurst, H. E. 4229. Toxicology of 3,5-butadiene, chloroprene, and isoprene. Review Environmental Contaminant Toxicology. :;:353-39;. 48. Fabiani, R., P. Rosignoli, A. De Bartolomeo, R. Fuccelli, and G. Morozzi. 4234. Genotoxicity of alkene epoxides in human peripheral blood mononuclear cells and HL60 leukaemia cells evaluated with the comet assay. Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 969:3-8. 49. Cornet, M., and V. Rogiers. 3;;9. Metabolism and Toxicity of 4-Methylpropene (Isobutene) — A Review. CRC Critical Reviews in Toxicology. 49:445-454. 4:. Gollapudi, B. B., V. Linscombe, and J. W. Wilmer. 3;;7. Clastogenicity of isoamylene oxide to rat lymphocytes in culture. Mutation Research Letters. 569:;-34.

Dissertation Judith Schuster 8. Diskussion ;9

4;. Malandain, C., F. Fayolle-Guichard, and T. M. Vogel. 4232. Cytochromes P672-mediated degradation of fuel oxygenates by environmental isolates. FEMS Microbiology Ecology. 94:4:;-4;8. 52. Kane, S. R., A. Y. Chakicherla, P. S. Chain, R. Schmidt, M. W. Shin, T. C. Legler, K. M. Scow, F. W. Larimer, S. M. Lucas, P. M. Richardson, and K. R. Hristova. 4229. Whole-genome analysis of the methyl tert-butyl ether-degrading beta-proteobacterium Methylibium petroleiphilum PM3. Journal of Bacteriology. 3:;:3;53-3;67. 53. Müller, R. H., T. Rohwerder, and H. Harms. 422:. Degradation of fuel oxygenates and their main intermediates by Aquincola tertiaricarbonis L32:. Microbiology. 376:3636-3643. 54. Schäfer, Franziska. 4234. Charakterisierung der tert.-Butylalkohol (TBA)-Monooxygenase als Enzym mit Potential zur Synthese von chiralen Produkten. Universität Leipzig. Institut für Biochemie. Dissertation. 55. Steinbüchel, A., and H. G. Schlegel. 3;;3. Physiology and molecular genetics of poly (β- hydroxyalkanoic acid) synthesis in Alcaligenes eutrophus. Molecular Microbiology. 7:757-764. 56. Streger, S. H., S. Vainberg, H. Dong, and P. B. Hatzinger. 4224. Enhancing transport of Hydrogenophaga flava ENV957 for bioaugmentation of aquifers contaminated with methyl tert-butyl ether. Applied and Environmental Microbiology. 8::7793-779;. 57. Smith, A. E., K. Hristova, I. Wood, D. M. Mackay, E. Lory, D. Lorenzana, and K. M. Scow. 4227. Comparison of biostimulation versus bioaugmentation with bacterial strain PM3 for treatment of groundwater contaminated with methyl tertiary butyl ether (MTBE). Environmental Health Perspectives. 335:539-544. 58. Karinen, R. S., and A. O. I. Krause. 4222. A novel tertiary ether. Synthesis of 5-methoxy- 5-methylheptane from 4-ethyl-3-hexene and methanol. Catalysis Letters. 89:95-9;. 59. Snelling, J., M. O. Barnett, D. Zhao, and J. S. Arey. 4228. Methyl tertiary hexyl ether and methyl tertiary octyl ether as gasoline oxygenates: assessing risks from atmospheric dispersion and deposition. Journal of the Air and Waste Management Association (3;;7). 78:36:6-36;4. 5:. Snelling, J., M. O. Barnett, D. Zhao, and J. S. Arey. 4229. Methyl-tert-hexyl ether and methyl-tert-octyl ether as gasoline oxygenates: Anticipating widespread risks to community water supply wells. Environmental Toxicology and Chemistry. 48:4475-447;. 5;. Bradley, P. M., L. B. Barber, D. W. Kolpin, P. B. McMahon, and F. H. Chapelle. 422:. Potential for 6-n-nonylphenol biodegradation in stream sediments. Environmental Toxicology and Chemistry. 49:482-487. 62. Corvini, P. F. X., A. Schäffer, and D. Schlosser. 4228. Microbial degradation of nonylphenol and other alkylphenols — our evolving view. Applied Microbiology and Biotechnology. 94:445-465. 63. Toyama, T., M. Murashita, K. Kobayashi, S. Kikuchi, K. Sei, Y. Tanaka, M. Ike, and K. Mori. 4233. Acceleration of nonylphenol and 6-tert-octylphenol degradation in sediment by Phragmites australis and associated Rhizosphere Bacteria. Environmental Science and Technology. 67:8746-8752. 64. Corvini, P. F. X., J. Hollender, R. Ji, S. Schumacher, J. Prell, G. Hommes, U. Priefer, R. Vinken, and A. Schäffer. 4228. The degradation of α-quaternary nonylphenol isomers by Sphingomonas sp. strain TTNP5 involves a type II ipso-substitution mechanism. Applied Microbiology and Biotechnology. 92:336-344.

Dissertation Judith Schuster Anhang

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Curriculum Vitæ

Persönliche Angaben Name Judith Christina Schuster. Geburt 22. Mai 1981, Dresden. Familienstand ledig. Beruflicher Werdegang 2013 Fertigstellung der Dissertation. 2007–2012 Helmholtzzentrum für Umweltforschung GmbH– UFZ, Wissenschaftliche Mitarbeit, Department Umwelt- mikrobiologie, Arbeitsgruppe Mikrobielle Physiologie. Projekte 2010–2012 Anfertigung der Dissertation. 08/2008–06/2010 Genom-Analyse zur Identifizierung relevanter Enzyme im Ether-Stoffwechsel des Stammes L108, Vektordesign zur gezielten Gen-Mutation, parallel dazu Transposon- Mutagenese. 09/2007–07/2008 Erforschung neuartiger Enzyme des Stammes L108 mit Potenzial zur spezifischen, heterologen Synthese industriell relevanter Basis-Chemikalien aus nachwachsenden Rohstoffen. 2000 Umweltzentrum der Handwerkskammer Leipzig, Freiwilliges Ökologisches Jahr, Mitarbeit an der Umwelt- bildung im Handwerk. Akademische Ausbildung 2006–2007 Helmholtzzentrum für Umweltforschung GmbH– UFZ, Diplomarbeit zum Thema „Molekularbiologische Analyse der mikrobiellen Besiedlung eines anaeroben in- situ Reaktors zur Dekontamination von BTEX-belastetem Grundwasser aus Zeitz.“. 2001–2007 Universität Leipzig, Biologiestudium, Hauptfächer Mikrobiologie, Genetik und Immunbiologie, Nebenfach Molekulare Medizin, Diplom, Note 2.3. Schulische Bildung 1991–1999 Leibnizschule–Gymnasium Leipzig, Abitur.

Leipzig, 3. Juli 2013

Dissertation Judith Schuster 322

Veröﬀentlichungen

Veröﬀentlichungen J. Schuster, J. Purswani, U. Breuer, C. Pozo, H. Harms, R. H. Müller, and T. Rohwerder, 2013, Constitutive expression of cytochrome P450 EthABCD monooxygenase system enables degradation of synthetic dialkyl ethers in Aquincola tertiaricarbonis L108. Applied and environmental microbiology. 79:2321–2327. J. Schuster, F. Schäfer, N. Hübler, A. Brandt, M. Rosell, C. Härtig, H. Harms, R. H. Müller, and T. Rohwerder, 2012, Bacte- rial degradation of tert-amyl alcohol proceeds via hemiterpene 2-methyl-3-buten-2-ol by employing the tertiary alcohol desaturase function of the Rieske nonheme mononuclear iron oxygenase MdpJ. Journal of bacteriology. 194:972-981. F. Schäfer, J. Schuster, B. Würz, C. Härtig, H. Harms, R. H. Müller, and T. Rohwerder, 2012, Synthesis of Short-Chain Diols and Unsaturated Alcohols from Secondary Alcohol Substrates by the Rieske Nonheme Mononuclear Iron Oxygenase MdpJ. Applied and environmental microbiology. 78:6280-6284. N. Yaneva, J. Schuster, F. Schäfer, V. Lede, D. Przybylski, T. Paproth, H. Harms, R. H. Müller, and T. Rohwerder, 2012, Bacterial Acyl-CoA Mutase Speciﬁcally Cata- lyzes Coenzyme B12-dependent Isomerization of 2- Hydroxyisobutyryl-CoA and (S)-3-Hydroxybutyryl-CoA. Journal of Biological Chemistry. 287:15502-15511. F. Schäfer, L. Muzica, J. Schuster, N. Treuter, M. Rosell, H. Harms, R. H. Müller, and T. Rohwerder, 2011, Formation of alkenes via degradation of tert-alkyl ethers and alcohols by Aquincola tertiaricarbonis L108 and Methylibium spp. Applied and environmental microbiology. 77:5981-5987.

Leipzig, 14. Juni 2013

Dissertation Judith Schuster 323

Tagungsbeiträge

Tagungsbeiträge J. Schuster, 2012 , Biodegradation of fuel oxygenates: one carbon atom makes the diﬀerence. Higrade Spring Conference 26.03.2012. Helmholtzzentrum für Umweltforschung GmbH – UFZ. Talk. J. Schuster, F. Schäfer, N. Yaneva, T. Rohwerder, R. H. Müller, and H. Harms, 2012, Key enzymes of fuel oxygenate ether degradation. VAAM-Jahrestagung Tübingen. Poster. J. Schuster, T. Rohwerder, R. H. Müller, and H. Harms, 2011, Key enzymes of fuel oxygenate ether degradation, 10th Leipzig Research Festival for Life Sciences. Universität Leipzig. Poster. F. Schäfer, J. Schuster, T. Rohwerder, H. Harms, and R. H. Müller, 2011, The tertiary alcohol monoxygenase/desaturase MdpJK – one enzyme: multiple possibilities. 10th Leipzig Research Festival for Life Sciences. Universität Leipzig. Poster.

Leipzig, 21. Mai 2013

Dissertation Judith Schuster 324

AnteileNachweis über der Anteile Co-Autoren der Co-Autoren, Judith Schuster

Nachweis über Anteile der Co-Autoren:

Titel: Constitutive expression of cytochrome P450 EIhABCD monooxygenase system enables degradation of synthetic dialkyl ethers in Aquincola tertiaricarbonis Ll08 Journal: submitted to AEM Autoren: Judith Schuster, Jessica Purswani, Uta Breuer, Clementina Pozo, Hauke Harms, Roland H. Müller, and Thore Rohwerder

Anteil Judith Schuster (Erstautor): - Zellkulturen auf MTBE, ETBE, TAME, TAEE und Glukose - Abbautests von MTBE aus genannten Kulturen - PCR, Primer-Design - qPCR - RT-qPCR - GC-Analytik MTBE - Analyse der Promotorregion von Ll08 - Analyse und Verarbeitung der Daten - Konzeption - Schreiben der Publikation

Anteil Jessica, Prrrswani (Autor 2): - Projektidee - PCR - Analyse der Promotorregion von Ll08 - Schreiben der Publikation

Anteil Uta Breuer (Autor 3): - Projektidee - Zellkulturen auf MTBE - PCR, Primer-Design - Sequenzierung der eth-Gene - Schreiben der Publikation

Anteil Clementina Pozo (Autor 4): - Konzeption - Schreiben der Publikation

Anteil Hauke Harms (Autor 5): - Konzeption - Schreiben der Publikation

Anteil Roland H. Müller (Autor 6): - Konzeption - Schreiben der Publikation

Dissertation Judith Schuster 325

Anteil Thore Rohwerder flrtztautor): - Projektidee - Konzeption - Zellkulturen auf Diethylether, Diisoptopylether, n-Hexylmethylether und den Aryl Alkyl Ethern - Abbautest dieser Ether - C€-Analytik - Analyse und Verarbeitung der Daten - SchreibenderPublikation

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Thore Rohwerder

Dissertation Judith Schuster 326

Nachweis über Anteile der Co-Autoren, Judith Schuster

Nachweis über Anteile der Co-Autoren:

Titel: Synthesis of short-chain diols and unsaturated alcohols from secondary alcoholic substrates by the Rieske non-heme mononuclear iron oxygenase MdpJ Journal: AppliedandEnvironmentalMicrobiology Autoren: Franziska Schäfer, Judith Schuster, Birgit Würz, Claus Härtig, Hauke Harmso Roland H. Müller and Thore Rohwerder

Anteil Franziska Schäfer (Erstautor): - Konzeption - KultivierungderBakterienkulturen - Substratabbau mit station?iren Zellen (2-Propanol, 2-Butanol, ungesättigter Alkohole) - Chirale Analytik von 1,2-Propandiol (Methodenentwicklung, Probenvorbereitung) - Analytik - Analyse und Verarbeitung der Daten - Schreiben der Publikation

Anteil Judith Schuster (Erstautor): - Konzeption - Generierung der mdpJ-Knockout Mutation von Aquincola tertiaricarbonis Ll08 - Kultivierung der Bakterienkulturen (WT und Mutante) - Substratabbau mit stationären Zellen (R)-2-Butanol und (S)-2-Butanol) - Analyse und Verarbeitung der Daten

Anteil Birgit Würz (Autor 3): - Analytik (GC-MS Analyse 2-Butanol-Stoffwechsel) - Analyse und Verarbeitung der Daten

Anteil Claus Härtig (Autor 4): - Chirale Analytik (GC-MS Analyse 1,2-Propandiol) - Analyse und Verarbeitung der Daten

Anteil Hauke Harms (Autor 5): - Konzeption - Schreiben der Publikation

Anteil Roland H. Müller (Autor 6): - Konzeption - Schreiben der Publikation

Anteil Thore Rohwerder (Letztautor): - Substratabbau mit stationären Zellen (Vergleich des Abbaus (R)-2-Butanol und (,9)-2-Butanol) - Analytik - Projektidee - Konzeption - Schreiben der Publikation

Dissertation Judith Schuster 327 I

Franziska Schäfer

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Dissertation Judith Schuster 328

Nachweis über Anteile der Co-Autoren, Judith Schuster

Nachweis über Anteile der Co-Autoren:

Titel: Bacterial Acyl-CoA Mutase Specifically Catalyzes Coenryme Bl2- dependent Isomerization of 2-Hydroxyisobutyryt-CoA and (^f)-3- Hydroxybutyryl-CoA Journal: Journal of Biological Chemistry Autoren: Nadya Yaneva, Judith Schuster, Franziska Schäfer, Vera Lede, Denise Przybylski, Torsten Paproth, Hauke Harms, Roland H. Müller, and Thore Rohwerder

Anteil Nadya Yaneva (Erstautor): - Konzeption - Klonierung und heterologe Expression von WT-HcmA sowie der HcmA Mutanten I90Y, I90F and I90V in diversen E. coli Expressionsstämmen - Kultivierung der diversen E. coli Bakterienkulturen - heterologe Expression von WT HcmA und HcmB, sowie der site-speci/ic HcmA- Mutanten in E. coli Stämmen - Proteinaufreinigung (Eluat) und Expressionsnachweis mittels SDS-PAGE - Synthese der benötigten Acyl-CoA-Ester - Etablierung der Enrymassays, Enrymkinetik, Substratspezifität - Methodenetablierung zur Analytik (ESI-MS/MS, HPLC) - Analyse und Verarbeitung der Daten - Schreiben der Publikation

Anteil Judith Schuster (Zweitautor): - Generierung der hcmA - lhcmB-Knoc kout Mutanten von l. tertiar ic arbon rs L I 08 - Kultivierung der Bakterienkulturen (1. tertiaricarborels L108 WT und Mutanten) - Substratabbau von TBA und 2-Methylpropan-1,2-diol mit L108 Mutanten - Analytik des Substratabbaus (GC, I{PLC) Sequenzierung des hcm-Operons von A. tertiaricarboreis Ll08 - multiple sequence alignments - Analyse und Verarbeitung der Daten - Schreiben der Publikation

Anteil Franziska Schäfer (Autor 3): - Kultivierung der Bakterienkulturen (1. tertiaricarbonrs L108 - WT und Mutanten) - Substratabbau von TBA und 2-Methylpropan-1,2-diol mit LIO8-WT - Analytik des Substratabbaus (GC,I{PLC) - Analyse und Verarbeitung der Daten

Anteil Vera Lede (Autor 4): - Synthese der CoA-Ester - Analyse und Verarbeitung der Daten

Anteil Denise Przfbylski (Autor 5): - Analyse und Verarbeitung der Daten - Schreiben der Publikation

Dissertation Judith Schuster 329

Anteil Torsten Paproth (Autor 6): Konzeption z.rr Knockout-Mutanten Erzeugung in Ll08 Analyse und Verarbeitung der Daten

Anteil Hauke Harms (Autor 7): Konzeption Schreiben der Publikation

Anteil Roland H. Müller (Autor 8): Konzeption Kinetische Berechnungen Schreiben der Publikation

Anteil Thore Rohwerder (Letztautor): - Projektidee - Konzeption - Hypothese zur biochemischen 2-HIBA Genese in Bakterien und zur Stereochemie - Stereochemie der Acyl-CoA-Ester - multiple sequence alignments - Analytik - Analyse und Verarbeitung der Daten - Schreiben der Publikation

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Hauke Harms Roland H. Müller

Thore Rohwerder

Dissertation Judith Schuster 32:

Nachweis über Anteile der Co-Autoren, Judith Schuster

Nachweis über Anteile der Co-Autoren:

Titel: Bacterial degradation of tert-amyl alcohol proceeds via hemiterpene 2-methyl-3-buten-2-ol by employing the tertiary alcohol desaturase function of the Rieske nonheme mononuclear iron oxygenase MdpJ Journal: Joumal of Bacteriology Autoren: Judith Schuster, Franziska Schäfer, Nora Hübler, Anne Brandt, Mönica Rosell, Claus Härtig, Hauke Harms, Roland H. Müller and Thore Rohwerder

Anteil Judith Schuster (Erstautor): - I(onzeption - Kultivierungder Bakterienkulturen - Substratabbau mit wachsenden und stationären Zellen (3-Methyl-3-pentanol, Vitaminabhängigkeit) - Analytik - Sequenzierung von I quincola tertiaricarborels Ll08 - Knockoul Mutanten von A. tertiaricarborzrs L108 - Analyse und Verarbeitung der Daten - Schreiben der Publikation

Anteil Franziska Schäfer (Erstautor): - Konzeption - KultivierungderBakterienkulturen - Substratabbau mit wachsenden und stationären Zellen (Abbau von TAA, TAME, TAEE) - Analytik - Knockoul Mutanten von A. tertiaricarbonis Ll08 - Analyse und Verarbeitung der Daten - Schreiben der Publikation

Anteil Nora Hübler (Autor 3): - Substratabbau mit wachsenden und stationärenZellen (Abbau von TAA, Vitaminabhängigkeit) - Analyse unci Verarbeitung der Daten

Anteil Anne Brandt (Autor 4): - Substratabbau mit wachsenden und stationärenZellen (Abbau von TAA, Vitaminabhängigkeit) - Analyse und Verarbeitung der Daten

Anteil Mönica Rosell (Autor 5): - TAA-Akkumulation beim TAME/TAEE-Abbau von PMl - Analytik (GC-MS) - Analyse und Verarbeitung der Daten - Schreiben der Publikation

Dissertation Judith Schuster 32;

Anteil Claus Härtig (Autor 6): - Analytik (GC-MS) - Analyse und Verarbeitung der Daten

Anteil Hauke Harms (Autor 7): - Konzeption - Schreiben der Publikation

Anteil Roland H. Müller (Autor 8): - Konzeption - Schreiben der Publikation

Anteil Thore Rohwerder (Letztautor): - Projektidee - Konzeption - Substratabbau mit wachsenden und stationärenZnllen(Alken-Bildung aus TAA und 2-Methyl-3-buten-2-ol) - Analytik - Analyse und Verarbeitung der Daten - Schreiben der Publikation

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Nora Hübler

Cleus Härtig

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- Dissertation Judith Schuster