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Styrol-Monooxygenasen (SMOs) spielen im bakteriellen Abbau von Styrol eine wichtige Rolle. Sie epoxidieren den Kohlenwasserstoff zu (S)-Styroloxid und waren bis vor kurzem vor allem aus Gram-negativen Vertretern wie Pseudomonaden bekannt. Das Grampositive nocardioforme Bodenbakterium Rhodococcus opacus 1CP kann Styrol als Energie- und Kohlenstoffquelle nutzen und verfügt über zwei Typen von SMOs. Neben StyA2B, einer fusionierten FAD:NADH-Oxidoreduktase (StyB) und Monooxygenase (StyA2) findet sich eine weitere Monooxygenase StyA1, deren Gen direkt stromaufwärts zu styA2B lokalisiert ist. Zusätzlich zum natürlichen Fusionsprotein StyA2B gelang kürzlich die Konstruktion künstlicher Fusionen StyAL1B und StyAL2B aus Pseudomonas fluorescens ST. Um sowohl StyA1/StyA2B als auch die künstlichen Fusionen StyAL1B und StyAL2B für eine biotechnologische Anwendung nutzen zu können, wurde im Rahmen dieser Arbeit angestrebt, ihre spezifische Oxygenierungsaktivität (StyA1/StyA2B: 0,24 U/mg) mit Hilfe der error prone PCR zu erhöhen. Um Veränderungen der katalytischen Aktivität in einer großen Zahl von Mutanten schnell zu erkennen, ist ein einfacher Screeningtest erforderlich. Die Fähigkeit von SMOs zur Oxidation von Indol zu blauem Indigo bietet diese Möglichkeit. Allerdings ist hierfür die Expression löslicher Proteine eine wesentliche Voraussetzung. Versuche zur Veränderung der Gene styA2B und styA1A2B mit Hilfe eines kommerziellen error prone PCR Kits lieferten ca. 300 bis 1.200 mutmaßlich veränderte Klone, welche jedoch keinerlei Aktivität für den Indolumsatz zeigten. Als Ursache wurde eine Expression der Proteine in Form inaktiver Inclusion Bodies vermutet. Die Fusionsproteine StyAL1B und StyAL2B bilden lösliches Protein, welche Indol zum blauen Farbstoff Indigo umsetzen. Verschiedene Kultivierungsbedingungen wurden auf den Umsatz von Indol untersucht. Dabei wurde erkannt, dass die Klone sich nicht identisch bezüglich ihrer Proteinlöslichkeit verhalten. Mit Hilfe dieser Ergebnisse wurde ein Test für das Aktivitätsscreening von Styrol-Monooxygenasen auf Platte entwickelt. Die Erhöhung der NaCl-Konzentration im Medium steigerte die Indoloxidation, welche sich jedoch durch zusätzliche physiologisch Faktoren schwer beeinflussen lassen. Auch für die Fusionsproteine erfolgte die Durchführung einer error prone PCR. Der Schritt der error prone PCR stellte kein Problem dar, jedoch die Einbindung des veränderten Genfragmentes in den Vektor, beziehungsweise dessen Transformation in E. coli. Alternative Strategien, wie die Nutzung alternativer DNA Polymerasen und eines konventionellen Konzepts, bei dem veränderte Gene in geschnittene Expressionsvektoren ligiert werden, führte zu keinen detektierbaren Klonen. Die Kultivierung von identischen Klonen auf Festmedium wirkte sich aufgrund nicht näher identifizierter Einflüsse auf das Verhalten bezüglich der Indoloxidation sehr unterschiedlich aus. Um diese Einflüsse zu minimieren, erfolgte die Untersuchung des Systems in einer Flüssigkultur. Im Blickpunkt stand hierbei die Indigoproduktion von E. coli BL21 (pET_StyAL2B) die in Abhängigkeit der optischen Dichte der Kultur untersucht wurde. / Styrene monooxygenases (SMOs) play an important role in the bacterial degradation of styrene. They epoxidize the hydrocarbon highly enantioselective to (S)-styrene oxide. Most of the styrene monooxygenases known so far were identified in Gram-negative microorganisms like pseudomonads. Rhodococcus opacus 1CP, a Gram-positive nocardioform actinobacterium, which uses styrene as energy and carbon source was recently found to possess a novel type of SMO, StyA2B. This protein represents a natural fusion between an FAD:NADH oxidoreductase (StyB) and a single monooxygenase subunit (StyA2) and might act in combination with another single oxygenase StyA1 in strain 1CP. Two artificial analogs to StyA2B, designated StyAL1B and StyAL2B, were recently prepared by a fusion of styA and styB of Pseudomonas fluorescens ST and both showed oxygenating activity. For StyA1/StyA2B as well as the artificial fusion proteins StyAL1B and StyAL2B, it was tried to enhance the specific oxygenation activity in order to support their biotechnological applicability. The method of error prone PCR was used for that purpose. In order to identify favorable modifications with increased catalytic activity from a high number of mutants, an easy and simple screening test is necessary. Therefore, it is reasonable to use the ability of SMOs to oxidize indole to the blue dye indigo. However, the expression of SMOs as soluble proteins is an important requirement for any activity screening. Attempts to modify the genes styA2B and styA1/styA2B by means of a commercial error prone PCR kit yielded 300 to 1,200 potential mutants. Unfortunately, none of the obtained colonies showed any indole-oxidizing activity and the formation of insoluble inclusion bodies was assumed to be a likely explanation. In contrast to StyA2B and StyA1, recombinant expression of the artificial fused SMOs StyAL1B und StyAL2B should yield detectable amounts of active proteins. In fact, cultivation of clones expressing both types of proteins showed a blue coloration. Since the coloration of clones from one single solid medium evolved in a non-uniform manner, cultivation conditions were varied in order to identify factors which promote a more uniform tendency for indole oxidation. Although a high NaCl concentration in the medium was shown to favor indole oxidation, the latter one seems to be influenced by additional physiological factors, hardly to control. For the artificially fused proteins an error prone PCR was carried out, too. Although the initial step of mutagenic PCR was found to be successful, completing the vector system by a second ll-up PCR reaction failed. Alternative strategies like the usage of alternative DNA polymerases as well as a conventional cloning approach of various genes into a digested expression vector did not lead to detectable clones. The cultivation of identical clones on petri dishes provided no uniform tendency for indole oxidation and thus did not allow the reliable comparison of mutants in respect of their specific SMO activities. Cultivation of mutants in liquid medium should lead to more reproducible conditions and for that purpose a method was successfully established to quantify indigo formation and cell density.

Styrol-Monooxygenasen (SMOs) spielen im bakteriellen Abbau von Styrol eine wichtige Rolle. Sie epoxidieren den Kohlenwasserstoff zu (S)-Styroloxid und waren bis vor kurzem vor allem aus Gram-negativen Vertretern wie Pseudomonaden bekannt. Das Grampositive nocardioforme Bodenbakterium Rhodococcus opacus 1CP kann Styrol als Energie- und Kohlenstoffquelle nutzen und verfügt über zwei Typen von SMOs. Neben StyA2B, einer fusionierten FAD:NADH-Oxidoreduktase (StyB) und Monooxygenase (StyA2) findet sich eine weitere Monooxygenase StyA1, deren Gen direkt stromaufwärts zu styA2B lokalisiert ist. Zusätzlich zum natürlichen Fusionsprotein StyA2B gelang kürzlich die Konstruktion künstlicher Fusionen StyAL1B und StyAL2B aus Pseudomonas fluorescens ST. Um sowohl StyA1/StyA2B als auch die künstlichen Fusionen StyAL1B und StyAL2B für eine biotechnologische Anwendung nutzen zu können, wurde im Rahmen dieser Arbeit angestrebt, ihre spezifische Oxygenierungsaktivität (StyA1/StyA2B: 0,24 U/mg) mit Hilfe der error prone PCR zu erhöhen. Um Veränderungen der katalytischen Aktivität in einer großen Zahl von Mutanten schnell zu erkennen, ist ein einfacher Screeningtest erforderlich. Die Fähigkeit von SMOs zur Oxidation von Indol zu blauem Indigo bietet diese Möglichkeit. Allerdings ist hierfür die Expression löslicher Proteine eine wesentliche Voraussetzung. Versuche zur Veränderung der Gene styA2B und styA1A2B mit Hilfe eines kommerziellen error prone PCR Kits lieferten ca. 300 bis 1.200 mutmaßlich veränderte Klone, welche jedoch keinerlei Aktivität für den Indolumsatz zeigten. Als Ursache wurde eine Expression der Proteine in Form inaktiver Inclusion Bodies vermutet. Die Fusionsproteine StyAL1B und StyAL2B bilden lösliches Protein, welche Indol zum blauen Farbstoff Indigo umsetzen. Verschiedene Kultivierungsbedingungen wurden auf den Umsatz von Indol untersucht. Dabei wurde erkannt, dass die Klone sich nicht identisch bezüglich ihrer Proteinlöslichkeit verhalten. Mit Hilfe dieser Ergebnisse wurde ein Test für das Aktivitätsscreening von Styrol-Monooxygenasen auf Platte entwickelt. Die Erhöhung der NaCl-Konzentration im Medium steigerte die Indoloxidation, welche sich jedoch durch zusätzliche physiologisch Faktoren schwer beeinflussen lassen. Auch für die Fusionsproteine erfolgte die Durchführung einer error prone PCR. Der Schritt der error prone PCR stellte kein Problem dar, jedoch die Einbindung des veränderten Genfragmentes in den Vektor, beziehungsweise dessen Transformation in E. coli. Alternative Strategien, wie die Nutzung alternativer DNA Polymerasen und eines konventionellen Konzepts, bei dem veränderte Gene in geschnittene Expressionsvektoren ligiert werden, führte zu keinen detektierbaren Klonen. Die Kultivierung von identischen Klonen auf Festmedium wirkte sich aufgrund nicht näher identifizierter Einflüsse auf das Verhalten bezüglich der Indoloxidation sehr unterschiedlich aus. Um diese Einflüsse zu minimieren, erfolgte die Untersuchung des Systems in einer Flüssigkultur. Im Blickpunkt stand hierbei die Indigoproduktion von E. coli BL21 (pET_StyAL2B) die in Abhängigkeit der optischen Dichte der Kultur untersucht wurde.:Eidesstattliche Erklärung II Danksagung III Zusammenfassung IV Abstract VI Abbildungsverzeichnis XI Tabellenverzeichnis XIII Abkürzungsverzeichnis XIV 1 Einleitung 1 1.1 Styrol - ein Produkt der Industrie . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Styrol-Monooxygenasen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.2.1 Abbauwege von Styrol . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.2.2 Struktur, Vorkommen und Eigenschaften klassischer Zweikomponenten Styrol-Monooxygenasen . . . . . . . . . . . . . . . . . . . . 4 1.2.3 Das neuartige Styrol-Monooxygenase-System StyA1/StyA2B aus Rhodococcus opacus 1CP . . . . . . . . . . . . . . . . . . . . . . . . 6 1.2.4 Künstlich verlinkte SMO aus Pseudomonas uorescens ST . . . . . 7 1.2.5 Biotechnologischer Einsatz von Styrol-Monooxygenasen . . . . . . . 8 1.3 Strategien des Protein-Engineering . . . . . . . . . . . . . . . . . . . . . . 9 1.3.1 Arbeitsmethoden zur Veränderung von DNA . . . . . . . . . . . . . 9 1.3.2 Error prone PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1.4 Arbeitsziele . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2 Material und Methoden 13 2.1 Bakterienstämme und Plasmide . . . . . . . . . . . . . . . . . . . . . . . . 13 2.2 Kultivierungsmedien und -bedingungen . . . . . . . . . . . . . . . . . . . . 14 2.2.1 Kultivierungsmedien . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2.2.2 Kultivierungstemperaturen . . . . . . . . . . . . . . . . . . . . . . . 14 2.3 Polymerase-Kettenreaktion (PCR) . . . . . . . . . . . . . . . . . . . . . . 16 2.3.1 Primer und Primerdesign . . . . . . . . . . . . . . . . . . . . . . . . 16 2.3.2 Standard-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2.4 Fehlerbehaftete Polymerase-Kettenreaktion (epPCR) . . . . . . . . . . . . 17 2.4.1 Synthese der mutagenen Megaprimer . . . . . . . . . . . . . . . . . 18 2.4.2 EZClone Reaktion . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.4.3 Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.4.4 Modi zierung des Protokolls des EZClone Reaktion Schrittes . . . . 20 2.5 Aufreinigung von PCR-Produkten aus der Lösung . . . . . . . . . . . . . . 20 2.6 TAE-Agarose-Gelelektrophorese . . . . . . . . . . . . . . . . . . . . . . . . 20 2.7 DNA-Extraktion aus Agarosegelen . . . . . . . . . . . . . . . . . . . . . . 21 2.8 Bestimmung der DNA-Konzentration . . . . . . . . . . . . . . . . . . . . . 21 2.9 Restriktionsverdau von DNA . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.10 Ligation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.11 Herstellung von kompetenten Zellen (E.coli DH5ff, E. coli BL21) . . . . . 23 2.11.1 Chemisch kompetente Zellen nach der CaCl2-Methode (42) . . . . . 23 2.11.2 TOP10 chemischkompetente Zellen . . . . . . . . . . . . . . . . . . 23 2.12 Transformation nach der Hitzeschock-Methode (19) . . . . . . . . . . . . . 24 2.13 Plasmidpräparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 2.14 Bestimmung der Indigobildung durch Klone mit mutmaÿlicher SMO-Aktivität 24 2.14.1 Abschätzung der Indigobildung durch Augenschein . . . . . . . . . 25 2.14.2 Quanti zierung der Indigobildung mittels UV/Vis-Spektrophotometrie 25 2.14.3 Quanti zierung der Indigobildung aus Flüssigkulturen . . . . . . . . 26 3 Ergebnisse 27 3.1 Versuche der error prone PCR von StyA2B aus Rhodococcus opacus 1CP . 27 3.1.1 Isolation von Templat-DNA und Durchführung der error prone PCR 28 3.1.2 Screening von Transformanden auf Fähigkeit zur Indol-Oxidation . 29 3.1.3 Herstellung und Aktivitätsscreening von E. coli DH5ff pET_StyA2B 30 3.2 Versuche der error prone PCR von styA1/styA2B aus Rhodococcus opacus 1CP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 3.2.1 Durchführung der error prone PCR und Aktivitätsscreening von StyA1/StyA2B in pBluescript KS(+) . . . . . . . . . . . . . . . . . 31 3.2.2 Durchführung des Aktivitätsscreening von StyA1/StyA2B in pET16bP 32 3.3 Fusionsproteine StyAL1B und StyAL2B aus Pseudomonas uorescens ST . 33 3.3.1 Optimierung der Zusammensetzung des LB-Mediums für das Aktivitätsscreenings von pET_StyAL2B in E. coli BL21 nach einer Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 3.3.2 Ein uss der Belüftung auf die Neigung von E. coli BL21 (pET_StyAL2B) Kolonien zur Oxidation von Indol . . . . . . . . . . . . . . . . . . . 38 3.3.3 Bestimmung der Indigobildung mittels UV/Vis-Spektroskopie . . . 40 3.3.4 Zeitliche Entwicklung der Indigokonzentration einer Flüssigkultur von E. coli BL21 (pET_StyAL2B) . . . . . . . . . . . . . . . . . . 42 3.3.5 Error prone PCR von pET_StyAL2B mit Gene Morph II EZ Clone Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 3.3.6 Error prone PCR nach der klassischen Methode mit pET_StyAL1B und pET_StyAL2B . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 4 Diskussion der Ergebnisse 49 4.1 Die error prone PCR als attraktive Methodik zur Optimierung von Styrol- Monooxygenasen hinsichtlich katalytischer Eigenschaften . . . . . . . . . . 49 4.2 Der Aktivitätsnachweis als mutmaÿlich limitierender Schritt in der Modi- zierung von StyA2B und StyA1/StyA2B mit Hilfe der error prone PCR . 51 4.3 Die künstlich fusionierten Styrol-Monooxygenasen StyAL2B und StyAL1B erlauben ein Aktivitätsscreening auf Platte . . . . . . . . . . . . . . . . . . 53 4.4 Die Entwicklung einer Methodik zur Quanti zierung der spezi schen Indigobildung eines Expressionsklons der Styrol-Monooxygenase StyAL2B . . . 58 4.5 Fehleranalyse zur error prone PCR . . . . . . . . . . . . . . . . . . . . . . 59 4.5.1 Fehler in der klassischen error prone PCR für pET_StyAL1B und pET_StyAL2B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Literaturverzeichnis 65 / Styrene monooxygenases (SMOs) play an important role in the bacterial degradation of styrene. They epoxidize the hydrocarbon highly enantioselective to (S)-styrene oxide. Most of the styrene monooxygenases known so far were identified in Gram-negative microorganisms like pseudomonads. Rhodococcus opacus 1CP, a Gram-positive nocardioform actinobacterium, which uses styrene as energy and carbon source was recently found to possess a novel type of SMO, StyA2B. This protein represents a natural fusion between an FAD:NADH oxidoreductase (StyB) and a single monooxygenase subunit (StyA2) and might act in combination with another single oxygenase StyA1 in strain 1CP. Two artificial analogs to StyA2B, designated StyAL1B and StyAL2B, were recently prepared by a fusion of styA and styB of Pseudomonas fluorescens ST and both showed oxygenating activity. For StyA1/StyA2B as well as the artificial fusion proteins StyAL1B and StyAL2B, it was tried to enhance the specific oxygenation activity in order to support their biotechnological applicability. The method of error prone PCR was used for that purpose. In order to identify favorable modifications with increased catalytic activity from a high number of mutants, an easy and simple screening test is necessary. Therefore, it is reasonable to use the ability of SMOs to oxidize indole to the blue dye indigo. However, the expression of SMOs as soluble proteins is an important requirement for any activity screening. Attempts to modify the genes styA2B and styA1/styA2B by means of a commercial error prone PCR kit yielded 300 to 1,200 potential mutants. Unfortunately, none of the obtained colonies showed any indole-oxidizing activity and the formation of insoluble inclusion bodies was assumed to be a likely explanation. In contrast to StyA2B and StyA1, recombinant expression of the artificial fused SMOs StyAL1B und StyAL2B should yield detectable amounts of active proteins. In fact, cultivation of clones expressing both types of proteins showed a blue coloration. Since the coloration of clones from one single solid medium evolved in a non-uniform manner, cultivation conditions were varied in order to identify factors which promote a more uniform tendency for indole oxidation. Although a high NaCl concentration in the medium was shown to favor indole oxidation, the latter one seems to be influenced by additional physiological factors, hardly to control. For the artificially fused proteins an error prone PCR was carried out, too. Although the initial step of mutagenic PCR was found to be successful, completing the vector system by a second ll-up PCR reaction failed. Alternative strategies like the usage of alternative DNA polymerases as well as a conventional cloning approach of various genes into a digested expression vector did not lead to detectable clones. The cultivation of identical clones on petri dishes provided no uniform tendency for indole oxidation and thus did not allow the reliable comparison of mutants in respect of their specific SMO activities. Cultivation of mutants in liquid medium should lead to more reproducible conditions and for that purpose a method was successfully established to quantify indigo formation and cell density.:Eidesstattliche Erklärung II Danksagung III Zusammenfassung IV Abstract VI Abbildungsverzeichnis XI Tabellenverzeichnis XIII Abkürzungsverzeichnis XIV 1 Einleitung 1 1.1 Styrol - ein Produkt der Industrie . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Styrol-Monooxygenasen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.2.1 Abbauwege von Styrol . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.2.2 Struktur, Vorkommen und Eigenschaften klassischer Zweikomponenten Styrol-Monooxygenasen . . . . . . . . . . . . . . . . . . . . 4 1.2.3 Das neuartige Styrol-Monooxygenase-System StyA1/StyA2B aus Rhodococcus opacus 1CP . . . . . . . . . . . . . . . . . . . . . . . . 6 1.2.4 Künstlich verlinkte SMO aus Pseudomonas uorescens ST . . . . . 7 1.2.5 Biotechnologischer Einsatz von Styrol-Monooxygenasen . . . . . . . 8 1.3 Strategien des Protein-Engineering . . . . . . . . . . . . . . . . . . . . . . 9 1.3.1 Arbeitsmethoden zur Veränderung von DNA . . . . . . . . . . . . . 9 1.3.2 Error prone PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1.4 Arbeitsziele . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2 Material und Methoden 13 2.1 Bakterienstämme und Plasmide . . . . . . . . . . . . . . . . . . . . . . . . 13 2.2 Kultivierungsmedien und -bedingungen . . . . . . . . . . . . . . . . . . . . 14 2.2.1 Kultivierungsmedien . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2.2.2 Kultivierungstemperaturen . . . . . . . . . . . . . . . . . . . . . . . 14 2.3 Polymerase-Kettenreaktion (PCR) . . . . . . . . . . . . . . . . . . . . . . 16 2.3.1 Primer und Primerdesign . . . . . . . . . . . . . . . . . . . . . . . . 16 2.3.2 Standard-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2.4 Fehlerbehaftete Polymerase-Kettenreaktion (epPCR) . . . . . . . . . . . . 17 2.4.1 Synthese der mutagenen Megaprimer . . . . . . . . . . . . . . . . . 18 2.4.2 EZClone Reaktion . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.4.3 Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.4.4 Modi zierung des Protokolls des EZClone Reaktion Schrittes . . . . 20 2.5 Aufreinigung von PCR-Produkten aus der Lösung . . . . . . . . . . . . . . 20 2.6 TAE-Agarose-Gelelektrophorese . . . . . . . . . . . . . . . . . . . . . . . . 20 2.7 DNA-Extraktion aus Agarosegelen . . . . . . . . . . . . . . . . . . . . . . 21 2.8 Bestimmung der DNA-Konzentration . . . . . . . . . . . . . . . . . . . . . 21 2.9 Restriktionsverdau von DNA . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.10 Ligation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.11 Herstellung von kompetenten Zellen (E.coli DH5ff, E. coli BL21) . . . . . 23 2.11.1 Chemisch kompetente Zellen nach der CaCl2-Methode (42) . . . . . 23 2.11.2 TOP10 chemischkompetente Zellen . . . . . . . . . . . . . . . . . . 23 2.12 Transformation nach der Hitzeschock-Methode (19) . . . . . . . . . . . . . 24 2.13 Plasmidpräparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 2.14 Bestimmung der Indigobildung durch Klone mit mutmaÿlicher SMO-Aktivität 24 2.14.1 Abschätzung der Indigobildung durch Augenschein . . . . . . . . . 25 2.14.2 Quanti zierung der Indigobildung mittels UV/Vis-Spektrophotometrie 25 2.14.3 Quanti zierung der Indigobildung aus Flüssigkulturen . . . . . . . . 26 3 Ergebnisse 27 3.1 Versuche der error prone PCR von StyA2B aus Rhodococcus opacus 1CP . 27 3.1.1 Isolation von Templat-DNA und Durchführung der error prone PCR 28 3.1.2 Screening von Transformanden auf Fähigkeit zur Indol-Oxidation . 29 3.1.3 Herstellung und Aktivitätsscreening von E. coli DH5ff pET_StyA2B 30 3.2 Versuche der error prone PCR von styA1/styA2B aus Rhodococcus opacus 1CP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 3.2.1 Durchführung der error prone PCR und Aktivitätsscreening von StyA1/StyA2B in pBluescript KS(+) . . . . . . . . . . . . . . . . . 31 3.2.2 Durchführung des Aktivitätsscreening von StyA1/StyA2B in pET16bP 32 3.3 Fusionsproteine StyAL1B und StyAL2B aus Pseudomonas uorescens ST . 33 3.3.1 Optimierung der Zusammensetzung des LB-Mediums für das Aktivitätsscreenings von pET_StyAL2B in E. coli BL21 nach einer Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 3.3.2 Ein uss der Belüftung auf die Neigung von E. coli BL21 (pET_StyAL2B) Kolonien zur Oxidation von Indol . . . . . . . . . . . . . . . . . . . 38 3.3.3 Bestimmung der Indigobildung mittels UV/Vis-Spektroskopie . . . 40 3.3.4 Zeitliche Entwicklung der Indigokonzentration einer Flüssigkultur von E. coli BL21 (pET_StyAL2B) . . . . . . . . . . . . . . . . . . 42 3.3.5 Error prone PCR von pET_StyAL2B mit Gene Morph II EZ Clone Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 3.3.6 Error prone PCR nach der klassischen Methode mit pET_StyAL1B und pET_StyAL2B . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 4 Diskussion der Ergebnisse 49 4.1 Die error prone PCR als attraktive Methodik zur Optimierung von Styrol- Monooxygenasen hinsichtlich katalytischer Eigenschaften . . . . . . . . . . 49 4.2 Der Aktivitätsnachweis als mutmaÿlich limitierender Schritt in der Modi- zierung von StyA2B und StyA1/StyA2B mit Hilfe der error prone PCR . 51 4.3 Die künstlich fusionierten Styrol-Monooxygenasen StyAL2B und StyAL1B erlauben ein Aktivitätsscreening auf Platte . . . . . . . . . . . . . . . . . . 53 4.4 Die Entwicklung einer Methodik zur Quanti zierung der spezi schen Indigobildung eines Expressionsklons der Styrol-Monooxygenase StyAL2B . . . 58 4.5 Fehleranalyse zur error prone PCR . . . . . . . . . . . . . . . . . . . . . . 59 4.5.1 Fehler in der klassischen error prone PCR für pET_StyAL1B und pET_StyAL2B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Literaturverzeichnis 65

Primary fatty acid amides (PFAMs) and N-acylglycines (NAGs) are important signaling molecules in the mammalian nervous system, binding to many drug receptors and demonstrating control over sleep, locomotor activity, angiogenesis, vasodilatation, gap junction communication, and many other processes. Oleamide is the best-studied of the PFAMs, while the in vivo activity of the others is largely unstudied. Even less is known about the NAGs, as their discovery as novel compounds is much more recent due to low endogenous levels. Herein is described extraction and quantification techniques for PFAMs and NAGs in cultured cells and media using solvent extraction combined with solid phase extraction (PFAM) or thin layer chromatography (NAG), followed by gas chromatography-mass spectroscopy to isolate and quantify these lipid metabolites. The assays were used to examine the endogenous amounts of a panel of PFAMs as well as the conversion of corresponding free fatty acids (FFAs) to PFAMs over time in several cell lines. The cell lines demonstrated the ability to convert all FFAs, including a non-natural FFA, and an ethanolamine to the corresponding PFAM. Different patterns of relative amounts of endogenous and FFA-derived PFAMs were observed in the cell lines tested. Essential to identifying therapeutic targets for the many disorders associated with PFAM signaling is understanding the mechanism(s) of PFAM and NAG biosynthesis. Enzyme expression studies were conducted to determine potential metabolic enzymes in the model cell lines in an attempt to understand the mechanism(s) of PFAM biosynthesis. It was found that two of the cell lines which show distinct metabolisms of PFAMs also demonstrate unique enzyme expression patterns, and candidate enzymes proposed to perform PFAM and NAG metabolism are described. RNAi knockdown studies revealed further information about the metabolism of PFAMs and calls into question the recently proposed involvement of cytochrome c. Isotopic labeling studies showed there are two pathways for PFAM formation. A novel enzyme is likely to be involved in formation of NAGs from acyl-CoA intermediates.

Biotechnological processes are distinguished from classical chemistry by employing bio-molecules or whole cells as the catalytic element, providing unique reaction mechanisms with unsurpassed specificity. Whole cells are the most versatile \'factories\' for natural or non-natural products, however, the conversion of e.g. hydrophobic substrates can quickly become cytotoxic. One host organism with the potential to handle such conditions is the gram-negative bacterium Pseudomonas putida, which distinguishes itself by solvent tolerance, metabolic flexibility, and genetic amenability. However, whole cell bioconversions are highly complex processes. A typical bottleneck compared to classical chemistry is lower yield and reproducibility owing to cell-to-cell variability. The intention of this work was therefore to characterize a model producer strain of P. putida KT2440 on the single cell level to identify non-productive or impaired subpopulations. Flow cytometry was used in this work to discriminate subpopulations regarding DNA content or productivity, and further mass spectrometry or digital PCR was employed to reveal differences in protein composition or plasmid copy number. Remarkably, productivity of the population was generally bimodally distributed comprising low and highly producing cells. When these two subpopulations were analyzed by mass spectrometry, only few metabolic changes but fundamental differences in stress related proteins were found. As the source for heterogeneity remained elusive, it was hypothesized that cell cycle state may be related to production capacity of the cells. However, subpopulations of one, two, or higher fold DNA content were virtually identical providing no clear hints for regulatory differences. On the quest for heterogeneity the loss of genetic information came into focus. A new work flow using digital PCR was created to determine the absolute number of DNA copies per cell and, finally, lack of expression could be attributed to loss of plasmid in non-producing cells. The average plasmid copy number was shown to be much lower than expected (1 instead of 10-20). In conclusion, this work established techniques for the quantification of proteins and DNA in sorted subpopulations, and by these means provided a highly detailed picture of heterogeneity in a microbial population.

A new Laser Induced Fluorescence Capillary Zone Electrophoresis (LIF-CZE) bioassay to detect and study the catalytic activity of the sulfur assimilating enzyme commonly found in E. coli species; alkanesulfonate monooxygenase (EC 1.14.14.5) is described for the first time. This technique enables the possibility for direct injection onto a capillary for detection without the need for pre-concentration of sample and with minimal sample preparative steps prior to analysis. In this bioassay, a group of Fischer based cyanine dyes and two Oxazine (Nile red) derivatives were designed for further optimization as key Vis-NIR fluorescent substrate. In developing this technique, the test dyes were first assessed for their photophysical properties, based on four criteria; (1) photostable (2) solvatochromism (3) binding affinity towards both the monooxygenase active site and serum albumin and (4) chemical stability in strong electric field strength. Applying key dye characterization procedures including; molar absorptivity determination, quantum yield determination, photostability, solvatochromism and protein interaction studies it was determined that the Fischer indolium cyanine dyes were most suitable for the method development. The data revealed that under the test conditions, reduced flavin, the oxidative monooxygenase catalytically specifically converts the alkylsulfonate substituted cyanine dyes to the corresponding aldehyde. This new bioassay has proven to be quick, portable, sensitive, reliable and the exhibit the possibility of ‘on-the-spot’ detection; advantages not readily realized with other commonly applied techniques such as PCR, SPR, ELISA and GC used to study bacterial sulfur assimilation processes. In addition, recent literature results proposed by other research groups developing similar techniques showed strong reliance on GC analyses. Those assays involve the use of low molecular weight straight chain non-emissive alkanesulfonate substrates. Once enzyme catalysis occurs the aldehyde is formed becomes rather volatile and requires complex and tedious headspace sampling for GC analyses. This feature limits the in vitro applicability and eliminated the possibility in vivo development. Our goal is to further develop, optimize and present this CZE based bioassay as a suitable alternative to the current trends in the field while creating a more robust and sensitive in vitro monooxygenase detection method with the possibilities of in vivo application.

Il suolo è un ambiente complesso che è alla base di molteplici servizi ecosistemici per la produzione agricola. Questa complessità è riflessa nella composizione e nelle funzioni degli organismi microbici coinvolti. Lo scopo di questa tesi è stato quello di esplorare le risposte della comunità microbica all’intervento dell’uomo in suoli agricoli. Lo studio ha coinvolto suoli di comune origine, modificatisi negli ultimi 6-700 anni a causa di differente uso e gestione agronomica del suolo. Per ottenere questo risultato, il DNA del suolo è stato analizzato con tecniche di sequenziamento avanzato. I risultati indicano che i suoli disturbati sono più diversi rispetto ai suoli naturali. I fattori che influenzano la comunità microbica sono il disturbo, la disponibilità di nutrienti e la dormienza microbica. In un altro esperimento, a carico di batteri ammonio ossidanti, si sono studiati gli effetti di diversi stress come l’umidità e la concentrazione di zinco nel suolo o la presenza di pesticidi nella lettiera. In tutte queste situazioni i batteri hanno mostrato una ridondanza e una variabilità che permettono di rispondere agli stress. In conclusione i risultati di questa tesi dimostrano che l’intervento umano è responsabile nel determinare la struttura e le funzioni della comunità procariotica dei suoli. / Soil is a complex environment comprising the basis for several ecosystem services, with many of them being connected to agricultural production. This complexity is reflected on the composition and functions of the hosted microbial life mainly responsible for the acquired services. Aim of the described studies was to explore microbial community responses to ecosystem services related human intervention in agricultural soils. Total prokaryotic diversity was studied in soils of common origin, which diverged in properties during the late 6-7 centuries due to different land use and management. For achieving this, related DNA markers were screened with high throughput sequencing. Cultivated environments had increased diversity compared to more natural soils. Factors potentially affecting the microbial community structure were: soil disturbance events; available nutrients; and microbial dormancy. In a second approach, ammonia oxidizing prokaryotes were used as biomarkers for studying stress effects caused by humidity and increased zinc concentrations and also the presence of organic pesticides in soil and litter respectively. In both referred cases the studied microbial guilds responded to the applied stresses showing strain or taxon level functional redundancy potentials, and tolerance variability. Overall, results show that human intervention is determining for the prokaryotic structure and functions in agricultural soils.

Alkane monooxygenases (encoded by the alkB gene) are a group of microbial enzymes that catalyze the first reaction of alkane degradation. Studies to determine the diversity and prevalence of alkB genotypes in the environment have focused on culturable organisms. The goal of this research was to use culture-independent methods (DGGE, clone library) to identify and characterize alkB genes, and to determine their prevalence in Arctic contaminated soils. General alkB PCR degenerate primers (alkB-Mc) were designed using the conserved nucleotide sequences of the Histidine I Box and Histidine III Box. General alkB-Mc and alkM (Acinetobacter spp. alkane monooxygenase genes) primers were used to screen the soils for the presence of alkane monooxygenase genotypes. A predominance of the Rhodococcus spp. alkB genotypes and the absence of alkM genotypes in these soils was found. alkB PCR fragments amplified from the soils were analyzed by DGGE (Denaturing Gradient Gel Electrophoresis). BlastN and blastX results of the DGGE bands sequences showed that they were similar to Rhodococcus spp. alkB genotypes (~80-90% DNA identity and ~80-90% amino acid homology). An alkB clone library was built using the general alkB-Mc primer set, screened by RFLP (Restriction Fragment Length Polymorphism) and characterized by sequencing of alkB clones. BlastN and blastX results of the alkB clone sequences showed the presence of divergent alkB genotypes (≤ 70% DNA identity and ≤ 67% of amino acid homology to data base sequences). The alignment of the clone-derived amino acid sequences to confirm functional alkane monooxygenase sequences revealed the presence of Histidine Box II and the HYG motif in all of the deduced amino acid clone sequences. These results indicate that the alkB sequences from the clone library represent novel alkB sequences. Both alkB DGGE and clone library techniques were independently able to identify alkB genotypes from High G+C microorganisms as predominant in the 1A03 soil sample. Nevertheless, only the clone library approach identified putative novel alkB sequences. Mineralization of hexadecane and naphthalene was clearly observed at subzero temperatures (-5ºC) in Arctic contaminated soils, proving that the indigenous microbial communities could mineralize these representative hydrocarbons at subzero temperatures in an environment that is predominantly frozen for most of the year.

Biotechnological processes are distinguished from classical chemistry by employing bio-molecules or whole cells as the catalytic element, providing unique reaction mechanisms with unsurpassed specificity. Whole cells are the most versatile \''factories\'' for natural or non-natural products, however, the conversion of e.g. hydrophobic substrates can quickly become cytotoxic. One host organism with the potential to handle such conditions is the gram-negative bacterium Pseudomonas putida, which distinguishes itself by solvent tolerance, metabolic flexibility, and genetic amenability. However, whole cell bioconversions are highly complex processes. A typical bottleneck compared to classical chemistry is lower yield and reproducibility owing to cell-to-cell variability. The intention of this work was therefore to characterize a model producer strain of P. putida KT2440 on the single cell level to identify non-productive or impaired subpopulations. Flow cytometry was used in this work to discriminate subpopulations regarding DNA content or productivity, and further mass spectrometry or digital PCR was employed to reveal differences in protein composition or plasmid copy number. Remarkably, productivity of the population was generally bimodally distributed comprising low and highly producing cells. When these two subpopulations were analyzed by mass spectrometry, only few metabolic changes but fundamental differences in stress related proteins were found. As the source for heterogeneity remained elusive, it was hypothesized that cell cycle state may be related to production capacity of the cells. However, subpopulations of one, two, or higher fold DNA content were virtually identical providing no clear hints for regulatory differences. On the quest for heterogeneity the loss of genetic information came into focus. A new work flow using digital PCR was created to determine the absolute number of DNA copies per cell and, finally, lack of expression could be attributed to loss of plasmid in non-producing cells. The average plasmid copy number was shown to be much lower than expected (1 instead of 10-20). In conclusion, this work established techniques for the quantification of proteins and DNA in sorted subpopulations, and by these means provided a highly detailed picture of heterogeneity in a microbial population.