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Exploring the Biosynthetic Pathways of Glutamate and Benzoate in Syntrophus Aciditrophicus

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Exploring the Biosynthetic Pathways of Glutamate and Benzoate in Syntrophus Aciditrophicus Exploring the biosynthetic pathways of glutamate and benzoate in Syntrophus aciditrophicus Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Biologie der Philipps-Universität Marburg Vorgelegt von Marie Kim aus Daegu, Republic of Korea Marburg/Lahn, Germany 2011 Die Untersuchungen zur vorliegenden Arbeit wurden von September 2007 bis Juni 2011 im Laboratorium für Mikrobiologie, Fachbereich Biologie, der Philipps-Universität Marburg unter der Leitung von Prof. Dr. W. Buckel durchgeführt. Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation am 06. 07. 2011 angenommen. Erstgutachter: Prof. Dr. Wolfgang Buckel Zweitgutachter: Prof. Dr. Johann Heider Tag der mündlichen Prüfung am: 07. 07. 2011 Contents Abbreviations ......................................................................................................................................... 5 Zusammenfassung ................................................................................................................................. 6 Summary ................................................................................................................................................ 7 Introduction ........................................................................................................................................... 8 1. Anaerobic food chain and syntrophic metabolism .............................................................................. 8 1.1 Anaerobic food chain ................................................................................................................ 8 1.2 Syntrophism and the global carbon cycle .............................................................................. 9 1.3 Syntrophus aciditrophicus SB ............................................................................................. 10 2. Fermentation and biosynthesis of glutamate ..................................................................................... 13 2.1 Fermentation of glutamate ................................................................................................... 13 2.2 2-Hydroxyglutarate pathway ............................................................................................... 14 2.3 Biosynthesis of glutamate and the TCA cycle..................................................................... 17 3. Mechanism of citrate synthase and its stereospecificity .................................................................... 19 4. Energy conservation and glutaconyl-CoA decarboxylase ................................................................. 20 4.1 Energy conservation via electrochemical ion gradients ...................................................... 20 4.2 Sodium ion-translocating decarboxylases ........................................................................... 21 4.3 Glutaconyl-CoA decarboxylases ......................................................................................... 22 5. Proposed pathways of glutamate and benzoate biosyntheses ............................................................ 27 5.1 Glutamate biosynthesis via glutaconyl-CoA ....................................................................... 27 5.2 Glutamate biosynthesis via the TCA cycle .......................................................................... 27 5.3 Benzoate biosynthesis by glutaconyl-CoA decarboxylase .................................................. 28 6. Aims of the work ............................................................................................................................... 29 Materials and Methods ....................................................................................................................... 30 1. Materials ............................................................................................................................................ 30 1.1 Chemicals and Reagents ...................................................................................................... 30 1.1.1 Acetyl-CoA synthesis ................................................................................................... 30 1.1.2 Glutaconyl-CoA synthesis ............................................................................................ 30 1.1.3 Preparation of NMR samples ....................................................................................... 31 1.1.4 Carbon isotope labeled compounds .............................................................................. 32 1.2 Instruments and columns ..................................................................................................... 32 1.3 Anaerobic work ................................................................................................................... 32 1.4 Bacteria and culture media .................................................................................................. 32 1.4.1 Syntrophus aciditrophicus SB ...................................................................................... 32 1.4.2 Escherichia coli ............................................................................................................ 35 1 1.5 Plasmids............................................................................................................................... 35 1.6 Antibiotics ........................................................................................................................... 36 2. Methods for DNA work .................................................................................................................... 36 2.1 Genomic DNA isolation from S. aciditrophicus SB ........................................................... 36 2.2 Plasmid DNA isolation ........................................................................................................ 36 2.3 DNA agarose gel electrophoresis ........................................................................................ 37 2.4 Elution of DNA fragments from agarose gel....................................................................... 37 2.5 DNA restriction and ligation ............................................................................................... 38 2.6 Dialysis of ligation mixtures ............................................................................................... 38 2.7 Preparation of competent E. coli cells for electrotransformation ........................................ 38 2.8 Electrotransformation .......................................................................................................... 38 2.9 Chemical transformation ..................................................................................................... 39 2.10 DNA concentration and purity determination ................................................................... 39 2.11 PCR reactions .................................................................................................................... 39 2.12 PCR primers ...................................................................................................................... 40 2.13 Cloning of the genes .......................................................................................................... 41 2.14 Sequencing of the cloned genes ......................................................................................... 41 3. Methods for protein work .................................................................................................................. 42 3.1 Gene expressions ................................................................................................................. 42 3.1.1 Expression in E. coli of the genes encoding Re-citrate synthase .................................. 42 3.1.2 Gene expression in E. coli of the genes encoding glutaconyl-CoA decarboxylase ...... 42 3.2 Protein purification .............................................................................................................. 43 3.2.1 Methods of cell disruption ............................................................................................ 43 3.2.2 Determination of protein concentration ........................................................................ 43 3.2.3 Polyacrylamide gel electrophoresis (PAGE) ................................................................ 44 3.2.4 Preparation of soluble membrane protein ..................................................................... 45 3.2.5 Purification of recombinant Re-citrate synthase from S. aciditrophicus ...................... 46 3.2.6 Purification of glutaconyl-CoA decarboxylase from S. aciditrophicus ........................ 46 3.2.7 Purification of the subunits of recombinant glutaconyl-CoA decarboxylase from ......... S. aciditrophicus .................................................................................................................... 47 3.2.8 Partial purification of recombinant glutaconate CoA-transferase from A. fermentans 47 3.2.9 Gel filtration ................................................................................................................. 48 3.3 N-terminal amino acid sequence analysis ........................................................................... 48 3.4 MALDI-TOF mass spectrometry ........................................................................................ 48 3.5 Chemical labeling studies ...................................................................................................
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