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 ...................................................................................................