Metabolic engineering of Corynebacterium glutamicum for the production of L-aspartate and its derivatives β-alanine and ectoine Dissertation zur Erlangung des akademischen Grades Doktor der Naturwissenschaften (Dr. rer. nat.) Fakultät für Biologie Universität Bielefeld vorgelegt von Christian Ziert aus Fulda Ebersberg, April 2014 Table of contents Table of figures .................................................................................................8 List of tables ......................................................................................................9 Abbreviations .................................................................................................. 10 Summary ......................................................................................................... 11 Zusammenfassung .......................................................................................... 12 1 Introduction .............................................................................................. 13 1.1 Characteristics of Corynebacterium glutamicum and its application in biotechnology ........... 13 1.2 The central metabolism of C. glutamicum ............................................................................... 14 1.2.1 Carbon source uptake ...................................................................................................... 16 1.2.2 Glycolysis .......................................................................................................................... 17 1.2.3 Pentose phosphate pathway ............................................................................................ 18 1.2.4 Anaplerosis ....................................................................................................................... 18 1.2.5 Tricarboxylic acid cycle ..................................................................................................... 19 1.3 Anaerobic conditions ................................................................................................................ 19 1.4 The amino acid aspartate ......................................................................................................... 20 1.4.1 Synthesis of aspartate under physiological conditions .................................................... 21 1.4.2 Synthesis of amino acids of the aspartate family ............................................................. 23 1.4.3 Export of amino acids ....................................................................................................... 24 1.5 The non proteinogenic amino acid β-alanine........................................................................... 24 1.6 Ectoine ...................................................................................................................................... 25 1.7 Aims of this work ...................................................................................................................... 27 2 Material and Methods............................................................................... 29 2.1 Chemicals.................................................................................................................................. 29 2.2 Bacterial strains ........................................................................................................................ 29 2.3 Plasmids and Oligonucleotides ................................................................................................. 30 2 2.4 Cultivation and conservation of strains .................................................................................... 34 2.4.1 E. coli strains ..................................................................................................................... 34 2.4.2 C. glutamicum strains ....................................................................................................... 34 2.4.2.1 Anaerobic cultivation of C. glutamicum strains ........................................................... 35 2.4.2.2 Cultivation of C. glutamicum in the Biolector .............................................................. 35 2.4.3 Conservation of strains ..................................................................................................... 36 2.4.4 Determination of cell dry weight ..................................................................................... 36 2.5 Generation of competent cells ................................................................................................. 36 2.5.1 E. coli DH5α and S-17 cells ............................................................................................... 36 2.5.2 C. glutamicum cells ........................................................................................................... 36 2.6 Transformation of bacterial cells.............................................................................................. 37 2.6.1 E. coli DH5α and S-17 cells ............................................................................................... 37 2.6.2 C. glutamicum cells ........................................................................................................... 37 2.6.3 Conjugation ( E. coli S-17/ C. glutamicum ) ......................................................................... 37 2.7 Generation of deletion strains ................................................................................................. 38 2.8 Isolation and purification of nucleic acids ................................................................................ 38 2.8.1 Isolation of genomic DNA ................................................................................................. 38 2.8.2 Isolation of plasmid DNA .................................................................................................. 39 2.8.3 Isolation of total RNA ....................................................................................................... 39 2.8.4 Purification of DNA ........................................................................................................... 39 2.8.5 Determination of nucleic acid concentrations ................................................................. 39 2.9 Polymerase chain reaction (PCR) methods .............................................................................. 39 2.9.1 PCR .................................................................................................................................... 39 2.9.2 Rapid amplification of cDNA ends with PCR (RACE-PCR) ................................................. 40 2.9.3 Agarose gel electrophoresis ............................................................................................. 40 2.10 Restriction, modification and ligation of DNA .......................................................................... 40 2.10.1 Restriction ........................................................................................................................ 40 2.10.2 Dephosphorylation of vector DNA ................................................................................... 41 3 2.10.3 Phosphorylation of nucleic acids ...................................................................................... 41 2.10.4 Ligation ............................................................................................................................. 41 2.11 Protein-biochemical methods .................................................................................................. 41 2.11.1 Preparation of crude protein extracts .............................................................................. 41 2.11.2 Determination of protein concentration according to Bradford ..................................... 41 2.12 Enzymatic assays ...................................................................................................................... 42 2.12.1 Aspartate aminotransferase (AspB) activity assay ........................................................... 42 2.12.2 Aspartate ammonium lyase (AspA) activity assay ............................................................ 42 2.12.3 Aspartate decarboxylase (PanD) activity assay ................................................................ 42 2.12.4 Aspartate dehydrogenase (AspDH) activity assay ............................................................ 43 2.12.5 Chloramphenicol acetyl-CoA (CAT) transferase assay ..................................................... 43 2.12.6 Diaminobutyrate acetylase (EctA) assay .......................................................................... 43 2.12.7 Diaminobutyrate transaminase (EctB) assay .................................................................... 44 2.12.8 Glyceraldehyde-3-phosphate dehydrogenase (GapA) assay ........................................... 44 2.12.9 Malate dehydrogenase (Mdh) activity assay ................................................................... 44 2.13 Quantification of amino acids and other organic acids............................................................ 44 2.13.1 Determination of pyruvate in culture supernatants ........................................................ 44 2.13.2 High performance liquid chromatography (HPLC) ........................................................... 45 3 Results ...................................................................................................... 47 3.1 Production of aspartate under