Automatisierte Schnellfiltration zur Untersuchung intrazellulärer Metabolite in Mikroorganismen

The analysis of intracellular metabolites in microorganisms requires fast and precise sampling and an effective quenching of the cells metabolism. To avoid leakage of intracellular metabolites during sampling while keeping the sampling time as short as possible, a method for automated fast filtration was developed in this work. With this leakage-free sampling method it was possible to reduce the time for sampling, filtration, washing of the filter cake and on-filter quenching of the cells with liquid nitrogen down to 6 seconds. This substantial reduction of sampling time was mainly due to the integration of the fast filtration system into an automated fast sampling system and thus an automation of the whole sampling procedure for fast filtration. The influence of cell density and the different growth phases during batch cultivation on the automated fast filtration were investigated. The comparison of automated fast filtration and sampling with cold methanol quenching led to 75 % higher intracellular amino acid concentrations in Escherichia coli with the automated fast filtration method. The application of the newly developed method for the investigation of the L-Lysine producion by Corynebacterium glutamicum clearly showed the influences of the genetic modifications in the producing strain on intracellular metabolite levels. It was shown, that the intracellular concentrations of all amino acids of the aspartate- and lysine- metabolism were increased in the production strain, while all other determined amino acids decreased. Furthermore it was shown, that the allosteric inhibition of the aspartate-kinase by lysine and threonine was reduced, but due to the elevated intracellular concentrations of the inhibitors it was still inhibiting the enzyme activity significantly, which shows the importance of further deregulation of the inhibition of this enzyme. The method for intracellular metabolite sampling developed in this work allows the detailed analysis of microbial metabolism and thus enables the targeted application of systems biology methods for strain and process development. In particular, the design of regulatory proteins and the dynamic regulation of metabolic pathways require precise information about intracellular metabolite concentrations in microorganisms.