Metabolische 13 C-Stoffflussanalyse : vom isotopisch stationären zum instationären Fall

With the established tools of 13C-Metabolic Flux Analysis (MFA) up to now it is not possible to analyse the metabolism of microorganisms in biotechnological production processes because of the long labelling time. Due to the proceeding development in mass spectrometry it
is now possible to measure the 13C-labelling directly in the intracellular metabolites of the central metabolism (glycolysis, pentose-phosphate-pathway and citric acid cycle). Here it is assumed that an isotopic steady state will be reached in a much shorter time. Main objective of this work was to use this data and reduce the labelling time to make it possible to analyse the cell metabolism in production processes that are mainly batch and fed batch processes.
In the first part of this work the isotopic stationary MFA based on the labelling data of intra cellular metabolites will be investigated for an E. coli K12 wildtype and a production strain. By adding 13C-labelled glucose at the beginning of the cultivation and labelling for several hours in a batch experiment comparable results with literature data are found for the wildtype. For investigation of metabolic changes in the production process of (3R,4R)-Di-hydroxy-3,4-di-hydrobenzoic acid the cells were labelled for different time periods of the fed batch process by using the sensor reactor (El Massaoudi 2004). The MFA showed big differences between the measured and fitted labelling data. By expansion of the metabolic model with unlabelled fluxes into the metabolic network the calculated data matched much better and biological meaningful metabolic fluxes are calculated. Based on the comparison with optimal fluxes (Flux Balance Analysis) potentials for genetic optimization are presented. In the second part of this work the use of isotopic instationary data for MFA is investigated. The development of a fast sampling device for mapping the labelling enrichment is presented. Different methods for metabolite extraction described in literature are tested and the results are discussed. Two labelling experiments were performed during the cultivation of the E. coli K12 wildtype and the labelling enrichment and the metabolite pool sizes of the central metabolism were measured. As expected in glycolysis an isotopic steady state is reached after a few seconds. In the citric acid cycle the labelling enrichment is much slower. As a possible reason for that a reflux of unlabelled metabolites from large pools outside of the central metabolism is discussed. After incorporation of unlabelled refluxes into the metabolic network model a good fitting of the labeling enrichment is reached. Several model variants were analyzed and are discussed here. In comparison with the isotopic stationary MFA the standard deviation of the calculated fluxes is generally lower. Particularly the anaplerotic fluxes and the exchange fluxes are better determined. In this work it is in principle shown that with the isotopic instationary MFA the metabolic fluxes of the central metabolism can be determined with a good reliability based on the data of a very short labelling experiment with a few seconds duration. The potential of this new method and the necessary further developments are discussed.