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| Dissertation / PhD Thesis/Book | PreJuSER-33319 |
2002
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/115
Report No.: Juel-3971
Abstract: The rational improvement of the productivity of cellular systems is one of the main goals of modern biotechnology and may be accomplished in two ways. One approach is the optimization of the production process ("bioprocess engineering"). Another possibility is the improvement of the cellular system itself by manipulatiog the metabolic pathways ("metabolic engineering") . A precise manipulation of microbial metabolism, and thereby a rational strain development, is possible today by using the tools of molecular biology. However, a prerequisite of this manipulation is a detailed and quantitative knowledge of the dynamics and regulation of metabolic fluxes in microbial metabolism. In this work a dynamic method is used for the quantification of microbial metabolism. The microorganism is cultivated in a substrate limited steady state and then rapidly shifted away from this metabolic equilibrium by application of a substrate pulse ("pulse experiment"). Samples are taken using a rapid sampling and quenching technique, which was developed in earlier works. The metabolites are then extracted and analyzed to determine the metabolic response ofthe microorganism. Using a novel system for injecting substrate pulses, a number of dynamic pulse experiments were performed. Intracellular metabolite dynamics were quantified using newly developed analytical techniques based an HPLC-MS, UV-HPLC, CE and enzymatic assays. . Pulse experiments were carried out to investigate the glucose uptake and utilisation via the PEP:PTS in $\textit{Escherichia coli}$ K12. The scope of these investigations was expanded using a genetically modified, PTS-deficient strain $\textit{E. coli}$ 3pMK7 in order to determine the metabolic changes taking place when the Substrate uptake depends an ATP instead of PEP. Additionally, the utilisation of glycerol by $\textit{E. coli}$ K12 and the dependency of glycerol uptake an ATP was investigated. In a further experiment, the switch from glycerol to glucose as carbon source during a pulse experiment showed a strong limitation of the available PEP concentration during uptake. The time courses of intracellular metabolite concentrations from these pulse experiments are the basis for the development of kinetic (structured) metabolic models. The modelling of the experimental data allowed a quantification of metabolic phenomena which up until now could only be explained phenomenologically.
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