Globale und lokale Untersuchung des Stoffwechsels von Gluconobacter oxydans mit Hilfe von Isotopen

The present work uses Substrates with isotope enrichment to answer fundamental questions of the metabolism of Gluconobacter oxydans. In a first step the growth was charcterized under usage of different cultivation Media and Substrates and a stable continuous cultivation was established. The focus was set on Glucose since its degradation occurs on a high level above and on a low levelbelow a benchmark concentraion of approximated 10 mM. The ration of cytoplasmicglucose and gluconate uptake stays constant at 1,8:1, independent of concentration and growth phase. Under usage of different substrates and deletion mutant strains, a high throughput method for elucidating the split ration of 2 metabolic pathways was established. With availability of glucose its carbon gets metabolised primary via the Entner-Doudoroff pathway, while depletion of glucose results in a upregualtion of the Pentose-Phosphate pathway. This observion fits to the enhancement of energetic positive reactions once the extracellular glucose is depleted completely. For the first time and for 2 different growth media the metabolom of Gluconobacteroxydans was quantified. Thereby an intracellular an accumulation of pyruvate and succinate could be observed. Further more an up to now unknown direct metabolic pathway connection between glycolysis and succinate was elucidated. Last, some hints were found for an in reductive direction active citric acid cycle. Due to the co-metabolisation of media components, low biomass yield and thus problems with the analysis Gluconobacter oxydans is not suitable for the global methodof 13C metabolic flux analysis. As a result differnt approaches were developed to use isotopes for elucidating specific metabolic hotspots. There it could be shown that 13C isotopes have no impact on the growth rate, while high concentration of 2H2O strongly reduce cellular activities. Finally a new internal standard enriched with high amounts of 13C, 15N and 2H isotopes was created. With this standard it is possible for the fist time to quantify directly the mass isotopomer concentration of metabolites