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|Language of final thesis:||English|
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|Title of the thesis:||Transport processes and metabolism of filamentous fungi|
|Summary:||Transport and metabolism in filamentous fungi is a very complex process. We have shown that transport of succinate and fumarate into A. nidulans is inducible not only by their presence in the medium but also by acetate. There have been differences in sensitivity of the transport to the proton uncoupler and the inhibitor of respiratory chain. Nutrient limitation causes variable excretion of organic acids. T. atroviride excretes mainly citrate and on nitrogen-, phosphorus- and sulfur-free media there is a delay in its excretion. P. ochrochloron excretes citrate only on complete medium but succinate and malate appeared also in limiting media. Fumarate was detected in most media but its concentration was too low to contribute significantly to the overall carbon balance. Although there is some difference in the excretion of organic acids, the primary reason for their efflux seems to be overflow metabolism as a mechanism for survival. Presence of inhibitory concentrations of 8-carbon oxylipins causes shift in metabolite concentrations toward anaerobic metabolism and GABA shunt. We speculate that at lower oxylipin concentration this may be the mechanism for conidiation induction. Under anaerobic conditions T. atroviride does not grow nor does it use fumarate for respiration. However, it can survive months in such environment, indicating a shift to a dormant state with low or no metabolic turnover. When there is a slow consumption of oxygen i.e. under hypoxic conditions small amounts of ethanol are formed that can be detected even after lyophilization because of complexes similar in function to cyclodextrins formed during medium autoclaving. Intracellular metabolism during germination is mainly characterized by the increase in amino acids concentrations. Choline and betaine increase seem to be indicative of membrane biosynthesis. Decrease in ethanol and lactate support published transcriptomic data that show a transfer from glycolysis to oxidative metabolism during germination, even though TCA intermediates did not increase during our study. Fungus probably keeps their concentrations in a steady state during the flow of nutrients into anabolic metabolites. The growth and aging of T. atroviride in batch culture showed changes in intracellular metabolites that were dependent on nutrient availability in the medium. Mannitol seems to serve as a carbon storage compound and decreases in later stages of cultivation in order to preserve viability. From the data we hypothesize that autophagy is one of the mechanisms for fungal survival because after nutrient depletion a second increase in many metabolites and fungal biomass was seen. Level of GABA copies to a good approximation the GAD activity observed earlier but its role in the metabolism cannot be ascertained from obtained results. The delta gad mutant besides absence of GABA does not show the influence of the gene disruption on the intracellular metabolites, questioning its role even more, but a search for its receptor and the research on involvement of calcineurin signaling pathway might shed more light in the future. Comparison to S. cerevisiae showed that this yeast does not form GABA and utilizes it mainly for catabolic purposes under physiological conditions. During conidiation induced by light, calcium and injury an increase in glutamate was the most prominent feature. GABA levels, contrary to our expectations, decreased, despite the increase in GAD activity observed after illumination of the mycelium. A transient increase of some amino acids has been observed that could indicate a preparatory phase for conidiation itself. Metabolism of 13C glucose showed surprisingly few metabolites in NMR spectra. Mannitol, glutamate, glutamine and alanine changes were in agreement with known metabolic pathways and the changes observed during growth and ageing. The involvement of phosphoenolpyruvate carboxykinase was implied from the label incorporation into position 5 of glutamate. Contrary to other metabolites, glutamine was shown to increase steadily over time hinting on its role in ammonium storage or detoxification. Since these were first experiments utilizing 13C labelled nutrients in our laboratory, future improvements in protocols and data processing show promise for more information-rich data including the elucidation of metabolic flows. Finally, a critical assessment of NMR-based metabolomics was presented. Despite quite a few limitations and drawbacks, it was shown to be an indispensable method, complementary to other metabolomic procedures.|
|Key words:||filamentous fungi, transport, metabolism|
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