Metabolic modelling and 13C flux analysis. Application to biotechnologically important yeasts and a fungus [Aineenvaihdunnan mallinnus ja 13C-vuoanalyysi. Sovellukset bioteknologisesti tärkeisiin hiivoihin ja homeeseen]
نویسنده
چکیده
All bioconversions in cells derive from metabolism. Microbial metabolisms contain potential for bioconversions from simple source molecules to unlimited number of biochemicals and for degradation of even detrimental compounds. Metabolic fluxes are rates of consumption and production of compounds in metabolic reactions. Fluxes emerge as an ultimate phenotype of an organism from an integrated regulatory function of the underlying networks of complex and dynamic biochemical interactions. Since the fluxes are time-dependent, they have to be inferred from other, measurable, quantities by modelling and computational analysis. C-labelling is crucial for quantitative analysis of fluxes through intracellular alternative pathways. Local flux ratio analysis utilises uniform C-labelling experiments, where the carbon source contains a fraction of uniformly C-labelled molecules. Carbon-carbon bonds are cleaved and formed in metabolic reactions depending on the in vivo fluxes. C-labelling patterns of metabolites or macromolecule components can be detected by mass spectrometry (MS) or nuclear magnetic resonance (NMR) spectroscopy. Local flux ratio analysis utilises directly the C-labelling data and metabolic network models to solve ratios of converging fluxes. In this thesis the local flux ratio analysis has been extended and applied to analysis of phenotypes of biotechnologically important yeasts Saccharomyces cerevisiae and Pichia pastoris, and a fungus Trichoderma reesei. Oxygen dependence of in vivo net flux distribution of S. cerevisiae was quantified by using local flux ratios as additional constraints to the stoichiometric model of the central carbon metabolism. The distribution of fluxes in the pyruvate branching point turned out to be most responsive to different oxygen availabilities. The distribution of fluxes was observed to vary not only between the fully respiratory, respiro-fermentative and fermentative metabolic states but also between different respiro-fermentative states. The local flux ratio analysis was extended
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