Microbial metabolism is strongly dependent on the environmental conditions. While these can be well controlled under laboratory conditions, large-scale bioreactors are characterized by inhomogeneities and consequently dynamic conditions for the organisms. How Saccharomyces cerevisiae response to frequent perturbations in industrial bioreactors is still not understood mechanistically. To study the adjustments to prolonged dynamic conditions, we used published repeated substrate perturbation regime experimental data, extended it with proteomic measurements and used both for modelling approaches. Multiple types of data were combined; including quantitative metabolome, ¹³C enrichment and flux quantification data. Kinetic metabolic modelling was applied to study the relevant intracellular metabolic response dynamics. An existing model of yeast central carbon metabolism was extended, and different subsets of enzymatic kinetic constants were estimated. A novel parameter estimation pipeline based on combinatorial enzyme selection supplemented by regularization was developed to identify and predict the minimum enzyme and parameter adjustments from steady-state to dynamic substrate conditions. This approach predicted proteomic changes in hexose transport and phosphorylation reactions, which were additionally confirmed by proteome measurements. Nevertheless, the modelling also hints at a yet unknown kinetic or regulation phenomenon. Some intracellular fluxes could not be reproduced by mechanistic rate laws, including hexose transport and intracellular trehalase activity during substrate perturbation cycles.

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Saccharomyces cerevisiae, also known as baker’s yeast, is a robust microorganism frequently used in industrial biotechnology. The scale of its applications ranges from several millilitres for research and process development in the lab to hundreds of cubic meters for cultivation in industrial production processes. In large-scale reactors mixing limitations inherently lead to physiochemical gradients in substrate and oxygen concentrations, pH or temperature. Such inhomogeneous environment in production processes can cause a reduced yield or titer compared to the small-scale development processes. Such scale performance differences can lead to significant worse process economics and increase costs and development time. The scope of this thesis is to study and understand the regulation of Saccharomyces cerevisiae metabolism under dynamic substrate conditions, using both experimental and modelling approaches.@en

Exposed to changes in their environment, microorganisms will adapt their phenotype, including metabolism, to ensure survival. To understand the adaptation principles, resource allocation-based approaches were successfully applied to predict an optimal proteome allocation under (quasi) steady-state conditions. Nevertheless, for a general, dynamic environment, enzyme kinetics will have to be taken into account which was not included in the linear resource allocation models. To this end, a resource-dependent kinetic model was developed and applied to the model organism Saccharomyces cerevisiae by combining published kinetic models and calibrating the model parameters to published proteomics and fluxomics datasets. Using this approach, we were able to predict specific proteomes at different dilution rates under chemostat conditions. Interestingly, the approach suggests that the occurrence of aerobic fermentation (Crabtree effect) in S. cerevisiae is not caused by space limitation in the total proteome but rather an effect of constraints on the mitochondria. When exposing the approach to repetitive, dynamic substrate conditions, the proteome space was allocated differently. Less space was predicted to be available for non-essential enzymes (reserve space). This could indicate that the perceived “overcapacity” present in experimentally measured proteomes may very likely serve a purpose in increasing the robustness of a cell to dynamic conditions, especially an increase of proteome space for the growth reaction as well as of the trehalose cycle that was shown to be essential in providing robustness upon stronger substrate perturbations. The model predictions of proteome adaptation to dynamic conditions were additionally evaluated against respective experimentally measured proteomes, which highlighted the model’s ability to accurately predict major proteome adaptation trends. This proof of principle for the approach can be extended to production organisms and applied for both understanding metabolic adaptation and improving industrial process design.

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Redox metabolism plays an essential role in the central metabolic network of all living cells, connecting, but at the same time separating, catabolic and anabolic pathways. Redox metabolism is inherently linked to the excretion of overflow metabolites. Overflow metabolism allows for higher substrate uptake rates, potentially outcompeting other microorganisms for the same substrate. Within dynamically changing environments, overflow metabolism can act as storage mechanism, as is shown in many recently described processes. However, for complete understanding of these mechanisms, the intracellular state of the metabolism must be elucidated. In recent years, progress has been made in the field of metabolomics to improve the accuracy and precision of measurements of intracellular and intercompartmental metabolites. This article highlights several of these recent advances, with focus on redox cofactor measurements, both fluorescence and mass spectrometry based.

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Natural habitats of microorganisms are dynamic environments with non-continuous supply of carbon and energy sources, in which intermediate storage of substrates can increase competitiveness. Plasticicumulans acidivorans are polyhydroxybutyrate (PHB) accumulating bacteria enriched from activated sludge using carbon feast-famine cycles as selective pressure. Despite growing slowly, P. acidivorans outcompetes other bacteria by quickly taking up acetate and storing it intracellularly as PHB to later use it for growth. As soon as acetate is depleted, these bacteria immediately ‘switch’ their metabolism from PHB production to consumption entailing a very interesting regulatory challenge as parallel activity could lead to significant losses (futile cycling). While the stoichiometry for both feast and famine phases has been extensively described in literature, the switch regulation is not yet fully understood. To elucidate the responsible regulatory processes, an enrichment of P. acidivorans was studied using targeted intracellular metabolite analysis over time, with emphasis on the feast to famine switch. In combination with extracellular rates, the measured intracellular metabolite pools are used to design a labelling experiment to obtain actual intracellular fluxes (dynamic 13C flux analysis). Here the challenge is to create an isotopically non-stationary state (usually mediated by changing the substrate’s isotopic composition) to study the metabolic response in the transition from presence-to-absence of substrate.In this way, we aim to unravel the responsible regulatory mechanism governing the metabolic switch from storage-to-consumption and use this knowledge not only to understand its ecological relevance, but to also propose novel metabolic strategies for microbial cell factory design.@en