The title of the first project is ‘Dynamics in the central plant cell metabolism’.

This research theme will focus on characterizing the fast dynamic metabolic changes occurring in tomato (Solanum lycopersicum L.) plant cells/tissue exposed to rapidly changing conditions. This information is crucial to developing metabolic pathway models for the central metabolism of the tomato plant cell. Steady state approaches can be used to quantify fluxes through metabolic pathways. However, to identify the actual regulation and control loops behind these pathways, data from dynamic experiments are required.

When using microorganisms, interpretation of the experimental data is facilitated by the relative low degree of organization and differentiation within the unicellular organism. Within plants, cell differentiation results in different cell types, each having their own function with different parts of the metabolism preferentially expressed. These cell types are arranged in 3D tissue structures with varying levels of communication between the individual cells. Within eukaryotic cells (like plant,fungal and yeast cells), differentiation has further resulted in a certain level of compartmentalization creating functional units that take their own role within the overall cell metabolism while in prokaryotic cells (like bacterial cells) this compartmentalization is non-existing. When going in plants from intact organs to pieces of tissue to isolated cells the barrier between environment and cell can be largely removed allowing for a more direct interaction between experimental conditions and the cellular metabolism. When working on isolated plant cell systems, experiments can be designed to rapidly induce changes in the cellular metabolism by feeding experiments.

By performing these feeding experiments with 13C labeled substrates the labeled carbon atoms are distributed all over the metabolic network until the isotopic enrichment in the intracellular metabolite pools can be measured by NMR or MS instruments. Since the early 90’s, the method has strongly evolved by the introduction of new experimental procedures, measurement techniques, and mathematical data evaluation methods. Many of these improvements require qdvanced skills in the application of experimental techniques, such as mass spectrometry techniques, on the one hand, and computational and statistical experience, on the other hand. The experimental task of identifying the different metabolites from complex biological samples has become more feasible over the years with the development of extended libraries. These isotope labeling techniques can be both used for steady state flux approximations and to follow small dynamic changes over time. For the latter, rapid sampling techniques need to be developed. In this way, the central metabolism will be studied collecting data capturing the rapid responses to changes in external gas conditions and various substrates, focusing on glycolysis and Krebs cycle.

Tasks in this research theme are: (i) experiment design, (ii) developing technical requirements like rapid sampling techniques, (iii) performing experiments to measure the response to step changes in various conditions (gas composition, type and concentration of substrates), (iv) performing experiments to measure the response to dynamic changes in various conditions (gas composition, type and concentration of substrates-.

The title of the second project is ‘Dynamics in the central metabolism of Streptomyces lividans producing heterologous proteins’.

This research focuses on the development of dynamic metabolic model for heterologous protein production by Streptomyces lividans consistent with cell metabolism but with manageable complexity to enable model-based optimization and control of the production process in bioreactors. Data and insights generated by metabolic flux analysis under steady state and dynamic conditions are exploited for model development but are also useful for improved strain engineering.

The potential of Streptomyces as an industrial host for production of heterologous proteins has been proven in the past decade. Heterologous genes are usually linked to signal peptides of strongly expressed/secreted endogenous Streptomyces proteins. Signal peptides act as address tags specifically recognizing the Sec-translocase positioned on the cell membrane. Within the secdependent secretion system, SecA (a precursor protein stimulated ATPase) takes a central role being the molecular motor driving translocation. Given that the central metabolism is the most important source of energy within a cell, a proper balancing of the energy flux towards growth and maintenance processes on the one hand, and towards protein secretion on the other hand, plays a crucial role in optimization of protein production. In this project, two target proteins are explored, namely, mouse Tumor Necrosis Factor alpha (mTNF ) and Jonesia sp. Xyloglucanase (Xeg). By studying two proteins, we can identify generic next to protein-specific phenomena.

In this project, we want to unravel the impact of the bioreactor environment on the central metabolism and protein production properties of S. lividans by means of metabolic flux analysis. Knowledge will gradually be built up by analyzing metabolic fluxes under steady state and subsequently under dynamic conditions. As such, the impact of protein synthesis and secretion on the carbon and energy metabolism and on the flux dynamics under changing process conditions can be deduced. As a start, the published metabolic reaction network of S. coelicolor is verified for S. lividans in well-designed 13C-labelling experiments. Based on the 13C-labelling distribution resulting from the consumption of labelled substrate, the intracellular fluxes and topology of the reaction network (in
particular the central metabolism) can be deduced. This knowledge will be further used in MFA based on the reaction stoichiometry and net conversion rates. Various limiting conditions (e.g., C, N, O) will be tested under steady state conditions. Conditions with significant impact on the metabolic balance between biomass formation and protein production are further investigated under
dynamic conditions. Dynamic experiments include, on the one hand, steady state experiments with various shifts in dilution rates (which results in shifts in the metabolic potential of the cell) and, on the other hand, fed-batch experiments during which the transition between substrate excess (feast)and limitation (famine) can be studied. The dynamics of the intracellular metabolites will be followed during transition zones by frequent sampling. Only metabolic dynamics with a direct impact on protein levels will be relevant. In all stages, techniques of optimal experimental design will be used to optimally design these experiments to guarantee identifiability and accurate flux estimation.

Tasks in this research theme are: (i) experiment design, (ii) developing technical requirements like rapid sampling techniques, (iii) performing dynamic steady state experiments to measure the metabolic response to various dilution rates, (iv) performing fed-batch experiments to measure the response to step changes in type and concentration of substrates.

These positions are targeted at candidates from non-EEA countries (European Economic Area = European Union + Iceland, Liechtenstein and Norway) Applications are invited from enthusiastic graduates with an excellent study track record. We offer a challenging research environment and an intense experience leading to a PhD degree.

To apply:

Apply via our website:

www.set.kuleuven.be/phd