Modifying the general stress response to improve yeast cell factories

Background

The yeast Saccharomyces cerevisiae is widely used as a microbial cell factory and especially known for the production of bread, beer and wine, but also the biofuel ethanol. It is also used and explored for a wide variety of other high- and low-added value compounds ranging from organic acids to pharmaceuticals. In such processes, most of the substrate should be converted into the product-of-interest and as little as possible in side-products including biomass. For this reason, we are interested in cultivating yeast in a non-growing but (highly) active, productive state.

Previous work has shown that under such non-growing conditions, yeast, and other microorganisms, activate a so-called general stress response. This general stress response can be triggered by a variety of stresses, and results, amongst others, in robustness and resistance against various stresses, not only the actually experienced stress. Why it is triggered in non-growing, nutrient-limited cells is not fully understood.

Hypothesis

Based on potential biological roles or origins, we formulated three hypotheses on the role of the GSR in non-growing cells:

1. The activation of the GSR is a purely evolutionary artifact. In nature, nutrient limitation often proceeds starvation and other stresses. Preparation to withstand these stresses offers an evolutionary advantage but must take place prior to exhaustion of nutrients.
2. The GSR has an energy-saving role. For example, heat-shock proteins can aid in refolding of misfolded, old proteins and thereby reduce the cost of protein turn-over. Similarly, proteins involved in ion-homeostasis and changes in membrane/cell wall composition can reduce the energy spend on maintaining ion-gradients.
3. The GSR serves to maintain an optimal protein density. Due to the reduced rates of biosynthesis, the levels of proteins involved therein, for example ribosomes, drop. To maintain an optimal protein density, important for reaction rates, cells should synthesize other proteins. This could be the main function of the GSR under non-growing conditions: to maintain protein density in an affordable way (many GSR-proteins are for example small) with a potential evolutionary benefit (hypothesis 1).

Aim

In this project, we set-out to test which hypothesis or a combination thereof may hold true. Based on this, we want to use the GSR to our advantage and increase heterologous protein production in non-growing cells.

Approach

For this we use a combination of techniques, of which the most important ones are:

• Advanced bioreactor cultivations, such as chemostats and retentostats to determine changes in substrate and energy usage.
• Synthetic biology tools, such as CRISPR-Cas, to generate mutants with reduced or increased stress responses or that express heterologous proteins.
• Cell biology analyses to assess the impact of mutations and cultivation conditions on cell survival, stress resistance and other aspects
• Omics analyses to gain deeper insight of intracellular changes in expression (transcript and protein levels) and metabolites to support findings at the macro-level.

For Master or Bachelor Thesis projects on this topic, you can contact PhD-candidate Nuran Temelli.