Characterising promoter heterogeneity for the production of novel wound healing proteins in S. cerevisiae

Abstract

In the last decade, we have seen a boom in the use of genetic modification toolkits for microorganisms, allowing us to expedite the manufacturing process for high-value products. Although toolkits are fast and easy to use, cell-to-cell heterogeneity, between cells of the same population, means that there is competition for resources between high and low-producers, so yield decreases. As a result, strains should be constructed in a way that reduces the formation of unwanted subpopulations. In this thesis, I provide an extensive characterisation of promoter heterogeneity in the Saccharomyces cerevisiae Toolkit and identified which promoters lead to the formation of subpopulations. I explore the effects of cell cycle arrest on subpopulation dynamics and demonstrate that heterogeneous promoters are detrimental for bioproduction during metabolic engineering. I then apply this knowledge to generate strains producing novel wound healing proteins to treat chronic wounds and test their effectiveness in vitro. In the process, I discover new secretion signals which can be applied for downstream bioprocessing in industrial settings. Altogether, the promoter characterisations and explorations of novel secretion signals presented here lay the groundwork for boosting the production of high-value metabolites and therapeutic proteins in yeast.