Use of the model yeast Saccharomyces cerevisiae to study the regulation of RNA processing enzyme, Rex1, and as a chassis for the development of biotechnological hosts

Saccharomyces cerevisiae (S. cerevisiae) is the most extensively studied eukaryote. It has been substantially used in research to elucidate many molecular mechanisms and as a host for heterologous genes. In this study, we have used S. cerevisiae as a model for studying the regulation of the rRNA processing enzyme, Rex1, in response to stress. Simultaneously we have explored the potential of using S. cerevisiae as a host for expression of a fungal glycolipid biosynthetic pathway. Eukaryotic cells are characterised by their sub-cellular compartmentalisation. This cellular segregation offers opportunities for specialised metabolic pockets as well as for metabolic regulation through altering the sub-cellular localisation of proteins. Additionally, cellular compartmentalisation can be used biotechnologically for targeting specialist metabolism to an organelle for biotechnological production, such as in the peroxisome. Rex1 is a nuclear 3’ exonuclease implicated in the processing of 5S rRNA and numerous species of tRNA. Cells exposed to heat stress temperatures show a pre-tRNA processing phenotype akin to rex1Δ cells. The implication of this is that Rex1 activity is downregulated in response to heat stress. This thesis describes two means of regulating Rex1 activity. In cells exposed to heat stress Rex1 is degraded by proteasome complexes and is delocalised from the nucleus where its substrates are found. Through in vivo phospholabelling, we have demonstrated that Rex1 is phosphorylated in response to heat stress and this may be required to regulate Rex1 degradation. Additionally, we have identified a nuclear localisation signal in the N-terminal of Rex1. Mannosylerythritol lipids (MELs) are fungal glycolipids with promising biosurfactant activity. Native producers are maize pathogens that require specific growth conditions and produce additional undesirable glycolipids. There is industrial demand for pure MELs, incentivising the production of MELs in a heterologous host. S. cerevisiae has been a model system for heterologous gene expression and for biotechnological manufacture. He we have demonstrated an impressive capacity for heterologous gene expression in S. cerevisiae through the heterologous expression of the 5-gene MEL biosynthetic cluster. We used nuclear magnetic resonance (NMR) and mass spectrometry to verify expression of the gene cluster and to explore limitations of foreign gene expression.