Foundational Tools for Synthetic Methylotrophy in Saccharomyces cerevisiae

The global expansion of biomanufacturing is currently limited by the availability of sugar-based microbial feedstocks. One-carbon feedstocks, like methanol, present an enticing alternative to sugar because they can be produced from organic waste, atmospheric carbon dioxide and hydrocarbons. The development of efficient industrial microorganisms which can convert one-carbon feedstocks into valuable products is therefore a research priority. This thesis will present work from three experiments and a literature review focused on the development of tools for engineering synthetic methanol assimilation in Saccharomyces cerevisiae, with the intent of laying a foundation for further projects. Experiment 1 tested multiple different methanol dehydrogenases as part of a synthetic ribulose-monophosphate cycle in S. cerevisiae to identify which enzymes could confer a growth advantage in the presence of methanol. Experiment 2 demonstrated a novel genome engineering technique known as Random Assembly and Integration, to show that promoters and methylotrophy associated genes from a pre-defined library could be randomly integrated into the repeated Ty1 retrotransposon loci in the S. cerevisiae genome. Experiment 3 used a previously reported synthetic xylose utilization pathway to establish a strain of S. cerevisiae which can grow on xylose that will be used in future projects to engineer synthetic methanol auxotrophy.