Global energetic and environmental concerns have generated interest in the biological systems for the production liquid biofuels. Butanol is one such biofuel, which can be naturally produced by some Clostridia species. However, possible limitations in Clostridial engineering and large-scale fermentation have led to an examination of other potential organisms that might house this pathway for butanol production. As a robust industrial host and key model organism in the study of fundamental biological processes, the yeast Saccharomyces cerevisiae has been used to house the Clostridial ABE-butanol pathway. However, butanol yields and titres in this yeast are relatively low. Therefore, in this thesis, three distinct strategies were carried out with the goal of optimising butanol production in the strain of yeast (previously constructed in the Ashe lab) bearing the ABE-butanol pathway: 1. Mutation of genes involved in the regulation of carbon source usage. 2. Deletion of genes where the product is involved in the consumption of cytosolic acetyl-CoA (the starting precursor for the butanol synthetic pathway). 3. Targeted mutagenesis to improve the efficiency of the thiolase enzyme, which catalyses the condensation of 2x acetyl-CoA to initiate the ABE-butanol synthesis pathway. The results showed the first two strategies did not lead to improvements in butanol yields. However, increases of intracellular acetyl-CoA were observed in some mutant strains, even though butanol production did not increase in these strains. In order to make maximum use of the accumulating cytosolic acetyl-CoA, thiolase engineering in the butanol production yeast strain was pursued. The introduced changes caused an increase in butanol (about two fold). Overall, this project has used a minimal engineering approach by modulation of associated pathways or optimisation of the heterologous enzyme with a view to improve butanol production in yeast. To achieve high and scalable butanol production in yeast, a robust approach involving whole synthetic biology - Design, Build Test, and Learn will need to be adopted to create a more efficient yeast-butanol system.