AbstractDoctor of Philosophy in the Faculty of Life sciences. 2016Genetic engineering of S. cerevisiae to confer xylose metabolism with a view to biofuel productionHassan Ahmed, The University of Manchester, FLS, Manchester, UKXylose is a pentose sugar that forms a substantial proportion of the monosaccharides released from lignocellulosic biomass after hydrolysis. Therefore, the economic and commercial viability of biofuel production from lignocellulosic material via microbial fermentation relies upon maximising the metabolism of monosaccharides like xylose. As such, second generation biofuels are becoming a focus of biofuel innovation because of the depleting fossil fuel reserves and increasing levels of carbon emissions. Even so the majority of current biofuel production uses glucose as a carbon source from corn, wheat or sugar cane. This conflicts with food production and has prompted the food versus fuel debate.The introduction of xylose metabolising pathways into current biofuel production microorganisms like yeast, which cannot utilize xylose, would allow xylose use from lignocellulosic biomass. The xylose-reductase (XR) pathway from fungi utilise the xylose reductase, xylose dehydrogenase and xylulokinase genes, whereas the xylose isomerase (XI) pathway from bacteria consists of xylose isomerase and xylulokinase. In this project plasmid constructs containing the two pathways were successfully introduced into yeast. The genes were further integrated into specific chromosomal sites for comparison. Depending on the type of media used, some xylose uptake and ethanol production could be demonstrated for some of these strains, but overall levels of xylose use did not reach a level likely to impact upon commercial biofuel production.As a result, several strategies were investigated with a view to increasing xylose metabolism and ethanol production from the strains. Alterations were made to the cassette design for the xylose enzyme genes, such as gene promoter replacement or removal of the epitope tag. A pentose specific transporter, GXF1, from Candida tropicalis was also introduced. However, none of these strategies improved xylose use. A further approach, which led to minor increases in xylose metabolism, was deletion of the PHO13 gene, which is thought to impact upon expression of pentose phosphate genes.One further goal of this work was to investigate whether xylose metabolism could be connected to butanol production, as butanol has superior properties as a biofuel in yeast. Unfortunately, butanol was not detected from heterologous butanol producing strains bearing the plasmid based XI pathway, presumably because the growth and health of these strains was quite poor.Overall this project has demonstrated that S. cerevisiae is able to metabolise hemicellulosic xylose to ethanol using heterologous pathways, however, the very low levels generated mean that a great deal of genetic and metabolic engineering would be required for optimisation of biofuel production for commercial viability.