Thesis (MSc. (Biochemistry)) --University of Limpopo, 2016 / The ever increasing cost of fossil-based fuels and the accompanying concerns about their impact on the environment is driving research towards clean and renewable sources of energy. Bioethanol has the potential to be a replacement for liquid transportation fuels. In addition to its near zero nett carbon dioxide emissions, bio-ethanol has a high energy to weight ratio and can easily be stored in high volumes. To produce bioethanol at economically competitive prices, the major cost in the production process needs to be addressed. The addition of enzymes to hydrolyse the lignocellulosic fraction of the agricultural waste to simple sugars is considered to be the major contributor to high production cost. A consolidated bioprocess (CBP) which ideally combines all the steps that are currently accomplished in different reactors by different microorganisms into a single process step would be a more economically feasible solution. In this study the potential of yeast hybridization with a CBP approach was used. In order to evaluate the reduction or elimination of the addition of cellulolytic and hemi-cellulolytic enzymes to the ethanol production process. High cellobiohydrolase I secreting progeny from hybridization of an industrial bioethanol yeast strain, S. cerevisiae M0341, and a laboratory strain S. cerevisiae Y294 were isolated. In order to determine if this characteristic was specific to cellobiohydrolase I secretion, these strains were evaluated for their ability to secrete other relevant recombinant hydrolase enzymes for CBP-based ethanol production. A total of seven S. cerevisiae strains were chosen from a progeny pool of 28 supersecreting hybrids and reconstructed to create two parental strains; S. cerevisiae M0341 and S. cerevisiae Y294, together with their hybrid segregants strains H3M1, H3M28, H3H29, H3K27 and H3O23. Three episomal plasmids namely pNS201, pNS202 and pNS203 were constructed; these plasmids together with two already available plasmids, namely pRDH166 and pRDH182 contained genes for different reporter enzymes, namely β-glucosidase I, xylanase II, endoglucanase lll, cellobiohydrolase l and α-glucuronidase. To allow for selection of the episomal plasmids, homologous recombination was used to replace the functional URA3 gene of selected strains, with the non-functional ura3 allele from the Y294 strain. Enzyme activity was used as an indicator of the amount of enzyme secreted. Fermentation studies in a bioreactor were used to determine the metabolic burden imposed on the segregants expressing the cellobiohydrolase at high levels. In addition all segregants were tested for resistance to inhibitors commonly found in pre-treated lignocellulosic material. The M28_Cel7A was found to be the best secretor of Cel7A (Cellobiohydrolase l); however it seems as though this phenomenon imposes a significant metabolic burden on the yeast. The supersecreting hybrid strains cannot tolerate lignocellulosic inhibitors at concentrations commonly produced during pretreatment / The National Research Foundation - Renewable Energy Scholarship (NRF-RSES)