AbstractsBiology & Animal Science

Expression of fungal b-glucosidases in Saccharomyces cerevisiae for enhanced growth on cellobiose

by Anathi Perseverence Njokweni




Institution: Stellenbosch University
Department: Microbiology
Year: 2011
Keywords: Microbiology; Dissertations  – Microbiology
Record ID: 1475013
Full text PDF: http://hdl.handle.net/10019.1/18113


Abstract

Thesis (MSc (Microbiology)) – Stellenbosch University, 2011. ENGLISH ABSTRACT: Bio-fuels have been considered an ideal substitute for fossil fuels due to their availability and renewable nature. Bio-ethanol is currently of great market interest as an alternative fuel with the potential of supplementing petroleum as transportation fuel. Lignocellulosic biomass, a renewable energy source, can be "readily" converted to bio-ethanol. The main impediment in the conversion process is the recalcitrance of the main lignocellulosic components (cellulose, hemicelluloses and lignin) to enzymatic hydrolysis as well as the lack of available low-cost technology. Consolidated Bioprocessing (CBP) is a single process step which offers a cost-effective and economically feasible strategy for bio-ethanol production. The process requires micro-organisms that produce ethanol at high rates and titres. Saccharomyces cerevisiae has potential as a CBP candidate due to its high ethanol yield, robustness in industrial processes, well-developed gene expression system and its safety status. Unfortunately S. cerevisiae does not degrade polysaccharides and therefore requires heterologous expression of cellulases. Genetic engineering of S. cerevisiae for cellulose hydrolysis serves as an important step in yeast strain development for CBP, and serves as a stepping stone for the commercialisation of lignocellulosic bio-ethanol. Although cellulose- utilising S. cerevisiae strains have been constructed, the cellobiose conversion is slow, hampering optimal ethanol production. β-glucosidases have been shown to be the major rate-limiting factors in cellulose saccharification as their activity determines the extent of cellulose hydrolysis, by removing excess cellobiose which causes feed-back inhibition on endoglucanase and cellobiohydrolase activities (Du Plessis et al. 2009;Lynd et al. 2002). Therefore, insufficient supply of β-glucosidase activity is detrimental to CBP and can be addressed by increasing the enzyme supply or using highly active β-glucosidases to enhance cellobiose hydrolysis. In this study, several cellobiose fermenting S. cerevisiae strains were constructed. Extracellular fungal β-glucosidase-encoding genes were successfully expressed in S. cerevisiae under the transcriptional control of the ENO1 (enolase) promoter and terminator sequences. The recombinant enzymes produced were characterised based on pH and temperature optima as well as kinetic parameters. Bio-fuels have been considered an ideal substitute for fossil fuels due to their availability and renewable nature. Bio-ethanol is currently of great market interest as an alternative fuel with the potential of supplementing petroleum as transportation fuel. Lignocellulosic biomass, a renewable energy source, can be „readily‟ converted to bio-ethanol. The main impediment in the conversion process is the recalcitrance of the main lignocellulosic components (cellulose, hemicelluloses and lignin) to enzymatic hydrolysis as well as the lack of available low-cost technology. Consolidated…