Genetic engineering of Saccharomyces cerevisiae for efficient polysaccharide utilisation
Thesis (PhD (Microbiology))--University of Stellenbosch, 2005.
Biomass is the sole foreseeable sustainable source of organic fuels, chemicals and materials. It is a rich and renewable energy source, which is abundant and readily available. Primary factors motivating the use of renewable enrgy sources include the growing concern over global climate change and the drastic depletion of non-renewable resources. Among various forms of biomass, cellulosic feedstocks have the greatest potential for energy production from. The biggest technological obstacle to large-scale utilisation of cellulosic feedstocks for the production of bioethanol as a cost-effective alternative to fossil fuels is the general absence of low-cost technology for overcoming the recalcitrance of cellulosic biomass. A promising strategy to overcome this impediment involves the production of cellulolytic enzymes, hydrolysis of biomass and fermentation of resulting sugars to ethanol in a single process step via a single microorganism or consortium. Such “consolidated bioprocessing” (CBP) offers very large cost reductions if microorganisms, such as the yeast Saccharomyces cerevisiae, can be developed that possess the required combination of efficient cellulose utilisation and high ethanol yields. Cellulose degradation in nature occurs in concert with a large group of bacteria and fungi. Cellulolytic microorganisms produce a battery of enzyme systems called cellulases. Most cellulases have a conserved tripartite structure with a large catalytic core domain linked by an O-glycosylated peptide to a cellulose-binding domain (CBD) that is required for the interaction with crystalline cellulose. The CBD plays a fundamental role in cellulose hydrolysis by mediating the binding of the cellulases to the substrate. This reduces the dilution effect of the enzyme at the substrate surface, possibly by helping to loosen individual cellulose chains from the cellulose surface prior to hydrolysis. Most information on the role of CBDs has been obtained from their removal, domain exchange, site-directed mutagenesis or the artificial addition of the CBD. It thus seems that the CBDs are interchangeable to a certain degree, but much more data are needed on different catalytic domain-CBD combinations to elucidate the exact functional role of the CBDs. In addition, the shortening, lengthening or deletion of the linker region between the CBD and the catalytic domain also affects the enzymatic activity of different cellulases. Enzymes such as the S. cerevisiae exoglucanases, namely EXG1 and SSG1, and the Saccharomycopsis fibuligera β-glucosidase (BGL1) do not exhibit the same architectural domain organisation as shown by most of the other fungal or bacterial cellulases. EXG1 and SSG1 display β-1,3-exoglucanase activities as their major activity and exhibit a significant β- 1,4-exoglucanase side activity on disaccharide substrates such as cellobiose, releasing a free glucose moiety. The BGL1 enzyme, on the other hand, displays β-1,4-exoglucanase activity on disaccharides. In this study, the domain engineering of EXG1, SSG1 and BGL1 was performed to link these enzymes to the CBD2 domain of the Trichoderma reesei CBHII cellobiohydrolase to investigate whether the CBD would be able to modulate these non-cellulolytic domains to function in cellulose hydrolysis. The engineered enzymes were constructed to display different modular organisations with the CBD, either at the N terminus or the C terminus, in single or double copy, with or without the synthetic linker peptide, to mimic the multi-domain organisation displayed by cellulases from other microorganisms. The organisation of the CBD in these recombinant enzymes resulted in enhanced substrate affinity, molecular flexibility and synergistic activity thereby improving their ability to act and hydrolyse cellulosic substrates, as characterised by adsorption, kinetics, thermostability and scanning electron microscopic (SEM) analysis. The chimeric enzyme of CBD2-BGL1 was also used as a reporter system for the development and efficient screening of mutagenised S. cerevisiae strains that overexpress CBD-associated enzymes such as T. reesei cellobiohydrolase (CBH2). A mutant strain WM91 was isolated showing up to 3-fold more cellobiohydrolase activity than that of the parent strain. The increase in the enzyme activity in the mutant strain was found to be associated with the increase in the mRNA expression levels. The CBH2 enzyme purified from the mutant strain did not show a significant difference in its characteristic properties in comparison to that of the parent strain. In summary, this research has paved the way for the improvement of the efficiency of the endogenous glucanases of S. cerevisiae, and the expression of heterologous cellulases in a hypersecreting mutant of S. cerevisiae. However, this work does not claim to advance the field closer to the goal of one-step cellulose processing in the sense of technological enablement; rather, its significance hinges on the fact that this study has resulted in progress towards laying the foundation for the possible development of efficient cellulolytic S. cerevisiae strains that could eventually be optimised for the one-step bioconversion of cellulosic materials to bioethanol.