Co-expression of cellulase genes in Saccharomyces cerevisiae for cellulose degradation

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dc.contributor.advisor Van Zyl, W. H. en_ZA
dc.contributor.advisor Rose, S. H. en_ZA
dc.contributor.author Du Plessis, Lisa en_ZA
dc.contributor.other Stellenbosch University. Faculty of Science. Dept. of Microbiology.
dc.date.accessioned 2008-11-20T08:16:59Z en_ZA
dc.date.accessioned 2010-06-01T08:27:45Z
dc.date.available 2008-11-20T08:16:59Z en_ZA
dc.date.available 2010-06-01T08:27:45Z
dc.date.issued 2008-12 en_ZA
dc.identifier.uri http://hdl.handle.net/10019.1/1577
dc.description Thesis (MSc (Microbiology))--Stellenbosch University, 2008. en_ZA
dc.description.abstract Complete degradation of cellulose produces mainly glucose, which can be fermented to ethanol. Therefore cellulose presents an abundant renewable energy resource for the production of an alternative, environmentally friendly, transportation fuel. Enzymatic degradation of cellulose is achieved by the synergistic action of three cellulase enzyme groups: endoglucanases, exoglucanases and -glucosidases. However, cellulolytic organisms do not produce significant amounts of ethanol. Therefore, a need has arisen to develop a recombinant microorganism with the ability to produce cellulolytic enzymes, hydrolyze cellulose and ferment the resulting sugars to ethanol in a single process step, referred to as “Consolidated Bioprocessing” (CBP). This would provide a cost-effective, economically feasible strategy for the production of bioethanol. The naturally fermentative yeast, Saccharomyces cerevisiae, is often used as host for the expression of recombinant proteins due to several characteristics, including its robustness in industrial processes, the well developed genetic tools available for manipulation and its proven safety status. A number of cellulase genes have previously been successfully expressed by recombinant S. cerevisiae strains. In this study, all three components of the cellulase system were co-expressed in S. cerevisiae to test the ability of the yeast to effectively produce the heterologous proteins, and consequently produce enough glucose for growth on an amorphous cellulosic substrate. The Trichoderma reesei endoglucanase gene egII (Cel5A) was successfully expressed by a S. cerevisiae Y294 strain. Recombinant EGII displayed activities of 19.6 nkat.ml-1 and 22.3 nkat.ml-1 towards CMC and barley -glucan, respectively. The major endoglucanase gene, egI (Cel7B) from T. reesei was subjected to random mutagenesis by propagating the egI-containing plasmid in an E. coli mismatch repair deficient strain. Screening of S. cerevisiae transformants revealed a strain, S. cerevisiae Y294[pLEM1], with improved levels of endoglucanase activity (21.8 nkat.ml-1), compared to S. cerevisiae Y294[pAZ40], expressing the wild type gene (10.3 nkat.ml-1). Through subcloning of the mutated ENO1 promoter region and the mutated egI gene fragment, it was established that the mutations located in both the promoter- and gene sequences were responsible for the improved levels of activity displayed by S. cerevisiae Y294[pLEM1]. The egII gene and the altered egI gene were co-expressed with a codon optimised T. reesei cellobiohydrolase (sCBHI) and a -glucosidase from Saccharomycopsis fibuligera. This resulted in a reduction in endoglucanase levels, possibly due to the metabolic burden placed on the yeast by co-expressing the different cellulases. The hydrolysis products produced by cellulase co-expressing strains were cellotriose, cellobiose and glucose, although the glucose yield was insufficient to enable growth on cellulose as sole carbon source. As the major hydrolysis product was cellobiose, it is likely that a bottleneck exists at its conversion to glucose, suggesting inadequate -glucosidase activity. This study has provided insight into co-expression of cellulase enzymes by the yeast S. cerevisiae. The knowledge obtained could be applied in optimizing cellulase cocktails for efficient cellulose degradation and eventual production of ethanol by recombinant yeast. It has also demonstrated the applicability of random mutagenesis for improving the activity of cellulases. en_ZA
dc.language.iso en en_ZA
dc.publisher Stellenbosch : Stellenbosch University
dc.subject Cellulose en_ZA
dc.subject Bioethanol en_ZA
dc.subject Saccharomyces cerevisiae en_ZA
dc.subject Trichoderma reesei en_ZA
dc.subject Dissertations -- Microbiology en_ZA
dc.subject Theses -- Microbiology en_ZA
dc.subject.other Microbiology en_ZA
dc.title Co-expression of cellulase genes in Saccharomyces cerevisiae for cellulose degradation en_ZA
dc.type Thesis en_ZA
dc.rights.holder Stellenbosch University


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