|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