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

[摘要] 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 analternative, environmentally friendly, transportation fuel. Enzymatic degradation of cellulose isachieved by the synergistic action of three cellulase enzyme groups: endoglucanases, exoglucanasesand-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 producecellulolytic enzymes, hydrolyze cellulose and ferment the resulting sugars to ethanol in a single processstep, 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 ofrecombinant proteins due to several characteristics, including its robustness in industrial processes, thewell developed genetic tools available for manipulation and its proven safety status. A number ofcellulase genes have previously been successfully expressed by recombinant S. cerevisiae strains. Inthis study, all three components of the cellulase system were co-expressed in S. cerevisiae to test theability of the yeast to effectively produce the heterologous proteins, and consequently produce enoughglucose for growth on an amorphous cellulosic substrate.The Trichoderma reesei endoglucanase gene egII (Cel5A) was successfully expressed by aS. cerevisiae Y294 strain. Recombinant EGII displayed activities of 19.6 nkat.ml-1 and 22.3 nkat.ml-1towards CMC and barley-glucan, respectively. The major endoglucanase gene, egI (Cel7B) fromT. reesei was subjected to random mutagenesis by propagating the egI-containing plasmid in an E. colimismatch 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). Throughsubcloning of the mutated ENO1 promoter region and the mutated egI gene fragment, it wasestablished that the mutations located in both the promoter- and gene sequences were responsible forthe 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. reeseicellobiohydrolase (sCBHI) and a-glucosidase from Saccharomycopsis fibuligera. This resulted in areduction in endoglucanase levels, possibly due to the metabolic burden placed on the yeast byco-expressing the different cellulases. The hydrolysis products produced by cellulase co-expressingstrains were cellotriose, cellobiose and glucose, although the glucose yield was insufficient to enablegrowth on cellulose as sole carbon source. As the major hydrolysis product was cellobiose, it is likelythat 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. Theknowledge obtained could be applied in optimizing cellulase cocktails for efficient cellulosedegradation and eventual production of ethanol by recombinant yeast. It has also demonstrated theapplicability of random mutagenesis for improving the activity of cellulases.

[发布机构] Stellenbosch University