Exploring improved endoglucanase expression in Saccharomyces cerevisiae strains.

Area of Science:

• Biotechnology
• Molecular Biology
• Enzyme Engineering

Background:

• Trichoderma reesei is a key source of industrial cellulases.
• Efficient expression of cellulase genes in heterologous hosts like Saccharomyces cerevisiae is crucial for biofuel production.
• Understanding the interplay between different cellulase components is vital for optimizing cellulose hydrolysis.

Purpose of the Study:

• To clone and express endoglucanase I and II genes from T. reesei in S. cerevisiae.
• To investigate the effect of random mutagenesis on endoglucanase I activity.
• To evaluate the co-expression of endoglucanases with cellobiohydrolase and beta-glucosidase genes in S. cerevisiae.

Main Methods:

• Gene cloning and expression in Saccharomyces cerevisiae using ENO1 promoter and terminator.
• Random mutagenesis of the endoglucanase I gene.
• Co-expression of endoglucanase genes with synthetic cellobiohydrolase (s-cbhI) and beta-glucosidase (bgl1) genes.
• Hydrolysis of phosphoric acid swollen cellulose and analysis of products.

Main Results:

• Successful cloning and expression of T. reesei endoglucanase I and II in S. cerevisiae.
• A twofold increase in extracellular endoglucanase I (EGI) activity was achieved through random mutagenesis.
• Co-expression with s-cbhI or bgl1 resulted in lower extracellular endoglucanase activity.
• Recombinant strains hydrolyzed cellulose into cellotriose, cellobiose, and glucose, with cellobiose accumulation indicating beta-glucosidase as a rate-limiting step.
• Insufficient glucose production for yeast growth on amorphous cellulose was observed.

Conclusions:

• Recombinant yeast strains expressing T. reesei endoglucanases show potential for cellulose degradation.
• Beta-glucosidase activity is a critical limiting factor in the complete hydrolysis of cellulose to glucose.
• Further optimization of cellulase combinations is necessary for efficient saccharification and fermentation of cellulose to ethanol.