S55: High level secretion of cellobiohydrolases by Saccharomyces cerevisiae

The main technological impediment to widespread utilization of lignocellulose for production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow conversion steps to be combined in a consolidated bioprocess (CBP). Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases. In this work we carried out a comprehensive study of expression of various cellobiohydrolases to identify enzymes that can be efficiently secreted by S. cerevisiae. Aiming for enhancing cellulose hydrolysis further, we engineered bi-modular derivatives of a well secreted CBM-less enzyme and constructed strains expressing various combinations of the cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, certain genes yielded ~0.3 g/liter CBH1 and ~1 g/liter CBH2 in high cell density fermentations. Furthermore, we examined the physiological impact of cellobiohydrolase production in S. cerevisiae expressing the different cbh genes and discovered activation of the unfolded protein response. The results pointed out that gene or protein specific features and compatibility with the host are important for efficient enzyme production. Finally, we report fermentation of microcrystalline Avicel cellulose to ethanol by CBH-producing S. cerevisiae strains with the aid of added β-glucosidase. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier of CBH sufficiency was overcome.