Abstract
BackgroundThe main technological impediment to widespread utilization of lignocellulose for the 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 separate 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.ResultsWe expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel™) to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase.ConclusionsGene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.
Highlights
The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance
Expression and secretion of CBH1 and CBH2 To identify enzymes that are efficiently secreted in an active form into the culture supernatant, we screened 14 cbh1 (Cel7A) and 10 cbh2 (Cel6A) genes from ascomycetes by functional expression in S. cerevisiae
All the cbh-expressing and the empty vector control strains were grown in yeast extract peptone dextrose (YPD) medium, and a subset of selected strains were grown in defined soybean casein digest without uracil (SCD-URA) medium with 2% glucose as the carbon source
Summary
The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. A low-cost bioprocess to produce bulk fuels and chemicals requires several changes to be made in the metabolism of S. cerevisiae. One of these is the utilization and fermentation of all biomass derived sugars. Since the conversion of lignocellulosic raw material into monomer sugars is limited by the rate and extent of conversion of the plant polysaccharides by enzymes, engineering of yeast to secrete rate limiting enzymes would complement extensive efforts undertaken to engineer existing enzymes, and further streamline the process towards a consolidated bioprocessing (CBP) and lower production costs [6,7]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
