Abstract
To permit direct cellulose degradation and ethanol fermentation, Saccharomyces cerevisiae BY4741 (Δsed1) codisplaying 3 cellulases (Trichoderma reesei endoglucanase II [EG], T. reesei cellobiohydrolase II [CBH], and Aspergillus aculeatus β-glucosidase I [BG]) was constructed by yeast cell-surface engineering. The EG used in this study consists of a family 1 carbohydrate-binding module (CBM) and a catalytic module. A comparison with family 1 CBMs revealed conserved amino acid residues and flexible amino acid residues. The flexible amino acid residues were at positions 18, 23, 26, and 27, through which the degrading activity for various cellulose structures in each biomass may have been optimized. To select the optimal combination of CBMs of EGs, a yeast mixture with comprehensively mutated CBM was constructed. The mixture consisted of yeasts codisplaying EG with mutated CBMs, in which 4 flexible residues were comprehensively mutated, CBH, and BG. The yeast mixture was inoculated in selection medium with newspaper as the sole carbon source. The surviving yeast consisted of RTSH yeast (the mutant sequence of CBM: N18R, S23T, S26S, and T27H) and wild-type yeast (CBM was the original) in a ratio of 1:46. The mixture (1 RTSH yeast and 46 wild-type yeasts) had a fermentation activity that was 1.5-fold higher than that of wild-type yeast alone in the early phase of saccharification and fermentation, which indicates that the yeast mixture with comprehensively mutated CBM could be used to select the optimal combination of CBMs suitable for the cellulose of each biomass.
Highlights
The production of energy without fossil resources has become necessary to establish a sustainable society because of expanded energy demand, limitations of oil drilling, and environmental pollution (Ge et al 2011; Gerngross and Slater 2000; Stöcker 2008)
Comparison of the sequences of family 1 carbohydrate-binding module (CBM) Conserved and flexible amino acid residues were determined by comparing the amino acid sequences of 92 types of family 1 CBMs (Table 1)
More than 99% of the 5th, 31st, and 32nd amino acids of the CBMs were aromatic amino acids that bind to the flat surface of crystalline cellulose; the 7th, 8th, 9th, 10th, 15th, 17th, 19th, 25th, 34th, and 35th amino acids were conserved as skeletal elements, where the 19th and 35th amino acids as well as the 8th and 25th amino acids formed disulfide bonds with each other without any exception
Summary
The production of energy without fossil resources has become necessary to establish a sustainable society because of expanded energy demand, limitations of oil drilling, and environmental pollution (Ge et al 2011; Gerngross and Slater 2000; Stöcker 2008). Biorefinery is an important concept because it uses the biomass in a natural cycle (Bouaid et al 2010; FitzPatrick et al 2010; López et al 2010). Bioethanol is a popular energy resource in biorefinery; current biorefineries mainly use grain biomass to produce biofuels, which competes with food supply (Ferreira et al 2010). Use of biomass garbage has attracted attention for biorefineries. The displaying yeast can be used as a whole-cell biocatalyst without requiring enzyme-separation and -purification processes, and it can immediately take in glucose degraded from cellulose; it is hardly contaminated by other organisms. Direct ethanol fermentation from cellulose could be conducted by using cellulase-displaying yeast (Fujita et al 2002, 2004; Murai et al 1998)
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