One strategy to decrease both the consumption of crude oil and environmental damage is through the production of bioethanol from biomass. Cellulolytic enzyme stability and enzymatic hydrolysis play important roles in the bioethanol process. However, the gradually increased ethanol concentration often reduces enzyme activity and leads to inactivation, thereby limiting the final ethanol yield. Herein, we employed an optimized Two-Gene Recombination Process (2GenReP) approach to evolve the exemplary cellulase CBHI for practical bioethanol fermentation. Two all-round CBHI variants (named as R2 and R4) were obtained with simultaneously improved ethanol resistance, organic solvent inhibitor tolerance, and enzymolysis stability in simultaneous saccharification and fermentation (SSF). Notably, CBHI R4 had a 7.0- to 34.5-fold enhanced catalytic efficiency (kcat/KM) in the presence/absence of ethanol. Employing the evolved CBHI R2 and R4 in the 1G bioethanol process resulted in up to 10.27% (6.7 g/L) improved ethanol yield (ethanol concentration) than non-cellulase, which was far more beyond than other optimization strategies. Besides bioenergy fields, this transferable protein engineering routine holds the potential to generate all-round enzymes that meet the requirement in biotransformation and bioenergy fields.
Keywords: bioethanol; cellulase; fermentation; optimized 2GenReP; robust enzyme.