Evolutionary engineering of cellobiose transporters for enhanced cellobiose fermentation

Direct fermentation of cellobiose by engineered yeast offers advantages for economic production of cellulosic biofuels. Enzyme costs can be reduced from elimination of β-glucosidase for cellulase cocktails, and more efficient and rapid production of a target biofuel can be achieved through simultaneous co-fermentation of cellobiose and xylose. While Saccharomyces cerevisiae cannot utilize cellobiose, this yeast can be engineered to ferment cellobiose by introducing heterologous cellobiose fermentation pathways. To this end, cellodextrin transporters and enzymes capable of degrading cellobiose into glucose intracellularly have been discovered from cellobiose fermenting microorganisms. Specifically, cellodextrin transporters (cdt-1, cdt-2, and HXT2.4), intracellular beta-glucosidase (gh1-1), and cellobiose phosphorylase (CBP) have been paired to implement either hydrolytic or phosphorolytic pathways for cellobiose fermentation in yeast. In order to improve cellobiose fermentation by engineered yeast, we obtained mutant cellodextin transporters enabling enhanced cellobiose fermentation through directed evolution. First, we isolated a mutant CDT-1 capable of increasing cellobiose fermentation rates by engineered strains with the phosphorolytic pathway. The mutant CDT-1 (F213L) exhibited higher Vmax than a wild type CDT-1 while it showed a reduced affinity for cellobiose. Second, we isolated a mutant of HXT2.4 which enabled improved cellobiose fermentation by engineered yeast with the hydrolytic pathway. The mutant HXT2.4 (A294D) also exhibited higher Vmax with a reduced affinity for cellobiose than a wild type HXT2.4. These results suggest that the kinetic properties of wild-type cellobiose transporters expressed in S. cerevisiae are suboptimal for cellobiose fermentation and mutant transporters with higher maximum velocities allow for faster cellobiose fermentation.