Single Amino Acid Substitutions in HXT2.4 from Scheffersomyces stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisiae

Saccharomyces cerevisiae cannot utilize cellobiose, but this yeast can be engineered to ferment cellobiose by introducing both cellodextrin transporter (cdt-1 ) and intracellular β-glucosidase (gh1-1 ) genes fromNeurospora crassa . Here, we report that an engineeredS. cerevisiae strain expressing the putative hexose transporter geneHXT2.4 fromScheffersomyces stipitis andgh1-1 can also ferment cellobiose. This result suggests that HXT2.4p may function as a cellobiose transporter whenHXT2.4 is overexpressed inS. cerevisiae . However, cellobiose fermentation by the engineered strain expressingHXT2.4 andgh1-1 was much slower and less efficient than that by an engineered strain that initially expressedcdt-1 andgh1-1 . The rate of cellobiose fermentation by theHXT2.4 -expressing strain increased drastically after serial subcultures on cellobiose. Sequencing and retransformation of the isolated plasmids from a single colony of the fast cellobiose-fermenting culture led to the identification of a mutation (A291D) in HXT2.4 that is responsible for improved cellobiose fermentation by the evolvedS. cerevisiae strain. Substitutions for alanine (A291) of negatively charged amino acids (A291E and A291D) or positively charged amino acids (A291K and A291R) significantly improved cellobiose fermentation. The mutant HXT2.4(A291D) exhibited 1.5-fold higherKm and 4-fold higherV max values than those from wild-type HXT2.4, whereas the expression levels were the same. These results suggest that the kinetic properties of wild-type HXT2.4 expressed inS. cerevisiae are suboptimal, and mutations of A291 into bulky charged amino acids might transform HXT2.4p into an efficient transporter, enabling rapid cellobiose fermentation by engineeredS. cerevisiae strains.