Tuesday, May 3, 2011
Scheffersomyces stipitis is the best-known yeast capable of fermenting hemicellulosic sugars to ethanol. It produces recoverable concentrations of ethanol, but fermentation rates, yields and inhibitor tolerance need to be improved. Acetic acid is a common byproduct of industrial hemicellulosic hydrolysis processes, and the fermentative capacity of S. stipitis is sensitive to acetic inhibition. For efficient fermentation of hemicellulosic hydrolysates, improvements must be made to increase tolerance to acetic acid and other inhibitors that are released during industrial pretreatment processes. Two non-auxotrophic strains of S. stipitis, NRRL Y-7124 and CBS 6054, were chosen for metabolic engineering based on desirable native characteristics. S. stipitis NRRL Y-7124 can ferment the hemicellulosic sugar xylose into ethanol at a faster rate than CBS 6054, while S. stipitis CBS 6054 demonstrates a higher resistance to acetic acid in hemicellulosic hydrolysates along with a higher capacity for cellobiose fermentation. Various approaches are being employed to modify the genotypes of these strains. Genes of interest have been cloned into shuttle vectors for integration into genomic DNA, using antibiotic markers to select for integrants. The resulting strains were cured of the antibiotic markers, screened for desired phenotypes, and further modified through subsequent rounds of transformation with other genes of interest. In addition, mutants derived from separate lines of development were mated to generate a pool of genetically diverse strains, from which to select for more resistant mutants. The resulting strains have demonstrated significant gains in the capacity of S. stipitis to ferment industrial hemicellulosic hydrolysates to ethanol.