17-36: Metabolic engineering of Scheffersomyces stipitis for improved fermentation of hemicellulosic hydrolysates

Tuesday, May 1, 2012
Napoleon Ballroom C-D, 3rd fl (Sheraton New Orleans)
Sarah D. Mahan and Thomas W. Jeffries, Forest Products Laboratory, USDA Forest Service, Madison, WI
The yeast Scheffersomyces stipitis has a high native capacity to ferment xylose and other hemicellulosic sugars into ethanol. It has become a model organism in which to study the native fermentation of xylose by yeasts. To further increase its efficiency we are genetically engineering it to improve the product formation rate, yields and its capacity to resist inhibitors found in hemicellulosic hydrolysates.  To enable these changes we have developed a highly efficient transformation system that includes three different antibiotic resistance markers and marker recovery mechanisms along with mating techniques.  Using these tools, we have engineered a line of strains based on the wild-type S. stipitis strain NRRL Y-7124 that contain overexpression cassettes for xylose reductase, xylitol dehydrogenase, D-xylulokinase, and a putative xylose transporter integrated into the genome.  We have screened multiple transformants at each step of development for their fermentative capacities, and have further modified them through subsequent rounds of gene integration and adaptation on industrial hemicellulosic hydrolysates. The resulting engineered S. stipitis strains have demonstrated increases in fermentation rate of up to 48% (0.41 g/L h-1 vs. 0.28 g/L h-1), and increases of up to 7.4% in specific ethanol yield (0.43 g ethanol/g xylose vs. 0.40 g ethanol/g xylose) as compared to the wild-type strain on industrial hemicellulosic hydrolysates.
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