17-04: Engineering an alternative glycolytic pathway that increases ethanol yield in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is the ideal host for ethanol production: It has a long history of robust performance under industrial fermentation conditions; produces ethanol up to 90% of theoretical maximum yield; tolerates ethanol up to 24% v/v; ferments hexose sugars at low-pH imparting resistance to bacterial contamination; is naturally resistant to phage infection; and is Generally Recognized as Safe (GRAS). Despite these advantages, there remains a need to improve ethanol yield, specific productivity, reduce fermentation by-products, uncouple growth and fermentation and enable anaerobic fermentation of the pentose sugars in cellulosic feedstocks. Yeast ferment hexose sugars via the Embden-Meyerhoff-Parnas pathway and commercial fermentations typically operate at 86-90% of the maximum theoretical yield of 0.51g ethanol/g sugar. We have engineered into yeast an alternative glycolytic pathway to increase ethanol yield from hexose sugars. A combinatorial library of genes encoding this alternative pathway was screened and the most active combination was integrated into the genome of both laboratory and commercial haploid yeast strains. We also used a fully-compartmentalized, constrained metabolic pathway model to predict the genotype that would maximize aerobic biomass flux and anaerobic ethanol flux. To evaluate the suggested mutations, ¹³C-labeled glucose was used to monitor carbon flux through the alternative glycolytic pathway. The combination of mutations that maximized carbon flux was then engineered into diploid yeast strains and fermentation performance compared in both laboratory and industrial fermentation media.