(211b) Integration of Metabolic Pathways to Develop Optimal Yeast Strains for Producing Biofuels and Chemicals

Plant cell wall hydrolysates contain fermentable sugars (e.g., glucose and xylose) and toxic fermentation inhibitors. Therefore, a fermenting microorganism will need to utilize both glucose and xylose under the presence of toxic levels of fermentation inhibitors. We engineered Saccharomyces cerevisiae for efficient and rapid fermentation of the cellulosic sugars. As S. cerevisiae cannot ferment xylose, a xylose-fermenting pathway from Scheffersomyces stipitis was introduced into S. cerevisiae and the resulting engineered strain was evolved to produce ethanol from xylose with a high yield and productivity. In addition to ethanol, we demonstrated that the xylose-fermenting yeast can be modified to produce polymer precursors, such as lactic acid and 2,3-butanediol. We further engineered the xylose-fermenting S. cerevisiae for assimilating cellobiose which is a disaccharide that can be produced from cellulose. When a cellobiose-fermenting pathway from Neurospora crassa was introduced into the xylose-fermenting yeast, the resulting strain was able to co-consume cellobiose and xylose simultaneously. While the engineered yeast can ferment cellobiose and xylose, the presence of acetic acid in lignocellulosic hydrolysates hindered efficient utilization of the cellulosic sugars. In order to alleviate the toxicity of acetate towards the xylose and cellobiose co-fermenting strain, we introduced an acetate reduction pathway which can enhance xylose fermentation through redox coupling. As a result, we constructed an optimal yeast strain capable of producing ethanol from a mixture of cellobiose, xylose, and acetate. Our results not only demonstrate the synergistic effects from integration of multiple metabolic pathways, but also exemplify the Design-Build-Test and Learn (DBTL) cycle of synthetic biology.