Simultaneous conversion of cellulosic sugars and acetic acid into biofuels and chemicals by engineered yeast

In order to overcome inefficient fermentation of cellulosic sugars, and the toxicity of acetic acid in plant cell wall hydrolysates, we constructed highly engineered Saccharomyces cerevisiae strains capable of converting mixed carbon components (cellobiose, xylose, and acetate) into biofuels and chemicals simultaneously. As S. cerevisiae cannot ferment xylose, we first constructed a rapid and efficient xylose fermenting S. cerevisiae (SR8) through a rational design that allows strong and balanced expression levels of the xylose metabolic genes (XYL1, XYL2, and XYL3), and evolution of the engineered strain for rapid xylose fermentation. Second, we introduced a cellobiose metabolic pathway into the efficient xylose fermenting strain (SR8) for co-fermentation of cellobiose and xylose. Again, a rational design for integrating genes (cdt-1 and gh1-1) coding for cellobiose transporter and intracellular β-glucosidase, and evolution of the engineered strain were combined to isolate an engineered strain (EJ4) co-fermenting cellobiose and xylose rapidly. After measuring the copy numbers of cdt-1 and gh1-1 in the evolved strain, we learned that cellobiose fermentation can be improved by amplification of cdt-1 and gh1-1. Finally, we introduced an acetate reduction pathway, which can enhance xylose fermentation through redox coupling, into the xylose and cellobiose co-fermenting strain (EJ4) for efficient co-utilization of cellobiose, xylose, and acetate. The resulting strain was able to produce ethanol from a mixture of cellobiose, xylose and acetate with a substantially higher yield and productivity than the control strains, demonstrating the synergistic effects from integration of multiple metabolic pathways.