Engineering high-gravity fermentations for ethanol production at elevated temperature with Saccharomyces cerevisiae.

Thermal damage, high osmolarity, and ethanol toxicity in the yeast Saccharomyces cerevisiae limit titer and productivity in fermentation to produce ethanol. We show that long-term adaptive laboratory evolution at 39.5°C generates thermotolerant yeast strains, which increased ethanol yield and productivity by 10% and 70%, in 2% glucose fermentations. From these strains, which also tolerate elevated-osmolarity, we selected a stable one, namely a strain lacking chromosomal duplications. This strain (TTY23) showed reduced mitochondrial metabolism and high proton efflux, and therefore lower ethanol tolerance. This maladaptation was bolstered by reestablishing proton homeostasis through increasing fermentation pH from 5 to 6 and/or adding potassium to the media. This change allowed the TTY23 strain to produce 1.3-1.6 times more ethanol than the parental strain in fermentations at 40°C with glucose concentrations ~300 g/L. Furthermore, ethanol titers and productivities up to 93.1 and 3.87 g·L ·hr were obtained from fermentations with 200 g/L glucose in potassium-containing media at 40°C. Albeit the complexity of cellular responses to heat, ethanol, and high osmolarity, in this study we overcome such limitations by an inverse metabolic engineering approach.