Simultaneous Co-Fermentation of Glucose and Xylose By Single and Mixed Cultures of Engineered Yeast

Glucose and xylose are two prevalent sugars in plant cell wall hydrolysates. Saccharomyces cerevisiae, extensively used for producing ethanol from corn or sugarcane, cannot ferment xylose. In order to enable xylose fermentation in S. cerevisiae, heterologous xylose metabolic pathways and additional genetic perturbations eliciting enhanced xylose fermentation have been introduced. The resulting engineered yeast was able to ferment xylose, but xylose fermentation was severely repressed by glucose, which causes inefficient and slow fermentation of plant wall hydrolysates. Endogenous hexose transporters in S. cerevisiae can transport xylose, but their xylose transport is highly inhibited by glucose, resulting in sequential fermentation of glucose and xylose rather than simultaneous fermentation that is more advantageous. Recently two mutants of hexose transporter that are not subject to glucose inhibition have been isolated. Using these transporters, we constructed single and co-culture systems for rapid and simultaneous co-fermentation of glucose and xylose by engineered yeast. In order to eliminate the glucose inhibition on xylose transport by hexose transporters, we deleted eight hexose transporters (HXT1-7 and GAL2) and overexpressed the mutant hexose sugar transporters in engineered yeast capable of fermenting xylose efficiently. Consistent to the reported kinetic properties of the mutant transporters, the engineered yeast expressing the mutant transporters individually was able to simultaneously co-ferment glucose and xylose. Overexpression of both mutant transporters in single engineered yeast showed faster simultaneous fermentation rates. Additionally, simultaneous co-fermentation of glucose and xylose can be achieved by co-culturing glucose-fermenting yeast and the xylose-fermenting yeast expressing the mutant transporter. Our results not only confirm that inhibition of xylose transport by glucose was a major reason why glucose and xylose are sequentially fermented in engineered yeast, but also demonstrate rapid simultaneous co-fermentation of glucose and xylose in plant cell wall hydrolysates by engineered yeast.