8-01: Rational and evolutionary engineering approaches uncover a small set of genetic changes efficient for rapid xylose fermentation in Saccharomyces cerevisiae

We developed a rapid and efficient xylose-fermenting S. cerevisiae through rational and inverse metabolic engineering strategies, comprising the optimization of the xylose-assimilating pathway and evolutionary engineering.  Strong and balanced expression levels of the XYL1, XYL2, and XYL3 genes constituting the xylose-assimilating pathway increased ethanol yields and the xylose consumption rates from a mixture of glucose and xylose with little xylitol accumulation.  The engineered strain, however, still exhibited a long lag time when metabolizing xylose above 10 g/l as a sole carbon source, defined here as xylose toxicity. Through serial-subcultures on xylose, we isolated evolved strains overcoming the xylose toxicity. As a result, the evolved strains exhibited a shorter lag time and improved xylose-fermenting capabilities than the parental strain.  Genome sequencing of the evolved strains revealed that mutations in PHO13 causing loss of the Pho13p function are associated with the improved phenotypes of the evolved strains.  Crude extracts of a PHO13-overexpressing strain showed a higher phosphatase activity on xylulose-5-phosphate (X-5-P), suggesting that the dephosphorylation of X-5-P by Pho13p can generate a futile cycle with xylulokinase overexpression.  Lastly, deletion of ALD6 coding for acetaldehyde dehydrogenase prevented acetate accumulation which prevented complete fermentation of high concentrations of xylose and mixed sugars by the evolved strain. Optimization of the expression levels of XYL1, XYL2, and XYL3, and disruption of PHO13 and ALD6 enabled efficient xylose fermentation by engineered S. cerevisiae. This finding provides direct guidance for developing industrial strains to produce cellulosic fuels and chemicals.