Identification of Mutations Responsible for Improved Xylose Utilization in an Adapted Xylose Isomerase Expressing Saccharomyces cerevisiae Strain

Economic conversion of biomass to biofuels and chemicals requires efficient and complete utilization of xylose. Saccharomyces cerevisiae strains engineered for xylose utilization are still considerably limited in their overall ability to metabolize xylose. In this study, we identified causative mutations resulting in improved xylose fermentation of an adapted S. cerevisiae strain expressing codon-optimized xylose isomerase and xylulokinase genes from the rumen bacterium Prevotella ruminicola. Genome sequencing identified single-nucleotide polymorphisms in seven open reading frames. Tetrad analysis showed that mutations in both PBS2 and PHO13 genes were required for increased xylose utilization. Single deletion of either PBS2 or PHO13 did not improve xylose utilization in strains expressing the xylose isomerase pathway. Saccharomyces can also be engineered for xylose metabolism using the xylose reductase/xylitol dehydrogenase genes from Scheffersomyces stipitis. In strains expressing the xylose reductase pathway, single deletion of PHO13 did show a significant increase xylose utilization, and further improvement in growth and fermentation was seen when PBS2 was also deleted. These findings will extend the understanding of metabolic limitations for xylose utilization in S. cerevisiae as well as understanding of how they differ among strains engineered with two different xylose utilization pathways.