Identification of mutations responsible for improved xylose utilization in an adapted xylose isomerase expressing Saccharomyces cerevisiae strain

Authors: Hector, Ronald - Ron; Mertens, Jeffrey; Nichols, Nancy

Submitted to: Fermentation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/17/2022
Publication Date: 11/23/2022
Citation: Hector, R.E., Mertens, J.A., Nichols, N.N. 2022. Identification of mutations responsible for improved xylose utilization in an adapted xylose isomerase expressing Saccharomyces cerevisiae strain. Fermentation. 8(12). Article 669. https://doi.org/10.3390/fermentation8120669.
DOI: https://doi.org/10.3390/fermentation8120669

Interpretive Summary: ARS has a long-term research commitment to developing an advanced biofuel industry that relies on use of cellulosic feedstocks in addition to corn. Cellulosic biomass includes agricultural residues such as corn stover and wheat straw or perennial grasses grown as a dedicated biofuel crop. The U.S. government estimated that a mature cellulosic biofuels industry would create 1.5 million jobs, supply up to 50 billion gallons of biofuels, and reduce annual carbon dioxide emissions by 550 million tons. It would also directly benefit farmers by creating new markets for crop residues. A cellulosic based industry has been slow to develop because cellulosic feedstocks contain multiple sugars and especially xylose, which is not fermented by Distillers’ Yeast. Yeast have been engineered to ferment xylose to ethanol but do so slowly and at reduced product yield compared to sugar from corn. ARS researchers have developed a new system that enables yeast to ferment xylose that is based on two genes mined from a bacterium isolated from a cow gut. The yeast system was improved by continuing to culture the yeast on xylose, thereby selecting for faster growing yeast. In this study, we identified mutations in two proteins that made the adapted yeast grow faster. From a commercial viewpoint, this discovery is another step forward to realizing a cellulosic biofuels industry in the USA.

Technical Abstract: 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 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 how they differ among strains engineered with two different xylose utilization pathways.