Submitted to: Biotechnology for Biofuels and Bioproducts
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/22/2013
Publication Date: 6/7/2013
Publication URL: https://handle.nal.usda.gov/10113/62051
Citation: Hector, R.E., Dien, B.S., Cotta, M.A., Mertens, J.A. 2013. Growth and fermentation of D-xylose by Saccharomyces cerevisiae expressing a novel D-xylose isomerase originating from the bacterium Prevotella ruminicola TC2-24. Biotechnology for Biofuels. 6:84.
Interpretive Summary: This research discovered a new xylose isomerase (XI) enzyme. Brewer’s yeast is commonly used for producing ethanol from corn grain. It is also the preferred microorganism for producing fuel ethanol from biomass feedstocks such as agricultural wastes. A significant portion of the available sugar present in most biomass is xylose, a sugar which Brewer’s yeast cannot use. This study identified a new XI from a bacterium found in the cow rumen. The new XI was expressed in yeast and the resulting strain was adapted to produce a strain with significantly increased ability to grow on and ferment xylose to ethanol. The new yeast strain has one of the fastest reported growth rates on xylose and produces ethanol at high yield.
Technical Abstract: Saccharomyces cerevisiae strains expressing xylose isomerase (XI) produce some of the highest reported ethanol yields from xylose. Unfortunately, most bacterial XIs that have been expressed in S. cerevisiae are not functional, require additional strain modification, and have low affinity for xylose. This study analyzed several XIs from rumen and intestinal microorganisms to identify enzymes with improved properties for engineering S. cerevisiae for xylose fermentation. Four XIs originating from rumen and intestinal bacteria were isolated and expressed in a S. cerevisiae CEN.PK2-1C parental strain primed for xylose metabolism by over expression of its native xylulokinase. Three of the XIs were functional in S. cerevisiae, based on the strain’s ability to grow in xylose medium. The most promising strain, expressing the XI mined from Prevotella ruminicola TC2-24 was further adapted for aerobic and fermentative growth by serial transfers of xylose cultures under aerobic, followed by microaerobic conditions. The evolved strain had a specific growth rate of 0.23 h^-1^ on xylose medium, which is comparable to the best reported results for analogous S. cerevisiae strains including those expressing the Piromyces sp. E2 XI. When used to ferment xylose, the adapted strain produced 13.6 g/l ethanol in 96 hr with a metabolic yield of 83% of theoretical. From analysis of the P. ruminicola XI, it was determined the enzyme possessed a Vmax of 0.81 µmole/min/mg protein and a Km of 34 mM. This study identifies a new xylose isomerase from the rumen bacterium Prevotella ruminicola TC2-24 that has one of the highest affinities and specific activities compared to other bacterial and fungal xylose isomerases expressed in yeast. When expressed in S. cerevisiae and used to ferment xylose, very high ethanol yield was obtained. This new XI should be a promising resource for constructing other xylose fermenting strains, including industrial yeast genetic backgrounds.