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Background   Saccharomyces cerevisiae  strains expressing D-xylose isomerase (XI) produce some of the highest reported ethanol yields from D-xylose. Unfortunately, most bacterial XIs that have been expressed in  S .  cerevisiae  are either not functional, require additional strain modification, or have low affinity for D-xylose. This study analyzed several XIs from rumen and intestinal microorganisms to identify enzymes with improved properties for engineering  S .  cerevisiae  for D-xylose fermentation.    Results  Four XIs originating from rumen and intestinal bacteria were isolated and expressed in a  S .  cerevisiae  CEN.PK2-1C parental strain primed for D-xylose metabolism by over expression of its native D-xylulokinase. Three of the XIs were functional in  S .  cerevisiae , based on the strain’s ability to grow in D-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 D-xylose cultures under aerobic, and followed by microaerobic conditions. The evolved strain had a specific growth rate of 0.23 h -1  on D-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 D-xylose, the adapted strain produced 13.6 g/L ethanol in 91 h with a metabolic yield of 83% of theoretical. From analysis of the  P .  ruminicola  XI, it was determined the enzyme possessed a  V   max   of 0.81 μmole/min/mg protein and a  K   m   of 34 mM.    Conclusion  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 D-xylose isomerases expressed in yeast. When expressed in  S .  cerevisiae  and used to ferment D-xylose, very high ethanol yield was obtained. This new XI should be a promising resource for constructing other D-xylose fermenting strains, including industrial yeast genetic backgrounds.

biotechnology for biofuels

Growth and fermentation of D-xylose by Saccharomyces cerevisiae expressing a novel D-xylose isomerase originating from the bacterium Prevotella ruminicola TC2-24