초록
<P><B>Background</B></P><P><I>Saccharomyces cerevisiae</I> 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 <I>S</I>. <I>cerevisiae</I> 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 <I>S</I>. <I>cerevisiae</I> for D-xylose fermentation.</P><P><B>Results</B></P><P>Four XIs originating from rumen and intestinal bacteria were isolated and expressed in a <I>S</I>. <I>cerevisiae</I> 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 <I>S</I>. <I>cerevisiae</I>, based on the strain’s ability to grow in D-xylose medium. The most promising strain, expressing the XI mined from <I>Prevotella ruminicola</I> 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<SUP>-1</SUP> on D-xylose medium, which is comparable to the best reported results for analogous <I>S</I>. <I>cerevisiae</I> strains including those expressing the <I>Piromyces</I> 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 <I>P</I>. <I>ruminicola</I> XI, it was determined the enzyme possessed a <I>V</I><SUB><I>max</I></SUB> of 0.81 μmole/min/mg protein and a <I>K</I><SUB><I>m</I></SUB> of 34 mM.</P><P><B>Conclusion</B></P><P>This study identifies a new xylose isomerase from the rumen bacterium <I>Prevotella ruminicola</I> 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 <I>S</I>. <I>cerevisiae</I> 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.</P>