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.2013 May 30;6(1):84.
doi: 10.1186/1754-6834-6-84.

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

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

Ronald E Hector et al. Biotechnol Biofuels..

Abstract

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 Vmax of 0.81 μmole/min/mg protein and a Km 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.

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Figures

Figure 1
Figure 1
Comparison ofSaccharomyces cerevisiaestrains engineered to express various D-xylose isomerase and D-xylulokinase genes. A) Strains were cultured under aerobic conditions using YP medium with 50 g/L D-xylose. Cultures were incubated at 30°C, shaking at 1000 rpm using a BioLector®. Cell density was measured every 30 minutes. Data shown are mean values from experiments performed in triplicate.B) Strains were cultured under microaerobic conditions using YP medium with 50 g/L D-xylose. Cultures were incubated at 30°C using a Bioscreen C™. Cell density was measured every 30 minutes. Data shown are the average values from three biological replicates. The standard deviation for most values was less than 5%. PanelB uses the same legend as in panelA.
Figure 2
Figure 2
Adaptation of strain YRH631 to create strain YRH1114. Strain YRH631, expressing thePrevotella ruminicola XI and XK genes was cultured under microaerobic conditions and passaged every seven days. Remaining D-xylose and the fermentation products ethanol and xylitol was measured prior to each passage. Data shown are from one of two replicates.
Figure 3
Figure 3
Growth curves for the adaptedSaccharomyces cerevisiaestrain engineered to express theP. ruminicolaD-xylose isomerase and D-xylulokinase genes. A) Strains were cultured under aerobic conditions in YP medium with 50 g/L D-xylose. Cultures were incubated at 30°C, shaking at 1000 rpm using a BioLector®. Cell density was measured every 30 minutes. Data shown are mean values from experiments performed in triplicate.B) Strains were cultured under microaerobic conditions using YP medium with 50 g/L D-xylose. Cultures were incubated at 30°C using a Bioscreen C™. Cell density was measured every 30 minutes. Data shown are the average values from three biological replicates. The standard deviation for most values was less than 5%. PanelB uses the same legend as in panelA.
Figure 4
Figure 4
Comparison of D-xylose fermentation usingSaccharomyces cerevisiaestrains engineered to express theP. ruminicolaD-xylose isomerase and D-xylulokinase genes vs. expression of theScheffersomyces stipitisD-xylose reductase and xylitol dehydrogenase genes. Fermentations were performed using YP medium with 50 g/L D-xylose. Pressure was measured every 15 minutes and converted to mmoles of CO2. Data shown are from a single representative fermentation from experiments performed in triplicate.
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