Xylose is a five-carbon monosaccharide that can be metabolized into useful products by a variety of organisms. One such organism that can naturally metabolize xylose is the yeast Pichia stipitis, which expresses the XYL1 and XYL2 genes that are necessary for the enzymatic breakdown of the xylose sugar. However, ethanol is one of the main products produced by P. stipitis during the catabolism of xylose and it happens to be sensitive to this byproduct. Therefore, Saccharomyces cerevisiae, another yeast strain known to be unaffected by ethanol, was genetically engineered to express the XYL1 and XYL2 genes so that ethanol could be effectively produced without harmful effects on the metabolizing yeast (Eliasson et al, 2000). Alternatively, investigations into the utility of bacterial Rhodococcus strains to produce oil as a byproduct of xylose metabolism are currently underway, which could potentially be a useful biodiesel energy source. However, most knowledge of xylose metabolism has been uncovered through studies involving yeast.
There are two main pathways of xylose metabolism, each unique in the characteristic enzymes they utilize. One pathway is called the “Xylose Reductase-Xylitol Dehydrogenase” or XR-XDH pathway. Xylose reductase (XR) and xylitol dehydrogenase (XDH) are the two main enzymes used in this method of xylose degradation. XR, encoded by the XYL1 gene, is responsible for the reduction of xylose to xylitol and is aided by cofactors NADH or NADPH. Xylitol is then oxidized to xylulose by XDH, which is expressed through the XYL2 gene, and accomplished exclusively with the cofactor NAD+. Because of the varying cofactors needed in this pathway and the degree to which they are available for usage, an imbalance can result in an overproduction of xylitol byproduct and an inefficient production of desirable ethanol. Varying expression of the XR and XDH enzyme levels have been tested in the laboratory in the attempt to optimize the efficiency of the xylose metabolism pathway.
The other pathway for xylose metabolism is called the “Xylose Isomerase” (XI) pathway. Enzyme XI is responsible for direct conversion of xylose into xylulose, and does not proceed via a xylitol intermediate. Both pathways create xylulose, although the enzymes utilized are different. After production of xylulose both the XR-XDH and XI pathways proceed through enzyme xylulokinase (XK), encoded on gene XKS1, to further modify xylulose into xylulose-5-P where it then enters the pentose phosphate pathway for further catabolism.
Studies on flux through the pentose phosphate pathway during xylose metabolism have revealed that limiting the speed of this step may be beneficial to the efficiency of fermentation to ethanol. Modifications to this flux that may improve ethanol production include a) lowering phosphoglucose isomerase activity, b) deleting the GND1 gene, and c) deleting the ZWF1 gene (Jeppsson et al, 2002). Since the pentose phosphate pathway produces additional NADPH during metabolism, limiting this step will help to correct the already evident imbalance between NAD(P)H and NAD+ cofactors and reduce xylitol byproduct. Another experiment comparing the two xylose metabolizing pathways revealed that the XI pathway was best able to metabolize xylose to produce the greatest ethanol yield, while the XR-XDH pathway reached a much faster rate of ethanol production (Karhumaa et al, 2007).
The aim of this genetic recombination in the laboratory is to develop a yeast strain that efficiently produces ethanol. However, the effectiveness of xylose metabolizing laboratory strains do not always reflect their metabolism abilities on raw xylose products in nature. Since xylose is mostly isolated from agricultural residues such as wood stocks then the genetically altered strains will need to be effective at metabolizing these less pure natural sources.
- Eliasson et al., “Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisiae Carrying XYL1, XYL2, and XKS1 in Mineral Medium in Chemostat Cultures.” Applied and Environmental Microbiology, 2000.
- Karhumaa et al., “Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae.” Microbial Cell Factories, 2007.
- Jeppsson et. al., “Reduced Oxidative Pentose Phosphate Pathway Flux in Recombinant Xylose-Utilizing Saccharomyces cerevisiae Strains Improves the Ethanol Yield from Xylose.” Applied and Environmental Microbiology, 2002.