Xylitol is a five-carbon sugar alcohol and has been used as a sugar substitute in the food industry because of its low caloric and anti-carcinogenic characteristics. In addition, xylitol is a building block for a variety of commodity chemicals. Biological routes for producing xylitol from cellulosic hydrolysates have been developed through metabolic engineering of Saccharomyces cerevisiae. S. cerevisiae cannot utilize xylose as a carbon source, but the expression of a xylose reductase (XR) gene from Pichia stipitis facilitates the production of xylitol from xylose with high yields. However, insufficient supply of NAD(P)H for XR, and inhibition of xylose transport by glucose, are major constraints for high productivity production of xylitol.
To overcome these problems, we engineered S. cerevisiae to convert xylose into xylitol through the simultaneous consumption of cellobiose and xylose. Specifically, xylose reductase from P. stipitis was chromosomally integrated into the S. cerevisiae genome, while a cellobiose transporter (cdt-1) and an intracellular β-glucosidase (gh1-1) from Neurospora crassa were introduced on a multi-copy plasmid. The resulting transformant was able to produce xylitol from a mixture of cellobiose and xylose. In contrast to glucose, cellobiose did not repress the transport of xylose, allowing the co-consumption of xylose and cellobiose. The co-consumption and metabolism of cellobiose regenerated NADPH for XR. As a result, an engineered S. cerevisiae strain (D-10-BT) exhibited 40% higher xylitol productivity when cellobiose was used as a co-substrate as compared to glucose. This result suggests that co-consumption of cellobiose and xylose allows efficient xylose uptake and cofactor regeneration without catabolic repression for the production of xylitol.
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