Xylitol is a five-carbon sugar alcohol (pentitol) used as a natural sweetener in food, pharmaceutical, and confectionery industries. It has garnered significant interest due to its anti-cariogenic properties as it cannot be metabolized by Streptococci, Lactobacilli and yeasts found in the mouth. In addition, its metabolism is insulin-independent, making it a desirable sugar substitute. The raw material for xylitol production, D-xylose, is available abundantly as it is the predominant sugar composing hemicellulose (up to 50% of corn fiber and 90% of birch wood) and is reduced readily to xylitol. Currently, xylitol is produced at the industrial scale by chemical reduction of purified D-xylose with elemental hydrogen over Raney-Ni catalyst at 135°C and 40atm pressure. In lieu of utilizing the pyrophoric and carcinogenic Raney-Ni catalyst, several attempts have been made at developing safer techniques such as yeast and recombinant bacterial fermentation, and in vitro enzymatic reduction, using xylose reductase (XR) for the same reaction. However, the major expense in producing xylitol arises due to the nonspecific nature of chemical reduction, which remains unaddressed by the biological techniques utilizing highly promiscuous XRs, thus requiring the expensive purification of D-xylose from its 4-epimer, L-arabinose, to avoid formation of undesirable byproducts.

Like most enzymes catalyzing the first step in sugar metabolism, the broad substrate acceptance of XR is evolutionarily advantageous to organisms, but disadvantageous for a chemical process attempting to minimize byproduct formation. The goal of this project is therefore to reverse the evolutionary advantage conferred by using protein engineering techniques to decrease the specificity of a XR toward L-arabinose, while maintaining high activity toward D-xylose. The Neurospora crassa XR (NcXR) was chosen for engineering work due to several favorable properties over other XRs, in addition to its innate >2-fold catalytic efficiency toward D-xylose than L-arabinose. A directed evolution strategy was developed that consists of a combined structure-function based semi-rational design involving active site residue mutagenesis followed by random mutagenesis and selection for desired substrate specificity. After the first round of evolution, a mutant was identified with fourteen-fold preference for D-xylose over L-arabinose. To our knowledge this is the most xylose-specific XR identified or engineered to date. Further engineering rounds are currently underway on this template to further reduce its promiscuity.