Monday, July 30, 2007 - 9:00 AM
S9

Evolution in Reverse: Engineering a Xylose-Specific Xylose Reductase

Huimin Zhao, Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL 61801

Xylitol is a five-carbon sugar alcohol with many industrial applications.  However, it is still relatively expensive to produce either by chemical synthesis or by fermentation. To address this limitation, we are using protein engineering techniques to create a xylose reductase (XR) mutant with decreased specificity toward L-arabinose, while maintaining its high activity toward D-xylose such that a mixture of pentose (D-xylose and L-arabinose) derived from renewable plant biomass can directly be used as substrates.  The Neurospora crassa XR was chosen for protein 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.

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