earticle

영어

Xylose reductase (XR) is a key enzyme in xylitol production, catalyzing the reduction of D-xylose to xylitol. However, the XRs in most yeast and filamentous fungi have evolved to have broad substrate specificity for efficient reduction of a number of pentoses and hexoses. The promiscuous property of these enzymes could adversely affect the biological production of xylitol. The hemicelluloses, major resources of xylose, also contain abundant L-arabinose and it can be reduced to arabitol, an undesirable byproduct. Arabitol, epimer of xylitol, is difficult to remove, and requires high cost in downstream process. Candida tropicalis XR also have a higher activity for L-arabinose than D-xylose, and this substrate specificity causes the formation of an arabitol byproduct. Neurospora crassa XR (NcXR) has significantly higher activity toward D-xylose than L-arabinose. However, because C. tropicalis and N. crassa have different patterns of codon usage, all NcXR codons were changed into preferred codons in C. tropicalis. In order to change substrate specificity of optimized NcXR (NXRG), point mutations were introduced by site-directed mutagenesis. Expression cassettes harboring mutated NXRGs were integrated into the genome of XYL1-disrupted C. tropicalis.
The resulting recombinant yeasts showed changed substrate specificity toward xylose and arabinose. In xylitol fermentation, they showed lower arabitol/xylitol ratio than that of the parental strain.