earticle

영어

Xylose reductase (XR) is a key enzyme in xylitol production, catalyzing the reduction of D-xylose to xylitol. However, most XRs from yeasts and fungi have higher activity for L-arabinose than D-xylose and this broad substrate acceptance causes formation of byproduct, arabitol.
Neurospora crassa XR(NcXR) is known to have high overall catalytic efficiency and higher activity toward D-xylose than L-arabinose significantly. C. tropicalis and N. crassa have different patterns in codon usage and three codons, CCC(P), CGC(R) and CTC(L) are particularly
expected to be problems in functional expression of NcXR. Consequently, all codons of NcXR were changed into preferred codons in C. tropicalis. A promoter region of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was isolated from the genomic DNA of C. tropicalis ATCC 20913 to construct a constitutive expression cassette for this strain. The codon-optimized NcXR(NXRG) gene was placed under the control of GAPDH promoter and integrated into the genome of C. tropicalis N43 which is xyl2-disrupted and xyl1-partially disrupted mutant. Expression of NXRG was confirmed by determining enzyme activity in cells grown on a medium containing glucose as a carbon source. The resulting recombinant yeast, C. tropicalis NG4, showed
higher XR activity (1287mU/mg of proteins) than that of the parental strain (26mU/mg of proteins) and 3.6 times higher substrate specificity for D-xylose than L-arabinose.