Molecular characterization of a gene for aldose reductase ( CbXYL1) from Candida boidinii and its expression in Saccharomyces cerevisiae.
Candida boidinii produces significant amounts of xylitol from xylose, and assays of crude homogenates for aldose (xylose) reductase (XYL1p) have been reported to show relatively high activity with NADH as a cofactor even though XYL1p purified from this yeast does not have such activity. A gene coding for XYL1p from C. boidinii (CbXYL1) was isolated by amplifying the central region using primers to conserved domains and by genome walking. CbXYL1 has an open reading frame of 966 bp encoding 321 amino acids. The C. boidinii XYL1p is highly similar to other known yeast aldose reductases and is most closely related to the NAD(P)H-linked XYL1p of Kluyveromyces lactis. Cell homogenates from C. boidinii and recombinant Saccharomyces cerevisiae were tested for XYL1p activity to confirm the previously reported high ratio of NADH:NADPH linked activity. C. boidinii grown under fully aerobic conditions showed an NADH:NADPH activity ratio of 0.76, which was similar to that observed with the XYL1p from Pichia stipitis XYL1, but which is much lower than what was previously reported. Cells grown under low aeration showed an NADH:NADPH activity ratio of 2.13. Recombinant S. cerevisiae expressing CbXYL1 showed only NADH-linked activity in cell homogenates. Southern hybridization did not reveal additional bands. These results imply that a second, unrelated gene for XYL1p is present in C. boidinii.
Kang
Min Hyung
MH
Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, USA.
Ni
Haiying
H
Jeffries
Thomas W
TW
D000449:Aldehyde Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002175:Candida / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014993:Xylitol / Q000378:metabolism; D014994:Xylose / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D014994:Xylose; D000449:Aldehyde Reductase; D014993:Xylitol