Cloning and expression in Saccharomyces cerevisiae of the NAD(P)H-dependent xylose reductase-encoding gene (XYL1) from the xylose-assimilating yeast Pichia stipitis.
The XYL1 gene of the yeast Pichia stipitis has been isolated from a genomic library using a specific cDNA probe, and its nucleotide (nt) sequence has been determined. In the 5' noncoding region of the P. stipitis XYL1 gene a TATAAA element (known to be necessary for transcription initiation in most yeast genes) is found at nt -81, and two CCAAT recognition motifs (often referred to as the CCAAT box) are present at nt -146 and -106. The XYL1 encodes a polypeptide of 35,927 Da that constitutes a NADH/NADPH-dependent xylose reductase (XR). The enzyme is part of the xylose-xylulose pathway that is absent or only weakly expressed in Saccharomyces cerevisiae. Extensive homology is found to the N terminus of the XR of Pachysolen tannophilus and Candida shehatae. None of the known cofactor binding domains found in many NAD-dependent dehydrogenases are present in the protein. Transformants of S. cerevisiae containing XYL1 of P. stipitis synthesize an active XR. Fusion of XYL1 with the prokaryotic tac promoter leads to a gene that can be expressed in S. cerevisiae and Escherichia coli.
Amore
R
R
Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, F.R.G.
Kötter
P
P
Küster
C
C
Ciriacy
M
M
Hollenberg
C P
CP
D000449:Aldehyde Reductase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002175:Candida / Q000235:genetics; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded; D015870:Gene Expression; D015723:Gene Library; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic
D004277:DNA, Single-Stranded; D011994:Recombinant Proteins; D009249:NADP; D000449:Aldehyde Reductase