Cloning, sequencing, and regulation of a xylanase gene from the fungus Aureobasidium pullulans Y-2311-1.
Aureobasidium pullulans Y-2311-1 growing on xylan secretes four major xylanases with different masses and isoelectric points. Two of these enzymes, named APX-I and APX-II, have been purified previously. Their N-terminal amino acid sequences are identical except that APX-I has Asp and APX-II has Asn at position 7. An 83-bp DNA region was amplified by PCR and used as a probe for the xylanase gene cloning. The longest cDNA (xynA) obtained by cDNA cloning and PCR amplification consisted of 895 bp. A. pullulans xynA had an open reading frame encoding a polypeptide of 221 amino acids with a calculated mass of 23,531 Da and contained a putative 34-amino-acid signal peptide in front of the amino terminus of the mature enzyme. Strong homology was found between the deduced amino acid sequence of XynA and some xylanases from bacterial and fungal sources. It is suggested that A. pullulans XynA belongs to the family G glycanases. Northern (RNA blot) analysis revealed that only one transcript of 900 bases was present in cultures grown in medium containing D-xylose or oat spelt xylan. Transcription was completely repressed in the presence of glucose in the medium. Southern blot analysis indicated that A. pullulans xynA was present as a single copy in the genome. Comparison between the genomic and cDNA sequences revealed that one intron of 59 bp was present in the coding region. The data presented suggest that the highly active xylanases, APX-I and APX-II, secreted by A. pullulans are encoded by the same gene.
Li
X L
XL
Department of Biochemistry, University of Georgia, Athens 30602-7229.
Ljungdahl
L G
LG
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D006026:Glycoside Hydrolases / Q000235:genetics*; D003904:Mitosporic Fungi / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D043325:Xylan Endo-1,3-beta-Xylosidase
D003062:Codon; D017931:DNA Primers; D018076:DNA, Complementary; D004271:DNA, Fungal; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase