Characterization and expression of a genomic pectin methyl esterase-encoding gene in Aspergillus niger.
The genomic pectin methylesterase (PME)-encoding gene (pmeA) from Aspergillus niger strain RH5344 was cloned by probing a genomic DNA library with a cDNA coding for PME. The recombinant phage clone was isolated and a 6-kb HindIII fragment was subcloned and characterized. The gene consists of seven exons and six introns. The nucleotide sequences of the coding regions were identical to those found in the pmeA cDNA. Cotransformation of A. niger was achieved with the vector, pAN7-1, and transformants were then tested for PME production. Transformants which produced more PME than the untransformed recipient strain were subjected to Southern-blot and Northern-blot analysis. The results show that there is a reasonable correlation between gene copy number, mRNA levels and PME production. PME was produced by A. niger transformants in an active 43-kDa form, which is similar to that of the mature protein isolated from the strain, RH5344. On the basis of the results of affinity labeling of PME with sugar-specific lectins and the amino acid sequence data, it has been revealed that PME is a glycoprotein and the protein-bound glycans are oligosaccharides with a high mannose content.
Institut für Biochemie, Technische Hochschule Darmstadt, F.R.G.
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Khanh
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Ruttkowski
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Leidinger
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Albrecht
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Gottschalk
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002265:Carboxylic Ester Hydrolases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic
D000345:Affinity Labels; D004271:DNA, Fungal; D012331:RNA, Fungal; D012333:RNA, Messenger; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase