Characterization of the Aspergillus niger pelB gene: structure and regulation of expression.
The nucleotide sequence of pelB, a member of the Aspergillus niger pectin lyase multigene family, has been determined. The pelB gene product, PLB, shares 65% amino acid identity with pectin lyase A (PLA) and 60% with pectin lyase D (PLD). Although growth of pelB multicopy transformants on pectin-containing media results in elevated pelB mRNA levels, pectin lyase B (PLB) is barely detectable. This is probably due to degradation of PLB by acid proteases, since multicopy transformants grown on pectin medium with a high concentration of phosphate, leading to a less rapid decline in pH, secrete detectable amounts of PLB. To produce PLB in high amounts under conditions where few other extracellular enzymes are present, we tried two strategies. Firstly, heterologous expression of the pelB gene in A. nidulans, and secondly, expression of the pelB gene under control of the constitutive A. niger pki promoter.
Kusters-van Someren
M
M
Department of Genetics, Wageningen Agricultural University, The Netherlands.
Flipphi
M
M
de Graaff
L
L
van den Broeck
H
H
Kester
H
H
Hinnen
A
A
Visser
J
J
D000595:Amino Acid Sequence; D001233:Aspergillus nidulans; D001234:Aspergillus niger / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004271:DNA, Fungal; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D007438:Introns; D008969:Molecular Sequence Data; D010957:Plasmids; D011133:Polysaccharide-Lyases / Q000235:genetics*; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics*; D011770:Pyruvate Kinase / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D014170:Transformation, Genetic
D004271:DNA, Fungal; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011770:Pyruvate Kinase; D011133:Polysaccharide-Lyases; C027349:pectin lyase; C074124:pectin lyase B