Characterization and overexpression of the Aspergillus niger gene encoding the cAMP-dependent protein kinase catalytic subunit.
The gene pkaC encoding the catalytic subunit of cAMP-dependent protein kinase has been isolated from the industrially important filamentous fungus Aspergillus niger. A probe for screening A. niger phage libraries was generated by a polymerase chain reaction using degenerate primers. cDNA and genomic DNA clones were isolated and sequenced. An open reading frame of 1440 bp, interrupted by three short introns, encodes a polypeptide of 480 amino acids with a calculated molecular mass of 53813 Da. The cAMP-dependent protein kinase catalytic subunit (PKA-C) from A. niger has a 126 amino acid extension at the N-terminus compared to the PKA-C of higher eukaryotes that-except for the first 15 amino acids, which are homologous to the Magnaporthe grisea PKA-C-shows no significant similarity to the N-terminal extension of PKA-C of other lower eukaryotes. The catalytic core of PKA-C of A. niger shows extensive homology with the PKA-C isolated from all other eukaryotes. Low-stringency hybridization did not reveal any other pkaC homologue in A. niger. The cloned pkaC was used for transformation of A. niger, leading to increased levels of pkaC mRNA and PKA-C activity. Transformants overexpressing pkaC were phenotypically different with respect to growth, showing a more compact colony morphology, accompanied by a more dense sporulation, especially on media containing trehalose and glycerol. A number of transformants also showed a strongly reduced or complete absence of sporulation. This phenotype was quickly lost upon propagation of the strains.
Bencina
Mojca
M
National Institute of Chemistry, Hajdrihova 19, SLO-61115 Ljubljana, Slovenia.|Wageningen Agricultural University, Section Molecular Genetics of Industrial Microorganisms, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands.
Panneman
Henk
H
Wageningen Agricultural University, Section Molecular Genetics of Industrial Microorganisms, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands.
Ruijter
George J G
GJG
Wageningen Agricultural University, Section Molecular Genetics of Industrial Microorganisms, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands.
Legiša
Matic
M
National Institute of Chemistry, Hajdrihova 19, SLO-61115 Ljubljana, Slovenia.
Visser
Jaap
J
Wageningen Agricultural University, Section Molecular Genetics of Industrial Microorganisms, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands.
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000166:cytology / Q000235:genetics*; D001483:Base Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D005800:Genes, Fungal; D015698:Genomic Library; D006019:Glycolysis; D008969:Molecular Sequence Data; D010641:Phenotype; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013172:Spores, Fungal; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D018076:DNA, Complementary; D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases