Molecular characterization of a PDI-related gene prpA in Aspergillus niger var. awamori

Abstract

A gene (prpA) homologous to the protein disulfide isomerase gene was isolated from Aspergillus niger by Southern hybridization using the pdi1 gene isolated from Trichoderma reesei as a DNA probe. The corresponding cDNA of the prpA gene has also been isolated from an A. niger var. awamori cDNA library. The prpA gene does not belong to any currently recognized family of protein disulfide isomerases since it contains only a single conserved thioredoxin domain at the N-terminus of the protein. The C-terminal two-thirds of the protein has no homology to any known proteins in the database. The PRPA protein contains an ER retention signal (HDEL) at its C-terminal end suggesting that it is located in the ER. Southern hybridization at high stringency showed that it was present as a single copy in the genome. Northern hybridization indicated that the transcript level of the prpA gene was higher if the cells were secreting a heterologous protein, bovine prochymosin. However, over-expression of the prpA gene from a multicopy integrated vector had little effect on chymosin secretion. A strain containing a deletion of the prpA gene was viable. However, deletion of the prpA gene appeared to cause a reduction of bovine chymosin production.

Cloning and Characterization of the Glucosidase II Alpha Subunit Gene of Trichoderma reesei : a Frameshift Mutation Results in the Aberrant Glycosylation Profile of the Hypercellulolytic Strain Rut-C30

“…In fungi, the production of heterologous proteins sometimes induces a UPR (52,70,91,92). As glucosidase II is involved in the quality control of protein folding in the ER, it might be required at elevated levels under such conditions.…”

“…The resulting mRNA is translated into a functional Hac1p that acts as a positive transcription factor for genes under control of the UPR (reviewed in references 43 and 61). Recent results indicate that a similar mechanism in filamentous fungi (74) induces elevated synthesis of chaperones (18,32,35,52,70,73,92) and other proteins involved in different processes in the secretion pathway (75). However, with the exception of one report on the Aspergillus niger calnexin gene (91), little is known about the sensitivity of several other partners of the calnexin-related quality control system (such as glucosidase II and GT) to the accumulation of unfolded proteins and to secretion stress in general.…”

Transcriptome Analysis of Recombinant Protein Secretion by Aspergillus nidulans and the Unfolded-Protein Response In Vivo

“…Disruption of prpA has been shown to reduce bovine chymosin production in A. niger, but overexpression had little effect (54). Perhaps coregulation is important, since constitutive expression of the entire UPR in a recombinant Aspergillus niger var.…”

Genomics ofAspergillus oryzae

“…The various genes encoding ER resident chaperones (Bip, Hsp70, and calnexin homologs) and foldases (protein disulfide isomerases and peptidylprolyl isomerase) for the quality control activity, apart from other secretion-pathway related genes, have been found in the genome of A. oryzae (Table 7). Among the three genes (AO090001000733, AO090001000733, and AO090003000829) classified as protein disulfide iso- merases, at least one of them (AO090003000829) appears to be unique to filamentous fungi, as reported, 144) with only a single thioredoxin domain in contrast to the two thioredoxin domains present in the others. In addition, the yeast ERO1 homolog reported to be essential for oxidative protein folding was also present in the A. oryzae genome (AO090005000630).…”