Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli.

The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of bipA, the BiP-encoding gene from Aspergillus niger and Aspergillus awamori. As this result could imply that BiP plays a role in protein overproduction, the effect of modulation of bipA gene expression on protein secretion was studied in several recombinant strains expressing glucoamylase (glaA) fusion genes. For overproduction of BiPA in these strains, extra copies of the bipA gene under the control of an inducible promoter were introduced. To allow analysis of the effect of a decreased bipA expression level on protein secretion, replacement of the wild-type gene for a bipA gene driven by the glaA promoter was attempted. However, this endeavour failed because of the lethality of this replacement. Although the final amount of secreted recombinant protein did not change significantly in strains with increased BiPA levels, increased levels of unprocessed fusion protein were detected in the total protein extracts of these strains.

Cloning, characterization and overexpression of the phytase-encoding gene (phyA) of Aspergillus niger.

Phytase catalyzes the hydrolysis of phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate. A gene (phyA) of Aspergillus niger NRRL3135 coding for extracellular, glycosylated phytase was isolated using degenerate oligodeoxyribonucleotides deduced from phytase amino acid (aa) sequences. Nucleotide (nt) sequence analysis of the cloned region revealed the presence of an open reading frame coding for 467 aa and interrupted once by an intron of 102 bp in the 5' part of the gene. The start codon is followed by a sequence coding for a putative signal peptide. Expression of phyA is controlled at the level of mRNA accumulation in response to inorganic phosphate levels. After cell growth in low-phosphate medium, a transcript of about 1.8 kb was visualized. Transcription of phyA initiates at at least seven start points within a region located 45-25 nt upstream from the start codon. In transformants of A. niger, expression of multiple copies of phyA resulted in up to more than tenfold higher phytase levels than in the wild-type strain.