Transcriptome analysis of recombinant protein secretion by Aspergillus nidulans and the unfolded-protein response in vivo

Abstract

Filamentous fungi have a high capacity for producing large amounts of secreted proteins, a property that has been exploited for commercial production of recombinant proteins. However, the secretory pathway, which is key to the production of extracellular proteins, is rather poorly characterized in filamentous fungi compared to yeast. We report the effects of recombinant protein secretion on gene expression levels in Aspergillus nidulans by directly comparing a bovine chymosin-producing strain with its parental wild-type strain in continuous culture by using expressed sequence tag microarrays. This approach demonstrated more subtle and specific changes in gene expression than those observed when mimicking the effects of protein overproduction by using a secretion blocker. The impact of overexpressing a secreted recombinant protein more closely resembles the unfolded-protein response in vivo.

FIG. 1.

Growth and chymosin production by recombinant and parental strains in batch shake flask culture. Samples were taken from both strains at times shown for comparative transcriptome analysis. ln (dry weight) and chymosin concentration are calculated from the average of three cultures, and error bars represent standard deviations.

FIG. 2.

Aspergillus genes in GenBank thought to be involved in recombinant protein secretion in batch shake flask culture. Red and green represent up- and down-regulation relative to white, which is equal to no change.

FIG. 3.

Venn diagrams showing the number of sequences with increased expression when the parental and recombinant protein-producing strains in shake flask and chemostat cultures are compared. The circle area is proportional to the number of sequences. ESTs and PCR products from the first-generation arrays are also included on the second-generation arrays. Gene lists are available at http://www.cogeme.man.ac.uk.

FIG. 4.

Venn diagrams showing the number of up-regulated spots when different approaches for induction of UPR are compared using the G2 array. The circle area is proportional to the number of spots. Gene lists are available at http://www.cogeme.man.ac.uk.

FIG. 5.

Model of the secretory pathway and UPR under different conditions. (A) Recombinant protein secretion induction of UPR. (B) Secretion blocker induction of UPR. Boxes represent known and putative secretion genes. Orthologs to yeast genes are in italics. Red, genes up-regulated; green, genes down-regulated; white, no significant change; grey, not present on the microarray. lE, late endosome, eE, early endosome.

FIG. 6.

Venn diagrams showing the number of transcripts with reduced levels when induction of the UPR is compared using the 2G array. (A) All spots. (B) Spots representing secreted proteins. The circle area is proportional to the number of spots. Gene lists are available at http://www.cogeme.man.ac.uk.