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

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.

Combining transcriptome data with genomic and cDNA sequence alignments to make confident functional assignments for Aspergillus nidulans genes.

Whole genome sequencing of several filamentous ascomycetes is complete or in progress; these species, such as Aspergillus nidulans, are relatives of Saccharomyces cerevisiae. However, their genomes are much larger and their gene structure more complex, with genes often containing multiple introns. Automated annotation programs can quickly identify open reading frames for hypothetical genes, many of which will be conserved across large evolutionary distances, but further information is required to confirm functional assignments. We describe a comparative and functional genomics approach using sequence alignments and gene expression data to predict the function of Aspergillus nidulans genes. By highlighting examples of discrepancies between the automated genome annotation and cDNA or EST sequencing, we demonstrate that the greater complexity of gene structure in filamentous fungi demands independent data on gene expression and the gene sequence be used to make confident functional assignments.

Glutamic protease distribution is limited to filamentous fungi.

Glutamic proteases are a distinct, and recently re-classified, group of peptidases that are thought to be found only in fungi. We have identified and analysed the distribution of over 20 putative glutamic proteases from all fungal species whose genomes have been sequenced so far. Although absent from the Saccharomycetales class, glutamic proteases appear to be present in all other ascomycetes species examined. A large number of coding regions for glutamic proteases were also found clustered together in the Phanerochaete chrysosporium genome, despite apparently being absent from three other species of Basidiomycota.

Use of expressed sequence tag analysis and cDNA microarrays of the filamentous fungus Aspergillus nidulans.

The use of microarrays in the analysis of gene expression is becoming widespread for many organisms, including yeast. However, although the genomes of a number of filamentous fungi have been fully or partially sequenced, microarray analysis is still in its infancy in these organisms. Here, we describe the construction and validation of microarrays for the fungus Aspergillus nidulans using PCR products from a 4092 EST conidial germination library. An experiment was designed to validate these arrays by monitoring the expression profiles of known genes following the addition of 1% (w/v) glucose to wild-type A. nidulans cultures grown to mid-exponential phase in Vogel's minimal medium with ethanol as the sole carbon source. The profiles of genes showing statistically significant differential expression following the glucose up-shift are presented and an assessment of the quality and reproducibility of the A. nidulans arrays discussed.

Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei.

The filamentous fungus Trichoderma reesei produces and secretes profuse quantities of enzymes that act synergistically to degrade cellulase and related biomass components. We partially sequenced over 5100 random T. reesei cDNA clones. Among the sequences whose predicted gene products had significant similarity to known proteins, 12 were identified that encode previously unknown enzymes that likely function in biomass degradation. Microarrays were used to query the expression levels of each of the sequences under different conditions known to induce cellulolytic enzyme synthesis. Most of the genes encoding known and putative biomass-degrading enzymes were transcriptionally co-regulated. Moreover, despite the fact that several of these enzymes are not thought to degrade cellulase directly, they were coordinately overexpressed in a cellulase overproducing strain. A variety of additional sequences whose function could not be ascribed using the limited sequence available displayed analogous behavior and may also play a role in biomass degradation or in the synthesis of biomass-degrading enzymes. Sequences exhibiting additional regulatory patterns were observed that might reflect roles in regulation of cellulase biosynthesis. However, genes whose products are involved in protein processing and secretion were not highly regulated during cellulase induction.