An inventory of the Aspergillus niger secretome by combining in silico predictions with shotgun proteomics data.

BACKGROUND: The ecological niche occupied by a fungal species, its pathogenicity and its usefulness as a microbial cell factory to a large degree depends on its secretome. Protein secretion usually requires the presence of a N-terminal signal peptide (SP) and by scanning for this feature using available highly accurate SP-prediction tools, the fraction of potentially secreted proteins can be directly predicted. However, prediction of a SP does not guarantee that the protein is actually secreted and current in silico prediction methods suffer from gene-model errors introduced during genome annotation. RESULTS: A majority rule based classifier that also evaluates signal peptide predictions from the best homologs of three neighbouring Aspergillus species was developed to create an improved list of potential signal peptide containing proteins encoded by the Aspergillus niger genome. As a complement to these in silico predictions, the secretome associated with growth and upon carbon source depletion was determined using a shotgun proteomics approach. Overall, some 200 proteins with a predicted signal peptide were identified to be secreted proteins. Concordant changes in the secretome state were observed as a response to changes in growth/culture conditions. Additionally, two proteins secreted via a non-classical route operating in A. niger were identified. CONCLUSIONS: We were able to improve the in silico inventory of A. niger secretory proteins by combining different gene-model predictions from neighbouring Aspergilli and thereby avoiding prediction conflicts associated with inaccurate gene-models. The expected accuracy of signal peptide prediction for proteins that lack homologous sequences in the proteomes of related species is 85%. An experimental validation of the predicted proteome confirmed in silico predictions.

Assessment of the pectin degrading enzyme network of Aspergillus niger by functional genomics.

The saprobic fungus Aspergillus niger is an efficient producer of a suite of extracellular enzymes involved in carbohydrate modification and degradation. Genome mining has resulted in the prediction of at least 39 genes encoding enzymes involved in the depolymerisation of the backbone of pectin. Additional genes,encoding enzymatic activities required for the degradation of the arabinan and arabinogalactan sidechains were predicted as well. DNA microarray analysis was used to study the condition-dependent expression of these genes, and to generate insights in possible synergistic interactions between the individual members of the pectin degrading enzyme network. For this purpose, A. niger was grown on sugarbeet pectin and on galacturonic acid, rhamnose and xylose, the main monomeric sugar constituents of pectin. An analysis of the corresponding transcriptomes revealed expression of 46 genes encoding pectinolytic enzymes. Their transcriptional profiles are discussed in detail and a cascade model of pectin degradation is proposed.

An evolutionary conserved d-galacturonic acid metabolic pathway operates across filamentous fungi capable of pectin degradation.

Transcriptome analysis of Aspergillus niger transfer cultures grown on galacturonic acid media identified a highly correlating cluster of four strongly induced hypothetical genes linked with a subset set of genes encoding pectin degrading enzymes. Three of the encoded hypothetical proteins now designated GAAA to GAAC are directly involved in further galacturonic acid catabolism. Functional and biochemical analysis revealed that GAAA is a novel d-galacturonic acid reductase. Two non-allelic Aspergillus nidulans strains unable to utilize galacturonic acid are mutated in orthologs of gaaA and gaaB, respectively. The A. niger gaaA and gaaC genes share a common promoter region. This feature appears to be strictly conserved in the genomes of plant cell wall degrading fungi from subphylum Pezizomycotina. Combined with the presence of homologs of the gaaB gene in the same set of fungi, these strongly suggest that a common d-galacturonic acid utilization pathway is operative in these species.

A new group of exo-acting family 28 glycoside hydrolases of Aspergillus niger that are involved in pectin degradation.

The fungus Aspergillus niger is an industrial producer of pectin-degrading enzymes. The recent solving of the genomic sequence of A. niger allowed an inventory of the entire genome of the fungus for potential carbohydrate-degrading enzymes. By applying bioinformatics tools, 12 new genes, putatively encoding family 28 glycoside hydrolases, were identified. Seven of the newly discovered genes form a new gene group, which we show to encode exoacting pectinolytic glycoside hydrolases. This group includes four exo-polygalacturonan hydrolases (PGAX, PGXA, PGXB and PGXC) and three putative exo-rhamnogalacturonan hydrolases (RGXA, RGXB and RGXC). Biochemical identification using polygalacturonic acid and xylogalacturonan as substrates demonstrated that indeed PGXB and PGXC act as exo-polygalacturonases, whereas PGXA acts as an exo-xylogalacturonan hydrolase. The expression levels of all 21 genes were assessed by microarray analysis. The results from the present study demonstrate that exo-acting glycoside hydrolases play a prominent role in pectin degradation.