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Filamentous fungi can be found in the majority of habitats of our planet. The wide-spread presence of filamentous fungi is related to their versatile metabolism, which allows them to grow on simple substrates, such as nitrate, acetate, ethanol, ammonia, or on complex matter such as biopolymers from plant or animal tissues. In order to grow on complex biopolymers such as plant cell wall polysaccharides, fungi must secret hydrolytic and modifying enzymes. These enzymes allow polysaccharide degradation and subsequent internalization of simpler molecules, such as sugar monomers. The filamentous fungus Aspergillus niger has been the subject of intense research in the past decades. This organism is responsible for the largest production of citric acid worldwide. In addition to this, A. niger produces high amounts of enzymes with important applications in the bioindustry, such as enzymes for food and feed processing, or enzymes used for the simultaneous saccharification and fermentation of cellulose for bioethanol production. The secretion of extracellular enzymes in A. niger has been mostly focused on the prediction of gene function based on genome annotation and on the analysis of gene expression. However, there is a gap in the knowledge of all the proteins present in cell, given by proteomics. The aim of the work presented in this thesis was to use a functional genomics approach to identify genes and proteins involved in protein secretion in A. niger and to investigate the dynamic changes of the secretory proteome under high-secretion conditions. For this purpose, we used a combination of gene expression profiling with shotgun proteomics of secretory organelles. Chapter 2 describes a method for gene silencing in filamentous fungi via RNA interference. This method makes use of vectors which express long hairpin RNAs. In A. niger, gene knock-out strategies have been the main method for the determination of gene function. These strategies have proven to be particularly useful when carried out in strains with defective pathways for non-homologous integration, such as the kusA mutant. Nevertheless, a gene knock-down strategy such as the one described in chapter 2 could be relevant for the study of gene function, for two reasons: a) essential genes could be studied as RNAi does not necessarily lead to loss-of-function, and b) multiple gene copies of a gene or paralogous genes could be targeted with a single construct. In our work, the gene coding the transcriptional activator of hemicellulases XlnR was silenced. Gene silencing resulted in various degrees of hemicellulase production depending on the different transformed fungal strains. In chapter 3, the effect of D-xylose on gene expression in A. niger was investigated. The inducer of (hemi)cellulases D-xylose was added to cultures of A. niger growing on the non-inducer sorbitol. Genes differentially expressed on D-xylose were identified as candidate genes involved in the response to this sugar. This study confirmed that D-xylose activates enzymes involved in xylan degradation and D-xylose utilisation, but also enzymes responsible for the removal of other monomers that occurr on arabinoxylan and cellulases. Statistical analysis of variance components was used to assess the contribution of each external factors affecting the measured gene expression. Such analysis of variance components is important for reproducible sample processing for microarray analysis. Chapter 4 describes the A. niger secretory pathway proteins that are involved in the production of (hemi)cellulases, via induction by D-xylose. For this, A. niger was grown under the same conditions as the ones described in chapter 3. After the isolation of microsomes, the corresponding proteins were analysed by shotgun proteomics. Induction by D-xylose was correlated with an increase in proteins related to protein secretion, namely small GTPases for vesicle transport and polarised growth. Most importantly, under induction by D-xylose, the complex for protein degradation 20S proteasome was associated with microsomes. These results indicate a novel mode of regulation in which the proteasome is recruited to secretory organelles upon the induction of extracellular enzymes. In chapter 5, the analysis of secretory proteins described in chapter 4 is now applied to a system in which D-maltose is an inducer of starch-degrading enzymes. This chapter also includes the study of the proteins secreted after D-maltose or D-xylose. After D-maltose addition, three starch-degrading enzymes were found more abundant and after D-xylose addition, several enzymes were more abundant and these enzymes were mostly related to arabinoxylan and cellulose degradation. The effects of D-maltose on the microsomal proteome are similar to the effects of D-xylose. Both the induction by D-maltose and by D-xylose resulted in increased amounts of mitochondrial proteins. Moreover, the 20S proteasome assembly is an ATP-dependent process. For this reason, it is hypothesised that the assembly and association of 20S proteasome upon induction is related to an increased ATP production in the vicinity of secretory organelles. |
