Project description:Filamentous fungi are confronted with changes and limitations of their carbon source during growth in their natural habitats and during industrial applications. To survive life-threatening starvation conditions, carbon from extra- and intracellular resources becomes mobilized to fuel fungal self-propagation. Key to understand the underlying cellular processes is the system-wide analysis of fungal starvation responses in a temporal and spatial resolution. The knowledge deduced is important for the development of optimized industrial production processes. This study describes the physiological, morphological and genome-wide transcriptional changes caused by prolonged carbon starvation during submerged batch cultivation of the filamentous fungus Aspergillus niger. Bioreactor cultivation supported highly reproducible growth conditions and monitoring of physiological parameters. Changes in hyphal growth and morphology were analyzed at distinct cultivation phases using automated image analysis. The Affymetrix GeneChip platform was used to establish genome-wide transcriptional profiles for three selected time points during prolonged carbon starvation. Compared to the exponential growth transcriptome, about 50% (7,292) of all genes displayed differential genes expression during at least one of the starvation time points. Enrichment analysis of Gene Ontology, Pfam domain and KEGG pathway annotations uncovered autophagy and asexual reproduction as major global transcriptional trends. Induced transcription of genes encoding hydrolytic enzymes was accompanied by increased secretion of hydrolases including chitinases, glucanases, proteases and phospholipases as identified by mass spectrometry. This study is the first system-wide analysis of the carbon starvation response in a filamentous fungus. Morphological, transcriptomic and secretomic analyses identified key events important for fungal survival and their chronology. The dataset obtained forms a comprehensive framework for further elucidation of the interrelation and interplay of the individual cellular events involved.
Project description:Filamentous fungi are confronted with changes and limitations of their carbon source during growth in their natural habitats and during industrial applications. To survive life-threatening starvation conditions, carbon from extra- and intracellular resources becomes mobilized to fuel fungal self-propagation. Key to understand the underlying cellular processes is the system-wide analysis of fungal starvation responses in a temporal and spatial resolution. The knowledge deduced is important for the development of optimized industrial production processes. This study describes the physiological, morphological and genome-wide transcriptional changes caused by prolonged carbon starvation during submerged batch cultivation of the filamentous fungus Aspergillus niger. Bioreactor cultivation supported highly reproducible growth conditions and monitoring of physiological parameters. Changes in hyphal growth and morphology were analyzed at distinct cultivation phases using automated image analysis. The Affymetrix GeneChip platform was used to establish genome-wide transcriptional profiles for three selected time points during prolonged carbon starvation. Compared to the exponential growth transcriptome, about 50% (7,292) of all genes displayed differential genes expression during at least one of the starvation time points. Enrichment analysis of Gene Ontology, Pfam domain and KEGG pathway annotations uncovered autophagy and asexual reproduction as major global transcriptional trends. Induced transcription of genes encoding hydrolytic enzymes was accompanied by increased secretion of hydrolases including chitinases, glucanases, proteases and phospholipases as identified by mass spectrometry. This study is the first system-wide analysis of the carbon starvation response in a filamentous fungus. Morphological, transcriptomic and secretomic analyses identified key events important for fungal survival and their chronology. The dataset obtained forms a comprehensive framework for further elucidation of the interrelation and interplay of the individual cellular events involved. for each post-exponential time point (Day1, Day3 and Day6 post-carbon depletion), biological duplicates were performed.
Project description:The genome of the filamentous fungus Aspergillus niger is rich in genes encoding pectinases, a broad class of enzymes that have been extensively studied due to their use in industrial applications. The sequencing of the A. niger genome provided more knowledge concerning the individual pectinolytic genes, but relatively little is still known about the regulatory genes involved in pectin degradation. Understanding regulation of the pectinolytic genes provides a tool to optimize the production of pectinases in this industrially important fungus. This study describes the identification and characterization of one of the activators of pectinase-encoding genes, RhaR. Inactivation of the gene encoding this regulator resulted in down-regulation of genes involved in the release and catabolism of L-rhamnose from the pectinolytic substructure rhamnogalacturonan I.
Project description:The genome of the filamentous fungus Aspergillus niger is rich in genes encoding pectinases, a broad class of enzymes that have been extensively studied due to their use in industrial applications. The sequencing of the A. niger genome provided more knowledge concerning the individual pectinolytic genes, but relatively little is still known about the regulatory genes involved in pectin degradation. Understanding regulation of the pectinolytic genes provides a tool to optimize the production of pectinases in this industrially important fungus. This study describes the identification and characterization of one of the activators of pectinase-encoding genes, RhaR. Inactivation of the gene encoding this regulator resulted in down-regulation of genes involved in the release and catabolism of L-rhamnose from the pectinolytic substructure rhamnogalacturonan I. We aim to discover differencial expressed genes in A.niger wild type strain N402 and M-NM-^TrhaR mutant while growing on rhamnose as carbon source. Biological duplicates were made for both strain at the growth of 2 hours, Affymetrix microarray experiments were performed on these samples.
Project description:Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry.
Project description:Genome-wide Transcriptional Responses of the Filamentous Fungus Aspergillus niger to Lignocellulose (Wheat straw) using RNA-sequencing
Project description:A. niger and A. oryzae are two filamentous fungi widely used in industry to produce various enzymes (e.g. pectinases, amylases) and metabolites (e.g. citric acid). Using proteomics, the co-cultivation of these two fungi in wheat bran showed an equal distribution of the two strains forming mixed colonies with a broad range of carbohydrate active enzymes produced. This stable mixed microbial system seems suitable for subsequent commercial processes such as enzyme production. XlnR knock-out strains for both aspergilli were used to study the influence of plant cell wall degrading enzyme production on the fitness of the mixed culture.