Project description:<i>Aspergillus niger</i> is an important filamentous fungus in industrial biotechnology for the production of citric acid and enzymes. In the late 1980s, the <i>A. niger</i> N400/NRRL3 strain was selected for both fundamental and applied studies in relation to several processes including gluconic acid and protein production. To facilitate handling of <i>A. niger</i>, the N400 wild-type strain was UV mutagenized in two consecutive rounds to generate N401 and N402. N402 was used as a reference laboratory strain and exhibits the phenotypes with reduced conidiophore stalk length and reduced radial growth. The conidiophore stalk length and radial growth of <i>A. niger</i> strain N400 were determined and compared to N401 and N402. The length of N400 conidiophore stalks (2.52 ± 0.40 mm) was reduced in N401 and N402 to 0.66 ± 0.14 mm and 0.34 ± 0.06 mm, respectively. Whereas N400 reached a colony diameter of 6.7 ± 0.2 cm after 7 days, N401 and N402 displayed reduced radial growth phenotype (4.3 ± 0.1 and 4.1 ± 0.1, respectively). To identify the mutations (dubbed <i>cspA</i> and <i>cspB</i>) responsible for the phenotypes of N401 and N402, the genomes were sequenced and compared to the N400 genome sequence. A parasexual cross was performed between N400 and N402 derivatives to isolate segregants which allowed cosegregation analysis of single nucleotide polymorphisms and insertions and deletions among the segregants. The shorter conidiophore stalk and reduced radial growth in N401 (<i>cspA</i>) was found to be caused by a 9-kb deletion on chromosome III and was further narrowed down to a truncation of NRRL3_03857 which encodes a kinesin-like protein homologous to the <i>A. nidulans</i> UncA protein. The mutation responsible for the further shortening of conidiophore stalks in N402 (<i>cspB</i>) was found to be caused by a missense mutation on chromosome V in a hitherto unstudied C2H2 transcription factor encoded by the gene NRRL3_06646. The importance of these two genes in relation to conidiophore stalk length and radial growth was confirmed by single and double gene deletion studies. The mutations in the laboratory strain N402 should be taken into consideration when studying phenotypes in the N402 background.
Project description:Conidia of Aspergillus niger are characterized by a dormant state and are moderate stress-resistant. Upon contact with a moist substrate, germination of conidia occurs by changing from a dormant stabilized state towards a growing vegetative cell. The RNA expression levels of dormant conidia and conidia that were in various stages of germination were studied. The RNA composition of dormant conidia was substantially different than all the subsequent stages of germination. This indicates that the distinct morphological changes that occur during germination are not correlated with the highest change in the transcriptome. Samples of germinating conidia of Aspergillus niger N402 were taken in triple at 0h (dormant), 2h, 4h, 6h and 8h after inoculation in CM.
Project description:AIMS:To create an Aspergillus niger mutant with increased tolerance against ferulic acid using evolutionary adaptation. METHODS AND RESULTS:Evolutionary adaptation of A. niger N402 was performed by consecutive growth on increasing concentrations of ferulic acid in the presence of 25 mmol l-1 d-fructose, starting from 0·5 mmol l-1 and ending with 5 mmol l-1 ferulic acid. The A. niger mutant obtained after six months, named Fa6, showed increased ferulic acid tolerance compared to the parent. In addition, Fa6 has increased ferulic acid consumption and a higher conversion rate, suggesting that the mutation affects aromatic metabolism of this species. Transcriptome analysis of the evolutionary mutant on ferulic acid revealed a distinct gene expression profile compared to the wild type. Further analysis of this mutant and the parent strain provided the first experimental confirmation that A. niger converts coniferyl alcohol to ferulic acid. CONCLUSIONS:The evolutionary adaptive A. niger mutant Fa6 has beneficial mutations that increase the tolerance, conversion rate and uptake of ferulic acid. SIGNIFICANCE AND IMPACT OF THE STUDY:This study demonstrates that evolutionary adaptation is a powerful tool to modify micro-organisms towards increased tolerance to harsh conditions, which is beneficial for various industrial applications.
Project description:Transcriptional profiling of the A. niger WT (N402) strain treated with xylan (1%, w/v) for 6, 12 and 24 h. The main objective was to identify genes related to cellulases and hemicellulases after treatment with the polysaccharide xylan. The experiment was further validated by enzymatic assays. Three-condition experiment: WT-Xylan for 6, 12 and 24 h at 30oC in batch culture. Firstly, the WT (N402) strain was grown in minimal medium with fructose as carbon source (control), and then transferred to 1% (w/v) xylan as carbon source. 2 biological replicates per time point.
Project description:Galactose catabolism in Aspergillus nidulans is regulated by at least two regulators, GalR and GalX. In Aspergillus niger only GalX is present, and its role in D-galactose catabolism in this fungus was investigated. Phenotypic and gene expression analysis of a wild type and a galX disruptant revealed that GalX does not substitute for the absence of GalR in A. niger, it regulates the D-galactose oxido-reductive pathway, but not the Leloir pathway. Four genes, including the recently characterized ladB (galactitol dehydrogenase) were found to have differencial expressions that are highly relevant to GalX , indicating a novel oxido-reductive pathway in A.niger . We aim to discover differentially expressed genes in A.niger wild type strain N402 and M-NM-^TgalX mutant while growing on galactose as carbon source. Biological duplicates were made for both strains. The strains were grown O/N in complete medium with 2% frunctose and mycelium was then washed and transferred to minimal medium with 25 mM D-galactose and incubated for 2 hours. Affymetrix microarray experiments were performed RNA isolated from these samples.
Project description:Ethanolic extracts of mycelia from Aspergillus niger (strain N402) grown in liquid media were observed to have haemolytic activity on bovine erythrocytes. This haemolytic activity decreased significantly during the time of growth (1-3 days). Moreover, when A. niger was grown on carbon-deprived medium, the efficiency of this haemolytic activity in the ethanolic extracts was much lower than when grown in carbon-enriched medium, and became almost undetectable after 3 days of growth in carbon-deprived medium. The lipid composition of these ethanolic extracts was analysed by liquid chromatography-electrospray ionisation tandem mass spectrometry. This haemolytic activity can be mainly linked to the relative levels of the molar ratios of the unsaturated fatty acids and lysophosphatidylcholines.
Project description:The Aspergillus niger genome contains a large repertoire of genes encoding carbohydrate active enzymes (CAZymes) that are targeted to plant polysaccharide degradation enabling A. niger to grow on a wide range of plant biomass substrates. Which genes need to be activated in certain environmental conditions depends on the composition of the available substrate. Previous studies have demonstrated the involvement of a number of transcriptional regulators in plant biomass degradation and have identified sets of target genes for each regulator. In this study, a broad transcriptional analysis was performed of the A. niger genes encoding (putative) plant polysaccharide degrading enzymes. Microarray data focusing on the initial response of A. niger to the presence of plant biomass related carbon sources were analyzed of a wild-type strain N402 that was grown on a large range of carbon sources and of the regulatory mutant strains ?xlnR, ?araR, ?amyR, ?rhaR and ?galX that were grown on their specific inducing compounds.The cluster analysis of the expression data revealed several groups of co-regulated genes, which goes beyond the traditionally described co-regulated gene sets. Additional putative target genes of the selected regulators were identified, based on their expression profile. Notably, in several cases the expression profile puts questions on the function assignment of uncharacterized genes that was based on homology searches, highlighting the need for more extensive biochemical studies into the substrate specificity of enzymes encoded by these non-characterized genes. The data also revealed sets of genes that were upregulated in the regulatory mutants, suggesting interaction between the regulatory systems and a therefore even more complex overall regulatory network than has been reported so far.Expression profiling on a large number of substrates provides better insight in the complex regulatory systems that drive the conversion of plant biomass by fungi. In addition, the data provides additional evidence in favor of and against the similarity-based functions assigned to uncharacterized genes.