Competition between homologous chromosomal DNA and exogenous donor DNA to repair CRISPR/Cas9-induced double-strand breaks in Aspergillus niger.

Background: Aspergillus niger is well-known for its high protein secretion capacity and therefore an important cell factory for homologous and heterologous protein production. The use of a strong promoter and multiple gene copies are commonly used strategies to increase the gene expression and protein production of the gene of interest (GOI). We recently presented a two-step CRISPR/Cas9-mediated approach in which glucoamylase (glaA) landing sites (GLSs) are introduced at predetermined sites in the genome (step 1), which are subsequently filled with copies of the GOI (step 2) to achieve high expression of the GOI.

Compatible solutes determine the heat resistance of conidia.

Background: Asexually developed fungal spores (conidia) are key for the massive proliferation and dispersal of filamentous fungi. Germination of conidia and subsequent formation of a mycelium network give rise to many societal problems related to human and animal fungal diseases, post-harvest food spoilage, loss of harvest caused by plant-pathogenic fungi and moulding of buildings. Conidia are highly stress resistant compared to the vegetative mycelium and therefore even more difficult to tackle.

Preservation stress resistance of melanin deficient conidia from Paecilomyces variotii and Penicillium roqueforti mutants generated via CRISPR/Cas9 genome editing.

Background: The filamentous fungi Paecilomyces variotii and Penicillium roqueforti are prevalent food spoilers and are of interest as potential future cell factories. A functional CRISPR/Cas9 genome editing system would be beneficial for biotechnological advances as well as future (genetic) research in P. variotii and P.

Loss of function of the carbon catabolite repressor CreA leads to low but inducer-independent expression from the feruloyl esterase B promoter in Aspergillus niger.

Objective: With the aim to decipher the mechanisms involved in the transcriptional regulation of feruloyl esterase encoded by faeB, a genetic screen was performed to isolate A. niger mutants displaying inducer-independent expression from the faeB promoter.

Result: PfaeB-amdS and PfaeB-lux dual reporter strains were constructed and used to isolate trans-acting mutants in which the expression of both reporters was increased, based on the ability to grow on acetamide plates and higher luciferase activity, respectively.

Aspergillus fumigatus establishes infection in zebrafish by germination of phagocytized conidia, while Aspergillus niger relies on extracellular germination.

Among opportunistically pathogenic filamentous fungi of the Aspergillus genus, Aspergillus fumigatus stands out as a drastically more prevalent cause of infection than others. Utilizing the zebrafish embryo model, we applied a combination of non-invasive real-time imaging and genetic approaches to compare the infectious development of A. fumigatus with that of the less pathogenic A.

Functional analysis of three putative galactofuranosyltransferases with redundant functions in galactofuranosylation in Aspergillus niger.

Galactofuranose (Galf)-containing glycostructures are important to secure the integrity of the fungal cell wall. Golgi-localized Galf-transferases (Gfs) have been identified in Aspergillus nidulans and Aspergillus fumigatus. BLASTp searches identified three putative Galf-transferases in Aspergillus niger.

Correction to: Mutations in AraR leading to constitutive expression of arabinolytic genes in Aspergillus niger under derepressing conditions.

The correct title is: Mutations in AraR leading to constitutive expression of arabinolytic genes in Aspergillus niger under derepressing conditions.

Mutations in AraR leading to constitutive expression of arabinolytic genes in Aspergillus niger under derepressing conditions [corrected].

The AraR transcription factor of Aspergillus niger encodes a Zn(II)Cys transcription factor required for the induction of genes encoding arabinolytic enzymes. One of the target genes of AraR is abfA, encoding an arabinofuranosidase. The expression of abfA as well as other L-arabinose-induced genes in A.

Parasexual Crossings for Bulk Segregant Analysis in Aspergillus niger to Facilitate Mutant Identification Via Whole Genome Sequencing.

The industrially important fungus Aspergillus niger is known to reproduce only asexually. The parasexual cycle of fungi can be used for crossing two different strains to produce segregants or progeny with combined mutations even in fungi without a known sexual cycle. In A.

Inducer-independent production of pectinases in Aspergillus niger by overexpression of the D-galacturonic acid-responsive transcription factor gaaR.

The transcription factor GaaR is needed for the expression of genes required for pectin degradation and transport and catabolism of the main degradation product, D-galacturonic acid (GA) in Aspergillus niger. In this study, we used the strong constitutive gpdA promoter of Aspergillus nidulans to overexpress gaaR in A. niger.

An Evolutionarily Conserved Transcriptional Activator-Repressor Module Controls Expression of Genes for D-Galacturonic Acid Utilization in Aspergillus niger.

The expression of genes encoding extracellular polymer-degrading enzymes and the metabolic pathways required for carbon utilization in fungi are tightly controlled. The control is mediated by transcription factors that are activated by the presence of specific inducers, which are often monomers or monomeric derivatives of the polymers. A D-galacturonic acid-specific transcription factor named GaaR was recently identified and shown to be an activator for the expression of genes involved in galacturonic acid utilization in Botrytis cinerea and Aspergillus niger Using a forward genetic screen, we isolated A.

The unconventional secretion of PepN is independent of a functional autophagy machinery in the filamentous fungus Aspergillus niger.

During unconventional protein secretion (UPS), proteins do not pass through the classical endoplasmic reticulum (ER)-Golgi-dependent pathway, but are transported to the cell membrane via alternative routes. One type of UPS is dependent on several autophagy-related (Atg) proteins in yeast and mammalian cells, but mechanisms for unconventional secretion are largely unknown for filamentous fungi. In this study, we investigated whether the autophagy machinery is used for UPS in the filamentous fungus Aspergillus niger An aspartic protease, which we called PepN, was identified as being likely to be secreted unconventionally, as this protein is highly abundant in culture filtrates during carbon starvation while it lacks a conventional N-terminal secretion sequence.

A set of isogenic auxotrophic strains for constructing multiple gene deletion mutants and parasexual crossings in Aspergillus niger.

To construct a set of isogenic auxotrophic strains in Aspergillus niger suited for creating multiple gene deletion mutants and executing parasexual crossings, we have combined mutations in genes involved in colour pigmentation (fwnA and olvA) with well-selectable auxotrophic markers (pyrG, nicB, argB, and adeA). All markers, except for the pyrG marker, were introduced by targeted deletion, omitting UV mutagenesis of the strains. Aspergillus oryzae orthologous genes of the argB, nicB, and adeA markers were used as heterologous selection markers, and all markers were shown to complement to respective auxotrophic A.

Identification of a Classical Mutant in the Industrial Host Aspergillus niger by Systems Genetics: LaeA Is Required for Citric Acid Production and Regulates the Formation of Some Secondary Metabolites.

The asexual filamentous fungus Aspergillus niger is an important industrial cell factory for citric acid production. In this study, we genetically characterized a UV-generated A. niger mutant that was originally isolated as a nonacidifying mutant, which is a desirable trait for industrial enzyme production.

Identification and functional analysis of two Golgi-localized UDP-galactofuranose transporters with overlapping functions in Aspergillus niger.

Background: Galactofuranose (Galf)-containing glycoconjugates are present in numerous microbes, including filamentous fungi where they are important for morphology, virulence and maintaining cell wall integrity. The incorporation of Galf-residues into galactomannan, galactomannoproteins and glycolipids is carried out by Golgi-localized Galf transferases. The nucleotide sugar donor used by these transferases (UDP-Galf) is produced in the cytoplasm and has to be transported to the lumen of the Golgi by a dedicated nucleotide sugar transporter.

I-SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei.

Targeted integration of expression cassettes for enzyme production in industrial microorganisms is desirable especially when enzyme variants are screened for improved enzymatic properties. However, currently used methods for targeted integration are inefficient and result in low transformation frequencies. In this study, we expressed the Saccharomyces cerevisiae I-SceI meganuclease to generate double-strand breaks at a defined locus in the Trichoderma reesei genome.

Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red.

The fungal cell wall is an essential organelle and represents a considerable metabolic investment. Its macromolecular composition, molecular organization and thickness can vary greatly depending on environmental conditions. Its construction is also tightly controlled in space and time.