Structure of the nucleus of 1928+738

Modeling of the trajectories of VLBI components ejected by the nucleus of 1928+738 shows the VLBI jet contains three families of trajectories, i.e. VLBI components are ejected from three different origins. The fit of components C1, C6 and C8 indicates that the nucleus of 1928+738 contains two binary black hole systems. The first binary black hole system is associated with the stationary components Cg and CS and is characterized by a radius $R_{bin,1} \approx 0.220$ mas; both black holes ejected VLBI components quasi regularly between 1990 and 2010. The second binary black hole system is not associated with stationary components and is characterized by a radius $R_{bin,2} \approx 0.140$ mas; it ejected only three VLBI components between 1994 and 1999. The two black hole sytems are separated by $\approx 1.35$ mas. We briefly discuss the consequences of the existence of binary black holes systems in radio quasars to make the link between radio quasars and GAIA.Comment: 18 pages, 24 figures, submitted to Astronomy & Astrophysics. arXiv admin note: text overlap with arXiv:1307.370

CRISPR/Cas9 facilitates rapid generation of constitutive forms of transcription factors in Aspergillus niger through specific on-site genomic mutations resulting in increased saccharification of plant biomass

The CRISPR/Cas9 system has been successfully applied for gene editing in filamentous fungi. Previous studies reported that single stranded oligonucleotides can be used as repair templates to induce point mutations in some filamentous fungi belonging to genus Aspergillus. In Aspergillus niger, extensive research has been performed on regulation of plant biomass degradation, addressing transcription factors such as XlnR or GaaR, involved in (hemi-)cellulose and pectin utilization, respectively. Single nucleotide mutations leading to constitutively active forms of XlnR and GaaR have been previously reported. However, the mutations were performed by the introduction of versions obtained through site-directed or UV-mutagenesis into the genome. Here we report a more time- and cost-efficient approach to obtaining constitutively active versions by application of the CRISPR/Cas9 system to generate the desired mutation on-site in the A. niger genome. This was also achieved using only 60-mer single stranded oligonucleotides, shorter than the previously reported 90-mer strands. In this study, we show that CRISPR/Cas9 can also be used to efficiently change functional properties of the proteins encoded by the target gene by on-site genomic mutations in A. niger. The obtained strains with constitutively active XlnR and GaaR versions resulted in increased production of plant biomass degrading enzymes and improved release of D-xylose and L-arabinose from wheat bran, and D-galacturonic acid from sugar beet pulp.Peer reviewe

Developments and opportunities in fungal strain engineering for the production of novel enzymes and enzyme cocktails for plant biomass degradation

Fungal strain engineering is commonly used in many areas of biotechnology, including the production of plant biomass degrading enzymes. Its aim varies from the production of specific enzymes to overall increased enzyme production levels and modification of the composition of the enzyme set that is produced by the fungus. Strain engineering involves a diverse range of methodologies, including classical mutagenesis, genetic engineering and genome editing. In this review, the main approaches for strain engineering of filamentous fungi in the field of plant biomass degradation will be discussed, including recent and not yet implemented methods, such as CRISPR/Cas9 genome editing and adaptive evolution.Peer reviewe

Non-homologous end-joining-deficient filamentous fungal strains mitigate the impact of off-target mutations during the application of CRISPR/Cas9

CRISPR/Cas9 genome editing technology has been implemented in almost all living organisms. Its editing precision appears to be very high and therefore could represent a big change from conventional genetic engineering approaches. However, guide RNA binding to nucleotides similar to the target site could result in undesired off-target mutations. Despite this, evaluating whether mutations occur is rarely performed in genome editing studies. In this study, we generated CRISPR/Cas9-derived filamentous fungal strains and analyzed them for the occurrence of mutations, and to which extent genome stability affects their occurrence. As a test case, we deleted the (hemi-)cellulolytic regulator-encoding gene xlnR in two Aspergillus niger strains: a wild type (WT) and a non-homologous end-joining (NHEJ)-deficient strain ΔkusA. Initial phenotypic analysis suggested a much higher prevalence of mutations in the WT compared to NHEJ-deficient strains, which was confirmed and quantified by whole-genome sequencing analysis. Our results clearly demonstrate that CRISPR/Cas9 applied to an NHEJ-deficient strain is an efficient strategy to avoid unwanted mutations. IMPORTANCE Filamentous fungi are commonly used biofactories for the production of industrially relevant proteins and metabolites. Often, fungal biofactories undergo genetic development (genetic engineering, genome editing, etc.) aimed at improving production yields. In this context, CRISPR/Cas9 has gained much attention as a genome editing strategy due to its simplicity, versatility, and precision. However, despite the high level of accuracy reported for CRISPR/Cas9, in some cases unintentional cleavages in non-targeted loci-known as off-target mutations-could arise. While biosafety should be a central feature of emerging biotechnologies to minimize unintended consequences, few studies quantitatively evaluate the risk of off-target mutations. This study demonstrates that the use of non-homologous end-joining-deficient fungal strains drastically reduces the number of unintended genomic mutations, ensuring that CRISPR/Cas9 can be safely applied for strain development

Detailed analysis of the D-galactose catabolic pathways in Aspergillus niger reveals complexity at both metabolic and regulatory level

Funding Information: TC was supported by a grant of the NWO ALWOP.233 to RPdV. RSK and SG were supported by a grant of the Applied Science division (TTW) of NWO and the Technology Program of the Ministry of Infrastructure and Water Management 15807 to RPdV. The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, was supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The Academy of Finland grant no. 308284 to MRM is also acknowledged. Publisher Copyright: © 2022 The Author(s)The current impetus towards a sustainable bio-based economy has accelerated research to better understand the mechanisms through which filamentous fungi convert plant biomass, a valuable feedstock for biotechnological applications. Several transcription factors have been reported to control the polysaccharide degradation and metabolism of the resulting sugars in fungi. However, little is known about their individual contributions, interactions and crosstalk. D-galactose is a hexose sugar present mainly in hemicellulose and pectin in plant biomass. Here, we study D-galactose conversion by Aspergillus niger and describe the involvement of the arabinanolytic and xylanolytic activators AraR and XlnR, in addition to the D-galactose-responsive regulator GalX. Our results deepen the understanding of the complexity of the filamentous fungal regulatory network for plant biomass degradation and sugar catabolism, and facilitate the generation of more efficient plant biomass-degrading strains for biotechnological applications.Peer reviewe

The transcriptional activator ClrB is crucial for the degradation of soybean hulls and guar gum in Aspergillus niger

Low-cost plant substrates, such as soybean hulls, are used for various industrial applications. Filamentous fungi are important producers of Carbohydrate Active enZymes (CAZymes) required for the degradation of these plant biomass substrates. CAZyme production is tightly regulated by several transcriptional activators and repressors. One such transcriptional activator is CLR-2/ClrB/ManR, which has been identified as a regulator of cellulase and mannanase production in several fungi. However, the regulatory network governing the expression of cellulase and mannanase encoding genes has been reported to differ between fungal species. Previous studies showed that Aspergillus niger ClrB is involved in the regulation of (hemi-)cellulose degradation, although its regulon has not yet been identified. To reveal its regulon, we cultivated an A. niger ΔclrB mutant and control strain on guar gum (a galactomannan-rich substrate) and soybean hulls (containing galactomannan, xylan, xyloglucan, pectin and cellulose) to identify the genes that are regulated by ClrB. Gene expression data and growth profiling showed that ClrB is indispensable for growth on cellulose and galactomannan and highly contributes to growth on xyloglucan in this fungus. Therefore, we show that A. niger ClrB is crucial for the utilization of guar gum and the agricultural substrate, soybean hulls. Moreover, we show that mannobiose is most likely the physiological inducer of ClrB in A. niger and not cellobiose, which is considered to be the inducer of N. crassa CLR-2 and A. nidulans ClrB

The Sugar Metabolic Model of Aspergillus niger Can Only Be Reliably Transferred to Fungi of Its Phylum

Fungi play a critical role in the global carbon cycle by degrading plant polysaccharides to small sugars and metabolizing them as carbon and energy sources. We mapped the well-established sugar metabolic network of Aspergillus niger to five taxonomically distant species (Aspergillus nidulans, Penicillium subrubescens, Trichoderma reesei, Phanerochaete chrysosporium and Dichomitus squalens) using an orthology-based approach. The diversity of sugar metabolism correlates well with the taxonomic distance of the fungi. The pathways are highly conserved between the three studied Eurotiomycetes (A. niger, A. nidulans, P. subrubescens). A higher level of diversity was observed between the T. reesei and A. niger, and even more so for the two Basidiomycetes. These results were confirmed by integrative analysis of transcriptome, proteome and metabolome, as well as growth profiles of the fungi growing on the corresponding sugars. In conclusion, the establishment of sugar pathway models in different fungi revealed the diversity of fungal sugar conversion and provided a valuable resource for the community, which would facilitate rational metabolic engineering of these fungi as microbial cell factories

The Amylolytic Regulator AmyR of Aspergillus niger Is Involved in Sucrose and Inulin Utilization in a Culture-Condition-Dependent Manner

Filamentous fungi degrade complex plant material to its monomeric building blocks, which have many biotechnological applications. Transcription factors play a key role in plant biomass degradation, but little is known about their interactions in the regulation of polysaccharide degradation. Here, we deepened the knowledge about the storage polysaccharide regulators AmyR and InuR in Aspergillus niger. AmyR controls starch degradation, while InuR is involved in sucrose and inulin utilization. In our study, the phenotypes of A. niger parental, ΔamyR, ΔinuR and ΔamyRΔinuR strains were assessed in both solid and liquid media containing sucrose or inulin as carbon source to evaluate the roles of AmyR and InuR and the effect of culture conditions on their functions. In correlation with previous studies, our data showed that AmyR has a minor contribution to sucrose and inulin utilization when InuR is active. In contrast, growth profiles and transcriptomic data showed that the deletion of amyR in the ΔinuR background strain resulted in more pronounced growth reduction on both substrates, mainly evidenced by data originating from solid cultures. Overall, our results show that submerged cultures do not always reflect the role of transcription factors in the natural growth condition, which is better represented on solid substrates. Importance: The type of growth has critical implications in enzyme production by filamentous fungi, a process that is controlled by transcription factors. Submerged cultures are the preferred setups in laboratory and industry and are often used for studying the physiology of fungi. In this study, we showed that the genetic response of A. niger to starch and inulin was highly affected by the culture condition, since the transcriptomic response obtained in a liquid environment did not fully match the behavior of the fungus in a solid environment. These results have direct implications in enzyme production and would help industry choose the best approaches to produce specific CAZymes for industrial purposes

CreA-mediated repression of gene expression occurs at low monosaccharide levels during fungal plant biomass conversion in a time and substrate dependent manner

Funding Information: The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, was supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231 . CK, EB was supported by a grant of the Applied and Engineering Sciences division of NWO , and the Technology Program of the Ministry of Economic Affairs 016.130.609 to RPdV. PD was supported by a grant of the Netherlands Scientific Organization NWO 824.15.023 to RPdV. The Academy of Finland grant no. 308284 to MRM is acknowledged. Publisher Copyright: © 2021 The Author(s)Carbon catabolite repression enables fungi to utilize the most favourable carbon source in the environment, and is mediated by a key regulator, CreA, in most fungi. CreA-mediated regulation has mainly been studied at high monosaccharide concentrations, an uncommon situation in most natural biotopes. In nature, many fungi rely on plant biomass as their major carbon source by producing enzymes to degrade plant cell wall polysaccharides into metabolizable sugars. To determine the role of CreA when fungi grow in more natural conditions and in particular with respect to degradation and conversion of plant cell walls, we compared transcriptomes of a creA deletion and reference strain of the ascomycete Aspergillus niger during growth on sugar beet pulp and wheat bran. Transcriptomics, extracellular sugar concentrations and growth profiling of A. niger on a variety of carbon sources, revealed that also under conditions with low concentrations of free monosaccharides, CreA has a major effect on gene expression in a strong time and substrate composition dependent manner. In addition, we compared the CreA regulon from five fungi during their growth on crude plant biomass or cellulose. It showed that CreA commonly regulated genes related to carbon metabolism, sugar transport and plant cell wall degrading enzymes across different species. We therefore conclude that CreA has a crucial role for fungi also in adapting to low sugar concentrations as occurring in their natural biotopes, which is supported by the presence of CreA orthologs in nearly all fungi.Peer reviewe

Unraveling the regulation of sugar beet pulp utilization in the industrially relevant fungus Aspergillus niger

Efficient utilization of agro-industrial waste, such as sugar beet pulp, is crucial for the bio-based economy. The fungus Aspergillus niger possesses a wide array of enzymes that degrade complex plant biomass substrates, and several regulators have been reported to play a role in their production. The role of the regulators GaaR, AraR, and RhaR in sugar beet pectin degradation has previously been reported. However, genetic regulation of the degradation of sugar beet pulp has not been assessed in detail. In this study, we generated a set of single and combinatorial deletion mutants targeting the pectinolytic regulators GaaR, AraR, RhaR, and GalX as well as the (hemi-)cellulolytic regulators XlnR and ClrB to address their relative contribution to the utilization of sugar beet pulp. We show that A. niger has a flexible regulatory network, adapting to the utilization of (hemi-)cellulose at early timepoints when pectin degradation is impaired