Harnessing the CRISPR/Cas9 system to study the regulatory network of plant biomass degradation in Aspergillus niger

Filamentous fungi are primary degraders of plant biomass, and therefore play a pivotal role in the bio-based economy. For this, the understanding of their abilities and limits regarding plant biomass degradation is essential. The regulation of plant biomass degradation in Aspergillus niger is controlled by a complex and intricate system involving the interaction of multiple transcription factors (TFs). In this thesis, marker-free CRISPR/Cas9 gene editing was performed to generate gene deletions (Chapters 5, 6, 7 and 8), precise point mutations (Chapter 3), domain swapping (Chapter 4), and gene tagging by fluorescent markers targeting several major TFs coordinating plant biomass degradation. In Chapter 3, by introducing single point mutations, we generated constitutively active forms of two key transcription factors, XlnR and GaaR, involved in (hemi-)cellulose and pectin utilization, respectively. In Chapter 4, we showed the generation of a chimeric GaaR-XlnR transcription factor mutant strain, that was able to control the expression of pectinolytic genes when induced by D-xylose, offering opportunities for the production of specific enzyme sets by cultivation on low cost agro-industrial substrates. The analysis of single and combinatorial gene deletion strains generated by CRISPR/Cas9 described in this thesis expanded the knowledge about the individual roles and interactions of several major TFs involved in the regulation of plant biomass utilization. In Chapter 5, using transcriptomic data, we identified the inducer and regulon of ClrB in A. niger and showed that ClrB influences the expression of clrA. Additional TF interactions have been revealed by the combined analysis of single and combinatorial (hemi-)cellulolytic and pectinolytic TF deletion strains in Chapter 7 and Chapter 8. In Chapter 7, proteomic data showed the relative role of XlnR, ClrB and ClrA in the degradation of wheat bran, and revealed several cases of gene co-regulation. In Chapter 8, transcriptomic data showed the upregulation of (hemi-)cellulolytic genes when major pectinolytic TFs were deleted, providing evidence for the antagonistic interaction between (hemi-)cellulose and pectin utilization, and the adaptation to the utilization of alternative components of sugar beet pulp. Finally, data described in Chapter 6 and Chapter 8 also support the observation that phenotypes of solid and liquid cultures cannot be directly compared. Although the characteristics of submerged fungal cultures is valuable for industrial applications, these do not represent the natural behavior of the fungus. Taken together, the results described in this thesis show various applications of the CRISPR/Cas9 system for the engineering of TFs involved in the regulation of plant biomass utilization and allowed for the analysis of several TFs on a network level in A. niger. Moreover, the deletion of major TFs involved in polysaccharide utilization can reveal putative back-up regulatory mechanisms, which can possibly indicate the action of novel TFs involved in this process. Additionally, the combined deletion of TFs regulating the expression of CAZy-encoding genes as well as the deletion of the protease regulator gene prtT can result in strains suitable for cell factories with impaired background protein production

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

CFRP-aluhab®-CFRP szendvics szerkezetű kompozit anyag fúrása = Drilling of CFRP-Aluhab®-CFRP Sandwich Structure

Napjainkban az ipar egyre több területén használnak különleges anyagokat. Ezek az anyagok általában kiváló mechanikai tulajdonságaikkal rendelkeznek. Az alumíniumhab is ezek közé az anyagok közé tartozik. Ez az anyag rendkívül könnyű és emellett nagy teherbírással rendelkezik. Az anyag rezgéselnyelő képessége, a cellás szerkezetének köszönhetően meglehetősen jó. Kutatásunk célja feltárni az alumíniumhab forgácsolásának lehetőségeit

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

Protein és glikán biomarkerek szerepe a krónikus obstruktív tüdőbetegség diagnosztikájában = The potential role of proteomic and glycomic biomarkers in chronic obstructive pulmonary disease diagnostics

Absztrakt: A krónikus obstruktív tüdőbetegség (COPD) világszerte előkelő helyet foglal el a morbiditási és mortalitási statisztikákban. A COPD megelőzhető és kezelhető betegség, kialakulásáért döntően a dohányzás tehető felelőssé. A prevenció kulcsfontosságú, de korlátozottan kivitelezhető, így a rizikó meghatározása és a korai noninvazív diagnosztika révén lehetne tovább csökkenteni a COPD miatti halálozást. A fejlődő diagnosztikus technikák ellenére az optimális szűrővizsgálat felfedezése még várat magára. Kellően szenzitív és specifikus biomarkerek felfedezése megfelelő eszközt adhat a klinikus kezébe a betegség korai diagnosztizálásához, a differenciáldiagnosztikához, a fenotipizáláshoz és a prognózis becsléséhez. Közleményünkben a COPD vonatkozásában a közelmúltban felfedezett potenciális fehérje és glikán biomarkereket foglaljuk össze. Orv Hetil. 2020; 161(4): 123–128. | Abstract: Chronic obstructive pulmonary disease (COPD) is worldwide a significant representative of morbidity and mortality statistics. COPD is a preventable and treatable disease and smoking is the main risk factor of disease development. Prevention is crucial, but it has its limitations, so risk estimation and early non-invasive diagnostics are essential to decrease COPD mortality. Although diagnostic techniques are evolving, the perfect screening tool is lacking. Discovery of properly sensitive and specific biomarkers is important. They could be effective diagnostic, differential diagnostic, phenotyping and prognostic tools to clinicians. The manuscript is focusing on recently discovered potential protein and glycan biomarkers for COPD. Orv Hetil. 2020; 161(4): 123–128

Examination of the Correlation Between Ethical Attitudes and Dark Triad Personality Traits Among University Students

The purpose of the present paper is to examine the relationship between the ethical attitudes of university students and their Dark Triad personality traits. Research has suggested that the juvenile attitudes of people predict their later behaviour. Therefore, it is worth exploring this area for future aspects. Both topics are at the center of research, both individually and in terms of the correlations between them. In the present paper, we examined the relationship between ethical attitudes and the Dark Triad personality traits through questionnaires used in international research with background variables. We found that the most unethical behaviour is ‘Divulging confidential information,’ ‘Passing blame for your errors to an innocent co-worker,’ and ‘Claiming credit for someone else's work.’ The least unethical behaviour is ‘Eating snacks while at your workstation’. Factor analysis and linear regression analysis were used. Despite our expectations, the revealed relationships between ethical attitudes and Dark Triad personality traits were not clearly positive. According to the regression model, the dark triad personality score is lower if the respondent is female, studies at the commerce-marketing major, and has higher values at the following factors: ‘physiological unethicalness’, ‘overcharge’, and ‘other ethical attitudes’ factors

Deletion of either the regulatory gene ara1 or metabolic gene xki1 in Trichoderma reesei leads to increased CAZyme gene expression on crude plant biomass.

BackgroundTrichoderma reesei is one of the major producers of enzymes for the conversion of plant biomass to sustainable fuels and chemicals. Crude plant biomass can induce the production of CAZymes in T. reesei, but there is limited understanding of how the transcriptional response to crude plant biomass is regulated. In addition, it is unknown whether induction on untreated recalcitrant crude plant biomass (with a large diversity of inducers) can be sustained for longer. We investigated the transcriptomic response of T. reesei to the two industrial feedstocks, corn stover (CS) and soybean hulls (SBH), over time (4 h, 24 h and 48 h), and its regulatory basis using transcription factor deletion mutants (Δxyr1 and Δara1). We also investigated whether deletion of a xylulokinase gene (Δxki1) from the pentose catabolic pathway that converts potential inducers could lead to increased CAZyme gene expression.ResultsBy analyzing the transcriptomic responses using clustering as well as differential and cumulative expression of plant biomass degrading CAZymes, we found that corn stover induced a broader range and higher expression of CAZymes in T. reesei, while SBH induced more pectinolytic and mannanolytic transcripts. XYR1 was the major TF regulating CS utilization, likely due to the significant amount of d-xylose in this substrate. In contrast, ARA1 had a stronger effect on SBH utilization, which correlates with a higher abundance of l-arabinose in SBH that activates ARA1. Blocking pentose catabolism by deletion of xki1 led to higher expression of CAZyme encoding genes on both substrates at later time points. Surprisingly, this was also observed for Δara1 at later time points. Many of these genes were XYR1 regulated, suggesting that inducers for this regulator accumulated over time on both substrates.ConclusionOur data demonstrates the complexity of the regulatory system related to plant biomass degradation in T. reesei and the effect the feedstock composition has on this. Furthermore, this dataset provides leads to improve the efficiency of a T. reesei enzyme cocktail, such as by the choice of substrate or by deleting xki1 to obtain higher production of plant biomass degrading CAZymes

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 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