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.

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.

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.

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.

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

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.

The Cultivation Method Affects the Transcriptomic Response of Aspergillus niger to Growth on Sugar Beet Pulp.

In nature, filamentous fungi are exposed to diverse nutritional sources and changes in substrate availability. Conversely, in submerged cultures, mycelia are continuously exposed to the existing substrates, which are depleted over time. Submerged cultures are the preferred choice for experimental setups in laboratory and industry and are often used for understanding the physiology of fungi. However, to what extent the cultivation method affects fungal physiology, with respect to utilization of natural substrates, has not been addressed in detail. Here, we compared the transcriptomic responses of Aspergillus niger grown in submerged culture and solid culture, both containing sugar beet pulp (SBP) as a carbon source. The results showed that expression of CAZy (Carbohydrate Active enZyme)-encoding and sugar catabolic genes in liquid SBP was time dependent. Moreover, additional components of SBP delayed the A. niger response to the degradation of pectin present in SBP. In addition, we demonstrated that liquid cultures induced wider transcriptome variability than solid cultures. Although there was a correlation regarding sugar metabolic gene expression patterns between liquid and solid cultures, it decreased in the case of CAZyme-encoding genes. In conclusion, the transcriptomic response of A. niger to SBP is influenced by the culturing method, limiting the value of liquid cultures for understanding the behavior of fungi in natural habitats. IMPORTANCE Understanding the interaction between filamentous fungi and their natural and biotechnological environments has been of great interest for the scientific community. Submerged cultures are preferred over solid cultures at a laboratory scale to study the natural response of fungi to different stimuli found in nature (e.g., carbon/nitrogen sources, pH). However, whether and to what extent submerged cultures introduce variation in the physiology of fungi during growth on plant biomass have not been studied in detail. In this study, we compared the transcriptomic responses of Aspergillus niger to growth on liquid and solid cultures containing sugar beet pulp (a by-product of the sugar industry) as a carbon source. We demonstrate that the transcriptomic response of A. niger was highly affected by the culture condition, since the transcriptomic response obtained in a liquid environment could not fully explain the behavior of the fungus in a solid environment. This could partially explain the differences often observed between the phenotypes on plates compared to liquid cultures.

The chimeric GaaR-XlnR transcription factor induces pectinolytic activities in the presence of D-xylose in Aspergillus niger.

Aspergillus niger is a filamentous fungus well known for its ability to produce a wide variety of pectinolytic enzymes, which have many applications in the industry. The transcriptional activator GaaR is induced by 2-keto-3-deoxy-L-galactonate, a compound derived from D-galacturonic acid, and plays a major role in the regulation of pectinolytic genes. The requirement for inducer molecules can be a limiting factor for the production of enzymes. Therefore, the generation of chimeric transcription factors able to activate the expression of pectinolytic genes by using underutilized agricultural residues would be highly valuable for industrial applications. In this study, we used the CRISPR/Cas9 system to generate three chimeric GaaR-XlnR transcription factors expressed by the xlnR promoter by swapping the N-terminal region of the xylanolytic regulator XlnR to that of the GaaR in A. niger. As a test case, we constructed a PpgaX-hph reporter strain to evaluate the alteration of transcription factor specificity in the chimeric mutants. Our results showed that the chimeric GaaR-XlnR transcription factor was induced in the presence of D-xylose. Additionally, we generated a constitutively active GaaR-XlnR V756F version of the most efficient chimeric transcription factor to better assess its activity. Proteomics analysis confirmed the production of several pectinolytic enzymes by ΔgaaR mutants carrying the chimeric transcription factor. This correlates with the improved release of D-galacturonic acid from pectin by the GaaR-XlnR V756F mutant, as well as by the increased L-arabinose release from the pectin side chains by both chimeric mutants under inducing condition, which is required for efficient degradation of pectin. KEY POINTS: • Chimeric transcription factors were generated by on-site mutations using CRISPR/Cas9. • PpgaX-hph reporter strain allowed for the screening of functional GaaR-XlnR mutants. • Chimeric GaaR-XlnR induced pectinolytic activities in the presence of D-xylose.

Blocking utilization of major plant biomass polysaccharides leads Aspergillus niger towards utilization of minor components.

Fungi produce a wide range of enzymes that allow them to grow on diverse plant biomass. Wheat bran is a low-cost substrate with high potential for biotechnological applications. It mainly contains cellulose and (arabino)xylan, as well as starch, proteins, lipids and lignin to a lesser extent. In this study, we dissected the regulatory network governing wheat bran degradation in Aspergillus niger to assess the relative contribution of the regulators to the utilization of this plant biomass substrate. Deletion of genes encoding transcription factors involved in (hemi-)cellulose utilization (XlnR, AraR, ClrA and ClrB) individually and in combination significantly reduced production of polysaccharide-degrading enzymes, but retained substantial growth on wheat bran. Proteomic analysis suggested the ability of A. niger to grow on other carbon components, such as starch, which was confirmed by the additional deletion of the amylolytic regulator AmyR. Growth was further reduced but not impaired, indicating that other minor components provide sufficient energy for residual growth, displaying the flexibility of A. niger, and likely other fungi, in carbon utilization. Better understanding of the complexity and flexibility of fungal regulatory networks will facilitate the generation of more efficient fungal cell factories that use plant biomass as a substrate.

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

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.

Genetic barcodes allow traceability of CRISPR/Cas9-derived Aspergillus niger strains without affecting their fitness.

Safe use of genetically modified organisms (GMOs) in biotechnology requires the ability to track the presence of these strains in any environment in which they are applied. For this, introduction of genetic barcodes within the editing site represents a valuable tool for the identification of microbial strains that have undergone genetic modifications. However, it is not known whether these barcodes would have any unexpected effect in the resulting strains or affect the efficiency of the genetic modification. CRISPR/Cas9 has become one of the fastest-growing technologies for genome editing in a range of organisms, including fungi. However, this technology enables the generation of scarless GMOs that are very difficult to distinguish from naturally occurring mutants or other modified organisms. In this study, we address this issue using the industrial workhorse Aspergillus niger as a test case. We applied CRISPR/Cas9 technology to delete the genes encoding the transcriptional regulators XlnR and AraR, involved in the production of plant biomass-degrading enzymes. We generated 20-bp barcoded and non-barcoded ΔxlnR and ΔaraR mutants and analyzed the traceability and fitness of the resulting strains, as well as the efficiency of the genetic modification. Results showed that both barcoded and non-barcoded mutants can be traced by routine PCR reactions when the specific CRISPR/Cas9 modification is known. Additionally, barcodes neither affected the efficiency of the genetic modification nor the growth or protein production of the resulting strains. These results confirm the suitability of genetic barcodes to trace CRISPR-derived GMOs without affecting the performance of the resulting strains.

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.

A comparative genomics study of 23 Aspergillus species from section Flavi.

Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.

CRISPR/Cas9 technology enables the development of the filamentous ascomycete fungus Penicillium subrubescens as a new industrial enzyme producer.

Penicillium subrubescens is an ascomycete fungus with an enriched content of specific carbohydrate-active enzyme families involved in plant biomass degradation, which makes this strain a promising industrial cell factory for enzyme production. The development of tools that allow genetic manipulation is crucial for further strain improvement and the functional characterization of its genes. In this context, the CRISPR/Cas9 system represents an excellent option for genome editing due to its high efficiency and versatility. To establish CRISPR/Cas9 genome editing in P. subrubescens, first a method for protoplast generation and transformation was developed, using hygromycin as selection marker. Then the CRISPR/Cas9 system was established in P. subrubescens by successfully deleting the ku70 gene, which is involved in the non-homologous end joining DNA repair mechanism. Phenotypic characterization of the mutants showed that ku70 mutation did not affect P. subrubescens growth at optimal temperature and Δku70 strains showed similar protein production pattern to the wild type.

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.

Blocking hexose entry into glycolysis activates alternative metabolic conversion of these sugars and upregulates pentose metabolism in Aspergillus nidulans.

BACKGROUND: Plant biomass is the most abundant carbon source for many fungal species. In the biobased industry fungi, are used to produce lignocellulolytic enzymes to degrade agricultural waste biomass. Here we evaluated if it would be possible to create an Aspergillus nidulans strain that releases, but does not metabolize hexoses from plant biomass. For this purpose, metabolic mutants were generated that were impaired in glycolysis, by using hexokinase (hxkA) and glucokinase (glkA) negative strains. To prevent repression of enzyme production due to the hexose accumulation, strains were generated that combined these mutations with a deletion in creA, the repressor involved in regulating preferential use of different carbon catabolic pathways. RESULTS: Phenotypic analysis revealed reduced growth for the hxkA1 glkA4 mutant on wheat bran. However, hexoses did not accumulate during growth of the mutants on wheat bran, suggesting that glucose metabolism is re-routed towards alternative carbon catabolic pathways. The creAΔ4 mutation in combination with preventing initial phosphorylation in glycolysis resulted in better growth than the hxkA/glkA mutant and an increased expression of pentose catabolic and pentose phosphate pathway genes. This indicates that the reduced ability to use hexoses as carbon sources created a shift towards the pentose fraction of wheat bran as a major carbon source to support growth. CONCLUSION: Blocking the direct entry of hexoses to glycolysis activates alternative metabolic conversion of these sugars in A. nidulans during growth on plant biomass, but also upregulates conversion of other sugars, such as pentoses.