Analysis of the molecular basis for the non-amylolytic and non-proteolytic nature of Aspergillus vadensis CBS 113365.

Aspergillus vadensis CBS 113365, a close relative of A. niger, has been suggested as a more favourable alternative for recombinant protein production as it does not acidify the culture medium and produces very low levels of extracellular proteases. The aim of this study was to investigate the underlying cause of the non-amylolytic and non-proteolytic phenotype of A. vadensis CBS 113365. Our results demonstrate that the non-functionality of the amylolytic transcription factor AmyR in A. vadensis CBS 113365 is primarily attributed to the lack of functionality of its gene's promoter sequence. In contrast, a different mechanism is likely causing the lack of PrtT activity, which is the main transcriptional regulator of protease production. The findings presented here not only expand our understanding of the genetic basis behind the distinct characteristics of A. vadensis CBS 113365, but also underscore its potential as a favourable alternative for recombinant protein production.

From 13C-lignin to 13C-mycelium: Agaricus bisporus uses polymeric lignin as a carbon source.

Plant biomass conversion by saprotrophic fungi plays a pivotal role in terrestrial carbon (C) cycling. The general consensus is that fungi metabolize carbohydrates, while lignin is only degraded and mineralized to CO2. Recent research, however, demonstrated fungal conversion of 13C-monoaromatic compounds into proteinogenic amino acids. To unambiguously prove that polymeric lignin is not merely degraded, but also metabolized, carefully isolated 13C-labeled lignin served as substrate for Agaricus bisporus, the world's most consumed mushroom. The fungus formed a dense mycelial network, secreted lignin-active enzymes, depolymerized, and removed lignin. With a lignin carbon use efficiency of 0.14 (g/g) and fungal biomass enrichment in 13C, we demonstrate that A. bisporus assimilated and further metabolized lignin when offered as C-source. Amino acids were high in 13C-enrichment, while fungal-derived carbohydrates, fatty acids, and ergosterol showed traces of 13C. These results hint at lignin conversion via aromatic ring-cleaved intermediates to central metabolites, underlining lignin's metabolic value for fungi.

Aspergillus nidulans gfdB, Encoding the Hyperosmotic Stress Protein Glycerol-3-phosphate Dehydrogenase, Disrupts Osmoadaptation in Aspergillus wentii.

The genome of the osmophilic Aspergillus wentii, unlike that of the osmotolerant Aspergillus nidulans, contains only the gfdA, but not the gfdB, glycerol 3-phosphate dehydrogenase gene. Here, we studied transcriptomic changes of A. nidulans (reference strain and ΔgfdB gene deletion mutant) and A. wentii (reference strain and An-gfdB expressing mutant) elicited by high osmolarity. A. nidulans showed a canonic hyperosmotic stress response characterized by the upregulation of the trehalose and glycerol metabolism genes (including gfdB), as well as the genes of the high-osmolarity glycerol (HOG) map kinase pathway. The deletion of gfdB caused only negligible alterations in the transcriptome, suggesting that the glycerol metabolism was flexible enough to compensate for the missing GfdB activity in this species. A. wentii responded differently to increased osmolarity than did A. nidulans, e.g., the bulk upregulation of the glycerol and trehalose metabolism genes, along with the HOG pathway genes, was not detected. The expression of An-gfdB in A. wentii did not abolish osmophily, but it reduced growth and caused much bigger alterations in the transcriptome than did the missing gfdB gene in A. nidulans. Flexible glycerol metabolism and hence, two differently regulated gfd genes, may be more beneficial for osmotolerant (living under changing osmolarity) than for osmophilic (living under constantly high osmolarity) species.

The bZIP transcription factor BIP1 of the rice blast fungus is essential for infection and regulates a specific set of appressorium genes.

The rice blast fungus Magnaporthe oryzae differentiates specialized cells called appressoria that are required for fungal penetration into host leaves. In this study, we identified the novel basic leucine zipper (bZIP) transcription factor BIP1 (B-ZIP Involved in Pathogenesis-1) that is essential for pathogenicity. BIP1 is required for the infection of plant leaves, even if they are wounded, but not for appressorium-mediated penetration of artificial cellophane membranes. This phenotype suggests that BIP1 is not implicated in the differentiation of the penetration peg but is necessary for the initial establishment of the fungus within plant cells. BIP1 expression was restricted to the appressorium by both transcriptional and post-transcriptional control. Genome-wide transcriptome analysis showed that 40 genes were down regulated in a BIP1 deletion mutant. Most of these genes were specifically expressed in the appressorium. They encode proteins with pathogenesis-related functions such as enzymes involved in secondary metabolism including those encoded by the ACE1 gene cluster, small secreted proteins such as SLP2, BAS2, BAS3, and AVR-Pi9 effectors, as well as plant cuticle and cell wall degrading enzymes. Interestingly, this BIP1 network is different from other known infection-related regulatory networks, highlighting the complexity of gene expression control during plant-fungal interactions. Promoters of BIP1-regulated genes shared a GCN4/bZIP-binding DNA motif (TGACTC) binding in vitro to BIP1. Mutation of this motif in the promoter of MGG_08381.7 from the ACE1 gene cluster abolished its appressorium-specific expression, showing that BIP1 behaves as a transcriptional activator. In summary, our findings demonstrate that BIP1 is critical for the expression of early invasion-related genes in appressoria. These genes are likely needed for biotrophic invasion of the first infected host cell, but not for the penetration process itself. Through these mechanisms, the blast fungus strategically anticipates the host plant environment and responses during appressorium-mediated penetration.

Genome Mining Reveals a Surprising Number of Sugar Reductases in Aspergillus niger.

Metabolic engineering of filamentous fungi has received increasing attention in recent years, especially in the context of creating better industrial fungal cell factories to produce a wide range of valuable enzymes and metabolites from plant biomass. Recent studies into the pentose catabolic pathway (PCP) in Aspergillus niger have revealed functional redundancy in most of the pathway steps. In this study, a closer examination of the A. niger genome revealed five additional paralogs for the three original pentose reductases (LarA, XyrA, XyrB). Analysis of these genes using phylogeny, in vitro and in vivo functional analysis of the enzymes, and gene expression revealed that all can functionally replace LarA, XyrA, and XyrB. However, they are also active on several other sugars, suggesting a role for them in other pathways. This study therefore reveals the diversity of primary carbon metabolism in fungi, suggesting an intricate evolutionary process that distinguishes different species. In addition, through this study, the metabolic toolkit for synthetic biology and metabolic engineering of A. niger and other fungal cell factories has been expanded.

Strategies for the Development of Industrial Fungal Producing Strains.

The use of microorganisms in industry has enabled the (over)production of various compounds (e.g., primary and secondary metabolites, proteins and enzymes) that are relevant for the production of antibiotics, food, beverages, cosmetics, chemicals and biofuels, among others. Industrial strains are commonly obtained by conventional (non-GMO) strain improvement strategies and random screening and selection. However, recombinant DNA technology has made it possible to improve microbial strains by adding, deleting or modifying specific genes. Techniques such as genetic engineering and genome editing are contributing to the development of industrial production strains. Nevertheless, there is still significant room for further strain improvement. In this review, we will focus on classical and recent methods, tools and technologies used for the development of fungal production strains with the potential to be applied at an industrial scale. Additionally, the use of functional genomics, transcriptomics, proteomics and metabolomics together with the implementation of genetic manipulation techniques and expression tools will be discussed.

Comparative Genomics and Transcriptomics Analyses Reveal Divergent Plant Biomass-Degrading Strategies in Fungi.

Plant biomass is one of the most abundant renewable carbon sources, which holds great potential for replacing current fossil-based production of fuels and chemicals. In nature, fungi can efficiently degrade plant polysaccharides by secreting a broad range of carbohydrate-active enzymes (CAZymes), such as cellulases, hemicellulases, and pectinases. Due to the crucial role of plant biomass-degrading (PBD) CAZymes in fungal growth and related biotechnology applications, investigation of their genomic diversity and transcriptional dynamics has attracted increasing attention. In this project, we systematically compared the genome content of PBD CAZymes in six taxonomically distant species, Aspergillus niger, Aspergillus nidulans, Penicillium subrubescens, Trichoderma reesei, Phanerochaete chrysosporium, and Dichomitus squalens, as well as their transcriptome profiles during growth on nine monosaccharides. Considerable genomic variation and remarkable transcriptomic diversity of CAZymes were identified, implying the preferred carbon source of these fungi and their different methods of transcription regulation. In addition, the specific carbon utilization ability inferred from genomics and transcriptomics was compared with fungal growth profiles on corresponding sugars, to improve our understanding of the conversion process. This study enhances our understanding of genomic and transcriptomic diversity of fungal plant polysaccharide-degrading enzymes and provides new insights into designing enzyme mixtures and metabolic engineering of fungi for related industrial applications.

Heterologous protein production in filamentous fungi.

Filamentous fungi are able to produce a wide range of valuable proteins and enzymes for many industrial applications. Recent advances in fungal genomics and experimental technologies are rapidly changing the approaches for the development and use of filamentous fungi as hosts for the production of both homologous and heterologous proteins. In this review, we highlight the benefits and challenges of using filamentous fungi for the production of heterologous proteins. We review various techniques commonly employed to improve the heterologous protein production in filamentous fungi, such as strong and inducible promoters, codon optimization, more efficient signal peptides for secretion, carrier proteins, engineering of glycosylation sites, regulation of the unfolded protein response and endoplasmic reticulum associated protein degradation, optimization of the intracellular transport process, regulation of unconventional protein secretion, and construction of protease-deficient strains. KEY POINTS: • This review updates the knowledge on heterologous protein production in filamentous fungi. • Several fungal cell factories and potential candidates are discussed. • Insights into improving heterologous gene expression are given.

The secretome of Agaricus bisporus: Temporal dynamics of plant polysaccharides and lignin degradation.

Despite substantial lignocellulose conversion during mycelial growth, previous transcriptome and proteome studies have not yet revealed how secretomes from the edible mushroom Agaricus bisporus develop and whether they modify lignin models in vitro. To clarify these aspects, A. bisporus secretomes collected throughout a 15-day industrial substrate production and from axenic lab-cultures were subjected to proteomics, and tested on polysaccharides and lignin models. Secretomes (day 6-15) comprised A. bisporus endo-acting and substituent-removing glycoside hydrolases, whereas ß-xylosidase and glucosidase activities gradually decreased. Laccases appeared from day 6 onwards. From day 10 onwards, many oxidoreductases were found, with numerous multicopper oxidases (MCO), aryl alcohol oxidases (AAO), glyoxal oxidases (GLOX), a manganese peroxidase (MnP), and unspecific peroxygenases (UPO). Secretomes modified dimeric lignin models, thereby catalyzing syringylglycerol-ß-guaiacyl ether (SBG) cleavage, guaiacylglycerol-ß-guaiacyl ether (GBG) polymerization, and non-phenolic veratrylglycerol-ß-guaiacyl ether (VBG) oxidation. We explored A. bisporus secretomes and insights obtained can help to better understand biomass valorization.

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.

Identification of an l-Arabitol Transporter from Aspergillus niger.

l-arabitol is an intermediate of the pentose catabolic pathway in fungi but can also be used as a carbon source by many fungi, suggesting the presence of transporters for this polyol. In this study, an l-arabitol transporter, LatA, was identified in Aspergillus niger. Growth and expression profiles as well as sugar consumption analysis indicated that LatA only imports l-arabitol and is regulated by the arabinanolytic transcriptional activator AraR. Moreover, l-arabitol production from wheat bran was increased in a metabolically engineered A. niger mutant by the deletion of latA, indicating its potential for improving l-arabitol-producing cell factories. Phylogenetic analysis showed that homologs of LatA are widely conserved in fungi.

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 transcriptomic response of two basidiomycete fungi to plant biomass is modulated by temperature to a different extent.

Many fungi show a strong preference for specific habitats and growth conditions. Investigating the molecular mechanisms of fungal adaptation to varying environmental conditions is of great interest to biodiversity research and is important for many industrial applications. In this study, we compared the transcriptome profiles of two previously genome-sequenced white-rot wood-decay fungi, Trametes pubescens and Phlebia centrifuga, during their growth on two common plant biomass substrates (wheat straw and spruce) at two temperatures (15 °C and 25 °C). The results showed that both fungi partially tailored their molecular responses to different types of carbon sources, differentially expressing genes encoding polysaccharide degrading enzymes, transporters, proteases and monooxygenases. Notably, more lignin modification related AA2 genes and cellulose degradation related AA9 genes were differentially expressed in the tested conditions of T. pubescens than P. centrifuga. In addition, we detected more remarkable transcriptome changes to different growth temperature in P. centrifuga than in T. pubescens, which reflected their different ability to adapt to the temperature fluctuations. In P. centrifuga, differentially expressed genes (DEGs) related to temperature response mainly encode protein kinases, trehalose metabolism, carbon metabolic enzymes and glycoside hydrolases, while the main temperature-related DEGs identified in T. pubescens are only the carbon metabolic enzymes and glycoside hydrolases. Our study revealed both conserved and species-specific transcriptome changes during fungal adaptation to a changing environment, improving our understanding of the molecular mechanisms underlying fungal plant biomass conversion at varying temperatures.

Species-specific effects of the introduction of Aspergillus nidulans gfdB in osmophilic aspergilli.

Industrial fungi need a strong environmental stress tolerance to ensure acceptable efficiency and yields. Previous studies shed light on the important role that Aspergillus nidulans gfdB, putatively encoding a NAD+-dependent glycerol-3-phosphate dehydrogenase, plays in the oxidative and cell wall integrity stress tolerance of this filamentous fungus model organism. The insertion of A. nidulans gfdB into the genome of Aspergillus glaucus strengthened the environmental stress tolerance of this xerophilic/osmophilic fungus, which may facilitate the involvement of this fungus in various industrial and environmental biotechnological processes. On the other hand, the transfer of A. nidulans gfdB to Aspergillus wentii, another promising industrial xerophilic/osmophilic fungus, resulted only in minor and sporadic improvement in environmental stress tolerance and meanwhile partially reversed osmophily. Because A. glaucus and A. wentii are phylogenetically closely related species and both fungi lack a gfdB ortholog, these results warn us that any disturbance of the stress response system of the aspergilli may elicit rather complex and even unforeseeable, species-specific physiological changes. This should be taken into consideration in any future targeted industrial strain development projects aiming at the fortification of the general stress tolerance of these fungi. KEY POINTS: • A. wentii c' gfdB strains showed minor and sporadic stress tolerance phenotypes. • The osmophily of A. wentii significantly decreased in the c' gfdB strains. • Insertion of gfdB caused species-specific phenotypes in A. wentii and A. glaucus.

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.

Comparative Analysis of Enzyme Production Patterns of Lignocellulose Degradation of Two White Rot Fungi: Obba rivulosa and Gelatoporia subvermispora.

The unique ability of basidiomycete white rot fungi to degrade all components of plant cell walls makes them indispensable organisms in the global carbon cycle. In this study, we analyzed the proteomes of two closely related white rot fungi, Obba rivulosa and Gelatoporia subvermispora, during eight-week cultivation on solid spruce wood. Plant cell wall degrading carbohydrate-active enzymes (CAZymes) represented approximately 5% of the total proteins in both species. A core set of orthologous plant cell wall degrading CAZymes was shared between these species on spruce suggesting a conserved plant biomass degradation approach in this clade of basidiomycete fungi. However, differences in time-dependent production of plant cell wall degrading enzymes may be due to differences among initial growth rates of these species on solid spruce wood. The obtained results provide insight into specific enzymes and enzyme sets that are produced during the degradation of solid spruce wood in these fungi. These findings expand the knowledge on enzyme production in nature-mimicking conditions and may contribute to the exploitation of white rot fungi and their enzymes for biotechnological applications.

Potential Fungi Isolated From Anti-biodegradable Chinese Medicine Residue to Degrade Lignocellulose.

Traditional Chinese medicine is one of the ancient medicines which is popular in Asian countries, among which the residue produced by the use of anti-biodegradables is endless, and causes significant adverse impacts on the environment. However, the high acidity of anti-biodegradable residues and some special biological activities make it difficult for microorganisms to survive, resulting in a very low degradation rate of lignocellulose in naturally stacked residues, which directly impedes the degradation of residues. We aimed to identify the fungal strains that efficiently biodegrade anti-biodegradable residue and see the possibility to improve the biodegradation of it and other agricultural wastes by co-cultivating these fungi. We isolated 302 fungal strains from anti-biodegradable residue to test hydrolysis ability. Finally, we found Coniochaeta sp., Fomitopsis sp., Nemania sp., Talaromyces sp., Phaeophlebiopsis sp. which inhabit the anti-biodegradable residues are capable of producing higher concentrations of extracellular enzymes. Synergistic fungal combinations (viz., Fomitopsis sp. + Phaeophlebiopsis sp.; Talaromyces sp. + Coniochaeta sp. + Fomitopsis sp.; Talaromyces sp. + Fomitopsis sp. + Piloderma sp. and Talaromyces sp. + Nemania sp. + Piloderma sp.) have better overall degradation effect on lignocellulose. Therefore, these fungi and their combinations have strong potential to be further developed for bioremediation and biological enzyme industrial production.

GH10 and GH11 endoxylanases in Penicillium subrubescens: Comparative characterization and synergy with GH51, GH54, GH62 α-L-arabinofuranosidases from the same fungus.

Penicillium subrubescens has an expanded set of genes encoding putative endoxylanases (PsXLNs) compared to most other Penicillia and other fungi. In this study, all GH10 and GH11 PsXLNs were produced heterologously in Pichia pastoris and characterized. They were active towards beech wood xylan (BWX) and wheat flour arabinoxylan (WAX), and showed stability over a wide pH range. Additionally, PsXLNs released distinct oligosaccharides from WAX, and showed significant cooperative action with P. subrubescens α-L-arabinofuranosidases (PsABFs) from GH51 or GH54 for WAX degradation, giving insight into a more diverse XLN and ABF system for the efficient degradation of complex hemicelluloses. Homology modeling analysis pointed out differences in the catalytic center of PsXLNs, which are discussed in view of the different modes of action observed. These findings facilitate understanding of structural requirements for substrate recognition to contribute to recombinant XLN engineering for biotechnological applications.

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.