Next to d-glucose, the pentoses l-arabinose and d-xylose are the main monosaccharide components of plant cell wall polysaccharides and are therefore of major importance in biotechnological applications that use plant biomass as a substrate. Pentose catabolism is one of the best-studied pathways of primary metabolism of Aspergillus niger, and an initial outline of this pathway with individual enzymes covering each step of the pathway has been previously established. However, although growth on l-arabinose and/or d-xylose of most pentose catabolic pathway (PCP) single deletion mutants of A. niger has been shown to be negatively affected, it was not abolished, suggesting the involvement of additional enzymes. Detailed analysis of the single deletion mutants of the known A. niger PCP genes led to the identification of additional genes involved in the pathway. These results reveal a high level of complexity and redundancy in this pathway, emphasizing the need for a comprehensive understanding of metabolic pathways before entering metabolic engineering of such pathways for the generation of more efficient fungal cell factories.

In fungi, L-rhamnose (Rha) is converted via four enzymatic steps into pyruvate and L-lactaldehyde, which enter central carbon metabolism. In Aspergillus niger, only the genes involved in the first three steps of the Rha catabolic pathway have been identified and characterized, and the inducer of the pathway regulator RhaR remained unknown. In this study, we identified the gene (lkaA) involved in the conversion of L-2-keto-3-deoxyrhamnonate (L-KDR) into pyruvate and L-lactaldehyde, which is the last step of the Rha pathway. Deletion of lkaA resulted in impaired growth on L-rhamnose, and potentially in accumulation of L-KDR. Contrary to ΔlraA, ΔlrlA and ΔlrdA, the expression of the Rha-responsive genes that are under control of RhaR, were at the same levels in ΔlkaA and the reference strain, indicating the role of L-KDR as the inducer of the Rha pathway regulator.

Lignocellulosic plant biomass is an important feedstock for bio-based economy. In particular, it is an abundant renewable source of aromatic compounds, which are present as part of lignin, as side-groups of xylan and pectin, and in other forms, such as tannins. As filamentous fungi are the main organisms that modify and degrade lignocellulose, they have developed a versatile metabolism to convert the aromatic compounds that are toxic at relatively low concentrations to less toxic ones. During this process, fungi form metabolites some of which represent high-value platform chemicals or important chemical building blocks, such as benzoic, vanillic, and protocatechuic acid. Especially basidiomycete white-rot fungi with unique ability to degrade the recalcitrant lignin polymer are expected to perform highly efficient enzymatic conversions of aromatic compounds, thus having huge potential for biotechnological exploitation. However, the aromatic metabolism of basidiomycete fungi is poorly studied and knowledge on them is based on the combined results of studies in variety of species, leaving the overall picture in each organism unclear. Dichomitus squalens is an efficiently wood-degrading white-rot basidiomycete that produces a diverse set of extracellular enzymes targeted for lignocellulose degradation, including oxidative enzymes that act on lignin. Our recent study showed that several intra- and extracellular aromatic compounds were produced when D. squalens was cultivated on spruce wood, indicating also versatile aromatic metabolic abilities for this species. In order to provide the first molecular level systematic insight into the conversion of plant biomass derived aromatic compounds by basidiomycete fungi, we analyzed the transcriptomes of D. squalens when grown with 10 different lignocellulose-related aromatic monomers. Significant differences for example with respect to the expression of lignocellulose degradation related genes, but also putative genes encoding transporters and catabolic pathway genes were observed between the cultivations supplemented with the different aromatic compounds. The results demonstrate that the transcriptional response of D. squalens is highly dependent on the specific aromatic compounds present suggesting that instead of a common regulatory system, fine-tuned regulation is needed for aromatic metabolism.

Here we report the genome sequence of the ascomycete saprobic fungus Penicillium subrubescens FBCC1632/CBS132785 isolated from a Jerusalem artichoke field in Finland. The 39.75Mb genome containing 14,188 gene models is highly similar for that reported for other Penicillium species, but contains a significantly higher number of putative carbohydrate active enzyme (CAZyme) encoding genes.

Heterologous production of fungal ligninolytic cocktails is challenging due to the low yields of catalytically active lignin modifying peroxidases. Production using a natural system, such as a wood-rotting fungus, is a promising alternative if specific or preferential induction of the ligninolytic activities could be achieved. Using transcriptomics, gene expression of the white-rot Dichomitus squalens during growth on mixtures of aromatic compounds, with ring structures representing the two major lignin sub-units, was compared to a wood substrate. Most of the genes encoding lignin modifying enzymes (laccases and peroxidases) categorised as highly or moderately expressed on wood were expressed similarly on aromatic compounds. Higher expression levels of a subset of manganese and versatile peroxidases was observed on di- compared to mono-methoxylated aromatics. The expression of polysaccharide degrading enzymes was lower on aromatic compounds compared to wood, demonstrating that the induction of lignin modifying enzymes became more specific. This study suggests potential for aromatic waste streams, e.g. from lignocellulose pretreatment, to produce a lignin-specific enzyme cocktail from D. squalens or other white-rot fungi.

The import of plant-derived small sugars by sugar transporters (STs) has received increasing interest due to its important biological role and great industrial potential. STs are important targets of genetic engineering to improve fungal plant biomass conversion. Comparatively analysis of the genome-wide prevalence and transcriptomics of STs was performed in four filamentous fungi: Aspergillus niger, Aspergillus nidulans, Penicillium subrubescens and Trichoderma reesei. Using phylogenetic analysis and literature mining, their predicted STs were divided into ten subfamilies with putative sugar specificities assigned. In addition, transcriptome analysis revealed complex expression profiles among different STs subfamilies and fungal species, indicating a sophisticated transcriptome regulation and functional diversity of fungal STs. Several STs showed strong co-expression with other genes involved in sugar utilization, encoding CAZymes and sugar catabolic enzymes. This study provides new insights into the diversity of STs at the genomic/transcriptomic level, facilitating their biochemical characterization and metabolic engineering.

The extent of carbon catabolite repression (CCR) at a global level is unknown in wood-rotting fungi, which are critical to the carbon cycle and are a source of biotechnological enzymes. CCR occurs in the presence of sufficient concentrations of easily metabolizable carbon sources (e.g., glucose) and involves downregulation of the expression of genes encoding enzymes involved in the breakdown of complex carbon sources. We investigated this phenomenon in the white-rot fungus Dichomitus squalens using transcriptomics and exoproteomics. In D. squalens cultures, approximately 7% of genes were repressed in the presence of glucose compared to Avicel or xylan alone. The glucose-repressed genes included the essential components for utilization of plant biomass-carbohydrate-active enzyme (CAZyme) and carbon catabolic genes. The majority of polysaccharide-degrading CAZyme genes were repressed and included activities toward all major carbohydrate polymers present in plant cell walls, while repression of ligninolytic genes also occurred. The transcriptome-level repression of the CAZyme genes observed on the Avicel cultures was strongly supported by exoproteomics. Protease-encoding genes were generally not glucose repressed, indicating their likely dominant role in scavenging for nitrogen rather than carbon. The extent of CCR is surprising, given that D. squalens rarely experiences high free sugar concentrations in its woody environment, and it indicates that biotechnological use of D. squalens for modification of plant biomass would benefit from derepressed or constitutively CAZyme-expressing strains.IMPORTANCE White-rot fungi are critical to the carbon cycle because they can mineralize all wood components using enzymes that also have biotechnological potential. The occurrence of carbon catabolite repression (CCR) in white-rot fungi is poorly understood. Previously, CCR in wood-rotting fungi has only been demonstrated for a small number of genes. We demonstrated widespread glucose-mediated CCR of plant biomass utilization in the white-rot fungus Dichomitus squalens This indicates that the CCR mechanism has been largely retained even though wood-rotting fungi rarely experience commonly considered CCR conditions in their woody environment. The general lack of repression of genes encoding proteases along with the reduction in secreted CAZymes during CCR suggested that the retention of CCR may be connected with the need to conserve nitrogen use during growth on nitrogen-scarce wood. The widespread repression indicates that derepressed strains could be beneficial for enzyme production.

In Section 3.1 of the Results and Discussion, it should have stated 100 μM and not 50 μM for the concentration of the aromatic compounds. The correct concentration was stated in the Materials and Methods and the error only occurred in the Results and Discussion section. The authors would like to apologize for any inconvenience caused.

The extent of carbon catabolite repression (CCR) at a global level is unknown in wood-rotting fungi, which are critical to the carbon cycle and are a source of biotechnological enzymes. CCR occurs in the presence of sufficient concentrations of easily metabolizable carbon sources (e.g., glucose) and involves downregulation of the expression of genes encoding enzymes involved in the breakdown of complex carbon sources. We investigated this phenomenon in the white-rot fungus Dichomitus squalens using transcriptomics and exoproteomics. In D. squalens cultures, approximately 7% of genes were repressed in the presence of glucose compared to Avicel or xylan alone. The glucose-repressed genes included the essential components for utilization of plant biomass-carbohydrate-active enzyme (CAZyme) and carbon catabolic genes. The majority of polysaccharide-degrading CAZyme genes were repressed and included activities toward all major carbohydrate polymers present in plant cell walls, while repression of ligninolytic genes also occurred. The transcriptome-level repression of the CAZyme genes observed on the Avicel cultures was strongly supported by exoproteomics. Protease-encoding genes were generally not glucose repressed, indicating their likely dominant role in scavenging for nitrogen rather than carbon. The extent of CCR is surprising, given that D. squalens rarely experiences high free sugar concentrations in its woody environment, and it indicates that biotechnological use of D. squalens for modification of plant biomass would benefit from derepressed or constitutively CAZyme-expressing strains.IMPORTANCE White-rot fungi are critical to the carbon cycle because they can mineralize all wood components using enzymes that also have biotechnological potential. The occurrence of carbon catabolite repression (CCR) in white-rot fungi is poorly understood. Previously, CCR in wood-rotting fungi has only been demonstrated for a small number of genes. We demonstrated widespread glucose-mediated CCR of plant biomass utilization in the white-rot fungus Dichomitus squalens This indicates that the CCR mechanism has been largely retained even though wood-rotting fungi rarely experience commonly considered CCR conditions in their woody environment. The general lack of repression of genes encoding proteases along with the reduction in secreted CAZymes during CCR suggested that the retention of CCR may be connected with the need to conserve nitrogen use during growth on nitrogen-scarce wood. The widespread repression indicates that derepressed strains could be beneficial for enzyme production.

Filamentous fungi produce a wide variety of enzymes in order to efficiently degrade plant cell wall polysaccharides. The production of these enzymes is controlled by transcriptional regulators, which also control the catabolic pathways that convert the released monosaccharides. Two transcriptional regulators, GalX and GalR, control d-galactose utilization in the model filamentous fungus Aspergillus nidulans, while the arabinanolytic regulator AraR regulates l-arabinose catabolism. d-Galactose and l-arabinose are commonly found together in polysaccharides, such as arabinogalactan, xylan and rhamnogalacturonan I. Therefore, the catabolic pathways that convert d-galactose and l-arabinose are often also likely to be active simultaneously. In this study, we investigated the interaction between GalX, GalR and AraR in d-galactose and l-arabinose catabolism. For this, we generated single, double and triple mutants of the three regulators, and analysed their growth and enzyme and gene expression profiles. Our results clearly demonstrated that GalX, GalR and AraR co-regulate d-galactose catabolism in A. nidulans. GalX has a prominent role on the regulation of genes of d-galactose oxido-reductive pathway, while AraR can compensate for the absence of GalR and/or GalX.