Closely related fungi employ diverse enzymatic strategies to degrade plant biomass.

BACKGROUND: Plant biomass is the major substrate for the production of biofuels and biochemicals, as well as food, textiles and other products. It is also the major carbon source for many fungi and enzymes of these fungi are essential for the depolymerization of plant polysaccharides in industrial processes. This is a highly complex process that involves a large number of extracellular enzymes as well as non-hydrolytic proteins, whose production in fungi is controlled by a set of transcriptional regulators. Aspergillus species form one of the best studied fungal genera in this field, and several species are used for the production of commercial enzyme cocktails. RESULTS: It is often assumed that related fungi use similar enzymatic approaches to degrade plant polysaccharides. In this study we have compared the genomic content and the enzymes produced by eight Aspergilli for the degradation of plant biomass. All tested Aspergilli have a similar genomic potential to degrade plant biomass, with the exception of A. clavatus that has a strongly reduced pectinolytic ability. Despite this similar genomic potential their approaches to degrade plant biomass differ markedly in the overall activities as well as the specific enzymes they employ. While many of the genes have orthologs in (nearly) all tested species, only very few of the corresponding enzymes are produced by all species during growth on wheat bran or sugar beet pulp. In addition, significant differences were observed between the enzyme sets produced on these feedstocks, largely correlating with their polysaccharide composition. CONCLUSIONS: These data demonstrate that Aspergillus species and possibly also other related fungi employ significantly different approaches to degrade plant biomass. This makes sense from an ecological perspective where mixed populations of fungi together degrade plant biomass. The results of this study indicate that combining the approaches from different species could result in improved enzyme mixtures for industrial applications, in particular saccharification of plant biomass for biofuel production. Such an approach may result in a much better improvement of saccharification efficiency than adding specific enzymes to the mixture of a single fungus, which is currently the most common approach used in biotechnology.

Comparative genomics of the white-rot fungi, Phanerochaete carnosa and P. chrysosporium, to elucidate the genetic basis of the distinct wood types they colonize.

BACKGROUND: Softwood is the predominant form of land plant biomass in the Northern hemisphere, and is among the most recalcitrant biomass resources to bioprocess technologies. The white rot fungus, Phanerochaete carnosa, has been isolated almost exclusively from softwoods, while most other known white-rot species, including Phanerochaete chrysosporium, were mainly isolated from hardwoods. Accordingly, it is anticipated that P. carnosa encodes a distinct set of enzymes and proteins that promote softwood decomposition. To elucidate the genetic basis of softwood bioconversion by a white-rot fungus, the present study reports the P. carnosa genome sequence and its comparative analysis with the previously reported P. chrysosporium genome. RESULTS: P. carnosa encodes a complete set of lignocellulose-active enzymes. Comparative genomic analysis revealed that P. carnosa is enriched with genes encoding manganese peroxidase, and that the most divergent glycoside hydrolase families were predicted to encode hemicellulases and glycoprotein degrading enzymes. Most remarkably, P. carnosa possesses one of the largest P450 contingents (266 P450s) among the sequenced and annotated wood-rotting basidiomycetes, nearly double that of P. chrysosporium. Along with metabolic pathway modeling, comparative growth studies on model compounds and chemical analyses of decomposed wood components showed greater tolerance of P. carnosa to various substrates including coniferous heartwood. CONCLUSIONS: The P. carnosa genome is enriched with genes that encode P450 monooxygenases that can participate in extractives degradation, and manganese peroxidases involved in lignin degradation. The significant expansion of P450s in P. carnosa, along with differences in carbohydrate- and lignin-degrading enzymes, could be correlated to the utilization of heartwood and sapwood preparations from both coniferous and hardwood species.

Aspergillus cibarius sp. nov., from traditional meju in Korea.

Aspergillus cibarius sp. nov. isolated from meju, a brick of dried fermented soybeans in Korea, is described. The species was also found from black bean, bread and salami in the Netherlands. It is characterized by abundant yellow to reddish brown ascomata and small lenticular ascospores (4.5-5.5 µm) with a wide furrow, low equatorial crests and tuberculate or reticulate convex surface. The species was resolved as phylogenetically distinct from the other reported Aspergillus species with an Eurotium teleomorph based on multilocus sequence typing using partial fragments of the ß-tubulin, calmodulin, ITS and RNA polymerase II genes.

D-Galactose uptake is nonfunctional in the conidiospores of Aspergillus niger.

The majority of black Aspergilli (Aspergillus section Nigri), including Aspergillus niger, as well as many other Ascomycetes fail to germinate on d-galactose as a sole carbon source. Here, we provide evidence that the ability of A. niger to transport D-galactose is growth stage dependent, being absent in the conidiospores but present in the mycelia. Despite earlier claims, we could identify galactokinase activity in growing cells and all genes of the Leloir pathway (responsible for channelling D-galactose into the EMP pathway) are well induced on D-galactose (and also on lactose, D-xylose and L-arabinose) in the mycelial stage. Expression of all Leloir pathway genes was also detectable in conidiospores, although galE (encoding a galactokinase) and galD (encoding a galactose-1-phosphate uridylyl transferase) were expressed poorly. These results suggest that the D-galactose-negative phenotype of A. niger conidiospores may be due to the lack of inducer uptake.

A broader role for AmyR in Aspergillus niger: regulation of the utilisation of D-glucose or D-galactose containing oligo- and polysaccharides.

AmyR is commonly considered a regulator of starch degradation whose activity is induced by the presence of maltose, the disaccharide building block of starch. In this study, we demonstrate that the role of AmyR extends beyond starch degradation. Enzyme activity assays, genes expression analysis and growth profiling on D-glucose- and D-galactose-containing oligo- and polysaccharides showed that AmyR regulates the expression of some of the Aspergillus niger genes encoding α- and ß-glucosidases, α- and ß- galactosidases, as well as genes encoding α-amlyases and glucoamylases. In addition, we provide evidence that D-glucose or a metabolic product thereof may be the inducer of the AmyR system in A. niger and not maltose, as is commonly assumed.

The molecular and genetic basis of conidial pigmentation in Aspergillus niger.

A characteristic hallmark of Aspergillus niger is the formation of black conidiospores. We have identified four loci involved in spore pigmentation of A. niger by using a combined genomic and classical complementation approach. First, we characterized a newly isolated color mutant, colA, which lacked pigmentation resulting in white or colorless conidia. Pigmentation of the colA mutant was restored by a gene (An12g03950) which encodes a putative 4'phosphopantetheinyl transferase protein (PptA). 4'Phosphopantetheinyl transferase activity is required for the activation of Polyketide Synthases (PKSs) and/or Non-Ribosomal Peptide Synthases (NRPSs). The loci whose mutation resulted in fawn, olive, and brown color phenotypes were identified by complementation. The fawn phenotype was complemented by a PKS protein (FwnA, An09g05730), the ovlA mutant by An14g05350 (OlvA) and the brnA mutant by An14g05370 (BrnA), the respective homologs of alb1/pksP, ayg1 and abr1 in A. fumigatus. Targeted disruption of the pptA, fwnA, olvA and brnA genes confirmed the complementation results. Disruption of the pptA gene abolished synthesis of all polyketides and non-ribosomal peptides, while the naphtho-γ-pyrone subclass of polyketides were specifically dependent on fwnA, and funalenone on fwnA, olvA and brnA. Thus, secondary metabolite profiling of the color mutants revealed a close relationship between polyketide synthesis and conidial pigmentation in A. niger.

Genome sequence of the model mushroom Schizophyllum commune.

Much remains to be learned about the biology of mushroom-forming fungi, which are an important source of food, secondary metabolites and industrial enzymes. The wood-degrading fungus Schizophyllum commune is both a genetically tractable model for studying mushroom development and a likely source of enzymes capable of efficient degradation of lignocellulosic biomass. Comparative analyses of its 38.5-megabase genome, which encodes 13,210 predicted genes, reveal the species's unique wood-degrading machinery. One-third of the 471 genes predicted to encode transcription factors are differentially expressed during sexual development of S. commune. Whereas inactivation of one of these, fst4, prevented mushroom formation, inactivation of another, fst3, resulted in more, albeit smaller, mushrooms than in the wild-type fungus. Antisense transcripts may also have a role in the formation of fruiting bodies. Better insight into the mechanisms underlying mushroom formation should affect commercial production of mushrooms and their industrial use for producing enzymes and pharmaceuticals.

Spatial and developmental differentiation of mannitol dehydrogenase and mannitol-1-phosphate dehydrogenase in Aspergillus niger.

The presence of a mannitol cycle in fungi has been subject to discussion for many years. Recent studies have found no evidence for the presence of this cycle and its putative role in regenerating NADPH. However, all enzymes of the cycle could be measured in cultures of Aspergillus niger. In this study we have analyzed the localization of two enzymes from the pathway, mannitol dehydrogenase and mannitol-1-phosphate dehydrogenase, and the expression of their encoding genes in nonsporulating and sporulating cultures of A. niger. Northern analysis demonstrated that mpdA was expressed in both sporulating and nonsporulating mycelia, while expression of mtdA was expressed only in sporulating mycelium. More detailed studies using green fluorescent protein and dTomato fused to the promoters of mtdA and mpdA, respectively, demonstrated that expression of mpdA occurs in vegetative hyphae while mtdA expression occurs in conidiospores. Activity assays for MtdA and MpdA confirmed the expression data, indicating that streaming of these proteins is not likely to occur. These results confirm the absence of the putative mannitol cycle in A. niger as two of the enzymes of the cycle are not present in the same part of A. niger colonies. The results also demonstrate the existence of spore-specific genes and enzymes in A. niger.

Post-genomic insights into the plant polysaccharide degradation potential of Aspergillus nidulans and comparison to Aspergillus niger and Aspergillus oryzae.

The plant polysaccharide degradative potential of Aspergillus nidulans was analysed in detail and compared to that of Aspergillus niger and Aspergillus oryzae using a combination of bioinformatics, physiology and transcriptomics. Manual verification indicated that 28.4% of the A. nidulans ORFs analysed in this study do not contain a secretion signal, of which 40% may be secreted through a non-classical method.While significant differences were found between the species in the numbers of ORFs assigned to the relevant CAZy families, no significant difference was observed in growth on polysaccharides. Growth differences were observed between the Aspergilli and Podospora anserina, which has a more different genomic potential for polysaccharide degradation, suggesting that large genomic differences are required to cause growth differences on polysaccharides. Differences were also detected between the Aspergilli in the presence of putative regulatory sequences in the promoters of the ORFs of this study and correlation of the presence of putative XlnR binding sites to induction by xylose was detected for A. niger. These data demonstrate differences at genome content, substrate specificity of the enzymes and gene regulation in these three Aspergilli, which likely reflect their individual adaptation to their natural biotope.

Mutations in genes encoding sorting nexins alter production of intracellular and extracellular proteases in Aspergillus nidulans.

XprG, a putative p53-like transcriptional activator, regulates production of extracellular proteases in response to nutrient limitation and may also have a role in programmed cell death. To identify genes that may be involved in the XprG regulatory pathway, xprG2 revertants were isolated and shown to carry mutations in genes which we have named sogA-C (suppressors of xprG). The translocation breakpoint in the sogA1 mutant was localized to a homolog of Saccharomyces cerevisiae VPS5 and mapping data indicated that sogB was tightly linked to a VPS17 homolog. Complementation of the sogA1 and sogB1 mutations and identification of nonsense mutations in the sogA2 and sogB1 alleles confirmed the identification. Vps17p and Vps5p are part of a complex involved in sorting of vacuolar proteins in yeast and regulation of cell-surface receptors in mammals. Protease zymograms indicate that mutations in sogA-C permit secretion of intracellular proteases, as in S. cerevisiae vps5 and vps17 mutants. In contrast to S. cerevisiae, the production of intracellular protease was much higher in the mutants. Analysis of serine protease gene expression suggests that an XprG-independent mechanism for regulation of extracellular protease gene expression in response to carbon starvation exists and is activated in the pseudorevertants.

Glucose uptake and growth of glucose-limited chemostat cultures of Aspergillus niger and a disruptant lacking MstA, a high-affinity glucose transporter.

This is a study of high-affinity glucose uptake in Aspergillus niger and the effect of disruption of a high-affinity monosaccharide-transporter gene, mstA. The substrate saturation constant (K(s)) of a reference strain was about 15 microM in glucose-limited chemostat culture. Disruption of mstA resulted in a two- to fivefold reduction in affinity for glucose and led to expression of a low-affinity glucose transport gene, mstC, at high dilution rate. The effect of mstA disruption was more subtle at low and intermediate dilution rates, pointing to some degree of functional redundancy in the high-affinity uptake system of A. niger. The mstA disruptant and a reference strain were cultivated in glucose-limited chemostat cultures at low, intermediate and high dilution rate (D=0.07 h(-1), 0.14 h(-1) and 0.20 h(-1)). Mycelium harvested from steady-state cultures was subjected to glucose uptake assays, and analysed for expression of mstA and two other transporter genes, mstC and mstF. The capacity for glucose uptake (v(max)) of both strains was significantly reduced at low dilution rate. The glucose uptake assays revealed complex uptake kinetics. This impeded accurate determination of maximum specific uptake rates (v(max)) and apparent affinity constants ( ) at intermediate and high dilution rate. Two high-affinity glucose transporter genes, mstA and mstF, were expressed at all three dilution rates in chemostat cultures, in contrast to batch culture, where only mstC was expressed. Expression patterns of the three transporter genes suggested differential regulation and functionality of their products.

The Aspergillus niger MADS-box transcription factor RlmA is required for cell wall reinforcement in response to cell wall stress.

In Aspergillus niger, the genes coding for glutamine:fructose-6-phosphate amidotransferase (gfaA) and alpha-1,3-glucan synthase (agsA) are induced in response to cell wall stress. In silico analysis of the promoter region of the two genes revealed the presence of putative DNA binding sites for transcription factors involved in stress responses, including sites identical to the Saccharomyces cerevisiae Rlm1p and Msn2p/Msn4p transcription factors. Promoter analysis indicated that the induction of the agsA gene in response to cell wall stress is fully dependent on a putative Rlm1p binding site in its promoter region. Database searches revealed the presence of S. cerevisiae Rlm1p homologues in most filamentous fungi examined, including A. niger. Deletion of the RLM1 homologue, named rlmA in A. niger, completely eliminated the induction of agsA and resulted in a twofold reduced induction of gfaA during Calcofluor White-induced cell wall stress. The rise in cell wall chitin in the presence of Calcofluor White was also affected in the rlmA deletion strain. In addition, the deletion strain was more sensitive towards cell wall stress agents. Our results indicate that A. niger responds to cell wall stress by transcriptional activation of cell wall reinforcing genes including agsA and gfaA through an Rlm1p-like transcription factor. We propose that such a cell wall salvage mechanism is wide spread in filamentous fungi.

Characterisation of CwpA, a putative glycosylphosphatidylinositol-anchored cell wall mannoprotein in the filamentous fungus Aspergillus niger.

Glycosylphosphatidylinositol (GPI)-anchored proteins in fungi are found at the cell surface, either as plasma membrane proteins (GPI-PMPs) or attached by a remnant of the GPI-anchor to the cell wall (GPI-CWPs). GPI-CWPs can be extracted from the cell wall by treatment with hydrofluoric acid (HF), which cleaves the phosphodiester bond that is present in the remnant of the GPI-anchor. The filamentous fungus Aspergillus niger contains at least seven HF-extractable cell wall mannoproteins. One gene encoding an HF-extractable cell wall mannoprotein, cwpA, was cloned and further characterised. The protein sequence of CwpA indicated the presence of two hydrophobic signal sequences both at the N-terminus and C-terminus of the protein, for entering the ER and the addition of a GPI-anchor, respectively. A CwpA-specific antiserum was raised and in combination with fractionation experiments, we show that this protein was abundantly present as an HF-extractable protein in the cell wall of A. niger.

Expression of agsA, one of five 1,3-alpha-D-glucan synthase-encoding genes in Aspergillus niger, is induced in response to cell wall stress.

1,3-alpha-D-Glucan is an important component of the cell wall of filamentous fungi. We have identified a family of five 1,3-alpha-D-glucan synthase-encoding genes in Aspergillus niger. The agsA gene was sequenced and the predicted protein sequence indicated that the overall domain structure of 1,3-alpha-D-glucan synthases is conserved in fungi. Using RT-PCR and Northern blot analysis, we found that expression of the agsA gene and to a lesser extent also of agsE were induced in the presence of the cell wall stress-inducing compounds such as Calcofluor White (CFW), SDS, and caspofungin. Loss of agsA function did not result in an apparent phenotype under normal growth conditions but rendered the cells more sensitive to CFW. The induction of 1,3-alpha-D-glucan synthase-encoding genes in response to cell wall stress was not limited to A. niger, but was also observed in Penicillium chrysogenum. We propose that this response to cell wall stress commonly occurs in filamentous fungi.

The cell wall stress response in Aspergillus niger involves increased expression of the glutamine : fructose-6-phosphate amidotransferase-encoding gene (gfaA) and increased deposition of chitin in the cell wall.

Perturbation of cell wall synthesis in Saccharomyces cerevisiae, either by mutations in cell wall synthesis-related genes or by adding compounds that interfere with normal cell wall assembly, triggers a compensatory response to ensure cell wall integrity. This response includes an increase in chitin levels in the cell wall. Here it is shown that Aspergillus niger also responds to cell wall stress by increasing chitin levels. The increased chitin level in the cell wall was accompanied by increased transcription of gfaA, encoding the glutamine : fructose-6-phosphate amidotransferase enzyme, which is responsible for the first and a rate-limiting step in chitin synthesis. Cloning and disruption of the gfaA gene in A. niger showed that it was an essential gene, but that addition of glucosamine to the growth medium could rescue the deletion strain. When the plant-pathogenic fungus Fusarium oxysporum and food spoilage fungus Penicillium chrysogenum were subjected to cell wall stress, the transcript level of their gfa gene increased as well. These observations suggest that cell wall stress in fungi may generally lead to activation of the chitin biosynthetic pathway.

Aspergillus niger mstA encodes a high-affinity sugar/H+ symporter which is regulated in response to extracellular pH.

A sugar-transporter-encoding gene, mstA, which is a member of the major facilitator superfamily, has been cloned from a genomic DNA library of the filamentous fungus Aspergillus niger. To enable the functional characterization of MSTA, a full-length cDNA was expressed in a Saccharomyces cerevisiae strain deficient in hexose uptake. Uptake experiments using 14C-labelled monosaccharides demonstrated that although able to transport D-fructose ( K(m), 4.5+/-1.0 mM), D-xylose ( K(m), 0.3+/-0.1 mM) and D-mannose ( K(m), 60+/-20 microM), MSTA has a preference for D-glucose (K(m), 25+/-10 microM). pH changes associated with sugar transport indicate that MSTA catalyses monosaccharide/H+ symport. Expression of mstA in response to carbon starvation and upon transfer to poor carbon sources is consistent with a role for MSTA as a high-affinity transporter for D-glucose, D-mannose and D-xylose. Northern analysis has shown that mstA is subject to CreA-mediated carbon catabolite repression and pH regulation mediated by PacC. A. niger strains in which the mstA gene had been disrupted are phenotypically identical with isogenic reference strains when grown on 0.1-60 mM D-glucose, D-mannose, D-fructose or D-xylose. This indicates that A. niger possesses other transporters capable of compensating for the absence of MSTA.

Mannitol is required for stress tolerance in Aspergillus niger conidiospores.

D-Mannitol is the predominant carbon compound in conidiospores of the filamentous fungus Aspergillus niger and makes up 10 to 15% of the dry weight. A number of physiological functions have been ascribed to mannitol, including serving as a reserve carbon source, as an antioxidant, and to store reducing power. In this study, we cloned and characterized the A. niger mpdA gene, which encodes mannitol 1-phosphate dehydrogenase (MPD), the first enzyme in the mannitol biosynthesis pathway. The mpdA promoter contains putative binding sites for the development-specific transcription factors BRLA and ABAA. Furthermore, increased expression of mpdA in sporulating mycelium suggests that mannitol biosynthesis is, to a certain extent, developmentally regulated in A. niger. Inactivation of mpdA abolished mannitol biosynthesis in growing mycelium and reduced the mannitol level in conidiospores to 30% that in the wild type, indicating that MPD and mannitol 1-phosphate phosphatase form the major metabolic pathway for mannitol biosynthesis in A. niger. The viability of spores after prolonged storage and germination kinetics were normal in an mpdA null mutant, indicating that mannitol does not play an essential role as a reserve carbon source in A. niger conidia. However, conidiospores of a DeltampdA strain were extremely sensitive to a variety of stress conditions, including high temperature, oxidative stress and, to a lesser extent, freezing and lyophilization. Since mannitol supplied in the medium during sporulation repaired this deficiency, mannitol appears to be essential for the protection of A. niger spores against cell damage under these stress conditions.

Isolation and characterization of two specific regulatory Aspergillus niger mutants shows antagonistic regulation of arabinan and xylan metabolism.

This paper describes two Aspergillus niger mutants (araA and araB) specifically disturbed in the regulation of the arabinanase system in response to the presence of L-arabinose. Expression of the three known L-arabinose-induced arabinanolytic genes, abfA, abfB and abnA, was substantially decreased or absent in the araA and araB strains compared to the wild-type when incubated in the presence of L-arabinose or L-arabitol. In addition, the intracellular activities of L-arabitol dehydrogenase and L-arabinose reductase, involved in L-arabinose catabolism, were decreased in the araA and araB strains. Finally, the data show that the gene encoding D-xylulose kinase, xkiA, is also under control of the arabinanolytic regulatory system. L-Arabitol, most likely the true inducer of the arabinanolytic and L-arabinose catabolic genes, accumulated to a high intracellular concentration in the araA and araB mutants. This indicates that the decrease of expression of the arabinanolytic genes was not due to lack of inducer accumulation. Therefore, it is proposed that the araA and araB mutations are localized in positive-acting components of the regulatory system involved in the expression of the arabinanase-encoding genes and the genes encoding the L-arabinose catabolic pathway.

The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds.

The faeB gene encoding a second feruloyl esterase from Aspergillus niger has been cloned and characterized. It consists of an open reading frame of 1644 bp containing one intron. The gene encodes a protein of 521 amino acids that has sequence similarity to that of an Aspergillus oryzae tannase. However, the encoded enzyme, feruloyl esterase B (FAEB), does not have tannase activity. Comparison of the physical characteristics and substrate specificity of FAEB with those of a cinnamoyl esterase from A. niger [Kroon, Faulds and Williamson (1996) Biotechnol. Appl. Biochem. 23, 255-262] suggests that they are in fact the same enzyme. The expression of faeB is specifically induced in the presence of certain aromatic compounds, but not in the presence of other constituents present in plant-cell-wall polysaccharides such as arabinoxylan or pectin. The expression profile of faeB in the presence of aromatic compounds was compared with the expression of A. niger faeA, encoding feruloyl esterase A (FAEA), and A. niger bphA, the gene encoding a benzoate-p-hydroxylase. All three genes have different subsets of aromatic compounds that induce their expression, indicating the presence of different transcription activating systems in A. niger that respond to aromatic compounds. Comparison of the activity of FAEA and FAEB on sugar-beet pectin and wheat arabinoxylan demonstrated that they are both involved in the degradation of both polysaccharides, but have opposite preferences for these substrates. FAEA is more active than FAEB towards wheat arabinoxylan, whereas FAEB is more active than FAEA towards sugar-beet pectin.