Abstract: Interactions between bacteria and fungi cover a wide range of incentives, mechanisms and outcomes. The genus Collimonas consists of soil bacteria that are known for their antifungal activity and ability to grow at the expense of living fungi. In non-contact confrontation assays with the fungus Aspergillus niger, Collimonas fungivorans showed accumulation of biomass concomitant with inhibition of hyphal spread. Through microarray analysis of bacterial and fungal mRNA from the confrontation arena, we gained new insights into the mechanisms underlying the fungistatic effect and mycophagous phenotype of collimonads. Collimonas responded to the fungus by activating genes for the utilization of fungal-derived compounds and for production of a putative antifungal compound. In A. niger, differentially expressed genes included those involved in lipid and cell wall metabolism and cell defense, which correlated well with the hyphal deformations that were observed microscopically. Transcriptional profiles revealed distress in both partners: downregulation of ribosomal proteins and upregulation of mobile genetic elements in the bacteria and expression of endoplasmic reticulum stress and conidia-related genes in the fungus. Both partners experienced nitrogen shortage in each other's presence. Overall, our results indicate that the Collimonas/Aspergillus interaction is a complex interplay between trophism, antibiosis and competition for nutrients.
Abstract: Filamentous fungi are widely known for their industrial applications, namely, the production of food-processing enzymes and metabolites such as antibiotics and organic acids. In the past decade, the full genome sequencing of filamentous fungi increased the potential to predict encoded proteins enormously, namely, hydrolytic enzymes or proteins involved in the biosynthesis of metabolites of interest. The integration of genome sequence information with possible phenotypes requires, however, the knowledge of all the proteins in the cell in a system-wise manner, given by proteomics. This review summarises the progress of proteomics and its importance for the study of biotechnological processes in filamentous fungi. A major step forward in proteomics was to couple protein separation with high-resolution mass spectrometry, allowing accurate protein quantification. Despite the fact that most fungal proteomic studies have been focused on proteins from mycelial extracts, many proteins are related to processes which are compartmentalised in the fungal cell, e.g. β-lactam antibiotic production in the microbody. For the study of such processes, a targeted approach is required, e.g. by organelle proteomics. Typical workflows for sample preparation in fungal organelle proteomics are discussed, including homogenisation and sub-cellular fractionation. Finally, examples are presented of fungal organelle proteomic studies, which have enlarged the knowledge on areas of interest to biotechnology, such as protein secretion, energy production or antibiotic biosynthesis.
Abstract: The fungus Aspergillus niger has been studied in considerable detail with respect to various industrial applications. Although its central metabolic pathways are established relatively well, the mechanisms that control the adaptation of its metabolism are understood rather poorly. In this study, clustering of co-expressed genes has been performed on the basis of DNA microarray data sets from two experimental approaches. In one approach, low amounts of inducer caused a relatively mild perturbation, while in the other approach the imposed environmental conditions including carbon source starvation caused severe perturbed stress. A set of conserved genes was used to construct gene co-expression networks for both the individual and combined data sets. Comparative analysis revealed the existence of modules, some of which are present in all three networks. In addition, experimental condition-specific modules were identified. Module-derived consensus expression profiles enabled the integration of all protein-coding A. niger genes to the co-expression analysis, including hypothetical and poorly conserved genes. Conserved sequence motifs were detected in the upstream region of genes that cluster in some modules, e.g., the binding site for the amino acid metabolism-related transcription factor CpcA as well as for the fatty acid metabolism-related transcription factors, FarA and FarB. Moreover, not previously described putative transcription factor binding sites were discovered for two modules: the motif 5'-CGACAA is overrepresented in the module containing genes encoding cytosolic ribosomal proteins, while the motif 5'-GGCCGCG is overrepresented in genes related to 'gene expression', such as RNA helicases and translation initiation factors.
Abstract: Protein secretion plays an eminent role in cell maintenance and adaptation to the extracellular environment of microorganisms. Although protein secretion is an extremely efficient process in filamentous fungi, the mechanisms underlying protein secretion have remained largely uncharacterized in these organisms. In this study, we analyzed the effects of the d-xylose induction of cellulase and hemicellulase enzyme secretion on the protein composition of secretory organelles in Aspergillus niger. We aimed to systematically identify the components involved in the secretion of these enzymes via mass spectrometry of enriched subcellular microsomal fractions. Under each condition, fractions enriched for secretory organelles were processed for tandem mass spectrometry, resulting in the identification of peptides that originate from 1,081 proteins, 254 of which-many of them hypothetical proteins-were predicted to play direct roles in the secretory pathway. d-Xylose induction led to an increase in specific small GTPases known to be associated with polarized growth, exocytosis, and endocytosis. Moreover, the endoplasmic-reticulum-associated degradation (ERAD) components Cdc48 and all 14 of the 20S proteasomal subunits were recruited to the secretory organelles. In conclusion, induction of extracellular enzymes results in specific changes in the secretory subproteome of A. niger, and the most prominent change found in this study was the recruitment of the 20S proteasomal subunits to the secretory organelles.
Abstract: The proper design of DNA microarray experiments requires knowledge of biological and technical variation of the studied biological model. For the filamentous fungus Aspergillus niger, a fast, quantitative real-time PCR (qPCR)-based hierarchical experimental design was used to determine this variation. Analysis of variance components determined the contribution of each processing step to total variation: 68% is due to differences in day-to-day handling and processing, while the fermentor vessel, cDNA synthesis, and qPCR measurement each contributed equally to the remainder of variation. The global transcriptional response to d-xylose was analyzed using Affymetrix microarrays. Twenty-four statistically differentially expressed genes were identified. These encode enzymes required to degrade and metabolize D-xylose-containing polysaccharides, as well as complementary enzymes required to metabolize complex polymers likely present in the vicinity of D-xylose-containing substrates. These results confirm previous findings that the d-xylose signal is interpreted by the fungus as the availability of a multitude of complex polysaccharides. Measurement of a limited number of transcripts in a defined experimental setup followed by analysis of variance components is a fast and reliable method to determine biological and technical variation present in qPCR and microarray studies. This approach provides important parameters for the experimental design of batch-grown filamentous cultures and facilitates the evaluation and interpretation of microarray data.
Abstract: The filamentous fungi Aspergillus niger and Hypocrea jecorina (Trichoderma reesei) have been the subject of many studies investigating the mechanism of transcriptional regulation of hemicellulase- and cellulase-encoding genes. The transcriptional regulator XlnR that was initially identified in A. niger as the transcriptional regulator of xylanase-encoding genes controls the transcription of about 20-30 genes encoding hemicellulases and cellulases. The orthologous xyr1 (xylanase regulator 1-encoding) gene product of H. jecorina has a similar function as XlnR, although at points, the mechanisms seems to be different. Specifically in H. jecorina, the interaction of Xyr1 and the co-regulators Ace1 and Ace2 in the regulation of transcription of xylanases and cellulases has been studied. This paper describes the similarities and differences in the transcriptional regulation of expression of hemicellulases and cellulases in A. niger and H. jecorina.
Abstract: The Aspergillus nidulans xlnR gene encodes a Zn(2)Cys(6) transcription activator necessary for the synthesis of the main xylanolytic enzymes, i.e. endo-xylanases X(22), X(24) and X(34), and beta-xilosidase XlnD. Expression of xlnR is not sufficient for induction of genes encoding the xylanolytic complex, the presence of xylose is absolutely required. It has been established previously that the wide-domain carbon catabolite repressor CreA indirectly represses xlnA (encodes X(22)) and xlnB (encodes X(24)) genes as well as exerting direct repression on xlnA. This work provides evidence that CreA-mediated indirect repression occurs through repression of xlnR: (i) the xlnR gene promoter is repressed by glucose and this repression is abolished in creA(d)30 mutant strains and (ii) deregulated expression of xlnR completely relieves glucose repression of xlnA and xlnB. Thus, CreA and XlnR form a transcriptional cascade regulating A. nidulans xylanolytic genes.
Abstract: An approach based on Gateway recombination technology to efficiently construct silencing vectors was developed for use in the biotechnologically important fungus Aspergillus niger. The transcription activator of xylanolytic and cellulolytic genes XlnR of A. niger was chosen as target for gene silencing. Silencing was based on the expression vector pXLNRir that was constructed and used in co-transformation. From all the strains isolated (N = 77), nine showed poor xylan-degrading activities in two semi-quantitative plate assays testing different activities for xylan degradation. Upon induction on D-xylose, transcript levels of xlnR were decreased in the xlnR-silenced strains, compared to a wild-type background. Under these conditions, the transcript levels of xyrA and xynB (two genes regulated by XlnR) were also decreased for these xlnR-silenced strains. These results indicate that the newly developed system for rapid generation of silencing vectors is an effective tool for A. niger, and this can be used to generate strains with a tailored spectrum of enzyme activities or product formation by silencing specific genes encoding, e.g., regulators such as XlnR.
Abstract: Systematic analyses of sequence features have resulted in a better characterisation of the organisation of the genome. A previous study in prokaryotes on the distribution of sequence repeats, which are notoriously variable and can disrupt the reading frame in genes, showed that these motifs are skewed towards gene termini, specifically the 5' end of genes. For eukaryotes no such intragenic analysis has been performed, though this could indicate the pervasiveness of this distribution bias, thereby helping to expose the selective pressures causing it.
Abstract: Protein EstA from Aspergillus niger was characterized through a multifaced approach involving molecular biology, bioinformatics, biophysical, biochemical and enzymatical analyses. EstA was identified as the lead member, within the superfamily of proteins with an alpha/beta-hydrolase fold, of a new class of fungal esterases that also contains predicted homologs from other fungus species of known broad host-range pathogenicity.
Abstract: Flavonoid glucosides have been reported to be more bioavailable than their rutinoside counterparts. The aim of this study is to describe a first step in the use of alpha-L-rhamnosidases (RhaA and RhaB) from Aspergillus aculeatus as a way to produce functional beverages based on their potentially increased flavonoid bioavailability. Blackcurrant juice (BCJ), orange juice (OJ), and green tea infusion (GT) were incubated with either RhaA or RhaB at 30 degrees C for 10 h. Aliquots of controls and enzyme-treated samples were taken at different time points and analyzed by high-performance liquid chromatography-photodiode-array detector-mass spectrometry of daughter fragments (HPLC-DAD-MS-MS). Both RhaA and RhaB selectively catalyze in situ the removal of terminal rhamnosyl groups in the three beverages despite the heterogeneity of assay conditions such as different rutinosides and pH. Incubation of the three beverages with the two rhamnosidases resulted in a hyperbolic decrease in the flavonoid rutinosides (anthocyanins in BCJ, flavanones in OJ, and flavonols in GT) and a concomitant increase in their flavonoid glucoside counterparts. The time required for conversion of 50% of the rutinoside into the corresponding flavonoid glucoside ranged from 30 min (RhaB-rutin in GT) to 6 h (RhaB-delphinidin 3-rutinoside in BCJ). The results presented in this paper are a step forward in the use of enzyme-treated beverages as a source of bioavailable flavonoid glucosides.
Abstract: From the fungus Aspergillus niger, we identified a new gene encoding protein EstA, a member of the alpha/beta-hydrolase fold superfamily but of unknown substrate specificity. EstA was overexpressed and its crystal structure was solved by molecular replacement using a lipase-acetylcholinesterase chimera template. The 2.1 A resolution structure of EstA reveals a canonical Ser/Glu/His catalytic triad located in a small pocket at the bottom of a large solvent-accessible, bowl-shaped cavity. Potential substrates selected by manual docking procedures were assayed for EstA activity. Consistent with the pocket geometry, preference for hydrolysis of short acyl/propyl chain substrates was found. Identification of close homologs from the genome of other fungi, of which some are broad host-range pathogens, defines EstA as the first member of a novel class of fungal esterases within the superfamily. Hence the structure of EstA constitutes a lead template in the design of new antifungal agents directed toward its pathogenic homologs.
Abstract: The transcriptional activator XlnR from Aspergillus niger is a zinc binuclear cluster transcription factor that belongs to the GAL4 superfamily. Several putative structural domains in XlnR were predicted using database and protein sequence analysis. Thus far, only the functionality of the N-terminal DNA-binding domain has been determined experimentally. Deletion mutants of the xlnR gene were constructed to localize the functional regions of the protein. The results showed that a putative C-terminal coiled-coil region is involved in nuclear import of XlnR. After deletion of the C-terminus, including the coiled-coil region, XlnR was found in the cytoplasm, while deletion of the C-terminus downstream of the coiled-coil region resulted in nuclear import of XlnR. The latter mutant also showed increased xylanase activity, indicating the presence of a region with an inhibitory function in XlnR-controlled transcription. Previous findings had already shown that a mutation in the XlnR C-terminal region resulted in transcription of the structural genes under non-inducing conditions. A regulatory model of XlnR is presented in which the C-terminus responds to repressing signals, resulting in an inactive state of the protein.
Abstract: The Aspergillus aculeatus rhaA gene encoding an alpha-L-rhamnosidase has been expressed in both laboratory and industrial wine yeast strains. Wines produced in microvinifications, conducted using a combination of the genetically modified industrial strain expressing rhaA and another strain expressing a beta-glucosidase, show increased content mainly of the aromatic compound linalool.
Abstract: By deletion across the promoter region of the xynF1 gene encoding the major Aspergillus oryzae xylanase, a 53-bp DNA fragment containing the XlnR binding sequence GGCTAAA as well as two similar sequences was shown to confer xylan inducibility on the gene. Complementary and genomic DNAs encoding the Aspergillus niger xlnR homologous gene, abbreviated AoxlnR, were cloned from A. oryzae and sequenced. AoXlnR comprised 971 amino acids with a zinc binuclear cluster domain at the N-terminal region and revealed 77.5% identity to the A. niger XlnR. Recombinant AoXlnR protein encompassing the zinc cluster region of the N-terminal part bound to both the consensus binding sequence and its cognate sequence, GGCTGA, with an approximately 10 times lower affinity. GGCTA/GA is more appropriate as the XlnR consensus binding sequence. Both sequences functioned independently in vivo in XlnR-mediating induction of the xynF1 gene. This was further confirmed by using an AoxlnR disruptant. Neither the xynF1 nor the xylA gene was expressed in the disruptant, suggesting that the xylan-inducible genes in A. oryzae may also be controlled in the same manner as described for A. niger.
Abstract: A novel gene, eglC, encoding an endoglucanase, was cloned from Aspergillus niger. Transcription of eglC is regulated by XlnR, a transcriptional activator that controls the degradation of polysaccharides in plant cell walls. EglC is an 858-amino-acid protein and contains a conserved C-terminal cellulose-binding domain. EglC can be classified in glycoside hydrolase family 74. No homology to any of the endoglucanases from Trichoderma reesei was found. In the plant cell wall xyloglucan is closely linked to cellulose fibrils. We hypothesize that the EglC cellulose-binding domain anchors the enzyme to the cellulose chains while it is cleaving the xyloglucan backbone. By this action it may contribute to the degradation of the plant cell wall structure together with other enzymes, including hemicellulases and cellulases. EglC is most active towards xyloglucan and therefore is functionally different from the other two endoglucanases from A. niger, EglA and EglB, which exhibit the greatest activity towards beta-glucan. Although the mode of action of EglC is not known, this enzyme represents a new enzyme function involved in plant cell wall polysaccharide degradation by A. niger.
Abstract: Two proteins exhibiting alpha-L-rhamnosidase activity, RhaA and RhaB, were identified upon fractionation and purification of a culture filtrate from Aspergillus aculeatus grown on hesperidin. Both proteins were shown to be N glycosylated and had molecular masses of 92 and 85 kDa, of which approximately 24 and 15%, respectively, were contributed by carbohydrate. RhaA and RhaB, optimally active at pH 4.5 to 5, showed K(m) and V(max) values of 2.8 mM and 24 U/mg (RhaA) and 0.30 mM and 14 U/mg (RhaB) when tested for p-nitrophenyl-alpha-L-rhamnopyranoside. Both enzymes were able to hydrolyze alpha-1,2 and alpha-1,6 linkages to beta-D-glucosides. Using polyclonal antibodies, the corresponding cDNA of both alpha-L-rhamnosidases, rhaA and rhaB, was cloned. On the basis of the amino acid sequences derived from the cDNA clones, both proteins are highly homologous (60% identity).
Abstract: Screening of an Aspergillus niger differential cDNA library, constructed by subtracting cDNA fragments of a xlnR loss-of-function mutant from wild-type cDNA fragments, resulted in the cloning of the gene encoding D-xylose reductase (xyrA). Northern blot analysis using an A. niger wild-type strain, a xlnR multiple-copy strain and a xlnR loss-of-function mutant confirmed that the xyrA gene is regulated by XlnR, the transcriptional activator of the xylanolytic enzyme system in A. niger. D-xylose reductase catalyses the NADPH-dependent reduction of D-xylose to xylitol, which is the first step in D-xylose catabolism in fungi. Until now, XlnR was shown to control the transcription of genes encoding extracellular hydrolytic enzymes involved in cellulose and xylan degradation. In the present study, we show that A. niger is able to harmonize its sugar metabolism and extracellular xylan degradation via XlnR by regulating the expression of XyrA.
Abstract: The expression of the feruloyl esterase gene faeA, the alpha-glucuronidase gene aguA, the endoxylanase gene xlnB, and the beta-xylosidase gene xlnD from Aspergillus niger on xylose was studied in a wild-type strain and in a CreA mutant. A decrease in expression of all four genes was observed with increasing xylose concentrations in the wild-type strain, whereas expression levels in the CreA mutant were not influenced. The results in the wild type indicated that xylose concentrations higher than 1 mM resulted in repression of the expression of the xylanolytic genes tested mediated by the carbon catabolite repressor protein CreA. On xylose, the expression levels of the xylanolytic genes were therefore not only determined by induction via XlnR, but also by repression via CreA. The genes tested were not influenced to the same extent by XlnR or CreA, resulting in specific expression levels and patterns for each individual gene.
Abstract: A gene encoding a third alpha-galactosidase (AglB) from Aspergillus niger has been cloned and sequenced. The gene consists of an open reading frame of 1,750 bp containing six introns. The gene encodes a protein of 443 amino acids which contains a eukaryotic signal sequence of 16 amino acids and seven putative N-glycosylation sites. The mature protein has a calculated molecular mass of 48,835 Da and a predicted pI of 4.6. An alignment of the AglB amino acid sequence with those of other alpha-galactosidases revealed that it belongs to a subfamily of alpha-galactosidases that also includes A. niger AglA. A. niger AglC belongs to a different subfamily that consists mainly of prokaryotic alpha-galactosidases. The expression of aglA, aglB, aglC, and lacA, the latter of which encodes an A. niger beta-galactosidase, has been studied by using a number of monomeric, oligomeric, and polymeric compounds as growth substrates. Expression of aglA is only detected on galactose and galactose-containing oligomers and polymers. The aglB gene is expressed on all of the carbon sources tested, including glucose. Elevated expression was observed on xylan, which could be assigned to regulation via XlnR, the xylanolytic transcriptional activator. Expression of aglC was only observed on glucose, fructose, and combinations of glucose with xylose and galactose. High expression of lacA was detected on arabinose, xylose, xylan, and pectin. Similar to aglB, the expression on xylose and xylan can be assigned to regulation via XlnR. All four genes have distinct expression patterns which seem to mirror the natural substrates of the encoded proteins.
Abstract: Two cellobiohydrolase-encoding genes, cbhA and cbhB, have been isolated from the filamentous fungus Aspergillus niger. The deduced amino acid sequence shows that CbhB has a modular structure consisting of a fungus-type cellulose-binding domain (CBD) and a catalytic domain separated by a Pro/Ser/Thr-rich linker peptide. CbhA consists only of a catalytic domain and lacks a CBD and linker peptide. Both proteins are homologous to fungal cellobiohydrolases in family 7 of the glycosyl hydrolases. Northern blot analysis showed that the transcription of the cbhA and cbhB genes is induced by D-xylose but not by sophorose and, in addition, requires the xylanolytic transcriptional activator XlnR.
Abstract: The xlnD gene encoding the 85-kDa beta-xylosidase was cloned from Aspergillus nidulans. The deduced primary structure of the protein exhibits considerable similarity to the primary structures of the Aspergillus niger and Trichoderma reesei beta-xylosidases and some similarity to the primary structures of the class 3 beta-glucosidases. xlnD is regulated at the transcriptional level; it is induced by xylan and D-xylose and is repressed by D-glucose. Glucose repression is mediated by the product of the creA gene. Although several binding sites for the pH regulatory protein PacC were found in the upstream regulatory region, it was not clear from a Northern analysis whether PacC is involved in transcriptional regulation of xlnD.
Abstract: Complementation by transformation of an Aspergillus niger mutant lacking xylanolytic activity led to the isolation of the xlnR gene. The xlnR gene encodes a polypeptide of 875 amino acids capable of forming a zinc binuclear cluster domain with similarity to the zinc clusters of the GAL4 superfamily of transcription factors. The XlnR-binding site 5'-GGCTAAA-3' was deduced after electrophoretic mobility shift assays, DNase I footprinting and comparison of various xylanolytic promoters. The importance of the second G within the presumed XlnR binding site 5'-GGCTAAA-3' was confirmed in vitro and in vivo. The 5'-GGCTAAA-3' consensus sequence is found within several xylanolytic promoters of various Aspergillus species and Penicillium chrysogenum. Therefore, this sequence may be an important and conserved cis-acting element in induction of xylanolytic genes in filamentous fungi. Our results indicate that XlnR is a transcriptional activator of the xylanolytic system in A. niger.
Abstract: An enzyme with beta-galactosidase activity and three proteins exhibiting alpha-galactosidase activity were purified from a culture filtrate of Aspergillus niger grown on arabinoxylan. beta-galactosidase, optimally active at pH 4 and 60-65 degrees C, was active against p-nitrophenyl-beta-D-galactopyranoside, lactose, and pectic galactan. It was not able to release galactose from sugar beet pectin or lemon pectin. Its action on pectic galactan was increased by the presence of beta-galactanase. The three forms of alpha-galactosidase activity that showed different molecular masses and pIs were found to have the same mass after deglycosylation with N-glycanase F and to be the same protein based on their N-terminal amino acid sequence data. The purified alpha-galactosidase was shown to be different from alpha-galactosidase A from A. niger. This confirmed the existence of at least two different alpha-galactosidases in A. niger. alpha-Galactosidase, optimally active at pH 4.5 and 50-55 degrees C, was active toward p-nitrophenyl-alpha-D-galactopyranoside, melibiose, raffinose, stachyose, and locust bean gum, on which substrate it exhibited synergism with beta-mannanase.
Abstract: The expression of genes encoding enzymes involved in xylan degradation and two endoglucanases involved in cellulose degradation was studied at the mRNA level in the filamentous fungus Aspergillus niger. A strain with a loss-of-function mutation in the xlnR gene encoding the transcriptional activator XlnR and a strain with multiple copies of this gene were investigated in order to define which genes are controlled by XlnR. The data presented in this paper show that the transcriptional activator XlnR regulates the transcription of the xlnB, xlnC, and xlnD genes encoding the main xylanolytic enzymes (endoxylanases B and C and beta-xylosidase, respectively). Also, the transcription of the genes encoding the accessory enzymes involved in xylan degradation, including alpha-glucuronidase A, acetylxylan esterase A, arabinoxylan arabinofuranohydrolase A, and feruloyl esterase A, was found to be controlled by XlnR. In addition, XlnR also activates transcription of two endoglucanase-encoding genes, eglA and eglB, indicating that transcriptional regulation by XlnR goes beyond the genes encoding xylanolytic enzymes and includes regulation of two endoglucanase-encoding genes.
Abstract: The genes encoding the enzyme arabinoxylan arabinofuranohydrolase, which releases L-arabinose from arabinoxylan, have been cloned from the closely related fungi Aspergillus niger and Aspergillus tubingensis and were shown to be functional in A. niger. Integration of multiple copies in the genome resulted in over-expression of the enzymes. The arabinofuranohydrolases encoded comprise 332 amino acids and have 94% amino acid identity. Their primary structure is not related to those of other alpha-L-arabinofuranosidases, except for a low similarity with XYLC, a bacterial alpha-L-arabinofuranosidase from Pseudomonas fluorescens which acts on oat spelt xylan. The axhA expression pattern in A. niger differed from that of abfB, since it was strongly induced by birchwood xylan and much less by L-arabitol or L-arabinose. Furthermore, Northern analysis revealed that axhA expression was de repressed in creAd mutants and carbon catabolite repressed by D-glucose.
Abstract: An enzyme with alpha-L-rhamnosidase activity was purified by anion exchange chromatography from an Aspergillus niger commercial preparation. The alpha-L-rhamnosidase was shown to be N-glycosylated, and had a molecular mass of 85 kD on sodium dodecylsulfate-polyacrylamide gel electrophoresis of which approximately 12% was contributed by carbohydrate. The enzyme was optimally active at pH 4.5 and 65 degrees C. When tested towards p-nitrophenyl-alpha-L-rhamnopyranoside it showed Km and Vmax values of 2.9 mM and 20.6 U mg-1, respectively whereas it was inhibited competitively by L-rhamnose (Ki 3.5 mM). Substrate specificity studies showed alpha-L-rhamnosidase to be active both on alpha-1,2 and alpha-1,6 linkages to beta-D-glucose. Moreover, the enzyme was able to release L-rhamnose from geranyl-beta-D-rutinoside and 2-phenylethyl-beta-D-rutinoside.
Abstract: Two proteins exhibiting beta-D-xylosidase activity were identified upon fractionation and purification of a culture filtrate of an arabinoxylan-grown Aspergillus niger. A single band of 110 kDa by SDS/PAGE was obtained in both cases and these were active on xylo-oligosaccharides and on xylan. Partial xlnD cDNA clones were immunochemically identified and isolated from a lambda cDNA expression library. Sequence analysis showed that all cDNA clones correspond to a single gene. A genomic clone was isolated and overexpressed in A. niger and A. nidulans. The xlnD gene has an ORF of 2412 nucleotides, encodes a protein of 804 amino acids and contains a potential signal peptide of 26 amino acids. This results in a mature protein of 778 amino acids with a predicted molecular mass of 85 kDa and an isoelectric point of 4.5. The protein is N-glycosylated and contains 15 potential N-glycosylation sites. Sequence similarity is found with beta-D-glucosidases both of bacterial and fungal origin. Both beta-xylosidase proteins purified have high activity on the artificial substrate p-nitrophenyl beta-D-xylopyranoside (XylNp) and a side activity on p-nitrophenyl alpha-L-arabinofuranoside and p-nitrophenyl beta-D-glucopyranoside. A niger strains in which the xlnD gene was disrupted accumulate mainly xylobiose and xylotriose when grown on xylan and have no significant beta-xylosidase activity in the culture medium, indicating that this gene encodes the major extracellular beta-xylosidase.
Abstract: Two Aspergillus nidulans genes, xlnA and xlnB, encoding the X22 and X24 xylanases from this fungus, respectively, have been cloned and sequenced. Their cDNAs have been expressed in a laboratory Saccharomyces cerevisiae strain under the control of a constitutive yeast promoter, resulting in the construction of recombinant xylanolytic yeast strains.
Abstract: Oligonucleotides, designed on the basis of conserved flanking amino acid sequence segments within the catalytic domain of eukaryotic protein kinase C (PKC) proteins, were used as primers for polymerase chain reactions to amplify a 427-bp chromosomal DNA fragment from the filamentous fungus Trichoderma reesei. This fragment was then used to isolate genes encoding PKC homologues of T. reesei and Aspergillus niger (pkc1 and pkcA, respectively). The genes contain six (T. reesei) and eight (A. niger) introns, which exhibit notable conservation in position with those found in the corresponding Schizosaccharomyces pombe pkc1+ and Drosophila melanogaster dPKC53Ebr genes. A single 4.2-kb transcript was detected in Northern analyses. The deduced PKC1 (T.reesei, 126 kDa) and PKCA (A. niger, 122 kDa) amino acid sequences reveal domains homologous to the C1 and C3/C4 domains of PKC-related proteins, but lack typical Ca(2+)-binding (C2) domains. Both contain a large, extended N-terminus, which shares a high degree of similarity with the corresponding regions of Saccharomyces cerevisiae PKC1 and S. pombe pkc1+ and pkc2+ proteins, but which is not present in PKCs of Dictyostelium or higher eukaryotes. This extended region can be divided into three subdomains; the N-terminal one contains a hydrophobic helix-turn-helix motif, whereas the C-terminal one contains potential targets for proteolytic processing. A polyclonal antiserum raised against the pseudosubstrate-binding domain of PKC1 recognizes in T. reesei a 115-120 kDa protein in Western blots. Expression of pkc1 cDNA in insect cells directs the synthesis of a PKC1 protein of similar size. The T. reesei PKC1 protein was partially purified and some of its properties examined: it is stimulated about twofold by phospholipids or phorbol esters but is not stimulated by Ca2+. We conclude that these PKC proteins from filamentous fungi represent the Ca(2+)-insensitive fungal homologues of the nPKC family.
Abstract: The xlnC gene encoding the 34-kDa xylanase (X34) of Aspergillus nidulans (An) has been cloned and sequenced, as has its corresponding cDNA. xlnC contains nine introns and shows considerable similarity to the xynA and xylP xylanase-encoding genes of A. kawachii (Ak) and Penicillium chrysogenum (Pc), respectively. Analysis of xylanase production in An multicopy transformants showed elevated levels of X34 and increased total xylanase activity, but no elevated production of other xylanases. Northern analysis demonstrated transcriptional induction by xylan and repression by glucose.
Abstract: A gene encoding an endo-1,4-beta-xylanase from Aspergillus tubigensis was cloned by oligonucleotide screening using oligonucleotides derived from amino acid sequence data obtained from the purified protein. The isolated gene was functional as it could be expressed in the very closely related fungus Aspergillus niger. The xylanase encoded by this gene is synthesized as a protein of 211 amino acids. After cleavage of the presumed prepropeptide this results in a mature protein of 184 amino acids with a molecular weight of 19 kDa and an isoelectric point of 3.6. The regulatory region of the xlnA gene was studied with respect to the response to xylan induction and carbon catabolite repression. By deletion analysis of the 5' upstream region of the gene a 158 bp region involved in the xylan specific induction was identified. To study this regulatory element a reporter system for transcriptional activating sequences was developed that is based on the A. niger glucose oxidase-encoding gene. From the results with this reporter system it is concluded that this 158 bp fragment not only contains the information required for induction of transcription but that it also plays a role in carbon catabolite repression of the xlnA gene. The region directly upstream of this fragment contains four potential CREA target sites; deletion of this region leads to an increase in the level of transcription. These results suggest that carbon catabolite repression of the xlnA gene is controlled at two levels, directly by repression of xlnA gene transcription and indirectly by repression of the expression of a transcriptional activator. This type of mechanism would be similar to the double lock mechanism for the regulation of gene expression of alcA in Aspergillus nidulans. The reporter system was also used to study the regulation of expression via the functions located on this fragment in A. niger and in A. nidulans. Essentially the same pattern of regulation was found in both of these hosts. Therefore, regulation of xylanase gene expression is basically conserved in all three aspergilli.
Abstract: Aspergillus niger secretes three glycosylated glycosyl hydrolases which are involved in degradation of the plant cell wall polysaccharide L-arabinan: alpha-L-arabinofuranosidases (ABF) A and B, and endo-1,5-alpha-L-arabinase (ABN) A. The nucleotide sequence of the previously cloned gene encoding ABF A (abfA) from A. niger was determined. The coding region contains seven introns. Mature ABF A comprises 603 amino acids with a molecular mass of 65.4 kDa as deduced from the nucleotide sequence. The secreted enzyme is N-glycosylated. The primary structures of the three A. niger arabinases characterized lack similarity. Regulation of arabinase expression upon induction by sugar beet pulp and by L-arabitol was studied as a function of time. This was done in wild-type A. niger as well as in transformants carrying multiple copies of either one of the ABF-encoding genes. Each arabinase gene responded differently upon a mycelial transfer to L-arabitol-containing medium. Extra copies of abfA or abfB led to a decreased expression level of ABN A, though the repression elicited by abfB is stronger and more persistent than that effected by abfA. Multiple copies of both abf genes influence expression of the other ABF similarly, but to a far less pronounced degree than they affect ABN A synthesis. Four putative promoter elements, shared by all three arabinase genes, could be involved in coordination of L-arabinan degradation by A. niger.
Abstract: Using L-arabitol as an inducer, simple induction conditions were established that resulted in high-level expression of alpha-L-arabinofuranosidase A by an Aspergillus niger D-xylulose kinase mutant strain. These conditions were adapted to construct a cDNA expression library from which an alpha-L-arabinofuranosidase A cDNA clone was isolated using specific antiserum. The corresponding gene encoding alpha-L-arabinofuranosidase A (abfA) was isolated from a genomic library and cloned into a high copy plasmid vector. By co-transformation of uridine auxotrophic mutants lacking orotidine-5-phosphate decarboxylase activity, the afbA gene was introduced both in A. niger and A. nidulans, using the A. niger pyrA gene as selection marker. The identity of the abfA gene was confirmed by overexpression of the gene product by A. niger and A. nidulans transformants, upon growth using sugar beet pulp as the carbon source.
Abstract: Secretion of endo-1,5-alpha-L-arabinase A (ABN A) by an Aspergillus niger xylulose kinase mutant upon mycelium transfer to medium containing L-arabitol was immunochemically followed with time to monitor its induction profile. A cDNA expression library was made from polyA+ RNA isolated from the induced mycelium. This library was immunochemically screened and one ABN A specific clone emerged. The corresponding abnA gene was isolated from an A. niger genomic library. Upon Southern blot analysis, a 3.1-kb HindIII fragment was identified and subcloned to result in plasmid pIM950. By means of co-transformation using the A. niger pyrA gene as selection marker, the gene was introduced in both A. niger and A. nidulans uridine auxotrophic mutants. Prototrophic A. niger and A. nidulans transformants overproduced A. niger ABN A upon growth in medium containing sugar beet pulp as the sole carbon source, thereby establishing the identity and functionality of the cloned gene. The DNA sequence of the complete HindIII fragment was determined and the structure of the abnA gene as well as of its deduced gene product were analysed. Gene abnA contains three introns within its structural region and codes for a protein of 321 amino acids. Signal peptide processing results in a mature protein of 302 amino acids with a deduced molecular mass of 32.5 kDa. A. niger abnA is the first gene encoding an ABN to be isolated and characterized.
Abstract: Based on amino-acid sequence data from Aspergillus niger alpha-L-arabinofuranosidase B (ABF B), and cyanogen bromide fragments derived thereof, deoxyoligonucleotide mixtures were designed to be employed as primers in a polymerase chain reaction (PCR) on A. niger genomic DNA. This resulted in amplification of three related PCR products. The abfB gene encoding ABF B was isolated from a genomic library using such an amplification product as a probe. A 5.1-kb BamHI fragment was subcloned to result in plasmid pIM991. Upon introduction by co-transformation into both A. niger and A. nidulans uridine auxotrophic strains, pIM991 was shown to contain the functional gene since prototrophic transformants overproduced ABF B upon growth on the inducing carbon source sugar beet pulp. A plate assay was developed enabling quick selection of ABF B-overproducing transformants. The sequence of a 4122-bp long BamHI/SstI fragment was determined. The abfB gene does not contain introns and codes for a protein of 499 amino acids. The mature ABF B, 481 amino acids in length, has a deduced molecular weight of 50.7 kDa. A. niger abfB is the first eukaryotic gene encoding an ABF to be characterized.
Abstract: The induction of glucose oxidase, catalase, and lactonase activities was studied both in wild-type and in glucose oxidase regulatory and structural mutants of Aspergillus niger. The structural gene for glucose oxidase was isolated and used for Northern analysis and in transformation experiments using various gox mutations. Wild-type phenotype could be restored in the glucose oxidase-negative mutant (goxC) by transformation with the structural gene. We conclude, therefore, that the goxC marker which is located on chromosome 2 represents the structural gene of glucose oxidase. Glucose and a high oxygen level are necessary for the induction of all three enzyme activities in the wild-type strain and it was shown that both glucose and oxygen effects reflect regulation at the transcriptional level. The goxB mutation results in constitutive expression of all three activities although modulated to some extent by the carbon source. The goxE mutation only has an effect on lactonase and glucose oxidase expression and does not relieve the necessity for a high oxygen level. Catalase and lactonase could not be induced in the glucose oxidase-negative strain (goxC). Addition of H2O2 resulted in the induction of all three enzymes in the wild-type without glucose being present. The H2O2 induction is probably mediated by the goxB product. Besides the H2O2 induction there is still an effect of the carbon source on the induction. A model for induction of glucose oxidase, catalase, and lactonase in A. niger is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Aspergillus niger produces several polygalacturonases that, with other enzymes, are involved in the degradation of pectin. One of the two previously characterized genes coding for the abundant polygalacturonases I and II (PGI and PGII) found in a commercial pectinase preparation was used as a probe to isolate five more genes by screening a genomic DNA library in phage lambda EMBL4 using conditions of moderate stringency. The products of these genes were detected in the culture medium of Aspergillus nidulans transformants on the basis of activity measurements and Western-blot analysis using a polyclonal antibody raised against PGI. These transformants were, with one exception, constructed using phage DNA. A. nidulans transformants secreted high amounts of PGI and PGII in comparison to the previously characterized A. niger transformants and a novel polygalacturonase (PGC) was produced at high levels by A. nidulans transformed with the subcloned pgaC gene. This gene was sequenced and the protein-coding region was found to be interrupted by three introns; the different intron/exon organization of the three sequenced A. niger polygalacturonase genes can be explained by the gain or loss of two single introns. The pgaC gene encodes a putative 383-amino-acid prepro-protein that is cleaved after a pair of basic amino acids and shows approximately 60% amino acid sequence similarity to the other polygalacturonases in the mature protein. The N-terminal amino acid sequences of the A. niger polygalacturonases display characteristic amino acid insertions or deletions that are also observed in polygalacturonases of phytopathogenic fungi. In the upstream regions of the A. niger polygalacturonase genes, a sequence of ten conserved nucleotides comprising a CCAAT sequence was found, which is likely to represent a binding site for a regulatory protein as it shows a high similarity to the yeast CYC1 upstream activation site recognized by the HAP2/3/4 activation complex.
Abstract: The filamentous fungus Aspergillus niger produces several endopolygalacturonases that are involved in the degradation of pectin. PGI, the enzyme representing the second most abundant activity in a commercial enzyme preparation, was further characterized and the corresponding gene was isolated. The nucleotide sequence of the pgaI gene was determined and the protein coding region was found to be interrupted by two short introns, one of which has a unusual donor splice site. The deduced 368 amino acids long protein with a putative prepropeptide of 31 amino acids shows 60% sequence identity to PGII in the mature protein. PGI overproducing A. niger strains were obtained by cotransformation with the cloned gene.
Abstract: Six uridine auxotroph mutants of Trichoderma reesei QM 9414 were isolated by resistance to 5-fluoroorotic acid and one strain was identified as OMP-decarboxylase negative (pyr-) by a radiometric enzyme assay. Transformation to uridine prototrophy was achieved with the pyr4 gene of Neurospora crassa (up to 1500 transformants/micrograms) and with pyrA of Aspergillus niger (700-800 transformants/micrograms). In many transformants the PYR+ function seems to be present as extrachromosomal DNA. There is evidence for a correlation between the stability of transformants and integration of the vector in the genome whereas unstable transformants are obtained when autonomous replication of the plasmid occurs.
Abstract: Pyruvate kinase was purified from the filamentous fungus Aspergillus nidulans with a 45-55% yield. The procedure involved dye-affinity chromatography and fast protein liquid chromatography, resulting in highly active and pure enzyme in milligram quantities within 2 days. The purified enzyme, a tetramer with a subunit molecular weight of 65,000 and an isoelectric point of 4.7, was used to determine the amino acid composition.
