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PubMed1999

Cloning and molecular characterization of a soluble epoxide hydrolase from Aspergillus niger that is related to mammalian microsomal epoxide hydrolase.

Aspergillus niger strain LCP521 harbours a highly processive epoxide hydrolase (EH) that is of particular interest for the enantioselective bio-organic synthesis of fine chemicals. In the present work, we report the isolation of the gene and cDNA for this EH by use of inverse PCR. The gene is composed of nine exons, the first of which is apparently non-coding. The deduced protein of the A. niger EH shares significant sequence similarity with the mammalian microsomal EHs (mEH). In contrast to these, however, the protein from A. niger lacks the common N-terminal membrane anchor, in line with the fact that this enzyme is, indeed, soluble in its native environment. Recombinant expression of the isolated cDNA in Escherichia coli yielded a fully active EH with similar characteristics to the fungal enzyme. Sequence comparison with mammalian EHs suggested that Asp(192), Asp(348) and His(374) constituted the catalytic triad of the fungal EH. This was subsequently substantiated by the analysis of respective mutants constructed by site-directed mutagenesis. The presence of an aspartic acid residue in the charge-relay system of the A. niger enzyme, in contrast to a glutamic acid residue in the respective position of all mEHs analysed to date, may be one important contributor to the exceptionally high turnover number of the fungal enzyme when compared with its mammalian relatives. Recombinant expression of the enzyme in E. coli offers a versatile tool for the bio-organic chemist for the chiral synthesis of a variety of fine chemicals.

Amino Acid Sequence↗

PubMed2002

Expression of the Pycnoporus cinnabarinus laccase gene in Aspergillus niger and characterization of the recombinant enzyme.

Pycnoporus cinnabarinus laccase lac1 gene was overexpressed in Aspergillus niger, a well-known fungal host producing a large amount of homologous or heterologous enzymes for industrial applications. The corresponding cDNA was placed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter as a strong and constitutive promoter. The laccase signal peptide or the glucoamylase preprosequence of A. niger was used to target the secretion. Both signal peptides directed the secretion of laccase into the culture medium as an active protein, but the A. niger preprosequence allowed an 80-fold increase in laccase production. The identity of the recombinant protein was further confirmed by immunodetection using Western blot analysis and N-terminal sequencing. The molecular mass of the mature laccase was 70 kDa as expected, similar to that of the native form, suggesting no hyperglycosylation. The recombinant laccase was purified in a three-step procedure including a fractionated precipitation using ammonium sulfate, and a concentration by ultrafiltration followed by a Mono Q column. All the characteristics of the recombinant laccase are in agreement with those of the native laccase. This is the first report of the production of a white-rot laccase in A. niger.

Amino Acid Sequence↗

PubMed2003

Selection of biochemical mutants of Aspergillus niger resistant to some abiotic stresses with increased inulinase production.

AIMS: The present work aimed at evaluating the usefulness of selecting different kinds of biochemical mutants of Aspergillus niger to increase inulinase production in submerged culture. METHODS AND RESULTS: Conidia of A. niger 13/36, an active producer of inulinase, were subjected to mutagenesis with both u.v. and N-methyl-N'-nitro-N-nitrosoguanidine (NTG), and the products were analysed for inulinase activity with our own diffusion plate method. As a result of mutagenization and selection for obtaining biochemical mutants (e.g. surviving conditions of certain abiotic stresses, good growing on basal medium at 15 and 40 degrees C), A. niger strains resistant to these agents were obtained. Studies of the relationship between a criterion of selection and the frequency of mutation showed that the highest frequency of positive mutations in the second selection (86%) was obtained in mutants characteristic of the best growth at the low temperature (15 degrees C), when compared with the parent culture (28%). The most active mutants grown under stress conditions showed significantly higher inulinase activity (about 1.2-4.5-fold), when compared with the parent strain. CONCLUSIONS: The studies presented seem to confirm a high effectiveness of selection in some kinds of biochemical mutants of A. niger with regard to increased inulinase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This screening strategy of mutants can be a contribution to modern commercial enzyme production.

Aspergillus niger↗

PubMed2000

The Aspergillus niger transcriptional activator XlnR, which is involved in the degradation of the polysaccharides xylan and cellulose, also regulates D-xylose reductase gene expression.

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.

Aldehyde Reductase↗

PubMed1998

pgaE encodes a fourth member of the endopolygalacturonase gene family from Aspergillus niger.

In the present study, the molecular and basic biochemical characterization of endopolygalacturonase E, the fourth Aspergillus niger N400 endopolygalacturonase, is reported. The entire endopolygalacturonase E gene consists of 1293 bp interrupted by three short introns (50, 50, and 59 bp, respectively) as concluded from the cDNA sequence. The deduced amino acid sequence comprises 378 residues that include 39 N-terminal amino acids of the prepropeptide. The calculated Mr and pI of the mature protein are 35,584 and 3.6, respectively. Compared with other endopolygalacturonases from A. niger N400, the mature protein endopolygalacturonase E has the highest sequence identity with endopolygalacturonase C (77.6%) followed by endopolygalacturonase I (57.6%) and endopolygalacturonase II (54.3%). For overproduction of endopolygalacturonase E, an A. niger multicopy strain was used that was transformed with a promoter gene fusion construct that directs expression from the glycolytic A. niger pyruvate kinase promoter. The enzyme was purified and characterized as an endopolygalacturonase based on product analysis after polygalacturonate hydrolysis and on bond cleavage frequencies of oligogalacturonates of different degree of polymerisation (n = 2-7). The pH optimum was 3.8. The Km and Vmax for polygalacturonate hydrolysis were 2.5 +/- 0.4 mg x ml(-1) and 1.3 +/- 0.2 microkat x mg(-1), respectively. A subsite map was calculated by the combination of the methods of Suganuma et al. [Suganuma, T., Matsuno, R., Ohnishi, M. & Hiromi, K. (1978) J. Biochem. (Tokyo) 84, 293-316] and Nitta et al. [Nitta, Y., Mizushima, M., Hiromi, K. & Ono, S. (1971) J. Biochem. (Tokyo) 69, 567-576]. This indicated that the enzyme was composed of at least five subsites.

Amino Acid Sequence↗

PubMed2001

Cloning and characterization of Aspergillus niger genes encoding an alpha-galactosidase and a beta-mannosidase involved in galactomannan degradation.

Alpha-galactosidase (EC 3.2.1.22) and beta-mannosidase (EC 3.2.1.25) participate in the hydrolysis of complex plant saccharides such as galacto(gluco)mannans. Here we report on the cloning and characterization of genes encoding an alpha-galactosidase (AglC) and a beta-mannosidase (MndA) from Aspergillus niger. The aglC and mndA genes code for 747 and 931 amino acids, respectively, including the eukaryotic signal sequences. The predicted isoelectric points of AglC and MndA are 4.56 and 5.17, and the calculated molecular masses are 79.674 and 102.335 kDa, respectively. Both AglC and MndA contain several putative N-glycosylation sites. AglC was assigned to family 36 of the glycosyl hydrolases and MndA was assigned to family 2. The expression patterns of aglC and mndA and two other genes encoding A. niger alpha-galactosidases (aglA and aglB) during cultivation on galactomannan were studied by Northern analysis. A comparison of gene expression on monosaccharides in the A. niger wild-type and a CreA mutant strain showed that the carbon catabolite repressor protein CreA has a strong influence on aglA, but not on aglB, aglC or mndA. AglC and MndA were purified from constructed overexpression strains of A. niger, and the combined action of these enzymes degraded a galactomanno-oligosaccharide into galactose and mannose. The possible roles of AglC and MndA in galactomannan hydrolysis is discussed.

Amino Acid Sequence↗

PubMed1996

Dietary Aspergillus niger phytase increases iron absorption in humans.

Phytate is an inhibitor of iron absorption that can be removed before the intestinal site of absorption by microbial phytase, thereby increasing iron absorption from a meal. The effects of two kinds of dietary phytase, cereal phytase and microbial phytase from Aspergillus niger, on iron absorption were investigated. Iron absorption was measured from single meals containing white wheat rolls supplemented with wheat bran with or without phytase activity (expt. 1) and phytase-deactivated wheat bran with or without addition of microbial phytase from A. niger (expt. 2). Each experiment had 10 subjects and two different radio iron tracers: 55Fe and 59Fe were used for comparison of the absorption from the test meals in each experiment. No differences in iron absorption were found between meals containing wheat brain with or without phytase activity. Addition of microbial phytase to the meal containing phytase-deactivated wheat bran increased iron absorption from 14.3 +/- 2.6% to 26.1 +/- 3.8% (P < 0.0001). Two pH optima, one at pH 2.0 and one at pH 6.0, were found for A. niger phytase at 37 degrees C, but activity occurred at all pH values between 1.0 and 7.5. The results suggest that effective and complete degradation of phytate occurred in the stomach when A. niger phytase was given with the meal. This may be explained by high activity of microbial phytase at physiological pH conditions of the stomach, whereas wheat phytase has a different pH optimum.

6-Phytase↗

PubMed1997

Isolation of Aspergillus niger creA mutants and effects of the mutations on expression of arabinases and L-arabinose catabolic enzymes.

Aspergillus niger mutants relieved of carbon repression were isolated from an areA parental strain by selection of colonies that exhibited improved growth on a combination of 4-aminobutanoic acid (GABA) and D-glucose. In addition to derepression of the utilization of GABA as a nitrogen source in the presence of D-glucose, three of the four mutants also showed derepression of L-alanine and L-proline utilization. Transformation of the mutants with the A. niger creA gene, encoding the repressor protein CREA, re-established the areA phenotype on GABA/D-glucose, identifying the mutations as creAd. The creA gene mapped on chromosome IV by linkage analysis and contour-clamped homogeneous electric field hybridization. The creA mutants obtained were used to study the involvement of CREA in repression by D-glucose of arabinases and L-arabinose catabolism in A. niger. In wild-type A. niger, alpha-L-arabinofuranosidase A, alpha-L-arabinofuranosidase B, endo-arabinase, L-arabinose reductase and L-arabitol dehydrogenase were induced on L-arabinose, but addition of D-glucose prevented this induction. Repression was relieved to varying degrees in the creA mutants, showing that biosynthesis of arabinases and L-arabinose catabolic enzymes is under control of CREA.

Arabinose↗

PubMed1993

The catR gene encoding a catalase from Aspergillus niger: primary structure and elevated expression through increased gene copy number and use of a strong promoter.

Synthetic oligonucleotide probes based on amino acid sequence data were used to identify and clone cDNA sequences encoding a catalase (catalase-R) of Aspergillus niger. One cDNA clone was subsequently used to isolate the corresponding genomic DNA sequences (designated catR). Nucleotide sequence analysis of both genomic and cDNA clones suggested that the catR coding region consists of five exons interrupted by four small introns. The deduced amino acid sequence of catalase-R spans 730 residues which show significant homology to both prokaryotic and eukaryotic catalases, particularly in regions involved in catalytic activity and binding of the haem prosthetic group. Increased expression of the catR gene was obtained by transformation of an A. niger host strain with an integrative vector carrying the cloned genomic DNA segment. Several of these transformants produced three- to fivefold higher levels of catalase than the untransformed parent strain. Hybridization analyses indicated that these strains contained multiple copies of catR integrated into the genome. A second expression vector was constructed in which the catR coding region was functionally joined to the promoter and terminator elements of the A. niger glucoamylase (glaA) gene. A. niger transformants containing this vector produced from three- to 10-fold higher levels of catalase-R than the untransformed parent strain.

Amino Acid Sequence↗

PubMed2005

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.

Amino Acid Sequence↗

PubMed2004

Aspergillus niger infection in patients with haematological diseases: a report of eight cases.

In this paper we analysed clinical, laboratory characteristics and outcome of patients with haematological diseases who developed an Aspergillus niger infection, in a multicentre study involving 14 Italian Haematological Divisions during a 10-year period. The study recorded 194 consecutive microbiologically documented aspergilloses, eight of which (4%) were due to A. niger, and were observed only in five of the participating centres. The primary localization of infection was lung in seven cases and paranasal sinus in one case. Seven patients died at the end of follow-up. The death was mainly attributable to A. niger progression in six of them. Our study that collected the largest number of cases of A. niger infection in haematological malignancies confirms that this infrequent complication is characterized by a high mortality rate.

Adult↗

PubMed2006

In vitro efficacy of 75 essential oils against Aspergillus niger.

Aspergillus niger is an opportunistic human pathogen and a strong air pollutant. A study was conducted with 75 different essential oils for the inhibition of hyphal growth and spore formation in Aspergillus niger. Cinnamomum zeylanicum (bark), Cinnamomum zeylanicum (leaf), Cinnamomum cassia, Syzygium aromaticum and Cymbopogon citratus were the top five essential oils which demonstrated marked inhibitory effect against hyphal growth and spore formation of A. niger. The chemical composition of these five most active essential oils was investigated by gas chromatography-mass spectra (GC-MS). Most of the other essential oils were found challenging to combat A. niger, suggesting their use as strong aroma therapeutic agents.

Aspergillus niger↗

PubMed1996

Trehalose-6-phosphate synthase A affects citrate accumulation by Aspergillus niger under conditions of high glycolytic flux.

Accumulation of citric acid by Aspergillus niger depends on a high flux through glycolysis. We have investigated the possibility of control of this flux by trehalose 6-phosphate, an inhibitor of hexokinase of Saccharomyces cerevisiae and other eukaryotes (Blasquez et al., FEBS Lett. (1993) 329, 51-54). Hexokinase of A. niger was shown in vitro to be only weakly inhibited by trehalose 6-phosphate (KI 1.5-2 mM). To investigate the in vivo relevance of this inhibition, we used isogenic strains of A. niger, carrying either a disruption or an amplification of the trehalose-6-phosphate synthase A (T6PSA)-encoding gene (ggsA) and exhibiting corresponding differences in T6PSA activity. These strains produced citric acid at comparable rates and with similar yields on 1 or 2.5% (w/v) sucrose. At 5-14% (w/v) sucrose, the ggsA disrupted strain initiated citric acid accumulation earlier, whereas the multicopy strain showed the reverse effect. When sucrose was replaced by lactose, which enabled only low rates of catabolism irrespective of its concentration (1-8%), no differences in the initiation or rate of citric acid accumulation were observed between the three strains. The possible mechanisms by which ggsA controls glycolytic flux in A. niger in the presence of high sugar concentrations are discussed.

Aspergillus niger↗

PubMed2001

Cloning of the Aspergillus niger pmrA gene, a homologue of yeast PMR1, and characterization of a pmrA null mutant.

The pmrA gene, a yeast PMR1 homologue, was isolated from Aspergillus niger. Sequence analysis of the pmrA cDNA and the genomic DNA revealed that two introns exist in the coding region, and that an open reading frame in the cDNA encodes a polypeptide of 1056 amino acids containing all the conserved regions present in P-type Ca2+-ATPases. The predicted pmrA protein exhibited a high degree of sequence similarity to the Pmr1 proteins from yeasts and mammalians (50-59% identity). The expression of the pmrA cDNA partially restored the growth defect of Yarrowia lipolytica pmr1 null mutant on EGTA-containing medium. This indicates that the A. niger pmrA gene encodes a functional homologue of the yeast P-type Ca2+-ATPase involved in the secretory pathway. An A. niger pmrA null mutant exhibited growth retardation on EGTA-containing medium and the growth defect was overcome by adding Ca2+ or Mn2+ into the medium. This suggests an involvement of the pmrA protein in Ca2+ and Mn2+ homeostasis in A. niger.

ATP-Binding Cassette Transporters↗

PubMed1998

[The correlation between tissue injury and calcium oxalate crystal production in rat's lung with experimental Aspergillus niger infection].

It is well known that calcium oxalate crystals (crystal) are commonly found in Aspergillus niger (A. niger) infection site. However, there have been no pathological reports of crystal production and tissue injury using an animal model. I experienced a case with pulmonary aspergilloma and infiltration shadow of the lung that I guessed resulted in crystal-induced inflammation. I performed animal experiments to investigate the possibility of crystals to cause tissue injury. Two different types of immunosuppressed rat models; i.e. cortisone acetate (CA) and CA + cyclophosphamide (CPM) induced-immunodeficiency models (CA model, CA + CPM model), were used for the experiments. In both models, rats were infected with A. niger. Then, the lungs were removed and examined histopathologically. The crystals were rarely found in both models on the third day after inoculation and it increased as time passed. However, more growth of hyphae and more production of crystals were observed in the CA + CPM model. In the CA model, crystal depositions in the bronchial epithelium and erosion of the bronchial epithelium adjacent to the crystals were observed. Crystal depositions in the bronchial epithelium were also seen in the CA + CPM model. In this lesion, severe erosions of the bronchial epithelium and invasion of A. niger mycelium into the tissue were observed. These findings suggest, the crystals in the bronchial epithelium may expand and destroy the cells around them. Therefore, I think that the crystals can induce tissue injury. However, in alveolar portions, depositions of crystals were seen mainly in the margin rather than in the central part of mycelial growth, but no findings indicating injury of the alveolar epithelial cells by crystals were seen. Therefore, possibility of crystals to cause tissue injury in the alveolar portion is not yet clear.

Animals↗

PubMed1999

Differential expression of three alpha-galactosidase genes and a single beta-galactosidase gene from Aspergillus niger.

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.

Amino Acid Sequence↗

PubMed2000

Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger.

Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes. A gene, pdiA, encoding PDIA was previously isolated from Aspergillus niger, and we report its functional characterization here. Functional analysis of PDIA showed that it catalyzes the refolding of denatured and reduced RNase A. pdiA also complemented PDI function in a Saccharomyces cerevisiae Deltapdi1 mutant in a yeast-based killer toxin assay. Levels of pdiA mRNA and PDIA protein were raised by the accumulation of unfolded proteins in the endoplasmic reticulum. This response of pdiA mRNA levels was slower and lower in magnitude than that of A. niger bipA, suggesting that the induction of pdiA is not part of the primary stress response. An increased level of pdiA transcripts was also observed in two A. niger strains overproducing a heterologous protein, hen egg white lysozyme (HEWL). Although overexpression of PDI has been successful in increasing yields of some heterologous proteins in S. cerevisiae, overexpression of PDIA did not increase secreted yields of HEWL in A. niger, suggesting that PDIA itself is not limiting for secretion of this protein. Downregulation of pdiA by antisense mRNA reduced the levels of microsomal PDIA activity by up to 50%, lowered the level of PDIA as judged by Western blots, and lowered the secreted levels of glucoamylase by 60 to 70%.

Actins↗

PubMed2004

The weak acid preservative sorbic acid inhibits conidial germination and mycelial growth of Aspergillus niger through intracellular acidification.

The growth of the filamentous fungus Aspergillus niger, a common food spoilage organism, is inhibited by the weak acid preservative sorbic acid (trans-trans-2,4-hexadienoic acid). Conidia inoculated at 10(5)/ml of medium showed a sorbic acid MIC of 4.5 mM at pH 4.0, whereas the MIC for the amount of mycelia at 24 h developed from the same spore inoculum was threefold lower. The MIC for conidia and, to a lesser extent, mycelia was shown to be dependent on the inoculum size. A. niger is capable of degrading sorbic acid, and this ability has consequences for food preservation strategies. The mechanism of action of sorbic acid was investigated using (31)P nuclear magnetic resonance (NMR) spectroscopy. We show that a rapid decline in cytosolic pH (pH(cyt)) by more than 1 pH unit and a depression of vacuolar pH (pH(vac)) in A. niger occurs in the presence of sorbic acid. The pH gradient over the vacuole completely collapsed as a result of the decline in pH(cyt). NMR spectra also revealed that sorbic acid (3.0 mM at pH 4.0) caused intracellular ATP pools and levels of sugar-phosphomonoesters and -phosphodiesters of A. niger mycelia to decrease dramatically, and they did not recover. The disruption of pH homeostasis by sorbic acid at concentrations below the MIC could account for the delay in spore germination and retardation of the onset of subsequent mycelial growth.

Aspergillus niger↗