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PubMed2003

Isolation and characterisation of a calnexin homologue, clxA, from Aspergillus niger.

We describe the isolation of a gene (clxA) encoding calnexin from laboratory and industrial strains of Aspergillus niger. Calnexin is a chaperone, which specifically recognises monoglucosylated glycoproteins in the endoplasmic reticulum, and is thus an essential component of the process that assesses the folded state of nascent secreted glycoproteins. Manipulation of chaperones has previously been adopted in attempts to overcome some of the problems associated with the secretion of heterologous proteins from filamentous fungi. The A. niger clxA gene encodes a 562-residue protein with strong homology to the calnexin of Schizosaccharomyces pombe. The clxAgene product complements a S. pombe cnx1 mutant. Motifs associated with genes controlled via the Unfolded Protein Response (UPR) were identified by sequence homology in the promoter of clxA. Steady-state levels of clxA mRNA were elevated in a strain expressing bovine prochymosin fused to the catalytic domain of glucoamylase. The ORF is punctuated by four introns, and contains two sets of four repeated peptide motifs that are characteristic of the calnexin family, together with a putative membrane-spanning domain. Deletion studies indicate that clxA is not an essential gene in A. niger.

Animals↗

PubMed2004

Ram horn peptone as a source of citric acid production by Aspergillus niger, with a process.

The present study deals with the production of citric acid from a ram horn peptone (RHP) by Aspergillus niger NRRL 330. A medium from RHP and a control medium (CM) were compared for citric acid production using A. niger in a batch culture. For this purpose, first, RHP was produced. Ram horns were hydrolyzed by treatment with acids (6 N H(2)SO(4), 6 N HCl) and neutralizing solutions. The amounts of protein, nitrogen, ash, some minerals, total sugars, total lipids and amino acids of the RHP were determined. RHP was compared with peptones with a bacto-tryptone from casein and other peptones. The results from RHP were similar to those of standard peptones. The optimal concentration of RHP for the production of citric acid was found to be 4% (w/w). A medium prepared from 4% RHP was termed ram horn peptone medium (RHPM). In comparison with CM, the content of citric acid in RHPM broth (84 g/l) over 6 days was 35% higher than that in CM broth (62 g/l). These results show that citric acid can be produced efficiently by A. niger from ram horn.

Animals↗

PubMed2026

Optimization of protoplast based DNA isolation and genome analysis in a gamma-irradiated Aspergillus niger mutant strain.

Aspergillus niger is an important industrial fungus widely used for citric acid production and a range of biotechnological applications. In this study, a protoplast-based DNA isolation protocol was optimized for a gamma-irradiated A. niger AN-L103_M1 mutant strain, followed by whole-genome sequencing and functional genome analysis. Protoplast yield was strongly influenced by enzyme concentration and the molarity of the osmotic stabilizer. The highest yield was achieved at an enzyme concentration of 50 mg/mL (2.487 ± 0.04 × 10⁸ cells/mL) and 0.8 M KCl (2.550 ± 0.06 × 10⁸ cells/mL), with both factors showing significant effects (p < 0.0001) in GraphPad Prism 11.0.0. Whole-genome sequencing performed using an Illumina NovaSeq 6000 platform yielded a 37.06 Mb draft genome assembled into 537 contigs, with an N50 of 363,084 bp and a GC content of 48.2%. BUSCO 14 analysis showed high completeness (97.95% complete BUSCOs). Functional annotation and KEGG pathway mapping identified genes involved in glycolysis, the tricarboxylic acid cycle, and citrate biosynthesis, while biosynthetic gene cluster analysis revealed diverse potential for secondary metabolite production. These findings provide an optimized workflow for protoplast-based DNA isolation and genome-scale functional analysis in A. niger, proposing a basis for future comparative genomics, transformation studies, and experimentally validated metabolic engineering.

Aspergillus niger↗

PubMed1988

Protein-mediated fusion of liposomes with microsomal membranes of Aspergillus niger: evidence for a complex mechanism dealing with membranous and cytosolic fusogenic proteins.

Membrane fusion is a fundamental and wide-spread phenomenon in the functioning of cells. Many studies were carried out concerning fusion of plasma membranes as for example cell-cell fusions or uptake by cells of lipid-enveloped viruses. The present study deals with the interaction of intracellular membranes of Aspergillus niger with artificial membranes (liposomes). Association is monitored by the uptake of radioactive liposomes by fungal microsomal membranes. The discrimination between aggregation and pure fusion is done by layering the liposomes-microsomes mixture on a continuous sucrose gradient. The accurate quantitation of the fusion phenomenon is monitored with a fluorescent assay based on resonance energy transfer (Struck, D.K. et al. (1981) Biochemistry 20, 4093-4099). Both methods show that, at physiological pH, there is a spontaneous fusion of microsomes with cholesterol-free liposomes. This phenomenon is protein dependent as trypsinized microsomal membranes are no longer able to fuse with liposomes. Biological significance of the fusion process has been demonstrated using microsomal intrinsic protein mannosylation assay; the enhancement of the lipid to protein ratio due to the fusion of liposomes with microsomes of A. niger results in an increase in the rate of endogenous proteins mannosylation. Moreover, cytosolic proteins of A. niger promote the fusion of any kind of liposomes with microsomes.

Aspergillus niger↗

PubMed1994

A simple and nondestructive method for the separation of polysaccharides from beta-glucosidase produced extracellularly by Aspergillus niger.

An apparatus based on electrophoresis has been devised that removes noncovalently bound polysaccharides from extracellular proteins of Aspergillus niger with concomitant partial beta-glucosidase purification and concentration. The apparatus consists of a series of three chambers separated by polyacrylamide gels. Dialyzed and concentrated crude extract of Aspergillus niger containing beta-glucosidase was poured into the middle chamber, while smaller anodic and cathodic chambers contained buffer. When electric current was applied, negatively charged protein-polysaccharide complexes moved toward the anode. Most of the negatively charged proteins, including beta-glucosidase, crossed the gel barrier into the anodic compartment, while neutral polysaccharides were either trapped in the gels or remained in the middle chamber. In this way, 125 ml of dialyzed and concentrated crude extract of Aspergillus niger was processed. Therefore, after 24 h of electrophoresis, 68% of the proteins and 90% of the beta-glucosidase activity, but only negligible amounts of polysaccharide, were transferred to the anodic chamber. The removal of high-molecular-weight polysaccharide from beta-glucosidase had a detrimental effect on the stability of the enzyme.

Aspergillus niger↗

PubMed1990

Changes in the concentration of fructose 2,6-bisphosphate in Aspergillus niger during stimulation of acidogenesis by elevated sucrose concentration.

The presence of fructose 2,6-bisphosphate (Fru-2,6-P2) and phosphofructokinase 2 (PFK 2) were established in the citric-acid-producing filamentous fungus Aspergillus niger. Fru-2,6-P2 levels were around 3.0 (+/- 0.8) nmol per g dry weight during growth on sucrose, and half of this in mycelia grown on citrate as a carbon source. PFK 2 was detected with a specific activity of 150 mU/mg protein and a Km for fructose 6-phosphate of 40 microM. Induction of citric acid accumulation (acidogenesis) in A. niger by cultivation on high concentrations of sucrose, or replacement on 14% (w/v) sucrose correlated with an increase in the intracellular concentration of Fru-2,6-P2. A similar correlation was obtained when A. niger was cultivated on different carbon sources, which induced different rates of acidogenesis. The increase in Fru-2,6-P2 during transfer to 14% (w/v) sucrose was not correlated with the behaviour of mycelial concentrations of cyclic AMP, a potential regulator of Fru-2,6-P2 formation in other organisms, nor with that of Fru-6-P and ATP, the precursors of its formation. The extracellular addition of cyclic AMP and theophylline, an inhibitor of cellular cyclic AMP breakdown, increased both Fru-2,6-P2 concentration and acidogenesis in mycelia cultivated in 1% (w/v) sucrose medium. It is concluded that Fru-2,6-P2 controls citric acid accumulation by enabling increased rates of glucolysis, a prerequisite to acidogenesis.

Adenosine Triphosphate↗

PubMed1985

Transformation of Aspergillus niger using the argB gene of Aspergillus nidulans.

A mutant of Aspergillus niger defective in ornithine transcarbamylase function was transformed with plasmids carrying a functional copy of the argB gene of Aspergillus nidulans after treatment of spheroplasts in the presence of polyethylene glycol and calcium ions. The plasmid pDG3 gave stable transformants at a frequency of 4 per microgram of input DNA. Southern blot analysis of DNA from transformants showed that pDG3 DNA had integrated into the A. niger chromosomes at a variety of locations. The transformants were phenotypically stable for many mitotic divisions. This procedure may potentially be used to insert any gene into the genome of A. niger. A cosmid shuttle vector, pDG1, for cloning in Aspergillus was also constructed.

Arginine↗

PubMed1989

Physical characterization of the aldehyde-dehydrogenase-encoding gene of Aspergillus niger.

To facilitate a better understanding of the regulation of aldA, the gene encoding aldehyde dehydrogenase (AldDH) in the ascomycete fungus, Aspergillus niger, the gene has been physically characterized. The complete nucleotide (nt) sequence of the gene and its flanking regions has been determined. Analysis of the gene has revealed the presence of three introns. The homologous gene in the related fungus, Aspergillus nidulans, contains only two introns. The coding regions of these genes, excluding the intron sequences, show 80% homology at the nt level and 82% homology at the amino acid (aa) level. The aa sequence of the A. niger enzyme is significantly homologous to mammalian sequences, particularly around one Cys residue, which is hypothesized to be adjacent to another Cys known to be at the active site of mammalian AldDHs. The 5' region of the gene shows one major and two minor transcription start points and a general structure common to highly expressed fungal genes. The 3' region shows four sites of polyadenylation. Sequence comparisons of the 5' region of the A. niger aldA gene to other Aspergillus genes has shown a common sequence in the 5' regions of several A. nidulans genes, all but one of which are subject to carbon catabolite repression. This sequence may be important for the regulatory mechanism.

Aldehyde Dehydrogenase↗

PubMed1991

Characterization and expression of a genomic pectin methyl esterase-encoding gene in Aspergillus niger.

The genomic pectin methylesterase (PME)-encoding gene (pmeA) from Aspergillus niger strain RH5344 was cloned by probing a genomic DNA library with a cDNA coding for PME. The recombinant phage clone was isolated and a 6-kb HindIII fragment was subcloned and characterized. The gene consists of seven exons and six introns. The nucleotide sequences of the coding regions were identical to those found in the pmeA cDNA. Cotransformation of A. niger was achieved with the vector, pAN7-1, and transformants were then tested for PME production. Transformants which produced more PME than the untransformed recipient strain were subjected to Southern-blot and Northern-blot analysis. The results show that there is a reasonable correlation between gene copy number, mRNA levels and PME production. PME was produced by A. niger transformants in an active 43-kDa form, which is similar to that of the mature protein isolated from the strain, RH5344. On the basis of the results of affinity labeling of PME with sugar-specific lectins and the amino acid sequence data, it has been revealed that PME is a glycoprotein and the protein-bound glycans are oligosaccharides with a high mannose content.

Affinity Labels↗

PubMed1992

The Aspergillus niger niaD gene encoding nitrate reductase: upstream nucleotide and amino acid sequence comparisons.

The Aspergillus niger niaD gene has been sequenced and the inferred nitrate reductase (NR) protein found to consist of 867 amino acid residues (97 kDa). The gene is interrupted by six small introns, as deduced by comparison with the niaD gene of Aspergillus nidulans. The positions of these putative introns are conserved between the two fungi, although the sequences are dissimilar. The niiA gene, encoding nitrite reductase, the second reductive step in the nitrate assimilation pathway, is tightly linked to niaD and divergently transcribed in A. niger, similar to the general organisation in the related fungi, Aspergillus oryzae and A. nidulans. The nucleotide (nt) sequences of the intergenic region between niiA and niaD (excluding the ATG translation start codon) of A. niger (1668 nt) and A. oryzae (1575 nt) were determined and compared with the previously determined A. nidulans (1262 nt) sequence. No striking extended nt regions of homology are observed in spite of the fact that transgenic strains with fungal niaD or the two control genes required for induction and repression show virtually normal regulation. Fungal NR shows considerable aa homology with higher plant NR, particularly within the co-factor domains for flavin adenoside dinucleotide, heme and molybdopterin cofactor.

Amino Acid Sequence↗

PubMed1993

Heterologous protein secretion directed by a repressible acid phosphatase system of Aspergillus niger.

A new expression-secretion system of Aspergillus niger which directs the secretion of heterologous proteins is described. The promoter and signal peptide-encoding region of the phosphate-repressible aphA gene of A. niger, when fused to the coding region of the human interferon alpha 2 (hIFN alpha 2)-encoding gene (hIFN alpha 2), drives the expression of this gene and the secretion of the hIFN alpha 2 protein. Synthesis of hIFN alpha 2 in either A. niger or A. nidulans transformants carrying these constructs was regulated by inorganic phosphate (Pi) present in the medium, so that derepression of heterologous protein expression can be attained by lowering Pi concentration.

Acid Phosphatase↗

PubMed1993

The Aspergillus niger carbon catabolite repressor encoding gene, creA.

In order to undertake a comparative analysis of carbon catabolite repression in two Aspergillus species, the creA gene has been isolated from A. niger by cross hybridization, using the cloned A. nidulans gene. The A. niger gene has been shown to be functional in A. nidulans by heterologous complementation of the creA204 mutation of A. nidulans. Overall, the genes show 90% sequence similarity (82% identity) at the amino acid (aa) level. There were some striking similarities between the aa sequences encoded by the two fungal creA genes and two genes involved in carbon catabolite repression in Saccharomyces cerevisiae. The zinc-finger regions showed 96% similarity (84% identity) with the zinc-finger region of the MIG1 gene of S. cerevisiae. The CREA protein contains a stretch of 42 aa that is identical in A. niger and A. nidulans, and these show 81% similarity (33% identity) with a region of the S. cerevisiae RGR1 gene.

Amino Acid Sequence↗

PubMed2005

The activity of barley alpha-amylase on starch granules is enhanced by fusion of a starch binding domain from Aspergillus niger glucoamylase.

High affinity for starch granules of certain amylolytic enzymes is mediated by a separate starch binding domain (SBD). In Aspergillus niger glucoamylase (GA-I), a 70 amino acid O-glycosylated peptide linker connects SBD with the catalytic domain. A gene was constructed to encode barley alpha-amylase 1 (AMY1) fused C-terminally to this SBD via a 37 residue GA-I linker segment. AMY1-SBD was expressed in A. niger, secreted using the AMY1 signal sequence at 25 mg x L(-1) and purified in 50% yield. AMY1-SBD contained 23% carbohydrate and consisted of correctly N-terminally processed multiple forms of isoelectric points in the range 4.1-5.2. Activity and apparent affinity of AMY1-SBD (50 nM) for barley starch granules of 0.034 U x nmol(-1) and K(d) = 0.13 mg x mL(-1), respectively, were both improved with respect to the values 0.015 U x nmol(-1) and 0.67 mg x mL(-1) for rAMY1 (recombinant AMY1 produced in A. niger). AMY1-SBD showed a 2-fold increased activity for soluble starch at low (0.5%) but not at high (1%) concentration. AMY1-SBD hydrolysed amylose DP440 with an increased degree of multiple attack of 3 compared to 1.9 for rAMY1. Remarkably, at low concentration (2 nM), AMY1-SBD hydrolysed barley starch granules 15-fold faster than rAMY1, while higher amounts of AMY-SBD caused molecular overcrowding of the starch granule surface.

Aspergillus niger↗

PubMed2004

Removal of multiple nitrogenous wastes by Aspergillus niger in a continuous fixed-slab reactor.

A biofilter reactor, to which is attached a large variety of microorganisms, can be employed to treat circulating water in an intensive aquaculture system. Some nitrogen-containing wastes, such as ammonium and nitrite, are toxic to the aquatic organisms. The removal rates of the nitrogenous wastes are regarded as indices for the efficiency of treatment by biofilters. In this study, a fungus that was characterized as being able to remediate multiple nitrogenous wastes was identified as Aspergillus niger NBG5. In a continuous fixed-slab reactor, the heterotrophic fungus utilized ammonium, nitrite, protein, and glucose simultaneously. The fungus assimilated ammonium, nitrite and protein at rates of 0.247, 0.07 and 0.096 g-N/g-cell/day, respectively, at 22 degrees C. The remediation rates of ammonium nitrogenous wastes decreased by a factor of eight at 35 degrees C, while the specific growth rates slightly increased. For nitrogenous wastes, ammonium was a preferred substrate but its rate of consumption declined significantly as temperature increased. The nitrogen consumption rates were inconsistent with the cell yields at high temperature. Further analysis of consumption ratios of C/N revealed that cells grew predominantly from the carbon at high temperature. The A. niger NBG5 consumed glucose rapidly at specific rates of 2-2.5 g-C/g-cell/day at 35 degrees C in the presence of ammonium and nitrite; while sluggish consumption of glucose was observed in the protein substrate. The protein could serve as an alternative carbon source. Further ANOVA statistical analysis with P < 0.05 revealed no significant effects of temperature on the specific growth rates of A. niger on the SG-NH4 and milk-protein substrates, whereas significant effects on the C/N ratio at culture temperatures higher than 25 degrees C were observed. These findings indicated that the carbon utilization rate increased with high temperature, whereas nitrogen utilization increased as temperature declined. A suitable operational temperature was suggested, depending upon the amount of waste contents of C/N. A high temperature stimulates the use of carbon waste, while a low temperature favors remediation of all nitrogenous wastes.

Analysis of Variance↗

PubMed2006

Production of vanillin from waste residue of rice bran oil by Aspergillus niger and Pycnoporus cinnabarinus.

A new technology of transforming ferulic acid, which was from waste residue of rice bran oil, into vanillin was developed by a combination of fungal strains Aspergillus niger CGMCC0774 and Pycnoporus cinnabarinus CGMCC1115. Various concentrations of ferulic acid were compared, and the highest yield reached 2.2 g l(-1) of vanillic acid by A. niger CGMCC0774 in a 25 l fermenter when concentration of ferulic acid was 4 g l(-1). The filtrate of A. niger CGMCC0774 culture was concentrated and vanillic acid in the filtrate was bio-converted into vanillin by P. cinnabarinus CGMCC1115. The yield of vanillin reached 2.8 g l(-1) when 5 g l(-1) of glucose and 25 g of HZ802 resin were supplemented in the bioconversion medium. The 13C isotope analysis indicated that delta13C(PDB) of vanillin prepared was much different from chemically synthesized vanillin.

Aspergillus niger↗

PubMed2006

Kinetic modeling for the biosorption of copper by pretreated Aspergillus niger biomass.

In this present work, a kinetic model for biosorption of copper was developed considering the possibility of different forms of functional groups being present on the surface of the biomass prepared from Aspergillus niger. Results showed that metal uptake by A. niger was a mass transfer driven process, requiring only 30min to achieve 70% adsorption efficiency. Copper sorption by A. niger was influenced by the biomass dose, initial metal ion concentration, and pH of the solution. The Langmuir and Freundlich adsorption isotherms were used to describe the behavior of the system at different pH. The retention capacity of the biomass was determined at pH 6.0 to be equal to 23.62mg/g of biomass. The pretreatment with formalin improved the uptake of metal ion.

Adsorption↗

PubMed2005

Discrimination of Aspergillus niger and other Aspergillus species belonging to section Nigri by PCR assays.

Aspergillus species included in section Nigri are common in plant products and processed food, such as grapes, cereals, coffee and derivatives, particularly in warm and tropical climates. Two of these species, A. carbonarius and A. niger, are known to produce ochratoxin A (OTA), a potent nephrotoxin and carcinogenic to human (group 2B). Recognition of the several species of this section is difficult and requires considerable expertise using conventional methods based on morphological features. In this work we describe rapid, sensitive and robust assays based on the PCR technique to discriminate the main species included in section Nigri: A. japonicus, A. heteromorphus, A. ellipticus and the two morphologically indistinguishable species of the A. niger aggregate: A. niger and A. tubingensis. The species-specific primers have been designed on the basis of ITS (internal transcribed spacers of rDNA units) sequence comparisons obtained from several Aspergillus strains and have been tested in a number of strains from different origins and hosts. These PCR assays, based on multi-copy sequences, are highly sensitive and specific and represent a good tool for an early detection of OTA-producing Aspergillus species in order to prevent OTA from entering the food chain.

Aspergillus↗

PubMed2006

Effect of water activity on ochratoxin A production by Aspergillus niger aggregate species.

The effect of water activity (a(w)) (0.82-0.99) on growth and ochratoxin A (OTA) production by twelve Aspergillus niger aggregate strains, cultured in Czapek Yeast Autolysate agar (CYA) and Yeast Extract Sucrose agar (YES), was studied for an incubation period of 30 days. The strains were selected to include diverse sources, different reported abilities to produce OTA and different ITS-5.8 S rDNA Restriction Fragment Length Polymorphism (RFLP) pattern. They were characterized by Random Amplification of Polymorphic DNA (RAPD) and ITS-5.8 S rDNA and 28 S rDNA (D1/D2) sequencing. Regardless of the a(w) value tested, YES was a better culture medium than CYA for OTA production. The a(w) range for OTA production was narrower than that for growth. OTA was produced from 0.90, 0.92, 0.94 or 0.96 to 0.99 a(w) depending on the strain and the culture medium. The molecular study differentiated strains into two groups which corresponded to the RFLP types N and T although it did not distinguish them by their source of isolation or OTA producing abilities. Our results show that A. niger aggregate strains are able to grow and produce OTA over a wide a(w) range. These results will lead to a better understanding of the contribution of A. niger aggregate in OTA contamination of food and feed.

Agar↗