The hydrolysis of xylo-oligosaccharides catalyzed by β-xylosidase plays an important role in the degradation of lignocellulose. However, the enzyme is easily inhibited by its catalytic product xylose, which severely limits its application. Based on molecular docking, this paper studied the xylose affinity of Aspergillus niger β-xylosidase An-xyl, which was significantly differentially expressed in the fermentation medium of tea stalks, through cloning, expression and characterization. The synergistic degradation effect of this enzyme and cellulase on lignocellulose in tea stems was investigated. Molecular docking showed that the affinity of An-xyl to xylose was lower than that of Aspergillus oryzae β-xylosidase with poor xylose tolerance. The K_i value of xylose inhibition constant of recombinant-expressed An-xyl was 433.2 mmol/L, higher than that of most β-xylosidases of the GH3 family. The K_m and V_max towards pNPX were 3.6 mmol/L and 10 000 μmol/(min·mL), respectively. The optimum temperature of An-xyl was 65 ℃, the optimum pH was 4.0, 61% of the An-xyl activity could be retained upon treatment at 65 ℃ for 300 min, and 80% of the An-xyl activity could be retained upon treatment at pH 2.0-8.0 for 24 h. The hydrolysis of tea stem by An-xyl and cellulase produced 19.3% and 38.6% higher reducing sugar content at 2 h and 4 h, respectively, than that of using cellulase alone. This study showed that the An-xyl mined from differential expression exhibited high xylose tolerance and higher catalytic activity and stability, and could hydrolyze tea stem lignocellulose synergistically, which enriched the resource of β-xylosidase with high xylose tolerance, thus may facilitate the advanced experimental research and its application.
Aspergillus niger/genetics* ; Xylose/metabolism* ; Molecular Docking Simulation ; Xylosidases/genetics* ; Cellulases ; Tea ; Hydrogen-Ion Concentration ; Substrate Specificity

Scopoletin is a coumarin compound with various biological activities including detumescence and analgesic, insecticidal, antibacterial and acaricidal effects. However, interference with scopolin and other components often leads to difficulties in purification of scopoletin with low extraction rates from plant resource. In this paper, heterologous expression of the gene encoding β-glucosidase An-bgl3 derived from Aspergillus niger were carried out. The expression product was purified and characterized with further structure-activity relationship between it and β-glucosidase analyzed. Subsequently, its ability for transforming scopolin from plant extract was studied. The results showed that the specific activity of the purified β-glucosidase An-bgl3 was 15.22 IU/mg, the apparent molecular weight was about 120 kDa. The optimum reaction temperature and pH were 55 ℃ and 4.0, respectively. Moreover, 10 mmol/L metal ions Fe²⁺ and Mn²⁺ increased the enzyme activity by 1.74-fold and 1.20-fold, respectively. A 10 mmol/L solution containing Tween-20, Tween-80 and Triton X-100 all inhibited the enzyme activity by 30%. The enzyme showed affinity towards scopolin and tolerated 10% methanol and 10% ethanol solution, respectively. The enzyme specifically hydrolyzed scopolin into scopoletin from the extract of Erycibe obtusifolia Benth with a 47.8% increase of scopoletin. This demonstrated that the β-glucosidase An-bgl3 from A. niger shows specificity on scopolin with good activities, thus providing an alternative method for increasing the extraction efficiency of scopoletin from plant material.
Aspergillus niger/genetics* ; beta-Glucosidase/chemistry* ; Scopoletin ; Polysorbates ; Coumarins

Aspergillus niger is an important industrial strain which has been widely used for production of enzymes and organic acids. Genome modification of A. niger is required to further improve its potential for industrial production. CRISPR/Cas9 is a widely used genome editing technique for A. niger, but its application in industrial strains modification is hampered by the need for integration of a selection marker into the genome or low gene editing efficiency. Here we report a highly efficient marker-free genome editing method for A. niger based on CRISPR/Cas9 technique. Firstly, we constructed a co-expression plasmid of sgRNA and Cas9 with a replication initiation region fragment AMA1 (autonomously maintained in Aspergillus) by using 5S rRNA promoter which improved sgRNA expression. Meanwhile, a strain deficient in non-homologous end-joining (NHEJ) was developed by knocking out the kusA gene. Finally, we took advantage of the instability of plasmid containing AMA1 fragment to cure the co-expression plasmid containing sgRNA and Cas9 through passaging on non-selective plate. With this method, the efficiency of gene editing reached 100% when using maker-free donor DNA with a short homologous arm of 20 bp. This method may facilitate investigation of gene functions and construction of cell factories for A. niger.
Gene Editing ; Aspergillus niger/genetics* ; CRISPR-Cas Systems/genetics* ; Plasmids/genetics*

Aspergillus niger is a vital industrial workhouse widely used for the production of organic acids and industrial enzymes. This fungus is a crucial cell factory due to its innate tolerance to a diverse range of abiotic conditions, high production titres, robust growth during industrial scale fermentation, and status as a generally recognized as safe (GRAS) organism. Rapid development of synthetic biology and systems biology not only offer powerful approaches to unveil the molecular mechanisms of A. niger productivity, but also provide more new strategies to construct and optimize the A. niger cell factory. As a new generation of genome editing technology, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas) system brings a revolutionary breakthrough in targeted genome modification for A. niger. In this review, we focus on current advances to the CRISPR/Cas genome editing toolbox, its application on gene modification and gene expression regulation in this fungal. Moreover, the future directions of CRISPR/Cas genome editing in A. niger are highlighted.
Aspergillus niger/genetics* ; CRISPR-Cas Systems/genetics* ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics* ; Gene Editing ; Genome

OBJECTIVE: A strain of Aspergillus niger (A. niger), capable of releasing bound phenolic acids from wheat bran, was isolated. This strain was identified by gene sequence identification. The antioxidant and anti-inflammatory capacity of ferulic acid released from wheat bran by this A. niger strain (FA-WB) were evaluated. METHODS: Molecular identification techniques based on PCR analysis of specific genomic sequences were conducted; antioxidant ability was examined using oxygen radical absorbance capacity (ORAC), cellular antioxidant activity (CAA) assays, and erythrocyte hemolysis assays. RAW264.7 cells were used as a model to detect anti-inflammatory activity. RESULTS: The filamentous fungal isolate was identified to be A. niger. ORAC and CAA assay showed that FA-WB had better antioxidant activity than that of the ferulic acid standard. The erythrocyte hemolysis assay results suggested that FA-WB could attenuate AAPH-induced oxidative stress through inhibition of reactive oxy gen species (ROS) generation. FA-WB could significantly restore the AAPH-induced increase in intracellular antioxidant enzyme activities to normal levels as well as inhibit the intracellular malondialdehyde formation. TNF-a, IL-6, and NO levels indicated that FA-WB can inhibit the inflammation induced by lipopolysaccharide (LPS). CONCLUSION Ferulic acid released from wheat bran by a new strain of A. niger had good anti-inflammatory activity and better antioxidant ability than standard ferulic acid.
Animals ; Anti-Inflammatory Agents ; metabolism ; pharmacology ; Antioxidants ; metabolism ; pharmacology ; Aspergillus niger ; genetics ; isolation & purification ; metabolism ; Coumaric Acids ; metabolism ; pharmacology ; DNA, Fungal ; analysis ; Dietary Fiber ; microbiology ; Erythrocytes ; drug effects ; metabolism ; Fermentation ; Hep G2 Cells ; Humans ; Interleukin-6 ; metabolism ; Lipopolysaccharides ; pharmacology ; Mice ; RAW 264.7 Cells ; Sheep ; Tumor Necrosis Factor-alpha ; metabolism

Aspergillus niger, as an important industrial fermentation strain, is widely applied in the production of organic acids and industrial enzymes. With the development of diverse omics technologies, the data of genome, transcriptome, proteome and metabolome of A. niger are increasing continuously, which declared the coming era of big data for the research in fermentation process of A. niger. The data analysis from single omics and the comparison of multi-omics, to the integrations of multi-omics based on the genome-scale metabolic network model largely extends the intensive and systematic understanding of the efficient production mechanism of A. niger. It also provides possibilities for the reasonable global optimization of strain performance by genetic modification and process regulation. We reviewed and summarized progress in omics research of A. niger, and proposed the development direction of omics research on this cell factory.
Aspergillus niger ; genetics ; Fermentation ; Genome, Fungal ; Metabolic Networks and Pathways ; Metabolome ; Proteome ; Transcriptome

The main commercial production of fructooligosaccharides (FOS) comes from enzymatic transformation using sucrose as substrate by microbial enzyme fructosyltransferase. A fructosyltransferase genomic DNA was isolated from Aspergillus niger QU10 by PCR. The nucleotide sequence showed a 1 941 bp size, and has been submitted to GenBank (KF699529). The cDNA of the fructosyltransferase, containing an open reading frame of 1 887 bp, was further cloned by RT-PCR. The fructosyltransferase gene from Aspergillus niger was functionally expressed both in Escherichia coli and Pichia pastoris GS 115. The highest activity value for the construction with the α-factor signal peptide reached 431 U/mL after 3 days of incubation. The recombinant enzyme is extensively glycosylated, and the active form is probably represented by a homodimer with an apparent molecular mass of 200 kDa as judged from mobility in seminative PAGE gels. The extracellular recombinant enzyme converted sucrose mostly to FOS, mainly 1-kestose and nystose, liberating glucose. FOS reached a maximal value and represented about 58% of total sugars present in the reaction mixture after 4 h reaction. The results suggest that the availability of recombinant Pichia pastoris as a new source of a FOS-producing enzyme might result of biotechnology interest for industrial application.
Aspergillus niger ; enzymology ; genetics ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; Fungal Proteins ; genetics ; metabolism ; Glycosylation ; Hexosyltransferases ; genetics ; metabolism ; Molecular Sequence Data ; Molecular Weight ; Pichia ; Sucrose ; metabolism ; Trisaccharides ; metabolism

The monoamine oxidase mutant A-1 (F210V/L213C) from Aspergillus niger showed some catalytic activity on mexiletine. To futher improve its activity, the mutant was subjected to directed evolution with MegaWHOP PCR (Megaprimer PCR of Whole Plasmid) and selection employing a high-throughput agar plate-based colorimetric screen. This approach led to the identification of a mutant ep-1, which specific activity was 189% of that for A-1. The ep-1 also showed significantly improved enantioselectivity, with the E value increased from 101 to 282; its kinetic k(cat)/K(m) value increased from 0.001 51 mmol/(L x s) to 0.002 89 mmol/(L x s), suggesting that catalytic efficiency of ep-1 had been improved. The mutant showed obviously higher specific activities on 7 of all tested 11 amines substrates, and the others were comparable. Sequence analysis revealed that there was a new mutation T162A on ep-1. The molecular dynamics simulation indicated that T162A may affect the secondary structure of the substrate channel and expand the binding pocket.
Aspergillus niger ; enzymology ; Catalysis ; Kinetics ; Monoamine Oxidase ; genetics ; metabolism ; Mutation ; Polymerase Chain Reaction ; Protein Engineering ; Protein Structure, Secondary ; Substrate Specificity

OBJECTIVETo evaluate the feasibility of PCR/reverse line blot hybridization (RLB) assay in the detection and identification of clinical pathogens in fungal sinusitis (FS).

METHODSTwenty-six formalin-fixed and paraffin-embedded tissues and 8 fresh tissues of FS were collected from Beijing Tongren Hospital, Capital Medical University from May 2009 to February 2010. Pathological examination, fungal culture and ITS2 region sequencing were carried out. The DNA of all samples was extracted by standard procedure and fungal universal primers ITS3 and ITS4 were used for PCR amplification of all tissues. Then the amplified products were used for RLB with five fungal species-specific probes. The results of PCR/RLB were compared with ITS region sequencing, fungal culture and pathological examination.

RESULTSFor the biopsy tissues, fungal cultures were positive in 14 cases (41.2%); pathologic examination demonstrated fungal hyphae in all cases; ITS2 region sequencing was successful in 16 cases (47.1%); PCR/RLB showed A. flavus in 14 cases, A. fumigatus in 10 cases, A. niger in four cases, A. nidulans in one case, A. flavus and A. fumigatus in three cases, and A. fumigatus and A. niger in two cases.

CONCLUSIONSThe PCR/RLB assay is suitable for rapid and accurate detection and identification of the pathogenic fungus of FS. Compared with the conventional fungal culture and microscopy, pathologic examination and DNA sequencing, the PCR/RLB has the advantages of more economy, time saving, and higher sensitivity, specificity and throughput.

Aspergillus ; classification ; genetics ; isolation & purification ; Aspergillus flavus ; genetics ; isolation & purification ; Aspergillus fumigatus ; genetics ; isolation & purification ; Aspergillus niger ; genetics ; isolation & purification ; DNA Primers ; DNA, Fungal ; genetics ; Humans ; Mycoses ; diagnosis ; microbiology ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Sinusitis ; diagnosis ; microbiology

Based on previous bioinformational analysis results, two Aspergillus niger lipase (ANL) mutants, ANL-Ser84Gly and ANL-Asp99Pro were constructed to screen ANL mutants with oil-water interface independence. ANL-Ser84Gly still displayed a pronounced interfacial activation, while ANL-Asp99Pro displayed no interfacial activation. The specific activity of ANL-Ser84Gly towards p-nitrophenyl palmitate (-myristate, -laurate and -decanoate) decreased by 29.8% (53.1, 60.1 and 77.1, respectively) than that of ANL, while the specific activity of ANL-Asp99Pro towards p-nitrophenyl palmitate increased by 2.2-fold. The mutation in the hinge region at both sides of the lid domain also destabilized various secondary structure factors of ANL-S84G and ANL-D99P, which resulted in a substantial decrease in thermostability. The achievement to construct oil-water interface-independent ANL mutants would help to further understand lipase interfacial activation mechanism.
Aspergillus niger ; enzymology ; genetics ; Base Sequence ; Enzyme Stability ; Fungal Proteins ; genetics ; metabolism ; Lipase ; genetics ; metabolism ; Molecular Sequence Data ; Mutant Proteins ; genetics ; Oils ; Substrate Specificity ; Water