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Essays in Biochemistry Jul 2021The filamentous ascomycete fungus Aspergillus niger is a prolific secretor of organic acids, proteins, enzymes and secondary metabolites. Throughout the last century,... (Review)
Review
The filamentous ascomycete fungus Aspergillus niger is a prolific secretor of organic acids, proteins, enzymes and secondary metabolites. Throughout the last century, biotechnologists have developed A. niger into a multipurpose cell factory with a product portfolio worth billions of dollars each year. Recent technological advances, from genome editing to other molecular and omics tools, promise to revolutionize our understanding of A. niger biology, ultimately to increase efficiency of existing industrial applications or even to make entirely new products. However, various challenges to this biotechnological vision, many several decades old, still limit applications of this fungus. These include an inability to tightly control A. niger growth for optimal productivity, and a lack of high-throughput cultivation conditions for mutant screening. In this mini-review, we summarize the current state-of-the-art for A. niger biotechnology with special focus on organic acids (citric acid, malic acid, gluconic acid and itaconic acid), secreted proteins and secondary metabolites, and discuss how new technological developments can be applied to comprehensively address a variety of old and persistent challenges.
Topics: Aspergillus niger; Biotechnology; Citric Acid; Gene Editing
PubMed: 33955461
DOI: 10.1042/EBC20200139 -
PloS One 2021A predicted metalloproteinase gene, HypZn, was cloned from Aspergillus niger CGMCC 3.7193 and expressed in Pichia pastoris GS115, and the physicochemical characteristics...
A predicted metalloproteinase gene, HypZn, was cloned from Aspergillus niger CGMCC 3.7193 and expressed in Pichia pastoris GS115, and the physicochemical characteristics of recombinant HypZn were investigated after separation and purification. The results showed that the specific activity of the purified HypZn reached 1859.2 U/mg, and the optimum temperature and pH value of HypZn were 35°C and 7.0, respectively. HypZn remained stable both at 40°C and at pH values between 5.0 and 8.0. The preferred substrate of HypZn was soybean protein isolates, and the Km and Vmax values were 21.5 μmol/mL and 4926.6 μmol/(mL∙min), respectively. HypZn was activated by Co2+ and Zn2+ and inhibited by Cu2+ and Fe2+. The degree of soybean protein isolate hydrolysis reached 14.7%, and the hydrolysates were of uniform molecular weight. HypZn could tolerate 5000 mM NaCl and completely lost its activity after 30 min at 50°C. The enzymological characterizations indicated that HypZn has great application potential in the food industry, especially in fermented food processing.
Topics: Aspergillus niger; Hydrolysis; Metalloproteases
PubMed: 34762700
DOI: 10.1371/journal.pone.0259809 -
MicrobiologyOpen Jan 2020The knowledge of how Aspergillus niger responds to ethanol can lead to the design of strains with enhanced ethanol tolerance to be utilized in numerous industrial...
The knowledge of how Aspergillus niger responds to ethanol can lead to the design of strains with enhanced ethanol tolerance to be utilized in numerous industrial bioprocesses. However, the current understanding about the response mechanisms of A. niger toward ethanol stress remains quite limited. Here, we first applied a cell growth assay to test the ethanol tolerance of A. niger strain ES4, which was isolated from the wall near a chimney of an ethanol tank of a petroleum company, and found that it was capable of growing in 5% (v/v) ethanol to 30% of the ethanol-free control level. Subsequently, the metabolic responses of this strain toward ethanol were investigated using untargeted metabolomics, which revealed the elevated levels of triacylglycerol (TAG) in the extracellular components, and of diacylglycerol, TAG, and hydroxy-TAG in the intracellular components. Lastly, stable isotope labeling mass spectrometry with ethanol-d showed altered isotopic patterns of molecular ions of lipids in the ethanol-d samples, compared with the nonlabeled ethanol controls, suggesting the ability of A. niger ES4 to utilize ethanol as a carbon source. Together, the studies revealed the upregulation of glycerolipid metabolism and ethanol utilization pathway as novel response mechanisms of A. niger ES4 toward ethanol stress, thereby underlining the utility of untargeted metabolomics and the overall approaches as tools for elucidating new biological insights.
Topics: Aspergillus niger; Diglycerides; Ethanol; Glycolipids; Metabolomics; Triglycerides
PubMed: 31646764
DOI: 10.1002/mbo3.948 -
Toxins Dec 2020Microbial degradation is an effective and attractive method for eliminating aflatoxin B1 (AFB1), which is severely toxic to humans and animals. In this study, RAF106...
Microbial degradation is an effective and attractive method for eliminating aflatoxin B1 (AFB1), which is severely toxic to humans and animals. In this study, RAF106 could effectively degrade AFB1 when cultivated in Sabouraud dextrose broth (SDB) with contents of AFB1 ranging from 0.1 to 4 μg/mL. Treatment with yeast extract as a nitrogen source stimulated the degradation, but treatment with NaNO and NaNO as nitrogen sources and lactose and sucrose as carbon sources suppressed the degradation. Moreover, RAF106 still degraded AFB1 at initial pH values that ranged from 4 to 10 and at cultivation temperatures that ranged from 25 to 45 °C. In addition, intracellular enzymes or proteins with excellent thermotolerance were verified as being able to degrade AFB1 into metabolites with low or no mutagenicity. Furthermore, genomic sequence analysis indicated that the fungus was considered to be safe owing to the absence of virulence genes and the gene clusters for the synthesis of mycotoxins. These results indicate that RAF106 and its intracellular enzymes or proteins have a promising potential to be applied commercially in the processing and industry of food and feed to detoxify AFB1.
Topics: Aflatoxin B1; Aspergillus niger; Proteolysis; Tandem Mass Spectrometry; Tea
PubMed: 33291337
DOI: 10.3390/toxins12120777 -
Applied and Environmental Microbiology Feb 2021Salicylic acid plays an important role in the plant immune response, and its degradation is therefore important for plant-pathogenic fungi. However, many nonpathogenic...
Salicylic acid plays an important role in the plant immune response, and its degradation is therefore important for plant-pathogenic fungi. However, many nonpathogenic microorganisms can also degrade salicylic acid. In the filamentous fungus , two salicylic acid metabolic pathways have been suggested. The first pathway converts salicylic acid to catechol by a salicylate hydroxylase (ShyA). In the second pathway, salicylic acid is 3-hydroxylated to 2,3-dihydroxybenzoic acid, followed by decarboxylation to catechol by 2,3-dihydroxybenzoate decarboxylase (DhbA). cleaves the aromatic ring of catechol catalyzed by catechol 1,2-dioxygenase (CrcA) to form ,-muconic acid. However, the identification and role of the genes and characterization of the enzymes involved in these pathways are lacking. In this study, we used transcriptome data of grown on salicylic acid to identify genes ( and ) involved in salicylic acid metabolism. Heterologous production in followed by biochemical characterization confirmed the function of ShyA and CrcA. The combination of ShyA and CrcA demonstrated that -muconic acid can be produced from salicylic acid. In addition, the roles of , , and were studied by creating deletion mutants which revealed the role of these genes in the fungal metabolism of salicylic acid. Nonrenewable petroleum sources are being depleted, and therefore, alternative sources are needed. Plant biomass is one of the most abundant renewable sources on Earth and is efficiently degraded by fungi. In order to utilize plant biomass efficiently, knowledge about the fungal metabolic pathways and the genes and enzymes involved is essential to create efficient strategies for producing valuable compounds such as ,-muconic acid. ,-Muconic acid is an important platform chemical that is used to synthesize nylon, polyethylene terephthalate (PET), polyurethane, resins, and lubricants. Currently, ,-muconic acid is mainly produced through chemical synthesis from petroleum-based chemicals. Here, we show that two enzymes from fungi can be used to produce ,-muconic acid from salicylic acid and contributes in creating alternative methods for the production of platform chemicals.
Topics: Aspergillus niger; Carboxy-Lyases; Catechol 1,2-Dioxygenase; Fungal Proteins; Mixed Function Oxygenases; Phylogeny; Salicylic Acid
PubMed: 33397706
DOI: 10.1128/AEM.02701-20 -
Bioprocess and Biosystems Engineering Feb 2018In its natural environment, the filamentous fungus Aspergillus niger grows on decaying fruits and plant material, thereby enzymatically degrading the lignocellulosic...
In its natural environment, the filamentous fungus Aspergillus niger grows on decaying fruits and plant material, thereby enzymatically degrading the lignocellulosic constituents (lignin, cellulose, hemicellulose, and pectin) into a mixture of mono- and oligosaccharides. To investigate the kinetics and stoichiometry of growth of this fungus on lignocellulosic sugars, we carried out batch cultivations on six representative monosaccharides (glucose, xylose, mannose, rhamnose, arabinose, and galacturonic acid) and a mixture of these. Growth on these substrates was characterized in terms of biomass yields, oxygen/biomass ratios, and specific conversion rates. Interestingly, in combination, some of the carbon sources were consumed simultaneously and some sequentially. With a previously developed protocol, a sequential chemostat cultivation experiment was performed on a feed mixture of the six substrates. We found that the uptake of glucose, xylose, and mannose could be described with a Michaelis-Menten-type kinetics; however, these carbon sources seem to be competing for the same transport systems, while the uptake of arabinose, galacturonic acid, and rhamnose appeared to be repressed by the presence of other substrates.
Topics: Aspergillus niger; Kinetics; Lignin; Monosaccharides
PubMed: 29052015
DOI: 10.1007/s00449-017-1854-3 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Dec 2022is an important industrial strain which has been widely used for production of enzymes and organic acids. Genome modification of . is required to further improve its...
is an important industrial strain which has been widely used for production of enzymes and organic acids. Genome modification of . is required to further improve its potential for industrial production. CRISPR/Cas9 is a widely used genome editing technique for . , 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 . 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 ) 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 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 . .
Topics: Gene Editing; Aspergillus niger; CRISPR-Cas Systems; Plasmids
PubMed: 36593207
DOI: 10.13345/j.cjb.220162 -
Microbiology (Reading, England) Apr 2019Arginase is the only fungal ureohydrolase that is well documented in the literature. More recently, a novel route for agmatine catabolism in Aspergillus niger involving...
Arginase is the only fungal ureohydrolase that is well documented in the literature. More recently, a novel route for agmatine catabolism in Aspergillus niger involving another ureohydrolase, 4-guanidinobutyrase (GBase), was reported. We present here a detailed characterization of A. niger GBase - the first fungal (and eukaryotic) enzyme to be studied in detail. A. niger GBase is a homohexamer with a native molecular weight of 336 kDa and an optimal pH of 7.5. Unlike arginase, the Mn enzyme from the same fungus, purified GBase protein is associated with Zn ions. A sensitive fluorescence assay was used to determine its kinetic parameters. GBase acted 25 times more efficiently on 4-guanidinobutyrate (GB) than 3-guanidinopropionic acid (GP). The Km for GB was 2.7±0.4 mM, whereas for GP it was 53.7±0.8 mM. While GB was an efficient nitrogen source, A. niger grew very poorly on GP. Constitutive expression of GBase favoured fungal growth on GP, indicating that GP catabolism is limited by intracellular GBase levels in A. niger. The absence of a specific GPase and the inability of GP to induce GBase expression confine the fungal growth on GP. That GP is a poor substrate for GBase and a very poor nitrogen source for A. niger offers an opportunity to select GBase specificity mutations. Further, it is now possible to compare two distinct ureohydrolases, namely arginase and GBase, from the same organism.
Topics: Agmatine; Arginase; Aspergillus niger; Butyrates; Cations; Culture Media; Fungal Proteins; Gene Expression; Guanidines; Kinetics; Molecular Weight; Mutation; Propionates; Protein Multimerization; Substrate Specificity; Ureohydrolases
PubMed: 30806615
DOI: 10.1099/mic.0.000782 -
Scientific Reports Jan 2017Despite a long and successful history of citrate production in Aspergillus niger, the molecular mechanism of citrate accumulation is only partially understood. In this...
Despite a long and successful history of citrate production in Aspergillus niger, the molecular mechanism of citrate accumulation is only partially understood. In this study, we used comparative genomics and transcriptome analysis of citrate-producing strains-namely, A. niger H915-1 (citrate titer: 157 g L), A1 (117 g L), and L2 (76 g L)-to gain a genome-wide view of the mechanism of citrate accumulation. Compared with A. niger A1 and L2, A. niger H915-1 contained 92 mutated genes, including a succinate-semialdehyde dehydrogenase in the γ-aminobutyric acid shunt pathway and an aconitase family protein involved in citrate synthesis. Furthermore, transcriptome analysis of A. niger H915-1 revealed that the transcription levels of 479 genes changed between the cell growth stage (6 h) and the citrate synthesis stage (12 h, 24 h, 36 h, and 48 h). In the glycolysis pathway, triosephosphate isomerase was up-regulated, whereas pyruvate kinase was down-regulated. Two cytosol ATP-citrate lyases, which take part in the cycle of citrate synthesis, were up-regulated, and may coordinate with the alternative oxidases in the alternative respiratory pathway for energy balance. Finally, deletion of the oxaloacetate acetylhydrolase gene in H915-1 eliminated oxalate formation but neither influence on pH decrease nor difference in citrate production were observed.
Topics: Aspergillus niger; Citric Acid; Fermentation; Gene Expression Profiling; Genomics; Metabolic Engineering; Transcriptome
PubMed: 28106122
DOI: 10.1038/srep41040 -
STAR Protocols Dec 2022This protocol describes procedures for quantifying Aspergillus niger growth in both solid and liquid culture. Firstly, by comparing radial growth between mutant and...
This protocol describes procedures for quantifying Aspergillus niger growth in both solid and liquid culture. Firstly, by comparing radial growth between mutant and progenitor isolates on solid agar supplemented with sublethal stressors, susceptibility coefficients can be calculated. Secondly, analysis of macromorphological growth types in liquid culture allows full quantification of how a gene of interest affects submerged growth. By combining these assays, an extensive and quantitative dataset of how a gene of interest impacts growth in this fungus is possible. For complete details on the use and execution of this protocol, please refer to Cairns et al. (2019) and Cairns et al. (2022)..
Topics: Aspergillus niger; Agar
PubMed: 36595891
DOI: 10.1016/j.xpro.2022.101883