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Microbial Cell Factories Aug 2022Itaconic acid (IA) is a versatile platform chemical widely used for the synthesis of various polymers and current methods for IA production based on Aspergillus terreus...
Synergistic effects on itaconic acid production in engineered Aspergillus niger expressing the two distinct biosynthesis clusters from Aspergillus terreus and Ustilago maydis.
BACKGROUND
Itaconic acid (IA) is a versatile platform chemical widely used for the synthesis of various polymers and current methods for IA production based on Aspergillus terreus fermentation are limited in terms of process efficiency and productivity. To construct more efficient IA production strains, A. niger was used as a chassis for engineering IA production by assembling the key components of IA biosynthesis pathways from both A. terreus and Ustilago maydis.
RESULTS
Recombinant A. niger S1596 overexpressing the A. terreus IA biosynthesis genes cadA, mttA, mfsA produced IA of 4.32 g/L, while A. niger S2120 overexpressing the U. maydis IA gene cluster adi1, tad1, mtt1, itp1 achieved IA of 3.02 g/L. Integration of the two IA production pathways led to the construction of A. niger S2083 with IA titers of 5.58 g/L. Increasing cadA copy number in strain S2083 created strain S2209 with titers of 7.99 g/L and deleting ictA to block IA degradation in S2209 created strain S2288 with IA titers of 8.70 g/L. Overexpressing acoA to enhance the supply of IA precursor in strain S2288 generated strain S2444 with IA titers of 9.08 g/L in shake flask.
CONCLUSION
Recombinant A. niger overexpressing the U. maydis IA biosynthesis pathway was capable of IA accumulation. Combined expression of the two IA biosynthesis pathways from A. terreus and U. maydis in A. niger resulted in much higher IA titers. Furthermore, increasing cadA copy number, deleting ictA to block IA degradation and overexpressing acoA to enhance IA precursor supply all showed beneficial effects on IA accumulation.
Topics: Aspergillus; Aspergillus niger; Basidiomycota; Succinates
PubMed: 35953829
DOI: 10.1186/s12934-022-01881-7 -
World Journal of Microbiology &... Jun 2019Siderophores are extra-cellular inducible compounds produced by aerobic microorganisms and plants to overcome iron insolubility via its chelation and then uptake inside...
Siderophores are extra-cellular inducible compounds produced by aerobic microorganisms and plants to overcome iron insolubility via its chelation and then uptake inside the cell. This work aims to study the characteristics of siderophore that is produced by a rhizosphere-inhabiting fungus. This fungus has been morphologically and molecularly identified as Aspergillus niger with the ability to produce 87% siderophore units. The obtained siderophore in PDB medium gave a positive result with tetrazolium test and a characteristic spectrum with a maximum absorbance at 450 nm in FeCl test that did not shift in response to different pH degrees (5-9). This indicates that the obtained siderophore is a trihydroxymate in nature. After purification, the FTIR and NMR analyses showed that the obtained siderophore is considered to be ferrichrome. The purified siderophore has been further evaluated as a tool to extract uranium, thorium and rare earth elements (REEs) from Egyptian phosphorites obtained from Abu Tartur Mine area. The inductively coupled plasma atomic emission spectroscopy analysis showed that the highest removal efficiency percentage was for uranium (69.5%), followed by samarium (66.7%), thorium (55%), lanthanum (51%), and cerium (50.1%). This result confirmed the ability of hydroxymate siderophores to chelate the aforementioned precious elements, a result that paves the way for bioleaching to replace abiotic techniques in order to save the cost of such elements in an environmentally friendly way.
Topics: Aspergillus niger; Egypt; Fatty Acids; Ferrichrome; Hydrogen-Ion Concentration; Iron; Minerals; Phosphates; Rhizosphere; Siderophores; Soil Microbiology
PubMed: 31187335
DOI: 10.1007/s11274-019-2666-1 -
Journal of Food Biochemistry Aug 2019α-l-Rhamnosidase is a biotechnologically important enzyme in food industry and in the preparation of drugs and drug precursors. To expand the functionality of our...
α-l-Rhamnosidase is a biotechnologically important enzyme in food industry and in the preparation of drugs and drug precursors. To expand the functionality of our previously cloned α-l-rhamnosidase from Aspergillus niger JMU-TS528, 14 mutants were constructed based on the changes of the folding free energy (ΔΔG), predicted by the PoPMuSiC algorithm. Among them, six single-site mutants displayed higher thermal stability than wild type (WT). The combinational mutant K573V-E631F displayed even higher thermostability than six single-site mutants. The spectra analyses displayed that the WT and K573V-E631F had almost similar secondary and tertiary structure profiles. The simulated protein structure-based interaction analysis and molecular dynamics calculation were further implemented to assess the conformational preferences of the K573V-E631F. The improved thermostability of mutant K573V-E631F may be attributed to the introduction of new cation-π and hydrophobic interactions, and the overall improvement of the enzyme conformation. PRACTICAL APPLICATIONS: The stability of enzymes, particularly with regards to thermal stability remains a critical issue in industrial biotechnology and industrial processing generally tends to higher ambient temperature to inhibit microbial growth. Most of the α-l-rhamnosidases are usually active at temperature from 30 to 60°C, which are apt to denature at temperatures over 60°C. To expand the functionality of our previously cloned α-l-rhamnosidase from Aspergillus niger JMU-TS528, we used protein engineering methods to increase the thermal stability of the α-l-rhamnosidase. Practically, conducting reactions at high temperatures enhances the solubility of substrates and products, increases the reaction rate thus reducing the reaction time, and inhibits the growth of contaminating microorganisms. Thus, the improvement on the thermostability of α-l-rhamnosidase on the one hand can increase enzyme efficacy; on the other hand, the high ambient temperature would enhance the solubility of natural substrates of α-l-rhamnosidase, such as naringin, rutin, and hesperidin, which are poorly dissolved in water at room temperature. Protein thermal resistance is an important issue beyond its obvious industrial importance. The current study also helps in the structure-function relationship study of α-l-rhamnosidase.
Topics: Algorithms; Aspergillus niger; Enzyme Stability; Fungal Proteins; Glycoside Hydrolases; Hot Temperature; Protein Engineering
PubMed: 31368575
DOI: 10.1111/jfbc.12945 -
Journal of Agricultural and Food... Apr 2022α-l-Arabinofuranosidase (Abf), a debranching enzyme that can remove arabinose substituents from arabinoxylan, promotes the hydrolysis of hemicellulose in plant biomass....
α-l-Arabinofuranosidase (Abf), a debranching enzyme that can remove arabinose substituents from arabinoxylan, promotes the hydrolysis of hemicellulose in plant biomass. However, the functional specificity of Abfs from different glycoside hydrolase (GH) families on the digestion of arabinoxylan and their synergistic interaction with xylanase have not been systematically studied. In this work, we characterized three Abfs (AxhA, AbfB, and AbfC) from GH62, GH54, and GH51 families in An76. Quantitative transcriptional analysis showed that expression of the gene was upregulated as a result of induction by xylose substrates, whereas expression of the gene was mainly induced by arabinose. Recombinant AxhA, AbfB, and AbfC exhibited different hydrolytic performances. AxhA showed the highest catalytic activity toward wheat arabinoxylan (WAX) and tended to hydrolyze monosubstituted arabinofuranose units, whereas AbfB had higher catalytic activity on AN and debranched arabinan (DAN), having the ability to cope with mono- and disubstituted arabinofuranose units. Furthermore, AbfC had greater arabinofuranosidase activity on -nitrophenyl-α-l-arabinofuranoside (NP-AraF) than on other substrates. Moreover, three Abfs displayed obvious synergistic action with GH11 xylanase XynB against WAX and barley husk residues. The elucidation of the degradation mechanisms of Abfs will lay a theoretical foundation for the efficient industrialized transformation of arabinoxylans.
Topics: Arabinose; Aspergillus niger; Glycoside Hydrolases; Humans; Hydrolysis; Substrate Specificity; Xylans
PubMed: 35420820
DOI: 10.1021/acs.jafc.1c08388 -
Biotechnology Letters Apr 2020To isolate a novel cis-epoxysuccinate hydrolase (CESH)-producing fungus for production of L( +)-tartaric acid, before this, all strains were selected from bacteria.
OBJECTIVES
To isolate a novel cis-epoxysuccinate hydrolase (CESH)-producing fungus for production of L( +)-tartaric acid, before this, all strains were selected from bacteria.
RESULTS
A CESH-producing fungus was first isolated from soil and identified as Aspergillus niger WH-2 based on its morphological properties and ITS sequence. The maximum activity of hyphaball and fermentation supernatants was 1278 ± 64 U/g and 5.6 ± 0.3 U/mL, respectively, in a 5 L fermenter based on the conditions optimized on the flask. Almost 70% of CESH was present in hyphaball, which maintained 40% residual activity at pH 4.0 and showed a good acid stability (pH 3.0-10.0), high conversion rate (> 98%), and enantioselectivity (EE > 99.6%). However, the reported CESHs from bacteria can't be catalyzed under acidic conditions.
CONCLUSIONS
The Aspergillus niger WH-2 was the first reported CESH-producing fungus, which could biosynthesize L ( +)-tartaric acid under acidic conditions and provide an alternative catalyst and process.
Topics: Acids; Aspergillus niger; Batch Cell Culture Techniques; Fermentation; Fungal Proteins; Hydrogen-Ion Concentration; Hydrolases; Phylogeny; Soil Microbiology; Tartrates
PubMed: 31955308
DOI: 10.1007/s10529-020-02799-z -
Biotechnology Letters Jun 2021To achieve continuous production of fructooligosaccharides (FOS) by recycling of the mycelial cells containing the thermal-stable β-fructofuranosidase in Aspergillus...
OBJECTIVE
To achieve continuous production of fructooligosaccharides (FOS) by recycling of the mycelial cells containing the thermal-stable β-fructofuranosidase in Aspergillus niger without immobilization.
RESULTS
The thermal-stable β-fructofuranosidase FopA-V1 was successfully expressed in A. niger ATCC 20611 under the control of the constitutive promoter PgpdA. The engineered A. niger strain FV1-11 produced the β-fructofuranosidase with improved thermostability, which remained 91.2% of initial activity at 50 °C for 30 h. Then its mycelial β-fructofuranosidase was recycled for the synthesis of FOS. It was found that the enzyme still had 79.3% of initial activity after being reused for six consecutive cycles, whereas only 62.3% β-fructofuranosidase activity was detected in the parental strain ATCC 20611. Meanwhile, the FOS yield of FV1-11 after six consecutive cycles reached 57.1% (w/w), but only 51.0% FOS yield was detected in ATCC 20611.
CONCLUSIONS
The thermal-stable β-fructofuranosidase produced by A. niger can be recycled to achieve continuous synthesis of FOS with high efficiency, providing a powerful and economical strategy for the industrial production of FOS.
Topics: Aspergillus niger; Enzyme Stability; Fungal Proteins; Mycelium; Oligosaccharides; Promoter Regions, Genetic; Protein Engineering; Thermodynamics; beta-Fructofuranosidase
PubMed: 33575897
DOI: 10.1007/s10529-021-03099-w -
FEMS Microbiology Letters Dec 1995The accumulation and excretion of polyols was investigated under various growth conditions and at different stages of the life cycle of Aspergillus niger. Glycerol was...
The accumulation and excretion of polyols was investigated under various growth conditions and at different stages of the life cycle of Aspergillus niger. Glycerol was found to be the major solute in osmotic adjustment of the hyphae. Conidiospores contain large amounts of mannitol, which are rapidly metabolized during early germination, leading to the accumulation of glycerol. Glycerol is the major polyol in young mycelium, whereas in older mycelium mannitol and erythritol predominate. In all experiments, polyols were also excreted. The mechanism and function of this process is unknown, but it might be a way to control the levels of the intracellular polyol pools. Polyols are rapidly taken up again upon starvation. In a glycerol kinase mutant the synthesis of glycerol is unaffected but the excreted level of the polyol is higher. This glycerol is taken up again upon starvation, and accumulates intracellularly as it can not be metabolized further.
Topics: Aspergillus niger; Glycerol; Glycerol Kinase; Mutation; Sugar Alcohols; Trehalose
PubMed: 8593956
DOI: 10.1111/j.1574-6968.1995.tb07914.x -
Angewandte Chemie (International Ed. in... Mar 2019Enzymes exist as an ensemble of conformational states, whose populations can be shifted by substrate binding, allosteric interactions, but also by introducing mutations...
Enzymes exist as an ensemble of conformational states, whose populations can be shifted by substrate binding, allosteric interactions, but also by introducing mutations to their sequence. Tuning the populations of the enzyme conformational states through mutation enables evolution towards novel activity. Herein, Markov state models are used to unveil hidden conformational states of monoamine oxidase from Aspergillus niger (MAO-N). These hidden conformations, not previously observed by any other technique, play a crucial role in substrate binding and enzyme activity. This reveals how distal mutations regulate MAO-N activity by stabilizing these hidden, catalytically important conformational states, but also by modulating the communication pathway between both MAO-N subunits.
Topics: Aspergillosis; Aspergillus niger; Fungal Proteins; Humans; Markov Chains; Molecular Dynamics Simulation; Monoamine Oxidase; Protein Conformation; Substrate Specificity
PubMed: 30600584
DOI: 10.1002/anie.201812532 -
Acta Biochimica Polonica 1986Aspergillus niger produces two extracellular glucoamylases (GAI of Mr 85 300 and GAII of Mr 77 600) separable on DEAE-cellulose. The enzymes differes in electrophoretic...
Aspergillus niger produces two extracellular glucoamylases (GAI of Mr 85 300 and GAII of Mr 77 600) separable on DEAE-cellulose. The enzymes differes in electrophoretic mobility, thermostability and substrate specificity. The GAI/GAII ratio depends on the concentration and form of nitrogen (nitrate or ammonium) in the culture medium. Proteinase VIII from Bacillus subtilis converts GAI to a form showing properties similar to those of GAII. Possible proteolytic degradation of GAI to GAII by Asp. niger endogenous proteinase(s) is suggested.
Topics: Aspergillus niger; Culture Media; Glucan 1,4-alpha-Glucosidase; Glucosidases; Kinetics
PubMed: 3087124
DOI: No ID Found -
Journal of Hazardous Materials Jan 2024Environmental hormones have attracted more attention because of their adverse impact on the health and ecological security of human. Biodegradation is still an efficient...
Degradation profile of environmental pollutant 17β-estradiol by human intestinal fungus Aspergillus niger RG13B1 and characterization of genes involved in its degradation.
Environmental hormones have attracted more attention because of their adverse impact on the health and ecological security of human. Biodegradation is still an efficient tactics to remove environmental hormones, but human intestinal microbes remain to be elucidated in the role of their degradation. In the present work, we intended to perform the in vitro experiment for investigating the degradation of 17β-estradiol, the main environmental estrogen, by human intestinal microflora Aspergillus niger RG13B1. Its degradation led to the production of eighteen metabolites characterized by H, C, and 2D NMR, and HRMS spectra, including nine new (1-9) and nine known metabolites (10-18). Based on their structures, the degradation pathway of 17β-estradiol mediated by A. niger RG13B1 involved hydroxylation, oxidation, methylation, acetylation, and dehydrogenation, especially infrequent lactylation, and the key degradation enzymes were found in the gene cluster of A. niger. In addition, we found that metabolite 12 interacted with amino acid residues Lys37, Gln39, Lys93, and Asn115 of NF-κB p65 to suppress expressions of inflammatory genes or proteins, exerting its anti-inflammatory effect. This study first illustrated the role of human gut microbe in 17β-estradiol degradation and provided new insights into its degradation mechanism by A. niger RG13B1.
Topics: Humans; Aspergillus niger; Environmental Pollutants; Estradiol; Estrogens; Biodegradation, Environmental
PubMed: 37774607
DOI: 10.1016/j.jhazmat.2023.132617