-
MBio Jun 2021Protocatechuic acid (3,4-dihydroxybenzoic acid) is a chemical building block for polymers and plastics. In addition, protocatechuic acid has many properties of great...
Protocatechuic acid (3,4-dihydroxybenzoic acid) is a chemical building block for polymers and plastics. In addition, protocatechuic acid has many properties of great pharmaceutical interest. Much research has been performed in creating bacterial protocatechuic acid production strains, but no protocatechuic acid-producing fungal cell factories have been described. The filamentous fungus Aspergillus niger can produce protocatechuic acid as an intermediate of the benzoic acid metabolic pathway. Recently, the -hydroxybenzoate--hydroxylase () and protocatechuate 3,4-dioxygenase () of A. niger have been identified. It has been shown that the deletion mutant is still able to grow on protocatechuic acid. This led to the identification of an alternative pathway that converts protocatechuic acid to hydroxyquinol (1,3,4-trihydroxybenzene). However, the gene involved in the hydroxylation of protocatechuic acid to hydroxyquinol remained unidentified. Here, we describe the identification of protocatechuate hydroxylase (decarboxylating) (PhyA) by using whole-genome transcriptome data. The identification of enabled the creation of a fungal cell factory that is able to accumulate protocatechuic acid from benzyl alcohol, benzaldehyde, benzoic acid, caffeic acid, cinnamic acid, cinnamyl alcohol, hydroxybenzoic acid, -hydroxybenzyl alcohol, hydroxybenzaldehyde, -hydroxybenzoic acid, -anisyl alcohol, -anisaldehyde, -anisic acid, -coumaric acid, and protocatechuic aldehyde. Aromatic compounds have broad applications and are used in many industries, such as the cosmetic, food, fragrance, paint, plastic, pharmaceutical, and polymer industries. The majority of aromatic compounds are synthesized from fossil sources, which are becoming limited. Plant biomass is the most abundant renewable resource on Earth and can be utilized to produce chemical building blocks, fuels, and bioplastics through fermentations with genetically modified microorganisms. Therefore, knowledge about the metabolic pathways and the genes and enzymes involved is essential to create efficient strategies for producing valuable aromatic compounds such as protocatechuic acid. Protocatechuic acid has many pharmaceutical properties but also can be used as a chemical building block to produce polymers and plastics. Here, we show that the fungus Aspergillus niger can be engineered to produce protocatechuic acid from plant-derived aromatic compounds and contributes to creating alternative methods for the production of platform chemicals. .
Topics: Aspergillus niger; Hydroxybenzoates; Metabolic Networks and Pathways
PubMed: 34154420
DOI: 10.1128/mBio.00391-21 -
Scientific Reports Apr 2021Nuts are the natural source of healthy lipids, proteins, and omega-3. They are susceptible to fungal and mycotoxins contamination because of their high nutritional...
Nuts are the natural source of healthy lipids, proteins, and omega-3. They are susceptible to fungal and mycotoxins contamination because of their high nutritional value. Twenty-five species comprising 12 genera were isolated from 80 samples of dried fruits and nuts using the dilution plate method. Peanut recorded the highest level of contamination followed by coconut; almond and raisin were the lowest. Aspergillus was the most prevalent genus and A. niger, was the most dominant species. The morphological identification of the selected A. niger isolates as they were detected in high frequency of occurrence was confirmed by using 18SrRNA sequence. Ochratoxin biosynthesis gene Aopks was detected in the tested isolates. Lipase production by the selected A. niger isolates was determined with enzyme activity index (EAI) ranging from 2.02 to 3.28. A. niger-26 was the highest lipase producer with enzyme activity of 0.6 ± 0.1 U/ml by the trimetric method. Lip2 gene was also detected in the tested isolates. Finally, the antibacterial and antibiofilm efficiency of crude lipase against some human pathogens was monitored. Results exhibited great antibacterial efficacy with minimum bactericidal concentration (MBC) of 20 to 40 µl/100 µl against Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, and Methicillin-resistant Staphylococcus aureus (MRSA). Interestingly, significant anti-biofilm efficacy with inhibition percentages of 95.3, 74.9, 77.1 and 93.6% was observed against the tested pathogens, respectively.
Topics: Anti-Bacterial Agents; Aspergillus niger; Bacteria; Base Sequence; Biofilms; Biosynthetic Pathways; Humans; Lipase; Microbial Sensitivity Tests; Mycobiome; Nuts; Ochratoxins; Phylogeny; Virulence
PubMed: 33846447
DOI: 10.1038/s41598-021-87079-0 -
Biochimica Et Biophysica Acta.... Mar 2021As the largest membrane organelle, the endoplasmic reticulum (ER) is the main location for protein preliminary processing and phospholipid synthesis. Phospholipid...
As the largest membrane organelle, the endoplasmic reticulum (ER) is the main location for protein preliminary processing and phospholipid synthesis. Phospholipid bilayer is the main component of the ER, so it plays an intuitively important role in the steady state of protein synthesis in the ER. Despite of their importance, relationship between phospholipid homeostasis and protein processing in Aspergillus niger remains poorly understood. In this study, phosphatidyl ethanolamine (PE)/phosphatidyl choline (PC) and phosphatidyl acid (PA) metabolic mutants and ER protein processing mutants were established by knockout the key genes in phospholipid synthesis or UPR effector hacA. Based on global transcriptome and lipidome analysis, the relationship between the phospholipids imbalance and ER protein secretory imbalance was revealed as followed: The cells compensate for the damage caused by ER protein secretory deficiency or phospholipid deficiency from enhancing the protein processing and the synthesis of phospholipids at the transcription level, therefore phospholipid deficiency (Δopi3) and continuous activation of UPR (hacAi) have a synergistic effect in promoting protein secretion and phospholipid biosynthesis. At the same time, the metabolic deficiencies of phospholipid homeostasis and the processing deficiencies of ER protein will also cause cells sensitive to oxidative stress, cell wall inhibition and DNA damage.
Topics: Aspergillus niger; Endoplasmic Reticulum; Fungal Proteins; Phospholipids
PubMed: 33309775
DOI: 10.1016/j.bbamem.2020.183530 -
Microbial Cell Factories Feb 2019Citric acid is the world's largest consumed organic acid and is widely used in beverage, food and pharmaceutical industries. Aspergillus niger is the main industrial... (Review)
Review
Citric acid is the world's largest consumed organic acid and is widely used in beverage, food and pharmaceutical industries. Aspergillus niger is the main industrial workhorse for citric acid production. Since the release of the genome sequence, extensive multi-omic data are being rapidly obtained, which greatly boost our understanding of the citric acid accumulation mechanism in A. niger to a molecular and system level. Most recently, the rapid development of CRISPR/Cas9 system facilitates highly efficient genome-scale genetic perturbation in A. niger. In this review, we summarize the impact of systems biology on the citric acid molecular regulatory mechanisms, the advances in metabolic engineering strategies for enhancing citric acid production and discuss the development and application of CRISPR/Cas9 systems for genome editing in A. niger. We believe that future systems metabolic engineering efforts will redesign and engineer A. niger as a highly optimized cell factory for industrial citric acid production.
Topics: Aspergillus niger; CRISPR-Cas Systems; Citric Acid; Gene Editing; Genome, Fungal; Genomics; Industrial Microbiology; Metabolic Engineering; Systems Biology
PubMed: 30717739
DOI: 10.1186/s12934-019-1064-6 -
Bioresource Technology Jan 2022Xylitol is widely used in the food and pharmaceutical industries as a valuable commodity product. Biotechnological production of xylitol from lignocellulosic biomass by...
Xylitol is widely used in the food and pharmaceutical industries as a valuable commodity product. Biotechnological production of xylitol from lignocellulosic biomass by microorganisms is a promising alternative option to chemical synthesis or bioconversion from D-xylose. In this study, four metabolic mutants of Aspergillus niger were constructed and evaluated for xylitol accumulation from D-xylose and lignocellulosic biomass. All mutants had strongly increased xylitol production from pure D-xylose, beechwood xylan, wheat bran and cotton seed hulls compared to the reference strain, but not from several other feed stocks. The triple mutant ΔladAΔxdhAΔsdhA showed the best performance in xylitol production from wheat bran and cotton seed hulls. This study demonstrated the large potential of A. niger for xylitol production directly from lignocellulosic biomass by metabolic engineering.
Topics: Aspergillus niger; Biomass; Fermentation; Metabolic Engineering; Xylitol; Xylose
PubMed: 34710597
DOI: 10.1016/j.biortech.2021.126199 -
Scientific Reports Oct 2021β-Glucanase has received great attention in recent years regarding their potential biotechnological applications and antifungal activities. Herein, the specific...
β-Glucanase has received great attention in recent years regarding their potential biotechnological applications and antifungal activities. Herein, the specific objectives of the present study were to purify, characterize and immobilize β-glucanase from Aspergillus niger using covalent binding and cross linking techniques. The evaluation of β-glucanase in hydrolysis of different lignocellulosic wastes with subsequent bioethanol production and its capability in biocontrol of pathogenic fungi was investigated. Upon nutritional bioprocessing, β-glucanase production from A. niger EG-RE (MW390925.1) preferred ammonium nitrate and CMC as the best nitrogen and carbon sources, respectively. The soluble enzyme was purified by (NH)SO, DEAE-Cellulose and Sephadex G with 10.33-fold and specific activity of 379.1 U/mg protein. Tyrosyl, sulfhydryl, tryptophanyl and arginyl were essential residues for enzyme catalysis. The purified β-glucanase was immobilized on carrageenan and chitosan with appreciable yield. However, the cross-linked enzyme exhibited superior activity along with remarkable improved thermostability and operational stability. Remarkably, the application of the above biocatalyst proved to be a promising candidate in liberating the associate lignocellulosic reducing sugars, which was utilized for ethanol production by Saccharomyces cerevisiae. The purified β-glucanase revealed an inhibitory effect on the growth of two tested phytopathogens Fusarium oxysporum and Penicillium digitatum.
Topics: Antifungal Agents; Aspergillus niger; Biological Control Agents; Biotechnology; Enzymes, Immobilized; Ethanol; Fermentation; Glycoside Hydrolases; Microbial Sensitivity Tests; Phylogeny
PubMed: 34697353
DOI: 10.1038/s41598-021-00237-2 -
Antimicrobial Agents and Chemotherapy Oct 2011Aspergillus niger is a common clinical isolate. Multiple species comprise the Aspergillus section Nigri and are separable using sequence data. The antifungal...
Aspergillus niger is a common clinical isolate. Multiple species comprise the Aspergillus section Nigri and are separable using sequence data. The antifungal susceptibility of these cryptic species is not known. We determined the azole MICs of 50 black aspergilli, 45 from clinical specimens, using modified EUCAST (mEUCAST) and Etest methods. Phylogenetic trees were prepared using the internal transcribed spacer, beta-tubulin, and calmodulin sequences to identify strains to species level and the results were compared with those obtained with cyp51A sequences. We attempted to correlate cyp51A mutations with azole resistance. Etest MICs were significantly different from mEUCAST MICs (P < 0.001), with geometric means of 0.77 and 2.79 mg/liter, respectively. Twenty-six of 50 (52%) isolates were itraconazole resistant by mEUCAST (MICs > 8 mg/liter), with limited cross-resistance to other azoles. Using combined beta-tubulin/calmodulin sequences, the 45 clinical isolates grouped into 5 clades, A. awamori (55.6%), A. tubingensis (17.8%), A. niger (13.3%), A. acidus (6.7%), and an unknown group (6.7%), none of which were morphologically distinguishable. Itraconazole resistance was found in 36% of the isolates in the A. awamori group, 90% of the A. tubingensis group, 33% of the A. niger group, 100% of the A. acidus group, and 67% of the unknown group. These data suggest that cyp51A mutations in section Nigri may not play as important a role in azole resistance as in A. fumigatus, although some mutations (G427S, K97T) warrant further study. Numerous cryptic species are found in clinical isolates of the Aspergillus section Nigri and are best reported as "A. niger complex" by clinical laboratories. Itraconazole resistance was common in this data set, but azole cross-resistance was unusual. The mechanism of resistance remains obscure.
Topics: Antifungal Agents; Aspergillosis; Aspergillus niger; Azoles; Base Sequence; Calmodulin; Cytochrome P-450 Enzyme System; DNA, Intergenic; Drug Resistance, Fungal; Fungal Proteins; Itraconazole; Microbial Sensitivity Tests; Mutation; Phylogeny; Sequence Alignment; Sequence Analysis, DNA; Tubulin
PubMed: 21768508
DOI: 10.1128/AAC.00304-11 -
MBio Feb 2023The fungus Aspergillus niger is among the most abundant fungi in the world and is widely used as a cell factory for protein and metabolite production. This fungus forms...
The fungus Aspergillus niger is among the most abundant fungi in the world and is widely used as a cell factory for protein and metabolite production. This fungus forms asexual spores called conidia that are used for dispersal. Notably, part of the spores and germlings aggregate in an aqueous environment. The aggregated conidia/germlings give rise to large microcolonies, while the nonaggregated spores/germlings result in small microcolonies. Here, it is shown that small microcolonies release a larger variety and quantity of secreted proteins compared to large microcolonies. Yet, the secretome of large microcolonies has complementary cellulase activity with that of the small microcolonies. Also, large microcolonies are more resistant to heat and oxidative stress compared to small microcolonies, which is partly explained by the presence of nongerminated spores in the core of the large microcolonies. Together, it is proposed that heterogeneity in germination and aggregation has evolved to form a population of different sized A. niger microcolonies, thereby increasing stress survival and producing a meta-secretome more optimally suited to degrade complex substrates. Aspergillus niger can form microcolonies of different size due to partial aggregation of spores and germlings. So far, this heterogeneity was considered a negative trait by the industry. We here, however, show that heterogeneity in size within a population of microcolonies is beneficial for food degradation and stress survival. This functional heterogeneity is not only of interest for the industry to make blends of enzymes (e.g., for biofuel or bioplastic production) but could also play a role in nature for effective nutrient cycling and survival of the fungus.
Topics: Aspergillus niger; Spores, Fungal; Hot Temperature; Fungal Proteins; Water
PubMed: 36629410
DOI: 10.1128/mbio.00870-22 -
Fungal Genetics and Biology : FG & B May 2017In both natural and man-made environments, microorganisms live in mixed populations, while in laboratory conditions monocultures are mainly used. Microbial interactions...
In both natural and man-made environments, microorganisms live in mixed populations, while in laboratory conditions monocultures are mainly used. Microbial interactions are often described as antagonistic, but can also be neutral or cooperative, and are generally associated with a metabolic change of each partner and cause a change in the pattern of produced bioactive molecules. A. niger and A. oryzae are two filamentous fungi widely used in industry to produce various enzymes (e.g. pectinases, amylases) and metabolites (e.g. citric acid). The co-cultivation of these two fungi in wheat bran showed an equal distribution of the two strains forming mixed colonies with a broad range of carbohydrate active enzymes produced. This stable mixed microbial system seems suitable for subsequent commercial processes such as enzyme production. XlnR knock-out strains for both aspergilli were used to study the influence of plant cell wall degrading enzyme production on the fitness of the mixed culture. Microscopic observation correlated with quantitative PCR and proteomic data suggest that the XlnR Knock-out strain benefit from the release of sugars by the wild type strain to support its growth.
Topics: Aspergillus niger; Aspergillus oryzae; Coculture Techniques; Dietary Fiber; Enzymes; Fermentation; Fungal Proteins; Microbial Interactions; Proteomics; Trans-Activators
PubMed: 28232095
DOI: 10.1016/j.fgb.2017.02.006 -
International Journal of Food... Feb 2019Ochratoxin A (OTA) is a nephrotoxic mycotoxin which may contaminate various foods and feed products worldwide. Aspergillus niger is one of the species responsible for...
Ochratoxin A (OTA) is a nephrotoxic mycotoxin which may contaminate various foods and feed products worldwide. Aspergillus niger is one of the species responsible for OTA contamination in grapes and derived products. This species has recently been split into A. niger and Aspergillus welwitschiae. Both species can not be distinguished by phenotypic or extrolite profiles and to date there is no ecophysiological information of A. welwitschiae. The aim of this study was to determine the effects of water activity (a) (0.90; 0.95 and 0.98-0.99), culture media (Yeast Extract Sucrose Broth (YESB); Synthetic Grape Juice Medium (SGM); White grape juice (WGJ)) and temperature (15 °C, 25 °C and 35 °C) on the growth and OTA production of four strains of A. niger and six strains of A. welwitschiae. The assay was performed in microtiter plates, determining the absorbance at 530 nm and the concentration of OTA at 1, 2, 4 and 10 days. No significant differences were observed in absorbance and OTA values between the two species under study. The highest absorbance values were recorded in YESB, followed by SGM and WGJ. Absorbance values increased with increasing a and temperature. The highest OTA values were obtained at 0.98-0.99 a and the best culture media for OTA production was YESB, followed by WGJ and SGM. The studied strains of A. niger produced the highest mean OTA level at 25 °C whereas A. welwitschiae strains produced the highest mean OTA concentration at 15 °C, although not differing significantly from concentration produced at 25 °C. To our knowledge, this is the first report on the impact of some environmental factors on growth and OTA production by A. welwitschiae.
Topics: Aspergillus; Aspergillus niger; Culture Media; Environment; Food Microbiology; Ochratoxins; Temperature; Vitis; Water
PubMed: 30419474
DOI: 10.1016/j.ijfoodmicro.2018.11.001