Aspergillus Niger Gene Research Articles

Functional analysis of FlbA-regulated transcription factor genes in Aspergillus niger using a multiplexed CRISPRoff system

FlbA of Aspergillus niger (indirectly) regulates 36 transcription factor (TF) genes. As a result, it promotes sporulation and represses vegetative growth, protein secretion and lysis. In this study, the functions of part of the FlbA-regulated TF genes were studied by using CRISPRoff. This system was recently introduced as an epigenetic tool for modulating gene expression in A. niger. A plasmid encompassing an optimized CRISPRoff system as well as a library of sgRNA genes that target the promoters of the 36 FlbA-regulated TF genes was introduced in A. niger. Out of 24 transformants that exhibited a sporulation phenotype, 12 and 18 strains also showed a biomass and secretion phenotype, respectively. The transforming sgRNAs, and thus the genes responsible for the phenotypes, were identified from five of the transformants. The results show that the genes dofA, dofB, dofC, dofD, and socA are involved in sporulation and extracellular enzyme activity, while dofA and socA also play roles in biomass formation. Overall, this study shows that the multiplexed CRISPRoff system can be effectively used for functional analysis of genes in a fungus.

Alternative splicing analysis of lignocellulose-degrading enzyme genes and enzyme variants in Aspergillus niger

Alternative splicing (AS) greatly expands the protein diversity in eukaryotes. Although AS variants have been frequently reported existing in filamentous fungi, it remains unclear whether lignocellulose-degrading enzyme genes in industrially important fungi undergo AS events. In this work, AS events of lignocellulose-degrading enzymes genes in Aspergillus niger under two carbon sources (glucose and wheat straw) were investigated by RNA-Seq. The results showed that a total of 23 out of the 56 lignocellulose-degrading enzyme genes had AS events and intron retention was the main type of these AS events. The AS variant enzymes from the annotated endo-β-1,4-xylanase F1 gene (xynF1) and the endo-β-1,4-glucanase d gene (eglD), noted as XYNF1-AS and EGLD-AS, were characterized compared to their normal splicing products XYNF1 and EGLD, respectively. The AS variant XYNF1-AS displayed xylanase activity whereas XYNF1 did not. As for EGLD-AS and EGLD, neither of them showed annotated endo-β-1,4-glucanase activity. Instead, both showed lytic polysaccharide monooxygenase (LPMO) activity with some differences in catalytic properties. Our work demonstrated that the AS variants in A. niger were good sources for discovering novel lignocellulose-degrading enzymes.Key points• AS events were identified in the lignocellulose-degrading enzyme genes of A. niger.• New β-1,4-xylanase and LPMO derived from AS events were characterized.

Aspergillus oryzae PrtR alters transcription of individual peptidase genes in response to the growth environment

Aspergillus oryzae PrtR is an ortholog of the transcription factor PrtT, which positively regulates the transcription of extracellular peptidase genes in Aspergillus niger and Aspergillus fumigatus. To identify the genes under the control of PrtR and elucidate its regulatory mechanism in A. oryzae, prtR gene disruption mutants were generated. The control strain clearly showed a halo on media containing skim milk as the nitrogen source, whereas the ΔprtR strain formed a smaller halo. Measurement of acid peptidase activity revealed that approximately 84% of acidic endopeptidase and 86% of carboxypeptidase activities are positively regulated by PrtR. As the transcription of the prtR gene varied depending on culture conditions, especially with or without a protein substrate, it was considered that its transcription would be regulated in response to a nitrogen source. In addition, contrary to previous expectations, PrtR was found to act both in promoting and repressing the transcription of extracellular peptidase genes. The mode of regulation varied from gene to gene. Some genes were regulated in the same manner in both liquid and solid cultures, whereas others were regulated in different ways depending on the culture conditions. Furthermore, PrtR has been suggested to regulate the transcription of peptidase genes that are closely associated with other transcription factors.Key points• Almost all peptidase genes in Aspergillus oryzae are positively regulated by PrtR• However, several genes are regulated negatively by PrtR• PrtR optimizes transcription of peptidase genes in response to culture conditions

CRISPR/Cas9-mediated Inactivation of the Pectinase Gene in Aspergillus niger and Evaluation of the Mutant Strain

CRISPR/Cas9-mediated Inactivation of the Pectinase Gene in Aspergillus niger and Evaluation of the Mutant Strain

Enhanced in vitro anticancer activity of yeast expressed recombinant glucose oxidase versus commercial enzyme.

Cancer treatments continue to have many disadvantages. Reactive oxygen species, such as H2O2, in high concentrations, can cause cytotoxicity to cells, being even greater in cancer cells. One of the H2O2-producing enzymes is glucose oxidase; its application in cancer treatment should be explored. In this work, the extracellular expression of the mutated recombinant enzyme glucose oxidase was carried out in the eukaryotic expression system Pichia pastoris SMD1168, through the modification and optimization of the gox gene of Aspergillus niger to improve its expression in yeast and its purification. Also, the secretion signal of the alpha-mating factor from Saccharomyces cerevisiae was added to the gene for extracellular expression, and it was inserted into the expression vector pPIC3.5k. The extracellular expression of the enzyme facilitated purification by anion exchange chromatography; the purification was corroborated by SDS-PAGE, with a molecular weight of its subunit between 63 kDa and 100 kDa. The mutated recombinant enzyme glucose oxidase showed greater anticancer activity compared to the commercial glucose oxidase and could have potential for cancer treatment. KEY POINTS: • Pichia pastoris is an excellent eukaryotic expression system for proteins that need post-translational modifications. • Extracellular expression facilitates protein purification. • Glucose oxidase has potential application in cancer treatment.

Expression of CAS9 Nuclease of the CRISPR/Cas Genome Editing System in the Filamentous Fungus Penicillium verruculosum

The cas9 gene of the Streptococcus pyogenes bacterium, which encodes Cas9 nuclease of the class 2 CRISPR/Cas system, is expressed in the filamentous fungus Penicillium verruculosum under the control of the constitutive autologous promoter of the glyceraldehyde-3-phosphate dehydrogenase gpdA gene, which is cloned and sequenced for the first time in the present work. The functionality of the gpdA promoter is confirmed by the expression of the heterologous β-glucosidase gene of Aspergillus niger in P. verruculosum. The relative copy number and expression level of the cas9 gene in recombinant strains are determined by real-time PCR. In order to detect and determine the localization of Cas9 nuclease in P. verruculosum fungal cells, the chimeric Cas9 nuclease with the fluorescent eGFP protein added to the C-terminus was obtained. The fluorescence microscopy of the growing mycelium confirms the intracellular expression of the chimeric construct in the cells of recombinant strains.

Genetic transformation of tropical maize (Zea mays L.) inbred line with a phytase gene from Aspergillus niger.

A full-length cDNA of phyA gene of Aspergillus niger, encoding phytase enzyme, was cloned and expressed in E. coli BL21 cells and assayed for its activity. The phyA cDNA consisted of 1404bp, which encoded 467 amino acid residues. The phytase activity of purified phytase was 826.33U/mL. The phyA gene under the control of endosperm-specific promoters was transformed into an Indian maize inbred line, UMI29, using particle bombardment-mediated transformation method to generate transgenic maize plants over-expressing phytase in seeds. PCR and GUS analyses demonstrated the presence of transgenes in T0 transgenic plants and their stable inheritance in the T1 progenies. Three transgenic events expressing detectable level of A. niger phytase were characterized by western blot analysis. Phytase activity of 463.158U/kg of seed was observed in one of the events, JB-UMI29-Z17/2. The phytase activity of transgenic maize seeds was 5.5- to 7-fold higher than the wild-type UMI29 seeds and, consequently, the seeds had 0.6- to 5-fold higher inorganic phosphorus content.

The mitochondrial citrate carrier in Yarrowia lipolytica: Its identification, characterization and functional significance for the production of citric acid

Mitochondrial citrate carrier plays a central role in exporting acetyl-CoA in the form of citrate from mitochondria to cytosol thereby connecting carbohydrate catabolism and lipogenesis. In this study, Yarrowia lipolytica mitochondrial citrate carrier was functionally defined and characterized. Firstly, deletion of Y. lipolytica YlCTP1 and YlYHM2 genes coding putative tricarboxylate mitochondrial carriers were performed. ΔYlctp1 strain did not differ significantly from wild type strain in terms of growth rate, organic acids and lipid production. In contrast, ΔYlyhm2 strain did not grow in liquid citrate-containing minimal medium. Moreover, in glucose-containing lipogenic medium YlYHM2 null mutant strain did not produce citric acid; the production of isocitric acid and lipids were decreased. Reintroduction of YlYHM2 gene as well as heterologous expression of Aspergillus niger gene AnYHM2 into ΔYlyhm2 strain restored the growth in minimal citrate medium and even enhanced citric acid production by 45% in both variants compared with wild type strain during test tube cultivation. Mitochondrial extracts isolated from YlYHM2 null mutant and wild type strain were incorporated into liposomes; citrate/citrate and α-ketoglutarate/α-ketoglutarate homoexchange activities were reduced by 87% and 40% in ΔYlyhm2 strain, respectively, compared with the wild type, whereas citratein/α-ketoglutarateout and α-ketoglutaratein/citrateout heteroexchanges were decreased by 87% and 95%, respectively. YlYhm2p was expressed in Escherichia coli, purified and reconstituted into liposomes. Besides high efficiency to citrate and α-ketoglutarate transport, YlYhm2p also transported oxaloacetate, succinate, fumarate, and to a much lesser extent, aconitate, malate, isocitrate, oxoadipate, and glutamate. The activity of reconstituted YlYhm2p was inhibited strongly by SH-blocking reagents, pyridoxal-5′-phosphate, and partly by N-ethylmaleimide. Co-expression of YlYHM2 and adenosine monophosphate deaminase YlAMPD genes resulted in the production of 49.7 g/L of citric acid during test tube cultivation, whereas wild type strain accumulated 30.1 g/L of citric acid. Large-scale cultivation in bioreactor of the engineered strain resulted in 97.1 g/L of citric acid production with a process selectivity of 94.2% and an overall citric acid yield of 0.5 g/g. The maximal specific rate of citric acid synthesis was 0.93 g/L/h. Therefore, the physiological role of YlYhm2p in glucose-containing medium is to catalyze both import of citrate into mitochondria for catabolic reactions and export of citrate as a source of acetyl-CoA from mitochondria. Possible shuttles for citrate exporting are discussed. Moreover, for the first time evidence has been given for the improvement of TCA cycle intermediate production by manipulation of a gene coding a mitochondrial carrier.

Correction to: Mutations in AraR leading to constitutive expression of arabinolytic genes in Aspergillus niger under derepressing conditions.

The correct title is: Mutations in AraR leading to constitutive expression of arabinolytic genes in Aspergillus niger under derepressing conditions.

Mutations in AraR leading to constitutive of arabinolytic genes in Aspergillus niger under derepressing conditions

The AraR transcription factor of Aspergillus niger encodes a Zn(II)2Cys6 transcription factor required for the induction of genes encoding arabinolytic enzymes. One of the target genes of AraR is abfA, encoding an arabinofuranosidase. The expression of abfA as well as other L-arabinose-induced genes in A. niger requires the presence of L-arabinose or its derivative L-arabitol as an inducer to activate AraR-dependant gene expression. In this study, mutants were isolated that express L-arabinose-induced genes independently of the presence of an inducer under derepressing conditions. To obtain these mutants, a reporter strain was constructed in a ΔcreA background containing the L-arabinose-responsive promoter (PabfA) fused to the acetamidase (amdS) gene. Spores of the ΔcreA PabfA-amdS reporter strain were UV-mutagenized and mutants were obtained by their ability to grow on acetamide without the presence of inducer. From a total of 164 mutants, 15 mutants were identified to contain transacting mutations resulting in high arabinofuranosidase activity in the medium after growth under non-inducing conditions. Sequencing of the araR gene of the 15 constitutive mutants revealed that 14 mutants carried a mutation in AraR. Some mutations were found more than once and in total nine different point mutations were identified in AraR. The AraRN806I point mutation was reintroduced into a parental strain and confirmed that this point mutation leads to inducer-independent expression of AraR target genes. The inducer independent of L-arabinose-induced genes in the AraRN806I mutant was found to be sensitive to carbon catabolite repression, indicating that the CreA-mediated carbon catabolite repression is dominant over the AraRN806I mutant allele. These mutations in AraR provide new opportunities to improve arabinase production in industrial fungal strains.

Genome Wide Identification and Analysis of <i>TRK</i> Gene Family in <i>Aspergillus niger</i

TRK gene is related to potassium uptake and transport, which plays an important role in regulating the life activity of living organisms. Based on Aspergillus niger genome database, using bioinformatic method predicted the members of TRK gene family, gene structure and phylogenetic relationships of Aspergillus nig er. The phylogenetic tree was constructed using MEGA 6.0.6 software with Maximum Likelihood Tree method. Using PAL2NAL software estimated da/ds. The structure of the genes was analyzed by GSDS 2.0 software. The motif was analyzed by MEME program. Four members of TRK gene family in the genome of Aspergillus niger were identified, which were AnTrk1 , AnTrk2 , AnTrk3 and AnTrk4 , respectively. Both the TRK feature domain and the conserved motif were found. The structure of different genes was different, but the structure of cluster was similar. TRK gene family members of Aspergillus niger in the genetic structure and other aspects of existed significant differences with the yeast. Genetic relationships and protein interaction network analysis have further verified this conclusion. Therefore, it can infer that TRK gene of Aspergillus niger has other regulation pathways. This conclusion could provide the basis for further research on the function and mechanism of TRK osmotic stress related protein, so as to lay a foundation for understanding the mechanism of the osmotic adjustment of Aspergillus niger .

Synthesis of Isomaltooligosaccharides by Saccharomyces cerevisiae Cells Expressing Aspergillus niger α-Glucosidase

The α-glucosidase encoded by the aglA gene of Aspergillus niger is a secreted enzyme belonging to family 31 of glycoside hydrolases. This enzyme has a retaining mechanism of action and displays transglycosylating activity that makes it amenable to be used for the synthesis of isomaltooligosaccharides (IMOs). We have expressed the aglA gene in Saccharomyces cerevisiae under control of a galactose-inducible promoter. Recombinant yeast cells expressing the aglA gene produced extracellular α-glucosidase activity about half of which appeared cell bound whereas the other half was released into the culture medium. With maltose as the substrate, panose is the main transglycosylation product after 8 h of incubation, whereas isomaltose is predominant after 24 h. Isomaltose also becomes predominant at shorter times if a mixture of maltose and glucose is used instead of maltose. To facilitate IMO production, we have designed a procedure by which yeast cells can be used directly as the catalytic agent. For this purpose, we expressed in S. cerevisiae gene constructs in which the aglA gene is fused to glycosylphosphatidylinositol anchor sequences, from the yeast SED1 gene, that determine the covalent binding of the hybrid protein to the cell membrane. The resulting hybrid enzymes were stably attached to the cell surface. The cells from cultures of recombinant yeast strains expressing aglA-SED1 constructions can be used to produce IMOs in successive batches.

Life cycle assessment of β-Galactosidase enzyme production

Many consumers are unable to enjoy the benefits of milk due to intolerance or even allergy to lactose. This inability to digest lactose is caused by a deficiency of the enzyme β-Galactosidase in the digestive tract, which performs the hydrolysis of lactose to form glucose and galactose, two sugars which are easily absorbed into the bloodstream. This study reports the Life Cycle Assessment (LCA) outcomes of the β-Galactosidase production in an industrial -scale facility from a cradle-to-gate perspective. Among the different alternatives of the production process of the β-Galactosidase enzyme, the use of recombinant Saccharomyces cerevisiae yeast expressing the lacA gene of Aspergillus niger was considered due to its higher production yield in comparison with wild strains. The primary source of environmental impact is the downstream processing related subsystem, specially attributed to the separation, concentration and purification of the enzyme as well as Cleaning-in-Place activities, required to maintain aseptic and sterile operational conditions. The use of alternative chemicals to the conventional ones in this latter stage could imply environmental improvements of the production process.

Engineering a chimeric acid-stable α-amylase-glucoamylase (Amy-Glu) for one step starch saccharification

For saccharifying starch in one step, a chimeric biocatalyst (Amy-Glu) was generated from engineered α-amylase (Ba-Gt-amy) of Bacillus acidicola and glucoamylase (Glu) gene of Aspergillus niger. In order to join two enzymes, a linker peptide of 25 amino acids was used. Chimeric Amy-Glu was expressed in E. coli. Glu is of 75kDa, while Amy-Glu is of 145kDa. Both Amy-Glu and Glu displayed similar pH profile with good activity in the acidic pH range like that of Ba-Gt-amy with optimum at pH 4.0. All three enzymes (Ba-Gt-amy, Amy-Glu and glucoamylase) exhibited activity in the temperature range between 40 and 70°C with optimum at 60°C. Amy-Glu and Glu have T1/2 of 90 and 70min at 60 and 70°C, respectively. The Km, Vmax and Kcat values of Glu (soluble starch) are 0.34mgmL−1, 606μmolmg−1min−1 and 727s−1, while for Amy-Glu are 0.84mgmL−1, 13,886μmolmg−1min−1 and 4.2×104s−1, respectively. The end product analysis suggested that Amy-Glu retains the activity of both parental enzymes and forms maltodextrins along with glucose as the major products. Amy-Glu saccharifies wheat and corn starches more efficiently than the Ba-Gt-amy and glucoamylase.

Cloning & expression of glucose oxidase fromAspergillusniger

Our aims is to clone the Glucose oxidase (GOx) gene which has been isolated from Aspergillus niger, in turn isolated from the fertile garden soil. Detection of the expression of gox gene in prokaryotic host. Aspergillus niger has been isolated from soil samples taken from Navsari region. Enzyme assay has been done to check enzyme. DNA extraction & PCR amplification was done using Banglore GeNei TM kits. 90% success has been achieved by obtaining the GOx protein from Escherichia coli BL21 in inclusion bodies. The protein has been purified onto Ni-NTA column. gox gene was successfully isolated and expressed in prokaryotic host, which would be purified by simple procedure. –We conclude that the by-product of gluconic acid fermentation has a great industrial importance. So, for greater production of GOx, the gene can be transferred from donor to the recipient. The gox gene of Aspergillus niger has been isolated and studied for the identification & its expression for improvement of enzyme production by establishing a process based on heterologous expression.

The Establishment of Real-Time PCR Detection System of Aspergillus Niger in Peanut

In this study, a set of real-time PCR primers for the detection of aflatoxin in peanut was successfully designed according to the AFLR gene of Aspergillus niger. Upstream primer was 5'-AACCTGATGACGACTGATAT-3'; Downstream primer was 5'-AGCACCTTGAGAACGATAA-3'; The real-time PCR detection system established in this study had good specificity, and the sensitivity of DNA detection can reach 3×10-13 ng/ul. The correlation coefficient between the contents of aflatoxin in peanut samples and PCR Ct of Real-time was-0.718, and the correlation coefficient was significantly negative.

The interaction of induction and repression mechanisms in the regulation of galacturonic acid-induced genes in Aspergillus niger

Aspergillus niger is an important industrial fungus expressing a broad spectrum of pectinolytic genes. The main constituent of pectin, polygalacturonic acid (PGA), is degraded into galacturonic acid (GA) by the combined activity of endo- and exo-polygalacturonases some of which are specifically induced by GA. The regulatory mechanisms that control the expression of genes encoding PGA-degrading enzymes are not well understood. Based on available genome-wide expression profiles from literature, we selected five genes that were specifically induced by GA. These genes include three exo-polygalacturonases (pgaX, pgxB and pgxC), a GA transporter (gatA), and an intracellular enzyme involved in GA metabolism (gaaB). These five genes contain a conserved motif (5′-TCCNCCAAT-3′) in their promoter regions, which we named GARE (galacturonic acid-responsive element). Promoter deletion studies and site-directed mutagenesis of the conserved motif of the pgaX gene showed that the conserved element is required for GA-mediated induction. A set of promoter reporter strains was constructed by fusing the promoter region of the five above-mentioned genes to the amdS reporter gene. Expression of the amdS gene is quantitatively correlated with ability to utilise acetamide as an N-source, hence higher expression of amdS improves growth of the strain on acetamide and therefore can be used as an in vivo reporter for gene expression. Growth analysis of the reporter strains indicated that four genes (pgaX, pgxB, pgxC, and gatA) are specifically induced by GA. The in vivo promoter reporter strains were also used to monitor carbon catabolite repression control. Except for gaaB, all promoter–reporter genes analysed were repressed by glucose in a glucose concentration-dependent way. Interestingly, the strength of glucose repression was different for the tested promoters. CreA is important in mediating carbon catabolite repression as deletion of the creA gene in the reporter strains abolished carbon catabolite repression for most promoters. Interestingly, the pgxC promoter was still repressed by glucose even in the creA null background, suggesting a role for alternative repression mechanisms. Finally, we showed that low concentrations of GA are required to induce gene expression of pgaX, pgxB, and pgxC even under derepressing conditions. The results obtained are consistent with a model in which a GA-specific transcription factor is activated by GA or a GA-derivative, which binds to the conserved motif, possibly in combination with the HAP-complex, to drive GA-specific gene expression.

Generation of Oxalic Acid Hyperproducers by Overexpressing the Oxaloacetate Hydrolase Gene in Aspergillus niger

Generation of Oxalic Acid Hyperproducers by Overexpressing the Oxaloacetate Hydrolase Gene in Aspergillus niger

Production of the Phanerochaete flavido-alba laccase in Aspergillus niger for synthetic dyes decolorization and biotransformation

We investigated the expression of Phanerochaete flavido-alba laccase gene in Aspergillus niger and the physical and biochemical properties of the recombinant enzyme (rLac-LPFA) in order to test it for synthetic dye biotransformation. A. niger was able to produce high levels of active recombinant enzyme (30mgL(-1)), whose identity was further confirmed by immunodetection using Western blot analysis and N-terminal sequencing. Interestingly, rLac-LPFA exhibited an improved stability at pH (2-9) and organic solvents tested. Furthermore, the percentage of decoloration and biotransformation of synthetic textile dyes, Remazol Brilliant Blue R (RBBR) and Acid Red 299 (NY1), was higher than for the native enzyme. Its high production, simple purification, high activity, stability and ability to transform textile dyes make rLac-LPFA a good candidate for industrial applications.

L-xylo-3-Hexulose Reductase Is the Missing Link in the Oxidoreductive Pathway for d-Galactose Catabolism in Filamentous Fungi

In addition to the well established Leloir pathway for the catabolism of d-galactose in fungi, the oxidoreductive pathway has been recently identified. In this oxidoreductive pathway, D-galactose is converted via a series of NADPH-dependent reductions and NAD(+)-dependent oxidations into D-fructose. The pathway intermediates include galactitol, L-xylo-3-hexulose, and d-sorbitol. This study identified the missing link in the pathway, the L-xylo-3-hexulose reductase that catalyzes the conversion of L-xylo-3-hexulose to D-sorbitol. In Trichoderma reesei (Hypocrea jecorina) and Aspergillus niger, we identified the genes lxr4 and xhrA, respectively, that encode the l-xylo-3-hexulose reductases. The deletion of these genes resulted in no growth on galactitol and in reduced growth on D-galactose. The LXR4 was heterologously expressed, and the purified protein showed high specificity for L-xylo-3-hexulose with a K(m) = 2.0 ± 0.5 mm and a V(max) = 5.5 ± 1.0 units/mg. We also confirmed that the product of the LXR4 reaction is D-sorbitol.