The Aspergillus niger multicopper oxidase family: analysis and overexpression of laccase-like encoding genes

<p>Abstract</p> <p>Background</p> <p>Many filamentous fungal genomes contain complex groups of multicopper oxidase (MCO) coding genes that makes them a good source for new laccases with potential biotechnological interest. A bioinformatics analysis of the <it>Aspergillus niger </it>ATCC 1015 genome resulted in the identification of thirteen MCO genes. Ten of them were cloned and homologously overexpressed.</p> <p>Results</p> <p>A bioinformatic analysis of the <it>A. niger </it>ATCC 1015 genome revealed the presence of 13 MCO genes belonging to three different subfamilies on the basis of their phylogenetic relationships: ascomycete laccases, fungal pigment MCOs and fungal ferroxidases. According to <it>in silico </it>amino acid sequence analysis, the putative genes encoding for functional extracellular laccases (<it>mcoA</it>, <it>mcoB</it>, <it>mcoC</it>, <it>mcoD</it>, <it>mcoE</it>, <it>mcoF</it>, <it>mcoG</it>, <it>mcoI</it>, <it>mcoJ </it>and <it>mcoM</it>) were placed under the control of the <it>glaA </it>promoter and overexpressed in <it>A. niger </it>N593. Enzyme activity plate assays with several common laccase substrates showed that all genes are actually expressed and code for active MCOs. Interestingly, expressed enzymes show different substrate specificities. In addition, optimization of fungal pigment MCOs extracellular production was investigated. The performance of the widely used glucoamylase signal sequence (ssGlaA) in McoA secretion was studied. Results obtained suggest that ssGlaA do not yield higher levels of secreted McoA when compared to its native secretion signal. Also, McoB synthesis was investigated using different nitrogen sources in minimal medium liquid cultures. Higher yields of extracellular McoB were achieved with (NH₄)₂tartrate.</p> <p>Conclusions</p> <p><it>Aspergillus niger </it>is a good source of new laccases. The different substrate specificity observed in plate assays makes them interesting to be purified and biochemically compared. The homologous signal sequence of McoA has been shown to be a good choice for its extracellular overexpression. From the nitrogen sources tested (NH₄)₂tartrate has been found to be the most appropriate for McoB production in <it>A. niger</it>.</p

Modeling and analysis of the dynamic behavior of the XlnR regulon in Aspergillus niger

Background: In this paper the dynamics of the transcription-translation system for XlnR regulon in Aspergillus niger is modeled. The model is based on Hill regulation functions and uses ordinary differential equations. The network response to a trigger of D-xylose is considered and stability analysis is performed. The activating, repressive feedback, and the combined effect of the two feedbacks on the network behavior are analyzed. Results: Simulation and systems analysis showed significant influence of activating and repressing feedback on metabolite expression profiles. The dynamics of the D-xylose input function has an important effect on the profiles of the individual metabolite concentrations. Variation of the time delay in the feedback loop has no significant effect on the pattern of the response. The stability and existence of oscillatory behavior depends on which proteins are involved in the feedback loop. Conclusions: The dynamics in the regulation properties of the network are dictated mainly by the transcription and translation degradation rate parameters, and by the D-xylose consumption profile. This holds true with and without feedback in the network. Feedback was found to significantly influence the expression dynamics of genes and proteins. Feedback increases the metabolite abundance, changes the steady state values, alters the time trajectories and affects the response oscillatory behavior and stability conditions. The modeling approach provides insight into network behavioral dynamics particularly for small-sized networks. The analysis of the network dynamics has provided useful information for experimental design for future in vitro experimental wor

Aspergillus niger Protein EstA Defines a New Class of Fungal Esterases within the α/β Hydrolase Fold Superfamily of Proteins

AbstractFrom the fungus Aspergillus niger, we identified a new gene encoding protein EstA, a member of the α/β-hydrolase fold superfamily but of unknown substrate specificity. EstA was overexpressed and its crystal structure was solved by molecular replacement using a lipase-acetylcholinesterase chimera template. The 2.1 Å resolution structure of EstA reveals a canonical Ser/Glu/His catalytic triad located in a small pocket at the bottom of a large solvent-accessible, bowl-shaped cavity. Potential substrates selected by manual docking procedures were assayed for EstA activity. Consistent with the pocket geometry, preference for hydrolysis of short acyl/propyl chain substrates was found. Identification of close homologs from the genome of other fungi, of which some are broad host-range pathogens, defines EstA as the first member of a novel class of fungal esterases within the superfamily. Hence the structure of EstA constitutes a lead template in the design of new antifungal agents directed toward its pathogenic homologs

До питання партійної діяльності М.І. Міхновського на початку ХХ ст.

Необхідність створення українських політичних партій на початку ХХ ст. відчувалася дуже гостро. Поставала потреба формування нової політичної партії, яка б стояла на самостійницьких засадах. З ініціативи М.І.Міхновського виникає Українська Народна Партія. Головним завданням політичної організації М.І.Міхновський вбачав в об’єднанні усіх, «кого визискують і кривдять». До цих верств політичний діяч відносив робітників, селян, ремісників, представників інтелігентних фахів, хліборобів

Proteomic Analysis of the Secretory Response of Aspergillus niger to D-Maltose and D-Xylose

Fungi utilize polysaccharide substrates through extracellular digestion catalyzed by secreted enzymes. Thus far, protein secretion by the filamentous fungus Aspergillus niger has mainly been studied at the level of individual proteins and by genome and transcriptome analyses. To extend these studies, a complementary proteomics approach was applied with the aim to investigate the changes in secretome and microsomal protein composition resulting from a shift to a high level secretion condition. During growth of A. niger on d-sorbitol, small amounts of d-maltose or d-xylose were used as inducers of the extracellular amylolytic and xylanolytic enzymes. Upon induction, protein compositions in the extracellular broth as well as in enriched secretory organelle (microsomal) fractions were analyzed using a shotgun proteomics approach. In total 102 secreted proteins and 1,126 microsomal proteins were identified in this study. Induction by d-maltose or d-xylose resulted in the increase in specific extracellular enzymes, such as glucoamylase A on d-maltose and β-xylosidase D on d-xylose, as well as of microsomal proteins. This reflects the differential expression of selected genes coding for dedicated extracellular enzymes. As expected, the addition of extra d-sorbitol had no effect on the expression of carbohydrate-active enzymes, compared to addition of d-xylose or d-maltose. Furthermore, d-maltose induction caused an increase in microsomal proteins related to translation (e.g., Rpl15) and vesicular transport (e.g., the endosomal-cargo receptor Erv14). Millimolar amounts of the inducers d-maltose and d-xylose are sufficient to cause a direct response in specific protein expression levels. Also, after induction by d-maltose or d-xylose, the induced enzymes were found in microsomes and extracellular. In agreement with our previous findings for d-xylose induction, d-maltose induction leads to recruitment of proteins involved in proteasome-mediated degradation

Second intravenous immunoglobulin dose in patients with Guillain-Barre syndrome with poor prognosis (SID-GBS):a double-blind, randomised, placebo-controlled trial

Background Treatment with one standard dose (2 g/kg) of intravenous immunoglobulin is insufficient in a proportion of patients with severe Guillain-Barre syndrome. Worldwide, around 25% of patients severely affected with the syndrome are given a second intravenous immunoglobulin dose (SID), although it has not been proven effective. We aimed to investigate whether a SID is effective in patients with Guillain-Barre syndrome with a predicted poor outcome. Methods In this randomised, double-blind, placebo-controlled trial (SID-GBS), we included patients (>= 12 years) with Guillain-Barre syndrome admitted to one of 59 participating hospitals in the Netherlands. Patients were included on the first day of standard intravenous immunoglobulin treatment (2 g/kg over 5 days). Only patients with a poor prognosis (score of >= 6) according to the modified Erasmus Guillain-Barre syndrome Outcome Score were randomly assigned, via block randomisation stratified by centre, to SID (2 g/kg over 5 days) or to placebo, 7-9 days after inclusion. Patients, outcome adjudicators, monitors, and the steering committee were masked to treatment allocation. The primary outcome measure was the Guillain-Barre syndrome disability score 4 weeks after inclusion. All patients in whom allocated trial medication was started were included in the modified intention-to-treat analysis. Findings Between Feb 16, 2010, and June 5, 2018, 327 of 339 patients assessed for eligibility were included. 112 had a poor prognosis. Of those, 93 patients with a poor prognosis were included in the modified intention-to-treat analysis: 49 (53%) received SID and 44 (47%) received placebo. The adjusted common odds ratio for improvement on the Guillain-Barre syndrome disability score at 4 weeks was 1.4 (95% CI 0.6-3.3; p=0.45). Patients given SID had more serious adverse events (35% vs 16% in the first 30 days), including thromboembolic events, than those in the placebo group. Four patients died in the intervention group (13-24 weeks after randomisation). Interpretation Our study does not provide evidence that patients with Guillain-Barre syndrome with a poor prognosis benefit from a second intravenous immunoglobulin course; moreover, it entails a risk of serious adverse events. Therefore, a second intravenous immunoglobulin course should not be considered for treatment of Guillain-Barre syndrome because of a poor prognosis. The results indicate the need for treatment trials with other immune modulators in patients severely affected by Guillain-Barre syndrome. Funding Prinses Beatrix Spierfonds and Sanquin Plasma Products. Copyright (C) 2021 Elsevier Ltd. All rights reserved

Proteomics of industrial fungi: trends and insights for biotechnology

Filamentous fungi are widely known for their industrial applications, namely, the production of food-processing enzymes and metabolites such as antibiotics and organic acids. In the past decade, the full genome sequencing of filamentous fungi increased the potential to predict encoded proteins enormously, namely, hydrolytic enzymes or proteins involved in the biosynthesis of metabolites of interest. The integration of genome sequence information with possible phenotypes requires, however, the knowledge of all the proteins in the cell in a system-wise manner, given by proteomics. This review summarises the progress of proteomics and its importance for the study of biotechnological processes in filamentous fungi. A major step forward in proteomics was to couple protein separation with high-resolution mass spectrometry, allowing accurate protein quantification. Despite the fact that most fungal proteomic studies have been focused on proteins from mycelial extracts, many proteins are related to processes which are compartmentalised in the fungal cell, e.g. β-lactam antibiotic production in the microbody. For the study of such processes, a targeted approach is required, e.g. by organelle proteomics. Typical workflows for sample preparation in fungal organelle proteomics are discussed, including homogenisation and sub-cellular fractionation. Finally, examples are presented of fungal organelle proteomic studies, which have enlarged the knowledge on areas of interest to biotechnology, such as protein secretion, energy production or antibiotic biosynthesis

Metabolic engineering of Rhizopus oryzae for the production of platform chemicals

Rhizopus oryzae is a filamentous fungus belonging to the Zygomycetes. It is among others known for its ability to produce the sustainable platform chemicals l-(+)-lactic acid, fumaric acid, and ethanol. During glycolysis, all fermentable carbon sources are metabolized to pyruvate and subsequently distributed over the pathways leading to the formation of these products. These platform chemicals are produced in high yields on a wide range of carbon sources. The yields are in excess of 85 % of the theoretical yield for l-(+)-lactic acid and ethanol and over 65 % for fumaric acid. The study and optimization of the metabolic pathways involved in the production of these compounds requires well-developed metabolic engineering tools and knowledge of the genetic makeup of this organism. This review focuses on the current metabolic engineering techniques available for R. oryzae and their application on the metabolic pathways of the main fermentation products

Optimization Strategies for Microbial Itaconic Acid Biosynthesis

Background: Itaconic acid is a C5 dicaboxylic acid that can serve as a building block to be used in industry to synthesize polymers that are currently based on petroleum-based components. Methods: An overview of the recent literature on microbial itaconic acid production is given in this minireview. The biosynthetic pathways as they are known in Aspergillus terreus and Ustilago maydis are described. Major advances have been made in the development of different microorganisms to serve as potential novel itaconic acid production hosts. Although fermentation strategies are discussed, our main focus is therefore on metabolic engineering strategies for optimal itaconic acid biosynthesis.Results: Itaconic acid is naturally produced by Aspergillus terreus, certain Ustilago and Candida species and Pseudozyma antarctica. Also in mammalian cells itaconic acid is found during macrophage activation. The biosynthetic pathway in A. terreus was well studied and the crucial enzyme for itaconic acid synthesis was found to be cis-aconitate decarboxylase (CadA) that converts cis-aconitate into itaconate. On one hand, optimization of itaconic acid production was done by optimizing fermentation processes and by applying metabolic engineering strategies to the natural producers, most of this was done with A. terreus. On the other hand, the identification of CadA allowed the exploration of heterologous expression of the gene in different hosts. Since citric acid is the metabolic precursor for itaconic acid biosynthesis, many research efforts have focused on Aspergillus niger as a potential itaconic acid producer. The results of this research showed that besides the heterologous expression of cadA, transport between different compartments and re-routing of the central carbon metabolism are important factors for the efficient biosynthesis of itaconic acid.Conclusion: Several microorganisms have been investigated in the past years as potential itaconic acid producing hosts. Titers obtained by metabolic engineering of non-producing hosts range between 14.5 mg/L and 7.8 g/L. Although substantial progress has been made, the titers are not yet competitive with the titers obtained with the natural producer A. terreus