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

Methods for identifying lipoxygenase producing microorganisms on agar plates

Plate assays for lipoxygenase producing microorganisms on agar plates have been developed. Both potassium iodide-starch and indamine dye formation methods were effective for detecting soybean lipoxygenase activity on agar plates. A positive result was also achieved using the β-carotene bleaching method, but the sensitivity of this method was lower than the other two methods. The potassium iodide-starch and indamine dye formation methods were also applied for detecting lipoxygenase production by Trichoderma reesei and Pichia pastoris transformants expressing the lipoxygenase gene of the fungus Gaeumannomyces graminis. In both cases lipoxygenase production in the transformants could be identified. For detection of the G. graminis lipoxygenase produced by Aspergillus nidulans the potassium iodide-starch method was successful. When Escherichia coli was grown on agar and soybean lipoxygenase was applied on the culture lipoxygenase activity could clearly be detected by the indamine dye formation method. This suggests that the method has potential for screening of metagenomic libraries in E. coli for lipoxygenase activity

Bone Health in Adults With Prader-Willi Syndrome : Clinical Recommendations Based on a Multicenter Cohort Study

Prader-Willi syndrome (PWS) is a rare complex genetic syndrome, characterized by delayed psychomotor development, hypotonia, and hyperphagia. Hormone deficiencies such as hypogonadism, hypothyroidism, and growth hormone deficiency are common. The combination of hypotonia, low physical activity, and hypogonadism might lead to a decrease in bone mass and increase in fracture risk. Moreover, one would expect an increased risk of scoliosis due to hypotonia and low physical activity. To study the prevalence and risk factors for skeletal problems (reduced bone mineral density, fractures, and scoliosis) in adults with PWS. We retrospectively collected patient characteristics, medical history, medication, biochemical measurements, dual-energy X-ray absorptiometry scans, and spinal X-rays and reviewed the current literature. We included 354 adults with PWS (median age 31 years; 43% males), of whom 51 (14%) had osteoporosis (T-score below −2.5) and 143 (54%) had osteopenia (T-score −1 to −2.5). The most prevalent modifiable risk factors for osteoporosis were hypogonadism, insufficient dairy intake, sedentary lifestyle, and corticosteroid use. Male sex was associated with osteoporosis (P =.005). Growth hormone treatment was not associated with osteoporosis. A history of vertebral fractures was present in 10 (3%) and nonvertebral fractures in 59 (17%). Scoliosis was present in 263 (80%), but no modifiable risk factors were identified. Besides scoliosis, osteoporosis is common in adults with PWS. Based on the literature and the risk factors for osteoporosis found in our cohort, we provide practical clinical recommendations to avoid skeletal complications in these vulnerable patients

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

Production of cyanophycin in Rhizopus oryzae through the expression of a cyanophycin synthetase encoding gene

Cyanophycin or cyanophycin granule peptide is a protein that results from non-ribosomal protein synthesis in microorganisms such as cyanobacteria. The amino acids in cyanophycin can be used as a feedstock in the production of a wide range of chemicals such as acrylonitrile, polyacrylic acid, 1,4-butanediamine, and urea. In this study, an auxotrophic mutant (Rhizopus oryzae M16) of the filamentous fungus R. oryzae 99-880 was selected to express cyanophycin synthetase encoding genes. These genes originated from Synechocystis sp. strain PCC6803, Anabaena sp. strain PCC7120, and a codon optimized version of latter gene. The genes were under control of the pyruvate decarboxylase promoter and terminator elements of R. oryzae. Transformants were generated by the biolistic transformation method. In only two transformants both expressing the cyanophycin synthetase encoding gene from Synechocystis sp. strain PCC6803 was a specific enzyme activity detected of 1.5 mU/mg protein. In one of these transformants was both water-soluble and insoluble cyanophycin detected. The water-soluble fraction formed the major fraction and accounted for 0.5% of the dry weight. The water-insoluble CGP was produced in trace amounts. The amino acid composition of the water-soluble form was determined and constitutes of equimolar amounts of arginine and aspartic acid

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

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