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Enhanced production of Aspergillus niger laccase-like multicopper oxidases through mRNA optimization of the glucoamylase expression system
Tamayo Ramos, J.A. ; Barends, S. ; Lange, D. ; Jel, A. de; Verhaert, R.M. ; Graaff, L.H. de - \ 2013
Biotechnology and Bioengineering 110 (2013)2. - ISSN 0006-3592 - p. 543 - 551.
5'-untranslated region - protein expression - gene - translation - transformation - promoter - nidulans - cloning - oryzae
In filamentous fungi, most of the strategies used for the improvement of protein yields have been based on an increase in the transcript levels of a target gene. Strategies focusing at the translational level have been also described, but are far less explored. Here the 5' untranslated sequence of the glaA mRNA, a widely used expression system for the expression of recombinant proteins, was modified by the introduction of different nucleotide elements that have positive role in the translation process. Five Aspergillus niger laccase-like multicopper oxidases (MCOs) coding genes were fused to the native glaA 5'UTR and the three synthetic versions (sUTR1, sUTR2, and sUTR3) as well, and placed under the control of the glucoamylase gene promoter. Afterwards, a total of 20 fungal transformations were done using A. niger N593 as a recipient strain and 50 transformants per transformation were isolated and analyzed. The result of the incorporation of the synthetic 5'UTRs on the overall productivity of the transformants was assessed, on one hand by monitoring the laccase activity of all the isolated transformants, and on the other hand by quantifying and comparing the activity of those secreting the highest level of each MCO. For this purpose, a high-throughput method for the screening and selection of the best producers was developed. Once the best transformants producing the highest yield of McoA, McoB, McoC, McoD, and McoJ laccases were selected, their production level was quantified in supernatants of liquid cultures. The results obtained in this work indicate that modifications in the native glaA 5'UTR can lead to improvements in protein yields. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc
Degradation of different pectins by fungi:correlations and contrasts between the pectinolytic enzyme sets identified in genomes and the growth on pectins of different origin
Benoit, I. ; Coutinho, P.M. ; Schols, H.A. ; Gerlach, G.F. ; Henrissat, B. ; Vries, R.P. de - \ 2012
BMC Genomics 13 (2012). - ISSN 1471-2164
aspergillus-niger - rhizopus-oryzae - podospora-anserina - trichoderma-reesei - sequence - protein - nidulans - acid - homogalacturonan - tryptoquivaline
Background: Pectins are diverse and very complex biomolecules and their structure depends on the plant speciesand tissue. It was previously shown that derivatives of pectic polymers and oligosaccharides from pectins havepositive effects on human health. To obtain specific pectic oligosaccharides, highly defined enzymatic mixes arerequired. Filamentous fungi are specialized in plant cell wall degradation and some produce a broad range ofpectinases. They may therefore shed light on the enzyme mixes needed for partial hydrolysis.Results: The growth profiles of 12 fungi on four pectins and four structural elements of pectins show that thepresence/absence of pectinolytic genes in the fungal genome clearly correlates with their ability to degradepectins. However, this correlation is less clear when we zoom in to the pectic structural elements.Conclusions: This study highlights the complexity of the mechanisms involved in fungal degradation of complexcarbon sources such as pectins. Mining genomes and comparative genomics are promising first steps towards theproduction of specific pectinolytic fractions.
Biocatalytic potential of laccase-like multicopper oxidases from Aspergillus niger
Tamayo Ramos, J.A. ; Berkel, W.J.H. van; Graaff, L.H. de - \ 2012
Microbial Cell Factories 11 (2012). - ISSN 1475-2859 - 11 p.
malachite green - fungal laccases - yellow laccase - phanerochaete-chrysosporium - pleurotus-ostreatus - filamentous fungi - blue - degradation - expression - nidulans
BACKGROUND: Laccase-like multicopper oxidases have been reported in several Aspergillus species but they remain uncharacterized. The biocatalytic potential of the Aspergillus niger fungal pigment multicopper oxidases McoA and McoB and ascomycete laccase McoG was investigated. RESULTS: The laccase-like multicopper oxidases McoA, McoB and McoG from the commonly used cell factory Aspergillus niger were homologously expressed, purified and analyzed for their biocatalytic potential. All three recombinant enzymes were monomers with apparent molecular masses ranging from 80 to 110 kDa. McoA and McoG resulted to be blue, whereas McoB was yellow. The newly obtained oxidases displayed strongly different activities towards aromatic compounds and synthetic dyes. McoB exhibited high catalytic efficiency with N,N-dimethyl-p-phenylenediamine (DMPPDA) and 2,2-azino-di(3-ethylbenzthiazoline) sulfonic acid (ABTS), and appeared to be a promising biocatalyst. Besides oxidizing a variety of phenolic compounds, McoB catalyzed successfully the decolorization and detoxification of the widely used textile dye malachite green. CONCLUSIONS: The A. niger McoA, McoB, and McoG enzymes showed clearly different catalytic properties. Yellow McoB showed broad substrate specificity, catalyzing the oxidation of several phenolic compounds commonly present in different industrial effluents. It also harbored high decolorization and detoxification activity with the synthetic dye malachite green, showing to have an interesting potential as a new industrial biocatalyst
The Aspergillus niger multicopper oxidase family: analysis and overexpression of laccase-like encoding genes
Tamayo Ramos, J.A. ; Barends, S. ; Verhaert, R.M. ; Graaff, L.H. de - \ 2011
Microbial Cell Factories 10 (2011)78. - ISSN 1475-2859
heterologous expression - recombinant enzyme - saccharomyces-cerevisiae - melanocarpus-albomyces - secreted proteins - filamentous fungi - multigene family - nidulans - fumigatus - sequence
BACKGROUND: 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 Aspergillus niger ATCC 1015 genome resulted in the identification of thirteen MCO genes. Ten of them were cloned and homologously overexpressed. RESULTS: A bioinformatic analysis of the A. niger 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 in silico amino acid sequence analysis, the putative genes encoding for functional extracellular laccases (mcoA, mcoB, mcoC, mcoD, mcoE, mcoF, mcoG, mcoI, mcoJ and mcoM) were placed under the control of the glaA promoter and overexpressed in A. niger 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)2 tartrate. CONCLUSIONS: Aspergillus niger 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)2 tartrate has been found to be the most appropriate for McoB production in A. niger
Shotgun proteomics of Aspergillus niger microsomes upon D-xylose induction
Ferreira de Oliveira, J.M.P. ; Passel, M.W.J. van; Schaap, P.J. ; Graaff, L.H. de - \ 2010
Applied and Environmental Microbiology 76 (2010)13. - ISSN 0099-2240 - p. 4421 - 4429.
multiple sequence alignment - endoplasmic-reticulum - organelle proteomics - saccharomyces-cerevisiae - actin organization - secretory pathway - protein - expression - yeast - nidulans
Protein secretion plays an eminent role in cell maintenance and adaptation to the extracellular environment of microorganisms. Although protein secretion is an extremely efficient process in filamentous fungi, the mechanisms underlying protein secretion have remained largely uncharacterized in these organisms. In this study, we analyzed the effects of the d-xylose induction of cellulase and hemicellulase enzyme secretion on the protein composition of secretory organelles in Aspergillus niger. We aimed to systematically identify the components involved in the secretion of these enzymes via mass spectrometry of enriched subcellular microsomal fractions. Under each condition, fractions enriched for secretory organelles were processed for tandem mass spectrometry, resulting in the identification of peptides that originate from 1,081 proteins, 254 of which-many of them hypothetical proteins-were predicted to play direct roles in the secretory pathway. d-Xylose induction led to an increase in specific small GTPases known to be associated with polarized growth, exocytosis, and endocytosis. Moreover, the endoplasmic-reticulum-associated degradation (ERAD) components Cdc48 and all 14 of the 20S proteasomal subunits were recruited to the secretory organelles. In conclusion, induction of extracellular enzymes results in specific changes in the secretory subproteome of A. niger, and the most prominent change found in this study was the recruitment of the 20S proteasomal subunits to the secretory organelles
The het-c heterokaryon incompatibility gene in Aspergillus niger
Diepeningen, A.D. van; Pal, K. ; Lee, T. ; Hoekstra, R.F. ; Debets, A.J.M. - \ 2009
Mycological Research 113 (2009)2. - ISSN 0953-7562 - p. 222 - 229.
programmed cell-death - neurospora-crassa - vegetative incompatibility - filamentous fungi - podospora-anserina - mating-type - locus - nidulans - sequence - protein
Heterokaryon incompatibility among Aspergillus niger strains is a widespread phenomenon that is observed as the inability to form stable heterokaryons. The genetic basis of heterokaryon incompatibility reactions is well established in some sexual filamentous fungi but largely unknown in presumed asexual species, such as A. niger. To test whether the genes that determine heterokaryon incompatibility in Neurospora crassa, such as het-c, vib-1 and pin-c, have a similar function in A. niger, we performed a short in silico search for homologues of these genes in the A. niger and several related genomes. For het-c, pin-c and vib-1 we did indeed identify putative orthologues. We then screened a genetically diverse worldwide collection of incompatible black Aspergilli for polymorphisms in the het-c orthologue. No size variation was observed in the variable het-c indel region that determines the specificity in N. crassa. Sequence comparison showed only minor variation in the number of glutamine coding triplets. However, introduction of one of the three N. crassa alleles (het-c2) in A. niger by transformation resulted in an abortive phenotype, reminiscent of the heterokaryon incompatibility in N. crassa. We conclude that although the genes required are present and the het-c homologue could potentially function as a heterokaryon incompatibility gene, het-c has no direct function in heterokaryon incompatibility in A. niger because the necessary allelic variation is absent
Analysis of variance components reveals the contribution of sample processing to transcript variation
Veen, D. van der; Oliveira, J.M. ; Berg, W.A.M. van den; Graaff, L.H. de - \ 2009
Applied and Environmental Microbiology 75 (2009)8. - ISSN 0099-2240 - p. 2414 - 2422.
time rt-pcr - aspergillus-niger - gene-expression - microarray experiments - quantification - information - standards - nidulans - cloning - growth
The proper design of DNA microarray experiments requires knowledge of biological and technical variation of the studied biological model. For the filamentous fungus Aspergillus niger, a fast, quantitative real-time PCR (qPCR)-based hierarchical experimental design was used to determine this variation. Analysis of variance components determined the contribution of each processing step to total variation: 68% is due to differences in day-to-day handling and processing, while the fermentor vessel, cDNA synthesis, and qPCR measurement each contributed equally to the remainder of variation. The global transcriptional response to d-xylose was analyzed using Affymetrix microarrays. Twenty-four statistically differentially expressed genes were identified. These encode enzymes required to degrade and metabolize D-xylose-containing polysaccharides, as well as complementary enzymes required to metabolize complex polymers likely present in the vicinity of D-xylose-containing substrates. These results confirm previous findings that the d-xylose signal is interpreted by the fungus as the availability of a multitude of complex polysaccharides. Measurement of a limited number of transcripts in a defined experimental setup followed by analysis of variance components is a fast and reliable method to determine biological and technical variation present in qPCR and microarray studies. This approach provides important parameters for the experimental design of batch-grown filamentous cultures and facilitates the evaluation and interpretation of microarray data
Glucose uptake and growth of glucose-limited chemostat cultures of Aspergillus niger and a disruptant lacking MstA, a high-affinity glucose transporter
Jorgensen, T.R. ; vanKuyk, P.A. ; Poulsen, B.R. ; Ruijter, G.J.G. ; Visser, J. ; Iversen, J.J.L. - \ 2007
Microbiology 153 (2007)6. - ISSN 1350-0872 - p. 1963 - 1973.
saccharomyces-cerevisiae - sugar-transport - candida-utilis - neurospora-crassa - hexose transport - nidulans - kinetics - genes - yeast - cultivations
This is a study of high-affinity glucose uptake in Aspergillus niger and the effect of disruption of a high-affinity monosaccharide-transporter gene, mstA. The substrate saturation constant (K-s) of a reference strain was about 15 mu M in glucose-limited chemostat culture. Disruption of mstA resulted in a two- to fivefold reduction in affinity for glucose and led to expression of a low-affinity glucose transport gene, mstC, at high dilution rate. The effect of mstA disruption was more subtle at low and intermediate dilution rates, pointing to some degree of functional redundancy in the high-affinity uptake system of A. niger. The mstA disruptant and a reference strain were cultivated in glucose-limited chemostat cultures at low, intermediate and high dilution rate (D=0.07 h(-1), 0.14 h(-1) and 0.20 h(-1)). Mycelium harvested from steady-state cultures was subjected to glucose uptake assays, and analysed for expression of mstA and two other transporter genes, mstC and mstF The capacity for glucose uptake (V-max) of both strains was significantly reduced at low dilution rate. The glucose uptake assays revealed complex uptake kinetics. This impeded accurate determination of maximum specific uptake rates (V-max) and apparent affinity constants (K-m(app)) at intermediate and high dilution rate. Two high-affinity glucose transporter genes, mstA and mstF, were expressed at all three dilution rates in chemostat cultures, in contrast to batch culture, where only mstC was expressed. Expression patterns of the three transporter genes suggested differential regulation and functionality of their products.
Regulation of pentose catabolic pathway genes of Aspergillus niger
Groot, M.J.L. de; Dool, C. van den; Wosten, H.A.B. ; Levisson, M. ; vanKuyk, P.A. ; Ruijter, G.J.G. ; Vries, R.P. de - \ 2007
Food Technology and Biotechnology 45 (2007)2. - ISSN 1330-9862 - p. 134 - 138.
transcriptional activator xlnr - d-xylose - l-arabinose - alpha-glucuronidase - degrading enzymes - kinase gene - expression - nidulans - polysaccharides - degradation
The aim of this study was to obtain a better understanding of the pentose catabolism in Aspergillus niger and the regulatory systems that affect it. To this end, we have cloned and characterised the genes encoding A. niger L-arabitol dehydrogenase (ladA) and xylitol dehydrogenase (xdhA), and compared the regulation of these genes to other genes of the pentose catabolic pathway. This demonstrated that activation of the pathway depends on two transcriptional regulators, the xylanolytic activator (XlnR) and an unidentified L-arabinose specific regulator (AraR). These two regulators affect those genes of the pentose catabolic pathway that are related to catabolic conversion of their corresponding inducers (D-xylose and L-arabinose, respectively).
Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88
Pel, H.J. ; Winde, J.H. de; Archer, D.B. ; Schaap, P.J. ; Benen, J.A.E. ; Debets, A.J.M. ; Kools, H.J. ; Pal, K. - \ 2007
Nature Biotechnology 25 (2007). - ISSN 1087-0156 - p. 221 - 231.
unfolded protein response - saccharomyces-cerevisiae - filamentous fungi - fumigatus - nidulans - dna - reconstruction - degradation - metabolism - autolysis
The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.
Dynamics of dsRNA mycoviruses in black Aspergillus population.
Diepeningen, A.D. van; Debets, A.J.M. ; Hoekstra, R.F. - \ 2006
Fungal Genetics and Biology 43 (2006)6. - ISSN 1087-1845 - p. 446 - 452.
double-stranded-rna - fragment-length-polymorphisms - chestnut blight fungus - niger aggregate - virus transfer - dna - cryphonectria - transmission - nidulans - elements
Approximately 10% of all examined 668 representatives of black Aspergillus species, independent of worldwide location, were infected with double-stranded RNA (dsRNA) mycoviruses. These isometric viruses (25-40 nm diameter) contained a variety of often multiple segments of different dsRNA sizes ranging from 0.8 to 4.4 kb in size. In one strain the virus shows clear visible effects on its host with non-sporulating sectors. We quantified the fitness costs of these and more 'cryptic' virus infections on mycelial growth rate and spore production, and on competitive ability with respect to other strains under different growth conditions. Mycovirus infection proved detrimental in all these measures. The reduced success in interference competition due to mycovirus infection belies co-evolution of mycovirus and host to a mutually beneficial symbiosis, like in killer virus systems in yeast and smut and agrees more to recent infections. For a stable virus infection frequency in the black Aspergillus population, fitness costs and spontaneous loss should be balanced with new infections. Implications of even small viral fitness effects combined with the observed transmission limits for host and mycovirus are discussed
Selection and characterisation of a xylitol-derepressed Aspergillus niger mutant that is apparently impaired in xylitol transport
Vondervoort, P.J.I. van de; Groot, M.J.L. de; Ruijter, G.J.G. ; Visser, J. - \ 2006
Applied Microbiology and Biotechnology 73 (2006)4. - ISSN 0175-7598 - p. 881 - 886.
metabolic-control analysis - saccharomyces-cerevisiae - l-arabinose - xylose catabolism - fermentation - xylan - construction - degradation - nidulans - genes
Aspergillus niger is known for its biotechnological applications, such as the use of xylanase enzyme for the degradation of hemicellulose. Depending on culture conditions, several polyols may also be accumulated, such as xylitol during D-xylose oxidation. Also during industrial fermentation of xylose for the production of fuel ethanol by recombinant yeast, xylitol is a by-product. We studied xylitol metabolism by isolating mutants that have impaired xylitol-mediated repression. Genetic and biochemical characterisation revealed that one of these mutants was affected not only in xylitol-mediated carbon repression, but also had impaired xylitol transport
Metabolic control analysis of Aspergillus niger L-arabinose catabolism
Groot, M.J.L. de; Prathumpai, W. ; Visser, J. ; Ruijter, G.J.G. - \ 2005
Biotechnology Progress 21 (2005)6. - ISSN 8756-7938 - p. 1610 - 1616.
alpha-l-arabinofuranosidase - enzyme-catalyzed reactions - d-xylose - extracellular arabinases - degrading enzymes - gene-expression - pichia-stipitis - reductase gene - nidulans - purification
A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography, and their kinetic properties were characterized. For the other enzymes of the pathway the kinetic data were available from the literature. The metabolic model was used to analyze flux and metabolite concentration control of the L-arabinose catabolic pathway. The model demonstrated that flux control does not reside at the enzyme following the intermediate with the highest concentration, L-arabitol, but is distributed over the first three steps in the pathway, preceding and following L-arabitol. Flux control appeared to be strongly dependent on the intracellular L-arabinose concentration. At 5 mM intracellular L-arabinose, a level that resulted in realistic intermediate concentrations in the model, flux control coefficients for L-arabinose reductase, L-arabitol dehydrogenase and L-xylulose reductase were 0.68, 0.17 and 0.14, respectively. The analysis can be used as a guide to identify targets for metabolic engineering aiming at either flux or metabolite level optimization of the L-arabinose catabolic pathway of A. niger. Faster L-arabinose utilization may enhance utilization of readily available organic waste containing hemicelluloses to be converted into industrially interesting metabolites or valuable enzymes or proteins.
Isolation of a fluffy mutant of Aspergillus niger from chemostat culture and its potential use as a morphologically stable host for protein production
Vondervoort, P.J.I. van de; Poulsen, B.R. ; Ruijter, G.J.G. ; Schuleit, T. ; Visser, J. ; Iversen, J.J.L. - \ 2004
Biotechnology and Bioengineering 86 (2004)3. - ISSN 0006-3592 - p. 301 - 307.
recombinant glucoamylase production - fusarium-graminearum a3/5 - penicillium-chrysogenum - genetic-analysis - argb gene - nidulans - transformation - growth - ph - fungus
Chemostat cultivation of Aspergillus niger and other filamentous fungi is often hindered by the spontaneous appearance of morphologic mutants. Using the Variomixing bioreactor and applying different chemostat conditions we tried to optimize morphologic stability in both ammonium- and glucose-limited cultures. In most cultivations mutants with fluffy (aconidial) morphology became dominant. From an ammonium-limited culture, a fluffy mutant was isolated and genetically characterized using the parasexual cycle. The mutant contained a single morphological mutation, causing an increased colony radial growth rate. The fluffy mutant was subjected to transformation and finally conidiospores from a forced heterokaryon were shown to be a proper inoculum for fluffy strain cultivation. (C) 2004 Wiley Periodicals, Inc.
Aspergillus niger mstA encodes a high affinity sugar/H+ symporter which is regulated in persponse to extracellular pH
Kuyk, P.A. van; Diderich, J.A. ; MacCabe, A.P. ; Hererro, O. ; Ruijter, G.J.G. ; Visser, J. - \ 2004
Biochemical Journal 379 (2004)2. - ISSN 0264-6021 - p. 375 - 383.
muscaria monosaccharide transporter - yeast saccharomyces-cerevisiae - individual hexose transporters - carbon catabolite repression - glucose-transport - neurospora-crassa - chromosome-iv - hxt genes - expression - nidulans
A sugar-transporter-encoding gene, mstA, which is a member of the major facilitator superfamily, has been cloned from a genomic DNA library of the filamentous fungus Aspergillus niger. To enable the functional characterization of MSTA, a full-length cDNA was expressed in a Saccharomyces cerevisiae strain deficient in hexose uptake. Uptake experiments using C-14-labelled monosaccharides demonstrated that although able to transport D-fructose (K-m, 4.5 +/- 1.0 mM), D-Xylose (K-m, 0.3 +/- 0.1 mM) and D-mannose (K-m, 60 +/- 20 muM), MSTA has a preference for D-glucose (K-m, 25 +/- 10 muM). pH changes associated with sugar transport indicate that MSTA catalyses monosaccharide/H+ symport. Expression of mstA in response to carbon starvation and upon transfer to poor carbon sources is consistent with a role for MSTA as a high-affinity transporter for D-glucose, D-mannose and D-xylose. Northern analysis has shown that mstA is subject to CreA-mediated carbon catabolite repression and pH regulation mediated by PacC. A. niger strains in which the mstA gene had been disrupted are phenotypically identical with isogenic reference strains when grown on 0.1-60 MM D-glucose, D-mannose, D-fructose or D-Xylose. This indicates that A. niger possesses other transporters capable of compensating for the absence of MSTA.
Functional analysis of the transcriptional activator XlnR from Aspergillus niger
Hasper, A.A. ; Trindade, L.M. ; Veen, D. van der; Ooyen, A.J.J. van; Graaff, L.H. de - \ 2004
Microbiology 150 (2004). - ISSN 1350-0872 - p. 1367 - 1375.
saccharomyces-cerevisiae - utilization pathway - importin beta - d-xylose - sequence - protein - regulator - gene - expression - nidulans
The transcriptional activator XlnR from Aspergillus niger is a zinc binuclear cluster transcription factor that belongs to the GAL4 superfamily. Several putative structural domains in XInR were predicted using database and protein sequence analysis. Thus far, only the functionality of the N-terminal DNA-binding domain has been determined experimentally. Deletion mutants of the xInR gene were constructed to localize the functional regions of the protein. The results showed that a putative C-terminal coiled-coil region is involved in nuclear import of XInR. After deletion of the C-terminus, including the coiled-coil region, XInR was found in the cytoplasm, while deletion of the C-terminus downstream of the coiled-coil region resulted in nuclear import of XlnR. The latter mutant also showed increased xylanase activity, indicating the presence of a region with an inhibitory function in XInR-controlled transcription. Previous findings had already shown that a mutation in the XlnR C-terminal region resulted in transcription of the structural genes under non-inducing conditions. A regulatory model of XInR is presented in which the C-terminus responds to repressing signals, resulting in an inactive state of the protein.
Polyol accumulation by Aspergillus oryzae at low water activity in solid-state fermentation
Ruijter, G.J.G. ; Visser, J. ; Rinzema, A. - \ 2004
Microbiology 150 (2004). - ISSN 1350-0872 - p. 1095 - 1101.
d-arabitol dehydrogenase - saccharomyces-cerevisiae - hyperosmotic stress - candida-albicans - glycerol - nidulans - pathway - growth - niger - expression
Polyol accumulation and metabolism were examined in Aspergillus oryzae cultured on whole wheat grains or on wheat dough as a model for solid-state culture. In solid-state fermentation (SSF), water activity (a(w)) is typically low resulting in osmotic stress. In addition to a high level of mannitol, which is always present in the cells, A. oryzae accumulated high concentrations of glycerol, erythritol and arabitol at relatively low a(w) (0(.)96-0(.)97) in SSF. Accumulation of such a mixture of polyols is rather unusual and might be typical for SSF. A. oryzae mycelium accumulating various polyols at low aw contained at least four distinct polyol dehydrogenases with highest activities toward glycerol, erythritol, D-arabitol and mannitol. NADP(+)-dependent glycerol dehydrogenase activity correlated very well with glycerol accumulation. A similar correlation was observed for erythritol and NADP(+)-erythritol dehydrogenase suggesting that NADP(+)-dependent glycerol and erythritol dehydrogenases are involved in biosynthesis of glycerol and erythritol, respectively, and that these enzymes are induced by osmotic stress.
Isolation and characterization of two specific regulatory Aspergillus niger mutants shows antagonistic regulation of arabinan and xylan metabolism
Groot, M.J.L. de; Vondervoort, P.J.I. van de; Vries, R.P. de; Kuyk, P.A. van; Ruijter, G.J.G. ; Visser, J. - \ 2003
Microbiology 149 (2003). - ISSN 1350-0872 - p. 1183 - 1191.
alpha-l-arabinofuranosidase - transcriptional activator xlnr - gene-expression - d-xylose - degrading enzymes - degradation - induction - nidulans - cloning - construction
This paper describes two Aspergillus niger mutants (araA and araB) specifically disturbed in the regulation of the arabinanase system in response to the presence of L-arabinose. Expression of the three known L-arabinose-induced arabinanolytic genes, abfA, abfB and abnA, was substantially decreased or absent in the araA and araB strains compared to the wild-type when incubated in the presence of L-arabinose or L-arabitol. In addition, the intracellular activities Of L-arabitol dehydrogenase and L-arabinose reductase, involved in L-arabinose catabolism, were decreased in the araA and araB strains. Finally, the data show that the gene encoding D-xylulose kinase, xkiA, is also under control of the arabinanolytic regulatory system. L-Arabitol, most likely the true inducer of the arabinanolytic and L-arabinose catabolic genes, accumulated to a high intracellular concentration in the araA and araB mutants. This indicates that the decrease of expression of the arabinanolytic genes was not due to lack of inducer accumulation. Therefore, it is proposed that the araA and araB mutations are localized in positive-acting components of the regulatory system involved in the expression of the arabinanase-encoding genes and the genes encoding the L-arabinose catabolic pathway.
Mannitol is required for stress tolerance in Aspergillus niger conidiospores
Ruijter, G.J.G. ; Bax, M. ; Patel, H. ; Flitter, S.J. ; Vondervoort, P.J.I. van de; Vries, R.P. de; Kuyk, P.A. van; Visser, J. - \ 2003
Eukaryotic Cell 2 (2003)4. - ISSN 1535-9778 - p. 690 - 698.
cryptococcus-neoformans - mannitol-1-phosphate dehydrogenase - agaricus-bisporus - pyruvate-kinase - nidulans - cycle - transformation - expression - nadph - gene
D-Mannitol is the predominant carbon compound in conidiospores of the filamentous fungus Aspergillus niger and makes up 10 to 15% of the dry weight. A number of physiological functions have been ascribed to mannitol, including serving as a reserve carbon source, as an antioxidant, and to store reducing power. In this study, we cloned and characterized the A. niger mpd4 gene, which encodes mannitol 1-phosphate dehydrogenase (MPD), the first enzyme in the mannitol biosynthesis pathway. The mpdA promoter contains putative binding sites for the development-specific transcription factors BRLA and ABAA. Furthermore, increased expression of mpd4 in sporulating mycelium suggests that mannitol biosynthesis is, to a certain extent, developmentally regulated in A. niger. Inactivation of mpdA abolished mannitol biosynthesis in growing mycelium and reduced the mannitol level in conidiospores to 30% that in the wild type, indicating that MPD and mannitol 1-phosphate phosphatase form the major metabolic pathway for mannitol biosynthesis in A. niger. The viability of spores after prolonged storage and germination kinetics were normal in an mpd4 null mutant, indicating that mannitol does not play an essential role as a reserve carbon source in A. niger conidia. However, conidiospores of a Deltampd4 strain were extremely sensitive to a variety of stress conditions, including high temperature, oxidative stress and, to a lesser extent, freezing and lyophilization. Since mannitol supplied in the medium during sporulation repaired this deficiency, mannitol appears to be essential for the protection of A. niger spores against cell damage under these stress conditions.