- L.H. Graaff de (3)
- M.J.L. Groot de (2)
- A.A. Hasper (1)
- B. Jovanovic (1)
- M. Juin (1)
- P.A. Kuyk van (1)
- M. Levisson (1)
- Qin Ling (1)
- A.R. Mach-Aigner (1)
- J.M. Oliveira (1)
- J. Omony (1)
- G.J.G. Ruijter (2)
- P.A. vanKuyk (2)
- D. Veen van der (1)
- J. Visser (1)
- J.A.G.M. Visser de (1)
- P.J.I. Vondervoort van de (1)
- R.P. Vries de (3)
- H.A.B. Wosten (2)
A broader role for AmyR in Aspergillus niger: regulation of the utilisation of d-glucose or d-galactose containing oligo- and polysaccharides
vanKuyk, P.A. ; Benen, J.A.E. ; Wosten, H.A.B. ; Visser, J.A.G.M. de; Vries, R.P. de - \ 2012
Applied Microbiology and Biotechnology 93 (2012)1. - ISSN 0175-7598 - p. 285 - 293.
heterologous protein-production - transcriptional activator xlnr - glucoamylase production - alpha-galactosidase - gene-expression - encoding genes - amylase genes - degradation - oryzae - growth
AmyR is commonly considered a regulator of starch degradation whose activity is induced by the presence of maltose, the disaccharide building block of starch. In this study, we demonstrate that the role of AmyR extends beyond starch degradation. Enzyme activity assays, genes expression analysis and growth profiling on d-glucose- and d-galactose-containing oligo- and polysaccharides showed that AmyR regulates the expression of some of the Aspergillus niger genes encoding alpha- and beta-glucosidases, alpha- and beta- galactosidases, as well as genes encoding alpha-amlyases and glucoamylases. In addition, we provide evidence that d-glucose or a metabolic product thereof may be the inducer of the AmyR system in A. niger and not maltose, as is commonly assumed.
D-Xylose Concentration-Dependent Hydrolase Expression Profiles and the Function of CreA and XlnR in Aspergillus niger
Mach-Aigner, A.R. ; Omony, J. ; Jovanovic, B. ; Boxtel, A.J.B. van; Graaff, L.H. de - \ 2012
Applied and Environmental Microbiology 78 (2012)9. - ISSN 0099-2240 - p. 3145 - 3155.
transcriptional activator xlnr - jecorina trichoderma-reesei - cell-wall polysaccharides - time rt-pcr - hypocrea-jecorina - encoding genes - xylanase expression - beta-xylosidase - enzyme-system - l-arabitol
Aspergillus niger is an important organism for the production of industrial enzymes such as hemicellulases and pectinases. The xylan-backbone monomer, d-xylose, is an inducing substance for the coordinate expression of a large number of polysaccharide-degrading enzymes. In this study, the responses of 22 genes to low (1 mM) and high (50 mM) d-xylose concentrations were investigated. These 22 genes encode enzymes that function as xylan backbone-degrading enzymes, accessory enzymes, cellulose-degrading enzymes, or enzymes involved in the pentose catabolic pathway in A. niger. Notably, genes encoding enzymes that have a similar function (e.g., xylan backbone degradation) respond in a similar manner to different concentrations of d-xylose. Although low d-xylose concentrations provoke the greatest change in transcript levels, in particular, for hemicellulase-encoding genes, transcript formation in the presence of high concentrations of d-xylose was also observed. Interestingly, a high d-xylose concentration is favorable for certain groups of genes. Furthermore, the repressing influence of CreA on the transcription and transcript levels of a subset of these genes was observed regardless of whether a low or high concentration of d-xylose was used. Interestingly, the decrease in transcript levels of certain genes on high d-xylose concentrations is not reflected by the transcript level of their activator, XlnR. Regardless of the d-xylose concentration applied and whether CreA was functional, xlnR was constitutively expressed at a low level
Efficient cloning system for construction of gene silencing vectors in Aspergillus niger
Oliveira, J.M. ; Veen, D. van der; Graaff, L.H. de; Qin Ling, - \ 2008
Applied Microbiology and Biotechnology 80 (2008)5. - ISSN 0175-7598 - p. 917 - 924.
transcriptional activator xlnr - double-stranded-rna - neurospora-crassa - caenorhabditis-elegans - functional-analysis - expression - interference - transformation - fungus - inactivation
An approach based on Gateway recombination technology to efficiently construct silencing vectors was developed for use in the biotechnologically important fungus Aspergillus niger. The transcription activator of xylanolytic and cellulolytic genes XlnR of A. niger was chosen as target for gene silencing. Silencing was based on the expression vector pXLNRir that was constructed and used in co-transformation. From all the strains isolated (N = 77), nine showed poor xylan-degrading activities in two semi-quantitative plate assays testing different activities for xylan degradation. Upon induction on D-xylose, transcript levels of xlnR were decreased in the xlnR-silenced strains, compared to a wild-type background. Under these conditions, the transcript levels of xyrA and xynB (two genes regulated by XlnR) were also decreased for these xlnR-silenced strains. These results indicate that the newly developed system for rapid generation of silencing vectors is an effective tool for A. niger, and this can be used to generate strains with a tailored spectrum of enzyme activities or product formation by silencing specific genes encoding, e.g., regulators such as XlnR
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).
Aspergillus niger protein estA defines a new class of fungal esterases within the alfa/beta hydrolase fold superfamily of proteins
Bourne, Y. ; Hasper, A.A. ; Chahinian, H. ; Juin, M. ; Graaff, L.H. de - \ 2004
Structure 12 (2004)8. - ISSN 0969-2126 - p. 677 - 687.
transcriptional activator xlnr - 3-dimensional structure - acetylxylan esterase - antifungal agents - gene-expression - vinyl esters - sequence - acetylcholinesterase - lipases - refinement
From the fungus Aspergillus niger, we identified a new gene encoding protein EstA, a member of the alpha/beta-hydrolase fold superfamily but of unknown substrate specificity. EstA was overexpressed and its crystal structure was solved by molecular replacement using a lipaseacetylcholinesterase chimera template. The 2.1 A 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.
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.