Transcriptional regulation of the xylanolytic enzyme system of Aspergillus

N.N.M.E. van Peij

Research output: Thesisinternal PhD, WU

Abstract

<p>Filamentous fungi, such as <em>Aspergillus niger</em> , produce high levels of polysaccharide degrading enzymes and are frequently used as production organisms for industrial enzyme preparations. The application of these polysaccharidases as xylanases and cellulases comprises <em>e.g.</em> the use in food and feed and in biopulping and bleaching in pulp- and paper-industry. In recent years many structural genes encoding cellulases and xylanases have been cloned. The expression of these genes is controlled at the level of transcription. In general, the expression of the genes encoding extracellular enzymes in <em>A. niger</em> is under dual control: by specific induction and by carbon catabolite repression.</p><p>At the start of this research, the transcriptional regulation of genes encoding the xylanolytic enzyme system was described in a (working-)model involving a number of individual steps (Fig. 1). Induction was described as a cascade which involved at least an inducer and a route-specific transcription factor. Carbon catabolite repression in this model was described to act at both the structural genes and the regulatory gene. The balance between the hypothetical transcription factor XlnR and the CreA repressor determines the level of transcription of the enzyme-encoding genes. Not much was known about the molecular processes in the signal transduction pathway of xylanolytic induction. Therefore, the aim of the research presented in this thesis, was to identify important factors within the signal transduction pathway of xylanolytic induction in <em>A. niger</em> .</p><div align="center"><img src="/wda/abstracts/i2719.gif" width="470" height="213" alt="Figure 1" border="0"/><br/><strong>Fig. 1.</strong> Schematic model for the double-lock regulation of genes encoding the xylanolytic enzyme system of <em>A. niger</em> . The model includes the transcriptional regulator XlnR and the carbon catabolite repressor CreA. Under conditions of carbon catabolite repression both the structural xylanolytic genes as well as the <em>xlnR</em> regulator gene are repressed by CreA.<br/></div><p>The results presented in this thesis have the model more defined at several steps. A first focus within the signal transduction pathway is the characterisation of inducers of the xylanolytic system and the way these inducers are formed. The xylanolytic system in <em>A. niger</em> is expressed upon growth on xylan and d-xylose as carbon sources. In Chapter two the role of the extracellularβ-xylosidase enzyme in the xylanolytic system is evaluated. In literature an important role is suggested forβ-xylosidase with respect to inducer formation. To address these questions the enzyme was purified and partially characterised and the <em>xlnD</em> gene, encoding theβ-xylosidase, was cloned. The use of strains with theβ-xylosidase-encoding gene <em>xlnD</em> disrupted, has excluded transglycosylation products ofβ-xylosidase as important for induction. d-Xylose is very likely the inducer of the xylanolytic enzyme system.</p><p>Another important focus within the signal transduction pathway is the regulation of xylanolytic genes. In the model, a route-specific transcription factor was postulated, which function was to regulate the transcription of the genes of the xylanolytic system. At the beginning of the research described in this thesis no fungal transcription factor of an extracellular enzyme system had been isolated. To be able to isolate the <em>A. niger</em><em>xlnR</em> gene encoding the route-specific xylanolytic activator, xylanase non-producing mutants were isolated. The mutants were used for cloning of the <em>xlnR</em> gene, after complementation by transformation of the mutants (Chapter four). The XlnR protein encodes a protein with a zinc binuclear cluster domain of the GAL4-type and is essential for the transcription of the genes encoding xylanolytic enzymes. This is the first gene isolated, encoding a fungal transcription factor involved in the transcriptional activation of genes encoding extracellular polysaccharide degrading enzymes.</p><p>The function and importance of XlnR within the whole process of polysaccharide degradation and gene induction in <em>A. niger</em> was established using the <em>xlnR</em> loss-of-function mutants. As originally envisaged, XlnR regulates the transcription of genes encoding xylanolytic enzymes. However, XlnR also has been shown to control the transcription of genes encoding enzymes involved in cellulose and hemicellulose degradation (Chapter five). Therefore, in <em>A. niger</em> , XlnR is a key-factor in transcriptional activation of hemicellulose- and cellulose-degrading enzymes.</p><p>Subsequently, the cloning of <em>xlnR</em> opened the possibility for a characterisation of the molecular mechanism of transcriptional activation for xylanolytic genes in <em>A. niger</em> . The transcriptional activator XlnR binds to 5' non-coding regions of xylanolytic structural genes. Comparison of promoters of XlnR-controlled genes allows the determination of a putative sequence for the XlnR-binding site. Site-directed mutagenesis, mobility shift assays and footprinting have been used to establish the XlnR-binding site to 5'-GGCTAA-3' which is present in the promoter of each XlnR-controlled gene (Chapter four).</p><p>Finally, different regulation mechanisms such as induction, carbon catabolite repression and pH regulation might influence the expression of the xylanolytic system. The influence of each of these mechanisms has been evaluated for the expression of the <em>A. nidulans</em> β-xylosidase-encoding <em>xlnD</em> gene (Chapter three). In <em>A. niger</em> the introduction of multiple copies of a structural gene, such as the <em>xlnA</em> gene having a strong promoter, results in a moderate increase of expression due to the introduction of a limitation at the level of a transcription factor (Chapter six). It also has been shown that the expression of the xylanolytic enzyme system in <em>A. niger</em> wild-type is limited at the level of the transcriptional activator XlnR. Introduction of multiple copies of the <em>xlnR</em> gene, relieves this limitation and results in an increased expression of the enzyme system. A simultaneous increase of both the copy number of the <em>xlnA</em> gene and the gene encoding the activator XlnR, shows no cumulative effect. A better understanding of the molecular mechanisms involved in the expression of this enzyme system, will allow its full exploitation in strain improvement.</p>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • van Ooyen, A.J.J., Promotor
  • de Graaff, L.H., Promotor, External person
Award date3 Dec 1999
Place of PublicationS.l.
Publisher
Print ISBNs9789058081544
Publication statusPublished - 1999

Keywords

  • aspergillus niger
  • xylanolytic microorganisms
  • transactivation

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