A molecular analysis of L-arabinan degradation in Aspergillus niger and Aspergillus nidulans

M.J.A. Flipphi

Research output: Thesisinternal PhD, WU

Abstract

<p>This thesis describes a molecular study of the genetics of<font size="-1">L</font>-arabinan degradation in <em>Aspergillus niger</em> and <em>Aspergillus nidulans.</em> These saprophytic hyphal fungi produce an extracellular hydrolytic enzyme system to depolymerize the plant cell wall polysaccharide<font size="-1">L</font>-arabinan. <strong>Chapter 1</strong> surveys the occurrence, properties and applications of<font size="-1">L</font>-arabinanolytic enzymes (arabinases). The <em>A.</em><em>niger</em> system, which constitutes an endolytic endo-1,5-α-<font size="-1">L</font>-arabinase (ABN A) and two distinct α-<font size="-1">L</font>-arabinofuranosidases (ABF A and ABF B), has been a frequent subject of investigation in the past and represents the best characterized<font size="-1">L</font>-arabinanolytic system to date. These three enzymes are all glycosylated. Current knowledge on the induction of fungal arabinase expression is summarized in this Chapter. Furthermore, the structure of the polysaccharide substrate and its function in the plant cell wall matrix are introduced.<p>In Chapters 2 to 5, the cloning and characterization of the structural genes coding for the three glycosyl hydrolases from the <em>A. niger</em><font size="-1">L</font>-arabinan-degrading complex are described. <em>A. niger abf</em> A <em></em> and <em>abf</em> B ar <em>e</em> the first eukaryotic ABF-encoding genes to be isolated and sequenced, <em>abn</em> A is <em></em> the first ABN-encoding gene published. <strong>Chapter 2</strong> reports on the isolation of the <em>abf</em> A gene encoding ABF A, the minor extracellular ABF. This gene could be cloned by utilizing ABF Aspecific cDNA as the probe. This cDNA was immunochemically identified from a cDNA library generated from<font size="-1">L</font>-arabitol-induced myceliurn of an <em>A. niger</em><font size="-1">D</font>-xylulose kinase mutant. This mutant is unable to grow on<font size="-1">L</font>-arabitol and features enhanced expression of all three arabinases when transferred to medium containing this pentitol as sole carbon source. In <strong>Chapter 3</strong> , the cloning of the ABN A-encoding gene <em>(abn</em> A) <em>is</em> described. This gene was isolated following the same strategy as with <em>abf</em> A, <em></em> although a second cDNA library had to be generated first. The induction process was immunochemically monitored in order to establish the proper induction conditions for the new library. The <em>abn</em> A gene <em></em> and the gene product were characterized by DNA sequence analyses of the cloned genomic DNA and the cDN <em>A.</em> The N-terminal amino acid sequences of ABN A and a CNBr-derived peptide were determined. Several transcription initiation sites and one polyadenylation site could be identified. The structural region codes for a protein of 321 amino acids and is interrupted by three introns. Extracellular ABN A consists of 302 amino acid residues with a deduced molecular weight of 32.5 kDa and a theoretical pl of 3.5. For the protein, an apparent pl of 3.0 and an apparent molecular weight of 43 kDa, determined upon SDS-PAGE, were previously reported. <strong>Chapter 4</strong> documents the isolation and characterization of the <em>abf</em> B gene, <em></em> coding for the major extracellular ABF. The determination of N-terminal amino acid sequences from ABF B and CNBr-generated peptides allowed the design of deoxyoligonucleotide mixtures which enabled the cloning of <em>abf</em> B. <em></em> When utilized as primers in a polymerase chain reaction (PCR), ABF B-specific amplification products emerged, one of which was used to probe the gene. The <em>abf</em> B gene <em></em> and the gene product were characterized by DNA sequence analyses of the cloned genomic DNA and of ABF B- specific cDNA isolated from the library described in Chapter 3. Several transcription initiation sites and one polyadenylation site could be identified. The structural region is a single open reading frame and codes for a protein of 499 amino acids. The mature enzyme consists of 481 amino acid residues with a deduced molecular weight of 50.7 kDa and a theoretical pl of 3.8. An apparent pl of 3.5 and an apparent molecular weight of 67 kDa, determined upon SDS-PAGE, were previously reported. The <em>abf</em> B gene <em></em> product was suggested to be identical to the ABF purified and characterized by Kaji and Tagawa (Biochim Biophys Acta <strong>207</strong> : 456-464 (1970)). Considering the non-amino acid content of the latter protein, a molecular weight of 64 kDa could be deduced for ABF B. In <strong>Chapter 5</strong> , the <em>abf</em> A gene and its gene product were characterized by DNA sequence analyses of the genomic DNA and of the cDNA for which the isolation was described in Chapter 2. The N-terminal amino acid sequences of ABF A and a CNBr-derived peptide were determined. One transcription initiation site and two polyadenylation sites could be identified. The structural region is interrupted by seven introns and codes for a protein of 628 amino acids. Mature ABF A consists of 603 amino acid residues with a deduced molecular weight of 65.4 kDa and a theoretical pl of 3.7. For this ABF, an apparent pi of 3.3 and an apparent molecular weight of 83 kDa, determined upon SDS-PAGE, were previously documented.<p>Although the three enzymes are all active against (1->5)-α-glycosidic bonds between<font size="-1">L</font>-arabinofuranosides, ABF A, ABF B and ABN A are genetically unrelated. ABF A was found to be <em>N</em> -glycosylated whereas ABF B and ABN A were not - these enzymes are only <em>O</em> -glycosylated. For each gene, arabinaseoverproducing strains were generated by introducing multiple gene copies in <em>A.</em><em>niger</em> or in <em>A.</em><em>nidulans</em> uridine auxotrophic strains through co-transformation. Transformants were isolated upon primary selection for uridine prototrophy. Subsequent overproduction of the genes introduced was demonstrated in these recombinant strains upon growth on sugar beet pulp, both immunochemically and by assaying enzyme activity. <em>abf</em> A <em></em> was shown to be expressed in the heterologous host <em>A.</em><em>nidulans,</em> despite the absence of an <em>abf</em> A <em></em> gene equivalent in this organism. High-copy number <em>A.</em><em>niger abf</em> B transformants featured impaired secretion of other extracellular proteins upon growth on sugar beet pulp. ABN A overproduction was found to be limited to approximately five times the wild-type level in <em>A.</em><em>niger abn</em> A transformants, but not in <em>A.</em><em>nidulans</em> transformants. Such a limitation was not observed in case of the ABFs.<p>In Chapters 5 and 6, the regulation of<font size="-1">L</font>-arabinan degradation is addressed. The structural genes seem to be regulated mainly at the transcriptional level. Additional copies of either A13F-encoding gene in <em>A.</em><em>niger</em> were shown to result in a reduction, but not in total silencing of the expression of the wild-type ABN Aencoding gene upon induction with either sugar beet pulp or<font size="-1">L</font>-arabitol ( <strong>Chapter 5</strong> ). The reduction of the expression level of <em>abn</em> A correlated <em></em> with the <em>abf</em> gene dosage. The repression effected by extra <em>abf</em> B gene <em></em> copies was more stringent and more persistent than that elicited by additional <em>abf</em> A copies. Although observed with both inducers, these phenomena were more outspoken and more persistent on sugar beet pulp. Similar, but more moderate effects were observed towards the expression of the other <em>abf</em> gene in multiple copy <em>abf</em> A- <em></em> and <em>abf</em> B-transformants. It was proposed that the <em>abf</em> genes titrate two distinct gene activators both involved in coordination of arabinase gene expression. However, the three genes were shown to respond differently upon a mycelial transfer to<font size="-1">L</font>-arabitol-containing medium, indicating that gene-specific factors are also involved. Four distinct sequence motifs were found in common in the promoter regions of the three genes. One of these elements is identical to the <em>A.</em><em>nidulans</em> CREA-motif, which has been shown to mediate carbon catabolite repression on several <em>A.</em><em>nidulans</em> enzyme systems. Arabinase expression in <em>A.</em><em>niger is</em> known to be repressed in the presence of<font size="-1">D</font>-glucose. Two other motifs are highly homologous to cAMP-responsive elements described in other organisms. For the fourth motif no functional analogues could be found, but the element was found to be present in several other fungal genes which are not involved in<font size="-1">L</font>-arabinan degradation at all. It is therefore likely that none of these common elements confer system-specific regulation.<p>The presumed involvement of<font size="-1">L</font>-arabitol in the induction process of fungal arabinases was further emphasized by the induction characteristics of an <em>A. nidulans</em> mutant unable to grow on the end-product of<font size="-1">L</font>-arabinan degradation,<font size="-1">L</font>-arabinose, nor on<font size="-1">L</font>-arabitol ( <strong>Chapter</strong> 6).<font size="-1">L</font>-Arabitol is an intermediate of<font size="-1">L</font>-arabinose catabolism in <em>Aspergilli.</em> This mutant was shown to lack NAD <sup><font size="-1">+</font></SUP>-dependent<font size="-1">L</font>-arabitol dehydrogenase activity resulting in<font size="-1">L</font>-arabitol accumulation, both intracellularly and in the culture medium, whenever<font size="-1">L</font>-arabinose is present. Upon submerged growth on various carbon sources in the presence of<font size="-1">L</font>-arabinose, the mutant featured enhanced expression of the enzymes involved in extracellular<font size="-1">L</font>-arabinan degradation, and of those of the intracellular<font size="-1">L</font>-arabinose catabolism. The co-substrates on which the mutant secreted large amounts of arabitol simultaneously exhibited high arabinase expression and featured reduced growth.<font size="-1">L</font>-Arabitol secretion and enzyme production were also observed on a mixed carbon source of<font size="-1">D</font>-glucose and<font size="-1">L</font>-arabinose, resulting in normal growth. Hence, in the presence of<font size="-1">L</font>- arabinose, the carbon catabolite repression conferred by<font size="-1">D</font>-glucose in the wild-type, is overruled in the mutant.<p>In <strong>Chapter 7</strong> , ABN A is shown to have remote sequence similarity with four bacterial xylanolytic glycosyl hydrolases (three β-<font size="-1">D</font>-xylosidases and an endo-1,4-β-<font size="-1">D</font>-xylanase), three of which feature activity against <em>para</em> -nitrophenyl-α-<font size="-1">L</font>-arabinofuranoside. This synthetic compound is commonly utilized to assay potential ABF activity, whereas it is known to be an inhibitor of the fourth enzyme. The homology became evident only after multi pie-sequence alignments and hydrophobic cluster analysis. It was proposed that these enzymes share a binding site for a terminal non-reducing α-linked<font size="-1">L</font>-arabinofuranosyl residue and that they all belong to glycosyl hydrolase family 43. Implications from these suggestions were discussed. The ABFs could not be assigned to an established glycosyl hydrolase family.<p>Based on the<font size="-1">L</font>-arabinolytic system of the brown-rot fungus <em>Monilinia fructigena,</em> the sequence similarity found amongst ABF A and bacterial pullulan-degrading enzymes, and ABF expression levels under carbon starvation conditions and on<font size="-1">D</font>-glucose as the carbon source, distinct functions in<font size="-1">L</font>-arabinan and plant cell-wall degradation were proposed for ABF A and ABF B. ABF A would be essentially cell-wall associated and act to degrade<font size="-1">L</font>-arabinan fragments generated by ABN A. ABF B activity would be important for the primary release of small amounts of<font size="-1">L</font>-arabinose which initiate induction of various endolytic systems to degrade plant cell walls, and thus function in substrate sensing. In line with these considerations, the involvement of other, not yet identified glycosyl hydrolases in<font size="-1">L</font>-arabinan degradation by <em>A.</em><em>niger</em> was suggested.<p>Induction and repression of arabinase gene expression are further discussed in <strong>Chapter 7</strong> . The results of the studies in <em>A.</em><em>niger</em> (Chapter 5) and <em>A.</em><em>nidulans</em> (Chapter 6) were interpreted in a mutual context. The identity of the lowmolecular-weight compound directly responsible for induction of arabinase gene expression, was addressed. Both<font size="-1">L</font>-arabinose and<font size="-1">L</font>-arabitol are likely candidates to fulfil such a role. However, it was not possible to weigh the actual inductive capacities of<font size="-1">L</font>-arabinose and<font size="-1">L</font>-arabitol due to their <em>in vivo</em> convertibility and the carbon catabolite repression elicited by the pentose. Competition for such a compound provides an alternative explanation for the phenomena observed in Chapter 5. The involvement of the transcriptional repressor CREA in arabinase gene expression is not limited to the direct repression of structural and regulatory genes of the<font size="-1">L</font>-arabinan-degrading system. It also plays a role in inducer exclusion and end-product repression, two processes shown to be eminently involved in the regulation of<font size="-1">L</font>-arabinan degradation in wild-type <em>A.</em><em>nidulans.</em> Fungal growth rate was suggested to be related to derepression of the<font size="-1">L</font>-arabinan-degrading system. The possible involvement of cAMP in arabinase gene expression, as suggested by the presence of potential <em>cis</em> -acting cAMP-responsive elements in the structural genes, was considered. Various ways by which cAMP might modulate arabinase synthesis were surveyed.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • van Ooyen, A.J.J., Promotor
  • Visser, J., Promotor
Award date2 May 1995
Place of PublicationS.l.
Publisher
Print ISBNs9789054853923
Publication statusPublished - 1995

Keywords

  • aspergillus
  • cell walls
  • carbohydrates
  • cellulose
  • cell membranes
  • fermentation
  • food biotechnology
  • glycosidases
  • polysaccharides
  • gene expression
  • pleiotropy
  • molecular genetics

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