Leo H. de Graaff

Regulation of transcription of cellulases- and hemicellulases-encoding genes in Aspergillus niger and Hypocrea jecorina (Trichoderma reesei)

The filamentous fungi Aspergillus niger and Hypocrea jecorina (Trichoderma reesei) have been the subject of many studies investigating the mechanism of transcriptional regulation of hemicellulase- and cellulase-encoding genes. The transcriptional regulator XlnR that was initially identified in A. niger as the transcriptional regulator of xylanase-encoding genes controls the transcription of about 20–30 genes encoding hemicellulases and cellulases. The orthologous xyr1 (xylanase regulator 1-encoding) gene product of H. jecorina has a similar function as XlnR, although at points, the mechanisms seems to be different. Specifically in H. jecorina, the interaction of Xyr1 and the co-regulators Ace1 and Ace2 in the regulation of transcription of xylanases and cellulases has been studied. This paper describes the similarities and differences in the transcriptional regulation of expression of hemicellulases and cellulases in A. niger and H. jecorina.

The Aspergillus niger transcriptional activator XlnR, which is involved in the degradation of the polysaccharides xylan and cellulose, also regulates d‐xylose reductase gene expression

Screening of an Aspergillus niger differential cDNA library, constructed by subtracting cDNA fragments of a xlnR loss‐of‐function mutant from wild‐type cDNA fragments, resulted in the cloning of the gene encoding d‐xylose reductase (xyrA). Northern blot analysis using an A. niger wild‐type strain, a xlnR multiple‐copy strain and a xlnR loss‐of‐function mutant confirmed that the xyrA gene is regulated by XlnR, the transcriptional activator of the xylanolytic enzyme system in A. niger. d‐xylose reductase catalyses the NADPH‐dependent reduction of d‐xylose to xylitol, which is the first step in d‐xylose catabolism in fungi. Until now, XlnR was shown to control the transcription of genes encoding extracellular hydrolytic enzymes involved in cellulose and xylan degradation. In the present study, we show that A. niger is able to harmonize its sugar metabolism and extracellular xylan degradation via XlnR by regulating the expression of XyrA.

EglC, a New Endoglucanase from Aspergillus niger with Major Activity towards Xyloglucan

ABSTRACT A novel gene, eglC, encoding an endoglucanase, was cloned from Aspergillus niger. Transcription of eglC is regulated by XlnR, a transcriptional activator that controls the degradation of polysaccharides in plant cell walls. EglC is an 858-amino-acid protein and contains a conserved C-terminal cellulose-binding domain. EglC can be classified in glycoside hydrolase family 74. No homology to any of the endoglucanases from Trichoderma reesei was found. In the plant cell wall xyloglucan is closely linked to cellulose fibrils. We hypothesize that the EglC cellulose-binding domain anchors the enzyme to the cellulose chains while it is cleaving the xyloglucan backbone. By this action it may contribute to the degradation of the plant cell wall structure together with other enzymes, including hemicellulases and cellulases. EglC is most active towards xyloglucan and therefore is functionally different from the other two endoglucanases from A. niger, EglA and EglB, which exhibit the greatest activity towards β-glucan. Although the mode of action of EglC is not known, this enzyme represents a new enzyme function involved in plant cell wall polysaccharide degradation by A. niger.

CreA mediates repression of the regulatory gene xlnR which controls the production of xylanolytic enzymes in Aspergillus nidulans

The Aspergillus nidulans xlnR gene encodes a Zn(2)Cys(6) transcription activator necessary for the synthesis of the main xylanolytic enzymes, i.e. endo-xylanases X(22), X(24) and X(34), and beta-xilosidase XlnD. Expression of xlnR is not sufficient for induction of genes encoding the xylanolytic complex, the presence of xylose is absolutely required. It has been established previously that the wide-domain carbon catabolite repressor CreA indirectly represses xlnA (encodes X(22)) and xlnB (encodes X(24)) genes as well as exerting direct repression on xlnA. This work provides evidence that CreA-mediated indirect repression occurs through repression of xlnR: (i) the xlnR gene promoter is repressed by glucose and this repression is abolished in creA(d)30 mutant strains and (ii) deregulated expression of xlnR completely relieves glucose repression of xlnA and xlnB. Thus, CreA and XlnR form a transcriptional cascade regulating A. nidulans xylanolytic genes.

Purification and Characterization of Two Different a-L-Rhamnosidases, RhaA and RhaB, from Aspergillus aculeatus

ABSTRACT Two proteins exhibiting α-l-rhamnosidase activity, RhaA and RhaB, were identified upon fractionation and purification of a culture filtrate from Aspergillus aculeatus grown on hesperidin. Both proteins were shown to be N glycosylated and had molecular masses of 92 and 85 kDa, of which approximately 24 and 15%, respectively, were contributed by carbohydrate. RhaA and RhaB, optimally active at pH 4.5 to 5, showedKm and Vmaxvalues of 2.8 mM and 24 U/mg (RhaA) and 0.30 mM and 14 U/mg (RhaB) when tested forp-nitrophenyl-α-l-rhamnopyranoside. Both enzymes were able to hydrolyze α-1,2 and α-1,6 linkages to β-d-glucosides. Using polyclonal antibodies, the corresponding cDNA of both α-l-rhamnosidases,rhaA and rhaB, was cloned. On the basis of the amino acid sequences derived from the cDNA clones, both proteins are highly homologous (60% identity).

Cloning of the Trichoderma reesei pyrG gene and its use as a homologous marker for a high-frequency transformation system

SummaryThe Trichoderma reesei orotidine-5′-phosphate decarboxylase gene was isolated by heterologous hybridization with the corresponding Neurospora gene as a probe. A 2.7 kb SalI fragment, which exclusively hybridized to the Neurospora gene, was subcloned in pGEM-5Zf(+). This subclone was termed pFG1 and was used to transform a Trichoderma reesei pyrG- negative mutant to PYR+. The transformation frequency in this homologous system was up to 12000 transformants per μg DNA. About one-fifth of the transformants tested were abortive. Perfect mitotic stability was found in half of the non-abortive transformants, correlating with vector integration at homologous and ectopic loci. In the unstable transformants the transforming DNA appears to be present in the form of extrachromosomal elements.

Cloning and characterization of the abfB gene coding for the major α-l-arabinofuranosidase (ABF B) of Aspergillus niger

Based on amino-acid sequence data from Aspergillus niger α-l-arabinofuranosidase B (ABF B), and cyanogen bromide fragments derived thereof, deoxyoligonucleotide mixtures were designed to the employed as primers in a polymerase chain reaction (PCR) on A. niger genomic DNA. This resulted in amplification of three related PCR products. The abfB gene encoding ABF B was isolated from a genomic library using such an amplification product as a probe. A 5.1-kb BamHI fragment was subcloned to result in plasmid pIM991. Upon introduction by co-transformation into both A. niger and A. nidulans uridine auxotrophic strains, pIM991 was shown to contain the functional gene since prototrophic transformants overproduced ABF B upon growth on the inducing carbon source sugar beet pulp. A plate assay was developed enabling quick selection of ABF B-overproducing transformants. The sequence of a 4122-bp long BamHI/SstI fragment was determined. The abfB gene does not contain introns and codes for a protein of 499 amino acids. The mature ABF B, 481 amino acids in length, has a deduced molecular weight of 50.7 kDa. A. niger abfB is the first eukaryotic gene encoding an ABF to be characterized.

Characterization of the Aspergillus niger pelB gene: structure and regulation of expression

SummaryThe nucleotide sequence of pelB, a member of the Aspergillus niger pectin lyase multigene family, has been determined. The pelB gene product, PLB, shares 65% amino acid identity with pectin lyase A (PLA) and 60% with pectin lyase D (PLD). Although growth of pelB multicopy transformants on pectin-containing media results in elevated pelB mRNA levels, pectin lyase B (PLB) is barely detectable. This is probably due to degradation of PLB by acid proteases, since multicopy transformants grown on pectin medium with a high concentration of phosphate, leading to a less rapid decline in pH, secrete detectable amounts of PLB. To produce PLB in high amounts under conditions where few other extracellular enzymes are present, we tried two strategies. Firstly, heterologous expression of the pelB gene in A. nidulans, and secondly, expression of the pelB gene under control of the constitutive A. niger pki promoter.

Dual transcriptional profiling of a bacterial/fungal confrontation: Collimonas fungivorans versus Aspergillus niger

Interactions between bacteria and fungi cover a wide range of incentives, mechanisms and outcomes. The genus Collimonas consists of soil bacteria that are known for their antifungal activity and ability to grow at the expense of living fungi. In non-contact confrontation assays with the fungus Aspergillus niger, Collimonas fungivorans showed accumulation of biomass concomitant with inhibition of hyphal spread. Through microarray analysis of bacterial and fungal mRNA from the confrontation arena, we gained new insights into the mechanisms underlying the fungistatic effect and mycophagous phenotype of collimonads. Collimonas responded to the fungus by activating genes for the utilization of fungal-derived compounds and for production of a putative antifungal compound. In A. niger, differentially expressed genes included those involved in lipid and cell wall metabolism and cell defense, which correlated well with the hyphal deformations that were observed microscopically. Transcriptional profiles revealed distress in both partners: downregulation of ribosomal proteins and upregulation of mobile genetic elements in the bacteria and expression of endoplasmic reticulum stress and conidia-related genes in the fungus. Both partners experienced nitrogen shortage in each others presence. Overall, our results indicate that the Collimonas/Aspergillus interaction is a complex interplay between trophism, antibiosis and competition for nutrients.

Construction of a Genetically Modified Wine Yeast Strain Expressing the Aspergillus aculeatus rhaA Gene, Encoding an -L-Rhamnosidase of Enological Interest

ABSTRACT The Aspergillus aculeatus rhaA gene encoding anα -l-rhamnosidase has been expressed in both laboratory and industrial wine yeast strains. Wines produced in microvinifications, conducted using a combination of the genetically modified industrial strain expressing rhaA and another strain expressing a β-glucosidase, show increased content mainly of the aromatic compound linalool.