Regulation of pentose utilisation by AraR, but not XlnR, differs in Aspergillus nidulans and Aspergillus niger

Abstract

Filamentous fungi are important producers of plant polysaccharide degrading enzymes that are used in many industrial applications. These enzymes are produced by the fungus to liberate monomeric sugars that are used as carbon source. Two of the main components of plant polysaccharides are L-arabinose and D-xylose, which are metabolized through the pentose catabolic pathway (PCP) in these fungi. In Aspergillus niger, the regulation of pentose release from polysaccharides and the PCP involves the transcriptional activators AraR and XlnR, which are also present in other Aspergilli such as Aspergillus nidulans. The comparative analysis revealed that the regulation of the PCP by AraR differs in A. nidulans and A. niger, whereas the regulation of the PCP by XlnR was similar in both species. This was demonstrated by the growth differences on L-arabinose between disruptant strains for araR and xlnR in A. nidulans and A. niger. In addition, the expression profiles of genes encoding L-arabinose reductase (larA), L-arabitol dehydrogenase (ladA) and xylitol dehydrogenase (xdhA) differed in these strains. This data suggests evolutionary changes in these two species that affect pentose utilisation. This study also implies that manipulating regulatory systems to improve the production of polysaccharide degrading enzymes, may give different results in different industrial fungi.

Fig. 1

Orthologous gene cluster of AraR. Graphic presentation of the genomic region of A. nidulans araR and comparison to other Aspergilli (Crabtree et al. 2007). AN0384 conserved serine rich protein, AN0385 putative branched-chain amino acid transaminase, AN0386 conserved hypothetical protein, AN0387 DNA photolyase, AN0388 AraR, AN0389 and AN0390 conserved hypothetical proteins, AN0391 NACHT domain-containing protein. The araR genes are connected by pink-shaded boxes. Nearby genes (in the 10-kb region on each side of araR) are shown, and those that are conserved are connected by grey-shaded boxes

Fig. 2

Multiple sequence alignment of AraR. The following amino acid sequences are aligned by ClustalW (Chenna et al. 2003): Neosartorya fischeri (NFIA023070), Aspergillus fumigatus (Afu1g01590), Aspergillus clavatus (ACLA032900), Aspergillus niger (An04g08600), Aspergillus nidulans (AN0388.3), Aspergillus oryzae (AO090003001292), Aspergillus flavus (AFL2G01779.2) and Aspergillus terreus (ATEG04909.1). Conserved amino acid residues are shown in grey. Single asterisk indicates identical residues. Semi-colon indicates conservative substitutions. Period indicate less conservative substitutions. Cysteine residues are indicated in green. Fungal-specific transcription-factor domain of unknown function (Suarez et al. 1995) is shown in yellow

Fig. 2

Multiple sequence alignment of AraR. The following amino acid sequences are aligned by ClustalW (Chenna et al. 2003): Neosartorya fischeri (NFIA023070), Aspergillus fumigatus (Afu1g01590), Aspergillus clavatus (ACLA032900), Aspergillus niger (An04g08600), Aspergillus nidulans (AN0388.3), Aspergillus oryzae (AO090003001292), Aspergillus flavus (AFL2G01779.2) and Aspergillus terreus (ATEG04909.1). Conserved amino acid residues are shown in grey. Single asterisk indicates identical residues. Semi-colon indicates conservative substitutions. Period indicate less conservative substitutions. Cysteine residues are indicated in green. Fungal-specific transcription-factor domain of unknown function (Suarez et al. 1995) is shown in yellow

Fig. 3

Growth of the A. niger and A. nidulans reference, ΔaraR, ΔxlnR and ΔaraR/ΔxlnR strains on 25 mM d-glucose, l-arabinose, l-arabitol, d-xylose and xylitol. The A. niger growth data was represented from our previous study (Battaglia et al. 2011) with permission from the publisher

Fig. 4

Expression analysis of the A. nidulans pentose catabolic pathway genes. All A. nidulans strains were transferred to 25 mM of the indicated carbon source for 2 h of growth. ladA l-arabitol dehydrogenase, larA l-arabinose reductase, xdhA xylitol dehydrogenase, xkiA xylulose kinase, xyrA d-xylose reductase, LC loading control. 1 d-fructose, 2 l-arabinose, 3 l-arabitol, 4 d-xylose, 5 xylitol

Fig. 5

The comparison of intracellular enzyme activities in A. nidulans and A. niger. l-2-keto-3-deoxyarabonatearabitol dehydrogenase (Lad), xylitol dehydrogenase (Xdh), l-2-keto-3-deoxyarabonatearabinose reductase (Lar), d-xylose reductase (Xyr) and l-2-keto-3-deoxyarabonatexylulose reductase (Lxr) activity were measured in the A. nidulans and A. niger reference, ΔaraR and ΔaraR/ΔxlnR strains. The A. niger enzyme activity data was represented from our previous study (Battaglia et al. 2011) with permission of the publisher. The enzyme activities are given in units per milligramme of the total protein