Analysis of regulation of pentose utilisation in Aspergillus niger reveals evolutionary adaptations in Eurotiales

Abstract

Aspergilli are commonly found in soil and on decaying plant material. D-xylose and L-arabinose are highly abundant components of plant biomass. They are released from polysaccharides by fungi using a set of extracellular enzymes and subsequently converted intracellularly through the pentose catabolic pathway (PCP).In this study, the L-arabinose responsive transcriptional activator (AraR) is identified in Aspergillus niger and was shown to control the L-arabinose catabolic pathway as well as expression of genes encoding extracellular L-arabinose releasing enzymes. AraR interacts with the D-xylose-responsive transcriptional activator XlnR in the regulation of the pentose catabolic pathway, but not with respect to release of L-arabinose and D-xylose.AraR was only identified in the Eurotiales, more specifically in the family Trichocomaceae and appears to have originated from a gene duplication event (from XlnR) after this order or family split from the other filamentous ascomycetes. XlnR is present in all filamentous ascomycetes with the exception of members of the Onygenales. Since the Onygenales and Eurotiales are both part of the subclass Eurotiomycetidae, this indicates that strong adaptation of the regulation of pentose utilisation has occurred at this evolutionary node. In Eurotiales a unique two-component regulatory system for pentose release and metabolism has evolved, while the regulatory system was lost in the Onygenales. The observed evolutionary changes (in Eurotiomycetidae) mainly affect the regulatory system as in contrast, homologues for most genes of the L-arabinose/D-xylose catabolic pathway are present in all the filamentous fungi, irrespective of the presence of XlnR and/or AraR.

Fig. 1. A.

Expression analysis of the three XlnR homologues (An04g08600 (araR), An11g00140 and An11g06290) on D-fructose (1), L-arabinose (2), L-arabitol (3), D-xylose (4) and xylitol (5). B. Growth of the reference (UU-A049.1), ΔaraR (UU-A033.21) and ΔaraR::araR (UU-A054.4) on D-glucose and L-arabitol.

Fig. 2.

Bootstrapped (1000 bs) Maximum Parsimony tree of of putative homologues of XlnR and AraR in fungi. Homologues of the A. nidulans acetate regulatory protein (FacB) were used as an outgroup.

Fig. 3.

Growth of the reference strain (Ref., UU-A049.1), and the ΔaraR (UU-A033.21), ΔxlnR (UU-A062.10) and ΔaraR/ΔxlnR (UU-A063.22) strains on a selection of mono- and polysaccharides. Concentrations of the substrates were 25 mM for D-glucose, D-xylose, L-arabinose, L-arabitol, xylitol and glycerol, and 1 % for birchwood xylan, Arabic gum, guar gum, arabinan, arabinogalactan and apple pectin.

Fig. 4.

Comparison of intracellular and extracellular enzyme activities in reference and disruption strains. The reference strains (UU-A049.1), ΔaraR (UU-A033.21), ΔxlnR (UU-A062.10) and ΔaraR/ΔxlnR (UU-A063.22) were transferred for 2 and 4 h on 25 mM L-arabinose or 25 mM D-xylose. Extracellular α-L-arabinofuranosidase (Abf), the ratio of intracellular L-arabinose reductase (ArdA) and D-xylose reductase (XyrA) activity, and the intracellular activities of xylitol dehydrogenase (Xdh) and L-arabitol dehydrogenase (Lad). Black bars: L-arabinose, 2 h; grey bars: L-arabinose, 4 h; dashed bars: D-xylose, 2 h; white bars: D-xylose, 4 h.

Fig. 5.

Regulatory model for release and utilisation of D-xylose and L-arabinose in A. niger. ArdA = L-arabinose reductase; LadA = L-arabitol dehydrogenase; LxrA = L-xylulose reductase; XdhA = xylitol dehydrogenase; XyrA = D-xylose reductase; XkiA = D-xylulose kinase; AbfA, AbfB = α-L-arabinofuranosidase A and B; AbnA= endo-1,5-alpha-L-arabinanase; AxhA = arabinoxylan arabinofuranohydrolase; XlnB, XlnC = endoxylanases B and C; XlnD = β-xylosidase. The square depicts the fungal cell wall. AraR regulated genes are in blue. XlnR regulated genes are in yellow. Genes regulated by AraR and XlnR are in green. Inclusion of axhA, abnA, xlnB, xlnC, xlnD was based on co-regulation with the other genes as reported previously (Gielkens et al. 1997, van Peij et al. 1998a, de Groot et al. 2003).