Revisiting a 'simple' fungal metabolic pathway reveals redundancy, complexity and diversity

Abstract

Next to d-glucose, the pentoses l-arabinose and d-xylose are the main monosaccharide components of plant cell wall polysaccharides and are therefore of major importance in biotechnological applications that use plant biomass as a substrate. Pentose catabolism is one of the best-studied pathways of primary metabolism of Aspergillus niger, and an initial outline of this pathway with individual enzymes covering each step of the pathway has been previously established. However, although growth on l-arabinose and/or d-xylose of most pentose catabolic pathway (PCP) single deletion mutants of A. niger has been shown to be negatively affected, it was not abolished, suggesting the involvement of additional enzymes. Detailed analysis of the single deletion mutants of the known A. niger PCP genes led to the identification of additional genes involved in the pathway. These results reveal a high level of complexity and redundancy in this pathway, emphasizing the need for a comprehensive understanding of metabolic pathways before entering metabolic engineering of such pathways for the generation of more efficient fungal cell factories.

Fig. 1

The links between the sugar catabolic pathways of A. niger and in more detail the pentose catabolic pathway (PCP). LarA = l‐arabinose reductase, LadA = l‐arabitol dehydrogenase, LxrA and LxrB = l‐xylulose reductases, SdhA = sorbitol dehydrogenase, XyrA and XyrB = d‐xylose reductases, XdhA = xylitol dehydrogenase, XkiA = d‐xylulose kinase. Enzymes identified in this work to be involved in each step of the pathway are indicated in magenta.

Fig. 2

(A) Growth profile of the A. niger reference strain (N593 ΔkusA) and the PCP deletion mutants on solid MM with or without addition of carbon source. Strains were grown for 5 days at 30°C. Variation in colony diameter between replicates is < 1 mm. (B) Intracellular accumulation of intermediate metabolites of the PCP, after 2 h transfer of the mycelia to 25 mM d‐xylose or l‐arabinose. (C) PCP enzyme activities (mmol/min per mg of mycelia) of the A. niger reference strain and the PCP deletion mutants, after 2 h transfer of the mycelia to 25 mM d‐xylose or l‐arabinose. The error bars represent the standard deviation between biological replicates. Statistically significant differences from the reference strain (based on T‐test, P < 0.05) are indicated by an asterisk.

Fig. 3

Presence of orthologues of the A. niger PCP genes in 28 fungal genome sequences covering the phyla Basidiomycota and Ascomycota. The figure is based on a BlastP analysis using the A. niger PCP genes as queries. The resulting hits were used in a phylogenetic analysis to identify orthologues. For the Agaricomycetes, no distinction could be made between the XdhA and SdhA orthologues. Mucor lusitanicus was used as an outlier.

Fig. 4

Hierarchical clustering of (A) the intracellular concentration (mM) of PCP intermediate metabolites, (B) PCP enzyme activities (mmol/min per mg of mycelia), (C) growth on pentose sugars and polyols (the colour code displayed represents the difference in colony diameter) and (D) the expression profiles (the colour code displayed represents averaged and logged expression values (FPKM + 1) of triplicates) of genes linked to pentose catabolism in the A. niger reference strain and PCP mutants, after 2 h transfer of the mycelia to 25 mM l‐arabinose (A) or D‐xylose (X). No significant differences were observed for the expression profile of the strains on d‐glucose (data not shown). LAR = l‐arabinose reductase, LAD = l‐arabitol dehydrogenase, LXR = l‐xylulose reductase, XYR = d‐xylose reductase, XDH = xylitol dehydrogenase.