Combinatorial control of gene expression in Aspergillus niger grown on sugar beet pectin

Abstract

Aspergillus niger produces an arsenal of extracellular enzymes that allow synergistic degradation of plant biomass found in its environment. Pectin is a heteropolymer abundantly present in the primary cell wall of plants. The complex structure of pectin requires multiple enzymes to act together. Production of pectinolytic enzymes in A. niger is highly regulated, which allows flexible and efficient capture of nutrients. So far, three transcriptional activators have been linked to regulation of pectin degradation in A. niger. The L-rhamnose-responsive regulator RhaR controls the production of enzymes that degrade rhamnogalacturonan-I. The L-arabinose-responsive regulator AraR controls the production of enzymes that decompose the arabinan and arabinogalactan side chains of rhamnogalacturonan-II. The D-galacturonic acid-responsive regulator GaaR controls the production of enzymes that act on the polygalacturonic acid backbone of pectin. This project aims to better understand how RhaR, AraR and GaaR co-regulate pectin degradation. For that reason, we constructed single, double and triple disruptant strains of these regulators and analyzed their growth phenotype and pectinolytic gene expression in A. niger grown on sugar beet pectin.

Figure 1

Phenotypic analysis of A. niger reference strain and regulatory mutants on pectin and pectin-related carbon sources.

Figure 2

Monomeric sugars released from 3% SBP with crude extracellular enzyme mix from 8 h cultures of A. niger reference strain and regulatory mutants. Significant changes (P-values ≤ 0.05) between the mutant and the reference strain were indicated by an asterisk.

Figure 3

Venn diagram showing an overlap between GaaR-, AraR- and RhaR-dependent genes in A. niger grown on SBP for 2 h (a) and 8 h (b). Genes with no biochemical validation available are marked by an asterisk. The gene abbreviations and CAZy family numbers are listed in Supplementary datasets S2 and S3. Genes that were previously identified as GaaR-dependent on SBP by Alazi et al. were marked in bold. Expression of genes under combinatorial control of two or three TFs was evaluated and, if possible, the predominant activator was indicated (violet letters behind the gene name).

Figure 4

Hierarchical clustering of genes linked to pectin utilization in the reference strain and regulatory mutants grown on SBP for 2 and 8 h. The color code displayed represents averaged and logged expression values (FPKM + 1) of duplicates. Genes classified as GaaR- (G), AraR- (A), RhaR-dependent (R) or not regulated after 2 and 8 h on SBP are indicated. Genes with no biochemical validation available are marked by an asterisk. The (putative) function of the genes can be found in Supplementary dataset S12.

Figure 5

RNA-seq analysis of genes involved in D-galacturonic acid (a), L-arabinose/D-xylose (b) and L-rhamnose (c) conversion in A. niger reference strain and regulatory mutants grown on SBP for 2 and 8 h. Expression values (FPKM) are averages of duplicates and presented as color gradient.

Figure 6

Hierarchical clustering of CAZy genes with significantly up-regulated expression levels in the triple mutant grown on SBP for 8 h. The color code displayed represents averaged and logged expression values (FPKM + 1) of duplicates. Clusters A-D can be distinguished. Genes with no biochemical validation available are marked by an asterisk. The (putative) function of the genes can be found in Supplementary dataset S12.