Molecular mechanisms shaping the plant biomass utilization potential in Aspergillus

Abstract

Coordination of the induction of genes encoding extracellular enzymes and sugar uptake systems in fungi are often mediated by transcription factors (TFs). The aim of this thesis was to study the transcriptional regulatory network in more detail by analysis of sugar-specific TFs in A. niger and A. nidulans. This research was focused on interactive aspects of regulation such as (1) combinatorial control of gene expression and (2) identification of novel transcriptional network motifs linking the TFs. In addition, a few previously characterized TFs involved in plant biomass degradation were re-evaluated in a broader context. Chapter 2 describes genetic interactions between A. nidulans AraR, XlnR and GalR in regulation of pentose and D-galactose catabolism. AraR and XlnR were previously shown to regulate genes encoding enzymes involved in L-arabinose and D-xylose conversion in both A. nidulans and A. niger, while GalR was linked to regulation of the Leloir D-galactose pathway. Our data showed that GalR is involved in the oxido-reductive D-galactose catabolic pathway in A. nidulans, but not in the pentose catabolic pathway. In contrast, AraR and XlnR not only control the pentose catabolic pathways, but also genes of the oxido-reductive D-galactose catabolic pathway. Chapter 3 describes characterization of D-galacturonic acid responsive TF GaaR in A. niger by studying the transcriptome response of the reference strain and ΔgaaR grown on D-galacturonic acid and pectin. GaaR was shown to mainly control expression of genes encoding enzymes involved in degradation of the homogalacturonan part of pectin as well as D-galacturonic acid transport and metabolism. Complete degradation of pectin might require co-operative action of several TFs in A. niger. Previous studies identified two TFs involved in degradation of pectin, AraR, responding to L-arabinose, and RhaR, responding to L-rhamnose. Chapter 4 describes combinatorial control of gene expression by AraR, RhaR and the recently characterized GaaR in A. niger grown on sugar beet pectin. The results of this study demonstrate that GaaR, AraR and RhaR are all involved in regulation of pectinolytic gene expression during growth on a complex carbon source, which is rich in pectin. Moreover, the study reveals genes under combined control of two or three TFs. Transcriptional regulatory networks are based on interactions between TFs and their target genes. In some cases, expression of a gene relies on two TFs, of which the ‘primary’ TF regulates the ‘modulating’ TF. The primary TF is activated by the presence of an inducer, while the modulating TF is transcriptionally activated by the primary TF. Chapter 5 describes the identification of two putative XlnR and AmyR-affected modulating TFs and asses their involvement in plant biomass degradation in A. niger. In Chapter 6, we studied the physiological role of XlnR and AraR in colonization of unprocessed wheat bran by high-resolution microscopy and exo-proteomics. Deletion of xlnR in A. niger reduced secretion of hemicellulolytic enzymes and resulted in reduced biofilm formation especially on the smooth surface of the wheat bran flake. This is the first in vivo demonstration of the physiological importance of these regulators. The results are summarized and discussed in Chapter 7.