Novel transcriptional activators of Aspergillus involved in plant biomass utilization

Fungi play an important role in the carbon cycle on Earth. Many fungi can degrade dead or living plant material that consist mainly of polysaccharides. These polysaccharides need to be degraded to monosaccharides before they can be taken up and used as nutrients by the fungal cell. Different polysaccharides, each with their unique composition, demand different enzymes for their complete hydrolysis. Fungi posses regulatory proteins which stimulate or repress the synthesis of specific enzyme sets depending on the available polysaccharides. Obtaining more knowledge about these regulatory proteins is also of applied interest as many industries use polysaccharide-degrading enzymes for a variety of applications. Specific regulatory proteins provide a tool to manipulate sets of enzymes rather than individual ones. This is particularly useful for applications where complete hydrolysis of the biomass is required (e.g. biofuel production). This thesis describes the identification and characterization of the regulatory proteins GalX, GalR and RhaR. GalX and GalR are regulators involved in D-galactose catabolism in Aspergilli. There are three pathways described for D-galactose catabolism within the Aspergilli: The Leloir pathway, the oxido-reductive D-galactose pathway and the DeLey-Doudoroff pathway. Regulation of D-galactose catabolism differs in Aspergilli.GalX regulates the expression of galR and ladB in A. nidulans. The ladB gene encodes a galactitol dehydrogenase that is part of the oxido-reductive pathway in A. niger and probably has a similar function in A. nidulans. GalR is unique to A. nidulans and regulates expression of genes of the Leloir pathway (galE and galD) and the oxido-reductive pathway (ladA). In A. niger, GalX regulates the oxido-reductive pathway genes ladB and sdhA, but does not substitute for the lack of GalR in this species as it does not regulate the expression of ladA and and genes from the Leloir pathway. In this thesis a redundancy of enzymes in the oxido-reductive pathway is suggested, and indications for the regulation of some of the genes by GalX were obtained. RhaR is a regulator of genes encoding L-rhamnose catabolic genes and genes encoding rhamnogalacturonan-I degrading enzymes. RhaR is not the only regulator involved in pectin degradation as genes encoding homogalacturonan, xylogalacturonan and pectinolytic side chain degrading enzymes are not under control of RhaR. In addition, this thesis describes a large scale transcriptional analysis of A. niger genes encoding (putative) plant polysaccharide degrading enzymes that identified potential target genes for known regulators. One of these regulators was AraR, for which 14 new potential target genes were identified in addition to the 3 target genes that were described previously. Some genes encoding enzymes that are active on the pectinolytic side chains were co-expressed and down-regulated in A. niger ΔaraR, suggesting that the arabinolytic regulator AraR also plays a role in pectin degradation. In A. nidulans, AraR appeared to be involved in D-galactose catabolism as A. nidulans ΔaraR showed reduced growth on D-galactose and galactitol. Although several regulators, target genes of regulators and interactions between regulators still need to be identified, the research described in this thesis adds new understanding of the complete regulatory network involved in the degradation of plant biomass