The Role of transcription factors XlnR, ClrA and ClrB and the protein kinases PkcA, SchA and SnfA in the regulation of cellulase-encoding genes in Aspergillus nidulans

Abstract

One of the most abundant natural resources is the plant cell wall structural component lignocellulose, which has gained importance for biofuel production as an environmentally friendlier alternative to petroleum-based energy resources. The degradation of plant biomass is catalysed in nature by enzymes from various different bacteria and from fungi such as Aspergillus nidulans and Trichoderma reesei. The genomes of both fungi encode many carbohydrate active enzymes (CAZy) which, through synergism, catalyse the deconstruction of cellulose and hemicelluloses, the main polysaccharides of lignocellulose. The regulation of the genes encoding these enzymes is governed by several transcription factors including the activator XlnR/XYR1 (A. nidulans/T. reesei) and the carbon catabolite repressor CreA/CRE1 (A. nidulans/T. reesei). Recently two additional transcription factors ClrA and ClrB were shown to be important for cellulase and hemicellulase gene expression in A. nidulans. These transcription factors are likely to respond to intracellular signals transduced and/or mediated by protein kinases in response to the presence of various different carbon sources. Indeed it was shown that deletion of the protein kinases PkcA, SnfA and SchA resulted in impaired cellulase activity in A. nidulans when incubated in the presence of cellulose. Despite an already great amount of knowledge on the regulation of genes encoding enzymes involved in lignocellulose degradation, there is substantial lack in the details of the mechanisms governing this regulation in Aspergilli spp. It has been reported that D-xylose, the main component of hemicelluloses, is absolutely required for the expression of hemicellulase (and cellulase)-encoding genes through triggering the phosphorylation of the main inducer XlnR. Several studies have indicated though that the regulation of the xylanolytic and cellulolytic system in Aspergilli spp. in the presence of cellulose and cellulose-derived inducers is likely to be regulated (at least partially) differently and mediated by additional regulatory factors than when compared to the regulatory mechanism in response to the presence of D-xylose. Furthermore, virtually nothing is known about how downstream components of signalling cascades, which are often initiated by G-proteins which sense the presence of a carbon source, mediate the response resulting in the deconstruction of the respective carbon source. The general aim of this project is therefore to further describe the regulation of cellulase-encoding genes by the transcription factors XlnR, ClrA and ClrB in A. nidulans and to investigate a potential relationship of these transcription factors with cellular signalling mechanisms. First, the binding of the transcription factors XlnR, ClrA, ClrB to the promoters of the main cellulase-encoding genes eglA and eglB will be described trough performing the appropriate EMSA experiments. Secondly, the effect of the protein kinases PkcA, SchA and SnfA, on the regulation of the transcription factors XlnR, ClrA and ClrB and consequently on the expression of cellulase-encoding genes will be described through determining the expression levels of the transcription factors in different strains. The cellular location of SchA, SnfA and PkcA will also be described. The results of this project will clarify regulatory mechanisms governing cellulase gene expression in A. nidulans. Furthermore, studying components of signal transduction mechanisms which are involved in various cellular processes including carbon source metabolism will provide novel insights into the regulation of cellulase-encoding genes and will identify new connections between transcription factors and signalling cascade components. The results of this study could be useful for strain improvement during industrial applications including using Aspergillus spp. for biofuel production.