A molecular analysis of (hemi-)cellulose degradation by Aspergilli

Glycosylhydrolases like cellulases and xylanases are of great importance for the ecological recycling of biomass. The saprophytic fungi, e.g Aspergillus niger , are capable of degrading plant cell wall material by secreting these enzymes. Because of their properties, a whole range of commercial enzyme preparations containing fungal polysaccharidases is used in industrial applications. For example, xylanases and cellulases are used in food and feed applications, and in pulp and paper applications, whereas cellulases are also used the textile industry in biostoning applications. The aim research described in this thesis is to clone genes encoding novel activities capable of degrading (hemi-)cellulose. Many of these novel activities are minor activities, which are difficult to detect and identify using standard conditions for growth. Several strategies will therefore be exploited to find these novel activities in Aspergilli. Firstly, the conditions of induction need to be optimal. This involves both the carbon source as well as the duration of growth (Chapter 4). Secondly, accumulation of the inducer often results in higher levels of expression (Chapter 3).Also the use of derepressed creA<sup>d</SUP>strains can result in elevated levels of expression as is shown in Chapters 2 and 3. Chapter 3 also shows that gene disruption of majorα-L-arabinofuranosidase can reveal minor activities which are otherwise difficult to identify. Finally, increasing the gene dosage of a specific transcriptional activator can increase the transcription levels of genes controlled by that activator (Chapters 5 and 6). Despite the fact that many glycosylhydrolases and their encoding genes have been isolated from a wide variety of microorganisms, little is known at the molecular level about the factors that are involved in the expression of these genes. The research described in this thesis gives a better understanding of the mechanisms underlying the regulation of expression of genes encoding cellulose- and hemicellulose-degrading enzymes produced by Aspergillus .Chapter 2 describes the isolation of A. niger creA<sup>d</SUP>mutants relieved of carbon repression and the effects of the mutations on the expression of arabinanases and L-arabinose catabolic enzymes. Carbon repression is a global regulatory mechanism by which in the presence of D-glucose or other rapidly metabolisable carbon sources the expression of genes involved in the utilisation of less-favoured carbon sources is repressed. The arabinanolytic system was selected to illustrate that the expression of genes involved in the utilisation of less-favoured carbon sources could be enhanced in derepressed creA<sup>d</SUP>mutant strains.Chapters 3 and 4 focus on genes encoding enzymes that are able to release L-arabinose from arabinoxylan. Chapter 3 describes the cloning and characterisation of the A. nidulans abfB gene, as well as the analysis of expression of the L-arabinofuranosidase encoding gene in an A. nidulans wild-type strain and several mutant strains. Super-induction of the abfB gene can be accomplished by combining a mutation leading to the intracellular accumulation of an inducer with a creA<sup>d</SUP>mutation, as is shown by expression analyses. This chapter also describes the identification of minor L-arabinose releasing activities when the majorα-L-arabinofuranosidase activity was disrupted in a derepressed creA<sup>d</SUP>genetic background.The cloning, characterisation and analysis of expression of the axhA genes from the closely related fungi A. niger and A. tubingensis is described in Chapter 4. These genes encode an arabinoxylan-arabinofuranohydrolase A enzyme which specifically releases L-arabinose substituents from arabinoxylan. This chapter also describes the transcriptional analysis of the axhA and abfB genes in A. niger . It demonstrates that the regulation of transcription of the two genes differs significantly, although both genes encode L-arabinose releasing activities.The transcription of genes encoding enzymes involved in xylan degradation and two endoglucanases involved in the degradation of cellulose in A. niger is studied in Chapter 5. In particular, the role of the xylanolytic transcriptional activator XlnR in the regulation of transcription of these genes was investigated. This analysis is extended in Chapter 6, which describes the cloning and characterisation of two cellobiohydrolase encoding genes in A. niger , cbhA and cbhB , which are also involved in the degradation of cellulose. The results described in Chapters 5 and 6 illustrate that the range of genes transcriptionally regulated by a specific activator is not necessarily limited to genes encoding pathway specific enzymes but that it also can include genes encoding non-pathway specific activities.Furthermore, the data described in these chapters give evidence that increasing the gene dosage of a specific transcriptional activator could elevate the expression of a broad range of genes controlled by that activator. Based on data described in this thesis a working model is proposed in which XlnR plays a central role in the degradation of plant cell walls