Harnessing the CRISPR/Cas9 system to study the regulatory network of plant biomass degradation in Aspergillus niger

Filamentous fungi are primary degraders of plant biomass, and therefore play a pivotal role in the bio-based economy. For this, the understanding of their abilities and limits regarding plant biomass degradation is essential. The regulation of plant biomass degradation in Aspergillus niger is controlled by a complex and intricate system involving the interaction of multiple transcription factors (TFs). In this thesis, marker-free CRISPR/Cas9 gene editing was performed to generate gene deletions (Chapters 5, 6, 7 and 8), precise point mutations (Chapter 3), domain swapping (Chapter 4), and gene tagging by fluorescent markers targeting several major TFs coordinating plant biomass degradation. In Chapter 3, by introducing single point mutations, we generated constitutively active forms of two key transcription factors, XlnR and GaaR, involved in (hemi-)cellulose and pectin utilization, respectively. In Chapter 4, we showed the generation of a chimeric GaaR-XlnR transcription factor mutant strain, that was able to control the expression of pectinolytic genes when induced by D-xylose, offering opportunities for the production of specific enzyme sets by cultivation on low cost agro-industrial substrates. The analysis of single and combinatorial gene deletion strains generated by CRISPR/Cas9 described in this thesis expanded the knowledge about the individual roles and interactions of several major TFs involved in the regulation of plant biomass utilization. In Chapter 5, using transcriptomic data, we identified the inducer and regulon of ClrB in A. niger and showed that ClrB influences the expression of clrA. Additional TF interactions have been revealed by the combined analysis of single and combinatorial (hemi-)cellulolytic and pectinolytic TF deletion strains in Chapter 7 and Chapter 8. In Chapter 7, proteomic data showed the relative role of XlnR, ClrB and ClrA in the degradation of wheat bran, and revealed several cases of gene co-regulation. In Chapter 8, transcriptomic data showed the upregulation of (hemi-)cellulolytic genes when major pectinolytic TFs were deleted, providing evidence for the antagonistic interaction between (hemi-)cellulose and pectin utilization, and the adaptation to the utilization of alternative components of sugar beet pulp. Finally, data described in Chapter 6 and Chapter 8 also support the observation that phenotypes of solid and liquid cultures cannot be directly compared. Although the characteristics of submerged fungal cultures is valuable for industrial applications, these do not represent the natural behavior of the fungus. Taken together, the results described in this thesis show various applications of the CRISPR/Cas9 system for the engineering of TFs involved in the regulation of plant biomass utilization and allowed for the analysis of several TFs on a network level in A. niger. Moreover, the deletion of major TFs involved in polysaccharide utilization can reveal putative back-up regulatory mechanisms, which can possibly indicate the action of novel TFs involved in this process. Additionally, the combined deletion of TFs regulating the expression of CAZy-encoding genes as well as the deletion of the protease regulator gene prtT can result in strains suitable for cell factories with impaired background protein production