Open Access

Hannes Blase

• Carbon catabolite repression (CCR) is a crucial regulatory mechanism facilitating the preferential utilization of the most favorable carbon source in the environment, and is mediated by a key regulator, CreA, in most fungi. The ascomycete Aspergillus niger (A. niger) is of particular in-terest for biotechnological applications due to its natural ability to secrete carbohydrate-active enzymesCarbon catabolite repression (CCR) is a crucial regulatory mechanism facilitating the preferential utilization of the most favorable carbon source in the environment, and is mediated by a key regulator, CreA, in most fungi. The ascomycete Aspergillus niger (A. niger) is of particular in-terest for biotechnological applications due to its natural ability to secrete carbohydrate-active enzymes (CAZymes) targeting plant biomass. The presence of easily metabolizable sugars such as glucose, which accumulate during the bioconversion of plant biomass, results in the repres-sion of CAZymes-encoding genes through CreA-mediated CCR. Consequently, CCR poses an economic challenge when easily metabolizable monosaccharides are present in the substrate. For this purpose, metabolic engineering strategies targeting CreA are employed to minimize the im-pact of CCR and enhance the enzymatic productivity of A. niger. Applying these strategies often results in reduced CreA-mediated CCR on one hand, but also lead to phenotypic side effects, including impaired growth and sporulation on the other hand. This study establishes that the conserved region (consv) is primarily responsible for the repressive properties of CreA, as the consv deletion of CreA leads to the strongest CC derepression of a luciferase CCR reporter compared to strains with different deletions and truncations of CreA. The consv deletion strain also exhibits no impairment of growth and sporulation compared to CreA wild type (WT) strain. The application of random mutagenesis to reduce CCR showed to be challenging, as no muta-tions were induced in the creA gene in mutants selected using a pyrG/hygR CCR reporter. The pyrG/hygR reporter, utilizing the conserved Cre-binding motif 5’-SYGGRG-3’ along with the selection markers pyrG and hph, could be an efficient tool with adjusted and further developed conditions to screen mutants less sensitive to CCR in future. Additionally, this study demon-strates that the expression of a CCR inactive truncation CreA*(1-162) under the inducible, non-CCR repressible, and cost-efficient sucA and bphA promoters of A. niger does not lead to a re-lease in CCR by displacement of native CreA. However, these promoters could be promising for further evaluating the mechanisms of CCR. In conclusion, this study anticipates that a com-bination of various metabolic engineering strategies in strain engineering for bioconversion of plant biomass, could efficiently alleviate CCR with minimal impact on phenotypical develop-ment as well lead to more knowledge about the mechanisms of CCR.…