Loss of function of the carbon catabolite repressor CreA leads to low but inducer‐independent expression from the feruloyl esterase B promoter in Aspergillus niger

• Published in: Biotechnology letters 2021-07, Vol.43 (7), p.1323-1336
• Main Authors: Reijngoud, Jos, Arentshorst, Mark, Ruijmbeek, Claudine, Reid, Ian, Alazi, Ebru Demirci, Punt, Peter J., Tsang, Adrian, Ram, Arthur F. J.
• Format: Article
• Language: English
• Subjects: Alcohol; Allyl alcohol; Applied Microbiology; Biochemistry; Biomedical and Life Sciences; Biotechnology; Carbon; Carbon sources; Catabolite repression; Esterase; Ferulic acid esterase; Fructose; Gene deletion; Gene regulation; Genetic screening; Genomes; Life Sciences; Microbiology; Mutants; Mutation; Original Research Paper; Point mutation; Single-nucleotide polymorphism
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-021-03104-2

Objective With the aim to decipher the mechanisms involved in the transcriptional regulation of feruloyl esterase encoded by faeB , a genetic screen was performed to isolate A. niger mutants displaying inducer-independent expression from the faeB promoter. Result PfaeB-amdS and PfaeB-lux dual reporter strains were constructed and used to isolate trans-acting mutants in which the expression of both reporters was increased, based on the ability to grow on acetamide plates and higher luciferase activity, respectively. The genetic screen on the non-inducing carbon source D-fructose yielded in total 111 trans-acting mutants. The genome of one of the mutants was sequenced and revealed several SNPs, including a point mutation in the creA gene encoding a transcription factor known to be involved in carbon catabolite repression. Subsequently, all mutants were analyzed for defects in carbon catabolite repression by determining sensitivity towards allyl alcohol. All except four of the 111 mutants were sensitive to allyl alcohol, indicating that the vast majority of the mutants are defective in carbon catabolite repression. The creA gene of 32 allyl alcohol sensitive mutants was sequenced and 27 of them indeed contained a mutation in the creA gene. Targeted deletion of creA in the reporter strain confirmed that the loss of CreA results in constitutive expression from the faeB promoter. Conclusion Loss of function of CreA leads to low but inducer-independent expression from the faeB promoter in A. niger.