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

Abstract

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.

Utilization of ferulic acid in Aspergillus niger requires the transcription factor FarA and a newly identified Far-like protein (FarD) that lacks the canonical Zn(II)2Cys6 domain

“…The A. niger strains used in this study are listed in Table 1. A. niger strain JR11.1 contains two reporter genes (PfaeB-amdS and PfaeB-lux 613) to monitor the activity of the faeB promoter via the ability to grow on acetamide plates or via luciferase assay (Reijngoud et al, 2021). Strains were grown on liquid or solidified (containing 1.5% (w/v) Scharlau agar (Scharlau, Barcelona, Spain)) minimal medium (MM) or on complete medium (CM) MM contains 7 mM KCl, 8 mM KH 2 PO 4 , 70 mM NaNO 3 , and 2 mM MgSO 4 (pH adjusted to 5.5) and spore elements as described (Arentshorst et al, 2012).…”

“…Several ferulyol esterases (FaeA-D) have been identified in A. niger , with expression of the genes being induced only under specific conditions. FaeA expression is induced on xylose, arabinose and several aromatics, whereas FaeB expression is only induced on aromatics ( de Vries et al., 2002 ; Reijngoud et al., 2021 ). FaeC is generally lowly expressed ( Dilokpimol et al., 2017 ; Oliveira et al., 2019 ; Lubbers et al., 2020 ) and no data are currently available for FaeD.…”

“…Both the genes encoding the enzymes involved in ferulic acid metabolism and the gene encoding feruloyl esterase B (FaeB) are induced in the presence of ferulic acid and other hydroxycinnamic acids ( de Vries et al., 2002 ; Reijngoud et al., 2021 ; Lubbers et al., 2021a ), suggesting that they might be controlled by the same transcription factor(s). We previously performed a screen to isolate A. niger mutants with constitutive expression of the faeB gene by using PfaeB-amdS and PfaeB-luciferase reporter strains ( Reijngoud et al., 2021 ). More than 100 of these mutants were isolated and all were found to be mutated in the creA gene encoding the major player in carbon catabolite repression.…”

Genome-wide transcription landscape of citric acid producing Aspergillus niger in response to glucose gradient

“…During fungal growth in both the natural niche and industrial bioprocesses, glucose is the preferred sugar over other carbon sources, which leads to the inactivation of the transport and metabolic pathway involved in the assimilation of alternative carbon sources. This preference was known as carbon catabolite repression (CCR) ( Reijngoud et al, 2021 ), resulting in the repression of catabolizing other alternative carbon sources until glucose was depleted ( Makela et al, 2018 ). In many filamentous fungi, the transcription repressor CreA is considered to be the major component of CCR, and its function and stability are regulated in response to glucose ( Reijngoud et al, 2021 ).…”

“…This preference was known as carbon catabolite repression (CCR) ( Reijngoud et al, 2021 ), resulting in the repression of catabolizing other alternative carbon sources until glucose was depleted ( Makela et al, 2018 ). In many filamentous fungi, the transcription repressor CreA is considered to be the major component of CCR, and its function and stability are regulated in response to glucose ( Reijngoud et al, 2021 ). Interestingly, however, Mäkelä et al found that A. niger takes up monomeric sugars in a sequential manner which is not mediated by CreA in some instances ( Makela et al, 2018 ).…”