Abstract
ObjectiveWith 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.ResultPfaeB-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.ConclusionLoss of function of CreA leads to low but inducer-independent expression from the faeB promoter in A. niger.
Highlights
As the most abundant organic carbon source available, plant biomass has a great potential to replace fossil fuels for renewable fuels and chemicals
Optimal enzyme cocktails are necessary for the efficient degradation of the lignocellulose into monosaccharides and aromatics
Ferulic acid is an important cross-linker in plant cell walls causing inefficient hydrolysis
Summary
As the most abundant organic carbon source available, plant biomass has a great potential to replace fossil fuels for renewable fuels and chemicals. The recalcitrant nature of the lignocellulose is mainly derived from cell wall composition and architecture, and comprises different factors such as cellulose crystallinity, hemicellulose polymerization and substitution pattern, lignin content and composition, and the occlusion of the cell wall by lignin-hydroxycinnamate-hemicellulose cross-linking (McCann and Carpita 2015; Oliveira et al 2015, 2019). Ferulic acid (4-hydroxy-3-methoxycinnamic acid) plays a key role in inter- and intra-polymer cross-linking (Terrett and Dupree 2019). Because ferulic acid plays such a crucial role in crosslinking, feruloyl esterases play a key role to remove those cross-links and make the cellulose, hemicellulose and pectin accessible for hydrolysis by cellulases, hemicellulases and pectinases. Fungal feruloyl esterases have been shown to act synergistically with cellulases and xylanases to facilitate the degradation of complex plant cell wall biomass (Selig et al 2008; Tabka et al 2006; Wong et al 2013)
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