W361R mutation in GaaR, the regulator of D‐galacturonic acid‐responsive genes, leads to constitutive production of pectinases in Aspergillus niger

Abstract

Polysaccharides present in plant biomass, such as pectin, are the main carbon source for filamentous fungi. Aspergillus niger naturally secretes pectinases to degrade pectin and utilize the released monomers, mainly D‐galacturonic acid. The transcriptional activator GaaR, the repressor of D‐galacturonic acid utilization GaaX, and the physiological inducer 2‐keto‐3‐deoxy‐L‐galactonate play important roles in the transcriptional regulation of D‐galacturonic acid‐responsive genes, which include the genes encoding pectinases. In this study, we described the mutations found in gaaX and gaaR that enabled constitutive (i.e., inducer‐independent) expression of pectinases by A. niger . Using promoter‐reporter strains (P pgaX ‐ amdS ) and polygalacturonic acid plate assays, we showed that W361R mutation in GaaR results in constitutive production of pectinases. Analysis of subcellular localization of C‐terminally eGFP ‐tagged GaaR/Gaa R W 361R revealed important differences in nuclear accumulation of N‐ versus C‐terminally eGFP ‐tagged GaaR.

CRISPR/Cas9 facilitates rapid generation of constitutive forms of transcription factors in Aspergillus niger through specific on-site genomic mutations resulting in increased saccharification of plant biomass

“…To obtain a constitutively active GaaR [21], a T  C transition in a TGG codon is required, resulting in a tryptophan-361-arginine (W361R) mutation. The gRNA sequence (P3-GaaR, P4-GaaR, Table S1) with the highest on-target activity was predicted by Geneious based on the experimentally determined predictive model proposed by Doench, et al [31].…”

“…The pectinolytic transcription factor GaaR was also reported to show constitutive activity caused by a single point mutation in A. niger [21]. The endogenous gaaR gene was deleted and replaced with a DNA construct carrying a W361R point mutation.…”

“…The endogenous gaaR gene was deleted and replaced with a DNA construct carrying a W361R point mutation. Alazi et al [21] proposed that this mutation disrupts the interaction between GaaR and its repressor, GaaX [22], under non-inducing conditions.…”

“…So far, attempts to generate constitutively active transcription factor mutants involved either in site-directed mutagenesis of the target gene and its insertion in a specific genomic loci [19], the deletion of entire C-terminal regions of the target genes [23], or the insertion of a mutant allele in the deleted locus of the endogenous gene [21]. These are relatively labor-intensive approaches, which also may cause subtle additional changes at the site of integration or deletion that could further effect the phenotype.…”

Modulating Transcriptional Regulation of Plant Biomass Degrading Enzyme Networks for Rational Design of Industrial Fungal Strains

“…Apart from identifying GaaX as a repressor for GA-induced genes expression, it also brought about the identification of a constitutive allele of GaaR (GaaR W361R ) resulting in constitutive expression of pectinases under even repressing conditions (on glucose or fructose). Within GaaR, W361 is situated in the fungal-specific TF domain and is highly conserved among Aspergillus species (Alazi et al, in press ).…”