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

Abstract

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 (PpgaX‐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/GaaRW361R revealed important differences in nuclear accumulation of N‐ versus C‐terminally eGFP‐tagged GaaR.

Keywords

• Aspergillus niger
• constitutively active transcription factor
• CRISPR‐Cas9
• missense mutation
• pectinase
• transcription factor localization