Abstract
Aspergillus niger is a filamentous fungus well known for its ability to produce a wide variety of pectinolytic enzymes, which have many applications in the industry. The transcriptional activator GaaR is induced by 2-keto-3-deoxy-L-galactonate, a compound derived from D-galacturonic acid, and plays a major role in the regulation of pectinolytic genes. The requirement for inducer molecules can be a limiting factor for the production of enzymes. Therefore, the generation of chimeric transcription factors able to activate the expression of pectinolytic genes by using underutilized agricultural residues would be highly valuable for industrial applications. In this study, we used the CRISPR/Cas9 system to generate three chimeric GaaR-XlnR transcription factors expressed by the xlnR promoter by swapping the N-terminal region of the xylanolytic regulator XlnR to that of the GaaR in A. niger. As a test case, we constructed a PpgaX-hph reporter strain to evaluate the alteration of transcription factor specificity in the chimeric mutants. Our results showed that the chimeric GaaR-XlnR transcription factor was induced in the presence of D-xylose. Additionally, we generated a constitutively active GaaR-XlnR V756F version of the most efficient chimeric transcription factor to better assess its activity. Proteomics analysis confirmed the production of several pectinolytic enzymes by ΔgaaR mutants carrying the chimeric transcription factor. This correlates with the improved release of D-galacturonic acid from pectin by the GaaR-XlnR V756F mutant, as well as by the increased L-arabinose release from the pectin side chains by both chimeric mutants under inducing condition, which is required for efficient degradation of pectin.Key points• Chimeric transcription factors were generated by on-site mutations using CRISPR/Cas9.• PpgaX-hph reporter strain allowed for the screening of functional GaaR-XlnR mutants.• Chimeric GaaR-XlnR induced pectinolytic activities in the presence of D-xylose.
Highlights
Filamentous fungi are efficient degraders of plant biomass, ensuring an essential role in the global carbon cycle
The xylanolytic transcription factor XlnR was the firstcellulolytic transcription factor described in A. niger, and it is the most studied transcriptional activator involved in the regulation of plant biomass degradation
We showed that the chimeric GaaR-XlnR transcription factor A. niger mutant was able to secrete enzymes required for the degradation of pectin when growing on pectin supplemented with D-xylose as inducer compound
Summary
Filamentous fungi are efficient degraders of plant biomass, ensuring an essential role in the global carbon cycle. Fungal Zn2Cys transcriptional activators play a key role in the regulation of enzyme production by activating the expression of genes encoding for enzymes required for the degradation of the substrates found in the environment. Aspergillus niger has a long history of safe application and is often used in industry for the production of valuable metabolites and enzymes (Cairns et al 2018) This fungus is well known to possess a large array of genes encoding pectinases and accessory enzymes (Martens-Uzunova and Schaap 2009). The xylanolytic transcription factor XlnR was the first (hemi-)cellulolytic transcription factor described in A. niger (van Peij et al 1998b), and it is the most studied transcriptional activator involved in the regulation of plant biomass degradation. We showed that the chimeric GaaR-XlnR transcription factor A. niger mutant was able to secrete enzymes required for the degradation of pectin when growing on pectin supplemented with D-xylose as inducer compound
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