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Aspergillus niger strain:EA21.6

ABSTRACT: Overexpression of GaaR, a D-galacturonic acid responsive transcription factor, enables inducer-independent production of pectinases in Aspergillus niger

PROVIDER: PRJNA396925 | ENA |

REPOSITORIES: ENA

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The chimeric GaaR::XlnR transcription factor induces pectinolytic activities in the presence of D-xylose in Aspergillus niger

Project description: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 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. Additionally, we generated a constitutively active GaaR::XlnR V756F version of the most efficient chimeric transcription factor to better assess its activity. Our results showed that the chimeric GaaR::XlnR transcription factor was no longer induced in the presence of D-galacturonic acid, but in the presence of D-xylose instead. Moreover, proteomics analysis and saccharification assays confirmed the production of enzymes involved in the release of L-arabinose from pectin, while the constitutive version of this chimeric transcription factor showed consistently improved D-galacturonic acid release from pectin in a gaaR deletion background.

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Evidence for inducer function of 2-keto-3-deoxy-L-galactonate in D-galacturonic acid induced gene expression in Aspergillus niger

Project description:In Aspergillus niger, the enzymes encoded by gaaA, gaaB, gaaC and gaaD catabolize D-galacturonic acid (GA) consecutively into L-galactonate, 2-keto-3-deoxy-L-galactonate, pyruvate and L-glyceraldehyde, and glycerol. We show here that deletion of gaaB or gaaC results in severely impaired growth on GA and accumulation of pathway intermediates L-galactonate and 2-keto-3-deoxy-L-galactonate, respectively. Expression levels of several GA-induced genes were specifically elevated in the ∆gaaC mutant on GA as compared to the reference strain or other GA catabolic pathway deletion mutants. The hyper-induction of GA-induced genes in ∆gaaC indicates that 2-keto-3-deoxy-L-galactonate is the inducer of genes required for GA utilization.

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The transcriptional activator GaaR of Aspergillus niger is required for release and utilization of D-galacturonic acid from pectin

Project description:We identified the D-galacturonic acid (GA) responsive transcriptional activator GaaR of the saprotrophic fungus Aspergillus niger, which was found to be essential for growth on GA and polygalacturonic acid (PGA). Growth of the ΔgaaR strain was reduced on complex pectins. Genome-wide expression analysis showed that GaaR is required for the expression of genes necessary to release GA from PGA and more complex pectins, to transport GA into the cell and to induce the GA-catabolic pathway. Residual growth of ΔgaaR on complex pectins is likely due to expression of pectinases acting on rhamnogalacturonan and subsequent metabolism of the monosaccharides other than GA.

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