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

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 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.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Aspergillus Niger Nrrl3

TISSUE(S): Cell Culture, Fungal Cell

DISEASE(S): Disease Free

SUBMITTER: Marcos Di Falco

LAB HEAD: Ronald P. deVries

PROVIDER: PXD025383 | Pride | 2021-11-18

REPOSITORIES: Pride

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Project description:Enzymatic degradation of plant biomass requires the coordinated action of various enzymes. In this study, the production of reducing sugars from pectic substrates and sugar beet pulp (SBP) was investigated and compared using commercial enzyme preparations, including M2, pectinase (E1) from Aspergillus niger, Viscozyme® L (V-L) and L-40. V-L, a cellulolytic enzyme mix produced by Aspergillus sp. was further evaluated as the most robust enzyme cocktail with the strongest SBP degradation ability in terms of dynamic release of monosaccharides, methanol, and acetate from substrate SBP. Glucose, the building block of cellulose, was the first product to reach its maximum concentration within 2.5 hours. In contrast, the major monosaccharides in pectin, including arabinose and galacturonic acid, were continuously released from the residue until reaching a peak of 2,092 mg L-1 and 7,069 mg L-1 after 19 hours of fermentation. Label-free proteomics analysis (MS/MS) of V-L revealed 151 individual proteins. Of these, 137 proteins were annotated as containing a carbohydrate-active enzymes (CAZyme) module. Notably, of the 50 most abundant proteins, which accounted for over 90% of the protein amount in V-L, ca. 40% were predicted to be involved in pectin degradation (belonging mainly to CAZyme families GH28, CE8, CE16, PL1 and PL3). To reveal the role of individual putative key enzymes of pectic substrate decomposition, two galacturonases (PglA and PglB), which are core of pectin-degrading enzymes of the GH28 family, were heterologously expressed in Pichia pastoris strain GB10 and further characterized. PglA and PglB demonstrated maximum activity at 57 °C and 67 °C, respectively. Both enzymes exhibited endo-cleavage patterns towards polygalacturonic acid (PGA) and released oligosaccharides with different degrees of polymerization (DP). In conclusion, our study identified two pectin-cleaving enzymes present in major amounts in a highly efficient SBP-saccharifying enzyme mix and characterized some of their properties. Further studies along this line may facilitate the understanding of SBP degradation and may help to design improved artificial enzyme mixtures with a lower complexity than V-L for future application in biotechnology.

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

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