Mutations in AraR leading to constitutive of arabinolytic genes in Aspergillus niger under derepressing conditions

Abstract

The AraR transcription factor of Aspergillus niger encodes a Zn(II) 2 Cys 6 transcription factor required for the induction of genes encoding arabinolytic enzymes. One of the target genes of AraR is abfA , encoding an arabinofuranosidase. The expression of abfA as well as other L-arabinose-induced genes in A. niger requires the presence of L-arabinose or its derivative L-arabitol as an inducer to activate AraR-dependant gene expression. In this study, mutants were isolated that express L-arabinose-induced genes independently of the presence of an inducer under derepressing conditions. To obtain these mutants, a reporter strain was constructed in a ΔcreA background containing the L-arabinose-responsive promoter ( PabfA ) fused to the acetamidase ( amdS ) gene. Spores of the ΔcreA PabfA-amdS reporter strain were UV-mutagenized and mutants were obtained by their ability to grow on acetamide without the presence of inducer. From a total of 164 mutants, 15 mutants were identified to contain transacting mutations resulting in high arabinofuranosidase activity in the medium after growth under non-inducing conditions. Sequencing of the araR gene of the 15 constitutive mutants revealed that 14 mutants carried a mutation in AraR. Some mutations were found more than once and in total nine different point mutations were identified in AraR. The AraR N806I point mutation was reintroduced into a parental strain and confirmed that this point mutation leads to inducer-independent expression of AraR target genes. The inducer independent of L-arabinose-induced genes in the AraR N806I mutant was found to be sensitive to carbon catabolite repression, indicating that the CreA-mediated carbon catabolite repression is dominant over the AraR N806I mutant allele. These mutations in AraR provide new opportunities to improve arabinase production in industrial fungal strains. Electronic supplementary material The online version of this article (10.1007/s00253-019-09777-0) contains supplementary material, which is available to authorized users.

Loss of function of the carbon catabolite repressor CreA leads to low but inducer‐independent expression from the feruloyl esterase B promoter in Aspergillus niger

“…We previously used forward genetic screens based on promoter- amdS reporter constructs, to isolate mutants showing constitutive expression of pectinase (Niu et al 2017 ) and arabinase encoding genes (Reijngoud et al 2019 ). To obtain insight into the transcriptional regulation of faeB , a similar strategy was used in which PfaeB-amdS and PfaeB-lux double reporter strains were constructed with the aim to identify mutants displaying constitutive, i.e.…”

Unravelling the genetic complexity and redundancy of Aspergillus niger sugar metabolism

“…In chapter 2, we have shown that accumulation of the inducers of XlnR and AraR does not automatically result in higher expression of the xylanolytic and arabinolytic genes compared to the reference strain, probably due to their repression mediated by the carbon catabolite repressor protein CreA. Nevertheless, the constitutively active XlnR was recently shown to increase production of Carbohydrate-Active enZymes (CAZymes) and pentose release from WB , while the constitutively active AraR resulted in constitutive production of arabinanolytic enzymes under derepressing conditions (Reijngoud et al, 2019). Therefore, combination of targeted metabolic mutations with manipulation of these two TFs could improve production of CAZymes, and subsequently the release of D-xylose and L-arabinose from WB to improve xylitol and L-arabitol production.…”

Fungal strain engineering from understanding towards applications

“…However, these processes require cautious control of growth conditions and long incubation times, while most microorganisms also consume cellulose and hemicellulose to support their growth [3]. These drawbacks prevent the industrial use of biological methods, although they are used in fields of biotechnology not covered in this review, such as bioremediation approaches [49][50][51].…”

“…Constitutively active XlnR leads to increased production of plant biomass degrading enzymes, and D-xylose and L-arabinose release from wheat bran [44]. The constitutively active AraR has also been shown to result in constitutive production of arabinanases under derepressing conditions [49]. Therefore, these two regulators could be manipulated to increase the production of plant biomass degrading enzymes, and subsequently the release of D-xylose and L-arabinose from…”

“…Moreover, regulatory systems are poorly conserved in fungi, most likely because of the need of different fungi to adapt to their specific biotope. D-galactose is a hexose sugar commonly found in plant cell wall polysaccharides, such as xylan, xyloglucan, arabinogalactan, and rhamnogalacturonan I and II, together with L-arabinose and D-xylose, suggesting that the metabolism of these sugars can be active simultaneously [49][50][51][52]. The pentoses L-arabinose and D-xylose are converted via the PCP, and D-galactose is converted by two main pathways in filamentous fungi, including the well-known Leloir pathway and the alternative D-galactose oxido-reductive pathway [53].…”