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Mutations in AraR leading to constitutive expression of arabinolytic genes in Aspergillus niger under derepressing conditions [corrected].

ABSTRACT: The AraR transcription factor of Aspergillus niger encodes a Zn(II)₂Cys₆ 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 ?creA background containing the L-arabinose-responsive promoter (PabfA) fused to the acetamidase (amdS) gene. Spores of the ?creA 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.

SUBMITTER: Reijngoud J

PROVIDER: S-EPMC6486530 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Mutations in AraR leading to constitutive expression of arabinolytic genes in Aspergillus niger under derepressing conditions [corrected].

Reijngoud Jos J Deseke Malte M Halbesma Elmar T M ETM Alazi Ebru E Arentshorst Mark M Punt Peter J PJ Ram Arthur F J AFJ

Applied microbiology and biotechnology 20190408 10

The AraR transcription factor of Aspergillus niger encodes a Zn(II)<sub>2</sub>Cys<sub>6</sub> 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 ...[more]

PMID: 30963207

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Project description:BACKGROUND:Glucoamylase is one of the most industrially applied enzymes, produced by Aspergillus species, like Aspergillus niger. Compared to the traditional ways of process optimization, the metabolic engineering strategies to improve glucoamylase production are relatively scarce. RESULTS:In the previous study combined multi-omics integrative analysis and amino acid supplementation experiment, we predicted four amino acids (alanine, glutamate, glycine and aspartate) as the limited precursors for glucoamylase production in A. niger. To further verify this, five mutants namely OE-ala, OE-glu, OE-gly, OE-asp1 and OE-asp2, derived from the parental strain A. niger CBS 513.88, were constructed respectively for the overexpression of five genes responsible for the biosynthesis of the four kinds of amino acids (An11g02620, An04g00990, An05g00410, An04g06380 and An16g05570). Real-time quantitative PCR revealed that all these genes were successfully overexpressed at the mRNA level while the five mutants exhibited different performance in glucoamylase production in shake flask cultivation. Notably, the results demonstrated that mutant OE-asp2 which was constructed for reinforcing cytosolic aspartate synthetic pathway, exhibited significantly increased glucoamylase activity by 23.5% and 60.3% compared to CBS 513.88 in the cultivation of shake flask and the 5 L fermentor, respectively. Compared to A. niger CBS 513.88, mutant OE-asp2 has a higher intracellular amino acid pool, in particular, alanine, leucine, glycine and glutamine, while the pool of glutamate was decreased. CONCLUSION:Our study combines the target prediction from multi-omics analysis with the experimental validation and proves the possibility of increasing glucoamylase production by enhancing limited amino acid biosynthesis. In short, this systematically conducted study will surely deepen the understanding of resources allocation in cell factory and provide new strategies for the rational design of enzyme production strains.

Dataset Information

Mutations in AraR leading to constitutive expression of arabinolytic genes in Aspergillus niger under derepressing conditions [corrected].

Publications

Mutations in AraR leading to constitutive expression of arabinolytic genes in Aspergillus niger under derepressing conditions [corrected].

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