Table_4_Utilization of ferulic acid in Aspergillus niger requires the transcription factor FarA and a newly identified Far-like protein (FarD) that lacks the canonical Zn(II)2Cys6 domain.docx

The feruloyl esterase B gene (faeB) is specifically induced by hydroxycinnamic acids (e.g. ferulic acid, caffeic acid and coumaric acid) but the transcriptional regulation network involved in faeB induction and ferulic acid metabolism has only been partially addressed. To identify transcription factors involved in ferulic acid metabolism we constructed and screened a transcription factor knockout library of 239 Aspergillus niger strains for mutants unable to utilize ferulic acid as a carbon source. The ΔfarA transcription factor mutant, already known to be involved in fatty acid metabolism, could not utilize ferulic acid and other hydroxycinnamic acids. In addition to screening the transcription factor mutant collection, a forward genetic screen was performed to isolate mutants unable to express faeB. For this screen a PfaeB-amdS and PfaeB-lux613 dual reporter strain was engineered. The rationale of the screen is that in this reporter strain ferulic acid induces amdS (acetamidase) expression via the faeB promoter resulting in lethality on fluoro-acetamide. Conidia of this reporter strain were UV-mutagenized and plated on fluoro-acetamide medium in the presence of ferulic acid. Mutants unable to induce faeB are expected to be fluoro-acetamide resistant and can be positively selected for. Using this screen, six fluoro-acetamide resistant mutants were obtained and phenotypically characterized. Three mutants had a phenotype identical to the farA mutant and sequencing the farA gene in these mutants indeed showed mutations in FarA which resulted in inability to growth on ferulic acid as well as on short and long chain fatty acids. The growth phenotype of the other three mutants was similar to the farA mutants in terms of the inability to grow on ferulic acid, but these mutants grew normally on short and long chain fatty acids. The genomes of these three mutants were sequenced and allelic mutations in one particular gene (NRRL3_09145) were found. The protein encoded by NRRL3_09145 shows similarity to the FarA and FarB transcription factors. However, whereas FarA and FarB contain both the Zn(II)2Cys6 domain and a fungal-specific transcription factor domain, the protein encoded by NRRL3_09145 (FarD) lacks the canonical Zn(II)2Cys6 domain and possesses only the fungal specific transcription factor domain.</p

Table_2_Utilization of ferulic acid in Aspergillus niger requires the transcription factor FarA and a newly identified Far-like protein (FarD) that lacks the canonical Zn(II)2Cys6 domain.xls

The feruloyl esterase B gene (faeB) is specifically induced by hydroxycinnamic acids (e.g. ferulic acid, caffeic acid and coumaric acid) but the transcriptional regulation network involved in faeB induction and ferulic acid metabolism has only been partially addressed. To identify transcription factors involved in ferulic acid metabolism we constructed and screened a transcription factor knockout library of 239 Aspergillus niger strains for mutants unable to utilize ferulic acid as a carbon source. The ΔfarA transcription factor mutant, already known to be involved in fatty acid metabolism, could not utilize ferulic acid and other hydroxycinnamic acids. In addition to screening the transcription factor mutant collection, a forward genetic screen was performed to isolate mutants unable to express faeB. For this screen a PfaeB-amdS and PfaeB-lux613 dual reporter strain was engineered. The rationale of the screen is that in this reporter strain ferulic acid induces amdS (acetamidase) expression via the faeB promoter resulting in lethality on fluoro-acetamide. Conidia of this reporter strain were UV-mutagenized and plated on fluoro-acetamide medium in the presence of ferulic acid. Mutants unable to induce faeB are expected to be fluoro-acetamide resistant and can be positively selected for. Using this screen, six fluoro-acetamide resistant mutants were obtained and phenotypically characterized. Three mutants had a phenotype identical to the farA mutant and sequencing the farA gene in these mutants indeed showed mutations in FarA which resulted in inability to growth on ferulic acid as well as on short and long chain fatty acids. The growth phenotype of the other three mutants was similar to the farA mutants in terms of the inability to grow on ferulic acid, but these mutants grew normally on short and long chain fatty acids. The genomes of these three mutants were sequenced and allelic mutations in one particular gene (NRRL3_09145) were found. The protein encoded by NRRL3_09145 shows similarity to the FarA and FarB transcription factors. However, whereas FarA and FarB contain both the Zn(II)2Cys6 domain and a fungal-specific transcription factor domain, the protein encoded by NRRL3_09145 (FarD) lacks the canonical Zn(II)2Cys6 domain and possesses only the fungal specific transcription factor domain.</p

Table_1_Utilization of ferulic acid in Aspergillus niger requires the transcription factor FarA and a newly identified Far-like protein (FarD) that lacks the canonical Zn(II)2Cys6 domain.docx

The feruloyl esterase B gene (faeB) is specifically induced by hydroxycinnamic acids (e.g. ferulic acid, caffeic acid and coumaric acid) but the transcriptional regulation network involved in faeB induction and ferulic acid metabolism has only been partially addressed. To identify transcription factors involved in ferulic acid metabolism we constructed and screened a transcription factor knockout library of 239 Aspergillus niger strains for mutants unable to utilize ferulic acid as a carbon source. The ΔfarA transcription factor mutant, already known to be involved in fatty acid metabolism, could not utilize ferulic acid and other hydroxycinnamic acids. In addition to screening the transcription factor mutant collection, a forward genetic screen was performed to isolate mutants unable to express faeB. For this screen a PfaeB-amdS and PfaeB-lux613 dual reporter strain was engineered. The rationale of the screen is that in this reporter strain ferulic acid induces amdS (acetamidase) expression via the faeB promoter resulting in lethality on fluoro-acetamide. Conidia of this reporter strain were UV-mutagenized and plated on fluoro-acetamide medium in the presence of ferulic acid. Mutants unable to induce faeB are expected to be fluoro-acetamide resistant and can be positively selected for. Using this screen, six fluoro-acetamide resistant mutants were obtained and phenotypically characterized. Three mutants had a phenotype identical to the farA mutant and sequencing the farA gene in these mutants indeed showed mutations in FarA which resulted in inability to growth on ferulic acid as well as on short and long chain fatty acids. The growth phenotype of the other three mutants was similar to the farA mutants in terms of the inability to grow on ferulic acid, but these mutants grew normally on short and long chain fatty acids. The genomes of these three mutants were sequenced and allelic mutations in one particular gene (NRRL3_09145) were found. The protein encoded by NRRL3_09145 shows similarity to the FarA and FarB transcription factors. However, whereas FarA and FarB contain both the Zn(II)2Cys6 domain and a fungal-specific transcription factor domain, the protein encoded by NRRL3_09145 (FarD) lacks the canonical Zn(II)2Cys6 domain and possesses only the fungal specific transcription factor domain.</p

Presentation_3_Utilization of ferulic acid in Aspergillus niger requires the transcription factor FarA and a newly identified Far-like protein (FarD) that lacks the canonical Zn(II)2Cys6 domain.pptx

The feruloyl esterase B gene (faeB) is specifically induced by hydroxycinnamic acids (e.g. ferulic acid, caffeic acid and coumaric acid) but the transcriptional regulation network involved in faeB induction and ferulic acid metabolism has only been partially addressed. To identify transcription factors involved in ferulic acid metabolism we constructed and screened a transcription factor knockout library of 239 Aspergillus niger strains for mutants unable to utilize ferulic acid as a carbon source. The ΔfarA transcription factor mutant, already known to be involved in fatty acid metabolism, could not utilize ferulic acid and other hydroxycinnamic acids. In addition to screening the transcription factor mutant collection, a forward genetic screen was performed to isolate mutants unable to express faeB. For this screen a PfaeB-amdS and PfaeB-lux613 dual reporter strain was engineered. The rationale of the screen is that in this reporter strain ferulic acid induces amdS (acetamidase) expression via the faeB promoter resulting in lethality on fluoro-acetamide. Conidia of this reporter strain were UV-mutagenized and plated on fluoro-acetamide medium in the presence of ferulic acid. Mutants unable to induce faeB are expected to be fluoro-acetamide resistant and can be positively selected for. Using this screen, six fluoro-acetamide resistant mutants were obtained and phenotypically characterized. Three mutants had a phenotype identical to the farA mutant and sequencing the farA gene in these mutants indeed showed mutations in FarA which resulted in inability to growth on ferulic acid as well as on short and long chain fatty acids. The growth phenotype of the other three mutants was similar to the farA mutants in terms of the inability to grow on ferulic acid, but these mutants grew normally on short and long chain fatty acids. The genomes of these three mutants were sequenced and allelic mutations in one particular gene (NRRL3_09145) were found. The protein encoded by NRRL3_09145 shows similarity to the FarA and FarB transcription factors. However, whereas FarA and FarB contain both the Zn(II)2Cys6 domain and a fungal-specific transcription factor domain, the protein encoded by NRRL3_09145 (FarD) lacks the canonical Zn(II)2Cys6 domain and possesses only the fungal specific transcription factor domain.</p

Presentation_2_Utilization of ferulic acid in Aspergillus niger requires the transcription factor FarA and a newly identified Far-like protein (FarD) that lacks the canonical Zn(II)2Cys6 domain.pptx

The feruloyl esterase B gene (faeB) is specifically induced by hydroxycinnamic acids (e.g. ferulic acid, caffeic acid and coumaric acid) but the transcriptional regulation network involved in faeB induction and ferulic acid metabolism has only been partially addressed. To identify transcription factors involved in ferulic acid metabolism we constructed and screened a transcription factor knockout library of 239 Aspergillus niger strains for mutants unable to utilize ferulic acid as a carbon source. The ΔfarA transcription factor mutant, already known to be involved in fatty acid metabolism, could not utilize ferulic acid and other hydroxycinnamic acids. In addition to screening the transcription factor mutant collection, a forward genetic screen was performed to isolate mutants unable to express faeB. For this screen a PfaeB-amdS and PfaeB-lux613 dual reporter strain was engineered. The rationale of the screen is that in this reporter strain ferulic acid induces amdS (acetamidase) expression via the faeB promoter resulting in lethality on fluoro-acetamide. Conidia of this reporter strain were UV-mutagenized and plated on fluoro-acetamide medium in the presence of ferulic acid. Mutants unable to induce faeB are expected to be fluoro-acetamide resistant and can be positively selected for. Using this screen, six fluoro-acetamide resistant mutants were obtained and phenotypically characterized. Three mutants had a phenotype identical to the farA mutant and sequencing the farA gene in these mutants indeed showed mutations in FarA which resulted in inability to growth on ferulic acid as well as on short and long chain fatty acids. The growth phenotype of the other three mutants was similar to the farA mutants in terms of the inability to grow on ferulic acid, but these mutants grew normally on short and long chain fatty acids. The genomes of these three mutants were sequenced and allelic mutations in one particular gene (NRRL3_09145) were found. The protein encoded by NRRL3_09145 shows similarity to the FarA and FarB transcription factors. However, whereas FarA and FarB contain both the Zn(II)2Cys6 domain and a fungal-specific transcription factor domain, the protein encoded by NRRL3_09145 (FarD) lacks the canonical Zn(II)2Cys6 domain and possesses only the fungal specific transcription factor domain.</p

Dynamic and functional profiling of xylan-degrading enzymes in <em>aspergillus</em> secretomes using activity-based probes

International audiencePlant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and endo-β-1,4-xylanases in the secretomes of Aspergillus niger, by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme–substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown

Impaired amino acid metabolism contributes to fasting-induced hypoglycemia in fatty acid oxidation defects

<p>The importance of mitochondrial fatty acid beta-oxidation (FAO) as a glucose-sparing process is illustrated by patients with inherited defects in FAO, who may present with life-threatening fasting-induced hypoketotic hypoglycemia. It is unknown why peripheral glucose demand outpaces hepatic gluconeogenesis in these patients. In this study, we have systematically addressed the fasting response in long-chain acyl-CoA dehydrogenase-deficient (LCAD KO) mice. We demonstrate that the fasting-induced hypoglycemia in LCAD KO mice was initiated by an increased glucose requirement in peripheral tissues, leading to rapid hepatic glycogen depletion. Gluconeogenesis did not compensate for the increased glucose demand, which was not due to insufficient hepatic glucogenic capacity but rather caused by a shortage in the supply of glucogenic precursors. This shortage in supply was explained by a suppressed glucose-alanine cycle, decreased branched-chain amino acid metabolism and ultimately impaired protein mobilization. We conclude that during fasting, FAO not only serves to spare glucose but is also indispensable for amino acid metabolism, which is essential for the maintenance of adequate glucose production.</p>