A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger

Roth, Andreas; Dersch, Petra

doi:10.1007/s00253-009-2252-9

Cited by 25 publications

(14 citation statements)

References 33 publications

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“…The angfus3 complementation plasmid was based on pARAn37 (34). A genomic fragment containing the ORF flanked by its 1-kb upstream and downstream sequences was amplified by PCR.…”

Section: Methodsmentioning

confidence: 99%

“…The replacement cassettes were assembled by yeast recombinational cloning as described in reference 32. Both constructs were integrated into strain AB1.13 by protoplast transformation as described previously (33,34). Protoplasts were embedded into minimal medium containing 0.6% (wt/vol) agar and 1.2 M sorbitol and plated onto solid minimal medium containing agar (1.2% [wt/vol]), sorbitol (1.2 M) and, if required, hygromycin (20 g/m).…”

Section: Methodsmentioning

confidence: 99%

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The AngFus3 Mitogen-Activated Protein Kinase Controls Hyphal Differentiation and Secondary Metabolism in Aspergillus niger

et al. 2015

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cAdaptation to a changing environment is essential for the survival and propagation of sessile organisms, such as plants or fungi. Filamentous fungi commonly respond to a worsening of their growth conditions by differentiation of asexually or sexually produced spores. The formation of these specialized cell types is, however, also triggered as part of the general life cycle by hyphal age or density. Spores typically serve for dispersal and, therefore, translocation but can also act as resting states to endure times of scarcity. Eukaryotic differentiation in response to environmental and self-derived signals is commonly mediated by threetiered mitogen-activated protein (MAP) kinase signaling cascades. Here, we report that the MAP kinase Fus3 of the black mold Aspergillus niger (AngFus3) and its upstream kinase AngSte7 control vegetative spore formation and secondary metabolism. Mutants lacking these kinases are defective in conidium induction in response to hyphal density but are fully competent in starvation-induced sporulation, indicating that conidiation in A. niger is triggered by various independent signals. In addition, the mutants exhibit an altered profile of volatile metabolites and secrete dark pigments into the growth medium, suggesting a dysregulation of the secondary metabolism. By assigning the AngFus3 MAP kinase pathway to the transduction of a potentially selfderived trigger, this work contributes to the unraveling of the intricate signaling networks controlling fungal differentiation. Moreover, our data further support earlier observations that differentiation and secondary metabolism are tightly linked in filamentous fungi.G rowth and propagation of filamentous fungi typically involve the differentiation of specialized cell types or multicellular structures, such as spores, spore-bearing hyphae, and fruiting bodies, which serve specific biological functions. Differentiation of sexual and vegetative spores, for example, allows the dispersal and, therefore, translocation of otherwise sessile fungi. In addition, spores commonly function as resting structures to endure hostile growth conditions, such as wintertime or droughts. The induction of fungal differentiation usually involves both internal and external signals. For example, conidiation in the red bread mold Neurospora crassa underlies an internal circadian rhythm but is also induced through environmental signals, including light and nutrient deprivation (1, 2). There is growing evidence that fungi also produce a wide range of chemical signals to govern the development and differentiation of individual cells or a mycelial colony within a population (3). It has, for example, long been appreciated that in sexually propagating, heterothallic fungi, cells polarize and direct their growth toward peptide pheromones secreted by the mating partner (4). Cell-to-cell signaling also occurs on the population level or within an individual colony. For example, the primarily unicellular fungus Candida albicans secretes the sesquiterpene alcohol farnesol and ...

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“…The angfus3 complementation plasmid was based on pARAn37 (34). A genomic fragment containing the ORF flanked by its 1-kb upstream and downstream sequences was amplified by PCR.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The AngFus3 Mitogen-Activated Protein Kinase Controls Hyphal Differentiation and Secondary Metabolism in Aspergillus niger

et al. 2015

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“…Fluorescence microscopy was also used to monitor RP-GFP localisation. Roth & Dersch (2010) used GFP fusions to test a novel expression system in Aspergillus niger. GFP fusions have also been successfully used to improve RPP production in mammalian cell culture (for example CHO cell selection; Pilbrough et al 2009).…”

Section: Fps As Reporters Of Recombinant Protein Productionmentioning

confidence: 99%

Fluorescent proteins in microbial biotechnology—new proteins and new applications

2011

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The recent advances over the past 5 years in the utilisation of fluorescent proteins in microbial biotechnology applications, including recombinant protein production, food processing, and environmental biotechnology, are reviewed. We highlight possible areas where fluorescent proteins currently used in other bioscience disciplines could be adapted for use in biotechnological applications and also outline novel uses for recently developed fluorescent proteins.

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“…The fragment was cloned in pJET1.2 and excised with Nhe I and Bsr GI. The fragment containing the abfA gene was subsequently cloned in Nhe I and Bsr GI digested pARAn19 (Roth and Dersch , 2010 ) to give pARAn19-AbfA. The fi nal expression plasmid was sequenced.…”

Section: Cloning Of Abfa and Abfbmentioning

confidence: 99%

Fungal α-arabinofuranosidases of glycosyl hydrolase families 51 and 54 show a dual arabinofuranosyl- and galactofuranosyl-hydrolyzing activity

Tefsen¹,

Lagendijk²,

Park

et al. 2012

Biological Chemistry

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Aspergillus niger possesses a galactofuranosidase activity, however, the corresponding enzyme or gene encoding this enzyme has never been identifi ed. As evidence is mounting that enzymes exist with affi nity for both arabinofuranose and galactofuranose, we investigated the possibility that α -l -arabinofuranosidases, encoded by the abfA and abfB genes, are responsible for the galactofuranosidase activity of A. niger . Characterization of the recombinant AbfA and AbfB proteins revealed that both enzymes do not only hydrolyze p -nitrophenyl-α -l -arabinofuranoside (pNp-α -Ara f ) but are also capable of hydrolyzing p -nitrophenyl-β -d -galactofuranoside (pNp-β -Gal f ). Molecular modeling of the AbfB protein with pNp-β -Gal f confi rmed the possibility for AbfB to interact with this substrate, similarly as with pNp-α -Ara f . We also show that galactomannan, a cell wall compound of A. niger , containing β -linked terminal and internal galactofuranosyl moieties, can be degraded by an enzyme activity that is present in the supernatant of inulin-grown A. niger . Interestingly, purifi ed AbfA and AbfB did not show this hydrolyzing activity toward A. niger galactomannan. In summary, our studies demonstrate that AbfA and AbfB, α -l -arabinofuranosidases from different families, both contain a galactofuranose (Galf) -hydrolyzing activity. In addition, our data support the presence of a Gal fhydrolase activity expressed by A. niger that is capable of degrading fungal galactomannan.

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A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger

Abstract: JournalApplied microbiology and biotechnology

Cited by 25 publications

References 33 publications

The AngFus3 Mitogen-Activated Protein Kinase Controls Hyphal Differentiation and Secondary Metabolism in Aspergillus niger

The AngFus3 Mitogen-Activated Protein Kinase Controls Hyphal Differentiation and Secondary Metabolism in Aspergillus niger

Fluorescent proteins in microbial biotechnology—new proteins and new applications

Fungal α-arabinofuranosidases of glycosyl hydrolase families 51 and 54 show a dual arabinofuranosyl- and galactofuranosyl-hydrolyzing activity

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