Construction of an improved Aspergillus niger platform for enhanced glucoamylase secretion
Abstract Background The lifestyle of filamentous fungi depends on the secretion of hydrolytic enzymes into the surrounding medium, which degrade polymeric substances into monomers that are then taken up to sustain metabolism. This feature has been exploited in biotechnology to establish platform str...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2018-06-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12934-018-0941-8 |
| _version_ | 1818384510114332672 |
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| author | Markus R. M. Fiedler Lars Barthel Christin Kubisch Corrado Nai Vera Meyer |
| author_facet | Markus R. M. Fiedler Lars Barthel Christin Kubisch Corrado Nai Vera Meyer |
| author_sort | Markus R. M. Fiedler |
| collection | DOAJ |
| description | Abstract Background The lifestyle of filamentous fungi depends on the secretion of hydrolytic enzymes into the surrounding medium, which degrade polymeric substances into monomers that are then taken up to sustain metabolism. This feature has been exploited in biotechnology to establish platform strains with high secretory capacity including Aspergillus niger. The accepted paradigm is that proteins become mainly secreted at the tips of fungal hyphae. However, it is still a matter of debate if the amount of growing hyphal tips in filamentous fungi correlates with an increase in secretion, with previous studies showing either a positive or no correlation. Results Here, we followed a systematic approach to study protein secretion in A. niger. First, we put the glaA gene encoding for glucoamylase (GlaA), the most abundant secreted protein of A. niger, under control of the tunable Tet-on system. Regulation of glaA gene expression by omitting or adding the inducer doxycycline to cultivation media allowed us to study the effect of glaA under- or overexpression in the same isolate. By inducing glaA expression in a fluorescently tagged v-SNARE reporter strain expressing GFP-SncA, we could demonstrate that the amount of post-Golgi carriers indeed depends on and correlates with glaA gene expression. By deleting the racA gene, encoding the Rho-GTPase RacA in this isolate, we generated a strain which is identical to the parental strain with respect to biomass formation but produces about 20% more hyphal tips. This hyperbranching phenotype caused a more compact macromorphology in shake flask cultivations. When ensuring continuous high-level expression of glaA by repeated addition of doxycycline, this hyperbranching strain secreted up to four times more GlaA into the culture medium compared to its parental strain. Conclusion The data obtained in this study strongly indicate that A. niger responds to forced transcription of secretory enzymes with increased formation of post-Golgi carriers to efficiently accommodate the incoming cargo load. This physiological adaptation can be rationally exploited to generate hypersecretion platforms based on a hyperbranching phenotype. We propose that a racA deletion background serves as an excellent chassis for such hypersecretion strains. |
| first_indexed | 2024-12-14T03:23:24Z |
| format | Article |
| id | doaj.art-84434a6ea7bc4df2bfdbc170aa4ed245 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-14T03:23:24Z |
| publishDate | 2018-06-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-84434a6ea7bc4df2bfdbc170aa4ed2452022-12-21T23:18:59ZengBMCMicrobial Cell Factories1475-28592018-06-0117111210.1186/s12934-018-0941-8Construction of an improved Aspergillus niger platform for enhanced glucoamylase secretionMarkus R. M. Fiedler0Lars Barthel1Christin Kubisch2Corrado Nai3Vera Meyer4Department Applied and Molecular Microbiology, Institute of Biotechnology, Technische Universität BerlinDepartment Applied and Molecular Microbiology, Institute of Biotechnology, Technische Universität BerlinDepartment Applied and Molecular Microbiology, Institute of Biotechnology, Technische Universität BerlinDepartment Applied and Molecular Microbiology, Institute of Biotechnology, Technische Universität BerlinDepartment Applied and Molecular Microbiology, Institute of Biotechnology, Technische Universität BerlinAbstract Background The lifestyle of filamentous fungi depends on the secretion of hydrolytic enzymes into the surrounding medium, which degrade polymeric substances into monomers that are then taken up to sustain metabolism. This feature has been exploited in biotechnology to establish platform strains with high secretory capacity including Aspergillus niger. The accepted paradigm is that proteins become mainly secreted at the tips of fungal hyphae. However, it is still a matter of debate if the amount of growing hyphal tips in filamentous fungi correlates with an increase in secretion, with previous studies showing either a positive or no correlation. Results Here, we followed a systematic approach to study protein secretion in A. niger. First, we put the glaA gene encoding for glucoamylase (GlaA), the most abundant secreted protein of A. niger, under control of the tunable Tet-on system. Regulation of glaA gene expression by omitting or adding the inducer doxycycline to cultivation media allowed us to study the effect of glaA under- or overexpression in the same isolate. By inducing glaA expression in a fluorescently tagged v-SNARE reporter strain expressing GFP-SncA, we could demonstrate that the amount of post-Golgi carriers indeed depends on and correlates with glaA gene expression. By deleting the racA gene, encoding the Rho-GTPase RacA in this isolate, we generated a strain which is identical to the parental strain with respect to biomass formation but produces about 20% more hyphal tips. This hyperbranching phenotype caused a more compact macromorphology in shake flask cultivations. When ensuring continuous high-level expression of glaA by repeated addition of doxycycline, this hyperbranching strain secreted up to four times more GlaA into the culture medium compared to its parental strain. Conclusion The data obtained in this study strongly indicate that A. niger responds to forced transcription of secretory enzymes with increased formation of post-Golgi carriers to efficiently accommodate the incoming cargo load. This physiological adaptation can be rationally exploited to generate hypersecretion platforms based on a hyperbranching phenotype. We propose that a racA deletion background serves as an excellent chassis for such hypersecretion strains.http://link.springer.com/article/10.1186/s12934-018-0941-8Aspergillus nigerTet-onProtein secretionGTPase RacAv-SNAREHyperbranching |
| spellingShingle | Markus R. M. Fiedler Lars Barthel Christin Kubisch Corrado Nai Vera Meyer Construction of an improved Aspergillus niger platform for enhanced glucoamylase secretion Microbial Cell Factories Aspergillus niger Tet-on Protein secretion GTPase RacA v-SNARE Hyperbranching |
| title | Construction of an improved Aspergillus niger platform for enhanced glucoamylase secretion |
| title_full | Construction of an improved Aspergillus niger platform for enhanced glucoamylase secretion |
| title_fullStr | Construction of an improved Aspergillus niger platform for enhanced glucoamylase secretion |
| title_full_unstemmed | Construction of an improved Aspergillus niger platform for enhanced glucoamylase secretion |
| title_short | Construction of an improved Aspergillus niger platform for enhanced glucoamylase secretion |
| title_sort | construction of an improved aspergillus niger platform for enhanced glucoamylase secretion |
| topic | Aspergillus niger Tet-on Protein secretion GTPase RacA v-SNARE Hyperbranching |
| url | http://link.springer.com/article/10.1186/s12934-018-0941-8 |
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