ATNT: an enhanced system for expression of polycistronic secondary metabolite gene clusters in Aspergillus niger
Background: Fungi are treasure chests for yet unexplored natural products. However, exploitation of their real potential remains difficult as a significant proportion of biosynthetic gene clusters appears silent under standard laboratory conditions. Therefore, elucidation of novel products requires...
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| Format: | Article |
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BioMed Central
2017
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| Online Access: | https://eprints.nottingham.ac.uk/49165/ |
| _version_ | 1848797936352231424 |
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| author | Geib, Elena Brock, Matthias |
| author_facet | Geib, Elena Brock, Matthias |
| author_sort | Geib, Elena |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | Background: Fungi are treasure chests for yet unexplored natural products. However, exploitation of their real potential remains difficult as a significant proportion of biosynthetic gene clusters appears silent under standard laboratory conditions. Therefore, elucidation of novel products requires gene activation or heterologous expression. For heterologous gene expression, we previously developed an expression platform in Aspergillus niger that is based on the transcriptional regulator TerR and its target promoter PterA.
Results: In this study, we extended this system by regulating expression of terR by the doxycycline inducible Tet-on system. Reporter genes cloned under the control of the target promoter PterA remained silent in the absence of doxycycline, but were strongly expressed when doxycycline was added. Reporter quantification revealed that the coupled system results in about five times higher expression rates compared to gene expression under direct control of the Tet-on system. As production of secondary metabolites generally requires the expression of several biosynthetic genes, the suitability of the self-cleaving viral peptide sequence P2A was tested in this optimised expression system. P2A allowed polycistronic expression of genes required for Asp-melanin formation in combination with the gene coding for the red fluorescent protein tdTomato. Gene expression and Asp-melanin formation was prevented in the absence of doxycycline and strongly induced by addition of doxycycline. Fluorescence studies confirmed the correct subcellular localisation of the respective enzymes.
Conclusion: This tightly regulated but strongly inducible expression system enables high level production of secondary metabolites most likely even those with toxic potential. Furthermore, this system is compatible with polycistronic gene expression and, thus, suitable for the discovery of novel natural products. |
| first_indexed | 2025-11-14T20:11:48Z |
| format | Article |
| id | nottingham-49165 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T20:11:48Z |
| publishDate | 2017 |
| publisher | BioMed Central |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-491652020-05-04T19:23:29Z https://eprints.nottingham.ac.uk/49165/ ATNT: an enhanced system for expression of polycistronic secondary metabolite gene clusters in Aspergillus niger Geib, Elena Brock, Matthias Background: Fungi are treasure chests for yet unexplored natural products. However, exploitation of their real potential remains difficult as a significant proportion of biosynthetic gene clusters appears silent under standard laboratory conditions. Therefore, elucidation of novel products requires gene activation or heterologous expression. For heterologous gene expression, we previously developed an expression platform in Aspergillus niger that is based on the transcriptional regulator TerR and its target promoter PterA. Results: In this study, we extended this system by regulating expression of terR by the doxycycline inducible Tet-on system. Reporter genes cloned under the control of the target promoter PterA remained silent in the absence of doxycycline, but were strongly expressed when doxycycline was added. Reporter quantification revealed that the coupled system results in about five times higher expression rates compared to gene expression under direct control of the Tet-on system. As production of secondary metabolites generally requires the expression of several biosynthetic genes, the suitability of the self-cleaving viral peptide sequence P2A was tested in this optimised expression system. P2A allowed polycistronic expression of genes required for Asp-melanin formation in combination with the gene coding for the red fluorescent protein tdTomato. Gene expression and Asp-melanin formation was prevented in the absence of doxycycline and strongly induced by addition of doxycycline. Fluorescence studies confirmed the correct subcellular localisation of the respective enzymes. Conclusion: This tightly regulated but strongly inducible expression system enables high level production of secondary metabolites most likely even those with toxic potential. Furthermore, this system is compatible with polycistronic gene expression and, thus, suitable for the discovery of novel natural products. BioMed Central 2017-12-19 Article PeerReviewed Geib, Elena and Brock, Matthias (2017) ATNT: an enhanced system for expression of polycistronic secondary metabolite gene clusters in Aspergillus niger. Fungal Biology and Biotechnology, 4 (1). e13. ISSN 2054-3085 Asp-melaninP2A ; Polycistronic mRNA ; Tet-on system ; Doxycycline ; Terrein biosynthetic gene cluster https://fungalbiolbiotech.biomedcentral.com/articles/10.1186/s40694-017-0042-1 doi:10.1186/s40694-017-0042-1 doi:10.1186/s40694-017-0042-1 |
| spellingShingle | Asp-melaninP2A ; Polycistronic mRNA ; Tet-on system ; Doxycycline ; Terrein biosynthetic gene cluster Geib, Elena Brock, Matthias ATNT: an enhanced system for expression of polycistronic secondary metabolite gene clusters in Aspergillus niger |
| title | ATNT: an enhanced system for expression of polycistronic secondary metabolite gene clusters in Aspergillus niger |
| title_full | ATNT: an enhanced system for expression of polycistronic secondary metabolite gene clusters in Aspergillus niger |
| title_fullStr | ATNT: an enhanced system for expression of polycistronic secondary metabolite gene clusters in Aspergillus niger |
| title_full_unstemmed | ATNT: an enhanced system for expression of polycistronic secondary metabolite gene clusters in Aspergillus niger |
| title_short | ATNT: an enhanced system for expression of polycistronic secondary metabolite gene clusters in Aspergillus niger |
| title_sort | atnt: an enhanced system for expression of polycistronic secondary metabolite gene clusters in aspergillus niger |
| topic | Asp-melaninP2A ; Polycistronic mRNA ; Tet-on system ; Doxycycline ; Terrein biosynthetic gene cluster |
| url | https://eprints.nottingham.ac.uk/49165/ https://eprints.nottingham.ac.uk/49165/ https://eprints.nottingham.ac.uk/49165/ |