A genetic tool to express long fungal biosynthetic genes
Abstract Background Secondary metabolites (SMs) from mushroom-forming fungi (Basidiomycota) and early diverging fungi (EDF) such as Mucoromycota are scarcely investigated. In many cases, production of SMs is induced by unknown stress factors or is accompanied by seasonable developmental changes on f...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-02-01
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| Series: | Fungal Biology and Biotechnology |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s40694-023-00152-3 |
| _version_ | 1811171864010031104 |
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| author | Leo Kirchgaessner Jacob M. Wurlitzer Paula S. Seibold Malik Rakhmanov Markus Gressler |
| author_facet | Leo Kirchgaessner Jacob M. Wurlitzer Paula S. Seibold Malik Rakhmanov Markus Gressler |
| author_sort | Leo Kirchgaessner |
| collection | DOAJ |
| description | Abstract Background Secondary metabolites (SMs) from mushroom-forming fungi (Basidiomycota) and early diverging fungi (EDF) such as Mucoromycota are scarcely investigated. In many cases, production of SMs is induced by unknown stress factors or is accompanied by seasonable developmental changes on fungal morphology. Moreover, many of these fungi are considered as non-culturable under laboratory conditions which impedes investigation into SM. In the post-genomic era, numerous novel SM genes have been identified especially from EDF. As most of them encode multi-module enzymes, these genes are usually long which limits cloning and heterologous expression in traditional hosts. Results An expression system in Aspergillus niger is presented that is suitable for the production of SMs from both Basidiomycota and EDF. The akuB gene was deleted in the expression host A. niger ATNT∆pyrG, resulting in a deficient nonhomologous end-joining repair mechanism which in turn facilitates the targeted gene deletion via homologous recombination. The ∆akuB mutant tLK01 served as a platform to integrate overlapping DNA fragments of long SM genes into the fwnA locus required for the black pigmentation of conidia. This enables an easy discrimination of correct transformants by screening the transformation plates for fawn-colored colonies. Expression of the gene of interest (GOI) is induced dose-dependently by addition of doxycycline and is enhanced by the dual TetON/terrein synthase promoter system (ATNT) from Aspergillus terreus. We show that the 8 kb polyketide synthase gene lpaA from the basidiomycete Laetiporus sulphureus is correctly assembled from five overlapping DNA fragments and laetiporic acids are produced. In a second approach, we expressed the yet uncharacterized > 20 kb nonribosomal peptide synthetase gene calA from the EDF Mortierella alpina. Gene expression and subsequent LC–MS/MS analysis of mycelial extracts revealed the production of the antimycobacterial compound calpinactam. This is the first report on the heterologous production of a full-length SM multidomain enzyme from EDF. Conclusions The system allows the assembly, targeted integration and expression of genes of > 20 kb size in A. niger in one single step. The system is suitable for evolutionary distantly related SM genes from both Basidiomycota and EDF. This uncovers new SM resources including genetically intractable or non-culturable fungi. |
| first_indexed | 2024-04-10T17:21:18Z |
| format | Article |
| id | doaj.art-1aa28ef0db804d1fa2968ab6b6bedeba |
| institution | Directory Open Access Journal |
| issn | 2054-3085 |
| language | English |
| last_indexed | 2024-04-10T17:21:18Z |
| publishDate | 2023-02-01 |
| publisher | BMC |
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| series | Fungal Biology and Biotechnology |
| spelling | doaj.art-1aa28ef0db804d1fa2968ab6b6bedeba2023-02-05T12:03:35ZengBMCFungal Biology and Biotechnology2054-30852023-02-0110111610.1186/s40694-023-00152-3A genetic tool to express long fungal biosynthetic genesLeo Kirchgaessner0Jacob M. Wurlitzer1Paula S. Seibold2Malik Rakhmanov3Markus Gressler4Institute of Pharmacy, Department Pharmaceutical Microbiology, Friedrich Schiller University JenaInstitute of Pharmacy, Department Pharmaceutical Microbiology, Friedrich Schiller University JenaInstitute of Pharmacy, Department Pharmaceutical Microbiology, Friedrich Schiller University JenaInstitute of Pharmacy, Department Pharmaceutical Microbiology, Friedrich Schiller University JenaInstitute of Pharmacy, Department Pharmaceutical Microbiology, Friedrich Schiller University JenaAbstract Background Secondary metabolites (SMs) from mushroom-forming fungi (Basidiomycota) and early diverging fungi (EDF) such as Mucoromycota are scarcely investigated. In many cases, production of SMs is induced by unknown stress factors or is accompanied by seasonable developmental changes on fungal morphology. Moreover, many of these fungi are considered as non-culturable under laboratory conditions which impedes investigation into SM. In the post-genomic era, numerous novel SM genes have been identified especially from EDF. As most of them encode multi-module enzymes, these genes are usually long which limits cloning and heterologous expression in traditional hosts. Results An expression system in Aspergillus niger is presented that is suitable for the production of SMs from both Basidiomycota and EDF. The akuB gene was deleted in the expression host A. niger ATNT∆pyrG, resulting in a deficient nonhomologous end-joining repair mechanism which in turn facilitates the targeted gene deletion via homologous recombination. The ∆akuB mutant tLK01 served as a platform to integrate overlapping DNA fragments of long SM genes into the fwnA locus required for the black pigmentation of conidia. This enables an easy discrimination of correct transformants by screening the transformation plates for fawn-colored colonies. Expression of the gene of interest (GOI) is induced dose-dependently by addition of doxycycline and is enhanced by the dual TetON/terrein synthase promoter system (ATNT) from Aspergillus terreus. We show that the 8 kb polyketide synthase gene lpaA from the basidiomycete Laetiporus sulphureus is correctly assembled from five overlapping DNA fragments and laetiporic acids are produced. In a second approach, we expressed the yet uncharacterized > 20 kb nonribosomal peptide synthetase gene calA from the EDF Mortierella alpina. Gene expression and subsequent LC–MS/MS analysis of mycelial extracts revealed the production of the antimycobacterial compound calpinactam. This is the first report on the heterologous production of a full-length SM multidomain enzyme from EDF. Conclusions The system allows the assembly, targeted integration and expression of genes of > 20 kb size in A. niger in one single step. The system is suitable for evolutionary distantly related SM genes from both Basidiomycota and EDF. This uncovers new SM resources including genetically intractable or non-culturable fungi.https://doi.org/10.1186/s40694-023-00152-3Early diverging fungiNonribosomal peptide synthetasePolyketide synthaseLaetiporic acidCalpinactam |
| spellingShingle | Leo Kirchgaessner Jacob M. Wurlitzer Paula S. Seibold Malik Rakhmanov Markus Gressler A genetic tool to express long fungal biosynthetic genes Fungal Biology and Biotechnology Early diverging fungi Nonribosomal peptide synthetase Polyketide synthase Laetiporic acid Calpinactam |
| title | A genetic tool to express long fungal biosynthetic genes |
| title_full | A genetic tool to express long fungal biosynthetic genes |
| title_fullStr | A genetic tool to express long fungal biosynthetic genes |
| title_full_unstemmed | A genetic tool to express long fungal biosynthetic genes |
| title_short | A genetic tool to express long fungal biosynthetic genes |
| title_sort | genetic tool to express long fungal biosynthetic genes |
| topic | Early diverging fungi Nonribosomal peptide synthetase Polyketide synthase Laetiporic acid Calpinactam |
| url | https://doi.org/10.1186/s40694-023-00152-3 |
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