CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932
Lophiotrema is a genus of ascomycetous fungi within the family Lophiotremataceae. Members of this genus have been isolated as endophytes from a wide range of host plants and also from plant debris within terrestrial and marine habitats, where they are thought to function as saprobes. Lophiotrema sp....
| Main Authors: | , , , , , , , , |
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| Format: | Journal Article |
| Language: | English |
| Published: |
2023
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| Online Access: | https://hdl.handle.net/10356/164545 |
| _version_ | 1824454719080235008 |
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| author | Gakuubi, Martin Muthee Ching, Kuan Chieh Munusamy, Madhaiyan Wibowo, Mario Lim, Chun Teck Ma, Guang-Lei Liang, Zhao-Xun Kanagasundaram, Yoganathan Ng, Siew Bee |
| author2 | School of Biological Sciences |
| author_facet | School of Biological Sciences Gakuubi, Martin Muthee Ching, Kuan Chieh Munusamy, Madhaiyan Wibowo, Mario Lim, Chun Teck Ma, Guang-Lei Liang, Zhao-Xun Kanagasundaram, Yoganathan Ng, Siew Bee |
| author_sort | Gakuubi, Martin Muthee |
| collection | NTU |
| description | Lophiotrema is a genus of ascomycetous fungi within the family Lophiotremataceae. Members of this genus have been isolated as endophytes from a wide range of host plants and also from plant debris within terrestrial and marine habitats, where they are thought to function as saprobes. Lophiotrema sp. F6932 was isolated from white mangrove (Avicennia officinalis) in Pulau Ubin Island, Singapore. Crude extracts from the fungus exhibited strong antibacterial activity, and bioassay-guided isolation and structure elucidation of bioactive constituents led to the isolation of palmarumycin C8 and a new analog palmarumycin CP30. Whole-genome sequencing analysis resulted in the identification of a putative type 1 iterative PKS (iPKS) predicated to be involved in the biosynthesis of palmarumycins. To verify the involvement of palmarumycin (PAL) gene cluster in the biosynthesis of these compounds, we employed ribonucleoprotein (RNP)-mediated CRISPR-Cas9 to induce targeted deletion of the ketosynthase (KS) domain in PAL. Double-strand breaks (DSBs) upstream and downstream of the KS domain was followed by homology-directed repair (HDR) with a hygromycin resistance cassette flanked by a 50 bp of homology on both sides of the DSBs. The resultant deletion mutants displayed completely different phenotypes compared to the wild-type strain, as they had different colony morphology and were no longer able to produce palmarumycins or melanin. This study, therefore, confirms the involvement of PAL in the biosynthesis of palmarumycins, and paves the way for implementing a similar approach in the characterization of other gene clusters of interest in this largely understudied fungal strain. |
| first_indexed | 2025-02-19T03:26:47Z |
| format | Journal Article |
| id | ntu-10356/164545 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2025-02-19T03:26:47Z |
| publishDate | 2023 |
| record_format | dspace |
| spelling | ntu-10356/1645452023-02-28T17:13:41Z CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932 Gakuubi, Martin Muthee Ching, Kuan Chieh Munusamy, Madhaiyan Wibowo, Mario Lim, Chun Teck Ma, Guang-Lei Liang, Zhao-Xun Kanagasundaram, Yoganathan Ng, Siew Bee School of Biological Sciences Singapore Institute of Food and Biotechnology Innovation (SIFBI), A*STAR Science::Biological sciences Ketosynthase Domain Palmarumycin Lophiotrema is a genus of ascomycetous fungi within the family Lophiotremataceae. Members of this genus have been isolated as endophytes from a wide range of host plants and also from plant debris within terrestrial and marine habitats, where they are thought to function as saprobes. Lophiotrema sp. F6932 was isolated from white mangrove (Avicennia officinalis) in Pulau Ubin Island, Singapore. Crude extracts from the fungus exhibited strong antibacterial activity, and bioassay-guided isolation and structure elucidation of bioactive constituents led to the isolation of palmarumycin C8 and a new analog palmarumycin CP30. Whole-genome sequencing analysis resulted in the identification of a putative type 1 iterative PKS (iPKS) predicated to be involved in the biosynthesis of palmarumycins. To verify the involvement of palmarumycin (PAL) gene cluster in the biosynthesis of these compounds, we employed ribonucleoprotein (RNP)-mediated CRISPR-Cas9 to induce targeted deletion of the ketosynthase (KS) domain in PAL. Double-strand breaks (DSBs) upstream and downstream of the KS domain was followed by homology-directed repair (HDR) with a hygromycin resistance cassette flanked by a 50 bp of homology on both sides of the DSBs. The resultant deletion mutants displayed completely different phenotypes compared to the wild-type strain, as they had different colony morphology and were no longer able to produce palmarumycins or melanin. This study, therefore, confirms the involvement of PAL in the biosynthesis of palmarumycins, and paves the way for implementing a similar approach in the characterization of other gene clusters of interest in this largely understudied fungal strain. Agency for Science, Technology and Research (A*STAR) Published version This work was funded by the Singapore Institute of Food and Biotechnology Innovation, Agency for Science, Technology, and Research (A*STAR), Singapore, through a Singapore Integrative Biosystems and Engineering Research (SIBER) Grant (#21719). 2023-01-31T08:13:22Z 2023-01-31T08:13:22Z 2022 Journal Article Gakuubi, M. M., Ching, K. C., Munusamy, M., Wibowo, M., Lim, C. T., Ma, G., Liang, Z., Kanagasundaram, Y. & Ng, S. B. (2022). CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932. Frontiers in Microbiology, 13, 1012115-. https://dx.doi.org/10.3389/fmicb.2022.1012115 1664-302X https://hdl.handle.net/10356/164545 10.3389/fmicb.2022.1012115 36246293 2-s2.0-85139929725 13 1012115 en #21719 Frontiers in Microbiology © 2022 Gakuubi, Ching, Munusamy, Wibowo, Lim, Ma, Liang, Kanagasundaram and Ng. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. application/pdf |
| spellingShingle | Science::Biological sciences Ketosynthase Domain Palmarumycin Gakuubi, Martin Muthee Ching, Kuan Chieh Munusamy, Madhaiyan Wibowo, Mario Lim, Chun Teck Ma, Guang-Lei Liang, Zhao-Xun Kanagasundaram, Yoganathan Ng, Siew Bee CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932 |
| title | CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932 |
| title_full | CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932 |
| title_fullStr | CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932 |
| title_full_unstemmed | CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932 |
| title_short | CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932 |
| title_sort | crispr cas9 rnp assisted validation of palmarumycin biosynthetic gene cluster in lophiotrema sp f6932 |
| topic | Science::Biological sciences Ketosynthase Domain Palmarumycin |
| url | https://hdl.handle.net/10356/164545 |
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