Establishment of a Genetic Transformation System in Guanophilic Fungus <em>Amphichorda guana</em>
Fungi from unique environments exhibit special physiological characters and plenty of bioactive natural products. However, the recalcitrant genetics or poor transformation efficiencies prevent scientists from systematically studying molecular biological mechanisms and exploiting their metabolites. I...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-02-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/7/2/138 |
| _version_ | 1797396565469429760 |
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| author | Min Liang Wei Li Landa Qi Guocan Chen Lei Cai Wen-Bing Yin |
| author_facet | Min Liang Wei Li Landa Qi Guocan Chen Lei Cai Wen-Bing Yin |
| author_sort | Min Liang |
| collection | DOAJ |
| description | Fungi from unique environments exhibit special physiological characters and plenty of bioactive natural products. However, the recalcitrant genetics or poor transformation efficiencies prevent scientists from systematically studying molecular biological mechanisms and exploiting their metabolites. In this study, we targeted a guanophilic fungus <i>Amphichorda guana</i> LC5815 and developed a genetic transformation system. We firstly established an efficient protoplast preparing method by conditional optimization of sporulation and protoplast regeneration. The regeneration rate of the protoplast is up to about 34.6% with 0.8 M sucrose as the osmotic pressure stabilizer. To develop the genetic transformation, we used the polyethylene glycol-mediated protoplast transformation, and the testing gene <i>AG04914</i> encoding a major facilitator superfamily transporter was deleted in strain LC5815, which proves the feasibility of this genetic manipulation system. Furthermore, a uridine/uracil auxotrophic strain was created by using a positive screening protocol with 5-fluoroorotic acid as a selective reagent. Finally, the genetic transformation system was successfully established in the guanophilic fungus strain LC5815, which lays the foundation for the molecular genetics research and will facilitate the exploitation of bioactive secondary metabolites in fungi. |
| first_indexed | 2024-03-09T00:52:11Z |
| format | Article |
| id | doaj.art-16acf49372a8475ebd793b5e7f2515d4 |
| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-09T00:52:11Z |
| publishDate | 2021-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj.art-16acf49372a8475ebd793b5e7f2515d42023-12-11T17:04:51ZengMDPI AGJournal of Fungi2309-608X2021-02-017213810.3390/jof7020138Establishment of a Genetic Transformation System in Guanophilic Fungus <em>Amphichorda guana</em>Min Liang0Wei Li1Landa Qi2Guocan Chen3Lei Cai4Wen-Bing Yin5Henan Academy of Science Institute of Biology, Zhengzhou 450008, ChinaState Key Laboratory of Mycology and CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, ChinaHenan Academy of Science Institute of Biology, Zhengzhou 450008, ChinaHenan Academy of Science Institute of Biology, Zhengzhou 450008, ChinaState Key Laboratory of Mycology and CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, ChinaState Key Laboratory of Mycology and CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, ChinaFungi from unique environments exhibit special physiological characters and plenty of bioactive natural products. However, the recalcitrant genetics or poor transformation efficiencies prevent scientists from systematically studying molecular biological mechanisms and exploiting their metabolites. In this study, we targeted a guanophilic fungus <i>Amphichorda guana</i> LC5815 and developed a genetic transformation system. We firstly established an efficient protoplast preparing method by conditional optimization of sporulation and protoplast regeneration. The regeneration rate of the protoplast is up to about 34.6% with 0.8 M sucrose as the osmotic pressure stabilizer. To develop the genetic transformation, we used the polyethylene glycol-mediated protoplast transformation, and the testing gene <i>AG04914</i> encoding a major facilitator superfamily transporter was deleted in strain LC5815, which proves the feasibility of this genetic manipulation system. Furthermore, a uridine/uracil auxotrophic strain was created by using a positive screening protocol with 5-fluoroorotic acid as a selective reagent. Finally, the genetic transformation system was successfully established in the guanophilic fungus strain LC5815, which lays the foundation for the molecular genetics research and will facilitate the exploitation of bioactive secondary metabolites in fungi.https://www.mdpi.com/2309-608X/7/2/138guanophilic fungusgenetic transformationsecondary metaboliteuridine/uracil auxotrophyprotoplast |
| spellingShingle | Min Liang Wei Li Landa Qi Guocan Chen Lei Cai Wen-Bing Yin Establishment of a Genetic Transformation System in Guanophilic Fungus <em>Amphichorda guana</em> Journal of Fungi guanophilic fungus genetic transformation secondary metabolite uridine/uracil auxotrophy protoplast |
| title | Establishment of a Genetic Transformation System in Guanophilic Fungus <em>Amphichorda guana</em> |
| title_full | Establishment of a Genetic Transformation System in Guanophilic Fungus <em>Amphichorda guana</em> |
| title_fullStr | Establishment of a Genetic Transformation System in Guanophilic Fungus <em>Amphichorda guana</em> |
| title_full_unstemmed | Establishment of a Genetic Transformation System in Guanophilic Fungus <em>Amphichorda guana</em> |
| title_short | Establishment of a Genetic Transformation System in Guanophilic Fungus <em>Amphichorda guana</em> |
| title_sort | establishment of a genetic transformation system in guanophilic fungus em amphichorda guana em |
| topic | guanophilic fungus genetic transformation secondary metabolite uridine/uracil auxotrophy protoplast |
| url | https://www.mdpi.com/2309-608X/7/2/138 |
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