Establishment of a selectable marker recycling system for iterative gene editing in Fusarium fujikuroi
Gibberellic acid (GA3) is a vital plant growth hormone widely used in agriculture. Currently, GA3 production relies on liquid fermentation by the filamentous fungus Fusarium fujikuroi. However, the lack of an effective selection marker recycling system hampers the application of metabolic engineerin...
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| Format: | Article |
| Language: | English |
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KeAi Communications Co., Ltd.
2024-03-01
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| Series: | Synthetic and Systems Biotechnology |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405805X24000103 |
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| author | Tian-Qiong Shi Cai-Ling Yang Dong-Xun Li Yue-Tong Wang Zhi-Kui Nie |
| author_facet | Tian-Qiong Shi Cai-Ling Yang Dong-Xun Li Yue-Tong Wang Zhi-Kui Nie |
| author_sort | Tian-Qiong Shi |
| collection | DOAJ |
| description | Gibberellic acid (GA3) is a vital plant growth hormone widely used in agriculture. Currently, GA3 production relies on liquid fermentation by the filamentous fungus Fusarium fujikuroi. However, the lack of an effective selection marker recycling system hampers the application of metabolic engineering technology in F. fujikuroi, as multiple-gene editing and positive-strain screening still rely on a limited number of antibiotics. In this study, we developed a strategy using pyr4-blaster and CRISPR/Cas9 tools for recycling orotidine-5′-phosphate decarboxylase (Pyr4) selection markers. We demonstrated the effectiveness of this method for iterative gene integration and large gene-cluster deletion. We also successfully improved GA3 titers by overexpressing geranylgeranyl pyrophosphate synthase and truncated 3-hydroxy-3-methyl glutaryl coenzyme A reductase, which rewired the GA3 biosynthesis pathway. These results highlight the efficiency of our established system in recycling selection markers during iterative gene editing events. Moreover, the selection marker recycling system lays the foundation for further research on metabolic engineering for GA3 industrial production. |
| first_indexed | 2024-03-07T14:29:00Z |
| format | Article |
| id | doaj.art-5dcfc3a148134623bdc963b71f71e3ec |
| institution | Directory Open Access Journal |
| issn | 2405-805X |
| language | English |
| last_indexed | 2025-03-22T04:16:43Z |
| publishDate | 2024-03-01 |
| publisher | KeAi Communications Co., Ltd. |
| record_format | Article |
| series | Synthetic and Systems Biotechnology |
| spelling | doaj.art-5dcfc3a148134623bdc963b71f71e3ec2024-04-28T08:07:57ZengKeAi Communications Co., Ltd.Synthetic and Systems Biotechnology2405-805X2024-03-0191159164Establishment of a selectable marker recycling system for iterative gene editing in Fusarium fujikuroiTian-Qiong Shi0Cai-Ling Yang1Dong-Xun Li2Yue-Tong Wang3Zhi-Kui Nie4Jiangxi New Reyphon Biochemical Co., Ltd, Salt & Chemical Industry, Xingan, Jiangxi, 331399, People’s Republic of China; School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, 2 Xuelin Road, Qixia District, Nanjing, 210023, People’s Republic of China; Corresponding author. Jiangxi New Reyphon Biochemical Co., Ltd, Salt & Chemical Industry, Xingan, Jiangxi, 331399, People's Republic of China.School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, 2 Xuelin Road, Qixia District, Nanjing, 210023, People’s Republic of ChinaSchool of Food Science and Pharmaceutical Engineering, Nanjing Normal University, 2 Xuelin Road, Qixia District, Nanjing, 210023, People’s Republic of ChinaSchool of Food Science and Pharmaceutical Engineering, Nanjing Normal University, 2 Xuelin Road, Qixia District, Nanjing, 210023, People’s Republic of China; Corresponding author.Jiangxi New Reyphon Biochemical Co., Ltd, Salt & Chemical Industry, Xingan, Jiangxi, 331399, People’s Republic of China; School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, 2 Xuelin Road, Qixia District, Nanjing, 210023, People’s Republic of China; Corresponding author. Jiangxi New Reyphon Biochemical Co., Ltd, Salt & Chemical Industry, Xingan, Jiangxi, 331399, People's Republic of China.Gibberellic acid (GA3) is a vital plant growth hormone widely used in agriculture. Currently, GA3 production relies on liquid fermentation by the filamentous fungus Fusarium fujikuroi. However, the lack of an effective selection marker recycling system hampers the application of metabolic engineering technology in F. fujikuroi, as multiple-gene editing and positive-strain screening still rely on a limited number of antibiotics. In this study, we developed a strategy using pyr4-blaster and CRISPR/Cas9 tools for recycling orotidine-5′-phosphate decarboxylase (Pyr4) selection markers. We demonstrated the effectiveness of this method for iterative gene integration and large gene-cluster deletion. We also successfully improved GA3 titers by overexpressing geranylgeranyl pyrophosphate synthase and truncated 3-hydroxy-3-methyl glutaryl coenzyme A reductase, which rewired the GA3 biosynthesis pathway. These results highlight the efficiency of our established system in recycling selection markers during iterative gene editing events. Moreover, the selection marker recycling system lays the foundation for further research on metabolic engineering for GA3 industrial production.http://www.sciencedirect.com/science/article/pii/S2405805X24000103F. fujikuroiGibberellic acidPlant growth hormoneSelection marker recycling systemCRISPR/Cas9pyr4-blaster |
| spellingShingle | Tian-Qiong Shi Cai-Ling Yang Dong-Xun Li Yue-Tong Wang Zhi-Kui Nie Establishment of a selectable marker recycling system for iterative gene editing in Fusarium fujikuroi Synthetic and Systems Biotechnology F. fujikuroi Gibberellic acid Plant growth hormone Selection marker recycling system CRISPR/Cas9 pyr4-blaster |
| title | Establishment of a selectable marker recycling system for iterative gene editing in Fusarium fujikuroi |
| title_full | Establishment of a selectable marker recycling system for iterative gene editing in Fusarium fujikuroi |
| title_fullStr | Establishment of a selectable marker recycling system for iterative gene editing in Fusarium fujikuroi |
| title_full_unstemmed | Establishment of a selectable marker recycling system for iterative gene editing in Fusarium fujikuroi |
| title_short | Establishment of a selectable marker recycling system for iterative gene editing in Fusarium fujikuroi |
| title_sort | establishment of a selectable marker recycling system for iterative gene editing in fusarium fujikuroi |
| topic | F. fujikuroi Gibberellic acid Plant growth hormone Selection marker recycling system CRISPR/Cas9 pyr4-blaster |
| url | http://www.sciencedirect.com/science/article/pii/S2405805X24000103 |
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