Optimized Linear DNA Recombineering for CRISPR-Cpf1 System in <i>Corynebacterium glutamicum</i>
<i>Corynebacterium glutamicum</i> is an important industrial production strain that is widely used in amino acid fermentation, biopharmaceuticals, and other fields. It is particularly important to develop efficient genome editing methods for the targeted modification of <i>C. gluta...
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MDPI AG
2023-12-01
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| Series: | Fermentation |
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| author | Ting Wang Xiaowan Jiang Shufang Lv Linfeng Hu Shuangcheng Gao Qingyang Xu Junhui Zhang Dianyun Hou |
| author_facet | Ting Wang Xiaowan Jiang Shufang Lv Linfeng Hu Shuangcheng Gao Qingyang Xu Junhui Zhang Dianyun Hou |
| author_sort | Ting Wang |
| collection | DOAJ |
| description | <i>Corynebacterium glutamicum</i> is an important industrial production strain that is widely used in amino acid fermentation, biopharmaceuticals, and other fields. It is particularly important to develop efficient genome editing methods for the targeted modification of <i>C. glutamicum</i> production strains. Currently, the gene editing system of <i>C. glutamicum</i> is inefficient and time-consuming. In this paper, we reported on a <i>Francisella novicida</i> (<i>Fn</i>) CRISPR-Cpf1-based system for genome editing. The system used linear DNA detached from the plasmid, and, with the assistance of the recombinase RecET, gene deletion was achieved by simultaneous electrotransformation of linear DNA with a plasmid carrying the <i>Fn</i>Cpf1 and crRNA expression cassette for double-strand breaks of the genome. Compared with previous all-in-one plasmids, this system reduced the time for one round of constructing recombinant plasmids and shortened the editing cycle by about 24 h. Finally, we successfully constructed an engineered strain (X−2) with high L-valine production by using the linear DNA-mediated gene deletion system. This method is of great importance for accelerating the process of metabolic engineering modification of <i>C. glutamicum</i> and its further application in high value-added products. |
| first_indexed | 2024-03-08T10:57:33Z |
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| issn | 2311-5637 |
| language | English |
| last_indexed | 2024-03-08T10:57:33Z |
| publishDate | 2023-12-01 |
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| series | Fermentation |
| spelling | doaj.art-b6ffd9e957784280b66795e822271cc12024-01-26T16:23:44ZengMDPI AGFermentation2311-56372023-12-011013110.3390/fermentation10010031Optimized Linear DNA Recombineering for CRISPR-Cpf1 System in <i>Corynebacterium glutamicum</i>Ting Wang0Xiaowan Jiang1Shufang Lv2Linfeng Hu3Shuangcheng Gao4Qingyang Xu5Junhui Zhang6Dianyun Hou7College of Agriculture, Henan University of Science & Technology, Luoyang 471023, ChinaCollege of Agriculture, Henan University of Science & Technology, Luoyang 471023, ChinaCollege of Agriculture, Henan University of Science & Technology, Luoyang 471023, ChinaCollege of Biotechnology, Tianjin University of Science & Technology, Tianjin 300459, ChinaCollege of Agriculture, Henan University of Science & Technology, Luoyang 471023, ChinaCollege of Biotechnology, Tianjin University of Science & Technology, Tianjin 300459, ChinaCollege of Agriculture, Henan University of Science & Technology, Luoyang 471023, ChinaCollege of Agriculture, Henan University of Science & Technology, Luoyang 471023, China<i>Corynebacterium glutamicum</i> is an important industrial production strain that is widely used in amino acid fermentation, biopharmaceuticals, and other fields. It is particularly important to develop efficient genome editing methods for the targeted modification of <i>C. glutamicum</i> production strains. Currently, the gene editing system of <i>C. glutamicum</i> is inefficient and time-consuming. In this paper, we reported on a <i>Francisella novicida</i> (<i>Fn</i>) CRISPR-Cpf1-based system for genome editing. The system used linear DNA detached from the plasmid, and, with the assistance of the recombinase RecET, gene deletion was achieved by simultaneous electrotransformation of linear DNA with a plasmid carrying the <i>Fn</i>Cpf1 and crRNA expression cassette for double-strand breaks of the genome. Compared with previous all-in-one plasmids, this system reduced the time for one round of constructing recombinant plasmids and shortened the editing cycle by about 24 h. Finally, we successfully constructed an engineered strain (X−2) with high L-valine production by using the linear DNA-mediated gene deletion system. This method is of great importance for accelerating the process of metabolic engineering modification of <i>C. glutamicum</i> and its further application in high value-added products.https://www.mdpi.com/2311-5637/10/1/31CRISPR-Cpf1linear DNA<i>Corynebacterium glutamicum</i>L-valine |
| spellingShingle | Ting Wang Xiaowan Jiang Shufang Lv Linfeng Hu Shuangcheng Gao Qingyang Xu Junhui Zhang Dianyun Hou Optimized Linear DNA Recombineering for CRISPR-Cpf1 System in <i>Corynebacterium glutamicum</i> Fermentation CRISPR-Cpf1 linear DNA <i>Corynebacterium glutamicum</i> L-valine |
| title | Optimized Linear DNA Recombineering for CRISPR-Cpf1 System in <i>Corynebacterium glutamicum</i> |
| title_full | Optimized Linear DNA Recombineering for CRISPR-Cpf1 System in <i>Corynebacterium glutamicum</i> |
| title_fullStr | Optimized Linear DNA Recombineering for CRISPR-Cpf1 System in <i>Corynebacterium glutamicum</i> |
| title_full_unstemmed | Optimized Linear DNA Recombineering for CRISPR-Cpf1 System in <i>Corynebacterium glutamicum</i> |
| title_short | Optimized Linear DNA Recombineering for CRISPR-Cpf1 System in <i>Corynebacterium glutamicum</i> |
| title_sort | optimized linear dna recombineering for crispr cpf1 system in i corynebacterium glutamicum i |
| topic | CRISPR-Cpf1 linear DNA <i>Corynebacterium glutamicum</i> L-valine |
| url | https://www.mdpi.com/2311-5637/10/1/31 |
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