Characterization, complete genome sequencing, and CRISPR/Cas9 system-based decontamination of a novel Escherichia coli phage TR1 from fermentation substrates
Phage contamination has become a major concern for industrial bacteria, such as Escherichia coli BL21(DE3), used in fermentation processes. Herein, we report a CRISPR/Cas9 defense system-based strategy to precisely prey and degrade phage DNA to decontaminate target phages. First, we isolated a novel...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-08-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1230775/full |
| _version_ | 1797744789193490432 |
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| author | Yuqi Dong Yunfei Huang Huahao Fan Lihua Song Xiaoping An Shan Xu Mengzhe Li Mengzhe Li Yigang Tong Yigang Tong |
| author_facet | Yuqi Dong Yunfei Huang Huahao Fan Lihua Song Xiaoping An Shan Xu Mengzhe Li Mengzhe Li Yigang Tong Yigang Tong |
| author_sort | Yuqi Dong |
| collection | DOAJ |
| description | Phage contamination has become a major concern for industrial bacteria, such as Escherichia coli BL21(DE3), used in fermentation processes. Herein, we report a CRISPR/Cas9 defense system-based strategy to precisely prey and degrade phage DNA to decontaminate target phages. First, we isolated a novel phage from fermentation substrates with BL21(DE3) as the host, named TR1. It showed a typical podovirus morphology with a head diameter of 51.46 ± 2.04 nm and a tail length of 9.31 ± 2.77 nm. The burst size of phage TR1 was 151 PFU/cell, suggesting its strong fecundity in the fermentation system. Additionally, whole-genome sequencing revealed that phage TR1 has a DNA genome of 44,099 bp in length with a 43.8% GC content, encoding a total of 68 open reading frames. Comparative genomics and phylogenetic analysis designated this phage to be a new species of the genus Christensenvirus. To counteract phage TR1, we employed the CRISPR/Cas9 system-based strategy and constructed two phage-resistant E. coli strains, BL21-C and BL21-T, based on conserved genes. Both EOP assays and growth curves indicated strong phage resistance of the recombinant strains, without affecting cell growth. Therefore, this study aimed to provide a resilient strategy to respond to ever-changing phages and ongoing phage–host arm race in industrial fermentation environments by the personalized design of spacers in the recombinant CRISPR/Cas system-containing plasmid. More importantly, our research sparks the use of phage defense mechanism to prevent phage contamination in extensive biotechnological applications. |
| first_indexed | 2024-03-12T15:14:16Z |
| format | Article |
| id | doaj.art-9e3d74b53c5443f19d1515d484947f31 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-03-12T15:14:16Z |
| publishDate | 2023-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-9e3d74b53c5443f19d1515d484947f312023-08-11T16:47:18ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2023-08-011410.3389/fmicb.2023.12307751230775Characterization, complete genome sequencing, and CRISPR/Cas9 system-based decontamination of a novel Escherichia coli phage TR1 from fermentation substratesYuqi Dong0Yunfei Huang1Huahao Fan2Lihua Song3Xiaoping An4Shan Xu5Mengzhe Li6Mengzhe Li7Yigang Tong8Yigang Tong9College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, ChinaCollege of Life Science and Technology, Beijing University of Chemical Technology, Beijing, ChinaCollege of Life Science and Technology, Beijing University of Chemical Technology, Beijing, ChinaCollege of Life Science and Technology, Beijing University of Chemical Technology, Beijing, ChinaCollege of Life Science and Technology, Beijing University of Chemical Technology, Beijing, ChinaCollege of Life Science and Technology, Beijing University of Chemical Technology, Beijing, ChinaCollege of Life Science and Technology, Beijing University of Chemical Technology, Beijing, ChinaQinhuangdao Bohai Biological Research Institute, Beijing University of Chemical Technology, Qinhuangdao, Hebei, ChinaCollege of Life Science and Technology, Beijing University of Chemical Technology, Beijing, ChinaBeijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Beijing, ChinaPhage contamination has become a major concern for industrial bacteria, such as Escherichia coli BL21(DE3), used in fermentation processes. Herein, we report a CRISPR/Cas9 defense system-based strategy to precisely prey and degrade phage DNA to decontaminate target phages. First, we isolated a novel phage from fermentation substrates with BL21(DE3) as the host, named TR1. It showed a typical podovirus morphology with a head diameter of 51.46 ± 2.04 nm and a tail length of 9.31 ± 2.77 nm. The burst size of phage TR1 was 151 PFU/cell, suggesting its strong fecundity in the fermentation system. Additionally, whole-genome sequencing revealed that phage TR1 has a DNA genome of 44,099 bp in length with a 43.8% GC content, encoding a total of 68 open reading frames. Comparative genomics and phylogenetic analysis designated this phage to be a new species of the genus Christensenvirus. To counteract phage TR1, we employed the CRISPR/Cas9 system-based strategy and constructed two phage-resistant E. coli strains, BL21-C and BL21-T, based on conserved genes. Both EOP assays and growth curves indicated strong phage resistance of the recombinant strains, without affecting cell growth. Therefore, this study aimed to provide a resilient strategy to respond to ever-changing phages and ongoing phage–host arm race in industrial fermentation environments by the personalized design of spacers in the recombinant CRISPR/Cas system-containing plasmid. More importantly, our research sparks the use of phage defense mechanism to prevent phage contamination in extensive biotechnological applications.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1230775/fullfermentationphage contaminationEscherichia coliCRISPR/Cas9defense system |
| spellingShingle | Yuqi Dong Yunfei Huang Huahao Fan Lihua Song Xiaoping An Shan Xu Mengzhe Li Mengzhe Li Yigang Tong Yigang Tong Characterization, complete genome sequencing, and CRISPR/Cas9 system-based decontamination of a novel Escherichia coli phage TR1 from fermentation substrates Frontiers in Microbiology fermentation phage contamination Escherichia coli CRISPR/Cas9 defense system |
| title | Characterization, complete genome sequencing, and CRISPR/Cas9 system-based decontamination of a novel Escherichia coli phage TR1 from fermentation substrates |
| title_full | Characterization, complete genome sequencing, and CRISPR/Cas9 system-based decontamination of a novel Escherichia coli phage TR1 from fermentation substrates |
| title_fullStr | Characterization, complete genome sequencing, and CRISPR/Cas9 system-based decontamination of a novel Escherichia coli phage TR1 from fermentation substrates |
| title_full_unstemmed | Characterization, complete genome sequencing, and CRISPR/Cas9 system-based decontamination of a novel Escherichia coli phage TR1 from fermentation substrates |
| title_short | Characterization, complete genome sequencing, and CRISPR/Cas9 system-based decontamination of a novel Escherichia coli phage TR1 from fermentation substrates |
| title_sort | characterization complete genome sequencing and crispr cas9 system based decontamination of a novel escherichia coli phage tr1 from fermentation substrates |
| topic | fermentation phage contamination Escherichia coli CRISPR/Cas9 defense system |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1230775/full |
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