Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti
Abstract Background As a very important coenzyme in the cell metabolism, Vitamin B12 (cobalamin, VB12) has been widely used in food and medicine fields. The complete biosynthesis of VB12 requires approximately 30 genes, but overexpression of these genes did not result in expected increase of VB12 pr...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2018-05-01
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| Series: | BMC Biotechnology |
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| Online Access: | http://link.springer.com/article/10.1186/s12896-018-0441-2 |
| _version_ | 1818200207222898688 |
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| author | Yingying Cai Miaomiao Xia Huina Dong Yuan Qian Tongcun Zhang Beiwei Zhu Jinchuan Wu Dawei Zhang |
| author_facet | Yingying Cai Miaomiao Xia Huina Dong Yuan Qian Tongcun Zhang Beiwei Zhu Jinchuan Wu Dawei Zhang |
| author_sort | Yingying Cai |
| collection | DOAJ |
| description | Abstract Background As a very important coenzyme in the cell metabolism, Vitamin B12 (cobalamin, VB12) has been widely used in food and medicine fields. The complete biosynthesis of VB12 requires approximately 30 genes, but overexpression of these genes did not result in expected increase of VB12 production. High-yield VB12-producing strains are usually obtained by mutagenesis treatments, thus developing an efficient screening approach is urgently needed. Result By the help of engineered strains with varied capacities of VB12 production, a riboswitch library was constructed and screened, and the btuB element from Salmonella typhimurium was identified as the best regulatory device. A flow cytometry high-throughput screening system was developed based on the btuB riboswitch with high efficiency to identify positive mutants. Mutation of Sinorhizobium meliloti (S. meliloti) was optimized using the novel mutation technique of atmospheric and room temperature plasma (ARTP). Finally, the mutant S. meliloti MC5–2 was obtained and considered as a candidate for industrial applications. After 7 d’s cultivation on a rotary shaker at 30 °C, the VB12 titer of S. meliloti MC5–2 reached 156 ± 4.2 mg/L, which was 21.9% higher than that of the wild type strain S. meliloti 320 (128 ± 3.2 mg/L). The genome of S. meliloti MC5–2 was sequenced, and gene mutations were identified and analyzed. Conclusion To our knowledge, it is the first time that a riboswitch element was used in S. meliloti. The flow cytometry high-throughput screening system was successfully developed and a high-yield VB12 producing strain was obtained. The identified and analyzed gene mutations gave useful information for developing high-yield strains by metabolic engineering. Overall, this work provides a useful high-throughput screening method for developing high VB12-yield strains. |
| first_indexed | 2024-12-12T02:33:59Z |
| format | Article |
| id | doaj.art-8f31a51b8de34594a3d2db83f507832b |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-12T02:33:59Z |
| publishDate | 2018-05-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-8f31a51b8de34594a3d2db83f507832b2022-12-22T00:41:19ZengBMCBMC Biotechnology1472-67502018-05-0118111110.1186/s12896-018-0441-2Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium melilotiYingying Cai0Miaomiao Xia1Huina Dong2Yuan Qian3Tongcun Zhang4Beiwei Zhu5Jinchuan Wu6Dawei Zhang7Tianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesCollege of Biotechnology, Tianjin University of Science & TechnologySchool of Food Science and Technology, Dalian Polytechnic University, National Engineering Research Center of SeafoodIndustrial Biotechnology Division, Institute of Chemical and Engineering SciencesTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesAbstract Background As a very important coenzyme in the cell metabolism, Vitamin B12 (cobalamin, VB12) has been widely used in food and medicine fields. The complete biosynthesis of VB12 requires approximately 30 genes, but overexpression of these genes did not result in expected increase of VB12 production. High-yield VB12-producing strains are usually obtained by mutagenesis treatments, thus developing an efficient screening approach is urgently needed. Result By the help of engineered strains with varied capacities of VB12 production, a riboswitch library was constructed and screened, and the btuB element from Salmonella typhimurium was identified as the best regulatory device. A flow cytometry high-throughput screening system was developed based on the btuB riboswitch with high efficiency to identify positive mutants. Mutation of Sinorhizobium meliloti (S. meliloti) was optimized using the novel mutation technique of atmospheric and room temperature plasma (ARTP). Finally, the mutant S. meliloti MC5–2 was obtained and considered as a candidate for industrial applications. After 7 d’s cultivation on a rotary shaker at 30 °C, the VB12 titer of S. meliloti MC5–2 reached 156 ± 4.2 mg/L, which was 21.9% higher than that of the wild type strain S. meliloti 320 (128 ± 3.2 mg/L). The genome of S. meliloti MC5–2 was sequenced, and gene mutations were identified and analyzed. Conclusion To our knowledge, it is the first time that a riboswitch element was used in S. meliloti. The flow cytometry high-throughput screening system was successfully developed and a high-yield VB12 producing strain was obtained. The identified and analyzed gene mutations gave useful information for developing high-yield strains by metabolic engineering. Overall, this work provides a useful high-throughput screening method for developing high VB12-yield strains.http://link.springer.com/article/10.1186/s12896-018-0441-2Vitamin B12CobalaminSinorhizobium melilotiRiboswitchHigh-throughputScreening |
| spellingShingle | Yingying Cai Miaomiao Xia Huina Dong Yuan Qian Tongcun Zhang Beiwei Zhu Jinchuan Wu Dawei Zhang Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti BMC Biotechnology Vitamin B12 Cobalamin Sinorhizobium meliloti Riboswitch High-throughputScreening |
| title | Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti |
| title_full | Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti |
| title_fullStr | Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti |
| title_full_unstemmed | Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti |
| title_short | Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti |
| title_sort | engineering a vitamin b12 high throughput screening system by riboswitch sensor in sinorhizobium meliloti |
| topic | Vitamin B12 Cobalamin Sinorhizobium meliloti Riboswitch High-throughputScreening |
| url | http://link.springer.com/article/10.1186/s12896-018-0441-2 |
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