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BMC Biotechnol. 2018 May 11;18(1):27. doi: 10.1186/s12896-018-0441-2.

Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti.

Cai Y1,2,3, Xia M1,2, Dong H1,2, Qian Y1,2, Zhang T3, Zhu B4, Wu J5, Zhang D6,7,8.

Author information

1
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308, China.
2
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308, China.
3
College of Biotechnology, Tianjin University of Science & Technology, No. 29, thirteenth Avenue Binhai District, Tianjin, 300457, China.
4
School of Food Science and Technology, Dalian Polytechnic University, National Engineering Research Center of Seafood, Dalian, 116034, People's Republic of China.
5
Industrial Biotechnology Division, Institute of Chemical and Engineering Sciences, 1 Pesek Road, Jurong Island, 627833, Singapore.
6
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308, China. zhang_dw@tib.cas.cn.
7
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308, China. zhang_dw@tib.cas.cn.
8
School of Food Science and Technology, Dalian Polytechnic University, National Engineering Research Center of Seafood, Dalian, 116034, People's Republic of China. zhang_dw@tib.cas.cn.

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.

KEYWORDS:

Cobalamin; High-throughputScreening; Riboswitch; Sinorhizobium meliloti; Vitamin B12

PMID:
29751749
PMCID:
PMC5948670
DOI:
10.1186/s12896-018-0441-2
[Indexed for MEDLINE]
Free PMC Article

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