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Background  As a very important coenzyme in the cell metabolism, Vitamin B 12  (cobalamin, VB 12 ) has been widely used in food and medicine fields. The complete biosynthesis of VB 12  requires approximately 30 genes, but overexpression of these genes did not result in expected increase of VB 12  production. High-yield VB 12 -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 VB 12  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 VB 12  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 VB 12  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 VB 12 -yield strains.    Electronic supplementary material  The online version of this article (10.1186/s12896-018-0441-2) contains supplementary material, which is available to authorized users.

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