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

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.