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

Abstract

BackgroundAs 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.ResultBy 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.ConclusionTo 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.Electronic supplementary materialThe online version of this article (10.1186/s12896-018-0441-2) contains supplementary material, which is available to authorized users.

Microbial and Genetic Resources for Cobalamin (Vitamin B12) Biosynthesis: From Ecosystems to Industrial Biotechnology

“…The production of cobalamin by these microorganisms has been extensively researched under specific culture conditions (supplementation of precursors and metal ions, carbon/nitrogen sources, oxygenic/anoxygenic conditions, cultivation time etc. ), with consequence improving their synthesis capacity by random mutagenesis (UV light, chemicals) and genetic manipulations (overexpression, modification, regulation) [ 6 , 20 , 23 ]. In addition, the reported cobalamin-producing natural or recombinant bacteria comprised genera Acetobacterium, Aerobacter, Agrobacterium, Alcaligenes, Arthrobacter, Azotobacter, Bacillus, Clostridium, Corynebacterium, Escherichia, Eubacterium, Flavobacterium, Methanobacillus, Methanosarcina, Mycobacterium, Propionibacterium, Proteus, Pseudomonas, Rhizobium, Rhodopseudomonas, Salmonella, Serratia, Streptococcus, Streptomyces, Xanthomonas and others, whose habitats are soil, ocean, and microflora in digestive tracts of humans and animals [ 7 , 12 , 24 , 25 ].…”

Contemporary Tools for Regulating Gene Expression in Bacteria

“…These include tight regulation of basal expression, large dynamic range, and high signal:noise ratio. Riboswitches have also been used as biosensors to improve the production of amino acids, such as tryptophan [102], lysine [103], and vitamin B 12 [104], and for the sensing and production of the flavonoid naringenin [105,106]. Given that riboswitches sense and respond to their cognate ligand without the need for any additional proteins, these regulatory devices yield a faster response time compared with protein-based systems, and should also place reduced burden upon the cellular machinery [10].…”

Advancement of Metabolic Engineering Assisted by Synthetic Biology

“…The diversity, specificity, and kinetics of regulatory RNAs contribute to the regulation of diverse physiological responses, including transcription, translation, and mRNA stability in living cells [96,97,[117][118][119][120][121][122]. The structures, functions, and mechanisms of regulatory RNAs have been elucidated in many bacterial species for decades.…”

“…Engineered riboswitches have been used as a genetic toolkit to obtain desired gene production or toxic product regulation in bacterial species. For example, the application of engineered VB12 riboswitches to develop high-yield VB12 production strains using a flow cytometry high-throughput screening system in Salmonella typhimurium has been reported [96]. Moreover, six different theophylline-responsive riboswitches have been used for intricate transcriptional regulation of the toxic protein SacB in several cyanobacterial species; these riboswitches have shown more efficient performances than isopropyl-D-thiogalactopyranoside (IPTG) induction of the lacI q -Ptrc promoter system [97].…”