Vitamin B2 overproduction in Lactiplantibacillus plantarum: new insights in the FMN-dependent regulatory activity of the rib operon riboswitch

Lactiplantibacillus plantarum is a metabolically versatile lactic acid bacterium found in matrices of animal and plant origin, where it contributes to fermentation. Many L. plantarum strains carry a complete riboflavin biosynthesis (rib) operon that is tightly regulated by an FMN riboswitch, which contains a sensing domain (aptamer) that, upon binding to the effector (FMN), induces an ON-to-OFF conformational change in the expression platform. Riboflavin-overproducing L. plantarum strains carrying mutations in the rib operon riboswitch have previously been selected by exposure to roseoflavin and used to produce vitamin-biofortified functional foods. Here, we have tested the real-time production of riboflavin by 11 mutants and found variable yields ranging from 0.9 to 6.6 mg/L. Using some of these mutants, we have developed an efficient screening method, based on imaging and quantifying the fluorescence of riboflavin in individual colonies, to identify the most effective riboflavin overproducers. Each of the 11 riboflavin-overproducing mutants had a single mutation in the aptamer of the rib operon riboswitch. To assess the riboswitch function, we have determined the transcription start site of the L. plantarum rib operon and constructed transcriptional fusions between the mCherry-encoding gene from plasmid pRCR and different riboswitch variants containing wild-type and mutant aptamers. The regulatory activity and effector responsiveness of the riboswitch variants were monitored by measuring mCherry fluorescence in L. plantarum host cells, carrying different plasmid constructs, that were cultivated with or without riboflavin. The results allowed the identification of nucleotides and secondary structures relevant for the effector-dependent regulatory activity of the riboswitch.