A single change in the aptamer of the Lactiplantibacillus plantarum rib operon riboswitch severely impairs its regulatory activity and leads to a vitamin B₂‐ overproducing phenotype

Abstract

Manufacturing of probiotics and functional foods using lactic acid bacteria (LAB) that overproduce vitamin B 2 has gained growing interest due to ariboflavinosis problems affecting populations of both developing and affluent countries. Two isogenic Lactiplantibacillus plantarum strains, namely a riboflavin-producing parental strain (UFG9) and a roseoflavin-resistant strain (B2) that carries a mutation in the FMN-aptamer of the potential rib operon riboswitch, were analysed for production and intraand extracellular accumulation of flavins, as well as for regulation of the rib operon expression. Strain B2 accumulated in the medium one of the highest levels of riboflavin+FMN ever reported for LAB, exceeding by ~25 times those accumulated by UFG9. Inside the cells, concentration of FAD was similar in both strains, while that of riboflavin+FMN was ~8-fold higher in B2. Mutation B2 could decrease the stability of the aptamer's regulatory P1 helix even in the presence of the effector, thus promoting the antiterminator structure of the riboswitch ON state. Although the B2-mutant riboswitch showed an impaired regulatory activity, it retained partial functionality being still sensitive to the effector. The extraordinary capacity of strain B2 to produce riboflavin, together with its metabolic versatility and probiotic properties, can be exploited for manufacturing multifunctional foods.

Weissella cibaria riboflavin-overproducing and dextran-producing strains useful for the development of functional bread

“…These results were expected due to an inhibitory effect of the FMN, upon internalization, in the riboflavin biosynthesis, proceeding to a latter expression of the rib operon when levels of the effector are exhausted in the cells. However, as expected the presence of the FMN had no influence on riboflavin production by the three mutant strains, since increase of fluorescence due to the presence of flavins was observed from the beginning of the exponential growth phase, as we have previously observed for riboflavin-overproducing L. plantarum mutants also carrying point mutations located in its corresponding aptamer of the FMN riboswitch ( 45 ). Consequently, these results and the fact that no mutations were found in the rib operon encoding the enzymes involved in the riboflavin biosynthetic pathway, strongly suggest that the mutations in the FMN-riboswitch are responsible for the riboflavin-overproducing phenotype of the W. cibaria B2 strains.…”

“…The Dsr from LAB produce extracellularly dextran by hydrolysis of sucrose molecules coupled to the transfer of glucose to the nascent α-glucan polymer ( 16 ). Moreover, we have shown that riboflavin produced by W. cibaria BAL3C-5, BAL3C-7, and BAL3C-22, as is the case in other LAB ( 12 , 45 ), can be detected and quantified in culture supernatants ( 32 ). Therefore, after growing the six LAB for 23 h in RAMS, the content of dextran and riboflavin present in the culture supernatants was determined, and the final biomass estimated by measuring the OD 600 nm and by plating ( Table 2 ).…”

“…This regulatory element consists of a sensor domain (the aptamer) that contains five hairpins (P2/L2 to P6/L6) and is closed by the P1 basal helix ( Figure 2A ), whose 3’-end is connected to the regulatory domain (or expression platform). The regulatory domain is predicted to adopt two alternative configurations that correspond to the ON state (characterized by an anti-terminator secondary structure) and to the OFF state, which includes an intrinsic transcriptional terminator preventing expression of the rib operon [ Figure 2B ; ( 45 )]. Binding of the effector (FMN) to the aptamer induces a conformational change in the expression platform from the ON to the OFF state, thus avoiding the metabolic burden of expressing the genes involved in riboflavin biosynthesis when not required.…”

“…Consequently, these results and the fact that no mutations were found in the rib operon encoding the enzymes involved in the riboflavin biosynthetic pathway, strongly suggest that the mutations in the FMN-riboswitch are responsible for the riboflavin-overproducing phenotype of the W. cibaria B2 strains. However, to prove this hypothesis a deep transcriptional analysis has to be performed in a future work, as has been already done for a L. plantarum riboflavin-overproducing mutant ( 45 ).…”

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“…To our knowledge, this is the first study describing an L. reuteri strain with a spontaneous riboflavin-overproducing phenotype. Besides the well-reported investigation and applications of overproducing L. plantarum strains (16,18,(52)(53)(54)(55)(56) only few other species of food-grade bacteria including L. lactis (50), Leuconostoc mesenteroides, Propionibacterium freudenreichii (43); Limosilactobacillus fermentum (57); Weissella cibaria, and Leuconostoc spp. (58) have been reported for the vitamin B2 bio-enrichment of fermented foods.…”