**Background:** Riboflavin (vitamin B2) is an essential micronutrient not synthesized by humans; deficiency is a global concern. Lactic acid bacteria (LAB) can produce riboflavin and dextran-type exopolysaccharides, making them attractive for in situ food biofortification. The rib operon, which encodes riboflavin biosynthesis enzymes, is regulated by an FMN riboswitch: when FMN binds, transcription is terminated. Roseoflavin, a toxic analog of riboflavin, binds the same riboswitch and kills bacteria unless mutations disrupt this regulation. Previous work identified three riboflavin- and dextran-producing W. cibaria strains (BAL3C-5, BAL3C-7, BAL3C-22) from rye sourdough and generated three mutants via stepwise roseoflavin exposure. The present study aimed to develop a more efficient method for detecting and isolating spontaneous riboflavin-overproducing mutants from these strains.
**Methods:** The three parental W. cibaria strains were grown in RAM medium and treated with roseoflavin at 100, 200, 300, or 400 μg/mL for ~60 h at 30°C. Genomic DNA was extracted from treated cultures, and the FMN riboswitch region was PCR-amplified and Sanger-sequenced to detect mutations. RNAfold was used to predict secondary structures and Gibbs free energy (ΔG) of mutant aptamers. Roseoflavin-treated cultures were plated on MRSS agar (containing sucrose to support dextran synthesis), and yellow mucous colonies were selected as putative riboflavin-overproducers. Eight mutant strains were isolated and characterized. Riboflavin production was monitored in real time by fluorescence (excitation 440 nm, emission 520 nm) in RAMS medium, with and without added FMN or riboflavin (3 μM). After 16 h growth, total and free riboflavin were quantified, and dextran was measured by the phenol-sulfuric method. RT-qPCR quantified ribG gene expression (first gene of the rib operon) and FMN riboswitch transcript levels, normalized to rpoB. Whole-genome sequencing of BAL3C-5 wild-type and BAL3C-5 C120T mutant was performed using Illumina MiSeq and Oxford Nanopore MiniION, assembled with Unicycler, and annotated with Prokka. Statistical analysis used one-way ANOVA with Dunnett or Tukey tests (p ≤ 0.05).
**Key Results:** DNA sequencing of roseoflavin-treated cultures revealed 10 distinct mutations in the FMN riboswitch aptamer: 9 point mutations (G14T, G15T, T16G, C23T, A59C, G87A, G109A, A115G, C120T) and one single-nucleotide deletion (ΔG15). Most mutations clustered in the P2/L2 and P6/L6 stem-loop regions. RNAfold predictions showed that mutations G14T, G15T, C23T, C120T, and ΔG15 increased ΔG (less stable structures; −45.0 to −47.0 kcal/mol vs. −47.9 kcal/mol for wild-type), while T16G and G109A decreased ΔG (−48.6 kcal/mol). Eight mutants were isolated: BAL3C-5 ΔG15, BAL3C-5 A59C, BAL3C-5 A115G, BAL3C-5 C120T, BAL3C-5 G15T, BAL3C-7 G14T, BAL3C-7 G109A, and BAL3C-22 C23T. All mutants produced significantly more riboflavin than wild-type strains (p < 0.01). In RAMS, total riboflavin ranged from 1.42 mg/L (BAL3C-5 A59C) to 6.78 mg/L (BAL3C-5 C120T), compared to 0.16–0.18 mg/L for wild-types. In RAM (without sucrose), the range was 0.73–5.10 mg/L vs. 0.03–0.04 mg/L. The BAL3C-5 C120T mutant showed a ~70-fold increase in RAMS and ~290-fold increase in RAM over its parental strain. Over 90% of riboflavin was released into the supernatant in mutant strains. Dextran production in RAMS ranged from 5.60 g/L (BAL3C-7 G14T) to 7.10 g/L (BAL3C-22 wild-type); only BAL3C-7 G14T showed significantly lower dextran than its parent (p < 0.05). Growth rates were similar across all strains (0.62–0.77 h⁻¹). RT-qPCR revealed that all mutants had significantly higher ribG expression than parental strains. In RAMS, fold changes ranged from 1.30 (BAL3C-5 A59C) to 7.42 (BAL3C-5 C120T). In RAMS + FMN, the increase was more pronounced: 10.9- to 161.2-fold, with BAL3C-5 C120T again highest. Wild-type strains showed near-complete repression of ribG in the presence of FMN (0.05–0.09-fold expression vs. without FMN). Mutants BAL3C-5 A59C and A115G showed partial repression (0.33- and 0.58-fold), while all other mutants showed no significant repression (0.95–1.18-fold), indicating deregulation. Whole-genome sequencing of BAL3C-5 and BAL3C-5 C120T revealed identical genomes (2,406,256 bp, 45.15% GC, 2,350 genes) except for a single C-to-T transition at position 446,494 (the C120T mutation in the FMN riboswitch). No antibiotic resistance genes or virulence factors were detected in either genome.
**Clinical Implications:** This study demonstrates that a single point mutation in the FMN riboswitch is sufficient to convert a wild-type W. cibaria strain into a high-level riboflavin overproducer, with production levels (6.78 mg/L) exceeding most previously reported LAB mutants. The BAL3C-5 C120T strain also maintains robust dextran production (~6.3 g/L), making it a promising candidate for in situ biofortification of fermented foods such as bread, potentially addressing riboflavin deficiency in vulnerable populations. The method described — roseoflavin treatment followed by sequencing-based screening and colony color selection on dextran-supporting medium — provides a reliable pipeline for generating and identifying overproducing strains. However, W. cibaria currently lacks QPS/GRAS status, and further safety and probiotic evaluation is required before industrial or clinical use.