Role of riboflavin and nicotinamide in vitamin B12 production by Propionibacterium freudenreichii

Riboflavin (RF) serves as a precursor for the biosynthesis of 5,6-dimethylbenzimidazole (DMBI), the lower ligand of vitamin B12, in Propionibacterium freudenreichii. Nicotinamide (NAM) has been reported to enhance this biosynthetic pathway. In this study, we investigated the effect of RF and NAM supplementation on vitamin B12 production by P. freudenreichii DSM 20271 in barley malt extract-based model medium. A medium containing 10 % (w/v) concentrated malt extract supplemented with lactate and tryptone supported superior growth and B12 production. RF (1, 3 and 40 μM with 27 mM NAM), and NAM (0.1, 0.6 and 27 mM with 3 μM RF) were evaluated for their impact, with 100 μM DMBI used as a reference. RF concentrations ≥3 μM significantly enhanced B12 production, while NAM was effective only at 27 mM. Growth and metabolite profiles were largely unaffected, suggesting that increased B12 synthesis resulted from improved precursor availability rather than enhanced biomass. Transcriptomic analysis revealed significant downregulation of RF biosynthesis genes, consistent with feedback inhibition from exogenous RF, while B12 biosynthesis genes remained transcriptionally stable, indicating that the observed increase in B12 production is primarily regulated at the metabolic rather transcriptional level. These findings demonstrate that B12 production in P. freudenreichii can be enhanced using RF and NAM precursors, but practical applications must balance efficacy with NAM safety limits for use in B12-enriched fermented foods.