Oxygen determines the requirement for cobalamin but not riboflavin in the growth of Propionibacterium freudenreichii.

Vitamin B (cobalamin, hereafter B) is essential for human health, particularly for neural function and DNA synthesis. It is synthesized exclusively by bacteria and archaea, with animal-derived foods serving as the primary sources for humans. Propionibacterium freudenreichii is notable for its long-standing use in food production, its efficient B biosynthesis, and its minimal production of inactive pseudovitamin B. This efficiency is largely attributed to its oxygen-dependent synthesis of 5,6-dimethylbenzimidazole (DMBI), the lower ligand of vitamin B, via the BluB enzyme. Additionally, the synthesis of another B-group vitamin, riboflavin (hereafter B), may influence DMBI production by providing precursor molecules. To clarify the roles of B and B in growth under different oxygen conditions, we generated P. freudenreichii DSM 4902 mutants with disrupted bluB and ribA genes, affecting B and B biosynthesis, respectively. The growth defects of both mutants were rescued by vitamin supplementation, indicating the presence of functional uptake systems for B and B. Riboflavin was essential under all tested conditions, especially during aerobic growth, while B was required for optimal growth only under anaerobic conditions (pO < 1%) and dispensable under aerobic conditions (pO ~ 20%). In the absence of B, the production of short-chain fatty acids (SCFAs) was significantly reduced. Titration experiments identified 0.1 µg/mL of B and 0.05 µg/mL of B as sufficient to support maximal growth. Our results also showed that external B₂ supplementation eliminates the influence of de novo B₂ synthesis on B₁₂ production, and that oxygen availability reduces the cellular requirement for B during growth. This study reveals how oxygen modulates the interplay between B₁₂ and B₂ metabolism in P. freudenreichii, emphasizing the importance of oxygen availability in regulating B₁₂ biosynthesis and utilization. These insights can inform the design of optimized fermentation processes for sustainable and efficient B₁₂ production in the food and supplement industries.