Abstract
Vitamin B(12) (cobalamin, hereafter B(12)) 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(12) biosynthesis, and its minimal production of inactive pseudovitamin B(12). This efficiency is largely attributed to its oxygen-dependent synthesis of 5,6-dimethylbenzimidazole (DMBI), the lower ligand of vitamin B(12), via the BluB enzyme. Additionally, the synthesis of another B-group vitamin, riboflavin (hereafter B(2)), may influence DMBI production by providing precursor molecules. To clarify the roles of B(12) and B(2) in growth under different oxygen conditions, we generated P. freudenreichii DSM 4902 mutants with disrupted bluB and ribA genes, affecting B(12) and B(2) biosynthesis, respectively. The growth defects of both mutants were rescued by vitamin supplementation, indicating the presence of functional uptake systems for B(12) and B(2). Riboflavin was essential under all tested conditions, especially during aerobic growth, while B(12) was required for optimal growth only under anaerobic conditions (pO(2) < 1%) and dispensable under aerobic conditions (pO(2) ~ 20%). In the absence of B(12), the production of short-chain fatty acids (SCFAs) was significantly reduced. Titration experiments identified 0.1 µg/mL of B(12) and 0.05 µg/mL of B(2) 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(12) 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.