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<p>Vitamin B<sub>12</sub> (cobalamin, hereafter B<sub>12</sub>) 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. <i>Propionibacterium freudenreichii</i> is notable for its long-standing use in food production, its efficient B<sub>12</sub> biosynthesis, and its minimal production of inactive pseudovitamin B<sub>12</sub>. This efficiency is largely attributed to its oxygen-dependent synthesis of 5,6-dimethylbenzimidazole (DMBI), the lower ligand of vitamin B<sub>12</sub>, via the BluB enzyme. Additionally, the synthesis of another B-group vitamin, riboflavin (hereafter B<sub>2</sub>), may influence DMBI production by providing precursor molecules. To clarify the roles of B<sub>12</sub> and B<sub>2</sub> in growth under different oxygen conditions, we generated <i>P. freudenreichii</i> DSM 4902 mutants with disrupted <i>bluB</i> and <i>ribA</i> genes, affecting B<sub>12</sub> and B<sub>2</sub> biosynthesis, respectively. The growth defects of both mutants were rescued by vitamin supplementation, indicating the presence of functional uptake systems for B<sub>12</sub> and B<sub>2</sub>. Riboflavin was essential under all tested conditions, especially during aerobic growth, while B<sub>12</sub> was required for optimal growth only under anaerobic conditions&#xa0;(pO<sub>2</sub> &lt; 1%) and dispensable under aerobic conditions (pO<sub>2</sub> ~ 20%). In the absence of B<sub>12</sub>, the production of short-chain fatty acids (SCFAs) was significantly reduced. Titration experiments identified 0.1&#xa0;µg/mL of B<sub>12</sub> and 0.05&#xa0;µg/mL of B<sub>2</sub> 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<sub>12</sub> during growth. This study reveals how oxygen modulates the interplay between B₁₂ and B₂ metabolism in <i>P. freudenreichii</i>, 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年7月29日

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  • Ruoxi Zhang,
  • Yuandong Sha,
  • Bhawani Chamlagain,
  • Minnamari Edelmann,
  • Kirsi Savijoki,
  • Vieno Piironen,
  • Paulina Deptula,
  • Pekka Varmanen

摘要

Vitamin B12 (cobalamin, hereafter B12) 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 B12 biosynthesis, and its minimal production of inactive pseudovitamin B12. This efficiency is largely attributed to its oxygen-dependent synthesis of 5,6-dimethylbenzimidazole (DMBI), the lower ligand of vitamin B12, via the BluB enzyme. Additionally, the synthesis of another B-group vitamin, riboflavin (hereafter B2), may influence DMBI production by providing precursor molecules. To clarify the roles of B12 and B2 in growth under different oxygen conditions, we generated P. freudenreichii DSM 4902 mutants with disrupted bluB and ribA genes, affecting B12 and B2 biosynthesis, respectively. The growth defects of both mutants were rescued by vitamin supplementation, indicating the presence of functional uptake systems for B12 and B2. Riboflavin was essential under all tested conditions, especially during aerobic growth, while B12 was required for optimal growth only under anaerobic conditions (pO2 < 1%) and dispensable under aerobic conditions (pO2 ~ 20%). In the absence of B12, the production of short-chain fatty acids (SCFAs) was significantly reduced. Titration experiments identified 0.1 µg/mL of B12 and 0.05 µg/mL of B2 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 B12 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.