Physiological Metabolic Analysis of VB₁₂ Accumulation in <i>Ensifer adhaerens Casida A</i> Enhanced by Oxygen Limitation

Cobalamin (VB₁₂) is in enormous demand across the fields of medicine, food, and feed additives. However, the oxygen supply plays a critical role in VB₁₂ biosynthesis by Ensifer adhaerens Casida A and has been identified as a bottleneck for economical substrate consumption. This study elucidates the relationship between oxygen limitation and VB₁₂ accumulation with transcriptomic and metabolomic analyses. Under oxygen limitation, E. adhaerens enhances oxygen transport and storage by increasing expression of flavin hemoglobin (Hmp), which was up-regulated 6-fold at 24 h of oxygen restriction compared to the oxygen restriction of 4 h (p < 0.01). Because of the cofactor of Hmp is heme, the demand for heme increases, leading to the upregulation of genes in the heme biosynthesis pathway. Similarly, genes involved in biosynthesis of its precursor, 5-ALA, were upregulated as well. 5-ALA is also a direct precursor of VB₁₂, further leading to the upregulation of genes in the VB₁₂ biosynthesis pathway. This process initiates biosynthesis and accumulation of VB₁₂. As VB₁₂ and heme biosynthesis progresses, genes associated with the biosynthesis and transportation pathways of compounds related to their biosynthesis were likewise upregulated, including genes involved in S-adenosyl methionine (SAM) biosynthesis, and the transport of Fe²⁺ and Co²⁺. Additionally, amino acids and organic acids associated with biosynthesis were also extensively consumed, such as methionine, which is used for synthesizing SAM, decreased by 310% after 24 h of oxygen limitation compared to 20% dissolved oxygen (p < 0.05). At the same time, genes related to growth-associated metabolic pathways, such as pentose phosphate pathway (PPP), were significantly downregulated. Therefore, the potential mechanism by which E. adhaerens accumulates VB₁₂ under oxygen-limited conditions by enhancing Hmp expression, which facilitates the porphyrin metabolic pathway and promotes VB₁₂ biosynthesis. This research provides valuable insights for increasing VB₁₂ production through metabolic engineering and process optimization.