State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
College of Life Science and Technology, Guangxi University, Nanning, Guangxi, China.
Biotechnol J. 2024 Sep;19(9):e202400305. doi: 10.1002/biot.202400305.
Cobalamin (VB) is in enormous demand across the fields of medicine, food, and feed additives. However, the oxygen supply plays a critical role in VB 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 VB 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 VB, further leading to the upregulation of genes in the VB biosynthesis pathway. This process initiates biosynthesis and accumulation of VB. As VB 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 Fe and Co. 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 VB under oxygen-limited conditions by enhancing Hmp expression, which facilitates the porphyrin metabolic pathway and promotes VB biosynthesis. This research provides valuable insights for increasing VB production through metabolic engineering and process optimization.
钴胺素 (VB) 在医学、食品和饲料添加剂等领域的需求巨大。然而，氧供应在恩氏固氮菌 Casida A 的 VB 生物合成中起着关键作用，并且已被确定为经济基质消耗的瓶颈。本研究通过转录组学和代谢组学分析阐明了氧限制与 VB 积累之间的关系。在氧限制下，E. adhaerens 通过增加黄素血红蛋白 (Hmp) 的表达来增强氧的运输和储存，与 4 小时氧限制相比，24 小时氧限制时 Hmp 的表达上调了 6 倍 (p < 0.01)。由于 Hmp 的辅因子是血红素，因此对血红素的需求增加，导致血红素生物合成途径中的基因上调。同样，其前体 5-ALA 的生物合成途径中的基因也上调。5-ALA 也是 VB 的直接前体，进一步导致 VB 生物合成途径中的基因上调。这个过程启动了 VB 的生物合成和积累。随着 VB 和血红素生物合成的进行，与它们生物合成相关化合物的生物合成和运输途径相关的基因也同样上调，包括涉及 S-腺苷甲硫氨酸 (SAM) 生物合成的基因，以及 Fe 和 Co 的运输。此外，与生物合成相关的氨基酸和有机酸也被广泛消耗，例如甲硫氨酸，在 24 小时氧限制后比 20%溶解氧时减少了 310% (p < 0.05)。同时，与生长相关的代谢途径相关的基因，如戊糖磷酸途径 (PPP)，显著下调。因此，E. adhaerens 通过增强 Hmp 表达积累 VB 的潜在机制是通过促进卟啉代谢途径和促进 VB 生物合成。这项研究为通过代谢工程和工艺优化提高 VB 产量提供了有价值的见解。
