Coenzyme B₁₂ can be produced by engineered Escherichia coli under both anaerobic and aerobic conditions

Abstract

Coenzyme B12 (Vitamin B12 ) is one of the most complex biomolecules and an essential cofactor required for the catalytic activity of many enzymes. Pseudomonas denitrificans synthesizes coenzyme B12 in an oxygen-dependent manner using a pathway encoded by more than 25 genes that are located in six different operons. Escherichia coli, a robust and suitable host for metabolic engineering was used to produce coenzyme B12 . These genes were cloned into three compatible plasmids and expressed heterologously in E. coli BL21 (DE3). Real-time PCR, SDS-PAGE analysis and bioassay showed that the recombinant E. coli expressed the coenzyme B12 synthetic genes and successfully produced coenzyme B12 . However, according to the quantitative determination by inductively coupled plasma-mass spectrometry, the amount of coenzyme B12 produced by the recombinant E. coli (0.21 ± 0.02 μg/g cdw) was approximately 13-fold lower than that by P. denitrificans (2.75 ± 0.22 μg/g cdw). Optimization of the culture conditions to improve the production of coenzyme B12 by the recombinant E. coli was successful, and the highest titer (0.65 ± 0.03 μg/g cdw) of coenzyme B12 was obtained. Interestingly, although the synthesis of coenzyme B12 in P. denitrificans is strictly oxygen-dependent, the recombinant E. coli could produce coenzyme B12 under anaerobic conditions.

Biobased organic acids production by metabolically engineered microorganisms

“…One drawback with this system is the requirement of adding the enzyme cofactor vitamin B 12 to the culture medium, which is not feasible at an industrial scale. Although E. coli has been reported to produce vitamin B 12 by heterologous expressing the biosynthetic pathway from Pseudomounas denitrificans [28], involving more than 25 genes represents a big challenge for incorporating and balancing this pathway with 3-HP production will require extensive engineering.…”

Biological Production of 3-Hydroxypropionic Acid: An Update on the Current Status

“…Another consideration when the dha operon is used is that the first step of the reaction requires coenzyme B12, which is produced naturally de novo by some bacterial strains, such as Klebsiella pneumoniae [15,16], whereas other bacteria, such as Escherichia coli, cannot produce it de novo [16]. When the host microorganism is not capable of producing this coenzyme, external addition of it should be included in the cultivation-which will affect the cost of the process.…”

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“…17–19 In contrast, E. coli requires nickel for the active sites of at least three anaerobically expressed hydrogenases, and thus has a dedicated nickel trafficking system for maintaining nickel homeostasis. 20–23 This trafficking system consists of an importer, NikABCDE, 24, 25 chaperones ( e.g .…”