Coenzyme B-12 can be produced by engineered Escherichia coli under both anaerobic and aerobic conditions

Abstract

Coenzyme B-12 (Vitamin B-12) is one of the most complex biomolecules and an essential cofactor required for the catalytic activity of many enzymes. Pseudomonas denitrificans synthesizes coenzyme B-12 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 B-12. 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 B-12 synthetic genes and successfully produced coenzyme B-12. However, according to the quantitative determination by inductively coupled plasma-mass spectrometry, the amount of coenzyme B-12 produced by the recombinant E. coli (0.21 +/- 0.02 mu g/g cdw) was approximately 13-fold lower than that by P. denitrificans (2.75 +/- 0.22 mu g/g cdw). Optimization of the culture conditions to improve the production of coenzyme B-12 by the recombinant E. coli was successful, and the highest titer (0.65 +/- 0.03 mu g/g cdw) of coenzyme B-12 was obtained. Interestingly, although the synthesis of coenzyme B-12 in P. denitrificans is strictly oxygen-dependent, the recombinant E. coli could produce coenzyme B-12 under anaerobic conditions.