title: 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.
authors.affiliation: School of Chemical and Biomolecular Engineering, Pusan National University, Busan, South Korea.
authors.forename: Yeounjoo
authors.initials: Y
authors.lastname: Ko
authors.forename: Somasundar
authors.initials: S
authors.lastname: Ashok
authors.forename: Satish Kumar
authors.initials: SK
authors.lastname: Ainala
authors.forename: Mugesh
authors.initials: M
authors.lastname: Sankaranarayanan
authors.forename: Ah Yeong
authors.initials: AY
authors.lastname: Chun
authors.forename: Gyoo Yeol
authors.initials: GY
authors.lastname: Jung
authors.forename: Sunghoon
authors.initials: S
authors.lastname: Park
mesh_terms: D000332:Aerobiosis; D000693:Anaerobiosis; D019149:Bioreactors; D003038:Cobamides / Q000032:analysis / Q000096:biosynthesis*; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial; D019344:Lactic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D011409:Propylene Glycols / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics*
chemical_list: D003038:Cobamides; D003470:Culture Media; D011409:Propylene Glycols; D019344:Lactic Acid; C041787:1,3-propanediol; C031601:hydracrylic acid; C000913:cobamamide
keywords: 1,3-Propanediol; 3-Hydroxypropionic acid; Coenzyme B12; Pseudomonas denitrificans; Quantitative bioassay