Coenzyme B12 can be produced by engineered Escherichia coli under both anaerobic and aerobic conditions.
Biotechnol J
; 9(12): 1526-35, 2014 Dec.
Article
em En
| MEDLINE
| ID: mdl-25146562
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.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Pseudomonas
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Cobamidas
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Escherichia coli
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Engenharia Metabólica
Idioma:
En
Revista:
Biotechnol J
Assunto da revista:
BIOTECNOLOGIA
Ano de publicação:
2014
Tipo de documento:
Article
País de afiliação:
Coréia do Sul