© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 1 Introduction Coenzyme B 12 , which is also known as cobamide coen- zyme, is the largest and structurally most complicated vitamin (Fig. 1). Coenzyme B 12 plays an important role in the normal functioning of the brain, nervous system and in the formation of blood cells in animals. The coenzyme plays an essential role in stimulating the folate synthesis needed for DNA synthesis [1]. Coenzyme B 12 consists of three parts: a central corrin ring, an a-axial ligand or nucleotide loop, and a β-axial ligand [2, 3]. The corrin ring or tetrapyrrole ring contains a central cobalt atom linked to four pyrrole nitrogen atoms. The β-axial ligand can be an adenosyl moiety (adenosylcobalamin; Coenzyme B 12 ), cyanide (cyanocobalamin; Vitamin B 12 ), hydroxyl group (hydroxocobalamin; aquacobalamin), or methyl group (methylcobalamin; met-Cbl). Adenosylcobalamin and methylcobalamin are two biologically active forms of vita- Research Article Coenzyme B 12 can be produced by engineered Escherichia coli under both anaerobic and aerobic conditions Yeounjoo Ko 1* , Somasundar Ashok 1* , Satish Kumar Ainala 1 , Mugesh Sankaranarayanan 1 , Ah Yeong Chun 1 , Gyoo Yeol Jung 2 and Sunghoon Park 1 1 School of Chemical and Biomolecular Engineering, Pusan National University, Busan, South Korea 2 Department of Chemical Engineering and School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Gyeongbuk, South Korea 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 coen- zyme 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 engi- neering was used to produce coenzyme B 12 . These genes were cloned into three compatible plas- mids 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 suc- cessfully produced coenzyme B 12 . However, according to the quantitative determination by induc- tively coupled plasma-mass spectrometry, the amount of coenzyme B 12 produced by the recom- binant 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 coen- zyme B 12 by the recombinant E. coli was successful, and the highest titer (0.65 ± 0.03 μg/g cdw) of coenzyme B 12 was obtained. Interestingly, although the synthesis of coenzyme B 12 in P. denitri- ficans is strictly oxygen-dependent, the recombinant E. coli could produce coenzyme B 12 under anaerobic conditions. Keywords: 1,3-Propanediol · 3-Hydroxypropionic acid · Coenzyme B 12 · Pseudomonas denitrificans · Quantitative bioassay See accompanying commentary by Yin Li DOI 10.1002/biot.201400472 Correspondence: Prof. Sunghoon Park, School of Chemical and Bio- molecular Engineering, Pusan National University, San 30, Jangjeon-dong, Geumjeong-gu, Busan 609-735, South Korea E-mail: parksh@pusan.ac.kr Abbreviations: met-Cbl, methylcobalamin; GRAS, generally recognized as safe; DMBI, 5,6-dimethylbenzimidazole group; ICP-MS, inductively coupled plasma-mass spectrometry; NEB, nebulizer gas flow; MCS, multiple cloning sites; IPTG, isopropyl β-D-1-thiogalactopyranoside; DEPC, diethylpyrocar- bonate; ALA, aminolevulinic acid; MS, mass spectrophotometer; 3-HP, 3-hydroxypropionic acid; 1,3-PDO, 1,3-propanediol; GDHt, glycerol dehy- dratase Biotechnol. J. 2014, 9 DOI 10.1002/biot.201400221 www.biotechnology-journal.com Biotechnology Journal Received 07 APR 2014 Revised 23 JUN 2014 Accepted 20 AUG 2014 Accepted article online 22 AUG 2014 Supporting information available online * These authors contributed equally to this work.