Metabolic engineering and optimization of the fermentation medium for vitamin B 12 production in Escherichia coli
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RESEARCH PAPER
Metabolic engineering and optimization of the fermentation medium for vitamin B12 production in Escherichia coli Dong Li1,2 · Huan Fang2,3 · Yuanming Gai2,3 · Jing Zhao2,3 · Pingtao Jiang2,3 · Lei Wang1 · Qun Wei1 · Dayu Yu1 · Dawei Zhang2,3,4 Received: 1 February 2020 / Accepted: 15 April 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Vitamin B12 is a crucial fine chemical that is widely used in the pharmaceutical, food and chemical industries, and its production solely dependents on microbial fermentation. We previously constructed an artificial vitamin B12 biosynthesis pathway in Escherichia coli, but the yield of the engineered strains was low. Here, we removed metabolic bottlenecks of the vitamin B12 biosynthesis pathway in engineered E. coli strains. After screening cobB genes from different sources, optimizing the expression of cobN and customizing the ribosome binding sites of cobS and cobT, the vitamin B 12 yield increased to 152.29 μg/g dry cell weight (DCW). Optimization of the downstream module, which converts co(II)byrinic acid a,c-diamide into adenosylcobinamide phosphate, elevated the vitamin B12 yield to 249.04 μg/g DCW. A comparison of a variety of equivalent components indicated that glucose and corn steep liquor are optimal carbon and nitrogen sources, respectively. Finally, an orthogonal array design was applied to determine the optimal concentrations of glucose and nitrogen sources including corn steep liquor and yeast extract, through which a vitamin B12 yield of 530.29 μg/g DCW was obtained. The metabolic modifications and optimization of fermentation conditions achieved in this study offer a basis for further improving vitamin B12 production in E. coli and will hopefully accelerate its industrial application. Keywords Metabolic engineering · Vitamin B12 · Escherichia coli · Orthogonal experimental design
Introduction Dong Li and Huan Fang contributed equally. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00449-020-02355-z) contains supplementary material, which is available to authorized users. * Qun Wei [email protected] * Dayu Yu [email protected] * Dawei Zhang [email protected] 1
Sci‑Tech Center for Clean Conversion and High‑Valued Utilization of Biomass, Northeast Electric Power University, Jilin 132012, Jilin, China
2
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
3
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
4
National Engineering Laboratory for Industrial Enzymes, Tianjin 300308, China
Vitamin B12 is an essential vitamin with a complex chemical structure. It is extensively used in the pharmaceutical, food and chemical industries. Vitamin B12 is synthesized de novo by some bacteria and archaea through an aerobic pathway or an anaerobic pathway [1], each of which include more than 40 steps from glucose to the end product. Industrial production of vitam
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