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Microbial Production of Vitamin B12: a Review and Future Perspectives Huan Fang1,2,3, Jie Kang1,4 and Dawei Zhang1,2*

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Microbial Production of Vitamin B12: a Review and Future Perspectives Huan Fang1,2,3, Jie Kang1,4 and Dawei Zhang1,2* Fang et al. Microb Cell Fact (2017) 16:15 DOI 10.1186/s12934-017-0631-y Microbial Cell Factories REVIEW Open Access Microbial production of vitamin B12: a review and future perspectives Huan Fang1,2,3, Jie Kang1,4 and Dawei Zhang1,2* Abstract Vitamin B12 is an essential vitamin that is widely used in medical and food industries. Vitamin B12 biosynthesis is con- fined to few bacteria and archaea, and as such its production relies on microbial fermentation. Rational strain engi- neering is dependent on efficient genetic tools and a detailed knowledge of metabolic pathways, regulation of which can be applied to improve product yield. Recent advances in synthetic biology and metabolic engineering have been used to efficiently construct many microbial chemical factories. Many published reviews have probed the vitamin B12 biosynthetic pathway. To maximize the potential of microbes for vitamin B12 production, new strategies and tools are required. In this review, we provide a comprehensive understanding of advances in the microbial production of vitamin B12, with a particular focus on establishing a heterologous host for the vitamin B12 production, as well as on strategies and tools that have been applied to increase microbial cobalamin production. Several worthy strategies employed for other products are also included. Keywords: Vitamin B12, Biosynthesis, Metabolic regulation, Synthetic biology, Escherichia coli, Metabolic engineering Background is regulated by a cobalamin riboswitch in the 5′ untrans- Vitamin B12, also known as cyanocobalamin, belongs lated regions (UTR) of the corresponding genes. to the cobalamin family of compounds, which are com- Large scale industrial production of vitamin B12 occurs posed of a corrinoid ring and an upper and lower ligand. via microbial fermentation, predominantly utilizing Pseu- The upper ligand can be adenosine, methyl, hydroxy, or a domonas denitrificans, Propionibacterium shermanii, or cyano group [1]. Vitamin B12 is synthesized by prokary- Sinorhizobium meliloti [7]. However, these strains have otes and inhibits the development of pernicious anemia several shortcomings, such as long fermentation cycles, in animals. complex and expensive media requirements, and a lack of Microbial de novo biosynthesis of vitamin B12 occurs suitable genetic systems for strain engineering. To date, through two alternative routes: the aerobic or anaero- most of the research on these producers has focused on bic pathway, in bacteria and archaea, respectively. Some traditional strategies, such as random mutagenesis and strains can also synthesize cobalamin by absorbing fermentation process optimization, with only limited corrinoids via a salvage pathway, as shown in Table 1. research on metabolic engineering. Recently, engineers Tetrapyrrole compounds including cobalamin, heme, and have shifted their attention to Escherichia coli as a plat- bacteriochlorophyll, are derived from δ-aminolevulinate form for vitamin B12 production. E. coli has become a (ALA) and a complex interdependent and interactional well-studied cell factory that has been extensively used relationship exists among these tetrapyrrole compounds for the production of various chemicals, such as ter- in numerous bacterial species [2]. To maintain vitamin penoids, non-natural alcohols, and poly-(lactate-co- B12 at stable levels, its biosynthesis and transportation glycolate) [8–10]. Furthermore, metabolic engineering and synthetic biology strategies have been extensively applied to improve the production of these compounds *Correspondence: [email protected] [11, 12]. Escherichia coli synthesizes ALA via the C5 path- 1 Tianjin Institute of Industrial Biotechnology, Chinese Academy way and has been used as a microbial cell factory to pro- of Sciences, Tianjin 300308, China Full list of author information is available at the end of the article duce ALA via C4 and C5 pathways [13, 14] and E. coli can © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fang et al. Microb Cell Fact (2017) 16:15 Page 2 of 14 Table 1 Cobalamin biosynthetic pathway in microbes investigation of several strategies for the development of Microorganisms De novo synthesis Salvage References microbial cell factories, including synthetic biology and pathway pathway metabolic engineering, among others. Since research on Aerobes the engineering of vitamin B12 producing strains remains Pseudomonas Yes Yes [3] limited, related strategies for other chemicals are also dentrificans outlined. Rhodobacter Yes Yes [3] capusulatus Cobalamin biosynthetic pathway Rhodobacter Yes Yes [3] De novo pathway sphaeroides As mentioned above, cobalamin can be synthesized Sinorhizobium Yes Yes [3] meliloti de novo in prokaryotes through two alternative routes Anaerobes according to the timing of cobalt insertion and the molecular oxygen requirement. These pathways are the Salmonella Yes Yes [4] typhimurium aerobic pathway, which has been best studied in P. deni- Bacillus megaterium Yes * [5] trificans, and the anaerobic pathway, which has been best Propionibacterium Yes * [5] studied in S. typhimurium, Bacillus megaterium, and shermanii P. shermanii, [19]. Both routes differ in terms of cobalt Escherichia coli No Yes [4] chelation (via hydrogenobyrinic a, c-diamide with the Thermotoga sp. RQ2 No No [6] CobNST complex in the aerobic pathway, and via precor- Thermotoga No No [6] rin-2 with CbiK in S. typhimurium) and oxygen require- maritima MSB8 ments (the aerobic pathway requires oxygen to promote Thermotoga No No [6] ring-contraction, while the anaerobic pathway does not neapolitana require oxygen in this step) (Fig. 1). Thermotoga No No [6] petrophila Biosynthetic pathways of tetrapyrrole compounds: Thermotoga No No [6] ALA is synthesized by either C4 or C5 pathway and aden- naphthophila osylcobalamin is synthesized either via de novo or salvage Thermotoga No Yes [6] pathways. The enzymes shown in the adenosylcobalamin thermarum biosynthetic pathway originate from P. denitrificans or S. Thermotoga No Yes [6] typhimurium, which use either the aerobic pathway or lettingae anaerobic pathway, respectively. Fervidobacterium No Yes [6] nodosum The first committed precursor of the tetrapyrrole syn- Thermosipho Yes Yes [6] thesis pathway is ALA. ALA is synthesized by either the melanesiensis C4 pathway or the C5 pathway. In the C4 pathway, the Thermosipho Yes Yes [6] enzyme ALA synthase from glycine and succinyl-CoA africanus catalyzes the formation of ALA. In the C5 pathway, ALA Kosmotoga olearia No Yes [6] is synthesized from glutamate through three enzymatic Mesotoga prima No No [6] reactions [20]. Two molecules of ALA are condensed to Petrotoga mobilis No No [6] form monopyrrole porphobilinogen by porphobilinogen Unidentified pathways are marked with “*” synthase and four porphobilinogen molecules are then polymerized and cyclized to form uroporphyrinogen III. This reaction is catalyzed by the enzymes porpho- also synthesize vitamin B12 via the salvage pathway. The closely related Salmonella typhimurium is able to synthe- bilinogen deaminase and uroporphyrinogen III synthase. Methylation of uroporphyrinogen III at C-2 and C-7 size vitamin B12 de novo. Many genes involved in vitamin results in the synthesis of precorrin-2 (which is a com- B12 biosynthesis in S. typhimurium have been shown to be functional in E. coli [15–17]. Transfer of 20 genes from mon precursor of cobalamin), siroheme, and coenzyme the S. typhimurium cob locus allowed the production of F430 [7, 21]. In P. denitrificans, CobA catalyzes this meth- ylation step. In S. typhimurium and E. coli, the fusion vitamin B12 in E. coli [18]. These advantages facilitate the enzyme CysG is shared between siroheme and cobalamin de novo production of vitamin B12 in E. coli. Due to the complexity of the pathway and its meta- synthesis. The N-terminus of CysG has dehydrogenase/ bolic regulation, numerous studies have been performed ferrochelatase activity and the C-terminus has uropor- phyrinogen III methyltransferase activity. In S. cerevisiae, by various groups on the vitamin B12 biosynthesis. This MET1p functions as a uroporphyrinogen III methyl- review provides an overview of the vitamin B12 bio- synthesis and its metabolic regulation, along with an transferase [22]. Fang et al. Microb Cell Fact (2017) 16:15 Page 3 of 14 Fang et al. Microb Cell Fact (2017) 16:15 Page 4 of 14 (See figure on previous page.) Fig. 1 Biosynthetic pathways of tetrapyrrole compounds. ALA is synthesized by either the C4 or the C5 pathway. Adenosylcobalamin is synthesized via the de novo or via salvage pathways. The enzymes shown in the adenosylcobalamin biosynthetic pathway originate from P. denitrificans or S. typhimurium, which either use the aerobic pathway or the anaerobic pathway, respectively The aerobic and anaerobic pathways diverge at precor- catalyzed by an AdoCbl-5-P phosphatase (CobC) [25]. rin-2 and converge at coby(II)rinic acid a, c-diamide. The nucleotide loop assembly pathway is the last char- Eight peripheral methylation
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