sign in sign up
<<
Home , Bacteriochlorophyll, Cobalamin biosynthesis, Corrinoid, Siroheme, Tetrapyrrole

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 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. is con- fined to few and , 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 , 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 ring and an upper and lower . via microbial fermentation, predominantly utilizing Pseu- The upper ligand can be , methyl, hydroxy, or a domonas denitrificans, 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 via a salvage pathway, as shown in Table 1. research on metabolic engineering. Recently, engineers compounds including cobalamin, , and have shifted their attention to Escherichia coli as a plat- , 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 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 through two alternative routes Anaerobes according to the timing of insertion and the molecular requirement. These pathways are the Yes Yes [4] typhimurium aerobic pathway, which has been best studied in P. deni- 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] (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 shown in the 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] 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 by porphobilinogen Unidentified pathways are marked with “*” synthase and four porphobilinogen molecules are then polymerized and cyclized to form 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. 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), , 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 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 loop assembly pathway is the last char- Eight peripheral methylation reactions occur during de acterized reaction in the vitamin B12 biosynthesis. novo , within identical temporal BluB from S. meliloti is a member of the reduced form and spatial orders in both the aerobic and anaerobic path- of nicotinamide-adenine dinucleotide (NADH)/flavin ways. Many of the methyltransferase enzymes involved in mononucleotide (FMN)-dependent nitroreductase fam- these reactions show high degrees of sequence similarity ily, which can convert FMNH2 to DMB (5, 6-dimeth- [23]. ylbenzimidazole) [26, 27]. In the anaerobic bacterium Cob(I)yrinic acid a,c-diamide is adenosylated to form E. limosum, 5-aminoimidazole ribotide is converted to adenosyl cobyrinic acid a,c-diamide. Cob(I)yrinic acid DMB by enzymes encoded in the bza operon [28] and a,c-diamide adenosyltransferase can also adenosylate subsequently, CobT can activate a range of lower ligand other corrinoids, where at least the a and c positions of substrates including DMB, which determine cobamide the carboxyl groups are amidated. Adenosyl cobyrinic diversity [29]. acid a,c-diamide is subjected to four stepwise amidation reactions at carboxyl groups at positions b, d, e, and g Salvage pathway to yield adenosyl cobyric acid. Two separate methods The salvage pathway is a cost-effective way (in terms of have evolved to attach (R)-1-amino-2-propanol or (R)- energy) for bacteria and archaea to obtain cobalamin. In 1-amino-2-propanol phosphate at the f position of the gram-negative bacteria, exogenous corrinoids are trans- carboxyl group of adenosyl cobyric acid in the aerobic ported into the cell via an ATP-binding cassette (ABC) and anaerobic pathways. In the anaerobic pathway, the transport system, consisting of BtuC, BtuD, and BtuF, linker between the corrinoid ring and the lower axial which are membrane permease, ATPase, and periplas- ligand is phosphorylated prior to attachment of the mic-binding protein components, respectively. BtuB is a corrinoid ring. The enzyme PduX from S. enterica is TonB-dependent transporter located in the outer mem- an l- kinase used in the de novo synthesis of brane, delivering corrinoid to the periplasmic corrinoid- coenzyme B12; however, it is not involved in the cobina- binding protein BtuF. The latter then delivers corrinoid mide salvage pathway [17]. l-threonine O-3-phosphate to the BtuCD complex located in the inner membrane is then decarboxylated to yield (R)-1-amino-2-propanol [30]. Archaea also use ABC transporters for corrinoid O-2-phosphate via CobD in S. typhimurium LT2 [15]. uptake. Archaeal orthologs of the bacterial BtuC, BtuD, However, in P. denitrificans it is most likely (although and BtuF have been found in sp. strain proof remains to be published), that (R)-1-amino- NRC-1 [31]. Subsequent to transport through the mem- 2-propanol is directly attached to the corrinoid ring brane, cobinamide is adenosylated by ATP:co(I)rrinoid via protein α and β. Currently, protein α remains as adenosyltransferases (ACATs). Three families of ACATs yet unknown and protein β is a complex of CobC and exist, namely: CobA, EutT, and PduO [32]. In bacte- CobD. The molecule is then phosphorylated by CobP, ria, AdoCbi is the substrate for a bifunctional enzyme which is a bifunctional enzyme possessing ATP:AdoCbi (CobU in S. typhimurium or CobP in P. denitrificans) (adenosylcobinamide) kinase and GTP:AdoCbi-P gua- with kinase and guanylyltransferase activities. In archaea, nylyltransferase activity [24]. Two additional reac- the cbiZ gene encodes an amidohydrolase that converts tions transfer lower axial onto AdoCbi-GDP, AdoCbi to adenosylcobyric acid, which is condensed thus producing adenosylcobalamin (AdoCbl). There with 1-aminopropanol-O-2-phosphate by an AdoCbi- are two alternative views of the last step in vitamin P synthase (CbiB) to yield AdoCbi-P. Since the archaeal B12 biosynthesis. One view is that the last reaction in enzyme lacks AdoCbi kinase activity, CobY, which has the biosynthesis of AdoCbl involves the addition of GTP:AdoCbi-P guanylyltransferase activity, is used to α-ribazole, catalyzed via cobalamine synthase. However, transfer guanylyl to AdoCbi-P [30, 33]. Identical to the in S. typhimurium, α-ribazole 5′-phosphate is added to de novo pathway, two additional reactions transfer lower AdoCbi-GDP and thus, the last reaction would be the axial ligands onto AdoCbi-GDP to produce AdoCbl in dephosphorylation of AdoCbl 5′-phosphate to AdoCbl, the salvage pathway. Fang et al. Microb Cell Fact (2017) 16:15 Page 5 of 14

Metabolic regulation of cobalamin synthesis the uroporphyrinogen III methyltransferase of Methano- The relationship between cobalamin, heme, bacterium ivanovii and Paracoccus pantotrophus were and not inhibited by uroporphyrinogen III, even at concen- Photosynthetic bacteria possess all three tetrapyrrole trations of up to 20 µM [42, 43]. The identification of new compounds including cobalamin, heme, and bacteri- SUMT enzymes that are insensitive to feedback inhibi- ochlorophyll (Fig. 1), which share the same biosynthetic tion to replace the native enzyme of industrial strains pathway from ALA to uroporphyrinogen III. Excess heme may be an effective method to improve vitamin 12B yield. inhibits HemA via feedback inhibition, reducing the flux of cobalamin and bacteriochlorophyll, while heme limita- Cobalamin riboswitches tion weakly regulates hemCD, hemH, and hemA expres- The cobalamin riboswitch is the predominant form of sion in E. coli [34]. However, not just a competitive metabolic regulation to control vitamin B12 concen- relationship exists among the tetrapyrrole compounds, tration in microbes. Riboswitches are evolutionarily as these compounds also share a complex interdepend- conserved non-coding regions that are situated in the ent and interactional relationship [2]. Hydrogenobyrinic 5′-untranslated region of mRNAs that regulate gene acid synthase from possesses expression in response to direct binding of intracellu- two Fe-S centers, a flavin and a heme group [35]. Rhodo- lar metabolites by the RNA itself [44]. Riboswitches are bacter capsulatus requires a cobalamin to form composed of two functional domains: one domain serves the isocyclic ring of chlorophyll [36]. Synthesis of heme as an evolutionarily conserved natural aptamer, bind- is also cobalamin dependent, as heme synthesis requires ing the target metabolite with high selectivity and affin- S-adenosylmethionine as a methyl group donor, while ity, while the other domain harnesses allosteric changes S-adenosylmethionine synthesis involves a cobalamin- in RNA structure, caused by aptamer-ligand complex dependent enzyme [37]. This complex relationship is formation, to control expression of an adjacent gene or also manifest at the transcriptional level. E.g., cobalamin operon. Johnson et al. solved the crystal structures of two participates in the regulation of bacteriochlorophyll syn- different classes of cobalamin riboswitches, which share a thesis via the AerR-CrtJ regulatory pair in R. capsulatus. common four-way junction (P3–P6 helices), forming the CrtJ is responsible for the repression of bacteriochloro- core receptor domain that is responsible for cobalamin phyll during aerobic growth [38], while AerR functions binding, but use distinct peripheral extensions (P8–P11) as an anti-repressor of CrtJ, inhibiting CrtJ binding to to recognize different cobalamin derivatives [45]. The P6 the bchC promoter when cobalamin is bound to the con- extension is present for the AdoCbl binding class, while served histidine (His145) of AerR [39]. In R. sphaeroides, it is absent for the and the aquoco- heme affects the ability of PpsR to regulate genes that are balamin binding classes [45–47]. A kissing-loop interac- involved in the tetrapyrrole biosynthesis by binding PpsR tion between loop L5 of the cobalamin-binding core and and changing its DNA binding pattern. Thus, PpsR func- L13 of the regulatory domain regulates the expression tions as both a and a heme sensor to coordinate machinery [46]. Holmstrom et al. identified the confor- cellular heme and bacteriochlorophyll levels [38]. This mational mechanism responsible for the regulation of complex relationship between tetrapyrrole compounds is by a binding ribos- a consequence of natural evolution. witch (env8HyCbl). The authors utilized single-molecule fluorescence resonance energy transfer techniques [48]. Regulation of the key enzyme S‑Adenosyl‑l‑methionine: Binding of hydroxocobalamin promotes the formation uroporphyrinogen III methyltransferase (SUMT) of the L5–L13 kissing-loop, which sequesters the Shine- Feedback inhibition of a key enzyme located at the Dalgarno sequence via base pairing, thus preventing branch point of a biosynthetic pathway is a common translation initiation. Cobalamin riboswitches participate method for metabolic regulation in microbes. In many in the regulation of cobalamin biosynthesis and trans- microorganisms, SUMT regulates cobalamin flux. In P. port at the transcriptional or translational levels, such as denitrificans, SUMT is sensitive to feedback inhibition by the btuB gene of E. coli and the cob operon of S. typh- cobalamin and corrinoid intermediates, and exhibits sub- imurium [49]. In case of transcription inhibition and for strate inhibition at uroporphyrinogen III concentrations high cobalamin concentrations, an alternative Rho-inde- above 2 µM [40]. Bacillus megaterium SUMT exhibits pendent termination hairpin or Rho binding site cause substrate inhibition at uroporphyrinogen concentrations premature transcriptional termination. High cobalamin above 0.5 µM [41], while P. denitrificans SUMT is inhib- concentrations can also promote sequestration of ribo- ited by uroporphyrinogen concentrations above 0.2 µM some binding sites (RBS) and blockage of translation [40]. Fortunately, uroporphyrinogen III methyltrans- initiation. When cobalamin concentrations are low, an ferase shows limited substrate inhibition. As an example, anti-terminator hairpin forms, enabling RNA polymerase Fang et al. Microb Cell Fact (2017) 16:15 Page 6 of 14

to complete transcription of the downstream gene. Low such as riboswitches, should be removed. After the addi- cobalamin concentrations facilitate anti-sequester hair- tion of transcriptional and translational elements, such pin formation that releases the RBS, thus enabling trans- as promoters, ribosome binding sequences, and termina- lation initiation [50, 51]. Cobalamin riboswitches also tors, the structural genes for cobalamin synthesis can be have a particular function in ethanolamine utilization. In expressed either monocistronically or polycistronically Listeria monocytogenes, a cobalamin riboswitch controls on plasmids or be integrated into the genome. The rapid the expression of a noncoding regulatory RNA named development of synthetic biology tools has facilitated the Rli55, which controls the expression of the eut genes that simplified construction of heterologous pathways. Het- requires vitamin B12 as a cofactor and determines ethan- erogeneous genes can be assembled by a number of dif- olamine utilization [52]. ferent techniques such as SLIC, CPEC, Gibson assembly, golden gate cloning, DNA assembler, and LCR [54]. When Synthetic biology to improve cobalamin too many heterogeneous genes need to be transferred to production a heterologous host, it is difficult to build the metabolic Design of a heterologous biosynthetic pathway for the pathway one gene at a time. The metabolic route can be vitamin B12 production split into multiple modules. After sequential validation of Apart from altering native microbial hosts or identifying the function of these modules, their assembly becomes novel microbial hosts to produce cobalamin, the con- possible. The final construct can then be transferred to the struction of heterologous biosynthetic pathways in model chosen host for heterologous expression, thus allowing organisms that can be easily genetically manipulated is a the host to synthesize cobalamin. To evaluate the capacity promising strategy. Synthetic biology is an efficient tool of vitamin B12 production, the engineered strains are then that can be used to reconstruct pathways or genetic net- cultured under optimal conditions. works to produce compounds in a heterologous host. Design of a heterologous biosynthetic pathway. (A) A The construction of a vitamin B12 biosynthetic pathway host for the heterologous biosynthetic pathway is selected in a heterologous host includes selection of a suitable considering capabilities of precursor and cofactor supply, host, building the biosynthetic pathway with functional tools, and industrial-scale fermenta- components, and pathway tuning (Fig. 2). Several points tion capability using cheap and readily available carbon should be noted in the selection of an ideal host. (1) The sources. (B) Enzyme activity is verified in vitro and there- host should have the ability to supply precursors (e.g. after in vivo. Products of in vitro assay or intracellular ALA) and cofactors (e.g. S-adenosylmethionine) for the reaction products are detected via spectroscopic analy- production of the desired chemical. E.g., the heterolo- sis, mass spectrometry, or microbiological assays. (C) gous C4 pathway in E. coli has been reported to generate Heterogeneous genes and other functional elements are high ALA production [13], avoiding addition of exog- assembled on plasmids via gene assembly methods such enous ALA to the medium; (2) there need to be suffi- as SLIC, CPEC, Gibson, golden gate, DNA assembler, cient genetic engineering tools such as transformation and LCR, or integrated into the genome. To decrease the protocols, expression vectors, and chromosomal gene difficulty of building the metabolic pathway, it is divided knockout/integration systems to manipulate the host into separate modules. These modules are sequentially [53]; (3) the host is suitable for industrial-scale fermenta- verified in a heterologous host, and then assembled. (D) tion, utilizing cheap and readily available carbon sources Based on the quantification of metabolites, bottlenecks such as glucose, xylose, and arabinose. When the host should be removed and metabolic flux to target com- is selected, various candidate genes from diverse native pound maximization. To optimize gene expression in vitamin B12 producers can be expressed in the host. The the metabolic pathway, promoters, RBS, and gene copy use of in vitro kinetic analysis is an efficient approach to number are designed and implemented at the transcrip- detect ideal enzymes. Most of the intermediates of the tional or translational levels. (E) The characteristics of vitamin B12 synthetic pathway are not available; conse- engineered strains are verified via fermentation. Various quently, to prepare substrates for in vitro kinetic analy- substrates (e.g., ALA, cobalt , betaine and DMB) and sis, the desired chemicals need to be separated from the varying conditions (e.g., dissolved oxygen concentration, products of corresponding strains or be prepared enzy- pH, and temperature) can be optimized to improve yield matically. The products of an in vitro assay can then be and productivity. detected by spectroscopic analysis, mass spectrometry, or microbiological assays. Sometimes, the heterologous pro- Pathway tuning duction of enzymes does not work and it is necessary to Biologists face the challenge to direct the flux of interme- screen for novel enzymes from various sources. For heter- diates to generate the desired product. To avoid the accu- ologous expression studies, all native forms of regulation, mulation of toxic intermediates and to drive flux to the Fang et al. Microb Cell Fact (2017) 16:15 Page 7 of 14

Fig. 2 Design of a heterologous biosynthetic pathway. a A host for the heterologous biosynthetic pathway is selected considering the capability of precursor and cofactor supply, genetic engineering tools, and industrial-scale fermentation capability, utilizing cheap and readily available carbon sources. b Enzyme activity is verified in vitro and subsequently in vivo. Products of the in vitro assay or intracellular reaction products are detected via spectroscopic analysis, mass spectrometry, or microbiological assays. c Heterogeneous genes and other functional elements are assembled on plasmids via gene assembly methods such as SLIC, CPEC, Gibson, golden gate, DNA assembler and LCR, or integrated into the genome. To decrease the difficulty of building the metabolic pathway, it is divided into separate modules. These modules are verified sequentially in a heterologous host and then assembled. d Based on the quantification of metabolites, bottlenecks should be removed and metabolic flux should be integrated to target compound maximization. To optimize gene expression in the metabolic pathway, promoters, RBS, and gene copy number are designed and implemented at the transcriptional or translational levels. e The characteristics of the engineered strains are verified via fermentation. Various substrates (e.g., ALA, cobalt ions, betaine and DMB) and varying conditions (e.g., dissolved oxygen concentration, pH, and temperature) can be optimized to improve yield and productivity

desired end product, each step in the pathway needs to Sometimes chromosomal expression of heterologous be balanced. In addition, gene overexpression may cause metabolic pathways can avoid plasmid instability and an undesirable metabolic burden on the host, and the thus reduce the metabolic burden on the cell. Vitamin B12 level of gene expression needs to be accurately adjusted biosynthesis is a highly evolved pathway, with more than to coordinate both metabolic flux and cell growth. twenty steps, including corrinoid ring construction and Fang et al. Microb Cell Fact (2017) 16:15 Page 8 of 14

peripheral modifications, and the enzymes involved have were co-localized in a protein scaffold, resulting in a three- high substrate specificity. Therefore, it is necessary to fold improvement in butyrate production [67]. Cobalamin optimize gene expression in existing metabolic pathways. synthesis is a complex pathway with several competing Gene expression in a given pathway can be altered at intermediates, such as heme and siroheme. Co-localization the level of transcription or translation [55]. Promoter of enzymes to the same subcellular organelle or compart- strength affects gene expression at the transcriptional ment can increase the local intermediate concentration and level, while DNA sequence of the RBS affects gene expres- exclude competing cytosolic pathways [68]. Substrate chan- sion at the translational level. The “Ribosome binding neling is a process of transferring the product of one enzyme site calculator” and RBSDesigner have been used to pre- to an adjacent enzyme within the cascade or cell without dict and design synthetic RBSs to yield the desired level complete mixing with the bulk phase [69]. This phenom- of protein expression in bacteria [56]. The expression of enon occurs for at least some of the enzymes involved in multiple genes in synthetic operons can be optimized to hydrogenobyrinic acid (HBA) biosynthesis. An enzyme-trap enhance production levels via engineering of promoters approach has been used to isolate intermediates of the cobal- [57] and RBSs [58], or via generation of libraries of tuna- amin biosynthetic pathway based on this mechanism [70]. ble intergenic regions [59]. Tuning operon expression can Therefore, protein scaffolds may offer a promising alternative be achieved via different plasmid replicons to control gene to balance multiple enzymes within the vitamin B12 biosyn- copy number, different promoters to control the rate of thetic pathway, thus improving flux to cobalamin. transcription initiation, and different RBSs for controlling All these approaches optimize gene expression or the level of translation [60]. Multiplex automated genome increase pathway flux in vivo. In vitro steady-state anal- engineering (MAGE) can be used for genome-scale opti- ysis is an efficient approach to identify bottlenecks in mization of gene expression on the chromosome [61]. metabolic pathways and to balance flux to the desired The translational efficiency of each gene in an operon compounds. It has been successfully used to increase the is affected by intergenic sequence regions and involves production of fatty acids, fatty acid short-chain esters, post-transcriptional processes such as transcription ter- fatty alcohols, farnesenes, alkenes, and alkanes [71–75]. mination, mRNA stability, and translation initiation. Previously, we have optimized the concentration of Therefore, achieving the desired expression level of each enzymes involved in precorrin-2 synthesis in vitro via gene within an operon remains challenging. Pfleger et al. response surface methodology [76]. This approach mini- generated libraries of tunable intergenic regions, recom- mizes the effects of substrate inhibition and feedback bining various post-transcriptional control elements to inhibition by SUMT and increases the production of optimize the expression of multiple genes in a synthetic precorrin-2 approximately five-fold. operon [62]. Tian et al. also designed synthetic operons that utilize translational coupling to achieve the desired Construction of a heterologous biosynthetic pathway expression level ratios [63]. for cobalamin in E. coli Scaffolding is a way to rapidly increase overall pathway Escherichia coli is a well-characterized that flux and is complementary to these conventional methods. has been used as a microbial cell factory for many chemi- Scaffolding can increase the effective concentration of inter- cals. Although it has lost the de novo cobalamin synthesis mediates in the pathway of interest, via the recruitment of pathway during evolution, E. coli can synthesize cobala- enzymes to synthetic protein scaffolds. Three mevalonate min through the salvage pathway, thus saving resources biosynthetic enzymes (acetoacetyl-CoA thiolase, hydroxy- and energy. Escherichia coli has been used to produce methylglutaryl-CoA synthase, and hydroxymethylglutaryl- ALA in many studies. A sufficient ALA supply is neces- CoA reductase) were recruited to a synthetic protein scaffold sary for the vitamin B12 biosynthesis. This implies that through interactions between the GTPase binding domain, E. coli may be a suitable host for vitamin B12 production. the SH3 domain, the PSD95/DlgA/Zo-1 domain and their Other common bacteria such as glu- respective ligands, increasing mevalonate yields 77-fold tamicum and Bacillus subtilis lack the genes involved in compared to cells without the scaffold [64]. Protein scaffolds the cobalamin synthesis pathway after precorrin-2, which simulate natural multienzyme complexes and have been may explain why the construction of a heterologous de used to solve problems of toxic pathway intermediate accu- novo cobalamin synthesis pathway in bacteria other than mulation, competing metabolic reactions, and imbalances E. coli has yet to be reported. in flux [65]. Protein scaffolds have been successfully applied For a long time, cobalamin production was restricted to in various other pathways, including the glucaric acid path- native bacterial producers, with the exception of several way, where three pathway enzymes were joined in a protein studies that focused on the determination of gene func- scaffold, increasing titers by approximately five-fold [66]. In tion in the cobalamin synthesis pathway in vivo [16, 35, another example, heterologous butyrate pathway enzymes 70, 77] or by using cell extracts from recombinant E. coli Fang et al. Microb Cell Fact (2017) 16:15 Page 9 of 14

Table 2 Research on the biosynthesis of vitamin B12 and its intermediates in vivo and in vitro in E. coli Strains Products Strategies References

In vivo E. coli Cobyric acid Expression of the cob I operon of B. megaterium and cbiP of S. typhimurium [77] E. coli Cobyrinic acid a,c-diamide Co-expression of the cobA gene from P. freudenreichii and the cbiAP, cbiCDETFGHJ, and cbiJKL [16] genes from S. typhimurium E. coli HBA Co-expression of cobA, cobI, cobJ, cobF, cobM, cobK, cobL, and cobH from R. capsulatus SB1003, [70] and cobG from B. melitensis 16M or P. denitrificans E. coli HBA Co-expression of cobA, cobI, cobJ, cobF, cobM, cobK, cobL, cobE, cobH and cobZ from R. capsulatus [35]

E. coli Vitamin B12 Expression of the operon pduBAF-pocR-cbiABCDETFGHJKLMNQOP-cobUST from S. typhimurium [18]

E. coli Vitamin B12 Co-expression of cobWNEMcbtAB, tonBcobOBRDCQchlID and cobGHIJLFK from P. denitrificans [83] In vitro E. coli HBA E. coli native genes hemB, hemC, hemD, and cobA, cobI, cobG, cobJ, cobF, cobM, cobK, cobL, and [78] cobH genes from P. denitrificans were heterologously expressed in E. coli. HBA was produced when ALA was added to the media and the cells were incubated aerobically in 200 ml Tris–HCl buffer, pH 8.0, containing SAM, NADH, and NADPH for 15 h at 30 °C with a mixture of twelve enzymes: HemB, HemC, HemD, crude enzyme extract of CobA, CobI, CobG, CobJ, CobM, CobF, CobK, CobL, and CobH E. coli Precorrin3b and precorrin-4 The cobG and cobJ genes from P. denitrificans were heterologously expressed in E. coli. Aerobic [18] incubation of precorrin-3A with the CobG enzyme from P. denitrificans alone yielded precor- rin3B. When CobJ from P. denitrificans and S-adenosyl-l-methionine were included in the reaction, the product precorrin-4 was formed E. coli Cob(II)yrinic acid a,c- The cobN, cobS, and cobT from B. melitensis were expressed and purified from E. coli. In the pres- [81] 2 diamide ence of ATP, Co +, and hydrogenobyrinic acid a,c diamide, these enzymes together produced cobyrinic acid E. coli Cob(I)yrinic acid a,c-diamide CobR from B. melitensis was expressed and purified from E. coli. This enzmye was confirmed to [82] have co(II)rrin reductase activity

in vitro (Table 2) [78–82]. After insertion of the B. mega- ligation into three compatible plasmids under the control terium cob I operon and S. typhimurium cbiP, E. coli was of a T7 promoter. The recombinant E. coli strain harbor- able to synthesize cobyric acid de novo [77]. Co-expression ing these three plasmids produced vitamin B12 under of the cobA gene from Propionibacterium freudenreichii both anaerobic and aerobic conditions. Via optimizing and the cbiAP, cbiCDETFGHJ and cbiJKL genes from S. the culture conditions, the engineered strain produced typhimurium equipped E. coli with the ability to produce 0.65 ± 0.03 μg/g cdw of coenzyme B12. These examples cobyrinic acid a,c-diamide [16]. Apart from the genes show that E. coli can be utilized to produce vitamin B12 involved in the anaerobic cobalamin biosynthetic pathway, or pathway intermediates de novo through the aerobic or genes involved in aerobic cobalamin biosynthetic pathway anaerobic pathway. have also been shown to work in E. coli. Furthermore, E. coli possesses enzymes that perform the transformation Metabolic engineering for cobalamin production of uroporphyrinogen III into HBA, thus potentially pro- Scheme of metabolic engineering ducing HBA [70]. McGoldrick et al. conducted a similar When a micro-organism possesses its own or a heter- experiment, where nine genes from R. capsulatus and Bru- ologous cobalamin synthesis pathway, efforts should cella melitensis, encoding enzymes for the transformation be directed towards engineering the metabolic network of uroporphyrinogen III into HBA, were constructed as an to enhance vitamin B12 production and yield. Classi- operon in pET14b and introduced into E. coli, allowing the cal metabolic engineering involves an iterative process host to produce HBA [35]. To the best of our knowledge, of synthesis and analysis, where increasingly refined there are only two reports on de novo cobalamin synthe- strains are designed and constructed based on gathered sis in E. coli: Firstly, an E. coli strain harboring a plasmid knowledge [84]. However, systems metabolic engineering with a 21.5-kb native operon (pduBAF-pocR-cbiABCDET- allows microbes to be engineered at the whole-organism FGHJKLMNQOP-cobUST) from S. typhimurium resulted level for the production of valuable chemicals far beyond in de novo cobalamin biosynthesis [18]. Recently, Ko et al. their native capabilities [85]. Metabolic design based on also accomplished cobalamin biosynthesis in E. coli [83]. in silico simulations and experimental validation of the Twenty-two genes located in six different operons of P. metabolic state in the engineered strain facilitates sys- denitrificans were cloned via traditional restriction and tematic metabolic engineering [86]. Many genome-scale Fang et al. Microb Cell Fact (2017) 16:15 Page 10 of 14

metabolic flux models have been developed to design production (up to 200 μg/l) [90]. All these strategies microbial cell factories. In silico simulations based on focus on the over-expression of structural genes that are genome-scale metabolic models have provided valid directly involved in the vitamin B12 biosynthesis. How- guidance for rational design. Computational tools used ever, to the best of our knowledge, no published reports in metabolic flux analysis and gene manipulation stud- exist on other genes, such as those responsible for the ies have been systematically reviewed [87]. Depending on S-adenosylmethionine (SAM) or DMB . This the design of the specific strain, experimental implemen- suggests that research into the metabolic engineering of tation can involve a combination of gene over-expres- vitamin B12 production is still in its infancy. sion, introduction of foreign enzymes, gene deletions, or knockdowns and the modification of enzyme properties. Inactivation or down‑regulation of genes A further general method of metabolic engineering is Gene over‑expression and heterologous expression to knockout genes to improve precursor supply, or to Gene over-expression is used to enhance or redirect eliminate by-products or competing chemical synthesis flux to the desired . Expression of the reactions. Genes that encode enzymes at specific branch target gene can be increased through the use of multi- points in the metabolic pathway are good targets for this copy plasmids, or the insertion of transcriptional and strategy. For essential genes that cannot be deleted, clus- translational control elements, such as strong promot- tered regularly interspaced short palindromic repeats ers, highly efficient RBSs, and terminator sequences. (CRISPR), CRISPR interference (CRISPRi), sRNA, or The introduction of heterologous genes is used to over- regulatory RNA can be used to repress the desired genes come the comparatively low activity of native genes [91, 92]. Many genome editing tools such as, traditional in a host. The structural genes required for cobalamin homologous recombination systems and ZFN (zinc fin- synthesis are typically targets for gene over-expression ger nucleases), TALEN (transcription activator-like effec- to boost cobalamin production. In a recombinant P. tor nucleases), and CRISPR/Cas-based methods readily freudenreichii strain, which harbors an expression vector enable genetic modifications. Heme and siroheme are containing cobA, cbiLF, or cbiEGH, vitamin B12 produc- competitive byproducts of the vitamin B12 synthesis. To tion was increased 1.7-, 1.9-, and 1.5-fold, respectively, reduce flux to the heme branch of the tetrapyrrole path- compared to P. freudenreichii harboring the expression way, antisense RNA was used to silence hemZ (which vector pPK705 [88]. Expression of cobU and cobS in encodes coproporphyrinogen III oxidase) in B. megate- the same P. freudenreichii strain led to a slight increase rium. This approach led to a 20% increase in the intra- in the production of vitamin B12. ALA is a precursor, cellular vitamin B12 concentration [93]. We also inhibited restricting cobalamin synthesis and a direct strategy hemE and hemH to improve flux to precorrin-2 via an to overcome this is to up-regulate the availability of sRNA approach (unpublished data). ALA. A recombinant P. freudenreichii strain with het- erologous hemA from R. sphaeroides, hemB, and cobA Protein engineering homologues, revealed a 2.2-fold increase in vitamin B12 During the engineering of native or heterologous hosts to production [88]. To improve the production of tetrapy- produce a desired chemical, a common scenario is that rrole compounds, the hemA gene from R. sphaeroides a substance or product inhibits an enzyme or it may not and the hemB gene from P. freudenreichii subsp. sherma- function heterologously. Protein engineering is a useful nii IFO12424 were expressed either monocistronically approach to improve the specificity, solubility, and sta- or polycistronically in the strain P. freudenreichii subsp. bility of enzymes. It includes directed evolution, semi shermanii IFO12426. The recombinant strains accumu- rational design, and rational design. The enzyme gluta- lated larger amounts of ALA and PBG, with a resultant myl-tRNA reductase (HemA) catalyzes the first commit- 28- to 33-fold increase in the production of porphy- ted step in the heme biosynthetic pathway. Expression rinogens, compared to strain P. freudenreichii subsp. of this gene is inhibited in a negative feedback loop by shermanii IFO12426, harboring the vector pPK705 excess heme. The addition of two positively charged [89]. A cobalamin-riboswitch can be used to inhibit lysine residues to the third and fourth positions of the N excess vitamin B12 in microbes. To bypass the effects terminus of HemA resulted in a complete stabilization of of the cobalamin-riboswitch, a cbi operon without the the protein. Cells expressing the stabilized HemA showed cobalamin-riboswitch was cloned. Growth of B. megate- a substantial decrease in heme inhibition [94]. Engineer- rium, harboring a plasmid expressing the cbi operon on ing of CobA to remove substrate inhibition or the identi- minimal media supplemented with glycerol as a carbon fication of novel genes without substrate inhibition may source, resulted in a significant increase in cobalamin improve the yield of vitamin B12. Fang et al. Microb Cell Fact (2017) 16:15 Page 11 of 14

13C‑metabolic flux analysis Biosensors 13C-metabolic flux analysis is an efficient method to Biosensors have been extensively applied in high- determine flux distribution based on experimental data throughput assays. For chemicals that cannot be easily and has been used to accurately estimate flux in the cen- detected via measurement of absorption or fluorescence, tral carbon metabolism of P. denitrificans in response to biosensors indirectly reflect chemical concentrations. different specific oxygen uptake rates under oxygen limit- A vitamin B12 riboswitch sensor has been used to study ing conditions [95]. Metabolic flux analysis revealed that synthesis and import of coenzyme B12, and was shown glucose was predominantly catabolized by the Entner– to detect intracellular vitamin B12 concentration using Doudoroff and pentose phosphate pathways. A higher colorimetric, fluorescent, or luminescent reporters with specific oxygen uptake rate accelerated the supply of pre- high sensitivity [98]. Reporter genes integrated with the cursors, methyl groups, and NADPH to increase vitamin btuB riboswitch have been used to monitor intracellular B12 production [95]. AdoCbl concentrations [98]. A combination of evolu- tionary strategies applied to metabolic pathways, whole Other strategies to improve cobalamin production genomes, or biosensors may be a useful approach to Random mutagenesis advance high-throughput screening for “ideal” strains. To produce strains with a high cobalamin yield, a con- ventional strategy is random mutagenesis. Specifically, Fermentation process optimization UV light or chemical mutagens can both be used to The addition of precursors of the vitamin 12B biosyn- treat the respective microorganisms and then, strains thetic pathway, such as cobalt ions, ALA, DMB, glycine, with the desired phenotype, such as improved pro- threonine, or compatible solutes like betaine and choline ductivity, genetic stability, reasonable growth rates, or has been frequently described to be beneficial [7]. Pro- resistance to high concentrations of toxic intermediates pionic acid is a byproduct of the vitamin B12 cultivation can be selected [7]. High throughput screening meth- process in P. freudenreichii and causes feedback inhibi- ods based on signals, such as survival and fluorescence, tion of microbial cell growth. production have been used to obtain desired mutants from large and DMB addition were controlled via expanded bed libraries. Moreover, random mutagenesis can also be adsorption bioreactors, which were shown to improve applied to inverse metabolic engineering for targeted vitamin B12 biosynthesis [99]. Mixed culture of Propioni- transformation. bacterium and Ralstonia eutropha have also been used to solve this problem, as the latter can assimilate propionic Genome‑scale engineering acid produced by the former [100]. Betaine is an impor- Genome shuffling, an approach that combines ran- tant methyl-group donor for the formation of methio- dom mutagenesis and protoplast fusion, has been used nine, which is further converted to SAM by the activity of to improve vitamin B12 production in P. shermanii. The methionine adenosyltransferase. S-adenosylmethionine engineered P. shermanii strain produced approximately is an important precursor during corrinoid ring forma- 61% more vitamin B12 than the parent strain after 96 h. tion. Despite the fact that betaine delays cell growth, Comparative proteome analysis revealed that the expres- betaine feeding during fermentation has been revealed as sion of 38 proteins varied significantly [96]. Compared an effective strategy to increase vitamin 12B production to genome shuffling, which results in random muta- [101]. To reduce medium and fermentation costs, cheap tions, MAGE provides an efficient method to simulta- carbon sources such as maltose syrup and corn steep liq- neously modify multiple genomic locations [61]. MAGE uor can be used to replace refined sucrose [102]. is based on the λ red recombination system. The repeti- tive introduction of ssDNA targeting multiple loci in the Conclusions E. coli genome results in various mutants. Combined Vitamin B12 is widely used in medical and food indus- with standard high-throughput screening methods, tries and microbes produce vitamin B12 via an intricate MAGE may represent a rapid and efficient tool to obtain pathway. Tetrapyrrole compounds, including heme, “ideal” bacterial producers. In addition, another versatile cobalamin, and siroheme are not simple competitive method, trackable multiplex recombineering (TRMR), compounds, but have both interdependent and interac- enables rapid and simultaneous modification and map- tive relationships in several bacterial species. To main- ping of thousands of genes. By changing functional tain stable vitamin B12 concentrations, its biosynthesis regions such as promoters, translation sites, switches, and transport are regulated at the transcriptional or oscillators, or sensors, a broad range of studies can be translational level via riboswitches. Vitamin B12 is pro- performed [97]. duced by microbial fermentation, using strains such as Fang et al. Microb Cell Fact (2017) 16:15 Page 12 of 14

P. denitrificans and P. shermanii. Since relatively few References 1. Roth JR, Lawrence JG, Rubenfield M, Kieffer-Higgins S, Church GM. genetic tools are available for these strains, and the fer- Characterization of the cobalamin (vitamin B12) biosynthetic genes of mentation process is complicated, strain engineering Salmonella typhimurium. J Bacteriol. 1993;175:3303–16. has focused on traditional strategies such as random 2. Yin L, Bauer CE. Controlling the delicate balance of tetrapyrrole biosyn- thesis. Philos Trans R Soc Lond B Biol Sci. 2013;368:20120262. mutagenesis and the optimization of the fermentation 3. Rodionov DA, Vitreschak AG, Mironov AA, Gelfand MS. Comparative process. It is imperative to introduce new engineer- genomics of the vitamin B12 metabolism and regulation in prokaryotes. ing tools, such as systems metabolic engineering, to J Biol Chem. 2003;278:41148–59. 4. Lawrence JG, Roth JR. Evolution of coenzyme B12 synthesis among manipulate these strains. Apart from native producers enteric bacteria: evidence for loss and reacquisition of a multigene of vitamin B12, E. coli has also been used as a heterolo- complex. Genetics. 1996;142:11–24. gous host. To provide guidance for the construction of 5. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28:27–30. microbial cell factories for vitamin B12 production, we 6. Swithers KS, Petrus AK, Secinaro MA, Nesbo CL, Gogarten JP, Noll KM, summarized synthetic biology and metabolic engineer- Butzin NC. Vitamin B12 synthesis and salvage pathways were acquired ing strategies, as well as other traditional strategies that by horizontal gene transfer to the Thermotogales. Genome Biol Evol. 2012;4:730–9. either have been or could be applied to vitamin B12 pro- 7. Martens JH, Barg H, Warren MJ, Jahn D. Microbial production of vitamin duction. These strategies have been extensively applied B12. Appl Microbiol Biotechnol. 2002;58:275–85. in microbial strain engineering to improve the produc- 8. Martin VJJ, Pitera DJ, Withers ST, Newman JD, Keasling JD. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. tion of many other chemicals. Based on a clear under- Nat Biotechnol. 2003;21:796–802. standing of the vitamin B12 metabolism in microbes, 9. Zhang K, Sawaya MR, Eisenberg DS, Liao JC. Expanding metabolism the utilization of these strategies should promote an for biosynthesis of nonnatural alcohols. Proc Natl Acad Sci USA. 2008;105:20653–8. improved microbial vitamin B12 production. 10. Choi SY, Park SJ, Kim WJ, Yang JE, Lee H, Shin J, Lee SY. One-step fermen- tative production of poly(lactate-co-glycolate) from carbohydrates in Abbreviations Escherichia coli. Nat Biotechnol. 2016;34:435–40. ALA: δ-aminolevulinate; UTR: untranslated regions; ACAT: ATP co(I)rrinoid 11. Lee SK, Chou H, Ham TS, Lee TS, Keasling JD. Metabolic engineering of adenosyltransferase; AdoCbi: adenosylcobinamide; AdoCbl: adenosyl- microorganisms for biofuels production: from bugs to synthetic biology cobalamin; NADH: reduced form of nicotinamide-adenine dinucleotide; to fuels. Curr Opin Biotechnol. 2008;19:556–63. FMN: ; NADPH: reduced nicotinamide adenine 12. Jarboe LR, Zhang X, Wang X, Moore JC, Shanmugam KT, Ingram LO. dinucleotide phosphate; ABC: ATP-binding cassette; SUMT: S-Adenosyl- Metabolic engineering for production of biorenewable fuels and l-methionine:uroporphyrinogen III methyltransferase; HBA: hydrogenobyrinic chemicals: contributions of synthetic biology. J Biomed Biotechnol. acid; RBS: ribosome-binding site; MAGE: multiplex automated genome engi- 2010;2010:761042. neering; CRISPR: clustered regularly interspaced short palindromic repeats; 13. Zhang L, Chen J, Chen N, Sun J, Zheng P, Ma Y. Cloning of two 5-ami- CRISPRi: CRISPR interference; TRMR: trackable multiplex recombineering; ZFN: nolevulinic acid synthase isozymes HemA and HemO from Rhodopseu- zinc finger nucleases; TALEN: transcription activator-like effector nucleases. domonas palustris with favorable characteristics for 5- production. Biotechnol Lett. 2013;35:763–8. Authors’ contributions 14. Kang Z, Wang Y, Gu P, Wang Q, Qi Q. Engineering Escherichia coli for HF and DZ conceived the manuscript. HF and DZ wrote the manuscript. JK efficient production of 5-aminolevulinic acid from glucose. Metab Eng. wrote the “Cobalamin riboswitches” section. All authors read and approved the 2011;13:492–8. final manuscript. 15. Brushaber KR. CobD, a novel enzyme with l-Threonine-O-3-phosphate decarboxylase activity, is responsible for the synthesis of (R)-1- Author details Amino-2-propanol O-2-phosphate, a proposed new intermediate in 1 Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, cobalamin biosynthesis in Salmonella typhimurium LT2. J Biol Chem. Tianjin 300308, China. 2 Key Laboratory of Systems Microbial Biotechnology, 1998;273:2684–91. Chinese Academy of Sciences, Tianjin 300308, China. 3 University of Chinese 16. Roessner CA, Williams HJ, Scott AI. Genetically engineered production Academy of Sciences, Beijing 100049, China. 4 College of Biotechnology of 1-desmethylcobyrinic acid, 1-desmethylcobyrinic acid a, c-diamide, and Food Science, Tianjin University of Commerce, Tianjin 300134, China. and cobyrinic acid a, c-diamide in Escherichia coli implies a role for CbiD in C-1 methylation in the anaerobic pathway to cobalamin. J Biol Chem. Acknowledgements 2005;280:16748–53. Not applicable. 17. Fan C, Bobik TA. The PduX enzyme of Salmonella enterica is an l-threonine kinase used for coenzyme B synthesis. J Biol Chem. Competing interests 12 2008;283:11322–9. The authors declare that they have no competing interests. 18. Raux E, Lanois A, Levillayer F, Warren MJ, Brody E, Rambach A, Thermes C. Salmonella typhimurium cobalamin (vitamin B12) biosynthetic genes: Funding functional studies in S. typhimurium and Escherichia coli. J Bacteriol. Funding was provided by the National Nature Science Foundation of China 1996;178:753–67. (31370089), the State Key Development 973 Program for Basic Research of 19. Moore SJ, Warren MJ. The anaerobic biosynthesis of vitamin B . Bio- China (2013CB733600), the Nature Science Foundation of Tianjin City (CN) 12 chem Soc Trans. 2012;40:581–6. (16JCYBJC23500), and the Key Projects in the Tianjin Science & Technology Pil- 20. Avissar Y, Ormerod J, Beale S. Distribution of δ-aminolevulinic acid lar Program (11ZCZDSY08400). All funding providers mentioned above had no biosynthetic pathways among phototrophic bacterial groups. Arch role in the design of the study, collection, analysis, and interpretation of data Microbiol. 1989;151:513–9. nor in the writing the manuscript. 21. Zappa S, Li K, Bauer CE. The tetrapyrrole biosynthetic pathway and its regulation in Rhodobacter capsulatus. Adv Exp Med Biol. Received: 18 September 2016 Accepted: 20 January 2017 2010;675:229–50. Fang et al. Microb Cell Fact (2017) 16:15 Page 13 of 14

22. Raux E, Mcveigh T, Peters SE, LeustekMcveigh T, Warren MJ. The role of 43. Zajicek RS, Bali S, Arnold S, Brindley AA, Warren MJ, Ferguson SJ. d(1) Saccharomyces cerevisiae Met1p and Met8p in sirohaem and cobalamin haem biogenesis—assessing the roles of three nir gene products. FEBS biosynthesis. Biochem J. 1999;338:701–8. J. 2009;276:6399–411. 23. Escalante-Semerena J, Warren M. Biosynthesis and use of cobalamin 44. Nahvi A, Barrick JE, Breaker RR. Coenzyme B12 riboswitches are wide- (B12). EcoSal Plus. 2008;3:1. spread genetic control elements in prokaryotes. Nucleic Acids Res. 24. Cohen GN. Biosynthesis of cobalamins including vitamin B12. In Micro- 2004;32:143–50. bial . Dordrecht: Springer; 2014. p. 555–565. 45. Johnson JE Jr, Reyes FE, Polaski JT, Batey RT. B12 cofactors directly stabi- 25. Zayas CL, Escalante-Semerena JC. Reassessment of the late steps of lize an mRNA regulatory switch. Nature. 2012;492:133–7. coenzyme B12 synthesis in Salmonella enterica: evidence that dephos- 46. Souliere MF, Haller A, Santner T, Micura R. New insights into gene regu- phorylation of adenosylcobalamin-5′-phosphate by the CobC phos- lation—high-resolution structures of cobalamin riboswitches. Angew phatase is the last step of the pathway. J Bacteriol. 2007;189:2210–8. Chem Int Ed Engl. 2013;52:1874–7. 26. Taga ME, Larsen NA, Howard-Jones AR, Walsh CT, Walker GC. BluB 47. Choudhary PK, Duret A, Rohrbach-Brandt E, Holliger C, Sigel RKO, cannibalizes flavin to form the lower ligand of vitamin 12B . Nature. Maillard J. Diversity of cobalamin riboswitches in the corrinoid-pro- 2007;446:449–53. ducing organohalide respirer Desulfitobacterium hafniense. J Bacteriol. 27. Campbell GR, Taga ME, Mistry K, Lloret J, Anderson PJ, Roth JR, 2013;195:5186–95. Walker GC. Sinorhizobium meliloti bluB is necessary for production of 48. Holmstrom ED, Polaski JT, Batey RT, Nesbitt DJ. Single-molecule con- 5,6-dimethylbenzimidazole, the lower ligand of B12. Proc Natl Acad Sci formational dynamics of a biologically functional hydroxocobalamin USA. 2006;103:4634–9. riboswitch. J Am Chem Soc. 2014;136:16832–43. 28. Mehta AP, Abdelwahed SH, Fenwick MK, Hazra AB, Taga ME, Zhang Y, 49. Vitreschak AG. Regulation of the vitamin B12 metabolism and Ealick SE, Begley TP. Anaerobic 5-hydroxybenzimidazole formation from transport in bacteria by a conserved RNA structural element. RNA. aminoimidazole ribotide: an unanticipated intersection of thiamin and 2003;9:1084–97. vitamin B12 biosynthesis. J Am Chem Soc. 2015;137:10444–7. 50. Mandal M, Breaker RR. Gene regulation by riboswitches. Nat Rev Mol 29. Hazra AB, Tran JL, Crofts TS, Taga ME. Analysis of substrate specificity in Cell Biol. 2004;5:451–63. CobT homologs reveals widespread preference for DMB, the lower axial 51. Serganov A, Nudler E. A decade of riboswitches. Cell. 2013;152:17–24. ligand of vitamin B12. Chem Biol. 2013;20:1275–85. 52. Mellin JR, Koutero M, Dar D, Nahori MA, Sorek R, Cossart P. Riboswitches. 30. Escalante-Semerena JC. Conversion of cobinamide into adenosylcoba- Sequestration of a two-component response regulator by a riboswitch- mide in bacteria and archaea. J Bacteriol. 2007;189:4555–60. regulated noncoding RNA. Science. 2014;345:940–3. 31. Woodson JD, Reynolds AA, Escalante-Semerena JC. ABC transporter for 53. Lee SY, Kim HU, Park JH, Park JM, Kim TY. Metabolic engineering of corrinoids in Halobacterium sp. strain NRC-1. J Bacteriol. 2005;187:5901–9. microorganisms: general strategies and drug production. Drug Discov 32. Moore TC, Newmister SA, Rayment I, Escalante-Semerena JC. Structural Today. 2009;14:78–88. insights into the mechanism of four-coordinate Cob(II)alamin formation 54. de Kok S, Stanton LH, Slaby T, Durot M, Holmes VF, Patel KG, Platt D, in the of the Salmonella enterica ATP:Co(I)rrinoid adenosyl- Shapland EB, Serber Z, Dean J, et al. Rapid and reliable DNA assembly transferase enzyme: critical role of residues Phe91 and Trp93. Biochemis- via ligase cycling reaction. ACS Synth Biol. 2014;3:97–106. try. 2012;51:9647–57. 55. Fong SS. Computational approaches to metabolic engineering utilizing 33. Newmister SA, Otte MM, Escalante-Semerena JC, Rayment I. Structure systems biology and synthetic biology. Comput Struct Biotechnol J. and mutational analysis of the archaeal GTP:AdoCbi-P guanylyltrans- 2014;11:28–34. ferase (CobY) from Methanocaldococcus jannaschii: insights into GTP 56. Salis HM. Chapter two—the ribosome binding site calculator. In: Chris- binding and dimerization. Biochemistry. 2011;50:5301–13. topher V, editor. Methods in enzymology volume, vol. 498. Cambridge: 34. McNicholas PM, Javor G, Darie S, Gunsalus RP. Expression of the heme Academic Press; 2011. p. 19–42. biosynthetic pathway genes hemCD, hemH, hemM and hemA of Escheri- 57. Alper H, Fischer C, Nevoigt E, Stephanopoulos G. Tuning genetic control chia coli. FEMS Microbiol Lett. 1997;146:143–8. through promoter engineering. Proc Natl Acad Sci. 2006;103:3006. 35. McGoldrick HM, Roessner CA, Raux E, Lawrence AD, McLean KJ, Munro 58. Salis HM, Mirsky EA, Voigt CA. Automated design of synthetic ribo- AW, Santabarbara S, Rigby SEJ, Heathcote P, Scott AI, Warren MJ. some binding sites to control protein expression. Nat Biotechnol. Identification and characterization of a novel vitamin 12B (cobalamin) 2009;27:946–50. biosynthetic enzyme (CobZ) from Rhodobacter capsulatus, containing 59. Pfleger BF, Pitera DJ, Smolke CD, Keasling JD. Combinatorial engineer- flavin, heme, and Fe-S cofactors. J Biol Chem. 2004;280:1086–94. ing of intergenic regions in operons tunes expression of multiple 36. Gough SP, Petersen BO, Duus JØ. Anaerobic chlorophyll isocyclic ring genes. Nat Biotech. 2006;24:1027–32. formation in Rhodobacter capsulatus requires a cobalamin cofactor. Proc 60. Arpino JA, Hancock EJ, Anderson J, Barahona M, Stan GB, Papachristo- Natl Acad Sci. 2000;97:6908–13. doulou A, Polizzi K. Tuning the dials of synthetic biology. Microbiology. 37. Layer G, Moser J, Heinz DW, Jahn D, Schubert W-D. Crystal structure of 2013;159:1236–53. coproporphyrinogen III oxidase reveals cofactor geometry of radical 61. Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, Forest CR, Church GM. SAM enzymes. EMBO J. 2003;22:6214–24. Programming cells by multiplex genome engineering and accelerated 38. Ponnampalam SN, Buggy JJ, Bauer CE. Characterization of an aerobic evolution. Nature. 2009;460:894–8. repressor that coordinately regulates bacteriochlorophyll, , 62. Pfleger BF, Pitera DJ, Smolke CD, Keasling JD. Combinatorial engineer- and light harvesting-II expression in Rhodobacter capsulatus. J Bacteriol. ing of intergenic regions in operons tunes expression of multiple 1995;177:2990–7. genes. Nat Biotechnol. 2006;24:1027–32. 39. Cheng Z, Li K, Hammad LA, Karty JA, Bauer CE. Vitamin B12 regulates 63. Tian T, Salis HM. A predictive biophysical model of translational cou- photosystem gene expression via the CrtJ antirepressor AerR in Rhodo- pling to coordinate and control protein expression in bacterial operons. bacter capsulatus. Mol Microbiol. 2014;91:649–64. Nucleic Acids Res. 2015;43:7137–51. 40. Blanche F, Debussche L, Thibaut D, Crouzet J, Cameron B. Purification 64. Dueber JE, Wu GC, Malmirchegini GR, Moon TS, Petzold CJ, Ullal AV, and characterization of S-adenosyl-l-methionine: uroporphyrinogen Prather KL, Keasling JD. Synthetic protein scaffolds provide modular III methyltransferase from Pseudomonas denitrificans. J Bacteriol. control over metabolic flux. Nat Biotechnol. 2009;27:753–9. 1989;171:4222–31. 65. Chen AH, Silver PA. Designing biological compartmentalization. Trends 41. Robin C, Blanche F, Cauchois L, Cameron B, Coude M, Crouzet J. Primary Cell Biol. 2012;22:662–70. structure, expression in Escherichia coli, and properties of S-adenosyl- 66. Moon TS, Dueber JE, Shiue E, Prather KL. Use of modular, synthetic l-methionine-uroporphyrinogen III methyltransferase from Bacillus scaffolds for improved production of glucaric acid in engineered E. coli. megaterium. J Bacteriol. 1991;173:4893–6. Metab Eng. 2010;12:298–305. 42. Blanche F, Robin C, Couder M, Faucher D, Cauchois L, Cameron B, 67. Baek JM, Mazumdar S, Lee SW, Jung MY, Lim JH, Seo SW, Jung GY, Oh Crouzet J. Purification, characterization, and molecular cloning of MK. Butyrate production in engineered Escherichia coli with synthetic S-adenosyl-l-methionine-uroporphyrinogen III methyltransferase from scaffolds. Biotechnol Bioeng. 2013;110:2790–4. Methanobacterium ivanovii. J Bacteriol. 1991;173:4637–45. Fang et al. Microb Cell Fact (2017) 16:15 Page 14 of 14

68. Boyle PM, Silver PA. Parts plus pipes: synthetic biology approaches to bypassing of the cobalamin riboswitch control elements. N Biotechnol. metabolic engineering. Metab Eng. 2012;14:223–32. 2014;31:553–61. 69. Zhang YH. Substrate channeling and enzyme complexes for biotechno- 91. Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS. CRISPR logical applications. Biotechnol Adv. 2011;29:715–25. interference (CRISPRi) for sequence-specific control of gene expression. 70. Deery E, Schroeder S, Lawrence AD, Taylor SL, Seyedarabi A, Waterman Nat Protoc. 2013;8:2180–96. J, Wilson KS, Brown D, Geeves MA, Howard MJ, et al. An enzyme-trap 92. Na D, Yoo SM, Chung H, Park H, Park JH, Lee SY. Metabolic engineering approach allows isolation of intermediates in cobalamin biosynthesis. of Escherichia coli using synthetic small regulatory RNAs. Nat Biotechnol. Nat Chem Biol. 2012;8:933–40. 2013;31:170–4. 71. Yu X, Liu T, Zhu F, Khosla C. In vitro reconstitution and steady-state 93. Biedendieck R, Malten M, Barg H, Bunk B, Martens JH, Deery E, Leech H, analysis of the fatty acid synthase from Escherichia coli. Proc Natl Acad Warren MJ, Jahn D. Metabolic engineering of cobalamin (vitamin B12) Sci USA. 2011;108:18643–8. production in Bacillus megaterium. Microb Biotechnol. 2010;3:24–37. 72. Guo D, Zhu J, Deng Z, Liu T. Metabolic engineering of Escherichia coli for 94. Wang L, Wilson S, Elliott T. A mutant HemA protein with positive charge production of fatty acid short-chain esters through combination of the close to the N terminus is stabilized against heme-regulated proteolysis fatty acid and 2-keto acid pathways. Metab Eng. 2014;22:69–75. in Salmonella typhimurium. J Bacteriol. 1999;181:6033–41. 73. Liu R, Zhu F, Lu L, Fu A, Lu J, Deng Z, Liu T. Metabolic engineering of 95. Wang Z-J, Wang P, Liu Y-W, Zhang Y-M, Chu J, Huang MZ, Zhuang YP, fatty acyl-ACP reductase-dependent pathway to improve fatty alcohol Zhang SL. Metabolic flux analysis of the central carbon metabolism of production in Escherichia coli. Metab Eng. 2014;22:10–21. the industrial vitamin B12 producing strain Pseudomonas denitrificans 74. Zhu F, Zhong X, Hu M, Lu L, Deng Z, Liu T. In vitro reconstitution of using 13C-labeled glucose. J Taiwan Inst Chem Eng. 2012;43:181–7. mevalonate pathway and targeted engineering of farnesene overpro- 96. Zhang Y, Liu J-Z, Huang J-S, Mao Z-W. Genome shuffling of Propionibac- duction in Escherichia coli. Biotechnol Bioeng. 2014;111:1396–405. terium shermanii for improving vitamin B12 production and comparative 75. Liu Q, Wu K, Cheng Y, Lu L, Xiao E, Zhang Y, Deng Z, Liu T. Engineering proteome analysis. J Biotechnol. 2010;148:139–43. an iterative polyketide pathway in Escherichia coli results in single-form 97. Warner JR, Reeder PJ, Karimpour-Fard A, Woodruff LB, Gill RT. Rapid alkene and alkane overproduction. Metab Eng. 2015;28:82–90. profiling of a microbial genome using mixtures of barcoded oligonu- 76. Fang H, Dong H, Cai T, Zheng P, Li H, Zhang D, Sun J. In vitro optimiza- cleotides. Nat Biotechnol. 2010;28:856–62. tion of enzymes involved in precorrin-2 synthesis using response 98. Fowler CC, Brown ED, Li Y. Using a riboswitch sensor to examine surface methodology. PLoS ONE. 2016;11:e0151149. coenzyme B12 metabolism and transport in E. coli. Chem Biol. 77. Evelyne RA, Rambach A, Warren MJ, Thermes C. Cobalamin (vitamin 2010;17:756–65. B12) biosynthesis: functional characterization of the Bacillus megate- 99. Wang P, Zhang Z, Jiao Y, Liu S, Wang Y. Improved propionic acid and rium cbi genes required to convert uroporphyrinogen III into cobyrinic 5,6-dimethylbenzimidazole control strategy for vitamin B12 fermenta- acid a, c-diamide. Biochem J. 1998;335:167–73. tion by Propionibacterium freudenreichii. J Biotechnol. 2015;193:123–9. 78. Roessner CA, Spencer JB, Stolowich NJ, Wang J, Nayar GP, Santander 100. Ken-ichiro Miyano KY, Shimizu K. Improvement of vitamin B12 fermenta- PJ, Pichon C, Min C, Holderman MT, Scott AI. Genetically engineered tion by reducing the inhibitory metabolites by cell recycle system and a synthesis of precorrin-6x and the complete corrinoid, hydrogenobyrinic mixed culture. Biochem Eng J. 2000;6:207–14. acid, an advanced precursor of vitamin B12. Chem Biol. 1994;1:119–24. 101. Li KT, Liu DH, Li YL, Chu J, Wang YH, Zhuang YP, Zhang SL. Improved 79. Roessner CA, Spencer JB, Ozaki S, Min CH, Atshaves BP, Nayar P, Anousis large-scale production of vitamin B12 by Pseudomonas denitrificans with N, Stolowich NJ, Holderman MT, Scott AI. Overexpression in Escheri- betaine feeding. Bioresour Technol. 2008;99:8516–20. chia coli of 12 vitamin B12 biosynthetic enzymes. Protein Expr Purif. 102. Xia W, Chen W, Peng WF, Li KT. Industrial vitamin B12 production by 1994;6:155–63. Pseudomonas denitrificans using maltose syrup and corn steep liquor 80. Stamford NPJ, Duggan S, Li Y, Alanine AID, Crouzet J, Battersby AR. as the cost-effective fermentation substrates. Bioprocess Biosyst Eng. Biosynthesis of vitamin B12: the multi-enzyme synthesis of precorrin-4 2015;38:1065–73. and factor IV. Chem Biol. 1997;4:445–51. 81. Lundqvist J, Elmlund D, Heldt D, Deery E, Soderberg CA, Hansson M, Warren M, Al-Karadaghi S. The AAA( ) motor complex of subunits CobS and CobT of cobaltochelatase+ visualized by single particle elec- tron microscopy. J Struct Biol. 2009;167:227–34. 82. Lawrence AD, Deery E, McLean KJ, Munro AW, Pickersgill RW, Rigby SE, Warren MJ. Identification, characterization, and structure/function analysis of a reductase involved in adenosylcobalamin biosyn- thesis. J Biol Chem. 2008;283:10813–21. 83. Ko Y, Ashok S, Ainala SK, Sankaranarayanan M, Chun AY, Jung GY, Park S. Coenzyme B12 can be produced by engineered Escherichia coli under both anaerobic and aerobic conditions. Biotechnol J. 2014;9:1526–35. 84. Tee TW, Chowdhury A, Maranas CD, Shanks JV. Systems metabolic engineering design: fatty acid production as an emerging case study. Biotechnol Bioeng. 2014;111:849–57. 85. Choi KR, Shin JH, Cho JS, Yang D, Lee SY. Systems metabolic engineer- Submit your next manuscript to BioMed Central ing of Escherichia coli. EcoSal Plus. 2016;7:1. 86. Toya Y, Shimizu H. Flux analysis and metabolomics for systematic meta- and we will help you at every step: bolic engineering of microorganisms. Biotechnol Adv. 2013;31:818–26. • We accept pre-submission inquiries 87. Park JM, Kim TY, Lee SY. Constraints-based genome-scale metabolic simulation for systems metabolic engineering. Biotechnol Adv. • Our selector tool helps you to find the most relevant journal 2009;27:979–88. • We provide round the clock customer support 88. Piao Y, Yamashita M, Kawaraichi N, Asegawa R, Ono H, Murooka Y. • Convenient online submission Production of vitamin B12 in genetically engineered Propionibacterium freudenreichii. J Biosci Bioeng. 2004;98:167–73. • Thorough peer review 89. Piao Y, Kiatpapan P, Yamashita M, Murooka Y. Effects of expression of • Inclusion in PubMed and all major indexing services hemA and hemB genes on production of in Propionibacte- • Maximum visibility for your research rium freudenreichii. Appl Environ Microbiol. 2004;70:7561–6. 90. Moore SJ, Mayer MJ, Biedendieck R, Deery E, Warren MJ. Towards Submit your manuscript at a cell factory for vitamin B12 production in Bacillus megaterium: www.biomedcentral.com/submit