Microbial Production of Vitamin B12 a Review and Future Perspectives

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• Published: 30 January 2017

Microbial production of vitamin B₁₂: a review and future perspectives

Microbial Jail cell Factories book xvi, Commodity number:15 (2017) Cite this article

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Abstract

Vitamin B₁₂ is an essential vitamin that is widely used in medical and food industries. Vitamin B₁₂ biosynthesis is confined to few bacteria and archaea, and as such its production relies on microbial fermentation. Rational strain engineering 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 chemic 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, nosotros provide a comprehensive agreement of advances in the microbial product of vitamin B₁₂, with a particular focus on establishing a heterologous host for the vitamin B₁₂ production, likewise as on strategies and tools that take been applied to increment microbial cobalamin production. Several worthy strategies employed for other products are also included.

Background

Vitamin B₁₂, also known equally cyanocobalamin, belongs to the cobalamin family of compounds, which are equanimous of a corrinoid ring and an upper and lower ligand. The upper ligand can be adenosine, methyl, hydroxy, or a cyano grouping [i]. Vitamin B₁₂ is synthesized by prokaryotes and inhibits the development of pernicious anemia in animals.

Microbial de novo biosynthesis of vitamin B₁₂ occurs through 2 alternative routes: the aerobic or anaerobic pathway, in leaner and archaea, respectively. Some strains can also synthesize cobalamin by absorbing corrinoids via a salvage pathway, as shown in Tabular array ane. Tetrapyrrole compounds including cobalamin, heme, and bacteriochlorophyll, are derived from δ-aminolevulinate (ALA) and a complex interdependent and interactional relationship exists among these tetrapyrrole compounds in numerous bacterial species [2]. To maintain vitamin B₁₂ at stable levels, its biosynthesis and transportation is regulated by a cobalamin riboswitch in the five′ untranslated regions (UTR) of the corresponding genes.

Table i Cobalamin biosynthetic pathway in microbes

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Big scale industrial production of vitamin B₁₂ occurs via microbial fermentation, predominantly utilizing Pseudomonas denitrificans, Propionibacterium shermanii, or Sinorhizobium meliloti [seven]. Even so, these strains have several shortcomings, such as long fermentation cycles, circuitous and expensive media requirements, and a lack of suitable genetic systems for strain engineering. To date, well-nigh of the research on these producers has focused on traditional strategies, such as random mutagenesis and fermentation process optimization, with only limited inquiry on metabolic technology. Recently, engineers have shifted their attention to Escherichia coli every bit a platform for vitamin B₁₂ product. E. coli has become a well-studied cell manufactory that has been extensively used for the product of diverse chemicals, such every bit terpenoids, non-natural alcohols, and poly-(lactate-co-glycolate) [8–x]. Furthermore, metabolic engineering and constructed biology strategies have been extensively applied to improve the production of these compounds [11, 12]. Escherichia coli synthesizes ALA via the C₅ pathway and has been used as a microbial jail cell manufactory to produce ALA via C_iv and C_five pathways [13, fourteen] and Due east. coli can likewise synthesize vitamin B₁₂ via the salvage pathway. The closely related Salmonella typhimurium is able to synthesize vitamin B₁₂ de novo. Many genes involved in vitamin B₁₂ biosynthesis in S. typhimurium have been shown to be functional in E. coli [15–17]. Transfer of 20 genes from the S. typhimurium cob locus immune the production of vitamin B₁₂ in Due east. coli [eighteen]. These advantages facilitate the de novo production of vitamin B₁₂ in E. coli.

Due to the complexity of the pathway and its metabolic regulation, numerous studies have been performed past diverse groups on the vitamin B₁₂ biosynthesis. This review provides an overview of the vitamin B₁₂ biosynthesis and its metabolic regulation, along with an investigation of several strategies for the development of microbial cell factories, including synthetic biology and metabolic engineering, amongst others. Since research on the technology of vitamin B₁₂ producing strains remains limited, related strategies for other chemicals are likewise outlined.

Cobalamin biosynthetic pathway

De novo pathway

Equally mentioned above, cobalamin can be synthesized de novo in prokaryotes through two alternative routes according to the timing of cobalt insertion and the molecular oxygen requirement. These pathways are the aerobic pathway, which has been all-time studied in P. denitrificans, and the anaerobic pathway, which has been best studied in S. typhimurium, Bacillus megaterium, and P. shermanii, [19]. Both routes differ in terms of cobalt chelation (via hydrogenobyrinic a, c-diamide with the CobNST complex in the aerobic pathway, and via precorrin-two with CbiK in South. typhimurium) and oxygen requirements (the aerobic pathway requires oxygen to promote ring-contraction, while the anaerobic pathway does not crave oxygen in this stride) (Fig. 1).

Fig. 1

Biosynthetic pathways of tetrapyrrole compounds. ALA is synthesized by either the C₄ or the C_five 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

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Biosynthetic pathways of tetrapyrrole compounds: ALA is synthesized by either C₄ or C₅ pathway and adenosylcobalamin is synthesized either via de novo or salvage pathways. The enzymes shown in the adenosylcobalamin biosynthetic pathway originate from P. denitrificans or Due south. typhimurium, which use either the aerobic pathway or anaerobic pathway, respectively.

The first committed precursor of the tetrapyrrole synthesis pathway is ALA. ALA is synthesized past either the C₄ pathway or the C₅ pathway. In the C₄ pathway, the enzyme ALA synthase from glycine and succinyl-CoA catalyzes the formation of ALA. In the C₅ pathway, ALA is synthesized from glutamate through iii enzymatic reactions [20]. Ii molecules of ALA are condensed to course monopyrrole porphobilinogen by porphobilinogen synthase and four porphobilinogen molecules are and so polymerized and cyclized to class uroporphyrinogen III. This reaction is catalyzed past the enzymes porphobilinogen deaminase and uroporphyrinogen III synthase. Methylation of uroporphyrinogen 3 at C-2 and C-7 results in the synthesis of precorrin-2 (which is a common precursor of cobalamin), siroheme, and coenzyme F₄₃₀ [7, 21]. In P. denitrificans, CobA catalyzes this methylation step. In Southward. typhimurium and E. coli, the fusion enzyme CysG is shared between siroheme and cobalamin synthesis. The North-terminus of CysG has dehydrogenase/ferrochelatase activity and the C-terminus has uroporphyrinogen III methyltransferase activity. In S. cerevisiae, MET1p functions equally a uroporphyrinogen III methyltransferase [22].

The aerobic and anaerobic pathways diverge at precorrin-2 and converge at coby(II)rinic acrid a, c-diamide. 8 peripheral methylation reactions occur during de novo cobalamin biosynthesis, within identical temporal and spatial orders in both the aerobic and anaerobic pathways. Many of the methyltransferase enzymes involved in these reactions bear witness high degrees of sequence similarity [23].

Cob(I)yrinic acid a,c-diamide is adenosylated to form adenosyl cobyrinic acrid a,c-diamide. Cob(I)yrinic acid a,c-diamide adenosyltransferase tin also adenosylate other corrinoids, where at least the a and c positions of the carboxyl groups are amidated. Adenosyl cobyrinic acid a,c-diamide is subjected to 4 stepwise amidation reactions at carboxyl groups at positions b, d, e, and 1000 to yield adenosyl cobyric acid. Two separate methods accept evolved to attach (R)-i-amino-two-propanol or (R)-one-amino-2-propanol phosphate at the f position of the carboxyl group of adenosyl cobyric acid in the aerobic and anaerobic pathways. In the anaerobic pathway, the linker betwixt the corrinoid ring and the lower axial ligand is phosphorylated prior to attachment of the corrinoid ring. The enzyme PduX from S. enterica is an 50-threonine kinase used in the de novo synthesis of coenzyme B₁₂; nevertheless, it is non involved in the cobinamide salvage pathway [17]. 50-threonine O-3-phosphate is then decarboxylated to yield (R)-1-amino-two-propanol O-ii-phosphate via CobD in S. typhimurium LT2 [15]. Still, in P. denitrificans it is most likely (although proof remains to be published), that (R)-i-amino-2-propanol is straight attached to the corrinoid ring via poly peptide α and β. Currently, poly peptide α remains as all the same unknown and poly peptide β is a complex of CobC and CobD. The molecule is and so phosphorylated by CobP, which is a bifunctional enzyme possessing ATP:AdoCbi (adenosylcobinamide) kinase and GTP:AdoCbi-P guanylyltransferase activity [24]. Two additional reactions transfer lower axial ligands onto AdoCbi-GDP, thus producing adenosylcobalamin (AdoCbl). There are two alternative views of the terminal stride in vitamin B₁₂ biosynthesis. Ane view is that the concluding reaction in the biosynthesis of AdoCbl involves the addition of α-ribazole, catalyzed via cobalamine synthase. However, in S. typhimurium, α-ribazole 5′-phosphate is added to AdoCbi-GDP and thus, the concluding reaction would exist the dephosphorylation of AdoCbl 5′-phosphate to AdoCbl, catalyzed past an AdoCbl-5-P phosphatase (CobC) [25]. The nucleotide loop associates pathway is the last characterized reaction in the vitamin B₁₂ biosynthesis. BluB from S. meliloti is a member of the reduced course of nicotinamide-adenine dinucleotide (NADH)/flavin mononucleotide (FMN)-dependent nitroreductase family, which tin convert FMNH₂ to DMB (5, half-dozen-dimethylbenzimidazole) [26, 27]. In the anaerobic bacterium E. limosum, 5-aminoimidazole ribotide is converted to DMB by enzymes encoded in the bza operon [28] and subsequently, CobT tin can activate a range of lower ligand substrates including DMB, which determine cobamide diversity [29].

Salvage pathway

The salvage pathway is a toll-effective fashion (in terms of free energy) for bacteria and archaea to obtain cobalamin. In gram-negative bacteria, exogenous corrinoids are transported into the cell via an ATP-binding cassette (ABC) transport arrangement, consisting of BtuC, BtuD, and BtuF, which are membrane permease, ATPase, and periplasmic-binding poly peptide components, respectively. BtuB is a TonB-dependent transporter located in the outer membrane, delivering corrinoid to the periplasmic corrinoid-binding protein BtuF. The latter then delivers corrinoid to the BtuCD complex located in the inner membrane [xxx]. Archaea also use ABC transporters for corrinoid uptake. Archaeal orthologs of the bacterial BtuC, BtuD, and BtuF have been found in Halobacterium sp. strain NRC-1 [31]. Subsequent to transport through the membrane, cobinamide is adenosylated by ATP:co(I)rrinoid adenosyltransferases (ACATs). Three families of ACATs be, namely: CobA, EutT, and PduO [32]. In bacteria, AdoCbi is the substrate for a bifunctional enzyme (CobU in S. typhimurium or CobP in P. denitrificans) with kinase and guanylyltransferase activities. In archaea, the cbiZ gene encodes an amidohydrolase that converts AdoCbi to adenosylcobyric acrid, which is condensed with ane-aminopropanol-O-two-phosphate by an AdoCbi-P synthase (CbiB) to yield AdoCbi-P. Since the archaeal enzyme lacks AdoCbi kinase activeness, CobY, which has GTP:AdoCbi-P guanylyltransferase activity, is used to transfer guanylyl to AdoCbi-P [xxx, 33]. Identical to the de novo pathway, two additional reactions transfer lower axial ligands onto AdoCbi-GDP to produce AdoCbl in the save pathway.

Metabolic regulation of cobalamin synthesis

The relationship between cobalamin, heme, and chlorophyll

Photosynthetic bacteria possess all three tetrapyrrole compounds including cobalamin, heme, and bacteriochlorophyll (Fig. i), which share the same biosynthetic pathway from ALA to uroporphyrinogen Iii. Excess heme inhibits HemA via feedback inhibition, reducing the flux of cobalamin and bacteriochlorophyll, while heme limitation weakly regulates hemCD, hemH, and hemA expression in Due east. coli [34]. However, not merely a competitive relationship exists among the tetrapyrrole compounds, as these compounds also share a complex interdependent and interactional relationship [2]. Hydrogenobyrinic acid synthase from Rhodobacter capsulatus possesses two Fe-S centers, a flavin and a heme group [35]. Rhodobacter capsulatus requires a cobalamin cofactor to form the isocyclic ring of chlorophyll [36]. Synthesis of heme is also cobalamin dependent, equally heme synthesis requires S-adenosylmethionine as a methyl group donor, while S-adenosylmethionine synthesis involves a cobalamin-dependent enzyme [37]. This complex relationship is also manifest at the transcriptional level. East.thou., cobalamin participates in the regulation of bacteriochlorophyll synthesis via the AerR-CrtJ regulatory pair in R. capsulatus. CrtJ is responsible for the repression of bacteriochlorophyll during aerobic growth [38], while AerR functions equally an anti-repressor of CrtJ, inhibiting CrtJ binding to the bchC promoter when cobalamin is bound to the conserved histidine (His145) of AerR [39]. In R. sphaeroides, heme affects the power of PpsR to regulate genes that are involved in the tetrapyrrole biosynthesis past binding PpsR and changing its Dna binding design. Thus, PpsR functions as both a redox and a heme sensor to coordinate cellular heme and bacteriochlorophyll levels [38]. This complex relationship between tetrapyrrole compounds is a consequence of natural evolution.

Regulation of the key enzyme S-Adenosyl-fifty-methionine: uroporphyrinogen III methyltransferase (SUMT)

Feedback inhibition of a cardinal enzyme located at the co-operative point of a biosynthetic pathway is a mutual method for metabolic regulation in microbes. In many microorganisms, SUMT regulates cobalamin flux. In P. denitrificans, SUMT is sensitive to feedback inhibition by cobalamin and corrinoid intermediates, and exhibits substrate inhibition at uroporphyrinogen III concentrations above ii µM [xl]. Bacillus megaterium SUMT exhibits substrate inhibition at uroporphyrinogen concentrations above 0.5 µM [41], while P. denitrificans SUMT is inhibited by uroporphyrinogen concentrations higher up 0.ii µM [xl]. Fortunately, uroporphyrinogen III methyltransferase shows limited substrate inhibition. As an instance, the uroporphyrinogen 3 methyltransferase of Methanobacterium ivanovii and Paracoccus pantotrophus were not inhibited by uroporphyrinogen III, even at concentrations of up to twenty µM [42, 43]. The identification of new SUMT enzymes that are insensitive to feedback inhibition to supervene upon the native enzyme of industrial strains may be an effective method to better vitamin B₁₂ yield.

Cobalamin riboswitches

The cobalamin riboswitch is the predominant form of metabolic regulation to control vitamin B₁₂ concentration in microbes. Riboswitches are evolutionarily conserved non-coding regions that are situated in the 5′-untranslated region of mRNAs that regulate cistron expression in response to direct binding of intracellular metabolites by the RNA itself [44]. Riboswitches are composed of two functional domains: 1 domain serves every bit an evolutionarily conserved natural aptamer, binding the target metabolite with loftier selectivity and affinity, while the other domain harnesses allosteric changes in RNA structure, caused by aptamer-ligand complex formation, to control expression of an side by side gene or operon. Johnson et al. solved the crystal structures of two unlike classes of cobalamin riboswitches, which share a mutual four-way junction (P3–P6 helices), forming the cadre receptor domain that is responsible for cobalamin bounden, but utilize distinct peripheral extensions (P8–P11) to recognize unlike cobalamin derivatives [45]. The P6 extension is present for the AdoCbl binding form, while it is absent-minded for the methylcobalamin and the aquocobalamin binding classes [45–47]. A kissing-loop interaction between loop L5 of the cobalamin-binding core and L13 of the regulatory domain regulates the expression machinery [46]. Holmstrom et al. identified the conformational mechanism responsible for the regulation of gene expression by a hydroxocobalamin binding riboswitch (env8HyCbl). The authors utilized unmarried-molecule fluorescence resonance energy transfer techniques [48]. Binding of hydroxocobalamin promotes the formation of the L5–L13 kissing-loop, which sequesters the Shine-Dalgarno sequence via base pairing, thus preventing translation initiation. Cobalamin riboswitches participate in the regulation of cobalamin biosynthesis and transport at the transcriptional or translational levels, such as the btuB gene of Due east. coli and the cob operon of South. typhimurium [49]. In example of transcription inhibition and for high cobalamin concentrations, an alternative Rho-independent termination hairpin or Rho bounden site cause premature transcriptional termination. High cobalamin concentrations can too promote sequestration of ribosome binding sites (RBS) and blockage of translation initiation. When cobalamin concentrations are depression, an anti-terminator hairpin forms, enabling RNA polymerase to complete transcription of the downstream gene. Low cobalamin concentrations facilitate anti-sequester hairpin formation that releases the RBS, thus enabling translation initiation [50, 51]. Cobalamin riboswitches also accept a particular role in ethanolamine utilization. In Listeria monocytogenes, a cobalamin riboswitch controls the expression of a noncoding regulatory RNA named Rli55, which controls the expression of the eut genes that requires vitamin B₁₂ every bit a cofactor and determines ethanolamine utilization [52].

Synthetic biology to improve cobalamin production

Design of a heterologous biosynthetic pathway for the vitamin B₁₂ production

Apart from altering native microbial hosts or identifying novel microbial hosts to produce cobalamin, the construction of heterologous biosynthetic pathways in model organisms that can exist easily genetically manipulated is a promising strategy. Constructed biology is an efficient tool that can be used to reconstruct pathways or genetic networks to produce compounds in a heterologous host. The construction of a vitamin B₁₂ biosynthetic pathway in a heterologous host includes selection of a suitable host, building the biosynthetic pathway with functional components, and pathway tuning (Fig. 2). Several points should be noted in the selection of an ideal host. (ane) The host should have the power to supply precursors (e.m. ALA) and cofactors (e.m. S-adenosylmethionine) for the production of the desired chemic. E.g., the heterologous C₄ pathway in Due east. coli has been reported to generate loftier ALA production [thirteen], avoiding addition of exogenous ALA to the medium; (two) there demand to exist sufficient genetic engineering tools such as transformation protocols, expression vectors, and chromosomal factor knockout/integration systems to dispense the host [53]; (3) the host is suitable for industrial-scale fermentation, utilizing cheap and readily bachelor carbon sources such as glucose, xylose, and arabinose. When the host is selected, various candidate genes from diverse native vitamin B₁₂ producers can be expressed in the host. The utilise of in vitro kinetic analysis is an efficient approach to detect platonic enzymes. Most of the intermediates of the vitamin B₁₂ synthetic pathway are not available; consequently, to prepare substrates for in vitro kinetic assay, the desired chemicals need to be separated from the products of corresponding strains or exist prepared enzymatically. The products of an in vitro assay can and then be detected by spectroscopic analysis, mass spectrometry, or microbiological assays. Sometimes, the heterologous production of enzymes does not work and it is necessary to screen for novel enzymes from diverse sources. For heterologous expression studies, all native forms of regulation, such as riboswitches, should be removed. Later the addition of transcriptional and translational elements, such every bit promoters, ribosome binding sequences, and terminators, the structural genes for cobalamin synthesis tin be expressed either monocistronically or polycistronically on plasmids or be integrated into the genome. The rapid development of synthetic biology tools has facilitated the simplified construction of heterologous pathways. Heterogeneous genes can be assembled past a number of unlike techniques such as SLIC, CPEC, Gibson assembly, golden gate cloning, Deoxyribonucleic acid assembler, and LCR [54]. When too many heterogeneous genes need to exist transferred to a heterologous host, it is difficult to build the metabolic pathway one gene at a fourth dimension. The metabolic route tin exist split into multiple modules. Afterward sequential validation of the function of these modules, their assembly becomes possible. The final construct can and so be transferred to the called host for heterologous expression, thus allowing the host to synthesize cobalamin. To evaluate the capacity of vitamin B₁₂ production, the engineered strains are then cultured under optimal conditions.

Fig. 2

Design of a heterologous biosynthetic pathway. a A host for the heterologous biosynthetic pathway is selected considering the adequacy of precursor and cofactor supply, genetic engineering tools, and industrial-scale fermentation capability, utilizing inexpensive 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 factor associates methods such equally 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 exist integrated to target compound maximization. To optimize factor expression in the metabolic pathway, promoters, RBS, and factor copy number are designed and implemented at the transcriptional or translational levels. e The characteristics of the engineered strains are verified via fermentation. Diverse substrates (eastward.1000., ALA, cobalt ions, betaine and DMB) and varying conditions (e.g., dissolved oxygen concentration, pH, and temperature) can be optimized to better yield and productivity

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Pattern of a heterologous biosynthetic pathway. (A) A host for the heterologous biosynthetic pathway is selected considering capabilities of precursor and cofactor supply, genetic engineering tools, and industrial-calibration fermentation capability using cheap and readily available carbon sources. (B) Enzyme activity is verified in vitro and thereafter in vivo. Products of in vitro assay or intracellular reaction products are detected via spectroscopic assay, mass spectrometry, or microbiological assays. (C) Heterogeneous genes and other functional elements are assembled on plasmids via gene associates methods such every bit SLIC, CPEC, Gibson, aureate gate, Deoxyribonucleic acid assembler, and LCR, or integrated into the genome. To subtract the difficulty of building the metabolic pathway, it is divided into separate modules. These modules are sequentially verified in a heterologous host, and and so assembled. (D) Based on the quantification of metabolites, bottlenecks should be removed and metabolic flux to target compound maximization. To optimize cistron expression in the metabolic pathway, promoters, RBS, and cistron re-create number are designed and implemented at the transcriptional or translational levels. (E) The characteristics of engineered strains are verified via fermentation. Diverse substrates (due east.g., ALA, cobalt ions, betaine and DMB) and varying conditions (e.g., dissolved oxygen concentration, pH, and temperature) tin be optimized to improve yield and productivity.

Pathway tuning

Biologists face the challenge to straight the flux of intermediates to generate the desired product. To avoid the accumulation of toxic intermediates and to drive flux to the desired end product, each step in the pathway needs to be counterbalanced. In improver, cistron overexpression may cause an undesirable metabolic burden on the host, and the level of cistron expression needs to be accurately adapted to coordinate both metabolic flux and prison cell growth. Sometimes chromosomal expression of heterologous metabolic pathways can avoid plasmid instability and thus reduce the metabolic burden on the prison cell. Vitamin B₁₂ biosynthesis is a highly evolved pathway, with more than than twenty steps, including corrinoid ring construction and peripheral modifications, and the enzymes involved have loftier substrate specificity. Therefore, information technology is necessary to optimize gene expression in existing metabolic pathways.

Gene expression in a given pathway can be altered at the level of transcription or translation [55]. Promoter strength affects factor expression at the transcriptional level, while Dna sequence of the RBS affects gene expression at the translational level. The "Ribosome bounden site figurer" and RBSDesigner take been used to predict and pattern constructed RBSs to yield the desired level of protein expression in leaner [56]. The expression of multiple genes in synthetic operons tin can be optimized to enhance product levels via engineering of promoters [57] and RBSs [58], or via generation of libraries of tunable intergenic regions [59]. Tuning operon expression can exist achieved via dissimilar plasmid replicons to control gene re-create number, unlike promoters to command the rate of transcription initiation, and different RBSs for controlling the level of translation [60]. Multiplex automatic genome engineering (MAGE) can be used for genome-calibration optimization of gene expression on the chromosome [61].

The translational efficiency of each cistron in an operon is affected by intergenic sequence regions and involves mail service-transcriptional processes such every bit transcription termination, mRNA stability, and translation initiation. Therefore, achieving the desired expression level of each gene within an operon remains challenging. Pfleger et al. generated libraries of tunable intergenic regions, recombining various postal service-transcriptional control elements to optimize the expression of multiple genes in a synthetic operon [62]. Tian et al. also designed synthetic operons that employ translational coupling to achieve the desired expression level ratios [63].

Scaffolding is a way to rapidly increase overall pathway flux and is complementary to these conventional methods. Scaffolding can increase the effective concentration of intermediates in the pathway of involvement, via the recruitment of enzymes to synthetic protein scaffolds. Iii mevalonate biosynthetic enzymes (acetoacetyl-CoA thiolase, hydroxy-methylglutaryl-CoA synthase, and hydroxymethylglutaryl-CoA reductase) were recruited to a synthetic poly peptide scaffold through interactions between the GTPase binding domain, the SH3 domain, the PSD95/DlgA/Zo-one domain and their respective ligands, increasing mevalonate yields 77-fold compared to cells without the scaffold [64]. Protein scaffolds simulate natural multienzyme complexes and take been used to solve bug of toxic pathway intermediate accumulation, competing metabolic reactions, and imbalances in flux [65]. Poly peptide scaffolds have been successfully practical in various other pathways, including the glucaric acid pathway, where iii pathway enzymes were joined in a poly peptide scaffold, increasing titers past approximately five-fold [66]. In another example, heterologous butyrate pathway enzymes were co-localized in a poly peptide scaffold, resulting in a threefold comeback in butyrate production [67]. Cobalamin synthesis is a complex pathway with several competing intermediates, such every bit heme and siroheme. Co-localization of enzymes to the aforementioned subcellular organelle or compartment tin can increase the local intermediate concentration and exclude competing cytosolic pathways [68]. Substrate channeling is a procedure of transferring the product of 1 enzyme to an next enzyme within the pour or jail cell without complete mixing with the bulk phase [69]. This phenomenon occurs for at to the lowest degree some of the enzymes involved in hydrogenobyrinic acid (HBA) biosynthesis. An enzyme-trap approach has been used to isolate intermediates of the cobalamin biosynthetic pathway based on this mechanism [70]. Therefore, poly peptide scaffolds may offer a promising culling to balance multiple enzymes within the vitamin B₁₂ biosynthetic pathway, thus improving flux to cobalamin.

All these approaches optimize gene expression or increase pathway flux in vivo. In vitro steady-country analysis is an efficient approach to identify bottlenecks in metabolic pathways and to residual flux to the desired compounds. It has been successfully used to increment the production of fatty acids, fatty acrid short-concatenation esters, fatty alcohols, farnesenes, alkenes, and alkanes [71–75]. Previously, we take optimized the concentration of enzymes involved in precorrin-ii synthesis in vitro via response surface methodology [76]. This approach minimizes the effects of substrate inhibition and feedback inhibition by SUMT and increases the production of precorrin-two approximately five-fold.

Construction of a heterologous biosynthetic pathway for cobalamin in East. coli

Escherichia coli is a well-characterized prokaryote that has been used every bit a microbial cell manufactory for many chemicals. Although it has lost the de novo cobalamin synthesis pathway during development, E. coli can synthesize cobalamin through the salvage pathway, thus saving resources and free energy. Escherichia coli has been used to produce ALA in many studies. A sufficient ALA supply is necessary for the vitamin B₁₂ biosynthesis. This implies that E. coli may exist a suitable host for vitamin B₁₂ production. Other common bacteria such as Corynebacterium glutamicum and Bacillus subtilis lack the genes involved in the cobalamin synthesis pathway subsequently precorrin-2, which may explain why the construction of a heterologous de novo cobalamin synthesis pathway in bacteria other than East. coli has nevertheless to exist reported.

For a long time, cobalamin production was restricted to native bacterial producers, with the exception of several studies that focused on the determination of factor part in the cobalamin synthesis pathway in vivo [16, 35, 70, 77] or by using cell extracts from recombinant E. coli in vitro (Table 2) [78–82]. After insertion of the B. megaterium cob I operon and S. typhimurium cbiP, Eastward. coli was able to synthesize cobyric acid de novo [77]. Co-expression of the cobA gene from Propionibacterium freudenreichii and the cbiAP, cbiCDETFGHJ and cbiJKL genes from S. typhimurium equipped E. coli with the ability to produce cobyrinic acid a,c-diamide [16]. Autonomously from the genes involved in the anaerobic cobalamin biosynthetic pathway, genes involved in aerobic cobalamin biosynthetic pathway accept also been shown to work in Due east. coli. Furthermore, E. coli possesses enzymes that perform the transformation of uroporphyrinogen III into HBA, thus potentially producing HBA [70]. McGoldrick et al. conducted a like experiment, where nine genes from R. capsulatus and Brucella melitensis, encoding enzymes for the transformation of uroporphyrinogen III into HBA, were synthetic as an operon in pET14b and introduced into East. coli, allowing the host to produce HBA [35]. To the all-time of our knowledge, there are only two reports on de novo cobalamin synthesis in E. coli: Firstly, an E. coli strain harboring a plasmid with a 21.five-kb native operon (pduBAF-pocR-cbiABCDETFGHJKLMNQOP-cobUST) from S. typhimurium resulted in de novo cobalamin biosynthesis [xviii]. Recently, Ko et al. also accomplished cobalamin biosynthesis in Due east. coli [83]. Twenty-2 genes located in six different operons of P. denitrificans were cloned via traditional restriction and ligation into three compatible plasmids under the command of a T7 promoter. The recombinant E. coli strain harboring these three plasmids produced vitamin B₁₂ nether both anaerobic and aerobic conditions. Via optimizing the culture conditions, the engineered strain produced 0.65 ± 0.03 μg/g cdw of coenzyme B₁₂. These examples show that E. coli can exist utilized to produce vitamin B₁₂ or pathway intermediates de novo through the aerobic or anaerobic pathway.

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

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Metabolic engineering for cobalamin production

Scheme of metabolic applied science

When a micro-organism possesses its ain or a heterologous cobalamin synthesis pathway, efforts should be directed towards engineering science the metabolic network to enhance vitamin B₁₂ production and yield. Classical metabolic engineering involves an iterative process of synthesis and assay, where increasingly refined strains are designed and constructed based on gathered knowledge [84]. However, systems metabolic applied science allows microbes to exist engineered at the whole-organism level for the production of valuable chemicals far beyond their native capabilities [85]. Metabolic design based on in silico simulations and experimental validation of the metabolic land in the engineered strain facilitates systematic metabolic applied science [86]. Many genome-calibration metabolic flux models have been adult to design microbial cell factories. In silico simulations based on genome-calibration metabolic models have provided valid guidance for rational design. Computational tools used in metabolic flux analysis and gene manipulation studies have been systematically reviewed [87]. Depending on the design of the specific strain, experimental implementation tin involve a combination of gene over-expression, introduction of foreign enzymes, gene deletions, or knockdowns and the modification of enzyme properties.

Gene over-expression and heterologous expression

Gene over-expression is used to enhance or redirect flux to the desired chemical reaction. Expression of the target gene can be increased through the utilise of multi-copy plasmids, or the insertion of transcriptional and translational control elements, such every bit strong promoters, highly efficient RBSs, and terminator sequences. The introduction of heterologous genes is used to overcome the insufficiently low activity of native genes in a host. The structural genes required for cobalamin synthesis are typically targets for gene over-expression to boost cobalamin production. In a recombinant P. freudenreichii strain, which harbors an expression vector containing cobA, cbiLF, or cbiEGH, vitamin B₁₂ product was increased ane.7-, 1.9-, and one.5-fold, respectively, compared to P. freudenreichii harboring the expression vector pPK705 [88]. Expression of cobU and cobS in the aforementioned P. freudenreichii strain led to a slight increment in the production of vitamin B₁₂. ALA is a precursor, restricting cobalamin synthesis and a direct strategy to overcome this is to upwards-regulate the availability of ALA. A recombinant P. freudenreichii strain with heterologous hemA from R. sphaeroides, hemB, and cobA homologues, revealed a two.2-fold increase in vitamin B₁₂ production [88]. To improve the production of tetrapyrrole compounds, the hemA gene from R. sphaeroides and the hemB gene from P. freudenreichii subsp. shermanii IFO12424 were expressed either monocistronically or polycistronically in the strain P. freudenreichii subsp. shermanii IFO12426. The recombinant strains accumulated larger amounts of ALA and PBG, with a resultant 28- to 33-fold increase in the production of porphyrinogens, compared to strain P. freudenreichii subsp. shermanii IFO12426, harboring the vector pPK705 [89]. A cobalamin-riboswitch can be used to inhibit backlog vitamin B₁₂ in microbes. To featherbed the effects of the cobalamin-riboswitch, a cbi operon without the cobalamin-riboswitch was cloned. Growth of B. megaterium, harboring a plasmid expressing the cbi operon on minimal media supplemented with glycerol every bit a carbon source, resulted in a significant increase in cobalamin production (up to 200 μg/50) [90]. All these strategies focus on the over-expression of structural genes that are straight involved in the vitamin B₁₂ biosynthesis. However, to the best of our knowledge, no published reports be on other genes, such as those responsible for the S-adenosylmethionine (SAM) or DMB metabolism. This suggests that inquiry into the metabolic engineering of vitamin B₁₂ production is still in its infancy.

Inactivation or downwards-regulation of genes

A further general method of metabolic engineering is to knockout genes to improve forerunner supply, or to eliminate past-products or competing chemical synthesis reactions. Genes that encode enzymes at specific co-operative points in the metabolic pathway are good targets for this strategy. For essential genes that cannot be deleted, clustered regularly interspaced brusk palindromic repeats (CRISPR), CRISPR interference (CRISPRi), sRNA, or regulatory RNA can exist used to repress the desired genes [91, 92]. Many genome editing tools such as, traditional homologous recombination systems and ZFN (zinc finger nucleases), TALEN (transcription activator-like effector nucleases), and CRISPR/Cas-based methods readily enable genetic modifications. Heme and siroheme are competitive byproducts of the vitamin B₁₂ synthesis. To reduce flux to the heme co-operative of the tetrapyrrole pathway, antisense RNA was used to silence hemZ (which encodes coproporphyrinogen III oxidase) in B. megaterium. This arroyo led to a 20% increase in the intracellular vitamin B₁₂ concentration [93]. Nosotros also inhibited hemE and hemH to improve flux to precorrin-ii via an sRNA approach (unpublished information).

Protein engineering

During the engineering of native or heterologous hosts to produce a desired chemic, a common scenario is that a substance or production inhibits an enzyme or it may not function heterologously. Poly peptide engineering is a useful approach to improve the specificity, solubility, and stability of enzymes. It includes directed development, semi rational design, and rational design. The enzyme glutamyl-tRNA reductase (HemA) catalyzes the first committed step in the heme biosynthetic pathway. Expression of this cistron is inhibited in a negative feedback loop by excess heme. The addition of 2 positively charged lysine residues to the third and quaternary positions of the N terminus of HemA resulted in a complete stabilization of the protein. Cells expressing the stabilized HemA showed a substantial decrease in heme inhibition [94]. Engineering of CobA to remove substrate inhibition or the identification of novel genes without substrate inhibition may improve the yield of vitamin B₁₂.

¹³C-metabolic flux analysis

¹³C-metabolic flux analysis is an efficient method to determine flux distribution based on experimental information and has been used to accurately gauge flux in the fundamental carbon metabolism of P. denitrificans in response to dissimilar specific oxygen uptake rates under oxygen limiting conditions [95]. Metabolic flux analysis revealed that glucose was predominantly catabolized by the Entner–Doudoroff and pentose phosphate pathways. A higher specific oxygen uptake charge per unit accelerated the supply of precursors, methyl groups, and NADPH to increase vitamin B₁₂ production [95].

Other strategies to improve cobalamin production

Random mutagenesis

To produce strains with a high cobalamin yield, a conventional strategy is random mutagenesis. Specifically, UV low-cal or chemical mutagens can both exist used to treat the respective microorganisms so, strains with the desired phenotype, such as improved productivity, genetic stability, reasonable growth rates, or resistance to high concentrations of toxic intermediates tin be selected [7]. High throughput screening methods based on signals, such as survival and fluorescence, take been used to obtain desired mutants from large libraries. Moreover, random mutagenesis tin likewise be practical to inverse metabolic engineering for targeted transformation.

Genome-scale engineering

Genome shuffling, an arroyo that combines random mutagenesis and protoplast fusion, has been used to ameliorate vitamin B₁₂ product in P. shermanii. The engineered P. shermanii strain produced approximately 61% more vitamin B₁₂ than the parent strain after 96 h. Comparative proteome analysis revealed that the expression of 38 proteins varied significantly [96]. Compared to genome shuffling, which results in random mutations, MAGE provides an efficient method to simultaneously modify multiple genomic locations [61]. MAGE is based on the λ red recombination organization. The repetitive introduction of ssDNA targeting multiple loci in the East. coli genome results in various mutants. Combined with standard high-throughput screening methods, MAGE may correspond a rapid and efficient tool to obtain "ideal" bacterial producers. In addition, another versatile method, trackable multiplex recombineering (TRMR), enables rapid and simultaneous modification and mapping of thousands of genes. By changing functional regions such as promoters, translation sites, switches, oscillators, or sensors, a broad range of studies tin be performed [97].

Biosensors

Biosensors have been extensively applied in high-throughput assays. For chemicals that cannot be hands detected via measurement of assimilation or fluorescence, biosensors indirectly reflect chemical concentrations. A vitamin B₁₂ riboswitch sensor has been used to study synthesis and import of coenzyme B₁₂, and was shown to detect intracellular vitamin B₁₂ concentration using colorimetric, fluorescent, or luminescent reporters with high sensitivity [98]. Reporter genes integrated with the btuB riboswitch have been used to monitor intracellular AdoCbl concentrations [98]. A combination of evolutionary strategies practical to metabolic pathways, whole genomes, or biosensors may be a useful approach to advance high-throughput screening for "platonic" strains.

Fermentation procedure optimization

The addition of precursors of the vitamin B₁₂ biosynthetic pathway, such as cobalt ions, ALA, DMB, glycine, threonine, or uniform solutes like betaine and choline has been frequently described to be benign [7]. Propionic acid is a byproduct of the vitamin B₁₂ cultivation process in P. freudenreichii and causes feedback inhibition of microbial jail cell growth. Propionic acid product and DMB addition were controlled via expanded bed adsorption bioreactors, which were shown to better vitamin B₁₂ biosynthesis [99]. Mixed civilisation of Propionibacterium and Ralstonia eutropha have also been used to solve this problem, every bit the latter tin can digest propionic acid produced by the former [100]. Betaine is an important methyl-group donor for the formation of methionine, which is further converted to SAM by the activity of methionine adenosyltransferase. Southward-adenosylmethionine is an important forerunner during corrinoid ring formation. Despite the fact that betaine delays cell growth, betaine feeding during fermentation has been revealed as an effective strategy to increment vitamin B₁₂ production [101]. To reduce medium and fermentation costs, cheap carbon sources such equally maltose syrup and corn steep liquor can be used to replace refined sucrose [102].

Conclusions

Vitamin B₁₂ is widely used in medical and food industries and microbes produce vitamin B₁₂ via an intricate pathway. Tetrapyrrole compounds, including heme, cobalamin, and siroheme are non uncomplicated competitive compounds, simply have both interdependent and interactive relationships in several bacterial species. To maintain stable vitamin B₁₂ concentrations, its biosynthesis and transport are regulated at the transcriptional or translational level via riboswitches. Vitamin B₁₂ is produced past microbial fermentation, using strains such as P. denitrificans and P. shermanii. Since relatively few genetic tools are available for these strains, and the fermentation process is complicated, strain engineering has focused on traditional strategies such as random mutagenesis and the optimization of the fermentation process. Information technology is imperative to introduce new applied science tools, such every bit systems metabolic engineering, to dispense these strains. Apart from native producers of vitamin B₁₂, Due east. coli has likewise been used as a heterologous host. To provide guidance for the construction of microbial prison cell factories for vitamin B₁₂ production, we summarized synthetic biology and metabolic technology strategies, too as other traditional strategies that either have been or could be applied to vitamin B₁₂ product. These strategies have been extensively applied in microbial strain technology to better the production of many other chemicals. Based on a clear agreement of the vitamin B₁₂ metabolism in microbes, the utilization of these strategies should promote an improved microbial vitamin B₁₂ production.

Abbreviations

ALA:

δ-aminolevulinate

UTR:

untranslated regions

ACAT:

ATP co(I)rrinoid adenosyltransferase

AdoCbi:

adenosylcobinamide

AdoCbl:

adenosylcobalamin

NADH:

reduced form of nicotinamide-adenine dinucleotide

FMN:

flavin mononucleotide

NADPH:

reduced nicotinamide adenine dinucleotide phosphate

ABC:

ATP-binding cassette

SUMT:

S-Adenosyl-50-methionine:uroporphyrinogen III methyltransferase

HBA:

hydrogenobyrinic acid

RBS:

ribosome-bounden site

MAGE:

multiplex automated genome engineering

CRISPR:

clustered regularly interspaced short palindromic repeats

CRISPRi:

CRISPR interference

TRMR:

trackable multiplex recombineering

ZFN:

zinc finger nucleases

TALEN:

transcription activator-similar effector nucleases

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Authors' contributions

HF and DZ conceived the manuscript. HF and DZ wrote the manuscript. JK wrote the "Cobalamin riboswitches" department. All authors read and approved the last manuscript.

Acknowledgements

Not applicable.

Competing interests

The authors declare that they take no competing interests.

Funding

Funding was provided by the National Nature Science Foundation of China (31370089), the State Key Development 973 Program for Basic Research of Mainland china (2013CB733600), the Nature Science Foundation of Tianjin City (CN) (16JCYBJC23500), and the Key Projects in the Tianjin Scientific discipline & Technology Colonnade Program (11ZCZDSY08400). All funding providers mentioned above had no part in the pattern of the written report, drove, analysis, and estimation of information nor in the writing the manuscript.

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Affiliations

1. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China

   Huan Fang, Jie Kang & Dawei Zhang

2. Central Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's republic of china

   Huan Fang & Dawei Zhang

3. Academy of Chinese Academy of Sciences, Beijing, 100049, China

   Huan Fang

4. College of Biotechnology and Nutrient Science, Tianjin Academy of Commerce, Tianjin, 300134, Mainland china

   Jie Kang

Corresponding author

Correspondence to Dawei Zhang.

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Fang, H., Kang, J. & Zhang, D. Microbial production of vitamin B₁₂: a review and future perspectives. Microb Prison cell Fact 16, 15 (2017). https://doi.org/10.1186/s12934-017-0631-y

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• Received: 18 September 2016

• Accepted: xx January 2017

• Published: xxx January 2017

• DOI : https://doi.org/ten.1186/s12934-017-0631-y

Keywords

• Vitamin B₁₂
• Biosynthesis
• Metabolic regulation
• Synthetic biology
• Escherichia coli
• Metabolic applied science

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