Vitamin B12 is involved in metabolic functions in all organisms, and is therefore an essential nutrient. However, it can only be synthesized by some bacteria and archaea, thus, animals have to obtain it through their diet. Thereby, only animal products like meat and dairy contain B12 in general. This B12 was synthesized by microorganisms colonizing the gastrointestinal tract of those animals and accumulated in the animals’ tissues.
is one of the most expensive biochemicals in the world, and its synthesis is extraordinarily complex. Since the chemical production of Vitamin B12
requires 70 synthesis steps, it is far too technically challenging and expensive. Therefore, its production is facilitated by genetically engineered microorganisms. These are able to produce Vitamin B12
in industrial amounts and achieve a high product quality. However, the produced Vitamin B12
is harvested by cell lysis, which prevents a continuous production. The efficiency of production could be increased by exporting Vitamin B12
outside the cells. To date, no natural cellular Vitamin B12
exporter is known.
An adult needs approximately 3.0 µg Vitamin B12 per day which is essential for certain functions: it is involved in processes concerning synthesis of DNA, hormones and neurotransmitters and also acts in the formation of the nervous system and blood. Hence, a B12 deficiency can cause diverse diseases like cancer, dementia, depression, pernicious anemia and polyneuropathy.
Nowadays, many people do not eat meat or animal products at all. Therefore, these people have a high risk to suffer from B12 deficiency if their diet does not provide enough B12.
Vitamin B12 is also used in the industry as it is needed in the biotechnical production of various organic substances. Furthermore, it is added to diverse daily products like toothpaste, fruit gum, non-diary milk or cleaning solution for contact lenses.
We intend to design, construct and introduce a synthetic Vitamin B12 exporter (“Synporter”) into a production organism. Thereby, we aim for facilitated and higher yields in the industrial Vitamin B12 production without requiring cell lysis.
The B12 Synporter consists of a signal for a Twin Arginine Transporter (Tat) mediated export that is linked to a B12-binding domain. This construct will be expressed in R. planticola and S. blattae. We will not only test these different production organisms but also different B12-binding domains.
However, Vitamin B12 can also be regarded as a prime example which is what we want to state with our work. This Synporter should work with basically any chemical which is desired to be exported out of a cell. We know from other iGEM Teams that they require bioactive compounds which are derived from nature, but are not synthesized by any organism (e.g. artificial amino acids). These are very expensive (< 1000 € per gram). However, finding novel enzymes for the synthesis of artificial compounds like drugs, for instance using metagenomics, turned out to be very promising and successful. Once one found the right enzymes, one is able to produce these compounds in an organism. Using our Synporter approach with an exchanged binding protein domain, these components can then be exported efficiently, making the production significantly easier and cheaper.
More Theory About Vitamin B12
Vitamin B12 – or Cobalamin – is a very complex compound (see Fig. 1), consisting of a corrin-macrocycle with a centrally incorporated Cobalt-ion, carrying varying upper ligands, and further a dimethylbenzimidazole group attached to the lower side of the corrin-ring and the Cobalt-ion. Depending on what ligand is attached to the Cobalt-ion, different forms of Cobalamin can be distinguished. Natural occuring forms are methyl- (CH3-), hydroxy- (OH-), cyano- (CN-) and adenosylcobalamin (5’-deoxyadenosyl-) (also called coenzyme B12), while cyanocobalamin (which is Vitamin B12 by definition) represents the industrial manufactured form. , , 
Fig. 1: The Structure of Cobalamin. Modified from 
Only certain microorganisms are able to synthesize Vitamin B12. Since these microorganisms colonize animals’ gastrointestinal tracts where the B12 accumulates, we humans uptake it via eating animal products like meat and dairy, with meat containing more B12 in general. Thereby, the B12 content varies between different types of animals and also between the diverse tissues. Ruminants’ entrails contain most B12. However, the type of cooking has further impact on the B12 content. 
Also some vegetarian B12 nourishments exist. But, again, the B12 was synthesized by microorganisms living in symbiosis with these products, e.g. algae and edible mushrooms, or being part of the production process as in tempe or beer. Nevertheless, the B12 content in plant-derived food is often biologically inactive in humans or only present in small quantities that an adult for example would have to drink several liters beer per day in order to cover the B12 requirement. , 
An adult needs approximately 3.0 µg Vitamin B12 per day . Vitamin B12 fulfills diverse functions in the human body. Thereby, only the two forms methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) are biologically active. MeCbl functions together with the methionine synthase in the methylation of homocysteine, resulting in methionine. Thus, MeCbl contributes to the one-carbon metabolism and the formation of S-adenosylmethionine, thereby being part of the synthesis of diverse molecules like DNA, RNA, hormones, proteins, lipids or myelin. AdoCbl on the other hand interacts with the methylmalonyl-coenzyme A mutase in forming succinate out of propionate, by that contributing to the fat and protein metabolism, hemoglobin synthesis and gluconeogenesis. Consequently, a Vitamin B12 deficiency can cause diverse disorders when important compounds cannot be synthesized and partially cytotoxic intermediates, like homocysteine, accumulate. Heart and vascular diseases, stroke, arteriosclerosis, megaloblastic anemia, depression, fatigue or neuropathy can amongst others result from a B12 deficiency. , , 
Especially vegetarians and vegans as well as elderly people have a higher risk to lack Vitamin B12. Therefore, these people could need to compensate the deficiency by taking B12 supplements.
Vitamin B12 is also added to diverse everyday products like vegetal milk, fruit gum, contact lense solution, tooth paste, energy drink etc. Just take a closer look on the products’ ingredients you use in your daily routine.
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More Theory About our Synporter
The blueprint of our B12 Synporter
Our B12 Synporter is built of a B12-binding protein domain linked to a signal for a Twin Arginine Transporter (Tat) mediated export. While the B12-binding domain (obviously) binds the vitamin, the export signal allows the protein-vitamin complex to leave the cytoplasm via the Tat system. In the following section, we will have a further look on these components.
The Tat export pathway and its signal peptide
In bacteria and archaea, proteins located outside the cytoplasm can reach their destination via either the Sec or the Tat (twin-arginine translocation) export pathway. While the Sec system translocates proteins in an unstructured state, the Tat apparatus has the unusual property of transporting fully folded proteins (Palmer and Berks, 2012). This system is very flexible in regard to the types of proteins that can be exported and the number of exported proteins highly differs between organisms. The E. coli Tat system is capable of transporting substrates up to 70 Å in diameter (Berks et al.., 2000). Many exported proteins containing non-covalently bound cofactors use this pathway, because the cofactor is held in place by the protein folding. The Tat pathway is only used by proteins containing certain types of cofactors that are classified as metal-sulphur clusters or nucleotide based cofactors, which include among others also cobalamins (Berks et al.., 2003). Therefore, our B12-bound Synporter should be able to be exported using this pathway.
Proteins are targeted to the Tat apparatus by N-terminal signal peptides that are normally cleaved by an externally facing signal peptidase (Lüke et al.., 2009). The overall architecture is similar to Sec signal peptides and includes a tripartite structure with a basic n region at the N terminus, a hydrophobic h region in the middle and a polar c region at the C terminus. The key element of a Tat signal peptide is the highly conserved twin-arginine motif, defined as SRRXFLK. The two arginines are almost always invariant, while the other residues occur with a frequency of > 50 %. The amino acid at position X is usually polar (Palmer and Berks, 2012). For our Synporter we chose the TorA signal peptide from the trimethylamine-N-oxide reductase from E. coli, a well characterized protein exported via the Tat system. Using this signal peptide, it has already been achieved to export a heterologous protein normally transported by the Sec pathway through the Tat system (Christóbal et al.., 1999). To connect the export signal with a B12-binding protein domain we use the five amino acids following the signal peptide in trimethylamine-N-oxide reductase as a linker.
Figure 1. Function of the Tat pathway. In contrast to the Sec pathway which translocates unfolded proteins, the Tat pathway translocates already folded proteins through a cellular membrane. (Rao and Anné, 2011)
Figure 2. Schematic view of the Tat signal peptide aligned with signal sequences from proteins exported via the tat pathway.
Residues that match the Tat consensus are shown in red, with the twin arginines underlined. (Palmer and Berks, 2012)
The B12 binding domain
For the binding of B12 to our Synporter, we use domains of proteins which are naturally capable of doing so. To increase our chances of success, we chose to test three different constructs.
MutB is the α-subunit of the methylmalonyl coenzyme A mutase from the Gram-positive bacterium Propionibacterium shermanii. The enzyme family it belongs to uses adenosylcobalamin to create a substrate radical that then rearranges to exchange a hydrogen atom with a group attached to a neighbouring carbon atom. Methylmalonyl CoA mutase catalyses the interconversion between linear succinyl CoA and branched methylmalonyl CoA and is thus involved in the fermentation of pyruvate to propionate (Allen et al..,1963). It is an αβ heterodimer and has a molecular weight of 150 kDa (80 kDa for the α chain and 70 kDa for the β chain). The active site as well as the cobalamin binding site are located in the α chain (Francalanci et al.., 1986). Crystal structures of the whole enzyme bound to cobalamin (PDB 1REQ; Mancia et al.., 1996) as well as bound to cobalamin and its substrate (PDB 4REQ; Manica and Evans, 1998) are available. Since a TorA-mutB fusion protein could already be produced in significant amounts and has been exported to the periplasm in E. coli BL21, C. freundii and S. blattae (Toeche-Mittler, 2002), MutB is our most promising candidate.
GlmS is the B12-binding subunit of glutamate mutase (Glm) from Clostridium cochlearium. The mechanism by which the enzyme uses adenosylcobalamin is highly similar to methylmalonyl coenzyme A mutase. Glm catalyzes the reversible rearrangement of (2S)-glutamate to (2S,3S)-3-methylaspartate (Leutbecher et al.., 1992). This reaction is the first step in the fermentation of glutamate to acetate and butyrate (Buckel et al.., 1974). The assembly of the active enzyme, an α2β2 tetramer, is mediated by coenzyme B12. While GlmS as the smaller subunit (14.8 kDa) binds B12, the larger subunit GlmE harbors the substrate binding site (Zelder et al.., 1994). Crystal structures of the whole enzyme in complex with its substrate and adenosylcobalamin (PDB 1I9C; Gruber et al.., 2001) as well as an NMR structure of only GlmS (PDB 1B1A; Hoffmann et al.., 1999) are available.
BtuF is the periplasmic binding protein for the Vitamin B12 transporter BtuCD from E. coli (Cadieux et al.., 2002). While some bacteria and archaea are capable of its synthesis, E. coli belongs to the majority of prokaryotes that contain transport systems to import B12 (Warren et al.., 2002). Its transmembrane transport is achieved by the Btu (B twelve uptake) system composed of BtuB, an outer membrane TonB-dependent transporter (Cadieux et al.., 1999), and the ABC transporter BtuCDF, which is located in the inner membrane. While BtuC and BtuD compose respectively the trans-membrane domain and the ABC (Bassford et al.., 1977), BtuF is the periplasmic binding protein. It has a size of 30.19 kDa and is composed of two globular domains, between which Vitamin B12 is bound, linked by a rigid interdomain α-helix (Karpowich et al.., 2003). A crystal structure of the protein bound to B12 is available (PDB1N4A; Karpowich et al.., 2003). For us it is highly interesting to see, if a protein that is naturally involved in B12 import can be converted to mediate its export. If this was possible, the concept could potentially be applied to other proteins involved in the import of small molecules.
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