Proof of Concept

Our aim was to transport Vitamin B12, a substance which has no known cellular exporter in nature, through the inner membrane out of the cytoplasm. Our different B12 Synporter proteins consist of a TorA signal peptide that mediates export through the Twin Arginine Translocation (Tat) system, linked to a B12-binding protein domain. This construct was produced, together with B12, by the B12 prototrophic enterobacteria R. planticola and S. blattae.

We were able to detect the Synporter proteins in the periplasm of the two organisms (see expression test, proving that the Tat mediated translocation was successful. Moreover, most important, we could also prove a significantly increased overall B12 production in the cells expressing the Synporter protein, compared to the cells containing an empty plasmid (see B12 assay). This increased production is mainly due to the fact that the cells have to compensate for the B12 leaving the cytoplasm bound to the Synporter. Thus, it proves the functionality of our system.

This is the first time reported that a compound of such a high-molecular weight as B12 was translocated through a cellular membrane without a native exporter. Therefore, our Synporter is truly innovative!

Vitamin B12 can also be regarded as a prime example for how highly valuable compounds can be gained from production organisms. In principle, a Synporter should work with basically any desired chemical compound, if a protein counterpart exists which can be used as Synporter binding domain. This concept could be applied for the production of all natural chemicals which are synthesized by microorganisms, but not exported due to the lack of a native cellular exporter. Furthermore, this principle could also be applied for the production of artificial molecules which can be produced by synthetic metabolic engineering in a cell. Using our Synporter approach, no cell lysis would be required any more for the industrial production. This concept has the potential to make the production process significantly easier and cheaper.

Fig. 5: Final results of the photometric assay. The plot shows the quantity of B12 per amount of protein, in the periplasmic fraction and the cytoplasmic fraction.