| Abstract |
In this era of synthetic biology, where it is possible to redesign and construct novel biological systems to perform new functions for useful purposes, it is often overlooked that such approaches are inherently flawed because of the accumulation of toxic materials. In using cells as factories the procurement of starting materials and the accumulation of intermediates/products can often prove detrimental to the bacterium. Such is the case with engineering the cobalamin (vitamin B12) pathway into E. coli. Here, it is the provision of cobalt, the metal centre of the vitamin, which is difficult for the bacterium to cope with. As the cell does not have a specific cobalt import system, high exogenous levels of the metal have to be added to the growth medium to permit transport of the metal to the site of vitamin synthesis and to allow production of the nutrient. However, the high level of cobalt compromises the viability of the cell as the cobalt interferes with Fe-S centre formation in redox proteins. To overcome this metal toxicity issue we have outlined a research plan to enhance cobalt uptake, increase its internal cellular concentration, and modulate its export. This will be accomplished by developing systems to allow improved uptake of the metal, increased internal binding capacity and enhanced delivery of the metal to where it is required within the metabolic pathway. We will monitor the effectiveness of these procedures by using internal reporters within the cell that will provide readouts on the level of cobalt and how it is being used internally. The research will also allow the characterisation of a number of metal transport and binding proteins. The results of these analyses will inform on how the cell can be engineered further so that cobalamin synthesis can be enhanced at lower metal concentrations. From this information we will develop a self contained, fully wired, metal circuit that will be able to control and regulate cellular cobalt supply. |
