Cell circuitry for metals: Integrative metabolism for cobalt uptake and cobalamin production

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.

The project deals with the advancement of synthetic biology and specifically with the design, introduction and adaption of regulatory control circuits for the uptake of metal ions. In this project we will be studying how cobalt can be controlled and regulated for its incorporation into adenosylcobalamin, the biologically active form of vitamin B12. We have shown that it is possible to engineer the cobalamin pathway into E. coli but that the cobalt uptake mechanism is very inefficient with less than 0.5% incorporation of cobalt from the medium into the nutrient. The yield of cobalamin improves as the level of exogenous cobalt in the medium is increased but this then leads to inhibition of cell growth due to the toxic effect of the cobalt on Fe-S formation. To overcome this problem we will engineer into E. coli a range of specific cobalt transporters to allow for the enhanced uptake of cobalt into the cell. The concentration of cobalt within the bacterium will be monitored by use of cobalt-sensitive reporter groups, allowing the concentration of total and buffered cobalt to be determined.
A number of different components of the cobalt transport and efflux system will be characterised in molecular detail. These will inform on how they can be modified to enhance the procurement of the metal from the medium and to maintain it within the cell. A key objective is to be able to take cobalt up at much lower concentrations to allow the metal to be incorporated into cobalamin. We have also identified a protein within the cobalamin pathway, CobW, which is a cobalt-binding GTPase that may act as a metal chaperone. We will undertake a detailed characterisation of this protein and determine whether the protein is able to deliver the metal to the chelatase for its incorporation into the vitamin. Finally, we will design and integrate a cobalamin-dependent riboswitch to control the amount of cobalt that enters the cell.

The research described in this application will have a major impact on several areas of science, including synthetic biology and the manipulation of metal-requiring pathways. It will permit the generation of bacterial strains into which metal uptake and utilisation can be tightly controlled through an increased understanding of the relationship between free and buffered metal pools. The research relates to how cells can be engineered to improve the uptake of potentially toxic metals and how the cells can be manipulated to make the metal available for biochemical pathways. This approach will be applicable to a broad range of natural products. With an increase in the interest especially of secondary metabolites such an approach is likely to prove popular with chemical biologists and medicinal chemists alike.
The research falls well within the remit of synthetic biology and is therefore addressing a key priority area. In this respect the project applies the engineering paradigm of systems design to metabolism. In essence, the project employs the re-design of existing, natural biological systems for useful purposes. The research also has the potential to engineer improvements in existing biological products and especially improve our understanding of biological systems through researching the role of modularity. The research will have application in the bioengineering and bioprocessing of pharmaceuticals and nutrients but also has the potential to be applied to the area of bioremediation.
The beneficiaries of this research will be researchers in academia and industry who are interested in synthetic biology and its applications. There is a current strong interest in this area and science needs to put forward a strong representation in terms of the positive contributions that it can make. The research will not only provide essential information about how pathways and enzymes can be investigated and modified, but it will also provide greater insight into the provision and procurement of pathway substrates. It will demonstrate how cells can be engineered to resource their nutrient components to allow for fast and efficient synthesis. We will ensure that our findings are widely disseminated through oral communication, research publications and reviews, and press releases.
The Kent and Durham groups are heavily involved in outreach programmes, through interactions with local schools and community groups. Kent is a member of the Authentic Biology Project, which is funded by a Wellcome Trust society award to bring real research into schools. Regular talks and demonstrations are given through organized events during science week and at other times by invitation via the biology4all website, ensuring there is good dissemination with the general public on a range of important issues.
The skills acquired by those involved in this project include not only a wide range of important biological techniques ranging from spectroscopy and structural biology through to microbiology and recombinant DNA technology but also the chance to make a significant contribution towards the development of biotherapeutics. The knowledge and techniques will provide those employed with skills that can be used across education and industry. The intellectual property resulting from this project will be protected and used via the Innovation and Enterprise Office. The research will be published in high impact journals and oral communications given at international conferences. Using the infrastructure of the new Centre for Molecular Processing within the University of Kent, the research will be brought to the attention of many leading industrial companies.