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

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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. 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 growing interest in 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 biomedicine 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 biosynthesis of cobalamin. 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, for example, short review articles. Furthermore, there is no doubt that the research will be of significance to those devising new strategies against disease and thus we will ensure that our findings are disseminated to those working in drug development.

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 biological techniques, ranging from spectroscopy and structural biology through to microbiology and recombinant DNA technology, but could also provide the capacity to make significant contributions 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 DBIS in Durham (in agreement with collaborators in Kent), the research will be brought to the attention of many leading industrial companies.