Engineering nickel supply to cyanobacterial hydrogenase to test the relationship between enzyme metallation and metal-sensing

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A large proportion of the proteins that biotechnology aims to exploit, need metals. Such proteins somehow acquire the correct metals inside cells. Efforts in synthetic biology (for example to engineer organisms for the sustainable manufacture of compounds for industry) must consider how to also engineer an adequate supply of metal cofactors, because so much of biological catalysis is driven by metals. Hypotheses about how cells help proteins to acquire the correct metals have been introduced for a general audience in a podcast which can be downloaded from the Nature web-site (search for 'nature podcast pick your partners') linked to review article (Nature (2009) 460, 823-830). This research will test these hypotheses. There is a tendency for proteins to form partnerships with unsuitable metals. It is thought that cells maintain each metal at optimal buffered concentrations to overcome this challenge. It is hypothesised that the most competitive metals are kept at the lowest buffered concentrations. Under this regime proteins compete with other proteins for the competitive metals, rather than metals competing with other metals for a limited pool of proteins. If this model is true, then the mechanisms which maintain the correct buffered concentrations of each metal are vital for the fidelity of metal co-factoring of a large proportion of proteins. It raises the possibility that the buffered metal levels may change from cell to cell with widespread implications for metabolism. Moreover, the model suggests an opportunity to engineer optimal metal-levels to assist co-factoring of useful proteins in, for example, synthetic biology.Metal sensor proteins detect fluctuations in the buffered concentrations of metals in the cytosol. Our previous BBSRC-funded research has discovered multiple metal-sensors and has identified factors that influence which metals bind and trigger their sensing-mechanisms. Critically, it is hypothesised that there is a direct relationship between the affinity of such sensors for the detected metal and the intracellular buffered metal concentration. This hypothesis will be tested for the detection of nickel. This research aims to engineer the supply of nickel to an enzyme relevant to bioenergy in a representative of a group of organisms significant for green technology.Hydrogenase is one of a small number (probably less than twenty types in the biosphere) of enzymes that require nickel. Under the correct conditions this enzyme can generate di-hydrogen gas and hence might contribute to a hydrogen economy. Photosynthetic organisms, including cyanobacteria, can be exploited to use energy from sunlight to produce useful compounds including carriers of energy such as hydrogen, and thus have potential to create sustainable processes. However, hydrogenase activity is limited by poor nickel supply in cyanobacteria. We have recently (with support from BBSRC) discovered a nickel sensor in the cytosol of a cyanobacterium (Journal of Biological Chemistry (2012) 287, 12142-12151). This discovery creates an opportunity to engineer metal-homeostasis with the aim of optimising nickel supply to hydrogenase in a cyanobacterium.