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

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.

We will weaken the Ni(II)-affinity of cytosolic Ni(II)-sensor InrS in Synechocystis PCC 6803. Cells will first be modified such that they cannot respond to periplasmic Ni(II) (via NrsRS). It is predicted that these cells will thus become dependent upon NrsD (and hence the InrS Ni(II)-sensor which regulates the nrsD gene encoding the Ni(II)-exporter) for maintaining the buffered concentration of Ni(II) in the cytosol. It is hypothesised that a Ni(II)-sensor with weaker Ni(II)-affinity should allow Ni(II) levels to rise to a higher level than normal before triggering Ni(II) efflux. Variant Synechocystis PCC 6803 will be tested for altered Ni(II) accumulation, for change in buffered concentration of Ni(II), for evidence of mal-occupancy of other proteins with Ni(II) (which it is proposed may dictate the limits within which buffered levels may be adjusted) and for enhanced activity of Ni(II) dependent enzymes, most importantly hydrogenase (due to its relevance to bioenergy).

Variant, recombinant InrS proteins will initially be expressed and purified to measure their Ni(II)-affinities and to test whether or not they remain allosterically competent to impair binding to a fragment of the nrsD operator-promoter in response to binding to Ni(II). Thus, in the course of this research we will increase knowledge of the mechanism of Ni(II)-sensing by InrS; an additional novelty since this family of metal-sensors was more recently discovered than most and the mechanism of Ni(II)-detection by InrS is unlike related RcnR.

This research will test the hypothesis that metal-sensors control the buffered levels of metals inside cells, that the set-point for buffering is determined by the affinity of the sensors, and that these buffered levels influence metal-speciation of metalloproteins, including enzymes relevant to biotechnology.

Understanding how cells assist proteins to acquire the correct metals has potential impact for synthetic biology. Efforts to engineer biological processes will commonly involve at least one metalloprotein creating a potential requirement to also optimise cofactor supply. The research thus has broad relevance to researchers across the biosciences and biotechnology. Impact from optimising the activity of hydrogenase in cyanobacteria relates to a potential hydrogen energy economy and the PI and CoIs are members of the Durham Energy Institute which provides a pathway to contact businesses engaged in the energy sector.

The manufacture of recombinant proteins using heterologous cells may also benefit from this research. We are currently developing links with Bioprocessing companies who wish to lower the intracellular availability of zinc when expressing recombinant proteins in eukaryotic cells, because zinc is a factor promoting cleavage of one class of proteins. Conversely, we have entered into discussions with other bioprocessing companies that express recombinant proteins that require metals. Several of the next generation of 'block-buster' biologic drugs are zinc-proteins. Here, it is possible to envisage the development of specific strains for the expression of each type of metalloprotein. If the hypothesis being tested in this programme is shown to be correct, then it would imply that a host strain for expressing zinc proteins should be engineered to switch to zinc sensors of weaker zinc-affinity at the point of protein induction to elevate the buffered cytosolic level of zinc; production of manganese proteins could use a strain that switched to a weaker affinity manganese sensor (and perhaps a tighter affinity iron sensor to keep iron out of the recombinant manganese sites), as examples. Moreover, such strains would be of considerable value to large numbers of researchers (based in industrial biotechnology companies and institutes, as well as in academia) who express recombinant metalloproteins which often become incorrectly, or inadequately, co-factored in standard expression hosts.

The work proposed here will test the hypothesis that the metal-affinities of metal-sensors control the buffered levels of metals available to proteins. This also has relevance to our industry-funded programs to subvert the cellular control of metal availability in the development of metal-related antimicrobials in collaborations with Syngenta (as agrochemicals) and with Procter and Gamble (as preservatives and in some antimicrobial products). Here the routes for exploitation are already well established.

Other forms of impact are the trained personnel (Andrew Foster) and outputs intended to engage the public. A case is made in the pathways to impact section that the PI is highly committed to both of these activities based on the subsequent employment of past RA's and on evidence of past press-releases and articles targeted to A-level students.