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

Lead Research Organisation: Durham University
Department Name: Biosciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Foster AW (2014) Metal preferences and metallation. in The Journal of biological chemistry

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Foster AW (2017) A tight tunable range for Ni(II) sensing and buffering in cells. in Nature chemical biology

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Schorsch M (2018) A unique ferredoxin acts as a player in the low-iron response of photosynthetic organisms. in Proceedings of the National Academy of Sciences of the United States of America

 
Description The structure of a nickel-sensing, DNA-binding, transcriptional-regulator was determined. Variants were engineered that require higher concentrations of nickel to trigger the sensory mechanism. Cells using these variant nickel-sensors were created and found to accumulate more nickel. This shows how the metal contents of cells can be engineered for synthetic biology by adjusting the set point of a metal sensor. To increase nickel-dependent hydrogenase activity, the metal-delivery metallochaperone pathway may also need to be engineered. The work established a range within which the set point of this sensor could be adjusted.

Intriguingly, although the metal affinities of sensors for different metals co-vary with buffered metal concentrations over more than twelve orders of magnitude, this relationship ceased to apply in the engineered variant strains. This led to the discovery that metal-sensors are attuned to buffered metal concentrations rather than the converse (buffered metal concentrations had previously been thought to be governed by the metal affinities of the respective metal-sensors). This insight was described in the major output from this programme (Nature Chemical Biology, 2017).

This knowledge enables the determination of the buffered set points for different metals within a cell, with implications for synthetic biology (when metalloenzymes are involved) and for the design of bioactive compounds that interfere with metals in cells.
Exploitation Route Some of the potential applications of this research are ongoing in projects supported by the metals in biology BBSRC NIBB.

The work shows how the metal contents of cells can be engineered, creating opportunities to enhance metal supply to metalloenzymes for example in Industrial Biotechnology.
Sectors Agriculture, Food and Drink,Chemicals,Energy,Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description This work has been described at workshops with Industrial partners and has contributed to on-going funded collaborations with Industry, and to the BBSRC Metals in Biology NIBB. The data obtained in this programme revealed that metal sensors are tuned to a buffered metal concentration inside cells, rather than dictating these metal concentrations. This has informed subsequent work enabling us to measure the 'tuning' of metal sensors with implications for engineering enzyme metalation in biotechnology.
Sector Chemicals,Energy,Manufacturing, including Industrial Biotechology
 
Description BBSRC NIBB phase II
Amount £1,360,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2019 
End 02/2024
 
Title Clones encoding proteins of metal homeostasis 
Description Clones distributed in 2014 include pETInrS, pETNmtR, pETKmtR, pETCucA, pBAD24-ssCucA-GFP-SsrA 
Type Of Material Biological samples 
Year Produced 2014 
Provided To Others? Yes  
Impact Supplied to reseachers in Bologna, UMass Boston, Universite de Riems 
 
Description Interaction with Industry partner 
Organisation Procter & Gamble
Country United States 
Sector Private 
PI Contribution Regular teleconferences and exchange visits, exchange of data and materials.
Collaborator Contribution Regular teleconferences and exchange visits, exchange of data and funding of multiple PhD students and other staff with only an approximate value given above relating to new activities in the most recent ca one year.
Impact Joint publication in 2014 and in subsequent years. Other outcomes are mostly subject to NDA's.
Start Year 2014
 
Description Metalloenzyme system for Hydrogen-driven NADPH recycling in pharmaceutical synthesis 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution Bacterial cells are miniature chemical factories, with enzymes as the key machinery for making molecules. Pharmaceutical companies have recognised the benefits of using enzymes instead of traditional chemical routes to make drug molecules. Enzymes generate less waste, make purer chemical products, and allow chemistry to be carried out in water rather than in toxic, polluting solvents. However, there are challenges to overcome before enzyme approaches can be widely adopted. Many enzymes only work in the presence of special helper-molecules called cofactors, which are used up during the production of chemicals. The cofactors are expensive and for enzyme processes to be economically viable, it is essential to have some way of recycling the cofactors. We have demonstrated a novel approach for recycling a common cofactor called NADH. In collaboration with GSK, this project extends the approach to a related cofactor, NADPH, and demonstrates NADPH recycling for enzyme synthesis of pharmaceutically-relevant molecules.
Collaborator Contribution We funded and administered a Business Interaction Voucher in a collaboration with Kylie Vincent of University of Oxford (BIVMiB004)
Impact Introduction. Biocatalysis is gaining increasing importance in synthesis of pharmaceuticals and other speciality chemicals, yet barriers remain - in particular the dependence of many redox enzymes on the expensive nicotinamide cofactor, NADH, and more costly NADPH. We have previously demonstrated a novel H2-driven NADH recycling system, and in this Business Interaction Voucher project we have extended this system to recycle NADPH. The project exploits genetic variants of an NAD+ reductase generated in the lab of Dr Oliver Lenz, Technical University of Berlin, which are designed to have higher affinity for the related cofactor NADP+. We have identified a suitable NADP+ reducing variant, and we have demonstrated NADPH recycling by coupling the cofactor recycling beads with a C=C bond reductase, PETNR, supplied by industrial collaborator GSK. Project outcomes: During the project we have recorded KM values for the most promising NAD+ reductase variants and identified one which functions as an NADP+ reductase with a KM of 1 mM NADP+, compared with >8 mM for the wild type. Data on the affinity constants for a series of variants will form the basis for the publication to be submitted in the coming months. The selected NADP+ reductase was then incorporated into cofactor recycling beads. A nickel-iron hydrogenase oxidises H2 and transfers electrons, via its internal relay chain of iron-sulphur clusters, to the electrically-conductive carbon bead. Electrons are then transferred through the bead to the co- immobilised NADP+-reductase which also possesses a relay chain of iron-sulphur clusters for fast electron transfer to its flavin active site. Thus H2 oxidation is coupled efficiently to NADP+ reduction. The imine reductase and ene reductase enzymes supplied by GSK were examined in biochemical assays with product detection by HPLC or GC. The C=C bond reductase PETNR was selected as the best enzyme for a first demonstration of NADPH recycling. In the presence of H2, NADPH was supplied to PETNR which was co-immobilised on the beads or handled in solution, for the reduction of cyclohexenone to cyclohexanone. Near-complete conversion (>99%) of the alkene to the alkane was observed by GC. Thus we have generated promising results on the feasibility of coupling H2-driven NADPH recycling to a NADPH-dependent hydrogenation. These results will be prepared for publication later this year as part of the demonstration of the modularity of the H2- driven cofactor recycling system which can be adapted for operation under different conditions by varying the hydrogenase or the NAD(P)+ reductase. Conference presentations: Dr Holly Reeve presented a Flash Talk and Poster that included these findings at the European Symposium on Biological and Organic Chemistry, Gregynog, Wales, 15th-17th May 2015. Prof Kylie Vincent gave an invited lecture at the Academic Day of GSK's Global Technologies Conference, Cambridge, 25 March 2015: 'Heterogeneous biocatalysts for H2-driven chemical synthesis', and included some of these results in her presentation.
Start Year 2014
 
Description AWF Biometals conference in North Carolina 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited lecture at conference on Metals in Biology.
Year(s) Of Engagement Activity 2014
 
Description AWF ICBIC 17 Bejing 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Presentation at International conference
Year(s) Of Engagement Activity 2015
 
Description AWF workshop on algal biotechnology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Industry-Academia workshop on algal biotechnology
Year(s) Of Engagement Activity 2014
 
Description Author profile associated with Journal of Biological Chemistry Review. 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Refer to URL to see the item

See next response
Year(s) Of Engagement Activity 2014
URL http://www.jbc.org/content/289/41/28095/suppl/DCAuthor_profile
 
Description Bite-sized chunks of Great British Bioscience Blog 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact BBSRC Blog in July 2014

Helped to promote the BBSRC network in industrial biotechnology on 'Metals in Biology: The elements of Biotechnology and Bioenergy'
Year(s) Of Engagement Activity 2014
URL http://bbsrc.tumblr.com/post/92833696876/researchers-have-engineered-cells-to-accumulate
 
Description EuroBIC bioinorganic chemistry conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Disseminated knowledge about the cell biology of metals which sparked questions and discussion.
Year(s) Of Engagement Activity 2018
URL https://www.birmingham.ac.uk/facilities/mds-cpd/conferences/eurobic/index.aspx
 
Description Invited speaker, BBSRC NIBB BioProNET 6th annual science meeting, Manchester 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact This aim of the event was to promote collaboration between industry and academia and advertsie the opportunities arising from the E3B BBSRC NIBB.
Year(s) Of Engagement Activity 2019
URL http://biopronetuk.org/6th-annual-science-meeting/
 
Description Invited talk at International Conference on BioInorganic Chemistry, Interlakken, Switzerland 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The results of our research were described which sparked questions and discussions immediately afterwards and ongoing by e-mail.
Year(s) Of Engagement Activity 2019
URL https://www.chem.uzh.ch/dam/jcr:d809e5d0-e81b-42d0-a1c9-175c8e13e958/ICBIC19_ScientificProgram_v5.pd...
 
Description Metal-related antimicrobials BBSRC NIBB workshop (November 2015) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact BBSRC NIBB event with representatives from agritechnology business, consumer goods industry, pharmaceutical companies, policy and standards agencies, industrial biotechnology companies to explore the opportunities for metal-related antimicrobials (PI and RA attended).
Year(s) Of Engagement Activity 2015
 
Description Metals in Biology Community Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact We organised a workshop involving academic and industry to show case funded projects as exemplars to instigate future collaborations.
Year(s) Of Engagement Activity 2016
URL http://prospect.rsc.org/MiB_NIBB/metals-in-biology-network-community-meeting/
 
Description Metals in Bioprocessing multiple BBSRC NIBB event (Hexham) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Interaction with Bio-processing industries. PI and RA both attended and PI gave a presentation.
Year(s) Of Engagement Activity 2015
 
Description NJR Bangalore 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited keynote speaker, 6th International conference on Metals in Genetics, Chemical Biology and Therapeutics, Bangalore, India.
Year(s) Of Engagement Activity 2016
 
Description NJR Biometals Dresden Invited Talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited speaker, Biometals Conference 2016, Dresden, Germany.
Year(s) Of Engagement Activity 2016
 
Description NJR Biophysical Society Invited Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited speaker, British Biophysical Society 2016 Biennial Meeting "Metals in Biology" microsymposium, Liverpool.
Year(s) Of Engagement Activity 2016
 
Description National Institutes of Health, Wshington DC, presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Disseminated knowledge of the cell biology of metals which sparked questions and discussion.
Year(s) Of Engagement Activity 2018
 
Description Poster presentation at Copper 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Conference poster discussion
Year(s) Of Engagement Activity 2014