Advancing Microbial Electrochemistry: Biophysical Characterisation of the Electron-Transfer Interactome in S. oneidensis MR-1

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Shewanella oneidensis MR-1 (MR-1) possesses a remarkably versatile respiratory system. Key to this versatility is the inner-membrane menaquinol dehydrogenase, CymA, that represents a hub for electron transfer between catabolic enzymes and terminal reductases. However, very little is known about the factors that determine electron exchange between CymA and the network of periplasmic and outer-membrane proteins with which it interacts. Here we propose to address this situation through recent advances in the applicants' laboratories. Specifically, we propose to use novel supported membrane technology with quartz-crystal microbalance and electrochemical measurements complemented by contemporary biochemistry with the aims of a) mapping out the periplasmic and outer-membrane proteins that interact with CymA, and, b) quantifying the factors that regulate protein-protein interaction and electron flux across this network. This will allow us to test our hypothesis that respiratory electron transfer by MR-1 may be regulated by the presence of terminal electron acceptors and predominantly controlled at the metabolic level through reconfiguration of the network of electron-transport proteins around CymA.
Key advances will include a) an understanding of the biophysical basis for respiratory flexibility in MR-1, and b) insight into the determinants of catalytic bias in CymA which belongs to a phylogenetically widespread family of quinol-dehydrogenases. Thus, we envisage our results will inform the understanding of electron flux and respiratory versatility in a number of bacteria. In addition, our results will define conditions where electron exchange between CymA and the outer-membrane electron transfer conduit MtrCAB is optimised. Thus, we envisage our results will underpin advances in microbial biotechnologies that exploit electrode-MtrCAB electron transfer such as microbial fuel cells and electrosynthesis for which MR-1 is a model organism.

Impact summary
The aim of this project is two-fold: (a) to use biochemical, biophysical and (bio)nanotechnological approaches to study Shewanella oneindensis MR-1 (MR-1) to optimise the exploitation of MR-1 and related microbes in biotechnology and (b) development of techniques in membrane biology. This work will impact the global society (timescale > 10 year) and research sectors in alternative energy sources (timescale > 3 year) and drug development (timescale > 2 years). Finally, public-engagement events organised during the life-time of the project will have a direct impact to the local community, while also the training of PDRAs and closely-involved research staff and PhD students will impact the scientific community.

We aim to increase our understanding of the bioenergetics of MR-1 and their respiratory proteins, which is urgently required to optimise the use of microbes to harvest energy and produce fuels using microbial electrochemistry, which includes microbial fuel cells and microbial electrosynthesis. Of particular interest for the studies proposed here are the multi-heme proteins in Shewanella which mediate electron transfer to the outside of the cell or to inorganic substrates. Shewanella bacteria serve as an important model system for mediator-less microbial fuel cells that run on waste carbon sources (such as in waste water) to produce electricity. This is a new area with much potential in the future. We propose that our work will contribute to the future design of such microbial electrochemistry, in particular where future work aims to genetically or synthetically modifying the microbes to enhance electron transfer rates to the electrodes (i.e., increase electrical current).

The study proposed here includes the further development of so-called solid-supported membranes. Recent pilot data has shown the latter tool to be very useful in the study of protein-protein interactions in cases where one or two of the proteins are membrane proteins. Currently, membrane biology is an extremely active and important research area. For instance, although only ~20% of the human genome, membrane proteins represent approximately 50% of today's drug targets. Having tools available that can screen protein-protein interactions of membrane proteins (this includes pharmaceutically important antibody mimetics) will be of high importance to the wider scientific community, including the industrial pharmaceutical sectors.

Societal impact
The search for alternative energy sources is of major importance to THE GLOBAL SOCIETY where conventional energy sources, based on burning of earth-stored carbon sources, are thought to be responsible for global warming. Furthermore, the extraction of these carbon sources themselves is becoming increasingly more expensive and, combined with the higher demand for energy world-wide, this has led to rapid increases in energy prices. A solution to this problem has to be sought by combining a multitude of 'alternative' energy sources; this research will contribute to this progress.
Timescale of likely impact: >10 years

The technology development of solid-supported membranes will also impact on the pharmaceutical industry, where improvements in drug development and screening of antibody mimetics will be of benefit to the THE GLOBAL SOCIETY on the longer timescale.
Timescale of likely impact: >10 years

As part of this grant we plan to contribute to a variety of events aimed to engage the public in alternative energies (see Pathways for Impact for more details). These events will contribute to the public understanding of communities in or near Leeds and Norwich.
Timescale of likely impact: During the lifetime of the project.

Commercial/industrial impact
Both the alternative energy industry sector and the biopharmaceutical sector may benefit from this research, as explained above.
Timescale of possible impact: >2-3 years after starting the project.