Creating and comprehending the circuitry of life: precise biomolecular design of multi-centre redox enzymes for a synthetic metabolism

A defining characteristic of life is the requirement of energy from an external source; we eat, plants absorb light. To maximize the energy gained from the food that we and all oxygen-breathing organisms consume, oxygen is converted to water as a final step and carbon dioxide is released. The oxygen in this equation arises from plants as they convert water, carbon dioxide and light, into oxygen and fuel. This cycle is not merely an auspicious result of billions of years of evolution. The molecular events that allow the processes of respiration and photosynthesis to happen are connected in deep ways, down to shared structures, molecules, and mechanisms.

At their most basic, respiration and photosynthesis are Nature's way to capture and convert energy from one form to another. To do this, Nature has evolved complex structures, termed oxidoreductases, that bind molecules that aid in this conversion. These molecules can both absorb light, imparting plants with their colours, and take and give electrons. The oxidoreductases have evolved to take energy from external sources and convert it into forms that can be used by living organisms to grow and survive. The evident complexity of this process belies a central feature of the oxidoreductases involved: evolution has yielded structures that are built from repeats of relatively simple modules. All of respiration and photosynthesis are built on these repeating modules. But despite nearly a century of investigation, where we have outlined how respiration and photosynthesis work in fine detail, we remain unable to construct our own models of these processes. This naturally leads to a question of whether we really understand how these processes occur.

Here we have assembled a team of researchers from multiple academic institutions and disciplines to address deficiencies in our knowledge, with the unified target of building completely new oxidoreductases from scratch. Through this work we will fill holes in our understanding of how Nature captures and converts energy.

Our work begins by combining powerful computational techniques that allow us to design and construct oxidoreductases with tailor made functions. Within a virtual reality framework that we are developing for this project, we will work together in a shared digital space to construct molecular binding sites, alter how molecules take and give electrons or catalyse reactions, and create oxidoreductase modules that, taking inspiration from Nature, we will join to produce more complex functions. With these designs, we will use an iterative 'build-test-learn' approach to construct new oxidoreductases that match the activities and actions of those Nature uses in respiration and photosynthesis. By pulling together our expertise in computational biophysical methods, oxidoreductase engineering, modular structure creation, molecular binding site assembly and their chemistry, and the analysis of very fast oxidoreductase functions, our team stands to make a substantial leap in our understanding of how to construct new oxidoreductases that has, so far, remained beyond our grasp.

The principles we establish through this work will help us to better understand the oxidoreductases of respiration and photosynthesis, finally clarifying architectural features that are essential for their assembly and function that have remained opaque for over a century. With our new sets of design principles, we will be able to create oxidoreductases that fulfil our needs in bioscience and biotechnology, from the creation of single structures that produce fuels from light, water and carbon dioxide akin to photosynthesis to biosensors that detect toxins in the environment or signs of disease.

Electron and captured energy flow through protein-based architectures is essential to life, underpinning cellular respiration and photosynthesis. While our understanding of this complex protein machinery has benefitted from the advances of the structural genomics era, we have yet to fully exploit the exceptional features and functions of these assemblies. Such exploitation will provide a clear route to test theory, clarifying unresolved details of these processes, and deepening fundamental understanding of the circuitry of photosynthesis, respiration, and metabolism. We are now at a pivotal moment where there is a convergence of empirical knowledge, computational power and spectroscopic tools, making such advances feasible.

Here we aim to apply a multifaceted approach to the precision de novo design and construction of single and multicentre redox and light-harvesting proteins. We will create diverse cofactor binding modules that can be assembled with a mix-and-match process into working biomolecular components for long-range electron and energy transfer, multielectron catalysis, broadband solar light-harvesting and electron bifurcation. To achieve this, we will use state-of-the-art experimental and computational methodologies supported by a collaborative VR platform for protein design and molecular analysis. Cutting-edge multidimensional and ultrafast spectroscopic techniques will also be used to map electron and energy transfer within our designs, helping us to refine their construction through an iterative process. This integrated approach will yield unprecedented access to directed and efficient energy, proton and electron flow within tailor-made biomolecular components. We anticipate that our unique approach will generate ground-breaking discoveries that will have lasting impact in fundamental biosciences, synthetic biology and industrial biotechnology.