Directed and adaptive evolution of photosynthetic systems

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Photosystems are the complex molecular assemblies of photosynthesis, they generate the energy that sustain nearly all the biosphere, directly powering the global fixation of about a hundred gigatons of carbon dioxide annually. Photosynthesis uses two photosystems working in series: photosystem II and photosystem I. Photosystem II harvests light to power the oxidation and decomposition of water into protons, electrons, and the oxygen we breathe. Photosystem I harvests light to generate the power needed to drive metabolic reactions, importantly, but not exclusively, carbon dioxide fixation. These properties make the photosystems amongst the most powerful enzymes in the history of life, both capable of driving their own difficult chemistry using light.My long-term vision is to use evolution-based methods to completely redesign the photosystems so that we can harness their properties to do useful chemistry beyond their naturally evolved function. I want to develop a technological platform that enables exquisite control of both photosystems to achieve bespoke multi-step light-driven oxidative or reductive biocatalysis. Eventually, I envision this platform linked to a research facility that allows for the rapid and high-throughput purification, characterisation, and production of these novel photosystems for a broad range of applications, from bioremediation to precision chemistry, all driven by light. Therefore, this technology can benefit and impact the biotechnology and chemical industry by creating new ways to perform clean chemical reactions.In this extension, I continue the work that was started in the first stage of the fellowship. My lab is currently developing, optimising, and characterising directed evolution approaches that target photosystem II to change its functional properties beyond its naturally evolved function. Directed evolution is an extremely versatile approach that is used to change the traits, or the activity of a given enzyme by exploiting evolution. It can be done simply by subjecting an organism through repeated cycles of selection under the conditions that favour the desired traits or by screening for the desired function, it can be enhanced by turbocharging mutational rates (hypermutation), it can be focused on a single gene of interest, parts of a gene, or multiple genes. Hypermutation can be done in vitro, where the genes are mutated in the test tube; or in vivo, where hypermutation occurs as the cells divide and replicate. My lab has a working in vitro system that we have already used to isolate several photosystem II variants harbouring a range of mutations and are about to deploy and in vivo CRISPR-based hypermutation system.The idea of applying directed evolution to engineer novel photosystems is without precedent. While directed evolution is a somewhat of a mature technology, it has not been extensively applied to complex enzymatic systems like the photosystems, if at all. In addition, the development of directed evolution approaches in photosynthetic organisms also lags compared with non-photosynthetic systems like E. coli and yeast. While an ambitious and challenging research endeavour, I've demonstrated that photosystem II remains evolvable and plastic in nature thanks to its structural modularity. Therefore, our approach harnesses this natural adaptability using directed evolution but accelerated to lab timescales.The overarching aim of this fellowship is to demonstrate that photosystem II is amenable to directed evolution, and to begin developing hypermutation and selection approaches to drive its evolution. The ultimate objective of this extension is to deliver proof-of-concept novel photosystems, or strains of cyanobacteria harbouring these novel photosystems, that could pave the wave towards scaling up and translating this vision into viable green biotechnologies.