Directed and adaptive evolution of photosynthetic systems

光系统是光合作用的复杂分子组合，它们产生的能量几乎维持了整个生物圈，每年直接为全球固定约100亿吨二氧化碳提供动力。光合作用利用两个串联工作的光系统：光系统II和光系统I。光系统II收集光，为水氧化和分解成质子、电子和我们呼吸的氧气提供动力。光系统I收集光以产生驱动代谢反应所需的能量，重要的是，但不是唯一的，二氧化碳固定。这些特性使光系统成为生命史上最强大的酶之一，两者都能够利用光来驱动它们自己的复杂化学反应。我的长期愿景是使用基于进化的方法来完全重新设计光系统，这样我们就可以利用它们的特性来进行有用的化学反应，而不仅仅是它们自然进化的功能。我想开发一个技术平台，能够精确控制两个光系统，以实现定制的多步光驱动氧化或还原生物催化。最终，我设想这个平台连接到一个研究设施，允许快速和高通量的纯化，表征和生产这些新的光系统，用于广泛的应用，从生物修复到精密化学，所有这些都由光驱动。因此，这项技术可以通过创造新的方法来进行清洁的化学反应，从而使生物技术和化学工业受益并产生影响。在此扩展中，我继续了在奖学金第一阶段开始的工作。我的实验室目前正在开发，优化和表征定向进化方法，这些方法针对光系统II，以改变其功能特性，超越其自然进化的功能。定向进化是一种非常通用的方法，用于通过利用进化来改变给定酶的特性或活性。它可以简单地通过在有利于所需性状的条件下对生物体进行重复的选择循环来完成，或者通过筛选所需的功能来完成，它可以通过加速突变率（超突变）来增强，它可以集中在单个感兴趣的基因，基因的部分或多个基因上。超突变可以在体外进行，其中基因在试管中突变;或者在体内，当细胞分裂和复制时发生超突变。我的实验室有一个工作在体外系统，我们已经用来分离几个光系统II变异携带一系列突变，并即将部署和体内基于CRISPR的超突变系统。应用定向进化工程师新的光系统的想法是没有先例的。虽然定向进化是一种成熟的技术，但它还没有广泛应用于复杂的酶系统，如光系统，如果有的话。此外，定向进化方法在光合生物中的发展也滞后于非光合系统，如大肠杆菌。大肠杆菌和酵母菌。虽然这是一项雄心勃勃且具有挑战性的研究工作，但我已经证明，由于其结构模块化，光系统II在自然界中仍然是可进化和可塑的。因此，我们的方法利用这种自然的适应性，使用定向进化，但加速到实验室timescales.The奖学金的首要目标是证明，光系统II是服从定向进化，并开始发展hypermutation和选择的方法，以推动其演变。这一扩展的最终目标是提供概念验证的新型光系统，或携带这些新型光系统的蓝藻菌株，这可以为扩大规模并将这一愿景转化为可行的绿色生物技术铺平道路。

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.