Organisation, dynamics and biogenesis of a photosynthetic membrane

Cyanobacteria are the oldest microorganisms that grow by photosynthesis in a similar way to plants. Cyanobacteria are widespread in our environment on Earth. For example, they are very abundant in rivers, lakes, and the oceans, and they make important contributions to the sustainable ecology of the planet. There are increasing interests in using cyanobacteria as possible sources of 'biofuels'. We may eventually be able to modify cyanobacteria to build new artificial cell "factories" that can use the energy of sunlight to produce fuels such as hydrogen.

Cyanobacteria have a more complex cell structure than most bacteria. Inside the cells are the thylakoid membranes, a complex internal membrane system which is the site of the 'light reactions' of photosynthesis. The thylakoid membranes contain the pigments that absorb energy from sunlight and the proteins that carry out the first steps in converting solar energy to stored chemical energy. Although we now have a great deal of knowledge about the photosynthetic protein modules, we know rather little about how the thylakoid membranes are generated in nature.

We propose to investigate this question using a 'model' cyanobacterium that can easily be genetically modified and have a regular shape of thylakoid membranes. We will first control the ability of cyanobacterial cells to produce thylakoid membranes. By switching the generation of thylakoid membranes in the cell and tagging the photosynthetic proteins with fluorescence, we will be able to watch in detail how proteins are synthesised and integrated into the thylakoid membrane during the membrane construction process using optical microscopy. To get more details on how the photosynthetic proteins are distributed in the thylakoid membrane, we will use a high-resolution microscope to scan the thylakoid membrane surface in order to determine individual proteins and their locations during the development of thylakoid membranes. The second section of this programme is to study how photosynthetic proteins interact with each other in the thylakoid membrane, which is important for their energy-transducing functions. For this purpose, we will label the proteins with different fluorescent tags and watch how different proteins move and assemble with others in the cell. We will also purify the protein complexes from cells and examine the protein composition of these complexes using biochemical techniques. Furthermore, we will also learn how the lipid molecules play roles in the formation and function of the thylakoid membrane, by extracting the lipids from the thylakoid membranes isolated from different development stages and identifying the lipid composition and content. If we can gain advanced understanding as to how thylakoid membranes are assembled we will be in a better position to modify the thylakoid membrane function, for example, to produce hydrogen from solar energy. In the long-term we may even be able to induce the production of similar membrane systems in different kinds of bacteria, giving us a new tool for the generation of microbial 'cell factories'.

Cyanobacteria are the oldest oxygenic phototrophs on Earth. They convert solar energy and CO2 into bioenergy and oxygen that is indispensable for sustaining aerobic life in the atmosphere. The cyanobacterial thylakoid membrane represents a system that performs both oxygenic photosynthesis and respiration. The dynamic formation and architecture of cyanobacterial thylakoid membranes in response to environmental changes are of fundamental importance to the metabolic robustness and plasticity of cyanobacteria. Current understanding of the biogenesis and regulation of thylakoid membranes is still insufficient. This project is aimed at unravelling the molecular basis governing the biosynthesis, organisation and dynamics of thylakoid membranes in the model cyanobacterium Synechococcus elongatus PCC7942 using multidisciplinary approaches, and elucidating the coordination between the dynamic organisation of thylakoid membranes and the regulation of bioenergetic electron flow. First, we will use fluorescent tagging combined with live-cell confocal microscopy, high-resolution AFM, Cryo-EM and proteomics to study the sequential synthesis, distribution and dynamics of photosynthetic/respiratory complexes in thylakoid membranes during thylakoid biogenesis. Secondly, we will determine the interactions of electron transport complexes and the dynamic assembly of photosynthetic/respiratory supercomplexes during thylakoid membrane biosynthesis and regulation using confocal/TIRF microscopy and quantitative proteomics. Moreover, we will explore the contributions of lipids to the thylakoid biogenesis and bioenergetic supercomplex formation using lipidome analysis, in specific the timing of lipid biosynthesis, the lipid composition and stoichiometry. This project will provide insights into the biogenesis and regulation of cyanobacterial thylakoid membranes and will empower synthetic biology tools to build artificial photosynthetic membranes and machinery for bioenergy development.

This project represents fundamental science addressing the molecular basis underlying the biosynthesis and organisation of cyanobacterial thylakoid membranes. Thus, the primary impact is to the broad scientific community. However, the live-cell microscopy and nanotechnology imaging and image analysis approaches developed will provide a resource that will be open to outside users and will be of interest to biotech industries. Moreover, advanced understanding of in vivo photosynthetic membrane biosynthesis will underpin the bottom-up design and engineering of artificial photosynthetic machinery using synthetic biology approaches for biofuel production.

- Academic and commercial communities of membrane biochemistry, microbiology, photosynthesis: We envisage considerable potential benefits of the fundamental outputs for those who work on cyanobacterial bioenergetics and membrane biogenesis. Knowledge derived from this project will be conceivably informative to the scientists who wish to engineer photosynthetic machinery for bioenergy development.

- Synthetic biology: We foresee that comprehensive knowledge of photosynthetic membrane biosynthesis will stimulate the design of synthetic biological strategies to construct bioenergetic modules in other organisms. The synthetic biology foundry - Liverpool GeneMill (BB/M00094X/1) has expressed interest in this project and will first conduct the engineering of photosynthetic complexes.

- Microscope manufacturers: The developed imaging technologies, including live-cell and time-lapse fluorescence imaging and high-resolution AFM imaging, can be widely used to image many biological samples. The PI currently has collaborative projects with JPK Instrument and Bruker Nano Surfaces Division, which will profit from the AFM imaging approaches developed in this work. The technical development will greatly enhance the imaging capacity of Liverpool Centre for Cell Imaging (CCI) and benefit other users. CCI has a long-term working relationship with Zeiss Microscope, which will facilitate us to build industrial links and define the potential applications of the technical developments in this project.

- Biotechnology industries: This project has potential societal impacts on renewable energy and energy security. The PI has established the contact with Dr. David Parker, the Platform Leader Bio-Fuels Group at Shell Global Solutions, for exchanging the idea of engineering microorganisms for bioenergy production. Likewise, Unilever has expressed strong interest in cyanobacterial metabolisms and using cyanobacteria as cell factories to produce biofuels, high-value chemicals and pharmaceuticals.

- Scientific community: We expect to provide extensive training to the PDRA/technician in the multidisciplinary skills during this work. We will ensure the academic impact of this work through timely seminars and publications. We will present the outputs at workshops and international meetings. This programme will promote the national and international collaborations by sharing data and expertise.

- Outreach activities: The PI has collaborated with Nuffield Foundation to host summer replacement students. During this project, he will continue to offer placements for Nuffield Bursaries students with related projects. The PDRAs will be involved in the Liverpool postdoc communities to deliver the scientific outputs. We will work with the Liverpool World Museum and local schools to develop exhibits showcasing this research programme.

- Intellectual property: The methodological and analytical approaches developed in this project may lead to the intellectual property. We will liaise with Liverpool Business Gateway to ensure the timely protection of intellectual property in this project.