Biosynthesis, Regulation and Engineering of Bacterial Carbon Fixation Machinery

The single-cell cyanobacteria are among the most abundant organisms on earth. They created and help to sustain our atmosphere, and account for an estimated 20-30 % of current global carbon fixation. To enhance carbon fixation, cyanobacteria develop small compartments, called carboxysomes, to absorb carbon dioxide and transform it to chemical energy by the process named photosynthesis. These highly efficient machines are structurally defined by an outer protein-based shell and internal highly concentrated CO2-fixing enzymes. The shell is composed of many distinct proteins, and serves as a selective "barrier" for the passage of specific molecules into and out of the compartments.

At present, there are great concerns over global food and energy security. How can we improve the food supply to keep pace with the world population? How can we develop environmentally sustainable solutions for food and energy production? Producing and engineering of synthetic carboxysomes and introducing them into other organisms, particularly plants, has significant potential for improving photosynthesis, carbon sequestration and crop yield. As the cyanobacteria is evolutionarily close to the plant chloroplast, lessons learned from the cyanobacteria will be very informative to plant sciences and engineering. Recent developments in synthetic biology have opened the door to generating artificial biological machines by providing the necessary strategies and approaches. However, producing functional carboxysomes in other organisms requires comprehensive knowledge about their development and physiological regulation in their natural hosts, the cyanobacteria.

The aims of this project are to elucidate comprehensively how cyanobacterial cells create these specialised compartments, how their activities are dynamically regulated within the cells in response to the changing environment, and how these machines function together with other cellular activities in the entire metabolic network within cells. In the first part of this research project, we will use a special optical microscopy to watch the development and distribution of carboxysomes in living cells, and study how these organelles are regulated within the cells grown under different environmental conditions. The second section will characterise how these organelles interact and function together with other cellular components to achieve their metabolic performance. Next we will find out how multiple proteins are organised in order to build the organelle shape. We will develop a computer programme to build a model of the compartment and simulate the protein dynamics and passage of molecules in and out of the compartment. Advanced understanding of the compartment structure, function and regulation derived from the three sections is important for genetic engineering of novel biological machines with appropriate functionality. In the last section, we will use the knowledge learned from the cyanobacterial cells to synthesise artificial biological machines with carbon fixation activities.

This work represents a model for studying the development of complex biological machines within cells. It will teach us about how thousands of proteins can assemble together by themselves to form a functional entity within cells, and what regulatory strategies are developed by the cells to lead the development and function of these machines. In translational terms, this work will provide an instructive example for the design and engineering of novel biological "factories" for specific cellular activities and physiology. If we can conduct genetic engineering to enable higher plants to develop synthetic cyanobacterial carbon-fixing machines, it will significantly enhance food and energy production.

To promote cell metabolism, many bacteria express proteinaceous microcompartments to encapsulate enzymes in a defined cytoplasmic environment. The first bacterial microcompartments discovered were carboxysomes (CBs). Their remarkable capacity of enhancing carbon fixation is ensured by specific protein assembly, and is regulated by the carbon flux pathways in the cell. Current understanding of the physiological regulation of CBs is still sparse. This project aims to survey extensively CB assembly and regulation by using a combination of molecular genetics, biochemistry, confocal microscopy, state-of-the-art AFM and EM imaging, as well as proteomics and computational modelling, and apply the knowledge to improve the synthetic strategies for engineering of CBs in other organisms. We will use live-cell fluorescence imaging and an imaging analysis method we have recently established to determine the spatial distribution of CBs in vivo, which are correlated with the carbon fixation. The developed imaging techniques will be also used to monitor the dynamic subcellular organisation of CBs under physiological regulation, and functional integration of CBs within the cellular metabolism. We will further explore the composition and stoichiometry of building blocks during CB biosynthesis using quantitative proteomic analysis. High-resolution AFM imaging will be used to determine the protein organisation in the shell. Based on advanced understanding of the shell structure, we will utilise computational modelling and molecular dynamics simulation to evaluate the molecular basis leading the assembly and permeability of the shell. In the last section, we will use synthetic biology approaches and recently developed systems for heterologous expression to produce functional synthetic CBs. This project will provide insights into the assembly and regulation of CBs, and will underpin the synthetic engineering of CBs in plants for supercharging photosynthesis and carbon fixation.

This project represents fundamental science addressing the molecular basis of the assembly and regulation of carboxysomes (CBs). Thus, the primary impact is to the broad scientific community. However, the live-cell microscope 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 biotechnology industries. In the long term, the significant potentials of CB biosynthesis in promoting cellular metabolism will attract the interests from synthetic biologists. It will help to develop environmentally sustainable solutions for food and energy production.

- Academic and commercial communities of protein biochemistry, microbiology, carbon fixation and plant sciences: We envisage considerable potential benefits of the fundamental outputs for those who work on cyanobacterial metabolism and photosynthetic carbon fixation. It will also contribute to the wider microbiology and protein science communities. Knowledge derived from this project will be conceivably informative to the plant scientists who wish to engineer chloroplasts for increasing crop productivity.

- Synthetic biology: We foresee the creation of new synthetic biological strategies. We have started to work with New England Biolabs for improving gene assembly strategies. Liverpool GeneMill has expressed strong interest to this project and will support the design and DNA synthesis of CB operons.

- Microscope manufacturers: The developed imaging technologies, including live-cell and time-lapse fluorescence imaging, high-resolution AFM imaging and affinity mapping, 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 will bring deep understanding of the self-assembling materials. It has potential societal impacts on renewable energy and food security issues. We have started the collaboration with Eppendorf-New Brunswick for optimising the growth of cyanobacteria using bioreactors. 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. Given the conceivable possibility of engineering CBs in plants to enhance photosynthesis, the PI will contact plant biotechnology industries for the feasibility of the translational work.

- 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 PDRA will be involved in the Liverpool PhD and postdoc communities to deliver the scientific outputs. We will work with the Liverpool World Museum to develop exhibits showcasing this work.

- 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.