Organisation, dynamics and biogenesis of a photosynthetic membrane

Lead Research Organisation: University of Liverpool
Department Name: Institute of Integrative Biology

Abstract

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'.

Technical Summary

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.

Planned Impact

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.
 
Description We have established a method to control the formation of photosynthetic machinery in bacteria. A few constructs have been generated for the study of photosynthetic membrane biogenesis and regulation, as well as membrane protein location and dynamics. A review articles has been published to summarise the current progress of photosynthetic protein structure, function and industrial applications (Biotechnology Advances, 2019, 37: 340-353).
Exploitation Route too early to say
Sectors Agriculture, Food and Drink,Energy,Manufacturing, including Industrial Biotechology

 
Description Organised a Halloween Science Outreach Day on 31 October 2018 to 200 school students, to disseminate the basis of protein self-assembly and biological shapes.
First Year Of Impact 2018
Sector Agriculture, Food and Drink,Energy,Manufacturing, including Industrial Biotechology
Impact Types Societal

 
Description A Dragonfly multimodal fast imaging platform with SRRF-stream (Super-Resolution Radial Fluctuation) in the Liverpool Centre for Cell Imaging (CCI)
Amount £290,246 (GBP)
Funding ID BB/R01390X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2018 
End 01/2019
 
Description Elucidating the organisation, activity and regulation of cyanobacterial bicarbonate transporters for engineering CO2 accumulation
Amount £330,756 (GBP)
Funding ID URF\R\180030 
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 10/2018 
End 09/2021
 
Description Engineering a new nanobioreactor for hydrogen production
Amount £10,000 (GBP)
Funding ID 201703780114 
Organisation British Council 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2018 
End 04/2019
 
Description Organisation, dynamics and biogenesis of a photosynthetic membrane
Amount £481,703 (GBP)
Funding ID BB/R003890/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2018 
End 01/2021
 
Description Unlocking the molecular stoichiometry of functional CO2-fixing organelles for synthetic engineering
Amount £200,000 (GBP)
Funding ID RGF\EA\181061 
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 05/2019 
End 04/2021
 
Title AFM 
Description atomic force microscopy imaging on biological samples 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact We have applied high-resolution AFM imaging on many biological samples. Recently we have established a hybrid AFM/confocal/TIRF microscopy for studying cell dynamics. AFM imaging on amyloid morphology provides further motivation to investigate the role of oxidative stress in AMA pathogenicity. The study has led to a paper published: Davies HA, Phelan MM, Wilkinson MC, Migrino RQ, Truran S, Franco DA, Liu LN, Longmore CJ, Madine J. Oxidative stress alters morphology and toxicity of aortic medial amyloid. Biophys J, 2015, 109(11): 2363-2370. AFM imaging on exosome structure has revealed that CLL cells secrete exosomes that alter the transcriptome and behaviour of recipient cells. Such communication with microenvironment is likely to have an important role in CLL disease biology. The study has led to a paper published: Farahani M, Rubbi C, Liu LN, Slupsky JR, Kalakonda N. CLL exosomes modulate the transcriptome and behaviour of recipient stromal cells and are selectively enriched in miR-202-3p. PLoS ONE, 2015, 10(10): e0141429. 
 
Description BMC - Bond 
Organisation Agency for Science, Technology and Research (A*STAR)
Department Bioinformatics institute (BII)
Country Singapore 
Sector Academic/University 
PI Contribution provide structural information of BMC proteins and metabolite molecules and data analysis.
Collaborator Contribution provide molecular dynamics simulations methods to determine molecule transport, structural changes and protein-protein interactions.
Impact developed a joint PhD studentship (2017-2021); submitted an EPSRC grant proposal in November 2017 based on the preliminary collaborative results.
Start Year 2016
 
Description BMC - Chiu 
Organisation Baylor College of Medicine
Country United States 
Sector Hospitals 
PI Contribution providing samples and constructs for microscopy imaging
Collaborator Contribution high resolution microscopy imaging
Impact this collaboration is multi-disciplinary. We have expertise in protein purification and mutagenesis. The collaborator is expert in microscopy imaging and data analysis. The preliminary results have helped to secure a BBSRC grant (BB/R003890/1)
Start Year 2015
 
Description BMC - Nixon 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution making cyanobacterial constructs and thylakoid membrane samples
Collaborator Contribution plant engineering, biochemical investigation of thylakoid membrane composition.
Impact the preliminary collaborative work has helped to secure a BBSRC grant (BB/R003890/1)
Start Year 2015
 
Description Cryo-EM - Shirouzu 
Organisation RIKEN
Country Japan 
Sector Private 
PI Contribution this is a PhD studentship funded by the University of Liverpool and Riken. The student will stay two years in each institute. My research group will provide cell culture and biochemical purification training for the student.
Collaborator Contribution The Riken group will provide training in cryoEM sample preparation and imaging and data analysis.
Impact The collaboration is multidisciplinary, based on the two groups expertise. The collaboration will focus on genetic modification, macromolecular complex isolation and cryo-EM imaging using molecular biology, biochemistry and structural biology approaches
Start Year 2017
 
Description TM - Mullineaux 
Organisation Queen Mary University of London
Department School of Biological and Chemical Science QMUL
Country United Kingdom 
Sector Academic/University 
PI Contribution generating cyanobacterial mutants
Collaborator Contribution electron transport activity and optical spectrum analysis
Impact The collaboration has led to a paper published (Casella S, Huang F, Mason D, Zhao GY, John GN, Mullineaux CW, Liu LN*. Dissecting the native architecture and dynamics of cyanobacterial photosynthetic machinery. Molecular Plant, 2017, 10: 1434-1448. DOI:http://dx.doi.org/10.1016/j.molp.2017.09.019). The preliminary collaborative work has helped to secure a BBSRC grant (BB/R003890/1).
Start Year 2015
 
Description TM - Schneider 
Organisation Johannes Gutenberg University of Mainz
Department Institute of Pharmacy and Biochemistry
PI Contribution conduct microscopic imaging
Collaborator Contribution providing mutants and procedures for imaging analysis
Impact The preliminary collaborative work has helped to secure a BBSRC grant (BB/R003890/1)
Start Year 2017
 
Description Halloween Science event - Outreach activity with school children 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Outreach activity entitled "Spooky Science" for Halloween. 200 primary and secondary school children attended activities at the Institute of Integrative Biology. The Liu group engaged in "Magnetic Madness" activities to exhibit the principles of protein self-assembly.
Year(s) Of Engagement Activity 2018
URL https://blogandlog.wordpress.com/2019/01/25/halloween-science-at-the-institute-of-integrative-biolog...
 
Description showcase at Meet the Scientists in Liverpool Word Museum January 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The event organised at Liverpool Word Museum by the University and Faculty was aimed at a family audience, with fun activities. We participated in the demonstration on cutting-edge research on bacterial organelles and biotechnology and nanotechnology to address the global impact of Liverpool's health and life sciences research.
Year(s) Of Engagement Activity 2018
URL http://www.liverpoolmuseums.org.uk/wml/events/displayevent.aspx?EventID=35294