Electrochemical and genetic tools for analysing and engineering bioenergetic processes in cyanobacteria

Oxygenic photosynthesis provides energy for the majority of Earth’s ecosystems. It is catalysed by photosynthetic electron transport chains: a collection of free and proteinbound redox cofactors found within the specialised thylakoid membranes of photosynthetic organisms. Cyanobacteria, to our knowledge, are the first organisms to have evolved oxygenic photosynthesis, and contribute approximately 25% of the Earth’s primary photosynthetic productivity. Cyanobacteria are also essential for maintaining important biogeochemical processes, such as the nitrogen cycle. Additionally, researchers have demonstrated how the electron transport chains of cyanobacteria can be ‘rewired’ for the sustainable production of electricity, fuels, pharmaceuticals, plastics, and high-value chemicals. However, research on cyanobacteria has been limited by the complex nature of their thylakoid membrane electron transport, which differs from that of plants and algae, not least because it includes a respiratory electron transport chain. Furthermore, the available tools for analysing and engineering cyanobacterial electron transport are scarce compared to those for model plant and green algal species. This thesis addresses this research challenge by developing a range of electrochemical and synthetic biology tools to analyse and engineer cyanobacterial electron transport. Firstly, a reproducible method of extracting cyanobacterial thylakoid membranes and wiring them to highly-structured electrodes was developed. By conducting electrochemistry experiments, electron transport pathways within these modified electrodes were determined, thereby establishing the technique as an analytical platform for studying cyanobacterial thylakoid membrane electron transport. The technique was used to answer biological questions inaccessible to other techniques, such as measuring plastoquinone reduction in different conditions. The technique was also utilised to engineer bio-photoelectrochemical systems for solar-powered electricity generation. An additional electrochemical platform was created and used alongside a series of analytical chemistry methods to study the role of outer membrane vesicles in cyanobacterial iron transport. The electron transport chains of cyanobacteria utilise numerous iron-containing redox cofactors, making iron availability essential for their assembly, function, and maintenance. This research revealed that outer membrane vesicles selectively uptake Fe³⁺ ions, answering a longstanding question on cyanobacterial physiology. Finally, in addition to these electrochemical platforms, a series of synthetic biology tools were created to aid the genetic manipulation of electron transport in cyanobacteria. These include improvements to an existing DNA assembly technique, and the creation of plasmids, selectable and counter-selectable markers, and CRISPR systems for use in different cyanobacterial species. This research provides crucial tools for advancing the understanding and engineering of bioenergetic processes in cyanobacteria.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Lawrence, Joshua

Advisors dc:contributor.advisor

• Howe, Christopher
• Zhang, Jenny

• Bioenergetics
• Electrochemistry
• Electron Transport
• Photosynthesis
• Synthetic Biology

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/faff261c-b051-495a-8be5-71b602c70fc9/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

Author Identifier

0000-0002-9250-8690

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/362880