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Title: Investigating the functional and structural modulation of carboxysomes in Synechococcus elongatus PCC7942
Author: Sun, Yaqi
ISNI:       0000 0004 7656 9877
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2019
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Organelle formation and compartmentalisation within eukaryotic and prokaryotic cells provide the structural foundation for modulation of metabolic reactions in space and time. Bacterial microcompartments (BMCs) are self-assembling organelles widespread among bacterial phyla. By physically sequestering specific enzymes key for metabolic processes from the cytosol, these organelles play essential roles in carbon fixation, microbial ecology and pathogenesis. Carboxysomes serve as the key CO2-fixing machinery in all cyanobacteria and some chemoautotrophs. The β-carboxysomes in the cyanobacterium Synechococcus elongatus PCC7942 (Syn7942) have been extensively characterised as the model carboxysomes. In Chapter 1, general research background centralised on carboxysomes was summarised. In Chapter 2, material and methods adopted in this thesis were documented. From Chapter 3 to Chapter 5, I addressed specific topics on functional and structural modulation of carboxysomes in Syn7942. In Chapter 6, I concluded the findings in previous chapters and showed the perspectives following this project. In Chapter 3, we used fluorescence tagging and live-cell confocal fluorescence imaging to explore the biosynthesis and subcellular localisation of β-carboxysomes within Syn7942 cell in response to light variation. We demonstrated that β-carboxysome synthesis on cellular level is re-modulated in response to increasing light intensity, thereby enhancing the carbon fixation activity of the cell. Inhibition of photosynthetic electron flow impairs the accumulation of carboxysomes, indicating close coordination between β-carboxysome biogenesis and photosynthetic electron transport. Likewise, the spatial organisation of carboxysomes in the cell correlates with the redox state of photosynthetic electron transport chain. In Chapter 4, we used live-cell single-molecule fluorescence microscopy, coupled with confocal and electron microscopy, to decipher the absolute protein stoichiometry and organisational variability of single β-carboxysomes in Syn7942 cell. I find that the protein stoichiometry, diameter, cellular localisation and mobility pattern of carboxysomes in cells depend sensitively on the microenvironmental levels of CO2 and light intensity during cell growth, revealing cellular strategies of dynamic regulation. In Chapter 5, I documented the biosynthesis and organisation of carboxysomes under diurnal dark-light cycles compare to constant light in Syn7942. We found the reoccurring carboxysome distribution changes and rhythmic carbon fixation capacities in diurnal condition. Also, we documented the localisation, enzymatic activity as well as the quantity of carboxysomes in circadian null background to provide preliminary evidence of circadian control in carboxysome biogenesis. The findings in this thesis provide essential knowledge for us to modulate the β-carboxysome biosynthesis and function in cyanobacteria. Furthermore, improving our understanding of carboxysome assembly principles will aid rational design of functional metabolic factories in heterologous organisms for metabolic engineering using synthetic biology.
Supervisor: Liu, Luning Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral