Repurposing Bacterial CO2-fixing Organelles Using Synthetic Engineering



Fang, Y
(2018) Repurposing Bacterial CO2-fixing Organelles Using Synthetic Engineering. PhD thesis, University of Liverpool.

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Abstract

Bacterial microcompartments (BMCs) are protein-based organelles widespread among bacterial phyla and provide a means for compartmentalization of specific metabolic pathways. They sequester catalytic enzymes from the cytoplasm, using an icosahedral proteinaceous shell with selective permeability to metabolic molecules and substrates, to enhance metabolic efficiency. Carboxysomes were the first BMCs discovered, and their unprecedented capacity of CO2 fixation allows cyanobacteria to make a significant contribution to global carbon fixation. The carboxysome was classified as α- and β-carboxysome according to the Rubisco form. The shell of the carboxysome ensures preferable transport of bicarbonate and CO2 assimilation, making the carboxysome as an ideal nanobioreactor. Assembly studies and functional characterization of synthetic carboxysomes are important for understanding the assembly of carboxysomes in native and heterologous hosts. Moreover, there is an increasing interest in utilizing synthetic biology to construct synthetic carboxysomes or carboxysome shells in new hosts, i.e., higher plants, to enhance carbon fixation and productivity. In Chapter 1, I summarized the research background and aims of the PhD project. In Chapter 3, I described the construction of a synthetic operon of β-carboxysomes from the cyanobacterium Synechococcus elongatus PCC7942 and the synthetic production of functional β-carboxysome structures in Escherichia coli. The structure, assembly, activity and interchangeability of synthetic β-carboxysomes were assessed using confocal, electron and atomic force microscopy, proteomics, immunoblot analysis, and enzymatic assays. To our knowledge, this is the first production of functional β-carboxysomes in heterologous organisms. In Chapter 4, the α-carboxysomes from the chemoautotroph Halothiobacillus neapolitanus were heterogeneous synthesized in E. coli. To improve the activity, the Rubisco activase CbbQO complex was introduced to the α-carboxysome expression system. We found that the addition of Rubisco activase CbbQ can improve the function of the synthetic carboxysomes and didn’t affect the assembly of synthetic carboxysomes. In Chapter 5, the synthetic α-carboxysome shell was constructed and characterized. In Chapter 6, I summarized the results and prospect the future perspectives for my projects. This study strengthens synthetic biology toolbox for generating not only functional carboxysome structures but also BMC-like organelles with tunable activities and BMC shell. It will inform the improvement of carboxysome engineering and the construction of functional CO2-fixing modules for plant engineering as well as new nanoreactors.

Item Type: Thesis (PhD)
Divisions: Fac of Health & Life Sciences > Institute of Integrative Biology
Depositing User: Symplectic Admin
Date Deposited: 21 Nov 2018 15:32
Last Modified: 03 Mar 2021 16:39
DOI: 10.17638/03025860
Supervisors:
  • Liu, Luning
URI: https://livrepository.liverpool.ac.uk/id/eprint/3025860