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Hendriks, Rudolf Martinus Antonius (2023) The development of efficient hemi-autotrophic carbon fixation in Escherichia Coli. PhD thesis, University of Nottingham.

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Abstract

Carbon fixation is a process vital to any life and as by far its most prevalent variant, the Calvin Benson Bassham (CBB) cycle is vital to virtually all known terrestrial life. Mostly occurring in plants, it uses light energy to sequester atmospheric carbon dioxide (CO2) and convert it into biomass. As the most inefficient natural carboxylation process and source of most biomass documented, even a small increase of its performance could have vast downstream effects. Such a development could assimilate the abundantly available atmospheric CO2 while generating minimal amounts of waste for any biosynthesized product. The Escherichia coli bacterium was previously shown to functionally express the CBB cycle upon the addition of phosphoribulokinase (PRK) and ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Further knock-outs severed its energetic metabolism from the carbon metabolism resulted CO2-dependent biomass accumulation. This carbon fixation is driven by the energy independently generated in the TCA cycle from a supply of pyruvate. This unique, split metabolism was dubbed hemi-autotrophy.

The hemi-autotrophic strain of E. coli serves as a model organism for the CBB cycle, but lacking any of the difficulties of light-dependent or multi-cellular organisms. A pyrophosphate-dependent 6-phosphofructokinase (PFP) originating from Methylococcus capsulatus Bath was characterised as catalyzing three reactions of the typical CBB cycle. Where PRK completes its catalysis with a dependency on energy-carrier adenosine triphosphate (ATP), PFP was shown to complete this reaction with the less energetic pyrophosphate (PPi) that is partially generated in its FBPase and SBPase-equivalent reactions. Successful integration of this synthetic CBB cycle would conserve

33% of all ATP expended in the native CBB cycle.

The hemi-autrophic E. coli strain’s unique culturing requirements proved challenging but methods with increased dependability were established. Transformations without the relief of these conditions remain elusive, requiring pre-cultures in rich media and heterotrophic metabolism. The consecutive sub-culturing of the strain to increase its hampered growth characteristics resulted in mild improvements. Despite observing modest culturing characteristic and a relatively high chromosomal mutation rate, the strain did not demonstrated an increase in transformation efficiency.

The attempted replacements of the plasmid-encoded prkA by pfp did not result in hemi-autotrophic growth in any of its constructs, despite modulation of their expression. Troubled by high mutation rates, it remains unknown whether the expression range of the significantly less efficient PFP was sufficient or if the cytoplasmic availability of PPi remained below its functionally required concentration. The putative H+-pyrophosphatase pump (HPP), natively expressed as the second gene in the pfp-hpp operon, remains uncharacterised but its co-expression did not manage to compensate for this deficiency either. Though native fbp was successfully knocked-out, the essential inorganic pyrophosphatase gene of E. coli remains.

Thorough analysis of the components in the CBB system led to several design improvements and pathway modelling indicates the proposed synthetic CBB cycle is a viable alternative to its natural variant. Thermodynamic feasibility of the synthetic pathway was confirmed and kinetic analysis also predicted it to perform at reduced efficiencies while still indicating culture viability. Growth rates approximating those of the hemi-autotrophic strain were produced in a kinetic model of the central carbon metabolism while incorporating minimal assumptions. Modifying it to support the synthetic CBB cycle suggested its viability at a nominal reduction of growth, while suggesting further directions of research for the system.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Soucaille, Philippe Kovacs, Katalin
Keywords:	Calvin cycle, CBB cycle, hemi-autotroph, Escherichia coli, metabolic engineering, genetic regulation, synthetic biology, carbon dioxide, CO2, kinetic modelling, thermodynamic analysis
Subjects:	Q Science > QR Microbiology > QR 75 Bacteria. Cyanobacteria
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	72927
Depositing User:	Hendriks, Rudolf
Date Deposited:	31 Jul 2023 04:40
Last Modified:	31 Jul 2023 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/72927

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