The development of human society, which is highly dependent on fossil fuels, is now facing a range of global issues, such as rising energy prices, energy security and climate changes. To successfully tackle the resultant issues, the energy transition from fossil fuels to renewable energy sources, such as solar energy, tide energy, hydroelectric power, geothermal heat and biofuels, is under way. Biodiesel, as an important type of biofuels, has been increasingly produced from vegetable oil or used cooking oil, especially in Europe. Nevertheless, considering the high production cost of biodiesel, there is still much to be done to improve the economics of biodiesel industry. Utilisation of crude glycerol, the main by-product of the biodiesel industry, to produce value-added products appears to be a promising solution. Poly(3-hydroxybutyric acid) (PHB), a biodegradable plastic, can be converted from glycerol by Cupriavidus necator DSM 545 under unbalanced growth conditions, such as nitrogen limitation. One way to enhance the batch production of PHB is to genetically engineer the strain of C. necator, which requires insights of the dynamic impact of extracellular environment on cell phenotypes. Hence in this thesis, we aim to perform metabolic modelling based on experimental measurements to gain a better understanding of the behaviour of the metabolic network of Cupriavidus necator DSM 545 and identify potential bottlenecks of the process. Initially, C. necator DSM 545 is a strain that hardly grows on glycerol, so in a preliminary study, we investigate the process by which the strain was adapted to consume glycerol through serial subcultivation. It is found that the adaptation can be achieved within 15 cell generations over three passages in basal mineral medium, and the acquired phenotype is sufficiently stable upon further passage. The study of metabolism started with the reconstruction of the cell's metabolic network, followed by a thermodynamic analysis to check the feasibility and reversibility of all the biochemical reactions included. Then the static flux balance analysis was extended and applied to analyse the shift of metabolic states during the microbial fermentation in different batch conditions. The resulting patterns of flux distribution reveal the TCA cycle to be the major competitor for PHB synthesis at the ACCoA node. Cells have the potential to enter an efficient PHB-production phase that features minimal TCA/PHB flux split ratio, and the length of the phase can be manipulated by aeration. Although low aeration rate favours optimal flux split ratio, such condition that limits respiration also limits nutrient uptake, leading to low PHB productivity overall. To identify the actual limiting factors of PHB synthesis in the system, we further performed metabolic control analysis based on the calculated flux distributions. The analysis demonstrated how the distribution of the metabolic control can vary widely, depending on the aeration conditions used and the flux split ratios. Glycerolipid pathway, glycolysis, PHB metabolism, as well as the electron transport chain are revealed to be potential engineering targets as they contribute to the great majority of the positive control of PHB flux.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:764611 |
| Date | January 2018 |
| Creators | Sun, Chenhao |
| Contributors | Webb, Colin ; Theodoropoulos, Konstantinos |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/dynamic-metabolic-studies-of-c-necator-producing-phb-from-glycerol(4b627cd4-c6c9-4f2c-9a2a-2e3da8d4f68a).html |
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