Metabolic engineering of Clostridium saccharoperbutylacetonicum for improved solvent production

In order to avert irreversible damage to the global climate, the global community has committed to reaching net zero carbon emission in the coming years. To meet this ambitious target, substantial changes will be needed. To minimise the disruption to people’s lives, there is a need for renewable technologies which are compatible with existing infrastructure, such as biofuels and drop in chemical compounds. A compound which can fulfil both roles is n-butanol. Clostridium are natural butanol producers but have fallen out of use as they have been unable to compete with fossil fuel-based production methods. The aim of this thesis was to improve the production of butanol in an asporogenic strain of Clostridium saccharoperbutylacetonicum N1-4. A systems scale approach was taken to improve butanol production. Combined analysis of Clostridium metabolism using flux balance analysis and 13C-metabolic flux analysis was used to guide metabolic engineering strategies, with the aim of increasing butanol production in asporogenic C. saccharoperbutylacetonicum N1-4 spo0AI261T. Flux balance analysis was used to gain an understanding of Clostridium metabolism and to explore manipulations that could lead to increased butanol production in wild type C. saccharoperbutylacetonicum N1-4. Key features of in silico engineered strains were compared to experimental data and identified an increase in NADH generation and key butanol synthesising genes as targets for increasing butanol production. A second round of flux balance analysis identified further manipulations relevant to C. saccharoperbutylacetonicum N1-4 spo0AI261T, mainly that the rate of glucose uptake appeared to be limiting butanol production. Simulations of strains with increased glucose uptake and butanol production suggested that ATP consuming enzymes would have to be engineered into the asporogenic strain to balance ATP. Additional investigation was performed using 13C-metabolic flux analysis, which was able to resolve intracellular fluxes of asporogenic C. saccharoperbutylacetonicum N1-4. It also identified a feature of C. saccharoperbutylacetonicum N1-4 spo0AI261T metabolism that was unexpected, that ATP was in excess to biomass synthesis requirements, resulting in a futile cycle. The results from this flux analysis confirmed the rationale of the flux balance analysis guided strategies. In the final chapter, the developed strategies were incorporated into the asporogenic strain of C. saccharoperbutylacetonicum N1-4. These mutant strains were analysed in fermentations. While none of the strains produced more butanol than the parent strain, this work incorporated several novel approaches to increase butanol production in Clostridium and will serve as a starting point for future metabolic engineering work