Harnessing the metabolic potential of thermophilic bacteria: Systems metabolic engineering tools for the production of volatile biochemicals

To remedy the escalating environmental damage resulting from global warming, sustainable alternatives to energy, materials, and chemicals are needed. To replace petrochemical processes, the catalytic power of cells can be used for microbial bioproduction as a sustainable alternative to assimilate carbon into valuable compounds. Current industrial bioproduction processes suffer from constant economic pressure, resulting in the oscillating introduction and closing of processing plants in part due to fluctuating oil and energy prices. To facilitate the implementation of industrial fermentation technologies, novel production modalities with robust and optimized performance are needed, in order to reduce their dependency on petrochemical prices. Thermophilic processing conditions carry significant advantages and have the potential to facilitate economically improved processes, even for products where profit margins are low. Although new gene editing tools have given improved access to thermophilic genomes, improved understanding and control of metabolism is required to exploit the potential of thermophilic fermentation further. This Ph.D. thesis focuses on the thermophile Parageobacillus thermoglucosidasius to create and deploy novel tools for the advancement of thermophiles as successful cell factories.

To improve the engineering of non-model hosts, this thesis provides a broad host range expression system to facilitate the screening of heterologous constructs across mesophilic and thermophilic hosts. Further modification of this system provides an industrially relevant genetic control element, where it is possible to control the expression of gene targets of interest through temperature shifting. To complement new experimental tools, the predictive and analytical capacity of computational tools far surpasses that of our human capacity. To enable this, a genome-scale metabolic model was built and used to gain new insights into the anaerobic metabolism of P. thermoglucosidasius. Combining the genome-scale metabolic model with process modelling to probe the landscape of process setup was used to identify improved conditions to facilitate CO2 fixation. This information can be valuable to further tailor the process and improve flux rewiring by keeping the final application in mind. To demonstrate the use of P. thermoglucosidasius as a suited production host, its metabolism has been modified for the production of 2-butanone, a volatile bulk chemical with significant environmental impact. Complementing the engineering efforts with a novel proposed high throughput colorimetric assay for the detection of ketones and aldehydes can further improve the iteration of the design-build-test-learn cycle and further the performance of these strains as suitable cell factories. With time and further technological advancement, thermophilic cell factories have the potential to contribute to transforming the way we produce our materials, and in doing so push society to the much-needed sustainable revolution.