@phdthesis{Dronsella2024,
  author    = {Dronsella, Beau B.},
  title     = {Overcoming natural biomass limitations in gram-negative bacteria through synthetic carbon fixation},
  doi       = {10.25932/publishup-64627},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-646273},
  school      = {Universit{\"a}t Potsdam},
  pages     = {174},
  year      = {2024},
  abstract  = {The carbon demands of an ever-increasing human population and the concomitant rise in net carbon emissions requires CO2 sequestering approaches for production of carbon-containing molecules. Microbial production of carbon-containing products from plant-based sugars could replace current fossil-based production. However, this form of sugar-based microbial production directly competes with human food supply and natural ecosystems. Instead, one-carbon feedstocks derived from CO2 and renewable energy were proposed as an alternative. The one carbon molecule formate is a stable, readily soluble and safe-to-store energetic mediator that can be electrochemically generated from CO2 and (excess off-peak) renewable electricity. Formate-based microbial production could represent a promising approach for a circular carbon economy. However, easy-to-engineer and efficient formate-utilizing microbes are lacking. Multiple synthetic metabolic pathways were designed for better-than-nature carbon fixation. Among them, the reductive glycine pathway was proposed as the most efficient pathway for aerobic formate assimilation. While some of these pathways have been successfully engineered in microbial hosts, these synthetic strains did so far not exceed the performance of natural strains. In this work, I engineered and optimized two different synthetic formate assimilation pathways in gram-negative bacteria to exceed the limits of a natural carbon fixation pathway, the Calvin cycle. The first chapter solidified Cupriavidus necator as a promising formatotrophic host to produce value-added chemicals. The formate tolerance of C. necator was assessed and a production pathway for crotonate established in a modularized fashion. Last, bioprocess optimization was leveraged to produce crotonate from formate at a titer of 148 mg/L. In the second chapter, I chromosomally integrated and optimized the synthetic reductive glycine pathway in C. necator using a transposon-mediated selection approach. The insertion methodology allowed selection for condition-specific tailored pathway expression as improved pathway performance led to better growth. I then showed my engineered strains to exceed the biomass yields of the Calvin cycle utilizing wildtype C. necator on formate. This demonstrated for the first time the superiority of a synthetic formate assimilation pathway and by extension of synthetic carbon fixation efforts as a whole. In chapter 3, I engineered a segment of a synthetic carbon fixation cycle in Escherichia coli. The GED cycle was proposed as a Calvin cycle alternative that does not perform a wasteful oxygenation reaction and is more energy efficient. The pathways simple architecture and reasonable driving force made it a promising candidate for enhanced carbon fixation. I created a deletion strain that coupled growth to carboxylation via the GED pathway segment. The CO2 dependence of the engineered strain and 13C-tracer analysis confirmed operation of the pathway in vivo. In the final chapter, I present my efforts of implementing the GED cycle also in C. necator, which might be a better-suited host, as it is accustomed to formatotrophic and hydrogenotrophic growth. To provide the carboxylation substrate in vivo, I engineered C. necator to utilize xylose as carbon source and created a selection strain for carboxylase activity. I verify activity of the key enzyme, the carboxylase, in the decarboxylative direction. Although CO2-dependent growth of the strain was not obtained, I showed that all enzymes required for operation of the GED cycle are active in vivo in C. necator. I then evaluate my success with engineering a linear and cyclical one-carbon fixation pathway in two different microbial hosts. The linear reductive glycine pathway presents itself as a much simpler metabolic solution for formate dependent growth over the sophisticated establishment of hard-to-balance carbon fixation cycles. Last, I highlight advantages and disadvantages of C. necator as an upcoming microbial benchmark organism for synthetic metabolism efforts and give and outlook on its potential for the future of C1-based manufacturing.},
  language  = {en}
}