Design, analysis and implementation of synthetic carbon fixation pathways

Biological carbon fixation is a fundamental process sustaining life on Earth, yet it is constrained by the efficiency and diversity of naturally occurring metabolic pathways. Rising global challenges such as climate change and the demand for sustainable bioproduction have renewed interest in rethinking carbon assimilation at the molecular level. This thesis explores the design, discovery, and contextualization of new and hidden metabolic routes for carbon fixation and assimilation. Through computational pathway design, experimental implementation, evolutionary discovery, and biotechnological assessment, it highlights how synthetic and natural metabolism can be expanded to unlock new opportunities for sustainable production of chemicals and food.Chapter 1 provides a general introduction to the topic of metabolism and carbon fixation starting from thermodynamics and biochemistry while also examining existing carbon fixation pathways and methods for pathway design.Chapter 2 begins with the rational design and experimental validation of the reductive methylaspartate cycle (rMASP), a novel ATP-efficient aerobic CO2 fixation pathway. Four rMASP variants producing glyoxylate or acetyl-CoA were computationally analyzed, and a modular engineering approach was used to reconstitute these modules in Escherichia coli. Growth-coupled selection and isotopic labeling confirmed module activity in vivo, building on and expanding previous research in the field.Chapter 3 expands the search space for new carbon fixation pathways. Using a bioretrosynthetic approach informed by enzyme promiscuity and the latent biochemical reaction space, eight novel pathway designs were identified and evaluated in comparison with both natural and previously proposed synthetic cycles. Thermodynamic, stoichiometric, and kinetic analyses revealed potential advantages over the Calvin-Benson-Bassham cycle, while also underscoring the importance of enzyme engineering for pathway realization. This work systematically broadens the landscape of candidate pathways for future CO2 fixation strategies.Chapter 4 then turns to evolutionary innovation in metabolism, showing how enzyme promiscuity can give rise to new biochemical functions. Using a strain of E. coli lacking canonical isoleucine biosynthesis, experimental evolution uncovered a previously unknown route dependent on the reactivation of acetohydroxyacid synthase II. This enzyme was shown to catalyze a novel condensation reaction between glyoxylate and pyruvate to form 2-ketobutyrate, establishing a recursive pathway to isoleucine. Unlike earlier “underground” isoleucine pathways, this one uses central metabolites under aerobic conditions, suggesting a potential role in natural E. coli metabolism and extending the known biosynthetic space of amino acids.Chapter 5 situates these findings within the emerging field of synthetic C1 metabolism, where methanol, formate, hydrogen, and CO2 are explored as sustainable feedstocks for microbial bioproduction. A comparative analysis of natural and synthetic pathway architectures highlights progress toward growth rates and biomass yields that rival or exceed those of natural C1-assimilating organisms. Remaining challenges for industrial application are identified, including the need to optimize product spectra, titers, and yields, as well as to expand host organism versatility.Chapter 6 examines the whole thesis work in the broader context of scientific, social, political and economic challenges.