Jerome Ari Satanowski

• Climate change, driven by increasing atmospheric levels of carbon dioxide (CO2), presents a significant societal challenge for the 21st century. Biotechnological approaches for microbial production of commodity chemicals and fuels offer possible solutions to re-fix CO2 from the atmosphere, thereby mitigating carbon emissions and contributing to a sustainable carbon-economy in the future. Biological CO2 fixation is also at the heart of agricultural productivity, where photosynthesis and the Calvin-Benson-Bassham cycle present promising biotechnological targets for crop improvement. Synthetic biology allows testing metabolic solutions not known to exist in nature, which may exceed their natural counterparts in terms of efficiency. In this thesis, I explore the design of such new-to-nature metabolic pathways for biological CO2 utilization and their implementation in living cells (in vivo). In the first chapter, I describe the development of a metabolic pathway that enables intracellular conversion of CO2 to formate, giving access toClimate change, driven by increasing atmospheric levels of carbon dioxide (CO2), presents a significant societal challenge for the 21st century. Biotechnological approaches for microbial production of commodity chemicals and fuels offer possible solutions to re-fix CO2 from the atmosphere, thereby mitigating carbon emissions and contributing to a sustainable carbon-economy in the future. Biological CO2 fixation is also at the heart of agricultural productivity, where photosynthesis and the Calvin-Benson-Bassham cycle present promising biotechnological targets for crop improvement. Synthetic biology allows testing metabolic solutions not known to exist in nature, which may exceed their natural counterparts in terms of efficiency. In this thesis, I explore the design of such new-to-nature metabolic pathways for biological CO2 utilization and their implementation in living cells (in vivo). In the first chapter, I describe the development of a metabolic pathway that enables intracellular conversion of CO2 to formate, giving access to highly efficient carbon fixation routes. In nature, CO2-reduction remains restricted to anaerobic organisms and low redox potentials. Here, we introduce the “CORE cycle”, a synthetic metabolic pathway that converts CO2 to formate under fully aerobic conditions and ambient CO2 levels, using only NADPH as a reductant. We leverage this synthetic, ATP-energized pathway to overcome the thermodynamic and kinetic barriers associated with CO2-reduction. Applying rational metabolic engineering and adaptive evolution, this work demonstrates that Escherichia coli can utilize ambient CO2 as the sole source of one-carbon units and serine, achieving a first step towards novel modes of synthetic autotrophy. We further apply computational modeling to showcase the potential of the CORE cycle as a photorespiratory bypass for enhancing photosynthesis. In the second chapter, I describe the development of the “LCM module”, a novel metabolic route for CO2-incorporating conversion of acetyl-CoA to pyruvate. This route relies on the newly uncovered, promiscuous activity of an adenosylcobalamin (B12)-dependent enzyme, which we significantly optimize through targeted hypermutation and in vivo selection strategies. The LCM module provides a shorter and more efficient pathway for acetyl-CoA assimilation compared to natural routes, offering novel opportunities for synthetic CO2 fixation. Overall, through theoretical pathway analysis, enzyme bioprospecting, and modular metabolic engineering in E. coli, this thesis expands the solution space for biological CO2 fixation.…