?Can E. coli be evolved to perform carbon fixation

The assimilation of carbon dioxide into organic material also known as carbon fixation is largest biosynthetic processes in the biosphere. While carbon fixation pathways offer a renewable alternative for biofuels and numerous chemicals, integrating a non-native carbon fixation pathway into a microbial host is still a standing challenge. I will present a metabolic engineering framework, which enforces a heterotrophic to be dependent upon carbon fixation even in the presence of a carbon source. By rewiring the native metabolic network of E.coli, expressing Calvin-Benson cycle components and selecting on specific growth conditions we engineered strains in which non-native RuBisCO dependent carbon fixation is essential for growth. Such selection systems are beneficial both as a direct selection tools for carbon fixation enzymes, as well as a metabolic engineering tool for the integration of non-native pathways. We analyze the metabolic and physiological effects of such RuBisCO dependent growth modes and explore the feasibility for expressing a fully functional non-native Calvin-Benson cycle using a directed evolution approach. Beyond practical implications for the design, construction and testing of synthetic carbon fixation pathways, we believe such an approach can be implemented for a variety of other metabolic pathways.