Engineering a biotechnological microorganism for growth on one-carbon (C1) intermediates, produced from the abiotic activation of CO2, is a key synthetic biology step towards the valorization of this greenhouse gas to commodity chemicals. Here, we redesign the central carbon metabolism of the model bacterium Escherichia coli for growth on C1 compounds using the reductive glycine pathway. Sequential genomic introduction of the four metabolic modules of the synthetic pathway resulted in a strain capable of growth on formate and CO2 with a doubling time of ~70 hours and growth yield of ~1.5 gCDW / molformate. Short-term evolution decreased doubling time to less than 8 hours and improved biomass yield to 2.3 gCDW / mol-formate. Growth on methanol and CO2 was achieved by the expression of methanol dehydrogenase in the evolved strain. Establishing synthetic formatotrophy and methylotrophy, as demonstrated here, paves the way for sustainable bioproduction rooted in CO2 and renewable energy.

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Assimilation of one-carbon compounds presents a key biochemical challenge that limits their use as sustainable feedstocks for microbial growth and production. The reductive glycine pathway is a synthetic metabolic route that could provide an optimal way for the aerobic assimilation of reduced C1 compounds. Here, we show that a rational integration of native and foreign enzymes enables the tetrahydrofolate and glycine cleavage/synthase systems to operate in the reductive direction, such that Escherichia coli satisfies all of its glycine and serine requirements from the assimilation of formate and CO. Importantly, the biosynthesis of serine from formate and CO does not lower the growth rate, indicating high flux that is able to provide 10% of cellular carbon. Our findings assert that the reductive glycine pathway could support highly efficient aerobic assimilation of C1-feedstocks.

Decoupling biorefineries from land use and agriculture is a major challenge. As formate can be produced from various sources, e.g., electrochemical reduction of CO, microbial formate-assimilation has the potential to become a sustainable feedstock for the bioindustry. However, organisms that naturally grow on formate are limited by either a low biomass yield or by a narrow product spectrum. The engineering of a model biotechnological microbe for growth on formate via synthetic pathways represents a promising approach to tackle this challenge. Here, we achieve a critical milestone for two such synthetic formate-assimilation pathways in Escherichia coli. Our engineering strategy involves the division of the pathways into metabolic modules; the activity of each module-providing at least one essential building block-is selected for in an appropriate auxotrophic strain. We demonstrate that formate can serve as a sole source of all cellular C1-compounds, including the beta-carbon of serine. We further show that by overexpressing the native threonine cleavage enzymes, the entire cellular glycine requirement can be provided by threonine biosynthesis and degradation. Together, we confirm the simultaneous activity of all pathway segments of the synthetic serine-threonine cycle. We go beyond the formate bioeconomy concept by showing that, under anaerobic conditions, formate produced endogenously by pyruvate formate-lyase can replace exogenous formate. The resulting prototrophic strain constitutes a substantial rewiring of central metabolism in which C1, glycine, and serine metabolism proceed via a unique set of pathways. This strain can serve as a platform for future metabolic-engineering efforts and could further pave the way for investigating the plasticity of metabolic networks.

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