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Abstract

Atmospheric CO2 poses a major threat to life on Earth by causing global warming and climate change. On the other hand, it can be considered as a resource that is scalable enough to establish a circular carbon economy. Accordingly, technologies to capture and convert CO2 into reduced one-carbon (C1) compounds (e.g. formic acid) are developing and improving fast. Driven by the idea of creating sustainable bioproduction platforms, natural and synthetic C1-utilization pathways are engineered into industrially relevant microbes. The realization of synthetic C1-assimilation cycles in living organisms is a promising but challenging endeavour. Here, we engineer the Serine Threonine Cycle, a synthetic C1-assimilation cycle in Escherichia coli to achieve growth on formic acid. Our stepwise engineering approach in tailored selection strains combined with adaptive laboratory evolution experiments enabled formatotrophic growth of the organism. Whole genome sequencing and reverse engineering allowed us to determine the key mutations linked to pathway activity. The Serine Threonine Cycle strains created in this work use formic acid as a carbon and energy source and can grow at ambient CO2 cultivation conditions. This work sets an example for the engineering of complex C1-assimilation cycles in heterotrophic microbes.