Evolution-assisted engineering of E. coli enables growth on formic acid at ambient CO via the Serine Threonine Cycle.

Atmospheric CO 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 CO into reduced one-carbon (C) compounds (e.g. formic acid) are developing and improving fast. Driven by the idea of creating sustainable bioproduction platforms, natural and synthetic C-utilization pathways are engineered into industrially relevant microbes. The realization of synthetic C-assimilation cycles in living organisms is a promising but challenging endeavour. Here, we engineer the Serine Threonine Cycle, a synthetic C-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 sole carbon and energy source and can grow at ambient CO cultivation conditions. This work sets an example for the engineering of complex C-assimilation cycles in heterotrophic microbes.