One-carbon fixation via the synthetic reductive glycine pathway exceeds yield of the Calvin cycle.

One-carbon feedstocks such as formate could be promising renewable substrates for sustainable microbial production of food, fuels and chemicals. Here we replace the native energy-inefficient Calvin-Benson-Bassham cycle in Cupriavidus necator with the more energy-efficient reductive glycine pathway for growth on formate and CO2. In chemostats, our engineered strain reached a 17% higher biomass yield than the wild type and a yield higher than any natural formatotroph using the Calvin cycle. This shows the potential of synthetic metabolism to realize sustainable, bio-based production.