One carbon compounds like formate provide a promising and sustainable feedstock for microbial bioproduction of fuels and chemicals, as an alternative to the petro-chemical production industry. Growth of Escherichia coli on formate was recently achieved by introducing the reductive glycine pathway (rGlyP) into its genome, which is theoretically the most efficient aerobic formate assimilation pathway. While adaptive laboratory evolution was used to enhance the growth rate and biomass yield significantly, still the best performing formatotrophic E. coli strain did not approach the theoretical optimal biomass yield of the rGlyP. In this study, we investigated the metabolism of these previously engineered formatotrophic E. coli strains in order to elucidate why the biomass yield was sub-optimal and how it may be improved. Through a combination of metabolic modelling and proteomic analysis, we identified several potential metabolic bottlenecks and targets for expression tuning. This characterization study provides insights that can inform future rational engineering efforts to improve the growth of E. coli on formate.