Abstract
One-carbon compounds, such as formate, are promising and sustainable feedstocks for microbial bioproduction of fuels and chemicals. Growth of Escherichia coli on formate was recently achieved by introducing the reductive glycine pathway (rGlyP) into its genome, which is theoretically the most energy-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 these previously engineered formatotrophic E. coli strains to find out why the biomass yield was sub-optimal and how it may be improved. Through a combination of metabolic modelling, genomic and proteomic analysis, we identified several potential metabolic bottlenecks and future targets for optimization. This study also reveals further insights in the evolutionary mutations and related changes in proteome allocation that supported the already substantially improved growth of formatotrophic E. coli strains. This systems-level analysis provides key insights to realize high-yield, fast growing formatotrophic strains for future bioproduction.