14Formate can be directly produced from CO2 and renewable electricity, making it a promising microbial 15 feedstock for sustainable bioproduction. Cupriavidus necator is one of the few biotechnologically-relevant 16 hosts that can grow on formate, but it uses the inefficient Calvin cycle. Here, we redesign C. necator 17 metabolism for formate assimilation via the highly efficient synthetic reductive glycine pathway. First, we 18 demonstrate that the upper pathway segment supports glycine biosynthesis from formate. Next, we explore 19 the endogenous route for glycine assimilation and discover a wasteful oxidation-dependent pathway. By 20 integrating glycine biosynthesis and assimilation we are able to replace C. necator's Calvin cycle with the 21 synthetic pathway and achieve formatotrophic growth. We then engineer more efficient glycine metabolism 22 and use short-term evolution to optimize pathway activity, doubling the growth yield on formate and 23 quadrupling the growth rate. This study thus paves the way towards an ideal microbial platform for realizing 24 the formate bioeconomy. 25 26 27 28 Microbial biosynthesis offers an environmentally friendly alternative to fossil-based production. However, the 31 limited availability and questionable sustainability of microbial feedstocks hamper the expansion of 32 biotechnological production and the establishment of a circular carbon economy. The common substrates for 33 microbial bioproduction are plant-based sugars, the utilization of which competes with food supply and 34 necessitates vast land use that negatively impacts the environment. Moreover, alternative feedstocks, such 35 as lignocellulosic biomass, suffer from crucial drawbacks, such as difficult and expensive processing 1 . A 36 fundamental limitation of all photosynthesis-based resources is the low energy conversion efficiency 37 associated with this process, typically below 1% 2,3 . 38Electromicrobial production has gained attention as an alternative route towards sustainable biotechnology 4,5 . 39 This strategy is based on the use of two key feedstocks: CO2-free electricitye.g. from solar, wind, hydro -40 the production of which is rapidly growing, and CO2, a virtually unlimited carbon source, captured either from 41 point sources or directly from air. Some microbes can grow by receiving electrons directly from a cathode; 42 however, low current densities limit the economic viability of this approach 6,7 . A more feasible option is the 43 electrochemical production of small reduced compounds 6 that are subsequently fed to microbes and then 44 converted into value-added chemicals. Among the possible mediator compounds, hydrogen, carbon 45 monoxide, and formate can be produced at high efficiency and rate 8 . Whereas hydrogen and carbon monoxide 46 are gases of low solubility, formate is completely miscible and can be readily introduced to microbial cells 47 without mass transfer limitations and without major safety concerns 9 . Hence, establishing a "formate bio-48 economy" has been proposed as a ro...
