Evolution-assisted engineering of formate assimilation via the formyl phosphate route in Escherichia coli

The transition towards a sustainable bioeconomy requires the use of alternative feedstocks, with CO₂-derived formate emerging as a promising candidate for industrial biotechnology. Despite its beneficial characteristics as a feedstock, microbial assimilation of formate is limited by the inefficiency of naturally evolved formate-fixing pathways. To overcome this limitation, synthetic formate reduction cascades could enable formate assimilation via formaldehyde, a key intermediate of several existing one carbon assimilation pathways. Recently, the formyl phosphate route, combining ATP-dependent activation of formate to formyl phosphate, followed by its reduction to formaldehyde, was developed through enzyme engineering and characterized in vitro. In this work, we successfully established the formyl phosphate route in vivo by developing a selection strategy that couples formate reduction to growth in a threonine/methionine auxotrophic Escherichia coli. Through adaptive laboratory evolution, we achieved formate-dependent growth via this novel pathway. Evolved strains were capable of growing robustly with formate concentrations between 20 mM and 100 mM with glucose in the co-feed. Genomic and proteomic analyses together with activity assays uncovered that formate activation was limiting in vivo. This discovery guided the rational engineering of a strain capable of efficient formate assimilation through the formyl phosphate route. By demonstrating that novel enzyme activities can link formate reduction to cell growth, our study shows how synthetic metabolic routes can be functionally established inside the cell, paving the way for the engineering of more complex synthetic pathways.