Paving the way for synthetic C1 - Metabolism in Pseudomonas putida through the reductive glycine pathway

One-carbon (C1) compounds such as methanol, formate, and CO₂ are alternative, sustainable microbial feedstocks for the biobased production of chemicals and fuels. In this study, we engineered the carbon metabolism of the industrially important bacterium Pseudomonas putida to modularly assimilate these three substrates through the reductive glycine pathway. First, we demonstrated the functionality of the C1-assimilation module by coupling the growth of auxotrophic strains to formate assimilation. Next, we extended the module in the auxotrophic strains from formate to methanol-dependent growth using both NAD and PQQ-dependent methanol dehydrogenases. Finally, we demonstrated, for the first time, engineered CO₂-dependent formation of part of the biomass through CO₂ reduction to formate by the native formate dehydrogenase, which required short-term evolution to rebalance the cellular NADH/NAD ⁺ ratio. This research paves the way to further engineer P. putida towards full growth on formate, methanol, and CO₂ as sole feedstocks, thereby substantially expanding its potential as a sustainable and versatile cell factory.