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

ABSTRACT One-carbon (C1) compounds such as methanol, formate, and CO 2 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 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 from formate to methanol using both NAD and PQQ – dependent methanol dehydrogenases. Finally, we demonstrated CO 2 -dependent growth through CO 2 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 engineer P. putida towards growth on formate, methanol, and CO 2 as sole feedstocks, thereby substantially expanding its potential as a sustainable and versatile cell factory.