Synthetic C ₁ metabolism in <i>Pseudomonas putida</i> enables strict formatotrophy and methylotrophy via the reductive glycine pathway

Formate and methanol are promising alternatives to sugar-based feedstocks for biotechnological applications. These one-carbon (C₁) substrates can be sustainably produced from CO₂ and renewable electricity and assimilated by both native and engineered microbial systems. However, their broader adoption is limited by the narrow range of bacterial hosts capable of efficient methanol and formate utilization. In this study, the industrially relevant soil bacterium <i>Pseudomonas putida</i> was metabolically engineered to assimilate formate and methanol as sole carbon and energy sources via the linear reductive glycine pathway. Initial strains were optimized for formate assimilation using acetate for energy conservation through adaptive laboratory evolution (ALE), leading to a substantial reduction in doubling time under mixotrophic conditions. Key mutations emerged in the promoter regions of synthetic pathway genes and within the native genome. Strictly formatotrophic growth, with a doubling time of ca. 28 h, was achieved by integrating a formate dehydrogenase gene either on a plasmid or chromosomally as a mini-Tn<i>5</i> module, combined with growth-coupled selection. The resulting strain, <i>P. putida</i> rG·F, was then re-engineered by replacing the formate dehydrogenase with an engineered methanol dehydrogenase from <i>Cupriavidus necator</i>. Following ALE, an isolate displaying full methylotrophy, <i>P. putida</i> rG·M, grew on methanol with a doubling time of ca. 24 h. These efforts demonstrate the feasibility of constructing robust C₁-assimilating <i>P. putida</i> strains and highlight the substrate versatility of this bacterium for bioproduction. Integrating evolutionary engineering with synthetic biology tools has expanded the range of viable microbial hosts for efficient C₁ feedstock utilization.IMPORTANCESoluble C₁ feedstocks, such as formate and methanol, have gained attention as sustainable substrates for biotechnology, with the potential to reduce greenhouse gas emissions and reliance on sugar-based resources. Despite their promise, the metabolic assimilation of these compounds remains uncharacterized in robust bacterial hosts beyond a few model species. <i>Pseudomonas putida</i>, known for its metabolic versatility and industrial relevance, has lacked the ability to grow solely on C₁ compounds. This study is a first-case example of strict synthetic formatotrophy and methylotrophy in any <i>Pseudomonas</i> species, enabling growth on formate and methanol as sole carbon and energy sources. Through pathway rewiring and adaptive laboratory evolution, key metabolic and regulatory adaptations were identified that enabled efficient C₁ assimilation. These findings not only expand the known capabilities of <i>P. putida</i> but also open directions for its deployment in carbon-efficient biomanufacturing. This study sets a precedent for leveraging non-model microorganisms in the development of scalable, carbon-efficient bioprocesses.