Seven mutations unlock strict synthetic methylotrophy in engineered Pseudomonas putida

Methanol is a reduced, soluble one-carbon (C ₁₎ feedstock for sustainable bioproduction, but converting this potential into robust microbial growth remains difficult. Several synthetic C ₁ assimilation routes depend on autocatalytic cycles, whose operation requires coordinated control of redox balance, toxic intermediates, substrate regeneration, and host regulation. Here, we implemented the serine-threonine cycle (STC) in the soil bacterium Pseudomonas putida and used growth-coupled selection with adaptive laboratory evolution (ALE) to transition from mixotrophic C ₁ incorporation to strict methylotrophy. The evolved strain grew with methanol as the sole carbon and energy source under atmospheric CO ₂ with a doubling time of ca. 40 h. Whole-genome sequencing, reverse genetics, biosensors, isotope labelling, and comparative RNA sequencing showed that evolution repeatedly targeted native pyrroloquinoline quinone (PQQ)-dependent methanol oxidation, membrane-bound transhydrogenase activity, glycine regeneration, STC enzyme balance, and global regulatory nodes. Additional ALE under glycine-methanol co-feeding increased growth rates and exposed further targets for improving cycle flux. These results establish P. putida as a chassis for strict synthetic methylotrophy and define actionable engineering routes toward C ₁ biomanufacturing.