Puiggene, O.; Fricano, M.; Rossi, R.; Jansen, L. F. M.; Ozdemir, E.; Kim, S. H.; Lenhard, C.; Mohamed, E. T.; Donati, S.; Foster, J.; Kandasamy, V.; Feist, A. F.; Orsi, E.; Nikel, P. I.

Methanol is a reduced, soluble one-carbon (C1) feedstock for sustainable bioproduction, but converting this potential into robust microbial growth remains difficult. Several synthetic C1 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 C1 incorporation to strict methylotrophy. The evolved strain grew with methanol as the sole carbon and energy source under atmospheric CO2 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 C1 biomanufacturing.