Systematic engineering of synthetic serine cycles in Pseudomonas putida uncovers emergent topologies for methanol assimilation

The urgent need for a circular carbon economy has driven research into sustainable substrates, including one-carbon (C₁) compounds. The non-pathogenic soil bacterium Pseudomonas putida is a promising host for exploring synthetic methylotrophy due to its versatile metabolism. In this research article we implemented synthetic serine cycle variants in P. putida for methanol assimilation, combining modular engineering and growth-coupled selection, whereby methanol assimilation supported biosynthesis of the essential amino acid serine. We adopted three synthetic variants (serine–threonine cycle, homoserine cycle, and modified serine cycle), divided these metabolic architectures into functional modules, and systematically compared their performance for in vivo implementation. Additionally, we harnessed native pyrroloquinoline quinone (PQQ)-dependent dehydrogenases for engineering methylotrophy. Recursive rewiring of synthetic and native activities revealed novel metabolic topologies for methanol utilization, termed enhanced serine–threonine cycle, providing a blueprint for engineering C₁ assimilation in non-model heterotrophic bacteria.