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Abstract

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 work, 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. The serine cycle forms acetyl-coenzyme A from C1 molecules without carbon loss but has bottlenecks that hinder engineering efforts. We adopted three synthetic variants (serine-threonine cycle, homoserine cycle, and modified serine cycle) that yield serine in a methanol-dependent fashion to overcome these challenges. By dividing these metabolic designs into functional modules, we systematically compared their performance for implementation in vivo. Additionally, we harnessed native pyrroloquinoline quinone-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.Competing Interest StatementThe authors have declared no competing interest.