Pseudomonas vancouverensis as a platform for one-carbon (C1) assimilation and polyhydroxyalkanoate accumulation.

Metabolic engineering calls for new bacterial chassis that combine robust metabolic and physiological traits with genetic tractability. We developed Pseudomonas vancouverensis DhA-51, a native one-carbon (C1)-trophic strain, into a platform that broadens chassis options beyond the usual Pseudomonas hosts while retaining native capacities relevant to bioproduction. We closed and annotated the chromosome to provide stable coordinates for genome editing and omics, established antibiotic susceptibility profiles, and explored replication of common broad-host-range plasmid toolsets. An expression toolkit, including three constitutive promoters and two chemically-inducible systems, was parameterized with fluorescent reporters. Clean markerless genome editing by homologous recombination was demonstrated through deletion of the benABCD cluster that mediates aromatic compound breakdown. Physiological tests showed growth on diverse carbon substrates and synthesis of medium-chain-length polyhydroxyalkanoates from glucose, octanoate, and C1 substrates, with polymer content and monomer composition tunable by the carbon-to-nitrogen ratio and feedstock type. Isotope tracing showed that methanol can be assimilated while formate is used as a reductant. The domestication sequence applied here shortens design-build-test-learn cycles and can be applied across the Pseudomonas genus to expand the portfolio of workable chassis for sustainable production of chemicals and materials from renewable feedstocks.