Multi-strain analysis of <i>Pseudomonas putida</i> reveals the metabolic and genetic diversity of the species

<i>Pseudomonas putida</i> is a gram-negative bacterial species increasingly utilized in biotechnology due to its robust growth, ability to degrade aromatic compounds, solvent tolerance, and genetic tractability. In this study, we report a comprehensive multi-strain analysis of 164 <i>P. putida</i> strains based on the reconstruction of a pan<i>-</i>putida metabolic network and the formulation of strain-specific genome-scale metabolic models (GEMs). We performed whole-genome sequencing and hybrid assembly for 40 strains, contributing a ~8% increase to the available genomic data for <i>P. putida</i>. Furthermore, high-throughput phenotypic profiling using the Biolog phenotype microarray system for 24 strains on 190 unique carbon sources, along with 15 aromatic compounds not present on Biolog plates, yielded 4,920 unique strain-phenotype measurements. These data were leveraged to curate GEMs for 24 representative strains, including a refined model for strain KT2440, which comprised 1,480 genes and 2,191 metabolites, achieving a prediction accuracy of 91.2% in carbon utilization. Systematic comparison of genomes and GEMs revealed both conserved core pathways and significant allelic and functional divergence across strains, highlighting strain-specific variation in aromatic degradation. While pathways for protocatechuate and phenylacetate degradation were widely conserved, metabolic capabilities for compounds such as ferulate, phenol, and cresols varied markedly, suggesting adaptation to distinct ecological niches. Alleleome analysis of enzymes, such as PcaI and PcaJ, revealed distinct, functionally similar clades, indicating possible convergent evolution or horizontal gene transfer. These results provide computable resources and informative models for selecting <i>P. putida</i> strains with desired traits for biomanufacturing and bioremediation and offer insights into the evolution and phylogeny of the <i>P. putida</i> species.IMPORTANCE<i>Pseudomonas putida</i> has become an organism of interest for biotechnological applications, but a species-level understanding of its metabolic diversity remains incomplete. In this study, we analyzed 164 <i>P. putida</i> strains using a combination of genome sequencing, phenotypic profiling, and metabolic modeling. Our results indicate that while many metabolic pathways are conserved, notable differences exist across strains, particularly in aromatic compound degradation. These observations may inform future strain selection and engineering strategies tailored to specific industrial or environmental goals. In addition, the genome-scale models and phenotypic data generated here can serve as a foundation for broader studies of metabolism and functional variation within this species.