Integrated genomic and experimental assessment of the biosafety of Paraburkholderia sacchari as a promising microbial chassis.

Paraburkholderia sacchari is an environmental member of the Burkholderiaceae with recognized biotechnological potential, yet its genomic diversity, biosafety profile, and evolutionary relationship to pathogenic Burkholderia species remain poorly understood. Here, we present a comprehensive genomic and phenotypic characterization of P. sacchari strain LMG 19,450T and compare it with three additional P. sacchari genomes and phylogenetically related Burkholderia species. A high-quality genome assembly confirmed its placement within the Paraburkholderia clade and species-level relatedness among P. sacchari strains despite detectable intraspecies genomic variability. Pangenome analysis revealed a large, conserved core genome and an extensive accessory gene repertoire, consistent with adaptation to plant-associated soil environments. Comparative analysis of virulence-associated genes showed a marked contrast between P. sacchari and pathogenic Burkholderia, with the former lacking most canonical pathogenicity determinants. Gene tree-species tree reconciliation further indicated a constrained horizontal gene transfer profile for virulence-associated gene families, while genes involved in transcriptional regulation displayed higher transfer propensity. Antimicrobial resistance profiling revealed a limited resistome dominated by efflux-associated homologs, consistent with broad phenotypic susceptibility. Notably, strain LMG 19,450T exhibited antibiotic susceptibility patterns comparable to those of the established microbial chassis Pseudomonas putida KT2440. Virulence assessment using the Galleria mellonella model demonstrated markedly attenuated pathogenicity relative to Burkholderia cepacia. Together, these findings support P. sacchari as an environmentally adapted bacterium with a favorable biosafety profile and highlight the value of integrating genomic and experimental approaches to evaluate emerging microbial chassis.