Identification and functional characterization of toluene degradation genes inAcinetobactersp. Tol 5

Microbial degradation of aromatic compounds provides sustainable solutions for environmental remediation and bioconversion.Acinetobactersp. Tol 5 is notable for its strong adhesiveness and potential as a biocatalyst for toluene degradation; however, its toluene metabolic pathway has not been fully elucidated. In this study, genomic analysis identified a cluster of genes in Tol 5 highly similar to the well-knowntodoperon ofPseudomonas putida, encoding enzymes responsible for toluene metabolism. Phylogenetic analyses indicated that thesetodgenes, unusual amongAcinetobacterspecies, were likely acquired through horizontal gene transfer. Transcriptomic analyses revealed thattodFandtodC1are co-transcribed, while the adjacentfadL2gene, encoding a putative outer membrane transporter corresponding toP. putida todX, is independently transcribed. Functional characterization using gene-knockout mutants revealed that TodC1, the large subunit of dioxygenase, is essential for growth on toluene, whereas FadL2 is not essential. Growth experiments further showed that thetodC1knockout mutant could metabolize benzoate, but not toluene or benzene, confirming that the TOD pathway is the primary route for toluene and benzene degradation in Tol 5. The identification of the functional TOD pathway, which is unique withinAcinetobacter, provides genetic and biochemical insights for the development of Tol 5 as an efficient immobilized biocatalyst for the bioremediation and bioconversion of aromatic compounds.