Identification and functional characterization of toluene degradation genes in Acinetobacter sp. Tol 5

Microbial degradation of aromatic compounds provides sustainable solutions for environmental remediation and bioconversion. Acinetobacter sp. 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-known tod operon of Pseudomonas putida , encoding enzymes responsible for toluene metabolism. Phylogenetic analyses indicated that these tod genes, unusual among Acinetobacter species, were likely acquired through horizontal gene transfer. Transcriptomic analyses revealed that todF and todC1 are co-transcribed, while the adjacent fadL2 gene, encoding a putative outer membrane transporter corresponding to P. 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 the todC1 knockout 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 within Acinetobacter , provides genetic and biochemical insights for the development of Tol 5 as an efficient immobilized biocatalyst for the bioremediation and bioconversion of aromatic compounds.