Insights into the Genomic Architecture and Improvement of the Capabilities of Acinetobacter calcoaceticus for the Biodegradation of Petroleum Hydrocarbons

Petroleum-contaminated terrestrial ecosystems require effective bioremediation strategies. In this study, genomic analysis revealed key biodegradation genes on the Acinetobacter calcoaceticus 21# chromosome: alkane hydroxylases (alkB, almA, LadA) and aromatic ortho-cleavage pathway genes (catABC). Phylogenetic and multiple sequence alignment analyses of the almA gene in strain 21# revealed the presence of signature motifs characteristic of Baeyer–Villiger monooxygenase. Functional annotation analysis demonstrated stronger phylogenetic affinity of this protein to previously characterized BVMOs than to hydroxylases. Therefore, it is suggested that the AlmA protein in 21# exhibits BVMO activity and participates in the subterminal oxidation pathway of alkane degradation. Wild-type 21# degraded both n-Octacosane (24.47%) and pyrene (34.03%). Engineered 21#-A3 showed significantly enhanced n-Octacosane degradation (28.68%). To validate AlmA function and assess impacts of exogenous gene integration, we expressed the almA gene from A. vivianii KJ-1 via pET-28a(+)-av-almA-BH vector. Enzymatic assays demonstrated no activity toward long-chain alkanes but high activity for 2-decanone (0.39 U/mg) and 2-dodecanone (0.37 U/mg). Metabolite analysis confirmed recombinant AlmA functions through subterminal oxidation. This study establishes a foundational framework for advancing the optimization of petroleum-degrading bacteria. To engineer more efficient hydrocarbon-degrading strains, future research should integrate meta-cleavage pathways to expand their substrate utilization range for polycyclic aromatic hydrocarbons.