Insights into the Genomic Architecture and Improvement of the Capabilities of 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 21# chromosome: alkane hydroxylases (, , ) and aromatic ortho-cleavage pathway genes (). Phylogenetic and multiple sequence alignment analyses of the 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 gene from KJ-1 via pET-28a(+)- 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.