Objective  Aflatoxin B₁ (AFB₁), one of the most toxic natural carcinogens known, poses severe threats to human health and food safety. Identifying and characterizing key enzyme genes responsible for efficient AFB₁ degradation are crucial for developing targeted biodetoxification technologies. This study aimed to isolate and identify AFB₁-degrading bacterial strains with high efficiency based on the structural features of AFB₁, explore potential biodegradation enzymes genes via genomics, thereby providing a reference for the research and application of AFB₁-degrading enzymes.

Methods  Potential AFB₁-degrading strains were isolated from sewage, fecal, and soil samples using coumarin (mimicking the lactone ring) and tetrahydrofuran (mimicking the furan ring) as the sole carbon sources. Secondary screening was performed under 2% tetrahydrofuran to mimic AFB₁-toxic environments. AFB₁ degradation efficiency was assessed via thin-layer chromatography (TLC). Whole-genome sequencing was conducted using the Illumina second-generation sequencing platform, followed by BLASTP homology alignment to identify potential degradation enzyme genes.

Results  Eight candidate degrading strains were isolated and preliminarily identified as Pseudomonas spp. through morphological and 16S rRNA gene sequence analysis. Among them, Pseudomonas promysalinigenes CT1-8 achieved a 55.23% degradation rate for 5 μg/mL AFB₁ after 5 days of cultivation. Genomic analysis revealed a genome size of 5 780 325 bp (GC content: 62.2%) encoding 5 464 proteins. Homology alignment indicated that the outer membrane porin OprF shared 38.3% sequence similarity with the known AFB₁-degrading enzyme OmpA. Functional annotation indicated that OprF gene belongs to an operon consisting of oprF, yiB, yiaD and rcpA, suggesting synergistic roles of this operon in AFB₁ transport and metabolism.

Conclusion  This study established a structural analog enrichment screening strategy and identified novel AFB₁-degrading strains and potential key enzymes, providing critical insights into AFB₁ biodegradation mechanisms and their engineering applications.