Novel Tetracycline-Degrading Enzymes from the Gut Microbiota of Black Soldier Fly: Discovery, Performance, Degradation Pathways, Mechanisms, and Application Potential

The antibiotic tetracycline (TC) is an emerging pollutant frequently detected in various environments. Although enzymatic remediation is a promising strategy for mitigating TC contamination, the availability of effective TC-degrading enzymes remains limited, and their mechanisms and applications are not fully understood. This study developed a comprehensive TC-degrading enzyme library from the gut microbiome of the highly TC-resistant saprophagous insect, black soldier fly larvae (BSFL), using an integrated metagenomic and comparative metatranscriptomic approach, identifying 105 potential novel TC-degradation genes. Bioinformatics analysis of 10 selected genes underscored the novelty of the identified enzymes. Among these, Trg2 demonstrated strong binding affinity and significant degradation capacity for TC. Key functional amino acid residues, including Thr231, Ala64, Ala82, Gly68, Gly79, and Ser81, were identified as essential for the interaction between TC and Trg2. Six TC degradation pathways were proposed, involving the transformation of TC into 19 metabolites through de-grouping, ring opening, oxidation, reduction, and addition reactions, effectively reducing TC toxicity. Furthermore, Trg2 exhibited resilience under harsh conditions, maintaining the capacity to remove about 45 % of the total TC in mariculture wastewater across eight successive batches. This study advances the understanding of TC degradation mechanisms and highlights the potential application of novel enzymes for bioremediation purposes.The antibiotic tetracycline (TC) is an emerging pollutant frequently detected in various environments. Although enzymatic remediation is a promising strategy for mitigating TC contamination, the availability of effective TC-degrading enzymes remains limited, and their mechanisms and applications are not fully understood. This study developed a comprehensive TC-degrading enzyme library from the gut microbiome of the highly TC-resistant saprophagous insect, black soldier fly larvae (BSFL), using an integrated metagenomic and comparative metatranscriptomic approach, identifying 105 potential novel TC-degradation genes. Bioinformatics analysis of 10 selected genes underscored the novelty of the identified enzymes. Among these, Trg2 demonstrated strong binding affinity and significant degradation capacity for TC. Key functional amino acid residues, including Thr231, Ala64, Ala82, Gly68, Gly79, and Ser81, were identified as essential for the interaction betwe