Pathway Elucidation and Key Enzymatic Processes in the Biodegradation of Difenoconazole by Pseudomonas putida A-3

The extensive agricultural use of the fungicide difenoconazole (DIF) and its associated toxicity increasingly damage ecosystems and human health. Thus, an urgent need is to develop environmentally friendly technological approaches capable of effectively removing DIF residues. In this study, strain Pseudomonas putida A-3 was isolated for the first time which can degrade DIF efficiently. After optimization of the degradation conditions, the degradation rate reached 75.98%. Moreover, a new DIF degradation pathway, including hydroxylation, hydrolysis, dechlorination, and ether bond breaking. The acute and chronic toxicity of DIF degradation products assessed using ECOSAR software showed lower toxicity than the parent compound. Furthermore, strain A-3 remarkably accelerated the degradation of DIF in contaminated water-sediment systems. We successfully predicted six potential key enzymes for DIF degradation based on the results of whole genome sequencing, RT-qPCR, and molecular docking. Overall, the results revealed novel pathways for DIF biodegradation and provide a strong candidate for bioremediation of DIF residue-polluted environments.