Engineering of a highly efficient quinone-dependent dehydrogenase for deoxynivalenol detoxification and evaluation of its in vivo protective efficacy

To effectively address contamination caused by deoxynivalenol (DON), a strain with high DON-degrading activity was isolated from the black soldier fly larvae gut (BSFLG) and identified as Klebsiella pneumoniae BSFLG-DON15. Results showed that BSFLG-DON15 achieved an 83.7% DON degradation rate within 7 d. Crude extraction was conducted on the extracellular enzymes from BSFLG-DON15, with a DON degradation rate of 28.9% detected within 12 h. After purification, the DON degradation rate of the target enzyme was increased to 35.3%, and the purified active protein was identified as quinone-dependent dehydrogenase (QDDH). A two-plasmid expression vector system was constructed to simultaneously express the pyrroloquinoline quinone (PQQ) and QDDH in E. coli , eliminating the dependence of QDDH on exogenous PQQ addition. Molecular docking and site-directed mutagenesis revealed that residues Q294 and R236 played a critical role in the DON oxidation activity of QDDH. The Q294N/R236K double-mutant enzyme increased its DON degradation rate to 53.1%. QDDH metabolized DON into the putative product 3-keto-DON, whereas its Q294N/R236K mutant generated the presumed low-toxicity product DON-15-glucuronide. By optimizing the dual-promoter system and ribosome binding site (RBS), the DON degradation rate of the double-mutant enzyme was increased to 90.8% within 12 h. The in vivo experimental results showed that this double-mutant enzyme effectively alleviated DON-induced intestinal injury, oxidative stress and inflammatory responses in mice, with no detectable adverse effects. • The enzyme’s DON degradation rate was increased from 28.9% to 90.8% via optimization. • A two-plasmid expression system was built to co-express PQQ and QDDH in E. coli . • QDDH-Q294N/R236K metabolized DON into the less toxic DON-15-glucuronide. • QDDH-Q294N/R236K effectively alleviated DON-induced systemic toxicity in mice. • QDDH-Q294N/R236K exhibited no detectable adverse reactions in mice.