Analysis of key enzymes and mechanisms involving aflatoxin B1 (AFB1) degradation in Tenebrio molitor using iTRAQ...

Aflatoxin B1 (AFB1) exhibits high toxicity and wide-ranging hazards, causing substantial economic losses to human society. The insect-based method, as an emerging biological detoxification technology, owes its core advantage to the efficient AFB1-degrading apacity of Tenebrio molitor. Currently, research on the mechanism underlying AFB1 degradation by T. molitor remains scarce and superficial globally. Thus, based on the efficient AFB1-transforming ability of T. molitor, this study uses isobaric tags for relative and absolute quantitation (iTRAQ) to identify the key proteins and metabolic pathways involved in AFB1 degradation by T. molitor. Proteins highly associated with AFB1 detoxification in T. molitor included: Phase I metabolism-related enzymes: Cytochrome P450 monooxygenases (P450s) such as P450 6k1, CYP4Q34, CYP4Q32, CYP6BK19, CYP6BQ36, P450 6a2, CYP9AD2, and CYP347A6; esterases (ESTs) and esterase family members (FE4, CXE3, CXE14); carbonyl reductases, dehydrogenases, and juvenile hormone epoxide hydrolases. Phase II metabolism-related enzymes: Glutathione S-transferases (GSTs) and UDP-glucuronosyltransferases (UGTs). Phase III metabolism-related proteins: ATP-binding cassette (ABC) transporter family members, including G20, A3, and MRP1BX1. Results from in vitro and in vivo experiments using enzyme inhibitors targeting P450s and GSTs demonstrated that P450s, GSTs, and ESTs are critical enzymes in the AFB1 detoxification process of T. molitor. Heterologous expression of CYP4C1 (a member of the P450 enzyme family) was performed in Pichia pastoris; microsomes extracted from the recombinant strain achieved an AFB1 degradation rate of 82 %. HPLC-MS/MS analysis identified the degradation product as Aflatoxin M1 (AFM1), whose toxicity is significantly reduced. This study provides a theoretical basis for using T. molitor as an animal bioreactor for the detoxification of mycotoxin-contaminated grains and the biotransformation of AFB1-polluted raw materials, offering a novel approach to address food and feed safety challenges in China.