Degradation of aflatoxin b1 and zearalenone by recombinant ligninase extracellularly produced from Lactococcus lactis

Aflatoxin B1 (AFB1) and zearalenone (ZEN) are two common mycotoxins found in grain feed, which cause substantial economic losses. This study employed molecular docking predictions, codon optimization, signal peptide modification, and a nisin-controlled gene expression system. The heterologous expression of acid ligninase (LigX) in food-grade Lactococcus lactis was achieved for the first time to degrade these toxins. Molecular docking experiments verified the high-affinity interaction between LigX and mycotoxins. Key residues within the enzyme engage the toxins via cation-π contacts, π-π stacking, and an extensive hydrogen-bond network. The lowest computed binding free energies are -7.5 kcal mol^-1 for AFB1 and - 7.1 kcal mol^-1 for ZEN, indicating highly favorable complex formation. Codon optimization and signal peptide engineering successfully increased the extracellular LigX expression efficiency by 34 % in Lactococcus lactis to a high level (up to 78 U/L). At 1 ng/mL nisin, the extracellular enzyme production of the nisin control system increased by 480 %. The SGM17 medium contained Mg²⁺ and Ca²⁺ (as coenzyme activator), which effectively increased the degradation rates of AFB1 (14.7 %) and ZEN (73.9 %) under 48 h. Tests revealed the optimized recombinant strain degraded 41 % of AFB1 and 97 % of ZEN under low pH 6 conditions. LC-MS analysis predicted key degradation products and pathways: AFB1 underwent furan ring epoxidation and lactone cleavage, while ZEN was metabolized via hydroxylation, lactone hydrolysis, and oxidative polymerization-pathways that disrupt toxic structures and reduce toxicity. This study demonstrates the potential of engineered L. lactis system as a promising biocontrol strategy for mycotoxin mitigation in feed and food.