Fusion strategy enables high-level expression of fungal laccase in Pichia pastoris for efficient detoxification of aflatoxin B1 and zearalenone

• A fusion expression strategy was employed to express fungal laccase in P. pastoris. • The fusion protein can transform AFB 1 and ZEN without the need for mediators. • The transformed products showed significantly reduced toxicity. • The fusion protein has potential applications in detoxifying feed ingredients. Detoxification of aflatoxin B 1 (AFB 1 ) and zearalenone (ZEN), which pose substantial threats to both agriculture and food safety, using fungal laccases is an efficient and environmentally friendly technology. However, the difficulty in heterologous expression of fungal laccases has hindered their practical application. In this study, the expression of a Steccherinum ochraceum -originated laccase in Pichia pastoris X33 was achieved through fusion with an Aspergillus niger xylanase ( An XynB). The volumetric activity of laccase was enhanced from an undetectable level to 9102.0 U/L in a 15-L fermenter. Molecular docking of fusion proteins with mycotoxins indicated that So Lac potentially interacts with AFB 1 and ZEN directly. Optimized conditions were established for the mediator-free catalytic transformation of AFB 1 and ZEN using the An XynB- So Lac fusion protein, achieving transformation efficiencies of 71.0% and 72.9%, respectively, under 50 °C and pH 7.0 for 20 h. The main AFB 1 and ZEN transformation products were AFQ 1 , 13-OH-ZEN-quinone, 15-OH-ZEN, and ZOM-1, respectively. Evaluation of toxicity revealed that the transformed products exhibited markedly reduced toxicity. Furthermore, An XynB- So Lac fusion protein effectively transformed AFB 1 and ZEN in raw feed materials, including soybean meal and maize flour. This study describes a novel and feasible strategy for the production of fungal laccases that are difficult to express in P. pastoris in a food-grade manner and illustrated its promise in addressing the detoxification of AFB 1 and ZEN residues in food and feed-related matrices.