Meichen Liu, Mingzhu Guo, Ye Tian, Huikang Lin, Zheng Yan, Bingxuan Jia, Xiyan Sun, Sarah De Saeger, Orphélie Lootens, Esther De Rycke, José Diana Di Mavungu, Dianzhen Yu, Aibo Wu
Deoxynivalenol (DON), a prevalent mycotoxin produced by Fusarium spp., represents a persistent threat to global food and feed safety. Two innovative strategies were developed herein for efficient DON detoxification. Coupling the genes encoding the aldo-keto reductase AKR13B2 and the pyrroloquinoline quinone-dependent DON dehydrogenase DepA using a flexible linker enabled rational engineering of the bifunctional fusion enzyme BGA (AKR13B2-[GGGGS]2-DepA). This design facilitated proximity-enhanced sequential conversion of DON to 3-keto-DON and the markedly less toxic 3-epi-DON. BGA exhibited significantly enhanced catalytic efficiency, thermal stability, and operational robustness. In parallel, food-grade Kluyveromyces marxianus was engineered for coexpression of codon-optimized DepA and AKR13B2 (termed as KmDepA and KmAKR13B2, respectively), yielding near-complete DON degradation in contaminated wheat grains. KmDepA displayed substantially improved solubility and specific activity relative to its Escherichia coli-expressed counterpart. Collectively, this integrated strategy provides a robust, scalable, and safe method for enzymatic DON remediation, with strong potential for industrial and agricultural applications.