Bifunctional Enzymes for Deoxynivalenol Degradation in Prokaryotic and Eukaryotic Chassis.

Abstract

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.