Abstract

Dye‐decolorizing peroxidases (DyPs) are heme‐dependent oxidoreductases with promising applications in mycotoxin detoxification by acting on the oxidizable conjugated structures of aflatoxin B 1 (AFB 1 ). The heme prosthetic group is critical for the catalytic process by mediating electron transfer and intermediate formation that govern overall enzymatic activity. However, intracellular heme biosynthesis in microbial hosts is tightly regulated, frequently leading to suboptimal catalytic performance of recombinant DyPs, thus highlighting the necessity to improve cofactor availability. To establish an efficient heme‐based expression system for DyPs, we developed a heme cofactor supplementation system in Escherichia coli by introducing a heterologous C4 pathway harboring 5‐aminolevulinic acid synthase from Caulobacter segnis together with the outer membrane heme transporter ChuA. Furthermore, intracellular heme supply was optimized by the integrated overexpression of the C4 and C5 pathways as well as the hemEFGH gene cluster, which significantly enhanced Rh DypB‐R80 activity. The engineered cells exhibited substantially higher heme content and Rh DypB‐R80 activity compared to those produced by conventional cultivation methods supplemented with exogenous 5‐ALA. Notably, heme‐based Rh DypB‐R80 achieved 91.03% AFB 1 degradation within 24 h, demonstrating its potential for enzymatic detoxification applications. This engineered heme‐producing host provides a practical platform for enhancing hemoprotein activity and supports the development of enzymatic strategies for mycotoxin control in food systems.