Efficient Expression of Lactone Hydrolase Cr2zen for Scalable Zearalenone Degradation in Pichia pastoris

Zearalenone (ZEN) is a thermostable, lipophilic, non-steroidal estrogenic mycotoxin produced by <i>Fusarium</i> spp. that persistently contaminates food and feed. Its strong estrogenic activity and resistance to conventional detoxification strategies pose significant threats to food safety and human and animal health. Conventional physical and chemical degradation methods often compromise nutritional quality and leave toxic residues. Here we report the engineering of a novel <i>Clonostachys rosea</i> lactone hydrolase, Cr2zen, for efficient ZEN degradation in <i>Pichia pastoris</i> under mild conditions. Native Cr2zen exhibited a protein concentration of 0.076 mg/mL, achieving a degradation rate of approximately 17.9% within 30 min, with kinetic parameters of <i>K_m</i> 75.9 µM and <i>V_max</i> 0.482 µmol/L/s at 30 °C and pH 8.0. By integrating signal peptide screening and codon optimization, we identified Ser-Cr2 as the most effective variant, achieving a rapid 81.53% degradation of 10 ppm ZEN under mild conditions. Fed-batch cultivation in a 7.5 L bioreactor resulted in high cell densities of OD₆₀₀ 332.8 for Ser-Cr2 and 310.8 for Oser-Cr2, with extracellular protein concentrations of 0.62 and 0.79 g/L, respectively. The results demonstrate that signal peptide engineering and codon optimization substantially improved the production of lactone hydrolase in <i>P. pastoris</i>. This study establishes a scalable ZEN degradation under mild conditions in <i>P. pastoris</i> and outlines a strategy to integrate protein and process engineering for enhanced enzymatic mycotoxin degradation.