Mutual interaction of the entomopathogenic and endophytic fungus Metarhizium anisopliae with zearalenone as a native component of crude Fusarium extract.

The present study revealed the consequences of the interaction of a widely used bioinsecticide and endophyte Metarhizium anisopliae with the hazardous mycotoxin zearalenone (ZEN) as a pure substance and with ZEN as a native component of a crude Fusarium extract. In the environment, microorganisms encounter a mixture of metabolites secreted by other organisms living in the same area, not single substances. The obtained results suggest that M. anisopliae, exposed to a variety of active substances produced by Fusarium graminearum, is able to eliminate ZEN. Within 14 days, M. anisopliae biotransformed 90.8% and 85.8% of ZEN as a pure substance and ZEN as a native component of the F. graminearum extract from Rice Medium (E-Fg-RM), respectively, through reduction predominantly to α-epimers of zearalenols and zearalanols, considered more estrogenic than ZEN, which can raise concerns. Compared to pure ZEN, E-Fg-RM significantly affected the production of Metarhizium secondary metabolites by increasing the destruxins amount by approximately 20-25% and reducing the swainsonine content by 96.2%. All these findings provide a possible picture of the interaction of M. anisopliae with ZEN in the wild, mainly as a result of the use of crude extract from Fusarium, which contained a mixture of different metabolites.

Enzymatic Hydrolysis of Resorcylic Acid Lactones by an Aeromicrobium sp.

Zearalenone and radicicol are resorcylic acid lactones produced by numerous plant pathogenic fungi. Zearalenone is a non-steroidal estrogen mimic that can cause serious reproductive issues in livestock that consume contaminated feed. Radicicol is a potent inhibitor of the molecular chaperone Hsp90, which, in plants, has an important role in coordinating the host's immune response during infection. Here, we describe the identification and characterization of a soil-borne strain of the Gram-positive bacterium Aeromicrobium sp. capable of hydrolyzing the macrolide ring of resorcylic acid lactones, including zearalenone and radicicol. Proteomic analysis of biochemically enriched fractions from the isolated and cultured bacterium identified an α/ß-hydrolase responsible for this activity. A recombinantly expressed and purified form of the hydrolase (termed RALH) was active against both zearalenone and radicicol. Interpretation of high-resolution mass spectrometry and NMR data confirmed the structures of the enzymatic products as the previously reported non-toxic metabolite hydrolyzed zearalenone and hydrolyzed radicicol. Hydrolyzed radicicol was demonstrated to no longer inhibit the ATPase activity of the Saccharomyces cerevisiae Hsp90 homolog in vitro. Enzymatic degradation of resorcylic acid lactones will enable insight into their biological functions.

Characteristics of a Novel Zearalenone Lactone Hydrolase ZHRnZ and Its Thermostability Modification.

Zearalenone (ZEN) is a toxic secondary metabolite produced by the Fusarium fungi, which widely contaminates grains, food, and feed, causing health hazards for humans and animals. Therefore, it is essential to find effective ZEN detoxification methods. Enzymatic degradation of ZEN is believed to be an eco-friendly detoxification strategy, specifically thermostable ZEN degradation enzymes are needed in the food and feed industry. In this study, a novel ZEN lactone hydrolase ZHRnZ from Rosellinia necatrix was discovered using bioinformatic and molecular docking technology. The recombinant ZHRnZ showed the best activity at pH 9.0 and 45 °C with more than 90% degradation for ZEN, α-zearalenol (α-ZOL), ß-zearalenol (ß-ZOL) and α-zearalanol (α-ZAL) after incubation for 15 min. We obtained 10 mutants with improved thermostability by single point mutation technology. Among them, mutants E122Q and E122R showed the best performance, which retained more than 30% of their initial activity at 50 °C for 2 min, and approximately 10% of their initial activity at 60 °C for 1 min. The enzymatic kinetic study showed that the catalytic efficiency of E122R was 1.3 times higher than that of the wild-type (WT). Comprehensive consideration suggests that mutant E122R is a promising hydrolase to detoxify ZEN in food and feed.

Conserved gatekeeper methionine regulates the binding and access of kinase inhibitors to ATP sites of MAP2K1, 4, and 7: Clues for developing selective inhibitors.

Mitogen-activated protein kinase kinases (MAP2Ks) 1, 4, and 7 are potential targets for treating various diseases. Here, we solved the crystal structures of MAP2K1 and MAP2K4 complexed with covalent inhibitor 5Z-7-oxozeaenol (5Z7O). The elucidated structures showed that 5Z7O was non-covalently bound to the ATP binding site of MAP2K4, while it covalently attached to cysteine at the DFG-1 position of the deep ATP site of MAP2K1. In contrast, we previously showed that 5Z7O covalently binds to MAP2K7 via another cysteine on the solvent-accessible edge of the ATP site. Structural analyses and molecular dynamics calculations indicated that the configuration and mobility of conserved gatekeeper methionine located at the central ATP site regulated the binding and access of 5Z7O to the ATP site of MAP2Ks. These structural features provide clues for developing highly potent and selective inhibitors against MAP2Ks. Abbreviations: ATP, adenosine triphosphate; FDA, Food and Drug Administration; MAP2Ks, mitogen-activated protein kinase kinases; MD, molecular dynamics; NSCLC, non-small cell lung cancer; 5Z7O, 5Z-7-oxozeaenol; PDB, protein data bank; RMSD, root-mean-square deviation.

Enhancing the thermal stability and activity of zearalenone lactone hydrolase to promote zearalenone degradation via semi-rational design.

Zearalenone (ZEN) is a fungal toxin produced by Fusarium exospore, which poses a significant threat to both animal and human health due to its reproductive toxicity. Removing ZEN through ZEN lactonase is currently the most effective method reported, however, all published ZEN lactonases suffer from the poor thermal stability, losing almost all activity after 10 min of treatment at 55℃. In this study, we heterologously expressed ZHD11A from Phialophora macrospora and engineered it via semi-rational design. A mutant I160Y-G242S that can retain about 40 % residual activity at 55℃ for 10 min was obtained, which is the most heat-tolerant ZEN hydrolase reported to date. Moreover, the specific activity of the I160Y-G242S was also elevated 2-fold compared to ZHD11A from 220 U/mg to 450 U/mg, which is one of the most active ZEN lactonses reported. Dynamics analysis revealed that the decreased flexibility of the main-chain carbons contributes to increased thermal stability and the improved substrate binding affinity and catalytic turnover contribute to enhanced activity of variant I160Y-G242S. In all, the mutant I160Y-G242S is an excellent candidate for the industrial application of ZEN degradation.

Mycotoxin production in different varieties of Dactylis glomerata L. silage in response to biological and chemical additives.

Silage has been identified as a source of different microbial toxins, that may impair farm animal health and productivity as human health can also be compromised. In this sense, the aim of this study was to determine the impact of silage additives on the concentrations of deoxynivalenol (DON) and zearalenone (ZEN) mycotoxins and, eventually, to evaluate the hygienic quality of orchardgrass (Dactylis glomerata L.) silage based on the concentration of them compared to control silage. This study evaluated the influence of biological and chemical additives used in six different varieties of orchardgrass silage on DON and ZEN mycotoxin contents for the first time. The content of both fusariotoxins (DON and ZEN) in fresh matter and grass silage were below the threshold stipulated by the European Commission. The concentration of DON ranges from ~21.86 to 37.26 ng/kg, ~10.21 to 15 ng/kg, ~20.72 to 29.14 ng/kg; and ZEN range from ~3.42 to 7.87 ng/kg, ~3.85 to 8.62 ng/kg and ~2.15 to 5.08 ng/kg, in control, biological and chemical silages, respectively. In general, the biological additive was more efficient for preventing DON contamination, whereas the chemical additive was more efficient for preventing ZEN contamination in grass silage. In summary, the results obtained in this work demonstrate that biological and chemical additives can inhibit fungal growth and mycotoxin production on Dactylis glomerata L. silage and whose use could prevent animal and human diseases.

Biochemical Characterization and Application of Zearalenone Lactone Hydrolase Fused with a Multifunctional Short Peptide.

Zearalenone (ZEN) is an estrogenic mycotoxin causing reproductive toxicity in livestock. Currently, lactone hydrolases are used in the enzymatic degradation of ZEN. However, most lactone hydrolases suffer from low degradation efficiency and poor thermal stability. ZHD518, as a documented neutral enzyme for ZEN degradation, exhibits high enzymatic activity under neutral conditions. In this study, a multifunctional peptide S1v1-(AEAEAHAH)2 was fused to the N-terminus of ZHD518. Compared with the wild-type enzyme, the peptide fusion significantly enhanced protein expression by 1.28 times, enzyme activity by 9.27 times, thermal stability by 37.08 times after incubation at 45 °C for 10 min and enzyme stability during long-term storage. Moreover, ZEN concentrations in corn bran, corn germ meal, and corn gluten powder decreased from 5.29 ± 0.04, 5.31 ± 0.03, and 5.30 ± 0.01 µg/g to 0.48 ± 0.05, 0.48 ± 0.06, and 0.21 ± 0.04 µg/g, respectively, following a 60 min treatment with S1v1-GS-ZHD518, resulting in degradation rates of 90.98, 91.00, and 95.32%, respectively. In conclusion, the properties of S1v1-GS-ZHD518, such as its efficient degradability, high temperature resistance and storage resistance, offer the possibility of its application in food or feed.

Actinobacteria as Promising Biocontrol Agents for In Vitro and In Planta Degradation and Detoxification of Zearalenone.

Zearalenone (ZEN) is a prevalent mycotoxin found in grains and grain-derived products, inducing adverse health effects in both animals and humans. The in-field application of microorganisms to degrade and detoxify ZEN is a promising strategy to enhance the safety of food and feed. In this study, we investigated the potential of three actinobacterial strains to degrade and detoxify ZEN in vitro and in planta on wheat ears. The residual ZEN concentration and toxicity in the samples were analysed with UHPLC-MS/MS and a bioluminescence BLYES assay, respectively. Streptomyces rimosus subsp. rimosus LMG19352 could completely degrade and detoxify 5 mg/L ZEN in LB broth within 24 h, along with significant reductions in ZEN concentration both in a minimal medium (MM) and on wheat ears. Additionally, it was the only strain that showed a significant colonisation of these ears. Rhodococcus sp. R25614 exhibited partial but significant degradation in LB broth and MM, whereas Streptomyces sp. LMG16995 degraded and detoxified ZEN in LB broth after 72 h by 39% and 33%, respectively. Although all three actinobacterial strains demonstrated the metabolic capability to degrade and detoxify ZEN in vitro, only S. rimosus subsp. rimosus LMG19352 showed promising potential to mitigate ZEN in planta. This distinction underscores the importance of incorporating in planta screening assays for assessing the potential of mycotoxin-biotransforming microorganisms as biocontrol agents.

Application of luminescent Photobacterium Phosphoreum T3 for the detection of zearalenone and estimating the efficiency of their enzymatic degradation.

Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin, causes enormous economic losses in the food and feed industries. Simple, rapid, low-cost, and quantitative analysis of ZEN is particularly urgent in the fields of food safety and animal husbandry. Using the bioluminescent bacterium Photobacterium phosphoreum T3, we propose a bioluminescence inhibition assay to evaluate ZEN levels quickly. The limit of detection (LOD), limit of quantification (LOQ), and quantitative working range of this bioluminescence inhibition assay were 0.1 µg/mL, 5 µg/mL, and 5-100 µg/mL, respectively. The concentration-response curve of the bioluminescence inhibition rate and ZEN concentration was plotted within the range 5 to 100 µg/mL, as follows: y = 0.0069x2 - 0.0190x + 7.9907 (R2 = 0.9943, y is luminescence inhibition rate, x is ZEN concentration). First, we used the bioluminescence inhibition assay to detect the remaining ZEN in samples treated with purified lactonohydrolase ZHD101. The bioluminescence inhibition assay results showed a strong correlation with the HPLC analysis. Furthermore, we successfully evaluated the overall toxicity of samples treated with purified peroxidase Prx and H2O2 using the P. phosphoreum T3 bioluminescence inhibition assay. The results indicate that the degradation products of ZEN created by purified peroxidase Prx and H2O2 showed little toxicity to P. phosphoreum T3. In this study, a simple, rapid, and low-cost assay method of zearalenone by bioluminescent P. phosphoreum T3 was developed. The bioluminescence inhibition assay could be used to estimate the efficiency of enzymatic degradation of ZEN.

Zearalenone degrading enzyme evolution to increase the hydrolysis efficiency under acidic conditions by the rational design.

Based on rational design, zearalenone degrading enzyme was evolved to improve the hydrolysis efficiency under acidic conditions. At pH 4.2 and 37 °C, the activity of the zearalenone degrading enzyme evolved with 8 mutation sites increased from 7.69 U/mg to 38.67 U/mg. Km of the evolved zearalenone degrading enzyme decreased from 283.61 µM to 75.33 µM. The evolved zearalenone degrading enzyme was found to effectively degrade zearalenone in pig stomach chyme. Molecular docking revealed an increase in the number of hydrogen bonds and π-sigma interactions between the evolved zearalenone degrading enzyme and zearalenone. The evolved zearalenone degrading enzyme was valuable for hydrolyzing zearalenone under acidic conditions.

Thermo-Alkali-Tolerant Recombinant Laccase from Bacillus swezeyi and Its Degradation Potential against Zearalenone and Aflatoxin B1.

The enzymatic biodegradation of mycotoxins in food and feed has attracted the most interest in recent years. In this paper, the laccase gene from Bacillus swezeyi was cloned and expressed in Escherichia coli BL 21(D3). The sequence analysis indicated that the gene consisted of 1533 bp. The purified B. swezeyi laccase was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis -12% with an estimated molecular weight of 56.7 kDa. The enzyme is thermo-alkali-tolerant, displaying the optimal degradation of zearalenone (ZEN) and aflatoxin B1 (AFB1) at pH 8 and 9, with incubation temperatures of 55 and 50 °C, respectively, within 24 h. The degradation potentials of the 50 µg of the enzyme against ZEN (5.0 µg/mL) and AFB1 (2.5 µg/mL) were 99.60 and 96.73%, respectively, within 24 h. To the best of our knowledge, this is the first study revealing the recombinant production of laccase from B. swezeyi, its biochemical properties, and potential use in ZEN and AFB1 degradation in vitro and in vivo.

Preparation of immunomagnetic composite nanostructures with bifunctional four-arm PEG derivatives as linkers for the ultrafast enrichment of zearalenone and its metabolites.

It is challenging to prepare sample pretreatment materials with simple use, strong selectivity and satisfactory enrichment performance. In this study, the antibody (3D4) that can specifically recognize zearalenone (ZEN) and its metabolites was immobilized on the surface of gold-coated magnetic Fe3O4 nanoparticles (GMN) by streptavidin (SA)-biotin interaction using GMN as the substrate and our designed four-arm PEG derivative (HS-4ARMPEG10K-(CM)3) as the linker. The immunomagnetic nanoparticles (GMN-4ARMPEG10K-SA-3D4) prepared by this strategy can achieve rapid enrichment (only 5 min) of analytes directly in the matrix, and higher enrichment capacity compared with the previous immunomagnetic particles. The sensitive and accurate analysis of ZEN and its metabolites can be achieved coupled with HPLC-MS/MS. The LODs and LOQs were 0.02-0.05 µg/kg and 0.05-0.10 µg/kg, respectively. The recoveries were 84.13%-112.67%, and the RSDs were 1.09%-9.39%. The method can provide a powerful tool for highly sensitive and rapid monitoring of mycotoxins in complex matrices due to its' strong selectivity and resistance to matrix interference.

A novel glycosyltransferase from Bacillus subtilis achieves zearalenone detoxification by diglycosylation modification.

Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin produced by Fusarium spp., contaminates cereals and threatens human and animal health by inducing hepatotoxicity, immunotoxicity, and genotoxicity. In this study, a new Bacillus subtilis strain, YQ-1, with a strong ability to detoxify ZEN, was isolated from soil samples and characterized. YQ-1 was confirmed to degrade more than 46.26% of 20 µg mL-1 ZEN in Luria-Bertani broth and 98.36% in fermentation broth within 16 h at 37 °C; one of the two resulting products was ZEN-diglucoside. Under optimal reaction conditions (50 °C and pH 5.0-9.0), the reaction mixture generated by YQ-1 catalyzing ZEN significantly reduced the promoting effect of ZEN on MCF-7 cell proliferation, effectively eliminating the estrogenic toxicity of ZEN. In addition, a new glycosyltransferase gene (yqgt) from B. subtilis YQ-1 was cloned with 98% similarity to Bs-YjiC from B. subtilis 168 and over-expressed in E. coli BL21 (DE3). ZEN glycosylation activity converted 25.63% of ZEN (20 µg mL-1) to ZEN-diG after 48 h of reaction at 37 °C. The characterization of ZEN degradation by B. subtilis YQ-1 and the expression of YQGT provide a theoretical basis for analyzing the mechanism by which Bacillus spp. degrades ZEN.

Zearalenone Decreases Food Intake by Disrupting the Gut-Liver-Hypothalamus Axis Signaling via Bile Acids.

Zearalenone (ZEN) is a mycotoxin that is harmful to humans and animals. In this study, female and male rats were exposed to ZEN, and the results showed that ZEN reduced the farnesoid X receptor (FXR) expression levels in the liver and disrupted the enterohepatic circulation of bile acids (BAs). A decrease in food intake induced by ZEN was negatively correlated with an increase in the level of total BAs. BA-targeted metabolomics revealed that ZEN increased glycochenodeoxycholic acid levels and decreased the ratio of conjugated BAs to unconjugated BAs, which further increased the hypothalamic FXR expression levels. Preventing the increase in total BA levels induced by ZEN via Lactobacillus rhamnosus GG intervention restored the appetite. In conclusion, ZEN disrupted the enterohepatic circulation of BAs to decrease the level of food intake. This study reveals a possible mechanism by which ZEN affects food intake and provides a new approach to decrease the toxic effects of ZEN.

Heme Oxygenase-1 Regulates Zearalenone-Induced Oxidative Stress and Apoptosis in Sheep Follicular Granulosa Cells.

Zearalenone (ZEA) is a common non-steroidal estrogenic mycotoxin found in a range of animal feeds and poses a serious threat to the reproductive health of farm animals and humans. However, the mechanism underlying ZEA-induced reproductive toxicity in sheep remains unknown. Granulosa cells are crucial for egg maturation and the fertility of female sheep. In this study, we aimed to examine the impact of different ZEA concentrations on sheep follicular granulosa cells and to elucidate the potential molecular mechanism underlying ZEA-induced toxicity using transcriptome sequencing and molecular biological approaches. Treating primary sheep follicular granulosa cells with different concentrations of ZEA promoted the overproduction of reactive oxygen species (ROS), increased lipid peroxidation products, led to cellular oxidative stress, decreased antioxidant enzyme activities, and induced cell apoptosis. Using transcriptome approaches, 1395 differentially expressed genes were obtained from sheep follicular granulosa cells cultured in vitro after ZEA treatment. Among them, heme oxygenase-1 (HMOX1) was involved in 11 biological processes. The protein interaction network indicated interactions between HMOX1 and oxidative and apoptotic proteins. In addition, N-acetylcysteine pretreatment effectively reduced the ZEA-induced increase in the expression of HMOX1 and Caspase3 by eliminating ROS. Hence, we suggest that HMOX1 is a key differential gene involved in the regulation of ZEA-induced oxidative stress and apoptosis in follicular granulosa cells. These findings provide novel insights into the prevention and control of mycotoxins in livestock.

Enhancing the activity of zearalenone lactone hydrolase toward the more toxic α-zearalanol via a single-point mutation.

Zearalenone (ZEN) and its derivatives are estrogenic mycotoxins known to pose significant health threats to humans and animals. Especially, the derivative α-zearalanol (α-ZAL) is over 10 times more toxic than ZEN. Simultaneous degradation of ZEN and its derivatives, especially α-ZAL, using ZEN lactone hydrolases (ZHDs) is a promising solution to eliminate their potential hazards to food safety. However, most available ZHDs exhibit limited activity toward the more toxic α-ZAL compared to ZEN. Here, we identified a broad-substrate spectrum ZHD, named ZHDAY3, from Exophiala aquamarina CBS 119918, which could not only efficiently degrade ZEN but also exhibited 73% relative activity toward α-ZAL. Through rational design, we obtained the ZHDAY3(N153H) mutant, which exhibited the highest specific activity (253.3 ± 4.3 U/mg) reported so far for degrading α-ZAL. Molecular docking, structural comparative analysis, and kinetic analysis collectively suggested that the shorter distance between the side chain of the catalytic residue His242 and the lactone bond of α-ZAL and the increased binding affinity to the substrate were mainly responsible for the improved catalytic activity of ZHDAY3(N153H) mutant. This mechanism was further validated through additional molecular docking of 18 mutants and experimental verification of six mutants.IMPORTANCEThe mycotoxins zearalenone (ZEN) and its derivatives pose a significant threat to food safety. Here, we present a highly promising ZEN lactone hydrolase (ZHD), ZHDAY3, which is capable of efficiently degrading both ZEN and the more toxic derivative α-ZAL. Next, the ZHDAY3(N153H) mutant obtained by single-point mutation exhibited the highest specific activity for degrading α-ZAL reported thus far. We further elucidated the molecular mechanisms underlying the enhanced hydrolytic activity of ZHDAY3(N153H) toward α-ZAL. These findings represent the first investigation on the molecular mechanism of ZHDs against α-ZAL and are expected to provide a significant reference for further rational engineering of ZHDs, which will ultimately contribute to addressing the health risks and food safety issues posed by ZEN-like mycotoxins.

Zearalenone Promotes Uterine Hypertrophy through AMPK/mTOR Mediated Autophagy.

Zearalenone (ZEN), a non-steroidal Fusarium graminearum with an estrogen effect, can cause damage to the gastrointestinal tract, immune organs, liver, and reproductive system. Further analysis of the mechanism of ZEN has become an important scientific issue. We have established in vivo and in vitro models of ZEN intervention, used AMPK/mTOR as a targeted pathway for ZEN reproductive toxicity, and explored the molecular mechanism by which ZEN may induce uterine hypertrophy in weaned piglets. Our study strongly suggested that ZEN can activate the phosphorylation of AMPK in uterine endometrial epithelium cells, affect the phosphorylation level of mTOR through TSC2 and Rheb, induce autophagy, upregulate the expression of proliferative genes PCNA and BCL2, downregulate the expression of apoptotic gene BAX, promote uterine endometrial epithelium cells proliferation, and ultimately lead to thickening of the endometrial and myometrium, increased density of uterine glands, and induce uterine hypertrophy.

Caspase-8 dependent apoptosis contributes to dyskinesia caused by muscle defects and neurotoxicity in zebrafish exposed to zearalenone.

Zearalenone (ZEA), one of the usual mycotoxins, has been recognized in many areas and crops, posing a significant threat to the living organisms even to human beings. However, the mechanisms of locomotive defects remain unknown. Herein, zebrafish larvae was employed to investigate ZEA effects on developmental indexes, muscle and neural toxicity, apoptosis, transcriptome and motor behaviors of zebrafish larvae. Zebrafish larvae exposed to ZEA (0, 0.5, 1, 2 and 4 µM) showed no change in survival rate, but the malformation rate of zebrafish larvae increased dramatically manifesting with severe body bending and accomplished with adverse effects on hatching rate and body length. Moreover, the larvae manifested with defective muscle and abnormal neural development, resulting in decreased swimming ability, which probably due to the abnormal overactivation of apoptosis. And this was confirmed by enriched caspase 8-mediated apoptosis signaling pathway in the following transcriptome analysis. Meanwhile, there was a recovery in swimming behaviors in the larvae co-exposed in ZEA and caspase 8 inhibitor. These findings provide an important evidence for risk assessment and potential treatment target of ZEA exposure.

Characterization, Structural Analysis, and Thermal Stability Mutation of a New Zearalenone-Degrading Enzyme Mined from Bacillus subtilis.

Zearalenone (ZEN) is a widespread mycotoxin that causes serious damage to animal husbandry and poses a threat to human health. A screen of ZEN-degrading soil bacteria yielded Bacillus subtilis YT-4, which yielded 80% ZEN degradation after 6 h and 95% after 36 h. The gene sequence encoding the degradative enzyme ZENY was mined from the genome of YT-4 and expressed in yeast. ZENY is an α/ß-hydrolase with an optimal enzyme activity at 37 °C and pH 8. By breaking the lactone ring of ZEN, it produces ZENY-C18H24O5 with a molecular weight of 320.16 g/mol. Sequence comparison and molecular docking analyses identified the catalytic ZENY triad 99S-245H-123E and the primary ZEN-binding mode within the hydrophobic pocket of the enzyme. To improve the thermal stability of the enzyme for industrial applications, we introduced a mutation at the N-terminus, specifically replacing the fifth residue N with V, and achieved a 25% improvement in stability at 45 °C. These findings aim to achieve ZEN biodegradation and provide insight into the structure and function of ZEN hydrolases.

Identification of the hub genes linked to zearalenone-induced hepatotoxicity in broiler chickens.

Zearalenone (ZEN) is a mycotoxin found in food and feed that impairs the function of multiple organs, especially the liver. However, the specific mechanisms through which ZEN induces liver damage in broiler chickens are not well understood. Therefore, this study aimed to identify the key genes linked to the hepatotoxicity induced by ZEN exposure in broiler chickens. Gene expression data from ZEN-treated and control chicken embryo primary hepatocytes (CEPHs) were used to implement differential expression analysis. Totally, 436 differentially expressed genes (DEGs) were detected, in which 223 and 213 genes were up- and down-regulated in ZEN-treated CEPHs, respectively. Gene ontology analysis suggested that these DEGs were involved in various biological processes, including chromosome segregation, mitotic cytokinesis, mitotic cell cycle, cell division, and mitotic spindle organization. Pathway analysis showed that the DEGs were associated with p53, FoxO, ubiquitin-mediated proteolysis, cell cycle, and mismatch repair signaling pathways. Furthermore, the hub genes, including BRCA1, CDC45, CDCA3, CDKN3, CENPE, CENPF, CENPI, CENPM, CENPU, and CEP55, potentially contributed to ZEN-induced hepatotoxicity. In conclusion, our study provides the valuable insight into the mechanism underlying ZEN-induced hepatotoxicity in broiler chickens.