Metabolism of Zearalenone by Genetically Modified Organisms Expressing the Detoxification Gene from Clonostachys rosea

Zearalenone (ZEN) is converted to a nontoxic product by a lactonohydololase encoded by zhd101. An enhanced green fluorescent protein (EGFP) gene was fused to zhd101 (i.e., egfp::zhd101) and expressed in Escherichia coli. Both recombinant ZHD101 and EGFP::ZHD101 were purified to homogeneity and characterized. Maximal activity of ZHD101 toward ZEN was measured at approximately 37 to 45°C and pH 10.5 (k_cat at 30°C, 0.51 s⁻¹). The enzyme was irreversibly inactivated at pH values below 4.5 or by treatment with serine protease inhibitors. ZHD101 was also active against five ZEN cognates, although the efficiencies were generally low; e.g., the k_cat was highest with zearalanone (1.5 s⁻¹) and lowest with β-zearalenol (0.075 s⁻¹). EGFP::ZHD101 had properties similar to those of the individual proteins with regard to the EGFP fluorescence and lactonohydrolase activity. Fortuitously, EGFP::ZHD101 exhibited a good correlation between the fluorescence intensity and reaction velocity under various pH conditions. We therefore used egfp::zhd101 to visually monitor the lactonohydrolase activity in genetically modified organisms and evaluated the usefulness of zhd101 for in vivo detoxification of ZEN. While recombinant E. coli and transgenic rice calluses exhibited strong EGFP fluorescence and completely degraded ZEN in liquid media, recombinant Saccharomyces cerevisiae gave poor fluorescence and did not eliminate all the toxicity of the mycotoxin in the medium; i.e., the rest of ZEN was transformed into an unfavorable substrate, β-zearalenol, by an as-yet-unidentified reductase and remained in the medium. Even so, as much as 75% of ZEN was detoxified by the yeast transformant, which is better than the detoxification system in which food-grade Lactobacillus strains are used (H. El-Nezami, N. Polychronaki, S. Salminen, and H. Mykkuäne, Appl. Environ. Microbiol. 68:3545-3549, 2002). An appropriate combination of a candidate host microbe and the codon-optimized synthetic gene may contribute significantly to establishing a mycotoxin detoxification system for food and feed.     

Metabolism of zearalenone by genetically modified organisms expressing the detoxification gene from Clonostachys rosea

Zearalenone (ZEN) is converted to a nontoxic product by a lactonohydololase encoded by zhd101. An enhanced green fluorescent protein (EGFP) gene was fused to zhd101 (i.e., egfp::zhd101) and expressed in Escherichia coli. Both recombinant ZHD101 and EGFP::ZHD101 were purified to homogeneity and characterized. Maximal activity of ZHD101 toward ZEN was measured at approximately 37 to 45 degrees C and pH 10.5 (k(cat) at 30 degrees C, 0.51 s(-1)). The enzyme was irreversibly inactivated at pH values below 4.5 or by treatment with serine protease inhibitors. ZHD101 was also active against five ZEN cognates, although the efficiencies were generally low; e.g., the k(cat) was highest with zearalanone (1.5 s(-1)) and lowest with beta-zearalenol (0.075 s(-1)). EGFP::ZHD101 had properties similar to those of the individual proteins with regard to the EGFP fluorescence and lactonohydrolase activity. Fortuitously, EGFP::ZHD101 exhibited a good correlation between the fluorescence intensity and reaction velocity under various pH conditions. We therefore used egfp::zhd101 to visually monitor the lactonohydrolase activity in genetically modified organisms and evaluated the usefulness of zhd101 for in vivo detoxification of ZEN. While recombinant E. coli and transgenic rice calluses exhibited strong EGFP fluorescence and completely degraded ZEN in liquid media, recombinant Saccharomyces cerevisiae gave poor fluorescence and did not eliminate all the toxicity of the mycotoxin in the medium; i.e., the rest of ZEN was transformed into an unfavorable substrate, beta-zearalenol, by an as-yet-unidentified reductase and remained in the medium. Even so, as much as 75% of ZEN was detoxified by the yeast transformant, which is better than the detoxification system in which food-grade Lactobacillus strains are used (H. El-Nezami, N. Polychronaki, S. Salminen, and H. Mykku?ne, Appl. Environ. Microbiol. 68:3545-3549, 2002). An appropriate combination of a candidate host microbe and the codon-optimized synthetic gene may contribute significantly to establishing a mycotoxin detoxification system for food and feed.     

Expression in Cereal Plants of Genes That Inactivate Fusarium Mycotoxins

Trichothecene 3-O-acetyltransferase (encoded by Tri101) inactivates the virulence factor of the cereal pathogen Fusarium graminearum. Zearalenone hydrolase (encoded by zhd101) detoxifies the oestrogenic mycotoxin produced by the same pathogen. These genes were introduced into a model monocotyledon rice plant to evaluate their usefulness for decontamination of mycotoxins. The strong and constitutive rice Act1 promoter did not cause accumulation of TRI101 protein in transgenic rice plants. In contrast, the same promoter was suitable for transgenic production of ZHD101 protein; so far, five promising T0 plants have been generated. Low transgenic expression of Tri101 was suggested to be increased by addition of an Ω enhancer sequence upstream of the start codon.     

Reduced Contamination by the Fusarium Mycotoxin Zearalenone in Maize Kernels through Genetic Modification with a Detoxification Gene

Maize is subject to ear rot caused by toxigenic Aspergillus and Fusarium species, resulting in contamination with aflatoxins, fumonisins, trichothecenes, and zearalenone (ZEN). The trichothecene group and ZEN mycotoxins are produced by the cereal pathogen Fusarium graminearum. A transgenic detoxification system for the elimination of ZEN was previously developed using an egfp::zhd101 gene (gfzhd101), encoding an enhanced green fluorescent protein fused to a ZEN-degrading enzyme. In this study, we produced a transgenic maize line expressing an intact copy of gfzhd101 and examined the feasibility of transgene-mediated detoxification in the kernels. ZEN-degrading activity has been detected in transgenic kernels during seed maturation (for a period of 6 weeks after pollination). The level of detoxification activity was unaltered after an additional storage period of 16 weeks at 6°C. When the seeds were artificially contaminated by immersion in a ZEN solution for 48 h at 28°C, the total amount of the mycotoxin in the transgenic seeds was uniformly reduced to less than 1/10 of that in the wild type. The ZEN in the transgenic maize kernels was also efficiently decontaminated under conditions of lower water activity (a_w) and temperature; e.g., 16.9 μg of ZEN was removed per gram of seed within 48 h at an a_w of 0.90 at 20°C. F. graminearum infection assays demonstrated an absence of ZEN in the transgenic maize seeds, while the mycotoxin accumulated in wild-type kernels under the same conditions. Transgene-mediated detoxification may offer simple solutions to the problems of mycotoxin contamination in maize.     

Expression in cereal plants of genes that inactivate Fusarium mycotoxins

Trichothecene 3-O-acetyltransferase (encoded by Tri101) inactivates the virulence factor of the cereal pathogen Fusarium graminearum. Zearalenone hydrolase (encoded by zhd101) detoxifies the oestrogenic mycotoxin produced by the same pathogen. These genes were introduced into a model monocotyledon rice plant to evaluate their usefulness for decontamination of mycotoxins. The strong and constitutive rice Act1 promoter did not cause accumulation of TRI101 protein in transgenic rice plants. In contrast, the same promoter was suitable for transgenic production of ZHD101 protein; so far, five promising T0 plants have been generated. Low transgenic expression of Tri101 was suggested to be increased by addition of an omega enhancer sequence upstream of the start codon.     

Efficient decontamination of zearalenone, the mycotoxin of cereal pathogen, by transgenic yeasts through the expression of a synthetic lactonohydrolase gene

Zearalenone (ZEN), an estrogenic mycotoxin produced by several Fusarium species, is converted to a non-estrogenic product by a detoxifying enzyme of Clonostachys rosea. Previously, we investigated whether recombinant Saccharomyces cerevisiae carrying this detoxification gene, zhd101, can remove 2 g ml^–1 of ZEN in a liquid culture. Although the transgenic yeasts eliminated most of the ZEN, they also converted a significant amount to a poor substrate, -zearalenol, which remained in the medium. In this study, we synthesized a codon-optimized zhd101 gene and investigated whether the transgenic yeast strain can overcome the problem of insufficient detoxification of ZEN. Importantly, within 48 h of incubation at 28°C or 8 h of incubation at 37°C, the transgenic yeasts completely eliminated 2 g ml^–1 of ZEN in the medium without accumulating even a trace amount of -zearalenol. The result suggests that incomplete ZEN detoxification attributed to the action of an endogenous yeast -reductase can be overcome by simply increasing the expression of the detoxifying gene.     

真菌毒素玉米赤霉烯酮生物降解的研究进展

玉米赤霉烯酮(Zearalenone,ZEN)及衍生物是一类主要由镰刀菌属的真菌产生的非甾体雌激素类真菌毒素,广泛存在于玉米、大麦、小麦和高粱等谷物饲料及其副产品中,严重危害牲畜及人类健康,迫切需要相关的技术对ZEN进行降解脱毒。传统的物理化学方法不能有效去除谷物中的毒素,并会破坏谷物的营养成分,影响食物口感,甚至造成二次污染,因此利用生物工程技术对ZEN及其衍生物进行脱毒是未来解决这一问题的主要方法。文中简要介绍了ZEN及衍生物和降解ZEN的微生物种类、降解特性,然后详细介绍了目前研究的ZEN降解酶种类、解析唯一的蛋白结构及其异源表达和应用情况,以期为通过分子酶工程和发酵工程等生物工程技术降低ZEN降解酶的成本提供指导,从而提高食品安全。     

Secretory expression and characterization of a novel peroxiredoxin for zearalenone detoxification in Saccharomyces cerevisiae

Zearalenone (ZEN) is a Fusarium mycotoxin, which is considered to be an oestrogenic endocrine disruptor found to cause severe morphological and functional disorders of reproductive organs in livestock. Increasing attention has been paid to the development of an effective strategy for ZEN decontamination. ZEN is oxidized into smaller estrogenic metabolites by a novel peroxiredoxin (Prx) isolated from Acinetobacter sp. SM04. The Prx coding gene was cloned in a secretory vector pYES2-alpha (pYα) with an alpha (α) signal peptide gene inserted into the multiple cloning site of pYES2. The recombinant Prx was secreted from Saccharomyces cerevisiae INVSc1 after inducing with 2% (w/v) galactose for 72 h, and was found to be nearly 20 kDa through 12% SDS-PAGE. The expressed amount of recombinant Prx was 0.24 mg/mL in the extracellular supernatant. Recombinant Prx showed a gradient increase at the beginning of ZEN degradation. The final ZEN degradation amount was 0.43 μg by one unit recombinant Prx after 12 h. Furthermore, the temperature, H₂O₂ concentration, and pH for highest peroxidase activity of recombinant Prx were 80 °C, 20 mM and 9.0, respectively. When compared with other peroxidases, the thermal stability and alkali resistance of recombinant Prx were much better. The results suggest that recombinant Prx is successfully expressed in S. cerevisiae.     

Catechol metabolites of the mycotoxin zearalenone are poor substrates but potent inhibitors of catechol-O-methyltransferase

The mycotoxin zearalenone (ZEN) elicits estrogenic effects and is biotransformed to two catechol metabolites, in analogy to the endogenous steroidal estrogen 17ß-estradiol (E2). Previous studies have shown that the catechol metabolites of ZEN have about the same potency to induce oxidative DNA damage as the catechol metabolites of E2, but are less efficiently converted to their methyl ethers by human hepatic catechol-O-methyltransferase (COMT). Here, we report that the two catechol metabolites of ZEN, i.e. 13-hydroxy-ZEN and 15-hydroxy-ZEN, are not only poor substrates of human COMT but are also able to strongly inhibit the O-methylation of 2-hydroxy-E2, the major catechol metabolite of E2. 15-Hydroxy-ZEN acts as a non-competitive inhibitor and is about ten times more potent than 13-hydroxy-ZEN, which is an uncompetitive inhibitor of COMT. The catechol metabolites of ZEN were also shown to inhibit the O-methylation of 2-hydroxy-E2 by hepatic COMT from mouse, rat, steer and piglet, although to a lesser extent than observed with human COMT. The powerful inhibitory effect of catechol metabolites of ZEN on COMT may have implications for the tumorigenic activity of E2, because catechol metabolites of E2 elicit genotoxic effects, and their impaired O-methylation may increase the tumorigenicity of steroidal estrogens.     

Degradation of zearalenone by the extracellular extracts of <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. SM04 liquid cultures

A bacterium (designated SM04) which can rapidly grow on zearalenone (ZEN) as sole carbon and energy source was isolated from agricultural soil. On the basis of 16S rDNA sequencing analysis, strain SM04 was classified as a bacterium belonging to the Acinetobacter genus. In this study, the biodegradation of ZEN by the extracellular extracts of strain SM04 liquid cultures in M1 medium and Nutrient Broth medium was examined using HPLC analysis, APCI-MS analysis, and MTT (tetrazolium salt) cell proliferation assay. Results showed no ZEN and other equally estrogenic metabolites were found after 12 h when ZEN was treated with the extracellular extracts of M1 cultures, but no significant (P < 0.01) reduction of ZEN was observed over the 12-h incubation period in the extracellular extracts of Nutrient Broth cultures. Results also indicated that some proteins in the extracellular extracts of M1 cultures were essential to ZEN degradation. The proteins in the extracellular extracts of M1 cultures and Nutrient Broth cultures were analyzed with SDS-PAGE, bands showing different intensities among the two extracellular extracts were processed for protein identification by MALDI-TOF/TOF/MS, and nine proteins from M1 cultures matched the database for Acinetobacter genus with great confidence. Furthermore, the function of some proteins identified is unknown or unconfirmed because of the lack of well-annotated genomic sequence data and protein data for Acinetobacter genus on the public database, but in further studies these data of proteins identified will be useful for screening the genes related to ZEN degradation.     

Isolation and characterization of a <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> strain capable of degrading zearalenone

The worldwide contamination of cereals, oilseeds, and other crops by mycotoxin-producing moulds is a significant problem. Mycotoxins have adverse effects on humans and animals that result in illnesses and economic losses. Reduction or elimination of mycotoxin contamination in food and feed is an important issue. This study aimed to screen soil bacteria for degradation of zearalenone (ZEN). A pure culture of strain CK1 isolated from soil samples showed most capable of degradation of ZEN. Using physiological, biochemical, and 16S rRNA gene sequence analysis methods, CK1 was identified as Bacillus licheniformis. Addition of 2 ppm of ZEN in Luria–Bertani (LB) medium, B. licheniformis CK1 decreased 95.8% of ZEN after 36 h of incubation. In ZEN-contaminated corn meal medium, B. licheniformis CK1 decreased more than 98% of ZEN after 36 h of incubation. In addition, B. licheniformis CK1 was non-hemolytic, non-enterotoxin producing, and displayed high levels of extracellular xylanase, cellulase, and protease activities. These findings suggest that B. licheniformis CK1 could be used to reduce the concentrations of ZEN and improve the digestibility of nutrients in feedstuffs simultaneously.     

Zearalenone (ZEN) detection by a single chain fragment variable (scFv) antibody

Zearalenone (ZEN) is a mycotoxin produced by members of the genus Fusarium. ZEN detection by ELISA using single chain fragment variable (scFv) represents a novel means of mycotoxin detection and diagnosis of fungal infection. To develop immunoassay of ZEN based on scFv, ZEN was conjugated to horseradish peroxidase (HRP). ELISA and SDS-PAGE analysis showed that the covalent attachment of ZEN to HRP was successful. Anti-ZEN scFv-E-tag fusion protein was used to detect ZEN by competitive direct-ELISA (CD-ELISA) method, and the results indicated that this scFv was appropriate in ZEN detection, meaning that scFv against fungal toxin could have an application in mycotoxin detection and diagnosis of fungal infection. Shi-Hua Wang and Xiao-Yu Du contributed to this work equally.     

Crocin and Quercetin protect HCT116 and HEK293 cells from Zearalenone-induced apoptosis by reducing endoplasmic reticulum stress

Mycotoxins are considered to be significant contaminants of food and animal feed. Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium in cereals and agricultural products. ZEN has been shown to be cytotoxic, genotoxic, and mutagenic in different cell types. In the present study, we investigated the involvement of endoplasmic reticulum (ER) stress in ZEN-mediated toxicity in human intestine (HCT116) and kidney (HEK293) cells and evaluated the effects of the two common dietary compounds Quercetin (QUER) and Crocin (CRO). We show that ZEN treatment induces ER stress and activates the unfolded protein response (UPR) as evidenced by XBP1 mRNA splicing and upregulation of GRP78, ATF4, GADD34, PDIA6, and CHOP. Activation of the ER stress response is associated with activation of the mitochondrial pathway of apoptosis. This apoptotic process is characterized by an increase in ROS generation and lipid peroxidation, a loss of mitochondrial transmembrane potential (ΔΨm), and an activation of caspases and DNA damages. We also demonstrate that the antioxidant properties of QUER and CRO help to prevent ER stress and reduce ZEN-induced apoptosis in HCT116 and HEK293 cells. Our results suggest that antioxidant molecule might be helpful to prevent ZEN-induced ER stress and toxicity.     

来源于Rhinocladiella mackenziei的玉米赤霉烯酮水解酶的表达纯化与酶学性质

【背景】玉米赤霉烯酮(Zearalenone,ZEN)是污染最广泛的霉菌毒素之一,对饲料行业和畜牧业造成了巨大的经济损失。目前研究最为广泛的玉米赤霉烯酮降解酶ZHD101因其热稳定性较差,无法满足工业应用上的要求。【目的】为实现玉米赤霉烯酮降解酶在工业上的应用,寻找酶学性质更突出的ZEN降解酶。【方法】基于对Gen Bank数据库的挖掘,发现一个来源于麦氏喙枝孢霉(Rhinocladiella mackenziei CBS 650.93)的Rmzhd基因,构建p ET-46-Rmzhd质粒。利用大肠杆菌表达体系和亲和层析、离子交换纯化体系对蛋白进行表达和纯化,通过高效液相凝胶色谱分析酶学性质。【结果】发现一个新的ZEN水解酶Rm ZHD,RmZHD在pH 8.6和45°C条件下的活性最高,而且具有较高的耐热性。结构分析表明,较高的盐桥数目和溶剂暴露脯氨酸含量可能是造成其高耐热性的原因。【结论】本研究为促进玉米赤霉烯酮降解酶在工业上的应用打下基础。     

Application in photocatalytic degradation of zearalenone based on graphitic carbon nitride

A semiconductor nano-material was prepared, and its degradation efficiency of zearalenone (ZEN) was studied. The photocatalytic material graphitic carbon nitride (g-C₃N₄) was synthesized by the traditional method of hot cracking. Its structure was characterized by X-ray diffraction (XRD), Fourier-transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The photocatalytic degradation experiment showed that under the irradiation of ultraviolet (UV) lamp (254 nm, including 185 nm), g-C₃N₄ could induce photocatalytic effect, which provided a new method for the degradation of ZEN in real powder samples. The experimental conditions of photocatalytic degradation of the primary reference material of ZEN and ZEN in real powder samples were explored. And the degradation products of ZEN were analyzed after high-performance liquid chromatography–mass spectrometry (HPLC–MS). Under each optimal experimental conditions, the degradation rate on primary reference material of ZEN and ZEN in real powder samples was 96.0% and 50.0%, respectively. The results in this work provide a theoretical reference and practical basis for the photocatalytic degradation of mycotoxin in real powder samples by g-C₃N₄.     

Mycotoxin Ochratoxin A-induced cell death and changes in oxidative metabolism of Arabidopsis thaliana

We evaluated the phytotoxicity of mycotoxin ochratoxin A (OTA) from Aspergillus and Penicillium strains on Arabidopsis thaliana. The results demonstrate that the growth of Arabidopsis thaliana on media containing OTA was inhibited significantly. Moreover, OTA induced necrotic lesions in detached leaves, which are reminiscent of hypersensitive response lesions that are activated during plant–pathogen interactions and other abiotic stress factors. From our study, we can see that OTA exposure stimulated a biphasic oxidative burst in the leaves, resulting in the generation of hydrogen peroxide (H₂O₂) and superoxide anion radicals (O₂^·−) and in the concomitant down-regulation of antioxidant enzyme defense responses and up-regulation of lipid peroxidation. These results suggested that OTA damage might result from reactive oxygen species pathways. Our experiments provide a useful model plant system for research on OTA-induced plant cell death.     

Influence of organically or conventionally produced wheat on health,performance and mycotoxin residues in tissues and bile of growing pigs

From 1999?–?2001 three different varieties of wheat [Contur (susceptible to Fusarium), Batis and Petrus (less susceptible to Fusarium)] were cultivated under organic and conventional conditions in order to determine mycotoxin burden. Soil quality, preceding crop and weather conditions were comparable in the different production systems. The wheat batches were analysed for moulds, and the contents of zearalenone (ZEN) and deoxynivalenol (DON). Feeding trials were carried out with growing pigs (n?=?96; average initial live weight 22.2 ±?1.5?kg [mean?±?SD]) to examine a possible influence on the animal performance and on mycotoxin residues. The data recorded were clinical conditions, performance, biochemical and hematological data. Residues of ZEN, α- and β-zearalenol (ZEL) and of DON were determined in bile, liver and muscle after slaughtering. Conventionally cultivated wheat was more frequently contaminated with Fusarium and contained more frequently ZEN and DON in higher concentrations than the organically produced wheat. Hematological and biochemical parameters of pigs fed with organically cultivated diets were not different from those of conventionally fed pigs. Pigs fed with organically produced wheat showed a slightly higher daily weight gain, but a lower carcass yield than the conventionally fed animals. The highest residues of DON and total-ZEN (ZEN + α-ZEL + β-ZEL) were found in bile. Bile samples of organically fed pigs contained lower concentrations of total-ZEN than those of conventionally fed pigs. Altogether, these data suggest that wheat from an organic farming does not have higher mycotoxin-contamination than wheat from the conventional farming system.     

In vitro degradation of zearalenone by Bacillus subtilis

Zearalenone (ZEN) is a non-steroidal estrogen produced by many Fusarium species in cereals and other plants, and is frequently implicated in safety of foods and feeds. A ZEN-degrading microorganism has been isolated and identified as a Bacillus subtilis subspecies. It degraded 99% ZEN (1 mg kg⁻¹) in liquid medium after 24 h and more than 95% of ZEN (0.25 mg kg⁻¹) could be degraded after 48 h in a solid-state fermentation. This isolate can thus be used to decontaminate raw materials, like grains, to reduce the mycotoxin concentration.