Enzymatic Degradation of Zearalenone in the Gastrointestinal Tract of Pigs, Chickens, and Rainbow Trout.

The estrogenic mycotoxin zearalenone (ZEN) is a common contaminant of animal feed. Effective strategies for the inactivation of ZEN in feed are required. The ZEN-degrading enzyme zearalenone hydrolase ZenA (EC 3.1.1.-, commercial name ZENzyme®, BIOMIN Holding GmbH, Getzersdorf, Austria) converts ZEN to hydrolyzed ZEN (HZEN), thereby enabling a strong reduction in estrogenicity. In this study, we investigated the efficacy of ZenA added to feed to degrade ZEN in the gastrointestinal tract of three monogastric animal species, i.e., pigs, chickens, and rainbow trout. For each species, groups of animals received (i) feed contaminated with ZEN (chickens: 400 µg/kg, pigs: 200 µg/kg, rainbow trout: 2000 µg/kg), (ii) feed contaminated with ZEN and supplemented with ZenA, or (iii) uncontaminated feed. To investigate the fate of dietary ZEN in the gastrointestinal tract in the presence and absence of ZenA, concentrations of ZEN and ZEN metabolites were analyzed in digesta of chickens and rainbow trout and in feces of pigs. Upon ZenA administration, concentrations of ZEN were significantly decreased and concentrations of the degradation product HZEN were significantly increased in digesta/feces of each investigated animal species, indicating degradation of ZEN by ZenA in the gastrointestinal tract. Moreover, upon addition of ZenA to the diet, the concentration of the highly estrogenic ZEN metabolite α-ZEL was significantly reduced in feces of pigs. In conclusion, ZenA was effective in degrading ZEN to HZEN in the gastrointestinal tract of chickens, pigs, and rainbow trout, and counteracted formation of α-ZEL in pigs. Therefore, ZenA could find application as a ZEN-degrading feed additive for these animal species.

Metabolism of Zearalenone in the Rumen of Dairy Cows with and without Application of a Zearalenone-Degrading Enzyme.

The mycotoxin zearalenone (ZEN) is a frequent contaminant of animal feed and is well known for its estrogenic effects in animals. Cattle are considered less sensitive to ZEN than pigs. However, ZEN has previously been shown to be converted to the highly estrogenic metabolite α-zearalenol (α-ZEL) in rumen fluid in vitro. Here, we investigate the metabolism of ZEN in the reticulorumen of dairy cows. To this end, rumen-fistulated non-lactating Holstein Friesian cows (n = 4) received a one-time oral dose of ZEN (5 mg ZEN in 500 g concentrate feed) and the concentrations of ZEN and ZEN metabolites were measured in free rumen liquid from three reticulorumen locations (reticulum, ventral sac and dorsal mat layer) during a 34-h period. In all three locations, α-ZEL was the predominant ZEN metabolite and ß-zearalenol (ß-ZEL) was detected in lower concentrations. ZEN, α-ZEL and ß-ZEL were eliminated from the ventral sac and reticulum within 34 h, yet low concentrations of ZEN and α-ZEL were still detected in the dorsal mat 34 h after ZEN administration. In a second step, we investigated the efficacy of the enzyme zearalenone hydrolase ZenA (EC 3.1.1.-, commercial name ZENzyme®, BIOMIN Holding GmbH, Getzersdorf, Austria) to degrade ZEN to the non-estrogenic metabolite hydrolyzed zearalenone (HZEN) in the reticulorumen in vitro and in vivo. ZenA showed a high ZEN-degrading activity in rumen fluid in vitro. When ZenA was added to ZEN-contaminated concentrate fed to rumen-fistulated cows (n = 4), concentrations of ZEN, α-ZEL and ß-ZEL were significantly reduced in all three reticulorumen compartments compared to administration of ZEN-contaminated concentrate without ZenA. Upon ZenA administration, degradation products HZEN and decarboxylated HZEN were detected in the reticulorumen. In conclusion, endogenous metabolization of ZEN in the reticulorumen increases its estrogenic potency due to the formation of α-ZEL. Our results suggest that application of zearalenone hydrolase ZenA as a feed additive may be a promising strategy to counteract estrogenic effects of ZEN in cattle.

Evaluation of the Efficacy of Mycotoxin Modifiers and Mycotoxin Binders by Using an In Vitro Rumen Model as a First Screening Tool.

Ruminal microbiota of cattle are not able to detoxify all mycotoxins. In addition, detoxification can be hampered by adverse ruminal conditions (e.g., low ruminal pH). Hence, in the cattle husbandry, mycotoxin binders and modifiers could be used to prevent animal exposure to mycotoxins. In this study, an in vitro rumen model, including feed matrix, was established as first screening tool to test the efficacy of five products claiming to detoxify mycotoxins. The detoxifiers had different modes of action: (a) binding (three products); (b) enzymatic detoxification of zearalenone (ZEN; one product, ZenA); and (c) bacterial transformation of trichothecenes (one product, BBSH 797). For the mycotoxin binders, the binding to the mycotoxins enniatin B (ENN B), roquefortine C (ROQ-C), mycophenolic acid (MPA), deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEN) were tested at a dose recommended by the manufacturers. The in vitro model demonstrated that all binders adsorbed ENN B to a certain extent, while only one of the binders also partially adsorbed ROQ-C. The binders did not change the concentrations of the other mycotoxins in the ruminal fluid. The enzyme ZenA detoxified ZEN very quickly and prevented the formation of the more toxic metabolite α-zearalenol (α-ZEL), both at normal (6.8) and low ruminal pH (5.8). The addition of BBSH 797 enhanced detoxification of DON and NIV, both at normal and low ruminal pH. The in vitro rumen model demonstrated that the addition of ZenA seems to be a very promising strategy to prevent estrogenic effects of ZEN contaminated feed, and BBSH 797 is efficient in the detoxification of trichothecenes.

Biotransformation of the Mycotoxin Zearalenone to its Metabolites Hydrolyzed Zearalenone (HZEN) and Decarboxylated Hydrolyzed Zearalenone (DHZEN) Diminishes its Estrogenicity In Vitro and In Vivo.

Zearalenone (ZEN)-degrading enzymes are a promising strategy to counteract the negative effects of this mycotoxin in livestock. The reaction products of such enzymes need to be thoroughly characterized before technological application as a feed additive can be envisaged. Here, we evaluated the estrogenic activity of the metabolites hydrolyzed zearalenone (HZEN) and decarboxylated hydrolyzed zearalenone (DHZEN) formed by hydrolysis of ZEN by the zearalenone-lactonase Zhd101p. ZEN, HZEN, and DHZEN were tested in two in vitro models, the MCF-7 cell proliferation assay (0.01-500 nM) and an estrogen-sensitive yeast bioassay (1-10,000 nM). In addition, we compared the impact of dietary ZEN (4.58 mg/kg) and equimolar dietary concentrations of HZEN and DHZEN on reproductive tract morphology as well as uterine mRNA and microRNA expression in female piglets (n = 6, four weeks exposure). While ZEN increased cell proliferation and reporter gene transcription, neither HZEN nor DHZEN elicited an estrogenic response, suggesting that these metabolites are at least 50-10,000 times less estrogenic than ZEN in vitro. In piglets, HZEN and DHZEN did not increase vulva size or uterus weight. Moreover, RNA transcripts altered upon ZEN treatment (EBAG9, miR-135a-5p, miR-187-3p and miR-204-5p) were unaffected by HZEN and DHZEN. Our study shows that both metabolites exhibit markedly reduced estrogenicity in vitro and in vivo, and thus provides an important basis for further evaluation of ZEN-degrading enzymes.

Disruption of genes involved in CORVET complex leads to enhanced secretion of heterologous carboxylesterase only in protease deficient Pichia pastoris.

The methylotrophic yeast Pichia pastoris (Komagataella spp.) is a popular microbial host for the production of recombinant proteins. Previous studies have shown that mis-sorting to the vacuole can be a bottleneck during production of recombinant secretory proteins in yeast, however, no information was available for P. pastoris. In this work the authors have therefore generated vps (vacuolar protein sorting) mutant strains disrupted in genes involved in the CORVET (class C core vacuole/endosome tethering) complex at the early stages of endosomal sorting. Both Δvps8 and Δvps21 strains contained lower extracellular amounts of heterologous carboxylesterase (CES) compared to the control strain, which could be attributed to a high proteolytic activity present in the supernatants of CORVET engineered strains due to rerouting of vacuolar proteases. Serine proteases were identified to be responsible for this proteolytic degradation by liquid chromatography-mass spectrometry and protease inhibitor assays. Deletion of the major cellular serine protease Prb1 in Δvps8 and Δvps21 strains did not only rescue the extracellular CES levels, but even outperformed the parental CES strain (56 and 80% higher yields, respectively). Further deletion of Ybr139W, another serine protease, did not show a further increase in secretion levels. Higher extracellular CES activity and low proteolytic activity were detected also in fed batch cultivation of Δvps21Δprb1 strains, thus confirming that modifying early steps in the vacuolar pathway has a positive impact on heterologous protein secretion.

Pun1p is a metal ion-inducible, calcineurin/Crz1p-regulated plasma membrane protein required for cell wall integrity.

Under conditions of environmental stress, the plasma membrane is involved in several regulatory processes to promote cell survival, like maintenance of signaling pathways, cell wall organization and intracellular ion homeostasis. PUN1 encodes a plasma membrane protein localizing to the ergosterol-rich membrane compartment occupied also by the arginine permease Can1. We found that the PUN1 (YLR414c) gene is transcriptionally induced upon metal ion stress. Northern blot analysis of the transcriptional regulation of PUN1 showed that the calcium dependent transcription factor Crz1p is required for PUN1 induction upon heavy metal stress. Here we report that mutants deleted for PUN1 exhibit increased metal ion sensitivity and morphological abnormalities. Microscopical and ultrastructural observations revealed a severe cell wall defect of pun1∆ mutants. By using chemical cross-linking, Blue native electrophoresis, and co-immunoprecipitation we found that Pun1p forms homo-oligomeric protein complexes. We propose that Pun1p is a stress-regulated factor required for cell wall integrity, thereby expanding the functional significance of lateral plasma membrane compartments.

Novel components of an active mitochondrial K(+)/H(+) exchange.

Defects of the mitochondrial K(+)/H(+) exchanger (KHE) result in increased matrix K(+) content, swelling, and autophagic decay of the organelle. We have previously identified the yeast Mdm38 and its human homologue LETM1, the candidate gene for seizures in Wolf-Hirschhorn syndrome, as essential components of the KHE. In a genome-wide screen for multicopy suppressors of the pet(-) (reduced growth on nonfermentable substrate) phenotype of mdm38Delta mutants, we now characterized the mitochondrial carriers PIC2 and MRS3 as moderate suppressors and MRS7 and YDL183c as strong suppressors. Like Mdm38p, Mrs7p and Ydl183cp are mitochondrial inner membrane proteins and constituents of approximately 500-kDa protein complexes. Triple mutant strains (mdm38Delta mrs7Delta ydl183cDelta) exhibit a remarkably stronger pet(-) phenotype than mdm38Delta and a general growth reduction. They totally lack KHE activity, show a dramatic drop of mitochondrial membrane potential, and heavy fragmentation of mitochondria and vacuoles. Nigericin, an ionophore with KHE activity, fully restores growth of the triple mutant, indicating that loss of KHE activity is the underlying cause of its phenotype. Mdm38p or overexpression of Mrs7p, Ydl183cp, or LETM1 in the triple mutant rescues growth and KHE activity. A LETM1 human homologue, HCCR-1/LETMD1, described as an oncogene, partially suppresses the yeast triple mutant phenotype. Based on these results, we propose that Ydl183p and the Mdm38p homologues Mrs7p, LETM1, and HCCR-1 are involved in the formation of an active KHE system.