Polypeptides for enzymatically detoxifying zearalenone, and isolated polynucleotides, and related additives, uses, and methods

Abstract

The present invention relates to polypeptides for enzymatically detoxifying zearalenone, which are monooxygenases that convert the keto group at position 7 of zearalenone into an ester group, in particular an amino acid sequence selected from the group consisting of SEQ ID No. 1, 2 and 3 or a functional variant thereof, wherein there is at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% sequence identity between the functional variant and at least one of said amino acid sequences.

Description

[0001] The present application relates to polypeptides for the enzymatic detoxification of zearalenone, isolated polynucleotides encoding polypeptides for the detoxification of zearalenone, transgenic host cells for the production of monooxygenases for the detoxification of zearalenone, additives for the enzymatic detoxification of zearalenone and uses thereof, and methods for the enzymatic detoxification of zearalenone.

[0002] Fungal toxins are secondary metabolites produced by filamentous fungi. One important representative is zearalenone (ZEN), formerly known as F-2 toxin, which is spread worldwide and is produced by a number of Fusarium fungi. These fungi infect, inter alia, cultivated plants, such as various cereals, where fungal infection usually occurs before harvest, where fungal growth or fungal toxin production can occur before harvest or also after harvest in the case of inadequate storage. The FAO estimates that 25% of agricultural products worldwide are contaminated with fungal toxins, which leads to enormous economic losses. In a recent worldwide study, a total of 23,781 samples were analyzed from January 2009 to 2011, 81% of which tested positive for at least one fungal toxin and 45% for ZEN. ZEN can be found in all regions of the world, likewise in all tested cereal and feed classes, such as corn, soybean meal, wheat, wheat bran, DDGS (dried distillers grains), and in premix feed mixtures, with a frequency of up to 100%.

[0003] ZEN is a non-steroidal, estrogenic, macrocyclic, lactone synthesized via a polyketide metabolite pathway, which has the following structural formula:

[0004]

[0005] and the IUPAC nomenclature name "(2E, 11S)-15,17-dihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraene-7,13-dione". For ZEN and ZEN metabolites, different nomenclatures exist, of which the nomenclature of Metzler (2011, Mycotox. Res., 27: 1-3) is used in this document as far as possible. In addition to ZEN, a number of ZEN derivatives exist in nature, which are formed by enzymatic or chemical modification of ZEN. In addition, ZEN metabolites are formed, inter alia, in the human or animal body.

[0006] ZEN, likewise also ZEN derivatives, such as a-ZEL or β-ZEL, can also be detected in processed food or feed, such as bread, beer or dried distillers grains, due to their high chemical and physical stability.

[0007] Although ZEN has a relatively low acute toxicity and has an oral LD50 value of up to 20,000 mg / kg body weight, in the case of longer-term ingestion subacute and / or subchronic toxic effects can occur in animals or humans, such as teratogenic, carcinogenic, immunosuppressive and oestrogenic effects. ZEN binds to oestrogen receptors and can cause hormonal disorders, so that the reduction of the metabolites formed in comparison to ZEN is generally understood as "detoxification or decontamination of zearalenone".

[0008] Ingestion of feed contaminated with ZEN leads to developmental disorders in mammals, with pigs, in particular young pigs, being extremely sensitive to ZEN. Concentrations of ZEN in feed of more than 0.5 ppm lead to developmental disorders, with concentrations of more than 1.5 ppm, for example, being able to cause hyperoestrogenic activity in pigs, and concentrations of 12 ppm ZEN being considered responsible for abortions in cattle. Since zearalenone is rapidly absorbed through the mucous membranes, in particular through the gastric mucous membranes, but also through the oral mucous membranes, immediate and in particular quantitative detoxification is necessary. They can already be detected in the blood 30 minutes after oral administration of ZEN. In order to achieve as complete a detoxification as possible, the use of isolated enzymes has proved advantageous over microorganisms, since they have a higher specific activity or act more quickly. Due to the harmful effects of ZEN, there is a mandatory upper limit for ZEN in food in the EU and a recommendation for an upper limit for ZEN in feed (EC NO: 1881 / 2006).

[0009] The first strategy for reducing ZEN contamination of food or feed is to limit fungal growth, for example by adhering to "good agricultural practice experience". This includes, inter alia, that the seed is free from pests and fungal infestation or that agricultural waste is removed from the field in time. In addition, fungal growth on the field can also be reduced by using fungicides. After harvesting, the harvest should be stored at a residual moisture content of less than 15% and at low temperatures in order to prevent fungal growth. Likewise, material contaminated by fungal infestation should be removed before further processing. Despite this series of measures, I. Rodriges and K. Naehrer (2012) report that even in regions with the highest agricultural standards, such as the USA and Central Europe, 29% and 39% of the corn samples tested were contaminated with ZEN in 2009 to 2011, respectively.

[0010] Other possibilities for the removal of ZEN from feed or food are the adsorption or transformation of the mycotoxin. Necessary for this is that the binding of the mycotoxin to the adsorbent is strong and specific over a broad pH range and remains stable over the entire digestion in the gastrointestinal area. While several abiotic adsorbents, such as activated carbon, silicates or synthetic polymers, such as cholecalciferol, can be used effectively for aflatoxins, they are limited for other mycotoxins. The main disadvantage of adsorbents is the non-specific binding of other molecules which are sometimes essential for the nutrient supply. In the literature also biological adsorbents, such as yeasts or yeast extracts, are described, however, biological adsorbents are similarly limited as abiotic adsorbents.

[0011] The detoxification of ZEN by physical and chemical treatments is also limited. By heat treatment ZEN cannot be effectively inactivated, but by extrusion and treatment with oxidizing agents, such as a 10% hydrogen peroxide solution at 80°C for 16 hours, the ZEN content can be reduced by 83.9%. The use of extrusion methods and oxidizing agents, such as ozone or hydrogen peroxide, in the preparation of feed and food is limited due to high costs, loss of quality, sometimes low efficiency and low specificity.

[0012] From EP 0 938 575 B1 the ZEN degrading properties of bacteria of the genus Rhodococcus and Nocardia, in particular R. globerulus, R. erythropolis and N. globerula, are known.

[0013] Vekiru et al. (Appl. and Environ. Microb., 2010, 76, 7, 2353-2359) describe the biological transformation of ZEN to the less estrogenic metabolite ZOM-1 by the microorganism Trichosporon mycotoxinivorans.

[0014] The terms used below are taken from the professional language and are always used in the conventional sense, unless otherwise indicated. Thus, the term "polynucleotide" relates to every type of genetic material of all lengths and sequences, such as single- and double-stranded DNA and RNA molecules, including regulatory elements, structural elements, gene pools, plasmids, whole genomes and fragments thereof. The designation "polypeptide" includes proteins, such as enzymes, antibodies, and polypeptides having up to 500 amino acids, such as peptide inhibitors, domains of proteins, but also short polypeptides having a small sequence length, such as less than 10 amino acids, such as receptors, ligands, peptide hormones, tags and the like. The designation "position in a polynucleotide or polypeptide" relates to a single specific base or amino acid in the sequence of the polynucleotide or polypeptide.

[0015] The present application now aims to provide such a polypeptide with which the rapid and reliable conversion of zearalenone into a zearalenone derivative can be successfully achieved, which zearalenone derivative is reduced in its toxicity and estrogenic effect to such an extent that it can remain in the feed or food without danger for the respective end user.

[0016] To solve this task, the present application is primarily characterized in that the polypeptide is a monooxygenase which converts the keto group at position 7 of zearalenone into an ester group. Monooxygenases are enzymes which belong to group I of the EC-classification and which catalyze the incorporation of an oxygen atom from O2 into the respective substrate, in this case zearalenone. More precisely, in the case of zearalenone, the oxygen atom is incorporated at position 7 of the ring structure of zearalenone, whereby a zearalenone derivative (iZOM) is obtained which has a lower toxicity intensity. It has surprisingly been proven that this detoxification takes place in only a single enzymatic reaction step; and the reaction product iZOM, despite the still present closed ring structure and the carbon-oxygen double bond at position 7, has a toxicity which is approximately 100 times lower and almost no longer exhibits an estrogenic effect.

[0017] It has been assumed so far that the cleavage of the lactone ring system is necessary for an effective detoxification of ZEN. However, it has surprisingly been proven that an extremely effective detoxification is also achieved by converting the keto group at position 7 of zearalenone into an ester group, in which rearrangement no cleavage of the ring system takes place.

[0018] According to a further aspect of the present application, the monooxygenase is selected such that it is a Baeyer-Villiger monooxygenase. The use of a Baeyer-Villiger monooxygenase system ensures that the reaction can take place only at the desired position 7, since only this position is available for the so-called Baeyer-Villiger oxidation, i.e. the conversion of a ketone into an ester by incorporation of an oxygen atom.

[0019] According to a further aspect of the present application, the polypeptide is selected such that it detoxifies at least 70% of the zearalenone in one step. This is successfully achieved, in particular, because the reaction product iZOM surprisingly has a toxicity which is approximately 100 times lower.

[0020] Due to the specific structure of the zearalenone molecule and in particular due to the ketone group present at position 7 of the zearalenone molecule, the targeted incorporation of the desired oxygen atom at position 7 of the zearalenone is successfully achieved by means of a subgroup of Baeyer-Villiger monooxygenases, namely the cyclohexanone monooxygenases, as this corresponds to a further aspect of the present application. The ester group resulting from this incorporation surprisingly leads to the fact that the formed cyclic metabolite (iZOM) has a significantly lower toxicity than zearalenone.

[0021]

[0022] When the polypeptide is an amino acid sequence selected from the group consisting of SEQ ID No. 1, 2 and 3 or a functional variant thereof, in which there is at least 60%, preferably at least 70%, more preferably at least 80% and most preferably at least 90% sequence identity between the functional variant and at least one of the amino acid sequences, the complete conversion of zearalenone into the less toxic derivative iZOM is successfully achieved. Due to the presence of at least one of these conserved amino acid sequences, such as the sequences of SEQ ID No. 1, 2 and 3 or a functional variant thereof, it is successfully achieved that such a polypeptide is provided for use, which, in addition to the rapid and complete conversion of ZEN, also has a particularly high activity value compared to the hitherto known polypeptides for converting ZEN.

[0023] The term "sequence identity" refers to the percentage of sequence identity. For amino acid sequences and nucleotide sequences, the sequence identity to each other can be determined visually, but preferably is calculated with a computer program. The amino acid sequences having SEQ ID No. 1, 2 and 3 and the nucleotide sequences having SEQ ID No. 3, 4 and 5 are defined as reference sequences. The sequence comparison is also carried out within sequence segments, wherein a consecutive sequence of the reference sequence is understood as a segment. For nucleotide sequences, the length of the sequence segment is usually 18 to 600 nucleotides, preferably 45 to 200 nucleotides, more preferably 100 to 150 nucleotides. For peptide sequences, the length of the sequence segment is usually 3 to 200 amino acids, more preferably 15 to 65 amino acids, most preferably 30 to 50 amino acids. There are a number of bioinformatics programs available for purchase or free of charge, which can be called upon for the determination of homology and are continuously further developed. Examples thereof are the GCG Wisconsin Bestfit software package (Devereux et al., 1984), BLAST (Altschul et al., 1990) or BLAST 2 (Tatusova and Madden, 1999). Since different setting possibilities of these algorithms are possible, it is possible that they give different results with the same input sequences, so that the search algorithm and the settings associated therewith must be defined. In the present case, the sequence identity is determined by means of the program NCBI BLAST (Basic Local Alignment Search Tool), in particular BLASTP for polypeptides and BLASTN for polynucleotides (in the version of 20 October 2014), which is provided for use on the homepage of the "National Center for Biotechnology Information" (NCBI; http: / / www.ncbi.nlm.nih.gov / ). It is thus possible to compare two or more sequences with each other according to the algorithm of Altschul et al., 1997 (Nucleic Acids Res., 25: 3389-3402). Here, the program of the version of 15 May 2013 is used.As program settings, the basic settings are considered, but in particular, for amino acid sequence comparison: "max target sequence" = 100; "expected threshold" = 10; "word size" = 3; "matrix" = BLOSUM62; "gap costs" = "Existence: 11; Extention: 1"; "computational adjustment" = "Conditional compositional score matrix adjustment"; and for nucleotide sequence comparison: word size: 11; Expect value: 10; Gap costs: Existence = 5, Extention = 2; Filter = low complexity activated; Match / Mismatch Scores: 2, -3; Filter String: L; m.

[0024] The term "functional polypeptide variant" or "functional variant" relates on the one hand to "allelic variants" of a polypeptide and "functional fragments" of a polypeptide and on the other hand to "modified polypeptides", wherein the enzymatic function is essentially not changed compared to the polypeptide having SEQ ID No. 1. The designation "allelic variant" relates to a polypeptide which is generated by a randomly occurring nucleotide sequence mutation in nature and which causes a change in the amino acid sequence, wherein the enzymatic function is not affected. The term "functional fragment" relates to a part or partial sequence of a polypeptide or a part or partial sequence of a functional variant thereof, wherein the enzymatic function is essentially retained. The "modified polypeptide" can be a C-terminal or N-terminal fusion protein or a polypeptide which is intentionally mutated, wherein the mutation can be obtained by substitution, insertion or deletion of at least one amino acid, in particular by site-specific mutagenesis or random mutagenesis, recombination and / or any other protein engineering method, wherein the enzymatic function is essentially retained. The terms "substitution", "insertion" and "deletion" are used in the meaning which is generally and familiar to the skilled person in genetic technology. The enzymatic function is essentially retained when the enzymatic reaction mechanism remains unchanged, i.e. the transformation of the keto group at position 7 of mycotoxin ZEN into the corresponding ester group (as shown in the reaction mechanism mentioned above) and the specific residual activity relative to the original polypeptide is at least 5%, preferably at least 10%, in particular at least 50%.

[0025] The polypeptides having the amino acid sequences of SEQ ID No. 1 and 3 are functional allelic variants, wherein the sequences each originate from different microorganisms. This is evident from the close mutual relationship measured by means of the percentage of sequence identity, and from the fact that the polypeptides act on ZEN by the same mechanism. The polypeptide having SEQ ID No. 1 is completely contained in the sequence having SEQ ID No. 2, whereas the polypeptide having SEQ ID No. 2 has a 28-amino-acid-long N-terminus.

[0026] The term "detoxification" or "detoxify" means reduction of the estrogenic activity of ZEN. Here, the measurement of the estrogenic activity is preferably carried out according to the method described in the examples. Other methods can also be employed, wherein the determining factor is always the reduction of the estrogenic activity of ZEN by its enzymatic conversion into iZOM. With the estrus performance assay described in the examples, iZOM has an estrogenic activity which is about 100 times lower than that of ZEN.

[0027] Furthermore, the present application aims at providing isolated polynucleotides for use with which the production of polypeptides for the rapid and reliable detoxification of ZEN can be successfully achieved.

[0028] To solve this task, the present application is primarily characterized in that the isolated polynucleotide has a nucleotide sequence which encodes a polypeptide which is a monooxygenase which converts the keto group at position 7 of zearalenone into an ester group, and / or which has a degree of sequence identity of at least 60% to at least one nucleotide sequence selected from the group consisting of SEQ ID No. 4, 5 and 6. Here, the nucleotide sequence can hybridize under conditions of medium stringency to at least one nucleotide sequence selected from the group consisting of SEQ ID No. 4 to 6, and / or to a partial sequence thereof having at least 200 nucleotides, in particular at least 100 nucleotides, and / or to the complementary strand of the aforementioned nucleotide sequence or partial sequence. By expression of such a polynucleotide, it can be ensured that the resulting polypeptide converts ZEN into iZOM.

[0029] The nucleotide sequence to be expressed, in particular its triplets (codons), generally varies with the host cell, so that the codon bias is optimized to the host cell. This leads to the fact that polynucleotides having a degree of sequence identity which is far below 80%, also below 70% or below 60%, can also encode the same polypeptide. The sequence comparison for determining the degree of sequence identity must also be carried out within sequence segments, wherein a segment refers to a contiguous sequence of the reference sequence. For nucleotide sequences, the length of the sequence segment is generally between 15 and 600.

[0030] By means of the existing isolated nucleotide sequences or sequence segments, nucleic acid probes having a length of usually at least 15, 30 or 40 nucleotides can be successfully produced. By means of such nucleotide sequences, which are referred to as probes (most of which are additionally labelled, for example, with 3 H, 32 P, 35 S, biotin or avidin), it is possible to identify nucleotide sequences encoding polypeptides having a degrading effect on ZEN by using standard methods. As starting material for identifying such sequences, it is possible to consider, for example, the DNA, RNA or cDNA of an individual microorganism, a genomic DNA library, or a cDNA library.

[0031] For nucleotide sequences having a length of at least 100 nucleotides, medium stringency conditions are defined as prehybridization and hybridization at 42°C in a solution containing 0.3% sodium dodecyl sulfate (SDS), 200 μg / ml sheared and denatured salmon sperm DNA, and 35% formamide in 5x NaCI Na-EDTA buffer (SSPE, 0.9 M NaCI, 60 mM NaH2PO4, 6 mM EDTA) followed by three washes in standard Southern blot conditions with 2x sodium chloride-citrate buffer (SSC, 300 mM NaCI and 30 mM trisodium citrate, 0.2% SDS) at 55°C for 15 min.

[0032] For nucleotide sequences having a length of 15 nucleotides to 100 nucleotides, medium stringency conditions are defined as prehybridization and hybridization at 42°C in 0.9 M NaCI, 0.09 M Tris-HCI pH=7.6, 6 mM EDTA, 0.5% NP-40, 1 x Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium bicarbonate, 0.1 mM ATP, and 0.2 mg / ml yeast RNA followed by washing once in 6x SCC buffer containing 0.1% SDS for 15 min and twice in 6x SSC buffer for 15 min each at a temperature 5 to 10°C lower than the calculated melting temperature (Tm) determined by calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA, 48: 1390). Subsequently, the assay is continued under standard Southern blotting conditions (J. Sambrook, E. F. Fritsch and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor, New York). Finally, the carrier material is washed with 6x SCC buffer containing 0.1% SDS for 15 min once and with 6x SSC buffer for 15 min twice each at a temperature 5 to 10°C lower than the calculated Tm.

[0033] It is another object of the present application to provide a transgenic host cell for the production of a monooxygenase detoxifying zearalenone for use.

[0034] To solve this task, the present application is mainly characterized in that the host cell expresses a polynucleotide having a nucleotide sequence encoding at least one polypeptide being a monooxygenase transforming a keto group at position 7 of zearalenone and / or having a degree of sequence identity of at least 60% to at least one nucleotide sequence selected from the group consisting of SEQ ID No. 4, 5 and 6, wherein the polynucleotide is integrated in a chromosome or is present extrachromosomally and the host cell is a prokaryotic or eukaryotic cell, in particular a yeast cell or a plant cell, and the host cell optionally additionally overexpresses an enzyme recycling a cofactor needed for the monooxygenase, in particular an enzyme transforming NADP + or NAD + into NADPH or NADH.

[0035] NADP + or NAD +Enzymes that convert NADPH or NADH and the cofactors required for this can be taken from the prior art, for example from Torres Pazmino et al., 2008 (Angew. Chem. 47(12), 2275-8). Preferably, the enzyme that converts NADP + or NAD + is selected in such a way that it can be used without problems in the food and / or feed industry, in particular in such a way that it is compatible with the provisions of the respective food and / or feed law. For example, as an enzyme that converts NADP + to NADPH, an NADPH-dependent xylose reductase, in particular from Pichia stipitis, can be used, which uses xylitol as a substrate that is allowed as a feed additive. As an alternative, a mannitol dehydrogenase from Lactobacillus sp. that converts NAD(P)H can also be used, in which mannitol as a substrate that is allowed as a feed additive can be used.

[0036] Preferably, it is possible to produce or express the polypeptide for detoxifying ZEN and the enzyme that enables the recycling of the cofactor as a fusion protein. Thereby, both enzyme activities can be produced in one common process, in particular in a biotechnological upstream and downstream process. Furthermore, in the case of using such a fusion protein, it is ensured that the cofactor that is recycled between the two oxidation steps remains in spatial proximity to the polypeptide for detoxifying ZEN.

[0037] Finally, the present application aims to provide an additive for use with which the successful detoxification of ZEN in a defined or complex matrix, for example in a feed or food, can be achieved quickly and reliably, the detoxification being defined as a reduction in the estrogenic activity.

[0038] To solve this task, an additive for detoxifying zearalenone for use is provided, wherein the additive comprises at least one polypeptide that is a monooxygenase that converts the keto group at position 7 of zearalenone into an ester group, the monooxygenase preferably comprising an amino acid sequence selected from the group consisting of SEQ ID No. 1, 2 and 3 or a functional variant thereof, wherein the sequence identity between the functional variant and at least one of the amino acid sequences is at least 60%; and optionally, at least one auxiliary agent. With such an additive, the one-step biochemical conversion of ZEN to a zearalenone derivative, namely iZOM, which is about 100 times less toxic than zearalenone, can be successfully achieved directly in a food or feed.

[0039] According to a preferred further aspect of the present application, the additive is produced such that the at least one auxiliary is selected from the group consisting of inert carriers, vitamins, minerals, enzymes, further components for detoxifying mycotoxins, and co-factors, in particular NADPH and / or NADH. By using such an additive, it can be ensured that a major part of the amount of ZEN contained, for example, in food or feed, is safely and reliably oxidized to iZOM, thereby guaranteeing no harmful effects on the organism of a subject ingesting these feeds or foods.

[0040] Here, the additive can comprise, in addition to the polypeptide according to the present application, for example, an enzyme preparation as auxiliary, which further comprises at least one enzyme, for example, which participates in the degradation of proteins, for example, proteases, or which participates in the metabolism of starch or fiber or fat or glycogen, for example, amylases, cellulases or glucanases, and, for example, hydrolytic enzymes, lipolytic enzymes, mannosidases, oxidases, oxidoreductases, phytases, xylanases, and / or combinations thereof.

[0041] Further possible auxiliaries which can be used according to the present application are enzyme preparations which, in addition to comprising at least one polypeptide according to the present application, comprise at least one component for detoxifying mycotoxins other than zearalenone, for example, enzymes for detoxifying mycotoxins, for example, aflatoxin oxidase, amatoxine hydrolase, amatoxine amide hydrolase, ochratoxin amide hydrolase, fusarinine carboxylate esterase, fusarinine amino transferase, aminopolylol amino oxidase, deoxynivalenol epoxide hydrolase; and / or at least one microorganism for detoxifying mycotoxins; and / or at least one component for binding mycotoxins, for example, microbial cell walls or inorganic materials, for example, bentonite.

[0042] According to a particularly preferred further aspect of the present application, the polypeptide is contained in the additive in such a concentration that a rapid conversion of ZEN into the non-toxic or significantly less toxic metabolite iZOM can be successfully achieved, in particular before its uptake by the body of a subject, in particular a mammal, consuming the contaminated feed or food.

[0043] Herein, the polypeptide can be present in the composition in encapsulated or coated form, wherein for the encapsulation or coating process standard methods can be considered, such as those described in WO 92 / 12645. By the encapsulation or coating process, it is possible to successfully transport the polypeptide to its site of use without change, in particular without degradation or impairment, so that after the protective shell has dissolved, for example in the digestive tract of an animal, the polypeptide only then begins to act, whereby a more targeted, faster and more complete conversion of ZEN is also possible in an acidic, protease-rich and oxygen-poor environment. Furthermore, by the encapsulation or coating process, it is also possible to successfully improve the temperature stability of the polypeptide in the additive.

[0044] The encapsulated or coated polypeptide can be further processed into a premix with the aid of auxiliaries that are customary in the feed industry.

[0045] Furthermore, the present application also aims at the use of the additive for detoxifying zearalenone in feed, in particular for pigs, poultry and aquaculture, in food or in distillers dried grains. By the use of the additive according to the present application, it is possible to successfully oxidize ZEN contained in food or feed or in distillers dried grains to iZOM by converting the keto group at position 7 into an ester group and thereby detoxifying it, wherein such detoxification has been successfully achieved with low polypeptide concentrations in contaminated feed or food.

[0046] Herein, preferably, the polypeptide is present in the range of about 1 g to about 500 g, the encapsulated or coated polypeptide is present in the range of about 20 g to about 3000 g, the premix is present in the range of about 200 g to about 10 kg or the additive is present in the range of about 5 mg to about 10 kg per ton of feed or food or distillers dried grains. Thereby, it is possible to convert the ZEN present in the feed or food, which can be present therein in concentrations of up to 10,000 pg / kg, predominantly into iZOM.

[0047] If the additive is used in the liquefaction of starch, in the saccharification, in the biogas process or in the fermentation process, for example in the mashing process or the fermentation process of bioethanol production, it is possible to ensure the conversion of ZEN in the products used for the process or resulting therefrom, for example distillers dried grains or starch, into iZOM, whereby no health-damaging amounts of ZEN remain intact.

[0048] Furthermore, the present application also aims at providing a method for use with which it is possible to rapidly and reliably convert ZEN into the nontoxic derivative iZOM.

[0049] To solve this task, the method is carried out in such a way that zearalenone is converted into iZOM and thus detoxified in one step enzymatic oxidation reaction by adding a polypeptide, in particular a monooxygenase, wherein the keto group at position 7 of zearalenone is converted into an ester group; and, optionally, the co-factor oxidized in this reaction, in particular NADP+ or NAD+, is reduced to NADPH or NADH by means of another enzyme. By this one-step enzymatic reaction, not only the rapid incorporation of an oxygen atom is ensured, which together with the keto group at position 7 forms an ester group, but especially it is ensured that the reaction proceeds to completion and zearalenone is completely converted into iZOM, which is about 100 times less toxic. At the same time, NADP + or NAD + can be converted or recycled into NADPH or NADH by the enzyme system already described.

[0050] According to a further aspect of the application, the method is carried out in such a way that a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID No. 1, 2 and 3 or a functional variant thereof is used as polypeptide for the enzymatic detoxification of zearalenone, wherein the sequence identity between the functional variant and at least one of the amino acid sequences is at least 60%. In the case of the use of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID No. 1, 2 and 3, a particularly complete detoxification of zearalenone is observed.

[0051] As this corresponds to a further aspect of the application, the method according to the application can be used here not only in the additive itself but also in transgenic host cells, in transgenic plants or in seeds and leads to consistently good results and in particular to the complete conversion of zearalenone into iZOM independent of the site of use.

[0052] Here, the method can be carried out in such a way that the polypeptide or the additive is mixed with a feed or food contaminated with zearalenone, the obtained mixture is brought into contact with moisture and the polypeptide or the additive detoxifies the zearalenone contained in the contaminated feed or food. In the case of moist feed or food, for example malt slurry or mash, the conversion of zearalenone into iZOM takes place in the feed or food before oral intake, whereby it can be ensured that the harmful effects of zearalenone on humans and animals are largely eliminated. Here, moisture means the presence of water or a water-containing liquid, wherein for example saliva or other liquids present in the digestive tract also belong to this category.

[0053] The method of the application can also be carried out in such a way that the feed or food is granulated before oral intake.

[0054] According to a further aspect of the present application, the method is carried out such that zearalenone is detoxified in the course of one reaction step to an extent of at least 70 %, preferably at least 80 %, in particular at least 90 %, as is shown in particular also in Example 3. Thereby, subacute and / or subchronic toxic effects in animals or humans, such as teratogenic, carcinogenic, estrual and immunosuppressive effects, can be prevented.

[0055] The present application will be explained in more detail below on the basis of examples and the accompanying figures. Therein:

[0056] Figure 1 Partial conversion of ZEN by S. cerevisiae in negative control.

[0057] Figure 2 Conversion of ZEN in test batch by S. cerevisiae expressing the polypeptide of SEQ ID No. 1.

[0058] Figure 3 Chemical structure of iZOM.

[0059] Figure 4 Estrogenic activity of ZEN (A) and iZOM (B). Figure 4 A) and iZOM (B). Figure 4 B).

[0060] All molecular biology or microbiology work was carried out by using standard methods if not more precisely indicated (Methods in Enzymology, Vol. 194, pages 3-933 (1991); Guide to Yeast Genetics and Molecular Biology. Edited by Christine Guthrie, Gerald R. Fink. ISBN: 978-0-12-182095-4; J. Sambrook et al., 2012, Molecular Cloning, A Laboratory Manual, 4thEdition, Cold Spring Harbor).

[0061] Example 1: Formation of iZOM from ZEN in one reaction step

[0062] The yeast strain YZGA515 (Saccharomyces cerevisiae, which was modified by inactivation of the ABC transporter gene PDR5 (Poppenberger et al., 2003, J. Biol. Chem., 278(48) 47905-14) for improved uptake of zearalenone (ZEN)) was transformed with the plasmid pCS57 by means of standard methods. pCS57 is a plasmid derived from the yeast expression plasmid pADH-FW (Mitterbauer et al., 2002, Appl. Environ. Microbiol., 68(3), 1336-1346) with LEU2 as selection marker and the ADH1 promoter.

[0063] In the test batch, the yeast strain YZGA515 was transformed with the pCS57 vector, which additionally comprises the polynucleotide having SEQ ID No. 4 as a BamHI-XhoI fragment behind the strong constitutive ADH1 promoter (alcohol dehydrogenase 1). This makes it possible for the polypeptide having SEQ ID No. 1 to be formed.

[0064] As a negative control, the yeast strain YZGA515 was transformed with the empty vector (pADH-FW without the polynucleotide having SEQ ID No. 4).

[0065] The transgenic yeasts (test batch and negative control) were cultivated in SC-LEU medium (Sherman 1991, Methods Enzymol., 194, 3-21), centrifuged and resuspended in fresh SC-LEU medium at a concentration of 2 mg / l ZEN, with the cell density being adjusted to OD600 = 4 in both cases. After different incubation times, samples were taken, 1 volume of methanol was added to the samples and the intracellular processes were thereby terminated. The cell suspensions were clarified by centrifugation and the resulting supernatants were used for the measurement by means of HPLC-MS. The starting substance zearalenone (ZEN) and the conversion product ZOM-1 formed by the mycotoxophilic Trichosporon asperellum (Vekuri et al., 2010, Appl. Environ. Microbiol. 76(7), 2353-9) were measured as described previously.

[0066] In the negative control transformed with the empty vector, no ZOM-1 and in particular no iZOM was formed. The corresponding HPLC analysis data are shown in Figure 1 , where the y-axis indicates nanograms per millilitre (ng / ml) and the x-axis indicates time in hours (h).

[0067] In contrast thereto, in the test batch zearalenone was significantly more rapidly converted and a new metabolite was identified, which has the expected mass of the intermediate postulated by Vekiru et al. (2010) (in the following referred to as iZOM). In addition, ZOM-1 was also detectable, which is a clear indication that the hydrolysis from iZOM to ZOM occurs to a small extent. The corresponding HPLC analysis data are shown in Figure 2

[0068] In another test batch, the metabolite iZOM was additionally quantitatively measured. The molar balance of the metabolism of ZEN is shown in Table 1. It is clearly apparent therefrom that after 6 hours, the majority of the used zearalenone (ZEN) has been converted into iZOM. The small deficit in the balance table can be explained by the formation of further metabolites and by the inaccuracy of the analysis, which is determined by the method. These results clearly show that the polypeptide having SEQ ID No. 1 is able to convert ZEN into the metabolite iZOM.

[0069] Table 1: Molar balance of the metabolism of ZEN in the test batch

[0070]

[0071] Example 2: Identification of the chemical structure of iZOM

[0072] Chemical structure of the metabolite iZOM Figure 3 ) can be determined by means of NMR measurements of the isolated and purified metabolite. For the measurement of the NMR spectrum, a sufficient amount of iZOM was prepared from the culture filtrate of several liters of yeast culture (SC-LEU, as described in Example 1). The iZOM was purified by means of solid-phase extraction and subsequent preparative HPLC.

[0073] By means of the isolated reference material for iZOM, the conversion of ZEN into iZOM (see Example 1) can be followed on the one hand and the chemical structure can be ascertained via NMR on the other hand.

[0074] The NMR spectrum for the identification of iZOM was obtained at room temperature (20°C) in a CD3OD solution with a 5 mm inverse broad-band z-gradient probe using a Bruker Avance DRX-400 FT-NMR spectrometer. The chemical shifts were established on the residual solvent resonance (about 1 H NMR, 3.31 ppm; about 13 ​on the basis of the 1H and13C NMR data (49.15 ppm). All pulse programs were taken from the Bruker software library. NMR data were evaluated with the help of TopSpin 1.3 (Bruker BioSpin GmbH). The complete structure determination and signal assignment were based on 1 H, 13 C-APT, 1 H 1 H correlation spectroscopy (COSY), 1 H 13 C heteronuclear single quantum correlation spectroscopy (HSQC), and 1 H 13 C heteronuclear multiple band correlation spectroscopy (HMBC).

[0075] The NMR spectroscopic data of iZOM show two major differences compared to ZEN. This confirms the transformation of the keto group into an ester group. The differences are mainly the shift of the carbonyl carbon atom from 212 ppm (characteristic for a ketone) in ZEN to 175 ppm (characteristic for a carboxylic acid derivative) and the shift of the adjacent CH2group from 36 ppm and 2.90 / 2.30 ppm (position 8 in ZEN) to 65 ppm and 4.20 / 4.05 (position 9 in iZOM) (respectively with regard to 13 C and 1 H), which is caused by the adjacent oxygen atom. In contrast to the ring opening of ZOM1, the macrocycle is still intact in iZOM as is evident from the long-range correlation between C7 and H9. The NMR data are given in Table 2. The chemical structure of iZOM is shown in Figure 3 .

[0076] Table 2: 1H and13C NMR data used for the identification of iZOM 1 H and 13 C

[0077]

[0078] Example 3: Strongly reduced estrogenic activity of iZOM compared to ZEN

[0079] For this, the purified metabolite iZOM (as described in Example 2) was used to determine its toxicity expressed in estrogenic activity and compared to the toxicity of zearalenone.

[0080] For the measurement of estrogenic activity a reporter yeast strain, YZHB817, was used, which was prepared specifically for this purpose (Bachmann, H.: Phenotypic detection of zearalenone in Saccharomyces cerevisiae. Master thesis BOKU Vienna, 2003). This strain was derived from the yeast two-hybrid strain PJ69-4a (MATa trp1-901 leu2-3, 112 ura3-52 his3-200 gal4-(deleted) gal80 (deleted) LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ) (James et al., 1996, Genetics, 144(4), 1425-1436). PJ69-4a was further developed by disruption of the ABC transporters PDR5 and SNQ2. The strain with the double mutation pdr5 and snq2 showed a particularly high uptake of ZEN (Mitterbauer et al., 2003, Appl. Environ. Microbiol., 69(2), 805-811). Subsequently, this strain was transformed with an expression vector (pTK103) which triggers the production of a fusion protein comprising the hormone-dependent activation domain of the human estrogen receptor alpha and the DNA binding domain of the yeast Gal4 protein to obtain strain YZHB817.

[0081] In the presence of estrogenic substances the yeast strain YZHB817 induces the expression of the GAL7 lacZ reporter gene, the product of which, beta-galactosidase, can be easily measured by hydrolysis of the chromogenic substrate ONPG (ortho-nitrophenyl-beta-galactoside) at 420 nm (Current Protocols in Molecular Biology, Chapter 13, Yeast (editors Lundblad V, Struhl, K.)).

[0082] 100 μl of the estrogen test solution (ZEN or iZOM) were dissolved in 50% ethanol and mixed with 1.9 ml of a yeast culture of strain YZGA817 in SC-TRP medium with a cell density of OD 600 = 0.1 and incubated for 18 hours at 30°C and 180 rpm. Thereafter, the OD 600and 1 ml of the cell pellet (10 min, 13 krpm) was resuspended in 500 ml Z-buffer and permeabilized with 25 μl chloroform. The enzymatic reaction was started by adding 100 μl ONPG solution (4 mg / ml) and terminated after yellow coloration by adding 250 μl of a 1 M Na2CO3solution. The supernatant was measured at 420 nm and "relative units" (relative to the amount of cells used) were calculated as follows:

[0083] RU = (OD 420 x 1000) / (OD 600 x incubation time in minutes).

[0084] As shown in Figure 4 , ZEN induces the expression of the GAL7-lacZ reporter gene in the low ppb range, so that for the expression of 7.5 relative units of β-galactosidase, approximately 45 ppb ZEN Figure 4 A) are required. In contrast thereto, iZOM shows a much lower estrogenic performance. In comparison, for approximately the same effect, approximately 500 ppb Figure 4 B) are required. It follows that iZOM exerts an estrogenic effect almost 100 times lower than ZEN.

[0085] Example 4: Determination of sequence identity

[0086] The determination of the percentage of sequence identity between the polypeptides having the amino acid sequences of SEQ ID No. 1, 2 and 3 is carried out by means of the mutual alignment of two sequences by means of the program BLAST (Basic Local Alignment Search Tool), in particular with BLASTP, which is available on the homepage of the "National Center for Biotechnology Information" (NCBI; http: / / www.ncbi.nlm.nih.gov / ). It is thus possible to compare two or more sequences with each other according to the algorithm of Altschul et al., 1997 (Nucleic Acids Res. (1997) 25: 3389-3402). As program settings, the basic settings are considered, but in particular: "Max. target sequences" = 100; "Expect threshold" = 10; "Word length" = 3; "Matrix" = BLOSUM62; "Gap costs" = "Existence: 11; Extension: 1"; "Calculation adjustment" = "Conditional composition-adjusted score matrix". The sequence identity between the sequence having SEQ ID No. 1 and the sequence having SEQ ID No. 3 is 73%.

Claims

1. Additive for the enzymatic detoxification of zearalenone, characterized in that, The additive comprises at least one polypeptide for the enzymatic detoxification of zearalenone, and optionally at least one auxiliary agent, wherein the polypeptide is a monooxygenase which converts the keto group at position 7 of zearalenone into an ester group and consists of the amino acid sequence of SEQ ID No.

1.

2. The additive of claim 1, wherein, The polypeptide detoxifies at least 70% of the zearalenone in one step reaction.

3. The additive of any one of claims 1 to 2, wherein The at least one auxiliary agent is selected from the group consisting of inert carriers, vitamins, minerals, enzymes, other components for the detoxification of mycotoxins, and co-factors.

4. The additive of claim 3, wherein, The co-factor is NADPH and / or NADH.

5. Use of the additive according to any one of claims 1 to 4 for the enzymatic detoxification of zearalenone in feed and in distillers dried grain or in food.

6. Use according to claim 5, characterized in that, The feed is feed for swine, poultry and aquaculture.

7. A method for detoxifying zearalenone enzymatically, characterized in that, In one step enzymatic oxidation reaction zearalenone is converted into iZOM and thereby detoxified by the addition of a polypeptide, wherein the keto group at position 7 of zearalenone is converted into an ester group; optionally the co-factor NADP+ or NAD+ oxidized in this reaction is reduced to NADPH or NADH by means of another enzyme; and a polypeptide consisting of the amino acid sequence of SEQ ID No. 1 is used as polypeptide for the enzymatic detoxification of zearalenone.

8. The method of claim 7, wherein, The polypeptide is used in an additive according to any one of claims 1 to 4, or in a transgenic host cell which expresses a polynucleotide encoding the polypeptide and optionally additionally overexpresses an enzyme which recycles the co-factor needed for the oxygenase.

9. The method of claim 7, wherein, The polypeptide is used in a transgenic plant or in the seeds of the transgenic plant, wherein the transgenic plant has transgenic plant cells for the production of a monooxygenase which detoxifies zearalenone, wherein the plant cells express a polynucleotide encoding the polypeptide, which is integrated in the chromosome or is present extrachromosomally, and the plant cells optionally additionally overexpress an enzyme which recycles the co-factor needed for the oxygenase.

10. The method of claim 9, wherein, The enzyme which recycles the co-factor needed for the oxygenase is an enzyme which converts NADP+ or NAD+ into NADPH or NADH.

11. The method of claim 7, wherein, During the one step reaction the zearalenone is detoxified to the extent of at least 70% of the zearalenone.

12. The method of claim 11, wherein, During the one step reaction the zearalenone is detoxified to the extent of at least 80% of the zearalenone.

13. The method of claim 12, wherein, During the one step reaction the zearalenone is detoxified to the extent of at least 90% of the zearalenone.

Images