Method for biotransformation of trichothecenes

Abstract

The present invention relates to novel methods for detoxifying trichothecene-contaminated materials. More specifically, the present invention relates to a method for bioconverting trichothecenes by contacting trichothecene-contaminated materials with an exogenous, non-animal glutathione-S-transferase (GST) that has substrate specificity for the epoxy ring of trichothecenes. The present invention also relates to recombinant GST and transgenic plants and animals expressing said GST.

Description

Technical Field

[0001] The present invention relates to novel methods for detoxifying trichothecene-contaminated materials. More specifically, the present invention relates to a method for bioconverting trichothecenes by contacting trichothecene-contaminated materials with an exogenous, non-animal glutathione-S-transferase (GST) that has substrate specificity for the epoxy ring of trichothecenes. The present invention also relates to recombinant GST and transgenic plants and animals expressing said GST. Background Art

[0002] Fungi produce a large number of metabolites that are not essential for life but can provide ecological advantages in certain environments. These metabolites are called secondary metabolites. Fungal secondary metabolites include plant growth regulators (such as gibberellins), pharmaceutically useful compounds (such as penicillin and lovastatin), pigments (such as carotenoids), and mycotoxins (such as trichothecenes, fumonisins, aflatoxins, and ochratoxins).

[0003] Trichothecene mycotoxins are a large family of chemically related mycotoxins that share a tricyclic 12,13-epoxytrichothecene-9-ene (EPT) core structure that is generally toxic to humans, animals, plants, and eukaryotic cells. Fusarium and other trichothecene-producing fungi and molds (such as Fusarium, Myrothecium, Trichoderma, Trichothecium, Cephalosporium, Verticimonosporium, and Stachybotrys) infect important crop plants, such as wheat, barley, or corn grains. As a result, they are associated with poisonings in humans and animals worldwide (Arunachalam C, Doohan FM., Toxicol Lett. 2013 Feb 27; 217(2):149-58. doi:10.1016 / j.toxlet.2012.12.003. Epub 2012 Dec 26. Review). Trichothecenes have potent cytotoxicity, as well as proinflammatory and emetogenic properties, and are deleterious to hematopoietic organs and immune function. Trichothecenes inhibit eukaryotic protein synthesis, in particular by preventing peptide bond formation in the peptidyl transferase center of the 60S ribosomal subunit, by inhibiting mitochondrial protein synthesis, and by interacting with protein sulfhydryl groups. Fusarium toxins can appear in many types of human and animal foods, including cereal grains such as barley, oats, rice, rye, teff, triticale, wheat, wild rice, finger millet, fonio, foxtail millet, Kodo millet, Japanese millet, Job's tears, maize (corn), pearl millet, proso millet, and sorghum. Toxins have also been found in hay, flax, peas, soybeans, rapeseed, and other oilseeds such as sunflower, hemp, and poppy. Trichothecenes can also be present in other types of foods, such as sugar beets grown in fields where residues of previous crops have been plowed into the soil.

[0004] Trichothecenes are sesquiterpenoids, small amphiphilic molecules consisting of a trichothecene core with epoxy rings at the C-12 and -13 positions. Trichothecenes are divided into four types (AD) based on the carbonyl group at the C-8 position, the macrolide rings at the 4- and 15-positions, and the number of epoxy rings. Of these types, type A trichothecenes and type B trichothecenes are primarily relevant in agriculture. One of the most widely studied trichothecenes is type A trichothecenes T-2 toxin. Important type B trichothecenes include deoxynivalenol (DON, vomitoxin) and nivalenol (NIV).

[0005] There are also macrocyclic trichothecenes (D-type trichothecenes). Examples of macrocyclic trichothecenes include verrucosporin, myrocin and stachybotrysin. Trichothecene mycotoxins are non-volatile, low molecular weight compounds that are generally relatively soluble in water and many organic solvents such as acetone, ethyl acetate, chloroform, dimethyl sulfoxide and ethanol.

[0006] Various enzymatic methods have been described in the literature concerning the detoxification of deoxynivalenol and other trichothecenes. Most of these detoxification reactions are targeted to side groups (e.g., reduction and epimerization of the C3-OH group, acetylation of C3-OH, glycosylation of C3-OH, hydroxylation of C16).

[0007] The reduction of epoxides to alkenes (C=C) by anaerobic bacteria has been described, but a hypothetical "epoxide reductase" is currently elusive and no gene has been cloned. The current state of research on enzymatic (microbial) detoxification has been reviewed (Karlovsky P., 2011, Appl. Microbiol. Biotechnol., 91(3), 491-504; McCormick SP., 2013, J. Chem. Ecol., 39(7), 907-18). The epoxides of trichothecenes are remarkably unreactive. There are no known epoxide hydrolases that can inactivate trichothecenes (opening the epoxide by adding water). Epoxide ring opening catalyzed by glutathione-S-transferase (GST) is known to occur under certain circumstances (de Vries EJ & Janssen DB, 2003, Curr Opin Biotechnol, 14(4), 414-429).

[0008] Gardiner SA et al. (Molecular Plant-Microbe Interactions, 2010, 23(7):962-976) reported that glutathione can form the so-called Michael adduct ( Figure 2, left structure) reacts spontaneously (non-enzymatically) with DON. DON-glutathione conjugates and processed products have been found in plants (Kluger B. et al., 2013, Analytical and Bioanalytical Chemistry, 405, 15, 5031-5036). It has been shown that the addition of a small S-methyl group (a putative degradation product) to the double bond significantly reduces toxicity (Fruhmann P. et al., 2014, Org. Biomol. Chem., 12 (28), 5144-5150), indicating that the addition of a large glutathione should also prevent interaction with the ribosomal target. However, the Michael adduct (especially under alkaline conditions) is quite unstable, and once glutathione is removed from the equilibrium, the original toxin can be regenerated.

[0009] Recently, it was described that epoxides can also be opened by spontaneous reaction with glutathione in a very slow reaction (after several weeks under alkaline conditions) (Stanic A. et al., 2016, J. Agric. Food Chem, 64(36), 6903-6910). Figure 2 A & B) are considered irreversible (but data on product stability are still insufficient). Further processing of epoxide-glutathione adducts in vivo is possible, but so far (Kluger B. et al., 2015, PLOS One 10(3):e0119656.) has only provided evidence for derivatives in which 2 hydrogens are missing in the DON part.

[0010] Adducts of C13 (epoxide) have been found in (bulk stored) wheat and it was proposed (Uhlig S. et al., 2016, Toxins 8(11):329) that this is the result of spontaneous (non-enzymatic) transfer of glutathione from the double bond (Michael adduct) to the epoxide.

[0011] WO 2015 / 169847 A1 reports the non-enzymatic detoxification of trichothecenes, which involves the opening of the trichothecene epoxy ring by reaction with thiols under alkaline conditions.

[0012] WO 2000 / 20573 A2 discloses the use of nucleic acids obtained by DNA shuffling for the detoxification of mycotoxins, for example nucleic acids encoding an enzyme having glutathione-S-transferase activity.

[0013] Wahibah NN, et al. reported a method for detoxifying reactive oxygen species and toxic compounds using exogenous non-animal glutathione S-transferase (Plant Biotechnology 2018, 35(1), 71-79).

[0014] He J., et al. provided a review on biotransformations for detoxification of trichothecenes in the food chain (Trends in Food Science and Technology 2010, 21(2), 67-76).

[0015] He Z., et al. reported China's demand for increased cereal food production (Journal of CerealScience 2014, 59(3), 235-244).

[0016] Borisjuk N., et al. described genetic modifications for increasing wheat yield (Biomed Research International 2019, 2019, 1-18).

[0017] Pan Y et al. disclosed transcriptome analysis and identification of differentially expressed genes, such as genes encoding glutathione S-transferase, membrane proteins, and different LRR-RKs, which are associated with resistance to Fusarium (BMC Genomics 2018, 19(1), 1-26).

[0018] Theodoulou F., et al. reported the co-induction of glutathione-S-transferase and multidrug resistance-related proteins by xenobiotics such as herbicides (Pest Management Science 2003, 59(2), 202-214).

[0019] Trichothecene mycotoxin contamination is a worldwide problem, with large quantities of agricultural products being processed and destroyed every year. As preventive measures have not been successful in preventing mycotoxin contamination, there is a need to find a way to provide detoxifying and decontaminating materials, as well as methods to decontaminate contaminated materials. Summary of the Invention

[0020] Therefore, one object of the present invention is to provide a method for the biotransformation of trichothecenes.

[0021] This object is achieved by the subject matter of the present invention.

[0022] The present invention provides a method for biotransforming trichothecenes, wherein the method is carried out by contacting a material contaminated with trichothecenes with an exogenous non-animal glutathione-S-transferase (GST) having substrate specificity for the epoxy ring of the trichothecenes, and the method comprises the following steps:

[0023] a) contacting the material with GST,

[0024] b) adding glutathione to the material, and

[0025] c) incubating the mixture in an aqueous solution having a pH ranging from about 6 to 9 under conditions wherein the glutathione reacts with the epoxide moiety, thereby forming an epoxide adduct.

[0026] The incubation is allowed to proceed for a period of time sufficient to allow the epoxide adduct formation to occur and thereby reduce the amount of trichothecenes in the material to an acceptable level, such as a level acceptable for human and / or animal consumption.

[0027] According to another embodiment, the trichothecene is selected from the group consisting of type A trichothecene, type B trichothecene, type C trichothecene and type D trichothecene, in particular, the trichothecene is selected from the group consisting of trichodermol, trichodermin, 4,15-diacetoxyfusarinol (DAS), neosolanol, T-2 toxin, HT-2 toxin, isotrichodermol, calonectrin, 7,8-dihydroxycalonectrin, harzianum A ( A), nivalenol (NIV), deoxynivalenol (DON), 3- and 15-acetyldeoxynivalenol, Fusarenon-X, trichothecenes, trichothecolone, trichothecinol A, planariacin, botrytiscine H, myclosan A, diacetin, and verrucosporin A.

[0028] More specifically, the trichothecene is selected from the group consisting of T-2 toxin, HT-2 toxin, solanol, deoxynivalenol (DON), nivalenol (NIV), 3- and 15-acetyldeoxynivalenol, roridin A and verrucarin A.

[0029] In general, the methods described herein can be used with any material or substance that may be contaminated with trichothecene mycotoxins.

[0030] According to a particular embodiment of the present invention, the method described herein is used for producing a decontaminated feed additive, feed material, food additive or food material.

[0031] According to another embodiment, the method described herein is used for decontaminating aerosols, liquid or solid materials or material surfaces, in particular textile materials, filter materials, gas masks, air conditioning systems, for purifying material surfaces, animal or human surfaces, such as skin, etc.

[0032] In another embodiment, the GST used in the methods described herein is recombinant GST.

[0033] According to a specific embodiment, the GST has the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 4, or a sequence having at least 80%, 85%, 90%, 95%, in particular at least 99% sequence identity with either SEQ ID NO. 2 or SEQ ID NO. 4 and having substrate specificity for the epoxy ring of trichothecene.

[0034] According to another specific embodiment, the GST comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications of SEQ ID NO. 2 or SEQ ID NO. 4.

[0035] In another embodiment, the GST is encoded by a polynucleotide sequence selected from the group consisting of a) SEQ ID NO. 1 or SEQ ID NO. 3, or a sequence having at least 80%, 85%, 90%, 95%, in particular at least 99% sequence identity with any one of SEQ ID NO. 1 or SEQ ID NO. 3, or b) an isolated nucleic acid molecule complementary to the polynucleotide sequence of a).

[0036] According to another specific embodiment, the GST is encoded by a polynucleotide sequence selected from the group consisting of: a) SEQ ID NO. 1 or SEQ ID NO. 3 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and up to 50 nucleotide modifications, or b) an isolated nucleic acid molecule complementary to the polynucleotide sequence in a).

[0037] According to one embodiment, the GST is expressed in a host cell.

[0038] The present invention also provides a feed additive or feed material comprising an exogenous non-animal GST having substrate specificity for the epoxy ring of trichothecenes, and optionally glutathione.

[0039] According to a specific embodiment, the feed additive or feed material comprises a transgenic plant part, a transgenic plant tissue, a transgenic plant cell, a seed or progeny thereof, in particular a leaf, a stem, a root, a cotyledon or a hypocotyl, each of which contains an exogenous GST having substrate specificity for the epoxy ring of a trichothecene.

[0040] According to a specific embodiment, the methods described herein are used for the enzymatic degradation of trichothecenes in animal feed or in the digestive tract of an animal.

[0041] According to an alternative embodiment, the method is used for the production of a feed or food additive.

[0042] The present invention also provides a feed or food material comprising the decontaminated plant material obtained by the method described herein.

[0043] The present invention further provides a host that overexpresses endogenous GST or is transformed with a vector expressing exogenous GST, wherein the GST has substrate specificity for the epoxy ring of trichothecenes. In particular, the host is a prokaryotic or eukaryotic organism, specifically a plant cell, an animal cell, a fungal cell, or a bacterial cell.

[0044] According to another embodiment, the host cell comprises GST, wherein the GST is encoded by:

[0045] a) a polynucleotide sequence selected from SEQ ID NO.1, SEQ ID NO.3, or a sequence having at least 80%, 85%, 90%, 95%, in particular at least 99% sequence identity with any one of SEQ ID NO.1, SEQ ID NO.3, or

[0046] b) an isolated nucleic acid molecule complementary to a).

[0047] According to a further embodiment, the host cell comprises an expression cassette. Specifically, the expression cassette comprises a polynucleotide sequence encoding an exogenous GST, which is operably linked to regulatory sequences functional in the host, such as but not limited to CaMV35S, NOS, OCS, AdhI, AdhII, Ubi-1, and malE.

[0048] In another embodiment, the expression cassette further comprises a scorable marker polynucleotide operably linked to a regulatory sequence functional in a plant, such as, but not limited to, GUS, GFP, CAT, LUC, sialidase. Additionally, a selectable marker polynucleotide, such as, but not limited to, nptII, hptII, pat, and bar, is operably linked to a regulatory sequence functional in the host.

[0049] In another embodiment, a host expressing the GST described herein is provided, wherein the GST has substrate specificity for a trichothecene selected from the group consisting of type A trichothecenes, type B trichothecenes, type C trichothecenes, and type D trichothecenes.

[0050] The present invention further provides a host expressing GST and having substrate specificity for a trichothecene selected from the group consisting of type A trichothecenes, type B trichothecenes, type C trichothecenes and type D trichothecenes, in particular, the trichothecene selected from the group consisting of trichoderma, trichodermin, 4,15-diacetoxystrychnol (DAS), solanol, T-2 toxin, HT-2 toxin, isotrichodermin, calonectrin, 7,8-dihydroxycalonectrin, Trichoderma harzianum A, nivalenol (NIV), deoxynivalenol (DON), 3- and 15-acetyldeoxynivalenol, fusarinone-X, trichothecenes, trichothecenes, trichothecinol A, planarian, botryocin H, myclocantin A, baccharin and verrucosin A.

[0051] More specifically, the trichothecene is selected from the group consisting of T-2 toxin, HT-2 toxin, neosolanol, deoxynivalenol (DON), nivalenol (NIV), 3- and 15-acetyldeoxynivalenol, myristocetin A and verrucosin A.

[0052] According to a specific embodiment, the exogenous GST of the host comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, or a sequence having at least 80%, 85%, 90%, 95%, and in particular at least 99% sequence identity with any one of SEQ ID NO. 2 and SEQ ID NO. 4 and having substrate specificity for the epoxy ring of trichothecene.

[0053] Also provided are transgenic plants having increased resistance to trichothecenes, comprising an exogenous GST having substrate specificity for the epoxy ring of the trichothecenes.

[0054] Furthermore, transgenic plant parts, transgenic plant tissues, transgenic plant cells, seeds or progeny thereof, in particular leaves, stems, roots, cotyledons and hypocotyls, are provided, which comprise an exogenous GST having substrate specificity for the epoxy ring of trichothecenes.

[0055] According to one embodiment, also provided herein is a transgenic animal having increased resistance to trichothecenes, comprising an exogenous GST having substrate specificity for the epoxy ring of trichothecenes.

[0056] The present invention also provides a method for producing a transgenic plant or animal, the method comprising transforming a plant or animal with a nucleic acid molecule encoding GST and expressing the nucleic acid molecule in the plant, wherein the nucleic acid molecule comprises a polynucleotide sequence selected from the following: a polynucleotide sequence comprising SEQ ID NO.1 or SEQ ID NO.3, or a polynucleotide sequence comprising a sequence having at least 80%, 85%, 90%, 95%, and in particular at least 99% sequence identity with either SEQ ID NO.1 or SEQ ID NO.3.

[0057] According to another embodiment, the plant is transformed by a method selected from the group consisting of Agrobacterium-mediated transformation, particle gun bombardment, vacuum infiltration, in situ transformation (planta transformation) and chemical methods.

[0058] The present invention also provides a method for expressing an exogenous GST having substrate specificity for the epoxy ring of a trichothecene in a host, the method comprising:

[0059] a) transforming a host cell with a vector containing a GST encoding gene sequence, and

[0060] b) growing the transformed cells under conditions suitable for the expression of the gene encoding the exogenous GST.

[0061] The transgenic plants described herein can be monocots or dicots.

[0062] In particular, the monocotyledonous plant is a grass, in particular selected from rice, corn, wheat, barley, sorghum, rye and oats.

[0063] Specifically, the dicotyledonous plant is selected from tobacco, tomato, pea, soybean, Brassica, chickpea, Arabidopsis and carrot. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 : The carbon numbering system for trichothecenes and the structures of different types of trichothecenes. The structures of trichothecenes and types A, B, C, and D.

[0065] Figure 2A) Deoxynivalenol (DON), epoxide ring opening. The arrow points to the carbon atom attacked by the SH group of glutathione. B) DON-glutathione adduct. The structure of the resulting DON-glutathione epoxide adduct is shown (in addition, the C15-OH group can also react with the C8-ketone to form a hemiketal). C) Structural requirements for Michael adduct formation, resulting in the DON-GSH adduct. Michael adduct formation is only possible when conjugated double bonds and a keto group are present (type B trichothecenes).

[0066] Figure 3 : Construction of the gsh T7-Express host strain. Schematic comparison of the wild-type (A) and disrupted strain (B).

[0067] Figure 4 Figure 2: SDS PAGE of 6xHIS-malE-(TEV)-GST fusion protein. Results of SDS PAGE 12% polyacrylamide gel electrophoresis to evaluate the expression of 6xHIS-malE-(TEV)-GST fusion protein. From each candidate, two samples were loaded onto the gel: total protein on the left and cell extract (clarified lysate) on the right.

[0068] Figure 5 : SDS-PAGE protein purification. Applied concentration: CE (cell extract) and FT (flow through): 0.75 mg / mL, protein: 0.23 mg / mL.

[0069] Figure 6 : Enzymatic formation of Michael adducts of DON (MH1) using different GSTs.

[0070] Figure 7 : Enzymatic formation of the epoxide adduct (MH2) of DON catalyzed by GST C02 and C12. Figure 6 DON-GSH_MH1 shown analyzes the same GSTs, but GSTs other than GST C02 and C12 show no detectable epoxide adduct formation.

[0071] Figure 8 : Extracted ion chromatogram of DON (reacted with wheat GST C02).

[0072] Figure 9 : Structure of T-2 toxin.

[0073] Figure 10 :[M+H] of A)T2, B)GSH, C)T2_GSH + Extracted ion chromatograms of ions (mass window: ±5 ppm).

[0074] Figure 11 : Enzyme-dependent formation of GSH adducts with T-2 toxin. T-2-GSH_MH; pCA02 is the empty vector, NC is the negative control (no protein), TaGSTs C02 / C12.

[0075] Figure 12 : Structure of trichodermin.

[0076] Figure 13 : A)TCM, B)GSH, C)TCM_GSH’s [M+H] + Extracted ion chromatogram of ions (mass window: ±5 ppm).

[0077] Figure 14 : Enzyme-dependent formation of GSH adducts with trichoderma. Trichoderma-GSH_MH; pCA02 is the empty vector, NC is the negative control (no protein), TaGSTs C02 / C12.

[0078] Figure 15 : Structure of myrocin A (ROA).

[0079] Figure 16 :A)ROA, B)GSH, C)ROA_GSH’s [M+H] + Extracted ion chromatograms of ions (mass window: ±5 ppm).

[0080] Figure 17 : Enzyme-dependent formation of GSH adducts with myrocin A; myrocin-GSH_MH; pCA02 is an empty vector, NC is a negative control (no protein), TaGSTs C02 / C12. DETAILED DESCRIPTION

[0081] Unless otherwise indicated or defined, all terms used herein have their ordinary meaning in the art, which will be clear to those skilled in the art. For example, reference may be made to standard manuals such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990), and Janeway et al., "Immunobiology" (5th Ed., or later versions, Garland Science, New York, 2001).

[0082] The claimed subject matter is specifically directed to artificial products, which may be variants of natural (wild-type) products, or methods of using or producing such artificial products. Although there may be a certain degree of sequence identity with natural structures, it is understood that the materials, methods and uses of the present invention, as particularly directed to isolated nucleic acid sequences, amino acid sequences, fusion constructs, expression constructs, transformed host cells and modified proteins (including enzymes), are "artificial" or synthetic and therefore cannot be considered to be the result of "the laws of nature."

[0083] As used herein, the terms "comprise," "contain," "have," and "include" are used synonymously and should be understood as open-ended definitions allowing for additional members, parts, or elements. "Consisting" is considered a closed definition with no additional elements beyond the defining features. Thus, "comprising" is broader and encompasses the definition of "consisting."

[0084] As used herein, the term "about" refers to the same value or a value that is + / - 5% different from a given value.

[0085] As used herein and in the claims, the singular forms such as "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0086] The present invention provides a novel method for biotransforming trichothecenes using a non-animal GST having substrate specificity for the epoxy ring of trichothecenes.

[0087] Glutathione S-transferases (GSTs) comprise a family of eukaryotic and prokaryotic Phase II metabolic isoenzymes known for their ability to catalyze the conjugation of reduced glutathione (GSH) to xenobiotic substrates for detoxification. The GST family consists of three superfamilies: cytosolic, mitochondrial, and microsomal proteins. The amino acid sequences of members of the GST superfamily are extremely diverse, and the functions of the majority of sequences deposited in public databases are unknown.

[0088] "Non-animal GST" refers to GST derived from any source other than animal sources (eg, rats or any other species of animal).

[0089] Type A trichothecenes include compounds with a hydroxyl group at the C-8 position (e.g., solanol), compounds with an ester functionality at the C-8 position (e.g., T-2 toxin), or compounds without oxygen substitution at the C-8 position (e.g., trichodermin, 4,15-diacetoxyfusarinol, and Trichoderma harzianum A).

[0090] Trichothecenes type A: trichoderma, trichodermin, 4,15-diacetoxyfusarium sclerotinol (DAS), neosolanol, T-2 toxin, HT-2 toxin, isotrichoderma, teicoplanin, 7,8-dihydroxyteicoplanin, Trichoderma harzianum A.

[0091] Type B trichothecenes have a keto (carbonyl) functionality at C-8 (eg, nivalenol, deoxynivalenol, and the trichothecenes).

[0092] In Fusarium, type B trichothecenes generally have a C-7 hydroxyl group, but this structural feature is absent in other genera.

[0093] Trichothecenes type B: nivalenol (NIV), deoxynivalenol (DON), 3- and 15-acetyldeoxynivalenol, fusarium-X, trichothecenes, trichothecinol A.

[0094] Type C trichothecenes have C-7 / C-8 epoxides (eg, planocetins).

[0095] Trichothecenes type C: planocene.

[0096] Type D trichothecenes have an additional ring connecting the C-4 and C-15 positions (eg, myrocin A, verrucosporin A, stachybotrysin H).

[0097] Trichothecenes type D: Stachybotrysin H, myclobutanil A, dithyroxine, and verrucosporin A.

[0098] All Fusarium species (including types A and B) have an oxygen functional group (i.e., hydroxyl or acetyl) at C-3. Trichothecenes produced by Trichoderma, Trichothecium, Myrothecium, or Stachybotrys (including types A, B, C, and D) lack an oxygen functional group at the C-3 position.

[0099] For the bioconversion methods described herein, contaminated material is contacted with GST and glutathione and incubated for a period of time sufficient to allow the detoxification reaction to occur, thereby reducing the amount of trichothecenes to an acceptable level, such as a level acceptable for human and / or animal consumption, by forming epoxide adducts in the sample. In particular, the trichothecenes are completely converted to epoxide adducts by the methods of the invention described herein.

[0100] For example, the bioconversion reaction can be carried out for about 1 hour to 24 hours, specifically, the material is incubated with GST for 1, 2, 3, 4, 5, 10, 15, 20, 24 hours. Specifically, the bioconversion reaction is stopped within 24 hours.

[0101] The reaction can take place at ambient temperature, such as room temperature. In addition, the reaction can be carried out at elevated temperatures, such as temperatures typically employed in food and feed production. These temperatures can be, for example, from about 30°C to about 40°C.

[0102] The bioconversion reaction can be carried out, for example, at a pH of about 6 to 9, such as in the pH range of about 6.5 to 8.5.

[0103] The biotransformation methods disclosed herein can be used for the detoxification of materials contaminated with any type of trichothecenes. The material can contain one of type A, B, C or D trichothecenes, or two or more of type A, B, C and / or D toxins. Typically, the material is a product intended to be used as food or feed, feed additives or food additives, such as agricultural products, either as is or after processing. In a further example, the sample is a food product or feed product. Exemplary materials to be treated include, but are not limited to, hay or straw, grains or seeds, flour and other ground products, livestock or fish feed. The material can be a product derived from or containing grains, such as grains or seeds for food or feed production. Typical grains include, but are not limited to, oats, barley, corn, rye, rice, sorghum, wheat, teff, triticale, wild rice, finger millet, fonio, foxtail millet, Kodo millet, Japanese millet, Job's tears, pearl millet and prosomillet. Other examples of materials that may be contaminated with trichothecenes include flax, peas, soybeans, rapeseed, and other oilseeds such as sunflower, hemp, and poppy. Trichothecenes may also be present in other types of food, such as sugar beets. Cereal-derived products include, but are not limited to, raw grains, flours, and cereals. In addition, grasses and animal feed products are suitable for detoxification according to the present invention. The method can also be used to decontaminate non-food materials or surfaces thereof, such as fabric materials for clothing, filter materials, gas masks, air conditioning systems, and the like.

[0104] The methods described herein can also be used to decontaminate materials and surfaces of materials from trichothecenes, and also to decontaminate or decontaminate animal and human surfaces (eg, skin, hair, fur, etc.).

[0105] Specifically, GST is recombinant GST.

[0106] As used herein, the term "recombination" refers to a molecule or construct that is not naturally present in a host cell. In some embodiments, a recombinant nucleic acid molecule contains two or more naturally occurring sequences that are linked together in a non-natural manner. Recombinant protein refers to a protein encoded and / or expressed by a recombinant nucleic acid. In some embodiments, "recombinant cells" express genes that are not found in the same form in cells of natural (i.e., non-recombinant) form and / or express natural genes that are otherwise abnormally overexpressed, underexpressed, and / or not expressed at all due to deliberate human intervention. Recombinant cells contain at least one recombinant polynucleotide or polypeptide. "Recombination (recombination)", "recombining (recomining)" and production of "recombined" nucleic acids generally encompass the assembly of at least two nucleic acid fragments. In some embodiments, recombinant proteins and recombinant nucleic acids retain functionality in host cells, i.e., retain their activity or show enhanced activity.

[0107] Specifically, the recombinant GST comprises one of the following nucleotide or amino acid sequences:

[0108]

[0109]

[0110]

[0111]

[0112]

[0113] The GST may have the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 4, or a sequence having at least 80%, 85%, 90%, 95%, or particularly at least 99% sequence identity to SEQ ID NO. 2 or SEQ ID NO. 4 and having substrate specificity for the epoxy ring of a trichothecene. Alternatively, the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 4 may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications, provided that the GST comprising SEQ ID NO. 2 or SEQ ID NO. 4 retains its substrate specificity for the epoxy ring of a trichothecene.

[0114] The GST may be encoded by the nucleic acid sequence of SEQ ID NO. 1 or SEQ ID NO. 3, or by a sequence having at least 80%, 85%, 90%, 95%, or particularly at least 99% sequence identity to SEQ ID NO. 1 or SEQ ID NO. 3 and having substrate specificity for the epoxy ring of a trichothecene. Alternatively, the nucleic acid sequence of SEQ ID NO. 1 or SEQ ID NO. 3 may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more modifications, provided that the GST encoded by SEQ ID NO. 1 or SEQ ID NO. 3 retains its substrate specificity for the epoxy ring of a trichothecene.

[0115] The amino acid or nucleotide modification herein refers to any modification known in the art, such as point mutation, deletion or insertion of an amino acid residue or nucleotide.

[0116] "Percent (%) identity" with respect to the amino acid sequences, homologs, and orthologs described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a specified polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared.

[0117] For the purposes described herein, sequence identity between two amino acid sequences is determined using the NCBI BLAST program version 2.2.29 (January 6, 2014), with blastp set at the following exemplary parameters: Program: blastp, Wordlength: 6, Expectation: 10, Hitlist size: 100, Gapcosts: 11.1, Matrix: BLOSUM62, Filter string: F, GeneticCode: 1, Window size: 40, Threshold: 21, Composition-based stats: 2.

[0118] "Percent (%) identity" with respect to, for example, a nucleotide sequence of a nucleic acid molecule or a portion thereof, particularly a coding DNA sequence, is defined as the percentage of nucleotides in the candidate DNA sequence that are identical to the nucleotides in the DNA sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent nucleotide sequence identity can be achieved in various ways within the skill in the art, for example using publicly available computer software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0119] Optimal alignment can be determined using any suitable algorithm for sequence alignment, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows WheelerAligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at Novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomis.orgy.cn), and Maq (available at maqsourceforge.net).

[0120] In order to express GST as described herein, any host cell suitable for expressing recombinant exogenous functional GST with substrate specificity for the epoxy ring of trichothecenes can be used. The host cell can be a prokaryotic or eukaryotic cell or cell culture. Such host cells can be, but are not limited to, fungal cells such as Saccharomyces sp., bacterial cells such as Escherichia coli, plant cells, or animal cells. Due to the recombinant expression of GST as described herein, the host cell transformed in this way can have substrate specificity for trichothecenes, particularly type A trichothecenes, type B trichothecenes, type C trichothecenes, and type D trichothecenes, particularly deoxynivalenol (DON), 3- and 15-acetyldeoxynivalenol, nivalenol (NIV), T2 toxin, HT-2 toxin, neosolanol, diacetoxystrychnol, trichothecenes, myristocetin A, and verrucosin A. Due to substrate specificity, host cells can better tolerate contamination with trichothecenes.

[0121] Specifically, transgenic plants or parts thereof (such as tissues, seeds, leaves, stems, roots, cotyledons or hypocotyls) are provided, which express exogenous GSTs with substrate specificity for the epoxy ring of trichothecenes. Such plants can be monocotyledonous or dicotyledonous plants, such as, but not limited to, rice, corn, wheat, barley and sorghum, rye, oats or tobacco, tomato, pea, soybean, Brassica, chickpea, Arabidopsis and carrot.

[0122] Specifically, the present invention also provides transgenic non-human animals with increased resistance to trichothecenes, comprising exogenous GSTs with substrate specificity for the epoxy ring of trichothecenes. Specifically, transgenic species (such as pigs, fish or insects) are thereby better able to tolerate toxin-contaminated feed.

[0123] As used herein, the term "exogenous non-animal glutathione-S-transferase" refers to a non-animal GST that is not naturally present in the material contaminated with the trichothecene or that is not originally expressed in the material. The term "exogenous" can be used interchangeably with the term "foreign." The non-animal GST is present in the material due to the addition of the GST protein or by expressing the GST coding sequence in a transgenic DNA sequence of a host cell or organism, thereby encoding the exogenous GST. Exogenous GST is any non-animal GST that is added to the material and / or expressed in an organism or (host) cell other than the material or organism, as opposed to the endogenous factor.

[0124] The term "expression" is understood as follows. As described herein, nucleic acid molecules containing the coding sequence of the desired expression product (such as GST as described herein) can be used for expression purposes. In particular, such nucleic acid molecules are referred to as "isolated nucleic acid molecules" or "isolated nucleotide sequences." Hosts transformed or transfected with these sequences are capable of producing the encoded protein. To achieve transformation, the expression system can be contained in a vector; however, the relevant DNA can also be integrated into the host chromosome. Specifically, the term refers to a host cell and a compatible vector under appropriate conditions, such as for expressing a protein encoded by an exogenous DNA carried by the vector and introduced into the host cell.

[0125] Coding DNA is a DNA sequence that encodes a specific amino acid sequence for a specific polypeptide or protein. Promoter DNA is a DNA sequence that starts, regulates, mediates, or controls the expression of coding DNA. Promoter DNA and coding DNA can be from the same gene or from different genes, and can be from the same or different organisms. Recombinant cloning vectors typically include one or more replication systems for cloning or expression, one or more markers for selection in a host (such as antibiotic resistance), one or more nuclear localization signals (NLS), and one or more expression cassettes.

[0126] "Expression vector" or "vector" as used herein is defined as a DNA sequence required for transcription of a recombinant nucleotide sequence (i.e., a recombinant gene) cloned in a suitable host organism and translation of its mRNA. In order to obtain expression, the sequence encoding the desired expression product (e.g., GST as described herein) is typically cloned into an expression vector containing a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art. The promoter used to direct nucleic acid expression depends on the specific application. For example, strong constitutive promoters are typically used for expression and purification of recombinant proteins. In contrast, when the expression product is administered in vivo for gene regulation, a constitutive or inducible promoter can be used, depending on the specific use of the expression product. In addition, preferred promoters for administration may be weak promoters. The expression vector comprises an expression cassette and generally also comprises an origin or genomic integration site for autonomous replication in the host cell, one or more selectable markers (e.g., amino acid synthesis genes or genes conferring antibiotic resistance, such as zeocin, kanamycin, G418 or hygromycin; or nptII, hptII, pat and bar), multiple restriction enzyme cleavage sites, a suitable promoter sequence and a transcription terminator, which are operably linked together. The vector may also contain regulatory elements, such as, but not limited to, CaMV, 35S, NOS, AdhI, AdhII, Ubi-1 and mal E.

[0127] An "expression cassette" is a DNA coding sequence or DNA segment that encodes an expression product and can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame. Typically, exogenous DNA is inserted into one or more restriction sites of a vector DNA and then carried by the vector into a host cell along with the transferable vector DNA. A construct with an inserted or added DNA segment or sequence, such as an expression vector, may also be referred to as a "DNA construct."

[0128] The term "vector" as used herein includes nucleotide sequences that replicate autonomously as well as nucleotide sequences that integrate into the genome. A common type of vector is a "plasmid," which is typically an independent double-stranded DNA molecule that can easily accept additional (exogenous) DNA and can be easily introduced into a suitable host cell. A plasmid vector typically contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting exogenous DNA. Specifically, the term "vector" or "plasmid" refers to a vector by which a DNA or RNA sequence (such as an exogenous gene) can be introduced into a host cell so as to transform the host and promote expression (such as transcription and translation) of the introduced sequence.

[0129] Any known method for importing exogenous nucleotide sequences into host cells can be used. These methods include using calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nuclear transfection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors (episomal and integrative), and any other well-known methods for importing cloned genomic DNA, cDNA, synthetic DNA or other exogenous genetic materials into host cells (see, e.g., Sambrook etc.). Plants can be transformed by Agrobacterium-mediated transformation, particle gun bombardment, vacuum infiltration, in situ transformation and chemical methods well known to those skilled in the art.

[0130] The following items are specific embodiments of the invention provided herein.

[0131] 1. A method for biotransforming trichothecenes, said method comprising contacting a material contaminated with trichothecenes with an exogenous non-animal glutathione-S-transferase (GST) having substrate specificity for the epoxy ring of trichothecenes, said method comprising the steps of:

[0132] a) contacting the material with GST,

[0133] b) optionally adding glutathione to said material, and

[0134] c) incubating the mixture in an aqueous solution having a pH in the range of 6 to 9 under conditions wherein the glutathione reacts with the epoxide moiety, thereby forming an epoxide adduct.

[0135] 2. The method according to item 1, wherein the trichothecene is selected from the group consisting of type A trichothecenes, type B trichothecenes, type C trichothecenes and type D trichothecenes.

[0136] 3. The method according to item 1 or 2, wherein the trichothecene is selected from T-2 toxin, HT-2 toxin, neosolanol, deoxynivalenol (DON), nivalenol (NIV), trichothecenes, 3- and 15-acetyldeoxynivalenol, myristocetin A and verrucosin A.

[0137] 4. The method according to any one of items 1 to 3, for producing a decontaminated feed additive, feed material, food additive or food material.

[0138] 5. The method according to any one of items 1 to 3 for decontaminating liquid or solid materials or material surfaces, in particular textile materials, filter materials, gas masks, air conditioning systems.

[0139] 6. The method according to any one of items 1 to 3, which is used for decontaminating material surfaces, animal or human surfaces.

[0140] 7. The method according to any one of items 1 to 6, wherein the GST is recombinant GST.

[0141] 8. The method according to any one of items 1 to 7, wherein the GST has the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 4, or a sequence having at least 80%, 85%, 90%, 95%, in particular at least 99% sequence identity with either SEQ ID NO. 2 or SEQ ID NO. 4 and having substrate specificity for the epoxy ring of a trichothecene.

[0142] 9. The method according to any one of items 1 to 8, wherein the GST is encoded by:

[0143] a) a polynucleotide sequence selected from SEQ ID NO.1 or SEQ ID NO.3, or a sequence having at least 80%, 85%, 90%, 95%, in particular at least 99% sequence identity with any one of SEQ ID NO.1 or SEQ ID NO.3, or

[0144] b) an isolated nucleic acid molecule complementary to the polynucleotide sequence of a).

[0145] 10. The method according to any one of items 1 to 9, wherein GST is expressed in the host cell.

[0146] 11. A feed additive or feed material comprising an exogenous non-animal GST having substrate specificity for the epoxy ring of a trichothecene.

[0147] 12. The feed additive or feed material according to item 11, which comprises a transgenic plant part, transgenic plant tissue, transgenic plant cell, seed or progeny thereof, in particular a leaf, stem, root, cotyledon or hypocotyl, each of which contains an exogenous GST having substrate specificity for the epoxy ring of a trichothecene.

[0148] 13. Use of the method according to any one of items 1 to 10 for enzymatic degradation of trichothecenes in animal feed or the digestive tract of animals.

[0149] 14. Use of the method according to any one of items 1 to 10 for producing feed or food additives.

[0150] 15. Feed or food material comprising decontaminated plant material obtained by the method of any one of items 1 to 10.

[0151] 16. The feed additive according to item 15, wherein the non-animal GST has the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 4, or has a sequence having at least 80%, 85%, 90%, 95%, in particular at least 99% sequence identity with either SEQ ID NO. 2 or SEQ ID NO. 4 and has substrate specificity for the epoxy ring of trichothecene.

[0152] 17. A host which overexpresses endogenous GST or is transformed with a vector expressing exogenous GST, wherein the GST has substrate specificity for the epoxy ring of trichothecene.

[0153] 18. The host cell according to item 17, wherein the exogenous GST comprises an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 4, or comprises a sequence having at least 80%, 85%, 90%, 95%, in particular at least 99% sequence identity with any one of SEQ ID NO. 2 and SEQ ID NO. 4 and having substrate specificity for the epoxy ring of trichothecenes.

[0154] 19. The host according to item 17 or 18, which is a prokaryotic organism or a eukaryotic organism, in particular, the host is a plant cell, an animal cell, a fungal cell or a bacterial cell.

[0155] 20. The host according to items 17 to 19, wherein the GST is encoded by:

[0156] a) a polynucleotide sequence selected from SEQ ID NO.1, SEQ ID NO.3, or a sequence having at least 80%, 85%, 90%, 95%, in particular at least 99% sequence identity with any one of SEQ ID NO.1, SEQ ID NO.3, or

[0157] b) an isolated nucleic acid molecule complementary to a).

[0158] 21. The host according to any one of items 17 to 20, wherein the vector comprises an expression cassette, in particular comprising a polynucleotide sequence encoding an exogenous GST, the polynucleotide sequence being operably linked to a regulatory sequence functional in the host.

[0159] 22. The host according to item 21, wherein the cassette further comprises a quantifiable marker polynucleotide operably linked to a regulatory sequence functional in a plant.

[0160] 23. The host according to item 21 or 22, wherein the cassette further comprises a selectable marker polynucleotide operably linked to a regulatory sequence functional in the host.

[0161] 24. The host according to any one of items 21 to 23, which has substrate specificity for a trichothecene selected from the group consisting of type A trichothecenes, type B trichothecenes, type C trichothecenes and type D trichothecenes.

[0162] 25. The host according to any one of items 21 to 24, wherein the trichothecene is selected from deoxynivalenol (DON), 3- and 15-acetyldeoxynivalenol, nivalenol (NIV), T2 toxin, HT-2 toxin, neosolanol, diacetoxystrypenol, trichothecenes, myristocetin A and verrucosin A.

[0163] 26. The host according to any one of items 21 to 25, wherein the exogenous GST comprises an amino acid sequence selected from SEQ ID NO. 2 and SEQ ID NO. 4, or comprises a sequence having at least 80%, 85%, 90%, 95%, in particular at least 99% sequence identity with any one of SEQ ID NO. 2 and SEQ ID NO. 4 and having substrate specificity for the epoxy ring of trichothecenes.

[0164] 27. A transgenic plant having increased resistance to trichothecenes, comprising an exogenous GST having substrate specificity for the epoxy ring of trichothecenes.

[0165] 28. A transgenic plant part, transgenic plant tissue, transgenic plant cell, seed or progeny thereof, in particular leaves, stems, roots, cotyledons and hypocotyls, comprising an exogenous GST having substrate specificity for the epoxy ring of a trichothecene.

[0166] 29. A transgenic animal having increased resistance to trichothecenes, comprising an exogenous GST having substrate specificity for the epoxy ring of trichothecenes.

[0167] 30. A method for producing a transgenic plant or animal according to items 27 to 29, the method comprising transforming a plant or animal with a nucleic acid molecule encoding GST, and expressing the nucleic acid molecule in the plant, wherein the nucleic acid molecule comprises a polynucleotide sequence selected from SEQ ID NO: 1 or SEQ ID NO. 3, or comprises a sequence having at least 80%, 85%, 90%, 95%, in particular at least 99% sequence identity with any one of SEQ ID NO. 1 or SEQ ID NO. 3.

[0168] 31. The method according to item 30, wherein the plant is transformed by a method selected from the group consisting of Agrobacterium-mediated transformation, particle gun bombardment, vacuum infiltration, in situ transformation and chemical methods.

[0169] 32. A method for expressing an exogenous GST having substrate specificity for the epoxy ring of a trichothecene in a host, the method comprising:

[0170] a) transforming host cells with a vector containing a GST encoding gene sequence,

[0171] b) growing the transformed cells under conditions suitable for the expression of the gene encoding the exogenous GST.

[0172] 33. The method for producing a transgenic plant according to any one of items 28 to 30, wherein the plant is a monocotyledonous plant or a dicotyledonous plant.

[0173] 34. The method according to item 31, wherein the monocotyledonous plant is a grass, in particular the monocotyledonous plant is selected from rice, corn, wheat, barley and sorghum, rye, oats.

[0174] 35. The method according to item 34, wherein the dicotyledonous plant is selected from tobacco, tomato, pea, soybean, Brassica, chickpea, Arabidopsis and carrot.

[0175] The following examples are provided to aid understanding of the present invention and are not intended to, nor should they be construed as, limit the scope of the present invention in any way. The examples do not include detailed descriptions of conventional methods. Such methods are well known to those of ordinary skill in the art.

[0176] Example

[0177] Example 1:

[0178] Construction of expression host:

[0179] In order to be able to detect the activity of plant GST in a standard colorimetric assay, a suitable E. coli host strain was constructed in which the endogenous GST gene (gstA) showing activity against the standard substrate for the colorimetric assay, chloro-2,4-dinitrobenzene (CDNB), was inactivated. The gstA gene in the T7 polymerase-based E. coli expression host "T7 Express" was disrupted as follows: First, plasmid pDK46 (with a temperature-sensitive origin of replication and from strain BW25113 / pKD46 ( http: / / cgsc2.biology.yale.edu / Strain.php?ID=68099) was introduced into the expression strain T7 Express using the arabinose-inducible phage lambda recombination system (γβexo). The PCR product obtained from the gstA mutant strain JW1627-1 (http: / / cgsc2.biology.yale.edu / Strain.php?ID=107667) from the system knockout collection of Escherichia coli was then used to transform the arabinose-inducible competent cells. The PCR product obtained using flanking primers (Del_gstA_fw#3818 and Del_gstA_rv#3819) contains the kanamycin resistance gene. Knockout mutants were selected on LB-KAN medium at 37°C, which also resulted in the loss of pKD46. Gene replacement was confirmed by PCR. Comparison of wild-type and disrupted strains is shown in Figure 2. Figure 3 As shown (A: wild type, B: after disruption of the endogenous gstA gene).

[0180] Table 1: Primer sequences used to generate gstA gene disruption mutants.

[0181]

[0182]

[0183] After disruption, the resulting strain "T7 ExpressΔgstA::Kan R ” has the following genotype:

[0184] ΔgstA785::kan fhuA2 lacZ::T7gene1[lon]ompT gal sulA11 R(mcr-73::miniTn10--Tet S )2[dcm]R(zgb-210::Tn10--Tet S )endA1Δ(mcrC-mrr)114::IS10; T7 polymerase (lacZ::T7 gene 1) in the chromosomal lac operon (no phage λDE3).

[0185] This host has no significant background in the CDNB assay and was used to test a number of constitutive and DON-inducible candidate GST genes (from barley, rice, Brachypodium, and wheat), most of which showed significant activity towards CDNB but no activity above background towards DON.

[0186] Example 2:

[0187] Cloning and expression of GST candidates:

[0188] The candidate was cloned into the E. coli expression vector pCA02 encoding an N-terminal 6x His tag and a maltose binding domain. This vector carries the ColE1 origin, an ampicillin marker, a copy of the lac repressor, a T7 promoter, and the lac operator sequence.

[0189] The empty vector pCA02 was used as a negative control.

[0190] Cloning of TrGST-C02:

[0191] The wheat glutathione S-transferase TRIAE_CS42_1DL_TGACv1_062916_AA0221650 was amplified from the genomic DNA of Chinese spring wheat using primers TrGST-C02_upstr_fw and TrGST-C02_3'UTR_rv and cloned into pMiniT( PCR cloning kit). Exon 1 was amplified using primers TrGST-C02_GA-E1-fw and TrGST-C02_GA-E1-rv, and exon 2 was amplified from pMiniT clone using primers TrGST-C02_GA-E2-fw and TrGST-C02_GA-E2-rv. Fusion PCR was performed using primers TrGST-C02_GA-E1-fw and TrGST-C02_GA-E2-rv. The PCR product was digested with NdeI / EcoRI and ligated into pCA02 digested with the same enzymes.

[0192] Cloning of TrGST-C12:

[0193] Wheat glutathione S-transferase TRIAE_CS42_1AL_TGACv1_001900_AA0036420 was amplified from Chinese spring wheat genomic DNA using primers TrGST-C12_5'UTR_fw and TrGST-C12_3'UTR_rv and cloned into pMiniT. Exon 1 was amplified using primers TrGST-C12_GA-E1_fw and TrGST-C12_GA-E1_rv, and exon 2 was amplified from pMiniT using primers TrGST-C12_GA-E2_fw and TrGST-C12_GA-E2_rv. Gibson assembly was performed using NdeI / EcoRI-digested pCA02 and the two PCR products.

[0194] Table 2: Primer sequences used for TrGST-C02 and TrGST-C12 cloning

[0195]

[0196]

[0197] Table 3

[0198]

[0199] The nucleotide and amino acid sequences of the expressed GST used herein are as follows:

[0200] TrGST-C01

[0201]

[0202]

[0203]

[0204] TrGST-C02: SEQ ID NO.1, SEQ ID NO.2

[0205] TrGST-C03

[0206]

[0207]

[0208] TrGST-C04

[0209]

[0210]

[0211]

[0212] TrGST-C06

[0213]

[0214]

[0215] TrGST-C07

[0216]

[0217]

[0218] TrGST-C08

[0219]

[0220]

[0221] TrGST-C09

[0222]

[0223]

[0224] TrGST-C11

[0225]

[0226]

[0227] TrGST-C12:SEQ ID NO.3,SEQ ID NO.4

[0228] TrGST-C15

[0229]

[0230]

[0231] HvGST15264

[0232]

[0233]

[0234] HvGST07634

[0235]

[0236]

[0237] HvGST07171

[0238]

[0239]

[0240]

[0241] HvGST21968

[0242]

[0243]

[0244] HvGST12776

[0245]

[0246]

[0247] Expression was carried out overnight at 20°C in 50 ml of Terrific Broth (TB + 100 ppm Amp) containing ampicillin, induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). The pellet was washed in 1 x phosphate-buffered saline (PBS: 50 mM sodium phosphate, 150 mM sodium chloride, pH 7) and resuspended in 2.5 ml of 100 mM sodium phosphate buffer (pH 6) containing 10% glycerol. The cells were disrupted by sonication (using a Brason sonicator W250D: big tip, 40% amplitude, 20 seconds total pulse time).

[0248] Table 4: Expression of GST candidate and control constructs (OD600 = optical density at 600 nm)

[0249]

[0250]

[0251] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) with 12% polyacrylamide gel was used to evaluate the expression of 6xHIS-malE-(TEV)-GST fusion protein. Figure 4 middle.

[0252] Dilute 2 μl of sample with water to 15 μl + 5 μl 4x SDS loading dye, incubate at 95°C for 5 minutes, and centrifuge. Load 12 μl (excluding 5 μl PageRuler TM Prestained Protein Ladder,ThermoFisher Scientific)

[0253] The 6xHIS-tagged protein was affinity purified by immobilized metal ion affinity chromatography (IMAC) and used for enzyme analysis. For comparison with Schistosoma japonicum, which does not produce DON-13-GSH, GST was also purified (which showed approximately 10-fold higher specific activity against CDNB but no activity against the epoxide of DON). The enzyme was expressed from the pGEX-4T3 vector lacking the His tag sequence and was therefore purified by glutathione agarose 4B affinity chromatography.

[0254] SDS-PAGE : (Expected sizes: TrGST-C02: 69.2 kDa; TrGST-C12: 69.2 kDa; SjGST: 26 kDa) Candidate GSTs were cloned into the vector pCA02 as described above and expressed with an N-terminal 6xHis tag and a maltose binding domain.

[0255] Figure 5 Shown are SDS-PAGE, applied concentrations: CE (cell extract) and FT (flow through): 0.75 mg / ml, protein: 0.23 mg / mL.

[0256] Enzymatic DON-GSH conjugation assay and analytical measurements:

[0257] Final concentrations in the assay: Toxin concentrations in the assay: 100 ppm DON (1000 ppm DON stock solution in H2O), 5 mM GSH, 0.1 M sodium phosphate buffer pH 6, 7.5% glycerol.

[0258] Time points: 0, 3, 6, 24h.

[0259] Orbitrap analysis

[0260] A 20 μl aliquot of the DON-GSH conjugation assay was mixed with 20 μl of acetonitrile in an eppendorf tube and centrifuged at 20,000 rcf for 7.5 minutes. 15 μl of the supernatant was removed and mixed with 135 μl of H₂O in a vial to provide a total of 20x dilution containing 5% acetonitrile and 5 ppm DON. Samples were measured within a few hours and stored at 4°C until measurement.

[0261] For DON detoxification analysis, HPLC-MS / MS was used. Samples were measured using LC-HRMS( / MS) using a Vanquish system (Thermo Scientific) coupled to a QExactive HF Orbitrap instrument (Thermo Scientific). For this purpose, 5 μL of the cooled diluted supernatant (10° C.) was injected into Gradient elution was performed on a dC18 column (3 μm, 2.1 x 1500 mm, water) at a flow rate of 0.3 mL / min. After equilibration for 1 minute, a double ramp gradient elution (eluent A: HO + 0.1% FA; eluent B: MeOH + 0.1% FA) was initiated, increasing eluent B from 3% to 20% over 5.5 minutes, followed by an increase from 20% to 100% B over 2.5 minutes. After elution with 100% B for 1.5 minutes, the system was reequilibrated for 3.5 minutes (total run time 14 minutes). Mass spectra were recorded at a resolution of 120,000 (at m / z 200) in full scan mode with rapid polarity switching and a scan range from m / z 100 to 1000. MS / MS fragmentation spectra were recorded in positive mode at a resolution of 30,000 (at m / z 200) and applying a stepped collision energy (25, 35, 45) using a data dependent method with inclusion tables.

[0262] In principle, both adducts of DON and glutathione are formed non-enzymatically. The formation of the Michael adduct (towards C10) occurs over a few days, so a background is visible within the 24-hour observation period. Most enzymes are unable to enhance the formation of Michael adducts above this background (see below). Plasmid pCA02 is an empty expression vector that is clustered with most (inactive) GST. Exceptions are two wheat enzymes, C02 and C12, that clearly enhance Michael adduct formation.

[0263] The formation of Michael adduct "MH1" is as follows Figure 6 shown.

[0264] In contrast, the formation of epoxide adducts ("MH2" with different retention times) did not occur to some extent within 24 hours. The empty vector and other enzymes tested above did not give a measurable background, while wheat GSTsC02 and C12 clearly catalyzed the formation of epoxide adducts ( Figure 8 ).

[0265] The peak identities were determined by comparison with standards generated by prolonged incubation (several weeks) of DON and GSH as described by Stanic et al. (https: / / www.ncbi.nlm.nih.gov / pubmed / 27548277). Compared to the human GST enzyme tested by these authors, C02 and C12 clearly catalyzed both the formation of the Michael adduct and the opening of the epoxide.

[0266] The sample chromatogram is shown in Figure 9 (Reacted with wheat GST C02).

[0267] In addition to the glutathione adduct, a smaller amount of a product lacking two hydrogen atoms in the DON moiety was also formed by the enzyme, which was most likely a GSH adduct of a C3-keto derivative of DON.

[0268] Example 3

[0269] TrGST-C02 and TrGST-C12 were shown to be capable of opening the epoxides of both type A and macrocyclic (type D) trichothecenes. The following compounds lack the conjugated C8-keto character of type B trichothecenes (required for Michael adduct formation), and thus adduct formation is due to epoxide ring opening. Calculations of the expected masses of the adducts with each compound are given in the table below.

[0270] Table 5

[0271] name Mode C H N O S Neutral quality [M+H]+ T-2 C24H34O9 24 34 0 9 466.2203 467.2276 GSH C10H17N3O6S 10 17 3 6 1 307.0838 308.0911 T-2-GSH 34 51 3 15 1 773.3041 774.3114

[0272] Table 6

[0273] name Mode C H N O S Neutral quality [M+H]+ Trichoderma C17H24O4 17 24 4 292.1675 293.1747 GSH C10H17N3O6S 10 17 3 6 1 307.0838 308.0911 TCM-GSH 27 41 3 10 1 599.2513 600.2585

[0274] Table 7

[0275] name Mode C H N O S Neutral quality [M+H]+ myclobutanin A C29H40O9 29 40 9 532.2672 533.2745 GSH C10H17N3O6S 10 17 3 6 1 307.0838 308.0911 ROA-GSH 39 57 3 15 1 839.3510 840.3403 Sequence Listing <110> DSM Austria GmbH <120> Method for biotransformation of trichothecenes <130> LC21310021P <150> EP19181392.2 <151> 2019-06-19 <160> 51 <170> PatentIn version 3.5 <210> 1 <211> 712 <212> DNA <213> wheat <400> 1 aatggccgga ggagatgact tgaagctgct gggcgcttgg gcaagtccat ttgtcaccag 60 ggtgaagctt gcgctgaact tcaagggcct gagcttcgag gatgtcgaag aggaccttag 120 caacaagagc gagctcctcc tcagctcgaa cccggtgcac aagaaggtgc ccgtgctcgt 180 ccacaacgga aaacccattt gcgagtcagt gatcatcgtt cagtacatcg atgaggcgtt 240 cgccggcatc ggccccgctc tccttccctc tgacccctac gaacgcgcca ttgcccgttt 300 ctgggccgcc tacgttgacg ataagctcgt cgccccatgg gtacagtcgt tgagggccaa 360 gacagaggag gagaagtccg aggggcttaa gcagacattt gccgcggtgg agacactgga 420 aggagccctg cgggagtgct ccaagggaga gggctacttt ggtggtgaga ccgtcgggct 480 tgtggacatt tcacttggga gcctgctctc ctggttgaac gcgacagaag tgatgtccgg 540 aaccaagata tttgatcctg ttaagactcc gctcctggca gcgtggatgg agcgctttag 600 caagctcgat gctgccaagg cggcgttgcc agaagttgat agggtggtcg aatttgccaa 660 gaagagacaa gcacaggctg ctgccgccgc cgctgcttca gagaccaagt aa 712 <210> 2 <211> 236 <212> PRT <213> Wheat <400> 2 Met Ala Gly Gly Asp Asp Leu Lys Leu Leu Gly Ala Trp Ala Ser Pro 1 5 10 15 Phe Val Thr Arg Val Lys Leu Ala Leu Asn Phe Lys Gly Leu Ser Phe 20 25 30 Glu Asp Val Glu Glu Asp Leu Ser Asn Lys Ser Glu Leu Leu Leu Ser 35 40 45 Ser Asn Pro Val His Lys Lys Val Pro Val Leu Val His Asn Gly Lys 50 55 60 Pro Ile Cys Glu Ser Val Ile Ile Val Gln Tyr Ile Asp Glu Ala Phe 65 70 75 80 Ala Gly Ile Gly Pro Ala Leu Leu Pro Ser Asp Pro Tyr Glu Arg Ala 85 90 95 Ile Ala Arg Phe Trp Ala Ala Tyr Val Asp Asp Lys Leu Val Ala Pro 100 105 110 Trp Val Gln Ser Leu Arg Ala Lys Thr Glu Glu Glu Lys Ser Glu Gly 115 120 125 Leu Lys Gln Thr Phe Ala Ala Val Glu Thr Leu Glu Gly Ala Leu Arg 130 135 140 Glu Cys Ser Lys Gly Glu Gly Tyr Phe Gly Gly Glu Thr Val Gly Leu 145 150 155 160 Val Asp Ile Ser Leu Gly Ser Leu Leu Ser Trp Leu Asn Ala Thr Glu 165 170 175 Val Met Ser Gly Thr Lys Ile Phe Asp Pro Val Lys Thr Pro Leu Leu 180 185 190 Ala Ala Trp Met Glu Arg Phe Ser Lys Leu Asp Ala Ala Lys Ala Ala 195 200 205 Leu Pro Glu Val Asp Arg Val Val Glu Phe Ala Lys Lys Arg Gln Ala 210 215 220 Gln Ala Ala Ala Ala Ala Ala Ala Ser Glu Thr Lys 225 230 235 <210> 3 <211> 708 <212> DNA <213> Wheat <400> 3 atggccggag gagatgactt gaagctgctc ggcgcttggg cgagtccatt tgtcgccagg 60 gtgaagcttg cgctgagctt caagggcctg agcttcgagg atgtcgagga ggacctcagc 120 aacaagagcg agctcctcct cagctcgaac ccggtgcaca agaaggtgcc cgtgctcgtc 180 cacaacggga aacccatttg cgagtcaatg atcatcgttc agtacatcga tgaggcgttc 240 cttgtcggcc cctctcttct tccctctgac ccctacaaac gtgcaattgc ccgtttttgg 300 gccgcctaca ttgacgataa gctcgtcacc ccatgggtac agtcgttgag ggccaagaca 360 gaggaggaga agtctgaggg ggttaagcag acatttgccg ctgtggaaac actggaagga 420 gccctgaggg agtgctccaa gggagagggc tactttggtg gtgagaccgt cgggcttgtg 480 gacatttcac ttgggagcct gctctcctgg ttgattgcga cagaagtgat gtctggaacc 540 aagatctttg atcctgttaa gactccgctc ctggcagcgt ggatggggcg ctttagcgag 600 ctcgacgctg ccaaggcggc gttgccagaa gttgataggg tggtcgaatt tgccaagaag 660 agacaggcac aggctgatgc cgccgccgct gcttcggaga ccaagtaa 708 <210> 4 <211> 235 <212> PRT <213> Wheat <400> 4 Met Ala Gly Gly Asp Asp Leu Lys Leu Leu Gly Ala Trp Ala Ser Pro 1 5 10 15 Phe Val Ala Arg Val Lys Leu Ala Leu Ser Phe Lys Gly Leu Ser Phe 20 25 30 Glu Asp Val Glu Glu Asp Leu Ser Asn Lys Ser Glu Leu Leu Leu Ser 35 40 45 Ser Asn Pro Val His Lys Lys Val Pro Val Leu Val His Asn Gly Lys 50 55 60 Pro Ile Cys Glu Ser Met Ile Ile Val Gln Tyr Ile Asp Glu Ala Phe 65 70 75 80 Leu Val Gly Pro Ser Leu Leu Pro Ser Asp Pro Tyr Lys Arg Ala Ile 85 90 95 Ala Arg Phe Trp Ala Ala Tyr Ile Asp Asp Lys Leu Val Thr Pro Trp 100 105 110 Val Gln Ser Leu Arg Ala Lys Thr Glu Glu Glu Lys Ser Glu Gly Val 115 120 125 Lys Gln Thr Phe Ala Ala Val Glu Thr Leu Glu Gly Ala Leu Arg Glu 130 135 140 Cys Ser Lys Gly Glu Gly Tyr Phe Gly Gly Glu Thr Val Gly Leu Val 145 150 155 160 Asp Ile Ser Leu Gly Ser Leu Leu Ser Trp Leu Ile Ala Thr Glu Val 165 170 175 Met Ser Gly Thr Lys Ile Phe Asp Pro Val Lys Thr Pro Leu Leu Ala 180 185 190 Ala Trp Met Gly Arg Phe Ser Glu Leu Asp Ala Ala Lys Ala Ala Leu 195 200 205 Pro Glu Val Asp Arg Val Val Glu Phe Ala Lys Lys Arg Gln Ala Gln 210 215 220 Ala Asp Ala Ala Ala Ala Ala Ser Glu Thr Lys 225 230 235 <210> 5 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 5 agaagaagct gcaatatgtg aa 22 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 6 ttgtaaatct ttgcccgcag 20 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 7 atctggacga agagcatcag 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 8 gcatcagcca tgatggatac 20 <210> 9 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 9 gctatggcct gcagagcatc gg 22 <210> 10 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 10 ggttaaacac ctggcgcgag ct 22 <210> 11 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 11 ccctgccgat gccaaccaag tt 22 <210> 12 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer sequence <400> 12 tggctatctg ggatcggcgg ag 22 <210> 13 <211> 714 <212> DNA <213> Wheat <400> 13 atggcagctc aaggagacct gaagttgcta ggcttgtcgg tgagcccatt cgtagttcgt 60 gttcgcatgg cgctccacat gaagggcttg agctacgagt atatcaaacg ggacctcttc 120 aacaagagtg agctcctcct caagtccaac ccggtggaga agaaggtgcc catactcatc 180 cacgatggca agaccgtact cgattcttcg gtcatcgtgc agtacatcga cgaagtctgg 240 gccgccatgg ggccgtcgat cctccctgtc gacccctacg agcgcgccac cgcccccttc 300 tgggccgcct acgtcgacga caaactattc tccgcttatg tcggtgttaa taaagcagtg 360 acggaggtgg agaggatgga gaaggttagc gagacgcttg cggtgctaga gcaacttgag 420 gaggcatttg ccaagcattc caacggaaag ggcttcttcg ccggggactc catcgggtac 480 cttgacctcg cagtaggatg ccacttgcac tggctcaagg cgcagtgtaa gatgttcggc 540 gtggtgttcc tcgacgccgg caagactccg ctcttagcga cctgggcgaa acggttcact 600 gagaccgatg cggcgaagga ggtggtacct gacacagacg tggtgatgga gtatgctaag 660 aagcgccagg cttatcgtgt tgctgttgct gcggcggcag cgagtgccaa gtga 714 <210> 14 <211> 237 <212> PRT <213> Wheat <400> 14 Met Ala Ala Gln Gly Asp Leu Lys Leu Leu Gly Leu Ser Val Ser Pro 1 5 10 15 Phe Val Val Arg Val Arg Met Ala Leu His Met Lys Gly Leu Ser Tyr 20 25 30 Glu Tyr Ile Lys Arg Asp Leu Phe Asn Lys Ser Glu Leu Leu Leu Lys 35 40 45 Ser Asn Pro Val Glu Lys Lys Val Pro Ile Leu Ile His Asp Gly Lys 50 55 60 Thr Val Leu Asp Ser Ser Val Ile Val Gln Tyr Ile Asp Glu Val Trp 65 70 75 80 Ala Ala Met Gly Pro Ser Ile Leu Pro Val Asp Pro Tyr Glu Arg Ala 85 90 95 Thr Ala Pro Phe Trp Ala Ala Tyr Val Asp Asp Lys Leu Phe Ser Ala 100 105 110 Tyr Val Gly Val Asn Lys Ala Val Thr Glu Val Glu Arg Met Glu Lys 115 120 125 Val Ser Glu Thr Leu Ala Val Leu Glu Gln Leu Glu Glu Ala Phe Ala 130 135 140 Lys His Ser Asn Gly Lys Gly Phe Phe Ala Gly Asp Ser Ile Gly Tyr 145 150 155 160 Leu Asp Leu Ala Val Gly Cys His Leu His Trp Leu Lys Ala Gln Cys 165 170 175 Lys Met Phe Gly Val Val Phe Leu Asp Ala Gly Lys Thr Pro Leu Leu 180 185 190 Ala Thr Trp Ala Lys Arg Phe Thr Glu Thr Asp Ala Ala Lys Glu Val 195 200 205 Val Pro Asp Thr Asp Val Val Met Glu Tyr Ala Lys Lys Arg Gln Ala 210 215 220 Tyr Arg Val Ala Val Ala Ala Ala Ala Ala Ser Ala Lys 225 230 235 <210> 15 <211> 651 <212> DNA <213> Wheat <400> 15 atggcgccgg tgaaggtgtt cgggcaggcc atgtcgccga acgtggcgcg ggtgctggtg 60 ttcctggagg aggccggcgc cgactacgag ctcgtcgacg tcgacttcca ggccaaggag 120 cacaagagcc ccgaccacct tgccagaaac ccgttcgggc aaatccccgc gttccaggac 180 ggtgacctcg ttctcttcga gtcacgagcg gtcgcaaagt acgtggcgcg caagtacaag 240 acggacgagg ccgacctgct gagggacggc gaccattcag aagccgccat ggtggacgtg 300 tggacggagg tggaggcgca cacgtacagc gcggccctct cgccgatcgt ctacgagtgt 360 ctcatcttcc ctctcatgca cggcaagccc accgacgaga aggtcgtcga cgagagcctc 420 gggaagctga ggaaagtgct cgaggtctat gaggagcggc tgtccaagca caggtacctg 480 gccggggatt tcctcagctt cgccgacctc aaccatttcc cctacacctt ctacttcatg 540 gcgacgccgc atggggccct gtttgagtcg tacccgcgcg tgaaggcgtg gtgggagagc 600 atcatgtcca ggccggcgat tcagaagctc agtgcaacca tgacaccatg a 651 <210> 16 <211> 216 <212> PRT <213> Wheat <400> 16 Met Ala Pro Val Lys Val Phe Gly Gln Ala Met Ser Pro Asn Val Ala 1 5 10 15 Arg Val Leu Val Phe Leu Glu Glu Ala Gly Ala Asp Tyr Glu Leu Val 20 25 30 Asp Val Asp Phe Gln Ala Lys Glu His Lys Ser Pro Asp His Leu Ala 35 40 45 Arg Asn Pro Phe Gly Gln Ile Pro Ala Phe Gln Asp Gly Asp Leu Val 50 55 60 Leu Phe Glu Ser Arg Ala Val Ala Lys Tyr Val Ala Arg Lys Tyr Lys 65 70 75 80 Thr Asp Glu Ala Asp Leu Leu Arg Asp Gly Asp His Ser Glu Ala Ala 85 90 95 Met Val Asp Val Trp Thr Glu Val Glu Ala His Thr Tyr Ser Ala Ala 100 105 110 Leu Ser Pro Ile Val Tyr Glu Cys Leu Ile Phe Pro Leu Met His Gly 115 120 125 Lys Pro Thr Asp Glu Lys Val Val Asp Glu Ser Leu Gly Lys Leu Arg 130 135 140 Lys Val Leu Glu Val Tyr Glu Glu Arg Leu Ser Lys His Arg Tyr Leu 145 150 155 160 Ala Gly Asp Phe Leu Ser Phe Ala Asp Leu Asn His Phe Pro Tyr Thr 165 170 175 Phe Tyr Phe Met Ala Thr Pro His Gly Ala Leu Phe Glu Ser Tyr Pro 180 185 190 Arg Val Lys Ala Trp Trp Glu Ser Ile Met Ser Arg Pro Ala Ile Gln 195 200 205 Lys Leu Ser Ala Thr Met Thr Pro 210 215 <210> 17 <211> 690 <212> DNA <213> Wheat <400> 17 atgtctccgg tgaaggtgtt cgggcacccg atgttgacaa acgtcgcacg ggtgctgctc 60 ttcctggagg aggtcggcgc tgagtacgag ctcgtgcccc tcgacttcgt agccggcgag 120[[ID=३०]] cacaagaggc cccaacacgt ccagttaaac ccgtttgcga agatgcctgg gttccaagat 180 ggggatctcg tcctgttcga gtcgcgcgcc atcgccaagt acatcctccg caagtacggg 240 gggacagccg gcctggacct cctcggagaa aacagtggaa tcgaagaatt agcaatggtg 300 gacatgtgga cggaggtgga ggcccagcag tactacccag ccatctcgcc ggtggtgttc 360 It should be noted that in the above translation, the Chinese character "३०" in line 30 seems to be an incorrect character. It should probably be "30" for correct reading and understanding. If this is an error in the original text, please check and correct it for a more accurate translation.gagtgcatca tcattccctt catcatccct ggcggtggcg cggcgccgaa ccggagcgtc 420 gtggacgaga gcctggagcg gctgaggggt gtactgggga tctacgaggc ccggctggag 480 aagagcagct acttggccgg ggactccatc agcttcgccg atctgaacca catcccgttc 540 accttctact tcatgaccac cccgtacgcc aaggtgtttg atgagtaccc caaggtgaag 600 gcctggtggg agatgctcat ggccaggccg gcggtgcaga gggtctgcaa gcatatgcct 660 accaagttta agctaggtgc gcagtactag 690 <210> 18 <211> 229 <212> PRT <213> Wheat <400> 18 Met Ser Pro Val Lys Val Phe Gly His Pro Met Leu Thr Asn Val Ala 1 5 10 15 Arg Val Leu Leu Phe Leu Glu Glu Val Gly Ala Glu Tyr Glu Leu Val 20 25 30 Pro Leu Asp Phe Val Ala Gly Glu His Lys Arg Pro Gln His Val Gln<00​​​​​​Leu Phe Glu Ser Arg Ala Ile Ala Lys Tyr Ile Leu Arg Lys Tyr Gly 65 70 75 80 Gly Thr Ala Gly Leu Asp Leu Leu Gly Glu Asn Ser Gly Ile Glu Glu 85 90 95 Leu Ala Met Val Asp Met Trp Thr Glu Val Glu Ala Gln Gln Tyr Tyr 100 105 110 Pro Ala Ile Ser Pro Val Val Phe Glu Cys Ile Ile Ile Pro Phe Ile 115 120 125 Ile Pro Gly Gly Gly Ala Ala Pro Asn Arg Ser Val Val Asp Glu Ser 130 135 140 Leu Glu Arg Leu Arg Gly Val Leu Gly Ile Tyr Glu Ala Arg Leu Glu 145 150 155 160 Lys Ser Ser Tyr Leu Ala Gly Asp Ser Ile Ser Phe Ala Asp Leu Asn 165 170 175 His Ile Pro Phe Thr Phe Tyr Phe Met Thr Thr Pro Tyr Ala Lys Val 180 185 190 Phe Asp Glu Tyr Pro Lys Val Lys Ala Trp Trp Glu Met Leu Met Ala 195 200 205 Arg Pro Ala Val Gln Arg Val Cys Lys His Met Pro Thr Lys Phe Lys 210 215 220 Leu Gly Ala Gln Tyr 225 <210> 19 <211> 669 <212> DNA <213> Wheat <400> 19 atggcgggcg agaagggtct ggtgctgctg aacttctggg tgagcccgtt cgggcagcgc 60 tgcctcatcg ctcttgcaga aaaaggcctc ccctacgagt acgtcgaaga gaacctcatg 120 gccggcaaga gcgaccgcct cctccgctcc aaccccatcc acaagaagat cccagtgctc 180 ctccacgacg gccgccccgt caacgagtct ctcatcatcc tcaactacct cgacgacgcc 240 ttcccggaca ccccgtccct cctcccctcc gacccctacg agcgctcgca ggctcgcttc 300 tgggccgact acgtcgacaa gaaggtctac gactgcggca cccggctctg gaagctcaag 360 ggcgagccgc acgcgcaggc gcgggccgag atggtggaaa tcctcaagaa tctggacggg 420 gcgctcgggg ataagtcctt cttcggcggc gatgccttcg ggttcgtcga cgccgcgttc 480 gcgcccttca cgtcgtggtt ccacagctac gagaagtacg gcgagttcag cgtggcggag 540 gtggcgccga agatcgcggc gtgggcaaag cggtgcggcg agcgggagag cgtcgccaag 600 agcctctact cgcctgacaa gatttacgag ttcatcggcg tgctcaagaa gatgcacggc 660 gtcgagtaa 669 <210> 20 <211> 222 <212> PRT <213> Wheat <400> 20 Met Ala Gly Glu Lys Gly Leu Val Leu Leu Asn Phe Trp Val Ser Pro 1 5 10 15 Phe Gly Gln Arg Cys Leu Ile Ala Leu Ala Glu Lys Gly Leu Pro Tyr 20 25 30 Glu Tyr Val Glu Glu Asn Leu Met Ala Gly Lys Ser Asp Arg Leu Leu 35 40 45 Arg Ser Asn Pro Ile His Lys Lys Ile Pro Val Leu Leu His Asp Gly 50 55 60 Arg Pro Val Asn Glu Ser Leu Ile Ile Leu Asn Tyr Leu Asp Asp Ala 65 70 75 80 Phe Pro Asp Thr Pro Ser Leu Leu Pro Ser Asp Pro Tyr Glu Arg Ser 85 90 95 Gln Ala Arg Phe Trp Ala Asp Tyr Val Asp Lys Lys Val Tyr Asp Cys 100 105 110 Gly Thr Arg Leu Trp Lys Leu Lys Gly Glu Pro His Ala Gln Ala Arg 115 120 125 Ala Glu Met Val Glu Ile Leu Lys Asn Leu Asp Gly Ala Leu Gly Asp 130 135 140 Lys Ser Phe Phe Gly Gly Asp Ala Phe Gly Phe Val Asp Ala Ala Phe 145 150 155 160 Ala Pro Phe Thr Ser Trp Phe His Ser Tyr Glu Lys Tyr Gly Glu Phe 165 170 175 Ser Val Ala Glu Val Ala Pro Lys Ile Ala Ala Trp Ala Lys Arg Cys 180 185 190 Gly Glu Arg Glu Ser Val Ala Lys Ser Leu Tyr Ser Pro Asp Lys Ile [[ID=​​​​​​​​​​​​​​​​​​​​​ttccaggacg gtgatctcat ccttttcgag tcgcgcgcga tttcgaggta cgtgctccgc 240 aaaggcgcat ccgatctact ccgagaaaac agcctcgccg agtcggcgac ggtggacgcg 300 tggctcgaag ctgagtccca caacttcgac agggccatgt cggcgatcac cttccagtgc 360 ttcgtcgtgc ccatgttcat gggcgggacg actgaccaca aaatcgtcga ggagaacctg 420 gagaagctta aggcggccct cggagtctac gaggagcgtc tgaccaggtt caaatacttg 480 gccggagatt tcatcagcct ggcggacctg agccattgcc ccatggctca ctacctgctg 540 gccagcccct gcgcgtcggt gctcgatgcg tatccgcgtg tgaaggactg ggttgatggg 600 atgatggatc gaccgagcgt gaaaaaggtc atggagctta tggatgcgtc atga 654 <210> 22 <211> 229 <212> PRT <213> Wheat <400> 22 Met Ser Pro Val Lys Val Phe Gly His Pro Met Leu Thr Asn Val Ala 1 5 10 15 Arg Val Leu Leu Phe Leu Glu Glu Val Gly Ala Glu Tyr Glu Leu Val 20 25 30 Pro Leu Asp Phe Val Ala Gly Glu His Lys Arg Pro Gln His Val Gln 35 40 45 Leu Asn Pro Phe Ala Lys Met Pro Gly Phe Gln Asp Gly Asp Leu Val 50 55 60 Leu Phe Glu Ser Arg Ala Ile Ala Lys Tyr Ile Leu Arg Lys Tyr Gly 65 70 75 80 Gly Thr Ala Gly Leu Asp Leu Leu Gly Glu Asn Ser Gly Ile Glu Glu 85 90 95 Leu Ala Met Val Asp Met Trp Thr Glu Val Glu Ala Gln Gln Tyr Tyr 100 105 110 Pro Ala Ile Ser Pro Val Val Phe Glu Cys Ile Ile Ile Pro Phe Ile 115 120 125 Ile Pro Gly Gly Gly Ala Ala Pro Asn Arg Ser Val Val Asp Glu Ser 130 135 140 Leu Glu Arg Leu Arg Gly Val Leu Gly Ile Tyr Glu Ala Arg Leu Glu 145 150 155 160 Lys Ser Ser Tyr Leu Ala Gly Asp Ser Ile Ser Phe Ala Asp Leu Asn 165 170 175 His Ile Pro Phe Thr Phe Tyr Phe Met Thr Thr Pro Tyr Ala Lys Val 180 185 190 Phe Asp Glu Tyr Pro Lys Val Lys Ala Trp Trp Glu Met Leu Met Ala 195 200 205 Arg Pro Ala Val Gln Arg Val Cys Lys His Met Pro Thr Lys Phe Lys 210 215 220 Leu Gly Ala Gln Tyr 225 <210> 23 <211> 795 <212> DNA <213> Wheat <400> 23 atgacgcatc gatcttttat atattccgca ctccacacta accacagaat cccacggaac 60 cttcaggcac acccacggca gaaaatggcc aacggaggag acgagctgaa gctgctgggc 120 atgtgggcga gcccatacgt ggtcagggtg cagctcgcgc tccacctcaa gggcgtgagc 180 tacgagtacg tcgaggagga cctcgccagc aagagcgagc tcttcctccg ctccaacccg 240 gtgcacaaga cagtcccgat cctcatccac aacggcaagc ccgtctgcga gtcccaggtc 300 atcctccagt acatcgacga ggccttcgcc ggcgtcggcc cgcccctcct ccccgctgac 360 ccctacgagc gcgccgtcgc gcgattctgg gccgcctacg tcgaggacaa gctgctggcg 420 ccgtggggga aggtgttcag ggtgaagacc gacgaggaga gggccgagtg gacgaggcag 480 acggcggcgg cgctgggtcc tctggaggat ggcctcaggg agtgctccaa ggggaagggc 540 ttcttcggcg gcgactgcgt cgggtacgtt gacgtcctgc tcggcagcat ggtgccgtgg 600 gtgcgcgcca ccgagaggct ctccggcgac aagttaatag acgccggcaa ggccccgctg 660 ctggcggcat ggatggagcg cattagcgag ctcgacgccg ccaaggcggt cttccaggac 720 gtcgacaggg tggttgagta cgccggggca atacaggccc ggctttccgc cgcggctgct 780 gcaagcaccc aataa 795 <210> 24 <211> 264 <212> PRT <213> Wheat <400> 24 Met Thr His Arg Ser Phe Ile Tyr Ser Ala Leu His Thr Asn His Arg 1 5 10 15 Ile Pro Arg Asn Leu Gln Ala His Pro Arg Gln Lys Met Ala Asn Gly 20 25 30 Gly Asp Glu Leu Lys Leu Leu Gly Met Trp Ala Ser Pro Tyr Val Val 35 40 45 Arg Val Gln Leu Ala Leu His Leu Lys Gly Val Ser Tyr Glu Tyr Val 50 55 60 Glu Glu Asp Leu Ala Ser Lys Ser Glu Leu Phe Leu Arg Ser Asn Pro 65 70 75 80 Val His Lys Thr Val Pro Ile Leu Ile His Asn Gly Lys Pro Val Cys 85 90 95 Glu Ser Gln Val Ile Leu Gln Tyr Ile Asp Glu Ala Phe Ala Gly Val 100 105 110 Gly Pro Pro Leu Leu Pro Ala Asp Pro Tyr Glu Arg Ala Val Ala Arg 115 120 125 Phe Trp Ala Ala Tyr Val Glu Asp Lys Leu Leu Ala Pro Trp Gly Lys 130 135 140 Val Phe Arg Val Lys Thr Asp Glu Glu Arg Ala Glu Trp Thr Arg Gln 145 150 155 160 Thr Ala Ala Ala Leu Gly Pro Leu Glu Asp Gly Leu Arg Glu Cys Ser 165 170 175 Lys Gly Lys Gly Phe Phe Gly Gly Asp Cys Val Gly Tyr Val Asp Val 180 185 190 Leu Leu Gly Ser Met Val Pro Trp Val Arg Ala Thr Glu Arg Leu Ser 195 200 205 Gly Asp Lys Leu Ile Asp Ala Gly Lys Ala Pro Leu Leu Ala Ala Trp 210 215 220 Met Glu Arg Ile Ser Glu Leu Asp Ala Ala Lys Ala Val Phe Gln Asp 225 230 235 240 Val Asp Arg Val Val Glu Tyr Ala Gly Ala Ile Gln Ala Arg Leu Ser 245 250 255 Ala Ala Ala Ala Ala Ser Thr Gln 260 <210> 25 <211> 714 <212> DNA <213> Wheat <400> 25 atgtcgtcgg ggaagcagga gacggcggcg gtgcgcgtgc tgggcaggtg gccgagcccg 60 ttcgtgatcc gggtgctgat agctcttggg ctcaagggcg tggaccacga gctcgtggag 120 gaggcggcgg gcaacaagag cgagctgctg ctcgcctcca accccgtgca caagaagatc 180 cccgtgctcc tgcaccacgg caggcccgtc tccgagtccc tcatcatcgt ccagtacgtc 240 gacgaggcct gggcctccca agccccggcg ctcatcccgt ccgaccccta cgcccgcgcg 300 gccgagcggt tctgggccca gtacgtcgac gacaagtttc ctacggcgat ccgggtcctg 360 aggggaaggc tggacggaga caaggaagaa gcggcggctc aggtgtgcgc cgctctgcag 420 cacctggagg tggccttcgt cgagtgcggc caagggaagg attacttcgg cggcgacggc 480 gtcggttacc tggacattgc tctcgggtcg cacctcggat gggtcagggc ggtagagagg 540 atcgctgaaa tcaggctgct cgacgcggcc aaggttccta agctggcggc gtgggcggat 600 cggttctgcg cccacccggc ggtggcgaac gccatgccta acgtggacag gttcgtggag 660 ttcagcgtca agaatgacgg cgttctgaag gcggctagtg ctaattccaa gtga 714 <210> 26 <211> 237 <212> PRT <213> Wheat <400> 26 Met Ser Ser Gly Lys Gln Glu Thr Ala Ala Val Arg Val Leu Gly Arg 1 5 10 15 Trp Pro Ser Pro Phe Val Ile Arg Val Leu Ile Ala Leu Gly Leu Lys 20 25 30 Gly Val Asp His Glu Leu Val Glu Glu Ala Ala Gly Asn Lys Ser Glu 35 40 45 Leu Leu Leu Ala Ser Asn Pro Val His Lys Lys Ile Pro Val Leu Leu 50 55 60 His His Gly Arg Pro Val Ser Glu Ser Leu Ile Ile Val Gln Tyr Val 65 70 75 80 Asp Glu Ala Trp Ala Ser Gln Ala Pro Ala Leu Ile Pro Ser Asp Pro 85 90 95 Tyr Ala Arg Ala Ala Glu Arg Phe Trp Ala Gln Tyr Val Asp Asp Lys 100 105 110 Phe Pro Thr Ala Ile Arg Val Leu Arg Gly Arg Leu Asp Gly Asp Lys 115 120 125 Glu Glu Ala Ala Ala Gln Val Cys Ala Ala Leu Gln His Leu Glu Val 130 135 140 Ala Phe Val Glu Cys Gly Gln Gly Lys Asp Tyr Phe Gly Gly Asp Gly 145 150 155 160 Val Gly Tyr Leu Asp Ile Ala Leu Gly Ser His Leu Gly Trp Val Arg 165 170 175 Ala Val Glu Arg Ile Ala Glu Ile Arg Leu Leu Asp Ala Ala Lys Val 180 185 190 Pro Lys Leu Ala Ala Trp Ala Asp Arg Phe Cys Ala His Pro Ala Val 195 200 205 Ala Asn Ala Met Pro Asn Val Asp Arg Phe Val Glu Phe Ser Val Lys 210 215 220 Asn Asp Gly Val Leu Lys Ala Ala Ser Ala Asn Ser Lys 225 230 235 <210> 27 <211> 693 <212> DNA <213> Wheat <400> 27 atggccggag cagcaaacga tctgaagcta ctgggcatgt gggcgagccc gtacgtcctg 60 cgggtgcgcc ttgctctcag catcaagagc atcagctacg agtacgcaga ggaggacctc 120 cggcacaaga gcgagctgct cctgcggtca aaccccgtcc acaacaaggt ccccgtgctg 180 atccacgccg gcaagcccgt ctgcgagtcg ctggtcatcc tacagtacat cgacgacgct 240 ttcggcggtg ccggccccgc cctcctcccg gccgatcccc acgagcgcgc cgtcgcccgg 300 ttctgggccg ccttcatcga ggacacgctc gtgaaggcga tgaaccaggc gtcatggagc 360 aagacggagg cggagaaggt ggaggggaac aaacgggcga ctgctgcgtt gaacaccctg 420 gaggcggccc tgagggatgt ctccaagggg aagcccttct tcgggggcga cagcaccggg 480 tatgtggaca tcgtgctcgg cggcctcctc gcgggggtgc gcgccatgga ggcgatgccg 540 ggcgtcaagg ccttcgaccc cgtcacgatg ccgctcctgg ccgcgtgggc ggaccacttc 600 ggcgcgctgg acgcggtggc ggccgtgatg ccggacgtga gcaagctcgt ggagctcttc 660 atcacgatgc acgctgctgt tgcggcaaac taa 693 <210> 28 <211> 230 <212> PRT <213> small <400> 28 Met Ala Gly Ala Ala Asn Asp Leu Lys Leu Leu Gly Met Trp Ala Ser 1 5 10 15 Pro Tyr Val Leu Arg Val Arg Leu Ala Leu Ser Ile Lys Ser Ile Ser 20 25 30 Tyr Glu Tyr Ala Glu Glu Asp Leu Arg His Lys Ser Glu Leu Leu Leu 35 40 45 Arg Ser Asn Pro Val His Asn Lys Val Pro Val Leu Ile His Ala Gly 50 55 60 Lys Pro Val Cys Glu Ser Leu Val Ile Leu Gln Tyr Ile Asp Asp Ala 65 70 75 80 Phe Gly Gly Ala Gly Pro Ala Leu Leu Pro Ala Asp Pro His Glu Arg 85 90 95 Ala Val Ala Arg Phe Trp Ala Ala Phe Ile Glu Asp Thr Leu Val Lys 100 105 110 Ala Met Asn Gln Ala Ser Trp Ser Lys Thr Glu Ala Glu Lys Val Glu 115 120 125 Gly Asn Lys Arg Ala Thr Ala Ala Leu Asn Thr Leu Glu Ala Ala Leu 130 135 140 Arg Asp Val Ser Lys Gly Lys Pro Phe Phe Gly Gly Asp Ser Thr Gly 145 150 155 160 Tyr Val Asp Ile Val Leu Gly Gly Leu Leu Ala Gly Val Arg Ala Met 165 170 175 Glu Ala Met Pro Gly Val Lys Ala Phe Asp Pro Val Thr Met Pro Leu 180 185 190 Leu Ala Ala Trp Ala Asp His Phe Gly Ala Leu Asp Ala Val Ala Ala 195 200 205 Val Met Pro Asp Val Ser Lys Leu Val Glu Leu Phe Ile Thr Met His 210 215 220 Ala Ala Val Ala Ala Asn 225 230 <210> 29 <211> 810 <212> DNA <213> Wheat <400> 29 atgacgcacg gaggtataca aacaaatgtg aagaatacgg tgtctatcag tgttgcactg 60 atagagcagg ggagaaaaaa aaagacggcg aacgaaacaa tggcagccga aggaggtctg 120 aagctgctcg gcttgacggt gagcccgttc gtgatccgtg tacgcatggc gctgcagatg 180 aaaggcgtcg gctacgagta cgtggagcag gacctgttca ccaagggcga gctcctccgc 240 aagtccaacc cggtgcacat gaaggtcccg gtgctcatcc acgacggcag acccgtctgc 300 gagtcgctgg ccatcgtgca gtacgtcgac gaggcctggg cggccgcggg cccctcgatc 360 ctccccgccg acccctacga ccgcgccgcc gctcgcttct gggccgccta cgccgacagc 420 aagctcttgc cggcgtgggt aggcatcatg tgggcggaga cggaggagga gagggcggag 480 aaggtcggcg acacgctcgc ggctatcggc cagttggagg aggcgttcgg gacgtgctcg 540 aacggtaagg ccttcttcgc cggcgactcc gtcgggtacc tagatctcgt cgtcggctcg 600 cagttgctct ggttcgaggt gctgcggaag atgttcggcg tcgtggtcgt tgaggtcggc 660 agggctctgc tcttggccgc gtgggtggag cggtttgggg agactgatac ggccaaggag 720 gtggtgccgg acgttgacac ggcggtggag tacctcaaga agcttcagtc tcgccgggct 780 ggttccacgg ttgcccagct gctgtcgtga 810 <210> 30 <211> 269 <212> PRT <213> Wheat <400> 30 Met Thr His Gly Gly Ile Gln Thr Asn Val Lys Asn Thr Val Ser Ile 1 5 10 15 Ser Val Ala Leu Ile Glu Gln Gly Arg Lys Lys Lys Thr Ala Asn Glu 20 25 30 Thr Met Ala Ala Glu Gly Gly Leu Lys Leu Leu Gly Leu Thr Val Ser 35 40 45 Pro Phe Val Ile Arg Val Arg Met Ala Leu Gln Met Lys Gly Val Gly 50 55 60 Tyr Glu Tyr Val Glu Gln Asp Leu Phe Thr Lys Gly Glu Leu Leu Arg 65 70 75 80 Lys Ser Asn Pro Val His Met Lys Val Pro Val Leu Ile His Asp Gly 85 90 95 Arg Pro Val Cys Glu Ser Leu Ala Ile Val Gln Tyr Val Asp Glu Ala 100 105 110 Trp Ala Ala Ala Gly Pro Ser Ile Leu Pro Ala Asp Pro Tyr Asp Arg 115 120 125 Ala Ala Ala Arg Phe Trp Ala Ala Tyr Ala Asp Ser Lys Leu Leu Pro 130 135 140 Ala Trp Val Gly Ile Met Trp Ala Glu Thr Glu Glu Glu Arg Ala Glu 145 150 155 160 Lys Val Gly Asp Thr Leu Ala Ala Ile Gly Gln Leu Glu Glu Ala Phe 165 170 175 Gly Thr Cys Ser Asn Gly Lys Ala Phe Phe Ala Gly Asp Ser Val Gly 180 185 190 Tyr Leu Asp Leu Val Val Gly Ser Gln Leu Leu Trp Phe Glu Val Leu 195 200 205 Arg Lys Met Phe Gly Val Val Val Val Glu Val Gly Arg Ala Leu Leu 210 215 220 Leu Ala Ala Trp Val Glu Arg Phe Gly Glu Thr Asp Thr Ala Lys Glu 225 230 235 240 Val Val Pro Asp Val Asp Thr Ala Val Glu Tyr Leu Lys Lys Leu Gln 245 250 255 Ser Arg Arg Ala Gly Ser Thr Val Ala Gln Leu Leu Ser 260 265 <210> 31 <211> 717 <212> DNA <213> Barley (Hordeum vulgare) <400> 31 atgtcgcagc agccagaaca ggcgccggtg aggctcatca cggcgttcgg cagcccgttc 60 gcgcaccggg tggaggtggc gctcacgctc aagggggtgc cgtacgagct gctcgtggag 120 gacctggcca gcaagagcga cctgctgctc gcccacaacc ccgtctacca gtcggtcccc 180 gtcctcctcc acggcgaccg cgccgtctgc gactccctcg tcatcgtcga gtacgtcgac 240 gaggccttcc acgacgacga cgggacggcg ccccggcgcc tcctcccggc ggacccctac 300 gaccgcgcca ccgcccgctt ctgggccgac ttcgtcgcca acaagtgctt gaagccgctg 360 tggcagtcga cgtggaccga cggcgaggag caggcgcggc tggcgaggga gaccaaggag 420 ggactggggg tactggaggc acagctcgac gggaagcggt tctttggggg cgaggccctc 480 ggcttcgtcg acctcgccgc ctgcacgctg gctcactggc tcggcgtgct gggggaagtc 540 ggcggggtgc ggctgatgga ggacggcgag taccctgctc tccgccggtg ggccaaggag 600 tacacttccc atgaggtcgt caggcggtcc ctgccggaca gggacgagct cgtcgcctac 660 ttcaccaaaa acaaggagaa gtaccggtcg tcgatgctca agccagcggt gaagtga 717 <210> 32 <211> 238 <212> PRT <213> Barley <400> 32 Met Ser Gln Gln Pro Glu Gln Ala Pro Val Arg Leu Ile Thr Ala Phe 1 5 10 15 Gly Ser Pro Phe Ala His Arg Val Glu Val Ala Leu Thr Leu Lys Gly 20 25 30 Val Pro Tyr Glu Leu Leu Val Glu Asp Leu Ala Ser Lys Ser Asp Leu 35 40 45 Leu Leu Ala His Asn Pro Val Tyr Gln Ser Val Pro Val Leu Leu His 50 55 60 Gly Asp Arg Ala Val Cys Asp Ser Leu Val Ile Val Glu Tyr Val Asp 65 70 75 80 Glu Ala Phe His Asp Asp Asp Gly Thr Ala Pro Arg Arg Leu Leu Pro 85 90 95 Ala Asp Pro Tyr Asp Arg Ala Thr Ala Arg Phe Trp Ala Asp Phe Val 100 105 110 Ala Asn Lys Cys Leu Lys Pro Leu Trp Gln Ser Thr Trp Thr Asp Gly 115 120 125 Glu Glu Gln Ala Arg Leu Ala Arg Glu Thr Lys Glu Gly Leu Gly Val 130 135 140 Leu Glu Ala Gln Leu Asp Gly Lys Arg Phe Phe Gly Gly Glu Ala Leu 145 150 155 160 Gly Phe Val Asp Leu Ala Ala Cys Thr Leu Ala His Trp Leu Gly Val 165 170 175 Leu Gly Glu Val Gly Gly Val Arg Leu Met Glu Asp Gly Glu Tyr Pro 180 185 190 Ala Leu Arg Arg Trp Ala Lys Glu Tyr Thr Ser His Glu Val Val Arg 195 200 205 Arg Ser Leu Pro Asp Arg Asp Glu Leu Val Ala Tyr Phe Thr Lys Asn 210 215 220[[ID=!2]] Lys Glu Lys Tyr Arg Ser Ser Met Leu Lys Pro Ala Val Lys 225 230 235 <210> 33 <211> 690 <212> DNA <213> Barley <400> 33 atggatcatc aagaggagaa ggtgaagctt tttggcatgt gggcgagccc ctacgtcctc 60 aaggtgaaat gggcgctgag catcaagggc gtggagtacg agtacctgga ggaggacctg 120 aggaacaaga gcgacgatct cctggaacac aaccccgtgc acaagaaggt ccccgtgctg 180 ctctaccacg gcaagccggt ggcagagtcc gacgtcatcg tcgagttcgt cgacgaggcg 240 tggagccacc gaggcggccg catcctcccc ggcgacccct acgagcgcgc catggctcgt 300 ttctgggtga ggtttgtgca cgacaagctc tcgccgccga tttggaagtg gttcacgacg 360 It should be noted that there seems to be a misspelling in "[[ID=!2]]", which should probably be "". If this is a real error in the original, it may affect the correct understanding and further processing of the content.gcgccaggcg aggaccagga ggccgcgcgg ggggcctccg ttgagcagct gcaggtcctg 420 gaggagttgc tcgccgtcgg cgggaaggag ttcttcgccg gggagagcgt tgggctcgtg 480 gacctgtcgc tcggcgcgat ggcgtacgtg gtcccgatgt acgaggagat cgtcggcgtg 540 aggctggtca ccgaggagag gtttccgtct ctgtcggcgt ggatggggcg gttcttgggc 600 tcgccgccgg tgaaggatca cccgccgcca gtggagaggc tgatacccag gtaccgagcc 660 atgcgcgaag cctttctgaa gatgggctag 690 <210> 34 <211> 229 <212> PRT <213> Barley <400> 34 Met Asp His Gln Glu Glu Lys Val Lys Leu Phe Gly Met Trp Ala Ser 1 5 10 15 Pro Tyr Val Leu Lys Val Lys Trp Ala Leu Ser Ile Lys Gly Val Glu 20 25 30 Tyr Glu Tyr Leu Glu Glu Asp Leu Arg Asn Lys Ser Asp Asp Leu Leu 35 40 45 Glu His Asn Pro Val His Lys Lys Val Pro Val Leu Leu Tyr His Gly 50 55 60 Lys Pro Val Ala Glu Ser Asp Val Ile Val Glu Phe Val Asp Glu Ala 65 70 75 80 Trp Ser His Arg Gly Gly Arg Ile Leu Pro Gly Asp Pro Tyr Glu Arg 85 90 95 Ala Met Ala Arg Phe Trp Val Arg Phe Val His Asp Lys Leu Ser Pro 100 105 110 Pro Ile Trp Lys Trp Phe Thr Thr Ala Pro Gly Glu Asp Gln Glu Ala 115 120 125 Ala Arg Gly Ala Ser Val Glu Gln Leu Gln Val Leu Glu Glu Leu Leu 130 135 140 Ala Val Gly Gly Lys Glu Phe Phe Ala Gly Glu Ser Val Gly Leu Val 145 150 155 160 Asp Leu Ser Leu Gly Ala Met Ala Tyr Val Val Pro Met Tyr Glu Glu 165 170 175 Ile Val Gly Val Arg Leu Val Thr Glu Glu Arg Phe Pro Ser Leu Ser 180 185 190 Ala Trp Met Gly Arg Phe Leu Gly Ser Pro Pro Val Lys Asp His Pro 195 200 205 Pro Pro Val Glu Arg Leu Ile Pro Arg Tyr Arg Ala Met Arg Glu Ala 210 215 220 Phe Leu Lys Met Gly 225 <210> 35 <211> 645 <212> DNA <213> Barley <400> 35 atggcactgc agatgaaacg cgtgggctac gagtacatgg agcaggacct gttcaccaag 60 ggcgagctcc tcctcaagtc caacccggtg cacatgaagg tcccggtgct catccacgac 120 ggcaaatcca tctgcgagtc gctggccatc gtgcagtacg tcgacgaggt ctgggccgcc 180 acgggcacct ccatcctccc cgccgacccc tacgaccgcg ccgccgctcg cttctgggcc 240[[ID=二十一]] [[ID=二十二]]gcctacgccg acagcaagct cttgcctgcg tgggtgggca tcatgtgggc ggcgacggag 300[[ID=二十三]] [[ID=二十四]]gaggagagag cggagaaggt cggcgacacg ctcgcggcta tcggccaact ggaggaggcg 360[[ID=二十五]] [[ID=二十六]]ttcggaaagt gctccaacgg aaagcccttc ttcgccggcg attccgtcgg ctacctcgat 420[[ID=二十七]] [[ID=二十八]]ctcgtcgtcg gttcgcagtt gctctggttc gaggtgctgc ggaagatgtt cggcgtcgtg 480[[ID=二十九]] [[ID=三十]]gtcattgagg cctgcagggc tcccttcttg gccgcgtggg tgaagcggtt ttgggagact 540[[ID=三十一]] [[ID=三十二]]gatacggcga aggcggtggt gccggacgtt ggcacggcgg cggagtacct gaagaagctt 600[[ID=三十三]] cagtctcatc gggctggtgc cacggttgcc cagttgctgt cgtga 645 <210> 36 <211> 214 <212> PRT <213> Barley <400> 36 Met Ala Leu Gln Met Lys Arg Val Gly Tyr Glu Tyr Met Glu Gln Asp 1 5 10 15 Leu Phe Thr Lys Gly Glu Leu Leu Leu Lys Ser Asn Pro Val His Met 20 25 30 Lys Val Pro Val Leu Ile His Asp Gly Lys Ser Ile Cys Glu Ser Leu 35 40 45 Ala Ile Val Gln Tyr Val Asp Glu Val Trp Ala Ala Thr Gly Thr Ser 50 55 60 Ile Leu Pro Ala Asp Pro Tyr Asp Arg Ala Ala Ala Arg Phe Trp Ala 65 70 75 80 Ala Tyr Ala Asp Ser Lys Leu Leu Pro Ala Trp Val Gly Ile Met Trp 85 90 95 Ala Ala Thr Glu Glu Glu Arg Ala Glu Lys Val Gly Asp Thr Leu Ala 100 105 110 Ala Ile Gly Gln Leu Glu Glu Ala Phe Gly Lys Cys Ser Asn Gly Lys 115 120 125 Pro Phe Phe Ala Gly Asp Ser Val Gly Tyr Leu Asp Leu Val Val Gly 130 135 140 Ser Gln Leu Leu Trp Phe Glu Val Leu Arg Lys Met Phe Gly Val Val 145 150 155 160 Val Ile Glu Ala Cys Arg Ala Pro Phe Leu Ala Ala Trp Val Lys Arg 165 170 175 Phe Trp Glu Thr Asp Thr Ala Lys Ala Val Val Pro Asp Val Gly Thr 180 185 190 Ala Ala Glu Tyr Leu Lys Lys Leu Gln Ser His Arg Ala Gly Ala Thr 195 200 205 Val Ala Gln Leu Leu Ser 210 <210> 37 <211> 93 <212> DNA <213> Barley <400> 37 gaggctgcaa agaaggcggc gccacccatg gaaagcatgt tggaggaggc cgagaagctg 60 cgggctatgt gggctgcggc ggctgccaag taa 93 <210> 38 <211> 232 <212> PRT <213> Barley <400> 38 Met Ala Ser Glu Gly Asp Asp Val Lys Val Leu Gly Thr Ala Ala Ser 1 5 10 15 Met Phe Ala Ile Arg Val Arg Met Ala Leu His Ala Lys Gly Val Ser 20 25 30 Tyr Glu Tyr Leu Glu Gln Asp Leu Phe His Lys Gly Glu Leu Leu Leu 35 40 45 Ala Ser Asn Pro Val Arg Lys Ala Val Pro Val Leu Ile His Ala Gly 50 55 60 Arg Pro Val Cys Glu Ser Leu Ala Ile Val Glu Tyr Ile Asp Glu Val 65 70 75 80 Trp Ala Gly Ala Ala Ser Leu Leu Pro Ala Asp Pro Tyr Asp Arg Ala 85 90 95 Val Ala Arg Phe Trp Ala Ala Tyr Val Asp Asp Lys Ala Val Pro Thr 100 105 110 Trp Ile Gly Ile Met Arg Ala Ala Thr Glu Glu Asp Arg Ala Glu Arg 115 120 125 Leu Ala Ala Ala Leu Ala Ala Val Ala Pro Leu Glu Asp Ala Phe Ala 130 135 140 Gln Cys Ser Gly Gly Lys Ala Phe Phe Ala Gly Asp Ser Ile Gly Tyr 145 150 155 160 Val Asp Leu Ala Leu Gly Cys Asn Leu Phe Trp Ile Glu Ala Leu Arg 165 170 175 His Met Phe Gly Ile Thr Val Ile Asp Ala Gly Arg Thr Pro Arg Leu 180 185 190 Ala Ala Trp Ala Glu Arg Phe Val Glu Thr Glu Ala Ala Lys Lys Ala 195 200 205 Ala Pro Pro Met Glu Ser Met Leu Glu Glu Ala Glu Lys Leu Arg Ala 210 215 220 Met Trp Ala Ala Ala Ala Ala Lys 225 230 <210> 39 <211> 708 <212> DNA <213> Barley <400> 39 atggcaggag gcggcgaggg cgagctgaag ctgctgggcg tgtggacgag cccgttcgtc 60 atccgggtgc gcgtggtgct caacctcaag tcgctgccgt acgagtacgt ggaggagaac 120 ctgggcagca agagcgcgct cctcctgggc tccaacccgg tgcaccagag cgtgccggtc 180 ctcctccacg gcggccgccc cgtgaacgag tcccaggtca tcgtgcagta catcgacgag 240 gtctgggcgg gggtcggccc gtcggtgctc ccggccgacc cctacgagcg cgccgtggcg 300 cgcttctggg cggcgtacgt cgacgacaag gtcgggtcgg cgtggacggg gatgctcttc 360 tcgtgcaaga cggaggagga gagggcggag gccgtgtccc gcgccgtggc ggcgctggag 420 accctggagg gcgcgctcgc ggagtgctcc ggggggaagc cgttcttcgg cggcgacgcc 480 atcgggttcg tcgacgtcgt gctcggcggc tacctcgggt ggttcggggc gatcgacaag 540 atcatcgggc gccggctgat cgacccggcg aggacgccgc tgctggccag gtgggaggag 600 tggttccgcg cggcggacgc ggccaagggc gtcgtgccgg acgacgccga caagatgctc 660 gacttcttgc ccaccctgct cgcttggatc gcctccaagg ccaagtga 708 <210> 40 <211> 235 <212> PRT <213> Barley <400> 40 Met Ala Gly Gly Gly Glu Gly Glu Leu Lys Leu Leu Gly Val Trp Thr 1 5 10 15 Ser Pro Phe Val Ile Arg Val Arg Val Val Leu Asn Leu Lys Ser Leu 20 25 30 Pro Tyr Glu Tyr Val Glu Glu Asn Leu Gly Ser Lys Ser Ala Leu Leu 35 40 45 Leu Gly Ser Asn Pro Val His Gln Ser Val Pro Val Leu Leu His Gly 50 55 60 Gly Arg Pro Val Asn Glu Ser Gln Val Ile Val Gln Tyr Ile Asp Glu 65 70 75 80 Val Trp Ala Gly Val Gly Pro Ser Val Leu Pro Ala Asp Pro Tyr Glu 85 90 95 Arg Ala Val Ala Arg Phe Trp Ala Ala Tyr Val Asp Asp Lys Val Gly 100 105 110 Ser Ala Trp Thr Gly Met Leu Phe Ser Cys Lys Thr Glu Glu Glu Arg 115 120 125 Ala Glu Ala Val Ser Arg Ala Val Ala Ala Leu Glu Thr Leu Glu Gly 130 135 140 Ala Leu Ala Glu Cys Ser Gly Gly Lys Pro Phe Phe Gly Gly Asp Ala 145 150 155 160 Ile Gly Phe Val Asp Val Val Leu Gly Gly Tyr Leu Gly Trp Phe Gly 165 170 175 Ala Ile Asp Lys Ile Ile Gly Arg Arg Leu Ile Asp Pro Ala Arg Thr 180 185 190 Pro Leu Leu Ala Arg Trp Glu Glu Trp Phe Arg Ala Ala Asp Ala Ala 195 200 205 Lys Gly Val Val Pro Asp Asp Ala Asp Lys Met Leu Asp Phe Leu Pro 210 215 220 Thr Leu Leu Ala Trp Ile Ala Ser Lys Ala Lys 225 230 235 <210> 41 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 41 ccggacaaaa tggggtcg 18 <210> 42 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 42 ttattcaatg gaagtcacgt c 21 <210> 43 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 43 aatctctact tccaaggcca tatggccgga ggagatgac 39 <210> 44 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 44 gggcgacgag cttatcgtca acgtaggcg 29 <210> 45 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 45 tgacgataag ctcgtcgccc catgggta 28 <210> 46 <211> 47 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 46 aagcttgtcg acggagctcg aattcttact tggtctctga agcagcg 47 <210> 47 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 47 ccttacacac acagatctag atg 23 <210> 48 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 48 caagaacaga aatacggatt tcc 23 <210> 49 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 49 gggtgacgag cttatcgtca atgtaggcgg 30 <210> 50 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 50 tgacgataag ctcgtcaccc catgggta 28 <210> 51 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 51 aagcttgtcg acggagctcg aattctactt ggtctccgaa gcagc 45

Claims

1. A method for biotransforming trichothecenes, said method comprising contacting a material contaminated with trichothecenes with an exogenous non-animal glutathione-S-transferase (GST) having substrate specificity for the epoxy ring of trichothecenes, said method comprising the steps of: a) contacting the material with GST, b) optionally adding glutathione to said material, and c) incubating the mixture in an aqueous solution having a pH in the range of 6 to 9 under conditions wherein the glutathione reacts with the epoxide moiety, thereby forming an epoxide adduct; The GST is a recombinant GST, the amino acid sequence of which is shown in SEQ ID NO: 2 or SEQ ID NO: 4, wherein the trichothecene is selected from the group consisting of deoxynivalenol (DON), myristocetin A, T-2 toxin and trichoderma.

2. The method according to claim 1 , wherein the method is used for producing a decontaminated feed additive, feed material, food additive or food material, for decontaminating liquid or solid materials or material surfaces, or textile materials, filter materials, gas masks, air conditioning systems, or for purifying material surfaces, animal or human surfaces. 3 . The method according to claim 1 , wherein the GST is encoded by a polynucleotide sequence selected from SEQ ID NO: 1 or SEQ ID NO:

3.

4. The method according to any one of claims 1 to 3, wherein GST is expressed in a host cell, wherein the host cell is a prokaryotic host cell or a fungal host cell.

5. A feed additive or feed material comprising an exogenous non-animal GST having substrate specificity for the epoxy ring of a trichothecene, wherein the amino acid sequence of the non-animal GST is shown in SEQ ID NO: 2 or SEQ ID NO: 4, wherein the trichothecene is selected from the group consisting of deoxynivalenol (DON), myristocetin A, T-2 toxin and trichoderma.

6. The feed additive or feed material according to claim 5, comprising a transgenic plant part containing an exogenous GST having substrate specificity for the epoxy ring of a trichothecene. 7 . The feed additive or feed material according to claim 5 , comprising transgenic plant tissue containing exogenous GST having substrate specificity for the epoxy ring of trichothecenes.

8. The feed additive or feed material according to claim 5, comprising a transgenic plant cell containing an exogenous GST having substrate specificity for the epoxy ring of a trichothecene. 9 . The feed additive or feed material according to claim 5 , comprising seeds or progeny thereof, wherein the seeds or progeny thereof contain exogenous GST having substrate specificity for the epoxy ring of trichothecenes.

10. The feed additive or feed material according to claim 5, comprising: leaves, stems, roots, cotyledons or hypocotyls, wherein the leaves, stems, roots, cotyledons or hypocotyls contain an exogenous GST having substrate specificity for the epoxy ring of trichothecenes.

11. Use of the method according to any one of claims 1 to 4 for the enzymatic degradation of trichothecenes in animal feed, wherein the trichothecenes are selected from deoxynivalenol (DON), myclocantin A, T-2 toxin and trichoderma.

12. Use of the method according to any one of claims 1 to 4 for producing feed or food additives.

13. A recombinant host cell overexpressing endogenous GST or transformed with a vector expressing exogenous GST, wherein the GST has substrate specificity for the epoxy ring of a trichothecene, wherein the host cell is a prokaryotic host cell or a fungal host cell, wherein the amino acid sequence of the GST is shown in SEQ ID NO: 2 or SEQ ID NO: 4, and wherein the trichothecene is selected from the group consisting of deoxynivalenol (DON), myristoctin A, T-2 toxin, and trichoderma. The recombinant host cell according to claim 13 , which is a bacterial cell. The recombinant host cell according to claim 13 or 14, wherein the GST is encoded by a polynucleotide sequence selected from SEQ ID NO: 1 and SEQ ID NO:

3.

16. The host cell according to any one of claims 14 to 15, comprising a GST having substrate specificity for a trichothecene, wherein the trichothecene is selected from the group consisting of deoxynivalenol (DON), myristocetin A, T-2 toxin and trichoderma. 17 . The host cell according to claim 13 , wherein the amino acid sequence of the exogenous GST is shown in SEQ ID NO: 2 or SEQ ID NO:

4.

18. A method for expressing an exogenous GST having substrate specificity for the epoxy ring of a trichothecene in a host cell, wherein the trichothecene is selected from deoxynivalenol (DON), myrosin A, T-2 toxin, and trichoderma, wherein the host cell is a prokaryotic host cell or a fungal host cell, the method comprising: a) transforming the host cell with a vector containing a GST encoding gene sequence, wherein the GST is encoded by a polynucleotide sequence selected from SEQ ID NO: 1 or SEQ ID NO: 3; b) growing the transformed cells under conditions suitable for the expression of the gene encoding the exogenous GST.

Images