Enzymatic degradation of a mycotoxin

Abstract

Enzymes having hydrolase, decarboxylase and Baeyer-Villiger oxidase activity are isolated from the black yeast-like fungus Exophiala sp. strain LRDC-1. The enzymes catalyze the degradation of resorcylic acid lactone mycotoxins such as zearalenone, and are useful in methods of detoxifying material contaminated with these mycotoxins.

Description

ENZYMATIC DEGRADATION OF A MYCOTOXIN Field

[0001] The present application is directed to compositions and methods for degrading toxic fungal metabolites. More specifically, the present application is directed to enzymatic methods of detoxifying resorcylic acid lactone mycotoxins such as zearalenone. Background

[0002] Resorcylic acid lactones are structurally related and functionally diverse polyketide secondary metabolites produced by numerous fungi, many of which are plant pathogens. In general, resorcylic acid lactones contain a β-resorcylic acid moiety (typically 2,4- dihydroxybenzoic acid) fused to a macrolactone ring of varying size, and include the hypothemycins, aigialomycins, pochonins and others. Resorcylic acid lactones have attracted attention as having a wide range of biological activities including antibacterial, antimalarial, antiviral, and anticancer activities. An important member of this group is zearalenone (Formula I).

[0003] Zearalenone was first discovered in 1962 in extracts from mould-infested maize, and is produced by several species of the fungal genus Fusarium, including F. graminearum, F. culmorum, F. cerealis, F. equiseti, F. crookwellense, and F. semitectum. Zearalenone is a common contaminant of maize and wheat, and is also detected in other grains including oats and barley. Zearalenone is a non-steroidal estrogen mimic that is capable of binding the mammalian estrogen receptor, and can cause serious reproductive issues in livestock when consumed, including enlargement and swelling of the ovaries and uterus along with reduced litter sizes and increased stillbirths in swine.

[0004] Reduction and / or elimination of zearalenone from agricultural products is an ongoing problem. Zearalenone detoxification efforts have included physical, chemical, and biological techniques with varying levels of success. Chemical treatments, including ozonation and oxidation, and the use of physical adsorbents that bind and sequester zearalenone can be effective but can significantly alter the palatability and alimentary qualities of the treated feed. Therefore, new methods of reducing toxicity caused by zearalenone contamination of grain are desirable.

[0005] Enzymatic detoxification of mycotoxins is a recognized mechanism to reduce or eliminate their toxicity and can be rapid, specific, and cost-effective. For example, hydrolase enzymes that hydrolyze the lactone moiety of zearalenone (ZEN, Formula I) to produce hydrolyzed zearalenone (HZEN, Formula II) are known. The first identified zearalenone hydrolase was produced by the fungus Clonostachys rosea and a zearalenone hydrolase is commercially available as an animal feed additive under the brand name ZENzymeTM. Under alkaline conditions, the hydrolyzed product spontaneously decarboxylates to produce decarboxylated hydrolyzed zearalenone (DHZEN, Formula III). Neither HZEN nor DHZEN bind the estrogen receptor and both compounds are non-toxic.Summary

[0006] In one aspect, the present application is directed to a protein active to degrade a mycotoxin, wherein the protein comprises one or more mycotoxin-degrading enzymes selected from a hydrolase enzyme, a decarboxylase enzyme and a Baeyer-Villiger oxidase enzyme. In at least one embodiment, the protein is active to reduce the toxicity of the mycotoxin. In at least one embodiment, the one or more mycotoxin-degrading enzymes are from Exophiala sp. strain LRDC-1. A deposit of Exophiala sp. strain LRDC-1 pursuant to the Budapest Treaty was received on July 31, 2024 and on September 5, 2024, by the International Depository Authority of Canada (IDAC), National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, Manitoba R3E 3R2, Canada (Accession number 310724-02). In at least one embodiment, the mycotoxin is a resorcylic acid lactone mycotoxin. In at least one embodiment, the mycotoxin is zearalenone.

[0007] In at least one embodiment, the hydrolase enzyme is active to catalyze hydrolysis of the mycotoxin to form a hydrolyzed mycotoxin. In at least one embodiment, the hydrolase enzyme is active to catalyze hydrolysis of a resorcylic acid lactone mycotoxin to form a hydrolyzed resorcylic acid lactone mycotoxin. In at least one embodiment, the resorcylic acid lactone mycotoxin is zearalenone. In at least one such embodiment, the hydrolase enzyme is active to catalyze hydrolysis of zearalenone to form hydrolyzed zearalenone as described herein.

[0008] In at least one embodiment, the hydrolase enzyme comprises an amino acid sequence indicated as SEQ ID NO:1, or a catalytically active portion thereof, or a variantamino acid sequence having at least 70% identity to SEQ ID NO:1, or to the catalytically active portion thereof. In at least one such embodiment, the hydrolase enzyme has an amino acid sequence indicated as SEQ ID NO:1. In at least one embodiment, the hydrolase enzyme is encoded by a polynucleotide comprising a nucleotide sequence indicated as SEQ ID NO:2 or by a polynucleotide comprising a variant nucleotide sequence having at least 70% identity to SEQ ID NO:2. In at least one embodiment, the hydrolase enzyme is isolated.

[0009] In at least one embodiment, the decarboxylase enzyme is active to catalyze decarboxylation of the hydrolyzed mycotoxin to form a decarboxylated hydrolyzed mycotoxin. In at least one embodiment, the decarboxylase enzyme is active to catalyze decarboxylation of a hydrolyzed resorcylic acid lactone mycotoxin to form a decarboxylated hydrolyzed resorcylic acid lactone mycotoxin. In at least one embodiment, the hydrolyzed resorcylic acid lactone mycotoxin is hydrolyzed zearalenone. In at least one such embodiment, the decarboxylase enzyme is active to catalyze decarboxylation of hydrolyzed zearalenone to form decarboxylated hydrolyzed zearalenone as described herein.

[0010] In at least one embodiment, the decarboxylase enzyme comprises an amino acid sequence indicated as SEQ ID NO:3, or a catalytically active portion thereof, or a variant amino acid sequence having at least 70% identity to SEQ ID NO:3, or to the catalytically active portion thereof. In at least one such embodiment, the decarboxylase enzyme has an amino acid sequence indicated as SEQ ID NO:3. In at least one embodiment, the decarboxylase enzyme is encoded by a polynucleotide comprising a nucleotide sequence indicated as SEQ ID NO:4 or by a polynucleotide comprising a variant nucleotide sequence having at least 70% identity to SEQ ID NO:4. In at least one embodiment, the decarboxylase enzyme is isolated.

[0011] In at least one embodiment, the Baeyer-Villiger oxidase enzyme is active to catalyze oxidation of the decarboxylated hydrolyzed mycotoxin to form an oxidized decarboxylated hydrolyzed mycotoxin. In at least one embodiment, the Baeyer-Villiger oxidase enzyme is active to catalyze oxidation of a decarboxylated hydrolyzed resorcylic acid lactone mycotoxin to form an oxidized decarboxylated hydrolyzed resorcylic acid lactone mycotoxin. In at least one embodiment, the decarboxylated hydrolyzed resorcylic acid lactone mycotoxin is decarboxylated hydrolyzed zearalenone. In at least one such embodiment, the Baeyer- Villiger oxidase enzyme is active to catalyze oxidation of decarboxylated hydrolyzed zearalenone to form oxidized decarboxylated hydrolyzed zearalenone as described herein.

[0012] In at least one embodiment, the Baeyer-Villiger oxidase enzyme comprises an amino acid sequence indicated as SEQ ID NO:5 or a catalytically active portion thereof, or a variant amino acid sequence having at least 70% identity to SEQ ID NO:5, or to the catalytically active portion thereof. In at least one such embodiment, the Baeyer-Villiger oxidase enzymehas an amino acid sequence indicated as SEQ ID NO:5. In at least one embodiment, the Baeyer-Villiger oxidase enzyme is encoded by a polynucleotide comprising a nucleotide sequence indicated as SEQ ID NO:6 or by a polynucleotide comprising a variant nucleotide sequence having at least 70% identity to SEQ ID NO:6. In at least one embodiment, the Baeyer-Villiger oxidase enzyme is isolated.

[0013] In at least one embodiment, the protein active to degrade the mycotoxin comprises one or more mycotoxin-degrading enzymes selected from: (i) a hydrolase enzyme comprising an amino acid sequence of SEQ ID NO:1, or a catalytically active portion thereof, or comprising a variant amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:1, or to the catalytically active portion of SEQ ID NO:1; or a hydrolase enzyme encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:2 or encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:2; (ii) a decarboxylase enzyme comprising an amino acid sequence of SEQ ID NO:3, or a catalytically active portion thereof, or comprising a variant amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:3, or to the catalytically active portion of SEQ ID NO:3; or a decarboxylase enzyme encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:4 or encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:4; and (iii) a Baeyer-Villiger oxidase enzyme comprising an amino acid sequence of SEQ ID NO:5, or a catalytically active portion thereof, or comprising a variant amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:5, or to the catalytically active portion of SEQ ID NO:5; or a Baeyer-Villiger oxidase enzyme encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:6 or encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:6.

[0014] In at least one embodiment, the protein active to degrade the mycotoxin comprises a fusion protein comprising a first polypeptide moiety fused to one or more additional polypeptide moieties. In at least one embodiment, the first polypeptide moiety comprises a hydrolase enzyme as described herein. In at least one embodiment, the one or more additional polypeptide moieties comprise a decarboxylase enzyme as described herein. In at least one embodiment, the one or more additional polypeptide moieties comprise a Baeyer- Villiger oxidase enzyme as described herein. In at least one embodiment, the fusion protein further includes one or more linker polypeptides. In at least one embodiment, the fusion protein is an isolated fusion protein.

[0015] In another aspect, the present application provides a cell expressing a protein active to degrade a mycotoxin as described herein. In at least one embodiment, the protein active to degrade the mycotoxin as described herein is native to the cell. In at least one embodiment, the cell is a recombinant host cell which has been genetically altered to express a heterologous protein active to degrade a mycotoxin as described herein. In at least one embodiment, the cell or the recombinant host cell is a microbial cell. In at least one embodiment, the microbial cell is a bacterial cell. In at least one embodiment, the bacterial cell is a cell of Bacillus subtilis, Lacticaseibacillus paracasei, Escherichia coli, Lactobacillus casei or Lactococcus lactis.

[0016] In at least one embodiment, the microbial cell or recombinant microbial host cell is a fungal cell. In at least one embodiment, the fungal cell is a yeast cell. In at least one embodiment, the yeast cell is a cell of Saccharomyces cerevisiae, Komagataella phaffii, Saccharomyces boulardii, Candida utilis or Kluyvermyces lactis. In at least one embodiment, the yeast cell is a cell of Saccharomyces cerevisiae or Komagataella phaffii.

[0017] A further aspect of the present application provides an autolysate or extract of the cell expressing the protein active to degrade the mycotoxin.

[0018] Another aspect of the present application provides a composition comprising a protein active to degrade a mycotoxin as described herein, a cell expressing the protein active to degrade the mycotoxin as described herein or an autolysate or extract of the cell expressing the protein active to degrade the mycotoxin as described herein. In at least one embodiment, the composition further comprises a carrier.

[0019] Another aspect of the present application provides a plant which is genetically altered to express a protein active to degrade a mycotoxin as described herein. In at least one embodiment, the plant is a crop plant at risk of being contaminated with a mycotoxin.

[0020] In another aspect, the present application is directed to a method of degrading a mycotoxin, the method comprising contacting the mycotoxin with a protein active to degrade the mycotoxin as described herein, with a cell expressing the protein active to degrade the mycotoxin as described herein, with an autolysate or extract of the cell expressing the protein active to degrade the mycotoxin as described herein, or with a composition as described herein.

[0021] A further aspect of the present application provides a method comprising contacting a material contaminated with a mycotoxin or at risk of being contaminated with the mycotoxin with a protein active to degrade the mycotoxin as described herein, with a cell expressing the protein active to degrade the mycotoxin as described herein, with an autolysate or extract of the cell expressing the protein active to degrade the mycotoxin as described herein, or with a composition as described herein. In at least one embodiment, the material is a food, a feed, a beverage, a food product, a feed product, a beverage product, a food ingredient, a feed ingredient or a beverage ingredient. In at least one embodiment, the material is a feedstock for a fermentation process. Brief Description of the Drawings

[0022] Further features of the present invention will become apparent from the following written description and the accompanying figures, in which:

[0023] Figure 1A is a graph showing the pH dependence of the activity of a hydrolase enzyme according to the present application;

[0024] Figure 1B is a graph showing the temperature dependence of the activity of the hydrolase enzyme of Figure 1A;

[0025] Figure 2A is an extracted ion ([M−H]−) chromatogram of zearalenone incubated in the presence of a fusion protein of a hydrolase enzyme and a decarboxylase enzyme according to the present application. The inset is an image of the result of a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the fusion protein enriched by Ni2+metal affinity chromatography;

[0026] Figure 2B is an extracted ion ([M−H]−) chromatogram of zearalenone incubated in the absence of the fusion protein of Figure 2A as a control;

[0027] Figure 3 is a MS / MS spectrum of a product formed by incubating decarboxylated hydrolyzed zearalenone with a Bayer-Villiger oxidase enzyme according to the present application;

[0028] Figure 4 is a graph showing the amount (in ppm) of zearalenone (ZEN), hydrolyzed zearalenone (HZEN) and decarboxylated hydrolyzed zearalenone (DHZEN) formed when the supernatant of a culture of an untransformed control strain of Saccharomyces cerevisiae (Control) or of a strain of Saccharomyces cerevisiae genetically modified to express ahydrolase enzyme according to the present application (Transformed) are incubated in the absence or presence of 5 ppm ZEN;

[0029] Figure 5 is a graph showing the amount (in ppm) of ZEN, HZEN and DHZEN when the supernatant of a culture of an untransformed control strain of Komagataella phaffii (Control) or of a strain of Komagataella phaffii genetically modified to express a hydrolase enzyme according to the present application (Transformed) are incubated in the absence or presence of 5 ppm ZEN;

[0030] Figure 6 is a graph showing the amount (in ppm) of ZEN and HZEN when supernatant (SUP) or whole cell broth (WCB) of the Transformed and Control strains of Figure 5 are incubated in the presence of 5 ppm ZEN; and

[0031] Figure 7 is a bar graph illustrating the relative activity of a hydrolase enzyme according to the present application (ZenH) compared to the activities of zearalenone hydrolases obtained from Aeromicrobium sp., Exophiala spinifera and Gliocladium roseum. Error bars represent standard deviation (n = 3) and unique letters above the bars represent statistically significant differences in relative activity as determined by Tukey’s test (p<0.05). Detailed Description

[0032] In one aspect, the present application is directed to a protein active to degrade a mycotoxin. In at least one embodiment, the protein is active to reduce the toxicity of the mycotoxin. In at least one embodiment, the protein active to degrade the mycotoxin comprises one or more mycotoxin-degrading enzymes selected from a hydrolase enzyme, a decarboxylase enzyme and a Baeyer-Villiger oxidase enzyme. In at least one embodiment, the protein active to degrade the mycotoxin comprises one or more mycotoxin-degrading enzymes from a species of Exophiala. In at least one embodiment, the species of Exophiala is Exophiala sp. strain LRDC-1. A deposit of Exophiala sp. strain LRDC-1 pursuant to the Budapest Treaty was received on July 31, 2024 and on September 5, 2024, by the International Depository Authority of Canada (IDAC), National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, Manitoba R3E 3R2, Canada (Accession number 310724-02). Exophiala is a genus of black yeast-like fungi known for their ability to remediate hydrocarbon contaminated sites, and which are commonly found in environmental samples, including soil, water, and decaying material.

[0033] In at least one embodiment, the mycotoxin is a resorcylic acid lactone mycotoxin. In at least one embodiment, the mycotoxin is zearalenone (Formula I), also referred to herein as “ZEN”.

[0034] As used herein with respect to the degradation of one or more mycotoxins, including but not limited to resorcylic acid lactone mycotoxins such as zearalenone, the terms “degrade”, “degradation” and grammatical forms thereof are intended to refer to chemical modification of the mycotoxins, including but not limited to chemical modification catalyzed by one or more enzymes, including but not limited to enzymes described herein, such that the concentration or amount of the mycotoxins present is decreased or eliminated. In at least one embodiment, the one or more mycotoxins can be chemically transformed to one or more products which cause fewer or less severe adverse effects in an organism, including but not limited to a plant, animal or human, than the adverse effects caused in that organism by the undegraded one or more mycotoxins. In such embodiments, the terms “detoxify”, “detoxification” and grammatical forms thereof may alternatively be used to refer to such chemical transformations or other processes by which the toxicity of a mycotoxin can be reduced, including but not limited to reactions catalyzed or mediated by a protein active to degrade or reduce the toxicity of a mycotoxin as described herein.

[0035] In at least one embodiment, the one or more mycotoxin-degrading enzymes comprise a hydrolase enzyme. In at least one embodiment, the hydrolase enzyme is active to catalyze hydrolysis of the mycotoxin to form a hydrolyzed mycotoxin. In at least one embodiment, the hydrolase enzyme is active to catalyze hydrolysis of a resorcylic acid lactone mycotoxin to form a hydrolyzed resorcylic acid lactone mycotoxin. In at least one embodiment, the hydrolase enzyme is active to catalyze the hydrolysis of zearalenone (ZEN, Formula I) to hydrolyzed zearalenone (HZEN Formula II)

[0036] In at least one embodiment, the hydrolase enzyme comprises the amino acid sequence indicated as SEQ ID NO:1 or a catalytically active portion thereof. As used herein, the term “catalytically active portion” of an amino acid sequence of a protein which catalyzes a chemical reaction is intended to mean any portion of the amino acid sequence whichretains the activity to catalyze the chemical reaction to at least some extent. The person skilled in the art would be able to determine the sequence of a catalytically active portion using techniques known in the art or described herein.

[0037] In at least one embodiment, the hydrolase enzyme has the amino acid sequence indicated as SEQ ID NO:1 (also referred to herein as “ZenH”). In at least one embodiment, the hydrolase enzyme comprises a variant amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:1 or to a catalytically active portion thereof.

[0038] In at least one embodiment, the hydrolase enzyme is encoded by a polynucleotide comprising a nucleotide sequence indicated as SEQ ID NO:2. In at least one embodiment, the hydrolase enzyme is encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:2.

[0039] As used herein, the term “percent identity” or “% identity” when used in reference to the sequence of a variant polypeptide or polynucleotide is intended to mean the percentage of the total number of amino acid or nucleotide residues, respectively, in the variant sequence which are identical to those at the corresponding position of a reference polypeptide or polynucleotide sequence. In at least one embodiment, when the length of the variant sequence and the length of the reference sequence are not identical, percent identity can be calculated based on the total number of residues in the variant sequence or based on the total number of residues in the reference sequence. Percent identity can be measured by various local or global sequence alignment algorithms well known in the art, including but not limited to the Smith- Waterman algorithm and the Needleman-Wunsch algorithm. Tools using these or other suitable algorithms include but are not limited to BLAST (Basic Local Alignment Search Tool) and other such tools well known in the art.

[0040] As used herein, the term “variant” when used in reference to a polynucleotide or a sequence thereof is intended to refer to a polynucleotide which differs in its nucleotide sequence from the sequence of a reference polynucleotide to which the variant is being compared by one or more nucleotide residues. The differences between the variant sequence and the sequence of the reference polynucleotide, also referred to herein as variations or mutations, can include substitution of one or more nucleotide residues with different nucleotide residues, insertion of additional nucleotide residues or deletion of nucleotide residues.

[0041] In at least one embodiment, the variant differs from the reference polynucleotide in that at least one codon of the reference polynucleotide has been replaced by a variant codon coding for the same amino acid as the replaced at least one codon. As will be understood in the art, when a polynucleotide is transcribed and / or translated to produce a protein in a cell, each group of three nucleotide residues following an origin of translation in the polynucleotide is a codon which determines the identity of the amino acid to be sequentially added to the translated protein. The degeneracy of the genetic code allows for some amino acids to be encoded by more than one codon. Thus, in certain embodiments, a variant can differ from a reference polynucleotide by substitution of one or more nucleotide residues with replacement nucleotide residues which do not alter the open reading frame(s) of the polynucleotide or the amino acid sequence of any polypeptide(s) or protein(s) encoded by the polynucleotide, but simply replace one codon encoding an amino acid with a different codon encoding the same amino acid. Thus, in certain embodiments, a variant sequence of a polynucleotide can be codon-optimized for expression in a particular recombinant host cell, such that the variant polynucleotide sequence includes codons which are preferentially used to encode amino acids by that host cell.

[0042] As used herein, the term “variant” when used in reference to a polypeptide or a sequence thereof is intended to refer to a polypeptide which differs in its amino acid sequence from the sequence of a reference polypeptide to which the variant is being compared by one or more amino acid residues. The differences between the sequence of the variant and the sequence of the reference polypeptide can include substitution of one or more amino acid residues with different amino acid residues, insertion of additional amino acid residues or deletion of amino acid residues. In certain embodiments, a variant can differ from a reference polypeptide by conservative substitution of one or more amino acid residues with replacement amino acid residues which may have similar properties, including but not limited to charge, size and hydrophilicity, to the amino acid residues which the new residues replace. In certain embodiments, variants may completely or partially retain one or more biological functions of the reference polypeptide. In certain embodiments, variants may not retain one or more biological functions of the reference polypeptide.

[0043] In view of the teaching of the present application, the person skilled in the art would be readily able to produce a protein as described herein that comprises or consists of a polypeptide having a specified sequence identity to a specified protein as described herein, or to a portion or variant thereof as described herein, by techniques well known in the art. For example, using techniques known in the art or described herein, a nucleic acid sequence encoding the polypeptide may be synthesized de novo or may be modified from another nucleic acid sequence through, for example, nucleotide exchanges, deletions and / or additions. The polypeptide encoded by such a synthesized or modified nucleic acidsequence can be produced by methods known in the art or described herein, including but not limited to heterologous expression of the nucleic acid sequence in a host cell and purification of the expressed polypeptide. Furthermore, the activity of a polypeptide produced by such methods may be readily determined using methods known in the art or described herein.

[0044] In at least one embodiment, the one or more mycotoxin-degrading enzymes comprise a decarboxylase enzyme. In at least one embodiment, the decarboxylase enzyme is active to catalyze decarboxylation of a hydrolyzed mycotoxin to form a decarboxylated hydrolyzed mycotoxin. In at least one embodiment, the decarboxylase enzyme is active to catalyze decarboxylation of a hydrolyzed resorcylic acid lactone mycotoxin to form a decarboxylated hydrolyzed resorcylic acid lactone mycotoxin. In at least one embodiment, the decarboxylase enzyme is active to catalyze the decarboxylation of hydrolyzed zearalenone (HZEN, Formula II) to decarboxylated hydrolyzed zearalenone (DHZEN, Formula III).

[0045] In at least one embodiment, the decarboxylase enzyme comprises the amino acid sequence indicated as SEQ ID NO:3 or a catalytically active portion thereof. The person skilled in the art would be able to determine the sequence of a catalytically active portion using techniques known in the art or described herein.

[0046] In at least one embodiment, the decarboxylase enzyme has the amino acid sequence indicated as SEQ ID NO:3 (also referred to herein as “ZenC”). In at least one embodiment, the decarboxylase enzyme comprises an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:3 or to a catalytically active portion thereof.

[0047] In at least one embodiment, the decarboxylase enzyme is encoded by a polynucleotide comprising the nucleotide sequence indicated as SEQ ID NO:4. In at least one embodiment, the decarboxylase enzyme is encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:4.

[0048] In at least one embodiment, the one or more mycotoxin-degrading enzymes comprise a Baeyer-Villiger oxidase enzyme. In at least one embodiment, the Baeyer-Villiger oxidase enzyme is active to catalyze oxidation of a decarboxylated hydrolyzed mycotoxin to form an oxidized decarboxylated hydrolyzed mycotoxin. In at least one embodiment, the Baeyer-Villiger oxidase enzyme is active to catalyze oxidation of a decarboxylated hydrolyzed resorcylic acid lactone mycotoxin to form an oxidized decarboxylated hydrolyzed resorcylic acid lactone mycotoxin. In at least one embodiment, the decarboxylase enzyme is active to catalyze the oxidation of decarboxylated hydrolyzed zearalenone (DHZEN, Formula III) to oxidized decarboxylated hydrolyzed zearalenone (oxyDHZEN, Formula IV).

[0049] In at least one embodiment, the Baeyer-Villiger oxidase enzyme comprises the amino acid sequence indicated as SEQ ID NO:5 or a catalytically active portion thereof. The person skilled in the art would be able to determine the sequence of a catalytically active portion using techniques known in the art or described herein.

[0050] In at least one embodiment, the Baeyer-Villiger oxidase enzyme has the amino acid sequence indicated as SEQ ID NO:5 (also referred to herein as “ZenB”). In at least one embodiment, the Baeyer-Villiger oxidase enzyme comprises a variant amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:5 or to a catalytically active portion thereof.

[0051] In at least one embodiment, the Baeyer-Villiger oxidase enzyme is encoded by a polynucleotide comprising the nucleotide sequence indicated as SEQ ID NO:6. In at least one embodiment, the Baeyer-Villiger oxidase enzyme is encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:6.

[0052] In at least one embodiment, the protein active to degrade the mycotoxin comprises one or more mycotoxin-degrading enzymes selected from: (i) a hydrolase enzyme comprising an amino acid sequence of SEQ ID NO:1, or a catalytically active portion thereof, or comprising a variant amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:1, or to the catalytically active portion of SEQ ID NO:1; or a hydrolase enzyme encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:2 or encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:2; (ii) a decarboxylase enzyme comprising an amino acid sequence of SEQ ID NO:3, or a catalytically active portion thereof, or comprising a variant amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:3, or to the catalytically active portion of SEQ ID NO:3; or a decarboxylase enzyme encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:4 or encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:4; and (iii) a Baeyer-Villiger oxidase enzyme comprising an amino acid sequence of SEQ ID NO:5, or a catalytically active portion thereof, or comprising a variant amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:5, or to the catalytically active portion of SEQ ID NO:5; or a Baeyer-Villiger oxidase enzyme encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:6 or encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:6.

[0053] In at least one embodiment, the protein active to degrade the mycotoxin can comprise a fusion protein comprising a first polypeptide moiety comprising a mycotoxin- degrading enzyme as described herein fused to one or more additional polypeptide moieties. As used herein, the term “fusion protein” is intended to mean a protein in which two or more polypeptide or protein moieties are fused to each other. As used herein, the term “fused”, when referring to polypeptide or protein moieties in a fusion protein as described herein, is intended to mean that the polypeptide or protein moieties are covalently bonded to each other by one or more peptide bonds, either directly or with one or more intervening linker polypeptides. In at least one embodiment, the polypeptide or protein moieties in the fusion protein each have individual functions when present as separate polypeptides or proteins,and the fusion protein retains, at least in part, the respective structure and / or functions of the individual polypeptides or proteins.

[0054] In at least one embodiment, the first polypeptide moiety comprises a hydrolase enzyme as described herein. In at least one such embodiment, the one or more additional polypeptide moieties can comprise one or more of a decarboxylase enzyme as described herein and a Baeyer-Villiger oxidase enzyme as described herein. In at least one embodiment, the first polypeptide moiety comprises a decarboxylase enzyme as described herein. In at least one embodiment, the first polypeptide moiety comprises a Baeyer-Villiger oxidase enzyme as described herein.

[0055] In at least one embodiment, the one or more additional polypeptide moieties can comprise one or more polypeptide moieties which facilitate expression of the fusion protein in a recombinant host cell or which facilitate the isolation or purification of the fusion protein after expression by the recombinant host cell. In at least one embodiment, the additional polypeptide moiety is a maltose-binding protein (MBP). In at least one embodiment, the additional polypeptide moiety comprises a polyhistidine sequence, also known as a “His- tag”, as will be understood by a person skilled in the art.

[0056] In at least one embodiment, the fusion protein further includes one or more linker polypeptides. As used herein, the term “linker polypeptide” is intended to refer to a polypeptide sequence which can be used to join two polypeptide or protein moieties in a fusion protein. In at least one embodiment, the linker polypeptide is selected to permit the fusion protein to retain, at least in part, the respective structure and / or functions of the individual polypeptides or proteins. Linker polypeptides are well known in the art and can be readily identified and used by the skilled person in view of the teaching of the present specification. In at least one embodiment, the linker polypeptide comprises one or more Gly- Gly-Ser (GGS) tripeptides. In at least one embodiment, the linker polypeptide comprises the polypeptide GGSGGSGGSGGS (SEQ ID NO:7).

[0057] In at least one embodiment, the fusion protein comprises a hydrolase enzyme as described herein fused to a decarboxylase enzyme as described herein. In at least one embodiment, the fusion protein has the sequence identified as SEQ ID NO:8.

[0058] In at least one embodiment, the protein active to degrade the mycotoxin is a recombinant protein. As used herein, the term “recombinant protein” or “recombinant polypeptide” is intended to mean a heterologous protein or polypeptide which is not native to a cell, but which the cell has been genetically altered to express. A cell expressing such a recombinant protein or polypeptide may be referred to herein as a “recombinant host cell”.

[0059] In at least one embodiment, the protein active to degrade the mycotoxin is in an isolated or purified form. In at least one embodiment, the isolated or purified protein active todegrade the mycotoxin is a recombinant protein active to degrade the mycotoxin. In at least one embodiment, the isolated or purified protein active to degrade the mycotoxin is at least partially purified. Methods of isolating and purifying the protein active to degrade the mycotoxin, either from an organism or microorganism expressing the native protein active to degrade the mycotoxin or from an organism or microorganism genetically engineered to express a recombinant form of the protein active to degrade the mycotoxin are well known in the art and would be apparent to the skilled person in view of the teaching of the present application. In such embodiments, an at least partially purified preparation containing the isolated or purified protein active to degrade the mycotoxin may also contain components of the host organism or microorganism expressing the protein active to degrade the mycotoxin, components of the culture media in which the host organism or microorganism is grown or components of any other material related to the expression and / or purification process. In at least one embodiment, the isolated or purified protein active to degrade the mycotoxin is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% purified.

[0060] A further aspect of the application provides a cell expressing a protein active to degrade a mycotoxin as described herein. In at least one embodiment, the protein active to degrade the mycotoxin as described herein is native to the cell. In at least one embodiment, the protein active to degrade the mycotoxin as described herein is heterologous to the cell. In such an embodiment, the cell is a recombinant host cell which has been genetically altered to express the protein active to degrade the mycotoxin as described herein.

[0061] In at least one embodiment, the cell, including but not limited to the recombinant host cell, is a microbial cell, including but not limited to a bacterial cell or a fungal cell. In at least one embodiment, the fungal cell is a yeast cell. In at least one embodiment, the bacterial cell is an Escherichia coli cell, a cell of a probiotic bacterial species, or a cell of a lactic acid bacterial species, including but not limited to a cell of a species of Lactobacillus. In at least one embodiment, the bacterial cell is a cell of Bacillus subtilis, Lacticaseibacillus paracasei, Escherichia coli, Lactobacillus casei or Lactococcus lactis. In at least one embodiment, the yeast cell is a cell of a species of Saccharomyces, a cell of a species of Komagataella, a cell of a species of Candida or a cell of a species of Kluyvermyces. In at least one embodiment, the yeast cell is a cell of Saccharomyces cerevisiae, Komagataella phaffii (Pichia pastoris), Saccharomyces boulardii, Candida utilis or Kluyvermyces lactis. In at least one embodiment, the cell is a recombinant plant host cell. A person of skill in the art would be well aware of methods for genetically altering a cell to express a recombinant protein active to degrade the mycotoxin as described herein, in view of the teaching of the present description.

[0062] A further aspect of the present application provides an autolysate or extract of the cell expressing the protein active to degrade the mycotoxin as described herein. Anotheraspect of the present application provides a composition comprising a protein active to degrade a mycotoxin as described herein, a cell, including but not limited to a recombinant host cell, which expresses the protein active to degrade the mycotoxin as described herein, or an autolysate or extract of the cell expressing the protein active to degrade the mycotoxin as described herein. As used herein, the term “autolysate” when used in relation to a cell expressing the protein active to degrade a mycotoxin as described herein, is intended to mean a product of autolysis of the cell as well known in the art and as described herein. As used herein, the term “extract” when used in relation to a cell expressing the protein active to degrade a mycotoxin as described herein, is intended to mean a material comprising the expressed protein active to degrade a mycotoxin in which the expressed protein active to degrade a mycotoxin has been at least partially separated from other components of the cell, or from other components of the medium containing the cell.

[0063] In at least one embodiment, the expressed protein active to degrade a mycotoxin may be located inside the cell after expression. In such embodiments, preparation of an extract comprising the expressed protein active to degrade a mycotoxin may require lysis of the cell. Such lysis methods are well known in the art and can include, but are not limited to, autolysis and other methods which disrupt or cause breakage of cell walls and / or cell or organelle membranes. In at least one embodiment, the cell or a lysis product thereof can be homogenized using techniques including but not limited to bead-milling, bead-beating or high-pressure homogenization.

[0064] In at least one embodiment, the expressed protein may be bound to one or more components of the cell or of a lysis product thereof. In such embodiments, preparation of an extract comprising the expressed protein active to degrade a mycotoxin may require further treatment of the cell or of a lysis product thereof. Such treatment would be known to the skilled person and can include, but is not limited to, treatment with detergents or with enzymes or other reagents which disrupt or disintegrate components of the cell to which the expressed enzyme may be bound. In at least one alternative embodiment, the extract can contain components of the cell or of a lysis product thereof which are bound to the expressed protein but which have been at least partially separated from other components of the cell or lysis product thereof or from other components of the medium containing the cell or lysis product thereof. In at least one such embodiment, the extract can comprise an inactivated fraction obtained by inactivating the cell.

[0065] In at least one embodiment, the expressed protein can be secreted by the cell as a soluble protein and can be isolated or recovered from the medium to which the cell secretes the expressed protein. In such embodiments, preparation of an extract may require using methods well known in the art for separating soluble proteins from other components of a mixture containing the soluble protein.

[0066] In at least one embodiment, a composition comprising a microbial cell, including but not limited to a recombinant microbial host cell which expresses the protein active to degrade the mycotoxin as described herein, or an autolysate or extract of the microbial cell, can be prepared by a process including propagating the microbial cell expressing the protein active to degrade a mycotoxin as described herein. In at least one embodiment, propagation of the microbial cell is carried out in a culture medium under conditions facilitating expression and accumulation of the protein active to degrade a mycotoxin. Such conditions include but are not limited to temperature, pH, nutrient content and degree of agitation, and can be readily identified and selected by one skilled in the art for a particular microbial cell in view of the teaching of the present specification. In at least one embodiment when the microbial cell is a yeast cell, the culture medium comprises molasses. In at least one embodiment when the microbial cell is a yeast cell or a bacterial cell, the culture medium comprises a yeast extract.

[0067] In at least one embodiment, the process further comprises separating the protein active to degrade a mycotoxin from at least one component of the propagated microbial cell to obtain an extract enriched in the protein active to degrade a mycotoxin. In embodiments where the protein active to degrade a mycotoxin is secreted in a soluble form by the microbial cell, separating the protein active to degrade a mycotoxin from at least one component of the propagated microbial cell can include, but is not limited to, filtering or centrifuging the mixture containing the propagated microbial cell to separate insoluble cell components, including but not limited to cell wall and membrane components, from the medium in which the protein active to degrade a mycotoxin is soluble.

[0068] In at least one embodiment, the process further comprises lysing the propagated microbial cell to obtain a lysed fraction. Such embodiments of the process can be useful when the protein active to degrade a mycotoxin is not secreted in a soluble form by the microbial cell but remains inside or bound to the cell after expression. In at least one such embodiment, an extract comprising the protein active to degrade a mycotoxin can be separated from at least one undesired component of the lysed propagated microbial cell by techniques including but not limited to filtration or centrifugation. The at least one undesired component may be more soluble or less soluble in the mixture being separated than the extract comprising the protein active to degrade a mycotoxin.

[0069] In at least one embodiment, the process further comprises drying, at least in part, the propagated microbial cell, the extract, the inactivated fraction or the lysed fraction to obtain a dried extract, by a method including not limited to roller-drying, spray-drying, freeze-drying, lyophilization, electrospray-drying or fluid-bed drying. In at least one embodiment, the process further comprises substantially purifying the protein active to degrade a mycotoxin from the extract, the inactivated fraction, the lysed fraction or the dried fraction to obtain asubstantially purified extract. In at least one embodiment, the process comprises formulating the propagated microbial cell, the extract, the inactivated fraction, the lysed fraction, the dried extract, or the substantially purified extract into a composition as described herein.

[0070] In at least one embodiment, the composition can include the propagated microbial cells, including but not limited to yeast cells and bacterial cells, in an active or semi-active form, such that the cells remain viable and capable of expressing the protein active to degrade a mycotoxin. In such embodiments, the process for preparing the composition can comprise a concentrating step, in which a portion of the propagation medium can be removed from the propagated cells by a method including but not limited to dialysis, filtration, or centrifugation followed by resuspension of the concentrated cells in the propagation medium or in fresh medium or water.

[0071] In at least one embodiment, the autolysate is prepared by autolysis of the propagated microbial cells. In at least one embodiment, when the microbial cell is a yeast cell, including but not limited to a recombinant yeast host cell, the autolysis can include subjecting the propagated yeast cells to a combined heat and pH treatment for a specific amount of time, including but not limited to for 24 hours. As the person of skill in the art will be aware, care should be taken when heating microbial cells or lysates thereof, including but not limited to autolysates thereof, to avoid damage to the cells and to avoid loss of activity of the protein active to degrade a mycotoxin.

[0072] In at least one embodiment, the propagated yeast cells can be subjected to a temperature of between about 40°C and about 70°C or between about 50°C and about 60°C. In at least one embodiment, the propagated yeast cells can be subjected to a temperature of at least about 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C or 70°C. Alternatively or in combination, the propagated yeast cells can be submitted to a temperature of no more than about 70°C, 69°C, 68°C, 67°C, 66°C, 65°C, 64°C, 63°C, 62°C, 61°C, 60°C, 59°C, 58°C, 57°C, 56°C, 55°C, 54°C, 53°C, 52°C, 51°C, 50°C, 49°C, 48°C, 47°C, 46°C, 45°C, 44°C, 43°C, 42°C, 41°C or 40°C.

[0073] In at least one embodiment, the propagated yeast cells can be subjected to a pH between about 4.0 and 8.5 or between about 5.0 and 7.5. In at least one embodiment, the propagated yeast cells can be submitted to a pH of at least about, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4 or 8.5. Alternatively or in combination, the propagated yeast cells can be submitted to a pH of no more than 8.5, 8.4, 8.3, 8.2, 8.1, 8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8,6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6 or 4.5.

[0074] In at least one embodiment, the autolysis can be performed in the presence of additional exogenous enzymes. In at least one embodiment, the autolysate can be dried with or without further purification or separation of the protein active to degrade a mycotoxin from other components of the autolysate. In at least one embodiment, the autolysate is formulated into the composition as described herein.

[0075] In at least one embodiment, the composition further comprises a carrier. Suitable carriers are well known in the art and can be identified and selected by the skilled person to be compatible with the application for which the composition is to be used. In at least one embodiment, the carrier is tolerable by, and does not typically produce harmful reactions in, an organism or microorganism with which the composition may come into contact.

[0076] In at least one embodiment, the carrier is an agriculturally acceptable carrier. As used herein, the term “agriculturally acceptable” is intended to refer to carriers and compositions containing such carriers that are tolerable and do not typically produce untoward reactions to a plant or crop being treated with such carriers and compositions, including but not limited to a plant or crop intended for use as food or feed for humans or animals or as feedstock for a fermentation process or other process by which the plant or crop is transformed to a product with which a human or animal may come into contact or which may be used as food or feed for humans or animals, or to a worker applying such carriers and compositions to a plant or crop under normal agricultural conditions, or to a human or animal ingesting or contacting a product formed from a plant or crop thus treated. In at least one embodiment, as used herein, the term “agriculturally acceptable” means approved by a regulatory agency of the federal or a state government for use in agricultural applications. Such agriculturally acceptable carriers are well known in the art.

[0077] In at least one embodiment, the carrier is a liquid carrier. In at least one embodiment, the liquid carrier comprises water. In at least one embodiment, the liquid carrier is an aqueous solution comprising components as described herein, including but not limited to components which are agriculturally acceptable or which are tolerable by an organism or microorganism with which the composition may come into contact. In at least one embodiment, the liquid carrier can be a medium in which a cell, including but not limited to a recombinant microbial host cell as described herein, can grow or remain viable or can express a protein active to degrade a mycotoxin as described herein.

[0078] In at least one embodiment, the carrier is a solid carrier. In at least one embodiment, the solid carrier is a powder. In at least one embodiment, the solid formulation is a lyophilized powder formulation. In at least one embodiment, the solid formulation is a spray-dried powder formulation. In at least one embodiment, the solid formulation is a granular formulation. In at least one embodiment, the solid carrier can include granules comprising organic and / or inorganic material which is agriculturally acceptable or which is tolerable by an organism or microorganism with which the composition may come into contact.

[0079] In at least one embodiment, the composition or formulation can contain one or more binders or other additives. As used herein, the term “binder” is intended to mean a component capable of physically associating (e.g., binding) to a mycotoxin to reduce its bioavailability. Suitable binders include but are not limited to mineral binders and organic binders. An exemplary mineral binder is a clay, such as a bentonite clay. An exemplary organic binder is a microbial component, such as, for example, a microbial extract, including but not limited to a bacterial extract, a yeast extract and / or a fungal extract. In at least one embodiment, the microbial component is a microbial cell and / or a component from a microbial cell. The microbial cell can be a bacterial cell, a yeast cell or a fungal cell. The microbial cell can be a recombinant microbial host cell having expressed and optionally accumulated the protein active to degrade a mycotoxin as described herein. The person of skill in the art would be aware of other additives suitable for inclusion in such a composition or formulation, in view of the teaching of the present specification.

[0080] Another aspect of the present application provides a plant genetically altered to express a protein active to degrade a mycotoxin as described herein. In at least one embodiment, the plant is a crop plant at risk of being contaminated with a mycotoxin as described herein. In at least one embodiment, the genetically altered plant can include but is not limited to corn, wheat, maize, barley, rye and other crops known in the art. It is contemplated that such crops will experience reduced contamination with a mycotoxin even if exposed to Fusarium species producing the mycotoxin during growth.

[0081] In another aspect, the present application provides a method of degrading a mycotoxin or a method of reducing the toxicity of a mycotoxin, the method comprising contacting the mycotoxin with a protein active to degrade the mycotoxin as described herein, with a cell expressing the protein active to degrade the mycotoxin as described herein, with an autolysate or extract of the cell expressing the protein active to degrade the mycotoxin as described herein, or with a composition as described herein. In at least one embodiment, contacting the mycotoxin with a protein active to degrade the mycotoxin as described herein, with a cell expressing the protein active to degrade the mycotoxin as described herein, with an autolysate or extract of the cell expressing the protein active to degrade the mycotoxin as described herein, or with a composition as described herein comprises contacting a material contaminated with the mycotoxin or at risk of being contaminated with the mycotoxin with the protein active to degrade the mycotoxin as described herein, with the cell expressing the protein active to degrade the mycotoxin as described herein, with the autolysate or extract ofthe cell expressing the protein active to degrade the mycotoxin as described herein, or with the composition as described herein. In at least one embodiment, the mycotoxin is a resorcylic acid lactone mycotoxin. In at least one embodiment, the mycotoxin is zearalenone.

[0082] A further aspect of the present application provides a method comprising contacting a material contaminated with a mycotoxin or at risk of being contaminated with the mycotoxin with a protein active to degrade the mycotoxin as described herein, with a cell expressing the protein active to degrade the mycotoxin as described herein, with an autolysate or extract of the cell expressing the protein active to degrade the mycotoxin as described herein, or with a composition as described herein. In at least one embodiment, the mycotoxin is a resorcylic acid lactone mycotoxin. In at least one embodiment, the mycotoxin is zearalenone.

[0083] As used herein, the expression “contaminated with the mycotoxin” is intended to refer to a material containing a level of the mycotoxin which is considered harmful to humans or animals ingesting or otherwise exposed to the contaminated material, or which would cause the contaminated material to be rejected for further use, including but not limited to further commercial use. As used herein, the expression “at risk of being contaminated with the mycotoxin” is intended to refer to a material which is not necessarily known to be contaminated with the mycotoxin but which is suspected of being contaminated with the mycotoxin or is in a context or situation where contamination with the mycotoxin can be expected to occur. In at least one embodiment where the mycotoxin is zearalenone, materials containing at least 0.25 µg / kg of zearalenone which are contemplated for use in animal feed can be considered to be contaminated with zearalenone. In at least one such embodiment, materials containing at least 0.5 µg / kg of zearalenone which are contemplated for use in animal feed can be considered to be contaminated with zearalenone. The person of skill in the art would be aware of other standards set by advisory or regulatory authorities setting levels of a mycotoxin at which material containing the set levels would be considered contaminated.

[0084] In at least one embodiment, the material contaminated with or at risk of being contaminated with the mycotoxin is a food, a feed, a beverage, a food product, a feed product, a beverage product, a food ingredient, a feed ingredient or a beverage ingredient. In at least one embodiment, the feed, feed product or feed ingredient includes but is not limited to silage, hay, straw, grains, grain by-products, legumes, cottonseed meal, vegetables, milk and / or milk by-products. In another embodiment, the feed, feed product or feed ingredient is or comprises grain by-products. In a further embodiment, the grain by- products are distillers’ products, including but not limited to distillers’ dried grains, distillers’ dried grains with solubles, distillers’ wet grains, distillers’ wet grains with solubles, dried solubles or syrup and condensed distillers’ solubles or concentrated syrup as described, forexample, in published patent application US 2022 / 0007683. In at least one embodiment, the food, food product or food ingredient includes but is not limited to cereals or grains and grain products such as, for example, wheat, barley and corn and products such as flour derived from such cereals or grains.

[0085] In at least one embodiment, contacting a food, a feed, a beverage, a food product, a feed product, a beverage product, a food ingredient, a feed ingredient or a beverage ingredient contaminated with or at risk of being contaminated with the mycotoxin with the protein active to degrade a mycotoxin as described herein, with a cell expressing the protein active to degrade the mycotoxin as described herein, with an autolysate or extract of the cell expressing the protein active to degrade the mycotoxin as described herein, or with a composition as described herein includes providing a composition as described herein as an additive for combination with at least one food ingredient, at least one feed ingredient or at least one beverage ingredient. In at least one embodiment, the composition can be included directly in a food product, a feed product or a beverage product. In at least one embodiment, the composition is provided in solid form. In at least one such embodiment, the solid form is a free-flowing powder. In at least one embodiment, the composition can be provided in liquid form. In at least one embodiment, the liquid form is a spray-dryable liquid form. In at least one such embodiment, the composition contains maltodextrin. In at least one embodiment, the at least one food ingredient or feed ingredient including the composition can be pelleted. In at least one embodiment, the resulting food or feed pellet can be coated with a suitable coating and / or subjected to heat treatment as would be understood by those skilled in the art.

[0086] In at least one embodiment, the material contaminated with or at risk of being contaminated with the mycotoxin is a feedstock for a fermentation process. Fermentation processes and feedstocks suitable for use with the present method are described, for example, in published patent application US 2022 / 0007683. In at least one such embodiment, the method further comprises fermenting the feedstock. In at least one such embodiment, the fermentation process comprises treating the feedstock with at least one cell of a yeast strain which is active to ferment the feedstock. In at least one embodiment, the fermentation process further comprises determining whether a feedstock is contaminated with a mycotoxin and, upon determining that the feedstock is contaminated, contacting the feedstock with the protein active to degrade the mycotoxin, with a recombinant microbial host cell expressing the protein active to degrade the mycotoxin as described herein, with an autolysate or extract of the cell expressing the protein active to degrade the mycotoxin as described herein, or with a composition as described herein.

[0087] In at least one embodiment, the at least one cell of the yeast strain which is active to ferment the feedstock is also the recombinant microbial host cell expressing the proteinactive to degrade a mycotoxin. In at least one embodiment, the at least one cell of the yeast strain is at least one cell of a species of Saccharomyces, at least one cell of a species of Komagataella, at least one cell of a species of Candida or at least one cell of a species of Kluyvermyces. In at least one embodiment, the at least one cell of the yeast strain is at least one cell of Saccharomyces cerevisiae, Komagataella phaffii (Pichia pastoris), Saccharomyces boulardii, Candida utilis or Kluyvermyces lactis. In at least one embodiment, the yeast strain is a strain of Saccharomyces cerevisiae. In at least one embodiment, the yeast strain is a strain of Komagataella phaffii. In at least one such embodiment, treatment with the at least one cell of the yeast strain occurs during fermentation of the contaminated feedstock.

[0088] In at least one embodiment, the recombinant microbial host cell expressing the protein active to degrade a mycotoxin is a bacterial cell expressing the protein active to degrade a mycotoxin. In at least one such embodiment, the bacterial cell is a cell of a probiotic bacterial species, or a cell of a lactic acid bacterial species, including but not limited to a cell of a species of Lactobacillus. In at least one embodiment, the bacterial cell is a cell of Bacillus subtilis, Lacticaseibacillus paracasei, Escherichia coli, Lactobacillus casei or Lactococcus lactis.

[0089] In at least one embodiment, the feedstock is a cereal or grain crop, including but not limited to corn, wheat, sorghum, and cereals or a residue thereof, including but not limited to bagasse, corn stover and straw. In at least one embodiment, the feedstock is maize. In at least one such embodiment, the fermentation process produces one or more of methanol, ethanol, 1-propanol (n-propanol), 2-propanol (iso-propanol), 1-butanol, 1,2-propanediol and acetone. In at least one such embodiment, the fermentation process further produces one or more distillers’ products, including but not limited to distillers’ dried grains, distillers’ dried grains with solubles, distillers’ wet grains, distillers’ wet grains with solubles, dried solubles or syrup and condensed distillers’ solubles or concentrated syrup. In at least one such embodiment, the one or more distillers’ products have a reduced level of the mycotoxin compared to the level of the mycotoxin in distillers’ products obtained from a fermentation process in which the contaminated feedstock is not contacted with the protein active to degrade a mycotoxin, with the cell expressing the protein active to degrade the mycotoxin as described herein, with the autolysate or extract of the cell expressing the protein active to degrade the mycotoxin as described herein, or with the composition as described herein. In at least one such embodiment, the one or more distillers’ products obtained from the present fermentation process are suitable for use as animal feed, including but not limited to swine feed or cattle feed.

[0090] In at least one embodiment, contacting the mycotoxin with a protein active to degrade a mycotoxin as described herein, with a cell expressing the protein active todegrade the mycotoxin as described herein, with an autolysate or extract of the cell expressing the protein active to degrade the mycotoxin as described herein, or with a composition as described herein comprises exposing a crop contaminated with or at risk of being contaminated with the mycotoxin to the protein active to degrade the mycotoxin as described herein, to the cell expressing the protein active to degrade the mycotoxin as described herein, to the autolysate or extract of the cell expressing the protein active to degrade the mycotoxin as described herein, or to the composition as described herein. In at least one embodiment, the composition is an agriculturally acceptable composition. In at least one embodiment, the agriculturally acceptable composition is suitable for application to the crop contaminated with or at risk of being contaminated with the mycotoxin, or to the surrounding soil or substrate, by methods well known in the art, including but not limited to spraying. In at least one embodiment, the crop contaminated with or at risk of being contaminated with the mycotoxin is being grown in a field or in a greenhouse.

[0091] Aspects of the present application are presented in the following numbered clauses. 1. A protein active to degrade a mycotoxin, the protein comprising one or more mycotoxin-degrading enzymes selected from a hydrolase enzyme, a decarboxylase enzyme and a Baeyer-Villiger oxidase enzyme. 2. The protein of clause 1 wherein the hydrolase enzyme: comprises an amino acid sequence of SEQ ID NO:1, or a catalytically active portion thereof, or comprises a variant amino acid sequence having at least 70% identity to SEQ ID NO:1, or to the catalytically active portion of SEQ ID NO:1; or is encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:2 or is encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70% identity to SEQ ID NO:2. 3. The protein of clause 1 or 2 wherein the decarboxylase enzyme: comprises an amino acid sequence of SEQ ID NO:3, or a catalytically active portion thereof, or comprises a variant amino acid sequence having at least 70% identity to SEQ ID NO:3, or to the catalytically active portion of SEQ ID NO:3; or is encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:4 or is encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70% identity to SEQ ID NO:4.4. The protein of any one of clauses 1 to 3 wherein the Baeyer-Villiger oxidase enzyme: comprises an amino acid sequence of SEQ ID NO:5, or a catalytically active portion thereof, or comprises a variant amino acid sequence having at least 70% identity to SEQ ID NO:5, or to the catalytically active portion of SEQ ID NO:5; or is encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:6 or is encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70% identity to SEQ ID NO:6. 5. The protein of clause 1 wherein the one or more mycotoxin-degrading enzymes are selected from: (i) a hydrolase enzyme comprising an amino acid sequence of SEQ ID NO:1, or a catalytically active portion thereof, or comprising a variant amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:1, or to the catalytically active portion of SEQ ID NO:1; or a hydrolase enzyme encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:2 or encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:2; (ii) a decarboxylase enzyme comprising an amino acid sequence of SEQ ID NO:3, or a catalytically active portion thereof, or comprising a variant amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:3, or to the catalytically active portion of SEQ ID NO:3; or a decarboxylase enzyme encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:4 or encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:4; and (iii) a Baeyer-Villiger oxidase enzyme comprising an amino acid sequence of SEQ ID NO:5, or a catalytically active portion thereof, or comprising a variant amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:5, or to the catalytically active portion of SEQ ID NO:5; or a Baeyer-Villiger oxidase enzyme encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:6 or encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:6. 6. The protein of clause 1 wherein the one or more mycotoxin-degrading enzymes are selected from: (i) a hydrolase enzyme comprising an amino acid sequence of SEQ ID NO:1, or a catalytically active portion thereof, or comprising a variant amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:1, or to the catalytically active portion of SEQ ID NO:1; or a hydrolase enzyme encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:2 or encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:2; and (ii) a decarboxylase enzyme comprising an amino acid sequence of SEQ ID NO:3, or a catalytically active portion thereof, or comprising a variant amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:3, or to the catalytically active portion of SEQ ID NO:3; or a decarboxylase enzyme encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:4 or encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:4. 7. The protein of any one of clauses 1 to 6, wherein the hydrolase enzyme is active to catalyze hydrolysis of the mycotoxin to form a hydrolyzed mycotoxin. 8. The protein of clause 7, wherein the decarboxylase enzyme is active to catalyze the decarboxylation of the hydrolyzed mycotoxin to form a decarboxylated hydrolyzed mycotoxin.9. The protein of clause 8 wherein the Baeyer-Villiger oxidase enzyme is active to catalyze the oxidation of the decarboxylated hydrolyzed mycotoxin to form an oxidized decarboxylated hydrolyzed mycotoxin. 10. The protein of any one of clauses 1 to 9, wherein the mycotoxin is a resorcylic acid lactone mycotoxin. 11. The protein of any one of clauses 1 to 10 wherein the mycotoxin is zearalenone. 12. The protein of clause 11 wherein the hydrolase enzyme is active to catalyze the hydrolysis of zearalenone to form a compound of Formula II:13. The protein of clause 12 wherein the decarboxylase enzyme is active to catalyze the decarboxylation of the compound of Formula II to form a compound of Formula III:14. The protein of clause 13 wherein the Baeyer-Villiger oxidase enzyme is active to catalyze the oxidation of the compound of Formula III to form a compound of Formula IV:15. The protein of any one of clauses 1 to 14 wherein the protein comprises the hydrolase enzyme. 16. The protein of any one of clauses 1 to 15 wherein the protein comprises the decarboxylase enzyme. 17. The protein of any one of clauses 1 to 16 wherein the protein comprises the Baeyer- Villiger oxidase enzyme.18. The protein of any one of clauses 1 to 17, wherein the protein is a fusion protein. 19. A cell, wherein the cell expresses a protein of any one of clauses 1 to 18. 20. The cell of clause 19, wherein the cell is a recombinant host cell genetically altered to express the protein of any one of clauses 1 to 18. 21. The cell of clause 20, wherein the recombinant host cell is a recombinant microbial host cell. 22. The cell of clause 21, wherein the recombinant microbial host cell is a recombinant bacterial host cell or a recombinant fungal host cell. 23. The cell of clause 22 wherein the recombinant microbial host cell is a recombinant Escherichia coli host cell or a recombinant yeast cell. 24. An autolysate or extract of the cell of any one of clauses 19 to 23. 25. A composition comprising a protein of any one of clauses 1 to 18, a cell of any one of clauses 19 to 23, or an autolysate or extract of clause 24. 26. A plant genetically altered to express a protein of any one of clauses 1 to 18. 27. A method of degrading a mycotoxin, the method comprising contacting the mycotoxin with a protein of any one of clauses 1 to 18, with a cell of any one of clauses 19 to 23, with an autolysate or extract of clause 24, or with a composition of clause 25. 28. A method comprising contacting a material contaminated with a mycotoxin or at risk of being contaminated with the mycotoxin with a protein of any one of clauses 1 to 18, with a cell of any one of clauses 19 to 23, with an autolysate or extract of clause 24, or with a composition of clause 25. 29. The method of clause 27 or 28, wherein the mycotoxin is a resorcylic acid lactone mycotoxin. 30. The method of clause 29, wherein the mycotoxin is zearalenone.

[0092] As used herein, the terms “a” and “an” are intended to mean “at least one”, and include both singular and plural, unless otherwise indicated.

[0093] As used herein, the terms “about” or “approximately” as applied to a numerical value or range of values are intended to mean that the recited values can vary within an acceptable degree of error for the quantity measured given the nature or precision of the measurements, such that the variation is considered in the art as equivalent to the recited values and provides the same function or result. For example, the degree of error can be indicated by the number of significant figures provided for the measurement, as is understood in the art, and includes but is not limited to a variation of ±1 in the most precise significant figure reported for the measurement. Typical exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given valueor range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" can mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated.

[0094] As used herein, the term “substantially” refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object that is “substantially” aligned would mean that the object is either completely aligned or nearly completely aligned. The exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained.

[0095] The use of “substantially” is equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result. For example, a composition that is “substantially free of” particles would either completely lack particles, or so nearly completely lack particles that the effect would be the same as if it completely lacked particles. In other words, a composition that is “substantially free of” an ingredient or element may still actually contain such item as long as there is no measurable effect thereof. EXAMPLES

[0096] Other features of the present invention will become apparent from the following non- limiting examples which illustrate, by way of example, the principles of the invention. Example 1: Liquid chromatography – mass spectrometry (LC-MS) and LC-MS / MS analysis

[0097] Samples were analyzed by high resolution LC-MS and LC-MS / MS analysis using an Agilent 1290 HPLC system coupled to a Thermo Q-Exactive™ Orbitrap™ mass spectrometer. Chromatographic separation being performed using an Eclipse™Plus rapid resolution high definition (RRHD) C-18 column (2.1 × 100 mm, 1.8 µm; Agilent). The mobile phase consisted of water with 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B) (Optima™ grade, Fisher Scientific, Lawn, NJ, USA) at a flow rate of 0.3 mL / min. The gradient was held at 0% B for 30 s, increased to 100% B over 3 min, held at 100% B for 2.5 min, decreased to 0% B over 30 s and finally held at 0% B for one minute. The injection volume was 5 µL and the column temperature was maintained at 35°C.

[0098] Full mass spectrometry (MS) data dependent MS / MS was used with heated electrospray ionization (HESI) in negative ionization mode. The spray voltage was set to 3.5 kV and the probe heater was held at 450°C. The capillary column temperature was400°C, while the nebulizing and auxiliary gases were held at 17 and 8 arbitrary units respectively. Samples were screened for degradation using a top-5 data dependent acquisition method, which consisted of a full scan between a mass range of 80-1200 m / z, resolution of 35,000, automatic gain control (AGC) of 3 × 106and the maximum injection time (maxIT) of 64 msec. This was followed by MS / MS scans using a 1.2 m / z isolation window, 22 normalized collision energy (NCE), 17,500 resolution, 5 x 106AGC target, minimum AGC target of 4 x 103, intensity threshold of 6.3 x 104, dynamic exclusion of 10.0 s and maximum IT of 64 ms. All data was analyzed using Thermo Xcalibur software. Example 2: Nuclear magnetic resonance (NMR) analysis

[0099] 1H and13C NMR spectra of native and hydrolyzed resorcylic acid lactones were recorded with a 400 MHz JEOL ECZS Spectrometer (Peabody, MA) with an auto-tuning probe. Resorcylic acid lactones and their hydrolyzed products were dissolved in approximately 500 μL of CD3OD (CDN Isotopes, Point Claire, QC, Canada) and referenced to the appropriate solvent peak (δH 3.31, δC 49.1). Example 3: Isolation and identification of Exophiala sp. LRDC-1 Isolation from soil samples

[0100] Soil samples were collected from various agricultural fields located in Southwestern Ontario, Canada. Crops grown on the fields included corn, wheat and ginseng. Core soil samples were frozen at −20°C in plastic bags prior to use. Each soil sample (0.5 g) was inoculated into 2.5 mL of various media spiked with 300 µM zearalenone (Triple Bond, Guelph, Canada), including yeast extract sucrose broth (YES), nutrient broth (NB), malt extract broth (MEB), Luria-Bertani broth (LB), and carbon free inorganic salt culture media (ISCM) (1.0 g of (NH4)2SO4, 6.0 g of K2HPO4·3H2O, 3.0 g of KH2PO4, 0.5 g of NaCl, 0.5 g of MgSO4·7H2O, 0.05 g of CaCl2, 1 L distilled water, pH 7.0) (He, C. et al, Int J Mol Sci (2008), 9: 2366-2375). Cultures were incubated aerobically and anaerobically for 4-7 days at 28°C with shaking at 150 rpm. After 4-7 days, cultures were refreshed by inoculating 100 µL of culture into fresh media spiked with 300 µM zearalenone. After four total refreshes, samples were extracted with methanol, filtered using a 0.45 µm polytetrafluoroethylene (PTFE) syringe filter, and analyzed by high resolution LC-MS / MS analysis (Example 1). Zearalenone degradation was detected by observing disappearance of the zearalenone [M−H]−peak at m / z 317.1394 (C18H22O5) compared to uninoculated culture media. Cultures showing significant zearalenone degradation were serially diluted and plated onto agar to obtain isolated colonies. Single colonies were then further tested for zearalenone degradation ability.

[0101] An isolated microbe (indicated herein as microbe A) which showed consistent degradation of zearalenone when grown in minimal media in the presence of 10 µMzearalenone as the sole carbon source was selected for further investigation. Colonies of microbe A had a distinct brown colour when grown on agar plates or in liquid media. Time course of zearalenone degradation

[0102] A 6 mL culture of microbe A was grown aerobically for 5 days at 28°C in malt extract broth (MEB, Difco) with shaking at 150 rpm in 15 mL conical tubes capped with foam plugs. Six one-mL aliquots were removed from the culture and centrifuged at 2000 × g at 4°C for 10 minutes, and the supernatant was discarded. Each cell pellet was resuspended in 1 mL of fresh carbon free inorganic salt culture media (ISCM) at pH 7.0.

[0103] Aliquots (30 µL) of a zearalenone stock solution (15 mg / mL in DMSO) were added to six separate conical tubes (labelled Day 0 – Day 5), each containing 2.5 mL ISCM.100 µL portions of the resuspended cell pellet were inoculated into each tube, and the conical tubes were capped with foam plugs and shaken at 28°C for from 1 to 5 days. Growth was monitored daily by absorbance (OD600) in 96 well plates.

[0104] Samples (100 µL) of the culture were transferred into 1.5 mL centrifuge tubes. The remaining liquid culture was used for proteomics analysis (Example 4). Each 100 µL sample was quenched 1:1 with methanol, and centrifuged at 10,000 × g at 4°C for 10 minutes. The supernatant was syringe filtered through a 0.45 µm PTFE into a 200 µL polypropylene snap cap HPLC vial (Agilent Technologies), briefly centrifuged at 2000 × g for 5 minutes and analyzed by LC-MS as described in Example 1.

[0105] Growth of microbe A continued gradually until Day 3, at which time zearalenone (ZEN) was no longer detectable in the culture medium. An [M−H]−ion peak at m / z 335.1501 (C18H24O6), corresponding to hydrolyzed zearalenone (HZEN), appeared and showed peak intensity at Day 1 of the growth, but was no longer observed beyond Day 3. An additional [M−H]−ion at m / z 291.1600 (C17H24O4) corresponding to the decarboxylated form of HZEN (DHZEN) showed peak intensity at Day 2 of the growth and was also no longer observed beyond Day 3. Genomic sequencing and identification of microbe A

[0106] Whole genome sequencing of the isolated microbe A was carried out using a combination of short reads (Illumina) and long reads (Oxford Nanopore Technologies (ONT)). Porechop software was used to trim residual adapter sequences from the ONT reads that may have been missed during base-calling and demultiplexing. De novo genome assemblies were generated from the ONT read data with Flye software (Lin, Y. et al, Proceedings of the National Academy of Sciences (2016), 113(52): E8396-E8405) under the nano-hq (ONT high-quality reads) model. Additional Flye options initiate the assembly by first using reads longer than an estimated N50 based on a genome size of 12 Mbp. Subsequent polishing used the Illumina read data with Pilon (Walker, B.J. et al, PloS one(2014), 9: e112963) under default parameters. To reduce erroneous assembly artifacts caused by low quality nanopore reads, long read contigs with an average short read coverage of 15× or less were removed from the assembly. Assembled genome sequences were annotated with funannotate software, and queried funannotate databases include: dbCAN, go, interpro, merops, mibig, pfam, repeats, and uniprot. Final assembly statistics were recorded with QUAST software (Gurevich, A., Bioinformatics (Oxford, England) (2013), 29: 1072-1075).

[0107] The sequenced, assembled, and annotated genome of microbe A consisted of 29,808,315 bp total in twelve distinct contigs. The GC content was 54.15%, with N50 and N75 values of 4,607,806 and 4,304,223 respectively, indicating a high quality assembly. The internal transcribed spacer (ITS) ITS2 region was identified in the sequenced genome and BLAST searched against the National Center for Biotechnology Information (NCBI) ITS database. The top hits from the search were to various strains of the dimorphic fungus Exophiala sp. and the ITS2 sequence of microbe A was 99.75% identical to that of Exophiala alcalophila strain CBS 520.82, and 92.56% identical to that of Exophiala halophila strain CBS 121512. Microbe A is therefore referenced as Exophiala sp. LRDC-1. Example 4: Proteomic analysis Protein sample extraction, trypsin digestion and solid phase extraction

[0108] Liquid cultures of Exophiala sp. LRDC-1 grown for 1, 2 or 3 days in carbon free inorganic salt culture media (ISCM) (Example 3) were centrifuged at 2000 × g at 4°C for 10 minutes and the supernatant was discarded. All microbial cell pellets were stored at −80°C and were resolubilized in 1 mL of lysis buffer (1% sodium deoxycholate (SDC), 100 mM Tris-HCl, pH 8.5, protease inhibitor (cOmplete™ protease inhibitor cocktail EDTA- free tablets, 1 tablet / 50 mL, Millipore Sigma)). Pellets were bead beaten twice in 2 mL bead tubes containing 0.5 mm glass beads at maximum speed (4340 rpm) for 60 seconds, with a 60 second incubation on ice in-between cycles (BeadBug™ benchtop microtube homogenizer). Samples in the bead tubes were centrifuged at 13,000 rpm for 3 minutes at 4°C, and protein was precipitated by adding 350 µL of the supernatant to 1.4 mL pre-cooled acetone with 1 hour incubation at −20°C. Samples were centrifuged at 13,000 × g at 4°C for 10 minutes, and the acetone was discarded.

[0109] Dried protein pellets were resolubilized in 200 µL digestion buffer (1% SDC, 100 mM Tris-HCl, pH 8.5) and quantified at OD480by BSA standard quantification (Pierce™ Rapid Gold BCA Protein Assay, ThermoFisher Scientific). For digestion, 25 µg total protein in 200 µL total volume was incubated at 60°C for 10 minutes at 800 rpm on a ThermoMixer™. 2.5 µL of 100 mM dithiothreitol (DTT) was added to protein samples, and the mixture was incubated at 60°C for 1 hour and returned to room temperature.3 µL of 200 mMiodoacetamide (IAA) was added, and the mixture was incubated for 30 minutes in darkness. 2.5 µL of 100 mM DTT was added, and the mixture was left at room temperature in the dark for five minutes.5 µL trypsin solution (100 ng / µL in 50 mM ammonium bicarbonate) was added and the mixture was incubated overnight at 37°C. Digested proteins were then mixed with 20 µL of 10% trifluoroacetic acid (TFA) and 200 µL ethyl acetate. The samples were then vortexed for 10 seconds prior to 3-minute centrifugation step at 13,000 × g at 20°C. Following centrifugation, the aqueous layer was transferred to a fresh 1.5 mL centrifuge tube, where it was mixed with 200 µL of Milli-Q™ water.

[0110] The peptides were then purified by solid phase extraction (SPE) prior to LC-MS analysis. The peptide samples were loaded into Oasis™ hydrophilic-lipophilic balance (HLB) 1 cc (30 mg) extraction cartridges that were pre-activated with 2 mL of methanol and equilibrated with 0.1% formic acid (FA). After the samples were allowed to slowly drip through, the cartridges were washed with 500 µL 0.1% FA. The peptides were eluted by the addition of 600 µL of 70% acetonitrile (ACN). The samples were then dried down in a CentriVap™ Centrifugal Vacuum Concentrator (Labconco Corporation, Kansas City, MO, United States) and the dried peptide pellets were reconstituted with 5% ACN, 0.1% FA solution. The reconstituted peptide mixtures were transferred into 250-µL snap cap polypropylene HPLC vials (Agilent Technologies, Santa Clara, CA, United States). Proteomic analysis by nanospray LC-MS (nLC-MS)

[0111] Peptide samples were analyzed using an EASY-nLC™ 1000 system (Thermo Fisher Scientific, Waltham, MA, United States) coupled with Q-Exactive™ Orbitrap™ mass spectrometer, which was fitted with a Nanospray Flex™ ion source (Thermo Fisher Scientific, Waltham, MA, United States). Samples were analyzed using a 70,000-resolution full MS analysis in the mass range from 340 m / z to 1,800 m / z, AGC target of 1 × 106and maximum IT of 256 ms. The MS / MS analysis was performed on the top 10 peptides with the resolution set to 17,500, 110 ms maximum IT, and AGC target of 1 × 106. The data- dependent acquisition mass spectrometry (DDA-MS) raw files were processed using MaxQuant software v.2.0.3.0. (Max Planck Institute of Biochemistry, Munich, Germany). To generate a protein group library, the identified peptide sequences were searched against the Exophiala sp. LRDC-1 proteome sequence (Example 3). The protein group IDs output file acquired from MaxQuant was imported into Perseus software for further data analysis. The protein groups identified by site, reverse sequences and potential contaminants were removed from the entry values and the label-free quantification (LFQ) intensity values were then transformed to log2(x) values. The final step of data processing involved imputation of missing values from normal distribution with width and down shift set up to default settings of 0.3 and 1.8, respectively.

[0112] Enzymes which were identified in the proteomic analysis as being significantly upregulated during growth of Exophiala sp. LRDC-1 on days 2 and 3 when levels of HZEN were elevated and DHZEN levels were at their maximum were investigated for potential involvement in zearalenone degradation. A putative α / β-hydrolase (termed ZenH) was identified having a sequence of 263 amino acids and sharing 64.39% identity with the zearalenone hydrolase produced by Clonostachys rosea (Takahashi-Ando, N. et al, Biochemical Journal (2002), 365: 1-6). ZenH also shares less than 70% identity with the closest matching putative α / β hydrolases or zearalenone hydrolases, including those from Fusarium decemcellulare (NCBI accession KAF5003705.1), Fusarium albosuccineum (NCBI accession KAF4460405.1), Exophiala bonariae (NCBI accession KAK5045548.1) and Exophiala aquamarina CBS 119918 (NCBI accession XP_013255140.1). The amino acid sequence of ZenH is shown below:The nucleotide sequence of the gene encoding ZenH is shown below:

[0113] Furthermore, the rapid appearance of elevated levels of decarboxylated hydrolyzed zearalenone (DHZEN) observed during the growth of Exophiala sp. LRDC-1 using zearalenone as the sole carbon source indicated that enzymatic production of DHZEN from HZEN was likely occurring. A 361 amino acid enzyme (termed ZenC) which is encoded by a gene immediately upstream of the zenH gene, and which shows some homology to sequences annotated as putative decarboxylases and / or amidohydrolases was also upregulated during growth of Exophiala sp. LRDC-1 and was selected for further investigation. The amino acid sequence of ZenC is shown below:The nucleotide sequence of the gene encoding ZenC is shown below:

[0114] In addition, a putative Baeyer-Villiger monooxygenase (BVMO; termed ZenB), encoded by a gene immediately downstream of the zenH gene, was also upregulated during growth of Exophiala sp. LRDC-1 and was selected for further study. Baeyer-Villiger monooxygenases are FAD- and NADPH-dependent enzymes that generate esters or lactones by the addition of an oxygen atom adjacent to a ketone moiety. The amino acid sequence of ZenB is shown below:The nucleotide sequence of the gene encoding ZenB is shown below:Example 5: Preparation and characterization of recombinant ZenH Recombinant protein production and isolation

[0115] The zenH gene was synthesized and codon optimized for expression in E. coli. Genes were inserted into the SspI site of the pET His6 MBP TEV LIC cloning vector (Addgene plasmid # 29656; RRID:Addgene_29656) and expressed in the E. coli BL21 (DE3) cell line as a N-terminal 6X His-tagged maltose-binding protein (MBP) fusion protein. In general, protein expression was induced with 500 µM IPTG at 16°C overnight with shaking, cells were harvested via centrifugation, and the expressed protein was isolated as described below. All protein concentrations were determined by absorbance at 280 nm using the extinction coefficient for each protein as calculated via the ProtParam tool (Expasy).

[0116] The 29-kDa ZenH protein was purified to homogeneity using a combination of nickel nitrilotriacetic acid (Ni-NTA) metal affinity and gel permeation chromatography steps. Harvested cell pellets were resuspended in 50 mM Tris HCl (pH 8.0), 500 mM NaCl, 10 mM β-mercaptoethanol, and lysed by sonication. The clarified lysate supernatant was subjected to nickel nitrilotriacetic acid (Ni-NTA) metal affinity chromatography. The sample was loaded and washed in 50 mM Tris HCl (pH 8.0), 300 mM NaCl, 5 mM imidazole (Buffer N) and batch-eluted using Buffer N + 300 mM imidazole. Fractions containing ZenH, as determined via sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) analysis, were pooled and subjected to tobacco etch virus (TEV) protease digestion to remove the maltose- binding protein (MBP) fusion tag. Following TEV digestion, the sample was brought to 1M (NH4)2SO4 and loaded onto a Phenyl-Sepharose™ HP column (Cytiva) equilibrated in 50 mM Tris HCl (pH 8) (Buffer T) and 1M (NH4)2SO4. Protein was eluted using a lineardecreasing (NH4)2SO4gradient (1 to 0 M) in Buffer T over 10 column volumes. Fractions containing ZenH were pooled and subjected to gel permeation chromatography using a HiLoad™ 16 / 600 Superdex™ 200 prep grade column (Cytiva) equilibrated in Buffer T + 150 mM NaCl. Fractions containing ZenH were pooled and concentrated to 3.0 mg / mL, flash frozen, and stored at −80°C until further use. Measurement of enzyme activity

[0117] All MS data were acquired with a Q-Exactive™ Quadrupole Orbitrap™ mass spectrometer (Thermo Scientific, MA, USA) coupled to an Agilent 1290 ultrahigh- performance liquid chromatography (UHPLC) system. Zearalenone and hydrolyzed zearalenone (HZEN) were resolved on a Zorbax™ Eclipse™ Plus RRHD C18 (2.1×50 mm, 1.8 μm; Agilent Technologies, CA, USA) column, maintained at 35°C. The mobile phase was comprised of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) (Optima™ grade, Fisher Scientific, NJ, USA). The gradient consisted of 0% B for 0.5 min before increasing to 100% over 3 min, held at 100% for 2.5 min and reduced to 0% over 0.5 min. Zearalenone and HZEN were detected in negative ionization mode using the following electrospray conditions: capillary voltage, 4.0 kV; capillary temperature, 400°C; sheath gas, 17.00 units; auxiliary gas, 8.00 units; probe heater temperature, 450°C; and S-Lens RF level, 45.00. The data-dependent acquisition method involved a full MS scan at 17,500 resolution over a scan range of 100–800 m / z; automatic gain control (AGC) target and maximum injection time (max IT) were 5 × 106and 64 ms, respectively. The five highest intensity ions from the full scan (excluding isotopes) were sequentially selected using a 1.2 m / z isolation window and analyzed at a resolution of 17,500; AGC target, 1 × 105; max IT, 64 ms; normalized collision energy (NCE) 30; threshold intensity 9.1 × 104; and dynamic exclusion of 1.5 s.

[0118] Reaction mixtures contained 50 mM Tris HCl (pH 8), 1% BSA, 60 µM zearalenone, and 20 nM ZenH. The enzyme reaction was performed at room temperature for 60 minutes and quenched via the addition of ice-cold 100% methanol. The zearalenone [M−H]−peak at m / z 317.1397 (C18H22O5) was observed to rapidly decrease and a corresponding increase in a new [M−H]−peak at m / z 335.1503 (C18H24O6) was observed. The 18.0106 Da increase in mass along with an earlier retention time (3.01 minutes vs.3.66 minutes for zearalenone) and the presence of product ions at m / z 317, 291, 161 and 149, as reported by Vekiru, E. et al, World Mycotoxin Journal (2016), 9: 353-363, indicated production of HZEN.

[0119] Identification of the enzymic reaction product as HZEN (Formula II) was confirmed by13C and1H NMR analysis, as seen from the data shown in Table 1. Hydrolyzed zearalenone (HZEN) is prepared for NMR structural characterization by incubating a mixture of 50 mM Tris HCl (pH 8), 1% bovine serum albumin, 0.4 µg / µL zearalenone, and 20 µM ZenH for72 hours at 37°C. HZEN is isolated by solid phase extraction (SPE) using an Oasis™ HLB 6 cc Vac Cartridge. Following a 1:1 dilution with water, the reaction mixture is passed through the cartridge, washed with 5 mL of water, then eluted stepwise via addition of 60% (v:v) MeOH:H2O and 100% MeOH. Samples are analyzed by LC-HRMS (Example 1) to confirm the presence of the hydrolyzed zearalenone, and are dried under a gentle stream of N2, desiccated for 48 h and weighed prior to NMR analysis. Table 1:1H and13C NMR data for zearalenone (Formula I) and hydrolyzed zearalenone (Formula II) in CD3OD

[0120] The pH dependence of ZenH hydrolysis activity was tested from pH 2.2 to 11. Reactions were conducted with 50 mM of each respective buffer, with 1% (w:v) BSA, 60 µM zearalenone, and 20 nM ZenH. As seen from the results presented in Figure 1A, ZenH was active across a broad pH range, displaying maximal activity at a pH of 8, but was inactive at pH values of 4 and below. Furthermore, as seen from the results presented in Figure 1B, the optimal temperature for zearalenone hydrolysis was determined to be 37°C, and the enzymedisplayed ca.10% of maximal activity at temperatures of 25°C and 50°C. The enzyme was inactive at temperatures above 55°C. Example 6: Preparation and characterization of recombinant ZenC Recombinant protein production and isolation

[0121] The zenC gene was synthesized and expressed in E. coli as described in Example 5 and the ZenC protein was purified to homogeneity with the maltose-binding protein (MBP) tag intact. Harvested cell pellets were resuspended in 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 100 µM ZnCl2, 5 mM β-mercaptoethanol, 20 mM imidazole, and 1X cOmplete™ protease inhibitor cocktail (Roche) (Buffer N + 100 µM ZnCl2.) The sample was loaded onto a Ni-NTA column, washed in lysis buffer and batch-eluted in lysis buffer + 400 mM imidazole. Fractions containing the MBP-ZenC fusion protein were pooled and subjected to gel permeation chromatography in buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, and 100 µM ZnCl2. Enzyme activity

[0122] Hydrolyzed zearalenone (HZEN) and decarboxylated hydrolyzed zearalenone (DHZEN) were detected by accurate mass (±3 ppm) and retention time (±0.01 min) and verified by MS / MS as described in Example 5.

[0123] 1 µM purified MBP-ZenC enzyme was incubated with 10 µM hydrolyzed zearalenone (HZEN) in 50 mM Tris-HCl, 150 mM NaCl, 100 µM ZnCl2in a reaction volume of 50 µL, and the reaction was allowed to proceed for 6 h at room temperature (RT). The reaction was stopped by adding 150 µL of 100% ice-cold methanol. Samples were centrifuged at 20,000 × g and 5 µL of the supernatant was analyzed by LC-MS.

[0124] Incubation of HZEN with ZenC resulted in depletion of the HZEN [M−H]−ion at m / z 335.1500 (C18H24O6) and the production of new [M−H]−peak at m / z 291.1600 (C17H24O4). The retention time of the HPLC peak for this compound was nearly identical to that of HZEN (3.02 mins. vs.3.03 mins.), however, the 43.99 Da decrease in mass between the two compounds is indicative of the loss of a CO2moiety. The new peak was therefore attributed to decarboxylated hydrolyzed zearalenone (DHZEN). ZenC showed no activity against intact zearalenone as a substrate. Example 7: Preparation and characterization of a ZenH / ZenC fusion protein Recombinant protein production and isolation

[0125] A ZenH / ZenC fusion protein was generated by placing ZenH upstream of ZenC and linking the two proteins with a 4x (Gly-Gly-Ser) linker (SEQ ID NO:7). The amino acid sequence of the fusion protein is shown below:

[0126] The gene encoding the fusion protein was synthesized and cloned into the NdeI / XhoI cut sites of the pET28a vector, placing a thrombin cleavable 6X His-tag at the N-terminus of the fusion protein. This vector was transformed into E. coli BL21(DE3) and grown at 37°C until the optical density of the culture at 600 nm (OD600) reached 0.8. The temperature was reduced to 16°C, and protein expression was induced with 500 μM isopropyl β-D-1- thiogalactopyranoside (IPTG) and allowed to proceed overnight at 16°C with shaking. Cells were harvested via centrifugation, resuspended in 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 100 μM ZnCl2, 5 mM β-mercaptoethanol, 20 mM imidazole, 1X complete protease inhibitor cocktail (Roche), and lysed via sonication. The lysate supernatant was clarified via centrifugation and subjected to nickel nitrilotriacetic acid (Ni-NTA) metal affinity chromatography. The sample was loaded and washed in lysis buffer and batch-eluted in lysis buffer + 400 mM imidazole. Fractions containing the fusion protein were then dialyzed exhaustively at 4°C against 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 100 μM ZnCl2. Enzyme activity

[0127] To monitor the ability of the fusion protein to hydrolyze and decarboxylate zearalenone (ZEN), 1 µM purified enzyme was incubated with 20 µM ZEN in 50 mM Tris-HCl, 150 mM NaCl, and 100 µM ZnCl2, and the reaction was allowed to proceed for 16 hrs at room temperature. The reaction was stopped by the addition of MeOH to a final concentration of 75% (v:v). The supernatant was analyzed by LC-MS as described in Example 1 and levels of ZEN and decarboxylated hydrolyzed zearalenone (DHZEN) were determined. Incubation of ZEN with the fusion protein resulted in complete depletion of the ZEN [M−H]−ion at m / z 317.1395 (C18H22O5) (Figure 2B) and the production of a new [M−H]−peak with a retention time of 3.01 minutes and m / z 291.1604 (C17H24O4) that corresponds to the mass of DHZEN (Figure 2A). No hydrolyzed zearalenone (HZEN) was observed at the end of the reaction, indicating complete conversion of ZEN to DHZEN.Example 8: Preparation and characterization of recombinant ZenB Recombinant protein production and isolation

[0128] The zenB gene was synthesized and expressed in E. coli as described in Example 5 and the MBP-tagged ZenB protein was purified to partial homogeneity. Harvested cell pellets were resuspended in 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10 μM FAD, 5 mM β-mercaptoethanol, 20 mM imidazole, and 1X cOmplete™ protease inhibitor cocktail (Roche). Clarified lysate supernatant was subjected to Ni-NTA metal affinity chromatography and sample was loaded and washed in lysis buffer and batch-eluted in lysis buffer + 400 mM imidazole. Fractions containing the MBP-ZenB fusion protein were then buffer exchanged by dialysis into 50 mM Tris (pH 8.0), 150 mM NaCl, and 10 µM FAD. Enzyme activity

[0129] Decarboxylated hydrolyzed zearalenone (DHZEN) and its reaction product were detected by accurate mass (±3 ppm) and retention time (±0.01 min) and verified by MS / MS as described in Example 5.

[0130] 1 µM purified MBP-ZenB enzyme was incubated with 10 µM DHZEN in 50 mM Tris- HCl, 150 mM NaCl, 10 µM FAD, 100 µM NADPH in a reaction volume of 50 µL, and the reaction was allowed to proceed for 16 hrs at RT. The reaction was stopped by adding 150 µL of 100% ice-cold methanol. Samples were centrifuged at 20,000 × g and 5 µL of the supernatant was analyzed by LC-MS.

[0131] Incubation of ZenB with DHZEN resulted in disappearance of the DHZEN [M−H]−ion at m / z 291.1607 (C17H24O4) and the appearance of a new [M−H]−peak at m / z 307.1550 (C17H24O5), as seen in Figure 3. The retention time of the HPLC peak for this compound, termed oxyDHZEN, was nearly identical to that of DHZEN (3.04 min vs.3.02 min), however, the 15.9943 Da increase in mass between the two compounds is indicative of the addition of an oxygen atom. Fragmentation of the newly formed ester bond readily occurred as observed by MS / MS analysis, allowing for the identification of an [M−H]−fragment at m / z 193.0862 (C11H13O3) indicative of addition of an oxygen atom adjacent to the DHZEN ketone and proximal to the dihydrobenzene ring, as seen in Figure 3. Additional fragmentation also occurred immediately adjacent to the HC=CH double bond in the hydrolyzed macrolactone ring of DHZEN, producing an [M−H]−fragment at m / z 165.0546 (C9H9O3) that is again consistent with oxygen atom addition adjacent to the ketone and proximal to the dihydrobenzene ring, as seen in Figure 3. ZenB is not active against intact zearalenone as the substrate. Example 9: Expression of recombinant ZenH in Saccharomyces cerevisiae

[0132] A strain of Saccharomyces cerevisiae was prepared which contains a heterologous expression cassette encoding ZenH (SEQ ID NO:1, Example 4), which is expressedextracellularly. The transformed strain and the control untransformed strain were grown at 30°C for 36 hours in yeast peptone dextrose broth containing 10 g / L yeast extract, 20 g / L peptone and 120 g / L dextrose (YPD120). The resulting cell free supernatants were incubated at 35°C for 60 minutes in the absence or presence of 5 ppm zearalenone (ZEN) in the presence of 20 mM Tris-HCl (pH 8.0). The reaction was quenched by boiling for 10 minutes at 99°C and addition of methanol to a final concentration of 66% (v / v) and the mixture was analyzed by LC-MS. As seen from the results shown in Figure 4, HZEN was formed by incubation of ZEN with the supernatant of the transformed strain expressing ZenH but not from incubation with the supernatant of the untransformed control strain. It is notable that conversion of HZEN to DHZEN is spontaneous in these samples. Example 10: Expression of recombinant ZenH in Komagataella phaffii

[0133] A strain of Komagataella phaffii was prepared which contains a heterologous expression cassette encoding ZenH (SEQ ID NO:1, Example 4), which is expressed extracellularly. The transformed strain and the control untransformed strain were grown at 30°C for 48 hours in yeast peptone ethanol broth containing 10 g / L yeast extract, 20 g / L peptone and 40 g / L dextrose (YPD40). Cells were transferred into inducing media containing 10 g / L yeast extract, 20 g / L peptone and 20 g / L ethanol (YPE20) and incubated at 30°C for 48 hours. The resulting cell free supernatants were incubated at 35°C for 15 minutes in the absence or presence of 5 ppm zearalenone (ZEN) in the presence of 20 mM Tris-HCl (pH 8.0). The reaction was quenched by boiling for 10 minutes at 99°C and addition of methanol to a final concentration of 66% (v / v) and the mixture was analyzed by LC-MS. As seen from the results shown in Figure 5, HZEN was formed by incubation of ZEN with the supernatant of the transformed strain expressing ZenH but not from incubation with the supernatant of the untransformed control strain. It is notable that conversion of HZEN to DHZEN is spontaneous in these samples.

[0134] The transformed and control strains of Komagataella phaffii described above were grown at 30°C for 48 hours in yeast peptone dextrose broth containing 10 g / L yeast extract, 20 g / L peptone and 40 g / L glucose (YPD40). The resulting cell suspension (40 mL) was added to 760 mL basal salts media containing 7.92 mL / L phosphoric acid, 0.793 g / L calcium chloride dihydrate, 18.2 g / L potassium sulfate, 7.45 g / L magnesium sulfate heptahydrate, 4.127 g / L potassium hydroxide and 40 g / L glucose for fed-batch propagation. Propagation was performed at 32°C for 70 hours at pH 5, with controlled ethanol feed and oxygen enrichment in a 2 liter reactor. The resulting cell-free supernatants (SUP) or whole cell broth (WCB, containing both the supernatant and the yeast cells) were incubated at 35°C for 15 minutes in the absence or presence of 5 ppm zearalenone (ZEN) in the presence of 20 mM Tris-HCl (pH 8.0). The reaction was quenched by boiling for 10 minutes at 99°C andaddition of methanol to a final concentration of 66% (v / v) and the mixture was analyzed by LC-MS. As seen from the results shown in Figure 6, more HZEN was formed with WCB than equivalent amount of WCB with cells removed via centrifugation (SUP) of the transformed strain, indicating that ZenH zearalenone hydrolase activity is present in both the culture supernatant as well as being cell-associated. No HZEN was formed by incubation of ZEN with WCB or SUP from the control, untransformed strain. Example 11: Comparison of activity of ZenH with other zearalenone hydrolases

[0135] Zearalenone hydrolases from Gliocladium roseum (Clonostachys rosea) (Takahashi- Ando, N. et al, Biochemical Journal (2002), 365: 1-6; National Centre for Biotechnology Information (NCBI) accession number AHB51772.1) and Aeromicrobium sp. (Hoogstra, S.J., et al, Toxins (2024), 16: 404), and a putative zearalenone hydrolase (NCBI accession number XP_016234729.1) from Exophiala spinifera identified via BLASTp search against the NCBI nr protein database were all codon optimized, synthesized, expressed, and purified to homogeneity as described in Example 5. The amino acid sequence of Gliocladium roseum zearalenone hydrolase is provided below:The amino acid sequence of Aeromicrobium sp. zearalenone hydrolase is provided below:The amino acid sequence of the putative Exophiala spinifera zearalenone hydrolase is provided below:

[0136] A multiple sequence alignment between the enzymes performed using the Clustal Omega software demonstrates that ZenH is 62% identical at the amino acid level to the putative E. spinifera hydrolase, 64% identical to the G. roseum hydrolase, and only 21% identical to the Aeromicrobium sp. hydrolase.

[0137] Reaction mixtures to determine hydrolase activity of each of the four enzymes contained 50 mM Tris HCl (pH 8), 1% (w:v) bovine serum albumin (BSA), 60 µM zearalenone, and 20 nM purified homogenous zearalenone hydrolase. A control reaction mixture including no enzyme was also included. Reactions were allowed to proceed at 37°C for 20 minutes and were quenched via the addition of ice-cold 100% methanol. The production of a [M−H]−peak at m / z 335.1503 (C18H24O6), attributable to hydrolyzed zearalenone, was monitored for each reaction via LC-MS analysis and relative activity was plotted by comparing to the most active enzyme in the assay. As seen from the results presented in Figure 7, under these conditions, the ZEN hydrolase from Gliocladium roseum displayed ca.31% relative activity compared to ZenH, while the Aeromicrobium sp. and Exophiala spinifera homologs displayed 82% and 5% relative activity, respectively.

[0138] The embodiments described herein are intended to be illustrative of the present compositions and methods and are not intended to limit the scope of the present invention. Various modifications and changes consistent with the description as a whole and which are readily apparent to the person of skill in the art are intended to be included. The appended claims should not be limited by the specific embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

CLAIMS 1. A protein active to degrade a mycotoxin, the protein comprising one or more mycotoxin-degrading enzymes selected from a hydrolase enzyme, a decarboxylase enzyme and a Baeyer-Villiger oxidase enzyme.

2. The protein of claim 1 wherein the hydrolase enzyme: comprises an amino acid sequence of SEQ ID NO:1, or a catalytically active portion thereof, or comprises a variant amino acid sequence having at least 70% identity to SEQ ID NO:1, or to the catalytically active portion of SEQ ID NO:1; or is encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:2 or is encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70% identity to SEQ ID NO:

2.

3. The protein of claim 1 or 2 wherein the decarboxylase enzyme: comprises an amino acid sequence of SEQ ID NO:3, or a catalytically active portion thereof, or comprises a variant amino acid sequence having at least 70% identity to SEQ ID NO:3, or to the catalytically active portion of SEQ ID NO:3; or is encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:4 or is encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70% identity to SEQ ID NO:

4.

4. The protein of claim 1 wherein the one or more mycotoxin-degrading enzymes are selected from: (i) a hydrolase enzyme comprising an amino acid sequence of SEQ ID NO:1, or a catalytically active portion thereof, or comprising a variant amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:1, or to the catalytically active portion of SEQ ID NO:1; or a hydrolase enzyme encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:2 or encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:2; and (ii) a decarboxylase enzyme comprising an amino acid sequence of SEQ ID NO:3, or a catalytically active portion thereof, or comprising a variant amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:3, or to the catalytically active portion of SEQ ID NO:3; or a decarboxylase enzyme encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO:4 or encoded by a polynucleotide comprising a variant nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:

4.

5. The protein of any one of claims 1 to 4, wherein the hydrolase enzyme is active to catalyze hydrolysis of the mycotoxin to form a hydrolyzed mycotoxin.

6. The protein of claim 5, wherein the decarboxylase enzyme is active to catalyze the decarboxylation of the hydrolyzed mycotoxin to form a decarboxylated hydrolyzed mycotoxin.

7. The protein of any one of claims 1 to 6 wherein the mycotoxin is zearalenone.

8. The protein of claim 7 wherein the hydrolase enzyme is active to catalyze the hydrolysis of zearalenone to form a compound of Formula II:

9. The protein of claim 8 wherein the decarboxylase enzyme is active to catalyze the decarboxylation of the compound of Formula II to form a compound of Formula III:

10. The protein of any one of claims 1 to 9 wherein the protein comprises the hydrolase enzyme.

11. The protein of any one of claims 1 to 10 wherein the protein comprises the decarboxylase enzyme.

12. The protein of any one of claims 1 to 11, wherein the protein is a fusion protein.

13. A cell, wherein the cell expresses a protein of any one of claims 1 to 12.

14. The cell of claim 13, wherein the cell is a recombinant host cell genetically altered to express the protein of any one of claims 1 to 12.

15. The cell of claim 14, wherein the recombinant host cell is a recombinant microbial host cell.

16. The cell of claim 15, wherein the recombinant microbial host cell is a recombinant bacterial host cell or a recombinant fungal host cell.

17. The cell of claim 16 wherein the recombinant microbial host cell is a recombinant Escherichia coli host cell or a recombinant yeast cell.

18. An autolysate or extract of the cell of any one of claims 13 to 17.

19. A composition comprising a protein of any one of claims 1 to 12, a cell of any one of claims 13 to 17, or an autolysate or extract of claim 18.

20. A method of degrading a mycotoxin, the method comprising contacting the mycotoxin with a protein of any one of claims 1 to 12, with a cell of any one of claims 13 to 17, with an autolysate or extract of claim 18, or with a composition of claim 19.

21. A method comprising contacting a material contaminated with a mycotoxin or at risk of being contaminated with the mycotoxin with a protein of any one of claims 1 to 12, with a cell of any one of claims 13 to 17, with an autolysate or extract of claim 18, or with a composition of claim 19.

22. The method of claim 20 or 21, wherein the mycotoxin is zearalenone.

Images