Enzymatic glycosylation of trichothecene mycotoxins

Abstract

A method of reducing the toxicity of a trichothecene mycotoxin such as deoxynivalenol (DON) comprises exposing the trichothecene mycotoxin to a UDP- glycosyltransferase enzyme originating from a strain of Bacillus subtilis. The enzyme catalyzes the conversion of the trichothecene mycotoxin to a non-toxic monoglycosylated metabolite. The enzyme is useful in reducing the toxicity of animal feed or food products which would otherwise be contaminated with the trichothecene mycotoxin.

Description

ENZYMATIC GLYCOSYLATION OF TRICHOTHECENE MYCOTOXINS Field

[0001] The present application is directed to an enzyme useful for detoxifying mycotoxins. More specifically, the present application is directed to an enzyme capable of catalyzing the conversion of trichothecene mycotoxins, such as deoxynivalenol, to products with reduced toxicity. Background

[0002] Fusarium head blight and ear rot are fungal diseases caused by multiple Fusarium species, including F. graminearum, that affect many crops (such as wheat, barley and corn). These diseases result in considerable economic losses due to reduced yields and the reduction in grain quality due to contamination with mycotoxins such as the trichothecene mycotoxins. The trichothecene mycotoxins are a family of compounds having the following general chemical formula:

[0003] During the course of plant infection, one of the most commonly detected trichothecene mycotoxins, deoxynivalenol (DON), also known as vomitoxin, or by its IUPAC name, (3α,7α)-3,7,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one, is secreted by the fungal pathogen and acts as a potent virulence factor, aiding in disease spread.Deoxynivalenol (DON)

[0004] Consumption of DON contaminated feed leads to a number of toxic effects in animals, including vomiting, feed refusal, immunosuppression, and reduced weight gain. At the molecular level, trichothecenes target the ribosome and interfere with protein synthesis.This interaction leads to the ribotoxic stress response that can alter cell growth and differentiation and induce apoptosis. Other mechanisms of DON toxicity include the upregulation of a large number of microRNAs. Although mitigation strategies exist across the feed chain such as the development of resistant plant varieties, crop rotation and chemical controls, climate change scenarios still predict an increase in disease epidemics and thus commodity contamination. Currently, when cereal grains are contaminated with high levels of DON, they are not suitable for use in food products or as animal feed.

[0005] Enzymatic biotransformation of mycotoxins represents the most promising avenue for remediation of contaminated food and feed. Enzymes can be rapid, specific, and cost- effective when applied at scale. Several enzymes that biotransform DON are known and the respective enzymatic biotransformation products have been characterized. For example, DON can be isomerized into the non-toxic 3-epi-DON in a two-step enzymatic process by the bacterium Devosia mutans. However, both enzymes in the isomerization process require costly co-factors. The reductive alkylation of DON to produce the non-toxic de-epoxy-DON (DOM-1), has been known for decades, however, the specific enzymatic mechanism responsible for this conversion remains unknown. Glycosylation, a phase II detoxification strategy in plants, is the most commonly observed enzymatic modification of DON that occurs in plantae.

[0006] Plants typically contain hundreds of glycosyltransferases that enable glycosylation of a diverse set of acceptor molecules, including secondary metabolites, hormones, and xenobiotics. Although there are 3 available hydroxyl positions on DON (the 3-, 7- and 15-hydroxyl positions) that could represent targets for glycosylation, glycosylation on the 3-hydroxyl position has been predominantly observed in naturally contaminated materials. DON-3-glucoside (D3G) is significantly less toxic than DON due to its inability to bind to the A-site of the ribosomal peptidyl transferase center. Unfortunately, although glycosylation at the 3-position reduces the toxicity of DON, this enzymatic detoxification strategy is not viable because efficient deglycosylation of D3G to release DON has been observed in both human and animal intestinal microbiota. Therefore, new strategies for detoxification of trichothecene mycotoxins are desirable. Summary

[0007] One aspect of the present application is directed to a method of reducing the toxicity of a trichothecene mycotoxin comprising exposing the trichothecene mycotoxin to a protein comprising UDP-glycosyltransferase activity. In at least one embodiment, the protein comprising UDP-glycosyltransferase activity is active to catalyze transformation of a trichothecene mycotoxin to a glycosylated product which is glycosylated at a position of the trichothecene mycotoxin other than the 3-position or the 15-position. In at least oneembodiment, the protein comprising UDP-glycosyltransferase activity is active to catalyze glycosylation of a hydroxyl group at a position of the trichothecene mycotoxin other than a 3-position or a 15-position. In at least one embodiment, the trichothecene mycotoxin is selected from deoxynivalenol, deoxynivalenol-3-glucoside, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, NX and 3-acetyl-NX. In at least one embodiment, the trichothecene mycotoxin is deoxynivalenol. In at least one embodiment, the trichothecene mycotoxin is HT-2.

[0008] In at least one embodiment, the protein comprising UDP-glycosyltransferase activity is a UDP-glycosyltransferase enzyme. In at least one embodiment, the UDP- glycosyltransferase enzyme is active to catalyze transformation of a trichothecene mycotoxin containing a 7-hydroxy group to a glycosylated product which is glycosylated at the 7-position of the trichothecene mycotoxin.

[0009] In at least one embodiment, the UDP-glycosyltransferase enzyme is isolated from a species of Bacillus. In at least one embodiment, the species of Bacillus is a strain of Bacillus subtilis. In at least one embodiment, the strain of Bacillus subtilis is Bacillus subtilis strain AAFC1. A deposit of Bacillus subtilis strain AAFC1 pursuant to the Budapest Treaty was received on July 31, 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 310724-01).

[0010] In at least one embodiment, the UDP-glycosyltransferase enzyme is an isoform of YjiC. In at least one embodiment, the UDP-glycosyltransferase enzyme has a 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%, at least 99% or 100% identity to SEQ ID NO:3 or SEQ ID NO:5.

[0011] In at least one embodiment, exposing the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity comprises exposing the trichothecene mycotoxin to a composition comprising the protein comprising UDP-glycosyltransferase activity as described herein In at least one embodiment, the composition further comprises a carrier. In at least one embodiment, the carrier is a solid carrier. In at least one embodiment, the carrier is a liquid carrier. In at least one embodiment, the carrier is an agriculturally acceptable carrier.

[0012] In at least one embodiment, exposing the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity comprises further exposing the trichothecene mycotoxin and the protein comprising UDP-glycosyltransferase activity to an enzyme active to catalyze the formation of UDP-glucose. In at least one embodiment, the enzyme active to catalyze the formation of UDP-glucose is a sucrose synthase. In at least one embodiment,the sucrose synthase is of a species of Arabidopsis. In at least one such embodiment, the species of Arabidopsis is Arabidopsis thaliana. In at least one embodiment, the sucrose synthase is encoded by the SUS1 gene of Arabidopsis thaliana. In at least one embodiment, the sucrose synthase has a 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%, at least 99% or 100% identity to SEQ ID NO:6.

[0013] In at least one embodiment, exposing the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity comprises exposing the trichothecene mycotoxin to a composition comprising the protein comprising UDP-glycosyltransferase activity as described herein and the enzyme active to catalyze the formation of UDP-glucose as described herein. In at least one embodiment, the composition further comprises a carrier as described herein.

[0014] In at least one embodiment, the protein comprising UDP-glycosyltransferase activity is a fusion protein comprising a first polypeptide moiety fused to a second polypeptide moiety, wherein the first polypeptide moiety comprises a UDP-glycosyltransferase enzyme as described herein. In at least one embodiment, the second polypeptide moiety comprises an enzyme active to catalyze the formation of UDP-glucose as described herein. In at least one embodiment, the fusion protein further includes a linker polypeptide inserted between the first polypeptide moiety and the second polypeptide moiety. In at least one embodiment, the first polypeptide moiety is located N-terminally with respect to the second polypeptide moiety. In at least one embodiment, the first polypeptide moiety is located C-terminally with respect to the second polypeptide moiety. In at least one embodiment, exposing the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity comprises exposing the trichothecene mycotoxin to a composition comprising a fusion protein as described herein. In at least one embodiment, the composition further comprises a carrier as described herein.

[0015] In at least one embodiment, exposing the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity as described herein comprises exposing the trichothecene mycotoxin to a microbial host cell expressing the protein comprising UDP- glycosyltransferase activity as described herein or to an autolysate thereof or an extract thereof. In at least one embodiment, the microbial host cell is a recombinant microbial host cell. In at least one embodiment, the microbial host cell is a bacterial host cell. In at least one embodiment, the bacterial host cell is a cell of a strain of a species of Bacillus or a cell of a strain of a species of Lactobacillus. In at least one embodiment, the microbial host cell is a fungal host cell. In at least one embodiment, the microbial host cell is a yeast host cell. In at least one embodiment, the yeast host cell is a cell of a strain of a species of Saccharomycesor a cell of a strain of a species of Komagataella. In at least one embodiment, exposing the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity as described herein comprises exposing the trichothecene mycotoxin to a composition comprising a recombinant microbial host cell as described herein, an autolysate thereof or an extract thereof. In at least one embodiment, the composition further comprises a carrier as described herein.

[0016] In at least one embodiment, exposing the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity as described herein comprises exposing the trichothecene mycotoxin to a plant expressing the protein comprising UDP-glycosyl- transferase activity as described herein. In at least one embodiment, the plant is a crop plant at risk of being contaminated with deoxynivalenol.

[0017] In at least one embodiment, exposing the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity as described herein comprises exposing a material contaminated with the trichothecene mycotoxin or at risk of being contaminated with the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity as described herein or to a microbial host cell expressing the protein comprising UDP-glycosyl- transferase activity as described herein or to 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 feed, feed product or feed ingredient is dried distiller’s grains or dried distiller’s grains with solubles. In at least one embodiment, the material is a cereal crop or grain. In at least one embodiment, the cereal crop or grain is corn or wheat.

[0018] In at least one embodiment, the material is a feedstock for a fermentation process. In at least one such embodiment, the method further comprises treating the feedstock with a yeast strain which is active to ferment the feedstock to produce one or more products, wherein the one or more products have a reduced level of the trichothecene mycotoxin compared to the level of the trichothecene mycotoxin in products obtained from a fermentation process in which the feedstock is not exposed to the protein comprising UDP- glycosyltransferase activity. In at least one such embodiment, the yeast strain expresses the protein comprising UDP-glycosyltransferase activity. In at least one embodiment, the method comprises exposing the feedstock to a bacterial strain expressing the protein comprising UDP-glycosyltransferase activity. In at least one such embodiment, the fermentation process produces one or more products selected from distillers’ dried grains, distillers’ dried grains with solubles, distillers’ wet grains, distillers’ wet grains with solubles, dried solubles and condensed distillers’ solubles.

[0019] A further aspect of the present application provides an isolated protein comprising UDP-glycosyltransferase activity as described herein. In at least one embodiment, the isolated protein comprising UDP-glycosyltransferase activity as described herein is at least partially purified. In at least one embodiment, the isolated protein comprising UDP-glycosyl- transferase activity is a fusion protein as described herein.

[0020] In another aspect, the present application provides a recombinant microbial host cell which has been genetically altered to express a heterologous protein comprising UDP- glycosyltransferase activity as described herein. In at least one embodiment, the microbial host cell is a bacterial host cell. In at least one embodiment, the bacterial host cell is a cell of a strain of a species of Bacillus or a cell of a strain of a species of Lactobacillus. In at least one embodiment, the microbial host cell is a fungal host cell. In at least one embodiment, the fungus is a yeast host cell. In at least one embodiment, the yeast host cell is a cell of a strain of a species of Saccharomyces or a cell of a strain of a species of Komagataella.

[0021] A further aspect of the present application provides a composition comprising one or more of a mixture comprising a UDP-glycosyltransferase enzyme as described herein and an enzyme active to catalyze the formation of UDP-glucose as described herein; a fusion protein as described herein and a recombinant microbial host cell as described herein, an autolysate thereof or an extract thereof. In at least one embodiment, the composition further comprises a carrier as described herein.

[0022] Another aspect of the present application provides a plant genetically altered to express a protein comprising UDP-glycosyltransferase activity as described herein. In at least one embodiment, the plant is a crop plant at risk of being contaminated with a trichothecene mycotoxin. Brief Description of the Drawings

[0023] Further features of the present invention will become apparent from the following written description and the accompanying figures, in which:

[0024] Figure 1A is a combined extracted ion chromatogram obtained from liquid chromatography – mass spectrometry (LC-MS) analysis of samples of a bacterial culture as described herein taken at 0 hours and 72 hours growth in the presence of deoxynivalenol (DON), showing relative intensities of peaks representing DON and mono- and diglycosylated DON products (+1 Glu, +2 Glu);

[0025] Figure 1B is a graph showing the change over time of optical density (OD) at 600 nm and relative LC-MS signal intensity of DON and the glycosylated DON products of Figure 1A in samples from the bacterial culture of Figure 1A. Error bars represent standard deviation (n=3);

[0026] Figure 2A is a bar graph showing the relative DON glycosylation activity of fractions obtained during biochemical enrichment of the activity from the bacterial culture of Figure 1A. Culture = live culture prior to sonication; sup. and pellet indicate supernatant and pellet post sonication and clarification; A.S.P. = ammonium sulfate ((NH4)2SO4) precipitation at various concentration ranges (%w:v) of ammonium sulfate. Error bars represent standard deviation (n=3);

[0027] Figure 2B is a graph showing Q-Sepharose™ enrichment of DON-glycosylation activity from the most active fraction of Figure 2A after ammonium sulfate precipitation. The solid line represents absorbance at 280 nm, and the dashed line represents LC-MS signal intensity levels (108) of glycosylated DON products following incubation of individual fractions with DON and UDP-glucose (n≥2);

[0028] Figure 2C is a graph showing gel permeation chromatography enrichment of DON glycosylation activity after Q-Sepharose™ ion exchange. The solid line represents absorbance at 280 nm, and the dashed line represents LC-MS signal intensity levels (108) of glycosylated DON-products following incubation of individual fractions with DON and UDP- glucose (n≥2). Vo and Vt represent the void and total volumes of the column respectively;

[0029] Figure 3A is a gel permeation chromatogram of a purified recombinant enzyme having DON glycosylation activity (Protein A). The black line represents absorbance at 280 nm, Vo = void volume, Vt = total volume of the column. The inset is a photograph of an SDS-PAGE gel, illustrating analysis of pooled fractions of Protein A following chromatographic separation. M = reference protein markers. Numbers represent the molecular weight (MW) of each marker;

[0030] Figure 3B is a combined extracted ion chromatogram from LC / MS analysis of products obtained from incubation of Protein A with DON and UDP-glucose or from incubation of DON and UDP-glucose under the same conditions in the absence of Protein A (control), showing relative intensities of peaks representing DON and mono- (DON+1Glu) and diglycosylated (DON+2Glu) DON products;

[0031] Figure 3C is a graph showing the LC-MS signal intensity of DON, DON+1Glu and DON-3-glucoside (DON-3-G) in culture supernatants of E. coli either over-expressing (+IPTG) or not expressing (−IPTG) Protein A and grown overnight at 37°C or 16°C in the presence of 30 ppm DON. S / N indicates supernatant;

[0032] Figure 4 is a series of LC chromatograms (left) and mass spectra (right) of the monoglycosylated product (DON+1Glu) obtained from incubation of Protein A with DON and UDP-glucose (panel (a)) and the reference compounds DON-3-glucoside (panel (b)) (D3G) and DON-15-glucoside (D15G) (panel (c));

[0033] Figure 5A is a series of photographs of duckweed (Lemna minor) grown in assay media only as a negative control (C), or in assay media with addition of DMSO alone, or of DMSO in addition to DON, D3G, DON+1Glu or an unpurified Protein A-catalyzed reaction mixture (mix), each at a final concentration of 20 µM;

[0034] Figure 5B is a bar graph showing the green pixel count of duckweed fronds from the assay of Figure 5A. Error bars represent standard deviation (n=4) and different letters (a, b) indicate statistical significance measured by single-factor AN VA with Tukey’s post hoc testing (p < 0.05);

[0035] Figure 6 is a series of graphs showing the stability of DON, D3G and DON+1Glu when incubated with cellulase (10 U / mL) (Panel A) or cellobiase (1.3 U / mL) (Panel B). Error bars represent standard deviation (n=3);

[0036] Figure 7 is a series of graphs showing the extent of conversion of D3G (Panel A) and DON+1Glu (Panel B) to DON when incubated with swine feces immediately prior to reaction termination (Control) or for 24 hours (Expt.). Panel C shows the results of incubation of DON itself with swine feces (DON-G represents any glycosylated product of DON). N.D. indicates value not detected. Error bars represent standard deviation (n=3);

[0037] Figure 8A is a graph showing the percentage of substrate remaining when Protein A is incubated with the trichothecenes deoxynivalenol (DON), DON+1Glu, deoxynivalenol-3- glucoside (DON3G), 15-acetyldeoxynivalenol (15-A-DON), 3-acetyldeoxynivalenol (3-A-DON), NX and 3-acetyl-NX (3-A-NX). ND indicates value not detected;

[0038] Figure 8B is a graph showing the percentage of substrate remaining when Protein A is incubated with the trichothecenes DON, HT-2 and T-2. ND indicates value not detected;

[0039] Figure 9A is a graph showing the growth of Saccharomyces cerevisiae strain A (measured as the optical density at 600 nm) in function of time (hours) in the presence of various concentrations (0, 50, 75 or 100 ppm) of deoxynivalenol (DON);

[0040] Figure 9B is a graph showing the growth of Saccharomyces cerevisiae strain B (measured as the optical density at 600 nm) in function of time (hours) in the presence of various concentrations (0, 50, 75 or 100 ppm) of DON;

[0041] Figure 9C is a graph showing the amount of DON (in ppm lower panel) or DON+1Glu (in ppm upper panel) found in the supernatant or cell lysate of S. cerevisiae strain A or B grown in the absence or presence of 50 ppm DON;

[0042] Figure 10A is a graph showing the percentage conversion of deoxynivalenol (DON) in reaction mixtures containing Protein A (or no Protein A as a control), sucrose and varying concentrations of UDP-glucose and sucrose synthase from Arabidopsis thaliana (SuS1).Measurement of the percent conversion of DON in the absence of SuS1 was not carried out at concentration of UDP-glucose of 10 mM;

[0043] Figure 10B is a graph showing the percentage conversion of deoxynivalenol (DON) in reaction mixtures containing DON, sucrose and UDP in the presence of fusion proteins (Protein A::AtSUS1 or MBP::Protein A::AtSUS1) or mixtures of Protein A and Arabidopsis thaliana sucrose synthase (Protein A + AtSUS1). N.D. indicates that the result was not determined; and

[0044] Figure 11 is a graph showing the percentage conversion of deoxynivalenol (DON) when dried distiller’s grains with solubles naturally contaminated with trichothecene mycotoxins are treated with Protein A at concentrations of 0 µM, 0.1 µM, 1 µM or 10 µM and at temperatures of 23°C, 30°C or 37°C. Detailed Description

[0045] One aspect of the present application is directed to a method of reducing the toxicity of a trichothecene mycotoxin comprising exposing the trichothecene mycotoxin to a protein comprising UDP-glycosyltransferase activity. In at least one embodiment, the trichothecene mycotoxin has a free hydroxyl group at a position other than the 3-position or the 15-position.In at least one embodiment, the trichothecene mycotoxin has a free hydroxyl group at the 7-position. In at least one embodiment, the trichothecene mycotoxin has a free hydroxyl group at the 4-position. In at least one embodiment, the trichothecene mycotoxin is selected from deoxynivalenol, deoxynivalenol-3-glucoside, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, NX and 3-acetyl-NX. In at least one embodiment, the trichothecene mycotoxin is deoxynivalenol. In at least one embodiment, the trichothecene mycotoxin is HT-2.

[0046] In at least one embodiment, the protein comprising UDP-glycosyltransferase activity is active to catalyze transformation of the trichothecene mycotoxin to a glycosylated product having a toxicity which is less than the toxicity of the trichothecene mycotoxin. In at least one embodiment, the glycosylated product bears a glucose moiety on a hydroxyl group at a position of the trichothecene mycotoxin other than a 3- position or a 15-position. In at least one embodiment, the glycosylated product bears a glucose moiety on the 7-hydroxyl group of the trichothecene mycotoxin.

[0047] In at least one embodiment, the protein comprising UDP-glycosyltransferase activity is a UDP-glycosyltransferase enzyme. In at least one embodiment, the UDP-glycosyl- transferase enzyme is active to catalyze transformation of a trichothecene mycotoxin to a glycosylated product which is glycosylated at a position other than the 3-position or the 15-position of the trichothecene mycotoxin. In at least one embodiment, the UDP-glycosyl-transferase enzyme is active to catalyze transformation of a trichothecene mycotoxin containing a 7-hydroxy group to a glycosylated product which is glycosylated at the 7-position of the trichothecene mycotoxin. In at least one embodiment, the UDP-glycosyl- transferase enzyme is active to catalyze transformation of deoxynivalenol to a glycosylated product which is glycosylated at the 7-position of deoxynivalenol.

[0048] In at least one embodiment, the UDP-glycosyltransferase enzyme is isolated from a microbe. In at least one embodiment, the microbe is a bacterium. In at least one embodiment, the bacterium is a species of Bacillus. In at least one embodiment, the species of Bacillus is a strain of Bacillus subtilis. In at least one embodiment, the strain of Bacillus subtilis is Bacillus subtilis strain AAFC1. A deposit of Bacillus subtilis strain AAFC1 pursuant to the Budapest Treaty was received on July 31, 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 310724-01).

[0049] In at least one embodiment, the UDP-glycosyltransferase enzyme is an isoform of YjiC. In at least one embodiment, the UDP-glycosyltransferase enzyme is the YjiC homolog found in B. subtilis strain 168 (GenBank reference number AAC46318.1; NCBI Protein database reference number NP_389104.1). In at least one embodiment, the UDP-glycosyl- transferase enzyme has the amino acid sequence represented by SEQ ID NO:5. MKKYHISMIN IPAYGHVNPT LALVEKLCEK GHRVTYATTE EFAPAVQQAG GEALIYHTSL NIDPKQIREM MEKNDAPLSL LKESLSILPQ LEELYKDDQP DLIIYDFVAL AGKLFAEKLN VPVIKLCSSY AQNESFQLGN EDMLKKIREA EAEFKAYLEQ EKLPAVSFEQ LAVPEALNIV FMPKSFQIQH ETFDDRFCFV GPSLGERKEK ESLLIDKDDR PLMLISLGTA FNAWPEFYKM CIKAFRDSSW QVIMSVGKTI DPESLEDIPA NFTIRQSVPQ LEVLEKADLF ISHGGMNSTM EAMNAGVPLV VIPQMYEQEL TANRVDELGL GVYLPKEEVT VSSLQEAVQA VSSDQELLSR VKNMQKDVKE AGGAERAAAE IEAFMKKSAV PQ (SEQ ID NO:5)

[0050] In at least one embodiment, the UDP-glycosyltransferase enzyme has 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.

[0051] In at least one embodiment, the UDP-glycosyltransferase enzyme contains one or more of a glycine (Gly, G) residue at position 97, a lysine (Lys, K) residue at position 148, a glutamine (Gln, Q) residue at position 210, a glycine (Gly, G) residue at position 212 and a threonine (Thr, T) residue at position 359. In at least one embodiment, the UDP-glycosyl- transferase enzyme is the YjiC homolog found in B. subtilis strain 168 (SEQ ID NO:5) having one or more mutations selected from Asp97Gly, Arg148Lys, Lys210Gln, Ser212Gly, andSer359Thr. In at least one embodiment, the UDP-glycosyltransferase enzyme has the amino acid sequence represented by SEQ ID NO:3. MKKYHISMIN IPAYGHVNPT LALVEKLCEK GHRVTYATTE EFAPAVQQAG GEALIYHTSL NIDPKQIREM MEKNDAPLSL LKESLSILPQ LEELYKGDQP DLIIYDFVAL AGKLFAEKLN VPVIKLCSSY AQNESFQLGN EDMLKKIKEA EAEFKAYLEQ EKLPAVSFEQ LAVPEALNIV FMPKSFQIQH ETFDDRFCFV GPSLGERKEQ EGLLIDKDDR PLMLISLGTA FNAWPEFYKM CIKAFRDSSW QVIMSVGKTI DPESLEDIPA NFTIRQSVPQ LEVLEKADLF ISHGGMNSTM EAMNAGVPLV VIPQMYEQEL TANRVDELGL GVYLPKEEVT VSSLQEAVQA VSSDQELLTR VKNMQKDVKE AGGAERAAAE IEAFMKKSAV PQ (SEQ ID NO:3)

[0052] In at least one embodiment, the UDP-glycosyltransferase enzyme has 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.

[0053] In at least one embodiment, the UDP-glycosyltransferase enzyme is encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO:4. atg aaa aag tac cat att tcg atg atc aat atc ccg gcg tac gga cat gtc aat cct acg ctt gct tta gta gag aag ctt tgt gag aaa ggg cac cgt gtc acg tac gcg acg act gag gag ttt gcg ccc gct gtt cag caa gcc ggt gga gaa gca ttg atc tat cat aca tcc ttg aat att gat cct aag caa atc agg gag atg atg gaa aag aat gac gcg ccc ctc agc ctt ttg aaa gaa tca ctc agc att ctg ccg cag ctt gag gag tta tat aag ggt gat cag cct gat ctg atc atc tat gac ttt gtt gcg ctg gct ggt aaa ttg ttt gct gaa aag ctt aat gtt ccg gtc att aag ctc tgt tcg tca tat gcc caa aat gaa tcc ttt cag tta gga aat gaa gac atg ctg aag aaa ata aaa gaa gca gag gct gaa ttt aaa gcc tac ttg gag caa gag aag ttg ccg gct gtt tca ttt gaa cag tta gct gtg ccg gaa gca tta aat att gtc ttt atg ccg aag tct ttt cag att cag cat gag acg ttc gat gac cgt ttc tgt ttt gtc ggc ccc tct ctc gga gaa cga aag gaa caa gaa ggc ctg ttg att gac aag gat gat cgc ccg ctt atg ctg att tct ttg ggt acg gcg ttt aac gca tgg ccg gaa ttt tac aag atg tgc atc aag gca ttt cgg gat tct tca tgg caa gtg atc atg tcg gtt ggg aaa acg att gat cca gaa agc ttg gag gat att cct gct aac ttt act att cgc caa agt gtg ccg cag ctt gag gtg tta gag aaa gct gat ttg ttc atc tct cat ggc ggg atg aac agt acg atg gaa gcg atg aac gca ggt gtg ccg ctt gtc gtc att ccg caa atg tat gag caa gag ctc act gca aat cgg gtt gat gaa tta ggc ctt ggc gtt tat ttg ccg aaa gag gaa gtg act gtt tcc agc ctg cag gaa gcg gtt cag gct gta tcc agt gat caa gag ctg ctc acc cgc gtc aag aat atg caa aag gat gta aaa gaa gct ggc gga gcg gag cgt gcg gca gct gag att gaa gcg ttt atg aaa aaa tcc gct gtc ccc cag taa (SEQ ID NO:4)

[0054] In at least one embodiment, the UDP-glycosyltransferase enzyme is encoded by a polynucleotide having a variant nucleotide sequence having at least 70%, at least 75%, atleast 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.

[0055] 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 or 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.

[0056] 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.

[0057] In at least one embodiment, the UDP-glycosyltransferase enzyme is encoded by a polynucleotide having a variant nucleotide sequence in which at least one codon 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 host 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 host cell, such that the variant polynucleotide sequence includes codons which are preferentially used to encode amino acids by that host cell.

[0058] 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.

[0059] As will be understood by the person of skill in the art, in at least one embodiment, exposing the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity comprises exposing the trichothecene mycotoxin to the UDP-glycosyltransferase enzyme in the presence of one or more cofactors or reactants required for the transformation of the trichothecene mycotoxin to a glycosylated product having a toxicity which is less than the toxicity of the trichothecene mycotoxin. In at least one embodiment, the one or more cofactors or reactants comprise UDP-glucose.

[0060] In at least one embodiment, the UDP-glucose is provided, or regenerated as it is consumed, by a system capable of forming, producing or generating UDP-glucose. In at least one embodiment, the system capable of forming, producing or generating UDP-glucose comprises an enzyme active to catalyze the formation of UDP-glucose. Without being bound by theory, it is contemplated that providing such an enzyme in combination with the UDP- glycosyltransferase enzyme can act to generate and / or replenish the UDP-glucose reactant consumed in the glycosylation reaction catalyzed by the UDP-glycosyltransferase enzyme while consuming a substrate which may be more readily available or less expensive than UDP-glucose. In at least one embodiment, the enzyme active to catalyze the formation of UDP-glucose is a sucrose synthase. Again, without being bound by theory, it is noted that sucrose synthase enzymes catalyze the cleavage of sucrose to produce fructose and UDP- glucose. In this way, the required UDP-glucose can be generated and replenished as it is used by consuming sucrose, which can be less expensive, more readily available, and / oreasier and safer to handle than UDP-glucose. In at least one embodiment, the sucrose synthase is a plant sucrose synthase. In at least one embodiment, the sucrose synthase is of a species of Arabidopsis. In at least one embodiment, the species of Arabidopsis is Arabidopsis thaliana. In at least one embodiment, the sucrose synthase is encoded by the SUS1 gene of Arabidopsis thaliana (AtSUS1; NCBI gene ID 832206; reference sequence NP_001031915.1). In at least one embodiment, the sucrose synthase has the sequence represented by SEQ ID NO:6. MANAERMITR VHSQRERLNE TLVSERNEVL ALLSRVEAKG KGILQQNQII AEFEALPEQT RKKLEGGPFF DLLKSTQEAI VLPPWVALAV RPRPGVWEYL RVNLHALVVE ELQPAEFLHF KEELVDGVKN GNFTLELDFE PFNASIPRPT LHKYIGNGVD FLNRHLSAKL FHDKESLLPL LKFLRLHSHQ GKNLMLSEKI QNLNTLQHTL RKAEEYLAEL KSETLYEEFE AKFEEIGLER GWGDNAERVL DMIRLLLDLL EAPDPCTLET FLGRVPMVFN VVILSPHGYF AQDNVLGYPD TGGQVVYILD QVRALEIEML QRIKQQGLNI KPRILILTRL LPDAVGTTCG ERLERVYDSE YCDILRVPFR TEKGIVRKWI SRFEVWPYLE TYTEDAAVEL SKELNGKPDL IIGNYSDGNL VASLLAHKLG VTQCTIAHAL EKTKYPDSDI YWKKLDDKYH FSCQFTADIF AMNHTDFIIT STFQEIAGSK ETVGQYESHT AFTLPGLYRV VHGIDVFDPK FNIVSPGADM SIYFPYTEEK RRLTKFHSEI EELLYSDVEN KEHLCVLKDK KKPILFTMAR LDRVKNLSGL VEWYGKNTRL RELANLVVVG GDRRKESKDN EEKAEMKKMY DLIEEYKLNG QFRWISSQMD RVRNGELYRY ICDTKGAFVQ PALYEAFGLT VVEAMTCGLP TFATCKGGPA EIIVHGKSGF HIDPYHGDQA ADTLADFFTK CKEDPSHWDE ISKGGLQRIE EKYTWQIYSQ RLLTLTGVYG FWKHVSNLDR LEARRYLEMF YALKYRPLAQ AVPLAQDD (SEQ ID NO:6)

[0061] In at least one embodiment, the sucrose synthase has 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:6.

[0062] In at least one embodiment, the protein comprising UDP-glycosyltransferase activity is a fusion protein including a first polypeptide moiety comprising a UDP-glycosyltransferase enzyme as described herein fused to one or more other polypeptides or proteins. 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. As used herein, the term “linker polypeptide” is intended to refer to a polypeptide sequence which can be used to join two polypeptide or proteinmoieties 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, a fusion protein can be encoded by a polynucleotide which can be expressed in a recombinant host as described herein.

[0063] In at least one embodiment, the one or more other polypeptides or proteins include but are not limited to an additional polypeptide moiety which facilitates expression of the fusion protein in a recombinant host cell or which facilitates 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. In at least one embodiment, the additional polypeptide moiety comprises a polyhistidine sequence, also known as a “ is- tag”, as will be understood by a person skilled in the art.

[0064] In at least one embodiment, the one or more other polypeptides or proteins include a second polypeptide moiety comprising an enzyme active to catalyze the formation of UDP- glucose as described herein. In at least one embodiment, the protein comprising UDP- glycosyltransferase activity is a fusion protein comprising a first polypeptide moiety fused to a second polypeptide moiety, wherein the first polypeptide moiety comprises a UDP- glycosyltransferase enzyme as described herein and the second polypeptide moiety comprises an enzyme active to catalyze the formation of UDP-glucose as described herein. In at least one embodiment of such a fusion protein, the first polypeptide moiety is located N-terminally of the second polypeptide moiety. In at least one embodiment of such a fusion protein, the first polypeptide moiety is located C-terminally of the second polypeptide moiety.

[0065] In at least one embodiment, the first polypeptide moiety comprises a UDP-glycosyl- transferase enzyme as described herein and the second polypeptide moiety comprises a sucrose synthase as described herein. In at least one embodiment, the fusion protein further comprises a linker sequence inserted between the first polypeptide moiety and the second polypeptide moieties. In at least one embodiment, the linker sequence comprises the sequence EAAAKEAAAK (SEQ ID NO:7). In at least one embodiment, the fusion protein has a sequence represented by SEQ ID NO:8. MKKYHISMIN IPAYGHVNPT LALVEKLCEK GHRVTYATTE EFAPAVQQAG GEALIYHTSL NIDPKQIREM MEKNDAPLSL LKESLSILPQ LEELYKGDQP DLIIYDFVAL AGKLFAEKLN VPVIKLCSSY AQNESFQLGN EDMLKKIKEA EAEFKAYLEQ EKLPAVSFEQ LAVPEALNIV FMPKSFQIQH ETFDDRFCFV GPSLGERKEQ EGLLIDKDDR PLMLISLGTA FNAWPEFYKM CIKAFRDSSW QVIMSVGKTI DPESLEDIPA NFTIRQSVPQ LEVLEKADLF ISHGGMNSTM EAMNAGVPLV VIPQMYEQEL TANRVDELGL GVYLPKEEVT VSSLQEAVQA VSSDQELLTR VKNMQKDVKE AGGAERAAAE IEAFMKKSAV PQEAAAKEAAAKMANAERMI TRVHSQRERL NETLVSERNE VLALLSRVEA KGKGILQQNQ IIAEFEALPE QTRKKLEGGP FFDLLKSTQE AIVLPPWVAL AVRPRPGVWE YLRVNLHALV VEELQPAEFL HFKEELVDGV KNGNFTLELD FEPFNASIPR PTLHKYIGNG VDFLNRHLSA KLFHDKESLL PLLKFLRLHS HQGKNLMLSE KIQNLNTLQH TLRKAEEYLA ELKSETLYEE FEAKFEEIGL ERGWGDNAER VLDMIRLLLD LLEAPDPCTL ETFLGRVPMV FNVVILSPHG YFAQDNVLGY PDTGGQVVYI LDQVRALEIE MLQRIKQQGL NIKPRILILT RLLPDAVGTT CGERLERVYD SEYCDILRVP FRTEKGIVRK WISRFEVWPY LETYTEDAAV ELSKELNGKP DLIIGNYSDG NLVASLLAHK LGVTQCTIAH ALEKTKYPDS DIYWKKLDDK YHFSCQFTAD IFAMNHTDFI ITSTFQEIAG SKETVGQYES HTAFTLPGLY RVVHGIDVFD PKFNIVSPGA DMSIYFPYTE EKRRLTKFHS EIEELLYSDV ENKEHLCVLK DKKKPILFTM ARLDRVKNLS GLVEWYGKNT RLRELANLVV VGGDRRKESK DNEEKAEMKK MYDLIEEYKL NGQFRWISSQ MDRVRNGELY RYICDTKGAF VQPALYEAFG LTVVEAMTCG LPTFATCKGG PAEIIVHGKS GFHIDPYHGD QAADTLADFF TKCKEDPSHW DEISKGGLQR IEEKYTWQIY SQRLLTLTGV YGFWKHVSNL DRLEARRYLE MFYALKYRPL AQAVPLAQDD (SEQ ID NO:8)

[0066] In at least one embodiment, exposing the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity comprises exposing the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity in an isolated or purified form. A related aspect of the present application provides an isolated or purified protein comprising UDP-glycosyltransferase activity . In at least one embodiment, the isolated or purified protein comprising UDP-glycosyltransferase activity is a recombinant protein comprising UDP-glycosyltransferase activity. In at least one embodiment, the isolated or purified protein comprising UDP-glycosyltransferase activity is at least partially purified. Methods of isolating and purifying the enzyme, either from an organism or microorganism expressing the native enzyme or from an organism or microorganism genetically engineered to express a recombinant form of the enzyme 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 comprising UDP-glycosyltransferase activity may also contain components of the host organism or microorganism expressing the protein comprising UDP- glycosyltransferase activity, 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 comprising UDP-glycosyltransferase activity 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.

[0067] In at least one embodiment, the protein comprising UDP-glycosyltransferase activity can be mixed with one or more carriers to form a composition or formulation active to catalyze the transformation of a trichothecene mycotoxin to a glycosylated product having atoxicity which is less than the toxicity of trichothecene mycotoxin. 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.

[0068] 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.

[0069] 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 recombinant microbe or microbial host cell as described herein can grow or remain viable or can express a protein comprising UDP-glycosyltransferase activity as described herein.

[0070] 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.

[0071] 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 itsbioavailability. 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 (e.g., a bacterial extract, a yeast extract and / or a fungal extract). In at least one embodiment, this further 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 expressing a protein comprising UDP-glycosyltransferase activity as described herein. The microbial host cell can be a recombinant microbial host cell having expressed and optionally accumulated the protein comprising UDP-glycosyl- transferase activity 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.

[0072] In at least one embodiment, exposing the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity comprises exposing the trichothecene mycotoxin to a microbe or microbial host cell expressing the protein comprising UDP- glycosyltransferase activity, including but not limited to a UDP-glycosyltransferase enzyme as described herein and a fusion protein comprising a polypeptide having a sequence of the UDP-glycosyltransferase enzyme fused to a polypeptide having a sequence of an enzyme active to catalyze the formation of UDP-glucose, as described herein. In at least one embodiment, the protein comprising UDP-glycosyltransferase activity is native to the microbe expressing the protein. In at least one such embodiment, the microbe is a strain of Bacillus subtilis expressing the UDP-glycosyltransferase enzyme native to the strain as described herein.

[0073] In at least one embodiment, the microbe is a recombinant microbe or microbial host cell expressing the protein comprising UDP-glycosyltransferase activity which is heterologous to the cell. As used herein, the term “recombinant host cell” is intended to refer to a cell, including but not limited to a microbial cell such as a bacterial, fungal or yeast cell, which has been genetically transformed to express one or more heterologous proteins not native to the cell. In at least one embodiment, the recombinant microbial host cell is a bacterial, fungal or yeast cell which has been genetically altered to express a recombinant form of the protein comprising UDP-glycosyltransferase activity.

[0074] In at least one embodiment, the recombinant microbial host cell is a yeast cell. In at least one embodiment, the recombinant microbial host cell is a Saccharomyces cerevisiae cell. In at least one embodiment, the recombinant microbial host cell is a Pichia pastoris (Komagataella phaffii) cell. In at least one embodiment, the recombinant microbial host cell is a bacterial cell. In at least one embodiment, the recombinant microbial host cell is anEscherichia coli cell. In at least one embodiment, the recombinant microbial host cell is a cell of a species of Bacillus. In at least one embodiment, the recombinant microbial host cell is a Bacillus subtilis cell. In at least one embodiment, the recombinant microbial host cell is a cell of a species of Lactobacillus.

[0075] A related aspect of the present application provides a recombinant microbial host cell genetically altered to express the protein comprising UDP-glycosyltransferase activity as described herein. Methods of preparing recombinant forms of the protein comprising UDP- glycosyltransferase activity and of genetically engineering a recombinant microbial host cell to express such a recombinant form of the protein are well known in the art and would be apparent to the skilled person in view of the teaching of the present application.

[0076] In at least one embodiment, exposing the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity comprises exposing the trichothecene mycotoxin to a composition comprising a carrier as described herein and a microbe or microbial host cell expressing the protein comprising UDP-glycosyltransferase activity as described herein or an autolysate thereof or an extract thereof, wherein the autolysate or extract comprises a protein comprising UDP-glycosyltransferase activity as described herein expressed by the microbe or microbial host cell. In at least one embodiment, the protein comprising UDP-glycosyltransferase activity is native to the microbe or microbial host cell. In at least one embodiment, the microbial host cell is a recombinant microbial host cell and the protein comprising UDP-glycosyltransferase activity is heterologous to the recombinant microbial host cell.

[0077] In at least one embodiment, the microbial host cell is a bacterial host cell as described herein. In at least one embodiment, the bacterial host cell is an Escherichia coli cell, a cell of a species of Bacillus or a cell of a species of Lactobacillus. In at least one embodiment, the microbial host cell is a fungal host cell. In at least one embodiment, the fungal host cell is a yeast host cell as described herein. In at least one embodiment, the yeast host cell is a cell of a species of Saccharomyces or a cell of a species of Komagataella.

[0078] As used herein, the term “extract” when used in relation to a microbe or microbial host cell expressing the protein comprising UDP-glycosyltransferase activity as described herein, is intended to mean a material comprising the expressed protein comprising UDP- glycosyltransferase activity in which the expressed protein comprising UDP-glycosyl- transferase activity has been at least partially separated from other components of the microbe or microbial host cell, or from other components of the medium containing the microbe or microbial host cell.

[0079] In at least one embodiment, the expressed protein comprising UDP-glycosyl- transferase activity may be located inside the host cell after expression. In such embodiments, preparation of an extract comprising the expressed protein comprising UDP- glycosyltransferase activity may require lysis of the host 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 host cell or a lysis product thereof can be homogenized using techniques including but not limited to bead-milling, bead-beating or high-pressure homogenization.

[0080] In at least one embodiment, the expressed protein may be bound to one or more components of the microbial host cell or of a lysis product thereof. In such embodiments, preparation of an extract comprising the expressed protein comprising UDP-glycosyl- transferase activity may require further treatment of the host 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 host cell to which the expressed enzyme may be bound. In at least one alternative embodiment, the extract can contain components of the host 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 microbial host cell or lysis product thereof or from other components of the medium containing the microbial host cell or lysis product thereof. In at least one such embodiment, the extract can comprise an inactivated fraction obtained by inactivating the propagated microbial host cell.

[0081] In at least one embodiment, the expressed protein can be secreted by the host cell as a soluble protein and can be isolated or recovered from the medium to which the host 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.

[0082] In at least one embodiment, the composition or extract thereof can be prepared by a process including propagating the microbial host cell expressing the protein comprising UDP-glycosyltransferase activity as described herein. In at least one embodiment, propagation of the microbial host cell is carried out in a culture medium under conditions facilitating expression and accumulation of the protein comprising UDP-glycosyltransferase activity. 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 host cell in view of the teaching of the present specification. In at least one embodiment when the microbial host cell is a yeast host cell, the culture mediumcomprises molasses. In at least one embodiment when the microbial host cell is a yeast host cell or a bacterial host cell, the culture medium comprises a yeast extract.

[0083] In at least one embodiment, the process further comprises separating the protein comprising UDP-glycosyltransferase activity from at least one component of the propagated microbial host cell to obtain an extract enriched in the protein comprising UDP-glycosyl- transferase activity. In embodiments where the protein comprising UDP-glycosyltransferase activity is secreted in a soluble form by the microbial host cell, separating the protein comprising UDP-glycosyltransferase activity from at least one component of the propagated microbial host cell can include, but is not limited to, filtering or centrifuging the mixture containing the propagated microbial host cell to separate insoluble cell components, including but not limited to cell wall and membrane components, from the medium in which the protein comprising UDP-glycosyltransferase activity is soluble.

[0084] In at least one embodiment, the process further comprises lysing the propagated microbial host cell to obtain a lysed fraction. Such embodiments of the process can be useful when the protein comprising UDP-glycosyltransferase activity is not secreted in a soluble form by the microbial host cell but remains inside or bound to the host cell after expression. In at least one such embodiment, an extract comprising the protein comprising UDP- glycosyltransferase activity can be separated from at least one undesired component of the lysed propagated microbial host 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 comprising UDP- glycosyltransferase activity.

[0085] In at least one embodiment, the process further comprises drying, at least in part, the propagated microbial host 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 comprising UDP-glycosyl- transferase activity from the extract, the inactivated fraction, the lysed fraction or the dried fraction to obtain a substantially purified extract. In at least one embodiment, the process comprises formulating the propagated microbial host cell, the extract, the inactivated fraction, the lysed fraction, the dried extract, or the substantially purified extract into a composition as described herein.

[0086] In at least one embodiment, the composition can include the propagated microbial host cells, including but not limited to yeast host cells and bacterial host cells, in an active or semi-active form, such that the host cells remain viable and capable of expressing the protein comprising UDP-glycosyltransferase activity. In such embodiments, the process forpreparing the composition can comprise a concentrating step, in which a portion of the propagation medium can be removed from the propagated host 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.

[0087] In at least one embodiment, the composition comprises an autolysate prepared by autolysis of the propagated microbial host cells. In at least one embodiment, when the microbial host cell is a yeast host cell, including but not limited to a recombinant yeast host cell, the autolysis can include subjecting the propagated yeast host 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 host 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 comprising UDP-glycosyltransferase activity.

[0088] In at least one embodiment, the propagated yeast host 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 host 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 host 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.

[0089] In at least one embodiment, the propagated yeast host 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 host 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 host 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.

[0090] 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 comprising UDP-glycosyl-transferase activity from other components of the autolysate. In at least one embodiment, the autolysate is formulated into the composition as described herein.

[0091] In at least one embodiment, exposing the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity comprises exposing a material contaminated with or at risk of being contaminated with the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity as described herein, to a recombinant microbe expressing the protein comprising UDP-glycosyltransferase activity as described herein, or to a composition as described herein. As used herein, the expression “contaminated with the trichothecene mycotoxin” is intended to refer to a material containing a level of the trichothecene 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 trichothecene mycotoxin” is intended to refer to a material which is not necessarily known to be contaminated with the trichothecene mycotoxin but which is suspected of being contaminated with the trichothecene mycotoxin or is in a context or situation where contamination with the trichothecene mycotoxin can be expected to occur. In at least one embodiment where the trichothecene mycotoxin is deoxynivalenol (DON), materials containing at least 5 ppm of DON which are contemplated for use in animal feed can be considered to be contaminated with DON. In at least one such embodiment, materials containing at least 1 ppm of DON which are contemplated for use in food for human consumption can be considered to be contaminated with DON. The person of skill in the art would be aware of other standards set by advisory or regulatory authorities setting levels of a trichothecene mycotoxin at which material containing the set levels would be considered contaminated.

[0092] In at least one embodiment, the composition further contains at least one further enzyme active to reduce the toxicity of one or more mycotoxins. Such embodiments may be advantageous for use in cases where a material to be treated is further contaminated with a mycotoxin or type of mycotoxin other than a trichothecene mycotoxin.

[0093] In at least one embodiment, the material contaminated with or at risk of being contaminated with the trichothecene 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, for example, 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.

[0094] In at least one embodiment, exposing 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 trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity 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.

[0095] In at least one embodiment, the material contaminated with the trichothecene 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 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 the trichothecene mycotoxin and, upon determining that the feedstock is contaminated, exposing the feedstock to the protein comprising UDP-glycosyltransferase activity or to a recombinant microbial host cell expressing the protein comprising UDP-glycosyltransferase activity. In at least one embodiment, the yeast strain which is active to ferment the feedstock is also the recombinant microbial host cell expressing the protein comprising UDP-glycosyltransferase activity. 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 Pichia pastoris(Komagataella phaffii). In at least one such embodiment, exposure to the yeast strain occurs during fermentation of the contaminated feedstock. In at least one embodiment, the recombinant microbial host cell expressing the protein comprising UDP-glycosyltransferase activity is a bacterial strain expressing the protein comprising UDP-glycosyltransferase activity. In at least one such embodiment, the bacterial host cell is a cell of a strain of a species of Bacillus or a cell of a strain of a species of Lactobacillus.

[0096] 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 trichothecene mycotoxin compared to the level of the trichothecene mycotoxin in distillers’ products obtained from a fermentation process in which the contaminated feedstock is not exposed to the protein comprising UDP-glycosyltransferase activity. 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.

[0097] In at least one embodiment, exposing the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity as described herein comprises exposing a crop contaminated with or at risk of being contaminated with the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity as described herein or to a 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 trichothecene 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 trichothecene mycotoxin is being grown in a field or in a greenhouse.

[0098] Another aspect of the present application provides a plant genetically altered to express a protein comprising UDP-glycosyltransferase activity 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 suchcrops will experience reduced contamination with a trichothecene mycotoxin even if exposed to Fusarium species producing the trichothecene mycotoxin during growth.

[0099] As used herein, the terms “a” and “an” are intended to mean “at least one”, and include both singular and plural, unless otherwise indicated.

[0100] 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 value or 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.

[0101] 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.

[0102] 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.

[0103] As used herein, terms indicating relative direction or orientation, including but not limited to “upper”, “lower”, “top”, “bottom”, “vertical”, “horizontal”, “outer”, “inner”, “front”, “back”, and the like, are intended to facilitate description of the present invention byindicating relative orientation or direction in usual use or as illustrated, and are not intended to limit the scope of the present invention in any way to such orientations or directions. EXAMPLES

[0104] 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: Identification of DON-glycosylation activity Microbial culturing and Isolation

[0105] Soil samples from fields in southwestern Ontario were collected in late fall of 2018, approximately 1 month after highly deoxynivalenol (DON) contaminated maize crops were plowed into the ground. Core samples were obtained using a soil sampler, placed in sealed plastic bags, and stored at −20°C prior to microbiological extraction. Nutrient Broth (NB), Luria-Bertani (LB) broth, Malt Extract Broth (MEB), and Yeast Extract Sucrose Broth (YES) (Millipore Sigma, St. Louis, MO) were used for bacterial enrichment. Soil samples (0.5 g) were incubated with 2.5 mL of culture media for one week. All cultures were incubated at 28°C and shaken at 200 rpm. Subsequently, 100 µL of the incubated cultures were transferred into fresh media each week for a total of 4 weeks. Cultures were incubated either aerobically with a foam cap or in an aerobic to anaerobic state with closed caps.300 µM DON (Triple Bond, Guelph, Canada) was added to each culture tube every week. After 4 weeks, 100 µL of each sample was quenched with 900 µL methanol, filtered using 0.45 µm PTFE syringe filter, and DON biotransformation assessed using LC-MS analysis as described below. Mixed bacterial cultures that displayed DON degradation capabilities were serially diluted and plated onto their respective media agar plates. Single colonies were inoculated into liquid media, incubated for one week at 28°C, and samples tested for DON degradation. LC-MS analysis for identification of DON glycosylation activity

[0106] Glycosylation activity was monitored using high resolution MS (HRMS) on a Q-Exactive™ Orbitrap™ mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with a heated electrospray ionization (HESI) source coupled to a 1290 Infinity™ ultra-high- performance liquid chromatography (UHPLC) system (Agilent). The samples were analyzed in negative ionization mode with DON being monitored as the formate adduct [DON+HCOO−] at m / z 341.1242. Singly glycosylated DON metabolites were screened as either the [M-H]−or [M+HCOO−] adducts at m / z 457.1715 and 503.1770, respectively. Chromatographic separation was performed using a Zorbax™ Eclipse Plus™ rapid resolution high definition (RRHD) C-18 column (2.1 × 50 mm, 1.8 µm; Agilent) maintained at 35°C with water with 0.1% formic acid (mobile phase A), and acetonitrile with 0.1% formicacid (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 30s and finally held at 0% B for one minute. For each sample, 5 µL was injected. HESI conditions were as follows: capillary temperature, 400°C; sheath gas 17 units; auxiliary gas, 8 units; probe heater temperature, 400°C or 450°C; S-Lens RF level, 45; capillary voltage 3.5 kV or 4.0 kV.

[0107] All samples were analyzed using a top 5, data-dependent acquisition method. The full scan was acquired between a mass range of m / z 175–900 at 35,000 resolution with an automatic gain control (AGC) of 3×106and a maximum injection time of 128 msec, or at 17,500 resolution with an AGC of 5×106and a maximum injection time of 64 msec. The MS / MS scans, including of the five highest intensity scans from the full scan (excluding isotopes), were acquired at 17,500 resolution, AGC of 1×105, maximum IT of 50 msec, intensity threshold of 4×103, normalized collision energy of 22 and an isolation window of 1.2 m / z or at maximum IT of 64 ms, intensity threshold of 9.1×104, normalized collision energy of 30 and dynamic exclusion of 1.5 s. All data was analyzed using Thermo Xcalibur™ Qual Browser software.

[0108] One isolated bacterial colony with an ability to reduce the concentration of DON in culture was identified and three polar transformation products were observed in samples taken from the bacterial culture and analyzed by LC-MS as described above. As seen from the data presented in Figure 1A, two transformation products had identical [M+HCOO−] pseudo-molecular ions at m / z 503.18 that eluted at 2.16 mins and 2.26 mins respectively, and a third [M+HCOO−] peak at m / z 665.23 that eluted at 2.08 mins. The increased mass at m / z 503.18 compared to intact [DON+HCOO−] corresponds to the addition of a single glucose moiety to the toxin (C6H12O6 -H2 ; Δ = 62.05 Da), while the product at m / z 665.23 corresponds to the addition of two glucose moieties. As seen from the data presented in Figure 1B, over a four-day growth of the bacterium in the presence of DON, gradual increases in the optical density at 600 nm (OD600) of the culture and a corresponding increase in the presence of the mono- (DON+1Glu) and di-glycosylated (DON+2Glu) products were observed, while the amount of unreacted DON decreased. Example 2: Bacterial identification and sequencing

[0109] Bacteria were identified through 16S ribosomal RNA Sanger sequencing. Genomic DNA was extracted using the DNeasy™ UltraClean™ Microbial Kit (QIAGEN). PCR amplification of the 16S gene was performed using the primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′; SEQ ID NO:1) and 1492R (5′-GGTTACCTTGTTACGACTT-3’, SEQ ID NO:2). The PCR reaction occurred as follows: 95°C for 3 min; 30 cycles of 95°C for 1 minute, 55°C for 30 seconds, 72°C for 1 minute; andfinally 72°C for 5 minutes. The PCR product was purified using the PureLink™ Quick PCR Purification Kit and sequenced. Genomic DNA was subjected to whole genome sequencing and assembly. The obtained genome was converted into a proteome FASTA format for subsequent proteomics analysis.

[0110] Complete sequencing of the 16S rRNA gene of the isolated bacterium indicated it was 99.94% identical to the 16S rRNA gene from Bacillus subtilis subsp. subtilis strain 168 (NCBI Reference Sequence: NC_000964.3), with only 1 base pair difference between the two genes across their entire lengths. The present isolated strain is therefore referred to herein as Bacillus subtilis_AAFC1 or B. subtilis strain AAFC1. The complete genome of B. subtilis_AAFC1 was sequenced and assembled in order to enable downstream proteomics analysis. The total genome length was 4,081,710 bp, consisting of one long (4,060,208 bp) and one short (21,502 bp) contig. The GC content of the genome was 43.9%. Example 3: Biochemical enrichment of DON glycosylation activity

[0111] All fractions obtained were mixed with 1 mM DON and 10 mM UDP-glucose and allowed to incubate overnight at room temperature prior to LC-MS analysis as described in Example 1 to test for DON glycosylation activity. B. subtilis_AAFC1 was inoculated into 25 mL of NB media and incubated at 37°C for 24 hours with shaking. Afterwards, the culture was inoculated into 1 L of NB media and grown at 37°C for 24 hours with shaking. The culture was centrifuged and the pellet resuspended in 50 mM 4-(2-hydroxyethyl)piperazine- 1-ethanesulfonic acid (HEPES) (pH 7.0), 150 mM NaCl, 1.43 mM β-mercaptoethanol, 2 mM ethylenediaminetetraacetic acid (EDTA), and 0.2 mg / mL lysozyme. The re-suspended pellet was lysed by sonication for a total of 2.5 minutes, and the lysate supernatant was clarified via centrifugation. As seen from the data presented in Figure 2A, the majority of DON glycosylation activity was soluble and found to be present within the lysate supernatant of the cultured bacterium.

[0112] The lysate supernatant was then subjected to a stepwise 30%, 30%-60%, and a 60%-90% (w:v) (NH4)2SO4 precipitation. Precipitated pellets were resuspended in 50 mM HEPES (pH 7.0) and dialyzed against 50 mM HEPES (pH 7.0) at 4°C overnight. As seen in Figure 2A, DON glycosylation activity was significantly enriched in the precipitated pellet following a 60%-90% (w:v) cut. The most active fraction was loaded onto a 1 mL HiTrap™ Q HP Column (GE Healthcare) equilibrated in 50 mM HEPES (pH 7.0), and 50 mM NaCl (Buffer A). Protein was eluted using a linear 50 - 1000 mM NaCl gradient in Buffer A over 10 column volumes (not displayed in Figure 2B). As seen in Figure 2B, DON glycosylation activity eluted discretely off the column at ca.400 mM NaCl. Fractions displaying DON- glycosylation activity were pooled and loaded onto an ENrich™ size exclusion chromatography (SEC) 650 column (BIO-RAD) equilibrated in 50 mM HEPES, and 150 mMNaCl (pH 7.0). DON glycosylation activity eluted discretely, located primarily in the shoulder of the main elution peak of the run, as seen in Figure 2C. Fractions displaying activity were pooled and dialyzed against 50 mM ammonium bicarbonate (pH 8.0). Example 4: Proteomics analysis

[0113] Dialyzed fractions showing glycosylation activity obtained from the procedure of Example 3 following size exclusion chromatography were subjected to LC-MS / MS proteomics analysis to identify candidate glycosylation enzymes. Thus, 5 mM dithiothreitol (DTT) was added to 200 µL aliquots of purified protein fractions and the mixtures were incubated at 60°C for 30 min. The reduced proteins were alkylated at room temperature in darkness using iodoacetamide at a concentration of 10 mM for 15 min, and additional DTT was added to a final concentration of 10 mM. The proteins were digested by incubating with 200 ng of trypsin (Thermo Scientific Pierce sequencing grade, Rockford, IL, USA) overnight at 34°C. The digestion was quenched by adding formic acid to a final concentration of 0.1%. The tryptic peptides were then passed through 1 ml Waters Oasis™ hydrophilic-lipophilic balance (HLB) solid phase extraction (SPE) cartridges containing 30 mg sorbent (Milford, MA, USA), which were activated with methanol and preconditioned with LC-MS grade H2O containing 0.1% formic acid. The cartridges were dried under vacuum for five minutes and the peptides were then eluted into fresh .5 ml microcentrifuge tubes by addition of 00 μl of 70% acetonitrile. The samples were subsequently dried by vacuum centrifugation. The samples were then reconstituted in 200 μl of water / acetonitrile / formic acid (95 / .9 / 0.1) and transferred to 250 µL polypropylene HPLC vials.

[0114] The purified peptide digests were separated using an Easy-nLC™ 000 nanoflow HPLC system fitted with a 2 cm Acclaim™ C18 PepMap™ trap column and a 75 µm x 25 cm Acclaim™ C18 PepMap™ analytical column (Thermo Scientific). Theflow rate was held at 300 nL min-1throughout the run and 10 µL of the digest was injected. The mobile phase A (LC / MS Optima™ water, 0.1% formic acid) began at 97% and was decreased to 90% over 4 min. Peptides were then eluted with a linear gradient of 10% to 40% mobile phase B (LC / MS Optima™ acetonitrile, 0.1% formic acid) over 150 min, followed by 40-90% over 10 min, and maintained constant for an additional 10 min. Each sample was then analyzed using a top 10, data-dependent acquisition method using a Thermo Q-Exactive™ Orbitrap™ mass spectrometer. The nanospray voltage was set at 2.8 kV, capillary temperature at 275°C, and the S-lens radio frequency (RF) level at 70. The full scan was acquired between a mass range of m / z 340-1800 at 70,000 resolution with an automatic gain control (AGC) of 1×106and a maximum injection time of 256 msec. The MS / MS scans were acquired at 17500 resolution, AGC of 1×106, maximum IT of 110 msec, intensity threshold of 2×105, normalized collision energy of 30 and an isolation window of 2 m / z. Unassigned, singlycharged, and >5 charged peptides were not selected for MS / MS, and a 15 sec dynamic exclusion was used.

[0115] The proteomic data was searched against the sequenced genome of Bacillus subtilis_AAFC1 as described in Example 2. Thermo .raw files were converted to .mgf with ProteoWizard v2 software and MS / MS scans were searched against the target proteome using the X! Tandem search algorithm operated from the SearchGUI v.3.3.3 interface and processed in PeptideShaker v1.16.26 software. A 3 ppm precursor ion mass error and a 0.02 Da product ion error were used along with oxidation of methionine as a variable modification. A 1% false discovery (FDR) rate was used at the protein, peptide, and peptide spectrum match level.

[0116] One of the top hits from the proteomic analysis as measured by spectral counts was to a putative UDP-glycosyltransferase (herein identified as Protein A), wherein 37 validated and unique peptides were observed that corresponded to 82% coverage of the entire enzyme. BLASTp searches against the National Center for Biotechnology Information (NCBI) nr (non-redundant) protein database indicated that Protein A was highly similar to YjiC, a 392 amino acid Bacillus UDP-glucosyltransferase capable of glycosylating a diverse array of plant secondary metabolites and xenobiotics. In particular, Protein A contained five conserved amino acid substitutions (Asp97Gly, Arg148Lys, Lys210Gln, Ser212Gly, and Ser359Thr) compared to the YjiC homolog found in B. subtilis strain 168 (NCBI Reference Sequence: NP_389104.1).

[0117] The amino acid sequence of Protein A is shown below: MKKYHISMIN IPAYGHVNPT LALVEKLCEK GHRVTYATTE EFAPAVQQAG GEALIYHTSL NIDPKQIREM MEKNDAPLSL LKESLSILPQ LEELYKGDQP DLIIYDFVAL AGKLFAEKLN VPVIKLCSSY AQNESFQLGN EDMLKKIKEA EAEFKAYLEQ EKLPAVSFEQ LAVPEALNIV FMPKSFQIQH ETFDDRFCFV GPSLGERKEQ EGLLIDKDDR PLMLISLGTA FNAWPEFYKM CIKAFRDSSW QVIMSVGKTI DPESLEDIPA NFTIRQSVPQ LEVLEKADLF ISHGGMNSTM EAMNAGVPLV VIPQMYEQEL TANRVDELGL GVYLPKEEVT VSSLQEAVQA VSSDQELLTR VKNMQKDVKE AGGAERAAAE IEAFMKKSAV PQ (SEQ ID NO:3)

[0118] The nucleotide sequence of the gene encoding Protein A is shown below: atg aaa aag tac cat att tcg atg atc aat atc ccg gcg tac gga cat gtc aat cct acg ctt gct tta gta gag aag ctt tgt gag aaa ggg cac cgt gtc acg tac gcg acg act gag gag ttt gcg ccc gct gtt cag caa gcc ggt gga gaa gca ttg atc tat cat aca tcc ttg aat att gat cct aag caa atc agg gag atg atg gaa aag aat gac gcg ccc ctc agc ctt ttg aaa gaa tca ctc agc att ctg ccg cag ctt gag gag tta tat aag ggt gat cag cct gat ctg atc atc tat gac ttt gtt gcg ctg gct ggt aaa ttg ttt gct gaa aag ctt aat gtt ccg gtc att aag ctc tgt tcg tca tat gcc caa aat gaa tcc ttt cag tta gga aat gaa gac atg ctg aag aaa ata aaa gaa gcagag gct gaa ttt aaa gcc tac ttg gag caa gag aag ttg ccg gct gtt tca ttt gaa cag tta gct gtg ccg gaa gca tta aat att gtc ttt atg ccg aag tct ttt cag att cag cat gag acg ttc gat gac cgt ttc tgt ttt gtc ggc ccc tct ctc gga gaa cga aag gaa caa gaa ggc ctg ttg att gac aag gat gat cgc ccg ctt atg ctg att tct ttg ggt acg gcg ttt aac gca tgg ccg gaa ttt tac aag atg tgc atc aag gca ttt cgg gat tct tca tgg caa gtg atc atg tcg gtt ggg aaa acg att gat cca gaa agc ttg gag gat att cct gct aac ttt act att cgc caa agt gtg ccg cag ctt gag gtg tta gag aaa gct gat ttg ttc atc tct cat ggc ggg atg aac agt acg atg gaa gcg atg aac gca ggt gtg ccg ctt gtc gtc att ccg caa atg tat gag caa gag ctc act gca aat cgg gtt gat gaa tta ggc ctt ggc gtt tat ttg ccg aaa gag gaa gtg act gtt tcc agc ctg cag gaa gcg gtt cag gct gta tcc agt gat caa gag ctg ctc acc cgc gtc aag aat atg caa aag gat gta aaa gaa gct ggc gga gcg gag cgt gcg gca gct gag att gaa gcg ttt atg aaa aaa tcc gct gtc ccc cag taa (SEQ ID NO:4) Example 5: Recombinant protein production and purification

[0119] All recombinant genes expressed in this study were codon optimized for expression in E. coli. Optimized genes were directly cloned into either pET-28a(+) for expression as N-terminally 6x His-tagged proteins or into pET His6-MBP TEV LIC cloning vector (Addgene plasmid #29653) to produce N-terminally 6x His-tagged maltose-binding protein (MBP) fusion proteins. Constructs were overexpressed in Escherichia coli BL21(DE3) and the resultant proteins were extracted in 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, and 5 mM imidazole. Clarified extracts were subsequently purified by immobilized metal affinity chromatography and gel permeation chromatography as previously described (Garnham, C.P. et al, J Agric Food Chem (2020), 68: 13779-13790). Proteins were separated via gel permeation chromatography using a HiLoad 16 / 600 Superdex 200™ prep grade column (Cytiva, USA) equilibrated in 50 mM Tris-Cl (pH 8.0), and 150 mM NaCl. Purified recombinant glycosyltransferases, with and without MBP fusions, were employed and compared in subsequent enzyme activity assays.

[0120] Protein A, identified from the analysis described in Example 4, was synthesized and cloned as a 6X-His-tagged N-terminal MBP fusion protein and was over-expressed in E. coli as described above. Protein A was purified to homogeneity using a combination of Ni-NTA metal affinity and gel permeation chromatography steps as described above. As seen from the results presented in Figure 3A, the protein eluted as an apparent monomer and was >99% pure as evidenced by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0121] Following purification, Protein A was incubated with DON and UDP-glucose as described in Example 3 and the reaction was analyzed by LC-MS. As seen from the resultspresented in Figure 3B, Protein A generated two mono-glycosylated product (DON+1Glu) ions ([M-H]−) at m / z = 503.18 that eluted at 2.16 and 2.26 minutes respectively, and one di- glycosylated (DON+2Glu) product ion ([M-H]−) at m / z = 665.23 that eluted at 2.08 minutes. The m / z values and retention times of the Protein A-catalyzed DON glycosylation products match those of the products observed from incubation of the B. subtilis_AAFC1 culture with DON and UDP-glucose (Figure 1A).

[0122] Alternatively, the gene for Protein A was synthesized and cloned into the NdeI / XhoI restriction cut sites of the pET28a expression vector and the vector was transformed into E. coli BL21(DE3), plated on LB-Agar containing 50 μg / mL kanamycin and incubated overnight at 37°C. One colony from the LB-agar plate was inoculated into 20 mL of LB (“Luria-Bertani” or “lysogeny broth”) medium containing 50 μg / mL kanamycin and incubated with shaking for 18 hrs. The following morning, 500 μL of the overnight culture was added to 20 mL of fresh LB-kanamycin broth and grown to an optical density (600 nm) of 0.8.2 ml aliquots of this culture were then transferred to 15 mL snap-cap culture tubes containing 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) and 30 ppm deoxynivalenol (DON) and grown for 18 h at either 37°C or 16°C to prepare induced cultures. Non-induced controls contained no added IPTG. Cultures were then subjected to centrifugation at 20,000 x g and the supernatant was transferred to a fresh tube. The centrifugation step was repeated once more and 100 μL of this supernatant was mixed with 200 μL 100% methanol. The cell pellets were then resuspended and washed 2x with 250 μL of 50 mM Tris pH 8.0, 100 mM NaCl and lysed by sonication.250 μL of 100% methanol was then added to each lysate. All samples (cell pellets and supernatants) were then centrifuged at 20,000 x g to remove cell debris and precipitated proteins prior to analysis of 5 μL portions of each sample by LC-MS as described in Example 1. Recombinant expression of Protein A in induced samples was confirmed by analyzing 20 μL of each cell pellet lysate by SDS-PAGE.

[0123] As can be seen from the results presented in Figure 3C, E. coli expressing Protein A intracellularly successfully converted DON into DON+1Glu. Following IPTG induction and incubation with 30 ppm DON overnight at 37°C, approximately 86% of the original DON was eliminated from the culture supernatant. A concomitant large increase in the signal of DON+1Glu appeared in the culture supernatant that was not present in the absence of Protein A expression. A minor amount of DON-3-G was also observed. With no Protein A expression, DON levels remained unchanged, and minimal DON+1Glu levels were observed via LC-MS. Similar observations were made after overnight incubation at 16°C. Example 6: Glycosyltransferase enzymatic activity

[0124] The steady state kinetics of the recombinant glycosyltransferases produced in E. coli were assessed using a coupled-continuous enzymatic assay modified from Wetterhorn,K.M., et al, Biochemistry (2017), 56(50): 6585-6596. Components of the reactions were divided into two parts and prepared as separate master mixes as described below. The two components were combined immediately before the reactions were incubated and measurements taken. The concentrations of each component in the master mixes below represent the final concentrations in the combined reaction. The first master mix consisted of 2 units of pyruvate kinase with 3 units of lactic dehydrogenase (Millipore Sigma, buffered aqueous glycerol solution), 1.5 mM phosphoenolpyruvate, 5 mM UDP-glucose, 0.5 mM β-NADH, 100 mM Tris-Cl (pH 8.0), and 100 nM of the glycosyltransferase being tested. The second master mix consisted of 50 mM KCl, 10 mM MnCl2, and deoxynivalenol (DON) ranging in concentration from 0 to 5.0 mM. The two component master mixes were combined for each reaction in 96-well plates and placed into a plate reader at 23°C. The progress of the reactions was monitored by the decrease in absorbance at 340 nm that resulted from the oxidation of β-NADH. Readings were taken every 2 minutes. The initial reaction rates were calculated for each of the concentrations of DON used with each of the glycosyltransferases. The rates were subsequently plotted against the concentration of DON using SigmaPlot and fit by non-linear regression with one-step saturation to obtain the Michaelis-Menten steady state kinetics values.

[0125] Values for KM and kcat were determined as 2.19 mM and 1.60 s-1respectively for Protein A towards DON. A purified recombinant form of a known isoform of YjiC from B. subtilis strain 168 was determined to have KM and kcat values of 2.16 mM and 1.42 s-1, respectively, using DON as substrate. These values closely approximate those of Protein A, indicating that the five amino acid differences between the two enzymes have a limited effect on activity towards DON. Example 7: Structural characterization of the major DON monoglycosylation product (DON+1Glu)

[0126] In order to determine the chemical structure of the predominant monoglycosylated DON product (DON+1Glu) produced by Protein A, its properties were compared with those of DON-3-glucoside (D3G) and DON-15-glucoside (D15G), whose chemical structures are shown below:D3G D15GPurification of DON+1Glu via HPLC

[0127] Recombinant Protein A (Example 5) was incubated with DON and UDP-glucose as described in Example 3 and the resulting mixture was purified by reverse-phase HPLC, performed on an Agilent 1200 system (CA, USA). An Eclipse XDB-C18 column (9.4 x 250 mm, 5 µm; Agilent Technologies) maintained at 35°C was used, with a mobile phase consisting of HPLC-grade water with 0.1% trifluoroacetic acid (TFA) (v / v) (A) and acetonitrile with 0.1% TFA (v / v) (B) (HPLC-grade; Sigma-Aldrich). The injection volume and flow rate were 100 µL and 4 mL / min, respectively. The analytes were monitored and collected based on absorbance signals at 254 nm. The collected fractions were tested for the presence of glycosylated DON products by LC-MS as described in Example 1 and the fraction containing DON+1Glu was dried down under a gentle stream of nitrogen. Synthesis of DON-15-glucoside (D15G)

[0128] DON-15-glucoside was prepared synthetically via a Koenigs-Knorr reaction as previously described (Fruhmann, P., et al, World Mycotoxin Journal (2012), 5(2): 127-132) for the production of DON-glucuronides using 3-acetyl-DON and acetobromo-α-D-glucose (Millipore Sigma, St Louis, MO) in place of DON and acetobromo-α-D-glucuronic acid methyl ester, respectively. After 144 h of reaction, 1 mL of the reaction supernatant was transferred to a 8 mL glass scintillation vial, dried under N2 and reconstituted in 1 mL 80% acetonitrile. The product was deacetylated by adding 5 µL of 6M KOH and incubating for 1 hour.10 µL of the basic solution containing the deacetylated product was transferred to a 2 mL amber glass HPLC vial containing 990 µL of 20% acetonitrile and analyzed by LC-MS / MS. LC-MS analysis of DON monoglucosides

[0129] The predominant monoglycosylated DON product (DON+1Glu) produced by Protein A and purified by reverse-phase LC as described above, commercially obtained DON-3- glucoside (D3G, Romer Labs; BC, Canada) and DON-15-glucoside (D15G) synthesized as described above were analyzed using targeted high-resolution LC-MS / MS as described in Example 1 with some modifications. The different glycosylated products were resolved chromatographically using a EclipsePlus™ RRHD C-18 column (2.1 × 150 mm, 1.8 µm; Agilent) maintained at 35°C. Mobile phase A was held at 100% for 45 s after which mobile phase B was increased to 15% over 30 s. Mobile phase B was then gradually increased from 15% to 32% over 5.75 min to obtain optimal resolution of the glycosylation products. Mobile phase B was increased to 100% over 1 min and held for 2.5 min before returning to 0% over 30 s. MS / MS was performed in negative ionization mode on both the [M-H]−or [M+HCOO−] adducts at m / z 457.1715 and 503.1770 respectively at a normalized collision energy of 25, 17500 resolution, AGC of 3×106, maximum IT of 64 msec and an isolation window of 1.2 m / z.NMR analysis

[0130] NMR spectra of HPLC purified metabolites (5.0 mg) were recorded with a 400 MHz JEOL ECZS Spectrometer (Peabody, MA) with an autotuning probe. The purified metabolites were dissolved in CD3OD (CDN Isotopes, Pointe-Claire, Quebec) and referenced to the appropriate solvent peak (δH3.31 and δC49.0). Structural elucidation of the major monoglycosylated DON product (DON+1Glu)

[0131] As seen from the data presented in Figure 4, the LC chromatogram (left) and mass spectrum (right) of the major Protein A-derived DON-glycosylation product (DON+1Glu, panel (a)) differ significantly in both retention time and fragmentation pattern from that of both D3G (panel (b)) and D15G (panel (c)). DON+1Glu elutes at 4.06 minutes and is thus found to be more polar than either of D3G, which elutes at 4.29 min, and D15G, which elutes at 4.32 minutes. Furthermore, the mass spectrum of D3G shows a significant peak due to a fragment ion at m / z 427.16, which is attributed to loss of the C-15 CH2O group. This peak is seen only to a very small extent in the mass spectrum of DON+1Glu.

[0132] The glycosylation product DON+1Glu was studied further by1H and13C NMR, and the data from this analysis is presented in Table 1 below. Table 1:1H (400 MHz;) and13C (100 MHz) NMR data for DON+1Glu in CD3OD Compound 1 Compound 2 Position δC,type δH(J in Hz) δC,type δH(J in Hz) 2 82.6, CH 3.43, d (4.6) 82.1, CH 3.49, d (4.6) 3 70.0, CH 4.38, dt (11.1, 4.6) 69.7, CH 4.38, dt (11.1, 4.6) 1.94, dd (14.4, 11.1) 2.32, dd (14.5, 4.2) 4 45.2, CH2 45.1, CH2 1.70, dd (14.4, 4.5) 1.94, dd (14.4, 4.5) 5 45.0, C 47.2, C 6 54.9, C 54.1, C 7 83.6, CH 4.16, s 78.7 CH 5.12, s 8 106.5, C 201.3, C 9 143.2, C 137.6, C 10 122.9, CH 5.44, dd (4.1, 1.8) 139.0, CH 6.56, dd (5.7, 1.6) 11 77.6, CH 4.78, dd (4.1, 1.8) 72.5, CH 4.95, d (5.5) 12 67.5, C 66.9, C 48.7, CH2 3.83, d (3.4) 49.0, CH2 3.78, d (3.6) 13 (overlapping signal) 2.95, d (3.4) (overlapping signal) 3.00, d (3.6) 14 15.3, CH3 1.11, s 15.0, CH3 1.08, s 4.16, d (8.5) 3.94, m 15 67.5, CH2 62.3, CH2 3.37, d (8.6) 3.74, mCompound 1 Compound 2 Position δC, type δH (J in Hz) δC, type δH (J in Hz) 16 16.2, CH3 1.77, bs 15.5, CH3 1.80, bs ’ 104.8, CH 4.73, d (8.0) 103.7, CH 4.96, d (7.9) 2’ 75.6, CH 3.25, m 76.1, CH 3.24, m 3’ 78.4, CH 3.40, m 78.5, CH 3.40, m ’ 71.5, CH 3.28, m 71.7, CH 3.26, m 5’ 78.5, CH 3.30, m 78.5, CH 3.30, m 3.94, m 3.94, m 6’ 62.7, CH2 62.8, CH2 3.72, m 3.72, m

[0133] The data in Table 1 confirms that DON+1Glu is not D3G or D15G. Rather, the data is consistent with a mixture of interconverting compounds (compounds 1 and 2) in a molar ratio of 3:2, in which the hydroxyl group at C-7 has formed a β linkage with a glucose moiety. The data for the major product in the mixture (compound 1) indicates that the C-8 carbon is in the form of a hemiketal, formed between the C-15 hydroxyl group and the C-8 carbonyl group, while the data for the minor product (compound 2) is consistent with the C-8 carbonyl group being in the form of a free carbonyl group, as seen in the chemical structures below:Example 8: Toxicity of DON glycosylation products Lemna minor toxicity assay

[0134] The L. minor strain CPCC 490 (Phycological Culture Collection, Kitchener, Ontario) was maintained in a growth cabinet using oagland’s E+ medium as described by Renaud, J.B. et al (Chemical Research in Toxicology (2021), 34: 1604-1611). Assay conditions were carried out in 24-well TC-treated Costar™ plates (Corning, United States) using 1.25 mL of oagland’s E+ without sucrose or tartaric acid. Stock concentrations of D N, (Triplebond) D3G (Romer Labs), DON+1Glu and the enzyme product mixture obtained from Protein A-catalyzed reaction of DON with UDP-glucose were prepared in Optima™ acetonitrile (Fisher Scientific). Test conditions included quadruplicate biological replicates of controls ( oagland’s E+ assay media only), DMS controls (assay media with 0. % DMS ), 20 µM DON, 20 µM D3G, 20 µM DON+1Glu and 20 µM mixed enzyme product.0.1% DMSO wasadded to each well as a solvent reservoir prior to the addition of the stock concentrations of DON, D3G, DON+1Glu and the mixed enzyme product. The acetonitrile in each well was dried before the addition of oagland’s assay media up to .25 mL. Duckweed plants with 3 fronds were selected and added to each well. The 24-well assay plate was put into a 3D printed plastic sleeve and incubated in a growth cabinet under consistent temperature and lighting conditions for 7 days. After 7 days, the assay plate was imaged and subjected to a Python pixel analysis pipeline previously adapted by Renaud, J.B. et al (Chemical Research in Toxicology (2021), 34: 1604-1611) to determine surface area of fronds as represented by green pixel count. Following image analysis, duckweed fronds were removed from each well, freeze dried and weighed for biomass. Single-factor AN VA with Tukey’s post hoc testing (p < 0.05) was used to evaluate significance from the green pixel count and dry biomass from each condition using R. Data were visualized using box and whisker plots prepared in R.

[0135] As seen from the results presented in Figures 5A and 5B, a significant reduction in the growth of the plant was observed in the presence of 20 µM DON compared to the controls. In contrast, no significant growth reduction was observed in the presence of D3G, DON+1Glu, or the YjiC-catalyzed mixture, indicating that neither D3G nor DON+1Glu is phytotoxic. Example 9: Stability / hydrolysis experiments Stability to cellulase or cellobiase treatment

[0136] It is known that cellulase and cellobiase are capable of catalyzing the deglycosylation of D3G to DON. Thus, the stability of DON+1Glu against enzymatic hydrolysis by cellulase (≥700 U / g, Trichoderma reesei, Millipore Sigma, St Louis, MO) and cellobiase (1300 U / g, Aspergillus niger, Millipore Sigma, St Louis, MO) was examined using the approach described by Berthiller, F. et al (Toxicology Letters (2011), 206: 264-267). D3G was similarly treated as a positive control. Reaction mixtures in 100 µL volumes containing 1 µM D3G or 1 µM DON+1Glu were incubated with either 10 U / mL cellulase (14.3 mg / mL; 0.1 M sodium acetate, pH 5) or 1.3 U / mL cellobiase (1 mg / mL; 50 mM sodium phosphate, pH 6, 5 mM EDTA) for 24 hours at 37°C with gentle shaking. The reactions were quenched by the addition of 100 µL MeOH, vortexed for 15 s and centrifuged.190 µL of the reaction supernatants were transferred to 250 µL polypropylene HPLC vials for analysis by LC-MS as described in Example 1.

[0137] As seen from the results presented in Figure 6, no significant hydrolysis of DON+1Glu occurred in the presence of either cellulase or cellobiase, while 98% of D3G was hydrolyzed by cellulase (panel A) and 86% of D3G was hydrolyzed by cellobiase (panel B) respectively.Fecal stability assay

[0138] DON glucoside hydrolysis stability assays were adapted from those described in Gratz, S.W., et al, Applied and Environmental Microbiology (2018), 84(2): e02106-02117. Briefly, feces from several pigs were collected directly after defecation. Feces were stored anaerobically at −80°C in 10 mL of 70% PBS (pH 7.0) / 30% glycerol until use. Samples were defrosted and centrifuged at 4000 x g for 15 minutes. The supernatant was discarded and the pellet was suspended with 1 mL of anaerobic M2 medium (Gratz, S.W., et al, Applied and Environmental Microbiology (2018), 84(2): e02106-02117). Tubes were incubated at 37°C and 200 rpm for one hour. Post incubation, 100 µL were collected and 2 mM DON+1Glu, D3G (DON-3G), and DON were added. Samples were analyzed immediately by LC-MS as described in Example 1 (Control) or were incubated at 37°C for 24 hours prior to analysis.

[0139] As seen from the results presented in Figure 7, a 95% reduction in total D3G levels was observed with a concomitant increase in the amount of free DON in the sample (panel A). In contrast, no significant decrease in the levels of DON+1Glu was observed, and accordingly no significant increase occurred in the presence of free DON during incubation (panel B). DON itself was not glycosylated to a detectable degree by exposure to swine feces (panel C). This data indicates that DON+1Glu is significantly more stable and resistant to hydrolysis than D3G. Example 10: Activity of Protein A against various trichothecenes

[0140] The activity of Protein A to catalyze glycosylation of the trichothecenes deoxynivalenol (DON), DON+1Glu (Example 7), deoxynivalenol-3-glucoside (D3G / DON3G; Example 7), 3-acetyldeoxynivalenol (3-A-DON), 15-acetyldeoxynivalenol (15-A-DON), T-2, HT-2, NX and 3-acetyl-NX (3-A-NX) was measured. The structural formulas of 3-A-DON, 15-A-DON, T-2, HT-2, NX and 3-A-NX are shown below.3-A-DON 15-A-DONNX 3-A-NX

[0141] Protein A (1 µM) was incubated for 22 h at 23°C with the trichothecene substrates (1 mM) in the presence of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 10 mM UDP- glucose. The reaction was quenched by addition of methanol to a final concentration of 90% (v / v) and the mixture was analyzed by LC-MS as described in Example 1. As seen from the results shown in Figures 8A and 8B, DON, DON3G and 3-A-DON are each good substrates for Protein A, showing a significant decrease in concentration. Protein A also shows strong activity against HT-2, the concentration of which was undetectable after exposure for 22 hours. By contrast, Protein A was unable to detectably glycosylate T-2 and showed little activity against DON+1Glu as a substrate, reducing its concentration by only 11%. However, Protein A showed activity against 15-A-DON, NX and 3-A-NX as substrates, reducing their concentrations by 58%, 48% and 24%, respectively. Example 11: Expression of Protein A in Saccharomyces cerevisiae Rescue of DON sensitivity in S. cerevisiae by expression of Protein A

[0142] A strain of Saccharomyces cerevisiae (referred to herein as strain A) was genetically engineered to accumulate deoxynivalenol (DON) intracellularly by deletion of the ATP- binding cassette (ABC) transporter genes yor1, snq2, pdr5 and pdr10 from S. cerevisiae strain CEN.PK113-13D (MATα ura3-52). Another strain of S. cerevisiae (referred to herein as strain B) was prepared which contains the same gene deletions as strain A but includes a heterologous expression cassette encoding Protein A, which is expressed intracellularly. Strains A and B were grown overnight in yeast peptone dextrose broth containing 10 g / L yeast extract, 20 g / L peptone and 40 g / L dextrose (YPD40), washed and diluted to0.13 OD600in water. The resulting cell suspension (10 µL) was added to 120 µL of yeast nitrogen base (YNB Ammonium sulfate) with amino acids (Sigma Y1250) containing uracil (0.076 g / L) and 0, 50, 75 or 100 ppm of deoxynivalenol (DON) in a 96-well round-bottom plate. Plates were incubated at 30°C for 92 hours, with OD600measurements taken every 10 minutes using a Biotek plate reader. As seen from the results shown in Figure 9A, the growth of Strain A is inhibited as the concentration of DON increases. In addition, as seen from the results shown in Figure 9B, the growth of Strain B, which expresses Protein A, is unaffected by increasing concentrations of DON. Glycosylation of DON by Protein A expressed in S. cerevisiae

[0143] Strains A and B of Saccharomyces cerevisiae are grown overnight in yeast peptone dextrose broth (YPD40), washed and inoculated at 0.1 OD600 in 2 mL of yeast nitrogen base (YNB) medium containing Synthetic Drop-out Medium supplements without leucine (Sigma Y1376) in the absence or presence of 50 ppm deoxynivalenol (DON) in a 10 mL serum vial. The samples were grown at 30°C for 48 hours and centrifuged. The supernatant (500 µL) was mixed with 500 µL of chilled (4°C) methanol, refrigerated for 30 minutes, filtered and analyzed by LC-MS as described in Example 1. The pellet from centrifuging 1.2 mL of sample is disrupted at 40 m / s for 20 seconds with a bead-beating homogenizer. Cell debris is removed and the supernatant is mixed with an equal volume of chilled (4°C) methanol, refrigerated for 30 minutes, filtered and analyzed by LC-MS as described in Example 1. As seen from the results shown in Figure 9C, DON+1Glu was formed by incubation of DON with Strain B expressing Protein A but not from incubation with Strain A. Example 12: Enhancement of Protein A-catalyzed glycosylation by Arabidopsis thaliana sucrose synthase (SuS1) Combination of Protein A and SuS1 as separate enzymes

[0144] The codon-optimized gene for Arabidopsis thaliana sucrose synthase (SuS1 or AtSUS1, NCBI reference sequence NP_001031915.1) was synthesized and expressed in Escherichia coli BL21(DE3). A mixture of Protein A (1 µM or 0 µM (no Protein A) as a control), UDP-glucose (0.1 mM, 1.0 mM or 10 mM), deoxynivalenol (DON, 1 mM), sucrose (100 mM) and SuS1 (0, 0.1 µM or 1 µM), was incubated at 23°C for 21 hours and the reaction was quenched by addition of methanol to a final concentration of 90% (v / v). The mixture was analyzed by reverse phase LC-MS, as described in Example 1.

[0145] As can be seen from the results shown in Figure 10A, at the lowest concentration of UDP-glucose (0.1 mM), increasing the concentration of SuS1 present in the reaction mixture increased the percent conversion of DON by Protein A from 6% in the absence of SuS1 to 14% at a SuS1 concentration of 0.1 µM and further to 38% at a SuS1 concentration of 1 µM.Similar results were observed at a higher concentration of UDP-glucose (1 mM), at which the percent conversion of DON by Protein A increased from 34% in the absence of SuS1 to 42% at a SuS1 concentration of 0.1 µM and further to 66% at a SuS1 concentration of 1 µM. At an even higher concentration of UDP-glucose (10 mM), increasing the SuS1 concentration from 0.1 µM to 1 µM had little effect, as the percent conversion was greater than 95% at both concentrations of SuS1. Measurement of the percent conversion of DON in the absence of SuS1 was not carried out at this concentration of UDP-glucose. Protein A - SuS1 fusion proteins

[0146] Constructs encoding a fusion protein containing Protein A fused at its C-terminal to the N-terminal of SuS1 with a linker comprising the sequence EAAAKEAAAK (SEQ ID NO:7) and including either an N-terminal 6X-His-tag (Protein A::AtSUS1) or an N-terminal 6X-His- tagged-maltose binding protein (MBP) N-terminal sequence (MBP::Protein A::AtSUS1) were designed for expression in Escherichia coli BL21(DE3). The sequence of the Protein A-SuS1 fusion is shown below. MKKYHISMIN IPAYGHVNPT LALVEKLCEK GHRVTYATTE EFAPAVQQAG GEALIYHTSL NIDPKQIREM MEKNDAPLSL LKESLSILPQ LEELYKGDQP DLIIYDFVAL AGKLFAEKLN VPVIKLCSSY AQNESFQLGN EDMLKKIKEA EAEFKAYLEQ EKLPAVSFEQ LAVPEALNIV FMPKSFQIQH ETFDDRFCFV GPSLGERKEQ EGLLIDKDDR PLMLISLGTA FNAWPEFYKM CIKAFRDSSW QVIMSVGKTI DPESLEDIPA NFTIRQSVPQ LEVLEKADLF ISHGGMNSTM EAMNAGVPLV VIPQMYEQEL TANRVDELGL GVYLPKEEVT VSSLQEAVQA VSSDQELLTR VKNMQKDVKE AGGAERAAAE IEAFMKKSAV PQEAAAKEAA AKMANAERMI TRVHSQRERL NETLVSERNE VLALLSRVEA KGKGILQQNQ IIAEFEALPE QTRKKLEGGP FFDLLKSTQE AIVLPPWVAL AVRPRPGVWE YLRVNLHALV VEELQPAEFL HFKEELVDGV KNGNFTLELD FEPFNASIPR PTLHKYIGNG VDFLNRHLSA KLFHDKESLL PLLKFLRLHS HQGKNLMLSE KIQNLNTLQH TLRKAEEYLA ELKSETLYEE FEAKFEEIGL ERGWGDNAER VLDMIRLLLD LLEAPDPCTL ETFLGRVPMV FNVVILSPHG YFAQDNVLGY PDTGGQVVYI LDQVRALEIE MLQRIKQQGL NIKPRILILT RLLPDAVGTT CGERLERVYD SEYCDILRVP FRTEKGIVRK WISRFEVWPY LETYTEDAAV ELSKELNGKP DLIIGNYSDG NLVASLLAHK LGVTQCTIAH ALEKTKYPDS DIYWKKLDDK YHFSCQFTAD IFAMNHTDFI ITSTFQEIAG SKETVGQYES HTAFTLPGLY RVVHGIDVFD PKFNIVSPGA DMSIYFPYTE EKRRLTKFHS EIEELLYSDV ENKEHLCVLK DKKKPILFTM ARLDRVKNLS GLVEWYGKNT RLRELANLVV VGGDRRKESK DNEEKAEMKK MYDLIEEYKL NGQFRWISSQ MDRVRNGELY RYICDTKGAF VQPALYEAFG LTVVEAMTCG LPTFATCKGG PAEIIVHGKS GFHIDPYHGD QAADTLADFF TKCKEDPSHW DEISKGGLQR IEEKYTWQIY SQRLLTLTGV YGFWKHVSNL DRLEARRYLE MFYALKYRPL AQAVPLAQDD (SEQ ID NO:8) The fusion proteins were expressed and purified to homogeneity using a combination of Ni2+metal affinity chromatography and gel permeation gel chromatography.

[0147] The purified fusion proteins were tested for their ability to catalyze the glycosylation of DON in the presence of sucrose and the absence of UDP-glucose. A mixture of isolatedProtein A and SuS1 as described above was tested as a comparison. Reaction mixtures contained the fusion protein at a concentration of 1 μM or 2 μM or a mixture of the individual enzymes at a concentration of μM, in addition to 1 mM DON, 100 mM sucrose, and 0.1 mM UDP, and were carried out in a buffer containing 50 mM Tris-HCl and 150 mM NaCl at pH 8.0. Reactions were allowed to proceed for 23 hours at 23°C and were quenched by addition of MeOH to a final concentration of 90% (v:v). All reactions were analyzed via LC-MS as described in Example 1.

[0148] As can be seen from the results presented in Figure 10B, reaction with the Protein A::AtSUS1 fusion protein at a concentration of 1 μM resulted in glycosylation of approximately 52% of total DON, increasing to 70% when the fusion protein was present at a concentration of 2 μM. The MBP::Protein A::AtSUS1 fusion protein showed similar activity, resulting in conversion of 40% of total DON at a concentration of 1 µM, increasing to 64% at a concentration of 2 µM. In comparison, reaction with the mixture of isolated enzymes (Protein A + AtSUS1) at a concentration of μM resulted in glycosylation of 73% of total DON in solution. Example 13: Detoxification of trichothecene-contaminated dried distiller’s grains with solubles (DDGS) by Protein A

[0149] Dried distiller’s grans with solubles (DDGS) naturally contaminated with approximately 20 ppm of trichothecenes (ca.100 mg) was resuspended in 500 µL of buffer solution (pH 8.0) containing 50 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl2 and 10 mM UDP- glucose, and Protein A was added to a final concentration of 0.1 µM, 1 µM or 10 µM. A mixture not containing Protein A (0 µM) provided a negative control. The mixture was incubated at a temperature of 23°C, 30°C or 37°C for 23 h, then extracted with 78% acetonitrile in water containing 2% acetic acid. The extracts were analyzed by LC-MS as described in Example 1. As seen from the results shown in Figure 11, the % conversion of deoxynivalenol (DON) was highest at 30°C for all concentrations of Protein A tested, and increased from 35% upon treatment with 0.1 µM Protein A to 49% upon treatment with 1 µM Protein A and to 57% upon treatment with 10 µM Protein A. The percentage conversion was lower at 23°C and even lower at 37°C for each concentration of Protein A tested.

[0150] 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 fusion protein comprising a first polypeptide moiety fused to a second polypeptide moiety, wherein the first polypeptide moiety comprises a UDP-glycosyltransferase enzyme and the second polypeptide moiety comprises an enzyme active to catalyze the formation of UDP-glucose, wherein the fusion protein is active to catalyze glycosylation of a hydroxyl group at a position of a trichothecene mycotoxin other than a 3-position or a 15-position.

2. The fusion protein of claim 1 wherein the UDP-glycosyltransferase enzyme is an isoform of YjiC.

3. The fusion protein of claim 1 wherein the UDP-glycosyltransferase enzyme is of a species of Bacillus.

4. The fusion protein of claim 3 wherein the species of Bacillus is Bacillus subtilis.

5. The fusion protein of claim 1 wherein the UDP-glycosyltransferase enzyme has a 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%, at least 99% or 100% identity to SEQ ID NO:3 or SEQ ID NO:

5.

6. The fusion protein of any one of claims 1 to 5 wherein the enzyme active to catalyze the formation of UDP-glucose is a sucrose synthase.

7. The fusion protein of claim 6 wherein the sucrose synthase is of a species of Arabidopsis.

8. The fusion protein of claim 7 wherein the species of Arabidopsis is Arabidopsis thaliana.

9. The fusion protein of claim 8 wherein the sucrose synthase is encoded by an SUS1 gene of Arabidopsis thaliana.

10. The fusion protein of claim 6 wherein the sucrose synthase has a 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%, at least 99% or 100% identity to SEQ ID NO:

6.

11. The fusion protein of any one of claims 1 to 10 further comprising a linker polypeptide inserted between the first polypeptide moiety and the second polypeptide moiety.

12. The fusion protein of any one of claims 1 to 11 wherein the first polypeptide moiety is located N-terminally with respect to the second polypeptide moiety.

13. The fusion protein of any one of claims 1 to 11 wherein the first polypeptide moiety is located C-terminally with respect to the second polypeptide moiety.

14. A recombinant microbial host cell expressing a heterologous protein comprising UDP-glycosyltransferase activity, wherein the heterologous protein comprising UDP- glycosyltransferase activity is active to catalyze glycosylation of a hydroxyl group at a position of a trichothecene mycotoxin other than a 3- position or a 15-position.

15. The recombinant microbial host cell of claim 14 wherein the heterologous protein comprising UDP-glycosyltransferase activity is a UDP-glycosyltransferase enzyme.

16. The recombinant microbial host cell of claim 15 wherein the UDP-glycosyltransferase enzyme is an isoform of YjiC.

17. The recombinant microbial host cell of claim 15 wherein the UDP-glycosyltransferase enzyme is of a species of Bacillus.

18. The recombinant microbial host cell of claim 17 wherein the species of Bacillus is Bacillus subtilis.

19. The recombinant microbial host cell of claim 15 wherein the UDP-glycosyltransferase enzyme has a 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%, at least 99% or 100% identity to SEQ ID NO:3 or SEQ ID NO:

5.

20. The recombinant microbial host cell of any one of claims 14 to 19, further expressing an enzyme active to catalyze the formation of UDP-glucose.

21. The recombinant microbial host cell of claim 20 wherein the enzyme active to catalyze the formation of UDP-glucose is a sucrose synthase.

22. The recombinant microbial host cell of claim 21 wherein the sucrose synthase is of a species of Arabidopsis.

23. The recombinant microbial host cell of claim 22 wherein the species of Arabidopsis is Arabidopsis thaliana.

24. The recombinant microbial host cell of claim 21 wherein the sucrose synthase is encoded by an SUS1 gene of Arabidopsis thaliana.

25. The recombinant microbial host cell of claim 21 wherein the sucrose synthase has a 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%, at least 99% or 100% identity to SEQ ID NO:6.

26. The recombinant microbial host cell of claim 14 wherein the heterologous protein comprising UDP-glycosyltransferase activity is a fusion protein as defined in any one of claims 1 to 9.

27. The recombinant microbial host cell of any one of claims 14 to 26, wherein the recombinant microbial host cell is a yeast host cell.

28. The recombinant microbial host cell of claim 27 wherein the yeast host cell is a cell of a strain of a species of Saccharomyces or a cell of a strain of a species of Komagataella.

29. The recombinant microbial host cell of any one of claims 14 to 26, wherein the recombinant microbial host cell is a bacterial host cell.

30. The recombinant microbial host cell of claim 29 wherein the bacterial host cell is a cell of a strain of a species of Bacillus or a cell of a strain of a species of Lactobacillus.

31. A composition comprising a carrier and one or more selected from: the fusion protein of any one of claims 1 to 13; the recombinant microbial host cell of any one of claims 14 to 30, an autolysate thereof or an extract thereof, wherein the recombinant microbial host cell is alive, inactivated or dead; and a mixture comprising a UDP-glycosyltransferase enzyme and an enzyme active to catalyze the formation of UDP-glucose, wherein the UDP-glycosyltransferase enzyme is active to catalyze glycosylation of a hydroxyl group at a position of a trichothecene mycotoxin other than a 3- position or a 15-position.

32. The composition of claim 31 wherein the UDP-glycosyltransferase enzyme is defined as in any one of claims 16 to 19, and / or the enzyme active to catalyze the formation of UDP-glucose is defined as in any one of claims 21 to 25.

33. The composition of claim 31 or 32 wherein the carrier is a solid carrier.

34. The composition of claim 31 or 32 wherein the carrier is a liquid carrier.

35. The composition of any one of claims 31 to 34 wherein the carrier is an agriculturally acceptable carrier.

36. A method of reducing the toxicity of a trichothecene mycotoxin comprising exposing the trichothecene mycotoxin to a protein comprising UDP-glycosyltransferase activity or to a composition comprising the protein comprising UDP-glycosyltransferase activity and a carrier, wherein the protein comprising UDP-glycosyltransferase activityis active to catalyze glycosylation of a hydroxyl group at a position of the trichothecene mycotoxin other than a 3- position or a 15-position.

37. The method of claim 36 wherein the protein comprising UDP-glycosyltransferase activity is a UDP-glycosyltransferase enzyme.

38. The method of claim 37 wherein the UDP-glycosyltransferase enzyme is defined as in any one of claims 16 to 19.

39. The method of any one of claims 36 to 38 comprising further exposing the trichothecene mycotoxin and the protein comprising UDP-glycosyltransferase activity to an enzyme active to catalyze the formation of UDP-glucose.

40. The method of claim 39 wherein the enzyme active to catalyze the formation of UDP- glucose is defined as in any one of claims 21 to 25.

41. The method of claim 36 wherein the protein comprising UDP-glycosyltransferase activity is a fusion protein according to any one of claims 1 to 13.

42. The method of claim 36 wherein exposing the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity comprises exposing the trichothecene mycotoxin to a recombinant microbial host cell according to any one of claims 14 to 30 or to a composition according to any one of claims 31 to 35.

43. The method of any one of claims 36 to 42 wherein the trichothecene mycotoxin is deoxynivalenol.

44. The method of any one of claims 36 to 43 wherein exposing the trichothecene mycotoxin to the protein comprising UDP-glycosyltransferase activity comprises exposing a material contaminated with the trichothecene mycotoxin or at risk of being contaminated with the trichothecene mycotoxin to the protein comprising UDP- glycosyltransferase activity.

45. The method of claim 44 wherein 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.

46. The method of claim 45 wherein the feed, feed product or feed ingredient is dried distiller’s grains or dried distiller’s grains with solubles.

47. The method of claim 44 wherein the material is a cereal crop.

48. The method of claim 47 wherein the cereal crop is selected from corn and wheat.

49. The method of claim 44 wherein the material is a feedstock for a fermentation process and the method further comprises treating the feedstock with a yeast strainwhich is active to ferment the feedstock to produce one or more products, wherein the one or more products have a reduced level of the trichothecene mycotoxin compared to the level of the trichothecene mycotoxin in products obtained from a fermentation process in which the feedstock is not exposed to the protein comprising UDP-glycosyltransferase activity.

50. The method of claim 49 wherein the fermentation process produces one or more products selected from distillers’ dried grains, distillers’ dried grains with solubles, distillers’ wet grains, distillers’ wet grains with solubles, dried solubles and condensed distillers’ solubles.

51. The method of claim 49 or 50 wherein the yeast strain expresses the protein comprising UDP-glycosyltransferase activity.

52. The method of claim 49 or 50 comprising exposing the feedstock to a bacterial strain expressing the protein comprising UDP-glycosyltransferase activity.

Images