# Patent

## Title
Enzymatic degradation of mycotoxins during grain processing

## Abstract

Methods, compositions, and systems for steeping, propagation and fermentation, particularly large-scale operations for production of starch and ethanol and fermentation product streams are provided. Addition of mycotoxin mitigating enzymes or microorganisms expressing mycotoxin mitigating enzymes to steeping, propagation, and/or fermentation tanks, and/or to post-fermentation product streams, mitigates mycotoxin levels in fermentation co-products obtained from mycotoxin contaminated feedstocks.

## Background
RELATED APPLICATIONS
This application is a Continuation of U.S. application Ser. No. 17/199,274 titled “Enzymatic Degradation of Mycotoxins During Grain Processing” filed Mar. 11, 2021, which claims the benefit of U.S. Provisional Application No. 62/988,670 titled “Enzymatic Degradation of Mycotoxins During Fermentation and Post-Fermentation” filed Mar. 12, 2020, which are both incorporated herein by reference.


TECHNICAL FIELD
Provided herein are methods, compositions, and genetically modified microorganisms for mitigating mycotoxin contamination of corn and other feedstocks used in bioprocessing and food and feed production.
SEQUENCE LISTING
An official copy of the sequence listing is submitted concurrently with the specification electronically via EFS-Web as an XML formatted sequence listing with a file name of PT-141-US04_Sequence_Listing, a creation date of Nov. 11, 2022, and a size of about 62 kilobytes. The sequence listing contained in this XML formatted document is part of the specification and is herein incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Mycotoxins are naturally occurring toxins produced by fungi resulting in contaminated food and feed, especially cereals, forages, grain, fruits, and manufactured products. Mycotoxin contamination is monitored, and increased mycotoxin loading results in lower prices, and/or loss of crops or food products with considerable economic consequences. Consumption of contaminated grains or feeds can result in decreased efficiency in weight gain and reproductivity in livestock. In humans, effects can range from mild gastrointestinal symptoms to liver damage, neurotoxicity, and cancer.
Many common mycotoxins are resistant to most forms of food and feed processing, and in fact, can become more concentrated during processing. For example, as components in the feedstock are separated or consumed the fraction of mycotoxin in the remaining material may increase. In one illustrative example, starch in the feedstock is converted via fermentation to a chemical and CO2 and the fraction of mycotoxin in the remaining unfermented material is greater than it was in the feedstock resulting in higher mycotoxin levels in fermentation by-products such as distillers' dried grains with solubles (DDGS). Processing grains into human foods also results in increased levels of mycotoxins. As such, it is important that mycotoxin contamination is mitigated in foods designed for human consumption and livestock feeds.
The present invention is directed toward overcoming one or more of the problems discussed above.
SUMMARY OF THE INVENTION
Provided herein are methods of mitigating mycotoxin contamination in a feed or food product produced in a dry mill or wet mill grain processing facility. In some aspects, the methods comprise (i) contacting a feedstock or process stream of a dry mill or wet mill grain processing facility with an enzyme selected from the group consisting of aflatoxin oxidase, 3-O acetyltransferase, peroxidase, F420H2-dependent reductases, Ery4, UDP-glycosyltransferase, laccase, manganese peroxidase, deoxynivalenol hydroxylase, lactono hydrolase, Zearalenone hydrolase, xylanase, DepA/DepB, and 2cys-peroxiredoxin; and (ii) producing a food or feed product from a process stream downstream of said contacting step.
In some aspects, the contacting comprises contacting whole grain, cracked grain, steeped grain, steep water, steep liquor, flour, slurry, beer, whole stillage, wet cake, thin stillage, concentrated thin stillage, defatted concentrated thin stillage (e.g. syrup), germ, oil, fiber, protein, starch, or combinations thereof, with a microorganism expressing the enzyme. In some aspects, the contacting comprises contacting whole grain, cracked grain, steeped grain, steep water, steep liquor, flour, slurry, beer, whole stillage, wet cake, thin stillage, concentrated thin stillage, defatted concentrated thin stillage (e.g. syrup), germ, oil, fiber, protein, starch, or combinations thereof, with a lysate obtained from a microorganism expressing the enzyme.
In some aspects, the contacting comprises contacting the feed stock or process stream with a microorganism expressing an enzyme selected from the group consisting of aflatoxin oxidase, 3-O acetyltransferase, peroxidase, F420H2-dependent reductases, Ery4, UDP-glycosyltransferase , laccase, manganese peroxidase, deoxynivalenol hydroxylase, lactono hydrolase, Zearalenone hydrolase, xylanase, DepA/DepB, and 2cys-peroxiredoxin, a broth containing a such a microorganism, a lysate formed by lysing a such a microorganism, a concentrate of such a microorganism or lysate, or combinations thereof.
In some embodiments, provided herein are grains and compositions sourced from mycotoxin contaminated grains wherein the contaminated grain or compositions are exposed to an enzyme to mitigate mycotoxin contamination. The resulting compositions have decreased levels of mycotoxin relative to compositions not exposed to the enzyme. Exposure to the enzyme can be through exposure to a microorganism expressing an enzyme to mitigate mycotoxin contamination. Exposure to the enzyme can be through exposure to materials obtained from lysing the microorganism which expresses the mycotoxin mitigating enzyme, i.e. a lysate.
In some embodiments, a human food composition sourced from mycotoxin contaminated grain exposed to a mycotoxin mitigating enzyme or a microorganism expressing an enzyme to mitigate mycotoxin contamination is provided. In some aspects, the levels of mycotoxin in the human food composition are decreased relative to the levels of mycotoxin in a human food composition sourced from mycotoxin contaminated grain without exposure to a mycotoxin mitigating enzyme or a microorganism expressing an enzyme to mitigate mycotoxin contamination.
In some embodiments, corn starch sourced from mycotoxin contaminated grain exposed to a mycotoxin mitigating enzyme or a microorganism expressing an enzyme to mitigate mycotoxin contamination is provided. In some aspects, the levels of mycotoxin in the corn starch is decreased relative to the levels of mycotoxin in corn starch sourced from mycotoxin contaminated grain without exposure to a mycotoxin mitigating enzyme or a microorganism expressing an enzyme to mitigate mycotoxin contamination.
In some embodiments, corn syrup sourced from mycotoxin contaminated grain exposed to a mycotoxin mitigating enzyme or a microorganism expressing an enzyme to mitigate mycotoxin contamination is provided. In some aspects, the levels of mycotoxin in the corn syrup is decreased relative to the levels of mycotoxin in corn syrup sourced from mycotoxin contaminated grain without exposure to a mycotoxin mitigating enzyme or a microorganism expressing an enzyme to mitigate mycotoxin contamination.
In some embodiments, a livestock feed composition comprising distiller's grain sourced from feedstock fermented in the presence of an enzyme to mitigate mycotoxin contamination or a microorganism expressing an enzyme to mitigate mycotoxin contamination is provided. In some aspects, the levels of mycotoxin in the livestock feed composition are decreased relative to the levels of mycotoxin in a livestock feed composition obtained from a feedstock fermented in the absence of a microorganism expressing an enzyme to mitigate mycotoxin contamination.
In some embodiments, a livestock feed composition comprising distiller's grain sourced from a post-fermentation whole stillage, wet cake, thin stillage and/or concentrated thin stillage (e.g. syrup) stream treated with an enzyme to mitigate mycotoxin contamination or a microorganism expressing an enzyme to mitigate mycotoxin contamination is provided. In some aspects, the levels of mycotoxin in the livestock feed composition are decreased relative to the levels of mycotoxin in a livestock feed composition obtained from an untreated post-fermentation whole stillage, wet cake, thin stillage and/or concentrated thin stillage (e.g. syrup) stream.
In some embodiments, a livestock feed composition comprising distiller's grain sourced from beer treated with an enzyme to mitigate mycotoxin contamination or a microorganism expressing an enzyme to mitigate mycotoxin contamination is provided. In some aspects, the levels of mycotoxin in the livestock feed composition are decreased relative to the levels of mycotoxin in a livestock feed composition obtained from untreated beer.
In some aspects, the mycotoxin is selected from the group consisting of aflatoxin, ochratoxin A, patulin, T-2, HT-2, beauvericin, neosolaniol, nivalenol, deoxynivalenol (DON), 3-ADON, 15-ADON, trichothecene, ochratoxin, and zearalenone.
In some aspects, the enzyme is selected from the group consisting of aflatoxin oxidase, 3-O acetyltransferase, peroxidase, F420H2-dependent reductases, Ery4, UDP-glycosyltransferase, laccase, manganese peroxidase, deoxynivalenol hydroxylase, and lactono hydrolase, xylanase, peroxidase, and 2cys-peroxiredoxin. In some aspects, the enzyme can be a combination of enzymes, for example, an enzyme system that works in two or more steps to detoxify a mycotoxin. For example, the enzyme system can be DepA/DepB, where DepA converts DON to 3-keto-DON and DepB, an NADPH dependent dehydrogenase, catalyzes the reduction of 3-keto-DON to 3-epi-DON.
In some aspects, the microorganism is a bacteria. In some aspects, the microorganism is a yeast. In some aspects, the microorganism is engineered to express and/or excrete a mycotoxin mitigating enzyme. In some aspects, the microorganism is a yeast such as S. cerevisiae or Pichia pastoris. In some aspects, the microorganism is a genetically modified commodity yeast or a genetically modified consolidated bioprocessing yeast, wherein the yeast is genetically modified to express a mycotoxin mitigating enzyme.
In some embodiments, the mycotoxin mitigating enzyme is expressed by a yeast host cell.
In some aspects, the human food composition or the livestock feed composition contains mycotoxins at levels less than about 10 ppm, less than about 8 ppm, less than about 5 ppm, less than about 3 ppm, or less than about 2 ppm.
In some embodiments, a method of producing corn starch is provided. In some aspects, the method comprises inoculating a mycotoxin contaminated corn with one or more yeast strains prior to or during steeping wherein at least one yeast strain expresses a mycotoxin mitigating enzyme. In some aspects, the mycotoxin mitigating enzyme is deoxynivalenol hydroxylase. In some aspects, the method further comprises processing the corn to produce corn starch. In some aspects, the method further comprises processing the corn starch to produce corn syrup.
In some embodiments, a method of producing corn starch is provided. In some aspects, the method comprises exposing a mycotoxin contaminated corn with a lysate obtained from a microorganism expressing a mycotoxin mitigating enzyme prior to or during steeping. In some aspects, the mycotoxin mitigating enzyme is deoxynivalenol hydroxylase. In some aspects, the method further comprises processing the corn to produce corn starch. In some aspects, the method further comprises processing the corn starch to produce corn syrup.
In some embodiments, a method of fermentation is provided. In some aspects, the method comprises inoculating a mycotoxin contaminated feedstock with one or more yeast strains, wherein at least one yeast strain expresses a mycotoxin mitigating enzyme. In some aspects, the mycotoxin mitigating enzyme is deoxynivalenol hydroxylase. The feedstock can be inoculated during yeast propagation and/or can be inoculated during fermentation. In some aspects, the method further comprises fermenting the feedstock to produce ethanol and distiller's grain.
In some embodiments, a method of fermentation is provided. In some aspects, the method comprises exposing a mycotoxin contaminated feedstock with a lysate obtained from a microorganism expressing a mycotoxin mitigating enzyme. In some aspects, the mycotoxin mitigating enzyme is deoxynivalenol hydroxylase. The exposure of the feedstock to the lysate can occur prior to or during fermentation. In some aspects, the method further comprises fermenting the feedstock to produce ethanol and distiller's grain.
In some aspects, the feedstock is contaminated with a mycotoxin, for example, deoxynivalenol. After exposure to the mycotoxin mitigating enzyme, for example, by a microorganism expressing a mycotoxin mitigating enzyme or to a lysate containing the enzyme, during fermentation, the levels of mycotoxin, e.g., deoxynivalenol, in the resulting fermentation by-products, e.g., distiller's grain, are decreased relative to the levels of mycotoxin in by-products obtained from a feedstock processed in the absence of enzyme exposure, e.g. such as exposure to a yeast expressing a mycotoxin mitigating enzyme such as deoxynivalenol hydroxylase or exposure to a yeast lysate containing the mycotoxin mitigating enzyme.
In some embodiments, a method of mitigating mycotoxin contamination in a human food composition is provided. In some aspects, the method comprises processing a grain in the presence of a mycotoxin mitigating enzyme or a microorganism expressing an enzyme, wherein the enzyme is selected from the group consisting of aflatoxin oxidase, 3-O acetyltransferase, peroxidase, F420H2-dependent reductases, Ery4, UDP-glycosyltransferase, laccase, manganese peroxidase, deoxynivalenol hydroxylase, lactono hydrolase, xylanase, and 2cys-peroxiredoxin. In some aspects, the enzyme can be a combination of enzymes, for example, an enzyme system that works in two or more steps to detoxify a mycotoxin. For example, the enzyme system can be DepA/DepB, where DepA converts DON to 3-keto-DON and DepB, an NADPH dependent dehydrogenase, catalyzes the reduction of 3-keto-DON to 3-epi-DON.
In some embodiments, a method of mitigating mycotoxin contamination in livestock feed is provided. In some aspects, the method comprises processing a feedstock in the presence of a mycotoxin mitigating enzyme or a microorganism expressing an enzyme, wherein the enzyme is selected from the group consisting of aflatoxin oxidase, 3-O acetyltransferase, peroxidase, F420H2-dependent reductases, Ery4, UDP-glycosyltransferase, laccase, manganese peroxidase, deoxynivalenol hydroxylase, lactono hydrolase, xylanase, and 2cys-peroxiredoxin. In some aspects, the method comprises processing a feedstock in the presence of two or more mycotoxin mitigating enzymes, for example, DepA and DepB. In some aspects, the method comprises processing a feedstock in the presence of a microorganism expressing two or more enzymes, for example, DepA and DepB.
In some embodiments, a method of mitigating mycotoxin contamination in livestock feed is provided. In some aspects, the method comprises contacting a post-fermentation whole stillage, wet cake, thin stillage and/or concentrated thin stillage (e.g. syrup) stream with an enzyme to mitigate mycotoxin contamination, wherein the levels of mycotoxin in the livestock feed composition are decreased relative to the levels of mycotoxin in a livestock feed composition obtained from an untreated post-fermentation whole stillage, wet cake, thin stillage and/or concentrated thin stillage (e.g. syrup) stream.
In some embodiments, a method of mitigating mycotoxin contamination in livestock feed is provided. In some aspects, the method comprises contacting beer with an enzyme to mitigate mycotoxin contamination, wherein the levels of mycotoxin in the livestock feed composition are decreased relative to the levels of mycotoxin in a livestock feed composition obtained from untreated beer.
In some embodiments, a vector comprising the nucleic acid sequence encoding a mycotoxin mitigating enzyme is provided. For example, a vector comprising a nucleic acid sequence of SEQ ID NO: 1, or nucleic acid sequence having at least about 95%, or at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 1, encodes deoxynivalenol hydroxylase. In some embodiments, the vector further comprises one or more nucleic acid sequences encoding a KdR gene or KdX gene. In some embodiments, the ddnA gene (SEQ ID NO: 1) is in one vector, the KdR gene is in another vector, and the KdX gene is in yet another vector. In some embodiments, all three genes are in one vector. In some embodiments, at least two of the ddnA gene, the KdR gene, and the KdX gene are in the same vector. In some embodiments, a vector comprises the nucleic acid sequences of one or both of DepA and DepB.
In some aspects, the vector is selected from the group consisting of pYEDP60 (S. cerevisiae) or pPINK (P. pastoris), though other commercially available vectors are known to those skilled in the art.
In some embodiments, a nucleic acid sequence of a mycotoxin mitigating enzyme is integrated into the genome of the microorganism, for example, via homologous recombination.
In some embodiments, a microorganism expressing one or more proteins encoded by the vector described herein is provided. In some aspects, the microorganism is selected from a yeast or a bacteria. In some aspects, the microorganism is engineered to express and/or excrete a mycotoxin mitigating enzyme. In some aspects, the microorganism is a genetically modified commodity yeast or a genetically modified consolidated bioprocessing yeast, wherein the yeast is genetically modified to express a mycotoxin mitigating enzyme. In some aspects, the microorganism is S. cerevisiae. In some aspects, the yeast is Pichia pastoris. 
In some embodiments, use of a microorganism engineered to comprise the nucleic acid sequence encoding a mycotoxin mitigating enzyme is provided. In some aspects, the nucleic acid sequence is provided in a vector described herein. In some aspects, the nucleic acid sequence is integrated into the genome of the microorganism. In some aspects, the use comprises expressing a mycotoxin mitigating enzyme during fermentation, in beer, and/or in distiller's grain sourced from post-fermentation stillage, wherein fermentation feedstock is contaminated with mycotoxin. In some aspects, the mycotoxin mitigating enzyme is deoxynivalenol hydroxylase and the feedstock is contaminated with deoxynivalenol.
In some embodiments, a method for expressing a mycotoxin mitigating enzyme in a yeast host cell is provided. In some aspects, the method comprises: transforming the host cell with an integration vector comprising a nucleic acid having one or more expression cassettes comprising a nucleic acid encoding a mycotoxin mitigating enzyme, and a targeting nucleic acid that directs insertion of the integration vector into a particular location of the genome of the host cell by homologous recombination, wherein the transformed host cell produces the mycotoxin modulating enzyme.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the surprising results achieved by purified enzyme addition as well as lysate addition to fermentation of a high DON corn, compared to a lack of change observed during fermentation in the absence of enzyme.
FIG. 2a is a schematic of an exemplary process showing examples of dry grind biorefinery process steps where the mycotoxin mitigating additions can be made.
FIG. 2b is a schematic of an exemplary process showing examples of dry grind biorefinery process steps, including optional separate cooking, liquefaction, and saccharification steps, where the mycotoxin mitigating additions can be made.
FIG. 2c is a schematic of an exemplary process showing examples of wet grind biorefinery process steps where the mycotoxin mitigating additions can be made. The fermenting step could be accompanied by one or more additional steps as shown in FIGS. 2a and 2b, e.g., cooking liquefaction, saccharification, and yeast propagation.
FIG. 3 shows DON levels (ppm) over time (0 to 88 hours fermentation) with yeast lysate containing deoxynivalenol hydroxylase compared to a control fermentation in the absence of enzyme.



DESCRIPTION
As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
The word “exemplary” is used to mean serving as an example, instance, or illustration. Any embodiment or design described as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments or designs, nor is it meant to preclude equivalent exemplary structures and techniques known to those of ordinary skill in the art. Rather, use of the word exemplary is intended to present concepts in a concrete fashion, and the disclosed subject matter is not limited by such examples.
The term “or” is intended to mean an inclusive “or” rather than an exclusive “or.” To the extent that the terms “comprises,” “has,” “contains,” and other similar words are used in either the detailed description or the claims, for the avoidance of doubt, such terms are intended to be inclusive in a manner similar to the term “comprising” as an open transition word without precluding any additional or other elements.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entireties.
Target chemicals are produced in biorefineries with biological catalysts, e.g. via fermentation. One example is corn to ethanol but fermentation of other feedstocks and the production of other products is also contemplated herein. In biorefineries, contaminants, e.g. mycotoxins, may be concentrated in the products of the biorefinery.
Mycotoxins are produced by fungi and are present in many agricultural crops but especially in grains including corn, barley, rye, triticale, and wheat. Contamination can occur in the field, after harvest and during storage, or even during processing into food and livestock feeds. Exemplary mycotoxins and the fungi that produce them are provided in Table 1.








TABLE 1





Mycotoxin
Fungal Species







Deoxynivalenol, nivalenol,
Fusarium graminearum, F.


3-ADON, 15-ADON
culmorum, F. crookwellense


Fumonisin B1, B2, B3
F. moniliform, F.



verticilliodes, F. proliferatum


Zearalenone
F. graminearum, F. culmorum,



F. equiseti


HT-2 toxin, T-2 toxin, Neosolaniol
F. equiseti, F. poae, F.



sporotrichioides


Aflatoxins Bj, B2; cyclopiazonic
Aspergillus flavus


acid


Aflatoxins Bi, B2, Gi, G2
A. parasiticus


Ochratoxin A; penicillic acid
A. ochraceus


Sterigmatocystin, cyclopiazonic
A. versicolor


acid


Ochratoxin B, citrinin
Penicillium verrucosum


Rubratoxins
P. purpurogenum


Patulin, citrinin
P. expansum


Beauvericin
Beauveria bassiana









Any mycotoxin of importance to human and animal food chains is contemplated as a target mycotoxin, including the mycotoxins included in Table 1. A “mycotoxin mitigating enzyme” or “mycotoxin modulating enzyme” includes any enzyme capable of detoxifying mycotoxins present in grains. It has been determined herein that mycotoxin mitigating enzymes can be expressed by microorganisms and that grains and/or intermediate grain processing streams can be exposed to such microorganisms and/or expressed enzymes during the processing of the grains into food or feed products, for example, before, during, or after such nonlimiting examples as storage, steeping, milling, mixing, cooking, liquifying, saccharifying, fermenting, distilling, pumping, separating, concentrating, evaporating, filtering, drying, refining, degerminating, and/or other processes used to process grain into food or feed products. Feedstocks, intermediate streams, and/or products may also be exposed to such microorganisms and/or expressed enzymes. Nonlimiting examples include whole grain, cracked grain, steeped grain, steep water, steep liquor, flour, slurry, beer, whole stillage, wet cake, thin stillage, concentrated thin stillage (e.g. syrup), defatted concentrated thin stillage (e.g. syrup), germ, oil, fiber, protein, starch, biochemicals, syrups, and dried distillers grains. Such exposure can be e.g. to the organism, a broth containing the organism, a lysate formed by lysing the organism, a concentrate of the organism or lysate, and/or to the isolated and/or concentrated enzyme. Depending on the heat tolerance of the organism or enzyme some streams may be less suitable for exposure.
Mycotoxin contamination of food and feed products can lead to adverse results. For example, in livestock feed, the presence of mycotoxins may cause animal feed refusal resulting in lack of weight gain. In processing grains into feed and food products, mycotoxins can become concentrated. For example, fermentation, solid liquid separation, evaporation, and drying processes in a biorefinery can result in concentration of mycotoxins in co-product streams like distillers grains, such as malt distillers grains, brewers grains, condensed distillers solubles, dried distillers solubles, distillers wet grains, distillers wet grains with solubles, gluten meal (e.g. protein), and gluten feed and the like used for animal feed. Similarly, in processing grains into human food products, such as corn into corn starch or corn syrup, mycotoxins may become concentrated and a threat to human health.
Deoxynivalenol (DON) is produced by several fungi in the Fusarium genus including F. graminearum and F. culmorum. Nivalenol (NIV), 3-ADON, 15-ADON, and T-2 toxin are also produced by the Fusarium fungi. The chemical structure of DON is shown below:




The three free hydroxy groups on the molecule are associated with its toxicity. In humans, DON elicits strong emetic effects after consumption, as the toxin reduces brain levels of serotonin and interacts with dopaminergic receptors in the brain. DON can be present in grains including oats, barley, wheat, corn, buckwheat, sorghum, rice, and popcorn, as well as flour, bread, breakfast cereals, noodles, infant foods, pancake mixes, noodles, beer, and malt.
Deoxynivalenol hydroxylase is an exemplary mycotoxin mitigating enzyme (encoded by DdnA, SEQ ID NO: 1). The enzyme modifies the C-16 carbon in DON, catalyzing the monooxygenation of the allylic methyl group of DON. This enzyme can also interact with NIV and 3-ADON, modifying the mycotoxins to a more tolerable, less toxic form. Methods of using this enzyme in decontaminating grains used for human or animal consumption are provided herein. Other mycotoxin mitigating enzymes include, but are not limited to, aflatoxin oxidase, 3-O acetyltransferase, peroxidase, F420H2-dependent reductases, Ery4, UDP-glycosyltransferase, laccase, manganese peroxidase, and lactono hydrolase, xylanase, DepA/DepB, and 2cys-peroxiredoxin. Microorganisms are engineered to express these enzymes are useful in the methods disclosed herein.
Aflatoxins are toxins produced by the mold Aspergillus flavus that can grow on food ingredients such as corn, peanuts, and other grains. At high levels, aflatoxins can cause illness (aflatoxicosis), liver damage, and death. The enzyme aflatoxin oxidase is useful in mitigating aflatoxin contamination of grains and food products produced using the contaminated grains.
Zearalenone is a potent estrogenic metabolite produced by some Fusarium and Gibberella species. The mycotoxin is heat-stable and is a contaminant of cereal crops such as maize, barley, oats, wheat, rice, and sorghum. Zearalenone concentrations are low in grain contaminated in the field, but levels increase under storage conditions with moisture greater than 30%-40%. Zearalenone is problematic for the swine industry, and particularly affects male and female reproduction. The enzymes lactonohydrolase or Zearalenone hydrolase detoxify zearalenone.
Compositions
Provided are compositions produced according to the methods disclosed herein, both for human consumption and animal consumption, which have decreased levels of mycotoxin contamination. The composition can be a human food product, e.g. any food containing grain products sourced from mycotoxin contaminated grains. Exemplary food products include most processed foods containing grains, including flour, bread, cereals, noodles, infant foods, pancake mixes, beer, and malt. The composition can be an animal feed, for example, a feed for livestock such as beef cattle, dairy cattle, swine, sheep, goats, poultry, etc., a feed for horses, donkeys, ponies, mules, etc., a feed for ruminating zoo animals, or can be a feed for companion animals such as dogs, cats, birds, etc. Acceptable levels of mycotoxins depend on the animal species and the mycotoxin. For example, maximum levels of DON are typically less than 20 ppm, for example, less than about 15 ppm, or less than 12 ppm, or less than 10 ppm, or less than 9 ppm, or less than 8 ppm, or less than 7 ppm, or less than 6 ppm, or less than 5 ppm, or less than 4 ppm, or less than 3 ppm, or less than 2 ppm, or less than 1 ppm. For example, in a feed composition for beef cattle, many sources recommend the levels of DON be 10 ppm or less; in a feed composition for dairy cattle, the levels of DON are recommended to be 2 ppm or less; in a feed composition for poultry, the levels of DON are recommended to be 2 ppm or less.
A livestock feed composition can comprise distiller's grain sourced from feedstock exposed to an enzyme to mitigate mycotoxin contamination or a microorganism expressing an enzyme to mitigate mycotoxin contamination during processing of the feedstock, e.g. during fermentation. A livestock feed composition can comprise distillers' grain sourced from stillage, wet cake, and/or a syrup stream treated with an enzyme to mitigate mycotoxin contamination or a microorganism expressing an enzyme to mitigate mycotoxin contamination. In some aspects, the levels of mycotoxin in the livestock feed composition are decreased relative to the levels of mycotoxin in a livestock feed composition obtained from untreated stillage, wet cake, and/or syrup stream. A livestock feed composition can comprise distiller's grains, such as malt distiller's grains, brewers grains, condensed distiller's solubles, dried distillers solubles, distiller's wet grains, distiller's wet grains with solubles, gluten meal, and gluten feed treated with an enzyme to mitigate mycotoxin contamination or a microorganism expressing an enzyme to mitigate mycotoxin contamination.
A typical feedstock can include any grain or cereal crop, for example, wheat, corn, sorghum, hops, oats, barley, rye, buckwheat, etc.
Systems and Methods
The microorganism expressing the mycotoxin mitigating enzyme can be inoculated into any suitable process streams and grown in situ. In an illustrative example, the microorganism can be inoculated into a mixing, steeping, propagation, saccharification, fermentation, stillage, syrup or other stream.
The microorganism expressing the mycotoxin mitigating enzyme can be grown separately and used as a whole broth to treat one or more or feed stock, process stream, or products. The culture may be concentrated prior to use, e.g., by filtering, evaporating, membrane separation, settling, centrifugation or other concentration method. The microorganism may be lysed to enhance enzyme exposure. Lysis can be accomplished by any suitable methodology known in the art. In an illustrative example, lysis is accomplished by homogenization. In an illustrative example, after growth and throughout further processing, the temperature is maintained below 150° F., even below 130° F. In an illustrative example, lysate is used or stored at or below 40° F.
Microorganisms can be grown in fed-batch or continuous fermentation methods depending on the organism used and the requirements for growth and robust enzyme expression (e.g. 1-10000 mg/L). In most cases, these organisms will be grown to late stationary phase, under conditions to promote expression of the enzymatic system. This is dependent upon the explicit system for expression, i.e. constitutive vs induced expression.
In an illustrative example, exposure of one or more of feedstock, process streams and/or products in a dry mill plant, e.g. as shown in FIGS. 2a and 2b, to one or more mycotoxin mitigating enzymes can result in reduced levels of contamination of plant products. Such exposure can be to a microorganism expressing the enzyme, a broth containing the organism, a lysate formed by lysing the organism, a concentrate of the organism, broth, or lysate, and/or to the isolated and/or concentrated enzyme. For example, in a dry grind ethanol plant, grain such as corn is used as a feedstock and ethanol is produced by fermentation of the starch contained within the grain. The fermentation product includes ethanol, water, and soluble components as well as residual unfermented particulate matter (among other things). The fermentation product is distilled and dehydrated to recover ethanol. The residual matter (e.g., whole stillage) comprising water, soluble components, oil, and unfermented solids can be further processed to separate out desirable fermentation by-products, for example, dried distillers' grains with solubles (DDGS), mash, beer, whole stillage, thin stillage, wet cake, syrup, etc., used for animal feed.
In an illustrative example, exposure of one or more of feedstock, process streams and/or products in a wet mill plant, e.g. as shown in FIG. 2c, to one or more mycotoxin mitigating enzymes can result in reduced levels of contamination of plant products. Such exposure can be to a microorganism expressing the enzyme, a broth containing the organism, a lysate formed by lysing the organism, a concentrate of the organism, broth, or lysate, and/or to the isolated and/or concentrated enzyme. For example, in a corn wet mill, steeping, actually a controlled fermentation, is essential for high yields and high starch quality. Cleaned corn is placed into large steeping tanks (steeps), where the corn is soaked in hot water to begin breaking the starch and protein bonds. The gluten bonds within the corn begin to loosen and release the starch. Addition of sulphur dioxide can improve the fermentation by enhancing growth of favorable microorganisms, such as lactobacillus, while suppressing detrimental bacteria, molds, fungi, and yeast. Solubles are extracted and the kernel softens. Exemplary products in addition to a biochemical such as ethanol, include corn fiber, corn and yeast protein, corn starch, corn syrup, and corn oil.
Systems for expressing enzymes useful in mitigating mycotoxin contamination of human and animal feed compositions, for example, by exposing feedstock and processing streams, include vectors containing a gene encoding a mycotoxin mitigating enzyme, and microorganisms expressing the gene. Microorganisms expressing these enzymes can be added to steep tanks, propagation and/or fermentation tanks, can be added to beer, and/or can be added to the post-fermentation stillage, wet cake, and/or syrup stream. Microorganisms expressing these enzymes can be added to mash used to prepare pellets, dog food, and cat food, for example. As an added benefit, the microorganism may add to the amino acid profile of the protein in the animal feed, e.g. the DDGS or high protein product.
In some aspects, microorganisms expressing the enzymes can be lysed and the enzyme added to steep tanks, propagation and/or fermentation tanks, can be added to beer, and/or can be added to the post-fermentation stillage, wet cake, and/or syrup stream. Lysate containing these enzymes can be added to mash used to prepare pellets, dog food, and cat food, for example. The lysed slurry can be added directly, or can be further purified to concentrate the mycotoxin mitigating enzymes before adding. In some aspects, the enzymes are purified and added to the process in a concentrated amount, for example, in the mash, beer, whole stillage, thin stillage, wet cake, syrup, DDGS, etc. Purified enzymes can be dosed, permitting controlled activity as desired at different parts of the fermentation process.
Relative to other approaches in mitigating mycotoxin contamination, the process disclosed herein is beneficial in that processing equipment is not exposed to corrosive agents typically used to reduce mycotoxin levels. Furthermore, this approach provides for a more nutritional product, with higher consistency in nutritional value, texture, taste, and appearance, as other systems used to mitigate mycotoxins can alter feed color, non-protein nitrogen, alter fat profiles, or in other ways result in a detriment to the final feed nutritional value.
Genes encoding the mycotoxin mitigating enzyme can be engineered into an ethanol production yeast so that it expresses the enzyme during propagation and/or fermentation to mitigate mycotoxin contamination present in the feedstock.
Genes encoding the mycotoxin mitigating enzyme can be engineered into a separate microorganism, e.g. yeast or bacteria, that is co-fermented with the production yeast.
Genes encoding the mycotoxin mitigating enzyme can be engineered into a separate microorganism that is independently propagated and then combined with a contaminated broth prior to distillation or combined with a post fermentation stillage, wet cake, and/or syrup stream.
Under any of the growth conditions useful in allowing the microorganism to express the mycotoxin mitigating enzyme, it can be helpful to add an iron source for improved function of the enzyme. Exemplary iron sources include FeCl3. 5-aminolevulinic acid is a heme precursor and can be used as a cofactor to assist in bacterial expression and activity of the Ddna enzyme.
Methods of fermentation can comprise the steps of any typical fermentation, whether in production of biofuels or foods and drinks for human and animal consumption. However, it is shown herein that an added step of adding a microorganism expressing a mycotoxin mitigating enzyme to the fermentation process decreases the levels of mycotoxins in the food and drink products and livestock feeds.
Methods of mitigating mycotoxin contamination in livestock feed composition and/or human foods can entail fermenting a feedstock in the presence of a microorganism expressing an enzyme selected from the group consisting of aflatoxin oxidase, 3-O acetyltransferase, peroxidase, F420H2-dependent reductases, Ery4, UDP-glycosyltransferase, laccase, manganese peroxidase, deoxynivalenol hydroxylase, lactono hydrolase, xylanase, DepA/DepB, and 2cys-peroxiredoxin.
Methods of mitigating mycotoxin contamination in livestock feed composition and/or human foods can comprise contacting post-fermentation products including post-fermentation beer, stillage, wet cake, and/or syrup stream with an enzyme to mitigate mycotoxin contamination. The levels of mycotoxin in the livestock feed composition and/or human foods are decreased relative to the levels of mycotoxin in a livestock feed composition obtained from untreated post-fermentation products.
Methods of mitigating mycotoxin contamination in livestock feed can comprise contacting beer with an enzyme to mitigate mycotoxin contamination. The levels of mycotoxin in the livestock feed composition are decreased relative to the levels of mycotoxin in a livestock feed composition obtained from untreated beer.
Lysate containing the mycotoxin mitigating enzyme can be used in excess as needed to decontaminate the particular mycotoxin. In some aspects, the mycotoxin contaminated composition can be treated with about 50 mg lysate to about 150 mg lysate per gram solids in the treated material. In some aspects, the composition can be treated with about 80 to about 120 mg lysate per gram solids. In some aspects, the composition can be treated with about 100 mg lysate per gram solids. In purified versions, the mycotoxin mitigating enzyme would be dosed at about 5 mg enzyme to about 15 mg enzyme per gram solids, even about 8 mg enzyme to about 12 mg enzyme per g solids, even about 10 mg enzyme per gram solids. The enzyme dose can also depend on yeast expression and activity of the particular enzyme. In some aspects, the dosage is from about 0 to 100 fold excess beyond the concentration of the contaminating mycotoxin.
In some aspects, the mycotoxin contaminated composition is treated with a high dose of mycotoxin mitigating enzyme, for example, about 100 to about 1000 mg lysate containing enzyme per gram solids of the contaminated composition. In some aspects, the mycotoxin contaminated composition is treated with a low dose of mycotoxin mitigating enzyme, for example, about 5 to about 50 mg enzyme per gram solids.
The dose can vary from the above parameters depending on the original concentration of mycotoxin in the contaminated composition. For example, highly contaminated material may require a correspondingly higher dose than less contaminated material. The level of contamination may be determined by measuring the contamination in the feedstock on a parts per million basis and a dose may be determined based on that level of contamination. For example, in treating a composition derived from a contaminated feedstock with a cell lysate, the dose may be stated as the number of milligrams of lysate per gram of solids in the treated composition per parts per million contaminant level of the feedstock. The dose may be 1-1000 mg of lysate, even 10-500 mg of lysate, even 50-500 mg lysate, even 10-50 mg of lysate per gram of solids in the treated composition per parts per million contamination in the feedstock. For example, a fermentation composition in a corn fermentation facility may be treated with a dose of 10 mg of lysate as follows. The contamination level, e.g, of DON in the corn feedstock may be measured as, e.g., 5 ppm. The dose would then be calculated as 10 mg/g solids/ppm contamination x #grams of solids x 5ppm contamination to yield a dose of 50 mg/g of solids in the fermenter.
Vectors and Host Cells
Methods for expressing a mycotoxin modulating enzyme in a yeast host cell can comprise transforming a host cell with an integration vector comprising a nucleic acid having one or more expression cassettes comprising a nucleic acid encoding a mycotoxin modulating enzyme, and a targeting nucleic acid that directs insertion of the integration vector into a particular location of the genome of the host cell by homologous recombination. The transformed host cell produces the mycotoxin modulating enzyme.
Vectors are provided for transforming a microorganism into an engineered microorganism for producing a mycotoxin mitigating enzyme. The vectors can comprise a nucleic acid sequence encoding one or more mycotoxin mitigating enzymes selected from the group consisting of aflatoxin oxidase, 3-O acetyltransferase, peroxidase, F420H2-dependent reductases, Ery4, UDP-glycosyltransferase, laccase, manganese peroxidase, deoxynivalenol hydroxylase, lactono hydrolase, xylanase, DepA/DepB, and 2cys-peroxiredoxin. For example, the vector can include the nucleic acid sequence of SEQ ID NO: 1 which encodes deoxynivalenol hydroxylase. In some embodiments, the vector further comprises one or more nucleic acid sequences encoding a KdX gene (SEQ ID NO: 2) or KdR gene (SEQ ID NO: 3). Alternatively, the vector can include a nucleic acid sequence which comprises the promoter, terminator, tag, and gene encoding the mycotoxin mitigating enzyme (SEQ ID NO: 10). In some aspects, the vector is selected from the group consisting of pYEDP60 (S. cerevisiae), pPINK (P. pastoris), or pCWori (bacterial).
Microorganisms useful herein are transformed with a vector comprising the nucleic acid sequence encoding a mycotoxin mitigating enzyme. The microorganism can be a yeast or a bacteria. The microorganism can be engineered to express and/or excrete a mycotoxin mitigating enzyme. The microorganism may be used solely to decontaminate a feedstock, process stream, or product. The microorganism may be an organism that is also used to carry out a primary conversion of a feedstock to a desired product. The microorganism may be an accessory microorganism that is cocultured with another microorganism so that it provides the decontaminating enzyme while the other organism produces a desired product. The microorganism can be separately cultured and added as a whole microorganism, whole broth, as a lysate, or isolated enzyme. Illustratively, the microorganism can be a yeast used in fermenting a feedstock in a bioethanol production facility or can be a yeast used in steeping corn for production of corn starch. The yeast can be, for example, a genetically modified commodity yeast or a genetically modified consolidated bioprocessing yeast. The yeast can be, for example, S. cerevisiae or P. pastoris. 
The microorganisms comprising such vectors are useful in expressing deoxynivalenol hydroxylase or another mycotoxin mitigating enzyme during fermentation, for example, in beer, and/or in distiller's grain sourced from post-fermentation stillage, wherein fermentation feedstock is contaminated with a mycotoxin such as deoxynivalenol.
EXAMPLES
The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1: Deoxynivalenol Hydroxylase Expression in P. Pastoris and S. Cerevisiae Yeast
DNA Resuspension:
DdnA DNA, KdX DNA, and KdR DNA, obtained from GenScript, is centrifuged at 6,000×g for 1 min at 4° C. 20 μL sterilized deionized water is added to dissolve DNA. The vial is vortexed for 1 min. The solution is then heated at 50° C. for 15 min to ensure DNA is dissolved in liquid, then stored at −20° C. until use.
Plasmid E. coli Transformation:
Chemically competent DH5a cells are thawed on ice for 20-30 min. DdnA DNA (1-2 μL) is added to chemically competent cells and gently mixed by flicking the bottom of the tube. The mixture is incubated on ice for 20-30 minutes, then heat shocked for 30-60 seconds at 42° C. The mixture is placed back on ice for 2 min and the cells are allowed to recover. 250-1000 μL of LB or SOC media without antibiotic is added to the mixture and incubated for 45-60 min at 37° C. Following incubation, the sample is gently centrifuged at 4,000×g for 1 min at 4° C. Three quarters of the remaining liquid is removed and the cell pellet is resuspended with the remainder liquid. The liquid is plated on room temperature LB agar plates with selective antibiotic and placed in a 37° C. incubator overnight (plates are pulled within 12-18 hours). Colonies are selected and grown in liquid culture. Freezer stock is made and DNA sequence is checked.
Transferring Gene from E. Coli Plasmid to Yeast Plasmid:
DdnA, KdX, and KdR sequences were codon biased for expression in a yeast host, and had a histidine or FLAG tag to the C-terminal side. A secretion signal can also be added to the 5′ side of the DNA to signal secretion in the yeast.
Restriction Enzyme Digestion: 5′ End (Start of Gene) Cut with EcoRI and 3′ End (End of Gene) Cut with RsrII, SphI, StuI, KpnI, NaeI, FseI, or SwaI. Enzyme and DNA are mixed (Both Yeast Plasmid and E. coli Plasmid [Individually]) at 37° C. for 2-24 hr.

    
    
        2 μL DNA
        1 μL 10× Restriction Enzyme Buffer
        5 μL Sterile Water
        1 μL EcoRI
        1 μL RE2* (*or any appropriate 2nd restriction enzyme)
    
    


A 1.5% agarose gel is run to separate cleaved gene from plasmid and to clean up plasmid to keep it from ligating back to itself.
The purified cut pieces are then ligated:

    
    
        2 μL 5× Ligation buffer
        0.5 μL T4 DNA Ligase
        1 μL Yeast Plasmid
        1 μL Insert Gene
        5.5 μL Sterile Water
    
    


Ingredients are mixed gently, then centrifuged to get all liquid to bottom of tube, and incubated at 25° C. for 1-2 hours or 16 C overnight. The yeast plasmid is transformed into E. coli, grown, sequenced, and purified for transformation of the plasmid DNA into Yeast.
Additional Methods for Construction of the Final Yeast Vector:
Gateway cloning protocol is used to combine all three genes into a single construct for placement in to a yeast vector to be integrated in to the yeast genome. Generally the protocol is as follows:
Following solutions are mixed:

    
    
        1 uL destination vector
        75 ng of each gene or insert in a 2:1 ratio dependent upon origination of insert solution
        2 uL T4 DNA Ligase Buffer
        1-2 uL Golden Gate Assembly Mix
        Nuclease Free Water to 20 uL
    
    


Once mixed well, the reaction undergoes the following protocol in a thermocycler: either: 37C for 1 hr, followed by 60° C. for 5 min or (37° C. for 1 min, 16° C. for 1 min) for 30-35 cycles followed by a 60° C. incubation for 5 min. These constructs are then transformed into E. coli for primary screening prior to transformation into yeast.
Plasmid Yeast Transformation:
Yeast from YPD plate are inoculated into 20 mL of YPD medium in 100 mL sterile flask and grown overnight with shaking (200-250 rpm) at 30° C. to reach stationary phase. The culture is diluted into 100 mL of YPD medium in 500 mL flask to OD600 of  ̃0.3, and grown with shaking at 30° C. for 3-6 hr, OD600. The culture should double and not surpass 1.5 (OD600). Cells are harvested by centrifugation at room temperature for 5 min at 5,000 rpm. Supernatant is discarded and cells resuspended in 50 mL of sterile water. Centrifugation is repeated.
Supernatant is discarded and the cell pellet resuspended in 1 mL of transformation buffer (cells are now ready for transformation). Salmon testes DNA is aliquoted at 10 μL of 10 mg/mL into each transformation tube, along with 0.1 μg of yeast plasmid DNA. 100 μL of competent cells are added and the mixture is vortexed. 600 μL of PLATE Buffer is added and the mixture is vortexed again. The mixtures are incubated for 30 min at 30° C. with shaking. 10% DMSO is added, and the mixture is heat shocked for 15 min at 42° C. The mixture is spun to pellet the cells, and the supernatant is removed. The cells are resuspended in 500 μL of sterile water and plated at 100 μL on appropriate SC selective plates without required supplement. Plates are then incubated at 30° C. for 2-3 days until colonies appear.
Western Blotting for Protein Expression Testing:
The deoxynivalenol hydroxylase enzyme is tagged with a C-terminal histidine or FLAG tag which is probed for in a Western blot. Western blot analysis is used for monitoring protein (enzyme) expression during bacterial or yeast growth.
Cellular growth is centrifuged and the cell pellet (solids) and the supernatant (aqueous) are separated and saved separately. Cell pellets are resuspended in a general low molar TRIS buffer and treated to break open cells (for example, by bead beating, sonicating, homogenizing, chemical treatments). The solution is centrifuged following cell rupture in an ultra-centrifuge to separate the cell debris and the soluble protein, and both fractions are kept.
The soluble aqueous phase can be run as is, or enriched further by running the soluble protein over an affinity column for a His-Tag or FLAG-Tag (Nickel or FLAG column). The protein is eluted off the column using increasing salt concentrations resulting in an enriched fraction. This fraction, or any of the previous fractions are then run on an SDS-PAGE protein gel-12% -18% acrylamide gels, for example. Once the gel has run under standard conditions, the proteins are transferred to a membrane (PVDF) and blocked with rehydrated milk powder. After blocking, a primary antibody to the His- or FLAG-Tag is added and allowed to incubate overnight at 16° C.
Following primary incubation, the membrane is washed several times before adding secondary antibodies which have a horseradish peroxidase attached. Secondary incubation is between 1 and 3 hours at room temperature.
The membrane is again washed, then signal compound is added to produce light at any band that the antibodies attached to. Tagged proteins are visualized on a gel box.
Example 2: Addition of Deoxynivalenol Hydroxylase Expressing P. Pastoris or S. Cerevisiae to Corn Steep
Whole corn contaminated with DON is filled into three large steeping tanks (steeps) and soaked in hot water 30 to 48 hours to begin breaking the starch and protein bonds. P. pastoris expressing deoxynivalenol hydroxylase and S. cerevisiae expressing deoxynivalenol hydroxylase are added, one to each of the steep tanks, and enzyme levels are monitored in each of the two steep tanks to ensure proper expression. The third steep tank is held under identical conditions but lacks addition of either microorganism. At 6 hour increments over the duration of the steep, water samples are obtained and DON levels are assessed in each sample. Over time, DON levels decrease in the steep water from treated tanks.
Example 3: Addition of Deoxynivalenol Hydroxylase Expressing S. Cerevisiae to Fermentation Tank
Ground corn or other feedstock is combined with water to form a slurry, and the pH of the slurry mixture is adjusted as needed. S. cerevisiae engineered to express deoxynivalenol hydroxylase is added to the fermenter along with an amount of yeast starter effective to produce a commercially significant quantity of ethanol. The fermentation is carried out for about 88 hours. Enzyme levels are monitored to ensure expression and proper levels to mitigate DON contamination. Samples of the solids are obtained at regular intervals to assess DON levels. Resulting post-fermentation products show commercially acceptable levels of DON (ppm).
Example 4: Addition of Deoxynivalenol Hydroxylase Via Lysate in Fermentation
A 72 hour fermentation was performed on DON contaminated corn in the presence or absence of a cell lysate of yeast genetically modified to produce deoxynivalenol hydroxylase (Ddna—the main catalytic enzyme), Kdr, or Kdx, the latter two of which are accessory recycling enzymes. The yeast cultures were grown to a late logarithmic growth phase or early stationary growth phase while producing mycotoxin mitigating enzymes, and then lysed. The yeast lysates were mixed to a 1:1:1 ratio, such that the final dosing was equal for each enzyme at 5-10 mg protein (which contained the enzyme) per gram solids, and added to the fermentation.
Results were analyzed for mycotoxin concentration and ethanol titer. DON in ppm was measured by LC/MS on all fermentation solids after 88h of fermentation and dry down. Comparative measurements were made based on untreated control fermentations done at the same time as the treated fermentations. The data in FIG. 1 shows that the enzymes are active in the yeast lysate and available to the corn, thus reducing DON contamination, and pose no harm to the final ethanol titer. Compared to the untreated control, DON levels in the treated samples were reduced by 94%. The % mitigated is relative to the untreated control fermentation, and not based on the starting to ending fermentation DON concentrations (ppm).
In a similar experiment, 88 hour fermentations were performed on DON contaminated corn in the presence or absence of a cell lysate obtained from a yeast genetically modified to produce deoxynivalenol hydroxylase. At various timepoints during the fermentation, DON levels were assessed in the untreated control and treated fermenters using LC/MS. FIG. 3 shows DON mitigation kinetics over the 88 hour fermentations. Over time, the levels of DON in the enzyme treated fermentations decrease to less than 2 ppm.
Example 5: Whole Stillage Treatment
Following distillation and removal of the ethanol fraction, mycotoxin-contaminated whole stillage is treated over a relatively shorter time with a high dose, e.g. 100-1000 mg whole cell lysate obtained from S. cerevisiae genetically modified to produce a mycotoxin mitigating enzyme containing enzyme per gram solids, or a relatively longer time at a lower dose, e.g. 5-50 mg whole cell lysate enzyme per gram solids. Samples are tested every 2-4 hours to determine mycotoxin concentration. Whole stillage after treatment exhibits commercially acceptable levels of DON (ppm).
Example 6: Secondary Whole Stillage Fermentation
Whole stillage obtained after ethanol distillation is subjected to a secondary fermentation with an ethanologen expressing one or more mycotoxin mitigating enzymes. Samples are obtained every 4-6 hours during the fermentation and assessed for mycotoxin levels. During the second fermentation, the mycotoxin is reduced. Resulting feed products show commercially acceptable levels of mycotoxin presence.
Example 7: Treatment of the Solubles and Syrup
In a first vat, lysate of microorganisms producing mycotoxin mitigating enzymes is dosed into the syrup tank and allowed to mitigate mycotoxin over the holding time and prior to dosing the feed in the drying process.
In a second vat, a live microorganism expressing mycotoxin mitigation enzymes is grown in the syrup or solubles. Both experiments demonstrate effectiveness of treatment of the partial stream containing mycotoxin which remains in the wet-cake or feed solids after separation of the two streams.
In further experiments, two vats containing mycotoxin contaminated thin stillage streams are treated with whole cell lysate from a mycotoxin mitigating enzyme producing microorganism or live microorganism expressing mycotoxin mitigation enzymes, respectively. A third vat is subjected to identical conditions but is not treated with enzyme. The three vats are incubated for 48-60 hours and samples are obtained from each vat every 4-8 hours to track mycotoxin levels. Over time, mycotoxin levels decrease to acceptable levels.
Example 8: Addition of Aflatoxin Oxidase Expressing S. Cerevisiae and P. Pastoris to Wheat Steep
Wheat contaminated with aflatoxin is filled into three large steeping tanks (steeps) and soaked in hot water 30 to 48 hours to begin breaking the starch and protein bonds. P. pastoris expressing aflatoxin oxidase and S. cerevisiae expressing aflatoxin oxidase are added, one to each of the steep tanks, and enzyme levels are monitored in each of the two steep tanks to ensure proper expression. The third steep tank is held under identical conditions but lacks addition of either microorganism. At 6 hour increments over the duration of the steep, water samples are obtained and aflatoxin levels are assessed in each sample. Over time, aflatoxin levels decrease in the steep water from treated tanks.
Example 9: Secondary Whole Stillage Fermentation
Whole stillage obtained after ethanol distillation is subjected to a secondary fermentation with an ethanologen expressing 3-O acetyltransferase. Samples are obtained every 4-6 hours during the fermentation and assessed for DON levels. During the second fermentation, the levels of DON are reduced. Resulting feed products show commercially acceptable levels of mycotoxin presence.
Example 10: Addition of DepA and DepB Expressing S. Cerevisiae to Fermentation Tank
Ground corn or other feedstock is combined with water to form a slurry, and the pH of the slurry mixture is adjusted as needed. S. cerevisiae engineered to express DepA and DepB is added to the fermenter along with an amount of yeast starter, i.e. ethanologen, effective to produce a commercially significant quantity of ethanol. Cofactors are added at the start of fermentation, including pyrroloquinoline quinone (PQQ) or methoxatin as purified powder or fruit extract. The fermentation is carried out for about 88 hours. Enzyme levels are monitored to ensure expression and proper levels to mitigate DON contamination. Samples of the solids are obtained at regular intervals to assess DON levels. Resulting post-fermentation products show commercially acceptable levels of DON (ppm).
Example 11: Addition of Yeast Lysate Containing DepA and DepB to Fermentation Tank
Ground corn or other feedstock is combined with water to form a slurry, and the pH of the slurry mixture is adjusted as needed. S. cerevisiae engineered to express DepA and DepB is lysed and the lysate is added to the fermenter along with an amount of a yeast starter effective to produce a commercially significant quantity of ethanol. Cofactors are added at the start of fermentation, including pyrroloquinoline quinone (PQQ) or methoxatin as purified powder or fruit extract. The fermentation is carried out for about 88 hours. Enzyme levels are monitored and additional lysate added as needed to decreased DON contamination. Samples of the solids are obtained at regular intervals to assess DON levels. Resulting post-fermentation products show commercially acceptable levels of DON (ppm).
ADDITIONAL EXAMPLES
1. A livestock feed composition comprising distiller's grain sourced from feedstock fermented in the presence of a microorganism expressing an enzyme to mitigate mycotoxin contamination, or a cell lysate comprising a microorganism expressing an enzyme to mitigate mycotoxin contamination, wherein the levels of mycotoxin in the livestock feed composition are decreased relative to the levels of mycotoxin in a livestock feed composition obtained from a feedstock fermented in the absence of a microorganism expressing an enzyme to mitigate mycotoxin contamination or a cell lysate comprising a microorganism expressing an enzyme to mitigate mycotoxin contamination.
2. A livestock feed composition comprising distiller's grain sourced from post-fermentation stillage, wet cake, and/or syrup stream treated with an enzyme to mitigate mycotoxin contamination, wherein the levels of mycotoxin in the livestock feed composition are decreased relative to the levels of mycotoxin in a livestock feed composition obtained from untreated post-fermentation stillage, wet cake, and/or syrup stream, and wherein the enzyme is obtained by inoculating post-fermentation stillage, wet cake, and/or syrup stream with a microorganism expressing an enzyme to mitigate mycotoxin contamination or by adding to the post-fermentation stillage, wet cake, and/or syrup stream a cell lysate comprising a microorganism expressing an enzyme to mitigate mycotoxin contamination.
3. A livestock feed composition comprising distiller's grain sourced from beer treated with an enzyme to mitigate mycotoxin contamination, wherein the levels of mycotoxin in the livestock feed composition are decreased relative to the levels of mycotoxin in a livestock feed composition obtained from untreated beer, and wherein the enzyme is obtained by inoculating beer with a microorganism expressing an enzyme to mitigate mycotoxin contamination or by adding to the beer a cell lysate comprising a microorganism expressing an enzyme to mitigate mycotoxin contamination.
4. The livestock feed composition of any one of examples 1-3, wherein the mycotoxin is selected from the group consisting of aflatoxin, Ochratoxin A, patulin, T-2, HT-2, beauvericin, neosolaniol, nivalenol, deoxynivalenol, 3-ADON, trichothecene, ochratoxin, and zearalenone.
5. The livestock feed composition of any one of examples 1-3, wherein the enzyme is selected from the group consisting of aflatoxin oxidase, 3-O acetyltransferase, peroxidase, F420H2-dependent reductases, Ery4, UDP-glycosyltransferase, laccase, manganese peroxidase, deoxynivalenol hydroxylase, xylanase, DepA/DepB, lactono hydrolase, Zearalenone hydrolase, and 2cys-peroxiredoxin.
6. The livestock feed composition of example 1, wherein the microorganism is a yeast.
7. The livestock feed composition of example 6, wherein the yeast is S. cerevisiae. 
8. The livestock feed composition of example 6, wherein the yeast is Pichia pastoris. 
9. The livestock feed composition of claim 6, wherein the yeast is a non-genetically modified commodity yeast or a consolidated bioprocessing yeast.
10. The livestock feed of any one of example 1-3, wherein the enzyme is expressed by a yeast host cell.
11. The livestock feed composition of any one of examples 1-10, wherein the levels of mycotoxin are less than about 10 ppm.
12. The livestock feed composition of any one of examples 1-10, wherein the levels of mycotoxin are less than about 5 ppm.
13. The livestock feed composition of any one of examples 1-10, wherein the levels of mycotoxin are less than about 2 ppm.
14. A method of fermentation comprising inoculating a feedstock with one or more yeast strains, wherein at least one yeast strain expresses one or more mycotoxin mitigating enzymes.
15. The method of example 14, further comprising fermenting the feedstock to produce ethanol and distiller's grain.
16. The method of example 14, wherein the mycotoxin mitigating enzyme is deoxynivalenol hydroxylase.
17. The method of example 16, wherein the feedstock is contaminated with deoxynivalenol.
18. The method of example 15, wherein the levels of deoxynivalenol in the distiller's grain are decreased relative to the levels of deoxynivalenol in distiller's grain obtained from a feedstock fermented in the absence of a yeast expressing deoxynivalenol hydroxylase.
19. A method of mitigating mycotoxin contamination in livestock feed, the method comprising fermenting a feedstock in the presence of a microorganism expressing an enzyme selected from the group consisting of aflatoxin oxidase, 3-O acetyltransferase, peroxidase, F420H2-dependent reductases, Ery4, UDP-glycosyltransferase, laccase, manganese peroxidase, deoxynivalenol hydroxylase, lactono hydrolase, Zearalenone hydrolase, xylanase, DepA/DepB, and 2cys-peroxiredoxin.
20. A method of mitigating mycotoxin contamination in livestock feed, the method comprising contacting post-fermentation stillage, wet cake, and/or syrup stream with an enzyme to mitigate mycotoxin contamination, wherein the levels of mycotoxin in the livestock feed composition are decreased relative to the levels of mycotoxin in a livestock feed composition obtained from untreated post-fermentation stillage, wet cake, and/or syrup stream, wherein the enzyme is obtained by inoculating post-fermentation stillage, wet cake, and/or syrup stream with a microorganism expressing an enzyme to mitigate mycotoxin contamination or by adding to the post-fermentation stillage, wet cake, and/or syrup stream a cell lysate comprising a microorganism expressing an enzyme to mitigate mycotoxin contamination.
21. A method of mitigating mycotoxin contamination in livestock feed, the method comprising contacting beer with an enzyme to mitigate mycotoxin contamination, wherein the levels of mycotoxin in the livestock feed composition are decreased relative to the levels of mycotoxin in a livestock feed composition obtained from untreated pre-distillation whole stillage, and wherein the enzyme is obtained by inoculating beer with a microorganism expressing an enzyme to mitigate mycotoxin contamination or by adding to the beer a cell lysate comprising a microorganism expressing an enzyme to mitigate mycotoxin contamination.
22. The method of example 21, wherein the step of contacting beer with an enzyme comprises lysing yeast expressing the enzyme and adding the lysate to the beer.
23. A vector comprising a nucleic acid sequence of SEQ ID NO: 1, or a nucleic acid having at least about 95% sequence identity to SEQ ID NO: 1, encoding deoxynivalenol hydroxylase (Ddna).
24. The vector of example 23, further comprising one or more nucleic acid sequences encoding a KdR gene, KdX gene, or Ddna gene.
25. The vector of example 23, selected from the group consisting of pYEDP60 (S. cerevisiae), pPINK (P. pastoris), or pCWori (bacterial) or other custom vector for integration into yeast genome.
26. A microorganism expressing a vector of example 23.
27. The microorganism of example 26 selected from a yeast and a bacteria.
28. The microorganism of example 26, wherein the yeast is S. cerevisiae. 
29. The microorganism of example 26, wherein the yeast is Pichia pastoris. 
30. The microorganism of example 26, wherein the bacteria is E. coli, lactobacillus, or zymomonas. 
31. The microorganism of example 26, wherein the yeast is a non-genetically modified commodity yeast or a consolidated bioprocessing yeast.
32. A vector comprising a nucleic acid sequence of SEQ ID NO: 10, or a nucleic acid having at least about 95% sequence identity to SEQ ID NO: 10.
33. A microorganism expressing a vector of claim 32.
34. The microorganism of example 33 selected from a yeast and a bacteria.
35. The microorganism of example 33, wherein the yeast is S. cerevisiae. 
36. The microorganism of example 33, wherein the yeast is Pichia pastoris. 
37. The microorganism of example 33, wherein the bacteria is E. coli, lactobacillus, or zymomonas. 
38. The microorganism of example 33, wherein the yeast is a non-genetically modified commodity yeast or a consolidated bioprocessing yeast.
39. Use of microorganism comprising a vector of any one of examples 23 to 26 in expressing deoxynivalenol hydroxylase during fermentation, in beer, and/or in distiller's grain sourced from post-fermentation stillage, wherein fermentation feedstock is contaminated with deoxynivalenol.
40. A method for expressing a mycotoxin modulating enzyme in a yeast host cell comprising: transforming the host cell with an integration vector comprising a nucleic acid having one or more expression cassettes comprising a nucleic acid encoding a mycotoxin modulating enzyme, and a targeting nucleic acid that directs insertion of the integration vector into a particular location of the genome of the host cell by homologous recombination, wherein the transformed host cell produces the mycotoxin modulating enzyme.
41. A microorganism expressing deoxynivalenol dehydroxylase, wherein the microorganism is Pichia pastoris or S. cerevisiae. 
42. A method of mitigating mycotoxin contamination in a feed or food product produced in a dry mill or wet mill grain processing facility, the method comprising: contacting a feedstock or process stream of a dry mill or wet mill grain processing facility with an enzyme selected from the group consisting of aflatoxin oxidase, 3-O acetyltransferase, peroxidase, F420H2-dependent reductases, Ery4, UDP-glycosyltransferase, laccase, manganese peroxidase, deoxynivalenol hydroxylase, lactono hydrolase, Zearalenone hydrolase, xylanase, DepA/DepB, and 2cys-peroxiredoxin; and producing a food or feed product from a process stream downstream of said contacting step.
43. The method of example 42, wherein contacting comprises contacting whole grain, cracked grain, steeped grain, steep water, steep liquor, flour, slurry, beer, whole stillage, wet cake, thin stillage, concentrated thin stillage, defatted concentrated thin stillage (e.g. syrup), germ, oil, fiber, protein, starch, or combinations thereof.
44. The method of example 42, wherein contacting comprises contacting the feed stock or process stream with a microorganism expressing an enzyme selected from the group consisting of aflatoxin oxidase, 3-O acetyltransferase, peroxidase, F420H2-dependent reductases, Ery4, UDP-glycosyltransferase, laccase, manganese peroxidase, deoxynivalenol hydroxylase, lactono hydrolase, Zearalenone hydrolase, xylanase, DepA/DepB, and 2cys-peroxiredoxin, a broth containing a such a microorganism, a lysate formed by lysing a such a microorganism, a concentrate of such a microorganism or lysate, or combinations thereof.
While the invention has been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details may be made to the various embodiments disclosed herein without departing from the spirit and scope of the invention and that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims.
SEQUENCE LISTING


    DdnA DNA SEQ ID NO: 1
    kdX Protein Modified DNA Sequence SEQ ID NO: 2
    kdR Protein Modified DNA Sequence SEQ ID NO: 3
    pUC57 Cloning Vector SEQ ID NO: 4
    pPink-HC (High Copy P. pastoris vector) SEQ ID NO: 5
    pPink-LC (Low Copy Vector for P. pastoris) SEQ ID NO: 6
    pPink-aHC (High Copy P. pastoris Vector) SEQ ID NO: 7
    pJKW1148 (same backbone as the pYEDP60 plasmid) SEQ ID NO: 8
    pCWori SEQ ID NO: 9
    Single Expression Cassette of Ddna, Kdx, Kdr expression SEQ ID NO: 10

## Claims

1. A method of mitigating deoxyniyalenol contamination in a food or feed product produced as a byproduct of fermentation in a dry mill or wet mill grain processing facility, the method comprising:
inoculating a feedstock comprising ground corn with an ethanologen to produce ethanol and disiller's grain;
contacting the feedstock or a fermentation process stream with deoxynivalenol hydroxylase, and
producing a food or feed product from the process stream downstream of said contacting step,
wherein the level of deoxynivalenol in the food or feed product is lower than the level of deoxynivalenol in the feedstock or process stream prior to exposure to the deoxynivalenol hydroxylase.

2. The method of claim 1, wherein the feedstock or process stream is selected from the group consisting of whole corn, cracked corn, steeped corn, steep water, steep liquor, flour, slurry, beer, whole stillage, wet cake, thin stillage, concentrated thin stillage, defatted concentrated thin stillage, germ, oil, fiber, protein, starch, and combinations thereof.

3. The method of claim 1, wherein the step of contacting comprises contacting the feedstock or process stream with a microorganism expressing deoxynivalenol hydroxylase, a broth containing such a microorganism, a lysate formed by lysing such a microorganism, a concentrate of such a microorganism or lysate, or combinations thereof.

4. The method of claim 3, wherein the microorganism is an ethanologen.

5. The method of claim 3, wherein the microorganism is selected from the group consisting of a yeast and a bacterium.

6. The method of claim 5, wherein the microorganism is a yeast selected from the group consisting of S. cerevisiae and Pichia pastoris.

7. The method of claim 5, wherein the microorganism is a bacterium selected from the group consisting of E. coli, Lactobacillus, and Zymomonas.

8. The method of claim 3, wherein the method further comprises contacting the feedstock or process stream with a microorganism expressing KdR and/or KdX, a broth containing such a microorganism, a lysate formed by lysing such a microorganism, a concentrate of such a microorganism or lysate, or combinations thereof.

9. The method of claim 8, wherein the gene encoding KdR, the gene encoding KdX and the gene encoding deoxynivalenol hydroxylase are provided on an expression cassette comprising a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO: 10, or a polynucleotide comprising a nucleotide sequence having at least about 95% sequence identity to SEQ ID NO: 10.

10. The method of claim 8, wherein the deoxynivalenol hydroxylase is expressed in a first microorganism and the KdR and/or KdX are expressed in a second microorganism.

11. The method of claim 8, wherein the deoxynivalenol hydroxylase and the KdR and/or KdX are expressed in the same microorganism.

12. The method of claim 1, wherein the level of mycotoxin in the food or feed product is less than about 20 ppm.

13. The method of claim 1, wherein the level of mycotoxin in the food or feed product is less than about 10 ppm.

14. The method of claim 1, wherein the level of mycotoxin in the food or feed product is less than about 5 ppm.

15. The method of claim 1, wherein the level of mycotoxin in the food or feed product is less than about 2 ppm.