Inhibition of methane production in ruminants

Abstract

The present disclosure provides a method for reducing methane emissions from a ruminant comprising administering to the ruminant a composition comprising a fungal strain, biomass from the fungal strain, a culture supernatant from the fungal strain wherein the fungal strain comprises at least one vanadium-dependent haloperoxidase (VHPO) with an amino acid sequence having at least 80% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-100 and a combination thereof. The present disclosure also provides a method for reducing methane emissions with a fungal strain belonging to a genus selected from the group consisting of Alternaria, Ascomycota, Bipolaris, Cadophora, Cladorrhinum, Clohesyomyces, Darksidea, Didymella, Dothideomycetes, Drechslera, Exserohilum, Epicoccum, Exophiala, Gaeumannomyces, Gliomastix, Helotiales, Keissleriella, Laburnicola, Lachnum, Leptodontidium, Magnaporthiopsis, Mollisia, Neocamarosporium, Oidiodendron, Ophiosphaerella, Penicillium, Phaeosphaeria, Phialophora, Phoma, Pleosporales, Poaceascoma, Septoriella, Slopeiomyces, Sordariales, Trametes, Trematosphaeria, Trichoderma, and Tricladium.

Description

[0001] INHIBITION OF METHANE PRODUCTION IN RUMINANTS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of Australian Patent Application No. 2023203976, filed on June 23, 2023, the contents of which are incorporated herein by reference in their entirety.

[0004] TECHNICAL FIELD

[0005] The present disclosure relates to the reduction of methane emissions from ruminants. Specifically, the present invention relates to methods and compositions involving several fungal species that, when administered to ruminants, can reduce the methane production of methanogens in the rumen and decrease methane emissions.

[0006] STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0007] A Sequence Listing in XML format, entitled LOAM-B003-00AU.xml, 139,286 bytes in size, generated on June 21, 2023, and filed herewith, is hereby incorporated by reference in its entirety for its disclosures.

[0008] BACKGROUND

[0009] Methane is a potent greenhouse gas, absorbing infrared radiation much more efficiently than CO2 and having a warming potential of about 86 times larger than its mass equivalent of CO2 on a 20-year timescale (IPCC, 2014). Methane is the second-most abundant greenhouse gas after carbon dioxide (CO2). CH4 is short-lived relative to other greenhouse gases (GHG), persisting in the atmosphere for about 10 years. Methane’s short lifespan means that taking steps now to significantly reduce methane will have a significant impact within our lifetimes.

[0010] Ruminants produce CH4 as a by-product of the anaerobic microbial fermentation of feeds in the rumen and, to a lesser extent, in the large intestine. The ruminal microbial community is highly diverse and composed of bacteria, protozoa, fungi, and bacteriophages that act collectively to ferment ingested organic matter (OM) to produce short chain fatty acids that are absorbed across the rumen wall into the blood stream. However, anaerobic microbial fermentation in the rumen also produces carbon dioxide and hydrogen but if hydrogen is allowed to accumulate there is inhibition of both forage digestion and microbial growth. Methanogens such as Archaea present in the rumen use these end-products and produce CH4. The production of CH4 reduces the partial pressure of H2 in the rumen, which could otherwise inhibit rumen fermentation, but it also reduces the amount of energy and carbon available for formation of the short chain fatty acids that are essential for ruminant nutrition. Furthermore, most of the CH4 produced in ruminants is exhaled and belched by the animal and so increases atmospheric CH4.

[0011] Agriculture contributes around 40-46% of global methane emissions, and because of rising food production, these emissions are on a path to increase roughly 40% by 2050. Of these emissions, two-thirds are from methanogenesis from livestock, primarily cattle, buffalo, sheep and goats and mostly released by eructation (in contrast to manure).

[0012] Numerous approaches for mitigating enteric methane emissions have been proposed and investigated over the last several decades, including rumen manipulation through feed supplements that indirectly or directly inhibit methanogenesis including administering seaweeds containing halogenated compounds. Seaweeds are producers of halogenated natural products. Biosynthesis of halogenated molecules is linked to reactive oxygen species (ROS) such as hydrogen peroxide that are substrates for haloperoxidase enzymes that promote the formation of halogenated molecules. It has been shown that vanadium-dependent haloperoxidases (VHPOs), halogenating enzymes that are present in seaweeds, are involved in bromoform biosynthesis. The antimethanogenic activity of bromoform has been studied in vitro by isolating bioactive compounds from the red seaweed Asparagopsis taxiformis.

[0013] The red macroalga Asparagopsis taxiformis has been linked to the reduction of methane (CH4) from beef cattle by up to 99%. A recent study shows that A. taxiformis is a highly efficient feed supplement for CH4 mitigation during enteric fermentation. The antimethanogenic compounds found in A. taxiformis are: bromoform, dibromochloromethane , bromochloroacetic acid, dibromoacetic acid and dichloromethane. However, bromoform, a halomethane, is the most abundant antimethanogenic compound found in A. taxiformis. Bromoform and dibromochloromethane had the highest activity at inhibiting methane production.

[0014] Bromoform is a halogen that has been found to interfere with the methanogenesis pathway by serving as a competitive inhibitor or analog of methyl-coenzyme M reductase (MCR), preventing the final catalysis step.

[0015] It would be attractive, practically and commercially, to be able to reduce the methane emissions produced by ruminants, and in particular, domestic livestock, by using a source of bromoform that is easily produced with limited impact on the environment. Fungal strains expressing VHPOs present an alternative to marine macroalgae for accomplishing this objective.

[0016] SUMMARY

[0017] In some aspects, the present disclosure relates to a method for reducing methane emissions from a ruminant comprising administering to the ruminant a composition comprising a fungal strain, biomass from the fungal strain, a culture supernatant from the fungal strain, or a combination thereof, wherein the fungal strain comprises at least one vanadium-dependent haloperoxidase (VHPO) with an amino acid sequence having at least 80% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-100 and a combination thereof.

[0018] In one aspect, the at least one VHPO comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-31 and a combination thereof.

[0019] In another aspect, the at least one VHPO comprises SEQ ID NO: 28, 29, or a combination thereof. In some aspects, a fungal strain of Leptodontidium orchidicola US-210 (ATCC Accession No. PT A- 127441 ), a mutant thereof having all identifying characteristics of the strain, or a biomass or a culture supernatant thereof is administered to the ruminant.

[0020] In other aspects, the at least one VHPO comprises SEQ ID NO: 30, 31, or a combination thereof. In one aspect, a fungal strain of Leptodontidium orchidicola US-70 (ATCC Accession No. PT A- 127439), a mutant thereof having all identifying characteristics of the strain, or a biomass or a culture supernatant thereof is administered to the ruminant.

[0021] In yet other aspects, the at least one VHPO comprises SEQ ID NO: 8, 25, 27, or a combination thereof. In one aspect, the at least one VHPO comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-80 and a combination thereof.

[0022] In another aspect, the at least one VHPO comprises SEQ ID NO: 43. In some aspects, a fungal strain of Altemaria chlamydospora AU-383 (NMI Accession No. V23 / 011105), a mutant thereof having all identifying characteristics of the strain, or a biomass or a culture supernatant thereof is administered to the ruminant.

[0023] In other aspects, the at least one VHPO comprises SEQ ID NO: 32. In one aspect, a fungal strain of Leptodontidium orchidicola AU-4873 (NMI Accession No. V22 / 003497), a mutant thereof having all identifying characteristics of the strain, or a biomass or a culture supernatant thereof is administered to the ruminant.

[0024] In certain aspects, the at least one VHPO comprises SEQ ID NO: 33. In another aspect, a fungal strain of Penicillium sp. AU-1850 (NMI Accession No. V23 / 011480), a mutant thereof having all identifying characteristics of the strain or a biomass or a culture supernatant thereof is administered to the ruminant.

[0025] In another aspect, a fungal strain of Penicillium griseolum AU-5435 (NMI Accession No. V24 / 010800), a mutant thereof having all identifying characteristics of the strain or a biomass or a culture supernatant thereof is administered to the ruminant. In other aspects, the at least one VHPO comprises SEQ ID NO: 34, 35, or a combination thereof.

[0026] In yet other aspects, the at least one VHPO comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 81-100 and a combination thereof.

[0027] In some aspects, the present disclosure provides a method for reducing methane emissions from a ruminant comprising administering to the ruminant a composition comprising a fungal strain, biomass from the fungal strain, a culture supernatant from the fungal strain, or a combination thereof, wherein the fungal strain belongs to a genus selected from the group consisting of Alternaria, Ascomycota, Bipolaris, Cadophora, Cladorrhinum, Clohesyomyces, Darksidea, Didymella, Dothideomycetes, Drechslera, Exserohilum, Epicoccum, Exophiala, Gaeumannomyces, Gliomastix, Helotiales, Keissleriella, Labumicola, Lachnum, Leptodontidium, Magnaporthiopsis, Mollisia, Neocamarosporium, Oidiodendron, Ophiosphaerella, Penicillium, Phaeosphaeria, Phialophora, Phoma, Pleosporales, Poaceascoma, Seploriella, Slopeiomyces, Sordariales, Trametes, Trematosphaeria, Trichoderma, and Tricladium.

[0028] In one aspect, the fungal strain belongs to a species selected from the group consisting of Alternaria chlamydospora, Cladorrhinum foecundissimum, Darksidea alpha, Darksidea zeta, Leptodontidium orchidicola, Magnaporthiopsis panicorum, Mollisia cinerea, Neocamarosporium phragmitis, Oidiodendron citrinum, Penicillium penarojense, Penicillium griseolum, Phaeosphaeria caricis, Phaeosphaeria microscopica, Phaeosphaeria poagena, Phaeosphaeria sinensis, Poaceascoma lochia, Septoriella leuchtmannii, Slopeiomyces cylindrosporus, Trametes versicolor, Trematosphaeria hydrela, Trematosphaeria terricola, Trichoderma lixii, and Tricladium terrestre.

[0029] In another aspect, the fungal strain is selected from the group consisting of Leptodontidium orchidicola US-210 (ATCC Accession No. PTA-127441), Leptodontidium orchidicola US-70 (ATCC Accession No. PTA- 127439), Alternaria chlamydospora AU-383 (NMI Accession No. V23 / 011105), Leptodontidium orchidicola AU-4873 (NMI Accession No. V22 / 003497), Penicillium sp. AU-1850 (NMI Accession No. V23 / 011480), Penicillium griseolum AU-5435 (NMI Accession No. V24 / 010800), and a mutant thereof having all identifying characteristics of the strain.

[0030] In some aspects, the ruminant is a member of the family Bovidae. In one aspect, the ruminant is Bos taurus or Ovis aries.

[0031] In other aspects, the present disclosure provides a cell or a biologically pure culture of a fungal strain comprising a VHPO with an amino acid sequence acid having at least 80% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-100. In yet other aspects, the present disclosure relates to a cell or a biologically pure culture of Alternaria chlamydospora AU-383 (NMI Accession No. V23 / 01 1105, Penicillium sp. AU-1850 (NMI Accession No. V23 / 011480), Penicillium griseolum AU-5435 (NMI Accession No. V24 / 010800), or a mutant thereof having all identifying characteristics of the strain.

[0032] In one aspect, the disclosure provides an agricultural composition comprising a cell or a biologically pure culture disclosed herein or biomass from the cell or biologically pure culture, a culture supernatant from the cell or biologically pure culture, or a combination thereof, and, optionally, an agriculturally acceptable carrier. In some aspects, the agricultural composition further comprises a cereal, starch, vegetable waste, vitamin, mineral, trace element, emulsifier, aromatizing product, binder, colorant, odorant, thickening agent, or a combination thereof.

[0033] In certain aspects, the disclosure provides animal feed comprising a cell or biologically pure culture disclosed herein or an agriculture composition disclosed herein.

[0034] In other aspects, the disclosure relates to the use of a cell or the biologically pure culture, an agriculture composition, or animal feed disclosed herein for reducing methane emissions from a ruminant.

[0035] The following description is intended only by way of example, and simply illustrates certain selected embodiments of devices, systems, and processes that are consistent with the disclosed subject matter as claimed herein.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1A depicts VHPO activity in equivalent volumes of fungal cell culture from thirty different strains of filamentous fungi. FIG. IB depicts VHPO activity in equivalent amounts of fungal cell biomass from the same thirty fungal strains.

[0038] FIG. 2 depicts VHPO activity in Curvularia sp. AU-4388 (NMI Accession No. V22 / 011149) and Alternaria chlamydospora AU-383 (NMI Accession No. V23 / 011105).

[0039] FIGs. 3A and 3B depict the geometry and distances between conserved amino acid residues, respectively, in the conserved vanadate binding site in the VHPO enzyme.

[0040] FIGs. 4A and 4B depict VHPO activities in several fungal strains measured with the phenol red assay with fungal cultures grown on agar plates and in liquid culture, respectively.

[0041] FIG. 5 depicts VHPO activities in several fungal strains measured with the dichlorodimedone assay.

[0042] FIG. 6A depicts VHPO activities in Curvularia sp. AU-4388 (NMI Accession No. V22 / 011149) and Alternaria chlamydospora AU-383 (NMI Accession No. V23 / 01 1105) under varying conditions. FIG. 6B depicts the dry weights (g / L) of these strains grown under the corresponding conditions.

[0043] FIG. 7 depicts the VHPO activity measured with the phenol red assay of Penicillium sp. AU-1850 (NMI Accession No. V23 / 011480).

[0044] FIG. 8 depicts the VHPO activity measured with the phenol red assay of Penicillium griseolum AU-5435 (NMI Accession No. V24 / 010800).

[0045] DETAILED DESCRIPTION

[0046] Preferred features, embodiments and variations of the invention may be discerned from the following detailed description which provides sufficient information for those skilled in the art to perform the invention. The detailed description is not to be regarded as limiting the scope of the preceding summary of the invention in any way.

[0047] In compliance with the statute, the invention has been described in language more or less specific to structural or methodical features. The term “comprises” and its variations, such as “comprising” and “comprised of’ is used throughout in an inclusive sense and not to the exclusion of any additional features. It is to be understood that the invention is not limited to specific features shown or described since the means herein described comprises preferred forms of putting the invention into effect. The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims appropriately interpreted by those skilled in the art.

[0048] Throughout the specification and claims (if present), unless the context requires otherwise, the term “substantially” or “about” will be understood to not be limited to the value for the range qualified by the terms.

[0049] It is to be understood that unless specifically stated otherwise, references to “a,” “an,” and / or “the” may include one or more than one and that reference to an item in the singular may also includes the item in the plural. Reference to an element by the indefinite article “a,” “an” and / or “the” does not exclude the possibility that more than one of the elements are present, unless the context clearly requires that there is one and only one of the elements. As used herein, the term “comprise,” and conjugations or any other variation thereof, are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

[0050] A “ruminant” is a mammal of the order Artiodactyla that digests plant-based food by initially softening and partially fermenting it within the animal's first stomach chambers, then regurgitating the semi-digested mass, now known as cud, and chewing it again. The process of rechewing the cud to further break down plant matter and stimulate digestion is called “ruminating”. Ruminants have a digestive tract with four chambers, namely the rumen, reticulum, omasum and abomasum. In the first two chambers, the rumen and the reticulum, the food is mixed with saliva and separates into layers of solid and liquid material. Solids clump together to form the cud, or bolus. The cud is then regurgitated, chewed slowly to completely mix it with saliva, which further breaks down fibers. Fiber, especially cellulose, is broken down into glucose in these chambers by symbiotic anaerobic bacteria, protozoa and fungi. The broken-down fiber, which is now in the liquid part of the contents, then passes through the rumen into the next stomach chamber, the omasum. The food in the abomasum is digested much like it would be in the monogastric stomach. Digested gut contents are finally sent to the small intestine, where the absorption of the nutrients occurs. Almost all the glucose produced by the breaking down of cellulose is used by the symbiotic bacteria. Ruminants get their energy from the volatile short chain fatty acids (VFAs) produced by the bacteria, namely acetate, propionate, butyrate, valerate, and isovalerate. Ruminants included cattle, goats, sheep, giraffes, yaks, deer, antelope, and others.

[0051] As used herein, the term “bovid” includes any member of the family Bovidae, which include hoofed mammals such as antelope, sheep, goats, and cattle, among others.

[0052] As used herein, the term “reducing” includes the reduction of amount of substance in comparison with a reference. For example, the reduction in the amount of total gas and / or methane produced by a ruminant animal or animals administered a composition comprising a fungal strain according to the present invention, relative to an animal or animals not administered a composition comprising a fungal strain of the present invention. The reduction can be measured in vitro with an artificial rumen system that simulates anaerobic fermentation, or in vivo with animals confined in respiration chambers. It is within the knowledge and skill of those trained in the art to assess enteric methanogenesis by a ruminant animal.

[0053] As used herein, the term “reducing methane production” refers to the reduction of methane produced in the gastro-intestinal tract. The term includes the specific volume of methane generated as a result of anaerobic fermentation, for example, in the systems described herein. Fermentation in the rumen and the gut of a ruminant gives rise to production of methane. The present invention aims to reduce this process, such as to reduce the total amount of methane produced in the gastrointestinal tract. It is within the knowledge and skill of those trained in the art to assess methane production by a ruminant animal.

[0054] Fungal Strains Expressing a Vanadium-Dependent Haloperoxidase In certain aspects, the present disclosure provides a method for reducing methane emissions from a ruminant comprising administering to the ruminant a composition comprising a fungal strain, biomass from the fungal strain, a culture supernatant from the fungal strain, or a combination thereof, wherein the fungal strain comprises a vanadium-dependent haloperoxidase (VHPO).

[0055] In some aspects, the VHPO comprises an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-100 and a combination thereof (see Table 1). In one aspect, the VHPO comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-31 and a combination thereof. In another aspect, the VHPO comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-80 and a combination thereof. In another aspect, the VHPO comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 81-100 and a combination thereof.

[0056] In other aspects, the VHPO is a variant protein of the protein comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 3-100. Variant proteins are within the scope of the invention as long as the resulting variant protein retains similar characteristics when compared to the parent peptide. Exemplary modifications are for example conservative substitutions that will result in VHPO variants with similar characteristics to those of the parent molecules. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids can be divided into four families: (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. Alternatively, the amino acid repertoire can be grouped as (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), with serine and threonine optionally grouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur-containing (cysteine and methionine) (Stryer (ed.), Biochemistry, 2nd ed, WH Freeman and Co., 1981). Nonconservative substitutions can be made to the VHPO variants that involve substitutions of amino acid residues between different classes of amino acids to improve properties of the VHPO variants. Whether a change in the amino acid sequence of a polypeptide or fragment thereof results in a functional homolog can be readily determined by assessing the ability of the modified polypeptide or fragment to produce a response in a fashion similar to the unmodified polypeptide or fragment using the assays described herein. Peptides, polypeptides or proteins in which more than one replacement takes place can readily be tested in the same manner.

[0057] In certain aspects, the VHPO or variant protein thereof demonstrates improved properties compared to a reference VHPO. The improved properties of the VHPO or variant protein thereof include increased substrate specificity, substrate selectivity, substrate binding, cofactor specificity, cofactor selectivity, cofactor binding, enzyme activity, enzyme expression, retention of the enzyme in the cell or sub-cellular component (e.g., cell membrane, cytoplasm, cytoskeleton, nucleus, centriole, lysosome, endoplasmic reticulum, ribosome, Golgi apparatus, mitochondria, peroxisome), secretion of the enzyme from the cell, and / or enzyme activity across a broader temperature range or pH range compared to the reference VHPO. In one aspect, the reference VHPO comprises SEQ ID NO: 1. In another aspect, the reference VHPO comprises SEQ ID NO: 2.

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] Deposited Fungal Strains

[0065] Biological deposits of each of the fungal strains listed in Table 2 were made on the dates shown at the American Type Culture Collection (ATCC®), located at 10801 University Blvd., Manassas, VA 20110, USA, or the National Measurement Institute (NMI), 1 / 153 Bertie Street, Port Melbourne, Victoria 3207, Australia, under the provisions of the Budapest Treaty and assigned by each International Depositary Authority (IDA) the accession numbers indicated. Upon issuance of a patent, all restrictions upon the deposits will be irrevocably removed. The deposits are intended to meet the requirements of 37 CFR §§ 1.801-1.809. The deposits will be maintained in the IDAs for a period of 30 years, or 5 years after the last request, or for the effective, enforceable life of the patent, whichever is longer, and will be replaced, if necessary, during that period; and the requirements of 37 CFR §§ 1.801-1.809 are met.

[0066] Table 2. Fungal strains deposited with the ATCC and NM1 International Depositary Authorities.

[0067] Animal Feed

[0068] Also disclosed herein is an animal feed comprising the compositions described herein (i.e., a composition comprising a fungal strain, biomass from the fungal strain, a culture supernatant from the fungal strain, or a combination thereof). The animal feed may be solid (e.g., powder, granules, pellets), semi-solid (e.g. gel, ointment, cream, paste) or liquid (e.g. solutions, suspensions, emulsions). The animal feed may independently be solid, semi-solid (e.g., gel, ointment, cream, paste) or liquid (e.g. solutions, suspensions, emulsions). For example, the animal feed may both be liquid or both be semi-solid or both be solid. Alternatively, the animal feed and composition may each be a different physical state. For example, the animal feed may be solid or semi-solid and the composition may be liquid. The composition may, for example, be used to "top- dress" (added on top) a ruminant feedlot ration or may be used to blend into a total mixed ration.

[0069] The composition may, for example, be added to the drinking water of the animal. In certain embodiments, the composition may be added to the drinking water of the animal immediately before ingestion, for example up to 1 hour before ingestion or up to 30 minutes before ingestion or up to 15 minutes before ingestion or up to 5 minutes before ingestion.

[0070] The three main types of animal feed include roughages, concentrates and mixed feeds. In general, roughages contain a higher percentage of crude fiber and a lower percentage of digestible nutrients than concentrates. For example, roughages may be defined as containing equal to or greater than 20 wt% crude fiber and equal to or less than 60 wt% total digestible nutrients. Roughages may include, for example, dry roughages (e.g., hay, straw, artificially dehydrated forages containing at least 90 wt% dry matter), silages (formed from green forages such as grass, alfalfa, sorghum and corn and preserved in a silo at dry matter contents of 20 to 50 %), and pastures (e.g. green growing pastures providing forage that has a high water content and generally less than 30 % dry matter). The two basic types of roughages include grasses and legumes. Grasses are generally higher in fiber and dry matter than legumes. Legumes are generally higher in proteins, metabolizable energy, vitamins and minerals. Concentrates contain a relatively lower percentage of crude fiber and a higher percentage of digestible nutrients than roughages. For example, concentrates may be defined as containing less than 20 wt% crude fiber and greater than 60 wt% total digestible nutrients. Concentrates may include, for example, energy-rich grains and molasses. Corn, wheat, oats, barley and milo (sorghum grain) are energy-rich grains, containing about 70 to 80 wt% total digestible nutrients.

[0071] Mixed feeds are generally a mixture of roughages and concentrates to provide "complete" balanced rations and may be either high or low in energy, protein or fiber. The disclosed compositions and fungal strains, for example, can be combined with animal feed in various amounts depending on the total amount of fungal strain, culture supernatant from the fungal strain, or combination thereof intended to be administered to the animal.

[0072] The animal feed may, for example, comprise from about 0.0001 wt% to about 10 wt% of disclosed compositions, based on the total dry weight of the animal feed. The animal feed may, for example, comprise from about 0.01 wt% to about 10 wt% of disclosed composition, based on the total dry weight of the animal feed. For example, the animal feed may comprise from about 0.001 wt% to about 9.5 wt%, or from about 0.005 wt% to about 9 wt%, or from about 0.01 wt% to about 8.5 wt%, or from about 0.05 wt% to about 8 wt%, or from about 0.1 wt% to about 7.5 wt%, or from about 0.9 wt% to about 7 wt%, or from about 1 wt% to about 6 wt%, or from about

[0073] 1.5 wt% to about 5.5 wt%, or from about 2 wt% to about 5 wt%, or from about 2.5 wt% to about

[0074] 4.5 wt%, or from about 3 wt% to about 4 wt% disclosed composition based on the total dry weight of the animal feed. For example, the animal feed may comprise from about 0.4 wt% to about 9.5 wt%, or from about 0.5 wt% to about 9 wt%, or from about 0.6 wt% to about 8.5 wt%, or from about 0.7 wt% to about 8 wt%, or from about 0.8 wt% to about 7.5 wt%, or from about 0.9 wt% to about 7 wt%, or from about 1 wt% to about 6 wt%, or from about 1 .5 wt% to about 5.5 wt%, or from about 2 wt% to about 5 wt%, or from about 2.5 wt% to about 4.5 wt%, or from about 3 wt% to about 4 wt% disclosed composition based on the total dry weight of the animal feed.

[0075] In one embodiment, the fungal strain, biomass from the fungal strain, a culture supernatant from the fungal strain, or a combination thereof is administered at a dose of preferably at least 16.67, 10, 5, 3, 2, 1, 0.5, 0.25 0.125 or 0.067% of the organic matter administered to the ruminant animal. For example, if a 450 kg ruminant animal (e.g., steer) consumes 2.5% to 3% of its body weight per day of feed, then the fungal strain is administered at a dose proportional to the amount of organic matter administered to the ruminant. In the case of a 450 kg ruminant animal, and where 80% of the feed is organic matter, if the animal consumes about 2.5% of its body weight per day, then the fungal strain, biomass from the fungal strain, a culture supernatant from the fungal strain, or a combination thereof is administered at a dose of about 0.27, 0. 18, 0.09, 0.045, 0.0225, 0.01125 or 0.00603 kg per day to result in a dose at least 3, 2, 1, 0.5, 0.25 0.125 or 0.067% of the organic matter administered to the ruminant animal.

[0076] Any embodiment of the invention is meant to be illustrative only and is not meant to be limiting to the invention. Therefore, it should be appreciated that various other changes and modifications can be made to any embodiment described without departing from the spirit and scope of the invention.

[0077] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0078] The present invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference in their entirety for all purposes. EXAMPLES

[0079] Example 1. Alternaria chlamydospora AU-383 (NMI Accession No. V23 / 011105) Demonstrates Relatively High Vanadium-Dependent Haloperoxidase (VHPO) Activity

[0080] Thirty strains of filamentous fungi were cultured at 28°C in a liquid medium containing 0.5% yeast extract; 0.2% mineral solution containing 4 mM K2HPO4, 2.5 mM CuSO4, 2.75 mM FeSO4, 4 mM MnCl2, 5 mM ZnSO4; and 8 g / L glucose. After 14 days growth, VHPO activity was determined using the phenol red assay (De Boer, E., et al. (1987). Vanadium containing bromoperoxidase: an example of an oxidoreductase with high operational stability in aqueous and organic media. Biotechnology and Bioengineering, 30(5), 607-610). Each isolate was grown in triplicate for the assay.

[0081] Alternaria chlamydospora AU-383 (NMI Accession No. V23 / 011105) showed the highest VHPO activity in equivalent culture volumes from each fungal strain (FIG. 1A) and in equivalent amounts of fungal cell biomass from each strain (FIG. IB).

[0082] Curvularia sp. AU-4388 (NMI Accession No. V22 / 011149) had previously been identified as a fungal strain demonstrating high VHPO activity (see Int. Pat. Pub. No. WO 2023 / 009899). The relative VHPO activities of Curvularia sp. AU-4388 (NMI Accession No. V22 / 011149) and Alternaria chlamydospora AU-383 (NMI Accession No. V23 / 011105) were compared in repeated sets of experiments.

[0083] Representative data from these experiments is shown in FIG.2. Alternaria chlamydospora AU-383 (NMI Accession No. V23 / 011105) demonstrated similar VHPO activity to Curvularia sp. AU-4388 (NMI Accession No. V22 / 011149) and in some cases outperformed Curvularia sp. AU- 4388 (NMI Accession No. V22 / 011149) in the assay.

[0084] Example 2. Identification of Additional Filamentous Fungi with VHPO Genes

[0085] Many of the filamentous fungal strains in the Loam Bio microbial library had previously had their genomes sequenced. These genomes were queried to identify fungal strains with one or more VHPO genes. Sequences were identified using BlastP to blast sequences against a model VHPO from Curvularia inaequalis (P49053 PRXC_CURIN, vanadium chloroperoxidase, available at https: / / www.uniprot.org / uniprotkb / P49053 / entry). Sequences were classified with either high homology (i.e., Evalue < IE-100) or moderate homology (i.e., Evalue < 1E-20 & > 1E- 100). The list of strains with their corresponding species classification and VHPO copy number are presented in Table 3.

[0086]

[0087]

[0088]

[0089] Example 3. Structural Alignment of VHPO Protein Structures

[0090] The VHPO proteins from C. inequalis (PDB: 1VNC) and Curvularia sp. AU-4388 (NMI Accession No. V22 / 011149) have demonstrated vanadium-dependent haloperoxidase activity. Moreover, the sequences of these VHPO proteins are known, and the structures of VHPO from C. inequalis (PDB: 1VNC) and Curvularia sp. AU-4388 (NMI Accession No. V22 / 011149) are available (see Messerschmidt A, Wever R. X-ray structure of a vanadium-containing enzyme: chloroperoxidase from the fungus Curvularia inaequalis. Proc Natl Acad Sci U S A. 1996 Jan 9;93( 1 ):392-6; de Macedo-Ribeiro S, Renirie R, Wever R, Messerschmidt A. Crystal structure of a trapped phosphate intermediate in vanadium apochloroperoxidase catalyzing a dephosphorylation reaction. Biochemistry. 2008 Jan 22;47(3):929-34; Messerschmidt A, Prade L, Wever R. Implications for the catalytic mechanism of the vanadium-containing enzyme chloroperoxidase from the fungus Curvularia inaequalis by X-ray structures of the native and peroxide form. Biol Chem. 1997 Mar-Apr;378(3-4):309-15). Thus, the protein structures of these VHPOs were used as models for structural comparisons with the predicted structures of the VHPOs identified in the fungal strains from the Loam Bio microbial library.

[0091] Hie amino acid sequence of the VHPO proteins found in strains in the Loam Bio microbial library were modeled by ESMfold (Evolutionary Scale Modeling) (see Lin Z, Akin H, Rao R, Hie B, Zhu Z, Lu W, Smetanin N, Verkuil R, Kabeli O, Shmueli Y, Dos Santos Costa A, FazeLZarandi M, Sercu T, Candido S, Rives A. Evolutionary-scale prediction of atomic-level protein structure with a language model. Science. 2023 Mar 17;379(6637): 1123-1130). The resulting three- dimensional structures were then aligned with the VHPO protein structures corresponding to C. inequalis (PDB: 1VNC) and Curvularia sp. AU-4388 (NMI Accession No. V22 / 011149). The structural alignment gives a root mean square deviation (RMSD) score that determines the degree of structural overlap between two protein structures and is an average measure of how far apart an a-carbon (Ca) atom is in one structure from the Ca atom from the other structure after they are aligned.

[0092] Hie RMSD values for the aligned structures are found in Tables 4. Structures with an RMSD lower than 3 Angstroms indicate VHPO structures that are highly similar to the VHPO structures from C. inequalis (PDB: 1VNC) and Curvularia sp. AU-4388 (NMI Accession No. V22 / 011149).

[0093]

[0094]

[0095] Example 4. Analysis of the Geometry of the VHPO Vanadium Coordination Site

[0096] The vanadium coordination site in the VHPO enzyme is composed of five amino acid residues (i.e., Lys353, Arg360, His404, Arg490 and His496 in C. inequalis (PDB:1VNC)). It is known that the coordination site is highly conserved in the family of chloroperoxidases and bromoperoxidases. A multiple sequence alignment of the fungal strains from the Loam Bio microbial library with VHPO genes confirmed a high degree of conservation of the amino acid residues in this coordination site (data not shown). While this coordination site is conserved, sequence diversity is observed among the members of the family of VHPOs.

[0097] The depictions presented in FIGs. 3A and 3B show the vanadate coordination site in the VHPO protein structure from C. inequalis (PDB:1VNC) with FIG. 3B indicating the distance between amino acids 353, 404 and 490: Lys353 / His404: 12.3 Å; Arg490 / His404: 7.7 Å; Arg490 / Lys353: 15.5 Å. These distances were used to explore the collection of filamentous fungal VHPOs as a model of conservation of the vanadate coordination site. VHPOs with a high degree of consistency to these amino acid distances are considered as similar to the model VHPO enzymes (i.e., those from C. inequalis (PDB: 1VNC) and Curvularia sp. AU-4388 (NMI Accession No. V22 / 011149) as shown in Table 5.

[0098]

[0099]

[0100]

[0101] Example 5. Measurement of VHPO Activity with the Phenol Red and Dichlorodimedone

[0102] Assays

[0103] Phenol Red Assays

[0104] Fungal cultures were prepared on potato dextrose agar (PDA) plates and grown for 2-4 weeks in a room temperature incubator. 9 mm circular sections were taken from the PDA and stored in 4 mL of deionized water, pH 7, or incubated in 15 mL of liquid minimal media (0.5% yeast extract; 0.2 % mineral solution containing 4 mM K2HPO4, 2.5 mM CuSO4, 2.75 mM FeSO4, 4 mM MnCh, 5 mM ZnSO4; 0.5 g / L glucose) for 12 days.

[0105] A sample of 1 mL was taken from the liquid minimal media on day 4, 8, and 12. The liquid minimal media with culture was then stored at 4°C. The samples in the deionized water and the samples in the liquid minimal media were spun at 9000 revolutions per minute (RPM) and the supernatant was assayed with the phenol red assay. Briefly, 50 μl of liquid fungal culture media was added to 50 pl of phenol red solution containing phosphate-citrate (100 mM, pH 5.0), NaBr (50 mM), phenol red (0.45 mM), Na3VO4(1 mM). The reaction was initiated with 20 mM H2O2and incubated over the course of 7 days at 30°C. The assay was repeated at pH 5, 6, and 7 for the phenol red solution. Readings were done every 24 hours and changes at an optical density of 590nm were used to determine VHPO activity. Two positive controls were used in the phenol red assay and were generated with increasing amounts of purified VHPO. Negative controls were generated with samples lacking any fungal culture.

[0106] The results of the phenol red assay performed on fungal samples taken from agar plates at pH 7 are presented in FIG. 4A. The related phenol red assay results from liquid fungal cultures at pH 7 are presented in FIG. 4B. Fungal cultures from the following strains demonstrated VHPO activity comparable to the positive control:

[0107] • Leptodontidium orchidicola US-210 (ATCC Accession No. PTA-127441),

[0108] • Leptodontidium orchidicola US-70 (ATCC Accession No. PTA- 127439),

[0109] • Leptodontidium orchidicola AU-4873 (NMI Accession No. V22 / 003497),

[0110] • Penicillium sp. AU-1850 (NMI Accession No. V23 / 011480),

[0111] • Penicillium penarojense US-133,

[0112] • Mollisia sp. AU-3419,

[0113] • Mollisia cinerea US- 1127,

[0114] • Phaeosphaeria caricis AU-3044, and

[0115] • Darksidea zeta US- 182. Dichlorodimedone Assays

[0116] Fungal samples were obtained from the previous phenol red process: the liquid cultures from Days 4, 8, 12 and circular agar sections stored in deionized water. 50 uL of the sample fungi were combined with 50 uL of dichlorodimedone assay solution containing phosphate-citrate (50 mM, pH 5.5), NaBr (50 mM), and monochlorodimedone (0.015 mM). The reaction was initiated with 20 mM H2O2 and the reading was done 24 hours after the initiation. Changes at an optical density of 290nm were used to determine haloperoxidase activity. The optical density of the negative control (i.e., lacking any fungal culture) was subtracted from the optical densities from each of the fungal samples to generate the values presented in FIG. 5. The results of the dichlorodimedone assay confirmed haloperoxidase activity in each of the strains identified with the phenol red assay except Darksidea zeta US-182.

[0117] Example 6. Confirmation of VHPO Activity with Alternaria chlamydospora AU-383 (NMI Accession No. V23 / 011105)

[0118] Previous studies had indicated that Alternaria chlamydospora AU-383 (NMI Accession No. V23 / 011105) produces considerable VHPO activity. To confirm these observations, a study was conducted to compare the VHPO activities of Alternaria chlamydospora AU-383 (NMI Accession No. V23 / 011105) and Curvularia sp. AU-4388 (NMI Accession No. V22 / 011149). In addition, experimental conditions contributing to enhanced VHPO activity were evaluated.

[0119] For medium preparation, a base medium of yeast extract (5g / L) and agar (0.2 g / L) was prepared and autoclaved. Glucose was prepared in stock of 100 g / L and autoclaved separately. A stock solution (1000X) of trace element was prepared and sterilized by filtration. The final medium was prepared by adding glucose into the base medium according to the defined concentrations. The final volume was adjusted with sterilized water. Finally, 1 mL of trace element solution was added into IL medium to have a final concentration of 9pM MnCL2, 10 pM K2HPO4, 11 pM ZnSO4, 5.5 pM FeSO4, and 6 pM CuSO4.

[0120] For flask culture setup, microparticles and talc were added to 500 mL Flasks and resuspended in 5mL of 100 mM citrate buffer (pH 6.5) or 5mL pure water. Two different forms of talc were evaluated: talc powder A (D50 of 9 pm and D98 of 30 p m; hydrous magnesium silicate 3MgO 4SiO2H2O; “talA”) and talc powder B (D50 of 6.8 pm; “talB”). The 500 mL Flasks were closed with cotton wool plugs and autoclaved. 100 mL of liquid medium was transferred into each 500 mL flask and then 2 mL of inoculum was added. The culture flasks were incubated at 28 °C, 180 rpm in a shaking incubator. In certain cultures, 8 g / L glucose was added on day 2 of the culture (“8gG”). Samples were collected on day 4, 9, 14, 18 and 25. Samples were filtered through 0.2pm (or 0.45pM) pore size for measuring VHPO activity using the phenol red assay. For measuring dry weight of biomass, a glass jar and Miracloth were prepared and weighed. All biomass in a flask was collected by filtration with the prepared Miracloth. Biomass in Miracloth was placed inside the jar and dried to a constant weight. The jar was removed from the oven and placed inside a desiccator before weighing.

[0121] Both Alternaria chlamydospora AU-383 (NMI Accession No. V23 / 011105) and Curvularia sp. AU-4388 (NMI Accession No. V22 / 011149) demonstrated observable VHPO activity which was enhanced by the addition of talc and inclusion of 8 g / L glucose added on day 2 of the cultures (see FIG. 6A). The addition of 8 g / L glucose on day 2 of the cultures also had a positive effect on biomass as evidenced by the dry mass measurements (see FIG. 6B).

[0122] Example 7. VHPO Activity of Penicillium sp. AU-1850 (NMI Accession No. V23 / 011480), Penicillium griseolum AU-5435 (NMI Accession No. V24 / 01088)

[0123] The phenol red assays was used to determine VHPO activities with cultures of Penicillium sp. AU-1850 (NMI Accession No. V23 / 011480), Penicillium griseolum AU-5435 (NMI Accession No. V24 / 01088) after about one week of growth in standard media. The VHPO assay was repeated with two separate cultures of each strain. As shown in FIGs. 7 and 8, both strains had observable VHPO activity compared to the water only negative control. With further optimization of the cultures and assay conditions, these initial results will be improved.

[0124] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

Claims

CLAIMSWhat is claimed is:

1. A method for reducing methane emissions from a ruminant comprising administering to the ruminant a composition comprising a fungal strain, biomass from the fungal strain, a culture supernatant from the fungal strain, or a combination thereof, wherein the fungal strain comprises at least one vanadium-dependent haloperoxidase (VHPO) with an amino acid sequence having at least 80% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-100 and a combination thereof.

2. The method of Claim 1 , wherein the at least one VHPO comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-31 and a combination thereof.

3. The method of Claim 2, wherein the at least one VHPO comprises SEQ ID NO: 28, 29, or a combination thereof.

4. The method of Claim 3 , wherein a fungal strain of Leptodontidium orchidicola US- 210 (ATCC Accession No. PTA-127441), a mutant thereof having all identifying characteristics of the strain, or a biomass or a culture supernatant thereof is administered to the ruminant.

5. The method of Claim 2, wherein the at least one VHPO comprises SEQ ID NO: 30, 31 , or a combination thereof.

6. The method of Claim 5, wherein a fungal strain of Leptodontidium orchidicola US- 70 (ATCC Accession No. PT A- 127439), a mutant thereof having all identifying characteristics of the strain, or a biomass or a culture supernatant thereof is administered to the ruminant.

7. The method of Claim 2, wherein the at least one VHPO comprises SEQ ID NO: 8, 25, 27, or a combination thereof.

8. The method of Claim 1, wherein the at least one VHPO comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-80 and a combination thereof.

9. The method of Claim 8, wherein the at least one VHPO comprises SEQ ID NO: 43.

10. The method of Claim 9, wherein a fungal strain of Alternaria chlamydospora AU-383 (NMI Accession No. V23 / 011105), a mutant thereof having all identifying characteristics of the strain, or a biomass or a culture supernatant thereof is administered to the ruminant.

11. The method of Claim 8, wherein the at least one VHPO comprises SEQ ID NO: 32.

12. The method of Claim 11, wherein a fungal strain of Leptodontidium orchidicola AU-4873 (NMI Accession No. V22 / 003497), a mutant thereof having all identifying characteristics of the strain, or a biomass or a culture supernatant thereof is administered to the ruminant.

13. The method of Claim 8, wherein the at least one VHPO comprises SEQ ID NO: 33.

14. The method of Claim 13, wherein a fungal strain of Penicil Hum sp. AU-1850 (NMI Accession No. V23 / 011480), a mutant thereof having all identifying characteristics of the strain or a biomass or a culture supernatant thereof is administered to the ruminant.

15. The method of Claim 1 , wherein a fungal strain of Penicillium griseolum AU-5435 (NMI Accession No. V24 / 010800), a mutant thereof having all identifying characteristics of the strain or a biomass or a culture supernatant thereof is administered to the ruminant.

16. The method of Claim 8, wherein the at least one VHPO comprises SEQ ID NO: 34, 35, or a combination thereof.

17. The method of Claim 1, wherein the at least one VHPO comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 81-100 and a combination thereof.

18. A method for reducing methane emissions from a ruminant comprising administering to the ruminant a composition comprising a fungal strain, biomass from the fungal strain, a culture supernatant from the fungal strain, or a combination thereof, wherein the fungal strain belongs to a genus selected from the group consisting of Alternaria, Ascomycota, Bipolaris, Cadophora, Cladorrhimim, Clohesyomyces, Darksidea, Didymella, Dothideomycetes, Drechslera, Exserohilum, Epicoccum, Exophiala,Gaeumannomyces, Gliomastix, Helotiales, Keissleriella, Laburnicola, Lachnum, Leptodontidium, Magnaporthiopsis, Mollisia, Neocamarosporium, Oidiodendron, Ophiosphaerella, Penicillium, Phaeosphaeria, Phialophora, Phoma, Pleosporales, Poaceascoma, Septoriella, Slopeiomyces, Sordariales, Trametes, Trematosphaeria, Trichoderma, and Tricladium.

19. The method of Claim 18, wherein the fungal strain belongs to a species selected from the group consisting of Alternaria chlamydospora, Cladorrhinum foecundissimum, Darksidea alpha, Darksidea zeta, Leptodontidium orchidicola, Magnaporthiopsis panicorum, Mollisia cinerea, Neocamarosporium phragmitis, Oidiodendron citrinum, Penicillium penarojense, Penicillium griseolum, Phaeosphaeria caricis, Phaeosphaeria microscopica, Phaeosphaeria poagena, Phaeosphaeria sinensis, Poaceascoma lochia, Septoriella leuchtmannii, Slopeiomyces cylindrosporus, Trametes versicolor, Trematosphaeria hydrela, Trematosphaeria terricola, Trichoderma lixii, and Tricladium terrestre.

20. The method of Claim 19, wherein the fungal strain is selected from the group consisting of Leptodontidium orchidicola US-210 (ATCC Accession No. PTA-127441), Leptodontidium orchidicola US-70 (ATCC Accession No. PTA-127439), Alternaria chlamydospora AU-383 (NMI Accession No. V23 / 011105), Leptodontidium orchidicola AU- 4873 (NMI Accession No. V22 / 003497), Penicillium sp. AU- 1850 (NMI Accession No. V23 / 011480), Penicillium griseolum AU-5435 (NMI Accession No. V24 / 010800), and a mutant thereof having all identifying characteristics of the strain.

21. The method of any one of Claims 1 to 20, wherein the ruminant is a member of the family Bovidae.

22. The method of Claim 21, wherein the ruminant is Bos taurus or Ovis aries.

23. A cell or a biologically pure culture of a fungal strain comprising a VHPO with an amino acid sequence having at least 80% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-100.

24. A cell or a biologically pure culture of Alternaria chlamydospora AU-383 (NMI Accession No. V23 / 011105, Penicillium sp. AU-1850 (NMI Accession No. V23 / 011480),Penicillium griseolum AU-5435 (NMI Accession No. V24 / 010800), or a mutant thereof having all identifying characteristics of the strain.

25. An agricultural composition comprising the cell or a biologically pure culture of Claim 23 or 24 or biomass from the cell or biologically pure culture, a culture supernatant from the cell or biologically pure culture, or a combination thereof, and, optionally, an agriculturally acceptable carrier.

26. The agricultural composition of Claim 25, further comprising a cereal, starch, vegetable waste, vitamin, mineral, trace element, emulsifier, aromatizing product, binder, colorant, odorant, thickening agent, or a combination thereof.

27. Animal feed comprising the cell or biologically pure culture of Claim 23 or 24 or the agricultural composition of Claim 25 or 26.

28. Use of the cell or the biologically pure culture of Claim 23 or 24, the agricultural composition of Claim 25 or 26, or the animal feed of Claim 27 for reducing methane emissions from a ruminant.

29. The use of Claim 28, wherein the ruminant is a member of the family Bovidae.

30. The method of Claim 29, wherein the ruminant is Bos taunts or Ovis aries.

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