INHIBITION OF METHANE PRODUCTION IN RUMINANTS
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) with an amino sequence having at least 80% identity with SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. The disclosure also 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.
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 a vanadium-dependent haloperoxidase (VHPO) with an amino sequence having at least 80% identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
2 . The method of claim 1 , wherein the VHPO comprises SEQ ID NO: 3.
3 . The method of claim 1 , wherein the VHPO comprises SEQ ID NO: 4.
4 . The method of claim 1 , wherein the VHPO comprises SEQ ID NO: 5.
5 . The method of claim 1 , wherein the VHPO comprises SEQ ID NO: 6.
6 . The method of claim 1 , wherein the VHPO comprises SEQ ID NO: 7.
7 . The method of claim 1 , wherein the VHPO comprises SEQ ID NO: 8.
8 . The method of any one of claims 1 to 7 , wherein the VHPO demonstrates an improved property compared to a reference VHPO comprising SEQ ID NO: 1 or SEQ ID NO: 2, and
the improved property is increased substrate specificity, substrate selectivity, substrate binding, cofactor specificity, cofactor selectivity, cofactor binding, enzyme activity, enzyme kinetics, enzyme expression, enzyme stability, enzyme biocatalysis, enzyme glycosylation, enzyme methylation, enzyme acetylation, retention of the enzyme in the cell or sub-cellular component, secretion of the enzyme from the cell, and/or enzyme activity across a broader temperature range or pH range compared to the reference VHPO compared to the reference VHPO.
9 . A method for reducing methane emissions from a ruminant comprising administering to the ruminant a halomethane produced with a vanadium-dependent haloperoxidase (VHPO) having an amino sequence with at least 80% identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8,
wherein the halomethane is selected from the group consisting of methyl bromide, methylene bromide, bromoform, methyl iodide, methylene iodide, iodoform, methyl chloride, methylene chloride, chloroform, and combinations thereof.
10 . The method of claim 9 , wherein the VHPO comprises SEQ ID NO: 3.
11 . The method of claim 9 , wherein the VHPO comprises SEQ ID NO: 4.
12 . The method of claim 9 , wherein the VHPO comprises SEQ ID NO: 5.
13 . The method of claim 9 , wherein the VHPO comprises SEQ ID NO: 6.
14 . The method of claim 9 , wherein the VHPO comprises SEQ ID NO: 7.
15 . The method of claim 9 , wherein the VHPO comprises SEQ ID NO: 8.
16 . The method of any one of claims 9 to 15 , wherein the halomethane is bromoform.
17 . The method of any one of claims 9 to 16 , wherein the halomethane is administered in an amount of between 1 mg/kg dry matter intake (DMI) and 1000 mg/kg DMI to the ruminant.
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 is Curvularia lunata US-991 (ATCC Accession No. PTA-127504), Curvularia pseudobrachyspora US-215 (ATCC Accession No. PTA-127509), Curvularia spicifera US-188 (ATCC Accession No. PTA-127510), Curvularia inaequalis US-998 (ATCC Accession No. PTA-127511), Curvularia intermedia US-429 (ATCC Accession No. PTA-127512), Curvularia hawaiiensis US-446 (ATCC Accession No. PTA-127601), Curvularia protuberata US-1111 (ATCC Accession No. PTA-127607), or a mutant thereof having all identifying characteristics of the strain.
19 . The method of claim 18 , wherein the fungal strain is Curvularia lunata US-991 (ATCC Accession No. PTA-127504) or a mutant thereof having all identifying characteristics of the strain.
20 . The method of claim 18 , wherein the fungal strain is Curvularia pseudobrachyspora US-215 (ATCC Accession No. PTA-127509) or a mutant thereof having all identifying characteristics of the strain.
21 . The method of claim 18 , wherein the fungal strain is Curvularia spicifera US-188 (ATCC Accession No. PTA-127510) or a mutant thereof having all identifying characteristics of the strain.
22 . The method of claim 18 , wherein the fungal strain is Curvularia inaequalis US-998 (ATCC Accession No. PTA-127511) or a mutant thereof having all identifying characteristics of the strain.
23 . The method of claim 18 , wherein the fungal strain is Curvularia intermedia US-429 (ATCC Accession No. PTA-127512) or a mutant thereof having all identifying characteristics of the strain.
24 . The method of claim 18 , wherein the fungal strain is Curvularia hawaiiensis US-446 (ATCC Accession No. PTA-127601) or a mutant thereof having all identifying characteristics of the strain.
25 . The method of claim 18 , wherein the fungal strain is Curvularia protuberata US-1111 (ATCC Accession No. PTA-127607) or a mutant thereof having all identifying characteristics of the strain.
26 . A method for reducing methane emissions from a ruminant comprising administering to the ruminant a halomethane produced by a fungal strain selected from the group consisting of Curvularia lunata US-991 (ATCC Accession No. PTA-127504), Curvularia pseudobrachyspora US-215 (ATCC Accession No. PTA-127509), Curvularia spicifera US-188 (ATCC Accession No. PTA-127510), Curvularia inaequalis US-998 (ATCC Accession No. PTA-127511), Curvularia intermedia US-429 (ATCC Accession No. PTA-127512), Curvularia hawaiiensis US-446 (ATCC Accession No. PTA-127601), Curvularia protuberata US-1111 (ATCC Accession No. PTA-127607), and a mutant thereof having all identifying characteristics of the strain,
wherein the halomethane is selected from the group consisting of methyl bromide, methylene bromide, bromoform, methyl iodide, methylene iodide, iodoform, methyl chloride, methylene chloride, chloroform, and combinations thereof.
27 . The method of claim 26 , wherein the fungal strain is Curvularia lunata US-991 (ATCC Accession No. PTA-127504) or a mutant thereof having all identifying characteristics of the strain.
28 . The method of claim 26 , wherein the fungal strain is Curvularia pseudobrachyspora US-215 (ATCC Accession No. PTA-127509) or a mutant thereof having all identifying characteristics of the strain.
29 . The method of claim 26 , wherein the fungal strain is Curvularia spicifera US-188 (ATCC Accession No. PTA-127510) or a mutant thereof having all identifying characteristics of the strain.
30 . The method of claim 26 , wherein the fungal strain is Curvularia inaequalis US-998 (ATCC Accession No. PTA-127511) or a mutant thereof having all identifying characteristics of the strain.
31 . The method of claim 26 , wherein the fungal strain is Curvularia intermedia US-429 (ATCC Accession No. PTA-127512) or a mutant thereof having all identifying characteristics of the strain.
32 . The method of claim 26 , wherein the fungal strain is Curvularia hawaiiensis US-446 (ATCC Accession No. PTA-127601) or a mutant thereof having all identifying characteristics of the strain.
33 . The method of claim 26 , wherein the fungal strain is Curvularia protuberata US-1111 (ATCC Accession No. PTA-127607) or a mutant thereof having all identifying characteristics of the strain.
34 . The method of any one of claims 26 to 33 , wherein the halomethane is bromoform.
35 . The method of any one of claims 26 to 34 , wherein the halomethane is administered in an amount of between 1 mg/kg dry matter intake (DMI) and 1000 mg/kg DMI to the ruminant.
36 . The method of any one of claims 1 to 35 , wherein the ruminant is a member of the family Bovidae.
37 . The method of claim 36 , wherein the ruminant is Bos taurus or Bos indicus.
38 . The method of claim 36 , wherein the ruminant is Ovis aries or Capra hircus.
39 . A cell or a biologically pure culture of a fungal strain comprising a VHPO with an amino sequence having at least 80% identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
40 . The cell or a biologically pure culture of claim 39 , wherein the fungal strain is a yeast belonging to the genus of Arxula, Candida, Ogataea, Kluyveromyces, Pichia, Saccharomyces , or Yarrowia.
41 . The cell or a biologically pure culture of claim 40 , wherein the yeast is Saccharomyces cerevisiae, Yarrowia lipolytica , or Pichia pastoris.
42 . A cell or a biologically pure culture of Curvularia lunata US-991 (ATCC Accession No. PTA-127504), Curvularia pseudobrachyspora US-215 (ATCC Accession No. PTA-127509), Curvularia spicifera US-188 (ATCC Accession No. PTA-127510), Curvularia inaequalis US-998 (ATCC Accession No. PTA-127511), Curvularia intermedia US-429 (ATCC Accession No. PTA-127512), Curvularia hawaiiensis US-446 (ATCC Accession No. PTA-127601), Curvularia protuberata US-1111 (ATCC Accession No. PTA-127607), or a mutant thereof having all identifying characteristics of the strain.
43 . An agricultural composition comprising the cell or a biologically pure culture of any one of claims 39 to 42 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.
44 . The agricultural composition of claim 43 , further comprising a cereal, starch, vegetable waste, vitamin, mineral, trace element, emulsifier, aromatizing product, binder, colorant, odorant, thickening agent, or a combination thereof.
45 . Animal feed comprising the biologically pure culture of any one of claims 39 to 42 or the agricultural composition of claim 43 or 44 .
46 . The animal feed of claim 45 , wherein the animal feed is in the form of a powder, granule, pellet, feed block, or lick block.
47 . Use of the cell or the biologically pure culture of any one of claims 39 to 42 , the agricultural composition of claim 43 or 44 , or the animal feed of claim 45 or 46 for reducing methane emissions from a ruminant.
48 . The use of claim 47 , wherein the ruminant is a member of the family Bovidae.
49 . The use of claim 48 , wherein the ruminant is Bos taurus or Bos indicus.
50 . The use of claim 48 , wherein the ruminant is Ovis aries or Capra hircus.