US20210084939A1 - Animal feed compositions and methods of use - Google Patents

Animal feed compositions and methods of use Download PDF

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Publication number
US20210084939A1
US20210084939A1 US16/603,621 US201816603621A US2021084939A1 US 20210084939 A1 US20210084939 A1 US 20210084939A1 US 201816603621 A US201816603621 A US 201816603621A US 2021084939 A1 US2021084939 A1 US 2021084939A1
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Prior art keywords
corn
plant
cattle
amylase
steam
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James S. Drouillard
Lucas Michael Horton
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Syngenta Participations AG
Kansas State University
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Syngenta Participations AG
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Assigned to KANSAS STATE UNIVERSITY RESEARCH FOUNDATION reassignment KANSAS STATE UNIVERSITY RESEARCH FOUNDATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DROUILLARD, JAMES S, HORTON, Lucas Michael
Publication of US20210084939A1 publication Critical patent/US20210084939A1/en
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K40/00—Shaping or working-up of animal feeding-stuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C12N9/242—Fungal source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

  • the present invention relates to animal feed compositions and methods of making and using the same.
  • Concentrates or compound feeds are high in energy value, including fat, cereal grains and their by-products (barley, corn, oats, rye, wheat), high-protein oil meals or cakes (soybean, canola, cottonseed, peanut and the like), and by-products from processing of sugar beets, sugarcane, animals, and fish, which can be produced in the form of pellets or crumbles.
  • Concentrates or compound feeds can be complete in that they can provide all the daily required food needs or they can provide a part of the ration, supplementing whatever else may be provided as a food ration.
  • Roughage includes pasture grasses, hays, silage, root crops, straw, and stover (cornstalks).
  • Feed constitutes the largest cost of raising animals for food production.
  • the present invention is directed to compositions and methods for improving the efficiency of animal feed utilization, thereby reducing the cost of production.
  • liver abscesses caused by pyogenic bacteria are common.
  • the prevalence of liver abscesses increases with low forage/high concentrate diets.
  • Reduction of liver abscesses in feedlot cattle results in improved live weight, carcass weight, dressing percentage and carcass trim.
  • Cattle with severe liver abscesses may require additional carcass trimming and in some instances, condemnation of entire viscera. Rupture of an abscess can lead to carcass contamination resulting in interrupted carcass flow, lost time, increased costs and increased labor.
  • the present invention provides an animal feed composition comprising microbial ⁇ -amylase and methods of using the animal feed composition to decrease liver abscesses in cattle.
  • the microbial ⁇ -amylase comprises a polypeptide having at least about 80% identity to the amino acid sequence of SEQ ID NO:1 or a polypeptide encoded by a nucleotide sequence having at least about 80% identity to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5.
  • the animal feed composition comprises steam-flaked corn expressing a microbial ⁇ -amylase. Also provided are methods of producing a steam flaked corn product by steam flaking corn kernels from a transgenic corn plant expressing a microbial ⁇ -amylase, and the steam flaked corn product produced thereby.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y.
  • phrases such as “between about X and Y” mean “between about X and about Y.”
  • phrases such as “from about X to Y” mean “from about X to about Y.”
  • the transitional phrase “consisting essentially of” means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of” when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”
  • the present invention is directed to compositions and methods for improving the efficiency of animal feed utilization, thereby reducing the cost of production.
  • the present inventors have made the surprising discovery that animals fed an animal feed composition comprising microbial ⁇ -amylase can have an increase in the average daily weight gain or growth rate, an increase in the efficiency of feed utilization or require a reduced number of days to achieve a desired weight as compared to animals not fed said feed composition.
  • an animal feed composition comprising microbial ⁇ -amylase.
  • the microbial ⁇ -amylase comprises a polypeptide having at least 80% identity to the amino acid sequence of SEQ ID NO:1 or a polypeptide encoded by a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5.
  • the ⁇ -amylase is a liquid.
  • an animal feed composition of the invention can be a supplement that comprises a liquid microbial ⁇ -amylase that can be added to the feed provided to an animal.
  • the present invention provides an animal feed composition comprising plant material, wherein the plant material comprises an expressed recombinant ⁇ -amylase.
  • the expressed recombinant ⁇ -amylase is encoded by a nucleotide sequence having at least about 80% identity to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5 or comprises a polypeptide having at least about 80% identity to the amino acid sequence of SEQ ID NO:1.
  • the invention provides an animal feed composition
  • plant material from a transgenic plant or plant part comprising a recombinant ⁇ -amylase encoded by a nucleotide sequence having at least about 80% identity to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5 or comprising a polypeptide having at least about 80% identity to the amino acid sequence of SEQ ID NO:1.
  • the transgenic plant or plant part can comprise about 1% to about 100% by weight of the plant material.
  • the transgenic plant or plant part can comprise about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
  • the plant material can comprise one or more different types of plants.
  • the plant material can be from a plant in which recombinant or heterologous (e.g., microbial) ⁇ -amylase is expressed.
  • the plant material comprises, consists essentially of, or consists of material from a plant in which recombinant or heterologous (e.g., microbial) ⁇ -amylase is expressed and material from a plant not expressing said recombinant or heterologous ⁇ -amylase (e.g., a commodity plant).
  • the plant material comprises material from a plant in which recombinant or heterologous (e.g., microbial) ⁇ -amylase is expressed and material from a plant not expressing said recombinant or heterologous ⁇ -amylase (e.g., a commodity plant)
  • the material from a plant in which recombinant or heterologous (e.g., microbial) ⁇ -amylase is expressed can comprise from about 1% to about 99% by weight of the plant material and the material from a plant not expressing said recombinant or heterologous ⁇ -amylase can comprise from about 99% to about 1% by weight of the plant material.
  • plant material can comprise from about 5% to about 100% by weight of the animal feed composition.
  • the plant material can comprise about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 8
  • the animal feed of the invention can be in any form that is useful with this invention.
  • the form of the animal feed can be, but is not limited to, pellets, grain including one or more types of grain mixed (i.e., mixed grain), a mixture of grain and pellets, silage, dry-rolled, steam flaked, whole kernel, coarsely cracked kernels (e.g., coarsely cracked corn), high moisture corn and/or any combination thereof.
  • the animal feed can comprise other components, including but not limited to coarsely cracked kernels, wet distillers grain, dry distillers grain, corn silage, supplements/liquid supplements, corn gluten feed, and/or ground hay.
  • plant material includes any plant part, including but not limited to endosperm, embryos (germ), pericarp (bran coat), pedicle (tip cap), pollen, ovules, seeds (grain), leaves, flowers, branches, stems, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, plant cells including plant cells that are intact in plants and/or parts of plants, plant protoplasts, plant tissues, plant cell tissue cultures, plant calli, plant clumps, and the like.
  • plant cell refers to a structural and physiological unit of the plant, which comprises a cell wall and also may refer to a protoplast.
  • a plant cell of the invention can be in the form of an isolated single cell or can be a cultured cell or can be a part of a higher-organized unit such as, for example, a plant tissue or a plant organ.
  • a “protoplast” is an isolated plant cell without a cell wall or with only parts of the cell wall.
  • a transgenic plant or plant part comprising a recombinant ⁇ -amylase encoded by a nucleotide sequence of the invention comprises a cell comprising said recombinant ⁇ -amylase encoded by a nucleotide sequence of the invention, wherein the cell is a cell of any plant or plant part including, but not limited to, a root cell, a leaf cell, a tissue culture cell, a seed cell, a flower cell, a fruit cell, a pollen cell, and the like.
  • the plant material can be a seed or grain.
  • the plant material can be from any plant.
  • the plant material is from a plant in which recombinant or heterologous (e.g., microbial) ⁇ -amylase can be expressed.
  • the plant material can be a mixture of plant material from a plant in which recombinant or heterologous (e.g., microbial) ⁇ -amylase is expressed and from a plant not expressing said recombinant or heterologous ⁇ -amylase (e.g., a commodity plant).
  • the plant material can be a mixture of normal “commodity” plant material (e.g., commodity corn) and plant material from a transgenic plant of the present invention expressing recombinant or heterologous ⁇ -amylase.
  • normal “commodity” plant material e.g., commodity corn
  • plant material from a transgenic plant of the present invention expressing recombinant or heterologous ⁇ -amylase.
  • the plant material can be from a corn plant, a sorghum plant, a wheat plant, a barley plant, a rye plant, an oat plant, a rice plant, and/or a millet plant.
  • the plant material can be from a corn plant.
  • the plant material can be a seed or grain from a corn plant.
  • the plant material can be a corn plant comprising corn event 3272 (see, U.S. Pat. No. 8,093,453).
  • the invention provides a “total mixed ration” comprising plant material from a transgenic corn plant or plant part stably transformed with a recombinant ⁇ -amylase encoded by a nucleotide sequence having about 80% identity to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5 or comprising a polypeptide having at least about 80% identity to the amino acid sequence of SEQ ID NO:1.
  • total mixed ration can mean the 24 hour feed allowance for an individual animal that includes, for example, plant material from a transgenic corn plant or plant part (e.g., corn kernels, coarsely cracked corn, and the like), supplements and additives, (e.g., vitamins and minerals), and/or “roughages” (e.g., pasture grasses, hays, silage, root crops, straw, and stover (cornstalks)).
  • plant material from a transgenic corn plant or plant part e.g., corn kernels, coarsely cracked corn, and the like
  • supplements and additives e.g., vitamins and minerals
  • roughages e.g., pasture grasses, hays, silage, root crops, straw, and stover (cornstalks)
  • the plant material from the transgenic corn plant or plant part comprises from about 1% to about 100% by weight of the total mixed ration.
  • the transgenic plant or plant part can comprise about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%
  • an animal feed composition that comprises a total mixed ration of the invention.
  • the total mixed ration can comprise about 5% to about 100% by weight of the animal feed composition.
  • the total mixed ration can comprise about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 7
  • the invention provides a corn ration comprising plant material from a transgenic corn plant or plant part stably transformed with a recombinant ⁇ -amylase encoded by a nucleotide sequence having about 80% identity to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5 or comprising a polypeptide having at least about 80% identity to the amino acid sequence of SEQ ID NO:1.
  • corn ration means the 24 hour corn allowance for an individual animal.
  • the plant material from the transgenic corn plant or plant part comprises from about 1% to about 100% by weight of the corn ration.
  • the transgenic plant or plant part can comprise about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,
  • an animal feed composition that comprises a corn ration of the invention.
  • the corn ration can comprise about 5% to about 100% by weight of the animal feed composition.
  • the corn ration can comprise about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 7
  • the total mixed ration can comprise wet corn gluten feed that can be about 10% to about 40% by weight of the animal feed composition.
  • the total mixed ration can comprise wet corn gluten feed that can be about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, by weight of the animal feed composition.
  • the total mixed ration can comprise modified distillers grains with solubles that can be about 5% to about 25% by weight of the animal feed composition. In further embodiments the total mixed ration can comprise modified distillers grains with solubles that can be about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, by weight of the animal feed composition.
  • the total mixed ration can comprise modified distillers grains with solubles that can be about 5% to about 25% by weight of the animal feed composition.
  • the total mixed ration can comprise dry distillers grains that can be about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, by weight of the animal feed composition.
  • the total mixed ration can comprise wet distillers grains with solubles that can be about 5% to about 25% by weight of the animal feed composition. In further embodiments the total mixed ration can comprise wet distillers grains with solubles that can be about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, by weight of the animal feed composition.
  • homologues Different nucleic acids or proteins having homology are referred to herein as “homologues.”
  • the term homologue includes homologous sequences from the same and other species and orthologous sequences from the same and other species.
  • “Homology” refers to the level of similarity between two or more nucleic acid and/or amino acid sequences in terms of percent of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties among different nucleic acids or proteins.
  • the compositions and methods of the invention further comprise homologues to the nucleotide sequences and polypeptide sequences of this invention (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5).
  • Orthologous refers to homologous nucleotide sequences and/or amino acid sequences in different species that arose from a common ancestral gene during speciation.
  • a homologue of this invention has a significant sequence identity (e.g., 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100%) to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • a homologue of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5 can be utilized with any feed composition or method of the invention, alone or in combination with one another and/or with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5.
  • sequence identity refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H.
  • percent sequence identity refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned.
  • percent identity can refer to the percentage of identical amino acids in an amino acid sequence.
  • nucleotide sequences or protein sequences refers to two or more sequences or subsequences that have at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms, described herein and as known in the art, or by visual inspection.
  • the substantial identity exists over a region of the sequences that is at least about 50 residues to about 200 residues, about 50 residues to about 150 residues, and the like, in length.
  • the substantial identity exists over a region of the sequences that is at least about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, or more residues in length.
  • the sequences are substantially identical over the entire length of the coding regions.
  • substantially identical nucleotide or protein sequences perform substantially the same function (e.g., ⁇ -amylase activity).
  • the sequences are substantially identical over at least about 150 residues and have ⁇ -amylase activity.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, Calif.).
  • An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence.
  • Percent sequence identity is represented as the identity fraction multiplied by 100.
  • the comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence.
  • percent identity may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always >0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • BLOSUM62 scoring matrix see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.1 to less than about 0.001.
  • the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.001.
  • Two nucleotide sequences can also be considered to be substantially identical when the two sequences hybridize to each other under stringent conditions.
  • two nucleotide sequences considered to be substantially identical hybridize to each other under highly stringent conditions.
  • Stringent hybridization conditions and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York (1993). Generally, highly stringent hybridization and wash conditions are selected to be about 5° C. lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Very stringent conditions are selected to be equal to the T m for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide with 1 mg of heparin at 42° C., with the hybridization being carried out overnight.
  • An example of highly stringent wash conditions is 0.15M NaCl at 72° C. for about 15 minutes.
  • An example of stringent wash conditions is a 0.2 ⁇ SSC wash at 65° C. for 15 minutes (see, Sambrook, infra, for a description of SSC buffer).
  • a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example of a medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1 ⁇ SSC at 45° C. for 15 minutes.
  • An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6 ⁇ SSC at 40° C. for 15 minutes.
  • stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30° C.
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide
  • a signal to noise ratio of 2 ⁇ or higher than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • Nucleotide sequences that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This can occur, for example, when a copy of a nucleotide sequence is created using the maximum codon degeneracy permitted by the genetic code.
  • a reference nucleotide sequence hybridizes to the “test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 2 ⁇ SSC, 0.1% SDS at 50° C.
  • SDS sodium dodecyl sulfate
  • the reference nucleotide sequence hybridizes to the “test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 1 ⁇ SSC, 0.1% SDS at 50° C. or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 0.5 ⁇ SSC, 0.1% SDS at 50° C.
  • SDS sodium dodecyl sulfate
  • the reference nucleotide sequence hybridizes to the “test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 0.1 ⁇ SSC, 0.1% SDS at 50° C., or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 0.1 ⁇ SSC, 0.1% SDS at 65° C.
  • SDS sodium dodecyl sulfate
  • a further indication that two nucleotide sequences or two polypeptide sequences are substantially identical can be that the protein encoded by the first nucleic acid is immunologically cross reactive with, or specifically binds to, the protein encoded by the second nucleic acid.
  • a polypeptide can be substantially identical to a second polypeptide, for example, where the two polypeptides differ only by conservative substitutions.
  • nucleotide sequences having significant sequence identity to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5 are provided.
  • “Significant sequence identity” or “significant sequence similarity” means at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% identity or similarity with another nucleotide sequence.
  • “significant sequence identity” or “significant sequence similarity” means a range of about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 81% to about 100%, about 82% to about 100%, about 83% to about 100%, about 84% to about 100%, about 85% to about 100%, about 86% to about 100%, about 87% to about 100%, about 88% to about 100%, about 89% to about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, and/or about 99% to about 100% identity or similarity with another nucleotide sequence.
  • a nucleotide sequence of the invention is a nucleotide sequence that has significant sequence identity to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 and encodes a polypeptide having ⁇ -amylase activity.
  • a nucleotide sequence of the invention is a nucleotide sequence that has 80% to 100% identity to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 and encodes a polypeptide having ⁇ -amylase activity.
  • a nucleotide sequence of the invention is a nucleotide sequence that has 95% identity to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5and encodes a polypeptide having ⁇ -amylase activity.
  • a polypeptide of the invention comprises, consists essentially of, or consists of an amino acid sequence that is at least 70% identical, e.g., at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% identical to the amino acid sequence of SEQ ID NO:1 and has a amylase activity.
  • a polypeptide or nucleotide sequence can be a conservatively modified variant.
  • conservatively modified variant refer to polypeptide and nucleotide sequences containing individual substitutions, deletions or additions that alter, add or delete a single amino acid or nucleotide or a small percentage of amino acids or nucleotides in the sequence, where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • a conservatively modified variant of a polypeptide is biologically active and therefore possesses the desired activity of the reference polypeptide (e.g., ⁇ -amylase activity) as described herein.
  • the variant can result from, for example, a genetic polymorphism or human manipulation.
  • a biologically active variant of the reference polypeptide can have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity or similarity (e.g., about 40% to about 99% or more sequence identity or similarity and any range therein) to the amino acid sequence for the reference polypeptide as determined by sequence alignment programs and parameters described elsewhere herein.
  • An active variant can differ from the reference polypeptide sequence by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • Naturally occurring variants may exist within a population. Such variants can be identified by using well-known molecular biology techniques, such as the polymerase chain reaction (PCR), and hybridization as described below. Synthetically derived nucleotide sequences, for example, sequences generated by site-directed mutagenesis or PCR-mediated mutagenesis which encode a polypeptide of the invention, are also included as variants. One or more nucleotide or amino acid substitutions, additions, or deletions can be introduced into a nucleotide or amino acid sequence disclosed herein, such that the substitutions, additions, or deletions are introduced into the encoded protein.
  • PCR polymerase chain reaction
  • additions may be made at the N-terminal or C-terminal end of the native protein, or at one or more sites in the native protein.
  • a substitution of one or more nucleotides or amino acids may be made at one or more sites in the native protein.
  • conservative amino acid substitutions may be made at one or more predicted preferably nonessential amino acid residues.
  • a “nonessential” amino acid residue is a residue that can be altered from the wild-type sequence of a protein without altering the biological activity, whereas an “essential” amino acid is required for biological activity.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue with a similar side chain. Families of amino acid residues having similar side chains are known in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • amino acid sequence variants of the reference polypeptide can be prepared by mutating the nucleotide sequence encoding the enzyme.
  • the resulting mutants can be expressed recombinantly in plants, and screened for those that retain biological activity by assaying for ⁇ -amylase activity using methods well known in the art.
  • Methods for mutagenesis and nucleotide sequence alterations are known in the art. See, e.g., Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; and Techniques in Molecular Biology (Walker & Gaastra eds., MacMillan Publishing Co.
  • deletions, insertions and substitutions in the polypeptides described herein are not expected to produce radical changes in the characteristics of the polypeptide (e.g., the activity of the polypeptide). However, when it is difficult to predict the exact effect of the substitution, deletion or insertion in advance of doing so, one of skill in the art will appreciate that the effect can be evaluated by routine screening assays that can screen for the particular polypeptide activities of interest (e.g., ⁇ -amylase activity).
  • compositions of the invention can comprise active fragments of the polypeptide.
  • fragment means a portion of the reference polypeptide that retains the polypeptide activity of ⁇ -amylase.
  • a fragment also means a portion of a nucleic acid molecule encoding the reference polypeptide.
  • An active fragment of the polypeptide can be prepared, for example, by isolating a portion of a polypeptide-encoding nucleic acid molecule that expresses the encoded fragment of the polypeptide (e.g., by recombinant expression in vitro), and assessing the activity of the fragment.
  • Nucleic acid molecules encoding such fragments can be at least about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 contiguous nucleotides, or any range therein, or up to the number of nucleotides present in a full-length polypeptide-encoding nucleic acid molecule.
  • polypeptide fragments can be at least about 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 525, 550, 600, 625, 650, 675, or 700 contiguous amino acid residues, or any range therein, or up to the total number of amino acid residues present in the full-length polypeptide.
  • the invention provides a polypeptide comprising, consisting essentially of, or consisting of at least about 150 contiguous amino acid residues of a polypeptide of the invention (e.g., SEQ ID NO:1) and having ⁇ -amylase activity.
  • nucleic acid molecule and/or a nucleotide sequence indicates that the nucleic acid molecule and/or nucleotide sequence is transcribed and, optionally, translated.
  • a nucleic acid molecule and/or nucleotide sequence may express or produce a polypeptide of interest or a functional untranslated RNA.
  • a “heterologous” or “recombinant” nucleotide sequence is a nucleotide sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleotide sequence.
  • a “native” or “wild type” nucleic acid, nucleotide sequence, polypeptide or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide or amino acid sequence.
  • a “wild type mRNA” is an mRNA that is naturally occurring in or endogenous to the organism.
  • a “homologous” nucleic acid sequence is a nucleotide sequence naturally associated with a host cell into which it is introduced.
  • nucleic acid can be used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA and chimeras of RNA and DNA.
  • polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain.
  • the nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand.
  • the nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
  • the present invention further provides a nucleic acid that is the complement (which can be either a full complement or a partial complement) of a nucleic acid, nucleotide sequence, or polynucleotide of this invention.
  • Nucleic acid molecules and/or nucleotide sequences provided herein are presented herein in the 5′ to 3′ direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth in the U.S. sequence rules, 37 CFR ⁇ 1.821-1.825 and the World Intellectual Property Organization (WIPO) Standard ST.25.
  • WIPO World Intellectual Property Organization
  • the recombinant nucleic acids molecules, nucleotide sequences and polypeptides of the invention are “isolated.”
  • An “isolated” nucleic acid molecule, an “isolated” nucleotide sequence or an “isolated” polypeptide is a nucleic acid molecule, nucleotide sequence or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • an isolated nucleic acid molecule, nucleotide sequence or polypeptide may exist in a purified form that is at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide.
  • the isolated nucleic acid molecule, the isolated nucleotide sequence and/or the isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more pure.
  • an isolated nucleic acid molecule, nucleotide sequence or polypeptide may exist in a non-native environment such as, for example, a recombinant host cell.
  • a non-native environment such as, for example, a recombinant host cell.
  • isolated means that it is separated from the chromosome and/or cell in which it naturally occurs.
  • a polynucleotide is also isolated if it is separated from the chromosome and/or cell in which it naturally occurs in and is then inserted into a genetic context, a chromosome and/or a cell in which it does not naturally occur (e.g., a different host cell, different regulatory sequences, and/or different position in the genome than as found in nature).
  • the recombinant nucleic acid molecules, nucleotide sequences and their encoded polypeptides are “isolated” in that, by the hand of man, they exist apart from their native environment and therefore are not products of nature, however, in some embodiments, they can be introduced into and exist in a recombinant host cell.
  • the nucleotide sequences and/or nucleic acid molecules of the invention can be operatively associated with a variety of promoters for expression in host cells (e.g., plant cells).
  • operatively associated with when referring to a first nucleic acid sequence that is operatively linked to a second nucleic acid sequence, means a situation when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operatively associated with a coding sequence if the promoter effects the transcription or expression of the coding sequence.
  • a DNA “promoter” is an untranslated DNA sequence upstream of a coding region that contains the binding site for RNA polymerase and initiates transcription of the DNA.
  • a “promoter region” can also include other elements that act as regulators of gene expression. Promoters can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters for use in the preparation of recombinant nucleic acid molecules, i.e., chimeric genes.
  • a “promoter” useful with the invention is a promoter capable of initiating transcription of a nucleotide sequence in a cell of a plant.
  • a “chimeric gene” is a recombinant nucleic acid molecule in which a promoter or other regulatory nucleotide sequence is operatively associated with a nucleotide sequence that codes for an mRNA or which is expressed as a protein, such that the regulatory nucleotide sequence is able to regulate transcription or expression of the associated nucleotide sequence.
  • the regulatory nucleotide sequence of the chimeric gene is not normally operatively linked to the associated nucleotide sequence as found in nature.
  • promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the host cell to be transformed.
  • expression of a nucleotide sequence can be in any plant and/or plant part, (e.g., in leaves, in stalks or stems, in ears, in inflorescences (e.g., spikes, panicles, cobs, etc.), in roots, seeds and/or seedlings, and the like).
  • dicotyledons have been shown to be operational in monocotyledons and vice versa
  • dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons.
  • there is no restriction to the provenance of selected promoters it is sufficient that they are operational in driving the expression of the nucleotide sequences in the desired cell.
  • Promoters useful with the invention include, but are not limited to, those that drive expression of a nucleotide sequence constitutively, those that drive expression when induced, and those that drive expression in a tissue- or developmentally-specific manner These various types of promoters are known in the art.
  • constitutive promoters include, but are not limited to, cestrum virus promoter (cmp) (U.S. Pat. No. 7,166,770), the rice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol. 12:3399-3406; as well as U.S. Pat. No. 5,641,876), CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812), CaMV 19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-324), nos promoter (Ebert et al. (1987) Proc. Natl. Acad.
  • the maize ubiquitin promoter (UbiP) has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926. Further, the promoter expression cassettes described by McElroy et al. ( Mol. Gen. Genet. 231: 150-160 (1991)) can be easily modified for the expression of nucleotide sequences and are particularly suitable for use in monocotyledonous hosts.
  • tissue specific/tissue preferred promoters can be used. Tissue specific or preferred expression patterns include, but are not limited to, green tissue specific or preferred, root specific or preferred, stem specific or preferred, and flower specific or preferred. Promoters suitable for expression in green tissue include many that regulate genes involved in photosynthesis and many of these have been cloned from both monocotyledons and dicotyledons.
  • a promoter useful with the invention is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12:579-589 (1989)).
  • tissue-specific promoters include those associated with genes encoding the seed storage proteins (such as ⁇ -conglycinin, cruciferin, napin and phaseolin), zein (e.g., gamma zein) or oil body proteins (such as oleosin), or proteins involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase and fatty acid desaturases (fad 2-1)), and other nucleic acids expressed during embryo development (such as Bce4, see, e.g., Kridl et al. (1991) Seed Sci. Res. 1:209-219; as well as EP Patent No. 255378).
  • seed storage proteins such as ⁇ -conglycinin, cruciferin, napin and phaseolin
  • zein e.g., gamma zein
  • oil body proteins such as oleosin
  • proteins involved in fatty acid biosynthesis including acyl carrier protein, stearoyl-ACP
  • Tissue-specific or tissue-preferential promoters useful for the expression of nucleotide sequences in plants, particularly maize include but are not limited to those that direct expression in root, pith, leaf or pollen. Such promoters are disclosed, for example, in PCT Publication WO 93/07278, herein incorporated by reference in its entirety.
  • tissue specific or tissue preferred promoters include the cotton rubisco promoter disclosed in U.S. Pat. No. 6,040,504; the rice sucrose synthase promoter disclosed in U.S. Pat. No.
  • tissue-specific/tissue preferred promoters include, but are not limited to, the root-specific promoters RCc3 (Jeong et al. Plant Physiol. 153:185-197 (2010)) and RB7 (U.S. Pat. No. 5,459,252), the lectin promoter (Lindstrom et al. (1990) Der. Genet. 11:160-167; and Vodkin (1983) Prog. Clin. Biol. Res. 138:87-98), corn alcohol dehydrogenase 1 promoter (Dennis et al. (1984) Nucleic Acids Res.
  • SAMS S-adenosyl-L-methionine synthetase
  • corn light harvesting complex promoter Bansal et al. (1992) Proc. Natl. Acad. Sci. USA 89:3654-3658
  • corn heat shock protein promoter O'Dell et al. (1985) EMBO J. 5:451-458; and Rochester et al. (1986) EMBO J.
  • RuBP carboxylase promoter (Cashmore, “Nuclear genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase” pp. 29-39 In: Genetic Engineering of Plants (Hollaender ed., Plenum Press 1983; and Poulsen et al. (1986) Mol. Gen. Genet. 205:193-200), Ti plasmid mannopine synthase promoter (Langridge et al. (1989) Proc. Natl. Acad. Sci. USA 86:3219-3223), Ti plasmid nopaline synthase promoter (Langridge et al.
  • petunia chalcone isomerase promoter van Tunen et al. (1988) EMBO J. 7:1257-1263
  • bean glycine rich protein 1 promoter Kerman et al. (1989) Genes Dev. 3:1639-1646
  • truncated CaMV 35S promoter O'Dell et al. (1985) Nature 313:810-812)
  • potato patatin promoter Wenzler et al. (1989) Plant Mol. Biol. 13:347-354
  • root cell promoter Yamamoto et al. (1990) Nucleic Acids Res. 18:7449
  • maize zein promoter Yama et al. (1987) Mol. Gen.
  • the promoter can be an endosperm-specific promoter including but not limited to a maize gamma-zein promoter or a maize ADP-gpp promoter.
  • Useful promoters for expression in mature leaves are those that are switched on at the onset of senescence, such as the SAG promoter from Arabidopsis (Gan et al. (1995) Science 270:1986-1988).
  • promoters functional in plastids can be used.
  • Non-limiting examples of such promoters include the bacteriophage T3 gene 9 5′ UTR and other promoters disclosed in U.S. Pat. No. 7,579,516.
  • Other promoters useful with the invention include but are not limited to the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).
  • inducible promoters can be used.
  • chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Regulation of the expression of nucleotide sequences via promoters that are chemically regulated enables the polypeptides of the invention to be synthesized only when the crop plants are treated with the inducing chemicals.
  • the promoter may be a chemical-inducible promoter, where application of a chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
  • Chemical inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1 a promoter, which is activated by salicylic acid (e.g., the PR1a system), steroid steroid-responsive promoters (see, e.g., the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88, 10421-10425 and McNellis et al.
  • inducible promoters include ABA- and turgor-inducible promoters, the auxin-binding protein gene promoter (Schwob et al. (1993) Plant J. 4:423-432), the UDP glucose flavonoid glycosyl-transferase promoter (Ralston et al. (1988) Genetics 119:185-197), the MPI proteinase inhibitor promoter (Cordero et al. (1994) Plant J. 6:141-150), and the glyceraldehyde-3-phosphate dehydrogenase promoter (Kohler et al. (1995) Plant Mol. Biol. 29:1293-1298; Martinez et al.
  • inducible promoters include ABA- and turgor-inducible promoters, the auxin-binding protein gene promoter (Schwob et al. (1993) Plant J. 4:423-432), the UDP glucose flavonoid glycosyl-transfer
  • a promoter for chemical induction can be the tobacco PR-1a promoter.
  • a polypeptide of this invention may or may not be targeted to a compartment within the plant through use of a signal sequence.
  • Numerous signal sequences are known to influence the expression or targeting of a polynucleotide to a particular compartment/tissue or outside a particular compartment/tissue.
  • Suitable signal sequences and targeting promoters are known in the art and include, but are not limited to, those provided herein (see, e.g., U.S. Pat. No. 7,919,681).
  • targets include, but are not limited to, the vacuole, endoplasmic reticulum (ER), chloroplast, amyloplast, starch granule, cell wall, seed, or to a particular tissue, e.g., endosperm.
  • a nucleotide sequence encoding a polypeptide of the invention can be operably linked to a signal sequence for targeting and/or retaining the polypeptide to a compartment within a plant.
  • the signal sequence may be an N-terminal signal sequence from waxy, an N-terminal signal sequence from gamma-zein, a starch binding domain, or a C-terminal starch binding domain.
  • the signal sequence can be an ER signal sequence, an ER retention sequence, an ER signal sequence and an additional ER retention sequence.
  • the ⁇ -amylase polypeptides can be fused with one or more signal sequences (and/or nucleotide sequences encoding said polypeptides can be operably linked to nucleotide sequences encoding said signal sequences).
  • expression cassette means a nucleic acid molecule comprising a nucleotide sequence of interest (e.g., the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5), wherein said nucleotide sequence is operatively associated with at least a control sequence (e.g., a promoter).
  • a control sequence e.g., a promoter
  • one or more plant promoters operatively associated with the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5 or a nucleotide sequence having substantial identity to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5 can be provided in an expression cassette for expression in an organism or cell thereof (e.g., a plant, plant part and/or plant cell).
  • An expression cassette comprising a nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • An expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular nucleic acid sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event.
  • an expression cassette can also include other regulatory sequences.
  • a “regulatory sequence” means a nucleotide sequence located upstream (5′ non-coding sequences), within and/or downstream (3′ non-coding sequences) of a coding sequence, and/or which influences the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include, but are not limited to, promoters, enhancers, introns, translation leader sequences, termination signals, and polyadenylation signal sequences.
  • an expression cassette can also include nucleotide sequences encoding signal sequences operably linked to a polynucleotide sequence of the invention.
  • the regulatory sequences or regions can be native/analogous to the plant, plant part and/or plant cell and/or the regulatory sequences can be native/analogous to the other regulatory sequences.
  • the regulatory sequences may be heterologous to the plant (and/or plant part and/or plant cell) and/or to each other (i.e., the regulatory sequences).
  • a promoter can be heterologous when it is operatively linked to a polynucleotide from a species different from the species from which the polynucleotide was derived.
  • a promoter can also be heterologous to a selected nucleotide sequence if the promoter is from the same/analogous species from which the polynucleotide is derived, but one or both (i.e., promoter and/or polynucleotide) are substantially modified from their original form and/or genomic locus, and/or the promoter is not the native promoter for the operably linked polynucleotide.
  • leader sequences derived from viruses are known to enhance gene expression. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the “ ⁇ -sequence”), Maize Chlorotic Mottle Virus (MCMV) and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (Gallie et al. (1987) Nucleic Acids Res. 15:8693-8711; and Skuzeski et al. (1990) Plant Mol. Biol. 15:65-79).
  • TMV Tobacco Mosaic Virus
  • MCMV Maize Chlorotic Mottle Virus
  • AMV Alfalfa Mosaic Virus
  • Other leader sequences known in the art include, but are not limited to, picornavirus leaders such as an encephalomyocarditis (EMCV) 5′ noncoding region leader (Elroy-Stein et al.
  • EMCV encephalomyocarditis
  • potyvirus leaders such as a Tobacco Etch Virus (TEV) leader (Allison et al. (1986) Virology 154:9-20); Maize Dwarf Mosaic Virus (MDMV) leader (Allison et al. (1986), supra); human immunoglobulin heavy-chain binding protein (BiP) leader (Macejak & Samow (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of AMV (AMV RNA 4; Jobling & Gehrke (1987) Nature 325:622-625); tobacco mosaic TMV leader (Gallie et al.
  • TMV Tobacco Etch Virus
  • MDMV Maize Dwarf Mosaic Virus
  • BiP human immunoglobulin heavy-chain binding protein
  • untranslated leader from the coat protein mRNA of AMV AMV RNA 4; Jobling & Gehrke (1987) Nature 325:622-625
  • tobacco mosaic TMV leader Gallie et al
  • An expression cassette also can optionally include a transcriptional and/or translational termination region (i.e., termination region) that is functional in plants.
  • a transcriptional and/or translational termination region i.e., termination region
  • a variety of transcriptional terminators are available for use in expression cassettes and are responsible for the termination of transcription beyond the heterologous nucleotide sequence of interest and correct mRNA polyadenylation.
  • the termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleotide sequence of interest, may be native to the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the nucleotide sequence of interest, the plant host, or any combination thereof).
  • Appropriate transcriptional terminators include, but are not limited to, the CAMV 35S terminator, the tm1 terminator, the nopaline synthase terminator and/or the pea rbcs E9 terminator. These can be used in both monocotyledons and dicotyledons. In addition, a coding sequence's native transcription terminator can be used.
  • An expression cassette of the invention also can include a nucleotide sequence for a selectable marker, which can be used to select a transformed plant, plant part and/or plant cell.
  • selectable marker means a nucleotide sequence that when expressed imparts a distinct phenotype to the plant, plant part and/or plant cell expressing the marker and thus allows such transformed plants, plant parts and/or plant cells to be distinguished from those that do not have the marker.
  • Such a nucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic, herbicide, or the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening (e.g., the R-locus trait).
  • a selective agent e.g., an antibiotic, herbicide, or the like
  • screening e.g., the R-locus trait
  • selectable markers include, but are not limited to, a nucleotide sequence encoding neo or nptII, which confers resistance to kanamycin, G418, and the like (Potrykus et al. (1985) Mol. Gen. Genet. 199:183-188); a nucleotide sequence encoding bar, which confers resistance to phosphinothricin; a nucleotide sequence encoding an altered 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, which confers resistance to glyphosate (Hinchee et al. (1988) Biotech.
  • a nucleotide sequence encoding neo or nptII which confers resistance to kanamycin, G418, and the like
  • a nucleotide sequence encoding bar which confers resistance to phosphinothricin
  • nucleotide sequence encoding a nitrilase such as bxn from Klebsiella ozaenae that confers resistance to bromoxynil (Stalker et al. (1988) Science 242:419-423); a nucleotide sequence encoding an altered acetolactate synthase (ALS) that confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP Patent Application No. 154204); a nucleotide sequence encoding a methotrexate-resistant dihydrofolate reductase (DHFR) (Thillet et al. (1988) J. Biol. Chem.
  • DHFR methotrexate-resistant dihydrofolate reductase
  • a nucleotide sequence encoding a dalapon dehalogenase that confers resistance to dalapon a nucleotide sequence encoding a mannose-6-phosphate isomerase (also referred to as phosphomannose isomerase (PMI)) that confers an ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629); a nucleotide sequence encoding an altered anthranilate synthase that confers resistance to 5-methyl tryptophan; and/or a nucleotide sequence encoding hph that confers resistance to hygromycin.
  • PMI phosphomannose isomerase
  • Additional selectable markers include, but are not limited to, a nucleotide sequence encoding ⁇ -glucuronidase or uidA (GUS) that encodes an enzyme for which various chromogenic substrates are known; an R-locus nucleotide sequence that encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., “Molecular cloning of the maize R-nj allele by transposon-tagging with Ac,” pp.
  • GUS uidA
  • tyrosinase an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to form melanin
  • ⁇ -galactosidase an enzyme for which there are chromogenic substrates
  • lux luciferase
  • a method of increasing the growth rate (weight gain) or the average daily weight gain of an animal comprising feeding to said animal an animal feed composition of the present invention, wherein the growth rate of the animal or the average daily weight gain of the animal is increased by about 0.05 lb/day to about 10 lbs/day as compared to the growth rate of a control animal that is not provided the animal feed composition of the invention.
  • the increase in growth rate or average daily weight gain can be about 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.1, 4.2, 4.21, 4.22, 4.23, 4.24, 4.25, 4.26, 4.27, 4.28, 4.29, 4.3, 4.31, 4.32, 4.33, 4.34, 4.35, 4.36, 4.37, 4.38, 4.39, 4.4, 4.41
  • the increase in growth rate or average daily weight gain can be from about 0.05 lb/day to about 0.5 lb/per day. In further embodiments, the increase in growth rate or average daily weight gain can be about 0.1 lb/day as compared to the growth of a control animal that is not provided said animal feed composition.
  • a method for reducing the number of days needed to achieve a desired weight in an animal comprising feeding to said animal an animal feed composition of the invention, thereby reducing the number of days needed to achieve a desired weight as compared to the number of days needed to achieve the same desired weight in a control animal that is not fed said animal feed composition.
  • a “desired weight” “or desired finished weight” can mean a live weight or a hot carcass weight.
  • a desired live weight can be between about 950 to about 1,600 lbs and a desired hot carcass weight can be between about 700 to about 1,000 lbs.
  • cattle Prior to entering a feedlot, cattle spend most of their life grazing on range or pasture land and then are transported to a feedlot for finishing where they are fed grain and other feed concentrates. Generally, cattle enter a feedlot at a weight of about 600 to about 750 lbs. Depending on weight at placement, the feeding conditions, and the desired finished weight, the period in a feedlot can be in a range from about 90 days to about 300 days. The average gain can be from about 2.5 to about 5 pounds per day.
  • the number of days needed to achieve a desired weight in an animal fed the animal feed compositions of the invention can be reduced by about 1 day to about 30 days as compared to a control animal that is not fed said animal feed composition.
  • the number of days needed to achieve a desired weight in an animal fed the animal feed compositions of the invention can be reduced by about 1 day to about 25 days, about 1 day to about 20 days, about 5 days to about 20 days, about 5 days to about 15 days, and the like, as compared to a control animal that is not fed said animal feed composition.
  • the number of days needed to achieve a desired weight in an animal fed an animal feed composition of the invention can reduced by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days and the like and/or any range therein.
  • a method of increasing the efficiency of feed utilization by an animal comprising feeding to said animal an animal feed composition of the invention in an amount effective to increase the efficiency of feed utilization by said animal as compared to a control animal that is not fed said animal feed composition.
  • Efficiency of feed utilization can be calculated as the gain in body weight of the animal per the amount of feed provided.
  • the body weight is the finished body weight prior to slaughter.
  • the feed provided is the amount of feed that is provided over a period of about 90 days to about 300 days.
  • the feed provided is the amount of feed that is provided over a period of about 100 days to about 275 days, about 125 days to about 250 days, about 150 days to about 225 days, about 180 days to about 200 days, and the like.
  • the time period (number of days) over which the weight gain is measured is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172
  • the feeding value of corn by the animal is increased by about 1% to about 25% as compared to a control animal that is not fed said animal feed composition.
  • the feeding value of corn equals the difference in feed efficiency of the feed composition of the present invention and the feed efficiency of a control animal that is not fed said feed composition, divided by the feed efficiency of said control animal that is not fed said feed composition, all of which is divided by the percent corn inclusion of said feed composition.
  • the increase in feeding value of corn can be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, and the like, and/or any range therein.
  • the increase in the feed value of corn is about 1% to about 10% as compared to a control.
  • the increase in the feed value is about 5% as compared to a control.
  • the efficiency of feed utilization by the animal is increased by about 0.005 to about 0.1 as compared to a control animal that is not fed said animal feed composition. Accordingly, in some embodiments, the increase in efficiency of feed utilization can be about 0.005, 0.006, 0.007, 0.008, 0.009,0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.02, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.03, and the like, and/or any range therein.
  • the increase in the efficiency of feed utilization is about 0.005 to about 0.01 as compared to a control. In a representative embodiment, the increase in the efficiency of feed utilization is about 0.06 as compared to a control.
  • the efficiency of feed utilization also known as “G:F”, is the average daily gain divided by the dry matter intake per day of the animal.
  • the animal is fed about 1 lb to about 30 lbs of an animal feed composition of the invention per animal per day. Accordingly, in some embodiments, the animal is fed about 1 lb, 2 lbs, 3 lbs, 4 lbs, 5 lbs, 6 lbs, 7 lbs, 8 lbs, 9 lbs, 10 lbs, 11 lbs, 12 lbs, 13 lbs, 14 lbs, 15 lbs, 16 lbs, 17 lbs, 18 lbs, 19 lbs, 20 lbs, 21 lbs, 22 lbs, 23 lbs, 24 lbs, 25 lbs, 26 lbs, 27 lbs, 28 lbs, 29 lbs, 30 lbs of the animal feed composition of the invention per animal per day, and the like, and/or any range therein.
  • the animal is fed about 9 lbs to about 21 lbs of the animal feed composition of the invention per animal per day.
  • an animal can be fed the animal feed composition of the invention ad libitum, or about one time to about three times per day (e.g., 1, 2, 3) or any combination thereof.
  • the invention also contemplates a method of reducing liver abscesses in harvested (e.g., slaughtered) cattle, the method comprising the steps of: a) feeding an animal feed composition to cattle, wherein the animal feed composition comprises plant material from a transgenic plant or plant part (e.g., a transgenic corn plant or plant part) expressing a recombinant thermotolerant (optionally, microbial) ⁇ -amylase; and b) harvesting the cattle.
  • a transgenic plant or plant part e.g., a transgenic corn plant or plant part
  • a recombinant thermotolerant optionally, microbial
  • ⁇ -amylase of the invention is as described herein.
  • the transgenic plant or plant part is a transgenic corn plant or plant part (e.g., steam flaked corn kernels), optionally comprising corn event 3272.
  • the transgenic plant or plant part comprises about 1% to about 100% by weight of the plant material.
  • the method can be practiced with any cattle, e.g., beef cattle (steers and/or heifers) and/or dairy cows. In embodiments, the cattle are feedlot cattle.
  • the method of reducing liver abscesses is practiced with cattle that are fed a high concentrate/low forage diet.
  • the diet comprises forage in an amount less than or equal to about 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% (dry matter basis).
  • the diet includes no or essentially no forage.
  • the diet comprises at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more feed concentrate (dry matter basis).
  • the overall frequency of liver abscess formation is reduced (e.g., by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60% or more).
  • the frequency of moderate and/or severe liver abscesses is reduced (e.g., by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60% or more).
  • a harvested (i.e., slaughtered) cattle carcass (or a plurality of harvested cattle carcasses) produced by a feeding method as described herein, wherein the harvested cattle carcass(es) comprises the liver of the harvested animal, and wherein there is a reduced incidence of liver abscesses in the liver of said harvested cattle carcass(es) as compared with carcasses from control cattle that are not fed plant material from a transgenic plant or plant part expressing a recombinant thermotolerant (e.g., microbial) ⁇ -amylase.
  • the overall frequency of liver abscess formation is reduced (e.g., by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60% or more).
  • the frequency of moderate and/or severe liver abscesses is reduced (e.g., by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60% or more).
  • steam flaking plant material e.g., corn kernels
  • a recombinant thermotolerant (e.g., microbial) ⁇ -amylase as described herein
  • the throughput rate of plant material e.g., corn kernels
  • the recombinant thermotolerant ⁇ -amylase can be increased as compared to plant material that does not express the ⁇ -amylase, e.g., under similar or even identical conditions (such as moisture, temperature and the like).
  • the method results in a steam flaked product with substantially the same (for example, within about 5% or 10%) or even improved (reduced) flake thickness, substantially the same (for example, within about 5% or 10%) or even improved (reduced) geometric mean particle size and/or substantially the same (for example, within 5% or 10%) or improved (increased) flake density.
  • An increased throughput rate has advantages as it may support the feeding of an increased number of animals and/or can translate into savings in terms of labor, water, energy (such as electricity and/or natural gas) and/or machinery costs.
  • the invention also provides methods of producing an animal feed by steam flaking a plant material comprising a recombinant thermotolerant (e.g., microbial) ⁇ -amylase.
  • the method comprises: a) providing transgenic corn kernels (as described herein, e.g., whole shelled corn kernels) comprising a recombinant thermotolerant (e.g., microbial) ⁇ -amylase; and b) steam flaking the corn kernels to produce a steam flaked corn product; optionally, wherein the throughput rate is increased as compared with suitable control corn kernels that do not comprise a recombinant thermotolerant (e.g., microbial) ⁇ -amylase, e.g., under similar or even identical conditions (such as moisture, temperature and the like).
  • transgenic corn kernels as described herein, e.g., whole shelled corn kernels
  • a recombinant thermotolerant e.g., microbial
  • the throughput rate is increased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40% or 50% or more.
  • the “throughput” rate generally refers to the rate at which the plant material is processed through the entire steam flaking process and apparatus, which may optionally include a preliminary cleaning chamber/step, a preliminary soaking chamber/step, a follow on cooling chamber/step, a follow on drying chamber/step, and the like.
  • the throughput rate refers specifically to the rate at which the plant material is processed through the steam chamber and flaking mill (e.g., rollers).
  • control plant material, corn kernel and the like refers to a plant material or corn kernel that does not express a recombinant thermotolerant ⁇ -amylase (as described herein), e.g., the control plant material or corn kernel otherwise has similar properties to the transgenic plant material or corn kernels.
  • a “control” steam flaked plant product or “control” steam flaked corn product, as used herein, is produced from a “control” plant material or corn kernel, respectively.
  • the transgenic corn kernels used in the steam flaking methods of the invention comprise corn event 3272.
  • the time to steam flake the transgenic plant material e.g., corn kernels
  • the recombinant thermotolerant (e.g., microbial) ⁇ -amylase can be reduced (e.g., by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more) as compared with the control plant material under otherwise similar conditions.
  • the digestability of starch in the steam flaked plant product is increased (e.g., by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20% or more) as compared with the digestability of starch in a control steam flaked corn product produced from the control corn kernels.
  • Starch digestability is a measure of how much of the starch in the feed is converted to energy and used by the animal and can be determined by any method known in the art, including whole animal (e.g., fecal starch) and laboratory methods (e.g., as described in the accompanying Examples).
  • the steam flaked plant product (e.g., steam flaked corn product) produced by the steam flaking methods of the invention has a decrease in geometric mean particle size (e.g., at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20% or more) as compared with a control steam flaked plant product produced from a control plant product.
  • geometric mean particle size e.g., at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20% or more
  • the steam flaking methods of the invention may result in an increase in browning of the steam flaked plant product (e.g., corn kernels) prepared from plant material expressing a recombinant thermotolerant (e.g., microbial) ⁇ -amylase as compared with a control steam flaked plant product.
  • the increase in browning in the steam-flaked product is observed during and/or after the cooling process.
  • thermotolerant e.g., microbial
  • ⁇ -amylase produces an increased concentration of soluble sugars, such as maltose and other reducing sugars, in the plant material.
  • Reducing sugars can participate in the Maillard reaction (a chemical reaction between amino acids and reducing sugars producing a brown color), which may result in increased browning in steam flaked plant material (e.g., corn kernels) expressing a recombinant thermotolerant (e.g., microbial) ⁇ -amylase as compared with a control steam flaked plant product produced from a control plant material (e.g., as determined by visual inspection or by laboratory analysis).
  • Maillard reaction a chemical reaction between amino acids and reducing sugars producing a brown color
  • steam flaked plant material e.g., corn kernels
  • a recombinant thermotolerant (e.g., microbial) ⁇ -amylase compared with a control steam flaked plant product produced from a control plant material (e.g., as determined by visual inspection or by laboratory analysis).
  • the invention also provides a steam flaked plant product (e.g., corn product) produced by a steam flaking method as described herein.
  • the steam flaked plant product can comprise one or more of the advantages discussed above (e.g., increased starch digestability, decreased geometric mean particle size, decreased flake thickness and/or increased flake density).
  • the steam flaked plant product is utilized more efficiency when fed to animals (e.g., cattle) as compared with the utilization of a control steam flaked plant product.
  • the feed efficiency as measured by gain-to-feed ratio can be increased. Methods of determining an increase in feed efficiency and gain-to-feed ratio are described herein.
  • the gain-to-feed ratio is increased by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15% or more as compared with a ration comprising a control steam flaked plant product.
  • an increase in feed efficiency is observed when feeding the steam flaked plant product of the invention, even as compared with a control steam flaked plant product that has a substantially similar (e.g., within about 5% or 10%) level of starch availability.
  • starch availability is in the range of equal to or greater than about 48%, 49%, 50% and/or equal to or less than about 51%, 52%, 53%, 54% or 55% (including any combination thereof, as long as the lower end of the range is less than the upper end of the range).
  • starch availability is in the range of about 48% to 55%, about 48% to 54%, about 48% to 53%, about 48% to 52%, about 49% to 55%, about 49% to 54%, about 49% to 53%, about 49% to 52%, about 50% to 55%, about 50% to 54%, about 50% to 53%, about 50% to 52%, about 51% to 55%, about 51% to 54%, about 51% to 53% or about 51% to 52%.
  • Starch availability reflects how much of the starch is gelatinized (e.g., during the steam flaking process) and/or otherwise broken out of the protein matrix that typically encapsulates the starch granules (e.g., in the corn kernel), and is therefore available for enzymatic digestion (e.g., in the rumen).
  • Starch availability can be measured by any method known in the art (e.g., Sindt et al., (2000) “Refractive Index: a rapid method for determination of starch availability in grains,” Kansas Agricultural Experiment Station Research Reports, Article 398: Vol. 0: Iss. 1, found on the internet at doi.org/10.4148/2378-5977.1801).
  • starch availability is highly correlated with flake density, which is often used in practice as an indirect measure of starch availability.
  • the animal feed composition of the present invention can be fed to any animal, for example, a farm animal, a zoo animal, a laboratory animal and/or a companion animal.
  • the animal can be, but is not limited to, a bovine (e.g., domestic cattle (cows (e.g., dairy and/or beef)), bison, buffalo), an equine (e.g., horse, donkey, zebra, and the like), an avian (e.g., a chicken, a quail, a turkey, a duck, and the like; e.g., poultry), a sheep, a goat, an antelope, a pig (e.g., swine), a canine, a feline, a rodent (e.g., mouse, rat, guinea pig); a rabbit, a fish, and the like.
  • a bovine e.g., domestic cattle (cows (e.g., dairy and/or
  • the animal can be a cow. In some embodiments the animal can be poultry. In other embodiments, the animal can be a chicken. In further embodiments, the animal can be swine. In still further embodiments, the animal can be a pig.
  • the present invention provides a method for increasing the volume of milk produced by a dairy animal (e.g., a cow, a goat, and the like), comprising feeding to said dairy animal an animal feed composition of the present invention, wherein the volume of milk produced by said animal is increased by about 5% to about 200% as compared to the volume of milk produced by a control animal that is not provided said animal feed composition of the invention.
  • the increase in the volume of milk is in over a time period from about 1 to about 72 hours.
  • the volume of milk produced by said animal is increased by about 25% to about 175%, about 50% to about 150%, and the like.
  • the volume of milk produced by said animal is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195% and/or 200% as compared to a control animal that has not been fed the animal feed composition of the invention.
  • the terms “increase,” “increasing,” “increased,” “enhance,” “enhanced,” “enhancing,” and “enhancement” (and grammatical variations thereof), as used herein, describe an increase in the specified parameter, e.g., the average daily weight gain of an animal or the growth rate (weight gain) of an animal by feeding to said animal an animal feed composition of the invention, wherein the average daily weight gain or growth rate of the animal is increased by about 0.05 lbs/day to about 10 lbs/day or an increase in the efficiency of feed utilization by an animal by feeding to said animal the animal feed composition of the invention in an amount effective to increase the efficiency of feed utilization by said animal.
  • the specified parameter e.g., the average daily weight gain of an animal or the growth rate (weight gain) of an animal by feeding to said animal an animal feed composition of the invention, wherein the average daily weight gain or growth rate of the animal is increased by about 0.05 lbs/day to about 10 lbs/day or an increase in the efficiency of feed utilization by an animal by
  • This increase in the average daily weight gain, in the growth rate (weight gain), or in the efficiency of feed utilization by an animal can be observed by comparing the average daily weight gain, the growth rate (weight gain) or increase in the efficiency of feed utilization by the animal to an animal not fed an animal feed composition of the invention (i.e., a control).
  • the terms “reduce,” “reduced,” “reducing,” “reduction,” “diminish,” “suppress,” and “decrease” describe, for example, a reduction of or decrease in the specified parameter, e.g., number and/or severity of liver abscess formation or the number of days needed to achieve a desired weight in an animal as compared to a control (e.g., a control animal that is not fed the animal feed composition).
  • Enogen® Feed Corn (EFC; Syngenta Seeds, LLC) is characterized by high-amylase expression in kernel endosperm. It was originally designed, and has been extensively used for the production of ethanol. Corn is well established as the most dominant ingredient fed to finishing cattle, as starch provides a majority of dietary energy. Ruminants have limited capacity for pancreatic-amylase secretion, and consequently are limited in post-ruminal digestion of starch (Harmon et al., 2004. Can. J. Anim. Sci. 84: 309). It is plausible that any ruminally undigested starch, could be further degraded in the small intestine by ⁇ -amylase produced by the grain. This would be an energetic advantage.
  • a randomized complete block design with 2 treatments was carried out using 700 crossbred heifers (394 kg ⁇ 8.5 initial BW). Two lots of cattle, blocked separately, were utilized in the trial. Three hundred fifty heifers received in June, were used previously in a receiving trial examining trace mineral supplementation. The second lot of cattle was received in November, targeting similar initial body weight (BW) between lots at study initiation. Heifers were blocked by lot, then BW, stratified, then randomly assigned to 1 of 28 dirt surfaced pens (25 animals/pen). Treatments randomly assigned within block, consisted of mill-run corn (CON) as control, steam-flaked to 360 g/L; and Enogen Feed Corn (EFC), steam-flaked to 390 g/L.
  • CON mill-run corn
  • EFC Enogen Feed Corn
  • Grain treatments were designed to target similar daily starch availability; based on preliminary work, a decision was made to flake EFC to a greater bulk density, and to flake with greater mill throughput to achieve this. Mill-run corn was flaked at approximately 6 tonne/h; EFC was flaked at approximately 9 tonne/h (50% increased mill throughput), decreasing steam-chest retention time.
  • Lot 2 vaccination was identical, except heifers from this lot were not treated with Micotil, and a topical parasiticide (Dectomax; Zoetis) was applied.
  • animals were ear-tagged with a unique number for identification, and BW was recorded.
  • BW was recorded on d 1 of trial initiation, starting BW was recorded as animals were sorted into pens, and received a trenbolone/estradiol implant (Component TE-IH with Tylan; Elanco Animal Health).
  • heifers were re-implanted (Component TE-200 with Tylan; Elanco Animal Health), and treated with a pour-on insecticide (Standguard; Elanco Animal Health).
  • Animals were housed in dirt surfaced pens that provided approximately 13 m 2 of surface area/animal; fences and gates were made of steel pipe, and divided in 2 by an additional electric fence. Automatic waterers allowing ad libitum access were shared between adjacent pens. Body weights were determined using a pen-scale and averaging pen-weight to determine mean BW for each pen.
  • Grain DM was determined by drying in a forced-air oven set to 105° C. for 24 h. If DM changes occurred, we would adjust the amount of moisture applied by the SarTec system accordingly to achieve equivalent moisture content between corn types.
  • Starch availability was determined by steeping 25 g corn flakes in 100 ml 2.5%-amyloglucosidase solution heated to 55° C. for 15 min (Sindt et al., 2006. J. Anim. Sci. 84: 424). The liquid fraction is then filtered, and percent soluble sugars are viewed on a handheld refractometer. Percent solubles and DM are then put into regression equations determining starch availability.
  • Particle size was determined by weighing approximately 200 g of flaked-corn poured onto a set of sieves, with decreasing screen sizes in the order: 4750, 3350, 2360, 1700, 1180, 850, 600 ⁇ m, and a solid pan. The stack is placed into a Ro-Tap orbital shaker, with a rotary tapping cycle run for 5 min Each individual sieve is cleaned, and particles weighed. Geometric particle size is calculated in a spreadsheet (Scott and Herrman, 2002. Evaluating Particle Size. Kansas State University Department of Grain Science and Industry. MF-2051, 1-6) using equations described by Pfost and Headley (Methods of determining and expressing particle size. In: H. Pfost (ed), Feed Manufacturing Technology II—Appendix C. Am. Feed Manufacturers Assoc. 1976:512-520).
  • liver abscess severity The relationship between liver abscess severity and HCW for each treatment is shown in FIG. 1 .
  • There were effects by diet (P ⁇ 0.01), liver abscess severity (P ⁇ 0.01), and tendency for a treatment ⁇ liver abscess interaction (P 0.09).
  • the EFC group maintained heavier carcasses, and did not display the same decrease in HCW when abscess severity increased that was observed for CON cattle. While mode of action for the effect steam-flaked EFC had on liver abscessation is unclear at this time, the implications could be substantial, as new methods for the prevention of liver abscesses are needed as use of antibiotics is increasingly discouraged.
  • Enogen Feed Corn could be used advantageously by producers to reduce production costs associated with steam-flaking. Reduced steam use, reduced grain processing, increased mill throughput (50% in this study), and therefore reduced costs and labor are all benefits that occur prior to feed being delivered to bunks. Improvements in mill throughput, ADG, feed efficiency, HCW, and liver abscess mitigation, appear to be substantial benefits from the use of steam-flaked EFC.
  • Volatile fatty acids were measured using an Aglient 7890 gas chromatograph (Aglient Technologies, Santa Clara, Calif.) equipped with a Nukol capillary column (15 m ⁇ 0.35 mm, d f 0.50 ⁇ m), yielding concentrations of acetate, propionate, isobutyrate, butyrate, isovalerate, valerate, isocaproate, caproate, and heptanoate.
  • the MIXED models procedure of the Statistical Analysis System (SAS version 9.4) was used to analyze data. Fixed effects included percent corn amylase, percent added moisture, steam conditioning time, and all 2- and 3-way interactions. Block was used as the random effect. Additional orthogonal contrasts allowed examination of linear and quadratic effects between treatments. Treatment effects were considered significant with a P-value less than 0.05.
  • Particle size plays a major role in the digestibility and fermentative properties of grains when fed to ruminants. There was a linear response (P ⁇ 0.01) to percent inclusion of amylase corn in grain mixtures. The higher the percentage of amylase corn the lower the mean particle size. Moisture treatments had no effects on particle size (P>0.10). Steam conditioning time of the grains induced a quadratic effect (P ⁇ 0.01), whereby 30 min of steam exposure resulted in the lowest mean particle size. This quadratic effect of steam mirrors that which is seen in subsequent assays.
  • starch availability assay is commonly used to characterize susceptibility of flaked grains to digestion. Pure amylase grain resulted in the greatest starch availability (52.7%), and starch availability declined linearly (P ⁇ 0.01) in response to dilution of amylase corn with mill-run corn, suggesting that amylase within the amylase corn fraction of mixtures had relatively little impact on non-amylase corn, mill-run grain component of mixtures.
  • Amylase corn content of grain mixtures had a notable impact (linear effect of amylase corn content; P ⁇ 0.01) on in situ digestibility of the flaked grain mixtures.
  • linear effect of amylase corn content P ⁇ 0.01
  • the absence of non-linear effects suggests that effects of amylase corn-derived amylase are essentially confined to the grain itself, and that there is no appreciable migration to non-amylase corn grains within the mixture.
  • Terminal substrate pH is a useful indicator of overall fermentative activity by the in vitro culture, with lower measurements indicating greater organic acid production, and therefore increased microbial digestion of grains.
  • Terminal pH of cultures decreases in direct proportion to the amount of amylase corn grain incorporated into the mixtures (linear effect P ⁇ 0.01).
  • a significant quadratic effect (P ⁇ 0.01) is observed for steam conditioning time, with 30-min yielding lowest culture pH. Moisture addition during the tempering phase had seemingly little effect on final pH of in vitro cultures.
  • VFA production is consistent with differences in terminal pH of cultures. Samples were analyzed for caproate, isocaproate, and heptanoate; however, none of these minor VFAs were detected. Table 7 summarizes VFA profiles for flaked grains comprised of varying proportions of amylase corn and mill-run corn.
  • VFA acetate, propionate, valerate, isovalerate, and total VFA
  • P ⁇ 0.01 acetate, propionate, valerate, isovalerate, and total VFA
  • the VFA contribute the vast majority of energy for maintenance and productive purposes in ruminants, and increased microbial digestion generally is consistent with increased total tract digestion of starch. Grains that are more susceptible to digestion by ruminal microbes are thus likely to greater total tract digestion, and thus may yield improvements in performance or efficiency.
  • Increasing the proportion of amylase corn grain also decreased acetate:propionate ratio (P ⁇ 0.01). This is a desirable effect, as downward shifts in acetate:propionate often are associated with decreases in methanogenesis, which is energetically more favorable.
  • In vitro gas production is a good indicator of total fermentative activity, with carbon dioxide and methane being the primary gaseous byproducts of ruminal fermentation.
  • Statistical differences between treatments were analyzed in 6hour intervals. At hour 6, the 0% amylase corn grain began to separate, being significantly lower than the 50, 75, and 100% treatments. At hour 12 amylase corn levels of 0% and 25% differed from all other treatments; 50% differed from 100%, while the difference between 75% and 100% was not significant. Hour 18 mirrored the treatment differences exactly to hour 12. At 24 hours of fermentation, the 50% amylase corn treatment closed the gap and could no longer be statistically distinguishable between 75% and 100%.
  • Amylase corn grain appears to be far more susceptible to digestion in comparison to the mill-run corn used as control, yielding substantial improvements in starch availability, in vitro and in situ digestion, and endproduct formation. Response to amylase corn grain in mixtures was in direct proportion to amylase corn content, suggesting that amylase corn-derived amylase has little or no impact on the other grain within mixtures. Amylase corn confers significant advantages for processing of grains by steam flaking.

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