Compositions and methods for protein production

Abstract

This specification discloses a food product containing animal-free protein and a method for producing the food product, wherein the animal-free protein can be a protein that is not obtained from animals, or a fragment or cleavage product of a protein.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Application No. 63 / 392,613, filed Jul. 27, 2022, and U.S. Provisional Application No. 63 / 402,544, filed Aug. 31, 2022, each of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Livestock consume the most land resources on Earth. Land used exclusively for meat and dairy production, including animal feed production, accounts for approximately 80% of the world's agricultural land. One-fifth of the world's meat production is consumed by companion animals (e.g., pets). Recombinant protein production can be utilized to supplement or offset meat production.

Summary of the Invention

Means for Solving the Problems

[0003] One aspect of the present disclosure provides a method for producing a food product, comprising: a) contacting a plurality of Pichia cells with a recombinant nucleic acid; b) expressing a protein encoded by the recombinant nucleic acid; and c) isolating the protein from the plurality of Pichia cells, wherein the protein comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 54 to 232.

[0004] In some embodiments, the food is a cat food product. In some embodiments, the protein is an albumin protein. In some embodiments, the albumin protein is a rabbit albumin protein. In some embodiments, the albumin protein is a duck albumin protein. In some embodiments, the duck albumin protein is a mallard albumin protein. In some embodiments, the duck albumin protein is a novalis albumin protein. In some embodiments, the albumin protein is a bovine albumin protein. In some embodiments, the albumin protein is a sheep albumin protein. In some embodiments, the albumin protein is a porcine albumin protein. In some embodiments, the albumin protein is a bison albumin protein. In some embodiments, the albumin protein is a deer albumin protein. In some embodiments, the albumin protein is a chicken albumin protein. In some embodiments, the albumin protein is a silkworm moth albumin protein. In some embodiments, the albumin protein is a salmon albumin protein. In some embodiments, the albumin protein is a cod albumin protein. In some embodiments, the albumin protein is a tuna albumin protein.

[0005] In some embodiments, the Pichia cell is a Pichia pastoris cell. In some embodiments, the protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 54 to 232. In some embodiments, the protein comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 54 to 232. In some embodiments, the food is substantially animal-free.

[0006] Another aspect of the present disclosure provides a food product containing a recombinant protein, wherein the recombinant protein comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 54 to 232. In some embodiments, the food product is substantially animal-free. In some embodiments, the recombinant protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 54 to 232. In some embodiments, the recombinant protein comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 54 to 232. In some embodiments, the food product is a cat food product.

[0007] In some embodiments, the recombinant protein is a recombinant albumin protein. In some embodiments, the recombinant albumin protein is a recombinant rabbit albumin protein. In some embodiments, the albumin protein is a recombinant duck albumin protein. In some embodiments, the recombinant duck albumin protein is a recombinant mallard albumin protein. In some embodiments, the recombinant duck albumin protein is a recombinant novalichen albumin protein. In some embodiments, the recombinant albumin protein is a recombinant bovine albumin protein. In some embodiments, the recombinant albumin protein is a recombinant ovine albumin protein. In some embodiments, the recombinant albumin protein is a recombinant porcine albumin protein. In some embodiments, the recombinant albumin protein is a recombinant bison albumin protein. In some embodiments, the recombinant albumin protein is a recombinant deer albumin protein. In some embodiments, the recombinant albumin protein is a recombinant chicken albumin protein. In some embodiments, the recombinant albumin protein is a recombinant silkworm moth albumin protein. In some embodiments, the recombinant albumin protein is a recombinant salmon albumin protein. In some embodiments, the recombinant albumin protein is a recombinant cod albumin protein. In some embodiments, the recombinant albumin protein is a recombinant tuna albumin protein.

[0008] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, which illustrates and describes only exemplary embodiments of the present disclosure. As will be understood, the present disclosure is capable of other and different embodiments, and some of the details thereof are capable of various obvious modifications in various respects without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0009] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that any incorporated publication and patent or patent application is inconsistent with the disclosure contained herein, this specification is intended to supersede and / or prevail over such inconsistent material.

Brief Description of the Drawings

[0010] The novel features of the present invention are set forth in detail in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description, which illustrates exemplary embodiments in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "figures" and "FIGs").

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0011] Although various embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and alternatives can be devised by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein are possible.

[0012] The production of animal-free protein is a way to reduce global meat production and offset the agricultural and environmental impacts of animal farming. Animal-free protein can be produced using microorganisms designed to produce meat proteins using recombinant protein expression. Pet foods (e.g., cat food, dog food, or food for other companion animals) and foods for human consumption can be developed from animal-free meat proteins as alternatives or supplements to conventional meat-based foods.

[0013] Using recombinant protein expression methods, microorganisms (e.g., yeast or bacteria) can be contacted with a DNA expression cassette. This DNA sequence can contain not only the gene of interest but also regulatory sequences that assist in the production of the gene of interest. Regulatory sequences (e.g., UTRs, promoters, signal sequences, etc.) can help, for example, with the efficiency of transcription or translation, the yield of the protein product, the expression of the protein, or the secretion of the protein.

[0014] Microorganisms can be induced to express and secrete the meat protein of a particular animal (e.g., rabbit protein or duck protein). Microorganisms can alternatively or additionally be induced to express and secrete a particular type of meat protein (e.g., albumin protein). Animal-free meat proteins can be purified and incorporated into foods (such as pet foods and foods for human consumption).

[0015] Definitions If the words "at least", "greater than", or "or more" are attached before the first numerical value of a series of two or more numerical values, the words "at least", "greater than", or "or more" are applied to each numerical value of that series of numerical values. For example, "1, 2, or 3 or more" is equivalent to "1 or more", "2 or more", or "3 or more".

[0016] If the words "no more than", "less than", or "less than or equal to" are attached before the first numerical value in a series of numerical values of 2 or more, the words "no more than", "less than", or "less than or equal to" shall apply to each numerical value in the series of numerical values. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0017] The terms "companion animal" and "pet" are used interchangeably in this specification. A pet is a domestic animal or a trained animal kept as a companion. Pets can be, but are not limited to, dogs, cats, pigs, rats, mice, snakes, turtles, cows, sheep, llamas, donkeys, goats, horses, chickens, parrots, ferrets, guinea pigs, hamsters, rabbits, fish, frogs, tree frogs, newts, iguanas, lizards, ducks, geese, or insects.

[0018] The term "meat" can be defined as skeletal muscle and associated tissues containing blood derived from mammals, birds, reptiles, amphibians, and aquatic organisms taken for consumption. Edible offal, including organs and non-skeletal muscle tissues, is also considered meat.

[0019] The term "animal-free" can be defined as not being taken directly from an animal, whether living or dead (e.g., meat taken from a dead animal).

[0020] Food This specification describes foods containing animal-free proteins. Animal-free proteins can be proteins that are not obtained from animals (e.g., by harvesting meat from animals), or fragments or cleavages of proteins. Animal-free proteins can be obtained by in vitro or ex vivo methods such as cell-free protein synthesis (CFPS). Alternatively, animal-free proteins can be obtained by in vivo methods such as genetic manipulation of cells for recombinant protein expression. The genetically engineered cells used for recombinant protein expression can be microbial cells (e.g., bacteria, yeast), plant cells, insect cells, or animal cells, but are not limited thereto.

[0021] In recombinant protein expression, cells are contacted with a DNA sequence. The cells take up the DNA sequence, and the cell's internal protein expression machinery transcribes and translates the DNA sequence into a peptide.

[0022] The cells used for recombinant protein expression can be microbial cells. Microorganisms include microorganisms such as bacteria, viruses, and fungi (e.g., yeast). The microbial cells used for recombinant protein expression can be bacterial cells. The bacterial cells can be cells from Escherichia coli, Bacillus licheniformis, Bacillus subtilis, or Lactococcus lactis, but are not limited thereto. The microbial cells used for recombinant protein expression may be yeast cells. The yeast cells can be cells from Schizosaccharomyces pombe, (Saccharomyces cerevisiae), Hansenula polymorpha (also known as Ogataea polymorpha), Pichia farinosa, Pichia anomala, Pichia heedii, Pichia guilliermondii, Pichia kluyveri, Pichia membranifaciens, Pichia norvegensis, Pichia ohmeri, Pichia Pastoris (also known as Komagataella phaffii), Yarrowia lipolytica, Pichia methanolica or Pichia subpelliculosa, but are not limited thereto. The microbial cells used for recombinant protein expression can be filamentous fungal cells.The filamentous fungal cells can be cells from Trichoderma reesei (also known as Hypocrea jecorina), Aspergillus niger, Aspergillus oryzae, Aspergillus tubingensis, or Ashbya gossypii (also referred to as Eremothecium gossypii), but are not limited thereto.

[0023] P. pastoris cells can be used in recombinant protein expression methods. P. pastoris cells have a strong protein secretion ability; have the ability to grow to high biomass density, enabling higher yields; the yield of secreted protein is as high as 35 g / L; have an endogenous secretome, requiring less downstream purification; grow on relatively inexpensive raw materials / media (e.g., glycerol or methanol); are genetically very easy to handle; have been demonstrated to be able to appropriately express and fold distantly related eukaryotic (e.g., plant or animal) proteins; have a doubling time of 90 minutes; have the ability to grow in large-scale fermenters; and have a fermentation time from start to harvest of about one week.

[0024] Cells used for recombinant protein expression can be selected because they exhibit desirable characteristics such as a short doubling time, the ability to properly fold the target recombinant protein; the ability to perform appropriate post-translational modifications on the target recombinant protein; the ability to efficiently secrete the target recombinant protein; the ability to grow inexpensively; the ability to grow in easily available media; or genetic tractability. Alternatively, cells used for recombinant protein expression may not secrete the target recombinant protein or may not secrete it efficiently. In one example, cells that do not secrete or do not secrete efficiently the target recombinant protein can be lysed to release the protein. Cell lysis can include mechanical disruption, liquid homogenization, sonication, freeze / thaw cycles, manual grinding, or any combination thereof. In some examples, cells that secrete the target recombinant protein can be further lysed to increase protein yield.

[0025] During recombinant protein expression, the cells are contacted with a DNA sequence that can contain an expression cassette. The expression cassette can be a component of vector DNA that can contain at least one gene and / or at least one regulatory sequence.

[0026] The expression cassette can contain at least one gene of interest. The expression cassette can contain at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more genes of interest. Alternatively or in addition, the expression cassette can contain additional genes other than at least one gene of interest. The expression cassette can contain at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more additional genes other than the gene(s) of interest. The expression cassette can be a linearized plasmid. Alternatively, the expression cassette can be a linear PCR product.

[0027] DNA sequences (e.g., vectors or expression cassettes) can be transfected into cells using a variety of chemical and physical methods such as electroporation, calcium phosphate exposure, spheroplasting, polyethylene glycol (PEG) exposure, alkali cation exposure, or liposome-based transfection.

[0028] The target gene can be an animal protein-coding gene. The animal protein-coding gene can encode the full length or a fragment or truncated version of a native protein. Alternatively or in addition, the animal protein-coding gene can encode a modified version of a native protein (e.g., a codon-optimized version). Alternatively, the animal protein-coding gene can encode a synthetic or novel protein.

[0029] The animal protein-coding gene can encode an antibody, a contractile protein, an enzyme, a hormone protein, a structural protein, a storage protein, or a transport protein. The animal protein-coding gene can be specific to a particular type of tissue such as, but not limited to, connective tissue, epithelial tissue, muscle tissue, or nerve tissue.

[0030] Animal protein-coding genes can encode proteins represented by the meat and other parts of edible animals. Examples of proteins found in meat include actin, myosin, myosin regulatory protein, albumin, carbonic anhydrase, myoglobin, collagen, elastin, titin, nebulin, sarcoplasmic, creatine kinase, phosphoglucose mutase, phosphoglycerate mutase, triose phosphate isomerase, α-actinin, glycogen phosphorylase, fructose diphosphatase, glyceraldehyde-3-phosphate dehydrogenase, L-lactate dehydrogenase, tropomyosin, myosin-binding protein, myomesin, β-enolase, pyruvate kinase, phosphoglycerate kinase, plectin, ATP-dependent 6-phosphofructokinase, malate dehydrogenase, hemoglobin, filamin, aconitate hydratase, parvalbumin, heat shock cognate 71, troponin, calsequestrin, obscurin, glycogen debranching enzyme, elongation factor 1-α2, aspartate aminotransferase, adenylate kinase isozyme, glucose-6-phosphate isomerase, desmin, AMP deaminase, and globulin, among others, but are not limited thereto.

[0031] Animal protein-coding genes can encode albumin. Albumin is a family of globular proteins, the most common of which is serum albumin. Albumin protein is water-soluble and is commonly found in plasma. Albumin protein differs from other blood proteins in that it is not glycosylated. Albumin proteins include, but are not limited to, serum albumin, α-fetoprotein, vitamin D-binding protein, afamin, ovalbumin, and extracellular matrix proteins.

[0032] Animal protein-coding genes can be genes from specific animals such as canines, felines, birds (e.g., songbirds, pigeons, chickens, ducks, geese, swans, crows, doves, parrots, turkeys), bovines (e.g., cows, bison), camels, goats, pigs, deer, sheep, horses, rodents (e.g., mice, rats, squirrels, hamsters, guinea pigs, chipmunks, gophers, voles), fish (e.g., tuna, salmon, trout, sardines, tilefish, halibut, cod, flounder), rabbits, lizards, snakes, or frogs.

[0033] The animal protein can include mouse protein (Table 1). The mouse protein can include mouse muscle protein. The mouse protein can include mouse blood protein. The mouse protein can include mouse albumin protein. The mouse protein can include a sequence having at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% sequence identity to any one of SEQ ID NOs: 1-53.

[0034] The food can contain fragments or cleavage products of mouse proteins. The food can contain a fragment or cleavage product of any one of SEQ ID NOs: 1 to 53. The sequence of the fragment or cleavage product can be identical or substantially identical to a portion of any one of SEQ ID NOs: 1 to 53. The sequence of the fragment or cleavage product can be at least about 70%, 80%, 90%, 95%, 98%, or more identical to a portion of the sequence of any one of SEQ ID NOs: 1 to 53. The length of the fragment or cleavage product can be at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the length of any one of SEQ ID NOs: 1 to 53. The length of the fragment or cleavage product can be about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10% or less, or less than the length of any one of SEQ ID NOs: 1 to 53.

[0035] Table 1: Mouse Protein

Table 1

[0036] Animal proteins can include rabbit proteins (Table 2). Rabbit proteins can include rabbit muscle proteins. Rabbit proteins can include rabbit blood proteins. Rabbit proteins can include rabbit albumin proteins. Rabbit proteins can include any one of SEQ ID NOs: 54-78 and at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% sequence identity. Rabbit albumin proteins can include SEQ ID NOs: 54-78 and at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% sequence identity.

[0037] Foods can include fragments or cleavages of rabbit proteins. The foods can include a fragment or cleavage of any one of SEQ ID NOs: 54-78. The sequence of the fragment or cleavage can be identical or substantially identical to a portion of any one of SEQ ID NOs: 54-78. The sequence of the fragment or cleavage can be at least about 70%, 80%, 90%, 95%, 98%, or more identical to a portion of the sequence of any one of SEQ ID NOs: 54-78. The length of the fragment or cleavage can be at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the length of any one of SEQ ID NOs: 54-78. The length of the fragment or cleavage can be about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10% or less, or less than that of any one of SEQ ID NOs: 54-78.

[0038] Table 2: Rabbit Protein

Table 2

[0039] Animal proteins can include bovine proteins (Table 3). Bovine proteins can include bovine muscle proteins. Bovine proteins can include bovine blood proteins. Bovine proteins can include bovine albumin proteins. Bovine proteins can include a sequence having at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99% or about 100% sequence identity with any one of SEQ ID NOs: 79-93.

[0040] The food can contain fragments or cleavage products of bovine protein. The food can contain a fragment or cleavage product of any one of SEQ ID NOs: 79 to 93. The sequence of the fragment or cleavage product can be identical or substantially identical to a portion of any one of SEQ ID NOs: 79 to 93. The sequence of the fragment or cleavage product can be at least about 70%, 80%, 90%, 95%, 98%, or more identical to a portion of the sequence of any one of SEQ ID NOs: 79 to 93. The length of the fragment or cleavage product can be at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the length of any one of SEQ ID NOs: 79 to 93. The length of the fragment or cleavage product can be about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10% or less, or less than the length of any one of SEQ ID NOs: 79 to 93.

[0041] Table 3: Bovine Protein [Table 3] TIFF2025524943000036.tif255166TIFF2025524943000037.tif255164TIFF2025524943000038.tif255162TIFF2025524943000039.tif255162TIFF2025524943000040.tif207170

[0042] Animal proteins can include sheep proteins (Table 4). Sheep proteins can include sheep muscle proteins. Sheep proteins can include sheep blood proteins. Sheep proteins can include sheep albumin proteins. Sheep proteins can include a sequence having at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99% or about 100% sequence identity with any one of SEQ ID NOs: 94-108.

[0043] Foods can include fragments or cleavage products of sheep proteins. The foods can include a fragment or cleavage product of any one of SEQ ID NOs: 94-108. The sequence of the fragment or cleavage product can be identical or substantially identical to a portion of any one of SEQ ID NOs: 94-108. The sequence of the fragment or cleavage product can be at least about 70%, 80%, 90%, 95%, 98% or more identical to a portion of the sequence of any one of SEQ ID NOs: 94-108. The length of the fragment or cleavage product can be at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the length of any one of SEQ ID NOs: 94-108. The length of the fragment or cleavage product can be about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10% or less, or less than the length of any one of SEQ ID NOs: 94-108.

[0044] Table 4: Sheep proteins

Table 4

[0045] Animal proteins can include porcine proteins (Table 5). The porcine proteins can include porcine muscle proteins. The porcine proteins can include porcine blood proteins. The porcine proteins can include porcine albumin proteins. The porcine proteins can include a sequence having at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% sequence identity with any one of SEQ ID NOs: 109-123.

[0046] The food can include a fragment or cleavage product of porcine protein. The food can include a fragment or cleavage product of any one of SEQ ID NOs: 109-123. The sequence of the fragment or cleavage product can be identical or substantially identical to a portion of any one of SEQ ID NOs: 109-123. The sequence of the fragment or cleavage product can be at least about 70%, 80%, 90%, 95%, 98%, or more identical to a portion of the sequence of any one of SEQ ID NOs: 109-123. The length of the fragment or cleavage product can be at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the length of any one of SEQ ID NOs: 109-123. The length of the fragment or cleavage product can be about 90%, 80%, 70%, 60%, 50%, 4%, 30%, 20%, 15%, 10% or less, or less than that of the length of any one of SEQ ID NOs: 109-123.

[0047] Table 5: Porcine Protein

Table 5

[0048] Animal proteins can include bison proteins (Table 6). Bison proteins can include bison muscle proteins. Bison proteins can include bison blood proteins. Bison proteins can include bison albumin proteins. Bison proteins can include a sequence having at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99% or about 100% sequence identity with any one of SEQ ID NOs: 124 - 138.

[0049] The food can contain a fragment or cleavage product of bison protein. The food can contain a fragment or cleavage product of any one of SEQ ID NOs: 124 to 138. The sequence of the said fragment or cleavage product can be identical or substantially identical to a portion of any one of SEQ ID NOs: 124 to 138. The sequence of the said fragment or cleavage product can be at least about 70%, 80%, 90%, 95%, 98%, or more identical to a portion of the sequence of any one of SEQ ID NOs: 124 to 138. The length of the said fragment or cleavage product can be at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the length of any one of SEQ ID NOs: 124 to 138. The length of the said fragment or cleavage product can be about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10% or less, or less than the length of any one of SEQ ID NOs: 124 to 138.

[0050] Table 6: Bison Protein

Table 6

[0051] Animal proteins can include deer proteins (Table 7). Deer proteins can include deer muscle proteins. Deer proteins can include deer blood proteins. Deer proteins can include deer albumin proteins. Deer proteins can include any one of SEQ ID NOs: 139 to 147 and at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% sequence identity.

[0052] Food can include fragments or cleavage products of deer proteins. The food can include a fragment or cleavage product of any one of SEQ ID NOs: 139 to 147. The sequence of the fragment or cleavage product can be identical or substantially identical to a portion of any one of SEQ ID NOs: 139 to 147. The sequence of the fragment or cleavage product can be at least about 70%, 80%, 90%, 95%, 98%, or more identical to a portion of the sequence of any one of SEQ ID NOs: 139 to 147. The length of the fragment or cleavage product can be at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the length of any one of SEQ ID NOs: 139 to 147. The length of the fragment or cleavage product can be about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10% or less, or less than the length of any one of SEQ ID NOs: 139 to 147.

[0053] Table 7: Deer Proteins

Table 7

[0054] Animal proteins can include chicken proteins (Table 8). Chicken proteins can include chicken muscle proteins. Chicken proteins can include chicken blood proteins. Chicken proteins can include chicken albumin proteins. Chicken proteins can include sequences having at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99% or about 100% sequence identity with any one of SEQ ID NOs: 148 - 161.

[0055] Food can include fragments or cleavage products of chicken proteins. The food can include a fragment or cleavage product of any one of SEQ ID NOs: 148 - 161. The sequence of the fragment or cleavage product can be identical or substantially identical to a portion of any one of SEQ ID NOs: 148 - 161. The sequence of the fragment or cleavage product can be at least about 70%, 80%, 90%, 95%, 98%, or more identical to a portion of the sequence of any one of SEQ ID NOs: 148 - 161. The length of the fragment or cleavage product can be at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the length of any one of SEQ ID NOs: 148 - 161. The length of the fragment or cleavage product can be about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10% or less, or less than the length of any one of SEQ ID NOs: 148 - 161.

[0056] Table 8: Chicken Proteins

Table 8

[0057] Animal proteins can include butterfly proteins (Table 9). Butterfly proteins can include butterfly muscle proteins. Butterfly proteins can include butterfly blood proteins. Butterfly proteins can include butterfly albumin proteins. Butterfly proteins can include any one of SEQ ID NOs: 162-174 and at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% sequence identity.

[0058] Food can include fragments or cleavage products of butterfly proteins. The food can include a fragment or cleavage product of any one of SEQ ID NOs: 162-174. The sequence of the fragment or cleavage product can be identical or substantially identical to a portion of any one of SEQ ID NOs: 162-174. The sequence of the fragment or cleavage product can be at least about 70%, 80%, 90%, 95%, 98%, or more identical to a portion of the sequence of any one of SEQ ID NOs: 162-174. The length of the fragment or cleavage product can be at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the length of any one of SEQ ID NOs: 162-174. The length of the fragment or cleavage product can be about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10% or less, or less than the length of any one of SEQ ID NOs: 162-174.

[0059] Table 9: Butterfly Proteins

Table 9

[0060] Animal proteins can include duck proteins (Tables 10 and 11). Duck proteins can include duck muscle proteins. Duck proteins can include duck blood proteins. Duck proteins can include duck albumin proteins. Duck proteins can include proteins from Anas platyrhynchos. Duck proteins can include proteins from Anas platyrhynchos platyrhynchos. Duck proteins can include proteins from Cairina moschata. Duck proteins can include proteins from Cairina moschata domestica. Duck proteins can include sequences having at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99% or about 100% sequence identity with any one of SEQ ID NOs: 175 to 200.

[0061] The food can contain fragments or cleavage products of mallard protein. The food can contain a fragment or cleavage product of any one of SEQ ID NOs: 175 to 200. The sequence of the fragment or cleavage product can be identical or substantially identical to a portion of any one of SEQ ID NOs: 175 to 200. The sequence of the fragment or cleavage product can be at least about 70%, 80%, 90%, 95%, 98%, or more identical to a portion of the sequence of any one of SEQ ID NOs: 175 to 200. The length of the fragment or cleavage product can be at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the length of any one of SEQ ID NOs: 175 to 200. The length of the fragment or cleavage product can be about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10% or less, or less than the length of any one of SEQ ID NOs: 175 to 200.

[0062] Table 10: Northern mallard protein

Table 10

[0063] Table 11: Bar-headed goose protein

Table 11

[0064] Animal proteins can include salmon proteins (Table 12). Salmon proteins can include salmon muscle proteins. Salmon proteins can include salmon blood proteins. Salmon proteins can include salmon albumin proteins. Salmon proteins can include a sequence having at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99% or about 100% sequence identity with any one of SEQ ID NOs: 201-216.

[0065] Foods can include fragments or cleavages of salmon proteins. The food can include a fragment or cleavage of any one of SEQ ID NOs: 201-216. The sequence of the fragment or cleavage can be identical or substantially identical to a portion of any one of SEQ ID NOs: 201-216. The sequence of the fragment or cleavage can be at least about 70%, 80%, 90%, 95%, 98%, or more identical to a portion of the sequence of any one of SEQ ID NOs: 201-216. The length of the fragment or cleavage can be at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the length of any one of SEQ ID NOs: 201-216. The length of the fragment or cleavage can be about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10% or less, or less than that of the length of any one of SEQ ID NOs: 201-216.

[0066] Table 12: Salmon Proteins

Table 12

[0067] Animal proteins can include pollack proteins (Table 13). The pollack proteins can include pollack muscle proteins. The pollack proteins can include pollack blood proteins. The pollack proteins can include pollack albumin proteins. The pollack proteins can include a sequence having at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99% or about 100% sequence identity with any one of SEQ ID NOs: 217-231.

[0068] The food can include a fragment or cleavage product of the pollack protein. The food can include a fragment or cleavage product of any one of SEQ ID NOs: 217-231. The sequence of the fragment or cleavage product can be identical or substantially identical to a portion of any one of SEQ ID NOs: 217-231. The sequence of the fragment or cleavage product can be at least about 70%, 80%, 90%, 95%, 98%, or more identical to a portion of the sequence of any one of SEQ ID NOs: 217-231. The length of the fragment or cleavage product can be at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the length of any one of SEQ ID NOs: 217-231. The length of the fragment or cleavage product can be about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10% or less, or less than the length of any one of SEQ ID NOs: 217-231.

[0069] Table 13: Pollack Proteins

Table 13

[0070] Animal proteins can include tuna proteins (Table 14). Tuna proteins can include tuna muscle proteins. Tuna proteins can include tuna blood proteins. Tuna proteins can include tuna albumin proteins. Tuna proteins can include a sequence having at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99% or about 100% sequence identity with SEQ ID NO: 232.

[0071] Food can include fragments or cleavages of tuna proteins. The food can include any one fragment or cleavage of SEQ ID NO: 232. The sequence of the fragment or cleavage can be identical or substantially identical to any one part of SEQ ID NO: 232. The sequence of the fragment or cleavage can be at least about 70%, 80%, 90%, 95%, 98%, or more identical to any one sequence part of SEQ ID NO: 232. The length of the fragment or cleavage can be at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of any one length of SEQ ID NO: 232. The length of the fragment or cleavage can be about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10% or less, or less than any one length of SEQ ID NO: 232.

[0072] Table 14: Tuna protein

Table 14

[0073] Additional genes that are not the target gene can be genes that regulate protein expression. Genes that regulate protein expression can enhance protein expression, decrease protein expression, stop protein expression, or maintain protein expression.

[0074] Genes that regulate protein expression can increase protein expression by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 5,000%, at least about 10,000% or more. Genes that regulate protein expression can increase protein expression by up to about 10,000%, up to about 5,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 150%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 5%, or less.

[0075] Genes that regulate protein expression can increase protein expression by at least or about 1-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 60-fold, at least or about 70-fold, at least or about 80-fold, at least or about 90-fold, at least or about 100-fold, at least or about 500-fold, at least or about 1,000-fold, at least or about 5,000-fold, or at least or about 10,000-fold. Genes that regulate protein expression can increase protein expression by up to or less than about 10,000-fold, up to or less than about 5,000-fold, up to or less than about 1,000-fold, up to or less than about 500-fold, up to or less than about 100-fold, up to or less than about 90-fold, up to or less than about 80-fold, up to or less than about 70-fold, up to or less than about 60-fold, up to or less than about 50-fold, up to or less than about 40-fold, up to or less than about 30-fold, up to or less than about 20-fold, up to or less than about 10-fold, up to or less than about 9-fold, up to or less than about 8-fold, up to or less than about 7-fold, up to or less than about 6-fold, up to or less than about 5-fold, up to or less than about 4-fold, up to or less than about 3-fold, up to or less than about 2-fold, or up to or less than about 1-fold.

[0076] Genes that regulate protein expression can reduce protein expression by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 5,000%, at least about 10,000% or more. Genes that regulate protein expression can reduce protein expression by up to about 10,000%, up to about 5,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 150%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 5%, or less.

[0077] Genes that regulate protein expression can reduce protein expression by at least or about 1-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 60-fold, at least or about 70-fold, at least or about 80-fold, at least or about 90-fold, at least or about 100-fold, at least or about 500-fold, at least or about 1,000-fold, at least or about 5,000-fold, or at least or about 10,000-fold. Genes that regulate protein expression can reduce protein expression by up to or less than about 10,000-fold, up to or less than about 5,000-fold, up to or less than about 1,000-fold, up to or less than about 500-fold, up to or less than about 100-fold, up to or less than about 90-fold, up to or less than about 80-fold, up to or less than about 70-fold, up to or less than about 60-fold, up to or less than about 50-fold, up to or less than about 40-fold, up to or less than about 30-fold, up to or less than about 20-fold, up to or less than about 10-fold, up to or less than about 9-fold, up to or less than about 8-fold, up to or less than about 7-fold, up to or less than about 6-fold, up to or less than about 5-fold, up to or less than about 4-fold, up to or less than about 3-fold, up to or less than about 2-fold, or up to or less than about 1-fold.

[0078] The additional target gene can be a gene that regulates protein secretion. Genes that regulate protein secretion can enhance protein secretion, decrease protein secretion, stop protein secretion, or maintain protein secretion.

[0079] Genes that regulate protein secretion can increase protein secretion by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 5,000%, at least about 10,000% or more. Genes that regulate protein secretion can increase protein secretion by up to about 10,000%, up to about 5,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 150%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 5%, or less.

[0080] Genes that regulate protein secretion can increase protein secretion by at least or about 1-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 60-fold, at least or about 70-fold, at least or about 80-fold, at least or about 90-fold, at least or about 100-fold, at least or about 500-fold, at least or about 1,000-fold, at least or about 5,000-fold, or at least or about 10,000-fold. Genes that regulate protein secretion can increase protein secretion by up to or less than about 10,000-fold, up to or less than about 5,000-fold, up to or less than about 1,000-fold, up to or less than about 500-fold, up to or less than about 100-fold, up to or less than about 90-fold, up to or less than about 80-fold, up to or less than about 70-fold, up to or less than about 60-fold, up to or less than about 50-fold, up to or less than about 40-fold, up to or less than about 30-fold, up to or less than about 20-fold, up to or less than about 10-fold, up to or less than about 9-fold, up to or less than about 8-fold, up to or less than about 7-fold, up to or less than about 6-fold, up to or less than about 5-fold, up to or less than about 4-fold, up to or less than about 3-fold, up to or less than about 2-fold, or up to or less than about 1-fold.

[0081] Genes that regulate protein secretion can reduce protein secretion by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 5,000%, at least about 10,000% or more. Genes that regulate protein secretion can reduce protein secretion by up to about 10,000%, up to about 5,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 150%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 5%, or less.

[0082] Genes that regulate protein secretion can reduce protein secretion by at least or about 1-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 60-fold, at least or about 70-fold, at least or about 80-fold, at least or about 90-fold, at least or about 100-fold, at least or about 500-fold, at least or about 1,000-fold, at least or about 5,000-fold, or at least or about 10,000-fold. Genes that regulate protein secretion can reduce protein secretion by up to or less than about 10,000-fold, up to or less than about 5,000-fold, up to or less than about 1,000-fold, up to or less than about 500-fold, up to or less than about 100-fold, up to or less than about 90-fold, up to or less than about 80-fold, up to or less than about 70-fold, up to or less than about 60-fold, up to or less than about 50-fold, up to or less than about 40-fold, up to or less than about 30-fold, up to or less than about 20-fold, up to or less than about 10-fold, up to or less than about 9-fold, up to or less than about 8-fold, up to or less than about 7-fold, up to or less than about 6-fold, up to or less than about 5-fold, up to or less than about 4-fold, up to or less than about 3-fold, up to or less than about 2-fold, or up to or less than about 1-fold.

[0083] The expression cassette can include at least one regulatory sequence. The regulatory sequence can include a promoter, a repressor, an enhancer, a terminator, a 5′UTR, a 3′UTR, or a combination thereof. The regulatory sequence can be part of the coding sequence. Alternatively, the regulatory sequence can be a non-coding sequence. The expression cassette can include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more regulatory sequences.

[0084] The promoter can be an endogenous P. pastoris promoter or a variant thereof. Alternatively, the promoter can be a yeast promoter from a different species (e.g., S. cerevisiae) or a variant thereof. Alternatively, the promoter can be a synthetic or novel promoter. The promoter can be the AOX1 promoter, the ADH3 promoter, the TEF1 promoter, the PGK1 promoter, the TPI1 promoter, the PHO89 promoter, the THI11 promoter, the AOD promoter, or the GAP promoter.

[0085] The enhancer can be an endogenous P. pastoris enhancer or a variant thereof. Alternatively, the enhancer can be a yeast enhancer from a different species (e.g., S. cerevisiae) or a variant thereof. Alternatively, the enhancer can be a synthetic or novel enhancer. The enhancer can be the AOX1 enhancer or the GAP enhancer.

[0086] The repressor can be an endogenous P. pastoris repressor or a variant thereof. Alternatively, the repressor can be a yeast repressor from a different species (e.g., S. cerevisiae) or a variant thereof. Alternatively, the repressor can be a synthetic or novel repressor. The repressor can be the AOX1 repressor or the GAP repressor.

[0087] The terminator can be an endogenous P. pastoris terminator or a variant thereof. Alternatively, the terminator can be a yeast terminator from a different species (e.g., S. cerevisiae) or a variant thereof. Alternatively, the terminator can be a synthetic or novel terminator. The terminator can be an AOX1 terminator, an RPL15A terminator, an RPL41B terminator, a DAS1 terminator, a VMA2 terminator, an SRP54 terminator, a FUR4 terminator, a PLB3 terminator, a GIC1 terminator, a FBP1 terminator, a CYC1 terminator, an IDP1 terminator, a PRC1 terminator, an RPL3 terminator, a PGK1 terminator, a YPT31 terminator, an ASP3-1 terminator, a TPO4 terminator, an RPS28A terminator, a VMA16 terminator, an LSC2 terminator, a DIT1 terminator, a MIG2 terminator, an ADH2 terminator, an ADH3 terminator, an ICY2 terminator, a PHO89 terminator, a THI11 terminator, an AOD terminator, or a GAP terminator.

[0088] The UTR can be a 5′UTR or a 3′UTR. The UTR can be an endogenous P. pastoris UTR or a variant thereof. Alternatively, the UTR can be a yeast UTR from a different species (e.g., S. cerevisiae) or a variant thereof. Alternatively, the UTR can be a synthetic or novel UTR. The UTR can be an AOX1 UTR, an RPL15A UTR, an RPL41B UTR, a DAS1 UTR, a VMA2 UTR, an SRP54 UTR, a FUR4 UTR, a PLB3 UTR, a GIC1 UTR, an FBP1 UTR, a CYC1 UTR, an IDP1 UTR, a PRC1 UTR, an RPL3 UTR, a PGK1 UTR, a YPT31 UTR, an ASP3-1 UTR, a TPO4 UTR, an RPS28A UTR, a VMA16 UTR, an LSC2 UTR, a DIT1 UTR, a MIG2 UTR, an ADH2 UTR, an ADH3 UTR, a TEF1 UTR, a PGK1 UTR, a TPI1 UTR, a PHO89 UTR, a THI11 UTR, an AOD UTR, or a GAP UTR.

[0089] The expression cassette can contain a signal sequence. The signal sequence (e.g., a secretion sequence or a nuclear localization sequence) can assist and / or induce protein secretion. The expression cassette can contain at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more signal sequences.

[0090] The signal sequence can be an endogenous P. pastoris signal sequence or a variant thereof. Alternatively, the signal sequence can be a yeast signal sequence from a different species (e.g., S. cerevisiae) or a variant thereof. Alternatively, the signal sequence can be a synthetic or novel promoter. The signal sequence can be an amylase signal sequence, an inulinase signal sequence, an invertase signal sequence, a lysozyme signal sequence, an albumin signal sequence, a glucoamylase signal sequence, or a killer protein signal sequence, an S. cerevisiae Ost1 signal sequence, an S. cerevisiae α - mating factor, a partial S. cerevisiae α - mating factor, or a Pichia ExgTp signal sequence.

[0091] The regulatory sequence can regulate protein yield. The regulatory sequence can increase, decrease, or maintain protein yield.

[0092] The regulatory sequences that regulate protein yield can increase the protein yield by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 5,000%, at least about 10,000% or more. The regulatory sequences that regulate protein yield can increase the protein yield by up to about 10,000%, up to about 5,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 150%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 5%, or less.

[0093] Regulatory sequences that regulate protein yield can increase the protein yield by at least or about 1-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 60-fold, at least or about 70-fold, at least or about 80-fold, at least or about 90-fold, at least or about 100-fold, at least or about 500-fold, at least or about 1,000-fold, at least or about 5,000-fold, or at least or about 10,000-fold. Regulatory sequences that regulate protein yield can increase the protein yield by up to or less than about 10,000-fold, up to or less than about 5,000-fold, up to or less than about 1,000-fold, up to or less than about 500-fold, up to or less than about 100-fold, up to or less than about 90-fold, up to or less than about 80-fold, up to or less than about 70-fold, up to or less than about 60-fold, up to or less than about 50-fold, up to or less than about 40-fold, up to or less than about 30-fold, up to or less than about 20-fold, up to or less than about 10-fold, up to or less than about 9-fold, up to or less than about 8-fold, up to or less than about 7-fold, up to or less than about 6-fold, up to or less than about 5-fold, up to or less than about 4-fold, up to or less than about 3-fold, up to or less than about 2-fold, or up to or less than about 1-fold.

[0094] The regulatory sequences that regulate protein yield can reduce the protein yield by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 5,000%, at least about 10,000% or more. The regulatory sequences that regulate protein yield can reduce the protein yield by up to about 10,000%, up to about 5,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 150%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 5%, or less.

[0095] Regulatory sequences that regulate protein yield can reduce the protein yield by at least or about 1-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 60-fold, at least or about 70-fold, at least or about 80-fold, at least or about 90-fold, at least or about 100-fold, at least or about 500-fold, at least or about 1,000-fold, at least or about 5,000-fold, or at least or about 10,000-fold. Regulatory sequences that regulate protein yield can reduce the protein yield by up to or less than about 10,000-fold, up to or less than about 5,000-fold, up to or less than about 1,000-fold, up to or less than about 500-fold, up to or less than about 100-fold, up to or less than about 90-fold, up to or less than about 80-fold, up to or less than about 70-fold, up to or less than about 60-fold, up to or less than about 50-fold, up to or less than about 40-fold, up to or less than about 30-fold, up to or less than about 20-fold, up to or less than about 10-fold, up to or less than about 9-fold, up to or less than about 8-fold, up to or less than about 7-fold, up to or less than about 6-fold, up to or less than about 5-fold, up to or less than about 4-fold, up to or less than about 3-fold, up to or less than about 2-fold, or up to or less than about 1-fold.

[0096] Regulatory sequences can regulate protein secretion. Regulatory sequences that regulate protein secretion can promote, reduce, stop, or maintain protein secretion.

[0097] Regulatory sequences that modulate protein secretion can increase protein secretion by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 5,000%, at least about 10,000% or more. Regulatory sequences that modulate protein secretion can increase protein secretion by up to about 10,000%, up to about 5,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 150%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 5%, or less.

[0098] Regulatory sequences that regulate protein secretion can increase protein secretion by at least or about 1-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 60-fold, at least or about 70-fold, at least or about 80-fold, at least or about 90-fold, at least or about 100-fold, at least or about 500-fold, at least or about 1,000-fold, at least or about 5,000-fold, or at least or about 10,000-fold. Regulatory sequences that regulate protein secretion can increase protein secretion by up to or less than about 10,000-fold, up to or less than about 5,000-fold, up to or less than about 1,000-fold, up to or less than about 500-fold, up to or less than about 100-fold, up to or less than about 90-fold, up to or less than about 80-fold, up to or less than about 70-fold, up to or less than about 60-fold, up to or less than about 50-fold, up to or less than about 40-fold, up to or less than about 30-fold, up to or less than about 20-fold, up to or less than about 10-fold, up to or less than about 9-fold, up to or less than about 8-fold, up to or less than about 7-fold, up to or less than about 6-fold, up to or less than about 5-fold, up to or less than about 4-fold, up to or less than about 3-fold, up to or less than about 2-fold, or up to or less than about 1-fold.

[0099] Regulatory sequences that regulate protein secretion can reduce protein secretion by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 5,000%, at least about 10,000% or more. Regulatory sequences that regulate protein secretion can reduce protein secretion by up to about 10,000%, up to about 5,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 150%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 5%, or less.

[0100] Regulatory sequences that regulate protein secretion can reduce protein secretion by at least or about 1-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 60-fold, at least or about 70-fold, at least or about 80-fold, at least or about 90-fold, at least or about 100-fold, at least or about 500-fold, at least or about 1,000-fold, at least or about 5,000-fold, or at least or about 10,000-fold. Regulatory sequences that regulate protein secretion can reduce protein secretion by up to or less than about 10,000-fold, up to or less than about 5,000-fold, up to or less than about 1,000-fold, up to or less than about 500-fold, up to or less than about 100-fold, up to or less than about 90-fold, up to or less than about 80-fold, up to or less than about 70-fold, up to or less than about 60-fold, up to or less than about 50-fold, up to or less than about 40-fold, up to or less than about 30-fold, up to or less than about 20-fold, up to or less than about 10-fold, up to or less than about 9-fold, up to or less than about 8-fold, up to or less than about 7-fold, up to or less than about 6-fold, up to or less than about 5-fold, up to or less than about 4-fold, up to or less than about 3-fold, up to or less than about 2-fold, or up to or less than about 1-fold.

[0101] A food or substance can be composed of a recombinant protein described herein. The food or substance can contain a recombinant protein described herein provided in isolated or purified form. Alternatively or in addition, the recombinant protein described herein can be provided in a food or substance as whole cells containing the recombinant protein. The food or substance can be a food for animals. The food or substance can be a food for pets (e.g., companion animals), farm or agricultural animals, wild animals, or humans. The food or substance can be a food for obligate carnivores (e.g., felines) that require animal protein as part of their diet.

[0102] The food can be a moist puree. Alternatively, the food can be a dry pellet. The food can substitute for the diet of animals or humans. Alternatively, the food can supplement the diet of animals or humans.

[0103] The food can include additional ingredients such as (but not limited to) animal meat, amino acid supplements (e.g., taurine), vitamins (e.g., vitamin E), fats, nutritional yeast, spirulina, agar, xanthan gum, kelp powder, pumpkin powder, sweet potato powder, papaya powder, flaxseed oil, coconut oil, probiotics, cranberry powder, purslane powder, turmeric, coconut powder, chitosan, pumpkin puree, inulin powder, guar gum, green tea extract, koji, stabilizers, vegetables, fruits, microorganisms, bulking agents, or flavor compounds.

Example

[0104] Example 1: Recombinant protein expression In this experiment, Pichia pastoris cells were transfected with a DNA sequence to express a recombinant protein.

[0105] The host cells were selected based on certain characteristics such as (but not limited to) doubling time; the ability to properly fold the recombinant protein and perform appropriate post-translational modifications; protein secretion; the ability to grow in a cost-effective and readily available medium / raw material; or genetic tractability. Host cells that do not secrete the protein efficiently can be lysed to release the target recombinant protein.

[0106] Pichia cells were grown overnight at 30 °C in YPD medium in a baffled flask to an optical density (OD600) of about 1 - 2.6 at a wavelength of 600 nm. The cells were harvested by centrifugation and resuspended at room temperature for 30 minutes in 100 mM lithium acetate (LiOAc), 10 mM dithiothreitol (DTT), 0.6 M sorbitol, and 10 mM Tris-HCl pH 7.5 to a concentration of 1e8 cells / mL. The cells were pelleted and washed four times with 1 mL of 1 M sorbitol. The cells were resuspended in 1 M sorbitol to a final concentration of 1e9 cells / mL. 40 μL of the cell suspension was mixed with 1 μL of DNA (100 - 1 μg), transferred to a 0.2 cm gap cuvette, and incubated on ice for 5 minutes.

[0107] The expression cassette used was composed of a promoter, 5′ untranslated region, protein secretion signal sequence, open reading frame (ORF) of an animal muscle protein, 3′ UTR, and transcription termination and 3′ end processing sequences. The construct used the P. pastoris AOX1 promoter, P. pastoris 5′ UTR, P. pastoris 3′ UTR, S. cerevisiae α - mating factor, rabbit serum albumin ORF, and P. pastoris terminator.

[0108] The above expression cassette was transfected into P. pastoris host cells using electroporation at 1.5 kV, 200 Ω, and 25 μF to integrate the expression cassette into the genome of P. pastoris. The electroporated cells were diluted with 1 mL of 1 M sorbitol. The cells were transferred to a 15 mL round - bottom tube containing 1 mL of YPD medium and incubated at 30 °C for 1 hour with shaking.

[0109] The cells were seeded on YPDS plates and incubated at 30°C. When the endogenous protein expression mechanism of the cells expressed the target protein, the cells secreted the target protein into the medium, which was then recovered and purified for further use.

[0110] Example 2: Analysis of Secreted Protein In this experiment, the target gene was cloned into a plasmid containing the GAP1 promoter, the Saccharomyces cerevisiae α - mating factor signal sequence, and the zeocin resistance gene. The plasmid was linearized and transformed into Pichia pastoris cells using the electroporation method. The transformed cells were plated on YPDS plates containing 100 μg / mL of zeocin and incubated at 30°C for 3 - 4 days. The colonies were re - cultured into single colonies twice. To produce the secreted protein, the cells were resuspended in 4 mL of buffered complex glycerol medium (BMGY) (high glycerol) and cultured with shaking at 250 revolutions per minute (rpm) at 30°C for 4 days. Every 24 hours, 200 μL of the culture broth was collected and centrifuged to separate the cells from the supernatant. 10 μL of the supernatant aliquot was analyzed for secreted protein on a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS - PAGE) gel.

[0111] Figure 1 is a diagram showing a Coomassie - stained SDS - PAGE gel of an exemplary medium from Pichia pastoris cells or wild - type cells containing the construct of secreted recombinant rabbit albumin. The wild - type cells lack the construct of secreted recombinant rabbit albumin. Lane 1 101 shows a 66 - kilodalton (kDa) band corresponding to recombinant rabbit albumin. Lane 2 102 corresponding to the wild - type medium lacks the 66 - kDa band. Lane 3 103 shows a bovine serum albumin marker having a 66 - kDa band.

[0112] Figure 2 shows a Coomassie stained sodium SDS-PAGE gel of an exemplary medium from Pichia pastoris cells or wild-type cells containing a secreted recombinant rabbit tropomyosin α-1 chain construct. The wild-type cells lack the construct. Lane 2201 shows the wild-type expressed protein from wild-type cells. Lanes 3-5202 show the secreted protein expression of tropomyosin α-1 chain. Lanes 3-5 show a 33 kDa band 203 corresponding to the tropomyosin α-1 chain.

[0113] For the protein produced in the bioreactor, the cells were resuspended in YPG medium and grown with shaking at 30 °C for 16 h. The cells were transferred to a seed fermenter and grown for an additional 16 h at 30 °C. The culture was transferred to a 100 liter (L) fermenter and batch fed at 30 °C for 92 h in a glycerol-based medium. The culture was then centrifuged to separate the solid and liquid phases, filtered, and spray dried to produce the final protein powder.

[0114] Figure 3 shows a Coomassie stained SDS-PAGE gel of an exemplary recombinant rabbit albumin. Lane 1301 shows the exemplary recombinant rabbit albumin (rALBU-RABIT) grown from the bioreactor. The rabbit albumin peptide was purified and spray dried. The protein powder was resuspended and electrophoresed on an SDS-PAGE gel. Lane 2302 shows the bovine serum albumin marker.

[0115] Example 3: Cat Food Containing Rabbit Protein In this example, rabbit protein is used to make cat food. Rabbits are hunted by wild felines in nature, and rabbit protein is hypoallergenic to cats. Recombinant rabbit protein is produced and used as an ingredient in both wet and dry cat food, snacks, and / or treats. In some formulations, the protein is in dry powder form. In some formulations, the protein is in solution. For dry food, the ingredients are mixed and extruded.

[0116] Produce 25 types of rabbit proteins using the recombinant protein expression method of Example 1. The 25 types of rabbit proteins tested are shown in Table 2.

[0117] Recover the rabbit protein from the recombinant protein expression method and produce it into cat snacks for wet puree. Further, recover the rabbit protein from the recombinant protein expression method and produce it into dry extruded cat food kibble.

[0118] Example 4: Pet Food Containing Bovine Protein In this example, the pet food is made using bovine protein. Recombinant bovine protein is produced and used as an ingredient in both wet and dry pet food, snacks, and / or treats. In some formulations, the protein is in dry powder form. In some formulations, the protein is in solution. For dry food, the ingredients are mixed and extruded.

[0119] Produce bovine protein using the recombinant protein expression method of Example 1. The bovine proteins tested are shown in Table 3.

[0120] Example 5: Pet Food Containing Sheep Protein In this example, the pet food is made using sheep protein. Recombinant sheep protein is produced and used as an ingredient in both wet and dry pet food, snacks, and / or treats. In some formulations, the protein is in dry powder form. In some formulations, the protein is in solution. For dry food, the ingredients are mixed and extruded.

[0121] Produce sheep protein using the recombinant protein expression method of Example 1. The sheep proteins tested are shown in Table 4.

[0122] Example 6: Pet Food Containing Porcine Protein In this example, the pet food is made using porcine protein. Recombinant porcine protein is produced and used as an ingredient in both wet and dry pet food, snacks, and / or treats. In some formulations, the protein is in dry powder form. In some formulations, the protein is a solution. In the case of dry food, the ingredients are blended and extruded.

[0123] Porcine protein is produced using the recombinant protein expression method of Example 1. The porcine proteins tested are shown in Table 5.

[0124] Example 7: Pet Food Containing Bison Protein In this example, the pet food is made using bison protein. Recombinant bison protein is produced and used as an ingredient in both wet and dry cat food, snacks, and / or treats. In some formulations, the protein is in dry powder form. In some formulations, the protein is a solution. In the case of dry food, the ingredients are blended and extruded.

[0125] Bison protein is produced using the recombinant protein expression method of Example 1. The bison proteins tested are shown in Table 6.

[0126] Example 8: Pet Food Containing Deer Protein In this example, the pet food is made using deer protein. Recombinant deer protein is produced and used as an ingredient in both wet and dry pet food, snacks, and / or treats. In some formulations, the protein is in dry powder form. In some formulations, the protein is a solution. In the case of dry food, the ingredients are blended and extruded.

[0127] Deer protein is produced using the recombinant protein expression method of Example 1. The deer proteins tested are shown in Table 7.

[0128] Example 9: Pet Food Containing Chicken Protein In this example, pet food is made using chicken protein. Recombinant chicken protein is produced and used as an ingredient in both wet and dry pet food, snacks, and / or treats. In some formulations, the protein is in dry powder form. In some formulations, the protein is a solution. In the case of dry food, the ingredients are mixed and extruded.

[0129] Manufacture chicken protein using the recombinant protein expression method of Example 1. The chicken proteins tested are shown in Table 8.

[0130] Example 10: Pet Food Containing Butterfly Protein In this example, cat food is made using butterfly protein. Recombinant butterfly protein is produced and used as an ingredient in both wet and dry pet food, snacks, and / or treats. In some formulations, the protein is in dry powder form. In some formulations, the protein is a solution. In the case of dry food, the ingredients are mixed and extruded.

[0131] Manufacture butterfly protein using the recombinant protein expression method of Example 1. The butterfly proteins tested are shown in Table 9.

[0132] Example 11: Cat Food Containing Duck Protein In this example, cat food is made using duck protein. Ducks are hunted by wild felines in nature, and duck protein is hypoallergenic for cats. Recombinant duck protein is produced and used as an ingredient in both wet and dry cat food, snacks, and / or treats. In some formulations, the protein is in dry powder form. In some formulations, the protein is a solution. In the case of dry food, the ingredients are mixed and extruded.

[0133] Manufacture duck protein using the recombinant protein expression method of Example 1. The duck proteins tested are shown in Tables 10 and 11.

[0134] Example 12: Pet Food Containing Salmon Protein In this example, the pet food is made using salmon protein. Recombinant salmon protein is produced and used as an ingredient in both wet and dry pet foods, snacks, and / or treats. In some formulations, the protein is in dry powder form. In some formulations, the protein is in solution. For dry food, the ingredients are blended and extruded.

[0135] The salmon protein is produced using the recombinant protein expression method of Example 1. The tested salmon proteins are shown in Table 12.

[0136] Example 13: Pet Food Containing Cod Protein In this example, the pet food is made using cod protein. Recombinant cod protein is produced and used as an ingredient in both wet and dry pet foods, snacks, and / or treats. In some formulations, the protein is in dry powder form. In some formulations, the protein is in solution. For dry food, the ingredients are blended and extruded.

[0137] The cod protein is produced using the recombinant protein expression method of Example 1. The tested cod proteins are shown in Table 13.

[0138] Example 14: Pet Food Containing Tuna Protein In this example, the pet food is made using tuna protein. Recombinant tuna protein is produced and used as an ingredient in both wet and dry pet foods, snacks, and / or treats. In some formulations, the protein is in dry powder form. In some formulations, the protein is in solution. For dry food, the ingredients are blended and extruded.

[0139] The tuna protein is produced using the recombinant protein expression method of Example 1. The tested tuna proteins are shown in Table 14.

[0140] Example 15: Food for Human Consumption In this example, any of the proteins described in Tables 2-14 is used to produce food for human consumption. Recombinant proteins from Tables 2-14 are produced and provided in food, beverage, or supplement. In some formulations, the protein is in dry powder form. In some formulations, the protein is a solution.

[0141] As described and illustrated hereinabove are the preferred embodiments of the present invention. However, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited to the specific examples provided herein. Although the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Those skilled in the art will envision many variations, modifications, and alternatives without departing from the present invention. Further, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions described herein, which are determined by various conditions and variables. It should be understood that various alternatives of the embodiments of the present invention described herein can be used in the practice of the present invention. Accordingly, it is contemplated that the present invention also encompasses such alternatives, modifications, variations, or equivalents. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims, as well as their equivalents, be covered thereby.

Claims

1. a) contacting a plurality of Pichia cells with a recombinant nucleic acid; b) expressing a protein encoded by the recombinant nucleic acid; and c) isolating the protein from the plurality of Pichia cells A method for producing a food product, comprising: The method, wherein the protein comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 54 to 232.

2. The method according to claim 1, wherein the food is a cat food product.

3. The method according to claim 1, wherein the protein is an albumin protein.

4. The method according to claim 3, wherein the albumin protein is a rabbit albumin protein.

5. The method according to claim 3, wherein the albumin protein is a duck albumin protein.

6. The method according to claim 5, wherein the duck albumin protein is a mallard albumin protein.

7. The method according to claim 5, wherein the duck albumin protein is a novariken albumin protein.

8. The method according to claim 3, wherein the albumin protein is a bovine albumin protein.

9. The method according to claim 3, wherein the albumin protein is a sheep albumin protein.

10. The method according to claim 3, wherein the albumin protein is a porcine albumin protein.

11. The method according to claim 3, wherein the albumin protein is a bison albumin protein.

12. The method according to claim 3, wherein the albumin protein is a deer albumin protein.

13. The method according to claim 3, wherein the albumin protein is a chicken albumin protein.

14. The method according to claim 3, wherein the albumin protein is a Japanese quail albumin protein.

15. The method according to claim 3, wherein the albumin protein is a salmon albumin protein.

16. The method according to claim 3, wherein the albumin protein is a cod albumin protein.

17. The method according to claim 3, wherein the albumin protein is a tuna albumin protein.

18. The method according to claim 1, wherein the Pichia cells are Pichia pastoris cells.

19. The method according to claim 1, wherein the protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 54 to 232.

20. The method according to claim 1, wherein the protein comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 54 to 232.

21. The method according to claim 1, wherein the food is substantially animal-free.

22. A food comprising a recombinant protein, wherein the recombinant protein comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 54 to 232.

23. The food according to claim 22, wherein the food is substantially animal-free.

24. The food according to claim 22, wherein the recombinant protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 54 to 232.

25. The food according to claim 22, wherein the recombinant protein comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 54 to 232.

26. The food according to claim 22, wherein the food is a cat food product.

27. The food according to claim 22, wherein the recombinant protein is a recombinant albumin protein.

28. The food according to claim 27, wherein the recombinant albumin protein is a recombinant rabbit albumin protein.

29. The food according to claim 27, wherein the recombinant albumin protein is a recombinant duck albumin protein.

30. The food according to claim 29, wherein the recombinant duck albumin protein is a recombinant mallard albumin protein.

31. The food according to claim 29, wherein the recombinant duck albumin protein is a recombinant novariken albumin protein.

32. The food according to claim 27, wherein the recombinant albumin protein is a recombinant bovine albumin protein.

33. The food according to claim 27, wherein the recombinant albumin protein is a recombinant ovine albumin protein.

34. The food according to claim 27, wherein the recombinant albumin protein is a recombinant porcine albumin protein.

35. The food according to claim 27, wherein the recombinant albumin protein is a recombinant bison albumin protein.

36. The food according to claim 27, wherein the recombinant albumin protein is a recombinant deer albumin protein.

37. The food according to claim 27, wherein the recombinant albumin protein is a recombinant chicken albumin protein.

38. The food according to claim 27, wherein the recombinant albumin protein is a recombinant silkworm albumin protein.

39. The food according to claim 27, wherein the recombinant albumin protein is a recombinant salmon albumin protein.

40. The food according to claim 27, wherein the recombinant albumin protein is a recombinant cod albumin protein.

41. The food according to claim 27, wherein the recombinant albumin protein is a recombinant tuna albumin protein.

Images