WO2025199485A1

WO2025199485A1 - Phosphorylated ovalbumin

Info

Publication number: WO2025199485A1
Application number: PCT/US2025/020990
Authority: WO
Inventors: Logan HURST; Dariya IGNATENKO; Frank Douglas IVEY; Lauren KOLYER; Joel Andrew KREPS; Ranjan Patnaik; Steve ROTHMAN; Giulio Salerno; Charles Albert TINDELL; Weixi ZHONG
Original assignee: Clara Foods Co
Current assignee: Every Co
Priority date: 2024-03-22
Filing date: 2025-03-21
Publication date: 2025-09-25
Anticipated expiration: 2026-09-22

Abstract

This disclosure describes methods and compositions related to the production of a recombinant phosphorylated ovalbumin protein.

Description

PHOSPHORYLATED OVALBUMIN

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/569,043, filed March 22, 2024, which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 21, 2025, is named 62395WO_CRF_sequencelisting.xml and is 192 kilobytes in size.

BACKGROUND OF THE INVENTION

[0003] Proteins are important dietary nutrients and food ingredients. They can serve as a fuel source or as sources of amino acids, including the essential amino acids that cannot be synthesized by the body. The daily recommended intake of protein for healthy adults is 10% to 35% of a person’s total calorie needs, and currently the majority of protein intake for most humans is from animal-based sources. In addition, proteins are used in a wide variety of foods and food ingredients. In many cases, these proteins are sourced from animals. With the world population growth and the coinciding growth in global food demand, there is a need to provide alternative sustainable, non-animal-based sources of proteins as useful source of protein for daily diet, food ingredients and food products.

SUMMARY

[0004] In a first aspect, provided herein is a method of producing a recombinant phosphorylated ovalbumin comprising: (a) co-expressing in a recombinant host cell a heterologous or exogenous ovalbumin protein and at least one heterologous or exogenous kinase, and (b) purifying or isolating the ovalbumin protein which is phosphorylated by the heterologous or exogenous kinase at least one site or residue.

[0005] In a second aspect, provided herein is a method of producing a recombinant phosphorylated ovalbumin comprising: (a) expressing in a recombinant host cell a heterologous or exogenous ovalbumin protein, (b) treating the ovalbumin protein with at least one heterologous or exogenous kinase, and (c) purifying or isolating the ovalbumin protein which is phosphorylated by the heterologous or exogenous kinase protein at least one site or residue. [0006] In another aspect, provided herein is an engineered host cell that recombinantly coexpresses a heterologous or exogenous ovalbumin protein and at least one heterologous or exogenous kinase protein.

[0007] In another aspect, provided herein is a protein composition comprising recombinant ovalbumin protein having a different ratio of diphosphorylated recombinant ovalbumin protein, monophosphorylated recombinant ovalbumin protein, and unphosphorylated recombinant ovalbumin protein than a ratio present in a protein composition obtained from an egg.

[0008] In another aspect, provided herein is a protein composition comprising recombinant ovalbumin protein having a different ratio of diphosphorylated recombinant ovalbumin protein, monophosphorylated recombinant ovalbumin protein, or unphosphorylated recombinant ovalbumin protein than a ratio present in a protein composition expressed by a recombinant cell that lacks heterologous or exogenous kinase.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0010] FIG. 1 is a phylogenetic tree of illustrative protein kinases. See Worby et al., The ABCs of the atypical Fam20 secretory pathway kinases. J Biol Chem. 2021 Jan-Jun; 296: 100267, the disclosure of which is incorporated by reference in its entirety.

[0011] FIG. 2 is an image of the 3D structure of chicken ovalbumin (OVA) with potential post-translational modification sites highlighted. See Yang et al., Analyzing protein microheterogeneity in chicken ovalbumin by high-resolution native mass spectrometry exposes qualitatively and semi-quantitatively 59 proteoforms. Anal Chem. 2013 Dec 17;85(24): 12037- 45, the disclosure of which is incorporated by reference in its entirety.

[0012] FIG. 3 is a native-PAGE gel showing examples of native (egg-derived) chicken OVA and recombinant (“B17” and “003”) OVA proteins before and after treatment with various phosphatases.

[0013] FIG. 4 is a native-PAGE gel showing examples of native OVA, recombinant OVA (rOVA) protein, and phosphorylated rOVA before and after treatment with phosphatase. [0014] FIG. 5 is a native-PAGE gel showing rOVA which was co-expressed in FAM20A- or FAM20C-expressing Pichia strains (“FAM20A in vivo” and “FAM20C in vivo”) compared to non-phosphorylated rOVA (‘Non-phos’), rOVA phosphorylated in vitro with a kinase, and native (egg-derived) ‘nOVA’.

[0015] FIG. 6 is a native-PAGE gel showing non-phosphorylated rOVA (“rOVA”), phosphorylated rOVA (“rOVA+Kinase”), control rOVA (control), rOVA co-expressed with FAM20A (“FAM20A”), and rOVA co-expressed with FAM20C (“FAM20C”).

[0016] FIG. 7 is a native-PAGE gel showing non-phosphorylated rOVA (“rOVA”), phosphorylated rOVA (“rOVA+Kinase”), rOVA expressed in the control strain (‘rOVA-con”), rOVA co-expressed with FAM20C isoform #2 (“rOVA-FAM20C”), and native OVA (“nOVA”).

[0017] FIG. 8A and FIG. 8B are native-PAGE gels showing migration of phosphorylated rOVA. FIG. 8A shows migration of rOVA treated with FAM20A and FAM20C isoform #1. FIG. 8B shows migration of rOVA treated with FAM20C isoform #2.

[0018] FIG. 9 is a chart showing the cell mass growth, and protein production of two strains with different protein moieties, control rOVA and rOVA expressed with the kinase (rOVA-con and rOVA+FAM20C). FIG. 9 shows that cell mass growth and protein production are consistent in two strains with different protein moieties.

[0019] FIG. 10 is a chart showing the protein elution profile following protein purification and chromatography across two strains with different protein moieties.

[0020] FIG. 11 is an alignment of FAM20C isoforms 1 and 2, highlighting the small sequence difference between the two proteins. Figure discloses SEQ ID NOs: 145-146, respectively, in order of appearance.

[0021] FIG. 12 is Native PAGE analysis of samples from small scale fermentations showing faster migration of phosphorylated rOVA produced by Pichia strains co-expressing FAM20C kinase isoform 1 or Isoform 2.

[0022] FIG. 13 shows the architecture of the FAM20C-2 kinase construct inserted into the Komagataella phaffii genome.

[0023] FIGs. 14A and 14B show dot plots comparing base strains (CS1512 and CS3042) to CS3042 clones expressing the FAM20C-2 kinase construct, with FIG. 14A showing the amount of protein produced by the base strains and the CS3042 clones as measured by Bradford assay and FIG. 14B showing the growth of the base strains and the CS3042 clones measured as OD600. [0024] FIG. 15 is Native PAGE analysis showing faster migration of phosphorylated rOVA produced by co-expressing FAM20C kinase isoform 2 in Komagatella phaffii strains. Lanes 1 & 2 are non-phosphorylated negative controls. Lanes 3 & 4 are OVA protein powder that has been both mono- and diphosphorylated (positive controls).

DETAILED DESCRIPTION

[0025] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0026] Provided herein are compositions and methods of making compositions for nonanimal-based sources of proteins which provide nutritional as well as functional properties to food ingredients and consumable products for ingestion by an animal, including a human, such as for daily diet, ingredients for human food and treats and for human and animal nutrition.

[0027] The compositions and methods provided herein contain fermentation-derived ovalbumin, produced through recombinant technology, i.e., a recombinant ovalbumin (rOVA). In some embodiments, the fermentation-derived recombinant ovalbumin (rOVA) produced herein is a mixture comprising rOVA phosphorylated at zero, one, or more phosphorylation sites (recombinant phosphorylated OVA or rpOVA). The compositions and methods for making compositions comprising the rpOVA mixture can increase the protein content of a consumable or food ingredient, and also provide functional features for use in the preparation of food ingredients and consumable food products for animal and human ingestion, including features similar to features found in native (egg-derived) OVA.

[0028] Notably, the present disclosure provides protein compositions comprising rOVA protein having a different ratio of diphosphorylated recombinant ovalbumin protein, monophosphorylated recombinant ovalbumin protein, or unphosphorylated recombinant ovalbumin protein than a ratio present in an OVA protein composition obtained from a natural egg-white. OVA derived from native egg whites are predominantly monophosphorylated and typically at Ser-68. Further, rOVA that is expressed by fungal cells, e.g., Pichia, which do not express the heterologous or exogenous kinases relevant to the present disclosure, is generally unphosphorylated. Together, an rOVA protein product produced by the cells and method disclosed herein is different from an rOVA protein product obtainable from standard fermentation from fungal cells, e.g., Pichia, or from native egg whites.

INTRODUCTION

[0029] Phosphorylation is a ubiquitous process that exists for many purposes including the regulation of cell cycle components, signal transduction pathways, and for activationdeactivation cycles of enzymes. Post-translational phosphorylation of proteins occurs by protein kinases that add phosphate groups to specific amino acid residues such as serine, threonine, and tyrosine. Often, these residues are contained within a signature or motif that is recognized by a specific kinase. Phosphorylation alters overall protein function by changing the protein’s charge or isoelectric point. The result of these changes can induce or disrupt protein-protein interactions or potentially protect against proteolysis.

[0030] Many proteins require phosphorylation for proper function. For example, native ovalbumin (nOVA) from chicken exists predominantly as a phosphoprotein with the phosphate group modification occurring at two positions, Ser68 and Ser344. These phosphate groups are thought to contribute to OVA’s thermal stability, as enzymatic removal with phosphatase reduces the mid-point denaturation temperature. In contrast to nOVA from chicken eggs, rOVA expressed in Pichia pastoris does not contain any phosphate groups and exhibits lower thermal stability. While Pichia pastoris has several kinase families including casein kinases that phosphorylate serine and threonine residues, these native kinases do not appear to recognize the phosphorylation consensus sites in recombinantly-expressed proteins, including rOVA.

[0031] As used herein “native” in the context of native egg white, native egg protein, native ovalbumin and native egg, refers to the egg white, egg protein, ovalbumin or whole egg, respectively, produced by an animal or collected from an animal, in particular an egg-laying animal such as a bird. The rOVA and/or rpOVA and compositions containing rOVA and/or rpOVA can be used in food ingredients and food products, such that the ingredient or product does not contain any native egg white, native egg protein, native ovalbumin or native egg. In some cases, the ingredients or food products made using rOVA and/or rpOVA do not include any egg-white proteins other than rOVA and/or rpOVA. The rOVA and/or rpOVA and compositions containing rOVA and/or rpOVA can be used in food ingredients and food products, such that the ingredient or product does not contain any animal products.

[0032] In some embodiments, the rpOVA mixture (comprising both unphosphorylated rOVA and rpOVA or rpOVA alone) can (alone or with other ingredients) substitute for the use of whole egg or egg white in the production of a food product. In various embodiments, an rpOVA mixture may comprise, consist essentially of, or consist of phosphorylated forms of rpOVA. In some embodiments, the characteristics provided by the rpOVA mixture is substantially the same or is better than the characteristics provided by a native egg white or native egg. For example, the rpOVA mixture and/or compositions containing the rpOVA mixture can have the same or better gelling, foaming, whipping, fluffing, binding, springiness, aeration, coating, film forming, emulsification, browning, thickening, texturizing, preserving moisture (humectant), clarification, and cohesiveness, improved color, such as a whiter color, as compared to native egg white or native whole egg and compositions made with native egg white.

Phosphorylated forms of r OVA in a rpOVA mixture

[0033] In some aspects, provided herein are consumable compositions which comprise rOVA. The addition of a phosphate group (PO3) to an amino acid residue (referred to here as “phosphorylation”) of rOVA expressed in a host cell results in unique physical and/or chemical properties as compared to “unphosphorylated” recombinant ovalbumin (“rOVA”). These differences are useful in commercial applications when provided as recombinant phosphorylated ovalbumin (rpOVA) alone or when combined with rOVA. The amount of rpOVA in a mixture can be quantified by chromatography, kinase activity assays, phosphospecific antibodies, Western blot, enzyme linked immunosorbent assays (ELISA), cell-based ELISA, flow cytometry, mass spectrometry, PAGE electrophoresis, or multi-analyte profiling. The term “phosphorylated” ovalbumin or “rpOVA” may refer to a protein with a modified or altered three-dimensional structure of the protein, such as through non-covalent interactions; yet comprises a contiguous amino acid backbone. In preferred embodiments, the phosphorylated form of the protein has an amino acid sequence identical to the native protein, i.e., nOVA. In some cases, the rpOVA is phosphorylated at one amino acid residue. In some cases, the rpOVA is phosphorylated at two (or more) amino acid residues. In some cases, a mixture of rpOVA comprises a mixture of unphosphorylated rOVA, rpOVA phosphorylated at two amino acid residues, and rpOVA phosphorylated at one amino acid residue.

[0034] In some embodiment, a consumable composition comprises a mixture of a recombinant ovalbumin (rOVA) protein and a recombinant phosphorylated ovalbumin (rpOVA) protein. In some embodiments, the rOVA protein has a single polypeptide chain or a continuous covalent peptide backbone or a continuous amino acid backbone. In some embodiments, the rpOVA protein has a single polypeptide chain or a continuous covalent peptide backbone or a continuous amino acid backbone. In some embodiments, the rpOVA protein is phosphorylated at one, two, three, four, or more amino acid residues in the contiguous amino acid backbone. In some embodiments, the rpOVA protein is phosphorylated at one, two, three, or more amino acid residues in the contiguous amino acid backbone. In some embodiments, the rpOVA protein is phosphorylated at one, two, or more amino acid residues in the contiguous amino acid backbone. In some embodiments, the rpOVA protein is phosphorylated at two amino acid residues in the contiguous amino acid backbone. In some embodiments, the rpOVA protein is phosphorylated at one amino acid residue in the contiguous amino acid backbone.

[0035] In some embodiments, the rpOVA protein is phosphorylated at one or more serine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more threonine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more tyrosine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine or threonine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine and threonine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine or tyrosine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine and tyrosine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine and histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more tyrosine or threonine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more tyrosine and threonine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more tyrosine or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more tyrosine and histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more threonine or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more threonine and histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, threonine, or tyrosine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, threonine, and tyrosine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, threonine, tyrosine, or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, threonine, or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, threonine, or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, tyrosine, or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, tyrosine, or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more threonine, tyrosine, or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more threonine, tyrosine, or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, threonine, tyrosine, or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, threonine, tyrosine, and histidine residues.

[0036] In some embodiments, the rpOVA comprises post-translation modifications. In some embodiments, the post-translation modification comprises glycosylation. In some embodiments, the rOVA protein is glycosylated before being phosphorylated. In some cases, the rOVA is not phosphorylated until the rOVA is glycosylated. In some embodiments, the glycosylation comprises a carbohydrate attached to an amino acid residue of rOVA. In some embodiments, the glycosylation is N-linked glycosylation. In some embodiments, the glycosylation is O-linked glycosylation. In some embodiments, the glycosylation is C-linked glycosylation. In some embodiments, the glycosylation is S-linked glycosylation. In some embodiments, the glycosylation is glypiation. In some embodiments, the glycosylation is phosphoglycosylation. In some embodiments, the glycosylation moiety is covalently attached to one or more amino acids in the rOVA protein. In some embodiments, the glycosylation moiety comprises a carbohydrate. In some embodiments, the glycosylation moiety comprises a glycan. In some embodiments, the glycosylation moiety comprises at least one mannose moiety. In some embodiments, the glycosylation moiety comprises at least one N- acetylglucosamine moiety. In some embodiments, the glycosylation moiety comprises at least one mannose moiety or at least one N-acetylglucosamine moiety. In some embodiments, the glycosylation moiety comprises at least one mannose moiety and at least one N- acetyl glucosamine moiety.

[0037] In some embodiments, an unglycosylated rOVA is unphosphorylated rOVA. In some embodiments, an rpOVA is glycosylated. In some embodiments, a glycosylated rOVA is unphosphorylated. In some embodiments, a glycosylated rpOVA is more stable than an unglycosylated rpOVA.

[0038] In some embodiments, the unphosphorylated rOVA is most abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rOVA is most abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rOVA at Ser-68 (with respect to SEQ ID NO: 2) is most abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rOVA at Ser-344 (with respect to SEQ ID NO: 2) is most abundant in the rpOVA mixture. In some embodiments, the diphosphorylated rOVA is most abundant in the rpOVA mixture. In some embodiments, the unphosphorylated rOVA is least abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rOVA is least abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rOVA at Ser-68 is least abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rOVA at Ser- 344 is least abundant in the rpOVA mixture. In some embodiments, the diphosphorylated rOVA is most abundant in the rpOVA mixture.

[0039] In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is about 1 : 1 to about 1:20. In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is about 1:20 to about 1:15, about 1:20 to about 1:10, about 1:20 to about 1:9, about 1:20 to about 1:8, about 1:20 to about 1:7, about 1:20 to about 1:6, about 1:20 to about 1:5, about 1:20 to about 1:4, about 1:20 to about 1:3, about 1:20 to about 1:2, about 1:20 to about 1:1, about 1:15 to about 1:10, about 1:15 to about 1:9, about 1:15 to about 1:8, about 1:15 to about 1:7, about 1:15 to about 1:6, about 1:15 to about 1:5, about 1:15 to about 1:4, about 1:15 to about 1:3, about 1:15 to about 1:2, about 1:15 to about 1:1, about 1:10 to about 1:9, about 1:10 to about 1:8, about 1:10 to about 1:7, about 1:10 to about 1:6, about 1:10 to about 1:5, about 1:10 to about 1:4, about 1:10 to about 1:3, about 1:10 to about 1:2, about 1:10 to about 1:1, about 1:9 to about 1:8, about 1 :9 to about 1 :7, about 1 :9 to about 1 :6, about 1 :9 to about 1:5, about 1 :9 to about 1:4, about 1:9 to about 1:3, about 1:9 to about 1:2, about 1:9 to about 1:1, about 1:8 to about 1:7, about 1:8 to about 1:6, about 1:8 to about 1:5, about 1:8 to about 1:4, about 1:8 to about 1:3, about 1:8 to about 1:2, about 1:8 to about 1:1, about 1:7 to about 1:6, about 1:7 to about 1:5, about 1:7 to about 1:4, about 1:7 to about 1:3, about 1:7 to about 1:2, about 1:7 to about 1:1, about 1:6 to about 1:5, about 1:6 to about 1:4, about 1:6 to about 1:3, about 1:6 to about 1:2, about 1:6 to about 1:1, about 1:5 to about 1:4, about 1:5 to about 1:3, about 1:5 to about 1:2, about 1:5 to about 1:1, about 1:4 to about 1:3, about 1:4 to about 1:2, about 1:4 to about 1:1, about 1:3 to about 1:2, about 1:3 to about 1:1, or about 1:2 to about 1:1. In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is about 1 :20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1. In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is at least about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, or about 1:2. In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is at most about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1.

[0040] In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is about 1 : 1 to about 20:1. In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is about 20:1 to about 15:1, about 20:1 to about 10:1, about 20:1 to about 9:1, about 20:1 to about 8:1, about 20 : 1 to about 7:1, about 20 to about 6:1, about 20 : 1 to about 5:1, about 20 : 1 to about 4:1, about 20:1 to about 3:1, about 20:1 to about 2:1, about 20:1 to about 1:1, about 15:1 to about 10:1, about 15:1 to about 9:1, about 15:1 to about 8:1, about 15:1 to about 7:1, about 15:1 to about 6:1, about 15:1 to about 5:1, about 15:1 to about 4:1, about 15:1 to about 3:1, about 15:1 to about 2:1, about 15:1 to about 1:1, about 10:1 to about 9:1, about 10:1 to about 8:1, about 10:1 to about 7:1, about 10:1 to about 6:1, about 10:1 to about 5:1, about 10:1 to about 4:1, about 10:1 to about 3:1, about 10:1 to about 2:1, about 10:1 to about 1:1, about 9:1 to about 8:1, about 9:1 to about 7:1, about 9:1 to about 6:1, about 9:1 to about 5:1, about 9:1 to about

4:1, about 9:1 to about 3:1, about 9:1 to about 2:1, about 9:1 to about 1:1, about 8:1 to about

7:1, about 8:1 to about 6:1, about 8:1 to about 5:1, about 8:1 to about 4:1, about 8:1 to about

3:1, about 8:1 to about 2:1, about 8:1 to about 1:1, about 7:1 to about 6:1, about 7:1 to about

5:1, about 7:1 to about 4:1, about 7:1 to about 3:1, about 7:1 to about 2:1, about 7:1 to about

1:1, about 6:1 to about 5:1, about 6:1 to about 4:1, about 6:1 to about 3:1, about 6:1 to about

2:1, about 6:1 to about 1:1, about 5:1 to about 4:1, about 5:1 to about 3:1, about 5:1 to about

2:1, about 5:1 to about 1:1, about 4:1 to about 3:1, about 4:1 to about 2:1, about 4:1 to about

1:1, about 3 : 1 to about 2:1, about 3 : 1 to about 1 : 1 , or about 2 : 1 to about 1 : 1. the ratio of rpOVA to unphosphorylated rOVA is about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is at least about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is at most about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2: 1, or about 1:1.

[0041] In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 (with respect to SEQ ID NO: 2) to monophosphorylated rOVA at Ser-344 (with respect to SEQ ID NO: 2) is about 1:1 to about 1:20. In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 to monophosphorylated rOVA at Ser-344 is about 1:20 to about 1:15, about 1:20 to about 1:10, about 1:20 to about 1:9, about 1:20 to about 1:8, about 1:20 to about 1:7, about 1:20 to about 1:6, about 1:20 to about 1:5, about 1:20 to about 1:4, about 1:20 to about 1:3, about 1:20 to about 1:2, about 1:20 to about 1:1, about 1:15 to about 1:10, about 1:15 to about 1:9, about 1:15 to about 1:8, about 1:15 to about 1:7, about 1:15 to about 1:6, about 1:15 to about 1:5, about 1:15 to about 1:4, about 1:15 to about 1:3, about 1:15 to about 1:2, about 1:15 to about 1:1, about 1:10 to about 1:9, about 1:10 to about 1:8, about 1:10 to about 1:7, about 1:10 to about 1:6, about 1:10 to about 1:5, about 1:10 to about 1:4, about 1:10 to about 1 :3, about 1 : 10 to about 1 :2, about 1 : 10 to about 1:1, about 1 :9 to about 1:8, about 1 :9 to about 1:7, about 1:9 to about 1:6, about 1:9 to about 1:5, about 1:9 to about 1:4, about 1:9 to about

1:3, about 1:9 to about 1:2, about 1:9 to about 1:1, about 1:8 to about 1:7, about 1:8 to about

1:6, about 1:8 to about 1:5, about 1:8 to about 1:4, about 1:8 to about 1:3, about 1:8 to about

1:2, about 1:8 to about 1:1, about 1:7 to about 1:6, about 1:7 to about 1:5, about 1:7 to about

1:4, about 1:7 to about 1:3, about 1:7 to about 1:2, about 1:7 to about 1:1, about 1:6 to about

1:5, about 1:6 to about 1:4, about 1:6 to about 1:3, about 1:6 to about 1:2, about 1:6 to about

1:1, about 1:5 to about 1:4, about 1:5 to about 1:3, about 1:5 to about 1:2, about 1:5 to about

1:1, about 1:4 to about 1:3, about 1:4 to about 1:2, about 1:4 to about 1:1, about 1:3 to about

1:2, about 1:3 to about 1:1, or about 1:2 to about 1:1. In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 to monophosphorylated rOVA at Ser-344 is about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1. In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 to monophosphorylated rOVA at Ser-344 is at least about 1:20, about 1:15, about 1:10, about 1 :9, about 1 :8, about 1 :7, about 1 :6, about 1:5, about 1 :4, about 1 :3, or about 1 :2. In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 to monophosphorylated rOVA at Ser-344 is at most about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1.

[0042] In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 (with respect to SEQ ID NO: 2) to monophosphorylated rOVA at Ser-344 (with respect to SEQ ID NO: 2) is about 1:1 to about 20:1. In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 to monophosphorylated rOVA at Ser-344 is about 20:1 to about 15:1, about 20:1 to about 10:1, about 20:1 to about 9:1, about 20:1 to about 8:1, about 20:1 to about 7:1, about 20 to about 6:1, about 20 : 1 to about 5:1, about 20 : 1 to about 4:1, about 20 : 1 to about 3:1, about 20: 1 to about 2:1, about 20: 1 to about 1:1, about 15:1 to about 10:1, about 15:1 to about 9:1, about 15:1 to about 8:1, about 15:1 to about 7:1, about 15:1 to about 6:1, about 15:1 to about 5:1, about 15:1 to about 4:1, about 15:1 to about 3:1, about 15:1 to about 2:1, about 15:1 to about 1:1, about 10:1 to about 9:1, about 10:1 to about 8:1, about 10:1 to about 7:1, about 10:1 to about 6:1, about 10:1 to about 5:1, about 10:1 to about 4:1, about 10:1 to about 3:1, about 10:1 to about 2:1, about 10:1 to about 1:1, about 9:1 to about 8:1, about 9:1 to about 7:1, about 9:1 to about 6:1, about 9:1 to about 5:1, about 9:1 to about 4:1, about 9:1 to about 3:1, about 9:1 to about 2:1, about 9:1 to about 1:1, about 8:1 to about 7:1, about 8:1 to about 6:1, about 8:1 to about 5:1, about 8:1 to about 4:1, about 8:1 to about 3:1, about 8:1 to about 2:1, about 8:1 to about 1:1, about 7:1 to about 6:1, about 7:1 to about 5:1, about 7:1 to about 4:1, about 7:1 to about 3:1, about 7:1 to about 2:1, about 7:1 to about 1:1, about 6:1 to about 5:1, about 6:1 to about 4:1, about 6:1 to about 3:1, about 6:1 to about 2:1, about 6:1 to about 1:1, about 5:1 to about 4:1, about 5:1 to about 3:1, about 5:1 to about 2:1, about 5:1 to about 1:1, about 4:1 to about 3:1, about 4:1 to about 2:1, about 4:1 to about 1:1, about 3:1 to about 2:1, about 3:1 to about 1:1, or about 2:1 to about 1:1. In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 to monophosphorylated rOVA at Ser-344 is about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 to monophosphorylated rOVA at Ser-344 is at least about 20: 1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 to monophosphorylated rOVA at Ser-344 is at most about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1.

[0043] In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is about 1:1 to about 1:20. In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is about 1 :20 to about 1:15, about 1 :20 to about 1:10, about 1 :20 to about 1 :9, about 1 :20 to about 1:8, about 1 :20 to about 1 :7, about 1 :20 to about 1 :6, about 1 :20 to about 1:5, about 1:20 to about 1:4, about 1:20 to about 1:3, about 1:20 to about 1:2, about 1:20 to about 1:1, about 1:15 to about 1:10, about 1:15 to about 1:9, about 1:15 to about 1:8, about 1:15 to about 1:7, about 1:15 to about 1:6, about 1:15 to about 1:5, about 1:15 to about 1:4, about 1:15 to about 1:3, about 1:15 to about 1:2, about 1:15 to about 1:1, about 1:10 to about 1 :9, about 1 : 10 to about 1:8, about 1 : 10 to about 1 :7, about 1 : 10 to about 1 :6, about 1:10 to about 1:5, about 1:10 to about 1:4, about 1:10 to about 1:3, about 1:10 to about 1:2, about 1:10 to about 1:1, about 1:9 to about 1:8, about 1:9 to about 1:7, about 1:9 to about 1:6, about 1:9 to about 1:5, about 1:9 to about 1:4, about 1:9 to about 1:3, about 1:9 to about 1:2, about 1:9 to about 1:1, about 1:8 to about 1:7, about 1:8 to about 1:6, about 1:8 to about 1:5, about

1:8 to about 1:4, about 1:8 to about 1:3, about 1:8 to about 1:2, about 1:8 to about 1:1, about

1:7 to about 1:6, about 1:7 to about 1:5, about 1:7 to about 1:4, about 1:7 to about 1:3, about

1:7 to about 1:2, about 1:7 to about 1:1, about 1:6 to about 1:5, about 1:6 to about 1:4, about

1:6 to about 1:3, about 1:6 to about 1:2, about 1:6 to about 1:1, about 1:5 to about 1:4, about

1:5 to about 1:3, about 1:5 to about 1:2, about 1:5 to about 1:1, about 1:4 to about 1:3, about

1 :4 to about 1 :2, about 1 :4 to about 1:1, about 1 :3 to about 1 :2, about 1 :3 to about 1 : 1, or about 1:2 to about 1:1. In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1. In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is at least about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, or about 1:2. In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is at most about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1.

[0044] In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is about 1:1 to about 20:1. In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is about 20: 1 to about 15:1, about 20: 1 to about 10:1, about 20: 1 to about 9:1, about 20:1 to about 8:1, about 20:1 to about 7:1, about 20 to about 6:1, about 20:1 to about 5:1, about 20:1 to about 4:1, about 20:1 to about 3:1, about 20:1 to about 2:1, about 20:1 to about 1:1, about 15:1 to about 10:1, about 15:1 to about 9:1, about 15:1 to about 8:1, about 15:1 to about 7:1, about 15:1 to about 6:1, about 15:1 to about 5:1, about 15:1 to about 4:1, about 15:1 to about 3:1, about 15:1 to about 2:1, about 15:1 to about 1:1, about 10:1 to about 9:1, about 10:1 to about 8:1, about 10:1 to about 7:1, about 10:1 to about 6:1, about 10:1 to about 5:1, about 10:1 to about 4:1, about 10:1 to about 3:1, about 10:1 to about 2:1, about 10:1 to about 1:1, about 9:1 to about 8:1, about 9:1 to about 7:1, about 9:1 to about 6:1, about 9:1 to about 5:1, about 9:1 to about 4:1, about 9:1 to about 3:1, about 9:1 to about 2:1, about

9:1 to about 1:1, about 8:1 to about 7:1, about 8:1 to about 6:1, about 8:1 to about 5:1, about

8:1 to about 4:1, about 8:1 to about 3:1, about 8:1 to about 2:1, about 8:1 to about 1:1, about

7:1 to about 6:1, about 7:1 to about 5:1, about 7:1 to about 4:1, about 7:1 to about 3:1, about

7:1 to about 2:1, about 7:1 to about 1:1, about 6:1 to about 5:1, about 6:1 to about 4:1, about

6:1 to about 3:1, about 6:1 to about 2:1, about 6:1 to about 1:1, about 5:1 to about 4:1, about

5:1 to about 3:1, about 5:1 to about 2:1, about 5:1 to about 1:1, about 4:1 to about 3:1, about 4: 1 to about 2:1, about 4: 1 to about 1:1, about 3 : 1 to about 2:1, about 3 : 1 to about 1 : 1, or about 2: 1 to about 1 : 1. the ratio of diphosphorylated rOVA to unphosphorylated rOVA is about 20: 1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is at least about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is at most about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2: 1, or about 1:1.

[0045] In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA (e.g., at Ser-68 or at Ser-344) is about 1 : 1 to about 1 :20. In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA is about 1:20 to about 1:15, about 1:20 to about 1:10, about 1:20 to about 1:9, about 1:20 to about 1:8, about 1:20 to about 1:7, about 1:20 to about 1:6, about 1:20 to about 1:5, about 1:20 to about 1:4, about 1:20 to about 1:3, about 1:20 to about 1:2, about 1:20 to about 1:1, about 1:15 to about 1:10, about 1:15 to about 1:9, about 1:15 to about 1:8, about 1:15 to about 1:7, about 1:15 to about 1:6, about 1:15 to about 1:5, about 1:15 to about 1:4, about 1:15 to about 1:3, about 1:15 to about 1:2, about 1:15 to about 1:1, about 1:10 to about 1:9, about 1:10 to about 1:8, about 1:10 to about 1:7, about 1:10 to about 1:6, about 1:10 to about 1:5, about 1:10 to about 1:4, about 1:10 to about 1 :3, about 1 : 10 to about 1 :2, about 1 : 10 to about 1:1, about 1 :9 to about 1:8, about 1 :9 to about 1:7, about 1:9 to about 1:6, about 1:9 to about 1:5, about 1:9 to about 1:4, about 1:9 to about

1:2, about 1:3 to about 1:1, or about 1:2 to about 1:1. In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA is about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1 : 1. In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA is at least about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, or about 1:2. In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA is at most about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1.

[0046] In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA (e.g., at Ser-68 or at Ser-344 and with respect to SEQ ID NO: 2) is about 1:1 to about 20:1. In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA is about 20: 1 to about 15:1, about 20: 1 to about 10:1, about 20: 1 to about 9:1, about 20: 1 to about 8:1, about 20: 1 to about 7: 1, about 20 to about 6:1, about 20: 1 to about 5:1, about 20: 1 to about 4:1, about 20:1 to about 3:1, about 20:1 to about 2:1, about 20:1 to about 1:1, about 15:1 to about 10:1, about 15:1 to about 9:1, about 15:1 to about 8:1, about 15:1 to about 7:1, about 15:1 to about 6:1, about 15:1 to about 5:1, about 15:1 to about 4:1, about 15:1 to about 3:1, about 15:1 to about 2:1, about 15:1 to about 1:1, about 10:1 to about 9:1, about 10:1 to about 8:1, about 10:1 to about 7:1, about 10:1 to about 6:1, about 10:1 to about 5:1, about 10:1 to about 4:1, about 10:1 to about 3:1, about 10:1 to about 2:1, about 10:1 to about 1:1, about 9:1 to about 8:1, about 9:1 to about 7:1, about 9:1 to about 6:1, about 9:1 to about 5:1, about

9:1 to about 4:1, about 9:1 to about 3:1, about 9:1 to about 2:1, about 9:1 to about 1:1, about

8:1 to about 7:1, about 8:1 to about 6:1, about 8:1 to about 5:1, about 8:1 to about 4:1, about

8:1 to about 3:1, about 8:1 to about 2:1, about 8:1 to about 1:1, about 7:1 to about 6:1, about

7:1 to about 5:1, about 7:1 to about 4:1, about 7:1 to about 3:1, about 7:1 to about 2:1, about

7:1 to about 1:1, about 6:1 to about 5:1, about 6:1 to about 4:1, about 6:1 to about 3:1, about

6:1 to about 2:1, about 6:1 to about 1:1, about 5:1 to about 4:1, about 5:1 to about 3:1, about

5:1 to about 2:1, about 5:1 to about 1:1, about 4:1 to about 3:1, about 4:1 to about 2:1, about

4:1 to about 1:1, about 3:1 to about 2:1, about 3:1 to about 1:1, or about 2:1 to about 1:1. the ratio of diphosphorylated rOVA to monophosphorylated rOVA is about 20: 1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA is at least about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA is at most about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1.

[0047] In some embodiments, the ovalbumin content of the consumable composition comprises 0.1% w/w to 30% w/w rpOVA (with one or more phosphorylated amino acids) while the rest of the ovalbumin is rOVA (full-length unphosphorylated ovalbumin with a single peptide chain). In some embodiments, the ovalbumin content of the consumable composition comprises at least 0.1% w/w rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises at most 30% w/w rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises 0.1% w/w to 0.5% w/w, 0.1% w/w to 1% w/w, 0.1% w/w to 5% w/w, 0.1% w/w to 10% w/w, 0.1% w/w to 15% w/w, 0.1% w/w to 20: 1% w/w, 0.1% w/w to 30% w/w, 0.5% w/w to 1% w/w, 0.5% w/w to 5% w/w, 0.5% w/w to 10% w/w, 0.5% w/w to 15% w/w, 0.5% w/w to 20: 1% w/w, 0.5% w/w to 30% w/w, 1% w/w to 5% w/w, 1% w/w to 10% w/w, 1% w/w to 15% w/w, 1% w/w to 20: 1% w/w, 1% w/w to 30% w/w, 5% w/w to 10% w/w, 5% w/w to 15% w/w, 5% w/w to 20: 1% w/w, 5% w/w to 30% w/w, 10% w/w to 15% w/w, 10% w/w to 20: 1% w/w, 10% w/w to 30% w/w, 15% w/w to 20: 1% w/w, 15% w/w to 30% w/w, or 20:1% w/w to 30% w/w rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises about 0.1% w/w, 0.5% w/w, 1% w/w, 5% w/w, 10% w/w, 15% w/w, 20: 1% w/w, or 30% w/w rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises at least 0.1% w/w, 0.5% w/w, 1% w/w, 5% w/w, 10% w/w, 15% w/w, 20: 1% w/w, or 30% w/w rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises at most 0.1% w/w, 0.5% w/w, 1% w/w, 5% w/w, 10% w/w, 15% w/w, 20:1% w/w, or 30% w/w rpOVA.

[0048] In some embodiments, the ovalbumin content of the consumable composition comprises a high concentration of rpOVA (with one or more phosphorylated amino acids), for instance from 35% w/w to 100% w/w rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises at least 35% w/w rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises 100% w/w rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises 35% w/w to 40% w/w, 35% w/w to 50% w/w, 35% w/w to 70% w/w, 35% w/w to 80% w/w, 35% w/w to 90% w/w, 35% w/w to 100% w/w, 40% w/w to 50% w/w, 40% w/w to 70% w/w, 40% w/w to 80% w/w, 40% w/w to 90% w/w, 40% w/w to 100% w/w, 50% w/w to 70% w/w, 50% w/w to 80% w/w, 50% w/w to 90% w/w, 50% w/w to 100% w/w, 70% w/w to 80% w/w, 70% w/w to 90% w/w, 70% w/w to 100% w/w, 80% w/w to 90% w/w, 80% w/w to 100% w/w, or 90% w/w to 100% w/w rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises about 35% w/w, 40% w/w, 50% w/w, 70% w/w, 80% w/w, 90% w/w, or 100% w/w rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises at least 35% w/w, 40% w/w, 50% w/w, 70% w/w, 80% w/w, or 90% w/w rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises at most 35% w/w, 40% w/w, 50% w/w, 70% w/w, 80% w/w, 90% w/w rpOVA.

[0049] In some embodiments, the ovalbumin content of the consumable composition comprises 0.1% w/w to 30% w/w of a mixture of rOVA and rpOVA (with one or more phosphorylated amino acids). In some embodiments, the ovalbumin content of the consumable composition comprises at least 0.1% w/w of a mixture of rOVA and rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises at most 30% w/w of a mixture of rOVA and rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises 0.1% w/w to 0.5% w/w, 0.1% w/w to 1% w/w, 0.1% w/w to 5% w/w, 0.1% w/w to 10% w/w, 0.1% w/w to 15% w/w, 0.1% w/w to 20:1% w/w, 0.1% w/w to 30% w/w, 0.5% w/w to 1% w/w, 0.5% w/w to 5% w/w, 0.5% w/w to 10% w/w, 0.5% w/w to 15% w/w, 0.5% w/w to 20: 1% w/w, 0.5% w/w to 30% w/w, 1% w/w to 5% w/w, 1% w/w to 10% w/w, 1% w/w to 15% w/w, 1% w/w to 20: 1% w/w, 1% w/w to 30% w/w, 5% w/w to 10% w/w, 5% w/w to 15% w/w, 5% w/w to 20:1% w/w, 5% w/w to 30% w/w, 10% w/w to 15% w/w, 10% w/w to 20 : 1 % w/w, 10% w/w to 30% w/w, 15% w/w to 20 : 1 % w/w, 15% w/w to 30% w/w, or 20: 1% w/w to 30% w/w of a mixture of rOVA and rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises about 0.1% w/w, 0.5% w/w, 1% w/w, 5% w/w, 10% w/w, 15% w/w, 20: 1% w/w, or 30% w/w of a mixture of rOVA and rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises at least 0.1% w/w, 0.5% w/w, 1% w/w, 5% w/w, 10% w/w, 15% w/w, 20: 1% w/w, or 30% w/w of a mixture of rOVA and rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises at most 0.1% w/w, 0.5% w/w, 1% w/w, 5% w/w, 10% w/w, 15% w/w, 20: 1% w/w, or 30% w/w of a mixture of rOVA and rpOVA.

[0050] In some embodiments, the ovalbumin content of the consumable composition comprises a high concentration of a mixture of rOVA and rpOVA (with one or more phosphorylated amino acids), for instance from 35% w/w to 100% w/w of a mixture of rOVA and rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises at least 35% w/w of a mixture of rOVA and rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises 100% w/w of a mixture of rOVA and rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises 35% w/w to 40% w/w, 35% w/w to 50% w/w, 35% w/w to 70% w/w, 35% w/w to 80% w/w, 35% w/w to 90% w/w, 35% w/w to 100% w/w, 40% w/w to 50% w/w, 40% w/w to 70% w/w, 40% w/w to 80% w/w, 40% w/w to 90% w/w, 40% w/w to 100% w/w, 50% w/w to 70% w/w, 50% w/w to 80% w/w, 50% w/w to 90% w/w, 50% w/w to 100% w/w, 70% w/w to 80% w/w, 70% w/w to 90% w/w, 70% w/w to 100% w/w, 80% w/w to 90% w/w, 80% w/w to 100% w/w, or 90% w/w to 100% w/w of a mixture of rOVA and rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises about 35% w/w, 40% w/w, 50% w/w, 70% w/w, 80% w/w, 90% w/w, or 100% w/w of a mixture of rOVA and rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises at least 35% w/w, 40% w/w, 50% w/w, 70% w/w, 80% w/w, or 90% w/w of a mixture of rOVA and rpOVA. In some embodiments, the ovalbumin content of the consumable composition comprises at most 35% w/w, 40% w/w, 50% w/w, 70% w/w, 80% w/w, 90% w/w of a mixture of rOVA and rpOVA.

[0051] In some embodiments, rOVA is phosphorylated by an exogenous kinase, e.g., a recombinantly-expressed or a kinase provided in vitro. In some embodiments, rOVA is phosphorylated by a heterologous kinase.

[0052] In some embodiments, a host cell may be engineered to express or over-express an ovalbumin gene. In some embodiments, a host cell may be engineered to express or overexpress a kinase. In some embodiments, the host cell is engineered to express or over-express an ovalbumin gene from a species of animal which is different from the species that naturally expresses the kinase. For example, a recombinant cell may express a chicken OVA and a duck kinase. In some embodiments, the host cell is engineered to express or over-express an ovalbumin gene from the species of animal that naturally expresses the kinase. As examples, a recombinant cell may express a chicken OVA and a chicken kinase, or a recombinant cell may express a recombinant duck OVA and a duck kinase. In some embodiments, more than one kinase may be expressed or overexpressed in the host cell which expresses recombinant ovalbumin. The expression or overexpression of kinases may be performed to modulate the amount of phosphorylated ovalbumin produced during fermentation. For instance, a host cell may overexpress one or more kinases to increase the amount of phosphorylated OVA produced during fermentation.

[0053] Kinases may be utilized to modulate the amount of phosphorylated ovalbumin produced during fermentation. Kinases may be added to the fermentation medium while recombinant ovalbumin is being produced by a host cell. Alternatively, kinases may be used to modulate the amount of phosphorylated ovalbumin in a purified ovalbumin protein preparation. In some embodiments, an exogenous gene encoding a kinase may be co-expressed with rOVA in a host cell.

[0054] In some embodiments, kinases may be kinases endogenously expressed in a range of organisms (FIG. 1). In some embodiments, the exogenous kinase protein is selected from the genera: Homo, Drosophila, Danio, Branchiostoma, Stronglycocentrotus, Caenorhabditis, Hydra, Amphimedon, any other animal, or any combination thereof. In certain embodiments, the kinase protein is selected from the Aves or Reptilia class. In various cases, the kinase protein is naturally expressed from a bird species which naturally expresses the OVA. As examples, a recombinant cell may express a chicken OVA and a chicken kinase or a recombinant cell may express a recombinant duck OVA and a duck kinase.

[0055] Illustrative kinases may be a FAM20 which includes but is not limited to: FAM20A, FAM20C isoform #1, FAM20C isoform #2, FAM20A, and FAM20B. In some embodiments, the kinase can have an amino acid sequence of FAM20 from a bird, a reptile, or another egglaying species. In some embodiments, the FAM20 having an amino acid sequence from an Aves species can be selected from the group consisting of: bam owl (Tyto alba), chicken (Gallus domesticus), chuck-will’s-willow (Antrostomus carolinensis), crow (Corvus brachyrhynchos), dove (Columba livia), emu (Dromaius novaehollandiae), golden manakin (Manacus vitellinus), goose (A user cygnoid), hummingbird (Calypte anna), Japanese quail (Coturnix japonica), little egret (Egretta garzetta), little spotted kiwi (Apteryx owenii), mallard (Anas platyrhynchos), ostrich (Struthio camelus), penguin (Aptenodytes forsteri), speckled mousebird (Colius striatus), turkey (Meleagris gallopavo), or zebra finch (Taeniopygia guttata), and any combination thereof. In some embodiments, the FAM20 can have an amino acid sequence derived from a single species, such as the chicken (Gallus Gallus, Gallus domesticus, or Gallus Gallus domesticus). In some embodiments, the heterologous or exogenous kinase protein is from G. gallus. In embodiments, the FAM20 has an amino acid sequence from an alga, e.g., Chloropicon primus.

[0056] In some embodiments, the kinase can have an amino acid sequence derived from two or more species, and as such can be a hybrid. In some embodiments, the kinase protein comprises a mixture of at least two or more distinct heterologous or exogenous kinase proteins. In some embodiments, the kinase or kinases can have an amino acid sequence derived from a single species. In some embodiments, the kinase can have an amino acid sequence derived from two or more species, and as such can be a hybrid. [0057] In some embodiments, a phosphorylated recombinant ovalbumin (rpOVA) can be produced by addition of exogenous kinase and/or by amplification of endogenous kinases of the host cell during or after the manufacturing process (e.g., during fermentation and expression of the rOVA or during downstream processing steps). In some cases, the phosphorylation of ovalbumin can be achieved by addition of exogenous, synthesized, and/or commercially- available kinase. In these cases, the phosphorylation may be considered “zw vitro". The kinase may be added (e.g., >4 units/mg) at a ratio within an order of 1 part kinase to 10,000 parts ovalbumin (mass/mass). The phosphorylation of rOVA may be performed at 37°C in a low- salt phosphate buffer near neutral pH. Phosphorylation may be performed within less than 48 hours. Other different time and temperature conditions extrapolated from the above conditions may be utilized for rOVA phosphorylation. In some cases, salt, pH, and other environmental conditions may be modified to increase phosphorylation and ovalbumin stability and activity.

[0058] In some embodiments, the in vitro kinase treatment of rOVA is performed at a kinase to OVA ratio of at least 1 : 100,000. In some embodiments, the kinase treatment of rOVA is performed at a kinase to OVA ratio of at most 1 :50. In some embodiments, the kinase treatment of rOVA is performed at a kinase to OVA ratio of at about 1 :50, 1 : 100, 1 : 1,000, 1 :2,000, 1 :5,000, 1 : 10,000 1 :20: 1,000, 1 :50,000, 1 : 100,000. In some embodiments, the kinase treatment of rOVA is performed at a kinase to OVA ratio from 1 :50 to 1 :1,000, 1 :50 to 1 : 10,000, 1 :50 to 1 : 100,000, 1 : 100 to 1 : 1,000, 1 : 100 to 1 : 10,000, 1 : 100 to 1 :50,000, 1 : 100 to 1 : 100,000, 1 : 1,0000 to 1 : 10,000, 1 : 1,000 to 1 :50,000, 1 : 1,000 to 1 :100,000, 1 : 10,000 to 1 :50,000, 1 :10,000 to 1 : 100,000. In some cases, the kinase may be a serine threonine kinase. In some cases, the kinase may be a tyrosine kinase.

[0059] In some embodiments, the in vitro kinase treatment of rOVA is performed at a temperature of 32 °C to 40 °C. In some embodiments, the kinase treatment of rOVA is performed at a temperature of at least 32 °C. In some embodiments, the kinase treatment of rOVA is performed at a temperature of at most 40 °C. In some embodiments, the kinase treatment of rOVA is performed at a temperature of 32 °C to 34 °C, 32 °C to 35 °C, 32 °C to 37 °C, 32 °C to 40 °C, 34 °C to 35 °C, 34 °C to 37 °C, 34 °C to 40 °C, 35 °C to 37 °C, 35 °C to 40 °C, or 37 °C to 40 °C. In some embodiments, the kinase treatment of rOVA is performed at a temperature of about 32 °C, 34 °C, 35 °C, 37 °C, or 40 °C.

[0060] In some embodiments, the in vitro kinase treatment of rOVA is performed at a pH of 5 to 9. In some embodiments, the kinase treatment of rOVA is performed at a pH of at least 5. In some embodiments, the kinase treatment of rOVA is performed at a pH of at most 9. In some embodiments, the kinase treatment of rOVA is performed at a pH of 5 to 6, 5 to 7, 5 to 8, 5 to 9, 6 to 7, 6 to 8, 6 to 9, 7 to 8, 7 to 9, or 8 to 9. In some embodiments, the kinase treatment of rOVA is performed at a pH of about 5, 6, 7, 8, or 9.

[0061] In some embodiments, the in vitro kinase treatment of rOVA is performed for 0.5 hours to 3 hours. In some embodiments, the kinase treatment of rOVA is performed for at least 0.5 hours. In some embodiments, the kinase treatment of rOVA is performed for at most 3 hours. In some embodiments, the kinase treatment of rOVA is performed for 0.5 hours to 0.7 hours, 0.5 hours to 1 hours, 0.5 hours to 1.2 hours, 0.5 hours to 1.5 hours, 0.5 hours to 1.7 hours, 0.5 hours to 2 hours, 0.5 hours to 2.5 hours, 0.5 hours to 3 hours, 0.7 hours to 1 hours, 0.7 hours to 1.2 hours, 0.7 hours to 1.5 hours, 0.7 hours to 1.7 hours, 0.7 hours to 2 hours, 0.7 hours to 2.5 hours, 0.7 hours to 3 hours, 1 hours to 1.2 hours, 1 hours to 1.5 hours, 1 hours to 1.7 hours, 1 hours to 2 hours, 1 hours to 2.5 hours, 1 hours to 3 hours, 1.2 hours to 1.5 hours, 1.2 hours to 1.7 hours, 1.2 hours to 2 hours, 1.2 hours to 2.5 hours, 1.2 hours to 3 hours, 1.5 hours to 1.7 hours, 1.5 hours to 2 hours, 1.5 hours to 2.5 hours, 1.5 hours to 3 hours, 1.7 hours to 2 hours, 1.7 hours to 2.5 hours, 1.7 hours to 3 hours, 2 hours to 2.5 hours, 2 hours to 3 hours, or 2.5 hours to 3 hours. In some embodiments, the protease treatment of rOVA is performed for 0.5 hours, 0.7 hours, 1 hours, 1.2 hours, 1.5 hours, 1.7 hours, 2 hours, 2.5 hours, or 3 hours. [0062] In some embodiments, the molecular weight of rpOVA is at least the molecular weight of the unphosphorylated rOVA. In some embodiments, the molecular weight of rpOVA is greater than the molecular weight of the unphosphorylated rOVA. In some embodiments, the molecular weight of the phosphorylated form of the rOVA is at least about 100%, about 105%, about 110%, about 120: 1%, about 130%, about 140%, or about 150% of the molecular weight of the full-length rOVA. In some embodiments, the molecular weight of rpOVA is about 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or 110% of the molecular weight of unphosphorylated rOVA.

METHODS

[0063] An aspect of the present disclosure is a method of producing a recombinant phosphorylated ovalbumin (rpOVA). The method comprising steps of co-expressing in a recombinant host cell a heterologous or exogenous ovalbumin protein and at least one heterologous or exogenous kinase and purifying or isolating the ovalbumin protein which is phosphorylated by the exogenous kinase protein on at least one site or residue. [0064] Another aspect of the present disclosure is a method of producing an rpOVA, the method comprising steps of expressing in a recombinant host cell a heterologous or exogenous ovalbumin protein, treating the ovalbumin protein with at least one kinase, and purifying or isolating the ovalbumin protein which is phosphorylated by the exogenous kinase protein on at least one site or residue.

[0065] In some embodiments, the recombinant host cell is a yeast host cell. In embodiments, the recombinant yeast host cell is Pichia. In some cases, the recombinant yeast host cell is Pichia pastoris. In various cases, the recombinant yeast host cell is Saccharomyces . In some cases, the recombinant yeast host cell is Saccharomyces cerevisiae. In some embodiments, the recombinant host cell is a filamentous fungal host cell. In some cases, the recombinant filamentous fungal host cell is Trichoderma. In some cases, the recombinant filamentous fungal host cell is Trichoderma. In various cases, the recombinant filamentous fungal host cell is Aspergillus. In various cases, the recombinant filamentous fungal host cell is Aspergillus niger. In various embodiments, the recombinant host cell is a bacterial host cell. In some cases, the recombinant bacterial host cell is E. coli. Additional illustrative hosts include fungi, such as filamentous fungi, as well as bacteria, yeast, plant, insect, and mammalian cells. A host cell may be Arxula spp., Arxula adeninivorans. Kluyveromyces spp., Kluyveromyces laclis. Komagataella phaffii, Pichia spp., Pichia angusla. Pichia pastoris, Saccharomyces spp., Saccharomyces cerevisiae, Schizosaccharomyces spp., Schizosaccharomyces pombe, Yarrowia spp., Yarrowia lipolytica, Agaricus spp., Agaricus bisporus, Aspergillus spp., Aspergillus awamori, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bacillus subtilis, Colletotrichum spp., Colletotrichum gloeosporiodes, Endothia spp., Endothia parasitica, Escherichia coli, Fusarium spp., Fusarium graminearum, Fusarium solani, Mucor spp., Mucor miehei, Mucor pusillus, Myceliophthora spp., Myceliophthora thermophila, Neurospora spp., Neurospora crassa, Penicillium spp., Penicillium camemberti, Penicillium canescens, Penicillium chrysogenum, Penicillium (Talaromyces) emersonii, P mcillium funiculo sum, Penicillium purpurogenum, Penicillium roqueforti, Pleurotus spp., Pleurotus ostreatus, Rhizomucor spp., Rhizomucor miehei, Rhizomucor pusillus, Rhizopus spp., Rhizopus arrhizus, Rhizopus oligosporus, Rhizopus oryzae, Trichoderma spp., Trichoderma altroviride, Trichoderma reesei, or Trichoderma vireus. A host cell can be an organism that is approved as generally regarded as safe by the U.S. Food and Drug Administration. [0066] An rpOVA protein and/or a kinase can be recombinantly expressed in yeast, filamentous fungi or a bacterium. In some embodiments, rpOVA protein is recombinantly expressed in a Pichia species (Komagataella phaffii and Komagataella pasloris). a Saccharomyces species, a Trichoderma species, a Pseudomonas species or an E. coli species.

[0067] In some embodiments, the method of producing rpOVA produces a higher protein titer than a method of producing unphosphorylated rOVA. In some embodiments, the method of producing rpOVA with the engineered host cell disclosed herein produces the same amount of recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell produces higher amounts of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell produces about 1% to about 200% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell produces about 1% to about 2%, about 1% to about 5%, about 1% to about 10%, about 1% to about 15%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 75%, about 1% to about 100%, about 1% to about 150%, about 1% to about 200%, about 2% to about 5%, about 2% to about 10%, about 2% to about 15%, about 2% to about 20%, about 2% to about 30%, about 2% to about 40%, about 2% to about 50%, about 2% to about 75%, about 2% to about 100%, about 2% to about 150%, about 2% to about 200%, about 5% to about 10%, about 5% to about 15%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 75%, about 5% to about 100%, about 5% to about 150%, about 5% to about 200%, about 10% to about 15%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 75%, about 10% to about 100%, about 10% to about 200%, about 15% to about 20%, about 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 75%, about 15% to about 100%, about 15% to about 150%, about 15% to about 200%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 75%, about 20% to about 100%, about 20% to about 200%, about 30% to about 40%, about 30% to about 50%, about 30% to about 75%, about 30% to about 100%, about 30% to about 150%, about 30% to about 200%, about 40% to about 50%, about 40% to about 75%, about 40% to about 100%, about 40% to about 150%, about 40% to about 200%, about 50% to about 75%, about 50% to about 100%, about 50% to about 200%, about 75% to about 100%, about 75% to about 200%, or about 100% to about 200% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell produces about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 100%, about 150%, or about 200% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell produces at least about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 150%, or about 100% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell produces at most about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 100%, about 150% or about 200% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein.

[0068] In some embodiments, the engineered host cell secretes the same amount of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell secretes more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell secretes about 1% to about 200% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell secretes about 1% to about 2%, about 1% to about 5%, about 1% to about 10%, about 1% to about 15%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 75%, about 1% to about 100%, about 1% to about 150%, about 1% to about 200%, about 2% to about 5%, about 2% to about 10%, about 2% to about 15%, about 2% to about 20%, about 2% to about 30%, about 2% to about 40%, about 2% to about 50%, about 2% to about 75%, about 2% to about 100%, about 2% to about 150%, about 2% to about 200%, about 5% to about 10%, about 5% to about 15%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 75%, about 5% to about 100%, about 5% to about 150%, about 5% to about 200%, about 10% to about 15%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 75%, about 10% to about 100%, about 10% to about 200%, about 15% to about 20%, about 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 75%, about 15% to about 100%, about 15% to about 150%, about 15% to about 200%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 75%, about 20% to about 100%, about 20% to about 200%, about 30% to about 40%, about 30% to about 50%, about 30% to about 75%, about 30% to about 100%, about 30% to about 150%, about 30% to about 200%, about 40% to about 50%, about 40% to about 75%, about 40% to about 100%, about 40% to about 150%, about 40% to about 200%, about 50% to about 75%, about 50% to about 100%, about 50% to about 200%, about 75% to about 100%, about 75% to about 200%, or about 100% to about 200% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell secretes about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 100%, about 150%, or about 200% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell secretes at least about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 150%, or about 100% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell secretes at most about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 100%, about 150% or about 200% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein.

Recombinant OVA

[0069] In some embodiments, the rpOVA can have an amino acid sequence from any species. For example, the rpOVA can have an amino acid sequence of OVA from a bird, a reptile, or another egg-laying species. In some embodiments, the rpOVA having an amino acid sequence from an avian species that can be selected from the group consisting of: poultry, fowl, waterfowl, game bird, chicken, quail, turkey, duck, ostrich, goose, gull, guineafowl, pheasant, emu, and any combination thereof. In some embodiments, the rpOVA can have an amino acid sequence derived from a single species, such as Gallus domesticus. In some embodiments, the rpOVA can have an amino acid sequence derived from two or more species, and as such can be a hybrid. [0070] Illustrative OVA amino acid sequences contemplated herein are provided in Table 1 below as SEQ ID NOs: 1-76.

Table 1. OVA Sequences

[0071] Expression of rpOVA in a host cell, for instance a Pichia species, a Saccharomyces species, a Trichoderma species, a Pseudomonas species may lead to an addition of one or more amino acids to the OVA sequence as part of post-transcriptional or post-translational modifications. Such amino acids may not be part of the native OVA sequences. For instance, expressing an OVA sequence in a Pichia species, such as Komagataella phaffii and Komagataella pastoris may lead to addition of one or more amino acids at the N-terminus or C-terminus. In some cases, four amino acids EAEA (SEQ ID NO: 77) is added to the N- terminus of the OVA sequence upon expression in a host cell as shown in SEQ ID NO:1. For example, chicken rpOVA may be provided encoding SEQ ID NO:2, which lacks the EAEA (SEQ ID NO: 77) peptide, and following expression and secretion, the rpOVA has the amino acid sequence of SEQ ID NO: 3 which comprises the EAEA (SEQ ID NO: 77) peptide. Any of the amino acid sequences recited in Table 1 which lacks an N-terminal EAEA (SEQ ID NO: 77) may be modified to comprise the N-terminal EAEA (SEQ ID NO: 77) peptide.

[0072] In some embodiments, the rpOVA can be a non-naturally occurring variant of an OVA. Such variants can comprise one or more amino acid insertions, deletions, or substitutions relative to a native OVA sequence. [0073] Such a variant can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 1-76. The term “sequence identity” as used herein in the context of amino acid sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a selected sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software, with BLAST being the preferable alignment algorithm. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.

[0074] In some embodiments, the rpOVA comprises post-translational modifications. In some embodiments, the post-translational modification comprises glycosylation. In some embodiments, the rOVA protein is glycosylated before being phosphorylated. In some cases, the rOVA is not phosphorylated until the rOVA is glycosylated. In some embodiments, the glycosylation comprises a carbohydrate attached to an amino acid residue of rOVA. In some embodiments, the glycosylation is N-linked glycosylation. In some embodiments, the glycosylation is O-linked glycosylation. In some embodiments, the glycosylation is C-linked glycosylation. In some embodiments, the glycosylation is S-linked glycosylation. In some embodiments, the glycosylation is glypiation. In some embodiments, the glycosylation is phosphoglycosylation. In some embodiments, the glycosylation moiety is covalently attached to one or more amino acids in the rOVA protein. In some embodiments, the glycosylation moiety comprises a carbohydrate. In some embodiments, the glycosylation moiety comprises a glycan. In some embodiments, the glycosylation moiety comprises at least one mannose moiety. In some embodiments, the glycosylation moiety comprises at least one N- acetylglucosamine moiety. In some embodiments, the glycosylation moiety comprises at least one mannose moiety or at least one N-acetylglucosamine moiety. In some embodiments, the glycosylation moiety comprises at least one mannose moiety and at least one N- acetyl glucosamine moiety.

[0075] In some embodiments, an unglycosylated rOVA is unphosphorylated. In some embodiments, a glycosylated rOVA is phosphorylated. In some embodiments, a glycosylated rOVA is unphosphorylated. In some embodiments, a phosphorylated recombinant ovalbumin is more stable than an unphosphorylated recombinant ovalbumin.

[0076] Depending on the host organism used to express the rpOVA, the rpOVA can have a glycosylation, acetylation, or phosphorylation pattern different from native OVA. For example, the rpOVA herein may or may not be glycosylated, acetylated, or phosphorylated. In some embodiments, the rpOVA may have an avian, non-avian, microbial, non-microbial, mammalian, or non-mammalian glycosylation, acetylation, or phosphorylation pattern.

[0077] In some cases, the rpOVA may be deglycosylated (e.g., chemically or enzymatically using one or more of Endo-H, PNGase F, O-Glycosidase, Neuraminidase, [31-4 Galactosidase, and P-N-acetylglucosaminidase), deacetylated (e.g., protein deacetylase, histone deacetylase, and sirtuin), or dephosphorylated (e.g., acid phosphatase, lambda protein phosphatase, calf intestinal phosphatase, and alkaline phosphatase). In some embodiments, deglycosylation, deacetylation or dephosphorylation may produce a protein product that is more uniform or is capable of producing a composition with less variation.

[0078] In some embodiments, the rpOVA protein is phosphorylated at one, two, three, four, or more amino acid residues in the contiguous amino acid backbone. In some embodiments, the rpOVA protein is phosphorylated at one, two, three, or more amino acid residues in the contiguous amino acid backbone. In some embodiments, the rpOVA protein is phosphorylated at one, two, or more amino acid residues in the contiguous amino acid backbone. In some embodiments, the rpOVA protein is phosphorylated at two amino acid residues in the contiguous amino acid backbone. In some embodiments, the rpOVA protein is phosphorylated at one amino acid residue in the contiguous amino acid backbone.

[0079] In some embodiments, the rpOVA protein is phosphorylated at one or more serine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more threonine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more tyrosine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine or threonine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine and threonine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine or tyrosine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine and tyrosine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine and histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more tyrosine or threonine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more tyrosine and threonine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more tyrosine or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more tyrosine and histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more threonine or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more threonine and histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, threonine, or tyrosine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, threonine, and tyrosine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, threonine, tyrosine, or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, threonine, or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, threonine, or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, tyrosine, or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, tyrosine, or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more threonine, tyrosine, or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more threonine, tyrosine, or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, threonine, tyrosine, or histidine residues. In some embodiments, the rpOVA protein is phosphorylated at one or more serine, threonine, tyrosine, and histidine residues.

Kinases

[0080] In certain embodiments, a kinase that rpOVA can be expressed in a host cell. In certain embodiments, the kinase can be co-expressed in a host cell with rOVA. In some embodiments, the rpOVA protein and kinase can be recombinantly co-expressed in yeast, filamentous fungi or a bacterium. In some embodiments, kinase protein is recombinantly expressed in a Pichia species (Komagataella phaffii and Komagataella pasloris). a Saccharomyces species, a Trichoderma species, a Pseudomonas species, an Aspergillus species or an E. coli species.

[0081] In certain embodiments, a kinase that rpOVA can be introduced to an unphosphorylated rOVA protein following secretion or isolation of rOVA. In certain embodiments, the rOVA can be treated with a kinase protein. In some embodiments, the rOVA protein can be recombinantly expressed in yeast, filamentous fungi or a bacterium, then treated with a kinase protein. In some embodiments, kinase protein is recombinantly expressed in a Pichia species (Komagataella phaffii and Komagataella pastoris), a Saccharomyces species, a Trichoderma species, a Pseudomonas species, or an E. coli species, then isolated and introduced to rOVA produced from another host cell.

[0082] In some embodiments, a kinase phosphorylates rOVA in a host cell. In some embodiments, a kinase phosphorylates rOVA in vitro. In some embodiments, a serine/threonine kinase phosphorylates rOVA. In some embodiments, a tyrosine kinase phosphorylates rOVA. In some embodiments, the kinase is a heterologous or exogenous kinase protein. In some embodiments, the kinase recognizes a recognition motif comprising a S-X- E/pS motif. In some embodiments, the kinase recognizes a recognition motif that comprises Glu at a +2 position.

[0083] In some embodiments, the exogenous kinase protein is selected from the genera: Homo, Drosophila, Danio, Branchiostoma, Stronglycocentrotus, Caenorhabditis, Hydra, Amphimedon, any other animal, or any combination thereof. In certain embodiments, the kinase protein is selected from the Aves or Reptilia class. In some embodiments, the kinase can have an amino acid sequence of FAM20 from a bird, a reptile, or another egg-laying species. In some embodiments, the FAM20 having an amino acid sequence from an Aves species can be selected from the group consisting of barn owl (Tyto alba), chicken (Gallus domesticus), chuck-will’s-willow (Antrostomus carolinensis), crow (Corvus brachyrhynchos), dove (columba livia), emu (Dromaius novaehollandiae), golden manakin (Manacus vitellinus), goose (Anser cygnoid), hummingbird (Calypte anna), Japanese quail (Cotumix japonica), little egret (Egretta garzetta), little spotted kiwi (Apteryx owenii), mallard (Anas platyrhynchos), ostrich (Struthio camelus), penguin (Aptenodytes forsteri), speckled mousebird (Colius striatus), turkey (Meleagris gallopavo), or zebra finch (Taeniopygia guttata), and any combination thereof. In some embodiments, the FAM20 can have an amino acid sequence derived from a single species, such as the chicken (Gallus gallus, Gallus domesticus, or Gallus gallus domesticus). In some embodiments, the heterologous or exogenous kinase protein is from G. gallus. In embodiments, the FAM20 has an amino acid sequence from an alga, e.g., Chloropicon primus.

[0084] In some embodiments, the kinase can have an amino acid sequence from any species. In various embodiments, the FAM20 kinase can have an amino acid sequence comprising FAM20A. In various embodiments, the FAM20 kinase can have an amino acid sequence comprising FAM20A from any species. In certain embodiments, the FAM20 kinase can have an amino acid sequence comprising FAM20C. In certain embodiments, the FAM20 kinase can have an amino acid sequence comprising FAM20C from any species. In some embodiments, the FAM20C kinase can have an amino acid sequence comprising any FAM20C isoform. In some embodiments, the FAM20C kinase can have an amino acid sequence comprising any FAM20C isoform from any species.

[0085] In certain embodiments, the kinase protein comprises an amino acid sequence with at least 70% homology to any one of SEQ ID NO: 80 to SEQ ID NO: 141, and maintains the enzymatic activity of any one of SEQ ID NO: 80 to SEQ ID NO: 141. In various embodiments, the heterologous or exogenous kinase protein comprises an amino acid sequence of any one of SEQ ID NO: 80 to SEQ ID NO: 141. In some embodiments, the heterologous or exogenous kinase protein comprises a nucleotide sequence encoding for an amino acid with at least 70% homology to any one of SEQ ID NO: 80 to SEQ ID NO: 141 and maintains the enzymatic activity of a kinase expressed by any one of SEQ ID NO: 80 to SEQ ID NO: 141. In various embodiments, the heterologous or exogenous kinase protein comprises a nucleotide sequence encoding for an amino acid of any one of SEQ ID NO: 80 to SEQ ID NO: 141.

[0086] In some embodiments, the kinase can have an amino acid sequence derived from two or more species, and as such can be a hybrid. In some embodiments, the kinase protein comprises a mixture of at least two or more distinct heterologous or exogenous kinase proteins. In some embodiments, the kinase or kinases can have an amino acid sequence derived from a single species. In some embodiments, the kinase can have an amino acid sequence derived from two or more species, and as such can be a hybrid.

[0087] Illustrative kinase amino acid sequences contemplated herein are provided in Table 2 below as SEQ ID NO: 80 to SEQ ID NO: 141.

Table 2. Kinase Sequences

[0088] As noted in the above Table 2, many of the amino acid sequences comprise an N- terminal methionine (“M”). In nature and when expressed by a host cell of the present disclosure, a protein’s N-terminal methionine is often cleaved by a peptidase. Thus, when in its active form, the amino acid sequences of Table 2 will lack the recited N-terminal methionine.

[0089] In certain embodiments, the kinase can be expressed in a host cell. In certain embodiments, the kinase can be co-expressed in a host cell with rOVA. In some embodiments, the rpOVA protein and kinase can be recombinantly co-expressed in yeast, filamentous fungi or a bacterium. In some embodiments, rpOVA protein and a kinase protein are recombinantly expressed in a Pichia species (Komagataella phaffii and Komagataella pasloris). a Saccharomyces species, a Trichoderma species, a Pseudomonas species or an E. coli species.

[0090] In various embodiments, the exogenous kinase protein is a FAM20: l or Xylosyl kinase. In certain embodiments, the heterologous or exogenous kinase protein may be a kinase from the FAM20: 1 kinase family. In some embodiments, the FAM20: 1 kinase family member may be FAM2O:1A, FAM2O: 1B, FAM2O: 1C, or any combination thereof. In some embodiments the heterologous or exogenous kinase protein is one heterologous or exogenous kinase protein. In some embodiments the heterologous or exogenous kinase protein is a mixture of at least two or more distinct heterologous or exogenous kinase proteins.

[0091] In some cases, the heterologous or exogenous kinase protein is from G. gallus. In some embodiments, the amino acid sequence encoding the heterologous or exogenous kinase protein has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% similar to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID

NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID

NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID

NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID

NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID

NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID

NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID

NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID

NO: 140 or SEQ ID NO: 141 and maintains the enzymatic activity of SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107,

SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112,

SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117,

SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122,

SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127,

SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132,

SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137,

SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140 or SEQ ID NO: 141. In these embodiments, an amino acid with at least 70% homology to SEQ ID NO: 80 maintains the enzymatic activity of a kinase expressed by the corresponding SEQ ID NO: 80; an amino acid with at least 70% homology to SEQ ID NO: 85 maintains the enzymatic activity of a kinase expressed by the corresponding SEQ ID NO: 85; an amino acid with at least 70% homology to SEQ ID NO: 97 maintains the enzymatic activity of a kinase expressed by the corresponding SEQ ID NO: 97; and so forth.

[0092] In some embodiments, the exogenous kinase protein is selected from the genera Homo, Drosophila, Danio, Branchiostoma, Stronglycocentrotus, Caenorhabditis, Hydra, Amphimedon, Avian, Reptile, any other egg-laying animal, or any combination thereof. In some cases, the Avian may be poultry, fowl, waterfowl, game bird, chicken, duck, ostrich, quail, goose, quail, turkey, gull, guineafowl, pheasant, emu, and any combination thereof.

[0093] In certain embodiments, the heterologous or exogenous kinase protein may be an amino acid sequence of a chicken ovalbumin, an ostrich ovalbumin, or a duck ovalbumin.

[0094] In certain embodiments, the kinase protein comprises an amino acid sequence with at least 70% homology to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID

NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID

NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID

NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID

NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID

NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID

NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID

NO: 140, or SEQ ID NO: 141 and maintains the enzymatic activity of SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107,

SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112,

SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117,

SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122,

SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127,

SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132,

SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137,

SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141.

[0095] In various embodiments, the kinase protein comprises an amino acid sequence of SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO:

106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO:

111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO:

116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO:

121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO:

126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO:

131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO:

136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141.

[0096] In some embodiments, the heterologous or exogenous kinase protein comprises a nucleotide sequence encoding for an amino acid with at least 70% homology to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID

NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID

NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID

NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID

NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID

NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID

NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID

NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141 and maintains the enzymatic activity of a kinase expressed by SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO:

108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO:

113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO:

118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO:

123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO:

128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO:

133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO:

138, SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141.

[0097] In various embodiments, the heterologous or exogenous kinase protein comprises a nucleotide sequence encoding for an amino acid of SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123,

SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128,

SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133,

SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138,

SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141.

[0098] In these embodiments, an amino acid with at least 70% homology to SEQ ID NO: 80 maintains the enzymatic activity of a kinase expressed by the corresponding SEQ ID NO: 80; an amino acid with at least 70% homology to SEQ ID NO: 85 maintains the enzymatic activity of a kinase expressed by the corresponding SEQ ID NO: 85; an amino acid with at least 70% homology to SEQ ID NO: 97 maintains the enzymatic activity of a kinase expressed by the corresponding SEQ ID NO: 97; and so forth.

Production of phosphorylated rOVA

[0099] In certain embodiments, the recombinant phosphorylated ovalbumin is more stable than an unphosphorylated recombinant ovalbumin. In some embodiments, the unphosphorylated rOVA is most abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rOVA is most abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rOVA at Ser-68 (with respect to SEQ ID NO: 2) is most abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rOVA at Ser-344 (with respect to SEQ ID NO: 2) is most abundant in the rpOVA mixture. In some embodiments, the diphosphorylated rOVA is most abundant in the rpOVA mixture. In some embodiments, the unphosphorylated rOVA is least abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rOVA is least abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rOVA at Ser-68 is least abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rOVA at Ser-344 is least abundant in the rpOVA mixture. In some embodiments, the diphosphorylated rOVA is most abundant in the rpOVA mixture.

[0100] In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is about 1 : 1 to about 1 :20. In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is about 1 :20 to about 1: 15, about 1 :20 to about 1 : 10, about 1 :20 to about 1 :9, about 1 :20 to about 1 :8, about 1 :20 to about 1 :7, about 1 :20 to about 1 :6, about 1 :20 to about 1 :5, about 1 :20 to about 1 :4, about 1 :20 to about 1 :3, about 1 :20 to about 1 :2, about 1 :20 to about 1 : 1, about 1 : 15 to about 1 : 10, about 1 : 15 to about 1 :9, about 1 : 15 to about 1 :8, about 1 : 15 to about 1 :7, about 1:15 to about 1:6, about 1:15 to about 1:5, about 1:15 to about 1:4, about 1:15 to about 1:3, about 1:15 to about 1:2, about 1:15 to about 1:1, about 1:10 to about 1:9, about 1:10 to about 1:8, about 1:10 to about 1:7, about 1:10 to about 1:6, about 1:10 to about 1:5, about 1:10 to about 1:4, about 1:10 to about 1:3, about 1:10 to about 1:2, about 1:10 to about 1:1, about 1:9 to about 1:8, about 1 :9 to about 1 :7, about 1 :9 to about 1 :6, about 1 :9 to about 1:5, about 1 :9 to about 1:4, about 1:9 to about 1:3, about 1:9 to about 1:2, about 1:9 to about 1:1, about 1:8 to about 1:7, about 1:8 to about 1:6, about 1:8 to about 1:5, about 1:8 to about 1:4, about 1:8 to about 1:3, about 1:8 to about 1:2, about 1:8 to about 1:1, about 1:7 to about 1:6, about 1:7 to about 1:5, about 1:7 to about 1:4, about 1:7 to about 1:3, about 1:7 to about 1:2, about 1:7 to about 1:1, about 1:6 to about 1:5, about 1:6 to about 1:4, about 1:6 to about 1:3, about 1:6 to about 1:2, about 1:6 to about 1:1, about 1:5 to about 1:4, about 1:5 to about 1:3, about 1:5 to about 1:2, about 1:5 to about 1:1, about 1:4 to about 1:3, about 1:4 to about 1:2, about 1:4 to about 1:1, about 1:3 to about 1:2, about 1:3 to about 1:1, or about 1:2 to about 1:1. In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is about 1 :20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1. In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is at least about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, or about 1:2. In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is at most about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1.

[0101] In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is about 1 : 1 to about 20:1. In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is about 20:1 to about 15:1, about 20:1 to about 10:1, about 20:1 to about 9:1, about 20:1 to about 8:1, about 20 : 1 to about 7:1, about 20 to about 6:1, about 20 : 1 to about 5:1, about 20 : 1 to about 4:1, about 20:1 to about 3:1, about 20:1 to about 2:1, about 20:1 to about 1:1, about 15:1 to about 10:1, about 15:1 to about 9:1, about 15:1 to about 8:1, about 15:1 to about 7:1, about 15:1 to about 6:1, about 15:1 to about 5:1, about 15:1 to about 4:1, about 15:1 to about 3:1, about 15:1 to about 2:1, about 15:1 to about 1:1, about 10:1 to about 9:1, about 10:1 to about 8:1, about 10:1 to about 7:1, about 10:1 to about 6:1, about 10:1 to about 5:1, about 10:1 to about 4:1, about 10:1 to about 3:1, about 10:1 to about 2:1, about 10:1 to about 1:1, about 9:1 to about

8:1, about 9:1 to about 7:1, about 9:1 to about 6:1, about 9:1 to about 5:1, about 9:1 to about

7:1, about 8:1 to about 6:1, about 8:1 to about 5:1, about 8:1 to about 4:1, about 8:1 to about 3:1, about 8:1 to about 2:1, about 8:1 to about 1:1, about 7:1 to about 6:1, about 7:1 to about

1:1, about 3 : 1 to about 2:1, about 3 : 1 to about 1 : 1 , or about 2 : 1 to about 1 : 1. the ratio of rpO VA to unphosphorylated rOVA is about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is at least about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of rpOVA to unphosphorylated rOVA is at most about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2: 1, or about 1:1.

[0102] In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 (with respect to SEQ ID NO: 2) to monophosphorylated rOVA at Ser-344 (with respect to SEQ ID NO: 2) is about 1:1 to about 1:20. In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 to monophosphorylated rOVA at Ser-344 is about 1:20 to about 1:15, about 1:20 to about 1:10, about 1:20 to about 1:9, about 1:20 to about 1:8, about 1:20 to about 1:7, about 1:20 to about 1:6, about 1:20 to about 1:5, about 1:20 to about 1:4, about 1:20 to about 1:3, about 1:20 to about 1:2, about 1:20 to about 1:1, about 1:15 to about 1:10, about 1:15 to about 1:9, about 1:15 to about 1:8, about 1:15 to about 1:7, about 1:15 to about 1:6, about 1:15 to about 1:5, about 1:15 to about 1:4, about 1:15 to about 1:3, about 1:15 to about 1:2, about 1:15 to about 1:1, about 1:10 to about 1:9, about 1:10 to about 1:8, about 1:10 to about 1:7, about 1:10 to about 1:6, about 1:10 to about 1:5, about 1:10 to about 1:4, about 1:10 to about 1 :3, about 1 : 10 to about 1 :2, about 1 : 10 to about 1:1, about 1 :9 to about 1:8, about 1 :9 to about 1:7, about 1:9 to about 1:6, about 1:9 to about 1:5, about 1:9 to about 1:4, about 1:9 to about

1:1, about 1:4 to about 1:3, about 1:4 to about 1:2, about 1:4 to about 1:1, about 1:3 to about 1:2, about 1:3 to about 1:1, or about 1:2 to about 1:1. In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 to monophosphorylated rOVA at Ser-344 is about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1. In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 to monophosphorylated rOVA at Ser-344 is at least about 1:20, about 1:15, about 1:10, about 1 :9, about 1 :8, about 1 :7, about 1 :6, about 1:5, about 1 :4, about 1 :3, or about 1 :2. In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 to monophosphorylated rOVA at Ser-344 is at most about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1.

[0103] In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 to monophosphorylated rOVA at Ser-344 is about 1:1 to about 20:1. In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 to monophosphorylated rOVA at Ser-344 is about 20: 1 to about 15:1, about 20: 1 to about 10:1, about 20: 1 to about 9:1, about 20: 1 to about 8:1, about 20 : 1 to about 7:1, about 20 to about 6:1, about 20 : 1 to about 5:1, about 20 : 1 to about 4:1, about 20:1 to about 3:1, about 20:1 to about 2:1, about 20:1 to about 1:1, about 15:1 to about 10:1, about 15:1 to about 9:1, about 15:1 to about 8:1, about 15:1 to about 7:1, about 15:1 to about 6:1, about 15:1 to about 5:1, about 15:1 to about 4:1, about 15:1 to about 3:1, about 15:1 to about 2:1, about 15:1 to about 1:1, about 10:1 to about 9:1, about 10:1 to about 8:1, about 10:1 to about 7:1, about 10:1 to about 6:1, about 10:1 to about 5:1, about 10:1 to about 4:1, about 10:1 to about 3:1, about 10:1 to about 2:1, about 10:1 to about 1:1, about 9:1 to about 8:1, about 9: 1 to about 7: 1, about 9: 1 to about 6:1, about 9: 1 to about 5:1, about 9: 1 to about 4:1, about 9:1 to about 3:1, about 9:1 to about 2:1, about 9:1 to about 1:1, about 8:1 to about 7:1, about 8:1 to about 6:1, about 8:1 to about 5:1, about 8:1 to about 4:1, about 8:1 to about 3:1, about 8:1 to about 2:1, about 8:1 to about 1:1, about 7:1 to about 6:1, about 7:1 to about 5:1, about 7:1 to about 4:1, about 7:1 to about 3:1, about 7:1 to about 2:1, about 7:1 to about 1:1, about 6:1 to about 5:1, about 6:1 to about 4:1, about 6:1 to about 3:1, about 6:1 to about 2:1, about 6:1 to about 1:1, about 5:1 to about 4:1, about 5:1 to about 3:1, about 5:1 to about 2:1, about 5:1 to about 1:1, about 4:1 to about 3:1, about 4:1 to about 2:1, about 4:1 to about 1:1, about 3:1 to about 2:1, about 3:1 to about 1:1, or about 2:1 to about 1:1. In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 to monophosphorylated rOVA at Ser-344 is about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 to monophosphorylated rOVA at Ser-344 is at least about20:l, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2: 1, or about 1 : 1. In some embodiments, the ratio of monophosphorylated rOVA at Ser-68 to monophosphorylated rOVA at Ser-344 is at most about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1.

[0104] In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is about 1:1 to about 1:20. In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is about 1 :20 to about 1:15, about 1 :20 to about 1:10, about 1 :20 to about 1 :9, about 1 :20 to about 1:8, about 1 :20 to about 1 :7, about 1 :20 to about 1 :6, about 1 :20 to about 1:5, about 1:20 to about 1:4, about 1:20 to about 1:3, about 1:20 to about 1:2, about 1:20 to about 1:1, about 1:15 to about 1:10, about 1:15 to about 1:9, about 1:15 to about 1:8, about 1:15 to about 1:7, about 1:15 to about 1:6, about 1:15 to about 1:5, about 1:15 to about 1:4, about 1:15 to about 1:3, about 1:15 to about 1:2, about 1:15 to about 1:1, about 1:10 to about 1 :9, about 1 : 10 to about 1:8, about 1 : 10 to about 1 :7, about 1 : 10 to about 1 :6, about 1:10 to about 1:5, about 1:10 to about 1:4, about 1:10 to about 1:3, about 1:10 to about 1:2, about 1:10 to about 1:1, about 1:9 to about 1:8, about 1:9 to about 1:7, about 1:9 to about 1:6, about 1:9 to about 1:5, about 1:9 to about 1:4, about 1:9 to about 1:3, about 1:9 to about 1:2, about

1:9 to about 1:1, about 1:8 to about 1:7, about 1:8 to about 1:6, about 1:8 to about 1:5, about

1 :4 to about 1 :2, about 1 :4 to about 1:1, about 1 :3 to about 1 :2, about 1 :3 to about 1 : 1, or about 1:2 to about 1:1. In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1. In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is at least about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, or about 1:2. In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is at most about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1. [0105] In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is about 1:1 to about 20:1. In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is about 20: 1 to about 15:1, about 20: 1 to about 10:1, about 20: 1 to about 9:1, about 20:1 to about 8:1, about 20:1 to about 7:1, about 20 to about 6:1, about 20:1 to about 5:1, about 20:1 to about 4:1, about 20:1 to about 3:1, about 20:1 to about 2:1, about 20:1 to about 1:1, about 15:1 to about 10:1, about 15:1 to about 9:1, about 15:1 to about 8:1, about 15:1 to about 7:1, about 15:1 to about 6:1, about 15:1 to about 5:1, about 15:1 to about 4:1, about 15:1 to about 3:1, about 15:1 to about 2:1, about 15:1 to about 1:1, about 10:1 to about 9:1, about 10:1 to about 8:1, about 10:1 to about 7:1, about 10:1 to about 6:1, about 10:1 to about 5:1, about 10:1 to about 4:1, about 10:1 to about 3:1, about 10:1 to about 2:1, about 10:1 to about 1:1, about 9:1 to about 8:1, about 9:1 to about 7:1, about 9:1 to about 6:1, about 9:1 to about 5:1, about 9:1 to about 4:1, about 9:1 to about 3:1, about 9:1 to about 2:1, about

5:1 to about 3:1, about 5:1 to about 2:1, about 5:1 to about 1:1, about 4:1 to about 3:1, about

4: 1 to about 2:1, about 4: 1 to about 1:1, about 3 : 1 to about 2:1, about 3 : 1 to about 1 : 1, or about 2: 1 to about 1 : 1. the ratio of diphosphorylated rOVA to unphosphorylated rOVA is about 20: 1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is at least about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of diphosphorylated rOVA to unphosphorylated rOVA is at most about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2: 1, or about 1:1.

[0106] In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA is about 1:1 to about 1:20. In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA is about 1 :20 to about 1:15, about 1 :20 to about 1:10, about 1 :20 to about 1:9, about 1:20 to about 1:8, about 1:20 to about 1:7, about 1:20 to about 1:6, about 1:20 to about 1:5, about 1:20 to about 1:4, about 1:20 to about 1:3, about 1:20 to about 1:2, about 1 :20 to about 1 : 1, about 1 : 15 to about 1 : 10, about 1 : 15 to about 1 :9, about 1 : 15 to about 1:8, about 1:15 to about 1:7, about 1:15 to about 1:6, about 1:15 to about 1:5, about 1:15 to about 1:4, about 1:15 to about 1:3, about 1:15 to about 1:2, about 1:15 to about 1:1, about 1:10 to about 1:9, about 1:10 to about 1:8, about 1:10 to about 1:7, about 1:10 to about 1:6, about 1:10 to about 1:5, about 1:10 to about 1:4, about 1:10 to about 1:3, about 1:10 to about 1:2, about 1:10 to about 1:1, about 1:9 to about 1:8, about 1:9 to about 1:7, about 1:9 to about 1:6, about 1:9 to about 1:5, about 1:9 to about 1:4, about 1:9 to about 1:3, about 1:9 to about 1:2, about 1:9 to about 1:1, about 1:8 to about 1:7, about 1:8 to about 1:6, about 1:8 to about 1:5, about 1:8 to about 1:4, about 1:8 to about 1:3, about 1:8 to about 1:2, about 1:8 to about 1:1, about 1:7 to about 1:6, about 1:7 to about 1:5, about 1:7 to about 1:4, about 1:7 to about 1:3, about 1:7 to about 1:2, about 1:7 to about 1:1, about 1:6 to about 1:5, about 1:6 to about 1:4, about 1:6 to about 1:3, about 1:6 to about 1:2, about 1:6 to about 1:1, about 1:5 to about 1:4, about 1:5 to about 1:3, about 1:5 to about 1:2, about 1:5 to about 1:1, about 1:4 to about 1:3, about 1 :4 to about 1 :2, about 1 :4 to about 1:1, about 1 :3 to about 1 :2, about 1 :3 to about 1 : 1, or about 1:2 to about 1:1. In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA is about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1. In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA is at least about 1 :20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, or about 1:2. In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA is at most about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1.

[0107] In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA is about 1:1 to about 20:1. In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA is about 20: 1 to about 15:1, about 20: 1 to about 10:1, about 20: 1 to about 9:1, about 20 : 1 to about 8:1, about 20 : 1 to about 7:1, about 20 to about 6:1, about 20 : 1 to about 5:1, about 20:1 to about 4:1, about 20:1 to about 3:1, about 20:1 to about 2:1, about 20:1 to about 1:1, about 15:1 to about 10:1, about 15:1 to about 9:1, about 15:1 to about 8:1, about 15:1 to about 7:1, about 15:1 to about 6:1, about 15:1 to about 5:1, about 15:1 to about 4:1, about 15:1 to about 3:1, about 15:1 to about 2:1, about 15:1 to about 1:1, about 10:1 to about 9:1, about 10:1 to about 8:1, about 10:1 to about 7:1, about 10:1 to about 6:1, about 10:1 to about 5:1, about 10:1 to about 4:1, about 10:1 to about 3:1, about 10:1 to about 2:1, about 10:1 to about 1:1, about 9:1 to about 8:1, about 9:1 to about 7:1, about 9:1 to about 6:1, about 9:1 to about 5:1, about 9:1 to about 4:1, about 9:1 to about 3:1, about 9:1 to about 2:1, about 9:1 to about 1:1, about 8:1 to about 7:1, about 8:1 to about 6:1, about 8:1 to about 5:1, about

4: 1 to about 2:1, about 4: 1 to about 1:1, about 3 : 1 to about 2:1, about 3 : 1 to about 1 : 1, or about 2:1 to about 1:1. the ratio of diphosphorylated rOVA to monophosphorylated rOVA is about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA is at least about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of diphosphorylated rOVA to monophosphorylated rOVA is at most about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1.

[0108] In this disclosure, phosphorylation of OVA at Ser-68 is identified as “with respect to SEQ ID NO: 2” and/or phosphorylation of OVA at Ser-344 is identified as “with respect to SEQ ID NO: 2”. SEQ ID NO: 2 relates to an OVA that is naturally expressed in one species of bird (chicken). However, this disclosure relates to phosphorylation of a number of recombinant OVAs, each having an amino acid sequence that is naturally expressed in one species, e.g., barn owl, crow, dove, hummingbird, and emu. These other species’ OVA may have serines that can be phosphorylated at the same location with respect to their SEQ ID NO, i.e., one of SEQ ID NO: 4 to SEQ ID NO: 76. Alternatively, these other species’ OVA may have serines that can be phosphorylated at a homologous serine, yet at a different specific location, or can be phosphorylated at homologous serines, yet at different specific locations (e.g., Ser-67, Ser- 69, Ser-343, and Ser-345) with respect to the other OVA’s SEQ ID NO, i.e., one of SEQ ID NO: 4 to SEQ ID NO: 76. Consequently, any herein disclosure that describes a Ser-68 or Ser- 344 applies to homologous serines in the other OVAs.

[0109] In some embodiments, the method further comprises combining the purified or isolated recombinant phosphorylated ovalbumin with food ingredients to make a food item. In certain embodiments, the recombinant phosphorylated ovalbumin improves functionality in a food product. In various embodiments, the recombinant phosphorylate ovalbumin provides to a food product at least one egg white characteristic selected from gelling, foaming, whipping, fluffing, binding, springiness, aeration, coating, film forming, emulsification, browning, thickening, texturizing, humectant, clarification, and cohesiveness, wherein the characteristic is the same or is improved as compared to recombinant ovalbumin that is not phosphorylated. In some embodiments, the food product is a cake, a pudding, a macaron, a bread, a roll, a pastry, a cracker, a muffin, a scone, a biscuit, a bagel, a pancake, a meringue, a choux pastry a souffle, a scramble, an omelet, a patty, a quiche, a frittata, a mouse, a custard, a popsicle, a frozen desert, an ice cream, or a cookie.

ENGINEERED HOST CELLS

[0110] A further aspect of the present disclosure is an engineered host cell that recombinantly co-expresses a heterologous or exogenous ovalbumin protein and at least one heterologous or exogenous kinase protein. In some embodiments, an rpOVA is recombinantly expressed in a host cell. As used herein, a “host” or “host cell” denotes here any protein production host selected or genetically modified to produce a desired product. Illustrative hosts include fungi, such as filamentous fungi, as well as bacteria, yeast, plant, insect, and mammalian cells. A host cell may be Arxula spp., Arxula adeninivorans, Kluyveromyces spp., Kluyveromyces laclis. Komagataella phaffii, Pi chi a spp., Pichia angusta, Pichia pastor is, Saccharomyces spp., Saccharomyces cerevisiae, Schizosaccharomyces spp., Schizosaccharomyces pombe, Yarrowia spp., Yarrowia lipolylica, Agaricus spp., Agaricus bisporus, Aspergillus spp., Aspergillus aw amori, Aspergillus fumigalus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bacillus sublilis, Colletotrichum spp., Colletotrichum gloeosporiodes, Endothia spp., Endothia parasitica, Escherichia coli, Fusarium spp., Fusarium graminearum, Fusarium solani, Mucor spp., Mucor miehei, Mucor pusillus, Myceliophthora spp., Myceliophthora thermophila, Neurospora spp., Neurospora crassa, Penicillium spp., Penicillium camemberti, Penicillium canescens, Penicillium chrysogenum, Penicillium (Talaromyces) emersonii, P mcillium funiculo sum, Penicillium purpurogenum, Penicillium roqueforti, Pleurotus spp., Pleurotus ostreatus, Rhizomucor spp., Rhizomucor miehei, Rhizomucor pusillus, Rhizopus spp., Rhizopus arrhizus, Rhizopus oligosporus, Rhizopus oryzae, Trichoderma spp., Trichoderma altroviride, Trichoderma reesei, or Trichoderma vireus. A host cell can be an organism that is approved as generally regarded as safe by the U.S. Food and Drug Administration.

[OHl] An rpOVA protein can be recombinantly expressed in yeast, filamentous fungi or a bacterium. In some embodiments, rpOVA protein is recombinantly expressed in a Pichia species (Komagataella phaffii and Komagataella pastoris), a Saccharomyces species, a Trichoderma species, a Pseudomonas species or an E. coll species.

[0112] In some embodiments, the engineered host cell produces the same amount of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell produces more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell produces about 1% to about 200% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell produces about 1% to about 2%, about 1% to about 5%, about 1% to about 10%, about 1% to about 15%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 75%, about 1% to about 100%, about 1% to about 150%, about 1% to about 200%, about 2% to about 5%, about 2% to about 10%, about 2% to about 15%, about 2% to about 20%, about 2% to about 30%, about 2% to about 40%, about 2% to about 50%, about 2% to about 75%, about 2% to about 100%, about 2% to about 150%, about 2% to about 200%, about 5% to about 10%, about 5% to about 15%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 75%, about 5% to about 100%, about 5% to about 150%, about 5% to about 200%, about 10% to about 15%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 75%, about 10% to about 100%, about 10% to about 200%, about 15% to about 20%, about 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 75%, about 15% to about 100%, about 15% to about 150%, about 15% to about 200%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 75%, about 20% to about 100%, about 20% to about 200%, about 30% to about 40%, about 30% to about 50%, about 30% to about 75%, about 30% to about 100%, about 30% to about 150%, about 30% to about 200%, about 40% to about 50%, about 40% to about 75%, about 40% to about 100%, about 40% to about 150%, about 40% to about 200%, about 50% to about 75%, about 50% to about 100%, about 50% to about 200%, about 75% to about 100%, about 75% to about 200%, or about 100% to about 200% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell produces about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 100%, about 150%, or about 200% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell produces at least about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 150%, or about 100% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell produces at most about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 100%, about 150% or about 200% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein.

[0113] In some embodiments, the engineered host cell secretes the same amount of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell secretes more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell secretes about 1% to about 200% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell secretes about 1% to about 2%, about 1% to about 5%, about 1% to about 10%, about 1% to about 15%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 75%, about 1% to about 100%, about 1% to about 150%, about 1% to about 200%, about 2% to about 5%, about 2% to about 10%, about 2% to about 15%, about 2% to about 20%, about 2% to about 30%, about 2% to about 40%, about 2% to about 50%, about 2% to about 75%, about 2% to about 100%, about 2% to about 150%, about 2% to about 200%, about 5% to about 10%, about 5% to about 15%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 75%, about 5% to about 100%, about 5% to about 150%, about 5% to about 200%, about 10% to about 15%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 75%, about 10% to about 100%, about 10% to about 200%, about 15% to about 20%, about 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 75%, about 15% to about 100%, about 15% to about 150%, about 15% to about 200%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 75%, about 20% to about 100%, about 20% to about 200%, about 30% to about 40%, about 30% to about 50%, about 30% to about 75%, about 30% to about 100%, about 30% to about 150%, about 30% to about 200%, about 40% to about 50%, about 40% to about 75%, about 40% to about 100%, about 40% to about 150%, about 40% to about 200%, about 50% to about 75%, about 50% to about 100%, about 50% to about 200%, about 75% to about 100%, about 75% to about 200%, or about 100% to about 200% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell secretes about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 100%, about 150%, or about 200% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell secretes at least about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 150%, or about 100% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein. In some embodiments, the engineered host cell secretes at most about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 100%, about 150% or about 200% more of the recombinant phosphorylated ovalbumin compared to a similar cell that does not express a heterologous or exogenous kinase protein.

[0114] In some embodiments, the rpOVA can have an amino acid sequence from any species. For example, the rpOVA can have an amino acid sequence of OVA from a bird, a reptile, or another egg-laying species. In some embodiments, the rpOVA having an amino acid sequence from an avian can be selected from the group consisting of poultry, fowl, waterfowl, game bird, chicken, quail, turkey, duck, ostrich, goose, gull, guineafowl, pheasant, emu, and any combination thereof. In some embodiments, the rpOVA can have an amino acid sequence derived from a single species, such as Gallus gallus domesticus. In some embodiments, the rpOVA can have an amino acid sequence derived from two or more species, and as such can be a hybrid.

[0115] Illustrative OVA amino acid sequences contemplated herein are provided in Table 1 as SEQ ID NOs: 1-76.

[0116] Expression of rpOVA in a host cell, for instance a Pichia species, a Saccharomyces species, a Trichoderma species, a Pseudomonas species may lead to an addition of one or more amino acids to the OVA sequence as part of post-transcriptional or post-translational modifications. Such amino acids may not be part of the native OVA sequences. For instance, expressing an OVA sequence in a Pichia species, such as Komagataella phaffii and Komagataella pastoris may lead to addition of one or more amino acids at the N-terminus or C-terminus. In some cases, four amino acids EAEA (SEQ ID NO: 77) is added to the N- terminus of the OVA sequence upon expression in a host cell as shown in SEQ ID NO:2. For example, chicken rpOVA may be provided encoding SEQ ID NO:2, which lacks the EAEA (SEQ ID NO: 77) peptide, and following expression and secretion, the rpOVA has the amino acid sequence of SEQ ID NO:3 which comprises the EAEA (SEQ ID NO: 77) peptide. Any of the amino acid sequences recited in Table 1 which lacks an N-terminal EAEA (SEQ ID NO: 77) may be modified to comprise the N-terminal EAEA (SEQ ID NO: 77) peptide.

[0117] Expression of rpOVA in a host cell, for instance a Pichia species, a Saccharomyces species, a Trichoderma species, a Pseudomonas species may lead to a deletion of one or more amino acids to the OVA sequence as part of post-transcriptional or post-translational modifications. As noted in the above Table 1, many of the amino acid sequences comprise an N-terminal methionine (“M”). In nature and when expressed by a host cell of the present disclosure, a protein’ ss N-terminal methionine is often cleaved by a peptidase. Thus, when secreted, the amino acid sequences of Table 1 will lack the recited N-terminal methionine. For example, when an rOVA having the amino acid sequence from Anas platyrhynchos, i.e., SEQ ID NO: 16, the secreted amino acid may start with GSIG (SEQ ID NO: 78) rather than MGSIG (SEQ ID NO: 79). Similarly, when an rOVA recited in Table 1 is identified as having the initial EAEA (SEQ ID NO: 77) amino acid sequence, e.g., SEQ ID NO: 74, the secreted amino acid may omit the EAEA (SEQ ID NO: 77).

[0118] In some embodiments, the rpOVA can be a non-naturally occurring variant of an OVA. Such variants can comprise one or more amino acid insertions, deletions, or substitutions relative to a native OVA sequence.

[0119] Such a variant can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 1-76. The term “sequence identity” as used herein in the context of amino acid sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a selected sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software, with BLAST being the preferable alignment algorithm. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.

[0120] Depending on the host organism used to express the rpOVA, the rpOVA can have a glycosylation, acetylation, or phosphorylation pattern different from native OVA. For example, the rpOVA herein may or may not be glycosylated, acetylated, or phosphorylated. In some embodiments, the rpOVA may have an avian, non-avian, microbial, non-microbial, mammalian, or non-mammalian glycosylation, acetylation, or phosphorylation pattern.

[0121] In some cases, the rpOVA may be deglycosylated (e.g., chemically, enzymatically, Endo-H, PNGase F, O-Glycosidase, Neuraminidase, [31-4 Galactosidase, -N- acetylglucosaminidase), deacetylated (e.g., protein deacetylase, histone deacetylase, sirtuin), or dephosphorylated (e.g., acid phosphatase, lambda protein phosphatase, calf intestinal phosphatase, alkaline phosphatase). In some embodiments, deglycosylation, deacetylation or dephosphorylation may produce a protein that is more uniform or is capable of producing a composition with less variation.

[0122] In some embodiments, the rpOVA comprises post-translational modifications. In some embodiments, the post-translational modification comprises glycosylation. In some embodiments, the rOVA protein is glycosylated before being phosphorylated. In some cases, the rOVA is not phosphorylated until the rOVA is glycosylated. In some embodiments, the glycosylation comprises a carbohydrate attached to an amino acid residue of rOVA. In some embodiments, the glycosylation is N-linked glycosylation. In some embodiments, the glycosylation is O-linked glycosylation. In some embodiments, the glycosylation is C-linked glycosylation. In some embodiments, the glycosylation is S-linked glycosylation. In some embodiments, the glycosylation is glypiation. In some embodiments, the glycosylation is phosphoglycosylation. In some embodiments, the glycosylation moiety is covalently attached to one or more amino acids in the rOVA protein. In some embodiments, the glycosylation moiety comprises a carbohydrate. In some embodiments, the glycosylation moiety comprises a glycan. In some embodiments, the glycosylation moiety comprises at least one mannose moiety. In some embodiments, the glycosylation moiety comprises at least one N- acetylglucosamine moiety. In some embodiments, the glycosylation moiety comprises at least one mannose moiety or at least one N-acetylglucosamine moiety. In some embodiments, the glycosylation moiety comprises at least one mannose moiety and at least one N- acetyl glucosamine moiety. [0123] In some embodiments, an unglycosylated rOVA is unphosphorylated. In some embodiments, a glycosylated rOVA is phosphorylated. In some embodiments, a glycosylated rOVA is unphosphorylated. In some embodiments, a phosphorylated recombinant ovalbumin is more stable than an unphosphorylated recombinant ovalbumin. In some embodiments, the rpOVA is more stable than an unphosphorylated rOVA.

[0124] In some embodiments, rOVA may be phosphorylated at multiple sites. In some embodiments, rOVA may be monophosphorylated at one site. In some embodiments, rOVA may be monophosphorylated at Ser-68 (with respect to SEQ ID NO: 2). In some embodiments, rOVA may be monophosphorylated at Ser-344 (with respect to SEQ ID NO: 2). In some embodiments, rOVA may be diphosphorylated at two sites. In some embodiments, rOVA may be diphosphorylated at Ser-68 and Ser-344. In some embodiments, rOVA may comprise a homogenous mixture. In certain embodiments, a homogenous rOVA mixture may comprise a single species of rOVA. In certain embodiments, a single species of rOVA may comprise unphosphorylated rOVA. In certain embodiments, a single species of rOVA may comprise monophosphorylated rOVA at Ser-68. In certain embodiments, a single species of rOVA may comprise monophosphorylated rOVA at Ser-344. In certain embodiments, a single species of rOVA may comprise diphosphorylated rOVA at Ser-68 and Ser-344.

[0125] In some embodiments, rOVA may comprise a heterogeneous mixture. In various embodiments, a heterogenous mixture may comprise two or more species of rOVA. In certain embodiments, a heterogenous rOVA mixture may comprise one or more of unphosphorylated rOVA, monophosphorylated rOVA at Ser-68, monophosphorylated rOVA at Ser-344, diphosphorylated rOVA at Ser-68 and Ser-344, rOVA phosphorylated at another site, or any combination thereof (each with respect to SEQ ID NO: 2). In some embodiments, the rOVA mixture comprises a mixture of rpOVA with varying amounts of rOVA and rpOVA species.

[0126] In some embodiments, the unphosphorylated rOVA is most abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rpOVA is most abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rpOVA at Ser-68 (with respect to SEQ ID NO: 2) is most abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rpOVA at Ser-344 (with respect to SEQ ID NO: 2) is most abundant in the rpOVA mixture. In some embodiments, the diphosphorylated rpOVA is most abundant in the rpOVA mixture. In some embodiments, the unphosphorylated rOVA is least abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rpOVA is least abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rpOVA at Ser-68 is least abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rOVA at Ser-344 is least abundant in the rpOVA mixture. In some embodiments, the diphosphorylated rpOVA is most abundant in the rpOVA mixture.

[0127] In some embodiments, the rOVA mixture may comprise a ratio of phosphorylated rpOVA species. In some embodiments, the ratio of phosphorylated rpOVA species may differ from the ratio of phosphorylated OVA found in substitute egg whites. In some embodiments, the ratio of phosphorylated rpOVA species may differ from the ratio of phosphorylated OVA to native OVA. In some embodiments, the ratio of phosphorylated rpOVA species may differ from the ratio found in an egg. In some embodiments, the ratio of phosphorylated rpOVA species may differ from the ratio of unphosphorylated OVA.

[0128] In certain embodiments, the ratio of rpOVA monophosphorylated at Ser-344 (with respect to SEQ ID NO: 2) is greater than the protein composition obtained from an egg. In various embodiments, the ratio of rOVA monophosphorylated at Ser-68 (with respect to SEQ ID NO: 2) is greater than the protein composition obtained from an egg. In various embodiments, the ratio of rpOVA diphosphorylated at Ser-68 and at Ser-344 is greater than the protein composition obtained from an egg. In certain embodiments, the ratio of rpOVA monophosphorylated at Ser-344 is less than the protein composition obtained from an egg. In some embodiments, the ratio of rpOVA monophosphorylated at Ser-68 is less than the protein composition obtained from an egg. In some embodiments, the ratio of rpOVA diphosphorylated at Ser-68 and at Ser-344 is less than the protein composition obtained from an egg.

[0129] In some embodiments, the ratio of phosphorylated rpOVA to unphosphorylated rOVA is about 1 : 1 to about 1 :20. In some embodiments, the ratio of phosphorylated rpOVA to unphosphorylated rOVA is about 1:20 to about 1:15, about 1:20 to about 1:10, about 1:20 to about 1 :9, about 1 :20 to about 1:8, about 1 :20 to about 1 :7, about 1 :20 to about 1 :6, about 1 :20 to about 1:5, about 1:20 to about 1:4, about 1:20 to about 1:3, about 1:20 to about 1:2, about 1:20 to about 1:1, about 1:15 to about 1:10, about 1:15 to about 1:9, about 1:15 to about 1:8, about 1:15 to about 1:7, about 1:15 to about 1:6, about 1:15 to about 1:5, about 1:15 to about 1:4, about 1:15 to about 1:3, about 1:15 to about 1:2, about 1:15 to about 1:1, about 1:10 to about 1 :9, about 1 : 10 to about 1:8, about 1 : 10 to about 1 :7, about 1 : 10 to about 1 :6, about 1:10 to about 1:5, about 1:10 to about 1:4, about 1:10 to about 1:3, about 1:10 to about 1:2, about 1:10 to about 1:1, about 1:9 to about 1:8, about 1:9 to about 1:7, about 1:9 to about 1:6, about 1:9 to about 1:5, about 1:9 to about 1:4, about 1:9 to about 1:3, about 1:9 to about 1:2, about 1:9 to about 1:1, about 1:8 to about 1:7, about 1:8 to about 1:6, about 1:8 to about 1:5, about

1 :4 to about 1 :2, about 1 :4 to about 1:1, about 1 :3 to about 1 :2, about 1 :3 to about 1 : 1, or about 1:2 to about 1:1. In some embodiments, the ratio of phosphorylated rpOVA to unphosphorylated rOVA is about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1. In some embodiments, the ratio of phosphorylated rpOVA to unphosphorylated rOVA is at least about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, or about 1:2. In some embodiments, the ratio of phosphorylated rpOVA to unphosphorylated rOVA is at most about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1.

[0130] In some embodiments, the ratio of phosphorylated rpOVA to unphosphorylated rOVA is about 1 : 1 to about 20: 1. In some embodiments, the ratio of phosphorylated rpOVA to unphosphorylated rOVA is about 20:1 to about 15:1, about 20:1 to about 10:1, about 20:1 to about 9:1, about 20:1 to about 8:1, about 20:1 to about 7:1, about 20 to about 6:1, about 20:1 to about 5:1, about 20:1 to about 4:1, about 20:1 to about 3:1, about 20:1 to about 2:1, about 20:1 to about 1:1, about 15:1 to about 10:1, about 15:1 to about 9:1, about 15:1 to about 8:1, about 15:1 to about 7:1, about 15:1 to about 6:1, about 15:1 to about 5:1, about 15:1 to about 4:1, about 15:1 to about 3:1, about 15:1 to about 2:1, about 15:1 to about 1:1, about 10:1 to about 9:1, about 10:1 to about 8:1, about 10:1 to about 7:1, about 10:1 to about 6:1, about 10:1 to about 5:1, about 10:1 to about 4:1, about 10:1 to about 3:1, about 10:1 to about 2:1, about 10:1 to about 1:1, about 9:1 to about 8:1, about 9:1 to about 7:1, about 9:1 to about 6:1, about 9:1 to about 5:1, about 9:1 to about 4:1, about 9:1 to about 3:1, about 9:1 to about 2:1, about

-n- 4: 1 to about 2:1, about 4: 1 to about 1:1, about 3 : 1 to about 2:1, about 3 : 1 to about 1 : 1, or about 2: 1 to about 1 : 1. the ratio of phosphorylated rpOVA to unphosphorylated rOVA is about 20: 1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of phosphorylated rpOVA to unphosphorylated rOVA is at least about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of phosphorylated rpOVA to unphosphorylated rOVA is at most about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2: 1, or about 1:1.

[0131] In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is about 1:1 to about 1:20 (each with respect to SEQ ID NO: 2). In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is about 1:20 to about 1:15, about 1:20 to about 1:10, about 1:20 to about 1:9, about 1:20 to about 1:8, about 1:20 to about 1:7, about 1:20 to about 1:6, about 1:20 to about 1:5, about 1:20 to about 1:4, about 1:20 to about 1:3, about 1:20 to about 1:2, about 1:20 to about 1:1, about 1:15 to about 1:10, about 1:15 to about 1:9, about 1:15 to about 1:8, about 1:15 to about 1:7, about 1:15 to about 1:6, about 1:15 to about 1:5, about 1:15 to about 1:4, about 1:15 to about 1:3, about 1:15 to about 1:2, about 1:15 to about 1:1, about 1:10 to about 1:9, about 1:10 to about 1:8, about 1:10 to about 1:7, about 1:10 to about 1 :6, about 1 : 10 to about 1:5, about 1 : 10 to about 1 :4, about 1 : 10 to about 1 :3, about 1:10 to about 1:2, about 1:10 to about 1:1, about 1:9 to about 1:8, about 1:9 to about 1:7, about 1:9 to about 1 :6, about 1 :9 to about 1:5, about 1 :9 to about 1 :4, about 1 :9 to about 1 :3, about 1 :9 to about 1:2, about 1:9 to about 1:1, about 1:8 to about 1:7, about 1:8 to about 1:6, about 1:8 to about 1:5, about 1:8 to about 1:4, about 1:8 to about 1:3, about 1:8 to about 1:2, about 1:8 to about 1:1, about 1:7 to about 1:6, about 1:7 to about 1:5, about 1:7 to about 1:4, about 1:7 to about 1:3, about 1:7 to about 1:2, about 1:7 to about 1:1, about 1:6 to about 1:5, about 1:6 to about 1:4, about 1:6 to about 1:3, about 1:6 to about 1:2, about 1:6 to about 1:1, about 1:5 to about 1:4, about 1:5 to about 1:3, about 1:5 to about 1:2, about 1:5 to about 1:1, about 1:4 to about 1:3, about 1:4 to about 1:2, about 1:4 to about 1:1, about 1:3 to about 1:2, about 1:3 to about 1:1, or about 1:2 to about 1:1. In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1. In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is at least about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, or about 1:2. In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is at most about 1:15, about 1 : 10, about 1 :9, about 1:8, about 1 :7, about 1 :6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1.

[0132] In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is about 1:1 to about 20:1 (each with respect to SEQ ID NO: 2). In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is about 20: 1 to about 15:1, about 20: 1 to about 10:1, about 20: 1 to about 9:1, about 20: 1 to about 8:1, about 20: 1 to about 7: 1, about 20 to about 6:1, about 20:1 to about 5:1, about 20:1 to about 4:1, about 20:1 to about 3:1, about 20:1 to about 2:1, about 20:1 to about 1:1, about 15:1 to about 10:1, about 15:1 to about 9:1, about 15:1 to about 8:1, about 15:1 to about 7:1, about 15:1 to about 6:1, about 15:1 to about 5:1, about 15:1 to about 4:1, about 15:1 to about 3:1, about 15:1 to about 2:1, about 15:1 to about 1:1, about 10:1 to about 9:1, about 10:1 to about 8:1, about 10:1 to about 7:1, about 10:1 to about 6:1, about 10:1 to about 5:1, about 10:1 to about 4:1, about 10:1 to about 3:1, about 10:1 to about 2:1, about 10:1 to about 1:1, about 9:1 to about 8:1, about 9:1 to about 7:1, about 9:1 to about 6:1, about 9:1 to about 5:1, about 9:1 to about 4:1, about 9:1 to about 3:1, about 9:1 to about

2:1, about 9:1 to about 1:1, about 8:1 to about 7:1, about 8:1 to about 6:1, about 8:1 to about

5:1, about 8:1 to about 4:1, about 8:1 to about 3:1, about 8:1 to about 2:1, about 8:1 to about

1:1, about 7:1 to about 6:1, about 7:1 to about 5:1, about 7:1 to about 4:1, about 7:1 to about

3:1, about 7:1 to about 2:1, about 7:1 to about 1:1, about 6:1 to about 5:1, about 6:1 to about

4:1, about 6:1 to about 3:1, about 6:1 to about 2:1, about 6:1 to about 1:1, about 5:1 to about

4:1, about 5:1 to about 3:1, about 5:1 to about 2:1, about 5:1 to about 1:1, about 4:1 to about

3:1, about 4:1 to about 2:1, about 4:1 to about 1:1, about 3:1 to about 2:1, about 3:1 to about

1 : 1, or about 2: 1 to about 1 : 1. In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is at least about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is at most about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about5:l, about 4:1, about3:l, about2:l, or about 1:1.

[0133] In some embodiments, the ratio of diphosphorylated rpOVA to unphosphorylated rOVA is about 1:1 to about 1:20. In some embodiments, the ratio of diphosphorylated rpOVA to unphosphorylated rOVA is about 1 :20 to about 1:15, about 1 :20 to about 1:10, about 1 :20 to about 1 :9, about 1 :20 to about 1:8, about 1 :20 to about 1 :7, about 1 :20 to about 1 :6, about 1 :20 to about 1:5, about 1:20 to about 1:4, about 1:20 to about 1:3, about 1:20 to about 1:2, about 1:20 to about 1:1, about 1:15 to about 1:10, about 1:15 to about 1:9, about 1:15 to about 1:8, about 1:15 to about 1:7, about 1:15 to about 1:6, about 1:15 to about 1:5, about 1:15 to about 1:4, about 1:15 to about 1:3, about 1:15 to about 1:2, about 1:15 to about 1:1, about 1:10 to about 1 :9, about 1 : 10 to about 1:8, about 1 : 10 to about 1 :7, about 1 : 10 to about 1 :6, about 1:10 to about 1:5, about 1:10 to about 1:4, about 1:10 to about 1:3, about 1:10 to about 1:2, about 1:10 to about 1:1, about 1:9 to about 1:8, about 1:9 to about 1:7, about 1:9 to about 1:6, about 1:9 to about 1:5, about 1:9 to about 1:4, about 1:9 to about 1:3, about 1:9 to about 1:2, about

1 :4 to about 1 :2, about 1 :4 to about 1:1, about 1 :3 to about 1 :2, about 1 :3 to about 1 : 1, or about 1:2 to about 1:1. In some embodiments, the ratio of diphosphorylated rpOVA to unphosphorylated rOVA is about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1. In some embodiments, the ratio of diphosphorylated rpOVA to unphosphorylated rOVA is at least about 1 :20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, or about 1:2. In some embodiments, the ratio of diphosphorylated rpOVA to unphosphorylated rOVA is at most about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1 :4, about 1:3, about 1 :2, or about 1:1.

[0134] In some embodiments, the ratio of diphosphorylated rpOVA to unphosphorylated rOVA is about 1 : 1 to about 20: 1. In some embodiments, the ratio of diphosphorylated rpOVA to unphosphorylated rOVA is about 20: 1 to about 15:1, about 20: 1 to about 10:1, about 20: 1 to about 9:1, about 20:1 to about 8:1, about 20:1 to about 7:1, about 20 to about 6:1, about 20:1 to about 5:1, about 20:1 to about 4:1, about 20:1 to about 3:1, about 20:1 to about 2:1, about 20:1 to about 1:1, about 15:1 to about 10:1, about 15:1 to about 9:1, about 15:1 to about 8:1, about 15:1 to about 7:1, about 15:1 to about 6:1, about 15:1 to about 5:1, about 15:1 to about 4:1, about 15:1 to about 3:1, about 15:1 to about 2:1, about 15:1 to about 1:1, about 10:1 to about 9:1, about 10:1 to about 8:1, about 10:1 to about 7:1, about 10:1 to about 6:1, about 10:1 to about 5:1, about 10:1 to about 4:1, about 10:1 to about 3:1, about 10:1 to about 2:1, about 10:1 to about 1:1, about 9:1 to about 8:1, about 9:1 to about 7:1, about 9:1 to about 6:1, about 9:1 to about 5:1, about 9:1 to about 4:1, about 9:1 to about 3:1, about 9:1 to about 2:1, about

4: 1 to about 2:1, about 4: 1 to about 1:1, about 3 : 1 to about 2:1, about 3 : 1 to about 1 : 1, or about 2:1 to about 1:1. the ratio of diphosphorylated rpOVA to unphosphorylated rOVA is about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2: 1, or about 1 : 1. In some embodiments, the ratio of diphosphorylated rpOVA to unphosphorylated rOVA is at least about 20: 1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of diphosphorylated rpOVA to unphosphorylated rOVA is at most about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1.

[0135] In some embodiments, the ratio of diphosphorylated rpOVA to monophosphorylated rOVA is about 1:1 to about 1:20. In some embodiments, the ratio of diphosphorylated rpOVA to monophosphorylated rOVA is about 1:20 to about 1:15, about 1:20 to about 1:10, about 1:20 to about 1:9, about 1:20 to about 1:8, about 1:20 to about 1:7, about 1:20 to about 1:6, about 1:20 to about 1:5, about 1:20 to about 1:4, about 1:20 to about 1:3, about 1:20 to about 1:2, about 1:20 to about 1:1, about 1:15 to about 1:10, about 1:15 to about 1:9, about 1:15 to about 1:8, about 1:15 to about 1:7, about 1:15 to about 1:6, about 1:15 to about 1:5, about 1:15 to about 1:4, about 1:15 to about 1:3, about 1:15 to about 1:2, about 1:15 to about 1:1, about 1:10 to about 1:9, about 1:10 to about 1:8, about 1:10 to about 1:7, about 1:10 to about 1:6, about 1:10 to about 1:5, about 1:10 to about 1:4, about 1:10 to about 1 :3, about 1 : 10 to about 1 :2, about 1 : 10 to about 1:1, about 1 :9 to about 1:8, about 1 :9 to about 1:7, about 1:9 to about 1:6, about 1:9 to about 1:5, about 1:9 to about 1:4, about 1:9 to about

1:2, about 1:3 to about 1:1, or about 1:2 to about 1:1. In some embodiments, the ratio of diphosphorylated rpOVAto monophosphorylated rOVA is about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1. In some embodiments, the ratio of diphosphorylated rpOVA to monophosphorylated rOVA is at least about 1 :20, about 1:15, about 1 : 10, about 1 :9, about 1:8, about 1 :7, about 1 :6, about 1:5, about 1:4, about 1:3, or about 1:2. In some embodiments, the ratio of diphosphorylated rpOVA to monophosphorylated rOVA is at most about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1.

[0136] In some embodiments, the ratio of diphosphorylated rpOVA to monophosphorylated rpOVA is about 1:1 to about 20:1. In some embodiments, the ratio of diphosphorylated rpOVA to monophosphorylated rpOVA is about 20:1 to about 15:1, about 20:1 to about 10:1, about 20:1 to about 9:1, about 20:1 to about 8:1, about 20:1 to about 7:1, about 20 to about 6:1, about 20 : 1 to about 5:1, about 20 : 1 to about 4:1, about 20 : 1 to about 3:1, about 20: 1 to about 2:1, about 20: 1 to about 1:1, about 15:1 to about 10:1, about 15:1 to about 9:1, about 15:1 to about 8:1, about 15:1 to about 7:1, about 15:1 to about 6:1, about 15:1 to about 5:1, about 15:1 to about 4:1, about 15:1 to about 3:1, about 15:1 to about 2:1, about 15:1 to about 1:1, about 10:1 to about 9:1, about 10:1 to about 8:1, about 10:1 to about 7:1, about 10:1 to about 6:1, about 10:1 to about 5:1, about 10:1 to about 4:1, about 10:1 to about 3:1, about 10:1 to about 2:1, about 10:1 to about 1:1, about 9:1 to about 8:1, about 9:1 to about 7:1, about 9:1 to about 6:1, about 9:1 to about 5:1, about 9:1 to about 4:1, about 9:1 to about 3:1, about 9:1 to about 2:1, about 9:1 to about 1:1, about 8:1 to about 7:1, about 8:1 to about 6:1, about 8:1 to about 5:1, about 8:1 to about 4:1, about 8:1 to about 3:1, about 8:1 to about 2:1, about 8:1 to about 1:1, about 7:1 to about 6:1, about 7:1 to about 5:1, about 7:1 to about 4:1, about 7:1 to about 3:1, about 7:1 to about 2:1, about 7:1 to about 1:1, about 6:1 to about 5:1, about 6:1 to about 4:1, about 6:1 to about 3:1, about 6:1 to about 2:1, about 6:1 to about 1:1, about 5:1 to about 4:1, about 5:1 to about 3:1, about 5:1 to about 2:1, about 5:1 to about 1:1, about 4:1 to about 3:1, about 4:1 to about 2:1, about 4:1 to about 1:1, about 3:1 to about 2:1, about 3:1 to about 1:1, or about 2:1 to about 1:1. the ratio of diphosphorylated rpOVA to monophosphorylated rpOVA is about 20: 1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of diphosphorylated rpOVA to monophosphorylated rpOVA is at least about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of diphosphorylated rpOVA to monophosphorylated rpOVA is at most about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1.

[0137] In some embodiments, rOVA is phosphorylated by a kinase. In some embodiments, rOVA is phosphorylated by a serine/threonine kinase. In some embodiments, rOVA is phosphorylated by a tyrosine kinase. In some embodiments, the kinase is a heterologous or exogenous kinase protein. In some embodiments, the kinase recognizes a recognition motif comprising a S-X-E/pS motif. In some embodiments, the kinase recognizes a recognition motif that comprises Glu at a +2 position.

[0138] In certain embodiments, the kinase can be expressed in a host cell. In certain embodiments, the kinase can be co-expressed in a host cell with rOVA or the kinase is expressed by a first host cell and the rOVA is expressed by a second host cell. The kinase may be secreted by a host cell such that phosphorylation occurs extracellularly. Extracellular phosphorylation will also occur when the kinase comprises or is linked to a domain that anchors the protein to the extracellular surface of the cell. Alternatively, the kinase may be anchored to a membrane within a cell such that phosphorylation occurs intracellularly. Here, the kinase comprises or is linked to a domain that anchors the protein to a membrane within the cell, e.g., the endoplasmic reticulum and/or Golgi apparatus. In some cases, the kinase’s amino acid sequence naturally includes an anchoring domain, e.g., VLILMVFLVACTMHIMI (SEQ ID NO: 142) or VLILMVFLVACTMHIMIDLL (SEQ ID NO: 143). In other cases, a kinase’s amino acid sequence does not include an anchoring domain of SEQ ID NO: 142 or of SEQ ID NO: 143, and in these cases, the present disclosure provides methods for replacing that kinase’s anchoring domain with one of SEQ ID NO: 142, SEQ ID NO: 143, or a variant thereof (which includes or more amino acid changes yet maintains the native anchoring ability). In various cases, a kinase’s anchoring domain may be replaced with an alternate anchoring domain, e.g., YTKIVKAVGIGFIAVGIIGYAIKLIHIPI (SEQ ID NO: 144) which anchors the kinase domain to the endoplasmic reticulum and/or Golgi apparatus. Any kinase recited herein may be modified to include an above-mentioned anchoring domain or may be modified to omit an anchoring domain and/or to add a signal sequence.

[0139] In some embodiments, the rpOVA protein can be recombinantly expressed in yeast, filamentous fungi or a bacterium. In some embodiments, rpOVA protein is recombinantly expressed in a Pichia species (Komagataella phaffii and Komagataella pasloris). a Saccharomyces species, a Trichoderma species, a Pseudomonas species or an E. coli species. In some embodiments, the kinase protein can be recombinantly expressed in yeast, filamentous fungi or a bacterium. In some embodiments, kinase protein is recombinantly expressed in a Pichia species (Komagataella phaffii and Komagataella pasloris). a Saccharomyces species, a Trichoderma species, a Pseudomonas species, or an E. coli species.

[0140] In some embodiments, the exogenous kinase protein is selected from the genera: Homo, Drosophila, Danio, Branchiostoma, Stronglycocentrotus, Caenorhabditis, Hydra, Amphimedon, any other animal, or any combination thereof. In certain embodiments, the kinase protein is selected from the Aves or Reptilia class. In some embodiments, the kinase can have an amino acid sequence of FAM20 from a bird, a reptile, or another egg-laying species. In some embodiments, the FAM20 having an amino acid sequence from an Aves species can be selected from the group consisting of poultry, fowl, waterfowl, game bird, chicken, quail, turkey, duck, ostrich, goose, gull, guineafowl, pheasant, emu, and any combination thereof. In some embodiments, the FAM20 can have an amino acid sequence derived from a single species, such as Gallus gallus domesticus. In some embodiments, the heterologous or exogenous kinase protein is from G. gallus.

[0141] In some embodiments, the kinase can have an amino acid sequence from any species. In various embodiments, the FAM20 kinase can have an amino acid sequence comprising FAM20A. In various embodiments, the FAM20 kinase can have an amino acid sequence comprising FAM20A from any species. In certain embodiments, the FAM20 kinase can have an amino acid sequence comprising FAM20C. In certain embodiments, the FAM20 kinase can have an amino acid sequence comprising FAM20C from any species. In some embodiments, the FAM20C kinase can have an amino acid sequence comprising any FAM20C isoform. In some embodiments, the FAM20C kinase can have an amino acid sequence comprising any FAM20C isoform from any species. [0142] In certain embodiments, the kinase protein comprises an amino acid sequence with at least 70% homology to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID

NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID

NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID

NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID

NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID

NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID

NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID

NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID

SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112,

SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117,

SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122,

SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127,

SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132,

SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137,

SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141. In various embodiments, the heterologous or exogenous kinase protein comprises an amino acid sequence of SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO:

121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO:

126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO:

131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO:

136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141. In some embodiments, the heterologous or exogenous kinase protein comprises a nucleotide sequence encoding for an amino acid with at least 70% homology to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID

NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID

NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID

NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID

NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID

NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID

108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO:

113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO:

123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO:

128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO:

133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO:

138, SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141. In various embodiments, the heterologous or exogenous kinase protein comprises a nucleotide sequence encoding for an amino acid of SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105,

SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110,

SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115,

SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120,

SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125,

SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130,

SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135,

SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141. In these embodiments, an amino acid with at least 70% homology to SEQ ID NO: 80 maintains the enzymatic activity of a kinase expressed by the corresponding SEQ ID NO: 80; an amino acid with at least 70% homology to SEQ ID NO: 85 maintains the enzymatic activity of a kinase expressed by the corresponding SEQ ID NO: 85; an amino acid with at least 70% homology to SEQ ID NO: 97 maintains the enzymatic activity of a kinase expressed by the corresponding SEQ ID NO: 97; and so forth.

[0143] In some embodiments, the kinase can have an amino acid sequence derived from two or more species, and as such can be a hybrid. In some embodiments, the kinase protein comprises a mixture of at least two or more distinct heterologous or exogenous kinase proteins. In some embodiments, the kinase or kinases can have an amino acid sequence derived from a single species. In some embodiments, the kinase can have an amino acid sequence derived from two or more species, and as such can be a hybrid.

[0144] Illustrative kinase amino acid sequences contemplated herein are provided in Table 2 as SEQ ID NOs: 80-141.

[0145] Expression of an rOVA or a kinase can be provided by an expression vector, a plasmid, a nucleic acid integrated into the host genome or other means. For example, a vector for expression can include: (a) a promoter element, (b) a signal peptide, (c) an OVA sequence heterologous to the host cell, and (d) a terminator element. Expression of a kinase can be provided by an expression vector, a plasmid, a nucleic acid integrated into the host genome or other means. For example, a vector for expression can include: (a) a promoter element, (b) a signal peptide, (c) a kinase sequence heterologous to the host cell, and (d) a terminator element. In some embodiments, the rOVA can be provided by the same expression vector, plasmid, nucleic acid integrated into the host genome or other means as the kinas.

[0146] Expression vectors that can be used for expression of OVA or a kinase include those containing an expression cassette with elements (a), (b), (c) and (d). In some embodiments, the signal peptide (b) need not be included in the vector. In general, the expression cassette is designed to mediate the transcription of the transgene when integrated into the genome of a cognate host microorganism.

[0147] To aid in the amplification of the vector prior to transformation into the host microorganism, a replication origin (e) may be contained in the vector (such as PUC ORIC and PUC (DNA2.0)). To aid in the selection of microorganisms stably transformed with the expression vector, the vector may also include a selection marker (f) such as URA3 gene and Zeocin resistance gene (ZeoR). The expression vector may also contain a restriction enzyme site (g) that allows for linearization of the expression vector prior to transformation into the host microorganism to facilitate the expression vector's stable integration into the host genome. In some embodiments the expression vector may contain any subset of the elements (b), (e), (f), and (g), including none of elements (b), (e), (f), and (g). Other expression elements and vector elements known to one of skill in the art can be used in combination or substituted for the elements described herein.

[0148] Illustrative promoter elements (a) may include, but are not limited to, a constitutive promoter, inducible promoter, and hybrid promoter. Promoters include, but are not limited to, acu-5, adhl+, alcohol dehydrogenase (ADH1, ADH2, ADH4), AHSB4m, AINV, alcA, a- amylase, alternative oxidase (AOD), alcohol oxidase I (A0X1), alcohol oxidase 2 (A0X2), AXDH, B2, CaMV, cellobiohydrolase I (cbhl), ccg-1, cDNAl, cellular filament polypeptide (cfp), cpc-2, ctr4+, CUP1, dihydroxyacetone synthase (DAS), enolase (ENO, ENO1), formaldehyde dehydrogenase (FLD1), FMD, formate dehydrogenase (FMDH), Gl, G6, GAA, GALI, GAL2, GAL3, GAL4, GAL5, GAL6, GAL7, GAL8, GAL9, GAL10, GCW14, gdhA, gla-1, a-glucoamylase (glaA), glyceraldehyde-3 -phosphate dehydrogenase (gpdA, GAP, GAPDH), phosphoglycerate mutase (GPM1), glycerol kinase (GUT1), HSP82, invl+, isocitrate lyase (ICL1), acetohydroxy acid isomeroreductase (ILV5), KAR2, KEX2, P-galactosidase (lac4), LEU2, melO, MET3, methanol oxidase (MOX), nmtl, NSP, pcbC, PET9, peroxin 8 (PEX8), phosphoglycerate kinase (PGK, PGK1), phol, PHO5, PHO89, phosphatidylinositol synthase (PIS1), PYK1, pyruvate kinase (pkil), RPS7, sorbitol dehydrogenase (SDH), 3- phosphoserine aminotransferase (SERI), SSA4, SV40, TEF, translation elongation factor 1 alpha (TEF1), THI11, homoserine kinase (THR1), tpi, TPS1, triose phosphate isomerase (TPI1), XRP2, YPT1, and any combination thereof.

[0149] A signal peptide (b), also known as a signal sequence, targeting signal, localization signal, localization sequence, signal peptide, transit peptide, leader sequence, or leader peptide, may support secretion of a protein or polynucleotide. Extracellular secretion of a recombinant or heterologously expressed protein from a host cell may facilitate protein purification. A signal peptide may be derived from a precursor (e.g., prepropeptide, preprotein) of a protein. Signal peptides can be derived from a precursor of a protein other than the signal peptides in native OVA or native kinases. An example of secretion protein is a S. cerevisiae alpha factor pre pro sequence shown bolded and underlined in SEQ ID NO: 1.

[0150] Any nucleic acid sequence that encodes OVA or a kinase can be used as (c). Preferably such sequence is codon optimized for the host cell.

[0151] Illustrative transcriptional terminator elements include, but are not limited to, acu- 5, adhl+, alcohol dehydrogenase (ADH1, ADH2, ADH4), AHSB4m, AINV, alcA, a-amylase, alternative oxidase (AOD), alcohol oxidase I (A0X1), alcohol oxidase 2 (A0X2), AXDH, B2, CaMV, cellobiohydrolase I (cbhl), ccg-1, cDNAl, cellular filament polypeptide (cfp), cpc-2, ctr4+, CUP1, dihydroxyacetone synthase (DAS), enolase (ENO, ENO1), formaldehyde dehydrogenase (FLD1), FMD, formate dehydrogenase (FMDH), Gl, G6, GAA, GALI, GAL2, GAL3, GAL4, GAL5, GAL6, GAL7, GAL8, GAL9, GAL10, GCW14, gdhA, gla-1, a- glucoamylase (glaA), glyceraldehyde-3 -phosphate dehydrogenase (gpdA, GAP, GAPDH), phosphoglycerate mutase (GPM1), glycerol kinase (GUT1), HSP82, invl+, isocitrate lyase (ICL1), acetohydroxy acid isomeroreductase (ILV5), KAR2, KEX2, P-galactosidase (lac4), LEU2, melO, MET3, methanol oxidase (MOX), nmtl, NSP, pcbC, PET9, peroxin 8 (PEX8), phosphoglycerate kinase (PGK, PGK1), phol, PHO5, PHO89, phosphatidylinositol synthase (PIS1), PYK1, pyruvate kinase (pki 1), RPS7, sorbitol dehydrogenase (SDH), 3 -phosphoserine aminotransferase (SERI), SSA4, SV40, TEF, translation elongation factor 1 alpha (TEF1), THI11, homoserine kinase (THR1), tpi, TPS1, triose phosphate isomerase (TPI1), XRP2, YPT1, and any combination thereof.

[0152] Illustrative selectable markers (f) may include, but are not limited to: an antibiotic resistance gene (e.g. zeocin, ampicillin, blasticidin, kanamycin, nourseothricin, chloroamphenicol, tetracycline, triclosan, ganciclovir, and any combination thereof), an auxotrophic marker (e.g. adel, arg4, his4, ura3, met2, and any combination thereof). [0153] In one example, a vector for expression in Pichia sp. can include an A0X1 promoter operably linked to a signal peptide (alpha mating factor) that is fused in frame with a nucleic acid sequence encoding OVA, and a terminator element (AOX1 terminator) immediately downstream of the nucleic acid sequence encoding OVA. In another example, a vector for expression in Pichia sp. can include an AOX1 promoter operably linked to a signal peptide that is fused in frame with a nucleic acid sequence encoding a kinase, and a terminator element (AOX1 terminator) immediately downstream of the nucleic acid sequence encoding a kinase.

[0154] In some embodiments, nucleic acid sequence encoding OVA is separate from the nucleic acid sequence encoding a kinase. In some embodiments, nucleic acid sequence encoding OVA is operably linked to the nucleic acid sequence encoding a kinase. In some embodiments, nucleic acid sequence encoding OVA is operably linked to the nucleic acid sequence encoding a kinase by a sequence encoding a self-cleaving peptide.

[0155] In another example, a vector comprising a DAS1 promoter is operably linked to a signal peptide (alpha mating factor) that is fused in frame with a nucleic acid sequence encoding OVA and a terminator element (AOX1 terminator) immediately downstream of OVA.

[0156] In some embodiments, the recombinant protein (rpOVA) described herein may be secreted from the one or more host cells. In some embodiments, rpOVA protein is secreted from the host cell. The secreted rpOVA may be isolated and purified by methods such as centrifugation, fractionation, filtration, ion exchange chromatography, affinity purification and other methods for separating protein from cells, liquid and solid media components and other cellular products and byproducts. In some embodiments, rpOVA is produced in a Pichia Sp. and secreted from the host cells into the culture media. The secreted rpOVA is then separated from other media components for further use.

[0157] In some embodiments, the rpOVA mixture described herein may be secreted from the one or more host cells. In some embodiments, the rpOVA mixture is secreted from the host cell. In some embodiments, the rpOVA is secreted from the host cell and further treated with an exogenous kinase. In some embodiments, the unphosphorylated rOVA is secreted from the host cell and further treated with an exogenous kinase that is not co-expressed in the host cell. The secreted rpOVA mixture may be isolated and purified by methods such as centrifugation, fractionation, filtration, ion exchange chromatography, affinity purification and other methods for separating protein from cells, liquid and solid media components and other cellular products and byproducts. In some embodiments, the rpOVA mixture is produced in a Pichia Sp. and secreted from the host cells into the culture media. The secreted rpOVA mixture is then separated from other media components for further use.

[0158] The molecular weight of rpOVA may be different as compared to nOVA. The molecular weight of the protein may be less than the molecular weight of nOVA. In certain embodiments, the molecular weight of an rpOVA may be between 40kDa and 55kDa. In some cases, an rOVA with glycosylation has a different molecular weight, such as compared to a native OVA (as produced by an avian host species) or as compared to a host cell that glycosylates the rOVA, such as where the rOVA includes N-linked mannosylation. In some cases, the molecular weight of rpOVA is greater than the molecular weight of the rOVA that is completely devoid of post-translational modifications or an rOVA that lacks all forms of N- linked glycosylation or that is unphosphorylated.

[0159] In some embodiments, the molecular weight of rpOVA is at least the molecular weight of the unphosphorylated rOVA. In some embodiments, the molecular weight of rpOVA is greater than the molecular weight of the unphosphorylated rOVA. In some embodiments, the molecular weight of the phosphorylated form of the rOVA is at least about 100%, about 105%, about 110%, about 120%, about 130%, about 140%, or about 150% of the molecular weight of the full-length rOVA. In some embodiments, the molecular weight of rpOVA is about 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or 110% of the molecular weight of unphosphorylated rOVA.

[0160] In various embodiments, the rpOVA is isolated from the engineered host cell. In some embodiments, the rpOVA can be used as part of a method of manufacturing a food product when isolated from a host cell. In some embodiments, the rpOVA isolated from a host cell improves functionality in a food product compared to an unphosphorylated rOVA, a substitute egg product, or nOVA. In some embodiments, the rpOVA isolated from a host cell provides to a food product at least one egg white characteristic selected from gelling, foaming, whipping, fluffing, binding, springiness, aeration, coating, film forming, emulsification, browning, thickening, texturizing, humectant, clarification, and cohesiveness, wherein the characteristic is improved as compared to recombinant ovalbumin that is not phosphorylated. In some embodiments, the food product is a cake, a pudding, a macaron, a bread, a roll, a pastry, a cracker, a muffin, a scone, a biscuit, a bagel, a pancake, a meringue, a choux pastry a souffle, a scramble, an omelet, a patty, a quiche, a frittata, a mouse, a custard, a popsicle, a frozen desert, an ice cream, or a cookie. [0161] In various embodiments, the recombinant ovalbumin protein may be isolated from the engineered host cell described herein. In further embodiments, a method for manufacturing a food item comprises pooling proteins isolated from the engineered host cell described herein.

PROTEIN COMPOSITIONS

[0162] A further aspect of the present disclosure is a protein composition comprising recombinant ovalbumin protein having a different ratio of diphosphorylated recombinant ovalbumin protein, monophosphorylated recombinant ovalbumin protein, or unphosphorylated recombinant ovalbumin protein than a ratio present in an OVA protein composition obtained from a natural egg-white. OVA derived from native egg whites are predominantly diphosphorylated at Ser-68 and at Ser-344. See, e.g., Kinoshita-Kikuta, etal., "Separation and identification of four distinct serine-phosphorylation states of ovalbumin by P hos-tag affinity electrophoresis." Electrophoresis 33.5 (2012): 849-855; the contents of which are incorporated herein by reference. Further, rOVA that is expressed by fungal cells, e.g., Pichia, which do not express the heterologous or exogenous kinases relevant to the present disclosure, is generally unphosphorylated. Together, an rOVA protein product produced by the cells and method disclosed herein is different from an rOVA protein product obtainable from standard fermentation from fungal cells, e.g., Pichia, or from native egg whites.

[0163] In some embodiments, rOVA may be phosphorylated at multiple sites. In some embodiments, rOVA may be monophosphorylated at one site. In some embodiments, rOVA may be monophosphorylated at Ser-68 (each with respect to SEQ ID NO: 2). In some embodiments, rOVA may be monophosphorylated at Ser-344 (each with respect to SEQ ID NO: 2). In some embodiments, rOVA may be diphosphorylated at two sites. In some embodiments, rOVA may be diphosphorylated at Ser-68 and Ser-344. In some embodiments, rOVA may comprise a homogenous mixture. In certain embodiments, a homogenous rOVA mixture may comprise a single species of rOVA. In certain embodiments, a single species of rOVA may comprise unphosphorylated rOVA. In certain embodiments, a single species of rOVA may comprise monophosphorylated rOVA at Ser-68. In certain embodiments, a single species of rOVA may comprise monophosphorylated rOVA at Ser-344. In certain embodiments, a single species of rOVA may comprise diphosphorylated rOVA at Ser-68 and Ser-344.

[0164] In some embodiments, rOVA may comprise a heterogeneous mixture. In various embodiments, a heterogenous mixture may comprise two or more species of rOVA. In certain embodiments, a heterogenous rOVA mixture may comprise one or more of unphosphorylated rOVA, monophosphorylated rOVA at Ser-68, monophosphorylated rOVA at Ser-344, diphosphorylated rOVA at Ser-68 and Ser-344, rOVA phosphorylated at another site, or any combination thereof. In some embodiments, the rOVA mixture comprises a mixture of rpOVA with varying amounts of rOVA and rpOVA species.

[0165] In some embodiments, the unphosphorylated rOVA is most abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rpOVA is most abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rpOVA at Ser-68 is most abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rpOVA at Ser-344 is most abundant in the rpOVA mixture. In some embodiments, the diphosphorylated rpOVA is most abundant in the rpOVA mixture. In some embodiments, the unphosphorylated rOVA is least abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rpOVA is least abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rpOVA at Ser-68 is least abundant in the rpOVA mixture. In some embodiments, the monophosphorylated rOVA at Ser-344 is least abundant in the rpOVA mixture. In some embodiments, the diphosphorylated rpOVA is most abundant in the rpOVA mixture.

[0166] In some embodiments, the rOVA mixture may comprise a ratio of phosphorylated rpOVA species. In some embodiments, the ratio of phosphorylated rpOVA species may differ from the ratio of phosphorylated OVA found in substitute egg whites. In some embodiments, the ratio of phosphorylated rpOVA species may differ from the ratio of phosphorylated native OVA. In some embodiments, the ratio of phosphorylated rpOVA species may differ from the ratio found in an egg. In some embodiments, the ratio of phosphorylated rpOVA species may differ from the ratio of a protein composition expressed by a recombinant cell that lacks heterologous or exogenous kinase. In some embodiments, the ratio of phosphorylated rpOVA species may differ from the ratio of a protein composition expressed by a recombinant cell that is not later treated with a heterologous or exogenous kinase.

[0167] In certain embodiments, the ratio of rpOVA monophosphorylated at Ser 344 is greater than the protein composition obtained from an egg, nOVA, substitute egg white, or of that expressed by a recombinant cell that lacks heterologous or exogenous kinase. In various embodiments, the ratio of rOVA monophosphorylated at Ser 68 is greater than the protein composition obtained from an egg, nOVA, substitute egg white, or of that expressed by a recombinant cell that lacks heterologous or exogenous kinase. In various embodiments, the ratio of rpOVA diphosphorylated at Ser 68 and at Ser 344 is greater than the protein composition obtained from an egg, nOVA, substitute egg white, or of that expressed by a recombinant cell that lacks heterologous or exogenous kinase. In certain embodiments, the ratio of rpOVA monophosphorylated at Ser 344 is less than the protein composition obtained from an egg, nOVA, substitute egg white, or of that expressed by a recombinant cell that lacks heterologous or exogenous kinase. In some embodiments, the ratio of rpOVA monophosphorylated at Ser 68 is less than the protein composition obtained from an egg, nOVA, substitute egg white, or of that expressed by a recombinant cell that lacks heterologous or exogenous kinase. In some embodiments, the ratio of rpOVA diphosphorylated at Ser 68 and at Ser 344 is less than the protein composition obtained from an egg, nOVA, substitute egg white, or of that expressed by a recombinant cell that lacks heterologous or exogenous kinase [0168] In some embodiments, the ratio of phosphorylated rpOVA to unphosphorylated rOVA is about 1 : 1 to about 1 :20. In some embodiments, the ratio of phosphorylated rpOVA to unphosphorylated rOVA is about 1:20 to about 1:15, about 1:20 to about 1:10, about 1:20 to about 1 :9, about 1 :20 to about 1:8, about 1 :20 to about 1 :7, about 1 :20 to about 1 :6, about 1 :20 to about 1:5, about 1:20 to about 1:4, about 1:20 to about 1:3, about 1:20 to about 1:2, about 1:20 to about 1:1, about 1:15 to about 1:10, about 1:15 to about 1:9, about 1:15 to about 1:8, about 1:15 to about 1:7, about 1:15 to about 1:6, about 1:15 to about 1:5, about 1:15 to about 1:4, about 1:15 to about 1:3, about 1:15 to about 1:2, about 1:15 to about 1:1, about 1:10 to about 1 :9, about 1 : 10 to about 1:8, about 1 : 10 to about 1 :7, about 1 : 10 to about 1 :6, about 1:10 to about 1:5, about 1:10 to about 1:4, about 1:10 to about 1:3, about 1:10 to about 1:2, about 1:10 to about 1:1, about 1:9 to about 1:8, about 1:9 to about 1:7, about 1:9 to about 1:6, about 1:9 to about 1:5, about 1:9 to about 1:4, about 1:9 to about 1:3, about 1:9 to about 1:2, about

[0169] In some embodiments, the ratio of phosphorylated rpOVA to unphosphorylated rOVA is about 1 : 1 to about 20: 1. In some embodiments, the ratio of phosphorylated rpOVA to unphosphorylated rOVA is about 20:1 to about 15:1, about 20:1 to about 10:1, about 20:1 to about 9:1, about 20:1 to about 8:1, about 20:1 to about 7:1, about 20 to about 6:1, about 20:1 to about 5:1, about 20:1 to about 4:1, about 20:1 to about 3:1, about 20:1 to about 2:1, about 20:1 to about 1:1, about 15:1 to about 10:1, about 15:1 to about 9:1, about 15:1 to about 8:1, about 15:1 to about 7:1, about 15:1 to about 6:1, about 15:1 to about 5:1, about 15:1 to about 4:1, about 15:1 to about 3:1, about 15:1 to about 2:1, about 15:1 to about 1:1, about 10:1 to about 9:1, about 10:1 to about 8:1, about 10:1 to about 7:1, about 10:1 to about 6:1, about 10:1 to about 5:1, about 10:1 to about 4:1, about 10:1 to about 3:1, about 10:1 to about 2:1, about 10:1 to about 1:1, about 9:1 to about 8:1, about 9:1 to about 7:1, about 9:1 to about 6:1, about 9:1 to about 5:1, about 9:1 to about 4:1, about 9:1 to about 3:1, about 9:1 to about 2:1, about

4: 1 to about 2:1, about 4: 1 to about 1:1, about 3 : 1 to about 2:1, about 3 : 1 to about 1 : 1, or about 2: 1 to about 1 : 1. the ratio of phosphorylated rpOVA to unphosphorylated rOVA is about 20: 1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of phosphorylated rpOVA to unphosphorylated rOVA is at least about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of phosphorylated rpOVA to unphosphorylated rOVA is at most about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2: 1, or about 1:1. [0170] In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is about 1:1 to about 1:20. In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is about 1 :20 to about 1:15, about 1 :20 to about 1 : 10, about 1 :20 to about 1 :9, about 1 :20 to about 1:8, about 1:20 to about 1:7, about 1:20 to about 1:6, about 1:20 to about 1:5, about 1:20 to about 1 :4, about 1 :20 to about 1 :3, about 1 :20 to about 1 :2, about 1 :20 to about 1 : 1, about 1:15 to about 1:10, about 1:15 to about 1:9, about 1:15 to about 1:8, about 1:15 to about 1:7, about 1:15 to about 1:6, about 1:15 to about 1:5, about 1:15 to about 1:4, about 1:15 to about 1:3, about 1:15 to about 1:2, about 1:15 to about 1:1, about 1:10 to about 1:9, about 1:10 to about 1:8, about 1:10 to about 1:7, about 1:10 to about 1:6, about 1:10 to about 1:5, about 1:10 to about 1:4, about 1:10 to about 1:3, about 1:10 to about 1:2, about 1:10 to about 1:1, about 1:9 to about 1:8, about 1 :9 to about 1 :7, about 1 :9 to about 1 :6, about 1 :9 to about 1:5, about 1 :9 to about 1:4, about 1:9 to about 1:3, about 1:9 to about 1:2, about 1:9 to about 1:1, about 1:8 to about 1:7, about 1:8 to about 1:6, about 1:8 to about 1:5, about 1:8 to about 1:4, about 1:8 to about 1:3, about 1:8 to about 1:2, about 1:8 to about 1:1, about 1:7 to about 1:6, about 1:7 to about 1:5, about 1:7 to about 1:4, about 1:7 to about 1:3, about 1:7 to about 1:2, about 1:7 to about 1:1, about 1:6 to about 1:5, about 1:6 to about 1:4, about 1:6 to about 1:3, about 1:6 to about 1:2, about 1:6 to about 1:1, about 1:5 to about 1:4, about 1:5 to about 1:3, about 1:5 to about 1:2, about 1:5 to about 1:1, about 1:4 to about 1:3, about 1:4 to about 1:2, about 1:4 to about 1:1, about 1:3 to about 1:2, about 1:3 to about 1:1, or about 1:2 to about 1:1. In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1 :6, about 1:5, about 1 :4, about 1 :3, about 1 :2, or about 1 : 1. In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is at least about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, or about 1:2. In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is at most about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1.

[0171] In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is about 1 : 1 to about 20: 1. In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is about 20: 1 to about 15:1, about 20: 1 to about 10:1, about 20: 1 to about 9:1, about 20: 1 to about 8:1, about 20 : 1 to about 7:1, about 20 to about 6:1, about 20 : 1 to about 5:1, about 20 : 1 to about 4:1, about 20:1 to about 3:1, about 20:1 to about 2:1, about 20:1 to about 1:1, about 15:1 to about 10:1, about 15:1 to about 9:1, about 15:1 to about 8:1, about 15:1 to about 7:1, about 15:1 to about 6:1, about 15:1 to about 5:1, about 15:1 to about 4:1, about 15:1 to about 3:1, about 15:1 to about 2:1, about 15:1 to about 1:1, about 10:1 to about 9:1, about 10:1 to about 8:1, about 10:1 to about 7:1, about 10:1 to about 6:1, about 10:1 to about 5:1, about 10:1 to about 4:1, about 10:1 to about 3:1, about 10:1 to about 2:1, about 10:1 to about 1:1, about 9:1 to about 8:1, about 9: 1 to about 7: 1, about 9: 1 to about 6:1, about 9: 1 to about 5:1, about 9: 1 to about 4:1, about 9:1 to about 3:1, about 9:1 to about 2:1, about 9:1 to about 1:1, about 8:1 to about 7:1, about 8:1 to about 6:1, about 8:1 to about 5:1, about 8:1 to about 4:1, about 8:1 to about 3:1, about 8:1 to about 2:1, about 8:1 to about 1:1, about 7:1 to about 6:1, about 7:1 to about 5:1, about 7:1 to about 4:1, about 7:1 to about 3:1, about 7:1 to about 2:1, about 7:1 to about 1:1, about 6:1 to about 5:1, about 6:1 to about 4:1, about 6:1 to about 3:1, about 6:1 to about 2:1, about 6:1 to about 1:1, about 5:1 to about 4:1, about 5:1 to about 3:1, about 5:1 to about 2:1, about 5:1 to about 1:1, about 4:1 to about 3:1, about 4:1 to about 2:1, about 4:1 to about 1:1, about 3:1 to about 2:1, about 3:1 to about 1:1, or about 2:1 to about 1:1. In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2 : 1 , or about 1 : 1. In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is at least about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2: 1, or about 1 : 1. In some embodiments, the ratio of monophosphorylated rpOVA at Ser-68 to monophosphorylated rpOVA at Ser-344 is at most about 20: 1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1.

[0172] In some embodiments, the ratio of diphosphorylated rpOVA to unphosphorylated rOVA is about 1:1 to about 1:20. In some embodiments, the ratio of diphosphorylated rpOVA to unphosphorylated rOVA is about 1 :20 to about 1:15, about 1 :20 to about 1:10, about 1 :20 to about 1 :9, about 1 :20 to about 1:8, about 1 :20 to about 1 :7, about 1 :20 to about 1 :6, about 1 :20 to about 1:5, about 1:20 to about 1:4, about 1:20 to about 1:3, about 1:20 to about 1:2, about 1:20 to about 1:1, about 1:15 to about 1:10, about 1:15 to about 1:9, about 1:15 to about 1:8, about 1:15 to about 1:7, about 1:15 to about 1:6, about 1:15 to about 1:5, about 1:15 to about 1:4, about 1:15 to about 1:3, about 1:15 to about 1:2, about 1:15 to about 1:1, about 1:10 to about 1 :9, about 1 : 10 to about 1:8, about 1 : 10 to about 1 :7, about 1 : 10 to about 1 :6, about 1:10 to about 1:5, about 1:10 to about 1:4, about 1:10 to about 1:3, about 1:10 to about 1:2, about 1:10 to about 1:1, about 1:9 to about 1:8, about 1:9 to about 1:7, about 1:9 to about 1:6, about 1:9 to about 1:5, about 1:9 to about 1:4, about 1:9 to about 1:3, about 1:9 to about 1:2, about

[0173] In some embodiments, the ratio of diphosphorylated rpOVA to unphosphorylated rOVA is about 1 : 1 to about 20: 1. In some embodiments, the ratio of diphosphorylated rpOVA to unphosphorylated rOVA is about 20: 1 to about 15:1, about 20: 1 to about 10:1, about 20: 1 to about 9:1, about 20:1 to about 8:1, about 20:1 to about 7:1, about 20 to about 6:1, about 20:1 to about 5:1, about 20:1 to about 4:1, about 20:1 to about 3:1, about 20:1 to about 2:1, about 20:1 to about 1:1, about 15:1 to about 10:1, about 15:1 to about 9:1, about 15:1 to about 8:1, about 15:1 to about 7:1, about 15:1 to about 6:1, about 15:1 to about 5:1, about 15:1 to about 4:1, about 15:1 to about 3:1, about 15:1 to about 2:1, about 15:1 to about 1:1, about 10:1 to about 9:1, about 10:1 to about 8:1, about 10:1 to about 7:1, about 10:1 to about 6:1, about 10:1 to about 5:1, about 10:1 to about 4:1, about 10:1 to about 3:1, about 10:1 to about 2:1, about 10:1 to about 1:1, about 9:1 to about 8:1, about 9:1 to about 7:1, about 9:1 to about 6:1, about 9:1 to about 5:1, about 9:1 to about 4:1, about 9:1 to about 3:1, about 9:1 to about 2:1, about

7:1 to about 6:1, about 7:1 to about 5:1, about 7:1 to about 4:1, about 7:1 to about 3:1, about 7:1 to about 2:1, about 7:1 to about 1:1, about 6:1 to about 5:1, about 6:1 to about 4:1, about

4: 1 to about 2:1, about 4: 1 to about 1:1, about 3 : 1 to about 2:1, about 3 : 1 to about 1 : 1, or about

2:1 to about 1:1. the ratio of diphosphorylated rpOVA to unphosphorylated rOVA is about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2: 1, or about 1 : 1. In some embodiments, the ratio of diphosphorylated rpOVA to unphosphorylated rOVA is at least about 20: 1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of diphosphorylated rpOVA to unphosphorylated rOVA is at most about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1.

[0174] In some embodiments, the ratio of diphosphorylated rpOVA to monophosphorylated rOVA is about 1:1 to about 1:20. In some embodiments, the ratio of diphosphorylated rpOVA to monophosphorylated rOVA is about 1:20 to about 1:15, about 1:20 to about 1:10, about 1:20 to about 1:9, about 1:20 to about 1:8, about 1:20 to about 1:7, about 1:20 to about 1:6, about 1:20 to about 1:5, about 1:20 to about 1:4, about 1:20 to about 1:3, about 1:20 to about 1:2, about 1:20 to about 1:1, about 1:15 to about 1:10, about 1:15 to about 1:9, about 1:15 to about 1:8, about 1:15 to about 1:7, about 1:15 to about 1:6, about 1:15 to about 1:5, about 1:15 to about 1:4, about 1:15 to about 1:3, about 1:15 to about 1:2, about 1:15 to about 1:1, about 1:10 to about 1:9, about 1:10 to about 1:8, about 1:10 to about 1:7, about 1:10 to about 1:6, about 1:10 to about 1:5, about 1:10 to about 1:4, about 1:10 to about 1 :3, about 1 : 10 to about 1 :2, about 1 : 10 to about 1:1, about 1 :9 to about 1:8, about 1 :9 to about 1:7, about 1:9 to about 1:6, about 1:9 to about 1:5, about 1:9 to about 1:4, about 1:9 to about

1:2, about 1:3 to about 1:1, or about 1:2 to about 1:1. In some embodiments, the ratio of diphosphorylated rpOVA to monophosphorylated rOVA is about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1. In some embodiments, the ratio of diphosphorylated rpOVA to monophosphorylated rOVA is at least about 1 :20, about 1:15, about 1 : 10, about 1 :9, about 1:8, about 1 :7, about 1 :6, about 1:5, about 1:4, about 1:3, or about 1:2. In some embodiments, the ratio of diphosphorylated rpOVA to monophosphorylated rOVA is at most about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1.

[0175] In some embodiments, the ratio of diphosphorylated rpOVA to monophosphorylated rpOVA is about 1:1 to about 20:1. In some embodiments, the ratio of diphosphorylated rpOVA to monophosphorylated rpOVA is about 20:1 to about 15:1, about 20:1 to about 10:1, about 20:1 to about 9:1, about 20:1 to about 8:1, about 20:1 to about 7:1, about 20 to about 6:1, about 20 : 1 to about 5:1, about 20 : 1 to about 4:1, about 20 : 1 to about 3:1, about 20: 1 to about 2:1, about 20: 1 to about 1:1, about 15:1 to about 10:1, about 15:1 to about 9:1, about 15:1 to about 8:1, about 15:1 to about 7:1, about 15:1 to about 6:1, about 15:1 to about 5:1, about 15:1 to about 4:1, about 15:1 to about 3:1, about 15:1 to about 2:1, about 15:1 to about 1:1, about 10:1 to about 9:1, about 10:1 to about 8:1, about 10:1 to about 7:1, about 10:1 to about 6:1, about 10:1 to about 5:1, about 10:1 to about 4:1, about 10:1 to about 3:1, about 10:1 to about 2:1, about 10:1 to about 1:1, about 9:1 to about 8:1, about 9:1 to about 7:1, about 9:1 to about 6:1, about 9:1 to about 5:1, about 9:1 to about 4:1, about 9:1 to about 3:1, about 9:1 to about 2:1, about 9:1 to about 1:1, about 8:1 to about 7:1, about 8:1 to about 6:1, about 8:1 to about 5:1, about 8:1 to about 4:1, about 8:1 to about 3:1, about 8:1 to about 2:1, about 8:1 to about 1:1, about 7:1 to about 6:1, about 7:1 to about 5:1, about 7:1 to about 4:1, about 7:1 to about 3:1, about 7:1 to about 2:1, about 7:1 to about 1:1, about 6:1 to about 5:1, about 6:1 to about 4:1, about 6:1 to about 3:1, about 6:1 to about 2:1, about 6:1 to about 1:1, about 5:1 to about 4:1, about 5:1 to about 3:1, about 5:1 to about 2:1, about 5:1 to about 1:1, about 4:1 to about 3:1, about 4:1 to about 2:1, about 4:1 to about 1:1, about 3:1 to about 2:1, about 3:1 to about 1:1, or about 2:1 to about 1:1. the ratio of diphosphorylated rpOVA to monophosphorylated rpOVA is about 20: 1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of diphosphorylated rpOVA to monophosphorylated rpOVA is at least about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, the ratio of diphosphorylated rpOVA to monophosphorylated rpOVA is at most about 20: 1, about 15: 1, about 10: 1, about 9: 1, about 8: 1, about 7:1, about 6: 1, about 5:1, about 4:1, about 3: 1, about 2: 1, or about 1 : 1.

[0176] In some embodiments, rOVA is phosphorylated by a kinase. In some embodiments, rOVA is phosphorylated by a serine/threonine kinase. In some embodiments, rOVA is phosphorylated by a tyrosine kinase. In some embodiments, the kinase is a heterologous or exogenous kinase protein. In some embodiments, the kinase recognizes a recognition motif comprising a S-X-E/pS motif. In some embodiments, the kinase recognizes a recognition motif that comprises Glu at a +2 position. In some embodiments, the kinase can have an amino acid sequence from any species. In various embodiments, the FAM20 kinase can have an amino acid sequence comprising FAM20A. In various embodiments, the FAM20 kinase can have an amino acid sequence comprising FAM20A from any species. In certain embodiments, the FAM20 kinase can have an amino acid sequence comprising FAM20C. In certain embodiments, the FAM20 kinase can have an amino acid sequence comprising FAM20C from any species. In some embodiments, the FAM20C kinase can have an amino acid sequence comprising any FAM20C isoform. In some embodiments, the FAM20C kinase can have an amino acid sequence comprising any FAM20C isoform from any species.

[0177] In some embodiments, the exogenous kinase protein is selected from the genera: Homo, Drosophila, Danio, Branchiostoma, Stronglycocentrotus, Caenorhabditis, Hydra, Amphimedon, Avian, Reptile, any other egg-laying animal, or any combination thereof. In some embodiments, the kinase can have an amino acid sequence of FAM20 from a bird, a reptile, or another egg-laying species. In some embodiments, the FAM20 having an amino acid sequence from an avian can be selected from the group consisting of poultry, fowl, waterfowl, game bird, chicken, quail, turkey, duck, ostrich, goose, gull, guineafowl, pheasant, emu, and any combination thereof. In some embodiments, the FAM20 can have an amino acid sequence derived from a single species, such as Gallus gallus domesticus. In some embodiments, the heterologous or exogenous kinase protein is from G. gallus.

[0178] In certain embodiments, the kinase protein comprises an amino acid sequence with at least 70% homology to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID

NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID

NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID

NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID

NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID

NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID

SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112,

SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117,

SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122,

SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127,

SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132,

SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137,

SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141. In various embodiments, the heterologous or exogenous kinase protein comprises an amino acid sequence of SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO:

106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO:

111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO:

116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO:

121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO:

126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO:

131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO:

136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141. In some embodiments, the heterologous or exogenous kinase protein comprises a nucleotide sequence with at least 70% homology to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108,

SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113,

SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118,

SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123,

SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128,

SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133,

SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138,

SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141 and maintains the enzymatic activity of a kinase expressed by SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID

NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID

NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID

NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID

NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID

NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID

NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID

NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID

NO: 140, or SEQ ID NO: 141. In various embodiments, the heterologous or exogenous kinase protein comprises a nucleotide sequence of SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID

NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID

NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID

NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID

NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID

NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141. In these embodiments, an amino acid with at least 70% homology to SEQ ID NO: 80 maintains the enzymatic activity of a kinase expressed by the corresponding SEQ ID NO: 80; an amino acid with at least 70% homology to SEQ ID NO: 85 maintains the enzymatic activity of a kinase expressed by the corresponding SEQ ID NO: 85; an amino acid with at least 70% homology to SEQ ID NO: 97 maintains the enzymatic activity of a kinase expressed by the corresponding SEQ ID NO: 97; and so forth.

[0179] In some embodiments, the FAM20 can have an amino acid sequence derived from two or more species, and as such can be a hybrid. In some embodiments, the kinase protein comprises a mixture of at least two or more distinct heterologous or exogenous kinase proteins. [0180] In some embodiments, the rpOVA protein can be recombinantly expressed in yeast, filamentous fungi or a bacterium. In some embodiments, rpOVA protein is recombinantly expressed in a Pichia species (Komagataella phaffii and Komagataella pasloris). a Saccharomyces species, a Trichoderma species, a Pseudomonas species or an E. coli species. In some embodiments, the protein composition is generated in an engineered host cell that recombinantly co-expresses a heterologous or exogenous ovalbumin protein and at least one heterologous or exogenous kinase protein. In some embodiments, the protein composition is generated in an engineered host cell that recombinantly expresses a heterologous or exogenous ovalbumin protein and is treated with at least one heterologous or exogenous kinase protein after the rOVA is isolated from the host cell.

[0181] In some embodiments, the rpOVA can have an amino acid sequence from any species. For example, the rpOVA can have an amino acid sequence of OVA from a bird, a reptile, or another egg-laying species. In some embodiments, the rpOVA having an amino acid sequence from an avian can be selected from the group consisting of poultry, fowl, waterfowl, game bird, chicken, quail, turkey, duck, ostrich, goose, gull, guineafowl, pheasant, emu, and any combination thereof. In some embodiments, the rpOVA can have an amino acid sequence derived from a single species, such as Gallus gallus domesticus. In some embodiments, the rpOVA can have an amino acid sequence derived from two or more species, and as such can be a hybrid. Illustrative OVA amino acid sequences contemplated herein are provided in Table 1 below as SEQ ID NOs: 1-76.

[0182] In some embodiments, the rpOVA comprises post-translation modifications. In some embodiments, the post-translation modification comprises glycosylation. In some embodiments, the rOVA protein is glycosylated before being phosphorylated. In some cases, the rOVA is not phosphorylated until the rOVA is glycosylated. In some embodiments, the glycosylation comprises a carbohydrate attached to an amino acid residue of rOVA. In some embodiments, the glycosylation is N-linked glycosylation. In some embodiments, the glycosylation is O-linked glycosylation. In some embodiments, the glycosylation is C-linked glycosylation. In some embodiments, the glycosylation is S-linked glycosylation. In some embodiments, the glycosylation is glypiation. In some embodiments, the glycosylation is phosphoglycosylation. In some embodiments, the glycosylation moiety is covalently attached to one or more amino acids in the rOVA protein. In some embodiments, the glycosylation moiety comprises a carbohydrate. In some embodiments, the glycosylation moiety comprises a glycan. In some embodiments, the glycosylation moiety comprises at least one mannose moiety. In some embodiments, the glycosylation moiety comprises at least one N- acetylglucosamine moiety. In some embodiments, the glycosylation moiety comprises at least one mannose moiety or at least one N-acetylglucosamine moiety. In some embodiments, the glycosylation moiety comprises at least one mannose moiety and at least one N- acetyl glucosamine moiety.

[0183] In some embodiments, an unglycosylated rOVA is unphosphorylated. In some embodiments, a glycosylated rOVA is phosphorylated. In some embodiments, a glycosylated rOVA is unphosphorylated. In some embodiments, a phosphorylated recombinant ovalbumin is more stable than an unphosphorylated recombinant ovalbumin. In some embodiments, the rpOVA is more stable than an unphosphorylated rOVA.

[0184] In some embodiments, the rpOVA described herein may be secreted from the one or more host cells. In some embodiments, the rpOVA described herein may be phosphorylated prior to being secreted from the one or more host cells. The secreted rpOVA may be isolated and purified by methods such as centrifugation, fractionation, filtration, ion exchange chromatography, affinity purification and other methods for separating protein from cells, liquid and solid media components and other cellular products and byproducts. In some embodiments, rpOVA is produced in a Pichia Sp. and secreted from the host cells into the culture media. The secreted rpOVA is then separated from other media components for further use.

[0185] In some embodiments, the rOVA described herein may be secreted from the one or more host cells. In some embodiments, the rOVA described herein may be unphosphorylated when it is secreted from the one or more host cells. The secreted rOVA may be isolated and purified by methods such as centrifugation, fractionation, filtration, ion exchange chromatography, affinity purification and other methods for separating protein from cells, liquid and solid media components and other cellular products and byproducts. In some embodiments, rOVA is produced in a Pichia Sp. and secreted from the host cells into the culture media. The secreted rOVA is then separated from other media components and can be phosphorylated in vitro by exogenous kinase protein. In some embodiments, the rOVA is phosphorylated by an exogenous kinase after the rOVA is secreted from the host cell. In certain embodiments, the rOVA is phosphorylated by an exogenous kinase after the rOVA is isolated from the host cell. In various embodiments, the rpOVA treated with an exogenous kinase is used in downstream applications.

[0186] In some embodiments, the rpOVA mixture comprising various ratios of unphosphorylated, monophosphorylated, and diphosphorylated rOVA described herein may be secreted from the one or more host cells. In some embodiments, the rpOVA mixture is secreted from the host cell. The secreted rpOVA mixture may be isolated and purified by methods such as centrifugation, fractionation, filtration, ion exchange chromatography, affinity purification and other methods for separating protein from cells, liquid and solid media components and other cellular products and byproducts. In some embodiments, the rpOVA mixture is produced in a Pichia Sp. and secreted from the host cells into the culture media. The secreted rpOVA mixture is then separated from other media components for further use. In certain embodiments, the rpOVA mixture is further treated with an exogenous kinase. In various embodiments, the rpOVA mixture treated with an exogenous kinase is used in downstream applications.

[0187] The molecular weight of rpOVA may be different as compared to nOVA or unphosphorylated rOVA. The molecular weight of the protein may be less than the molecular weight of nOVA or unphosphorylated rOVA. In certain embodiments, the molecular weight of an rpOVA may be between 40kDa and 55kDa. In some cases, an rOVA with glycosylation has a different molecular weight, such as compared to a native OVA (as produced by an avian host species) or as compared to a host cell that glycosylates the rOVA, such as where the rOVA includes N-linked mannosylation but does not phosphorylate the rOVA. In some cases, the molecular weight of rpOVA is greater than the molecular weight of the rOVA that is completely devoid of post-translational modifications or an rOVA that lacks all forms of N- linked glycosylation or that is unphosphorylated.

[0188] In some embodiments, the molecular weight of rpOVA is at least the molecular weight of the unphosphorylated rOVA. In some embodiments, the molecular weight of rpOVA is greater than the molecular weight of the unphosphorylated rOVA. In some embodiments, the molecular weight of the phosphorylated form of the rOVA is at least about 100%, about 105%, about 110%, about 120%, about 130%, about 140%, or about 150% of the molecular weight of the full-length rOVA. In some embodiments, the molecular weight of rpOVA is about 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or 110% of the molecular weight of unphosphorylated rOVA.

[0189] The phosphate groups also lower the isoelectric point of OVA, which can impact the nature of the gel formed upon OVA denaturation. At neutral pH, gels formed from an OVA with a lower isoelectric point are expected to exhibit greater translucence and altered viscoelasticity (J. Agric. Food Chem. 20: 102, 50, 1636-1642). These two features can impact the appearance and functional properties of food products.

[0190] In some embodiments, the rpOVA protein composition described herein provides an equivalent functionality or an improved functionality in a food product. In various embodiments, the rpOVA protein composition disclosed herein provides to a food product at least one egg white characteristic selected from gelling, foaming, whipping, fluffing, binding, springiness, aeration, coating, film forming, emulsification, browning, thickening, texturizing, humectant, clarification, and cohesiveness, wherein the characteristic is the same or is improved as compared to recombinant ovalbumin that is not phosphorylated. In certain embodiments, the food product may be a cake, a pudding, a macaron, a bread, a roll, a pastry, a cracker, a muffin, a scone, a biscuit, a bagel, a pancake, a meringue, a choux pastry a souffle, a scramble, an omelet, a patty, a quiche, a frittata, a mouse, a custard, a popsicle, a frozen desert, an ice cream, or a cookie, or any other food product.

FOOD PRODUCTS

[0191] In embodiments, the rpOVA improves functionality in a food product. In embodiments, the rpOVA comprises a mixture that improves functionality in a food product. In some cases, the rpOVA provides to a food product at least one egg white characteristic selected from gelling, foaming, whipping, fluffing, binding, springiness, aeration, coating, film forming, emulsification, browning, thickening, texturizing, humectant, clarification, and cohesiveness, wherein the characteristic is improved as compared to an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In various cases, the food product is a cake, a pudding, a macaron, a bread, a roll, a pastry, a cracker, a muffin, a scone, a biscuit, a bagel, a pancake, a meringue, a choux pastry a souffle, a scramble, an omelet, a patty, a quiche, a frittata, a mouse, a custard, a popsicle, a frozen desert, an ice cream, or a cookie.

[0192] The rpOVA containing compositions herein can provide one or more functional features to food ingredients and food products, including features similar to features found in native (egg-derived) OVA.

[0193] In some embodiments, the rpOVA provides a nutritional feature such as protein content, protein fortification and amino acid content to a food ingredient or food product. The nutritional feature provided by rpOVA in the composition may be comparable or substantially similar to an egg, egg white, or native OVA (nOVA) or recombinant ovalbumin that is not phosphorylated. The nutritional feature provided by rpOVA in the composition may be better than that provided by a native whole egg or native egg white. In some cases, rpOVA provides the one or more functional features of egg-white in absence of any other egg-white proteins, including features similar to features found in native (egg-derived) OVA.

[0194] In some embodiments, the rpOVA compositions disclosed herein can provide foaming and foam capacity to a composition. For example, rpOVA can be used for forming a foam to use in baked products, such as cakes, for meringues and other foods where rpOVA can replace egg white to provide foam capacity. In some cases, rpOVA provides foaming and foam capacity of egg-white in absence of any other egg-white proteins.

[0195] A composition comprising rpOVA may have a foam height greater than a foam height of an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, a composition comprising rpOVA may have a foam height of about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120: 1%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 20:10%, 210%, 220: 1%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450%, or 500% relative to an egg white, nOVA compositions, unphosphorylated rOVA, or a substitute egg white. In some cases, a composition comprising rpOVA may have a foam height of up to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120: 1%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 20: 10%, 210%, 220: 1%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450%, or 500% relative to an egg white, nOVA compositions, unphosphorylated rOVA, or a substitute egg white. Substitute egg whites may include products such as aquafaba, chia seeds, flax seeds, starches; apple sauce, banana puree; condensed milk, etc. which are commonly used as egg white substitutes.

[0196] In some embodiments, the composition provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) may have a foam height greater than a foam height of an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, the composition provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) may have a foam height of about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120: 1%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 20: 10%, 210%, 220: 1%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450%, or 500% relative to an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, the composition provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) may have a foam height of up to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120: 1%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 20: 10%, 210%, 220: 1%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450%, or 500% relative to the corresponding OVA of an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. Substitute egg whites may include products such as aquafaba, chia seeds, flax seeds, starches; apple sauce, banana puree; condensed milk, etc. which are commonly used as egg white substitutes.

[0197] In some embodiments, the composition provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) may have a foam stability greater than a foam stability of an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, the composition provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) may have a foam stability of about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120: 1%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 20: 10%, 210%, 220: 1%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450%, or 500% relative to an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, the composition provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) may have a foam stability of up to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120: 1%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 20: 10%, 210%, 220: 1%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450%, or 500% relative to an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some embodiments, foam stability may be calculated by measuring drainage of a foamed solution. The drainage may be measured in 10-minute increments for 30 minutes to gather data for foam stability. The drained volume after 30 minutes may be compared to the initial liquid volume (5 mL) for instance, foam Stability (%): (Initial volume - drained volume) / initial volume* 100.

[0198] In some embodiments, the composition provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) may have a foam stability greater than a foam stability of the corresponding OVA an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, the composition provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) may have a foam stability of about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120: 1%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 20: 10%, 210%, 220: 1%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450%, or 500% relative to the an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, the composition provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) may have a foam stability of up to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120: 1%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 20: 10%, 210%, 220: 1%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450%, or 500% relative to the corresponding OVA of an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some embodiments, foam stability may be calculated by measuring drainage of a foamed solution. The drainage may be measured in 10-minute increments for 30 minutes to gather data for foam stability. The drained volume after 30 minutes may be compared to the initial liquid volume (5 mL) for instance, foam Stability (%): (Initial volume - drained volume) / initial volume* 100.

[0199] In some embodiments, the composition disclosed herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) may have a foam capacity greater than a foam capacity of an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, a composition comprising rpOVA may have a foam capacity of about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120: 1%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 20: 10%, 210%, 220: 1%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450%, or 500% relative to an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, a composition comprising rpOVA may have a foam capacity of up to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120: 1%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 20:10%, 210%, 220: 1%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450%, or 500% relative to an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. Foam capacity may be determined by measuring the initial volume of foam following the whipping and compare against the initial volume of 5mL. Foam Capacity (%) = (volume of foam / initial volume)* 100.

[0200] In some embodiments, the composition disclosed herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) may have a foam capacity greater than a foam capacity of the corresponding OVA of an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, a composition comprising rpOVA may have a foam capacity of about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120: 1%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 20: 10%, 210%, 220: 1%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450%, or 500% relative to the corresponding OVA of an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, a composition comprising rpOVA may have a foam capacity of up to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120: 1%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 20: 10%, 210%, 220: 1%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450%, or 500% relative to the corresponding OVA of an egg, egg white, native OVA

-I l l- (nOVA), or recombinant ovalbumin that is not phosphorylated. Foam capacity may be determined by measuring the initial volume of foam following the whipping and compare against the initial volume of 5mL. Foam Capacity (%) = (volume of foam / initial volume)* 100.

[0201] In some embodiments, the liquid composition disclosed herein comprising the rpOVA mixture may foam faster than a composition comprising an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, an rOVA composition foams at least 1%, 5%, 10%, 15%, 20: 1%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, faster relative to an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, an rpOVA composition foams up to 1%, 5%, 10%, 15%, 20: 1%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% faster relative to an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated.

[0202] In some embodiments, the liquid composition disclosed herein comprising the rpOVA mixture may foam faster than the corresponding OVA of an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, the rpOVA mixture containing composition foams at least 1%, 5%, 10%, 15%, 20: 1%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, faster than the corresponding OVA of an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, the rpOVA mixture containing composition foams up to 1%, 5%, 10%, 15%, 20: 1%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% faster than the corresponding OVA of an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated.

[0203] In some embodiments, the liquid composition disclosed herein comprising the rpOVA mixture may form a homogenous solution of the rpOVA at a higher concentration than the corresponding rOVA containing composition that does not comprise any rpOVA. In some cases, the rpOVA mixture has higher solubility than the rOVA in a composition that does not comprise any rpOVA, or as compared to an egg, egg white, or native OVA (nOVA).

[0204] In some embodiments, the composition disclosed herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) may have a gel strength greater than a gel strength of an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, the rpOVA mixture containing composition may have a gel strength within the range from 100 g to 1500 g, from 500 g to 1500 g, or from 700 g to 1500 g. In some cases, the rpOVA mixture containing composition has a gel strength of about or at least 10, 50, 100, 150, 20: 10, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 120: 10, 1250, 1300, 1350, 1400, 1450, or 1500 g. In some cases, the rpOVA mixture containing composition has a gel strength of up to 10, 50, 100, 150, 20: 10, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 120: 10, 1250, 1300, 1350, 1400, 1450, or 1500 g. In some cases, the rpOVA mixture containing composition has a gel strength of about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% relative to an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, an rpOVA composition has a gel strength of up to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% relative to an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated.

[0205] In some embodiments, the composition disclosed herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) may have a reduced gel strength than the gel strength of an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, the rpOVA mixture containing composition may have a gel strength within the range from 100 g to 1500 g, from 500 g to 1500 g, or from 700 g to 1500 g. In some cases, the rpOVA mixture containing composition has a gel strength of about or less than 10, 50, 100, 150, 20: 10, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 120: 10, 1250, 1300, 1350, 1400, 1450, or 1500 g. In some cases, the rpOVA mixture containing composition has a gel strength of up to 10, 50, 100, 150, 20: 10, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 120: 10, 1250, 1300, 1350, 1400, 1450, or 1500 g. In some cases, the rpOVA mixture containing composition has a gel strength of about or less than 10%, 20:1%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% relative to an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, the rOVA mixture containing composition has a gel strength of up to 5%, 10%, 15%, 20: 1%, 25%, 30%, 35%, 40%, 45%, or 50% relative to an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. In some cases, the rpOVA mixture containing composition has no gel strength. In some cases, the rpOVA mixture containing composition provides not gelation.

[0206] In some embodiments, the composition provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) can provide structure, texture or a combination of structure and texture. In some embodiments, the rpOVA mixture is added to a food ingredient or food product for baking and the rpOVA mixture provides structure, texture or a combination of structure and texture to the baked product. In some embodiments, the rpOVA mixture can be used in such baked products in place of an egg, egg white, native OVA (nOVA), or recombinant ovalbumin that is not phosphorylated. The addition of the rpOVA mixture to baked products can also provide protein fortification to improve the nutritional content. In some embodiments, the rpOVA mixture is used in a baked product in an amount between 0.1% and 25% on a weight/weight or weight/volume basis. In some embodiments, the rpOVA mixture is used in a baked product in an amount between 0.1% and 5%. In some cases, the rpOVA mixture provides the structure and/or texture of egg-white in absence of any other egg-white proteins.

[0207] In some embodiments, the composition provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) can be compatible with gluten formation, such that the rpOVA mixture can be used where gluten formation provides structure, texture and/or form to a food ingredient or food product.

[0208] Illustrative baked products in which the rpOVA mixture can be used as an ingredient include, but are not limited to cake, a pudding, a macaron, a bread, a roll, a pastry, a cracker, a muffin, a scone, a biscuit, a bagel, a pancake, a meringue, a choux pastry, a souffle, a scramble, an omelet, a patty, a quiche, a frittata, a mouse, a custard, a popsicle, a frozen desert, an ice cream, or a cookie. For example, the rpOVA mixture can be used as an ingredient to make cakes such as pound cake, sponge cake, yellow cake, or angel food cake, where such cakes do not contain any native egg white, native whole egg or native egg protein. Along with the rpOVA mixture, baked products may contain additional ingredients such as flour, sweetening agents, gum, hydrocolloids, starches, fibers, flavorings (such as flavoring extracts) and other protein sources. In some embodiments, a baked product may comprise the rpOVA mixture and at least one fat or oil, at least one grain starch, and optionally at least one sweetener. Grain starch for use in such compositions include flours such as wheat flour, rice flour, corn flour, millet flour, spelt flour, and oat flour, and starches such as from com, potato, sorghum, and arrowroot. Oil and fat for use in such compositions include plant-derived oils and fats, such as olive oil, corn oil, avocado oil, nut oils (e.g., almond, walnut and peanut) and safflower oil. R In some embodiments, the OVA mixture may provide such baked goods with at least one characteristic of an egg white such as binding, springiness, aeration, browning, texturizing, humectant, and cohesiveness of the baked product. In some cases, the baked product does not comprise any natural egg white or natural egg, and/or does not include any other egg white derived or egg white related proteins except full length or clipped forms of rpOVA. In some cases, the rpOVA mixture is provided to the baked composition as an ingredient, such as starting with a concentrate, isolate or powder form of the rpOVA mixture. In some cases, the rpOVA mixture provided as an ingredient for baked products is at a pH range between about 3.5 and 7.0. In some cases, a sweetener is included in the baked product such as a sugar, plant- derived syrup, honey or sugar- substitute, e.g., an artificial sweetener.

[0209] In some embodiments, the compositions provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) can also be used to prepare egg-less food products, such as food products made where native whole egg or native egg white is a primary or featured ingredient such as scramble, omelet, patty, souffle, quiche, and frittata. In some embodiments, the rpOVA mixture provides one or more functional features to the preparation including foaming, coagulation, binding, structure, texture, film-formation, nutritional profile, absence of cholesterol (i.e., cholesterol free), and protein fortification. Such egg-less preparations can be vegan, vegetarian, halal, or kosher, or a combination thereof. An egg-less preparation (also referred to as an egg-white substitute) may comprise the rpOVA mixture and at least one fat or oil, a polysaccharide or polysaccharide-containing ingredient, and a starch. In some cases, the egg-less preparation may also include a flavoring agent (such as to provide a salty, sulfur-like, or umami flavor), and/or a coloring agent (for example to provide yellow-like or off-white color to the baked product). In some cases, the inclusion of the rpOVA mixture in the egg-less preparation provides a characteristic of natural (native) egg white such as hardness, adhesiveness, fracturability, cohesiveness, gumminess, and chewiness when the composition is heated or cooked. Illustrative polysaccharide or polysaccharide-containing ingredients for such compositions include but are not limited to gellan gum, sodium alginate, and psyllium. Oil and fat for use in such compositions include plant-derived oils and fats, such as olive oil, corn oil, avocado oil, and safflower oil.

[0210] In some embodiments, the compositions provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) can be used for a processed meat product or meat-like product, or for fish-like or shell-fish-like products. In such products, the rpOVA mixture can provide one or more functional characteristics such as protein content and protein supplementations as well as binding, and texturizing properties. Illustrative meat and meat-like products include burger, patty, sausage, hot dog, sliced deli meat, jerky, bacon, nugget and ground meat-like mixtures. Meat-like products can resemble beef, pork, chicken, lamb, and other edible and consumed meats for humans and for other animals. Fish-like and shell-fish like products can resemble, for example, fish cakes, crab cakes, shrimp, shrimp balls, fish sticks, seafood meat, crab meat, fish filets and clam strips. In some embodiments, the rpOVA mixture is present in an amount between about 0.1% and 30% w/w/ or w/v in the meat or meat-like product. In some embodiments, the rpOVA mixture is used for a meat-like product (also referred to as a meat-analog and includes at least one fat or oil; and a plant-derived protein. Oil and fat for use in such compositions include plant-derived oils and fats, such as olive oil, corn oil, avocado oil, and safflower oil. Plant- derived proteins for use in meat analogs include soy protein, nut proteins, pea protein, lentil and other pulse proteins, and whey protein. In some cases, such plant protein is extruded, in other cases, such plant protein is non-extruded protein. In some cases, a meat analog includes the rpOVA mixture at about 2% to 15% (w/w). In some cases, for meat analog compositions, the rpOVA mixture acts as a binding agent, a gelling agent or a combination of a binding and gelling agent for such compositions.

[0211] In some embodiments, the compositions provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) can be employed in coatings for food products. For example, the rpOVA mixture can provide binding or adhesion characteristics to adhere batter or breading to another food ingredient. In certain embodiments, the rpOVA mixture can be used as an “egg-less egg wash” where the rpOVA protein provides appearance, color, or texture when coated onto other food ingredients or food products, such as baked products. In one example, the “egg-less egg wash” may be used to coat a baked good such that the baked good adheres to a coating (e.g., seed, salt, spice, and herb). The addition of the rpOVA mixture as a coating to a food product can provide a crunchy texture or increase the hardness, for example, of the exterior of a food product such as when the product is cooked, baked or fried.

[0212] In some embodiments, the compositions provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) include sauces and dressings, such as an eggless mayonnaise, commercial mayonnaise substitutes, gravy, sandwich spread, salad dressing or food sauce. In some embodiments, the inclusion of the rpOVA mixture in a sauce or dressing, and the like, can provide one or more characteristics such as binding, emulsifying, odor neutrality, and mouthfeel. In some embodiments, the rpOVA mixture is present in such sauces and dressing in an amount between 0.1% and 3% or between about 3% and about 5% w/w/ or w/v. In some cases, the amount of the rpOVA mixture in a sauce or dressing may be substantially similar to the amount of whole egg, egg-white or nOVA used in a commercially available or commonly used recipe. Illustrative sauces and dressing include mayonnaise, commercial mayonnaise substitutes, alfredo sauce, and hollandaise sauce. In some embodiments, the rpOVA-containing sauce or dressing does not contain whole egg, egg white, or any other protein derived from egg or related to a native egg. In some cases, the sauce, dressing or other emulsified product made with the rpOVA mixture includes at least one fat or oil and water. Illustrative fats and oils for such compositions include corn oil, safflower oil, nut oils, and avocado oil.

[0213] In some embodiments, the compositions provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) can be used to prepare confectionaries such as eggless, animal-free, vegetarian, and vegan confectionaries. In some embodiments, the rpOVA mixture can provide one or more functional features to the confectionary including odor neutrality, flavor, mouthfeel, texture, gelling, cohesiveness, foaming, frothiness, nutritional value, and protein fortification. In some embodiments, the prepared confectionery containing the rpOVA mixture does not contain any native egg protein or native egg white. In some embodiments, the rpOVA mixture in such confectionaries can provide a firm or chewy texture. In some embodiments, the rpOVA mixture is present between about 0.1% and 15% in a confectionary. Illustrative confectionaries include a gummy, a taffy, a divinity candy, meringue, marshmallow, and a nougat. In some embodiments, a confectionery includes rpOVA, at least one sweetener and optionally a consumable liquid. Illustrative sweeteners include sugar, honey, sugar-substitutes, and plant- derived syrups. In some cases, the rpOVA mixture is provided as an ingredient for making a confectionery at a pH between about 3.5 and about 7. In some cases, the rpOVA mixture is present in the confectionary composition at about 2% to about 15% (w/v). In some embodiments, the confectionery is a food product such as a meringue, a whipped dessert, or a whipped topping. In some embodiments, the rpOVA mixture in the confectionary provides foaming, whipping, fluffing or aeration to the food product, and/or provides gelation. In some cases, the confectionery is a liquid, such as a foamed drink. In some cases, the liquid may include a consumable alcohol (such as in a sweetened cocktail or after-dinner drink).

[0214] In some embodiments, the compositions provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) can be used in dairy products, dairy-like products or dairy containing products. For example, the rOVA mixture can be used in preparations of beverages such as a smoothie, milkshake, “eggnog”, and coffee beverage. In some embodiments, the rpOVA mixture is added to additional ingredients where at least one ingredient is a dairy ingredient or dairy-derived ingredient (such as milk, cream, whey, and butter). In some embodiments, the rpOVA mixture is added to additional ingredients to create a beverage that does not contain any native egg protein, native egg white or native egg. In some embodiments, the rpOVA mixture is an ingredient in a beverage that does not contain any animal-derived ingredients, such as one that does not contain any native egg-derived or any dairy-derived ingredients. Examples of such non-dairy derived drinks include nut milks, such as soy milk, cashew milk, macadamia milk, or almond milk, oat milk, and coconut milk. In some embodiments, the rpOVA mixture can also be used to create beverage additions, such as creamer or “milk” to provide protein, flavor, texture and mouthfeel to a beverage such as a coffee, tea, alcohol-based beverages or cocoa. In some embodiments, the rpOVA mixture is present in a beverage ingredient or beverage addition in an amount between about 0.1% and 20: 1% w/w or w/v.

[0215] In some embodiments herein, the rpOVA mixture can be used to prepare a dairylike product such as yogurt, cheese, or butter. Dairy products with the rpOVA mixture can include other animal-based dairy components or proteins. In some embodiments, dairy products prepared with rpOVA do not include any animal-based ingredients.

[0216] Preparations of dessert products can be prepared using the rpOVA mixture. In some embodiments of the dessert products, the rpOVA mixture can provide one or more characteristics such as creamy texture, low fat content, odor neutrality, flavor, mouthfeel, texture, binding, and nutritional value. In some embodiments, the rpOVA mixture may be present in an ingredient or set of ingredients that is used to prepare a dessert product. Illustrative dessert products suitable for preparation with the rpOVA mixture include a mousse, a cheesecake, a custard, a pudding, a popsicle, and a frozen confectionery (e.g., a sherbet, a sorbet, or an ice cream). In some embodiments, dessert products prepared to include the rpOVA are vegan, vegetarian, or dairy-free. Dessert products that include rOVA can have an amount of the rpOVA mixture that is between about 0.1% and about 10% unphosphorylated or phosphorylated forms of rOVA w/w or w/v.

[0217] In some embodiments, the rOVA mixture can be used to prepare a snack food, such as a protein bar, an energy bar, a nutrition bar or a granola bar. The rpOVA mixture can provide characteristics to the snack food including one or more of binding, protein supplementation, flavor neutrality, odor neutrality, coating and mouth feel. In some embodiments, the rpOVA mixture is added to a preparation of a snack food in an amount between about 0.1% and 30% w/w or w/v.

[0218] In some embodiments, the rpOVA mixture can be used for nutritional supplements such as in parenteral nutrition, protein drink supplements, and protein shakes where the rpOVA mixture provides a high protein supplement. In some embodiments, the rpOVA mixture can be added to such compositions in an amount between about 10% and 30% w/w or w/v.

[0219] In some embodiments, the compositions provided herein comprising the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) can be used as an egg-replacer or an egg white-replacer. In some embodiments, the rpOVA mixture can be mixed or combined with at least one additional component to form the egg white replacer. In some embodiments, the rpOVA mixture can provide one or more characteristics to the egg-replacer or egg white-replacer, such as gelling, foaming, whipping, fluffing, binding, springiness, aeration, creaminess and cohesiveness. In some embodiments, the characteristic is the same or better than a native egg or native egg white provided in the same amount or concentration (w/w or w/v). In some embodiments, the egg-replacer or egg white-replacer, does not contain any egg, egg white, protein extracted or isolated from egg.

[0220] The rpOVA-containing food ingredient and food products, such as described herein, can contain additional ingredients or components. For example, the compositions provided herein comprising the rpOVA mixture can be prepared with an additional component such as one or more of a sweetener, a gum, a flavoring, a thickener, an acidulant and an emulsifier. Other ingredients such as flour, grains, oils and fats, fiber, fruit and vegetables can be combined with the rpOVA mixture. Such rpOVA compositions comprising the rpOVA mixture can be vegan, vegetarian, halal, kosher and animal-free, or a combination thereof. In some embodiments, the rpOVA mixture can be a food ingredient or prepared for a food product that is normally animal based or normally contains animal-derived components, such as meat, dairy or eggs.

[0221] Compositions comprising the rpOVA mixture such as food ingredients and food products can be compatible with one or more steps a of consumables preparation such as heated, baked, grilled, roasted, braised, microwaved, broiled, boiled, steamed, extruded, deep fried, or pan-fried, or processed using ohmic heating, Sous Vide, freezing, chilling, blanching, packaging, canning, bleaching, enriching, drying, pressing, grinding, mixing, par cooking, cooking, proofing, marinating, cutting, slicing, dicing, crushing, shredding, chopping, shaking, coring, spiralizing, rollingjuicing, straining, filtering, kneading, whisking, beating, whipping, grating, stuffing, peeling, smoking, curing, salting, preserving, pickling, fermenting, homogenizing, pasteurizing, sterilizing, irradiating, cold plasma processing, high pressure processing, pulse electric field processing, microwave assisted thermal sterilization, stabilizing, blending, pureeing, fortifying, refining, hydrogenating, aging, extending shelf life, or adding enzymes.

[0222] In some embodiments, the composition provided herein is treated with heat. In some embodiments, the composition provided herein is not treated with heat. In some cases, the composition is treated with heat at 45°C to 70°C. In some cases, the composition is treated at 45°C to 65 °C. In some cases, the composition is treated at 45°C to 60°C. In some cases, the composition is treated at 50°C to 70°C. In some case, the composition is treated at 50°C to 65°C. In some cases, the composition is treated at 50°C to 60°C. In some cases, the composition is treated at 55°C to 65 °C. In some embodiments, the composition is treated for 1, 2, 3, 4, 5, 6,7 8, 9, or 10 hours. In some embodiments, the composition is treated for 1-2 hours, 1-3 hours, 1-4 hours, 1-5 hours, 1-6 hours, 1-7 hours, 1-8 hours, 1-9 hours, or 1-10 hours. In one embodiment, the composition is treated for 1-3 hours. In a specific embodiment, the composition is treated at 50°C to 70°C for 1-3 hours.

[0223] Food ingredients and food products prepared with the rpOVA mixture can be essentially free of any microbial cells or microbial cell debris. For instance, rpOVA may be secreted from a microbial host cell and isolated from microbial cells, culture media and/or microbial cell debris.

[0224] In some embodiments, the rpOVA mixture may be prepared as a whole cell extract or fractionated extract such that an rpOVA composition contains microbial cells and/or microbial cell components.

[0225] In one embodiment, an rpOVA composition is prepared for animal consumption where the rpOVA mixture is present in a whole cell extract or fractionated extract such that an rpOVA composition contains microbial cells and/or microbial cell components. In some embodiments, an rpOVA composition is prepared for animal consumption where the rpOVA mixture is isolated from microbial cells, culture media and microbial cell debris. Illustrative compositions for animal consumption can include a pet food, an animal feed, a chewy treat, bone broth, smoothie or other liquid for animal nutrition and a solid nutritional supplement suitable for animal consumption. In these cases, the microbial cell extract or microbial cell debris may provide additional nutritional value. [0226] Animals which may consume rpOVA compositions can include companion animals (e.g., dog, cat, horse), farm animals, exotic animals (lion, tiger, zebra) as well as livestock (such as cow, pig, sheep, goat). The rpOVA compositions comprising the rpOVA mixture as described herein can also be used for aquaculture (such as for fish and shellfish) and for avian nutrition (such as for bird pets, zoo birds, wild birds, fowl and birds raised for human and animal food).

[0227] In some embodiments of the consumable food compositions described herein, the composition is essentially free of animal-derived components, whey protein, caseinate, fat, lactose, hydrolyzed lactose, soy protein, collagen, hydrolyzed collagen, or gelatin, or any combination thereof. In some embodiments, the composition described herein may be essentially free of cholesterol, glucose, fat, saturated fat, trans fat, or any combination thereof. In some cases, a composition described herein comprises less than 10%, 5%, 4%, 3%, 2%, 1%, or 0.5% fat by dry weight. In some embodiments, the composition may be fat-containing (e.g., such as a mayonnaise and commercial mayonnaise substitutes) and such composition may include up to about 60% fat or a reduced-fat composition (e.g., reduced fat mayonnaise and commercial mayonnaise substitutes) and such composition may include lesser percentages of fat. In some embodiments, the composition that is free of an animal-derived component can be considered vegetarian and/or vegan.

[0228] In some embodiments, the rpOVA powder composition comprises less than 5% ash. The term “ash” is an art-known term and represents inorganics such as one or more ions, elements, minerals, and/or compounds. In some cases, the rpOVA powder composition comprises less than 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.75%, 0.5%, 0.25% or 0.1% ash weight per total weight (w/w) and/or weight per total volume (w/v).

[0229] In some embodiments, the moisture content of the rpOVA powder composition may be less than 15%. The rpOVA powder composition may have less than 15%, 12%, 10%, 8%, 6%, 5%, 3%, 2% or 1% moisture weight per total weight (w/w) and/or weight per total volume (w/v). In some embodiments, the carbohydrate content of the rpOVA powder composition may be less than 30%. The rpOVA powder composition may have less than 30%, 27%, 25%, 22%, 21%, 20%, 17%, 15%, 12%, 10%, 8%, 5%, 3% or 1% carbohydrate content w/w or w/v.

1. Additional components of compositions

[0230] The consumable food compositions containing the rpOVA mixture (e.g., the rOVA mixture comprising one or more phosphorylated forms of the rOVA) disclosed herein and the methods of making such compositions may including adding or mixing the rpOVA mixture with one or more ingredients. For example, food additives may be added in or mixed with the compositions. In some embodiments, food additives can add volume and/or mass to a composition. In some embodiments, the food additive may improve functional performance and/or physical characteristics. For example, a food additive may prevent gelation or increased viscosity due to the lipid portion of the lipoproteins in the freeze-thaw cycle. In some embodiments, the anticaking agent may be added to make a free-flowing composition. In some embodiments, carbohydrates can be added to increase resistance to heat damage, e.g., less protein denaturation during drying and improve stability and flowability of dried compositions. Food additives include, but are not limited to, food coloring, pH adjuster, natural flavoring, artificial flavoring, flavor enhancer, batch marker, food acid, filler, anticaking agent (e.g., sodium silico aluminate), anti greening agent (e.g., citric acid), food stabilizer, foam stabilizer or binding agent, antioxidant, acidity regulatory, bulking agent, color retention agent, whipping agent (e.g., ester-type whipping agent, triethyl citrate, sodium lauryl sulfate), emulsifier (e.g., lecithin), humectant, thickener, excipient, solid diluent, salts, nutrient, sweetener, glazing agent, preservative, vitamin, dietary elements, carbohydrates, polyol, gums, starches, flour, oil, or bran.

[0231] Food coloring includes, but is not limited to, FD&C Yellow #5, FD&C Yellow #6, FD&C Red #40, FD&C Red #3, FD&C Blue No. 1, FD&C Blue No. 2, FD&C Green No. 3, carotenoids (e.g., saffron, P-carotene), anthocyanins, annatto, betanin, butterfly pea, caramel coloring, chlorophyllin, elderberry juice, lycopene, carmine, pandan, paprika, turmeric, curcuminoids, quinoline yellow, carmoisine, Ponceau 4R, Patent Blue V, and Green S.

[0232] Ingredients for pH adjustment include, but are not limited to, Tris buffer, potassium phosphate, sodium hydroxide, potassium hydroxide, citric acid, sodium citrate, sodium bicarbonate, and hydrochloric acid.

[0233] Salts include, but are not limited, to acid salts, alkali salts, organic salts, inorganic salts, phosphates, chloride salts, sodium salts, sodium chloride, potassium salts, potassium chloride, magnesium salts, magnesium chloride, magnesium perchlorate, calcium salts, calcium chloride, ammonium chloride, iron salts, iron chlorides, zinc salts, and zinc chloride. [0234] Nutrient includes, but is not limited to, macronutrient, micronutrient, essential nutrient, non-essential nutrient, dietary fiber, amino acid, essential fatty acids, omega-3 fatty acids, and conjugated linoleic acid. [0235] Sweeteners include, but are not limited to, sugar substitute, artificial sweetener, acesulfame potassium, advantame, alitame, aspartame, sodium cyclamate, dulcin, glucin, neohesperidin dihydrochalcone, neotame, P-4000, saccharin, aspartame-acesulfame salt, sucralose, brazzein, curculin, glycyrrhizin, glycerol, inulin, mogroside, mabinlin, maltooligosaccharide, mannitol, miraculin, monatin, monellin, osladin, pentadin, stevia, trilobatin, and thaumatin.

[0236] Carbohydrates include, but are not limited to, sugar, sucrose, glucose, fructose, galactose, lactose, maltose, mannose, allulose, tagatose, xylose, arabinose, high fructose corn syrup, high maltose com syrup, com syrup (e.g., glucose-free corn syrup), sialic acid, monosaccharides, disaccharides, polysaccharides (e.g., polydextrose, maltodextrin), and starch.

[0237] Polyols include, but are not limited to, xylitol, maltitol, erythritol, sorbitol, threitol, arabitol, hydrogenated starch hydrolysates, isomalt, lactitol, mannitol, and galactitol (dulcitol). [0238] Gums include, but are not limited to, gum arabic, gellan gum, guar gum, locust bean gum, acacia gum, cellulose gum, and xanthan gum.

[0239] Vitamins include, but are not limited to, niacin, riboflavin, pantothenic acid, thiamine, folic acid, vitamin A, vitamin B6, vitamin B12, vitamin D, vitamin E, lutein, zeaxanthin, choline, inositol, and biotin.

[0240] Dietary elements include, but are not limited to, calcium, iron, magnesium, phosphorus, potassium, sodium, zinc, copper, manganese, selenium, chlorine, iodine, sulfur, cobalt, molybdenum, nickel, and bromine.

Definitions

[0241] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0242] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. [0243] As used herein, the phrases “at least one”, “one or more”, and “and/or” are open- ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and/or C” mean A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

[0244] As used herein, “or” may refer to “and”, “or,” or “and/or” and may be used both exclusively and inclusively. For example, the term “A or B” may refer to “A or B”, “A but not B”, “B but not A”, and “A and B”. In some cases, context may dictate a particular meaning.

[0245] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0246] The terms “comprise”, “comprising”, “contain,” “containing,” “including”, “includes”, “having”, “has”, “with”, or variants thereof as used in either the present disclosure and/or in the claims, are intended to be inclusive in a manner similar to the term “comprising.” [0247] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0248] The terms “increased”, “increasing”, or “increase” are used herein to generally mean an increase by a statically significant amount relative to a reference level. In some aspects, the terms “increased,” or “increase,” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level. Other examples of “increase” include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.

[0249] The terms “decreased”, “decreasing”, or “decrease” are used herein generally to mean a decrease in a value relative to a reference level. In some aspects, “decreased” or “decrease” means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level.

[0250] Sequence identity, such as for the purpose of assessing percent complementarity, may be measured by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see e.g., the EMBOSS Needle aligner available at the World Wide Web at ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html, optionally with default settings), the BLAST algorithm (see e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionally with default settings), and the Smith-Waterman algorithm (see e.g., the EMBOSS Water aligner available at the World Wide Web at ebi.ac.uk/Tools/psa/emboss_water/nucleotide.htrnl, optionally with default settings). Optimal alignment may be assessed using any suitable parameters of a chosen algorithm, including default parameters.

[0251] In this disclosure, phosphorylation of OVA at Ser-68 is identified as “with respect to SEQ ID NO: 2” and/or phosphorylation of OVA at Ser-344 is identified as “with respect to SEQ ID NO: 2”. SEQ ID NO: 2 relates to an OVA that is naturally expressed in one species of bird (chicken). However, this disclosure relates to phosphorylation of a number of recombinant OVAs, each having an amino acid sequence that is naturally expressed in one species, e.g., barn owl, crow, dove, hummingbird, and emu. These other species’ OVA may have serines that can be phosphorylated at the same location with respect to their SEQ ID NO, i.e., one of SEQ ID NO: 4 to SEQ ID NO: 76. Alternatively, these other species’ OVA may have serines that can be phosphorylated at a homologous serine, yet at a different specific location, or can be phosphorylated at homologous serines, yet at different specific locations (e.g., Ser-67, Ser- 69, Ser-343, and Ser-345) with respect to the other OVA’s SEQ ID NO, i.e., one of SEQ ID NO: 4 to SEQ ID NO: 76. Consequently, any herein disclosure that describes a Ser-68 or Ser- 344 applies to homologous serines in the other OVAs.

[0252] The term “bird” includes both domesticated birds and non-domesticated birds such as wildlife and the like. Birds include, but are not limited to, poultry, fowl, waterfowl, game bird, ratite (e.g., flightless bird), chicken (Gallus Gallus, Gallus domesticus, or Gallus Gallus domeslicus), quail, turkey, duck, ostrich (Struthio camelus), Somali ostrich (Struthio molybdophanes), goose, gull, guineafowl, pheasant, emu (Dromaius novaehollandiae), American rhea (Rhea americana), Darwin’s rhea (Rhea pennata), and kiwi. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. A bird may lay eggs.

INCORPORATION BY REFERENCE

[0253] All publications, patents, and patent applications mentioned in this specification are herein incorporated 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.

[0254] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

EXAMPLES

[0255] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0256] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Example 1: Preparation of recombinant ovalbumin

[0257] A Gallus gallus OVA coding sequence was fused in-frame with the alpha mating factor signal sequence downstream of the promoter sequence (SEQ ID NO: 1). A promoter was placed upstream of the signal sequence OVA coding sequence and a transcriptional terminator was placed downstream of the OVA sequence. The expression construct was placed into a Kpas-URA 3 vector.

[0258] The expression constructs were transformed into Pichia pastoris. Successful integration was confirmed by genomic sequencing.

[0259] Fermentation'. Recombinant OVA was produced in a bioreactor at ambient conditions. A seed train for the fermentation process begins with the inoculation of shake flasks with liquid growth broth using 2ml cryovials of Pichia pastoris which are stored at -80°C and thawed at room temperature prior to inoculation.

[0260] The inoculated shake flasks were kept in a shaker at 30°C for 24 hours, after which the grown Pichia pastoris was transferred to a production scale reactor.

[0261] The culture was grown at 30°C, at a set pH and dissolved oxygen (DO). The culture was fed with a carbon source. At the end of the fermentation, the target OVA protein was harvested from the supernatant.

[0262] Cell debris was removed, protein was purified and lyophilized to a dry powder. The OVA produced was used in the examples described below.

Example 2: Native OVA is phosphorylated while recombinant OVA produced in Pichia is unphosphorylated

[0263] Native OVA from chicken can be phosphorylated at Serine 68 and/or Serine 344 (FIG. 2). To determine the phosphorylation status of recombinant OVA expressed in Pichia, native and recombinant OVA from Pichia were treated with different phosphatases and examined on a native-PAGE gel (FIG. 3). The native and recombinant OVA were examined under three conditions: control (no phosphatase), Lambda (treated with Lambda Phosphatase), and CIP (treated with Calf Intestinal Phosphatase). The native OVA from chicken (native lane in FIG. 3) and two batches of recombinant OVA from Pichia (Batch 17 (“B17”) and RC 003/1 FP (“003”) lanes in FIG. 3) were treated with control, Lambda phosphatase, or CIP for 30 minutes at 30° C. Phosphorylation was expected to enhance migration of the protein due to the presence of negatively charged phosphates. The migration of native OVA was altered when treated with phosphatases, indicating that phosphate groups were removed from the native OVA when treated with Lambda phosphatase or CIP. The migration was not altered for either the B17 or 003 batch of recombinant OVA from Pichia, indicating the recombinant OVA is not phosphorylated.

Example 3: Recombinant ovalbumin is phosphorylated by FAM2O:1C in vitro

[0264] This example provides a method for phosphorylating recombinant OVA through treatment with an exogenous kinase protein in vitro.

[0265] Phosphorylated recombinant OVA was prepared in vitro as follows: 10 mg/ml recombinant OVA was incubated in a 200 microliter reaction containing 0.0238 mg/ml human Fam20C (Catalog #: 9265-FM, R&D Systems), 1 mM ATP, 25 mM HEPES pH 7.3, and 1 mM manganese chloride. The reaction was allowed to proceed for 24 h at 30 C after which an additional 0.00476 mg/ml human Fam20C was added to reaction and incubated 1 hr at 30 C. The sample was desalted to quench the phosphorylation reaction with a Zeba column (789882, Thermo Scientific) that had been equilibrated with 10 mM sodium phosphate pH 7.

[0266] Phosphatase assays were performed in 20 microliter reactions containing 0.5 mg/ml OVA (rOVA, rOVA-phos, nOVA), 50 units Calf Intestinal Phosphatase (M0525S, New England Biolabs), and rCutSmart™ Buffer (New England Biolabs). Reactions were performed at 37C for 1 h. Native PAGE analyses were performed with 4 to 16% Bis-Tris gels from Thermo Scientific (BN1002BOX).

[0267] To determine if the recombinant OVA from Pichia could be phosphorylated in vitro, recombinant OVA from Pichia (rOVA in FIG. 4) and native OVA from chicken (nOVA) were treated with human kinase FAM20: 1C to add at least one phosphate group and examined on a native-PAGE gel. Recombinant OVA from Pichia was treated with human FAM20: 1C (rOVA- Phos) and compared to recombinant OVA from Pichia without kinase (rOVA) and native OVA from chicken (nOVA). An aliquot of rOVA-phos, rOVA, and nOVA were further treated with Calf Intestinal Phosphatase (+phosphatase column).

[0268] Consistent with native OVA, the migration on native-PAGE of the phosphorylated recombinant OVA (rOVA-Phos) is sensitive to phosphatase treatment (FIG. 4). These results indicate that the recombinant OVA can be phosphorylated by an exogenous kinase in vitro. Example 4: Recombinant ovalbumin is phosphorylated by FAM2O:1C in vivo

[0269] This example provides a method for phosphorylating recombinant OVA through genetic co-expression with an exogenous kinase. To determine if the recombinant OVA from Pichia could be phosphorylated in vivo, recombinant OVA was co-expressed in strains expressing FAM20: 1 kinases and examined on a native-PAGE gel (FIG. 5). Unphosphorylated recombinant OVA from Pichia showed a nonphosphorylation pattern (‘Non-phos rOVA,’). Recombinant OVA from Pichia was phosphorylated in vitro (‘rOVA+kinase in vitro) as described in Example 3. To genetically phosphorylate recombinant OVA from Pichia, FAM2O: 1A or FAM2O: 1C isoform #1 were co-expressed constitutively in Pichia expressing recombinant OVA (FAM2O: 1A in vivo and FAM2O: 1C isoform #1 in vivo, respectively). Native OVA (nOVA) was run for comparison. The FAM2O: 1C isoform #1 band pattern indicated about 75% phosphorylation of recombinant OVA.

[0270] To further demonstrate the in vitro and in vivo phosphorylation of recombinant OVA in Pichia, recombinant OVA treated under various conditions was run on a native-PAGE gel (FIG. 6). A non-phosphorylated sample or batch of ovalbumin (“rOVA”) is shown. The non-phosphorylated recombinant OVA was phosphorylated in vitro using the recombinant FAM2O: 1C enzyme preparation described above (“rOVA+kinase”). Another non- phosphorylated ovalbumin expressed in vivo from the control strain is shown (“control”). Recombinant OVA was co-expressed with the pseudo-kinase FAM2O: 1A and mimics a nonphosphorylation pattern (“FAM20: 1 A”). Recombinant OVA was co-expressed with the kinase FAM20: 1C isoform 1 (“FAM20: 1C”) and exhibits a phosphorylated pattern similar to in vitro phosphorylated recombinant OVA (“rOVA+kinase”). This experiment demonstrated that in vivo phosphorylation of recombinant ovalbumin occurs when it is co-expressed with FAM2O: 1C isoform 1 (“rOVA-FAM20C-l”).

[0271] To determine if recombinant OVA in Pichia can be phosphorylated in vivo with an additional FAM20: 1C isoform, recombinant OVA was treated with FAM20: 1C isoform #2 and run on a native-PAGE gel (FIG. 7). A non-phosphorylated sample or batch of ovalbumin (“rOVA”) is shown. The non-phosphorylated recombinant rOVA was phosphorylated in vitro using the recombinant FAM2O: 1C enzyme preparation described above (“rOVA+kinase”). Another non-phosphorylated ovalbumin expressed in vivo from the control strain is shown (“rOVA-con”). Recombinant OVA was co-expressed with the kinase FAM2O: 1C isoform 2 (“rOVA-FAM20C-2”) and exhibits a phosphorylated pattern similar to in vitro phosphorylated recombinant OVA (“rOVA+kinase”). Native OVA (nOVA) was run for comparison. [0272] Additional replicates were completed to further demonstrate the robust phosphorylation patterns of the methods and compositions disclosed herein (FIG. 8A and FIG. 8B). The rOVA from a control line (“control” in FIG. 8A and FIG. 8B) was run on a native- PAGE gel. Phosphorylated rOVA from strains expressing FAM20A (FIG. 8A), FAM20C isoform #1 (FIG. 8 A), and FAM20C isoform #2 (FIG. 8B) were also run on the native-PAGE gel and show shifted bands due to a greater negative charge due to phosphorylation. Taken together, these data indicate that the co-expression of an exogenous kinase can phosphorylate rOVA expressed in Pichia.

Example 5: Phosphorylation of rOVA by FAM2O:1C isoforms in vivo

[0273] To further evaluate in vivo phosphorylation of OVA in Pichia expression strains, recombinant OVA was co-expressed with either kinase FAM2O: 1C isoform 1 (‘isoform 1’) or FAM2O: 1C isoform 2 (‘isoform 2’) in small scale fermentations. The resultants supernatants were run on native PAGE together with controls for the non-phosphorylated rOVA (‘Non-phos rOVA,’), in vitro phosphorylated rOVA (‘rOVA+kinase in vitro’), and native OVA (‘nOVA’). As shown in Figure 12, rOVA co-expressed with the kinase FAM2O: 1C isoform 1 migrates similar to in vitro phosphorylated rOVA. Both migrate further than the nonphosphorylated recombinant protein. This is consistent with the result for FAM2O: 1C isoform 1 with batch growth as described in Example 4. In contrast, rOVA co-expressed with the kinase FAM20: 1C isoform 2 migrates on native PAGE more closely to native OVA than either in vitro phosphorylated rOVA or nonphosphorylated rOVA. The findings suggest that rOVA coexpressed with FAM2O: 1C isoform 2 is di-phosphorylated, while the rOVA co-expressed with FAM2O: 1C isoform 1 is mono-phosphorylated. To confirm this, proteins were run on SDS-PAGE and the resultant bands were excised and submitted for peptide mapping. LC-MS on peptide fragments of the samples were performed to detect the presence of phosphates covalently attached to protein residues. Consistent with the native PAGE results, a single phosphorylation modification at the serine of position 344 was observed for both in vitro phosphorylated rOVA and rOVA coexpressed with FAM2O:1C isoform 1. For rOVA coexpressed with FAM20: 1C isoform 2, phosphorylation modifications were found for serines at positions 68 and 344. Phosphorylation modifications were not evident for nonphosphorylated rOVA. These results demonstrate that dephosphorylated rOVA or monophosphorylated rOVA can be generated in a manner that is dependent on the isoform of co-expressed Fam20:lC and the growth conditions employed for rOVA expression. [0274] FAM20C-1 and FAM20C-2 share 98% sequence similarity but have observable differences in the degree of phosphorylation (see FIG.8). It therefore indicates that a string of 10 amino acids that flank the membrane localization sequence that is the only difference between the two isoforms (FIG.11) accounts for the change in efficiency of P2 phosphorylation.

Example 6: Ratios of diphosphorylated recombinant ovalbumin protein, monophosphorylated recombinant ovalbumin protein, or unphosphorylated recombinant ovalbumin protein

[0275] This example illustrates the phosphorylation patterns of recombinant ovalbumin protein as compared to the phosphorylation patterns of native OVA. Native OVA (nOVA) is found as a mixture of diphosphorylated OVA protein, monophosphorylated OVA protein, or unphosphorylated OVA protein. Diphosphorylated nOVA is phosphorylated at Serine 68 and Serine 344. Monophosphorylated nOVA is phosphorylated at Serine 68 or Serine 344, but not both. In nOVA samples, the ratio of dephosphorylated to monophosphorylated to unphosphorylated OVA is 85:12:3. In nOVA samples, there is more monophosphorylated OVA at Ser-68 than at Ser- 344. The phosphorylated recombinant OVA disclosed herein exhibits a ratio of di-phosphorylated to mono-phosphorylated to unphosphorylated OVA that is different than the ratio found in egg of nOVA of 85: 12:3.

Example 7: Ovalbumin expression in Pichia pastoris

[0276] A Pichia pastoris strain derived from the historic Phillips Petroleum strain (NRRL

Y-11430) was designed to generate a non-methanol-utilization (mutM) phenotype with transformants that express ovalbumin under the control of a strong methanol inducible promoter. The transformant strains were further modified to phosphorylate the serine residues on the ovalbumin moieties. The strains with and without this specific modification were grown in fermentation conditions in high-density growth conditions at about a pH of 5. A batch phase was carried out for 15 hours using a batch media comprising 10 g/L glucose and then in a fed- batch mode for 20 hours to gain biomass. After about 36 hours of growth under fermentation conditions, the pH of the culture was raised to about a pH of 6, and expression of ovalbumin was induced by the addition of methanol to the culture. Example 8: Optimized ovalbumin expression in Pichia pastoris

[0277] A Pichia pastoris strain derived from the historic Phillips Petroleum strain (NRRL Y-11430) was designed to generate a non-methanol-utilization (mutM) phenotype with transformants that express ovalbumin under the control of a strong methanol inducible promoter. The transformant strains were further modified to phosphorylate the serine residues on the ovalbumin moieties. The strains with and without this specific modification were grown in fermentation conditions suitable for high-density growth, then grown in conditions suitable for methanol-induced ovalbumin expression. This example compares two strains, rOVA-con and rOVA+FAM20C.

[0278] The two strains were specifically grown in accordance with run conditions comprising three stages: (1) a batch phase, (2) a fed-batch phase, and (3) an induction phase (Table 3)

[0279] Batch phase: Fermentation started with 81 mL of batch media (comprising 10 g/L glucose). After inoculation with 10% culture, the batch phase ran for 15 hours where the glucose in the batch media was metabolized by the culture.

Fed-batch phase

[0280] In the fed-batch phase, glucose feed was triggered following the feed strategy as outlined in Table 3.

Induction phase:

[0281] In the induction phase, glucose feed was continued following the feed rates outlined in Table 3. In addition to the glucose feed, a methanol feed was also triggered as outlined in Table 4.

Table 4: Methanol feed schedule for optimized ovalbumin expression by Pichia pastoris.

[0282] The fermentation process described herein resulted in robust and consistent cell mass growth and protein production, reproducible across two strains rOVA-con and rOVA- FAM20C expressing very different protein moieties (FIG. 9). The fermentation process described herein also resulted in consistent nitrogen and glucose yields, reproducible across two strains OVA-con and rOVA-FAM20C expressing very different protein moieties.

[0283] The fermentation broth may optionally be centrifuged using a bench centrifuge (Avant JI 8 Rotor; Beckman Coulter) to remove cells. This may be followed by filtration of the supernatant using a 0.2 micrometer hollow fiber membrane filter to remove host proteins and cell debris. The protein solution may then be concentrated using a 10 kDa ultrafiltration membrane to a concentration of 50 g/L protein. The protein solution may further be diafiltered extensively to remove most of the organic and inorganic impurities. The diafiltered concentrate may then be microfiltered to eliminate any bioburden accumulated during the process and dried.

Example 9: Expression and purification of recombinant ovalbumin

[0284] A Pichia pastoris strain derived from the historic Phillips Petroleum strain (NRRL

Y-11430) was designed to generate a non-methanol-utilization (mutM) phenotype with transformants that express ovalbumin under the control of a strong methanol inducible promoter. A resulting strain was grown in fermentation conditions in high-density growth conditions at about a pH of 5. A batch phase was carried out for 15 hours using a batch media comprising 10 g/L glucose and then in a fed-batch mode for 20 hours to gain biomass. After about 36 hours of growth under fermentation conditions, the pH was raised to about a pH of 6, and expression of ovalbumin was induced by the addition of methanol to the culture.

[0285] The fermentation broth was centrifuged using a bench centrifuge (Avant JI 8 Rotor; Beckman Coulter) to remove cells. This was followed by filtration of the supernatant using a 0.2 micrometer hollow fiber membrane filter to remove host protein and cell debris. The protein solution was then concentrated using a 10 kDa ultrafiltration membrane to a concentration of over 30 g/L protein and diafiltered extensively to remove most of the organic and inorganic impurities. The resulting protein concentrate was adjusted for pH and loaded onto a chromatography column.

[0286] This column was packed with cation exchange resin (SP400; Mitsubishi Chemicals, Japan) The chromatography steps were carried out with an AKTA Explorer 900 (GE Healthcare Life Sciences) and the Unicorn interface software (version 5.11) at approximately 22 °C in a down-flow mode. The chromatography method comprised an equilibration step, a load (flowthrough) step, a wash step to remove unbound proteins, an elution step to remove the desired product, a cleaning in place (CIP) step, and a regeneration step.

[0287] The pH-adjusted protein concentrate was flowed through the cation exchange column following Table 5, which highlights the column volumes of elution at each step and the buffers used. The elution profile is shown in FIG. 10.

Table 5: List of buffers and the column volumes used for cation exchange chromatography to purify recombinant ovalbumin. Example 10: Expression and purification of phosphorylated recombinant ovalbumin

[0288] A Pichia pastoris strain derived from the historic Phillips Petroleum strain (NRRL Y-11430) was designed to generate a non-methanol-utilization (mutM) phenotype with transformants that express ovalbumin under the control of a strong methanol inducible promoter. These transformant strains were further modified to phosphorylate serine residues on the ovalbumin moieties. The strains with and without this specific modification were grown in fermentation conditions in high-density growth conditions at about a pH of 5. A batch phase was carried out for 15 hours using a batch media comprising 10 g/L glucose and then in a fed- batch mode for 20 hours to gain biomass. After about 36 hours of growth under fermentation conditions, the pH of the culture was raised to about a pH of 6, and expression of ovalbumin was induced by the addition of methanol to the culture.

[0289] The fermentation broth is centrifuged using a bench centrifuge (Avant JI 8 Rotor; Beckman Coulter) to remove cells. This is followed by filtration of the supernatant using a 0.2 micrometer hollow fiber membrane filter to remove host protein and cell debris. The protein solution then is concentrated using a 10 kDa ultrafiltration membrane to a concentration of over 30 g/L protein and diafiltered extensively to remove most of the organic and inorganic impurities. The resulting protein concentrate is adjusted for pH and loaded onto a chromatography column.

[0290] This column is packed with cation exchange resin (SP400; Mitsubishi Chemicals, Japan) The chromatography steps is carried out with an AKTA Explorer 900 (GE Healthcare Life Sciences) and the Unicorn interface software (version 5.11) at approximately 22 °C in a down-flow mode. The chromatography method comprises an equilibration step, a load (flowthrough) step, a wash step to remove unbound proteins, an elution step to remove the desired product, a cleaning in place (CIP) step, and a regeneration step.

[0291] The pH-adjusted protein concentrate is flowed through the cation exchange column following Table 6, which highlights the column volumes of elution at each step and the buffers used. Table 6: List of buffers and the column volumes used for cation exchange chromatography to purify phosphorylated recombinant ovalbumin.

Example 11: Purification of recombinant ovalbumin with anion exchange column

[0292] A protein concentrate comprising recombinant ovalbumin is prepared for loading onto an anion exchange column following the methods outlined in Example 9. In the instant example, the chromatography column is packed with anionic exchange capto Q resin (Cytiva Chemicals). Notably, no pH adjustment to the protein concentrate is needed in this example, thereby simplifying the purification process significantly.

[0293] The protein concentrate is flowed through the anion exchange column following Table 7, which highlights the column volumes of elution at each step and the buffers used.

Table 7: List of buffers and the column volumes that are used for anion exchange chromatography to purify recombinant ovalbumin.

Example 12: Purification of phosphorylated recombinant ovalbumin with anion exchange column

[0294] A protein concentrate comprising phosphorylated recombinant ovalbumin is prepared for loading onto an anion exchange column following the methods outlined in Example 10. In the instant example, the chromatography column is packed with anionic exchange capto Q resin (Cytiva Chemicals). Notably, no pH adjustment to the protein concentrate is needed in this example, thereby simplifying the purification process significantly.

[0295] The protein concentrate is flowed through the anion exchange column following Table 8, which highlights the column volumes of elution at each step and the buffers used.

Table 8: List of buffers and the column volumes that are used for anion exchange chromatography to purify phosphorylated recombinant ovalbumin. Example 13: FAM20C-2 kinase from Gallus gallus expressed in Komagatella phaffii strains for producing recombinant Gallus gallus ovalbumin (OVA)

[0296] Codon optimized nucleotide sequence (SEQ ID NO: 147, below) of FAM20C-2 kinase from Gallus gallus (SEQ ID NO : 82) was inserted into the Komagataella phaffii genome at a specified location with the construct shown in FIG. 13.

[0297] Codon optimized nucleotide sequence of FAM20C-2 kinase from Gallus gallus'.

ATGAAAATGTTACTTGTTAGAAAATTTAGAGTTCTGATCTTGATGGTATTTCTAGT GGCATGTACCATGCACATTATGATAGACTTACTACCCAGGTTGGAGAGACGTGG AGCTGAAGGCAGACCCGGTTGTTCCTGTCCACCACCTGCCGCTCCCCCTAGGGCC GCTCCACGTTGGCCTTCTAAACACACCTTGAGAATCCTACAAGATTTTTCTGCAG AGCCCGCCTCCAACTTATCATCACAGAGTAGGGAGGCTGCTGAAAGAGCAGCTG GCGGTGGAGGAGATGCTGCCGCCGCTGCAGCCGCTGCTGGTGGTGAGGGCGCTG CTGCAGGTAGACCTAGGCGTCTAATAGCACCAGGCGCTCCCAGACCACCTCCACC TGCTCACGCCGCACCCTTGGCCGCTCTTTTCCAGCACCCACTTTACAGAGCTGCTC TTCCTCCCTTAGCCGATGGCGATTTACTGTTCAACGTTAATAGTGACATACGTTTC AATCCTAGAGCAGCCGAGCAAGGCGAATGGCATAACGAGGAAAACGAGGAGTT CCTACCAACTGGCGAAACGAGTATAGACTCCTACCCTAATTGGCTAAAGTTTCAC ATCGGAATTAACAGATACGAGCTTTATTCAAGGCACAACCCTGCTATAGAGGCTC TGCTACAAGAcCTAGTATCCCAGAAAATCACCAGTGTCGCTATGAAATCAGGTGG AACACAGCTGAAGCTAATAATGACGTTCCAAAATTATGGACAGGCCCTATTTAA GCCCATGAAGCAGACAAGGGAACAAGAAACCCCCCCCGACTTTTTCTATTTTTCC GACTATGAACGTCACAACGCAGAAATAGCTGCATTTCACCTTGACAGAATCCTAG ACTTCAGAAGGGTTCCCCCCGTTGCTGGCAGGCTGGTGAACATGACTAGGGAGA TTCGTGACGTCACTAGAGACAAGAAGCTGTGGAGAACTTTTTTTATATCACCCGC TAATAACATCTGCTTCTATGGTGAGTGTTCCTATTACTGCTCAACTGAACATGCTC TTTGCGGTAAGCCAGACCAAATCGAGGGtTCCTTAGCAGCCTTTTTGCCCGATCTA AGTTTAGCCAAACGTAAAACATGGAGGAACCCCTGGCGTAGAAGTTACCATAAA AGGAAGAAAGCCGAATGGGAGGTTGACCCCGATTATTGTGAGGAGGTTAAGCAG ACTCCTCCTTATGACAGTGGAACCAGAATTTTGGATATAATGGATATGACTGTTT TTGATTTTTTAATGGGAAATATGGACAGGCACCATTACGAAACGTTTGAGAAATT TGGTAATGAAACTTTCATTATCCACCTGGACAATGGACGTGGATTCGGTAAGTAT AGTCATGATGAGCTTTCTATCCTGGTACCCCTAAACCAGTGTTGCAGGATACGTA AATCTACCTACCTACGTCTACAACTGTTAGCTAAAGAGGAGTATAAGTTGTCTTT ATTAATGAAAGAGAGTTTACTGAAGGACAAGATCGCTCCTATATTATACCAGCCA CACCTTGAGGCTATGGATAGACGTTTAAGAATTGTCTTAAAGGCTGTGTCAGATT GTATCGAAAAAGATGGATACGACAACGTCGTGGAGAATGACTTCAACACTGACG TGAACACAGTCGCCACCGAACGTTGA (SEQ ID NO: 147).

[0298] The construct was transformed into OVA producing Komagatella phaffii strains and tested in standard High Throughput Screen (HTS) process. The process utilized OD600 to assess the impact of an edit on the cell’s ability to grow and Bradford assay to assess the amount of protein made in a reaction. FIGs. 14A and 14B show dot plot graphs comparing the base strains (CS1512 and CS3042) to the CS3042 clones expressing the kinase construct. The expression of the kinase construct did not change the CS3042 clones’ ability to grow and the amount of protein produced compared to the base strains.

[0299] FIG. 15 shows the results of a Native PAGE experiment, in which proteins migrated farther in the gel if they had a stronger negative charge because phosphorylation increases the negative charge of a protein and decreases the pKa/pI value. Lanes 1 & 2 in FIG. 15 represent non-phosphorylated negative controls. Lanes 3 & 4 represent OVA protein powder that has been both mono- and diphosphorylated (MALDI-TOF data not shown), as positive controls. Lanes 5 through 14 represent CS3042 clones that co-express OVA and the kinase. The protein bands in lanes 5 through 14 were downshifted compared to the negative controls (lanes 1 & 2) and were closer to the in vitro phosphorylated positive controls (lanes 3 & 4).

[0300] The results indicate that FAM20C-2 kinase from Gallus gallus functioned to catalyze the phosphorylation of the recombinant OVA in Komagatella phaffii strains.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of producing a recombinant phosphorylated ovalbumin comprising:

(a) co-expressing in a recombinant host cell a heterologous or exogenous ovalbumin protein and at least one heterologous or exogenous kinase, and

(b) purifying or isolating the ovalbumin protein, which is phosphorylated by the heterologous or exogenous kinase at at least one site or residue.

2. A method of producing a recombinant phosphorylated ovalbumin comprising:

(a) expressing in a recombinant host cell a heterologous or exogenous ovalbumin protein,

(b) treating the ovalbumin protein with at least one heterologous or exogenous kinase, and

(c) purifying or isolating the ovalbumin protein, which is phosphorylated by the heterologous or exogenous kinase protein at at least one site or residue.

3. The method of either claim 1 or 2, wherein the recombinant host cell is a yeast host cell.

4. The method of claim 3, wherein the recombinant yeast host cell is Pichia.

5. The method of claim 3, wherein the recombinant yeast host cell is Saccharomyces .

6. The method of either claim 1 or 2, wherein the recombinant host cell is a fungal host cell.

7. The method of claim 6, wherein the recombinant fungal host cell is Trichoderma.

8. The method of claim 6, wherein the recombinant fungal host cell is Aspergillus .

9. The method of either claim 1 or 2, wherein the recombinant host cell is a bacterial host cell.

10. The method of claim 9, wherein the recombinant bacterial host cell is E. coli.

11. The method of any one of claims 1-10 wherein the exogenous kinase protein is selected from the genera: Homo, Drosophila, Danio, Branchiostoma, Stronglycocentrotus, Caenorhabditis, Hydra, Amphimedon, Avian, Reptile, any other egg-laying animal, or any combination thereof.

12. The method of claim 11, wherein the Avian is poultry, fowl, waterfowl, game bird, chicken, duck, ostrich, quail, goose, quail, turkey, gull, guineafowl, pheasant, emu, and any combination thereof.

13. The method of any one of claims 1-12, wherein the heterologous or exogenous kinase protein comprises a FAM20 kinase.

14. The method of claim 13, wherein the FAM20 kinase comprises FAM20A.

15. The method of claim 13, wherein the FAM20 kinase comprises FAM20C.

16. The method of claim 15, wherein the FAM20C kinase comprises any FAM20C isoform.

17. The method of any of claims 1-16, wherein the heterologous or exogenous kinase protein is from G. gallus.

18. The method of any of claims 1-17, wherein the heterologous or exogenous kinase protein comprises an amino acid sequence with at least 70% identity to any one of SEQ ID NO: 80 to SEQ ID NO: 141 and maintains the enzymatic activity of a kinase comprising the amino acid sequence of the corresponding SEQ ID NO: 80 to SEQ ID NO: 141.

19. The method of any of claims 1-18, wherein the heterologous or exogenous kinase protein comprises an amino acid sequence with at least 80% identity to any one of SEQ ID NO: 80 to SEQ ID NO: 141 and maintains the enzymatic activity of a kinase comprising the amino acid sequence of the corresponding SEQ ID NO: 80 to SEQ ID NO: 141.

20. The method of any of claims 1-19, wherein the heterologous or exogenous kinase protein comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NO: 80 to SEQ ID NO: 141 and maintains the enzymatic activity of a kinase comprising the amino acid sequence of the corresponding SEQ ID NO: 80 to SEQ ID NO: 141.

21. The method of any of claims 1-20, wherein the heterologous or exogenous kinase protein comprises an amino acid sequence with at least 95% identity to any one of SEQ ID NO: 80 to SEQ ID NO: 141 and maintains the enzymatic activity of a kinase comprising the amino acid sequence of the corresponding SEQ ID NO: 80 to SEQ ID NO: 141.

22. The method of any of claims 1-21, wherein the heterologous or exogenous kinase protein comprises an amino acid sequence with at least 99% identity to any one of SEQ ID NO: 80 to SEQ ID NO: 141 and maintains the enzymatic activity of a kinase comprising the amino acid sequence of the corresponding SEQ ID NO: 80 to SEQ ID NO: 141.

23. The method of any of claims 1-22, wherein the heterologous or exogenous kinase protein comprises an amino acid sequence of any one of SEQ ID NO: 80 to SEQ ID NO: 141.

24. The method of any one of claims 1-23, wherein the heterologous or exogenous kinase protein comprises a mixture of at least two or more distinct heterologous or exogenous kinase proteins.

25. The method of any one of claims 1-24, wherein the heterologous or exogenous ovalbumin protein comprises an amino acid sequence with at least 70% (e.g., 80%, 90%, 95%, and 99%) identity to any one of SEQ ID NO: 1 to SEQ ID NO: 76.

26. The method of any one of claims 1-25, wherein the heterologous or exogenous ovalbumin protein comprises an amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 76.

27. The method of any one of claims 1-26, wherein the heterologous or exogenous kinase protein recognizes a recognition motif comprising a S-X-E/pS motif.

28. The method of claim 27, wherein the recognition motif comprises Glu at a +2 position.

29. The method of claim 1, wherein the heterologous or exogenous ovalbumin protein is glycosylated prior to phosphorylation.

30. The method of any one of claims 1-29 wherein the recombinant phosphorylated ovalbumin is glycosylated, e.g., the recombinant phosphorylated ovalbumin comprises a glycosylation moiety.

31. The method of claim 30, wherein the glycosylation moiety comprises at least one mannose moiety or one N-acetylglucosamine moiety.

32. The method of any one of claims 1-31, wherein an unglycosylated recombinant ovalbumin is unphosphorylated.

33. The method of any one of claims 1-32, wherein the method further comprises combining the purified or isolated recombinant phosphorylated ovalbumin with food ingredients to make a food item.

34. The method of any one of claims 1-33, wherein the recombinant phosphorylated ovalbumin is more stable than an unphosphorylated recombinant ovalbumin.

35. The method of any one of claims 1-34, wherein the recombinant phosphorylated ovalbumin improves functionality in a food product.

36. The method of claim 35, wherein the recombinant phosphorylate ovalbumin provides to a food product at least one egg white characteristic selected from gelling, foaming, whipping, fluffing, binding, springiness, aeration, coating, film forming, emulsification, browning, thickening, texturizing, humectant, clarification, and cohesiveness, wherein the characteristic is the same as or is improved when compared to recombinant ovalbumin that is not phosphorylated.

37. The method of claim 35 or 36 wherein the food product is a cake, a pudding, a macaron, a bread, a roll, a pastry, a cracker, a muffin, a scone, a biscuit, a bagel, a pancake, a meringue, a choux pastry a souffle, a scramble, an omelet, a patty, a quiche, a frittata, a mouse, a custard, a popsicle, a frozen desert, an ice cream, or a cookie.

38. The method of any one of claims 1 to 37, wherein the kinase comprises or is linked to a domain that anchors the kinase to a membrane, e.g., endoplasmic reticulum and/or Golgi apparatus, of the host cell.

39. An isolated recombinant ovalbumin protein, isolated from the recombinant host cell used in the method of claims 1-38.

40. An engineered host cell that recombinantly co-expresses a heterologous or exogenous ovalbumin protein and at least one heterologous or exogenous kinase protein.

41. The engineered host cell of claim 40, wherein the engineered host cell is a yeast host cell.

42. The engineered host cell of claim 41, wherein the engineered yeast host cell is Pichia.

43. The engineered host cell of claim 41, wherein the engineered yeast host cell is Saccharomyces.

44. The engineered host cell of claim 40, wherein the engineered host cell is a fungal host cell.

45. The engineered host cell of claim 44, wherein the engineered fungal host cell is Trichoderma.

46. The engineered host cell of claim 44, wherein the engineered fungal host cell is Aspergillus .

47. The engineered host cell of claim 40, wherein the engineered host cell is a bacterial host cell.

48. The engineered host cell of claim 47, wherein the engineered bacterial host cell is E. coli.

49. The engineered host cell of any one of claims 40-48, wherein the exogenous kinase protein is selected from the genera: Homo, Drosophila, Danio, Branchiostoma, Stronglycocentrotus, Caenorhabditis, Hydra, Amphimedon, Avian, Reptile, any other egg-laying animal, or any combination thereof.

50. The engineered host cell of claim 49, wherein the Avian is poultry, fowl, waterfowl, game bird, chicken, duck, ostrich, quail, goose, quail, turkey, gull, guineafowl, pheasant, emu, and any combination thereof.

51. The engineered host cell of any one of claims 40-50, wherein the heterologous or exogenous kinase protein comprises a FAM20 kinase.

52. The engineered host cell of claim 51, wherein the FAM20 kinase comprises FAM20A.

53. The engineered host cell of claim 51, wherein the FAM20 kinase comprises FAM20C.

54. The engineered host cell of claim 53, wherein the FAM20C kinase comprises any FAM20C isoform.

55. The engineered host cell of any of claims 40-54, wherein the heterologous or exogenous kinase protein is from G. gallus.

56. The engineered host cell of any of claims 40-55, wherein the heterologous or exogenous kinase protein comprises an amino acid sequence with at least 70% homology to any one of SEQ ID NO: 80 to SEQ ID NO: 141 and maintains the enzymatic activity of the corresponding SEQ ID NO: 80 to SEQ ID NO: 141.

57. The engineered host cell of any of claims 40-56, wherein the heterologous or exogenous kinase protein comprises an amino acid sequence of any one of SEQ ID NO: 80 to SEQ ID NO: 141.

58. The engineered host cell of any one of claims 40-57, wherein the heterologous or exogenous kinase protein comprises a mixture of at least two or more distinct heterologous or exogenous kinase proteins.

59. The engineered host cell of any one of claims 40-58, wherein the heterologous or exogenous ovalbumin protein comprises an amino acid sequence with at least 70% homology to any one of SEQ ID NO: 1 to SEQ ID NO: 76.

60. The engineered host cell of any one of claims 40-59 wherein the heterologous or exogenous ovalbumin protein comprises an amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 76.

61. The engineered host cell of any one of claims 40-60, wherein the heterologous or exogenous ovalbumin protein comprises an amino acid sequence of a chicken ovalbumin, an ostrich ovalbumin, or a duck ovalbumin.

62. The engineered host cell of any one of claims 40-61, wherein the heterologous or exogenous kinase protein recognizes a recognition motif comprising a S-X-E/pS motif.

63. The engineered host cell of claim 62, wherein the recognition motif comprises Glu at a +2 position.

64. The engineered host cell of any one of claims 40-63, wherein the heterologous or exogenous ovalbumin protein is glycosylated prior to phosphorylation.

65. The engineered host cell of any one of claims 40-64, wherein the recombinant phosphorylated ovalbumin is glycosylated.

66. The engineered host cell of claim 65, wherein the glycosylation moiety comprises at least one mannose moiety or one N-acetylglucosamine moiety.

67. The engineered host cell of any one of claims 40-66, wherein an unglycosylated recombinant phosphorylated ovalbumin is unphosphorylated.

68. The engineered host cell of any one of claims 40-67, wherein the method further comprises combining the purified or isolated recombinant phosphorylated ovalbumin with food ingredients to make a food item.

69. The engineered host cell of any one of claims 40-68, wherein the recombinant phosphorylated ovalbumin is more stable than an unphosphorylated recombinant ovalbumin.

70. The engineered host cell of any one of claims 40-69, wherein the recombinant phosphorylated ovalbumin improves functionality in a food product.

71. The engineered host cell of claim 70, wherein the recombinant phosphorylate ovalbumin provides to the food product at least one egg white characteristic selected from gelling, foaming, whipping, fluffing, binding, springiness, aeration, coating, film forming, emulsification, browning, thickening, texturizing, humectant, clarification, and cohesiveness, wherein the characteristic is improved as compared to recombinant ovalbumin that is not phosphorylated.

72. The engineered host cell of claim 70 or 71, wherein the food product is a cake, a pudding, a macaron, a bread, a roll, a pastry, a cracker, a muffin, a scone, a biscuit, a bagel, a pancake, a meringue, a choux pastry a souffle, a scramble, an omelet, a patty, a quiche, a frittata, a mouse, a custard, a popsicle, a frozen desert, an ice cream, or a cookie.

73. An isolated recombinant ovalbumin protein, isolated from the engineered host cell of claims 40-72.

74. A method for manufacturing a food item comprising a pool of proteins isolated from the engineered host cell of claims 40-73.

75. A protein composition comprising recombinant ovalbumin protein having a ratio of diphosphorylated recombinant ovalbumin protein, monophosphorylated recombinant ovalbumin protein, and unphosphorylated recombinant ovalbumin protein that is different from a ratio of diphosphorylated ovalbumin protein, monophosphorylated ovalbumin protein, and unphosphorylated ovalbumin protein present in a protein composition obtained from an egg.

76. The protein composition of claim 75, wherein the ratio of recombinant ovalbumin protein monophosphorylated at Ser 344 is greater than the ratio of ovalbumin protein monophosphorylated at Ser 344 in a protein composition obtained from an egg.

77. The protein composition of claim 75, wherein the ratio of recombinant ovalbumin protein monophosphorylated at Ser 68 is greater than the ratio of ovalbumin protein monophosphorylated at Ser 68 in a protein composition obtained from an egg.

78. The protein composition of claim 75, wherein the ratio of recombinant ovalbumin protein diphosphorylated at Ser 68 and at Ser 344 is greater than the ratio of ovalbumin protein diphosphorylated at Ser 68 and at Ser 344 in a protein composition obtained from an egg.

79. The protein composition of claim 75, wherein the ratio of recombinant ovalbumin protein monophosphorylated at Ser 344 is less than the ratio of ovalbumin protein monophosphorylated at Ser 344 in a protein composition obtained from an egg.

80. The protein composition of claim 75, wherein the ratio of recombinant ovalbumin protein monophosphorylated at Ser 68 is less than the ratio of ovalbumin protein monophosphorylated at Ser 68 in a protein composition obtained from an egg.

81. The protein composition of claim 75, wherein the ratio of recombinant ovalbumin protein diphosphorylated at Ser 68 and at Ser 344 is less than the ratio of ovalbumin protein diphosphorylated at Ser 68 and at Ser 344 in a protein composition obtained from an egg.

82. The protein composition of any one of claims 75-81, wherein the recombinant phosphorylated ovalbumin improves functionality in a food product.

83. The protein composition of claim 82, wherein the recombinant phosphorylate ovalbumin provides to a food product at least one egg white characteristic selected from gelling, foaming, whipping, fluffing, binding, springiness, aeration, coating, film forming, emulsification, browning, thickening, texturizing, humectant, clarification, and cohesiveness, wherein the characteristic is the same as or improved when compared to recombinant ovalbumin that is not phosphorylated.

84. The protein composition of claim 81 or 82, wherein the food product is a cake, a pudding, a macaron, a bread, a roll, a pastry, a cracker, a muffin, a scone, a biscuit, a bagel, a pancake, a meringue, a choux pastry a souffle, a scramble, an omelet, a patty, a quiche, a frittata, a mouse, a custard, a popsicle, a frozen desert, an ice cream, or a cookie.

85. The protein composition of any one of claims 75-84, wherein the recombinant ovalbumin protein comprises an amino acid sequence with at least 70% homology to any one of SEQ ID NO: 1 to SEQ ID NO: 76.

86. The protein composition of any one of claims 75-85, wherein the recombinant ovalbumin protein comprises an amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 76.

87. The protein composition of any one of claims 75-86, wherein the recombinant ovalbumin protein comprises an amino acid sequence of a chicken ovalbumin, an ostrich ovalbumin, or a duck ovalbumin.

88. The protein composition of any one of claims 75-87, wherein a heterologous or exogenous kinase protein recognizes a recognition motif comprising a S-X-E/pS motif.

89. The protein composition of claim 88, wherein the recognition motif comprises Glu at a +2 position.

90. The protein composition of any one of claims 75-89, wherein the recombinant ovalbumin protein is glycosylated prior to phosphorylation.

91. The protein composition of any one of claims 75-90, wherein the recombinant ovalbumin is phosphorylated and glycosylated.

92. The protein composition of any one of claims 75-91, wherein the glycosylation moiety comprises at least one mannose moiety or one N-acetylglucosamine moiety.

93. The protein composition of any one of claims 75-92, wherein an unglycosylated recombinant phosphorylated ovalbumin is unphosphorylated.

94. The protein composition of any one of claims 75-93, wherein a phosphorylated recombinant ovalbumin is more stable than an unphosphorylated recombinant ovalbumin.

95. A protein composition comprising recombinant ovalbumin protein having a ratio of diphosphorylated recombinant ovalbumin protein, monophosphorylated recombinant ovalbumin protein, and unphosphorylated recombinant ovalbumin protein that is different from a ratio of diphosphorylated recombinant ovalbumin protein, monophosphorylated recombinant ovalbumin protein, and unphosphorylated recombinant ovalbumin protein present in a protein composition expressed by a recombinant cell that lacks heterologous or exogenous kinase.

96. The protein composition of claim 95, wherein the ratio of recombinant ovalbumin protein monophosphorylated at Ser 344 is greater than the ratio of recombinant ovalbumin protein monophosphorylated at Ser 344 in a protein composition expressed by a recombinant cell that lacks heterologous or exogenous kinase.

97. The protein composition of claim 95, wherein the ratio of recombinant ovalbumin protein monophosphorylated at Ser 68 is greater than the ratio of recombinant ovalbumin protein monophosphorylated at Ser 68 in a protein composition expressed by a recombinant cell that lacks heterologous or exogenous kinase.

98. The protein composition of claim 95, wherein the ratio of recombinant ovalbumin protein diphosphorylated at Ser 68 and at Ser 344 is greater than the ratio of recombinant ovalbumin protein diphosphorylated at Ser 68 and at Ser 344 in a protein composition expressed by a recombinant cell that lacks heterologous or exogenous kinase.

99. The protein composition of claim 95, wherein the ratio of recombinant ovalbumin protein monophosphorylated at Ser 344 is less than the ratio of recombinant ovalbumin protein monophosphorylated at Ser 344 in a protein composition expressed by a recombinant cell that lacks heterologous or exogenous kinase.

100. The protein composition of claim 95, wherein the ratio of recombinant ovalbumin protein monophosphorylated at Ser 68 is less than the ratio of recombinant ovalbumin protein monophosphorylated at Ser 68 in a protein composition expressed by a recombinant cell that lacks heterologous or exogenous kinase.

101. The protein composition of claim 95, wherein the ratio of recombinant ovalbumin protein diphosphorylated at Ser 68 and at Ser 344 is less than the ratio of recombinant ovalbumin protein diphosphorylated at Ser 68 and at Ser 344 in a protein composition expressed by a recombinant cell that lacks heterologous or exogenous kinase.

102. The protein composition of any one of claims 95-101, wherein the recombinant phosphorylated ovalbumin improves functionality in a food product.

103. The protein composition of claim 102, wherein the recombinant phosphorylate ovalbumin provides to a food product at least one egg white characteristic selected from gelling, foaming, whipping, fluffing, binding, springiness, aeration, coating, film forming, emulsification, browning, thickening, texturizing, humectant, clarification, and cohesiveness, wherein the characteristic is improved as compared to recombinant ovalbumin that is not phosphorylated.

104. The protein composition of claim 102 or 103, wherein the food product is a cake, a pudding, a macaron, a bread, a roll, a pastry, a cracker, a muffin, a scone, a biscuit, a bagel, a pancake, a meringue, a choux pastry a souffle, a scramble, an omelet, a patty, a quiche, a frittata, a mouse, a custard, a popsicle, a frozen desert, an ice cream, or a cookie.

105. The protein composition of any one of claims 95-104, wherein the recombinant ovalbumin protein comprises an amino acid sequence with at least 70% homology to any one of SEQ ID NO: 1 to SEQ ID NO: 76

106. The protein composition of any one of claims 95-105, wherein the recombinant ovalbumin protein comprises an amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 76.

107. The protein composition of any one of claims 95-106, wherein the recombinant ovalbumin protein comprises an amino acid sequence of a chicken ovalbumin, an ostrich ovalbumin, or a duck ovalbumin.

108. The protein composition of any one of claims 95-107, wherein a heterologous or exogenous kinase protein recognizes a recognition motif comprising a S-X-E/pS motif.

109. The protein composition of claim 108, wherein the recognition motif comprises Glu at a +2 position.

110. The protein composition of any one of claims 95-109, wherein the recombinant ovalbumin protein is glycosylated prior to phosphorylation.

111. The protein composition of any one of claims 95-110, wherein the recombinant ovalbumin is phosphorylated and glycosylated.

112. The protein composition of any one of claims 95-111, wherein the glycosylation moiety comprises at least one mannose moiety or one N-acetylglucosamine moiety.

113. The protein composition of any one of claims 95-112, wherein an unglycosylated recombinant phosphorylated ovalbumin is unphosphorylated.

114. The protein composition of any one of claims 95-112, wherein a phosphorylated recombinant ovalbumin is more stable than an unphosphorylated recombinant ovalbumin.