Proteins, methods and systems for subcellular localization of proteins for post-translational modifications

Abstract

The present disclosure provides proteins, methods, and systems for targeting enzymes to specific subcellular locations to produce post-translational modifications on proteins. Specifically, kinases for phosphorylating proteins containing one or more sites susceptible to phosphorylation are disclosed, along with methods and systems for phosphorylating proteins of interest, such as proteins with nutritional and therapeutic uses. Recombinant host cells engineered to express a non-naturally occurring polypeptide comprising a heterologous serine / threonine kinase, wherein the heterologous serine / threonine kinase is anchored in an intracellular membrane, are disclosed herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 436,041, filed December 29, 2022, the entire contents of which are incorporated by reference. [Background technology]

[0002] background Kinases that act to phosphorylate proteins can be used to produce therapeutic and nutritional protein products. For example, the assembly and stabilization of protein micelles (protein aggregates that provide structural properties to protein products) often involve protein phosphorylation by one or more kinases. These protein micelles may also contain one or more minerals, such as calcium and phosphorus (e.g., calcium phosphate). The formation of protein micelles, and the phosphorylation of proteins by protein kinases that results in micelle formation, can significantly contribute to the therapeutic and nutritional properties of protein products. The use of recombinant non-animal host cells to produce animal-derived phosphorylated proteins, which boasts advantages in speed, scale, and cost, may be limited in part by the absence of suitable kinases in the secretory pathway of recombinant non-animal cells. Furthermore, the expression of animal-derived kinases in recombinant non-animal cells that retain their full phosphorylation function can be difficult. Therefore, the use of recombinant non-animal cells to produce animal-derived phosphorylated proteins has met with limited success. Summary of the Invention

[0003] overview Disclosed herein is a recombinant host cell engineered to express a non-naturally occurring polypeptide comprising a heterologous serine / threonine kinase, wherein the heterologous serine / threonine kinase is anchored in an intracellular membrane. The heterologous serine / threonine kinase can be a human, bovine, primatial, avian, or reptilian serine / threonine kinase or a biologically active portion thereof. The heterologous serine / threonine kinase can be a human serine / threonine kinase or a biologically active portion thereof. The heterologous serine / threonine kinase can be a lemur serine / threonine kinase or a biologically active portion thereof. The heterologous serine / threonine kinase can comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:7. The heterologous serine / threonine kinase can consist of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:7.

[0004] Disclosed herein is a non-naturally occurring polypeptide comprising a serine / threonine kinase coupled to a heterologous domain capable of anchoring the serine / threonine kinase to an intracellular membrane.

[0005] In some embodiments, the serine / threonine kinase comprises a human, bovine, primate, avian, or reptilian serine / threonine kinase. In some embodiments, the serine / threonine kinase comprises a human serine / threonine kinase. In some embodiments, the human serine / threonine kinase comprises Fam20c kinase. In some embodiments, the human serine / threonine kinase is Fam20c kinase. In some embodiments, the serine / threonine kinase comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:7.

[0006] In some embodiments, the serine / threonine kinase comprises a truncation at the N-terminal end of up to 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids, hi some embodiments, the serine / threonine kinase comprises a truncation at the N-terminal end of up to 92 amino acids.

[0007] In some embodiments, the serine / threonine kinase consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the serine / threonine kinase consists of the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the serine / threonine kinase comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 142. In some embodiments, the serine / threonine kinase consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 142. In some embodiments, the serine / threonine kinase consists of the amino acid sequence set forth in SEQ ID NO: 142. In some embodiments, the serine / threonine kinase comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 143. In some embodiments, the serine / threonine kinase consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 143. In some embodiments, the serine / threonine kinase consists of the amino acid sequence set forth in SEQ ID NO: 143. In some embodiments, the serine / threonine kinase comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 144. In some embodiments, the serine / threonine kinase consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 144. In some embodiments, the serine / threonine kinase consists of the amino acid sequence set forth in SEQ ID NO:144.

[0008] In some embodiments, the heterologous domain is coupled to the N-terminus of the serine / threonine kinase. In some embodiments, the heterologous domain is coupled to the C-terminus of the serine / threonine kinase. In some embodiments, the heterologous domain comprises a fragment of a type I or type II transmembrane protein. In some embodiments, the fragment of a type I or type II transmembrane protein comprises a type I or type II transmembrane domain. In some embodiments, the heterologous domain comprises a fragment of a type II transmembrane protein. In some embodiments, the fragment of a type II transmembrane protein comprises a type II transmembrane domain. In some embodiments, the fragment of a type II transmembrane protein comprises a type II transmembrane domain. In some embodiments, the fragment of a type II transmembrane protein comprises a type II transmembrane protein with a truncation at the C-terminus. In some embodiments, the fragment of a type II transmembrane protein is 160 amino acids or less, 100 amino acids or less, or 60 amino acids or less. In some embodiments, the fragment of a type II transmembrane protein is derived from Saccharomyces cerevisiae or Pichia pastoris. In some embodiments, the fragment of a type II transmembrane protein is derived from Saccharomyces cerevisiae. In some embodiments, the fragment of a type II transmembrane protein is derived from Pichia pastoris. In some embodiments, the heterologous domain comprises a multipass transmembrane domain.

[0009] In some embodiments, the heterologous domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 23-85. In some embodiments, the heterologous domain consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 23-85. In some embodiments, the heterologous domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 71. In some embodiments, the heterologous domain consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 71. In some embodiments, the non-naturally occurring polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 93, 95-98, 100-109, 111-114, 116, 117, 119-125, 127-130, 132, 133, 136-141, and 154. In some embodiments, the non-naturally occurring polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 133. In some embodiments, the non-naturally occurring polypeptide consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 93, 95-98, 100-109, 111-114, 116, 117, 119-125, 127-130, 132, 133, 136-141, and 154. In some embodiments, the non-naturally occurring polypeptide consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 133.

[0010] In some embodiments, the non-naturally occurring polypeptide further comprises a tag. In some embodiments, the tag comprises a FLAG tag. In some embodiments, the tag comprises a MYC tag.

[0011] In some embodiments, the heterologous domain comprises a Golgi retention sequence.

[0012] In some embodiments, the intracellular membrane comprises a Golgi apparatus membrane, an endoplasmic reticulum membrane, a nuclear membrane, or a mitochondrial membrane. In some embodiments, the intracellular membrane comprises an endoplasmic reticulum membrane. In some embodiments, the intracellular membrane is a Golgi apparatus membrane. In some embodiments, the serine / threonine kinase is anchored such that the kinase domain of the serine / threonine kinase is located on the luminal side of the Golgi apparatus membrane.

[0013] Described herein are nucleic acid molecules that encode any one of the non-naturally occurring polypeptides described herein.

[0014] Described herein are expression vectors that encode (i) any one of the non-naturally occurring polypeptides described herein and (ii) a secretory protein.

[0015] Described herein is a recombinant host cell comprising (i) any one of the non-naturally occurring polypeptides described herein, wherein the serine / threonine kinase is heterologous to the recombinant host cell, and (ii) any one of the nucleic acid molecules described herein.

[0016] In some embodiments, the recombinant host cell comprises a fungal host cell, a bacterial host cell, an algal host cell, or a plant host cell. In some embodiments, the recombinant host cell comprises a bacterium. In some embodiments, the bacterium is Escherichia coli or Bacillus subtilis.

[0017] In some embodiments, the recombinant host cell comprises a eukaryotic host cell. In some embodiments, the eukaryotic host cell comprises a fungus. In some embodiments, the fungus comprises Aspergillus, Candida, Fusarium, Hansenula, Kluyveromyces, Pichia, Penicillium, Saccharomyces, Tetrahymena, Thermothelomyces, Trichoderma, Yarrowia, or Zygosaccharomyces. In some embodiments, the fungus comprises a yeast cell. In some embodiments, the fungus comprises Kluyveromyces lactis. In some embodiments, the fungus comprises Pichia pastoris. In some embodiments, the fungus comprises a filamentous fungus. In some embodiments, the filamentous fungus comprises Aspergillus or Thermothelomyces heterothallica.

[0018] In some embodiments, the serine / threonine kinase is anchored to an intracellular membrane via a lipid. In some embodiments, the lipid is covalently attached to the serine / threonine kinase. In some embodiments, the serine / threonine kinase and the lipid form a prenylated protein. In some embodiments, the lipid covalently attached to the serine / threonine kinase comprises a fatty acyl group. In some embodiments, the lipid and the serine / threonine kinase form a glycosylphoshaditylinositol-linked protein.

[0019] Described herein are kits that include a nucleic acid molecule encoding any one of the non-naturally occurring polypeptides described herein and a nucleic acid molecule encoding a secreted protein. In some embodiments, the kit further includes a host cell.

[0020] Described herein are methods that include using any one of the serine / threonine kinases of the recombinant host cells described herein to phosphorylate a secreted protein, thereby producing a phosphorylated secreted protein. In some embodiments, the method further includes recovering the phosphorylated secreted protein from the recombinant host cell, thereby producing a recovered phosphorylated secreted protein. In some embodiments, the method further includes using the recovered phosphorylated secreted protein for the production of a food product.

[0021] In some embodiments, the food product is a dairy product. In some embodiments, the food product is a dairy substitute. In some embodiments, the food product is cheese.

[0022] Described herein are fungal cells comprising a heterologous kinase, wherein the heterologous kinase is a Fam20c member. In some embodiments, the heterologous kinase is a human Fam20c member. In some embodiments, the Fam20c member is a truncated Fam20c member.

[0023] Described herein are caseins phosphorylated at one or more amino acid residues that are not naturally phosphorylated. In some embodiments, the casein is αS1-casein. In some embodiments, the αS1-casein is phosphorylated at S56, S61, S63, T64, S79, S81, S82, S83, S90, S103, or S130. In some embodiments, the αS1-casein is phosphorylated at S56, S79, S81, S82, S83, or S103. In some embodiments, the αS1-casein is phosphorylated at S56. In some embodiments, the αS1-casein is phosphorylated at S56, S61, S63, T64, S79, S81, S82, S83, S90, S103, and S130. In some embodiments, the phosphorylated casein is αS2-casein. In some embodiments, αS2-casein is phosphorylated at S23, S24, S25, S46, S52, S71, S72, S73, S76, T145, S146, S150, S158, T159, or T163. In some embodiments, αS2-casein is phosphorylated at S23, S24, S25, S52, S71, S72, S73, or S76. In some embodiments, αS2-casein is phosphorylated at S23, S52, S71, S72, S73, or S76. In some embodiments, αS2-casein is phosphorylated at S23, S52, S71, S72, S73, and S76. In some embodiments, αS2-casein is phosphorylated at S23, S24, S25, S46, S52, S71, S72, S73, S76, T145, S146, S150, S158, and T159. In some embodiments, the phosphorylated casein is β-casein. In some embodiments, β-casein is phosphorylated at S30, S32, S33, S34, S37, T39, S50, or T56. In some embodiments, β-casein is phosphorylated at S30, S32, S33, S34, S37, or T39. In some embodiments, β-casein is phosphorylated at S37.In some embodiments, the β-casein is phosphorylated at S30, S32, S33, S34, S37, T39, S50, and T56. In some embodiments, the phosphorylated casein is κ-casein.

[0024] In some embodiments, the casein is phosphorylated at two or more amino acid residues that are not naturally phosphorylated. In some embodiments, the casein is phosphorylated at three or more amino acid residues that are not naturally phosphorylated. In some embodiments, the casein is phosphorylated at four or more amino acid residues that are not naturally phosphorylated. In some embodiments, the casein is phosphorylated at five or more amino acid residues that are not naturally phosphorylated. In some embodiments, the casein is phosphorylated at six or more amino acid residues that are not naturally phosphorylated.

[0025] Described herein are engineered food products comprising the caseins described herein. In some embodiments, the food product is a dairy product. In some embodiments, the food product is a dairy substitute. In some embodiments, the food product is cheese. Described herein are methods for producing phosphorylated caseins described herein. In some embodiments, the methods comprise phosphorylating casein using a non-naturally occurring polypeptide described herein.

[0026] The heterologous serine / threonine kinase can be a primate serine / threonine kinase or a biologically active portion thereof. The heterologous serine / threonine kinase can be a lemur serine / threonine kinase or a biologically active portion thereof. The heterologous serine / threonine kinase can comprise an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO:6. The heterologous serine / threonine kinase can consist of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO:6.

[0027] The heterologous serine / threonine kinase can be an avian serine / threonine kinase or a biologically active portion thereof. The heterologous serine / threonine kinase can be a sandgrouse serine / threonine kinase or a biologically active portion thereof. The heterologous serine / threonine kinase can comprise an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO:8. The heterologous serine / threonine kinase can consist of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO:8.

[0028] In some embodiments, the heterologous serine / threonine kinase is less than 400 amino acids in length. In some embodiments, the heterologous serine / threonine kinase is less than 360 amino acids in length. In some embodiments, the heterologous serine / threonine kinase is less than 350 amino acids in length. In some embodiments, the heterologous serine / threonine kinase is less than 300 amino acids in length. In some embodiments, the heterologous serine / threonine kinase is less than 200 amino acids in length. In some embodiments, the heterologous serine / threonine kinase is less than 140 amino acids in length.

[0029] In some embodiments, the heterologous serine / threonine kinase or biologically active portion thereof is a heterologous reptilian serine / threonine kinase or biologically active portion thereof. In some embodiments, the heterologous reptilian serine / threonine kinase is a reptilian Fam20c protein or biologically active portion thereof. In some embodiments, the reptilian Fam20c protein is a snake Fam20c protein or biologically active portion thereof. The snake Fam20c protein or biologically active portion thereof can comprise an amino acid sequence having at least about 80%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:3. The snake Fam20c protein can comprise the amino acid sequence of SEQ ID NO:3. The heterologous serine / threonine kinase can comprise a sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO:3, but does not include a sequence longer than 20 amino acids in length having 90% sequence identity to any region of the sequence set forth in SEQ ID NO:9.

[0030] In some embodiments, the heterologous serine / threonine kinase disclosed herein can be anchored to an intracellular membrane via a polypeptide. The heterologous serine / threonine kinase can be linked to the polypeptide at the N-terminus or C-terminus of the heterologous serine / threonine kinase. The polypeptide can be a type 1, type 2, or multi-pass transmembrane domain. The polypeptide can include a Golgi retention sequence. The polypeptide can include a sequence set forth in any one of SEQ ID NOS: 21-22. In some embodiments, the polypeptide set forth in any one of SEQ ID NOS: 21-22 is at the C-terminus of the polypeptide. The polypeptide can include an amino acid sequence having at least about 75%, 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in any one of SEQ ID NOS: 23-85. The polypeptide can include the amino acid sequence set forth in any one of SEQ ID NOS: 23-85.

[0031] In some embodiments, the heterologous serine / threonine kinase disclosed herein can be anchored to an intracellular membrane via a lipid. In some embodiments, the lipid is covalently attached to the heterologous serine / threonine kinase. In some embodiments, the heterologous serine / threonine kinase and the lipid form a prenylated protein. The lipid can include a fatty acyl group. In some embodiments, the lipid attached to the heterologous serine / threonine kinase can form a glycosylphoshaditylinositol-linked protein. The intracellular membrane can be the endoplasmic reticulum (ER) membrane, nuclear membrane, mitochondrial membrane, or Golgi apparatus membrane. The heterologous serine / threonine kinase can be anchored to the Golgi apparatus membrane such that the kinase domain of the heterologous serine / threonine kinase is located on the luminal side of the Golgi apparatus membrane.

[0032] The serine / threonine kinase can further comprise a purification, identification, or solubility enhancer tag. The purification and / or identification tag can be a FLAG tag. The solubility enhancer tag can be a SUMO tag, a TRX tag, a MBP tag, a MISTIC tag, a NusA tag, a FLAG-SUMO tag, a FLAG-TRX tag, a FLAG-MBP tag, a FLAG-MISTIC tag, or a FLAG-NusA tag. The purification and / or identification tag can be a FLAG tag comprising the sequence set forth in SEQ ID NO: 10. The solubility enhancer tag can be a SUMO tag comprising the sequence set forth in SEQ ID NO: 11. The solubility enhancer tag can be a TRX tag comprising the sequence set forth in SEQ ID NO: 12. The solubility enhancer tag can be a MBP tag comprising the sequence set forth in SEQ ID NO: 13. The solubility enhancer tag can be a MISTIC tag comprising the sequence set forth in SEQ ID NO: 14. The solubility enhancer tag can be a NusA tag comprising the sequence set forth in SEQ ID NO: 15. The solubility enhancer tag may be a FLAG-SUMO tag comprising the sequence set forth in SEQ ID NO: 16. The solubility enhancer tag may be a FLAG-TRX tag comprising the sequence set forth in SEQ ID NO: 17. The solubility enhancer tag may be a FLAG-MBP tag comprising the sequence set forth in SEQ ID NO: 18. The solubility enhancer tag may be a FLAG-MISTIC tag comprising the sequence set forth in SEQ ID NO: 19. The solubility enhancer tag may be a FLAG-NusA tag comprising the sequence set forth in SEQ ID NO: 20.

[0033] In some embodiments, described herein are recombinant host cells that can be configured to express a heterologous secretory protein that is susceptible to phosphorylation by a serine / threonine kinase. The heterologous secretory protein can be a therapeutic protein or a nutritive protein. The nutritive protein can be a dairy protein. The dairy protein can be casein or a portion thereof. The casein can be αs1-casein, αs2-casein, β-casein, or κ-casein or a portion thereof. The dairy protein can be glycomacropeptide or a portion thereof. The dairy protein can be osteopontin or a portion thereof. The dairy protein can be lactoferrin or a portion thereof. The nutritive protein can be an egg white protein. The egg white protein can be ovalbumin or a portion thereof.

[0034] The heterologous secreted protein containing one or more phosphorylation sites can be a component of a protein complex. The protein complex can include a first protein and a second protein. The first protein can be a heterologous protein containing one or more phosphorylation sites susceptible to phosphorylation by a serine / threonine kinase. The second protein can be a heterologous protein containing one or more phosphorylation sites susceptible to phosphorylation by a serine / threonine kinase. The protein complex can further include four proteins, each of which contains one or more phosphorylation sites susceptible to phosphorylation by a serine / threonine kinase.

[0035] The recombinant host cell can be a fungal recombinant host cell, a bacterial recombinant host cell, an algal recombinant host cell, or a plant recombinant host cell. The recombinant host cell can be a bacterium. The bacterium can be Escherichia coli or Bacillus subtilis. The recombinant host cell can be a eukaryotic cell. The recombinant host cell can be a fungus. The fungus can be Aspergillus, Candida, Fusarium, Hansenula, Kluyveromyces, Pichia (synonym Komagataella), Penicillium, Saccharomyces, Tetrahymena, Trichoderma, Yarrowia, or Zygosaccharomyces. The fungus can be a yeast. The yeast cell can be Kluyveromyces lactis or Pichia pastoris (synonym Komagataella phaffi). The fungus can be a filamentous fungus. The filamentous fungus may be Aspergillus.

[0036] Recombinant host cells comprising expression vectors are described herein.The expression vectors can comprise nucleic acid sequences encoding heterologous serine / threonine kinases.In some embodiments, recombinant host cells comprising expression vectors can comprise nucleic acid sequences encoding (1) heterologous serine / threonine kinases and (2) polypeptides linked to heterologous serine / threonine kinases are described herein.

[0037] Described herein are compositions comprising a heterologous serine / threonine kinase conjugated to a domain for anchoring the serine / threonine kinase to an intracellular membrane. In some embodiments, the composition can be expressed in a recombinant host cell as described above. The domain can be a polypeptide. The polypeptide can be a type 1, type 2, or multi-pass transmembrane domain. The polypeptide can include a Golgi retention sequence. The polypeptide can include a sequence set forth in any one of SEQ ID NOS: 21-22. In some embodiments, the polypeptide set forth in any one of SEQ ID NOS: 21-22 is at the C-terminus of the polypeptide. The polypeptide can include an amino acid sequence having at least about 75%, 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in any one of SEQ ID NOS: 23-85. The polypeptide can include the amino acid sequence set forth in any one of SEQ ID NOS: 23-85. The domain can be a lipid. In some embodiments, the lipid is covalently attached to the heterologous serine / threonine kinase. In some embodiments, the heterologous serine / threonine kinase and the lipid form a prenylated protein. The lipid can include a fatty acyl group. In some embodiments, the lipid binding to the heterologous serine / threonine kinase can form a glycosylphoshaditylinositol-linked protein. The intracellular membrane can be the endoplasmic reticulum (ER) membrane, the nuclear membrane, the mitochondrial membrane, or the Golgi apparatus membrane. The heterologous serine / threonine kinase can be anchored to the Golgi apparatus membrane such that the kinase domain of the heterologous serine / threonine kinase is located on the luminal side of the Golgi apparatus membrane.

[0038] Described herein are secreted proteins phosphorylated by the composition. In some embodiments, described herein are food products comprising secreted proteins phosphorylated by the composition. The food product can be a dairy product or dairy substitute. The food product can be cheese.

[0039] Described herein previously is the use of recombinant host cells to produce a food product. Described herein previously is the use of recombinant host cells to produce a food product, wherein the food product can be a dairy product or a dairy substitute. Described herein previously is the use of recombinant host cells to produce a food product, wherein the food product can be cheese.

[0040] Described herein is a method for producing a phosphorylated secreted protein, comprising expressing in a cell population comprising a plurality of recombinant host cells: (1) a heterologous serine / threonine kinase conjugated to a domain for anchoring the serine / threonine kinase to an intracellular membrane, and (2) a heterologous secreted protein comprising one or more phosphorylation sites accessible to phosphorylation by the serine / threonine kinase. The method may further comprise: (1) transforming the cell population with a first vector encoding the serine / threonine kinase under the control of a first inducible promoter; and (2) transforming the cell population with a second vector encoding the secreted protein comprising one or more phosphorylation sites accessible to phosphorylation by the serine / threonine kinase under the control of a second inducible promoter. The method may further include transforming the cell population with a vector encoding both (1) a serine / threonine kinase under the control of a first inducible promoter and (2) a secreted protein comprising one or more phosphorylation sites susceptible to phosphorylation by the serine / threonine kinase under the control of a second inducible promoter. The method may further include integrating into the recombinant host cell genome a first fragment of DNA encoding the serine / threonine kinase under the control of the first promoter and a second fragment of DNA encoding the secreted protein comprising one or more phosphorylation sites susceptible to phosphorylation by the serine / threonine kinase under the control of a second promoter. The method may further include integrating into the recombinant host cell genome fragments of DNA encoding both (1) a serine / threonine kinase under the control of the first promoter and (2) a secreted protein comprising one or more phosphorylation sites susceptible to phosphorylation by the serine / threonine kinase under the control of the second promoter.

[0041] The recombinant host cells used in the methods described herein can include any of the recombinant host cells described above. The domain of the methods described herein can be a polypeptide. The polypeptide of the methods described herein can be a type 1, type 2, or multi-pass transmembrane domain. The polypeptide of the methods described herein can include a Golgi retention sequence. The polypeptide of the methods described herein can include a sequence set forth in any one of SEQ ID NOs: 21-22. In some embodiments, the polypeptide of any one of SEQ ID NOs: 21-22 is C-terminal to the polypeptide of the methods described herein. The polypeptide of the methods described herein can include an amino acid sequence having at least about 75%, 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in any one of SEQ ID NOs: 23-85. The polypeptide of the methods described herein can include the amino acid sequence of any one of SEQ ID NOs: 23-85. The domain of the methods described herein can be a lipid. In some embodiments, the lipid of the methods described herein is covalently attached to the heterologous serine / threonine kinase. In some embodiments, the heterologous serine / threonine kinase and lipid of the methods described herein form a prenylated protein. The lipid of the methods described herein can include a fatty acyl group. In some embodiments, the lipid binding to the heterologous serine / threonine kinase of the methods described herein can form a glycosylphoshaditylinositol-linked protein. The intracellular membrane of the methods described herein can be the endoplasmic reticulum (ER) membrane, nuclear membrane, mitochondrial membrane, or Golgi apparatus membrane. The heterologous serine / threonine kinase of the methods described herein can be anchored to the Golgi apparatus membrane such that the kinase domain of the heterologous serine / threonine kinase is located on the luminal side of the Golgi apparatus membrane.

[0042] In the method described herein, expressing a heterologous serine / threonine kinase can include culturing a recombinant host cell in a culture medium. The culture medium can be BMGY medium. The first promoter of the method described herein can be an inducible promoter or a constitutive promoter. The second promoter of the method described herein can be an inducible promoter or a constitutive promoter. The first promoter and the second promoter of the method described herein can be the same.

[0043] The inducible promoter can be AOX1, ADH3, DAS, FLD1, THI11, GTH1, CUP1, LRA3, LRA4, amyB, bphA, catA, gloaA, or thiA. The constitutive promoter can be GAP, TEF1, YPT1, PGK1, adhA, gdhA, pkgA, or pkiA. Expressing the heterologous serine / threonine kinase can include adding an inducer to the culture medium. The inducer can be methanol, IPTG, ethanol, maltose, starch, xylose, thiamine, copper, quinic acid, nitrate, glucose, galactose, sugars, H2O2, CaCO3, or benzoate. The heterologous serine / threonine kinase can be induced by light, blue light, low pH, iron starvation, or copper depletion. The method can further include culturing the plurality of recombinant cells at a temperature between 20°C and 40°C. The method may further include recovering the phosphorylated protein by centrifuging the plurality of recombinant host cells and collecting the supernatant.

[0044] Described herein are expression vectors encoding (1) a heterologous serine / threonine kinase conjugated to a domain for anchoring the serine / threonine kinase to an intracellular membrane, and (2) a heterologous secreted protein comprising one or more phosphorylation sites susceptible to phosphorylation by the serine / threonine kinase. In some embodiments, the heterologous serine / threonine kinase conjugated to a domain for anchoring the serine / threonine kinase to an intracellular membrane comprises the aforementioned composition. In some embodiments, the secreted protein of the expression vector is a therapeutic protein. In some embodiments, the secreted protein of the expression vector is a nutritive protein. In some embodiments, the nutritive protein is a dairy protein. The nutritive protein can be a dairy protein. The dairy protein can be casein or a portion thereof. The casein can be αs1-casein, αs2-casein, β-casein, or κ-casein or a portion thereof. The dairy protein can be glycomacropeptide or a portion thereof. The dairy protein can be osteopontin or a portion thereof. The dairy protein can be lactoferrin or a portion thereof. The nutritive protein can be egg white protein. The egg white protein can be ovalbumin or a portion thereof.

[0045] The heterologous secreted protein containing one or more phosphorylation sites of the expression vector can be a component of a protein complex. The protein complex can include a first protein and a second protein. The first protein can be a heterologous protein containing one or more phosphorylation sites susceptible to phosphorylation by a serine / threonine kinase. The second protein can be a heterologous protein containing one or more phosphorylation sites susceptible to phosphorylation by a serine / threonine kinase. The protein complex can further include four proteins, each of which contains one or more phosphorylation sites susceptible to phosphorylation by a serine / threonine kinase.

[0046] Incorporation by Reference 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. [Brief explanation of the drawings]

[0047] [Figure 1] Figure 1 shows a cartoon illustrating the alignment of Fam20c protein kinases from snake (SEQ ID NO: 3), lemur (SEQ ID NO: 6), and sandgrouse (SEQ ID NO: 8) compared to the human Fam20c protein kinase sequence (top, SEQ ID NO: 7). The putative Fam20c kinase domain annotated by NCBI is shown second from the bottom.

[0048] [Figure 2] Figure 2 shows an alignment of Fam20c protein kinase sequences from human (top row, SEQ ID NO: 7), bovine (second row, SEQ ID NO: 2), mouse (third row, SEQ ID NO: 1), sandgrouse (fourth row, SEQ ID NO: 8), lemur (fifth row, SEQ ID NO: 6) and snake (sixth row, SEQ ID NO: 3).

[0049] [Figure 3] Figure 3 shows an image of a Western blot of proteins secreted by transgenic P. pastoris CBS7435 expressing the snake Fam20c Common Garter Snake (FCGS)-3, FCGS-5, or FCGS-8 mutants, probed with an anti-FLAG primary antibody conjugated to HRP.

[0050] [Figure 4A-E] Figure 4A shows an image of a Western blot used to detect extracellular mouse Fam20c protein (SEQ ID NO: 1) extracted from the supernatant portion of transgenic Pichia pastoris CBS7435, probed with an anti-FLAG primary antibody conjugated to HRP.

[0051] Figure 4B shows an image of a Western blot used to detect extracellular bovine Fam20c protein (SEQ ID NO: 2) extracted from the supernatant of transgenic Pichia pastoris CBS7435, probed with an anti-FLAG primary antibody conjugated to HRP.

[0052] Figure 4C shows an image of a Western blot used to detect intracellular mouse Fam20c protein (SEQ ID NO: 1) extracted from cell lysates of transgenic Pichia pastoris CBS7435, probed with an anti-FLAG primary antibody conjugated to HRP.

[0053] Figure 4D shows an image of a Western blot used to detect intracellular bovine Fam20c protein (SEQ ID NO: 2) extracted from cell lysates of transgenic Pichia pastoris CBS7435, probed with an anti-FLAG primary antibody conjugated to HRP.

[0054] Figure 4E shows an image of a Western blot used to detect intracellular snake Fam20c protein (SEQ ID NO: 3) extracted from cell lysates of transgenic Pichia pastoris CBS7435, probed with an anti-FLAG primary antibody conjugated to HRP.

[0055] [Figure 5] FIG. 5 shows an image of a Western blot used to detect intracellular expression of soluble and insoluble bovine αS1 casein protein (SEQ ID NO: 4) fractions under the control of the pAOXI promoter extracted from transgenic PichiaPink™ yeast, probed with an anti-FLAG primary antibody conjugated to HRP.

[0056] [Figure 6]Figure 6 shows an image of a Western blot used to detect extracellular expression of human osteopontin protein (SEQ ID NO: 5) under the control of the pAOXI promoter in extracts from transgenic Pichia pastoris CBS7435 yeast, probed with an anti-FLAG primary antibody conjugated to HRP.

[0057] [Figure 7A-D] Figures 7A-7D show a schematic of the approach to engineering the human serine / threonine protein kinase Fam20c (huFam20c) for fungal expression. Figure 7A shows the structure of the type II transmembrane domain protein. Figure 7B shows an illustration of huFam20c and three truncations. Figure 7C shows three truncations of five different fungal type II transmembrane proteins. "SP" refers to signal peptide, "TM" refers to transmembrane domain, "Sc" refers to Saccharomyces cerevisiae, and "Pp" refers to Pichia pastoris. Numbers indicate amino acid sequence. KRE2 is a glycolipid 2-alpha-mannosyltransferase, MNN2 is an alpha 1,2-mannosyltransferase, MNN1 is an alpha 1,3-mannosyltransferase, and MNN6 is a mannosyltransferase, also referred to as KTR6. Figure 7D shows the generation of engineered mutants by combining three truncations of huFam20c (Figure 7B) with a fungal localization sequence (Figure 7C).

[0058] [Figure 8A-D]Figures 8A-8D show the expression of the human serine / threonine protein kinase Fam20c (huFam20c) in Pichia pastoris. Expression of Fam20c(M1-R584)_FLAG and four fragments (R32-R584_FLAG, R64-R854_FLAG, D93-R584_FLAG, and Q289-R584_FLAG) using PichiaPink™ strain 4 (Δade2, Δprb1, Δpep4). The strain was expressed at 20°C and 30°C for 48 hours in BMMY medium, where methanol induces recombinant production. Supernatant samples were separated by denaturing electrophoresis, transferred to nitrocellulose blots, and probed with an HRP-conjugated anti-DYDDDDK mouse monoclonal antibody. Figure 8A shows a schematic diagram of the huFam20c structure and various truncated forms of huFam20c. Four truncations (R31, R64, D93, and Q289) are shown. Figure 8B shows that the native transmembrane domain of Fam20c(M1-R584)_FLAG exhibits leaky secretion in yeast. Removal of this sequence (Fam20c(R32-R584)_FLAG) abolishes this effect. Leaky secretion is not the result of insufficient target expression, as demonstrated by increased expression with the yeast signal peptide (α_Fam20c(R32-R584)_FLAG). Figure 8C and Figure 8D show that Fam20c(M1-R584)_FLAG can be truncated at the N-terminus (α_Fam20c(R32-R584)_FLAG, α_Fam20c(R64-R584)_FLAG, and α_Fam20c(D93-R584)_FLAG) to generate mutants for localization, but the minimal mutant α_Fam20c(Q289-R584)_FLAG exhibits poor expression and degradation. The image inset in Figure 8D shows the blot after long exposure.

[0059] [Figure 9A-C]Figures 9A-9C show in vivo phosphorylation by engineered human serine / threonine protein kinase Fam20c, as well as the corresponding unengineered and delocalized controls. Coexpression of FLAG_TRX_SPP1(G158-N314)_HiBiT with engineered huFam20c and the corresponding unengineered and delocalized controls was tested in PichiaPink™ strain 4 (Δade2, Δprb1, Δpep4). Strains were expressed in YPD medium (expression in this medium is constitutive) at 30°C for 48 hours. Figure 9A shows supernatant samples separated by denaturing electrophoresis, transferred to a nitrocellulose blot, and probed with an anti-DYDDDDK mouse monoclonal antibody conjugated to HRP. Protein migration in the gel is affected by phosphorylation. Figure 9B shows quantification of phosphorylation; supernatant samples were acetone precipitated, and intact isoforms were identified by LC-MS. Figure 9C shows the abundance of each phosphorylated isoform, quantified by integrating the spectral peak associated with each. "ND" indicates not detected.

[0060] [Figure 10] Figure 10 shows the phosphorylation of non-recombinant and recombinant ovalbumin. Co-expression of FLAG_SERPINB14_HiBiT with unengineered and engineered human serine / threonine protein kinase Fam20c (huFam20c) in PichiaPink™ strain 4 (Δade2, Δprb1, Δpep4). Strains were expressed in YPD medium (expression is constitutive in this medium) at 30°C for 72 hours. Phosphorylation sites were detected in non-recombinant ovalbumin (Sigma-Aldrich A5503; amino acids underlined), recombinant ovalbumin co-expressed with Fam20c(M1-R584) (triangles), and recombinant ovalbumin(M1-S36)_Fam20c(D93-R584)_FLAG co-expressed with ScMNN2 (circles).

[0061] [Figure 11]Figure 11 shows the phosphorylation of non-recombinant and recombinant αS-1 casein (I), αS-2 casein (II), and β-casein (III). Co-expression of casein with unengineered and engineered human serine / threonine protein kinase Fam20c (huFam20c) in Kluyveromyces lactis (Δku80, Δyps1). Strains were expressed in YPD medium (expression is constitutive in this medium) at 30°C for 72 hours. Phosphorylation sites were detected in non-recombinant casein (amino acids underlined), in recombinant casein co-expressed with Fam20c(M1-R584) (triangles), and in recombinant casein co-expressed with ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG (circles).

[0062] [Figure 12A-D] Figures 12A-12D show expression of recombinant Fam20c protein in P. pastoris. Figure 12A is a Western blot showing lemur Fam20c extracted from the extracellular portion of transgenic P. pastoris CBS7435. Figure 12B is a Western blot showing sandgrouse Fam20c extracted from the extracellular portion of transgenic P. pastoris CBS7435. Figure 12C is a Western blot showing lemur Fam20c extracted from the intracellular portion of transgenic P. pastoris CBS7435. Figure 12D is a Western blot showing sandgrouse Fam20c extracted from the intracellular portion of transgenic P. pastoris CBS7435. DETAILED DESCRIPTION OF THE INVENTION

[0063] Detailed Description I. Introduction Protein phosphorylation by protein kinases can contribute therapeutic and nutritional value to protein products. However, the challenge of expressing functional animal-derived protein kinases in non-animal cells presents a bottleneck that can hinder the production of such phosphorylated proteins in a cost-effective, humane, and environmentally friendly manner. Efficient production of natural animal-derived phosphorylated proteins in non-animal cells can provide a cost-effective, humane, and environmentally friendly source for therapeutic and nutritional protein products. In certain embodiments, described herein are recombinant host cells (e.g., yeast) engineered to express a kinase (e.g., a serine / threonine kinase) and a heterologous protein (e.g., a casein protein). The kinase can be chimeric and / or heterologous relative to the recombinant host cell. The heterologous protein can be therapeutic and / or nutritional and can be composed of one or more post-translational modification (e.g., phosphorylation) sites. The expressed heterologous protein can be susceptible to phosphorylation by the expressed kinase.

[0064] The kinase may be a serine / threonine kinase. In some embodiments, the kinase may be engineered to have features (e.g., the inclusion of a solubility enhancer) that improve the efficiency of phosphorylation of therapeutic and nutritive protein products. In some examples, the kinase may be 400 or less, or 300 or less amino acids in length, and in some embodiments, less than 140 amino acids in length. Kinases with shorter amino acid lengths may increase the efficiency of phosphorylation of heterologous protein substrates in several ways. Reducing kinase length may reduce the number of proteolytic and post-translational modification motifs, which may contribute to increased protein stability. This may enhance the efficiency of kinase phosphorylation by reducing the risk of kinase proteolysis by enzymes endogenously expressed by the recombinant host. This may further enhance the efficiency of kinase phosphorylation by reducing the risk of unproductive post-translational modifications made to the kinase by enzymes endogenously expressed by the recombinant host. Furthermore, kinases with shorter sequences may require fewer amino acids for translation, resulting in stronger kinase expression due to reduced metabolic demands for protein production.

[0065] The kinases described herein can be targeted to a subcellular location of interest (e.g., the Golgi apparatus or the endoplasmic reticulum). In some embodiments, the kinase can be localized to a subcellular location of interest. In some embodiments, the kinase can be anchored to an intracellular membrane (e.g., the nuclear membrane, the Golgi apparatus membrane, the endoplasmic reticulum membrane). In some embodiments, the anchoring mechanism can be provided in part by an anchoring domain. In some embodiments, the anchoring domain can be covalently attached to the kinase. Targeted subcellular localization of the kinase can increase the phosphorylation efficacy of its cognate substrate. Targeted localization of the kinase can increase the concentration of the kinase in a particular subcellular location. Targeted localization of the kinase can increase the half-life of the kinase. Targeted colocalization of the kinase and the substrate can maximize the opportunity for kinase-substrate interaction for enzymatic catalysis. Targeted colocalization of the kinase and the substrate can result in higher efficiency of phosphorylation of the target protein during secretion.

[0066] In some embodiments, described herein is a bioreactor system comprising a plurality of recombinant host cells, a reaction vessel (e.g., a shake flask), and a medium capable of promoting expression of a kinase and a heterologous protein. In some examples, the recombinant host cells and medium may be disposed within the reaction vessel. Also described herein are methods for producing phosphorylated heterologous proteins that may have therapeutic and nutritional value. Such methods may use cell populations comprising a plurality of recombinant host cells and a bioreactor system. The resulting phosphorylated heterologous proteins described herein may contribute to the generation of therapeutic, nutritional, or food products. Collectively, the embodiments described herein may provide a solution that enables the efficient production of naturally occurring animal-derived phosphorylated proteins by non-animal cells in a cost-effective, humane, and / or environmentally friendly manner.

[0067] The following definitions are provided to facilitate explanation of the subject matter of this disclosure.

[0068] All technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art, unless otherwise defined below. References to techniques used herein are intended to refer to techniques commonly understood in the art, including variations of those techniques and / or equivalent technique substitutions that would be apparent to those skilled in the art.

[0069] Any range recited herein is intended to be inclusive, for example, the range 2 to 4 includes 2 and 4.

[0070] As used herein, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. For example, the term "a sample" includes multiple samples, including mixtures thereof.

[0071] The term "about" or "approximately," when immediately preceding a numerical value, refers to ±10% of the provided value. Additionally, the phrases "less than about" a value or "greater than about" a value should be understood in light of the definition of the term "about" provided herein. Similarly, the term "about," when preceding a series of numerical values ​​or ranges of values ​​(e.g., "about 10, 20, 30," or "about 10-30%"), refers to the endpoints of all values ​​in the series or ranges, respectively.

[0072] Unless the context clearly indicates otherwise, as used herein, the terms "including," "includes," "having," "has," "with," or variations thereof, are intended to be inclusive in the same sense as the term "comprising."

[0073] The term "percent sequence identity" with respect to a reference polypeptide sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. The term "percent sequence identity" with respect to a reference nucleic acid sequence refers to the percentage of nucleic acid residues in a candidate sequence that are identical to those in the reference polynucleotide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity. Alignment to determine percent amino acid or nucleic acid sequence identity can be achieved in a variety of known ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment across the entire length of the sequences being compared. The % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it can be expressed as a given amino acid sequence A having or containing a particular % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored by a sequence alignment program as identical matches in that program's alignment of A with B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid or nucleic acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program.When assessing the percent sequence identity of a serine / threonine kinase to a designated sequence, all amino acids of the polypeptide chain containing the serine / threonine kinase are aligned with the designated sequence, except that amino acids associated with tags whose primary purpose is to provide a functional handle for enhancing purification, identification, or solubility are omitted. Such purification and / or identification tags may include FLAG or MYC. Examples of such solubility enhancer tags include SUMO, TRX, MBP, MISTIC, NusA, FLAG-SUMO, FLAG-TRX, FLAG-MBP, FLAG-MISTIC, or FLAG-NusA tags.

[0074] The term "intact" when used in reference to a protein refers to the full-length protein.

[0075] The terms "heterologous" and "foreign" refer to something not normally present in the context being described. When used in reference to a protein expressed in a host cell, the term means that the protein is not naturally produced by the host cell.

[0076] The term "transformation" refers to a process by which nucleic acid is introduced into a cell, either transiently or stably. Transformation can rely on any known method for inserting nucleic acid sequences into prokaryotic or eukaryotic host cells, including Agrobacterium-mediated transformation protocols, viral infection, electroporation, heat shock, lipofection, polyethylene glycol treatment, microinjection, and protoplasting.

[0077] The term "signal peptide" is used herein to refer to a peptide sequence that directs a protein to a specific cellular location or pathway. Signal peptides are often cleaved from proteins during translation or transport and therefore may not be present in the mature protein.

[0078] The term "purifying" is used interchangeably with the term "isolating" and generally refers to the separation of a particular component from other components (e.g., membrane lipids, chromosomes, proteins) of the environment in which it is found or produced. The term allows, but does not require, that the purified or isolated component be separated from all other chemical components.

[0079] The terms "dairy protein" or "milk protein" refer to any protein, or fragment or variant thereof, that can be found in one or more mammalian milk. In some embodiments, the dairy proteins described herein are casein proteins, such as αs1-casein, αs2-casein, β-casein, and κ-casein.

[0080] The term "secreted protein" is used herein to refer to any protein or fragment or variant thereof that is secreted by recombinant host cells into the cell culture medium.

[0081] All publications, patent applications, issued patents, and other documents mentioned herein are incorporated by reference herein as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in the text incorporated by reference are excluded to the extent they conflict with definitions in this disclosure.

[0082] II. Recombinant Host Cells The recombinant host cells described herein can be engineered to express (1) a protein (e.g., a kinase) capable of catalyzing a post-translational modification of a protein, and (2) a heterologous protein containing one or more post-translational modification (e.g., phosphorylation) sites. Such proteins capable of catalyzing protein phosphorylation are referred to herein as kinases. The recombinant host cells herein can be derived from a variety of taxonomic cell types (e.g., eukaryotic cells). In some embodiments, the recombinant host cell can be a fungal recombinant host cell (e.g., yeast), a bacterial recombinant host cell (e.g., Escherichia coli (E. coli)), an algal recombinant host cell, or a plant recombinant host cell. In some embodiments, the fungal recombinant host cell can be a yeast cell, and the yeast cell can be Kluyveromyces lactis or Pichia pastoris. In some embodiments, the fungal recombinant host cell can be a filamentous fungus, such as Aspergillus or Thermocellomyces heterothalica. In some examples, the fungal recombinant host cell can be Candida, Fusarium, Hansenula, Penicillium, Saccharomyces, Tetrahydromina, Thermocellomyces, Trichoderma, Yarrowia, or Zygosaccharomyces.

[0083] In some embodiments, the fungal recombinant host cell can comprise a heterologous kinase, wherein the heterologous kinase is a Fam20c member. In some embodiments, the heterologous kinase can be a human Fam20c member. In some embodiments, the Fam20c member can be a truncated Fam20c member.

[0084] The fungal recombinant host cell can comprise any one of the non-naturally occurring polypeptides described herein, and can comprise any non-naturally occurring polypeptide, including a Fam20c protein.

[0085] Expressed kinases The recombinant host cells described herein can be engineered to express a kinase (e.g., a serine / threonine kinase) capable of phosphorylating a protein substrate of interest. The kinases described herein can be heterologous with respect to the recombinant host cell, can be derived in part from a serine / threonine kinase, and can have multiple characteristics. In some embodiments, the expressed kinase can be a reptilian serine / threonine kinase, and the reptilian serine / threonine kinase can be a snake serine / threonine kinase. In some embodiments, the expressed kinase can have at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:3. In some embodiments, the expressed kinase can have at least 80% sequence identity to SEQ ID NO:3, but does not include a sequence longer than 20 amino acids in length having 90%, 95%, or 100% sequence identity to SEQ ID NO:9. In some embodiments, the expressed kinase may be less than 140, 200, or 300 amino acids in length and may have at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 3. In some embodiments, the expressed kinase may be less than 140, 200, or 300 amino acids in length and may have at least 80% sequence identity to SEQ ID NO: 3, but does not include a sequence longer than 20 amino acids in length that has 90% sequence identity to SEQ ID NO: 9. In some examples, the expressed kinase is snake Fam20c kinase or a biologically active portion thereof.

[0086] In some embodiments, the expressed kinase can be a primate serine / threonine kinase, and the primate kinase can be a lemur serine / threonine kinase. In some embodiments, the expressed kinase can have at least 75%, 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO:6.

[0087] In some embodiments, the expressed kinase can be an avian serine / threonine kinase, and the avian serine / threonine kinase can be a salmon serine / threonine kinase. In some embodiments, the expressed kinase can have at least 75%, 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO:8.

[0088] In some embodiments, the expressed kinase can be a human serine / threonine kinase. In some embodiments, the human serine / threonine kinase can be Fam20c kinase. In some embodiments, the expressed kinase can have at least 75%, 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO:7.

[0089] In some embodiments, the human serine / threonine kinase can be Fam20c kinase having the amino acid sequence of SEQ ID NO: 145. In some embodiments, the expressed kinase can have at least 75%, 80%, 90%, 95% or 100% sequence identity to SEQ ID NO: 145.In some embodiments, the human serine / threonine kinase is at most 1 amino acid, at most 2 amino acids, at most 3 amino acids, at most 4 amino acids, at most 5 amino acids, at most 6 amino acids, at most 7 amino acids, at most 8 amino acids, at most 9 amino acids, at most 10 amino acids, at most 11 amino acids, at most 12 amino acids, at most 13 amino acids, at most 14 amino acids, at most 15 amino acids, at most 16 amino acids, at most 17 amino acids, at most 18 amino acids, at most 19 amino acids, at most 20 amino acids, at most 21 amino acids, at most 22 amino acids, at most 23 amino acids, at most 24 amino acids, at most 25 amino acids, at most 26 amino acids, at most 27 amino acids, at most 28 amino acids, at most 29 amino acids, at most 30 amino acids, at most 31 amino acids, at most 32 amino acids, at most 33 amino acids, at most 34 amino acids, at most 35 amino acids, at most 36 amino acids, at most 37 amino acids, at most 38 amino acids, at most 39 amino acids, at most 40 amino acids, at most 41 amino acids, at most 42 amino acids, at most 43 amino acids, at most 44 amino acids, at most 45 amino acids, at most 46 amino acids, at most 47 amino acids, at most 48 amino acids, at most 49 amino acids, at most 50 amino acids, at most 51 amino acids, at most 52 amino acids, at most 53 amino acids, at most 54 amino acids, at most 55 amino acids, at most 56 amino acids, at most 57 amino acids, at most 58 amino acids, at most 59 amino acids, at most 60 amino acids, at most 61 amino acids, at most 62 amino acids, at most 63 amino acids, at The Fam20c kinase may comprise a truncation at the N-terminal end of up to 49 amino acids, up to 50 amino acids, up to 51 amino acids, up to 52 amino acids, up to 53 amino acids, up to 54 amino acids, up to 55 amino acids, up to 56 amino acids, up to 57 amino acids, up to 58 amino acids, up to 59 amino acids, up to 60 amino acids, up to 61 amino acids, up to 62 amino acids, up to 63 amino acids, up to 64 amino acids, up to 65 amino acids, up to 66 amino acids, up to 67 amino acids, up to 68 amino acids, up to 69 amino acids, up to 70 amino acids, up to 71 amino acids, up to 72 amino acids, up to 73 amino acids, up to 74 amino acids, up to 75 amino acids, up to 76 amino acids, up to 77 amino acids, up to 78 amino acids, up to 79 amino acids, up to 80 amino acids, up to 81 amino acids, up to 82 amino acids, up to 83 amino acids, up to 84 amino acids, up to 85 amino acids, up to 86 amino acids, up to 87 amino acids, up to 88 amino acids, up to 89 amino acids, up to 90 amino acids, up to 91 amino acids, up to 92 amino acids, or up to 93 amino acids. In some embodiments, the human serine / threonine kinase can be Fam20c kinase, which has an amino acid sequence that is 584 amino acids in length. [ka]

[0090] In some embodiments, the human serine / threonine kinase can be Fam20c kinase, further comprising a FLAG tag having the amino acid sequence of SEQ ID NO: 146. In some embodiments, the expressed kinase can further comprise a FLAG tag and have at least 75%, 80%, 90%, 95% or 100% sequence identity to the sequence set forth in SEQ ID NO: 146. [ka]

[0091] In some embodiments, the human serine / threonine kinase can be Fam20c kinase, further comprising a MYC tag having the amino acid sequence of SEQ ID NO: 147. In some embodiments, the expressed kinase can further comprise a MYC tag and can have at least 75%, 80%, 90%, 95% or 100% sequence identity to the sequence set forth in SEQ ID NO: 147. [ka]

[0092] In some embodiments, the human serine / threonine kinase can be Fam20c kinase having the amino acid sequence of SEQ ID NO: 148. In some embodiments, the expressed kinase can have at least 75%, 80%, 90%, 95% or 100% sequence identity according to the amino acid sequence set forth in SEQ ID NO: 148. [ka]

[0093] In some embodiments, the human serine / threonine kinase can be Fam20c kinase having an endogenous secretion signal according to the amino acid sequence set forth in SEQ ID NO:149. [ka]

[0094] In some embodiments, the human serine / threonine kinase can be Fam20c kinase having a propeptide according to the amino acid sequence set forth in SEQ ID NO:150. [ka]

[0095] In some embodiments, the human serine / threonine kinase can be a Fam20c kinase having a transmembrane domain according to the amino acid sequence set forth in SEQ ID NO:151. [ka]

[0096] In some embodiments, the human serine / threonine kinase can be a Fam20c kinase having a stem region according to the amino acid sequence set forth in SEQ ID NO:152. [ka]

[0097] In some embodiments, the human serine / threonine kinase can be Fam20c kinase having a catalytic kinase domain according to the amino acid sequence set forth in SEQ ID NO:153. [ka]

[0098] In some embodiments, the expressed serine / threonine kinase can be a Fam20c kinase having at least 75%, 80%, 90%, 95%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 93-144 and 154. In some embodiments, the expressed serine / threonine kinase can be encoded by a nucleic acid sequence having at least 75%, 80%, 90%, 95%, or 100% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 185-238 and 302-303. In some embodiments, the expressed serine / threonine kinase can be encoded by a vector having at least 75%, 80%, 90%, 95%, or 100% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 241-295 and 309-310.

[0099] Table 1 provides exemplary full-length Fam20c and Fam20c fragment sequences. [Table 1-1] [Table 1-2]

[0100] In some embodiments, the expressed kinase may further comprise a purification and / or identification tag. The purification and / or identification tag may be a FLAG tag comprising SEQ ID NO: 10. The purification and / or identification tag may be a MYC tag comprising SEQ ID NO: 311. In some embodiments, the expressed kinase may further comprise a solubility enhancer tag, which may be a SUMO tag comprising SEQ ID NO: 11, a TRX tag comprising SEQ ID NO: 12, a MBP tag comprising SEQ ID NO: 13, a MISTIC tag comprising SEQ ID NO: 14, a NusA tag comprising SEQ ID NO: 15, a FLAG-SUMO tag comprising SEQ ID NO: 16, a FLAG-TRX tag comprising SEQ ID NO: 17, a FLAG-MBP tag comprising SEQ ID NO: 18, a FLAG-MISTIC tag comprising SEQ ID NO: 19, or a FLAG-NusA tag comprising SEQ ID NO: 20. In some embodiments, an N-terminal methionine may be added to the purification and / or identification tags of SEQ ID NOs: 10-20 and the solubility enhancer tag.

[0101] Table 2 provides sequences of exemplary purification tags, identification tags and solubility enhancer tags. [Table 2-1] [Table 2-2] [Table 2-3]

[0102] Targeted subcellular localization The recombinant host cells described herein can be engineered to express a kinase (e.g., a serine / threonine kinase) that can be targeted to a subcellular location of interest (e.g., the Golgi apparatus or the ER). In some examples, the kinase can be localized to the subcellular location of interest. In some examples, the kinase can be anchored to an intracellular membrane (e.g., the plasma membrane). In some examples, the anchoring mechanism can be provided in part by an anchoring domain. In some embodiments, the anchoring domain can be covalently attached to the kinase. The intracellular membrane described herein can be the Golgi apparatus membrane, the ER membrane, or the mitochondrial membrane. In some embodiments, the kinase can be anchored such that the kinase domain is located on the luminal side of the Golgi apparatus membrane. In some embodiments, the kinase can be anchored to an intracellular membrane via a polypeptide. In some embodiments, the polypeptide can be covalently attached to the kinase. In some embodiments, the polypeptide can be attached to the N-terminus or C-terminus of the kinase.

[0103] In some embodiments, the polypeptide comprises a type I or type II transmembrane protein. In some embodiments, the polypeptide comprises a type II transmembrane protein. In some embodiments, the type II transmembrane protein may have at least 75%, 80%, 90%, 95%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 155-159.

[0104] In some embodiments, the polypeptide comprises a fragment of a type I or type II transmembrane protein. In some embodiments, the polypeptide comprises a fragment of a type II transmembrane protein. In some embodiments, the polypeptide comprises a type I, type II, or multi-pass transmembrane domain. In some embodiments, the polypeptide comprises a type II transmembrane domain. In some embodiments, the type II transmembrane domain may have at least 75%, 80%, 90%, 95%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 181-184. In some embodiments, the polypeptide comprises a fragment of a type II transmembrane protein. In some embodiments, the type II transmembrane protein may have at least 75%, 80%, 90%, 95%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 160-174. In some embodiments, the polypeptide comprises a type II transmembrane domain.

[0105] In some embodiments, the fragment of a type II transmembrane protein comprises a type II transmembrane protein comprising a truncation at the C-terminus, hi some embodiments, the fragment of a type II transmembrane protein comprises a type II transmembrane protein comprising a truncation of at most 50 amino acids, at most 100 amino acids, at most 150 amino acids, at most 200 amino acids, at most 250 amino acids, at most 300 amino acids, at most 350 amino acids, at most 400 amino acids, at most 450 amino acids, at most 500 amino acids, at most 550 amino acids, at most 600 amino acids, at most 650 amino acids, at most 700 amino acids, or at most 750 amino acids.

[0106] In some embodiments, the polypeptide comprises a Golgi retention sequence. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 21-22. In some embodiments, the amino acid sequence of any one of SEQ ID NOs: 21-22 is at the C-terminus of the polypeptide.

[0107] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% sequence identity to any one of SEQ ID NOs: 23-85. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 23-85. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 71.

[0108] In some instances, the kinase may be anchored to an intracellular membrane via a lipid. In some instances, the lipid may be covalently attached to the kinase. In some instances, the kinase and the lipid may form a prenylated protein. In some instances, the lipid bound to the kinase may include a fatty acyl group. In some instances, the lipid and the kinase form a glycosylphoshaditylinositol-linked protein.

[0109] Table 3 provides exemplary Golgi apparatus retrieval sequences. [Table 3]

[0110] Table 4 provides exemplary Golgi apparatus and endoplasmic reticulum retention sequences. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0111] Table 5 provides exemplary promoter sequences. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5]

[0112] Table 6 provides exemplary type II transmembrane proteins. [Table 6-1] [Table 6-2] [Table 6-3]

[0113] Table 7 provides exemplary type II transmembrane domains. [Table 7]

[0114] Table 8 provides exemplary type II transmembrane protein truncates. [Table 8-1] [Table 8-2]

[0115] Expressed heterologous proteins The recombinant host cells described herein can be engineered to express heterologous proteins containing one or more motifs that are susceptible to post-translational modification (e.g., phosphorylation). In some embodiments, such proteins are secreted proteins. The heterologous protein can be therapeutic or nutritional, and the nutritive protein can be a dairy protein. In some embodiments, the dairy protein is casein or a portion thereof, and the casein is αs1-casein or a portion thereof, αs2-casein or a portion thereof, β-casein or a portion thereof, or κ-casein or a portion thereof. In some embodiments, the dairy protein is glycomacropeptide or a portion thereof, osteopontin or a portion thereof, or lactoferrin or a portion thereof. In some embodiments, the nutritive protein is an egg white protein, and the egg white protein is ovalbumin or a portion thereof.

[0116] In some embodiments, the heterologous protein may comprise, consist of, or consist essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 4. In some embodiments, the heterologous protein may comprise, consist of, or consist essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 5. In some embodiments, the heterologous protein may comprise, consist of, or consist essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 175. In some embodiments, the heterologous protein may comprise, consist of, or consist essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 176. In some embodiments, the heterologous protein may comprise, consist of, or consist essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the SEQ ID sequence set forth in NO. 177. In some embodiments, the heterologous protein may comprise, consist of, or consist essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 178. In some embodiments, the heterologous protein may comprise, consist of, or consist essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 179.In some embodiments, the heterologous protein may comprise, consist of, or consist essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 180. In some embodiments, the heterologous protein may comprise, consist of, or consist essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 416, 422, 417, 423, 418, 424, 419, 425, 420, 421, or 435.

[0117] The expressed heterologous protein can be a component of a protein complex, the protein complex comprising a first protein and a second protein. In some embodiments, the first protein, the second protein, or both the first and second proteins of the protein complex can be expressed heterologous proteins that can contain one or more phosphorylation sites that are susceptible to phosphorylation by a serine / threonine kinase. The protein complex can further comprise four proteins, each of which contains one or more phosphorylation sites that are susceptible to phosphorylation by a serine / threonine kinase.

[0118] Table 9 provides exemplary heterologous proteins. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

[0119] Polynucleotide Expression Kinase and heterologous protein expression by the recombinant host cells described herein can be regulated by various polynucleotide cassette formats. Kinase and heterologous polynucleotide expression can each be regulated by individual promoters. In some embodiments, kinase expression is under the control of a first promoter, and heterologous protein expression (e.g., expression of a protein susceptible to phosphorylation by the kinase) is under the control of a second promoter. In some embodiments, the first promoter is an inducible promoter (e.g., pAOX1) or a constitutive promoter (e.g., pGAP). In some embodiments, the second promoter is an inducible promoter (e.g., pAOX1) or a constitutive promoter (e.g., pGAP). In some embodiments, the first promoter and the second promoter are the same. In some embodiments, DNA fragments encoding the kinase and / or heterologous protein can be site-specifically integrated into the recombinant host cell genome such that the kinase and / or heterologous protein is linked to its native promoter and secretion signal.

[0120] Table 12 shows 1) codon-optimized nucleic acid sequences encoding bovine αS-1 casein, αS-2 casein, β-casein, and κ-casein proteins lacking their native signal sequences and having an N-terminal alpha-mating factor (α) secretion signal (SEQ ID NO: 426), and 2) codon-optimized nucleic acid sequences encoding bovine αS-1 casein, αS-2 casein, β-casein, and κ-casein proteins lacking their native signal sequences and lacking the N-terminal alpha-mating factor (α) secretion signal (SEQ ID NO: 426). The nucleic acid molecules may comprise, consist of, or consist essentially of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 431, 427, 432, 428, 433, 429, 434, or 430. [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5]

[0121] III. Bioreactor Described herein is a bioreactor for obtaining phosphorylated secreted proteins. The bioreactor may include a plurality of recombinant host cells, a reaction vessel (e.g., shake flasks), and a medium (e.g., YPD and BMGY), wherein the medium and recombinant host cells are disposed within the reaction vessel. The plurality of recombinant host cells may be engineered to express a heterologous serine / threonine kinase such that 24 hours after expression of the heterologous serine / threonine kinase, at least 60% of the serine / threonine kinase in the reaction vessel is intact. In some embodiments, the plurality of recombinant host cells within the bioreactor are further engineered to express a heterologous secreted protein containing one or more phosphorylation sites susceptible to phosphorylation by the serine / threonine kinase. The bioreactor may be maintained at an internal temperature of 20° Celsius to 40° Celsius. Various embodiments of the recombinant host cells described herein may be used as the plurality of recombinant host cells used in the bioreactor system. In some embodiments, at least 70%, 80%, or 90% of the serine / threonine kinase in the reaction vessel is intact 24 hours after initiation of expression. In some examples, expression of the serine / threonine kinase is initiated by adding an inducer to the culture medium. Inducing agents for initiating expression of the heterologous serine / threonine kinase described herein may include ethanol, maltose, starch, xylose, thiamine, copper, quinic acid, nitrate, glucose, sugars, H2O2, CaCO3, or benzoic acid. In some examples, expression of the serine / threonine kinase is initiated by light, blue light, low pH, iron starvation, or copper depletion. In some examples, expression of the serine / threonine kinase is initiated by culturing the recombinant host cell in the culture medium.

[0122] IV. Method for Producing Phosphorylated Proteins Described herein are methods for producing a phosphorylated heterologous protein that can be secreted. The method includes expressing (1) a heterologous serine / threonine kinase and (2) a heterologous secreted protein containing one or more phosphorylation sites susceptible to phosphorylation by the serine / threonine kinase in a cell population comprising a plurality of recombinant host cells, and culturing the cell population in a reaction vessel containing a medium (e.g., YPD and BMGY) for at least 24 hours after expression of the heterologous serine / threonine kinase and the heterologous secreted protein, wherein at least 60% of the serine / threonine kinase in the reaction vessel is intact. In some embodiments, at least 70%, 80%, or 90% of the serine / threonine kinase in the reaction vessel is intact 24 hours after the initiation of expression. In some examples, the heterologous serine / threonine kinase expressed by the recombinant host cell can be in a modified form as described herein, such as by incorporation of an intracellular membrane-anchoring domain. In some embodiments, expression of the heterologous serine / threonine kinase can include culturing the recombinant host cell in the medium. In some embodiments, expression of the heterologous serine / threonine kinase comprises adding an inducer to the medium, where the inducer can be methanol, IPTG, ethanol, maltose, starch, xylose, thiamine, copper, quinic acid, nitrate, glucose, sugars, HO, CaCO, or benzoate. In some embodiments, expression of the heterologous serine / threonine kinase can comprise induction by light, blue light, low pH, iron starvation, or copper depletion. In some embodiments, the method further comprises culturing the plurality of recombinant cells at a temperature between 20 degrees Celsius and 40 degrees Celsius. In some embodiments, the method further comprises recovering the phosphorylated protein by centrifuging the plurality of recombinant host cells and collecting the supernatant.

[0123] Generation of recombinant host cells The recombinant host cells described in this method may further include additional steps described herein for expressing a serine / threonine kinase of interest and a heterologous protein. The recombinant host cells may be generated by transforming a cell population with a polynucleotide expression cassette encoding a serine / threonine kinase under the control of a first promoter and a secreted protein containing one or more phosphorylation sites susceptible to phosphorylation by the serine / threonine kinase under the control of a second promoter. In some examples, the recombinant cell population may be sequentially transformed with (1) a first vector encoding a serine / threonine kinase under the control of a first promoter and (2) a second vector encoding a secreted protein containing one or more phosphorylation sites susceptible to phosphorylation by the serine / threonine kinase under the control of a second promoter. In some examples, the recombinant cell population may be transformed with vectors encoding both (1) a serine / threonine kinase under the control of a first promoter and (2) a secreted protein containing one or more phosphorylation sites susceptible to phosphorylation by the serine / threonine kinase under the control of a second promoter.

[0124] In some examples, separate fragments of DNA encoding (1) a serine / threonine kinase under the control of a first promoter and (2) a secreted protein containing one or more phosphorylation sites susceptible to phosphorylation by a serine / threonine kinase under the control of a second promoter can be integrated into a recombinant host cell genome. In some examples, fragments of DNA encoding both (1) a serine / threonine kinase under the control of a first promoter and (2) a secreted protein containing one or more phosphorylation sites susceptible to phosphorylation by a serine / threonine kinase under the control of a second promoter can be integrated into a recombinant host cell genome.

[0125] Phosphorylated Protein Products The phosphorylated proteins produced by the methods described herein, which may be produced by the use of heterologous serine / threonine kinases or modified serine / threonine kinases, can be used to produce one or more therapeutic and / or nutritional products. In other words, the therapeutic and / or nutritional products can include the phosphorylated proteins produced by the methods described herein. In some embodiments, the phosphorylated proteins produced by the methods described herein can be used in food products. In some examples, the food product is a dairy product, a dairy substitute, or cheese. In some embodiments, the phosphorylated proteins produced by the methods described herein can also be used in dietary supplements or therapeutic compositions comprising a therapeutically effective amount of the phosphorylated proteins.

[0126] In some embodiments, a phosphoprotein can have a distinct phosphorylation pattern compared to its native state (i.e., as found in nature). In some embodiments, a phosphoprotein can be more phosphorylated compared to its native state phosphoprotein. In some embodiments, a phosphoprotein can be less phosphorylated compared to its native state phosphoprotein. In some embodiments, a phosphoprotein can have an increased number of phosphorylation sites compared to its native state phosphoprotein. In some embodiments, a phosphoprotein can have a decreased number of phosphorylation sites compared to its native state phosphoprotein. In some embodiments, a phosphoprotein can have an increased phosphorylation abundance at a particular phosphorylation site compared to its native state phosphoprotein. In some embodiments, a phosphoprotein can have a decreased phosphorylation abundance at a particular phosphorylation site compared to its native state phosphoprotein.

[0127] In some embodiments, the phosphoprotein has at least one more phosphorylation site than the phosphoprotein in its native state, hi some embodiments, the phosphoprotein has at least two, at least three, at least five, at least 10, at least 15, or at least 20 more phosphorylation sites than the phosphoprotein in its native state.

[0128] In some embodiments, the phosphoprotein has at least one fewer phosphorylation site than the phosphoprotein in its native state, hi some embodiments, the phosphoprotein has at least two, at least three, at least five, at least 10, at least 15, or at least 20 fewer phosphorylation sites than the phosphoprotein in its native state.

[0129] In some embodiments, phosphorylated proteins can be generated by the use of a heterologous serine / threonine kinase. In some embodiments, phosphorylated proteins can be generated by the use of a modified heterologous serine / threonine kinase. In some embodiments, phosphorylated proteins generated by the use of a modified heterologous serine / threonine kinase can have a distinct phosphorylation pattern compared to an unmodified heterologous serine / threonine kinase. In some embodiments, phosphorylated proteins generated by the use of a modified heterologous serine / threonine kinase can be more phosphorylated compared to an unmodified heterologous serine / threonine kinase. In some embodiments, phosphorylated proteins generated by the use of a modified heterologous serine / threonine kinase can be less phosphorylated compared to an unmodified heterologous serine / threonine kinase. In some embodiments, phosphorylated proteins generated by the use of a modified heterologous serine / threonine kinase can have an increased number of phosphorylation sites compared to an unmodified heterologous serine / threonine kinase. In some embodiments, phosphorylated proteins produced by the use of modified heterologous serine / threonine kinases may have a reduced number of phosphorylation sites compared to unmodified heterologous serine / threonine kinases. In some embodiments, phosphorylated proteins produced by the use of modified heterologous serine / threonine kinases may have increased phosphorylation abundance at specific phosphorylation sites compared to unmodified heterologous serine / threonine kinases. In some embodiments, phosphorylated proteins produced by the use of modified heterologous serine / threonine kinases may have decreased phosphorylation abundance at specific phosphorylation sites compared to unmodified heterologous serine / threonine kinases.

[0130] In some embodiments, the phosphorylated protein can be a therapeutic protein or a nutritive protein. In some embodiments, the nutritive protein can be a dairy protein. In some embodiments, the dairy protein can be casein or a portion thereof. In some embodiments, the casein can be αs1-casein, αs2-casein, β-casein, or κ-casein, or a portion thereof. In some embodiments, the dairy protein can be glycomacropeptide or a portion thereof. In some embodiments, the dairy protein can be osteopontin or a portion thereof. In some embodiments, the dairy protein can be lactoferrin or a portion thereof. In some embodiments, the nutritive protein can be egg white protein. In some embodiments, the egg white protein can be ovalbumin or a portion thereof.

[0131] In some embodiments, the phosphorylated protein may be ovalbumin. In some embodiments, ovalbumin may be phosphorylated at residues including S69, T76, T92, S99, S165, T202, S206, and S345. In some embodiments, ovalbumin may be phosphorylated at residues including T92, S99, S165, T202, S206, S222, S271, and S345.

[0132] In some embodiments, the phosphorylated protein may be casein or a portion thereof, hi some embodiments, the casein may be αs1-casein, αs2-casein, β-casein, or κ-casein, or a portion of any of them.

[0133] In some embodiments, the casein is αS1-casein (e.g., bovine αS-1 casein (variant C)). The αS1-casein (e.g., bovine αS-1 casein (variant C)) may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 416 or a fragment of full-length αS1-casein (e.g., bovine αS-1 casein (variant C)), such as the sequence set forth in SEQ ID NO: 177, and the αS1-casein (e.g., bovine αS-1 casein (variant C)) may be coupled to an N-terminal alpha mating factor (α) secretion signal and may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 422. In some embodiments, αS1-casein (e.g., bovine αS-1 casein (variant C)) is phosphorylated at 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 15 or more, or 20 or more amino acids (e.g., serine and / or threonine). In some embodiments, αS1-casein (e.g., bovine αS-1 casein (variant C)) is phosphorylated at one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven of S56, S61, S63, T64, S79, S81, S82, S83, S90, S103 (numbering relative to the bovine αS-1 casein (variant C) sequence in SEQ ID NO: 416) or S130.In some embodiments, αS1-casein (e.g., bovine αS-1 casein (variant C)) is phosphorylated at one or more, two or more, three or more, four or more, or five or more of S56, S79, S81, S82, S83, or S103. In some embodiments, αS1-casein (e.g., bovine αS-1 casein (variant C)) is phosphorylated at S56. In some embodiments, αS1-casein (e.g., bovine αS-1 casein (variant C)) is phosphorylated at one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten of S61, S63, T64, S79, S81, S82, S83, S90, S103, or S130. In some embodiments, αS1-casein (e.g., bovine αS-1 casein (variant C)) is phosphorylated at S61, S63, T64, S79, S81, S82, S83, S90, S103, and S130. In some embodiments, the αS1-casein (e.g., bovine αS-1 casein (variant C)) comprises, consists of, or consists essentially of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 177, 416 or 422. In some embodiments, the αS1-casein (e.g., bovine αS-1 casein (variant C)) comprises, consists of, or consists essentially of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 177.

[0134] In some embodiments, the phosphorylated casein is αS2-casein (e.g., bovine αS2-casein), which may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO:417, or may be a fragment of full-length αS2-casein (e.g., bovine αS2-casein), e.g., comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:178, which may be coupled to an N-terminal alpha-mating factor (α) secretion signal and may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO:423. In some embodiments, αS2-casein (e.g., bovine αS2-casein) is phosphorylated at 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 15 or more, or 20 or more amino acids (e.g., serine and / or threonine). In some embodiments, αS2-casein (e.g., bovine αS2-casein) is phosphorylated at one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more of S23, S24, S25, S46, S52, S71, S72, S73, S76, T145, S146, S150, S158, T159, or T163.In some embodiments, αS2-casein (e.g., bovine αS2-casein) is phosphorylated at one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight of S23, S24, S25, S52, S71, S72, S73, or S76. In some embodiments, αS2-casein (e.g., bovine αS2-casein) is phosphorylated at one or more, two or more, three or more, four or more, five or more, or six of S23, S52, S71, S72, S73, or S76. In some embodiments, αS2-casein (e.g., bovine αS2-casein) is phosphorylated at S23, S52, S71, S72, S73, and S76. In some embodiments, αS2-casein (e.g., bovine αS2-casein) is phosphorylated at S23, S24, S25, S46, S52, S71, S72, S73, S76, T145, S146, S150, S158, and T159. In some embodiments, the αS2-casein (e.g., bovine αS2-casein) comprises, consists of, or consists essentially of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 178, 417 or 423. In some embodiments, the αS2-casein (e.g., bovine αS2-casein) comprises, consists of, or consists essentially of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 178.

[0135] In some embodiments, the phosphorylated casein is β-casein (e.g., bovine β-casein (variant A2)). The β-casein (e.g., bovine β-casein (variant A2)) may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO:418, or may be a fragment of β-casein (e.g., bovine β-casein (variant A2)), such as comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:179, and the β-casein (e.g., bovine β-casein (variant A2)) may be coupled to an N-terminal alpha mating factor (α) secretion signal and may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO:424. In some embodiments, the β-casein (e.g., bovine β-casein (variant A2)) comprises S30, S32, S33, S34, S37, T39, S50, and T56 (at In some embodiments, beta-casein (e.g., bovine beta-casein (variant A2)) is phosphorylated at one or more, two or more, three or more, four or more, or eight of S30, S32, S33, S34, S37, T39. In some embodiments, beta-casein (e.g., bovine beta-casein (variant A2)) is phosphorylated at one or more, two or more, three or more, four or more, or five ... In some embodiments, the β-casein (e.g., bovine β-casein (variant A2)) is phosphorylated at S37. In some embodiments, the β-casein (e.g., bovine β-casein (variant A2)) is phosphorylated at S30, S32, S33, S34, S37, T39, S50, and T56. In some embodiments, the β-casein (e.g., bovine β-casein (variant A2)) comprises, consists of, or consists essentially of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 179, 418, or 424.In some embodiments, the β-casein (e.g., bovine β-casein (variant A2)) comprises, consists of, or consists essentially of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 179.

[0136] In some embodiments, the phosphorylated casein is κ-casein (e.g., bovine κ-casein). The κ-casein (e.g., bovine κ-casein) may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO:419, or may be a fragment of κ-casein (e.g., bovine κ-casein) comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:180, which may be coupled to an N-terminal alpha-mating factor (α) secretion signal and may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO:425. In some embodiments, the κ-casein (e.g., bovine κ-casein) comprises, consists of, or consists essentially of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 180, 419 or 425. In some embodiments, the κ-casein (e.g., bovine κ-casein) comprises, consists of, or consists essentially of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 180.

[0137] In some embodiments, casein is phosphorylated at one or more amino acid residues that are not naturally phosphorylated. In some embodiments, casein is phosphorylated at two or more amino acid residues that are not naturally phosphorylated. In some embodiments, casein is phosphorylated at three or more amino acid residues that are not naturally phosphorylated. In some embodiments, casein is phosphorylated at four or more amino acid residues that are not naturally phosphorylated. In some embodiments, casein is phosphorylated at five or more amino acid residues that are not naturally phosphorylated. In some embodiments, casein is phosphorylated at six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, or twenty or more amino acid residues that are not naturally phosphorylated. [Example]

[0138] Example The following illustrative examples are representative of embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0139] Example 1: Expression of the reptilian serine / threonine protein kinase Fam20c in the yeast Pichia pastoris using a non-constitutive promoter Vector Design For recombinant protein expression in yeast (P. pastoris), a recombinant vector encoding snake Ser / Thr Fam20c kinase (SEQ ID NO: 3) was generated and codon-optimized for expression in P. pastoris. DNA encoding the Ser / Thr kinase of SEQ ID NO: 3 was cloned into the pD902 vector, an integration vector targeting the AOX1 or GAP locus of P. pastoris. The expression cassette contained an N-terminal alpha-mating factor secretion signal, a FLAG-SUMO tag, and a C-terminal HiBiT tag. In one mutant, expression of the recombinant snake Ser / Thr Fam20c protein was controlled by the pAOX1 promoter, a non-constitutive inducible promoter activated by the presence of methanol in the growth medium (mutant Fam20C Common Garter Snake (FCGS)-3). In the second mutant, expression of the recombinant snake Ser / Thr Fam20c protein was controlled by the constitutive promoter pGAP (mutant FCGS-8). In the third mutant, expression of recombinant snake Ser / Thr Fam20c protein (with an N-terminal FLAG tag) was controlled by the pGAP promoter (mutant FCGS-5).

[0140] Expression of recombinant snake Ser / Thr Fam20c protein in eukaryotic host cells The snake Fam20c vector was transformed into P. pastoris CBS7435, and putative strains were confirmed by colony PCR. Strains were grown overnight in BMGY (non-inducing medium) and diluted to an OD600 of 1.0 in BMMY (inducing medium). Expression of the recombinant snake Ser / Thr Fam20c protein was monitored from 0 to 48 hours after induction. At each 24-hour time point, the culture was supplemented with additional methanol to maintain induction conditions. The culture was harvested, and the supernatant was retained for analysis.

[0141] The extracellular expression of snake Ser / Thr Fam20c FCGS-3, FCGS-5, and FCGS-8 mutants from P. pastoris CBS7435 was probed during the 0-48 h period after induction of protein expression at the 24 h time point (Figure 3). Supernatant samples were separated by denaturing protein gel. Samples were then transferred to nitrocellulose membranes and probed with anti-FLAG primary antibody conjugated to HRP.

[0142] Figure 3 shows Western blots of proteins secreted from transgenic P. pastoris CBS7435 expressing the snake Fam20c FCGS-3, FCGS-5, or FCGS-8 mutants, probed with an anti-FLAG primary antibody conjugated to HRP. Expression time points are indicated at the top of each lane. The expected weight for the FCGS-3 mutant is 30.2 kDa (Figure 3, left). The expected weight for the FCGS-5 mutant is 19 kDa (Figure 3, center). The expected weight for the FCGS-8 mutant is 30.2 kDa (Figure 3, right). Expression in the FCGS-8 mutant, whose expression is driven by the constitutive pGAP promoter, is reduced from that seen in the FCGS-3 mutant, whose expression is driven by the inducible pAOX1 promoter. FCGS-5, in which the N-terminal SUMO tag has been removed, significantly reduces snake Fam20c expression. Taken together, these data demonstrate that robust expression of snake Fam20c with a solubility enhancer tag can be achieved.

[0143] Example 2: Expression and stability of mouse, bovine, and snake serine / threonine protein kinase (Fam20c) in yeast cells For recombinant protein expression in yeast (Pichia pastoris (P. pastoris)), recombinant vectors encoding the mouse serine / threonine protein kinase Fam20c (mouse Ser / Thr Fam20c; SEQ ID NO: 1), bovine Ser / Thr Fam20c kinase (SEQ ID NO: 2), and snake Ser / Thr Fam20c kinase (SEQ ID NO: 3) were generated and codon-optimized for expression in P. pastoris. DNA encoding the Ser / Thr kinases SEQ ID NOs: 1, 2, and 3 was cloned into the pD902 vector, an integration vector targeting the AOX1 locus of P. pastoris. The expression cassette contained an N-terminal alpha-mating factor secretion signal, a FLAG-SUMO tag, and a C-terminal protein detection HiBiT tag. Expression of the recombinant Ser / Thr Fam20c protein was controlled by the AOX1 promoter, an inducible promoter activated by the presence of methanol in the growth medium.

[0144] The resulting mouse, bovine, and snake Fam20c vectors were transformed into P. pastoris CBS7435, and putative recombinant strains were confirmed by colony PCR. Strains were grown overnight in BMGY (non-inducing medium) and diluted to an OD600 of 1.0 in BMMY (inducing medium). Expression of the recombinant Ser / Thr Fam20c protein was monitored from 0 to 96 hours after induction. At each 24-hour time point, the cultures were supplemented with additional methanol to maintain induction conditions. The cultures were harvested. The cell pellets were lysed by bead-beating. The cell lysates and supernatants were retained for analysis.

[0145] The extracellular expression of mouse and bovine Ser / Thr Fam20c from P. pastoris CBS7435 and the intracellular expression of mouse, bovine, and snake Ser / Thr Fam20c were probed from 0 to 96 hours after induction of protein expression at 24 hours. Samples were separated by denaturing protein gels. Samples were then transferred to nitrocellulose membranes and probed with anti-FLAG primary antibody conjugated to HRP.

[0146] Figures 4A and 4B show Western blots of proteins extracted from the supernatant of transgenic P. pastoris CBS7435 expressing mouse or bovine Fam20c, respectively, probed with an anti-FLAG primary antibody conjugated to HRP. Figures 4C, 4D, and 4E show Western blots of proteins extracted from cell lysates of transgenic P. pastoris CBS7435 expressing mouse, bovine, or snake Fam20c, respectively, probed with an anti-FLAG primary antibody conjugated to HRP. The expression time points are indicated above each lane. For mouse Ser / Thr Fam20c, the expected weight of the full-length product is 74.5 kDa. For bovine Ser / Thr Fam20c, the expected weight of the full-length product is 77.8 kDa. For snake Ser / Thr Fam20c, the expected weight of the full-length product is 30.2 kDa. Mouse and bovine Fam20c extracted from both the extracellular and intracellular portions of the cultures were fragmented (Figure 4A-D). In contrast, the snake gene construct (Figure 4E) produced full-length (intact) serine / threonine kinase Fam20c.

[0147] Example 3: Expression of primate and avian serine / threonine protein kinase Fam20c in Pichia pastoris yeast Vector design for constitutive expression of the primate and avian serine / threonine protein kinase Fam20c For recombinant protein expression in yeast (P. pastoris), recombinant vectors encoding a primate serine / threonine protein kinase (lemur Ser / Thr Fam20c; SEQ ID NO: 6) or an avian Ser / Thr Fam20c (sandwich Ser / Thr Fam20c; SEQ ID NO: 8) were generated and codon-optimized for expression in P. pastoris. DNA encoding the Ser / Thr kinases of SEQ ID NOs: 6 and 8 (without the N-terminal native secretion signal) was cloned into the pD902 vector, an integrative vector targeting the GAP locus in P. pastoris. The expression cassette contained an N-terminal α-mating factor secretion signal, a FLAG-SUMO tag, and a C-terminal HiBiT tag. In both mutants, expression of the recombinant lemur or sandwich Ser / Thr Fam20c protein was controlled by the constitutive promoter, pGAP.

[0148] Constitutive expression of recombinant Ser / Thr Fam20c protein in P. pastoris The vector was transformed into P. pastoris CBS7435, and the putative strain was confirmed by colony PCR. Strains were grown overnight in YPD and diluted to an OD600 of 1.0 in YPD. Expression of the recombinant Ser / Thr Fam20c protein was monitored between 0 and 48 hours post-inoculation. At each 24-hour time point, the culture was supplemented with additional glucose to maintain growth conditions. The culture was harvested. The cell pellet was lysed by bead-beating. The cell lysate and supernatant were retained for analysis.

[0149] The extracellular and intracellular expression of the lemur and sandgrouse Ser / Thr Fam20c variants from P. pastoris CBS7435 was probed from 0 to 48 h postinoculation at the 24 h time point. Samples were separated by denaturing protein gels. Samples were then transferred to nitrocellulose membranes and probed with anti-FLAG primary antibodies conjugated to HRP.

[0150] Figures 12A and 12B show Western blots of proteins extracted from the supernatant of transgenic P. pastoris CBS7435 expressing lemur or sandgrouse Fam20c, respectively, probed with an anti-FLAG primary antibody conjugated to HRP. Figures 12C and 12D show Western blots of proteins extracted from the cell lysates of transgenic P. pastoris CBS7435 expressing lemur or sandgrouse Fam20c, respectively, probed with an anti-FLAG primary antibody conjugated to HRP. The expression time points are indicated above each lane. For lemur Ser / Thr Fam20c, the expected weight of the full-length product is 43.3 kDa. For sandgrouse Ser / Thr Fam20c, the expected weight of the full-length product is 48.6 kDa. Lemur Fam20c and sandgroyne Fam20c extracted from the extracellular portion of the cultures were fragmented (Figure 12A-B). In contrast, the intracellular portion of the cultures produced full-length (intact) serine / threonine kinase Fam20c for both constructs, lemur and sandgroyne (Figure 12C-D).

[0151] Vector design for inducible expression of the primate and avian serine / threonine protein kinase Fam20c For recombinant protein expression in yeast (P. pastoris), recombinant vectors encoding primate serine / threonine protein kinase (lemur Ser / Thr Fam20c; SEQ ID NO: 6) or avian Ser / Thr Fam20c (sandwich Ser / Thr Fam20c; SEQ ID NO: 8) were generated and codon-optimized for expression in P. pastoris. DNA encoding the Ser / Thr kinases of SEQ ID NOs: 6 and 8 (without the N-terminal native secretion signal) was cloned into the pD902 vector, an integrative vector targeting the AOX1 locus in P. pastoris. The expression cassette contained an N-terminal alpha-mating factor secretion signal, a FLAG-SUMO tag, and a C-terminal HiBiT tag. Expression of the recombinant Ser / Thr Fam20c protein was controlled by the pAOX1 promoter, an inducible promoter activated by the presence of methanol in the growth medium.

[0152] Inducible expression of recombinant Ser / Thr Fam20c protein in P. pastoris The vector was transformed into P. pastoris CBS7435, and the putative strain was confirmed by colony PCR. Strains were grown overnight in BMGY (non-inducing medium) and diluted to an OD600 of 1.0 in BMMY (inducing medium). Expression of the recombinant Ser / Thr Fam20c protein was monitored from 0 to 48 hours after induction. At each 24-hour time point, the culture was supplemented with additional methanol to maintain induction conditions. The culture was harvested, and the supernatant was retained for analysis.

[0153] Example 4: Intracellular expression of bovine αS1 casein in PichiaPink™ yeast Vector Design For recombinant protein expression in yeast (P. pastoris), a recombinant vector encoding bovine αS1 casein, variant B (SEQ ID NO: 4), was generated and codon-optimized for expression in P. pastoris. DNA encoding αS1 casein (SEQ ID NO: 4) (without the N-terminal native secretion signal) was cloned into the pPINKα-HC vector, an integrative vector targeted to the ADE2 locus in P. pastoris. In one variant, the expression cassette contained an N-terminal α-mating factor secretion signal, a detectable FLAG tag, and a C-terminal HiBiT tag, and expression was controlled by the pAOX1 promoter, an inducible promoter activated by the presence of methanol in the growth medium. In the second variant, the expression cassette contained an N-terminal Ost1 α-mating factor hybrid secretion signal, a FLAG tag, and a C-terminal HiBiT tag, and expression was controlled by the pAOX1 promoter. In the third variant, the expression cassette contained an N-terminal α-mating factor secretion signal, a FLAG tag, and a C-terminal HiBiT tag, and expression was controlled by the constitutive pGAP promoter. In the fourth variant, the expression cassette contained an N-terminal Ost1 α mating factor hybrid secretion signal, a FLAG tag, and a C-terminal HiBiT tag, and expression was controlled by the pGAP promoter.

[0154] Intracellular expression of αS1 casein in PichiaPink yeast The above vector, expression controlled by the pGAP promoter, was transformed into the PichiaPink™ (Strain 4) yeast strain, and putative recombinant strains were confirmed by colony PCR. Strains were grown overnight in YPD and diluted to an OD600 of 1.0 in YPD. Expression of the αS1 casein protein was monitored between 0 and 48 h postinoculation. At each 24-h time point, the culture was supplemented with additional glucose to maintain growth conditions. The culture was harvested. The cell pellet was lysed by bead-beating. Soluble and insoluble intracellular protein fractions were analyzed by denaturing protein gel. The gel was transferred to a nitrocellulose membrane and probed with an anti-FLAG primary antibody conjugated to HRP.

[0155] Figure 5 shows a Western blot detecting the intracellular expression of soluble and insoluble αS1 casein protein fractions under the control of the pGAP promoter extracted from transgenic PichiaPink™ yeast. The expression time points are indicated at the top of each lane. The expected weight for both soluble and insoluble αS1 casein protein is 25.1 kDa. Figure 5 appears to show full-length (intact) bovine αS1 casein protein expressed intracellularly in yeast cells.

[0156] Example 5: Extracellular expression of human osteopontin protein in Escherichia coli bacteria and Pichia pastoris yeast Vector design and osteopontin protein expression in E. coli A recombinant vector encoding human osteopontin (SEQ ID NO: 5) was generated. The expression cassette contained a C-terminal 6xHis tag for detection and purification. Osteopontin protein expression was controlled by the T7 promoter, an inducible promoter activated by the presence of IPTG in the culture medium. The vector was transformed into BL21 competent E. coli strain, and putative recombinant strains were confirmed by colony PCR.

[0157] Vector design and osteopontin protein expression in P. pastoris yeast For recombinant protein expression in the yeast Pichia pastoris (P. pastoris), a recombinant vector encoding human osteopontin (SEQ ID NO: 5) was generated and codon-optimized for expression in P. pastoris. DNA encoding human osteopontin (SEQ ID NO: 5) (without the N-terminal native secretion signal) was cloned into the pET24b vector for episomal expression in Escherichia coli. The described exemplary constructs were cloned into the yeast pD902 and pD915 vectors, which contain the inducible pAOX1 promoter or the constitutive pGAP promoter, respectively. Both vectors contain the N-terminal alpha-mating factor secretion signal (SEQ ID NO: 312), a detectable FLAG tag, and a C-terminal HiBiT tag, and expression was controlled by either the inducible pAOX1 promoter or the constitutive pGAP promoter.

[0158] The vector was transformed into P. pastoris CBS7435, and putative recombinant strains were confirmed by colony PCR. Strains were grown overnight in BMGY (non-inducing medium) and diluted to an OD600 of 1.0 in BMMY (inducing medium). Expression of recombinant human osteopontin protein was monitored from 0 to 48 hours after induction. At each 24-hour time point, the culture was supplemented with additional methanol to maintain induction conditions. The culture was harvested, and the supernatant was retained for analysis.

[0159] Figure 6 shows a Western blot detecting extracellular expression of human osteopontin protein under the control of the pAOX1 promoter extracted from transgenic P. pastoris CBS7435 yeast. The expression time points are indicated at the top of each lane. Figure 6 appears to show full-length (intact) human osteopontin protein extracellularly expressed by yeast cells.

[0160] Example 6: Engineered human serine / threonine protein kinase Fam20c mutants for fungal expression Described herein is an approach to engineer the human serine / threonine protein kinase Fam20c (Fam20c), a type II transmembrane protein, for fungal expression (Figures 7A-7D). Type II transmembrane proteins can include a transmembrane domain, a catalytic domain, and a stem region located between the transmembrane and catalytic domains (Figure 7A).

[0161] This approach exploits the ability to enhance protein activity by increasing the stability of huFam20c in fungal hosts. This can be achieved by fusing different lengths of the human Fam20c (huFam20c) stem region and catalytic domain (Figure 7B) with different lengths of the fungal transmembrane domain and stem region (Figure 7C), since membrane protein stability can be directly related to interactions between amino acid side chains and phospholipids. This can result in a fused stem region containing fungal and human sequences (Figure 7D). In some embodiments, the engineered huFam20c variants can contain a C-terminal FLAG tag.

[0162] HuFam20c of different lengths, including the stem region and catalytic domain, can be generated by truncating huFam20c from the N-terminus (Figure 7B). The native signal peptide and predicted transmembrane domain can be removed from the N-terminus of huFam20c, thereby generating the huFam20c truncant R32-R584. The native signal peptide, predicted transmembrane domain, and a section of the propeptide region can be removed from the N-terminus of huFam20c, thereby generating the huFam20c truncant R64-R584. The native signal peptide, predicted transmembrane domain, and propeptide region can be removed from the N-terminus of huFam20c, thereby generating the huFam20c truncant D93-R584.

[0163] Fungal transmembrane domains and stem regions of different lengths can be generated by truncating various fungal type II transmembrane proteins from their C-terminal ends (Figure 7C). Three lengths of glycolipid 2-alpha-mannosyltransferase (ScKRE2) from Saccharomyces cerevisiae were generated by C-terminal truncation to obtain ScKRE2 containing the first 102 amino acids (ScKRE_M1-D102), ScKRE2 containing the first 80 amino acids (ScKRE_M1-S80), and ScKRE2 containing the first 58 amino acids (ScKRE_M1-I58). Three lengths of Pichia pastoris glycolipid 2-alpha-mannosyltransferase (PpKRE2) were generated by C-terminal truncation: PpKRE2 containing the first 150 amino acids (PpKRE_M1-H150), PpKRE2 containing the first 84 amino acids (PpKRE_M1-D84), and PpKRE2 containing the first 31 amino acids (PpKRE_M1-G31). Three lengths of Saccharomyces cerevisiae alpha 1,2-mannosyltransferase (ScMNN2) were generated by C-terminal truncation: ScMNN2 containing the first 150 amino acids (ScMNN2_M1-S150), ScMNN2 containing the first 97 amino acids (ScMNN2_M1-P97), and ScMNN2 containing the first 36 amino acids (ScMNN2_M1-S36). Three lengths of alpha alpha 1,3-mannosyltransferase (ScMNN1) from Saccharomyces cerevisiae were generated by truncation at the C-terminus to obtain ScMNN1 containing the first 153 amino acids (ScMNN1_M1-G153), ScMNN1 containing the first 93 amino acids (ScMNN1_M1-Q93), and ScMNN1 containing the first 42 amino acids (ScMNN1_M1-A42).Three lengths of Saccharomyces cerevisiae mannosyltransferase (ScMNN6) were generated by truncation at the C-terminus to obtain ScMNN6 containing the first 160 amino acids (ScMNN6_M1-E160), ScMNN6 containing the first 85 amino acids (ScMNN6_M1-V85), and ScMNN6 containing the first 30 amino acids (ScMNN6_M1-P30).

[0164] Membrane-anchored huFam20c variants were engineered by combinatorial fusion of different lengths of huFam20c containing stem and catalytic domains with different lengths of fungal transmembrane and stem regions (Figure 7D). The membrane-anchored huFam20c variants generated are shown in Table 10. The polynucleic acid sequences encoding the engineered kinases are shown in Table 11.

[0165] Table 10 provides the engineered kinases. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7] [Table 10-8] [Table 10-9] Table 10-10 Table 10-11 Table 10-12 Table 10-13 Table 10-14 Table 10-15 Table 10-16 Table 10-17 Table 10-18 Table 10-19 Table 10-20 Table 10-21 Table 10-22 Table 10-23 Table 10-24 Table 10-25 Table 10-26 Table 10-27 Table 10-28 Table 10-29 Table 10-30 Table 10-31 Table 10-32 Table 10-33 Table 10-34 Table 10-35 Table 10-36 Table 10-37 Table 10-38 Table 10-39 Table 10-40 Table 10-41 Table 10-42 Table 10-43 Table 10-44 Table 10-45 Table 10-46 Table 10-47 Table 10-48 Table 10-49 Table 10-50 Table 10-51 Table 10-52 Table 10-53 Table 10-54 Table 10-55 Table 10-56 Table 11-1 Table 11-2 Table 11-3 Table 11-4 Table 11-5 Table 11-6 Table 11-7 Table 11-8 Table 11-9 Table 11-10 Table 11-11 Table 11-12 Table 11-13 Table 11-14 Table 11-15 Table 11-16 Table 11-17 Table 11-18 Table 11-19 Table 11-20 Table 11-21 Table 11-22 Table 11-23 Table 11-24 Table 11-25 Table 11-26 Table 11-27 Table 11-28 Table 11-29 Table 11-30 Table 11-31 Table 11-32 Table 11-33 Table 11-34 Table 11-35 Table 11-36 Table 11-37 Table 11-38 Table 11-39 Table 11-40 Table 11-41 Table 11-42 Table 11-43 Table 11-44 Table 11-45 Table 11-46 Table 11-47 Table 11-48 Table 11-49 Table 11-50 Table 11-51 Table 11-52 Table 11-53 [Table 11-54] [Table 11-55] [Table 11-56] [Table 11-57] [Table 11-58] [Table 11-59] [Table 11-60] [Table 11-61] [Table 11-62] [Table 11-63] [Table 11-64] [Table 11-65] [Table 11-66]

[0166] Example 7: Secretion of human serine / threonine protein kinase Fam20c by Pichia pastoris We generated recombinant vectors encoding various forms of the human serine / threonine protein kinase Fam20c (huFam20c) (Figure 8I). The DNA sequence of huFam20c was ordered as a DNA fragment (SEQ ID NO: 185) and codon-optimized for expression in Pichia pastoris. From this, we used PCR to amplify four fragments: (i) Fam20c(R32-R584), (ii) Fam20c(R64-R584), (iii) Fam20c(D93-R584), and (iv) Fam20c(Q289-R584).

[0167] Each was cloned in frame with an N-terminal alpha mating factor (α) secretion signal (SEQ ID NO: 312) and a C-terminal FLAG tag (SEQ ID NO: 10) into the pPINKα-HC commercially available vector to generate the vectors: (i) pPINKα_Fam20c(R32-R584)_FLAG, (ii) pPINKα_Fam20c(R64-R584)_FLAG, (iii) pPINKα_Fam20c(D93-R584)_FLAG, and (iv) pPINKα_Fam20c(Q289-R584)_FLAG. Furthermore, M1-R584 and R32-R584 were cloned into the pPINKα expression vector, which removes the N-terminal alpha mating factor secretion signal but retains the C-terminal FLAG tag, to generate the vectors pPINK_Fam20c(M1-R584)_FLAG and pPINK_Fam20c(R32-R584)_FLAG. For pPINK_Fam20c(R32-R584)_FLAG, an additional N-terminal methionine was included to allow protein translation. MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 312).

[0168] The vector was transformed into PichiaPink™ strain 4 (Δade2, Δprb1, Δpep4), and the putative strain was confirmed by colony PCR. For each strain, clones were grown overnight in BMGY (non-inducing medium), diluted to an OD of 1.0 in BMMY (inducing medium), and expressed for 48 hours at 20°C and 30°C. Supernatant samples were taken at 0, 24, and 48 hours. At 24 and 48 hours, the cultures were supplemented with additional methanol to maintain inducing conditions.

[0169] Supernatant samples were TCA precipitated, electrophoresed under denaturing conditions using NuPAGE 4-12% Bis-Tris protein gels, transferred to nitrocellulose blots, blocked with 5% milk in TBS-T, and probed with an HRP-conjugated anti-DYKDDDDK mouse monoclonal antibody. Chemiluminescence detection was performed using an ECL substrate kit (High Sensitivity). A positive control of FLAG-tagged recombinant protein was included in each blot to confirm the functionality of the Western blot.

[0170] Figures 8B-8D show Western blots detecting the extracellular expression of various forms of the human serine / threonine protein kinase Fam20c after 24 and 48 hours of methanol induction at 20°C and 30°C. The expression time points and induction temperatures are indicated above each lane. Figure 8 shows the secretion of various forms of the human serine / threonine protein kinase Fam20c by Pichia pastoris cells.

[0171] Example 8: In vivo phosphorylation by engineered human serine / threonine protein kinase Fam20c Three fragments of the human serine / threonine protein kinase Fam20c (huFam20c), codon-optimized for expression in Pichia pastoris: (i) R32-R584, (ii) R64-R584, and (iii) D93-R584, were amplified using PCR. These were cloned into the commercially available pd915 vector, which lacks the N-terminal alpha mating factor but retains a C-terminal FLAG tag, generating the vectors: (i) pd915_Fam20c(R32-R584)_FLAG, (ii) pd915_Fam20c(R64-R584)_FLAG, and (iii) pd915_Fam20c(D93-R584)_FLAG. For each of these vectors, three truncations of five different fungal localization sequences were cloned into the N-terminus of Fam20c, as described in Example 7. Additionally, a mutant with an N-terminal methionine was generated to allow translation of the delocalization control. Finally, the complete M1-R584 sequence was cloned into pd915, resulting in an in-frame deletion of the N-terminal alpha mating factor with a C-terminal MYC tag, generating the vector pd915_Fam20c(M1-R584)_MYC. A list of the engineered huFam20c constructs generated is shown in Table 10. A C-terminal MYC tag can be added to any of the engineered huFam20c constructs in Table 10.

[0172] The DNA sequence for human osteopontin (SPP1) was ordered as a codon-optimized DNA fragment for expression in Pichia pastoris. From this, a single truncation (G158-N314) was amplified using PCR. Furthermore, the TRX DNA sequence was ordered as a codon-optimized DNA fragment for expression in Pichia pastoris. These two fragments were cloned in-frame with an N-terminal alpha mating factor (α) secretion signal, an N-terminal FLAG tag, and a C-terminal HiBiT tag into the pPINKα-HC commercially available vector (in which the AOX1 promoter has been replaced with the GAP promoter) to generate the vector pPINKα_FLAG_TRX_SPP1(G158-N314)_HiBiT.

[0173] The pPINKα_FLAG_TRX_SPP1(G158-N314)_HiBiT vector was transformed into PichiaPink™ strain 4 (Δade2, Δprb1, Δpep4), and the putative strains were confirmed by colony PCR. For each strain, clones were grown overnight in YPD, diluted to an OD of 1.0 in fresh YPD, and expressed for 48 hours at 30°C. Protein expression was confirmed using electrophoresis and Western blotting as described in Example 7.

[0174] High-expressing strains of TRX_SPP1(G158-N314)_HiBiT were selected and competent cells were prepared. A panel of vectors containing engineered Fam20c proteins and corresponding delocalized controls were transformed into the strains, and putative strains were confirmed by colony PCR. For each strain, clones were grown overnight in YPD, diluted to an OD600 of 1.0 in fresh YPD, and expressed for 48 hours at 30°C.

[0175] Phosphorylation screening was performed as follows: Supernatant samples were electrophoresed under denaturing conditions using NuPAGE 10% Bis-Tris protein gels, transferred to nitrocellulose blots, blocked with 5% milk in TBS-T, and probed with HRP-conjugated anti-DYKDDDDK mouse monoclonal antibody. Chemiluminescence detection was performed using an ECL substrate kit (High Sensitivity). Since phosphorylation alters the migration behavior of proteins during electrophoresis, the degree of phosphorylation was estimated semiquantitatively. In relation to the specific assay described in this example, a downward shift of the SPP1 phosphorylation reporter construct indicates phosphorylation, as gel migration is affected by both protein mass and charge. This trend was confirmed by in vitro phosphorylation treatment with phosphatase (data not shown). A representative gel from the Western blot-based phosphorylation screening is shown in Figure 9A. A summary of the results from the Western blot-based phosphorylation screening for the engineered huFam20c panel is summarized in Table 13.

[0176] To validate the screening, LC-MS detection of the intact isoform was performed to verify the number of phosphorylation sites occupied on TRX_SPP1 (G158-N314) when coexpressed with either the engineered Fam20c or the native huFam20c control (Figure 9B and Figure 9C). Supernatant samples were acetone precipitated, and proteins in the supernatant samples were identified using a quadrupole time-of-flight (QTOF) mass spectrometer (MS). An automated workflow (Bioconfirm software) was applied by including the following steps: spectral preprocessing, peak detection, peak deconvolution, and charge state assignment. Finally, the mass and abundance of molecular species (proteins) were reported. To demonstrate improvements in population phosphorylation, the integrated area of ​​the deconvoluted spectral peaks was matched with the corresponding number of phosphorylation sites. LC-MS analysis was performed on a subset of the engineered huFam20c constructs, and the results are summarized in Table 13.

[0177] Table 13 provides a summary of the phosphorylation data for the engineered kinases. [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4]

[0178] Example 9: Secretion of phosphorylated ovalbumin co-expressed with ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG in Pichia pastoris The chicken ovalbumin (SERPINB14) DNA sequence was ordered as a codon-optimized DNA fragment for expression in Pichia pastoris. The sequence was amplified using PCR and cloned in-frame with an N-terminal alpha mating factor (α) secretion signal, an N-terminal FLAG tag, and a C-terminal HiBiT tag into the pPINKα-HC commercially available vector (in which the AOX1 promoter has been replaced with the GAP promoter) to generate the vector pPINKα_FLAG_SERPINB14_HiBiT.

[0179] The pPINKα_FLAG_SERPINB14_HiBiT vector was transformed into PichiaPink™ strain 4 (Δade2, Δprb1, Δpep4), and the putative strains were confirmed by colony PCR. For each strain, clones were grown overnight in YPD, diluted to an OD of 1.0 in fresh YPD, and expressed at 30°C for 72 hours. Supernatant samples were taken at 0, 24, 48, and 72 hours. Growth conditions were maintained by supplementing the cultures with additional glucose at 24 and 48 hours.

[0180] Supernatant samples were electrophoresed under denaturing conditions using NuPAGE 4-12% Bis-Tris protein gels, transferred to nitrocellulose blots, blocked with 5% milk in TBS-T, and probed with HRP-conjugated anti-DYKDDDDK mouse monoclonal antibody. Chemiluminescence detection was performed using an ECL substrate kit (High Sensitivity). A positive control of FLAG-tagged recombinant protein was included in each blot to confirm the functionality of the Western blot. High-expressing strains of FLAG_SERPINB14_HiBiT were selected, and competent cells were prepared.

[0181] The pd915 vectors containing the unengineered (Fam20c(M1-R584)_MYC) and engineered (ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG) Fam20c kinases were transformed into the FLAG_SERPINB14_HiBiT strain, and the putative strains were confirmed by colony PCR.

[0182] To confirm phosphorylation, the strain was cultured at a 200 mL scale, and recombinant ovalbumin was purified from the supernatant using affinity chromatography targeting the FLAG tag. The supernatant was incubated with anti-DYKDDDDK G1 affinity resin overnight at 4°C with gentle agitation. The resin was separated from the supernatant and washed with a Pierce centrifugal column. The target protein was eluted by competitive binding with 0.5 mg / mL of the FLAG peptide (MDYKDHDGDYKDHDIDYKDDDDK).

[0183] To increase sample concentration, the eluate was processed using a VivaSpin 10 kDa MWCO column and an acetone precipitation step. Samples were electrophoresed under denaturing conditions using NuPAGE 4-12% Bis-Tris protein gels and visualized with InstantBlue Coomassie stain. Bands were extracted, and samples for LC-MS / MS analysis were prepared using in-gel tryptic digests. Briefly, proteins were extracted from gel bands and enzymatically digested into peptides using trypsin. Peptides were analyzed using an Orbitrap MS mass spectrometer operating in MS / MS mode. Raw MS / MS fragmentation data were processed using the database search engine software CHYMERIS, and the following modifications were applied to peptides: phosphorylation of Ser(S), Thr(T), and Tyr(Y), oxidation of Met(M), and carbamidomethylation of Cys. Specifically, phosphorylation was reported for each protein sequence. The software Scaffold was used to generate amino acid coverage maps of tryptic peptides sequenced by LC-MS / MS.

[0184] Phosphorylation of recombinant ovalbumin by engineered and native huFam20c when coexpressed in Pichia pastoris is shown in Figure 10. The phosphorylation sites detected in non-recombinant ovalbumin (amino acids underlined), in recombinant ovalbumin coexpressed with native huFam20c, Fam20c(M1-R584) (triangles), and in recombinant ovalbumin coexpressed with engineered huFam20c, ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG (circles) are annotated in the recombinant ovalbumin sequence shown in Figure 10. The data show that native huFam20c phosphorylates recombinant ovalbumin at S69, T76, T92, S99, S165, T202, S206, and S345 (Figure 10). The data show that the engineered huFam20c, ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG, phosphorylates recombinant ovalbumin at T92, S99, S165, T202, S206, S222, S271, and S345 (Figure 10). The data show that co-expression of native and engineered huFam20c results in distinct phosphorylation patterns of the substrate, recombinant ovalbumin.

[0185] Example 10: Secretion of phosphorylated casein co-expressed with ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG in Kluyveromyces lactis DNA sequences for bovine αS-1 casein, αS-2 casein, β-casein, and κ-casein were ordered as codon-optimized DNA fragments for expression in Kluyveromyces lactis (see Table 12 for codon-optimized nucleic acid sequences). The sequences were amplified using PCR and cloned into a unique vector (pBDα) containing sequences homologous to the NTS2 locus flanking regions, allowing multicopy integration into the host genome. The amplified sequences were cloned in frame with sequences encoding the N-terminal alpha mating factor (α) secretion signal (SEQ ID NO: 426), generating the vectors: (i) pBDα_αS1, (ii) pBDα_αS2, (iii) pBDα_β, and (iv) pBDα_κ. The sequence of the N-terminal alpha mating factor (α) secretion signal (SEQ ID NO: 426) (pre-Ost1 + pro-alpha-factor hybrid mating signal) is MRQVWFSWIVGLFLCFFNVSSAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKR.

[0186] The expression cassettes of Fam20c(M1-R584)_FLAG and ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG were amplified from the vectors pPINK_Fam20c(M1-R584)_FLAG and pd915_ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG, respectively, and cloned into vectors containing sequences with homology to the ku80 locus flanking regions. In a second step, the GAP1 promoter from Kluyveromyces lactis (SEQ ID NO: 415) was inserted in frame with the kinase expression cassette to generate the vectors: pKL_Fam20c(M1-R584)_FLAG (see open reading frame nucleic acid sequence in SEQ ID NO: 361) and pKL_ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG (see open reading frame nucleic acid sequence in SEQ ID NO: 406).

[0187] Vectors pBDα_αS1, pBDα_αS2, pBDα_β, and pBDα_κ were cotransformed with vectors pKL_Fam20c(M1-R584)_FLAG or pKL_ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG into Kluyveromyces lactis GG799 ΔKLLA0D01507g Δku80 to generate two putative strains expressing casein and kinase, which were confirmed by colony PCR.

[0188] To increase protein titer in the secretome, a fragment knocking out the protease gene YPS1 was generated by PCR amplification. The fragment contained a Zeocin expression cassette flanked by 500-bp sequences with homology to the flanking regions of the YPS1 locus. The purified PCR product was transformed into Kluyveromyces lactis GG799ΔKLLA0D01507gΔku80 strains expressing casein αS1, αS2, β, and κ and either unengineered (Fam20c(M1-R584)_FLAG) or engineered (ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG) Fam20c kinase. Putative strains were confirmed by colony PCR. For each strain, clones were diluted to an OD600 of 0.2 in fresh YPD medium and expressed at 30°C for 70 hours.

[0189] To increase the sample concentration, the supernatant sample was loaded onto a VivaSpin 10 kDa MWCO column. The sample was electrophoresed under denaturing conditions on a NuPAGE 12% Bis-Tris protein gel and visualized with InstantBlue Coomassie stain. To increase the sample concentration, the supernatant sample was loaded onto a VivaSpin 10 kDa MWCO column. The sample was electrophoresed under denaturing conditions on a NuPAGE 12% Bis-Tris protein gel and visualized with InstantBlue Coomassie stain.

[0190] Bands were extracted, and samples for LC-MS / MS analysis were prepared using in-gel tryptic digests. Briefly, proteins were extracted from gel bands and enzymatically digested into peptides with trypsin. Peptides were analyzed using an Orbitrap MS mass spectrometer operating in MS / MS mode. Raw MS / MS fragmentation data was processed using the database search engine software CHYMERIS, and the following modifications were applied to peptides: phosphorylation on Ser (S), Thr (T), and Tyr (Y), oxidation on Met (M), and carbamidomethylation on Cys (C). Specifically, phosphorylation was reported for each protein sequence. The software Scaffold was used to generate amino acid coverage maps of tryptic peptides sequenced by LC-MS / MS.

[0191] All four recombinant caseins were expressed: αS-1 casein (SEQ ID NO: 422), αS-2 casein (SEQ ID NO: 423), β-casein (SEQ ID NO: 424), and κ-casein (SEQ ID NO: 425). The secretory sequence is cleaved during secretion from yeast cells, resulting in bovine αS1 casein (variant C) (no signal peptide) (SEQ ID NO: 177), bovine αS2 casein (no signal peptide) (SEQ ID NO: 178), bovine β-casein (variant A2) (no signal peptide) (SEQ ID NO: 179), and bovine κ-casein (variant B) (no signal peptide) (SEQ ID NO: 180).

[0192] The phosphorylation of recombinant casein by engineered and native huFam20c when coexpressed in Kluyveromyces lactis is shown in Figure 11. The phosphorylation status of non-recombinant αS1, αS2, β, and κ-casein was also examined. Non-recombinant casein was obtained from Bacarel Express (Micellar Casein Concentrate Powder (MicCC85) product specification number PS-13-01 / EN (a powdered micellar casein concentrate of whole milk proteins processed by ultrafiltration and spray drying processes)).

[0193] The sequence coverage for non-recombinant αS1, αS2, β, and κ-casein was 85, 75, 100, and 42%, respectively. The sequence coverage for recombinant αS1, αS2, β, and κ-casein coexpressed with Fam20c(M1-R584) was 71, 67, 93, and 36%, respectively. The sequence coverage for recombinant αS1, αS2, β, and κ-casein coexpressed with ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG was 72, 94, 98, and 35%, respectively. Data for κ-casein are not presented due to the low sequence coverage for this protein.

[0194] The phosphorylation sites detected in non-recombinant αS-1 casein (amino acids underlined), recombinant αS-1 casein coexpressed with native huFam20c, Fam20c(M1-R584) (triangles), and recombinant αS-1 casein coexpressed with engineered huFam20c, ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG (circles), are annotated in the recombinant αS-1 casein sequence shown in Figure 11(I). The numbering of amino acid positions is relative to bovine αS-1 casein (variant C) with the native signal peptide (SEQ ID NO: 416). Non-recombinant αS-1 casein obtained as described above was phosphorylated at S61, S63, S64, S90, S103, and S130. The data show that native huFam20c phosphorylates recombinant αS-1 casein at S61, S63, T64, S79, S81, S82, S83, S90, S103, and S130 (Figure 11(I)). The data show that engineered huFam20c, ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG, phosphorylates recombinant αS-1 casein at S56, S61, S63, T64, S79, S81, S82, S83, S90, S103, and S130 (Figure 11(I)).

[0195] The phosphorylation sites detected in non-recombinant αS-2 casein (amino acids underlined), recombinant αS-2 casein co-expressed with native huFam20c, Fam20c(M1-R584) (triangles), and recombinant αS-2 casein co-expressed with engineered huFam20c, ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG (circles), are annotated in the recombinant αS-2 casein sequence shown in Figure 11(II). Amino acid position numbering is relative to bovine αS2 casein with the native signal peptide (SEQ ID NO: 417). Non-recombinant αS-2 casein obtained as described above was phosphorylated at S46, S144, T145, S146, S150, S158, T159, and T163. The data show that native huFam20c phosphorylates recombinant αS-2 casein at S24, S25, S46, T145, S146, S150, S158, T159, and T163 (Figure 11(II)). The data show that engineered huFam20c, ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG, phosphorylates recombinant αS-2 casein at S23, S24, S25, S46, S52, S71, S72, S73, S76, T145, S146, S150, S158, and T159 (Figure 11(II)).

[0196] The phosphorylation sites detected in non-recombinant β-casein (amino acids underlined), recombinant β-casein coexpressed with native huFam20c, Fam20c(M1-R584) (triangles), and recombinant β-casein coexpressed with engineered huFam20c, ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG (circles), are annotated in the recombinant β-casein sequences shown in Figure 11(III). The numbering of amino acid positions is relative to bovine β-casein (variant A2) with the native signal peptide (SEQ ID NO: 418). Non-recombinant β-casein obtained as described above was phosphorylated at S50 and T56. The data show that native huFam20c phosphorylates recombinant β-casein at S30, S32, S33, S34, T39, S50, and T56 (Figure 11(III)). The data show that the engineered huFam20c, ScMNN2(M1-S36)_Fam20c(D93-R584)_FLAG, phosphorylates recombinant β-casein at S30, S32, S33, S34, S37, T39, S50 and T56 (at T56) (Figure 11(III)).

[0197] The data show that co-expression of native and engineered huFam20c results in distinct phosphorylation patterns of the substrates recombinant αS-1 casein, recombinant αS-2 casein and recombinant β-casein.

[0198] While preferred embodiments have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments of the present disclosure described herein may be used in practicing the present disclosure. 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.

Claims

1. A non-naturally occurring polypeptide comprising a serine / threonine kinase coupled to a heterologous domain capable of anchoring said serine / threonine kinase to an intracellular membrane.

2. 2. The non-naturally occurring polypeptide of claim 1, wherein the serine / threonine kinase comprises a human, bovine, primate, avian, or reptilian serine / threonine kinase.

3. 2. The non-naturally occurring polypeptide of claim 1, wherein the serine / threonine kinase comprises a human serine / threonine kinase.

4. 4. The non-naturally occurring polypeptide of claim 3, wherein the human serine / threonine kinase comprises Fam20c kinase.

5. 2. The non-naturally occurring polypeptide of claim 1, wherein the human serine / threonine kinase is Fam20c kinase.

6. 2. The non-naturally occurring polypeptide of claim 1, wherein the serine / threonine kinase comprises amino acids having at least 75%, 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

7.

7. 7. The non-naturally occurring polypeptide of claim 6, wherein the serine / threonine kinase comprises a truncation at the N-terminal end of at most 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids.

8. 7. The non-naturally occurring polypeptide of claim 6, wherein the serine / threonine kinase comprises a truncation at the N-terminal end of up to 92 amino acids.

9. 2. The non-naturally occurring polypeptide of claim 1, wherein the serine / threonine kinase consists of amino acids having at least 75%, 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

7.

10. 2. The non-naturally occurring polypeptide of claim 1, wherein the serine / threonine kinase consists of the amino acid sequence set forth in SEQ ID NO:

7.

11. 2. The non-naturally occurring polypeptide of claim 1, wherein the serine / threonine kinase comprises amino acids having at least 75%, 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

142.

12. 2. The non-naturally occurring polypeptide of claim 1, wherein the serine / threonine kinase consists of amino acids having at least 75%, 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

142.

13. 2. The non-naturally occurring polypeptide of claim 1, wherein the serine / threonine kinase consists of the amino acids set forth in SEQ ID NO:

142.

14. 2. The non-naturally occurring polypeptide of claim 1, wherein the serine / threonine kinase comprises amino acids having at least 75%, 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

143.

15. 2. The non-naturally occurring polypeptide of claim 1, wherein the serine / threonine kinase consists of amino acids having at least 75%, 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

143.

16. 2. The non-naturally occurring polypeptide of claim 1, wherein the serine / threonine kinase consists of the amino acid sequence set forth in SEQ ID NO:

143.

17. 2. The non-naturally occurring polypeptide of claim 1, wherein the serine / threonine kinase comprises amino acids having at least 75%, 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

144.

18. 2. The non-naturally occurring polypeptide of claim 1, wherein the serine / threonine kinase consists of amino acids having at least 75%, 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

144.

19. 2. The non-naturally occurring polypeptide of claim 1, wherein the serine / threonine kinase consists of the amino acid sequence set forth in SEQ ID NO:

144.

20. 20. The non-naturally occurring polypeptide of any one of claims 1 to 19, wherein the heterologous domain is coupled to the N-terminus of the serine / threonine kinase.

21. 20. The non-naturally occurring polypeptide of any one of claims 1 to 19, wherein the heterologous domain is coupled to the C-terminus of the serine / threonine kinase.

22. 22. The non-naturally occurring polypeptide of any one of claims 1 to 21, wherein the heterologous domain comprises a fragment of a type I or type II transmembrane protein.

23. 23. The non-naturally occurring polypeptide of claim 22, wherein the fragment of the type I or type II transmembrane protein comprises a type I or type II transmembrane domain.

24. 22. The non-naturally occurring polypeptide of any one of claims 1 to 21, wherein the heterologous domain comprises a fragment of a type II transmembrane protein.

25. 25. The non-naturally occurring polypeptide of claim 24, wherein said fragment of said type II transmembrane protein comprises a type II transmembrane domain.

26. 26. The non-naturally occurring polypeptide of any one of claims 1 to 25, wherein said fragment of said type II transmembrane protein comprises said type II transmembrane protein having a truncation at its C-terminal end.

27. 27. The non-naturally occurring polypeptide of any one of claims 1-26, wherein the fragment of the type II transmembrane protein is 160 amino acids or less, 100 amino acids or less, or 60 amino acids or less.

28. 28. The non-naturally occurring polypeptide of any one of claims 1 to 27, wherein said fragment of said type II transmembrane protein is derived from Saccharomyces cerevisiae or Pichia pastoris.

29. 28. The non-naturally occurring polypeptide of any one of claims 1 to 27, wherein said fragment of said type II transmembrane protein is derived from Saccharomyces cerevisiae.

30. 28. The non-naturally occurring polypeptide of any one of claims 1 to 27, wherein said fragment of said type II transmembrane protein is derived from Pichia pastoris.

31. 22. The non-naturally occurring polypeptide of any one of claims 1 to 21, wherein the heterologous domain comprises a multipass transmembrane domain.

32. 2. The non-naturally occurring polypeptide of claim 1, wherein the heterologous domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:23-85.

33. 2. The non-naturally occurring polypeptide of claim 1, wherein the heterologous domain consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:23-85.

34. 2. The non-naturally occurring polypeptide of claim 1, wherein the heterologous domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

71.

35. 2. The non-naturally occurring polypeptide of claim 1, wherein the heterologous domain consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

71.

36. 2. The non-naturally occurring polypeptide of claim 1, wherein the non-naturally occurring polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 93, 95-98, 100-109, 111-114, 116, 117, 119-125, 127-130, 132, 133, 136-141, 154, and 313-360.

37. 2. The non-naturally occurring polypeptide of claim 1, wherein the non-naturally occurring polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

133.

38. 2. The non-naturally occurring polypeptide of claim 1, wherein the non-naturally occurring polypeptide consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 93, 95-98, 100-109, 111-114, 116, 117, 119-125, 127-130, 132, 133, 136-141, 154, and 313-360.

39. 2. The non-naturally occurring polypeptide of claim 1, wherein the non-naturally occurring polypeptide consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

133.

40. 40. The non-naturally occurring polypeptide of any one of claims 1 to 39, wherein the non-naturally occurring polypeptide further comprises a tag.

41. 41. The non-naturally occurring polypeptide of claim 40, wherein the tag comprises a FLAG tag.

42. 41. The non-naturally occurring polypeptide of claim 40, wherein the tag comprises a MYC tag.

43. 43. The non-naturally occurring polypeptide of any one of claims 1 to 42, wherein the heterologous domain comprises a Golgi retention sequence.

44. 44. The non-naturally occurring polypeptide of any one of claims 1 to 43, wherein the intracellular membrane comprises a Golgi apparatus membrane, an endoplasmic reticulum membrane, a nuclear membrane, or a mitochondrial membrane.

45. 45. The non-naturally occurring polypeptide of any one of claims 1 to 44, wherein the intracellular membrane comprises the endoplasmic reticulum membrane.

46. 45. The non-naturally occurring polypeptide of any one of claims 1 to 44, wherein the intracellular membrane is a Golgi apparatus membrane.

47. 47. The non-naturally occurring polypeptide of any one of claims 1 to 44 or 46, wherein the serine / threonine kinase is anchored such that the kinase domain of the serine / threonine kinase is located on the luminal side of the Golgi apparatus membrane.

48. A nucleic acid molecule encoding the non-naturally occurring polypeptide of any one of claims 1 to 47.

49. 49. An expression vector comprising the nucleic acid molecule of claim 48.

50. 48. An expression vector encoding (i) a non-naturally occurring polypeptide of any one of claims 1 to 47, and (ii) a secreted protein.

51. 1. A recombinant host cell comprising: (i) the non-naturally occurring polypeptide of any one of claims 1 to 47, wherein the serine / threonine kinase is heterologous to the recombinant host cell; and (ii) a nucleic acid molecule according to claim 48; and A recombinant host cell comprising:

52. 52. The recombinant host cell of claim 51, wherein the recombinant host cell comprises a fungal host cell, a bacterial host cell, an algal host cell, or a plant host cell.

53. 52. The recombinant host cell of claim 51, wherein the recombinant host cell comprises a bacterium.

54. The bacterium is Escherichia coli or Bacillus subtilis.

54. The recombinant host cell of claim 53.

55. 52. The recombinant host cell of claim 51, wherein the recombinant host cell comprises a eukaryotic host cell.

56. 56. The recombinant host cell of claim 55, wherein the eukaryotic host cell comprises a fungus.

57. 57. The recombinant host cell of claim 56, wherein the fungus comprises Aspergillus, Candida, Fusarium, Hansenula, Kluyveromyces, Pichia, Penicillium, Saccharomyces, Tetrahymena, Thermocellomyces, Trichoderma, Yarrowia, or Zygosaccharomyces.

58. 57. The recombinant host cell of claim 56, wherein the fungus comprises a yeast cell.

59. 59. The recombinant host cell of claim 58, wherein the fungus comprises Kluyveromyces lactis.

60. 59. The recombinant host cell of claim 58, wherein the fungus comprises Pichia pastoris.

61. 57. The recombinant host cell of claim 56, wherein the fungus comprises a filamentous fungus.

62. 62. The recombinant host cell of claim 61 , wherein the filamentous fungus comprises Aspergillus.

63. 62. The recombinant host cell of claim 61, wherein the filamentous fungus comprises Thermocellomyces heterothalica.

64. 64. The recombinant host cell of any one of claims 51 to 63, wherein the serine / threonine kinase is anchored in the intracellular membrane via a lipid.

65. 65. The recombinant host cell of claim 64, wherein the lipid is covalently attached to the serine / threonine kinase.

66. 66. The recombinant host cell of any one of claims 64 or 65, wherein the serine / threonine kinase and the lipid form a prenylated protein.

67. 66. The recombinant host cell of claim 65, wherein the lipid covalently attached to the serine / threonine kinase comprises a fatty acyl group.

68. 66. The recombinant host cell of claim 65, wherein the lipid and the serine / threonine kinase form a glycosylphoshaditylinositol-linked protein.

69. 48. A kit comprising a nucleic acid molecule encoding the non-naturally occurring polypeptide of any one of claims 1 to 47 and a nucleic acid molecule encoding a secreted protein.

70. 70. The kit of claim 69, wherein the kit further comprises a host cell.

71. 69. A method comprising using the serine / threonine kinase of any one of claims 51 to 68 to phosphorylate a secreted protein, thereby producing a phosphorylated secreted protein.

72. 72. The method of claim 71, further comprising recovering the phosphorylated secreted protein from the recombinant host cell, thereby producing a recovered phosphorylated secreted protein.

73. 73. The method of claim 72, further comprising using the recovered phosphorylated secretory protein for the manufacture of a food product.

74. 74. The method of claim 73, wherein the food product is a dairy product.

75. 74. The method of claim 73, wherein the food product is a dairy substitute.

76. 74. The method of claim 73, wherein the food product is cheese.

77. A fungal cell comprising a heterologous kinase, wherein said heterologous kinase is a Fam20c member.

78. 78. The fungal cell of claim 77, wherein the heterologous kinase is a human Fam20c member.

79. 79. The fungal cell of claim 78, wherein the Fam20c member is a truncated Fam20c member.

80. Casein that is phosphorylated at one or more amino acid residues that are not naturally phosphorylated.

81. The casein of claim 80, wherein the casein is αS1-casein.

82. 82. The casein of claim 81, wherein the αS1-casein is phosphorylated at S56, S61, S63, S64, S79, S81, S82, S83, S90, S103 or S130.

83. 82. The casein of claim 81, wherein the αS1-casein is phosphorylated at S56, S79, S81, S82, S83 or S103.

84. 82. The casein of claim 81, wherein the αS1-casein is phosphorylated at S56.

85. 82. The casein of claim 81, wherein the αS1-casein is phosphorylated at S56, S61, S63, S64, S79, S81, S82, S83, S90, S103 and S130.

86. 81. The casein of claim 80, wherein the phosphorylated casein is αS2-casein.

87. 87. The casein of claim 86, wherein the αS2-casein is phosphorylated at S23, S24, S25, S46, S52, S71, S72, S73, S76, T145, S146, S150, S158, T159 or T163.

88. 87. The casein of claim 86, wherein the αS2-casein is phosphorylated at S23, S24, S25, S52, S71, S72, S73 or S76.

89. 87. The casein of claim 86, wherein the αS2-casein is phosphorylated at S23, S52, S71, S72, S73 or S76.

90. 87. The casein of claim 86, wherein the αS2-casein is phosphorylated at S23, S52, S71, S72, S73 and S76.

91. 87. The casein of claim 86, wherein the αS2-casein is phosphorylated at S23, S24, S25, S46, S52, S71, S72, S73, S76, T145, S146, S150, S158 and T159.

92. 81. The casein of claim 80, wherein the phosphorylated casein is β-casein.

93. 93. The casein of claim 92, wherein the beta-casein is phosphorylated at S30, S32, S33, S34, S37, T39, S50 or T56.

94. 93. The casein of claim 92, wherein the β-casein is phosphorylated at S30, S32, S33, S34, S37 or T39.

95. 93. The casein of claim 92, wherein the β-casein is phosphorylated at S37.

96. 93. The casein of claim 92, wherein the beta-casein is phosphorylated at S30, S32, S33, S34, S37, T39, S50 and T56.

97. 81. The casein of claim 80, wherein the phosphorylated casein is κ-casein.

98. 98. The casein of any one of claims 80 to 97, wherein the casein is phosphorylated at two or more amino acid residues that are not naturally phosphorylated.

99. 98. The casein of any one of claims 80 to 97, wherein the casein is phosphorylated at three or more amino acid residues that are not naturally phosphorylated.

100. 98. The casein of any one of claims 80 to 97, wherein the casein is phosphorylated at 4 or more amino acid residues that are not naturally phosphorylated.

101. 98. The casein of any one of claims 80 to 97, wherein the casein is phosphorylated at 5 or more amino acid residues that are not naturally phosphorylated.

102. 98. The casein of any one of claims 80 to 97, wherein the casein is phosphorylated at 6 or more amino acid residues that are not naturally phosphorylated.

103. 103. An engineered food product comprising the casein of any one of claims 80 to 102.

104. 104. The engineered food product of claim 103, wherein the food product is a dairy product.

105. 104. The engineered food product of claim 103, wherein the food product is a dairy substitute.

106. 104. The engineered food product of claim 103, wherein the food product is cheese.

107. 103. A method of producing a phosphorylated casein according to any one of claims 80 to 102, the method comprising phosphorylating casein using a non-naturally occurring polypeptide according to any one of claims 1 to 47.

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