Method to produce phosphorylated milk proteins in microbes

Abstract

The present disclosure relates to methods for production of phosphorylated recombinant animal proteins, phosphorylated phosphomimetic animal proteinsand phosphomimetic animal proteins, as well as the use of such animal proteins.

Description

[0001] Method to produce phosphorylated milk proteins in microbes

[0002] Technical field

[0003] The present disclosure relates to methods for production of phosphorylated casein, phosphorylated phosphomimetic casein and phosphomimetic casein, as well as the use of such caseins.

[0004] Background

[0005] Milk proteins, such as caseins and whey proteins are commonly found in mammalian milk. Caseins are some of the most valued protein sources, for their superior nutritional and functional attributes, and has a wide variety of uses, from being a major component of cheese, to the use of casein as a food additive for emulsification, texture and stabilization.

[0006] Methods for production of recombinant caseins have been known for many years (Jimenez-Flores et al, 1990, Thurmond et al, 1997), however these methods generally suffer from limitations such as poor yield ranges and expression of milk protein without the valued functional attributes. To this date, no method allowing for industrial production of recombinant caseins, which can overcome the expression limitations has been developed.

[0007] Summary

[0008] The invention is as defined in the claims.

[0009] In a first aspect the present disclosure concerns a system for producing one or more phosphorylated recombinant animal proteins, said system comprising: i. a microbial host cell expressing: a. at least one recombinant animal protein comprising a phosphorylation motif being Serine (S) - X - Glutamic acid (E)ZAspartic acid (D), wherein X is any amino acid, or a continuous stretch of at least two serines, and b. at least one microbial kinase; or ii. a microbial host cell expressing at least one animal protein comprising a phosphorylation motif being S-X-E / D or a continuous stretch of at least two serines, and a composition comprising at least one microbial kinase, whereby said system produces one or more phosphorylated recombinant animal proteins.

[0010] In a second aspect the present disclosure concerns a system for producing one or more phosphorylated caseins, said system comprising: i. a microbial host cell expressing: a. at least one recombinant casein, wherein said casein is alpha-casein S1 (aS1), alpha-casein S2 (aS2), beta-casein and kappa-casein, and b. at least one microbial kinase; or ii. a microbial host cell expressing at least one casein selected from alphacasein (aS1 , aS2), beta-casein or kappa-casein, and a composition comprising at least one microbial kinase, whereby said system is capable of producing one or more phosphorylated recombinant caseins.

[0011] In a third aspect the present disclosure concerns a system for producing one or more phosphomimetic recombinant caseins, said system comprising a microbial host cell expressing: i. at least one recombinant casein, wherein said casein is alpha-casein S1 (aS1), alpha-casein S2 (aS2), beta-casein and kappa-casein, wherein one or more amino acid residues that are phosphorylated in a native mammalian casein are substituted with one or more phosphomimetic amino acids; and ii. optionally at least one microbial kinase capable of phosphorylating said phosphomimetic casein at residues which are phosphorylated in a native mammalian casein, and which have not been substituted with phosphomimetic amino acids, whereby said system is capable of producing one or more phosphomimetic recombinant caseins.

[0012] In a fourth aspect the present disclosure concerns phosphorylated, recombinant casein. In a fifth aspect the present disclosure concerns phosphomimetic recombinant casein.

[0013] In a sixth aspect the present disclosure concerns phosphorylated, phosphomimetic recombinant casein.

[0014] In a seventh aspect the present disclosure concerns a micelle comprising phosphorylated and / or phosphomimetic recombinant casein.

[0015] In an eighth aspect the present disclosure concerns a food product comprising phosphorylated and / or phosphomimetic recombinant casein.

[0016] Description of Drawings

[0017] Figure 1. Expression of Human Fam20C and Bovine Fam20C in E. coli. The proteins are expressed as aggregates in the insoluble fraction. A. Insoluble fraction. B. Soluble fraction. V: Vector; aCN: asiON ; Hf: Human Fam20C ; Bf: Bovine Fam20C domain ; Bdp: Bovine Fam20C whole gene ; M: Marker.

[0018] Figure 2. Co-expression of casein aSiand p, and PrkD in E. coli. The arrows indicate PrkD and the caseins aSiand p.

[0019] Figure 3. Co-expression of casein aSiand YabT in E. coli. The arrows indicate YabT and asi and p caseins.

[0020] Figure 4. Phosphomimetic variants of cxsiand p casein having serine replaced with aspartic acid.

[0021] Figure 5. Calcium binding.

[0022] Bar graph depicts calcium binding affinity of the protein samples, a-casein (asi& as2) sample from Sigma are used as a positive control. Y-axis represents calcium binding affinity as a function of pg / well per mg of protein. In vivo phosphorylated asivariants were used in the study; Dp - Dephosphorylated. Data are presented as mean = SD, n =3. Statistical analysis: two-way analysis of variance (ANOVA) followed by Sidak's multiple comparisons test, *p <0.05, **p < 0.01 , ***p <0.001 , ****p <0.0001 , ns - no significance (compared to control a-casein and different variants; compared to Rasiand other recombinant variants; and compared to asvariants and its dephosphorylated counterparts).

[0023] Figure 6. Clustal O (1.2.4) multiple sequence alignment of alpha s1 casein from camel, human, goat, sheep, bovine and buffalo. Amino acids in bold (S) are phosphorylated.

[0024] Figure 7. Clustal O (1.2.4) multiple sequence alignment of alpha s2 casein from camel, human, goat, sheep, bovine and buffalo. Amino acids in bold (S) are phosphorylated.

[0025] Figure 8. Clustal O (1.2.4) multiple sequence alignment of beta casein from camel, human, goat, sheep, bovine and buffalo. Amino acids in bold (S, T) are phosphorylated.

[0026] Figure 9. Clustal O (1.2.4) multiple sequence alignment of kappa casein from camel, human, goat, sheep, bovine and buffalo. Amino acids in bold (S, T) are phosphorylated.

[0027] Figure 10. pH 4-7 immobilized pH gradient (IPG) strips were used for isoelectric focusing, followed by separation on a second-dimension gel and staining with Pro-Q diamond and SYPRO Ruby for mapping phosphorylation levels and total proteome by the different kinases; a-casein (a-Cn) from Sigma was used as a positive control. Rasi: asi-casein sample without any kinase activity, Rasi-PD: co-expressed with PrkD, Rasi-YT: coexpressed with YabT,

[0028] Detailed description

[0029] Definitions

[0030] The term ‘milk protein’ as used herein refers to whey protein and casein protein, which are two types of protein found in milk, such as in milk from cows, goats, sheep, horse, camel, deer, reindeer and buffalo, humans, or other mammals.

[0031] The term ‘whey protein’ refers generally to a group of milk proteins that remain soluble when liquid milk is acidified to a pH of 4.6 or lower, whereas ‘casein proteins’ are milk proteins that coagulate at acidic pH to become cheese, yogurt, or another solidified or semi-solidified milk product.

[0032] The term ‘casein’ as used herein refers to alpha Slcasein, alpha S2 -casein, beta casein or kappa casein (aS1 , aS2, [3-casein and K-casein) which can be differentiated from one another by their amino acids composition, their charge distribution and their tendency to form aggregates in the presence of calcium.

[0033] The term ‘native casein’ as well as the term ‘native mammalian casein’ are used interchangeably, and refers to casein which can be obtained from mammalian milk.

[0034] The term ‘recombinant casein’ as used herein refers to casein produced by recombinant techniques, such as by microbial host cells. For example, a recombinant casein may be a mammalian (human, bovine or other) casein recombinantly expressed in a microbial host cell. The term ‘phosphorylated recombinant casein’ as used herein refers to recombinant casein which has been phosphorylated at one or more specific, native and / or non-native phosphorylation sites. The term ‘phosphomimetic recombinant casein’ as used herein refers to recombinant casein wherein specific native phosphorylation sites have been replaced with phosphomimetic amino acid. The term ‘phosphorylated, phosphomimetic recombinant casein’ refers to phosphomimetic casein, which has been phosphorylated at specific, native phosphorylation sites.

[0035] The term ‘in vivo phosphorylation’ as used herein refers to methods where one or more kinase genes and one or more casein genes are co-expressed in the same host cell, and where the expressed kinase(s) phosphorylates the expressed casein(s) either inside the host cell, or in the medium after secretion of the kinase(s) and casein(s).

[0036] The term ‘in vitro phosphorylation’ as used herein refers to methods where one or more casein genes are expressed in one host cell, and where the recombinantly produced caseins are phosphorylated outside the host cell after being put in contact with a kinase. The term ‘co-expression’ as used herein should be understood as a simultaneous expression of casein and kinase, either in the same host cell or in different host cells in the same medium.

[0037] The term ‘host cell’ as used herein, refers to an organism selected from the group consisting of bacteria, yeast and filamentous fungi. Preferred bacteria are selected from the group consisting of, but are not limited to, lactic acid bacteria, for example Lactococcus spp. and Lactobacillus spp.; Corynebacterium spp., Pseudomonas spp., Streptomyces spp. Escherichia coli, Geobacillus spp., and Bacillus spp., for example Bacillus subtilis, Bacillus thuringiensis and Bacillus licheniformis. Preferred fungi from the group consisting of, but are not limited to are Trichoderma reesei, Aspergillus vadensis, Aspergillus oryzae and Aspergillus niger and preferred yeast cells are selected from the group consisting of Kluyveromyces spp., Pichia spp., Saccharomyces spp., Tetrahymena spp., Yarrowia spp., Hansenula spp., Blastobotrys spp., Aspergillus spp., Candida spp., Zygosaccharomyces spp., Trichoderma spp., Chrysosporium spp., Fusarium spp., and Neurospora spp. and Debaryomyces sp.

[0038] The term ‘kinase’ as used herein refers to class of enzymes which catalyse transfer of y- phosphate from ATP to a hydroxyl group on a side chain of Ser / Thr or Tyr in proteins and peptides. The term ‘microbial kinase’ as used herein refers to kinases of microbial origin, such as bacterial kinases and / or fungal kinases.

[0039] The term ‘sequence identity’ as used herein refers to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the NCBI BLAST program.

[0040] The term ‘phosphomimetic amino acid’ as used herein refers to non-phosphorylated amino acids which mimic phosphorylated amino acids, without being phosphorylated. Those nonphosphorylated amino acids appear chemically similar to phosphorylated amino acids. As an example, aspartic acid (D) is chemically similar to phospho-serine (both carries negative charges at physiological pH). The replacement of a phosphor-serine with e.g. aspartic acid, is refers to as a phosphomimetic substitution. The substitution has the effect, that the phosphomimetic amino acid always will appear in its phosphorylated form, and thus mimic a phosphorylated amino acid.

[0041] The term ‘phosphomimetic protein’ as used herein refers to proteins in with amino acids which are phosphorylated in the native protein, referred to herein as ‘native phosphorylation sites’, are substituted with phosphomimetic amino acids. A phosphomimetic protein could be a casein, where a serine and / or a threonine, which is commonly phosphorylated, is substituted with a phosphomimetic amino acid, e.g., an aspartic acid (D), or a glutamic acid (E).

[0042] The term ‘degree of phosphorylation’ as used herein refers to the efficiency of the phosphorylation process performed by the kinase. A degree of phosphorylation of 80% at a native phosphorylation site X, means that 80% of the recombinant casein proteins are phosphorylated at the given native site X.

[0043] The term ‘functional recombinant casein’ as used herein refers to phosphorylated recombinant casein, phosphomimetic recombinant casein and phosphorylated, phosphomimetic recombinant casein, which has functional properties which allows for the use of the recombinant casein as a substitute for native casein. Examples of functional properties include calcium-binding properties, which is of importance for the formation of micelles.

[0044] The term “animal protein” or “animal-derived protein” as used herein refers to proteins and / or protein components derived from higher multicellular non-plant eukaryotes, including but not limited to proteins derived from dairy, such as milk, eggs, meat, fish, and other animal-derived products. Animal Protein

[0045] Animal proteins have a wide variety of uses. Animal proteins may be useful as food components; animal proteins may also be useful as drug components, for example as vaccine components, as drug delivery vehicle and / or for encapsulation of drugs; and as polymer for synthetic materials, and cosmetic products. Particularly useful animal proteins may be proteins from milk, such as caseins, and proteins from egg, such as ovalbumins or phosvitins.

[0046] In some embodiments, a recombinant animal protein is a recombinant variant of a native animal protein, wherein said native animal protein is produced by an animal of the kingdom Animalia.

[0047] Without being bound by theory, phosphorylation may play an important role in determining the functional properties of said animal proteins. For example, phosphorylation may play a role in the stabilisation and binding properties of animal proteins, such as caseins, ovalbumins, and phosvitins.

[0048] Provided herein is a system for producing one or more phosphorylated recombinant animal proteins, said system comprising: i. a microbial host cell expressing: a. at least one recombinant animal protein comprising a phosphorylation motif being S-X-E / D or a continuous stretch of at least two serines, and b. at least one microbial kinase; or ii. a microbial host cell expressing at least one animal protein comprising a phosphorylation motif being S-X-E / D or a continuous stretch of at least two serines, and a composition comprising at least one microbial kinase, whereby said system produces one or more phosphorylated recombinant animal protein.

[0049] Further provided herein is a method of producing recombinant animal protein in a microbial host cell, comprising the steps of: i. providing the system as described herein, wherein said host cell is capable of producing recombinant animal protein; ii. incubating said host cell in a medium, under conditions allowing growth of the host cell , whereby at least one recombinant animal protein is produced; and iii. incubating the at least one animal protein with at least one microbial kinase, whereby phosphorylated recombinant animal protein is produced, preferably wherein said recombinant animal protein is a casein or egg protein.

[0050] In some embodiments, said recombinant animal protein comprises a continuous stretch of at least three serines, such as at least four serines, such as at least five serines, such as at least six serines, such as at least seven serines, such as at least eight serines, such as at least nine serines, such as a continuous stretch of at least ten serines.

[0051] In some embodiments, a serine or threonine residue is phosphorylated. In some embodiments, a serine residue may be phosphorylated. In some embodiments, a threonine residue may be phosphorylated. In some embodiments, said phosphorylated serine residue may be comprised within said continuous stretch of serines. In some embodiments, said phosphorylated serine residue may not be comprised within said continuous stretch of serines.

[0052] In some embodiments, said phosphorylated serine residue may be comprised within said S-X-E / D phosphorylation motif. In some embodiments, said phosphorylated serine residue may not be comprised within said S-X-E / D phosphorylation motif.

[0053] In some embodiments, at least a serine residue located upstream of the continuous stretch of serines is phosphorylated. In some embodiments, at least a threonine residue located upstream of the continuous stretch of serines is phosphorylated. In some embodiments, at least a serine residue located downstream of the continuous stretch of serines is phosphorylated. In some embodiments, at least a threonine residue located downstream of the continuous stretch of serines is phosphorylated.

[0054] In some embodiments, at least a serine residue located upstream of the S-X-E / D phosphorylation motif is phosphorylated. In some embodiments, at least a threonine residue located upstream of the S-X-E / D phosphorylation motif is phosphorylated. In some embodiments, at least a serine residue located downstream of the S-X-E / D phosphorylation motif is phosphorylated. In some embodiments, at least a threonine residue located downstream of the S-X-E / D phosphorylation motif is phosphorylated.

[0055] The present invention moreover allows for phosphorylation of non-native phosphorylation sites, including serines, threonines and tyrosines sites different from / he above-mentioned phosphorylation motifs, on a peptide or protein comprising the above-mentioned phosphorylation motifs.

[0056] Casein

[0057] Casein has a wide variety of uses, being a major component of food products such as cheese and yoghurt, as well as being a valuable food additive due to its emulsifying properties.

[0058] In milk, native caseins form large colloidal particles between 50 to 600 nm in diameter (approximately 150 nm on average) referred to as casein micelles. The micelles form a very stable colloidal system in milk, being one of the main causes for its colour, heat stability and coagulation by acid and / or rennin

[0059] Phosphorylation plays an important role in the formation of casein micelles as the micelles are formed by calcium phosphate complexation by phosphoserine residues present in the casein structure. Phosphorylation of caseins enhances their ability to form and stabilize the casein micelle structure, which is essential for the texture, viscosity, and stability of dairy products like cheese and yogurt. Moreover the phosphorylation is important for gelling and emulsification properties.

[0060] When preparing recombinant casein, it is highly important that the caseins are phosphorylated at the corrects sites, with an appropriate degree of phosphorylation, as this is crucial for the structural and functional properties of the protein, e.g. for recombinant casein to be able to efficiently form micelles. Native casein phosphorylation sites

[0061] Native mammalian caseins, including alpha S1 casein, alpha s2 casein, beta casein and kappa casein, are typically synthesized and phosphorylated in the lactating mammary gland. The native phosphorylation sites of caseins, are typically phosphorylated after translation by casein kinases, such as casein kinase 1 (CK1) and Fam20C.

[0062] Native caseins produced by mammals typically present one or more native phosphorylation sites including both serine and threonine residues, which are well conserved between the different mammals as depicted in Figure 6 (Alpha s1 casein, sequence alignment for camel, human, goat, sheep, bovine and buffalo), Figure 7 (Alpha s2 casein, sequence alignment for camel, human, goat, sheep, bovine and buffalo), Figure 8 (Beta casein, sequence alignment for camel, human, goat, sheep, bovine and buffalo), and Figure 9 (Kappa casein, sequence alignment for camel, human, goat, sheep, bovine and buffalo).

[0063] Human alpha-s1 casein (SEQ ID NO: 10) is known to have the following native phosphorylation sites: S31 , S33, S41 , S71 , S85, S86, S88, S89, S90, S91.

[0064] Camel alpha-s1 casein (SEQ ID NO: 11) is known to have the following native phosphorylation sites: S33, S83, S85, S86, S87, S88.

[0065] Sheep alpha-s1 casein (SEQ ID NO: 12) is known to have the following native phosphorylation sites: S27, S56, S61 , S63, S79, S80, S81 , S82, S83, S90, S130. Goat alpha-s1 casein (SEQ ID NO: 13) is known to have the following native phosphorylation sites: S61 , S63, S79, S80, S81 , S82, S83, S90, S130.

[0066] Bovine alpha-s1 casein (SEQ ID NO: 14) is known to have the following native phosphorylation sites: S56, S61 , S63, S79, S81 , S82, S83,S90.

[0067] Buffalo alpha-s1 casein (SEQ ID NO: 15) is known to have the following native phosphorylation sites: S63, S79, S80, S81 , S82, S83, S90. Camel alpha-s2 casein (SEQ ID NO: 17) is known to have the following native phosphorylation sites: S23, S24, S25, S28, S47, S68, S123, S125, S128, S136.

[0068] Sheep alpha-s2 casein (SEQ ID NO: 18) is known to have the following native phosphorylation sites: S23, S24, S25, S72, S73, S74, S77, S145, S147, S151 , S159.

[0069] Goat alpha-s2 casein (SEQ ID NO: 19) is known to have the following native phosphorylation sites: S23, S24, S25, S72, S73, S74, S77, S145, S147, S151 , S159. Bovine alpha-s2 casein (SEQ ID NO: 20) is known to have the following native phosphorylation sites: S23, S24, S25, S28, S46, S71 , S72, S73, S76, S144, S146, S150, S158.

[0070] Buffalo alpha-s2 casein (SEQ ID NO: 21) is known to have the following native phosphorylation sites:

[0071] Human beta-casein (SEQ ID NO: 22) is known to have the following native phosphorylation sites: T18, S21 , S23, S24, S25.

[0072] Camel beta-casein (SEQ ID NO: 23) is not known to have native phosphorylation sites.

[0073] Sheep beta-casein (SEQ ID NO: 24) is known to have the following native phosphorylation sites:T27, S30, S32, S33, S34.

[0074] Goat beta-casein (SEQ ID NO: 25) is known to have the following native phosphorylation sites:T27, S30, S32, S33, S34.

[0075] Bovine beta-casein (SEQ ID NO: 26) is known to have the following native phosphorylation sites: S30, S32, S33, S34, S50.

[0076] Buffalo beta-casein (SEQ ID NO: 27) is known to have the following native phosphorylation sites: S30, S32, S33, S34.

[0077] Human kappa-casein (SEQ ID NO: 28) is known to have the following native phosphorylation sites: T157. Camel kappa-casein (SEQ ID NO: 29) is not known to have native phosphorylation sites.

[0078] Sheep kappa-casein (SEQ ID NO: 30) is known to have the following native phosphorylation sites: S148, S172, S189.

[0079] Goat kappa-casein (SEQ ID NO: 31) is known to have the following native phosphorylation sites: S148, T166, S170, S187.

[0080] Bovine kappa-casein (SEQ ID NO: 32) is known to have the following native phosphorylation sites: S148, T166, S170, S187.

[0081] Buffalo kappa-casein (SEQ ID NO: 33) is not known to have native phosphorylation sites.

[0082] Systems and methods for production of recombinant casein

[0083] The current disclosure relates to different strategies to produce recombinant caseins in microorganisms such as bacteria, yeasts, fungi.

[0084] In one embodiment, the system of the current disclosure relates to recombinant casein, wherein the recombinant casein is a recombinant variant of a native mammalian casein, optionally wherein said mammalian casein is that found in milk from a mammal selected from the group consisting of human, cow, buffalo, zebu, yak, equines, sheep, goat, horse, donkey, camel, deer, and reindeer.

[0085] In one embodiment the native mammalian casein is a casein natively produced in a mammal, such as a casein natively produced in human, cow, buffalo, zebu, yak, equines, sheep, goat, horse, donkey, camel, deer, and reindeer, and wherein said native mammalian casein is naturally phosphorylated at one or more native phosphorylation sites.

[0086] In one embodiment the recombinant casein is recombinant bovine casein.

[0087] In one embodiment the recombinant bovine casein is recombinant alpha-casein (aS1 , aS2), beta-casein and / or kappa-casein. In one embodiment the recombinant bovine casein is alpha-casein S1 (aS1). In one embodiment the recombinant bovine casein is alphacasein S2 (aS2). In one embodiment the recombinant bovine casein is beta-casein. In one embodiment the recombinant bovine casein is kappa-casein.

[0088] In one embodiment the recombinant casein is recombinant human casein.

[0089] In one embodiment the recombinant casein is recombinant human alpha-casein (aS1 , aS2), beta-casein or kappa-casein.

[0090] In one embodiment the recombinant casein of the current disclosure is a casein selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33 or a variant thereof, wherein said variant has at least 70% sequence identity, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 88% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, to any one of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, or SEQ ID NO: 33.

[0091] Phosphorylated recombinant casein

[0092] For recombinant casein to become an alternative to casein obtained from mammalian milk, it is important that the recombinant casein holds at least some of the functional properties of casein. The phosphorylation pattern of casein plays an important role in the formation of casein micelles, as the micelle formation is supported by complexation between phosphoserine residues and calcium. Thus it is important that the recombinant casein is sufficiently phosphorylated, both in terms of phosphorylated sites, and degree of phosphorylation at the phosphorylated sites.

[0093] The present disclosure presents methods for production of phosphorylated recombinant casein. The present disclosure moreover presents methods for production of phosphomimetic casein, and methods for production of phosphorylated, phosphomimetic recombinant casein.

[0094] In one embodiment the current disclosure relates to a system for producing one or more phosphorylated recombinant caseins.

[0095] In one embodiment the system comprises: i. a microbial host cell expressing: a. at least one recombinant casein selected from the group consisting of alpha-casein aS1 , alpha-casein aS2, beta-casein and kappa-casein and b. at least one microbial kinase; or ii. a microbial host cell expressing at least one casein selected from the group consisting of alpha-casein aS1 , alpha-casein aS2, beta-casein or kappa- casein, and a composition comprising at least one microbial kinase, whereby said microbial host cell is capable of producing one or more phosphorylated recombinant casein.

[0096] In one embodiment the present disclosure relates to a method of producing recombinant casein in a microbial host cell, comprising the steps of: i. Providing a microbial host cell according to the present disclosure, ii. incubating said microbial host cell in a medium, under conditions allowing growth of the microbial host cell , whereby at least one recombinant casein is produced; and iii. incubating the at least one casein with at least one microbial kinase, whereby phosphorylated recombinant casein is produced. In one embodiment the host cell according to the present disclosure is engineered to express a casein gene, such as a mammalian casein gene, wherein said gene is inserted into an expression vector and transformed into the host cell.

[0097] In one embodiment the kinase is capable of phosphorylating one or more amino acid residues of said recombinant casein at its native phosphorylation sites.

[0098] In one embodiment, the method of producing recombinant casein, comprises a step of recovering the recombinant casein.

[0099] In one embodiment the recombinant casein is phosphorylated at one or more native phosphorylation sites. In one embodiment the recombinant casein is phosphorylated at two native phosphorylation sites. In one embodiment the recombinant casein is phosphorylated at three native phosphorylation sites. In one embodiment the recombinant casein is phosphorylated at four native phosphorylation sites. In one embodiment the recombinant casein is phosphorylated at five native phosphorylation sites. In one embodiment the recombinant casein is phosphorylated at six native phosphorylation sites. In one embodiment the recombinant casein is phosphorylated at seven native phosphorylation sites. In one embodiment the recombinant casein is phosphorylated at eight native phosphorylation sites. In one embodiment the recombinant casein is phosphorylated at nine native phosphorylation sites. In one embodiment the recombinant casein is phosphorylated at ten native phosphorylation sites. In one embodiment the recombinant casein is phosphorylated at more than ten native phosphorylation sites.

[0100] In one embodiment the recombinant casein is phosphorylated at all its native phosphorylation sites. Native phosphorylation sites are serine and / or threonine residues and are described in detail herein in a dedicated section.

[0101] In one embodiment one or more serine and / or threonine residues of the recombinant casein, such as of a casein selected from the group consisting of: alpha casein (aS1) as set forth in SEQ ID NO 10, 11 , 12, 13 14 and 15, or a variant thereof, wherein said variant has at least 70% sequence identity to any one of SEQ ID NO 10, 11 , 12, 13 14 and 15; alpha casein (aS2) as set forth in SEQ ID NO 16, 17, 18, 19, 20 and 21 , or a variant thereof, wherein said variant has at least 70% sequence identity to any one of SEQ ID NO 16, 17, 18, 19, 20 and 21 ; beta casein as set forth in SEQ ID NO 22, 23, 24, 25, 26, and 27, or a variant thereof, wherein said variant has at least 70% sequence identity to any one of SEQ ID NO 22, 23, 24, 25, 26, and 27; and kappa casein as set forth in SEQ ID NO 28, 29, 30, 31 , 32 and 33, or a variant thereof, wherein said variant has at least 70% sequence identity to any one of SEQ ID NO 28, 29, 30, 31 , 32 and 33, are phosphorylated.

[0102] In one embodiment the recombinant bovine alpha casein (aS1) as set forth in SEQ ID NO: 1 , or a variant thereof having at least 70% sequence identity to in SEQ ID NO: 1 , is phosphorylated at least at one of the following positions: S46, S48, S64, S66, S67, S68, and S75.

[0103] In one embodiment the recombinant bovine alpha-S1 casein as set forth in SEQ ID NO: 14, or a variant thereof having at least 70% sequence identity to in SEQ ID NO: 14, is phosphorylated at least at one of the following positions: S56, S61 , S63, S79, S81 , S82, S83, and S90.

[0104] In one embodiment the recombinant bovine alpha-S2 casein as set forth in SEQ ID NO: 20, or a variant thereof having at least 70% sequence identity to in SEQ ID NO: 20, is phosphorylated at least at one of the following positions: S23, S24, S25, S28, S46, S71 , S72, S73, S76, S144, S146, S150, or S158.

[0105] In one embodiment the recombinant bovine beta casein as set forth in SEQ ID NO: 3, or a variant thereof having at least 70% sequence identity to in SEQ ID NO: 3, is phosphorylated at least at one of the following positions: S15, S17, S18, S19 and S35. In one embodiment the recombinant bovine beta casein as set forth in SEQ ID NO: 26, or a variant thereof having at least 70% sequence identity to in SEQ ID NO: 26, is phosphorylated at least at one of the following positions: S30, S32, S33, S34 and S50.

[0106] In one embodiment the recombinant bovine kappa casein as set forth in SEQ ID NO: 32, or a variant thereof having at least 70% sequence identity to in SEQ ID NO: 32, is phosphorylated at least at one of the following positions: S148, T166, S170, or S187.

[0107] In one embodiment the recombinant human alpha-S1 casein as set forth in SEQ ID NO: 10, or a variant thereof having at least 70% sequence identity to in SEQ ID NO: 10, is phosphorylated at least at one of the following positions: S31 , S33, S41 , S71 , S85, S86, S88, S89, S90, or S91.

[0108] In one embodiment the recombinant human beta casein as set forth in SEQ ID NO: 22, or a variant thereof having at least 70% sequence identity to in SEQ ID NO: 22, is phosphorylated at least at one of the following positions: T18, S21 , S23, S24, or S25.

[0109] In one embodiment the recombinant human kappa casein as set forth in SEQ ID NO: 28 or a variant thereof having at least 70% sequence identity to in SEQ ID NO: 28, is phosphorylated at least at one of the following positions: T157.

[0110] In one embodiment the phosphorylated recombinant casein of the current disclosure is a casein having a sequence selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, and SEQ ID NO: 33, or a variant thereof, wherein said variant has at least 70% sequence identity to any one of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, and wherein one or more amino acid residues of said casein or variant thereof are phosphorylated at : one or more positions selected from the group consisting of S46, S48, S64, S66, S67, S68, and S75 of SEQ ID NO: 1 ; one or more positions selected from the group consisting of S15, S17, S18, S19 and S35 of SEQ ID NO: 3; one or more positions selected from the group consisting of S31 , S33, S41 , S71 , S85, S86, S88, S89, S90, and S91 of SEQ ID NO: 10; one or more positions selected from the group consisting of S33, S83, S85, S86, S87, and S88 of SEQ ID NO: 11 ; one or more positions selected from the group consisting of S27, S56, S61 , S63, S79, S80, S81 , S82, S83, S90, and S130 of SEQ ID NO: 12; one or more positions selected from the group consisting of S61 , S63, S79, S80, S81 , S82, S83, S90, and S130 of SEQ ID NO: 13; one or more positions selected from the group consisting of S56, S61 , S63, S79, S81 ,

[0111] 582, S83, and S90 of SEQ ID NO: 14; one or more positions selected from the group consisting of S63, S79, S80, S81 , S82,

[0112] 583, and S90 of SEQ ID NO: 15; one or more positions selected from the group consisting of S23, S24, S25, S28, S47, S68, S123, S125, S128, and S136 of SEQ ID NO: 17; one or more positions selected from the group consisting of S23, S24, S25, S72, S73, S74, S77, S145, S147, S151 , and S159 of SEQ ID NO:18; one or more positions selected from the group consisting of S23, S24, S25, S72, S73, S74, S77, S145, S147, S151 , and S159 of SEQ ID NO:19; one or more positions selected from the group consisting of S23, S24, S25, S28, S46, S71 , S72, S73, S76, S144, S146, S150, and S158 of SEQ ID NQ:20; one or more positions selected from the group consisting of T18, S21 , S23, S24, and S25 of SEQ ID NO:22; one or more positions selected from the group consisting of T27, S30, S32, S33, and S34 of SEQ ID NO:24; one or more positions selected from the group consisting of T27, S30, S32, S33, and S34 of SEQ ID NO:25; one or more positions selected from the group consisting of S30, S32, S33, S34 and S50 of SEQ ID NO:26; one or more positions selected from the group consisting of S30, S32, S33, and S34 of SEQ ID NO:27; or position T157 of SEQ ID NO:28; one or more positions selected from the group consisting of S148, S172, and S189 SEQ ID NQ:30; one or more positions selected from the group consisting of S148, T166, S170, and S187 of SEQ ID NO:31 ; one or more positions selected from the group consisting of S148, T166, S170, and S187 of SEQ ID NO:32.

[0113] The degree of phosphorylation can be determined using MS (Beausoleil, et al, 2006).

[0114] In one embodiment the phosphorylated recombinant casein has a degree of phosphorylation at each native phosphorylation site of at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as about 100%.

[0115] Phosphomimetic recombinant casein

[0116] Phosphorylation as presented above, introduces negative charges to the phosphorylated amino acid residues. Another feasible way of introducing negative charges to casein, is the introduction of phosphomimetic amino acids, and thus the preparation of phosphomimetic recombinant casein.

[0117] The current disclosure presents methods for production of recombinant casein comprising phosphomimetic amino acids.

[0118] In one embodiment the current disclosure relates to a system for producing one or more phosphomimetic recombinant caseins. In one embodiment the system comprises a microbial host cell expressing: i. at least one recombinant casein selected from the group consisting of alpha-casein (aS1), alpha-casein (aS2), beta-casein and kappa-casein, wherein one or more amino acid residues that are phosphorylated in a native mammalian casein are substituted with one or more phosphomimetic amino acids; and ii. optionally at least one microbial kinase capable of phosphorylating said phosphomimetic casein at residues which are phosphorylated in a native mammalian casein, and which have not been substituted with phosphomimetic amino acids, whereby said host cell is capable of producing one or more phosphomimetic recombinant caseins.

[0119] In one embodiment one or more amino acids have been substituted with a phosphomimetic amino acid.

[0120] In one embodiment one or more serine and / or threonine residues have been substituted with a phosphomimetic amino acid.

[0121] In one embodiment one or more amino acid residues which are phosphorylated in the native mammalian casein have been substituted with one or more phosphomimetic amino acids.

[0122] In one embodiment the phosphomimetic amino acid is a natural amino acid. In one embodiment the phosphomimetic amino acid is an unnatural amino acid.

[0123] In one embodiment the phosphomimetic amino acid carries negatively charged functional groups.

[0124] In one embodiment the phosphomimetic amino acid carries carboxylic acid groups.

[0125] In one embodiment the phosphomimetic amino acid is independently selected from the group consisting of aspartic acid, glutamic acid and / or combinations thereof. In one embodiment the phosphomimetic amino acid is aspartic acid.

[0126] In one embodiment one or more serine and / or threonine residues of the recombinant casein, such as of: alpha casein (aS1) as set forth in any one of SEQ ID NOs: 1 , 10, 11 , 12, 13, 14 and 15, alpha casein (aS2) as set forth in any one of SEQ ID NOs: 16, 17, 18, 19, 20 and 21 , beta casein as set forth in any one of SEQ ID NOs: 3, 22, 23, 24, 25, 26, and 27, and kappa casein as set forth in any one of SEQ ID NOs: 28, 29, 30, 31 , 32 and 33, have been substituted with one or more phosphomimetic amino acids.

[0127] In one embodiment the phosphomimetic recombinant casein of the current disclosure is a casein having a sequence selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 3, of SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, and SEQ ID NO: 33, or a variant thereof, wherein said variant has at least 70% sequence identity to any one of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, and wherein one or more amino acid residues of said variant are individually substituted by a phosphomimetic amino acids, wherein the substituted amino acid residues correspond to: one or more positions selected from the group consisting of S46, S48, S64, S66, S67, S68, and S75 of SEQ ID NO: 1 ; one or more positions selected from the group consisting of S15, S17, S18, S19 and S35 of SEQ ID NO: 3; one or more positions selected from the group consisting of S31 , S33, S41 , S71 , S85, S86, S88, S89, S90, and S91 of SEQ ID NO: 10; one or more positions selected from the group consisting of S33, S83, S85, S86, S87, and S88 of SEQ ID NO: 11 ; one or more positions selected from the group consisting of S27, S56, S61 , S63, S79, S80, S81 , S82, S83, S90, and S130 of SEQ ID NO: 12; one or more positions selected from the group consisting of S61 , S63, S79, S80, S81 , S82, S83, S90, and S130 of SEQ ID NO: 13; one or more positions selected from the group consisting of S56, S61 , S63, S79, S81 ,

[0128] 582, S83, and S90 of SEQ ID NO: 14; one or more positions selected from the group consisting of S63, S79, S80, S81 , S82,

[0129] 583, and S90 of SEQ ID NO: 15; one or more positions selected from the group consisting of S23, S24, S25, S28, S47, S68, S123, S125, S128, and S136 of SEQ ID NO: 17; one or more positions selected from the group consisting of S23, S24, S25, S72, S73, S74, S77, S145, S147, S151 , and S159 of SEQ ID NO: 18; one or more positions selected from the group consisting of S23, S24, S25, S72, S73, S74, S77, S145, S147, S151 , and S159 of SEQ ID NO: 19; one or more positions selected from the group consisting of S23, S24, S25, S28, S46, S71 , S72, S73, S76, S144, S146, S150, and S158 of SEQ ID NO: 20; one or more positions selected from the group consisting of T18, S21 , S23, S24, and S25 of SEQ ID NO: 22; one or more positions selected from the group consisting of T27, S30, S32, S33, and S34 of SEQ ID NO: 24; one or more positions selected from the group consisting of T27, S30, S32, S33, and S34 of SEQ ID NO: 25; one or more positions selected from the group consisting of S30, S32, S33, S34 and S50 of SEQ ID NO: 26; one or more positions selected from the group consisting of S30, S32, S33, and S34 of

[0130] SEQ ID NO: 27; or position T157 of SEQ ID NO: 28; one or more positions selected from the group consisting of S148, S172, and S189 SEQ

[0131] ID NO: 30; one or more positions selected from the group consisting of S148, T166, S170, and S187 of SEQ ID NO: 31 ; one or more positions selected from the group consisting of S148, T166, S170, and S187 of SEQ ID NO: 32. In one embodiment the recombinant bovine alpha casein (aS1) as set forth in SEQ ID NO: 1 , or a variant thereof having at least 70% sequence identity to SEQ ID NO: 1 , comprises a phosphomimetic amino acid at least at one of the following positions: S46, S48, S64, S66, S67, S68, and S75.

[0132] In one embodiment the recombinant bovine alpha-S1 casein as set forth in SEQ ID NO: 14, or a variant thereof having at least 70% sequence identity to SEQ ID NO: 14, comprises a phosphomimetic amino acid at least at one of the following positions: S56, S61 , S63, S79, S81 , S82, S83, or S90.

[0133] In one embodiment the recombinant bovine alpha-S2 casein as set forth in SEQ ID NO: 20, or a variant thereof having at least 70% sequence identity to SEQ ID NO: 20, comprises a phosphomimetic amino acid at least at one of the following positions: S23, S24, S25, S28, S46, S71 , S72, S73, S76, S144, S146, S150, or S158.

[0134] In one embodiment the recombinant bovine beta casein as set forth in SEQ ID NO: 3, or a variant thereof having at least 70% sequence identity to SEQ ID NO: 3, comprises a phosphomimetic amino acid at least at one of the following positions: S15, S17, S18, S19 and S35.

[0135] In one embodiment the recombinant bovine beta casein as set forth in SEQ ID NO: 26, or a variant thereof having at least 70% sequence identity to SEQ ID NO: 26, comprises a phosphomimetic amino acid at least at one of the following positions: S30, S32, S33, S34 and S50.

[0136] In one embodiment the recombinant bovine kappa casein as set forth in SEQ ID NO: 32, or a variant thereof having at least 70% sequence identity to SEQ ID NO: 32, comprises a phosphomimetic amino acid at least at one of the following positions: S148, T166, S170, or S187.

[0137] In one embodiment the recombinant human alpha-S1 casein as set forth in SEQ ID NO:

[0138] 10, or a variant thereof having at least 70% sequence identity to SEQ ID NO: 10, comprises a phosphomimetic amino acid at least at one of the following positions: S31 , S33, S41 , S71 , S85, S86, S88.S89, S90, or S91.

[0139] In one embodiment the recombinant human beta casein as set forth in SEQ ID NO: 22, or a variant thereof having at least 70% sequence identity to SEQ ID NO: 22, comprises a phosphomimetic amino acid at least at one of the following positions: T18, S21 , S23, S24, or S25.

[0140] In one embodiment the recombinant human kappa casein as set forth in SEQ ID NO: 28, or a variant thereof having at least 70% sequence identity to SEQ ID NO: 28, comprises a phosphomimetic amino acid at least at one of the following positions: T 157.

[0141] In one embodiment the phosphomimetic, recombinant casein comprises one phosphomimetic amino acid. In one embodiment the phosphomimetic, recombinant casein comprises two phosphomimetic amino acids. In one embodiment the phosphomimetic, recombinant casein comprises three phosphomimetic amino acids. In one embodiment the phosphomimetic, recombinant casein comprises four phosphomimetic amino acids. In one embodiment the phosphomimetic, recombinant casein comprises five phosphomimetic amino acids. In one embodiment the phosphomimetic, recombinant casein comprises six phosphomimetic amino acids. In one embodiment the phosphomimetic, recombinant casein comprises seven phosphomimetic amino acids. In one embodiment the phosphomimetic, recombinant casein comprises eight phosphomimetic amino acids. In one embodiment the phosphomimetic, recombinant casein comprises nine phosphomimetic amino acids. In one embodiment the phosphomimetic, recombinant casein comprises ten phosphomimetic amino acids. In one embodiment the phosphomimetic, recombinant casein comprises eleven phosphomimetic amino acids. In one embodiment the phosphomimetic, recombinant casein comprises twelve phosphomimetic amino acids. In one embodiment the phosphomimetic, recombinant casein comprises thirteen phosphomimetic amino acids. In one embodiment the phosphomimetic, recombinant casein comprises fourteen phosphomimetic amino acids. In one embodiment the phosphomimetic, recombinant casein comprises fifteen phosphomimetic amino acids. In one embodiment the present disclosure relates to a method of producing recombinant casein in a microbial host cell, comprising the steps of: i. Providing a microbial host cell encoding a recombinant casein as described in the present disclosure; and ii. incubating said microbial host cell in a medium, under conditions allowing growth of the host cell of at least one phosphomimetic casein, whereby at least one phosphomimetic recombinant casein is produced.

[0142] In one embodiment of the present disclosure, the one or more phosphomimetic recombinant casein is as set forth in SEQ ID NO: 2 or SEQ ID NO. 4, or a variant thereof having at least 90% sequence identity, similarity or homology thereto.

[0143] Phosphorylated, phosphomimetic recombinant casein

[0144] The current disclosure presents methods for production of phosphorylated recombinant casein comprising phosphomimetic amino acids.

[0145] In one embodiment the phosphorylated, phosphomimetic recombinant casein has a degree of phosphorylation at each phosphorylated native phosphorylation site of at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as about 100%.

[0146] In one embodiment the phosphomimetic, phosphorylated recombinant casein of the current disclosure is a casein having a sequence selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, and SEQ ID NO: 33, or a variant thereof, wherein said variant has at least 70% sequence identity to any one of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, and wherein: i) one or more amino acid residues of said casein or variant thereof are phosphorylated at; one or more positions selected from the group consisting of S46, S48, S64, S66, S67, S68, and S75 of SEQ ID NO: 1 ; one or more positions selected from the group consisting of S15, S17, S18, S19 and S35 of SEQ ID NO: 3; one or more positions selected from the group consisting of S31 , S33, S41 , S71 , S85, S86, S88, S89, S90, and S91 of SEQ ID NO: 10; one or more positions selected from the group consisting of S33, S83, S85, S86, S87, and S88 of SEQ ID NO: 11 ; one or more positions selected from the group consisting of S27, S56, S61 , S63, S79, S80, S81 , S82, S83, S90, and S130 of SEQ ID NO: 12; one or more positions selected from the group consisting of S61 , S63, S79, S80, S81 , S82, S83, S90, and S130 of SEQ ID NO: 13; one or more positions selected from the group consisting of S56, S61 , S63, S79, S81 ,

[0147] 582, S83, and S90 of SEQ ID NO: 14; one or more positions selected from the group consisting of S63, S79, S80, S81 , S82,

[0148] 583, and S90 of SEQ ID NO: 15; one or more positions selected from the group consisting of S23, S24, S25, S28, S47, S68, S123, S125, S128, and S136 of SEQ ID NO: 17; one or more positions selected from the group consisting of S23, S24, S25, S72, S73, S74, S77, S145, S147, S151 , and S159 of SEQ ID NO: 18; one or more positions selected from the group consisting of S23, S24, S25, S72, S73, S74, S77, S145, S147, S151 , and S159 of SEQ ID NO: 19; one or more positions selected from the group consisting of S23, S24, S25, S28, S46, S71 , S72, S73, S76, S144, S146, S150, and S158 of SEQ ID NO: 20; one or more positions selected from the group consisting of T18, S21 , S23, S24, and S25 of SEQ ID NO: 22; one or more positions selected from the group consisting of T27, S30, S32, S33, and S34 of SEQ ID NO: 24; one or more positions selected from the group consisting of T27, S30, S32, S33, and S34 of SEQ ID NO: 25; one or more positions selected from the group consisting of S30, S32, S33, S34 and S50 of SEQ ID NO: 26; one or more positions selected from the group consisting of S30, S32, S33, and S34 of

[0149] SEQ ID NO: 27; or position T157 of SEQ ID NO: 28; one or more positions selected from the group consisting of S148, S172, and S189 SEQ

[0150] ID NO: 30; one or more positions selected from the group consisting of S148, T166, S170, and S187 of SEQ ID NO: 31 ; one or more positions selected from the group consisting of S148, T166, S170, and S187 of SEQ ID NO: 32 and ii) one or more amino acid residues of said variant are individually substituted by a phosphomimetic amino acids, wherein the substituted amino acid residues correspond to: one or more positions selected from the group consisting of S46, S48, S64, S66, S67, S68, and S75 of SEQ ID NO: 1 ; one or more positions selected from the group consisting of S15, S17, S18, S19 and S35 of SEQ ID NO: 3; one or more positions selected from the group consisting of S31 , S33, S41 , S71 , S85, S86, S88, S89, S90, and S91 of SEQ ID NO: 10; one or more positions selected from the group consisting of S33, S83, S85, S86, S87, and S88 of SEQ ID NO: 11 ; one or more positions selected from the group consisting of S27, S56, S61 , S63, S79, S80, S81 , S82, S83, S90, and S130 of SEQ ID NO: 12; one or more positions selected from the group consisting of S61 , S63, S79, S80, S81 , S82, S83, S90, and S130 of SEQ ID NO: 13; one or more positions selected from the group consisting of S56, S61 , S63, S79, S81 , S82, S83, and S90 of SEQ ID NO: 14; one or more positions selected from the group consisting of S63, S79, S80, S81 , S82, S83, and S90 of SEQ ID NO: 15; one or more positions selected from the group consisting of S23, S24, S25, S28, S47, S68, S123, S125, S128, and S136 of SEQ ID NO: 17; one or more positions selected from the group consisting of S23, S24, S25, S72, S73, S74, S77, S145, S147, S151 , and S159 of SEQ ID NO: 18; one or more positions selected from the group consisting of S23, S24, S25, S72, S73, S74, S77, S145, S147, S151 , and S159 of SEQ ID NO: 19; one or more positions selected from the group consisting of S23, S24, S25, S28, S46, S71 , S72, S73, S76, S144, S146, S150, and S158 of SEQ ID NO: 20; one or more positions selected from the group consisting of T18, S21 , S23, S24, and S25 of SEQ ID NO: 22; one or more positions selected from the group consisting of T27, S30, S32, S33, and S34 of SEQ ID NO: 24; one or more positions selected from the group consisting of T27, S30, S32, S33, and S34 of SEQ ID NO: 25; one or more positions selected from the group consisting of S30, S32, S33, S34 and S50 of SEQ ID NO: 26; one or more positions selected from the group consisting of S30, S32, S33, and S34 of SEQ ID NO: 27; or position T157 of SEQ ID NO: 28; one or more positions selected from the group consisting of S148, S172, and S189 SEQ ID NO: 30; one or more positions selected from the group consisting of S148, T166, S170, and S187 of SEQ ID NO: 31 ; one or more positions selected from the group consisting of S148, T166, S170, and S187 of SEQ ID NO: 32.

[0151] In one embodiment the recombinant bovine alpha casein (aS1) as set forth in SEQ ID NO: 1 , or a variant thereof having at least 70% sequence identity to SEQ ID NO: 1 , i) comprises a phosphomimetic amino acid at least at one position selected from the group consisting of: S46, S48, S64, S66, S67, S68, and S75, and ii) is phosphorylated at least at one position selected from the group consisting of:

[0152] S46, S48, S64, S66, S67, S68, and S75, which is not substituted by a phosphomimetic amino acid.

[0153] In one embodiment the recombinant bovine alpha-S1 casein as set forth in SEQ ID NO:

[0154] 14, or a variant thereof having at least 70% sequence identity to SEQ ID NO: 14 i) comprises a phosphomimetic amino acid at least at one position selected from the group consisting of: S56, S61 , S63, S79, S81 , S82, S83, and S90, and ii) is phosphorylated at least at one position selected from the group consisting of: S56, S61 , S63, S79, S81 , S82, S83, and S90, which is not substituted by a phosphomimetic amino acid.

[0155] In one embodiment the recombinant bovine alpha-S2 casein as set forth in SEQ ID NO:

[0156] 20, or a variant thereof having at least 70% sequence identity to SEQ ID NO: 20 i) comprises a phosphomimetic amino acid at least at one position selected from the group consisting of: S23, S24, S25, S28, S46, S71 , S72, S73, S76, S144, S146, S150, and S158, and ii) is phosphorylated at least at one position selected from the group consisting of: S23, S24, S25, S28, S46, S71 , S72, S73, S76, S144, S146, S150, and S158, which is not substituted by a phosphomimetic amino acid.

[0157] In one embodiment the recombinant bovine beta casein as set forth in SEQ ID NO: 3, or a variant thereof having at least 70% sequence identity to SEQ ID NO: 3 i) comprises a phosphomimetic amino acid at least at a position selected from the group consisting of: S15, S17, S18, S19 and S35, and ii) is phosphorylated at least at one position selected from the group consisting of: S15, S17, S18, S19 and S35, which is not substituted by a phosphomimetic amino acid.

[0158] In one embodiment the recombinant bovine beta casein as set forth in SEQ ID NO: 26, or a variant thereof having at least 70% sequence identity to SEQ ID NO: 26 i) comprises a phosphomimetic amino acid at least at one position selected from the group consisting of: S30, S32, S33, S34 and S50, and ii) is phosphorylated at least at one position selected from the group consisting of: S30, S32, S33, S34 and S50, which is not substituted by a phosphomimetic amino acid.

[0159] In one embodiment the recombinant bovine kappa casein as set forth in SEQ ID NO: 32, or a variant thereof having at least 70% sequence identity to SEQ ID NO: 32 i) comprises a phosphomimetic amino acid at least at one position selected from the group consisting of: S148, T166, S170, and S187, and ii) is phosphorylated at least at one position selected from the group consisting of: S148, T166, S170, and S187, which is not substituted by a phosphomimetic amino acid.

[0160] In one embodiment the recombinant human alpha-S1 casein as set forth in SEQ ID NO:

[0161] 10, or a variant thereof having at least 70% sequence identity to SEQ ID NO: 10 i) comprises a phosphomimetic amino acid at least at one position selected from the group consisting of: S31 , S33, S41 , S71 , S85, S86, S88,S89, S90, and S91 , and ii) is phosphorylated at least at one position selected from the group consisting of: S31 , S33, S41 , S71 , S85, S86, S88,S89, S90, and S91 , which is not substituted by a phosphomimetic amino acid.

[0162] In one embodiment the recombinant human beta casein as set forth in SEQ ID NO: 22, or a variant thereof having at least 70% sequence identity to SEQ ID NO: 22 i) comprises a phosphomimetic amino acid at least at one position selected from the group consisting of: T18, S21 , S23, S24, and S25, and ii) is phosphorylated at least at one position selected from the group consisting of: T18, S21 , S23, S24, and S25, which is not substituted by a phosphomimetic amino acid.

[0163] In one embodiment the present disclosure relates to a method of producing recombinant casein in a microbial host cell, comprising the steps of: i. Providing a microbial host cell encoding a recombinant casein as described in the present disclosure; and ii. incubating said microbial host cell in a medium, under conditions sufficient to allow for production of at least one phosphomimetic casein, whereby at least one phosphomimetic casein is produced; iii. incubating the at least one casein with at least one microbial kinase, whereby phosphorylated and / or phosphomimetic recombinant casein is produced.

[0164] Expression system

[0165] In one embodiment the present disclosure relates to an expression system for expression of phosphorylated recombinant casein in a microbial host cell, comprising: i. a first nucleic acid encoding at least one recombinant casein selected from the group consisting of alpha S1 casein (aS1), alpha-S2 casein (aS2), beta-casein and kappa-casein; and ii. a second nucleic acid encoding a kinase.

[0166] In one embodiment the nucleic acid encoding a casein and the nucleic acid encoding a kinase are expressed in a microbial host cell.

[0167] In one embodiment the nucleic acid encoding at least one casein is expressed in a first microbial host cell, and the nucleic acid encoding at least one kinase is expressed in a second microbial host cell.

[0168] In one embodiment the present disclosure relates to the use of an expression system as defined elsewhere in the description, for production of at least one recombinant casein of interest.

[0169] In one embodiment the at least one recombinant casein expressed by an expression system, is selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, and SEQ ID NO: 33, or a variant thereof having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, sequence identity, similarity, or homology thereto.

[0170] Host cell

[0171] The present disclosure also relates to a microbial host cell genetically engineered to express at least one casein. The present disclosure also relates to a microbial host cell genetically engineered to express at least one egg protein. The host cells described in this section may also usefully be used to produce egg proteins.

[0172] In one embodiment the microbial host cell is a bacterial cell, a fungal cell or a yeast cell. In one embodiment the microbial host cell comprises at least one protease deficiency rendering said microbial host cell completely or partially deficient in the production and / or function of one or more proteases.

[0173] In one embodiment the microbial host cell expresses at least one chaperone protein.

[0174] In one embodiment the present disclosure relates to a microbial host cell comprising a system as defined elsewhere in the description, wherein said host cell is capable of producing a recombinant casein.

[0175] In one embodiment the microbial host cell is a bacterial cell, which belongs to a genus selected from the group consisting of Bacillus spp., Escherichia spp., Corynebacterium spp., Pseudomonas spp., and Streptomyces spp.. For example, the microbial host cell may belong to a species selected from Bacillus subtilis, Escherichia coli,

[0176] In one embodiment the microbial host cell is a bacterial cell, in particular a lactic acid bacteria cell, for example a bacterial cell belonging to Lactococcus spp. and Lactobacillus spp..

[0177] In one embodiment the microbial host cell is a fungal host cell, which belongs to a genus selected from the group consisting of Trichoderma spp., Aspergillus spp., Kluyveromyces spp., Pichia spp., Saccharomyces spp., Tetrahymena spp., Yarrowia spp., Hansenula spp., Blastobotrys spp., Candida spp., Zygosaccharomyces spp., Chrysosporium spp., Fusarium spp., Neurospora spp. and Debaryomyces spp..

[0178] In one embodiment the microbial host cell is a fungal host cell, which belongs to a species selected from selected from the group consisting Trichoderma reesei, Aspergillus vadensis, Aspergillus oryzae and Aspergillus niger.

[0179] Kinase

[0180] The kinases described in this section may be useful for the phosphorylation of animal proteins. In particular, the kinases described in this section may be useful for the phosphorylation of caseins. Furthermore, the kinases described in this section may be useful for the phosphorylation of egg proteins, such as ovalbumin, phosvitin, ovotransferrin, ovoglobulin, ovomucoid, lysozyme, ovomucin, livetins, lipovitellins, and lipovitellenin.

[0181] In one embodiment the microbial kinase is a prokaryotic kinase or a fungal kinase.

[0182] In one embodiment the microbial kinase is a serine / threonine kinase.

[0183] In one embodiment the microbial kinase is a bacterial kinase.

[0184] In one embodiment the microbial kinase is a yeast kinase.

[0185] In one embodiment the microbial kinase is of the Hanks family.

[0186] In one embodiment the kinase is selected from the group consisting of YabT (SEQ ID NO: 5 or SEQ ID NO: 6), PrkD (SEQ ID NO: 7), PrkC (SEQ ID NO: 8) and PKNB1 (SEQ ID NO: 9) or functional variants thereof having at least 60% sequence identity, similarity or homology thereto, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity to any one of YabT (SEQ ID NO: 5), PrkD (SEQ ID NO: 7), PrkC (SEQ ID NO: 8) and PKNB1 (SEQ ID NO: 9). In one embodiment the microbial kinase is YabT as set forth in SEQ ID NO: 5, or a functional variant thereof having at least 60% sequence identity, similarity or homology thereto, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity.

[0187] In one embodiment the microbial kinase is YabT as set forth in SEQ ID NO: 6, or a functional variant thereof having at least 60% sequence identity, similarity or homology thereto, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity.

[0188] In one embodiment the microbial kinase is PrkD as set forth in SEQ ID NO: 7, or a functional variant thereof having at least 70% sequence identity, similarity or homology thereto, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity.

[0189] In one embodiment the microbial kinase is PrkC as set forth in SEQ ID NO: 8, or a functional variant thereof having at least 60% sequence identity, similarity or homology thereto, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity.

[0190] In one embodiment the microbial kinase is PKNB1 as set forth in SEQ ID NO: 9, or a functional variant thereof having at least 60% sequence identity, similarity or homology thereto, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity.

[0191] In vitro phosphorylation

[0192] In one embodiment the recombinant casein is phosphorylated outside the microbial host cell by a microbial kinase that is obtained separately and put in contact with the recombinant casein. In one embodiment the recombinant casein is phosphorylated after the casein has been secreted.

[0193] In one embodiment the recombinant casein is phosphorylated in vitro.

[0194] In vivo phosphorylation

[0195] In one embodiment the recombinant casein is phosphorylated inside the microbial host cell. This may be possible if the same microbial host cell has been engineered to recombinantly produce a casein according to the present disclosure and a microbial kinase according to the present disclosure.

[0196] In one embodiment the recombinant casein is phosphorylated in vivo.

[0197] Functional properties

[0198] For recombinant caseins to become a valuable substitute for casein obtained from mammalian milk, it is important that the recombinant casein holds at least some of the functional properties which casein obtained from mammalian milk holds. One of the most important properties is the ability to form micelles. The ability to form micelles is dependent on the ability of the casein to bind calcium, and the phosphorylation or presence of phosphomimetic residues in a casein is crucial for said casein to be able to bind calcium ions.

[0199] In one embodiment, the phosphorylated recombinant casein of the present disclosure binds calcium ions to comparable levels as casein obtained from mammalian milk, or from bovine milk.

[0200] In one embodiment, the phosphomimetic recombinant casein of the present disclosure binds calcium ions to comparable levels as casein obtained from mammalian milk or from bovine milk. In one embodiment, the phosphorylated, phosphomimetic recombinant casein of the present disclosure binds calcium ions to comparable levels as casein obtained from mammalian milk or from bovine milk.

[0201] Other functional properties include for example coagulation, size of micelles, stability of micelles, gelling properties and emulsification properties. These parameters are critical for developing identical dairy products with functionality and structural properties similar to bovine caseins.

[0202] In one embodiment, the phosphorylated recombinant casein of the present disclosure, the phosphomimetic recombinant casein of the present disclosure and / or the phosphorylated, phosphomimetic recombinant casein of the present disclosure allows coagulation, gelation, emulsification and / or formation of stable micelles comparably to casein obtained from mammalian milk or from bovine milk.

[0203] Micelle

[0204] The method of the current disclosure invention enables production of recombinant caseins, which are phosphorylated and / or phosphomimetic caseins, capable of forming micelles.

[0205] In one embodiment the present disclosure relates to a micelle comprising the recombinant phosphorylated and / or phosphomimetic casein produced as defined elsewhere in the description.

[0206] Use of recombinant casein

[0207] The methods of the current disclosure provide recombinant casein, which is an alternative to casein obtained from mammalian milk. The recombinant casein can be used as an alternative to milk proteins in products which typically are derived from milk, that is dairy products, such as cheese and yoghurt. Moreover, the recombinant casein of the current invention will provide useful as food additives, for example in meat analogues to improve nutritional profile, texture, taste and flavour. A casein product comprising casein of the current invention is advantageous compared to casein obtained from mammalian milk, e.g. as it does not contain lactose, which is not tolerated well by people suffering form lactose intolerance. Casein products obtained from mammalian milk may in fact comprise lactose as an impurity. Moreover, the casein can be produced by greener methods.

[0208] In one embodiment the present disclosure relates to the use of a recombinant casein produced as defined elsewhere in the description, as a food ingredient / for preparing cheese, yogurt, or other liquid, solid or semi-solid milk products.

[0209] In one embodiment the present disclosure relates to a food product (including drinks) or a functional food product or a dairy product comprising recombinant casein produced as defined elsewhere in the description.

[0210] Egg protein

[0211] Egg proteins include, but are not limited to, ovalbumin, phosvitin, ovotransferrin, ovoglobulin, ovomucoid, lysozyme, ovomucin, livetins, lipovitellins, and lipovitellenin. Phosphorylation of recombinant egg proteins may play an important role in ensuring said recombinant egg proteins can effectively recapitulate native egg protein characteristics and functions. For example, phosphorylation may play a role in mineral binding, protein stability and alter protein binding properties. These proteins may be used as ingredients in foods and drinks as they can act as an emulsifier, coagulant, binding agent, leavening agent, thickening agent, moisturizing agent, adhesive, browning agent, clarification agent, gelation agent, and could also be used as a preservative and antimicrobial agent.

[0212] Egg proteins may be useful as food additives, food ingredients and / or drug components, for example as components of vaccines.

[0213] Ovalbumin

[0214] Ovalbumin, also known as OVA, is the primary protein found in egg whites and a phosphoglycoprotein. Ovalbumin may be useful as a food component, particularly for use in products traditionally comprising egg or egg whites. Ovalbumin may be used to stimulate immune responses, thus ovalbumin may be a useful as a drug component, particular those intended to stimulate immune responses, such as vaccines. Thus, recombinant ovalbumin may be useful as a vaccine component.

[0215] Ovalbumin (SEQ ID NO: 34) is known to have the following phosphorylation sites: S68 and S344.

[0216] Phosvitin

[0217] Phosvitin is a protein found in egg yolks. Phosvitin is a phosphoprotein, and in its native state is it known to be the most phosphorylated protein found in nature. In egg yolks, phosvitin plays a role in metal chelation, emulsification and nutrition sequestration. Several Phosvitins exist, including both Phosvitin 1 (SEQ ID NO: 35), Phosvitin 2 (SEQ ID NO: 36) and Phosvitin 3 (SEQ ID NO: 37).

[0218] Phosvitin 1 (SEQ ID NO: 35) is known to have the following phosphorylation sites: S11 , S12, S13, S14, S16, S37, S43, S45, S46, S47, S48, S51 , S52, S69, S70, S71 , S73, S75, S79, T110, S117, S124, S180, S184, S186, S187, S240, and S244.

[0219] Phosvitin 2 (SEQ ID NO: 36) is known to have the following phosphorylation sites: T5, T11 , S12, S13, S14, S15, S16, S17, S19, S20, T21 , T23, S24, S31 , S82, S89, S91 , S96, S98, S99, S100, S104, S124, S125, S126, S130, S134, S135, S137, S197, S198, S199, S200, S201 , S202, S203, S204, or S207.

[0220] Other egg proteins

[0221] Other egg proteins include ovotransferrin (SEQ ID NO: 38), ovoglobulin (SEQ ID NO: 39), ovomucoid (SEQ ID NO: 40), ovomucin (SEQ ID NO: 41), lipovitellin-1 (SEQ ID NO: 42), lipovitellin-2 (SEQ ID NO: 43), livetin (SEQ ID NO: 44) and lysozyme (SEQ ID NO: 45). While these proteins are not natively phosphorylated, phosphorylation of one or more of their Serine, Threonine and / or Tyrosine residues may improve their properties and functionalities, for example may improve their thermostability, tolerance to physical stress.

[0222] Systems and methods for production of recombinant egg proteins

[0223] The current disclosure relates to different strategies to produce recombinant egg proteins in microorganisms such as bacteria, yeasts, fungi. In one embodiment the eggs proteins are avian egg proteins. In other words, said egg proteins may be proteins found in bird eggs, or found in the eggs of animals of the class Aves. In some embodiments, the recombinant ovalbumin is avian recombinant ovalbumin. In some embodiments, the recombinant phosvitin is avian recombinant phosvitin. In some embodiments, the recombinant phosvitin 1 is avian recombinant phosvitin 1. In some embodiments, the recombinant phosvitin 2 is avian recombinant phosvitin 2.

[0224] In one embodiment the system of the current disclosure relates to recombinant egg protein, wherein the recombinant egg protein is a recombinant variant of a native avian egg protein. Said native avian egg protein may be found in eggs from a bird, such as from chicken, duck, goose, quail, guineafowl, turkey, emu, pheasant, gull, partridge or ostrich. In one embodiment the native egg protein is an egg protein found in a bird egg, such as an egg protein found in a chicken, duck, goose, quail, guineafowl, turkey, emu, pheasant, gull, partridge or ostrich egg, and wherein said native avian egg protein is naturally phosphorylated at one or more native phosphorylation sites.

[0225] In one embodiment of the current disclosure, the recombinant egg protein of the current disclosure is one of: ovalbumin as set forth in SEQ ID NO: 34, phosvitin 1 as set forth in SEQ ID NO: 35, phosvitin 2 as set forth in SEQ ID NO: 36, phosvitin 3 as set forth in SEQ ID NO: 37, phosvitin 3 as set forth in SEQ ID NO: 37, ovotransferrin as set forth in SEQ ID NO: 38, ovoglobulin as set forth in SEQ ID NO: 39, ovomucoid as set forth in SEQ ID NO: 40, ovomucin as set forth in SEQ ID NO: 41 , lipovitellin-1 as set forth in SEQ ID NO: 42, lipovitellin -2 as set forth in SEQ ID NO: 43, livetin as set forth in SEQ ID NO: 44, and lysozyme as set forth in SEQ ID NO: 45, or a functional variant thereof, wherein said variant has at least 70% sequence identity, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 88% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, to any one of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO: 45. Phosphorylated recombinant egg protein

[0226] For recombinant egg proteins to become an alternative to native egg proteins, such as those obtained from bird eggs, it is important that the recombinant egg proteins hold at least some of the functional properties of the native egg proteins.

[0227] The present disclosure presents methods for production of phosphorylated recombinant egg proteins. Egg proteins include ovalbumin, phosvitin 1 , phosvitin 2, phosvitin 3, ovotransferrin, ovoglobulin, ovomucoid, ovomucin, lipovitellin-1 , lipovitellin-2, livetin, and lysozyme. Preferably said egg proteins are ovalbumin, phosvitin 1 , and phosvitin 2.

[0228] In one embodiment the current disclosure relates to a system for producing one or more phosphorylated egg proteins. In one embodiment, said egg protein is ovalbumin. In one embodiment, said egg protein is phosvitin, such as phosvitin 1 , phosvitin 2 or phosvitin 3. . In one embodiment said egg protein is ovotransferrin, ovoglobulin, ovomucoid, ovomucin, lipovitellin-1 , lipovitellin-2, livetin, and lysozyme.

[0229] In one embodiment the system comprises: i. a microbial host cell expressing: a. at least one recombinant egg protein selected from the group consisting of ovalbumin, phosvitin 1 , phosvitin 2 and phosvitin 3, and b. at least one microbial kinase; or ii. a microbial host cell expressing at least one egg protein selected from the group consisting of ovalbumin, phosvitin 1 , phosvitin 2 and phosvitin 3, and a composition comprising at least one microbial kinase, whereby said system produces one or more phosphorylated recombinant egg protein.

[0230] In one embodiment the system comprises: i. a microbial host cell expressing: a. at least one recombinant egg protein selected from the group consisting of ovotransferrin, ovoglobulin, ovomucoid, ovomucin, lipovitellin-1 , lipovitellin- 2, livetin, and lysozyme, and b. at least one microbial kinase; or ii. a microbial host cell expressing at least one egg protein selected from the group consisting of ovotransferrin, ovoglobulin, ovomucoid, ovomucin, lipovitellin-1 , lipovitellin-2, livetin, and lysozyme, and a composition comprising at least one microbial kinase, whereby said system produces one or more phosphorylated recombinant egg protein.

[0231] In one embodiment the present disclosure relates to a method of producing recombinant egg protein in a microbial host cell, comprising the steps of: i. Providing a microbial host cell according to the present disclosure, ii. incubating said microbial host cell in a medium, under conditions allowing growth of the microbial host cell, whereby at least one recombinant egg protein is produced; and iii. incubating the at least one egg protein with at least one microbial kinase, whereby phosphorylated recombinant egg protein is produced. Preferably, said recombinant egg protein is ovalbumin, phosvitin 1 , phosvitin 2 and / or phosvitin 3. Preferably, said recombinant egg protein is ovotransferrin, ovoglobulin, ovomucoid, ovomucin, lipovitellin-1 , lipovitellin-2, livetin, and / or lysozyme.

[0232] In one embodiment the host cell according to the present disclosure is engineered to express an ovalbumin, phosvitin 1 , phosvitin 2, or phosvitin 3 gene, such as an avian ovalbumin, phosvitin 1 , or phosvitin 2 gene, wherein said gene is inserted into an expression vector and transformed into the host cell.

[0233] In one embodiment the host cell according to the present disclosure is engineered to express an ovotransferrin, ovoglobulin, ovomucoid, ovomucin, lipovitellin-1 , lipovitellin-2, livetin or lysozyme gene, such as an avian ovotransferrin, ovoglobulin, ovomucoid, ovomucin, lipovitellin-1 , lipovitellin-2, livetin, and / or lysozyme gene, wherein said gene is inserted into an expression vector and transformed into the host cell.

[0234] In one embodiment the kinase is capable of phosphorylating one or more amino acid residues of said recombinant egg protein, such as ovalbumin, phosvitin 1 , phosvitin 2 or phosvitin 3, at its native phosphorylation sites.

[0235] In one embodiment the kinase is capable of phosphorylating one or more amino acid residues of said recombinant egg protein, such as ovotransferrin, ovoglobulin, ovomucoid, ovomucin, lipovitellin-1 , lipovitellin-2, livetin, or lysozyme, at its native phosphorylation sites.

[0236] In one embodiment, the method of producing recombinant egg protein, such as ovalbumin, phosvitin 1 , phosvitin 2, phosvitin 3, ovotransferrin, ovoglobulin, ovomucoid, ovomucin, lipovitellin-1 , lipovitellin-2, livetin, or lysozyme comprises a step of recovering the recombinant egg protein.

[0237] In one embodiment the recombinant egg protein, such as ovalbumin, phosvitin 1 , phosvitin 2, phosvitin 3, ovotransferrin, ovoglobulin, ovomucoid, ovomucin, lipovitellin-1 , lipovitellin- 2, livetin, or lysozyme, is phosphorylated at one or more native phosphorylation sites.

[0238] In one embodiment the recombinant ovalbumin is phosphorylated at one or more native phosphorylation sites. In one embodiment the recombinant ovalbumin is phosphorylated at two native phosphorylation sites. In one embodiment the recombinant ovalbumin is phosphorylated at one or more non-native phosphorylation sites.

[0239] In one embodiment the recombinant phosvitin 1 is phosphorylated at one or more native phosphorylation sites, such as two, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as fifteen or more, such as twenty or more, such as twenty-five or more native phosphorylation sites. In one embodiment the recombinant phosvitin 1 is phosphorylated at one or more non-native phosphorylation sites. In one embodiment the recombinant phosvitin 2 is phosphorylated at one or more native phosphorylation sites, such as two, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as fifteen or more, such as twenty or more, such as twenty-five or more native phosphorylation sites. In one embodiment the recombinant phosvitin 2 is phosphorylated at one or more non-native phosphorylation sites.

[0240] In one embodiment the recombinant phosvitin 3 is phosphorylated at one or more native phosphorylation sites, such as two, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as fifteen or more, such as twenty or more, such as twenty-five or more native phosphorylation sites. In one embodiment the recombinant phosvitin 3 is phosphorylated at one or more non-native phosphorylation sites.

[0241] In one embodiment the recombinant ovotransferrin is phosphorylated at one or more native phosphorylation sites, such as two, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as fifteen or more, such as twenty or more, such as twenty-five or more native phosphorylation sites. In one embodiment the recombinant ovotransferrin is phosphorylated at one or more non-native phosphorylation sites.

[0242] In one embodiment the recombinant ovoglobulin is phosphorylated at one or more native phosphorylation sites, such as two, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as fifteen or more, such as twenty or more, such as twenty-five or more native phosphorylation sites. In one embodiment the recombinant ovoglobulin is phosphorylated at one or more non-native phosphorylation sites.

[0243] In one embodiment the recombinant ovomucoid is phosphorylated at one or more native phosphorylation sites, such as two, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as fifteen or more, such as twenty or more, such as twenty-five or more native phosphorylation sites. In one embodiment the recombinant ovomucoid is phosphorylated at one or more non-native phosphorylation sites.

[0244] In one embodiment the recombinant ovomucin is phosphorylated at one or more native phosphorylation sites, such as two, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as fifteen or more, such as twenty or more, such as twenty-five or more native phosphorylation sites. In one embodiment the recombinant ovomucin is phosphorylated at one or more non-native phosphorylation sites.

[0245] In one embodiment the recombinant lipovitellin-1 is phosphorylated at one or more native phosphorylation sites, such as two, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as fifteen or more, such as twenty or more, such as twenty-five or more native phosphorylation sites. In one embodiment the recombinant lipovitellin-1 is phosphorylated at one or more non-native phosphorylation sites.

[0246] In one embodiment the recombinant lipovitellin-2 is phosphorylated at one or more native phosphorylation sites, such as two, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as fifteen or more, such as twenty or more, such as twenty-five or more native phosphorylation sites. In one embodiment the recombinant lipovitellin-2 is phosphorylated at one or more non-native phosphorylation sites.

[0247] In one embodiment the recombinant livetin is phosphorylated at one or more native phosphorylation sites, such as two, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as fifteen or more, such as twenty or more, such as twenty-five or more native phosphorylation sites. In one embodiment the recombinant livetin is phosphorylated at one or more non-native phosphorylation sites.

[0248] In one embodiment the recombinant lysozyme is phosphorylated at one or more native phosphorylation sites, such as two, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as fifteen or more, such as twenty or more, such as twenty-five or more native phosphorylation sites. In one embodiment the recombinant lysozyme is phosphorylated at one or more non-native phosphorylation sites.

[0249] In one embodiment the recombinant egg protein is phosphorylated at all its native phosphorylation sites. Native phosphorylation sites are serine and / or threonine residues and are described in detail herein. Preferably the egg proteins are ovalbumin, phosvitin 1 , phosvitin 2 or phosvitin 3. Preferably the egg proteins are ovotransferrin, ovoglobulin, ovomucoid, ovomucin, lipovitellin-1 , lipovitellin-2, livetin, or lysozyme

[0250] In one embodiment one or more serine and / or threonine residues of the recombinant egg protein, such as: a recombinant ovalbumin as set forth in SEQ ID NO: 34, is phosphorylated at least at one of the following position: S68, or S344; a recombinant phosvitin 1 as set forth in SEQ ID NO: 35, is phosphorylated at least at one of the following positions: S11 , S12, S13, S14, S16, S37, S43, S45, S46, S47, S48, S51 , S52, S69, S70, S71 , S73, S75, S79, T110, S117, S124, S180, S184, S186, S187, S240, or S244; and / or a recombinant phosvitin 2 as set forth in SEQ ID NO: 36, is phosphorylated at least at one of the following positions: T5, T11 , S12, S13, S14, S15, S16, S17, S19, S20, T21 , T23, S24, S31 , S82, S89, S91 , S96, S98, S99, S100, S104, S124, S125, S126, S130, S134, S135, S137, S197, S198, S199, S200, S201 , S202, S203, S204, or S207.

[0251] In one embodiment the recombinant ovalbumin as set forth in SEQ ID NO: 34, or a variant thereof having at least 70% sequence identity to in SEQ ID NO: 34, is phosphorylated at least at one of the following position: S68, or S344.

[0252] In one embodiment the recombinant phosvitin 1 as set forth in SEQ ID NO: 35, or a variant thereof having at least 70% sequence identity to in SEQ ID NO: 35, is phosphorylated at least at one of the following positions: S11 , S12, S13, S14, S16, S37, S43, S45, S46, S47, S48, S51 , S52, S69, S70, S71 , S73, S75, S79, T110, S117, S124, S180, S184, S186, S187, S240, or S244. In one embodiment the recombinant phosvitin 2 as set forth in SEQ ID NO: 36, or a variant thereof having at least 70% sequence identity to in SEQ ID NO: 36, is phosphorylated at least at one of the following positions: T5, T11 , S12, S13, S14, S15, S16, S17, S19, S20, T21 , T23, S24, S31 , S82, S89, S91 , S96, S98, S99, S100, S104, S124, S125, S126, S130, S134, S135, S137, S197, S198, S199, S200, S201 , S202, S203, S204, or S207.

[0253] The degree of phosphorylation can be determined using MS (Beausoleil, et al, 2006).

[0254] In one embodiment the phosphorylated recombinant egg protein has a degree of phosphorylation at each native phosphorylation site of at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as about 100%.

[0255] Expression system

[0256] In one embodiment the present disclosure relates to an expression system for expression of phosphorylated recombinant egg proteins in a microbial host cell, comprising: iii. a first nucleic acid encoding at least one recombinant egg protein, wherein the egg protein is ovalbumin, phosvitin 1 or phosvitin 2; and iv. a second nucleic acid encoding a kinase.

[0257] In one embodiment the nucleic acid encoding an egg protein and the nucleic acid encoding a kinase are expressed in a microbial host cell.

[0258] In one embodiment the nucleic acid encoding at least one egg protein is expressed in a first microbial host cell, and the nucleic acid encoding at least one kinase is expressed in a second microbial host cell.

[0259] In one embodiment the present disclosure relates to the use of an expression system as defined elsewhere in the description, for production of at least one recombinant egg protein of interest. In one embodiment the at least one recombinant egg protein expressed by an expression system, is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or a variant thereof having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, sequence identity, similarity, or homology thereto.

[0260] In one embodiment the at least one recombinant egg protein expressed by an expression system, is selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 or a variant thereof having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, sequence identity, similarity, or homology thereto.

[0261] Host cell

[0262] Useful host cell are described elsewhere herein, such as in the section “Casein > Host cell”.

[0263] Kinase

[0264] Useful kinases are described elsewhere herein, such as in the section “Casein > Kinase”.

[0265] In vitro phosphorylation

[0266] In one embodiment the recombinant egg protein is phosphorylated outside the microbial host cell by a microbial kinase that is obtained separately and put in contact with the recombinant casein. Preferably the egg protein is ovalbumin or phosvitin.

[0267] In one embodiment the recombinant egg protein is phosphorylated after the egg protein has been secreted. Preferably the egg protein is ovalbumin or phosvitin. In one embodiment the recombinant egg protein is phosphorylated in vitro. Preferably the egg protein is ovalbumin or phosvitin.

[0268] In vivo phosphorylation

[0269] In one embodiment the recombinant egg protein is phosphorylated inside the microbial host cell. This may be possible if the same microbial host cell has been engineered to recombinantly produce an egg protein according to the present disclosure and a microbial kinase according to the present disclosure.

[0270] In one embodiment the recombinant egg protein is phosphorylated in vivo. Preferably the egg protein is ovalbumin or phosvitin.

[0271] Use of recombinant egg protein

[0272] The methods of the current disclosure provide recombinant egg protein, which is an alternative to egg protein obtained from avian eggs. The recombinant egg proteins can be used as an alternative to egg proteins in products which typically are derived from egg, that is egg products, such as egg whites and egg yolk. Moreover the recombinant egg protein of the current invention will provide useful food additives.

[0273] The egg protein of the current invention is advantageous compared to egg protein obtained from avian eggs, as the egg protein may be produced by greener methods.

[0274] In one embodiment the present disclosure relates to the use of a recombinant egg proteins produced as defined elsewhere in the description, as a food ingredient.

[0275] In one embodiment the present disclosure relates to a food product or a functional food product comprising recombinant egg protein produced as defined elsewhere in the description.

[0276] Furthermore, said recombinant egg protein may be useful as a drug component, for example as a vaccine component. Items

[0277] 1 . A system for producing one or more phosphorylated recombinant animal proteins, said system comprising: i. a microbial host cell expressing: a. at least one recombinant animal protein comprising a phosphorylation motif being S-X-E / D or a continuous stretch of at least two serines, and b. at least one microbial kinase; or ii. a microbial host cell expressing at least one animal protein comprising a phosphorylation motif being S-X-E / D or a continuous stretch of at least two serines, and a composition comprising at least one microbial kinase, whereby said system produces one or more phosphorylated recombinant animal protein.

[0278] 2. The system of item 1 , wherein a recombinant animal protein is a recombinant variant of a native animal protein, wherein said native animal protein is produced by an animal of the kingdom Animalia.

[0279] 3. The system of any of the preceding items, wherein said recombinant animal protein comprises a continuous stretch of at least three serines, such as at least four serines, such as at least five serines, such as at least six serines, such as at least seven serines, such as at least eight serines, such as at least nine serines, such as a continuous stretch of at least ten serines.

[0280] 4. A system for producing one or more phosphorylated recombinant caseins, said system comprising: i. a microbial host cell expressing: a. at least one recombinant casein selected from the group consisting of alpha-casein S1 (aS1), alpha-casein S2 (aS2), beta-casein and kappacasein and b. at least one microbial kinase; or ii. a microbial host cell expressing at least one casein selected from the group consisting of alpha-casein S1 (aS1), alpha-casein S2 (aS2), beta-casein and kappa-casein, and a composition comprising at least one microbial kinase, whereby said system produces one or more phosphorylated recombinant casein.

[0281] 5. A system for producing one or more phosphorylated recombinant egg proteins, said system comprising: i. a microbial host cell expressing: a. at least one recombinant egg protein selected from the group consisting of ovalbumin, phosvitin 1 , phosvitin 2 and phosvitin 3, and b. at least one microbial kinase; or ii. a microbial host cell expressing at least one egg protein selected from the group consisting of ovalbumin, phosvitin 1 , phosvitin 2 and phosvitin 3, and a composition comprising at least one microbial kinase, whereby said system produces one or more phosphorylated recombinant egg protein.

[0282] 6. A system for producing one or more phosphomimetic recombinant caseins, said system comprising a microbial host cell expressing: i. at least one recombinant casein selected from the group consisting of alpha-casein S1 (aS1), alpha-casein S2 (aS2), beta-casein and kappa- casein, wherein one or more amino acid residues that are phosphorylated in a native mammalian casein are substituted with one or more phosphomimetic amino acids; and ii. optionally at least one microbial kinase capable of phosphorylating said phosphomimetic casein at residues which are phosphorylated in a native mammalian casein, and which have not been substituted with phosphomimetic amino acids, whereby said system is capable of producing one or more phosphomimetic recombinant casein. The system according to any one of the preceding items, wherein the recombinant casein is a recombinant variant of a native mammalian casein, optionally wherein said mammalian casein is that found in milk from a mammal selected from the group consisting of human, cow, buffalo, zebu, yak, equines, sheep, goat, horse, donkey, camel, deer, and reindeer. The system according to any one of the preceding items, wherein a native mammalian casein is a casein produced in a mammal, such as a casein produced in human, cow, buffalo, zebu, yak, equines, sheep, goat, horse, donkey, camel, deer, and reindeer, and wherein said native mammalian casein is naturally phosphorylated at one or more native phosphorylation sites. The system according to any one of the preceding items, wherein the recombinant egg protein is a recombinant variant of a native avian egg protein, optionally wherein said avian egg protein is that found in egg from a bird selected from the group consisting of chicken, duck, goose, quail, guineafowl, turkey, emu, pheasant, gull, partridge and ostrich. The system according to any one of the preceding items, wherein a native avian egg protein is an egg protein found in a bird egg, such as an egg protein found in a chicken, duck, goose, quail, guineafowl, turkey, emu, pheasant, gull, partridge, and ostrich egg, and wherein said native avian egg protein is naturally phosphorylated at one or more native phosphorylation sites. The system according to any one of the preceding items, wherein the kinase is capable of phosphorylating one or more amino acid residues of said recombinant animal protein, such as recombinant casein or recombinant egg protein, at its native phosphorylation sites. The system according to any one of the preceding items, wherein the recombinant animal protein, such as recombinant casein or recombinant egg protein, is phosphorylated inside the microbial host cell. 13. The system according to any one of the preceding items, wherein the recombinant animal protein, such as recombinant casein or recombinant egg protein, is phosphorylated outside the microbial host cell.

[0283] 14. The system according to any one of the preceding items, wherein the recombinant animal protein, such as recombinant casein or recombinant egg protein, is phosphorylated after the casein has been secreted.

[0284] 15. The system according to any one of the preceding items, wherein the recombinant animal protein, such as recombinant casein or recombinant egg protein, is phosphorylated at one or more native phosphorylation sites, such as phosphorylated at least at two native phosphorylation sites, such as phosphorylated at least at three native phosphorylation sites, such as phosphorylated at least at four native phosphorylation sites, such as phosphorylated at least at five native phosphorylation sites, such as phosphorylated at least at six native phosphorylation sites, such as phosphorylated at least at seven native phosphorylation sites, such as phosphorylated at least at eight native phosphorylation sites, such as phosphorylated at least at nine native phosphorylation sites, such as phosphorylated at ten native phosphorylation sites, such as phosphorylated at eleven native phosphorylation sites, such as phosphorylated at twelve native phosphorylation sites, such as phosphorylated at thirteen native phosphorylation sites, such as phosphorylated at fourteen native phosphorylation sites, such as phosphorylated at fifteen native phosphorylation sites.

[0285] 16. The system according to any one of the preceding items, wherein the recombinant animal protein, such as recombinant casein or recombinant egg protein, is phosphorylated at all its native phosphorylation sites.

[0286] 17. The animal protein, such as casein or egg protein, according to any one of the preceding items, wherein the recombinant animal protein, such as recombinant casein or recombinant egg protein, has a degree of phosphorylation at each native phosphorylation site of at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as about 100%.

[0287] 18. The system according to any one of the preceding items, wherein the microbial host cell is a bacterial cell, a fungal cell or a yeast cell.

[0288] 19. The system according to any one of the preceding items, wherein the microbial host cell is a bacterial cell, which belongs to a genus selected from the group consisting of lactic acid bacteria, for example Lactococcus spp. and Lactobacillus spp., Escherichia spp., Corynebacterium spp., Pseudomonas spp., Streptomyces spp., Geobacillus spp., and Bacillus spp.

[0289] 20. The system according to any one of the preceding items, wherein the microbial host cell is a bacterial host cell, which belongs to a species selected from the group consisting of species selected from Bacillus subtilis and Escherichia coli.

[0290] 21 . The system according to any one of the preceding items, wherein the microbial host cell is a fungal host cell, which belongs to a genus selected from the group consisting of of Kluyveromyces spp., Pichia spp., Saccharomyces spp., Tetrahymena spp., Yarrowia spp., Hansenula spp., Blastobotrys spp., Aspergillus spp., Candida spp., Zygosaccharomyces spp., Trichoderma spp., Chrysosporium spp., Fusarium spp., and Neurospora spp. and Debaryomyces spp..

[0291] 22. The system according to any one of the preceding items, wherein the microbial host cell is a fungal host cell, which belongs to a species selected from the group consisting of Trichoderma reesei, Aspergillus vadensis, Aspergillus oryzae and Aspergillus niger.

[0292] 23. The system according to any one of the preceding items, wherein the microbial kinase is a prokaryotic kinase or a fungal kinase. The system according to any one of the preceding items, wherein the microbial kinase is a serine / threonine kinase. The system according to any one of the preceding items, wherein the microbial kinase is a bacterial kinase. The system according to any one of the preceding items, wherein the microbial kinase is a yeast kinase. The system according to any one of the preceding items, wherein the microbial kinase is of the Hanks family. The system according to any one of the preceding items, wherein the kinase is selected from the group consisting of YabT (SEQ ID NO:5 or SEQ ID NO: 6), PrkD (SEQ ID NO: 7), PrkC (SEQ ID NO: 8) and PKNB1 (SEQ ID NO: 9) or functional variants thereof having at least 60% sequence identity, similarity or homology thereto, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity. The system according to any one of the preceding items, wherein the microbial kinase is YabT as set forth in SEQ ID NO: 5, or a functional variant thereof having at least 70% sequence identity, similarity or homology thereto. The system according to any one of the preceding items, wherein the microbial kinase is PrkD as set forth in SEQ ID NO: 7, or a functional variant thereof having at least 70% sequence identity, similarity or homology thereto. The system according to any one of the preceding items, wherein the recombinant casein is selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ

[0293] ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ

[0294] ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ

[0295] ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, and SEQ ID NO: 33, or a variant thereof having at least 90% sequence identity, similarity or homology thereto.

[0296] 32. The system according to any one of the preceding items, wherein the recombinant casein is recombinant bovine casein.

[0297] 33. The system according to item 32, wherein the recombinant bovine casein is selected from the group consisting of alpha-casein S1 (aS1), alpha-casein S2 (aS2), beta-casein and kappa-casein.

[0298] 34. The system according to items 32 and 33, wherein the recombinant bovine casein is alpha-casein S1 (aS1).

[0299] 35. The system according to items 32 to 34, wherein the recombinant bovine casein is alpha-casein S2 (aS2).

[0300] 36. The system according to items 32 to 35, wherein the recombinant bovine casein is beta-casein.

[0301] 37. The system according to items 32 to 36, wherein the recombinant bovine casein is kappa-casein.

[0302] 38. The system according to items 1 to 30, wherein the recombinant egg protein is recombinant avian egg protein.

[0303] 39. The system according to items 1 to 30, and 38, wherein the recombinant avian egg protein is avian ovalbumin, phosphovitin 1 and / or phosphovitin 2.

[0304] 40. The system according to any one of items 32 to 33, wherein the recombinant bovine alpha-S1 casein (aS1) as set forth in SEQ ID NO: 1 , is phosphorylated at least at one of the following positions: S46, S48, S64, S66, S67, S68, and S75. 41 . The system according to any one of items 32 to 33, wherein the recombinant bovine alpha-S1 casein (aS1) as set forth in SEQ ID NO: 14, is phosphorylated at least at one of the following positions: S56, S61 , S63, S79, S81 , S82, S83, or S90.

[0305] 42. The system according to any one of items 32 to 33, wherein the recombinant bovine alpha-S2 casein (aS2) as set forth in SEQ ID NO: 20, is phosphorylated at least at one of the following positions: S23, S24, S25, S28, S46, S71 , S72, S73, S76, S144, S146, S150, or S158.

[0306] 43. The system according to any one of items 32 to 33, wherein the recombinant bovine beta casein as set forth in SEQ ID NO: 3, is phosphorylated at least at one of the following positions: S15, S17, S18, S19 and S35.

[0307] 44. The system according to any one of items 32 to 33, wherein the recombinant bovine beta casein as set forth in SEQ ID NO: 26, is phosphorylated at least at one of the following positions: S30, S32, S33, S34 and S50.

[0308] 45. The system according to any one of items 32 to 33, wherein the recombinant bovine kappa casein as set forth in SEQ ID NO: 32, is phosphorylated at least at one of the following positions: S148, T166, S170, or S187.

[0309] 46. The system according to any one of the preceding items wherein the recombinant casein is selected from the group consisting of human alpha-casein S1 (aS1), human alpha-casein (aS2), human beta-casein and human kappa-casein.

[0310] 47. The system according to any one of the preceding items wherein the recombinant human alpha-S1 casein as set forth in SEQ ID NO: 10 is phosphorylated at least at one of the following positions: S31 , S33, S41 , S71 , S85, S86, S88,S89, S90, or S91.

[0311] 48. The system according to any one of the preceding items wherein the recombinant human beta casein as set forth in SEQ ID NO: 22, is phosphorylated at least at one of the following positions: T18, S21 , S23, S24, or S25. 49. The system according to any one of the preceding items wherein the recombinant human kappa casein as set forth in SEQ ID NO: 28 is phosphorylated at positions T157.

[0312] 50. The system according to any one of the preceding items, wherein the recombinant ovalbumin as set forth is SEQ ID NO: 34, is phosphorylated at least at one of the following positions: S68, or S344.

[0313] 51 . The system according to any one of the preceding items, wherein the recombinant phosvitin 1 as set forth in SEQ ID NO: 35, is phosphorylated at least at one of the following positions: S11 , S12, S13, S14, S16, S37, S43, S45, S46, S47, S48, S51 , S52, S69, S70, S71 , S73, S75, S79, T110, S117, S124, S180, S184, S186, S187, S240, or S244.

[0314] 52. The system according to any one of the preceding items, wherein the recombinant phosvitin 2 as set forth in SEQ ID NO: 36, is phosphorylated at least at one of the following positions: T5, T11 , S12, S13, S14, S15, S16, S17, S19, S20, T21 , T23, S24, S31 , S82, S89, S91 , S96, S98, S99, S100, S104, S124, S125, S126, S130, S134, S135, S137, S197, S198, S199, S200, S201 , S202, S203, S204, or S207.

[0315] 53. The system according to any one of the preceding items, wherein said host cell comprises at least one protease deficiency rendering said host cell completely or partially deficient in the production and / or function of one or more proteases.

[0316] 54. The system according to any one of the preceding items, wherein said host cell expresses at least one chaperone protein.

[0317] 55. The system according to any one of the preceding items, wherein the recombinant animal protein, such as recombinant casein or recombinant egg protein, is phosphorylated in vivo. 56. The system according to any one of the preceding items, wherein the recombinant animal protein, such as recombinant casein or recombinant egg protein, is phosphorylated in vitro.

[0318] 57. The system according to any one of the preceding item, wherein one or more amino acids have been substituted with a phosphomimetic amino acid.

[0319] 58. The system according to any one of the preceding items, wherein one or more serine and / or threonine residues have been substituted with a phosphomimetic amino acid.

[0320] 59. The system according to any one of the preceding items wherein one or more amino acid residues which are phosphorylated in the native mammalian casein have been substituted with one or more phosphomimetic amino acids.

[0321] 60. The system according to any one of the preceding items, wherein one or more serine and / or threonine residues of the recombinant casein, such as of including alpha casein (aS1) as set forth in any one of SEQ ID NOs: 1 , 10, 11 , 12, 13 14 and 15, alpha casein (aS2) as set forth in any one of SEQ ID NOs: 16, 17, 18, 19, 20 and 21 , beta casein as set forth in any one of SEQ ID NOs: 3, 22, 23, 24, 25, 26, and 27, and kappa casein as set forth in any one of SEQ ID NO 28, 29, 30, 31 , 32 and 33, have been substituted with one or more phosphomimetic amino acids.

[0322] 61 . The system according to any one of the preceding items, wherein the recombinant casein is bovine alpha casein (aS1) as set forth in SEQ ID NO: 1 , comprising a phosphomimetic amino acid at least at one of the following positions: S46, S48, S64, S66, S67, S68, and S75.

[0323] 62. The system according to any one of the preceding items, wherein the recombinant casein is bovine alpha-S1 casein as set forth in SEQ ID NO: 14, comprising a phosphomimetic amino acid at least at one of the following positions: S56, S61 , S63, S79, S81 , S82, S83, or S90. The system according to any one of the preceding items, wherein the recombinant casein is bovine alpha-S2 casein as set forth in SEQ ID NO: 20, comprising a phosphomimetic amino acid at least at one of the following positions: S23, S24, S25, S28, S46, S71 , S72, S73, S76, S144, S146, S150, or S158. The system according to any one of the preceding items, wherein the recombinant casein is bovine beta casein as set forth in SEQ ID NO: 3, comprising a phosphomimetic amino acid at least at one of the following positions: S15, S17, S18, S19 and S35. The system according to any one of the preceding items, wherein the recombinant casein is bovine beta casein as set forth in SEQ ID NO: 26, comprising a phosphomimetic amino acid at least at one of the following positions: S30, S32, S33, S34 and S50. The system according to any one of the preceding items, wherein the recombinant casein is bovine kappa casein as set forth in SEQ ID NO: 32, comprising a phosphomimetic amino acid at least at one of the following positions: S148, T166, S170, or S187. The system according to any one of the preceding items, wherein the recombinant casein is human alpha-S1 casein as set forth in SEQ ID NO: 10, comprising a phosphomimetic amino acid at least at one of the following positions: S31 , S33, S41 , S71 , S85, S86, S88, S89, S90, or S91. The system according to any one of the preceding items, wherein the recombinant casein is human beta casein as set forth in SEQ ID NO: 22, comprising a phosphomimetic amino acid at least at one of the following positions: T18, S21 , S23, S24, or S25. The system according to any one of the preceding items, wherein the recombinant casein is human kappa casein as set forth in SEQ ID NO: 28, comprising a phosphomimetic amino acid at position T157. 70. The system according to any one of the preceding items 2 to 60, wherein the phosphomimetic amino acid is an unnatural amino acid.

[0324] 71 . The system according to any one of items 2 to 70, wherein the phosphomimetic amino acid carries negatively charged functional groups.

[0325] 72. The system according to any one of items 2 to 71 , wherein the phosphomimetic amino acid carries carboxylic acid groups.

[0326] 73. The system according to any one of items 6 to 72, wherein each phosphomimetic amino acid is independently selected from the group consisting of aspartic acid, glutamic acid and / or combinations thereof.

[0327] 74. The system according to any one of items 6 to 73, wherein each phosphomimetic amino acid is aspartic acid.

[0328] 75. A phosphorylated recombinant animal protein, such as recombinant casein or recombinant egg protein, obtained using a system according to any one of items 1 to 74.

[0329] 76. Phosphomimetic casein represented by the following sequences: SEQ ID NO: 1 ,

[0330] SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,

[0331] SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,

[0332] SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,

[0333] SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, or a variant thereof, wherein said variant has at least 70% sequence identity to any one of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ

[0334] ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ

[0335] ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ

[0336] ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, and wherein said variant comprises at least one or more phosphomimetic amino acids at one or more of positions corresponding to positions S46, S48, S64, S66, S67, S68, and S75 of SEQ ID NO: 1 ; or positions of S15, S17, S18, S19 and S35 of SEQ ID NO: 3; or positions S31 , S33, S41 , S71 , S85, S86, S88.S89, S90, S91 of SEQ ID NO: 10; or positions S33, S83, S85, S86, S87, S88 of SEQ ID NO: 11 ; or positions S27, S56, S61 , S63, S79, S80, S81 , S82, S83, S90, S130 of SEQ ID NO: 12; or positions S61 , S63, S79, S80, S81 , S82, S83, S90, S130 of SEQ ID NO: 13; or positions S56, S61 , S63, S79, S81 , S82, S83, S90 of SEQ ID NO: 14; or positions S63, S79, S80, S81 , S82, S83, S90 of SEQ ID NO: 15; or positions S23, S24, S25, S28, S47, S68, S123, S125, S128, S136 of SEQ ID NO: 17; or positions S23, S24, S25, S72, S73, S74, S77, S145, S147, S151 , S159 of SEQ ID NO:18; or positions S23, S24, S25, S72, S73, S74, S77, S145, S147, S151 , S159 of SEQ ID NO:19; or positions S23, S24, S25, S28, S46, S71 , S72, S73, S76, S144, S146, S150, S158 of SEQ ID NO:20; or positions T18, S21 , S23, S24, S25 of SEQ ID NO:22;or positions T27, S30, S32, S33, S34 of SEQ ID NO:24; or positions T27, S30, S32, S33, S34 of SEQ ID NO:25; or positions S30, S32, S33, S34, S50 of SEQ ID NO:26; or positions S30, S32, S33, S34 of SEQ ID NO:27; or position T157 of SEQ ID NO: 28; or positions of S148, S172, S189 SEQ ID NQ:30; or positions S148, T166, S170, S187 of SEQ ID NO:31 ; or positions S148, T166, S170, S187 of SEQ ID NO: 32. An expression system for expression of phosphorylated recombinant animal protein in a microbial host cell, comprising: i. a first nucleic acid encoding at least one recombinant animal protein such as a mammalian casein or an avian egg protein; and ii. a second nucleic acid encoding a kinase. An expression system for expression of phosphorylated recombinant casein in a microbial host cell, comprising: i. a first nucleic acid encoding at least one recombinant casein selected from the group consisting of alpha-casein S1 (aS1), alpha-casein (aS2), beta-casein and kappa-casein; and ii. a second nucleic acid encoding a kinase. The expression system according to item 77, wherein the at least one recombinant casein is SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID

[0337] NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID

[0338] NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID

[0339] NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID

[0340] NO: 33, or a variant thereof having at least 90% sequence identity, similarity, or homology thereto. The expression system according to item 77, wherein the nucleic acid encoding said at least one recombinant avian egg protein encodes a polypeptide as set forth in SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or a variant thereof having at least 90% sequence identity, similarity or homology thereto. The expression system according to item 77, wherein the nucleic acid encoding said at least one recombinant avian egg protein encodes a polypeptide as set forth in SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO: 45, or a variant thereof having at least 90% sequence identity, similarity or homology thereto. The expression system according to any one of items 77 to 79, wherein the kinase is YabT as set forth in SEQ ID NO: 5 or SEQ ID NO: 6, PrkD as set forth in SEQ ID NO: 7, PrkC as set forth in SEQ ID NO: 8, PKNB1 as set forth in SEQ ID NO: 9 or functional variants thereof having at least 70% sequence identity, similarity, or homology to the sequences. The expression system according to any one of items 77 to 80, wherein the nucleic acid encoding a casein and the nucleic acid encoding a kinase are expressed in a microbial host cell. The expression system according to any one of items 77 to 83, wherein the nucleic acid encoding at least one casein is expressed in a first microbial host cell, and wherein the nucleic acid encoding at least one kinase is expressed in a second microbial host cell. A host cell comprising the system according to any one of items 1 to 84, wherein said host cell is capable of producing recombinant animal protein, such as recombinant casein and / or recombinant egg protein. A host cell comprising the system according to any one of items 1 to 84, wherein said host cell is capable of producing recombinant casein. A method of producing recombinant animal protein, such as a recombinant casein and / or a recombinant egg protein, in a microbial host cell, comprising the steps of: i. Providing a host cell according to item 85 or 86; ii. incubating said host cell in a medium, under conditions allowing growth of the host cell, whereby at least one recombinant animal protein, such as recombinant casein or recombinant egg protein, is produced; and iii. incubating the at least one animal protein with at least one microbial kinase, whereby phosphorylated recombinant animal protein is produced, preferably wherein said recombinant animal protein is either a casein or an egg protein, such as ovalbumin, phosvitin, ovotransferrin, ovoglobulin, ovomucoid, ovomucin, lipovitellin, livetin, or lysozyme. A method of producing recombinant casein in a microbial host cell, comprising the steps of: i. Providing a host cell according to item 85; ii. incubating said host cell in a medium, under conditions allowing growth of the host cell , whereby at least one recombinant casein is produced; and iii. incubating the at least one casein with at least one microbial kinase, whereby phosphorylated recombinant casein is produced.

[0341] 89. A method of producing recombinant casein in a microbial host cell, comprising the steps of: i. Providing a host cell according to item 85; and ii. incubating said host cell in a medium, under conditions allowing growth of the host cell of at least one phosphomimetic casein, whereby at least one phosphomimetic recombinant casein is produced.

[0342] 90. A method of producing recombinant casein in a microbial host cell, comprising the steps of: i. Providing a host cell according to item 85; and ii. incubating said host cell in a medium, under conditions sufficient to allow for production of at least one phosphomimetic casein, whereby at least one phosphomimetic casein is produced; iii. incubating the at least one casein with at least one microbial kinase, whereby phosphorylated and / or phosphomimetic recombinant casein is produced.

[0343] 91 . The method according to any one of items 87 to 90, further comprising a step of recovering the recombinant casein.

[0344] 92. Use of the system according to any one of items 1 to 74 for production of at least one recombinant animal protein, such as casein or egg protein, of interest.

[0345] 93. Use of the expression system according to any one of items 77 to 84, for production of at least one recombinant animal protein, such as casein or egg protein, of interest.

[0346] 94. Use of the methods according to any one of items 87 to 91 for production of at least one recombinant animal protein, such as casein or egg protein, of interest.

[0347] 95. Use of the system according to any one of items 1 to 74 for production of at least one recombinant casein of interest. 96. Use of the expression system according to any one of items 77 to 84, for production of at least one recombinant casein of interest.

[0348] 97. Use of the methods according to any one of items 87 to 90 for production of at least one recombinant casein of interest.

[0349] 98. A food product (including drinks) or a functional food product or a dairy product comprising recombinant casein produced according to any one of items 1 to 91 .

[0350] 99. Use of the recombinant casein produced according to any one of items 1 to 91 as a food ingredient / for preparing yoghurt / cheese.

[0351] 100. A micelle comprising the recombinant casein produced according to any one of items 1 to 91.

[0352] Examples

[0353] Example 1. In vitro phosphorylation of caseins Phosphorylation by bovine and human kinase Fam20C

[0354] Aim

[0355] Golgi-enriched fraction casein kinases with the consensus motif S-X-E / ps from the lactating mammary gland has been suggested in the prior art to be responsible for phosphorylating caseins (Moore et al, 1985). Fam20C from the GEF-CK was identified to be the one that specifically phosphorylates at S-X-E motifs (Tagliabracci et al, 2012).

[0356] The aim of the experiment was to express Fam20C from both bovine and human origin to test for phosphorylation of the caseins both in vitro and in vivo.

[0357] Materials and Methods

[0358] Expression vectors pET15_BF20C and pET15_HF20C were generated by introducing the gene coding for bovine and human Fam20C respectively under the control of T7 promoter. Expression vector pREP4 GroESL was generated by introducing the gene coding for GroES / L under the control of T5 promoter. The expression vector containing the kinase (pET15_BF20C / pET15_HF20C) and the chaperone (pREP4 GroESL) was introduced into the host E. coli BL21 (DE3).

[0359] A colony was cultured overnight in LB broth with respective antibiotics of the vectors at 37 degrees with 200 rpm. A subculture was prepared after 16-18 hours and grown for 2-3 hours until the OD6oo reached between 0.6-0.8. Then 0.4mM of IPTG (final concentration) was added and the culture was grown for 6 hours at 30 degrees to induce expression of the proteins. The cells were harvested, lysed using a sonicator and analyzed on SDS PAGE for identifying the target proteins from the soluble fraction. For recovering the insoluble proteins, 8M urea was used. The insoluble fraction was also analyzed on SDS- PAGE.

[0360] Results

[0361] The kinases were produced as aggregates as shown in Figure 1 and there was no visible expression of proteins from the soluble fraction of the cell lysate.

[0362] Conclusions

[0363] The expression of bovine and human kinase Fam20C in soluble form was difficult to achieve. These proteins when expressed in E. coli formed inclusion bodies, and therefore we were unable to test activity and specificity (Figure 1).

[0364] Example 2. In vitro phosphorylation of caseins - Phosphorylation of a-casein by microbial kinases

[0365] Aim

[0366] To investigate the in vitro phosphorylation of a-casein by PKNB-CGL0041-BAB97434 from C. glutamicunr, PrkC, YabT, and PrkD from B. subtilis. The specific objectives were to identify the phosphorylation sites on asi-casein catalyzed by these kinases, compare the phosphorylation patterns with native bovine phosphorylation sites, and assess the efficiency and specificity of each kinase in phosphorylating a-casein. Materials and Methods

[0367] Bovine asi-casein (SEQ ID NO: 1) and the kinases were expressed individually and purified from E. coli BL21 (DE3). Casein was constructed using the pET15b vector and kinase using pQE30 vector. The strain with kinase plasmid is also transformed with pREP4 GroESL vector with the chaperones. The proteins were expressed in separate strains. asi-casein phosphorylation reactions were carried out using 3 pM PKNB-CGL0041- BAB97434 from C. glutamicunr, PrkC, YabT, and PrkD from B. subtilis, along with 15 pM asi-casein. The reaction mixture, totaling 35 pl, consisted of the following components: 10 mM ATP, 5 mM MgCI2, and 50 mM Tris-HCI at pH 7.4. Following a 2-hour incubation at 37°C, the sample was stored at -80°C and sent for mass spectrometry (MS) analysis.

[0368] Results

[0369] In vitro phosphorylation MS results, obtained by following the protocol of Beausoleil et al, 2006, demonstrated that PKNB and PrkC phosphorylated 6 sites in asi-casein each, of which only one and two sites, respectively, were similar to native bovine phosphorylation sites (showed with * in the table below). On the other hand, PrkD and YabT phosphorylated 13 and 10 sites, respectively, with 9 and 8 sites showing similarity to native phosphorylation sites.

[0370] Conclusions

[0371] PrkD exhibited the capability to phosphorylate all 9 native phosphorylation sites, while YabT could phosphorylate 8 out of the 9 native phosphorylation sites with a higher intensity.

[0372] Example 3. In vitro phosphorylation of caseins - Phosphorylation of (3-casein by microbial kinases

[0373] Aim

[0374] To investigate the in vitro phosphorylation of p -casein by PKNB-CGL0041-BAB97434 from C. glutamicunr, PrkC, YabT, and PrkD from B. subtilis. The specific objectives were to identify the phosphorylation sites on [3-casein catalyzed by these kinases, compare the phosphorylation patterns with native bovine phosphorylation sites, and assess the efficiency and specificity of each kinase in phosphorylating [3-casein.

[0375] Materials and Methods

[0376] Bovine [3-casein (SEQ ID NO: 3) and the kinases were expressed individually and purified from E. coli BL21 (DE3). Casein was constructed using the pET15b vector and kinase using pQE30 vector. The strain with kinase plasmid is also transformed with pREP4 GroESL vector with the chaperones. The proteins were expressed in separate strains. [3- casein phosphorylation reactions were carried out using 3 pM PKNB-CGL0041-BAB97434 from C. glutamicunr, PrkC, YabT, and PrkD from B. subtilis, along with 15 pM [3-casein. The reaction mixture, in total 35 pl, consisted of the following components: 10 mM ATP, 5 mM MgCI2, and 50 mM Tris-HCI at pH 7.4. Following a 2-hour incubation at 37°C, the sample was stored at -80°C and sent for Mass spectrometry analysis.

[0377] Results In vitro phosphorylation MS results, obtained by following the protocol of Beausoleil et al, 2006, demonstrated that PKNB and PrkC phosphorylated 2 sites in [3-casein each, of which only one site were similar to native bovine phosphorylation sites (showed with * in the table below). On the other hand, PrkD and YabT phosphorylated 5 sites each, with 3 sites showing similarity to native phosphorylation sites.

[0378] Conclusions

[0379] PrkD and YabT exhibited the capability to phosphorylate 3 native phosphorylation sites in [3-casein, while YabT showed higher intensity.

[0380] Example 4. Co-expression of kinase (B. subtilis PrkD) and casein

[0381] Aim

[0382] To facilitate phosphorylation of caseins in vivo through co-expression of caseins and PrkD in E. coli BL21 (DE3).

[0383] Materials and Methods

[0384] The expression vector pET asi_PrkD I pET[3_PrkD was generated by inserting the casein (bovine asi-casein (SEQ ID NO: 1) or bovine [3-casein (SEQ ID NO: 3)) and PrkD (SEQ ID NO: 7) under the control of T7 promoter. The expression vector containing the casein (pET asi_PrkD I pET [3_PrkD) and the chaperone (pREP4 GroESL) was introduced into the host E. coli BL21 (DE3).

[0385] A colony was cultured overnight in LB broth with respective antibiotics of the vectors at 37 degrees with 200 rpm. A subculture was prepared after 16-18 hours and grown for 2-3 hours until the OD6oo reached between 0.6-0.8. Then 0.4mM of IPTG (final concentration) was added and the culture was grown for 6 hours at 30 degrees to induce expression of the proteins. The cells were harvested, lysed using a sonicator and analyzed on SDS PAGE for identifying the target proteins from the soluble fraction.

[0386] Results

[0387] Both casein and PrkD were expressed in soluble form. The expression levels of PrkD was low, however, for phosphorylation even low amounts of kinase is sufficient.

[0388] Conclusions

[0389] B. subtilis PrkD and caseins aSiand p were successfully co-expressed (Figure 2).

[0390] Example 5. Co-expression of kinase (B. subtilis YabT) and casein

[0391] Aim

[0392] To facilitate in vivo phosphorylation of asi-casein through co-expression of caseins and YabT in E. coli BL21 (DE3).

[0393] Materials and Methods

[0394] The expression vector pET asi_YabT was generated by inserting the casein and YabT under the control of T7 promoter. The expression vector containing the casein (bovine asi-casein (SEQ ID NO: 1) or bovine [3-casein (SEQ ID NO: 3)) and YabT (SEQ ID NO: 6)), (expression vector pET asi_YabT) and the chaperone (pREP4 GroESL) was introduced into the host E. coli BL21 (DE3).

[0395] A colony was cultured overnight in LB broth with respective antibiotics of the vectors at 37 degrees with 200 rpm. A subculture was prepared after 16-18 hours and grown for 2-3 hours until the OD6oo reached between 0.6-0.8. Then 0.4mM of IPTG (final concentration) was added and the culture was grown for 6 hours at 30 degrees to induce expression of the proteins. The cells were harvested, lysed using a sonicator and analyzed on SDS PAGE for identifying the target proteins from the soluble fraction.

[0396] Results Both asi casein and YabT were expressed in soluble form.

[0397] Conclusions

[0398] B. subtilis YabT and casein aSiwere successfully co-expressed (Figure 3).

[0399] Example 6. Phosphomimetic modification

[0400] Aim

[0401] To express phosphomimetic variants of caseins in E. coli BL21 (DE3) as an alternative to phosphorylation.

[0402] Materials and Methods

[0403] The expression vector pET asi_Asp I pET[3_Asp was generated by inserting the casein phosphomimetic variants (asi-casein (SEQ ID NO: 2) or bovine [3-casein (SEQ ID NO: 4)) under the control of T7 promoter. The caseins were synthesized by replacing the potential serine sites into aspartic acid. The expression vector containing the casein (pET asi_Asp I pET P_Asp) and the chaperone (pREP4 GroESL) was introduced into the host E. coli BL21 (DE3).

[0404] A colony was cultured overnight in LB broth with respective antibiotics of the vectors at 37 degrees with 200 rpm. A subculture was prepared after 16-18 hours and grown for 2-3 hours until the OD6oo reached between 0.6-0.8. Then 0.4mM of IPTG (final concentration) was added and the culture was grown for 6 hours at 30 degrees to induce expression of the proteins. The cells were harvested, lysed using a sonicator and analyzed on SDS PAGE for identifying the target proteins from the soluble fraction.

[0405] Results

[0406] The phosphomimetic variants of the caseins were successfully expressed in soluble form.

[0407] Conclusions

[0408] Phosphomimetic variants- cxsi& [3- casein having serine replaced with aspartic acid were successfully expressed (Figure 4). Example 7. Phosphorylation

[0409] Aim

[0410] To test the phosphorylation levels of the casein qualitatively.

[0411] Materials and Methods

[0412] For protein separation by SDS-PAGE, 10 pL of cell lysate was combined with 4x Laemmli sample buffer containing 100 mM DTT and heated at 95°C for 5 minutes. The prepared samples were loaded onto a 4-20% Tris-HCI precast gel (Bio-Rad) and separated by electrophoresis.

[0413] Isoelectric focusing (IEF) was performed using 11 cm IPG strips (pH 4-7, Bio-Rad) with the IPGPhor system (Amersham). Strips were rehydrated overnight in 200 pL of buffer containing 50 pg protein, 8 M urea, 2% CHAPS, 2% Bio-Lyte, and 50 mM DTT, under a 300 pL mineral oil overlay. IEF was conducted in stages: an initial 500 V for 1 hour, followed by a gradient to 1000 V in the next hour, then up to 6000 V over 2.5 hours, and finally held at 6000 V for 50 minutes.

[0414] After focusing, strips were equilibrated in two buffers. Buffer I contained 6 M urea, 30% glycerol, 2% SDS, 0.05 M Tris-HCI (pH 8.8), and 1 % DTT for 10 minutes. In Buffer II, 4% iodoacetamide replaced DTT, with strips equilibrated for 10 minutes.

[0415] The IPG strips were positioned on a 4-20% Tris-HCI gel with a peppermint phosphoprotein ladder and sealed with low-melting agarose. The gel ran at 180 V for 60 minutes, then was stained with Pro-Q Diamond for phosphoproteins, followed by SYPRO Ruby for total proteins, and visualized using the ChemiDoc MP imager.

[0416] Results

[0417] When co-expressed with either PrkD or YabT (Rasi-PD / Rasi-YT), asi-casein was heavily phosphorylated, yielding a signal comparable to that of commercial native / phosphorylated a-casein while, no signal was observed for asi-casein sample without any kinase activity (Rds-i) (Figure 10).

[0418] Conclusion

[0419] High levels of phosphorylation was achieved when co-expressed with microbial kinases

[0420] PrkD and YabT. Example 8. Calcium binding assay

[0421] Aim

[0422] To test the calcium binding properties of unmodified and modified caseins.

[0423] Materials and Methods

[0424] 1 mg of bovine casein (asi-casein, p-casein phosphorylated in vitro by microbial kinases YabT or PrkD) was incubated with 2.29 pM of Cacl2in 20mM of Tris buffer, pH 8 at 37 °C for 1 hour. Commercially available bovine asi-casein and [3-casein were used as positive controls. Dephosphorylated commercially available bovine asi-casein and [3-casein were used as negative controls. The samples were centrifuged at 15000 rpm for 30 minutes. The supernatant was filtered using 10kDa centrifugal filters. The flow through contained buffer with unbound ca ions which was then measured using Ca calorimetry assay kit from Sigma Aldrich. Caseins from Sigma was dephosphorylated were used as negative controls, along with Tris buffer with Cacl2.

[0425] 1.0 mg / ml of in vivo phosphorylated aSi-casein and [3-casein samples were dephosphorylated using FastAP (Thermo Scientific). Phosphorylated and dephosphorylated asi-casein samples were mixed at a final concentration of 1 mg / ml with 1.145 mM CaCI2in 100 mM Tris-HCI (pH 8.0), 50 mM MgCI2, 1 M KCI, 0.2% Triton X-100. The solutions were agitated at 37°C for 2 hours, with the pH maintained at 8.0. The reaction mixtures were then filtered using Amicon Ultra centrifugal filters (Sigma-Aldrich) at 10,000 g at room temperature for 30 minutes. Calcium content was measured using the Calcium Colorimetric Assay (Sigma-Aldrich). A CaCI2mixture without protein was used as a negative control for calculating the bound calcium.

[0426] Results

[0427] The results are presented in Figure 5. The positive controls ‘a-casein (Sigma)’ and ‘p- casein (Sigma)’ from Sigma Aldrich, show maximum binding of the calcium ions, and when the samples are dephosphorylated (Dp), they lose the ability to bind calcium. This trend is observed in the phosphorylated samples as well, respectively ‘asi-PrkD’, ‘asi-YabT’, ‘ - PrkD’ and ‘p-YabT’ . The unmodified asiand caseins (respectively ‘asi -unmodified’ and ‘P-casein-unmodified’ bind low levels of calcium and upon dephosphorylation, the binding affinity is not affected. The phosphomimetic casein, respectively ‘asi-Asp’ and ‘p-Asp’ have similar binding affinity as the commercial bovine caseins. Their binding affinity is not affected upon dephosphorylation.

[0428] Conclusion

[0429] Commercial caseins, phosphorylated and the phosphomimetic samples bind calcium ions to comparable levels.

[0430] Sequence overview

[0431] Bovine Alpha-s1 -casein (SEQ ID NO: 1)

[0432] RPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNELSKDIGSESTEDQAMEDIKQM EAESISSSEEIVPNSVEQKHIQKEDVPSERYLGYLEQLLRLKKYKVPQLEIVPNSAEERLHS MKEGIHAQQKEPMIGVNQELAYFYPELFRQFYQLDAYPSGAWYYVPLGTQYTDAPSFSDI PNPIGSENSEKTTMPLW

[0433] Alpha-s1 -casein (Ser->Asp) (SEQ ID NO: 2)

[0434] RPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNELSKDIGDEDTEDQAMEDIKQM EAEDIDDDEEIVPNDVEQKHIQKEDVPSERYLGYLEQLLRLKKYKVPQLEIVPNDAEERLH SMKEGIHAQQKEPMIGVNQELAYFYPELFRQFYQLDAYPSGAWYYVPLGTQYTDAPSFS DIPNPIGSENSEKTTMPLW

[0435] Bovine Beta-casein (SEQ ID NO: 3)

[0436] RELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQTQSLVYPF PGPIPNSLPQNIPPLTQTPVWPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFTES QSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSLSQSKVLPVPQKAVPYP QRDMPIQAFLLYQEPVLGPVRGPFPIIV

[0437] Beta-casein (Ser->Asp) (SEQ ID NO: 4)

[0438] RELEELNVPGEIVEDLDDDEESITRINKKIEKFQDEEQQQTEDELQDKIHPFAQTQSLVYPF

[0439] PGPIPNSLPQNIPPLTQTPVWPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFTES QSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSLSQSKVLPVPQKAVPYP

[0440] QRDMPIQAFLLYQEPVLGPVRGPFPIIV

[0441] YabT (GenBank: SPU13583.1) (SEQ ID NO: 5)

[0442] MLNWTPKLQSLSTPFAKLSTAAITPTGPAIREATLQFEKYAQEGACWQMMNDALTSLACS

[0443] LKPGTTIKGKWNGNTYTLRKQLGKGANGIVYLAETSDGHVALKVSDDSLSITSEVNVLKSF

[0444] SKAQSVTMGPSFFDTDDAYIPSANTKVSFYAMEYIKGPLLLKYVSDKGAEWIPVLMIQLLS

[0445] SLSVLHQQGWIFGDLKPDNLIVTGPPARIRCIDVGGTTKEGRAIKEYTEFYDRGYWGYGT

[0446] RKAEPSYDLFAVAMIMINSVHKKEFKKTNQPKEQLRSLIEGNPLLQKYKKALFSALNGDYQ

[0447] SADEMKKDMLDAGQKAAQRKQPIKASPQPATRQRQQKPRQGKITKTRYTPKQKPAKSG

[0448] GLFETTLIVISVLALYFAYIIFFLI

[0449] Truncated YabT- used for co-expression (SEQ ID NO: 6)

[0450] MMNDALTSLACSLKPGTTIKGKWNGNTYTLRKQLGKGANGIVYLAETSDGHVALKVSDD

[0451] SLSITSEVNVLKSFSKAQSVTMGPSFFDTDDAYIPSANTKVSFYAMEYIKGPLLLKYVSDK

[0452] GAEWIPVLMIQLLSSLSVLHQQGWIFGDLKPDNLIVTGPPARIRCIDVGGTTKEGRAIKEYT

[0453] EFYDRGYWGYGTRKAEPSYDLFAVAMIMINSVHKKEFKKTNQPKEQLRSLIEGNPLLQKY

[0454] KKALFSALNGDYQSADEMKKDMLDAGQKAAQRKQPIKASPQPATRQRQQKPRQGKITK

[0455] TRYTPKQKPAKSGGLFETTLI

[0456] PrkD (Bacillus subtilis 168 | BSU_02030) (SEQ ID NO: 7)

[0457] MALKLLKKLLFDRPLKNGVILNHQYKIEECLGMGGYGLVYLCTDILAQTPYVLKQLRPTKA

[0458] KKEKEKVRFQQEIKLLKNIHHPQIPGFIDEFIIDGQAYYVMQFIEGENIEELLFFRKQPFTEL

[0459] MALQLISQLLEIIEYLHDRLIFHSDIRTPNIIINDGRLCLIDFGLAKQLTPEEMEEIKVRKQDDF

[0460] FDLGETLLFLLYSQYKGKKKKNGTWLEELTLTKEVTLLLKRLLGIEEEYQHTASIREDLNRA

[0461] IQSVT

[0462] PrkC (Bacillus subtilis) (SEQ ID NO: 8)

[0463] MLIGKRISGRYQILRVIGGGGMANVYLAEDIILDREVAIKILRFDYANDNEFIRRFRREAQSA

[0464] SSLDHPNIVSIYDLGEEDDIYYIVMEYVEGMTLKEYITANGPLHPKEALNIMEQIVSAIAHAH

[0465] QNQIVHRDIKPHNILIDHMGNIKVTDFGIATALSSTTITHTNSVLGSVHYLSPEQARGGLATK

[0466] KSDIYALGIVLFELLTGRIPFDGESAVSIALKHLQAETPSAKRWNPSVPQSVENIILKATAKD PFHRYETAEDMEADIKTAFDADRLNEKRFTIQEDEEMTKAIPIIKDEELAKAAGEKEAEVTT

[0467] AQENKTKKNGKRKKWPWVL

[0468] PKNB1_Cgl2751 (Corynebacterium glutamicum) (SEQ ID NO: 9)

[0469] MYEVLGVIAHGGMGWIYLANDRNVSGRIVVLKGMMAQSSVQDQGTAEAEREFLADITHP

[0470] GIVKAYNFIDDPRVPGGFIVMEYVNGPSLKDRCKAQPDGVLRVDLAIGYILELLPAMDYLH

[0471] QRGWYNDLKPENVIATEDQVKLIDLGAVTGIGAFGYIYGTKGFQAPEVATHGPSISSDIFT

[0472] IGRTLAALTMPLPVEDGVLAPGIPSPKNSPLLRRHLSFYRLLQRATADDPQHRFRNVSELR

[0473] TQLYGVLREILAVRDGKQYPPQHSLFSPQRSTFGTKHLVF

[0474] Human Alpha-s1 casein: P47710 CASA1_HUMAN (SEQ ID NO: 10)

[0475] MRLLILTCLVAVALARPKLPLRYPERLQNPSESSEPIPLESREEYMNGMNRQRNILREKQT

[0476] DEIKDTRNESTQNCWAEPEKMESSISSSSEEMSLSKCAEQFCRLNEYNQLQLQAAHAQ

[0477] EQIRRMNENSHVQVPFQQLNQLAAYPYAVWYYPQIMQYVPFPPFSDISNPTAHENYEKN NVMLQW

[0478] Camel Alpha-s1 casein: 097943 CASA1_CAMDR (SEQ ID NO: 11)

[0479] MKLLILTCLVAVALARPKYPLRYPEVFQNEPDSIEEVLNKRKILELAWSPIQFRQENID

[0480] ELKDTRNEPTEDHIMEDTERKESGSSSSEEWSSTTEQKDILKEDMPSQRYLEELHRLNK

[0481] YKLLQLEAIRDQKLIPRVKLSSHPYLEQLYRINEDNHPQLGEPVKWTQEQAYFHLEPFPQ

[0482] FFQLGASPYVAWYYPPQVMQYIAHPSSYDTPEGIASEDGGKTDVMPQWW

[0483] Sheep Alpha-s1 casein: P04653 CAS A1_SHEEP (SEQ ID NO: 12)

[0484] MKLLILTCLVAVALARPKHPIKHQGLSSEVLNENLLRFWAPFPEVFRKENINELSKDIG

[0485] SESIEDQAMEDAKQMKAGSSSSSEEIVPNSAEQKYIQKEDVPSERYLGYLEQLLRLKKYN

[0486] VPQLEIVPKSAEEQLHSMKEGNPAHQKQPMIAVNQELAYFYPQLFRQFYQLDAYPSGAW

[0487] YYLPLGTQYTDAPSFSDIPNPIGSENSGKITMPLW

[0488] Goat Alpha-s1 casein: P18626 CAS A1_CAPHI (SEQ ID NO: 13)

[0489] MKLLILTCLVAVALARPKHPINHRGLSPEVPNENLLRFWAPFPEVFRKENINELSKDIG

[0490] SESTEDQAMEDAKQMKAGSSSSSEEIVPNSAEQKYIQKEDVPSERYLGYLEQLLRLKKYN

[0491] VPQLEIVPKSAEEQLHSMKEGNPAHQKQPMIAVNQELAYFYPQLFRQFYQLDAYPSGAW

[0492] YYLPLGTQYTDAPSFSDIPNPIGSENSGKTTMPLW Bovine Alpha-s1 casein: P02662 CAS A1_BOVIN (SEQ ID NO: 14) (including bovine signal peptide, highlighted)

[0493] MKLLILTCLVAVALARPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNELSKDIGSE

[0494] STEDQAMEDIKQMEAESISSSEEIVPNSVEQKHIQKEDVPSERYLGYLEQLLRLKKYKVPQ LEIVPNSAEERLHSMKEGIHAQQKEPMIGVNQELAYFYPELFRQFYQLDAYPSGAWYYVP LGTQYTDAPSFSDIPNPIGSENSEKTTMPLW

[0495] Buffalo Alpha-s1 casein: 062823 CAS A1_BUBBU (SEQ ID NO: 15)

[0496] MKLLILTCLVAVALARPKQPIKHQGLPQGVLNENLLRFFVAPFPEVFGKEKVNELSTDIGSE

[0497] STEDQAMEDIKQMEAESISSSEEIVPISVEQKHIQKEDVPSERYLGYLEQLLRLKKYNVPQ LEIVPNLAEEQLHSMKEGIHAQQKEPMIGVNQELAYFYPQLFRQFYQLDAYPSGAWYYVP LGTQYPDAPSFSDIPNPIGSENSGKTTMPLW

[0498] Human Alpha-s2 casein: Tr Q8IUJ1 CASA2_HUMAN (SEQ ID NO: 16)

[0499] MKFFIFTCLLAVALAHHEIKHSSSSSE

[0500] Camel Alpha-s2 casein: Sp 097944 CASA2_CAMDR (SEQ ID NO: 17)

[0501] MKFFIFTCLLAWLAKHEMDQGSSSEESINVSQQKFKQVKKVAIHPSKEDICSTFCEEAVR

[0502] NIKEVESAEVPTENKISQFYQKWKFLQYLQALHQGQIVMNPWDQGKTRAYPFIPTVNTEQ

[0503] LSISEESTEVPTEESTEVFTKKTELTEEEKDHQKFLNKIYQYYQTFLWPEYLKTVYQYQKT MTPWNHIKRYF

[0504] Sheep Alpha-s2 casein: Sp P04654 CAS A2_SHEEP (SEQ ID NO: 18)

[0505] MKFFIFTCLLAVALAKHKMEHVSSSEEPINISQEIYKQEKNMAIHPRKEKLCTTSCEEW RNADEEEYSIRSSSEESAEVAPEEVKITVDDKHYQKALNEINQFYQKFPQYLQYLYQGPIV

[0506] LNPWDQVKRNAGPFTPTVNREQLSTSEENSKKTIDMESTEVFTKKTKLTEEEKNRLNFLK KISQYYQKFAWPQYLKTVDQHQKAMKPWTQPKTNAIPYVRYL

[0507] Goat Alpha-s2 casein: Sp P33049 CAS A2_CAPHI (SEQ ID NO: 19)

[0508] MKFFIFTCLLAVALAKHKMEHVSSSEEPINIFQEIYKQEKNMAIHPRKEKLCTTSCEEW RNANEEEYSIRSSSEESAEVAPEEIKITVDDKHYQKALNEINQFYQKFPQYLQYPYQGPIV

[0509] LNPWDQVKRNAGPFTPTVNREQLSTSEENSKKTIDMESTEVFTKKTKLTEEEKNRLNFLK

[0510] KISQYYQKFAWPQYLKTVDQHQKAMKPWTQPKTNAIPYVRYL

[0511] Bovine Alpha-s2 casein: Sp P02663 CAS A2_BOVIN (SEQ ID NO: 20)

[0512] MKFFIFTCLLAVALAKNTMEHVSSSEESIISQETYKQEKNMAINPSKENLCSTFCKEVVRN

[0513] ANEEEYSIGSSSEESAEVATEEVKITVDDKHYQKALNEINQFYQKFPQYLQYLYQGPIVLN

[0514] PWDQVKRNAVPITPTLNREQLSTSEENSKKTVDMESTEVFTKKTKLTEEEKNRLNFLKKIS

[0515] QRYQKFALPQYLKTVYQHQKAMKPWIQPKTKVIPYVRYL

[0516] Buffalo Alpha-s2 casein: Tr E9NZN2 CAS A2_BUBBU (SEQ ID NO: 21)

[0517] MKFFIFTCLLAVALAKHTMEHVSSSEESIISQETYKQEKNMAIHPSKENLCSTFCKEVIR

[0518] NANEEEYSIGSSSEESAEVATEEVKITVDDKHYQKALNEINQFYQKFPQYLQYLYQGPIVL

[0519] NPWDQVKRNAVPITPTLNREQLSTSEENSKKTVDMESTEVFTKKTKLTEEDKNRLNFLKKI

[0520] SQHYQKFAWPQYLKTVYQYQKAMKPWTQPKTNVIPYVRYL

[0521] Human Beta-casein: Sp P05814 CASB_human (SEQ ID NO: 22)

[0522] MKVLILACLVALALARETIESLSSSEESITEYKQKVEKVKHEDQQQGEDEHQDKIYPSFQP

[0523] QPLIYPFVEPIPYGFLPQNILPLAQPAWLPVPQPEIMEVPKAKDTVYTKGRVMPVLKSPTI

[0524] PFFDPQIPKLTDLENLHLPLPLLQPLMQQVPQPIPQTLALPPQPLWSVPQPKVLPIPQQW

[0525] PYPQRAVPVQALLLNQELLLNPTHQIYPVTQPLAPVHNPISV

[0526] Camel Beta-casein: Tr M1E4K4 CASB_CAMDR (SEQ ID NO: 23)

[0527] MKVLILACLVALALAREKEEFKTAGEALESISSSEESITHINKQKIEKFKIEEQQQTEDE

[0528] QQDKIYTFPQPQSLVYSHTEPIPYPILPQNFLPPLQPAVMVPFLQPKVMDVPKTKETIIP

[0529] KRKEMPLLQSPWPFTESQSLTLTDLENLHLPLPLLQSLMYQIPQPVPQTPMIPPQSLLSL

[0530] SQFKVLPVPQQMVPYPQRAMPVQAVLPFQEPVPDPVRGLHPVPQPLVPVIA

[0531] Sheep Beta-casein: Sp P11839 CASB_SHEEP (SEQ ID NO: 24)

[0532] MKVLILACLVALALAREQEELNWGETVESLSSSEESITHINKKIEKFQSEEQQQTEDEL

[0533] QDKIHPFAQAQSLVYPFTGPIPNSLPQNILPLTQTPWVPPFLQPEIMGVPKVKETMVPKH

[0534] KEMPFPKYPVEPFTESQSLTLTDVEKLHLPLPLVQSWMHQPPQPLPPTVMFPPQSVLSLS

[0535] QPKVLPVPQKAVPQRDMPIQAFLLYQEPVLGPVRGPFPILV Goat Beta-casein: Sp P33048 CASB_CAPHI (SEQ ID NO: 25)

[0536] MKVLILACLVALAIAREQEELNWGETVESLSSSEESITHINKKIEKFQSEEQQQTEDEL

[0537] QDKIHPFAQAQSLVYPFTGPIPNSLPQNILPLTQTPWVPPFLQPEIMGVPKVKETMVPKH

[0538] KEMPFPKYPVEPFTESQSLTLTDVEKLHLPLPLVQSWMHQPPQPLSPTVMFPPQSVLSLS

[0539] QPKVLPVPQKAVPQRDMPIQAFLLYQEPVLGPVRGPFPILV

[0540] Bovine Beta-casein: Sp P02666 CASB_BOVIN (SEQ ID NO: 26) (including bovine signal Peptide, highlighted) used for numbering of phosphorylation sites

[0541] MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDK

[0542] IHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPWVPPFLQPEVMGVSKVKEAMAPKHKE

[0543] MPFPKYPVEPFTESQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSLSQ

[0544] SKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPIIV

[0545] Buffalo Beta-casein: Sp Q9TSI0 CASB_BUBBU (SEQ ID NO: 27)

[0546] MKVLILACLVALALARELEELNVPGEIVESLSSSEESITHINKKIEKFQSEEQQQMEDEL

[0547] QDKIHPFAQTQSLVYPFPGPIPKSLPQNIPPLTQTPWVPPFLQPEIMGVSKVKEAMAPKH

[0548] KEMPFPKYPVEPFTESQSLTLTDVENLHLPLPLLQSWMHQPPQPLPPTVMFPPQSVLSLS

[0549] QSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPIIV

[0550] Human Kappa-casein: Sp P07498 CASK_human (SEQ ID NO: 28)

[0551] MKSFLLWNALALTLPFLAVEVQNQKQPACHENDERPFYQKTAPYVPMYYVPNSYPYYG

[0552] TNLYQRRPAIAINNPYVPRTYYANPAWRPHAQIPQRQYLPNSHPPTWRRPNLHPSFIAI

[0553] PPKKIQDKIIIPTINTIATVEPTPAPATEPTVDSWTPEAFSESIITSTPETTTVAVTPPTA

[0554] Camel Kappa-casein: Sp P79139 CASK_CAMDR (SEQ ID NO: 29)

[0555] MKSFFLWTILALTLPFLGAEVQNQEQPTCFEKVERLLNEKTVKYFPIQFVQSRYPSYGINY

[0556] YQHRLAVPINNQFIPYPNYAKPVAIRLHAQIPQCQALPNIDPPTVERRPRPRPSFIAI

[0557] PPKKTQDKTVNPAINTVATVEPPVIPTAEPAVNTWIAEASSEFITTSTPETTTVQITST

[0558] El

[0559] Sheep Kappa-casein: Sp P02669 CASK_SHEEP (SEQ ID NO: 30)

[0560] MMKSFFLWTILALTLPFLGAQEQNQEQRICCEKDERFFDDKIAKYIPIQYVLSRYPSYG LNYYQQRPVALINNQFLPYPYYAKPVAVRSPAQTLQWQVLPNAVPAKSCQDQPTAMARH

[0561] PHPHLSFMAIPPKKDQDKTEIPAINTIASAEPTVHSTPTTEAVVNAVDNPEASSESIASAPE

[0562] TNTAQVTSTEV

[0563] Goat Kappa-casein: Sp P02670 CASK_CAPHI (SEQ ID NO: 31)

[0564] MMKSFFLVVTILALTLPFLGAQEQNQEQPICCEKDERFFDDKIAKYIPIQYVLSRYPSYG LNYYQQRPVALINNQFLPYPYYAKPVAVRSPAQTLQWQVLPNTVPAKSCQDQPTTLARH PHPHLSFMAIPPKKDQDKTEVPAINTIASAEPTVHSTPTTEAIVNTVDNPEASSESIASASE

[0565] TNTAQVTSTEV

[0566] Bovine Kappa-casein: Sp P02668 CASK_BOVIN (SEQ ID NO: 32)

[0567] MMKSFFLVVTILALTLPFLGAQEQNQEQPIRCEKDERFFSDKIAKYIPIQYVLSRYPSYG

[0568] LNYYQQKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLSNTVPAKSCQAQPTTMARH

[0569] PHPHLSFMAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINT VQVTSTAV

[0570] Buffalo Kappa-casein: Tr K9ZU53 CASK_BUBBU (SEQ ID NO: 33)

[0571] YGLNYYQQKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLPNTVPAKSCQAQPTTMT

[0572] RHPHPHLSFMAIPPKKNQDKTEIPTINTIVSVEPTSTPTTEAIENTVATLEASSDVIESVPET NTAQVTSTVV

[0573] Ovalbumin (SEQ ID NO: 34) (Chicken)

[0574] GSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDK

[0575] LPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKE

[0576] LYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLW

[0577] EKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLV

[0578] LLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITD VFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFL FCIKHIATNAVLFFGRCVSP

[0579] Phosvitin 1 (SEQ ID NO: 35) (Chicken)

[0580] NEKKFYPFGDSSSSGSSSSSSSSSSSSSDSSSSSRSSSSSDSSSSSSSSSSSSSSKSKS SSRSSKSNRSSSSSNSKDSSSSSSKSNSKGSSSSSSKASGTRQKAKKQSKTTSFPHASA AEGERSVHEQKQETQSSSSSSSRASSNSRSTSSSTSSSSESSGVSHRQWKQDREAETK RVKSQFNSHSSYDIPNEWETYLPKVYRLRFRSAHTHWHSGHRTSSSSSSSSSESGSSHS

[0581] NSSSSDSSSRRSHMSDS

[0582] Phosvitin 2 (SEQ ID NO: 36) (Chicken)

[0583] AEFGTEPDAKTSSSSSSASSTATSSSSSSASSPNRKKPMDEEENDQVKQARNKDASSSS

[0584] RSSKSSNSSKRSSSKSSNSSKRSSSSSSSSSSSSRSSSSSSSSSSNSKSSSSSSKSSSS

[0585] SSRSRSSSKSSSSSSSSSSSSSSKSSSSRSSSSSSKSSSHHSHSHHSGHLNGSSSSSSS

[0586] SRSVSHHSHEHHSGHLEDDSSSSSSSSVLSKIWGRHEIYQ

[0587] Phosvitin 3 (SEQ ID NO: 37) (Chicken)

[0588] TDLWEEPSATPLSSSSSTDSSAEGEEPGNKRDKDEIWQFGKKYGADSSSSSSSSSTGS

[0589] GSSKTCSSSREDSSRDKHCSVDSEYFNQQADLPIYQ

[0590] Ovotransferrin (SEQ ID NO: 38) (Chicken)

[0591] APPKSVIRWCTISSPEEKKCNNLRDLTQQERISLTCVQKATYLDCIKAIANNEADAISLDGG

[0592] QAFEAGLAPYKLKPIAAEVYEHTEGSTTSYYAVAWKKGTEFTVNDLQGKTSCHTGLGRS

[0593] AGWNIPIGTLLHRGAIEWEGIESGSVEQAVAKFFSASCVPGATIEQKLCRQCKGDPKTKC

[0594] ARNAPYSGYSGAFHCLKDGKGDVAFVKHTTVNENAPDQKDEYELLCLDGSRQPVDNYK

[0595] TCNWARVAAHAWARDDNKVEDIWSFLSKAQSDFGVDTKSDFHLFGPPGKKDPVLKDLL

[0596] FKDSAIMLKRVPSLMDSQLYLGFEYYSAIQSMRKDQLTPSPRENRIQWCAVGKDEKSKC

[0597] DRWSWSNGDVECTWDETKDCIIKIMKGEADAVALDGGLVYTAGVCGLVPVMAERYDD

[0598] ESQCSKTDERPASYFAVAVARKDSNVNWNNLKGKKSCHTAVGRTAGWVIPMGLIHNRT

[0599] GTCNFDEYFSEGCAPGSPPNSRLCQLCQGSGGIPPEKCVASSHEKYFGYTGALRCLVEK

[0600] GDVAFIQHSTVEENTGGKNKADWAKNLQMDDFELLCTDGRRANVMDYRECNLAEVPTH

[0601] AWVRPEKANKIRDLLERQEKRFGVNGSEKSKFMMFESQNKDLLFKDLTKCLFKVREGTT

[0602] YKEFLGDKFYTVISSLKTCNPSDILQMCSFLEGK

[0603] Ovoglobulin (SEQ ID NO: 39) (Chicken)

[0604] STSTSHLILALSEHFYTSLYFTLERAGAFNMTIPSMLTTATLAQKITQVGSLYHEDLPITLSA

[0605] ALRSSPRWLEEGRAALKLFLTVHIGAGSPDFQSFLSVSADVTAGLQLSVSDTRMMISTAV

[0606] IEDAELSLAASNVGLVRAALLEELFLAPVCQQVPAWMDDVLREGVHLPHLSHFTYTDVSV VVHKDYVLVPC Ovomucoid (SEQ ID NO: 40) (Chicken)

[0607] AEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISKEHDG

[0608] ECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVTYDNECLLCAHKVEQGAS

[0609] VDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNG TLTLSHFGKC

[0610] Ovomucin (SEQ ID NO: 41) (Chicken)

[0611] KEPVQIVQVSTVGRSECTTWGNFHFHTFDHVKFTFPGTCTYVFASHCNDSYQDFNIKIRR

[0612] SDKNSHLIYFTVTTDGVILEVKETGITVNGNQIPLPFSLKSILIEDTCAYFQVTSKLGLTLKW

[0613] NWADTLLLDLEETYKEKICGLCGNYDGNKKNDLILDGYKMHPRQFGNFHKVEDPSEKCP

[0614] DVRPDDHTGRHPTEDDNRCSKYKKMCKKLLSRFGNCPKVVAFDDYVATCTEDMCNCVV

[0615] NSSHSDLVSSCICSTLNQYSRDCVLSKGDPGEWRTKELCYQECPSNMEYMECGNSCAD

[0616] TCADPERSKICKAPCTDGCFCPPGTILDDLGGKKCVPRDSCPCMFQGKVYSSGGTYSTP

[0617] CQNCTCKGGHWSCISLPCSGSCSIDGGFHITTFDNKKFNFHGNCHYVLAKNTDDTFWIG

[0618] EIIQCGTSKTMTCLKNVLVTLGRTTIKICSCGSIYMNNFIVKLPVSKDGITIFRPSTFFIKILSS

[0619] TGVQIRVQMKPVMQLSITVDHSYQNRTSGLCGNFNNIQTDDFRTATGAVEDSAAAFGNS

[0620] WKTRASCFDVEDSFEDPCSNSVDKEKFAQHWCALLSNTSSTFAACHSVVDPSVYIKRCM

[0621] YDTCNAEKSEVALCSVLSTYSRDCAAAGMTLKGWRQGICDPSEECPETMVYNYSVKYC

[0622] NQSCRSLDEPDPLCKVQIAPMEGCGCPEGTYLNDEEECVTPDDCPCYYKGKIVQPGNSF

[0623] QEDKLLCKCIQGRLDCIGETVLVKDCPAPMYYFNCSSAGPGAIGSECQKSCKTQDMHCY

[0624] VTECVSGCMCPDGLVLDGSGGCIPKDQCPCVHGGHFYKPGETIRVDCNTCTCNKRQWN

[0625] CTDSPCKGTCTVYGNGHYMSFDGEKFDFLGDCDYILAQDFCPNNMDAGTFRIVIQNNAC

[0626] GKSLSICSLKITLIFESSEIRLLEGRIQEIATDPGAEKNYKVDLRGGYIVIETTQGMSFMWDQ

[0627] KTTWVHVTPSFQGKVCGLCGDFDGRSRNDFTTRGQSVEMSIQEFGNSWKITSTCSNIN

[0628] MTDLCADQPFKSALGQKHCSIIKSSVFEACHSKVNPIPYYESCVSDFCGCDSVGDCECFC

[0629] TSVAAYARSCSTAGVCINWRTPAICPVFCDYYNPPDKHEWFYKPCGAPCLKTCRNPQGK

[0630] CGNILYSLEGCYPECSPDKPYFDEERRECVSLPDCTSCNPEEKLCTEDSKDCLCCYNGK

[0631] TYPLNETIYSQTEGTKCGNAFCGPNGMIIETFIPCSTLSVPAQEQLMQPVTSAPLLSTEAT

[0632] PCFCTDNGQLIQMGENVSLPMNISGHCAYSICNASCQIELIWAECKWQTEALETCEPNS

[0633] EACPPTAAPNATSLVPATALAPMSDCLGLIPPRKFNESWDFGNCQIATCLGEENNIKLSSI

[0634] TCPPQQLKLCVNGFPFMKHHDETGCCEVFECQCICSGWGNEHYVTFDGTYYHFKENCT

[0635] YVLVELIQPSSEKFWIHIDNYYCGAADGAICSMSLLIFHSNSLVILTQAKEHGKGTNLVLFN

[0636] DKKVVPDISKNGIRITSSGLYIIVEIPELEVYVSYSRLAFYIKLPFGKYYNNTMGLCGTCTNQ KSDDARKRNGEVTDSFKEMALDWKAPVSTNRYCNPGISEPVKIENYQHCEPSELCKIIWN

[0637] LTECHRVVPPQPYYEACVASRCSQQHPSTECQSMQTYAALCGLHGICVDWRGQTNGQ

[0638] CEATCARDQVYKPCGEAKRNTCFSREVIVDTLLSRNNTPVFVEGCYCPDGNILLNEHDGI

[0639] CVSVCGCTAQDGSVKKPREAWEHDCQYCTCDEETLNISCFPRPCAKSPPINCTKEGFVR

[0640] KIKPRLDDPCCTETVCECDIKTCIINKTACDLGFQPWAISEDGCCPIFSCIPKGVCVSEGV

[0641] EFKPGAVVPKSSCEDCVCTDEQDAVTGTNRIQCVPVKCQTTCQQGFRYVEKEGQCCSQ

[0642] CQQVACVANFPFGSVTIEVGKSYKASYDNCTQYTCTESGGQFSLTSTVKVCLPFEESNC

[0643] VPGTVDVTSDGCCKTCIDLPHKCKRSMKEQYIVHKHCKSAAPVPVPFCEGTCSTYSVYSF

[0644] ENNEMEHKCICCHEKKSHVEKVELVCSEHKTLKFSYVHVDECGCVETKCPMRRT

[0645] Lipovitellin-1 (SEQ ID NO: 42) (Chicken)

[0646] QHLNYQPDFGENKVYTYNYESILFSGIPEKGLARTGIRIRSEVEISGIGPKLCLIRIHSIEAAE

[0647] YNGIWPTSSFSRSLKLTQALTGQLSIPIKFEYSNGHVGNLMAPDSVSDDGLNIYRGILNILE

[0648] LSLKKMQHSYSIQEAGIGGICNTTYAIQENKKANLVDVTKSKDLNSCEEKVQWTGSAYTQ

[0649] PCQTCQQRNKNSRATATYNYKIKYTHNEAVITQAEVEEVHQFTPFHEITGGNAIVEARQKL

[0650] ALIEVQKQVAEVPPKEFQKRGSLQYQFGSELLQLPVHLFKIKDVERQIEERLQDLVETTYE

[0651] QLPSDAPAKALKLMHLLRAANEENYESVWKQFSSRPAYRRYLLDLLPAAASHRSLRFLRH

[0652] KMERQELTNWEIAQTVLVALHSSSPTQEVMEEATLIVKKHCPRSSSVLRKVCLLSYASLC

[0653] HKRCSSPYSCSECLQVFHVFAGEALGKSNIEEVLLALKALGNVGHPASIKHIKKFLPGYAA

[0654] GASELPLKVHETAVMALKSIGMRDPQMVQAITLEIFLNHKIHPRIRMLAAWLLETKPGLPIL

[0655] MILVDAVLKEPSMQVASFIYSHLRALGRSTAPDLQMMASACRMAVRALSPKFDRSGYQF

[0656] SKVFRFSMFKEFLMSGLAAKYFVLNNAGSLIPTMAVSQLRTHFLGRVADPIEVGIAAEGLQ

[0657] EMFVRGYSPDKDWETNYDFREILKKLSDWKALPRDKPFASGYLKMFGQELLFGRLDKDT

[0658] LQNVLQVWYGPDEKIPSIRRLISSLQTGIGRQWTKALLLSEIRCIVPTCVGFPMETSFYYSS

[0659] VTKVAGNVQAQITPSPRSDFRLTELLNSNVRLRSKMSLSMAKHMTFVIGINTNMIQAGLEA

[0660] HTKVNAHVPVNWATIQMKEKSIKAEIPPCKEETNLIIVSSKTFAVTRNIEDLAASKMTPVLL

[0661] PEAVPDIMKMSFDSDSASGETDNIRDRQSVEDVSSGNSFSFGHPSSGKEPFIQSMCSNA

[0662] STFGVQVCIEKKSVHAAFIRNVPLYNAIGEHALRMSFKPVYSDVPIEKIQVTIQAGDQAPTK

[0663] MVRLVTFEDPERQESSRKEVMKRVKKILDDTDNQATRNSRSSSSSASSISESSESTTSTP

[0664] SSSDSDNRASQGDPQINLKSRQSKA

[0665] Lipovitellin -2 (SEQ ID NO: 43) (Chicken) SSSSSSHRHGEKAAHSSRRSPTSRAASAHHRPGSSLTRERNFLGDVIPPGITIVAQAVRS DNRNQGYQATAYVRSDAAKVDVQLWVQLAETNWKACADAVILPLKAQARMRWGKECR DYRIAALATTGQMARKLAVQLKVQWGIIPSWIKKTSTALMRYVPGVALVLGFSEAHQRNP SRELIVRAVATSPRSIDTVIKVPGVTLYYQGLRVPFTLALGASSSSYETRDITAWNFL

[0666] Livetin (SEQ ID NO: 44) (Chicken)

[0667] FARDAEHKSEIAHRYNDLKEETFKAVAMITFAQYLQRCSYEGLSKLVKDWDLAQKCVAN

[0668] EDAPECSKPLPSIILDEICQVEKLRDSYGAMADCCSKADPERNECFLSFKVSQPDFVQPY QRPASDVICQEYQDNRVSFLGHFIYSVARRHPFLYAPAILSFAVDFEHALQSCCKESDVG ACLDTKEIVMREKAKGVSVKQQYFCGILKQFGDRVFQARQLIYLSQKYPKAPFSEVSKFV HDSIGVHKECCEGDMVECMDDMARMMSNLCSQQDVFSGKIKDCCEKPIVERSQCIMEA EFDEKPADLPSLVEKYIEDKEVCKSFEAGHDAFMAEFVYEYSRRHPEFSIQLIMRIAKGYE SLLEKCCKTDNPAECYANAQEQLNQHIKETQDWKTNCDLLHDHGEADFLKSILIRYTKKM

[0669] PQVPTDLLLETGKKMTTIGTKCCQLGEDRRMACSEGYLSIVIHDTCRKQETTPINDNVSQ

[0670] CCSQLYANRRPCFTAMGVDTKYVPPPFNPDMFSFDEKLCSAPAEEREVGQMKLLINLIKR KPQMTEEQIKTIADGFTAMVDKCCKQSDINTCFGEEGANLIVQSRATLGIGA

[0671] Lysozyme (SEQ ID NO: 45) (Chicken)

[0672] KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQIN SRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKG TDVQAWIRGCRL

[0673] References

[0674] Beausoleil, SA et al.: A probability-based approach for high-throughput protein phosphorylation analysis and site localization, Nature Biotechnology 24, 1285 - 1292 (2006)

[0675] Jimenez-Flores R, Richardson T, and Bisson L.F. Expression of bovine .beta. -casein in Saccharomyces cerevisiae and characterization of the protein produced in vivo. Journal of Agricultural and Food Chemistry 1990 38 (4), 1134-1141. Moore A, Boulton AP, Heid HW, Jarasch ED, Craig RK. Purification and tissue-specific expression of casein kinase from the lactating guinea-pig mammary gland. EurJ Biochem. 1985 Nov 4;152(3):729-37. doi: 10.1111 / j.1432-1033.1985.tb09254.x. PMID: 3863754. Tagliabracci VS, Engel JL, Wen J, Wiley SE, Worby CA, Kinch LN, Xiao J, Grishin NV, Dixon JE. Secreted kinase phosphorylates extracellular proteins that regulate biomineralization. Science. 2012 Jun 1 ;336(6085): 1150-3.

[0676] Thurmond JM, Hards RG, Seipelt CT, Leonard AE, Hansson L, Strdmqvist M, Bystrom M, Enquist K, Xu BC, Kopchick J J, Mukerji P. Expression and characterization of phosphorylated recombinant human beta-casein in Escherichia coli. Protein ExprPurif.

[0677] 1997 Jul; 10(2):202-8.

[0678] Tubb, C., and T. Seba. 2019. Rethinking food and agriculture 2020-2030: The second domestication of plants and animals, the disruption of the cow, and the collapse of industrial livestock farming.

Claims

Claims1 . A system for producing one or more phosphorylated recombinant animal proteins, said system comprising: i. a microbial host cell expressing: a. at least one recombinant animal protein comprising a phosphorylation motif being: Serine (S) - X - Glutamic acid (E)ZAspartic acid (D), wherein X is any amino acid; or a continuous stretch of at least two serines, and b. at least one microbial kinase; or ii. a microbial host cell expressing at least one animal protein comprising a phosphorylation motif being: Serine (S) - X - Glutamic acid (E)ZAspartic acid (D), wherein X is any amino acid; or a continuous stretch of at least two serines, and a composition comprising at least one microbial kinase, whereby said system produces one or more phosphorylated recombinant animal proteins.

2. The system according to claim 1 , wherein said recombinant animal protein comprises a continuous stretch of at least three serines, such as at least four serines, such as at least five serines, such as at least six serines, such as at least seven serines, such as at least eight serines, such as at least nine serines, such as a continuous stretch of at least ten serines.

3. The system according to any one of the preceding claims, wherein the at least one recombinant animal protein is a casein, wherein said casein is alpha-casein S1 (aS1), alpha-casein S2 (aS2), beta-casein or kappa-casein, or wherein the at least one recombinant animal protein is an egg protein, wherein said egg protein is ovalbumin, phosvitin 1 , phosvitin 2, phosvitin 3, ovotransferrin, ovoglobulin, ovomucoid, lysozyme, ovomucin, livetins, lipovitellins, or lipovitellenin.

4. The system according to any one of the preceding claims, wherein the recombinant casein is a recombinant variant of a native mammalian casein, optionally wherein said mammalian casein is that found in milk from a mammal, wherein said mammal is ahuman, cow, buffalo, zebu, yak, equines, sheep, goat, horse, donkey, camel, deer, or reindeer, and wherein said native mammalian casein is naturally phosphorylated at one or more native phosphorylation sites.

5. The system according to any one of the preceding claims, wherein the recombinant egg protein is a recombinant variant of a native avian egg protein, optionally wherein said avian egg protein is that found in egg from a bird selected from the group consisting of chicken, duck, goose, quail, guineafowl, turkey, emu, pheasant, gull, partridge and ostrich.

6. The system according to any one of the preceding claims, wherein a native avian egg protein is an egg protein found in a bird egg, such as an egg protein found in a chicken, duck, goose, quail, guineafowl, turkey, emu, pheasant, gull, partridge, and ostrich egg, and wherein said native avian egg protein is naturally phosphorylated at one or more native phosphorylation sites.

7. The system according to according to any one of the preceding claims, wherein the microbial kinase is capable of phosphorylating one or more amino acid residues of said recombinant animal protein corresponding to one or more of the native phosphorylation sites.

8. The system according to according to any one of the preceding claims, wherein the microbial kinase is capable of phosphorylating one or more amino acid residues of said recombinant animal protein corresponding to one or more non-native phosphorylation sites.

9. The system according to any one of the preceding claims, wherein the recombinant animal protein, such as recombinant casein or recombinant egg protein, is phosphorylated inside the microbial host cell.

10. The system according to any one of the preceding claims, wherein the recombinant animal protein, such as recombinant casein or recombinant egg protein, is phosphorylated outside the microbial host cell.11 . The system according to any one of the preceding claims, wherein the recombinant animal protein, such as recombinant casein or recombinant egg protein, is phosphorylated after the casein has been secreted.

12. The system according to any one of the preceding claims, wherein the recombinant animal protein, such as recombinant casein or recombinant egg protein, is phosphorylated in vivo.

13. The system according to any one of the preceding claims, wherein the recombinant animal protein, such as recombinant casein or recombinant egg protein, is phosphorylated in vitro.

14. The system according to any one of the preceding claims, wherein the recombinant animal protein, such as recombinant casein or recombinant egg protein, is phosphorylated at one or more native phosphorylation sites, such as phosphorylated at least at two native phosphorylation sites, such as phosphorylated at least at three native phosphorylation sites, such as phosphorylated at least at four native phosphorylation sites, such as phosphorylated at least at five native phosphorylation sites, such as phosphorylated at least at six native phosphorylation sites, such as phosphorylated at least at seven native phosphorylation sites, such as phosphorylated at least at eight native phosphorylation sites, such as phosphorylated at least at nine native phosphorylation sites, such as phosphorylated at ten native phosphorylation sites, such as phosphorylated at eleven native phosphorylation sites, such as phosphorylated at twelve native phosphorylation sites, such as phosphorylated at thirteen native phosphorylation sites, such as phosphorylated at fourteen native phosphorylation sites, such as phosphorylated at fifteen native phosphorylation sites.

15. The system according to any one of the preceding claims, wherein the recombinant animal protein, such as recombinant casein or recombinant egg protein, is phosphorylated at all its native phosphorylation sites.

16. The animal protein, such as casein or egg protein, according to any one of the preceding claims, wherein the recombinant animal protein, such as recombinant casein or recombinant egg protein, has a degree of phosphorylation at each native phosphorylation site of at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as about 100%.

17. The casein according to according to any one of the preceding claims, wherein the recombinant animal protein has a degree of phosphorylation at each native phosphorylation site of at least 5%.

18. The system according to according to any one of the preceding claims, wherein the microbial host cell is a bacterial cell, a fungal cell or a yeast cell.

19. The system according to according to any one of the preceding claims, wherein the microbial host cell is a bacterial cell, which belongs to a genus, wherein said genus is Lactococcus spp., Lactobacillus spp., Escherichia spp., Corynebacterium spp., Pseudomonas spp., Streptomyces spp., Geobacillus spp., or Bacillus spp.; or wherein the microbial host cell is a fungal host cell, which belongs to a genus, wherein said genus is Kluyveromyces spp., Pichia spp., Saccharomyces spp., Tetrahymena spp., Yarrowia spp., Hansenula spp., Blastobotrys spp., Aspergillus spp., Candida spp., Zygosaccharomyces spp., Trichoderma spp., Chrysosporium spp., Fusarium spp., Neurospora spp. or Debaryomyces spp..

20. The system according to any one of the preceding claims, wherein the microbial kinase is a prokaryotic kinase or a fungal kinase.21 . The system according to according to any one of the preceding claims, wherein the microbial kinase is a bacterial serine / threonine kinase or a yeast serine / threonine kinase.

22. The system according to according to any one of the preceding claims, wherein the microbial kinase is a bacterial kinase of the Hanks family or a yeast kinase of the Hanks family.

23. The system according to according to any one of the preceding claims, wherein the microbial kinase is YabT (SEQ ID NO:5 or SEQ ID NO: 6), PrkD (SEQ ID NO: 7), PrkC (SEQ ID NO: 8) or PKNB1 (SEQ ID NO: 9), or functional variants thereof having at least 60% sequence identity, similarity or homology thereto, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity.

24. The system according to any one of the preceding claims, wherein the microbial kinase is YabT as set forth in SEQ ID NO: 5, or a functional variant thereof having at least 70% sequence identity, similarity or homology thereto.

25. The system according to any one of the preceding claims, wherein the microbial kinase is PrkD as set forth in SEQ ID NO: 7, or a functional variant thereof having at least 70% sequence identity, similarity or homology thereto.

26. The system according to according to any one of the preceding claims, wherein the recombinant casein is SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO:32, or SEQ ID NO: 33, or a variant thereof having at least 90% sequence identity, similarity or homology thereto.

27. The system according to according to any one of the preceding claims, wherein the recombinant casein is recombinant bovine casein or a recombinant human casein.

28. The system according to claim 27, wherein the recombinant bovine casein is alphacasein S1 (aS1), alpha-casein S2 (aS2), beta-casein or kappa-casein.

29. The system according to anyone of the preceding claims, wherein the recombinant egg protein is recombinant avian egg protein.

30. The system according to anyone of the preceding claims, wherein the recombinant avian egg protein is avian ovalbumin, phosphovitin 1 and / or phosphovitin 2.31 . The system according to according to any one of the preceding claims, wherein the recombinant animal protein is: a. a recombinant bovine alpha-S1 casein (aS1) as set forth in SEQ ID NO: 1 , is phosphorylated at least at one of the following positions: S46, S48, S64, S66, S67, S68, and S75; b. a recombinant bovine alpha-S1 casein (aS1) as set forth in SEQ ID NO: 14, is phosphorylated at least at one of the following positions: S56, S61 , S63, S79, S81 , S82, S83, or S90; c. a recombinant bovine alpha-S2 casein (aS2) as set forth in SEQ ID NO: 20, is phosphorylated at least at one of the following positions: S23, S24, S25, S28, S46, S71 , S72, S73, S76, S144, S146, S150, or S158; d. a recombinant bovine beta casein as set forth in SEQ ID NO: 3, is phosphorylated at least at one of the following positions: S15, S17, S18, S19 and S35; e. a recombinant bovine beta casein as set forth in SEQ ID NO: 26, is phosphorylated at least at one of the following positions: S30, S32, S33, S34 and S50; f. a recombinant bovine kappa casein as set forth in SEQ ID NO: 32, is phosphorylated at least at one of the following positions: S148, T166, S170, or S187.g. a recombinant casein, wherein said recombinant casein is human alphacasein S1 (aS1), human alpha-casein (aS2), human beta-casein or human kappa-casein. h. a recombinant human alpha-S1 casein as set forth in SEQ ID NO: 10 is phosphorylated at least at one of the following positions: S31 , S33, S41 , S71 , S85, S86, S88.S89, S90, or S91. i. a recombinant human beta casein as set forth in SEQ ID NO: 22, is phosphorylated at least at one of the following positions: T18, S21 , S23, S24, or S25. j. a recombinant human kappa casein as set forth in SEQ ID NO: 28 is phosphorylated at positions T157. k. a recombinant ovalbumin as set forth in SEQ ID NO: 34, is phosphorylated at least at one of the following position: S68, or S344. l. a recombinant phosvitin 1 as set forth in SEQ ID NO: 35, is phosphorylated at least at one of the following positions: S11 , S12, S13, S14, S16, S37, S43, S45, S46, S47, S48, S51 , S52, S69, S70, S71 , S73, S75, S79, T110, S117, S124, S180, S184, S186, S187, S240, or S244. m. a recombinant phosvitin 2 as set forth in SEQ ID NO: 36, is phosphorylated at least at one of the following positions: T5, T11 , S12, S13, S14, S15, S16, S17, S19, S20, T21 , T23, S24, S31 , S82, S89, S91 , S96, S98, S99, S100, S104, S124, S125, S126, S130, S134, S135, S137, S197, S198, S199, S200, S201 , S202, S203, S204, or S207.

32. The system according to according to any one of the preceding claims, wherein said host cell comprises at least one protease deficiency rendering said host cell completely or partially deficient in the production and / or function of one or more proteases, and / or wherein said host cell expresses at least one chaperone protein.

33. The system according to any one of the preceding claims, wherein said host cell expresses at least one chaperone protein.

34. A system for producing one or more phosphomimetic recombinant caseins, said system comprising a microbial host cell expressing: i. at least one recombinant casein, wherein said casein is alpha-casein S1 (aS1), alpha-casein S2 (aS2), beta-casein or kappa-casein, wherein one or more amino acid residues that are phosphorylated in a native mammalian casein are substituted with one or more phosphomimetic amino acids; and ii. optionally at least one microbial kinase capable of phosphorylating said phosphomimetic casein at residues which are phosphorylated in a native mammalian casein, and which have not been substituted with phosphomimetic amino acids, whereby said system is capable of producing one or more phosphomimetic recombinant casein.

35. The system according to claim 34, wherein one or more amino acids have been substituted with a phosphomimetic amino acid.

36. The system according to any one of claims 34 and 35, wherein one or more serine and / or threonine residues have been substituted with a phosphomimetic amino acid.

37. The system according to any one of claims 34 to 36, wherein one or more amino acid residues which are phosphorylated in the native mammalian casein have been substituted with one or more phosphomimetic amino acids.

38. The system according to any one of claims 34 to 37, wherein one or more serine and / or threonine residues of the recombinant casein, such as of including alpha casein (aS1) as set forth in any one of SEQ ID NOs: 1 , 10, 11 , 12, 13 14 and 15, alpha casein (aS2) as set forth in any one of SEQ I D NOs: 16, 17, 18, 19, 20 and 21 , beta casein as set forth in any one of SEQ ID NOs: 3, 22, 23, 24, 25, 26, and 27, and kappa casein as set forth in any one of SEQ ID NO 28, 29, 30, 31 , 32 and 33, have been substituted with one or more phosphomimetic amino acids.

39. The system according to claim 34 to 38, wherein the recombinant casein is:a. a recombinant casein is bovine alpha casein (aS1) as set forth in SEQ ID NO: 1 , comprising a phosphomimetic amino acid at least at one of the following positions: S46, S48, S64, S66, S67, S68, and S75; b. a recombinant casein is bovine alpha-S1 casein as set forth in SEQ ID NO: 14, comprising a phosphomimetic amino acid at least at one of the following positions: S56, S61 , S63, S79, S81 , S82, S83, or S90; c. a recombinant casein is bovine alpha-S2 casein as set forth in SEQ ID NO: 20, comprising a phosphomimetic amino acid at least at one of the following positions: S23, S24, S25, S28, S46, S71 , S72, S73, S76, S144, S146, S150, or S158; d. a recombinant casein is bovine beta casein as set forth in SEQ ID NO: 3, comprising a phosphomimetic amino acid at least at one of the following positions: S15, S17, S18, S19 and S35; e. a recombinant casein is bovine beta casein as set forth in SEQ ID NO: 26, comprising a phosphomimetic amino acid at least at one of the following positions: S30, S32, S33, S34 and S50; f. a recombinant casein is bovine kappa casein as set forth in SEQ ID NO: 32, comprising a phosphomimetic amino acid at least at one of the following positions: S148, T166, S170, or S187; g. a recombinant casein is human alpha-S1 casein as set forth in SEQ ID NO: 10, comprising a phosphomimetic amino acid at least at one of the following positions: S31 , S33, S41 , S71 , S85, S86, S88, S89, S90, or S91 ; h. a recombinant casein is human beta casein as set forth in SEQ ID NO: 22, comprising a phosphomimetic amino acid at least at one of the following positions: T18, S21 , S23, S24, or S25; i. a recombinant casein is human kappa casein as set forth in SEQ ID NO: 28, comprising a phosphomimetic amino acid at position T157.

40. The system according to claim 34 to 39, wherein each phosphomimetic amino acid is aspartic acid, glutamic acid and / or combinations thereof.41 . The system according to claim 34 to 40, wherein the one or more phosphomimetic recombinant casein is as set forth in SEQ ID NO: 2 or SEQ ID NO:4, or a variant thereof having at least 90% sequence identity, similarity or homology thereto.

42. A phosphorylated recombinant animal protein, such as recombinant casein or recombinant egg protein, obtained using a system according to any one of claims 1 to 41.

43. Phosphomimetic casein represented by the following sequences: SEQ ID NO: 1 ,SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, or a variant thereof, wherein said variant has at least 70% sequence identity to any one of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, and wherein said variant comprises at least one or more phosphomimetic amino acids at one or more of positions corresponding to positions S46, S48, S64, S66, S67, S68, and S75 of SEQ ID NO: 1 ; or positions of S15, S17, S18, S19 and S35 of SEQ ID NO: 3; or positions S31 , S33, S41 , S71 , S85, S86, S88.S89, S90, S91 of SEQ ID NO: 10; or positions S33, S83, S85, S86, S87, S88 of SEQ ID NO: 11 ; or positions S27, S56, S61 , S63, S79, S80, S81 , S82, S83, S90, S130 of SEQ ID NO: 12; or positions S61 , S63, S79, S80, S81 , S82, S83, S90, S130 of SEQ ID NO: 13; or positions S56, S61 , S63, S79, S81 , S82, S83, S90 of SEQ ID NO: 14; or positions S63, S79, S80, S81 , S82, S83, S90 of SEQ ID NO: 15; or positions S23, S24, S25, S28, S47, S68, S123, S125, S128, S136 of SEQ ID NO: 17; or positions S23, S24, S25, S72, S73, S74, S77, S145, S147, S151 , S159 of SEQ ID NO:18; or positions S23, S24, S25, S72, S73, S74, S77, S145, S147, S151 , S159 of SEQ ID NO:19; or positions S23, S24, S25, S28, S46, S71 , S72, S73, S76, S144, S146,S150, S158 of SEQ ID NO:20; or positions T18, S21 , S23, S24, S25 of SEQ ID NO:22;or positions T27, S30, S32, S33, S34 of SEQ ID NO:24; or positions T27, S30, S32, S33, S34 of SEQ ID NO:25; or positions S30, S32, S33, S34, S50 of SEQ ID NO:26; or positions S30, S32, S33, S34 of SEQ ID NO:27; or position T157 of SEQ ID NO: 28; or positions of S148, S172, S189 SEQ ID NQ:30; or positions S148, T166, S170, S187 of SEQ ID NO:31 ; or positions S148, T166, S170, S187 of SEQ ID NO: 32.

44. An expression system for expression of a phosphorylated recombinant animal protein in a microbial host cell, comprising: iii. a first nucleic acid encoding at least one recombinant animal protein such as a mammalian casein or an avian egg protein; and iv. a second nucleic acid encoding a kinase.

45. An expression system for expression of phosphorylated recombinant casein in a microbial host cell, comprising: iii. a first nucleic acid encoding at least one recombinant casein selected from the group consisting of alpha-casein S1 (aS1), alpha-casein (aS2), beta-casein and kappa-casein; and iv. a second nucleic acid encoding a kinase.

46. The expression system according to any one of claims 44 and 45, wherein the at least one recombinant casein is SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 ,SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 ,SEQ ID NO: 32, SEQ ID NO: 33, or a variant thereof having at least 90% sequence identity, similarity, or homology thereto.

47. The expression system according to claim 45, wherein the nucleic acid encoding said at least one recombinant avian egg protein encodes a polypeptide as set forthin SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or a variant thereof having at least 90% sequence identity, similarity or homology thereto.

48. The expression system according to claim 45, wherein the nucleic acid encoding said at least one recombinant avian egg protein encodes a polypeptide as set forth in SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO: 45, or a variant thereof having at least 90% sequence identity, similarity or homology thereto.

49. The expression system according to any one of claims 45 to 48, wherein the kinase is YabT as set forth in SEQ ID NO: 5 or SEQ ID NO: 6, PrkD as set forth in SEQ ID NO: 7, PrkC as set forth in SEQ ID NO: 8, PKNB1 as set forth in SEQ ID NO: 9 or functional variants thereof having at least 70% sequence identity, similarity, or homology to the sequences.

50. The expression system according to any one of claims 45 to 49, wherein the nucleic acid encoding a casein and the nucleic acid encoding a kinase are expressed in a microbial host cell.51 . The expression system according to any one of claims 45 to 50, wherein the nucleic acid encoding at least one casein is expressed in a first microbial host cell, and wherein the nucleic acid encoding at least one kinase is expressed in a second microbial host cell.

52. A host cell comprising the system according to any one of claims 1 to 51 , wherein said host cell is capable of producing recombinant animal protein, such as recombinant casein and / or recombinant egg protein.

53. A method of producing recombinant animal protein in a microbial host cell, comprising the steps of: i. providing the system according to any one of claims 44 to 51 , wherein said host cell is capable of producing recombinant animal protein;ii. incubating said host cell in a medium, under conditions allowing growth of the host cell, whereby at least one recombinant animal protein is produced; and iii. incubating the at least one animal protein with at least one microbial kinase, whereby phosphorylated recombinant animal protein is produced, preferably wherein said recombinant animal protein is casein or egg protein.

54. A method of producing recombinant casein in a microbial host cell, comprising the steps of: iv. Providing a host cell according to claim 53; and v. incubating said host cell in a medium, under conditions sufficient to allow for production of at least one phosphomimetic casein, whereby at least one phosphomimetic casein is produced; vi. incubating the at least one casein with at least one microbial kinase, whereby phosphorylated and / or phosphomimetic recombinant casein is produced.

55. The method according to any one of claims 53 to 54, further comprising a step of recovering the recombinant casein.

56. Use of the system according to any one of claims 1 to 41 for production of at least one recombinant animal protein, such as casein or egg protein, of interest.

57. Use of the expression system according to any one of claims 44 to 51 , for production of at least one recombinant animal protein, such as casein or egg protein, of interest.

58. Use of the methods according to any one of claims 53 to 55 for production of at least one recombinant animal protein, such as casein or egg protein, of interest.

59. Use of the system according to any one of claims 1 to 41 for production of at least one recombinant casein of interest.

60. Use of the expression system according to any one of claims 44 to 5184, for production of at least one recombinant casein of interest.61 . Use of the methods according to any one of claims 53 to 55 for production of at least one recombinant casein of interest.

62. A food product (including drinks) or a functional food product or a dairy product comprising recombinant casein produced according to any one of claims 1 to 55.

63. Use of the recombinant casein produced according to any one of claims 1 to 55 as a food ingredient / for preparing yoghurt / cheese.

64. A micelle comprising the recombinant casein produced according to any one of claims 1 to 55.

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