Methods and compositions for the generation of dairy proteins in microbial systems

Abstract

Methods and compositions are disclosed for a precision fermentation system that provides efficient production of secreted dairy proteins. Disclosed are high yielding expression constructs, microbial cells, resulting dairy proteins, and products obtained therefrom.

Description

METHODS AND COMPOSITIONS FOR THE GENERATIONOF DAIRY PROTEINS IN MICROBIAL SYSTEMSRelated application

[0001] This application claims the benefit of United States provisional patent application number 63 / 535,043, filed 28 August 2023, the entire contents of which are incorporated by reference herein.Ffeki

[0002] The present disclosure relates generally to the generation of dairy proteins in microbial-based expression systems. Further related are methods and compositions employed in the production these proteins, the isolated proteins, and the products obtained therefrom.Background

[0003] The environmental impact of the dairy industry is significant. In 2015, the industry's emissions were equivalent to more than 1,700 million tons of CO2 and made up 3.4% of the world's total.1Dairy's contribution to global greenhouse gas emissions is close to that from aviation and shipping combined (which are 1.9% and 1.7% respectively)2. In New Zealand, it contributes nearly a quarter (22.4%) of greenhouse gas emissions.3

[0004] As the global demand for dairy continues to increase, the need for a more sustainable approach to dairy production becomes more important. Generating bovine milk proteins, such as caseins and whey proteins, using synthetic biology and precision fermentation has the potential to drastically reduce the greenhouse gas emissions per unit of protein. It is the aim of the inventors to meet these needs or at least to provide the public with a useful alternative.Summary

[0005] All of the embodiments described in the following clause list are contemplated for use in accordance with the present disclosure. For all of the embodiments described in the following clauses, any applicable combination of embodiments is considered to be in accordance with the present disclosure. Any embodiment described in the following clause list is also contemplated for use with any embodiment described in the summary section or in the detailed description section of this application.

[0006] Clause 1. A recombinant yeast or filamentous fungal strain, wherein the strain has a genome comprising one or more polynucleotides that encode a P-lactoglobulin protein, wherein the one or more polynucleotides encode a P-lactoglobulin protein that shares at least 95% identity with a mammalian P-lactoglobulin protein, wherein the strain allows expression and secretion of the P-lactoglobulin protein, and wherein at least 80% of the P-lactoglobulin protein that is expressed and secreted is correctly processed at both N and C terminal ends.

[0007] Clause 2. The recombinant yeast or filamentous fungal strain of clause 1, wherein said strain comprises multiple copies of a polynucleotide that encodes the P-lactoglobulin protein.

[0008] Clause 3. The recombinant yeast or filamentous fungal strain of clause 2, wherein said strain comprises one, two, three, four, five, six, seven, eight, or nine copies of the polynucleotide that encodes the P-lactoglobulin protein.

[0009] Clause 4. The recombinant yeast or filamentous fungal strain of clauses 1-3, wherein said strain further comprises one, two, three, or more promoter sequences which are operably linked to the one or more polynucleotides that encode the P-lactoglobulin protein.

[0010] Clause 5. The recombinant yeast or filamentous fungal strain of clause 4, wherein said one, two, three, or more promoter sequences comprise one or more inducible promoters.

[0011] Clause 6. The recombinant yeast or filamentous fungal strain of clause 5, wherein said one, two, three, or more promoter sequences comprise one or more inducible promoters selected from the group consisting of: AOX1, FDH, and HpFMD.

[0012] Clause 7. The recombinant yeast or filamentous fungal strain of clause 6, wherein said one or more inducible promoters comprise the AOX1 promoter.

[0013] Clause 8. The recombinant yeast or filamentous fungal strain of clause 6, wherein said one or more inducible promoters comprise the FDH promoter.

[0014] Clause 9. The recombinant yeast or filamentous fungal strain of clause 6, wherein said one or more inducible promoters comprise the HpFMD promoter.

[0015] Clause 10. The recombinant yeast or filamentous fungal strain of clause 6, wherein said one or more inducible promoters comprise the AOX1 promoter and at least one of the following promoters: FDH and HpFMD.

[0016] Clause 11. The recombinant yeast or filamentous fungal strain of clause 6, wherein said one or more inducible promoters comprise the FDH promoter and at least one of the following promoters: AOX1 and HpFMD.

[0017] Clause 12. The recombinant yeast or filamentous fungal strain of clause 6, wherein said one or more inducible promoters comprise the HpFMD promoter and at least one of the following promoters: FDH and AOXl.

[0018] Clause 13. The recombinant yeast or filamentous fungal strain of clause 6, wherein said one or more inducible promoters comprise all of the following promoters: AOX1, FDH and HpFMD.

[0019] Clause 14. The recombinant yeast or filamentous fungal strain of clause 4, wherein said one, two, three, or more promoter sequences comprise a constitutive promoter.

[0020] Clause 15. The recombinant yeast or filamentous fungal strain of clause 14, wherein said constitutive promoter comprises the UPP promoter.

[0021] Clause 16. The recombinant yeast or filamentous fungal strain of clause 4, wherein said wherein said one, two, three, or more promoter sequences comprise a combination of a constitutive promoter and one or more inducible promoters.

[0022] Clause 17. The recombinant yeast or filamentous fungal strain of clause 16, wherein said one, two, three, or more promoter sequences comprise an UPP constitutive promoter and an AOX1 inducible promoter.

[0023] Clause 18. The recombinant yeast or filamentous fungal strain of clause 16, wherein said one, two, three, or more promoter sequences comprise a combination of an UPP constitutive promoter and an FDH inducible promoter.

[0024] Clause 19. The recombinant yeast or filamentous fungal strain of clause 16, wherein said one, two, three, or more promoter sequences comprise a combination of an UPP constitutive promoter and a HpFMD inducible promoter.

[0025] Clause 20. The recombinant yeast or filamentous fungal strain of clause 16, wherein said one, two, three, or more promoter sequences comprise a combination of an UPP constitutive promoter and an inducible promoter selected from the group consisting of: FDH, HpFMD, AOX1.

[0026] Clause 21. The recombinant yeast or filamentous fungal strain of clause 16, wherein said one, two, three, or more promoter sequences comprise a combination of an UPP constitutive promoter and an inducible promoter selected from the group consisting of: FDH, AOX1.

[0027] Clause 22: The recombinant yeast or filamentous fungal strain of clause 16, wherein said one, two, three, or more promoter sequences comprise a combination of an UPP constitutive promoter and an inducible promoter comprising: AOX1.

[0028] Clause 23. The recombinant yeast or filamentous fungal strain of clauses 1-22, wherein said strain further comprises one or more polynucleotide sequences encoding one or more transcription factors, chaperones, or other helper factors.

[0029] Clause 24. The recombinant yeast or filamentous fungal strain of clause 23, wherein said one or more transcription factors, chaperones or other helper factors are selected from the group consisting of: MXR1, Sec53, RPP0, KAR2, PDH, and Had.

[0030] Clause 24.1. The recombinant yeast or filamentous fungal strain of clauses 23-24, wherein said strain further comprises one or more promoter sequences which are operably linkedto the one or more polynucleotide sequences encoding one or more transcription factors, chaperones or other helper factors.

[0031] Clause 24.2. The recombinant yeast or filamentous fungal strain of clause 24.1, wherein said one or more promoter sequences are selected from the group consisting of: AOX2, PEX11, and FLD1.

[0032] Clause 25. The recombinant yeast or filamentous fungal strain of clauses 1-24.2, wherein said strain further comprises one or more polynucleotide sequences encoding one or more secretion signals which are operably linked to the one or more polynucleotides that encode the P- lactoglobulin protein.

[0033] Clause 26. The recombinant yeast or filamentous fungal strain of clause 25, wherein said one or more secretion signals are selected from the group consisting of: sO, si, si.5, s3, s4, s5, s6, and s9.

[0034] Clause 27. The recombinant yeast or filamentous fungal strain of clause 25, wherein said one or more secretion signals comprise sO.

[0035] Clause 28. The recombinant yeast or filamentous fungal strain of clause 25, wherein said one or more secretion signals comprise sl.5.

[0036] Clause 29. The recombinant yeast or filamentous fungal strain of clause 25, wherein said one or more secretion signals comprise s6 .

[0037] Clause 30. The recombinant yeast or filamentous fungal strain of clause 25, wherein said one or more secretion signals comprise s9.

[0038] Clause 31. The recombinant yeast or filamentous fungal strain of clauses 2-30, which has a genome comprising multiple copies of the polynucleotide that encodes a P -lactoglobulin protein, wherein each copy is operably linked to: a promoter sequence, a signal sequence (e.g., secretion signal), and a transcription terminator.

[0039] Clause 32. The recombinant yeast or filamentous fungal strain of clause 31, wherein said multiple copies are integrated at multiple loci of the genome.

[0040] Clause 33. The recombinant yeast or filamentous fungal strain of clause 2-31 which has a genome comprising multiple copies of the polynucleotide that encodes a P-lactoglobulin protein integrated at multiple loci within the genome.

[0041] Clause 33.1. The recombinant yeast or filamentous fungal strain of clauses 32-33, wherein the multiple loci are selected from the group consisting of: A0X1, FDH, UPP, Tl, T6, and T9.

[0042] Clause 33.2. The recombinant yeast or filamentous fungal strain of clause 33.1, wherein T-sites comprise any one of SEQ ID NOS: 9, 12, or 14.

[0043] Clause 34. The recombinant yeast or filamentous fungal strain of clauses 1-33.2, wherein said strain is a Trichoderma, a Pichia, a Kluyveromyces, or a Komagataella strain.

[0044] Clause 35. The recombinant yeast or filamentous fungal strain of clauses 1-33.2, wherein said strain is a Trichoderma, a Pichia, or a Kluyveromyces strain.

[0045] Clause 36. The recombinant yeast or filamentous fungal strain of clauses 1-34, wherein said strain is Trichoderma reesei.

[0046] Clause 37. The recombinant yeast or filamentous fungal strain of clauses 1-34, wherein said strain is Kluyveromyces lactis.

[0047] Clause 38. The recombinant yeast or filamentous fungal strain of clauses 1-34, wherein said strain is Pichia pastoris.

[0048] Clause 39. A recombinant expression vector comprising a polynucleotide that encodes a P -lactoglobulin protein, wherein the vector allows for insertion of the polynucleotide into a genome of a host cell, wherein the polynucleotide encodes a P -lactoglobulin protein that shares at least 95% identity with a mammalian P-lactoglobulin protein, wherein the vector allows expression and secretion of the P-lactoglobulin protein from the host cell, and wherein at least 80% of the P- lactoglobulin protein that is expressed and secreted is correctly processed at both N and C terminal ends.

[0049] Clause 40. The recombinant expression vector of clause 39, further comprising multiple copies of the polynucleotide that encodes the P-lactoglobulin protein.

[0050] Clause 41. The recombinant expression vector of clause 40, wherein said multiple copies comprise 1 to 3 copies or 2 to 4 copies.

[0051] Clause 42. The recombinant expression vector of clauses 39-41, wherein said recombinant expression vector further comprises one, two, three, or more promoter sequences which are operably linked to the one or more polynucleotides that encode the P -lactoglobulin protein.

[0052] Clause 43. The recombinant expression vector of clause 42, wherein said one, two, three, or more promoter sequences comprise one or more inducible promoters.

[0053] Clause 44. The recombinant expression vector of clause 43, wherein said one, two, three, or more promoter sequences comprise one or more of the following inducible promoters: AOX1, FDH, and HpFMD.

[0054] Clause 45. The recombinant expression vector of clause 43, wherein said one or more inducible promoters comprise the AOX1 promoter.

[0055] Clause 46. The recombinant expression vector of clause 43, wherein said one or more inducible promoters comprise the FDH promoter.

[0056] Clause 47. The recombinant expression vector of clause 43, wherein said one or more inducible promoters comprise the HpFMD promoter.

[0057] Clause 47.1. The recombinant expression vector of clause 43, wherein said one or more inducible promoters comprise the AOX1 promoter and at least one of the following promoters: FDH and HpFMD.

[0058] Clause 47.2. The recombinant expression vector of clause 43, wherein said one or more inducible promoters comprise the FDH promoter and at least one of the following promoters: AOX1 and HpFMD.

[0059] Clause 47.3. The recombinant expression vector of clause 43, wherein said one or more inducible promoters comprise the HpFMD promoter and at least one of the following promoters: FDH and AOX1.

[0060] Clause 47.4. The recombinant expression vector of clause 43, wherein said one or more inducible promoters comprise all of the following promoters: AOX1, FDH and HpFMD.

[0061] Clause 48. The recombinant expression vector of clause 42, wherein said one, two, three, or more promoter sequences comprise a constitutive promoter.

[0062] Clause 49. The recombinant expression vector of clause 48, wherein said constitutive promoter comprises the UPP promoter.

[0063] Clause 50. The recombinant expression vector of clause 42, wherein said one, two, three, or more promoter sequences comprise a combination of a constitutive promoter and one or more inducible promoters.

[0064] Clause 51. The recombinant expression vector of clause 50, wherein said one, two, three, or more promoter sequences comprise an UPP constitutive promoter and an AOX1 inducible promoter.

[0065] Clause 52. The recombinant expression vector of clause 50, wherein said one, two, three, or more promoter sequences comprise an UPP constitutive promoter and an FDH inducible promoter.

[0066] Clause 53. The recombinant expression vector of clause 50, wherein one, two, three, or more promoter sequences comprise an UPP constitutive promoter and a HpFMD inducible promoter.

[0067] Clause 54. The recombinant expression vector of clause 50, wherein said one, two, three, or more promoter sequences comprise a UPP constitutive promoter and an inducible promoter selected from the group consisting of: FDH, HpFMD, AOX1.

[0068] Clause 55. The recombinant expression vector of clause 50, wherein said one, two, three, or more promoter sequences comprise a UPP constitutive promoter and an inducible promoter selected from the group consisting of: FDH, AOX1.

[0069] Clause 56. The recombinant expression vector of clause 39-55, wherein said recombinant expression vector further comprises one or more polynucleotide sequences encodingone or more secretion signals which are operably linked to the one or more polynucleotides that encode the P -lactoglobulin protein.

[0070] Clause 57. The recombinant expression vector of clause 56, wherein the one or more secretion signals are selected from the group consisting of: sO, si, si.5, s3, s4, s5, s6 and s9.

[0071] Clause 58. The recombinant expression vector of clause 57, wherein the one or more secretion signals comprise sO.

[0072] Clause 59. The recombinant expression vector of clause 57, wherein the one or more secretion signals comprise si.5.

[0073] Clause 60. The recombinant expression vector of clause 57, wherein the one or more secretion signals comprise s9.

[0074] Clause 61. The recombinant expression vector of clause 57, wherein the one or more secretion signals comprise s6.

[0075] Clause 62. The recombinant expression vector of clauses 39-61, wherein said recombinant expression vector further comprises a polynucleotide sequence homologous to a locus in the genome of the host cell.

[0076] Clause 63. The recombinant expression vector of clause 62, wherein said host cell is selected from the group consisting of: Pichia pastoris (Komagataella phaffii), Trichoderma reesei, and Kluyveromyces lactis.

[0077] Clause 63.1. The recombinant expression vector of clauses 62-63, wherein said host cell is a Pichia pastoris (Komagataella phaffii) cell.

[0078] Clause 64. The recombinant expression vector of clauses 62-63.1., wherein said locus is selected from the group consisting of: A0X1, FDH, UPP, Tl, T6, and T9 .

[0079] Clause 65. A yeast or filamentous fungal strain, wherein the strain has a genome comprising one or more polynucleotide insertions at one or more loci of said genome, and wherein said one or more polynucleotide insertions are obtained from one or more expression vectors of clauses 39-64.

[0080] Clause 66. A yeast or filamentous fungal strain, wherein the strain expresses and secretes a mammalian P -lactoglobulin protein, wherein the strain has a genome comprising one or more polynucleotide insertions at one or more loci of said genome, and wherein said one or more polynucleotide insertions are obtained from one or more expression vectors of clauses 39-64.

[0081] Clause 66.1. A yeast or filamentous fungal strain with a recombinant genome, wherein said genome comprises one or more targeted polynucleotide inserts, wherein said one or more targeted polynucleotide inserts are obtained from one or more expression vectors of clauses 39-64 and wherein each targeted polynucleotide insert is located at a locus selected from the group consisting of: AOX1, FDH, UPP, Tl, T6, and T9.

[0082] Clause 66.2. A yeast or filamentous fungal strain with a recombinant genome, wherein said genome comprises one or more heterologous polynucleotide inserts, wherein said one or more heterologous polynucleotide inserts are obtained from one or more expression vectors of clauses 39-64 and wherein each heterologous polynucleotide insert is located at a locus selected from the group consisting of: AOX1, FDH, UPP, Tl, T6, and T9.

[0083] Clause 66.3. A yeast or filamentous fungal strain with a recombinant genome of Clause 66.1. or 66.2. 2, wherein the T-sites comprise any one of SEQ ID NOS: 9, 12, or 14.

[0084] Clause 66.4. The yeast or filamentous fungal strain of clauses 65-66.3, wherein said strain is a Trichoderma, a Pichia, a Kluyveromyces, or a Komagataella strain.

[0085] Clause 66.5. The yeast or filamentous fungal strain of clause 66.4, wherein said strain is selected from the group consisting of: Pichia pastoris (Komagataella phaffii), Trichoderma reesei, and Kluyveromyces lactis.

[0086] Clause 66.6. The yeast or filamentous fungal strain of clauses 66.5., wherein said strain is a Pichia pastoris ( Komagataella phaffii ) strain.

[0087] Clause 67. A recombinant host cell that contains integrated within its genome one or more heterologous polynucleotides obtained from one or more expression vectors of clauses 39- 64, wherein said host cell is a yeast or filamentous fungal cell.

[0088] Clause 67.1. A recombinant host cell that contains one or more heterologous polynucleotides obtained from one or more expression vectors of clauses 39-64, wherein said host cell is a yeast or filamentous fungal cell, and wherein said one or more heterologous polynucleotides are integrated at multiple loci within the host cell’s genome.

[0089] Clause 67.2. A recombinant host cell of clauses 67.1, wherein each heterologous polynucleotide is located at a locus selected from the group consisting of: A0X1, FDH, UPP, Tl, T6, and T9.

[0090] Clause 68. The recombinant host cell of clauses 67-67.2, wherein said host cell is a Trichoderma reesei cell.

[0091] Clause 69. The recombinant host cell of clauses 67-67.2, wherein said host cell is a Kluyveromyces lactis cell.

[0092] Clause 71. The recombinant host cell of clauses 67-67.2, wherein said host cell is a Pichia pastoris (Komagataella phaffii) cell.

[0093] Clause 72. A recombinant host cell comprising a genome, integrated within which is one or more sequences of a recombinant expression vector of clauses 39-64.

[0094] Clause 73. A recombinant host cell comprising a genome, in which, integrated at multiple loci is one, two, three, four, or more sequences of a recombinant expression vectors of clauses 39-64.

[0095] Clause 73.1. A recombinant host cell of clause 73, wherein each sequence of a recombinant expression vector of clauses 39-64 is located at a locus selected from the group consisting of: A0X1, FDH, UPP, Tl, T6, and T9.

[0096] Clause 74. The recombinant host cell of clauses 72-73.1, wherein said host cell is a fungal or yeast cell.

[0097] Clause 75. The recombinant host cell of clause 74, wherein said host cell is aTrichoderma reesei cell.

[0098] Clause 76. The recombinant host cell of clause 74, wherein said host cell is a Kluyveromyces lactis cell.

[0099] Clause 78. The recombinant host cell of clause 74, wherein said host cell is a Pichia pastoris ( Komagataella phaffii) cell.

[0100] Clause 79. A P-lactoglobulin protein produced by a recombinant yeast or filamentous fungal strain of clauses 1-38 or 65-66.6, or by a recombinant host cell of clauses 67-78, that is at least 95%, at least 98%, at least 99% or 100% identical in its amino acid sequence to a P- lactoglobulin protein produced by a mammal.

[0101] Clause 80. A P -lactoglobulin protein produced by a recombinant yeast or filamentous fungal strain of clauses 1-38 or 65-66.6, or by a recombinant host cell of clauses 67-78, that is at least 95%, at least 98% at least 99%, or 100% identical in its amino acid sequence to a P- lactoglobulin protein produced by a bovine.

[0102] Clause 81. A P -lactoglobulin protein produced by a recombinant yeast or filamentous fungal strain or host cell of clauses 1-38 or 65-66.6, or by a recombinant host cell of clauses 67- 78, that is at least 95%, at least 98% or at least 99% identical in its amino acid sequence to a P- lactoglobulin protein produced by a goat or sheep.

[0103] Clause 83. A P -lactoglobulin protein produced by a recombinant yeast or filamentous fungal strain, wherein the strain comprises: one or more copies of a P -lactoglobulin encoding nucleic acid sequence operably linked with: one or more promoter sequences; one or more secretion sequences; and one or more transcription terminators.

[0104] Clause 84. A P-lactoglobulin protein produced by a recombinant yeast or filamentous fungal strain or host cell of clauses 1-38 or 65-66.6, or by a recombinant host cell of clauses 67- 78, wherein said protein is secreted into the culture medium.

[0105] Clause 85. A P-lactoglobulin protein produced by a recombinant yeast or filamentous fungal strain or host cell of clauses 1-38 or 65-66.6, or by a recombinant host cell of clauses 67- 78, wherein said protein has the amino acid sequence of SEQ ID NO: 8.

[0106] Clause 86. A P-lactoglobulin protein produced by a recombinant yeast or filamentous fungal strain or host cell of clauses 1-38 or 65-66.6, or by a recombinant host cell of clauses 67- 78, wherein said protein is secreted into the culture medium at a concentration from 0.4 g / L to 20 g / L.

[0107] Clause 87. The P-lactoglobulin protein of clause 86, which is secreted into the culture medium at a concentration selected from 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35 or 40 g / L.

[0108] Clause 88. The P-lactoglobulin protein of clauses 84-87, which is secreted into culture medium during expression, and that is 95-100% identical to a native P-lactoglobulin protein sequence.

[0109] Clause 88.1. The P-lactoglobulin protein of clauses 84-88, which is secreted into culture medium during expression, and that constitutes up to 99.9% of the total protein in the culture medium prior to concentration or purification.

[0110] Clause 88.1. The P-lactoglobulin protein of clauses 86-88.1, which is secreted into culture medium during expression, where at least 80% is correctly processed at both N and C terminal ends.

[0111] Clause 89. A dairy protein produced by a recombinant yeast or filamentous fungal strain, wherein the strain has a genome comprising one or more polynucleotides that encode a dairy protein, wherein the one or more polynucleotides encode a dairy protein that shares at least 95% identity with a mammalian milk protein, wherein the strain allows expression and secretion of the dairy protein, and wherein at least 80% of the dairy protein that is expressed and secreted is correctly processed at both N and C terminal ends.

[0112] Clause 89.1. A dairy protein produced by a recombinant yeast or filamentous fungal strain of clause 89, wherein the one or more polynucleotides encode a P-lactoglobulin protein, and the one or more polynucleotides include the sequence of SEQ ID NO: 7.

[0113] Clause 90. A dairy protein of clauses 89-89.1, wherein the dairy protein is a P- lactoglobulin protein, and wherein the P-lactoglobulin protein is produced from multiple copies of a polynucleotide encoding the P-lactoglobulin protein.

[0114] Clause 90.1. A method of producing a protein, said method comprising the step of culturing in a culture medium a recombinant yeast or filamentous fungal strain of Clauses 1-38 or 65-66.6, or a recombinant host cell of Clauses 67-78, wherein P-lactoglobulin is secreted into the culture medium.

[0115] Clause 91. The dairy protein of clauses 79-90.1, which is used to produce a dairy product selected from the group consisting of: milk, evaporated milk, and milk powder.

[0116] Clause 92. The dairy protein of clauses 79-90.1, which is used to produce a dairy product selected from the group consisting of: milk substitute, evaporated milk substitute, and milk substitute powder.

[0117] Clause 93. The dairy protein of clauses 79-90.1, which is used to produce a dairy product selected from the group consisting of cream, creamer, cocktail mixer, custard, cheese, cream cheese, dessert topping, gelato, ghee, ice cream, milkshake, pudding, butter, spread, sauce, sorbet, smoothies, snack food, yoghurt, yoghurt drink, and frozen yoghurt.

[0118] Clause 94. The dairy protein of clauses 79-90.1, which is used to produce a dairy product selected from the group consisting of infant formula, toddler formula, nutritional supplement, energy supplement, sports supplement, weight gain supplement, weight loss supplement, energy drink, sports drink, weight gain drink, weight loss drink, energy food, sports food, weight gain food, and weight loss food.

[0119] Clause 95. The dairy protein of clauses 79-90.1, which is used to produce a dairy ingredient for one or more of baked goods, beverages, cooked goods, frozen goods, cooking mixes, baking mixes, beverage mixes, and sauce mixes.

[0120] Clause 96. The dairy protein of clauses 79-90.1, which is used to produce a dairy ingredient selected from the group consisting of milk protein concentrate and milk protein isolate.

[0121] Clause 97. The dairy protein of clause 96, wherein the milk protein concentrate is selected from the group consisting of whey protein concentrate, lactoferrin concentrate, a- lactalbumin concentrate, P-lactoglobulin concentrate, glycomacropeptide concentrate, and casein concentrate.

[0122] Clause 98. The dairy protein of clause 96, wherein the milk protein isolate is selected from the group consisting of whey protein isolate, lactoferrin protein isolate, a- lactalbumin protein isolate, P -lactoglobulin protein isolate, glycomacropeptide protein isolate, and casein protein isolate.Brief description of the drawings

[0123] Figures 1A-1D. Effect of gene encodings and secretion signals on P -lactoglobulin yield. Expression trials of three strains per encoding / secretion signal pair were carried out in 96 deep well plates and the supernatants were analyzed by SDS PAGE with Coomassie Blue staining. An example SDS PAGE is shown (A). Equal volumes of supernatant were loaded across all wells. Band intensities were averaged for each encoding / secretion signal pair (B), across all strains for each encoding (C), and across all strains for each secretion signal (D).

[0124] Figures 2A-2B. Effect of secretion signal on P-lactoglobulin yield. Expression trials of 4-6 transformants per secretion signal were carried out in snap top tubes and the supernatants were analyzed by SDS PAGE with Coomassie Blue staining. A representative 1-2 samples per secretion signal are shown for sO, s4, s5 and s6 (A) and for sO, sl.5, and s9 (B). Within each gel, equal volumes of supernatant were loaded across all wells.

[0125] Figures 3 A-3B. Effect of promoter on P-lactoglobulin yield. Expression under the control of the HpFMD promoter vs the A0X1 promoter (A): expression trials were carried out in 96 deep well plates. Expression under the control of the FDH and UPP promoters vs the A0X1 promoter (B): expression trials were carried out in snap top tubes. Supernatants of a representative 1-2 transformants per promoter were analyzed by SDS PAGE with Coomassie Blue staining. Within each gel, equal volumes of supernatant were loaded across all wells.

[0126] Figures 4A-4F. Effect of copy number and curing on P-lactoglobulin yield. Expression trials were carried out in snap top tubes and the supernatants were analyzed by SDS PAGE with Coomassie Blue staining. 2.25 pL (A) or 0.9375 pL (B, C, D, E, F) of supernatant sample was loaded per well. See Tables 9 and 9A for strain descriptions.

[0127] Figures 5A-5E. Effect of different transcription factors, chaperones, and other helper factors on protein yield in 4 copy strains (A and B), 7 copy strains (C and D) and 9 copy strains (E). Expression trials of 4-6 transformants per chaperone were carried out in snap top tubes and the supernatants were analyzed by SDS PAGE with Coomassie Blue staining. Within each gel, equal volumes of supernatant were loaded across all wells. See Tables 9 and 9A for strain descriptions.

[0128] Figures 6A-6G: Parent vector plasmid maps. pJAG (A), pJUG (B), pR-H (C), pGAN (D), pSAH-Tl (E), pSAZ-T6 (F), pSAN-T9 (G).

[0129] Figures 7A-7G: Chaperone and helper factor plasmid maps. pGFsH HAC1 (A), pGPUH PDI1 (B), pGFsH KAR2 (C), pGA2sH KAR2 (D), pGPllsH RPP0 (E), pGPUH SEC53 (F), pGA2sH MXR1 (G).

[0130] Figure 8: pJ(FDH)H plasmid map.

[0131] Figure 9: pJTG-Cre ARS plasmid map.Detailed descriptionDefinitions

[0132] The various examples, embodiments, and aspects as set out herein may be readily combined, without departing from the scope or spirit of the invention. Thus, the phrase “in one example”, “in one embodiment”, or “in one aspect” is not necessarily exclusive of other examples, embodiments, or aspects that are also described. In the same way, the phrase “in another example”, “in another embodiment”, or “in another aspect” is not necessarily exclusive of other examples, embodiments, or aspects that are described.

[0133] As used herein, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0134] As used herein and in the claim(s), the use of the term “or” is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein, “another” may mean at least a second or more.

[0135] As used herein, the term “about” or “approximately” means up to 10% greater than or up to 10% lesser than a particular value, or up to 5% greater than or up to 5% lesser than a particular value.

[0136] Where a set of values or a range is given in the specification, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. Thus, each set of values (e.g., 1, 2, 3, 4, 5) in a range that is specified (e.g., 1 to 5) includes all possible combinations of numerical values between the lowest value and the highest value enumerated and also any range of rational numbers within that range, and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. The numeric values provided in parentheses here are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure in a similar manner.

[0137] As used herein, an “isolated” component (e.g., isolated protein such as isolated P- lactoglobulin) refers to a component that has been purified from (e.g., separated from) other components. An isolated component may have: about 70% purity or greater, about 80% purity or greater, about 90% purity or greater; or, in particular aspects, about 99% purity or greater.

[0138] In each instance herein, in descriptions, embodiments, and examples of the present disclosure, the terms “comprising”, “including”, etc., are to be read expansively, without limitation. Thus, unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as to opposed to an exclusive sense, that is to say in the sense of “including but not limited to”.

[0139] The term “consisting essentially of’, as used herein, may refer to the presence of a component (e.g., a protein such as P -lactoglobulin) in a composition. For example, the component may be at least 80% by weight of the composition, or at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% by weight of the composition (% w / w). For liquids, the component may be at least 80% by volume of the composition volume, or at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% by volume of the composition volume (%v / v).

[0140] As used herein, and unless otherwise indicated, the term “nucleotide” broadly in the present disclosure as a unit consisting of a sugar, base, and one or more phosphate groups, for which the sugar, for example, and without limitation, consists of a ribose, a modified ribose with additional chemical groups attached to one or more atoms of the ribose group, a 2'-deoxyribose, or a modified 2'-deoxyribose with additional chemical groups attached to one or more atoms of the 2'-deoxyribose group, and for which the base, for example, and without limitation, consists of a canonical base, hypermodified base, or non-natural base, as described in the nucleic acid molecule definition above. As used herein, and unless otherwise indicated, the term “polynucleotide” is defined as more than three nucleotides.

[0141] As used herein, and unless otherwise indicated, the term “nucleic acid”, “nucleic acid molecule”, or “nucleic acid sequence”, or “polynucleotide”, refers to any single-stranded or double- stranded nucleic acid molecule including standard canonical bases, hypermodified bases, non-natural bases, or any combination of the bases thereof. For example, and without limitation, the nucleic acid molecule contains the four canonical DNA bases - adenine, cytosine, guanine, and thymine, and / or the four canonical RNA bases - adenine, cytosine, guanine, and uracil. The nucleic acids of the present disclosure may be single-stranded or double- stranded, as specified, or contain portions of both double- stranded or single- stranded sequence.

[0142] As used herein, and unless otherwise indicated, the term “recombinant” is known to the art and when referring to a nucleic acid that has been removed from its naturally occurringenvironment, is substantially free of sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived. The term “recombinant” can be used, e.g., to describe cloned DNA isolates, or a nucleic acid including a chemically synthesized nucleotide analogue. When “recombinant” is used to describe a protein, it can refer to, e.g., a protein that is produced in a cell of a different species or type, as compared to the species or type of cell that produces the protein in nature. “Recombinant” can also be used to describe cells and strains. In illustrative embodiments, recombinant yeast or fungal cells are constructed to contain one or more expression cassettes comprising a heterologous nucleic acid encoding P-lactoglobulin operably linked to one of the promoters described herein using available procedures.

[0143] As used herein, and unless otherwise indicated, the term “expression” means transcription and / or translation of a nucleic acid in a host cell and / or secretion of the resulting translated protein. A fungal or yeast expression system may include, but is not limited to, the yeast or filamentous fungal host cell and the expression vector(s) (e.g., a DNA construct) used to express the protein. The expression vector can contain a promoter described herein and as is available, the promoter is heterologous to the expression vector (i.e., the combination does not occur in nature).

[0144] As used herein, and unless otherwise indicated, the term “expression cassette” means the elements of an expression vector that direct the yeast cell to make RNA. An expression cassette comprises at least regulatory sequences (e.g., a promoter and / or terminator) and a coding sequence for the RNA and protein (i.e., an open reading frame). The open reading frame encodes a coding sequence from the start codon to the stop codon, and may further comprise a secretion signal and a gene of interest (e.g., P-lactoglobulin),

[0145] As used herein, and unless otherwise indicated, the term “transformation” means the transfer of a nucleic acid, or a nucleic acid fragment, into a host cell. Such transformation protocols include, but, are not limited to: electroporation, lithium acetate methods, use of spheroplasts, and inter alia. In some embodiments, the expressed nucleic acid coding sequence can be a heterologous nucleic acid coding sequence. As used herein, a heterologous coding sequence linked to a promoter described herein does not occur in nature.

[0146] As used herein, and unless otherwise indicated, the term “promoter” means a nucleotide sequence typically located upstream from the 5' end of a protein coding sequence thatcontrols the transcription of RNA from DNA, in part, by interacting with various regulatory factors that control transcription. In one embodiment, the promoter may be derived from the same species of fungus or yeast as the fungal or yeast host cell being used for protein expression. In another embodiment, the promoter may be derived from a different fungus or yeast species than the fungal or yeast host cell used for protein expression. In some embodiments, a promoter may include a TATA box sequence that acts as a recognition site to direct initiation of transcription, including, but not limited to one or more transcriptional enhancer elements. The enhancer elements may be proximal or distal to the TATA box sequence and may be in a normal 5' to 3' orientation or may be in a 3' to 5' orientation.

[0147] As used herein, and unless otherwise indicated, the term “operably linked” means functionally linked. As described herein, the promoter can be a constitutive or an inducible promoter, such as a methanol inducible promoter.

[0148] As used herein, and unless otherwise indicated, the term “constitutive promoter” means a promoter that regulates expression of a gene of interest such that expression is continuous and at a relatively constant level.

[0149] As used herein, and unless otherwise indicated, the term “inducible promoter” means a regulated promoter that is turned on in a cell by an external stimulus, such as a chemical, light, a change in temperature, a change in cell density, or a protein, such as a hormone. A methanol inducible promoter can have some constitutive activity, although a methanol inducible promoter has maximal activity when induced in the presence of methanol. Likewise, a constitutive promoter can have some inducible activity, but a “constitutive promoter” as used herein does not have maximal activity when induced in the presence of methanol.

[0150] As used herein, and unless otherwise indicated, the term “regulatory control sequences” refer to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operably linked. Such regulatory control sequences can control the transcription, post-transcriptional events, translation of nucleic acid sequences, and secretion of amino acid sequences. At a minimum, regulatory control sequences comprise all components whose presence is essential for expression, and generally can include promoter, ribosomal binding site, and transcription termination sequence, but can also include additional components whosepresence is advantageous, for example, secretion signals, leader sequences, enhancer sequences; efficient RNA processing signals and fusion partner sequences. In some illustrative embodiments constructs may comprise additional regulatory control sequences, as needed to obtain optimal expression.

[0151] The term “polypeptide”, as used herein, encompasses amino acid chains of any length, wherein the amino acid residues are linked by covalent peptide bonds. “Polypeptide” may refer to a polypeptide that is a purified natural product, or that has been produced partially or wholly using recombinant or synthetic techniques. The term may refer to an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment (e.g., peptide), a native polypeptide, a polypeptide variant, modification, fragment, or derivative thereof. The term “polypeptide” is used interchangeably herein with the term “protein”.

[0152] A “native” polypeptide or protein will have the same amino acid sequence as the naturally occurring polypeptide or protein (e.g., P-lactoglobulin in cow’s milk). A “modified” polypeptide or protein may have one or more amino acid sequence modifications, e.g., deletions, additions, substitutions, or any combination of these. “Modified” polypeptides or proteins may also refer to polypeptides or proteins having one or more post-translational modifications that are not naturally occurring.

[0153] A “dairy protein” as used herein refers to a protein that is normally produced in a mammal’s milk. The mammal may include, for example, a human, cow, sheep, goat, buffalo, bison, oxen, camel, horse, donkey, inter alia. The mammal may be a bovine animal, such as a cow, bison, buffalo, beefalo, or yak. Illustrated embodiments include P-lactoglobulin protein (e.g., P-lactoglobulin B), whey protein, lactoferrin protein, a-lactalbumin protein, glycomacropeptide protein, casein protein, amongst others.

[0154] As used herein, and unless otherwise indicated, the term “dairy identical” protein refers to a protein encoded by a polynucleotide, where the encoded protein has at least 95% identity to the amino acid sequence of a native mammalian milk protein, and where at least 80% of the cell expressed protein is correctly processed at both N and C terminal ends (e.g., not elongated). Dairy identical proteins can be used in food formulations such as, but not limited to, ice cream, yogurts, milks, cheeses, and specialized nutrition products.

[0155] It is understood that, for any DNA molecule disclosed herein, the corresponding RNA molecule and peptide / polypeptide molecules are also encompassed and disclosed. Likewise, for any peptide / polypeptide molecule disclosed herein, the corresponding RNA and DNA sequences are also considered to be encompassed and disclosed. In addition, where there are multiple sequence identifiers, e.g., “SEQ ID NO: 40-47” or “SEQ ID NO: 40 to SEQ ID NO: 47”, this format may be understood as referring to each sequence individually, or any combination thereof.Compositions, methods, and systems

[0156] The present disclosure is related to isolated nucleic acids, strains, host cells, expression methods, expression vectors, and DNA constructs for producing proteins, including P- lactoglobulin (BLG), to the proteins that are produced using the described methods, and to the food products prepared with said proteins.

[0157] While the benefits of microbially-produced dairy proteins are well recognized, the optimum method for production remains undefined. This is true even for P-lactoglobulin, the most common whey protein in bovine milk. Described herein are compositions and methods utilizing a super- secreting recombinant strains, recombinant cells, and cell expression systems for yielding purified dairy proteins, including P-lactoglobulin. The highly effective strains, cells, and systems as described herein lead to cost-effective manufacturing and downstream processing. The resulting products can be used in food formulations such as ice cream, yogurts, milks, cheeses, specialized nutrition, amongst others.

[0158] Described herein, are inter alia, recombinant strains, recombinant cells, and expression systems that secrete a dairy protein (e.g., P-lactoglobulin) that is at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% and up to 100% identical in sequence to the native protein (e.g., bovine P-lactoglobulin). In particular aspects, the recombinant strains, recombinant cells, and expression systems can include multiple copies of the nucleic acid encoding the dairy protein that have been incorporated at multiple loci, under the control of multiple promoters, with or without one or more copies of a transcription factor. Specific systems are described in more detail, below.

[0159] In certain embodiments, the expression system utilizes a recombinant yeast or fungal strain, for example, a filamentous fungus strain. In specific embodiments, the yeast or fungal strain is of the genus Trichoderma, Pichia (Komagataella), or Kluyveromyces. Specifically noted are Trichoderma reesei, Pichia pastoris (Komagataella phaffii), and Kluyveromyces lactis. In an exemplary embodiment, the expression system utilizes the yeast Pichia pastoris.

[0160] In some embodiments, the P. pastoris strain may be Muts(e.g., aoxl-Al), Mut+, or Mutnu11, may be pep4- (e.g., pep4-Al), prbl- (e.g., prbl-Al), sub2- (e.g., sub2-Al), ypsl- (e.g., ypsl-Al), or wildtype for proteases. In some embodiments, the strain may also include nucleic acid sequences encoding one or more additional factors, such as helper factors. These include but are not limited to those encoding transcription factors, chaperones, kinases, foldases, initiation factors, ribosomal components, post translational modification factors. Specific exemplifications include, for example, PDI, PDI1, RPP0, KAR2 / BiP, SLY1, SEC1, HAC1, Erol, Hsp70, MXR1, GCN4, SEC53, Cenl, inter alia.

[0161] To construct the illustrative yeast or fungal strain, a series of plasmids may be constructed and propagated in a microbial cell such as E. coli. Specific construction methodologies are described in more detail in the examples below. Once constructed, one, two, three, four, five or six plasmids, each containing 1, 2, 3 or 4 expression cassettes and each targeting a different locus (e.g., Tl, T6, T9, A0X1, FDH or UPP), may be successively or simultaneously transformed into the selected strain (e.g., P. pastoris). As a result, recombinant strains containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and up to 12 or more copies of the dairy protein encoding gene (e.g., P-lactoglobulin gene) may be constructed.

[0162] In some embodiments, the recombinant strain may be constructed to include one or more polynucleotides encoding a dairy protein (e.g., P-lactoglobulin) that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical in sequence to a native protein present in milk (e.g., bovine P-lactoglobulin protein). In some embodiments the encoded dairy protein has up to 99.1%, up to 99.2%, up to 99.3%, up to 99.4%, up to 99.5%, up to 99.6%, up to 99.7%, up to 99.8%, up to 99.9% sequence identity to a native protein present in milk.

[0163] In some embodiments, the recombinant strain may be constructed to include one or more polynucleotides comprising operably linked promoter sequences. For example, a recombinant strain can include polynucleotides which comprise one or more promoter sequences that direct the expression of the encoded dairy protein. In some embodiments, the promoter sequence comprises an inducible promoter. In some embodiments, the inducible promoter sequence comprises a methanol-inducible promoter. In other embodiments, the inducible promoter sequence comprises the AOX1, FDH or HpFMD promoter. In some embodiments, the promoter sequence comprises a constitutive promoter. In some embodiments, the constitutive promoter sequence comprises the UPP promoter. In some embodiments, the promoter sequences comprise a combination of constitutive promoters. In some embodiments, the promoter sequences comprise a combination of inducible promoters. In some embodiments, the promoter sequences comprise a combination of constitutive and inducible promoters.

[0164] In specific embodiments, the promoter combinations can comprise the AOX1 and the FDH inducible promoters. In some embodiments, the promoter combinations can comprise the AOX1 and the HpFMD inducible promoters. In some embodiments, the promoter combinations can comprise the FDH and the HpFMD inducible promoters. In some embodiments, the promoter combinations can comprise the AOX1, FDH and HpFMD inducible promoters. In some embodiments, the promoter combinations can comprise the UPP constitutive promoter and the AOX1 inducible promoter. In some embodiments, the promoter combinations can comprise the UPP constitutive promoter and the FDH inducible promoter. In some embodiments, the promoter combinations can comprise the UPP constitutive promoter and the HpFMD inducible promoter. In other embodiments, the promoter combinations can comprise the UPP constitutive promoter and one or more of the following inducible promoters: AOX1, FDH, HpFMD.

[0165] In some embodiments, the recombinant strain may be constructed to include one or more polynucleotides with operably linked secretion signals. For example, a recombinant strain can include polynucleotides which comprise one or more signal sequences (e.g., secretion signals) that direct the secretion of the expressed protein. In some embodiments, the secretion signal comprises: sO, si, si.5, s3, s4, s5, s6, s9, or any combination thereof.

[0166] In some embodiments, the recombinant strain may be constructed to include one or more polynucleotides with transcription terminators. For example, a recombinant strain can include polynucleotides which comprise one or more transcription terminators. In some embodiments, the transcription terminator comprises the AOX1 or FDH (e.g., FDH1) transcription terminator.

[0167] In some embodiments, the recombinant strain may be constructed to include one or more one or more transcription factors. For example, a polynucleotide encoding one or more transcription factors can be integrated into a strain along with integration of the expression cassette(s). In some embodiments, the transcription factor comprises MXR1.

[0168] In some embodiments, the recombinant strain may be constructed to further comprise one or more additional factors, such as helper factors. Such one or more additional factors include, but are not limited to, transcription factors, chaperones, kinases, foldases, initiation factors, ribosomal components, post translational modification factors, and other processing related proteins that have a positive effect on dairy protein production. In some embodiments, the one or more additional factors may upregulate or downregulate cellular machinery, such as vacuoles. In some embodiments, the one or more additional factors may be introduced by additional plasmids / vectors with nucleic acid sequences encoding these factors. In some embodiments, the one or more additional factors are integrated into the host’s genome. In some embodiments, the one or more additional factors are integrated into the host’s genome at any of the following loci: AOX2, PEX11, FLD1, or any combination thereof. In some embodiments, the other factors may comprise one or more of the following: MXR1, SEC53, RPP0, KAR2, PDI1, HAC1. In further embodiments, these one or more additional factors may each be operably linked to a promoter sequence. In some embodiments, these one or more additional factors may each be operably linked to one of the following promoter sequences: FLD1, PEX11, or AOX2.

[0169] Certain combinations of copies in a selected strain (e.g., P. pastoris) include, for example, any one or combination of the following: (1) one or more dairy protein (e.g., P- lactoglobulin protein) expression cassettes at the FDH locus; (2) one or more dairy protein expression cassettes at the AOX1 locus; (3) one or more dairy protein expression cassettes at the UPP locus; (4) one to three, or more, dairy protein expression cassettes at the T1 locus; (5) one totwo, or more, dairy protein expression cassettes at the T6 locus; (6) one to three, or more, dairy protein expression cassettes at the T9 locus; wherein each expression cassette contains a promoter (e.g., FDH, HpFMD, UPP or A0X1), a secretion signal (e.g., sO, si.5, s9, or s6), the nucleic acid sequence encoding a dairy protein (e.g., P -lactoglobulin), which may be native or modified, and a transcription terminator (e.g., FDH1 or A0X1).

[0170] Plasmids may be constructed comprising an antibiotic resistance gene and one or more expression cassettes, wherein each expression cassette contains a promoter (e.g., FDH, HpFMD, UPP or AOX1), a secretion signal (e.g., sO, si.5, s9, or s6), the nucleic acid sequence encoding a dairy protein (e.g., P -lactoglobulin), which may be native or modified, and a transcription terminator (e.g., FDH1 or AOX1). A plurality of plasmids (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, up to 10 or more) may be utilized for strain construction, whereby each plasmid is capable of integration into a particular locus in the genome of the strain. These plasmids may be incorporated into the genome using targeted integrated via homologous recombination. The transformants, in which successful integration has been achieved, can be confirmed by selection in media containing the appropriate antibiotic(s).

[0171] At this time, it is possible for the antibiotic resistance marker(s) and any other unwanted DNA remaining to be removed. For example, a cre-lox system may be utilized for sequence removal. Specifically, plasmids used for the strain construction and genomic integration may be designed to contain “lox” sites flanking either side of the unwanted region of DNA. Then, an episomal plasmid may be introduced transiently to express ere, which will loop out the unwanted DNA fragment. In this way, certain integrated plasmid sequences (e.g., one or more plasmid markers) may be removed from the strain. Once the now extraneous sequences are removed, the recombinant strains may be confirmed and confirmed to meet generally regarded as safe (GRAS) guidelines.

[0172] The construction and use of expression vectors and expression cassettes is described, for example, in Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 3rd and 4th Editions, Cold Spring Harbor Laboratory Press, (2001 and 2012, respectively), incorporated herein by reference. In another embodiment, the expression vector, or a fragment thereof, can be synthesized de novo or PCR can be used to amplify sections of expression vectors. PCR fragments,synthesized fragments, and / or backbone vectors can subsequently be joined by cloning techniques, including Gibson Assembly and Golden Gate Assembly.

[0173] As demonstrated herein, select target sites (T-sites) such as those developed and supplied by BioGrammatics, Inc. may be used to facilitate the targeted insertion of DNA into the Pichia genome. In various embodiments, a desired T-site may be targeted by flanking the DNA for insertion by 300-700 bp of DNA that is homologous to the right (5’) and left (3’) ends of the T-site, respectively. For example, this targeting can be used to obtain insertion of an expression cassette or expression cassettes into the host cell. Insertion for integration can happen anywhere within this homologous DNA region, including those specific locations listed in Tables 8 and 8 A. In some embodiments, this may be accomplished by linearizing a plasmid such that the ends of the linearized plasmid contain T-site DNA that is homologous to the right (5’) of the T-site on one side and DNA that is homologous to the left (3’) of the T-site on the other side.

[0174] In some embodiments, these T-sites can be used as insertion sites for expression vectors comprising heterologous dairy protein genes (e.g., P -lactoglobulin). For example, a gene foreign to the host cell can be utilized, although such sequences may also comprise some elements from host cell. In some embodiments, the targeted insertion of DNA at these T-sites may be facilitated by sequence homology in a targeting vector to the T-site. For example, this is illustrated in the plasmid DNA maps for pSAH-Tl, pSAZ-T6, or pSAN-T9 (Figures 6E-6G), which were supplied by BioGrammatics, Inc. Some of the advantages that the use of these T-sites for insertion of DNA into a host cell include, but are not limited to, integration of inserted DNA for a specific application (e.g., heterologous expression of P-lactoglobulin) while minimizing interference with host cell host cell functions, as well as, facilitating proper function of the inserts.

[0175] It should be noted that the Pichia chromosome is numbered uniquely in the Pichia reference genome for BglO (~9.4 million bp total: Chromosome #1 has 2,895,354 bp, #2 has 2,396,458 bp, #3 has 2,263,458 bp and #4 as 1,827,941 bp). The Chromosome and integration site numbers provided in Tables 8 and 8 A identify the base immediately before the T-site integration site used in vectors designed by BioGrammatics, Inc. The broader T-sites encompass 600-1400 bp of DNA that include those identified in Tables 8 and 8A and integration can happen anywhere within this DNA, including at the precise locations listed in Tables 8 and 8 A.

[0176] In specific embodiments, it is possible to obtain vectors that are targeted to three or more T-sites (e.g., from BioGrammatics, Inc.), and allow the expression of different proteins, with or without secretion signals. Particular plasmids may be constructed, e.g., using pSAH-Tl, pSAZ- T6 and pSAN-T9 as exemplified herein. In this way, it is possible to make expression vectors which can contain one, two, three, or more expression cassettes for the expression and secretion of a dairy protein (e.g., P -lactoglobulin) and which can allow for targeted integration at any of the three or more sites (e.g., T6, T9, and Tl). For example, the plasmids may enable integration of the expression cassettes after linearization of the plasmids (e.g., by digestion with Nrul) and a recombination event (e.g., “double” recombination). Using this approach, it is possible to obtain expression of one or more dairy proteins in higher copy numbers.

[0177] In particular aspects, an expression vector can add one, two, three, or more copies of an expression cassette at a first T-site, and at a second T-site, and at a third T-site, and at the A0X1, FDH or UPP loci. Different expression cassettes can be under the control of the same or different regulatory control sequences. This includes, for example, various promoters, secretion signals and transcription terminators. Thus, multiple expression cassettes can be inserted at multiple sites in the genome. For example, multiple copies of a polynucleotide (e.g., P- lactoglobulin polynucleotide) can be inserted at multiple sites in the genome and each copy can be under the control of the same or different regulatory control sequences, including a promoter (e.g., A0X1, FDH, UPP or HpFMD), a secretion signal (e.g., sO, si.5, s6, or s9), and a transcription terminator (e.g., FDH1 or A0X1).

[0178] In specific embodiments, the T-site may comprise a nucleotide sequence selected from the group consisting of SEQ ID NO: 9, 12, 14, or other sequences as described herein. In specific embodiments, the T-site may consist of a nucleotide sequence selected from the group consisting of SEQ ID NO: 9, 12, 14, or other sequences as described herein.

[0179] In some illustrative embodiments, yeast or fungal strains (e.g., P. pastoris) comprise DNA obtained from expression vectors. In some illustrative embodiments, expression vectors may comprise one or multiple expression cassettes, wherein each expression cassette contains a promoter (e.g., FDH, HpFMD, UPP, or A0X1), a secretion signal (e.g., sO, si.5, s9, or s6), the nucleic acid sequence encoding a dairy protein (e.g., P-lactoglobulin), which may be nativeor modified, and a transcription terminator (e.g., FDH1 or A0X1). In some embodiments, expression vectors comprise multiple copies of the polynucleotide sequence that encodes an amino acid sequence that encodes a dairy protein (e.g., P-lactoglobulin protein); one or more promoter sequences, one or more secretion signal sequences (e.g., secretion signals), and one or more transcription terminators.

[0180] In some embodiments, yeast or fungal strains (e.g., in P. pastoris) comprise multiple copies of expression cassettes, wherein each expression cassette contains a promoter (e.g., FDH, HpFMD, UPP, or AOX1), a secretion signal (e.g., sO, si.5, s9, or s6), the nucleic acid sequence encoding a dairy protein (e.g., P -lactoglobulin), which may be native or modified, and a transcription terminator (e.g., FDH1 or AOX1).

[0181] In some embodiments, the yeast or fungal strains (e.g., P. pastoris) comprise multiple copies of the polynucleotide that encodes an amino acid sequence of the dairy protein (e.g., P-lactoglobulin). In some embodiments, the strains comprise multiple copies of the polynucleotide of the dairy protein. In some embodiments, the strains comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or less than 10 copies.

[0182] The recombinant strains of this disclosure may be prepared utilizing the expression vectors and expression cassettes as described herein. In some embodiments, the expression cassette encodes dairy protein (e.g., P-lactoglobulin) that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical in sequence to a native protein present in milk (e.g., bovine P-lactoglobulin protein). In some embodiments the encoded dairy protein has up to 99.1%, up to 99.2%, up to 99.3%, up to 99.4%, up to 99.5%, up to 99.6%, up to 99.7%, up to 99.8%, up to 99.9% sequence identity to a native protein present in milk.

[0183] In some embodiments, the expression cassettes comprise operably linked promoter sequences. For example, an expression cassette can include one or more promoter sequences that direct the expression of the encoded protein. In some embodiments, the promoter sequence comprises an inducible promoter. In some embodiments, the inducible promoter sequence comprises a methanol-inducible promoter. In other embodiments, the inducible promoter sequence comprises the AOX1, FDH or HpFMD promoter. In some embodiments, the promotersequence comprises a constitutive promoter. In some embodiments, the constitutive promoter sequence comprises the UPP promoter. In some embodiments, the promoter sequences comprise a combination of constitutive promoters. In some embodiments, the promoter sequences comprise a combination of inducible promoters. In some embodiments, the promoter sequences comprise a combination of constitutive and inducible promoters.

[0184] In specific embodiments, the promoter combinations can comprise the AOX1 and the FDH inducible promoters. In some embodiments, the promoter combinations can comprise the AOX1 and the HpFMD inducible promoters. In some embodiments, the promoter combinations can comprise the FDH and the HpFMD inducible promoters. In some embodiments, the promoter combinations can comprise the AOX1, FDH and HpFMD inducible promoters. In some embodiments, the promoter combinations can comprise the UPP constitutive promoter and the AOX1 inducible promoter. In some embodiments, the promoter combinations can comprise the UPP constitutive promoter and the FDH inducible promoter. In some embodiments, the promoter combinations can comprise the UPP constitutive promoter and the HpFMD inducible promoter. In other embodiments, the promoter combinations can comprise the UPP constitutive promoter and one or more of the following inducible promoters: AOX1, FDH, HpFMD.

[0185] In some embodiments, the expression cassettes may comprise operably linked secretion signals. For example, an expression cassette can encode one or more secretion signals that direct the secretion of the expressed protein. In some embodiments, the secretion signal comprises: sO, si, si.5, s3, s4, s5, s6, s9, or any combination thereof.

[0186] In some embodiments, expression cassettes may comprise polynucleotides that include transcription terminators. For example, an expression cassette can include one or more transcription terminators. In some embodiments, the transcription terminator comprises the AOX1 or FDH (e.g., FDH1) transcription terminator.

[0187] In some embodiments, the expression cassettes may be used in conjunction with one or more transcription factors. For example, a polynucleotide encoding one or more transcription factors can be integrated into a strain along with integration of the expression cassette(s). In some embodiments, the transcription factor comprises MXR1.

[0188] In some embodiments, the expression vector may comprise a polynucleotide homologous to a polynucleotide in the host’s genome that is used to target directed insertion or integration to that specific locus. In some embodiments, the homologous polynucleotide comprises: a AOX1 promoter sequence, a AOX2 promoter sequence, a FDH promoter sequence, a UPP promoter sequence, a PEX11 promoter sequence, a FLD1 promoter sequence, a T1 locus sequence, a T6 locus sequence, a T9 locus sequence, or any combination thereof. In some embodiments, the expression vector or the expression cassette comprises an insertion site. In some embodiments, a polynucleotide encoding the protein of interest is inserted into the insertion site. In some embodiments, the expression vector facilitates expression of the protein of interest.

[0189] In some embodiments, an insertion site is a site in a nucleic acid which is suitable for directed insertion of a polynucleotide (e.g., a synthetic or exogenous polynucleotide), including but not limited to: an expression cassette, a promoter of interest, or a gene of interest. In some embodiments, an insertion site comprises one or more restriction enzyme sites. In some embodiments, an insertion site is a multi-cloning site. In some embodiments, a multi-cloning site is a short span of a nucleic acid which comprises one or more restriction sites (e.g., Bsal, EcoRI, Xhol, Eco3 II, Sall, Bsal, BstBI, Bsp \ 191, Xmal, BamHI, Swal, AsiSI, Notl, SacII, Nhe , AccI, etc.). In some embodiments, an insertion site is a landing pad. In some embodiments, an insertion site is a landing pad, wherein the landing pad is suitable for recombinase -mediated insertion of a synthetic or exogenous polynucleotide as described in Sambrook et al., ibid, incorporated herein by reference.

[0190] In some embodiments, an insertion site is a 600-1400 bp target site (e.g., T-site as described herein). In some embodiments, the desired T-site is targeted by flanking the DNA for insertion by 300-700 bp of DNA that is homologous to the right (5’) and left (3’) ends of the T- site, respectively. In some embodiments, insertion for integration can happen anywhere within this homologous DNA region. In some embodiments, the plasmids containing various expression cassettes also contain antibiotic resistance genes that are also incorporated into the host cell genome using targeted integrated via homologous recombination. In some embodiments, the successfully transformed cells, in which integration and expression have been achieved can be confirmed by selection in media containing the appropriate antibiotic(s) . In some embodiments,the antibiotic resistance marker and any additional unwanted DNA remaining can be removed. For example, a cre-lox system may be utilized as described herein. Once the extraneous sequences are removed, the transformed strains can be confirmed and confirmed to meet generally regarded as safe (GRAS) guidelines.

[0191] In some embodiments, the expression vector is utilized with a host cell selected from P. pastoris, T. reesei, and K. lactis.

[0192] In some illustrative embodiments, the recombinant yeast or fungal cells may comprise one or more copies of the polynucleotide that encodes a dairy protein of interest (e.g., P- lactoglobulin). In some embodiments, the cells may comprise multiple copies of the polynucleotide that encodes this protein. In some embodiments, the cells may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or less than 10 copies. In specific embodiments, the cells may comprise 4 to 6 copies, 4 to 7 copies, 4 to 8 copies, 4 to 9 copies, 4 to 10 copies, 4 to 11 copies, or more. In specific embodiments, the cells may comprise 2 to 3 copies, 2 to 4 copies, 3 to 4 copies, 3 to 5 copies, 4 to 5 copies, 4 to 6 copies, 5 to 6 copies, 5 to 7 copies, 6 to 7 copies, 6 to 8 copies, 6 to 9 copies, 7 to 9 copies, 7 to 10 copies, 7 to 11 copies, 9 to 11 copies, or more.

[0193] In some embodiments, the recombinant cells may comprise one or more operably linked promoter sequences to achieve expression of the dairy protein (e.g., P -lactoglobulin). For example, the cells may comprise multiple integrated expression cassettes, where each expression cassette may include one or more promoter sequences that direct the expression of the encoded protein. In some embodiments, the promoter sequence comprises an inducible promoter. In some embodiments, the cells comprise an inducible promoter sequence that comprises a methanolinducible promoter. In other embodiments, the inducible promoter sequence comprises the A0X1, FDH or HpFMD promoter. In some embodiments, a promoter sequence comprises a constitutive promoter. In some embodiments, the constitutive promoter sequence comprises the UPP promoter. In some embodiments, the cells comprise a combination of promoter sequences. In some embodiments, the promoter sequences comprise a combination of constitutive promoters. In some embodiments, the promoter sequences comprise a combination of inducible promoters. In someembodiments, the promoter sequences comprise a combination of constitutive and inducible promoters.

[0194] In specific embodiments, the promoter combinations can comprise the AOX1 and the FDH inducible promoters. In some embodiments, the promoter combinations can comprise the AOX1 and the HpFMD inducible promoters. In some embodiments, the promoter combinations can comprise the FDH and the HpFMD inducible promoters. In some embodiments, the promoter combinations can comprise the AOX1, FDH and HpFMD inducible promoters. In some embodiments, the promoter combinations can comprise the UPP constitutive promoter and the AOX1 inducible promoter. In some embodiments, the promoter combinations can comprise the UPP constitutive promoter and the FDH inducible promoter. In some embodiments, the promoter combinations can comprise the UPP constitutive promoter and the HpFMD inducible promoter. In other embodiments, the promoter combinations can comprise the UPP constitutive promoter and one or more of the following inducible promoters: AOX1, FDH, HpFMD.

[0195] In some embodiments, the recombinant cells may comprise at least one polynucleotide that encodes a transcription factor. For example, a polynucleotide encoding one or more transcription factors can be integrated into a cell along with integration of the expression cassette(s). In some embodiments, the transcription factor comprises MXR1.

[0196] In some embodiments, the recombinant cells may comprise at least one polynucleotide that encodes a secretion signal. For example, an expression cassette that is integrated into the cell can include one or more secretion signals. In some embodiments, the secretion signal comprises: sO, si, si.5, s3, s4, s5, s6, or s9, or any combination thereof.

[0197] In some embodiments, the recombinant cells may comprise at least one polynucleotide that comprises a transcription terminator. For example, an expression cassette that is integrated into the cell can include one or more transcription terminators. In some embodiments, the transcription terminator comprises the AOX1 or FDH (e.g., FDH1) transcription terminator.

[0198] In some embodiments, the recombinant cells may further comprise one or more additional factors, such as helper factors. These can assist with production of the dairy protein (e.g., P -lactoglobulin). Such factors include, but are not limited to, transcription factors,chaperones, kinases, foldases, initiation factors, ribosomal components, post translational modification factors, and other processing related proteins that have a positive effect on dairy protein production. In some embodiments, the one or more additional factors may upregulate or downregulate cellular machinery, such as vacuoles. In some embodiment, the one or more additional factors may be introduced by additional plasmids / vectors with nucleic acid sequences encoding these factors. In some embodiments, the one or more additional factors are integrated into the host’s genome. In some embodiments, the one or more additional factors are integrated into the host’s genome at any of the following loci: A0X2, PEX11, FLD1, or any combination thereof. In some embodiments, the one or more additional factors may include one or more of the following: MXR1, SEC53, RPPO, KAR2, PDI1, HAC1. In further embodiments, the one or more additional factors may each be operably linked to a promoter sequence. In some embodiments, the one or more additional factors may each be operably linked to one of the following promoter sequences : FLD 1 , PEX 11 , or A0X2.

[0199] In some embodiments, any bacterial DNA, unwanted DNA, and / or antibiotic resistance markers may be removed from integrated plasmids / vectors in the host’s genome using a cre-lox system as described herein. Other available systems may also be used.

[0200] In some embodiments, a bioreactor, fermenter, or other vessel includes a sensor and / or a control system to measure and / or adjust reaction parameters. Non-limiting examples of reaction parameters include biological parameters (e.g., growth rate, cell size, cell number, cell density, cell type, or cell state, etc.), chemical parameters (e.g., pH, redox-potential, feed rates, concentration of reaction substrate and / or product, concentration of dissolved gases, nutrient concentrations, metabolite concentrations, etc.), physical / mechanical parameters (e.g., density, conductivity, degree of agitation, pressure, and flow rate, stir rate, etc.).

[0201] The cell expression systems of this disclosure may be prepared utilizing the expression vectors, expression cassettes, and host cells as described herein. In some illustrative embodiments, a dairy protein (e.g., P-lactoglobulin) expressing and secreting system can include recombinant cells (e.g., cells from a recombinant yeast or fungal strain) that comprise one or multiple copies of a polynucleotide that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% identical insequence to a native dairy protein encoding gene. In some embodiments the polynucleotide has up to 99.1%, up to 99.2%, up to 99.3%, up to 99.4%, up to 99.5%, up to 99.6%, up to 99.7%, up to 99.8%, up to 99.9% sequence identity to a native dairy protein encoding gene.

[0202] In specific embodiments, a dairy protein that is secreted comprises the amino acid sequence of SEQ ID NO: 8. In specific embodiments, a dairy protein that is secreted consists of the amino acid sequence of SEQ ID NO: 8. In specific embodiments, a dairy protein that is secreted is encoded by a nucleotide sequence comprising SEQ ID NO: 7. In specific embodiments, a dairy protein that is secreted is encoded by a nucleotide sequence consisting of or consisting essentially of SEQ ID NO: 7.

[0203] In some illustrative embodiments, the cell expression system can utilize multiple copies of the polynucleotide, which can be integrated at different loci within the genome. In some embodiments, a dairy protein encoding sequence is part of an expression cassette, wherein each expression cassette contains a promoter (e.g., FDH, HpFMD, UPP, or A0X1), a secretion signal (e.g., sO, si.5, s9, or s6), the gene sequence encoding the dairy protein (e.g., P -lactoglobulin), which may be native or modified, and a transcription terminator (e.g., FDH1 or A0X1).

[0204] In some embodiments, the cell expression system can utilize a dairy protein encoding gene sequence (e.g., P -lactoglobulin), which may be part of plasmid / vector, wherein each plasmid / vector contains one or more expression cassettes. In some embodiments, each gene is expressed under the control of one of the following promoters: FDH, UPP, HpFMD, A0X1. In some embodiments, the expression of all of the gene sequences is under the control of the FDH promoter alone or in combination with any of the following promoters: UPP, HpFMD and AOX1. In alternative embodiments, the expression of all of the gene sequences is under the control of the UPP promoter alone or in combination with any of the following promoters: FDH, HpFMD and AOX1. In alternative embodiments, the expression of all of the gene sequences is under the control of the HpFMD promoter alone or in combination with any of the following promoters: UPP, FDH and AOX1. In yet other embodiments, the expression of all of the gene sequences is under the control of the AOX1 promoter alone or in combination with any of the following promoters: UPP, HpFMD and FDH.

[0205] In other illustrative embodiments, the cell expression system can utilize expression of each integrated gene sequence (e.g., P -lactoglobulin gene sequence), which is under the control of one of the following promoters, and at least two of the following promoters are used: FDH, UPP, HpFMD and A0X1. In some embodiments, the expression of all of the gene sequences is under the control of the FDH promoter and one or more of the following promoters: UPP, HpFMD and AOX1. In other embodiments, the expression of all of the gene sequences is under the control of the UPP promoter and one or more of the following promoters: FDH, HpFMD and AOX1. In additional embodiments, the expression of all of the gene sequences is under the control of the HpFMD promoter and one or more of the following promoters: UPP, FDH and AOX1. In more embodiments, the expression of all of the gene sequences is under the control of the AOX1 promoter and one or more of the following promoters: UPP, HpFMD and FDH. In additional embodiments, the expression of all of the gene sequences is under the control of the HpFMD and AOX1 promoters. In additional embodiments, the expression of all of the gene sequences is under the control of the FDH and AOX1 promoters. In additional embodiments, the expression of all of the gene sequences is under the control of the HpFMD, FDH, and AOX1 promoters.

[0206] In specific embodiments, where multiple copies of the gene sequence (e.g., P- lactoglobulin gene sequence) are integrated in the genome of the cell (e.g., at least 4, at least 5, at least 6, at least 7 copies), the expression of the P -lactoglobulin protein may be obtained by at least two different promoters selected from the group consisting of: AOX1, FDH, and HpFMD, or the expression of the P-lactoglobulin protein may be obtained by at least three different promoters selected from the group consisting of: AOX1, FDH, and HpFMD.

[0207] In specific embodiments, a promoter that is utilized comprises the nucleotide sequence of SEQ ID NO: 33, 34, 35, 36, 48 or 49. In specific embodiments, a promoter that is utilized consists of the nucleotide sequence of SEQ ID NO: 33, 34, 35, 36, 48, or 49.

[0208] In specific embodiments, a secretion signal that is utilized comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 40-47. In specific embodiments, a secretion signal that is utilized consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 40-47. In specific embodiments, a secretion signal that is utilized comprises or consists of the amino acid sequence of SEQ ID NO: 40.

[0209] In specific embodiments, a secretion signal that is utilized is encoded by a nucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 25- 32. In specific embodiments, a secretion signal that is utilized is encoded by a nucleotide sequence consisting of a sequence selected from the group consisting of SEQ ID NO: 25-32. In specific embodiments, a secretion signal that is utilized is encoded by a nucleotide sequence comprising or consisting of SEQ ID NO: 25.

[0210] In some illustrative embodiments, the cell expression system can utilize one, two, three, or more copies of the gene sequence (e.g., P -lactoglobulin gene sequence), which are distributed at any one, or combination of, the following loci in the cell expression system (e.g., in P. pastoris): FDH, UPP, AOX1, Tl, T6 and T9. In some embodiments, each cell has a total of between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more, copies of the gene sequence. In some embodiments, the cells may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or less than 10 copies. In specific embodiments, the cells may comprise 4 to 6 copies, 4 to 7 copies, 4 to 8 copies, 4 to 9 copies, 4 to 10 copies, 4 to 11 copies, or more. In specific embodiments, the cells may comprise 2 to 3 copies, 2 to 4 copies, 3 to 4 copies, 3 to 5 copies, 4 to 5 copies, 4 to 6 copies, 5 to 6 copies, 5 to 7 copies, 6 to 7 copies, 6 to 8 copies, 6 to 9 copies, 7 to 9 copies, 7 to 10 copies, 7 to 11 copies, 9 to 11 copies, or more.

[0211] In specific embodiments, where multiple copies of the gene sequence (e.g., P- lactoglobulin gene sequence) are integrated in the genome of the cell (e.g., at least 4, at least 5, at least 6, at least 7 copies), each copy of the gene sequence may be integrated in different loci in the genome; or the copies of the gene sequence may be integrated in at least two different loci in the genome; or the copies of the gene sequence may be integrated in at least three different loci in the genome; or the copies of the gene sequence may be integrated in at least four different loci in the genome, or more.

[0212] In some embodiments, the cell expression system may contain one or more copies of a transcription factor. For example, a polynucleotide encoding one or more transcription factors can be integrated into a cell along with integration of the expression cassette(s). In some embodiments, the transcription factor comprises the MXR1 transcription factor. In furtherembodiments, the transcription factor is under the control of the A0X2 promoter or the A0X1 promoter. In further embodiments, the transcription factor is located at the A0X2 locus or the A0X1 locus.

[0213] In some embodiments, the cell expression system may comprise at least one polynucleotide that encodes a secretion signal. For example, an expression cassette that is integrated into the cell can include one or more secretion signals. In some embodiments, the secretion signal comprises: sO, si, si.5, s3, s4, s5, s6, or s9, or any combination thereof.

[0214] In some embodiments, the cell expression system may comprise at least one polynucleotide that comprises a transcription terminator. For example, an expression cassette that is integrated into the cell can include one or more transcription terminators. In some embodiments, the transcription terminator comprises the AOX1 or FDH (e.g., FDH1) transcription terminator.

[0215] In some embodiments, the cell expression system may also comprise one or more additional factors to assist with production of the dairy protein (e.g., P -lactoglobulin). Such factors include, but are not limited to, additional plasmids / vectors with nucleic acid sequences encoding transcription factors, chaperones, kinases, foldases, initiation factors, ribosomal components, post translational modification factors, and other processing related proteins that have a positive effect on dairy protein expression. In some embodiments, the one or more additional factors may upregulate or downregulate cellular machinery, such as vacuoles. In some embodiments, the one or more additional factors are integrated into the host’s genome. In some embodiments, the one or more additional factors are integrated into the host’s genome at any of the following loci: AOX2, PEX11, FLD1, or any combination thereof. In some embodiments, the other factors may comprise one or more of the following: MXR1, SEC53, RPP0, KAR2, PDI1, HAC1. In further embodiments, the one or more additional factors may each be operably linked to a promoter sequence. In some embodiments, the one or more additional factors may each be operably linked to one of the following promoter sequences: FLD1, PEX11, and AOX2.

[0216] In some embodiments, the cell expression system has multiple copies of the corresponding gene integrated at different loci within its genome, and it secretes dairy protein (e.g., P -lactoglobulin) that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%,at least 99%, or 100% identical in sequence to the native protein present in milk (e.g., bovine P- lactoglobulin protein). In some embodiments the secreted dairy protein has up to 99.1%, up to 99.2%, up to 99.3%, up to 99.4%, up to 99.5%, up to 99.6%, up to 99.7%, up to 99.8%, up to 99.9% sequence identity to a native dairy protein, e.g., a bovine, a goat, a sheep, or a human dairy protein (e.g., bovine P -lactoglobulin protein).

[0217] In some embodiments, the expression system secretes dairy protein (e.g., P- lactoglobulin) that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% properly processed at the N-terminus; for example, with no additional amino acids present in the secreted protein when compared to the native amino acid sequence (e.g., mature bovine P- lactoglobulin protein).

[0218] In other embodiments, the expression system secretes dairy protein (e.g., P- lactoglobulin) that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% properly processed at the C-terminus, for example, with no additional amino acids present in the secreted protein when compared to the native amino acid sequence (e.g., mature bovine P- lactoglobulin). Thus in some embodiments, the expression system secretes dairy protein that is not elongated and is dairy identical.

[0219] In specific embodiments, the expression system (or recombinant strain or recombinant cell) may secrete a pool of dairy proteins where 0.5% to 2%, 0.5% to 3%, 0.5% to 5%, 0.5% to 10%, 0.5% to 15%, or 0.5% to 20%, or more of the dairy proteins in the pool have one or more sequence alterations (e.g., deletions, insertions, and / or substitutions) and / or one or more post-translational modifications (e.g., additions or deletions for glycations, glycosylations, phosphorylations, methylations, lauroylations, etc.) which differ from the native protein in milk. For example, 0.5% to 5% or more of the P-lactoglobulin proteins secreted in the pool may include one or more deletions (e.g., missing LIVTQ (SEQ ID NO: 50) at the N-terminal end) and / or one or more post-translational modifications (e.g., glycation with a monosaccharide and / or disaccharide) which differ from the native protein in milk (e.g., bovine P-lactoglobulin).

[0220] In some embodiments, the cell expression system that has multiple copies of the dairy protein encoding gene integrated at different loci within its genome, secretes the dairy protein (e.g., P-lactoglobulin) at a concentration of (titer) of equal to or more than 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 , 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40 grams, or more, per (liter) liter of culture media prior to concentration or purification.

[0221] In some embodiments, the cell expression system that has multiple copies of the corresponding gene integrated at different loci within its genome, secretes dairy protein that constitutes equal to or more than 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the protein present in culture media prior to concentration or purification.

[0222] In specific embodiments, the secreted dairy protein (e.g., P-lactoglobulin protein) constitutes at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of total protein that is secreted by the cell expression system (or the recombinant strain or the recombinant cell). In specific embodiments, the P-lactoglobulin protein as isolated from cell expression system (or the recombinant strain or the recombinant cell) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90% purity, at least 95% purity, or at least 99% purity.

[0223] In some embodiments, the cell expression system is utilized with a host cell selected from P. pastoris, T. reesei, and K. lactis.

[0224] In some embodiments, the host cell is Mut+, Muts(aoxl-Al), or Mutnu11. In some embodiments the secreting host cell is wildtype for proteases or has any or may have all of the following genotypes: pep4- (e.g., pep4-Al), prbl- (e.g., prbl-Al), sub2- (e.g., sub2-Al), ypsl- (e.g., ypsl-Al). In some embodiments, the host cell may or may not contain additional sequences encoding proteins of any or all of the following heterologous or homologous factors, which include but are not limited to, transcription factors, chaperones, kinases, foldases, initiation factors, ribosomal components, post translational modification factors, PDI, PDI1, RPP0, KAR2 / BiP, SLY1, SEC1, HAC1, Erol, Hsp70, MXR1, GCN4, SEC53, Cenl, and inter alia.

[0225] In various illustrative embodiments, the cells may be grown by techniques including culturing in deep well plates, snap top test tubes, shake flasks. In other illustrative embodiments, the cells may be grown by techniques including, for example, batch, fed-batch andcontinuous fermentation in a liquid medium or on a semi-solid medium, or a solid medium. In other illustrative embodiments, the cells may be fermented in bioreactors. Typically, conditions permitting expression of the protein refers to conditions for batch, fed-batch or continuous fermentation in a liquid medium, but growth on a semi- solid medium, such as agar, is not excluded.

[0226] Culture media for yeast and fungal cells are widely available and are typically supplemented with a carbon source (e.g., glucose). A typical culture medium is YPD broth (Sunrise Science Products, Inc.) comprising yeast extract (10 grams), Bacto™ peptone (20 grams), and dextrose (20 grams). In one illustrative aspect, the transformed cells can be grown aerobically at 28-32° C. in a controlled pH environment (e.g., a pH of about 5-6) and with the carbon source (e.g., glucose, glycerol, or methanol) maintained continuously at a predetermined level known to support growth of the cells to a desired density within a specific period of time.

[0227] A large part of the popularity of dairy milk stems from its usefulness in derivative products, such as, but not limited to cheese, yogurt, cream, butter, or ice cream. In some illustrative embodiments are dairy like substitute compositions that have desirable flavor and performance characteristics, e.g., a composition that replicates dairy flavors while providing a similar nutritional profile as milk protein produced by a mammal. In some embodiments such compositions have a similar flavor, a similar appearance, a similar nutritional value, a similar aroma, and a similar mouth feel of mammal-produced milk. In some embodiments, these compositions may further comprise one or more color balancing agents, one or more sweetening agent, one or more carbohydrate, one or more lipid (e.g., fats, fatty acids, etc.), additional proteins and one or more flavor compound, among other components. In some embodiments, these components can be plant based in origin.

[0228] Therefore, in some illustrative embodiments, are methods of producing dairy like substitute compositions such as, but not limited to, milk, evaporated milk, milk powder, cheeses, cream cheese, cream, creamer, cocktail mixer, custard, dessert topping, gelato, ghee, ice cream, milkshake, pudding, butter, spread, sauce, sorbet, smoothies, snack food, yoghurt, yoghurt drink and frozen yoghurt. In additional illustrative embodiments, are methods and compositions of producing dairy product substitute compositions such as, but not limited to infant formula, toddler formula, nutritional supplement, energy supplement, sports supplement, weight gain supplement,weight loss supplement, energy drink, sports drink, weight gain drink, weight loss drink, energy food, sports food, weight gain food, and weight loss food. In other embodiments, said dairy product substitutes can be used in compositions such as, of baked goods, beverages, cooked goods, frozen goods, cooking mixes, baking mixes, beverage mixes, and sauce mixes.

[0229] The illustrative exemplifications described herein refer to the production of bovine P-lactoglobulin. However, those of skill in the art should, in light of the present disclosure, appreciate that similar results could be obtained with a sequence that encodes P-lactoglobulin protein from other mammals. Such mammals include, but are not limited to, a sheep, goat, bison, camels, horse, donkey, inter alia. In addition to P-lactoglobulin protein, the described methods and compositions can be used to generate other dairy proteins. These other dairy proteins can be of a bovine or other mammal, including but not limited to, a human, sheep, goat, bison, camel, horse, donkey, inter alia. These other dairy proteins include but are not limited to whey protein, lactoferrin protein, a- lactalbumin protein, glycomacropeptide protein, and casein protein. Isolates of these proteins can be used to generate concentrates of said proteins.

[0230] In some embodiments, described are host cells with a recombinant genome, wherein said genome comprises a polynucleotide that encodes a dairy protein (e.g., P-lactoglobulin protein) and results in the expression of secreted protein that does not contain elongated amino acid sequence and is dairy identical as described herein. In certain embodiments the cell is a yeast or fungal cell (e.g., filamentous fungi). In some embodiments the recombinant yeast or fungal cell (e.g., filamentous fungi) is of the genus Trichoderma, Pichia (Komagataella), or Kluyveromyces.

[0231] In some embodiments, the host cell comprises multiple copies of the polynucleotide that encodes the dairy protein. In additional embodiments, the cell comprises at least one at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or up to or greater than 9 copies of the polynucleotide that encodes a dairy protein. In specific embodiments, the cell may comprise 4 to 6 copies, 4 to 7 copies, 4 to 8 copies, 4 to 9 copies, 4 to 10 copies, 4 to 11 copies, or more. In specific embodiments, the cell may comprise 2 to 3 copies, 2 to 4 copies, 3 to 4 copies, 3 to 5 copies, 4 to 5 copies, 4 to 6 copies, 5 to 6 copies, 5 to 7 copies, 6 to 7 copies, 6 to 8 copies, 6 to 9 copies, 7 to 9 copies, 7 to 10 copies, 7 to 11 copies, 9 to 11 copies, or more.

[0232] In some embodiments, the host cells comprise multiple copies of a polynucleotide that encodes a dairy protein (e.g., P-lactoglobulin protein), wherein each copy is operably linked to: one or more promoter sequences, a signal sequence (e.g., secretion signal), and a transcription terminator. In some aspects, the multiple copies are integrated at multiple loci of the genome. In some aspects, the yeast or fungal cell is a yeast or fungal cell with a recombinant genome, wherein said genome comprises multiple copies of the polynucleotide that encodes the dairy protein integrated at multiple loci within the genome.

[0233] In some embodiments, the host cells may comprise one or more operably linked promoter sequences to achieve expression of the dairy protein (e.g., P-lactoglobulin). For example, the cells may comprise multiple integrated expression cassettes, where each expression cassette may include one or more promoter sequences that direct the expression of the encoded protein. In some embodiments, the promoter sequence comprises an inducible promoter. In some embodiments, the cells comprise an inducible promoter sequence that comprises a methanolinducible promoter. In other embodiments, the inducible promoter sequence comprises the AOX1, FDH or HpFMD promoter. In some embodiments, a promoter sequence comprises a constitutive promoter. In some embodiments, the constitutive promoter sequence comprises the UPP promoter. In some embodiments, the cells comprise a combination of promoter sequences. In some embodiments, the promoter sequences comprise a combination of constitutive promoters. In some embodiments, the promoter sequences comprise a combination of inducible promoters. In some embodiments, the promoter sequences comprise a combination of constitutive and inducible promoters.

[0234] In specific embodiments, the promoter combinations can comprise the AOX1 and the FDH inducible promoters. In some embodiments, the promoter combinations can comprise the AOX1 and the HpFMD inducible promoters. In some embodiments, the promoter combinations can comprise the FDH and the HpFMD inducible promoters. In some embodiments, the promoter combinations can comprise the AOX1, FDH and HpFMD inducible promoters. In some embodiments, the promoter combinations can comprise the UPP constitutive promoter and the AOX1 inducible promoter. In some embodiments, the promoter combinations can comprise the UPP constitutive promoter and the FDH inducible promoter. In some embodiments, thepromoter combinations can comprise the UPP constitutive promoter and the HpFMD inducible promoter. In other embodiments, the promoter combinations can comprise the UPP constitutive promoter and one or more of the following inducible promoters: A0X1, FDH, HpFMD.

[0235] In some embodiments, the host cells may comprise at least one polynucleotide that encodes a transcription factor. For example, a polynucleotide encoding one or more transcription factors can be integrated into the cell along with integration of the expression cassette(s). In some embodiments, the transcription factor comprises MXR1.

[0236] In some embodiments, the host cells may comprise at least one polynucleotide that encodes a secretion signal. For example, an expression cassette that is integrated into the cell can include one or more secretion signals. In some embodiments, the secretion signal comprises: sO, si, si.5, s3, s4, s5, s6, or s9, or any combination thereof.

[0237] In some embodiments, the host cells may comprise at least one polynucleotide that comprises a transcription terminator. For example, an expression cassette that is integrated into the cell can include one or more transcription terminators. In some embodiments, the transcription terminator comprises the AOX1 or FDH (e.g., FDH1) transcription terminator.

[0238] In some embodiments, the host cells may further comprise one or more additional factors to assist with production of the dairy protein (e.g., P-lactoglobulin). Such factors include, but are not limited to, transcription factors, chaperones, kinases, foldases, initiation factors, ribosomal components, post translational modification factors, and other processing related proteins that have a positive effect on dairy protein expression. In some embodiments, the one or more additional factors may upregulate or downregulate cellular machinery, such as vacuoles. In some embodiment, the one or more additional factors may be introduced by additional plasmids / vectors with nucleic acid sequences encoding these factors. In some embodiments, the one or more additional factors are integrated into the host’s genome. In some embodiments, the one or more additional factors are integrated into the host’s genome at any of the following loci: AOX2, PEX11, FLD1, or any combination thereof. In some embodiments, the one or more additional factors may include one or more of the following: MXR1, SEC53, RPP0, KAR2, PDI1, HAC1. In further embodiments, the one or more additional factors may each be operably linkedto a promoter sequence. In some embodiments, the one or more additional factors may each be operably linked to one of the following promoter sequences: FLD1, PEX11, or A0X2.

[0239] In some embodiments, are host cells that express and secrete a mammalian dairy protein (e.g., P -lactoglobulin protein), wherein said cells comprise one or more insertions at one or more loci of the genome, these insertions comprising the expression cassettes / expression vectors described herein. In some embodiments are yeast or fungal cells, with a recombinant genome comprising inserted sequences comprising one or more insertions at one or more loci of the genome, these insertions comprising an expression cassette / expression vector described herein. In other embodiments are yeast or fungal cells, with a recombinant genome, which express and secrete a mammalian dairy protein, wherein said cells comprise one or more insertions at one or more loci of the genome, these insertions comprising an expression cassette / expression vector described herein.

[0240] In other embodiments are recombinant host cells that contain integrated within its genome heterologous sequences described herein, wherein said host cell is a yeast or fungal cell (e.g., filamentous fungal cell). In some aspects the host cell is a Trichoderma reesei cell, a Kluyveromyces lactis cell, or a Pichia pastoris (Komagataella phaffii ) cell. In some embodiments these recombinant host cells comprise a genome integrated within which are the sequences of the recombinant expression vectors described herein. In other embodiments are recombinant host cells comprising a genome, in which, integrated at multiple loci is one, two, three, four, five, six, seven, or more sequences of a recombinant expression vector described herein.

[0241] In some embodiments are isolated dairy proteins (e.g., P -lactoglobulin protein), produced by a recombinant yeast or fungal cell described herein, that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical in its amino acid sequence to that produced in a mammal. In other embodiments these isolated dairy proteins are at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical in its amino acid sequence to that produced in by a bovine, a goat, a sheep, or a human. In some embodiments are isolated dairy proteins that are up to 99.1%, up to 99.2%, up to 99.3%, up to 99.4%, up to 99.5%, up to 99.6%, up to 99.7%, up to 99.8%, up to 99.9% identical to a mammalian dairy protein, e.g., a bovine, a goat, a sheep, or a human dairy protein. In some embodiments are isolated dairy proteins produced by a recombinantyeast or fungal cell that comprises: one or more copies of a dairy protein encoding nucleic acid sequence operably linked with: one or more promoter sequences; one or more secretion sequences; and one or more transcription terminators.

[0242] In some embodiments, the dairy protein (e.g., P-lactoglobulin protein) produced is secreted into the culture medium. In some aspects, the dairy protein is secreted into the culture medium at a concentration from 0.4 g / L to 4 g / L, or 0.4 g / L to 8 g / L, or 0.4 g / L to 10 g / L, 0.4 g / L to 12 g / L, 0.4 g / L to 14 g / L, 0.4 g / L to 20 g / L, or 0.4 g / L to 24 g / L, or 0.4 g / L to 30 g / L or 0.4 g / L to 34 g / L or 0.4 g / L to 40 g / L. In other aspects the dairy protein is secreted into the culture medium at a concentration selected from at least 0.05, at least 0.1, at least 0.5, at least 1.0, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, at least 4.5, at least 5.5, at least 6.0, at least 6.5, at least 7.0, at least 7.5, at least 8.0, at least 8.5, at least 9.0, at least 9.5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35 or at least 40 g / L. In some embodiments the dairy protein produced and secreted into the culture medium is 70-100% identical, or 80-100% identical, 90-100% identical, or 95-100% identical to a native mammalian sequence.

[0243] In some embodiments, the dairy protein (e.g., P-lactoglobulin protein) that is secreted is used to produce a dairy product that can selected from the group consisting of milk, evaporated milk, milk powder, cream, creamer, cocktail mixer, custard, cheese, cream cheese, desert topping, gelato, ghee, ice cream, milkshake, pudding, butter, spread, sauce, sorbet, smoothies, snack food, yoghurt, yoghurt drink, and frozen yoghurt. In additional embodiments, the dairy protein can be used to produce a dairy product to be used in the formulation of products selected from the group consisting of infant formula, toddler formula, nutritional supplement, energy supplement, sports supplement, weight gain supplement, weight loss supplement, energy drink, sports drink, weight gain drink, weight loss drink, energy food, sports food, weight gain food, and weight loss food. In some embodiments, the dairy protein can be used to produce a dairy ingredient for use in one or more of baked goods, beverages, cooked goods, frozen goods, cooking mixes, baking mixes, beverage mixes, and sauce mixes.

[0244] In some embodiments, the dairy protein can be used to produce a dairy ingredient selected from the group consisting of milk protein concentrate and milk protein isolate. In some aspects, the milk protein isolate may be selected from the group consisting of whey protein isolate,lactoferrin protein isolate, a-lactalbumin protein isolate, P-lactoglobulin protein isolate, glycomacropeptide protein isolate, and casein protein isolate. In additional aspects, the milk protein concentrate is selected from the group consisting of whey protein concentrate, lactoferrin concentrate, a-lactalbumin concentrate, P-lactoglobulin concentrate, glycomacropeptide concentrate, and casein concentrate.

[0245] The expression vectors, expression cassettes, strains, cells, and cell expression systems of this disclosure may be prepared in accordance with standard techniques. In various embodiments, any expression vector known to the skilled artisan (e.g., a vector that replicates autonomously or integrates into the host genome) and compatible with an expression system can be used. As used herein, the term “vector” means any plasmid, or other vector, in double-stranded or single- stranded form or in linear or circular form that can transform a host cell by integration into the host cell genome or by existing episomally or extrachromosomally (e.g., an autonomously replicating plasmid). It will be understood that a vector (e.g., expression vector or expression cassette) is a nucleic acid construct used to transform a host cell for expression of the dairy protein, or fragment thereof, and the vector is not found in nature in the host cell it transforms.

[0246] In additional illustrative embodiments, the expression vector has restriction endonuclease cleavage sites for the insertion of DNA fragments (e.g., one or more cloning sites and / or a multiple cloning site), genetic markers for selection of transformants. For example, the genetic markers for selection of transformants can include a selection marker such as antibiotic resistance or a needed nutrient that allows a transformed cells to grow on a medium in the presence of antibiotics or medium devoid of a necessary nutrient that cannot be produced by a deficient strain, a selection marker that encodes an enzyme for which chromogenic substrates are known, or a selection marker that provides resistance to a drug, including, but not limited to, G418, nourseothricin (Nat), zeocin, blasticidin, or hygromycin. In some embodiments, the expression vector has lox cites to facilitate the “loop out” of bacterial and / or resistance marker DNA.

[0247] In one illustrative embodiment, protein coding sequence can be operably linked in the expression vector to the promoter sequence capable of directing the expression of the protein, for example, in yeast. Yeast expression systems are described in, but not limited to, U.S. Pat. Nos. 6,451,572, 6,841,370, 6,974,690, 7,320,876, 7,078,035, 7,138,260, 10,676,750, 11,555,194 andPCT Publication No. WO 2007112739 and WO 2014138679 which are all incorporated herein by reference.

[0248] In some illustrative embodiments, the constructs described herein comprise expression vectors with recombinant nucleic acids encoding a dairy protein (e.g., P-lactoglobulin protein) and further comprising one or more operably linked promoter sequence. In some embodiments, the promoter sequence encodes an inducible promoter. In some embodiments, the promoter sequence encodes the inducible promoter sequence of the AOX1 promoter. In other embodiments, the promoter sequence encodes the inducible promoter sequence of the FDH promoter. In other embodiments, the promoter sequence encodes the inducible promoter sequence of the FMD promoter from related methylotrophic yeasts, such as Hansenula polymorpha (HpFMD). Other promoters or combinations of promoters may be utilized in accordance with the present disclosure.

[0249] In another embodiment, a method of producing a dairy protein using these promoter sequences is provided. The method comprises the steps of culturing in a culture medium a host cell comprising a first expression cassette comprising any of the above promoter sequences operably linked to a heterologous coding sequence encoding a protein, wherein the culturing is done under conditions permitting expression of the protein. In a specific illustrative embodiment, an isolated P-lactoglobulin protein produced according to this method is provided.

[0250] In specific embodiments, recombinant nucleic acids are provided comprising a polynucleotide that encodes a P-lactoglobulin protein and results in secreted P-lactoglobulin protein that does not contain an elongated amino acid sequence and is dairy identical.

[0251] In specific embodiments, recombinant expression vectors are provided comprising a polynucleotide that encodes a P-lactoglobulin protein and results in secreted P-lactoglobulin protein that does not contain an elongated amino acid sequence and is dairy identical.

[0252] In specific embodiments, the recombinant expression vector further comprises multiple copies of the polynucleotide that encodes a P-lactoglobulin protein. In specific embodiments, the multiple copies comprise at least 2 copies, at least 3 copies, at least 4 copies, at least 5 copies, at least 6 copies, at least 7 copies, or at least 8 copies. In specific embodiments, themultiple copies comprises 2 to 3 copies, 2 to 4 copies, 2 to 5 copies, 2 to 6 copies, 2 to 7 copies, 2 to 8 copies, but less than 10 copies.

[0253] As specific exemplifications, one, two, three, four, five, six or more plasmids, each containing one, two, three or four P-lactoglobulin expression cassettes and each targeting one or more loci (e.g., Tl, T6, T9, A0X1, FDH or UPP), may be successively transformed into the selected strain. As a result, these strains can contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve copies, or more of the P-lactoglobulin gene.

[0254] The specific constructs can include, for example: (1) one or more P-lactoglobulin expression cassettes at the FDH locus; (2) one or more P-lactoglobulin expression cassettes at the A0X1 locus; (3) one or more P-lactoglobulin expression cassettes at the UPP locus; (4) one to four, or more, P-lactoglobulin expression cassettes at the Tl locus; (5) one to four, or more, P- lactoglobulin expression cassettes at the T6 locus; (6) one to four, or more, P-lactoglobulin expression cassettes at the T9 locus; wherein each expression cassette contains a promoter (e.g., FDH, the FMD promoter from Hansenula polymorpha (HpFMD), UPP, or A0X1), a secretion signal (e.g., sO, si.5, s9, or s6), a nucleic acid sequence encoding bovine P-lactoglobulin protein (e.g., native or modified sequence) and a transcription terminator (e.g., FDH1 or A0X1).

[0255] As specific exemplifications, the yeast or fungal strains are modified to include multiple copies of the polynucleotide that encodes an amino acid sequence that encodes a P- lactoglobulin protein; one or more promoter sequences, and one or more signal sequences (e.g., secretion signals). For example, the strain may comprise two to three, two to four, three to four, three to five, four to five, four to six, five to six, five to seven, six to seven, six to eight, seven to nine, or seven to ten copies of the P-lactoglobulin gene.

[0256] In specific embodiments, the yeast strains are recombinant P. pastoris strains that comprise multiple copies of the polynucleotide that encodes an amino acid sequence that encodes a P-lactoglobulin protein; one or more promoter sequences, and a signal sequence (e.g., secretion signal).

[0257] In specific illustrative embodiments, the resulting recombinant yeast or fungal strains include multiple P-lactoglobulin encoding nucleic acid sequences that are integrated atmultiple loci and operably linked to one or more promoter sequences. The strains constructed using the described methodology results in cells that secrete high levels of P-lactoglobulin protein that does not contain an elongated amino acid sequence and is therefore dairy identical.

[0258] In specific embodiments, the nucleic acid sequence consists of, consists essentially of, or comprises a P-lactoglobulin encoding nucleic acid sequence that is SEQ ID NO: 7. In further specific embodiments, the amino acid sequence consists of, consists essentially of, or comprises a P-lactoglobulin amino acid sequence that is SEQ ID NO: 8.Examples

[0259] The following examples are included to demonstrate preferred embodiments of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques determined by the inventors to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Example 1 - Materials and MethodologyOverview

[0260] The present disclosure provides methods and compositions that allow the production of P-lactoglobulin as a microbial protein that has an amino acid sequence that is the same as native bovine milk protein. Specifically provided are fungal cell-based expression systems that can be used to provide dairy identical proteins, such as P-lactoglobulin, that are secreted into culture media at high concentrations.

[0261] The inventors initially established P-lactoglobulin expression in several organisms, including K. lactis, T. reesei, and P. pastoris (Komagataella phaff'ii) (data not shown). Expression in P. pastoris produced secreted P-lactoglobulin at reasonable yields with very few endogenous protein contaminants. Described are exemplary recombinant P. pastoris cells, strains, andexpression systems. These yeast strains were constructed using a series of plasmids that were generated as set out in more detail below.Codon Optimization

[0262] Various codon optimization strategies were employed and tested. The native DNA sequence of P-lactoglobulin from Bos taurus (NCBI Reference Sequence: XM_027556706.1, GenBank: BC108213.i l; see Table 1A) was modified as follows: 1) the native DNA sequence was used (Nat); 2) the amino acid sequence was back-translated using Benchling Software10optimized for Pichia pastoris (DL); or 3) using custom strategy employed by BioGrammatics, Inc. (unpublished) (BioG). Any sequences without a TAA stop codon were further modified to replace the existing stop codon with TAA. Additionally, any sequences that contained a Pmel, Bsal and B5M36I restriction enzyme recognition site were modified to remove the recognition sites but maintain the original amino acid sequences (see Table 1). Descriptions of the plasmids employed to test these encodings can be found in Table 3.Secretion Signals

[0263] Various secretion signals were tested for their ability to effectively secrete P- lactoglobulin out of the cell. Secretion signals used are listed in Table 2 and are briefly described here. The native Bos taurus secretion signal (here called sO) was taken from NCBI Reference Sequence: XM_027556706.1, GenBank: BC108213.i l (see Table 1A). The S. cerevisiae a- mating factor pre-pro peptide (si), the Ostl / a-mating factor fusion (s3), the Ostl (s4), and the Aga2 (s5) signal sequences were provided by BioGrammatics, Inc. The human serum albumin (s6) signal sequence was taken from NCBI Reference Sequence: NM_000477.7 (see Table 1A). Additional modifications were made to S. cerevisiae a-mating factor pre-pro peptide: a deletion was made of the Glu-Ala repeats after the Kex2 cleavage site (sl.5); a deletion was made of residues 57 to 70 and the Glu-Ala repeats after the Kex2 cleavage site (s9). Descriptions of the plasmids employed to test these secretion signals can be found in Tables 3 and 4.Promoters

[0264] Various promoters were evaluated for their potential to express P-lactoglobulin: the Pichia pastoris alcohol oxidase 1 promoter (A0X1); the methanol inducible FDH promoter which natively drives expression of formate dehydrogenase in Pichia; the constitutive UPPpromoter, a commercial variant of the P. pastoris glycosyl phosphatidyl inositol-anchored protein promoter13;and the formate dehydrogenase promoter from Hansenula polymorpha (HpFMD)16The A0X1, FDH and UPP sequences were provided by BioGrammatics, Inc. and the HpFMD promoter sequence was ordered as a clonal gene in the pTwist Chlor Medium Copy vector from Twist Bioscience (San Francisco, CA, USA). The sequences of the A0X1, FDH, HpFMD and UPP promoters and a description of the plasmids employed to test these promoters can be found in Table 5 and Table 6.Multiple Copy Integration

[0265] Various copy numbers of P-lactoglobulin inserted at multiple genomic integration sites were tested under the control of methanol inducible promoters. Multiple copy plasmids with up to three expression cassettes were designed, with each expression cassette comprising a promoter, the sO secretion signal, the Nat encoding, and a transcription terminator. Multiple copy plasmids (Tables 7 and 7A) were employed along with pKC45P (see Table 5) to test various copy numbers. Several genomic integration sites were used for genomic integration of these plasmids, which have been abbreviated to Tl, T6 and T9. These are shown in Tables 8 and 8A. In regard to Tables 8 and 8 A, it is noted that the Pichia chromosome is numbered uniquely in the Pichia reference genome for strain BglO (~9.4 million bp total: Chromosome #1 has 2,895,354 bp, #2 has 2,396,458 bp, #3 has 2,263,458 bp and #4 as 1,827,941 bp). The chromosome and integration site numbers identify the base immediately before the T-site integration site used in vectors designed by BioGrammatics, Inc. The broader T-sites encompass 600-1400 bp of DNA that include those identified and integration can happen anywhere within this DNA, including at the precise locations listed.Chaperones and Helper Factors

[0266] To evaluate the impact of chaperones and other helper factors on the expression and secretion of P-lactoglobulin, the vectors in Figures 7A-7G were employed.Parent Strains and Vectors

[0267] The MutsP. pastoris strain BG11 (genotype aoxl-Al) and vectors pJAG, pJUG, pGAN, pR-H, pSAH-Tl, pSAN-T9, pSAZ-T6, pGPUH PDI1, pGFsH HAC1, pGFsH KAR2, pGA2sH KAR2, pGPl lsH RPP0, pGPUH SEC53, pGA2sH MXR1, pJ(FDH)H, and pJTG-Cre-ARS were supplied by BioGrammatics, Inc;. (Carlsbad, CA, USA). See Figures 6A-6G, Figures 7A-7G, Figure 8 and Figure 9~ for the plasmid maps of these vectors.

[0268] The sO, si, si.5 and s3 secretion signals in Table 2 and the Nat, DL and BioG P- lactoglobulin gene encodings in Table 1 were ordered from Twist Bioscience (San Francisco, CA, USA) as clonal genes in the Bsal insertion site of the pJAG or pJUG vectors (see Table 3 for resulting plasmids and plasmid names). All other vectors were sub-cloned using Gibson Assembly (Cloning Kit from NEB) or Golden Gate Assembly (Reagents purchased separately from NEB) (see Tables 4, 5, 7, 7A for resulting plasmids).

[0269] Plasmids were chemically transformed into E. coli Top 10 or HST08 strains, which were cultured at 37°C in LB medium (1% peptone, 0.5% yeast extract and 1% NaCl) with 100 pg / mL ampicillin or 50 pg / mL chloramphenicol, where appropriate. Transformants were screened by colony PCR or plasmid extraction and restriction digest. The plasmids were extracted from E. coli transformants and digested with restriction enzymes from NEB prior to transformation into P. pastoris.

[0270] Select plasmids were sequence-confirmed using Oxford Nanopore Technology through the PlasmidSeq Express Service: Whole plasmid sequencing by the Auckland Genomics Centre, The University of Auckland, Auckland, New Zealand. Other select plasmids were sequence-confirmed by Plasmidsaurus (Whole Plasmid Sequencing was performed by Plasmidsaurus using Oxford Nanopore Technology with custom analysis and annotation).Construction of P. pastoris Strains

[0271] Linearized vectors were transformed into electrocompetent Pichia pastoris using electroporation. Distinct transformant colonies were streaked on selective media for single colonies to ensure clonality.

[0272] Prior to transformation, pJAG-based vectors and pKC45P were linearized with Pmel, pKC57P and pKC23P were linearized with Nrul, pKC47P was linearized with B5«36I, and pKC48P was linearized with spAI. These linearized vectors were each transformed into electrocompetent P. pastoris BG11 cells.

[0273] Multiple copy vectors pKC50P, pKC56P, pKC59P, pKC61P, pKC63P and pKC64P were linearized with Nrul. The chaperone vector pGPUH SEC53 was digested with Smal. The chaperone vector pGPl 1H PDI1 was digested with Bsal and the chaperone and helper factor vectors pGFsH HAC1, pGFsH KAR2, pGA2sH KAR2, pGPl lsH RPPO, and pGA2sH MXR1 were digested with BbsI and the resulting linear DNA was blunted with Q5 High-Fidelity DNA Polymerase (NEB) before transformation. The transformations of multiple copy plasmids, and transformations of chaperone and helper factor plasmids, and the resulting strains are summarized in Tables 9 and 9A.

[0274] When desired, antibiotic resistance markers were removed using a cre / lox system. Cells were transformed with pJTG-Cre-ARS and transformants were plated on YPD G418 media then transformant colonies were streaked on selective YPD G418 media for single colonies. The single colonies were then plated on YPD and the appropriate antibiotic(s) corresponding to the resistance marker(s) to be removed. Cells that grew on YPD plates but were sensitive to the antibiotic plates were streaked for single colonies on YPD plates. Those single colonies were plated on YPD and YPD G418. Resulting strains that grew on YPD but were sensitive to G418 no longer contain antibiotic resistance markers or the pJTG-Cre-ARS plasmid and are considered “cured”.P. pastoris Culture and Expression Trial Media

[0275] P. pastoris was grown in YPD (yeast peptone dextrose: 1% yeast extract, 2% peptone, 2% dextrose) or on YPD agar with 800 pg / mL G418 (Gold Biotechnology, Inc., Saint Louis, MO), 80 pg / mL Nourseothricin (Gold Biotechnology, Inc.), 100 pg / mL hygromycin (Sigma-Aldrich, Burlington, MA) or zeocin (Thermo Fisher Scientific, Waltham, MA) for selection.

[0276] The following media were used for expression trials: BMGY 1.5% GY (buffered glycerol-complex medium: 1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 x 10-5 % biotin, 1.5% glycerol), BMMY 2% MeOH (buffered methanol-complex medium: 1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 x 10-5 % biotin, 2% methanol), BMMY20 (100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 x 10-5 % biotin, 10% methanol).Methanol Induced Expression Trials

[0277] For 96 deep well plate trials, seed cultures were prepared in a 96 well plate or a 96 deep well plate. In the former, 200 pL YPD was inoculated with cells from a single colony and incubated for ~24 hr at 29-30°C and 650-700 rpm. In the latter, 600 pL YPD was inoculated with cells from a single colony and incubated for ~24 hr at 29-30°C and 650-700 rpm. The seed cultures were used to inoculate 430 pL or 480 pL of BMGY 1.5% GY in 96 deep well plate wells, which were then incubated for ~24 hr at 29-30°C and 850-1000 rpm. If 480 pL of BMGY 1.5% GY was used, 50 pL of culture was removed prior to induction to measure the ODeoo. The strains were then induced by adding 120 pL BMMY20 for a final concentration of 2% methanol and cultured for a further ~48 hr at 29-30°C and 850-1000 rpm. Culture supernatants were harvested by centrifugation at ~48 hr post-induction.

[0278] For snap top tube trials, seed cultures were sometimes prepared in 96 well plates (200 pL YPD was inoculated with cells from a single colony and incubated for ~24 hr at 29-30°C and 650-700 rpm) or in 10 mL snap top tubes (~2 mL YPD was inoculated with cells from a single colony and incubated for ~24 hr at 29-30°C and 650-700 rpm). Cells from a single colony or 50- 100 pL of seed culture were used to inoculate 2 mL BMGY 1.5% GY in 10 mL snap top tubes, which was then incubated on a ~45-degree slant for ~24 hr at 29-30°C and 250 rpm. The strains were then centrifuged and resuspended in BMMY 2% MeOH and cultured for a further ~48 hr at 29-30°C and 250 rpm. Culture supernatants were harvested by centrifugation at ~48 hr postinduction.Constitutive Expression Trials

[0279] Snap top tube trials were used to evaluate strains utilizing the UPP promoter, where methanol induction is not needed. Cells from a single colony were used to inoculate 2 mL BMGY 1.5% GY in 10 mL snap top tubes, which were then incubated on a ~45-degree slant for ~24 hr at 29-30°C and 250 rpm. Glycerol was added to 1.5% 2-3 times over the next ~48 hr. Culture supernatants were harvested at ~48 hr by centrifugation.Sample Analysis

[0280] Culture supernatants were analyzed by SDS PAGE with Coomassie Brilliant Blue staining. Where appropriate, band intensity was analyzed using Image Lab software (Bio-Rad, Hercules, CA).

[0281] Select supernatant samples were prepared as follows then “SBS: LC-MS analysis for proteins / peptides” was performed by Deanna Shea and Martin Middleditch, who belong to the Technical Services, Faculty of Science, University of Auckland, and who operate within the ShaRE Mass Spectrometry Facility, Faculty of Science, University of Auckland, Auckland, New Zealand. Samples were passed through a 0.22 pm syringe filter then optionally concentrated with a Pierce Protein Concentrator PES 10K MWCO (Catalog Number 88516; Thermo Fisher Scientific, Waltham, MA). Resulting LC-MS data was used to identify the relative abundance / purity of P-lactoglobulin and to confirm correct processing of the N and C terminal ends of the protein.Table 1. P-lactoglobulin gene encoding sequences.Table 1A: NCBI and GenBank reference sequencesSecretion signals shown in bold with underlining.Table 2. Signal sequences / secretion signals.Table 3. Plasmids ordered from Twist Bioscience: Secretion signal / encoding pairs in pJAG or pJUG.Table 4. Subcloned plasmids: Nat encoding with different secretion signals in pGAN.Table 5. Subcloned plasmids: Different promoters with sO secretion signal and Nat encoding in pR-H.Table 6. AOX1 promoters present in select plasmids.Table 7. Subcloned plasmids: Multiple copy plasmids in pSAH-Tl, pSAZ-T6 and pSAN-T9.Table 7A. Plasmids used for construction of select multicopy strains.Table 8. Target Site (T-Site) Identification and Location of Integration Sites.Table 8A. Select Target Site (T-Site) Identification and Location of Integration Sites.Table 9. Multi-copy strain names, construction, and descriptions.*Plasmid was transiently present and was expressed to remove resistance markers, then selection was dropped to achieve plasmid loss in the described strain(s).Table 9A. Select multi-copy strains, construction, and descriptions.* Plasmid was transiently present and was expressed to remove resistance markers, then selection was dropped to achieve plasmid loss in the described strain(s).Example 2 - Results and DiscussionComparison of Codon Optimization

[0282] The advantages of codon optimization for heterologous protein expression in P. pastoris and other organisms have been widely demonstrated.4-9Codon optimization algorithms often produce significantly different sequences, which can affect expression levels. The inventors tested two codon-optimized encodings of the Bos taurus P-lactoglobulin variant B gene, the Daisy Lab encoding (DL) and the BioGrammatics encoding (BioG). The native encoding (Nat) was also tested as a control for baseline expression. See Table 1 for these encoding sequences, which were tested with several secretion signals as described below.

[0283] Gene encoding was shown to significantly impact P-lactoglobulin yield and, contrary to what was expected, the native encoding outperformed the DL and BioG codon optimized encodings for every secretion signal (Figures 1A-1D). The average protein yield for the native encoding was about 31% and 16% greater than the average protein yield for the BioG and DL encodings, respectively. This unexpected result indicates that the Nat encoding is superior. While not wishing to be limited to any particular mechanism, this is perhaps due to some influence on transcription rates and thus translation rates. Consequently, the Nat encoding was used when testing further secretion signals, different promoters and different copy numbers.Comparison of Secretion SignalsOverview

[0284] Secretory signal peptides are sequence motifs that target proteins for secretion. The secretion of a recombinant protein into culture media makes the protein much easier to harvest and purify. The efficiency of secretion signals is often protein specific and can have a significant impact on protein expression and secretion out of the cell. To determine what secretion signal would be most efficient for the secretion of P-lactoglobulin, each of the encodings described above (Nat, DL, and BioG) were initially paired and tested with four secretion signals with disparate properties (see Initial Comparison of Secretion Signals). The most promising two secretion signals from these tests and an additional four secretion signals were then paired and tested with the best encoding (see Further Comparison of Secretion Signals). See Table 2 for all secretion signal sequences.Initial Comparison of Secretion Signals

[0285] The Nat, DL and BioG encodings were each initially paired with the S. cerevisiae a-mating factor pre-pro peptide sequence (si), the Ostl / a-mating factor fusion wherein the pre region of the a-mating factor is replaced with the Ostl signal sequence (s3), and the native Bos taurus secretion signal (sO). si was selected as it is most widely used in heterologous protein expression in P. pastoris and is often most effective for the secretion of many proteins.18The pre region of si directs translocation into the endoplasmic reticulum and the pro region directs packaging into ER-derived COPII transport vesicles.11The replacement of the pre region with the Ostl signal sequence in s3 promotes co-translational translocation with this signal sequence, halting protein folding until the protein reaches the ER lumen. s3 has been shown to improve secretion for several proteins when compared to si.

[0286] In contrast to both si and s3, the native Bos taurus secretion signal, sO, is only a short signal sequence that lacks a pro domain altogether. sO was included as a control for baseline expression. Because P-lactoglobulin may be left with residual Glu-Ala repeats on the N-terminus when the full si secretion signal is used for secretion in P. pastoris12, a version of the S. cerevisiae a-mating factor pre-pro peptide sequence with a deletion of the Glu-Ala repeats after the Kex2 cleavage site (si.5) was also paired and tested with the DL encoding.

[0287] Secretion signals were shown to have a significant impact on P-lactoglobulin yield (Figures 1A-1D). sO and s3 performed similarly, indicating that P-lactoglobulin does not require a pro domain for efficient secretion, si outperformed both sO and s3, indicating that co- translational translocation is not required for efficient secretion, si also outperformed sl.5, indicating that the Glu-Ala repeats might be required for better secretion with the a-mating factor secretion signal.

[0288] Supernatant samples from test tube expression trials of sO Nat, si Nat and sl.5 Nat strains were analyzed to see if the secretion signals were being properly processed. SBS: LC-MS analysis for proteins / peptides was performed by Deanna Shea and Martin Middleditch, with the Technical Services, Faculty of Science, University of Auckland, and operating within the ShaRE Mass Spectrometry Facility, Faculty of Science, University of Auckland, Auckland, New Zealand. The results revealed that sO and sl.5 strains secreted P-lactoglobulin that was up to 100% correctlyprocessed at both the N and C terminal ends while si left unwanted Glu-Ala repeats on the N- terminus of P-lactoglobulin (Table 10). These unwanted Glu-Ala will likely be present on P- lactoglobulin produced by any strains with the si or s3 secretion signals as they share the same pro domain.Table 10. N and C terminal ends of P-lactoglobulin secreted with different secretion signals, results from LC-MSFurther Comparison of Secretion Signals

[0289] After determining that the sO and si.5 secretion signals gave high yields of secreted and correctly processed P-lactoglobulin, the following six secretion signals were paired with Nat encoding and compared: sO, si.5, Ostl (s4) and Aga2 (s5) signal sequences provided by BioGrammatics, Inc., the human serum albumin signal sequence (s6), and a version of the S. cerevisiae a-mating factor pre-pro peptide sequence with a deletion of residues 57 to 70 and the Glu-Ala repeats after the Kex2 cleavage site (s9). The deletion of residues 57 to 70 has been shown to increase expression for certain target proteins.17The other new secretion signals (s4, s5 and s6) were all selected because they have been used for efficient secretion of select heterologous proteins and / or, like sO, are shorter signal sequences / secretion signals.

[0290] Once again, secretion signal had a significant impact on P-lactoglobulin yield. sO, s6 and s9 secretion signals outperformed sl.5, s4 and s5 considerably (Figures 2A-2B). While using any of these four signals alone or in combination with the others would likely result in high protein yields, the sO secretion signal was selected for future work as it yields properly processed P-lactoglobulin (Table 10) and, being the native secretion signal, it would be innocuous if some ended up in a precision fermentation product.Comparison of Promoters

[0291] Heterologous expression of target proteins can also vary when different promoters are used to drive expression. Determining which promoters work best for heterologous expressionof P-lactoglobulin in Pichia pastoris is an important step toward developing a strain for commercially viable precision fermentation of P-lactoglobulin. Constructs were designed to test the expression of the sO Nat open reading frame under the control of the following promoters: the widely used methanol inducible P. pastoris alcohol oxidase 1 promoter (A0X1), the methanol inducible promoter that natively drives expression of formate dehydrogenase in P. pastoris (FDH), the methanol inducible and de-repressible formate dehydrogenase promoter from Hansenula polymorpha (HpFMD)16, and a commercial variant of the P. pastoris glycosyl phosphatidyl inositol-anchored protein promoter obtained from BioGrammatics, Inc. (UPP).13Each of these promoters has performed well for the expression of heterologous proteins in the literature.13,16,18See Table 5 and Table 6 for promoter sequences.

[0292] The inventors found expression levels under the control of the A0X1, HpFMD, FDH and UPP promoters all yield high levels of secreted protein (Figures 3A-3B). Fermentation trials are needed to optimize expression conditions and confirm which promoter performs the best, but all four promoters are good candidates for the high expression of secreted P-lactoglobulin.Multiple Copy Integration

[0293] Integrating multiple copies of a gene of interest has been shown to increase heterologous protein expression levels in P. pastoris for certain proteins.14,15Often, expression increases as copy number increases until bottlenecks cause expression to plateau and / or the cells become over-burdened and expression starts decreasing.14The inventors have combined a number of methods to overcome such cellular limitations and maximize expression with multiple copies of P-lactoglobulin. In addition to the method used for generating multiple copy strains, where to genomically integrate those copies and what promoter(s), secretion signal(s) and encoding(s) to use were considered.

[0294] There are many ways to generate multi-copy strains, including the use of random integration, targeting the same locus repeatedly, targeting different loci, etc. While not wishing to be limited to any particular theory or mechanism, inserting copies at different loci in the genome may increase the cellular machinery available for the expression of each copy, increasing yields. Additionally, the location that heterologous genes are integrated into the genomes of host organisms is known to affect transcription levels and expression levels. BioGrammatics, Inc. hascreated unique Target-sites (T-sites) to facilitate targeted insertion of expression vectors into the Pichia genome, without interference of host cell functions, for enhanced expression and activity of gene sequences. Vectors pSAZ-T6, pSAN-T9 and pSAH-Tl were purchased from BioGrammatics, Inc. and used for sub-cloning to generate plasmids containing multiple P- lactoglobulin expression cassettes, which were used to construct multi-copy strains. See Figures 6E-6G for plasmid maps of the parent vectors pSAZ-T6, pSAN-T9 and pSAH-Tl and Tables 8 and 8A for the identification and location of the target integration sites corresponding to these vectors.

[0295] The inventors used the sO secretion signal and Nat encoding in their multiple copy strains as these components demonstrated great promise for expression and secretion of P- lactoglobulin in P. pastoris. While not wishing to be limited to any particular theory or mechanism, regulating the expression of multiple copies of P-lactoglobulin using a combination of promising promoters that showed similar strength for P-lactoglobulin expression may help reduce the burden on cellular machinery and bottlenecks in a strain. Specifically, it is possible that simultaneously regulating separate expression cassettes with a mix of A0X1, FDH and HpFMD promoters may stagger their activation upon induction with methanol, maximizing expression and making precision fermentation of P-lactoglobulin more economical.

[0296] Therefore, plasmids targeting different loci, each comprising up to three sO Nat P- lactoglobulin expression cassettes under the control of different combinations of promoters (A0X1 and / or FDH and / or HpFMD), were constructed and used in successive transformations to construct multiple copy strains. Additionally, the inventors used cre / lox to remove resistance markers. See Table 9 and Table 9 A for the strain names and descriptions of the resulting four, seven, and nine copy strains. At each stage, transformants were screened using 96 deep well plate expression trials and / or by test tube expression trials and resulting samples were analyzed by SDS PAGE to select the strain transformant(s) with the highest reproducible yield for each copy number and cured status (data not shown). The secretion levels of the best strains are shown in Figures 4A-4F.

[0297] SDS PAGE results from expression trials comparing strains with different copy numbers showed that secretion levels increased with copy number. The greatest increase was observed as copy number increased from one to four copies, with yield increasing approximately400%. Further increases of up to about 50% were observed between four and seven copies and between seven and nine copies. The removal of resistance markers did not significantly affect yields for any given copy number. BSA standards were used to quantify yields of nine-copy strains based on band intensities; up to 0.9 g / L of P-lactoglobulin was secreted by these strains.

[0298] Fermentation trials were conducted in 1-3 L bioreactors with several of the best secreting strains, including sDLP265_25 (data not shown). Sample supernatants were analyzed by SDS PAGE to determine yield. The results showed that these 9 copy strains secreted up to 7 g / L of P-lactoglobulin. To determine relative abundance / purity and proper processing of P- lactoglobulin at the C and N terminal ends “SBS: LC-MS analysis for proteins / peptides” was performed by Deanna Shea and Martin Middleditch, who belong to the Technical Services, Faculty of Science, University of Auckland, and who operate within the ShaRE Mass Spectrometry Facility, Faculty of Science, University of Auckland, Auckland, New Zealand. The results showed that, without purification, P-lactoglobulin already comprises up to 99.13% of the total protein in the fermentation supernatant sample. Further, analysis of the intact species showed that at least 80% of the P-lactoglobulin is mature, full-length P-lactoglobulin that is correctly processed at the C and N terminal ends.Chaperones and Helper Factors

[0299] Transcription factors, chaperones, kinases, foldases, initiation factors, ribosomal components, post translational modification factors, and other helper factors, inter alia, can help reduce bottlenecks and increase expression levels for a variety of proteins. Select cured four copy, uncured seven copy, and uncured nine copy strains were transformed with plasmids purchased from BioGrammatics, Inc. containing transcription factors, chaperones, and other helper factors (see Figures 7A-7G for plasmid maps). Transformants were screened by test tube expression trials and resulting samples were analyzed by SDS PAGE to determine if any of the chaperones impacted yield (Figures 5A-5E).

[0300] There was no significant increase in expression levels of P-lactoglobulin for any of the chaperones or helper factors tested at four copies, seven copies, or nine copies. Coupled with a measurable increase in yield at each higher copy number, this implies that the cellular machinery is well equipped to express and secrete P-lactoglobulin. However, these or other chaperones couldincrease yields at higher copy numbers or under different conditions, such as in bioreactors. Additionally, they may be useful for increasing yields for heterologous expression of other dairy proteins. Further, since secretion can be improved to different levels by different helper factors (e.g., transcription factors, chaperones, kinases, foldases, initiation factors, ribosomal components, post translational modification factors, inter alia) other helper factors and combinations of helper factors could be tested and utilized in conjunction with P-lactoglobulin. The upregulation or downregulation of other cellular machinery, such as vacuoles, could also improve secretion of P- lactoglobulin or other dairy proteins.Example 3 - Conclusions

[0301] The inventors tested various expression strategies to optimize the expression and secretion of bovine P-lactoglobulin B. The inventors tested the effect of codon optimization, signal sequence, promoter, gene copy number, and helper factors in Pichia pastoris strains. Protein expression was evaluated through expression trials in various growth media, followed by analysis using SDS-PAGE and LC-MS.

[0302] Several factors were found to be involved in increased bovine P-lactoglobulin B expression and secretion. The native gene sequence outperformed gene sequences that were codon-optimized for P. pastoris. The native bovine secretion signal outperformed other secretion signals commonly used for secretion of heterologous proteins in P. pastoris. Further, the promoters UPP, FDH, HpFMD and A0X1 all showed promise for driving high expression. Additionally, a large increase in protein yield was achieved when moving from one to four copies of the P-lactoglobulin gene, with additional increases when moving to seven and nine copies of the gene. Gene copies were distributed at different loci under the control of different methanol inducible promoters.

[0303] All of the recombinant strains successfully expressed and secreted P-lactoglobulin, and the best performing strains were identified as sDLP264_19, sDLP265_2, sDLP265_25, and sDLP268_l. These strains produced the highest yields of secreted P-lactoglobulin in snap top tube expression trials (up to 0.9 g / L) and in fermentation trials (up to 7 g / L). LC-MS analysis of arepresentative sDLP265_25 fermentation trial supernatant sample showed that P-lactoglobulin comprised 99.13% of the total protein in the sample and at least 80% of the P-lactoglobulin secreted was mature, full-length P-lactoglobulin that was correctly processed at the C and N terminal ends.

[0304] sDLP264_19 was constructed using vectors pKC45P, pKC50P, pKC59P and pKC63P. Briefly, the parent strain BG11 was transformed with pKC45P to create strain sDLP009- 2, which was transformed with pKC50P to create strain sDLP021_82, which was transformed with pJTG-Cre-ARS and cured to create sDLP138_12, which was transformed with pKC59P to create sDLP185_51, which was transformed with pKC63P to create sDLP200_6, which was transformed with pJTG-Cre-ARS and cured to create the final strain sDLP264_19

[0305] sDLP265_2 and sDLP265_25 were each constructed using vectors pKC45P, pKC50P, pKC59P and pKC64P. Briefly, the parent strain BG11 was transformed with pKC45P to create strain sDLP009-2, which was transformed with pKC50P to create strain sDLP021_82, which was transformed with pJTG-Cre-ARS and cured to create sDLP138_12, which was transformed with pKC59P to create sDLP185_51, which was transformed with pKC64P to create sDLP201_62 and sDLP201_74, which were each transformed with pJTG-Cre-ARS and cured to create the final strains sDLP265_2 and sDLP265_25, respectively (which are different transformants with the same strain construction).

[0306] sDLP268_l was constructed using vectors pKC45P, pKC50P, pKC61P and pKC64P. Briefly, the parent strain BG11 was transformed with pKC45P to create strain sDLP009- 2, which was transformed with pKC50P to create strain sDLP021_82, which was transformed with pJTG-Cre-ARS and cured to create sDLP138_12, which was transformed with pKC61P to create sDLP186_5, which was transformed with pJTG-Cre-ARS and cured to create sDLP216_13, which was transformed with pKC64P to create sDLP226_70, which was transformed with pJTG-Cre- ARS and cured to create the final strain sDLP268_l.

[0307] The strains sDLP264_19, sDLP265_2, sDLP265_25, and sDLP268_l all comprise nine copies of sO Nat integrated into their genomes. Strain sDLP264_19 comprises: one copy under the control of an A0X1 promoter at the A0X1 locus; three copies, each under the control of an A0X1 promoter, at the T1 locus; two copies, where one copy is under the control of an FDHpromoter and one copy is under the control of an A0X1 promoter, at the T6 locus; and three copies, where one copy is under the control of a HpFMD promoter and two copies are each under the control of an A0X1 promoter, at the T9 locus.

[0308] Strains sDLP265_2 and sDLP265_25 were constructed similarly, but are different transformants. These comprise: one copy under the control of an AOX1 promoter at the AOX1 locus; three copies, each under the control of an AOX1 promoter, at the T1 locus; two copies, where one copy is under the control of a HpFMD promoter and one copy is under the control of an AOX1 promoter, at the T6 locus; and three copies, where one copy is under the control of a HpFMD promoter and two copies are each under the control of an AOX1 promoter, at the T9 locus.

[0309] Strain sDLP268_l comprises: one copy under the control of an AOX1 promoter at the AOX1 locus; three copies, each under the control of an AOX1 promoter, at the T1 locus; two copies, where one copy is under the control of a HpFMD promoter and one copy is under the control of an AOX1 promoter, at the T6 locus; and three copies, where one copy is under the control of a HpFMD promoter, one copy is under the control of an FDH promoter and one copy is under the control of an AOX1 promoter, at the T9 locus.

[0310] Importantly, the constructed strains enabled expression and secretion of significant amounts of very pure bovine P-lactoglobulin B. The results highlight the effectiveness of the genetic constructs, which were designed and employed by the inventors, in enhancing recombinant protein production and secretion of bovine P-lactoglobulin, providing insights for future biotechnological applications.

[0311] Further to this, the bovine P-lactoglobulin recovered from fermentation trials with the strains listed in Tables 9 and 9A was successfully used as an ingredient in prototype food products, including cheese, yoghurt, and ice cream. This demonstrated the effectiveness of the microbial dairy protein for food industry applications.

[0312] In this specification, where reference has been made to external sources of information, including patent specifications and other documents, this is generally for the purpose of providing a context for discussing the features of this disclosure. Unless stated otherwise,reference to such sources of information is not to be construed, in any jurisdiction, as an admission that such sources of information are prior art or form part of the common general knowledge in the art.

[0313] While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present disclosure.

[0314] The description herein may contain subject matter that falls outside of the scope of the claimed invention. This subject matter is included to aid understanding of the invention.References

[0315] The following references, as well as those cited above are incorporated by reference to the extent that they support the present disclosure and provide exemplary procedural or other details supplementary to those set forth herein.

[0316] U.S. Patent Nos. 6,451,572, 6,841,370, 6,974,690, 7,320,876, 7,078,035, 7,138,260, 9,924,728, 10,676,750 and 11,555,194. U.S. Patent Document Nos. US20210329941 A, US20220195000A. PCT Publication Nos. W02007112739, WO2014138679, WO2018115520, WO2022051696, W02022239000, WO2022269549.

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[0318] 2Ritchie, H. Sector by sector: where do global greenhouse gas emissions come from? Our World in Data. 2020. https: / / ourworldindata.org / ghg-emissions-by-sector.

[0319] 3Key findings of the 1990-2019 Inventory. Ministry for the Environment. 2021. https: / / environment.govt.nz / publications / new-zealands-greenhouse-gas-inventory-1990-2019- snapshot / key-findings-of-the-2021 -inventory / .

[0320] 4Sinclair, G.; Choy, F. Y. M. Synonymous Codon Usage Bias and the Expression of Human Glucocerebrosidase in the Methylotrophic Yeast, Pichia Pastoris. Protein Expr Purif. 2002, 26 (1), 96-105. https: / / doi.Org / 10.1016 / S 1046-5928(02)00526-0.

[0321] 5Outchkourov, N. S.; Stiekema, W. J.; Jongsma, M. A. Optimization of the Expression of Equistatin in Pichia Pastoris. Protein Expr Purif. 2002, 24 (1), 18-24. https: / / doi.org / 10.1006 / prep.2001.1523.

[0322] 6Mellitzer, A.; Flicker, K.; Weis, R.; Glieder, A. High Level Expression of Lignocellulolytic Enzymes in Pichia Pastoris. J Biotechnol. 2010, 150, 175. https: / / doi.Org / 10.1016 / j.jbiotec.2010.08.456.

[0323] 7Woo, J. H.; Liu, Y. Y.; Mathias, A.; Stavrou, S.; Wang, Z.; Thompson, J.; Neville, D. M. Gene Optimization Is Necessary to Express a Bivalent Anti-Human Anti-T Cell Immunotoxin in Pichia Pastoris. Protein Expr Purif. 2002, 25 (2), 270-282. https: / / doi.org / 10.1016 / S 1046-5928(02)00009-8.

[0324] 8Gouy, M.; Gautier, C. Codon Usage in Bacteria: Correlation with Gene Expressivity. Nucleic Acids Res. 1982, 10 (22), 7055-7074. https: / / doi.org / 10.1093 / nar / 10.22.7055.

[0325] 9Sharp, P. M.; Emery, L. R.; Zeng, K. Forces That Influence the Evolution of Codon Bias. Philos Trans R Soc Eond B Biol Sci. 2010, 365 (1544), 1203-1212. http s : / / doi. org / 10.1098 / rstb .2009.0305.

[0326] 10Benchling [Biology Software]. 2023. Retrieved from https: / / benchling.com.

[0327] 1Carrero, Juan J., Jason C. Casler, Francisco Valero, Pau Ferrer, and Benjamin S.Glick. An improved secretion signal enhances the secretion of model proteins from Pichia pastoris. Microbial cell factories. 2018, 17, 1-13.

[0328] 12Kim, T. R., Goto, Y., Hirota, N., Kuwata, K., Denton, H., Wu, S. Y., Sawyer, L, Batt, C. A. High-level expression of bovine beta-lactoglobulin in Pichia pastoris and characterization of its physical properties. Protein engineering. 1997, 70(11), 1339-1345.

[0329] 13Garrigds-Martmez, J., Vuoristo, K., Nieto-Taype, M.A., Tahtiharju, J., Uusitalo, J., Tukiainen, P., Schmid, C., Tolstorukov, I., Madden, K., Penttila, M. and Montesinos-Seguf, J.L. Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris. Microbial cell factories. 2021, 20, 1-12.

[0330] 14Macauley-Patrick, S., Fazenda, M. L., McNeil, B., & Harvey, L. M. Heterologous protein production using the Pichia pastoris expression system. Yeast. 2005, 22(4), 249-270.

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Claims

What, is claimed is:

1. A recombinant yeast or filamentous fungal strain, wherein the strain comprises at least 4 polynucleotides that are integrated in a genome of the strain, wherein:(i) each polynucleotide encodes a P-lactoglobulin protein that shares at least 95% sequence identity with a mammalian P-lactoglobulin protein;(ii) each polynucleotide includes an inducible promoter for obtaining expression of the P- lactoglobulin protein in the strain;(iii) each polynucleotide encodes a secretion signal for obtaining secretion of the P-lactoglobulin protein by the strain;(iv) optionally, each polynucleotide includes a transcription terminator; wherein, optionally, the genome further comprises at least 1 polynucleotide that encodes a helper factor selected from the group consisting of: transcription factors, chaperones, kinases, foldases, initiation factors, ribosomal components, post translational modification factors, and any combination thereof, wherein at least 80% of the P-lactoglobulin protein that is secreted by the strain is correctly processed at both N and C terminal ends, and wherein the P-lactoglobulin protein that is secreted by the strain constitutes at least 90% of total protein that is secreted by the strain.

2. The recombinant yeast or filamentous fungal strain of claim 1, wherein:(a) the at least 4 polynucleotides are integrated in at least two different loci in the genome;(b) the at least 4 polynucleotides are integrated in at least three different loci in the genome; or(c) the at least 4 polynucleotides are integrated in at least four different loci in the genome.

3. The recombinant yeast or filamentous fungal strain of claim 1 or claim 2, wherein:(a) the strain comprises 4 to 9 polynucleotides that encode the P-lactoglobulin protein;(b) the strain comprises 6 to 9 polynucleotides that encode the P-lactoglobulin protein;(c) the strain comprises 7 to 9 polynucleotides that encode the P-lactoglobulin protein; or(d) the strain comprises 9 to 11 polynucleotides that encode the P-lactoglobulin protein.

4. The recombinant yeast or filamentous fungal strain of any one of claims 1 to 3, wherein:(a) the inducible promoter comprises a FDH, HpFMD, or A0X1 promoter; or(b) the expression of the P-lactoglobulin protein is obtained by at least two different inducible promoters;(c) the expression of the P-lactoglobulin protein is obtained by at least three different inducible promoters;(d) the expression of the P-lactoglobulin protein is obtained by at least two different inducible promoters selected from the group consisting of: A0X1, FDH, and HpFMD; or(e) the expression of the P-lactoglobulin protein is obtained by at least three different inducible promoters selected from the group consisting of: A0X1, FDH, and HpFMD.

5. The recombinant yeast or filamentous fungal strain of any one of claims 1 to 4, wherein:(a) the transcription terminator comprises a FDH or A0X1 transcription terminator; and / or(c) the helper factor is selected from the group consisting of: PDI, PDI1, RPPO, KAR2 / BiP, SLY1, SEC1, HAC1, Erol, Hsp70, MXR1, GCN4, SEC53, Cenl, and any combination thereof.

6. The recombinant yeast or filamentous fungal strain of any one of claims 1 to 5, wherein:(a) the secretion signal comprises a native P-lactoglobulin secretion signal sequence;(b) the secretion signal comprises a secretion signal sequence selected from the group consisting of sO, si, si.5, s3, s4, s5, s6, and s9 (SEQ ID NO: 40-47); or(c) the secretion signal comprises a sO secretion signal sequence (SEQ ID NO: 40).

7. The recombinant yeast or filamentous fungal strain of any one of claims 1 to 6, wherein:(a) the strain is selected from the group consisting of a Trichoderma, a Pichia, a Kluyveromyces, and a Komagataella strain;(b) the strain is selected from the group consisting of Trichoderma reesei, Pichia pastoris, and Kluyveromyces lactis', or(c) the strain is Pichia pastoris.

8. The recombinant yeast or filamentous fungal strain of any one of claims 1 to 7, wherein:(a) the P-lactoglobulin protein shares at least 98% sequence identity with a mammalian P- lactoglobulin protein;(b) the P-lactoglobulin protein shares at least 99% sequence identity with a mammalian P- lactoglobulin protein;(c) the P-lactoglobulin protein shares up to 99.1%, up to 99.2%, up to 99.3%, up to 99.4%, up to 99.5%, up to 99.6%, up to 99.7%, up to 99.8%, up to 99.9% sequence identity with a mammalian P-lactoglobulin protein; or(d) the P-lactoglobulin protein shares 100% sequence identity with a mammalian P-lactoglobulin protein.

9. The recombinant yeast or filamentous fungal strain of any one of claims 1 to 8, wherein:(a) the mammalian P-lactoglobulin protein is selected from group consisting of bovine, sheep, and goat P-lactoglobulin protein; or(b) the mammalian P-lactoglobulin protein is a bovine P-lactoglobulin protein.

10. The recombinant yeast or filamentous fungal strain of any one of claims 1 to 9, wherein:(a) the P-lactoglobulin protein comprises the amino acid sequence of SEQ ID NO: 8;(b) the P-lactoglobulin protein consists of the amino acid sequence of SEQ ID NO: 8;(c) the P-lactoglobulin protein is encoded by a nucleotide sequence comprising SEQ ID NO: 7; or(d) the P-lactoglobulin protein is encoded by a nucleotide sequence consisting of SEQ ID NO: 7.

11. A recombinant yeast or filamentous fungal cell, wherein the cell comprises at least 4 polynucleotides that are integrated in a genome of the cell, wherein:(i) each polynucleotide encodes a P-lactoglobulin protein that shares at least 95% sequence identity with a mammalian P-lactoglobulin protein;(ii) each polynucleotide includes an inducible promoter for obtaining expression of the P- lactoglobulin protein in the cell;(iii) each polynucleotide encodes a secretion signal for obtaining secretion of the P-lactoglobulin protein by the cell;(iv) optionally, each polynucleotide includes a transcription terminator; wherein, optionally, the genome further comprises at least 1 polynucleotide that encodes a helper factor selected from the group consisting of: transcription factors, chaperones, kinases, foldases,initiation factors, ribosomal components, post translational modification factors, and any combination thereof, wherein at least 80% of the P-lactoglobulin protein that is secreted by the cell is correctly processed at both N and C terminal ends, and wherein the P-lactoglobulin protein that is secreted by the cell constitutes at least 90% of total protein that is secreted by the cell.

12. The recombinant yeast or filamentous fungal cell of claim 11, wherein:(a) the at least 4 polynucleotides are integrated in at least two different loci in the genome;(b) the at least 4 polynucleotides are integrated in at least three different loci in the genome; or(c) the at least 4 polynucleotides are integrated in at least four different loci in the genome.

13. The recombinant yeast or filamentous fungal cell of claim 11 or claim 12, wherein:(a) the cell comprises 4 to 9 polynucleotides that encode the P-lactoglobulin protein;(b) the cell comprises 6 to 9 polynucleotides that encode the P-lactoglobulin protein;(c) the cell comprises 7 to 9 polynucleotides that encode the P-lactoglobulin protein; or(d) the cell comprises 9 to 11 polynucleotides that encode the P-lactoglobulin protein.

14. The recombinant yeast or filamentous fungal cell of any one of claims 11 to 13, wherein:(a) the inducible promoter comprises a FDH, HpFMD, or A0X1 promoter;(b) the expression of the P-lactoglobulin protein is obtained by at least two different inducible promoters;(c) the expression of the P-lactoglobulin protein is obtained by at least three different inducible promoters;(d) the expression of the P-lactoglobulin protein is obtained by at least two different inducible promoters selected from the group consisting of: A0X1, FDH, and HpFMD; or(e) the expression of the P-lactoglobulin protein is obtained by at least three different inducible promoters selected from the group consisting of: A0X1, FDH, and HpFMD.

15. The recombinant yeast or filamentous fungal cell of any one of claims 11 to 14, wherein:(a) the transcription terminator comprises a FDH or A0X1 transcription terminator; and / or(b) the helper factor is selected from the group consisting of: PDI, PDI1 , RPPO, KAR2 / BiP, SLY 1 , SEC1, HAC1, Erol, Hsp70, MXR1, GCN4, SEC53, Cenl, and any combination thereof.

16. The recombinant yeast or filamentous fungal cell of any one of claims 11 to 15, wherein:(a) the secretion signal comprises a native P-lactoglobulin secretion signal sequence;(b) the secretion signal comprises a secretion signal sequence selected from the group consisting of sO, si, si.5, s3, s4, s5, s6, and s9 (SEQ ID NO: 40-47); or(c) the secretion signal comprises a sO secretion signal sequence (SEQ ID NO: 40).

17. The recombinant yeast or filamentous fungal cell of any one of claims 11 to 16, wherein:(a) the cell is selected from the group consisting of a Trichoderma, a Pichia, a Kluyveromyces, or a Komagataella cell;(b) the cell is selected from the group consisting of Trichoderma reesei, Pichia pastoris, and Kluyveromyces lactis cells; or(c) the cell is a Pichia pastoris cell.

18. The recombinant yeast or filamentous fungal cell of any one of claims 11 to 17, wherein:(a) the P-lactoglobulin protein shares at least 98% sequence identity with a mammalian P- lactoglobulin protein;(b) the P-lactoglobulin protein shares at least 99% sequence identity with a mammalian P- lactoglobulin protein;(c) the P-lactoglobulin protein shares up to 99.1%, up to 99.2%, up to 99.3%, up to 99.4%, up to 99.5%, up to 99.6%, up to 99.7%, up to 99.8%, up to 99.9% sequence identity with a mammalian P-lactoglobulin protein; or(d) the P-lactoglobulin protein shares 100% sequence identity with a mammalian P-lactoglobulin protein.

19. The recombinant yeast or filamentous fungal cell of any one of claims 11 to 18, wherein:(a) the mammalian P-lactoglobulin protein is selected from group consisting of bovine, sheep, and goat P-lactoglobulin protein; or(b) the mammalian P-lactoglobulin protein is a bovine P-lactoglobulin protein.

20. The recombinant yeast or filamentous fungal cell of any one of claims 11 to 19, wherein:(a) the P-lactoglobulin protein comprises the amino acid sequence of SEQ ID NO: 8;(b) the P-lactoglobulin protein consists of the amino acid sequence of SEQ ID NO: 8;(c) the P-lactoglobulin protein is encoded by a nucleotide sequence comprising SEQ ID NO: 7; or(d) the P-lactoglobulin protein is encoded by a nucleotide sequence consisting of SEQ ID NO: 7.

21. A plurality of expression vectors for constructing a recombinant yeast or filamentous fungal strain, wherein each expression vector comprises at least 1 polynucleotide that is capable of integration into a genome of the strain, wherein:(i) each polynucleotide encodes a P-lactoglobulin protein that shares at least 95% sequence identity with a mammalian P-lactoglobulin protein;(ii) each polynucleotide includes an inducible promoter for obtaining expression of the P- lactoglobulin protein in the strain;(iii) each polynucleotide encodes a secretion signal for obtaining secretion of the P-lactoglobulin protein by the strain; wherein, optionally, each polynucleotide further comprises a transcription terminator sequence, and wherein the plurality of expression vectors allows integration of at least 4 of the polynucleotides in at least two different loci in the genome of the strain.

22. The plurality of expression vectors of claim 21, wherein:(a) each expression vector comprises 2 to 4 polynucleotides that encode a P-lactoglobulin protein; or(b) each expression vector comprises 2 to 3 polynucleotides that encode a P-lactoglobulin protein.

23. The plurality of expression vectors of claim 21 or claim 22, wherein:(a) the plurality of expression vectors allows integration of the at least 4 polynucleotides in at least three different loci in the genome; or(b) the plurality of expression vectors allows integration of the at least 4 polynucleotides in at least four different loci in the genome.

24. The plurality of expression vectors of any one of claims 21 to 23, wherein:(a) at least 80% of the P-lactoglobulin protein that is secreted by the strain is correctly processed at both N and C terminal ends, and / or(b) wherein the P-lactoglobulin protein that is secreted by the strain constitutes at least 90% of total protein that is secreted by the strain.

25. The plurality of expression vectors of any one of claims 21 to 24, wherein:(a) the inducible promoter comprises a FDH, HpFMD, or A0X1 promoter; or(b) at least one expression vector in the plurality includes at least two different inducible promoters for obtaining the expression of the P-lactoglobulin protein; or(c) at least one expression vector in the plurality includes at least three different inducible promoters for obtaining the expression of the P-lactoglobulin protein; or(d) at least one expression vector in the plurality includes at least two different inducible promoters for obtaining the expression of the P-lactoglobulin protein, such promoters being selected from the group consisting of: A0X1, FDH, and HpFMD; or(e) at least one expression vector in the plurality includes at least three different inducible promoters for obtaining the expression of the P-lactoglobulin protein, such promoters being selected from the group consisting of: A0X1, FDH, and HpFMD; and / or(f) the transcription terminator comprises a FDH or A0X1 transcription terminator.

26. The plurality of expression vectors of any one of claims 21 to 25, wherein:(a) the section signal comprises a native P-lactoglobulin secretion signal sequence;(b) the secretion signal comprises a secretion signal sequence selected from the group consisting of sO, si, si.5, s3, s4, s5, s6, and s9 (SEQ ID NO: 40-47); or(c) the secretion signal comprises a sO secretion signal sequence (SEQ ID NO: 40).

27. The plurality of expression vectors of any one of claims 21 to 26, wherein:(a) the strain is selected from the group consisting of a Trichoderma, a Pichia, a Kluyveromyces, or a Komagataella strain;(b) the strain is selected from the group consisting of Trichoderma reesei, Pichia pastoris, and Kluyveromyces lactis', or(c) the strain is Pichia pastoris.

28. The plurality of expression vectors of any one of claims 21 to 27, wherein:(a) the P-lactoglobulin protein shares at least 98% sequence identity with a mammalian P- lactoglobulin protein;(b) the P-lactoglobulin protein shares at least 99% sequence identity with a mammalian P- lactoglobulin protein;(c) the P-lactoglobulin protein shares up to 99.1%, up to 99.2%, up to 99.3%, up to 99.4%, up to 99.5%, up to 99.6%, up to 99.7%, up to 99.8%, up to 99.9% sequence identity with a mammalian P-lactoglobulin protein; or(d) the P-lactoglobulin protein shares 100% sequence identity with a mammalian P-lactoglobulin protein.

29. The plurality of expression vectors of any one of claims 21 to 28, wherein:(a) the mammalian P-lactoglobulin protein is selected from group consisting of bovine, sheep, and goat P-lactoglobulin protein; or(b) the mammalian P-lactoglobulin protein is a bovine P-lactoglobulin protein.

30. The plurality of expression vectors of any one of claims 21 to 29, wherein:(a) the P-lactoglobulin protein comprises the amino acid sequence of SEQ ID NO: 8;(b) the P-lactoglobulin protein consists of the amino acid sequence of SEQ ID NO: 8;(c) the P-lactoglobulin protein is encoded by a nucleotide sequence comprising SEQ ID NO: 7; or(d) the P-lactoglobulin protein is encoded by a nucleotide sequence consisting of SEQ ID NO: 7.

31. A P-lactoglobulin protein that is secreted by a recombinant yeast or filamentous fungal strain of any one of claims 1 to 10 or by a recombinant yeast or filamentous fungal cell of any one of claims 11 to 20, wherein:(a) the P-lactoglobulin protein shares at least 98% sequence identity with a mammalian P- lactoglobulin protein;(b) the P-lactoglobulin protein shares at least 99% sequence identity with a mammalian P- lactoglobulin protein;(c) the P-lactoglobulin protein shares up to 99.1%, up to 99.2%, up to 99.3%, up to 99.4%, up to 99.5%, up to 99.6%, up to 99.7%, up to 99.8%, up to 99.9% sequence identity with a mammalian P-lactoglobulin protein; or(d) the P-lactoglobulin protein shares 100% sequence identity with a mammalian P-lactoglobulin protein.

32. The P-lactoglobulin protein of claim 31, wherein:(a) the strain is selected from the group consisting of a Trichoderma, a Pichia, a Kluyveromyces, or a Komagataella strain;(b) the strain is selected from the group consisting of Trichoderma reesei, Pichia pastoris, and Kluyveromyces lactis', or(c) the strain is Pichia pastoris.

33. The P-lactoglobulin protein of claim 31, wherein:(a) the cell is selected from the group consisting of a Trichoderma, a Pichia, a Kluyveromyces, or a Komagataella cell;(b) the cell is selected from the group consisting of Trichoderma reesei, Pichia pastoris, and Kluyveromyces lactis cells; or(c) the cell is a Pichia pastoris cell.

34. The P-lactoglobulin protein of any one of claims 31 to 33, wherein:(a) the mammalian P-lactoglobulin protein is selected from group consisting of bovine, sheep, and goat P-lactoglobulin protein; or(b) the mammalian P-lactoglobulin protein is a bovine P-lactoglobulin protein.

35. The P-lactoglobulin protein of any one of claims 31 to 34, wherein:(a) the P-lactoglobulin protein comprises the amino acid sequence of SEQ ID NO: 8; or(b) the P-lactoglobulin protein consists of the amino acid sequence of SEQ ID NO: 8.

36. The P-lactoglobulin protein of any one of claims 31 to 35, wherein:(a) the P-lactoglobulin that is secreted constitutes at least 90% of total protein secreted by the strain or the cell; or(b) the P-lactoglobulin that is secreted constitutes at least 99% of total protein secreted by the strain or the cell.

37. The P-lactoglobulin protein of any one of claims 31 to 36, which is used to produce a dairy product selected from the group consisting of: milk, evaporated milk, milk powder, milk substitute, evaporated milk substitute, and milk substitute powder.

38. The P-lactoglobulin protein of any one of claims 31 to 36, which is used to produce a dairy product selected from the group consisting of cream, creamer, cocktail mixer, custard, cheese, cream cheese, dessert topping, gelato, ghee, ice cream, milkshake, pudding, butter, spread, sauce, sorbet, smoothies, snack food, yoghurt, yoghurt drink, and frozen yoghurt.

39. The P-lactoglobulin protein of any one of claims 31 to 36, which is used to produce a dairy product selected from the group consisting of infant formula, toddler formula, nutritional supplement, energy supplement, sports supplement, weight gain supplement, weight loss supplement, energy drink, sports drink, weight gain drink, weight loss drink, energy food, sports food, weight gain food, and weight loss food.

40. The P-lactoglobulin protein of any one of claims 31 to 36, which is used to produce a dairy ingredient for one or more of: baked goods, beverages, cooked goods, frozen goods, cooking mixes, baking mixes, beverage mixes, and sauce mixes.

41. A recombinant Pichia pastoris strain, wherein the strain comprises at least 4 polynucleotides that are integrated in a genome of the strain,wherein:(i) each polynucleotide encodes a dairy protein that shares at least 95% sequence identity with a mammalian dairy protein selected from the group consisting of: P-lactoglobulin, lactoferrin, a- lactalbumin, glycomacropeptide, casein, and whey;(ii) each polynucleotide includes an inducible promoter for obtaining expression of the dairy protein in the strain(iii) each polynucleotide encodes a secretion signal for obtaining secretion of the dairy protein by the strain;(iv) optionally, each polynucleotide includes a transcription terminator; wherein, optionally, the genome further comprises at least 1 polynucleotide that encodes a helper factor selected from the group consisting of: transcription factors, chaperones, kinases, foldases, initiation factors, ribosomal components, post translational modification factors, and any combination thereof, and wherein at least 80% of the dairy protein that is secreted by the strain is correctly processed at both N and C terminal ends, and wherein the dairy protein that is secreted by the strain constitutes at least 90% of total protein that is secreted by the strain.

42. The recombinant Pichia pastoris strain of claim 41, wherein:(a) the at least 4 polynucleotides are integrated in at least two different loci in the genome;(b) the at least 4 polynucleotides are integrated in at least three different loci in the genome; or(c) the at least 4 polynucleotides are integrated in at least four different loci in the genome.

43. The recombinant Pichia pastoris strain of claim 41 or claim 42, wherein:(a) the strain comprises 4 to 9 polynucleotides that encode the P-lactoglobulin protein;(b) the strain comprises 6 to 9 polynucleotides that encode the P-lactoglobulin protein;(c) the strain comprises 7 to 9 polynucleotides that encode the dairy protein; or(d) the strain comprises 9 to 11 polynucleotides that encode the dairy protein.

44. The recombinant Pichia pastoris strain of any one of claims 41 to 43, wherein:(a) the inducible promoter comprises a FDH, HpFMD, or A0X1 promoter;(b) the expression of the dairy protein is obtained by at least two different inducible promoters;(c) the expression of the dairy protein is obtained by at least three different inducible promoters;(d) the expression of the dairy protein is obtained by at least two different inducible promoters selected from the group consisting of: A0X1, FDH, and HpFMD; or(e) the expression of the dairy protein is obtained by at least three different inducible promoters selected from the group consisting of: A0X1, FDH, and HpFMD.

45. The recombinant Pichia pastoris strain of any one of claims 41 to 44, wherein:(a) the secretion signal comprises a native dairy protein secretion signal sequence;(b) the secretion signal comprises a secretion signal sequence selected from the group consisting of sO, si, si.5, s3, s4, s5, s6, and s9 (SEQ ID NO: 40-47); or(c) the secretion signal comprises a sO secretion signal sequence (SEQ ID NO: 40).

46. The recombinant Pichia pastoris strain of any one of claims 41 to 45, wherein:(a) the transcription terminator comprises a FDH or A0X1 transcription terminator; and / or(b) the helper factor is selected from the group consisting of: PDI, PDI1 , RPPO, KAR2 / BiP, SLY 1 , SEC1, HAC1, Erol, Hsp70, MXR1, GCN4, SEC53, Cenl, and any combination thereof.

47. The recombinant Pichia pastoris strain of any one of claims 41 to 46, wherein:(a) the strain does not contain expression vector derived drug resistance markers; and / or(b) the strain does not contain expression vector derived bacterial DNA.

48. The recombinant Pichia pastoris strain of any one of claims 41 to 47, wherein the dairy protein is P-lactoglobulin protein and:(a) the P-lactoglobulin protein shares at least 98% sequence identity with a mammalian P- lactoglobulin protein;(b) the P-lactoglobulin protein shares at least 99% sequence identity with a mammalian P- lactoglobulin protein;(c) the P-lactoglobulin protein shares up to 99.1%, up to 99.2%, up to 99.3%, up to 99.4%, up to 99.5%, up to 99.6%, up to 99.7%, up to 99.8%, up to 99.9% sequence identity with a mammalian P-lactoglobulin protein; or(d) the P-lactoglobulin protein shares 100% sequence identity with a mammalian P-lactoglobulin protein.

49. The recombinant Pichia pastoris strain of any one of claims 41 to 48, wherein the dairy protein is P-lactoglobulin protein and:(a) the mammalian dairy protein is selected from group consisting of bovine, sheep, and goat P- lactoglobulin protein; or(b) the mammalian dairy protein is a bovine P-lactoglobulin protein.

50. The recombinant Pichia pastoris strain of any one of claims 41 to 49, wherein the dairy protein is P-lactoglobulin protein and:(a) the P-lactoglobulin protein comprises the amino acid sequence of SEQ ID NO: 8; or(b) the P-lactoglobulin protein consists of the amino acid sequence of SEQ ID NO: 8;(c) the P-lactoglobulin protein is encoded by a nucleotide sequence comprising SEQ ID NO: 7; or(d) the P-lactoglobulin protein is encoded by a nucleotide sequence consisting of SEQ ID NO: 7.

51. A recombinant Pichia pastoris cell, wherein the cell comprises at least 4 polynucleotides that are integrated in a genome of the cell, wherein:(i) each polynucleotide encodes a dairy protein that shares at least 95% identity with a mammalian dairy protein selected from the group consisting of: P-lactoglobulin, lactoferrin, a-lactalbumin, glycomacropeptide, casein, and whey;(ii) each polynucleotide includes an inducible promoter for obtaining expression of the dairy protein in the cell;(iii) each polynucleotide encodes a secretion signal for obtaining secretion of the dairy protein by the cell;(iv) optionally, each polynucleotide includes a transcription terminator; wherein, optionally, the genome further comprises at least 1 polynucleotide that encodes a helper factor selected from the group consisting of: transcription factors, chaperones, kinases, foldases, initiation factors, ribosomal components, post translational modification factors, and any combination thereof, and wherein at least 80% of the dairy protein that is secreted by the cell is correctly processed at both N and C terminal ends, andwherein the dairy protein that is secreted by the cell constitutes at least 90% of total protein that is secreted by the cell.

52. The recombinant Pichia pastoris cell of claim 51, wherein:(a) the at least 4 polynucleotides are integrated in at least two different loci in the genome;(b) the at least 4 polynucleotides are integrated in at least three different loci in the genome; or(c) the at least 4 polynucleotides are integrated in at least four different loci in the genome.

53. The recombinant Pichia pastoris cell of claim 51 or claim 52, wherein:(a) the strain comprises 4 to 9 polynucleotides that encode the dairy protein;(b) the strain comprises 6 to 9 polynucleotides that encode the dairy protein;(c) the cell comprises 7 to 9 polynucleotides that encode the dairy protein; or(d) the cell comprises 9 to 11 polynucleotides that encode the dairy protein.

54. The recombinant Pichia pastoris cell of any one of claims 51 to 53, wherein:(a) the cell does not contain expression vector derived drug resistance markers; and / or(b) the cell does not contain expression vector derived bacterial DNA.

55. The recombinant Pichia pastoris cell of any one of claims 51 to 54, wherein:(a) the inducible promoter comprises a FDH, HpFMD, or A0X1 promoter;(b) the expression of the dairy protein is obtained by at least two different inducible promoters;(c) the expression of the dairy protein is obtained by at least three different inducible promoters;(d) the expression of the dairy protein is obtained by at least two different inducible promoters selected from the group consisting of: A0X1, FDH, and HpFMD; or(e) the expression of the dairy protein is obtained by at least three different inducible promoters selected from the group consisting of: A0X1, FDH, and HpFMD.

56. The recombinant Pichia pastoris cell of any one of claims 51 to 55, wherein:(a) the transcription terminator comprises a FDH or A0X1 transcription terminator; and / or(b) the helper factor is selected from the group consisting of: PDI, PDI1 , RPPO, KAR2 / BiP, SLY 1 , SEC1, HAC1, Erol, Hsp70, MXR1, GCN4, SEC53, Cenl, and any combination thereof.

57. The recombinant Pichia pastoris cell of any one of claims 51 to 56, wherein:(a) the secretion signal comprises a native dairy protein secretion signal sequence;(b) the secretion signal comprises a secretion signal sequence selected from the group consisting of sO, si, si.5, s3, s4, s5, s6, and s9 (SEQ ID NO: 40-47); or(c) the secretion signal comprises a sO secretion signal sequence (SEQ ID NO: 40).

58. The recombinant Pichia pastoris cell of any one of claims 51 to 57, wherein the dairy protein is P-lactoglobulin protein and:(a) the P-lactoglobulin protein shares at least 98% sequence identity with a mammalian P- lactoglobulin protein;(b) the P-lactoglobulin protein shares at least 99% sequence identity with a mammalian P- lactoglobulin protein;(c) the P-lactoglobulin protein shares up to 99.1%, up to 99.2%, up to 99.3%, up to 99.4%, up to 99.5%, up to 99.6%, up to 99.7%, up to 99.8%, up to 99.9% sequence identity with a mammalian P-lactoglobulin protein; or(d) the P-lactoglobulin protein shares 100% sequence identity with a mammalian P-lactoglobulin protein.

59. The recombinant Pichia pastoris cell of any one of claims 51 to 58, wherein the dairy protein is P-lactoglobulin protein and:(a) the mammalian dairy protein is selected from group consisting of bovine, sheep, and goat P- lactoglobulin protein; or(b) the mammalian dairy protein is a bovine P-lactoglobulin protein.

60. The recombinant Pichia pastoris cell of any one of claims 51 to 59, wherein the dairy protein is P-lactoglobulin protein and:(a) the P-lactoglobulin protein comprises the amino acid sequence of SEQ ID NO: 8;(b) the P-lactoglobulin protein consists of the amino acid sequence of SEQ ID NO: 8;(c) the P-lactoglobulin protein is encoded by a nucleotide sequence comprising SEQ ID NO: 7; or(d) the P-lactoglobulin protein is encoded by a nucleotide sequence consisting of SEQ ID NO: 7.

61. A dairy protein that is secreted by a recombinant Pichia pastoris strain of any one of claims 41 to 50 or by a recombinant Pichia pastoris cell of any one of claims 51 to 60, wherein:(a) the dairy protein shares at least 98% sequence identity with a mammalian P-lactoglobulin, lactoferrin, a-lactalbumin, glycomacropeptide, casein, or whey protein;(b) the dairy protein shares at least 99% sequence identity with a mammalian P-lactoglobulin, lactoferrin, a-lactalbumin, glycomacropeptide, casein, or whey protein;(c) the dairy protein shares up to 99.1%, up to 99.2%, up to 99.3%, up to 99.4%, up to 99.5%, up to 99.6%, up to 99.7%, up to 99.8%, up to 99.9% sequence identity with a mammalian P- lactoglobulin, lactoferrin, a-lactalbumin, glycomacropeptide, casein, or whey protein; or(d) the dairy protein shares 100% sequence identity with a mammalian P-lactoglobulin, lactoferrin, a-lactalbumin, glycomacropeptide, casein, or whey protein.

62. The dairy protein of claim 61, wherein the mammalian P-lactoglobulin, lactoferrin, a- lactalbumin, glycomacropeptide, casein, or whey protein is a human, bovine, sheep, or goat protein.

63. The dairy protein of claim 61, wherein the mammalian P-lactoglobulin, lactoferrin, a- lactalbumin, glycomacropeptide, casein, or whey protein protein is a bovine protein.

64. The dairy protein of claim 61, wherein the mammalian P-lactoglobulin, lactoferrin, a- lactalbumin, glycomacropeptide, casein, or whey protein is a Bos taurus protein.

65. The dairy protein of claim 61, wherein the dairy protein is a P-lactoglobulin protein and:(a) the P-lactoglobulin protein comprises the amino acid sequence of SEQ ID NO: 8; or(b) the P-lactoglobulin protein consists of the amino acid sequence of SEQ ID NO: 8.

66. The dairy protein of any one of claims 61 to 65, wherein:(a) the dairy protein that is secreted constitutes at least 90% of total protein secreted by the strain or the cell; or(b) the dairy protein that is secreted constitutes at least 99% of total protein secreted by the strain or the cell.

67. The dairy protein of any one of claims 61 to 66, which is used to produce a dairy product selected from the group consisting of: milk, evaporated milk, milk powder, milk substitute, evaporated milk substitute, and milk substitute powder.

68. The dairy protein of any one of claims 61 to 66, which is used to produce a dairy product selected from the group consisting of cream, creamer, cocktail mixer, custard, cheese, cream cheese, dessert topping, gelato, ghee, ice cream, milkshake, pudding, butter, spread, sauce, sorbet, smoothies, snack food, yoghurt, yoghurt drink, and frozen yoghurt.

69. The dairy protein of any one of claims 61 to 66, which is used to produce a dairy product selected from the group consisting of infant formula, toddler formula, nutritional supplement, energy supplement, sports supplement, weight gain supplement, weight loss supplement, energy drink, sports drink, weight gain drink, weight loss drink, energy food, sports food, weight gain food, and weight loss food.

70. The dairy protein of any one of claims 61 to 66, which is used to produce a dairy ingredient for one or more of: baked goods, beverages, cooked goods, frozen goods, cooking mixes, baking mixes, beverage mixes, and sauce mixes.

71. An expression vector for constructing a recombinant yeast or filamentous fungal strain,wherein each expression vector comprises at least 2 polynucleotides that are capable of integration into a genome of the strain, wherein:(i) each polynucleotide encodes a P-lactoglobulin protein that shares at least 95% sequence identity with a mammalian P-lactoglobulin protein;(ii) each polynucleotide includes an inducible promoter for obtaining expression of the P- lactoglobulin protein in the strain;(iii) each polynucleotide encodes a secretion signal for obtaining secretion of the P-lactoglobulin protein by the strain; wherein, optionally, each polynucleotide further comprises a transcription terminator sequence, and wherein the expression vector allows integration of at least 2 polynucleotides in a locus in the genome of the strain.

72. The expression vectors of claim 71, wherein:(a) the expression vector comprises 2 to 4 polynucleotides that encode a P-lactoglobulin protein; or(b) the expression vector comprises 2 to 3 polynucleotides that encode a P-lactoglobulin protein.

73. The expression vector of claim 71 or claim 72, wherein:(a) the expression vector allows integration of the at least 4 polynucleotides in at least three different loci in the genome; or(b) the expression vector allows integration of the at least 4 polynucleotides in at least four different loci in the genome.

74. The expression vector of any one of claims 71 to 73, wherein:(a) at least 80% of the P-lactoglobulin protein that is secreted by the strain is correctly processed at both N and C terminal ends, and / or(b) wherein the P-lactoglobulin protein that is secreted by the strain constitutes at least 90% of total protein that is secreted by the strain.

75. The expression vector of any one of claims 71 to 74, wherein:(a) the inducible promoter comprises a FDH, HpFMD, or A0X1 promoter; or(b) the expression vector includes at least two different inducible promoters for obtaining the expression of the P-lactoglobulin protein; or(c) the expression vector includes at least three different inducible promoters for obtaining the expression of the P-lactoglobulin protein; or(d) the expression vector includes at least two different inducible promoters for obtaining the expression of the P-lactoglobulin protein, such promoters being selected from the group consisting of: A0X1, FDH, and HpFMD; or(e) the expression vector includes at least three different inducible promoters for obtaining the expression of the P-lactoglobulin protein, such promoters being selected from the group consisting of: A0X1, FDH, and HpFMD; and / or(f) the transcription terminator comprises a FDH or A0X1 transcription terminator.

76. The expression vector of any one of claims 71 to 75, wherein:(a) the secretion signal comprises a native P-lactoglobulin secretion signal sequence;(b) the secretion signal comprises a secretion signal sequence selected from the group consisting of sO, si, si.5, s3, s4, s5, s6, and s9 (SEQ ID NO: 40-47); or(c) the secretion signal comprises a sO secretion signal sequence (SEQ ID NO: 40).

77. The expression vector of any one of claims 71 to 76, wherein:(a) the strain is selected from the group consisting of a Trichoderma, a Pichia, a Kluyveromyces, or a Komagataella strain;(b) the strain is selected from the group consisting of Trichoderma reesei, Pichia pastoris, and Kluyveromyces lactis', or(c) the strain is Pichia pastoris.

78. The expression vector of any one of claims 71 to 77, wherein:(a) the P-lactoglobulin protein shares at least 98% sequence identity with a mammalian P- lactoglobulin protein;(b) the P-lactoglobulin protein shares at least 99% sequence identity with a mammalian P- lactoglobulin protein;(c) the P-lactoglobulin protein shares up to 99.1%, up to 99.2%, up to 99.3%, up to 99.4%, up to 99.5%, up to 99.6%, up to 99.7%, up to 99.8%, up to 99.9% sequence identity with a mammalian P-lactoglobulin protein; or(d) the P-lactoglobulin protein shares 100% sequence identity with a mammalian P-lactoglobulin protein.

79. The expression vector of any one of claims 71 to 78, wherein:(a) the mammalian P-lactoglobulin protein is selected from group consisting of bovine, sheep, and goat P-lactoglobulin protein; or(b) the mammalian P-lactoglobulin protein is a bovine P-lactoglobulin protein.

80. The expression vector of any one of claims 71 to 79, wherein:(a) the P-lactoglobulin protein comprises the amino acid sequence of SEQ ID NO: 8;(b) the P-lactoglobulin protein consists of the amino acid sequence of SEQ ID NO: 8;(c) the P-lactoglobulin protein is encoded by a nucleotide sequence comprising SEQ ID NO: 7; or(d) the P-lactoglobulin protein is encoded by a nucleotide sequence consisting of SEQ ID NO: 7.

81. A plurality of expression vectors for constructing a recombinant Pichia pastoris strain, wherein each expression vector comprises at least 1 polynucleotide that is capable of integration into a genome of the strain, wherein:(i) each polynucleotide encodes a dairy protein that shares at least 95% sequence identity with a mammalian dairy protein selected from the group consisting of: P-lactoglobulin, lactoferrin, a- lactalbumin, glycomacropeptide, casein, and whey;(ii) each polynucleotide includes an inducible promoter for obtaining expression of the dairy protein in the strain;(iii) each polynucleotide encodes a secretion signal for obtaining secretion of the dairy protein by the strain; wherein, optionally, each polynucleotide further comprises a transcription terminator sequence, and wherein the plurality of expression vectors allows integration of at least 4 of the polynucleotides in at least two different loci in the genome of the strain.

82. The plurality of expression vectors of claim 81, wherein:Ill(a) each expression vector comprises 2 to 4 polynucleotides that encode the dairy protein; or(b) each expression vector comprises 2 to 3 polynucleotides that encode the dairy protein.

83. The plurality of expression vectors of claim 81 or claim 82, wherein:(a) the plurality of expression vectors allows integration of the at least 4 polynucleotides in at least three different loci in the genome; or(b) the plurality of expression vectors allows integration of the at least 4 polynucleotides in at least four different loci in the genome.

84. The plurality of expression vectors of any one of claims 81 to 83, wherein:(a) at least 80% of the dairy protein that is secreted by the strain is correctly processed at both N and C terminal ends, and / or(b) wherein the dairy protein that is secreted by the strain constitutes at least 90% of total protein that is secreted by the strain.

85. The plurality of expression vectors of any one of claims 81 to 84, wherein:(a) the inducible promoter comprises a FDH, HpFMD, or A0X1 promoter;(b) at least one expression vector in the plurality includes at least two different inducible promoters for obtaining the expression of the dairy protein;(c) at least one expression vector in the plurality includes at least three different inducible promoters for obtaining the expression of the dairy protein;(d) at least one expression vector in the plurality includes at least two different inducible promoters for obtaining the expression of the dairy protein, such promoters being selected from the group consisting of: A0X1, FDH, and HpFMD; or(e) at least one expression vector in the plurality includes at least three different inducible promoters for obtaining the expression of the dairy protein, such promoters being selected from the group consisting of: A0X1, FDH, and HpFMD.

86. The plurality of expression vectors of any one of claims 81 to 85, wherein:(a) the section signal comprises a native dairy protein secretion signal sequence;(b) the secretion signal comprises a secretion signal sequence selected from the group consisting of sO, si, si.5, s3, s4, s5, s6, and s9 (SEQ ID NO: 40-47); or(c) the secretion signal comprises a sO secretion signal sequence (SEQ ID NO: 40).

87. The plurality of expression vectors of any one of claims 81 to 86, wherein: the transcription terminator comprises a FDH or A0X1 transcription terminator.

88. The plurality of expression vectors of any one of claims 81 to 87, wherein the dairy protein is P-lactoglobulin protein and:(a) the P-lactoglobulin protein shares at least 98% sequence identity with a mammalian P- lactoglobulin protein;(b) the P-lactoglobulin protein shares at least 99% sequence identity with a mammalian P- lactoglobulin protein;(c) the P-lactoglobulin protein shares up to 99.1%, up to 99.2%, up to 99.3%, up to 99.4%, up to 99.5%, up to 99.6%, up to 99.7%, up to 99.8%, up to 99.9% sequence identity with a mammalian P-lactoglobulin protein; or(d) the P-lactoglobulin protein shares 100% sequence identity with a mammalian P-lactoglobulin protein.

89. The plurality of expression vectors of any one of claims 81 to 88, wherein the dairy protein is 0-lactoglobulin protein and:(a) the mammalian dairy protein is selected from group consisting of bovine, sheep, and goat 0- lactoglobulin protein; or(b) the mammalian dairy protein is a bovine P-lactoglobulin protein.

90. The plurality of expression vectors of any one of claims 81 to 89, wherein the dairy protein is P-lactoglobulin protein and:(a) the -lactoglobulin protein comprises the amino acid sequence of SEQ ID NO: 8;(b) the -lactoglobulin protein consists of the amino acid sequence of SEQ ID NO: 8;(c) the -lactoglobulin protein is encoded by a nucleotide sequence comprising SEQ ID NO: 7; or(d) the -lactoglobulin protein is encoded by a nucleotide sequence consisting of SEQ ID NO: 7.

91. A method of producing a P-lactoglobulin protein, the method comprising: incubating (i) the recombinant yeast or filamentous fungi strain of any one of claims 1 to 10; or (ii) the recombinant yeast or filamentous fungi cell of any one of claims 11 to 20; under conditions to allow expression and secretion of the P-lactoglobulin protein.

92. A method of producing a dairy protein, wherein the dairy protein is selected from the group consisting of a P-lactoglobulin, lactoferrin, a-lactalbumin, glycomacropeptide, casein, and whey protein, the method comprising: incubating (i) the recombinant Pichia pastoris strain of any one of claims 41 to 50; or (ii) the recombinant Pichia pastoris cell of any one of claims 51 to 60, under conditions to allow expression and secretion of the dairy protein.

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