Proteins and products containing them

Abstract

The present disclosure relates to modified proteins, which comprise an amino acid sequence having one or more amino acid modifications relative to a reference protein.

Description

[Technical field]

[0001] The present invention relates to a novel protein and a food product containing the same. [Background technology]

[0002] References which are deemed relevant as background to the subject matter of this disclosure are listed below. GB2123672 WO8402450 WO2019215730 Liu Yang, et al. Chemical Senses, 2019, Vol 44, 607-614 and CN109627307

[0003] The inclusion of the above references in this specification should not be inferred as implying that they are in any way relevant to the patentability of the subject matter of the present disclosure.

[0004] Excessive sugar consumption is becoming recognized as a root cause of metabolic syndrome, and there has been growing interest in sugar alternatives.

[0005] Sweet proteins have been proposed as alternatives to sugar.

[0006] GB2123672 describes sweet proteins such as thaumatin and monellin, with weakly acidic polysaccharide gums incorporated therein, and optionally further edible acids or bulking agents, in various beverages, mouthwashes or as pharmaceutical bases.

[0007] WO8402450 describes coating the surface of a chewing gum composition containing a gum base, a sweetener, and a flavor with thaumatin or monellin.

[0008] WO2019215730 discloses modified proteins with improved food-related properties.

[0009] Liu Yang, et al. Chemical Senses, 2019, Vol 44, 607-614 and CN109627307 describe modifications in the loop region of single-chain monellin (MNEI). Summary of the Invention

[0010] According to some aspects, there is provided a modified protein comprising an amino acid sequence comprising at least one amino acid modification in at least one of: (i) an alpha helix of a reference protein, (ii) a core of a reference protein, (iii) a sweet loop of a reference protein, (iv) a linker region of a reference protein, or (v) any combination thereof, wherein the reference protein has an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

[0011] According to some embodiments, a modified protein is provided comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56 compared to a reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0012] According to some aspects, a food product is provided that comprises a protein of the present disclosure.

[0013] Embodiment Certain embodiments of the present disclosure are described in the following numbered paragraphs. The following description is intended to supplement the general description above and is not intended to be limiting thereof in any way.

[0014] 1. A modified protein comprising an amino acid sequence comprising at least one amino acid modification in at least one of: (i) an alpha helix of a reference protein, (ii) a core of a reference protein, (iii) a sweet loop of a reference protein, (iv) a linker region of a reference protein, or (v) any combination thereof, wherein the reference protein has an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

[0015] 2. The modified protein of embodiment 1, having an energy expressed in REU of less than about -315.

[0016] 3. The modified protein of embodiment 2, having an energy expressed in REU of less than about -321.

[0017] 4. A modified protein according to any one of embodiments 1 to 3, comprising an amino acid sequence that is 90% to 98% identical to a reference protein.

[0018] 5. A modified protein comprising an amino acid sequence having at least three amino acid deletions in amino acids located between amino acid T46 and amino acid I56 compared to a reference protein, wherein the reference protein has an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.

[0019] 6. The modified protein according to any one of embodiments 1 to 5, comprising amino acid deletions compared to the reference protein at least at amino acids E50, F52 and R53.

[0020] 7. The modified protein according to any one of embodiments 1 to 6, wherein the reference protein is as set forth in SEQ ID NO:8.

[0021] 8. A modified protein according to any one of embodiments 1 to 7, comprising at least one amino acid substitution compared to a reference protein.

[0022] 9. A modified protein according to any one of embodiments 1 to 7, comprising at least five amino acid substitutions compared to a reference protein.

[0023] 10. The modified protein according to embodiment 9, which comprises substitutions at amino acids E2, E23 and Y65 compared to the reference protein, wherein the reference protein is as set forth in SEQ ID NO:8.

[0024] 11. The modified protein according to any one of embodiments 1 to 10, wherein the reference protein is as set forth in SEQ ID NO:9.

[0025] 12. The modified protein according to any one of embodiments 9 to 11, comprising a substitution at amino acid L70 compared to the reference protein.

[0026] 13. The modified protein according to any one of embodiments 9 or 12, comprising substitutions at amino acids E2, E23, Y65 and L70 compared to the reference protein.

[0027] 14. The modified protein according to any one of embodiments 1 to 13, wherein the reference protein is as set forth in SEQ ID NO: 10.

[0028] 15. The modified protein according to any one of embodiments 9 to 14, comprising an amino acid substitution, relative to a reference protein, in at least one amino acid selected from the group consisting of E4, T12, A19, V20, K25, I26, Q28, R31, T33, N35, C41, Q61, V64, D68, A73, I75, R84 and F89.

[0029] 16. The modified protein according to embodiment 15, comprising an amino acid substitution, relative to a reference protein, at least one amino acid selected from the group consisting of Q28, C41 and Q68.

[0030] 17. The modified protein of embodiment 15, comprising an amino acid substitution, relative to a reference protein, in at least one amino acid selected from the group consisting of E4, A19, V20, K25, I26, T33, N35, A73, and R84.

[0031] 18. The modified protein according to embodiment 15, comprising an amino acid substitution, relative to a reference protein, in at least one amino acid selected from the group consisting of T12, R31, Q61, V64, I75 and F89.

[0032] 19. The modified protein of embodiment 15, comprising an amino acid substitution, relative to a reference protein, at least one amino acid selected from the group consisting of A19, V20, K25, I26, Q28, T33, C41 and D68.

[0033] 20. The modified protein of embodiment 15, comprising an amino acid substitution compared to a reference protein at least one amino acid selected from the group consisting of A19, V20, K25, I26, Q28, T33, C41 and D68.

[0034] 21. The modified protein according to embodiment 9 or 10, comprising a substitution in at least one amino acid in the reference protein, the modified protein or both that results in (i) stabilization of an alpha-helical structure, (ii) reduction in aggregation, (iii) an effect on core repacking, (iv) an effect on electrostatics, or (v) any combination thereof.

[0035] 22. The modified protein according to embodiment 21, comprising a substitution in at least one amino acid that affects core repacking of the reference protein.

[0036] 23. The modified protein according to embodiment 9 or 10, comprising a substitution in at least one amino acid located in (i) the alpha helix, (ii) the core, (iii) the sweet loop, or (iv) any combination thereof of the reference protein.

[0037] 24. The modified protein according to embodiment 23, comprising a substitution in at least one amino acid located in the core of the reference protein.

[0038] 25. The modified protein according to embodiment 15 or 24, comprising at least one amino acid substitution compared to a reference protein in at least one amino acid residue selected from the group consisting of T12, C41, A19, V20, A73, I75, F89, G16, L32, V37, L62 and V64.

[0039] 26. The modified protein according to embodiment 25, comprising at least one amino acid substitution, relative to a reference protein, in at least one amino acid residue selected from the group consisting of T12, C41, A19, V20, A73, I75 and F89.

[0040] 27. The modified protein according to embodiment 26, comprising at least one amino acid substitution in at least one amino acid residue selected from the group consisting of A19 and V20, compared to the reference protein.

[0041] 28. The modified protein according to embodiment 21, comprising a substitution in at least one amino acid that stabilizes the alpha-helical structure of the reference protein.

[0042] 29. The modified protein according to embodiment 15 or 28, comprising a substitution in at least one of the amino acids K25, I26, Q28 or any combination thereof compared to the reference protein.

[0043] 30. The modified protein according to embodiment 23, comprising a substitution in at least one amino acid located in the sweet loop of the reference protein.

[0044] 31. The modified protein according to embodiment 15 or 30, which comprises, compared to the reference protein, a substitution in at least one amino acid selected from the group consisting of T33 and D68.

[0045] 32. The modified protein according to embodiment 21, comprising a substitution in at least one amino acid that reduces aggregation of the reference protein, the modified protein, or both.

[0046] 33. The modified protein according to embodiment 15 or 32, which comprises a substitution at amino acid C41 compared to the reference protein.

[0047] 34. The modified protein according to embodiment 21, comprising a substitution in at least one amino acid that affects the electrostatics of the reference protein.

[0048] 35. The modified protein according to embodiment 15 or 34, comprising a substitution in at least one of the amino acid residues T33, E4 or any combination thereof compared to the reference protein.

[0049] 36. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid E4 compared to the reference protein.

[0050] 37. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid T12 compared to the reference protein.

[0051] 38. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid A19 compared to the reference protein.

[0052] 39. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid V20 compared to the reference protein.

[0053] 40. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid K25 compared to the reference protein.

[0054] 41. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid I26 compared to the reference protein.

[0055] 42. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid Q28 compared to the reference protein.

[0056] 43. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid R31 compared to the reference protein.

[0057] 44. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid T33 compared to the reference protein.

[0058] 45. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid N35 compared to the reference protein.

[0059] 46. ​​The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid C41 compared to the reference protein.

[0060] 47. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid Q61 compared to the reference protein.

[0061] 48. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid V64 compared to the reference protein.

[0062] 49. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid D68 compared to the reference protein.

[0063] 50. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid A73 compared to the reference protein.

[0064] 51. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid I75 compared to the reference protein.

[0065] 52. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid R84 compared to the reference protein.

[0066] 53. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution at least at amino acid F89 compared to the reference protein.

[0067] 54. The modified protein according to any one of embodiments 1 to 35, comprising, compared to a reference protein, an amino acid substitution at amino acid Q28 and at least an amino acid substitution at an amino acid selected from the group consisting of V20, K25, I26 and R31.

[0068] 55. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions at amino acids Q28 and V20 compared to the reference protein.

[0069] 56. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions at amino acids Q28 and K25 compared to the reference protein.

[0070] 57. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions at amino acids Q28 and R31 compared to the reference protein.

[0071] 58. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions at amino acids Q28 and I26 compared to the reference protein.

[0072] 59. The modified protein according to any one of embodiments 1 to 35, comprising, compared to a reference protein, an amino acid substitution at amino acid D68 and at least an amino acid substitution at an amino acid selected from the group consisting of V20, R31, R84.

[0073] 60. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions at amino acids V20 and D68 compared to the reference protein.

[0074] 61. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions at amino acids D68 and R84 compared to the reference protein.

[0075] 62. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions at amino acids D68 and R31 compared to the reference protein.

[0076] 63. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions at amino acids R84 and R31 compared to the reference protein.

[0077] 64. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions at amino acids A73 and F89 compared to the reference protein.

[0078] 65. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions at amino acids C41 and T12 compared to the reference protein.

[0079] 66. The modified protein according to any one of embodiments 1 to 35, comprising, compared to a reference protein, an amino acid substitution at amino acid V20 and at least an amino acid substitution selected from the group consisting of K25, V64 and D68.

[0080] 67. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions at amino acids V20 and V64 compared to the reference protein.

[0081] 68. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions at amino acids V20 and K25 compared to the reference protein.

[0082] 69. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions at amino acids V20, C41, A73, I75 and F89 compared to the reference protein.

[0083] 70. The modified protein according to embodiment 10, comprising at least an amino acid substitution selected from the group consisting of E2N, E23A and Y65R.

[0084] 71. The modified protein of embodiment 10, 13 or 70, comprising at least an amino acid substitution selected from the group consisting of E2N, E23A, Y65R and L70I.

[0085] 72. The modified protein according to embodiment 10, comprising at least an amino acid substitution selected from the group consisting of E2N, E23V and Y65K.

[0086] 73. The modified protein of embodiment 10, 13 or 72, comprising at least an amino substitution selected from the group consisting of E2N, E23V, Y65K and L70I.

[0087] 74. The modified protein according to any one of the preceding embodiments, comprising at least one amino acid substitution selected from the group consisting of E4Q, T12V, A19V, V20I, K25R, I26T, I26V, I26W, Q28K, Q28R, R31T, T33R, N35T, C41T, C41V, C41A, Q61N, V64I, V64L, D68Q, D68N, A73V, A73F, I75L, R84L, R84K, and F89M compared to a reference protein, wherein the reference protein is selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

[0088] 75. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least one amino acid selected from the group consisting of Q28K, Q28R, C41T, C41V, C41A, D68Q and D68N compared to a reference protein.

[0089] 76. The modified protein according to embodiments 1 to 73, comprising an amino acid substitution in at least one amino acid selected from the group consisting of E4Q, A19V, V20I, K25R, I26T, I26V, I26W, T33R, N35T, A73V, A73F, and R84L, R84K, compared to a reference protein.

[0090] 77. The modified protein according to embodiments 1 to 73, comprising an amino acid substitution in at least one amino acid selected from the group consisting of T12V, R31T, Q61N, V64I, V64L, I75L, and F89M compared to a reference protein.

[0091] 78. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid E4Q compared to the reference protein.

[0092] 79. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid T12V compared to the reference protein.

[0093] 80. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at amino acid A19V compared to the reference protein.

[0094] 81. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid V20I compared to the reference protein.

[0095] 82. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid K25R compared to the reference protein.

[0096] 83. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid I26W compared to the reference protein.

[0097] 84. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid I26T compared to the reference protein.

[0098] 85. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid I26V compared to the reference protein.

[0099] 86. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid Q28K compared to the reference protein.

[0100] 87. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid Q28R compared to the reference protein.

[0101] 88. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid R31T compared to the reference protein.

[0102] 89. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid T33R compared to the reference protein.

[0103] 90. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid N35T compared to the reference protein.

[0104] 91. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid C41T compared to the reference protein.

[0105] 92. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid C41A compared to the reference protein.

[0106] 93. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid C41V compared to the reference protein.

[0107] 94. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid Q61N compared to the reference protein.

[0108] 95. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid V64I compared to the reference protein.

[0109] 96. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid V64L compared to the reference protein.

[0110] 97. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid D68N compared to the reference protein.

[0111] 98. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid D68Q compared to the reference protein.

[0112] 99. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid A73V compared to the reference protein.

[0113] 100. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at amino acid A73F compared to the reference protein.

[0114] 101. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid I75L compared to the reference protein.

[0115] 102. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid R84K compared to the reference protein.

[0116] 103. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid R84L compared to the reference protein.

[0117] 104. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution at least at the amino acid F89M compared to the reference protein.

[0118] 105. SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:41, The modified protein according to any one of embodiments 1 to 104, comprising an amino acid sequence that is about 90% to about 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:71.

[0119] 106. SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, 105. The modified protein according to any one of the preceding embodiments, comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:71.

[0120] 107. The modified protein of any one of embodiments 1 to 106, comprising at least one amino acid sequence as set forth in Table 20.

[0121] 108. A modified protein according to any one of embodiments 1 to 106, having improved sweetness compared to SEQ ID NO:8.

[0122] 109. The modified protein according to any one of embodiments 1 to 106, having improved sweetness compared to SEQ ID NO: 9.

[0123] 110. A modified protein according to any one of embodiments 1 to 106, having improved sweetness compared to SEQ ID NO: 10.

[0124] 111. The modified protein according to any one of embodiments 1-106, having improved sweetness compared to SEQ ID NO:7.

[0125] 112. A modified protein according to any one of embodiments 1 to 106, which has improved heat treatment compared to SEQ ID NO:8.

[0126] 113. A modified protein according to any one of embodiments 1 to 106, which has improved heat treatment compared to SEQ ID NO: 9.

[0127] 114. A modified protein according to any one of embodiments 1 to 106, which has improved heat treatment compared to SEQ ID NO: 10.

[0128] 115. A modified protein according to any one of embodiments 1 to 106, which has improved heat treatment compared to SEQ ID NO: 7.

[0129] 116. A modified protein according to any one of embodiments 1 to 106, having an improved Tm compared to SEQ ID NO:8.

[0130] 117. A modified protein according to any one of embodiments 1 to 106, having an improved Tm compared to SEQ ID NO:9.

[0131] 118. A modified protein according to any one of embodiments 1 to 106, having an improved Tm compared to SEQ ID NO: 10.

[0132] 119. A modified protein according to any one of embodiments 1 to 106, having an improved Tm compared to SEQ ID NO:7.

[0133] 120. A modified protein selected from the group consisting of DM65, DM57, DM43, DM96, DM87, DM77, DM150, DM151, DM46, DM66, DM69, DM41, DM72, DM42, DM70, DM85, DM75 and DM47.

[0134] 121. A food product comprising any one of embodiments 1 to 120.

[0135] In order to better understand the subject matter disclosed herein, and to illustrate how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, the description of which is now given. [Brief description of the drawings]

[0136] [Figure 1] 1 is a spider graph showing that the energy bar with 40% reduced added sugar is less sweet than the energy bar with the DM protein prototype. [Diagram 2] FIG. 2 is a spider graph showing that marzipan containing sweet protein DM31 (70% less added sugars) is sweeter than marzipan with 70% less added sugars. [Diagram 3] 1 is a graph showing the stability of DM31 in citrate buffer. The Y-axis is sweetness intensity on a scale of 0-100, with 100 defined as 8Bx equivalents. The sweetness intensity of DM31 is stable after 12 weeks at both 21° C. and 32° C. The black line on the graph indicates a 25% decrease in sweetness intensity. [Figure 4]1 is a histogram showing the stability of sweetness intensity of DM31 in rice flour with 40% moisture content over 24 hours at 60° C. in a sealed jar. The y-axis is sweetness intensity on a scale of 0 to 100, with 100 defined as 8 Bx equivalents. [Diagram 5] 1 is a histogram showing the thermal stability of powdered DM31 at high temperatures. The sweetness intensity of powdered DM31 is stable at up to 120°C for 5 minutes. [Figure 6A] The hydrogen bonds of I26 and T26 are shown before (FIG. 6A) and after (FIGS. 6B and 6C) the I26T substitution, with backbone hydrogen bonds represented by yellow dashed lines. FIGS. 6B and 6C show two optional hydrogen bonds between the T26-E22 backbone or between T26-Q28 that are formed following the substitution. [Figure 6B] Same as above [Figure 6C] Same as above [Figure 7] The duration of certain hydrogen bonds based on the measured distance between the acceptor and donor during molecular dynamics simulations is shown. Percentages for both DM31 and DM42 are shown for comparison purposes. The larger the bar, the more frequently the bond was detected during the simulation. A distance of 3.5 Å was used as the threshold. Importantly, after the I26T substitution in DM42, the bonds between residue 26 and residues 22 and 23, which support the helix, are filled and protected, and therefore present for a longer period of time. [Figure 8] The hydrogen bonds of K25 before substitution (i.e., in MNEI) are shown. Backbone hydrogen bonds are represented by yellow dashed lines. The hydrogen bonds between K25 and E22 are indicated by the upper arrow (between the backbone) and the lower arrow (between the side chains). These bonds are important for helix stability and are further stabilized by the substitution in DM43, K25R. [Figure 9] Shown is C41 in stick (A) versus T41 in stick (B). The internal cavity is shown as the purple shape. The substitution C41T reduces the cavity volume within the core of the protein. [Figure 10] 1 is a spider graph showing that DM31 sweetened protein-containing granola with 70% less sugar is sweeter than no protein added granola with 70% less sugar. [Figure 11] 1 is a spider graph showing that 75% reduced sugar peanut butter spread combined with Amai sweetened protein is sweeter than 75% reduced sugar peanut butter spread with no added protein. [Figure 12] Average root mean square fluctuation (RMSF) plot of helix capping variants: DM42 (DM31+I26T), DM43 (DM31+K25R), DM65 (DM31+Q28K), DM115 (DM31+I26W+Q28E), DM116 (DM31+I26W+Q28K), DM144 (DM31+I26W). Results obtained using GROMACS2022.1. [Figure 13] Average RMSF (root mean square fluctuation) plot of core repacking variants: DM96 (DM31+V20I), DM77 (DM31+A19V), DM84 (DM31+V64I), and DM85 (DM31+A73V). Results obtained with GROMACS2022.1. [Figure 14] Average RMSF (root mean square variation) plot of variants related to the C41 position: DM91 (DM31+C41V), DM150 (DM31+C41A), DM151 (DM31+C41S), DM143 (DM31+C41V+T12V), DM152 (DM31+T12V), and DM46 (DM31+C41T). Results obtained with GROMACS2022.1. [Figure 15] Average RMSF (Root Mean Square Fluctuation) of DM31, MNEI, DM70(R84L), DM420(R84Y) and DM424(R84I). Each variant is shown in a different color. DM70 and DM424 are expected to be as stable as DM31. Results obtained with GROMACS2022.1. [Figure 16] Average root mean square fluctuation (RMSF) plots for MNEI, DM31, and DM72 (DM31+N35T). Results obtained using GROMACS2022.1. [Figure 17]Average root mean square fluctuation (RMSF) plots for MNEI, DM31, and DM75 (DM31+E4Q). Results obtained using GROMACS2022.1. [Figure 18] Average root mean square fluctuation (RMSF) plots for MNEI, DM31, and DM57 (DM31+T33R). Results obtained using GROMACS2022.1. [Figure 19] Spider graph showing that Savarina with 50% less sugar combined with Amai sweetened protein is as sweet as the full sugar product. [Figure 20] 1 is a spider graph showing that a formulation of Halva spread with 80% reduced sugar and containing DM31 is sweeter than a Halva spread without DM31 and with 80% reduced sugar. [Figure 21A] VoroMQA analysis of the packing and molecular dynamics of DM42 (I26T), DM65 (Q28K) and DM92 (I26T+Q28K), highlighting variants predicted to be more stable due to improved packing. (A) Calculated scores for packing are represented by the residue color, with red representing better (more sufficient) packing and blue representing less good (less sufficient) packing. The green color at the ends of the α-helices indicates improved packing in DM42 and DM92 compared to DM31 (corresponding regions in dark blue). Lysine and serine at position 28 pack better than wild-type glutamine, and threonine at position 26 packs better than wild-type isoleucine. (Graphic translation?) [Figure 21B] (B) Molecular dynamics average RMSF of DM31, MNEI, DM65 (Q28K), DM42 (I26T) and DM92 (I26T+Q28K). Lower values ​​represent less variation. Each variant is shown in a different color. DM92 is expected to be more stable than DM31, DM42 and DM65 due to its decreased RMSF. Results were obtained with GROMACS2022.1. [Figure 22]Average RMSF (root mean square variation) of DM31, MNEI, DM87 (D68N), DM43 (K25R), and DM464 (K25R+D68N). Each variant is shown in a different color. Comparing the RMSF of the helix-terminal region (25-30 aa) of the different DMs with DM31 (black line), DM43 and DM464 show lower RMSF. [Diagram 23] VoroMQA analysis of packing. The calculated scores for packing are indicated by the color of the residues, with red representing better (more adequate) packing and blue representing worse (less adequate) packing. Note that the color change in the side chain of residue 28 indicates that the substitution of Q28 to K increases local packing. [Figure 24] Electrostatic potential surfaces of DM31 and DM57(T33R) (using APBS-Adaptive Poisson-Boltzmann Solver). Position 33 is indicated by a red circle. DM57 is sweeter than DM31. Substitution of arginine at position 33 increases the positive charge in this region. [Diagram 25] Average RMSF (Root Mean Square Fluctuation) of DM31, MNEI, DM87 (D68N), DM108 (D68T) and DM61 (D68S). DM61, DM108 are predicted to be more stable than DM31 in the region of loop A2 (residues 26-35). Each variant is shown in a different color. D68S and D68T substitutions are predicted to be more stable than D68N substitution. Results obtained by GROMACS2022.1. [Figure 26] Electrostatic potential surfaces (using APBS) for DM61 (D68S), DM87 (D68N), DM31, and M108 (D68T). Position 68 is indicated by a red circle. DM87 is sweeter than DM31. DM61 and DM108 are predicted to be as sweet as DM87. [Figure 27] Average RMSF (Root Mean Square Fluctuation) of DM31, MNEI, DM46(C41T), DM150(C41A) and DM151(C41S). DM46 is expected to be more stable than DM31. Results obtained with GROMACS2022.1. [Figure 28]Average root mean square fluctuations (RMSFs) for DM31, MNEI, DM508(V20I+Q28K), DM509(V20I+K25R), DM96(V20I), DM117(V20I+V64I), DM43(K25R), DM84(V64I), and DM65(Q28K). All DMs are predicted to be as stable as DM31. Results obtained with GROMACS2022.1. [Figure 29] Average root mean square fluctuation (RMSF) of DM31, MNEI, DM498 (C41SV64I+A73F+I75L+F89V), DM77 (A19V), and DM491 (V20I+C41A+A73F+I75L+F89M). All DMs are expected to be at least as stable as DM31. Results obtained with GROMACS2022.1. [Diagram 30] 1 is a spider graph showing that a 50% reduced sugar vinaigrette combined with Amai sweetened protein is sweeter than a 50% reduced sugar vinaigrette with no added protein. [Diagram 31] Average RMSF (Root Mean Square Fluctuation) of DM31, MNEI, DM508 (V20I+Q28K), DM65 (Q28K) and DM96 (V20I). Each variant is shown in a different color. DM508 is expected to be more stable within loop A2 (26-30 aa). Results obtained by GROMACS2022.1. [Diagram 32] Electrostatic potentials of DM31, DM508 (V20I+Q28K) compared to DM96 (V20I) (containing Q28) and DM65 (Q28K). Position 28 is indicated by a red circle. DM508 is predicted to be as sweet as DM65. [Diagram 33] Electrostatic potential surface (using APBS) for DM87 (D68N) and DM506 (V20I+D68N). Position 68 is indicated by a red circle. DM87 is sweeter than DM31. DM506 is expected to be as sweet as DM87. See also Figure 1. [Diagram 34]Average RMSF of DM31, MNEI, DM65 (Q28K), DM69 (K25R+Q28K) and DM43 (K25R). Each variant is shown in a different color. DM69 shows less variation overall along the protein sequence, especially in the helix cap region. DM43 shows less variation in the helix cap region. Results obtained with GROMACS2022.1. [Diagram 35] Electrostatic potential surfaces (using APBS) for DM31, DM65 (Q28K) and DM69 (K25R+Q28K). Q28K is shown with a red circle. DM69 and DM65 are expected to be more lenient than DM31 in light of the electrostatic changes shown. [Diagram 36] Average RMSF (root mean square variation) of DM31, MNEI, DM65 (Q28K), DM330 (Q28S) and DM66 (Q28R). Each variant is shown in a different color. Arg and Ser at position 28 can be equivalent to Lys. Results were obtained by GROMACS2022.1. [Figure 37] Electrostatic potential surfaces (using APBS) of DM31, DM65 (Q28K), DM330 (Q28S) and DM66 (Q28R). Position 28 is indicated by a red circle. All these DMs are predicted to be sweeter than DM31 based on electrostatic analysis. Figure 38. Ratio of sweetness intensity of DM65 (Q28K) to that of its precursor (DM31). A sensory panel tasted both proteins and ranked their sweetness. [Figure 38] Figure 1 shows the relative sweetness at 10 Brix of DM65 and DM66 compared to DM31. According to the sensory panel, both proteins showed a 15% and 19% increase in sweetness, respectively. [Figure 39] Average RMSF (Root Mean Square Fluctuation) of DM31, MNEI, DM452 (C41A+V64L+I75V), DM432 (G16A+C41A+V64L+I75V), DM84 (V64I), DM103 (I75L) and DM150 (C41A). Each variant is shown in a different color. DM452 is expected to be more stable in the loop. Results obtained with GROMACS2022.1. [Diagram 40] Average RMSF (Root Mean Square Fluctuation) of DM31, MNEI, DM506 (D68N+V20I), DM87 (D68N), and DM96 (V20I). Each variant is shown in a different color. DM452 is expected to be more stable in the loop. Results obtained with GROMACS2022.1. [Diagram 41] APBS analysis is shown. [Diagram 42] 1 is a spider graph showing that 70% reduced sugar teriyaki sauce combined with Amai sweet protein is sweeter than 70% reduced sugar teriyaki sauce with no added protein. [Diagram 43] 1 is a spider graph showing that 75% reduced sugar peanut butter spread combined with Amai sweetened protein is sweeter than 75% reduced sugar peanut butter spread with no added protein in a chocolate peanut butter cup. [Diagram 44] Spider graph showing that sweet chili sauce with 50% less sugar combined with Amai sweet protein is just as sweet as the full sugar version. [Diagram 45] 1 is a spider graph showing that 62% reduced sugar Thousand Island Sauce combined with Amai Sweetened Protein is sweeter than 62% reduced sugar Thousand Island Sauce with no added protein. [Figure 46] 1 is a spider graph showing that dark chocolate containing sweet protein DM-31 with 70% less sugar is sweeter than dark chocolate with 70% less sugar. [Figure 47] FIG. 11 is a histogram showing that the sweetness intensity of DM31 in rice flour with 40% moisture content is stable after heat treatment at 60° C. and 70° C. for 18 hours in an open plate and reconstitution in water. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0137] Although artificial low-calorie sweeteners are readily available on the market, many have serious side effects. For example, saccharin, which is widely used to sweeten food and beverages without adding calories or carbohydrates, has been linked to cancers, such as bladder cancer. Therefore, there is a significant need for replacements for currently available artificial low-calorie sweeteners that provide optimal sensory profiles and are suitable for use in food and beverages.

[0138] The present disclosure relates to novel proteins that have been shown to be sweet, while also being stable. As described herein, the novel proteins have been identified by a variety of computational methods.

[0139] Surprisingly, the inventors have discovered that introducing a variety of specific modifications, in particular modifications including amino acid deletions or substitutions, into the amino acid sequence of a reference protein results in a protein having at least one improved food-related property.

[0140] Specifically, as shown in the Examples below, the new proteins (herein referred to as "modified proteins" or "designer proteins") exhibited improved sensory profile and / or thermal stability and / or reduced hydrophobicity compared to the reference proteins. Sensory profile, as described herein, relates to the taste profile (e.g., sweetness potency) of the modified proteins.

[0141] Based on the results, it is suggested that the new protein can be used in food and beverage applications to prepare a variety of food products.

[0142] Thus, in its broadest aspect, the present disclosure relates to modified proteins comprising an amino acid sequence having at least one amino acid deletion, replacement and / or insertion compared to the amino acid sequence of a reference protein, wherein the modified protein has at least one improved food-related property compared to the reference protein, as described herein.

[0143] In the present disclosure, modified proteins are sometimes also called designer proteins and may be considered as variants of reference proteins. As used herein, the term "variant" refers to a sequence that contains at least one amino acid modification compared to the reference protein. The present disclosure also encompasses modified nucleic acid sequences that contain the corresponding codon substitutions, deletions or insertions found in modified proteins.

[0144] The present disclosure is not limited to a particular number of amino acid modifications in a reference protein that ultimately result in the formation of a modified protein.

[0145] As used herein, a modification refers to a modification of one or more amino acids of a reference protein and includes an amino acid substitution (replacement), an amino acid deletion, an amino acid insertion, or any combination thereof.

[0146] In some embodiments, the modification may be an amino acid insertion.

[0147] In some embodiments, the modified protein comprises an amino acid sequence having at least 2, at least 3, at least 4, at least 5 amino acid insertions compared to the reference protein.

[0148] In some examples, the modification may be an amino acid deletion.

[0149] In some embodiments, the modified protein comprises an amino acid sequence having at least 2, at least 3, at least 4, at least 5 amino acid deletions compared to the reference protein.

[0150] In some examples, the modification may be an amino acid substitution.

[0151] In some embodiments, the modified protein comprises an amino acid sequence having at least two, at least three, at least four, at least five amino acid substitutions compared to the reference protein.

[0152] As described herein, modified proteins can result from amino acid modifications (substitutions or deletions) in various regions of a protein. As used herein, a "region of a protein" refers to an amino acid sequence or structural motif that is part of a protein sequence (amino acid sequence) or structure. Non-limiting examples of protein regions include a protein surface, a protein core, a protein loop, a secondary structure element, a secondary structure capping, a disulfide bond site, a linker, a hydrophobic patch, or a protein hydrophobic region.

[0153] The amino acid modification in the reference protein is not limited to a particular protein region or sequence. Regions of the reference protein that may contain amino acid modifications include the reference protein surface, the hydrophobic core, or regions called loop regions (also referred to as regions lacking secondary structure), edges of secondary structure (also referred to as secondary structure capping regions), disulfide regions, binding site regions, linker regions, and hydrophobic patch regions.

[0154] As used herein, a "reference surface region," "reference core region," or "reference disulfide bond or loop region" may refer to a corresponding region of a reference protein.

[0155] In some embodiments, the reference protein may be modified (e.g., substituted) within a limited region within the reference protein structure and / or sequence. In some embodiments, the reference protein may be modified (e.g., substituted) in a surface region. In some embodiments, the reference protein may be modified (e.g., substituted) in a core region. In some embodiments, the reference protein may be modified (e.g., substituted) by a disulfide bond. In some embodiments, the reference protein may be modified (e.g., substituted) in a loop region. In some embodiments, the amino acid modifications (replacements) are located on the surface of the reference protein.

[0156] In some embodiments, the reference protein may be replaced by a limited region that is not adjacent to the known or predicted binding site of the reference protein for the receptor, in this context "adjacent" may mean 4-7 Å from the binding interface.

[0157] In some embodiments, the reference protein may be substituted in different regions within the reference protein structure and / or sequence, hi some embodiments, the reference protein may be substituted at least in the surface region, in the core region, in the disulfide bond or in the loop region, or any combination thereof.

[0158] As used herein, a protein surface region is an area that has partial or complete solvent accessibility (SASA: Solvent Accessible Surface Area). A protein core region, as used herein, is an area that is not accessible to solvent with less than 50% of the amino acids relative SASA (solvent accessible surface area), or less than 20% for the inner core.

[0159] As shown in the examples below, the amino acid sequence of the reference protein may be modified in at least one of: (i) an alpha helix of the reference protein, (ii) a core of the reference protein, (iii) a sweet loop of the reference protein, (iv) a linker region of the reference protein, or (v) any combination thereof.

[0160] The amino acid sequence of the reference protein can be modified such that the modifications affect the core packing and / or electrostatics of the reference protein, the modified protein, or both.

[0161] The amino acid sequence of the reference protein may be modified in the linker region of the reference protein.

[0162] The amino acid sequence of the reference protein may be modified in the linker region of the reference protein such that at least one amino acid is deleted.

[0163] The modified protein contains at least one, sometimes at least two, and sometimes at least three amino acid deletions in the reference protein.

[0164] Thus, according to some aspects, the present disclosure provides a protein comprising an amino acid sequence comprising at least one amino acid modification in at least one of: (i) an alpha helix of a reference protein, (ii) a core of a reference protein, (iii) a sweet loop of a reference protein, (iv) a linker region of a reference protein, or (v) any combination thereof, wherein the reference protein has an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

[0165] In the following text, reference to modified proteins should be understood to also refer to the food products disclosed herein, and therefore, whenever characteristics are provided with reference to modified proteins, the same characteristics should be understood to be defined mutatis mutandis with respect to the food products.

[0166] As shown in Example 1 below, molecular dynamics tools were used to design new modified proteins based on the optimization of various regions of a reference protein. Specifically, Rosetta Energy Unit (REU) scores were used for the newly designed modified proteins to predict those proteins that exhibit improved properties, in particular at least one improved food-related property as described herein.

[0167] Energy calculations can be applied to the entire amino acid sequence, or alternatively, can be restricted to specific regions or selected amino acids within the protein, in which case the information can be combined to measure the entire protein.

[0168] Calculations for each amino acid sequence (e.g., modified protein) alone can be performed by a combination of physics-based (also known as biophysical methods) and statistically-based potentials (also known as knowledge-based potentials or informatics methods), such as using the Rosetta Energy Unit (REU). The Rosetta Energy Unit (REU) is an algorithm in the Rosetta software, a package of algorithms for computational modeling and protein structure analysis. The Rosetta software enables remarkable scientific advances in computational biology, including de novo protein design, enzyme design, ligand docking, and structure prediction of biological macromolecules and macromolecular complexes. The Rosetta Energy function is a combination of physics-based and statistically-based potentials that does not correspond to any actual physical energy unit. The Rosetta Energy is an arbitrary measure and is sometimes referred to as REU ("Rosetta Energy Unit").

[0169] In some embodiments, the REU may be calculated for the entire protein sequence that contains at least one amino acid modification. In some other embodiments, the REU may be calculated for at least one region in the entire protein sequence that contains at least one amino acid modification. In some other embodiments, the REU may be calculated for at least one amino acid modification in the entire protein sequence.

[0170] In some embodiments, the modified protein has an energy expressed in REU of less than about -315, sometimes less than about -317, sometimes less than about -319, sometimes less than about -321, sometimes less than about -322, and sometimes less than about -324.

[0171] In some embodiments, the modified protein has an energy expressed in REU of about -315. In some embodiments, the modified protein has an energy expressed in REU of about -317. In some embodiments, the modified protein has an energy expressed in REU of about -319. In some embodiments, the modified protein has an energy expressed in REU of about -321. In some embodiments, the modified protein has an energy expressed in REU of about -324. In some embodiments, the modified protein has an energy expressed in REU of about -326.

[0172] In some embodiments, the modified protein comprises an amino acid sequence that is 40% to 98% identical to the amino acid sequence of the reference protein. In some embodiments, the modified protein comprises an amino acid sequence that is 90% to 98% identical to the amino acid sequence of the reference protein.

[0173] In some embodiments, the modified protein comprises an amino acid sequence that is 60%-90% identical to the reference amino acid sequence. In some embodiments, the modified protein comprises an amino acid sequence that is 70%-90% identical to the reference amino acid sequence.

[0174] In some embodiments, the modified protein comprises an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to a reference amino acid sequence.

[0175] In some embodiments, the modified protein comprises an amino acid sequence having 90%-98% identity to a reference amino acid sequence.

[0176] The percent identity between two or more amino acid sequences is determined by comparing and aligning the two or more sequences to obtain the maximum correspondence. In the context of the present disclosure, sequences (amino acids) described herein that have a percent identity are considered to have the same function / activity as the reference sequence to which the identity is calculated.

[0177] In some embodiments, the modified protein comprises an amino acid sequence that is 40%-98% similar to the amino acid sequence of the reference protein. In some embodiments, the modified protein comprises an amino acid sequence that is 90%-98% similar to the amino acid sequence of the reference protein.

[0178] In some embodiments, the modified protein comprises an amino acid sequence that is 60%-90% similar to the reference amino acid sequence. In some embodiments, the modified protein comprises an amino acid sequence that is 70%-90% similar to the reference amino acid sequence.

[0179] In some embodiments, the modified protein comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% similarity to a reference amino acid sequence.

[0180] In some embodiments, the modified protein comprises an amino acid sequence having 90%-98% similarity to a reference amino acid sequence.

[0181] In some embodiments, the reference protein is set forth in SEQ ID NO: 8, also referred to as MNEI. SEQ ID NO: 8 referred to herein has the following amino acid sequence: GEWEIIDIGPFTQNLGKFAVDEENKIGQYGRLTFNKVIRPCMKKTIYENEGFREIKGYEYQLYVYASDKLFRADISEDYKTRGRKLLRFNGPVPPP

[0182] As will be appreciated, MNEI is a synthetic protein made from the combination of monellin A chain (GenBank entry no. P02881) and monellin B chain (GenBank entry no. P02882).

[0183] The main difference between wild-type monellin and MNEI is the region where the two monellin subunits are linked together into a single chain monellin called MNEI. The amino acid sequence between amino acid T46 and amino acid I56, referred to as the loop region, links the two monellin subunits.

[0184] Modified proteins, in some embodiments, refer to variants of MNEI, and in some further embodiments, to variants of MNEI having modifications in the linker (loop) region.

[0185] As shown in the examples below, modifications in the loop regions of the modified proteins improve the sweetness and / or stability of the reference protein. Specifically, deletion of amino acid residues in protein loops and beta strand edges results in a more stable modified protein compared to the reference protein, which in some examples is MNEI.

[0186] Thus, according to some embodiments, the present disclosure provides a protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56 compared to a reference protein, wherein the reference protein has an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.

[0187] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56 compared to a reference protein, wherein the reference protein has an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, and the modified protein has at least one improved food-related property compared to the reference protein.

[0188] According to the present disclosure, the reference protein is a synthetic protein. When referring to a synthetic protein, it should be understood that the protein is considered a synthetic protein because it is not found in nature.

[0189] As noted above, in some embodiments, the reference protein is MNEI, represented herein by SEQ ID NO:8.

[0190] Thus, according to some embodiments, the present disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56 compared to a reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0191] As shown in the Examples below which form part of this patent application, deletion of three amino acids, E50, F52 and R53, from the amino acid sequence of MNEI (DM29, referred to herein as SEQ ID NO:11) suggested improved stability of the modified protein based on an REU value of -319.65 compared to an REU value of -315.35 for MNEI.

[0192] Thus, according to some aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion of at least amino acids E50, F52 and R53 compared to a reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0193] According to some embodiments, the modified protein comprises an amino acid substitution compared to the reference protein.

[0194] In some embodiments, the modified protein comprises an amino acid sequence having at least two, at least three, at least four, or at least five amino acid substitutions compared to the reference protein.

[0195] In some embodiments, the modified protein comprises an amino acid sequence having at least five amino acid substitutions, at least six, at least seven, at least eight, at least nine amino acid substitutions compared to the reference protein.

[0196] In some embodiments, the modified protein contains 5 to 20 amino acid substitutions, and sometimes 5 to 10 amino acid substitutions, compared to a reference protein (reference amino acid sequence).

[0197] According to some other embodiments, the modified protein comprises at least five amino acid substitutions compared to a reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0198] Thus, according to some embodiments, the present disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56 and amino acid substitutions at at least five amino acids compared to a reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0199] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0200] In some embodiments, the at least five amino acid substitutions include substitutions at amino acids E2, E23, and Y65 relative to the reference protein.

[0201] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56 compared to a reference protein and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23 and Y65 compared to the reference protein, and the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0202] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23 and Y65 compared to the reference protein, and the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0203] In some embodiments, the at least five amino acid substitutions include a substitution at amino acid L70 compared to the reference protein.

[0204] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56 compared to a reference protein and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include a substitution at amino acid L70 compared to the reference protein, and the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0205] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include a substitution at amino acid L70 compared to the reference protein, and the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0206] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56 compared to a reference protein, and at least five amino acid substitutions at amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70 compared to the reference protein, and wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0207] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70 compared to the reference protein, and wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0208] As will be appreciated, amino acid substitutions are not limited to a particular region of the reference protein, but can be located throughout the reference protein structure / sequence described herein.

[0209] In some embodiments, the modified protein comprises an amino acid substitution at at least one amino acid selected from the group consisting of E4, T12, A19, V20, K25, I26, Q28, R31, T33, N35, C41, Q61, V64, D68, A73, I75, R84 and F89 compared to the reference protein.

[0210] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions at at least one amino acid selected from the group consisting of E4, T12, A19, V20, K25, I26, Q28, R31, T33, N35, C41, Q61, V64, D68, A73, I75, R84 and F89 compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0211] According to some other aspects, the disclosure provides modified proteins comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions at at least one amino acid selected from the group consisting of E4, T12, A19, V20, K25, 126, Q28, R31, T33, N35, C41, Q61, V64, D68, A73, 175, R84 and F89 compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0212] In some embodiments, the modified protein comprises an amino acid substitution relative to the reference protein at at least one amino acid selected from the group consisting of Q28, C41, and D68.

[0213] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56, and amino acid substitutions at at least five amino acids, compared to a reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions at at least one amino acid selected from the group consisting of Q28, C41 and D68, compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0214] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions at at least one amino acid selected from the group consisting of Q28, C41, and F68 compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0215] In some embodiments, the modified protein comprises an amino acid substitution relative to the reference protein at at least one amino acid selected from the group consisting of A19, V20, K25, I26, T33, N35 and R84.

[0216] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56, and amino acid substitutions at at least five amino acids, compared to a reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions at at least one amino acid selected from the group consisting of A19, V20, K25, I26, Q28, T33, and N35, compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0217] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions at at least one amino acid selected from the group consisting of A19, V20, K25, I26, Q28, T33, and N35 compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0218] In some embodiments, the modified protein comprises an amino acid substitution relative to the reference protein at at least one amino acid selected from the group consisting of E4, T12, R31, V64, and A73.

[0219] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56, and amino acid substitutions at at least five amino acids, compared to a reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions at at least one amino acid selected from the group consisting of E4, T12, R31, V64, A73, and R84, compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0220] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions at at least one amino acid selected from the group consisting of E4, T12, R31, V64, A73, and R84 compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0221] In some embodiments, the modified protein comprises an amino acid substitution relative to the reference protein at at least one amino acid selected from the group consisting of A19, V20, K25, I26, Q28, T33, C41 and D68.

[0222] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions at at least one amino acid selected from the group consisting of A19, V20, K25, I26, Q28, T33, C41 and D68 compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0223] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions in at least one amino acid selected from the group consisting of A19, V20, K25, I26, Q28, T33, C41 and D68 compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0224] In some embodiments, the modified protein comprises an amino acid substitution relative to the reference protein at at least one amino acid selected from the group consisting of A19, V20, K25, I26, Q28, T33, C41 and D68.

[0225] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions at at least one amino acid selected from the group consisting of A19, V20, K25, I26, Q28, T33, C41 and D68 compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0226] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions at at least one amino acid selected from the group consisting of A19, V20, K25, I26, Q28, T33, C41 and D68 compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0227] As noted above, modifications, particularly including amino acid substitutions, are not limited to particular regions of the reference protein, but according to some embodiments are made in regions predicted to improve the properties of the reference protein.

[0228] In some embodiments, the at least five amino acid substitutions include substitutions in at least one amino acid that (i) stabilizes alpha-helical structure, (ii) reduces aggregation, (iii) affects core repacking, (iv) affects electrostatics, or (v) any combination thereof, of the reference protein, the modified protein, or both.

[0229] In some embodiments, the at least five amino acid substitutions include substitutions in at least one amino acid located in (i) an alpha helix, (ii) a core, (iii) a sweet loop, or (iv) any combination thereof of the reference protein.

[0230] In some embodiments, the at least five amino acid substitutions include substitutions in at least one amino acid that affects core repacking of the reference protein, the modified protein, or both.

[0231] In some embodiments, the at least five amino acid substitutions include substitutions in at least one amino acid located in the core of the reference protein.

[0232] The core of the reference protein, for example, MNEI, is partially (half) exposed to the surface and is susceptible to interference from water. It was suggested that there is a hole that allows the core to be accessible to surface water (since the protein is small, the core may be half exposed). Therefore, it was suggested that modifications in the core region that affect core repacking reduce accessibility to water, thereby stabilizing the modified protein. Therefore, the term modification in core repacking as used herein refers to any modification that affects core repacking and thereby reduces accessibility to water.

[0233] In some embodiments, the modified protein comprises an amino acid substitution at at least one amino acid residue selected from the group consisting of T12, C41, A19, V20, A73, I75, F89, G16, L32, V37, L62 and V64 compared to the reference protein.

[0234] In some embodiments, the modified protein comprises an amino acid substitution relative to the reference protein at at least one amino acid residue selected from the group consisting of T12, C41, A19, V20, A73, I75, and F89.

[0235] In some embodiments, the modified protein comprises an amino acid substitution relative to the reference protein at at least one amino acid residue selected from the group consisting of A19 and V20.

[0236] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56, and amino acid substitutions at at least five amino acids, compared to a reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions at at least one amino acid selected from the group consisting of A19 and V20, compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0237] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions at at least one amino acid selected from the group consisting of A19 and V20 compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0238] In some embodiments, the at least five amino acid substitutions include substitutions in at least one amino acid that stabilizes an alpha-helical structure of the reference protein, the modified protein, or both.

[0239] As used herein, the term stabilizing α-helical structure refers to any modification that affects the α-helical structure of the reference protein, the modified protein, or both, including modifications at any of amino acid residues K25, I26, and Q28 of the reference protein.

[0240] In some embodiments, the modified protein comprises a substitution at least one of amino acids K25, I26, Q28, or any combination thereof, compared to the reference protein.

[0241] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56, and amino acid substitutions at at least five amino acids, compared to a reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65, and L70, and substitutions at at least one amino acid selected from the group consisting of K25, I26, and Q28, compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0242] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions at at least one amino acid selected from the group consisting of K25, I26 and Q28 compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0243] In some embodiments, the at least five amino acid substitutions include substitutions in at least one amino acid located in the sweet loop of the reference protein.

[0244] As used herein, the sweet loop refers to the region in the reference protein between residues 63 and 68.

[0245] In some embodiments, the modified protein comprises a substitution at least one amino acid selected from the group consisting of T33 and D68 compared to the reference protein.

[0246] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56, and amino acid substitutions at at least five amino acids, compared to a reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65, and L70, and substitutions at at least one amino acid selected from the group consisting of T33 and D68, compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0247] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions at at least one amino acid selected from the group consisting of T33 and D68 compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0248] In some embodiments, the at least five amino acid substitutions include a substitution in at least one amino acid that reduces aggregation of the reference protein, the modified protein, or both.

[0249] The term reducing protein aggregation as used herein refers to any modification that affects protein aggregation, including modifications at amino acid residue C41.

[0250] In some embodiments, the modified protein comprises a substitution at amino acid C41 compared to the reference protein.

[0251] Based on computational analysis, it was suggested that amino acid C41 is prone to disulfide bonding and promotes aggregation, and therefore any modification at this residue may reduce protein aggregation.

[0252] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65, L70 and C41 compared to the reference protein, and wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0253] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65, L70 and C41 compared to the reference protein, and wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0254] In some embodiments, the at least five amino acid substitutions include substitutions in amino acids that affect the electrostatics of the reference protein, the modified protein, or both.

[0255] The term electrostatic as used herein refers to any modification that affects electrostatics, including modifications at at least one of amino acid residues T33, E4, or any combination thereof.

[0256] In some embodiments, the modified protein comprises a substitution at least one amino acid residue, T33, E4, or any combination thereof, compared to the reference protein.

[0257] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56, and amino acid substitutions at at least five amino acids, compared to a reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions at at least one amino acid selected from the group consisting of T33 and E4, compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0258] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2, E23, Y65 and L70, and substitutions at at least one amino acid selected from the group consisting of T33 and E4 compared to the reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0259] In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid E4 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid T12 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid A19 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid V20 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid K25 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid I26 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid Q28 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid R31 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid T33 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid N35 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid C41 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid Q61 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid V64 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid D68 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid A73 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid I75 compared to the reference protein.In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid R84 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acid F89 compared to the reference protein.

[0260] In some embodiments, the modified protein comprises an amino acid substitution at amino acid Q28 and at least an amino acid substitution at an amino acid selected from the group consisting of V20, K25, 126 and R31, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at amino acid Q28 and V20, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at amino acid Q28 and K25, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at amino acid Q28 and 126, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at amino acid Q28 and R31, as compared to the reference protein.

[0261] In some embodiments, the modified protein comprises, relative to the reference protein, an amino acid substitution at amino acid D68 and at least an amino acid substitution at an amino acid selected from the group consisting of V20, R31, R84. In some embodiments, the modified protein comprises, relative to the reference protein, an amino acid substitution at amino acids V20 and D68. In some embodiments, the modified protein comprises, relative to the reference protein, an amino acid substitution at amino acids D68 and R84.

[0262] In some embodiments, the modified protein comprises amino acid substitutions at amino acids D68 and R31 compared to the reference protein. In some embodiments, the modified protein comprises amino acid substitutions at amino acids R84 and R31 compared to the reference protein.

[0263] In some embodiments, the modified protein comprises amino acid substitutions at amino acids A73 and F89 compared to the reference protein.

[0264] In some embodiments, the modified protein comprises amino acid substitutions at amino acids C41 and T12 compared to the reference protein.

[0265] In some embodiments, the modified protein comprises an amino acid substitution at amino acid V20 and at least an amino acid substitution at an amino acid selected from the group consisting of K25, V64, and D68, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acids V20 and V64, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution at least at amino acids K25 and V20, as compared to the reference protein. In some embodiments, the modified protein comprises amino acid substitutions at amino acids V20 and C41 and A73 and I75 and F89, as compared to the reference protein.

[0266] As previously described by the inventors, modifications of amino acids E2, E23 and Y65, specifically the amino acid substitutions E2N, E23A and Y65R of the reference protein set forth herein as SEQ ID NO:8, have provided a modified protein, designated DM9, as provided by SEQ ID NO:9, characterized by improved sweetness and stability compared to the reference protein.

[0267] As shown in the Examples below, modifications of residues E2, E23, Y65, and L70, specifically substitutions E2N, E23A, Y65R, L70I of the reference protein set forth herein as SEQ ID NO:8, provided a modified protein designated DM14, provided herein as SEQ ID NO:10, which is suggested to have improved stability as indicated by an REU value of -321.07 compared to the REU value of -315.35 of the reference protein.

[0268] As mentioned above, it was discovered that the deletion of three amino acids, specifically E50, F52 and R53, improved the stability of the modified protein.

[0269] In some embodiments, the modified protein comprises at least an amino acid substitution selected from the group consisting of E2N, E23A, and Y65R.

[0270] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2N, E23A and Y65R, and the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0271] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2N, E23A and Y65R compared to the reference protein, and wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0272] As described herein, the modified protein designated DM28, described herein by the amino acid sequence of SEQ ID NO:40, contains amino acid deletions at amino acids E50, F52 and R53 and substitutions at amino acids E2N, E23A and Y65R, as compared to the reference protein, which has the amino acid sequence set forth in SEQ ID NO:8. DM28 has a Tm of 91°C, which is an improvement over the reference protein MNEI (SEQ ID NO:8), which has a Tm of 71°C. m It has been shown to have

[0273] In some embodiments, the modified protein comprises at least an amino acid substitution selected from the group consisting of E2N, E23A, Y65R, and L70I.

[0274] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2N, E23A, Y65R and L70I, and wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0275] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2N, E23A, Y65R and L70I compared to the reference protein, and wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0276] As described herein, the modified protein designated DM31, described herein by the amino acid sequence of SEQ ID NO:7, contains amino acid deletions at amino acids E50, F52 and R53 and substitutions at amino acids E2N, E23A, Y65R and L70I, as compared to the reference protein, which has the amino acid sequence set forth in SEQ ID NO:8. DM31 has a Tm of 91°C, which is an improvement over the reference protein MNEI (SEQ ID NO:8), which has a Tm of 71°C. m Furthermore, DM31 has a sweetness intensity of 3953, whereas the reference protein has a sweetness intensity of 1008.

[0277] In some embodiments, the modified protein comprises at least an amino acid substitution selected from the group consisting of E2N, E23V, and Y65K.

[0278] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2N, E23V and Y65K, and wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0279] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2N, E23V and Y65K compared to the reference protein, and wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0280] In some embodiments, the modified protein comprises at least an amino acid substitution selected from the group consisting of E2N, E23V, Y65K, and L70I.

[0281] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having at least three amino acid deletions at amino acids located between amino acid T46 and amino acid I56 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to a reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2N, E23V, Y65K and L70I, and wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0282] According to some other aspects, the disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions at amino acids E50, F52 and R53 compared to a reference protein, and amino acid substitutions at at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions include substitutions at amino acids E2N, E23V, Y65K and L70I compared to the reference protein, and wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8.

[0283] As described herein, the modified protein designated DM32, described herein by the amino acid sequence of SEQ ID NO:46, contains amino acid deletions at amino acids E50, F52 and R53 and substitutions at amino acids E2N, E23V, Y65K and L70I, as compared to the reference protein, which has the amino acid sequence set forth in SEQ ID NO:8. DM32 has a Tm of >90°C, which is an improvement over the reference protein MNEI (SEQ ID NO:8), which has a Tm of 71°C. m It has been shown to have

[0284] Without wishing to be bound by any particular theory, it has been suggested that loop remodeling increases the stability of the modified protein. It has further been suggested that the remodeling results in extra hydrogen bonds, elongated and more ordered beta strands, and loops 2 and 3 converted into beta turns. Although the loops are located in specific parts of the beta sheet, their role is very important since they are located at the weak points of the beta.

[0285] In some embodiments, the modified protein comprises at least one of the amino acid substitutions (i) E2N, (ii) E23A or E23V, and (iii) Y65R or Y65K, as compared to the reference protein, and at least one amino acid substitution selected from the group consisting of E4Q, T12V, A19V, V20I, K25R, I26S, I26T, I26W, Q28K, Q28R, Q28K, Q28E, Q28S, R31T, T33R, N35T, C41T, C41V, C41A, C41S, Q61N, V64I, D68S, D68N, D68T, A73V, A73F, I75L, R84L, F89V, and F89M.

[0286] In some embodiments, the modified protein comprises, relative to a reference protein, amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R, Y65K, L70I, and at least one amino acid substitution selected from the group consisting of E4Q, T12V, A19V, V20I, K25R, I26S, I26T, I26W, Q28K, Q28R, Q28K, Q28E, Q28S, R31T, T33R, N35T, C41T, C41V, C41A, C41S, Q61N, V64I, D68S, D68N, D68T, A73V, A73F, I75L, R84L, F89V, and F89M.

[0287] In some embodiments, the modified protein comprises, relative to a reference protein, amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K and L70I, and at least one amino acid substitution selected from the group consisting of E4Q, T12V, A19V, V20I, K25R, I26S, I26T, I26W, Q28K, Q28R, Q28K, Q28E, Q28S, R31T, T33R, N35T, C41T, C41V, C41A, C41S, Q61N, V64I, D68S, D68N, D68T, A73V, A73F, I75L, R84L, F89V, and F89M.

[0288] In some embodiments, the modified protein comprises at least one of the amino acid substitutions (i) E2N, (ii) E23A or E23V, and (iii) Y65R or Y65K, as well as at least one amino acid substitution selected from the group consisting of A19V, V20I, K25R, I26S, I26T, I26W, Q28K, Q28R, Q28K, Q28E, Q28S, T33R, N35T, C41T, C41V, C41A, C41S, D68S, D68N, and D68T, relative to the reference protein.

[0289] In some embodiments, the modified protein comprises, relative to a reference protein, amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K and L70I, and at least one amino acid substitution selected from the group consisting of A19V, V20I, K25R, I26S, I26T, I26W, Q28K, Q28R, Q28K, Q28E, Q28S, T33R, N35T, C41T, C41V, C41A, C41S, D68S, D68N, and D68T.

[0290] In some embodiments, the modified protein comprises, relative to a reference protein, at least one of the amino acid substitutions (i) E2N, (ii) E23A or E23V, and (iii) Y65R or Y65K, and at least one amino acid substitution selected from the group consisting of A19V, V20I, K25R, Q28K, T33R, C41A, C41S, and D68N.

[0291] In some embodiments, the modified protein comprises, relative to a reference protein, amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K and L70I, and at least one amino acid substitution selected from the group consisting of A19V, V20I, K25R, Q28K, T33R, C41A, C41S, and D68N.

[0292] In some embodiments, the modified protein comprises, relative to a reference protein, at least one of the amino acid substitutions (i) E2N, (ii) E23A or E23V, and (iii) Y65R or Y65K, and at least one amino acid substitution selected from the group consisting of A19V, V20I, K25R, Q28K, T33R, C41A, C41S, and D68N.

[0293] In some embodiments, the modified protein comprises, relative to a reference protein, amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K and L70I, and at least one amino acid substitution selected from the group consisting of A19V, V20I, K25R, Q28K, T33R, C41A, C41S, and D68N.

[0294] In some embodiments, the modified protein comprises, relative to the reference protein, amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K, L70I and Q28R, and at least an amino acid selected from the group consisting of V20, K25R, I26 and R31T.

[0295] In some embodiments, the modified protein comprises, relative to the reference protein, amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K, L70I and Q28K, and at least an amino acid selected from the group consisting of V20, K25R, I26T, I26W and R31.

[0296] In some embodiments, the modified protein comprises, relative to the reference protein, amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K, L70I, and Q28E, and at least an amino acid selected from the group consisting of V20, K25, I26W, and R31.

[0297] In some embodiments, the modified protein comprises, relative to the reference protein, amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K, L70I and D68N, and at least an amino acid selected from the group consisting of V20, K25R, R31T and R84L.

[0298] In some embodiments, the modified protein comprises, relative to the reference protein, amino acid substitutions: (i) E2N; (ii) at least one of E23A or E23V; (iii) at least one of Y65R or Y65K, L70I, R84L, and R31T.

[0299] In some embodiments, the modified protein comprises, relative to the reference protein, amino acid substitutions: (i) E2N; (ii) at least one of E23A or E23V; (iii) at least one of Y65R or Y65K, L70I, A73F, and F89M.

[0300] In some embodiments, the modified protein comprises, relative to the reference protein, amino acid substitutions: (i) E2N; (ii) at least one of E23A or E23V; (iii) at least one of Y65R or Y65K, L70I, C41V, and T12V.

[0301] In some embodiments, the modified protein comprises, relative to the reference protein, amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K, L70I, and V20I, and at least one amino acid selected from the group consisting of K25, V64I, and D68.

[0302] In some embodiments, the modified protein comprises, relative to the reference protein, amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K, L70I, V20I, and at least two amino acids selected from the group consisting of C41A, A73F and I75L and F89M.

[0303] In some embodiments, the modified protein comprises, relative to the reference protein, amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K, L70I, and at least two amino acids selected from the group consisting of C41S, V64I, A73F, I75L and F89V.

[0304] In some embodiments, the modified protein is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO: 5, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:71.

[0305] In some embodiments, the modified protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, The amino acid sequence is about 90% to about 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:71.

[0306] In some embodiments, the modified protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:41, The present invention relates to a method for producing a nucleic acid sequence comprising the steps of: (a) administering a nucleic acid sequence comprising: a) administering a nucleic acid sequence comprising: a) administering a nucleic acid sequence comprising: a) administering a nucleic acid sequence comprising: a) administering a nucleic acid sequence comprising: a) administering a nucleic acid sequence comprising: a) administering a nucleic acid sequence comprising:

[0307] As used herein, sequence similarity or sequence identity refers to the amount (%) of conserved amino acids that have similar physicochemical properties, such as, for example, leucine and isoleucine.

[0308] In determining sequence identity, gaps are not counted and sequence identity is determined for the shorter of the two sequences. Note that in this context, the length of the reference MNEI protein (amino acid sequence) may be the same as or different from the modified MNEI protein (amino acid sequence).

[0309] The terms "amino acid sequence" and / or "polypeptide chain" are used to describe a protein having an amino acid sequence or a polypeptide chain. Thus, the term "reference protein" is equivalent to the term "reference amino acid sequence" and the term "modified protein" is equivalent to the term "modified amino acid sequence." It should be noted that the terms "amino acid sequence" and / or "polypeptide chain" encompass sequences with and without a 3D structure.

[0310] The term "fragment" as used herein in connection with the present disclosure relates to a protein or peptide that is truncated from the full-length protein, i.e. lacking at least one amino acid. Such a fragment may comprise at least more than 10, for example 20, 30 or more consecutive amino acids of the primary sequence of the protein.

[0311] In some embodiments, the modified protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:71, or a fragment or variant thereof.

[0312] In some embodiments, the modified protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:49, or a fragment or variant thereof.

[0313] In some embodiments, the modified protein is or comprises an amino acid sequence represented by any of DM42 (SEQ ID NO:4), DM43 (SEQ ID NO:5), DM41 (SEQ ID NO:12), DM65 (SEQ ID NO:14), DM66 (SEQ ID NO:15), DM68 (SEQ ID NO:16), DM69 (SEQ ID NO:17), DM73 (SEQ ID NO:18), DM92 (SEQ ID NO:19), DM115 (SEQ ID NO:21), DM116 (SEQ ID NO:22), DM144 (SEQ ID NO:23) or DM145 (SEQ ID NO:24).

[0314] In some embodiments, the modified protein is or includes an amino acid sequence represented by any of DM46 (SEQ ID NO:1), DM91 (SEQ ID NO:28), DM143 (SEQ ID NO:44), DM150 (SEQ ID NO:32), or DM151 (SEQ ID NO:33).

[0315] In some embodiments, the modified protein is or comprises an amino acid sequence represented by any of DM46 (SEQ ID NO:1), DM77 (SEQ ID NO:25), DM84 (SEQ ID NO:26), DM85 (SEQ ID NO:27), DM91 (SEQ ID NO:28), DM96 (SEQ ID NO:38), DM103 (SEQ ID NO:20), DM104 (SEQ ID NO:29), DM131 (SEQ ID NO:30), DM132 (SEQ ID NO:31), DM143 (SEQ ID NO:44), DM150 (SEQ ID NO:32), DM151 (SEQ ID NO:33), DM152 (SEQ ID NO:45).

[0316] In some embodiments, the modified protein is or includes an amino acid sequence represented by either DM57 (SEQ ID NO: 34), DM75 (SEQ ID NO: 35).

[0317] In some embodiments, the modified protein is or includes an amino acid sequence represented by any of DM87 (SEQ ID NO: 36), DM93 (SEQ ID NO: 37), DM157 (SEQ ID NO: 39, used as a reference).

[0318] In some embodiments, the modified protein is or includes an amino acid sequence represented by either DM47 (SEQ ID NO: 2), DM72 (SEQ ID NO: 6).

[0319] In some embodiments, the modified protein is or comprises the amino acid sequence represented by DM70 (SEQ ID NO:3).

[0320] In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:1 (DM46). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:2 (DM47). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:3 (DM70). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:4 (DM42). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:5 (DM43). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:6 (DM72). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:12 (DM41). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:14 (DM65). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:15 (DM66). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:16 (DM68). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 17 (DM69). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 18 (DM73). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 19 (DM92). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 20 (DM103). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 21 (DM115). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 22 (DM116).In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:23 (DM144). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:24 (DM145). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:25 (DM77). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:26 (DM84). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:27 (DM85). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:28 (DM91). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:29 (DM104). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:30 (DM131). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:31 (DM132). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 32 (DM150). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 33 (DM151). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 34 (DM57). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 35 (DM75). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 36 (DM87). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 37 (DM93). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 38 (DM96).In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 41 (DM94). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 42 (DM97). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 43 (DM99). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 44 (DM143). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 45 (DM152). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 46 (DM32). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 47 (DM33). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 48 (DM61). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 50 (DM108). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:51 (DM117). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:52 (DM330). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:54 (DM164). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:56 (DM491). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:57 (DM498). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:58 (DM506).In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:59 (DM508). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:60 (DM509). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:63 (DM341). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:64 (DM420). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:65 (DM424). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:66 (DM432). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:67 (DM452). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:68 (DM489). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO:69 (DM505). In some embodiments, the modified protein is or comprises the amino acid sequence set forth in SEQ ID NO: 71 (DM510).

[0321] In some examples, the modified protein is at least one of the proteins set forth in Table 20. It is noted that the present disclosure encompasses combinations of two or more of the modified proteins set forth in Table 20. It is further noted that each row in Table 20 constitutes an embodiment of the present disclosure.

[0322] [Table 1] TIFF2025514608000002.tif242162TIFF2025514608000003.tif247162

[0323] As described herein, the design of modified proteins is performed by computational tools or specialized protein design and structural biology methods, such as site-directed mutagenesis, protein engineering, or directed evolution, as further described below. The inventors have developed computational methodologies based on sequence, structural, and / or evolutionary data of reference flavor proteins and other proteins that have local or global similarity in sequence and / or structural features to the reference flavor proteins. The computational methods developed and applied herein have allowed the inventors to design proteins with specific amino acid substitutions that are predicted to be energetically favorable and therefore have improved traits such as thermostability, halostability, pH stability, shelf life, folding, and solubility characteristics. In particular, Computational Protein Design (CPD) has been applied to specific sites or regions within the reference protein structure and / or sequence that are not required for functional binding to the receptor. In addition, CPD has allowed the substitutions to be set to a pre-determined set of amino acids that fit the required improved characteristics. The set of predetermined amino acids is both the input data, i.e., the regions of the protein that are subjected to CPD, and the output data, i.e., the positions and types of amino acids that are present in the resulting modified protein.

[0324] For example, the use of CPDs allows for the replacement of "non-ideal" amino acids (e.g., hydrophilic amino acids in the hydrophobic core or hydrophobic amino acids on the exterior surface region) with "ideal" amino acids (e.g., hydrophilic amino acids in the exterior surface region and hydrophobic amino acids in the hydrophobic core).

[0325] Without wishing to be bound by any particular theory, the inventors suggest that replacement of hydrophobic amino acids on the exterior surface region with hydrophilic amino acids reduces non-specific binding to the oral cavity and reduces residual aftertaste.

[0326] The methodology developed herein involves searching for "stabilizing substitutions", e.g., amino acid substitutions that reduce the overall energy of the protein structure. The overall energy can be calculated by applying algorithms known in the art. Non-limiting examples of such algorithms include Rosetta, OSPREY (M. Hallen, J. Martin, et al., Journal of Computational Chemistry 2018; 39(30): 2494-2507), or EnCoM (Frappier V, Chartier M, Najmanovich RJ. Nucleic Acids Res. 2015;43(W1): W395-400). These CPD methods are focused and filtered by a series of orthogonal methods, such as evolutionary sequence and structural consensus, conventional and high temperature molecular dynamics (MD) and other dynamic simulations, correlated mutation analysis (CMA), surface electrostatic analysis, visual inspection, but also analysis of cavities, hydrophobic patches, unsatisfied hydrogen bonds, etc.

[0327] Amino acid substitutions are based on the following considerations: (a) surface electrostatic potential and (lack of) hydrophobic patches on the surface, (b) retention of the isoelectric point (pI) of the protein in a particular range, (c) analysis of intra-protein cavities, (d) dynamic stability including correlated mutation analysis, normal mode analysis, and root mean square fluctuation (RMSF) at high or room temperature kinetics, (e) entropy and / or enthalpy components of substitution energetics, (f) visualization of specific substitutions, (g) amino acid types tolerated in families of related proteins as reflected by evolutionary conservation analysis of curated multiple sequence alignments (MSA), and (h) frequency of substitutions as reflected in low pseudo-energy CPD calculations.

[0328] The calculation method includes one or more of the following steps.

[0329] (1) Multiple sequence alignment (MSA) or multiple structural alignment. In this step, DNA and / or protein sequences with similarity to the target reference protein or its fragments are queried in public databases. Based on the results obtained, a multiple sequence alignment (MSA) or multiple structural alignment is constructed and the conservation rate is calculated. According to the MSA result, a decision is made regarding the level of CPD to be performed. In non-conserved positions, all amino acids (with or without cysteine) are allowed in the CPD, while in more conserved positions, the CPD is restricted to residues with similar properties (charge, size, internal dynamics, etc.). This step involves restricting the substitutions at each position based on biophysical knowledge and conservation data. MSA can result in a Position Specific Substitution Matrix (PSSM) in which each position along the sequence is described in a manner that correlates with the relative abundance of each amino acid, possibly taking into account the potential probability of amino acid substitution or deletion or insertion.

[0330] (2) Protein function analysis and analysis of structure-function-dynamics relationships. In this step, a database of substitutions whose effects (on activity, structure, binding, etc.) are known is constructed using prior knowledge. Based on prior knowledge, the CPD is restricted to substitutions and adjacent positions (e.g., 0.5-1 nm distance) known to disrupt protein stability and / or function and are not substituted.

[0331] (3) CPD. This step is partly performed by designated software such as ROSETTA, OSPREY, SCWRL, PyMol, AlphaFold, etc. Before performing deterministic CPD, the energy of the 3D structure / model of the reference protein is minimized. CPD may involve site-specific amino acid substitution, where one amino acid is replaced by another, or replacement of protein regions by other amino acid sequences, resulting in a protein with a different length. The latter can be done by reconstructing regions such as loops by ab initio methods, or by taking regions from other proteins, a method that may be called "grafting". For each reference protein, multiple models are considered.

[0332] (4) Selection: Collect the lowest energy models of the protein. MSA is built on these models and conserved sequences are determined. Subsets of substitutions are selected based on biochemical and biophysical prior knowledge. These subsets represent substitutions at one or more positions that occur frequently in the CPD. Each subset is then modeled on the 3D structure of the protein and energy minimized. The lowest energy subset is then selected for further computational and experimental validation.

[0333] One of the considerations used in CPD is whether to replace amino acids in the receptor binding site and in the binding region and its vicinity. The determination of the amino acid residues important for taste receptor binding can generally be performed by single substitution of various amino acids. As detailed in the examples herein, the inventors used computational analysis to characterize the putative binding site for taste receptors. The inventors identified several novel binding sites in taste receptors that bind to reference proteins and modified proteins.

[0334] Another consideration in CPD is to improve thermal stability while retaining the functional plasticity required for binding to receptor, which is often intrinsically related to protein rigidification. Proteins must undergo some conformational changes (also known as "functional plasticity") to activate receptors. Therefore, we focus on regions that can be rigidified while preserving the regions where functional plasticity must be maintained.

[0335] As shown herein, CPD analysis has identified specific amino acid residues / regions in the MNEI protein that can be modified to yield modified proteins with improved properties, particularly stability and taste.

[0336] For example, MD simulations of MNEI suggested that the beta-sheet of MNEI is less stable. MNEI loses its structure at 70°C (Kim et al., 1989, Protein Eng Des Sel, 2(8), 571-575). In general, high temperatures reveal the following weaknesses of MNEI and MNEI-based variants:

[0337] 1. The beta loops, especially the loop between beta strands 2 and 3, do not have a defined secondary structure and their movements reduce stability (Spadaccini et al., 2001, J Mol Biol., 305(3), 505-514).

[0338] 2. Beta backbone exposed to water. Simulation analysis of MNEI at high temperatures shows that the main weak points of MNEI are the edges of the interface and the backbone between the 3rd and 4th strands. Herein we find that atoms of the backbone are exposed to water. Simulations also show water intrusion into that area and interference with hydrogen bonding. (You could probably make a diagram of this.)

[0339] As described herein, the modified proteins described herein have improved food-related properties. The sweetness profile of the protein, such as the sweetness potency (sugar-like flavor), lack of off-taste, reduced onset time, and reduced aftertaste of the modified protein, can be determined by any taste test known in the art. For example, a comparison of the sweetness of sucrose or other sweeteners can be performed by a taste panel, and the sweetness potency can be graded as detailed in the following examples.

[0340] The comparison can be made, for example, by determining the minimum concentration required to elicit a sweet taste sensation, or a sweetness profile assessment including characteristics such as sweetness profile, sweetness onset time, lingering taste, mouthfeel, aftertaste, off-taste, and masking of undesirable tastes, to determine the threshold value of the modified protein compared to known sweeteners such as sucrose.

[0341] As used herein, the term sweetness affecting characteristics includes a sweet taste sensation determined by at least one of a sweetness threshold of about 0.28 mg / L, about 0.5 mg / L or greater, and a sweetness duration of about 1-20 seconds, sometimes 2-18 seconds, and sometimes 2-4 seconds.

[0342] The modified protein binds to the sweet receptor like the reference protein.

[0343] In some embodiments, the modified protein has a perceived sweetness threshold that is 300-16,000 higher than sugar on a weight basis.

[0344] The sensory profile includes taste kinetics, which indicates taste intensity over time, i.e., onset duration (time to taste sensation), taste duration, and aftertaste time (corresponding to the Gaussian tail). Additional characteristics include off-tastes (e.g., due to binding to other receptors), taste roundness, metallic and other minor tastes, synergy with other ingredients (e.g., masking and enhancing other flavors or undesirable tastes such as stevia), mouthfeel, astringency, etc.

[0345] In some embodiments, the modified protein is characterized by equal or improved at least one of the following relative to the reference protein: (1) structural thermal stability, (2) functional thermal stability, (3) pH stability, (4) halostability, (5) solubility in water or a partially aqueous environment (e.g., a food product containing fat), or (6) shelf-life stability.

[0346] The modified proteins described herein are characterized by sweet taste as well as other taste effects (masking of undesirable tastes, less aftertaste, less lingering taste, less off-taste, umami, better mouthfeel) that may be used as sweeteners in the preparation of products for oral delivery.

[0347] The modified proteins may be used as flavor modifiers or flavor enhancers.

[0348] The modified proteins described herein are for use as oral products. In some embodiments, the product is a food or beverage, a dietary supplement, or a pharmaceutical product. For preparation of the product, the proteins described herein may be combined with any food grade additives. The food or beverage may be provided and used in any solid or dry form, including, but not limited to, fine powder, lyophilisate, granules, tablets, etc. In some embodiments, the composition is provided in liquid form, for example, as a solute in water (aqueous solution).

[0349] Products comprising modified proteins may have a variety of uses, including, but not limited to, as sweeteners, flavorants, enhancers, masking agents, and proteins with flavor characteristics in the food and beverage industry (fruit and vegetable juices and nectars, soft drinks, ready-to-drink beverages, syrups, functional drinks, sports drinks, etc.), the dairy industry, i.e., dairy products, yogurt, and puddings, the pharmaceutical industry, the naturopathic industry, the functional food industry (e.g., functional food bars), and other health care products (e.g., toothpaste and mouthwash), the confectionery, candy, and gum industry, vegetables (e.g., ketchup or sauces), or any other application requiring the use of a flavor modifying composition as an excipient or additive.

[0350] According to some embodiments, the additional food ingredient is selected from the group consisting of sucrose, fructose, glucose, agave nectar, brown rice syrup, date sugar, honey, maple syrup, molasses, Monk fruit, sugar alcohols, rare sugars, steviol glycosides, aspartame, sucralose, acesulfame potassium, and dietary fiber.

[0351] In some embodiments, the modified protein has equal or improved structural thermal stability relative to the reference protein.

[0352] The term "structural thermostability" or "thermal stability" as used herein refers to the ability of a modified protein to retain its 3D structure at a temperature higher than that at which a reference protein retains its 3D structure. The 3D structural stability of a protein can be measured by any method known in the art, such as Circular Dichroism (CD), or thermal shift assays such as Differential Scanning Fluorimetry (DSF) or Differential Scanning Calorimetry (DSC), or titration with a protein denaturant such as guanidinium chloride. 3D protein structure can affect protein function. Of note, the shelf life and thermal stability required for food and beverages can be related to structural thermostability and are composed of different measurables, for example, pasteurization (or heat treatment during preparation of the final product for commercial packaging products) can be applied by different protocols and are related to the heat resistance that retains protein structure for very short periods of time.

[0353] In some embodiments, the modified protein has equal or greater functional thermostability relative to the reference protein. As used herein, the term "functional thermostability" refers to the ability of a modified protein to retain its function after exposure to elevated temperatures compared to a reference protein.

[0354] In some embodiments, the modified proteins herein can maintain their sweetening effect at higher temperatures or after exposure to higher temperatures for a limited time. In other words, there is no obvious change in sweetness or sensory profile after the product is exposed to temperatures above room temperature, sometimes up to 50°C, sometimes up to 100°C, or even up to 150°C. The functionality of the protein, e.g., sweetness, can be measured by sensory testing after cooling to a temperature at which the protein can be tasted.

[0355] In some embodiments, the modified protein has equal or greater pH stability than the reference protein. pH stability refers to the long-term stability of the modified protein over a wider pH range than the reference protein, i.e., the modified protein maintains its 3D structure and / or function after exposure of the product to any pH between 3 and 8, and sometimes to a pH between 4 and 8. For example, soda, such as cola, has a pH between 2.3 and 2.5, at which pH some of the sweet proteins are unstable and lose functionality immediately or after a time shorter than the normal shelf life of the beverage.

[0356] In some embodiments, the modified proteins have greater solubility than the reference MNEI protein, which may be in aqueous, partially aqueous, or non-aqueous environments, such as foods containing fat.

[0357] In some embodiments, the modified protein has an improved shelf life relative to the reference protein. Improved shelf life refers to no perceptible change in sweetness (functionality) or physical deterioration (e.g., color change, phase separation, etc.) of a product comprising the composition after the product is exposed to any temperature up to 150° C., sometimes any temperature between 4° C. and 150° C., or even to 100° C.

[0358] In some other embodiments, the modified protein is characterized by at least one of the following that is comparable to or improved from the reference protein: (1) folding kinetics; (2) a post-translational modification (e.g., glycosylation or acetylation) pattern of the protein that differs from the reference protein.

[0359] In some embodiments, the modified protein has folding kinetics that are comparable to or greater than the reference protein, i.e., a faster rate of protein folding from an unfolded or partially folded structure (e.g., assessed in silico by molecular dynamics or by experimental in vitro or in vivo methods). Alternatively, faster folding kinetics refers to slower unfolding kinetics in denaturation experiments, e.g., by denaturant titration (e.g., guanidinium chloride and / or high concentration urea) or other methods.

[0360] In some embodiments, the modified protein is characterized by an expression yield that is equal to or higher than the reference protein in the host organism being evaluated.

[0361] In some embodiments, the modified protein has a pI value of 8.6 to 9.5.

[0362] The modified proteins described herein are characterized by sweet taste and other taste effects (masking of undesirable tastes, less aftertaste, less lingering taste, less off-taste, shorter lingering taste duration, and umami) that may be used as sweeteners in the preparation of products for oral delivery.

[0363] The modified proteins may be used as flavor modifiers or flavor enhancers.

[0364] The modified proteins described herein are for use as oral products. In some embodiments, the product is a food product, a dietary supplement, or a pharmaceutical product. For the preparation of the product, the proteins described herein can be combined with any food grade additives. The food product can be provided and used in any solid dry form, including, but not limited to, fine powder, lyophilisate, granules, tablets, etc. In some embodiments, the composition is provided in liquid form, for example, as a solute in water (aqueous solution).

[0365] Products containing modified proteins may have a variety of uses, including, but not limited to, as sweeteners, flavorings, enhancers, or masking agents in the food and beverage industry (fruit and vegetable juices and nectars, soft drinks, ready-to-drink beverages, syrups, functional drinks, sports drinks, etc.), the dairy industry, i.e., dairy products, yogurt, and puddings, the pharmaceutical industry, the naturopathic industry, the functional food industry, and other health care products (e.g., toothpaste and mouthwash), the confectionery, candy, and gum industry, vegetables (e.g., ketchup or sauces), or any other application requiring the use of a flavor modifying composition as an excipient or additive (each of the following constitutes a separate embodiment of the present disclosure).

[0366] The product may include additional food ingredients. In some embodiments, the food ingredient is a sweetener, such as steviol glycosides. The combination of the modified proteins described herein with steviol glycosides produces a synergistic effect. Thus, in some embodiments, the product includes at least one modified protein shown in Table 10 and steviol glycosides.

[0367] When stevia (herein referred to as steviol glycosides or mixtures thereof) and / or variants thereof are combined with the modified proteins of the present invention in the range of 0.5Bx-8Bx sucrose equivalents, the modified proteins represent 30%-70% replacement of sucrose sweetness. The perceived sweetness intensity is at least 100% of the stevia solution at 0.5Bx-8Bx sucrose equivalents. The perceived residual sensory profile is better than 100% of the stevia solution at 0.5Bx-8Bx sucrose equivalents. The perceived sourness sensory profile is better than 100% of the stevia solution at 0.5Bx-8Bx sucrose equivalents.

[0368] According to some embodiments, the additional food ingredient is selected from the group consisting of sucrose, agave nectar, brown rice syrup, date sugar, honey, maple syrup, molasses, Monk fruit, sugar alcohols, rare sugars, aspartame, sucralose, acesulfame potassium, and dietary fiber.

[0369] In some embodiments, the formulations described herein provide a sugar-like taste profile with reduced, eliminated or masked aftertastes or off-flavors (e.g., metallic or licorice tastes), reduced, eliminated or masked bitterness, or reduced, eliminated or masked sweet linger.

[0370] It should be noted that modified proteins according to the present invention may be produced by any method known in the art, for example, the proteins may be produced synthetically, by recombinant DNA technology, or by protein production in microorganisms via fermenters, plants, plant calli, or other bioreactors. In some embodiments, the modified proteins may be produced in bacteria, for example, E. coli. In some other embodiments, the modified proteins may be produced in producer yeasts, such as Saccharomyces cerevisiae or Pichia pastoris. In some other embodiments, the modified proteins may be produced in filamentous fungi, such as Trichoderma or Aspergillus.

[0371] The term "yeasts and filamentous fungi" includes any Kluyveromyces sp., e.g. Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces sp., e.g. Saccharomyces cerevisiae, Pichia sp., e.g. Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pyperi, Pichia stiptis, Pichia methanolica, Hansenula polymorpha, Candida albicans, any Aspergillus sp., e.g. Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, Myceliopthora, and Neurospora crassa.

[0372] In some embodiments, the DNA sequence of the selected amino acid sequence is optimized at the RNA and DNA levels. At the RNA level, this includes minimizing RNA secondary structures to ensure rapid insertion into the ribosome. At the DNA level, this includes codon optimization for the host organism (taking into account the RNA level optimization). Optimization of codon usage prioritizes the use of the most abundant tRNA in the host organism for each amino acid expressed.

[0373] The term "about" as used herein refers to values ​​that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the stated value, the range of deviation being inclusive of integer values, where applicable, non-integer values ​​also constitute a continuous range. In some embodiments, the term "about" refers to ±10%.

[0374] It should be noted that various embodiments of the invention may be presented in a range format. The description of a range should be considered to specifically disclose all the possible subranges and individual numerical values ​​within that range. For example, description of a range such as 1-6 or 1 to 6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., and individual numerical values ​​within that range, for example, 1, 2, 3, 4, 5, and 6.

[0375] As used herein, the word forms "a," "an," and "the" are inclusive of the singular and plural unless the context clearly dictates otherwise.

[0376] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination or in any other described embodiment of the invention as suitable. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment can be practiced without those elements.

[0377] It should be noted that various embodiments and examples detailed herein in relation to various aspects of the invention may be applicable to one or more aspects disclosed herein. It should be further noted that any embodiment described herein, for example relating to a method, may be applied separately or in various combinations. Various embodiments and aspects of the invention as described herein above and claimed in the claims section below are experimentally supported in the following examples. As used herein, the phrase "in another embodiment" or any reference to an embodiment does not necessarily refer to a different embodiment, although that may be the case. Thus, various embodiments of the invention may be combined (from the same or different aspects) without departing from the scope of the invention.

[0378] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below, find experimental support in the following examples.

[0379] Although disclosed and described, it should be understood that the invention is not limited to the specific examples, method steps, and reactors disclosed herein, as such method steps and reactors may vary somewhat. It should also be understood that the terminology used herein is used solely for the purpose of describing particular embodiments, and is not intended to be limiting, since the scope of the invention is limited only by the appended claims and equivalents.

[0380] The following examples are representative of techniques employed by the inventors to carry out aspects of the present invention. While these techniques exemplify preferred embodiments for carrying out the invention, those skilled in the art will recognize in light of this disclosure that numerous modifications are possible without departing from the spirit and intended scope of the invention. EXAMPLES

[0381] Non-limiting examples Example 1: Computational design of novel proteins Polypeptides (proteins) were designed by using the amino acid sequence of MNEI as a reference protein for modification. MNEI, the single-chain monellin (referred to herein as SEQ ID NO:8), is a 96 amino acid polypeptide with a molecular weight of approximately 11 kD and a pI of approximately 8.7.

[0382] method Molecular Dynamics (MD)-System Setup and Simulation: Protonation states for all simulated protein residues were appropriately set by PROPKA (Olsson et al., 2011, J Chem Theory Comput, 7(22), 525-537) version 3.4 at pH 7.0. MD simulations were performed by GROMACS software (Abraham et al., 2015, SoftwareX(1-2), 19-25) version 2022.1. The system was parameterized by Amber99SB all-atom field (Hornak et al., 2006, Proteins, 65, 712-725). Independent iterations of each system were performed at 433 K for 50 ns each.

[0383] Molecular Dynamics (MD) - Measurements: 1. Native contacts were calculated by the Best-Hummer native contact fraction function (Best et al., 2013, PNAS, 110(44), 17874-17879) as implemented in MDTraj version 1.9.6 (McGibbon et al., 2015, Biophys J., 109(8), 1528-32). Native contacts are contacts between two amino acids that are not adjacent in the amino acid sequence but are spatially close in the native state tertiary structure of the protein. These contacts are used to measure deviations in molecular dynamics. A beta constant of 50 1 / nm, a lambda constant of 1.8, and a native contact cutoff of 0.45 nm were used. The fraction of native contacts was averaged over the time course of the simulation. The more contacts between the heavy atoms of a protein, the more stable it is during the simulation.

[0384] 2. To detect the unfolding of the secondary structure, the helical H-bonds during the simulation were calculated by the GROMACS hbond utility with intra-backbone mediated hydrogen bonds. The helical residues are defined as residues 10–26.

[0385] 3. Beta sheet H-bonds during the simulation were calculated by the GROMACS hbond utility with intra-backbone mediated hydrogen bonds. Beta sheet residues are typically defined as residues 2-6, 35-37, 42-48, 54-66, 69-78, 82-90 numbered according to the sequence and structure of MNEI (SEQ ID NO: 8, 2O9U).

[0386] 4. Average H-bonds Helix Measures the average number of mediated H-bonds within the helical backbone for a helix. This value indicates the stability of the helix during the simulation. Higher values ​​represent more canonical (and likely favored) helices.

[0387] 5. Average H-bonds beta Measures the average number of interstrand mediated H-bonds for the beta sheets. This value indicates the stability of the beta sheets during the simulation. The more stable the beta sheets are, the higher the value is expected, resulting in a correspondingly more stable protein.

[0388] 6. Water-Beta Sheet Backbone H-bonds Measures the average number of water molecules around the beta sheets of a protein that are stable enough to allow binding to the protein backbone in segments consisting of beta strand structures.

[0389] 7. Proportion of canonical helices This is the proportion of H-bonds of type i → i+4 in the helix. There are three types of helical H-bonds: i:i+3, i:i+4 and i:i+5, which indicate 3-10 helices, α-helices and pi-helices, respectively. The α-helices with i:i+4 bonds are the most canonical. This value indicates that the α-helices are maintained during the simulation. The higher the proportion of canonical H-bonds, the more stable the helices are expected.

[0390] 8.Sum RMSD The average of the RMSDs of the C-alpha atoms of the protein in the helical regions (residues 10-26), loops (residues 38-42, 49-53, 67-68, 79-81, 7-9, 27-34), the C-terminus of the protein (residues 90-96) and the beta-sheets (residues 2-6, 35-37, 42-48, 54-66, 69-78, 82-90) were calculated by the GROMACS rms utility. The averages were then summed to create a single measurement. Residues are numbered according to the sequence and structure of MNEI (SEQ ID NO: 8, PDB ID: 2O9U).

[0391] 9. Root-mean-square fluctuations (RMSFs) of the protein backbone atoms over the MD simulations. These were calculated by the GROMACS rmsf utility and averaged per residue.

[0392] Molecular graphics: Structure analyses, measurements, figures and movie clips were performed in either VMD (Humphrey, W., Dalke, A. and Schulten, K., VMD - Visual Molecular Dynamics, J. Molec. Graphics 1996, 14.1, 33-38.) version 1.9.4. Figures and movies were rendered by VMD or PyMol (The PyMOL Molecular Graphics System, Version 2.5.2, Schrodinger, LLC.).

[0393] Structure prediction and energy calculation: The new variants were modeled on the template of MNEI or DM31 crystal structures (MNEI, ID 2O9U, based on structure from the Protein Data Bank (PDB); DM31 determined by the Weizmann Institute of Science Structural Proteomics Unit).

[0394] The resulting structures were subjected to Rosetta software for energy minimization and energy score calculation with Rosetta 3.8 (Schueler-Furman et al., 2005, Science, 310, 638-642; Baker, 2006, Philos Trans R Soc Lond B Biol Sci., 361, 459-63; Kaufmann et al., 2010, Biochemistry, 49, 2987-2998). Minimization and scoring were performed with Rosetta's FastRelax protocol and the REF2015 energy function (Park et al., 2016, J Chem Theory Comput, 12, 6201-6212). For each input sequence, the protocol was repeated at least 30K times to obtain multiple structures and their Rosetta Energy Function (REU) scores. For each variant, the 30K scores were sorted and the 500 lowest scoring structures were used for further inspection and analysis. For MNEI and DM31, the input structure was the corresponding crystal structure (2O9U for MNEI) and the protocol was repeated 100K times. Table 1 shows the new variants selected for expression in the laboratory and their corresponding Rosetta Energy Unit (REU) scores. For some analyses, the lowest energy structure of each new variant was used.

[0395] Additional Protein Features: Visualization and analysis of hydrogen bonds were performed with PyMol. In addition, hydrogen bonds were examined by the Baker-Hubbard method (Baker and Hubbard, 1984, Progress in Biophysics and Molecular Biology, 44.2, 97-179) as implemented in the MDTraj Python package (McGibbon et al., 2015, Biophys J., 109(8),1528-1532) version 1.9.6.

[0396] An additional measure of hydrophobicity was used: the SAP (Spatial Aggregation Propensity) score calculated by Rosetta (Lauer et al., 2012, J Pharm Sci, 101(1), 102-115). This score measures the local hydrophobicity of a surface patch. Such surface areas can form hydrophobic interactions with other patches on other proteins, thus increasing the risk of aggregation. The higher the score, the greater the aggregation tendency.

[0397] An additional measure was used for estimating protein structure packing: VoroMQA (Olechnovic and Venclovas, 2017, Proteins, 85, 1131-1145). This method combines statistical potentials and the use of contact areas between atoms instead of distances. Contact areas derived from a Voronoi partition of the protein structure are used to describe and integrate both explicit interactions between protein atoms and implicit interactions between protein atoms and the solvent. VoroMQA produces scores at the atom, residue, and global levels, all in a fixed range between 0 and 1.

[0398] result Table 1 shows the calculated Rosetta Energy Unit (REU) scores of the new protein variants selected based on modeling.

[0399] For both minimization and calculation, the REF2015 energy function in Rosetta was used. The table also shows the SAP score, which indicates hydrophobicity and aggregation tendency. The residue numbers are according to MNEI in SEQ ID NO:8, and ΔE50 / ΔF52 / ΔR53 indicated deletion of E50, F52 and R53.

[0400] [Table 2] TIFF2025514608000005.tif214144

[0401] Table 2 gives an overview of the results obtained from molecular simulations of the novel protein. The average native contacts were calculated by the fraction of native contacts. The average RMSD was calculated by the backbone atoms of the protein. The average H-bonds in the helices and beta-sheet backbone were calculated with GROMACS (see molecular dynamics under Methods section). The average RMSD of specific secondary structure element loops was calculated by the C-alpha atoms of the protein. Two RMSD results are described here. Please see Methods section for a detailed explanation.

[0402] [Table 3] TIFF2025514608000007.tif89164

[0403] MD considerations for novel modified proteins (variants) As described herein, the purpose of the MD analysis was to evaluate different regions in the protein (MNEI) and its variants to predict novel proteins with improved properties. Modifications were evaluated, for example, in the alpha helix region, the beta sheet region, and the core region.

[0404] Example 1A: Helix Capping Variants To identify targets for the design, molecular dynamics were performed by MNEI simulations at high temperature. Lα2 was identified as a target region for stabilization by molecular dynamics. Thus, the aim is to identify modified proteins that stabilize the loop between the helix and the second beta strand.

[0405] Table 3 shows the computational data analysis of novel variants, which are considered to be helix-capping variants. The difference in AUC (area under the curve) (ΔAUC) was calculated by the average RMSF of three MD simulation repeats for each DM as ΔAUC=(AUC(variant)-AUC(DM31)).

[0406] [Table 4]

[0407] DM42(I26T): predicted to be favorable based on helix capping analysis (using CAPS-DB database, Segura et al., 2012, Nucleic Acids Res. 40(Database issue)). The substitution removes hydrophobic residues from the surface of the protein, resulting in a lower SAP score (Table 1). MD simulations show high stability, with backbone-mediated hydrogen bonds between 26 and E22 and between position 26 and A23 being more stable in DM42 compared to DM31 (Figure 21B, Table 2 and Figure 7). Hydrogen bonds are seen in the model (Figure 6). VoroMQA analysis shows more favorable packing of residue 26 and its surroundings (Figure 21A). Furthermore, MD analysis shows indications of improved stability (Table 2). Experimental DSF analysis for DM42 measured a very high melting temperature (99°C) (Table 7A).

[0408] DM43(K25R): The SAP score of this DM indicates a decrease in hydrophobicity (Table 1). The native contacts and backbone-mediated H-bonds within the beta-sheets and within the helices of DM43 are more stable during simulation at 433 K compared to MNEI (Table 2), suggesting an increased stability of this variant. The side chain of K25 exhibits hydrogen bonding with the side chain of E21 in DM31 (Figure 8). Simulations show that in DM31, K25 attacks the E22 backbone, decreasing helix stability, whereas in DM43, R25 maintains the R25-E21 inter-residue H-bond. The substitution K25R is expected to increase the strength of the bond with more positive charges. The hydrogen bond between K25 and E22 contributes to the stability of the helix (Figure 8, indicated by an arrow) and is further stabilized by the substitution in DM43, i.e., K25R. Experimental DSF analysis for DM43 measured a high melting temperature (94° C.) (Table 7A), reflecting an improved ability to maintain sweetness despite extreme temperatures.

[0409] DM69(K25R+Q28K): A combination of DM43(K25R) and DM65(Q28K). The SAP score of this DM is relatively low due to these highly polar substitutions (Table 1). Molecular dynamics simulations and RMSF of this DM determined it to be favorable (Table 3 and Figure 34). DM69 is expected to be sweeter than DM31 in light of the electrostatic changes shown in Figure 35. Experimental DSF analysis for DM69 measured a very high melting temperature (97.5°C) (Table 7A).

[0410] DM508(V20I+Q28K): combines the core-directed DM96(V20I) and DM65(Q28K), both of which affect the helices of the protein. The RMSF also shows high stability, especially in the Lα2 loop, as evident from molecular dynamics simulations (Figures 28 and 31). This is embodied in an improved ability to maintain sweetness at high temperature conditions (Table 7A). According to the SAP scores, DM508 is predicted to be less hydrophobic (Table 1), as seen from the APBS (Figure 32).

[0411] DM509(K25R+V20I): combines the core-directed substitutions DM96(V20I) with DM43(K25R), both of which affect the helix of the protein. The resulting RMSF shows improved stability as evidenced by molecular dynamics simulations (Figure 28 and Table 3). This DM has a low SAP score indicating reduced hydrophobicity (Table 1).

[0412] DM92 (I26T+Q28K): The Lα2 region was further stabilized by substituting a lysine residue (Q28K) at position 28. MD simulations show that wild-type Gln does not show a stabilizing interaction. Substitution with lysine at this position allows hydrophobic interactions with the region around position 28, so that this region remains fixed with increased rigidity. This is possible in combination with I26T, which brings the surrounding backbone closer to the protein. The combination of substitutions improves the packing of both residues compared to their presence alone (Figures 21A and 21B). In addition, this combination reduces the SAP score of this DM (Table 1), likely due to the replacement of a hydrophobic residue with a polar one. The RMSF shows high stability (Table 3, Figure 21B).

[0413] DM464 (K25R+D68N): The combination of K25R+D68N is predicted to contribute to both stability and sweetness by reducing the negative charge and increasing the positive local surface charge (D68N). The substitution also reduces the hydrophobicity of the protein, as evidenced by the very low SAP score (Table 1). The RMSF plot indicates high stability (Figure 22).

[0414] Figure 22 shows the average RMSF (root mean square variation) of DM31, MNEI, DM87 (D68N), DM43 (K25R), and DM464 (K25R+D68N). Each variant is shown in a different color. Comparing the RMSF of the helix terminal region (25-30aa) of different DMs with DM31 (black line), DM43 and DM464 show lower RMSF.

[0415] DM330(Q28S): DM330 was designed to have improved packing. VoroMQA analysis indicates favorable packing (Figure 21A) and this variant shows high stability in RMSF and MD (Tables 2 and 3, Figure 36). DM330 is predicted to be sweeter than DM31 based on electrostatic analysis (Figure 37).

[0416] DM66 (Q28R): Molecular dynamics suggest high stability (Tables 2 and 3, Figure 36). DM66 is predicted to be sweeter than DM31 based on electrostatic analysis (Figure 37) and was proven to be extremely sweet by the sensory panel with a strong ability to maintain sweetness at high temperatures (Table 7A and Figure 38). Experimental DSF analysis for DM66 measured a high melting temperature (95°C) (Table 7A). In addition to these improved properties, DM66 has a low SAP score (Table 1).

[0417] DM65(Q28K): This variant was proven to have a very high sweetness by the sensory panel, with high sweetness persistence at high temperatures as reflected by heat treatment (Table 7A and Figure 38). As the modification is close to position 26, we tested its effect on packing, which shows improved packing (Figure 21A). This link between sweetness and packing of monellin has not been suggested before. Experimental DSF analysis for DM65 measured a high melting temperature (93°C) (Table 7A). DM65 shows high stability by MD simulations at high temperatures, as also shown by RMSF plots (Table 3 and Figure 21B, Figure 34).

[0418] DM41(I26S): predicted to be favorable based on helix capping analysis (using CAPS-DB database, Segura et al., 2012, Nucleic Acids Res. 40(Database issue)). The substitution removes hydrophobic residues from the surface of the protein, thus decreasing the SAP score (Table 1). In addition, the AUC indicates high overall stability (Table 3). Experimental DSF analysis for DM41 measured a high melting temperature (94°C) (Table 7A).

[0419] Figure 12 shows that DM65, DM42 and DM43 have higher stability compared to MNEI. DM145, DM144, DM115, DM116 are more stable than MNEI. The native contacts of DM42, DM43, DM115, DM116, DM144 are increased compared to MNEI (Table 2). DM42, DM43 and DM144 have increased number of helical backbone mediated H-bonds compared to MNEI (Table 2). DM65, DM145, DM115 and DM116 have more helical backbone mediated H-bonds than MNEI (Table 2). DM42 helix is ​​more canonical on average during MD simulation (Table 2). As shown by MD simulation, the helices of DM43, DM115, DM116, DM144, DM145 and DM65 are more canonical on average than MNEI (Table 2). DM115 and DM43 also have low RMSF ΔAUC, and DM144, DM145, and DM330 have low SAP scores (Tables 1 and 2).

[0420] Figure 38 shows the relative sweetness of DM65 and DM66 at 10 Brix compared to DM31. According to the sensory panel, both proteins had a 15% and 19% increase in sweetness, respectively.

[0421] Example 1B: Sweet variants

[0422] [Table 5]

[0423] DM57(T33R): APBS analysis (Figure 24) shows an increase in the size of the positive surface patch around residue 33. Similarly, hydrophobicity was decreased by substitution with highly polar residues, as evident from the SAP scores (Table 1). RMSF plots showed high stability (Figure 18) and MD analysis showed good stability index (Table 2). Experimental DSF analysis for DM57 measured a high melting temperature (91°C) (Table 7A) and the sensory panel showed high sweetness and very high sweetness after heat treatment (Table 7A).

[0424] DM87 (D68N): The melting temperature of DM87 is 90, it is sweet and maintains its sweetness after heat treatment (see Table 7A). The number of native contacts increased during the simulation, as well as the secondary structure backbone mediated H-bonds in the helices and beta sheets (Table 2). The helices of DM87 are on average more canonical than those of MNEI (Table 2). This substitution of charged residues with neutral and polar residues reduces the hydrophobicity as shown by the SAP score (Table 1). Experimental DSF analysis for DM57 measured a high melting temperature (90°C) (Table 7A) and the sensory panel showed high sweetness and very high sweetness after heat treatment (Table 7A).

[0425] DM75(E4Q): The RMSF of this variant indicates high stability (Figure 17). Based on MD simulations, DM75 shows good stability indices (Table 2), e.g. its helices are on average more canonical than MNEI (Table 2). The hydrophobicity of this protein is reduced as indicated by the SAP score (Table 1). Experimental DSF analysis indicates a high melting temperature (Table 7A).

[0426] DM108(D68T): The modification results in a more positive charge on the protein - Figure 26. MD simulations at 433K show that the protein keeps its structure intact and still shows higher stability than MNEI (Figure 25). Surprisingly, the substitution of charged residues with neutral and polar residues leads to a decrease in hydrophobicity as evident from the SAP scores (Table 1).

[0427] DM61(D68S): MD simulations suggest that the RMSF of this DM is also better than DM87 and closer to DM31 with a more stabilized Lα2 loop (Figure 25). The Asp to Ser substitution at position 68 reduces the negative charge of the protein, which is predicted to improve long-range interaction / attraction to the sweet receptor (Figure 26).

[0428] D68N - See results in Example 1A.

[0429] DM65 (Q28K), DM66 (Q28R) and DM69 (Q28K+K25R) are 20% sweeter than DM31 (Table 7A). They also have high melting temperatures up to 97.5°C (DM69) and their sweetness remains high after heat treatment, up to 75% higher for DM66 than for DM31. The sweetness of DM65 and DM66 at 10 Bx is higher than that of DM31 (Table 7B and Figure 38).

[0430] DM341 - Figure 41 shows the APBS analysis of DM341.

[0431] Example 1C: Core Repacking

[0432] [Table 6]

[0433] Core variants: Another region of the protein that was recommended as a design target by computational protein design is the core of the protein. Various proteins were designed with novel substitutions that improve the packing of the core, thereby decreasing its accessibility to water and making the protein more stable. These positions were the result of the Rosetta design protocol performed on the core residues, rational design and visual analysis of the core, additional packing and solvent accessibility analysis and MD simulations. In addition, the C41 position was also targeted.

[0434] DM46(C41T): C41T removes a cysteine ​​residue that has several unfavorable properties. Cysteine ​​at position 41 is at risk of disulfide bond formation and nucleophilic attack that causes the release of a sulfur atom, which may reduce the shelf life of the protein. In addition, cysteine ​​is the second rarest amino acid (after tryptophan), so a reduction in available tRNA for the codon of this amino acid leads to lower expression levels. Substitution of cysteine ​​solves these problems. This replacement also results in a reduction of the proximal cavity found in the hydrophobic core of the protein (Figure 9, A: C41 in sticks, B: T41 in sticks, internal cavity shown in pink). The better packed core is expected to be more stable due to increased rigidity and reduced access to water. DM46 also improves thermostability (Table 7A) and is predicted to be stable during MD simulations at 433 K (Figure 27).

[0435] DM150(C41A): DSF analysis shows improved thermostability and excellent melting temperature (Table 7A) and is predicted to be stable during MD simulations at 433 K (Figure 27). Surprisingly, this protein shows a significantly reduced hydrophobicity despite the substitution of polar residues for hydrophobic residues (see SAP scores in Table 1).

[0436] DM151(C41S): has improved thermal stability (Table 7A) and is predicted to be as stable as DM31 during MD simulations at 433 K (Figure 27). This DM and DM150 show significantly reduced hydrophobicity (SAP score, Table 1).

[0437] DM85 (A73V): Experimental DSF analysis for DM85 measured a high melting temperature of 94.5° C. (Table 7A). Surprisingly, substitution of small hydrophobic residues for larger ones reduced hydrophobicity as measured by SAP score (Table 1).

[0438] DM77(A19V): predicted to have improved thermostability (Table 7A) and improved packing compared to DM31 (higher vuroMQA global score, Table 5). Surprisingly, substitution of small hydrophobic residues for large hydrophobic residues reduced hydrophobicity as measured by SAP score (Table 1).

[0439] DM96 (V20I): Increased sweetness at 6brix, improved heat resistance, and excellent melting temperature (Table 7A). RMSF plot shows high stability (Figure 31). Figure 32 shows APBS analysis of DM96.

[0440] DM117 (V20I+V64I): This variant shows a lower RMSF compared to DM84, DM96 and DM31 (Figure 32). It is more packed and has a higher vuroMQA score compared to DM84 (Table 5). DM117 has a lower REU than DM31 (Table 1). Surprisingly, these substitutions also have a lower hydrophobicity (SAP score, Table 1), despite the substitution of large hydrophobic residues at both positions.

[0441] DM498 (C41S+V64I+A73F+I75L+F89V): is the result of the core design described above. The variant shows a lower RMSF compared to DM31 (Figure 29). DM498 shows improved packing compared to DM31 (Table 5). The SAP score of this DM is significantly lower compared to the DMs analyzed in Table 1.

[0442] DM491 (V20I+C41A+A73F+I75L+F89M): is the result of the core design described above. DM491 shows improved packing compared to DM31 (Table 5). It has a lower RMSF compared to DM31 (Figure 28, Table 5). DM492 has a lower REU than DM31 (Table 1). These substitutions reduced the overall hydrophobicity of the protein as evidenced by the SAP score (Table 1).

[0443] DM452 (C41A+V64L+I75V): This is an improvement of DM432, and removes G16A, which is known to impair stability. This variant shows high stability in RMSF plot (Figure 39). In addition, this DM shows lower hydrophobicity, as evidenced by its SAP score (Table 1).

[0444] DM489 (V20I+C41V+A73V): Shows improved packing compared to DM31 (Table 5) and lower REU values ​​indicating stability (Table 1).

[0445] DM509 (K25R+V20I): Described in the Helix-Capping section. It is a combination of K25R and V20I. Surprisingly, it shows lower hydrophobicity despite being chemically similar substitutions (Table 1).

[0446] DM508 (V20I+Q28K): Described in the Helix-Capping section. It is a combination of V20I and Q28K. The variant shows high stability in the RMSF plot (Figure 31). Q28K is expected to add sweetness due to an increase in the positive surface local electrostatic potential as shown by APBS analysis (see Figure 32). In addition, the protein shows a low SAP score (Table 1) and increased packing about position 28 according to VoroMQA analysis (Figure 23).

[0447] DM506 (V20I + D68N): The electrostatic potentials shown by APBS (Figure 33) indicate that DM506 is expected to be as sweet as DM87. MD plots and analysis show high stability (Figure 40, Tables 2, 4, 5). In addition, the protein has a reduced SAP score (Table 1). Figure 40 shows the average RMSF (root mean square fluctuation) of DM31, MNEI, DM506 (V20I + D68N), DM87 (D68N), and DM96 (V20I), indicating the stability of DM506.

[0448] DM505 (A19V + Q28K): A combination of DM77 (A19V) and DM65 (Q28K). It is expected to have better packing than DM31 (Table 5).

[0449] DM47 (R31T): The Rosetta Energy Unit (REU) scoring energy of this substitution results in a lower energy (-322.6 REU) than DM31, and also a lower SAP score (Table 1). The substitution results in a shorter hydrophobic "neck" at residue 31 (stretching between the C-beta and C-delta atoms). As the position is solvent accessible, R31T was also suggested to reduce the overall hydrophobicity and risk of aggregation (Samish I., 2017, Methods Mol Biol., 1529, 3-19). The Tm of DM47 was experimentally determined by DSF, and showed improved stability compared to DM31 (Table 7A).

[0450] Figure 13 shows RMSF plots of proteins DM84, DM96, DM77, DM85, illustrating their stability. Table 2 shows additional MD-based measurements of these core variants, indicating high stability.

[0451] Figure 14 shows RMSF plots of proteins DM91, DM150, DM151, DM143, DM152 and DM46, indicating their stability. Table 2 shows additional MD-based measurements of these core variants, indicating high stability.

[0452] Example 1D: Additional Variants: DM72: N35T replaces the asparagine with a smaller amino acid while maintaining polarity, thus avoiding the effect of exposed hydrophobic atoms. As can be seen from Table 1, Rosetta predicts that N35T is favored. Molecular dynamics simulations of MNEI and DM72 showed that DM72 is generally more stable and rigid than MNEI. At 433 K (Figure 16, Tables 2 and 4), all regions of DM72 are more stable than MNEI. Experimental DSF analysis for DM72 measured a high melting temperature (99°C) (Table 7A).

[0453] DM70 (R84L): As a positively charged long amino acid, arginine has a hydrophobic section in its side chain (from C-beta to C-delta atoms). Based on the crystal data, it was suggested that in MNEI and DM31, R84L is mostly exposed, resulting in an exposed hydrophobic stretch of atoms. It was also suggested that the substitution R84L is expected to allow for more adequate protein packing rather than stretching the hydrophobic "neck" (one that stretches between the C-beta and C-delta atoms). Rosetta analysis suggests that, indeed, R84L significantly improves the stability of the protein (Table 1). Figure 15 shows the RMSF of DM70 in molecular dynamics simulations at 433 K, demonstrating the stability. In addition, the number of mediated H-bonds within the secondary structure backbone of the helices and beta sheets of DM70 is increased (Table 2). The RMSD values ​​of the loops (loop A2 (residues 26-35) and L23) and helices in DM70 indicate stability. The DM70 helices are, on average, more canonical than those of MNEI (Table 2). Furthermore, the melting temperature (Tm) of DM70 was experimentally determined by DSF, indicating high thermal stability (94°C, see Table 7A).

[0454] DM420: R84Y is a mutation predicted to increase protein packing while maintaining hydrogen bonding with the original residue. The Rosetta REU score indicates high stability (Table 1). Surprisingly, this substitution reduced hydrophobicity as shown by the SAP score (Table 1) despite the substitution with a more hydrophobic residue (Y).

[0455] DM424: R84I is a mutation designed for better protein packing. Figure 15 shows the RMSF of DM424 and DM31 in molecular dynamics simulations at 433K, indicating that the loops of DM424 are more stable and rigid. The protein is predicted to pack better than DM31 (Table 5).

[0456] Figure 15 shows the average RMSF (root mean square fluctuation) of DM31, MNEI, DM70 (R84L), DM420 (R84Y) and DM424 (R84I), all of which contain a substitution at residue 84. The RMSFs indicate high stability.

[0457] Example 2: Cloning, Expression and Characterization of MNEI Designer Proteins Recombinant MNEI protein was produced in E. coli BL21(DE3+) under the T7 promoter induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). Using this system, MNEI protein was expressed as a cytoplasmic protein (soluble fraction) in a high-density fermentation process. Designer MNEI (DM) is a designed protein with up to 11% amino acid substitutions.

[0458] All DMs were produced in E. coli fermentations and purified to levels >95%.

[0459] Cloning Site-directed mutagenesis (SDM) was used to generate DMs.

[0460] [Table 7]

[0461] [Table 8] TIFF2025514608000013.tif168150

[0462] [Table 9]

[0463] fermentation All DM clones were subjected to fermentation in 3 L vessels using the Sartorius BioStatB system or in 2 L vessels in the Solaris Jupiter system. Some DM clones were generated by outsourcing at VTT (Finland) and SciVac (Israel). All fermentations followed a protocol based on "High cell-density fermentation of Escherichia coli" by Arie Geerlof - EMBL Hamburg 29 January 2008.

[0464] purification All DM samples were purified by the following steps: 1. Dissolution by pressure homogenizer. 2. Capture of proteins on a multimode resin and elution with increasing NaCl concentrations in the same buffer. 3. At least one polishing step using a resin from the following group: 1. Ion exchange. 2. Hydrophobic interactions. 3. Size exclusion. 4. Final microbial filtration (0.2um) and storage at -20°C.

[0465] Characterization Refining Level Purification levels were assessed by SEC-HPLC using gel electrophoresis followed by Coomassie staining and densitometric analysis. Densitometric analysis was performed by running 20 μg / lane of each protein using a pure DM standard curve of 50-500 ng / lane. In all samples, maximum contamination reached 3% (i.e., 97% purity).

[0466] Example 3: Characterization of modified proteins: This example included sensory evaluation of sweetness intensity and stability.

[0467] The shelf-life stability of DM31 was tested (5Bx equivalent in citrate buffer).

[0468] As shown in FIG. 3, the sweetness intensity of DM31 stabilizes after 12 weeks at both 21° C. and 32° C.

[0469] Heat stability of DM31 in rice flour base (40% moisture) The heat stability of DM31 was tested on rice flour with 40% moisture content dissolved in water. DM31 was added to the water at a concentration of 30 Bx equivalents. The rice flour containing sweet protein was dried in a sealed bottle at 60°C for 1, 3, 6, 9, 12, 15, 18 and 24 hours. Amai's expert panel compared the sweetness intensity on a scale of 0-100, where 100 is defined as 8 Bx equivalents, with the fresh product defined as a scale of 60 by the panel. As shown in Figure 4, the sweetness intensity of DM31 is stable at 60°C for 24 hours.

[0470] Sweetness intensity of DM31 after heat treatment of rice flour with 40% moisture content The heat stability of DM31 was tested in rice flour with 40% moisture content in water. DM31 was added to the water at a concentration of 30 Bx equivalents. The rice flour containing sweet protein was dried in an open jar at 60°C and 70°C for 18 hours.

[0471] As shown in Figure 47, the sweetness intensity of DM31 in rice flour with 40% moisture content is stable after heat treatment at 60°C and 70°C for 18 hours in an open plate and reconstitution with water.

[0472] Thermal stability of powdered DM31 The heat stability of powdered DM31 was tested at elevated temperatures for 1-5 minutes. The sweet protein powder was dried at 90°C, 100°C, 110°C and 120°C for 1, 2, 3, 4 and 5 minutes. Samples were suspended in water and tested by Amai's expert sensory panel, who rated the sweetness intensity against the fresh product on a scale of 0-100. As shown in Figure 5, the sweetness intensity of powdered DM31 is stable up to 120°C for 5 minutes.

[0473] Sweetness Evaluation The professional sensory panel included expert supertaster panelists (as described below) who were first calibrated with sugar solutions on a 0-100 scale (magnitude estimate), with 0 = not at all sweet and 100 = very sweet. After calibration, the tasters blindly graded the test samples on the same scale following a validated tasting protocol. A linear scale of sucrose was obtained for concentrations of 2Bx, 4Bx, 6Bx, and 8Bx. Brix (Bx) = gr / 100 ml.

[0474] An initial sweetness evaluation was performed in each test at the selected potency X4000 by comparing with sugar at 6Bx and the newly selected DM. All dilutions were performed with water only. Samples were evaluated by an expert panel in various cases using sucrose at 6°Bx as a control.

[0475] DSF and DSC Analysis - Thermal Sensitivity Tm values ​​were determined by differential scanning fluorimetry (DSF) using a Nanotemper Prometheus Panta. DSF is an easy, rapid and accurate method for the analysis of protein stability and aggregation. DSF detects changes in the fluorescence of tryptophan and tyrosine residues in proteins. The fluorescence of tryptophan and tyrosine residues strongly depends on their close environment. Conformational changes in proteins are reflected as fluorescence changes. The first derivative of the fluorescence ratio (330 nm / 350 nm) is used to determine the inflection point. Since no secondary reporter fluorophore is required, protein solutions can be analyzed over a concentration range from 250 mg / ml to 10 μg / ml, independent of the buffer composition. DM was analyzed at a concentration of 0.5 mg / ml in 5 mM citrate buffer pH 6.

[0476] result:

[0477] [Table 10]

[0478] [Table 11]

[0479] Example 4: Energy bar formulation containing designer-MNEI (DM) protein The energy bar formulation containing DM protein is shown in Table 8.

[0480] [Table 12]

[0481] Testing Methodology - Amai's Expert Super Taste Panel All sensory evaluations were determined using a trained expert panel for analytical discrimination. The sensory expert panel was established by a screening process of potential tasters. Screening tests conducted according to ISO standards (IS 8586-1) examined the sensory sensitivity, consistency, and sensory memory of tasters. The panel is well trained and calibrated. The selected panel is regularly trained to maintain high performance results.

[0482] Sensory profile of energy bars with DM protein prototypes - Test procedure: For the energy bar category, a sensory vocabulary was determined by an expert panel. The sensory vocabulary was constructed by tasting a wide range of products from the category and raising all relevant sensory attributes that describe the category. As many different languages ​​as possible were used to also describe the products well.

[0483] Once the sensory vocabulary has been determined, select the key attributes that will be used to describe the product in the questionnaire.

[0484] Construction of a sensory profile of an energy bar containing DM protein: Panelists rated each test product against a reference product on a binary scale (-3 to +3) with a fixed reference point (0) for all attributes selected from the glossary. If the test product was rated "more" than the reference product for a particular attribute (e.g. sweeter, richer, etc.), a positive rating (+1, +2, or +3) was obtained, and if it was rated "less" than the reference product for a particular attribute (e.g. less sweet, less rich, etc.), a negative rating (-1, -2, or -3) was obtained. Before and between each attribute rating, tasters were asked to rinse their mouths with mineral water, eat an unsalted cracker and a cucumber, and drink water again.

[0485] result Energy Bar Prototype As demonstrated in Figure 1, the 40% reduced added sugar energy bar is less sweet than the DM protein prototype energy bar.

[0486] Example 5: Marzipan formulations containing designer-MNEI (DM) proteins Marzipan is a confectionery made mainly from ground almonds and sugar. It is widely used in the bakery industry and in the manufacture of various kinds of confectionery.

[0487] Marzipan formulations containing DM protein are shown in Table 9.

[0488] [Table 13]

[0489] Preparation method: The almonds were ground and all the powders were gradually added to the mixer and finely ground. All the liquid ingredients and sweet protein were added to the mixture and grinding was continued until a crystalline dough was obtained.

[0490] 0.01-0.05% protein by weight was added to the recipe. The sweetening potency of the sweetening protein DM-31 is 1000-3000, which corresponds to 25-45 brix.

[0491] result As demonstrated in Figure 2, marzipan containing the sweet protein DM-31 (70% less added sugar) is sweeter than marzipan with 70% less added sugar.

[0492] Example 6: Non-dairy milk prototypes containing designer monellin (DM) protein Non-dairy milk formulations containing DM protein are shown in Table 10.

[0493] [Table 14]

[0494] Preparation method: Sugar and AMAI sweet protein DM31 were added to unsweetened non-dairy milk and mixed to form a homogenous solution.

[0495] result Non-dairy milk with 50% reduced added sugars is less sweet than non-dairy milk with DM protein prototype (50% reduced added sugars).

[0496] Example 7: Granola Preparation The formulation of the granola containing DM protein is shown in Table 11.

[0497] [Table 15]

[0498] Preparation method: Bake the oatmeal, crispy rice, pecans, and almonds in a freezer for 15 minutes at 160°C.

[0499] Glucose syrup, powdered sugar, sunflower oil, glycerol, lecithin, and salt are heated to form a homogeneous syrup.

[0500] Toasted pecans and almonds are finely ground. The dry ingredients (oatmeal, crispy rice, pecans, and almonds) are added to the syrup and mixed. Maltodextrin and DM31 are added last.

[0501] Place mixture in baking dish.

[0502] Bake the granola mixture at 90°C for 10 minutes, then remove, cool and store in an airtight container in a cool place.

[0503] result: According to FIG. 10, the AMAI sweetened protein-containing granola formulation with 70% reduced sugar is sweeter than the non-protein-containing granola with 70% reduced sugar.

[0504] Example 8: Peanut Butter Spread Peanut butter spread formulations containing DM protein for various filling applications are shown in Table 12.

[0505] [Table 16]

[0506] Preparation method: 1. Grind the peanuts. 2. Slowly add ingredients 2-7 to the mixer and grind until a uniform dough is obtained. 3. Add sweet protein DM-31 to the mixture and continue grinding at low speed until completely assimilated.

[0507] result: As shown in FIG. 11, in chocolate peanut butter cups, the Amai sweet protein-containing peanut butter spread with 75% less sugar is sweeter than the no protein peanut butter spread with 75% less sugar.

[0508] Example 9: Halva (Sesame) Spread The halva (sesame) spread formulation containing DM protein is shown in Table 13.

[0509] [Table 17]

[0510] Preparation method: Mix all ingredients until a uniform mixture is obtained.

[0511] Results: As shown in FIG. 20, the halva spread formulation containing DM31 with 80% reduced sugar is sweeter than the halva spread without DM31 with 80% reduced sugar.

[0512] Preparation method: 1. Mix all the ingredients and grind them gradually in a Chocolate Refiner for 6 hours until a fine mixture is obtained. 2. Tempering with stirring: first heat to 45°C, then cool to 27°C and reheat to 29°C. 3. Pour the chocolate into molds and let it harden.

[0513] result: As demonstrated in Figure 43, milk chocolate containing sweet protein DM-31 with 50% reduced sugar is sweeter than milk chocolate with 50% reduced sugar.

[0514] Example 11: Chocolate Peanut Butter Cups This dessert is a combination of two ingredients: milk chocolate and peanut butter spread.

[0515] The formulation of the milk chocolate containing DM protein is shown in Table 15.

[0516] [Table 18]

[0517] Preparation method: 1. Melt the chocolate, cocoa mass and cocoa butter at 70°C for 3 to 5 minutes. 2. Add glucose and fiber and mix at 70°C for 1-3 minutes. 3. Add milk powder, whey and lecithin and mix for 10 minutes at 75-80 degrees. 4. Cool the mixture to 70 degrees, add sweet protein DM-31 and mix for 3-5 minutes until fully assimilated. 5. Pour the chocolate into molds and allow the mixture to cool.

[0518] Peanut Butter Spread Peanut butter spread was mentioned above.

[0519] result: As shown in FIG. 43, in chocolate peanut butter cups, the Amai sweetened protein-containing peanut butter spread with 75% less sugar is sweeter than the no protein added peanut butter spread with 75% less sugar.

[0520] Example 12: Sweet Chili Sauce: The formulation of sweet chili sauce containing DM protein is shown in Table 16.

[0521] [Table 19]

[0522] Preparation method: 1. Add vinegar, lemon juice, chili powder, garlic powder, salt and cornstarch. 2. In a separate bowl, weigh the sugar and xanthan gum and mix thoroughly. 3. Mix both bowls and add water. 4.Cook at 114°C for 10 minutes. 5. Cook the other one at 95°C for 30 minutes. 6. Add the Sweelin™ and mix thoroughly. 7. After cooling, pass through a fine sieve.

[0523] result: As demonstrated in Figure 44, sweet chili sauce with 50% reduced sugar combined with Amai sweet protein is just as sweet as the full sugar product.

[0524] Example 13: Sugar syrup for Savarina cake The formulation of sugar syrup containing DM protein is shown in Table 17.

[0525] [Table 20]

[0526] Preparation method: 1. Prepare Savarina cake according to manufacturer's instructions and allow to cool completely. 2. Bring the water to a boil and add the sugar while stirring. 3. After boiling, add rose water, vanilla flavor extract, and sweet protein DM31 and mix thoroughly. 4. Pour into a bowl and leave the Savarina cake to soak for 10 minutes. 5. Serve the cake with the prepared whipped cream.

[0527] result: As demonstrated in Figure 19, Savarina with 50% reduced sugar combined with Amai sweet protein is as sweet as the full sugar product.

[0528] Example 14: Vinaigrette Salad Dressing The formulation of a vinaigrette salad dressing containing DM protein is shown in Table 18.

[0529] [Table 21]

[0530] Preparation method: 1. Weigh out all ingredients except sweet protein. 2. Blend them all together in a blender for 1 minute. 3. After blending, add the sweet protein and mix thoroughly.

[0531] result: As demonstrated in Figure 30, the vinaigrette with 50% less sugar combined with Amai sweet protein is sweeter than the vinaigrette with 50% less sugar and no added protein.

[0532] Example 15: Teriyaki The formulation of teriyaki containing DM protein is shown in Table 19.

[0533] [Table 22]

[0534] Preparation method: 1. Mix cornstarch with water until dissolved. 2. Weigh out the glucose, sugar, and soy sauce into a saucepan and add the cornstarch dissolved in water. 3. Heat the mixture to 100° C. for 5 minutes. Then mix until the temperature drops to 60° C. (approximately 10 minutes). 4. When the temperature reaches 60°C, add the sweet protein DM-31. 5. Strain the sauce through a fine sieve.

[0535] result: As demonstrated in Figure 42, teriyaki sauce with 70% less sugar combined with Amai sweet protein is sweeter than teriyaki sauce with 70% less sugar and no added protein.

[0536] Example 16: Thousand Island Sauce: The formulation of the Thousand Island sauce is shown in Table 20.

[0537] [Table 23]

[0538] Preparation method: 1. Weigh out tomato paste, ketchup spice, fiber, salt, sugar, vinegar, water, and xanthan. Mix for 2.5 minutes, then mix in the opposite direction for 5 minutes until mixture is smooth. 3. Check to see if all the xanthan has dissolved. If not, repeat the mixing procedure. 4. After the mixture is uniform, add the sweet protein DM-31 and mix slowly. 5. Add mayonnaise and water and mix thoroughly.

[0539] result: As shown in Figure 45, Thousand Island Sauce with 62% less sugar combined with Amai sweet protein is sweeter than Thousand Island Sauce with 62% less sugar and no added protein.

[0540] Example 17: Dark Chocolate: The dark chocolate formulation is shown in Table 21.

[0541] Preparation method 1. Mix all the ingredients and grind them in a Chocolate Refiner for 4 hours until a uniform mixture is obtained. 2. Tempering with stirring: First heat to 45°C, then cool to 27°C and reheat to 31°C. 3. Pour the chocolate into molds and let it harden.

[0542] result: As shown in FIG. 46, the dark chocolate formulation with sweet protein DM-31 and 70% reduced sugar is sweeter than the dark chocolate with 70% reduced sugar.

Claims

1. A modified protein comprising an amino acid sequence having at least three amino acid deletions in the amino acids located between amino acid T46 and amino acid I56 compared to a reference protein, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:

8.

2. 2. The modified protein of claim 1, wherein the amino acid deletions comprise deletion of amino acids E50, F52 and R53 compared to the reference protein.

3. 3. The modified protein of claim 1 or 2, comprising amino acid substitutions in at least five amino acids compared to the reference protein.

4. 4. The modified protein of claim 3, wherein the amino acid substitutions comprise substitutions at amino acids E2, E23 and Y65 compared to the reference protein.

5. 4. The modified protein of claim 3, wherein the amino acid substitution comprises a substitution at L70 compared to the reference protein.

6. 6. The modified protein of any one of claims 3 to 5, wherein the amino acid substitution comprises a substitution in at least one amino acid selected from the group consisting of E4, T12, A19, V20, K25, I26, Q28, R31, T33, N35, C41, Q61, V64, D68, A73, I75, R84 and F89 compared to the reference protein.

7. 6. The modified protein of any one of claims 3 to 5, wherein the amino acid substitution comprises a substitution at at least one amino acid selected from the group consisting of A19, V20, K25, I26, Q28, T33, C41 and D68 compared to the reference protein.

8. 6. The modified protein of any one of claims 3 to 5, wherein the amino acid substitution comprises a substitution at at least one amino acid selected from the group consisting of A19, V20, K25, I26, Q28, T33 and D68 compared to the reference protein.

9. 6. The modified protein of any one of claims 3 to 5, wherein the amino acid substitution comprises an amino acid substitution at at least one amino acid selected from the group consisting of Q28, C41 and Q68 compared to the reference protein.

10. 6. The modified protein of any one of claims 3 to 5, wherein the amino acid substitution comprises a substitution at at least one amino acid selected from the group consisting of A19 and V20 compared to the reference protein.

11. 6. The modified protein of any one of claims 3 to 5, wherein the amino acid substitution comprises a substitution at at least one amino acid selected from the group consisting of K25, I26 and Q28 compared to the reference protein.

12. 6. The modified protein of any one of claims 3 to 5, wherein the amino acid substitution comprises a substitution at least one amino acid selected from the group consisting of T33 and D68 compared to the reference protein.

13. 13. The modified protein of any one of claims 3 to 12, wherein the amino acid substitutions include the amino acid substitutions E2N, E23A, Y65R and L70I.

14. 14. The modified protein of claim 13, comprising at least one amino acid substitution selected from the group consisting of E4Q, T12V, A19V, V20I, K25R, I26S, I26T, I26W, Q28K, Q28R, Q28K, Q28E, Q28S, R31T, T33R, N35T, C41T, C41V, C41A, C41S, Q61N, V64I, D68S, D68N, D68T, A73V, A73F, I75L, R84L, F89V, and F89M compared to the reference protein.

15. 14. The modified protein of claim 13, comprising at least one amino acid substitution selected from the group consisting of A19V, V20I, K25R, I26S, I26T, I26W, Q28K, Q28R, Q28K, Q28E, Q28S, T33R, N35T, C41T, C41V, C41A, C41S, D68S, D68N, and D68T compared to the reference protein.

16. 14. The modified protein of claim 13, comprising at least one amino acid substitution selected from the group consisting of A19V, V20I, K25R, Q28K, T33R, C41A, C41S, and D68N, compared to the reference protein.

17. SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:4 2. The modified protein of any one of claims 1 to 16, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:71, or a fragment or variant thereof.

18. 17. The modified protein of any one of claims 1 to 16, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38 and SEQ ID NO:49, or a fragment or variant thereof.

19. 19. The modified protein of any one of claims 1 to 18, having at least one improved property compared to the reference protein, the at least one improved property being selected from the group consisting of a better sweetness profile (shortened sweetness aftertaste), better sweetness potency, better sweetness kinetics, improved thermal stability, increased pressure stability, improved pH stability, reduced binding to hydrophobic regions, improved freeze-thaw stability, improved reconstitution after drying, improved solubility, a sensory profile closer to sugar, and improved shelf-life stability.

20. 20. The modified protein of claim 19, wherein the improved properties are sweetness profile and stability.

21. 21. A modified protein according to any one of claims 1 to 20, or any combination of two or more of said proteins, for use in the preparation of a product for oral delivery.

22. 22. The modified protein of claim 21, wherein the product is a food product, a dietary supplement, or a pharmaceutical product.

23. 21. A modified protein according to any one of claims 1 to 20, or any combination of two or more of said proteins, for use as a flavour modifier, flavour enhancer or flavour masking agent.

24. 21. A modified protein according to any one of claims 1 to 20, or any combination of two or more of said modified proteins, for use as a sweetener.

25. 21. A food product comprising a modified protein according to any one of claims 1 to 20, or any combination thereof.

26. 26. The food product of claim 25, which is a low-sugar or no-added-sugar food product.

27. 26. The food product of claim 25, which is a beverage selected from the group consisting of carbonated soft drinks, non-carbonated soft drinks, fountain drinks, frozen ready-to-drink beverages, coffee beverages, tea beverages, dairy beverages, non-dairy milks, fruit beverages, flavored waters, enhanced waters, sports drinks, energy drinks, isotonic drinks, low calorie drinks, and alcoholic beverages.

28. 26. The food product of claim 25, wherein the non-dairy milk is selected from the group consisting of almond milk, cashew milk, soy milk, coconut milk, pea milk, macadamia milk, tiger nut milk, chickpea milk, rice milk, oat milk and flax milk.

29. 26. The food product of claim 25, which is a food product selected from the group consisting of bakery products, cookies, biscuits, baking mixes, cereals, energy bars, marzipan, confectionery, candy, toffee, chewing gum, bubble gum, dairy products, yogurt, flavored yogurt, peanut butter, soy sauce, soy-based products, non-dairy products, salad dressings, ketchup, mayonnaise, vinegar, frozen desserts, meat products, fish products, bottled and canned foods, tabletop sweeteners, chocolate, fruit, dried fruit, and vegetables.

30. 26. The food product of claim 25 which is an energy bar, marzipan.

31. 31. A food product according to any one of claims 25 to 30 comprising at least one food ingredient.

32. 32. The food product of claim 31, wherein the food ingredient is at least one of an artificial flavor, a food additive, a food coloring, a preservative, or a sweetener enhancer.

33. 32. The food product of claim 31, wherein the food ingredient is selected from the group consisting of stevia, sucrose, agave nectar, brown rice syrup, date sugar, honey, maple syrup, molasses, monk fruit, sugar alcohols, rare sugars, aspartame, sucralose, acesulfame potassium, saccharin, neotame, advantame, and dietary fiber.

34. 32. The food product of claim 31, wherein the food ingredient is selected from the group consisting of flavorings, food additives, food colorings, preservatives, and sweetener enhancers.

35. 21. A sweetening composition comprising a protein according to any one of claims 1 to 20 or any combination thereof.

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