Protein Sweetener

Abstract

The present disclosure relates to modified proteins having an amino acid sequence that includes at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, and having improved stability.

Description

[Technical Field]

[0001] The present disclosure relates to protein sweeteners and uses thereof. [Background technology]

[0002] References considered relevant as background to the presently disclosed subject matter are listed below. -Weiffert T,Linse S.Protein stabilization with retained function of monellin using a split GFP system.Sci Rep.2018 Aug 24;8(1):12763.doi:10.1038 / s41598-018-31177-z.PMID:30143736;PMCID:PMC6109104. -Delfi,M.;Emendato,A.;Leone,S.;Lampitella,EA;Porcaro,P.;Cardinale,G.;Petraccone,L.;Picone,DA Super Stable Mutant of the Plant Protein Monellin Endowed with Enhanced Sweetness.Life 2021,11,236.https: / / doi.org / 10.3390 / life11030236 -Zhao,M.et al.Structure basis of the improved sweetness and thermostability of a unique double-sites single-chain sweet-tasting protein monellin(MNEI)mutant.Biochimie 2018,154,156-163.doi:https: / / doi.org / 10.1016 / j.biochi.2018.08.010 -Somoza, JRet al.The Taste-active Regions of Monellin, a Potently Sweet Protein.Chem Senses.1995,20(1),61-68.https: / / doi.org / 10.1093 / chemse / 20.1.61 -Yang et al.The flexible loop is a new sweetness determinant site of the sweet-tasting protein:Characterization of novel sweeter mutants of the single-chain Monellin(MNEI).Chemical Senses 2019,44(8),607-614..doi:10.1093 / chemse / bjz057 -Liu et al.,Functional characterization of the heterodimeric sweet taste receptor T1R2 and T1R3 from a New World monkey species(squirrel monkey)and its response to sweet-tasting proteins.Biochemical and Biophysical Research Communications 2012,427(2),431-437.doi:10.1016 / j.bbrc.2012.09.083. -Leone et al.,Sweeter and stronger:enhancing sweetness and stability of the single chain monellin MNEI through molecular design.Scientific Reports 2016,6(1).doi:10.1038 / srep34045. Acknowledgment of the above references herein should not be inferred as meaning that they are in any way relevant to the patentability of the presently disclosed subject matter.

[0003] Background technology Sweet proteins are a class of proteins that interact with the sweet taste receptors T1R2 / T1R3 and stimulate the sweet taste sensation. Single-chain monellin, MNEI, is an example of such a sweet protein; however, its use in food and beverage products is limited due to its low stability.

[0004] Weiffert T. et al. describe functional studies showing that the S76Y substitution retains sweet taste and has potential use within the food industry.

[0005] Delfi, M. et al. describe a new construct of MNEI that has increased sweetness and stability compared to MNEI.

[0006] Zhao, M. et al. describe the effects of E2N and E23A substitutions on MNEI: the effect of the substitutions on thermostability, protein structure, and sweetness.

[0007] Somoza, J.R. et al. describe mutagenesis studies on monellin, suggesting at least four residues involved in the taste-active region of monellin.

[0008] Yang et al. describe the effect of substitutions in the flexible loop (L23) of MNEI on the sweetness of MNEI.

[0009] Liu et al. described hybridization of human and squirrel monkey sweet taste receptor subunits. These hybrids highlight the interactions between the native receptor subunits and the importance of electrostatics involved in MNEI:receptor interactions.

[0010] Leone et al. describe substitutions in MNEI that increase sweetness and thermal stability. Summary of the Invention

[0011] The present disclosure provides, according to some aspects, a modified protein having an amino acid sequence that includes at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence set forth in SEQ ID NO:1, and wherein the at least one amino acid deletion is at one or more amino acids located between T46 and I56 of the reference protein and the at least one amino acid substitution is at amino acid S76 of the reference protein.

[0012] According to some other aspects, the present disclosure provides a modified protein having an amino acid sequence that includes at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence set forth in SEQ ID NO:1, and wherein the at least one amino acid deletion is at one or more amino acids that are at least E50, F52, and R53 of the reference protein, and the at least one amino acid substitution is at amino acid S76 of the reference protein.

[0013] According to some further aspects, the present disclosure provides engineered proteins having an amino acid sequence that includes at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence set forth in SEQ ID NO:1, and wherein the at least one amino acid deletion is at one or more of amino acids E50, F52, and R53 of the reference protein, and the at least one amino acid substitution is at least amino acids E2, E23, Y65, L70, S76 of the reference protein.

[0014] According to some further aspects, the present disclosure provides engineered proteins having an amino acid sequence that includes at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence set forth in SEQ ID NO:1, and wherein the at least one amino acid deletion is at amino acids E50, F52, and R53 of the reference protein, and the at least one amino acid substitution is at least E2N, E23A, Y65R, L70I, and at amino acid S76 of the reference protein.

[0015] The present disclosure provides, according to some aspects, engineered proteins having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution relative to a reference protein, wherein the reference protein is represented by the amino acid sequence set forth in SEQ ID NO:1, wherein the at least one amino acid deletion is at one or more amino acids located between T46 and I56 of the reference protein and the at least one amino acid substitution is at amino acid S76 of the reference protein, and wherein the engineered protein has at least increased stability relative to the reference protein.

[0016] The present disclosure, according to some other aspects, provides engineered proteins having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution relative to a reference protein, wherein the reference protein is represented by the amino acid sequence set forth in SEQ ID NO:1, wherein the at least one amino acid deletion is at one or more amino acids that are at least E50, F52, and R53 of the reference protein and the at least one amino acid substitution is at amino acid S76 of the reference protein, and wherein the engineered protein has at least increased stability relative to the reference protein.

[0017] The present disclosure, according to some further aspects, provides engineered proteins having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution relative to a reference protein, wherein the reference protein is represented by the amino acid sequence set forth in SEQ ID NO:1, wherein the at least one amino acid deletion is at one or more of amino acids E50, F52, and R53 of the reference protein and the at least one amino acid substitution is at least at amino acids E2, E23, Y65, L70, S76 of the reference protein, and wherein the engineered protein has at least increased stability relative to the reference protein.

[0018] The present disclosure, according to some further aspects, provides engineered proteins having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution relative to a reference protein, wherein the reference protein is represented by the amino acid sequence set forth in SEQ ID NO:1, wherein the at least one amino acid deletion is at amino acids E50, F52, and R53 of the reference protein, and the at least one amino acid substitution is at least at E2N, E23A, Y65R, L70I, and at amino acid S76 of the reference protein, and wherein the engineered protein has at least increased stability relative to the reference protein.

[0019] The present disclosure, according to some aspects, provides stabilized engineered proteins as described herein.

[0020] The present disclosure, according to some aspects, provides food products comprising the engineered proteins described herein.

[0021] The present disclosure provides, according to some embodiments, a method for increasing the stability of a reference protein, the method comprising deleting at least one amino acid from the reference protein and substituting at least one amino acid from the reference protein, wherein the reference protein is represented by the amino acid sequence set forth in SEQ ID NO: 1, the at least one deleted amino acid is one or more amino acids located between T46 and I56 of the reference protein, and the at least one substituting amino acid is amino acid S76 of the reference protein.

[0022] 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, in which: [Brief explanation of the drawings]

[0023] [Figure 1A] The structure of the DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) protein is presented. Figure 1A: Amino acids Y76 (cyan) and E59 (yellow) are adjacent to the loop (magenta). Figure 1B: Amino acids Y76 (cyan) and R84 (orange) are adjacent to the loop (magenta). [Figure 1B] The structure of the DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) protein is presented. Figure 1A: Amino acids Y76 (cyan) and E59 (yellow) are adjacent to the loop (magenta). Figure 1B: Amino acids Y76 (cyan) and R84 (orange) are adjacent to the loop (magenta). [Figure 2] The structure of the DM652 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59V, S76V) protein is presented. Colors indicate residues V59 (yellow) and V76 (turquoise). The loop is also shown (magenta), and the modification of residues 59 and 76 to valine prevents water molecules from interacting with the backbone. [Figure 3]We present an anisotropic network model (ANM) analysis of the global motion of DM31. The movements of residue Cαs are shown as arrows, and their lengths represent the movement size. The most mobile part of the protein is the β-hairpin, composed of β-strands 2 and 3, and β-strands 4 and 5. These drive the movements of the β-sheet, the helix, and the C-terminal loop of the helix. [Figure 4A] Electrostatic potential contour maps representing equipotential surfaces of DM31-based variants with positive (blue) and negative (red) potentials of + / - 1 kT / e are presented. Figure 4A shows DM222 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, S76Y), Figure 4B shows DM479 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, D68N, S76Y), Figure 4C shows DM31, and Figure 4D shows DM224 ( 4E shows DM606 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76R), and Figure 4F shows DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y). Specific DM substitutions, as well as additional residues known to be important for sweetness, are labeled and presented as spheres. [Figure 4B]Electrostatic potential contour maps representing equipotential surfaces of DM31-based variants with positive (blue) and negative (red) potentials of + / - 1 kT / e are presented. Figure 4A shows DM222 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, S76Y), Figure 4B shows DM479 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, D68N, S76Y), Figure 4C shows DM31, and Figure 4D shows DM224 ( 4E shows DM606 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76R), and Figure 4F shows DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y). Specific DM substitutions, as well as additional residues known to be important for sweetness, are labeled and presented as spheres. [Figure 4C] Electrostatic potential contour maps representing equipotential surfaces of DM31-based variants with positive (blue) and negative (red) potentials of + / - 1 kT / e are presented. Figure 4A shows DM222 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, S76Y), Figure 4B shows DM479 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, D68N, S76Y), Figure 4C shows DM31, and Figure 4D shows DM224 ( 4E shows DM606 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76R), and Figure 4F shows DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y). Specific DM substitutions, as well as additional residues known to be important for sweetness, are labeled and presented as spheres. [Figure 4D]Electrostatic potential contour maps representing equipotential surfaces of DM31-based variants with positive (blue) and negative (red) potentials of + / - 1 kT / e are presented. Figure 4A shows DM222 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, S76Y), Figure 4B shows DM479 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, D68N, S76Y), Figure 4C shows DM31, and Figure 4D shows DM224 ( 4E shows DM606 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76R), and Figure 4F shows DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y). Specific DM substitutions, as well as additional residues known to be important for sweetness, are labeled and presented as spheres. [Figure 4E] Electrostatic potential contour maps representing equipotential surfaces of DM31-based variants with positive (blue) and negative (red) potentials of + / - 1 kT / e are presented. Figure 4A shows DM222 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, S76Y), Figure 4B shows DM479 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, D68N, S76Y), Figure 4C shows DM31, and Figure 4D shows DM224 ( 4E shows DM606 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76R), and Figure 4F shows DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y). Specific DM substitutions, as well as additional residues known to be important for sweetness, are labeled and presented as spheres. [Figure 4F]Electrostatic potential contour maps representing equipotential surfaces of DM31-based variants with positive (blue) and negative (red) potentials of + / - 1 kT / e are presented. Figure 4A shows DM222 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, S76Y), Figure 4B shows DM479 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, D68N, S76Y), Figure 4C shows DM31, and Figure 4D shows DM224 ( 4E shows DM606 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76R), and Figure 4F shows DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y). Specific DM substitutions, as well as additional residues known to be important for sweetness, are labeled and presented as spheres. [Figure 5] RMSF plots of variants DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y), DM653 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59I, S76V), DM656 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59I, S76I), DM663 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76V), DM31, and MNEI. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present disclosure relates to novel sweet proteins characterized by high stability. The novel proteins were designed using a series of computer optimization methods. Specifically, the computer methods are based on the computer identification of specific regions and amino acid residues in proteins that may affect protein characteristics, including stability and sweetness, among others, thus obtaining novel proteins with improved characteristics, as further described herein.

[0025] As shown below, the inventors have found that introducing certain modifications, such as deletions or substitutions, in the amino acid sequence of a reference protein results in novel proteins (referred to herein as "modified proteins" or "designer proteins") that have at least improved stability compared to the reference protein. Based on these results, it has been suggested that the new proteins with improved stability may be used in food and beverage applications for the preparation of various edible products.

[0026] Thus, in its broadest aspect, the present disclosure relates to a modified protein comprising an amino acid sequence that has at least one of: (i) at least one amino acid deletion, (ii) at least one amino acid replacement (substitution), (iii) at least one amino acid insertion, or (iv) any combination thereof, relative to the amino acid sequence of a reference protein. As described herein, the modified protein has at least improved thermal stability relative to the reference protein.

[0027] In the following text, reference to engineered proteins should be understood as a reference to the food products disclosed herein as well, and therefore whenever characteristics are provided with respect to engineered proteins, they should be understood as defining the same characteristics with respect to the food products mutatis mutandis.

[0028] In the present disclosure, modified proteins, sometimes referred to as designer proteins, can be considered variants of the reference protein. As used herein, the term "variant" refers to a sequence that contains at least one amino acid modification (e.g., at least one substitution, at least one deletion, at least one insertion, or any combination thereof) compared to the reference protein. Note that the modification results in a modified / variant sequence.

[0029] The present disclosure also encompasses modified nucleic acid sequences that contain substitutions, deletions, or insertions of corresponding codons that are similar to the codons in the modified protein, in other words, the same modifications are applied to the nucleic acid sequence, resulting in a modified sequence that contains the corresponding substitutions, deletions, or insertions of codons.

[0030] The present disclosure is not limited to a particular number of amino acid modifications made in a reference protein that ultimately result in the formation of a variant protein.

[0031] As used herein, a modification refers to an alteration in 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.

[0032] In some instances, the modification may be an amino acid insertion.

[0033] In some embodiments the variant protein comprises an amino acid sequence that has an insertion of at least 1, at least 2, at least 3, at least 4, at least 5 amino acids compared to the reference protein.

[0034] In some instances, the modification may be an amino acid deletion.

[0035] In some embodiments the variant protein comprises an amino acid sequence that has at least 2, at least 3, at least 4, at least 5 amino acid deletions compared to the reference protein.

[0036] In some instances, the modification may be an amino acid substitution.

[0037] In some embodiments the variant protein comprises an amino acid sequence that has at least one, at least two, at least three, at least four, or at least five amino acid substitutions compared to the reference protein.

[0038] 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 the protein sequence (amino acid sequence) or structure. Non-limiting examples of protein regions include the protein surface, protein core, protein loops, secondary structure elements, secondary structure capping, disulfides, binding sites, linkers, hydrophobic patches, or protein hydrophobic regions.

[0039] As shown in the examples below, molecular dynamics tools were used to design new engineered proteins based on the optimization of various regions of a reference protein. In particular, Rosetta Energy Unit (REU) scores for the newly designed engineered proteins were used to predict those proteins that would exhibit improved properties, particularly at least the stability of the engineered protein. As part of the computational optimization process, engineered proteins can be selected from a large output collection of amino acid sequences after computational bioinformatics or structural biology analysis based on energetic considerations, i.e., sequences with low energy.

[0040] 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.

[0041] Calculations for each amino acid sequence (e.g., engineered proteins) can be performed by combining physics-based (also known as biophysical methods) and statistical-based potentials (also known as knowledge-based potentials or informatics methods), such as by using Rosetta Energy Units (REUs). Rosetta Energy Units (REUs) are an algorithm in the Rosetta software, a package of algorithms for computer modeling and protein structure analysis. 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 statistical-based potentials that does not correspond to any actual physical energy unit. Rosetta energy is an arbitrary measure and is sometimes referred to as REUs ("Rosetta Energy Units").

[0042] In some embodiments, the REU can be calculated for the entire protein sequence consisting of at least one amino acid modification. In some other embodiments, the REU can be calculated for at least one region consisting of at least one amino acid modification in the entire protein sequence. In some other embodiments, the REU can be calculated for at least one amino acid modification in the entire protein sequence.

[0043] In some embodiments, the engineered protein has an energy given by REU of less than about -315.

[0044] In some embodiments, the engineered protein has an energy given by REU of less than about -321.

[0045] In some embodiments, the engineered protein has an energy lower than -316 as assigned by REU. In some embodiments, the engineered protein has an energy lower than -317 as assigned by REU. In some embodiments, the engineered protein has an energy lower than -318 as assigned by REU. In some embodiments, the engineered protein has an energy lower than -319 as assigned by REU. In some embodiments, the engineered protein has an energy lower than -320 as assigned by REU. In some embodiments, the engineered protein has an energy lower than -321 as assigned by REU. In some embodiments, the engineered protein has an energy lower than -322 as assigned by REU. In some embodiments, the engineered protein has an energy lower than -323 as assigned by REU. In some embodiments, the engineered protein has an energy lower than -324 as assigned by REU. In some embodiments, the engineered protein has an energy lower than -325 as assigned by REU. In some embodiments, the engineered protein has an energy lower than -326 as assigned by REU. In some embodiments, the engineered protein has an energy lower than -327 as assigned by REU. In some embodiments, the engineered protein has an energy lower than −328 given by REU. In some embodiments, the engineered protein has an energy lower than −329 given by REU. In some embodiments, the engineered protein has an energy lower than −330 given by REU.

[0046] In some embodiments, the engineered protein has an energy given in REU of about -315 and about -330.

[0047] In some embodiments, the engineered protein comprises an amino acid sequence that is 40% to 98% identical to the amino acid sequence of the reference protein, hi some embodiments, the engineered protein comprises an amino acid sequence that is 90% to 98% identical to the amino acid sequence of the reference protein.

[0048] In some embodiments, the engineered protein comprises an amino acid sequence that is 60% to 90% identical to a reference amino acid sequence, hi some embodiments, the engineered protein comprises an amino acid sequence that is 70% to 90% identical to a reference amino acid sequence.

[0049] In some embodiments the engineered 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% identity to a reference amino acid sequence.

[0050] In some embodiments, the modified protein comprises an amino acid sequence that has between 90% and 98% identity to a reference amino acid sequence.

[0051] The percent identity between two or more amino acid sequences is determined when the two or more sequences are compared and aligned for maximum correspondence. In the context of this 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.

[0052] In some embodiments, the engineered protein comprises an amino acid sequence that is 40% to 98% similar to the amino acid sequence of a reference protein, hi some embodiments, the engineered protein comprises an amino acid sequence that is 90% to 98% similar to the amino acid sequence of a reference protein.

[0053] In some embodiments, the engineered protein comprises an amino acid sequence that is 60% to 90% similar to a reference amino acid sequence, hi some embodiments, the engineered protein comprises an amino acid sequence that is 70% to 90% similar to a reference amino acid sequence.

[0054] In some embodiments the variant 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.

[0055] In some embodiments, the variant protein comprises an amino acid sequence having 90% to 98% similarity to a reference amino acid sequence.

[0056] As used herein, sequence similarity or sequence homology refers to the amount (%) of conserved amino acids with similar physicochemical properties, such as leucine and isoleucine.

[0057] In determining sequence identity, gaps are not counted and the sequence identity is to the shorter of the two sequences. In this regard, it should be noted that the length of the reference protein (amino acid sequence) may be the same as or different from the length of the variant protein (amino acid sequence).

[0058] As described herein, the reference protein that serves as the basis for the computational analysis is a sweet protein. The reference sweet protein can be a naturally occurring protein or a synthetic protein.

[0059] In some instances, the reference protein is a synthetic protein. When referring to a synthetic protein, it should be understood as a protein that is not found in nature and is therefore considered a synthetic protein.

[0060] According to the present disclosure, the reference protein is MNEI or a modified protein based on MNEI.

[0061] In some embodiments, the reference protein is MNEI.

[0062] As recognized, MNEI is a synthetic protein made from the combination of A-chain monellin (GenBank Accession No. P02881) and B-chain monellin (GenBank Accession No. P02882).

[0063] MNEI is represented herein by SEQ ID NO:1 and has the following amino acid sequence: GEWEIIDIGPFTQNLGKFAVDEENKIGQYGRLTFNKVIRPCMKKTIYENEGFREIKGYEYQLYVYASDKLFRADISEDYKTRGRKLLRFNGPVPPP

[0064] In some embodiments, the reference protein is represented herein by SEQ ID NO:1.

[0065] The modified proteins according to the present disclosure may be considered variants of MNEI. Note that a variant sequence (modified) is considered a variant sequence as long as it contains a different sequence compared to a reference sequence, in this case, MNEI, regardless of how it was synthesized.

[0066] As shown in the Examples below, substitutions in the reference protein at least at amino acid S76, located in the solvent-exposed beta-strand, resulted in modified proteins with improved stability. As further shown in the Examples below, combining the substitution at S76 with one or more amino acid modifications, including, inter alia, amino acid deletions and / or amino acid substitutions, improved the stability of the modified proteins.

[0067] In some embodiments the engineered protein comprises an amino acid substitution at least at amino acid S76 of SEQ ID NO:1.

[0068] Thus, according to some aspects, the present disclosure provides a modified protein comprising an amino acid sequence having at least one amino acid substitution at least at amino acid S76 compared to a reference protein, wherein the reference protein is set forth as SEQ ID NO:1.

[0069] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having at least one amino acid substitution at least at amino acid S76 compared to a reference protein, wherein the modified protein has at least improved stability compared to the reference protein, wherein the reference protein is set forth as SEQ ID NO:1.

[0070] As used herein, the term stability refers to the ability of a protein to maintain its three-dimensional structure. In the context of the present disclosure, the term stability encompasses thermal stability, short-term and long-term functional stability (structure and / or function), long-term functional stability (shelf-life stability), pH stability, salt concentration stability, ionic strength stability or stability in fat-containing or protein-containing matrices, and stability in the presence of different preservatives.

[0071] In some embodiments, the stability is thermostability.

[0072] As used herein, the term "thermal stability" refers to the ability of a modified protein to retain its 3D structure at temperatures above that of a reference protein. 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). The 3D structure of a protein can affect the function of the protein.

[0073] In some embodiments, the variant protein has equal or greater thermal stability than the reference protein.

[0074] As used herein, the term "functional stability" refers to the ability of a variant protein to retain its function after exposure to elevated temperatures compared to a reference protein.

[0075] In some embodiments, the engineered proteins herein may maintain their sweetening effect at higher temperatures or after exposure to higher temperatures for periods that may be limited. In other words, there is a noticeable change in sweetness or sensory profile after exposing the product to temperatures above room temperature, sometimes up to 50° C., sometimes up to 100° C., or even up to 150° C. Protein function, e.g., sweetness, may be measured by sensory testing.

[0076] In some embodiments, the engineered protein has equal or greater pH stability than the reference protein. pH stability refers to the long-term stability of the engineered protein over a wider pH range than the reference protein, i.e., the engineered protein maintains its 3D structure (and / or function) after exposure of the product to any pH between 3 and 8, sometimes even between 4 and 8. For example, sodas such as cola have a pH between 2.3 and 2.5, at which pH some sweet proteins are not stable and lose functionality quickly or after a time shorter than the normal shelf life of the beverage.

[0077] In some embodiments, the engineered protein has greater solubility relative to the reference protein. Solubility can be in aqueous, partially aqueous, or non-aqueous environments, such as foods containing fat.

[0078] In some embodiments, the engineered protein has an improved shelf life relative to a 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 exposure of the product to any temperature up to 45°C, sometimes any temperature between 4°C and 32°C or 45°C.

[0079] In some embodiments, the modified protein has a PI value of 7.8 to 8.4.

[0080] In some other embodiments, the variant protein is characterized by at least one of the following being equal to or improved compared to 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.

[0081] In some embodiments, the variant protein has equal or higher folding kinetics relative to a reference protein, i.e., the rate of protein folding from an unfolded or partially folded structure is faster (as assessed in silico, e.g., by molecular dynamics, or by experimental in vitro or in vivo methods). Alternatively, faster folding kinetics also 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.

[0082] In some embodiments, the engineered protein is characterized by an expression yield that is equal to or higher than that of a reference protein in the host organism being evaluated.

[0083] In particular, the shelf life and functional stability required for food and beverage products may be related to structural heat stability and consist of different measurables; for example, pasteurization may be applied by different protocols and heat resistance and functional properties may be measured over very short or long periods of time.

[0084] As described herein, in some examples, the reference protein is an MNEI protein.

[0085] In some embodiments, the amino acid substitution comprises amino acid S76 compared to MNEI.

[0086] In some embodiments, the modified protein is the MNEI protein having SEQ ID NO:1, which contains a substitution at amino acid residue S76.

[0087] In some embodiments the engineered protein comprises the amino acid substitution S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, which has improved stability compared to SEQ ID NO:1.

[0088] In some embodiments the engineered protein is represented herein by the amino acid designated as DM129. In some embodiments the engineered protein is represented herein by the amino acid designated as DM147. In some embodiments the engineered protein is represented herein by the amino acid designated as DM89. In some embodiments the engineered protein is represented herein by the amino acid designated as DM658. In some embodiments the engineered protein is represented herein by the amino acid designated as DM137. In some embodiments the engineered protein is represented herein by the amino acid designated as DM114. In some embodiments the engineered protein is represented herein by the amino acid designated as DM606. In some embodiments the engineered protein is represented herein by the amino acid designated as DM663. In some embodiments the engineered protein is represented herein by the amino acid designated as DM659. In some embodiments the engineered protein is represented herein by the amino acid designated as DM690.

[0089] In some embodiments the engineered protein comprises the amino acid substitution S76F or S76Y which has improved stability compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented herein by the amino acid designated as DM129. In some embodiments the engineered protein is represented herein by the amino acid designated as DM147.

[0090] As can be seen from Tables 2 and 4, the modification at amino acid S76 increased the Tm value. For example, the experimental Tm value for DM89 was 101° C., the experimental Tm value for DM137 was 97° C., the experimental Tm value for DM114 was 101.5° C., and the experimental Tm value for DM89 was 101° C. Furthermore, the predicted Tm value for DM658 was 96° C., the predicted Tm value for DM663 was 98° C., the predicted Tm value for DM664 was 99° C., the predicted Tm value for DM659 was 93.6° C., and the predicted Tm value for DM690 was 98.5° C.

[0091] Based on these results, it was suggested that various modifications at this amino acid would be highly effective in significantly increasing the stability of the protein.

[0092] As shown in Table 2, substitution of S76 and at least one additional amino acid resulted in a modified protein with at least improved stability.

[0093] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1 containing a substitution at amino acid residue S76 and at least one other amino acid substitution.

[0094] According to some other aspects, the present disclosure provides engineered proteins comprising an amino acid sequence having a substitution at least at amino acid S76 compared to SEQ ID NO:1 and an additional modification to at least one amino acid residue of SEQ ID NO:1, wherein the engineered protein has at least improved stability compared to SEQ ID NO:1.

[0095] According to some aspects, the present disclosure provides engineered proteins comprising an amino acid sequence having a substitution at least at amino acid S76 compared to SEQ ID NO:1 and having an additional modification at least one amino acid residue of SEQ ID NO:1, wherein the at least one additional modification is present 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.

[0096] According to some aspects, the disclosure provides engineered proteins comprising an amino acid sequence having a substitution at least at amino acid S76 compared to SEQ ID NO:1 and an additional modification to at least one amino acid residue of SEQ ID NO:1, wherein the at least additional modification is a modification that stabilizes the alpha-helical structure and the helix terminus, and wherein the engineered protein has at least improved stability compared to SEQ ID NO:1.

[0097] As used herein, the term "stabilizing alpha-helical structure" refers to any modification that affects the MNEI alpha-helical structure, including modifications at any one of amino acid residues K25, 126, and Q28 of MNEI.

[0098] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1, comprising a substitution at amino acid residue S76, and comprising at least one additional substitution at least one of amino acid residues K25, 126, Q28, or any combination thereof.

[0099] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO:1, comprising a substitution at amino acid residue S76 and at least one additional substitution at at least one amino acid residue E23.

[0100] According to some aspects, the present disclosure provides engineered proteins comprising an amino acid sequence having a substitution at least at amino acid S76 compared to SEQ ID NO:1 and an additional modification to at least one amino acid residue of SEQ ID NO:1, wherein the at least additional modification is a modification that stabilizes the linker region, and wherein the engineered protein has at least one improved food-related property compared to SEQ ID NO:1.

[0101] As used herein, the term stabilizing the linker region refers to any modification that affects the MNEI linker region.

[0102] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1, comprising a substitution at amino acid residue S76 and a deletion at least one of E50, F52, R53, or any combination thereof.

[0103] According to some aspects, the present disclosure provides engineered proteins comprising an amino acid sequence having a substitution at least at amino acid S76 compared to SEQ ID NO:1 and having an additional modification at least one amino acid residue of SEQ ID NO:1, wherein the at least additional modification is a modification that affects aggregation, and wherein the engineered protein has at least improved stability compared to SEQ ID NO: 1. In some embodiments, the at least one modification is a modification that reduces protein aggregation.

[0104] As used herein, the term "reducing protein aggregation" refers to any modification that affects protein aggregation.It is suggested that the two chains of MNEI can form a disulfide bond between two cysteine residues (C41).Therefore, such modifications include modifications at amino acid residue C41.

[0105] In some embodiments, the modified protein is the MNEI protein having SEQ ID NO: 1, which contains a substitution at amino acid residue S76 and at least one additional substitution at C41. Based on computational analysis, it was suggested that C41 is prone to disulfide bonding and promotes aggregation, and therefore any modification at this residue may reduce protein aggregation.

[0106] According to some aspects, the present disclosure provides engineered proteins comprising an amino acid sequence having a substitution at least at amino acid S76 compared to SEQ ID NO:1 and an additional modification to at least one amino acid residue of SEQ ID NO:1, wherein the at least additional modification is a modification that affects core repacking, and wherein the engineered protein has at least improved stability compared to SEQ ID NO:1.

[0107] As used herein, the term core repacking refers to any modification that affects core repacking, thus reducing water accessibility and stabilizing the modified protein. It is suggested that there is a hole that makes the core accessible to surface water (because the protein is small, the core may be semi-exposed).

[0108] The MNEI core is partially (semi) exposed to the surface and susceptible to water interference, and therefore the suggested modifications stabilize the protein, including modifications at at least one of amino acid residues T12, C41, A19, V20, V64, A73, I75, F89, or any combination thereof.

[0109] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1, which comprises a substitution at amino acid residue S76 and which comprises a substitution at at least one amino acid residue of T12, C41, A19, V20, V64, A73, I75, F89, or any combination thereof.

[0110] According to some aspects, the disclosure provides engineered proteins comprising an amino acid sequence having a substitution at least at amino acid S76 compared to SEQ ID NO:1 and an additional modification to at least one amino acid residue of SEQ ID NO:1, wherein the at least additional modification is a modification that affects electrostatics and hydrophobicity, and wherein the engineered protein has at least improved stability compared to SEQ ID NO:1.

[0111] As used herein, the term electrostatic refers to any modification that affects electrostatic charge, including modifications in at least one amino acid residue at T33, E4, or any combination thereof.

[0112] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1, comprising a substitution at amino acid residue S76 and comprising a substitution at at least one amino acid residue of T33, E4, or any combination thereof.

[0113] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1, comprising a substitution at amino acid residue S76 and comprising a substitution in at least one of E2, Y65, D68, or any combination thereof.

[0114] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1, comprising a substitution at amino acid residue S76 and comprising a substitution at at least one of R31, Q61, N35, or any combination thereof.

[0115] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1, which comprises a substitution at amino acid residue S76 and which comprises a substitution at least at R84.

[0116] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1 comprising a substitution at amino acid residue S76 and comprising an alteration in at least one amino acid residue selected from E23, K25, 126, Q28, E50, F52, R53, C41, T12, C41, A19, V20, A73, 175, F89, T33, E4, E2, Y65, D68, R31, Q61, N35, R84, V64, or any combination thereof.

[0117] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1 comprising a substitution at amino acid residue S76 and at least one amino acid substitution in at least one residue selected from E23, K25, 126, Q28, T12, C41, A19, V20, V64, A73, 175, F89, E2, Y65, D68, R84, T33, E4, R31, Q61, N35, or any combination thereof.

[0118] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1 comprising a substitution at amino acid residue S76 and at least one amino acid substitution at E23, K25, I26, Q28, or any combination thereof.

[0119] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1 comprising a substitution at amino acid residue S76 and at least one amino acid substitution at T12, C41, A19, V20, V64, A73, I75, F89, or any combination thereof.

[0120] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1, comprising a substitution at amino acid residue S76 and at least one amino acid substitution in E2, Y65, D68, or any combination thereof.

[0121] As shown in Table 2, computer analysis suggested that substitution of S76 and at least one other amino acid in the amino acid sequence of MNEI would result in a modified protein with at least improved stability compared to MNEI.

[0122] In some embodiments, the modified protein comprises a substitution at amino acid S76 and at least one other amino acid substitution relative to MNEI, and the modified protein has at least improved stability relative to MNEI.

[0123] In some embodiments the amino acid substitutions include amino acids S76 and R84. In some embodiments the engineered protein comprises an amino acid sequence having at least two amino acid substitutions compared to SEQ ID NO:1 at least at amino acids R84 and S76.

[0124] In some embodiments the engineered protein having at least two amino acid substitutions R84L and S76Y has improved stability compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid designated herein as DM298.

[0125] The main difference between wild-type monellin and MNEI is the region where the two monellin subunits are connected to form a single-chain monellin called MNEI. The amino acid sequence between amino acid T46 and amino acid I56 represents the loop region connecting the two monellin subunits.

[0126] As shown herein, CPD analysis identified specific amino acid residues / regions in the MNEI protein that can be modified to obtain modified proteins with improved properties, including, inter alia, stability and taste.

[0127] For example, MD simulations of MNEI suggested that the beta sheet of MNEI is less stable. Without being bound by theory, it was suggested that the beta loops, especially the loop between beta strands 2 and 3, do not have a clear secondary structure, and their movement reduces stability. Furthermore, it was suggested that the beta backbone is exposed to water. Simulation analysis of MNEI at high temperatures showed that the main weak points of MNEI are the interface between strands 3 and 4 and the edges of the backbone. The backbone atoms are exposed to water. The simulations also indicate that water can intrude and interfere with hydrogen bonding in that region.

[0128] As shown in the examples below, modifications in the loop regions of the variant proteins increased the sweetness and / or stability of the reference protein. In particular, deletion of amino acid residues in the edges of protein loops and beta strands resulted in variant proteins that were more stable than the reference protein, which in some instances was MNEI.

[0129] In some embodiments, the modified protein comprises a modification in an amino acid located in the linker (loop) region compared to SEQ ID NO:1.

[0130] In some embodiments, the modified protein is the MNEI protein having SEQ ID NO: 1, comprising a substitution at amino acid residue S76 and at least one amino acid deletion.

[0131] In some embodiments, the modified protein comprises a modification at one or more amino acids located between amino acid T46 and amino acid 156 compared to SEQ ID NO:1.

[0132] In some embodiments, the amino acid sequence of the reference protein may be modified in the linker region of the reference protein.

[0133] 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.

[0134] The variant protein contains at least one, sometimes at least two, and sometimes at least three amino acid deletions in the reference protein.

[0135] Thus, according to some aspects, the present disclosure provides a modified protein having an amino acid sequence that comprises at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence set forth in SEQ ID NO: 1, and wherein the at least one amino acid deletion is at one or more amino acids located between amino acid T46 and amino acid I56 of the reference protein, and the at least one amino acid substitution is at amino acid S76 of the reference protein.

[0136] As noted above, in some examples, the reference protein is MNEI, represented herein by SEQ ID NO:1.

[0137] Thus, according to some embodiments, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three 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:1.

[0138] 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, designated herein as SEQ ID NO: 33), suggested that the modified protein had increased stability based on an REU value of -319.65 compared to the REU value of MNEI of -315.35. The deletion of amino acids E50, F52, and R53 is sometimes referred to herein as ΔE50ΔF52, and ΔR53.

[0139] In some embodiments, the at least one amino acid deletion is at one or more of E50, F52, R53, or any combination thereof of SEQ ID NO:1.

[0140] Thus, according to some aspects, the present disclosure provides modified proteins having an amino acid sequence that comprises at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence set forth in SEQ ID NO:1, and wherein the at least one amino acid deletion is at one or more amino acids that are at least E50, F52, and R53 of the reference protein, and the at least one amino acid substitution is at amino acid S76 of the reference protein.

[0141] As shown in Table 2 below, the amino acid substitution at S76 combined with amino acid deletions at least at amino acids E50, F52, and R53 relative to SEQ ID NO: 1 resulted in an engineered protein with at least improved stability. In some embodiments, the engineered protein comprises the amino acid substitution S76Y and amino acid deletions at amino acids E50, F52, and R53 relative to SEQ ID NO: 1 and has improved stability relative to SEQ ID NO: 1. In some embodiments, the engineered protein is represented by the amino acid designated herein as DM148.

[0142] As shown in Table 2 below, substitution of at least amino acid residue S76 of the MNEI protein, in combination with other amino acid substitutions and deletions in the proteins described herein, significantly increases the stability of the protein, as shown by computer modeling and optimization methods.

[0143] In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R53, an amino acid substitution at S76, and at least one amino acid substitution in one or more of G1, E2, E4, T12, A19, V20, E23, K25, 126, G27, Q28, T33, N35, C41, Q61, V64, Y65, D68, L70, A73, 175, R84, F89, or any combination thereof.

[0144] In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R53, an amino acid substitution at S76, and at least one amino acid substitution in one or more of K25, 126, Q28, C41, E23, D68, E2, L70, Y65, E4, G1, or any combination thereof.

[0145] In some embodiments the engineered protein comprises the amino acid substitution S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, which has improved stability compared to SEQ ID NO: 1. In some embodiments the engineered protein comprises the amino acid substitution S76F or S76Y, which has improved stability compared to SEQ ID NO: 1.

[0146] In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R53, an amino acid substitution at S76, and at least one amino acid substitution that is one or more of the following: (i) K25R, (ii) at least one of I26W, I26V, I26T, or any combination thereof, (iii) at least one of Q28K, Q28R, Q28S, Q28E, or any combination thereof, (iv) at least one of C41A, C41V, C41T, C41S, or any combination thereof, (v) E23A or E23Q, (vi) at least one of D68N or D68T, (vii) E2M or E2N, (viii) L70I, (ix) Y65R, (x) E4Q, (xi) G1M, or (xii) any combination thereof. In some embodiments, the amino acid substitution at S76 comprises one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, hi some embodiments, the amino acid substitution at S76 is S76Y.

[0147] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, Q28, and E23, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1. In some embodiments the engineered protein having at least three amino acid substitutions E23A, Q28K, and S76Y, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1, has improved stability compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid designated herein as DM335.

[0148] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, K25, D68, E2, L70, and E23, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, K25R, D68N, E2N, L70I, and E23A, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid sequence designated herein as DM480.

[0149] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, D68, and E23, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, D68N, and E23A, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid designated herein as DM362.

[0150] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, E2, and E23, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, E2N, and E23A, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid designated herein as DM364.

[0151] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, E23, C41, Y65, and deletions at amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, E23A, C41A, Y65R, and deletions at amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid designated herein as DM126.

[0152] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, E4, and E23, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, E23A, E4Q, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1, and has improved stability, compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid designated herein as DM365.

[0153] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, D68, E2, and E23, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, D68N, E2N, E23A, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1, and has improved stability, compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid designated herein as DM397.

[0154] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, E23, Y65, and L70, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, E23A, Y65R, L70I, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1, and has improved stability, compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid sequence designated herein as DM122.

[0155] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, E2, E23, and Y65, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, E2N, E23A, Y65R, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1, and has improved stability, compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid sequence designated herein as DM124.

[0156] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, E2, E23, and L70, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, E2N, E23A, L70I, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1, and has improved stability, compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid sequence designated herein as DM123.

[0157] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, D68, E2, E23, and L70, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, D68N, E2N, E23A, L70I, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1, and has improved stability, compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid sequence designated herein as DM361.

[0158] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, Q28, E2, E23, and L70, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, Q28K, E2N, E23A, L70I, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1, and has improved stability, compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid sequence designated herein as DM473.

[0159] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, Q28, D68, E2, E23, and L70, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, Q28R, D68N, E2N, E23A, L70I, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1, and has improved stability, compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid sequence designated herein as DM521.

[0160] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, C41, D68, E2, E23, and L70, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, C41T, D68N, E2N, E23A, L70I, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid sequence designated herein as DM477.

[0161] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, 126, Q28, D68, E2, E23, and L70, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, 126T, Q28K, D68N, E2N, E23A, L70I, and deletions at amino acids E50, F52, and R53, compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid designated herein as DM486.

[0162] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, Q28, E23, and deletions at amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, Q28K, E23Q, and deletions at amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid designated herein as DM334.

[0163] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, D68, and deletions at amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, D68N, and deletions at amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid designated herein as DM333.

[0164] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, E2, and deletions at amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, E2N, and deletions at amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid designated herein as DM338.

[0165] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, L70, and deletions at amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, L70I, and deletions at amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid designated herein as DM125.

[0166] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, E4, and deletions at amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, E4Q, and deletions at amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid designated herein as DM337.

[0167] In some embodiments the engineered protein comprises amino acid substitutions at amino acids S76, G1, E2, and deletions at amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments the engineered protein has at least amino acid substitutions S76Y, G1M, E2M, and deletions at amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments the engineered protein is represented by the amino acid designated herein as DM332.

[0168] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R53, an amino acid substitution at S76, and at least one amino acid substitution at one or more of E2, E23, Y65, L70.

[0169] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R53, and amino acid substitutions at least at E2, E23, Y65, L70, and S76, compared to MNEI (SEQ ID NO: 1).

[0170] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R5, and amino acid substitutions at least at E2, E23, Y65, L70, and S76 compared to MNEI (SEQ ID NO: 1), wherein the amino acid substitution in E2 is E2M or E2N, the amino acid substitution in E23 is E23A or E23Q, the amino acid substitution at Y65 is Y65R, the amino acid substitution at L70 is L70I, and the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A.

[0171] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions of at least E2M, E23A, Y65R, L70I, and S76Y, compared to MNEI (SEQ ID NO: 1). In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions of E2M, E23A, Y65R, L70I, and S76Y, compared to MNEI (SEQ ID NO: 1), and is designated herein as DM396.

[0172] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions at E2, E23, Y65, L70, and S76, and at one or more amino acids G1, A19, V20, K25, 126, Q28, R31, T33, N35, C41, E59, D68, A73, D74, 175, E77, R84, compared to MNEI (SEQ ID NO: 1).

[0173] In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions in E2, E23, Y65, L70, and S76, and at one or more amino acids G1, E2, A19, V20, K25, 126, Q28, R31, T33, N35, C41, E59, D68, A73, D74, 175, E77, R84, relative to MNEI (SEQ ID NO: 1), wherein the amino acid substitution in E2 is E2M or E2N, and the amino acid substitution in E23 is E2M or E2N. the amino acid substitution at Y65 is Y65R; the amino acid substitution at L70 is L70I; the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is the amino acid substitution at I26 is one or more of I26W, I26V, I26T; the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, C41S; The amino acid substitution at 59 is one or more of E59T, E59V, E59I, E59Y, E59F, E59W, and E59R; the amino acid substitution at D68 is D68N or D68T; the amino acid substitution at A73 is A73F or A73V; the amino acid substitution at D74 is D74V; the amino acid substitution at I75 is I75V or I75L; the amino acid substitution at E77 is E77V; and the amino acid substitution at R84 is R84L.

[0174] In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions at least G1, E2, E23, Y65, L70, Y76, compared to SEQ ID NO: 1. In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R5, and amino acid substitutions at least G1M, E2M, E23A, Y65R, L70I, S76Y, compared to SEQ ID NO: 1. In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R5, and amino acid substitutions G1M, E2M, E23A, Y65R, L70I, S76Y, compared to SEQ ID NO: 1. In some examples, the engineered protein is represented by SEQ ID NO: 360.

[0175] In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions at least at E2, E23, Y65, L70, compared to SEQ ID NO: 1. In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R5, and amino acid substitutions at least at E2N, E23A, Y65R, L70I, compared to SEQ ID NO: 1.

[0176] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions of at least E2N, E23A, Y65R, L70I, and S76Y, compared to MNEI (SEQ ID NO: 1).

[0177] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions of at least E2N, E23A, Y65R, L70I compared to SEQ ID NO: 1, and is designated herein as DM31 and represented herein by SEQ ID NO: 34.

[0178] According to some aspects, there are provided engineered proteins having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence set forth in SEQ ID NO:1, and wherein the at least one amino acid deletion is at one or more of amino acids E50, F52, and R53 of the reference protein, and the at least one amino acid substitution is at least at amino acids E2, E23, Y65, L70, S76 of the reference protein.

[0179] According to some aspects, there are provided engineered proteins having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence set forth in SEQ ID NO: 1, wherein the at least one amino acid deletion is at amino acids E50, F52, and R53 of the reference protein, and the at least one amino acid substitution is at amino acids E2, E23, Y65, L70, and S76 of the reference protein. In some examples, the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A.

[0180] According to some aspects, there are provided engineered proteins having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence set forth in SEQ ID NO: 1, wherein the at least one amino acid deletion is at amino acids E50, F52, and R53 of the reference protein, and the at least one amino acid substitution is at least E2N, E23A, Y65R, L70I, and at S76 of the reference protein. In some examples, the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A.

[0181] As shown in the Examples below, substitution of amino acid S76 in DM31 improved stability compared to DM31.

[0182] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions at amino acids E50, F52, and R54, and amino acid substitutions at E2N, E23A, Y65R, L70I, S76Y, compared to SEQ ID NO: 1. In some examples, the modified protein is designated herein as DM89.

[0183] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions at amino acids E50, F52, and R54, and amino acid substitutions of E2N, E23A, Y65R, L70I, S76F, compared to SEQ ID NO: 1. In some examples, the modified protein is designated herein as DM114.

[0184] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions at amino acids E50, F52, and R54, and amino acid substitutions at E2N, E23A, Y65R, L70I, S76W, compared to SEQ ID NO: 1. In some examples, the modified protein is designated herein as DM137.

[0185] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions at amino acids E50, F52, and R54, and amino acid substitutions at E2N, E23A, Y65R, L70I, S76T, compared to SEQ ID NO: 1. In some examples, the modified protein is designated herein as DM658.

[0186] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions at amino acids E50, F52, and R54, and amino acid substitutions at E2N, E23A, Y65R, L70I, S76R, compared to SEQ ID NO: 1. In some examples, the modified protein is designated herein as DM606.

[0187] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions at amino acids E50, F52, and R54, and amino acid substitutions at E2N, E23A, Y65R, L70I, S76I, compared to SEQ ID NO: 1. In some examples, the modified protein is designated herein as DM664.

[0188] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions at amino acids E50, F52, and R54, and amino acid substitutions of E2N, E23A, Y65R, L70I, S76V, compared to SEQ ID NO: 1. In some examples, the modified protein is designated herein as DM663.

[0189] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions at amino acids E50, F52, and R54, and amino acid substitutions at E2N, E23A, Y65R, L70I, S76H, compared to SEQ ID NO: 1. In some examples, the modified protein is designated herein as DM659.

[0190] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions at amino acids E50, F52, and R54, and amino acid substitutions at E2N, E23A, Y65R, L70I, S76A, compared to SEQ ID NO: 1. In some examples, the modified protein is designated herein as DM690.

[0191] In some instances, the modified protein is represented by one or more of DM89, DM114, DM137, DM658, DM606, DM664, DM663, DM659, or DM690. In some instances, the modified protein is represented by one or more of DM89, DM114, or DM137.

[0192] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions at E2N, E23A, Y65R, L70I, an amino acid substitution at S76, and amino acid substitutions at one or more of amino acids G1, A19, V20, K25, 126, Q28, R31, T33, N35, C41, E59, D68, A73, D74, 175, E77, R84, compared to MNEI (SEQ ID NO: 1).

[0193] In some examples, the engineered protein contains, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, and amino acid substitutions at E2N, E23A, Y65R, L70I, an amino acid substitution at S76, and amino acid substitutions at one or more of amino acids G1, E2, A19, V20, K25, 126, Q28, R31, T33, N35, C41, E59, D68, A73, D74, 175, E77, R84. the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; and the amino acid substitution at I26 is I26W, I26V, or I26T. the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at E59 is E59T, E5 the amino acid substitution at D68 is D68N or D68T; the amino acid substitution at A73 is A73F or A73V; the amino acid substitution at D74 is D74V; the amino acid substitution at I75 is I75V or I75L; the amino acid substitution at E77 is E77V; and the amino acid substitution at R84 is R84L.

[0194] In some examples, the modified protein includes, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, an amino acid substitution at S76, and an amino acid substitution at one or more of amino acids G1, E2, A19, V20, K25, 126, Q28, R31, T33, N35, C41, E59, D68, A73, D74, 175, E77, R84. the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; and the amino acid substitution at I26 is I26W, I26V, or I26T. the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at E59 is E59T, E5 the amino acid substitution at D68 is D68N or D68T; the amino acid substitution at A73 is A73F or A73V; the amino acid substitution at D74 is D74V; the amino acid substitution at I75 is I75V or I75L; the amino acid substitution at E77 is E77V; and the amino acid substitution at R84 is R84L.

[0195] In some examples, the engineered protein contains deletions of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76, A19, and amino acid substitutions at one or more of the following amino acids: G1, E2, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, D68, A73, D74, I75, E77, R84, as compared to MNEI (SEQ ID NO: 1). the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; and the amino acid substitution at I26 is I26W, I26V, or I26T. the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at E59 is E59T, E5 the amino acid substitution at D68 is D68N or D68T; the amino acid substitution at A73 is A73F or A73V; the amino acid substitution at D74 is D74V; the amino acid substitution at I75 is I75V or I75L; the amino acid substitution at E77 is E77V; and the amino acid substitution at R84 is R84L.

[0196] In some examples, the modified protein comprises, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76, A19, and amino acid substitutions at one or more amino acids Q28, T33, N35, R84, wherein the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at A19 is A19V; the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, Q28E; the amino acid substitution at T33 is T33R; and the amino acid substitution at R84 is R84L.

[0197] In some examples, the engineered protein contains, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76, V20, and amino acid substitutions at one or more of the following amino acids: G1, E2, A19, V20, K25, 126, Q28, R31, T33, N35, C41, E59, D68, A73, D74, 175, E77, R84. the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; and the amino acid substitution at I26 is I26W, I26V, I26I. the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at E59 is E59T, E the amino acid substitution at D68 is D68N or D68T; the amino acid substitution at A73 is A73F or A73V; the amino acid substitution at D74 is D74V; the amino acid substitution at I75 is I75V or I75L; the amino acid substitution at E77 is E77V; and the amino acid substitution at R84 is R84L.

[0198] In some examples, the engineered protein comprises, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76, V20, and one or more amino acids at Q28, R31, T33, N35, D68, R84, and the amino acid substitution at S76 is S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution at V20 is V20I, the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E, the amino acid substitution at R31 is R31T, the amino acid substitution at T33 is T33R, the amino acid substitution at N35 is N35T, the amino acid substitution at D68 is D68N or D68T, and the amino acid substitution at R84 is R84L.

[0199] In some examples, the modified protein includes, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76, K25, and amino acid substitutions at one or more of the following amino acids: G1, E2, A19, V20, 126, Q28, R31, T33, N35, C41, E59, D68, A73, D74, 175, E77, R84. the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; and the amino acid substitution at I26 is I26W, I26V, or I26T. the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at E59 is E59T, E5 the amino acid substitution at D68 is D68N or D68T; the amino acid substitution at A73 is A73F or A73V; the amino acid substitution at D74 is D74V; the amino acid substitution at I75 is I75V or I75L; the amino acid substitution at E77 is E77V; and the amino acid substitution at R84 is R84L.

[0200] In some examples, the engineered protein comprises, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76 and K25, and amino acid substitutions at one or more of Q28, T33, C41, D68, and the amino acid substitution at S76 is S76Y, S76F, S76T, S76W, S76R, S76V, S the amino acid substitution at K25 is K25R; the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at T33 is T33R; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at D68 is D68N or D68T.

[0201] In some examples, the modified protein includes, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76, I26, and amino acid substitutions at one or more of the following amino acids: G1, E2, A19, V20, K25, Q28, R31, T33, N35, C41, E59, D68, A73, D74, I75, E77, R84. the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; and the amino acid substitution at I26 is I26W, I26V, or I26T. the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at E59 is E59T, E5 the amino acid substitution at D68 is D68N or D68T; the amino acid substitution at A73 is A73F or A73V; the amino acid substitution at D74 is D74V; the amino acid substitution at I75 is I75V or I75L; the amino acid substitution at E77 is E77V; and the amino acid substitution at R84 is R84L.

[0202] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at S76, I26, and Q28 relative to MNEI (SEQ ID NO: 1), wherein the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at I26 is one or more of I26W, I26V, I26T; and the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, Q28E.

[0203] In some examples, the engineered protein contains, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76 and Q28, and amino acid substitutions at one or more of the following amino acids: G1, E2, A19, V20, K25, 126, R31, T33, N35, C41, E59, D68, A73, D74, 175, E77, R84. the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; and the amino acid substitution at I26 is I26W, I26V, or I26T. the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at E59 is E59T, E5 the amino acid substitution at D68 is D68N or D68T; the amino acid substitution at A73 is A73F or A73V; the amino acid substitution at D74 is D74V; the amino acid substitution at I75 is I75V or I75L; the amino acid substitution at E77 is E77V; and the amino acid substitution at R84 is R84L.

[0204] In some examples, the engineered protein comprises, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76 and Q28, and amino acid substitutions at one or more of A19, V20, K25, I26, R31, T33, C41, D68, wherein the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at A19 is A19V; and the amino acid substitution at V20 is A19V. the amino acid substitution at K25 is K25R; the amino acid substitution at I26 is one or more of I26W, I26V, and I26T; the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at D68 is D68N or D68T.

[0205] In some examples, the engineered protein contains, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76 and R31, and amino acid substitutions at one or more of amino acids G1, E2, A19, V20, K25, 126, Q28, R31, T33, N35, C41, E59, D68, A73, D74, 175, E77, R84. the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; and the amino acid substitution at I26 is I26W, I26V, I26I ... the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at E59 is E59T, E the amino acid substitution at D68 is D68N or D68T; the amino acid substitution at A73 is A73F or A73V; the amino acid substitution at D74 is D74V; the amino acid substitution at I75 is I75V or I75L; the amino acid substitution at E77 is E77V; and the amino acid substitution at R84 is R84L.

[0206] In some examples, the engineered protein comprises, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76 and R31, and amino acid substitutions at one or more amino acids V20, Q28, T33, D68, wherein the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at V20 is V20I; the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; and the amino acid substitution at D68 is D68N or D68T.

[0207] In some examples, the engineered protein has, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76 and T33, and substitutions at one or more of the following amino acids: G1, E2, A19, V20, K25, 126, Q28, R31, N35, C41, E59, D68, A73, D74, 175, E77, R84. the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; and the amino acid substitution at I26 is I26W, I26 the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at E59 is E59T. , E59V, E59I, E59Y, E59F, E59W, E59R, the amino acid substitution at D68 is D68N or D68T, the amino acid substitution at A73 is A73F or A73V, the amino acid substitution at D74 is D74V, the amino acid substitution at I75 is I75V or I75L, the amino acid substitution at E77 is E77V, and the amino acid substitution at R84 is R84L.

[0208] In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76 and T33, and amino acid substitutions at one or more amino acids A19, V20, K25, Q28, D68, relative to MNEI (SEQ ID NO: 1), including the amino acid substitution at S76 being S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, the amino acid substitution at G1 is G1M, the amino acid substitution at A19 is A19V, the amino acid substitution at V20 is V20I, the amino acid substitution at K25 is K25R, the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, Q28E, the amino acid substitution at T33 is T33R, and the amino acid substitution at D68 is D68N or D68T.

[0209] In some examples, the engineered protein comprises, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76 and N35, and one or more of amino acids G1, E2, A19, V20, K25, 126, Q28, R31, T33, N35, C41, E59, D68, A73, D74, 175, E77, R84. the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; and the amino acid substitution at I26 is I26W, I26I ... the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at E59 is E59 the amino acid substitution at D68 is D68N or D68T; the amino acid substitution at A73 is A73F or A73V; the amino acid substitution at D74 is D74V; the amino acid substitution at I75 is I75V or I75L; the amino acid substitution at E77 is E77V; and the amino acid substitution at R84 is R84L.

[0210] In some examples, the modified protein comprises, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76 and N35, and amino acid substitutions at one or more amino acids at A19, V20, wherein the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, the amino acid substitution at G1 is G1M, the amino acid substitution at A19 is A19V, the amino acid substitution at V20 is V20I, and the amino acid substitution at N35 is N35T.

[0211] In some examples, the engineered protein contains, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76 and C41, and amino acid substitutions at one or more of amino acids G1, E2, A19, V20, K25, 126, Q28, R31, T33, N35, C41, E59, D68, A73, D74, 175, E77, R84. the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; and the amino acid substitution at I26 is I26W, I26V, I26I ... the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at E59 is E59T, E the amino acid substitution at D68 is D68N or D68T; the amino acid substitution at A73 is A73F or A73V; the amino acid substitution at D74 is D74V; the amino acid substitution at I75 is I75V or I75L; the amino acid substitution at E77 is E77V; and the amino acid substitution at R84 is R84L.

[0212] In some examples, the modified protein comprises, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76 and C41, and amino acid substitutions at one or more amino acids at K25, Q28, wherein the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at K25 is K25R; the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, Q28E; and the amino acid substitution at C41 is one or more of C41A, C41V, C41T, C41S.

[0213] In some examples, the engineered protein contains, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76 and E59, and amino acid substitutions at one or more of the following amino acids: G1, E2, A19, V20, K25, 126, Q28, R31, T33, N35, C41, D68, A73, D74, 175, E77, R84. the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; and the amino acid substitution at I26 is I26W, I26V, or I26T. the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at E59 is E59T, E5 the amino acid substitution at D68 is D68N or D68T; the amino acid substitution at A73 is A73F or A73V; the amino acid substitution at D74 is D74V; the amino acid substitution at I75 is I75V or I75L; the amino acid substitution at E77 is E77V; and the amino acid substitution at R84 is R84L.

[0214] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at S76, E59, and D74 compared to MNEI (SEQ ID NO: 1), wherein the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at E59 is one or more of E59T, E59V, E59I, E59Y, E59F, E59W, E59R; and the amino acid substitution at D74 is D74V.

[0215] In some examples, the engineered protein contains, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76 and D68, and amino acid substitutions at one or more of the following amino acids: G1, E2, A19, V20, K25, 126, Q28, R31, T33, N35, C41, E59, A73, D74, 175, E77, R84. the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; and the amino acid substitution at I26 is I26W, I26V, or I26T. the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at E59 is E59T, E5 the amino acid substitution at D68 is D68N or D68T; the amino acid substitution at A73 is A73F or A73V; the amino acid substitution at D74 is D74V; the amino acid substitution at I75 is I75V or I75L; the amino acid substitution at E77 is E77V; and the amino acid substitution at R84 is R84L.

[0216] In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76 and D68, and amino acid substitutions at one or more amino acids V20, K25, Q28, T33, relative to MNEI (SEQ ID NO: 1), including amino acid substitutions at S76, such as S76Y, S76F, S76T, S76W, S76R, S76V, ... the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, Q28E; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; and the amino acid substitution at D68 is D68N or D68T.

[0217] In some examples, the engineered protein contains, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at S76, D74, and amino acid substitutions at one or more of amino acids G1, E2, A19, V20, K25, 126, Q28, R31, T33, N35, C41, E59, D68, A73, 175, E77, R84. the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; and the amino acid substitution at I26 is I26W, I26V, I26I. the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at E59 is E59T, E the amino acid substitution at D68 is D68N or D68T; the amino acid substitution at A73 is A73F or A73V; the amino acid substitution at D74 is D74V; the amino acid substitution at I75 is I75V or I75L; the amino acid substitution at E77 is E77V; and the amino acid substitution at R84 is R84L.

[0218] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at S76, D74, and E59 relative to MNEI (SEQ ID NO: 1), wherein the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at E59 is one or more of E59T, E59V, E59I, E59Y, E59F, E59W, E59R; and the amino acid substitution at D74 is D74V.

[0219] In some examples, the modified protein includes a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76, I75, and amino acid substitutions at one or more of the following amino acids: G1, E2, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, D68, A73, D74, E77, R84, relative to MNEI (SEQ ID NO: 1). the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; and the amino acid substitution at I26 is I26W, I26V, or I26T. the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at E59 is E59T, E5 the amino acid substitution at D68 is D68N or D68T; the amino acid substitution at A73 is A73F or A73V; the amino acid substitution at D74 is D74V; the amino acid substitution at I75 is I75V or I75L; the amino acid substitution at E77 is E77V; and the amino acid substitution at R84 is R84L.

[0220] In some examples, the modified protein includes, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76, E77, and amino acid substitutions at one or more of the following amino acids: G1, E2, A19, V20, K25, 126, Q28, R31, T33, N35, C41, E59, D68, A73, D74, 175, R84. the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; and the amino acid substitution at I26 is I26W, I26V, or I26T. the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at E59 is E59T, E5 the amino acid substitution at D68 is D68N or D68T; the amino acid substitution at A73 is A73F or A73V; the amino acid substitution at D74 is D74V; the amino acid substitution at I75 is I75V or I75L; the amino acid substitution at E77 is E77V; and the amino acid substitution at R84 is R84L.

[0221] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at S76, I75, and E77 compared to MNEI (SEQ ID NO: 1), wherein the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, the amino acid substitution at I75 is I75V or I75L, and the amino acid substitution at E77 is E77V.

[0222] In some examples, the modified protein includes a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, amino acid substitutions at S76, R84, and amino acid substitutions at one or more of the following amino acids: G1, E2, A19, V20, K25, 126, Q28, R31, T33, N35, C41, E59, D68, A73, D74, 175, E77, relative to MNEI (SEQ ID NO: 1). the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at K25 is K25R; and the amino acid substitution at I26 is I26W, I26V, or I26T. the amino acid substitution at Q28 is one or more of Q28R, Q28K, Q28S, and Q28E; the amino acid substitution at R31 is R31T; the amino acid substitution at T33 is T33R; the amino acid substitution at N35 is N35T; the amino acid substitution at C41 is one or more of C41A, C41V, C41T, and C41S; and the amino acid substitution at E59 is E59T, E5 the amino acid substitution at D68 is D68N or D68T; the amino acid substitution at A73 is A73F or A73V; the amino acid substitution at D74 is D74V; the amino acid substitution at I75 is I75V or I75L; the amino acid substitution at E77 is E77V; and the amino acid substitution at R84 is R84L.

[0223] In some examples, the modified protein has, relative to MNEI (SEQ ID NO: 1), a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at S76, R84, and amino acid substitutions at one or more of the following amino acids: G1, E2, A19, V20, K25, 126, Q28, R31, T33, N35, C41, E59, D68, A73, D74, 175, E77. wherein the amino acid substitution at S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A; the amino acid substitution at G1 is G1M; the amino acid substitution at A19 is A19V; the amino acid substitution at V20 is V20I; the amino acid substitution at D68 is D68N or D68T; and the amino acid substitution at R84 is R84L.

[0224] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at G1 and S76, compared to MNEI (SEQ ID NO: 1).

[0225] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, G1M, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0226] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, G1M, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM413.

[0227] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at A19 and S76, relative to MNEI (SEQ ID NO: 1). In some examples, the modified protein comprises A19V and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0228] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, A19V, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid sequence designated herein as DM202.

[0229] In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at V20 and S76 relative to MNEI (SEQ ID NO: 1). In some examples, the engineered protein comprises V20I and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A relative to MNEI (SEQ ID NO: 1).

[0230] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, V20I, and S76Y, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM206.

[0231] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, K25, and S76, compared to MNEI (SEQ ID NO: 1).

[0232] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, K25R, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, compared to MNEI (SEQ ID NO: 1).

[0233] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, K25R, and S76W compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM162.

[0234] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, K25R, and S76F, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM160.

[0235] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, K25R, and S76Y, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid sequence designated herein as DM101.

[0236] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at I26 and S76, compared to MNEI (SEQ ID NO: 1).

[0237] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, compared to MNEI (SEQ ID NO: 1).

[0238] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, I26V, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0239] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, I26W, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0240] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, and S76F compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM159.

[0241] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, and S76W compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid sequence designated herein as DM161.

[0242] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid sequence designated herein as DM100.

[0243] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at Q28 and S76, compared to MNEI (SEQ ID NO: 1).

[0244] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, Q28S, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0245] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28S, and S76Y, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM520.

[0246] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0247] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid sequence designated herein as DM363.

[0248] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0249] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, and S76Y, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM517.

[0250] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at R31 and S76, compared to MNEI (SEQ ID NO: 1).

[0251] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, R31T, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0252] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, R31T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM286.

[0253] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at T33 and S76, compared to MNEI (SEQ ID NO: 1).

[0254] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, T33R, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0255] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, T33R, and S76W compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM234.

[0256] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, T33R, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM222.

[0257] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, T33R, and S76F, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM228.

[0258] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at N35 and S76, compared to MNEI (SEQ ID NO: 1).

[0259] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, N35T, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0260] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, N35T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM289.

[0261] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at C41 and S76, compared to MNEI (SEQ ID NO: 1).

[0262] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, C41V, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0263] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, C41V, and S76F compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM249.

[0264] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, C41V, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM146.

[0265] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, C41V, and S76W compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM269.

[0266] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, C41T, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0267] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, C41T, and S76W compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid sequence designated herein as DM291.

[0268] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, C41T, and S76F compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM294.

[0269] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, C41T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM287.

[0270] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, C41S, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0271] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, C41S, and S76Y, compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid sequence designated herein as DM346. [DS instead of DM211]

[0272] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, C41A, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0273] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, C41A, and S76Y, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM210.

[0274] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at E59 and S76, compared to MNEI (SEQ ID NO: 1).

[0275] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, E59T, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0276] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, E59T, and S76I compared to MNEI (SEQ ID NO: 1), represented herein by the amino acid designated DM657.

[0277] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, E59T, and S76V, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM654.

[0278] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, E59T, and S76T compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid sequence designated herein as DM651.

[0279] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, E59V, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0280] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, E59V, and S76I, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM655.

[0281] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, E59V, and S76V compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM652.

[0282] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, E59V, and S76T compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM605.

[0283] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, E59I, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0284] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, E59I, and S76I, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM656.

[0285] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, E59I, and S76V, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM653.

[0286] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, E59I, and S76T, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM650.

[0287] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, E59Y, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0288] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, E59Y, and S76V, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM662.

[0289] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, E59F, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0290] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, E59F, and S76M compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM660.

[0291] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, E59W, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0292] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, E59W, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid sequence designated herein as DM661.

[0293] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, E59R, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0294] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, E59R, and S76T compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM604.

[0295] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, D68, and S76, compared to MNEI (SEQ ID NO: 1).

[0296] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, D68N, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0297] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, D68N, and S76W, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM236.

[0298] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, D68N, and S76F, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM230.

[0299] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, D68N, and S76Y, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM224.

[0300] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, D68T, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0301] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, D68T, and S76Y, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM649.

[0302] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, A73, and S76, compared to MNEI (SEQ ID NO: 1).

[0303] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, A73V, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0304] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, A73V, and S76F compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM248.

[0305] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, A73V, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM204.

[0306] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, A73F, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0307] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I75, and S76, which is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0308] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, I75V, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0309] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I75V, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid sequence designated herein as DM600.

[0310] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I75L, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0311] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, E77, and S76, compared to MNEI (SEQ ID NO: 1).

[0312] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, E77V, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0313] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, E77V, and S76Y, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM594.

[0314] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at R84 and S76, compared to MNEI (SEQ ID NO: 1).

[0315] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, R84L, and an amino acid substitution at S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, relative to MNEI (SEQ ID NO: 1).

[0316] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, R84L, and S76Y, relative to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM90.

[0317] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at A19, Q28, and S76 compared to MNEI (SEQ ID NO: 1).

[0318] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, A19V, Q28K, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM610.

[0319] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, A19V, Q28R, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid sequence designated herein as DM621.

[0320] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at A19, T33, and S76 compared to MNEI (SEQ ID NO: 1).

[0321] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, A19V, T33R, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM617.

[0322] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at A19, N35, and S76 compared to MNEI (SEQ ID NO: 1).

[0323] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, A19V, N35T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM625.

[0324] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at A19, R84, and S76, compared to MNEI (SEQ ID NO: 1).

[0325] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, A19V, R84L, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM626.

[0326] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at V20, Q28, and S76 compared to MNEI (SEQ ID NO: 1).

[0327] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, V20I, Q28R, and S76F compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid sequence designated herein as DM681.

[0328] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, V20I, Q28R, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM620.

[0329] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, V20I, Q28K, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM613.

[0330] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at V20, R31, and S76 compared to MNEI (SEQ ID NO: 1).

[0331] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, V20I, R31T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM633.

[0332] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at V20, T33, and S76 compared to MNEI (SEQ ID NO: 1).

[0333] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, V20I, T33R, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM616.

[0334] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at V20, N35, and S76 compared to MNEI (SEQ ID NO: 1).

[0335] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, V20I, N35T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM634.

[0336] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at V20, D68, and S76 compared to MNEI (SEQ ID NO: 1).

[0337] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, V20I, D68N, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM611.

[0338] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, V20, R84, and S76 compared to MNEI (SEQ ID NO: 1).

[0339] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, V20I, R84L, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM635.

[0340] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at K25, Q28, and S76 compared to MNEI (SEQ ID NO: 1).

[0341] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70, K25R, Q28K, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM166.

[0342] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70, K25R, Q28K, and S76F compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM168.

[0343] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, K25R, Q28R, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM165.

[0344] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70, K25R, Q28R, and S76F compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM167.

[0345] In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at K25, 126, Q28, and S76, relative to MNEI (SEQ ID NO: 1). In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, K25R, 126T, Q28K, and S76Y, relative to MNEI (SEQ ID NO: 1). In some embodiments, the engineered protein is represented by the amino acid designated herein as DM288.

[0346] In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at K25, T33, and S76, relative to MNEI (SEQ ID NO: 1). In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, K25R, T33R, and S76Y, relative to MNEI (SEQ ID NO: 1). In some embodiments, the engineered protein is represented by the amino acid designated herein as DM618.

[0347] In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at K25, C41, and S76, relative to MNEI (SEQ ID NO: 1). In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, K25R, C41T, and S76Y, relative to MNEI (SEQ ID NO: 1). In some embodiments, the engineered protein is represented by the amino acid sequence designated herein as DM481.

[0348] In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at K25, D68, and S76, relative to MNEI (SEQ ID NO: 1). In some examples, the engineered protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, K25R, D68N, and S76Y, relative to MNEI (SEQ ID NO: 1). In some embodiments, the engineered protein is represented by the amino acid designated herein as DM479.

[0349] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at I26, Q28, and S76 compared to MNEI (SEQ ID NO: 1).

[0350] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I26W, Q28K, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM128.

[0351] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I26W, Q28E, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM127.

[0352] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I26V, Q28K, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM455.

[0353] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, Q28K, and S76W compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM164.

[0354] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, Q28R, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM526.

[0355] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, Q28K, and S76F compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM163.

[0356] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, Q28K, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM158.

[0357] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, Q28S, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM456.

[0358] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at I26, Q28, C41, and S76 compared to MNEI (SEQ ID NO: 1).

[0359] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, Q28K, C41T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM483.

[0360] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at I26, Q28, D68, and S76 compared to MNEI (SEQ ID NO: 1).

[0361] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, Q28K, D68N, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM484.

[0362] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at Q28, R31, and S76 compared to MNEI (SEQ ID NO: 1).

[0363] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, R31T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid sequence designated herein as DM171.

[0364] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, R31T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM630.

[0365] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at Q28, T33, and S76 compared to MNEI (SEQ ID NO: 1).

[0366] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, T33R, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM619.

[0367] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, T33R, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM622.

[0368] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at Q28, N35, and S76 compared to MNEI (SEQ ID NO: 1).

[0369] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, N35T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid sequence designated herein as DM631.

[0370] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, N35T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM640.

[0371] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at Q28, C41, and S76 compared to MNEI (SEQ ID NO: 1).

[0372] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, C41A, and S76W compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM682.

[0373] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, C41A, and S76F compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM683.

[0374] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, C41S, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM534.

[0375] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, C41A, and S76F compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM685.

[0376] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, C41A, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM532.

[0377] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, C41T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM528.

[0378] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, C41S, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM530.

[0379] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, C41A, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM536.

[0380] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, C41T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM478.

[0381] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at Q28, D68, and S76 compared to MNEI (SEQ ID NO: 1).

[0382] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, D68T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM607.

[0383] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, D68N, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM472.

[0384] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, D68T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM608.

[0385] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, D68N, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM542.

[0386] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at Q28, R84, and S76 compared to MNEI (SEQ ID NO: 1).

[0387] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, R84L, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM632.

[0388] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, R84L, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid sequence designated herein as DM641.

[0389] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at R31, T33, and S76 compared to MNEI (SEQ ID NO: 1).

[0390] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, R31T, T33R, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM636.

[0391] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at T33, N35, and S76 compared to MNEI (SEQ ID NO: 1).

[0392] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, T33R, N35T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM637.

[0393] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at T33, D68, and S76 compared to MNEI (SEQ ID NO: 1).

[0394] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, T33R, D68N, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM614.

[0395] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at T33, R84, and S76 compared to MNEI (SEQ ID NO: 1).

[0396] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, T33R, R84L, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM638.

[0397] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at N35, D68, and S76 compared to MNEI (SEQ ID NO: 1).

[0398] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, N35T, D68T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM643.

[0399] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, N35T, D68N, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM628.

[0400] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at E59, D74, and S76 compared to MNEI (SEQ ID NO: 1).

[0401] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, E59V, D74V, and S76V compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM602.

[0402] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at D68, R31, and S76 compared to MNEI (SEQ ID NO: 1).

[0403] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, D68T, R31T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM642.

[0404] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at D68, R31, and S76 compared to MNEI (SEQ ID NO: 1).

[0405] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, D68N, R31T, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM627.

[0406] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at D68, R84, and S76 compared to MNEI (SEQ ID NO: 1).

[0407] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, D68N, R84L, and S76F compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM242.

[0408] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, D68N, R84L, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM240.

[0409] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, D68T, R84L, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM644.

[0410] In some instances, the modified protein comprises a deletion of amino acids E50, F52, and R54, as well as amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions at I75, E77, and S76, compared to MNEI (SEQ ID NO: 1).

[0411] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, I75V, E77V, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid designated herein as DM595.

[0412] In some examples, the modified protein is one or more of DM89, DM114 (Tm=101.5°C), DM137 (Tm=97°C), or any combination thereof.

[0413] In some examples, the engineered protein is one or more of DM456 (Tm>104°C), DM517 (Tm=100°C), DM100 (Tm=100°C), DM165 (Tm=99°C), DM361 (Tm=97°C), DM224 (Tm=95°C), DM346 (Tm=97°C), DM101 (Tm=96°C), DM206 (Tm=96°C), DM209 (Tm=96°C), DM202 (Tm=96°C), DM479 (Tm=97°C), or any combination thereof. As described herein, Tm can be measured by any method known in the art, for example, using DSF as described in the Examples below.

[0414] As shown herein and in the Examples below, the predicted Tm values correlated highly with the experimentally determined Tm values. In some examples, the modified protein is DM635 (basal Tm 105.7°C), DM644 (basal Tm 105.43°C), DM242 (basal Tm 105.06°C), DM632 (predicted Tm 104°C), DM90 (predicted Tm 104°C), DM240 (predicted Tm 104°C), DM641 (predicted Tm 103.8°C), DM162 (predicted Tm 102°C), DM630 (predicted Tm 101.9°C), DM657 (predicted Tm 101.8°C), DM655 (predicted Tm 101.8°C), DM642 (predicted Tm 101.7°C), DM633 (predicted Tm 101.6°C), DM595 (predicted Tm 101.5°C), DM656 (predicted Tm 101.3°C), DM638 (predicted Tm 101.2°C), DM335 (predicted Tm 101.1°C), DM286 (predicted Tm 101.1°C).

[0415] Interestingly, as shown in the examples below, the substitutions have been shown to be synergistic. As used herein, the term synergistic substitution refers to the simultaneous modification of multiple amino acids within a protein sequence that collectively enhance or alter its function. In other words, the combined effect of these substitutions is greater than the sum of their individual effects.

[0416] In some examples, the modified protein is one or more of DM479, DM472, DM346, DM165, DM361, DM456, or any combination thereof.

[0417] In some examples, the modified protein is DM240, DM641, DM633, DM627, DM171, DM480, DM362, DM236, DM396, DM479, DM167, DM165, DM397, DM679, DM230, DM681, DM361, DM224, DM611, DM620, DM473, DM517, DM472, DM685, DM608, DM 628, DM521, DM542, DM477, DM640, DM532, DM528, DM621, DM530, DM360, DM73, DM614, DM526, DM622, DM94, DM486, DM484, DM457, DM209, DM540, DM333, DM511, DM539, DM524, DM148, or any combination thereof.

[0418] In some examples, the engineered protein is one or more of DM202, DM207, DM289, DM210, DM89, DM287, DM224, DM346, DM209, DM203, DM274, DM273, DM222, DM206, DM160, DM292, DM253, DM241, DM204, DM128, DM158, DM250, DM240, DM247, DM127, DM178, DM174, DM248, DM254, DM208, DM179, DM242, or any combination thereof.

[0419] Table 1 shows proteins relevant to this disclosure and their corresponding SEQ ID NOs, including modified proteins encompassed by this disclosure.

[0420] [Table 1-1]

[0421] [Table 1-2]

[0422] [Table 1-3]

[0423] [Table 1-4]

[0424] [Table 1-5]

[0425] [Table 1-6]

[0426] [Table 1-7]

[0427] [Table 1-8]

[0428] [Table 1-9]

[0429] [Table 1-10]

[0430] In some forms, the engineered protein comprises the amino acid sequence set forth in one or more of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:221, SEQ ID NO:108, SEQ ID NO:9, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:52, SEQ ID NO:182, SEQ ID NO:10, SEQ ID NO:69, SEQ ID NO:157, SEQ ID NO:114, SEQ ID NO:174, SEQ ID NO:162, SEQ ID NO:231, SEQ ID NO:57, SEQ ID NO:192, SEQ ID NO:68, SEQ ID NO:106, SEQ ID NO:8, SEQ ID NO:79, SEQ ID NO:76, SEQ ID NO:22, SEQ ID NO:194, SEQ ID NO:262, SEQ ID NO:84, SEQ ID NO:239, SEQ ID NO:135, SEQ ID NO:211, SEQ ID NO:237, SEQ ID NO:198, SEQ ID NO:179, SEQ ID NO:172, or any combination thereof.

[0431] As described herein, the variant protein has at least improved stability compared to the reference protein.

[0432] According to the present disclosure, the engineered protein has at least one improved food-related property characteristic compared to the reference protein.

[0433] As used herein, the term food-related property encompasses properties that enable the suitability of the engineered protein in food and beverage applications, such as flavor, texture, taste, sweetness threshold, sweetness level, sweetness profile, sensory profile, sweetness kinetics, stability (structural and functional), heat resistance, compatibility with food matrices, shelf life, masking and / or enhancement of other flavors, off-taste, taste onset, lingering taste, roundness of taste, or sugar-like taste.

[0434] In some embodiments, at least one food-related property is a sensory-affecting property. As used herein, the term "sensory-affecting property" refers to a change in sensory impression, for example, determined by taste. Sensory-affecting properties include, for example, sweetness profile, such as sweetness potency (sugar-like taste), sweetness kinetics (onset time, lingering time, taste duration), lack of off-taste, and masking or enhancing other tastes, off-tastes (e.g., metallic taste). For example, improved properties relate to increased sweetness, reduced onset time, or reduced aftertaste.

[0435] According to some embodiments, the engineered protein may be considered a sugar substitute, in which at least one property is a sensory-affecting property, hi some embodiments, the at least one food-related property is at least one of sweetness potency, reduced onset time, or reduced aftertaste.

[0436] In some examples, the engineered protein has an improved aftertaste. In some examples, the engineered protein is DM114. In some examples, the engineered protein is DM137. In some examples, the engineered protein is DM157. In some examples, the engineered protein is DM224. In some examples, the engineered protein is DM157. In some examples, the engineered protein is DM02.

[0437] In some examples, the modified protein is DM165.

[0438] As detailed above, the engineered protein may be used in combination with at least one additional food ingredient. In some embodiments, the at least one food-related property may refer to a synergistic effect between the engineered protein and at least one food ingredient. Non-limiting examples of food ingredients include artificial or natural flavors, food additives, food dyes, preservatives, or additional sugar additives. The food ingredient may have a taste-masking or taste-enhancing effect.

[0439] As described herein, the engineered proteins described herein have improved food-related properties. The sweetness profile of the engineered proteins, such as sweetness potency (sugar-like flavor), lack of off-taste, reduced onset time, and reduced aftertaste, can be determined by any known taste test known in the art. For example, a comparison with the sweetness of sucrose or other sweeteners can be made by a taste panel, and sweetness potency can be ranked as detailed in the Examples below.

[0440] Comparisons can be made, for example, by determining the minimum concentration required to elicit a sweet taste sensation or an evaluation of the sweetness profile, 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 engineered protein compared to a known sweetener, such as sucrose.

[0441] 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 or greater, a sweetness duration of about 1 to 20 seconds, sometimes 2 to 18 seconds, and sometimes 2 to 4 seconds.

[0442] The variant protein, which is similar to the reference protein, binds to the sweet taste receptor.

[0443] In some embodiments the engineered protein has a perceived sweetness threshold that is 300 to 16,000 higher than that of sugar on a weight basis.

[0444] The sensory profile typically includes taste kinetics, i.e., onset duration (time to taste sensation), taste duration, and aftertaste time (corresponding to the tail of the Gaussian distribution), which is a Gaussian distribution that indicates taste intensity over time. Additional characteristics include off-tastes (e.g., due to binding to other receptors), roundness of taste, metallic and other minor tastes, synergistic effects with other ingredients (e.g., masking and enhancing other flavors or undesirable tastes, such as stevia).

[0445] As described herein, computer analysis was performed to identify regions and / or amino acid residues based on MNEI or MNEI-based proteins.

[0446] The variant proteins described herein can be designed by a variety of methods.

[0447] In some embodiments, protein design is performed using computational tools or by specialized protein design and structural biology methods (e.g., site-directed mutagenesis, protein engineering, or directed evolution), as further described below. The inventors developed a computational methodology based on sequence, structural, and / or evolutionary data of a reference flavor protein and other proteins whose sequence and / or structural features show local or global similarity to the reference flavor protein. The computational method developed and applied herein allowed the inventors to design proteins with specific amino acid substitutions predicted to be energetically favorable and therefore have improved traits such as thermal stability, halostability, pH stability, shelf life, folding, and solubility characteristics. Specifically, computational protein design (CPD) was applied to specific sites or regions within the structure and / or sequence of the reference protein that are not required for functional binding to the receptor. In addition, CPD allowed the inventors to limit substitutions to a predetermined set of amino acids that meet the required improved characteristics. The predetermined set of amino acids is found both in the input data, i.e., the regions of the protein subjected to CPD, and in the output data, i.e., the positions and types of amino acids present in the resulting engineered protein.

[0448] For example, by using CPD, it is possible to replace "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).

[0449] Without being bound by theory, the inventors suggest that replacing hydrophobic amino acids with hydrophilic amino acids on the exterior surface region reduces non-specific binding to the oral cavity and reduces lingering aftertaste.

[0450] The methodology developed herein involves searching for "stabilizing substitutions," e.g., amino acid substitutions that reduce the overall energy of a 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 perform focusing and filtering through a series of orthogonal methods, such as evolutionary sequence and structural consensus, regular and high-temperature molecular dynamics (MD) and other dynamic simulations, correlated mutational analysis (CMA), surface electrostatic analysis, visual inspection, and analysis of voids, hydrophobic patches, unsatisfied hydrogen bonds, etc.

[0451] 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 specific range, (c) analysis of the intraprotein cavity, (d) dynamic stability including correlated mutation analysis, normal mode analysis, and root mean square fluctuation (RMSF) in high-temperature or room-temperature kinetics, (e) entropy and / or enthalpy components in substitution energetics, (f) visualization of specific substitutions, (g) amino acid species tolerated in families of related proteins as reflected by evolutionary conservation analysis of curated multiple sequence alignments (MSAs), and (h) frequency of substitutions as reflected in low-pseudo-energy CPD calculations.

[0452] The computer method includes one or more of the following steps.

[0453] (1) Multiple sequence alignment (MSA) or multiple structural alignment. In this step, public databases are queried for DNA and / or protein sequences that share similarity with the target reference protein or its fragment. Based on the results obtained, a multiple sequence alignment (MSA) or multiple structural alignment is constructed, and the conservation ratio is calculated. According to the MSA results, a decision is made regarding the level of CPD to be performed. At non-conserved positions, all amino acids (with or without cysteine) are allowed by CPD, whereas at more conserved positions, CPD is limited to residues with similar properties (charge, size, internal dynamics, etc.). This step involves limiting substitutions at each position based on biophysical knowledge and conservation data. MSA can generate a position-specific substitution matrix (PSSM), in which each position along the sequence is described in a way that correlates with the relative abundance of each amino acid, possibly taking into account the potential probability of amino acid substitution, deletion, or insertion.

[0454] (2) Protein function analysis and analysis of structure-function-dynamics relationships. In this step, a database of substitutions with known effects (on activity, structure, binding, etc.) is constructed using prior knowledge. Based on previous knowledge, substitutions known to disrupt protein stability and / or function and their neighboring positions (e.g., 0.5-1 nm distance) are limited in the CPD and not substituted.

[0455] (3) CPD. This step is partially performed by designated software such as ROSETTA, OSPREY, SCWRL, PyMol, and AlphaFold. Before performing deterministic CPD, the energy of the 3D structure / model of the reference protein is minimized. CPD can involve site-specific amino acid replacement, where one amino acid is replaced by another, or replacing protein regions with other amino acid sequences, resulting in a protein with a different length. The latter can be performed by reconstructing regions such as loops using ab initio methods or by extracting regions from other proteins, a method sometimes called "grafting." For each reference protein, multiple models are considered.

[0456] Another consideration in CPD is to increase thermal stability while maintaining the functional plasticity required for receptor binding, which is often inherently associated with protein rigidification. Proteins must undergo several conformational changes (also known as "functional plasticity") to activate receptors. Therefore, we focused on regions that can be rigidified while preserving the regions that must maintain functional plasticity.

[0457] The terms "amino acid sequence" and / or "polypeptide chain" are used to describe proteins having an amino acid sequence or 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 a 3D structure as well as sequences without a 3D structure.

[0458] Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. As used herein, an amino acid substitution (replacement) refers to a change from one amino acid to a different amino acid. This typically results from a point mutation in the DNA sequence caused by a nonsynonymous missense mutation that alters the codon sequence to encode another amino acid rather than the reference amino acid. Amino acid substitutions can affect protein function or structure, and generally depend on how similar or dissimilar the replaced amino acids are and their position in the sequence or structure. For example, amino acid substitutions may be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, bulk (or flexibility), beta-branching, aromaticity, ability to confer specific binding interactions (hydrogen bonds, salt bridges, polar and nonpolar interactions), pK, ability to bind sugars and other post-translational modifications, and / or the amphipathic nature of the residues involved.

[0459] In some embodiments, amino acid substitutions may be conservative substitutions. Such substitutions involve changing an amino acid for another amino acid that exhibits similar properties. A conservative amino acid substitution (also referred to as a conservative amino acid "substitution" or conservative amino acid mutation) is an amino acid substitution in a protein that changes a given amino acid for a different amino acid with similar biochemical, structural, and / or chemical properties.

[0460] For example, amino acids can be divided into six major classes based on their structure and the general chemical characteristics of their side chains (R groups).

[0461] Aliphatic: isoleucine (I), leucine (L), glycine (G), alanine (A), valine (V), Hydroxyl or sulfur / selenium containing: serine (S), cysteine (C), threonine (T), methionine (M), Cyclic: proline (P), Aromatic: phenylalanine (F), tyrosine (Y), tryptophan (W), Basic: histidine (H), lysine (K), arginine (R), Acids and their amides: aspartate (D), glutamate (E), asparagine (N), glutamine (Q).

[0462] In addition, each of the following groups contains other exemplary amino acids that are conservative substitutions for one another. 1) Very small: alanine (A), glycine (G), 2) Negative charge: aspartic acid (D), glutamic acid (E), 3) Polar (amidated carboxyl side chain): asparagine (N), glutamine (Q), 4) Positive charge: arginine (R), lysine (K), 6) Aromatic: phenylalanine (F), tyrosine (Y), tryptophan (W), and occasionally histidine (H); 7) Small polarity: serine (S), threonine (T), 8) Sulfur-containing: cysteine (C), methionine (M) 9) Small: Ala (A), Glycine (G), Serine (S). 10) beta-branched: valine (V), isoleucine (I), and optionally threonine (T); 11) Polar: Asparagine (N), Glutamine (Q), Serine (S), Threonine (T).

[0463] Nevertheless, there are many clusters of amino acids that result in numerous amino acid indexes, each emphasizing a different aspect of the amino acid's characteristics. For example, hundreds of such indexes can be found in the aa index database https: / / www.genome.jp / aaindex / . As a result, some of the conservative substitutions may actually represent other characteristics that are important for protein suitability for industrial use in the food and beverage industry, such as non-specific binding to the tongue or other aspects of the sensory profile.

[0464] In addition, additional conservation analyses are based on the following. the non-polar "hydrophobic" amino acids are selected from the group consisting of valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tryptophan (W), cysteine (C), alanine (A), tyrosine (Y), histidine (H), threonine (T), serine (S), proline (P), glycine (G), arginine (R), and lysine (K); - the "polar" amino acids are selected from the group consisting of arginine (R), lysine (K), aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q); - the "positively charged" amino acid is selected from the group consisting of arginine (R), lysine (K), and histidine (H); - "Acidic" amino acids are selected from the group consisting of aspartic acid (D), asparagine (N), glutamic acid (E), and glutamine (Q).

[0465] In some embodiments, the replacement is a radical replacement. A radical replacement (substitution) is the exchange of an amino acid for another amino acid with different properties.

[0466] The degree of sequence similarity and / or sequence identity between a reference protein and a modified protein can generally affect the properties of the modified protein. For example, multiple substitutions can affect binding kinetics, folding kinetics, solubility, thermal stability, halostability, pH stability, shelf life, binding to non-aqueous particles (e.g., proteins or fats in food matrices, or hydrophobic regions in the oral cavity), 3D structure, and its activity and related properties. The computational methods developed and applied herein provide a thorough understanding of predicted amino acid residues for substitution that will result in improved modified proteins.

[0467] The variant proteins can be used as flavor modifiers or flavor enhancers.

[0468] The proteins described herein are for use as oral products. In some embodiments, the product is a food product, a dietary supplement, or a pharmaceutical. To prepare 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 or dry form, including, but not limited to, a fine powder, a lyophilized product, a granule, a tablet, or the like. In some embodiments, the composition is provided in a liquid form, for example, as a solute in water (aqueous solution).

[0469] Products comprising the proteins may have a variety of uses, including, but not limited to, as sweeteners, flavoring agents, enhancers, or masking agents in the food and beverage industry (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 naturopathy industry, the functional food industry, and other health care products (e.g., toothpaste and mouthwash), the candy and gum industry, 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).

[0470] The product may include an additional food ingredient. In some embodiments, the food ingredient is a sweetener, such as stevia. As shown in the examples below, the combination of an engineered protein described herein with stevia produced a synergistic effect. Thus, in some embodiments, the product includes at least one engineered protein described herein and stevia.

[0471] It should be noted that engineered proteins according to the present invention can be produced by any method known in the art, for example, proteins can be produced synthetically, by recombinant DNA technology, or by protein production in microorganisms via fermenters, or plants, or plant calli, or other bioreactors. In some embodiments, engineered proteins can be produced in bacteria, such as E. coli. In some other embodiments, engineered proteins can be produced in production yeasts, such as Saccharomyces cerevisiae or Pichia pastoris. In some embodiments, the DNA sequence of a selected amino acid sequence is optimized at the RNA and DNA levels. At the RNA level, this includes minimizing RNA secondary structure to ensure rapid insertion into the ribosome. At the DNA level, this includes codon optimization for the host organism (taking into account RNA-level optimization). Codon usage optimization prioritizes the use of the most abundant tRNA in the host organism for each expressed amino acid.

[0472] As used herein, the term "about" 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, and the deviation range includes integer values, and where applicable, non-integer values also constitute a continuous range. As used herein, the term "about" refers to ±10%.

[0473] As described herein, the variant protein has at least improved stability compared to the reference protein. As used herein, the term "improved" in reference to the stability or other characteristic of a variant protein refers to an increase or increase in stability of about 1% to 100%, particularly 5% to 100%, more particularly about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85%, about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, 100% or more in the indicated parameter compared to the stability of a reference protein.

[0474] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are intended to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0475] As used herein, the term "about" 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, and the range of deviation includes integer values, and where applicable, non-integer values also constitute a continuous range. Thus, as used herein, the term "about" refers to ±10%.

[0476] It should be noted that various embodiments of the present invention may be presented in a range format. The description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as 1 to 6 or 1-6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6.

[0477] As used herein, the forms "a," "an," and "the" include the singular and plural unless the context clearly dictates otherwise.

[0478] It is understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination, or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0479] It should be noted that various embodiments and examples detailed herein in connection with various aspects of the present invention may be applicable to one or more of the aspects disclosed herein. It should be further noted that any embodiment described herein, e.g., method-related embodiments, may be applied separately or in various combinations. The various embodiments and aspects of the present invention as described above and claimed below find experimental support 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 present invention may be combined (either from the same aspect or from different aspects) without departing from the scope of the present invention.

[0480] 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.

[0481] Although disclosed and described, it is to be understood that the invention is not limited to the particular examples, process steps, and reactors disclosed herein, as such process steps and reactors may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

[0482] The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be understood that while these techniques are exemplary of preferred embodiments for carrying out the invention, those skilled in the art will recognize that, in light of this disclosure, many modifications may be made without departing from the spirit and intended scope of the invention. [Example]

[0483] Non-limiting examples Example 1: Design of MNEI-based proteins MNEI-based proteins were designed as follows: Single-chain monellin, MNEI (SEQ ID NO: 1), is a 96 amino acid polypeptide with a molecular weight of approximately 11 kD and a pI of approximately 8.7.

[0484] method ANM (Anisotropic Network Model) ANM is a computational method used to study protein dynamics and motion. It assumes that proteins can be approximated as networks of interacting atoms or residues.

[0485] ANM considers interactions between neighboring atoms in a protein and calculates force constants between them. By representing a protein as a network of nodes (representing atoms) connected by edges (representing interactions), ANM analyzes the vibrational modes and collective motions of the protein structure.

[0486] This analysis represents the structure as a network of nodes and springs. ANM dynamics is based exclusively on an N × N harmonic potential with uniform force constant γ for all interacting residues, and the inter-residue contact topology represented by a 3N × 3N Hessian H of the second derivative of the potential for N nodes / residues. A mode spectrum of N-1 (or 3N-6) non-zero modes is obtained by eigenvalue decomposition of H. The modes are rank-ordered by increasing frequency, with mode 1 being the slowest and softest (most easily accessible) mode. Soft modes are highly cooperative. The general inverse of H is scaled by the cross-correlations between residue fluctuations, organized into an N × N (or 3N × 3N) covariance matrix (Hongchun Li et al., 2017, Nucleic Acids Research 45(W1), W374-W380).

[0487] molecular dynamics Molecular dynamics (MD) system setup and simulation. For all simulated proteins, residue protonation states were appropriately set at pH 7.0 using PROPKA (Olsson et al., 2011, Journal of Chemical Theory and Computation, 7(22), 525-537) version 3.4. All systems were placed in a dodecahedral box with TIP4P water (Jorgensen et al., 1983, Chem. Phys., 79, 926-935), with a minimum distance of 1.5 nm from the protein to the box wall. Each system was neutralized with randomly placed sodium or chloride counterions with a concentration of 150 mM NaCl. MD simulations were performed using GROMACS software (Abraham et al., 2015, SoftwareX, 1-2, 19-25) version 20221. The system was parameterized using the Amber99SB all-atom force field (Hornak et al., 2006, Proteins, 65, 712-725).

[0488] The system was relaxed with 50,000 steps of steepest descent energy minimization and subsequently equilibrated using the NVT ensemble. In the equilibration, the initial velocities of the atoms were randomly distributed according to the Maxwell-Boltzmann distribution at 300 K. The initial velocity was 1,000 kJ / mol. -1 nm -2 Harmonic positional restraints were applied to the protein atoms, and the temperature was steadily increased to 300 K over 1000 ps steps using V-Rescale (a modified Berendsen thermostat), then increased to 433 K over 1000 ps. The pressure was then equilibrated to 1 atm using the Parrinello-Rahman method (Parrinello and Rahman, 1981, J. Appl. Phys., 52, 7182-7190), and the restraints were steadily removed over five steps of 1000 ps each. The production simulations were performed in the NPT ensemble without positional restraints, using an integration time step of 2 fs and saving snapshots every 10 ps for analysis. Independent replicates of each system were run for 50 ns each. MD simulations were performed using periodic boundary conditions, and long-range interactions were calculated using PME (T. Darden, D. York, L. Pedersen, Particle mesh Ewald: An N log(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089-10092 (1993)) with a radius cutoff of 1.5 nm.

[0489] The MD analysis included the following components: The average native contacts were calculated using the Best-Hummer ratio of the native contact function (Best et al., 2013, PNAS, 110(44), 17874-17879) 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 protein's native-state tertiary structure. These contacts are used in molecular dynamics to measure deviations. 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 percentage of native contacts was averaged over the simulation time course. More contacts between heavy atoms of a protein imply higher stability during the simulation.

[0490] 1. The average helix H-bonds during the simulation were calculated using the GROMACS hbond utility, which uses intra-chain hydrogen bonds to detect unfolded secondary structures. Helix residues are defined as residues 10–26.

[0491] 2. Average beta-sheet H-bonds during the simulation were calculated using the GROMACS hbond utility, which uses intra-chain hydrogen bonds. Beta-sheet residues are defined as residues 2–6, 35–37, 42–48, 54–66, 69–78, and 82–90, numbered according to the sequence and structure of MNEI(2O9U).

[0492] 3. Water-Beta Sheet Backbone H-bonds - measures the average number of water molecules around the beta sheet of a protein that are stable enough to be able to connect the protein backbone in the segment with the beta strand secondary structure.

[0493] 4. Canonical Helix Fraction: This is the fraction of H-bonds in the helix that are of type i:i+4. There are three types of helix H-bonds, i:i+3, i:i+4, and i:i+5, which represent 3-10 helices, alpha helices, and pi helices, respectively. Alpha helices with i:i+4 bonds are the most canonical. This value indicates that alpha helices are maintained during the simulation. The higher the fraction of canonical H-bonds, the more stable the helix is expected to be.

[0494] 5. Total root mean square deviations (RMSD) 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-sheet (residues 2-6, 35-37, 42-48, 54-66, 69-78, 82-90). The average of the RMSDs was calculated using the GROMACS rms utility. The averages were then summed to obtain a single measurement. Residues are numbered according to the sequence and structure of MNEI (SEQ ID NO: 8, PDBID: 2O9U).

[0495] 6. Root mean square fluctuations (RMSF) of the protein backbone atoms throughout the MD simulation. These were calculated using the GROMACS rmsf utility and averaged over each residue.

[0496] 7. RMSF AUC (Area Under Curve) difference (ΔAUC) was calculated using the average RMSF of three replicates of MD simulations for each DM as ΔAUC = (AUC(variant) - AUC(reference), where reference is DM31 or MNEI).

[0497] Molecular graphics: Structural analysis, measurements, figures, and video clips were performed using 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 videos were rendered using either VMD or PyMOL (The PyMOL Molecular Graphics System, version 2.5.2, Schrodinger, LLC.).

[0498] Structure prediction and energy calculation New variants of MNEI were modeled using the DM31 and / or MNEI crystal structure templates (DM31 was determined by the Weizmann Institute's Crystallization Unit, and MNEI was based on the Protein Data Bank (PDB) structure ID 2O9U). The resulting structures were submitted to Rosetta software for further energy minimization and energy score calculation using 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 using 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, and multiple structures were then obtained along with their Rosetta Energy Unit (REU) scores. For each variant, the 30K scores were sorted, and the 500 lowest-scoring structures were used for further testing and analysis. For MNEI and DM31, the input structures were the corresponding crystal structures (2O9U for MNEI), and the protocol was repeated 100K times. Table 2 presents the new variants selected for laboratory expression along with their corresponding Rosetta Energy Unit (REU) scores. For some analyses, the lowest-energy structure of each new variant was used.

[0499] Additional protein features Hydrogen bonds were visualized and analyzed using PyMol. Additionally, hydrogen bonds were examined using the Baker-Hubbard method (Baker and Hubbard, 1984, Progress in Biophysics and Molecular Biology, 44.2, 97-179) implemented in the MDTraj Python package (McGibbon et al., 2015, Biophys J., 109(8), 1528-1532) version 1.9.6. An additional measure of hydrophobicity—the SAP (Spatial Aggregation Propensity) score calculated using Rosetta (Lauer et al., 2012, J Pharm Sci, 101(1), 102-115)—was used. This score measures the local hydrophobicity of a surface patch. Such surface areas could potentially form hydrophobic interactions with other patches on other proteins, thus increasing the risk of aggregation.

[0500] To estimate protein structural packing, we used an additional measure—VoroMQA (Olechnovic & Venclovas, 2017, Proteins, 85, 1131–1145). This method combines statistical potentials with the use of interatomic contact areas instead of distances. Contact areas, derived using a Voronoi tessellation of the protein structure, are used to account for and integrate both explicit interactions between protein atoms and implicit interactions between protein atoms and the solvent. VoroMQA generates scores at the atom level, residue level, and global level, all within a fixed range from 0 to 1.

[0501] result Table 1, presented above, provides examples of proteins designed using the methods described herein. Table 1 also includes the sequence of MNEI (a known protein).

[0502] Table 2 presents the calculated Rosetta Energy Unit (REU) scores for MNEI, variant y. For both minimization and calculation, Rosetta's REF2015 energy function was used. The table also presents the SAP score, which refers to hydrophobicity and indicates aggregation tendency.

[0503] The average RMSD was calculated using the protein's backbone atoms. The average H-bonds in the helices and beta-sheet backbones were calculated in GROMACS (see Methods, Molecular Dynamics) colored according to a green-yellow-red gradient (green indicates a higher number of H-bonds). The average RMSD of specific secondary structure elements (helices, La2, and L23) was calculated using the protein's C-alpha atoms and colored according to a green-yellow-red gradient (green indicates a lower RMSD).

[0504] [Table 2-1]

[0505] [Table 2-2]

[0506] [Table 2-3]

[0507] [Table 2-4]

[0508] [Table 2-5]

[0509] [Table 2-6]

[0510]

Table 2-7

[0511]

Table 2-8

[0512]

Table 2-9

[0513]

Table 2-10

[0514]

Table 2-11

[0515]

Table 2-12

[0516]

Table 2-13

[0517]

Table 2-14

[0518]

Table 2-15

[0519]

Table 2-16

[0520] In terms of Rosetta energy scores for the MNEI-based variants, DM212 (A19V, S76Y) has a score of -304.306 REU. DM217 (I75L, S76Y) has a score of -308.118 REU. DM299 (N35T, S76Y) has a score of -312.195 REU. DM276 (A19V, S76W) has a score of -307.017 REU. DM256 (A19V, S76F) has a score of -307.977 REU. DM220 (C41A, S76Y) has a score of -313.722 REU. DM281 (I75L, S76W) has a score of -308.084 REU. DM129 (S76Y) has a score of -313.148 REU. DM297 (C41T, S76Y) has a score of -312.022 REU. DM221 (C41S, S76Y) has a score of -312.548 REU. DM227 (D68N, S76Y) has a score of -314.09 REU. DM219 (A73F, F89M, S76Y) has a score of -315.41 REU. DM213 (V64I, S76Y) has a score of -313.15 REU. DM183 (S76W, I26T) has a score of -310.896 REU. DM284 (C41A, S76W) has a score of -312.41 REU. DM283 (A73F, F89M, S76W) has a score of -312.802 REU. DM225 (T33R, S76Y) has a score of -314.501 REU. DM307 (N35T, S76F) has a score of -312.43 REU. DM216 (V20I, S76Y) has a score of -316.389 REU. DM182 (S76F, K25R) has a score of -313.12 REU. DM303 (N35T, S76W) has a score of -312.963 REU. DM237 (T33R, S76W) has a score of -311.444 REU. DM263 (A73F, F89M, S76F) has a score of -315.016 REU. DM244 (D68N, R84L, S76W) has a score of -316.843 REU. DM214 (A73V, S76Y) has a score of -316.179 REU.DM201 (I26W, Q28K, S76Y) has a score of -319.166 REU. DM261 (I75L, S76F) has a score of -311.468 REU. DM180 (Q28K, I26T, S76Y) has a score of -315.492 REU. DM260 (V20I, S76F) has a score of -314.026 REU. DM243 (D68N, R84L, S76Y) has a score of -317.997 REU. DM285 (C41S, S76W) has a score of -311.278 REU. DM257 (V64I, S76F) has a score of -313.764 REU. DM280 (V20I, S76W) has a score of -313.281 REU. DM239 (D68N, S76W) has a score of -313.781 REU. DM200 (I26W, Q28E, S76Y) has a score of -319.278 REU. DM198 (I26W, Q28K, S76W) has a score of -316.895 REU. DM181 (S76F, I26T) has a score of -312.985 REU. DM265 (C41S, S76F) has a score of -312.498 REU. DM187 (K25R, Q28R, S76Y) has a score of -313.041 REU. DM302 (R84L, S76W) has a score of -317.531 REU. DM258 (A73V, S76F) has a score of -317.64 REU. DM233 (D68N, S76F) has a score of -314.319 REU. DM264 (C41A, S76F) has a score of -315.808 REU. DM147 (S76F) has a score of -314.468 REU. DM282 (F89M, S76W) has a score of -312.956 REU. DM231 (T33R, S76F) has a score of -313.647 REU. DM218 (F89M, S76Y) has a score of -317.154 REU.

[0521] In terms of Rosetta energy scores for DM31-based variants, DM202 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, S76Y) has a score of -326.104 REU, DM207 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I75L, S76Y) has a score of -325.596 REU, and DM289 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, N35T, S76Y) has a score of -327.695 REU. DM266 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, S76W) has a score of -322.122 REU. DM246 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, S76F) has a score of -322.969 REU. DM210 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41A, S76Y) has a score of -328.185 REU. DM271 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I75L, S76W) has a score of -322.428 REU. DM287 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T, S76Y) has a score of -325.849 REU. DM211 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41S, S76Y) has a score of -326.053 REU. DM224 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76Y) has a score of -327.546 REU. DM203 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V64I, S76Y) has a score of -326.036 REU. DM161 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76W, I26T) has a score of -323.585 REU. DM274 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41A, S76W) has a score of −324.868 REU.DM273 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A73F, F89M, S76W) has a score of -324.977 REU. DM222 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, S76Y) has a score of -326.421 REU. DM295 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, N35T, S76F) has a score of -324.151 REU. DM206 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, S76Y) has a score of -327.962 REU. DM160 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76F, K25R) has a score of -324.536 REU. DM292 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, N35T, S76W) has a score of -324.361 REU. DM234 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, S76W) has a score of -322.785 REU. DM241 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76W) has a score of -327.95 REU. DM204 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A73V, S76Y) has a score of -327.016 REU. DM128 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28K, S76Y) has a score of -329.989 REU. DM251 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I75L, S76F) has a score of -322.262 REU. DM158 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K, I26T, S76Y) has a score of -326.098 REU. DM250 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, S76F) has a score of −324.555 REU.DM240 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76Y) has a score of -328.51 REU. DM275 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41S, S76W) has a score of -321.76 REU. DM247 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V64I, S76F) has a score of -324.213 REU. DM270 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, S76W) has a score of -323.622 REU. DM236 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76W) has a score of -324.065 REU. DM127 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28E, S76Y) has a score of -329.421 REU. DM178 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28K, S76W) has a score of -326.997 REU. DM159 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76F, I26T) has a score of -323.01 REU. DM255 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41S, S76F) has a score of -322.513 REU. DM165 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, Q28R, S76Y) has a score of -322.968 REU. DM174 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R84L, S76W) has a score of -327.282 REU. DM248 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A73V, S76F) has a score of -327.375 REU. DM230 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76F) has a score of −324.008 REU.DM254 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41A, S76F) has a score of -325.464 REU. DM272 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, F89M, S76W) has a score of -322.55 REU. DM228 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, S76F) has a score of -323.104 REU. DM208 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, F89M, S76Y) has a score of -326.518 REU. DM679 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, D68N, S76F) has a score of -324.319 REU. DM687 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T, Q28K, S76F) has a score of -323.70 REU. DM688 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T, Q28R, S76W) has a score of -320.87 REU. DM689 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T, Q28R, S76F) has a score of -322.01 REU.

[0522] Example 2: Cloning, expression, and characterization of MNEI designer proteins Recombinant MNEI protein was produced in T7-promoted E. coli BL21 (DE3+) 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 engineered protein with up to 11% amino acid substitutions.

[0523] All DMs were produced in E. coli culture and purified to levels greater than 90%.

[0524] Cloning Site-directed mutagenesis (SDM) was used to generate DM.

[0525] culture All DM clones were produced by bacterial fermentation in flasks with horizontal steering and incubated at 37° C. Production of the protein variants was induced by IPTG.

[0526] purification All DM samples are purified using the following steps. 1. Dissolution using a pressure homogenizer. 2. Capture of proteins on a multimode resin and elution with increasing NaCl concentrations in the same buffer.

[0527] 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 (0.2 um) and storage at 2-8°C.

[0528] Characterization Refinement Level The level of purity is assessed by SDS-page followed by Coomassie blue staining and RP-HPLC.

[0529] Sweetness Evaluation Amai Proteins uses a routine, biweekly expert panel for sensory evaluation. The sensory panel includes panelists calibrated with sugar solutions on a 0-100 scale (magnitude estimation), with 0 = not sweet at all and 100 = very sweet. A linear scale of sucrose is obtained at concentrations of 2°Bx, 4°Bx, 6°Bx, and 8°Bx.

[0530] Brix (Bx) = gr / 100 ml. After calibration, tasters graded the test samples on the same scale according to a validated tasting protocol.

[0531] The newly selected DMs were tested in aqueous solution at a potency of 6°Bx equivalent of X3500.

[0532] All test products were provided using a code number.

[0533] Before and between samples, tasters were asked to rinse their mouths with mineral water, eat an unsalted cracker and a cucumber, and drink water again.

[0534] Sweetness after heat treatment The sweetness intensity of the new DM is measured after heat treatment at 95°C for 30 minutes.

[0535] The new DM was tested in citrate buffer (pH ∼3.1) at a potency of 5°Bx equivalent X5000.

[0536] The citrate buffer was heated to 95°C in a Thermomix, then DM was added to the Thermomix and the temperature was maintained at 95°C for 30 minutes. The heated buffer solution with DM was then poured into a bottle and immediately cooled. Sweetness intensity was measured at room temperature.

[0537] All test products were provided using a code number.

[0538] Before and between samples, tasters were asked to rinse their mouths with mineral water, eat an unsalted cracker and a cucumber, and drink water again.

[0539] Time-Intensity Time Intensity = measurement of sweetness intensity over time. 1. The newly selected DMs were tested in aqueous solution at a potency of 6°Bx equivalent X3500. 2. Panelists were initially calibrated using sugar solutions (in water) on a scale of 0 to 100, with 0 = not sweet at all. The calibration solutions used were 4°Bx sucrose (defined as 50 sweetness) and 8°Bx (defined as 100 sweetness grade). 3. After calibration, tasters performed a time-intensity evaluation for each sample according to the following steps: a. After the cue, all tasters placed the sample in their mouths, rolled it around in their mouths for 2 seconds, and then swallowed it. b. After swallowing, the guide measured the time and at each time point the taster was asked to rate the currently perceived sweetness intensity on the same scale (0-100) until the taste sensation disappeared. c. Times tested were 1, 2, 5, 7, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 135, 150, 180, 210, 240, 270, and 300 seconds. 4. All test products were provided using a code number. 5. Before and between samples, tasters were asked to rinse their mouths with mineral water, eat an unsalted cracker and a cucumber, and drink water again.

[0540] DSF and DSC analysis - thermal sensitivity Relative Tm was determined by differential scanning fluorimetry (DSF) using the Nanotemper Prometheus. DSF is a simple, rapid, and accurate method for analyzing protein stability and aggregation. DSF detects changes in the fluorescence of tryptophan and tyrosine residues in proteins. The fluorescence of tryptophan and tyrosine residues is strongly dependent on their immediate environment. Changes in protein conformation are reflected as fluorescence changes. The first derivative of the fluorescence ratio (330 / 350) is used to determine the inflection point. Because no secondary reporter fluorophore is required, protein solutions can be analyzed over a concentration range from 250 mg / ml to 10 μg / ml, regardless of buffer composition. DM was analyzed at a concentration of 0.5 mg / ml in 10 mM phosphate buffer, pH 7.

[0541] [Table 3-1]

[0542] [Table 3-2]

[0543] Delta Time / Max Intensity vs. DM31 is the ratio between delta time and maximum sweetness intensity of the DM. Delta time is the difference between the sweetness point equal to zero and the start of the experiment. Max Sweetness Intensity is the maximum sweetness value of the DM. For each new variant, the ratio delta time / max intensity of the variant is divided by the same ratio calculated for DM31 from the same sensory panel.

[0544] Table 4 shows the predicted and experimental Tm values of modified proteins with substitutions at amino acid S76.

[0545] [Table 4]

[0546] As can be seen, the predictions are in line with the experimental data. This suggested that the predicted Tm values can be used to predict the experimental Tm. As can be seen from this table, modifications at amino acid S76 on MNEI or comparted to DM31 increased the Tm value. Based on these results, it was suggested that various modifications at this amino acid are very effective in significantly increasing protein stability.

[0547] Combining substitution at S76 with loop remodeling: It was suggested that the L23 loop of MNEI is close to amino acid S76, and therefore, it was suggested that substitution at amino acid S76 may improve stability.

[0548] Several modified proteins with substitutions at S76 were prepared. Table 3 shows a list of modified proteins with substitutions at amino acid S76 and the experimental results for these proteins. Some variants were based on S76, which is specific to DM31.

[0549] Figure 1A shows DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) with the L23 loop colored magenta and S76Y colored turquoise. The proximity of the loop and S76 is clearly visible, indicating potential interactions and possible synergistic effects. The results show that adding the substitution at residue S76 to DM31 did indeed increase stability, as can be seen from the DSF results (Table 3). DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) has a Tm of 101°C and is as sweet as or slightly sweeter than DM31. In addition, it has a reduced lingering effect compared to DM31, as indicated by the fact that DM89 has an approximately 10% reduction in delta time / maximum sweetness intensity (Table 3). DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y, Tm 101°C), DM114 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76F, Tm 101.5°C), and DM137 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76W, Tm 97°C) are more stable than DM31. Their sweetness is comparable to that of DM31, but all three proteins exhibit reduced aftertaste (more than 25% reduction for DM114 and approximately 10% reduction for DM89 and DM137; see Table 3, Delta Time / Maximum Sweetness Intensity).

[0550] Amino acid R84: The minimized model of DM89 (Figure 1B) reveals that R84 is relatively close to S76. Therefore, modified proteins containing substitutions at R84 were analyzed.

[0551] DM90 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R84L, S76Y) has more native contacts, better REU scores, and lower RMSF AUC than DM70 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R8RL), DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y), and DM31. In addition, DM90 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R84L, S76Y) has a predicted Tm of 104°C, which is higher than DM70 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R84L), which has an experimental Tm of 94°C.

[0552] As can be seen from the table above, combinations of one or more substitutions provide increased stability, which is greater than each alone.

[0553] [Table 5-1]

[0554] [Table 5-2]

[0555] The following modified proteins were predicted to benefit from the combination of modifications: DM240, DM641, DM633, DM627, DM171, DM480, DM362, DM236, DM396, DM479, DM167, DM165, DM397, DM679, DM230, DM681, DM361, DM224, DM611, DM620, DM473, DM517, DM472, DM685, DM608 , DM628, DM521, DM542, DM477, DM640, DM532, DM528, DM621, DM530, DM360, DM73, DM614, DM 526, DM622, DM94, DM486, DM484, DM457, DM209, DM540, DM333, DM511, DM539, DM524, DM148.

[0556] Predicted Tm values are calculated relative to DM31.

[0557] All DMs below are based on MNEI (SEQ ID NO: 1) and have substitutions of E2N, Y65R, L70I, and E23A, and deletions at amino acids E50, F52, and R53, unless otherwise indicated.

[0558] DM635 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, R84L, S76Y): The predicted Tm of this DM is 106°C, higher than that of DM31. The overall VoroMQA packing score for this DM (0.51) is higher than that of DM31 (0.49) and DM89 (0.49), and the residue-specific packing score at position 76 (0.60) is much higher than that for DM31 (0.52) and DM89 (0.53).

[0559] DM286 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R31T, S76Y): A surprisingly high predicted Tm of 101°C compared to predicted Tm values of 95°C (DM47) and 98°C (DM89).

[0560] DM combining substitution at S76 with substitutions at D68, T33, G1M, G2M: Taking into account the computational analysis and predictions made with S76, the inventors designed additional proteins with additional substitutions that were suggested to have improved properties, such as improved stability.

[0561] We first examined the effect of substitutions at D68. D68N was previously shown to increase sweetness (Kohmura 1992), but its effect on stability was not described.

[0562] To predict the effect of substitutions in D68, the following modified proteins were designed.

[0563] Modification of DM31 with the substitution D68T (DM108) was shown to increase sweetness (Table 3).

[0564] As can be seen from Table 2, the Rosetta energy score for the substitution D68N+S76Y for MNEI (DM227) is 1.3 REU lower, while the same substitution for DM31 (DM224) improves the energy score by -5.9 REU. The REU score of DM224 suggested its high stability. This indicates that there is a synergistic effect when D68N+S76Y is combined with the substitution made in DM31.

[0565] These computational conclusions were experimentally verified, and as can be seen in Table 3, DM224 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76Y) has a higher sweetness and higher stability compared to DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) (Table 3).

[0566] DM361 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, D68N, S76Y) has a higher Tm (97°C) than DM224 (D68N, S76Y) (95°C) (Table 3), and its stability prediction is better (Table 2). The experimental Tm of DM361 is 97°C, which is 3 degrees higher than DM31. In addition, the Tm of the substitutions included in DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, D68N, Tm 90°C), DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y, Tm 101°C), and DM591 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I), Tm 91°C). Therefore, the combination of D68N, S76Y, and R65Y (reverse) is synergistic.

[0567] DM397 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y, D68N) with a predicted Tm of 99.06°C and a ΔTm of 7.74°C It exhibits synergistic effects because it has a better ΔTm than the sum of the ΔTms of the following DMs (3.4°C): DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) with a predicted Tm of 87.09°C, DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) with a predicted Tm of 98.19°C, DM142 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R) with a predicted Tm of 91.52°C, and DM591 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I) with a predicted Tm of 91.89°C.

[0568] DM362 (ΔE50, ΔF52, ΔR53, E23A, S76Y, D68N) has a predicted Tm of 100°C, which is higher than DM224 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76Y). It has a predicted Tm of 100.32°C and a ΔTm of 9.0°C, which is better than the sum of the ΔTms of the following DMs (5.44°C), indicating a synergistic effect. DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, D68N, predicted Tm of 87.09°C), DM122 (ΔE50ΔF52, ΔR53, E23A, Y65R, L70I, S76Y), predicted Tm of 100.23°C), DM142 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R), predicted Tm of 91.52°C), DM591 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I), predicted Tm of 91.89°C).

[0569] replacement DM627 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R31T, S76Y), with a predicted Tm of 100.65°C and a ΔTm of 9.33°C, exhibits a synergistic effect because it has a better ΔTm than the sum of the ΔTms of the following DMs (5.55°C): DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N), with a predicted Tm of 87.09°C, and DM286 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R31T, S76Y), with a predicted Tm of 101.1°C.

[0570] DM642 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68T, R31T, S76Y) has a predicted Tm of 101°C, while DM567 (D68T, R31T) has a predicted Tm of 94°C.

[0571] DM614 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, T33R, S76Y) has a predicted Tm of 94 °C, which is higher than those of DM31, DM511 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, T33R), DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N), and DM57 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R). Molecular dynamics analysis showed that it has better REU and more native contacts than DM31. This DM has a ΔTm of 2.6°C, which is better than the sum of the ΔTms of the following DMs (-0.77°C): DM57 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R) with a predicted Tm of 87.92°C, DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) with a predicted Tm of 87.09°C, and DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) with a predicted Tm of 98.19°C, demonstrating a synergistic effect.

[0572] DM542 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, Q28R, S76Y) is based on the sweeter DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R). DM542 has a higher predicted Tm than DM540 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, Q28R). With a predicted Tm of 96.7°C and a ΔTm of 5.38°C, this DM exhibits a synergistic effect because it has a better ΔTm than the sum of the ΔTms of the following DMs (-11.49°C). DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R) with a predicted Tm of 77.2°C, DM87 (D68N) with a predicted Tm of 87.09°C, and DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) with a predicted Tm of 98.19°C.

[0573] DM240 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76Y), with a predicted Tm of 104.03°C and a ΔTm of 12.71°C, exhibits a synergistic effect because it has a better ΔTm than the sum of the ΔTm of the following DMs (8.85°C). DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N), with a predicted Tm of 87.09°C, and DM90 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y, R84L), with a predicted Tm of 104.4°C. Additionally, there is a synergistic effect in the Rosetta energy scores. These combinations resulted in an improvement of 6.85 REU over DM31 and a smaller improvement of only 2.65 REU over MNEI, indicating that there is a synergistic effect between these substitutions and the modifications in DM31.

[0574] DM472 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, Q28K, S76Y) has a higher Tm than DM31 (100°C). DM472 is similar to DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y), DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N), and DM65 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K). DM479 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, D68N, S76Y) has a low RMSF, which also indicates high stability. DM479 has a Tm of 97, which is 3 degrees better than DM31. DM479 exhibits synergy, as it has a delta Tm that is 3 degrees better than the sum of the delta Tms of DM101 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, S76Y), DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N), or DM224 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76Y) and DM43 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R). DM240 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76Y) has a high predicted Tm, a 6.9 REU improvement over the improvement of DM243 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76Y) from MNEI (2.6 REU), indicating synergy of its combination with DM31, REU-329, and a better RMSF AUC.

[0575] DM241 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76W) has a lower REU than DM137 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76W), DM70 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R84L), DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N), and DM31. The Rosetta score of DM241 was -327.9 REU, a higher improvement of 6 REU from DM31 when compared to DM244 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76W against MNEI) which had an improvement of -1.50 against MNEI. These data indicated that the combination with DM31 acted synergistically.

[0576] The Rosetta energy score of these substitutions (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76W) in MNEI (DM236) was reduced by 1.6 REU compared to MNEI, whereas the same substitutions (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76W) in DM31 improved the energy score by -2.4 REU compared to DM31, indicating a synergistic effect when the D68N and S76W substitutions (in DM236) were combined with the DM31 modifications. This DM has a Tm of 99.75 and a ΔTm of 8.43, which is better than the sum of the ΔTms (5.21) of the following DMs: DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) with a Tm of 87.09, and DM137 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76W) with a Tm of 97.

[0577] The Rosetta energy score of these substitutions (DM233) for MNEI (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76F) was reduced by 1.0 REU compared to MNEI, whereas the same substitutions (D68N, S76F) for DM31 improved the energy score by -2.3 REU. This indicates a synergistic effect when the DM230 substitutions (D68N, S76F) are combined with the DM31 modifications. This DM has a Tm of 98.95 and a ΔTm of 7.63, which is better than the sum of the ΔTms of the following DMs (3.83). DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) with a Tm of 87.09, and DM114 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76F) with a Tm of 99.39.

[0578] DM608 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68T, Q28R, S76Y) has a higher predicted Tm than DM108 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68T) and DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R). DM608 has a ΔTm of 5.74°C, which is better than the sum of the ΔTms of the following DMs (-7.05°C), demonstrating a synergistic effect. DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R) with a predicted Tm of 77.2°C, and DM649 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68T, S76Y) with a predicted Tm of 98.4°C.

[0579] DM644 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68T, R84L, S76Y) has a predicted Tm of 105. The RMSF AUC-0.56 is similar to that of DM31, and the packing (using VoroMQA) is very good at 76th place.

[0580] The combination of G1M and E2M is known to increase sweetness (Somoza et al., 1995). When these substitutions were combined with S76Y, DM360 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, G1M, E2M, S76Y) had lower beta-sheet RMSD and lower REU than DM413 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, G1M, S76Y). DM360 also exhibits synergistic effects, with a predicted Tm of 94.48°C and a ΔTm of 3.16°C, better than the combined ΔTm of the following DMs (1.09°C). DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) with a predicted Tm of 98.19°C, DM395 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E2M) with a predicted Tm of 90.63°C, and DM412 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, G1M) with a predicted Tm of 86.23°C.

[0581] DM396 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E2M, S76Y) contains E2M, which Zhao et al. (2018) cited as increasing sweetness. Its sweetness is three times higher than that of MNEI, but its Tm is 4°C lower than that of MNEI (see Zhao et al.), indicating a significant decrease in stability. Based on a predictive model, we predicted a 9°C increment for the combination of E2M and S76Y on the DM31 template. The predicted Tm for DM396 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E2M, S76Y) is 99.6°C. DM396 exhibits synergy because it has a better ΔTm than the sum of the ΔTms of the following DMs (6.18°C): DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) with a predicted Tm of 98.19°C, and DM395 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E2M) with a predicted Tm of 90.63°C.

[0582] DM222 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, S76Y) has a higher predicted Tm (98.19°C) than DM57 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R) (87.92°C). The RMSF is similar to DM57 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R) and DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y). The Rosetta energy score for these substitutions on MNEI (DM225) is 0.8 REU lower, while the same substitutions on DM31 improve the energy score by −4.8 REU, indicating a synergistic effect between the substitutions on DM222 and the modifications on DM31.

[0583] DM638 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, R84L, S76Y) has a predicted Tm of 101.2

[0584] The predicted Tm values for DM622 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, Q28R, S76Y) are higher than those for DM539 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, Q28R), DM57 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R), and DM65 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K). Both Q28R and T33R have been shown to increase sweetness (Table 3). With a predicted Tm of 93.72°C and a ΔTm of 2.4°C, this DM has a better ΔTm than the sum of the ΔTms of the following DMs (-10.65°C): DM57 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R) with a predicted Tm of 87.92°C, DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R) with a predicted Tm of 77.2°C, and DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) with a predicted Tm of 98.19°C.

[0585] DM242 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76F). The Tm of this DM was predicted to be 105°C, which is higher than DM70 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R84L), DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N), and DM114 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76F). DM242 has an REU of -328, a difference of 6.6 units from DM31, which is a higher improvement than DM245 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76F against MNEI, which improves by 3.8 REU), indicating that this combination has a synergistic effect against DM31.

[0586] DM628 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, N35T, S76Y), with a predicted Tm of 96.88°C and a ΔTm of 5.56°C, exhibits a synergistic effect because it has a better ΔTm than the sum of the ΔTms of the following DMs (3.04°C): DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N), with a Tm of 87.09°C, and DM289 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, N35T, S76Y), with a Tm of 98.6°C.

[0587] Figure 4 shows the results of the DM31 (Figure 4C), DM89 (Figure 4F) (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y), DM222 (Figure 4A) (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, S76Y), and DM224 (Figure 4D) ( The electrostatic potentials of DM479 (Figure 4B) (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, D68N, S76Y), DM479 (Figure 4B) (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, D68N, S76Y), and DM606 (Figure 4E) (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76R) are shown. Residues known to be important for sweet taste are labeled and presented as spheres. R88, R72, R39, K36, and D7 are known to be important for receptor binding and sweet taste (Somoza et al., 1995; Yang et al., 2020; Liu et al., 2012). Specific DM substitutions are labeled and presented as spheres. S76Y does not interfere with the positive electrostatic surfaces of R88, R72, and R39. T33R and D68N increase the positive surface imbalance and therefore have a positive effect on sweetness.

[0588] Core replacement combined with S76Y Additional modified proteins were designed in which the MNEI core region was substituted. The Rosetta energy score of these substitutions (DM257) relative to MNEI (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V64I, S76F) is 1.6 REU worse, while the same substitutions in DM31 improve the energy score by -2.6 REU, indicating a synergistic effect between the DM247 substitutions and the DM31 modifications.

[0589] DM202 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, S76Y) has a higher experimental Tm of 96 °C than DM31. The RMSF AUC of this variant also indicates stability. Additionally, the overall VoroMQA packing score of this variant (0.52) is better than those of DM31 (0.49) and DM89 (0.49). Notably, DM202 has a better REU than DM31, decreasing it by 4 points (-326.1 for DM202 vs. -321.66 for DM31), while the substitution A19V, S76Y on MNEI (DM212) increases the REU by 11 points (-304.31 for DM212 vs. -315 for MNEI). This suggests that A19V, S76Y have a synergistic effect with the DM31 substitution and deletion. DM276 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, S76W) and DM256 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, S76F) reduced REU by 8 points compared to MNEI. However, the same substitutions in DM31, i.e., DM266 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, S76W) and DM246 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, S76F), increase REU by 1 point. In conclusion, the interaction of residues 19 and 76 is synergistic with the other substitutions in DM31.

[0590] DM204 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A73V, S76Y). This DM has a predicted Tm of 93°C, indicating increased stability. The overall VoroMQA packing score (0.51) is higher than its predecessors DM31 (0.49), DM89 (0.49), and DM85 (0.5) (A73V for DM31). The Rosetta energy score (REU) of this variant is similar to that of DM89 and higher than DM31 and DM85. Its Rosetta score was -327 REU, a 5 REU improvement from DM31, which was greater than that of DM214 (A73V, S76Y for MNEI), which showed no improvement from MNEI, indicating that the combination with DM31 acts synergistically.

[0591] DM206 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, S76Y) shows an exemplary Tm of 96°C, higher than DM31. The Rosetta Energy Score (REU) of this variant is -6 REU better than that of DM31, and also better than DM89 and DM96 (V20I relative to DM31). The same substitutions in MNEI (DM216) improve the REU by -1, indicating a synergistic effect between the substitutions in DM206 and the modifications in DM31.

[0592] DM250 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, S76F) reduced REU by 3 points relative to DM31. However, DM260 (V20I, S76F relative to MNEI) increased REU by 1 point relative to MNEI, indicating a synergistic effect between the DM250 substitutions and the DM31 alterations.

[0593] DM209 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A73F, F89M, S76Y). Its experimental Tm is 96°C, higher than that of DM31. This combination of substitutions improves the REU by 8 points (-328.41 for DM209 vs. -321.66 for DM31). However, the same combination for MNEI (DM219) does not improve the REU (-315 for both MNEI and DM219). This means that A73F, F89M, S76Y are synergistic with the substitutions and deletions of DM31.

[0594] DM253 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A73F, F89M, S76F) improves -REU by 6 points (-326.13 for DM253 vs. -321.66 for DM31). However, the same combination for MNEI (DM263) does not improve REU (-315 for both MNEI and DM263). This means that A73F, F89M, S76F are synergistic with the substitutions in DM31. This DM has a ΔREU of -4.47, thus demonstrating a synergistic effect, as it has a better ΔREU than the sum of the ΔREUs of the following DMs that contain it (-0.86). DM131 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, A73F) has an REU of -320.83, and DM252 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, F89M, S76F) has an REU of -323.36. Additionally, DM253 has a better ΔREU than the sum of the ΔREUs of the following DMs (0.06), demonstrating a synergistic effect. DM104 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, F89M) with an REU of -319.99, DM114 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, S76F) with an REU of -324.1, and DM131 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, A73F) with an REU of -320.83.

[0595] DM273 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A73F, F89M, S76W) improves REU by 3.3 points, while the same combination for MNEI (DM283) increases REU by 2.5 points. This means that A73F, F89M, and S76W are synergistic with the DM31 substitutions. This DM has a ΔREU of -3.32, which is better than the sum of the ΔREUs of the following DMs (-0.06), demonstrating a synergistic effect. DM131 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, A73F) has an REU of -320.83, and DM272 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, F89M, S76W) has an REU of -322.55. Additionally, DM273 exhibits a synergistic effect, as it has a better ΔREU than the sum of the ΔREUs of the following DMs (0.21). DM104 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, F89M) with an REU of -319.99, DM131 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, A73F) with an REU of -320.83, and DM137 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, S76W) with an REU of -323.95.

[0596] DM248 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A73V, S76F). The predicted Tm of this DM is 95°C, indicating good stability. The Rosetta energy score (REU) for this DM is nearly identical to that of DM89 and is greater than the combined effect of the A73V (DM85) and S76F (DM114) substitutions alone. Its Rosetta score was -327 REU, a 5.7 REU improvement from DM31, which is greater than that of DM258 (A73V, S76F for MNEI), which had only a 2.3 REU improvement from MNEI, indicating that the combination with DM31 acts synergistically. The average native contacts for this DM are higher than those of DM31. The A73V substitution (DM85) has a higher overall VoroMQA packing score (0.5) than DM31 and DM89 (0.49), and this effect carries over to DM248 and is not seen in DM114 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76F), where residue-specific packing reductions are observed at positions 73 and 76. The phenylalanine substitution at position 76 is found in structural homologs with a similar fold to this stem and loop structure.

[0597] DM210 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41A, S76Y). The Tm of this DM is predicted to be 96 °C, higher than DM31. The Rosetta Energy score for this DM is much better than the Rosetta score of DM31 of -328, an REU improvement of 6.5 compared to DM31. The same combination (DM220) against MNEI resulted in a poorer Rosetta score of 1.6 REU from MNEI, indicating a synergistic effect of the combination with DM31.

[0598] DM208 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41A, S76F) reduces REU by 4 points compared to DM31, whereas DM264 (C41A, S76F to MNEI) has the same REU as MNEI, suggesting a synergistic effect of C41A, S76F with the DM31 substitutions and deletions.

[0599] DM274 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41A, S76W) reduces REU by 3 points compared to DM31. However, DM284 (C41A, S76W to MNEI) increases REU of MNEI by 2.9 points, suggesting a synergistic effect of C41A+S76W and the DM31 substitutions and deletions.

[0600] DM346 (ΔE5ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41S, S76Y). The Tm measured by DSF for this DM is 97 °C, higher than DM31 and DM151 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, C41S). The RMSF AUC for this DM is better than DM31, DM89, and DM151. The average native contacts are also better than DM31. The Rosetta score is better than DM31 and DM151. The residue-specific VoroMQA packing score is better at position 76 (0.54) compared to DM31 (0.52) and DM89 (0.53). DM221 (C41S, S76Y to MNEI) increased the REU of MNEI by 2.8 points, whereas DM346 decreased the REU by 4.3 points, suggesting a synergistic effect between the C41S, S76Y, and DM31 substitutions and deletions.

[0601] DM287 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T, S76Y). The predicted Tm of this DM is 96 °C, higher than that of DM31. The VoroMQA global packing score (0.5) of this DM is better than that of DM31 (0.49), DM89 (0.49), and DM46 (C41T for DM31), which have identical scores. Surprisingly, the RMSF AUC of this DM is significantly worse than that of DM31, DM89, and DM46, while the values of DM89 and DM46 are similar and better than that of DM31. Despite this predicted instability, the Tm is higher than that of DM31. DM287 lowers the REU of DM31 by 4 points. However, the same substitution for MNEI (DM297) increases the REU by 3 points. This suggests a synergistic effect between C41T, S76Y and the DM31 substitution and deletion.

[0602] DM477 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, C41T, D68N, S76Y). The predicted Tm of this DM is 97°C, higher than that of DM31, with a ΔTm of 5.3°C, which is better than the sum of the ΔTm of the following DMs (1.38°C), indicating a synergistic effect. DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, D68N) with a predicted Tm of 87.09°C, DM287 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, C41T, S76Y) with a predicted Tm of 96.37°C, and DM591 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I) with a predicted Tm of 91.89°C.

[0603] DM203 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V64I, S76Y). The Rosetta energy score of these substitutions relative to MNEI (DM213) is 2.2 REU worse, but the same substitutions relative to DM31 improve the energy score by -4.4 REU. This indicates a synergistic effect between the DM203 and DM31 substitutions.

[0604] DM207 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, I75L, S76Y) has an REU of −325.6, a 3-point improvement over DM31. However, DM217 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, I75L, S76Y vs. MNEI) has an REU of −308.12, which increases the REU of MNEI (−315) by 11 points. This suggests a synergistic effect between the I75L, S76Y, and DM31 substitutions and deletions.

[0605] DM595 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I75V, S76Y, E77V). The predicted Tm of this DM is 102 °C, higher than that of all DMs containing it: DM31, DM89, DM593, DM594, DM600, and DM601. The average β-sheet RMSD is also lower than that of the DMs containing it, indicating that the β-sheet is stabilized by these substitutions.

[0606] DM611 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, D68N, S76Y). The predicted Tm of this DM is 98°C, higher than that of DM31. DM611 has a ΔTm of 6.78°C, which is better than the sum of the ΔTms of the following DMs (4.08°C), demonstrating a synergistic effect: DM87 (D68N) with a predicted Tm of 87.09°C, DM89 (S76Y) with a predicted Tm of 98.19°C, and DM96 (V20I) with a predicted Tm of 92.76°C.

[0607] DM679 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, D68N, S76F), with a predicted Tm of 98.98°C and a ΔTm of 7.66°C, exhibits a synergistic effect because it has a better ΔTm than the sum of the ΔTms of the following DMs (5.28°C): DM87 (D68N), with a predicted Tm of 87.09°C; DM96 (V20I), with a predicted Tm of 92.76°C; and DM114 (S76F), with a predicted Tm of 99.39°C.

[0608] DM620 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, Q28R, S76Y). The predicted Tm of this DM is 98°C, higher than that of DM31. Both V20I and Q28R have been shown to increase sweetness (Table 3). This DM has a ΔTm of 6.58°C, which is better than the sum of the ΔTms of the following DMs (-5.81°C), demonstrating a synergistic effect: DM66 (Q28R) with a predicted Tm of 77.2°C, DM89 (S76Y) with a predicted Tm of 98.19°C, and DM96 (V20I) with a predicted Tm of 92.76°C.

[0609] DM681 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, Q28R, S76F), with a predicted Tm of 98.64°C and a ΔTm of 7.32°C, exhibits a synergistic effect because it has a better ΔTm than the sum of the ΔTms of the following DMs (-4.61°C): DM66 (Q28R), with a predicted Tm of 77.2°C; DM96 (V20I), with a predicted Tm of 92.76°C; and DM114 (S76F), with a predicted Tm of 99.39°C.

[0610] DM621 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, Q28R, S76Y). The predicted Tm of this DM is 95°C, higher than that of DM31. Both A19V and Q28R have been shown to increase sweetness (Table 3). DM621 has a ΔTm of 3.93°C, which is better than the sum of the ΔTms of the following DMs (-9.92°C), demonstrating a synergistic effect: DM66 (Q28R) with a predicted Tm of 77.2°C, and DM202 (A19V, S76Y) with a predicted Tm of 95.52°C.

[0611] DM289 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, N35T, S76Y) has a predicted Tm of 98°C. The RMSF_AUC is better than DM31. DM289 also improves REU by 6 points. However, DM299 (N35T, S76Y to MNEI) increases REU by 3 points, suggesting a synergistic effect between N35T, S76Y and the DM31 substitutions and deletions.

[0612] DM292 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, N35T, S76W). This combination of substitutions for DM31 improves the Rosetta energy score by 2.7 REU, while these substitutions for MNEI result in a Rosetta score that is 2.38 REU worse. These results indicate a synergistic effect between these substitutions and the DM31 modifications on MNEI.

[0613] DM626 (ΔE50, ΔF52, (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, R84L, S76Y). The predicted Tm of this DM is 101 °C, higher than that of DM31 and equal to that of DM89. The Rosetta energy score of this DM is better than that of DM31 (approximately -4 ΔREU).

[0614] DM633 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, ((ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, R31T, S76Y). The predicted Tm of this DM is 102°C, higher than DM31 and DM89 and all of their precursors (DM96, DM47, DM286, DM206). With a ΔTm of 10.36°C, DM633 is better than the sum of the ΔTm of the following DMs (8.13°C). These DMs have favorable ΔTm values, thus demonstrating synergistic effects. DM89(S76Y) has a predicted Tm of 98.19°C, and DM555 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, R31T) has a predicted Tm of 92.58°C. The Rosetta energy score for this DM was higher than DM31 and DM89. The average native contacts of this DM are better than all of its predecessors except DM89.

[0615] DM600 (ΔE50, ΔF52, ((ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I75V, S76Y)) has a predicted Tm (99.6°C) that is surprisingly and synergistically greater than the sum of the substitutions that contain it (DM601 and DM89). The RMSF AUC of this DM is better than DM31. The I75V substitution correlates with S76Y compared to spatially similar stem and loop structures found in other proteins. DM600 also has a better overall VoroMQA packing score (0.5) than its predecessors DM31 (0.49), DM89 (0.49), and DM601.

[0616] DM208 (ΔE50, ΔF52, ((ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, F89M, S76Y) had a Rosetta score of -326 REU, a 4.8 REU improvement from DM31, compared to DM218 (F89M, S76Y to MNEI) which only had a 1.8 REU improvement from MNEI, indicating that the combination with DM31 acts synergistically.

[0617] Helix Related S76Y was also combined with two helix-capping substitutions, DM100 and DM101. DM100 was highly stable, with an experimental Tm of 100°C, 6°C higher than DM31. DM101 was highly stable, with an experimental Tm of 96°C, 2°C higher than DM31. These DMs with DM31 changes, including loop remodeling, S76Y, and helix-capping substitutions, were suggested to have the following synergistic effects: Beta is stabilized by the stiffening of the loop Beta is water protected by S76Y Helices are stabilized with helix-capping substitutions · Native contacts of DM100 and DM101 are higher than DM31 and DM89 (DM130 is not).

[0618] DM100 exhibits a higher number of backbone H-bonds that are intra-beta sheet and intra-helical than MNEI, DM31, and DM130. These H-bonds maintain the secondary structure of these segments during the simulation. The proportion of canonical helices in DM100 is also higher in DM100 compared to these references.

[0619] Combining beta sheet stability with alpha helices for synergistic stability increases: ANM analysis (Figure 3) indicates that the overall movement of MNEI is primarily driven by two flexible regions in the protein: the β-hairpin composed of β-strands 2 and 3 and β-strands 4 and 5, which drive the movement of the β-sheet; the 2-helix; and its C-terminal loop. Joint stiffening of these two key regions is predicted to result in a synergistic increase in stability. This is achieved by combining the S76Y modification with modifications at the C-terminus of the helix: Q28K, Q28R, Q28S, and K25R.

[0620] Q28K: This substitution was first discovered by Leone et al. (Leone 2016) to increase sweetness. Q28K was found to strengthen the helix terminus through improved packing in this region (ref PCT march). We investigated the effect of DM363 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I) on the sweetness of the DM363 mutant. 、 Combining these two substitutions in DM89(Q28K, S76Y), MD simulations show that it is better at maintaining native contacts and has an RMSD that is somewhat better than that of DM89(S76Y) (Table 2, partial RMSDs and ). This supports the hypothesis that the beta sheets and helices are related.

[0621] DM632 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K, R84L, S76Y) has a predicted Tm of 104.5, higher than DM31.

[0622] DM335 (ΔE50, ΔF52, ΔR53, E23A, Q28K, S76Y), a Q28K, S76Y relative to MNEI, also showed better average native contacts and significantly improved RMSD (Table 2).

[0623] DM630 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K, R31T, S76Y) vs. DM552 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K, R31T) added later.

[0624] DM158 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K, I26T, S76Y) has a predicted Tm of 93.98. Its Rosetta score was −326 REU, a 4.4 REU improvement from DM31, which was greater than DM180 (Q28K, I26T, S76Y on S76Y to MNEI), which showed no improvement from MNEI, indicating that the combination with DM31 acts synergistically.

[0625] DM486 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, I26T, Q28K, D68N, S76Y) has a predicted Tm of 93.48°C and a similar ΔTm to DM31, which has a ΔTm of 2.16°C, and has a better ΔTm than the sum of the ΔTms of the following DMs (-1.0°C), indicating a synergistic effect. DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) with a predicted Tm of 87.09°C, DM158 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K, I26T, S76Y) with a predicted Tm of 93.98°C, and DM591 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I) with a predicted Tm of 91.89°C.

[0626] DM456 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28S, I26T, S76Y). DM456 has better native contacts (Table 2). Its experimental Tm is 104 °C, 10 degrees higher than DM31. DM456 exhibits synergy because it has a better Tm delta than the sum of the deltas of DM100 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26T, S76Y) with a Tm of 100 and DM330 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28S) with a Tm of 94.1, DM31, and DM455 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K, I26V, S76Y), which has a better predicted Tm than DM31. DM456 also has a better proportion of canonical helices than DM89, DM31, and DM65.

[0627] DM473 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, D68N, Q28K, S76Y) has a predicted Tm of 97.75 and a ΔTm of 6.43°C, which is better than the sum of the ΔTms of the following DMs (2.85°C), indicating a synergistic effect. DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) with a predicted Tm of 87.09°C, DM363 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K, S76Y) with a predicted Tm of 97.83°C, and DM591 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I,) with a predicted Tm of 91.89°C.

[0628] Helix and core combinations to S76Y: Because the region connecting the beta sheets and alpha helices is the core of the protein, we added a core modification to the above combination to improve the interaction between these regions and thereby combine the effects of the two stabilizing modifications.

[0629] Residue C41 is located in the core of the protein, and a C41S substitution has previously been shown to increase sweetness (Liu 2015, Zhao 2019). C41A was predicted to increase sweetness without affecting stability (Tang 2020).

[0630] Q28R In March 2023, we proposed Q28R, following the activation mode of Q28K, which showed a stronger positive effect on packing.

[0631] DM517 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R, S76Y). Its experimental Tm is 100°C, 6 degrees higher than DM31. Its sweetness intensity is similar to DM31. Its sweetness intensity after heat treatment is similar to DM31. MD simulations show better H-bonding of the beta sheet and better protection of H-bonds from water (Table 2).

[0632] DM641 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R, R84L, S76Y) has a predicted Tm of 103.8°C, which is better than the Tm of DM31. DM641 has a ΔTm of 12.52°C, which is better than the sum of the ΔTms of the following DMs (-1.04°C), demonstrating synergy: DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R) with a predicted Tm of 77.2°C, and DM90 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y, R84L) with a predicted Tm of 104.4°C.

[0633] DM640 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R, N35T, S76Y), with a predicted Tm of 96.57°C and a ΔTm of 5.25°C, exhibits a synergistic effect because it has a better ΔTm than the sum of the ΔTms of the following DMs (-6.84°C). DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R), with a Tm of 77.2°C, and DM289 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, N35T, S76Y), with a Tm of 98.6°C.

[0634] To further improve protein stability and RMSF parameters through core modifications, we tested the combination of DM517 with C41A, C41S, and C41T. All of these DMs had predicted Tm values better than DM31 and similar to DM89. On the other hand, DM532 ((ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41A, Q28R, S76Y)) had a predicted Tm of 96.43°C and a ΔTm of 5.11°C, which is better than the sum of the ΔTm of the following DMs (-9.79°C), indicating a synergistic effect. DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A) had a predicted Tm of 77.2°C. , Y65R, L70I, (Q28R), DM210 (C41A, S76Y) with a predicted Tm of 95.65°C. DM528 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, (C41T, Q28R, S76Y) and DM530 ((ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41S, Q28R, S76Y)) are similar to DM66, DM31, and DM Compared to DM89, DM530 had a better RMSF AUC (Table 2). DM530 also had a better RMSD than DM66, DM31, and DM89. Both criteria showed increased stability (Table 2). DM530 had a predicted Tm of 94.79°C and a ΔTm of 3.47°C, demonstrating a synergistic effect, as it has a better ΔTm than the sum of the ΔTms of the following DMs (-9.65°C): DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, (Q28R)) with a predicted Tm of 77.2°C; DM89 (S76Y) with a predicted Tm of 98.19°C; and DM702 (C41S) with a predicted Tm of 88.93°C.

[0635] DM685 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41A, Q28R, S76F), with a predicted Tm of 97.31°C and a ΔTm of 5.99°C, exhibits a synergistic effect because it has a better ΔTm than the sum of the ΔTms of the following DMs (-6.47°C). DM66 ((ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R)) with a predicted Tm of 77.2°C, DM114 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76F) with a predicted Tm of 99.39°C, and DM150 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41A) with a predicted Tm of 90.9°C.

[0636] DM528 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T, Q28R, S76Y), with a predicted Tm of 96.31°C and a ΔTm of 4.99°C, exhibits a synergistic effect because it has a better ΔTm than the sum of the ΔTms of the following DMs (-9.07°C): DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R), with a predicted Tm of 77.2°C, and DM287 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T, S76Y), with a predicted Tm of 96.37°C.

[0637] DM165 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, Q28R, S76Y) has a good predicted Tm against DM31 and DM89. Its Tm was experimentally evaluated (99°C). This Tm is synergistic compared to the combination of DM101 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, S76Y) and DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R), because DM165 has a delta of 5 degrees relative to DM31, while DM66 and DM101 have a delta of only 3 degrees. DM165 also exhibits a synergistic effect in sweetness intensity due to increased sweetness compared to the combination of DM101 ((ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, S76Y) and DM66 ((ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R)). DM165 has better RMSD than DM66, DM31, and DM89, suggesting a closer match to the native components of the starting structures. DM165 maintains tacticity better (Table 2, average native contacts). DM165 has a Rosetta energy score 1 REU lower than DM31 (-322.97 vs. -321.66), but the same substitution for MNEI (DM187) increased the REU (-313.04 for DM187 vs. -315 for MNEI). This may suggest a synergistic effect of K25R, Q28K, and S76Y with the DM31 substitution.

[0638] DM167 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, Q28R, S76F), with a predicted Tm of 99.37°C and a ΔTm of 8.05°C, exhibits a synergistic effect because it has a better ΔTm than the sum of the ΔTms of the following DMs (-5.1°C): DM66 (Q28R), with a predicted Tm of 77.2°C, and DM160 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76F, K25R), with a predicted Tm of 100.34°C.

[0639] DM171 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R, R31T, S76Y) has a better predicted Tm than DM31 and DM89. DM171 has a predicted Tm of 100.46°C and a ΔTm of 9.14°C, which is better than the sum of the ΔTms of the following DMs (-4.34°C), demonstrating a synergistic effect: DM66 ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, with a predicted Tm of 77.2°C. DM286 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R31T, S76Y) with a predicted Tm of 101.1°C. DM171 has a better RMSF AUC compared to DM31.

[0640] DM521 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, D68N, Q28R, S76Y) exhibits synergy, as it has a predicted Tm that is better than DM31 and similar to DM89, and a ΔTm of 5.41°C, which is better than the sum of the ΔTms of the following DMs (-10.7°C). DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R) with a predicted Tm of 77.2°C, DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) with a predicted Tm of 87.09°C, and DM123 (S76Y, R65Y (reverse)) with a predicted Tm of 98.98°C.

[0641] DM526 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R, I26T, S76Y) has a predicted Tm of 93.89°C, similar to that of DM31, and a ΔTm of 2.57°C, but better than the sum of the ΔTm of the following DMs (-9.87°C), demonstrating synergy: DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R) with a predicted Tm of 77.2°C; and DM100 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y, I26T) with a predicted Tm of 95.57°C.

[0642] K25R The inventors submitted the K25R substitution as part of a PCT application in March 2023. This substitution had the effect of stronger H-bonding and backbone protection between positions 21 and 25, resulting in increased stability. The complex of K25R and S76Y (DM101: (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, S76Y) yielded better RMSF than DM31, DM43 (K25R), and DM89.

[0643] DM160 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, S76F) has a predicted Tm of 100°C. Its REU is better than DM31 (-324.54 vs. -321.66). However, K25R, S76F relative to MNEI (DM182) has a worse REU than MNEI (-313.12 for DM182 vs. -315 for MNEI). This indicates a synergistic effect between the K25R, S76F, and DM31 substitutions.

[0644] DM480 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, K25R, D68N, S76Y) has a better predicted Tm than DM31 and DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) and a ΔTm of 9.01°C, which is better than the sum of the ΔTms of the following DMs (5.32°C), indicating a synergistic effect. DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) with a predicted Tm of 87.09 ° C., DM101 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y, K25R) with a predicted Tm of 100.3 ° C., and DM591 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I) with a predicted Tm of 91.89 ° C. DM480 has a better RMSF AUC compared to DM31 (Table 2) and a better RMSD than DM31, DM43 (K25R), and DM89, indicating a stronger increase in stability.

[0645] DM687 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T, Q28K, S76F), with an REU of -323.7 and a ΔREU of -2.04, exhibits a synergistic effect because it has a better ΔREU than the sum of the ΔREUs (-0.01) of the following DMs: DM65 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K), with an REU of -320.54, and DM294 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T, S76F), with an REU of -322.79. Additionally, DM687 exhibits synergistic effects, as it has a better ΔREU than the sum (0.07) of the ΔREUs of the following DMs that contain it: DM46 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T) with an REU of −320.28, DM65 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K) with an REU of −320.54, and DM114 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76F) with an REU of −324.1.

[0646] DM127 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28E, S76Y) and DM128 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28K, S76Y) had Tm predictions as good as DM89 and had better protected H-bonds (water-beta sheet backbone H-bonds, Table 2) with favorable minimum REU of -329.421 (ΔREU -7.76) and -329.989 (ΔREU -8.33), respectively. The combinatorial improvement over REU from DM31 was DM200 ((ΔE50, ΔF52, ΔR53, E2N, Y65R, L70I, I26W, Q28K, S76Y)) with a ΔREU of -3.93. The efficacy of DM127 and DM128 was higher than that of DM31, DM89, DM115 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28E), and DM116 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28K, S76Y), which has the same combination against MNEI.

[0647] DM162 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76W, K25R) has better Tm predictions than DM31 and DM89. Its average native contacts are better than DM31 and similar to DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y). Its helix is more standard. Its RMSF AUC is better than DM43 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R).

[0648] DM159 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76F, I26T) has a Tm prediction value of 96 and a better RMSF AUC compared to DM31 and DM89 (Table 2).

[0649] The Rosetta score for DM178 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28K, S76W) was -327 REU, a 5 REU improvement from DM31, which was greater than that of DM198 (I26W, Q28K, S76W to MNEI), which only had a 1.5 REU improvement from MNEI, indicating that the combination with DM31 acts synergistically. Additionally, this DM has a ΔREU of -5.34, which is better than the sum of the ΔREUs of the following DMs (-2.98), indicating a synergistic effect. DM65 ((ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K)) with an REU of 320.54, DM137 ((ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76W)) with an REU of -323.95, and DM144 (I26W) with an REU of -323.48.

[0650] The Rosetta score for DM179 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28E, S76W) was −326 REU, a 4.4 REU improvement from DM31, which was greater than that of DM199 (I26W, Q28E, S76W to MNEI), which only had a 1.5 REU improvement from MNEI, indicating that the combination with DM31 acts synergistically.

[0651] DM100 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y, I26T) has an experimental Tm of 100, 10 degrees higher than DM31. It exhibits a higher number of backbone H-bonds that are intra-beta sheet and intra-helical than MNEI, DM31, and DM130. These H-bonds maintain the secondary structure of these segments during simulation. The proportion of canonical helices in DM100 is also higher when compared to the reference designs above.

[0652] The Rosetta score for DM174 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R84L, S76W) was −327 REU, a 5.6 REU improvement from DM31, which was greater than that of DM302 (R84L, S76W to MNEI), which only had a 2.2 REU improvement from MNEI, indicating that the combination with DM31 acts synergistically.

[0653] Substitution of S76Y - Suggesting a new title: Stabilization of beta-sheet through protection of backbone H-bonds (59aa-76aa) To stabilize the beta-sheet of the protein, we made modifications that protect the backbone H-bonds between beta-strands 3 and 4. This is primarily achieved by substituting residues at positions 59 and 76 with beta-branched or bulky residues. Because position 59 is close to position 76, substitutions at this position can protect the hydrogen bonds within the beta-sheet (Figure 1A). For example, DM652 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59V, S76Y) has beta-branched substitutions, and structural analysis reveals that the backbone is not exposed to solvent (Figure 2).

[0654] DM652 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59V, S76V). The predicted Tm of this DM is 101°C, similar to that of DM89 and much higher than that of DM31. The Rosetta energy score is better than that of DM31 (approximately -1.5ΔREU). The average native contacts for this DM are much higher than those of DM31 and DM89. The E59V substitution correlates with S76V in the homologous structure of this stem-loop, and together, this pair is dominant in these homologous protein substructures.

[0655] DM653 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59I, S76V). The predicted Tm of this DM is 100 °C, similar to that of DM89 and much higher than that of DM31. The Rosetta energy score is better than that of DM31 (approximately -2ΔREU). The RMSF AUC of this DM is better than that of DM31 (Figure 5) and DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y). The average native contacts of this DM are higher than those of DM31 and slightly lower than those of DM89. The E59I substitution correlates with S76V in the homologous structure of this stem-loop.

[0656] DM654 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59T, S76V). The predicted Tm of this DM is 101°C, similar to that of DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) and much higher than that of DM31. The E59T substitution correlates with S76V in the homologous structure of this stem-loop.

[0657] DM655 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59V, S76I). The predicted Tm of this DM is 102 °C, higher than that of DM89 and DM31. The Rosetta energy score of this DM is better than that of DM31 (approximately -2 ΔREU) but worse than that of DM89. The overall VoroMQA packing score (0.55) is better than that of DM31 (0.49) and DM89 (0.49), and the residue packing score at position 76 (0.66) is higher than that of DM31 (0.52) and DM89 (0.53). The RMSF AUC is lower than that of its precursor DM664 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76I). The Tm values obtained here are surprising as the RMSF AUC values are much worse than those of DM31 and DM89, and the same can be said for the average native contacts.

[0658] DM656 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59I, S76I). The predicted Tm of this DM is 101 °C, similar to that of DM89 and much higher than that of DM31. The Rosetta energy score is better than that of DM31 (approximately -3 ΔREU). The RMSF AUC is better than that of DM31 and DM89 (Figure 5). The average RMSD of the β-sheet is lower than that of DM31 and DM89, indicating a stabilized sheet. The residue VoroMQA scores at positions 59 (-0.58) and 76 (-0.55) are better than those of DM31 (0.57 and 0.52), and the value at position 76 is also better than that of DM89 (0.53). The E59I substitution correlates with S76I in the homologous structure of this stem-loop.

[0659] DM657 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59T, S76I). The predicted Tm of this DM is 102°C, higher than DM89 and DM31. The RMSF AUC is better than DM31 and DM89. The average RMSD of the β-sheets is lower than that of DM31 and DM89, indicating a stabilized sheet. The average native contacts are lower than DM31 and DM89. The Rosetta energy score is similar to DM31. Surprisingly, despite the lower average native contacts and lack of improvement in the Rosetta energy score compared to DM31, the predicted Tm is very high.

[0660] DM662 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59Y, S76V). The predicted Tm of this DM is approximately 100 °C higher than that of DM31. The Rosetta energy score of this DM is approximately -2 REU better than that of DM31. The average native contact of this DM is better than that of DM31, DM89, and DM663 (S76V). The overall VoroMQA packing score (0.51) for this DM is also better than that of DM31 (0.49) and DM89 (0.49), and similar to that of DM661.

[0661] DM663 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76V). The predicted Tm of this DM is 98°C, higher than that of DM31. The Rosetta energy score of this DM is better than that of DM31. The RMSF AUC of this DM is better than that of DM31 and DM89 (Figure 5). Structures homologous to stem-loop structures in this region often contained valine at position 76.

[0662] DM664 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76I). The predicted Tm of this DM is 100°C, much higher than that of DM31 and similar to that of DM89. The average native contact of this DM is higher than that of DM31 and DM89. The Rosetta energy score of this DM is better than that of DM31 but worse than that of DM89. Structures homologous to stem-loop structures in this region often contained isoleucine at position 76.

[0663] Example 3: Energy bar formulation containing Designer MNEI (DM) protein The formula for the energy bar containing DM protein is presented in Table 6.

[0664] [Table 6]

[0665] Example 4: Formulation of marzipan containing Designer MNEI (DM) protein Marzipan is a confectionery made mainly from ground almonds and sugar. It is widely used in the baking industry and for the preparation of different types of confectionery.

[0666] The marzipan formulation containing DM protein is presented in Table 7.

[0667] [Table 7]

[0668] Preparation method: Grind the almonds and gradually add all the powder to the mixer and grind thoroughly. Add all the liquid ingredients and sweeteners to the mix and continue grinding until a crystalline dough is obtained. Add 0.01-0.05% protein by weight to the recipe. The DM potency can be 1000-3000, which corresponds to 25-45 Brix.

[0669] Example 5: Non-dairy milk prototype containing designer monellin (DM) protein The formulation of non-dairy milk containing DM protein is presented in Table 8.

[0670] [Table 8]

[0671] Preparation method: Sugar and AMAI Sweet Protein DM are added to unsweetened non-dairy milk and mixed to form a homogenous solution.

[0672] Example 6: Granola Preparation The formulation of the granola containing DM protein is presented in Table 9.

[0673] [Table 9]

[0674] Preparation method Bake the oatmeal, rice crisps, pecans, and almonds in a pan at 160°C for 15 minutes.

[0675] Glucose syrup, powdered sugar, sunflower oil, glycerol, lecithin, and salt are heated to form a homogeneous syrup.

[0676] The toasted pecans and almonds are ground into small pieces. The dry ingredients (oatmeal, crisp rice, pecans, and almonds) are added to the syrup and mixed. The maltodextrin and DM31 are added last.

[0677] Pour the mixture into a baking dish.

[0678] The granola mix is baked at 90°C for 10 minutes, then removed, cooled and stored tightly closed in a cool place.

[0679] Example 7: Peanut Butter Spread Peanut butter spread formulations with various filling options containing DM protein are presented in Table 10.

[0680] [Table 10]

[0681] Preparation method: 1. Grind the peanuts. 2. Gradually add ingredients 2 to 7 to the mixer and grind thoroughly until a homogeneous dough is obtained. 3. Add the sweet protein DM to the mass and continue grinding at a slow speed until completely assimilated.

Claims

1. A modified protein having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence described in Sequence ID No. 1, the at least one amino acid deletion is located in one or more amino acids between T46 and I56 of the reference protein, and the at least one amino acid substitution is located in amino acid S76 of the reference protein.

2. The modified protein according to claim 1, wherein the at least one amino acid deletion is one or more of the amino acids E50, F52, R53, or any combination thereof of the reference protein.

3. The modified protein according to claim 1 or 2, wherein the at least one amino acid substitution includes at least one amino acid substitution in addition to the amino acid substitution at amino acid S76 of the reference protein.

4. The modified protein according to claim 3, wherein the at least one amino acid substitution comprises one or more amino acid substitutions of amino acids G1, E2, E4, T12, A19, V20, E23, K25, I26, G27, Q28, T33, N35, C41, Q61, V64, Y65, D68, L70, A73, I75, R84, F89, or any combination thereof.

5. The modified protein according to claim 4, wherein the at least one amino acid substitution comprises one or more of the following amino acid substitutions. (i) G1M, (ii) A19V, (iii) V20I, (iv) K25R, (v) I26W, I26V, or I26T, one or more of (vi) Q28R, Q28K, Q28S, Q28E, one or more of (vii) R31T, (viiii) T33R, (ix) N35T, (x) C41A, C41V, C41T, Or one or more of C41S, one or more of (xi)E59T, E59V, E59I, E59Y, E59F, E59W, or E59R, (xi)D68N or D68T, (xiiii)A73F or A73V, (xiv)D74V, (xv)I75V or I75L, (xvi)E77V, (xvii)R84L.

6. The modified protein according to claim 1, wherein the at least one amino acid substitution comprises one or more amino acid substitutions of amino acids E2, E23, Y65, L70, or any combination thereof.

7. The modified protein according to claim 1, wherein the amino acid substitution in amino acid S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A.

8. The modified protein according to claim 1, wherein the at least one amino acid substitution comprises one or more of the following amino acid substitutions: (i) E2N, E23A, Y65R, L70I, (ii) E2M, E23A, Y65R, L70I, (iii) E2N, E23Q, Y65R, L70I, or (iv) E2M, E23Q, Y65R, L70I.

9. The modified protein according to claim 1, comprising one or more amino acid sequences from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 221, SEQ ID NO: 108, SEQ ID NO: 9, SEQ ID NO: 102, SEQ ID NO: 105, SEQ ID NO: 52, SEQ ID NO: 182, SEQ ID NO: 10, SEQ ID NO: 69, SEQ ID NO: 157, SEQ ID NO: 114, SEQ ID NO: 174, SEQ ID NO: 162, SEQ ID NO: 231, SEQ ID NO: 57, SEQ ID NO: 192, SEQ ID NO: 68, SEQ ID NO: 106, SEQ ID NO: 8, SEQ ID NO: 79, SEQ ID NO: 76, SEQ ID NO: 22, SEQ ID NO: 194, SEQ ID NO: 262, SEQ ID NO: 84, SEQ ID NO: 239, SEQ ID NO: 135, SEQ ID NO: 211, SEQ ID NO: 237, SEQ ID NO: 198, SEQ ID NO: 179, SEQ ID NO: 172, or any combination thereof.

10. The modified protein according to claim 1, having increased stability compared to the reference protein.

11. The modified protein according to claim 10, wherein the stability is one or more of the following: thermal stability, chemical stability (within a specific pH range, various hydrophobic levels, various food and beverage matrices, and various levels of proteins, lipids, and preservatives in the matrices), and functional stability (including short-term or long-term sensory profiles and shelf-life studies).

12. (i) for preparing an oral delivery product, (ii) Flavor modifiers, flavor enhancers, or flavor masking agents, (iii) Sweetener The modified protein according to claim 1 for use as a protein.

13. A modified protein according to claim 1 for use as a stabilizer.

14. A food product comprising the modified protein described in Claim 1.

15. A method for increasing the stability of a reference protein, the method comprising deleting at least one amino acid of the reference protein and substituting at least one amino acid of the reference protein, wherein the reference protein is represented by the amino acid sequence described in SEQ ID NO: 1, the at least one amino acid to be deleted is one or more amino acids located between T46 and I56 of the reference protein, and the at least one amino acid to be substituted is amino acid S76 of the reference protein.

16. The method according to claim 15, wherein the at least one amino acid to be deleted is one or more of the amino acids E50, F52, R53 of the reference protein, or any combination thereof.

17. The at least one amino acid substitution includes one or more amino acid substitutions from among amino acids G1, E2, E4, T12, A19, V20, E23, K25, I26, G27, Q28, T33, N35, C41, Q61, V64, Y65, D68, L70, A73, I75, R84, F89, or any combination thereof. The above at least one amino acid substitution is one or more of the following amino acid substitutions: (i) G1M, (ii) A19V, (iii) V20I, (iv) K25R, (v) I26W, I26V, or I26T, (vi) one or more of Q28R, Q28K, Q28S, Q28E, (vii) R31T, (viiii) T33R, (ix) N35T, (x) C41A, One or more of C41V, C41T, or C41S, one or more of (xi)E59T, E59V, E59I, E59Y, E59F, E59W, or E59R, one or more of (xi)D68N or D68T, (xiiii)A73F or A73V, (xiv)D74V, (xv)I75V or I75L, (xvi)E77V, (xvii)R84L, The method according to claim 15 or 16.

18. The method according to claim 15, wherein the at least one amino acid substitution comprises one or more amino acid substitutions of amino acids E2, E23, Y65, L70, or any combination thereof.

19. The method according to claim 15, wherein the amino acid substitution in amino acid S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A.

20. The method according to claim 15, comprising one or more amino acid sequences described in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 221, SEQ ID NO: 108, SEQ ID NO: 9, SEQ ID NO: 102, SEQ ID NO: 105, SEQ ID NO: 52, SEQ ID NO: 182, SEQ ID NO: 10, SEQ ID NO: 69, SEQ ID NO: 157, SEQ ID NO: 114, SEQ ID NO: 174, SEQ ID NO: 162, SEQ ID NO: 231, SEQ ID NO: 57, SEQ ID NO: 192, SEQ ID NO: 68, SEQ ID NO: 106, SEQ ID NO: 8, SEQ ID NO: 79, SEQ ID NO: 76, SEQ ID NO: 22, SEQ ID NO: 194, SEQ ID NO: 262, SEQ ID NO: 84, SEQ ID NO: 239, SEQ ID NO: 135, SEQ ID NO: 211, SEQ ID NO: 237, SEQ ID NO: 198, SEQ ID NO: 179, SEQ ID NO: 172, or any combination thereof.

Images