Sweet protein mutants from truffles

Abstract

A newly identified fungal sweetness-modifying protein, named Honey Truffle Sweetener (HTS), and a polynucleotide encoding the protein are described. Specifically, a Myd protein having sweetness-modulating activity and a cDNA encoding the same are described, along with methods for isolating such cDNA, and methods for isolating and expressing such protein. Sweetener compositions comprising the protein of the invention and methods for providing improved flavor to products for oral administration are also disclosed.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 295,200, filed December 30, 2021, the entire contents of which are incorporated by reference herein. Reference to Electronic Sequence Listing

[0002] The contents of the electronic sequence listing (0640-44_WO.xml; size: 321,784 bytes; and creation date: December 27, 2022) are incorporated herein by reference in their entirety.

[0002] FIELD OF THEINVENTION

[0003] The present invention includes embodiments of sweet proteins (Honey Truffle Sweetener (HTS)), genes and cDNAs encoding said proteins, and methods of using such proteins, genes, and cDNAs in modulating food taste. More particularly, the present invention includes embodiments of newly identified fungal sweet proteins, and genes and cDNAs encoding such proteins. [Background technology]

[0003]

[0004] Excessive intake of nutritive sweeteners has long been associated with food-related health problems, such as obesity, heart disease, metabolic disease and dental problems. As a result, consumers are increasingly seeking ways to reduce the amount of nutritive sweeteners in their foods, and manufacturers are responding to this demand by attempting to replace nutritive sweeteners with substitutes that mimic the desired taste and functional properties of nutritive sweeteners.

[0004]

[0005] To limit the negative effects of high sugar consumption (e.g., diabetes and obesity, among others), zero or low calorie sweeteners, preferably those derived from natural sources, are desirable, although commonly known zero or low calorie sweetener substitutes, such as aspartame, acesulfame potassium, monk fruit extract, neotame, saccharin, stevia and sucralose, have undesirable taste defects, such as bitterness.

[0005]

[0006] Zero or low calorie sweeteners from natural sources may be preferred to limit the negative effects of high sugar consumption. So far, only seven sweetness and taste modifying proteins are known, namely monellin, thaumatin, brazzein, curculin, mabinlin, miraculin and pentadin. For any of these proteins, the key residues on the protein surface involved in biological activity have not yet been identified with any certainty. Monellin was found to be 100,000 times sweeter than sucrose on a molar basis, followed by thaumatin and brazzein, which are 3000 and 500 times sweeter than sucrose, respectively, on a gram basis. Most of them have no sequence homology or structural similarity; thaumatin shares extensive similarity at the protein sequence level with certain non-sweet proteins found in other plants.

[0006]

[0007] International Patent Application PCT / US2020 / 012955, filed 01 / 09 / 2020, published as WO2020 / 146650 on 07 / 16 / 2020, relates to a sweetening composition comprising (i) mycelium or an aqueous extract thereof of an ascomycete fungus, or (ii) an aqueous extract of the fruiting body of an ascomycete fungus, and the use of such a composition to provide an improved flavor in a product for oral administration. This application also relates to a composition comprising the combination of the sweetening composition and a product for oral administration.

[0007]

[0008] International Patent Application PCT / US2021 / 039176, filed June 25, 2021, published as WO2021 / 263158 on December 30, 2021, relates to newly identified fungal sweetness modifying proteins and cDNAs encoding the proteins. The application further relates to Myd proteins active in sweetness activation and cDNAs encoding same, as well as methods for isolating such cDNAs, and methods for isolating and expressing such proteins. The application also relates to sweetener compositions comprising the proteins and methods for providing improved flavor to products for oral administration. Summary of the Invention [Problem to be solved by the invention]

[0008]

[0009] Thus, there remains a need for new low or zero calorie sweeteners with improved taste from natural sources, particularly from fungal species of Ascomycetes. There is also a need to produce and use fungal-derived low or zero calorie sweeteners with improved taste. [Means for solving the problem]

[0009] Summary of the Invention

[0010] The present invention meets these and other needs by providing the first newly identified fungal sweet protein, identified herein as Myd1, and more generally as the Myd family of proteins, as well as genes and cDNAs encoding such proteins, and methods of using such proteins, genes, and cDNAs in modulating food taste. The present invention provides, in particular, a DNA sequence, identified herein as MYD1, that encodes the corresponding sweet polypeptide Myd1 (also referred to as mycodulcein and honey truffle sweetener (HTS)), the first sweet protein identified from a fungus. The polypeptides of the present invention modulate sweetness and sensation, either alone or in combination with foods, beverages, dietary supplements, or pharmaceuticals. Myd1 reduces the sour, bitter, or astringent taste of foods and beverages; Myd1 also has taste-enhancing, i.e., taste-modifying, activity in foods and beverages.

[0010]

[0011] Accordingly, one aspect of the invention provides a polynucleotide (e.g., an isolated polynucleotide) that encodes a polypeptide, wherein the encoded polypeptide has sweet taste modulating activity, and optionally differs in amino acid sequence from the polypeptide of SEQ ID NO:3.In an embodiment, the encoded polypeptide is selected from the group consisting of (a) SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, (b) a polypeptide sequence selected from the group consisting of SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:140 and SEQ ID NO:141; (b) a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140; and (c) a polypeptide sequence modified from a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140 by deletion, insertion, substitution, or addition of not more than 24 amino acids, wherein the polypeptide sequence optionally differs from the polypeptide sequence of SEQ ID NO:3.

[0011]

[0012] Another aspect of the present invention provides a polynucleotide (e.g., an isolated polynucleotide) encoding a polypeptide having sweet taste modulating activity. The polynucleotide is selected from the group consisting of: (a) a polynucleotide comprising SEQ ID NO:2, optionally with at least one modification; (b) a polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:2, optionally different from SEQ ID NO:2; (c) a polynucleotide comprising (i) SEQ ID NO:2 and (ii) a nucleotide sequence encoding a histidine tag. In one embodiment, the polynucleotide encoding a polypeptide having sweet taste modulating activity is a polynucleotide other than the polynucleotide of SEQ ID NO:2.

[0012]

[0013] Another aspect of the present invention provides a polynucleotide (e.g., an isolated polynucleotide) encoding a polypeptide having sweetness modulating activity. The polynucleotide may be selected from the group consisting of (a) SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:1 (b) a polynucleotide comprising the nucleotides of SEQ ID NO:12, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:139; a polynucleotide comprising the nucleotide of sequence number 79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:139, having at least one substitution or modification and optionally differing from SEQ ID NO:2;(c) SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, a polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity to nucleotides of SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:139, optionally differing from SEQ ID NO:2; (d)(i) SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, a polynucleotide comprising any one of the nucleotides set forth in SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:139;and (e) SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:1 11, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:139;

[0013]

[0014] In other embodiments, the polynucleotide encoding the polypeptide having sweet taste modulating activity is optionally operably linked to a heterologous regulatory element. Additionally or alternatively, the polynucleotide sequence further encodes a protein tag or label. The protein tag is optionally an affinity tag. The protein tag is optionally a histidine tag. In specific embodiments, the polynucleotide encoding the polypeptide having sweet taste modulating activity is the nucleotide of SEQ ID NO: 2 and further encodes a protein tag or label. In specific embodiments, the polynucleotide encoding the polypeptide having sweet taste modulating activity is the nucleotide of SEQ ID NO: 2, optionally operably linked to a heterologous regulatory element. Additionally or alternatively, the nucleotide of SEQ ID NO: 2, operably linked to a heterologous regulatory element, further encodes a protein tag or label. The protein tag is optionally an affinity tag. The protein tag is optionally a histidine tag. The protein tag is optionally a (His) 6 It is.

[0014]

[0015] In particular embodiments, a polynucleotide encoding a polypeptide having sweetness modulating activity is selected from the group consisting of SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, SEQ ID NO:303, SEQ ID NO:304, SEQ ID NO:305, SEQ ID NO:306, SEQ ID NO:307, SEQ ID NO:308, SEQ ID NO:309, SEQ ID NO:401, SEQ ID NO:402, SEQ ID NO:403, SEQ ID NO:404, SEQ ID NO:405, SEQ ID NO:406, SEQ ID NO:407, SEQ ID NO:408, SEQ ID NO:409, SEQ ID NO:501, SEQ ID NO:502, S 10, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:139, optionally operably linked to a heterologous regulatory element. Additionally or alternatively, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO: Nucleotides of SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:139 are excluded from the sequence encoding the histidine tag in each of the listed SEQ IDs.Additionally or alternatively, the sequences encoding the histidine tag in each of the listed SEQ ID NOs are omitted: SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, The nucleotides of sequence number 110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:139 are optionally operably linked to a heterologous regulatory element. Additionally or alternatively, the sequence encoding the histidine tag in each of the listed SEQ ID NOs: 76, 77, 79, 80, 82, 83, 85, 86, 88, 89, 90, 91, 93, 94, 96, 97, 99, 100, 102, 103, 104, 105, 107, 108, 109, 110, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194 8, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:139 further encodes a protein tag or label. The protein tag is optionally an affinity tag. The protein tag is optionally (His). 6 Other than histidine tag.

[0015]

[0016] Another aspect of the invention provides a polynucleotide comprising the nucleotides of SEQ ID NO:20, which corresponds to the coding sequence of a His-tagged mycodulcein in E. coli, where residues 364-381 correspond to the optional His-tag sequence. SEQ ID NO:20 is codon-optimized for expression in E. coli. In a related aspect, the invention provides a polynucleotide having at least 80% sequence identity to the nucleotides of SEQ ID NO:20. In yet another aspect, the polynucleotide comprises SEQ ID NO:22, which corresponds to the coding sequence of a His-tagged mycodulcein in S. cerevisiae, where residues 364-381 correspond to the optional His-tag sequence. SEQ ID NO:22 is codon-optimized for expression in S. cerevisiae. The corresponding polypeptide of SEQ ID NO:21 corresponds to a His-tagged mycodulcein protein (wherein residues 122-127 correspond to the optional His-tag sequence), which polypeptide sequence is the same when expressed in E. coli and when expressed in S. cerevisiae. In a related embodiment, the invention provides a polynucleotide having at least 80% sequence identity to the nucleotide of SEQ ID NO:22.

[0016]

[0017] In additional aspects, the present invention provides an expression cassette comprising the polynucleotide and a vector comprising the polynucleotide, as well as a host cell transformed with the vector. Also provided is a method of producing a protein having sweet taste modulating activity, comprising culturing the transformed host cell in a medium under conditions that result in the production of the protein having sweet taste modulating activity.

[0017]

[0018] Another aspect of the invention is a polypeptide sequence having sweetness modulating activity and selected from the sequences of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:140 and SEQ ID NO:141, and optionally different from SEQ ID NO:3; or ii) providing a polypeptide (e.g., an isolated polypeptide), comprising a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140, wherein optionally the polypeptide sequence differs from SEQ ID NO:3. In additional embodiments, the polypeptide (a) contains at least one modification compared to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140, and the polypeptide optionally differs from the polypeptide of SEQ ID NO:3, or (b) further comprises a protein tag, in particular a histidine tag, and the polypeptide has sweet taste modulating activity.

[0018]

[0019] Another aspect of the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:141, which is optionally different from SEQ ID NO:3. ...

[0019]

[0020] In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified by at least one mutation selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by two mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by three mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by four mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by five mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by six mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by seven mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by eight mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by nine mutations selected from those listed in Table 7.In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 10 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 11 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 12 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 13 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 14 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 15 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 16 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 17 mutations selected from those listed in Table 7.In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 18 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 19 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 20 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 21 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 22 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by 23 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by 24 mutations selected from those listed in Table 7. The present invention also provides polynucleotides encoding the aforementioned mutant polypeptides of SEQ ID NO: 3. The present invention further provides the aforementioned mutant polypeptides further comprising a protein tag, more specifically a histidine tag. The present invention also provides polynucleotides encoding the aforementioned mutant polypeptides of SEQ ID NO: 3 further comprising a protein tag, more specifically a histidine tag.

[0020]

[0021] In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one mutation selected from the mutations Q37K, Q37E, Q37N, V74A, V76A, V87A, V100A or Q102K. In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by two mutations selected from the mutations Q37K, Q37E, Q37N, V74A, V76A, V87A, V100A or Q102K. In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by three mutations selected from the mutations Q37K, Q37E, Q37N, V74A, V76A, V87A, V100A or Q102K. In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by four mutations selected from the mutations Q37K, Q37E, Q37N, V74A, V76A, V87A, V100A or Q102K. In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by five mutations selected from the mutations Q37K, Q37E, Q37N, V74A, V76A, V87A, V100A or Q102K. In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by six mutations selected from the mutations Q37K, Q37E, Q37N, V74A, V76A, V87A, V100A or Q102K. The present invention also provides a polynucleotide encoding the aforementioned mutant polypeptide of SEQ ID NO: 3. The present invention further provides the aforementioned mutant polypeptide further comprising a protein tag, more particularly a histidine tag.The present invention also provides a polynucleotide encoding the aforementioned mutant polypeptide of SEQ ID NO:3, further comprising a protein tag, more particularly a histidine tag.

[0021]

[0022] In another embodiment, the polypeptide of the present invention is optionally isolated and / or optionally purified. In another embodiment, the polynucleotide of the present invention is optionally isolated and / or purified. In another embodiment, the polypeptide of the present invention optionally comprises a protein tag. In another embodiment, the polypeptide of the present invention optionally comprises a histidine tag.

[0022]

[0023] Another aspect of the present invention provides a composition comprising a combination of (a) a product for oral administration other than a truffle of Mattirolomyces terfezioides and (b) a sweetener composition comprising a polypeptide, the combination having enhanced sweetness compared to the product for oral administration. The polypeptide comprises one or more amino acid sequences, either individually or in combination of two or more thereof, and has sweet taste modulating activity, and is selected from (i) SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:140 and SEQ ID NO:141, and optionally different from SEQ ID NO:3. or (ii) a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140, optionally wherein the polypeptide sequence differs from SEQ ID NO:3; or (iii) where the polypeptide of the SEQ ID NOs listed above further comprises a protein tag, the protein tag is optionally a histidine tag.

[0023]

[0024] Another more specific aspect of the present invention provides a composition comprising a combination of (a) a product for oral administration other than a truffle of Mattiromyces terfesioides and (b) a sweetener composition comprising a polypeptide, wherein the combination has an enhanced sweetness compared to the product for oral administration. The polypeptide comprises (a) one or more amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140, (b) one or more amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or (c) one or more amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:141. In an embodiment, the polypeptide is not the polypeptide of SEQ ID NO:3.

[0024]

[0025] Another aspect of the present invention provides a method for modulating the taste of a product for oral administration. The method includes combining a product for oral administration with an effective amount of a sweetener composition comprising a polypeptide, wherein the product for oral administration is different from Mattiromyces terfesioides truffles, and the combination has an enhanced sweetness compared to the product for oral administration. The polypeptide comprises one or more sequences as enumerated herein, either individually or in combination of two or more thereof.

[0025]

[0026] Another aspect of the present invention provides a method for purifying a polypeptide having sweet taste modulating activity, comprising the steps of (a) subjecting a composition comprising the polypeptide to hydrophobic interaction chromatography (HIC), and (b) then subjecting the composition comprising the polypeptide to size exclusion chromatography (SEC). The polypeptide has sweetness modulating activity and is selected from (i) SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:140 and SEQ ID NO:141, optionally different from SEQ ID NO:3; or (ii) SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or (iii) a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140, wherein the polypeptide sequence is optionally different from SEQ ID NO:3; or (iii) a polypeptide sequence of the SEQ ID NOs listed above further comprising a protein tag, wherein the protein tag is optionally a histidine tag, either individually or in combination of two or more thereof.

[0026]

[0027] Other aspects and embodiments of the invention will be apparent from consideration of the drawings, detailed description and non-limiting examples provided herein. [Brief description of the drawings]

[0027] [Figure 1]

[0028] Figure 1 shows the predicted three-dimensional structure of Myd1 (honey truffle sweetener (HTS)) based on sequence data using the PHYRE2.0 protein fold prediction tool. [Diagram 2]

[0029] FIG. 2 shows a Coomassie stained SDS-PAGE gel of proteins from partially purified M. terfesioides gleba fractions. [Diagram 3]

[0030] Figure 3 shows a Coomassie stained SDS-PAGE gel of the purification steps of SEQ ID NO: 21 expressed in E. coli. Lane 1: molecular weight standards; lane 3, crude product lysate; lane 4, flow-through fraction from HisPur™ Ni-NTA; lane 5, wash 1; lane 6, wash 2; lane 7, wash 3; lane 8, elution fraction. [Figure 4]

[0031] FIG. 4 shows the concentration-response functions for sweetness of mycodulcein (honey truffle sweetener (HTS)), aspartame, thaumatin, and rebaudioside A. Data are plotted as the ratio (p) of the response produced to the 200 mM sucrose-related ("sweet") target. Each data point in the curves for mycodulcein, aspartame, thaumatin, and rebaudioside A was calculated as the average over 32 replicates, and for the sucrose curve, averaged over 16 replicates; error bars are SEM. Points for water and sucrose controls were similarly calculated as the average over 128 and 64 replicates, respectively. Curves were fitted by nonlinear regression. [Figure 5A]

[0032] FIG. 5A shows a comparison of the predicted tertiary structure of mycodulcein (honey truffle sweetener (HTS)) to the known crystal structures of thaumatin (PDB:1RQW), monellin (PDB:2O9U), brazzein (PDB:1BRZ), and hen egg white lysozyme (PDB:1LSN) (Protein Database), indicating that mycodulcein has similar predicted tertiary structure to other known sweet proteins, all containing antiparallel beta sheets with alpha helices parallel to the beta sheets. [Figure 5B]

[0033] FIG. 5B shows the predicted secondary structure of SEQ ID NO:3 superimposed on the putative secondary structure motifs and a representation of the locations of point mutations within each motif. [Figure 6]

[0034] FIG. 6 shows the results of comparing mycodulceins with mutated his-tags with each other and with non-mutated mycodulceins with his-tags normalized to equal protein concentrations in terms of sweetness intensity, time to onset of sweetness perception, and duration of sweetness perception, as measured by ELISA. [Figure 7A]

[0035] Figure 7A shows SDS-PAGE analysis of fractions eluted from Capto MMC, Coomassie stained. M: protein marker; lane 1: eluted fraction showing low purity after cation exchange. Arrow indicates mycodulcein band. [Figure 7B]

[0036] FIG. 7B shows SDS-PAGE analysis, Coomassie staining, of two eluted fractions collected during gradient elution from a HiScreen Capto Butyl column analyzed by SDS-PAGE. Lane 1 shows eluted fraction 1 containing no mycodulcein, and lane 2 shows eluted mycodulcein. The purity of the eluted fractions was determined by GelAnalyzer to be approximately 86%. The arrow indicates the mycodulcein band. [Figure 7C]

[0037] FIG. 7C shows SDS-PAGE analysis of proteins eluted from the HIC column after chromatography on HiPrep 26 / 60 Sephacryl S-200, Coomassie staining. Lane 1 shows purified his-tagged mycodulcein and lane 2 shows purified native mycodulcein. The purity of the eluted fraction was determined by GelAnalyzer to be approximately 98%. The arrow indicates the mycodulcein band. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028]

[0038] The present invention provides embodiments of an isolated nucleic acid molecule (MYD1) encoding a new and unexpectedly discovered fungal sweet protein, Myd1 (also called mycodulcein, also known as honey truffle sweetener (HTS)), which is the first sweet protein identified from a fungus. The present invention provides embodiments of isolated nucleotides encoding Myd1 polypeptides, the encoded Myd1 polypeptides capable of modulating sweet taste and perception, and methods of making and using them. The present invention provides embodiments of MYD1 and Myd1 polypeptides, either alone or in combination with foods, beverages, dietary supplements, or pharmaceuticals; and methods of modifying the taste of such foods, beverages, dietary supplements, or pharmaceutical compositions with the isolated polynucleotides and polypeptides of the present invention.

[0029]

[0039] An embodiment of the present invention provides Myd polypeptides (also referred to as honey truffle sweeteners (HTS)). The term "Myd polypeptide" is used herein to identify any of the polypeptides according to the present invention having at least 80% sequence identity, for example to SEQ ID NO:3, and also having sweet taste modifying activity. Myd polypeptides also include polypeptides of SEQ ID NO:8-17, which have sweet taste modifying activity. Myd polypeptides further include polypeptides of SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140, which have sweet taste modifying activity. A partially purified extract of sweet-tasting Terfesioides gleba was subjected to de novo amino acid sequencing to identify the 20-mer N-terminal sequence (SEQ ID NO:4). After de novo assembly of the entire transcriptome of M. terfeziodes gleba using RNAseq reads, a Myd1 coding sequence (putatively from the MYD1 gene) was identified. By screening the entire transcriptome of M. terfeziodes using a 20-mer N-terminal sequence, a transcript predicted to code for a protein with 100% identity at the N-terminus was identified. The identified transcript is predicted to code for a protein of 121 amino acids. This method identified a polynuclotide of SEQ ID NO:1. Start and stop codons were identified in the transcript to identify a putative coding sequence of SEQ ID NO:2. SEQ ID NO:3 is a putative protein, a protein of 121 amino acids. The identity of the predicted protein of SEQ ID NO:3 to other protein sequences in GENBANK was 31% or less.The coding sequences of native mycodulcein codon-optimized for expression in Escherichia coli and Saccharomyces cerevisiae correspond to the nucleic acid sequences of SEQ ID NO:20 and SEQ ID NO:22, respectively (both of which encode the amino acid sequence of SEQ ID NO:3 with an optional six-residue histidine tag, i.e., the amino acid sequence of SEQ ID NO:21).

[0030] Polynucleotides

[0040] The present invention also includes polynucleotides (e.g., isolated polynucleotides) encoding polypeptides having sweet taste modulating activity. Examples of polynucleotides encoding polypeptides having sweet taste modulating activity include, but are not limited to, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO: No. 90, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:139, or a polypeptide such as SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, No. 85, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 125,Examples of such nucleic acid sequences include those having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:139.

[0031]

[0041] In one embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97, No. 99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:139, or SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:1 00, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:139;The nucleic acid sequence of the present invention comprises, consists essentially of, or consists of a polynucleotide selected from the group consisting of nucleic acid sequences having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of the present invention.

[0032]

[0042] In one embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:139, or SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:11 7, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:139. Each of the polynucleotides of SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:139 encodes a histidine tag. It will be understood that polynucleotides excluding sequences encoding histidine tags are also provided and useful in the present invention. A given histidine tag (e.g., (His) 6 It will further be understood that for any specific polynucleotide indicated as encoding a histidine tag, the sequence encoding a histidine tag can be replaced with a sequence encoding a different His tag or a sequence encoding a different protein tag.

[0033]

[0043] In embodiments, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, or SEQ ID NO:67, excluding the sequence encoding the histidine tag. In further embodiments, the polynucleotide comprises a polynucleotide having 80% sequence identity to the polynucleotide sequence of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, or SEQ ID NO:67, excluding the sequence encoding the histidine tag in each recited SEQ ID NO. In one embodiment, the polynucleotide comprises SEQ ID NO:23 (encoding a protein of SEQ ID NO:24 having a D3E mutation, i.e., an Asp to Glu substitution at amino acid 3, compared to the wild-type protein of SEQ ID NO:3), or a polynucleotide having at least 80% sequence identity to SEQ ID NO:23. In one embodiment, the polynucleotide comprises SEQ ID NO:25 (encoding a protein of SEQ ID NO:26 having a K11R mutation, i.e., a Lys to Arg substitution at amino acid 11, compared to the wild-type protein of SEQ ID NO:3), or a polynucleotide having at least 80% sequence identity to SEQ ID NO:25. In one embodiment, the polynucleotide comprises SEQ ID NO:29 (encoding a protein of SEQ ID NO:30 having a K26R mutation, i.e., a Lys to Arg substitution at amino acid 26, compared to the wild-type protein of SEQ ID NO:3), or a polynucleotide having at least 80% sequence identity to SEQ ID NO:29. In one embodiment, the polynucleotide comprises SEQ ID NO:37 (corresponding to the protein of SEQ ID NO:38 having a K51R mutation, i.e., a Lys to Arg substitution at amino acid 51, compared to the wild-type protein of SEQ ID NO:3) or a polynucleotide having at least 80% sequence identity to SEQ ID NO:37.In one embodiment, the polynucleotide comprises SEQ ID NO:41 (corresponding to the protein of SEQ ID NO:42 having an R57K mutation, i.e., an Arg to Lys substitution at amino acid 57, compared to the wild-type protein of SEQ ID NO:3) or a polynucleotide having at least 80% sequence identity with SEQ ID NO:41. In one embodiment, the polynucleotide comprises SEQ ID NO:43 (corresponding to the protein of SEQ ID NO:44 having an R66K mutation, i.e., an Arg to Lys substitution at amino acid 66, compared to the wild-type protein of SEQ ID NO:3) or a polynucleotide having at least 80% sequence identity with SEQ ID NO:43. In one embodiment, the polynucleotide comprises SEQ ID NO:45 (corresponding to the protein of SEQ ID NO:46 having a D69E mutation) or at least 80% sequence identity with SEQ ID NO:45. In one embodiment, the polynucleotide comprises SEQ ID NO:49 (corresponding to the protein of SEQ ID NO:50 having a D85E mutation) or at least 80% sequence identity with SEQ ID NO:49. In one embodiment, the polynucleotide comprises SEQ ID NO:51 (corresponding to the protein of SEQ ID NO:52 with an E86D mutation) or at least 80% sequence identity with SEQ ID NO:51. In one embodiment, the polynucleotide comprises SEQ ID NO:53 (corresponding to the protein of SEQ ID NO:54 with an E89D mutation) or at least 80% sequence identity with SEQ ID NO:53. In one embodiment, the polynucleotide comprises SEQ ID NO:57 (corresponding to the protein of SEQ ID NO:58 with a D97E mutation) or at least 80% sequence identity with SEQ ID NO:57. In one embodiment, the polynucleotide comprises SEQ ID NO:59 (corresponding to the protein of SEQ ID NO:60 with a K103R mutation) or at least 80% sequence identity with SEQ ID NO:59. In one embodiment, the polynucleotide comprises SEQ ID NO:61 (corresponding to the protein of SEQ ID NO:62 with an R106K mutation) or at least 80% sequence identity with SEQ ID NO:61. In one embodiment, the polynucleotide comprises SEQ ID NO:63 (corresponding to the protein of SEQ ID NO:64 with an R110K mutation) or at least 80% sequence identity with SEQ ID NO:63.In one embodiment, the polynucleotide comprises SEQ ID NO:65 (corresponding to the protein of SEQ ID NO:66 having an E117D mutation) or at least 80% sequence identity to SEQ ID NO:65. In one embodiment, the polynucleotide comprises SEQ ID NO:67 (corresponding to the protein of SEQ ID NO:68 having a K120R mutation) or at least 80% sequence identity to SEQ ID NO:67. Each of the polynucleotides of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, or SEQ ID NO:67 encodes a histidine tag.

[0034]

[0044] In embodiments, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, or SEQ ID NO:67, excluding the sequence encoding the histidine tag. In further embodiments, the polynucleotide comprises a polynucleotide having 80% sequence identity to the polynucleotide sequence of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, or SEQ ID NO:67, excluding the sequence encoding the histidine tag in each recited SEQ ID NO.

[0035]

[0045] In yet another embodiment, the polynucleotide comprises SEQ ID NO:20, which corresponds to the coding sequence of His-tagged mycodulcein in E. coli, where residues 364-381 correspond to the optional His-tag sequence. SEQ ID NO:20 is codon-optimized for expression in E. coli. In another aspect, the polynucleotide comprises SEQ ID NO:22, which corresponds to the coding sequence of His-tagged mycodulcein in S. cerevisiae, where residues 364-381 correspond to the optional His-tag sequence. SEQ ID NO:22 is codon-optimized for expression in S. cerevisiae. The corresponding polypeptide of SEQ ID NO:21 corresponds to a His-tagged mycodulcein protein, where residues 122-127 correspond to the optional His-tag sequence, and the polypeptide sequence is the same for expression in E. coli and S. cerevisiae. Thus, the present invention also provides a polypeptide comprising the amino acid sequence of SEQ ID NO:21. The present invention also provides the polynucleotide of SEQ ID NO:20, excluding the sequence encoding the histidine tag, as well as polynucleotide sequences having 80% sequence identity to the polynucleotide sequence of SEQ ID NO:20, excluding the sequence encoding the histidine tag.

[0036]

[0046] Another embodiment includes a polynucleotide (e.g., an isolated polynucleotide) selected from the group consisting of: (a) a polynucleotide comprising a nucleic acid sequence set forth in SEQ ID NO:2, optionally containing at least one and up to 72 alterations (e.g., deletions, insertions, substitutions, or additions); and (b) a polynucleotide comprising a nucleic acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the nucleic acid sequence set forth in SEQ ID NO:2, wherein optionally the polynucleotide is not the polynucleotide of SEQ ID NO:2. Where a polynucleotide sequence has multiple modifications, the number of modifications the polynucleotide sequence has can range from at least 1 to up to 72 modifications (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 modifications, as desired). Furthermore, unless expressly stated otherwise, when a polynucleotide sequence has multiple nucleotide modifications, each modification can be independently modified with a selected modification, such as deletion, insertion, substitution, or addition, regardless of what the other modifications are.In addition, multiple modifications in a polynucleotide sequence can be the same or different from each other; the modification options of each modification can be independently varied, such as deletion, insertion, substitution, or addition, unless expressly stated otherwise.When multiple modifications are present, each modification can be independently substituted, added, inserted, or deleted, regardless of what the other modifications are.In one embodiment, the polynucleotide sequence has at least one substitution modification.In certain embodiments, the polynucleotide sequence has multiple substitution modifications; each substitution can be independently selected substitution regardless of what other substitutions are.In addition, multiple substitutions in a polynucleotide sequence can be the same or different from each other; the nucleotide substitution choice of each substitution can be independently varied unless expressly stated otherwise.When a polynucleotide sequence has multiple substitutions, each substitution can be independently selected regardless of what other substitutions are.In an embodiment, the polynucleotide described herein is optionally isolated and / or optionally purified.

[0037]

[0047] In embodiments, at least 80% sequence identity for polynucleotides includes, but is not limited to, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity. In embodiments, at least 80% sequence identity for polynucleotides also includes, but is not limited to, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity.

[0038]

[0048] Another embodiment of the present invention comprises, consists essentially of, or consists of a polynucleotide (e.g., an isolated polynucleotide) selected from the group consisting of: (a) a polynucleotide comprising SEQ ID NO:2 having at least 1 and up to 72 substitution modifications; (b) a polynucleotide having at least 80% (at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%) sequence identity to SEQ ID NO:2, optionally other than a polynucleotide of SEQ ID NO:2; and (c) a polynucleotide comprising (i) SEQ ID NO:2 and (ii) a nucleotide sequence encoding a histidine tag, wherein the polynucleotide encodes a polypeptide having sweet taste modulating activity.

[0039]

[0049] In another embodiment, the polynucleotide comprises SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:47, or SEQ ID NO:55, or a nucleic acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:47, or SEQ ID NO:55.

[0040]

[0050] In one embodiment, the polynucleotide comprises a polynucleotide sequence of SEQ ID NO:27 (corresponding to a protein of SEQ ID NO:28 with an R20K mutation compared to the wild-type protein of SEQ ID NO:3) or a polynucleotide with at least 80% sequence identity to SEQ ID NO:27. In one embodiment, the polynucleotide comprises SEQ ID NO:31 (corresponding to a protein of SEQ ID NO:32 with an E35D mutation compared to the wild-type protein) or at least 80% sequence identity to SEQ ID NO:31. In one embodiment, the polynucleotide comprises SEQ ID NO:33 (corresponding to a protein of SEQ ID NO:34 with a K44R mutation compared to the wild-type protein) or at least 80% sequence identity to SEQ ID NO:33. In one embodiment, the polynucleotide comprises SEQ ID NO:35 (corresponding to a protein of SEQ ID NO:36 with a D46E mutation) or at least 80% sequence identity to SEQ ID NO:35. In one embodiment, the polynucleotide comprises SEQ ID NO:39 (corresponding to a protein of SEQ ID NO:40 with a D52E mutation) or at least 80% sequence identity to SEQ ID NO:39. In one embodiment, the polynucleotide comprises SEQ ID NO: 47 (corresponding to the protein of SEQ ID NO: 48 having a R75K mutation) or at least 80% sequence identity to SEQ ID NO: 47. In one embodiment, the polynucleotide comprises SEQ ID NO: 55 (corresponding to the protein of SEQ ID NO: 56 having a D94E mutation) or at least 80% sequence identity to SEQ ID NO: 55.

[0041]

[0051] Each of the polynucleotides of SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:47, and SEQ ID NO:45 encodes a histidine tag.

[0042]

[0052] In embodiments, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:47, or SEQ ID NO:45, excluding the sequence encoding the histidine tag. In further embodiments, the polynucleotide comprises a polynucleotide having 80% sequence identity to the polynucleotide sequence of SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:47, and SEQ ID NO:45, excluding the sequence encoding the histidine tag in each recited SEQ ID NO.

[0043]

[0053] In one embodiment, the polynucleotide comprises: (a) an amino acid sequence as set forth in SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140; The amino acid sequence set forth in sequence number 3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140, and a sequence which is at least 80% (e.g., at least 81%, at least 82%, at least 84%, at least 86%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 109 ...5%, at least 106%, at least 109%, at least 109%, at least 109%, at least 105 2%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, by deletion, insertion, substitution, or addition of 24 or less amino acids; and , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140.In the above recited embodiments, the polypeptide encoded by the polynucleotide is any polypeptide other than the polypeptide encoded by SEQ ID NO:3. When an amino acid sequence has multiple modifications, the number of modifications that an amino acid has may range from at least 1 to up to 24 modifications, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 modifications, as required. Furthermore, unless expressly stated otherwise, when an amino acid sequence has multiple modifications, each modification may be independently modified with a selected modification, such as deletion, insertion, substitution, or addition, regardless of what other modifications are. In addition, the multiple modifications in an amino acid sequence may be the same or different from each other; the modification options for each modification may be independently varied, such as deletion, insertion, substitution, or addition, unless expressly stated otherwise. When an amino acid sequence has multiple modifications, each modification may be independently substituted, added, inserted, or deleted, regardless of what the other modifications are. In one embodiment, the amino acid sequence has at least one substitution modification. In a particular embodiment, the amino acid sequence has multiple substitution modifications; each substitution modification may be independently replaced with a selected substitution, regardless of what the other substitutions are. In addition, the multiple substitutions in an amino acid sequence may be the same or different from each other; the substitution options of each amino acid may be independently varied, unless expressly stated otherwise. When an amino acid sequence has multiple substitutions, each substitution may be independently selected, regardless of what the other substitutions are. In some embodiments, the encoded polypeptide having sweet taste modulating activity includes the amino acid sequence of SEQ ID NO: 3, a sequence having at least 80% sequence identity with SEQ ID NO: 3, or an amino acid modified from the sequence of SEQ ID NO: 3 by deletion, insertion, substitution, or addition of 24 or less amino acids.

[0044]

[0054] Particular embodiments include: (a) a polypeptide sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140; (b) a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO: and (c) a polynucleotide (e.g., an isolated polynucleotide) encoding a polypeptide selected from the group consisting of a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140, modified by deletion, insertion, substitution, or addition of 24 or less amino acids. The encoded polypeptide has sweet taste modulating activity and is different from the polypeptide of SEQ ID NO:3.

[0045]

[0055] In one embodiment, the polynucleotide comprises: (a) an amino acid sequence set forth in SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140; (b) an amino acid sequence set forth in SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140, and / or a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%) identical to the amino acid sequence set forth in SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO %, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and (c) a polypeptide sequence comprising, consisting essentially of, or consisting of a polypeptide selected from the group consisting of an amino acid sequence modified from SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140 by deletion, insertion, substitution, or addition of 24 or less amino acids. In the above-listed embodiments, the polypeptide encoded by the polynucleotide is any polypeptide other than the polypeptide encoded by SEQ ID NO:3. In an embodiment, the polypeptide encoded by the polynucleotide has sweet taste modulating activity.When an amino acid sequence has multiple modifications, the number of modifications that the amino acid has may range from at least 1 to up to 24 modifications, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 modifications, as desired. Furthermore, unless expressly stated otherwise, when an amino acid sequence has multiple modifications, each modification may be independently modified with a selected modification, such as deletion, insertion, substitution, or addition, regardless of what the other modifications are. In addition, the multiple modifications in an amino acid sequence may be the same or different from each other; the modification options for each modification may be independently varied, such as deletion, insertion, substitution, or addition, unless expressly stated otherwise. When an amino acid sequence has multiple modifications, each modification may be independently modified with a selected modification, such as deletion, insertion, substitution, or deletion, regardless of what the other modifications are. In one embodiment, the amino acid sequence has at least one substitution modification. In certain embodiments, the amino acid sequence has multiple substitution modifications; each substitution modification may be independently substituted with a selected substitution, regardless of what other substitutions are. In addition, multiple substitutions in an amino acid sequence may be the same or different from each other; the substitution options of each amino acid may be independently varied, unless expressly stated otherwise. When an amino acid sequence has multiple substitutions, each substitution may be independently selected, regardless of what other substitutions are. In some embodiments, the encoded polypeptide with sweet taste modulating activity includes the amino acid sequence of SEQ ID NO: 3, a sequence having at least 80% sequence identity with SEQ ID NO: 3, or an amino acid modified from the sequence of SEQ ID NO: 3 by deletion, insertion, substitution, or addition of 24 or less amino acids.

[0046]

[0056] Particular embodiments include: (a) a polypeptide sequence selected from the group consisting of SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140; (b) a polypeptide sequence selected from the group consisting of SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140. and (c) a polypeptide sequence modified from a polypeptide sequence selected from the group consisting of SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140 by deletion, insertion, substitution, or addition of 24 or fewer amino acids. The encoded polypeptide has sweet taste modulating activity and is distinct from the polypeptide of SEQ ID NO:3.

[0047]

[0057] In one embodiment, the polynucleotide comprises: (a) the amino acid sequence set forth in SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140; (b) the amino acid sequence set forth in SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140; and (c) an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and (c) a polypeptide sequence comprising, consisting essentially of, or consisting of a polypeptide selected from the group consisting of an amino acid sequence modified from SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140 by deletion, insertion, substitution, or addition of 24 or less amino acids. In the above-listed embodiments, the polypeptide encoded by the polynucleotide is any polypeptide other than the polypeptide encoded by SEQ ID NO:3. In the above-listed embodiments, the polynucleotide optionally further encodes a protein tag, in particular a histidine tag.

[0048]

[0058] In certain embodiments of the polynucleotides described herein, the polynucleotides code for the polypeptides described herein and also code for histidine tags.In all cases where such certain polynucleotides code for histidine tags, the present invention also provides polynucleotides that exclude the sequence that codes for histidine tags.In additional embodiments, in the polynucleotides described herein that code for histidine tags, the sequence that codes for histidine tags can be deleted or eliminated, or can be replaced with the coding sequence of a different protein tag, such as a different histidine tag.

[0049]

[0059] Particular embodiments include (a) a polynucleotide (e.g., an isolated polynucleotide) encoding a polypeptide selected from the group consisting of a polypeptide sequence selected from the group consisting of SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:141.

[0050]

[0060] The invention provides isolated and / or purified embodiments of each polynucleotide, where applicable. The invention provides isolated or non-isolated, purified or non-purified embodiments of each polynucleotide of the SEQ ID NOs described herein, where applicable. The invention provides embodiments of each polynucleotide of the SEQ ID NOs described herein, where applicable, having one or more mutations.

[0051]

[0061] The invention also provides host cells transformed with expression cassettes and vectors containing one or more polynucleotides encoding Myd polypeptides.

[0052]

[0062] The polynucleotides encoding Myd of the present invention may be in the form of single-stranded or double-stranded DNA, RNA, or artificial nucleic acids, or may be cDNA or chemically synthesized DNA that does not contain any introns. The term "MYD family" may refer to (1) naturally occurring alleles, mutants, alleles, and polymorphic variants, including interspecies homologs, that encode polypeptides having at least about 35-50% amino acid sequence identity, optionally about 60, 75, 80, 85, 90, 95, 96, 97, 98, or 99% amino acid sequence identity with SEQ ID NO:3, over a range of about 25 amino acids, optionally over a range of 50-100 amino acids. In one embodiment, the term "isolated" encompasses products that have been removed from a biological environment (e.g., cells, tissues, culture medium, body fluids, etc.) or that have otherwise been subjected to any degree of increased purity (e.g., products isolated from synthetic media). Thus, isolated products may be synthetic or naturally produced.

[0053]

[0063] The term "nucleic acid" or "nucleic acid sequence" refers to a deoxy-ribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides. The term also encompasses nucleic acid-like structures having synthetic backbones (see, e.g., Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, Oxford Univ. Press (1991); Antisense Strategies, Annals of the NYAcademy of Sciences, Vol. 600, Baserga et al. (NYAS 1992); Milligan J. Med. Chem. 36:1923-1937 (1993); Antisense Research and Applications (1993, CRC Press), WO97 / 03211; WO96 / 39154; Mata, Toxicol. Appl. Pharmacol. 144:189-197 (1997); Strauss-Soukup, Biochemistry 36:8692-8698 (1997); Samstag, Antisense Nucleic Acid Drug Dev, 6:153-156 (1996).

[0054]

[0064] A "nucleic acid probe or oligonucleotide" as used herein is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through the formation of hydrogen bonds. A probe as used herein may contain natural bases (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe may be joined by linkages other than phosphodiester bonds, so long as they do not interfere with hybridization. Thus, for example, a probe may be a peptide nucleic acid in which the constituent bases are joined by peptide bonds rather than phosphodiester bonds. Those skilled in the art will understand that a probe may bind to a target sequence lacking complete complementarity with the probe sequence, depending on the stringency of the hybridization conditions. A probe may optionally be directly labeled, for example, with an isotope, chromophore, lumiphore, chromogen, or indirectly labeled, such as with biotin, to which a streptavidin complex may subsequently bind. By assaying for the presence or absence of the probe, the presence or absence of a selected sequence or subsequence can be detected.

[0055]

[0065] A polynucleotide or polypeptide may be naturally occurring or non-naturally occurring (e.g., synthetic, recombinant, modified, and / or variant products). In one embodiment, a naturally occurring or non-naturally occurring product is isolated or purified. In another embodiment, a naturally occurring or non-naturally occurring product is not isolated or purified.

[0056]

[0066] "Recombinant," as used herein, refers to a polynucleotide that has been synthesized or otherwise manipulated in vitro (e.g., a "recombinant polynucleotide"), a method of using a recombinant polynucleotide to produce a gene product in a cell or other biological system, or a polypeptide encoded by a recombinant polynucleotide (a "recombinant protein"). "Recombinant means" also encompasses the ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into an expression cassette or vector for, e.g., inducible or constitutive expression, for expression of a fusion protein comprising a translocation domain of the invention and a nucleic acid sequence amplified using the primers of the invention.

[0057]

[0067] The terms "amplifying" and "amplification," as used herein, refer to the use of any suitable amplification technique to generate or detect recombinant or naturally expressed nucleic acids, as described in detail below. For example, the invention provides methods and reagents (e.g., specific degenerate oligonucleotide primer pairs) for amplifying (e.g., by polymerase chain reaction, PCR) naturally expressed (e.g., genomic or mRNA) or recombinant (e.g., cDNA) nucleic acids of the invention (e.g., taste stimuli-binding sequences of the invention) in vivo or in vitro.

[0058]

[0068] As used herein, the term "isolated" refers to a situation of purification or enrichment different from that found in nature when referring to a nucleic acid or polypeptide. Any degree of purification or enrichment greater than that found in nature, including (1) purification from other naturally occurring associated structures or compounds, or (2) association with structures or compounds not normally associated in the body, is within the meaning of "isolated" as used herein. The nucleic acids or polypeptides described herein may be isolated or otherwise associated with structures or compounds with which they are not normally associated in nature, according to various methods and processes known to those of skill in the art. In one embodiment, the polypeptides described herein contain up to 5% (e.g., up to 4%, up to 3%, up to 2%, up to 1%) of other fungal proteins by weight.

[0059]

[0069] "Modified" or "variant" products refer to products (e.g., polynucleotides or polypeptides) that are altered from the original (e.g., naturally occurring) structure. Variants, as described herein, include polynucleotides or polypeptides that have one or more alterations to the nucleic acid or amino acid sequence, respectively. Alterations include alterations to the nucleic acid or amino acid sequence, such as additions, deletions, insertions, and substitutions. Modified or variant products can also include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling moiety, compared to the original structure.

[0060]

[0070] Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences in addition to the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved, for example, by generating sequences in which the third position of one or more selected codons is replaced with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The term nucleic acid is used synonymously with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0061]

[0071] It will be understood that where a particular polynucleotide is indicated herein as encoding a histidine tag, polynucleotides are also provided which exclude the sequence encoding the histidine tag. 6 It will further be understood that for any specific polynucleotide indicated as encoding a histidine tag, the sequence encoding a histidine tag can be replaced with a sequence encoding a different His tag or a sequence encoding a different protein tag.

[0062] Polypeptides

[0072] It is to be understood that embodiments of the present invention also encompass Myd polypeptides encoded by one or more polynucleotides. The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term also applies to naturally occurring and non-naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids.

[0063]

[0073] In some embodiments of the invention, a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140 is provided. Optionally, the polypeptide comprises, consists essentially of, or consists of a polypeptide sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the polypeptide. Optionally, the polypeptide is not the polypeptide of SEQ ID NO: 3. Optionally, the amino acid sequence has at least 1 and up to 24 modifications. When an amino acid sequence has multiple modifications, the number of modifications that the amino acid has may range from at least 1 to up to 24 modifications, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 modifications, as desired. Furthermore, unless expressly stated otherwise, when an amino acid sequence has multiple modifications, each modification may be independently modified with a selected modification, such as deletion, insertion, substitution, or addition, regardless of what the other modifications are. In addition, the multiple modifications in an amino acid sequence may be the same or different from each other; the modification options for each modification may be independently varied, such as deletion, insertion, substitution, or addition, unless expressly stated otherwise. When an amino acid sequence has multiple modifications, each modification may be independently modified with a selected modification, such as deletion, insertion, substitution, or deletion, regardless of what the other modifications are.In one embodiment, the amino acid sequence has at least one substitution modification. In certain embodiments, the amino acid sequence has multiple substitution modifications; each substitution modification may be independently substituted with a selected substitution, regardless of what the other substitutions are. In addition, multiple substitutions in an amino acid sequence may be the same or different from each other; the substitution options for each amino acid may be independently varied, unless expressly stated otherwise. When an amino acid sequence has multiple substitutions, each substitution may be independently selected, regardless of what the other substitutions are. The term "consists essentially of" allows for the inclusion of ingredients that are not essential to and do not significantly affect the function or activity of the product, such as anti-caking agents, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltodextrose, gum arabic, etc.).

[0064]

[0074] In one embodiment, the polypeptide comprises SEQ ID NO:3 or a polypeptide sequence having at least 80% sequence identity to SEQ ID NO:3. In one embodiment, the polypeptide comprises SEQ ID NO:8 or a polypeptide having at least 80% sequence identity to SEQ ID NO:8. In one embodiment, the polypeptide comprises SEQ ID NO:9 or a polypeptide having at least 80% sequence identity to SEQ ID NO:9. In one embodiment, the polypeptide comprises SEQ ID NO:10 or a polypeptide having at least 80% sequence identity to SEQ ID NO:10. In one embodiment, the polypeptide comprises SEQ ID NO:12 or a polypeptide having at least 80% sequence identity to SEQ ID NO:12. In one embodiment, the polypeptide comprises SEQ ID NO:13 or a polypeptide having at least 80% sequence identity to SEQ ID NO:13. In one embodiment, the polypeptide comprises SEQ ID NO:14 or a polypeptide having at least 80% sequence identity to SEQ ID NO:14. In one embodiment, the polypeptide comprises SEQ ID NO:15 or a polypeptide having at least 80% sequence identity to SEQ ID NO:15. In one embodiment, the polypeptide comprises SEQ ID NO:16 or comprises at least 80% sequence identity to SEQ ID NO:16. In one embodiment, the polypeptide comprises SEQ ID NO:17 or at least 80% sequence identity to SEQ ID NO:17.

[0065]

[0075] In one embodiment, the polypeptide comprises SEQ ID NO:78 or a polypeptide sequence having at least 80% sequence identity to SEQ ID NO:78. In one embodiment, the polypeptide comprises SEQ ID NO:81 or a polypeptide having at least 80% sequence identity to SEQ ID NO:81. In one embodiment, the polypeptide comprises SEQ ID NO:84 or a polypeptide having at least 80% sequence identity to SEQ ID NO:84. In one embodiment, the polypeptide comprises SEQ ID NO:87 or a polypeptide having at least 80% sequence identity to SEQ ID NO:87. In one embodiment, the polypeptide comprises SEQ ID NO:92 or a polypeptide having at least 80% sequence identity to SEQ ID NO:92. In one embodiment, the polypeptide comprises SEQ ID NO:95 or a polypeptide having at least 80% sequence identity to SEQ ID NO:95. In one embodiment, the polypeptide comprises SEQ ID NO:98 or a polypeptide having at least 80% sequence identity to SEQ ID NO:98. In one embodiment, the polypeptide comprises SEQ ID NO:101 or a polypeptide having at least 80% sequence identity to SEQ ID NO:101. In one embodiment, the polypeptide comprises SEQ ID NO:106 or comprises at least 80% sequence identity to SEQ ID NO:106. In one embodiment, the polypeptide comprises SEQ ID NO: 109 or comprises at least 80% sequence identity to SEQ ID NO: 109. In one embodiment, the polypeptide comprises SEQ ID NO: 114 or comprises at least 80% sequence identity to SEQ ID NO: 114. In one embodiment, the polypeptide comprises SEQ ID NO: 119 or comprises at least 80% sequence identity to SEQ ID NO: 119. In one embodiment, the polypeptide comprises SEQ ID NO: 124 or comprises at least 80% sequence identity to SEQ ID NO: 124. In one embodiment, the polypeptide comprises SEQ ID NO: 127 or comprises at least 80% sequence identity to SEQ ID NO: 127. In one embodiment, the polypeptide comprises SEQ ID NO: 132 or comprises at least 80% sequence identity to SEQ ID NO: 132. In one embodiment, the polypeptide comprises SEQ ID NO: 137 or comprises at least 80% sequence identity to SEQ ID NO: 137.In one embodiment, the polypeptide comprises SEQ ID NO:140 or at least 80% sequence identity to SEQ ID NO:140.

[0066]

[0076] In one embodiment, the polypeptide sequence is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140. Additionally, the polypeptide is optionally different from the polypeptide of SEQ ID NO:3.

[0067]

[0077] In one embodiment, the polypeptide comprises amino acid residues 1 to 11, 17 to 32, 39, 40, 45 to 67, 73 to 100, and 110 to 121 of SEQ ID NO: 3. Additionally, the polypeptide may differ from the polypeptide of SEQ ID NO: 3.

[0068]

[0078] In another embodiment, the polypeptide comprises amino acid residues 1 to 121 of SEQ ID NO: 3 and has at least 1 and up to 24 amino acid substitutions, additions, insertions, or deletions at amino acid residues 12 to 16, 33 to 38, 41 to 44, 68 to 72, or 101 to 109 compared to SEQ ID NO: 3. In another embodiment, the polypeptide having sweetness regulating activity comprises amino acid residues 1 to 121 of SEQ ID NO: 3 and has at least 1 and up to 24 amino acid substitutions, additions, insertions, or deletions at amino acid residues 12 to 16, 33 to 38, 41 to 44, 68 to 72, or 101 to 109 compared to SEQ ID NO: 3.

[0069]

[0079] Another embodiment of the present invention provides a recombinant polypeptide having sweet taste modulating activity, the polypeptide having at least 80% (e.g., 100% or more) affinity for SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140. For example, recombinant polypeptides comprising, consisting essentially of, or consisting of a sequence having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a heterologous signal peptide or transport peptide. The term "consisting essentially of" allows for the inclusion of ingredients that are not essential to and do not significantly affect the function or activity of the product, such as anti-caking agents, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltodextrose, gum arabic, etc.).

[0070]

[0080] Another embodiment of the present invention includes a polypeptide having sweet taste modulating activity. The polypeptide is selected from the group consisting of SEQ ID NO:3 (or SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140) and at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%). 9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140).In some embodiments, the polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 23, 24, 25, 26, 27, and having 1-24 amino acid substitutions at positions 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or 121. The term "consisting essentially of" allows for the inclusion of ingredients that are not essential to and do not significantly affect the function or activity of the product, such as anti-caking agents, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltodextrose, gum arabic, etc.).

[0071]

[0081] In certain embodiments, the polypeptide having sweet taste modulating activity comprises, consists essentially of, or consists of a sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140. The polypeptide may optionally be different from SEQ ID NO:3.

[0072]

[0082] In other particular embodiments, the polypeptide having sweetness modulating activity is selected from the group consisting of (i) the polypeptide sequences of SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140; These include polypeptides having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the polypeptide sequences of SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140, where the polypeptide is optionally different from SEQ ID NO:3.

[0073]

[0083] In one embodiment, the polypeptide having sweet taste modulating activity comprises a modified SEQ ID NO: 3, wherein the peptide has at least one to up to 24 amino acid modifications as shown in Table 6 or Table 7. In another embodiment, the polypeptide has at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with a modified polypeptide of SEQ ID NO: 3, wherein the polypeptide optionally differs from SEQ ID NO: 3.

[0074]

[0084] In one embodiment, a polypeptide (e.g., an isolated polypeptide) comprises, consists essentially of, or consists of a polypeptide sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to a sequence selected from the group consisting of the sequences: SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140. The polypeptide optionally further comprises a protein tag, in particular a histidine tag, and differs from the polypeptide of SEQ ID NO:3.

[0075]

[0085] In one embodiment, the polypeptide (e.g., isolated polypeptide) comprises, consists essentially of, or consists of a polypeptide sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to a sequence selected from the group consisting of the sequences of SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140. The polypeptide optionally further comprises a protein tag, in particular a histidine tag. The polypeptide is optionally different from the polypeptide of SEQ ID NO:3. In one embodiment, the polypeptide is not the polypeptide of SEQ ID NO:3.

[0076]

[0086] The present invention also provides a polypeptide comprising the amino acid sequence of SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:48, or SEQ ID NO:56, or an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:48, or SEQ ID NO:56.

[0077]

[0087] In one embodiment, the polypeptide comprises SEQ ID NO:28 (having an R20K mutation compared to the polypeptide of SEQ ID NO:3) or a polypeptide having at least 80% sequence identity to SEQ ID NO:28. In one embodiment, the polypeptide comprises SEQ ID NO:32 (having an E35D mutation) or at least 80% sequence identity to SEQ ID NO:32. In one embodiment, the polypeptide comprises SEQ ID NO:34 (having a K44R mutation) or at least 80% sequence identity to SEQ ID NO:34. In one embodiment, the polypeptide comprises SEQ ID NO:36 (having a D46E mutation) or at least 80% sequence identity to SEQ ID NO:36. In one embodiment, the polypeptide comprises SEQ ID NO:40 (having a D52E mutation) or at least 80% sequence identity to SEQ ID NO:40. In one embodiment, the polypeptide comprises SEQ ID NO:48 (having an R75K mutation) or at least 80% sequence identity to SEQ ID NO:48. The polypeptide comprises SEQ ID NO:56 (having a D94E mutation) or at least 80% sequence identity to SEQ ID NO:56.

[0078]

[0088] In one embodiment, the polypeptide comprises SEQ ID NO:78 or a polypeptide having at least 80% sequence identity to SEQ ID NO:78. In one embodiment, the polypeptide comprises SEQ ID NO:81 or a polypeptide having at least 80% sequence identity to SEQ ID NO:81. In one embodiment, the polypeptide comprises SEQ ID NO:84 or a polypeptide having at least 80% sequence identity to SEQ ID NO:84. In one embodiment, the polypeptide comprises SEQ ID NO:87 or a polypeptide having at least 80% sequence identity to SEQ ID NO:87. In one embodiment, the polypeptide comprises SEQ ID NO:92 or a polypeptide having at least 80% sequence identity to SEQ ID NO:92. In one embodiment, the polypeptide comprises SEQ ID NO:95 or a polypeptide having at least 80% sequence identity to SEQ ID NO:95. In one embodiment, the polypeptide comprises SEQ ID NO:98 or a polypeptide having at least 80% sequence identity to SEQ ID NO:98. In one embodiment, the polypeptide comprises SEQ ID NO:101 or a polypeptide having at least 80% sequence identity to SEQ ID NO:101. In one embodiment, the polypeptide comprises SEQ ID NO:106 or a polypeptide having at least 80% sequence identity to SEQ ID NO:106. In one embodiment, the polypeptide comprises SEQ ID NO:109 or a polypeptide having at least 80% sequence identity to SEQ ID NO:109. In one embodiment, the polypeptide comprises SEQ ID NO:114 or a polypeptide having at least 80% sequence identity to SEQ ID NO:114. In one embodiment, the polypeptide comprises SEQ ID NO:119 or a polypeptide having at least 80% sequence identity to SEQ ID NO:119. In one embodiment, the polypeptide comprises SEQ ID NO:124 or a polypeptide having at least 80% sequence identity to SEQ ID NO:124. In one embodiment, the polypeptide comprises SEQ ID NO:127 or a polypeptide having at least 80% sequence identity to SEQ ID NO:127. In one embodiment, the polypeptide comprises SEQ ID NO:132 or a polypeptide having at least 80% sequence identity to SEQ ID NO:132.In one embodiment, the polypeptide comprises SEQ ID NO: 137 or a polypeptide having at least 80% sequence identity to SEQ ID NO: 137. In one embodiment, the polypeptide comprises SEQ ID NO: 140 or a polypeptide having at least 80% sequence identity to SEQ ID NO: 140.

[0079]

[0089] In certain embodiments described herein, certain polypeptides are described as having a histidine tag or as having an optional histidine tag (represented in the sequence listing described herein as XXXXXX, where X is His).The present invention provides all of the specific polypeptides that include or optionally include a histidine tag, in addition to the corresponding polypeptides that exclude the histidine tag.The present invention also provides all of the specific polypeptides that include a histidine tag or an optional histidine tag, in addition to the polypeptides that exclude the histidine tag or that replace the histidine tag with a different protein tag, such as a different histidine tag.

[0080]

[0090] In one embodiment, the Myd peptides described above may have sweet taste modulating activity. The Myd polypeptides of the present invention may have, for example, functional, physical and chemical actions on taste receptors, such as sweet taste receptors. "Sweet taste modulating activity" may refer to the inhibition, activation, e.g., agonist or antagonist properties of the polypeptides of the present invention identified using in vitro and in vivo assays related to taste transduction. Proteins with inhibitory activity can bind to partially or completely block stimulation, reduce, prevent, delay activation, inactivate, desensitize or downregulate taste transduction, e.g., can be antagonists. Activating polypeptides can bind to stimulate, increase, open, activate, promote, enhance activity, sensitize or upregulate taste transduction, e.g., can be agonists. Activating polypeptides are preferred.

[0081]

[0091] Sweetness modulation also refers to enhancing the taste, eg, sweetness, of a particular product for oral administration when administered in combination.

[0082]

[0092] In some embodiments, Myd polypeptides of the invention include polypeptides that are at least as sweet (on a w / w basis) as sugar (e.g., 1x), or alternatively, 2x, 5x, 10x, 50x, 100x, 200x, 400x, 600x, 800x, 1000x, 1500x, 2000x, 3000x, 5000x, 10,000x, 20,000x or more sweeter than sugar, as measured by any of the methods described above or known in the art. In other embodiments, the Myd polypeptide is at least 1% (at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) of the sweetness of the sugar.

[0083]

[0093] In an embodiment, the polypeptide having sweet taste modulating activity comprises a modified SEQ ID NO:3 having from 1 up to 24 different amino acid modifications as shown in Table 3 or Table 6.

[0084]

[0094] In some embodiments, the polypeptide having sweet taste modulating activity comprises a modified SEQ ID NO: 3 having from 1 to up to 24 different amino acid modifications, as set forth in SEQ ID NO: 24, 26, 30, 38, 42, 44, 46, 50, 52, 54, 58, 60, 62, 64, 66, 68, 78, 81, 84, 87, 92, 95, 98, 101, 106, 109, 114, 119, 124, 127, 132, 137, or 140 in Table 3.

[0085]

[0095] In an embodiment, the polypeptide having sweet taste modulating activity comprises a modified SEQ ID NO: 3 having one or more different amino acid modifications, as shown in SEQ ID NO: 24, 26, 30, 38, 42, 44, 46, 50, 52, 54, 58, 60, 62, 64, 66, or 68 in Table 3, or SEQ ID NO: 78, 81, 84, 87, 92, 95, 98, 101, 106, 109, 114, 119, 124, 127, 132, 137, or 140 in Table 6.

[0086]

[0096] In an embodiment, the polypeptide having sweet taste modulating activity comprises a modified SEQ ID NO:3 having from 1 up to 24 different amino acid modifications as shown in Table 6.

[0087]

[0097] In certain embodiments, the polypeptide having sweet taste modulating activity is selected from the group consisting of (i) a polypeptide as set forth in SEQ ID NO: 24, 26, 30, 38, 42, 44, 46, 50, 52, 54, 58, 60, 62, 64, 66, or 68 of Table 3, or SEQ ID NO: 78, 81, 84, 87, 92, 95, 98, 101, 106, 109, 114, 119, 124, 127, 132, 137, or 14 of Table 6. or (ii) a modified polypeptide of SEQ ID NO: 3 having from 1 to 24 amino acid modifications, as indicated by SEQ ID NO: 24, 26, 30, 38, 42, 44, 46, 50, 52, 54, 58, 60, 62, 64, 66, or 68 in Table 3, or SEQ ID NO: 78, 81, 84, 87, 92, 95, 98, 101, 106, 109, 114, 119, 120, 122, 124, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 19 3 having 1 up to 24 amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 modifications), as set forth in SEQ ID NO: 24, 127, 132, 137, or 140, and polypeptides having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity. The polypeptide may optionally vary from SEQ ID NO:3 and may optionally further comprise a protein tag, in particular a histidine tag.

[0088]

[0098] In certain embodiments, the polypeptide having sweet taste modulating activity includes (i) a polypeptide of SEQ ID NO: 95, SEQ ID NO: 98, SEQ ID NO: 101, SEQ ID NO: 119, SEQ ID NO: 124, SEQ ID NO: 132, SEQ ID NO: 137, or SEQ ID NO: 140, (ii) a polypeptide having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with a polypeptide of SEQ ID NO: 95, SEQ ID NO: 98, SEQ ID NO: 101, SEQ ID NO: 119, SEQ ID NO: 124, SEQ ID NO: 132, SEQ ID NO: 137, or SEQ ID NO: 140. The polypeptide may optionally be different from SEQ ID NO: 3 and may optionally further include a protein tag, in particular a histidine tag.

[0089]

[0099] In embodiments, at least 80% sequence identity for a polypeptide includes, but is not limited to, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity. In further embodiments, at least 80% sequence identity for a polypeptide also includes, but is not limited to, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity.

[0090] Comparison of sweetness and heat stability of modified and unmodified peptides

[0100] The Myd proteins described herein also include "analogs," or "conservative variants" and "mimetics" ("peptide mimetics") that have structure and activity substantially corresponding to the exemplary sequences. Thus, the term "conservative variant" or "analog" or "mimetics" refers to a polypeptide having an amino acid sequence that has been modified such that the change does not substantially alter the structure and / or activity of the polypeptide (of the conservative variant) as defined herein. Examples of these include conservatively modified variations of the amino acid sequence, i.e., amino acid substitutions, additions or deletions of residues that are not important for protein activity, or substitutions of important amino acids with residues that have similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitution does not substantially alter the structure and / or activity.

[0091]

[0101] More specifically, "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.

[0092]

[0102] For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.

[0093]

[0103] Such nucleic acid variations are "silent variations", which are a type of conservatively modified variation. All nucleic acid sequences described herein that code for a polypeptide also describe all possible silent variations of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid that codes for a polypeptide is implicitly included in each sequence described.

[0094]

[0104] Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, one exemplary guideline for selecting conservative substitutions includes (original residue followed by exemplary substitution): ala / gly or ser; arg / lys; asn / gln or his; asp / glu; cys / ser; gln / asn; gly / asp; gly / ala or pro; his / asn or gln; ile / leu or val; leu / ile or val; lys / arg or gln or glu; met / leu or tyr or ile; phe / met or leu or tyr; ser / thr; thr / ser; trp / tyr; tyr / trp or phe; val / ile or leu. An alternative exemplary guideline uses six groups containing amino acids that are conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (I); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W); (see also, e.g., Creighton, Proteins, W. H. Freeman and Company (1984); Schultz and Schimer, Principles of Protein Structure, Springer-Vrlag (1979)). Another alternative exemplary guideline uses the following six groups, in which proline is unique: 1) Gly (G), Ala (A), Val (V), Leu (L), Ile (I); 2) Ser (S), Cys (C), Thr (T), Met (M); 3) Pro (P); 4) Phe (F), Tyr (Y), Try (W); 5) His (H), Lys (K), Arg (R); and 6) Asp (D), Glu (E), Gln (N). Those skilled in the art will understand that the substitutions identified above are not the only possible conservative substitutions. For example, for some purposes, all charged amino acids can be considered as conservative substitutions for each other, regardless of whether they are positive or negative.In addition, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence can also be considered "conservatively modified variations." One of skill in the art will be familiar with the codon choices in a given host expressing a protein of interest.

[0095]

[0105] Nucleotide and amino acid sequence information for MYD family members can also be used to build models of the polypeptide that regulates sweetness in computer systems and how it interacts with the sweet taste receptor, as well as computer system models of the same. The sweet taste receptor is composed of a heterodimer of taste 1 receptor member 2 (T1R2) and taste 1 receptor member 3 (T1R3). These models are subsequently used to identify variants and mutations of Myd that can increase sweet taste receptor activation, and to identify more active versions of Myd.

[0096]

[0106] It is envisaged that various conservative mutations and substitutions are within the scope of the present invention.For example, it is expected that it is within the level of a person skilled in the art to carry out amino acid substitution using known protocols of recombinant gene technology such as PCR, gene cloning, site-directed mutagenesis of cDNA, transfection of host cells and in vitro transcription.Then, variants can be screened for the functional activity of taste receptor agonist.

[0097]

[0107] In embodiments, modified polypeptides of SEQ ID NO: 3 having one or more amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 modifications) as set forth in SEQ ID NO: 24, 26, 30, 38, 42, 44, 46, 50, 52, 54, 58, 60, 62, 64, 66, or 68 in Table 3 or as set forth in Table 6 exhibit enhanced sweetness compared to the unmodified polypeptide of SEQ ID NO: 3. In some embodiments, the sweetness of each modified SEQ ID NO:3 is enhanced by at least 10% (or enhanced by 10% to 100%, or enhanced by 10% to 200%, or enhanced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% or more) compared to the unmodified polypeptide of SEQ ID NO:3. In other embodiments, the modified polypeptide of SEQ ID NO:3 exhibits an equivalent sweetness compared to the unmodified polypeptide of SEQ ID NO:3. The equivalent sweetness of the modified polypeptide is at least 10% (or at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%) of the unmodified polypeptide of SEQ ID NO:3 compared to the unmodified polypeptide of SEQ ID NO:3, as described above. Sweetness was measured by any method known in the art for comparing sweetness, and more particularly, by the method for assessing sweetness as described herein.

[0098]

[0108] Sweet taste regulating activity may be detected by methods known in the art, for example, by in vitro methods or in vivo by animal or human sensory tests.Without wishing to be bound by any particular theory, Myd is involved in sweet taste activation, for example, it is an agonist of taste 1 receptor member 2 (T1R2) and / or taste 1 receptor member 3 (T1R3).However, Myd agonizes other taste receptors, such as bitter, umami, sour and salty tastes. Such functional effects can be measured by any means known to those of skill in the art, including measurements of binding to the taste receptor T1R via spectroscopic (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties, patch clamp techniques, voltage-sensitive dyes, whole-cell currents, efflux of radioisotopes, inducible markers, changes in transcriptional activation of the T1R gene; ligand binding assays; changes in voltage, membrane potential and conductance; ion flux assays; changes in intracellular second messengers such as cAMP, cGMP, and inositol triphosphate (IP3); changes in intracellular calcium levels; neurotransmitter release, and the like.

[0099]

[0109] Sensory testing (human or animal) can also be employed to determine whether a Myd candidate polypeptide has sweet taste modulating activity. Sensory evaluation is the scientific field that analyzes and measures human responses to the composition of food or beverage, such as appearance, feel, smell, texture, temperature and taste. Sometimes measurements using humans as instruments are necessary. The selection of an appropriate method for determining sweeteners can be determined by those skilled in the art, and examples include, for example, discrimination tests or discrimination tests designed to measure the likelihood that two products are perceptually different. Responses from evaluators are recorded for accuracy and statistically analyzed to see if any are more accurate than would be expected by mere chance.

[0100]

[0110] Sensory evaluation is the scientific field that analyzes and measures human responses to food and beverage composition, e.g., appearance, feel, smell, texture, temperature and taste. Measurements using humans as instruments are sometimes necessary. The sensory characteristics of flavor and texture were obvious properties that could not be easily measured by instruments, and the food industry first needed to develop this measurement tool. The selection of an appropriate method for determining the quality of a sensory stimulus, e.g., the sweetness of the proteins disclosed in the present invention, can be determined by one of skill in the art, and examples include, for example, discrimination tests or discrimination tests designed to measure the possibility that two products are sensorily different. In the case of sweetness perception, for example, one or more samples of 5% sucrose, 6% sucrose, 7% sucrose, 8% sucrose, 9% sucrose, 10% sucrose, and test samples can be ranked in order of sweetness intensity from low to high sweetness by trained judges. In the present invention, it is to be understood that there are many ways that one of skill in the art can measure the sensory differences.

[0101]

[0111] Brix measurement (or Brix scale) is a well-known application in the food and beverage industry to determine the pure sucrose content in water: 1 degree Brix (°Bx) = 1 g sucrose / 100 g solution, which expresses the strength of the solution as a mass percentage. 8°Bx is equivalent to approximately 8% sugar solution. As described in the examples, the purified polypeptide corresponding to SEQ ID NO: 21 was tasted (0.2 mL aliquot) at 0.03 mg / ml by trained sensory scientists and found to have a sweetness equivalent to 8°Bx (approximately 8% sugar solution) (see Examples 4, 5, 9, and 10). thermal stability

[0112] The thermostability of the sweetness-modifying polypeptide may affect the possible application of the polypeptide, for example, food applications at high temperatures. In an embodiment, the modified polypeptide of SEQ ID NO: 3 having one or more amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 modifications) as shown in SEQ ID NO: 24, 26, 30, 38, 42, 44, 46, 50, 52, 54, 58, 60, 62, 64, 66 or 68 in Table 3 or as shown in Table 6 or Table 7, shows comparable thermostability compared to the unmodified polypeptide of SEQ ID NO: 3. The equivalent thermal stability of a modified polypeptide is, as described above, substantially the same as compared to the unmodified polypeptide of SEQ ID NO:3, which in this specification means a change in thermal stability of less than or equal to 4.5% (including less than or equal to 1%, less than or equal to 2%, less than or equal to 3%, less than or equal to 4%) as compared to the thermal stability of the unmodified polypeptide of SEQ ID NO:3.

[0102]

[0113] In some embodiments, modified SEQ ID NO:3 having one or more amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 modifications) exhibits enhanced thermostability compared to unmodified SEQ ID NO:3, as shown by SEQ ID NO:24, 26, 30, 38, 42, 44, 46, 50, 52, 54, 58, 60, 62, 64, 66, or 68 in Table 3, or as shown in Table 6 or Table 7. In other embodiments, the enhanced thermal stability of the modified polypeptide is greater than 4.5% enhanced compared to the thermal stability of the unmodified polypeptide of SEQ ID NO: 3, as described above, including 4.5-10% enhanced, greater than 5% enhanced, greater than 6% enhanced, greater than 7% enhanced, greater than 8% enhanced, greater than 9% enhanced, greater than 5% to 10% enhanced, greater than 6% to 10% enhanced, greater than 7% to 10% enhanced, greater than 8% to 10% enhanced, or enhanced by 10%. Thermal stability is measured by any method known in the art for assessing thermal stability, more particularly by the methods for assessing thermal stability described herein.

[0103]

[0114] In embodiments, the sweetness modifying polypeptides described herein exhibit equivalent sweetness and equivalent thermostability compared to that of the unmodified polypeptide of SEQ ID NO:3.

[0104]

[0115] In embodiments, the sweetness modifying polypeptides described herein exhibit comparable sweetness and enhanced thermostability compared to that of the unmodified polypeptide of SEQ ID NO:3.

[0105]

[0116] In embodiments, the sweet taste modifying polypeptides described herein exhibit enhanced sweetness and comparable thermostability compared to that of the unmodified polypeptide of SEQ ID NO:3.

[0106]

[0117] In embodiments, the sweet taste modifying polypeptides described herein exhibit enhanced sweetness and enhanced thermostability compared to that of the unmodified polypeptide of SEQ ID NO:3.

[0107]

[0118] In some embodiments of modified polypeptides of SEQ ID NO:3 having one or more amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 modifications), as set forth in SEQ ID NOs:24, 26, 30, 38, 42, 44, 46, 50, 52, 54, 58, 60, 62, 64, 66, or 68 in Table 3, or as set forth in Table 6 or Table 7, the modified polypeptides exhibit comparable sweetness and comparable thermostability compared to that of the unmodified polypeptide of SEQ ID NO:3. In some embodiments of modified polypeptides of SEQ ID NO:3 having one or more amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 modifications), as shown in SEQ ID NO:24, 26, 30, 38, 42, 44, 46, 50, 52, 54, 58, 60, 62, 64, 66, or 68 in Table 3, or as shown in Table 6 or Table 7, the modified polypeptides exhibit enhanced sweetness and comparable thermostability compared to that of the unmodified polypeptide of SEQ ID NO:3. In some embodiments of modified polypeptides of SEQ ID NO: 3 having one or more amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 modifications), as shown in SEQ ID NO: 24, 26, 30, 38, 42, 44, 46, 50, 52, 54, 58, 60, 62, 64, 66, or 68 in Table 3, or as shown in Table 6 or Table 7, the modified polypeptides exhibit comparable sweetness and enhanced thermostability compared to that of the unmodified polypeptide of SEQ ID NO: 3.In some embodiments of modified polypeptides of SEQ ID NO:3 having one or more amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 modifications), as set forth in SEQ ID NOs:24, 26, 30, 38, 42, 44, 46, 50, 52, 54, 58, 60, 62, 64, 66, or 68 in Table 3, or as set forth in Table 6 or Table 7, the modified polypeptides exhibit enhanced sweetness and enhanced thermostability compared to that of the unmodified polypeptide of SEQ ID NO:3.

[0108]

[0119] In certain embodiments, the polypeptide sequence of the polypeptide having sweet taste modulating activity is (i) a modified polypeptide of SEQ ID NO: 3, wherein the modification is one or more amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 modifications) as shown in Table 6 or Table 7, or (ii) a modified polypeptide of SEQ ID NO: 3 and a polypeptide sequence that is at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%) of the modified polypeptide of SEQ ID NO: 3. %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3, wherein the modification is one or more amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 modifications) as shown in Table 6 or Table 7, wherein the modified polypeptide optionally differs from SEQ ID NO: 3 and optionally further comprises a histidine tag, and the modified polypeptide has sweet taste modulating activity.

[0109]

[0120] In one embodiment, modified polypeptides of SEQ ID NO: 3 having one or more amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 modifications) as shown in Table 6 or Table 7 exhibit enhanced sweetness compared to the unmodified polypeptide of SEQ ID NO: 3. In an embodiment, the sweetness is enhanced by at least 10% (or an enhancement in the range of 10% to 100%, or an enhancement in the range of 10% to 200%, or an enhancement of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% or more) compared to the sweetness of the unmodified polypeptide of SEQ ID NO: 3.

[0110]

[0121] In one embodiment, a modified polypeptide of SEQ ID NO: 3 having one or more amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 modifications) as shown in Table 6 or Table 7 exhibits equivalent sweetness compared to an unmodified polypeptide of SEQ ID NO: 3. The equivalent sweetness of a modified polypeptide is at least 10% (or at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%) sweeter than the unmodified polypeptide of SEQ ID NO: 3, as described above.

[0111]

[0122] In one embodiment, a modified polypeptide of SEQ ID NO: 3 having one or more amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 modifications) as shown in Table 6 or Table 7 exhibits comparable thermal stability compared to an unmodified polypeptide of SEQ ID NO: 3. Comparable thermal stability of a modified polypeptide is substantially the same as compared to an unmodified polypeptide of SEQ ID NO: 3, as described above, which herein means a change in thermal stability of less than or equal to 4.5% (including less than or equal to 1%, less than or equal to 2%, less than or equal to 3%, less than or equal to 4%) compared to the thermal stability of an unmodified polypeptide of SEQ ID NO: 3.

[0112]

[0123] In one embodiment, a modified polypeptide of SEQ ID NO:3 having one or more amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 modifications) as shown in Table 6 or Table 7 exhibits enhanced thermostability compared to an unmodified polypeptide of SEQ ID NO:3. The enhanced thermal stability of the modified polypeptide, as described above, compared to the unmodified polypeptide of SEQ ID NO:3, is enhanced by greater than 4.5% (including 4.5-10% enhanced, greater than 5% enhanced, greater than 6% enhanced, greater than 7% enhanced, greater than 8% enhanced, greater than 9% enhanced, greater than 5% up to 10% enhanced, greater than 6% up to 10% enhanced, greater than 7% up to 10% enhanced, greater than 8% up to 10% enhanced, or up to 10%) compared to the thermal stability of the unmodified polypeptide of SEQ ID NO:3.

[0113]

[0124] In one embodiment, a modified polypeptide of SEQ ID NO:3 having one or more amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 modifications) as shown in Table 6 or Table 7 exhibits equivalent sweetness and equivalent thermostability compared to that of the unmodified polypeptide of SEQ ID NO:3.

[0114] In one embodiment, a modified polypeptide of SEQ ID NO: 3 having one or more amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 modifications) as shown in Table 6 or Table 7 exhibits enhanced sweetness and equivalent thermostability compared to that of the unmodified polypeptide of SEQ ID NO: 3. In one embodiment, a modified polypeptide of SEQ ID NO: 3 having one or more amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 modifications) as shown in Table 6 or Table 7 exhibits equivalent sweetness and enhanced thermostability compared to that of the unmodified polypeptide of SEQ ID NO: 3. In one embodiment, a modified polypeptide of SEQ ID NO:3 having one or more amino acid modifications (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 modifications) as shown in Table 6 or Table 7 exhibits enhanced sweetness and enhanced thermostability compared to that of the unmodified polypeptide of SEQ ID NO:3.

[0115]

[0125] In some embodiments, the polypeptide exhibits sweetness regulating activity.Non-limiting examples of sweetness regulating activity include providing sweetness.In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75.

[0116]

[0126] SEQ ID NO:71 (consensus sequence 1) corresponds to SEQ ID NO:3, except that any amino acid can be selected from positions 3, 11 to 16, 26, 33, 34, 36 to 38, 41 to 43, 51, 57, 66, 68 to 72, 85, 86, 89, 97, 101 to 110, 117, and 120. The present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO:71.

[0117]

[0127] SEQ ID NO:72 (consensus sequence 2) corresponds to SEQ ID NO:3, except that positions 3, 11-16, 26, 33, 37, 38, 41, 43, 51, 57, 66, 68-70, 72, 85, 86, 89, 97, 101-103, 105-110, 117, and 120 are any amino acid (i.e., the prolines at positions 34, 36, 42, 71, and 104 of SEQ ID NO:3 are maintained). The present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO:72.

[0118]

[0128] SEQ ID NO:73 (consensus sequence 3) and SEQ ID NO:74 (consensus sequence 4) correspond to SEQ ID NO:3, except that positions 3, 11-16, 26, 33, 37, 38, 41, 43, 51, 57, 66, 68-70, 72, 85, 86, 89, 97, 101-103, 105-110, 117, and 120 may contain conservative modifications as described herein. The present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO:73. The present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO:74.

[0119]

[0129] SEQ ID NO:75 (consensus sequence 5) corresponds to SEQ ID NO:3, except that positions 3, 11, 26, 51, 57, 66, 69, 85, 86, 89, 97, 103, 106, 110, 117, and 120 may contain conservative modifications as described herein. The present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO:75.

[0120]

[0130] In a specific embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO:141.

[0121]

[0131] In one embodiment, the polypeptide having sweet taste modulating activity comprises SEQ ID NO: 3 having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16) modifications at residues 3, 11, 26, 51, 57, 66, 69, 85, 86, 89, 97, 103, 106, 110, 117, and 120 of SEQ ID NO: 3. Exemplary modifications (for polypeptides) related to SEQ ID NO: 3 are described herein (see Example 8; Table 3). In one illustrative example, but not limited to, the polypeptide sequence of the polypeptide having sweet taste modulating activity includes SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, or SEQ ID NO:68, or a sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, or SEQ ID NO:68.

[0122]

[0132] In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one mutation selected from those listed in Tables 3 and 7. In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one and up to 24 mutations at different amino acid positions selected from those listed in Tables 3 and 7.

[0123]

[0133] In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one mutation selected from those listed in Table 7. In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one and up to 24 mutations at different amino acid positions selected from those listed in Table 7.

[0124]

[0134] In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by two mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by three mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by four mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by five mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by six mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by seven mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by eight mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by nine mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by ten mutations selected from those listed in Table 7.In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 11 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 12 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 13 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 14 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 15 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 16 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 17 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 18 mutations selected from those listed in Table 7.In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 19 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 20 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 21 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 22 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweetness modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 23 mutations selected from those listed in Table 7. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by 24 mutations selected from those listed in Table 7. The present invention also provides polynucleotides encoding the aforementioned mutant polypeptides of SEQ ID NO: 3. The present invention further provides the aforementioned mutant polypeptides further comprising a protein tag, more specifically a histidine tag. The present invention also provides polynucleotides encoding the aforementioned mutant polypeptides of SEQ ID NO: 3 further comprising a protein tag, more specifically a histidine tag.

[0125]

[0135] In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one mutation selected from the mutations Q37K, Q37E, Q37N, V74A, V76A, V87A, V100A, or Q102K. In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different amino acid positions by at least one to up to six mutations selected from the mutations Q37K, Q37E, Q37N, V74A, V76A, V87A, V100A, or Q102K.

[0126]

[0136] In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by two mutations selected from the mutations Q37K, Q37E, Q37N, V74A, V76A, V87A, V100A or Q102K. In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by three mutations selected from the mutations Q37K, Q37E, Q37N, V74A, V76A, V87A, V100A or Q102K. In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by four mutations selected from the mutations Q37K, Q37E, Q37N, V74A, V76A, V87A, V100A or Q102K. In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by five mutations selected from the mutations Q37K, Q37E, Q37N, V74A, V76A, V87A, V100A or Q102K. In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by six mutations selected from the mutations Q37K, Q37E, Q37N, V74A, V76A, V87A, V100A or Q102K. The present invention also provides a polynucleotide encoding the aforementioned mutant polypeptide of SEQ ID NO: 3. The present invention further provides the aforementioned mutant polypeptide further comprising a protein tag, more specifically a histidine tag. The present invention also provides a polynucleotide encoding the aforementioned mutant polypeptide of SEQ ID NO: 3 further comprising a protein tag, more specifically a histidine tag.

[0127]

[0137] In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one mutation selected from those listed in Table 3. In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different amino acid positions by at least one to up to 24 mutations selected from those listed in Table 3. In another embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one mutation selected from those listed in Table 3. In another aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by one to 16 mutations selected from D3E, K11R, K26R, K51R, R57K, R66K, D69E, D85E, E86D, E89D, D97E, K103R, R106K, R110K, E117D, or K120R.

[0128]

[0138] In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by two mutations selected from D3E, K11R, K26R, K51R, R57K, R66K, D69E, D85E, E86D, E89D, D97E, K103R, R106K, R110K, E117D, or K120R. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by three mutations selected from D3E, K11R, K26R, K51R, R57K, R66K, D69E, D85E, E86D, E89D, D97E, K103R, R106K, R110K, E117D, or K120R. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by four mutations selected from D3E, K11R, K26R, K51R, R57K, R66K, D69E, D85E, E86D, E89D, D97E, K103R, R106K, R110K, E117D, or K120R. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by five mutations selected from D3E, K11R, K26R, K51R, R57K, R66K, D69E, D85E, E86D, E89D, D97E, K103R, R106K, R110K, E117D, or K120R. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by six mutations selected from D3E, K11R, K26R, K51R, R57K, R66K, D69E, D85E, E86D, E89D, D97E, K103R, R106K, R110K, E117D, or K120R.In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by seven mutations selected from D3E, K11R, K26R, K51R, R57K, R66K, D69E, D85E, E86D, E89D, D97E, K103R, R106K, R110K, E117D, or K120R. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by eight mutations selected from D3E, K11R, K26R, K51R, R57K, R66K, D69E, D85E, E86D, E89D, D97E, K103R, R106K, R110K, E117D, or K120R. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by nine mutations selected from D3E, K11R, K26R, K51R, R57K, R66K, D69E, D85E, E86D, E89D, D97E, K103R, R106K, R110K, E117D, or K120R. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by ten mutations selected from D3E, K11R, K26R, K51R, R57K, R66K, D69E, D85E, E86D, E89D, D97E, K103R, R106K, R110K, E117D, or K120R. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by eleven mutations selected from D3E, K11R, K26R, K51R, R57K, R66K, D69E, D85E, E86D, E89D, D97E, K103R, R106K, R110K, E117D, or K120R.In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by 12 mutations selected from D3E, K11R, K26R, K51R, R57K, R66K, D69E, D85E, E86D, E89D, D97E, K103R, R106K, R110K, E117D, or K120R. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by 13 mutations selected from D3E, K11R, K26R, K51R, R57K, R66K, D69E, D85E, E86D, E89D, D97E, K103R, R106K, R110K, E117D, or K120R. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by 14 mutations selected from D3E, K11R, K26R, K51R, R57K, R66K, D69E, D85E, E86D, E89D, D97E, K103R, R106K, R110K, E117D, or K120R. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by 15 mutations selected from D3E, K11R, K26R, K51R, R57K, R66K, D69E, D85E, E86D, E89D, D97E, K103R, R106K, R110K, E117D, or K120R. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 16 mutations selected from D3E, K11R, K26R, K51R, R57K, R66K, D69E, D85E, E86D, E89D, D97E, K103R, R106K, R110K, E117D, or K120R. The present invention also provides polynucleotides encoding the aforementioned mutant polypeptides of SEQ ID NO:3.The present invention further provides a mutant polypeptide as described above, which further comprises a protein tag, more particularly a histidine tag.The present invention also provides a polynucleotide encoding the mutant polypeptide as described above of SEQ ID NO:3, which further comprises a protein tag, more particularly a histidine tag.

[0129]

[0139] In another aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO:3, which is modified by at least one mutation selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R. In another aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified by 1 to 22 mutations at different positions selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R.

[0130]

[0140] In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by two mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by three mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by four mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by five mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R.In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by six mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by seven mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by eight mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by nine mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R.In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 10 mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by eleven mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 12 mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 13 mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R.In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 14 mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 15 mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 16 mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 17 mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R.In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 18 mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 19 mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 20 mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO:3 modified at different positions by 21 mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R.In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO: 3, which is modified at different positions by 22 mutations selected from D3E, K11R, K26R, Q37K, Q37E, Q37N, K51R, R57K, R66K, D69E, V74A, V76A, D85E, E86D, V87A, E89D, D97E, V100A, Q102K, K103R, R106K, R110K, E117D, or K120R. The present invention also provides a polynucleotide encoding the aforementioned mutant polypeptide of SEQ ID NO: 3. The present invention further provides the aforementioned mutant polypeptide further comprising a protein tag, more specifically a histidine tag. The present invention also provides a polynucleotide encoding the aforementioned mutant polypeptide of SEQ ID NO: 3, which further comprises a protein tag, more specifically a histidine tag.

[0131]

[0141] The following examples are illustrative and not limiting. A polypeptide having sweet taste modulating activity comprises SEQ ID NO:24 (having a D3E mutation) or at least 80% sequence identity with SEQ ID NO:24. A polypeptide having sweet taste modulating activity comprises SEQ ID NO:26 (having a K11R mutation) or at least 80% sequence identity with SEQ ID NO:26. A polypeptide having sweet taste modulating activity comprises SEQ ID NO:30 (having a K26R mutation) or at least 80% sequence identity with SEQ ID NO:30. A polypeptide having sweet taste modulating activity comprises SEQ ID NO:38 (having a K51R mutation) or at least 80% sequence identity with SEQ ID NO:38. A polypeptide having sweet taste modulating activity comprises SEQ ID NO:42 (having an R57K mutation) or at least 80% sequence identity with SEQ ID NO:42. A polypeptide having sweet taste modulating activity comprises SEQ ID NO:44 (having an R66K mutation) or at least 80% sequence identity with SEQ ID NO:44. A polypeptide having sweet taste modulating activity comprises SEQ ID NO: 46 (having a D69E mutation) or at least 80% sequence identity with SEQ ID NO: 46. A polypeptide having sweet taste modulating activity comprises SEQ ID NO: 50 (having a D85E mutation) or at least 80% sequence identity with SEQ ID NO: 50. A polypeptide having sweet taste modulating activity comprises SEQ ID NO: 52 (having an E86D mutation) or at least 80% sequence identity with SEQ ID NO: 52. A polypeptide having sweet taste modulating activity comprises SEQ ID NO: 54 (having an E89D mutation) or at least 80% sequence identity with SEQ ID NO: 54. A polypeptide having sweet taste modulating activity comprises SEQ ID NO: 58 (having a D97E mutation) or at least 80% sequence identity with SEQ ID NO: 58. A polypeptide having sweet taste modulating activity comprises SEQ ID NO: 60 (having a K103R mutation) or at least 80% sequence identity with SEQ ID NO: 60. A polypeptide having sweet taste modulating activity comprises SEQ ID NO: 62 (having an R106K mutation) or at least 80% sequence identity to SEQ ID NO: 62. A polypeptide having sweet taste modulating activity comprises SEQ ID NO: 64 (having an R110K mutation) or at least 80% sequence identity to SEQ ID NO: 64.A polypeptide having sweet taste modulating activity comprises SEQ ID NO: 66 (having an E117D mutation) or at least 80% sequence identity to SEQ ID NO: 66. A polypeptide having sweet taste modulating activity comprises SEQ ID NO: 68 (having a K120R mutation) or at least 80% sequence identity to SEQ ID NO: 68.

[0132]

[0142] Aspect 1: (a) a polypeptide sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:140, and SEQ ID NO:141; (b) a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140; and (c) a polypeptide sequence modified by deletion, insertion, substitution, or addition of 24 or fewer amino acids from a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140. An isolated polynucleotide encoding a polypeptide selected from the group consisting of: A polynucleotide wherein the encoded polypeptide has sweetness modulating activity and differs from the polypeptide of SEQ ID NO:3.

[0133]

[0143] Embodiment 2: The isolated polynucleotide according to embodiment 1, wherein the encoded polypeptide sequence comprises SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:141.

[0134]

[0144] Embodiment 3: The isolated polynucleotide according to embodiment 1, wherein the encoded polypeptide sequence comprises SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140.

[0135]

[0145] Embodiment 4: The isolated polynucleotide according to any one of embodiments 1 to 3, operably linked to a heterologous regulatory element.

[0136]

[0146] Embodiment 5. The isolated polynucleotide according to any one of embodiments 1 to 4, wherein the polynucleotide sequence further encodes a histidine tag.

[0137]

[0147] Embodiment 6: An expression cassette comprising the isolated polynucleotide according to any one of embodiments 1 to 5.

[0138]

[0148] Embodiment 7: A vector comprising the isolated polynucleotide according to any one of embodiments 1 to 6.

[0139]

[0149] Embodiment 8: A host cell transformed with a vector according to embodiment 7.

[0140]

[0150] Embodiment 9: A method for producing a protein having sweet taste modulating activity, comprising: Cultivating the host cells transformed with the vector in a medium under conditions for protein expression. Including, The vector comprises an isolated polynucleotide encoding a polypeptide sequence, the polypeptide sequence being (a) a polypeptide sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:140, and SEQ ID NO:141; (b) a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140; or (c) a polypeptide sequence modified by deletion, insertion, substitution, or addition of 24 or fewer amino acids from a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140. and The method, wherein the encoded polypeptide has sweet taste modulating activity and differs from the polypeptide of SEQ ID NO:3.

[0141]

[0151] Embodiment 10: A polypeptide having sweet taste modulating activity, (a) a polypeptide sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:140, and SEQ ID NO:141; (b) a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140; or (c) a polypeptide sequence modified by deletion, insertion, substitution, or addition of 24 or fewer amino acids from a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, and SEQ ID NO:140. Including, (a) the polypeptide is different from the polypeptide of SEQ ID NO:3; or (b) the polypeptide further comprises a protein tag.

[0142]

[0152] Embodiment 11: The polypeptide according to embodiment 10, comprising SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75 or SEQ ID NO:141; and which is different from SEQ ID NO:3.

[0143]

[0153] Embodiment 12: The polypeptide according to embodiment 10, comprising SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140.

[0144]

[0154] Embodiment 13: (a) SEQ ID NO:2 having at least one substitution alteration; (b) a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:2, wherein the polynucleotide is a nucleic acid sequence different from SEQ ID NO:2; (c) a polynucleotide comprising: (i) SEQ ID NO: 2 or a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 2; and (ii) a nucleotide sequence encoding a histidine tag, the polynucleotide encoding a polypeptide having sweetness modulating activity. 2. An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of:

[0145]

[0155] Embodiment 14: A polypeptide encoding a polypeptide having a sweetness modulating activity selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:140, and SEQ ID NO:141, or a polypeptide encoding a polypeptide having a sweetness modulating activity selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11 14. The polynucleotide of embodiment 13, encoding a polypeptide having sweetening activity having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:140, and SEQ ID NO:141.

[0146]

[0156] Embodiment 15: The polynucleotide sequence is selected from the group consisting of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99 , SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO: No. 139, or the polynucleotide sequence is SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97 , SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138,or SEQ ID NO: 139.

[0147]

[0157] Embodiment 16: The polynucleotide according to any one of embodiments 13 to 15, operably linked to a heterologous regulatory element.

[0148]

[0158] Embodiment 17: The polynucleotide according to any one of embodiments 13 to 16, further comprising a nucleotide sequence encoding a protein tag, optionally a histidine tag.

[0149]

[0159] Embodiment 18: An expression cassette or vector comprising a polynucleotide according to any one of embodiments 13 to 17.

[0150]

[0160] Embodiment 19: A host cell transformed with a vector according to embodiment 18.

[0151]

[0161] Embodiment 20: A method for producing a protein having sweet taste modulating activity, comprising culturing a host cell according to embodiment 19 in a medium under conditions that result in the production of a protein having sweet taste modulating activity.

[0152]

[0162] Aspect 21: (a) a product for oral administration, different from the truffles of Mattiromyces terfesioides, and (b) a sweetener composition comprising an isolated polypeptide having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140. A composition comprising a combination of (c) A composition, wherein the combination has enhanced sweetness compared to the product for oral administration.

[0153]

[0163] Embodiment 22: The composition according to embodiment 21, comprising a plurality of isolated polypeptides.

[0154]

[0164] Aspect 23: The composition according to any one of aspects 21 to 22 or aspect 29, wherein the product for oral administration is a food product selected from the group consisting of baked goods; sweet bakery products, pre-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings, gelatins and puddings; frozen desserts; yogurt; snack bars; bread products; pre-made bread mixes for preparing bread products; sauces, syrups and dressings; sweet spreads; confectionery products; and sweetened breakfast cereals.

[0155]

[0165] Embodiment 24: A method for modulating the taste of a product for oral administration, comprising: combining the product for oral administration with an effective amount of a sweetener composition comprising an isolated polypeptide having at least 80% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140. Including, The method, wherein the product for oral administration is different from the Mattiromyces terfesioides truffles, the isolated polypeptide is not the polypeptide of SEQ ID NO: 3, and the combination has an enhanced sweetness compared to the product for oral administration.

[0156]

[0167] Aspect 25: The method of aspect 24 or 30, wherein the product for oral administration is a food product selected from the group consisting of baked goods; sweet bakery products, pre-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings, gelatins and puddings; frozen desserts; yogurt; snack bars; bread products; pre-made bread mixes for preparing bread products; sauces, syrups and dressings; sweet spreads; confectionery products; and sweetened breakfast cereals.

[0157]

[0168] Embodiment 26: The method according to embodiment 24 or 25 or 30, wherein the product for oral administration is a beverage product selected from the group consisting of carbonated beverages; non-carbonated beverages; and beverage concentrates.

[0158]

[0169] Embodiment 27: A method for purifying a polypeptide having sweet taste modulating activity, comprising: (a) subjecting a polypeptide to hydrophobic interaction chromatography (HIC); and (b) then subjecting the polypeptide to size exclusion chromatography (SEC) Including, The method of claim 1, wherein the polypeptide comprises an amino acid sequence having at least 80% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140.

[0159]

[0170] Embodiment 28: A method for producing a protein having sweet taste modulating activity, comprising: Cultivating a host cell transformed with a vector according to aspect 7 in a medium under conditions for protein expression. The method includes:

[0160]

[0172] Aspect 29: (a) a product for oral administration, different from the truffles of Mattiromyces terfesioides, and (b) a sweetener composition comprising the isolated polypeptide of any preceding aspect. A composition comprising a combination of:

[0161]

[0173] Embodiment 30: A method for modulating the taste of a product for oral administration, comprising: combining the product for oral administration with an effective amount of a sweetener composition comprising the isolated polypeptide of any preceding embodiment. Including, The method, wherein the product for oral administration is different from the Mattiromyces terfesioides truffles and the isolated polypeptide is not the polypeptide of SEQ ID NO: 3, and the combination has an enhanced sweetness compared to the product for oral administration.

[0162]

[0174] The term "expression vector" or "expression cassette" refers to any recombinant expression system intended for expressing the nucleic acid sequences of the present invention in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cells, constitutively or inducibly, in vitro or in vivo. The term includes linear or circular expression systems. The term includes expression systems that remain episomal or that integrate into the genome of the host cell. The expression system may or may not have the ability to replicate autonomously, i.e., drive only transient expression in the cell. The term includes recombinant expression "cassettes" that contain only the minimal elements required for transcription of the recombinant nucleic acid.

[0163]

[0175] "Host cell" refers to a cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells can be prokaryotic cells, such as E. coli, or eukaryotic cells, such as yeast, insect, amphibian, or mammalian cells, such as CHO, HeLa, HEK-293, and can be, for example, cultured cells, explants, and in vivo cells.

[0164]

[0176] In one embodiment, the host cell is selected from the group consisting of Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Agrobacterium tumefaciens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, and the like. mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, Pseudomonas putida, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizopus oryzaeoryzae, Yarrowia lipolytica, Candida albicans, Issatchenkia orientalis, Scheffersomyces stipitis, Yarrowia lipolytica, Ogataea polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus polymorphus, and Schwanniomyces occidentalis.

[0165]

[0177] In another embodiment, the host cell is selected from the group consisting of gram-positive non-spore-forming bacteria, gram-positive spore-forming bacteria, gram-negative bacteria, yeast, and protists / algae.

[0166]

[0178] Non-limiting examples of gram-positive non-spore-forming bacteria include Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Carnobacterium divergens, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus spp. ... amylolyticus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus alimentarius, Lactobacillus aviaries, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus collinoides, Lactobacillus coryniformis, Lactobacillus crispatus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus dextrinixdextrinicus, Lactobacillus diolivorans, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus mucosae, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus parafarraginis, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei sakei, Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactococcus lactislactis, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Microbacterium imperial, Oenococcus oeni, Pasteuria nishizawae, Pediococcus acidilactic, Pediococcus parvulus, Pediococcus pentosaceus pentosaceus, Propionibacterium acidipropioni, Propionibacterium freudenreichii, and Streptococcus thermophiles.

[0167]

[0179] Non-limiting examples of gram-positive spore-forming bacteria include Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus flexus, Bacillus fusiformis, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus mojavensis, Bacillus pumilus, Bacillus Examples of such bactericidal bacteria include Bacillus pumilus, Bacillus smithii, Bacillus subtilis, Bacillus vallismortis, Bacillus velezensis, Geobacillus stearothermophilus, Paenibacillus illinoisensis, and Parageobacillus thermoglucosidasius. Non-limiting examples of gram-negative bacteria include Cupriavidus necator, Gluconobacter oxydans, Komagataeibacter sucrofermentans, and Xanthomonas campestris.

[0168]

[0180] Non-limiting examples of yeast include Candida cylindracea, Debaryomyces hansenii, Hanseniaspora uvarum, Kluyveromyces lactis, Kluyveromyces marxianus, Komagataella pastoris, Komagataella phaffi, Lindnera jadinii, Ogataea angusta, Saccharomyces bayanus, Saccharomyces cerevisiae, and the like. cerevisiae, Saccharomyces pastorianus, Schizosaccharomyces pombe, Wickerhamomyces anomalus, Xanthophyllomyces dendrorhous, Yarrowia lipolytica, and Zygosaccharomyces rouxii.

[0169]

[0181] Non-limiting examples of protists / algae include Aurantiochytrium limacinum, Euglena gracilis, and Tetraselmis chuii.

[0170]

[0182] The terms "mimetic" and "peptidomimetics" refer to synthetic chemicals having substantially the same structural and / or functional characteristics of a polypeptide of the invention, e.g., a translocation domain, a ligand binding domain, or a chimeric receptor. Mimetics can be either entirely composed of synthetic, non-natural analogs of amino acids, or can be chimeric molecules of partially natural peptide amino acids and partially non-natural analogs of amino acids. Mimetics can also incorporate naturally occurring amino acid conservative substitutions in any amount so long as such substitutions do not also substantially alter the mimetic's structure and / or activity.

[0171]

[0183] As with the polypeptides of the invention that are conservative variants, routine experimentation will be expected to determine whether a mimetic falls within the scope of the invention, i.e., whether its structure and / or function is substantially unchanged. A polypeptide mimetic composition may contain any combination of non-natural structural components, typically from the following three structural groups: a) residue linkages other than natural amide bonds ("peptide bonds") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues that induce secondary structural mimicry, i.e., induce or stabilize secondary structures, such as beta turns, gamma turns, beta sheets, alpha helix conformations, etc. A polypeptide can be characterized as a mimetic if all or some of its residues are joined by chemical means other than natural peptide bonds. The individual peptidomimetic residues can be joined by peptide bonds or other chemical bonds or coupling means such as, for example, glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC). Linking groups that can be alternatives to the traditional amide bond ("peptide bond") linkage include, for example, ketomethylene (e.g., --C(O)--NH-- versus --C(O)--CH). 2 --), aminomethylene (CH 2--NH), ethylene, olefins (CH=CH), ethers (CH 2 --O), thioether (CH--S), tetrazole (CN 4 ), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp. 267-357, "Peptide Backbone Modifications", Marcell Dekker, NY (1983)). Polypeptides can also be characterized as mimetics by containing all or some non-natural residues in place of naturally occurring amino acid residues; non-natural residues are well described in the scientific and patent literature. Phyre2 is a web-available suite of tools for predicting and analyzing protein structure, function, and mutations.

[0172]

[0184] Protein folding analysis is performed using tools such as PHYRE Protein Homology / analogY Recognition Engine V2.0 and JPred, Protein Secondary Structure Prediction server, which reveal that SEQ ID NO:3 predicts a globular protein with predominantly β-sheet structure, with significant sections of β-sheet and some short sections of α-helices. Specifically, the Jpred tool predicts putative β-sheets from about residues 5-11, 16-19, 28-29, 39-40, 61-67, 71-77, and 98-101; and α-helices from about residues 20-25, 45-55, 111-119 of SEQ ID NO:3; the PHYRE tool predicts β-sheets from about residues 5-12, 17-32, 38-40, 45-57, 62-66, 73-81, 98-104, and 112-116 of SEQ ID NO:3, and α-helices from about residues 84-89 and 116-118 of SEQ ID NO:3. See FIG. 1.

[0173]

[0185] Examples of conservatively modified variations of Myd1 protein structure can be obtained using homology modeling algorithms: SWISS-MODEL, PHYRE2.0, and Jpred to identify sequence-based consensus loop regions, as known in the art.See, for example, Pechmann, S. and Frydman, J. Interplay between Chaperones and Protein Disorder Promotes the Evolution of Protein Networks.PLoS Computational Biology 10, e1003674 (2014).

[0174]

[0186] Alternative protein sequences with similar structure and function to putative Myd1 are provided herein as SEQ ID NO:8-17. To obtain SEQ ID NO:8-17, consensus loop regions were chosen as mutation sites because most insertions and deletions are usually found in regions between secondary structure elements, where they can be more easily accommodated without causing significant distortions to the overall fold of the protein. The core of this protein has a higher degree of sequence conservation, as found within 4JOX.

[0175]

[0187] Of the 29 possible amino acid positions within the consensus loop region, 12 amino acids were replaced using conservative substitutions. When selected for mutation, the wild-type amino acid was given equal probability among the conservative amino acid residues (Gly can be replaced with Ala, Cys, Asp, Glu, and Arg with equal 20% probability).

[0176]

[0188] Specific regions of MYD / Myd nucleotide and amino acid sequences can be used to identify polymorphic variants, interspecies homologs, and alleles of Myd family members. This identification can be done in vitro, for example, under stringent hybridization conditions, or by PCR (for example, using primers that code for the Myd sequences identified herein), or by using sequence information in computer systems for comparison with other nucleotide sequences. Different alleles of the MYD gene within a single species population are also expected to be useful in determining whether differences in allelic sequences correlate with differences in taste perception between members of the population. Classical PCR-type amplification and cloning techniques are useful for isolating orthologs, for example, when degenerate primers are sufficient to detect related genes across species.

[0177]

[0189] For example, primers designed using sequences disclosed herein can be used to amplify and clone MYD-related genes from different fungal genomes. In contrast, genes within a single species related to MYD are best identified using sequence pattern recognition software to look for related sequences. Typically, identification of polymorphic variants and alleles of MYD family members can be made by comparing amino acid sequences of about 25 amino acids or more, e.g., 50-100 amino acids. Approximately at least 35-50%, optionally 60%, 70%, 75%, 80%, 85%, 90%, 95-99% amino acid identity, or higher, typically demonstrates that the protein is a polymorphic variant, interspecies homolog, or allele of a MYD family member. Sequence comparison can be performed using any of the sequence comparison algorithms discussed below. Antibodies that specifically bind to Myd polypeptides or conserved regions thereof can also be used to identify alleles, interspecies homologs, and polymorphic variants.

[0178]

[0190] In one embodiment, sequences encoding hybrid proteins may be constructed that contain nucleic acids encoding Myd fusion proteins. These nucleic acid sequences may be operably linked to transcriptional or translational control elements, such as transcriptional and translational initiation sequences, promoters and enhancers, transcriptional and translational terminators, polyadenylation sequences, and other sequences useful for transcribing DNA into RNA. The fusion proteins may include C-terminal or N-terminal translocation sequences. In addition, the fusion proteins may include additional elements, such as additional elements for protein detection, purification, or other applications. Domains that facilitate detection and purification include, for example, peptides that chelate metals, such as polyhistidine tracts, histidine-tryptophan modules, or other domains that allow purification on immobilized metals; maltose binding proteins; protein A domains that allow purification on immobilized immunoglobulins; or domains utilized in the FLAGS extension / affinity purification system (Immunex Corp, Seattle Wash.).

[0179]

[0191] In one embodiment, the fusion protein comprises a peptide or protein tag (e.g., for purification or detection of the protein). Peptide / protein tags are known in the art and are described, for example, in Johnson, "Protein / Peptide Tags," in J. Am. Soc., 1999, 143:131-132, and ...4:131-132, and in J. Am. Soc., 1999, 145:131-132, and in J. Am. Soc., 1999, 146:131-132, and in J. Am. Soc., 1999, 147:131-132, and in J. Am. Soc., 1999, 148:1 and examples thereof include, but are not limited to, green fluorescent protein (GFP), FLAG, Myc epitope, polyhistidine, glutathione-S-transferase (GST), HA, V5, ABDz1-tag, adenylate kinase (AK-tag), BC2-tag, calmodulin-binding peptide, CusF, Fc, Fh8, Halo tag, heparin-binding peptide (HB-tag), ketosteroid isomerase (KSI), maltose-binding protein (MBP), thioredoxin, PA (NZ-1), polyArg, polyLys, S-tag, SBP / streptavidin-binding peptide, SNAP, Strep-II (Twin-Strep), and SUMO / SUMO2.

[0180]

[0192] Affinity tags are a type of protein tag attached to proteins so that they can be purified from their crude biological sources using affinity techniques. Affinity tags are known in the art and include those described in Kimple et al., Curr Protoc Protein Sci.;73:Unit-9.9.doi:10.1002 / 0471140864.ps0909s73. Examples of these include polyhistidine, GST, MBP, calmodulin binding peptide, intein-chitin binding domain, streptavidin / biotin-based tag, and His-Patch ThioFusion (thioredoxin). Affinity tags include small (e.g., 20 or fewer amino acid residues) or large affinity tags. Examples of small affinity tags include His, FLAG, Strep II, and S-peptide, and examples of large affinity tags include MBP, GST, cellulose binding domain, calmodulin binding peptide, and His-patch thioredoxin.

[0181]

[0193] Affinity tags include epitope tags and reporter tags. Reporter tags serve as reporters of protein expression and protein-protein interactions. Reporter tags include, but are not limited to, enzymes such as β-galactosidase (β-gal), alkaline phosphatase (AP), chloramphenicol acetyltransferase (CAT), and horseradish peroxidase (HRP).

[0182]

[0194] Epitope tags include FLAG, hemagglutinin (HA), c-myc, T7, and Glu-Glu, which are used for the detection of fusion proteins in vitro and in cell culture. Their short linear recognition motifs rarely affect the properties of the protein of interest and are usually very specific for their respective primary antibodies. When anti-myc antibodies are used, specificity can be increased by detecting the conjugated anti-myc primary antibody using an enzyme-linked secondary antibody instead of using HRP or AP anti-myc conjugates alone.

[0183]

[0195] The tag may be present at either end of the target protein. Some epitope tags, such as FLAG, are often used in tandem or in combination with another tag to increase their desired properties, for example in His-Myc and His-V5 constructs.

[0184]

[0196] Tandem affinity purification (TAP) is a dual affinity purification method based on fusing two affinity tags to a protein of interest, which allows for the purification of the tagged protein and the isolation of protein complexes that interact with the protein of interest. The use of TAP is encompassed within the scope of the present invention.

[0185]

[0197] In one embodiment, the fusion protein comprises a histidine tag comprising 2 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) histidine residues. For example, the histidine tag may comprise 6 histidine residues.

[0186]

[0198] To facilitate purification, it may be useful to include a cleavable linker sequence between the translocation domain (for efficient plasma membrane expression) and the remainder of the newly translated polypeptide, such as, for example, Factor Xa (see, e.g., Ottavi, Biochimie 80:289-293 (1998)), a subtilisin protease recognition motif (see, e.g., Polyak, Protein Eng. 10:615-619 (1997)); enterokinase (Invitrogen, San Diego, Calif.). For example, one construct may include a nucleic acid sequence linked to six histidine residues, followed by a polypeptide encoding thioredoxin, an enterokinase cleavage site (see, e.g., Williams, Biochemistry 34:1787-1797 (1995)), and a C-terminal translocation domain. The enterokinase cleavage site provides a means for purifying the desired protein from the remainder of the fusion protein, while the histidine residues facilitate detection and purification. Technology relating to vectors encoding fusion proteins and applications of fusion proteins are well described in the scientific and patent literature; see, for example, Kroll, DNA Cell. Biol. 12:441-53 (1993).

[0187]

[0199] A fusion protein may contain one or more linkers (e.g., flexible linkers, rigid linkers, and in vivo cleavable linkers). Besides their fundamental role in linking functional domains together (as in the case of flexible and rigid linkers) or releasing free functional domains in vivo (as in the case of in vivo cleavable linkers), linkers provide many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yields, and achieving desired pharmacokinetic profiles. Linkers are known in the art (see, for example, Chen et al., Adv Drug Deliv Rev. 65(10):1357-1369 (2013)).

[0188]

[0200] Flexible linkers are used when the combined domains require a certain degree of movement or interaction. They are generally composed of small non-polar amino acids (e.g., Gly) or polar amino acids (e.g., Ser or Thr). The small size of these amino acids provides flexibility and allows mobility of the connected functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thereby reducing unfavorable interactions between the linker and the protein moieties.

[0189]

[0201] The most commonly used flexible linkers have a sequence consisting of a stretch of primarily Gly and Ser residues (the "GS" linker). An example of the most widely used flexible linker is (Gly-Gly-Gly-Gly-Ser). n(SEQ ID NO: 69). By adjusting the copy number "n", the length of this GS linker can be optimized to achieve proper separation of functional domains or to maintain necessary interdomain interactions. Besides the GS linker, many other flexible linkers for recombinant fusion proteins have been designed. These flexible linkers are also rich in small or polar amino acids such as Gly and Ser, but may also contain amino acids such as Thr and Ala to maintain flexibility, as well as polar amino acids such as Lys and Glu to improve solubility.

[0190]

[0202] Rigid linkers maintain a fixed distance between the domains, allowing them to function independently. An example of a rigid linker is (EAAAK). n (SEQ ID NO: 70) an alpha-helix-forming linker having the sequence of (XP) n wherein X represents any amino acid, preferably Ala, Lys, or Glu.

[0191]

[0203] Polypeptides of the invention may also contain signal peptides (i.e., signal sequences, localization signals, localization sequences, transit peptides, leader sequences, or leader peptides that are short peptides that are present at the N-terminus, or occasionally the C-terminus, of most newly synthesized proteins that are destined for the secretory pathway. These proteins include those that are either present inside a specific organelle (endoplasmic reticulum, Golgi, or endosome), those that are secreted from the cell, or those that are inserted into most cell membranes. Exemplary signal peptides are known in the art, and one of skill in the art would know how to select a particular signal peptide for use in the present invention.

[0192]

[0204] As used herein, "at least 80% identity" relative to an amino acid or nucleotide sequence refers to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater identity.

[0193]

[0205] As used herein, examples of "amino acid sequences modified by the deletion, insertion, substitution, or addition of one or more amino acids" include amino acid sequences modified by the deletion, insertion, substitution, or addition of one or more or 30 or less, preferably 20 or less, more preferably 10 or less, and even more preferably 5 or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any range therein) of amino acids. As used herein, examples of "nucleotide sequences modified by deletion, insertion, substitution, or addition of one or more nucleotides" include sequences having 1 or more or 90 or less, preferably 60 or less, more preferably 30 or less, even more preferably 15 or less, and even more preferably 10 or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108 4, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or any range therein).

[0194]

[0206] For example, in sequence comparison, typically one sequence serves as a reference sequence to which test sequence is compared.Using sequence comparison algorithm, input test and reference sequences into computer, specify subsequence coordinates, and if necessary, specify sequence algorithm program parameters.For BLASTN and BLASTP programs, default program parameters can be used as described below, or alternative parameters can be specified.The sequence comparison algorithm then calculates the percent sequence identity of test sequence compared to reference sequence based on program parameters.

[0195]

[0207] "Comparison window", as used herein, includes reference to any one segment of the number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, and more usually about 100 to about 150, within which a sequence can be compared to a reference sequence of the same number of contiguous positions after optimal alignment of the two sequences. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison may be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds., 1995 supplement)).

[0196]

[0208] Preferred examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc.Acids Res.25:3389-3402 (1977) and Altschul et al., J Mol.Biol.215:403-410 (1990), respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that either match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J Mol. Biol. 215:403-410 (1990)). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is discontinued if the cumulative alignment score falls by the amount of X from its maximum achieved value; if the cumulative score becomes zero or less due to the accumulation of one or more negatively scored residue alignments; or if either sequence reaches its end. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad Sci. USA 89:10915 (1989)) of 50 alignment (B), an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0197]

[0209] Another example of a useful algorithm is PILEUP. PILEUP produces multiple sequence alignments from a group of related sequences using progressive pairwise alignments to show the relationships and percent sequence identity. It also plots a so-called "tree" or "dendrogram" showing the clustering relationships used to produce the alignment (see, for example, FIG. 2). PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle, J Mol. Evol. 35:351-360 (1987). The method used is similar to that described by Higgins and Sharp, CABIOS 5:151-153 (1989). The program can align up to 300 sequences, each with a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with a pairwise alignment of the two most similar sequences and produces a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for the region of sequence comparison, and by designating the program parameters. Using PILEUP, a reference sequence is compared to other test sequences to determine percent sequence identity relationships using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, for example, version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395 (1984)), and was obtained by conceptual translation of the corresponding open reading frame encoded by the gene.

[0198]

[0210] The polynucleotide encoding the polypeptide of the present invention can be synthesized chemically or by genetic engineering based on the amino acid sequence of Myd. For example, the polynucleotide can be chemically synthesized based on the amino acid sequence of the polypeptide of the present invention or its precursor protein. For chemical synthesis of the polynucleotide, a nucleic acid custom synthesis service (e.g., provided by Medical & Biological Laboratories Co., Ltd., Genscript, etc.) can be used. Furthermore, the synthesized polynucleotide can be amplified by PCR, cloning, etc.

[0199]

[0211] The polypeptide of the present invention can be produced, for example, by expressing a gene encoding the Myd polypeptide of the present invention. Preferably, the Myd polypeptide of the present invention can be produced from a transformed strain into which a polynucleotide encoding the Myd polypeptide of the present invention has been introduced. For example, a polynucleotide encoding the Myd polypeptide of the present invention or a vector containing the same is introduced into a host to obtain a transformed strain, which is then cultured in an appropriate medium, and the Myd polypeptide of the present invention is produced in the transformed strain from the polynucleotide encoding the Myd polypeptide of the present invention introduced therein. The protein of the present invention can be obtained by isolating or purifying the produced Myd polypeptide from the culture.

[0200]

[0212] Therefore, the present invention further provides a polynucleotide encoding a Myd polypeptide of the present invention and a vector comprising the same. The present invention further provides a method for producing a transformed strain, the method comprising the step of introducing a polynucleotide encoding a Myd polypeptide of the present invention or a vector comprising the same into a host. The present invention further provides a transformed strain comprising a polynucleotide encoding a Myd polypeptide of the present invention or a vector comprising the same introduced from outside the cell. The present invention further provides a method for producing a Myd polypeptide of the present invention, the method comprising the step of culturing a transformed strain.

[0201]

[0213] The present invention also includes a polynucleotide of the present invention operably linked to a heterologous regulatory element. The present invention can include an expression cassette or vector comprising a polynucleotide of the present invention, and a host cell transformed with a vector of the present invention.

[0202]

[0214] Alternatively, a polynucleotide encoding a Myd polypeptide of the present invention can be produced by introducing a mutation into a polynucleotide synthesized according to a procedure using known mutagenesis methods such as ultraviolet radiation irradiation and site-directed mutagenesis. For example, a polynucleotide encoding a polypeptide of the present invention can be obtained by introducing a mutation into a polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 2 using known methods, expressing the resulting polynucleotide, examining the sweetness-modifying activity of the expressed protein, and selecting a polynucleotide encoding a protein having the desired sweetness-modifying activity.

[0203]

[0215] Site-directed mutagenesis of polynucleotides can be carried out using any method, such as inverse PCR and annealing (Muramatsu et al., eds., "Revised 4th edition New genetic engineering handbook", YODOSHA, pp. 82-88). Various commercially available kits for site-directed mutagenesis, such as the QuickChange II Site-Directed Mutagenesis Kit from Stratagene, and the QuickChange Multi Site-Directed Mutagenesis Kit, can be used as needed.

[0204]

[0216] Examples of vector types that contain a polynucleotide encoding a polypeptide of the present invention include, but are not limited to, vectors that are commonly used for gene cloning, such as plasmids, cosmids, phages, viruses, YACs, and BACs. Examples of vectors include plasmids (e.g., DNA plasmids), yeast (e.g., Saccharomyces), and viral vectors, such as poxviruses, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, polioviruses, alphaviruses, baculoviruses, Sindbis viruses, plant viruses (e.g., Alphaflexiviridae or Potyviridae), and insect viruses (e.g., baculoviruses).

[0205]

[0217] Among these, plasmid vectors are preferred, and for example, commercially available plasmid vectors for protein expression, such as pUC19, pUC118, pUC119, pBR322, etc. (all available from TAKARA BIO INC.) can be used.

[0206]

[0218] The vector may contain a DNA region containing a DNA replication origin or replication origin. Alternatively, a regulatory sequence such as a promoter region for initiating transcription of a gene, a terminator region for secreting an expressed protein outside a cell, or a secretion signal region can be operably linked upstream of a polynucleotide encoding a protein of the present invention (i.e., the MYD gene of the present invention) in a vector. As used herein, the phrase "operably linked" between a gene and a regulatory sequence refers to a state in which the gene and the regulatory region are arranged so that the gene can be expressed under the regulation of the regulatory region.

[0207]

[0219] The types of regulatory sequences such as promoter regions, terminators, and secretory signal regions are not specifically limited, and the usual promoters and secretory signal sequences used can be selected as necessary to be used depending on the host into which the sequence is to be introduced. For example, a preferred example of a regulatory sequence that can be incorporated into the vector of the present invention is the cbh1 promoter sequence derived from Trichoderma reesei (Curr. Genet., 1995, 28(1):71-79).

[0208]

[0220] Alternatively, a marker gene (e.g., a resistance gene to drugs such as ampicillin, neomycin, kanamycin, and chloramphenicol) for selecting a host into which the vector is appropriately introduced may be further incorporated into the vector of the present invention. Alternatively, when an auxotrophic strain is used as a host, a gene encoding a synthase of a necessary nutrient may be incorporated into the vector as a marker gene. Alternatively, when a selective medium that requires a specific metabolism for growth is used, a metabolic related gene may be incorporated into the vector as a marker gene. An example of such a metabolic related gene includes an acetamidase gene for using acetamide as a nitrogen source.

[0209]

[0221] Ligation between the polynucleotide encoding the Myd polypeptide of the present invention, the regulatory sequence, and the marker gene can be carried out by methods known in the art, such as SOE (splicing by overlap extension)-PCR (Gene, 1989, 77:61-68). Procedures for introducing the ligated fragment into a vector are known in the art.

[0210]

[0222] Examples of hosts for transformed strains into which vectors are introduced include microorganisms such as bacteria and filamentous fungi. Examples of bacteria include Escherichia coli and bacteria belonging to the genera Staphylococcus, Enterococcus, Listeria and Bacillus, among which Escherichia coli and bacteria of the genus Bacillus (e.g., Bacillus subtilis or mutants thereof) are preferred. Examples of Bacillus subtilis mutants include KA8AX, a protease 9 double-deficient strain described in J.Biosci.Bioeng., 2007, 104(2):135-143, and DBPA, a mutant from a protease 8 double-deficient strain described in Biotechnol.Lett., 2011, 33(9):1847-1852, and have improved protein folding efficiency. Examples of filamentous fungi include Trichoderma, Aspergillus and Rhizopus. Also suitable expression hosts include, for example, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowia lipolytica, Schizosaccharomyces pombe, and Kluyveromyces lactis. In yet another aspect, the invention includes a host cell comprising one or more of the expression cassettes described herein operably linked to control elements compatible with expression in the cell. The cell may be, for example, a mammalian cell (e.g., BHK, VERO, HT1080, 293, RD, COS-7, or CHO cell), an insect cell (e.g., Trichoplusia ni (Tn5) or Sf9), a bacterial cell, a plant cell, or a yeast cell.

[0211] purification

[0223] The polypeptide recombinantly expressed from an expression cassette encoding Myd is typically isolated from lysed cells or culture medium. Purification can be accomplished by methods known in the art, such as salt partitioning, ion exchange chromatography, gel filtration, size exclusion chromatography, size partitioning, and affinity chromatography. For example, immunoaffinity chromatography using antibodies generated against the Gag antigen can be employed.

[0212]

[0224] The present invention provides a method for purifying a polypeptide having sweet taste modulating activity, comprising: (a) obtaining a composition comprising the polypeptide; and (b) purifying the composition by hydrophobic interaction chromatography (HIC) followed by size exclusion chromatography (SEC). In one embodiment, the polypeptide comprises an amino acid sequence having at least 80% sequence identity with a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and the sequences shown in Table 6 or Table 7. In another embodiment, the polypeptide has a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and the sequences shown in Table 6 or Table 7; (a) the polypeptide contains at least one substitution modification compared to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and the sequences shown in Table 6 or Table 7, and the polypeptide is different from the polypeptide of SEQ ID NO:3, or (b) the polypeptide further comprises a histidine tag and has sweet taste modulating activity.

[0213]

[0225] Those skilled in the art are familiar with the purification techniques of hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC), such as selecting appropriate columns, buffers, and elution solutions.Exemplary HIC and SEC purification techniques are described herein in Example 11.In exemplary embodiments, the purity of the polypeptide after purification by HIC and SEC is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or any range of values ​​therebetween.

[0214] plant

[0226] The present invention also contemplates transgenic plants comprising the heterologous polynucleotides and / or heterologous polypeptides of the present invention described herein. The plants have an altered phenotype due to expression of the heterologous nucleic acid sequences. The altered phenotype can include an increased sweetness phenotype in any plant part, such as fruit. The transgenic plant can contain an expression cassette as defined herein as part of the plant, the cassette being introduced by transformation of the plant with the vector of the present invention. Such an expression cassette includes regulatory sequences for expression of the heterologous coding sequence in the plant, such as a promoter and terminator expressible in the plant. The transgenic plant can be any type of plant capable of expressing the heterologous nucleic acid sequences described herein. The term "plant" includes whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and their progeny. The classes of plants that can be used in the methods of the present invention are generally broader than the classes of higher plants that are amenable to transformation techniques, including both monocotyledonous plants (monocots) and dicotyledonous plants (dicots). Examples include plants of various ploidy levels, such as polyploid, diploid and haploid, etc. For example, the transgenic plant may be an apple or strawberry.

[0215]

[0227] Techniques for transforming various plant species are well known in the art and described in technical and scientific literature. See, for example, Weising et al. (1988) Ann. Rev. Genet., 22:421-477, and Joung et al. (2015) "Plant Transformation Methods and Applications" in Current Technology in Plant Molecular Breeding, (eds. Koh et al.) Springer Dordrecht Heidelberg New York London, Chapter 9, pp. 297-344. Any method known in the art for transformation of plant cells, including plant protoplasts or plant tissues, can be employed for plant transformation. Specific methods for plant transformation include, among others, bolistic methods (gene gun), electroporation, microinjection, protoplast fusion and Agrobacterium-mediated transformation. Agrobacterium-mediated transformation may employ, for example, binary vectors that replicate in E. coli and Agrobacterium tumefaciens or other Agrobacterium strains. A variety of such binary vectors are known in the art and can be employed to introduce heterologous polynucleotides into plant cells and plant tissues. Plant expression vectors containing regulatory sequences for expression of heterologous coding sequences, such as plant-expressible promoter sequences and other plant regulatory sequences, in plant cells and plant tissues are known in the art and can be employed to transform plants to express the polypeptides described herein.

[0216]

[0228] A variety of plant-expressible promoters are known in the art and are available for use in the heterologous constructs, vectors and transformed plant materials described herein that contain a polynucleotide encoding a protein having sweet taste modulating activity. Plant-expressible promoters can be obtained from natural plant sources, plant viral sources, and from bacteria, such as Agrobacterium strains that have plant-expressible promoters. Plant-expressible promoters include, among others, the cauliflower mosaic virus promoter (CaMV35S), octopine and nopaline synthase promoters (e.g., the nos promoter), plant ubiquitin promoter (Ubi), rice actin promoter (Act-1), and corn alcohol dehydrogenase (Adh-1). Plant-expressible promoters include constitutive promoters, inducible promoters, tissue-specific promoters, and developmental stage-specific promoters, with examples of each type of promoter known in the art. Tissue-specific promoters include those that direct expression in plant roots, plant leaves, fruits, flowers, pollen, or cells involved in active photosynthesis (e.g., the phosphoenolpyruvate promoter (PEP)). Developmental stage-specific promoters include those that directly direct expression during fruit ripening, flowering, or fruit set. Synthetic plant promoters are also known in the art and are useful in heterologous constructs, vectors, and transformed plant materials (see, e.g., Ali S. and Kim WC (2019) Frontiers in Plant Science, 10, Paper 1433).

[0217]

[0229] Techniques for the regeneration of plants from transformed protoplasts, plant cells, callus, or other plant tissues are well known in the art and can be employed to regenerate whole plants and plant parts from such transformed plant material. Regeneration methods include organogenesis and embryogenesis. See Handbook of plant cell culture. Vol. 1: Techniques for propagation and breeding (1983) eds. DAEvans et al., Macmillan, New York; RH Smith, Plant Tissue Culture: Techniques and Experiments, 3rd Edition (2012) Academic Press, New York; M. R. Davey and P. Anthony, Plant Cell Culture: Essential Methods (2010) John Wiley & Sons, New York, especially Chapters 3 and 9.

[0218]

[0230] Methods commonly used in the field, such as the protoplast method and electroporation, can be used to introduce a vector into a host. The desired transformed strain can be obtained by selecting a strain into which the vector has been appropriately introduced using indicators such as marker gene expression and / or auxotrophy.

[0219]

[0231] Alternatively, a fragment into which a polynucleotide encoding a Myd polypeptide of the present invention, a regulatory sequence, and a marker gene are ligated can be directly introduced into the genome of a host. For example, a polynucleotide encoding a Myd polypeptide of the present invention is introduced into the genome of a host by constructing a DNA fragment having sequences complementary to the genome of the host added to both ends of the ligated fragment, introducing the fragment into the host, and inducing homologous recombination between the host genome and the DNA fragment by SOE-PCR.

[0220]

[0232] Culturing the thus obtained transformant into which a polynucleotide encoding the Myd polypeptide of the present invention or a vector containing the same has been introduced in an appropriate medium results in expression of the MYD cDNA in the vector, which then results in production of the Myd polypeptide of the present invention. The medium used to culture such a transformant can be selected by those skilled in the art according to the type of microorganism of the transformant, as necessary.

[0221]

[0233] Alternatively, the Myd polypeptides of the invention can be expressed from polynucleotides encoding the Myd polypeptides of the invention or transcription products thereof using a cell-free translation system. A "cell-free translation system" refers to an in vitro transcription-translation system or an in vitro translation system that is constructed by adding reagents, such as amino acids, required for protein translation to a suspension obtained by mechanically disrupting the host cells.

[0222]

[0234] The Myd polypeptide of the present invention produced in the culture or cell-free translation system can be isolated or purified, if necessary, by using a general method used for purifying a protein, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, and affinity chromatography, either alone or in combination as necessary. Here, when a gene encoding the Myd polypeptide of the present invention and a secretion signal sequence are operably linked on a vector in a transformant strain, the Myd polypeptide is secreted outside the cell, and therefore the produced Myd polypeptide can be more easily collected from the culture. The Myd polypeptide collected from the culture can be further purified by known means. Method for Producing Proteins Having Sweetness Modulating Activity

[0235] The present invention also includes a method for producing a protein having sweet taste modulating activity, the method comprising culturing a host cell of the present invention in a medium under conditions that result in the production of a protein having sweet taste modulating activity similar to a known sweet taste enhancer or compound.

[0223]

[0236] As used herein, "sweet flavor enhancer," "sweet compound," or "compound that activates sweet taste receptors" refers to a composition that elicits a detectable sweet flavor in a subject, such as sucrose, fructose, glucose, and other known natural sugar-based sweeteners, or known artificial sweeteners such as saccharin, cyclamate, aspartame, as further discussed herein, or materials that activate T1R2 / T1R3 receptors in vitro. The subject may be a human or an animal.

[0224]

[0237] The sweetening seasoning or sweetener composition may be used in an effective amount, which refers to an amount of the sweetener composition of the present invention sufficient to induce a sweet taste in a subject when present in a product for oral administration.

[0225] Food, beverages, supplements, medicines

[0238] An embodiment of the present invention includes a composition. In one embodiment, the composition comprises, consists essentially of, or consists of a combination of a product for oral administration and one or more sweetener compositions comprising an isolated Myd polypeptide according to the present invention, as described herein. In one embodiment, the combination has an enhanced sweetness compared to a product for oral administration lacking the Myd polypeptide (control). In one embodiment, the product for oral administration is not a Mattiromyces terfesioides truffle. The term "consisting essentially of" allows for the inclusion of ingredients that are not essential to and do not significantly affect the function or activity of the product, such as anti-caking agents, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltodextrose, gum arabic, etc.). In one embodiment, the composition comprises multiple isolated Myd polypeptides. In certain embodiments, the composition comprises multiple isolated Myd polypeptides that are different from each other to enhance taste. Compositions comprising one or more Myd polypeptides of the invention are not limited by form, shape, and means of administration, and are understood to include solid, liquid, powder, and other forms, either individually, or in combinations of two or more thereof. Further, compositions can be administered or consumed orally, by injection, etc.

[0226]

[0239] In another embodiment, a composition comprising an isolated Myd protein of the invention comprises a formulation that provides enhanced functionality to the isolated Myd protein. For example, the composition may comprise a formulation that stabilizes the Myd protein against thermal, osmotic, pH, or other types of degradation. In one embodiment, the formulation stabilizes the Myd protein against thermal degradation. Exemplary compounds for stabilizing Myd proteins include, for example, L-arginine glycine, L-proline, L-histidine, β-alanine, L-serine, L-arginine ethyl ester dihydrochloride, L-arginine amide dihydrochloride, 6-aminohexanoic acid, gly-gly, gly-gly-gly, tryptone, betaine monohydrate, D-(+)-trehalose dihydrate, xylitol, D-sorbitol, sucrose, hydroxyectoine, trimethylamine n-oxide dihydrate, methyl-α-d-glucopyranoside, triethylene glycol, spermine tetrahydrochloride, spermidine, 5-aminovaleric acid, glutaric acid, adipic acid, ethylenediamine dihydrochloride, guanidinium hydrochloride, and the like. dine, urea, N-methyl urea, N-ethyl urea, N-methylformamide, hypotaurine, TCEP hydrochloride, GSH (reduced l-glutathione), benzamidine hydrochloride, ethylenediaminetetraacetic acid disodium salt dihydrate, magnesium chloride hexahydrate, cadmium chloride hydrate, non-surfactant sulfobetaine 195 (NDSB-195), non-surfactant sulfobetaine 201 (NDSB-201), non-surfactant sulfobetaine 211 (NDSB-211), non-surfactant sulfobetaine 221 (NDSB-221), non-surfactant sulfobetaine 256 (NDSB-256), taurine, acetamide, oxalic acid dihydrate, sodium malonate pH 7.0, succinic acid pH 7.0, tacsimate pH 7.0, tetraethylammonium bromide, choline acetate, 1-ethyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazolium chloride, ethylammonium nitrate, ammonium sulfate, ammonium chloride, magnesium sulfate hydrate, potassium thiocyanate, gadolinium(III) chloride hexahydrate, cesium chloride, 4-aminobutyric acid (GABA), lithium sulfate, DL-malic acid pH 7.0, lithium citrate tribasic tetrahydrate, ammonium acetate, sodium benzenesulfonate, sodium p-toluenesulfonate, sodium chloride, potassium chloride, sodium phosphate monobasic monohydrate, sodium sulfate decahydrate, lithium chloride, sodium bromide, glycerol Examples of suitable cyclodextrins include ethylene glycol, polyethylene glycol 200, polyethylene glycol monomethyl ether 550, polyethylene glycol monomethyl ether 750, formamide, polyethylene glycol 400, pentaerythritol ethoxylate (15 / 4EO / OH), 1,2-propanediol, polyethylene glycol monomethyl ether 1,900, polyethylene glycol 3,350, polyethylene glycol 8,000, polyvinylpyrrolidone k15, polyethylene glycol 20,000, (2-hydroxypropyl)-β-cyclodextrin, α-cyclodextrin, β-cyclodextrin, and methyl-β-cyclodextrin.

[0227]

[0240] In an embodiment, a sweetener composition comprises one or more Myd polypeptides as described above. In one embodiment, a sweetener composition comprises more than one Myd polypeptide as described above. In certain embodiments, the Myd polypeptides are different from one another.

[0228]

[0241] The present invention also includes a method for modulating the taste of a product for oral administration, comprising combining the product for oral administration with an effective amount of an isolated Myd polypeptide described herein. In one aspect, the combination has an enhanced sweetness compared to a product for oral administration lacking the Myd polypeptide (control). In one embodiment, the product for oral administration is not a Mattiromyces terfesioides truffle.

[0229]

[0242] The product for oral administration may be a food, a beverage, a dietary supplement composition, or a pharmaceutical composition.

[0230]

[0243] The term "product for oral administration" may refer to edible products, such as food, beverage products; drug (medicinal) products, or dietary supplement products, such as herbal supplements. As used herein, the term "medicinal product" includes both solid and liquid compositions that are ingestible non-toxic materials of medical value or contain medicamentously active agents, such as cough syrup, cough drops, aspirin, and chewable pharmaceutical tablets. Oral hygiene products are also products for oral administration, examples of which include solids and liquids, such as toothpaste or mouthwash.

[0231]

[0244] In general terms, the present invention contemplates that a food or beverage product may contain an effective amount of an isolated sweet protein of the present invention, for example, in an amount up to about 99% by weight, for example, from about 0.01% to about 99% by weight, based on the total weight of the food or beverage product. All intermediate amounts by weight (i.e., 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, ... 90%, 95%, 99%) based on the total weight of the food or beverage product are contemplated, as are all intermediate ranges based on these amounts.

[0232]

[0245] The compositions of the present invention may include "edible, biologically, or pharma- ceutically acceptable carriers or excipients," which may include solid or liquid media and / or compositions used to prepare a desired dosage form of the Myd polypeptide for administration of the Myd polypeptide in a dispersed / diluted form such that the biological effectiveness of the Myd polypeptide is maximized. Edible, biologically, or pharma- ceutically acceptable carriers include many common food ingredients, such as water of neutral, acidic, or basic pH, fruit or vegetable juices, vinegar, marinades, beer, wine, natural water / fat emulsions such as milk or condensed milk, edible oils and shortenings, fatty acids, low molecular weight oligomers of propylene glycol, glyceryl esters of fatty acids, and dispersions or emulsions of such hydrophobic materials in aqueous media, salts such as sodium chloride, solvents such as flour, ethanol, solid edible diluents such as vegetable powders or flours, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents; thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like.

[0233]

[0246] Pharmaceutically acceptable carriers or excipients can include excipients that allow for microencapsulation of Myd polypeptides to enhance functionality, such as protecting or extending sweetness perception. Indeed, microencapsulation is known in the art to be a technique that can facilitate regular use in addition to creating many potential new uses for sweeteners. See, for example, Favaro-Trindade, Carmen & Rocha-Selmi, Glaucia & dos Santos, Milla. (2015). Microencapsulation of Sweeteners. 10.1016 / B978-0-12~800350-3.00022-4. In one embodiment, microencapsulation methods known in the art to stabilize and / or modify (e.g., extend) the sweetness release of Myd. For example, sugar-free chewing gums and crumbly confections typically have sweeteners encapsulated in their formulations to extend their sweetness during chewing.

[0234]

[0247] Food or beverage products and compositions comprising one or more of the described Myd polypeptides are not limited by form and shape, and are understood to encompass solid, liquid, powder, and other forms, either individually or in combination of two or more thereof. Examples of food or beverage products of the present invention include, for example, but are not limited to, baked goods; sweet bakery products (such as, but not limited to, rolls, cakes, pies, pastries, and cookies); pre-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings (such as, but not limited to, fruit pie fillings and nut pie fillings, such as pecan pie fillings, as well as fillings for cookies, cakes, pastries, confectionery products, such as fat-based cream fillings); desserts, gelatins, and puddings; frozen desserts (such as, but not limited to, frozen dairy desserts, such as ice cream, including regular ice cream, soft ice cream, and all other types of ice cream, and frozen non-dairy desserts, such as non-dairy ice cream, sorbets, and the like; carbonated beverages (such as, but not limited to, non-alcoholic carbonated beverages); non-carbonated beverages (such as, but not limited to, non-alcoholic non-carbonated beverages, such as flavored waters and sweet tea or coffee based beverages); beverage concentrates (such as, but not limited to, liquid concentrates and syrups, as well as non-liquid concentrates, such as freeze-dried and / or powder preparations); yogurts (such as, but not limited to, full fat, low fat and non-fat dairy yogurts, as well as non-dairy and lactose-free yogurts, and all frozen equivalents thereof); snack bars (such as, but not limited to, cereal, nut, seed and / or fruit bars);bread products (such as, but not limited to, leavened and unleavened breads, yeast-raised and non-yeast-raised breads, e.g., soda bread, breads containing any type of wheat flour, breads containing any type of flour other than wheat (e.g., potato flour, rice flour and rye flour), gluten-free breads, and the like); pre-manufactured bread mixes for preparing bread products; sauces, syrups and dressings; sweet spreads (such as, but not limited to, jellies, jams, butters, nut spreads and other spreadable preserves, confectioneries, and the like); confectionery products (such as, but not limited to, jelly candies, soft candies, hard candies, chocolates and gums, and the like); sweetened breakfast cereals (such as, but not limited to, extruded (kix-type) breakfast cereals, flaked breakfast cereals and puffed breakfast cereals, and the like); and cereal coating compositions for use in the preparation of sweetened breakfast cereals. Other types of food and beverage products not mentioned herein but which customarily contain one or more nutritious sweeteners are also contemplated in the context of the present invention.

[0235]

[0248] As a result of the complete or partial replacement of nutritional sweeteners in the food or beverage products of the present invention, the food or beverage products of the present invention may be useful as low calorie or diet products, medical foods / products (such as pills and tablets), and sports nutrition products, and may be particularly suitable for food or beverage products that require lower sweetness at a given soluble solids level.

[0236]

[0249] In some embodiments, the sweetener composition of the present invention may be supplemented with other nutritive or non-nutritive sweeteners to form a sweetener system. The sweetener system may include the sweetener composition of the present invention, a bulking agent, such as maltodextrose, gum arabic, and at least one high intensity sweetener. The composition may be provided as a liquid composition or a dry blend.

[0237]

[0250] In one embodiment, the invention includes a process for enhancing the sweetness of a product for oral administration comprising the addition of a Myd polypeptide of the invention.

[0238]

[0251] In another embodiment, the method of the present invention includes a method for improving the sweet flavor of a product for oral administration comprising adding to the product for oral administration a sweetener composition made by the method of the present invention. The amount to be added can be determined by methods known in the art, for example, using sensory testing as a guide.

[0239]

[0252] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLES

[0240]

[0253] Example 1

[0254] Fresh Mattiromyces terfesioides truffles were obtained in situ within their natural range using appropriate procedures and permits. Fresh samples (29 in total) were shipped to the MycoTechnology, Inc. facility, gently washed in RO water, then frozen in liquid nitrogen and stored at -80°C. The average moisture content of the truffles was 83.6% plus or minus 4.6%.

[0241]

[0255] Aqueous extraction of truffles was carried out as follows: Eight different samples of truffles were ground to a powder in liquid nitrogen, then 5:1 v / w of 4°C water was added to the mass of truffle and allowed to incubate at 4°C for 30 minutes. The extracted material was then subjected to a short low speed centrifugation and the filtrate was tasted and called "neat". Sweetness intensity was rated from 0 for no sweetness to 10 for very sweet. The samples were rated for sweetness as follows:

[0256]

[0242] [Table 1]

[0257] The aqueous extract was stored in sodium phosphate buffer at 4° C., pH 7 and pH 2, and little change in sweetness was observed over a period of 8 days.

[0243]

[0258] Example 2

[0259] Purification of sweet protein Myd from M. terfezoides. Fresh Mattiromyces terfezoides truffles were obtained ex situ using appropriate procedures and permits within their natural range and stored at -80 °C. A 16.3 g sample was removed from the freezer and ground in liquid nitrogen using a mortar and pestle (white ceramic). Grinding continued for 15 min to completely pulverize the tissue and obtain a fine frozen powder. The powder was added to a 50 mL Falcon tube and diluted with 20 mL of RO-H. 2 HO was added and vortexed to mix the tissue until no ice crystals were observed. The fragments were broken down using a rotor-stator setting of 20 for 2 × 1 min at 4 °C to create a homogenous solution (H1). The volume of the slurry was reduced to RO-H 2The volume was adjusted to 53 mL with 0 and centrifuged at 7500 x G for 30 min at 4 °C. The supernatant from this step (S1) was collected in a 2 mL Eppendorf tube and centrifuged in a 5417R and Eppendorf centrifuge at 20,000 x G for 15 min at 4 °C. The pellet (P1) was discarded. A sweet taste (mediated by human perception) was noted in the supernatant. The supernatant from this step was collected and pooled. The supernatant was then filtered through a 0.45 micrometer syringe filter (Cellulose, VWR International), 25 mm, which was referred to as S1 + 0.22um filtration. The filtrate was then washed twice with hexane (38 mL to 50 mL of hexane) and the aqueous phase was collected. S1FH is the S1F + hexane wash. The hexane phase was saved and dried. The aqueous phase was then precipitated with acetone (50 mL was added to 33 ml of fraction S1FH at -20°C and left for 30 min at -20°C). The sample was centrifuged at 3,000 x g and the precipitate was collected. The precipitate was resuspended in 10 mM sodium phosphate at pH 6. The supernatant from this step was called S2 and the precipitate was called P2. The supernatant S2 was first applied to an AMICON centrifugal filter unit with a molecular weight cutoff of 100 kD to obtain a filtrate (flow-through) portion (called 100F) and a retentate (called 100R); 100F was subsequently applied to a unit with a molecular weight cutoff of 30 kD to obtain a filtrate (30F) and a retentate (30R). As the sweet fraction passed through the 100 kD column it emerged in 100F, which was retained in the 30 kD column (30R). In the SDS-PAGE of Figure 2, a band was observed at approximately 13 kDa, indicated by an arrow, and this band was excised and subjected to N-terminal sequence analysis by Edman degradation using standard methods of detection, e.g., liquid chromatography and mass spectrometry, to identify the residues for each cycle. A polypeptide was detected, SEQ ID NO:4.

[0244]

[0260] Example 3 (RNA Identification)

[0261] Sample collection

[0262] Fresh Mattiromyces terfesioides truffles were obtained in situ using proper procedures and permits within their natural range. A wild isolate (BDP2_18) of Mattiromyces terfesioides truffle (Gleba) was obtained from the natural environment. BDP2_18 was the largest wild truffle collected, and the truffle had sweet characteristics that were "sweet upfront, more mushroom-like and earthy, with a weak sweet flavor that lingers."

[0245]

[0263] Sample Identification

[0264] A wild isolate of M. gleba was sent to GeneWiz and frozen for Internal Transcribed Spacer (ITS) sequencing. The genomic loci sequenced were the 1 and 2 regions. The resulting Sanger sequencing reads were then aligned and low quality bases were trimmed. Each sequence was then subjected to an individual Basic Local Alignment Search Tool (BLAST) (Altschul, Gish, Miller, Myers, & Lipman, 1990) search to verify identity. BLASTn searches were employed using a nucleotide collection (nr / nt) from which unpublished sample sequences were excluded. The entry with the highest percent identity to the wild isolate is Mattiromyces terfesioides strain rib02.

[0246]

[0265] Sample preparation

[0266] The exterior of the wild isolate, BPD2_18, was washed with sterile water, cut into cubes of approximately 100 mg, and soaked in liquid N 2 The samples were flash frozen in a centrifuge and stored at -80° C. Shipped to GeneWiz on dry ice.

[0247]

[0267] RNA sequencing

[0268] The following samples were submitted via GeneWiz for standard RNASeq performed via Illumina HiSeq, 2 x 150bp single index per lane, with approximately 350M as-measured paired-end reads per lane. This RNASeq study included polyA+ selection for transcriptome profiling.

[0248]

[0269] RNA was extracted using the Qiagen RNeasy Plus Universal Mini Kit according to the manufacturer's instructions. RNA library preparation was performed using the NEBNext Ultra RNA Library Prep Kit for Illumina. Illumina adapter sequences are outlined below.

[0249]

[0270] 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 18)

[0271] 5'-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC[i7 barcode]ATCTCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 19)

[0272] Fastq sequence files (three replicates each) from an RNA-seq study in two strains of Mattiromyces terfesioides were trimmed and cleaned with HTStream to remove the following contaminants: PhiX (a common contaminant from sequencing), rRNA reads, sequencing adapters, low quality and "N" bases, polyA tracts, primers and reads <50 bp. rnaSPAdes (Bushmanova E, Antipov D, Lapidus A, Prjibelski AD. rnaSPAdes: a de novo transcriptome assembler and its application to RNA-Seq data. Gigascience. 2019;8(9):giz100. doi:10.1093 / gigascience / giz100) was then used for de novo assembly of transcriptomes for each strain of M. terfesioides from the cleaned files.We visualized the assemblies using Bandage (a Bioinformatics Application for Navigating De novo Assembly Graphs Easily) (Ryan R. Wick, Mark B. Schultz, Justin Zobel, Kathryn E. Holt, Bandage: interactive visualization of de novo genome assemblies. Bioinformatics, Vol. 31, No. 20, October 15, 2015, pp. 3350-3352) and performed tBLASTn searches (Gertz EM, Yu YK, Agarwala R, Schaffer AA, Altschul SF. Composition-based statistics and translated nucleotide searches: improving the TBLASTN module of BLAST. BMC Biol. 2006;4:41. Published December 7, 2006, doi:10.1186 / 1741-7007-4-41) was used to search the assemblies for the target peptide. Contigs that contained a perfect match with the query protein sequence were analyzed for open reading frames (ORFs) and finally used to construct a full-length mRNA linked to the target peptide.

[0250]

[0273] RNA transcripts were identified and their DNA copies have the sequence shown as SEQ ID NO:1, of which SEQ ID NO:2 is the predicted coding sequence based on the start and stop codons. Also provided is SEQ ID NO:3, a predicted protein having 121 amino acids and a predicted size of 13.3 kDa. Length: 122 aa, molecular weight: 13.381 kDa, predicted isoelectric point: 8.64, and predicted charge at pH 7: 1.01.

[0251]

[0274] Blast analysis. The predicted protein SEQ ID NO:3 has 31% or less identity with other protein sequences in GENBANK. A hypothetical protein from Pisolithus tincturius was found to have approximately 31% homology with SEQ ID NO:3 (GenBank: KIN98154.1; SEQ ID NO:7); it is called hypothetical protein M404DRAFT_1005519 [Pisolithus tinctorius Marx 270]. It is hypothesized that SEQ ID NO:7 may also have sweet taste modulating activity. The full-length cDNA copy of the RNA transcript is SEQ ID NO:5, and the coding sequence is given as SEQ ID NO:6.

[0252]

[0275] Example 4 (Cloning and heterologous expression of mycodulcein in E. coli; confirmation of sweetness.)

[0276] Based on the nucleotide sequence identified as SEQ ID NO:3, three expression vectors for expressing SEQ ID NO:20 containing a histidine tag were synthesized and cloned by Atum, Inc. (Newark, CA) into three different Atum vector backbones: pD454-SR (plasmid pMy_3000), pD454-MR (plasmid pMy_3001), and pD454-WR (plasmid pMy_3002), all of which are E. coli IPTG-inducible T7 promoter expression vectors with ampicillin-r, lacl, Lac01, Ori_pUC, and medium (M), strong (S), and weak (W) ribosome binding sites on the plasmid. E. coli BL21DE3 (Studier et al. (1986) J. Mol. Biol. 189:113-130) (New England Biolabs) was transformed with pMy_3000, pMy_3001, and pMy_3002 using the manufacturer's protocol. Briefly, previously frozen competent cells were thawed, mixed with 1 pg-100 ng of plasmid DNA, and kept on ice for 30 min. The mixture was subjected to a heat shock at 42°C for 45 s. Immediately afterwards, the mixture was placed in an ice bath for 10 min. 950 μL of pre-warmed LB medium was added and then subjected to shaking at 225 rpm at 37°C for 1 h. Dilutions of cells 1:10 and 1:100 and plating of 100 μL on antibiotic plates were performed for each transformation reaction. Overnight growth at 37°C allowed sufficient recovery of colonies to become visible. To confirm successful transformation, csPCR (colony screen PCR) was performed to interrogate the cDNA region of the plasmid, and expression was confirmed by SDS-PAGE of the lysates. Induced expression at shake flask scale was used to confirm heterologous expression in E. coli host. This process yielded strains Z14CE, Z15CE, and Z16CE, containing plasmids pMy_3000, pMy_3001, and pMy_3002, respectively. Three strains (Z14CE, Z15CE, Z16CE) were maintained on LB + ampicillin 100 μg / mL agar plates, while the negative control (Eco_0001) was maintained on LB agar plates.Overnight cultures were grown in 50 mL of LB liquid medium for each strain in 250 mL unbaffled culture shake flasks with appropriate antibiotics overnight at 37°C, shaking at 150 rpm. Each overnight culture was then inoculated into 200 mL of TB liquid medium and the next day inoculated into 1000 mL baffled culture shake flasks, shaking at 200 rpm at 37°C, and adjusted to an OD600 of 0.1 with the appropriate antibiotic. When the OD600 reached 0.8, IPTG supplements were added to the medium to a final concentration of 0.66 mM. Shaking was continued for an additional 5 hours at 37°C. After expression, the cells were centrifuged at 4000 g for 20 minutes. The supernatant was discarded and the pellet was suspended in approximately 20 mL of wash buffer (10 mM sodium phosphate buffer, pH 7.0). The suspended cells were disrupted with a high-pressure homogenizer (C3 Emulsiflex, Avestin, Inc., Ottawa, ON, Canada) operating at up to 2,000 bar. The disrupted cells were centrifuged at 13,000 g (30 min), the supernatant was collected, and the pellet was discarded. The supernatant containing the solubilized proteins was filtered through a 0.22 μm PES membrane unit (Millipore, Burlington, MA, USA). When run on SDS PAGE, a 13.1 kD band (Coomassie staining) was observed, confirming expression.

[0253]

[0277] The his-tagged protein of SEQ ID NO:20 was purified using validated immobilized metal affinity chromatography (IMAC) using Thermo Scientific™ HisPur™ Ni-NTA resin. SEQ ID NO:20 was purified using a nickel-charged nitrilotriacetic acid (NTA) chelate immobilized on 6% cross-linked agarose resin. The lysate was loaded onto a prepared IMAC column, equilibrated in binding buffer: 20 mM monobasic sodium phosphate, 0.5 M sodium chloride, 0.1 M imidazole, pH 7.4, and eluted using elution buffer: 20 mM monobasic sodium phosphate, 0.5 M sodium chloride, 0.5 M imidazole. The column was washed three times with binding buffer, followed by elution of the his-tagged mycodulcein four times with elution buffer, followed by ultrafiltration of the eluted fraction using a 30 kDa MWCO filter, followed by filtration of the filtrate through a 10 kD filter at 4000×G for 15 min. The retentate was diluted and spun again for a washing step, which was repeated three times. Analysis of the purification steps by SDS-PAGE is shown in FIG. 3.

[0254]

[0278] The purified fraction (shown in lane 8 from FIG. 3, containing highly purified SEQ ID NO:21) was tasted (0.2 mL aliquot) at 0.03 mg / ml by trained sensory scientists and found to have a sweetness equivalent to 8° Brix (approximately 8% sugar solution), confirming that mycodulcein isolated from M. terfesioides is responsible for the sweetening activity observed in Examples 1 and 2. The sweetness was very significantly sweet, with a "clean" sweetness (sugar-like taste) lacking other flavors, a slightly slow onset, and a sweet aftertaste.

[0255]

[0279] Example 5 (Pilot-scale production of mycodulcein (HIS-tag))

[0280] The E. coli strain Z14CE containing the coding sequence of SEQ ID NO:20 prepared in Example 4 was tested for its performance during fermentation in a laboratory-scale bioreactor. The bioreactor cultivation was carried out in a 10.0 L Bioflo 320 round-bottom stirred fermenter (BioFlo / CelliGen 310, New Brunswick Scientific, Edison, NJ, USA). The fermenter was fitted with pH and dissolved oxygen sensors (Mettler Toledo, OH, USA). Temperature was controlled via a water-filled stainless steel base. Agitation was provided by two mounted six-blade Rushton turbines spaced 47 mm apart, with the lowest impeller located just above the bottom of the shaft. Aeration was provided through a perforated pipe sparger ring. Dissolved oxygen (DO) was controlled at 20% air saturation by using sequential trains of agitation at 500–800 rpm and aeration at 5–8 L / min with air sparging at high cell densities. pH was controlled at 7.0 using 5.0 M ammonium hydroxide. Foaming was controlled by automatic addition of Antifoam 204 (Sigma, St Louis, MO, USA). The latter was sensed using a conductivity probe mounted 10 cm above the culture level. The main fermentation medium consisted of (per liter) 24 g yeast extract, 12 g tryptone, 5.42 mL glycerol, 100 mL phosphate buffer stock (0.17 M KH 2 PO 4 , 0.72M K 2 HPO 4 ). The medium was adjusted to pH 7.0 using 2 M HCl. Once the original glucose supply was exhausted (indicated by a rise in pH), 200 g glucose, 21.1 g (NH 4 ) 2 SO 4 and 19.7 g MgSO 4The feed medium, consisting of 1.00 mL / min, was pumped into the fermenter at an initial flow rate of 1.00 mL / min. The initial volume of medium in the vessel was 4.0 L unless otherwise specified. The inoculum (200 ml) consisted of a culture grown in a 1 L baffled shake flask (37 °C, 200 rpm) in initial LB culture medium for 16 h. The temperature of the fermenter was 37 °C. After the optical density reached 10-20, the fermentation was induced with 0.66 mM IPTG and continued for 24 h thereafter. The liquid medium was subsequently centrifuged at 4000 g for 20 min after the 24-h induction period. The supernatant was discarded and the pellet was suspended in 1 L of wash buffer (10 mM sodium phosphate buffer, pH 7.0). The suspended cells were disrupted with a high-pressure homogenizer (C3 Emulsiflex, Avestin, Inc., Ottawa, ON, Canada) operating up to 1,500 bar. The disrupted cells were centrifuged at 13,000 g (30 min), the supernatant was collected, and the pellet was discarded. The supernatant containing the solubilized proteins was filtered through a 0.22 μm PES membrane unit (Millipore, Burlington, MA, USA).

[0256]

[0281] The clarified supernatant prepared in this example was tasted (0.2 mL aliquots) at 0.03 mg / ml by trained sensory scientists and found to have a sweetness equivalent to 8° Brix (approximately 8% sugar solution). The sweetness was very significantly sweet, with a "clean" sweetness (tastes like sugar) without other flavors, a slightly slow onset, and a sweet aftertaste.

[0257]

[0282] The supernatant was stored in aliquots at -20°C and used for further experiments.

[0258]

[0283] Example 6 (Mycodulcein from E. coli ELISA quantification)

[0284] A direct ELISA was developed to quantify His-tagged mycodulcein (SEQ ID NO:21) using a horseradish peroxidase (HRP)-conjugated antibody to the 6xHis tag sequence on the carboxy terminus of SEQ ID NO:21. The ELISA allows for the measurement of mycodulcein concentrations in complex lysates and purified proteins. Recombinant 6xHis-tagged E. coli mycodulcein has a molecular weight of 14.2 kDa and a molecular weight of 27,960 M. -1 ·cm -1 The molar extinction coefficient was calculated from the amino acid sequence by methods known in the art. Purity was assessed by SDS-PAGE and was ≧98%. Mycodulcein protein concentration was determined by absorbance at 280 nm using the Beer-Lambert law, and a standard curve was then generated using known mycodulcein concentrations (μg / ml) for ELISA.

[0259]

[0285] ELISA procedure: Proteins were bound to the walls of a high protein binding 96-well plate in a coating buffer of 50 mM carbonate buffer, pH 9.4, for 30 min at room temperature. The plate was washed three times with phosphate-buffered saline (PBS), pH 7.4, containing 0.02% Tween-20. Nonspecific binding sites on the microplate were blocked with 5% BSA in PBS, pH 7.4, for 15 min at room temperature and washed three times with PBS containing 0.02% Tween-20. Primary antibodies were diluted (1:1000) in blocking buffer, and the microplate was incubated for 1 h and washed three times with PBS containing 0.02% Tween-20. The HRP reaction was allowed to proceed for 8 min using the colorimetric substrate 3,3',5,5'-tetramethylbenzidine (TMB) and stopped with 2N sulfuric acid.

[0260]

[0286] Example 7 (Quantitative characterization of mycodulcein concentration dependence)

[0287] Opertech Bio (Philadelphia, PA) performed a quantitative characterization of the concentration dependence of the taste properties of purified his-tagged mycodulcein (SEQ ID NO: 21), obtained from E. coli as described in Example 5 and purified as described in Example 4. The sweetness potency and relative efficacy of mycodulcein was compared to sucrose and other sweeteners thaumatin, rebaudioside A, and aspartame. The control was a solution of 200 mM sucrose, 100 mM NaCl, 0.5 mM quinine, and 10 mM citric acid. Figure 4 shows the concentration response functions for the sweetness of mycodulcein, aspartame, thaumatin, and rebaudioside A. The data are plotted as the ratio (p) of the response generated to the 200 mM sucrose-related ("sweet") target. Each data point in the curves for mycodulcein, aspartame, thaumatin, and rebaudioside A was calculated as the average over 32 replicates, and for the sucrose curve it was averaged over 16 replicates; error bars are SEM. Points for the water and sucrose controls were similarly calculated as the average over 128 and 64 replicates, respectively. Curves were fitted by nonlinear regression.

[0261]

[0288] The concentrations eliciting a half-maximal sweet response (EC50, or potency) were derived from nonlinear regression. Table 2 shows the EC50 (and 95% confidence intervals) for mycodulcein (MYC), sucrose (SUC), aspartame (ASP), rebaudioside A (REB), and thaumatin (THN). Equivalence to sucrose based on molar concentration and weight is also provided.

[0262]

[0289]

[0263] [Table 2]

[0290] Evaluation of thaumatin, sucrose, and SEQ ID NO: 21 was performed using CATA (click all that apply) time-intensity of sweetness perception of named analytes in water at 0.045 mg / ml protein and 10% sucrose. Method: time-intensity technique; Data collection software: EyeQuestion, responses were recorded every 2.57 seconds; Scaling method: 15-point sweetness scale, e.g. score 5=5% sucrose, score 10=10% sucrose; Evaluation protocol: sip, up and down, and spit out the test article. Performed with 3-6 judges and 2 replicates. All samples were blinded and presented with a randomized 3-digit code.

[0264]

[0291] Training strategy: Intensive training on the sweetness scale over 6 weeks on a 15-point sweetness scale to confidently assign sweetness values. Due to the unique sweetness behavioral patterns, training on the time-intensity principle over 3 weeks is necessary. Samples were tasted using a stopwatch to record the time and help reach a consensus. Number of judges: 3-6, Number of replicates: 2. All samples were blinded and presented with a randomized 3-digit code. Statistical analysis: Due to the small number of judges, statistical analysis cannot be performed.

[0265]

[0292] The maximum intensity (Imax) of mycodulcein and thaumatin at the amounts tested indicates approximately one point higher than sucrose on a 15 point scale. Thaumatin and mycodulcein have flatter slopes, indicating a longer peak time and a more gradual / longer decay. Sucrose reaches threshold sweetness (intensity <1) at 162 seconds, sooner than thaumatin and mycodulcein. When sucrose reaches threshold levels, thaumatin and mycodulcein are perceived as low to moderate intensities. In this experiment, mycodulcein and thaumatin appear to be of similar potency, approximately 3000 times sweeter than sucrose on a weight basis, or approximately 120,000 times sweeter than sucrose on a molar basis. These two experiments indicate that mycodulcein is a high intensity sweet protein with a sweetness potency ranging from 400 times that of sucrose to 3000 times that of sucrose on a weight basis.

[0266]

[0293] Example 8 (Production of mycodulcein variants and testing for sweetness and heat stability)

[0294] Methods for identifying potential key residues in mycodulcein. Sweet proteins have no primary sequence identity, but their overall tertiary structure has a sweet finger motif (Tancredi, T., Pastore, A., Salvadori, S., Esposito, V. and Temussi, PA Interaction of sweet proteins with their receptor: A conformational study of peptides corresponding to loops of brazzein, monellin and thaumatin. European Journal of Biochemistry 271, (2004):2231-2240.). Sweet proteins have antiparallel beta sheets and alpha helices perpendicular to the beta sheets. The tertiary structures of the sweet proteins thaumatin, monellin, brazzein, and lysozyme were analyzed using PyMOL2.0 (The PyMOL Molecular Graphics System, version 2.0 Schrodinger, LLC) and compared to a model of mycodulcein generated using Phyre2 (Kelley LA et al., The Phyre2 web portal for protein modeling, prediction, and analysis Nature Protocols 10, (2015):845-858) (Figure 5A). Twenty-three conservative single point mutations of ionizable amino acid residues were made. The negatively charged glutamine and aspartic acid differ in having an additional carbon in the fatty chain. Substituting the positively charged lysine and arginine would be considered a conservative substitution, but the guanidinium of arginine may form additional interactions with amino acids, including hydrogen bonds, aromatic, and aliphatic contacts. The ionic amino acid mutations were lysine to arginine, arginine to lysine, aspartic acid to glutamic acid, and glutamic acid to aspartic acid. The relative positions of each mutant were modeled by PyMOL2.0 and categorized as the N-terminus, three loop regions, five beta sheets, one alpha helix, and the C-terminus (see Figure 5B).

[0267]

[0295] Specifically, the following single mutants were generated, and their predicted locations are shown in Table 3. See also FIG. 5B, which shows the predicted secondary structure of SEQ ID NO:3 superimposed on the putative secondary structure motifs and a representation of the locations of the point mutations in Table 3 within each motif.

[0268]

[0296] Cloning: Eco_0001, commonly known as E. coli BL21DE3 (E. coli strain BF - ompT gal dcm lon hsdS B (r B - m B - )λ(DE3[lacI lacUV5-T7p07 ind1 sam7 nin5])[malB + ] K-12 (λ S )) (obtained from New England Biolabs, #C2527I) was transformed with 23 plasmids (pMy_3018-pMy_3040) using the manufacturer's protocol. Briefly, previously frozen competent cells were thawed, mixed with 1 pg-100 ng of plasmid DNA, and kept on ice for 30 min. The mixture was subjected to a heat shock at 42°C for 45 s. Immediately after, the mixture was placed in an ice bath for 10 min. 950 μL of pre-warmed LB medium was added and then subjected to shaking at 225 rpm at 37°C for 1 h. For each transformation reaction, cells were diluted 1:10 and 1:100 and 100 μL was plated on antibiotic plates. Overnight growth at 37°C allowed colonies to recover sufficiently to become visible. This process yielded strains Z18CE-Z40CE containing plasmids pMy_3018-pMy_3040 sequentially. Heterologous expression in the E. coli host was confirmed using induced expression at shake flask scale. Mutants after transformation were plated and maintained on LB + ampicillin 100 μg / mL agar plates.

[0269]

[0297] Plates were incubated at 37°C for 16 hours. Colony screening PCR was performed to confirm the genotype using colony screening primers designed to interrogate the flanking regions along with the cDNA region of the plasmid. Successful transformation yielded DNA fragments of specific sizes, while negative and no template controls yielded no PCR bands. Successful transformation was observed for all mutants.

[0270]

[0298] Shake flask scale induced expression was used to confirm heterologous expression in the E. coli host.

[0271]

[0299] Twenty-three strains (Z38CE-Z60CE) were maintained on LB + ampicillin 100 μg / mL agar plates, while a negative control (Eco_0001) was maintained on LB agar plates. Overnight cultures were grown in 50 mL of LB liquid medium for each strain in unbaffled 250 mL culture shake flasks with the appropriate antibiotic overnight at 37 °C, shaking at 150 rpm. Each overnight culture was then inoculated into 200 mL of TB liquid medium and the following day inoculated into baffled 1000 mL culture shake flasks, shaking at 200 rpm at 37 °C, and grown to an OD of 0.1 with the appropriate antibiotic. 600 The OD was adjusted to 600 When the β-actin concentration reached 0.8, IPTG supplement was added to the medium to a final concentration of 0.66 mM. Shaking was continued for an additional 5 h at 37° C. Afterwards, the cells were harvested by centrifugation at 5000 g for 5 min at 4° C. The E. coli cells were then resuspended in cold dH2O. 2 The cells were washed once with 2× HO and centrifuged again at 5000 g for 10 min at 4° C. For confirmation of successful expression, cell lysates were made using liquid nitrogen and a mortar and pestle. The cell pellet was dissolved in 10 mL of cold dHO. 2The crude lysates were resuspended in 0 and spun at 20,000 g for 5 min at 4° C. Finally, the supernatant was aspirated, filtered through a 0.2 μm PES filter, and run on SDS-PAGE protein electrophoresis. The crude lysates were tasted to identify sweet tasting mutants. Table 3 shows the results of the study.

[0272]

[0300]

[0273] [Table 3-1]

[0274] [Table 3-2]

[0301] Sixteen of the variants, all sweet (Z38CE, Z39CE, Z41CE, Z45CE, Z47CE, Z48CE, Z49CE, Z51CE, Z52CE, Z53CE, Z55CE, Z56CE, Z57CE, Z58CE, Z59CE, Z60CE), were added and expressed again using 200 mL of medium. After expression, the cells were centrifuged at 4000 g for 20 min after a 24-h induction period. The supernatant was discarded and the pellet was suspended in approximately 20 mL of wash buffer (10 mM sodium phosphate buffer, pH 7.0). The suspended cells were disrupted with a high-pressure homogenizer (C3 Emulsiflex, Avestin, Inc., Ottawa, ON, Canada) operating at up to 2,000 bar. The disrupted cells were centrifuged at 13,000 g (30 min), the supernatant was collected and the pellet was discarded. The supernatant containing the solubilized protein was filtered through a 0.22 μm PES membrane unit (Millipore, Burlington, Mass., USA). The material was then isolated by IMAC purification as described in Example 4.

[0275]

[0302] The purified samples were tasted by trained sensory scientists. Mycodulcein stocks were diluted to a protein concentration equal to that measured by ELISA. Subjects followed a drink-and-spit protocol approved by the Institutional Review Board. 0.2 ml of each purified mutant was placed on the tongue and the intensity of sweetness perception, the time of onset of sweetness perception, and the duration of sweetness perception were recorded. The results are shown in Figure 6 and discussed herein below.

[0276]

[0303] Effect of conservative point mutations on the sweetness of mycodulcein.

[0304] To correlate the effect of conservative single point mutations on sweetness, published mutations of thaumatin, brazzein, monellin and lysozyme were compared with mycodulcein. The aim was to match the time and intensity profile of mycodulcein with that of sucrose, so we measured sucrose equivalence, onset and total duration by sensory analysis. Sucrose has a fast onset, high intensity and fast duration. Therefore, mutations that reduce onset and duration are desirable, as are mutations that match or improve sucrose equivalence. Mutations that increase onset and total duration are undesirable, as are mutations that reduce sucrose equivalence. See Figures 5B and 6.

[0277]

[0305] External N-terminal D3E

[0306] A conservative change of a single mutation of D3E at the external N-terminus resulted in loss of sucrose equivalence and duration; however, generation was only slightly reduced. These results suggest that the charge, size, and polarity of the N-terminus of sweet proteins are important for sweetness and protein stability.

[0278]

[0307] Beta sheet 1 external K11R

[0308] A conservative single mutation at K11R resulted in a small increase in sucrose equivalence and a moderate reduction in onset; however, the duration of sweetness increased dramatically. These results suggest that K11 is a critical residue for binding to the sweet taste receptor T1R2 / T1R3 and may affect the off-rate of mycodulcein from the receptor.

[0279]

[0309] K26R-linker region outside the region between beta sheets 2 and 3

[0310] A conservative mutation at K26 resulted in a slight decrease in sucrose equivalents, indicating that this conservative mutation does not have a significant effect on the functionality of the protein.

[0280]

[0311] K51R outside the loop 2 region

[0312] Molecular modeling showed that all putative loop region mutations were solvent exposed. Except for a moderate decrease in onset, only slight decreases in sucrose equivalence and total duration were observed in sensory studies, indicating that the conservative mutations have no significant effect on protein functionality.

[0281]

[0313] R57K outside loop 2 region

[0314] The mutation R57K has a significant inhibitory effect on onset, sucrose equivalence and total duration.

[0282]

[0315] Beta sheet 4 external R66K

[0316] The mutation R66K results in a significant decrease in sucrose equivalence and total duration, while slightly increasing onset. The R66K mutation may be a key residue for affinity and off-rate to the sweet taste receptor.

[0283]

[0317] D69E outside the loop 3 region

[0318] Mutations at D69E result in a small decrease in sucrose equivalence, duration, and a small increase in duration. This area in the region is predicted to be relatively insensitive to conservative mutations.

[0284]

[0319] D85E and E86D outside the alpha helix region

[0320] Both D85E and E86D have similar effects on the sensory stimulation properties of mycodulcein. The sucrose equivalence and duration of D85E and E86D was reduced. Onset was similar to the control.

[0285]

[0321] D97E, K103R, R106K, and E117D outside the C-terminus had minimal effects compared to controls, suggesting that these areas are relatively insensitive to conservative mutations. However, R110K and K120R showed reduced sweetness and duration, but similar onset.

[0286]

[0322] As shown in Table 3, conservative single point mutations at R20K, E35D, K44R, D46E, D52E, R75K, and D94E resulted in loss of protein expression. These data suggest that these residues may be involved in protein folding or expression in the E. coli host. The predicted tertiary structure model discussed above in this example supports the misfolding of the protein resulting from these changes, as all these residues are contained in predicted beta sheets.

[0287]

[0323] References:

[0324] Korz, D. J., Rinas, U., Hellmuth, K., Sanders, E. A. & Deckwer, W.-D. Simple fed-batch technique for high cell density cultivation of Escherichia coli. Journal of Biotechnology 39, 59-65 (1995).

[0325] Norsyahida, A., Rahmah, N. & Ahmad, R. M. Y. Effects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coli. Letters in Applied Microbiology 49, 544-550 (2009). Example 9.

[0288]

[0326] Thermal stability was performed on protein samples from the IMAC purification of the 16 sweet mutants described above in Example 8 using the GloMelt™ Thermal Shift Protein Stability Kit (Biotium, Inc. Fremont, CA) using the instructions provided by the manufacturer and normalized to 0.04 mg / mL. Briefly, the individual reactions to measure the thermal shift relied on the following: mixing mycodulcein, 36 μg / ml, in 25 mM sodium phosphate buffer at pH 7.4, with the reagents provided in the kit, according to the manufacturer's instructions. For thermal shift measurements, a Bio-Rad CFX96 Touch system was used using BR Clear plates, scan mode SYBER / FAM only, 25°C for 30 seconds, melting curve 25°C to 95°C, 0.5°C increments in 10 seconds, plus plate read. Tm is determined based on the midpoint determined for a curve fitted to the experimental data with a five parameter equation using techniques such as those described in Schulz, MN, Landstrom, J. and Hubbard, REMTSA-A Matlab program to fit thermal shift data. Analytical Biochemistry 433, 43-47 (2013). The results (Table 4) show that thermal stability was minimally affected by the amino acid changes in the mutants tested.

[0289] [Table 4]

[0327] Example 10 (Cloning and heterologous expression of mycodulcein in Saccharomyces cerevisiae; confirmation of sweetness)

[0328] Based on the nucleotide sequence identified as SEQ ID NO:3, two expression vectors for expressing mycodulcein, SEQ ID NO:21 (pMy_4003) (with his tag) and SEQ ID NO:3 (pMy_4002) (native), were synthesized by Atum, Inc. (Newark, CA) and cloned into the non-secretory backbone pD1234 of the Atum vector containing the URA3 marker and the strong constitutive promoter GPD. The transformation procedure involves making electrocompetent cells and then introducing the expression vector by electroporation. Briefly, electrocompetent cells are made by first growing the cells to between early and mid-logarithmic phase with multiple washes to remove salt from the growth medium. After mixing 1-5 μg of expression vector, the sample is subjected to the following settings in a Gene Pulser II electroporator (charging voltage: 1.5 kV, capacitance: 25 μF, resistance: 200 Ω), 1 mL of pre-warmed 30 °C YPD is added immediately, and the suspension is incubated at 30 °C for 1-2 h with shaking at 200-250 rpm. The transformed mutants were plated and maintained on SC-ura agar plates. This process yielded strains Z19ES, Z20ES, containing plasmids pMy_4002 and pMy_4003, respectively. csPCR (colony screening PCR) was performed to interrogate the cDNA region of the plasmids. Thus, successful transformation was expected to result in a 303 bp DNA fragment, while the negative control and no template control were expected to result in no PCR band, and expression was confirmed.

[0290]

[0329] Two strains (Z19ES, Z20ES) were maintained on SC-ura agar plates, while the negative control was maintained on SC agar plates. Overnight cultures were grown in 50 mL of SC-ura / SC liquid medium for each strain in 250 mL culture shake flasks without baffles, shaking at 150 rpm overnight at 37°C. Each overnight culture was inoculated into 200 mL of SC-ura / SC (O-RDL-R10_TB medium) liquid medium at 30°C in 1000 mL culture shake flasks with baffles, shaking at 200 rpm, and adjusted to an OD600 of 0.02. Shaking was continued at 30°C for an additional 48 hours. Cells were then harvested by centrifugation at 5000g for 5 minutes at 4°C. S. cerevisiae cells were then washed with cold dH2O and centrifuged again at 5000g for 5 minutes at 4°C. To confirm successful expression, cells were lysed using liquid nitrogen and a mortar and pestle. The cell pellet was dissolved in 10 mL of cold dHO. 2 The cultures were resuspended in 0 and the lysates were spun at 20,000 g for 5 min at 4° C. The supernatants were filtered using 0.2 μm PES filters. The filtrates (for both strains) were confirmed to taste sweet by the method described in Example 3.

[0291]

[0330] The his-tagged protein of SEQ ID NO:20 was purified from S. cerevisiae using a validated immobilized metal affinity chromatography (IMAC) using Thermo Scientific™ HisPur™ Ni-NTA resin. SEQ ID NO:20 was purified using a nickel-charged nitrilotriacetic acid (NTA) chelate immobilized on a 6% cross-linked agarose resin. The lysate was loaded onto a prepared IMAC column, the column was washed three times with 0.02 M imidazole in PBS, the his-tagged mycodulcein was subsequently eluted four times with 0.3 M imidazole in PBS, and the eluted fractions were subsequently ultrafiltered using a 50 kDa MWCO filter, followed by concentration and desalting using a 3 kDa MWCO filter.

[0292]

[0331] Example 11 (Purification of native mycodulcein from strain Z19ES (S. cerevisiae))

[0332] Three chromatographic techniques were evaluated for their effectiveness in purifying native mycodulcein (SEQ ID NO: 3) expressed in S. cerevisiae: cation exchange (CIEX), hydrophobic interaction (HIC), and size exclusion chromatography (SEC).

[0293]

[0333] Cation exchange evaluation.

[0294]

[0334] The isoelectric point of native mycodulcein was determined by isoelectric focusing to be about 9.5, suggesting that the cation exchange column could be successful in purifying mycodulcein. The clarified cell lysate prepared as described in Example 10 was mixed with 2x starting buffer to obtain cell lysate in 50 mM sodium phosphate, 1 M ammonium sulfate at pH 7.0, which was stored at 4°C for future use. The purification procedure was carried out on an AKTA Explorer 100 system (GE Healthcare, Sweden), and the eluted protein was monitored at 280 nm and 215 nm in a UV detector UV-900 (GE Healthcare, Sweden). The binding conditions of native mycodulcein were screened using a predictor plate (GE Healthcare, Sweden) pre-filled with CIEX resin. The pre-packed plates contain the three main resins: Capto S (strong CIEX), Capto MMC (weak CIEX), and SP Sepharose Fast Flow (strong CIEX). The lysate was dialyzed against 20 mM dibasic sodium phosphate, various pH values ​​ranging from 4 to 9 were screened, and the optimal conditions were then scaled up using a HiScreen column. Equilibration was performed using 25 mM dibasic sodium phosphate at pH 5 at a flow rate of 3 mL / min. Bound proteins were eluted by a sodium chloride gradient increasing from 0 to 1 M using 25 mM dibasic sodium phosphate at pH 7, 1 M sodium chloride. Various binding and elution conditions were screened, and the weak cation exchanger Capto MMC showed the best binding performance at pH 5. However, the low purity (25%) after this step led to the exploration of alternative purification steps. Figure 7A shows the PAGE analysis of fractions eluted from Capto MMC. M: protein marker; lane 1: eluted fraction showing low purity after cation exchange. The arrow indicates the mycodulcein band.

[0295]

[0335] Evaluation by HIC.

[0296]

[0336] The cell lysate was also subjected to hydrophobic interaction chromatography (HIC) using a HiScreen CaptoButyl column (Cytiva, Sweden); equilibration was performed using 5 column volumes of 50 mM sodium phosphate, 1 M ammonium sulfate at pH 7.0. The cell lysate was then loaded onto the column at a flow rate of 3 mL / min. Elution of bound proteins was performed with a decreasing ammonium sulfate gradient from 1 M to 0 M using 50 mM sodium phosphate at pH 7.0. All resulting fractions were analyzed by SDS-PAGE.

[0297]

[0337] Figure 7B shows two eluted fractions collected during gradient elution from a HiScreen Capto Butyl column analyzed by SDS-PAGE. Lane 1 shows eluted fraction 1 containing no mycodulcein, and lane 2 shows eluted mycodulcein. The purity of the eluted fractions was determined by GelAnalyzer to be approximately 86%.

[0298]

[0338] SEC rated.

[0299]

[0339] The eluted fractions containing native mycodulcein were then further purified using a size-exclusion chromatography (SEC) HiPrep 26 / 60 Sephacryl S-200 HR column (Cytiva, Sweden) and eluted with a buffer containing 50 mM sodium phosphate and 150 mM NaCl at pH 7.0. The fractions were collected, then concentrated and desalted using a 3 kDa molecular weight cut-off (MWCO) centrifugal filter (Millipore-Sigma, Germany) and then analyzed by SDS-PAGE.

[0300]

[0340] Summary: Native mycodulcein binds well to Capto MMC, a weak cation exchanger, but the relatively low purity of the eluted fractions made CIEX a less convenient capture / intermediate purification step. Meanwhile, fractions of higher purity were obtained from HIC, a Capto Butyl column. By SDS-PAGE analysis, the impurities appeared to have a relatively high molecular weight, making SEC a good candidate to obtain highly pure native mycodulcein.

[0301]

[0341] The purity after HIC / SEC is approximately 98% by GelAnalyzer on SDS-PAGE. Figure 7C shows proteins eluted from the HIC column after chromatographic analysis on HiPrep 26 / 60 Sephacryl S-200. Lane 1 shows purified his-tagged mycodulcein and lane 2 shows purified native mycodulcein.

[0302]

[0342] Native purified protein from S. cerevisiae was tasted (0.2 mL aliquots) at 0.03 mg / ml by trained sensory scientists and found to have a sweetness equivalent to 8° Brix (approximately an 8% sugar solution). The sweetness was very significantly sweet, with a "clean" sweetness (tastes like sugar) without other flavors, a slightly slow onset, and a sweet aftertaste.

[0303]

[0343] Example 12 (Application Data)

[0344] The His-tagged mycodulcein prepared in Example 5 and purified as described in Example 5 was tested in a yogurt base, which had the recipe in Table 5 below.

[0304]

[0345]

[0305] [Table 5]

[0346] Sucrose is added as a carbon source for the yogurt cultures and is at least partially consumed by the cultures. Mycodulcein is added to approximate the sweetness of 8°-10° Brix sugar, with a final concentration of 0.05 mg / ml in the yogurt base. Taste tests were performed by trained sensory scientists and the yogurt was found to have a sweetness equivalent to 8° Brix (approximately 8% sugar solution). The sweetness was very significantly sweet, with a "clean" sweetness (tastes like sugar) lacking other flavors, a slightly slow onset and a sweet aftertaste.

[0306]

[0347] The His-tagged mycodulcein prepared as described in Example 5 and purified as described in Example 5 was tested in whole milk; non-dairy pea-based milk (water, 93.75%, pea protein, 4.2%, canola oil, 1.7%, TIC Gum Blend Pro 181 AG (acacia + gellan), 0.3%, sunflower lecithin, 0.05%); cold coffee; and water (control) at a final concentration of 0.04 mg / ml, predicted to provide a sweetness level of 8°-10° Brix. Taste testing confirmed that the sweet protein provided a sweetness level of 8°-10° Brix in all samples, and all samples had similar sweetness intensity, onset, and duration to the water control.

[0307]

[0348] Example 13 (Production and testing of additional variants of mycodulcein for sweetness and heat stability)

[0349] Methods for identifying possible key residues in mycodulcein. Specifically, the following single mutants were generated. Their predicted positions are listed in Table 6. These mutants were generated in the background of mycodulcein with a his tag, e.g., SEQ ID NO: 21. Thus, all Table 6 sequences are mutants with a single amino acid change as shown in Table 6, e.g., compared to SEQ ID NO: 21. Thus, Z88CE is SEQ ID NO: 21, but with a mutation of S to Y (serine to tyrosine) at position 25.

[0308]

[0350] Cloning: Eco_0001, commonly known as E. coli BL21DE3 (obtained from New England Biolabs, #C2527I), was transformed with 20 plasmids (pMy_3083-pMy_3103) using the manufacturer's protocol. Competent cells were thawed and mixed with 1ng-100ng of plasmid DNA and kept on ice for 30 minutes. The mixture was subjected to a heat shock at 42°C for 45 seconds. Immediately after, the suspension was placed in an ice bath for 10 minutes. Approximately 950 μL of pre-warmed LB medium was added and then placed in a 37°C incubator with shaking at 225 rpm for 1 hour. For each transformation reaction, 1:10 and 1:100 dilutions were made and 100 μL was plated on antibiotic plates. These plates were grown overnight at 37°C, allowing colonies to recover and become visible. This transformation process resulted in strains Z83CE-Z103CE containing plasmids pMy_3083-pMy_3103 sequentially. Heterologous expression in the E. coli host was confirmed using induced expression at shake flask scale. Post-transformation mutants were plated and maintained on LB + ampicillin 100 μg / mL agar plates.

[0309]

[0351] Plates were incubated at 37°C for 16 hours. Colony screening PCR was performed to confirm the genotype using colony screening primers designed to interrogate the flanking regions along with the cDNA region of the plasmid. Successful transformation yielded DNA fragments of specific sizes, while negative and no template controls yielded no PCR bands. Successful transformation was observed for all mutants.

[0310]

[0352] Induced expression at shake flask scale was used to confirm heterologous expression in the E. coli host.

[0311]

[0353] Twenty strains (Z83CE-Z103CE; Z93CE was not transformed) were maintained on LB + ampicillin 100 μg / mL agar plates, while negative controls were maintained simply on LB plates. Overnight cultures were grown in 50 mL of LB liquid medium for each strain in baffled 250 mL culture shake flasks. These flasks were shaken overnight at 37°C at 150 rpm with the appropriate antibiotic. Each overnight culture was then inoculated into 200 mL of TB liquid medium after 16 hours of growth. These larger culture flasks were placed at 37°C with shaking at 200 rpm and incubated at an OD of 0.1 with the appropriate antibiotic. 600 The OD was adjusted to 600 When the NA reached 0.8, IPTG supplement was added to the medium to a final concentration of 0.66 mM. The flask was then left shaking for an additional 5 hours. After this time, the cells were harvested by centrifugation at 4000 rpm for 5 minutes. The E. coli cells were then resuspended in cold dHO. 2 The cells were washed once with 0 and centrifuged again at 4000 rpm for 10 min. For confirmation of successful expression, cell lysates were made by sonication of the cell membrane. Cell lysates and dead cell debris were separated by centrifugation at 10,000 rpm for 5 min. Finally, the supernatant was aspirated, filtered through a 0.2 μm PES filter, and run on SDS-PAGE protein electrophoresis. The crude lysates were tasted to identify sweet-tasting mutants.

[0312]

[0354] The results of the tests are shown in Table 6. The thermal stability was tested according to the method of Example 9. The sensory test was carried out according to Example 8.

[0313]

[0355]

[0314] [Table 6-1]

[0315] [Table 6-2]

[0356] The purified samples were tasted by trained sensory scientists. Mycodulcein stocks were diluted to a protein concentration equal to that measured by ELISA. Subjects followed a drink-and-spit protocol approved by the Institutional Review Board. 0.2 ml of each purified mutant was placed on the tongue and the intensity of sweetness perception, the time of onset of sweetness perception, and the duration of sweetness perception were recorded. The results are shown in Figure 6 and discussed herein below.

[0316]

[0357] The results (Table 6) show that thermostability was minimally affected by the amino acid changes in the mutants tested.

[0317]

[0358] Additional single-site mutants for testing were generated by the method described in this example. Table 7 shows the mutants that were made. Mutations are identified relative to SEQ ID NO:3. The first column in Table 7 shows the amino acid position, the second column shows the amino acid at that position in WT (SEQ ID NO:3), and the third column shows the mutation (either none or in the form of the original amino acid, position, and new amino acid using one-letter code). The single-site mutations in Table 7 are embodied in the consensus sequence of SEQ ID NO:141.

[0318]

[0359] Table 7: Summary of single-site mutations

[0360] 1 M is

[0361] 2 P is

[0362] 3 D D3A, D3F, D3H, D3I, D3K, D3L, D3N, D3Q, D3R, D3V, D3W, D3Y

[0363] 4 L L4D, L4E, L4F, L4H, L4K, L4N, L4Q, L4R, L4W, L4Y

[0364] 5 S is

[0365] 6 S is

[0366] 7 F F7A, F7D, F7E, F7H, F7I, F7K, F7L, F7N, F7Q, F7R, F7V, F7W, F7Y

[0367] 8 I I8D, I8E, I8F, I8H, I8I, I8K, I8N, I8Q, I8R, I8V, I8W, I8Y

[0368] 9 T is

[0369] 10 I I10D, I10E, I10F, I10H, I10K, I10L, I10N, I10Q, I10R, I10V, I10W, I10Y

[0370] 11 K K11A, K11D, K11E, K11F, K11H, K11I, K11L, K11N, K11Q, K11V, K11W, K11Y

[0371] 12 N N12A, N12D, N12E, N12F, N12H, N12I, N12K, N12L, N12Q, N12R, N12V, N12W, N12Y

[0372] 13 N N13A, N13D, N13E, N13F, N13H, N13I, N13K, N13L, N13Q, N13R, N13V, N13W, N13Y

[0373] 14 S is

[0374] 15 N N15A, N15D, N15E, N15F, N15H, N15I, N15K, N15L, N15Q, N15R, N15V, N15W, N15Y

[0375] 16 H H16A、H16D、H16E、H16F、H16I、H16K、H16L、H16N、H16Q、H16R、H16V、H16W、H16Y

[0376] 17 V V17D、V17E、V17F、V17H、V17K、V17N、V17Q、V17R、V17W、V17Y

[0377] 18 F F18A、F18D、F18E、F18H、F18I、F18K、F18L、F18N、F18Q、F18R、F18V、F18W

[0378] 19 T なし

[0379] 20 R R20A、R20D、R20E、R20F、R20H、R20I、R20L、R20N、R20Q、R20V、R20W、R20Y

[0380] 21 T なし

[0381] 22 A A22D、A22E、...

Claims

1. 1. An isolated polynucleotide encoding a polypeptide having at least 90% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140, A polynucleotide wherein the encoded polypeptide has sweet taste modulating activity and is not the polypeptide represented in SEQ ID NO:

3.

2. 2. The polynucleotide of claim 1, wherein the encoded polypeptide is a polypeptide represented in SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:

140.

3. having a nucleotide sequence represented in SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:139; 2. The polynucleotide of claim 1, wherein the sequence encoding the histidine tag in each of the listed SEQ ID NOs is excluded.

4. 2. The polynucleotide of claim 1, encoding a polypeptide selected from the group consisting of SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:

140.

5. 2. The polynucleotide of claim 1, which encodes a polypeptide selected from the group consisting of SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:

140.

6. The polynucleotide of any one of claims 1 to 5, operably linked to a heterologous regulatory element.

7. The polynucleotide of any one of claims 1 to 5, which encodes or further encodes a protein tag.

8. The polynucleotide of any one of claims 1 to 5, which encodes or further encodes a histidine tag.

9. An expression cassette comprising the polynucleotide of any one of claims 1 to 5.

10. A vector comprising the polynucleotide according to any one of claims 1 to 5.

11. A host cell transformed with a vector containing the polynucleotide of any one of claims 1 to 5.

12. An isolated polypeptide comprising a polypeptide having at least 90% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140, wherein the isolated polypeptide has sweet taste modulating activity.

13. 13. The polypeptide of claim 12, wherein the polypeptide sequence is selected from the group consisting of SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:

140.

14. 13. The polypeptide of claim 12, which is a polypeptide represented in SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:

140.

15. 13. The polypeptide of claim 12, which is a polypeptide represented by SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:

140.

16. 13. The polypeptide of claim 12, wherein the N-terminal amino acid sequence of the polypeptide is the sequence represented in SEQ ID NO:

4.

17. The polypeptide of claim 12, comprising a histidine tag.

18. the sweet taste modulating activity is produced by culturing a host cell in a medium under conditions that result in the production of a polypeptide having sweet taste modulating activity, wherein the host cell has been transformed with a vector comprising a polynucleotide encoding a polypeptide having at least 90% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:132, SEQ ID NO:137, or SEQ ID NO:140; 13. The polypeptide of claim 12, which has sweet taste modulating activity and is not a polypeptide represented in SEQ ID NO:

3.

19. A method for producing a protein having sweetness regulating activity, comprising culturing a host cell transformed with a vector comprising the polynucleotide of any one of claims 1 to 5 in a medium under conditions that result in the production of a protein having sweetness regulating activity.

20. (a) a product for oral administration, and (b) a sweetener composition comprising the isolated polypeptide of any one of claims 12 to 18.

1. A composition comprising the combination of the above, which does not contain Mattiromyces terfesioides truffles and has an enhanced sweetness compared to a product for oral administration.

21. 21. The composition of claim 20, wherein the product for oral administration is a food product selected from the group consisting of baked goods; sweet bakery products, pre-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings, gelatins and puddings; frozen desserts; yogurt; snack bars; bread products; pre-made bread mixes for preparing bread products; sauces, syrups and dressings; sweet spreads; confectionery products; and sweetened breakfast cereals, or a beverage product selected from the group consisting of carbonated beverages; non-carbonated beverages; and beverage concentrates.

22. 21. The composition of claim 20, wherein the sweetener composition further comprises a nutritive sweetener, a non-nutritive sweetener, or a high intensity sweetener different from the polypeptide having sweetness modulating activity.

23. 20. A method for preparing the taste of a product for oral administration, comprising combining an effective amount of a sweetener composition comprising an isolated polypeptide according to any one of claims 12 to 18 with a food product for oral administration, wherein the sweetener composition does not contain Mattiromyces terfesioides truffles, and the combination has an enhanced sweetness compared to the product for oral administration.

24. 24. The method of claim 23, wherein the product for oral administration is a food product selected from the group consisting of baked goods; sweet bakery products, pre-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings, gelatins and puddings; frozen desserts; yogurt; snack bars; bread products; pre-made bread mixes for preparing bread products; sauces, syrups and dressings; sweet spreads; confectionery products; and sweetened breakfast cereals, or a beverage product selected from the group consisting of carbonated beverages; non-carbonated beverages; and beverage concentrates.

25. 24. The method of claim 23, wherein the sweetener composition further comprises a nutritive sweetener, a non-nutritive sweetener, or a high intensity sweetener different from the polypeptide having sweetness modulating activity.

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