Honey truffle sweetener (HTS) variants

Abstract

Fungal sweetness regulating protein variants (designated as honey truffle sweetener (HTS) protein variants) and polynucleotides encoding the protein variants are described. In particular, Myd (HTS) protein variants having sweetness modulating activity and cDNA encoding the same are described, as well as methods for isolating such cDNA and for isolating and expressing such proteins. Sweet compositions comprising the proteins of the invention are also disclosed, as well as methods for providing improved flavor for orally administered products.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 444,185, filed February 8, 2023, U.S. Provisional Application No. 63 / 524,794, filed July 3, 2023, U.S. Provisional Application No. 63 / 541,591, filed September 29, 2023, and U.S. Provisional Application No. 63 / 618,616, filed January 8, 2024, each of which is incorporated herein by reference in its entirety.

[0003] Electronically submitted materials are incorporated herein by reference

[0004] The following computer-readable nucleotide / amino acid sequence listing identified as follows is incorporated herein by reference: a 282KB XML file named "0640-47_WO.xml" created on February 8, 2024. Field of the Invention

[0005] The present invention includes embodiments of sweet (honey truffle (truffle) sweetener (HTS)) proteins, genes and cDNAs encoding the proteins, and methods of using these proteins, genes and cDNAs to modulate the taste of foods. More particularly, the present invention includes embodiments of HTS variant sweet proteins, and genes and cDNAs encoding such proteins. Background of the Invention

[0007] Excessive intake of nutritive sweeteners has long been linked to diet-related health problems such as obesity, heart disease, metabolic disorders, and dental problems. Consequently, consumers are increasingly looking for ways to reduce the amount of nutritive sweeteners in their diets; and manufacturers are responding to this demand by attempting to replace nutritive sweeteners with alternatives that mimic the desirable taste and functional properties of nutritive sweeteners.

[0008] It is desirable to preferably use zero-calorie or low-calorie sweeteners derived from natural sources to limit the negative effects of high sugar consumption (e.g., diabetes and obesity, etc.) However, well-known zero-calorie or low-calorie sweetener substitutes such as aspartame, acesulfame potassium, monk fruit extract, neotame, saccharin, stevia, and sucralose have unpleasant taste defects such as bitterness.

[0009] Zero-calorie or low-calorie sweeteners derived from natural sources may be preferred to limit the negative effects of high sugar intake. To date, only seven sweet-tasting proteins are known: monellin, thaumatin, brazzein, curculin, mabinin, miraculin, and pentadin. For any of these proteins, the key residues on the protein surface responsible for biological activity have not yet been determined. Monellin was found to be 100,000 times sweeter than sucrose on a molar basis, followed by thaumatin and brazzein, which are 3000 times and 500 times sweeter than sucrose on a gram basis, respectively. Most of them have no sequence homology or structural similarity; and thaumatin has extensive similarities to certain non-sweet proteins found in other plants at the protein sequence level.

[0010] International patent application PCT / US2020 / 012955, filed on September 1, 2020, and published as WO2020 / 146650 on July 16, 2020, relates to sweetening compositions comprising (i) mycelium of an ascomycete or an aqueous extract thereof, or (ii) an aqueous extract of an ascomycete fruiting body, and the use of such compositions in providing improved flavor to products for oral administration. The application also relates to compositions comprising a combination of a sweetening composition and a product for oral administration.

[0011] International patent application PCT / US2021 / 039176, filed on June 25, 2021 and published as WO2021 / 263158 on December 30, 2021, relates to newly identified fungal sweet taste modifier proteins and cDNAs encoding the proteins. Sweet taste modulator proteins were identified in the truffle, Mattirolomyces terfeziodes. The application also relates to Myd proteins as sweeteners and sweet taste activators / regulators and cDNAs encoding the proteins, as well as methods for isolating such cDNAs and for isolating and expressing such proteins. The application also relates to sweet compositions comprising the protein and methods for providing improved flavor for products for oral administration.

[0012] International patent application PCT / US2022 / 82443, filed on December 27, 2022, and published on July 6, 2023 as WO2023 / 129938, relates to sweet protein variants obtained from truffles, such as truffles, including proteins, genes encoding the proteins, cDNAs and combinations thereof, and is hereby incorporated by reference in its entirety.

[0013] There remains a need for new low-calorie or zero-calorie sweeteners with improved taste from natural sources, particularly Ascomycetes species. There remains a need for methods of producing fungal-derived low-calorie or zero-calorie sweeteners with improved taste, and for using these ingredients in finished food products without compromising taste and with superior taste characteristics. Summary of the Invention

[0014] The present invention satisfies these and other needs by providing sweet proteins of fungal origin identified herein as the Myd protein family, as well as genes and cDNAs encoding these proteins, and methods of using these proteins, genes and cDNAs to modulate the taste of foods. The present invention provides variants of MYD1 (also known as mycodulcein and honey truffle sweetener proteins (HTS proteins)). The polypeptides of the present invention exhibit flavor-modulating properties and, in particular, modulate sweetness and perception, alone or in combination with foods, beverages, dietary supplements, or pharmaceuticals. In embodiments, the Myd1 variants of the present invention can provide sweetness to ingested substances such as foods, beverages, dietary supplements, or pharmaceuticals. In embodiments, the Myd1 variants of the present invention can reduce the sourness, bitterness, or astringency of foods and beverages. In embodiments, the Myd1 variants of the present invention can exhibit enhancement of the taste of foods and beverages, i.e., flavor-modulating activity. In embodiments, the Myd1 variants can exhibit, in addition to sweetness-modulating activity, enhanced thermal stability compared to the native form of the HTS protein. In embodiments, the Myd1 variants can exhibit flavor-modulating properties. In embodiments, Myd1 variants may exhibit enhanced thermostability in addition to flavor modulating properties compared to the native form of the HTS protein. In embodiments, the invention provides naturally occurring Myd proteins. In embodiments, the invention provides variant Myd proteins other than the naturally occurring proteins.

[0015] Naturally occurring Myd proteins exist in two isoforms; HTS-1 (SEQ ID NO: 3) and HTS-2 (SEQ ID NO: 141), such as isolated from the fungus Truffle. The relative amounts of HTS-1 and HTS-2 isolated from the fungus Truffle range from about 40% to 60% by weight for HTS-2 and about 60% to 40% by weight for HTS-1. In an embodiment, the present invention provides a non-naturally occurring sweet protein comprising a non-naturally occurring mixture of two isoforms, HTS-1 and HTS-2. In an embodiment, the present invention provides a sweet protein produced by recombinant expression of the coding sequence of SEQ ID NO: 2 or a codon-optimized version of the coding sequence of SEQ ID NO: 2 in a non-natural host (e.g., a host other than the fungus Truffle), and a method for producing a sweet protein by recombinant expression.

[0016] In an embodiment, the encoded polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 3 (also referred to as an HTS-1 isoform). In an embodiment, the encoded polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 3, except that the methionine at position 1 in SEQ ID NO: 3 is absent, which is hereinafter referred to as a polypeptide having the amino acid sequence of SEQ ID NO: 141 (also referred to as an HTS-2 isoform). In an embodiment, the encoded polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 5, which is an HTS-1 isoform with (His-tag) 6. In an embodiment, the encoded polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 142, which is an HTS-2 isoform with (His-tag) 6.

[0017] Thus, one aspect of the present invention provides a polynucleotide (e.g., an isolated polynucleotide) encoding a polypeptide, wherein the encoded polypeptide has sweet taste modulating activity and has an amino acid sequence that is different from the polypeptide of SEQ ID NO: 3, or optionally different from the polypeptide of SEQ ID NO: 141. In embodiments, the encoded polypeptide is selected from the group consisting of: (a) a polypeptide sequence selected from the group consisting of the amino acid sequences of the variants listed in Table 8, Table 9, or Table 10; (b) a polypeptide having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of the amino acid sequences of the variants listed in Table 8, Table 9, or Table 10; and (c) a polypeptide sequence modified from the group consisting of the amino acid sequences of the variants listed in Table 8, Table 9, or Table 10 by deletion, insertion, substitution, or addition of no more than 24 amino acids, and wherein the polypeptide sequence is different from the polypeptide sequence of SEQ ID NO: 3, and optionally different from the polypeptide sequence of SEQ ID NO: 141. In embodiments, the polynucleotide encoding the polypeptide is not the polynucleotide of SEQ ID NO: 2. In an embodiment, the polynucleotide encoding the polypeptide is a polynucleotide of SEQ ID NO: 2, which has been codon-optimized for expression in bacteria, yeast, or fungi. In an embodiment, the polynucleotide encoding the polypeptide is a polynucleotide of SEQ ID NO: 6 with an optional His tag, which has been codon-optimized for expression in E. coli. In an embodiment, the polynucleotide encoding the polypeptide is a polynucleotide of SEQ ID NO: 7 with an optional His tag, which has been codon-optimized for expression in Saccharomyces cerevisiae.

[0018] In an embodiment, the encoded polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO:3, except that the methionine at position 1 in SEQ ID NO:3 is absent, which is represented herein as a polypeptide having the amino acid sequence of SEQ ID NO:141. In an embodiment, the encoded polypeptide is a polypeptide other than a polypeptide having the amino acid sequence of SEQ ID NO:141. In an embodiment, the encoded polypeptide exhibits a sweet taste. In an embodiment, the encoded polypeptide exhibits a sweet taste when present in a composition at a concentration of 30 ppm or greater. In an embodiment, the encoded polypeptide exhibits a sweet taste when present in a composition at a concentration of 40 ppm or greater.

[0019] In a related aspect, the present invention provides a polynucleotide (e.g., an isolated polynucleotide) encoding a polypeptide, wherein the encoded polypeptide has flavor-modulating properties and has an amino acid sequence that is different from the polypeptide of SEQ ID NO: 3 and, optionally, different from SEQ ID NO: 141. In embodiments, the encoded polypeptide is selected from the group consisting of: (a) a polypeptide sequence selected from the group consisting of the amino acid sequences of the variants listed in Table 8; (b) a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of the amino acid sequences of the variants listed in Table 8; and (c) a polypeptide sequence modified from the group consisting of the amino acid sequences of the variants listed in Table 8 by deletion, insertion, substitution, or addition of no more than 24 amino acids, and wherein the polypeptide sequence is different from the polypeptide sequence of SEQ ID NO: 3 and, optionally, different from the polypeptide sequence of SEQ ID NO: 141. In embodiments, the encoded polypeptide is a polypeptide selected from the group consisting of the amino acid sequences of the variants listed in Table 8, which is not a variant listed in Table 9 or Table 10. In embodiments, the polynucleotide encoding the polypeptide is a polynucleotide other than the polynucleotide of SEQ ID NO: 2. In an embodiment, the encoded polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 141. In an embodiment, the encoded polypeptide is a polypeptide other than a polypeptide having the amino acid sequence of SEQ ID NO: 141. In an embodiment, the encoded polypeptide does not exhibit a sweet taste. In an embodiment, the encoded polypeptide does not exhibit a sweet taste when present in the composition at a concentration of 30 ppm or more. In an embodiment, the encoded polypeptide does not exhibit a sweet taste when present in the composition at a concentration of 40 ppm or more. In an embodiment, the encoded polypeptide exhibits a sweet taste only when present in the composition at a concentration of 30 ppm or more.

[0020] In another aspect of the invention, amino acid mutations of HTS polypeptides and protein variants are summarized in Table 8. The mutations in Table 8 are identified relative to the amino acid sequence of SEQ ID NO:3. Thus, a polypeptide having the amino acid sequence of a variant listed in Table 8 will have the amino acid sequence of SEQ ID NO:3, but with one or more site mutations identified in Table 8. The mutations in Table 8 also include mutants in which the methionine at position 1 of the protein is deleted. In a related aspect, the present invention provides HTS polypeptide and protein variants, wherein the variant has a single site mutation of Table 8 relative to the amino acid sequence of SEQ ID NO:3, and further, wherein the methionine at position 1 in SEQ ID NO:3 is deleted.

[0021] In another aspect of the invention, amino acid mutations of HTS polypeptide and protein variants are summarized in Table 9. The mutations in Table 9 are identified relative to the amino acid sequence of SEQ ID NO: 3. Thus, a polypeptide having the amino acid sequence of a variant listed in Table 9 will have the amino acid sequence of SEQ ID NO: 3, but with one or more site mutations identified in Table 9. The mutations in Table 9 also include mutants in which the methionine at position 1 of the protein is deleted. In a related aspect, the present invention provides HTS polypeptide and protein variants, wherein the variant has a single site mutation of Table 9 relative to the amino acid sequence of SEQ ID NO: 3, and further, wherein the methionine at position 1 in SEQ ID NO: 3 is deleted.

[0022] In another aspect of the invention, amino acid mutations of HTS polypeptide and protein variants are summarized in Table 10. The mutations in Table 10 are identified relative to the amino acid sequence of SEQ ID NO:3. Thus, a polypeptide having the amino acid sequence of a variant listed in Table 10 will have the amino acid sequence of SEQ ID NO:3, but with one or more site mutations identified in Table 10. The mutations in Table 10 also include mutants in which the methionine at position 1 of the protein is deleted. In a related aspect, the present invention provides HTS polypeptide and protein variants, wherein the variant has a single site mutation of Table 8 relative to the amino acid sequence of SEQ ID NO:3, and further, wherein the methionine at position 1 in SEQ ID NO:3 is deleted.

[0023] In another aspect of the invention, HTS polypeptide and protein variants are those having a single site mutation listed in Table 7 relative to the amino acid sequence of SEQ ID NO: 3. In a related aspect of the invention, HTS polypeptide and protein variants are those having a single site mutation listed in Table 7 relative to the amino acid sequence of SEQ ID NO: 3, and additionally, wherein the methionine at position 1 in SEQ ID NO: 3 is deleted.

[0024] In another aspect of the invention, the HTS polypeptides and protein variants are not those having the single site mutations listed in Table 7, as identified relative to the amino acid sequence of SEQ ID NO: 3. In a related aspect, the invention does not include polynucleotides encoding HTS polypeptides and protein variants having the single site mutations listed in Table 7. In a related aspect of the invention, the HTS polypeptides and protein variants are not those having the single site mutations listed in Table 7, and wherein the methionine at position 1 of SEQ ID NO: 3 is deleted. In a related aspect, the invention does not include polynucleotides encoding HTS polypeptides and protein variants having the single site mutations listed in Table 7, and wherein the methionine of SEQ ID NO: 3 is deleted.

[0025] In another aspect of the invention, the HTS polypeptide or protein variant does not include the mutations of Table 7. In a related aspect, the invention does not include HTS polypeptide or protein variants comprising the mutations listed in Table 7. In a related aspect, the invention does not include polynucleotides encoding HTS polypeptide or protein variants comprising the mutations listed in Table 7.

[0026] In another aspect of the invention, the HTS polypeptide or protein variant does not include the mutations listed in Table 3 or Table 6. In a related aspect, the invention does not include HTS polypeptide or protein variants comprising the mutations listed in Table 3 or Table 6. In a related aspect, the invention does not include polynucleotides encoding HTS polypeptide or protein variants comprising the mutations listed in Table 3 or Table 6.

[0027] In another aspect of the invention, an HTS polypeptide or protein variant does not include a mutation of Table 8, Table 9, or Table 10, but includes one or more conservative mutations of the amino acid sequence of SEQ ID NO: 3 or, optionally, SEQ ID NO: 141, wherein in the variant, the methionine at position 1 is present or absent. In a related aspect, the invention includes a polynucleotide encoding an HTS polypeptide or protein variant that does not include a mutation of Table 8, Table 9, or Table 10, but includes one or more conservative mutations of the amino acid sequence of SEQ ID NO: 3, wherein in the variant, the methionine at position 1 is present or absent.

[0028] In other aspects, the polynucleotide encoding the polypeptide having flavor modulation or sweet taste modulation activity is optionally operably linked to a heterologous regulatory element. Additionally or alternatively, the polynucleotide sequence also encodes a protein / peptide tag or marker. The protein / peptide tag is optionally an affinity tag. The protein tag is optionally a histidine tag (His tag). Optionally, the protein tag is (His) 6. In an embodiment, the polynucleotide encoding the polypeptide having sweet taste modulation activity is not a nucleotide of SEQ ID NO: 2 that further encodes a protein / peptide tag or marker.

[0029] One aspect of the invention provides a polynucleotide (e.g., an isolated polynucleotide) encoding a polypeptide, wherein the encoded polypeptide is selected from the group consisting of: (a) a polypeptide sequence selected from the group consisting of the amino acid sequences of the variants listed in Table 8, Table 9 or Table 10, wherein the polypeptide further consists of a protein / peptide tag, optionally an affinity tag or optionally a histidine tag; (b) a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of the amino acid sequences of the variants listed in Table 8, Table 9 or Table 10, wherein the polypeptide further consists of a protein / peptide tag, optionally an affinity tag or optionally a histidine tag; and (c) a polypeptide sequence modified from the group consisting of the amino acid sequences of the variants listed in Table 8, Table 9 or Table 10 by deletion, insertion, substitution or addition of no more than 24 amino acids, and wherein the polypeptide further consists of a protein / peptide tag, optionally an affinity tag or optionally a histidine tag, and wherein the polypeptide sequence is different from the polypeptide sequence of SEQ ID NO: 3 containing a histidine tag. In a related embodiment, the encoded polypeptide is different from the polypeptide of SEQ ID NO: 142 containing a histidine tag.

[0030] In particular aspects, the polynucleotide encoding the polypeptide having sweet taste modulating activity is not 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:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125 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, SEQ ID NO:139 or SEQ ID NO:140.

[0031] Another aspect of the present invention provides a polynucleotide other than the polynucleotide of SEQ ID NO: 4, which corresponds to a coding sequence optimized for a His-tagged HTS in E. coli (wherein residues 364-381 correspond to an optional His-tag sequence). Another aspect of the present invention provides a polynucleotide other than the polynucleotide of SEQ ID NO: 6, which corresponds to a coding sequence of an HTS optimized for expression in S. cerevisiae (to which a His-tag coding sequence has been added, e.g., (His)6).

[0032] In a specific embodiment, the present invention provides Myd variants that exhibit enhanced thermal stability relative to the natural isoforms of the Myd protein (HTS-1 and HTS-2). In a specific embodiment, the present invention provides

[0033] In other aspects, the present invention provides expression cassettes comprising the polynucleotides described herein and vectors comprising the polynucleotides, as well as host cells transformed with the vectors. In addition, a method for producing a protein having sweet taste modulating activity is provided, comprising culturing the transformed host cell in a culture medium under conditions that result in the production of a protein having sweet taste modulating activity.

[0034] In another aspect, the present invention provides a host cell for expressing a polypeptide as described herein. In an embodiment, the host cell is an Escherichia coli. In an embodiment, the host cell is a plant cell.

[0035] Another aspect of the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity and comprising: (i) a polypeptide sequence selected from those of the amino acid sequences of the variants listed in Table 8, Table 9, or Table 10; wherein the polypeptide sequence is different from SEQ ID NO: 3 and optionally different from SEQ ID NO: 141, or (ii) a polypeptide having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of those of the amino acid sequences of the variants listed in Table 8, Table 9, or Table 10, wherein the polypeptide sequence is different from SEQ ID NO: 3 and optionally different from SEQ ID NO: 141. In a further aspect, the polypeptide (a) contains at least one modification relative to the polypeptide sequence selected from the group consisting of the amino acid sequences of the variants listed in Table 8, Table 9, or Table 10 by deletion, insertion, substitution, or addition of no more than 24 amino acids, wherein the polypeptide is different from the polypeptide of SEQ ID NO: 3 and optionally different from SEQ ID NO: 141, and / or (b) further comprises a protein / peptide tag, optionally an affinity tag, and particularly a histidine tag, and wherein the polypeptide has sweet taste modulating activity.

[0036] In another aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide) comprising an amino acid sequence of SEQ ID NO: 3 modified by at least one mutation selected from those listed in Table 8, Table 9, or Table 10. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide) comprising an amino acid sequence of SEQ ID NO: 3 modified by two mutations at different positions selected from those listed in Table 8, Table 9, or Table 10. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide) comprising an amino acid sequence of SEQ ID NO: 3 modified by three mutations at different positions selected from those listed in Table 8, Table 9, or Table 10. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide) comprising an amino acid sequence of SEQ ID NO: 3 modified by four mutations at different positions selected from those listed in Table 8, Table 9, or Table 10. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste regulating activity, comprising an amino acid sequence of SEQ ID NO:3 modified by five mutations selected from those listed in Table 8, Table 9 or Table 10 at different positions. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste regulating activity, comprising an amino acid sequence of SEQ ID NO:3 modified by six mutations selected from those listed in Table 8, Table 9 or Table 10 at different positions. In other embodiments of the foregoing embodiments, the 1-6 modifications are not to remove the methionine at position 1. In an embodiment, in a polypeptide having 1-6 amino acid modifications, the methionine at position 1 is present or absent. The present invention also provides a polynucleotide encoding the above-mentioned mutant (variant) polypeptide of SEQ ID NO:3. The present invention also provides the above-mentioned mutant polypeptide, which further comprises a protein / peptide tag, optionally an affinity tag, and more specifically, a histidine tag. The present invention also provides a polynucleotide encoding the above mutant polypeptide of SEQ ID NO: 3, wherein the mutant further comprises a protein / peptide tag, optionally an affinity tag, and more specifically, a histidine tag. In an embodiment, the polypeptide does not include a polypeptide comprising a mutation listed in Table 7.

[0037] In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by seven mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by eight mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by nine mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by ten mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO: 3 modified by eleven mutations selected from those listed in Table 8, Table 9 or Table 10 at different positions. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO: 3 modified by twelve mutations selected from those listed in Table 8, Table 9 or Table 10 at different positions. In other embodiments of the foregoing embodiments, the 1-12 modifications are not removal of the methionine at position 1. In an embodiment, in the polypeptide having 1-12 amino acid modifications, the methionine at position 1 is present or absent. The present invention also provides a polynucleotide encoding the mutant polypeptide of the aforementioned SEQ ID NO: 3. The present invention also provides the above-mentioned mutant polypeptide, which further comprises a protein / peptide tag, optionally an affinity tag, and more specifically, a histidine tag. The present invention also provides polynucleotides encoding the above mutant polypeptide of SEQ ID NO: 3, wherein the mutant polypeptide further comprises a protein / peptide tag, optionally an affinity tag, and more specifically, a histidine tag. In one aspect, the polypeptide does not include a polypeptide comprising the mutations listed in Table 7.

[0038] In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by thirteen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by fourteen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by fifteen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by sixteen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) with sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO:3 modified by seventeen mutations selected from those listed in Table 8, Table 9 or Table 10 at different positions. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) with sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO:3 modified by eighteen mutations selected from those listed in Table 8, Table 9 or Table 10 at different positions. In other embodiments of the foregoing embodiments, the 1-18 modifications are not to remove the methionine at position 1. In an embodiment, in a polypeptide with 1-18 amino acid modifications, the methionine at position 1 is present or absent. The present invention also provides a polynucleotide encoding the mutant polypeptide of the aforementioned SEQ ID NO:3. The present invention also provides the above-mentioned mutant polypeptide, which further comprises a protein / peptide tag, optionally an affinity tag, and more specifically, a histidine tag. The present invention also provides polynucleotides encoding the above mutant polypeptide of SEQ ID NO: 3, wherein the mutant polypeptide further comprises a protein / peptide tag, optionally an affinity tag, and more specifically, a histidine tag. In one aspect, the polypeptide does not include a polypeptide comprising the mutations listed in Table 7.

[0039] In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by nineteen mutations at different positions selected from those listed in Table 8, Table 9, or Table 10. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by twenty mutations at different positions selected from those listed in Table 8, Table 9, or Table 10. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by twenty-one mutations at different positions selected from those listed in Table 8, Table 9, or Table 10. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by twenty-two mutations at different positions selected from those listed in Table 8, Table 9, or Table 10. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO: 3 modified by twenty-three mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO: 3 modified by twenty-four mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In other embodiments of the foregoing embodiments, the 1-24 modifications are not removal of the methionine at position 1. In an embodiment, in the polypeptide having 1-24 amino acid modifications, the methionine at position 1 is present or absent. The present invention also provides a polynucleotide encoding the mutant polypeptide of the aforementioned SEQ ID NO: 3. The present invention also provides the above-mentioned mutant polypeptide, which further comprises a protein / peptide tag, optionally an affinity tag, and more specifically, a histidine tag. The present invention also provides polynucleotides encoding the above mutant polypeptide of SEQ ID NO: 3, wherein the mutant polypeptide further comprises a protein / peptide tag, optionally an affinity tag, and more specifically, a histidine tag. In one aspect, the polypeptide does not include a polypeptide comprising the mutations listed in Table 7.

[0040] Another aspect of the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO: 3 modified by at least one mutation, wherein the at least one mutation is selected from the mutations listed in Table 8, Table 9 or Table 10, and is not a mutation listed in Table 7, and wherein the polypeptide is further modified by one or more additional mutations, wherein the one or more additional mutations are selected from the mutations listed in Table 7. In other aspects, the polypeptide is modified by two or more mutations selected from the mutations listed in Table 8, Table 9 or Table 10. In one aspect, the polypeptide is further modified by two or more additional mutations, wherein the two or more additional mutations are selected from the mutations listed in Table 7. In one aspect, the polypeptide has at least 80% sequence identity to a polypeptide sequence selected from the group consisting of those amino acid sequences of the variants listed in Table 8, Table 9 or Table 10. The above polypeptide optionally further comprises a protein / peptide tag, an optional affinity tag, or an optional histidine tag.

[0041] In another aspect, the polypeptides of the invention are optionally isolated and / or optionally purified. In another aspect, the polynucleotides of the invention are optionally isolated and / or purified. In another aspect, the polypeptides of the invention optionally include a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag. In another aspect, the polypeptides of the invention optionally include an affinity tag. In another aspect, the polypeptides of the invention optionally include a histidine tag. In another aspect, the polypeptides of the invention optionally have a methionine at position 1. In another aspect, the polypeptides of the invention are optionally derivatized, as described herein. In another aspect, the polypeptides of the invention are acylated at the N-terminus and optionally acetylated. In another aspect, the polypeptides of the invention are derivatized at the N-terminus. In another aspect, the polypeptides of the invention have a methionine at position 1, and the methionine is derivatized, and optionally the S of the methionine is oxidized. In another aspect, the polypeptides of the invention have a methionine at position 1, and the methionine is derivatized, wherein the S of the methionine is oxidized. In another aspect, the polypeptides of the invention are pegylated at the N-terminus. In another aspect, the polypeptides of the invention are derivatized at the C-terminus, as described herein. In another aspect, the polypeptides of the invention are esterified at the C-terminus.

[0042] In other aspects, certain polypeptides of the present invention exhibit enhanced sweet taste modulating activity compared to the polypeptide of SEQ ID NO: 3. In other aspects, certain polypeptides of the present invention exhibit enhanced thermostability compared to the polypeptide of SEQ ID NO: 3.

[0043] Another aspect of the present invention provides a composition comprising one or more polypeptides of the present invention, wherein the one or more polypeptides exhibit flavor-modulating and / or sweetness-modulating activity and / or exhibit sweetness. The composition comprises a combination of (a) an orally administered product other than a Truffle and (b) a sweetening composition comprising the polypeptides, wherein the combination has an enhanced sweetness and / or a change or enhancement in flavor compared to the orally administered product. The polypeptide includes one or more polypeptide sequences having flavor-modulating and / or sweetness-modulating activity, the one or more polypeptide sequences being present alone or in combinations of two or more and comprising: (i) a polypeptide sequence selected from the amino acid sequence of a variant of Table 8, Table 9 or Table 10, wherein the polypeptide sequence is different from SEQ ID NO: 3 and optionally different from SEQ ID NO: 141; or (ii) a polypeptide having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of the amino acid sequence of a variant of Table 8, Table 9 or Table 10, wherein the polypeptide sequence is different from SEQ ID NO: 3 and optionally different from SEQ ID NO: 141; or (iii) the polypeptide of the variant of Table 8, Table 9 or Table 10 further comprises a protein / peptide tag or an affinity tag, wherein the protein / peptide tag is optionally a histidine tag.

[0044] Another aspect of the invention is a sweetening composition comprising one or more polypeptides of the invention that exhibit sweetness (e.g., HTS variants). A related aspect of the invention is a flavor-modulating composition comprising one or more polypeptides of the invention that exhibit flavor-modulating activity (e.g., HTS variants). In embodiments, the sweetening composition contains 30 ppm or more of the total HTS variant polypeptides. In embodiments, the flavor-modulating composition contains 30 ppm or less of the total HTS variant polypeptides.

[0045] Another more specific aspect of the present invention provides a composition comprising the following combination: (a) an orally administered product other than a Truffle Truffle, and (b) a sweetening composition comprising a polypeptide, wherein the combination has an enhanced sweetness compared to the orally administered product. In embodiments, the polypeptide comprises (a) one or more amino acid sequences selected from the group consisting of amino acid sequences of variants of Table 8, Table 9, or Table 10. In embodiments, the polypeptide is not a variant listed in Table 7, Table 3, or Table 6.

[0046] Another aspect of the present invention provides a method for adjusting the taste of an orally administered product. The method comprises combining the orally administered product with an effective amount of a flavor-modulating composition or a sweetening composition comprising a polypeptide (e.g., an HTS variant), wherein the orally administered product is different from the truffle Truffle Truffle and wherein the combination has an enhanced flavor or sweetness, respectively, compared to the orally administered product. In embodiments, the polypeptide comprises one or more sequences as listed herein, particularly those having one or more mutations in Table 8, Table 9, or Table 10, either alone or in combination of two or more. In embodiments, the polypeptide is not a variant listed in Table 7, Table 3, or Table 6.

[0047] Another aspect of the present invention provides a method for purifying a polypeptide having flavor modulating activity or sweet modulating activity. The method comprises: (a) subjecting a composition comprising the polypeptide to hydrophobic interaction chromatography (HIC), and / or (b) subjecting a composition comprising the polypeptide to size exclusion chromatography (SEC). In embodiments, the method comprises (a) subjecting a composition comprising the polypeptide to hydrophobic interaction chromatography (HIC), or (b) subjecting a composition comprising the polypeptide to size exclusion chromatography (SEC). In embodiments, the method comprises (a) subjecting a composition comprising the polypeptide to hydrophobic interaction chromatography (HIC) to produce a purified composition, and then (b) subjecting the purified composition comprising the polypeptide to size exclusion chromatography (SEC). In an embodiment, the polypeptide comprises one or more sequences having sweet taste modulating activity, the one or more sequences being present alone or in a combination of two or more and comprising: (i) a polypeptide sequence selected from the group consisting of variants of Table 8, Table 9 or Table 10, wherein the polypeptide sequence is different from SEQ ID NO: 3 and optionally different from SEQ ID No: 141; or (ii) a polypeptide having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of variants of Table 8, Table 9 or Table 10, wherein the polypeptide sequence is different from SEQ ID NO: 3 and optionally different from SEQ ID NO: 141; (iii) wherein the polypeptide sequence of the variant of Table 8, Table 9 or Table 10 further comprises a protein / peptide tag, wherein the protein tag is optionally an affinity tag or more particularly a histidine tag; (iv) wherein the polypeptide contains 1-24 mutations selected from those listed in Table 8, Table 9 or Table 10, and optionally further comprises a protein / peptide tag, wherein the protein tag is optionally an affinity tag, and more particularly a histidine tag. In a related aspect, the present invention provides a polypeptide purified by the method described herein.

[0048] Another aspect of the present invention provides a sweetener composition. In some embodiments, the sweetener composition comprises: (a) one or more polypeptides, wherein the one or more polypeptides comprise: (i) a polypeptide sequence selected from the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10; (ii) a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10; (iii) a polypeptide sequence containing at least one modification by deletion, insertion, substitution or addition of no more than 24 amino acids relative to a polypeptide sequence selected from the group consisting of the amino acid sequence of the variant listed in Table 8, Table 9 or Table 10; or (iv) a polypeptide of any one of (i)-(iii), wherein the methionine at position 1 is deleted; and / or the polypeptide further comprises a protein / peptide tag, optionally an affinity tag or optionally a histidine tag or optionally a (His)6 tag; and (b) at least one additional sweetener other than the Myd sweet protein.

[0049] The additional sweetener is optionally selected from steviol glycoside sweeteners, mogroside sweeteners, sucrose, psicose, sucralose, polyols and high fructose corn syrup (HFCS). In other embodiments, the sweetener composition comprises: (a) one or more polypeptides, wherein the one or more polypeptides comprise: (i) a polypeptide sequence selected from the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10; (ii) a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10; (iii) a polypeptide sequence selected from the group consisting of the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10 by deletion, insertion, substitution or A polypeptide sequence having at least one modification by the addition of no more than 24 amino acids; or (iv) the polypeptide of any one of (i) to (iii), wherein the methionine at position 1 is deleted; and / or the polypeptide further comprises a protein / peptide tag, optionally an affinity tag or optionally a histidine tag or optionally a (His)6 tag; and (b) at least one additional component selected from the group consisting of a monosaccharide, a disaccharide (e.g., sucrose), a sugar alcohol (e.g., mannitol), an amino acid (e.g., leucine), an organic acid (e.g., citric acid), hypoxanthine, theophylline, a vitamin, and a combination thereof.

[0050] In embodiments, the sweetener compositions herein include at least one additional component selected from sucrose, mannitol, citric acid, hypoxanthine, theophylline, leucine, and combinations thereof.

[0051] In a specific embodiment, the polypeptide of the sweetener composition has the amino acid sequence of SEQ ID NO: 3. In a specific embodiment, the polypeptide of the sweetener composition has the amino acid sequence of SEQ ID NO: 3, wherein the methionine at position 1 is deleted. In an embodiment, the polypeptide of the sweetener is a mixture of a polypeptide having the amino acid sequence of SEQ ID NO: 3 and a polypeptide having the amino acid sequence of SEQ ID NO: 3, wherein the methionine at position 1 is deleted.

[0052] In an embodiment, the polypeptide of the sweetener composition is present in an amount of about 1ppm to about 50ppm. In some embodiments, the polypeptide is present in an amount of about 1ppm to about 40ppm. In other embodiments, the polypeptide is present in an amount selected from the group consisting of: about 1ppm to about 30ppm, about 1ppm and about 25ppm, about 1ppm to about 20ppm, or about 1ppm to about 15ppm. In some embodiments, the polypeptide is present in an amount of about 5ppm to about 50ppm. In some embodiments, the polypeptide is present in an amount of about 10ppm to about 50ppm. In some embodiments, the polypeptide is present in an amount of about 15ppm to about 50ppm. In some embodiments, the polypeptide is present in an amount of about 20ppm to about 50ppm. In some embodiments, the polypeptide is present in an amount of about 25ppm to about 50ppm. In some embodiments, the polypeptide is present in an amount of about 30ppm to about 50ppm. In some embodiments, the polypeptide is present in an amount of about 35ppm to about 50ppm. In some embodiments, the polypeptide is present in an amount of about 40ppm to about 50ppm. In some embodiments, the polypeptide is present in an amount of about 45 ppm to about 50 ppm. In some embodiments, the polypeptide is present in an amount of about 10 ppm to about 40 ppm. In some embodiments, the polypeptide is present in an amount of about 20 ppm to about 30 ppm. In some embodiments, the steviol glycoside sweetener is selected from rebaudioside M ("Reb M"), Reb M80, rebaudioside D ("Reb D"), Reb A95, and rebaudioside A ("Reb A"). In embodiments, the mogroside sweetener is selected from siamenoside I and mogroside V.

[0053] On the other hand, the present invention provides a combination of an orally administered product and a sweetening composition as described herein, wherein the product is not a truffle of the genus Matrimony Truffle. In some embodiments, the orally administered product has at least one improved organoleptic property compared to an orally administered product without the sweetener composition, wherein the organoleptic property is selected from the group consisting of: fragrance, flavor, basic taste (sweet, sour, salty, bitter or umami), aftertaste or linger, time characteristics, mouthfeel or a combination thereof. In an embodiment, the orally administered product is a solid food product or a beverage product. In an embodiment, the orally administered product is a yogurt product. In an embodiment, the orally administered product is a chewing gum product.

[0054] On the other hand, the present invention provides beverages or beverage products, which include sweetener compositions as described herein. In some embodiments, compared to beverages or beverage products without the sweetener compositions, the beverages or beverage products have at least one improved organoleptic properties, wherein the organoleptic properties are selected from the group consisting of: fragrance, local flavor, basic taste (sweetness, sourness, saltiness, bitterness or umami), aftertaste or aftertaste, time characteristics, mouthfeel or the combination thereof. In some embodiments, the beverages or beverage products are selected from low-calorie or calorie-free beverages or beverage products. In embodiments, the beverages are selected from cola, ginger ale, soft drinks, root beer, fruit juice, fruit juice, vegetable juice, vegetable juice, sports drinks, energy drinks, plant protein drinks, near-water beverages (e.g., water with natural or synthetic flavorings), teas (e.g., black tea (black tea), green tea, black tea leaf type tea (red tea), oolong tea), coffee, cocoa drinks, beverages containing milk components (e.g., milk beverages, coffee containing milk components, au lait, milk tea, fruit milk beverages). In an embodiment, the beverage or beverage product comprises at least one organic acid additive salt which is a sodium, calcium, potassium or magnesium salt of an organic acid. In an embodiment, the organic acid is selected from citric acid, malic acid, tartaric acid, fumaric acid, lactic acid, alginic acid, ascorbic acid, benzoic acid and adipic acid.

[0055] On the other hand, the present invention provides a method for improving at least one organoleptic characteristic of a product administered orally as herein described. In embodiments, the product administered orally is a solid food product or a liquid beverage product. In embodiments, the present invention provides a method for improving at least one organoleptic characteristic of a product administered orally as herein described. Said method comprises adding a sweetener composition as herein described to a solid or liquid matrix, thereby providing a product with at least one improved organoleptic characteristic. In embodiments, the organoleptic characteristic of said improvement is selected from the group consisting of: fragrance, local flavor, basic taste (sweetness, sourness, saltiness, bitterness or umami), aftertaste or aftertaste, time characteristics, mouthfeel or the combination thereof. In embodiments, the amount of the polypeptide present in the product improved by said method is the amount of about 1ppm to about 50ppm, about 1ppm and about 40ppm, about 1ppm to about 30ppm, about 1ppm to about 25ppm, about 1ppm to about 20ppm or about 1ppm to about 15ppm. In embodiments, said product is a solid food product, and said sweetener composition is added to, combined with or mixed into a solid food product.

[0056] In embodiments, the invention provides a method for improving at least one organoleptic characteristic of beverage as herein described or beverage product. Said method comprises adding sweetener composition as herein described to a liquid matrix, thereby providing a beverage or beverage product with the organoleptic characteristic of at least one improvement. In embodiments, the organoleptic characteristic of said improvement is selected from the group consisting of: fragrance, local flavor, basic taste (sweetness, sourness, saltiness, bitterness or umami), aftertaste or aftertaste, time signature, mouthfeel or above combination. In embodiments, the amount of the polypeptide present in the beverage improved by said method or beverage product is the amount of about 1ppm to about 50ppm, about 1ppm and about 40ppm, about 1ppm to about 30ppm, about 1ppm to about 25ppm, about 1ppm to about 20ppm or about 1ppm to about 15ppm.

[0057] On the other hand, the invention provides a method for changing / adjusting at least one organoleptic characteristic of a product administered orally as herein described. Said method comprises adding a sweetener composition as herein described to said product, thereby providing a product with the organoleptic characteristic of at least one change / adjustment. In an embodiment, the organoleptic characteristic of said change / adjustment is selected from the group consisting of: fragrance, local flavor, basic taste (sweetness, sourness, saltiness, bitterness or umami), aftertaste or aftertaste, time characteristics, mouthfeel or the combination thereof. In an embodiment, the amount of the polypeptide present in the product changed / adjusted by said method is the amount of about 1ppm to about 50ppm, about 1ppm and about 40ppm, about 1ppm to about 30ppm, about 1ppm to about 25ppm, about 1ppm to about 20ppm or about 1ppm to about 15ppm. In an embodiment, the product administered orally is a food product or a beverage product. In an embodiment, said product is a solid food product, and said sweetener composition is added to, combined with or mixed into a solid food product.

[0058] On the other hand, the invention provides the method for at least one organoleptic characteristic of change / adjustment beverage or beverage product as herein described.Described method comprises adding sweetener composition as herein described in liquid matrix, thereby provides beverage or beverage product with the organoleptic characteristic of at least one change / adjustment.In embodiments, the organoleptic characteristic of described change is selected from the group consisting of: fragrance, local flavor, basic taste (sweetness, sourness, saltiness, bitterness or umami), aftertaste or aftertaste, time characteristic, mouthfeel or above combination.In embodiments, the amount of the polypeptide existing in the beverage or beverage product changed / adjusted by described method is the amount of about 1ppm to about 50ppm, about 1ppm and about 40ppm, about 1ppm to about 30ppm, about 1ppm to about 25ppm, about 1ppm to about 20ppm or about 1ppm to about 15ppm.

[0059] Other aspects and embodiments of the invention will become apparent upon review of the drawings, detailed description, and non-limiting examples herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Shown is a Coomassie-stained SDS-PAGE gel of proteins obtained from fractions of partially purified sporulating tissue of M. agaricoides.

[0061] Figure 2 Coomassie Brilliant Blue stained SDS-PAGE gel showing the purification steps of a His-tagged sweet polypeptide from the coding region of SEQ ID NO: 4 expressed in E. coli, lane 1: molecular weight standard; lane 3, crude lysate; lane 4, purified protein from HisPur TMNi-NTA flow-through fraction; lane 5, 1st wash; lane 6, 2nd wash; lane 7, 3rd wash; lane 8, elution fraction. The His-tagged sweet protein expressed in E. coli is a His-tagged HTS-2 version in which the methionine is deleted (SEQ ID NO: 142).

[0062] Figure 3 Concentration response functions for the sweetness of mycodulcein (honey truffle sweetener (HTS)), aspartame, thaumatin, and rebaudioside A are shown. Data are plotted as the proportion of responses (p) occurring at a 200 mM sucrose-related ("sweet") target. Each data point in the curves for mycodulcein, aspartame, thaumatin, and rebaudioside A was calculated as the mean of 32 replicates and, for the sucrose curve, as the mean of 16 replicates; error bars are SEM. Points for the water and sucrose controls were similarly calculated as the mean of 128 and 64 replicates, respectively. Curves were fitted by nonlinear regression.

[0063] Figure 4A Coomassie-stained SDS-PAGE analysis of elution fractions from Capto MMC multimodal weak cation exchange resin is shown. M: protein marker; Lane 1: elution fraction showing low purity after cation exchange. Arrow indicates mycodulcein (native HTS) band.

[0064] Figure 4B Coomassie-stained SDS-PAGE analysis of two elution fractions collected during gradient elution from a HiScreen Capto Butyl (Cytiva Sweden AB, Upsala, Sweden) column for SDS-PAGE analysis is shown. Lane 1 shows elution fraction 1, which does not contain mycodulcein, and lane 2 shows eluted mycodulcein. The purity of the elution fractions was determined to be ~86% by GelAnalyzer. The arrow indicates the mycodulcein (native HTS) band.

[0065] Figure 4C Shows the 26 / 60 Coomassie-stained SDS-PAGE analysis of proteins eluted from the HIC column after S-200 (Cytiva Sweden AB, Upsala, Sweden) chromatography. Lane 1 shows purified His-tagged mycodulcein, and lane 2 shows purified native mycodulcein. The purity of the eluted fractions was determined to be ~98% by Gel Analyzer. The arrow indicates the mycodulcein (native HTS) band.

[0066] Figure 5 This is a map of the pD44-CH vector induced by isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0067] Figure 6A and 6B Included were GloMelt assays comparing native HTS (1) and the HTS variant S33C_I49C (double mutant, 2). TM Graph of exemplary melting temperature results obtained in a thermostability assay. Figure 6A The melting curves of the native HTS (1) and the double mutant (2) were compared. Figure 6B The melting peak curves of 1 and 2 were compared. The relative fluorescence (RFU) was measured as a function of temperature. Compared to the native HTS protein, an increase of 7.5°C in the average melting temperature of the double mutant was observed.

[0068] Figure 7 GloMelt analysis of the native HTS protein and the HTS variant with the double mutation S33C_I49C is shown. TM Melting temperature results obtained in the thermostability assay. In this assay, samples were placed at a low pasteurization temperature (63°C) for one hour. DETAILED DESCRIPTION

[0069] The present invention provides embodiments of isolated nucleotides encoding Myd1 polypeptide variants, encoded Myd1 polypeptide variants capable of modulating flavor and / or modulating sweetness and perception (also known as Honey Truffle Sweetener (HTS) proteins and variants thereof), and methods of making and using the same. The present invention provides MYD1 variants and Myd1 polypeptide variants, alone or in combination with foods, beverages, dietary supplements, or pharmaceuticals; and embodiments of methods of using the isolated polynucleotides and polypeptides of the present invention to modulate the taste of such foods, beverages, dietary supplements, or pharmaceutical compositions. The present invention provides naturally occurring Myd1 polypeptides and nucleic acids encoding these polypeptides. The present invention provides non-naturally occurring Myd1 polypeptide variants and nucleic acids encoding these polypeptides.

[0070] Embodiments of the present invention provide Myd polypeptide variants (also referred to as HTS variants). The term "Myd polypeptide variant" is used herein to identify non-naturally occurring HTS polypeptides and includes any polypeptide according to the present invention that has, for example, at least 80% sequence identity with a variant of Table 8, Table 9, or Table 10 and further has flavor modulation (or tailoring) and / or sweetness modulation activity.

[0071] De novo amino acid sequencing was performed on a purified extract of the sweet portion of the spore-forming tissue of Terfezioides gleba to identify a 20-mer N-terminal sequence. After de novo assembly of the entire transcriptome of the spore-forming tissue of M. terfeziodes using RNAseq reads, the Myd1 coding sequence (putatively derived from the MYD1 gene) was identified. Screening the entire transcriptome of M. terfeziodes using 20-mer N-terminal sequences identified a transcript that is expected to encode a protein with 100% identity at the N-terminus. The identified transcript is expected to encode a protein of 121 amino acids. The method identified the polynucleotide of SEQ ID NO: 1. The start and stop codons were identified in the transcript to identify the putative coding sequence SEQ ID NO: 2. SEQ ID NO: 3 is the putative encoded protein, i.e., a 121 amino acid protein. Subsequently, it was discovered that the naturally occurring protein isolated from the sporulating tissue of the fungus Glomerella glomerata is a mixture of two isoforms: the protein of SEQ ID NO: 3 and the mature protein of SEQ ID NO: 3 with a deletion of the methionine (Met) residue at amino acid position 1 (hereinafter referred to as SEQ ID NO: 141). The predicted protein of SEQ ID NO: 3 shares 31% or less identity with other protein sequences in GENBANK.

[0072] Natural HTS appears to be a mixture of two isoforms (HTS-1 and HTS-2). HTS-1 is the polypeptide of SEQ ID NO:3. HTS-2 is the polypeptide of SEQ ID NO:141. As described herein, natural HTS isolated from Truffles is a mixture of approximately 40%-60% HTS-2 by weight and 60%-40% HTS-1 by weight. The relative amounts of HTS-1 and HTS-2 isolated from Truffles vary at least depending on the source of the truffle. After isolation, approximately 80% of HTS-1 is acetylated at the N-terminal amine group. No significant differences in sweetness have been observed between the two sequence isoforms of HTS. Acetylation of the N-terminus of the HTS polypeptide HTS-1 also has no significant effect on sweetness.

[0073] Polynucleotides encoding the polypeptide of SEQ ID NO:3 have been successfully expressed in a variety of hosts. In some cases, the polynucleotide sequence encoding the polypeptide of SEQ ID NO:3 has been codon-optimized, as known in the art, for expression in a given host. For example, in E. coli, optimized codons were used for intracellular expression to express the polynucleotide encoding SEQ ID NO:3 using the nucleotide coding sequence of SEQ ID NO:4, producing a sweet protein. For example, in E. coli, optimized codons were used for intracellular expression to express the polynucleotide encoding SEQ ID NO:3 and a (His)6 tag, producing a sweet protein. Subsequently, proteomic analysis determined that the sweet protein with a His tag expressed from SEQ ID NO:4 in E. coli was SEQ ID NO:141 (SEQ ID NO:3 without methionine in position 1), and no expression of the His tag (SEQ ID NO:148) of the HTS-1 isoform of the protein was observed. The sweet protein of SEQ ID NO: 141 (without a His tag) is a protein expressed based on the expression of an optimized E. coli coding region (SEQ ID NO: 148) without a His tag.

[0074] For example, intracellular expression in Saccharomyces cerevisiae using optimized codons (SEQ ID NO: 6) to express a polynucleotide encoding SEQ ID NO: 3 with an additional His-tag coding sequence produces a sweet protein. The expressed protein is a mixture of HTS-1 and HTS-2 isoforms, each with a His-tag. The relative amount of HTS-2 to HTS-1 is approximately 60% to 40% by weight. Extracellular expression in Saccharomyces cerevisiae using optimized codons and an appropriate signal peptide at the N-terminus to promote secretion into the fermentation medium produces a non-sweet expression product.

[0075] For example, using optimized codons, intracellular expression in Yarrowia lipolytica to express a polynucleotide encoding SEQ ID NO: 3 produces a sweet protein. The expressed protein is a mixture of HTS-1 and HTS-2 isoforms. The relative amount of HTS-2 to HTS-1 is approximately 20% to 80% by weight.

[0076] For example, using optimized codons, intracellular expression in Pichia pastoris to express a polynucleotide encoding SEQ ID NO: 3 produced a sweet protein. The expressed protein was a mixture of HTS-1 and HTS-2 isoforms. The relative amount of HTS-2 to HTS-1 was approximately 90% to 10% by weight.

[0077] The coding sequences of the codon-optimized native mycodulcein (HTS) for expression in Escherichia coli and Saccharomyces cerevisiae correspond to the nucleic acid sequences of SEQ ID NO: 4 and SEQ ID NO: 6, respectively (both encoding the amino acid sequence of SEQ ID NO: 3 with an optional 6-residue histidine tag, i.e., the amino acid sequence of SEQ ID NO: 5).

[0078] In an embodiment, the present invention provides a non-naturally occurring sweet protein comprising a non-naturally occurring mixture of two isoforms, HTS-1 and HTS-2. In an embodiment, the present invention provides HTS-2 (SEQ ID NO: 141) that is substantially free of another isoform, HTS-1, wherein substantially free means that HTS-1 (SEQ ID NO: 3) is less than 10% by weight. In an embodiment, the present invention provides HTS-2 (SEQ ID NO: 141) that is free of another isoform, HTS-1, wherein free means that HTS-1 (SEQ ID NO: 3) is less than 1% by weight. In an embodiment, the present invention provides HTS-1 (SEQ ID NO: 3) that is substantially free of another isoform, HTS-2, wherein substantially free means that HTS-1 (SEQ ID NO: 141) is less than 10% by weight. In an embodiment, the present invention provides HTS-1 (SEQ ID NO: 3) that is substantially free of another isoform, HTS-2, wherein free means that HTS-2 (SEQ ID NO: 141) is less than 1% by weight. In an embodiment, the present invention provides non-naturally occurring mixtures of HTS-1 and HTS-2, in particular, those enriched in HTS-2, wherein the amount of HTS-2 is greater than 60% by weight, and for example, 65% or more by weight, 70% or more by weight, 75% or more by weight, 80% or more by weight, 85% or more by weight, 90% or more by weight, or 95% or more by weight. In an embodiment, the present invention provides non-naturally occurring mixtures of HTS-1 and HTS-2, in particular, those enriched in HTS-1, wherein the amount of HTS-1 is greater than 60% by weight, and for example, 65% or more by weight, 70% or more by weight, 75% or more by weight, 80% or more by weight, 85% or more by weight, 90% or more by weight, or 95% or more by weight.

[0079] In an embodiment, the present invention provides a sweet protein produced by recombinant expression of the coding sequence of SEQ ID NO: 2 or a codon-optimized version of the coding sequence of SEQ ID NO: 2 in a non-natural host (e.g., a host other than the fungus truffle). In a specific embodiment, the sweet protein is produced by recombinant expression in bacteria, yeast, or fungi. In a specific embodiment, the non-natural host is Escherichia coli. In a specific embodiment, the non-natural host is Saccharomyces cerevisiae. In a specific embodiment, the non-natural host is Yarrowia lipolytica. In a specific embodiment, the non-natural host is Pichia pastoris. In an embodiment, recombinant expression in a non-natural host produces a non-naturally occurring mixture of naturally occurring isoforms of HTS-1 and HTS-2 of mycodulcein (Myd).

[0080] polynucleotides

[0081] Embodiments of the present invention include polynucleotides (e.g., isolated polynucleotides) encoding polypeptide variants with sweet taste modulating activity. Examples of polynucleotides encoding polypeptides with sweet taste modulating activity include polynucleotides capable of encoding polypeptides, such as, but not limited to, variants of Table 8, Table 9, or Table 10, or polypeptides that also have a histidine tag, or 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 polynucleotides encoding variants of Table 8, Table 9, or Table 10, or polypeptides that also have a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag. It should be understood that polynucleotides including or not including sequences encoding protein / peptide tags, affinity tags, or histidine tags have been provided and can be used in the present invention. It is further understood that for any specific polynucleotide indicated as encoding a given histidine tag (e.g., (His)6), the sequence encoding the histidine tag can be replaced with a sequence encoding a different His tag or a sequence encoding a different protein / peptide tag or affinity tag. In embodiments, the protein / peptide, affinity or histidine tag is one encoded by 3-30 or 3-18 nucleotides. In embodiments, the variants described above include a methionine at position 1 or a deletion of a methionine at position 1. In embodiments, the variant HTS polypeptide is not the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 141.

[0082] In embodiments, the polynucleotide comprises, consists essentially of, or consists of a polynucleotide selected from the group consisting of: a polynucleotide encoding a variant of Table 8, Table 9, or Table 10, or a nucleic 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 a polynucleotide capable of encoding a variant of Table 8, Table 9, or Figure 10. In embodiments, the encoded variant polypeptide is not the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 141.

[0083] Unless otherwise expressly stated, when a polynucleotide sequence has multiple nucleotide modifications, each modification can be independently modified with a selected modification, such as a 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; and unless otherwise expressly stated, the modification options for each modification can be independently different, such as a deletion, insertion, substitution, or addition, etc. When multiple modifications are present, each modification can be independently a substitution, addition, insertion, or deletion, regardless of what the other modifications are. In an embodiment, the polynucleotide sequence has at least one substitution modification. In a specific embodiment, the polynucleotide sequence has multiple substitution modifications; and each substitution can be independently substituted by a selected substitution, regardless of what the other substitutions are. In addition, multiple substitutions in a polynucleotide sequence can be the same or different from each other; and unless otherwise expressly stated, the nucleotide substitution options for each substitution can be independently different. When a polynucleotide sequence has multiple substitutions, each substitution can be independently selected, regardless of what the other substitutions are. In an embodiment, the polynucleotides herein are optionally isolated and / or optionally purified.

[0084] In embodiments, at least 80% sequence identity relative to a polynucleotide 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 relative to a polynucleotide 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.

[0085] In embodiments, the polynucleotide encodes a polypeptide sequence comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of: (a) an amino acid sequence of a variant of Table 8, Table 9, or Table 10, or a polypeptide further having a protein / peptide tag, affinity tag, or histidine tag; (b) an amino acid 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%, at least 91%, at least 92%, or more similar to an amino acid sequence of a variant of Table 8, Table 9, or Table 10;

[0015] The present invention also provides an amino acid sequence having 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 any of the above-described polypeptides, or a polypeptide wherein the variant further comprises a protein / peptide tag, an affinity tag or a histidine tag; and (c) an amino acid sequence modified from an amino acid sequence of a variant of Table 8, Table 9 or Table 10 by deletion, insertion, substitution or addition of no more than 24 amino acids (e.g., modification of 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 amino acids), or a polypeptide wherein the variant further comprises a histidine tag. In the embodiments listed above, the polypeptide encoded by the polynucleotide is optionally a polypeptide other than SEQ ID NO: 3, and optionally a polypeptide other than SEQ ID NO: 141. When an amino acid sequence has multiple modifications, the number of modifications an amino acid has can range from at least one to up to 24 modifications, such as 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 desired. Furthermore, unless otherwise explicitly stated, when an amino acid sequence has multiple modifications, each modification can independently be modified with a selected modification, such as a deletion, insertion, substitution, or addition, regardless of the other modifications. Furthermore, multiple modifications in an amino acid sequence can be the same or different from one another; and unless otherwise explicitly stated, the modification options for each modification can independently be different, such as a deletion, insertion, substitution, or addition. When an amino acid sequence has multiple modifications, each modification can independently be a substitution, addition, insertion, or deletion, regardless of the other modifications. In an embodiment, the amino acid sequence has at least one substitution modification. In a specific embodiment, the amino acid sequence has multiple substitution modifications; and each substitution modification can independently be replaced with a selected substitution, regardless of the other substitutions. In an embodiment, the amino acid sequence has one substitution modification (single mutant) or two substitution modifications (double mutant) compared to SEQ ID NO: 3 or SEQ ID NO: 141.In addition, multiple substitutions in an amino acid sequence may be the same or different from each other; and unless otherwise expressly stated, the substitution options for each amino acid may be independently different. When an amino acid sequence has multiple substitutions, each substitution may be selected independently, regardless of the other substitutions. In an embodiment, the encoded polypeptide may include 1-6, 1-12, 1-18, or 1-24 substitutions compared to the polypeptide of SEQ ID NO: 3, wherein the substitutions are those listed in Table 8. In an embodiment, the encoded polypeptide may include 1-6, 1-12, 1-18, or 1-24 substitutions compared to the polypeptide of SEQ ID NO: 3, wherein the substitutions are those listed in Table 9. In an embodiment, the encoded polypeptide may include 1-6, 1-12, 1-18, or 1-24 substitutions compared to the polypeptide of SEQ ID NO: 3, wherein the substitutions are those listed in Table 10. In embodiments, the encoded polypeptide may include 1-6, 1-12, 1-18, or 1-24 substitutions compared to the polypeptide of SEQ ID NO: 3, wherein the substitutions are any one or more of those listed in Table 3, Table 6, Table 7, Table 8, Table 9, or Table 10. In embodiments, the encoded polypeptide may include 1-6, 1-12, 1-18, or 1-24 substitutions compared to the polypeptide of SEQ ID NO: 3, wherein the substitutions are not those listed in Table 7. In embodiments, the encoded polypeptide may include 1-6, 1-12, 1-18, or 1-24 substitutions compared to the polypeptide of SEQ ID NO: 3, wherein the substitutions are not those listed in Table 3 or Table 6. In embodiments, the encoded polypeptide may include 1-6, 1-12, 1-18, or 1-24 substitutions compared to the polypeptide of SEQ ID NO: 3, wherein the substitutions are not those listed in Table 3 or Table 6. In an embodiment, the encoded polypeptide may include 1-6, 1-12, 1-18, or 1-24 substitutions compared to the polypeptide of SEQ ID NO: 3, wherein the substitutions are those listed in Table 9 and Table 10 and are not those listed in Table 3 or Table 6 or Table 7.

[0086] In certain embodiments of the polynucleotides herein, the polynucleotides encode the polypeptides herein and further encode protein / peptide tags, affinity tags, or histidine tags. In such cases where all specific polynucleotides encode such tags, the present invention also provides polynucleotides other than the sequence encoding the tag. In other embodiments, in those polynucleotides herein encoding a histidine tag, the sequence encoding the histidine tag can be deleted or excluded, or can be replaced with the coding sequence of a different protein / peptide tag or a different affinity tag, including a different histidine tag.

[0087] The present invention provides embodiments of separation and / or purification of corresponding polynucleotides, where applicable. The present invention provides embodiments of each polynucleotide capable of encoding the variants of Table 8, Table 9 or Table 10 herein, which, where applicable, may be separated or not separated and may be purified or not purified. The present invention provides embodiments of each polynucleotide capable of encoding the variants of Table 8, Table 9 or Table 10 herein, which, where applicable, have one or more mutations.

[0088] The present invention also provides expression cassettes comprising one or more polynucleotides encoding Myd polypeptide variants (HTS variants) and host cells transformed with the vectors.

[0089] The polynucleotide encoding the HTS variant of the present invention may be in the form of single-stranded or double-stranded DNA, RNA or artificial nucleic acid, or may be cDNA or chemically synthesized DNA that does not include any introns. The term "MYD family" may refer to polymorphic variants, including natural alleles, mutants, alleles and interspecies homologs encoding polypeptides that: (1) have at least about 35% to 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 within a window of about 25 amino acids, optionally 50-100 amino acids. In one aspect, the term "isolated" includes products that have been removed from a biological environment (e.g., cells, tissues, culture medium, body fluids, etc.), or in other cases, increased to any degree of purity (e.g., isolated from a synthetic culture medium). Thus, the isolated product may be synthetic or naturally occurring. In an embodiment, the term "isolated" includes a product that has been separated from other proteins present in its natural environment. In an embodiment, the term "isolated" includes a product that has been separated from carbohydrates present in its natural environment.

[0090] The term "nucleic acid" or "nucleic acid sequence" refers to a deoxyribonucleotide or ribonucleotide oligonucleotide in single- or double-stranded form. The term includes nucleic acids containing known analogs of natural nucleotides, i.e., oligonucleotides. The term also includes nucleic acid-like structures with synthetic backbones (see, e.g., Oligonucleotides and Analogues, a Practical Approach, ed. F. Eckstein, Oxford Univ. Press (1991); Antisense Strategies, Annals of the NY Academy of Sciences, Vol. 600, Baserga et al. (NYAS 1992); Milligan J. Med. Chem. 36: 1923-1937 (1993); Antisense Research and Applications (1993, CRC Press), WO 97 / 03211; WO 96 / 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)).

[0091] As used herein, "nucleic acid probe or oligonucleotide" is defined as a nucleic acid that can be combined with a target nucleic acid of a complementary sequence by one or more types of chemical bonds, typically by complementary base pairing, typically by hydrogen bond formation. As used herein, probes can include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanine, inosine, etc.). In addition, the bases in the probe can be connected by connections other than phosphodiester bonds, as long as they do not interfere with hybridization. Therefore, for example, the probe can be a peptide nucleic acid, in which the constituent bases are connected by peptide bonds rather than phosphodiester bonds. It will be understood by those skilled in the art that, depending on the stringency of hybridization conditions, the probe can be combined with a target sequence lacking complete complementarity with the probe sequence. The probe can optionally be directly labeled with an isotope, chromophore, lumiphores, chromogen, or indirectly labeled with a biotin that may be combined with it later, such as a streptavidin complex. By determining the presence or absence of the probe, the presence or absence of a selected sequence or subsequence can be detected.

[0092] The polynucleotides or polypeptides can be naturally occurring or non-naturally occurring (e.g., synthetic, recombinant, modified and / or variant products). In one aspect, the naturally occurring or non-naturally occurring products are isolated or purified. In another aspect, the naturally occurring or non-naturally occurring products are not isolated or purified. In embodiments herein, the polynucleotides and polypeptides are non-naturally occurring. In embodiments herein, the polynucleotides and polypeptides are naturally occurring mycodulcein (HTS) polypeptides and non-naturally occurring variants of naturally occurring polynucleotide sequences encoding naturally occurring mycobactin (HTS).

[0093] As used herein, "recombinant" refers to polynucleotides synthesized or otherwise manipulated in vitro (e.g., "recombinant polynucleotides"), methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or polypeptides encoded by recombinant polynucleotides ("recombinant proteins"). "Recombinant means" also includes ligating nucleic acids having various coding regions or domains or promoter sequences from different sources into an expression cassette or vector for expression, e.g., inducible or constitutive expression, of a fusion protein comprising a translocation domain of the invention and a nucleic acid sequence amplified using primers of the invention.

[0094] As used herein, the terms "amplifying" and "amplification" refer to the use of any suitable amplification method to produce or detect recombinant or naturally expressed nucleic acids, as described in detail below. For example, the present 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 present invention (e.g., taste stimulant binding sequences of the present invention) in vivo or in vitro.

[0095] As used herein, the term "isolated" when referring to a nucleic acid or polypeptide refers to a state of purification or concentration different from that found in nature. Any degree of purification or concentration greater than that found in nature, including (1) purification from other naturally occurring related structures or compounds (e.g., other proteins, carbohydrates), or (2) association with structures or compounds with which they are not normally associated in the body, is within the meaning of "isolated" as used herein. The nucleic acids or polypeptides described herein can 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 skilled 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%) by weight of other fungal proteins, such as fungal proteins other than Myd proteins.

[0096] A "modified" or "variant" product refers to a product (e.g., a polynucleotide or polypeptide) that is altered from an original (e.g., naturally occurring) structure. As described herein, variants include polynucleotides or polypeptides having one or more changes from the nucleic acid or amino acid sequence, respectively. Changes include modifications of the nucleic acid or amino acid sequence, including additions, deletions, insertions, and substitutions. Modified or variant products may also include those modified to include disulfide bond formation relative to the original structure, as well as those derived by using post-translational modification methods, lipidation, acylation, acetylation, phosphorylation, or any other manipulation such as glycosylation in conjunction with a labeling component. As is known in the art, derivatization can be achieved chemically or enzymatically after the polypeptide is translated. Derivatization can also be achieved by post-translational modifications during expression of the polypeptide in a selected host.

[0097] Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions (Batzer et al., Nucleic Acid Res., 19: 5081 (1991) can be achieved by generating, for example, a sequence in which the third position of one or more selected codons is replaced by mixed bases and / or deoxyinosine residues; Ohtsuka et al., J. Biol. Chem., 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8: 91-98 (1994)). The term nucleic acid can be used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0098] In this document, when a particular polynucleotide is indicated as encoding a histidine tag, it is understood that polynucleotides other than sequences encoding said histidine tag are also provided. It is further understood that for a polynucleotide encoding a given histidine tag (e.g., (His )6 )) of any specific polynucleotide, the sequence encoding the histidine tag can be replaced with a sequence encoding a different His tag or a sequence encoding a different protein / peptide tag or affinity tag.

[0099] peptides

[0100] It should be understood that embodiments of the present invention also include Myd polypeptide variants (HTS variants) 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. These terms also apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0101] Embodiments of the invention include a polypeptide comprising, consisting essentially of, or consisting 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 a variant of Table 8 or a variant of Table 8 further having a histidine tag. In embodiments, the polypeptide is not the polypeptide of SEQ ID NO: 3. In embodiments, the polypeptide is not the polypeptide of SEQ ID NO: 141. In embodiments, the polypeptide is the polypeptide of SEQ ID NO: 3 that is substantially free (less than 95%, or more particularly less than 99%, by weight) of the polypeptide of SEQ ID NO: 141. In an embodiment, the polypeptide is a polypeptide of SEQ ID NO: 141, which is substantially free of (less than 95% by weight, or more particularly less than 99% by weight) a polypeptide of SEQ ID NO: 3. Optionally, the amino acid sequence has at least one and up to 24 modifications. When an amino acid sequence has multiple modifications, the number of modifications that an amino acid has can be as needed within the range of at least one to a maximum of 24 modifications, such as 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. In addition, unless otherwise expressly stated, when an amino acid sequence has multiple modifications, each modification can be independently modified with the modification selected, such as deletion, insertion, substitution or addition, regardless of what the other modifications are. In addition, multiple modifications in an amino acid sequence can be the same or different from each other; and unless otherwise expressly stated, the modification options for each modification can be independently different, such as deletion, insertion, substitution or addition, etc. When an amino acid sequence has multiple modifications, each modification can independently be a substitution, addition, insertion or deletion, regardless of the other modifications. In an embodiment, the amino acid sequence has at least one substitution modification. In a specific embodiment, the amino acid sequence has multiple substitution modifications; and each substitution modification can be independently replaced by a selected substitution, regardless of the other substitutions. In addition, multiple substitutions in an amino acid sequence can be the same or different from each other; and unless otherwise expressly stated, the substitution options for each amino acid can be independently different. When an amino acid sequence has multiple substitutions, each substitution can be independently selected, regardless of the other substitutions.The term "consisting essentially of" allows for the inclusion of components that are not essential to the function or activity of the product and do not materially affect the function or activity, such as anti-caking agents, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltodextrin, gum arabic, etc.).

[0102] Another embodiment of the present invention includes a recombinant polypeptide having sweet taste modulating activity comprising, consisting essentially of, or consisting of a 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%, 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 variant of Table 8, Table 9, or Table 10, or a variant of these tables that also has a histidine tag or is fused to a heterologous signal peptide or transit peptide. The term "consisting essentially of" allows for the inclusion of components that are not essential to the function or activity of the product and do not substantially affect the function or activity, such as anti-caking agents, fillers, stabilizers (e.g., thermal stabilizers), and bulking agents (e.g., maltodextrin, gum arabic, etc.).

[0103] In an embodiment, the polypeptide having sweet taste modulating activity comprises, consists essentially of, or consists of a modified SEQ ID NO: 3, wherein the peptide has at least one and up to 24 amino acid modifications as shown in Table 8, Table 9, or Table 10. 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 to a modified polypeptide of SEQ ID NO: 3 having at least one and up to 24 amino acid modifications as shown in Table 8, Table 9, or Table 10; and the polypeptide is different from the polypeptide of the amino acid sequence of SEQ ID NO: 3, and optionally different from the polypeptide of the amino acid sequence of SEQ ID NO: 141.

[0104] In an embodiment, the polypeptide having sweet taste modulating activity comprises, consists essentially of, or consists of a modified SEQ ID NO: 3, wherein the peptide has at least one and up to 24 amino acid modifications as shown in Table 8 and further has 1-24 amino acid modifications as shown in Table 7, wherein the total number of modifications is 1-24. 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 to a modified polypeptide of SEQ ID NO: 3 having at least one and up to 24 amino acid modifications as shown in Table 8 and 1-24 amino acid modifications as shown in Table 7.

[0105] In an embodiment, the polypeptide having sweet taste modulating activity comprises, consists essentially of, or consists of a modified SEQ ID NO: 3, wherein the peptide has at least 1 and up to 12 amino acid modifications as shown in Table 8 and further has 1-12 amino acid modifications as shown in 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 to a modified polypeptide of SEQ ID NO: 3 having at least 1 and up to 12 amino acid modifications as shown in Table 8 and 1-12 amino acid modifications as shown in Table 7.

[0106] In an embodiment, the polypeptide having sweet taste modulating activity comprises, consists essentially of, or consists of a modified SEQ ID NO: 3, wherein the peptide has at least one and up to six amino acid modifications as shown in Table 8 and further has 1-6 amino acid modifications as shown in 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 to a modified polypeptide of SEQ ID NO: 3 having at least one and up to six amino acid modifications as shown in Table 8 and 1-6 amino acid modifications as shown in Table 7.

[0107] In embodiments, the polypeptide having sweet taste modulating activity comprises, consists essentially of, or consists of a modified SEQ ID NO: 3, wherein the peptide has at least one and up to 24 amino acid modifications as shown in Table 8, Table 9, Table 10, Table 7, Table 6, or Table 3. 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 to a modified polypeptide of SEQ ID NO: 3 having at least one and up to 24 amino acid modifications as shown in Table 8, Table 9, Table 10, Table 7, Table 6, or Table 3.

[0108] In an embodiment, the polypeptide having sweet taste modulating activity comprises, consists essentially of, or consists of a modified SEQ ID NO: 3, wherein the peptide has at least one and up to 24 amino acid modifications as shown in Table 8, Table 9, or Table 10, and no modifications as shown in Table 7, Table 3, or Table 6. 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 to a modified polypeptide of SEQ ID NO: 3 having at least one and up to 24 amino acid modifications as shown in Table 8, Table 9, Table 10, and no modifications as shown in Table 7, Table 3, or Table 6.

[0109] In an embodiment, the polypeptide having sweet taste modulating activity comprises, consists essentially of, or consists of a modified SEQ ID NO: 3, wherein the peptide has at least one and up to six amino acid modifications as shown in Table 8, Table 9, or Table 10. 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 to a modified polypeptide of SEQ ID NO: 3 having at least one and up to six amino acid modifications as shown in Table 8, Table 9, or Table 10.

[0110] In certain embodiments herein, a specific polypeptide is described as having a histidine tag or optionally having a histidine tag (denoted as XXXXXX in the sequence listing herein, where X is His). The present invention provides all specific polypeptides that include or optionally include a histidine tag, as well as corresponding polypeptides that exclude the histidine tag. The present invention also provides all specific polypeptides that have a histidine tag or optionally a histidine tag, as well as polypeptides that exclude the histidine tag, or polypeptides in which the histidine tag is replaced by a different protein tag, including a different histidine tag.

[0111] In an embodiment, the Myd peptide (HTS) variants described herein possess the ability of sweet taste modulating activity. The HTS polypeptide variants of the present invention can have, for example, functional, physical and chemical effects at taste receptors such as sweet taste receptors. "Sweet taste modulating activity" can refer to inhibition, activation, such as agonist or antagonist properties of the polypeptide of the present invention identified using in vitro and in vivo taste transduction assays. Proteins with inhibitory activity can bind to, partially or completely block stimulation, reduce, prevent, delay activation, inactivate, desensitize or turn down taste conduction, such as antagonists. Activating polypeptides can bind to, stimulate, increase, turn on, activate, promote, enhance activation, sensitize or turn up taste conduction, such as agonists. Preferably, activating polypeptides.

[0112] Sweet taste modulation also refers to enhancing the taste, such as sweetness, of a particular orally administered product when administered as a combination. In addition to sweetness, HTS variants can also exhibit differences in sweetness intensity and sweetness duration. These attributes of sweetness can be evaluated in taste tests described herein.

[0113] Sweet taste modulating also refers herein to polypeptide variants that, when added to a composition or formulation, exhibit sweetness or impart sweetness to a composition or formulation that does not itself exhibit sweetness.

[0114] In some embodiments, Myd(HTS) polypeptide variants of the invention include polypeptides that are at least as sweet as sucrose (on a w / w basis) (e.g., 1X), or alternatively, 2X, 5X, 10X, 50X, 100X, 200X, 400X, 600X, 800X, 1000X, 1500X, 2000X, 3000X, 5000X, 10,000X, 20,000X or sweeter than sucrose as measured by any method described above or known in the art. In other embodiments, the Myd polypeptide variant 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 sucrose.

[0115] In embodiments, the polypeptide having sweet taste modulating activity comprises a modified SEQ ID NO: 3 having 1 up to 24 different amino acid modifications as shown in Table 8, Table 9, or Table 10. In embodiments, the polypeptide having sweet taste modulating activity comprises a modified SEQ ID NO: 141 having 1 up to 24 different amino acid modifications as shown in Table 8, Table 9, or Table 10.

[0116] In embodiments, the Myd (HTS) peptide variants described herein have a flavor modulating property (FMP) or activity. A compound with an FMP is a compound comprising a protein or polypeptide that causes a change in any attribute of the perceived flavor of a formulation relative to a formulation without the compound, but does not directly provide the flavor attribute. A compound may have flavor modulating properties at concentrations below the threshold for perceivable sweetness and provide a direct flavor sensation above that threshold. The HTS protein / polypeptide or variant can exhibit a FMP threshold of the wild-type protein in a formulation, below which the FMP is observed, and above which the HTS protein or variant directly contributes to the perception of sweetness. The exact threshold for this perceptual change is HTS protein and application specific.

[0117] In an embodiment, a composition or formulation containing about 1 to about 50 ppm of an HTS protein or variant exhibits a flavor adjustment, but does not provide additional sweetness to the composition or formulation. In an embodiment, a composition or formulation containing about 1 to about 40 ppm of an HTS protein or variant exhibits a flavor change, but does not provide additional sweetness to the composition or formulation. In an embodiment, a composition or formulation containing about 1 to about 30 ppm of an HTS protein or variant exhibits a flavor change, but does not provide additional sweetness to the composition or formulation. In an embodiment, a composition or formulation containing about 1 to about 25 ppm of an HTS protein or variant exhibits a flavor change, but does not provide additional sweetness to the composition or formulation. In an embodiment, a composition or formulation containing about 1 to about 20 ppm of an HTS protein or variant exhibits a flavor change, but does not provide additional sweetness to the composition or formulation. In an embodiment, a composition or formulation containing about 1 to about 15 ppm of an HTS protein or variant exhibits a flavor change, but does not provide additional sweetness to the composition or formulation. In an embodiment, a composition or formulation containing about 5 to about 50 ppm of an HTS protein or variant exhibits a flavor change, but does not provide additional sweetness to the composition or formulation. In an embodiment, a composition or formulation containing about 10 to about 50 ppm of an HTS protein or variant exhibits a change in flavor, but does not provide additional sweetness to the composition or formulation. In an embodiment, a composition or formulation containing about 15 to about 50 ppm of an HTS protein or variant exhibits a change in flavor, but does not provide additional sweetness to the composition or formulation. In an embodiment, a composition or formulation containing about 20 to about 50 ppm of an HTS protein or variant exhibits a change in flavor, but does not provide additional sweetness to the composition or formulation. In an embodiment, a composition or formulation containing about 25 to about 50 ppm of an HTS protein or variant exhibits a change in flavor, but does not provide additional sweetness to the composition or formulation. In an embodiment, a composition or formulation containing about 30 to about 50 ppm of an HTS protein or variant exhibits a change in flavor, but does not provide additional sweetness to the composition or formulation. In an embodiment, a composition or formulation containing about 35 to about 50 ppm of an HTS protein or variant exhibits a change in flavor, but does not provide additional sweetness to the composition or formulation. In an embodiment, a composition or formulation containing about 40 to about 50 ppm of an HTS protein or variant exhibits a change in flavor, but does not provide additional sweetness to the composition or formulation. In embodiments, compositions or formulations containing about 45 to about 50 ppm of an HTS protein or variant exhibit a flavor change without providing additional sweetness to the composition or formulation. In embodiments, compositions or formulations containing about 10 to about 40 ppm of an HTS protein or variant exhibit a flavor change without providing additional sweetness to the composition or formulation.In embodiments, a composition or formulation containing about 20 to about 30 ppm of an HTS protein or variant exhibits a flavor change without providing additional sweetness to the composition or formulation.

[0118] In an embodiment, a composition or formulation containing an HTS protein or variant at a concentration greater than 30 ppm, and particularly greater than 40 ppm, provides a sweet taste to the composition or formulation. In an embodiment, the composition or formulation may contain 1-100 ppm of an HTS protein or variant. In an embodiment, the composition or formulation may contain 1-5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 1718, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 ppm of total HTS protein or variant. In an embodiment, the composition or formulation may contain 1-5, 5-10, 1-10, 1-30 or 1-30 ppm of total HTS protein or variant. In embodiments, the composition or formulation may contain 30-100, 30-40, 40-50, 50-60, 70-80, 80-90, 90-100, 40-100, or 50-100 ppm of total HTS protein or variant.

[0119] In an embodiment, at least 80% sequence identity relative to 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 an embodiment, at least 80% sequence identity relative to 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.

[0120] Comparison of sweetness and thermal stability of modified and unmodified peptides

[0121] The Myd (HTS) protein variants described herein also include "analogs" or "conservative variants" and "mimetics" ("peptide mimetics") that substantially correspond in structure and activity to the exemplary sequences. Thus, the term "conservative variant" or "analog" or "mimetics" refers to a polypeptide having a modified amino acid sequence such that one or more of the changes does not substantially alter the structure and / or activity of the polypeptide (of the conservative variant), as defined herein. These include conservatively modified variants of the amino acid sequence, i.e., amino acid substitutions, additions or deletions of residues that are not critical for protein activity, or substitutions of amino acids with residues having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.), such that even substitutions of key amino acids do not substantially alter the structure and / or activity.

[0122] 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 refers to those 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.

[0123] For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at each 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.

[0124] Such nucleic acid variations are "silent variations," which are a type of conservatively modified variation. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variation of the nucleic acid. The skilled artisan will recognize that each codon in a nucleic acid (except AUG and TGG, which are typically the only codons for methionine and TGG, which are typically the only codons for tryptophan) can be modified to produce functionally identical molecules. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

[0125] When needing to express coding sequence in heterologous host (i.e. non-naturally occurring host), codon optimization known in the art can be adopted to improve the expression level in the selected heterologous host. Codon optimization relates to codon replacement of given naturally occurring coding sequence with codons having higher usage level in selected heterologous host. In general, codon optimization is to compare the codon frequency in the naturally occurring coding sequence with the codon frequency in the selected heterologous host, and when a given codon is not the codon that is usually adopted in the selected heterologous host, the codon is replaced with the codon that is more frequently used by the selected heterologous host. One to all codons in a given naturally occurring coding sequence can be optimized. Depending on the codon frequency in naturally occurring coding sequence and heterologous host and the specific coding sequence, at least 50%, at least 75%, at least 85% or at least 95% of the codons can be replaced. Many codon optimization tools are known in the art and are easily obtained from various sources. For example, OPTIMIZER is an online application for codon optimization (P. Puigbo et al. (2007) “OPTIMIZER; a web server for optimizing the codonusage of DNA sequences” Nucleic Acids Res. 35:W126-W131). A recent review of codon optimization methods is provided in H. Fu et al. “Codon optimization with deep learning to enhance protein expression” Nature Research Scientific Reports (2020) 10:17617.Additional references for codon optimization methods include, among others: NM Marlatt et al. (2010) “Codon optimization for enhanced Escherichia coil expression of human S100A11 and S100A1 proteins” Protein Expr. Purif. 73(1):58-64; A. Mellitzer et al. (2012) “Expression of lignocellulolytic enzymes in Pichia pastoris” Microb. Cell Fact. 11(1)61; and E. Angov et al. (2008) “Heterologous protein expression is enhanced by harmonizing the codon usage frequencies of the target gene with those of the expression host” PLoS ONE 3(5):e21899).

[0126] Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, an exemplary guide to selecting conservative substitutions includes (original residue followed by exemplary substitutions): 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. Optional exemplary guidelines use the following six groups, each 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, WH 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). One skilled in the art will appreciate that the substitutions identified above are not the only possible conservative substitutions. For example, for some purposes, one might consider all charged amino acids to be conservative substitutions for one another, regardless of whether they are positively or negatively charged. Additionally, single substitutions, deletions, or additions that alter, add, or delete a single amino acid or a small number of amino acids in a coding sequence can also be considered "conservatively modified variations." One skilled in the art will be familiar with the codon usage in a given host for expressing a protein of interest.

[0127] The nucleotide and amino acid sequence information of MYD family members can also be used to construct in silico models of sweet taste modulating peptides and how they interact with the sweet taste receptor, which is composed of a heterodimer of taste 1 receptor member 2 (T1R2) and taste 1 receptor member 3 (T1R3). These models can then be used to identify variants and mutations of Myd that increase sweet taste receptor activation and identify more active forms of Myd.

[0128] Various conservative mutations as listed in Table 8, Table 9, Table 10, Table 7, Table 6 and Table 3 and various less conservative mutations and substitutions are considered to be within the scope of the present invention. The mutations that show sweet taste in Table 9, Table 10, Table 7, Table 3 and Table 6 are currently preferred mutants. For example, it is within the technical level of this area to use known recombinant gene technology schemes, including PCR, gene cloning, cDNA site-directed mutagenesis, host cell transfection and in vitro transcription to carry out amino acid replacement. The sweet taste of the variant is then screened for activity, and particularly for sweet taste and organoleptic properties or the variation of such activity or properties. For example, the variant produced can be screened using sensory tests as described herein and understood in this area. For example, the taste receptor agonist functional activity of the variant produced can be screened as known in the art.

[0129] In embodiments, the HTS variant polypeptides herein exhibit enhanced sweetness compared to the unmodified polypeptide SEQ ID NO: 3. In some embodiments, the sweetness of the corresponding HTS variant is increased by at least 10% (or in the range of 10%-100%, or in the range of 10%-200%, 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% or more) compared to the unmodified polypeptide of SEQ ID NO: 3. In other embodiments, the HTS variants exhibit comparable sweetness compared to the unmodified polypeptide of SEQ ID NO: 3. As described above, the comparable 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 sweetness of the unmodified polypeptide of SEQ ID NO: 3. Sweetness is measured by any method known in the art for comparing sweetness, and more particularly, by the methods for assessing sweetness described herein.

[0130] Flavor modulating activity and sweet taste modulating activity can be detected by methods known in the art, such as in vitro methods, or in vivo by animal or human sensory testing. Although not wishing to be bound by any particular theory, Myd (HTS) is involved in sweet taste activation, for example, being an agonist of taste receptor 1, member 2 (Tas1R2) and / or taste receptor 1, member 3 (Tas1R3). However, Myd (HTS) can stimulate other taste receptors, such as bitter, umami, sour, and salty. Such functional effects can be measured by any means known to those skilled in the art, for example, by measuring binding to the taste receptor Tas1R via spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), fluid dynamics (e.g., shape), chromatographic or solubility properties, patch clamping, voltage-sensitive dyes, whole-cell currents, radioisotope efflux, inducible labeling, transcriptional activation of the Tas1R 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, etc.

[0131] Sensory testing (human or animal) can also be used to determine whether a Myd(HTS) candidate polypeptide possesses sweetness-modulating activity. Sensory evaluation is a scientific discipline that analyzes and measures human responses to food and beverage components, such as appearance, touch, smell, texture, temperature, and taste. Sometimes, human instruments are used for measurement. The selection of appropriate methods for determining sweetness can be determined by those skilled in the art and include, for example, discrimination tests or difference tests, which aim to measure the likelihood that two products are perceived differently. The evaluators' responses are summed for accuracy and statistically analyzed to determine whether they are more accurate than expected by chance alone. The food industry is primarily in need of developing such measurement tools, as sensory characteristics of flavor and texture are distinct attributes that cannot be easily measured instrumentally. For example, for sweetness perception, samples and test samples containing one or more of 5% sucrose, 6% sucrose, 7% sucrose, 8% sucrose, 9% sucrose, and 10% sucrose can be ranked by sweetness intensity from least to most sweet by trained panelists. In the present invention, it should be understood that those skilled in the art can measure sensory properties (e.g., sensory differences) in any number of ways. For example, sensory responses can be measured using trained panelists who rate their responses on a recognized scale or rating system such as a hedonic rating system. Alternatively or in addition, when the subject's (panel member's) response to sensory properties can be linked to the properties measured by an instrumental method, the instrumental method is available or can be easily adjusted. See, for example, Ray, S. (2221) "Sensory Properties of Foods and Their Measurement Methods." Khan, MS, Shafiur Rahman, M. (eds) Techniques to Measure Food Safety and Quality. Springer, Cham. https: / / doi.org / 10.1007 / 978-3-030-68636-9_15.

[0132] The Brix measurement (or Brix scale) is a well-known application in the food and beverage industry for determining the pure sucrose content in water: 1 degree Brix (°Bx) = 1 g sucrose / 100 g solution, and the strength of the solution is expressed as a mass percentage. 8°Bx is equivalent to an approximately 8% sucrose solution. As described in the Examples, a purified polypeptide corresponding to SEQ ID NO: 5 (0.2 mL aliquot) was tasted at 0.03 mg / ml by trained sensory scientists and found to have a sweetness equivalent to 8°Bx (approximately 8% sucrose solution) (see Examples 4, 5, 9, and 10).

[0133] Thermal stability

[0134] The thermostability of sweet taste-modulating polypeptides may affect the potential applications of the polypeptides, such as for food applications at elevated temperatures. In an embodiment, the HTS variants herein exhibit comparable thermostability compared to the unmodified polypeptide of SEQ ID NO: 3. As described above, the comparable thermostability of the modified polypeptides compared to the unmodified polypeptide of SEQ ID NO: 3 is substantially the same, which herein means that the change in thermostability is 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 thermostability of the unmodified polypeptide of SEQ ID NO: 3.

[0135] In some embodiments, the HTS variants herein exhibit enhanced thermostability compared to unmodified SEQ ID NO:3. In other embodiments, the enhanced thermostability of the modified polypeptide is enhanced by greater than 4.5% (including an increase of 4.5% to 15%, an increase of greater than 5%, an increase of greater than 6%, an increase of greater than 7%, an increase of greater than 8%, an increase of greater than 9%, an increase of greater than 10%, an increase of greater than 11%, an increase of greater than 12%, an increase of greater than 13%, an increase of greater than 14%, an increase of greater than 5% to up to 15%, an increase of greater than 6% to up to 15%, an increase of greater than 7% to up to 15%, an increase of greater than 8% to up to 15%, an increase of greater than 9% to up to 15%, an increase of greater than 10% to up to 15%, an increase of greater than 11% to up to 15%, an increase of greater than 12% to up to 15%, an increase of greater than 13% to up to 15%, an increase of greater than 14% to up to 15%, or an increase of up to 15%) compared to the thermostability of the unmodified polypeptide of SEQ ID NO: 3, as described above. In some embodiments, the present invention relates to a method for evaluating thermostability of a sweet taste regulator polypeptide. In some embodiments, the present invention relates to a method for evaluating thermostability of a sweet taste regulator polypeptide. In some embodiments, the present invention relates to a method for evaluating thermostability of a sweet taste regulator polypeptide. In some embodiments, the present invention relates to a method for evaluating thermostability of a sweet taste regulator polypeptide. In some embodiments, the present invention relates to a method for evaluating thermostability of a sweet taste regulator polypeptide. In some embodiments, the present invention relates to a method for evaluating thermostability of a sweet taste regulator polypeptide. In some embodiments, the present invention relates to a method for evaluating thermostability of a sweet taste regulator polypeptide. In some embodiments, the present invention relates to a method for evaluating thermostability of a sweet taste regulator polypeptide. In some embodiments, the present invention relates to a method for evaluating thermostability of a sweet taste regulator polypeptide. In some embodiments, the present invention relates to a method for evaluating thermostability of a sweet taste regulator polypeptide. In some embodiments, the present invention relates to a method for evaluating thermostability of a sweet taste regulator polypeptide.

[0136] In some embodiments, the HTS variants herein exhibit comparable sweetness and comparable thermostability compared to those of the unmodified polypeptide of SEQ ID NO: 3. In some embodiments, the HTS variants herein exhibit enhanced sweetness and comparable thermostability compared to those of the unmodified polypeptide of SEQ ID NO: 3. In some embodiments, the HTS variants herein exhibit comparable sweetness and enhanced thermostability compared to those of the unmodified polypeptide of SEQ ID NO: 3. In some embodiments, the HTS variants herein exhibit enhanced sweetness and enhanced thermostability compared to those of the unmodified polypeptide of SEQ ID NO: 3.

[0137] As described above, the comparable thermal stability of the modified polypeptide is substantially the same as that of the unmodified polypeptide of SEQ ID NO: 3, which means herein that the change in thermal stability is 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 the unmodified polypeptide of SEQ ID NO: 3.

[0138] As compared to the unmodified polypeptide of SEQ ID NO: 3, as described above, the enhanced thermostability of the modified polypeptide is enhanced by greater than 4.5% (including an increase of 4.5% to 15%, an increase of greater than 5%, an increase of greater than 6%, an increase of greater than 7%, an increase of greater than 8%, an increase of greater than 9%, an increase of greater than 10%, an increase of greater than 11%, an increase of greater than 12%, an increase of greater than 13%, an increase of greater than 15%, an increase of greater than 5% to up to 15%, an increase of greater than 6% to up to 15%, an increase of greater than 7% to up to 15%, an increase of greater than 8% to up to 15%, an increase of greater than 9% to up to 15%, an increase of greater than 10% to up to 15%, an increase of greater than 11% to up to 15%, an increase of greater than 12% to up to 15%, an increase of greater than 13% to up to 15%, an increase of greater than 14% to up to 15%, or an increase of up to 15%) compared to the thermostability of the unmodified polypeptide of SEQ ID NO: 3.

[0139] In an embodiment, the polypeptide variants herein exhibit sweet taste modulating activity. Non-limiting examples of sweet taste modulating activity include providing a sweet taste.

[0140] In another aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide) comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one mutation selected from those listed in Table 8 and optionally one deletion, such as a deletion of Met at position 1. In another aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide) comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one mutation to up to 24 mutations at different amino acid positions selected from those listed in Table 8.

[0141] In another aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide) comprising an amino acid sequence of SEQ ID NO: 3 modified by at least one mutation selected from those listed in Table 8 and at least one mutation selected from Table 7, and optionally one deletion, such as a deletion of Met at position 1. In another aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide) comprising an amino acid sequence of SEQ ID NO: 3 modified by at least one mutation to up to 24 mutations at different amino acid positions selected from those listed in Table 8 and one to 24 mutations selected from Table 7.

[0142] In another aspect, the present invention provides polypeptides (e.g., isolated polypeptides) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one mutation to up to 24 mutations selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion, such as a deletion of Met at position 1. In another aspect, the present invention provides polypeptides (e.g., isolated polypeptides) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one mutation to up to 24 mutations at different amino acid positions selected from those listed in Table 8, Table 9 or Table 10.

[0143] In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO: 3 modified by two mutations at different positions selected from those listed in Table 8, Table 9 or Table 10, and optionally one deletion, such as a deletion of Met at position 1. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO: 3 modified by three mutations at different positions selected from those listed in Table 8, Table 9 or Table 10, and optionally one deletion, such as a deletion of Met at position 1. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO: 3 modified by four mutations at different positions selected from those listed in Table 8, Table 9 or Table 10, and optionally one deletion, such as a deletion of Met at position 1. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO: 3 modified by five mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion, such as a deletion of Met at position 1. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO: 3 modified by six mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion, such as a deletion of Met at position 1.

[0144] In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising an amino acid sequence of SEQ ID NO: 3 modified by seven mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion, such as a Met deletion at position 1. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising an amino acid sequence of SEQ ID NO: 3 modified by eight mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion, such as a Met deletion at position 1. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising an amino acid sequence of SEQ ID NO: 3 modified by nine mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion, such as a Met deletion at position 1. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by ten mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion, such as a deletion of Met at position 1. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by eleven mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion, such as a deletion of Met at position 1. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by twelve mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion, such as a deletion of Met at position 1.

[0145] In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by thirteen mutations at various positions selected from those listed in Table 8, Table 9, or Table 10, and optionally one deletion, such as a deletion of Met at position 1. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by fourteen mutations at various positions selected from those listed in Table 8, Table 9, or Table 10, and optionally one deletion, such as a deletion of Met at position 1. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by fifteen mutations at various positions selected from those listed in Table 8, Table 9, or Table 10, and optionally one deletion, such as a deletion of Met at position 1. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by sixteen mutations at various positions selected from those listed in Table 8, Table 9, or Table 10, and optionally one deletion, such as a deletion of Met at position 1. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by seventeen mutations at various positions selected from those listed in Table 8, Table 9, or Table 10, and optionally one deletion, such as a deletion of Met at position 1. In a related aspect, the present invention provides a polypeptide having sweet taste modulating activity (e.g., an isolated polypeptide), comprising an amino acid sequence of SEQ ID NO: 3 modified by eighteen mutations at various positions selected from those listed in Table 8, Table 9, or Table 10, and optionally one deletion, such as a deletion of Met at position 1.

[0146] In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO: 3 modified by nineteen mutations at various positions selected from those listed in Table 8, Table 9, or Table 10, and optionally one deletion, such as a deletion of Met at position 1. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO: 3 modified by twenty mutations at various positions selected from those listed in Table 8, Table 9, or Table 10, and optionally one deletion, such as a deletion of Met at position 1. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO: 3 modified by twenty-one mutations at various positions selected from those listed in Table 8, Table 9, or Table 10, and optionally one deletion, such as a deletion of Met at position 1. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO: 3 modified by twenty-two mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion, such as a Met deletion at position 1. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO: 3 modified by twenty-three mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion, such as a Met deletion at position 1. In a related aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising an amino acid sequence of SEQ ID NO: 3 modified by twenty-four mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion, such as a Met deletion at position 1. The present invention also provides a polynucleotide encoding the above-mentioned mutant polypeptide of SEQ ID NO: 3 or SEQ ID NO: 141. The present invention also provides the above mutant polypeptides, which further include a protein tag, and more particularly, a histidine tag. The present invention also provides polynucleotides encoding the above mutant polypeptides of SEQ ID NO: 3 or SEQ ID NO: 141, which further include a protein tag, and more particularly, a histidine tag. In embodiments, the HTS variant does not include any one or more of the mutations listed in Table 3, Table 6, or Table 7.

[0147] In some embodiments, the polypeptide having sweet taste modulating activity further has enhanced thermostability compared to the polypeptide of SEQ ID NO: 3. In embodiments, the polypeptide having sweet taste modulating activity and enhanced thermostability comprises a modified SEQ ID NO: 3, wherein the polypeptide has at least one and up to six amino acid modifications as shown in Table 8, Table 9 or Table 10 and optionally one deletion, such as a Met deletion at position 1. In embodiments, the polypeptide having sweet taste modulating activity and enhanced thermostability comprises a modified SEQ ID NO: 3, wherein the polypeptide has at least one and up to six amino acid modifications as shown in Table 7 and optionally one deletion, such as a Met deletion at position 1. In embodiments, the polypeptide having sweet taste modulating activity and enhanced thermostability comprises a modified SEQ ID NO: 3, wherein the polypeptide has at least one and up to six amino acid modifications as shown in Table 8, Table 9 or Table 10 and further has 1-6 amino acid modifications as shown in Table 7 and optionally one deletion, such as a Met deletion at position 1. In other embodiments of the foregoing embodiments, the polypeptide has sweet taste modulating activity (including sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 141.

[0148] In another embodiment, the polypeptide has sweet taste modulating activity (and particularly sweet taste) and enhanced thermostability compared to a polypeptide of SEQ ID NO: 3, which 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 to a modified polypeptide of SEQ ID NO: 3 having at least one and up to six amino acid modifications as shown in Table 8, Table 9, or Table 10 and 1-6 amino acid modifications as shown in Table 7, and optionally one deletion, such as a deletion of Met at position 1. In other embodiments of the foregoing embodiments, the polypeptide has sweet taste modulating activity (including sweet taste) and enhanced thermostability compared to a polypeptide of SEQ ID NO: 141.

[0149] In another embodiment, the polypeptide having sweet taste modulating activity (and in particular sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 has both mutations I49C and S33C (compared to SEQ ID NO: 3) and optionally 1, 2, 3, 4, 5 or 6 other mutations as shown in Table 8, Table 9, Table 10, Table 3, Table 6 or Table 7, and in particular optionally a deletion such as a deletion of Met at position 1. In a related embodiment, the polypeptide having sweet taste modulating activity (and particularly sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 has both mutations I49C and S33C (compared to SEQ ID NO: 3) and 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 to the modified polypeptide of SEQ ID NO: 3. In other embodiments of the foregoing embodiments, the polypeptide has sweet taste modulating activity (including sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 141.

[0150] In another embodiment, the polypeptide having sweet taste modulating activity (and in particular sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 has both mutations Y23C and Y61C (compared to SEQ ID NO: 3) and optionally 1, 2, 3, 4, 5 or 6 other mutations as shown in Table 8, Table 9, Table 10, Table 3, Table 6 or Table 7, and optionally a deletion such as a deletion of Met at position 1. In a related embodiment, the polypeptide having sweet taste modulating activity (and particularly sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 has both mutations I49C and S33C (compared to SEQ ID NO: 3) and 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 to the modified polypeptide of SEQ ID NO: 3. In other embodiments of the foregoing embodiments, the polypeptide has sweet taste modulating activity (including sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 141.

[0151] In an embodiment, a polypeptide having sweet taste modulating activity and enhanced thermostability comprises a modified SEQ ID NO: 3, wherein the polypeptide has two modifications as shown in Table 7 and / or Table 8, Table 9 or Table 10 and optionally one deletion, such as a Met deletion at position 1. In an embodiment, a polypeptide having sweet taste modulating activity and enhanced thermostability comprises a modified SEQ ID NO: 3, wherein the polypeptide has two modifications as shown in Table 7 and / or Table 8, Table 9 or Table 10, and optionally one deletion, such as a Met deletion at position 1, that result in the formation of a disulfide bond between the two modified amino acids. In one embodiment, a polypeptide having sweet taste modulating activity and enhanced thermostability comprises a modified SEQ ID NO: 3, wherein the polypeptide has at least two modifications as shown in Table 7 and / or Table 8, Table 9 or Table 10, and optionally one deletion, such as a Met deletion at position 1, that result in the formation of a disulfide bond between at least two modified amino acids. In other embodiments of the foregoing embodiments, the polypeptide has sweet taste modulating activity (including sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 141.

[0152] In an embodiment, the polypeptide having sweet taste modulating activity (and in particular sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 includes the polypeptide of SEQ ID NO: 143 (double mutant S33C_I49C) or SEQ ID NO: 145 (double mutant Y24C_Y62C). In a related embodiment, the polypeptide having sweet taste modulating activity (and in particular sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 includes the polypeptide of SEQ ID NO: 142 or SEQ ID NO: 145, and further includes SEQ ID NO: 146 and SEQ ID NO: 147 in which the methionine at position 1 is deleted. In an embodiment, the polypeptide having sweet taste modulating activity (and in particular sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 is the polypeptide of SEQ ID NO: 143. In an embodiment, the polypeptide having sweet taste modulating activity (and in particular sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 is the polypeptide of SEQ ID NO: 145. In an embodiment, the polypeptide having sweet taste modulating activity (and particularly sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 is a polypeptide of SEQ ID NO: 146. In an embodiment, the polypeptide having sweet taste modulating activity (and particularly sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 is a polypeptide of SEQ ID NO: 147.

[0153] In some embodiments, the polypeptides of the present invention exhibit enhanced stability at low pH (<pH 7) compared to the polypeptide of SEQ ID NO:3. In some embodiments, the polypeptides of the present invention exhibit enhanced stability at high pH (>pH 7) compared to the polypeptide of SEQ ID NO:3. In one embodiment, the polypeptides of the present invention exhibit greater stability at a pH of about 2 compared to the polypeptide of SEQ ID NO:3. In another embodiment, the polypeptides of the present invention exhibit greater stability at a pH of about 10 compared to the polypeptide of SEQ ID NO:3. In one embodiment, the polypeptides of the present invention having methionine at position 1 exhibit greater stability at a pH of about 2 compared to the polypeptide of SEQ ID NO:3. In another embodiment, the polypeptides of the present invention having methionine at position 1 exhibit greater stability at a pH of about 10 compared to the polypeptide of SEQ ID NO:3.

[0154] The term "expression vector" or "expression cassette" refers to any recombinant expression system for expressing the nucleic acid sequences of the present invention in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cells. The term includes linear or circular expression systems. The term includes expression systems that remain free or integrate into the host cell genome. The expression system may have the ability to self-replicate or not, i.e., only drive transient expression in the cell. The term includes recombinant expression cassettes that include only the minimal elements required for transcription of recombinant nucleic acids.

[0155] A recent review of recombinant protein expression methods can be found in Tripathi & Shrivastava (2019) "Recent Developments in Bioprocessing of Recombinant Proteins; Expression Hosts and Process Development," Frontiers in Bioeng. Biotech. 7:420, doi:10.3389 / fbio.2019.00420. This reference is incorporated herein by reference in its entirety for details of host expression systems and recombinant protein expression methods.

[0156] By "host cell" is meant a cell that contains an expression vector and supports replication or expression of the expression vector. In one embodiment, the host cell is a prokaryotic cell. In one embodiment, the host cell is a eukaryotic cell. The host cell can be a prokaryotic cell such as E. coli, or a eukaryotic cell such as yeast, insect, amphibian, or mammalian cell such as CHO, HeLa, HEK-293, etc., for example, cultured cells, explants, and in vivo cells.

[0157] In an embodiment, the host cell is selected from the group consisting of: Escherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Agrobacterium tumefaciens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, coelicolor), Clostridium acetobutylicum, Pseudomonas fluorescens, Pseudomonas putida, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizopus oryzae, Yarrowia lipolytica, Candida albicans, Issatchenkia orientalis), Pichia stipitis (Scheffersomycesstipitis), Yarrowia lipolytica (Yarrowia lipolytica), Ogataea polymorpha (Ogataea polymorpha), Phaffia rhodozyma (Phaffiarhodozyma), Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, and Schwanniomyces occidentalis.

[0158] In an embodiment, the host cell is selected from the group consisting of biological agents recommended by the Qualified Presumption of Safety (QPS). A list of such hosts can be found at the following website: efsa.europa.eu / efsajournal EFSA Journal 2021;19(7):6689. In an embodiment, the host organism is selected from the group consisting of: Bacillus megaterium, Trichoderma reesei, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Carnobacterium divergens, Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus alimentarius, Lactobacillus aviaries, Lactobacillus breve, Lactobacillus longum, Lactobacillus divergens, Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus alimentarius, Lactobacillus aviaries, Lactobacillus breve ... brevis), Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus collinoides, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus farciminis, Lactobacillus fermentumfermentum), 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 mucosa pontis), Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus Sanfranciscensis, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc Pseudomesenteroides, Microbacterium imperial, Oenococcus oeni, Pasteuria nishizawae, Pediococcus acidilactici, Pediococcus parvulus, Pediococcus pentosaceus pentosaceus, Propionibacterium acidi-producing bacteriaacidipropionic), Propionibacterium freudenreichii, Streptococcus thermophilus, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus flexus, Bacillus fusiformis, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus mojavensis, Bacillus paralicheniformis, Bacillus pumilus pumilus), Bacillus smithii, Bacillus subtilis, Bacillus vallismortis, Bacillus velezensis, Geobacillus stearothermophilus, Paenibacillus sillinoisensis, Parageobacillus thermoglucosidasius, Gluconobacter oxydans, Komagataeibacter sucrofermentans, Xanthomonas campestris, Candidacylindracea, Cyberlindnera jadinii, Debaryomyces hansenii hansenii), Hanseniaspora uvarum, Kluyveromyces lactis, Kluyveromyces marxianusmarxianus, Komagataella pastoris, Komagataella phaffi, Lindnera jadinii, Ogataea angusta, Saccharomyces bayanus, Schizosaccharomyces pombe, Wickerhamomyces anomalus, Xanthophyllomyces dendrorhous, or Zygosaccharomyces rouxii.

[0159] In another aspect, 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. In other aspects, the host cell is selected from plant cells. In other aspects, the host cell is selected from insect cells. For insect cells, baculovirus insect expression systems are available. Insect cells that can be used as hosts for the production of recombinant proteins include, among others, Spodoptera frugiperda cells (e.g., Sf9, Sf21), Drosophila cells (e.g., S2), Trichoplusia ni cells (e.g., Tn-368, High-Five TM (Thermo Fisher Scientific, Waltham, MA). Various host cells are known in the art and can be obtained from commercial sources, etc.

[0160] 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 amylovorus, Lactobacillus animalis, Lactobacillus alimentarius, Lactobacillus avium, Lactobacillus spp. aviaries), Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus collinoides, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticushelveticus), 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 sake sakei), Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactococcus lactis, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Microbacterium imperial, Oenococcus oeni, Pasteuria nishizawae, Pediococcus acidilactic, Pediococcus parvulus, Pediococcus pentosaceus, Propionibacterium acidipropioni, Propionibacterium freudenreichii freudenreichii) and Streptococcus thermophiles.

[0161] 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 smithii, Bacillus subtilis, Bacillus valerianus, and Bacillus thunbergii. vallismortis), Bacillus velezensis, Geobacillus stearothermophilus, Paenibacillus sillinoisensis, and Parageobacillus thermoglucosidasius. Non-limiting examples of Gram-negative bacteria include Cupriavidus necator, Gluconobacter oxydans, Komagataeibacter sucrofermentans, and Xanthomonas campestris.

[0162] 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, Saccharomyces pastorianus, Schizosaccharomyces pombe, Wickerhamomyces anomalus, Xanthophyllomyces dendrorhous, Yarrowia lipolytica, and Zygosaccharomyces rouxii.

[0163] Non-limiting examples of protists / algae include Aurantiochytrium limacinum, Euglena gracilis, and Tetraselmis chuii.

[0164] In certain embodiments, the recombinant HTS is produced by a transgenic mammal, ie, in milk.

[0165] The expression of HTS (or its variants) can be stable or transient. In a stable expression system, the foreign DNA is integrated into the chromosome or as an episome (a separate piece of nuclear DNA) and passed on to the progeny of the host cell.

[0166] The terms "mimetic" and "peptide mimetic" refer to synthetic compounds that have substantially the same structural and / or functional characteristics as a polypeptide (e.g., a translocation domain, a ligand binding domain, or a chimeric receptor) of the invention. A mimetic can be composed entirely of synthetic non-natural amino acid analogs, or can be a chimeric molecule composed partially of natural peptide amino acids and partially of non-natural amino acid analogs. A mimetic can also incorporate any amount of conservative substitutions of natural amino acids, as long as such substitutions do not significantly alter the structure and / or activity of the mimetic.

[0167] For polypeptides of the invention that are conservative variants, routine experimentation will determine whether the mimetic is within the scope of the invention, i.e., whether its structure and / or function is not substantially altered. A polypeptide mimetic composition can include any combination of non-natural structural components, which generally come from three structural groups: a) residue linkages other than natural amide bonds ("peptide bonds"); b) non-natural residues that replace naturally occurring amino acid residues; c) residues that induce secondary structure mimicry, i.e., induce or stabilize secondary structure, such as beta turns, gamma turns, beta sheets, alpha helical conformations, etc. A polypeptide can be characterized as a mimetic when all or some of its residues are linked by chemical means other than natural peptide bonds. Individual peptide mimetic residues can be linked by peptide bonds, 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 replace traditional amide ("peptide bond") linkages include, for example, ketomethylene (e.g., --C(O)--CH2-- instead of --C(O)--NH--), aminomethylene (CH2--NH), ethylene, olefin (CH=CH), ether (CH2--O), thioether (CH--S), tetrazole (CN4), thiazole, retroamide, thioamide, or ester (see, for example, 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 including 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 suite of web-available tools for predicting and analyzing protein structure, function, and mutations.

[0168] Examples of conservatively modified variations in Myd1 protein structure can be derived 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. & Frydman, J. Interplay between Chaperones and Protein Disorder Promotes the Evolution of Protein Networks. PLoS Computational Biology 10, e1003674 (2014).

[0169] Specific regions of the MYD / Myd nucleotide and amino acid sequences can be used to identify polymorphic variants, interspecies homologs, and alleles of Myd family members. Such identification can be performed in vitro, for example, under stringent hybridization conditions or PCR (e.g., using primers encoding the Myd sequences identified herein), or by comparing the sequence information in a computer system with other nucleotide sequences. Different alleles of the MYD gene in a population of a single species will also help to determine whether differences in allelic sequence are associated with taste differences between members of the population. Classical PCR-type amplification and cloning techniques can be used to isolate orthologs, for example, where degenerate primers are sufficient to detect related genes across species.

[0170] For example, primers designed using the sequences disclosed herein can be used to amplify and clone MYD-related genes from different fungal genomes. In contrast, genes associated with MYD within a single species are best identified using sequence pattern recognition software to find related sequences. Typically, polymorphic variants and alleles of MYD family members can be identified by comparing amino acid sequences of about 25 amino acids or more, for example 50-100 amino acids. Amino acid identity of approximately at least 35% to 50% and optionally 60%, 70%, 75%, 80%, 85%, 90%, 95-99% or more generally indicates that the protein is a polymorphic variant, interspecies homolog or allele of a MYD family member. Sequence comparisons can be performed using any sequence comparison algorithm 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.

[0171] In an embodiment, a hybrid protein coding sequence comprising a nucleic acid encoding a Myd variant fusion protein can be constructed. These nucleic acid sequences can be operably linked to the following: transcription or translation control elements, such as transcription and translation initiation sequences, promoters and enhancers, transcription and translation terminators, polyadenylation sequences, and other sequences for transcribing DNA into RNA. The fusion protein can include a C-terminal or N-terminal translocation sequence. In addition, the fusion protein can include additional elements, for example, for protein detection, purification, or other applications. Detection and purification facilitating domains include, for example, metal chelating peptides, such as polyhistidine bundles, 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 used in the FLAGS extension / affinity purification system (Immunex Corp, Seattle Wash.).

[0172] In embodiments, the fusion protein includes a peptide or protein tag (eg, for protein purification or detection). A protein / peptide tag is a peptide sequence genetically grafted into a recombinant (eg, fusion) protein. Peptide / protein tags are known in the art, such as those described in Johnson, “Protein / Peptide Tags,” DOI / / dx.doi.org / 10.13070 / mm.en.2.116, and include, but are not limited to, green fluorescent protein (GFP), FLAG, Myc epitope, polyhistidine, glutathione-S-transferase (GST), HA, V5, ABDzl-tag, adenylate kinase (AK-tag), BC2-tag, calmodulin binding peptide, CusF, Fc, Fh8, halogen tag, heparin binding peptide (HB-tag), ketosteroid isomerase (KSI), maltose binding protein (MBP), thioredoxin, PA (NZ-1), Poly-Arg, Poly-Lys, S-tag, SBP / streptavidin binding peptide, SNAP, Strep-II (Twin-Strep), and SUMO / SUMO2.

[0173] An affinity tag is a protein tag that is attached to a protein so that it can be purified from its original biological source using affinity techniques. Affinity tags are known in the art, such as those described in Kimple et al. Curr Protoc Protein Sci.; 73: Unit-9.9. doi: 10.1002 / 0471140864.ps0909s73. These include, among others, polyhistidine, GST, MBP, calmodulin binding peptides, intein-chitin binding domains, streptavidin / biotin-based tags, 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 peptides, and examples of large affinity tags include MBP, GST, cellulose binding domains, calmodulin binding peptides, and His-patch ThioFusion.

[0174] Protein / peptide tags include epitope tags and reporter tags. Reporter gene 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).

[0175] Epitope tags, including FLAG, hemagglutinin (HA), c-myc, T7, and Glu-Glu, are used to detect fusion proteins in vitro and in cell culture. Their short, linear recognition motifs rarely affect the properties of the target protein and are generally very specific for their respective primary antibodies. If an anti-myc antibody is used, specificity can be improved by detecting the conjugated anti-myc primary antibody with an enzyme-linked secondary antibody, rather than using an HRP- or AP-anti-myc conjugate alone.

[0176] Protein / peptide tags also include solubilization tags, which are used to help proteins fold correctly and prevent them from aggregating in inclusion bodies. In an embodiment, solubilization tags are used for proteins expressed in E. coli. Solubilization tags include thioredoxin and poly (NANP), etc. Some affinity tags may also help solubilization, such as MBP and GST.

[0177] Protein / peptide tags can be located at either end of the protein of interest. Some tags, such as FLAG, are often used in tandem to enhance their desired characteristics, or in combination with another tag, such as in constructs like His-Myc and His-V5.

[0178] Tandem affinity purification (TAP) is a dual affinity purification method based on fusing two affinity tags to a protein of interest, which allows purification of the tagged protein and isolation of protein complexes interacting with the protein of interest. The use of TAP is encompassed by the present invention.

[0179] In one embodiment, the fusion protein comprises a histidine tag comprising 2-10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) histidine residues. For example, the histidine tag can comprise 6 histidine residues.

[0180] The newly translated polypeptide includes a cleavable linker sequence between the translocation domain (for efficient plasma membrane expression) and the rest of the polypeptide, such as factor Xa (see, e.g., Ottavi, Biochimie 80: 289-293 (1998)), a subtilisin recognition motif (see, e.g., Polyak, Protein Eng. 10: 615-619 (1997)); enterokinase (Invitrogen, San Diego, Calif.) and the like can be used to facilitate purification. For example, a construct can include a polypeptide encoding a nucleic acid sequence linked to six histidine residues, followed by thioredoxin, an enterokinase cleavage site (see, e.g., Williams, Biochemistry 34: 1787-1797 (1995)) and a C-terminal translocation domain. The histidine residues facilitate detection and purification, while the enterokinase cleavage site provides a method for purifying one or more desired proteins from the rest of the fusion protein. Technology relating to vectors encoding fusion proteins and the use of fusion proteins is well described in the scientific and patent literature; see, eg, Kroll, DNA Cell. Biol. 12:441-53 (1993).

[0181] The fusion protein can include one or more linkers (e.g., flexible linkers, rigid linkers, and in vivo cleavable linkers). In addition to the basic role of connecting functional domains together (e.g., in flexible and rigid linkers) or releasing free functional domains in vivo (e.g., in vivo cleavable linkers), linkers also provide many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yield, and achieving desired pharmacokinetic characteristics. Linkers are known in the art (see, e.g., Chen et al., Adv Drug Deliv Rev. 65(10): 1357-1369(2013)).

[0182] Flexible linkers are used when the domains connected require a certain degree of mobility or interaction. They are generally composed of small non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The small size of these amino acids provides flexibility and allows the 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, and thereby reduce the adverse interactions between the linker and the protein portion.

[0183] The most commonly used flexible linkers have a sequence consisting primarily of stretches of Gly and Ser residues ("GS" linkers). An example of the most widely used flexible linker has the sequence (Gly-Gly-Gly-Gly-Ser) n(SEQ ID NO: 7). By adjusting the copy number "n", the length of the GS linker can be optimized to achieve proper separation of functional domains or maintain necessary inter-domain interactions. In addition to the GS linker, many other flexible linkers have been designed for recombinant fusion proteins. These flexible linkers are also rich in small or polar amino acids, such as Gly and Ser, but can include additional amino acids (such as Thr and Ala) to maintain flexibility, as well as polar amino acids (such as Lys and Glu) to improve solubility.

[0184] Rigid linkers maintain a fixed distance between domains and preserve their independent functions. Examples of rigid linkers include those with (EAAAK) n (SEQ ID NO: 8) sequence α helix forming linker and having Pro-rich sequence (XP) n wherein X represents any amino acid, preferably Ala, Lys or Glu.

[0185] Polypeptides of the present invention may also include signal peptides (i.e., signal sequences, targeting signals, localization signals, localization sequences, transit peptides, leader sequences, or leader peptides), which are short peptides present at the N-terminus or occasionally the C-terminus of most newly synthesized proteins that are intended to enter the secretory pathway. These proteins include those that reside in certain organelles (endoplasmic reticulum, Golgi apparatus, or endosomes), are secreted from cells, or are inserted into most cell membranes. Exemplary signal peptides are known in the art, and those of ordinary skill in the art will recognize how to select a specific signal peptide for use in the present invention.

[0186] Protein derivatization

[0187] HTS proteins / polypeptides and sequence variants thereof can be further derivatized or modified rather than by replacing one or more amino acids. The HTS wild-type (native) proteins / polypeptides and HTS protein / polypeptide variants disclosed herein can be derivatized at the N-terminus, C-terminus or amino acid side chains without losing taste regulating activity. Derivatization typically includes acylation, esterification, glycosylation, oxidation, methylation, reductive alkylation, phosphorylation (such as phospho-amino acids), sulfonylation, sulfonylation or oxidation or reduction of side chain heteroatoms. Derivatization can be achieved by chemical methods using reagents and methods well known in the art. Alternatively, derivatization can be achieved by biological methods, such as treatment with one or more enzymes, or by post-translational methods (post-translational modification). One of ordinary skill in the art can select one or more enzymes to achieve the desired derivatization of the HTS protein / polypeptide or its variants. The isolated HTS protein or polypeptide is preferably treated with the selected enzyme to achieve the desired derivatization. Post-translational modification (PTM) is the covalent modification of a protein / polypeptide by proteolytic cleavage (e.g., removal of the N-terminal methionine) and / or the addition of modifying groups such as acetyl (more broadly acyl), phosphoryl, glycosyl, and / or methyl groups to one or more amino acids. Other chemical modifications of the amino acids of a protein / polypeptide (e.g., oxidation of the sulfur of the methionine group) can be achieved by those skilled in the art through chemical and biological means. Post-translational modifications can occur during the natural expression of a protein / polypeptide, or can be controlled during protein expression using recombinant methods in a non-natural host. Such recombinant methods rely on the use of specially constructed expression vectors, including, for example, coding sequences for expressing one or more enzymes to initiate selected post-translational modifications. One of ordinary skill in the art is aware of chemical or recombinant techniques for derivatizing proteins at one or more positions on a given protein. In an embodiment, preferred protein / polypeptide derivatizations are those that do not significantly affect the sweet taste modulating activity of the HTS protein / polypeptide variants disclosed herein. In embodiments, preferred protein / polypeptide derivatizations are those that do not significantly adversely affect the sweetness of the HTS protein / polypeptide variants of the present disclosure (e.g., do not significantly reduce the sweetness of the HTS variants herein). In embodiments, derivatization of HTS variants can enhance the flavor modulation, sweetness modulation, or sweetening activity of the HTS variants.

[0188] Derivatization of the HTS protein / polypeptide or variant can occur at the N-terminus, C-terminus or one or more amino acid side groups (e.g., side chain sulfur, side chain amine or side chain carboxylic acid) by any known method. In an embodiment, the HTS protein / polypeptide or HTS variant of the present disclosure can have 1, 2, 3, 4, 5 or 6 different derivatizations. Preferably, the HTS protein or HTS variant has a single derivatization. For example, the N-terminus of the HTS protein / polypeptide or variant can be derivatized by acylation, and more particularly by acetylation. In a more specific embodiment, the N-terminus of the protein / polypeptide or variant is an N-acetylated methionine. In an embodiment, the derivatization is the derivatization of the N-terminal amino acid or amine side chain of the amino acids of the HTS protein / polypeptide. N-terminal amino acid or amine side chains (e.g., lysine side chains) can be derivatized by acylation; glycosylation; methylation; reductive amination to form –NH-CH2-R, where R is an alkyl group (e.g., an alkyl group having 1-19 carbon atoms); phosphorylation of amino acid side chains such as serine or threonine (-OH) with kinases to form phospho-peptides; reaction of the sulfur in the amino acid side chain with a monooxygenase to form a sulfoxide derivative. The N-terminal amine or amine side chain can be derivatized chemically or by biological processes after the polypeptide is translated, resulting in post-translational modification of the nitrogen. The reactive side chains of other amino acids can be derivatized chemically or by biological processes after the polypeptide is translated, resulting in post-translational modification of those side chains.

[0189] In a specific embodiment, derivatization is the addition, removal, or substitution of one or more of the following chemical moieties on the amine nitrogen: acyl (R-CO-, where R is a straight or branched chain alkyl group having 1-20 carbon atoms); acetyl (CH3CO), formyl (HCO), saccharide (e.g., CH6H 11 O6-), hydroxyl (HO-), methyl (CH3-) or other alkyl groups, phosphatidyl (PO4), phosphonyl (PO2), sulfhydryl (SH-) or sulfonyl (HSO2-).

[0190] In a specific embodiment, wherein the first amino acid of the HTS protein / polypeptide is methionine, after translation of the polypeptide, the first amino acid's δ position sulfur is modified chemically, enzymatically (in vitro or in vivo), or by a biological process, resulting in a post-translational modification of the sulfur. In an embodiment, the modification of the δ position sulfur of the first amino acid is the addition, removal, or substitution of any of the following chemical moieties on the δ sulfur: an acyl group (R-CO-, wherein R is a linear or branched alkyl group having 1-20 carbon atoms); an acetyl group (CH3CO), a formyl group (HCO), a glycosyl group (e.g., C6H11O6-), a hydroxyl group (HO-), a methyl group (CH3-) or other alkyl groups, a phosphatidyl group (PO4), a phosphonyl group (PO2), a sulfhydryl group (SH-), or a sulfonyl group (HSO2-). In an embodiment, the modification of the δ position sulfur of the first amino acid is oxidation of the sulfur, resulting in the production of a sulfoxide derivative.

[0191] As used herein, "at least 80% identity" with respect to an amino acid sequence or a 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 more identity.

[0192] As used herein, examples of “an amino acid sequence 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 1 or more to 30 or less, preferably 20 or less, more preferably 10 or less, and further preferably 5 or less amino acids (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 thereof). As used herein, examples of “a nucleotide sequence modified by deletion, insertion, substitution or addition of one or more nucleotides” include a nucleotide sequence modified by deletion, insertion, substitution or addition of 1 or more nucleotides (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 85, 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, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135

[0193] For example, in sequence comparison, a sequence is usually used as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are specified (if necessary), and sequence algorithm program parameters are specified. Default program parameters can be used, as described below for BLASTN and BLASTP programs, or alternative parameters can be specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters.

[0194] As used herein, "comparison window" includes a segment referring to any one of a plurality of contiguous positions selected from the group consisting of 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48:443 (1970), by the search similarity method of Pearson & 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, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

[0195] 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 at al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J Mol. Biol. 215:403-410 (1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy a positive-valued threshold score T when compared to a word of the same length in a database sequence. T is called 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 that include them. Word hits are extended in both directions along each sequence for as long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is 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 amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension hits in each direction are stopped when: the cumulative alignment score drops by the number X from its maximum achieved value; the cumulative score becomes zero or below due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the comparison. The BLASTN program (for nucleotide sequences) uses a default word length (W) of 11, an expected value (E) of 10, M=5, N=-4, and a comparison of two chains. For amino acid sequences, the BLASTP program uses a default word length of 3, an expected value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad Sci. USA 89:10915 (1989)) alignment (B) of 50, an expected value (E) of 10, M=5, N=-4, and a comparison of two chains.

[0196] Another example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationships and percentages of sequence identity. It also draws a so-called "tree" or "dendrogram" showing the clustering relationships used to create the alignment (see, e.g., Figure 1 ). PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J Mol. Evol. 35: 351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5: 151-153 (1989). The program can align up to 300 sequences, with a maximum length of 5,000 nucleotides or amino acids for each sequence. The multiple alignment program starts with a pairwise alignment of the two most similar sequences, generating a cluster of two aligned sequences. The cluster is then aligned with the next most related sequence or aligned sequence cluster. The two sequence clusters are aligned by a simple extension of the pairwise alignment of the two individual sequences. The final alignment is achieved by a series of progressive pairwise alignments. The program is run by specifying specific sequences and their amino acid or nucleotide coordinates for the sequence comparison region and by specifying program parameters. Using PILEUP, the reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP is available from the GCG sequence analysis software package, for example, version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395 (1984), where the sequences encoded by the gene are translated from the concept of the corresponding open reading frame.

[0197] The polynucleotide encoding the polypeptide of the present invention can be chemically synthesized based on the amino acid sequence of Myd or synthesized by genetic engineering. 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. Contract synthesis services for nucleic acids (such as those provided by Medical & Biological Laboratories Co., Ltd., Genscript, etc.) can be used for chemical synthesis of polynucleotides. In addition, the synthesized polynucleotide can be amplified by methods such as PCR and cloning.

[0198] The polypeptides of the present invention can be produced, for example, by expressing a gene encoding a Myd polypeptide variant of the present invention. Preferably, the Myd polypeptide variant of the present invention can be produced from a transformant into which a polynucleotide encoding a Myd polypeptide variant of the present invention has been introduced. For example, after introducing a polynucleotide encoding a Myd polypeptide variant of the present invention or a vector comprising the same into a host to obtain transformants and culturing the transformants in an appropriate culture medium, the Myd polypeptide variant of the present invention can be produced from the polynucleotide encoding the Myd polypeptide variant of the present invention introduced into the transformants. The protein of the present invention can be obtained by isolating or purifying the produced Myd polypeptide variant from the culture.

[0199] Therefore, the present invention further provides polynucleotides encoding the Myd polypeptide variants of the present invention and vectors comprising the same. The present invention also provides methods for producing transformants, comprising introducing a polynucleotide encoding the Myd polypeptide variants of the present invention or a vector comprising the same into a host. The present invention also provides transformants comprising a polynucleotide encoding the Myd polypeptide variants of the present invention or a vector comprising the same introduced from outside the cell. The present invention also provides methods for producing the Myd polypeptide variants of the present invention, comprising culturing the transformants.

[0200] The present invention also includes a polynucleotide of the present invention operably linked to a heterologous regulatory element. The present invention may 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.

[0201] Alternatively, polynucleotides encoding Myd polypeptide variants of the present invention can be generated by introducing one or more mutations into a polynucleotide synthesized according to a procedure using known mutagenesis methods, such as ultraviolet irradiation and site-directed mutagenesis. For example, polynucleotides encoding the polypeptides of the present invention can be obtained by introducing one or more mutations into the polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 2 using known methods, expressing the obtained polynucleotide, examining the sweet taste-modifying activity of the expressed protein, and selecting a polynucleotide encoding a protein having the desired sweet taste-modifying activity.

[0202] Site-directed mutagenesis of a polynucleotide can be performed using any method, such as, for example, inverse PCR and annealing (Muramatsu et al. edit., "Revised 4th edition New genetic engineering handbook", YODOSHA, p. 82-88). Various commercially available kits for site-directed mutagenesis can be used as needed, such as the QuickChange II Site-Directed Mutagenesis Kit and the QuickChange Multi Site-Directed Mutagenesis Kit from Stratagene.

[0203] Examples of vector types that include polynucleotides encoding polypeptides of the present invention include, but are not limited to, vectors 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, herpesviruses, polioviruses, alphaviruses, baculoviruses, Sindbis viruses, plant viruses (e.g., Alphaflexiviridae or Potyviridae), and insect viruses (e.g., Baculoviridae).

[0204] Among these, plasmid vectors are preferred, and for example, commercially available plasmid vectors for protein expression such as pUC19, pUC118, pUC119, pBR322, etc. (all of these from TAKARA BIO INC.) can be used.

[0205] The vector can include a DNA region containing a replication initiation region or a replication origin of DNA. Alternatively, regulatory sequences such as promoter regions, terminator regions, or secretion signal regions for secreting expressed proteins into the extracellular space for initiating gene transcription can be operably connected upstream of the polynucleotide encoding protein of the present invention (i.e., the MYD gene of the present invention) in the vector. As used herein, gene and regulatory sequence are "operably connected" to refer to conditions in which the gene and regulatory region are positioned so that the gene can be expressed under the regulation of the regulatory region.

[0206] The types of regulatory sequences such as the promoter region, terminator, and secretion signal region are not particularly limited, and commonly used promoters and secretion signal sequences can be selected and used as appropriate, depending on the host into which the sequence is introduced. For example, preferred examples of regulatory sequences that can be incorporated into the vector of the present invention include the cbh1 promoter sequence derived from Trichoderma reesei (Curr, Genet, 1995, 28(1):71-79).

[0207] Alternatively, a marker gene (e.g., a resistance gene to agents such as ampicillin, neomycin, kanamycin, and chloramphenicol) for selecting a host to which the vector is suitable for introduction can be incorporated into the vector of the present invention. Alternatively, when an auxotrophic strain is used as the host, a gene encoding a synthase of the desired nutrient can be incorporated into the vector as a marker gene. Alternatively, when a selective medium requiring specific metabolism for growth is used, a metabolically related gene can be incorporated into the vector as a marker gene. Examples of such metabolically related genes include acetamidase genes for use as a nitrogen source using acetamide.

[0208] The polynucleotide encoding the Myd polypeptide variant of the present invention can be linked to the regulatory sequence and marker gene by methods known in the art, such as SOE (Splicing by Overlap Extension)-PCR (Gene, 1989, 77: 61-68). The procedures for introducing the linked fragments into the vector are known in the art.

[0209] The example of the host of the transformant introduced into the vector includes microorganisms such as bacteria or filamentous fungi. The example of bacteria includes Escherichia coli and bacteria belonging to Staphylococcus, Enterococcus, Listeria and Bacillus, wherein preferably Escherichia coli and Bacillus bacteria (for example, Bacillus subtilis or its mutant). The example of Bacillus subtilis mutant can include J.Biosci.Bioeng., 2007, 104 (2): Protease 9 double defective strain KA8AX described in 135-143, and Biotechnol.Lett., 2011, 33 (9): Protease 8 double defective strain DBPA strain described in 1847-1852, which improves protein folding efficiency. The example of filamentous fungi includes Trichoderma, Aspergillus and Rhizopus. In addition, for example, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowia lipolytica, Schizosaccharomyces pombe, Kluyveromyces lactis are suitable expression hosts. In embodiments, the host cell is a fungal cell other than Matsutsuga gloeosporioides. In embodiments, the HTS protein variant can be expressed in mycelia.

[0210] In embodiments, the HTS protein variant may be expressed in plant cells, plant organs, leaves, roots, or whole plants.

[0211] In yet another aspect, the present invention includes host cells comprising one or more of the expression cassettes described herein operably linked to control elements compatible with expression in the cell. The cell can 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.

[0212] In certain embodiments, the HTS (or variants thereof) is produced in a yeast expression system (i.e., a yeast-derived HTS or variants thereof), for example, in Kluyveromyces (e.g., K. lactis), Lactococcus (e.g., L. lactis), Lactobacillus, Saccharomyces (e.g., S. cerevisiae), Pichia (e.g., P. pastoris), Hansenula (e.g., H. polymorpha), or Yarrowia (e.g., Y. lipolytica).

[0213] In another specific embodiment, the HTS (or variant thereof) is produced in a bacterial expression system (i.e., bacterial-derived HTS or variant thereof), for example, in E. coli or Bacillus subtilis. In one embodiment, the HTS (or variant thereof) is not produced in E. coli.

[0214] In other specific embodiments, the HTS (or variants thereof) is produced in an insect expression system (ie, insect-derived HTS or variants thereof), for example, in baculovirus-infected or non-lytic insect cells (eg, sf9, Sf21).

[0215] In another embodiment, the HTS (or variant thereof) is produced in a fungal expression system (i.e., a fungal-derived HTS or variant thereof), for example, in Chrysosporium, Thielavia, Talaromyces, Trichoderma, Thermomyces, or Thermoascus.

[0216] In another embodiment, the HTS (or its variant) is produced in a mammalian expression system (i.e., a mammalian-derived HTS or its variant), for example, in Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK), COS, and baby hamster kidney (BHK) cells. Alternatively, the HTS (or its variant) can be produced in vitro using a cell-free expression system such as an E. coli S30 extract.

[0217] purification

[0218] The recombinantly expressed polypeptide from the expression cassette encoding Myd is typically isolated from lysed cells or culture medium. Purification can be performed by methods known in the art, including salt fractionation, ion exchange chromatography, gel filtration, size exclusion chromatography, size fractionation, affinity chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing, and differential precipitation or solubilization. Immunoaffinity chromatography can be employed using antibodies raised against, for example, the Gag antigen.

[0219] 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 via hydrophobic interaction chromatography (HIC) followed by size exclusion chromatography (SEC).

[0220] Those skilled in the art are familiar with purification techniques of hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC), including the selection of appropriate columns, buffers, and elution solutions. Exemplary HIC and SEC purification techniques are described in Example 11 herein. In exemplary aspects, 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 ​​thereof.

[0221] In cell-based systems, the first step in the protein purification process is to extract the protein from the cells by lysing or disrupting the cells. Any suitable cell lysis method can be used, such as mechanical disruption, chemical decomposition, freeze-thaw cycles, or enzymatic digestion. The protein can then be purified by any suitable protein purification method, such as affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing, and differential precipitation or solubilization.

[0222] The yield of HTS (or its variant) produced in vivo may vary. In a specific embodiment, HTS accounts for at least about 1% of the total cellular protein. In a specific embodiment, HTS accounts for about 1% to about 5% of the total cellular protein. In another embodiment, HTS accounts for between about 5% and about 10%, or the total cellular protein. In other embodiments, HTS accounts for about 10% to about 20% of the total cellular protein. In certain embodiments, HTS accounts for more than 20% of the total cellular protein.

[0223] In another specific embodiment, HTS HTS is purified to provide a yield of about 1 mg / mL to about 200 mg / mL, more particularly, about 5 mg / mL to about 195 mg / mL, about 10 mg / mL to about 190 mg / mL, about 15 mg / mL to about 185 mg / mL, about 20 mg / mL to about 180 mg / mL, about 25 mg / mL to about 175 mg / mL, about 30 mg / mL to about 170 mg / mL, or about 35 mg / mL to about 165 mg / mL. In one embodiment, the HTS is purified to provide a yield of about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 75 mg / mL, about 100 mg / mL, about 125 mg / mL, about 150 mg / mL, about 175 mg / mL, or about 200 mg / L or more. Optionally, the HTS is produced as a fusion protein that also includes a tag, and the above yields reflect the purification and removal of the tag.

[0224] In certain embodiments, the HTS (or variant thereof) is substantially pure. In one embodiment, the HTS (or variant thereof) is at least about 80% pure, at least 85% pure, at least about 90% pure, at least about 95% pure, or at least about 99% pure. In another embodiment, the HTS (or variant thereof) is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% pure.

[0225] plant

[0226] The present invention also contemplates transgenic plants comprising heterologous polynucleotides and / or heterologous polypeptides of the present invention as described herein. Due to the expression of heterologous nucleic acid sequences, the plant has an altered phenotype. The altered phenotype may include a phenotype in which the sweetness of any plant part (including fruit) is increased. Transgenic plants may include an expression cassette as defined herein as part of the plant, which has been introduced by transforming the plant with a vector of the present invention. Such an expression cassette includes regulatory sequences for expressing heterologous coding sequences in the plant, including plant-expressible promoters and terminators. Transgenic plants may be any type of plant that can express 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. Plant species that can be used in the methods of the present invention are generally as broad as those of higher plants suitable for transformation techniques, including monocotyledonous (monocots) plants and dicotyledonous (dicots). They include plants of various ploidy levels, including polyploids, diploids, and haploids. For example, the transgenic plant may be an apple or a strawberry. The HTS proteins / polypeptides or variants of the present disclosure can be produced in plants or plant cultures.

[0227] The techniques for transforming a variety of plant species are well known in the art and are described in the 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, (Koh et al., eds) Springer Dordrecht Heidelberg New York London, Chapter 9, pages 297-344. Any method known in the art for transforming plant cells (including plant protoplasts or plant tissues) can be used for plant transformation. Specific methods for plant transformation include, in particular, bolistic methods (gene guns), electroporation, microinjection, protoplast fusion, and Agrobacterium-mediated transformation. Agrobacterium-mediated transformation can, for example, use binary vectors that replicate in Escherichia coli and Agrobacterium tumefaciens or other Agrobacterium strains. A variety of such binary vectors are known in the art and can be used to introduce heterologous polynucleotides into plant cells and plant tissues. Plant expression vectors comprising regulatory sequences (including plant-expressible promoter sequences and other plant regulatory sequences) for expressing heterologous coding sequences in plant cells and plant tissues are known in the art and can be used to transform plants to express polypeptides as described herein.

[0228] The promoter that can express of multiple plants is known in the art and can be used for the heterologous constructs herein, carriers and the plant material of transformation of the polynucleotide of coding with the protein of sweet taste regulating activity.The promoter that can express of plant can derive from natural plant origin, plant virus origin and the bacterium with the promoter that can express of plant, such as Agrobacterium strain.The promoter that can express of plant especially includes cauliflower mosaic virus promoter (CaMV 35S), octopine and nopaline synthase promoter (for example nos promoter), plant ubiquitin promoter (Ubi), rice actin promoter (Act-1) and corn alcohol dehydrogenase (Adh-1).The promoter that can express of plant includes constitutive promoter, inducible promoter, tissue-specific promoter, developmental stage specific promoter, and the example of every type of promoter is known in the art.Tissue-specific promoter is especially included in the promoter (for example, phosphoenolpyruvate promoter (PEP)) that directly expresses in the cell of plant root, plant leaf, fruit, flower, pollen or participation active photosynthesis. Developmental stage specific promoters include those that direct expression during fruit ripening, flowering or seed setting.Synthetic plant promoters are also known in the art and can be used for heterologous constructs, vectors and transformed plant materials (see, e.g., Ali S. & Kim WC (2019) Frontiers in Plant Science, 10, article 1433).

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

[0230] In an embodiment, the HTS (or variants thereof) is produced in an algal expression system (ie, an algal-derived HTS or variants thereof).

[0231] In an embodiment, the HTS (or variant thereof) is produced in a plant expression system (i.e., a plant-derived HTS or variant thereof), for example, in maize, corn, tobacco, melon (e.g., watermelon), potato, strawberry, duckweed, or sugarcane. In one embodiment, the plant expression system is a plant cell culture expression system.

[0232] In a specific embodiment, HTS (or its variant) is produced in maize, and more specifically, in maize seeds. In another specific embodiment, HTS (or its variant) is produced in corn, and more specifically, in corn seeds. According to these embodiments, HTS (or its variant) can be used as germ meal containing HTS.

[0233] Method for producing proteins with sweet taste modulating activity (in a host or cell-free expression system)

[0234] Methods commonly used in the art such as protoplast methods and electroporation can be used as methods for introducing the vector into the host. Target transformants can be obtained by selecting strains appropriately introduced with the vector using indicators such as marker gene expression and / or auxotrophy.

[0235] Alternatively, a fragment in which the polynucleotide encoding the Myd polypeptide variant of the present invention, the regulatory sequence, and the marker gene are linked can be directly introduced into the host's genome. For example, a DNA fragment with sequences complementary to the host genome appended to both ends of the linked fragment can be constructed, the fragment can be introduced into the host, and homologous recombination between the host genome and the DNA fragment can be induced by SOE-PCR to introduce the polynucleotide encoding the Myd polypeptide variant of the present invention into the host's genome.

[0236] The transformants thus obtained, into which the polynucleotide encoding the Myd polypeptide variant of the present invention or a vector comprising the same has been introduced, are cultured in an appropriate culture medium, resulting in expression of the MYD cDNA on the vector, and then producing the Myd polypeptide variant of the present invention. The culture medium for culturing such transformants can be appropriately selected by those skilled in the art according to the type of microorganism of the transformant.

[0237] Alternatively, a cell-free translation system can be used to express the Myd polypeptide variants of the present invention from a polynucleotide encoding the Myd polypeptide variants of the present invention or its transcription product. A "cell-free translation system" refers to an in vitro transcription and translation system or an in vitro translation system constructed by adding reagents required for protein translation, such as amino acids, to a suspension obtained by mechanically disrupting host cells.

[0238] Cell-free systems that can be used to produce HTS for use in the compositions described herein include, but are not limited to, protein expression components from eukaryotic, prokaryotic, and / or viral sources. For example, the cell-free systems used herein can include mammalian and / or bacterial protein expression systems derived from mammalian and / or bacterial lysates.

[0239] If desired, the Myd polypeptide variants of the present invention produced in culture or a cell-free translation system can be isolated or purified by using, alone or in combination, general methods for purifying proteins, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, and affinity chromatography. Here, when a gene encoding a Myd polypeptide variant of the present invention and a secretion signal sequence are operably linked to a vector within a transformant, the Myd polypeptide variant produced can be more easily collected from the culture because the Myd polypeptide variant is secreted outside the cells. The Myd polypeptide variant collected from the culture can be further purified by any known means.

[0240] In an embodiment, the Myd protein is dissolved in a liquid solution, such as, for example, a buffer solution or any solution that readily dissolves the Myd protein in solution. In an embodiment, the Myd protein contained in the liquid solution is lyophilized to form a powder. In an embodiment, the Myd protein contained in the liquid solution is dried to form a powder, such as, for example, by using a spray dryer. In an embodiment, spray drying comprises using a carrier known in the art. In an embodiment, the carrier used for spray drying is maltodextrin, gum arabic, or whey protein concentrate.

[0241] The present invention also includes methods for producing a protein having sweet taste modulating activity, comprising culturing a host cell of the present invention in a culture medium under conditions that result in the production of a protein having sweet taste modulating activity similar to a known sweet flavoring or compound.

[0242] Sweetened compositions—foods, beverages, supplements, pharmaceuticals

[0243] Disclosed herein are sweetener compositions and flavor-modulating compositions that each contain Myd (HTS) or a variant thereof. In certain embodiments, the sweetener compositions and flavor-modulating compositions alter (e.g., improve) one or more sensory experiences of a subject consuming them. In specific embodiments, the sweetener compositions and flavor-modulating compositions disclosed herein include HTS variants. In one embodiment, the HTS variant differs from wild-type HTS at at least one amino acid position, and more particularly, at one, two, three, or more amino acid positions.

[0244] As used herein, "sweet flavoring," "sweet compound," or "sweet receptor activating compound" refers to a composition that induces a detectable sweet taste in a subject, such as sucrose, fructose, glucose, and other known natural sugar-based sweeteners, or known artificial sweeteners such as saccharin, cyclamate, aspartame, etc., as discussed elsewhere herein, or a substance that activates T1R2 / T1R3 receptors in vitro. The subject can be a human or an animal.

[0245] Sweet flavoring agents or sweetening compositions can be used in an effective amount, which refers to an amount of the sweetening composition of the present invention sufficient to induce a sweet taste in a subject when present in a product for oral administration.

[0246] Embodiments of the present invention include compositions. In embodiments, the composition comprises, consists essentially of, or consists of: a product for oral administration and a combination of one or more sweetening compositions comprising an isolated Myd polypeptide variant according to the present invention as described herein. In embodiments, the combination has enhanced sweetness compared to an orally administered product lacking the Myd polypeptide variant (control). In one embodiment, the product for oral administration is not a truffle of the species Mydius spp. The term "consisting essentially of" allows for the inclusion of components that are not essential to and do not substantially affect the function or activity of the product, such as anti-caking agents, fillers, stabilizers (e.g., thermal stabilizers), and bulking agents (e.g., maltodextrin, gum arabic, etc.). In embodiments, the composition comprises a plurality of isolated Myd polypeptides. In specific embodiments, the composition comprises a plurality of isolated Myd polypeptides that are different from each other to enhance taste. It should be understood that compositions comprising one or more Myd polypeptides of the present invention are not limited by form, shape, or mode of administration, and encompass solid, liquid, powder, and other forms, individually or in combination with two or more thereof. Furthermore, the composition can be administered orally, by injection, etc., or eaten.

[0247] In another embodiment, a composition comprising an isolated Myd protein of the present invention comprises a formulation that provides enhanced functionality to the isolated Myd protein. For example, a composition can include 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 the Myd protein include, for example, L-arginine glycine, L-proline, L-histidine, β-alanine, L-serine, L-arginine ethyl ester dihydrochloride, L-arginine amide dihydrochloride, 6-aminocaproic acid, gly-gly, gly-gly-gly, tryptone, betaine monohydrate, D-(+)-trehalose dihydrate, xylitol, D-sorbitol, sucrose, hydroxyectoine, trimethylamine n-oxide dihydrate, methyl-α-d-pyranoside, triethylene glycol, spermine tetrahydrochloride, spermidine, 5-aminovaleric acid, glutaric acid, adipic acid, ethyl dihydrochloride, 1,2-di ... Diamine, guanidine hydrochloride, urea, N-methylurea, N-ethylurea, N-methylformamide, hypotaurine, TCEP hydrochloride, GSH (reduced l-glutathione), benzamidine hydrochloride, ethylenediaminetetraacetic acid disodium salt dihydrate, magnesium chloride hexahydrate, cadmium chloride hydrate, non-detergent sulfobetaine 195 (NDSB-195), non-detergent sulfobetaine 201 (NDSB-201), non-detergent sulfobetaine 211 (NDSB-211), non-detergent sulfobetaine 221 (NDSB-221), non-detergent 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, hydrated magnesium sulfate, potassium thiocyanate, gadolinium (III) chloride hexahydrate, cesium chloride, 4-aminobutyric acid (GABA), lithium nitrate, DL-malic acid pH 7.0, lithium citrate tetrahydrate, ammonium acetate, sodium benzenesulfonate, sodium p-toluenesulfonate, sodium chloride, potassium chloride, sodium dihydrogen phosphate monohydrate, sodium sulfate decahydrate, lithium chloride, bromide Sodium, glycerin, 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-propylene glycol, polyethylene glycol monomethyl ether 1,900, polyethylene glycol 3,350, polyethylene glycol 8,000, polyvinyl pyrrolidone k15, polyethylene glycol 20,000, (2-hydroxypropyl)-β-cyclodextrin, α-cyclodextrin, β-cyclodextrin, methyl-β-cyclodextrin.

[0248] In an embodiment, the sweetening composition comprises one or more Myd polypeptides as described above. In an embodiment, the sweetening composition comprises a plurality of Myd polypeptides as described above. In a specific embodiment, the plurality of Myd polypeptides are different from each other.

[0249] The present invention also includes a method for modifying the taste of an orally administered product comprising combining the orally administered product with an effective amount of an isolated Myd polypeptide variant, as described herein. In one aspect, the combination has an enhanced sweet taste compared to a product for oral administration lacking the Myd polypeptide variant (control). In one embodiment, the product for oral administration is not a Truffle.

[0250] The product for oral administration may be a food, a beverage, a dietary supplement composition, or a pharmaceutical composition.

[0251] The term "product for oral administration" can refer to an edible (comestible) product (consumable), such as a food, a beverage product; a pharmaceutical (drug) product, or a dietary supplement product such as an herbal supplement. As used herein, the term "consumable" can be used interchangeably with the term "one or more products for oral administration." As used herein, the term "pharmaceutical product" includes both solid and liquid compositions, which are ingestible non-toxic materials with pharmaceutical value or include pharmaceutically active agents such as cough syrups, cough drops, aspirin, and chewable pharmaceutical tablets. Oral hygiene products are also products for oral administration and include solids and liquids such as toothpaste or mouthwash. In general, the present invention contemplates that a food or beverage product can include an effective amount of the isolated sweet protein of the present invention, for example, in an amount of up to about 99% by weight relative to the gross weight of the food or beverage product, for example, in an amount of about 0.01% by weight to about 99% by weight. All intermediate weights, relative to the total weight of the food or beverage product, by weight (i.e., 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, ... 90%, 95%, 99%), and all intermediate ranges based on these amounts are contemplated. The compositions of the present invention may include an "edible, biologically or medically acceptable carrier or excipient," which may include a solid or liquid medium and / or composition for preparing the Myd polypeptide variant in the desired dosage form, so that the Myd polypeptide variant is administered in a dispersed / diluted form to maximize the biological efficacy of the Myd polypeptide variant. Edible, biologically or medically acceptable carriers include many common food ingredients, such as water at a neutral, acidic or alkaline pH, fruit or vegetable juice, 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, glycerides of fatty acids, and dispersions or emulsions of such hydrophobic substances in aqueous media, salts such as sodium chloride, wheat flour, solvents such as ethanol, solid edible diluents such as vegetable powders or powdered substances, or other liquid carriers, dispersing or suspending aids, surfactants, isotonic agents; thickeners or emulsifiers, preservatives, solid binders, lubricants, etc.

[0252] Medically acceptable carriers or excipients may include excipients that allow microencapsulation of Myd polypeptide variants to enhance functionality such as protection and prolonged sweetness perception. In fact, microencapsulation known in the art is a technology that, in addition to creating many possible new uses for sweeteners, can also promote conventional use. 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 are used to stabilize and / or alter (e.g., extend) the sweetness release of Myd. For example, sugar-free chewing gum and chewing candies typically have encapsulated sweeteners in their formulas to prolong their sweetness during chewing. It should be understood that food or beverage products and compositions comprising the one or more Myd polypeptides are not limited by form or shape and include solid, liquid, powder and other forms alone or in combination of two or more thereof.Examples of food or beverage products of the present invention include, but are not limited to, baked goods; sweet baked products (including but not limited to rolls, cakes, pies, pastries and cookies); pre-made sweet baking mixes for preparing sweet baked products; pie fillings and other sweet fillings (including but not limited to fruit pie fillings and nut pie fillings, such as pecan pie fillings, and fillings for cookies, cakes, pastries, confectionery products, etc., such as fat-based cream fillings); desserts, gelatin and puddings; frozen desserts (including 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, sorbet, etc.); carbonated beverages (including but not limited to soft carbonated beverages); non-carbonated beverages (including but not limited to soft non-carbonated beverages such as flavored waters and sweetened tea or coffee-based beverages); beverage concentrates (including but not limited to liquid concentrates and syrups and non-liquid concentrates and / or powdered preparations); yogurt (including but not limited to whole fat, low fat and skim yogurt, as well as non-dairy and lactose-free yogurt and frozen equivalents of all of these yogurts); snacks (including but not limited to cereal, nut, seed and / or fruit bars); bread products (including but not limited to leavened and unleavened bread, yeast and yeast-free bread, such as soda bread, bread comprising any type of wheat flour, bread comprising any type of non-wheat flour (such as potato, rice and rye flour), gluten-free bread); pre-made bread mixes for making bread products; spreads, syrups and condiments; sweet spreads (including but not limited to jellies, jams, butters, nut butters and other spreadable preserves, candied fruits, etc.); confectionery products (including but not limited to jelly candies, soft candies, hard candies, chocolates and chewing gums); sweetened breakfast cereals (including but not limited to extruded (KIX-type) breakfast cereals, flaked breakfast cereals and puffed breakfast cereals); cereal coating compositions for making sweetened breakfast cereals. Other types of food and beverage products not mentioned here but which typically include one or more nutritive sweeteners are also contemplated in the context of the present invention.

[0253] In an embodiment, the product for oral administration is a food product that is warmed or heated before consumption, or a food product for consumption in a warm or hot state. In an embodiment, the polypeptide variants exhibiting enhanced thermal stability as compared to the polypeptide of SEQ ID NO: 3 are preferably used in orally administered compositions (e.g., food products) that are to be cooked, warmed or heated before administration or consumption, or to be administered or consumed in a warm or hot state.

[0254] Due to the complete or partial replacement of nutritive sweeteners in the food or beverage products of the present invention, the food or beverage products of the present invention can be used as low-calorie or weight loss products, medical foods / products (including 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.

[0255] In some embodiments, the sweetening composition of the present invention can be supplemented with other nutritional or non-nutritional sweeteners to form a sweetener system. The sweetener system can include a sweetening composition of the present invention, a filler such as maltose glucose, gum arabic, etc., and at least one high-intensity sweetener. Said composition can be provided as a liquid composition or a dry blend.

[0256] As used herein, the term "high intensity sweetener" refers to any synthetic or semi-synthetic sweetener or sweetener found in nature. A high intensity sweetener is a compound or mixture of compounds that is sweeter than sucrose. A high intensity sweetener is typically many times sweeter than sucrose (e.g., 20 times and more, 30 times and more, 50 times and more, or 100 times and more).

[0257] In an embodiment, the invention includes a method for enhancing the sweetness of a product for oral administration comprising adding a Myd polypeptide variant of the invention.

[0258] In another embodiment, the method of the present invention includes a method for improving the sweetness of a product for oral administration, the method comprising adding a sweetening composition prepared by the method of the present invention to the product for oral administration. The amount added can be determined by methods known in the art, for example, using sensory testing as a guide.

[0259] In another embodiment, the method of the present invention includes a method for adjusting the product local flavor of oral administration, including adding a flavor adjustment composition prepared by the method of the present invention to the product of oral administration. The amount added can be determined by methods known in the art, for example, using sensory testing as a guide. The flavor adjustment composition can regulate (for example, enhance, suppress or change) a given composition, for example, taste, fragrance and / or texture of edibles. In a specific embodiment, the flavor adjustment composition regulates (for example, enhances, suppresses or changes) one or more specific tastes. In another embodiment, the flavor adjustment composition regulates (for example, enhances, suppresses or changes) a given texture. In certain embodiments, the flavor adjustment composition regulates (for example, enhances, suppresses or changes) one or more given tastes and textures.

[0260] The flavor-modulating composition can be sweetened or non-sweetened. Thus, in some embodiments, the addition of a flavor-modulating composition can be used to both add a flavor modifier and further sweeten the composition selected for flavor modulation. A sweetening flavor-modulating composition can be added in addition to or in lieu of another sweetening composition.

[0261] Sweetener compositions and flavor-modifying compositions disclosed herein contain Myd (HTS) and variants thereof. In certain embodiments, HTS (or its variants) is the only sweetening component in the sweetener composition or flavor-modifying composition. In certain embodiments, the sweetener composition or flavor-modifying composition further comprises one or more additional sweetening components (i.e., additional sweeteners or high-intensity sweeteners). In embodiments, the additional sweeteners are polypeptide or protein sweeteners other than HTS or its variants. In embodiments, the sweeteners are carbohydrate sweeteners. In embodiments, the additional sweeteners are synthetic sweeteners. In specific embodiments, the one or more sweetening components comprise steviol glycosides (e.g., Reb M, Reb A) and high fructose corn syrup (HFCS).

[0262] High fructose corn syrup (HFCS), also known as glucose-fructose, isoglucose, and glucose-fructose syrup, is a sweetener made from corn starch. Like regular corn syrup, the starch is broken down into glucose using enzymes.

[0263] Steviosides are compounds that are responsible for the sweet taste of the leaves of the plant Stevia rebaudiana and several related plants. Certain steviol glycosides are ingredients or precursors of ingredients in stevia sweeteners. Steviosides can be single compounds or mixtures of compounds. Steviosides also include, among others, stevioside, dulcoside A, rebaudioside A (Reb A), rebaudioside M (Reb M), rebaudioside B (Reb B), rebaudioside C (Reb C), rebaudioside D (Reb D), rebaudioside E, rebaudioside F, rubusoside, steviolbioside, and combinations thereof.

[0264] Mogrosides are glycosides of cucurbitane derivatives, including mogrol, which is associated with the sweet taste of Siraitia grosvenorii (monk fruit or luo han guo) extracts. Certain mogrosides are components of monk fruit sweeteners. Mogrosides include, among others, mogroside II A1, mogroside II B, 7-oxomogroside II E, 11-oxomogroside A1, mogroside III A2, 11-deoxymogroside III, 11-oxomogroside IV A, mogroside V, 7-oxomogroside V, 11-oxyylidene-mogroside V, mogroside VI, simenoside I, and combinations thereof. Preferred mogrosides are mogroside V, mogroside VI, and simenoside.

[0265] In one embodiment, the one or more other sweeteners can be carbohydrate sweeteners. The limiting examples of suitable carbohydrate sweeteners include sucrose, fructose, glucose, erythritol, maltitol, lactitol, sorbitol, mannitol, xylitol, D-tagatose, trehalose, galactose, rhamnose, cyclodextrin (e.g., α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin), ribulose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, trans- saccharide, isotrehalose, neotrehalose, palatinose or isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, glucosamine, mannosamine, fucose, fucose, glucuronic acid, gluconic acid, gluconolactone, abequose, galactosamine, oligoxylose (

[0014] Examples of the present invention include, but are not limited to, sucrose, maltotriose, maltobiose, maltohexaose, maltoheptaose, and maltotriose. These sugars may include, but are not limited to, oligosaccharides (e.g., xylotriose, xylobiose, etc.), gentiooligosaccharides (gentiobiose, gentiotriose, gentiotetraose, etc.), galacto-oligosaccharides, sorbose, ketotriose (dihydroxyacetone), aldotrose (glyceraldehyde), nigero-oligosaccharides, oligofructose (kestose, nesose, etc.), maltotetraose, maltotriitol, tetrasaccharides, oligomannose, isomalto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, etc.), dextrin, lactulose, melibiose, raffinose, rhamnose, ribose, isomerized liquid sugars such as high fructose corn / starch syrup (HFCS / HFSS) (e.g., HFCS55, HFCS42, or HFCS90), coupling sugars, soy oligosaccharides, glucose syrup, and combinations thereof.

[0266] In other embodiments, the at least one additional sweetener is a synthetic sweetener. As used herein, the term "synthetic sweetener" refers to any composition that is not naturally found in nature and characteristically has a sweetness greater than sucrose, fructose, or glucose but has fewer calories. Non-limiting examples of synthetic high-potency sweeteners suitable for use in embodiments of the present disclosure include sucralose, acesulfame potassium, aspartame, alitame, saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, advantame, glycosylated steviol glycosides (GSG), and combinations thereof.

[0267] In other embodiments, the at least one additional sweetener is a protein sweetener (i.e., a polypeptide or protein that tastes sweet). Typically, such protein sweeteners are extracted from plants. Non-limiting examples of protein sweeteners are monellin, thaumatin, brazilin, curculin, pentadin, and mabinlin.

[0268] In certain embodiments, the sweetener compositions and flavor-modulating compositions disclosed herein include a Myd (HTS) variant. In one embodiment, the HTS variant differs from wild-type HTS (with or without methionine at position 1) at at least one amino acid position, and more specifically, at one, two, three, or more amino acid positions.

[0269] In one embodiment, the sweetener compositions and flavor-modulating compositions disclosed herein include an HTS variant having a sweetness equal to or greater than that of a wild-type HTS having the amino acid sequence of SEQ ID NO:3.

[0270] In one embodiment, the sweetener compositions and flavor-modulating compositions disclosed herein include an HTS variant having a stability equal to or greater than that of a wild-type HTS having the amino acid sequence of SEQ ID NO:3.

[0271] In one embodiment, the sweetener compositions and flavor-modulating compositions disclosed herein include an HTS variant having a sweetness equal to or greater than that of a wild-type HTS having the amino acid sequence of SEQ ID NO: 3 without the methionine at position 1.

[0272] In one embodiment, the sweetener compositions and flavor-modulating compositions disclosed herein include an HTS variant having a stability equal to or greater than that of a wild-type HTS having the amino acid sequence of SEQ ID NO: 3 without the methionine at position 1.

[0273] HTS (or variants thereof) suitable for use in the compositions disclosed herein (e.g., sweetener compositions, flavor and / or taste-modifying compositions, edibles) can be produced in any suitable manner as described above herein. Representative production methods include extraction, chemical synthesis (i.e., solid-state synthesis), or recombinant production (i.e., in vivo or in vitro).

[0274] In one embodiment, the HTS used in the compositions disclosed herein is isolated from (i) the mycelial truffle of the edible family Terfeziaceae or an aqueous extract thereof, or (ii) an aqueous extract of the fruiting body of a truffle of the family Terfeziaceae. In one embodiment, the HTS is isolated from the mycelial truffle or the fruiting body of the truffle Terfeziaceae.

[0275] In another embodiment, the HTS (or variants thereof) used in the compositions disclosed herein is produced in vivo. In one embodiment, the nucleic acid coding sequence of the HTS isolated from the optionally optimized Truffle Truffle is introduced into a suitable vector and then cloned into a host cell in a suitable growth system / environment—resulting in recombinant expression of the protein. Suitable host cells and expression systems are previously described herein.

[0276] The amount of HTS (or variant thereof) in the sweetener compositions and flavor-modulating compositions disclosed herein can vary. In one embodiment, the HTS (or variant thereof) is present in the sweetener composition above its sweetness threshold concentration.

[0277] In one embodiment, HTS (or a variant thereof) is present in a sweetener composition or flavor-modifying composition in any amount to impart a desired sweetness when the sweetener composition or flavor-modifying component is added to a comestible (e.g., a beverage), either alone or in combination with one or more additional sweetening components (e.g., steviol glycosides, HFCS) present in the sweetener composition or flavor-modifying composition, i.e., before these compositions are added to the comestible.

[0278] In particular embodiments, the desired sweetness of the edible is the same as the sweetness of a sucrose-sweetened edible, having a sweetness of at least about 8 Brix, such as, for example, about 9 Brix, about 10 Brix, about 11 Brix, about 12 Brix, about 13 Brix, about 14 Brix, or about 15 Brix.

[0279] In another embodiment, the desired sweetness of the edible is the same as the sweetness of a sucrose-sweetened edible having a sweetness of about 10 to about 15 Brix, such as, for example, about 10 to about 14 Brix, about 10 to about 13 Brix, about 10 to about 12 Brix, about 10 to about 11 Brix, about 11 to about 15 Brix, about 1 1 Brix to about 14 Brix, about 11 Brix to about 13 Brix, about 11 Brix to about 12 Brix, about 12 Brix to about 15 Brix, about 12 Brix to about 14 Brix, about 12 Brix to about 13 Brix, about 13 Brix to about 15 Brix, about 13 Brix to about 14 Brix, and about 14 Brix to about 15 Brix.

[0280] In one embodiment, the Myd polypeptide (or variant thereof) is present in the sweetener composition or flavor-modifying composition in an amount that, alone or in combination with one or more additional sweet components (e.g., steviol glycosides, HFCS) present in the sweetener composition or flavor-modifying composition, enhances the sweetness of the comestible to which the compositions are added by about 1.0% (w / v) sucrose equivalent (SE) or more, prior to addition of the compositions to the comestible.

[0281] In particular embodiments, the Myd polypeptide (or variant thereof) is present in the sweetener composition in an amount, alone or in combination with one or more additional sweet components (e.g., steviol glycosides, HFCS) present in the sweetener composition or flavor-modifying composition, that enhances the sweetness of the comestible to which the compositions are added by about 1.0% to about 3.0% (w / v) sucrose equivalent (SE), such as, for example, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9% or about 3.0% sucrose equivalent, prior to addition of the compositions to the comestible.

[0282] In another specific embodiment, the Myd polypeptide (or variant thereof) is present in the sweetener composition or flavor-modifying composition in an amount, alone or in combination with one or more additional sweet components (e.g., steviol glycosides, HFCS) present in the sweetener composition or flavor-modifying composition, that enhances the sweetness of the comestible to which the compositions are added by about 3.0% to about 5% (w / v) sucrose equivalent (SE), e.g., about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9% or about 5.0%, prior to addition of the compositions to the comestible.

[0283] The sweetness of a given composition is typically measured with reference to a sucrose solution. See generally, "A Systematic Study of Concentration-Response Relationships of Sweeteners," G.E. DuBois, D.E. Walters, S.S. Schiffman, Z.S. Warwick, B.J. Booth, S.D. Pecore, K. Gibes, B.T. Carr, and L.M. Brands, Sweeteners: Discovery, Molecular Design and Chemoreception, D.E. Walters, F.T. Orthoefer and G.E. DuBois, Eds., American Chemical Society, Washington, D.C. (1991), pp 261-276.

[0284] The amount of sucrose in a reference solution can be described in degrees Brix (°Bx). One degree Brix is ​​1 gram of sucrose in 100 grams of solution and represents the strength of the solution expressed as a percentage by weight (% w / w) (strictly speaking, by mass).

[0285] In one embodiment, a sweetener composition is provided containing an amount of a Myd polypeptide (or variant thereof) that, when added to an edible, is effective to provide, alone or in combination with one or more sweet components (e.g., steviol glycosides, HFCS) present in the sweetener composition or flavor-modifying composition or edible, a sweetness equivalent of sugar of about 1 to about 12 degrees Brix, such as, for example, about 2 to about 9 degrees Brix, about 3 to about 8 degrees Brix, about 4 to about 7 degrees Brix, or about 5 degrees Brix.

[0286] In another embodiment, the Myd polypeptide (or variant thereof) is present in an amount effective to provide a sweetness equivalent of about 10 degrees Brix when added to a sweetenable composition, alone or in combination with the sweetener composition, flavor-modifying composition, or one or more sweet components (e.g., steviol glycosides, HFCS) present in an added comestible.

[0287] The sweetness of non-sucrose sweeteners can also be measured against sucrose reference by determining their sucrose equivalent. Typically, taste panelists are trained to detect the sweetness of reference sucrose solutions containing 1%-15% sucrose (w / v). Other non-sucrose sweeteners are then tasted with a series of dilutions to determine the concentration of the non-sucrose sweetener that is as sweet as a given sucrose reference percentage. For example, if a 1% sweetener solution is as sweet as a 10% sucrose solution, the sweetener is said to be 10 times as potent as sucrose.

[0288] In one embodiment, the amount of Myd polypeptide (or variant thereof) present in a sweetener composition or flavor-modifying composition disclosed herein is any amount that contributes to one or more improved sensory properties of an edible (e.g., a beverage) to which the sweetener composition or flavor-modifying composition is added. In a specific embodiment, the improved sensory properties are related to a base taste. In one embodiment, improving one or more sensory properties results in an improvement in the taste profile. The overall taste profile of a composition is an interaction of several different tastes, such as sweetness, sourness, saltiness, bitterness, umami, etc.

[0289] As used herein, "organoleptic properties" are aspects of food, water, or other substances that create a personal experience through the senses, including taste, sight, smell, and touch. Organoleptic properties include, for example, appearance, texture, color, smell, size, shape, and flavor. It is a qualitative assessment based on studies of the morphological and sensory characteristics of food, water, or other substances (such as, for example, sweetener compositions).

[0290] Examples of improved organoleptic properties can include, for example, a reduction in bitterness, a reduction in astringency and licorice, a slow onset of sweetness, a reduction in sweet aftertaste, a reduction in bitter aftertaste, a reduction in bitter aftertaste, a reduction in metallic aftertaste, a reduction in chemical and synthetic aftertaste, and combinations thereof. In particular embodiments, the term "improved organoleptic properties" means that a sweetened or flavor-modulated composition (e.g., a beverage) will have one or more improved organoleptic properties for most users. The improvement can be expressed qualitatively or quantitatively, for example, as a percentage improvement.

[0291] Improved sensory properties can be measured by or using technologies such as taste sensing systems (TSS), which refers to analytical sensor array units (e.g., electrochemical, gravimetric, optical or biosensors) that can detect specific substances Sliwi'nska, M et al. J. Agric. Food Chem. (2014), 62, 1423-1448.

[0292] In certain embodiments, the Myd polypeptide (or variant thereof) is present in any amount in the sweetener composition or flavor-modifying composition, alone or in combination with one or more sweet components (e.g., steviol glycosides, HFCS) in the sweetener composition or flavor-modifying composition, i.e., before addition to the sweetener or flavor-modifying composition, to reduce, suppress, or mask the bitterness of an edible (e.g., a beverage) to which the sweetener or flavor-modifying composition is added, as compared to an edible to which the sweetener or flavor-modifying composition is not added.

[0293] In certain embodiments, the Myd polypeptide (or variant thereof) is present in the sweetener composition or flavor-modifying composition in an amount, alone or in combination with one or more sweet components (e.g., steviol glycosides, HFCS) in the sweetener composition or flavor-modifying composition, such that, prior to addition to the edible (e.g., a beverage), the bitterness of the edible to which it is added is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, or at least about 25% or more. In one embodiment, the majority of subjects experienced a reduction in bitterness, as compared to an edible to which the sweetener or flavor-modifying composition was not added.

[0294] In certain embodiments, the Myd polypeptide (or variant thereof) is present in any amount in the sweetener composition or flavor-modifying composition, alone or in combination with one or more sweet components (e.g., steviol glycosides, HFCS) in the sweetener composition or flavor-modifying composition, that reduces the bitter aftertaste of an edible (e.g., a beverage) to which the sweetener or flavor-modifying composition is added, i.e., before it is added to the edible. In certain embodiments, the Myd polypeptide (or variant thereof) is present in an amount in the sweetener composition or flavor-modifying composition that reduces the bitter aftertaste of the edible (e.g., a beverage) to which it is added by at least about 5%, at least about 10%, at least about 15%, at least about 20%, or at least about 25% or more. In one embodiment, the majority of subjects experienced a reduction in the bitter aftertaste, as compared to an edible to which the sweetener or flavor-modifying composition was not added.

[0295] In another embodiment, the Myd polypeptide (or its variant) is present in the sweetener composition or taste-modifying composition in any amount that reduces the sweet aftertaste of an edible (e.g., a beverage) to which the sweetener or taste-modifying composition is added. Sucrose exhibits a sweetness in which the maximum response is perceived quickly and in which the perceived sweetness disappears relatively quickly upon swallowing the food or beverage. In contrast, the sweetness of essentially all high-potency sweeteners reaches its maximum response somewhat more slowly than with sucrose, and its intensity subsequently decreases more slowly. This decrease in sweetness is commonly referred to as "sweet aftertaste" and is a major limitation of high-potency sweeteners, including NHPSs. A slow onset of sweetness can also be a problem. However, generally speaking, the sweet aftertaste is a more serious problem. Therefore, preferred embodiments of the present invention exhibit a significant reduction in the sweet aftertaste.

[0296] In certain embodiments, the Myd polypeptide (or variant thereof) is present in the sweetener composition or flavor-modifying composition in an amount that, alone or in combination with one or more sweet components (e.g., steviol glycosides, HFCS) in the sweetener composition or flavor-modifying composition prior to addition to an edible, reduces the sweet aftertaste of the edible (e.g., a beverage) to which it is added by at least about 5%, at least about 10%, at least about 15%, at least about 20%, or at least about 25% or more. In one embodiment, the reduction in sweet aftertaste is perceived by a majority of subjects. In certain embodiments, the comparison is made to an edible to which the sweetener composition or flavor-modifying composition has not been added.

[0297] In certain embodiments, the Myd polypeptide (or variant thereof) is present in the sweetener composition or flavor-modulating composition in an amount that results in at least one change / modification in a sensory property of an edible (e.g., a beverage) compared to an edible not containing the sweetener composition, wherein the sensory property is selected from the group consisting of: aroma, flavor, base taste (sweet, sour, salty, bitter, or umami), aftertaste or aftertaste, temporal characteristics, mouthfeel, or a combination thereof. In this embodiment, the change or modification can be any perceived difference in one or more sensory properties, which may or may not be considered an improvement. For example, if the presence of a Myd polypeptide in an edible results in a flavor change from chocolate to caramel, this would be considered a "change," but not necessarily an "improvement."

[0298] In some embodiments, the sweetener composition or local flavor and / or taste adjustment composition contains one or more other sweeteners. In one embodiment, the presence of the other sweetener exceeds its sweetness threshold concentration. In some embodiments, the sweetener composition containing Myd and the one or more other sweeteners synergistically enhances the sweetness of the edibles to which the sweetener composition has been added. In one embodiment, the sweetness of the edibles is enhanced in a manner unexpected by those skilled in the art.

[0299] The additional sweetener can be any type of sweetener, such as a natural, non-natural or synthetic sweetener.

[0300] In at least one embodiment, the at least one additional sweetener is selected from natural sweeteners other than Stevia sweeteners. In another embodiment, the at least one additional sweetener is selected from synthetic high-potency sweeteners (SHPS).

[0301] In particular embodiments, the one or more additional sweeteners may be natural high-potency sweeteners (NHPS). Suitable natural high-potency sweeteners include, but are not limited to, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, dulcoside A, dulcoside B, rubusoside, stevia, steviol glycosides, mogroside IV, mogroside V, monk fruit sweetener, simenoside, monatin and its salts (monatin SS, RR, RS, SR), curculin, glycyrrhizic acid and its salts, thaumatin, monellin, mabinin, brazilin, hernandulcin, phyllodulcin, glycyphyllin, phloridzin, trilobatin, baiyunoside, osladin, polypodoside A, pterocaryoside A, The natural high-potency sweeteners include rebaudioside A, pterocaryoside B, mukurozioside, phlomisoside I, periandrin I, abrusoside A, steviolbioside, and cyclocarioside I. The natural high-potency sweeteners can be provided as pure compounds or, alternatively, as part of an extract. For example, rebaudioside A can be provided as a single compound or as part of a Stevia extract.

[0302] In one embodiment, the one or more additional sweeteners are selected from the group consisting of rebaudioside M, rebaudioside A, simanoside I, and mogroside V.

[0303] In certain embodiments, the sweetener compositions and / or flavor-modulating compositions of the present invention include a Myd polypeptide (or a variant thereof) and simenoside I.

[0304] In another specific embodiment, the sweetener composition and / or flavor-modulating composition of the present invention comprises a Myd polypeptide (or a variant thereof) and mogroside V.

[0305] In another embodiment, the one or more additional sweeteners are selected from the group consisting of rebaudioside D, rebaudioside N, rebaudioside O, rebaudioside E, steviol glycosides, steviolbiosides, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside I, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M2, rebaudioside D2, rebaudioside S, rebaudioside T, rebaudioside U, rebaudioside V, rebaudioside W, rebaudioside Z1, rebaudioside Z2, rebaudioside IX, enzymatically saccharified steviol glycosides, and combinations thereof.

[0306] In other embodiments, the one or more additional sweeteners are selected from the group consisting of mogroside IA, mogroside IE, 11-oxomogroside IA, mogroside II, mogroside II A, mogroside II B, mogroside II E, 7-oxomogroside II E, mogroside III, mogroside IIIe, 11-deoxymogroside III, mogroside IV, 11-oxomogroside IV, 11-oxomogroside IV A, 11-deoxymogroside V, 7-oxomogroside V, 11-oxomogroside V, isomogroside V, mogroside VI, mogroside alcohol, 11-oxomogroside alcohol, the 1,6-alpha isomer of simenoside I, Luo Han Guo extract, and combinations thereof.

[0307] In a particular embodiment, the one or more additional sweeteners are rebaudioside M (13-[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-ene-19-oleic acid-[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl) ester having the formula (I):

[0308]

[0309] Reb M can be provided in a purified or unpurified form, i.e., as part of a naturally occurring mixture containing Reb M. In one embodiment, Reb M can be obtained from a Stevia extract by any suitable purification method. Suitable purification methods are known in the art and include, but are not limited to, column chromatography, recrystallization, phase separation, extraction, high performance liquid chromatography, and combinations thereof.

[0310] In another embodiment, the additional sweetener is a steviol glycoside composition. An exemplary steviol glycoside composition is A95, which primarily contains Reb D and Reb M, and contains small amounts of one or more of the following: Reb E, Reb O, Reb N, Reb A, steviol glycosides, Reb C, and Reb B. Methods for obtaining A95 are provided in WO 2017 / 059414, which is incorporated herein by reference.

[0311] The amount of Reb M in a sweetener composition or taste-modifying composition can vary. In one embodiment, Reb M is present in the sweetener composition in any amount to impart a desired sweetness when the sweetener composition is added to an edible (e.g., a beverage). In a particular embodiment, the desired sweetness of the edible is greater than about 10 degrees Brix.

[0312] In one embodiment, the sweetener composition contains an amount of Reb M effective to provide a sweetness equivalent of about 1-12 Brix when added to a comestible (e.g., a beverage), such as, for example, about 2 to about 9 Brix, about 3 to about 8 Brix, about 4 to about 7 Brix, or about 5 Brix.

[0313] In particular embodiments, Reb M is present in an effective amount to provide a sucrose equivalence (SE) of about 8 or less, such as, for example, about 7, about 6.5, about 6, about 5.5, or about 5 SE.

[0314] In another specific embodiment, Reb M is present in an effective amount to provide a sucrose equivalence of about 8 or greater, such as, for example, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, or about 15.

[0315] In one embodiment, Reb M is present in the flavor-adjusting composition in any amount to impart a desired flavor when the flavor-adjusting composition is added to a flavor-adjustable composition (e.g., a beverage). In a particular embodiment, the desired flavor is a more sugar-like temporal or taste profile.

[0316] In one embodiment, Reb M and Myd polypeptide (or variants thereof) produce a synergistic effect, such as synergistic sweetness, i.e., the sweetness of the combination is greater than the sum of the individual sweeteners. In a specific embodiment, Reb M and Myd polypeptide (or variants thereof) produce an effect that would be unexpected to one skilled in the art.

[0317] Reb M can be provided in a purified form or as a component of a mixture containing Reb M and one or more additional components. In one embodiment, Reb X is provided as a component of a mixture. In a specific embodiment, the mixture is a Stevia extract. The Stevia extract can contain Reb M in an amount ranging from about 5% to about 100% by weight on a dry basis, such as, for example, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, and about 90% to about 100%. In other embodiments, the Stevia extract includes Reb M in an amount greater than about 90%, e.g., greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, and greater than about 99% by weight on a dry basis.

[0318] In one embodiment, Reb M is provided as a component of a steviol glycoside mixture, i.e., a mixture of steviol glycosides, wherein the remainder of the mixture other than the Reb M portion consists entirely of steviol glycosides. The identity of steviol glycosides is known in the art and includes, but is not limited to, steviol, steviol glycoside, rubososide, steviolbioside, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, and dulcoside A. The steviol glycoside mixture may contain from about 5% to about 100% Reb M by weight, on a dry basis. For example, the steviol glycoside mixture may contain from about 10% to about 100% by weight on a dry basis, such as, for example, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, and from about 90% to about 100% Reb M. In other embodiments, the steviol glycoside mixture may contain greater than about 90% by weight on a dry basis, such as greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, and greater than about 99% Reb M.

[0319] Reb M80 refers to a Stevia extract or steviol glycoside composition having about 80% Reb M by weight.

[0320] In particular embodiments, the one or more additional sweeteners is rebaudioside A.

[0321] Reb A can be provided in a purified or unpurified form, i.e., as part of a naturally occurring mixture containing Reb A. In one embodiment, Reb A can be obtained from a Stevia extract by any suitable purification method. Suitable purification methods are known in the art and include, but are not limited to, column chromatography, recrystallization, phase separation, extraction, high performance liquid chromatography, and combinations thereof.

[0322] Reb A can be provided in a purified form or as a component of a mixture containing Reb A and one or more additional components. In one embodiment, Reb A is provided as a component of a mixture. In a particular embodiment, the mixture is a Stevia extract. The Stevia extract can contain Reb A in an amount ranging from about 5% to about 100% by weight on a dry basis, such as, for example, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, and about 90% to about 100%. In other embodiments, the Stevia extract includes Reb A in an amount greater than about 90%, e.g., greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, and greater than about 99% by weight on a dry basis.

[0323] In one embodiment, Reb A is provided as a component of a steviol glycoside mixture, i.e., a mixture of steviol glycosides, wherein the remainder of the mixture other than the Reb A portion is composed entirely of steviol glycosides. The steviol glycoside mixture may contain from about 5% to about 100% by weight of Reb A on a dry basis. For example, the steviol glycoside mixture may contain from about 10% to about 100% by weight of Reb A on a dry basis, such as, for example, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, and from about 90% to about 100%. In other embodiments, the steviol glycoside mixture may contain greater than about 90%, e.g., greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, and greater than about 99% Reb A by weight on a dry basis.

[0324] The amount of Reb A in a sweetener composition or taste-modifying composition can vary. In one embodiment, Reb A is present in the sweetener composition in any amount to impart a desired sweetness when the sweetener composition is added to a composition that can increase the sweetness. In a particular embodiment, the desired sweetness of the sweetened composition is greater than about 10 degrees Brix.

[0325] In one embodiment, Reb A and a Myd polypeptide (or a variant thereof) produce a synergistic effect, such as synergistic sweetness, i.e., the sweetness of the combination is greater than the sum of the individual sweeteners. In certain embodiments, Reb A and the Myd polypeptide (or a variant thereof) produce an effect that would not be expected by one skilled in the art.

[0326] In particular embodiments, Reb A is present in an effective amount to provide a sucrose equivalence (SE) of about 8 or less, such as, for example, about 7, about 6.5, about 6, about 5.5, or about 5 SE.

[0327] In another specific embodiment, Reb A is present in an effective amount to provide a sucrose equivalent (SE) of about 8 or greater, such as, for example, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, or about 15.

[0328] In another specific embodiment, Reb A is present in an effective amount to provide a sucrose equivalent of about 8 SE, e.g., about 9 SE, about 9.5 SE, about 10 SE.

[0329] In one embodiment, Reb A is present in the flavor-adjusting composition in any amount to impart a desired taste when the flavor-adjusting composition is added to a taste-adjustable composition (e.g., a beverage). In a particular embodiment, the desired taste is a sugar-like taste.

[0330] In a specific embodiment, the Myd polypeptide (or variant thereof) and Reb A produce a synergistic effect. In one embodiment, the Myd polypeptide (or variant thereof) and Reb A produce an effect that is unexpected to one skilled in the art.

[0331] The sweetener compositions can be tailored to achieve a desired calorie content. For example, the sweetener compositions can be "high calorie" such that when added to a composition that can increase sweetness (such as, for example, a beverage), they impart a desired sweetness and have approximately 120 calories per 8 oz serving.

[0332] The sweetener compositions can be tailored to achieve a desired calorie content. For example, the sweetener compositions can be "mid-calorie" such that when added to a composition that can be sweetened (such as, for example, a beverage), they impart a desired sweetness and have approximately 80 calories per 8 oz serving.

[0333] For example, the sweetener compositions can be "low calorie" such that when added to a sweetenable composition (such as, for example, a beverage), they impart a desired sweetness and have less than 40 calories per 8 oz serving.

[0334] In other embodiments, the sweetener compositions may be "zero calorie," such that when added to a sweetenable composition (such as, for example, a beverage), they impart a desired sweetness and have less than 5 calories per 8 oz serving.

[0335] additive

[0336] In addition to the Myd (HTS) polypeptide (or variant thereof) and optionally one or more additional sweeteners (e.g., one or more steviol glycosides), the sweetener compositions or flavor-modifying compositions disclosed herein may optionally include additional additives, as described below. In some embodiments, the sweetener compositions contain additives including, but not limited to, carbohydrates, polyols, amino acids and their corresponding salts, polyamino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavorings and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers, and combinations thereof. In some embodiments, the additives are used to improve the temporal and flavor profile of the sweetener to provide a sweetener composition that tastes similar to sucrose.

[0337] In one embodiment, the sweetener composition or flavor adjustment composition contains one or more polyols. The term "polyol" as used herein refers to a molecule containing more than one hydroxyl group. A polyol can be a diol, triol or tetraol containing 2, 3 and 4 hydroxyl groups, respectively. A polyol can also contain more than 4 hydroxyl groups, such as amyl alcohol, hexanol, heptanol, etc., which contain 5, 6 or 7 hydroxyl groups, respectively. In addition, a polyol can also be a sugar alcohol, a polyhydroxy alcohol or a polyol, which is a reduced form of a carbohydrate, wherein the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary hydroxyl group or a secondary hydroxyl group.

[0338] In some embodiments, non-limiting examples of polyols include erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, isomalt, propylene glycol, glycerol (glycerin), threitol, galactitol, palatinose, reduced isomaltooligosaccharides, reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup and sugar alcohols, or any other carbohydrate that can be reduced without adversely affecting the taste.

[0339] Suitable amino acid additives for improving sweet taste include but are not limited to aspartic acid, arginine, glycine, glutamic acid, proline, threonine, theanine, cysteine, cystine, alanine, valine, tyrosine, leucine, arabinose, trans-4-hydroxyproline, isoleucine, asparagine, serine, lysine, histidine, ornithine, methionine, carnitine, aminobutyric acid (α-isomer, β-isomer and / or δ-isomer), glutamine, hydroxyproline, taurine, norvaline, sarcosine, and above salt forms, such as sodium salt or potassium salt or acid salt. The amino acid additives for improving sweet taste can also be D or L configuration, and the single, two or three forms of identical or different amino acids. In addition, if suitable, amino acid can be α-isomer, β-isomer, γ-isomer and / or δ-isomer. In some embodiments, the combination of the above-mentioned amino acid and its corresponding salt (for example, its sodium, potassium, calcium, magnesium salt or other alkali metal or alkaline earth metal salt, or acid salt) is also a suitable additive for improving sweet taste.Amino acid can be natural or synthetic.Amino acid can also be modified.The amino acid of modification refers to any amino acid (for example, N-alkyl amino acid, N-acyl amino acid or N-methyl amino acid) in which at least one atom is added, removed, replaced or the above combination.The limiting examples of the amino acid of modification include amino acid derivatives, such as trimethylglycine, N-methylglycine and N-methylalanine.As used herein, the amino acid of modification includes modified and unmodified amino acid.As used herein, amino acid also includes peptides and polypeptides (for example, dipeptides, tripeptides, tetrapeptides and pentapeptides), such as glutathione and L-alanyl-L-glutamine. Suitable polyamino acid additives for improving sweet taste include poly-L-aspartic acid, poly-L-lysine (e.g., poly-L-α-lysine or poly-L-ε-lysine), poly-L-ornithine (e.g., poly-L-□α-ornithine or poly-L-□ε-ornithine), poly-L-arginine, other polymer forms of amino acids and the above salt forms (e.g., calcium, potassium, sodium or magnesium salts, such as monosodium L-glutamate). The polyamino acid additives for improving sweet taste can also be D-configuration or L-configuration. In addition, if appropriate, the polyamino acid can be an α-isomer, β-isomer, γ-isomer, δ-isomer and ε-isomer. In some embodiments, a combination of the above-mentioned polyamino acids and their corresponding salts (e.g., sodium, potassium, calcium, magnesium salts or other alkali metal or alkaline earth metal salts, or acid salts) is also a suitable additive for improving sweet taste. The polyamino acids described herein can also include copolymers of different amino acids. The polyamino acids can be natural or synthetic. The polyamino acid can also be modified such that at least one atom is added, removed, substituted, or a combination thereof (eg, N-alkyl polyamino acids or N-acyl polyamino acids). As used herein, polyamino acids include modified and unmodified polyamino acids.For example, modified polyamino acids include, but are not limited to, polyamino acids of various molecular weights (MW), such as poly-L-α-lysine of MW 1,500, MW 6,000, MW 25,200, MW 63,000, MW 83,000, or MW 300,000.

[0340] Suitable sugar acid additives include, but are not limited to, aldonic acid, uronic acid, saccharic acid, alginic acid, gluconic acid, glucuronic acid, glucaric acid, galactaric acid, galacturonic acid, and salts thereof (e.g., sodium, potassium, calcium, magnesium salts or other physiologically acceptable salts), and combinations thereof.

[0341] Suitable nucleotide additives include, but are not limited to, inosine monophosphate ("IMP"), guanosine monophosphate ("GMP"), adenosine monophosphate ("AMP"), cytosine monophosphate (CMP), uracil monophosphate (UMP), inosine diphosphate, guanosine diphosphate, adenosine diphosphate, cytosine diphosphate, uracil diphosphate, inosine triphosphate, guanosine triphosphate, adenosine triphosphate, cytosine triphosphate, uracil triphosphate and the above alkali metal or alkaline earth metal salts, and the above combinations. The nucleotides described herein can also include nucleotide-related additives, such as nucleosides or nucleic acid bases (e.g., guanine, cytosine, adenine, thymine, uracil). In a particular embodiment, the nucleotides are present in the sweetener composition in an amount of about 5ppm to about 1000ppm.

[0342] Suitable organic acid additives include any compound containing a -COOH moiety, such as, for example, C2-C30 carboxylic acids, substituted hydroxy C2-C30 carboxylic acids, benzoic acid, substituted benzoic acid (e.g., 2,4-dihydroxybenzoic acid), substituted cinnamic acids, hydroxy acids, substituted hydroxybenzoic acids, substituted cyclohexylcarboxylic acids, tannic acid, lactic acid, tartaric acid, citric acid, gluconic acid, glucoheptonic acid, adipic acid, hydroxycitric acid, malic acid, a mixture of fruitaric acid (malic acid, fumaric acid, and tartaric acid), fumaric acid, maleic acid, succinic acid, chlorogenic acid, salicylic acid, creatine, caffeic acid, bile acid, acetic acid, ascorbic acid, alginic acid, isoascorbic acid, polyglutamic acid, glucono delta lactone, and alkali metal or alkaline earth metal salt derivatives thereof. Additionally, the organic acid additive may also be in the D- or L- configuration.

[0343] Suitable organic acid additive salts include, but are not limited to, sodium, calcium, potassium, and magnesium salts of all organic acids such as citric acid, malic acid, tartaric acid, fumaric acid, lactic acid (e.g., sodium lactate), alginic acid (e.g., sodium alginate), ascorbic acid (e.g., sodium ascorbate), benzoic acid (e.g., sodium benzoate or potassium benzoate), and adipic acid. The examples of the described organic acid additives for improving sweet taste may optionally be substituted with at least one group selected from hydrogen, alkyl, alkenyl, alkynyl, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amide, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, sulfhydryl, imine, sulfonyl, sulfenyl, sulfinyl, sulfamyl, carboxyalkoxy, carboxamido, phosphono, phosphinyl, phosphoryl, phosphino, thioether, thioether, anhydride, oxime, hydrazine, carbamoyl, phosphorus or phosphonato. In a specific embodiment, the organic acid additive is present in the sweetener composition in an amount of about 10 ppm to about 5,000 ppm.

[0344] Suitable inorganic acid additives include, but are not limited to, phosphoric acid, phosphorous acid, polyphosphoric acid, hydrochloric acid, sulfuric acid, carbonic acid, sodium dihydrogen phosphate, and alkali metal or alkaline earth metal salts thereof (eg, inositol hexaphosphate Mg / Ca).

[0345] Suitable bitter compound additives include, but are not limited to, caffeine, quinine, urea, bitter orange oil, naringin, quassia, and salts thereof.

[0346] Suitable flavorings and flavoring ingredient additives include, but are not limited to, vanillin, vanilla extract, mango extract, cinnamon, citrus, coconut, ginger, viridiflorol, almonds, menthol (including mint-free menthol), grape skin extract, and grape seed extract. "Flavoring" and "flavoring ingredient" are synonymous and may include natural or synthetic substances or combinations thereof. Flavorings also include any other flavor-imparting substance and may include natural or non-natural (synthetic) substances that are safe for humans or animals when used within generally accepted limits. Non-limiting examples of proprietary flavorings include Natural Flavoring Sweetness Enhancer K14323( Darmstadt, Germany), Symrise TMNatural Flavor Mask for Sweeteners 161453 and 164126 (Symrise TM ,Holzminden,Germany),Natural Advantage TM Bitterness Blockers 1, 2, 9 and 10 (Natural Advantage TM ,Freehold,NJ,USA), and Sucramask TM (Creative Research Management, Stockton, Calif., USA).

[0347] Suitable polymer additives include, but are not limited to, chitosan, pectin, pectic acid, pectinic acid, polyuronic acid, polygalacturonic acid, starch, food hydrocolloids or crude extracts thereof (e.g., gum acacia senegal (Fibergum TM ), gum acacia seyal, carageenan), poly-L-lysine (e.g., poly-L-α-lysine or poly-L-ε-lysine), poly-L-ornithine (e.g., poly-L-α-ornithine or poly-L-ε-ornithine), polypropylene glycol, polyethylene glycol, poly(ethylene glycol methyl ether), polyarginine, polyaspartic acid, polyglutamic acid, polyethyleneimine, alginic acid, sodium alginate, propylene glycol alginate and polyethylene glycol sodium alginate, sodium hexametaphosphate and its salts, and other cationic and anionic polymers.

[0348] Other suitable polymer additives that also provide gelling and thickening properties include conventional low methoxyl (LMC) pectin. LMC pectin can also be used as a food stabilizer. LMC apple pectin is a conventional low methoxyl pectin extracted from apple pomace and standardized with sucrose. It is used in low-calorie jams and jellies because it relies on calcium rather than sugar for solidification. As calcium is added, LMC pectin becomes harder and harder until it reaches its saturation point. At that point, the process reverses and it becomes less hard. In some embodiments, the orally administered products described herein may include one or more food products selected from the group consisting of: pie fillings and other sweet fillings, gelatin and puddings; yogurt; sauces, syrups and condiments; and sweet spreads. In one embodiment, the orally administered product also includes low methoxyl pectin.

[0349] Suitable protein or protein hydrolysate additives include, but are not limited to, bovine serum albumin (BSA), whey protein (including fractions or concentrates thereof, such as 90% instant whey protein isolate, 34% whey protein, 50% hydrolyzed whey protein, and 80% whey protein concentrate), soluble rice protein, soy protein, protein isolate, protein hydrolysate, reaction products of protein hydrolysates, glycoproteins and / or proteoglycans containing amino acids (e.g., glycine, alanine, serine, threonine, asparagine, glutamine, arginine, valine, isoleucine, leucine, norvaline, methionine, proline, tyrosine, hydroxyproline, etc.), collagen (e.g., gelatin), partially hydrolyzed collagen (e.g., hydrolyzed fish collagen), and collagen hydrolysate (e.g., porcine collagen hydrolysate).

[0350] Suitable surfactant additives include, but are not limited to, polysorbates (e.g., polyoxyethylene sorbitan monooleate (polysorbate 80), polysorbate 20, polysorbate 60), sodium dodecylbenzenesulfonate, dioctyl sulfosuccinate or sodium dioctyl sulfosuccinate, sodium lauryl sulfate, cetylpyridinium chloride (hexadecylpyridinium chloride), cetyltrimethylammonium bromide, sodium cholate, carbamoyl, choline chloride, sodium glycocholate, sodium taurodeoxycholate, lauroyl arginate, sodium stearoyl lactylate, sodium taurocholate, lecithin, sucrose oleate, sucrose stearate, sucrose palmitate, sucrose laurate, and other emulsifiers.

[0351] Suitable flavonoid additives are classified as flavonols, flavones, flavanones, flavan-3-ols, isoflavones or anthocyanidins. Non-limiting examples of flavonoid additives include, but are not limited to, catechins (e.g., green tea extracts such as Polyphenon TM 60. Polyphenon TM 30 and Polyphenon TM 25 (Mitsui Norin Co., Ltd., Japan), polyphenols, rutin (e.g., enzyme-modified rutin Sanmelin TM AO (San-fi Gen FFI, Inc., Osaka, Japan)), neohesperidin, naringin, neohesperidin dihydrochalcone, etc.

[0352] Suitable alcohol additives include, but are not limited to, ethanol.

[0353] Suitable astringent compound additives include, but are not limited to, tannic acid, europium chloride (EuCl3), gadolinium chloride (GdCl3), terbium chloride (TbCl3), alum, tannic acid, and polyphenols (e.g., tea polyphenols). In a particular embodiment, the astringent additive is present in an amount of about 10 ppm to about 5,000 ppm.

[0354] In a particular embodiment, a sweetener composition or a flavor-modulating composition comprises the Myd polypeptide (or variant thereof), optionally in combination with one or more steviol glycosides (e.g., Reb M, Reb A), a polyol selected from erythritol, maltitol, mannitol, xylitol, sorbitol, and combinations thereof; and optionally at least one additional sweetener and / or functional ingredient. In a particular embodiment, the polyol is erythritol. The steviol glycoside (e.g., Reb M, Reb A) can be provided as a pure compound or as part of a stevia extract or steviol glycoside mixture, as described above. The steviol glycoside (e.g., Reb M, Reb A) is present in the steviol glycoside mixture or stevia extract in an amount of about 5% to about 100% by weight, on a dry basis.

[0355] In certain embodiments, the sweetener composition or flavor-modifying composition comprises the Myd polypeptide (or variant thereof), optionally in combination with one or more steviol glycosides (e.g., Reb M, Reb A); a carbohydrate sweetener selected from sucrose, fructose, glucose, maltose, and combinations thereof; and optionally at least one additional sweetener and / or functional ingredient. The steviol glycosides (e.g., Reb M, Reb A) can be provided as pure compounds or as part of a stevia extract or steviol glycoside mixture, as described above. The steviol glycosides (e.g., Reb M, Reb A) are present in the steviol glycoside mixture or stevia extract in an amount of about 5% to about 100% by weight, on a dry basis.

[0356] In certain embodiments, the sweetener composition or flavor-modulating composition comprises the Myd polypeptide (or variant thereof), optionally in combination with one or more steviol glycosides (e.g., Reb M, Reb A); an amino acid selected from glycine, alanine, proline, and combinations thereof; and optionally at least one additional sweetener and / or functional ingredient. The steviol glycoside can be provided as a pure compound or as part of a stevia extract or steviol glycoside mixture, as described above. The steviol glycoside (e.g., Reb M, Reb A) is present in the steviol glycoside mixture or stevia extract in an amount of about 5% to about 100% by weight, on a dry basis.

[0357] In certain embodiments, a sweetener composition or flavor-modulating composition comprises the Myd polypeptide (or variant thereof), optionally in combination with one or more steviol glycosides (e.g., Reb M, Reb A), a salt selected from sodium chloride, magnesium chloride, potassium chloride, calcium chloride, and combinations thereof; and optionally at least one additional sweetener and / or functional ingredient. The steviol glycosides (e.g., Reb M, Reb A) can be provided as pure compounds or as part of a Stevia extract or steviol glycoside mixture, as described above.

[0358] Functional ingredients

[0359] The sweetener compositions or flavor-modulating compositions disclosed herein may also contain one or more functional ingredients that provide real or perceived health benefits to the compositions. Functional ingredients include, but are not limited to, saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydrating agents, probiotics, prebiotics, weight management agents, osteoporosis management agents, phytoestrogens, long-chain aliphatic saturated primary alcohols, phytosterols, and combinations thereof.

[0360] Examples of suitable antioxidants for use in embodiments of the present invention include, but are not limited to, vitamins, vitamin cofactors, minerals, hormones, carotenoids, carotenoid terpenes, non-carotenoid terpenes, flavonoids, flavonoid polyphenols (e.g., bioflavonoids), flavonols, flavonoids, phenols, polyphenols, phenolic esters, polyphenolic esters, non-flavonoid phenols, isothiocyanates, and combinations thereof. In some embodiments, the antioxidant is vitamin A, vitamin C, vitamin E, ubiquinone, the mineral selenium, manganese, melatonin, α-carotene, β-carotene, lycopene, lutein, zeanthin, cryptoxanthin, resveratol, eugenol, quercetin, catechins, gossypol, hesperetin, curcumin, ferulic acid, thymol, hydroxytyrosol, turmeric, thyme, olive oil, lipoic acid, glutathione, glutamine, oxalic acid, a compound derived from tocopherols, Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediaminetetraacetic acid (EDTA), butylhydroquinone, acetic acid, pectin, tocotrienols, tocopherol, coenzyme Q10, zeaxanthin, astaxanthin, canthaxanthin, saponins, limonoids, kaempfedrol, myricetin, isorhamnetin, proanthocyanidin, quercetin, rutin, luteolin, apigenin, tangeritin, hesperetin, naringenin, erodichol tyol), flavan-3-ols (e.g., anthocyanidins), gallocatechin, epicatechin and its gallate forms, epigallocatechin and its gallate forms (ECGC), theaflavins and its gallate forms, thearubigins, isoflavone phytoestrogens, genistein, daidzein, glycitein, anthocyanidins, cyaniding, delphinidin, malvidin, pelargonidin, peonidin, petunidin In some embodiments, the antioxidant is a synthetic antioxidant such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHT), citric acid, lignans, anti-nutrients, bilirubin, uric acid, R-α-lipoic acid, N-acetylcysteine, emblicanin, and phytic acid or a combination thereof.Other sources of suitable antioxidants for use in embodiments of the present invention include, but are not limited to, fruits, vegetables, tea, cocoa, chocolate, spices, herbs, rice, organ meats from livestock, yeast, whole grains or cereals.

[0361] Specific antioxidants belong to the class of phytonutrients known as polyphenols (also known as "polyphenolics"), which are a group of chemicals found in plants characterized by the presence of more than one phenolic group per molecule. Polyphenols suitable for use in embodiments of the present invention include catechins, proanthocyanidins, proanthocyanidins, anthocyanidin glycosides, quercetin, rutin, resveratrol, isoflavones, curcumin, punicalagin, ellagitannins, hesperidin, naringin, citrus flavonoids, chlorogenic acid, other similar substances, and combinations thereof.

[0362] In a specific embodiment, the antioxidant is a catechin, such as, for example, epigallocatechin gallate (EGCG). Suitable sources of catechins for embodiments of the present invention include, but are not limited to, green tea, white tea, black tea, oolong tea, chocolate, cocoa, red wine, grape seeds, red grape skins, purple grape skins, red grape juice, purple grape juice, berries, pycnogenol, and red apple peels.

[0363] In some embodiments, the antioxidant is selected from proanthocyanidins, proanthocyanidins, or a combination thereof. Suitable sources of proanthocyanidins and proanthocyanidins for use in embodiments of the present invention include, but are not limited to, red grapes, purple grapes, cocoa, chocolate, grape seeds, red wine, cocoa beans, cranberries, apple peels, plums, blueberries, black currants, chokeberries, green tea, sorghum, cinnamon, barley, red kidney beans, pinto beans, hops, almonds, hazelnuts, pecans, pistachios, pycnogenol, and colored berries.

[0364] In a particular embodiment, the antioxidant is an anthocyanin. Suitable sources of anthocyanins for use in embodiments of the present invention include, but are not limited to, red raspberries, blueberries, bilberries, cranberries, raspberries, cherries, pomegranates, strawberries, elderberries, chokeberries, red grape skins, purple grape skins, grape seeds, red wine, black currants, red currants, cocoa, plums, apple peels, peaches, red pears, red cabbage, red onions, red oranges, and blackberries.

[0365] In some embodiments, the antioxidant is selected from quercetin, rutin, or a combination thereof. Suitable sources of quercetin and rutin for embodiments of the present invention include, but are not limited to, red apple, onion, kale, bog bilberry, lingonberry, rowan berry, cranberry, blackberry, blueberry, strawberry, raspberry, blackcurrant, green tea, black tea, plum, apricot, parsley, leek, broccoli, pepper, berry wine, and ginkgo.

[0366] In some embodiments, the antioxidant is resveratrol. Suitable sources of resveratrol for use in embodiments of the present invention include, but are not limited to, red grapes, peanuts, cranberries, blueberries, bilberries, mulberries, Japanese knotweed tea, and red wine.

[0367] In a particular embodiment, the antioxidant is an isoflavone. Suitable sources of isoflavones for use in embodiments of the present invention include, but are not limited to, soybeans, soy products, beans, alfalfa sprouts, chickpeas, peanuts, and red clover.

[0368] In some embodiments, the antioxidant is curcumin. Suitable sources of curcumin for use in embodiments of the present invention include, but are not limited to, turmeric and mustard.

[0369] In a particular embodiment, the antioxidant is selected from punicalagin, ellagitannin, or a combination thereof. Suitable sources of punicalagin and ellagitannin for use in embodiments of the present invention include, but are not limited to, pomegranate, raspberry, strawberry, walnut, and oak-aged red wine.

[0370] In some embodiments, the antioxidant is a citrus flavonoid, such as hesperidin or naringin. Suitable sources of citrus flavonoids such as hesperidin or naringin for embodiments of the present invention include, but are not limited to, oranges, grapefruits, and citrus juice.

[0371] In a specific embodiment, the antioxidant is chlorogenic acid. Suitable sources of chlorogenic acid for use in embodiments of the present invention include, but are not limited to, green coffee, yerba mate, red wine, grape seeds, red grape skins, purple grape skins, red grape juice, purple grape juice, apple juice, cranberries, pomegranates, blueberries, strawberries, sunflowers, Echinacea, pycnogenol, and apple peels.

[0372] Suitable dietary fibers include, but are not limited to, non-starch polysaccharides, lignin, cellulose, methylcellulose, hemicellulose, beta-glucan, pectin, gums, mucilages, waxes, inulin, oligosaccharides, oligofructose, cyclodextrins, chitin, and combinations thereof.

[0373] Food sources of dietary fiber include, but are not limited to, cereals, beans, fruits, and vegetables. Cereals that provide dietary fiber include, but are not limited to, oats, rye, barley, and wheat. Legumes that provide fiber include, but are not limited to, peas and beans such as soybeans. Fruits and vegetables that provide a source of fiber include, but are not limited to, apples, oranges, pears, bananas, berries, tomatoes, green beans, broccoli, cauliflower, carrots, potatoes, and celery. Plant foods such as bran, nuts, and seeds (such as flaxseed) are also sources of dietary fiber. Parts of plants that provide dietary fiber include, but are not limited to, stems, roots, leaves, seeds, pulp, and skin.

[0374] Fatty acid includes any straight or branched monocarboxylic acid, and includes saturated fatty acids, unsaturated fatty acids, long-chain fatty acids, medium-chain fatty acids, short-chain fatty acids, fatty acid precursors (including ω-9 fatty acid precursors), and esterified fatty acids. In an embodiment, the fatty acid is a straight-chain monocarboxylic acid. As used herein, "long-chain polyunsaturated fatty acids" refers to any polyunsaturated carboxylic acid or organic acid with a long aliphatic tail. Suitable ω-3 fatty acids include but are not limited to linolenic acid, α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, stearic acid, eicosatetraenoic acid and combinations thereof.

[0375] Suitable ω-6 fatty acids include, but are not limited to, linoleic acid, γ-linolenic acid, dihommo-gamma-linolenic acid, arachidonic acid, eicosadienoic acid, docosadienoic acid, adrenic acid, docosapentaenoic acid, and combinations thereof. Suitable esterified fatty acids for use in embodiments of the present invention include, but are not limited to, monoacylglycerols containing ω-3 and / or ω-6 fatty acids, diacylglycerols containing ω-3 and / or ω-6 fatty acids, or triacylglycerols containing ω-3 and / or ω-6 fatty acids, and combinations thereof.

[0376] Suitable vitamins include vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12 and vitamin C. Various other compounds are classified as vitamins by some authoritative agencies. These compounds can be referred to as pseudo vitamins, and include but are not limited to compounds such as ubiquinone (coenzyme Q10), pancreatic acid, dimethylglycine, taestrile, amygdaline (amygdaline), flavonoids, p-aminobenzoic acid, adenine, adenylic acid and s-methylmethionine. As used herein, the term vitamin includes pseudo vitamins.

[0377] Minerals are selected from macrominerals, trace minerals, or combinations thereof. Non-limiting examples of macrominerals include calcium, chloride, magnesium, phosphorus, potassium, sodium, and sulfur. Non-limiting examples of trace minerals include chromium, cobalt, copper, fluorine, iron, manganese, molybdenum, selenium, zinc, and iodine. Although iodine is generally classified as a trace mineral, its requirement is greater than other trace minerals and is generally classified as a macromineral.

[0378] In other specific embodiments of the invention, the mineral is a trace mineral believed to be essential for human nutrition, non-limiting examples of which include bismuth, boron, lithium, nickel, rubidium, silicon, strontium, tellurium, tin, titanium, tungsten, and vanadium.

[0379] The preservative is selected from an antimicrobial, an antioxidant, an antienzyme or a combination thereof. Non-limiting examples of antimicrobials include sulfites, propionates, benzoates, sorbates, nitrates, nitrites, bacteriocins, salts, sugars, acetic acid, dimethyl dicarbonate (DMDC), ethanol and ozone. Sulfites include but are not limited to sulfur dioxide, sodium bisulfite and potassium bisulfite. Propionates include but are not limited to propionic acid, calcium propionate and sodium propionate. Benzoates include but are not limited to sodium benzoate and benzoic acid. Sorbic acids include but are not limited to potassium sorbate, sodium sorbate, calcium sorbate and sorbic acid. Nitrates and nitrites include but are not limited to sodium nitrate and sodium nitrite. In another specific embodiment, the at least one preservative is a bacteriocin, such as, for example, nisin. In another specific embodiment, the preservative is ethanol. In another specific embodiment, the preservative is ozone. Antienzymatic agents suitable for use as preservatives in certain embodiments of the invention include ascorbic acid, citric acid, and metal chelators, such as ethylenediaminetetraacetic acid (EDTA).

[0380] The hydration product can be an electrolyte, non-limiting examples of which include sodium, potassium, calcium, magnesium, chloride, phosphate, bicarbonate, and combinations thereof. Suitable electrolytes for specific embodiments of the present invention are also described in U.S. Patent No. 5,681,569, the disclosure of which is expressly incorporated herein by reference. Non-limiting examples of salts for specific embodiments include chloride, carbonate, sulfate, acetate, bicarbonate, citrate, phosphate, hydrogen phosphate, tartrate, sorbate, citrate, benzoate, or combinations thereof.

[0381] In a specific embodiment of the present invention, the hydrated product is a carbohydrate, to supplement the energy storage of muscle burning. Suitable electrolytes for specific embodiments of the present invention are described in No. 4,312,856, No. 4,853,237, No. 5,681,569 and No. 6,989,171 United States Patents, the disclosure of which is expressly incorporated herein by reference. The limiting examples of suitable carbohydrates include monosaccharides, disaccharides, oligosaccharides, complex polysaccharides or their combinations. The limiting examples of monosaccharides for the suitable type of specific embodiments include trioses, tetroses, pentoses, hexoses, heptoses, octose and nonoses. The limiting examples of suitable monosaccharides of specific types include glyceraldehyde, dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose, ribose, xylose, ribulose, xylulose, allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose, mannoheptulose, sedoheltulose, octolose and sialose. The limiting examples of suitable disaccharides include sucrose, lactose and maltose. The limiting examples of suitable oligosaccharides include sucrose, maltotriose and maltodextrin. In other specific embodiments, the carbohydrate is provided by corn syrup, beet sugar, sucrose, fruit juice or tea. In another specific embodiment, the hydration is a flavanol that provides cell rehydration. Non-limiting examples of suitable flavanols for use in certain embodiments of the present invention include catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, epigallocatechin 3-gallate, theaflavin, theaflavin 3-gallate, theaflavin 3'-gallate, theaflavin 3,3' gallate, thearubigins, or combinations thereof. In certain embodiments, the hydration product is a glycerol solution to enhance athletic endurance.

[0382] Probiotics include microorganisms that are beneficial to health when taken in an effective amount. Probiotics can include, but are not limited to, bacteria, yeasts, and fungi. Examples of probiotics include, but are not limited to, bacteria of the genus Lactobacilli, Bifidobacteria, Streptococci, or combinations thereof. In a specific embodiment of the present invention, the at least one probiotic is selected from the genus Lactobacilli. Lactobacillus (i.e., bacteria of the genus Lactobacillus, hereinafter referred to as "L."). Non-limiting examples of Lactobacillus species found in the human intestine include Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus saliva roes, Lactobacillus brevis, Lactobacillus leichmannii, Lactobacillus plantarum, Lactobacillus cellobiosus, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus GG, Lactobacillus bulgaricus, and Lactobacillus thermophilus. According to other specific embodiments of the present invention, the probiotic is selected from the genus Bifidobacterium.Non-limiting species of Bifidobacteria found in the human gastrointestinal tract include: B. angulata, B. animalis, B. asteroides, B. bifidum, B. boum, B. breve, B. catenulatum, B. choerinum, B. coryneforme, B. cuniculi, B. dentium, B. gallicum, B. gallinarum, B. indica, B. In some embodiments, the probiotics are selected from the group consisting of Streptococcus, Streptococcus spp., Streptococcus spp., B. indicum, B. longum, B. magnum, B. merycicum, B. minimum, B. pseudocatenulatum, B. pseudolongum, B. psychraerophilum, B. pullorum, B. ruminantium, B. saeculare, B. scardovia, B. simiae, B. subtile, B. thermocidophilum, B. thermophilum, B. urinalis, and B. sp. According to other specific embodiments of the present invention, the probiotics are selected from the genus Streptococcus. Streptococcus thermophilus is a Gram-positive, facultative anaerobic bacterium. Other non-restricted probiotic species of this bacterium include Streptococcus salivarus and Streptococcus cremoris.

[0383] Prebiotics are components that promote the growth of beneficial bacteria in the intestinal tract. Prebiotics include but are not limited to mucopolysaccharides, oligosaccharides, polysaccharides, amino acids, vitamins, nutrient precursors, proteins and combinations thereof. According to specific embodiments, the prebiotics are selected from dietary fiber, including but not limited to polysaccharides and oligosaccharides. According to a specific embodiment of the present invention, the limiting examples of the oligosaccharides classified as prebiotics include oligofructose, inulin, isomaltooligosaccharides, lactitol (lactilol), lactosucrose (lactosucrose), lactulose, pyrodextrins (pyrodextrins), soybean oligosaccharides, transgalacto-oligosaccharides and xylo-oligosaccharides. According to other specific embodiments, the prebiotics are amino acids.

[0384] As used herein, "weight management agents" include appetite suppressants and / or thermogenic agents. As used herein, the phrases "appetite suppressants," "appetite satisfying compositions," "satiating agents," and "satiating ingredients" are synonymous. The phrase "appetite suppressants" describes macronutrients, herbal extracts, exogenous hormones, anorectics, anorexigenics, drugs, and combinations thereof, which, when delivered in effective amounts, suppress, inhibit, reduce, or otherwise limit a person's appetite. The phrase "thermogenic agent" describes macronutrients, herbal extracts, exogenous hormones, anorectics, anorexigenics, drugs, and combinations thereof, which, when delivered in effective amounts, activate or otherwise enhance a person's thermogenesis or metabolism.

[0385] Suitable weight management agents include macronutrients selected from the group consisting of proteins, carbohydrates, dietary fats, and combinations thereof. Carbohydrates typically include sugars, starches, cellulose, and gums, which the body converts into glucose for energy. Non-limiting examples of carbohydrates include polydextrose; inulin; polyols from monosaccharide sources such as erythritol, mannitol, xylitol, and sorbitol; alcohols from disaccharide sources such as isomalt, lactitol, and maltitol; and hydrogenated starch hydrolysates. Carbohydrates will be described in more detail below. Dietary fats are lipids that include a combination of saturated and unsaturated fatty acids. Polyunsaturated fatty acids have been shown to have greater satiety than monounsaturated fatty acid esters. Therefore, the dietary fats included herein ideally include polyunsaturated fatty acids, non-limiting examples of which include triacylglycoside oils.

[0386] In certain embodiments, the weight management agent is an herbal extract. Non-limiting examples of plants whose extracts have appetite suppressant properties include Hoodia, Trichocaulon, Caralluma, Stapelia, Orbea, Asclepias, and Camelia. Other embodiments include extracts from Gymnema sylvestre, Kola Nut, Aurantium, Yerba Mate, Griffonia simplicifolia, Guarana, Myrrh, Guggul Lipid, and Blackcurrant Seed Oil. In certain embodiments, the herbal extract is derived from plants of the genus Hoodia, including species such as H. alstonii, H. currorii, H. dregei, H. flava, H. gordonii, H. jutatae, H. mossamedensis, H. officinalis, H. parviflorai, H. pedicellata, H. pifera, H. Ruschii, and H. triebneri. Hoodia plants are stem succulent plants native to southern Africa.

[0387] In another specific embodiment, the herbal extract is derived from plants of the genus C. indica, C. fimbriata, C. attenuate, C. tuberculata, C. edulis, C. adscendens, C. stalagmifera, C. umbellate, C. penicillata, C. russeliana, C. retrospicens, C. arabica, and C. lasiantha. C. genus C. arabica belongs to the same subfamily as the genus Hoodia in the Asclepiadaceae family.

[0388] In another specific embodiment, the at least one herbal extract is derived from a plant in the genus Amur. Amur plants are succulents typically native to southern Africa, similar to the genus Pieris, and include the species T. piliferum and T. officinale. In another specific embodiment, the herbal extract is derived from a plant in the genus Stapelia or Stapelia, including species S. gigantean and O. variegate, respectively. Both Stapelia and Stapelia belong to the same subfamily as the genus Pieris in the Apocynaceae family.

[0389] In another specific embodiment, the herbal extract is a plant derived from the genus Asclepiadaceae. Asclepiadaceae plants also belong to the Asclepiadaceae family. Non-limiting examples of Asclepiadaceae plants include A. incarnate, A...

Claims

1. A polynucleotide encoding a Myd variant polypeptide selected from the group consisting of: (a) a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10 relative to the polypeptide of SEQ ID NO: 3, or a polypeptide having the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10 in which the methionine at position 1 is additionally deleted; (b) a polypeptide having at least 80% sequence identity to a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10 relative to the polypeptide of SEQ ID NO: 3, or a polypeptide having the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10 in which the methionine at position 1 is additionally deleted; as well as (c) a polypeptide having an amino acid sequence modified by deletion, insertion, substitution or addition of no more than 24 amino acids from a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10 relative to the polypeptide of SEQ ID NO: 3, or a polypeptide having the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10 in which the methionine at position 1 is additionally deleted; The encoded polypeptide has sweet taste modulating activity or optionally has a sweet taste, and is different from the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO:

141.

2. The polynucleotide of claim 1 , operably linked to one or more heterologous regulatory elements.

3. The polynucleotide according to claim 1 or 2, wherein The polynucleotide further encodes a protein / peptide tag, optionally an affinity tag or optionally a histidine tag.

4. The polynucleotide according to any one of claims 1 to 3, wherein The polynucleotide encodes a polypeptide modified by deletion, insertion, substitution or addition of no more than 12 amino acids from a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10, or a polypeptide having the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10 in which the methionine at position 1 is additionally lacking.

5. The polynucleotide according to any one of claims 1 to 4, wherein The polynucleotide encodes a polypeptide modified by deletion, insertion, substitution or addition of no more than 6 amino acids from a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10, or a polypeptide having the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10 in which the methionine at position 1 is additionally deleted.

6. The polynucleotide according to any one of claims 1 to 5, wherein The encoded polypeptide has at least 90% sequence identity to a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10, or a polypeptide having the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10 wherein the methionine at position 1 is additionally deleted.

7. The polynucleotide according to any one of claims 1 to 6, wherein The encoded polypeptide has at least 95% sequence identity to a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10, or a polypeptide having the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10 in which the methionine at position 1 is additionally deleted.

8. The polynucleotide according to any one of claims 1 to 7, wherein The encoded polypeptide does not contain a methionine at position 1 of its amino acid sequence, and wherein the encoded polypeptide is different from the polypeptide of SEQ ID NO:

141.

9. The polynucleotide according to any one of claims 1 to 8, wherein The variants are the variants listed in Table 8 or the variants listed in Table 8 wherein the methionine at position 1 is additionally deleted.

10. The polynucleotide according to any one of claims 1 to 8, wherein The variants are the variants listed in Table 9 or the variants listed in Table 8 wherein the methionine at position 1 is additionally deleted.

11. The polynucleotide according to any one of claims 1 to 8, wherein The variants are the variants listed in Table 10 or the variants listed in Table 8 wherein the methionine at position 1 is additionally deleted.

12. The polynucleotide according to any one of claims 1 to 11, encoding a polypeptide selected from the group consisting of: (a) a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of a variant listed in Table 8, which further has a protein / peptide tag, optionally an affinity tag or optionally a histidine tag, or which does not have a methionine at amino acid position 1; (b) a polypeptide having at least 80% sequence identity to a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of a variant listed in Table 8, which further has a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag; or which does not have a methionine at amino acid position 1; and (c) a polypeptide modified by deletion, insertion, substitution or addition of no more than 24 amino acids to a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of a variant listed in Table 8, which further has a protein / peptide tag, optionally an affinity tag or optionally a histidine tag, or which does not have a methionine at amino acid position 1; The polypeptide sequence is different from SEQ ID NO: 3, the polypeptide sequence of SEQ ID NO: 3 containing a protein / peptide tag, affinity tag or histidine tag, and optionally different from the polypeptide of SEQ ID NO:

141.

13. The polynucleotide according to any one of claims 1 to 11, encoding a polypeptide selected from the group consisting of: (a) a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of a variant listed in Table 9, which further has a protein / peptide tag, optionally an affinity tag or optionally a histidine tag, or which does not have a methionine at amino acid position 1; (b) a polypeptide having at least 80% sequence identity to a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of a variant listed in Table 9, which further has a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag; or which does not have a methionine at amino acid position 1; and (c) a polypeptide modified by deletion, insertion, substitution or addition of no more than 24 amino acids to a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of a variant listed in Table 9, which further has a protein / peptide tag, optionally an affinity tag or optionally a histidine tag, or which does not have a methionine at amino acid position 1; The polypeptide sequence is different from SEQ ID NO: 3, the polypeptide sequence of SEQ ID NO: 3 containing a protein / peptide tag, an affinity tag or a histidine tag, and optionally different from the polypeptide of SEQ ID NO:

141.

14. The polynucleotide according to any one of claims 1 to 11, encoding a polypeptide selected from the group consisting of: (a) a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of a variant listed in Table 8, which further has a protein / peptide tag, optionally an affinity tag or optionally a histidine tag, or which does not have a methionine at amino acid position 1; (b) a polypeptide having at least 80% sequence identity to a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of a variant listed in Table 8, which further has a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag; or which does not have a methionine at amino acid position 1; and (c) a polypeptide modified by deletion, insertion, substitution or addition of no more than 24 amino acids to a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of a variant listed in Table 8, which further has a protein / peptide tag, optionally an affinity tag or optionally a histidine tag, or which does not have a methionine at amino acid position 1; The polypeptide sequence is different from SEQ ID NO: 3, the polypeptide sequence of SEQ ID NO: 3 containing a protein / peptide tag, an affinity tag or a histidine tag, and optionally different from the polypeptide of SEQ ID NO:

141.

15. The polynucleotide according to any one of claims 1 to 14, wherein The encoded polypeptide does not include the mutations listed in Table 7.

16. The polynucleotide according to any one of claims 1 to 14, wherein The encoded polypeptide does not include the mutations listed in Table 3.

17. The polynucleotide according to any one of claims 1 to 14, wherein The encoded polypeptide does not include the mutations listed in Table 6.

18. The polynucleotide of any one of claims 12-17, operably linked to one or more heterologous regulatory elements.

19. An expression cassette comprising the polynucleotide according to any one of claims 1 to 18.

20. A vector comprising the polynucleotide according to any one of claims 1-18.

21. A host cell transformed with the vector according to claim 18.

22. A method for producing a protein having sweet taste modulating activity (and in particular sweet taste), comprising: The host cell transformed with the vector according to claim 18 is cultured in a culture medium under protein expression conditions.

23. A method for producing a protein having sweet taste modulating activity and / or having a sweet taste, comprising: Host cells transformed with the vector are cultured in a culture medium under protein expression conditions, wherein the vector comprises an isolated polynucleotide encoding a polypeptide selected from the group consisting of: (a) a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10, or a polypeptide of a variant thereof in which the methionine at position 1 is deleted; (b) a polypeptide having at least 80% sequence identity to a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of a variant listed in Table 8, Table 9 or Table 10, or a polypeptide of a variant thereof in which the methionine at position 1 is deleted; as well as (c) a polypeptide modified by deletion, insertion, substitution or addition of no more than 24 amino acids to a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of a variant listed in Table 8, Table 9 or Table 10, or a polypeptide of a variant thereof in which the methionine at position 1 is deleted; wherein the encoded polypeptide has sweet taste modulating activity and optionally sweet taste, and is different from the polypeptide of SEQ ID NO: 3, and is different from the polypeptide of SEQ ID NO:

141.

24. The method according to claim 23, wherein The variants are those of Table 8.

25. The method according to claim 23, wherein The variants are those of Table 9.

26. The method according to claim 23, wherein The variants are those of Table 10.

27. The method according to claim 23, wherein The variant is a variant other than the variant of Table 3, a variant other than the variant of Table 6, or a variant other than the variant of Table 7.

28. The method for producing a protein according to any one of claims 22 to 27, wherein 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, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, coelicolor), Clostridium acetobutylicum, Pseudomonas fluorescens, Pseudomonas putida, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizopus oryzae, Yarrowia lipolytica, Candida albicans, Issatchenkia orientalis), Pichia stipitis (Scheffersomycesstipitis), Yarrowia lipolytica (Yarrowia lipolytica), Ogataea polymorpha (Ogataea polymorpha), Phaffia rhodozyma (Phaffiarhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schwanniomyces occidentalis, Bacillus megaterium, Trichoderma reesei, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Carnobacterium divergens, Lactobacillus acidophilus, Lactobacillus amyloliquefaciens amylolyticus), Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus alimentarius, Lactobacillus aviaries, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus collinoides, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus dextrinicus, Lactobacillus diolivorans), Lactobacillusfarciminis), 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 plantarum), Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus Sanfranciscensis, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc Pseudomesenteroides, Microbacterium imperial, Oenococcus oeni, Pasteuria nishizawae, Pediococcus acidilactici, Pediococcus parvum parvulus), Pediococcus pentosaceuspentosaceus, Propionibacterium acidipropionic, Propionibacterium freudenreichii, Streptococcus thermophilus, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus flexus, Bacillus fusiformis, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus mojavensis, Bacillus paralicheniformis paralicheniformis), Bacillus pumilus, Bacillus smithii, Bacillus subtilis, Bacillus vallismortis, Bacillus velezensis, Geobacillus stearothermophilus, Paenibacillus illinoisensis, Parageobacillus thermoglucosidase, Gluconobacter oxydans, Komagataeibacter sucrofermentans, Xanthomonas campestris, Candida cylindracea, Cyberlindnera jadinii), Debaryomyces hansenii, Hanseniaspora uvarum, Kluyveromyces lactislactis, Kluyveromyces marxianus, Komagataella pastoris, Komagataella phaffi, Lindnera jadinii, Ogataea angusta, Saccharomyces bayanus, Schizosaccharomyces pombe, Wickerhamomyces anomalus, Xanthophyllomyces dendrorhous, and Zygosaccharomyces rouxii cells.

29. The method for producing a protein according to any one of claims 22 to 27, wherein 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.

30. The method for producing a protein according to any one of claims 22 to 27, wherein The host cell is a plant cell.

31. The method for producing a protein according to any one of claims 22 to 27, wherein The host cell is a fungal cell other than Mattirolomyces terfezioides.

32. The method for producing a protein according to any one of claims 22 to 27, wherein The host cell is a cell of Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris or Yarrowia lipolytica.

33. A method for producing a non-naturally occurring mixture of two isoforms of a Myd sweet protein, comprising recombinantly expressing a nucleic acid coding sequence encoding SEQ ID NO: 3 in a heterologous host.

34. The method according to claim 33, wherein The nucleic acid coding sequence also encodes a protein tag.

35. The method of claim 33, wherein: The nucleic acid coding sequence also encodes a His tag.

36. The method according to any one of claims 33 to 35, wherein The heterologous host is selected from an Escherichia coli strain, a Saccharomyces cerevisiae strain, a Pichia pastoris strain, a Trichoderma reesei strain or a Yarrowia lipolytica strain.

37. The method according to any one of claims 33 to 36, wherein The sweet protein mixture is enriched in HST-1 or protein-tagged HST-1.

38. The method according to any one of claims 33 to 36, wherein The sweet protein mixture is enriched in HST-2 or protein-tagged HST-2.

39. A polypeptide having sweet taste modulating activity (particularly sweet taste), comprising: (i) a polypeptide sequence selected from the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10, or a variant listed in Table 8, Table 9 or Table 10 further having a deletion of methionine at position 1; (ii) a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of the amino acid sequences of any variant listed in Table 8; Table 9 or Table 10, or a variant listed in Table 8, Table 9 or Table 10 further having a deletion of methionine at position 1; or (iii) a polypeptide sequence containing at least one modification by deletion, insertion, substitution or addition of no more than 24 amino acids relative to a polypeptide sequence selected from the group consisting of the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10, or a variant listed in Table 8, Table 9 or Table 10 further having a deletion of methionine at position 1, The polypeptide has sweet taste modulating activity (especially sweet taste), and: (a) the polypeptide is different from the polypeptide of SEQ ID NO: 3; and / or (b) the polypeptide is different from the polypeptide of SEQ ID NO: 141; and / or (c) the polypeptide is different from the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 141 which further comprises a protein / peptide tag, optionally an affinity tag or optionally a histidine tag or optionally a (His)6 tag.

40. The polypeptide according to claim 39, further comprising a protein / peptide tag, optionally an affinity tag or optionally a histidine tag or optionally a (His)6 tag.

41. The polypeptide according to claim 39 or 40, wherein The polypeptide comprises the amino acid sequence of SEQ ID NO: 3, which is modified by at least one and up to 24 amino acid mutations shown in Table 8, Table 9 or Table 10.

42. The polypeptide according to claim 39 or 40, wherein The polypeptide comprises the amino acid sequence of SEQ ID NO: 3, which is modified by at least one and up to 12 amino acid mutations shown in Table 8, Table 9 or Table 10.

43. The polypeptide according to claims 39-40, wherein The polypeptide comprises the amino acid sequence of SEQ ID NO: 3, which is modified by at least one and up to six amino acid mutations shown in Table 8, Table 9 or Table 10.

44. The polypeptide according to claims 39-40, wherein The polypeptide comprises the amino acid sequence of SEQ ID NO: 3 modified by at least one mutation, wherein the at least one mutation is selected from the mutations listed in Table 8, and wherein the polypeptide is further modified by one or more additional mutations, wherein the one or more additional mutations are selected from the mutations listed in Table 7.

45. The polypeptide according to claim 39 or 40, wherein The polypeptide comprises the amino acid sequence of SEQ ID NO: 3, which is modified by two or more mutations selected from the mutations listed in Table 8.

46. ​​The polypeptide according to claim 39 or 40, wherein The polypeptide comprises the amino acid sequence of SEQ ID NO: 3, which is further modified by two or more additional mutations, wherein the two or more additional mutations are selected from the mutations listed in Table 7.

47. The polypeptide according to claim 39 or 40, wherein The polypeptide comprises the amino acid sequence of SEQ ID NO: 3, which is modified by two or more mutations selected from the mutations listed in Table 9.

48. The polypeptide according to claim 39 or 40, wherein The polypeptide comprises the amino acid sequence of SEQ ID NO: 3, which is modified by two or more mutations selected from the mutations listed in Table 10.

49. The polypeptide according to claim 39 or 40, wherein The polypeptide includes an amino acid sequence other than the amino acid sequence of the variant of Table 3 or Table 6.

50. The polypeptide according to claim 39 or 40, wherein The polypeptide includes an amino acid sequence other than the amino acid sequence of the variant of Table 7.

51. The polypeptide according to claim 39 or 40, wherein The polypeptide includes the amino acid of SEQ ID NO: 3; or the amino acid sequence of SEQ ID NO: 3, wherein the methionine at position 1 is deleted, and it further has one or more or two or more and up to 24 different mutations listed in Table 9 or Table 10.

52. The polypeptide according to claim 51, wherein The polypeptide further comprises a protein / peptide tag, optionally an affinity tag or optionally a histidine tag or optionally a (His)6 tag.

53. The polypeptide according to any one of claims 39-52, which exhibits a sweet taste.

54. The polypeptide according to any one of claims 39 to 53, wherein The polypeptide is an isolated polypeptide or a purified polypeptide.

55. The polypeptide according to any one of claims 39 to 54, wherein The polypeptide has at least 90%, at least 95% or at least 99% sequence identity to a polypeptide sequence selected from the group consisting of the amino acid sequences of any variant listed in Table 8, Table 9 or Table 10, or a variant listed in Table 8, Table 9 or Table 10 in which the methionine at position 1 is additionally deleted.

56. The polypeptide according to any one of claims 39 to 54, wherein The polypeptide has at least 90%, at least 95% or at least 99% sequence identity to a polypeptide sequence selected from the group consisting of the amino acid sequences of any variant listed in Table 9 or Table 10, or a variant listed in Table 8, Table 9 or Table 10 in which the methionine at position 1 is additionally deleted.

57. The polypeptide according to any one of claims 39 to 54, wherein The polypeptide has at least 90%, at least 95% or at least 99% sequence identity with a polypeptide sequence selected from the group consisting of the amino acid sequences of any variant listed in Table 9 or the variants listed in Table 8, Table 9 or Table 10 in which the methionine at position 1 is additionally lacking.

58. The polypeptide according to any one of claims 39 to 54, wherein The polypeptide has at least 90%, at least 95% or at least 99% sequence identity to a polypeptide sequence selected from the group consisting of the amino acid sequences of any variant listed in Table 10 or a variant listed in Table 10 in which the methionine at position 1 is additionally deleted.

59. The polypeptide according to any one of claims 39 to 58, wherein The polypeptide sequence does not contain the amino acid substitutions listed in Table 3 or Table 4.

60. The polypeptide according to any one of claims 39 to 59, wherein The polypeptide further comprises a protein / peptide tag, optionally an affinity tag or optionally a histidine tag.

61. The polypeptide according to any one of claims 39-60, wherein The polypeptide contains a histidine tag, which is (His)6.

62. A sweet taste polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 141, having two or more mutations listed in Table 8, Table 9 or Table 10, and which further exhibits greater thermal stability than a native (wild-type) HTS protein.

63. The polypeptide of claim 62, comprising two mutations to introduce two cysteine ​​amino acids, such that the two cysteines are capable of forming an -SS- bond in the polypeptide.

64. The polypeptide according to claim 63, wherein The two mutations introducing cysteine ​​are located at amino acids 20-70 (inclusive) of SEQ ID NO:

3.

65. The polypeptide according to claim 63, wherein The mutation introducing cysteine ​​is located at any two of amino acids 24, 33, 49 and 62 of SEQ ID NO:

3.

66. The polypeptide according to claim 63, wherein The mutations introducing cysteine ​​are located at amino acids 33 and 49 of SEQ ID NO:

3.

67. The polypeptide according to any one of claims 62 to 66, wherein The polypeptide does not have a methionine at position 1.

68. The polypeptide according to any one of claims 62 to 67, wherein The polypeptide further comprises a protein tag, an affinity tag or optionally a histidine tag or optionally a (His)6 tag.

69. The polypeptide according to claim 62, wherein The polypeptide is selected from the group consisting of a polypeptide having an amino acid sequence of SEQ ID No: 143, SEQ ID NO: 145, SEQ ID No: 146 or SEQ ID NO:

147.

70. A sweet protein having the amino acid sequence of SEQ ID NO: 141, which is substantially free of a protein having the amino acid sequence of SEQ ID NO:

3.

71. The sweet protein of claim 70, comprising less than 1% of a protein having the amino acid sequence of SEQ ID NO:

3.

72. A sweet protein having the amino acid sequence of SEQ ID NO: 3, which is substantially free of a protein having the amino acid sequence of SEQ ID NO:

141.

73. The sweet protein of claim 72, comprising less than 1% of a protein having the amino acid sequence of SEQ ID NO:

141.

74. A sweet protein, which is a non-naturally occurring mixture of a sweet protein having the amino acid sequence of SEQ ID NO: 3 and a sweet protein having the amino acid sequence of SEQ ID NO:

141.

75. The sweet protein according to claim 74, which is a mixture of a sweet protein having the amino acid sequence of SEQ ID NO: 3 and a sweet protein having the amino acid sequence of SEQ ID NO: 141, wherein the amount of the sweet protein having the sequence of SEQ ID NO: 3 is greater than 60% by weight of the mixture.

76. The sweet protein according to claim 74, which is a mixture of a sweet protein having the amino acid sequence of SEQ ID NO: 3 and a sweet protein having the amino acid sequence of SEQ ID NO: 141, wherein the amount of the sweet protein having the sequence of SEQ ID NO: 141 is greater than 60% by weight of the mixture.

77. The sweet protein according to claim 74, which is a mixture of a sweet protein having the amino acid sequence of SEQ ID NO: 3 and a sweet protein having the amino acid sequence of SEQ ID NO: 141, wherein the amount of the sweet protein having the sequence of SEQ ID NO: 141 is 90% by weight or more of the mixture.

78. A composition comprising a combination of: (a) a product for orally administration, wherein the product is other than the Truffle Truffle, and (b) a flavor modulating composition comprising one or more isolated polypeptides or one or more sweet proteins of any one of claims 39-77, or one or more isolated polypeptides produced by expressing a polynucleotide of any one of claims 1-18 in a heterologous host cell; wherein the combination exhibits flavor modulation compared to an orally administered product.

79. The composition of claim 78, wherein The flavor-modulating composition is a sweet composition, and the combination has a sweet taste or an enhanced sweet taste compared to the orally administered product.

80. The composition according to claim 78 or 79, wherein The composition includes a plurality of isolated polypeptides.

81. The composition of any one of claims 78-80, wherein The orally administered product is a food product selected from the group consisting of: baked goods; sweet baked products, pre-made sweet baking mixes for preparing sweet baked products; pie fillings and other sweet fillings, gelatin and puddings; frozen desserts; yogurt; snack bars; bread products; pre-made bread mixes for preparing bread products; sauces, syrups and condiments; sweet spreads; confectionery products; Sweet breakfast cereals are also available.

82. The composition of any one of claims 78-81, wherein The orally administered product is a food product that is warmed or heated before consumption, or a food product for consumption in a warm or hot state.

83. The composition of claim 82, wherein At least one of the polypeptides exhibits enhanced thermostability compared to a native HTS.

84. The composition of any one of claims 78-80, 82 or 83, wherein The orally administered product is a beverage product selected from the group consisting of a carbonated beverage; a non-carbonated beverage; a beverage concentrate, a coffee-based beverage or a tea-based beverage.

85. A method for modifying the taste of an orally administered product, comprising: combining the orally administered product with an effective amount of one or more isolated polypeptides having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of those amino acid sequences of the variants listed in Table 8, Table 9 or Table 10 or the variants listed in Table 8, Table 9 or Table 10 in which the methionine at position 1 is deleted, or having at least 80% sequence identity to a polypeptide thereof or a polypeptide further having a protein / peptide tag, optionally an affinity tag or optionally a histidine tag or optionally a (His)6 tag, wherein the orally administered product is other than Truffle Truffle, wherein the one or more isolated polypeptides is not the polypeptide of SEQ ID NO: 3, the polypeptide of SEQ ID NO: 141, or the polypeptide of SEQ ID NO: 3 containing a protein / peptide tag, an affinity tag, or a histidine tag, and wherein the combination has a flavor modulation compared to the orally administered product.

86. The composition of claim 85, wherein The at least one polypeptide has a sweet taste, and the composition has a sweet taste or an enhanced sweet taste compared to the orally administered product.

87. The method according to claim 85 or 86, wherein The orally administered product is a food product selected from the group consisting of: baked goods; sweet baked products, pre-made sweet baking mixes for making sweet baked products; pie fillings and other sweet fillings, gelatin and puddings; frozen desserts; yogurt; snack bars; bread products; pre-made bread mixes for making bread products; sauces, syrups and condiments; sweet spreads; confectionery products; and Sweet breakfast cereal.

88. The method according to any one of claims 85 to 87, wherein The orally administered product is a food product that is warmed or heated before consumption, or a food product for consumption in a warm or hot state.

89. A method as claimed in any one of claims 85, 86 or 88, wherein The orally administered product is a beverage product selected from the group consisting of carbonated beverages; non-carbonated beverages and beverage concentrates.

90. The method of claim 89, wherein The orally administered product is a heated or warmed beverage product, which is optionally consumed warm or hot, or is optionally a coffee or tea based product.

91. A method for purifying a polypeptide having sweet taste modulating activity, comprising: (a) performing hydrophobic interaction chromatography (HIC) on the polypeptide, and (b) then subjecting the polypeptide to size exclusion chromatography (SEC), wherein the polypeptide comprises an amino acid sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of: an amino acid sequence of any variant listed in Table 8, Table 9 or Table 10, or an amino acid sequence having at least 80% sequence identity thereto, or wherein the polypeptide further comprises a protein / peptide tag, optionally an affinity tag, optionally a histidine tag, or optionally a (His)6 tag.

92. A polypeptide purified by the method of claim 91.

93. The polypeptide of any preceding claim, which exhibits enhanced sweet taste modulating activity compared to a sweet protein having the amino acid sequence of SEQ ID NO:

3.

94. A polypeptide according to any preceding claim, wherein The polypeptide has enhanced thermal stability compared to the sweet protein having the amino acid sequence of SEQ ID NO:

3.

95. A polypeptide according to any preceding claim, wherein The polypeptide has enhanced sweet taste regulating activity and enhanced thermal stability compared to the sweet protein having the amino acid sequence of SEQ ID NO:

3.

96. A polynucleotide encoding the polypeptide of claim 93 or 95.

97. An expression cassette or vector comprising the polynucleotide of claim 96.

98. A host cell transformed with the expression cassette or vector of claim 97.

99. A method for producing a protein having sweet taste modulating activity, comprising: The host cell transformed with the expression cassette or vector of claim 97 is cultured in a culture medium under protein expression conditions.

100. A host cell expressing the polypeptide of any preceding claim.

101. The polypeptide of any preceding claim which is isolated.

102. The polynucleotide of any preceding claim which is isolated.

103. The polynucleotide of any preceding claim operably linked to a heterologous regulatory element.

104. A composition according to any preceding claim, wherein At least one of the one or more isolated polypeptides exhibits enhanced sweet taste modulating activity compared to the polypeptide of SEQ ID NO:

3.

105. A composition according to any preceding claim, wherein At least one of the one or more isolated polypeptides exhibits enhanced thermostability compared to the polypeptide of SEQ ID NO:

3.

106. A polypeptide according to any preceding claim, further comprising derivatization of one or more of the N-terminal amino acid, the C-terminal amino acid or an amino acid side chain of the polypeptide.

107. The polypeptide according to claim 106, wherein The derivatization is selected from the addition, removal or substitution of any of the following chemical moieties on the amine nitrogen: acyl, acetyl (CH3CO), formyl (HCO), glycosyl (C6H 11 O6), hydroxyl (HO), methyl (CH3), phosphatidyl (PO4), phosphono (PO2), sulfhydryl (SH) or sulfonyl (HSO2).

108. The polypeptide according to claim 106, wherein The derivatization is selected from the group consisting of adding, removing or substituting any one of the following chemical moieties to the delta-sulfur of methionine: acyl, acetyl (CH3CO), formyl (HCO), glycosyl (C6H 11 O6), hydroxyl (HO), methyl (CH3), phosphatidyl (PO4), phosphono (PO2), sulfhydryl (SH) or sulfonyl (HSO2).

109. The polypeptide according to claim 106, wherein The derivatization is selected from acylation, acetylation, esterification, glycosylation, oxidation, methylation, reductive alkylation, phosphorylation, sulfurization or sulfonylation.

110. The polypeptide according to claim 106, wherein The one or more derivatizations are acylations of one or more different amino groups.

111. The polypeptide according to claim 106, wherein The derivatization is acylation, and optionally acetylation of the N-terminal amino acid.

112. The polypeptide according to claim 106, wherein The derivatization is the oxidation of the delta sulfur of methionine.

113. The polypeptide according to claim 106, wherein The derivatization is the esterification of the C-terminal carboxyl (carboxylate) group.

114. The polypeptide according to any one of claims 106-113, wherein The one or more derivatizations are accomplished chemically or enzymatically in vitro.

115. The polypeptide according to any one of claims 106-113, wherein The one or more derivatizations are accomplished by post-translational processing.

116. A sweetener composition comprising: (a) one or more polypeptides, wherein the one or more polypeptides comprise: (i) a polypeptide sequence selected from the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10; (ii) a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of the amino acid sequences of any variant listed in Table 8; Table 9 or Table 10; (iii) a polypeptide sequence comprising at least one modification by deletion, insertion, substitution or addition of no more than 24 amino acids relative to a polypeptide sequence selected from the group consisting of the amino acid sequences of the variants listed in Table 8, Table 9 or Table 10; or (iv) The polypeptide of any one of (i) to (iii), wherein the methionine at position 1 is deleted; and / or The polypeptide further comprises a protein / peptide tag, optionally an affinity tag or optionally a histidine tag or optionally a (His)6 tag; as well as (b) at least one additional sweetener other than Myd sweet protein.

117. The sweetener composition of claim 116, wherein The additional sweetener is selected from the group consisting of steviol glycoside sweeteners, mogroside sweeteners, sucrose, psicose, sucralose, polyols, and high fructose corn syrup (HFCS).

118. The sweetener composition of claim 117, wherein The steviol glycoside sweetener is selected from the group consisting of rebaudioside M ("Reb M"), Reb M80, rebaudioside D ("Reb D"), Reb A95, and rebaudioside A ("Reb A").

119. The sweetener composition of claim 117, wherein The mogroside sweetener is selected from simenoside I and mogroside V.

120. The sweetener composition of claim 117, wherein The mogroside sweetener is mogroside V.

121. The sweetener composition of claim 118, wherein The Reb M has a purity greater than about 95%.

122. The sweetener composition of any one of claims 116-121, wherein The polypeptide has the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO:

141.

123. An edible or edible product, optionally a beverage or beverage product, comprising the sweetener composition of any one of claims 116-122.

124. The comestible or comestible product, optionally a beverage or beverage product, of claim 123, wherein the polypeptide is present in an amount of about 1 ppm to about 50 ppm.

125. The comestible or comestible product, optionally a beverage or beverage product, of claim 123, wherein the polypeptide is present in an amount of about 1 ppm to about 40 ppm.

126. The comestible or comestible product, optionally a beverage or beverage product, of claim 123, wherein the polypeptide is present in an amount selected from the group consisting of: about 1 ppm to about 30 ppm, about 1 ppm to about 25 ppm, about 1 ppm to about 20 ppm, or about 1 ppm to about 15 ppm.

127. The edible or edible product, optionally a beverage or beverage product, of any one of claims 123-126, wherein the edible or edible product, optionally the beverage or beverage product, has at least one improved organoleptic characteristic compared to the edible or edible product or beverage or beverage product without the sweetener composition, wherein the organoleptic characteristic is selected from the group consisting of aroma, flavor, base taste (sweet, sour, salty, bitter or umami), aftertaste or aftertaste, temporal characteristics, mouthfeel or a combination thereof.

128. An edible or edible product, optionally a beverage or beverage product, according to claim 127, wherein the at least one improved organoleptic property is a reduction in bitterness, an aftertaste or an improved mouthfeel.

129. The comestible or comestible product, optionally a beverage or beverage product, of claim 127, wherein the at least one improved organoleptic property is improved mouthfeel and the polypeptide is in an amount of about 1 ppm to about 40 ppm.

130. An edible or edible product, optionally a beverage or beverage product, according to any one of claims 123 to 129, which is a beverage or beverage product selected from low-calorie or no-calorie beverages or beverage products.

131. An edible article or edible product according to any one of claims 123-129, optionally a beverage or beverage product, which is a beverage or beverage product selected from the group consisting of: cola, ginger ale, soft drink, root beer, fruit juice, fruit juice, vegetable juice, vegetable flavored juice, sports drink, energy drink, plant protein drink, near water beverage (e.g., water with natural or synthetic flavoring), tea (e.g., black tea, green tea, black tea type tea, oolong tea), coffee, cocoa drink, beverage containing milk ingredients (e.g., milk beverage, coffee containing milk ingredients, au lait, milk tea, fruit milk beverage).

132. The edible article or edible product, optionally a beverage or beverage product, of any one of claims 123 to 131 , further comprising at least one organic acid additive salt, the at least one organic acid additive salt being a sodium, calcium, potassium or magnesium salt of an organic acid.

133. An edible or edible product, optionally a beverage or beverage product, according to claim 132, wherein the organic acid is selected from the group consisting of citric acid, malic acid, tartaric acid, fumaric acid, lactic acid, alginic acid, ascorbic acid, benzoic acid and adipic acid.

134. An edible article or edible product, optionally a beverage or beverage product, according to any one of claims 123-133, which is a beverage or beverage product having at least one change in organoleptic characteristic compared to the beverage or beverage product without the sweetener composition, wherein the organoleptic characteristic is selected from the group consisting of aroma, flavor, base taste (sweet, sour, salty, bitter or umami), aftertaste or aftertaste, temporal characteristics, mouthfeel or a combination thereof.

135. A method of improving at least one organoleptic property of an edible or edible product, optionally a beverage or a beverage product, comprising adding the sweetener composition of any one of claims 116-122 to an edible or edible product, optionally a beverage or a beverage product, thereby improving at least one organoleptic property thereof.

136. The method of claim 135, wherein The comestible or comestible product is a beverage or beverage product, and the sweetener composition is added to a liquid matrix to provide the beverage or beverage product with at least one improved organoleptic property.

137. The method of claim 135 or 136, wherein: The added sweetener of the sweetener composition is a steviol glycoside sweetener selected from the group consisting of rebaudioside M ("Reb M"), Reb M80, rebaudioside D ("Reb D"), Reb A95, and rebaudioside A ("Reb A").

138. The method of any one of claims 135-137, wherein: The improved organoleptic properties are selected from the group consisting of aroma, flavor, base taste, aftertaste or aftertaste, temporal characteristics, mouthfeel, or combinations thereof.

139. The method according to any one of claims 135-138, wherein The improved organoleptic properties are reduced bitterness, aftertaste or improved mouthfeel.

140. The method according to any one of claims 135-139, wherein The improved organoleptic property is improved mouthfeel, and the polypeptide is present in the beverage or beverage product in an amount between about 1 ppm and about 40 ppm.

141. The method of any one of claims 135-140, wherein The polypeptide is present in the comestible or comestible product, optionally the beverage or beverage product, in an amount between about 1 ppm and about 50 ppm.

142. The method of any one of claims 135-140, wherein The polypeptide is present in the comestible or comestible product, optionally a beverage or beverage product, in an amount selected from an amount between about 1 ppm to about 40 ppm, about 1 ppm to about 30 ppm, about 1 ppm to about 25 ppm, about 1 ppm to about 20 ppm, or about 1 ppm to about 15 ppm.

143. The method according to any one of claims 135-142, wherein The added sweetener is a mogroside sweetener selected from simenoside I and mogroside V.

144. A method of regulating / altering at least one organoleptic characteristic of a beverage or beverage product, comprising adding the sweetener composition of any preceding claim to a liquid matrix, thereby providing a beverage or beverage product having at least one adjusted / altered organoleptic characteristic.

145. The method of claim 144, wherein The organoleptic properties are selected from the group consisting of aroma, flavor, base taste (sweet, sour, salty, bitter or umami), aftertaste or aftertaste, temporal characteristics, mouthfeel or combinations thereof.

146. A sweetener composition comprising: (a) one or more polypeptides, wherein the one or more polypeptides comprise: (i) a polypeptide sequence selected from the amino acid sequence of any variant listed in Table 8, Table 9 or Table 10; (ii) a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of the amino acid sequences of any variant listed in Table 8; Table 9 or Table 10; (iii) a polypeptide sequence comprising at least one modification by deletion, insertion, substitution or addition of no more than 24 amino acids relative to a polypeptide sequence selected from the group consisting of the amino acid sequences of the variants listed in Table 8, Table 9 or Table 10; or (iv) The polypeptide of any one of (i) to (iii), wherein the methionine at position 1 is deleted; and / or The polypeptide further comprises a protein / peptide tag, optionally an affinity tag or optionally a histidine tag or optionally a (His)6 tag; as well as (b) at least one additional component selected from the group consisting of sucrose, mannitol, citric acid, hypoxanthine, theophylline, leucine, and combinations thereof.

147. The composition of claim 146, wherein The orally administered product is selected from pie fillings and other sweet fillings, gelatin and pudding; yogurt; sauces, syrups and dressings; and food products from the group consisting of sweet spreads.

148. The composition of claim 147, wherein The orally administered product also includes low methoxyl pectin.

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