Honey Truffle Sweetener (HTS) Variant

Abstract

Protein variants that alter fungal sweetness (referred to as honey truffle sweetener (HTS) protein variants) and polynucleotides encoding the protein variants are described. Specifically, Myd (HTS) protein variants with sweetness-modulating activity and cDNAs encoding them are described, along with methods for isolating such cDNAs and methods for isolating and expressing such proteins. Sweetener compositions containing the proteins of the invention and methods for providing improved flavor to products for oral administration are also disclosed.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 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 by reference in its entirety herein.

[0002] Incorporation by Reference of Electronically Submitted Material

[0002] A list of computer-readable nucleotide / amino acid sequences identified as follows: One 282 KB XML file entitled "0640-47_WO.xml", created on February 8, 2024, is incorporated herein by reference in its entirety.

[0003] This invention includes embodiments of sweet (honey truffle sweetener (HTS)) proteins, genes and cDNAs encoding said proteins, and methods of using such proteins, genes, and cDNAs in modulating the taste of foods. More particularly, the invention includes embodiments of HTS variant sweet proteins, and genes and cDNAs encoding such proteins. [Background technology]

[0004]

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

[0005]

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

[0006]

[0006] Zero- or low-calorie sweeteners derived from natural sources may be desirable to limit the negative effects of high sugar consumption. To date, only seven proteins that modify sweetness and taste are known: monellin, thaumatin, brazzein, curculin, mabinlin, miraculin, and pentadin. For none of these proteins have the key residues on the protein surface responsible for biological activity been identified with certainty. Monellin was found to be 100,000 times sweeter than sucrose on a molar basis, followed by thaumatin and brazzein, which are 3,000 and 500 times sweeter than sucrose, respectively, on a gram basis. Most of them share no sequence homology or structural similarity; thaumatin shares extensive similarity at the protein sequence level with certain nonsweet proteins found in other plants.

[0007]

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

[0008]

[0008] International Patent Application PCT / US2021 / 039176, filed June 25, 2021, and published as WO2021 / 263158 on December 30, 2021, relates to a newly identified fungal sweetness-modifying protein and a cDNA encoding the protein. A sweetness-modifying protein was identified in the truffle, Mattiromyces terfesioides. The application further relates to Myd proteins as sweeteners and in activating / regulating sweetness, cDNAs encoding them, and methods for isolating such cDNAs and for isolating and expressing such proteins. The application also relates to sweetener compositions containing the proteins and methods for providing improved flavor to products for oral administration.

[0009]

[0009] International patent application PCT / US2022 / 82443, filed on December 27, 2022 and published as WO2023 / 129938 on July 6, 2023, relates to sweet protein variants derived from truffles, such as Mattiromyces terfesioides, including proteins, genes encoding the proteins, cDNAs, and compositions thereof, which are incorporated herein by reference in their entirety. Summary of the Invention [Problem to be solved by the invention]

[0010]

[0010] There remains a need for new low- or zero-calorie sweeteners with improved taste from natural sources, particularly from the fungal species Ascomycetes. There is also a need to provide fungal-derived low- or zero-calorie sweeteners with improved taste and methods for using such ingredients in final food products with a superior taste profile without compromising taste. [Means for solving the problem]

[0011]

[0011] The present invention fulfills these and other needs by providing fungal sweet proteins identified herein as members of the Myd family of proteins, as well as genes and cDNAs encoding such proteins, and methods for using such proteins, genes, and cDNAs in modulating food taste. The present invention provides variants of MYD1 (also referred to as mycodulcein and honey truffle sweetener protein (HTS protein)). The polypeptides of the present invention, either alone or in combination with foods, beverages, dietary supplements, or pharmaceuticals, exhibit flavor-modifying properties, particularly modulating sweetness and perception. In embodiments, Myd1 variants of the present invention can provide sweetness to ingested materials such as foods, beverages, dietary supplements, or pharmaceuticals. In embodiments, Myd1 variants of the present invention can reduce the sour, bitter, or astringent taste of foods and beverages. In embodiments, Myd1 variants of the present invention can exhibit taste-enhancing, i.e., taste-modifying, activity in foods and beverages. In embodiments, the Myd1 variants may exhibit enhanced thermostability compared to the native form of the HTS protein in addition to sweet taste modulating activity. In embodiments, the Myd1 variants may exhibit flavor-modifying properties. In embodiments, the Myd1 variants may exhibit enhanced thermostability compared to the native form of the HTS protein in addition to flavor-modifying properties. In one embodiment, the present invention provides a naturally occurring Myd protein. In one embodiment, the present invention provides a variant Myd protein other than a naturally occurring protein.

[0012] Naturally occurring Myd proteins exist as two isoforms: HTS-1 (SEQ ID NO: 3) and HTS-2 (SEQ ID NO: 141), which were isolated from Mattiromyces terfeizioides. The relative amounts of HTS-1 and HTS-2 isolated from Mattiromyces terfeizioides range from approximately 40% to 60% by weight of HTS-2 to 60% to 40% by weight of HTS-1. In embodiments, the present invention provides a non-naturally occurring sweet protein comprising a non-naturally occurring mixture of the two isoforms, HTS-1 and HTS-2. In embodiments, 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-native host (e.g., a host other than the truffle Mattiromyces terfeizioides), as well as a method for producing the sweet protein by recombinant expression.

[0013] In one 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 one embodiment, the encoded polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 3 except for the absence of methionine at position 1 in SEQ ID NO: 3, which is referred to below 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 a (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 a (His tag)6.

[0014]

[0014] Accordingly, 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 wherein the amino acid sequence differs from that of the polypeptide of SEQ ID NO: 3 or, optionally, differs from that of SEQ ID NO: 141. In embodiments, the encoded polypeptide is (a) a polypeptide sequence selected from the group consisting of the amino acid sequences of variants listed in Table 8, Table 9, or Table 10, (b) a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of the amino acid sequences of variants listed in Table 8, Table 9, or Table 10; and (c) a polypeptide sequence modified from a polypeptide sequence selected from the group consisting of the amino acid sequences of variants listed in Table 8, Table 9, or Table 10 by deletion, insertion, substitution, or addition of 24 or fewer amino acids, wherein the polypeptide sequence differs from that of SEQ ID NO: 3 and, optionally, differs from that of SEQ ID NO: 141. In one embodiment, the polynucleotide encoding the polypeptide is other than the polynucleotide of SEQ ID NO: 2. In one embodiment, the polynucleotide encoding the polypeptide is the polynucleotide of SEQ ID NO: 2, codon-optimized for expression in bacteria, yeast, or fungi. In one embodiment, the polynucleotide encoding the polypeptide is the polynucleotide of SEQ ID NO: 6, codon-optimized for expression in Escherichia coli (E. coli), with an optional His-tag. In one embodiment, the polynucleotide encoding the polypeptide is the polynucleotide of SEQ ID NO: 7, codon-optimized for expression in Saccharomyces cerevisiae, with an optional His-tag.

[0015] In one embodiment, the encoded polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 3, except for the absence of methionine at position 1 in SEQ ID NO: 3, and is referred to herein as a polypeptide having the amino acid sequence of SEQ ID NO: 141. In one 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.

[0016] In a related aspect, the invention provides a polynucleotide (e.g., an isolated polynucleotide) encoding a polypeptide, wherein the encoded polypeptide has a flavor-modifying property and has an amino acid sequence that differs from the polypeptide of SEQ ID NO: 3, and optionally differs 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 a polypeptide sequence selected from the group consisting of the amino acid sequences of the variants listed in Table 8 by the deletion, insertion, substitution, or addition of 24 or fewer amino acids, wherein the polypeptide sequence differs from that of SEQ ID NO: 3, and optionally differs from that 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, other than a variant listed in Table 9 or Table 10. In one embodiment, the polynucleotide encoding the polypeptide is a polynucleotide other than the polynucleotide of SEQ ID NO: 2. In one embodiment, the encoded polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 141. In one embodiment, the encoded polypeptide is a polypeptide other than the polypeptide having the amino acid sequence of SEQ ID NO: 141. In an embodiment, the encoded polypeptide does not taste sweet. In an embodiment, the encoded polypeptide does not taste sweet when present in a composition at a concentration of 30 ppm or higher. In an embodiment, the encoded polypeptide does not taste sweet when present in a composition at a concentration of 40 ppm or higher. In an embodiment, the encoded polypeptide only tastes sweet when present in a composition at a concentration of 30 ppm or higher.

[0017] In another embodiment of the invention, amino acid mutations of HTS polypeptide 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 is expected to have the amino acid sequence of SEQ ID NO:3 but with one or more site mutations identified in Table 8. Mutants in Table 8 also include those in which the methionine at position 1 of the protein is absent. In a related embodiment, the invention provides HTS polypeptide and protein variants that have a single site mutation in Table 8 compared to the amino acid sequence of SEQ ID NO:3, and in addition, the methionine at position 1 in SEQ ID NO:3 is absent.

[0018] In another embodiment 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 is expected to have the amino acid sequence of SEQ ID NO:3 but with one or more site mutations identified in Table 9. Mutants in Table 9 also include those in which the methionine at position 1 of the protein is absent. In a related embodiment, the invention provides HTS polypeptide and protein variants that have a single site mutation in Table 9 compared to the amino acid sequence of SEQ ID NO:3, and in addition, the methionine at position 1 in SEQ ID NO:3 is absent.

[0019] In another embodiment 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 is expected to have the amino acid sequence of SEQ ID NO:3 but with one or more site mutations identified in Table 10. Mutants in Table 10 also include those in which the methionine at position 1 of the protein is absent. In a related embodiment, the invention provides HTS polypeptide and protein variants that have a single site mutation in Table 8 compared to the amino acid sequence of SEQ ID NO:3, and in addition, the methionine at position 1 in SEQ ID NO:3 is absent.

[0020] In another embodiment of the invention, HTS polypeptide and protein variants have a single-site mutation listed in Table 7 relative to the amino acid sequence of SEQ ID NO: 3. In a related embodiment of the invention, HTS polypeptide and protein variants have a single-site mutation listed in Table 7 relative to the amino acid sequence of SEQ ID NO: 3, and in addition, the methionine at position 1 in SEQ ID NO: 3 is absent.

[0021] In another embodiment of the invention, HTS polypeptide and protein variants are other than those with a single-site mutation listed in Table 7 identified relative to the amino acid sequence of SEQ ID NO:3. In a related embodiment, the invention excludes polynucleotides encoding HTS polypeptide and protein variants with a single-site mutation listed in Table 7. In a related embodiment of the invention, HTS polypeptide and protein variants are other than those with a single-site mutation listed in Table 7, wherein the methionine at position 1 in SEQ ID NO:3 is absent. In a related embodiment, the invention excludes polynucleotides encoding HTS polypeptide and protein variants with a single-site mutation listed in Table 7, wherein the methionine at position 1 in SEQ ID NO:3 is absent.

[0022] In another embodiment of the invention, the HTS polypeptide or protein variant does not comprise a mutation in Table 7. In a related embodiment, the invention excludes HTS polypeptide or protein variants that comprise a mutation listed in Table 7. In a related embodiment, the invention excludes polynucleotides that encode HTS polypeptide or protein variants that comprise a mutation listed in Table 7.

[0023] In another embodiment of the invention, the HTS polypeptide or protein variant does not comprise a mutation in Table 3 or Table 6. In a related embodiment, the invention excludes HTS polypeptide or protein variants that comprise a mutation listed in Table 3 or Table 6. In a related embodiment, the invention excludes polynucleotides that encode HTS polypeptide or protein variants that comprise a mutation listed in Table 3 or Table 6.

[0024] In yet another embodiment of the invention, the HTS polypeptide or protein variant does not contain a mutation in Table 8, Table 9, or Table 10, but contains one or more conservative mutations of the amino acid sequence of SEQ ID NO: 3, or optionally SEQ ID NO: 141, wherein the methionine at position 1 is either present or absent in the variant. In a related embodiment, the invention includes polynucleotides encoding HTS polypeptide or protein variants that do not contain a mutation in Table 8, Table 9, or Table 10, but contain one or more conservative mutations of the amino acid sequence of SEQ ID NO: 3, wherein the methionine at position 1 is either present or absent in the variant.

[0025]

[0025] In another aspect, the polynucleotide encoding the polypeptide having flavor-modulating or sweetness-modulating activity is optionally operably linked to a heterologous regulatory element. Additionally or alternatively, the polynucleotide sequence further encodes a protein / peptide tag or label. 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 one embodiment, the polynucleotide encoding the polypeptide having sweetness-modulating activity is other than the nucleotide sequence of SEQ ID NO: 2, which further encodes a protein / peptide tag or label.

[0026]

[0026] One aspect of the present invention is a polynucleotide (e.g., an isolated polynucleotide) encoding a polypeptide, wherein the encoded polypeptide is: (a) a polypeptide sequence selected from the group consisting of a variant amino acid sequence listed in Table 8, Table 9, or Table 10, wherein the polypeptide further comprises a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag; or (b) a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of a variant amino acid sequence listed in Table 8, Table 9, or Table 10, wherein the polypeptide is and (c) a polypeptide sequence modified from a polypeptide sequence selected from the group consisting of the amino acid sequences of a variant listed in Table 8, Table 9 or Table 10 by deletion, insertion, substitution or addition of 24 or fewer amino acids, wherein the polypeptide further comprises a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag, and the polypeptide sequence differs from that of SEQ ID NO: 3 containing the histidine tag. In a related embodiment, the encoded polypeptide differs from that of SEQ ID NO: 142 containing the histidine tag.

[0027] In certain embodiments, the polynucleotide encoding the polypeptide having sweetness modulating activity is selected from the group consisting of SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, and SEQ ID NO:103, identified in International Patent Application PCT / US2022 / 82443 and U.S. Patent Application Serial No. 18 / 146,958. 3, other than SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:139 or SEQ ID NO:140.

[0028] Another aspect of the invention provides polynucleotides other than the polynucleotide of SEQ ID NO: 4 corresponding to a coding sequence optimized for an HTS with a His tag in E. coli (wherein residues 364-381 correspond to the optional His tag sequence). Another aspect of the invention provides polynucleotides other than the polynucleotide of SEQ ID NO: 6 corresponding to a coding sequence for an HTS optimized for expression in S. cerevisiae (to which the coding sequence for a His tag, e.g., (His)6, has been added).

[0029] In a specific embodiment, the present invention provides Myd variants that exhibit enhanced thermostability compared to the native isoforms of the Myd protein (HTS-1 and HTS-2).

[0030] In an additional aspect, the present invention provides expression cassettes comprising the polynucleotides and vectors comprising the polynucleotides described herein, as well as host cells transformed with the vectors. Also provided is a method for producing a protein having sweet taste modulating activity, the method comprising culturing the transformed host cell in a medium under conditions that result in the production of the protein having sweet taste modulating activity.

[0031]

[0031] In an additional aspect, the present invention provides a host cell that expresses a polypeptide described herein. In an embodiment, the host cell is Escherichia coli. In an embodiment, the host cell is a plant cell.

[0032]

[0032] Another aspect of the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising: (i) a polypeptide sequence selected from the amino acid sequences of a variant 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 to a polypeptide sequence selected from the group consisting of the amino acid sequences of a variant 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 additional embodiments, polypeptide (a) contains at least one modification by deletion, insertion, substitution, or addition of 24 or fewer amino acids compared 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 differs from the polypeptide of SEQ ID NO: 3, and optionally differs from SEQ ID NO: 141, and / or (b) further comprises a protein / peptide tag, optionally an affinity tag, particularly a histidine tag, and wherein the polypeptide has sweet taste modulating activity.

[0033] In another aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified by 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 (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the 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 (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the 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 (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the 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 modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by five mutations selected from those listed in Table 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 the amino acid sequence of SEQ ID NO: 3 modified at different positions by six mutations selected from those listed in Table 8, Table 9, or Table 10. In further embodiments of the above embodiments, the one to six modifications are other than removal of the methionine at position 1. In embodiments, in the polypeptides having one to six amino acid modifications, the methionine at position 1 is present or absent. The present invention also provides polynucleotides encoding the aforementioned mutant (variant) polypeptides of SEQ ID NO: 3. The present invention further provides the aforementioned mutant polypeptides, further comprising a protein / peptide tag, optionally an affinity tag, more particularly a histidine tag.The present invention also provides a polynucleotide encoding the aforementioned mutant polypeptide of SEQ ID NO: 3, wherein the mutant further comprises a protein / peptide tag, optionally an affinity tag, more particularly a histidine tag. In one embodiment, the polypeptide excludes polypeptides comprising a mutation listed in Table 7.

[0034] In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by seven mutations selected from those listed in Table 8, Table 9, or Table 10. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by eight mutations selected from those listed in Table 8, Table 9, or Table 10. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by nine mutations selected from those listed in Table 8, Table 9, or Table 10. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by ten mutations selected from those listed in Table 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 the amino acid sequence of SEQ ID NO: 3 modified at different positions by 11 mutations 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 the amino acid sequence of SEQ ID NO: 3 modified at different positions by 12 mutations selected from those listed in Table 8, Table 9, or Table 10. In further embodiments of the above embodiments, the 1 to 12 modifications are other than removal of the methionine at position 1. In embodiments, in the polypeptides having 1 to 12 amino acid modifications, the methionine at position 1 is present or absent. The present invention also provides polynucleotides encoding the aforementioned mutant polypeptides of SEQ ID NO: 3. The present invention further provides the aforementioned mutant polypeptides, further comprising a protein / peptide tag, optionally an affinity tag, more particularly a histidine tag.The present invention also provides polynucleotides encoding the aforementioned mutant polypeptides of SEQ ID NO: 3, which further comprise a protein / peptide tag, optionally an affinity tag, more particularly a histidine tag. In one embodiment, the polypeptides exclude those comprising a mutation listed in Table 7.

[0035] In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by 13 mutations selected from those listed in Table 8, Table 9, or Table 10. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by 14 mutations selected from those listed in Table 8, Table 9, or Table 10. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by 15 mutations selected from those listed in Table 8, Table 9, or Table 10. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by 16 mutations selected from those listed in Table 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 the amino acid sequence of SEQ ID NO: 3 modified at different positions by 17 mutations 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 the amino acid sequence of SEQ ID NO: 3 modified at different positions by 18 mutations selected from those listed in Table 8, Table 9, or Table 10. In further embodiments of the above embodiments, the 1-18 modifications are other than removal of the methionine at position 1. In embodiments, in the polypeptides having 1-18 amino acid modifications, the methionine at position 1 is present or absent. The present invention also provides polynucleotides encoding the aforementioned mutant polypeptides of SEQ ID NO: 3. The present invention further provides the aforementioned mutant polypeptides, further comprising a protein / peptide tag, optionally an affinity tag, more particularly a histidine tag.The present invention also provides polynucleotides encoding the aforementioned mutant polypeptides of SEQ ID NO: 3, which further comprise a protein / peptide tag, optionally an affinity tag, more particularly a histidine tag. In one embodiment, the polypeptides exclude those comprising a mutation listed in Table 7.

[0036] In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by 19 mutations selected from those listed in Table 8, Table 9, or Table 10. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by 20 mutations selected from those listed in Table 8, Table 9, or Table 10. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by 21 mutations selected from those listed in Table 8, Table 9, or Table 10. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO: 3 modified at different positions by 22 mutations 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 the amino acid sequence of SEQ ID NO: 3 modified at different positions by 23 mutations 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 the amino acid sequence of SEQ ID NO: 3 modified at different positions by 24 mutations selected from those listed in Table 8, Table 9, or Table 10. In further embodiments of the foregoing embodiments, the 1-24 modifications are other than removal of the methionine at position 1. In embodiments, in the polypeptides having 1-24 amino acid modifications, the methionine at position 1 is present or absent. The present invention also provides polynucleotides encoding the aforementioned mutant polypeptides of SEQ ID NO: 3. The present invention further provides the aforementioned mutant polynucleotides, further comprising a protein / peptide tag, optionally an affinity tag, more particularly a histidine tag.The present invention also provides polynucleotides encoding the aforementioned mutant polypeptides of SEQ ID NO: 3, which further comprise a protein / peptide tag, optionally an affinity tag, more particularly a histidine tag. In one embodiment, the polypeptides exclude those comprising a mutation listed in Table 7.

[0037] Another aspect of the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity comprising the amino acid sequence of SEQ ID NO:3 modified by at least one mutation, wherein the at least one mutation is selected from the mutations listed in Table 8, Table 9, or Table 10, and wherein the polypeptide is also modified by one or more additional mutations, wherein the one or more additional mutations are selected from the mutations listed in Table 7. In a further aspect, 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 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, a polynucleotide has 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. The aforementioned polypeptides optionally further comprise a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag.

[0038]

[0038] In another embodiment, the polypeptides of the present invention are optionally isolated and / or optionally purified. In another embodiment, the polynucleotides of the present invention are optionally isolated and / or purified. In another embodiment, the polypeptides of the present invention optionally comprise a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag. In another embodiment, the polypeptides of the present invention optionally comprise an affinity tag. In another embodiment, the polypeptides of the present invention optionally comprise a histidine tag. In another embodiment, the polypeptides of the present invention optionally have a methionine at position 1. In another embodiment, the polypeptides of the present invention are optionally derivatized as described herein. In another embodiment, the polypeptides of the present invention are acylated at the N-terminus and optionally acetylated. In another embodiment, the polypeptides of the present invention are acylated at the N-terminus. In another embodiment, the polypeptides of the present invention have a methionine at position 1, the methionine is derivatized, and optionally the S of the methionine is oxidized. In another embodiment, a polypeptide of the present invention has a methionine at position 1, wherein the methionine is derivatized and the S of the methionine is oxidized. In another embodiment, a polypeptide of the present invention is pegylated at the N-terminus. In another embodiment, a polypeptide of the present invention is derivatized at the C-terminus as described herein. In another embodiment, a polypeptide of the present invention is esterified at the C-terminus.

[0039] In other embodiments, certain polypeptides of the present invention exhibit enhanced sweet taste modulating activity compared to the polypeptide of SEQ ID NO: 3. In other embodiments, certain polypeptides of the present invention exhibit enhanced thermostability compared to the polypeptide of SEQ ID NO: 3.

[0040] Another aspect of the present invention provides compositions comprising one or more polypeptides of the present invention that exhibit flavor-modulating and / or sweetness-modulating activity and / or exhibit sweetness, including a combination of (a) a product for oral administration other than the truffle Mattiromyces terfesioides and (b) a sweetener composition comprising the polypeptide, wherein the combination has an enhanced sweetness and / or flavor alteration or enhancement compared to the product for oral administration. The polypeptides comprise one or more polypeptide sequences, either individually or in combination of two or more thereof, that have flavor-modulating and / or sweetness-modulating activity, and include (i) a polypeptide sequence selected from the amino acid sequences 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 to a polypeptide sequence selected from the group consisting of the amino acid sequences 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) a polypeptide of a variant of Table 8, Table 9 or Table 10, further comprising a protein / peptide tag or affinity tag, wherein the protein / peptide tag is optionally a histidine tag.

[0041] Another aspect of the present invention is a sweetener composition comprising one or more sweet-tasting polypeptides (e.g., HTS variants) of the present invention. A related aspect of the present invention is a flavor modulating composition comprising one or more polypeptides (e.g., HTS variants) of the present invention that exhibit flavor modulating activity. In embodiments, the sweetener composition contains 30 ppm or more total HTS variant polypeptides. In embodiments, the flavor modulating composition contains 30 ppm or less total HTS variant polypeptides.

[0042]

[0042] Another more specific aspect of the present invention provides a composition comprising a combination of (a) a product for oral administration other than truffles of Mattiromyces terfesioides and (b) a sweetener composition comprising a polypeptide, wherein the combination has an enhanced sweetness compared to the product for oral administration. In an embodiment, the polypeptide comprises one or more amino acid sequences selected from the group consisting of (a) the amino acid sequences of variants of Table 8, Table 9 or Table 10. In an embodiment, the polypeptide is not a variant listed in Table 7, Table 3 or Table 6.

[0043] Another aspect of the present invention provides a method for modulating the taste of a product for oral administration. The method includes combining the product for oral administration with an effective amount of a flavor-modulating composition or sweetener composition comprising a polypeptide (e.g., an HTS variant), wherein the product for oral administration is different from the truffle, Mattiromyces terfesioides, and the combination has enhanced flavor or sweetness, respectively, compared to the product for oral administration. 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 individually or in combination with two or more thereof. In embodiments, the polypeptide is not a variant listed in Table 7, Table 3, or Table 6.

[0044] Another aspect of the present invention provides a method for purifying a polypeptide having flavor-modulating activity or sweetness-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, either individually or in combination with two or more thereof, that have sweet taste modulating activity, wherein the polypeptide is (i) a polypeptide sequence selected from the group consisting of a polypeptide sequence of a variant of Table 8, Table 9 or Table 10, wherein the polypeptide sequence differs from SEQ ID NO: 3 and optionally differs from SEQ ID NO: 141; or (ii) a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of a polypeptide sequence of a variant of Table 8, Table 9 or Table 10, wherein the polypeptide sequence differs from SEQ ID NO: 3. (iii) a polypeptide sequence of a variant of Table 8, Table 9 or Table 10, further comprising a protein / peptide tag, wherein the protein tag is optionally an affinity tag, or more particularly a histidine tag; (iv) a polypeptide containing 1 to 24 mutations selected from those listed in Table 8, Table 9 or Table 10, optionally further comprising a protein / peptide tag, wherein the protein tag is optionally an affinity tag, or more particularly a histidine tag.In a related aspect, the invention provides a polypeptide that has been purified by the methods described herein.

[0045] Another aspect of the present invention provides a sweetener composition. In some embodiments, the sweetener composition comprises: (a) one or more polypeptides, the polypeptide sequences being: (i) a polypeptide sequence selected from the amino acid sequences 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 containing at least one modification by deletion, insertion, substitution, or addition of 24 or fewer amino acids compared 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 (iv) a polypeptide comprising any of the polypeptides (i) to (iii), wherein the methionine at position 1 is absent and / or further comprises a protein / peptide tag, optionally an affinity tag, optionally a histidine tag, or optionally a (His)6 tag; and (b) at least one additional sweetener other than a Myd sweet protein.

[0046]

[0046] The additional sweetener is optionally selected from steviol glycoside sweeteners, mogroside sweeteners, sucrose, allulose, sucralose, polyols, and high fructose corn syrup (HFCS). In another embodiment, the sweetener composition comprises (a) one or more polypeptides, wherein: (i) a polypeptide sequence selected from the amino acid sequences 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 sequences of any variant listed in Table 8, Table 9, or Table 10; or (iii) a polypeptide sequence having at least 24 amino acid deletions, insertions, substitutions, or additions compared 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 (iv) a polypeptide comprising any of polypeptides (i) to (iii), wherein methionine at position 1 is absent and / or 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 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 combinations thereof.

[0047] In embodiments, the sweetener compositions described herein comprise at least one additional ingredient selected from sucrose, mannitol, citric acid, hypoxanthine, theophylline, leucine, and combinations thereof.

[0048] 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 with the methionine missing at position 1. 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 with the methionine missing at position 1.

[0049] In embodiments, the polypeptide of the sweetener composition is present in an amount of about 1 ppm to about 50 ppm. In some embodiments, the polypeptide is present in an amount of about 1 ppm to about 40 ppm. In further embodiments, the polypeptide is present in an amount selected from 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. In some embodiments, the polypeptide is present in an amount of about 5 ppm to about 50 ppm. In some embodiments, the polypeptide is present in an amount of about 10 ppm to about 50 ppm. In some embodiments, the polypeptide is present in an amount of about 15 ppm to about 50 ppm. In some embodiments, the polypeptide is present in an amount of about 20 ppm to about 50 ppm. In some embodiments, the polypeptide is present in an amount of about 25 ppm to about 50 ppm. In some embodiments, the polypeptide is present in an amount of about 30 ppm to about 50 ppm. In some embodiments, the polypeptide is present in an amount of about 35 ppm to about 50 ppm. In some embodiments, the polypeptide is present in an amount of about 40 ppm to about 50 ppm. 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.

[0050] In another aspect, the present invention provides a combination of a product for oral administration that is not the truffle Mattiromyces terfesioides and a sweetener composition described herein. In some embodiments, the product for oral administration has at least one improved sensory characteristic compared to a product for oral administration that does not contain the sweetener composition, the sensory characteristic being selected from the group consisting of aroma, flavor, base taste (sweet, sour, salty, bitter, or umami), aftertaste or lingering taste, temporal profile, texture, or a combination thereof. In embodiments, the product for oral administration is a solid food or beverage product. In embodiments, the product for oral administration is a yogurt product. In embodiments, the product for oral administration is a chewing gum product.

[0051] In another aspect, the present invention provides a beverage or beverage product comprising the sweetener composition described herein. In some embodiments, the beverage or beverage product has at least one improved sensory characteristic compared to a beverage or beverage product not containing the sweetener composition, the sensory characteristic being selected from the group consisting of aroma, flavor, base taste (sweet, sour, salty, bitter, or umami), aftertaste or lingering taste, temporal profile, texture, or a combination thereof. In some embodiments, the beverage or beverage product is selected from low-calorie or calorie-free beverages or beverage products. In embodiments, the beverage is selected from cola, ginger ale, soft drinks, root beer, fruit juice, fruit-flavored juice, vegetable juice, vegetable-flavored juice, sports drinks, energy drinks, plant protein drinks, water-like beverages (e.g., water containing natural or synthetic flavorings), certain teas (e.g., black tea, green tea, red tea, oolong tea), coffee, cocoa drinks, and dairy-containing beverages (e.g., dairy drinks, dairy-containing coffee, cafe au lait, milk tea, and fruit-dairy drinks). In an embodiment, the beverage or beverage product comprises at least one organic acid addition salt that 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.

[0052] In another aspect, the present invention provides a method for improving at least one sensory characteristic of a product for oral administration described herein. In an embodiment, the product for oral administration is a solid food or liquid beverage product. In one embodiment, the present invention provides a method for improving at least one sensory characteristic of a product for oral administration described herein. The method comprises adding a sweetener composition described herein to a solid or liquid matrix, thereby providing a product having at least one improved sensory characteristic. In an embodiment, the improved sensory characteristic is selected from the group consisting of aroma, flavor, base taste (sweet, sour, salty, bitter, or umami), aftertaste or lingering taste, temporal profile, texture, or a combination thereof. In an embodiment, the amount of polypeptide present in a product improved by the method is an amount of about 1 ppm to about 50 ppm, 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. In an embodiment, the product is a solid food product and the sweetener composition is added to, combined with, or mixed into the solid food product.

[0053] In an embodiment, the present invention provides a method for improving at least one sensory characteristic of a beverage or beverage product described herein. The method comprises adding a sweetener composition described herein to a liquid matrix, thereby providing a beverage or beverage product having at least one improved sensory characteristic. In an embodiment, the improved sensory characteristic is selected from the group consisting of aroma, flavor, base taste (sweet, sour, salty, bitter, or umami), aftertaste or lingering taste, temporal profile, texture, or a combination thereof. In an embodiment, the amount of polypeptide present in the beverage or beverage product improved by the method is from about 1 ppm to about 50 ppm, from about 1 ppm to about 40 ppm, from about 1 ppm to about 30 ppm, from about 1 ppm to about 25 ppm, from about 1 ppm to about 20 ppm, or from about 1 ppm to about 15 ppm.

[0054] In another aspect, the present invention provides a method for changing / modifying at least one sensory characteristic of a product for oral administration described herein. The method comprises adding a sweetener composition described herein to the product, thereby providing a product having at least one changed / modified sensory characteristic. In embodiments, the changed / modified sensory characteristic is selected from the group consisting of aroma, flavor, base taste (sweet, sour, salty, bitter, or umami), aftertaste or lingering taste, temporal profile, texture, or a combination thereof. In embodiments, the amount of polypeptide present in the product changed / modified by the method is an amount of about 1 ppm to about 50 ppm, 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. In embodiments, the product for oral administration is a food or beverage product. In an embodiment, the product is a solid food product and the sweetener composition is added to, combined with, or mixed into the solid food product.

[0055] In another aspect, the present invention provides a method for changing / modifying at least one sensory characteristic of a beverage or beverage product described herein. The method comprises adding a sweetener composition described herein to a liquid matrix, thereby providing a beverage or beverage product having at least one changed / modified sensory characteristic. In embodiments, the changed sensory characteristic is selected from the group consisting of aroma, flavor, base taste (sweet, sour, salty, bitter, or umami), aftertaste or lingering taste, temporal profile, texture, or a combination thereof. In embodiments, the amount of polypeptide present in a beverage or beverage product changed / modified by the method is from about 1 ppm to about 50 ppm, from about 1 ppm to about 40 ppm, from about 1 ppm to about 30 ppm, from about 1 ppm to about 25 ppm, from about 1 ppm to about 20 ppm, or from about 1 ppm to about 15 ppm.

[0056]

[0056] Other aspects and embodiments of the present invention will be apparent from a consideration of the drawings, detailed description and non-limiting examples set forth herein. [Brief explanation of the drawings]

[0057] [Figure 1] FIG. 1 shows a Coomassie-stained SDS-PAGE gel of proteins obtained from a partially purified M. terfesioides gleba fraction. [Figure 2]

[0058] 2 shows a Coomassie-stained SDS-PAGE gel of the purification steps of a His-tagged sweet polypeptide expressed in E. coli from the coding region of SEQ ID NO: 4. Lane 1: molecular weight standard; Lane 3: crude lysate; Lane 4: flow-through fraction from HisPur™ Ni-NTA; Lane 5: wash 1; Lane 6: wash 2; Lane 7: wash 3; Lane 8: elution fraction. The His-tagged sweet protein expressed in E. coli is a His-tagged version of HTS-2 (SEQ ID NO: 142) lacking methionine. [Figure 3]

[0059] Figure 3 shows the concentration-response functions for sweetness of mycodulcein (honey truffle sweetener (HTS)), aspartame, thaumatin, and rebaudioside A. Data are plotted as the ratio (p) of the response produced to the 200 mM sucrose-related ("sweet") target. Each data point in the curves for mycodulcein, aspartame, thaumatin, and rebaudioside A was calculated as the average over 32 replicates, and for the sucrose curve it was averaged over 16 replicates; error bars are SEM. Points for the water and sucrose controls were similarly calculated as the average over 128 and 64 replicates, respectively. Curves were fitted by nonlinear regression. [Figure 4A]

[0060] Figure 4A shows SDS-PAGE analysis, Coomassie staining, of fractions eluted from Capto MMC, a multimodal weak cation exchange resin. M: protein marker; lane 1: eluted fraction showing low purity after cation exchange. The arrow indicates the mycodulcein (native HTS) band. [Figure 4B]

[0061] Figure 4B shows SDS-PAGE analysis, Coomassie staining, of two eluted fractions collected during gradient elution from a HiScreen Capto Butyl (Cytiva Sweden AB, Upsala, Sweden) column analyzed by SDS-PAGE. Lane 1 shows eluted fraction 1, which does not contain mycodulcein, and lane 2 shows eluted mycodulcein. The purity of the eluted fractions was determined by GelAnalyzer to be approximately 86%. The arrow indicates the mycodulcein (native HTS) band. [Figure 4C]

[0062] Figure 4C shows an SDS-PAGE analysis, Coomassie stain, of proteins eluted from the HIC column after chromatography on HiPrep® 26 / 60 Sephacryl® S-200 (Cytiva Sweden AB, Upsala, Sweden). Lane 1 shows purified his-tagged mycodulcein, and lane 2 shows purified native mycodulcein. The purity of the eluted fractions was determined by GelAnalyzer to be approximately 98%. The arrow indicates the mycodulcein (native HTS) band. [Figure 5]

[0063] FIG. 5 is a map of the pD44-CH vector, which is isopropyl beta-D-1-thiogalactopyranoside (IPTG) inducible. [Figure 6A]

[0064] Figures 6A and 6B contain graphs of exemplary melting temperature results obtained from a GloMelt™ thermostability assay comparing the native HTS (1) and HTS variant S33C_I49C (double mutant, 2). Figure 6A compares the melting curves of the native HTS (1) and the double mutant (2). [Figure 6B] Figures 6A and 6B contain graphs of exemplary melting temperature results obtained from a GloMelt™ thermostability assay comparing the native HTS (1) and HTS variant S33C_I49C (double mutant, 2). Figure 6B compares the melting peak curves of 1 and 2. Relative fluorescence (RFU) is measured as a function of temperature. A 7.5°C increase in the average melting temperature is observed for the double mutant compared to the native HTS protein. [Figure 7]

[0065] Figure 7 shows melting temperature results from a GloMelt™ thermostability assay comparing the native HTS protein with the HTS variant double mutant S33C_I49C, in which samples were brought to pasteurization temperature (63°C) and held for 1 hour. DETAILED DESCRIPTION OF THE INVENTION

[0058]

[0066] The present invention provides embodiments of isolated nucleotides encoding Myd1 polypeptide variants, the encoded Myd1 polypeptide variants capable of modulating flavor and / or sweetness and perception (also referred to as honey truffle sweetener (HTS) proteins and variants thereof), and methods of making and using the same. The present invention provides embodiments of MYD1 variants and Myd1 polypeptide variants, either alone or in combination with foods, beverages, dietary supplements, or pharmaceuticals; and methods of modifying the taste of such foods, beverages, dietary supplements, or pharmaceutical compositions with the isolated polynucleotides and polypeptides of the present invention. 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.

[0059]

[0067] 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 a non-naturally occurring HTS polypeptide, including, for example, any of the polypeptides according to the present invention having at least 80% sequence identity to a variant in Table 8, Table 9, or Table 10, which also have flavor-modulating (or altering) and / or sweetness-altering activity.

[0060]

[0068] Partially purified extracts of sweet-tasting Terfeizioides gleba were subjected to de novo amino acid sequencing to identify a 20-mer N-terminal sequence. After de novo assembly of the entire transcriptome of Mattiromyces terfeizioides gleba using RNA-seq reads, the Myd1 coding sequence (putatively derived from the MYD1 gene) was identified. Screening the entire transcriptome of M. terfeizioides using the 20-mer N-terminal sequence identified a transcript predicted to encode a protein with 100% identity at the N-terminus. The identified transcript is predicted to encode a 121-amino acid protein. This method identified the polynucleotide sequence shown in SEQ ID NO:1. Identification of the start and stop codons in the transcript led to the identification of the putative coding sequence shown in SEQ ID NO:2. SEQ ID NO:3 is the predicted encoded protein, a 121-amino acid protein. The naturally occurring protein isolated from M. terfesioides gleba was subsequently found to be a mixture of two isoforms: the protein of SEQ ID NO:3 and the mature protein of SEQ ID NO:3 (referred to below as SEQ ID NO:141) lacking the methionine (Met) residue at amino acid position 1. The predicted protein of SEQ ID NO:3 shared 31% or less identity with other protein sequences in GENBANK.

[0061]

[0069] Native HTS is believed 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. Native HTS isolated from M. terfeizioides, as described herein, 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 M. terfeizioides vary, at least depending on the truffle source. In the as-isolated state, approximately 80% of HTS-1 is acetylated at the N-terminal amine group. No significant difference in sweetness has been observed between the two sequence isoforms of HTS. N-terminal acetylation of the HTS polypeptide HTS-1 also had no significant effect on sweetness.

[0062]

[0070] Polynucleotides encoding the polypeptide of SEQ ID NO: 3 have been successfully expressed in multiple hosts. In some cases, polynucleotide sequences encoding the polypeptide of SEQ ID NO: 3 have been codon-optimized for expression in a given host, as is known in the art. For example, intracellular expression in E. coli using codons optimized to express a polynucleotide encoding SEQ ID NO: 3 using the nucleotide coding sequence of SEQ ID NO: 4 results in a protein with a sweet taste. For example, intracellular expression in E. coli using a polynucleotide encoding SEQ ID NO: 3 and codons optimized to express a (His)6 tag results in a protein with a sweet taste. Subsequently, the sweet protein with a His tag expressed from SEQ ID NO: 4 in E. coli was determined by proteomic analysis to be SEQ ID NO: 141 (SEQ ID NO: 3 without a methionine at position 1), and expression of the His tag of the HTS-1 isoform of the protein (SEQ ID NO: 148) was not observed. The sweet protein of SEQ ID NO: 141 (without a His tag) is the protein expressed upon expression of the optimized E. coli coding region without a His tag (SEQ ID NO: 148).

[0063]

[0071] For example, intracellular expression in Saccharomyces cerevisiae using codons optimized to express a polynucleotide encoding SEQ ID NO:3 with an additional His-tag coding sequence (SEQ ID NO:6) resulted in a sweet-tasting protein. The expressed protein is a mixture of HTS-1 and HTS-2 isoforms, each with a His-tag. The relative amounts of HTS-2 to HTS-1 are approximately 60% to 40% by weight. Extracellular expression in Saccharomyces cerevisiae using codons optimized to facilitate secretion into the fermentation medium and an appropriate signal peptide at the N-terminus resulted in a non-sweet expression product.

[0064]

[0072] For example, intracellular expression in Yarrowia lipolytica using codon-optimized sequences to express a polynucleotide encoding SEQ ID NO:3 results in a sweet-tasting protein. The expressed protein is a mixture of HTS-1 and HTS-2 isoforms. The relative amounts of HTS-2 to HTS-1 are approximately 20% to 80% by weight.

[0065]

[0073] For example, intracellular expression in Pichia pastoris using codon-optimized sequences to express a polynucleotide encoding SEQ ID NO:3 results in a sweet-tasting protein. The expressed protein is a mixture of the HTS-1 and HTS-2 isoforms. The relative amounts of HTS-2 to HTS-1 are approximately 90% to 10% by weight.

[0066]

[0074] The coding sequences of native mycodulcein (HTS) codon-optimized 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).

[0067]

[0075] In embodiments, the present invention provides a non-naturally occurring sweet protein comprising a non-naturally occurring mixture of the two isoforms, HTS-1 and HTS-2. In embodiments, the present invention provides HTS-2 (SEQ ID NO: 141) substantially free of the other isoform, HTS-1, where substantially free means less than 10% by weight of HTS-1 (SEQ ID NO: 3). In embodiments, the present invention provides HTS-2 (SEQ ID NO: 141) substantially free of the other isoform, HTS-1, where free means less than 1% by weight of HTS-1 (SEQ ID NO: 3). In embodiments, the present invention provides HTS-1 (SEQ ID NO: 3) substantially free of the other isoform, HTS-2, where substantially free means less than 10% by weight of HTS-1 (SEQ ID NO: 141). In embodiments, the present invention provides HTS-1 (SEQ ID NO: 3) substantially free of the other isoform, HTS-2, where free means less than 1% by weight of HTS-2 (SEQ ID NO: 141). In embodiments, the present invention provides non-naturally occurring mixtures of HTS-1 and HTS-2, particularly those enriched in HTS-2, wherein the amount of HTS-2 is greater than 60% by weight, e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more by weight. In embodiments, the present invention provides non-naturally occurring mixtures of HTS-1 and HTS-2, particularly those enriched in HTS-1, wherein the amount of HTS-1 is greater than 60% by weight, e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more by weight.

[0068]

[0076] In embodiments, the present invention provides a sweet protein or a codon-optimized version of the coding sequence of SEQ ID NO:2 produced by recombinant expression of the coding sequence of SEQ ID NO:2 in a non-native host (e.g., a host other than the truffle Mattiromyces terfesioides). In specific embodiments, the sweet protein is produced by recombinant expression in bacteria, yeast, or fungi. In specific embodiments, the non-native host is Escherichia coli. In specific embodiments, the non-native host is Saccharomyces cerevisiae. In specific embodiments, the non-native host is Yarrowia lipolytica. In specific embodiments, the non-native host is Pichia pastoris. In specific embodiments, recombinant expression in a non-native host results in a non-naturally occurring mixture of HTS-1 and HTS-2, the naturally occurring isoforms of mycodulcein (Myd).

[0069]

[0077] Polynucleotides

[0078] 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, but are not limited to, polynucleotides that can encode polypeptides such as variants of Table 8, Table 9, or Table 10, or those polypeptides that further have a histidine tag, or nucleic acid sequences that have 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 with the polynucleotides encoding variants of Table 8, Table 9, or Table 10, or those polypeptides that further have a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag. It will be understood that polynucleotides are provided and useful in the present invention, both with and without sequences encoding protein / peptide tags, affinity tags, or histidine tags. Furthermore, for any particular polynucleotide shown to encode a given histidine tag (e.g., (His)), it will be understood that 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 encoded by 3-30 or 3-18 nucleotides. In embodiments, the variants enumerated herein above include a methionine at position 1 or are absent at position 1. In embodiments, the variant HTS polypeptide is other than the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 141.

[0070]

[0079] In one embodiment, the polynucleotide comprises, consists essentially of, or consists of a polynucleotide selected from the group consisting of 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 encoding, or capable of encoding, a variant of Table 8, Table 9, or Table 10. In an embodiment, the encoded variant polypeptide is other than the polypeptide of SEQ ID NO:3 or SEQ ID NO:141.

[0071]

[0080] Unless expressly stated otherwise, 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 the other modifications. In addition, multiple modifications in a polynucleotide sequence can be the same or different from each other; the modification options for each modification can be independently varied, such as a deletion, insertion, substitution, or addition, unless expressly stated otherwise. When multiple modifications are present, each modification can be independently substituted, added, inserted, or deleted, regardless of the other modifications. In one embodiment, a polynucleotide sequence has at least one substitution modification. In certain embodiments, a polynucleotide sequence has multiple substitution modifications; each substitution can be independently substituted with a selected substitution, regardless of the other substitutions. In addition, multiple substitutions in a polynucleotide sequence can be the same or different from each other; the nucleotide substitution options for each substitution can be independently varied, unless expressly stated otherwise. Where a polynucleotide sequence has multiple substitutions, each substitution may be independently selected, regardless of what the other substitutions are. In embodiments, the polynucleotides described herein are optionally isolated and / or optionally purified.

[0072]

[0081] In embodiments, at least 80% sequence identity for polynucleotides includes, but is not limited to, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity. In embodiments, at least 80% sequence identity for polynucleotides also includes, but is not limited to, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity.

[0073]

[0082] In one embodiment, the polynucleotide comprises: (a) an amino acid sequence of a variant of Table 8, Table 9, or Table 10, or a polypeptide thereof further comprising a protein / peptide tag, affinity tag, or histidine tag; (b) an amino acid sequence of a variant of Table 8, Table 9, or Table 10, or a polypeptide thereof further comprising a protein / peptide tag, affinity tag, or histidine tag, and at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least and (c) an amino acid sequence modified from the amino acid sequence of Table 8, Table 9, or Table 10 by the deletion, insertion, substitution, or addition of 24 or fewer 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, or 24 amino acid modifications). In the above-listed embodiments, the polypeptide encoded by the polynucleotide is optionally a polypeptide other than that of SEQ ID NO: 3, and optionally other than that of SEQ ID NO: 141. When an amino acid sequence has multiple modifications, the number of modifications the amino acid has can range from at least 1 to a maximum of 24 modifications, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 modifications, as desired. Furthermore, unless expressly stated otherwise, when an amino acid sequence has multiple modifications, each modification 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 an amino acid sequence may be the same or different from each other; the modification options for each modification may vary independently, such as deletion, insertion, substitution, or addition, unless explicitly stated otherwise. When an amino acid sequence has multiple modifications, each modification may be a substitution, addition, insertion, or deletion, independently of the other modifications. In one embodiment, an amino acid sequence has at least one substitution modification. In a specific embodiment, an amino acid sequence has multiple substitution modifications; each substitution modification may be independently replaced with a selected substitution, independently of the other substitutions. In one embodiment, an 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; the substitution options for each amino acid may vary independently, unless explicitly stated otherwise. When an amino acid sequence has multiple substitutions, each substitution may be independently selected, independently of the other substitutions. In embodiments, the encoded polypeptide may comprise 1 to 6, 1 to 12, 1 to 18, or 1 to 24 substitutions compared to the polypeptide of SEQ ID NO: 3, wherein the substitutions are listed in Table 8. In embodiments, the encoded polypeptide may comprise 1 to 6, 1 to 12, 1 to 18, or 1 to 24 substitutions compared to the polypeptide of SEQ ID NO: 3, wherein the substitutions are listed in Table 9. In embodiments, the encoded polypeptide may comprise 1 to 6, 1 to 12, 1 to 18, or 1 to 24 substitutions compared to the polypeptide of SEQ ID NO: 3, wherein the substitutions are listed in Table 10. In embodiments, the encoded polypeptide may comprise 1 to 6, 1 to 12, 1 to 18, or 1 to 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 comprise 1 to 6, 1 to 12, 1 to 18, or 1 to 24 substitutions compared to the polypeptide of SEQ ID NO: 3, wherein the substitutions are other than those listed in Table 7. In embodiments, the encoded polypeptide may comprise 1 to 6, 1 to 12, 1 to 18, or 1 to 24 substitutions compared to the polypeptide of SEQ ID NO: 3, wherein the substitutions are other than those listed in Table 3 or 6. In embodiments, the encoded polypeptide may comprise 1 to 6, 1 to 12, 1 to 18, or 1 to 24 substitutions compared to the polypeptide of SEQ ID NO: 3, wherein the substitutions are other than those listed in Table 3 or Table 6. In embodiments, the encoded polypeptide may comprise 1 to 6, 1 to 12, 1 to 18, or 1 to 24 substitutions compared to the polypeptide of SEQ ID NO: 3, wherein the substitutions are those listed in Tables 9 and 10, and other than those listed in Table 3 or Table 6 or Table 7.

[0074]

[0083] In certain embodiments of the polynucleotides described herein, the polynucleotides encode the polypeptides described herein and also encode a protein / peptide tag, affinity tag, or histidine tag. In all cases where such specific polynucleotides encode such tags, the present invention also provides polynucleotides that exclude the sequence encoding the tag. In additional embodiments, in the polynucleotides described herein that encode a histidine tag, the sequence encoding the histidine tag may be deleted or eliminated, or may be replaced with a coding sequence for a different protein / peptide tag or a different affinity tag, such as a different histidine tag.

[0075]

[0084] The invention provides isolated and / or purified embodiments of each polynucleotide, where applicable. The invention provides isolated or non-isolated, purified or non-purified embodiments of each polynucleotide capable of encoding a variant of Table 8, Table 9, or Table 10 described herein, where applicable. The invention provides isolated or non-isolated, purified or non-purified embodiments of each polynucleotide capable of encoding a variant of Table 8, Table 9, or Table 10 described herein, where applicable, having one or more mutations.

[0076]

[0085] The present invention also provides host cells transformed with expression cassettes and vectors comprising one or more polynucleotides encoding Myd polypeptide variants (HTS variants).

[0077]

[0086] Polynucleotides encoding the HTS variants of the present invention may be in the form of single- or double-stranded DNA, RNA, or artificial nucleic acids, or may be cDNA or chemically synthesized DNA without any introns. The term "MYD family" may refer to (1) naturally occurring alleles, mutants, alleles, and polymorphic variants, including interspecies homologs, that encode polypeptides spanning a range of approximately 25 amino acids, optionally spanning a range of 50-100 amino acids, and having at least about 35-50% amino acid sequence identity, and optionally about 60, 75, 80, 85, 90, 95, 96, 97, 98, or 99% amino acid sequence identity, to SEQ ID NO: 3. In one embodiment, the term "isolated" encompasses products that have been removed from a biological environment (e.g., a cell, tissue, culture medium, body fluid, etc.) or otherwise increased in purity to any degree (e.g., a product isolated from a synthetic medium). Thus, an isolated product may be synthetic or naturally produced. In one embodiment, the term "isolated" encompasses a product that is isolated from other proteins in its natural environment. In an embodiment, the term "isolated" encompasses a product that is isolated from carbohydrates in its natural environment.

[0078]

[0087] The term "nucleic acid" or "nucleic acid sequence" refers to a deoxyribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogs of natural nucleotides. The term also encompasses nucleic acid-like structures with synthetic backbones (e.g., Oligonucleotides and Analogues, a Practical Approach, F. Eckstein, ed., Oxford University Press (1991); Antisense Strategies, Annals of the NYA 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), WO97 / 03211; WO96 / 39154; Mata, Toxicol. Appl. Pharmacol. 144:189-197 (1997); Strauss-Soukup, Biochemistry 36:8692-8698 (1997); Samstag, Antisense Nucleic Acid Drug Dev, 6:153-156 (1996).

[0079]

[0088] As used herein, a "nucleic acid probe or oligonucleotide" is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bond, typically through complementary base pairing, typically through the formation of hydrogen bonds. As used herein, a probe may contain natural bases (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe may be joined by linkages other than phosphodiester bonds, as long as they do not interfere with hybridization. Thus, for example, a probe may be a peptide nucleic acid in which the constituent bases are joined by peptide bonds rather than phosphodiester bonds. Those skilled in the art will understand that a probe may bind to a target sequence lacking complete complementarity with the probe sequence, depending on the stringency of the hybridization conditions. The probe may optionally be directly labeled, for example, with an isotope, chromophore, lumiphore, or chromogen, or indirectly labeled, such as with biotin, to which a streptavidin complex can subsequently bind. By assaying for the presence or absence of the probe, the presence or absence of a selected sequence or subsequence can be detected.

[0080]

[0089] The polynucleotide or polypeptide may be naturally occurring or non-naturally occurring (e.g., synthetic, recombinant, modified, and / or variant product). In one aspect, the naturally occurring or non-naturally occurring product is isolated or purified. In another aspect, the naturally occurring or non-naturally occurring product is not isolated or purified. In the embodiments described herein, the polynucleotides and polypeptides are not naturally occurring. In the embodiments described herein, the polynucleotides and polypeptides are naturally occurring mycodulcein (HTS) polypeptides and non-naturally occurring variants of naturally occurring polynucleotide sequences encoding naturally occurring mycodulcein (HTS).

[0081]

[0090] "Recombinant," as used herein, refers to a polynucleotide that is synthesized or otherwise manipulated in vitro (e.g., a "recombinant polynucleotide"), a method of using a recombinant polynucleotide to produce a gene product in a cell or other biological system, or a polypeptide encoded by a recombinant polynucleotide (a "recombinant protein"). "Recombinant means" also encompasses the ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into an expression cassette or vector, e.g., for inducible or constitutive expression, for expression of a fusion protein comprising a translocation domain of the invention and a nucleic acid sequence amplified using the primers of the invention.

[0082]

[0091] The terms "amplifying" and "amplification," as used herein, refer to the use of any suitable amplification technique to generate or detect recombinant or naturally expressed nucleic acids, as described in detail below. For example, the 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 invention (e.g., taste stimuli-binding sequences of the invention) in vivo or in vitro.

[0083]

[0092] 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, such as (1) purification from other naturally occurring associated 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 may be isolated or otherwise associated with structures or compounds with which they are not normally associated in nature, according to various methods and processes known to those of skill in the art. In one embodiment, the polypeptides described herein contain up to 5% by weight (e.g., up to 4%, up to 3%, up to 2%, up to 1%) of other fungal proteins, e.g., fungal proteins other than Myd proteins.

[0084]

[0093] A "modified" or "variant" product refers to a product (e.g., a polynucleotide or polypeptide) that has been altered from its original (e.g., naturally occurring) structure. Variants, as used herein, encompass polynucleotides or polypeptides having one or more alterations to the nucleic acid or amino acid sequence, respectively. Alterations include modifications to the nucleic acid or amino acid sequence, such as additions, deletions, insertions, and substitutions. Modified or variant products can also include those modified to include disulfide bond formation, as well as those derivatized by glycosylation, lipidation, acylation, acetylation, phosphorylation, or any other manipulation using post-translational modification methods, such as conjugation with a labeling component, compared to the original structure. As is known in the art, derivatization can be achieved by chemical or enzymatic methods after translation of the polypeptide. Derivatization can also be achieved by post-translational modification during polypeptide expression in a selected host.

[0085]

[0094] Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences in addition to the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved, for example, by generating sequences in which the third position of one or more selected codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0086]

[0095] It will be understood that where a specific polynucleotide is herein indicated as encoding a histidine tag, polynucleotides excluding the sequence encoding the histidine tag are also provided. It will be 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.

[0087]

[0096] Polypeptides

[0097] It should be understood that embodiments of the present invention also encompass 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. The terms also apply to naturally occurring and non-naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid.

[0088]

[0098] Embodiments of the present invention include polypeptides 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 comprising 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 (present less than 95%, or more particularly less than 99% by weight) of the polypeptide of SEQ ID NO: 141. In embodiments, the polypeptide is a polypeptide of SEQ ID NO: 141 that is substantially free (present at less than 95%, or more particularly, less than 99% by weight) of the polypeptide of SEQ ID NO: 3. Optionally, the amino acid sequence has at least one and up to 24 modifications. If the amino acid sequence has multiple modifications, the number of modifications the amino acids have can range from at least one and up to 24 modifications, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 modifications, as desired. Furthermore, unless expressly stated otherwise, if the amino acid sequence has multiple 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 an amino acid sequence may be the same or different from one another; the modification options for each modification may independently vary, e.g., deletion, insertion, substitution, or addition, unless explicitly stated otherwise.When an amino acid sequence has multiple modifications, each modification may be independently a substitution, addition, insertion, or deletion, regardless of the other modifications. In one embodiment, the amino acid sequence has at least one substitution modification. In certain embodiments, the amino acid sequence has multiple substitution modifications; each substitution modification may be independently substituted with a selected substitution, regardless of the other substitutions. In addition, multiple substitutions in an amino acid sequence may be the same or different from each other; the substitution options for each amino acid may be independently varied, unless explicitly stated otherwise. When an amino acid sequence has multiple substitutions, each substitution may be independently selected, regardless of the other substitutions. The term "consisting essentially of" allows for the inclusion of ingredients that are not essential to and do not significantly affect the function or activity of the product, such as anti-caking agents, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltodextrose, gum arabic, etc.).

[0089]

[0099] Another embodiment of the present invention includes a recombinant polypeptide having sweet taste modulating activity that comprises, consists essentially of, or consists of a sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to a variant of Table 8, Table 9, or Table 10, or a variant of these tables further comprising a histidine tag or fused to a heterologous signal or transit peptide. The term "essentially consisting of" allows for the inclusion of ingredients that are not essential to and do not significantly affect the function or activity of the product, such as anti-caking agents, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltodextrose, gum arabic, etc.).

[0090]

[0100] In one 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; 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.

[0091]

[0101] In one 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 up to 24 amino acid modifications set forth in Table 8 and further has 1-24 amino acid modifications set forth in Table 7, the total number of modifications being 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 up to 24 amino acid modifications set forth in Table 8 and 1-24 amino acid modifications set forth in Table 7.

[0092]

[0102] In one 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 up to 12 amino acid modifications set forth in Table 8 and further has 1-12 amino acid modifications set forth 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 up to 12 amino acid modifications set forth in Table 8 and 1-12 amino acid modifications set forth in Table 7.

[0093]

[0103] In one 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 to up to six amino acid modifications set forth in Table 8 and further has one to six amino acid modifications set forth 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 to up to six amino acid modifications set forth in Table 8 and one to six amino acid modifications set forth in Table 7.

[0094]

[0104] In one 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, 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.

[0095]

[0105] In one 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 to up to 24 amino acid modifications shown in Table 8, Table 9, or Table 10, and does not have a modification 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 to up to 24 amino acid modifications shown in Table 8, Table 9, Table 10, and not having a modification shown in Table 7, Table 3, or Table 6.

[0096]

[0106] In one 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 to up to six amino acid modifications 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 SEQ ID NO: 3 polypeptide having at least one to up to six amino acid modifications shown in Table 8, Table 9, or Table 10.

[0097]

[0107] In certain embodiments described herein, certain polypeptides are described as having a histidine tag or optionally having a histidine tag (represented in the sequence listing described herein as XXXXXX, where X is His). The present invention provides all specific polypeptides that include or optionally include a histidine tag, in addition to corresponding polypeptides in which the histidine tag has been omitted. The present invention also provides all specific polypeptides that include a histidine tag or an optional histidine tag, in addition to polypeptides in which the histidine tag has been omitted or in which the histidine tag has been replaced with a different protein tag, such as a different histidine tag.

[0098]

[0108] In one embodiment, the Myd peptide (HTS) variants described herein are capable of sweet taste-modulating activity. The HTS polypeptide variants of the present invention can have, for example, functional, physical, and chemical effects on taste receptors, such as sweet taste receptors. "Sweet taste-modulating activity" can refer to the inhibitory, activating, e.g., agonist or antagonist properties of the polypeptides of the present invention identified using in vitro and in vivo assays related to taste transduction. Proteins with inhibitory activity can bind to and partially or completely block stimulation, reduce, prevent, delay activation, inactivate, desensitize, or down-regulate taste transduction, e.g., be antagonists. Activating polypeptides can bind to and stimulate, increase, open, activate, promote, enhance activity, sensitize, or up-regulate taste transduction, e.g., be agonists. Activating polypeptides are preferred.

[0099]

[0109] Sweetness modulation also refers to enhance the taste, for example, sweetness, of a particular product for oral administration when administered as a combination.In addition to sweetness, HTS variants may also show differences in sweetness intensity and sweetness duration.These sweetness characteristics can be evaluated by taste test as described herein.

[0100]

[0110] Modulation of sweetness also refers to the polypeptide variants described herein either exhibiting a sweet taste or, when added to a composition or formulation in which the polypeptide variant does not itself exhibit a sweet taste, imparting a sweet taste to the composition or formulation.

[0101]

[0111] In some embodiments, the Myd (HTS) polypeptide variants of the present invention comprise a polypeptide that is at least as sweet (w / w basis) as sucrose (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 of the methods described above or known in the art. In other embodiments, the Myd polypeptide variant has 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.

[0102]

[0112] In an embodiment, the polypeptide having sweet taste modulating activity comprises a modified SEQ ID NO: 3 with from 1 to up to 24 different amino acid modifications shown in Table 8, Table 9 or Table 10. In an embodiment, the polypeptide having sweet taste modulating activity comprises a modified SEQ ID NO: 141 with from 1 to up to 24 different amino acid modifications shown in Table 8, Table 9 or Table 10.

[0103]

[0113] In one embodiment, the Myd (HTS) peptide variants described herein have flavor-modifying properties (FMP) or activity. A compound with FMP is a compound containing a protein or polypeptide that alters one of the perceived flavor attributes of a formulation compared to a formulation without the compound, but does not directly contribute to the flavor attribute. The compound has flavor-modifying properties below the perceptible sweetness threshold concentration, and above that threshold provides a direct flavor perception. The HTS protein / polypeptide or variant can exhibit the FMP threshold of the wild-type protein in the formulation, below which 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 depends on the HTS protein and the specific application.

[0104]

[0114] In one embodiment, a composition or formulation containing an HTS protein or variant at about 1 to about 50 ppm exhibits a flavor modification but does not provide added sweetness to the composition or formulation. In one embodiment, a composition or formulation containing an HTS protein or variant at about 1 to about 40 ppm exhibits a flavor modification but does not provide added sweetness to the composition or formulation. In one embodiment, a composition or formulation containing an HTS protein or variant at about 1 to about 30 ppm exhibits a flavor modification but does not provide added sweetness to the composition or formulation. In one embodiment, a composition or formulation containing an HTS protein or variant at about 1 to about 25 ppm exhibits a flavor modification but does not provide added sweetness to the composition or formulation. In one embodiment, a composition or formulation containing an HTS protein or variant at about 1 to about 20 ppm exhibits a flavor modification but does not provide added sweetness to the composition or formulation. In one embodiment, a composition or formulation containing an HTS protein or variant at about 1 to about 15 ppm exhibits a flavor modification but does not provide added sweetness to the composition or formulation. In one embodiment, a composition or formulation containing an HTS protein or variant at about 5 to about 50 ppm exhibits a flavor modification but does not provide added sweetness to the composition or formulation. In one embodiment, a composition or formulation containing an HTS protein or variant at about 10 to about 50 ppm exhibits a flavor modification but does not provide added sweetness to the composition or formulation. In one embodiment, a composition or formulation containing an HTS protein or variant at about 15 to about 50 ppm exhibits a flavor modification but does not provide added sweetness to the composition or formulation. In one embodiment, a composition or formulation containing an HTS protein or variant at about 20 to about 50 ppm exhibits a flavor modification but does not provide added sweetness to the composition or formulation. In one embodiment, a composition or formulation containing about 25 to about 50 ppm of an HTS protein or variant exhibits flavor modification but does not provide added sweetness to the composition or formulation.In one embodiment, a composition or formulation containing an HTS protein or variant at about 30 to about 50 ppm exhibits a flavor modification but does not provide added sweetness to the composition or formulation. In one embodiment, a composition or formulation containing an HTS protein or variant at about 35 to about 50 ppm exhibits a flavor modification but does not provide added sweetness to the composition or formulation. In one embodiment, a composition or formulation containing an HTS protein or variant at about 40 to about 50 ppm exhibits a flavor modification but does not provide added sweetness to the composition or formulation. In one embodiment, a composition or formulation containing an HTS protein or variant at about 45 to about 50 ppm exhibits a flavor modification but does not provide added sweetness to the composition or formulation. In one embodiment, a composition or formulation containing an HTS protein or variant at about 10 to about 40 ppm exhibits a flavor modification but does not provide added sweetness to the composition or formulation. In one embodiment, a composition or formulation containing about 20 to about 30 ppm of an HTS protein or variant exhibits flavor modification but does not provide added sweetness to the composition or formulation.

[0105]

[0115] In embodiments, compositions or formulations containing an HTS protein or variant at a concentration greater than 30 ppm, particularly greater than 40 ppm, provide a sweet taste to the composition or formulation. In embodiments, the composition or formulation may contain 1-100 ppm of HTS protein or variant. In embodiments, the composition or formulation may contain 1 ppm to 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, or 30 ppm of total HTS protein or variant. In embodiments, 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.

[0106]

[0116] In embodiments, at least 80% sequence identity for polypeptides includes, but is not limited to, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity. In further embodiments, at least 80% sequence identity for polypeptides 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.

[0107]

[0117] Comparison of sweetness and thermal stability of modified and unmodified peptides

[0118] The Myd (HTS) protein variants described herein also include "analogs," or "conservative variants" and "mimetics" ("peptidomimetics") that have structure and activity substantially corresponding to the exemplary sequences. Thus, the term "conservative variant" or "analog" or "mimetics" refers to a polypeptide having an amino acid sequence that has been modified such that the change does not substantially alter the structure and / or activity of the polypeptide (of the conservative variant), as defined herein. Examples of these include conservatively modified variations of the amino acid sequence, i.e., amino acid substitutions, additions, or deletions of residues that are not critical to protein activity, or substitutions of important amino acids with residues that have similar properties (e.g., acidic, basic, positively or negatively charged, polar or nonpolar, etc.), such that the substitution does not substantially alter the structure and / or activity.

[0108]

[0119] More specifically, "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to nucleic acids that encode identical or essentially identical amino acid sequences, or, if 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.

[0109]

[0120] For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.

[0110]

[0121] Such nucleic acid variations are "silent variations," which are a type of conservatively modified variation. Every nucleic acid sequence described herein that encodes a polypeptide also describes all possible silent variations of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, every silent variation of a nucleic acid that encodes a polypeptide is implicitly included in each sequence described.

[0111]

[0122] When it is desired to express a coding sequence in a heterologous host (i.e., a non-naturally occurring host), codon optimization, as known in the art, can be employed to enhance expression levels in the selected heterologous host. Codon optimization involves replacing codons in a given naturally occurring coding sequence with codons that have a higher usage level in the selected heterologous host. Generally, codon optimization is performed by comparing the codon frequency in the naturally occurring coding sequence with the codon frequency in the selected heterologous host, and if a given codon is not typically used in the selected heterologous host, replacing that codon with one 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 the naturally occurring coding sequence, the 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. Numerous codon optimization tools are known and readily available in the art 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 codon usage of DNA sequences," Nucleic Acids Res. 35:W126-W131). A review of recent 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 regarding codon optimization methods include, among others: N. M. Marlatt et al. (2010) "Codon optimization for enhanced Escherichia coli 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).

[0112]

[0123] Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, one exemplary guideline for selecting conservative substitutions includes (original residue followed by exemplary substitution): ala / gly or ser; arg / lys; asn / gln or his; asp / glu; cys / ser; gln / asn; gly / asp; gly / ala or pro; his / asn or gln; ile / leu or val; leu / ile or val; lys / arg or gln or glu; met / leu or tyr or ile; phe / met or leu or tyr; ser / thr; thr / ser; trp / tyr; tyr / trp or phe; val / ile or leu. An alternative exemplary guideline uses six groups containing amino acids that are conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (I); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W); (see also, e.g., Creighton, Proteins, W.H. Freeman and Company (1984); Schultz and Schimer, Principles of Protein Structure, Springer-Vrlag (1979)). Another alternative exemplary guideline uses the following six groups, in which proline is unique: 1) Gly (G), Ala (A), Val (V), Leu (L), Ile (I); 2) Ser (S), Cys (C), Thr (T), Met (M); 3) Pro (P); 4) Phe (F), Tyr (Y), Try (W); 5) His (H), Lys (K), Arg (R); and 6) Asp (D), Glu (E), Gln (N). Those skilled in the art will understand that the substitutions identified above are not the only possible conservative substitutions. For example, for some purposes, all charged amino acids can be considered conservative substitutions for each other, regardless of whether they are positive or negative.In addition, individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a small percentage of amino acids in an encoded sequence can also be considered "conservatively modified variations." One of skill in the art will be familiar with codon choices in a given host in which to express a protein of interest.

[0113]

[0124] Nucleotide and amino acid sequence information for MYD family members can also be used to construct computer models of the polypeptides that regulate sweet taste and how they interact with the sweet taste receptor. The sweet taste receptor is composed of a heterodimer of taste 1 receptor member 2 (T1R2) and taste 1 receptor member 3 (T1R3). These models can then be used to identify Myd variants and mutations that can increase sweet taste receptor activation and to identify more active versions of Myd.

[0114]

[0125] The various conservative mutations listed in Tables 8, 9, 10, 7, 6, and 3, as well as various less conservative mutations and substitutions, are contemplated within the scope of the present invention. The mutations in Tables 9, 10, 7, 3, and 6 exhibit sweetness and are currently preferred mutants. For example, it is within the skill level of those in the art to perform amino acid substitutions using known recombinant gene technology protocols, such as PCR, gene cloning, site-directed mutagenesis of cDNA, transfection of host cells, and in vitro transcription. The variants are then screened for sweetness-modulating activity, particularly for sensory stimuli or changes in such activity or properties in addition to sweetness. For example, the resulting variants are screened using sensory tests as described herein and as understood in the art. For example, the resulting variants are screened for functional activity of taste receptor agonists, as known in the art.

[0115]

[0126] In embodiments, the HTS variant polypeptides described herein exhibit enhanced sweetness compared to the unmodified polypeptide of SEQ ID NO: 3. In some embodiments, the sweetness of each HTS variant is enhanced by at least 10% (or an enhancement ranging from 10% to 100%, or an enhancement ranging from 10% to 200%, or an enhancement of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% or more) compared to the unmodified polypeptide of SEQ ID NO: 3. In other embodiments, the HTS variants exhibit equivalent sweetness compared to the unmodified polypeptide of SEQ ID NO: 3. The equivalent sweetness of the modified polypeptide is at least 10% (or at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) sweeter than the unmodified polypeptide of SEQ ID NO: 3, as described above. Sweetness is measured by any method known in the art for comparing sweetness, and more particularly by the method for assessing sweetness as described herein.

[0116]

[0127] Flavor-modifying activity and sweetness-modulating activity can be detected by methods known in the art, for example, by in vitro methods, or by animal or human sensory tests in vivo. Without wishing to be bound by any particular theory, Myd (HTS) is involved in sweet taste activation, and is, for example, an agonist of taste 1 receptor member 2 (Tas1R2) and / or taste 1 receptor member 3 (Tas1R3). However, Myd (HTS) also agonizes 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, including measurements of binding to the taste receptor Tas1R via spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties, patch clamp techniques, voltage-sensitive dyes, whole-cell currents, radioisotope efflux, inducible markers, changes in 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, and the like.

[0117]

[0128] Sensory testing (human or animal) can also be used to determine whether a Myd (HTS) candidate polypeptide has sweet taste-modulating activity. Sensory evaluation is the scientific field that analyzes and measures human responses to the composition of foods and beverages, such as appearance, feel, smell, texture, temperature, and taste. Sometimes, measurements using humans as instruments are necessary. The selection of an appropriate method for determining sweeteners can be determined by those skilled in the art, and examples include discrimination or discrimination tests designed to measure the likelihood that two products are perceptually different. Responses from the human evaluators are recorded for accuracy and statistically analyzed to determine whether any are more accurate than would be expected by mere chance. The food industry first needed to develop tools to measure the sensory characteristics of flavor and texture because these are obvious properties that cannot be easily measured by instruments. In the case of sweetness perception, for example, one or more samples of 5% sucrose, 6% sucrose, 7% sucrose, 8% sucrose, 9% sucrose, and 10% sucrose, and test samples, can be ranked by trained judges in order of sweetness intensity from low to high sweetness.In the present invention, it is understood that there are various methods that can be used by those skilled in the art to measure sensory stimulus properties (e.g., sensory difference).For example, sensory stimulus response can be measured using trained judges who rate their responses according to an accepted scale or rating system, such as a hedonic rating system.Alternatively or additionally, if a method is available or can be easily adapted, the subject's (judge's) response to sensory stimulus properties can be related to the property measured by the method. See, for example, Ray, S. (2221) "Sensory Properties of Foods and Their Measurement Methods." In: 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.

[0118]

[0129] 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 of sucrose / 100 g of solution, which expresses the strength of the solution as a mass percentage. 8°Bx is equivalent to approximately an 8% sucrose solution. As described in the Examples, purified polypeptide corresponding to SEQ ID NO: 5 was tasted (0.2 mL aliquot) at 0.03 mg / ml by trained sensory scientists and found to have a sweetness equivalent to 8°Bx (approximately an 8% sucrose solution) (see Examples 4, 5, 9, and 10).

[0119]

[0130] thermal stability

[0131] The thermal stability of a sweetness-modifying polypeptide may affect the potential application of the polypeptide, for example, food applications at high temperatures. In embodiments, the HTS variants described herein exhibit equivalent thermal stability compared to the unmodified polypeptide of SEQ ID NO: 3. As described above, equivalent thermal stability of a modified polypeptide is substantially the same as that of the unmodified polypeptide of SEQ ID NO: 3, which, as used herein, means a change in thermal stability of less than or equal to 4.5% (including less than or equal to 1%, less than or equal to 2%, less than or equal to 3%, and less than or equal to 4%) compared to the thermal stability of the unmodified polypeptide of SEQ ID NO: 3.

[0120]

[0132] In some embodiments, the HTS variants described herein exhibit enhanced thermal stability compared to the unmodified SEQ ID NO: 3. In other embodiments, as described above, the enhanced thermal stability of the modified polypeptide compared to the unmodified polypeptide of SEQ ID NO: 3 is greater than 4.5% enhanced (e.g., 4.5-15% enhanced, greater than 5% enhanced, greater than 6% enhanced, greater than 7% enhanced, greater than 8% enhanced, greater than 9% enhanced, greater than 10% enhanced, greater than 11% enhanced, greater than 12% enhanced, greater than 13% enhanced, greater than 14% enhanced, greater than 15% enhanced, greater than 16% enhanced, greater than 17% enhanced, greater than 18% enhanced, greater than 19% enhanced, greater than 20% enhanced, greater than 21% enhanced, greater than 22% enhanced, greater than 23% enhanced, greater than 24% enhanced, greater than 25% enhanced, greater than 26% enhanced, greater than 27% enhanced, greater than 28% enhanced, greater than 29% enhanced, greater than 30% enhanced, greater than 31% enhanced, greater than 32% enhanced, greater than 33% enhanced, greater than 34% enhanced, greater than 35% enhanced, greater than 36% enhanced, greater than 37% enhanced, greater than 38% enhanced, greater than 39% enhanced, greater than 40% enhanced, greater than 41% enhanced, greater than 42% enhanced, greater than 43% enhanced, greater than 44% enhanced, greater than 45% enhanced, greater than 46% enhanced, greater than 47% enhanced, greater than 48% enhanced, greater than 49% enhanced, greater than 50% enhanced, greater than 50% enhanced, greater than 51% enhanced, greater than 52% enhanced, greater than 53% enhanced, greater than 54% enhanced, greater than 55% enhanced, greater than 56% enhanced, greater than 57% enhanced % enhanced, greater than 14% enhanced, greater than 5% and up to 15% enhanced, greater than 6% and up to 15% enhanced, greater than 7% and up to 15% enhanced, greater than 8% and up to 15% enhanced, greater than 9% and up to 15% enhanced, greater than 10% and up to 15% enhanced, greater than 11% and up to 15% enhanced, greater than 12% and up to 15% enhanced, greater than 13% and up to 15% enhanced, greater than 14% and up to 15% enhanced, or up to 15% enhanced, etc.). Thermal stability can be measured by any method known in the art for assessing thermal stability, more particularly by the methods for assessing thermal stability described herein. In an embodiment, the sweetness-modifying polypeptides described herein exhibit comparable sweetness and comparable thermal stability compared to the unmodified polypeptide of SEQ ID NO: 3. In embodiments, the sweetness-modifying polypeptides described herein exhibit comparable sweetness and enhanced thermostability compared to the unmodified polypeptide of SEQ ID NO: 3. In embodiments, the sweetness-modifying polypeptides described herein exhibit enhanced sweetness and comparable thermostability compared to the unmodified polypeptide of SEQ ID NO: 3. In embodiments, the sweetness-modifying polypeptides described herein exhibit enhanced sweetness and enhanced thermostability compared to the unmodified polypeptide of SEQ ID NO: 3.

[0121]

[0133] In some embodiments, the HTS variants described herein exhibit comparable sweetness and comparable thermostability compared to that of the unmodified polypeptide of SEQ ID NO: 3. In some embodiments, the HTS variants described herein exhibit enhanced sweetness and comparable thermostability compared to that of the unmodified polypeptide of SEQ ID NO: 3. In some embodiments, the HTS variants described herein exhibit comparable sweetness and enhanced thermostability compared to that of the unmodified polypeptide of SEQ ID NO: 3. In some embodiments, the HTS variants described herein exhibit enhanced sweetness and enhanced thermostability compared to that of the unmodified polypeptide of SEQ ID NO: 3.

[0122]

[0134] The equivalent thermal stability of the modified polypeptide is, as described above, substantially the same as compared to the unmodified polypeptide of SEQ ID NO: 3, which in this specification means a change in thermal stability of less than or equal to 4.5% (including less than or equal to 1%, less than or equal to 2%, less than or equal to 3%, less than or equal to 4%) as compared to the thermal stability of the unmodified polypeptide of SEQ ID NO: 3.

[0123]

[0135] The enhanced thermal stability of the modified polypeptide, as described above, compared to the unmodified polypeptide of SEQ ID NO: 3, is enhanced by more than 4.5% (e.g., 4.5-15% enhanced, greater than 5% enhanced, greater than 6% enhanced, greater than 7% enhanced, greater than 8% enhanced, greater than 9% enhanced, greater than 10% enhanced, greater than 11% enhanced, greater than 12% enhanced, greater than 13% enhanced) compared to the thermal stability of the unmodified polypeptide of SEQ ID NO: 3. (greatly enhanced, greater than 15% enhanced, greater than 5% up to 15% enhanced, greater than 6% up to 15% enhanced, greater than 7% up to 15% enhanced, greater than 8% up to 15% enhanced, greater than 9% up to 15% enhanced, greater than 10% up to 15% enhanced, greater than 11% up to 15% enhanced, greater than 12% up to 15% enhanced, greater than 13% up to 15% enhanced, greater than 14% up to 15% enhanced, or up to 15% enhanced, etc.).

[0124]

[0136] In embodiments, the polypeptide variants described herein exhibit sweet taste modulating activity, a non-limiting example of which is the provision of a sweet taste.

[0137] In another aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one mutation selected from those listed in Table 8, and optionally by one deletion, e.g., deletion of Met at position 1. In another aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one and up to 24 mutations at different amino acid positions selected from those listed in Table 8.

[0125]

[0138] In another aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one mutation selected from those listed in Table 8, and at least one mutation selected from Table 7, and optionally one deletion, e.g., a deletion of Met at position 1. In another aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one mutation at different amino acid positions selected from those listed in Table 8, and from 1 to 24 mutations selected from Table 7.

[0126]

[0139] In another aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one to up to 24 mutations selected from those listed in Table 8, Table 9, or Table 10, optionally modified by one deletion, e.g., the deletion of Met at position 1. In another aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one to up to 24 mutations at different amino acid positions selected from those listed in Table 8, Table 9, or Table 10.

[0127]

[0140] In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the 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, optionally modified by one deletion, e.g., the deletion of Met at position 1. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the 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, optionally modified by one deletion, e.g., the deletion of Met at position 1. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the 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, optionally modified by one deletion, e.g., the deletion of Met at position 1. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the 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, optionally modified by one deletion, e.g., the deletion of Met at position 1. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the 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, optionally modified by one deletion, e.g., the deletion of Met at position 1.

[0128]

[0141] In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the 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, optionally modified by one deletion, e.g., the deletion of Met at position 1. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the 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, optionally modified by one deletion, e.g., the deletion of Met at position 1. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the 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, optionally modified by one deletion, e.g., the deletion of Met at position 1. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 modified by 10 mutations at different positions selected from those listed in Table 8, Table 9, or Table 10, optionally modified by one deletion, e.g., the deletion of Met at position 1. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 modified by 11 mutations at different positions selected from those listed in Table 8, Table 9, or Table 10, optionally modified by one deletion, e.g., the deletion of Met at position 1. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 modified by 12 mutations at different positions selected from those listed in Table 8, Table 9, or Table 10, optionally modified by one deletion, e.g., the deletion of Met at position 1.

[0129]

[0142] In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 modified by 13 mutations at different positions selected from those listed in Table 8, Table 9, or Table 10, optionally modified by one deletion, e.g., the deletion of Met at position 1. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 modified by 14 mutations at different positions selected from those listed in Table 8, Table 9, or Table 10, optionally modified by one deletion, e.g., the deletion of Met at position 1. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 modified by 15 mutations at different positions selected from those listed in Table 8, Table 9, or Table 10, optionally modified by one deletion, e.g., the deletion of Met at position 1. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 modified by 16 mutations at different positions selected from those listed in Table 8, Table 9, or Table 10, optionally modified by one deletion, e.g., the deletion of Met at position 1. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 modified by 17 mutations at different positions selected from those listed in Table 8, Table 9, or Table 10, optionally modified by one deletion, e.g., the 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, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 modified by 18 mutations at different positions selected from those listed in Table 8, Table 9, or Table 10, optionally modified by one deletion, e.g., a deletion of Met at position 1.

[0130]

[0143] In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 modified by 19 mutations at different positions selected from those listed in Table 8, Table 9, or Table 10, optionally modified by one deletion, e.g., the deletion of Met at position 1. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 modified by 20 mutations at different positions selected from those listed in Table 8, Table 9, or Table 10, optionally modified by one deletion, e.g., the deletion of Met at position 1. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 modified by 21 mutations at different positions selected from those listed in Table 8, Table 9, or Table 10, optionally modified by one deletion, e.g., the deletion of Met at position 1. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 modified by 22 mutations at different positions selected from those listed in Table 8, Table 9, or Table 10, optionally modified by one deletion, e.g., the deletion of Met at position 1. In a related embodiment, the present invention provides a polypeptide (e.g., an isolated polypeptide) having sweet taste modulating activity, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 modified by 23 mutations at different positions selected from those listed in Table 8, Table 9, or Table 10, optionally modified by one deletion, e.g., the 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, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 modified by 24 mutations at different positions selected from those listed in Table 8, Table 9, or Table 10, and optionally modified by one deletion, e.g., a deletion of Met at position 1. The present invention also provides polynucleotides encoding the aforementioned mutant polypeptides of SEQ ID NO: 3 or SEQ ID NO: 141. The present invention further provides the aforementioned mutant polypeptides further comprising a protein tag, more specifically a histidine tag. The present invention also provides polynucleotides encoding the aforementioned mutant polypeptides of SEQ ID NO: 3 or SEQ ID NO: 141, further comprising a protein tag, more specifically a histidine tag. In embodiments, the HTS variant does not comprise any one or more of the mutations listed in Table 3, Table 6, or Table 7.

[0131]

[0144] In some embodiments, the polypeptide having sweet taste modulating activity also has enhanced thermostability compared to the polypeptide of SEQ ID NO: 3. In one embodiment, the polypeptide having sweet taste modulating and enhanced thermostability activity comprises a modified SEQ ID NO: 3, wherein the polypeptide has at least one to up to six amino acid modifications shown in Table 8, Table 9, or Table 10, and optionally has one deletion, e.g., a deletion of Met at position 1. In one embodiment, the polypeptide having sweet taste modulating and enhanced thermostability activity comprises a modified SEQ ID NO: 3, wherein the polypeptide has at least one to up to six amino acid modifications shown in Table 7, and optionally has one deletion, e.g., a deletion of Met at position 1. In one embodiment, the polypeptide having sweet taste modulating and enhanced thermostability activity comprises a modified SEQ ID NO: 3, wherein the polypeptide has at least one to up to six amino acid modifications shown in Table 8, Table 9, or Table 10, and further has one to six amino acid modifications shown in Table 7, and optionally has one deletion, e.g., a deletion of Met at position 1. In further embodiments of the above embodiments, the polypeptide has sweet taste modulating activity (including sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO:141.

[0132]

[0145] In another embodiment, a polypeptide having sweet taste modulating activity (particularly sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 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 polypeptide of SEQ ID NO: 3 modified with at least one and up to six amino acid modifications as shown in Table 8, Table 9, or Table 10, or with one to six amino acid modifications as shown in Table 7, and optionally one deletion, such as a deletion of Met at position 1. In a further embodiment of the above embodiment, the polypeptide has sweet taste modulating activity (including sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 141.

[0133]

[0146] In another embodiment, a polypeptide having sweet taste modulating activity (particularly sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 has both the mutations I49C and S33C (compared to SEQ ID NO: 3), and optionally one, two, three, four, five or six other mutations shown in Table 8, Table 9, Table 10, Table 3, Table 6 or Table 7, and in particular optionally has one deletion, for example a deletion of Met at position 1. In a related embodiment, the polypeptide having sweet taste modulating activity (especially sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 has both the 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 with the modified polypeptide of SEQ ID NO: 3. In a further embodiment of the above embodiment, the polypeptide has sweet taste modulating activity (including sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 141.

[0134]

[0147] In another embodiment, a polypeptide having sweet taste modulating activity (particularly sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 has both the mutations Y23C and Y61C (compared to SEQ ID NO: 3), optionally one, two, three, four, five or six other mutations shown in Table 8, Table 9, Table 10, Table 3, Table 6 or Table 7, and optionally one deletion, for example a deletion of Met at position 1. In a related embodiment, the polypeptide having sweet taste modulating activity (especially sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 has both the 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 with the modified polypeptide of SEQ ID NO: 3. In a further embodiment of the above embodiment, the polypeptide has sweet taste modulating activity (including sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 141.

[0135]

[0148] In one embodiment, a polypeptide having sweet taste modulating and enhanced thermostability activity comprises a modified SEQ ID NO:3, wherein the polypeptide has two modifications shown in Table 7 and / or Table 8, Table 9 or Table 10, and optionally one deletion, such as the deletion of Met at position 1. In one embodiment, a polypeptide having sweet taste modulating and enhanced thermostability activity comprises a modified SEQ ID NO:3, wherein the polypeptide has two modifications shown in Table 7 and / or Table 8, Table 9 or Table 10 that result in disulfide bond formation between the two modified amino acids, and optionally one deletion, such as the deletion of Met at position 1. In one embodiment, a polypeptide having sweet taste modulating and enhanced thermostability activity comprises a modified SEQ ID NO:3, wherein the polypeptide has at least two modifications shown in Table 7 and / or Table 8, Table 9 or Table 10 that result in disulfide bond formation between the at least two modified amino acids, and optionally one deletion, such as the deletion of Met at position 1. In further embodiments of the above embodiments, the polypeptide has sweet taste modulating activity (including sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO:141.

[0136]

[0149] In one embodiment, a polypeptide having sweet taste modulating activity (particularly sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 comprises the polypeptide of SEQ ID NO: 143 (double mutant S33C_I49C) or SEQ ID NO: 145 (double mutant Y24C_Y62C). In a related embodiment, a polypeptide having sweet taste modulating activity (particularly sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 comprises the polypeptide of SEQ ID NO: 142 or SEQ ID NO: 145, and additionally the polypeptides of SEQ ID NO: 146 and SEQ ID NO: 147, in which methionine at position 1 is absent. In one embodiment, a polypeptide having sweet taste modulating activity (particularly sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 is the polypeptide of SEQ ID NO: 143. In one embodiment, a polypeptide having sweet taste modulating activity (particularly sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 is the polypeptide of SEQ ID NO: 145. In one embodiment, a polypeptide having sweet taste modulating activity (particularly sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 is the polypeptide of SEQ ID NO: 146. In one embodiment, the polypeptide having sweet taste modulating activity (particularly sweet taste) and enhanced thermostability compared to the polypeptide of SEQ ID NO:3 is the polypeptide of SEQ ID NO:147.

[0137]

[0150] In some embodiments, the polypeptide of the present invention exhibits enhanced stability against low pH (<pH7) compared to the polypeptide of SEQ ID NO: 3. In some embodiments, the polypeptide of the present invention exhibits enhanced stability against high pH (>pH7) compared to the polypeptide of SEQ ID NO: 3. In one embodiment, the polypeptide of the present invention exhibits higher stability compared to the polypeptide of SEQ ID NO: 3 at a pH of about 2. In another embodiment, the polypeptide of the present invention exhibits higher stability compared to the polypeptide of SEQ ID NO: 3 at a pH of about 10. In one embodiment, the polypeptide of the present invention having methionine at position 1 exhibits higher stability compared to the polypeptide of SEQ ID NO: 3 at a pH of about 2. In another embodiment, the polypeptide of the present invention having methionine at position 1 exhibits higher stability compared to the polypeptide of SEQ ID NO: 3 at a pH of about 10.

[0138]

[0151] The term "expression vector" or "expression cassette" refers to any recombinant expression system for the purpose of expressing the nucleic acid sequences of the present invention constitutively or inducibly, in vitro or in vivo, in any cell including prokaryotic, yeast, fungal, plant, insect or mammalian cells. This term includes linear or circular expression systems. This term includes expression systems that remain episomal or are integrated into the genome of the host cell. The expression system may or may not have the ability to self-replicate, i.e., it may drive only transient expression in the cell. This term includes recombinant expression cassettes containing only the minimal elements necessary for transcription of the recombinant nucleic acid.

[0139]

[0152] A review of recent methods for recombinant protein expression can be found in Tripathi and Shrivastava (2019) "Recent Developments in Bioprocessing of Recombinant Proteins; Expression Hosts and Process Development," Frontiers in Bioeng. Biotech. Biotech. 7:420, doi:10.3389 / fbio.2019.00420, which is incorporated herein by reference in its entirety for details of host expression systems and methods for recombinant protein expression.

[0140]

[0153] "Host cell" refers to a cell that contains an expression vector and supports the 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 may be a prokaryotic cell such as E. coli, or a eukaryotic cell such as a yeast, insect, amphibian, or mammalian cell, e.g., CHO, HeLa, HEK-293, etc., including cultured cells, explants, and in vivo cells.

[0141]

[0154] In one embodiment, the host cell is selected from the group consisting of Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Agrobacterium tumefaciens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, and the like. mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, Pseudomonas putida, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizopus oryzaeoryzae, Yarrowia lipolytica, Candida albicans, Issatchenkia orientalis, Scheffersomyces stipitis, Yarrowia lipolytica, Ogataea polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus polymorphus, and Schwanniomyces occidentalis.

[0142]

[0155] In one embodiment, the host cell is selected from the group consisting of biological substances recommended by the Qualified Presumption of Safety (QPS). A list of such hosts is available on the website: efsa.europa.eu / efsajournal EFSA Journal 2021;19(7):6689. In one 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, and the like. amylovorus, Lactobacillus animalis, Lactobacillus alimentarius, Lactobacillus aviaries, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus collinoides, Lactobacillus coryniformiscoryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii hilgardii, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus mucosae, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus parafarraginis, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis pontis), Lactobacillus reuteri, Lactobacillus rhamnosusrhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus Sanfranciscensis, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc Pseudomesenteroides, Microbacterium imperial, Oenococcus oeni, Pasteuria nishizawae, Pediococcus acidilactici acidilactici, Pediococcus parvulus, Pediococcus pentosaceus, Propionibacterium acidipropionic, Propionibacterium freudenreichii, Streptococcus thermophilus, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans, Bacillus clausii, Bacillus coagulans coagulans, Bacillus flexus, Bacillus fusiformis, Bacillus lentus, Bacillus licheniformislicheniformis, Bacillus megaterium, Bacillus mojavensis, Bacillus paralicheniformis, Bacillus pumilus, Bacillus smithii, Bacillus subtilis, Bacillus vallismortis, Bacillus velezensis, Geobacillus stearothermophilus, Paenibacillus illinoisensis, Parageobacillus thermoglucosidasius thermoglucosidasius, Gluconobacter oxydans, Komagataeibacter sucrofermentans, Xanthomonas campestris, Candida cylindracea, Cyberlindnera jadinii, Debaryomyces hansenii, Hanseniaspora uvarum, Kluyveromyces lactis, Kluyveromyces marxianus, Komagataella pastoris pastoris, Komagataella phaffi, Lindnera jadinii, Ogataea angusta, Saccharomyces bayanusbayanus, Schizosaccharomyces pombe, Wickerhamomyces anomalus, Xanthophyllomyces dendrorhous, or Zygosaccharomyces rouxii.

[0143]

[0156] In another embodiment, the host cell is selected from the group consisting of Gram-positive non-spore-forming bacteria, Gram-positive spore-forming bacteria, Gram-negative bacteria, yeast, and protists / algae. In another embodiment, the host cell is selected from plant cells. In another embodiment, the host cell is selected from insect cells. For insect cells, baculovirus insect expression systems are useful. Insect cells useful as hosts for recombinant protein production include, among others, Spodoptera frugiperda cells (e.g., Sf9, Sf21), Drosophila cells (e.g., S2), and Trichoplusia ni cells (e.g., Tn-368, High-Five™ (Thermo Fisher Scientific, Waltham, MA)). A variety of host cells are known in the art and are available from commercial sources, among others.

[0144]

[0157] 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, and the like. amylolyticus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus alimentarius, Lactobacillus aviaries, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus collinoides, Lactobacillus coryniformis, Lactobacillus crispatus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus dextrinixdextrinicus, Lactobacillus diolivorans, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus mucosae, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus parafarraginis, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei sakei, Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactococcus lactislactis, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Microbacterium imperial, Oenococcus oeni, Pasteuria nishizawae, Pediococcus acidilactic, Pediococcus parvulus, Pediococcus pentosaceus pentosaceus, Propionibacterium acidipropioni, Propionibacterium freudenreichii, and Streptococcus thermophiles.

[0145]

[0158] Non-limiting examples of Gram-positive spore-forming bacteria include Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus flexus, Bacillus fusiformis, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus mojavensis, Bacillus pumilus, Bacillus Examples of bacteria that may be causing bacterial infection include Bacillus pumilus, Bacillus smithii, Bacillus subtilis, Bacillus vallismortis, Bacillus velezensis, Geobacillus stearothermophilus, Paenibacillus illinoisensis, and Parageobacillus thermoglucosidasius. Non-limiting examples of gram-negative bacteria include Cupriavidus necator, Gluconobacter oxydans, Komagataeibacter sucrofermentans, and Xanthomonas campestris.

[0146]

[0159] Non-limiting examples of yeast include Candida cylindracea, Debaryomyces hansenii, Hanseniaspora uvarum, Kluyveromyces lactis, Kluyveromyces marxianus, Komagataella pastoris, Komagataella phaffi, Lindnera jadinii, Ogataea angusta, Saccharomyces bayanus, Saccharomyces cerevisiae, and the like. cerevisiae, Saccharomyces pastorianus, Schizosaccharomyces pombe, Wickerhamomyces anomalus, Xanthophyllomyces dendrorhous, Yarrowia lipolytica, and Zygosaccharomyces rouxii.

[0147]

[0160] Non-limiting examples of protists / algae include Aurantiochytrium limacinum, Euglena gracilis, and Tetraselmis chuii.

[0148]

[0161] In certain embodiments, the recombinant HTS is produced by a transgenic mammal, ie, produced in its milk.

[0162] Expression of the HTS (or its variants) can be stable or transient. In stable expression systems, the exogenous DNA is integrated into a chromosome or passed on to future generations of the host cell as an episome (a separate piece of nuclear DNA).

[0149]

[0163] The terms "mimetic" and "peptidomimetics" refer to synthetic chemicals that have substantially the same structural and / or functional characteristics of a polypeptide of the invention, e.g., a translocation domain, a ligand-binding domain, or a chimeric receptor. Mimetics can be either entirely composed of synthetic, non-natural analogs of amino acids, or can be chimeric molecules of partially natural peptide amino acids and partially non-natural analogs of amino acids. Mimetics can also incorporate naturally occurring amino acid conservative substitutions in any amount so long as such substitutions do not also substantially alter the mimetic's structure and / or activity.

[0150]

[0164] As with polypeptides of the present invention that are conservative variants, routine experimentation will be expected to determine whether a mimetic is within the scope of the present invention, i.e., whether its structure and / or function are substantially unchanged. Polypeptide mimetic compositions may contain any combination of non-natural structural components, typically from the following three structural groups: a) residue linkages other than natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues that induce secondary structural mimicry, i.e., that 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 if all or some of its residues are joined by chemical means other than natural peptide bonds. The individual peptidomimetic residues can be joined by peptide bonds or other chemical bonds or coupling means such as, for example, glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC). Linking groups that can be alternatives to the traditional amide bond ("peptide bond") linkage include, for example, ketomethylene (e.g., --C(O)--NH-- versus --C(O)--CH2--), aminomethylene (CH2--NH), ethylene, olefin (CH=CH), ether (CH2--O), thioether (CH--S), tetrazole (CN4), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp. 267-357, "Peptide Backbone Modifications," Marcell Dekker, NY (1983)). Polypeptides can also be characterized as mimetics by containing all or some non-natural residues in place of naturally occurring amino acid residues; non-natural residues are well described in the scientific and patent literature.Phyre2 is a web-enabled suite of tools for predicting and analyzing protein structure, function, and mutations.

[0151]

[0165] Examples of conservatively modified variations in Myd1 protein structure can be obtained using homology modeling algorithms such as SWISS-MODEL, PHYRE2.0, and Jpred to identify sequence-based consensus loop regions, as known in the art. See, for example, Pechmann, S. and Frydman, J. Interplay between Chaperones and Protein Disorder Promotes the Evolution of Protein Networks. PLoS Computational Biology 10, e1003674 (2014).

[0152]

[0166] Specific regions of MYD / Myd nucleotide and amino acid sequences can be used to identify polymorphic variants, interspecies homologs, and alleles of Myd family members. This identification can be performed in vitro, for example, under stringent hybridization conditions, or by PCR (for example, using primers encoding the Myd sequences identified herein), or by using sequence information in a computer system for comparison with other nucleotide sequences. Different alleles of the MYD gene within a single species population are also expected to be useful in determining whether differences in allelic sequences correlate with differences in taste perception between members of the population. Classical PCR-type amplification and cloning techniques are useful for isolating orthologs, for example, when degenerate primers are sufficient to detect related genes across species.

[0153]

[0167] 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 related to MYD within a single species are best identified using sequence pattern recognition software to search for related sequences. Typically, identification of polymorphic variants and alleles of MYD family members can be achieved by comparing amino acid sequences of about 25 amino acids or more, e.g., 50-100 amino acids. Approximately at least 35-50%, optionally 60%, 70%, 75%, 80%, 85%, 90%, 95-99%, or higher, amino acid identity typically demonstrates that a protein is a polymorphic variant, interspecies homolog, or allele of a MYD family member. Sequence comparison can be performed using any of the sequence comparison algorithms discussed below. Antibodies that specifically bind to Myd polypeptides or conserved regions thereof can also be used to identify alleles, interspecies homologs, and polymorphic variants.

[0154]

[0168] In one embodiment, sequences encoding hybrid proteins containing nucleic acids encoding Myd variant fusion proteins may be constructed. These nucleic acid sequences may be operably linked to transcriptional or translational control elements, such as transcriptional and translational initiation sequences, promoters and enhancers, transcriptional and translational terminators, polyadenylation sequences, and other sequences useful for transcribing DNA into RNA. The fusion protein may also include a C-terminal or N-terminal translocation sequence. Furthermore, the fusion protein may contain additional elements, such as additional elements for protein detection, purification, or other applications. Domains that facilitate detection and purification include, for example, metal-chelating peptides, such as polyhistidine tracts, histidine-tryptophan modules, or other domains that allow purification on immobilized metals; maltose-binding proteins; protein A domains that allow purification on immobilized immunoglobulins; or domains utilized in the FLAGS extension / affinity purification system (Immunex Corp, Seattle, Wash.).

[0155]

[0169] In one embodiment, the fusion protein comprises a peptide or protein tag (e.g., for protein purification or detection). Protein / peptide tags are peptide sequences that are genetically grafted onto a recombinant (e.g., fusion) protein. Peptide / protein tags are known in the art and are described, for example, in Johnson, "Protein / Peptide Tags," in and DOI / / dx.doi.org / 10.13070 / mm.en.2.116, examples of which include, but are not limited to, green fluorescent protein (GFP), FLAG, Myc epitope, polyhistidine, glutathione-S-transferase (GST), HA, V5, ABDz1-tag, adenylate kinase (AK-tag), BC2-tag, calmodulin-binding peptide, CusF, Fc, Fh8, Halo tag, heparin-binding peptide (HB-tag), ketosteroid isomerase (KSI), maltose-binding protein (MBP), thioredoxin, PA (NZ-1), poly-Arg, poly-Lys, S-tag, SBP / streptavidin-binding peptide, SNAP, Strep-II (Twin-Strep), and SUMO / SUMO2.

[0156]

[0170] Affinity tags are a type of protein tag attached to proteins to enable their purification from crude biological sources using affinity techniques. Affinity tags are known in the art, including those described in Kimple et al., Curr Protoc Protein Sci.; 73:Unit-9.9, doi:10.1002 / 0471140864.ps0909s73. Examples of affinity tags include, among others, polyhistidine, GST, MBP, calmodulin-binding peptide, intein-chitin binding domain, streptavidin / biotin-based tags, and His-Patch ThioFusion (thioredoxin). Affinity tags can be small (e.g., 20 or fewer amino acid residues) or large. Examples of small affinity tags include His, FLAG, Strep II, and S-peptide, while examples of large affinity tags include MBP, GST, cellulose-binding domain, calmodulin-binding peptide, and His-patch thioredoxin.

[0157]

[0171] Protein / peptide tags include epitope tags and reporter tags. Reporter tags serve as reporters of protein expression and protein-protein interactions. Reporter tags include, but are not limited to, enzymes such as β-galactosidase (β-gal), alkaline phosphatase (AP), chloramphenicol acetyltransferase (CAT), and horseradish peroxidase (HRP).

[0158]

[0172] Epitope tags, including FLAG, hemagglutinin (HA), c-myc, T7, and Glu-Glu, are used for detecting fusion proteins in vitro and in cell culture. Their short, linear recognition motifs rarely affect the properties of the protein of interest and are usually highly specific for their respective primary antibodies. When anti-myc antibodies are used, specificity can be increased by detecting the conjugated anti-myc primary antibody using an enzyme-linked secondary antibody instead of using HRP or AP anti-myc conjugates alone.

[0159]

[0173] Protein / peptide tags also include solubilization tags, which are used to aid protein proper folding and prevent them from aggregating into inclusion bodies.In embodiments, solubilization tags are employed in proteins that are expressed in E. coli.Solubilization tags include, among others, thioredoxin and poly(NANP).Some affinity tags can also aid solubilization, such as MBP and GST.

[0160]

[0174] The protein / peptide tag may be at either end of the target protein. Some tags, such as FLAG, are often used in tandem or in combination with other tags, for example, in His-Myc and His-V5 constructs, to enhance their desired characteristics.

[0161]

[0175] Tandem affinity purification (TAP) is a dual affinity purification method based on fusing two affinity tags to a protein of interest, which allows for the purification of the tagged protein and the isolation of protein complexes that interact with the protein of interest. The use of TAP is encompassed within the scope of the present invention.

[0162]

[0176] In one embodiment, the fusion protein comprises a histidine tag containing 2 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) histidine residues. For example, the histidine tag may contain 6 histidine residues.

[0163]

[0177] To facilitate purification, it may be useful to include a cleavable linker sequence, such as that found in Factor Xa (see, e.g., Ottavi, Biochimie 80:289-293 (1998)), a subtilisin protease recognition motif (see, e.g., Polyak, Protein Eng. 10:615-619 (1997)), or enterokinase (Invitrogen, San Diego, Calif.), between the translocation domain (for efficient plasma membrane expression) and the remainder of the newly translated polypeptide. For example, one construct may include a nucleic acid sequence linked to six histidine residues, followed by a thioredoxin, an enterokinase cleavage site (see, e.g., Williams, Biochemistry 34:1787-1797 (1995)), and a polypeptide encoding a C-terminal translocation domain. The histidine residues facilitate detection and purification, while the enterokinase cleavage site provides a means for purifying the desired protein from the remainder of the fusion protein. Technology relating to vectors encoding fusion proteins and applications of fusion proteins are well described in the scientific and patent literature; see, eg, Kroll, DNA Cell. Biol. 12:441-53 (1993).

[0164]

[0178] Fusion proteins may contain one or more linkers (e.g., flexible linkers, rigid linkers, and in vivo cleavable linkers). In addition to their essential role in linking functional domains together (as in the case of flexible and rigid linkers) or releasing free functional domains in vivo (as in the case of in vivo cleavable linkers), linkers offer many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yields, and achieving desired pharmacokinetic profiles. Linkers are known in the art (see, for example, Chen et al., Adv Drug Deliv Rev. 65(10):1357-1369 (2013)).

[0165]

[0179] Flexible linkers are used when the combined domains require a certain degree of movement or interaction. These are generally composed of small, nonpolar amino acids (e.g., Gly) or polar amino acids (e.g., Ser or Thr). The small size of these amino acids provides flexibility, allowing for 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, reducing unfavorable interactions between the linker and the protein moiety.

[0166]

[0180] The most commonly used flexible linkers have a sequence consisting primarily of a stretch of Gly and Ser residues ("GS" linker). An example of the most widely used flexible linker is (Gly-Gly-Gly-Gly-Ser). n(SEQ ID NO: 7). By adjusting the copy number "n", the length of this GS linker can be optimized to achieve appropriate separation of functional domains or to maintain necessary interdomain interactions. In addition to the GS linker, many other flexible linkers for recombinant fusion proteins have been designed. These flexible linkers are also rich in small or polar amino acids such as Gly and Ser, but may also contain amino acids such as Thr and Ala to maintain flexibility, as well as polar amino acids such as Lys and Glu to improve solubility.

[0167]

[0181] Rigid linkers maintain a fixed distance between domains, preserving their independent function. An example of a rigid linker is (EAAAK). n an alpha-helix-forming linker having the sequence of (SEQ ID NO: 8), and a Pro-rich sequence, (XP) n (wherein X represents any amino acid, preferably Ala, Lys, or Glu).

[0168]

[0182] Polypeptides of the invention may also contain 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 sometimes the C-terminus, of most newly synthesized proteins destined for the secretory pathway. These proteins include those that are either present inside specific organelles (endoplasmic reticulum, Golgi, or endosomes), secreted from the cell, or inserted into most cell membranes. Exemplary signal peptides are known in the art, and one of skill in the art would know how to select a particular signal peptide for use in the present invention.

[0169]

[0183] Protein derivatization

[0184] HTS proteins / polypeptides and their sequence variants may be further derivatized or modified other than by substitution of one or more amino acids. The HTS wild-type (native) proteins / polypeptides and HTS protein / polypeptide variants of this disclosure may be derivatized at the N-terminus, C-terminus, or amino acid side chain without impairing taste-modulating activity. Derivatization generally includes acylation, esterification, glycosylation, oxidation, methylation, reductive alkylation, phosphorylation (as a phosphoamino acid), sulfurylation, sulfonylation, or oxidation or reduction of a side chain heteroatom. 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 skilled in the art can select one or more enzymes to achieve the desired derivatization of an HTS protein / polypeptide or its variant. The HTS protein or polypeptide, preferably isolated, is treated with the selected enzyme to achieve the desired derivatization. A post-translational modification (PTM) is a covalent modification of a protein / polypeptide by proteolytic cleavage (e.g., removal of N-terminal methionine) and / or by the addition of modifying groups, such as acetyl (or more broadly, acyl), phosphoryl, glycosyl, and / or methyl, to one or more amino acids. Other chemical modifications of amino acids of proteins / polypeptides (e.g., oxidation of the sulfur of methionine groups) can be achieved by both chemical and biological means by those skilled in the art. Post-translational modifications can occur during expression of the native protein / polypeptide, or they can be controlled during protein expression in a non-native host using recombinant methods. Such recombinant methods rely, for example, on the use of specially constructed expression vectors containing coding sequences for the expression of one or more enzymes to effect the selected post-translational modification. Those skilled in the art are aware of chemical or recombinant techniques for derivatizing proteins at one or more positions on a given protein.In one embodiment, preferred protein / polypeptide derivatization is one that does not significantly affect the sweetness modulating activity of the HTS protein / polypeptide variants of this disclosure. In one embodiment, preferred protein / polypeptide derivatization is one that does not significantly adversely affect the sweetness of the HTS protein / polypeptide variants of this disclosure (e.g., does not significantly reduce the sweetness of the HTS variants described herein). In an embodiment, derivatization of the HTS variants can modify the flavor, modify the sweetness, or enhance the sweetness activity of the HTS variants.

[0170]

[0185] Derivatization of the HTS protein / polypeptide or variant by any known method can be at the N-terminus, C-terminus, or at one or more amino acid side groups (e.g., side chain sulfur, side chain amine, or side chain carboxylic acid). In embodiments, the HTS protein / polypeptide or HTS variant of this disclosure may have one, two, three, four, five, or six 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 may be derivatized by acylation, more specifically by acetylation. In a more specific embodiment, the N-terminus of the protein / polypeptide or variant is an N-acetylated methionine. In embodiments, the derivatization is derivatization of the N-terminal amino acid or derivatization of the amine side chain of an amino acid 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-CH-R (where R is an alkyl group (e.g., an alkyl group having 1 to 19 carbon atoms)); phosphorylation of amino acid side chains such as serine or threonine (-OH) with kinases to form phosphopolypeptides; or reaction of sulfur in amino acid side chains with monooxygenases to produce sulfoxide derivatives. N-terminal amine or amine side chains can be derivatized by chemical methods or biological processes after polypeptide translation, resulting in post-translational modification of the nitrogen. Reactive side chains of other amino acids can be derivatized by chemical methods or biological processes after polypeptide translation, resulting in post-translational modification of these side chains.

[0171]

[0186] In specific embodiments, derivatization is performed using the following chemical moieties: acyl (R—CO—, where R is a straight or branched chain alkyl group having 1 to 20 carbon atoms); acetyl (CHCO), formyl (HCO), glycosyl (e.g., CH11 The addition, removal, or substitution of one or more of the following groups to the amine nitrogen: O6-), hydroxyl (HO-), methyl (CH3-) or other alkyl groups, phosphatidyl (PO4), phosphonyl (PO2), sulfhydryl (SH-), or sulfonyl (HSO2-).

[0172]

[0187] In specific embodiments, when the first amino acid of an HTS protein / polypeptide is methionine, the sulfur at the delta position of the first amino acid is modified after translation of the polypeptide by chemical, enzymatic (in vitro or in vivo), or biological processes, resulting in a post-translational modification of the sulfur. In embodiments, the modification of the sulfur at the delta position of the first amino acid is the addition, elimination, or substitution of any of the following chemical moieties at the sulfur at the delta position: acyl (R-CO-, where R is a straight or branched chain alkyl group having 1 to 20 carbon atoms); acetyl (CHCO), formyl (HCO), glycosyl (e.g., CHO-), hydroxyl (HO-), methyl (CH-) or other alkyl groups, phosphatidyl (PO), phosphonyl (PO), sulfhydryl (SH-), or sulfonyl (HSO-). In embodiments, the modification of the sulfur at the delta position of the first amino acid is oxidation of the sulfur, resulting in a sulfoxide derivative.

[0173]

[0188] As used herein, "at least 80% identity" relative to an amino acid or nucleotide sequence refers to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater identity.

[0174]

[0189] As used herein, examples of "amino acid sequences modified by the deletion, insertion, substitution, or addition of one or more amino acids" include amino acid sequences modified by the deletion, insertion, substitution, or addition of at least one or up to 30, preferably up to 20, more preferably up to 10, and even more preferably up to 5 (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) amino acids. As used herein, examples of "a nucleotide sequence modified by deletion, insertion, substitution, or addition of one or more nucleotides" include those having 1 or more and 90 or less, preferably 60 or less, more preferably 30 or less, even more preferably 15 or less, and even more preferably 10 or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 1 4, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or any range thereof).

[0175]

[0190] For example, in sequence comparison, typically one sequence serves as the reference sequence with which test sequence is compared.Using sequence comparison algorithm, test and reference sequences are input into computer, and subsequence coordinates are designated, and if necessary, the parameters of sequence algorithm program are designated.For BLASTN and BLASTP program, as described below, default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence with reference sequence based on program parameters.

[0176]

[0191] "Comparison window," as used herein, refers to any segment of the number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, and more usually about 100 to about 150, within which a sequence can be compared to a reference sequence of the same number of contiguous positions after optimal alignment of the two sequences. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison may be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds., 1995 supplement)).

[0177]

[0192] Preferred examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that either match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J Mol. Biol. 215:403-410 (1990)). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is discontinued if the cumulative alignment score falls by the amount X from its maximum achieved value; if the cumulative score becomes zero or less due to the accumulation of one or more negatively scored residue alignments; or if either sequence reaches its end. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0178]

[0193] Another example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive pairwise alignment to show the relationship and percent sequence identity. It also plots a so-called "tree" or "dendrogram" showing the clustering relationships used to create the alignment (see, for example, Figure 1). PILEUP uses a simplified version of the progressive alignment method of Feng and Doolittle, J Mol. Evol. 35:351-360 (1987). The method used is similar to that described by Higgins and Sharp, CABIOS 5:151-153 (1989). This program can align up to 300 sequences, each with a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with a pairwise alignment of the two most similar sequences and produces a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive pairwise alignments. The program is run by specifying specific sequences and their amino acid or nucleotide coordinates for the region of sequence comparison, as well as by specifying program parameters. PILEUP is used to compare a reference sequence with other test sequences to determine percent sequence identity relationships using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395 (1984)), and is obtained by conceptual translation of the corresponding open reading frame encoded by the gene.

[0179]

[0194] Polynucleotides encoding the polypeptides of the present invention can be synthesized chemically or by genetic engineering based on the amino acid sequence of Myd. For example, polynucleotides can be chemically synthesized based on the amino acid sequence of the polypeptides of the present invention or their precursor proteins. For chemical synthesis of polynucleotides, custom nucleic acid synthesis services (e.g., those provided by Medical & Biological Laboratories Co., Ltd., Genscript, etc.) may be used. Furthermore, synthesized polynucleotides may be amplified by PCR, cloning, etc.

[0180]

[0195] Polypeptides of the present invention can be produced, for example, by expressing a gene encoding a Myd polypeptide variant of the present invention. Preferably, Myd polypeptide variants of the present invention can be produced from a transformed strain into which a polynucleotide encoding a Myd polypeptide variant of the present invention has been introduced. For example, a polynucleotide encoding a Myd polypeptide variant of the present invention or a vector containing the same is introduced into a host to obtain a transformed strain. After culturing the transformed strain in an appropriate medium, the Myd polypeptide variant of the present invention is produced in the transformed strain from the polynucleotide encoding the Myd polypeptide variant of the present invention introduced therein. Proteins of the present invention can be obtained by isolating or purifying the produced Myd polypeptide variant from the culture.

[0181]

[0196] Therefore, the present invention further provides polynucleotides encoding Myd polypeptide variants of the present invention and vectors comprising the same. The present invention further provides methods for producing transformed strains, the methods comprising the step of introducing a polynucleotide encoding a Myd polypeptide variant of the present invention or a vector comprising the same into a host. The present invention further provides transformed strains containing a polynucleotide encoding a Myd polypeptide variant of the present invention or a vector comprising the same introduced from outside the cell. The present invention further provides methods for producing a Myd polypeptide variant of the present invention, the methods comprising the step of culturing a transformed strain.

[0182]

[0197] The present invention also includes a polynucleotide of the present invention operably linked to a heterologous regulatory element. The present invention can include an expression cassette or vector comprising a polynucleotide of the present invention, and a host cell transformed with a vector of the present invention.

[0183]

[0198] Alternatively, polynucleotides encoding the Myd polypeptide variants of the present invention can be produced by introducing one or more mutations into a polynucleotide synthesized according to known mutagenesis procedures, 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 resulting polynucleotide, examining the sweetness-modifying activity of the expressed protein, and selecting a polynucleotide encoding a protein having the desired sweetness-modifying activity.

[0184]

[0199] Site-directed mutagenesis of polynucleotides can be carried out using any method, such as inverse PCR and annealing (Muramatsu et al., eds., "Revised 4th Edition New Genetic Engineering Handbook," YODOSHA, pp. 82-88). Various commercially available kits for site-directed mutagenesis, such as the QuickChange II Site-Directed Mutagenesis Kit and the QuickChange Multi Site-Directed Mutagenesis Kit from Stratagene, can be used as needed.

[0185]

[0200] Examples of types of vectors comprising a polynucleotide encoding a polypeptide 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., baculoviruses).

[0186]

[0201] Among these, plasmid vectors are preferred, and for example, commercially available plasmid vectors for protein expression, such as pUC19, pUC118, pUC119, and pBR322 (all available from TAKARA BIO INC.), can be used.

[0187]

[0202] A vector may contain a DNA region containing a DNA replication origin or replication origin. Alternatively, a regulatory sequence such as a promoter region for initiating gene transcription, a terminator region for secreting an expressed protein outside the cell, or a secretion signal region can be operably linked upstream of a polynucleotide encoding a protein of the present invention (i.e., the MYD gene of the present invention) in a vector. As used herein, "operably linked" of a gene and a regulatory sequence refers to a state in which the gene and the regulatory region are arranged so that the gene can be expressed under the regulation of the regulatory region.

[0188]

[0203] The types of regulatory sequences, such as promoter regions, terminators, and secretory signal regions, are not particularly limited, and the promoter and secretory signal sequence used can be selected as needed depending on the host into which the sequence will be introduced. For example, a preferred example of a regulatory sequence that can be incorporated into the vector of the present invention is the cbh1 promoter sequence derived from Trichoderma reesei (Curr. Genet., 1995, 28(1):71-79).

[0189]

[0204] Alternatively, a marker gene (e.g., a resistance gene to drugs such as ampicillin, neomycin, kanamycin, and chloramphenicol) for selecting a host into which the vector is appropriately introduced may be further incorporated into the vector of the present invention. Alternatively, when an auxotrophic strain is used as a host, a gene encoding a synthase of a necessary nutrient may be incorporated into the vector as a marker gene. Alternatively, when a selective medium requiring a specific metabolism for growth is used, a metabolically related gene may be incorporated into the vector as a marker gene. An example of such a metabolically related gene is the acetamidase gene for using acetamide as a nitrogen source.

[0190]

[0205] Ligation of a polynucleotide encoding a Myd polypeptide variant of the present invention with a regulatory sequence and a marker gene can be carried out by methods known in the art, such as splicing by overlap extension (SOE)-PCR (Gene, 1989, 77:61-68). Procedures for introducing the ligated fragment into a vector are known in the art.

[0191]

[0206] Examples of hosts for transformants into which vectors can be introduced include microorganisms such as bacteria or filamentous fungi. Examples of bacteria include Escherichia coli and bacteria belonging to the genera Staphylococcus, Enterococcus, Listeria, and Bacillus. Among these, Escherichia coli and bacteria belonging to the genus Bacillus (e.g., Bacillus subtilis or mutants thereof) are preferred. Examples of Bacillus subtilis mutant strains include KA8AX, a protease 9 double-deficient strain described in J. Biosci. Bioeng., 2007, 104(2):135-143, and DBPA, a mutant from the protease 8 double-deficient strain described in Biotechnol. Lett., 2011, 33(9):1847-1852, which have improved protein folding efficiency. Examples of filamentous fungi include Trichoderma, Aspergillus, and Rhizopus. Suitable expression hosts include, for example, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowia lipolytica, Schizosaccharomyces pombe, and Kluyveromyces lactis. In some embodiments, the host cell is a fungal cell other than Mattiromyces terfesioides. In some embodiments, the HTS protein variant can be expressed in mycelia.

[0192]

[0207] In embodiments, the HTS protein variants can be expressed in plant cells, plant organs, leaves, roots, or in the whole plant.

[0208] In yet another aspect, the invention includes a host cell comprising one or more of the expression cassettes described herein operably linked to control elements compatible with expression in the cell. The cell may be, for example, a mammalian cell (e.g., a BHK, VERO, HT1080, 293, RD, COS-7, or CHO cell), an insect cell (e.g., a Trichoplusia ni (Tn5) or Sf9), a bacterial cell, a plant cell, or a yeast cell.

[0193]

[0209] In certain embodiments, the HTS (or variant thereof) is produced in a yeast expression system (i.e., a yeast-derived HTS or variant thereof), for example, produced 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).

[0194]

[0210] In other specific embodiments, the HTS (or variant thereof) is produced in a bacterial expression system (i.e., a bacterially derived HTS or variant thereof), e.g., in Escherichia coli or Bacillus subtilis. In one embodiment, the HTS (or variant thereof) is not produced in E. coli.

[0195]

[0211] In further specific embodiments, the HTS (or variant thereof) is produced in an insect expression system (i.e., an insect-derived HTS or variant thereof), for example, in baculovirus-infected or non-lytic insect cells (e.g., sf9, Sf21).

[0196]

[0212] In another embodiment, the HTS (or variant thereof) is produced in a fungal expression system (i.e., an HTS or variant thereof derived from a fungus), for example, produced in the genus Chrysosporium, Thielavia, Talaromyces, Trichoderma, Thermomyces, or Thermoascus.

[0197]

[0213] In yet another embodiment, the HTS (or variant thereof) is produced in a mammalian expression system (i.e., a mammalian-derived HTS or variant thereof), such as in Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK), COS, and baby hamster kidney (BHK) cells. Alternatively, the HTS (or variant thereof) may be produced in vitro using a cell-free expression system, such as an E. coli S30 extract.

[0198]

[0214] purification

[0215] Polypeptides recombinantly expressed from an expression cassette encoding Myd are typically isolated from lysed cells or culture medium. Purification can be achieved by methods known in the art, such as 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. For example, immunoaffinity chromatography using antibodies raised against the Gag antigen can be employed.

[0199]

[0216] The present invention provides a method for purifying a polypeptide having sweet taste modulating activity, the method comprising the steps of (a) obtaining a composition comprising the polypeptide, and (b) purifying the composition by hydrophobic interaction chromatography (HIC) followed by size exclusion chromatography (SEC).

[0200]

[0217] Those skilled in the art are familiar with hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC) purification techniques, such as selecting appropriate columns, buffers, and elution solutions. Exemplary HIC and SEC purification techniques are described herein in Example 11. In exemplary embodiments, the purity of the polypeptide after purification by HIC and SEC is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or any range of values ​​therebetween.

[0201]

[0218] In cell-based systems, the first step in the protein purification process is to extract proteins from cells by lysing or breaking them open. Any suitable cell lysis method can be used, such as mechanical disruption, chemical disruption, freeze-thaw cycles, or enzymatic digestion. The proteins 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, reversed-phase chromatography, concanavalin A chromatography, chromatofocusing, and differential precipitation or solubilization.

[0202]

[0219] The yield of in vivo production of HTS (or variants thereof) can vary. In certain embodiments, the HTS represents at least about 1% of total cellular protein. In certain embodiments, the HTS represents about 1% to about 5% of total cellular protein. In other embodiments, the HTS comprises about 5% to about 10% of total cellular protein. In further embodiments, the HTS comprises 10% to about 20% of total cellular protein. In certain embodiments, the HTS comprises more than 20% of total cellular protein.

[0203]

[0220] In other specific embodiments, the 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 higher. Optionally, the HTS is produced as a fusion protein further comprising a tag, and the yields stated above reflect both purification and tag removal.

[0204]

[0221] 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 about 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.

[0205]

[0222] plant

[0223] The present invention also contemplates transgenic plants comprising the heterologous polynucleotides and / or heterologous polypeptides of the present invention described herein. The plants have an altered phenotype due to expression of the heterologous nucleic acid sequences. The altered phenotype can include increased sweetness in any plant part, such as fruit. The transgenic plant can contain an expression cassette as defined herein as part of the plant, the cassette being introduced by transforming the plant with a vector of the present invention. Such an expression cassette contains regulatory sequences for expression of the heterologous coding sequence in the plant, such as a plant-expressible promoter and terminator. The transgenic plant can be any type of plant capable of expressing the heterologous nucleic acid sequences described herein. The term "plant" includes whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, and plant cells, as well as their progeny. The classes of plants that can be used in the methods of the present invention generally span a wide range of higher plant classes amenable to transformation techniques, including both monocotyledonous plants (monocots) and dicotyledonous plants (dicots). Examples include plants of various ploidy levels, such as polyploid, diploid and haploid. For example, the transgenic plant may be an apple or strawberry. The HTS protein / polypeptide or variant of this disclosure may be produced in plants or plant cultures.

[0206]

[0224] Techniques for transforming various plant species are well known in the art and are described in technical and scientific literature. See, for example, Weising et al. (1988) Ann. Rev. Genet., 22:421-477, and Joung et al. (2015) "Plant Transformation Methods and Applications" in Current Technology in Plant Molecular Breeding, (eds. Koh et al.), Springer Dordrecht Heidelberg, New York, London, Chapter 9, pp. 297-344. Any method known in the art for transforming plant cells, including plant protoplasts or plant tissues, can be used for plant transformation. Specific methods for plant transformation include, among others, biolistic methods (gene guns), electroporation, microinjection, protoplast fusion, and Agrobacterium-mediated transformation. Agrobacterium-mediated transformation may employ, for example, a binary vector that replicates in E. coli and Agrobacterium tumefaciens or other Agrobacterium strains. A variety of such binary vectors are known in the art and can be employed to introduce heterologous polynucleotides into plant cells and tissues. Plant expression vectors containing regulatory sequences for expression of heterologous coding sequences, such as plant-expressible promoter sequences and other plant regulatory sequences, in plant cells and tissues are known in the art and can be employed to transform plants to express the polypeptides described herein.

[0207]

[0225] A variety of plant-expressible promoters are known in the art and are available for use in the heterologous constructs, vectors, and transformed plant materials described herein that contain a polynucleotide encoding a protein with sweet taste-modulating activity. Plant-expressible promoters can be obtained from natural plant sources, plant viral sources, and bacteria, such as Agrobacterium strains that contain plant-expressible promoters. Plant-expressible promoters include, among others, the cauliflower mosaic virus promoter (CaMV35S), octopine and nopaline synthase promoters (e.g., the nos promoter), the plant ubiquitin promoter (Ubi), the rice actin promoter (Act-1), and the maize alcohol dehydrogenase (Adh-1). Plant-expressible promoters include constitutive promoters, inducible promoters, tissue-specific promoters, and developmental stage-specific promoters; examples of each type of promoter are known in the art. Tissue-specific promoters include those that direct expression in plant roots, plant leaves, fruits, flowers, pollen, or cells involved in active photosynthesis (e.g., the phosphoenolpyruvate promoter (PEP)). Developmental stage-specific promoters include those that direct expression during fruit ripening, flowering, or fruit set. Synthetic plant promoters are also known in the art and are useful in heterologous constructs, vectors, and transformed plant materials (see, e.g., Ali S. and Kim WC (2019) Frontiers in Plant Science, 10, paper 1433).

[0208]

[0226] Techniques for the regeneration of plants from transformed protoplasts, plant cells, callus, or other plant tissues are well known in the art and can be employed to regenerate whole plants and plant parts from such transformed plant material. Regeneration methods include organogenesis and embryogenesis. See Handbook of Plant Cell Culture, Vol. 1: Techniques for Propagation and Breeding (1983) by D.A. Evans et al. (eds.), Macmillan, New York; R.H. Smith, Plant Tissue Culture: Techniques and Experiments, 3rd Edition (2012) by Academic Press, New York; M.R. Davey and P. Anthony, Plant Cell Culture: Essential Methods (2010) by John Wiley & Sons, New York, especially Chapters 3 and 9.

[0209]

[0227] In an embodiment, the HTS (or variant thereof) is produced in an algal expression system (ie, an algal-derived HTS or variant thereof).

[0228] 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, produced in corn, maize, tobacco, melon (e.g., watermelon), potato, strawberry, duckweed, or sugarcane. In one embodiment, the plant expression system is a plant cell culture expression system.

[0210]

[0229] In certain embodiments, the HTS (or variant thereof) is produced in corn, more particularly in corn seeds. In other certain embodiments, the HTS (or variant thereof) is produced in corn, more particularly in corn seeds. According to these embodiments, the HTS (or variant thereof) may be utilized as an HTS-containing germ flour.

[0211]

[0230] Methods for producing proteins with sweetness-modulating activity (in a host or cell-free expression system)

[0231] Methods commonly used in the field, such as the protoplast method and electroporation, can be used to introduce vectors into hosts. The desired transformed strain can be obtained by selecting strains into which the vector has been appropriately introduced using indicators such as marker gene expression and / or auxotrophy.

[0212]

[0232] Alternatively, a fragment to which a polynucleotide encoding a Myd polypeptide variant of the present invention, a regulatory sequence, and a marker gene are ligated can be directly introduced into the genome of a host. For example, a polynucleotide encoding a Myd polypeptide variant of the present invention can be introduced into the genome of a host by constructing a DNA fragment in which sequences complementary to the host genome are added to both ends of the ligated fragment, introducing the fragment into the host, and inducing homologous recombination between the host genome and the DNA fragment by SOE-PCR.

[0213]

[0233] Culturing the thus obtained transformed strain into which a polynucleotide encoding a Myd polypeptide variant of the present invention or a vector containing the same has been introduced in an appropriate medium results in expression of the MYD cDNA in the vector, which in turn results in production of the Myd polypeptide variant of the present invention. The medium used to culture such a transformed strain can be selected by those skilled in the art, as needed, depending on the type of microorganism of the transformed strain.

[0214]

[0234] Alternatively, the Myd polypeptide variants of the present invention can be expressed from polynucleotides encoding the Myd polypeptide variants of the present invention or their transcription products using a cell-free translation system. A "cell-free translation system" refers to an in vitro transcription-translation system or an in vitro translation system, which is constructed by adding reagents, such as amino acids, necessary for protein translation to a suspension obtained by mechanically disrupting host cells.

[0215]

[0235] 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 elements from eukaryotic, prokaryotic, and / or viral sources. For example, cell-free systems used herein can include mammalian and / or bacterial protein expression systems derived from mammalian and / or bacterial lysates.

[0216]

[0236] The Myd polypeptide variants of the present invention produced in culture or in a cell-free translation system can be isolated or purified, as desired, by using common methods used for protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, and affinity chromatography, either individually or in combination as needed. When a gene encoding the Myd polypeptide variant of the present invention and a secretory signal sequence are operably linked on a vector in a transformed strain, the Myd polypeptide variant is secreted outside the cells, making it easier to collect the produced Myd polypeptide variant from the culture. The Myd polypeptide variant collected from the culture can be further purified by any known means.

[0217]

[0237] In embodiments, the Myd protein is solubilized in a liquid solution, such as a buffered solution or any solution in which the Myd protein is readily dissolved. In embodiments, the Myd protein contained in the liquid solution is freeze-dried to form a powder. In embodiments, the Myd protein contained in the liquid solution is dried to form a powder, for example, by using a spray dryer. In embodiments, spray drying includes the use of carriers known in the art. In embodiments, the carrier used for spray drying is maltodextrin, gum arabic, or whey protein concentrate.

[0218]

[0238] The present invention also includes a method for producing a protein having sweet taste modulating activity, the method comprising culturing a host cell of the present invention in a culture medium under conditions that result in the production of a protein having sweet taste modulating activity similar to that of a known sweet taste enhancer or compound.

[0219]

[0239] Sweetening compositions - foods, beverages, supplements, pharmaceuticals

[0240] In each case, sweetener compositions and flavor-modifying compositions containing Myd (HTS) or their variants are disclosed herein. In certain embodiments, the sweetener compositions and flavor-modifying compositions alter (e.g., improve) one or more sensory experiences of the subject consuming them. In certain embodiments, the sweetener compositions and flavor-modifying compositions disclosed herein comprise an HTS variant. In one embodiment, the HTS variant differs from the wild-type HTS at at least one amino acid position, more particularly at one, two, three, or more amino acid positions.

[0220]

[0241] As used herein, "sweet flavor enhancer," "sweet compound," or "compound that activates sweet taste receptors" refers to a composition that elicits a detectable sweet flavor in a subject, such as sucrose, fructose, glucose, and other known natural sugar-based sweeteners, or, as further discussed herein, known artificial sweeteners such as saccharin, cyclamate, aspartame, or materials that activate T1R2 / T1R3 receptors in vitro. The subject may be a human or an animal.

[0221]

[0242] The sweetening seasoning or sweetener composition can be used in an effective amount, which refers to an amount of the sweetener composition of the present invention sufficient to induce a sweet taste in a subject when present in a product for oral administration.

[0222]

[0243] Embodiments of the present invention include compositions. In one embodiment, the composition comprises, consists essentially of, or consists of a combination of a product for oral administration and one or more sweetener compositions comprising an isolated Myd polypeptide variant according to the present invention, as described herein. In one embodiment, the combination has an enhanced sweetness compared to a product for oral administration lacking the Myd polypeptide variant (control). In one embodiment, the product for oral administration is not a Mattiromyces terfesioides truffle. The term "essentially consisting of" allows for the inclusion of ingredients that are not essential to or do not significantly affect the function or activity of the product, such as anti-caking agents, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltodextrose, gum arabic, etc.). In one embodiment, the composition comprises multiple isolated Myd polypeptides. In a specific embodiment, the composition comprises multiple isolated Myd polypeptides that are different from each other to enhance taste. Compositions comprising one or more Myd polypeptides of the invention are not limited by form, shape, and means of administration, and should be understood to encompass solid, liquid, powder, and other forms, either individually or in combination of two or more thereof. Furthermore, compositions can be administered or consumed orally, by injection, etc.

[0223]

[0244] In another embodiment, a composition comprising an isolated Myd protein of the present invention includes a formulation that provides enhanced functionality to the isolated Myd protein. For example, the composition may 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 Myd proteins include, for example, L-arginine glycine, L-proline, L-histidine, β-alanine, L-serine, L-arginine ethyl ester dihydrochloride, L-arginine amide dihydrochloride, 6-aminohexanoic acid, gly-gly, gly-gly-gly, tryptone, betaine monohydrate, D-(+)-trehalose dihydrate, xylitol, D-sorbitol, sucrose, hydroxyectoine, trimethylamine n-oxide dihydrate, methyl-α-d-glucopyranoside, triethylene glycol, spermine tetrahydrochloride, spermidine, 5-aminovaleric acid, glutaric acid, adipic acid, ethylenediamine dihydrochloride, guanidinium chloride, and the like. Ingredients: 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-surfactant sulfobetaine 195 (NDSB-195), non-surfactant sulfobetaine 201 (NDSB-201), non-surfactant sulfobetaine 211 (NDSB-211), non-surfactant sulfobetaine 221 (NDSB-221), non-surfactant sulfobetaine 256 (NDSB-256), taurine, acetamide, oxalic acid dihydrate, sodium malonate pH 7.0, succinic acid pH 7.0, and taximate pH 7.0, tetraethylammonium bromide, choline acetate, 1-ethyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazolium chloride, ethylammonium nitrate, ammonium sulfate, ammonium chloride, magnesium sulfate hydrate, potassium thiocyanate, gadolinium(III) chloride hexahydrate, cesium chloride, 4-aminobutyric acid (GABA), lithium sulfate, DL-malic acid pH 7.0, lithium citrate tribasic tetrahydrate, ammonium acetate, sodium benzenesulfonate, sodium p-toluenesulfonate, sodium chloride, potassium chloride, monobasic sodium phosphate monohydrate, sodium sulfate decahydrate, lithium chloride, sodium bromide, glycerol ethanol, ethylene glycol, polyethylene glycol 200, polyethylene glycol monomethyl ether 550, polyethylene glycol monomethyl ether 750, formamide, polyethylene glycol 400, pentaerythritol ethoxylate (15 / 4EO / OH), 1,2-propanediol, polyethylene glycol monomethyl ether 1,900, polyethylene glycol 3,350, polyethylene glycol 8,000, polyvinylpyrrolidone K15, polyethylene glycol 20,000, (2-hydroxypropyl)-β-cyclodextrin, α-cyclodextrin, β-cyclodextrin, methyl-β-cyclodextrin.

[0224]

[0245] In embodiments, the sweetener composition comprises one or more Myd polypeptides described above. In one embodiment, the sweetener composition comprises multiple Myd polypeptides described above. In certain embodiments, the multiple Myd polypeptides are different from one another.

[0225]

[0246] The present invention also includes a method for modulating the taste of a product for oral administration, comprising combining the product for oral administration with an effective amount of an isolated Myd polypeptide variant described herein. In one aspect, the combination has an enhanced sweetness compared to a product for oral administration lacking the Myd polypeptide variant (control). In one embodiment, the product for oral administration is not a Mattiromyces terfesioides truffle.

[0226]

[0247] The product for oral administration can be a food, a beverage, a dietary supplement composition, or a pharmaceutical composition.

[0248] The term "product for oral administration" may refer to edible products (consumables), such as foods, beverage products, drug (medicinal) products, or dietary supplement products, such as herbal supplements. The term "consumables," as used herein, can be used synonymously with the term "product for oral administration." The term "medicinal product," as used herein, includes both solid and liquid compositions that are ingestible, non-toxic materials of pharmaceutical value or contain pharmaceutically active agents, such as cough syrup, cough drops, aspirin, and chewable pharmaceutical tablets. Oral hygiene products are also products for oral administration, and examples include solids and liquids, such as toothpaste or mouthwash. Generally speaking, the present invention contemplates that a food or beverage product may contain an isolated sweet protein of the present invention in an effective amount, for example, in an amount of up to about 99% by weight, e.g., from about 0.01% to about 99% by weight, based on the total weight of the food or beverage product. All intermediate amounts by weight (i.e., 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 90%, 95%, 99%) of the total weight of the food or beverage product are contemplated, as are all intermediate ranges based on these amounts. The compositions of the present invention may include an "edible, biologically, or pharmaceutically acceptable carrier or excipient," which may include solid or liquid media and / or compositions used to prepare a desired dosage form of the Myd polypeptide variant for administration of the Myd polypeptide variant in a dispersed / diluted form that maximizes the biological effectiveness of the Myd polypeptide variant.Edible, biologically, or pharmaceutically acceptable carriers include many common food ingredients, such as water of neutral, acidic, or basic pH, fruit or vegetable juices, vinegar, marinades, beer, wine, natural water / fat emulsions such as milk or condensed milk, edible oils and shortenings, fatty acids, low molecular weight oligomers of propylene glycol, glyceryl esters of fatty acids, and dispersions or emulsions of such hydrophobic materials in aqueous media, salts such as sodium chloride, flour, solvents such as ethanol, solid edible diluents such as vegetable powders or flours or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents; thickeners or emulsifiers, preservatives, solid binders, lubricants, and the like.

[0227]

[0249] Pharmaceutically acceptable carriers or excipients can include excipients that allow for microencapsulation of Myd polypeptide variants to enhance functionality, such as protecting or extending sweetness perception. Indeed, microencapsulation is known in the art as a technology that can facilitate regular use in addition to creating many potential new uses for sweeteners. See, for example, Favaro-Trindade, Carmen & Rocha-Selmi, Glaucia & dos Santos, Milla. (2015). Microencapsulation of Sweeteners. 10.1016 / B978-0-12~800350-3.00022-4. In one embodiment, microencapsulation methods known in the art stabilize and / or modify (e.g., extend) the release of Myd sweetness. For example, sugar-free chewing gums and chewable confections typically have encapsulated sweeteners in their formulations to extend their sweetness during chewing. It is understood that food or beverage products and compositions comprising one or more of the described Myd polypeptides are not limited by form or shape, and include solid, liquid, powder, and other forms, either individually or in combination of two or more thereof. Examples of food or beverage products of the present invention include, but are not limited to, baked goods; sweet bakery products (such as, but not limited to, rolls, cakes, pies, pastries, and cookies); pre-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings (such as, but not limited to, fruit pie fillings and nut pie fillings, e.g., pecan pie fillings, as well as fillings for cookies, cakes, pastries, confectionery products, e.g., fat-based cream fillings); desserts, gelatins, and puddings;Frozen desserts (e.g., 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 (e.g., but not limited to, non-alcoholic carbonated beverages); non-carbonated beverages (e.g., but not limited to, non-alcoholic non-carbonated beverages, such as flavored water and sweet tea or coffee-based beverages); beverage concentrates (e.g., but not limited to, liquid concentrates and syrups, as well as non-liquid concentrates, such as freeze-dried and / or powder preparations); yogurt (e.g., but not limited to, full-fat, low-fat, and non-fat dairy yogurt, as well as non-dairy and lactose-free yogurt, and all frozen equivalents thereof); snack bars (e.g., but not limited to, syrupy bread products (such as, but not limited to, leavened and unleavened breads, yeast-raised and non-leavened breads, e.g., soda bread, breads containing any type of wheat flour, breads containing any type of non-wheat flour (e.g., potato flour, rice flour, and rye flour), gluten-free breads, etc.); pre-made bread mixes for preparing bread products; sauces, syrups, and dressings; sweet spreads (such as, but not limited to, jellies, jams, butters, nut spreads and other spreadable preserves, confectioneries, etc.); confectionery products (such as, but not limited to, jelly candy, soft candy, hard candy, chocolate, and gum); sweetened breakfast cereals (such as, but not limited to, extruded (KIX-type) breakfast cereals, flaked breakfast cereals, and puffed breakfast cereals, etc.);and cereal coating compositions for use in preparing sweetened breakfast cereals. Other types of food and beverage products not mentioned here but which conventionally contain one or more nutritious sweeteners are also contemplated within the context of the present invention.

[0228]

[0250] In embodiments, the product for oral administration is a food that is warmed or heated before eating, or is served in a warm or hot state for consumption. In embodiments, the polypeptide variants described herein that exhibit enhanced thermostability compared to the polypeptide of SEQ ID NO: 3 are preferred for application in compositions for oral administration (e.g., foods) that are expected to be cooked, warmed, or heated before administration or consumption, or that are expected to be administered or consumed in a warm or hot state.

[0229]

[0251] As a result of the complete or partial replacement of nutritional sweeteners in the food or beverage products of the present invention, the food or beverage products of the present invention may be useful as low-calorie or diet products, medical foods / products (such as pills and tablets), and sports nutrition products, and may be particularly suitable for food or beverage products requiring lower sweetness at a given soluble solids level.

[0230]

[0252] In some embodiments, the sweetener composition of the present invention may be supplemented with other nutritive or non-nutritive sweeteners to form a sweetener system. The sweetener system may include the sweetener composition of the present invention, a bulking agent such as maltodextrose or gum arabic, and at least one high-intensity sweetener. The composition may be provided as a liquid composition or a dry blend.

[0231]

[0253] The term "high-intensity sweetener" as used herein refers to any synthetic or semi-synthetic sweetener or naturally occurring sweetener. A high-intensity sweetener is a compound or mixture of compounds that is sweeter than sucrose. A high-intensity sweetener is typically several times sweeter than sucrose (e.g., 20 times or more, 30 times or more, 50 times or more, or 100 times or more).

[0232]

[0254] In one embodiment, the invention comprises a process for enhancing the sweetness of a product for oral administration comprising the addition of a Myd polypeptide variant of the invention.

[0255] In another embodiment, the method of the present invention includes a method for improving the sweet flavor of a product for oral administration, comprising adding to the product for oral administration a sweetener composition made by the method of the present invention, The amount to be added can be determined by methods known in the art, for example, using sensory tests as a guide.

[0233]

[0256] In another embodiment, the method of the present invention includes a method for modifying the flavor of a product for oral administration, comprising adding a flavor-modifying composition made by the method of the present invention to the product for oral administration. The amount to be added can be determined by methods known in the art, for example, using sensory testing as a guide. The flavor-modifying composition can modify (e.g., enhance, inhibit, or change) the taste, aroma, and / or texture of a given composition, for example, a consumable product. In certain embodiments, the flavor-modifying composition modifies (e.g., enhances, inhibits, or changes) a particular taste. In another embodiment, the flavor-modifying composition modifies (e.g., enhances, inhibits, or changes) a given texture. In certain embodiments, the flavor-modifying composition modifies (e.g., enhances, inhibits, or changes) both a given taste and texture.

[0234]

[0257] The flavor-modifying composition may be sweetened or sugar-free. Thus, in some embodiments, the addition of a flavor-modifying composition can serve both to add a flavor modifier and also to provide sweetness to the selected composition for taste adjustment. The addition of a sweetened flavor-modifying composition can be used in addition to, or as a substitute for, the addition of another sweetener composition.

[0235]

[0258] The sweetener compositions and flavor-modifying compositions disclosed herein contain Myd (HTS) and variants thereof. In certain embodiments, HTS (or its variant) is the only sweetening ingredient 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 ingredients (i.e., additional sweeteners or high-intensity sweeteners). In certain embodiments, the additional sweetener is a polypeptide or protein sweetener other than HTS or its variant. In certain embodiments, the sweetener is a carbohydrate sweetener. In certain embodiments, the additional sweetener is a synthetic sweetener. In certain embodiments, the one or more sweetening ingredients include steviol glycosides (e.g., Reb M, Reb A) and high fructose corn syrup (HFCS).

[0236]

[0259] High fructose corn syrup (HFCS), also known as glucose-fructose, isoglucose, and glucose-fructose syrup, is a sweetener made from corn starch. As in the production of conventional corn syrup, the starch is enzymatically broken down into glucose.

[0237]

[0260] Steviol glycosides are compounds responsible for the sweetness of the leaves of the Stevia rebaudiana plant and several related plants. Specific steviol glycosides are components in stevia sweeteners or precursors to components in stevia sweeteners. Steviol glycosides can be single compounds or mixtures of compounds. Steviol glycosides 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.

[0238]

[0261] Mogrosides are glycosides of cucurbitane derivatives such as mogrol, and are responsible for the sweetness of extracts of Siraitia grosvenorii (monk fruit or monk fruit). Specific mogrosides are components of monk fruit sweeteners. Among the mogrosides are mogroside II A1, mogroside II B, 7-oxomogroside II E, and 11-oxomogroside A. 1、 Mogroside III A 2、 These include 11-deoxymogroside III, 11-oxomogroside IV A, mogroside V, 7-oxomogroside V, 11-oxomogroside V, mogroside VI, siamenoside I, and combinations thereof. Preferred mogrosides are mogroside V, mogroside VI, and siamenoside.

[0239]

[0262] In one embodiment, the one or more additional sweeteners may be carbohydrate sweeteners. Non-limiting examples of suitable carbohydrate sweeteners include sucrose, fructose, glucose, erythritol, maltitol, lactitol, sorbitol, mannitol, xylitol, D-tagatose, trehalose, galactose, rhamnose, cyclodextrins (e.g., α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin), ribulose, threose, arabinose, xylose, lyxose, and aryl. Sugar, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, palatinose or isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, glucosamine, mannosamine, fucose, fuculose, glucuronic acid, gluconic acid, gluconolactone, abequase, galactosamine, chitin Scirtooligosaccharides (xylotriose, xylobiose, etc.), gentiooligosaccharides (gentiobiose, gentiotriose, gentiotetraose, etc.), galactooligosaccharides, sorbose, ketotriose (dehydroxyacetone), aldotriose (glyceraldehyde), nigerooligosaccharides, fructooligosaccharides (kestose, nystose, etc.), maltotetraose, maltotriol, tetrasaccharides, mannanoligosaccharides, maltooligosaccharides (maltotriose, maltotriose, etc.), Examples of sugars include maltose, 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 sugar, soybean oligosaccharides, glucose syrup, and combinations thereof.

[0240]

[0263] In other embodiments, at least one additional sweetener is a synthetic sweetener.The term "synthetic sweetener" as used herein refers to any composition that is not naturally found in nature and has a sweetness potency greater than that of sucrose, fructose, or glucose, but characteristically has fewer calories.Non-limiting examples of synthetic high-potency sweeteners suitable for embodiments of this disclosure include sucralose, potassium acesulfame, aspartame, alitame, saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, advantame, glucosylated steviol glycoside (GSG), and combinations thereof.

[0241]

[0264] In another embodiment, the at least one additional sweetener is a protein sweetener (i.e., a sweet-tasting polypeptide or protein). Typically, such protein sweeteners are extracted from plants. Non-limiting examples of protein sweeteners include monellin, thaumatin, brazzein, curculin, pentadin, and mabinlin.

[0242]

[0265] In certain embodiments, the sweetener compositions and flavor-modifying compositions disclosed herein comprise a Myd (HTS) variant. In one embodiment, the HTS variant differs from the wild-type HTS (with or without methionine at position 1) at at least one amino acid position, more particularly at one, two, three, or more amino acid positions.

[0243]

[0266] In one embodiment, the sweetener compositions and flavor modifying compositions disclosed herein comprise an HTS variant that has a sweetness equal to or greater than that of a wild-type HTS having the amino acid sequence of SEQ ID NO:3.

[0244]

[0267] In one embodiment, the sweetener compositions and flavor modifying compositions disclosed herein comprise an HTS variant that has equal or greater stability than the wild-type HTS having the amino acid sequence of SEQ ID NO:3.

[0245]

[0268] In one embodiment, the sweetener compositions and flavor-altering compositions disclosed herein comprise an HTS variant that has 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.

[0246]

[0269] In one embodiment, the sweetener compositions and flavor modifying compositions disclosed herein comprise an HTS variant that has equal or greater stability than a wild-type HTS having the amino acid sequence of SEQ ID NO: 3 without the methionine at position 1.

[0247]

[0270] An HTS (or variant thereof) suitable for use in the compositions disclosed herein (e.g., sweetener compositions, flavor and / or taste modifying compositions, consumables) can be produced in any suitable manner as previously described herein. Exemplary production methods include extraction, chemical synthesis (i.e., solid phase synthesis), or recombinant production (i.e., in vivo or in vitro production).

[0248]

[0271] In one embodiment, the HTS used in the compositions disclosed herein is isolated from the mycelium or aqueous extract of an edible (i) truffle of the family Terfeziaceae, 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 mycelium or fruiting body of the truffle Mattiroromyces terfezioides.

[0249]

[0272] 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 and optionally optimized from the truffle Mattiromyces terfesioides is introduced into a suitable vector, which is then cloned into a host cell in an appropriate growth system / environment, resulting in recombinant expression of the protein. Suitable host cells and expression systems have been previously described herein.

[0250]

[0273] The amount of HTS (or variant thereof) in the sweetener compositions and flavor modifying compositions disclosed herein can vary. In one embodiment, HTS (or variant thereof) is present in the sweetener composition above its sweetness threshold concentration.

[0251]

[0274] In one embodiment, the HTS (or a variant thereof) is present in the sweetener or flavor modifying composition in any amount that imparts a desired sweetness when the sweetener or flavor modifying composition is added to a consumable (e.g., a beverage), either alone or in combination with one or more additional sweet ingredients (e.g., steviol glycosides, HFCS) present in the sweetener or flavor modifying composition, i.e., combined with such composition before it is added to the consumable.

[0252]

[0275] In certain embodiments, the desired sweetness of the consumable is equivalent to a sucrose-sweetened consumable having a sweetness of at least about 8 degrees Brix, for example, equivalent to a sucrose-sweetened consumable having a sweetness of about 9 degrees Brix, about 10 degrees Brix, about 11 degrees Brix, about 12 degrees Brix, about 13 degrees Brix, about 14 degrees Brix, or about 15 degrees Brix.

[0253]

[0276] In another embodiment, the desired sweetness of the consumable is equivalent to a sweetness of a sucrose-sweetened consumable having a sweetness of about 10 degrees Brix to about 15 degrees Brix, e.g., about 10 degrees Brix to about 14 degrees Brix, about 10 degrees Brix to about 13 degrees Brix, about 10 degrees Brix to about 12 degrees Brix, about 10 degrees Brix to about 11 degrees Brix, about 11 degrees Brix to about 15 degrees Brix, about 11 degrees Brix to about 14 degrees Brix. , about 11 degrees Brix to about 13 degrees Brix, about 11 degrees Brix to about 12 degrees Brix, about 12 degrees Brix to about 15 degrees Brix, about 12 degrees Brix to about 14 degrees Brix, about 12 degrees Brix to about 13 degrees Brix, about 13 degrees Brix to about 15 degrees Brix, about 13 degrees Brix to about 14 degrees Brix, and about 14 degrees Brix to about 15 degrees Brix.

[0254]

[0277] In one embodiment, the Myd polypeptide (or variant thereof), either alone or in combination with one or more additional sweet ingredients (e.g., steviol glycosides, HFCS) present in the sweetener or flavor modifying composition, i.e., combined with such composition before it is added to a consumable, is present in an amount that enhances the sweetness of the consumable to which it is added, i.e., about 1.0% (w / v) sucrose equivalent (SE) or more.

[0255]

[0278] In certain embodiments, the Myd polypeptide (or variant thereof) is added to the consumable product to which it is added, either alone or in combination with one or more additional sweet ingredients (e.g., steviol glycosides, HFCS) present in the sweetener composition or flavor-modifying composition, i.e., before such composition is added to the consumable product. It is present in an amount to enhance the sweetness of the product, i.e., about 1.0% to about 3.0% (w / v) sucrose equivalent (SE), e.g., 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.

[0256]

[0279] In certain other embodiments, the Myd polypeptide (or variant thereof) is present in a sweetener or flavor modifying composition, either alone or in combination with one or more additional sweet ingredients (e.g., steviol glycosides, HFCS) present in the sweetener or flavor modifying composition, i.e., before such composition is added to a consumable product. It is present in an amount that enhances the sweetness of the consumable to which it is added, i.e., 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% sucrose equivalent.

[0257]

[0280] The sweetness of a given composition is typically measured with reference to a solution of sucrose. 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.P. Ecore, K. Gibes, B.T. Carr, and L.M. Brands, in Sweeteners: Discovery, Molecular Design and Chemoreception, D.E. Walters, F.T. Orthoefer, and G.E. DuBois, eds., American Chemical Society, Washington, DC (1991), pp. 261-276.

[0258]

[0281] The amount of sucrose in a reference solution can be stated in degrees Brix (°Bx): 1 degree Brix is ​​1 gram of sucrose in 100 grams of solution, and represents the strength of the solution as a weight percentage (% w / w) (strictly speaking, by mass).

[0259]

[0282] In one embodiment, a sweetener composition is provided that contains a Myd polypeptide (or a variant thereof) in an amount effective to provide a sweetness equivalent to about 1 to about 12 degrees Brix of sugar, e.g., 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 of sugar, either alone or in combination with a sweetener composition or a flavor modifying composition or one or more sweet ingredients (e.g., steviol glycosides, HFCS) present in the consumable, when added to a consumable.

[0260]

[0283] In another embodiment, the Myd polypeptide (or variant thereof) is present in an amount effective to provide a sweetness equivalent to about 10 degrees Brix when added to a sweetened composition, either alone or in combination with one or more sweet ingredients (e.g., steviol glycosides, HFCS) present in a sweetener composition, flavor modifying composition, or consumable product to which it is added.

[0261]

[0284] The sweetness of a non-sucrose sweetener can also be measured against a sucrose reference by determining the sucrose equivalent of the non-sucrose sweetener. Typically, taste panelists are trained to detect the sweetness of a reference sucrose solution containing 1-15% sucrose (w / v). Other non-sucrose sweeteners are then tasted at a series of dilutions to determine the concentration of the non-sucrose sweetener that is as sweet as a given percentage of the sucrose reference. For example, if a 1% solution of a sweetener is as sweet as a 10% sucrose solution, the sweetener is said to be 10 times more potent than sucrose.

[0262]

[0285] In one embodiment, the amount of Myd polypeptide (or variant thereof) present in the sweetener or flavor-modifying compositions disclosed herein is any amount that contributes to one or more improved sensory characteristics of the consumable product (e.g., a beverage) to which the sweetener or taste-modifying composition is added. In certain embodiments, the improved sensory characteristics relate to the base taste. In one embodiment, improving one or more sensory characteristics results in an improved taste profile. The overall taste profile of a composition is an interaction of several different tastes, such as sweet, sour, salty, bitter, umami, etc.

[0263]

[0286] "Organoleptic properties," as used herein, are aspects of food, water, or other substances that are experienced by an individual through the senses, including taste, sight, smell, and touch. Organoleptic properties include, for example, appearance, texture, color, odor, size, shape, and flavor. This is a qualitative assessment based on the study of the morphological and sensory profile of food, water, or other substances (such as, for example, sweetener compositions).

[0264]

[0287] Examples of improved sensory properties can include, for example, reduced bitterness, reduced astringency and licorice-like aroma, slower sweetness onset, reduced sweetness lingering, reduced bitterness lingering, reduced bitter aftertaste, reduced metallic aftertaste, reduced chemical or synthetic aftertaste, and combinations thereof. In certain embodiments, the term "improved sensory properties" means that a sweetened or taste-modified composition (e.g., a beverage) is expected to have one or more improved sensory properties for a majority of users. Improvements can be expressed qualitatively or quantitatively, for example, as a percentage improvement.

[0265]

[0288] Improved sensory properties can be measured by or using technological means such as taste sensing systems (TSS), a term that refers to analytical sensory array units (e.g., electrochemical, gravimetric, visual, or biosensors) that can detect specific substances. Sliwińska, M. et al., J. Agric. Food Chem. (2014), 62, 1423–1448.

[0266]

[0289] In certain embodiments, the Myd polypeptide (or variant thereof) is present in the sweetener or flavor modifying composition, either alone or together with one or more sweet-tasting ingredients (e.g., steviol glycosides, HFCS) in the sweetener or flavor modifying composition, i.e., before it is added to the consumable, in any amount that reduces, inhibits, or masks the bitter taste of the consumable (e.g., beverage) to which the sweetener or flavor modifying composition is added. Comparisons are made to consumables to which the sweetener or flavor modifying composition has not been added.

[0267]

[0290] In certain embodiments, the Myd polypeptide (or variant thereof), either alone or combined with one or more sweetening ingredients (e.g., steviol glycosides, HFCS) in a sweetener or flavor-modifying composition, i.e., before it is added to a consumable, is present in the sweetener or flavor-modifying composition in an amount that reduces the bitterness of the consumable (e.g., 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 bitterness is experienced by a majority of subjects. Comparison is made to a consumable to which no sweetener or flavor-modifying composition has been added.

[0268]

[0291] In certain embodiments, the Myd polypeptide (or variant thereof), either alone or combined with one or more sweetening ingredients (e.g., steviol glycosides, HFCS) in the sweetener or flavor-modifying composition, i.e., combined therewith before it is added to the consumable, is present in the sweetener or flavor-modifying composition in any amount that reduces the bitter aftertaste of the consumable (e.g., beverage) to which the sweetener or flavor-modifying composition is added. In certain embodiments, the Myd polypeptide (or variant thereof) is present in the sweetener or flavor-modifying composition in an amount that reduces the bitter aftertaste of the consumable (e.g., 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 bitter aftertaste is experienced by a majority of subjects. Comparison is made to a consumable to which the sweetener or flavor-modifying composition has not been added.

[0269]

[0292] In another embodiment, the Myd polypeptide (or a variant thereof) is present in the sweetener or taste-modifying composition in any amount that reduces the sweetness aftertaste of a consumable product (e.g., a beverage) to which the sweetener or taste-modifying composition is added. Sucrose exhibits a rapid peak perceived sweetness response, where the perceived sweetness dissipates relatively quickly upon swallowing the food or beverage. In contrast, the sweet taste of essentially all high-potency sweeteners reaches its peak response somewhat later than that of sucrose and then its intensity declines more slowly. This reduction in sweetness is often referred to as "sweetness aftertaste" and is a major disadvantage for high-potency sweeteners such as NHPSs. Slow onset of sweetness can also be problematic. However, sweetness aftertaste is generally the more significant problem. Thus, preferred embodiments of the present invention exhibit a significant reduction in sweetness aftertaste.

[0270]

[0293] In certain embodiments, the Myd polypeptide (or variant thereof), either alone or in combination with one or more sweet-tasting ingredients (e.g., steviol glycosides, HFCS) in a sweetener or flavor-modifying composition, i.e., before it is added to a consumable, is present in the sweetener or flavor-modifying composition in an amount that reduces the sweetness aftertaste of a consumable (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, a majority of subjects perceive a reduction in sweetness aftertaste. In certain embodiments, the comparison is made to a consumable to which the sweetener or flavor composition has not been added.

[0271]

[0294] In certain embodiments, the Myd polypeptide (or a variant thereof) is present in a sweetener composition or flavor-altering composition in an amount that results in at least one change / alteration in the sensory characteristics of the consumable (e.g., a beverage) compared to a consumable not containing the sweetener composition, where the sensory characteristic is selected from the group consisting of aroma, flavor, base taste (sweet, sour, salty, bitter, or umami), aftertaste or lingering taste, temporal profile, texture, or a combination thereof. In this embodiment, the change or modification can be any perceived difference in a sensory characteristic that may or may not be considered an improvement. For example, if the Myd polypeptide present in a consumable results in a change in flavor from chocolate to caramel, this would be expected to be considered a "change," but not necessarily an "improvement."

[0272]

[0295] In certain embodiments, the sweetener composition or flavor and / or taste modifying composition contains one or more additional sweeteners. In one embodiment, the additional sweetener is present at a concentration above its sweetness threshold. In certain embodiments, the sweetener composition containing Myd and one or more additional sweeteners synergistically enhances the sweetness of the consumable to which the sweetener composition is added. In one embodiment, the sweetness of the consumable is enhanced in a manner that would be unexpected for a person skilled in the art.

[0273]

[0296] The additional sweetener may be any type of sweetener, for example, natural, non-natural, or synthetic sweeteners.

[0297] In at least one embodiment, the at least one additional sweetener is selected from natural sweeteners other than stevia sweeteners, hi another embodiment, the at least one additional sweetener is selected from synthetic high-potency sweeteners (SHPS).

[0274]

[0298] In certain embodiments, the one or more additional sweeteners may be natural high potency sweeteners (NHPSs). 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, stevioside, mogroside IV, mogroside V, monak fruit sweetener, siamenoside, monatin and its salts (monatin SS, RR, RS, SR), curculin, glycyrrhizic acid and its salts, tamarind, thiamin mononitrate ... Examples of natural high-potency sweeteners include umatin, monellin, mabinlin, brazzein, hernandulcin, phyllodulcin, glycyphyllin, phloridzin, trilobatin, bayounoside, osladin, polypodoside A, pterocaryoside A, pterocaryoside B, mukuroziosid, phlomisoside I, periandrin I, abrusoside A, steviolbioside, and cyclocaryoside I. The natural high-potency sweeteners may be provided as pure compounds, or alternatively, as part of an extract. For example, rebaudioside A may be provided as a single compound or as part of a stevia extract.

[0275]

[0299] In one embodiment, the one or more additional sweeteners are selected from the group consisting of rebaudioside M, rebaudioside A, siamenoside I, and mogroside V.

[0300] In certain embodiments, the sweetener and / or flavor modifying compositions of the present invention comprise a Myd polypeptide (or a variant thereof) and siamenoside I.

[0276]

[0301] In other specific embodiments, the sweetener and / or flavor modifying compositions of the present invention comprise a Myd polypeptide (or a variant thereof) and mogroside V.

[0277]

[0302] In another embodiment, the one or more additional sweeteners are rebaudioside D, rebaudioside N, rebaudioside O, rebaudioside E, steviolmonoside, steviolbioside, 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 glucosylated steviol glycosides, and combinations thereof.

[0278]

[0303] In further 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, mogrol, 11-oxomogrol, the 1,6-alpha isomer of siamenoside I, monk fruit extract, and combinations thereof.

[0279]

[0304] In certain embodiments, the one or more additional sweeteners is rebaudioside M (13-[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]entokaur-16-en-19-olic acid (-[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl) ester), having the following formula (I):

[0280] [ka]

[0305] Reb M can be provided in 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.

[0281]

[0306] In another embodiment, the additional sweetener is a steviol glycoside composition. An exemplary steviol glycoside composition is A95, which contains primarily reb D and reb M with minor amounts of one or more of the following: Reb E, Reb O, Reb N, Reb A, stevioside, Reb C, and Reb B. Methods for obtaining A95 are provided in WO2017 / 059414, which is incorporated herein by reference.

[0282]

[0307] The amount of Reb M in a sweeter or taste-altering composition may vary. In one embodiment, Reb M is present in the sweetener composition in any amount that imparts a desired sweetness when the sweetener composition is added to a consumable (e.g., a beverage). In certain embodiments, the desired sweetness of the consumable is greater than about 10 degrees Brix.

[0283]

[0308] In one embodiment, the sweetener composition contains Reb M in an amount effective to provide a sweetness equivalent to about 1 to 12 degrees Brix when added to a consumable product (e.g., a beverage), such as 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.

[0284]

[0309] In certain embodiments, Reb M is present in an amount effective to provide about 8 or less sucrose equivalents (SE), such as about 7, about 6.5, about 6, about 5.5, or about 5 SE.

[0285]

[0310] In other specific embodiments, Reb M is present in an amount effective to provide about 8 or more sucrose equivalents, such as 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.

[0286]

[0311] In one embodiment, Reb M is present in the flavor modifying composition in any amount that imparts a desired flavor when the flavor modifying composition is added to a flavor-modifying composition (e.g., a beverage). In certain embodiments, the desired flavor is a temporal or taste profile that is more sugar-like.

[0287]

[0312] In one embodiment, the Reb M and Myd polypeptides (or variants thereof) produce a synergistic effect, e.g., synergistic sweetness, i.e., the sweetness of the combination is greater than the sum of the individual sweeteners. In certain embodiments, the Reb M and Myd polypeptides (or variants thereof) produce an effect that would be unexpected to one of skill in the art.

[0288]

[0313] Reb M may 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 the mixture. In a specific embodiment, the mixture is a stevia extract. The stevia extract may contain Reb M in an amount ranging from about 5% to about 100% by weight on a dry basis, such as 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 still further embodiments, the stevia extract contains Reb M in an amount greater than about 90% by weight on a dry basis, 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 M.

[0289]

[0314] In one embodiment, Reb M is provided as a component of a steviol glycoside mixture, i.e., a mixture of steviol glycosides, with the remainder of the mixture consisting entirely of steviol glycosides, non-Reb M portions of the mixture. The identities of steviol glycosides are known in the art and include, but are not limited to, steviol, steviolmonoside, rubusoside, 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, a steviol glycoside mixture may contain, by weight on a dry basis, 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% Reb M. In still further embodiments, a steviol glycoside mixture may contain greater than about 90% Reb M 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.

[0290]

[0315] Reb M80 refers to a stevia extract or steviol glycoside composition having about 80% Reb M by weight.

[0316] In certain embodiments, the one or more additional sweeteners is rebaudioside A.

[0291]

[0317] Reb A can be provided in 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 the stevia extract by any suitable purification method. Suitable purification methods are known in the art, and examples include, but are not limited to, column chromatography, recrystallization, phase separation, extraction, high performance liquid chromatography, and combinations thereof.

[0292]

[0318] Reb A may 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 specific embodiment, the mixture is a stevia extract. The stevia extract may contain Reb A in an amount ranging from about 5% to about 100% by weight on a dry basis, such as 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 still further embodiments, the stevia extract contains Reb A in an amount greater than about 90% by weight on a dry basis, 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%.

[0293]

[0319] In one embodiment, Reb A is provided as a component of a steviol glycoside mixture, i.e., a mixture of steviol glycosides, and the remainder of the mixture (i.e., the non-Reb A portion) is entirely composed of steviol glycosides. The steviol glycoside mixture may contain about 5% to about 100% Reb A by weight on a dry basis. For example, the steviol glycoside mixture may contain 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%, or about 90% to about 100% Reb A by weight on a dry basis. In still further embodiments, the steviol glycoside mixture may contain greater than about 90% Reb A by weight on a dry basis, 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.

[0294]

[0320] The amount of Reb A in a sweetener or taste-modifying composition may vary. In one embodiment, Reb A is present in the sweetener composition in any amount that imparts a desired sweetness when the sweetener composition is added to a sweetened composition. In certain embodiments, the desired sweetness of the sweetened composition is greater than about 10 degrees Brix.

[0295]

[0321] In one embodiment, the Reb A and Myd polypeptides (or variants thereof) produce a synergistic effect, e.g., synergistic sweetness, i.e., the sweetness of the combination is greater than the sum of the individual sweeteners. In certain embodiments, the Reb A and Myd polypeptides (or variants thereof) produce an effect that would not be expected by one of ordinary skill in the art.

[0296]

[0322] In certain embodiments, Reb A is present in an amount effective to provide about 8 or less sucrose equivalents (SE), such as about 7, about 6.5, about 6, about 5.5, or about 5 SE.

[0297]

[0323] In other specific embodiments, Reb A is present in an amount effective to provide about 8 or more sucrose equivalents (SE), such as 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 sucrose equivalents.

[0298]

[0324] In other specific embodiments, Reb A is present in an amount effective to provide a sucrose equivalent of greater than about 8 SE, for example, about 9 SE, about 9.5 SE, about 10 SE.

[0299]

[0325] In one embodiment, Reb A is present in the flavor-modifying composition in any amount that imparts a desired taste when the taste-modifying composition is added to a taste-modifying composition (e.g., a beverage). In certain embodiments, the desired taste is a sugar-like taste.

[0300]

[0326] In certain embodiments, 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 would be unexpected to one of skill in the art.

[0301]

[0327] Sweetener compositions can be customized to provide a desired calorie content. For example, sweetener compositions can be "high-calorie," such that when added to a sweetened composition (e.g., a beverage), they impart a desired sweetness and have about 120 calories per 8 oz serving.

[0302]

[0328] Sweetener compositions can be customized to achieve a desired calorie content. For example, sweetener compositions can be "medium-calorie," such that when added to a sweetened composition (e.g., a beverage), they impart a desired sweetness and have about 80 calories per 8 oz serving.

[0303]

[0329] For example, sweetener compositions may be "low-calorie," such that when added to a sweetened composition (such as a beverage), they impart a desired sweetness and have less than 40 calories per 8 ounce serving.

[0304]

[0330] In other embodiments, sweetener compositions may be "zero-calorie," such that when added to a sweetened composition (e.g., a beverage), they impart a desired sweetness and have less than 5 calories per 8 ounce serving.

[0305]

[0331] additives

[0332] In addition to a 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 contain additional additives, as described in more detail herein below. In some embodiments, the sweetener compositions contain additives such as, 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, flavorants and flavor components, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers, and combinations thereof. In some embodiments, the additives act to improve the temporal and flavor profile of the sweetener, providing a sweetener composition with a taste similar to sucrose.

[0306]

[0333] In one embodiment, the sweetener composition or flavor-modifying composition contains one or more polyols. The term "polyol" as used herein refers to a molecule containing more than one hydroxyl group. A polyol may be a diol, triol, or tetraol, which contain 2, 3, or 4 hydroxyl groups, respectively. A polyol may also contain more than four hydroxyl groups, such as a pentaol, hexaol, or heptaol, which contain 5, 6, or 7 hydroxyl groups, respectively. In addition, a polyol may also be a sugar alcohol, polyhydric alcohol, or polyalcohol, which are reduced forms of carbohydrates in which the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group.

[0307]

[0334] Non-limiting examples of polyols in some embodiments include erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, isomalt, propylene glycol, glycerol (glycerin), threitol, galactitol, palatinose, reduced isomaltooligosaccharides, reduced xylooligosaccharides, reduced gentiooligosaccharides, reduced maltose syrup, reduced glucose syrup, and sugar alcohols, or any other carbohydrate that can be reduced without adversely affecting taste.

[0308]

[0335] Suitable sweet taste improving amino acid additives 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-, β-, and / or δ-isomers), glutamine, hydroxyproline, taurine, norvaline, sarcosine, and their salt forms, such as sodium or potassium salts or acid salts.Sweet taste improving amino acid additives may also be D- or L-structure, and may be mono-, di-, or tri-forms of the same or different amino acids.In addition, amino acids may optionally be α-, β-, γ-, and / or δ-isomers. In some embodiments, combinations of the aforementioned amino acids and their corresponding salts (e.g., sodium, potassium, calcium, magnesium salts, or other alkali or alkaline earth metal salts thereof, or acid salts) are also suitable sweet taste improving additives. Amino acids may be natural or synthetic. Amino acids may also be modified. Modified amino acids refer to any amino acid in which at least one atom has been added, removed, substituted, or a combination thereof (e.g., N-alkylamino acids, N-acylamino acids, or N-methylamino acids). Non-limiting examples of modified amino acids include amino acid derivatives such as trimethylglycine, N-methyl-glycine, and N-methyl-alanine. Modified amino acids, as used herein, encompass both modified and unmodified amino acids. Amino acids, as used herein, also encompass both peptides and polypeptides (e.g., dipeptides, tripeptides, tetrapeptides, and pentapeptides), such as glutathione and L-alanyl-L-glutamine.Suitable sweet taste improving polyamino acid additives 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 polymeric forms of amino acids, and their salt forms (e.g., calcium, potassium, sodium, or magnesium salts, e.g., monosodium L-glutamate). Sweet taste improving polyamino acid additives may also be D- or L-configuration. In addition, polyamino acids may optionally be α-, β-, γ-, δ-, and ε-isomers. Combinations of the foregoing polyamino acids and their corresponding salts (e.g., their sodium, potassium, calcium, magnesium, or other alkali or alkaline earth metal or acid salts) are also suitable sweet taste improving additives in some embodiments. The polyamino acids described herein may also include copolymers of different amino acids. The polyamino acids may be natural or synthetic. Polyamino acids can also be modified by adding, removing, substituting, or a combination thereof, such as N-alkyl polyamino acids or N-acyl polyamino acids. As used herein, polyamino acids include both modified and unmodified polyamino acids. For example, modified polyamino acids include, but are not limited to, polyamino acids with various molecular weights (MW), such as poly-L-α-lysine with MW of 1,500, MW of 6,000, MW of 25,200, MW of 63,000, MW of 83,000, or MW of 300,000.

[0309]

[0336] Suitable sugar acid additives include, but are not limited to, aldonic acid, uronic acid, aldaric 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.

[0310]

[0337] 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, alkali or alkaline earth metal salts thereof, and combinations thereof. The nucleotides described herein may also include nucleotide-related additives, such as nucleosides or nucleic acid bases (e.g., guanine, cytosine, adenine, thymine, uracil). In certain embodiments, the nucleotide is present in the sweetener composition in an amount of about 5 ppm to about 1,000 ppm.

[0311]

[0338] Suitable organic acid additives include any compound containing a -COOH moiety, such as C2-C30 carboxylic acids, substituted hydroxyl C2-C30 carboxylic acids, benzoic acid, substituted benzoic acids (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, frutic acid (a blend of malic, fumaric, and tartaric acids), fumaric acid, maleic acid, succinic acid, chlorogenic acid, salicylic acid, creatine, caffeic acid, bile acid, acetic acid, ascorbic acid, alginic acid, erythorbic acid, polyglutamic acid, glucono-delta-lactone, and alkali or alkaline earth metal salt derivatives thereof. In addition, the organic acid additive may be in either the D- or L-configuration.

[0312]

[0339] Suitable organic acid addition 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 described exemplary sweet taste improving organic acid additives may optionally be substituted with at least one group selected from hydrogen, alkyl, alkenyl, alkynyl, halo, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, thiol, imine, sulfonyl, sulfenyl, sulfinyl, sulfamyl, carboxyalkoxy, carboxyamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, anhydride, oximino, hydrazino, carbamyl, phosphoro, or phosphonato. In certain embodiments, the organic acid additive is present in the sweetener composition in an amount of from about 10 ppm to about 5,000 ppm.

[0313]

[0340] 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 or alkaline earth metal salts thereof (e.g., inositol hexaphosphate Mg / Ca).

[0314]

[0341] Suitable bitter compound additives include, but are not limited to, caffeine, quinine, urea, bitter orange oil, naringin, cassia, and salts thereof.

[0342] Suitable flavoring substances and flavoring ingredient additives include, but are not limited to, vanillin, vanilla extract, mango extract, cinnamon bark, citrus fruits, coconut, ginger, viridiflorol, almond, menthol (including non-mint menthol), grape skin extract, and grape seed extract. "Flavoring substance" and "flavoring ingredient" are synonymous and examples include natural or synthetic substances or combinations thereof. Flavoring substances also include any other substance that imparts flavor, and examples include natural or non-natural (synthetic) substances that are safe for humans or animals when used within generally accepted standards. Non-limiting examples of proprietary flavorings include Dohler™ Natural Flavoring Sweetness Enhancer K14323 (Dohler™, Darmstadt, Germany), Symrise™ Natural Flavor Mask for Sweeteners 161453 and 164126 (Symrise™, Holzminden, Germany), Natural Advantage™ Bitterness Blockers 1, 2, 9 and 10 (Natural Advantage™, Freehold, NJ, USA), and Sucramask™ (Creative Research Management, Stockton, Calif., USA).

[0315]

[0343] Suitable polymer additives include, but are not limited to, chitosan, pectin, pectinic acid, pectinic acid, polyuronic acid, polygalacturonic acid, starch, edible hydrocolloids or crude extracts thereof (e.g., Senegal gum arabic (Fibergum™), Seyal gum arabic, carrageenan), 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 sodium polyethylene glycol alginate, sodium hexametaphosphate and its salts, and other cationic and anionic polymers.

[0316]

[0344] Other suitable polymer additives that also provide gelling and thickening properties include conventional low-methoxyl (LMC) pectin. LMC pectin also acts 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 they rely on calcium instead of sugar for setting. As calcium is added until a saturation point is reached, the LMC pectin becomes increasingly firm. At that point, the process is reversed and the LMC pectin becomes less firm. In some embodiments, the oral product described herein may comprise one or more foods selected from the group consisting of pie fillings and other sweet fillings, gelatins, and puddings; yogurt; sauces, syrups, and dressings; and sweet spreads. In one embodiment, the oral product further comprises low-methoxyl pectin.

[0317]

[0345] 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% concentrated whey protein), 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 hydrolysates (e.g., porcine collagen hydrolysate).

[0318]

[0346] 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 dioctyl sodium sulfosuccinate, sodium dodecyl sulfate, cetylpyridinium chloride (hexadecylpyridinium chloride), hexadecyltrimethylammonium bromide, sodium cholate, carbamoyl, choline chloride, sodium glycocholate, sodium taurodeoxycholate, lauric arginate, sodium stearoyl lactylate, sodium taurocholate, lecithin, sucrose oleate, sucrose stearate, sucrose palmitate, sucrose laurate, and other emulsifiers.

[0319]

[0347] 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™ 60, Polyphenon™ 30, and Polyphenon™ 25 (Mitsui Norin Co., Ltd., Japan)), polyphenols, rutin (e.g., enzyme-modified rutin, Sammelin™ AO (San-Ei Gen F.F.I., Osaka, Japan)), neohesperidin, naringin, neohesperidin dihydrochalcone, and the like.

[0320]

[0348] Suitable alcohol additives include, but are not limited to, ethanol.

[0349] Suitable astringent compound additives include, but are not limited to, tannic acid, europium chloride (EuCl), gadolinium chloride (GdCl), terbium chloride (TbCl), alum, tannic acid, and polyphenols (e.g., tea polyphenols). In certain embodiments, the astringent additive is present in an amount of about 10 ppm to about 5,000 ppm.

[0321]

[0350] In certain embodiments, a sweetener composition or flavor-modifying composition comprises a Myd polypeptide (or a 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 certain embodiments, the polyol is erythritol. The steviol glycosides (e.g., Reb M, Reb A) may 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) may be present in either the steviol glycoside mixture or the stevia extract in an amount of about 5% to about 100% by weight on a dry basis.

[0322]

[0351] In certain embodiments, a sweetener composition or flavor-modifying composition comprises a Myd polypeptide (or a 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) may 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) may be present in either the steviol glycoside mixture or the stevia extract in an amount of about 5% to about 100% by weight on a dry basis.

[0323]

[0352] In certain embodiments, a sweetener composition or flavor-modifying composition comprises a Myd polypeptide (or a 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 glycosides may 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) may be present in either the steviol glycoside mixture or the stevia extract in an amount of about 5% to about 100% by weight on a dry basis.

[0324]

[0353] In certain embodiments, a sweetener composition or flavor-modifying composition comprises a Myd polypeptide (or a 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) may be provided as pure compounds or as part of a stevia extract or steviol glycoside mixture, as described above.

[0325]

[0354] functional ingredients

[0355] The sweetener or flavor modifying compositions disclosed herein may contain one or more functional ingredients that provide the composition with a real or perceived health benefit, including, but 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 primary aliphatic saturated alcohols, phytosterols, and combinations thereof.

[0326]

[0356] Examples of suitable antioxidants for embodiments of this invention include, but are not limited to, vitamins, vitamin cofactors, inorganic substances, hormones, carotenoids, carotenoid terpenoids, non-carotenoid terpenoids, flavonoids, flavonoid polyphenols (e.g., bioflavonoids), flavonols, flavones, phenols, polyphenols, esters of phenols, esters of polyphenols, non-flavonoid phenols, isothiocyanates, and combinations thereof.In some embodiments, the antioxidant is selected from the group consisting of vitamin A, vitamin C, vitamin E, ubiquinone, inorganic selenium, manganese, melatonin, alpha-carotene, beta-carotene, lycopene, lutein, zeaxanthin, cryptoxanthin, resveratrol, eugenol, quercetin, catechin, gossypol, hesperetin, curcumin, ferulic acid, thymol, hydroxytyrosol, turmeric, thyme, olive oil, lipoic acid, gluta-onone, glutamine, oxalic acid, tocopherol-derived compounds, butylated hydroxypropyl ... Hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediaminetetraacetic acid (EDTA), tert-butylhydroquinone, acetic acid, pectin, tocotrienols, tocopherol, coenzyme Q10, zeaxanthin, astaxanthin, canthaxanthin, saponin, limonoids, kaempferol, myricetin, isorhamnetin, proanthocyanidins, quercetin, rutin, luteolin, apigenin, tangeritin, hesperetin, naringenin, eriodictyol, flava catechin, epicatechin and its gallate form, epigallocatechin and its gallate form (ECGC), theaflavin and its gallate form, thearubigins, phytoestrogenic isoflavones, genistein, daidzein, glycitein, anthocyanins, cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin, ellagic acid, gallic acid, salicylic acid, rosemarinic acid, cinnamic acid and its derivatives (e.g., ferulic acid), chlorogenic acid, chicoric acid, gallotannins, ellagitannins, anthoxanthins, betacyanins and other plant pigments, silymarin, citric acid, lignans, nutrient anti-absorption drugs, bilirubin, uric acid, R-α-lipoic acid, N-acetylcysteine, emblicanin, and phytic acid, or combinations thereof. In alternative embodiments, the antioxidant is a synthetic antioxidant, such as butylated hydroxytoluene or butylated hydroxyanisole.Other sources of suitable antioxidants for embodiments of this invention include, but are not limited to, fruits, vegetables, tea, cocoa, chocolate, spices, herbs, rice, offal from livestock, yeast, whole grains, or cereals.

[0327]

[0357] Certain antioxidants belong to a class of phytonutrients called polyphenols (also known as "polyhydric phenols"), which are a group of chemicals found in plants characterized by the presence of more than one phenolic group per molecule. Suitable polyphenols for embodiments of this invention include catechins, proanthocyanidins, procyanidins, anthocyanins, quercetin, rutin, resveratrol, isoflavones, curcumin, punicalagins, ellagitannins, hesperidin, naringin, citrus flavonoids, chlorogenic acid, other similar materials, and combinations thereof.

[0328]

[0358] In certain embodiments, the antioxidant is a catechin, such as epigallocatechin gallate (EGCG). Suitable sources of catechins for embodiments of this invention include, but are not limited to, green tea, white tea, black tea, oolong tea, chocolate, cocoa, red wine, grapeseed, red grape skin, purple grape skin, red grape juice, purple grape juice, berries, Pycnogenol, and red apple skin.

[0329]

[0359] In some embodiments, the antioxidant is selected from proanthocyanidins, procyanidins, or combinations thereof. Suitable sources of proanthocyanidins and procyanidins for embodiments of this invention include, but are not limited to, red grapes, purple grapes, cocoa, chocolate, grape seeds, red wine, cocoa beans, cranberries, apple skin, plums, blueberries, black currants, chokeberries, green tea, sorghum, cinnamon bark, barley, red kidney beans, pinto beans, hops, almonds, hazelnuts, pecans, pistachios, pycnogenol, and colorful berries.

[0330]

[0360] In certain embodiments, the antioxidant is an anthocyanin. Suitable sources of anthocyanins for embodiments of this invention include, but are not limited to, red berries, blueberries, bilberries, cranberries, raspberries, cherries, pomegranates, strawberries, elderberries, chokeberries, red grape skin, purple grape skin, grape seeds, red wine, black currants, red currants, cocoa, plums, apple skin, peaches, red pears, red cabbage, red onions, red oranges, and blackberries.

[0331]

[0361] In some embodiments, the antioxidant is selected from quercetin, rutin, or a combination thereof. Suitable sources of quercetin and rutin for embodiments of this invention include, but are not limited to, red apples, onions, kale, black beans, bilberries, chokeberries, cranberries, blackberries, blueberries, strawberries, raspberries, black currants, green tea, black tea, plums, apricots, parsley, leeks, broccoli, chili peppers, berry wine, and ginkgo biloba.

[0332]

[0362] In some embodiments, the antioxidant is resveratrol. Suitable sources of resveratrol for embodiments of this invention include, but are not limited to, red grapes, peanuts, cranberries, blueberries, bilberries, mulberry, Japanese knotweed tea, and red wine.

[0333]

[0363] In certain embodiments, the antioxidant is an isoflavone. Suitable sources of isoflavones for embodiments of this invention include, but are not limited to, soybeans, soybean products, legumes, alfalfa sprouts, chickpeas, peanuts, and red clover.

[0334]

[0364] In some embodiments, the antioxidant is curcumin. Suitable sources of curcumin for embodiments of this invention include, but are not limited to, turmeric and mustard.

[0335]

[0365] In certain embodiments, the antioxidant is selected from punicalagins, ellagitannins, or combinations thereof. Suitable sources of punicalagins and ellagitannins for embodiments of this invention include, but are not limited to, pomegranates, raspberries, strawberries, walnuts, and red wine aged in oak barrels.

[0336]

[0366] 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 this invention include, but are not limited to, oranges, grapefruit, and citrus juices.

[0337]

[0367] In certain embodiments, the antioxidant is chlorogenic acid. Suitable sources of chlorogenic acid for embodiments of this invention include, but are not limited to, unroasted coffee, yerba mate, red wine, grape seeds, red grape skin, purple grape skin, red grape juice, purple grape juice, apple juice, cranberries, pomegranates, blueberries, strawberries, sunflowers, echinacea, pycnogenol, and apple peel.

[0338]

[0368] Suitable dietary fibers include, but are not limited to, non-starch polysaccharides, lignin, cellulose, methylcellulose, hemicellulose, β-glucans, pectins, gums, mucilages, waxes, inulin, oligosaccharides, fructooligosaccharides, cyclodextrins, chitin, and combinations thereof.

[0339]

[0369] Food sources of dietary fiber include, but are not limited to, grains, legumes, fruits, and vegetables. Grains 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 pulses such as soybeans. Fruits and vegetables that provide fiber sources 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 (e.g., flaxseed) are also sources of dietary fiber. Plant parts that provide dietary fiber include, but are not limited to, stems, roots, leaves, seeds, pith, and skin.

[0340]

[0370] Fatty acids include any straight-chain or branched monocarboxylic acid, examples of which include saturated fatty acids, unsaturated fatty acids, long-chain fatty acids, medium-chain fatty acids, short-chain fatty acids, fatty acid precursors (including omega-9 fatty acid precursors), and esterified fatty acids.In one embodiment, the fatty acid is a straight-chain monocarboxylic acid.As used herein, "long-chain polyunsaturated fatty acid" refers to any polyunsaturated carboxylic acid or organic acid with a long aliphatic tail.Suitable omega-3 fatty acids include, but are not limited to, linolenic acid, alpha-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, stearidonic acid, eicosatetraenoic acid, and combinations thereof.

[0341]

[0371] Suitable omega-6 fatty acids include, but are not limited to, linoleic acid, gamma-linolenic acid, dihomogamma-linolenic acid, arachidonic acid, eicosadienoic acid, docosadienoic acid, adrenic acid, docosapentaenoic acid, and combinations thereof.Suitable esterified fatty acids for embodiments of the present invention include, but are not limited to, monoacylglycerols containing omega-3 and / or omega-6 fatty acids, diacylglycerols containing omega-3 and / or omega-6 fatty acids, or triacylglycerols containing omega-3 and / or omega-6 fatty acids, and combinations thereof.

[0342]

[0372] 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 organizations. These compounds are sometimes referred to as pseudovitamins, and examples include, but are not limited to, compounds such as ubiquinone (coenzyme Q10), pangamic acid, dimethylglycine, taestrile, amygdalin, flavanoids, para-aminobenzoic acid, adenine, adenylic acid, and s-methylmethionine. The term vitamin as used herein includes pseudovitamins.

[0343]

[0373] The inorganic material is selected from bulk inorganic materials, trace inorganic materials, or a combination thereof. Non-limiting examples of bulk inorganic materials include calcium, chlorine, magnesium, phosphorus, potassium, sodium, and sulfur. Non-limiting examples of trace inorganic materials include chromium, cobalt, copper, fluorine, iron, manganese, molybdenum, selenium, zinc, and iodine. Although iodine is generally classified as a trace inorganic material, it is required in greater amounts than other trace inorganic materials and is often classified as a bulk inorganic material.

[0344]

[0374] In other specific embodiments of this invention, the inorganic substances are trace inorganic substances considered necessary for human nutrition, non-limiting examples of which include bismuth, boron, lithium, nickel, rubidium, silicon, strontium, tellurium, tin, titanium, tungsten, and vanadium.

[0345]

[0375] The preservative is selected from an antimicrobial agent, an antioxidant, an antienzyme agent, or a combination thereof. Non-limiting examples of antimicrobial agents 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 hydrogen sulfite. 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. Sorbates 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 yet another specific embodiment, at least one preservative is a bacteriocin, such as nisin. In other particular embodiments, the preservative is ethanol. In yet another particular embodiment, the preservative is ozone. In particular embodiments of the present invention, antienzyme agents suitable for use as preservatives include ascorbic acid, citric acid, and metal chelators, such as ethylenediaminetetraacetic acid (EDTA).

[0346]

[0376] The hydration product may be an electrolyte, non-limiting examples of which include sodium, potassium, calcium, magnesium, chloride, phosphate, bicarbonate, and combinations thereof. Suitable electrolytes for use in certain embodiments of this 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 use in certain embodiments include chloride, carbonate, sulfate, acetate, bicarbonate, citrate, phosphate, hydrogen phosphate, tartrate, sorbate, citrate, benzoate, or combinations thereof.

[0347]

[0377] In certain embodiments of this invention, the hydration product is a carbohydrate for supplementing the energy reserves burned by muscles. Suitable carbohydrates for use in certain embodiments of this invention are described in U.S. Patent Nos. 4,312,856, 4,853,237, 5,681,569, and 6,989,171, the disclosures of which are expressly incorporated herein by reference. Non-limiting examples of suitable carbohydrates include monosaccharides, disaccharides, oligosaccharides, complex polysaccharides, or combinations thereof. Non-limiting examples of suitable types of monosaccharides for use in certain embodiments include triose, tetrose, pentose, hexose, heptose, octose, and nonose. Non-limiting examples of specific types of suitable monosaccharides 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, sedoheptulose, octolose, and sialose. Non-limiting examples of suitable disaccharides include sucrose, lactose, and maltose. Non-limiting examples of suitable oligosaccharides include sucrose, maltotriose, and maltodextrin. In certain other embodiments, the carbohydrate is provided by corn syrup, sugar beet sugar, sucrose, juice, or tea. In certain other embodiments, the hydrating agent is a flavanol that causes cellular rehydration. Non-limiting examples of suitable flavanols for use in certain embodiments of this invention include catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, epigallocatechin 3-gallate, theaflavin, theaflavin 3-gallate, theaflavin 3'-gallate, theaflavin 3,3' gallate, thearubigin, or combinations thereof. In certain embodiments, the hydration product is a glycerol solution for enhancing athletic endurance.

[0348]

[0378] Probiotics include microorganisms that provide health benefits when consumed in effective amounts. Probiotics can include, but are not limited to, bacteria, yeast, and fungi. Examples of probiotics include, but are not limited to, bacteria from the genus Lactobacillus, Bifidobacterium, Streptococcus, or combinations thereof. In certain embodiments of the present invention, at least one probiotic is selected from the genus Lactobacillus. Lactobacillus (i.e., bacteria from the genus Lactobacillus, hereinafter referred to as "L.") Non-limiting examples of Lactobacillus species found in the human intestinal tract include L. acidophilus, L. casei, L. fermentum, L. saliva roes, L. brevis, L. leichmannii, L. plantarum, L. cellobiosus, L. reuteri, L. rhamnosus, L. GG, L. bulgaricus, and L. thermophilus. According to other specific embodiments of the invention, the probiotic is selected from the genus Bifidobacterium.Non-limiting species of the genus Bifidobacterium found in the human gastrointestinal tract include B. angulatum, B. animalis, B. asteroides, Bifidobacterium, B. boum, B. breve, B. catenulatum, B. choerinum, B. coryneforme, B. cuniculi, B. dentium, B. gallicum, B. gallinarum, B. indicum, B. longum, B. magnum, B. merycicum, and B. minisum. Examples of suitable probiotics include B. minimum, B. pseudocatenulatum, B. pseudolongum, B. psychraerophilum, B. pullorum, B. ruminantium, B. saeculare, B. scardovii, B. simiae, B. subtile, B. thermocidophilum, B. thermophilum, B. urinalis, and B. species. According to another specific embodiment of the invention, the probiotic is selected from the genus Streptococcus. Streptococcus thermophilus is a gram-positive facultative anaerobic bacterium. Other non-limiting probiotic species of this bacterium include Streptococcus salivarius and Streptococcus cremoris.

[0349]

[0379] Prebiotics are compositions that promote the growth of beneficial bacteria in the intestine. Prebiotics include, but are not limited to, mucopolysaccharides, oligosaccharides, polysaccharides, amino acids, vitamins, nutrient precursors, proteins, and combinations thereof. According to certain embodiments, the prebiotic is selected from dietary fibers, including, but not limited to, polysaccharides and oligosaccharides. Non-limiting examples of oligosaccharides that are classified as prebiotics according to certain embodiments of this invention include fructooligosaccharides, inulin, isomaltooligosaccharides, lactitol, lactulose, pyrodextrin, soybean oligosaccharides, transgalactooligosaccharides, and xylooligosaccharides. According to other specific embodiments, the prebiotic is an amino acid.

[0350]

[0380] "Weight management agents," as used herein, include appetite suppressants and / or thermogenic agents. The terms "appetite suppressant," "appetite-satiating composition," "satiety agent," and "satiety component" are synonymous as used herein. The term "appetite suppressant" describes macronutrients, herbal extracts, exogenous hormones, anorectic agents, appetite suppress...

Claims

1. A polynucleotide encoding a Myd variant polypeptide, (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 as described 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, wherein, in addition, the methionine at position 1 is absent; (b) a polypeptide having at least 80% sequence identity with 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 as described 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, wherein the methionine at position 1 is additionally absent; and (c) A polypeptide selected from the group consisting of polypeptides having the amino acid sequence of any variant listed in Table 8, Table 9, or Table 10 as compared to the polypeptide of SEQ ID NO: 3, or a polypeptide having an amino acid sequence modified by deletion, insertion, substitution, or addition of 24 or less amino acids from a polypeptide having the amino acid sequence of any variant listed in Table 8, Table 9, or Table 10, in which methionine at position 1 is additionally absent. is selected from the group consisting of 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. The above polynucleotide.

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

3. 3. The polynucleotide of claim 1 or 2, further encoding a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag.

4. 4. The polynucleotide of any one of claims 1 to 3, wherein the polynucleotide encodes 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, in addition, the methionine at position 1 is absent, by deletion, insertion, substitution, or addition of 12 or fewer amino acids.

5. 5. The polynucleotide of any one of claims 1 to 4, wherein the polynucleotide encodes 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, in addition, the methionine at position 1 is absent, by deletion, insertion, substitution, or addition of 6 or fewer amino acids.

6. 6. The polynucleotide of 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, additionally, the methionine at position 1 is absent.

7. 7. The polynucleotide of 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, wherein, in addition, the methionine at position 1 is absent.

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

141.

9. 9. The polynucleotide of any one of claims 1 to 8, wherein the variant is a variant listed in Table 8 or a variant listed in Table 8 in addition to which the methionine at position 1 is absent.

10. 9. The polynucleotide of any one of claims 1 to 8, wherein the variant is a variant listed in Table 9 or a variant listed in Table 8 in which the methionine at position 1 is additionally absent.

11. 9. The polynucleotide of any one of claims 1 to 8, wherein the variant is a variant listed in Table 10 or a variant listed in Table 8 in addition to which the methionine at position 1 is absent.

12. (a) a polypeptide selected from the group consisting of polypeptides having an amino acid sequence of a variant listed in Table 8, those polypeptides further comprising a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag, or those polypeptides that do 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 an amino acid sequence of a variant listed in Table 8, those polypeptides further comprising a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag; or those polypeptides that do not have a methionine at amino acid position 1; and (c) A polypeptide modified by deletion, insertion, substitution, or addition of 24 or fewer amino acids from a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of a variant listed in Table 8, those polypeptides further comprising a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag, or those polypeptides that do not have a methionine at amino acid position 1. and encoding a polypeptide selected from the group consisting of:

12. The polynucleotide of any one of claims 1 to 11, wherein the polypeptide sequence differs from that of SEQ ID NO: 3, SEQ ID NO: 3 containing a protein / peptide tag, affinity tag or histidine tag, and optionally the polypeptide sequence differs from the polypeptide of SEQ ID NO:

141.

13. (a) a polypeptide selected from the group consisting of polypeptides having an amino acid sequence of a variant listed in Table 9, those polypeptides further comprising a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag, or those polypeptides that do 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 an amino acid sequence of a variant listed in Table 9, those polypeptides further comprising a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag; or those polypeptides that do not have a methionine at amino acid position 1; and (c) A polypeptide modified by deletion, insertion, substitution, or addition of 24 or fewer amino acids from a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of a variant listed in Table 9, those polypeptides further comprising a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag, or those polypeptides that do not have a methionine at amino acid position 1. and encoding a polypeptide selected from the group consisting of:

12. The polynucleotide of any one of claims 1 to 11, wherein the polypeptide sequence differs from that of SEQ ID NO: 3, SEQ ID NO: 3 containing a protein / peptide tag, affinity tag or histidine tag, and optionally the polypeptide sequence differs from the polypeptide of SEQ ID NO:

141.

14. (a) a polypeptide selected from the group consisting of polypeptides having an amino acid sequence of a variant listed in Table 8, those polypeptides further comprising a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag, or those polypeptides that do 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 an amino acid sequence of a variant listed in Table 8, those polypeptides further comprising a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag; or those polypeptides that do not have a methionine at amino acid position 1; and (c) A polypeptide modified by deletion, insertion, substitution, or addition of 24 or fewer amino acids from a polypeptide selected from the group consisting of a polypeptide having the amino acid sequence of a variant listed in Table 8, those polypeptides further comprising a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag, or those polypeptides that do not have a methionine at amino acid position 1. and encoding a polypeptide selected from the group consisting of:

12. The polynucleotide of any one of claims 1 to 11, wherein the polypeptide sequence differs from that of SEQ ID NO: 3, SEQ ID NO: 3 containing a protein / peptide tag, affinity tag or histidine tag, and optionally the polypeptide sequence differs from the polypeptide of SEQ ID NO:

141.

15. 15. The polynucleotide of any one of claims 1 to 14, wherein the encoded polypeptide does not contain a mutation listed in Table 7.

16. 15. The polynucleotide of any one of claims 1 to 14, wherein the encoded polypeptide does not contain a mutation listed in Table 3.

17. 15. The polynucleotide of any one of claims 1 to 14, wherein the encoded polypeptide does not contain a mutation listed in Table 6.

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

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

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

21. A host cell transformed with the vector of claim 18.

22. A method for producing a protein having sweet taste modulating activity (especially sweetness), comprising culturing a host cell transformed with the vector described in claim 18 in a medium under conditions for protein expression.

23. 1. A method for producing a protein having sweet taste modulating activity and / or having a sweet taste, comprising culturing a host cell transformed with a vector in a medium under conditions for protein expression, the vector comprising: (a) a polypeptide selected from the group consisting of a polypeptide having an amino acid sequence of any variant listed in Table 8, Table 9 or Table 10, or a variant thereof in which methionine at position 1 is missing; (b) a polypeptide having at least 80% sequence identity with a polypeptide selected from the group consisting of a polypeptide having an amino acid sequence of a variant listed in Table 8, Table 9 or Table 10, or a variant thereof in which methionine at position 1 is missing; and (c) A polypeptide modified by deletion, insertion, substitution, or addition of 24 or fewer amino acids from 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 variant thereof in which methionine at position 1 is missing. an isolated polynucleotide encoding a polypeptide selected from the group consisting of: the encoded polypeptide has sweet taste modulating activity and optionally has a sweet taste; different from the polypeptide of SEQ ID NO: 3 and different from the polypeptide of SEQ ID NO: 141; The above method.

24. 24. The method of claim 23, wherein the variant is a variant of Table 8.

25. 24. The method of claim 23, wherein the variant is a variant of Table 9.

26. 24. The method of claim 23, wherein the variant is a variant of Table 10.

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

28. The host cell may be Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Agrobacterium tumefaciens, Rhizobium etori, or the like. etli), Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, acetobutylicum, Pseudomonas fluorescens, Pseudomonas putida, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger niger), Pichia pastoris, Rhizopus arrhizus, Rhizopus oryzaeoryzae), Yarrowia lipolytica, Candida albicans, Issatchenkia orientalis, Scheffersomyces stipitis, Yarrowia lipolytica, Ogataea polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans ... adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schwanniomyces occidentalis, Bacillus megaterium, Trichoderma reesei, Bifidobacterium adolescentis, Bifidobacterium animalis, animalis), Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Carnobacterium divergens, Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amylovorus amylovorus), Lactobacillus animalis, Lactobacillus alimentariusLactobacillus almentarius, Lactobacillus aviaries, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus collinoides, Lactobacillus coeliformis Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus farciminis, Lactobacillus fermentum fermentum, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus kefiri kefiri), Lactobacillus mucosae, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus parafaraginisparafarraginis), Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei sakei), Lactobacillus salivarius, Lactobacillus sanfranciscensis, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Microbacterium imperial, Oenococcus oeni, oeni), Pasteuria nishizawae, Pediococcus acidilactici, Pediococcus parvulus, Pediococcus pentosaceus, Propionibacterium acidipropionic, Propionibacterium freudenreichii ... freudenreichii), Streptococcus thermophilus, Bacillus amyloliquefaciensamyloliquefaciens, Bacillus atrophaeus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus flexus, Bacillus fusiformis, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium megaterium), Bacillus mojavensis, Bacillus paralicheniformis, Bacillus pumilus, Bacillus smithii, Bacillus subtilis, Bacillus vallismortis, Bacillus velezensis, Geobacillus stearothermophilus stearothermophilus, Paenibacillus illinoisensis, Parageobacillus thermoglucosidasius, Gluconobacter oxydans, Komagataebacter sucrose, Xanthomonas campestris, Candida cylindracea, Cyberlindracea, Cyberlindnera jadinii, Debaryomyces hansenii, Hanseniaspora uvarumuvarum), Kluyveromyces lactis, Kluyveromyces marxianus, Komagataella pastoris, Komagataella phaffi, Lindnera jadinii, Ogataea angusta, Saccharomyces bayanus, 28. A method for producing the protein of any one of claims 22 to 27, wherein the cell is selected from the group consisting of cells of Schizosaccharomyces pombe, Wickerhamomyces anomalus, Xanthophyllomyces dendrorhous, and Zygosaccharomyces rouxii.

29. 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 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. 28. The method for producing a protein according to any one of claims 22 to 27, wherein the host cell is a cell of a fungus other than Mattiromyces terfesioides.

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

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

34. 34. The method of claim 33, wherein the nucleic acid coding sequence further encodes a protein tag.

35. 34. The method of claim 33, wherein the nucleic acid coding sequence further encodes a His tag.

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

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

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

39. A polypeptide having sweetness-modulating activity (especially sweetness), (i) a polypeptide sequence selected from 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 wherein the methionine at position 1 is also missing; (ii) a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of any variant listed in Table 8, Table 9 or Table 10, or the amino acid sequence of a variant listed in Table 8, Table 9 or Table 10 in which the methionine at position 1 is also missing; or (iii) a polypeptide sequence selected from the group consisting of the amino acid sequences of a variant listed in Table 8, Table 9 or Table 10, or a polypeptide sequence containing at least one modification by deletion, insertion, substitution, or addition of 24 or fewer amino acids compared to a variant listed in Table 8, Table 9 or Table 10 in which the methionine at position 1 is also missing. Including, The polypeptide has sweetness-modulating activity (especially sweetness): (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 tagged with a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag, or optionally (His) ６ different from the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 141, which further comprises a tag; The above polypeptide.

40. Protein / peptide tag, optionally affinity tag, or optionally histidine tag, or optionally (His) ６ 40. The polypeptide of claim 39, further comprising a tag.

41. 41. The polypeptide of claim 39 or 40, comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one and up to 24 amino acid mutations as set forth in Table 8, Table 9 or Table 10.

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

43. 41. The polypeptide of any one of claims 39 to 40, comprising the amino acid sequence of SEQ ID NO: 3 modified by at least one and up to six amino acid mutations as shown in Table 8, Table 9 or Table 10.

44. 41. The polypeptide of any one of 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. 41. The polypeptide of claim 39 or 40, comprising the amino acid sequence of SEQ ID NO: 3 modified by two or more mutations selected from the mutations listed in Table 8.

46. 41. The polypeptide of claim 39 or 40, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 3 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. 41. The polypeptide of claim 39 or 40, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 3 modified by two or more mutations selected from the mutations listed in Table 9.

48. 41. The polypeptide of claim 39 or 40, comprising the amino acid sequence of SEQ ID NO: 3 modified by two or more mutations selected from the mutations listed in Table 10.

49. 41. The polypeptide of claim 39 or 40, comprising an amino acid sequence other than the amino acid sequence of a variant of Table 3 or Table 6.

50. 41. The polypeptide of claim 39 or 40, comprising an amino acid sequence other than the amino acid sequence of a variant of Table 7.

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

52. The polypeptide may be tagged with a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag, or optionally (His) ６ 52. The polypeptide of claim 51, further comprising a tag.

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

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

55. 55. The polypeptide of any one of claims 39 to 54, having 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 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, wherein the methionine at position 1 is additionally missing.

56. 55. The polypeptide of any one of claims 39 to 54, having 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, wherein the methionine at position 1 is additionally missing.

57. 55. The polypeptide of any one of claims 39 to 54, having 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 sequence of any variant listed in Table 9, or a variant listed in Table 8, Table 9, or Table 10, wherein the methionine at position 1 is additionally missing.

58. 55. The polypeptide of any one of claims 39-54, having 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 sequence of any variant listed in Table 10, or a variant listed in Table 10 wherein the methionine at position 1 is additionally missing.

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

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

61. (His) ６ The polypeptide of any one of claims 39 to 60, comprising a histidine tag represented by the formula:

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

63. 63. The polypeptide of claim 62, comprising two mutations that introduce two cysteine ​​amino acids such that the two cysteines can form -S-S- bonds in the polypeptide.

64. 64. The polypeptide of claim 63, wherein the two mutations to introduce cysteines are at amino acids 20 to 70 (inclusive) of SEQ ID NO:

3.

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

3.

66. 64. The polypeptide of claim 63, wherein the mutation to introduce cysteine ​​is at amino acids 33 and 49 of SEQ ID NO:

3.

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

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

69. 63. The polypeptide of claim 62, selected from polypeptides having the 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. 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. 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 that 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. 75. The sweet protein of 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 more than 60% by weight of the mixture.

76. 75. The sweet protein of 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 more than 60% by weight of the mixture.

77. 75. The sweet protein of 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) a product for oral administration that is different from the truffle Mattiromyces terfesioides, and (b) a flavor modulating composition comprising one or more isolated polypeptides, or one or more sweet proteins, or one or more isolated polypeptides, according to any one of claims 39 to 77, produced by expression of a polynucleotide according to any one of claims 1 to 18 in a heterologous host cell. A composition comprising a combination of The composition, wherein the combination exhibits flavor modulation compared to a product for oral administration.

79. 79. The composition of claim 78, wherein the flavor modifying composition is a sweetener composition and the combination has a sweet taste or enhanced sweetness compared to a product for oral administration.

80. 80. The composition of claim 78 or 79, comprising a plurality of isolated polypeptides.

81. 81. The composition of any one of claims 78 to 80, wherein the product for oral administration is a food product selected from the group consisting of baked goods; sweet bakery products, pre-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings, gelatins and puddings; frozen desserts; yogurt; snack bars; bread products; pre-made bread mixes for preparing bread products; sauces, syrups and dressings; sweet spreads; confectionery products; and sweetened breakfast cereals.

82. 82. The composition of any one of claims 78 to 81, wherein the product for oral administration is a food product that is warmed or heated before eating or that is presented warm or hot for consumption.

83. 83. The composition of claim 82, wherein at least one of the polypeptides exhibits enhanced thermal stability compared to native HTS.

84. 84. The composition of any one of claims 78 to 80, 82 or 83, wherein the product for oral administration 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. 1. A method for modifying the taste of a product for oral administration, comprising: A product for oral administration is provided comprising one or more isolated polypeptides having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of a 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 missing, or one or more isolated polypeptides having at least 80% sequence identity to said polypeptides, or a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag, or optionally a (His) ６ with an effective amount of one or more isolated polypeptides further comprising a tag; the product for oral administration differs from the truffle Mattiromyces terfesioides, and the one or more isolated polypeptides are 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 this combination has flavor modulation properties compared to the product for oral administration; The above method.

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

87. 87. The method of claim 85 or 86, wherein the product for oral administration is a food product selected from the group consisting of baked goods; sweet bakery products, pre-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings, gelatins and puddings; frozen desserts; yogurt; snack bars; bread products; pre-made bread mixes for preparing bread products; sauces, syrups and dressings; sweet spreads; confectionery products; and sweetened breakfast cereals.

88. 88. The method of any one of claims 85 to 87, wherein the product for oral administration is a food product that is warmed or heated before eating or that is presented warm or hot for consumption.

89. 89. The method of any one of claims 85, 86, or 88, wherein the product for oral administration is a beverage product selected from the group consisting of carbonated beverages; non-carbonated beverages; and beverage concentrates.

90. 90. The method of claim 89, wherein the product for oral administration is a heated or warmed beverage product, optionally consumed warm or hot, or optionally a coffee or tea based product.

91. 1. A method for purifying a polypeptide having sweet taste modulating activity, comprising: (a) subjecting the polypeptide to hydrophobic interaction chromatography (HIC); and (b) then subjecting the polypeptide to size exclusion chromatography (SEC); Including, The polypeptide comprises an amino acid sequence having at least 80% sequence identity 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 an amino acid sequence having at least 80% sequence identity thereto, or the polypeptide comprises a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag, or optionally a (His) ６ further including tags, The above method.

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

93. 93. The polypeptide of any one of claims 39 to 69 and 92, 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 one of claims 39 to 69 and 92 to 93, having enhanced thermostability compared to a sweet protein having the amino acid sequence of SEQ ID NO:

3.

95. A polypeptide described in any one of claims 39 to 69 and 92 to 94, which has enhanced sweet taste regulating activity and enhanced thermostability compared to a sweet protein having the amino acid sequence of SEQ ID NO:

3.

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

97. 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. 1. A method for producing a protein having sweetness modulating activity, comprising:

98. The method comprising culturing a host cell transformed with the expression cassette or vector of claim 97 in a medium under conditions for protein expression.

100. A host cell expressing a polypeptide according to any one of claims 39 to 69 and 92 to 95.

101. 96. The polypeptide of any one of claims 39 to 69 and 92 to 95, which is isolated.

102. 97. The polynucleotide of any one of claims 1 to 18 and 96, which is isolated.

103. The polynucleotide of any one of claims 1 to 18, 96 and 102 operably linked to a heterologous regulatory element.

104. 87. The composition of any one of claims 78-84 and 86, 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. 105. The composition of any one of claims 78-84, 86 and 104, wherein at least one of the one or more isolated polypeptides exhibits enhanced thermostability compared to the polypeptide of SEQ ID NO:

3.

106. 102. The polypeptide of any one of claims 39-69, 92-95 and 101, further comprising one or more derivatizations of the N-terminal amino acid, the C-terminal amino acid or an amino acid side chain of the polypeptide.

107. Derivatization can be with the following chemical moieties: acyl, acetyl (CH ３ CO), formyl (HCO), glycosyl (C ６ H １１ O ６ ), hydroxyl (HO), methyl (CH ３ ), phosphatidylcholine (PO ４ ), phosphonyl (PO ２ ), sulfhydryl (SH), or sulfonyl (HSO ２ 107. The polypeptide of claim 106, wherein the amino acid is selected from the group consisting of an addition to, an exclusion from, or a substitution of an amine nitrogen of any of the amino acids.

108. Derivatization can be with the following chemical moieties: acyl, acetyl (CH ３ CO), formyl (HCO), glycosyl (C ６ H １１ O ６ ), hydroxyl (HO), methyl (CH ３ ), phosphatidylcholine (PO ４ ), phosphonyl (PO ２ ), sulfhydryl (SH), or sulfonyl (HSO ２ 107. The polypeptide of claim 106, wherein the amino acid is selected from the group consisting of an addition to, deletion from, or substitution of a sulfur at the delta position of the methionine of either of the amino acid sequences.

109. 107. The polypeptide of claim 106, wherein the derivatization is selected from acylation, acetylation, esterification, glycosylation, oxidation, methylation, reductive alkylation, phosphorylation, sulfurylation, or sulfonylation.

110. 107. The polypeptide of claim 106, wherein the derivatization is acylation of one or more different amino groups.

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

112. 107. The polypeptide of claim 106, wherein the derivatization is oxidation of the sulfur at the delta position of methionine.

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

114. 114. The polypeptide of any one of claims 106 to 113, wherein derivatization is achieved by chemical or in vitro enzymatic methods.

115. A polypeptide according to any one of claims 106 to 113, wherein the derivatisation is achieved by post-translational processing.

116. (a) one or more polypeptides, (i) a polypeptide sequence selected from the amino acid sequences 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 that contains at least one alteration by deletion, insertion, substitution, or addition of 24 or fewer amino acids compared 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 methionine at position 1 is absent; and / or the polypeptide is tagged with a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag, or optionally (His) ６ Any of the polypeptides (i) to (iii) further comprising a tag. a polypeptide comprising: and (b) at least one additional sweetener other than Myd sweet protein; A sweetener composition comprising:

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

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

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

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

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

122. 122. The sweetener composition of any one of claims 116 to 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. 123. A consumable or consumable product, optionally a beverage or beverage product, comprising the sweetener composition of any one of claims 116 to 122.

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

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

126. 124. The consumable or consumable product, optionally a beverage or beverage product, of claim 123, wherein the polypeptide is present in an amount selected from an amount 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. 127. The consumable or consumable product, optionally a beverage or beverage product, of any one of claims 123 to 126, wherein the consumable or consumable product, optionally a beverage or beverage product, has at least one improved sensory characteristic compared to a consumable or consumable product or beverage or beverage product that does not contain the sweetener composition, the sensory characteristic being selected from the group consisting of aroma, flavor, base taste (sweet, sour, salty, bitter or umami), aftertaste or lingering taste, temporal profile, texture or a combination thereof.

128. 128. A consumable or consumable product, optionally a beverage or beverage product, according to claim 127, wherein the at least one improved organoleptic property is reduced bitterness, aftertaste or improved texture.

129. 128. A consumable or consumable product, optionally a beverage or beverage product, according to claim 127, wherein the at least one improved organoleptic property is improved mouthfeel and the amount of polypeptide is from about 1 ppm to about 40 ppm.

130. 130. A consumable or consumable 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. 130. A consumable or consumable product according to any one of claims 123 to 129, optionally a beverage or beverage product, which is a beverage or beverage product selected from the group consisting of cola, ginger ale, soft drinks, root beer, fruit juice, fruit flavoured juice, vegetable juice, vegetable flavoured juice, sports drinks, energy drinks, plant protein drinks, water-like drinks (e.g. water with natural or synthetic flavourings), certain teas (e.g. black tea, green tea, red tea, oolong tea), coffee, cocoa drinks, dairy drinks (e.g. dairy drinks, dairy coffee, cafe au lait, milk tea, fruit dairy drinks).

132. 132. A consumable or consumable product, optionally a beverage or beverage product, according to any one of claims 123 to 131, further comprising at least one organic acid addition salt which is a sodium, calcium, potassium, or magnesium salt of an organic acid.

133. 133. A consumable or consumable 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. 134. A consumable or consumable product, optionally a beverage or beverage product, according to any one of claims 123 to 133, having at least one change in sensory characteristic compared to a beverage or beverage product not containing the sweetener composition, wherein the sensory characteristic is selected from the group consisting of aroma, flavor, base taste (sweet, sour, salty, bitter or umami), aftertaste or lingering taste, temporal profile, texture or a combination thereof.

135. 123. A method for improving at least one sensory property of a consumable or consumable product, optionally a beverage or beverage product, comprising the step of adding to the consumable or consumable product, optionally a beverage or beverage product, a sweetener composition according to any one of claims 116 to 122, thereby improving at least one sensory property thereof.

136. 136. The method of claim 135, wherein the consumable or consumable product is a beverage or beverage product and the sweetener composition is added to a liquid matrix, thereby providing the beverage or beverage product with at least one improved sensory stimulus characteristic.

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

138. 138. The method of any one of claims 135 to 137, wherein the improved organoleptic property is selected from the group consisting of aroma, flavor, base taste, aftertaste or lingering taste, temporal profile, texture, or a combination thereof.

139. 139. The method of any one of claims 135 to 138, wherein the improved organoleptic property is reduced bitterness, aftertaste, or improved mouthfeel.

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

141. 141. The method of any one of claims 135 to 140, wherein the polypeptide is present in the consumable or consumable product, optionally the beverage or beverage product, in an amount of from about 1 ppm to about 50 ppm.

142. 141. The method of any one of claims 135-140, wherein the polypeptide is present in the consumable or consumable product, optionally the beverage or beverage product, in an amount selected from an amount of 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. 143. The method of any one of claims 135 to 142, wherein the added sweetener is a mogroside sweetener selected from siamenoside I and mogroside V.

144. 123. A method for modifying / altering at least one sensory characteristic of a beverage or beverage product, comprising the step of adding a sweetener composition according to any one of claims 116 to 122 to a liquid matrix, thereby providing a beverage or beverage product having at least one modified / altered sensory characteristic.

145. 145. The method of claim 144, wherein the sensory stimulus characteristic is selected from the group consisting of aroma, flavor, base taste (sweet, sour, salty, bitter or umami), aftertaste or lingering taste, temporal profile, texture or a combination thereof.

146. (a) one or more polypeptides, (i) a polypeptide sequence selected from the amino acid sequences 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 that contains at least one alteration by deletion, insertion, substitution, or addition of 24 or fewer amino acids compared 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 methionine at position 1 is absent; and / or the polypeptide is tagged with a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag, or optionally (His) ６ Any of the polypeptides (i) to (iii) further comprising a tag. a polypeptide comprising: (b) at least one additional component selected from sucrose, mannitol, citric acid, hypoxanthine, theophylline, leucine, and combinations thereof; A sweetener composition comprising:

147. 147. The composition of claim 146, wherein the product for oral administration is a food product selected from the group consisting of pie fillings and other sweet fillings, gelatins and puddings; yogurts; sauces, syrups and dressings; and sweet spreads.

148. 148. The composition of claim 147, wherein the product for oral administration further comprises low methoxyl pectin.

Images