Honey truffle sweetener (HTS) variants

Abstract

Fungal sweet-taste modifying protein variants (designated Honey Truffle Sweetener (HTS) protein variants), and polynucleotides encoding the protein variants are described. Specifically, Myd (HTS) protein variants with sweet taste modulation activity, and the cDNA encoding the same, are described, along with methods for isolating such cDNA and for isolating and expressing such proteins. Also disclosed are sweetening compositions which include the proteins of the invention, and methods to provide improved flavor to a product for oral administration.

Description

Atty Docket: 340391: 0640.49WO HONEY TRUFFLE SWEETENER (HTS) VARIANTS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional application 63 / 680,777 filed August 8, 2024, which is incorporated by reference in its entirety herein. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing identified as follows: One 288,327-byte XML file named “0640-49_WO_seq.xml” created on August 7, 2025. FIELD OF THE INVENTION

[0003] This invention includes embodiments of sweet-tasting (Honey Truffle Sweetener (HTS)) proteins, genes, and cDNA encoding said proteins, and to methods of using such proteins, genes, and cDNA in the modulation of the taste of foods. More particularly, the invention includes embodiments of HTS variant sweet proteins, and to the genes and cDNA encoding such proteins, as well as consumable formulations and non-consumable formulations for oral hygiene or therapeutics comprising same. BACKGROUND OF THE INVENTION333

[0004] Excess intake of nutritive sweeteners has long been associated with diet-related health issues, such as obesity, heart disease, metabolic disorders and dental problems. Consequently, consumers are increasingly looking for ways to decrease the amount of nutritive sweeteners in their diets; and manufacturers are trying to respond to this demand by attempting to replace nutritive sweeteners with substitutes to mimic the desirable taste and functional properties of the nutritive sweeteners.

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

[0006] Zero or low-calorie sweeteners derived from natural sources may be preferred to limit the negative effects of high sugar consumption. So far there are only seven known sweet and taste- modifying proteins, namely monellin, thaumatin, brazzein, curculin, mabinlin, miraculin and pentadin. The key residues on the protein surface responsible for biological activity have not yet been identified with certainty for any of these proteins. Monellin was found to be 100,000 times 1Atty Docket: 340391: 0640.49WO sweeter than sucrose on a molar basis, followed by thaumatin and brazzein which are 3000 times and 500 times sweeter than sucrose, respectively, on a gram basis. Most of them share no sequence homology or structural similarity; and thaumatin shares extensive similarity at the protein sequence level with certain non-sweet proteins found in other plants.

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

[0008] International patent application PCT / US2021 / 039176, filed June 25, 2021, published as WO2021 / 263158 on December 30, 2021, relates to newly identified fungal sweet-taste modifying proteins and the cDNA encoding the proteins. Sweet-taste modifying proteins were identified in the truffle, Mattirolomyces terfezioides. The application further relates to Myd proteins as sweeteners and in sweet taste activation / modulation and the cDNA encoding the same and methods for isolating such cDNA and for isolating and expressing such proteins. The application also relates to a sweetening composition which includes the proteins and methods to provide improved flavor to a product for oral administration.

[0009] International patent application PCT / US2022 / 82443, filed December 27, 2022, and published as WO2023 / 129938, on July 6, 2023, relates to sweet protein variants from truffle, e.g., Mattirolomyces terfezioides, including proteins, genes, cDNA encoding said proteins, and compositions thereof, and are each hereby incorporated by reference in its entirety.

[0010] International patent application PCT / US24 / 15023, filed February 8, 2024, and published as WO2024 / 168164, on August 15, 2024, relates to additional sweet protein variants from truffle, e.g., Mattirolomyces terfezioides, including proteins, genes, cDNA encoding said proteins, and compositions thereof, and are each hereby incorporated by reference in its entirety.

[0011] A need still remains for new low or zero calorie sweeteners with improved taste from natural sources, particularly from ascomycetes fungal species. A need also remains for producing fungally- derived low or zero calorie sweeteners with improved taste and methods of using such ingredients in final food products without compromising on taste and with superior taste profiles. A further need remains for consumables and consumable products with improved taste from natural resources, particularly for such products that are lower calorie products. A particular need remains for consumables and consumable products containing one or more sweet protein of the mycoduclein (MYD) family of proteins and one or more additional sweetener other than a sweet 2Atty Docket: 340391: 0640.49WO protein of the MYD family of proteins wherein the combination provides improved taste and in particular provides improved sweet taste. SUMMARY OF THE DISCLOSURE

[0012] The invention meets these and other needs by providing fungally-derived sweet-tasting proteins identified herein as the MYD family of proteins, as well as the genes and cDNA encoding such proteins, and methods of using such proteins, genes, and cDNA in the modulation of the taste of foods. In embodiments, the invention provides consumables and consumable products comprising one or more sweet protein of the mycoduclein (MYD) family of proteins in combination with another sweetener or sweetening agent other than a MYD family protein.

[0013] The invention provides naturally-occurring MYD1 (also referred to as mycodulcein, and Honey Truffle Sweetener protein (HTS protein)) and variants thereof. Polypeptides of the invention exhibit flavor-modifying properties and, in particular, modulate sweet taste and perception, either alone, or in combination with food, beverages, dietary supplements, or pharmaceuticals. In embodiments, Myd1 variants of the invention can provide sweet-taste to materials, such as food, beverages, dietary supplements, or pharmaceuticals that are ingested or can provide improved sweet taste. In embodiments, Myd1 variants of the invention can reduce the sourness, bitterness or astringency of foods and drinks. In embodiments, Myd1 variants of the invention can exhibit enhancement of the taste of foods and drinks, namely a taste-modifying activity. In embodiments, Myd1 variants, can exhibit in addition to sweet-taste modulation activity, enhanced thermal stability compared to native forms of HTS protein. In embodiments, Myd1 variants can exhibit flavor-modifying properties. In embodiments, Myd1 variants, can exhibit in addition to flavor modifying properties, enhanced thermal stability compared to native forms of HTS protein. In an embodiment, the invention provides naturally-occurring Myd proteins. In an embodiment, the invention provides variant Myd proteins other than naturally-occurring proteins.

[0014] Naturally-occurring Myd protein exists as two isoforms; HTS-1 (SEQ ID NO:3) and HTS-2 (SEQ ID NO:141), as isolated from Mattirolomyces terfezioides. The relative amounts of HTS-1 and HTS-2 isolated from Mattirolomyces terfezioides range from about 40% to 60% by weight HTS-2 to 60% to 40% by weight HTS-1. In embodiments, the invention provides non-naturally occurring sweet proteins which include non-naturally occurring mixtures of the two isoforms HTS- 1 and HTS-2. In embodiments, the invention provides sweet protein generated 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 Mattirolomyces terfezioides truffle), as well as methods of generating sweet protein by recombinant expression. 3Atty Docket: 340391: 0640.49WO

[0015] In an embodiment the encoded polypeptide is the polypeptide having the amino acid sequence of SEQ ID NO:3 (also designated as the HTS-1 isoform). In an embodiment the encoded polypeptide is the polypeptide having the amino acid sequence of SEQ ID NO:3, except that the methionine at position 1 in SEQ ID NO:3 is absent, designated hereinafter as the polypedptide having the amino acid sequence of SEQ ID NO:141(also designated as the HTS-2 isoform). In embodiments, the encoded polypeptide is the polypeptide having amino acid sequence SEQ ID NO:5 which is the HTS-1 isoform with a (His-tag)6.In embodiments, the encoded polypeptide is the polypeptide having amino acid sequence SEQ ID NO:142 which is the HTS-2 isoform with a (His-tag)6.

[0016] Accordingly, one aspect of the invention provides a polynucleotide (e.g., isolated polynucleotide) encoding a polypeptide, wherein the encoded polypeptide has a sweet-taste modulation activity and which is different in amino acid sequence from the polypeptide of SEQ ID NO:3 or optionally different from the polypeptide of SEQ ID NO:141. In embodiments, the encoded polypeptide is selected from the group consisting of: (a) a polypeptide sequence selected from the group consisting of the amino acid sequences of a variant 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 a variant listed in Table 8, Table 9 or Table 10; and (c) a polypeptide sequence modified from the 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 no more than 24 amino acids, and wherein the polypeptide sequence is different from that of SEQ ID NO:3 and optionally different from that of SEQ ID NO:141. In an embodiment, the polynucleotide encoding the polypeptide is other than the polynucleotide of SEQ ID NO:2. In an embodiment, the polynucleotide encoding the polypeptide is the polynucleotide of SEQ ID NO:2 which is codon optimized for expression in a bacterium, yeast or fungus. In an embodiment, the polynucleotide encoding the polypeptide is the polynucleotide of SEQ ID NO:6 with optional His-tag which is codon optimized for expression in Escherichia coli. In an embodiment, the polynucleotide encoding the polypeptide is the polynucleotide of SEQ ID NO:7 with optional His-tag which is codon optimized for expression in Saccharomyces cerevisiae.

[0017] In an embodiment, the encoded polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO:3, except that the methionine at position 1 in SEQ ID NO:3 is absent, presented herein as the polypeptide having the amino acid sequence of SEQ ID NO:141. In an embodiment, the encoded polypeptide is a polypeptide other than the polypeptide having the amino acid sequence of SEQ ID NO:141. In embodiments, the encoded polypeptide exhibits sweet taste. In embodiments, the encoded polypeptide exhibits sweet taste, when present in a composition at a 4Atty Docket: 340391: 0640.49WO concentration of 30 ppm or higher. In embodiments, the encoded polypeptide exhibits sweet taste, when present in a composition at a concentration of 40 ppm or higher.

[0018] In a related aspect, the invention provides a polynucleotide (e.g., isolated polynucleotide) encoding a polypeptide, wherein the encoded polypeptide has flavor modifying properties, and which is different in amino acid sequence from polypeptide SEQ ID NO:3 and optionally 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 a variant 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 a variant listed in Table 8; and (c) a polypeptide sequence modified from the polypeptide sequence selected from the group consisting of the amino acid sequences of a variant listed in Table 8, by deletion, insertion, substitution, or addition of no more than 24 amino acids, and wherein the polypeptide sequence is different from that of SEQ ID NO:3 and optionally from SEQ ID NO:141. In embodiments, the encoded polypeptide is a polypeptide selected from the group consisting of the amino acid sequences of a variant listed in Table 8, that is other than a variant listed in Table 9 or Table 10. In an embodiment, the polynucleotide encoding the polypeptide is a polynucleotide other than the polynucleotide of SEQ ID NO:2. In an embodiment, the encoded polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO:141. In an embodiment, the encoded polypeptide is a polypeptide other than the polypeptide having the amino acid sequence of SEQ ID NO:141. In embodiments, the encoded polypeptide does not exhibit sweet taste. In embodiments, the encoded polypeptide does not exhibit sweet taste when present in a composition at a concentration of 30 ppm or higher. In embodiments, the encoded polypeptide does not exhibit sweet taste when present in a composition at a concentration of 40 ppm or higher. In embodiments, the encoded polypeptide only exhibits sweet taste when present in a composition at a concentration of 30 ppm or higher.

[0019] In another aspect of the invention, amino acid mutations of HTS polypeptide and protein variants are summarized in Table 8. The mutations of Table 8 are identified relative to the amino acid sequence of SEQ ID NO:3. Thus, a polypeptide having an amino acid sequence of a variant listed in Table 8 will have the amino acid sequence of SEQ ID NO:3, but with one or more site mutations identified in Table 8. Mutants of Table 8 also include those in which the methionine at position 1 of the protein is absent. In a related aspect, the invention provides HTS polypeptide and protein variants, wherein the variants have the single site mutations of Table 8 relative to the amino acid sequence of SEQ ID NO:3 and in addition wherein the methionine at position 1 in SEQ ID NO:3 is absent. 5Atty Docket: 340391: 0640.49WO

[0020] In another aspect of the invention, amino acid mutations of HTS polypeptide and protein variants are summarized in Table 9. The mutations of Table 9 are identified relative to the amino acid sequence of SEQ ID NO:3. Thus, a polypeptide having an amino acid sequence of a variant listed in Table 9 will have the amino acid sequence of SEQ ID NO:3, but with one or more site mutations identified in Table 9. Mutants of Table 9 also include those in which the methionine at position 1 of the protein is absent. In a related aspect, the invention provides HTS polypeptide and protein variants, wherein the variants have the single site mutations of Table 9 relative to the amino acid sequence of SEQ ID NO:3 and in addition wherein the methionine at position 1 in SEQ ID NO:3 is absent.

[0021] In another aspect of the invention, amino acid mutations of HTS polypeptide and protein variants are summarized in Table 10. The mutations of Table 10 are identified relative to the amino acid sequence of SEQ ID NO:3. Thus, a polypeptide having an amino acid sequence of a variant listed in Table 10 will have the amino acid sequence of SEQ ID NO:3, but with one or more site mutations identified in Table 10. Mutants of Table 10 also include those in which the methionine at position 1 of the protein is absent. In a related aspect, the invention provides HTS polypeptide and protein variants, wherein the variants have the single site mutations of Table 8 relative to the amino acid sequence of SEQ ID NO:3 and in addition wherein the methionine at position 1 in SEQ ID NO:3 is absent.

[0022] In another aspect of the invention, the HTS polypeptide and protein variants are those having the single-site mutations listed in Table 7, which are identified relative to the amino acid sequence of SEQ ID NO:3. In a related aspect of the invention, the HTS polypeptide and protein variants are those having the single-site mutations listed in Table 7 relative to the amino acid sequence of SEQ ID NO:3, and in addition wherein the methionine at position 1 in SEQ ID NO:3 is absent.

[0023] In another aspect of the invention, the HTS polypeptide and protein variants are other than those having the single-site mutations listed in Table 7 which are identified relative to the amino acid sequence of SEQ ID NO:3. In a related aspect, the present invention excludes polynucleotides encoding the HTS polypeptide and protein variants having the single-site mutations listed in Table 7. In a related aspect of the invention, the HTS polypeptide and protein variants are other than those having the single-site mutations listed in Table 7 and wherein the methionine at position 1 in SEQ ID NO:3 is absent. In a related aspect, the present invention excludes polynucleotides encoding the HTS polypeptide and protein variants having the single-site mutations listed in Table 7 and wherein the methionine of SEQ ID NO:3 is absent.

[0024] In another aspect of the invention, the HTS polypeptide or protein variants do not include a mutation of Table 7. In a related aspect, the present invention excludes HTS polypeptide or protein 6Atty Docket: 340391: 0640.49WO variants comprising a mutation listed in Table 7. In a related aspect, the present invention excludes polynucleotides encoding a HTS polypeptide or protein variant comprising a mutation listed in Table 7.

[0025] In another aspect of the invention, the HTS polypeptide or protein variants do not include a mutation of Table 3 or Table 6. In a related aspect, the present invention excludes HTS polypeptide or protein variants comprising a mutation listed in Table 3 or Table 6. In a related aspect, the present invention excludes polynucleotides encoding a HTS polypeptide or protein variant comprising a mutation listed in Table 3 or Table 6.

[0026] In yet another aspect of the invention, the HTS polypeptide or protein variant does not include a mutation of Table 8, Table 9 or Table 10, but does include 1 or more conservative mutations of the amino acid sequence of SEQ ID NO:3 or optionally of SEQ ID NO:141, wherein, in the variant, the methionine at position 1 is present or absent. In a related aspect, the present invention includes polynucleotides encoding a HTS polypeptide or protein variant that does not include a mutation of Table 8, Table 9 or Table 10, but does include 1 or more conservative mutations of the amino acid sequence of SEQ ID NO:3, wherein, in the variant, the methionine at position 1 is present or absent.

[0027] In other aspects, the polynucleotide encoding the polypeptide having flavor-modulation or sweet-taste modulation activity is optionally operably linked to a heterologous regulatory element. Additionally or alternatively, the polynucleotide sequence 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 an embodiment, the polynucleotide encoding the polypeptide having sweet-taste modulation activity is other than the nucleotide of SEQ ID NO:2 further encoding a protein / peptide tag or label.

[0028] An aspect of the invention provides a polynucleotide (e.g., isolated polynucleotide) encoding a polypeptide, where 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 a variant listed in Table 8, Table 9 or Table 10, where the polypeptide further consists of a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag; (b) a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of the amino acid sequences of a variant listed in Table 8, Table 9 or Table 10, where the polypeptide further consists of a protein / peptide tag, optionally an affinity tag, or optionally a histidine tag; and (c) a polypeptide sequence modified from the 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 no more than 24 amino acids and wherein the polypeptide 7Atty Docket: 340391: 0640.49WO further consists of a protein / peptide tag, optionally an affinity tag or optionally a histidine tag and where the polypeptide sequence is different from that of SEQ ID NO:3 containing the histidine tag. In a related embodiment, the encoded polypeptide is different from that of SEQ ID NO:142 containing a histidine tag.

[0029] In specific aspects, the polynucleotide encoding the polypeptide having sweet-taste modulation activity is other than SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:139 or SEQ ID NO:140 as identified in international patent application PCT / US2022 / 82443 and U.S. patent application 18 / 146,958.

[0030] Another aspect of the invention provides a polynucleotide other than the polynucleotide of SEQ ID NO:4, which corresponds to the coding sequence optimized for His tagged HTS in E. coli (wherein residues 364-381 correspond to an optional His tag sequence). Another aspect of the invention provides a polynucleotide other than the polynucleotide of SEQ ID NO:6, which corresponds to the coding sequence of HTS optimized for expression in S. cerevisiae (to which a coding sequence for a His-tag, e.g., (His)6is added).

[0031] In a specific embodiment, the invention provides Myd variants which exhibit enhanced thermostabilty with respect to native isofomrs of Myd proteins (HTS-1 and HTS-2). In specific embodiment, the invention provides a

[0032] In additional aspects, the invention provides an expression cassette comprising a polynucleotide herein and a vector comprising the polynucleotide, as well as a host cell transformed with the vector. Additionally, a method of producing a protein having sweet-taste modulation activity, comprising culturing the transformed host cell in a medium under conditions that result in producing the protein having sweet-taste modulation activity also is provided.

[0033] In additional aspects, the invention provides a host cell which expresses a polypeptide as described herein. In embodiments, the host cell is E. coli. In embodiments, the host cell is a plant cell. 8Atty Docket: 340391: 0640.49WO

[0034] Another aspect of the invention provides a polypeptide (e.g., isolated polypeptide) having sweet-taste modulation activity and comprising (i) a polypeptide sequence selected from those 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 those 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 aspects, the polypeptide (a) contains at least one modification relative to a polypeptide sequence selected from the group consisting of an amino acid sequence of a variant listed in Table 8, Table 9 or Table 10 by deletion, insertion, substitution, or addition of no more than 24 amino acids, wherein the polypeptide is different from polypeptide SEQ ID NO:3 and optionally different from SEQ ID NO:141, and / or (b) further comprises a protein / peptide tag, optionally an affinity tag, and particularly a histidine tag, and wherein the polypeptide has sweet- taste modulation activity.

[0035] In another aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by at least one mutation selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by two mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by three mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by four mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by five mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by six mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In further embodiments of the preceding embodiments, the 1-6 modifications are other than removal of the methionine at position 1. In embodiments, in the polypeptides having 1-6 amino acid modifications, the methionine at position 1 is present or absent. The invention also provides 9Atty Docket: 340391: 0640.49WO polynucleotides encoding the foregoing mutant (variant) polypeptides of SEQ ID NO:3. The invention further provides the foregoing mutant polypeptides which further comprise a protein / peptide tag, optionally an affinity tag and more specifically a histidine tag. The invention also provides polynucleotides encoding the foregoing mutant polypeptides of SEQ ID NO:3 which mutants further comprise a protein / peptide tag, optionally an affinity tag and more specifically a histidine tag. In an embodiment, the polypeptide excludes polypeptides comprising a mutation listed in Table 7.

[0036] In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by seven mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by eight mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by nine mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by ten mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by eleven mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by twelve mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In further embodiments of the preceding embodiments, the 1-12 modifications are other than removal of the methionine at position 1. In embodiments, in the polypeptides having 1-12 amino acid modification, the methionine at position 1 is present or absent. The invention also provides polynucleotides encoding the foregoing mutant polypeptides of SEQ ID NO:3. The invention further provides the foregoing mutant polypeptides which further comprise a protein / peptide tag, optionally an affinity tag and more specifically a histidine tag. The invention also provides polynucleotides encoding the foregoing mutant polypeptides of SEQ ID NO:3 which further comprise a protein / peptide tag, optionally an affinity tag and more specifically a histidine tag. In an aspect, the polypeptide excludes polypeptides comprising a mutation listed in Table 7. 10Atty Docket: 340391: 0640.49WO

[0037] In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by thirteen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by fourteen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by fifteen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by sixteen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by seventeen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by eighteen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In further embodiments of the preceding 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 modification, the methionine at position 1 is present or absent. The invention also provides polynucleotides encoding the foregoing mutant polypeptides of SEQ ID NO:3. The invention further provides the foregoing mutant polypeptides which further comprise a protein / peptide tag, optionally an affinity tag and more specifically a histidine tag. The invention also provides polynucleotides encoding the foregoing mutant polypeptides of SEQ ID NO:3 which further comprise a protein / peptide tag, optionally an affinity tag and more specifically a histidine tag. In an aspect, the polypeptide excludes polypeptides comprising a mutation listed in Table 7.

[0038] In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by nineteen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by twenty mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is 11Atty Docket: 340391: 0640.49WO modified by twenty-one mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by twenty-two mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by twenty-three mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by twenty-four mutations at different positions selected from those listed in Table 8, Table 9 or Table 10. In further embodiments of the preceding 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 modification, the methionine at position 1 is present or absent. The invention also provides polynucleotides encoding the foregoing mutant polypeptides of SEQ ID NO:3. The invention further provides the foregoing mutant polypeptides which further comprise a protein / peptide tag, optionally an affinity tag, and more specifically a histidine tag. The invention also provides polynucleotides encoding the foregoing mutant polypeptides of SEQ ID NO:3 which further comprise a protein / peptide tag, optionally an affinity tag and more specifically a histidine tag. In an aspect, the polypeptide excludes polypeptides comprising a mutation listed in Table 7.

[0039] Another aspect of the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by at least one mutation, where the at least one mutation is selected from the mutations listed in Table 8, Table 9 or Table 10 and is not a mutation listed in Table 7, and where the polypeptide is also modified by one or more additional mutations, where 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 an aspect, the polypeptide is also modified by two or more additional mutations, where the two or more additional mutations are selected from the mutations listed in Table 7. In an aspect, the polypeptide has at least 80% sequence identity to a polypeptide sequence selected from the group consisting of those amino acid sequences of variants listed in Table 8, Table 9 or Table 10. The forgoing polypeptides optionally further comprise a protein / peptide tag, optionally an affinity tag or optionally a histidine tag. 12Atty Docket: 340391: 0640.49WO

[0040] In a related aspect, the invention provides a protein which is expressed from a polynucleotide encoding a MYD family protein in a host cell. In embodiments, the protein is expressed in a host cell from the polynucleotide of SEQ ID NO:2, a polynucleotide having 90% sequence identity with the polynucleotide of SEQ ID NO:2, or any variant thereof that is codon-optimized for expression in the host cell. In embodiments, the host cell is a non-native host cell wherein the host cell is a cell other than a cell of the truffle Mattirolomyces terfezioides. In embodiments, the protein is expressed in a host cell from the polynucleotide of SEQ ID NO:2 or a variant of SEQ ID NO:2 codon- optimized for expression in the host cell. In embodiments, the host cell is a bacterium or yeast. In embodiments, the host cell is Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris.

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

[0042] In other aspects, certain polypeptides (MYD family proteins) of the invention exhibit sweet- taste modulation activity that is enhanced compared to the polypeptide of SEQ ID NO:3. In other aspects certain polypeptides of the invention exhibit thermal stability that is enhanced compared to the polypeptide of SEQ ID NO:3.

[0043] Another aspect of the invention provides a composition which comprises one or more polypeptides (MYD family proteins) of this invention which exhibit flavor modulation and / or sweet taste modulation activity and / or exhibit sweet taste. The composition includes a combination of (a) a product for oral administration other than Mattirolomyces terfezioides truffle, and (b) a sweetening composition comprising the polypeptide, wherein the combination has enhanced sweet 13Atty Docket: 340391: 0640.49WO taste and / or a change or enhancement in flavor compared to the product for oral administration. The polypeptide includes one or more polypeptide sequences, either individually or in a combination of two or more thereof having flavor modulation and / or sweet-taste modulation activity and comprising (i) a polypeptide sequence selected from an amino acid sequence of a variant of Table 8, Table 9 or Table 10 , wherein the polypeptide sequence is different from SEQ ID NO:3 and optionally different from SEQ ID NO:141; or (ii) a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of an amino acid sequence of a variant of Table 8, Table 9 or Table 10, wherein the polypeptide sequence is different from SEQ ID NO:3 and optionally different from SEQ ID NO:141; or (iii) wherein the polypeptide of a variant of Table 8, Table 9 or Table 10 further comprises a protein / peptide tag, or affinity tag, wherein the protein / peptide tag is optionally a histidine tag. In embodiments, the proteins are expressed from a polynucleotide encoding a MYD family protein in a host cell.

[0044] Another aspect of the invention is a sweetening composition which comprises one or more polypeptides (MYD family proteins) of the invention (e.g., HTS variants) exhibiting sweet taste. A related aspect of the invention is a flavor modulation composition which comprises one or more polypeptides of the invention (MYD family proteins) exhibiting flavor modulation activity. In embodiments, sweetening compositions contain 30 ppm or more of total MYD family protein. In embodiments, flavor modulation compositions contain 30 ppm or less of total MYD family protein.

[0045] Another more specific aspect of the invention provides a composition including a combination of (a) a product for oral administration other than Mattirolomyces terfezioides truffle, and (b) a sweetening composition comprising the polypeptide, wherein the combination has enhanced sweet taste compared to the product for oral administration. In embodiments, the polypeptide includes (a) one or more amino acid sequences selected from the group consisting of the amino acid sequences of a variant of Table 8, Table 9 or Table 10. In embodiments, the polypeptide is not a variant listed in Table 7, Table 3 or Table 6. In embodiments, the HST polypeptide is a peptide of SEQ ID NO:3 or SEQ ID NO:141 or a sequence variant thereof. Further embodiments include sequence variants having 1-6, 1-3, 1-2, 2 or 1 amino acid variations (e.g., substitutions, deletions or additions) from the HTS peptide of SEQ ID NO:3 or SEQ ID NO:141. Additional embodiments include sequence variants having amino acid substitutions of the HTS peptide of SEQ ID NO:3 or SEQ ID NO: 141. Additional embodiments include sequence variants having 1 or 2 amino acid substitutions of the HTS peptide of SEQ ID NO:3 or SEQ ID NO: 141. In embodiments, HTS peptides for use in compositions are other than a naturally-occurring Myd (mycodulcein) polypeptide of Mattirolomyces terfezioides. 14Atty Docket: 340391: 0640.49WO

[0046] Another aspect of the invention provides a method for modulating the taste of a product for oral administration. The method comprises combining the product for oral administration with an effective amount of a flavor modulation composition or a sweetening composition comprising one or more polypeptide of the MYD family of proteins (e.g., HTS-1, HTS-2 or a HTS variant), wherein the product for oral administration differs from Mattirolomyces terfezioides truffle, and wherein the combination has enhanced flavor or sweet taste, respectively, compared to the product for oral administration. In embodiments, the polypeptide includes one or more sequences as listed herein, particularly those having one or more mutations of Table 8, Table 9 or Table 10, either individually or in a combination of two or more thereof. In embodiments, the polypeptide is not a variant listed in Table 7, Table 3 or Table 6. In embodiments, the HST polypeptide is a peptide of SEQ ID NO:3 or SEQ ID NO:141 or a sequence variant thereof. Further embodiments include sequence variants having 1-6, 1-3, 1-2, 2 or 1 amino acid variations (e.g., substitutions, deletions or additions) from the HTS peptide of SEQ ID NO:3 or SEQ ID NO:141. Additional embodiments include sequence variants having amino acid substitutions of the HTS peptide of SEQ ID NO:3 or SEQ ID NO: 141. Additional embodiments include sequence variants having 1 or 2 amino acid substitutions of the HTS peptide of SEQ ID NO:3 or SEQ ID NO: 141. In embodiments, HTS peptides for use in modulating taste are other than a naturally occurring Myd (mycodulcein) polypeptide of Mattirolomyces terfezioides. In embodiments, the one or more polypeptide is expressed from a polynucleotide encoding a MYD family protein.

[0047] In related aspects, the invention provides a product for oral administration, optionally a beverage or beverage product, comprising a sweetener composition, the sweetener composition comprising one or more protein of the mycoduclein (MYD) family of proteins and one or more additional sweetener other than a protein of the MYD family of proteins. The additional sweetener is optionally selected from a steviol glycoside sweetener, a mogroside sweetener, sucrose, glucose, fructose, galactose, xylose, lactose, ribose, lactulose, maltose, trehalose, allulose, sucralose, a polyol, honey, aspartame, erythritol, xylitol, sorbitol, monk fruit extract, dextrose, maltitol, acesulfame potassium (Ace-K), agave extract, tapioca syrup, polyglycitol syrup, sodium saccharin, or high fructose corn syrup (HFCS).

[0048] Another aspect of the invention provides a method of purifying a polypeptide having flavor modulation activity or sweet-taste modulation activity. The method includes: (a) conducting hydrophobic interaction chromatography (HIC) on a composition comprising a polypeptide, and / or (b) conducting size exclusion chromatography (SEC) on the composition comprising the polypeptide. In embodiments, the method includes (a) conducting hydrophobic interaction chromatography (HIC) on a composition comprising a polypeptide, or (b) conducting size exclusion chromatography (SEC) on the composition comprising the polypeptide. In embodiments, 15Atty Docket: 340391: 0640.49WO the method includes (a) conducting hydrophobic interaction chromatography (HIC) on a composition comprising a polypeptide to generate a purified composition, and thereafter (b) conducting size exclusion chromatography (SEC) on the purified composition comprising the polypeptide. In embodiments, the polypeptide includes one or more sequences, either individually or in a combination of two or more thereof having sweet-taste modulation activity and comprising (i) a polypeptide sequence selected from that 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 that 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; (iii) wherein the polypeptide sequence of the variants of Table 8, Table 9 or Table 10 further comprises a protein / peptide tag, wherein the protein tag is optionally an affinity tag or more specifically a histidine tag; (iv) wherein the polypeptide contains 1-24 mutations selected from those listed in Table 8, Table 9 or Table 10 and optionally further comprises a protein / peptide tag, wherein the protein tag is optionally an affinity tag and more specifically a histidine tag. In a related aspect, the invention provided polypeptides that are purified by the method described herein.

[0049] Another aspect of the invention provides sweetener compositions comprising one or more MYD family proteins. In some embodiments, the sweetener compositions include: (a) one or more polypeptide, wherein the one or more polypeptide comprises: (i) a polypeptide sequence selected from 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 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 no more than 24 amino acids, relative to a polypeptide sequence selected from the group consisting of an amino acid sequence of a variant listed in Table 8, Table 9 or Table 10; or (iv) a polypeptide of any of (i)-(iii), wherein the methionine at position 1 is absent; and / or the polypeptide further comprises a protein / peptide tag, optionally an affinity tag or optionally a histidine tag or optionally a (His)6tag; and (b) at least one additional sweetener other than a Myd sweet protein. In embodiments, the HST polypeptide of such sweetener compositions is a peptide of SEQ ID NO:3 or SEQ ID NO:141 or a sequence variant thereof. Further embodiments include sequence variants having 1-6, 1-3, 1-2, 2 or 1 amino acid variations (e.g., substitutions, deletions or additions) from the HTS peptide of SEQ ID NO:3 or SEQ ID NO:141. Additional embodiments include sequence variants having amino acid substitutions of the HTS peptide of SEQ ID NO:3 or SEQ ID NO: 141. Additional embodiments include sequence variants having 1 or 2 amino acid substitutions of the HTS peptide of SEQ ID NO:3 or SEQ ID NO: 141. In embodiments, HTS peptides for use in 16Atty Docket: 340391: 0640.49WO sweetener compositions are other than a naturally-occurring Myd (mycodulcein) polypeptide of Mattirolomyces terfezioides. In additional embodiments, the MYD family protein is a protein expressed from a polynucleotide encoding the MYD family protein in a non-native host cell. In additional embodiments, the polynucleotide encoding the Myd family protein is codon-optimized for expression in the non-native host.

[0050] The additional sweetener is optionally selected from a steviol glycoside sweetener, a mogroside sweetener, sucrose, glucose, fructose, galactose, xylose, lactose, ribose, lactulose, maltose, trehalose, allulose, sucralose, a polyol, honey, aspartame, erythritol, xylitol, sorbitol, monk fruit extract, dextrose, maltitol, acesulfame potassium (Ace-K), agave extract, tapioca syrup, polyglycitol syrup, sodium saccharin, or high fructose corn syrup (HFCS).

[0051] In embodiments, the additional sweetener is sugar or honey. In other embodiments, the sweetener compositions include: (a) one or more polypeptide, wherein the one or more polypeptide comprises: (i) a polypeptide sequence selected from 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 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 no more than 24 amino acids, relative to a polypeptide sequence selected from the group consisting of an amino acid sequence of a variant listed in Table 8, Table 9 or Table 10; or (iv) a polypeptide of any of (i)- (iii), wherein the methionine at position 1 is absent; and / or the polypeptide further comprises a protein / peptide tag, optionally an affinity tag or optionally a histidine tag or optionally a (His)6tag; and (b) at least one additional component selected from monosaccharides, disaccharides (e.g., sucrose), sugar alcohols (e.g., mannitol), amino acids (e.g., leucine), organic acids (e.g., citric acid), hypoxanthine, theophylline, vitamins and combinations thereof.

[0052] In embodiments, sweetener composition herein comprise at least one additional component selected from sucrose, mannitol, citric acid, hypoxanthine, theophylline, leucine, and combinations thereof.

[0053] In specific embodiments, the polypeptide of the sweetener compositions has the amino acid sequence of SEQ ID NO:3. In specific embodiments, the polypeptide of the sweetener composition has the amino acid sequence of SEQ ID NO:3 where the methionine at position 1 is missing. In embodiments, the polypeptide of the sweetener is a mixture of a polypeptide having amino acid sequence of SEQ ID NO:3 and a polypeptide having amino acid sequence o SEQ ID NO:3 where the methionine at position 1 is missing. 17Atty Docket: 340391: 0640.49WO

[0054] In embodiments, the polypeptide of the sweetener compositions is present in an amount between about 1 ppm and about 50 ppm. In some embodiments, the polypeptide is present in an amount between about 1 ppm and about 40 ppm. In further embodiments, the polypeptide is present in an amount selected from an amount between about 1 ppm and about 30 ppm, about 1 ppm and about 25 ppm, about 1 ppm and about 20 ppm or about 1 ppm and about 15 ppm. In some embodiments, the polypeptide is present in an amount between about 5 ppm and about 50 ppm. In some embodiments, the polypeptide is present in an amount between about 10 ppm and about 50 ppm. In some embodiments, the polypeptide is present in an amount between about 15 ppm and about 50 ppm. In some embodiments, the polypeptide is present in an amount between about 20 ppm and about 50 ppm. In some embodiments, the polypeptide is present in an amount between about 25 ppm and about 50 ppm. In some embodiments, the polypeptide is present in an amount between about 30 ppm and about 50 ppm. In some embodiments, the polypeptide is present in an amount between about 35 ppm and about 50 ppm. In some embodiments, the polypeptide is present in an amount between about 40 ppm and about 50 ppm. In some embodiments, the polypeptide is present in an amount between about 45 ppm and about 50 ppm. In some embodiments, the polypeptide is present in an amount between about 10 ppm and about 40 ppm. In some embodiments, the polypeptide is present in an amount between about 20 ppm and about 30 ppm. In some embodiments, the steviol glycoside sweetener is selected from rebaudioside M (“Reb M”), RebM80, rebaudioside D (“Reb D”), Reb A95 and rebaudioside A (“Reb A”). In embodiments, the mogroside sweetener is selected from siamenoside I and mogroside V.

[0055] In another aspect, the invention provides a combination of a product for oral administration, wherein the product is not Mattirolomyces terfezioides truffle, and a sweetening composition described herein. In some embodiments, the product for oral administration has at least one improved organoleptic property compared to a product for oral administration that does not contain the sweetener composition, wherein the organoleptic property is selected from the group consisting of aroma, flavor, basic taste (sweetness, sour, saltiness, bitterness or umami), aftertaste or linger, temporal profile, mouthfeel or a combination thereof.

[0056] In embodiments, the product for oral administration is a solid food product or a beverage product. In embodiments, the product for oral administration is a yogurt product. In embodiments, the product for oral administration is an ice cream product. In embodiments, the product for oral administration is a dairy product. In embodiments, the product for oral administration is an alternative diary product. In embodiments, the product for oral administration is a chewing gum product. 18Atty Docket: 340391: 0640.49WO

[0057] In embodiments, the product for oral administration is a solid, liquid or gel product for oral hygiene or oral therapeutic applications. In embodiments, the product for oral administration is a toothpaste or tooth powder, including a tooth whitening toothpaste or composition. In embodiments, the product for oral administration is a mouthwash or a mouth rinse.

[0058] In another aspect, the 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 organoleptic property compared to a beverage or beverage product that does not contain the sweetener composition, wherein the organoleptic property is selected from the group consisting of aroma, flavor, basic taste (sweetness, sour, saltiness, bitterness or umami), aftertaste or linger, temporal profile, mouthfeel or a combination thereof. In some embodiments, the beverage or beverage product is selected from a low-calorie or no-calorie beverage or beverage product. 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 beverage, near water drinks (e.g., water with natural or synthetic flavorants), tea type (e.g. black tea, green tea, red tea, oolong tea), coffee, cocoa drink, beverage containing milk components (e.g. milk beverages, coffee containing milk components, café au lait, milk tea, fruit milk beverages). In embodiments, the beverage or beverage product includes at least one organic acid additive salt that is a sodium, calcium, potassium, or magnesium salt of an organic acid. In embodiments, 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.

[0059] In another aspect, the invention provides methods of improving at least one organoleptic property of the product for oral administration described herein. In embodiments the product for oral administration is a solid food product or a liquid beverage product. In an embodiment, the invention provides methods of improving at least one organoleptic property of the product for oral administration described herein. The methods include adding a sweetener composition as described herein to a solid or liquid matrix, thereby providing the product having at least one improved organoleptic property. In embodiments, the improved organoleptic property is selected from the group consisting of aroma, flavor, basic taste (sweetness, sourness, saltiness, bitterness or umami), aftertaste or linger, temporal profile, mouthfeel or a combination thereof. In embodiments, the amount of polypeptide present in the product improved by the methods is an amount between about 1 ppm and about 50 ppm, about 1 ppm and about 40 ppm, about 1 ppm and about 30 ppm, about 1 ppm and about 25 ppm, about 1 ppm and about 20 ppm or about 1 ppm and about 15 ppm. In embodiments, the product is a solid food product and the sweetener composition is added to, combined with or mixed into the solid food product. 19Atty Docket: 340391: 0640.49WO

[0060] In embodiments, the invention provides methods of improving at least one organoleptic property of the beverage or beverage product described herein. The methods include adding a sweetener composition as described herein to a liquid matrix, thereby providing a beverage or beverage product having at least one improved organoleptic property. In embodiments, the improved organoleptic property is selected from the group consisting of aroma, flavor, basic taste (sweetness, sourness, saltiness, bitterness or umami), aftertaste or linger, temporal profile, mouthfeel or a combination thereof. In embodiments, the amount of polypeptide present in the beverage or beverage product improved by the methods is an amount between about 1 ppm and about 50 ppm, about 1 ppm and about 40 ppm, about 1 ppm and about 30 ppm, about 1 ppm and about 25 ppm, about 1 ppm and about 20 ppm or about 1 ppm and about 15 ppm.

[0061] In another aspect, the invention provides methods of changing / modifying at least one organoleptic property of a product for oral administration as described herein. The methods include adding a sweetener composition as described herein to the product, thereby providing a product having at least one changed / modified organoleptic property. In embodiments, the changed / modified organoleptic property is selected from the group consisting of aroma, flavor, basic taste (sweetness, sour, saltiness, bitterness or umami), aftertaste or linger, temporal profile, mouthfeel or a combination thereof. In embodiments, the amount of polypeptide present in the product changed / modified by the methods is an amount between about 1 ppm and about 50 ppm, about 1 ppm and about 40 ppm, about 1 ppm and about 30 ppm, about 1 ppm and about 25 ppm, about 1 ppm and about 20 ppm or about 1 ppm and about 15 ppm. In embodiments, the product for oral administration is a food product or a beverage product. In embodiments, the product is a solid food product and the sweetener composition is added to, combined with or mixed into the solid food product.

[0062] In another aspect, the invention provides methods of changing / modifying at least one organoleptic property of the beverage or beverage product described herein. The methods include adding a sweetener composition as described herein to a liquid matrix, thereby providing a beverage or beverage product having at least one changed / modified organoleptic property. In embodiments, the changed organoleptic property is selected from the group consisting of aroma, flavor, basic taste (sweetness, sour, saltiness, bitterness or umami), aftertaste or linger, temporal profile, mouthfeel or a combination thereof. In embodiments, the amount of polypeptide present in the beverage or beverage product changed / modified by the methods is an amount between about 1 ppm and about 50 ppm, about 1 ppm and about 40 ppm, about 1 ppm and about 30 ppm, about 1 ppm and about 25 ppm, about 1 ppm and about 20 ppm or about 1 ppm and about 15 ppm. 20Atty Docket: 340391: 0640.49WO

[0063] Other aspects and embodiments of the invention will be apparent on review of the figures, detailed description and non-limiting examples herein. BRIEF DESCRIPTION OF THE FIGURES

[0064] Figure 1 shows a Coomassie-stained SDS-PAGE gel of proteins obtained from fractions of partially purified M. terfezioides gleba.

[0065] Figure 2 shows a SDS-PAGE gel, Coomassie stained, of purification steps of His-tagged sweet polypeptide expressed in E. coli from the coding region of SEQ ID NO:4 lane 1: molecular weight standards; 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 Hist-tagged sweet protein expressed in E. coli is the His-tagged version of HTS-2 in which methionine is absent (SEQ ID NO:142)

[0066] Figure 3 shows the concentration-response functions for sweet taste for mycodulcein (Honey Truffle Sweetener (HTS)), aspartame, thaumatin, rebaudioside A. Data are plotted as the proportion (p) of responses occurring on the 200 mM sucrose-associated (“sweet”) target. Each data point in the curves for mycodulcein, aspartame, thaumatin, rebaudioside A was calculated as the average across 32 replicates and averaged across 16 replicates for the sucrose curve; error bars are SEM. Points for water and sucrose controls were similarly calculated as the average across 128 and 64 replicates, respectively. Curves were fit by non-linear regression.

[0067] Figure 4A shows SDS-PAGE analysis, Coomassie stain, of the eluted fraction from Capto MMC a multimodal weak cation exchange resin. M: protein marker; lane 1: eluted fraction, showing low purity after cation exchange. Arrow indicates mycodulcein (native HTS) band.

[0068] Figure 4B shows SDS-PAGE analysis, Coomassie stain, of two eluted fractions collected during the gradient elution from the HiScreen Capto Butyl (Cytiva Sweden AB, Upsala, Sweden) column analyzed on SDS-PAGE. Lane 1 shows eluted fraction 1 not containing mycodulcein and Lane 2 shows eluted mycodulcein. The purity of the eluted fraction was determined by GelAnalyzer to be ~86%. Arrow indicates mycodulcein (native HTS) band.

[0069] Figure 4C shows SDS-PAGE analysis, Coomassie stain, the eluted protein from the HIC column after chromatographing on HiPrep® 26 / 60 Sephacryl® S-200 (Cytiva Sweden AB, Upsala, Sweden). Lane 1 shows purified his-tag mycodulcein and Lane 2 shows purified native mycodulcein. The purity of the eluted fraction was determined by GelAnalyzer to be ~98%. Arrow indicates mycodulcein (native HTS) band.

[0070] Figure 5 is a map of the pD44-CH vector which is isopropyl beta-D-1-thiogalactopyranoside (IPTG) inducible. 21Atty Docket: 340391: 0640.49WO

[0071] Figures 6A and 6B include graphs of exemplary melting temperature results obtained from GloMelt™ thermostability assays comparing native HTS (1) and HTS variant S33C_I49C (double mutant, 2). Fig.6A compares melt curves for native HTS (1) and the double mutant (2). Fig.6B compares melt peak curves for 1 and 2. Relative fluorescence (RFU) is measured as a function of temperature. An increase in average melting temperature of 7.5 °C is observed with the double mutant compared to native HTS protein.

[0072] Figure 7 shows melting temperature results obtained from GloMelt™ thermostability assays comparing native HTS protein with HTS variant double mutation S33C_I49C. In this assay, the sample was brought to a low pasteurization temperature (63 °C) and held for an hour. DETAILED DESCRIPTION OF THE INVENTION

[0073] The invention provides embodiments of isolated polynucleotides encoding MYD family proteins. MYD family proteins include Myd proteins isolated from Mattirolomyces. Terfezioides and those expressed in a host cell from isolated polynucleotide encoding MYD family proteins. MYD family proteins include Myd polypeptide variants, the encoded Myd1 polypeptide variants (also called Honey Truffle Sweetener (HTS) proteins and variants thereof) and proteins expressed from polynucleotides encoding MYD family proteins including those that are codon-optimized for expression in a selected host and that are capable of flavor modulation and / or modulating sweet taste and perception. The invention includes methods of making and using such MYD family proteins and the polynucleotides encoding them. The invention provides embodiments of MYD family proteins (including those encoded by MYD1 variants and Myd1 polypeptide variants) either alone, or in combination with food, beverage, dietary supplement, 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 invention. The invention provides naturally occurring Myd1 polypeptides and nucleic acids encoding these polypeptides. In embodiments, the invention provides non-naturally occurring Myd1 polypeptide variants and polynucleotides encoding these polypeptides. Polynucleotides encoding MYD family proteins may be codon-optimized for expression in a selected host cell. MYD family proteins include proteins expressed in a selected host cell which are optionally modified by post translational modification.

[0074] An embodiment of the invention provides Myd polypeptide variants (also referred to as HTS variants). The term “Myd polypeptide variants” is used herein to identify non-naturally occurring HTS polypeptides and includes any of the polypeptides according to the present invention which e.g., have at least 80% sequence identity to a variant of Table 8, Table 9 or Table 10 and also have flavor modulation (or modifying) and / or sweet-taste modifying activity. 22Atty Docket: 340391: 0640.49WO

[0075] A sweet tasting partially purified extract of terfezioides gleba was subjected to de novo amino acid sequencing to identify a 20-mer N-terminal sequence. The Myd1 coding sequence (putatively derived from the MYD1 gene) was identified after the whole transcriptome of the Mattirolomyces. terfezioides gleba was de novo assembled using RNAseq reads. Screening the M. terfezioides whole transcriptome using the 20-mer N-terminus sequences 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 of SEQ ID NO:1. Start and stop codons were identified in the transcript to identify putative coding sequence SEQ ID NO:2. SEQ ID NO:3 is the putative encoded protein, a 121 amino acid protein. The naturally-occurring protein isolated from M. terfezioides gleba was subsequently found to be a mixture of two isoforms the protein of SEQ ID NO:3 and a mature protein of SEQ ID NO: 3 with the methionine (Met) residue at amino acid position 1 deleted (designated SEQ ID NO:141 hereinafter). Identity between the predicted protein SEQ ID NO:3, and other protein sequences in GENBANK were 31% or less.

[0076] Native HTS appears to be a mixture of two isoforms (HTS-1 and HTS-2). HTS-1 is the polypeptide of SEQ ID NO:3. HTS-2 is the polypeptide of SEQ ID NO:141. Native HTS isolated from M. terfezioides, as described herein, is a mixture of about 40-60% by weight HTS-2 and 60 to 40% by weight HTS-1. The relative amount of HTS-1 and HTS-2 isolated from M. terfezioides varies at least by truffle source. As isolated, about 80% of HTS-1 is acetylated at the N-terminal amine group. No significant difference in sweet taste has as yet been observed for the two sequence isoforms of HTS. Acetylation of the N-terminus of the HTS polypeptide HTS-1 also does not have a significant effect on sweet taste.

[0077] The polynucleotide encoding the polypeptide of SEQ ID NO:3 has been successfully expressed in multiple hosts. In some cases, the polynucleotide sequence encoding the polypeptide of SEQ ID NO:3 has been codon-optimized, as known in the art, for expression in a given host. For example, intracellular expression in E. coli using optimized codons to express the polynucleotide encoding SEQ ID NO:3 using nucleotide coding sequence of SEQ ID NO:4 results in a sweet tasting protein. For example, intracellular expression in E. coli using optimized codons to express the polynucleotide encoding SEQ ID NO:3 and a (His)6tag results in a sweet tasting protein. The sweet protein with His-tag expressed from SEQ ID NO:4 in E. coli has subsequently been determined by proteome analysis to be SEQ ID NO:141 (SEQ ID NO:3 without methionine in position 1) with a His-tag (SEQ ID NO: 148) expression of the HTS-1 isoform of the protein is not observed. The sweet protein of SEQ ID NO:141 (without His-tag) is the protein expressed on expression of the optimized E. coli coding region without the His-tag (SEQ ID NO:148). 23Atty Docket: 340391: 0640.49WO

[0078] For example, intracellular expression in Saccharomyces cerevisiae using optimized codons (SEQ ID NO:6) to express the polynucleotide encoding SEQ ID NO:3 with an additional His-tag coding sequence results in sweet tasting protein. The protein expressed is a mixture of HTS-1 and HTS-2 isoforms each with His-tags. The relative amounts of HTS-2 to HTS-1 is about 60% to 40% by weight. Extracellular expression in Saccharomyces cerevisiae using optimized codons and an appropriate signal peptide at the N-terminal to facilitate secretion into the fermentation media resulted in an expression product that was not sweet.

[0079] For example, intracellular expression in Yarrowia lipolytica using optimized codons to express the polynucleotide encoding SEQ ID NO:3 results in sweet tasting protein. The protein expressed is a mixture of HTS-1 and HTS-2 isoforms. The relative amounts of HTS-2 to HTS-1 is about 20% to 80% by weight.

[0080] For example, intracellular expression in Pichia pastoris using optimized codons to express the polynucleotide encoding SEQ ID NO:3 results in sweet tasting protein. The protein expressed is a mixture of HTS-1 and HTS-2 isoforms. The relative amounts of HTS-2 to HTS-1 is about 90% to 10% by weight.

[0081] The coding sequences for native mycodulcein (HTS), which have been codon-optimized for expression in E. coli and Saccharomyces cerevisiae correspond to the nucleic acid sequences of SEQ ID NO:4 and SEQ ID NO:6, respectively (which both encode 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).

[0082] In embodiments, the invention provides non-naturally occurring sweet proteins which include non-naturally occurring mixtures of the two isoforms HTS-1 and HTS-2. In embodiments, the 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 invention provides HTS-2 (SEQ ID NO:141) 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 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 invention provides HTS-1 (SEQ ID NO:3) 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 invention provides non-naturally occurring mixtures of HTS-1 and HTS-2, specifically those enriched in HTS-2 where the amount of HTS-2 is greater than 60% by weight and for example is 65% or more by weight, 70% or more by weight, 75% or more by weight, 80% or more by wight, 85% or more by weight, 90% or more by weight, or 95% or more by weight. In embodiments, the invention provides non-naturally occurring mixtures of HTS-1 and HTS-2, specifically those enriched in HTS-1 where the amount of HTS-1 is 24Atty Docket: 340391: 0640.49WO greater than 60% by weight and for example is 65% or more by weight, 70% or more by weight, 75% or more by weight, 80% or more by weight, 85% or more by weight, 90% or more by weight, or 95% or more by weight.

[0083] In embodiments, the invention provides sweet protein generated 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 Mattirolomyces terfezioides truffle). In specific embodiment, the sweet protein is generated by recombinant expression in a bacterium, yeast or fungus. 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 embodiments, recombinant expression in a non-native host results in non-naturally occurring mixtures of the HTS-1 and HTS-2 naturally-occurring isoforms of mycodulcein (Myd).

[0084] POLYNUCLEOTIDES

[0085] An embodiment of the invention comprises a polynucleotide (e.g., isolated polynucleotide) encoding a polypeptide variant having sweet-taste modulation activity. Examples of polynucleotides encoding a polypeptide having sweet-taste modulation activity include polynucleotides able to encode polypeptides such as, but not limited to, a variant of Table 8, Table 9 or Table 10, or a polypeptide thereof further having a histidine tag, or an nucleic acid sequence with 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 polynucleotides encoding a variant of Table 8, Table 9, or Table 10 or a polypeptide thereof further having a protein / peptide tag, optionally an affinity tag or optionally a histidine tag. It will be understood that the polynucleotides, including or excluding the sequence encoding the protein / peptide tag, affinity tag or histidine tag, are both provided and useful in this invention. It will be further understood that for any specific polynucleotide indicated to encode a given histidine tag (e.g., (His)6), the sequence encoding the histidine tag can be substituted with a sequence encoding a different His tag or a sequence encoding a different protein / peptide tag or affinity tag. In embodiments, the protein / peptide, affinity or histidine tag is one that is encoded by 3-30 or 3-18 nucleotides. In embodiments, the variants recited herein above include a methionine at position 1 or the methionine at position 1 is absent. In embodiments, variant HTS polypeptides are other than the polypeptides of SEQ ID NO:3 or SEQ ID NO:141.

[0086] In an embodiment, the polynucleotide comprises, consists essentially of, or consists of a polynucleotide selected from the group consisting of polynucleotide encoding a variant of Table 8, Table 9 or Table 10, or a nucleic acid sequence with at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 25Atty Docket: 340391: 0640.49WO 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a polynucleotide able to encode a variant of Table 8, Table 9 or Table 10. In embodiments, the encoded variant polypeptide is other than the polypeptides of SEQ ID NO:3 or SEQ ID NO:141.

[0087] Unless expressly stated otherwise, when a polynucleotide sequence has a plurality of nucleotide modifications, each modification may independently be modified with a modification of choice such as deletion, insertion, substitution, or addition, regardless of what the other modifications are. Additionally, the plurality of modifications in the polynucleotide sequence may be the same or differ from each other; and the modification options for each modification may independently vary such deletion, insertion, substitution, or addition, etc. unless expressly stated otherwise. When there are a plurality of modifications, each modification may independently be a substitution, addition, insertion, or deletion regardless of what the other modification is or are. In an embodiment, the polynucleotide sequence has at least one substitute modification. In a particular embodiment, the polynucleotide sequence has a plurality of substitution modifications; and each substitution may independently be substituted with a substitution of choice regardless of what the other substitutions are. Additionally, the plurality of substitutions in the polynucleotide sequence may be the same or differ from each other; and the options for nucleotide substitutions for each substitution may independently vary unless expressly stated otherwise. When the polynucleotide sequence has a plurality of substitutions, each substitution may independently be chosen regardless of what the other substitutions is or are. In embodiments, polynucleotides herein are optionally isolated and / or optionally purified.

[0088] In embodiments, at least 80% sequence identity with respect to a polynucleotide includes, without limitation, 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 with respect to a polynucleotide also includes, without limitation, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity.

[0089] In an embodiment, the polynucleotide encodes a polypeptide sequence comprising, consisting essentially of, or consisting of a polypeptide selected from the group consisting of: (a) the amino acid sequence of a variant of Table 8, Table 9 or Table 10, or a polypeptide thereof further having a protein / peptide tag, an affinity tag or a histidine tag; (b) an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 26Atty Docket: 340391: 0640.49WO 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of a variant of Table 8, Table 9 or Table 10, or a polypeptide thereof further having a protein / peptide tag, an affinity tag or a histidine tag; and (c) an amino acid sequence modified from an amino acid sequence of a variant of Table 8, Table 9 or Table 10 by deletion, insertion, substitution, or addition of no more than 24 amino acids (e.g., modification of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids), or a polypeptide thereof further having a histidine tag. 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 is other than that of SEQ ID NO:141. When the amino acid sequence has a plurality of modifications, the number of modifications the amino acid has may range from at least one and up to 24 modifications, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 modifications, as desired. Furthermore, unless expressly stated otherwise, when an amino acid sequence has a plurality of modifications, each modification may independently be modified with a modification of choice such as deletion, insertion, substitution, or addition, regardless of what the other modifications are. Additionally, the plurality of modifications in the amino acid sequence may be the same or differ from each other; and the modification options for each modification may independently vary such deletion, insertion, substitution, or addition, etc. unless expressly stated otherwise. When the amino acid sequence has a plurality of modifications, each modification may independently be a substitution, addition, insertion, or deletion regardless of what the other modification is or are. In an embodiment, the amino acid sequence has at least one substitute modification. In a particular embodiment, the amino acid sequence has a plurality of substitution modifications; and each substitution modification may independently be substituted with a substitution of choice regardless of what the other substitutions are. In an embodiment, the amino acid sequence has one substitution modification (a single mutant) or two substitution modifications (a double mutant) compared to SEQ ID NO:3 or SEQ ID NO:141. Additionally, the plurality of substitutions in the amino acid sequence may be the same or differ from each other; and the options for substitutions for each amino acid may independently vary unless expressly stated otherwise. When the amino acid sequence has a plurality of substitutions, each substitution may independently be chosen regardless of what the other substitutions is or are. In embodiments, the polypeptide encoded may include 1-6, 1-12, 1-18 or 1-24 substitutions compared to the polypeptide of SEQ ID NO:3, wherein the substitutions are those listed in Table 8. In embodiments, the polypeptide encoded may include 1-6, 1-12, 1-18 or 1-24 substitutions compared to the polypeptide of SEQ ID NO:3, wherein the substitutions are those listed in Table 9. In embodiments, the polypeptide encoded may include 1-6, 1-12, 1-18 or 1-24 substitutions compared to the polypeptide of SEQ ID NO:3, wherein the substitutions are those listed in Table 10. In embodiments, the 27Atty Docket: 340391: 0640.49WO polypeptide encoded may include 1-6, 1-12, 1-18 or 1-24 substitutions compared to the polypeptide of SEQ ID NO:3, wherein the substitutions are any one or more of those listed in Table 3, Table 6, Table 7, Table 8, Table 9 or Table 10. In embodiments, the polypeptide encoded may include 1-6, 1-12, 1-18 or 1-24 substitutions compared to the polypeptide of SEQ ID NO:3, wherein the substitutions are other than those listed in Table 7. In embodiments, the polypeptide encoded may include 1-6, 1-12, 1-18 or 1-24 substitutions compared to the polypeptide of SEQ ID NO:3, wherein the substitutions are other than those listed in Table 3 or Table 6. In embodiments, the polypeptide encoded may include 1-6, 1-12, 1-18 or 1-24 substitutions compared to the polypeptide of SEQ ID NO:3, wherein the substitutions are other than those listed in Table 3 or Table 6. In embodiments, the polypeptide encoded may include 1-6, 1-12, 1-18 or 1-24 substitutions compared to the polypeptide of SEQ ID NO:3, wherein the substitutions are those listed in Tables 9 and 10 and are other than those listed in Table 3, Table 6 or Table 7.

[0090] In certain embodiments of polynucleotides herein, the polynucleotide encodes a polypeptide herein and also encodes a protein / peptide tag, an affinity tag or a histidine tag. In all such cases in which a specific polynucleotide encodes such a tag, the invention also provides the polynucleotide excluding the sequence encoding the tag. In additional embodiments, in those polynucleotides herein encoding a histidine tag, the sequence encoding the histidine tag can be deleted or excluded, or can be replaced with a coding sequence for a different protein / peptide tag or a different affinity tag, including a different histidine tag.

[0091] The invention provides isolated and / or purified embodiments of the respective polynucleotides, when applicable. The invention provides embodiments of each of the polynucleotides able to encode the variants of Table 8, Table 9 or Table 10 herein with or without being isolated and with or without being purified, when applicable. The invention provides embodiments of each of the polynucleotides able to encode the variants of Table 8, Table 9 or Table 10 herein with one or more mutations, when applicable.

[0092] The invention also provides an expression cassette comprising one or more polynucleotides encoding a Myd polypeptide variant (HTS variant) and a host cell transformed with the vector.

[0093] The polynucleotide encoding the HTS variant of the present invention can be in the form of a single-stranded or double-stranded DNA, RNA or an artificial nucleic acid, or can be a cDNA or a chemically synthesized DNA which does not comprise any intron. The term “MYD family” can refer to polymorphic variants, including natural alleles, mutants, alleles, and interspecies homologs that encode polypeptides that: (1) have at least about 35 to 50% amino acid sequence identity, optionally about 60, 75, 80, 85, 90, 95, 96, 97, 98, or 99% amino acid sequence identity to SEQ ID NO:3 over a window of about 25 amino acids, optionally 50-100 amino acids. In one aspect, the 28Atty Docket: 340391: 0640.49WO 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., isolated from a synthesis medium). Isolated products, thus, can be synthetic or naturally produced. In an embodiment, the term “isolated” encompasses products that have been isolated from other proteins that are present in the natural environment. In embodiments, the term “isolated” encompasses products that have been isolated from carbohydrates that are present in the natural environment.

[0094] The term "nucleic acid" or "nucleic acid sequence" refers to a deoxy-ribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogs of natural nucleotides. The term also encompasses nucleic-acid-like structures with synthetic backbones (see e.g., Oligonucleotides and Analogues, a Practical Approach, ed. F. Eckstein, Oxford Univ. Press (1991); Antisense Strategies, Annals of the N.Y. Academy of Sciences, Vol.600, Eds. Baserga et al. (NYAS 1992); Milligan J. Med. Chem.36:1923-1937 (1993); Antisense Research and Applications (1993, CRC Press), WO 97 / 03211; WO 96 / 39154; Mata, Toxicol. Appl. Pharmacol.144:189-197 (1997); Strauss-Soukup, Biochemistry 36:8692-8698 (1997); Samstag, Antisense Nucleic Acid Drug Dev, 6:153-156 (1996)).

[0095] 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 bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (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 a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, for example, probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. The probes are optionally directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.

[0096] The polynucleotide or polypeptide can be naturally occurring or non-naturally occurring (e.g., synthetic, recombinant, modified, and / or variant products). In one aspect, the naturally occurring or non-naturally occurring products are isolated or purified. In another aspect, the naturally occurring or non-naturally occurring products are not isolated or are not purified. In 29Atty Docket: 340391: 0640.49WO embodiments herein, the polynucleotides and polypeptides are non-naturally occurring. In embodiments herein, the polynucleotides and polypeptides are non-naturally occurring variants of the naturally-occurring mycoduclein (HTS) polypeptide and the naturally-occurring polynucleotide sequence that encodes the naturally-occurring mycoduclein (HTS).

[0097] As used herein, "recombinant" refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., "recombinant polynucleotide"), to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a polypeptide ("recombinant protein") encoded by a recombinant polynucleotide. "Recombinant means" also encompass the ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into an expression cassette or vector for expression of, e.g., inducible or constitutive expression of a fusion protein comprising a translocation domain of the invention and a nucleic acid sequence amplified using a primer of the invention.

[0098] As used herein, the terms "amplifying" and "amplification" refer to the use of any suitable amplification methodology for generating or detecting recombinant or naturally expressed nucleic acid, as described in detail, below. For example, the invention provides methods and reagents (e.g., specific degenerate oligonucleotide primer pairs) for amplifying (e.g., by polymerase chain reaction, PCR) naturally expressed (e.g., genomic or mRNA) or recombinant (e.g., cDNA) nucleic acids of the invention (e.g., taste stimulus-binding sequences of the invention) in vivo or in vitro.

[0099] As used herein, the term "isolated," when referring to a nucleic acid or polypeptide refers to a state of purification or concentration different than that which occurs naturally. Any degree of purification or concentration greater than that which occurs naturally, including (1) the purification from other naturally occurring associated structures or compounds (e.g., other proteins, carbohydrates), or (2) the association with structures or compounds to which it is not normally associated in the body are 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 to which they are not normally associated in nature, according to a variety of methods and processes known to those of skill in the art. In an embodiment, the polypeptides described herein contain at most 5% (e.g., at most 4%, at most 3%, at most 2% at most 1%) by weight of other fungal proteins, e.g., fungal proteins other than Myd proteins.

[0100] “Modified” or “variant” products refer to products (e.g., polynucleotides or polypeptides) that have been altered from the original (e.g., naturally occurring) structure. As described herein, variants encompass polynucleotides or polypeptides with one or more changes to the nucleic acid or amino acid sequences, respectively. Changes include modifications to the nucleic acid or amino acid sequence, including additions, deletions, insertions, and substitutions. Modified or variant 30Atty Docket: 340391: 0640.49WO products also can encompass those modified to include disulfide bond formation, as well as those that are derivatized by glycosylation using post-translation modification methods, lipidation, acylation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component, relative to the original structure. As is known in the art, derivatization can be accomplished by chemical or enzymatic methods after translation of a polypeptide. Derivatization can also be accomplished with post-translation modification during expression of the polypeptide in a selected host.

[0101] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating, e.g., 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.

[0102] Herein where a specific polynucleotide is indicated to encode a histidine tag, it will be understood that the polynucleotide excluding the sequence encoding the histidine tag is also provided. It will be further understood that for any specific polynucleotide indicated to encode a given histidine tag (e.g., (His)6), the sequence encoding the histidine tag can be substituted with a sequence encoding a different His tag or a sequence encoding a different protein / peptide tag or affinity tag.

[0103] POLYPEPTIDES

[0104] It should be appreciated that embodiments of the 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 amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0105] An embodiment of the invention includes a polypeptide comprising, consisting essentially of, or consisting of a polypeptide sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to a sequence selected from the group consisting of a variant of Table 8 or a variant of Table 8 further having a histidine tag. In embodiments, the 31Atty Docket: 340391: 0640.49WO 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 which is substantially free (less than 95% or more specifically less than 99% by weight being present) of the polypeptide of SEQ ID NO:141. In embodiments, the polypeptide is the polypeptide of SEQ ID NO:141 which is substantially free (less than 95% or more specifically less than 99% by weight being present) of the polypeptide of SEQ ID NO:3. Optionally, the amino acid sequence has at least one and up to 24 modifications. When the amino acid sequence has a plurality of modifications, the number of modifications the amino acid has may range from at least one and up to 24 modifications such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 modifications, as desired. Furthermore, unless expressly stated otherwise, when an amino acid sequence has a plurality of modifications, each modification may independently be modified with a modification of choice such as deletion, insertion, substitution, or addition, regardless of what the other modifications are. Additionally, the plurality of modifications in the amino acid sequence may be the same or differ from each other; and the modification options for each modification may independently vary such deletion, insertion, substitution, or addition, etc. unless expressly stated otherwise. When the amino acid sequence has a plurality of modifications, each modification may independently be a substitution, addition, insertion, or deletion regardless of what the other modification is or are. In an embodiment, the amino acid sequence has at least one substitute modification. In a particular embodiment, the amino acid sequence has a plurality of substitution modifications; and each substitution modification may independently be substituted with a substitution of choice regardless of what the other substitutions are. Additionally, the plurality of substitutions in the amino acid sequence may be the same or differ from each other; and the options for substitutions for each amino acid may independently vary unless expressly stated otherwise. When the amino acid sequence has a plurality of substitutions, each substitution may independently be chosen regardless of what the other substitutions is or are. The term “consisting essentially of” allows for the inclusion of components that are not essential to the function or activity of the product and do not materially affect the function or activity, such as an anti-caking agent, filler, stabilizer (e.g., thermal stabilizer), and bulking agent (e.g., maltodextrose, gum acacia and the like).

[0106] Another embodiment of the invention includes a recombinant polypeptide having sweet modulation activity comprising, consisting essentially of, or consisting 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 having a histidine tag, or being 32Atty Docket: 340391: 0640.49WO fused to a heterologous signal peptide or transit peptide. The term “consisting essentially of” allows for the inclusion of components that are not essential to the function or activity of the product and do not materially affect the function or activity, such an anti-caking agent, filler, stabilizer (e.g., thermal stabilizer), and bulking agent (e.g., maltodextrose, gum acacia and the like).

[0107] In an embodiment, the polypeptide having sweet-taste modulation activity comprises, consists essentially of or consists of a modified SEQ ID NO:3 wherein the peptide has at least 1 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 with at least 1 and up to 24 amino acid modifications as shown in Table 8, Table 9 or Table 10; and the polypeptide differs from the polypeptide of amino acid sequence SEQ ID NO:3 and optionally differs from the polypeptide of amino acid sequence SEQ ID NO:141.

[0108] In an embodiment, the polypeptide having sweet-taste modulation activity comprises, consists essentially of or consists of a modified SEQ ID NO:3 wherein the peptide has at least 1 and up to 24 amino acid modifications as shown in Table 8, and further has 1-24 amino acid modifications as shown in Table 7, wherein the total number of modifications is 1-24. In another embodiment, the polypeptide has at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to a modified polypeptide of SEQ ID NO: 3 with at least 1 and up to 24 amino acid modifications as shown in Table 8 and 1-24 amino acid modifications as shown in Table 7.

[0109] In an embodiment, the polypeptide having sweet-taste modulation activity comprises, consists essentially of or consists of a modified SEQ ID NO:3 wherein the peptide has at least 1 and up to 12 amino acid modifications as shown in Table 8, and further has 1-12 amino acid modifications as shown in Table 7. In another embodiment, the polypeptide has at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to a modified polypeptide of SEQ ID NO: 3 with at least 1 and up to 12 amino acid modifications as shown in Table 8 and 1-12 amino acid modifications as shown in Table 7. 33Atty Docket: 340391: 0640.49WO

[0110] In an embodiment, the polypeptide having sweet-taste modulation activity comprises, consists essentially of or consists of a modified SEQ ID NO:3 wherein the peptide has at least 1 and up to 6 amino acid modifications as shown in Table 8, and further has 1-6 amino acid modifications as shown in Table 7. In another embodiment, the polypeptide has at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to a modified polypeptide of SEQ ID NO: 3 with at least 1 and up to 6 amino acid modifications as shown in Table 8 and 1-6 amino acid modifications as shown in Table 7.

[0111] In an embodiment, the polypeptide having sweet-taste modulation activity comprises, consists essentially of or consists of a modified SEQ ID NO:3 wherein the peptide has at least 1 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 with at least 1 and up to 24 amino acid modifications as shown in Table 8, Table 9, Table 10, Table 7, Table 6 or Table 3.

[0112] In an embodiment, the polypeptide having sweet-taste modulation activity comprises, consists essentially of or consists of a modified SEQ ID NO:3 wherein the peptide has at least 1 and up to 24 amino acid modifications as shown in Table 8, Table 9 or Table 10 and no modifications as shown in Table 7, Table 3 or Table 6. In another embodiment, the polypeptide has at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to a modified polypeptide of SEQ ID NO: 3 with at least 1 and up to 24 amino acid modifications as shown in Table 8, Table 9, Table 10 and no modifications as shown in Table 7, Table 3 or Table 6.

[0113] In an embodiment, the polypeptide having sweet-taste modulation activity comprises, consists essentially of or consists of a modified SEQ ID NO:3 wherein the peptide has at least 1 and up to 6 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%) 34Atty Docket: 340391: 0640.49WO sequence identity to a modified polypeptide of SEQ ID NO:3 with at least 1 and up to 6 amino acid modifications as shown in Table 8, Table 9 or Table 10.

[0114] In certain embodiments herein, a specific polypeptide is described as having a histidine tag or as optionally having a histidine tag (noted as XXXXXX, where X is His, in the sequence listing herein). The invention provides all specific polypeptides including or optionally including a histidine tag as well as the corresponding polypeptides excluding the histidine tag. The invention also provides all specific polypeptides having a histidine tag or an optional histidine tag as well as the polypeptides excluding the histidine tag or wherein the histidine tag is replaced with a different protein tag including a different histidine tag.

[0115] In an embodiment, the Myd peptide (HTS) variants as described herein are capable of sweet- taste modulation activity. HTS polypeptide variants of the invention may have e.g., functional, physical and chemical effects at taste receptors, such as sweet taste receptors. “Sweet-taste modulation activity” may refer to inhibitory, activating, e.g., agonist or antagonist properties of a polypeptide of the invention, identified using in vitro and in vivo assays for taste transduction. Proteins with inhibitory activity may bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate taste transduction, e.g., antagonists. Activating polypeptides may bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize, or up regulate taste transduction, e.g., agonists. Activating polypeptides are preferred.

[0116] Sweet taste modulation also refers to enhancing the taste, such as a sweet taste, of a particular product for oral administration when administered as a combination. In addition to sweet taste, HTS variants, may also exhibit differences in intensity of sweetness and duration of sweetness. These attributes of sweetness can be assessed in taste tests as described herein.

[0117] Sweet taste modulation also refers to polypeptide variants herein which exhibit sweet taste or convey sweet taste to compositions or formulations that do not themselves exhibit sweet taste when the polypeptide variant is added to the compositions or formulations.

[0118] In some embodiments, the Myd (HTS) polypeptide variants of the invention include polypeptides that are at least as sweet as sucrose (on a w / w basis) (e.g., 1X), or alternatively, are 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 variants are 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%) as sweet as sucrose. 35Atty Docket: 340391: 0640.49WO

[0119] In embodiments, the polypeptides having sweet-taste modulation activity include modified SEQ ID NO:3 with 1 to up to 24 different amino acid modifications as shown in Table 8, Table 9 or Table 10. In embodiments, the polypeptides having sweet-taste modulation activity include modified SEQ ID NO:141, with 1 to up to 24 different amino acid modifications as shown in Table 8, Table 9 or Table 10.

[0120] In an embodiment, the Myd (HTS) peptide variants as described herein have flavor modifying properties (FMP) or activity. A compound having FMP is a compound, including a protein or polypeptide that causes a change in any attributes of perceived flavor of a formulation relative to a formulation without the compound, but does not directly provide a flavor attribute. Compounds may have flavor modifying properties below a perceptible sweetness threshold concentration and provide a direct flavor sensation above that threshold. HTS proteins / polypeptide or variants can exhibit a FMP threshold for the wild-type protein in a formulation, where below the threshold, FMP are observed, and above the threshold the HTS proteins or variants directly causes sweetness perception. The exact threshold for this perception change is HTS protein and application specific.

[0121] In an embodiment, the HTS peptide variant exhibits flavor modifying properties when used in sub-sweet inclusion levels (e.g. < 9ppm) in food and beverage applications. Embodiments herein include use of HTS peptides of SEQ ID NO:3 or SEQ ID NO:141 or sequence variants thereof at sub-sweet inclusion levels in food and beverage applications. Embodiments include sequence variants having 1-6, 1-3, 1-2, 2 or 1 amino acid variations (e.g., substitutions, deletions or additions) from the HTS peptide so SEQ ID NO:3 or SEQ ID NO:141. Embodiments include sequence variants having 1 or 2 amino acid substitutions from the HTS peptide so SEQ ID NO:3 or SEQ ID NO:141. In embodiments, HTS peptides for use in sub-sweet inclusion levels applications are other than a naturally occurring Myd (mycodulcein) polypeptide of Mattirolomyces terfezioides.

[0122] In an embodiment, a composition or formulation containing HTS proteins or variants at about 1 to about 50 ppm exhibits flavor modification, but does not provide additive sweetness to the composition or formulation. In an embodiment, a composition or formulation containing HTS proteins or variants at about 1 to about 40 ppm exhibits flavor modification, but does not provide additive sweetness to the composition or formulation. In an embodiment, a composition or formulation containing HTS proteins or variants at about 1 to about 30 ppm exhibits flavor modification, but does not provide additive sweetness to the composition or formulation. In an embodiment, a composition or formulation containing HTS proteins or variants at about 1 to about 25 ppm exhibits flavor modification, but does not provide additive sweetness to the composition or formulation. In an embodiment, a composition or formulation containing HTS proteins or variants 36Atty Docket: 340391: 0640.49WO at about 1 to about 20 ppm exhibits flavor modification, but does not provide additive sweetness to the composition or formulation. In an embodiment, a composition or formulation containing HTS proteins or variants at about 1 to about 15 ppm exhibits flavor modification, but does not provide additive sweetness to the composition or formulation. In an embodiment, a composition or formulation containing HTS proteins or variants at about 5 to about 50 ppm exhibits flavor modification, but does not provide additive sweetness to the composition or formulation. In an embodiment, a composition or formulation containing HTS proteins or variants at about 10 to about 50 ppm exhibits flavor modification, but does not provide additive sweetness to the composition or formulation. In an embodiment, a composition or formulation containing HTS proteins or variants at about 15 to about 50 ppm exhibits flavor modification, but does not provide additive sweetness to the composition or formulation. In an embodiment, a composition or formulation containing HTS proteins or variants at about 20 to about 50 ppm exhibits flavor modification, but does not provide additive sweetness to the composition or formulation. In an embodiment, a composition or formulation containing HTS proteins or variants at about 25 to about 50 ppm exhibits flavor modification, but does not provide additive sweetness to the composition or formulation. In an embodiment, a composition or formulation containing HTS proteins or variants at about 30 to about 50 ppm exhibits flavor modification, but does not provide additive sweetness to the composition or formulation. In an embodiment, a composition or formulation containing HTS proteins or variants at about 35 to about 50 ppm exhibits flavor modification, but does not provide additive sweetness to the composition or formulation. In an embodiment, a composition or formulation containing HTS proteins or variants at about 40 to about 50 ppm exhibits flavor modification, but does not provide additive sweetness to the composition or formulation. In an embodiment, a composition or formulation containing HTS proteins or variants at about 45 to about 50 ppm exhibits flavor modification, but does not provide additive sweetness to the composition or formulation. In an embodiment, a composition or formulation containing HTS proteins or variants at about 10 to about 40 ppm exhibits flavor modification, but does not provide additive sweetness to the composition or formulation. In an embodiment, a composition or formulation containing HTS proteins or variants at about 20 to about 30 ppm exhibits flavor modification, but does not provide additive sweetness to the composition or formulation.

[0123] In each of the embodiments of the previous paragraph, the HTS peptides is a peptide of SEQ ID NO:3 or SEQ ID NO:141 or a sequence variant thereof. Furhter embodiments include sequence variants having 1-6, 1-3, 1-2, 2 or 1 amino acid variations (e.g., substitutions, deletions or additions) from the HTS peptide so SEQ ID NO:3 or SEQ ID NO:141. Additional embodiments, include sequence variants having amino acid substitutions of the HTS peptide of SEQ ID NO:3 or SEQ ID No: 141. Additional embodiments, include sequence variants having 1 or 2 amino acid 37Atty Docket: 340391: 0640.49WO substitutions of the HTS peptide of SEQ ID NO:3 or SEQ ID NO: 141. In embodiments, HTS peptides for use in sub-sweet inclusion levels applications are other than a naturally occurring Myd (mycodulcein) polypeptide of Mattirolomyces terfezioides.

[0124] In embodiments, a composition or formulation containing HTS proteins or variants at concentrations above 30 ppm, and particularly at concentrations above 40 ppm, provides sweetness to the composition or formulation. In embodiments, compositions or formulations can contain from 1-100 ppm of HTS proteins or variants. In embodiments, compositions or formulations can contain from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 1718, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 ppm of total HTS protein or variant. In embodiments, compositions or formulations can contain from 1-5, 5-10, 1-10, 1-30, or 1-30 ppm of total HTS protein or variant. In embodiments, compositions or formulations can contain from 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.

[0125] In embodiments, at least 80% sequence identity with respect to a polypeptide includes, without limitation, 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 with respect to a polypeptide also includes, without limitation, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity.

[0126] SWEETNESS AND THERMAL STABILITY COMPARISON OF MODIFIED PEPTIDE TO UNMODIFIED POLYPEPTIDE

[0127] The MYD family of proteins and specifically the Myd (HTS) protein variants described herein also include "analogs," or "conservative variants" and "mimetics" ("peptidomimetics") with structures and activity that substantially correspond to the exemplary sequences. Thus, the terms "conservative variant" or "analog" or "mimetic" refer to a polypeptide which has a modified amino acid sequence, such that the change(s) do not substantially alter the polypeptide's (the conservative variant's) structure and / or activity, as defined herein. These include conservatively modified variations of an amino acid sequence, i.e., amino acid substitutions, additions or deletions of those residues that are not critical for protein activity, or substitution of amino acids with residues having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids does not substantially alter structure and / or activity.

[0128] More particularly, "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants 38Atty Docket: 340391: 0640.49WO refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.

[0129] For instance, 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.

[0130] Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of ordinary skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0131] When it is desired to express a coding sequence in a heterologous host (i.e., a non-naturally occurring host), codon optimization can be employed as is known in the art to enhance expression levels in a 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. In general, 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 where a given codon is not one typically employed in the selected heterologous host replacing that codon with one that is used by the selected heterologous host more frequently. One to all codons in a given naturally-occurring coding sequence can be optimized. Dependent upon the codon frequencies in the naturally-occurring coding sequence and 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. A number of codon optimization tools are known and readily available in the art from various sources. For example, OPTIMIZER is an on-line 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 recent review of codon optimization methods is provided in H. Fu et al. “Codon optimization with deep learning to enhance protein expression” Nature Research Scientific Reports (2020) 10:17617. Additional references regarding methods of codon optimization include, among others: N. M. Marlatt et al. (2010) “Codon optimization for enhanced Escherichia coil expression of human S100A11 and S100A1 proteins” Protein Expr. Purif.73(1):58-64; A. Mellitzer et al. (2012) “Expression of lignocellulolytic enzymes in Pichia 39Atty Docket: 340391: 0640.49WO 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).

[0132] Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, one exemplary guideline to select 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 the following six groups, each containing amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (I); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); (see also, e.g., Creighton, Proteins, W.H. Freeman and Company (1984); Schultz and Schimer, Principles of Protein Structure, Springer-Vrlag (1979)). Another alternative exemplary guidelines uses the following six groups, where proline is unique: 1) Gly (G), Ala (A), Val (V), Leu (L), Ile (I); 2) Ser (S), Cys (C), Thr (T), Met (M); 3) Pro (P); 4) Phe (F), Tyr (Y), Try (W); 5) His (H), Lys (K), Arg (R); and 6) Asp (D), Glu (E), Gln (N). One of skill in the art will appreciate that the above-identified substitutions are not the only possible conservative substitutions. For example, for some purposes, one may regard all charged amino acids as conservative substitutions for each other 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 would be familiar with codon selection in a given host that is expressing the protein of interest.

[0133] Nucleotide and amino acid sequence information for MYD family members may also be used to construct models of sweet modulating polypeptides in a computer system and how they interact with sweet receptors and computer system models of same. Sweet taste receptors are composed of a heterodimer of taste 1 receptor member 2 (T1R2) and taste 1 receptor member 3 (T1R3). These models can be subsequently used to identify variants and mutations of MYD / Myd that can increase activation of sweet receptors and identify more active versions of MYD / Myd.

[0134] Various conservative mutations as well as the various less conservative mutations, as listed in Table 8, Table 9, Table 10, Table 7, Table 6 and Table 3, and substitutions are envisioned to be within the scope of the invention. Mutations of Table 9, Table 10, Table 7, Table 3 and Table 6, which exhibit sweet taste, are currently preferred mutants. For instance, it is within the level of skill 40Atty Docket: 340391: 0640.49WO in the art to perform amino acid substitutions using known protocols of recombinant gene technology including PCR, gene cloning, site-directed mutagenesis of cDNA, transfection of host cells, and in-vitro transcription. The variants are then be screened for sweet-taste modulation activity and particularly for sweet taste as well as for organoleptic properties or changes in such activities or properties. For example, variants generated are screened employing sensory tested as described herein and as understood in the art. For example, variants generated are screened for taste receptor agonist functional activity as is known in the art.

[0135] In embodiments, HTS variant polypeptides herein exhibit enhanced sweetness compared to the unmodified polypeptide SEQ ID NO:3. In some embodiments, the sweetness of respective HTS variants is enhanced at least 10% (or an enhancement ranging from 10% to 100%, or an enhancement ranging from 10 % to 200%, or at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% or more) compared to the unmodified polypeptide of SEQ ID NO:3. In other embodiments, HTS variants exhibit comparable sweetness compared to the unmodified polypeptide of SEQ ID NO:3. Comparable sweetness of a modified polypeptide, as described above, compared to the unmodified polypeptide SEQ ID NO:3 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%) as sweet as the unmodified polypeptide of SEQ ID NO:3. Sweetness is measured by any method known in the art for comparison of sweetness, and more particularly by a method for assessing sweetness as described herein.

[0136] Flavor modification activity and sweet-taste modulation activity may be detected by methods known in the art, e.g., in vitro methods, or in vivo by animal or human sensory testing. While not wishing to be bound to any particular theory, Myd (HTS) is involved in sweet taste activation e.g., is an agonist of taste 1 receptor member 2 (Tas1R2) and / or taste 1 receptor member 3 (Tas1R3). However, Myd (HTS) 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, e.g., measurement of binding to taste receptors Tas1Rs via changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties, patch clamping, voltage-sensitive dyes, whole cell currents, radioisotope efflux, inducible markers, transcriptional activation of Tas1R genes; ligand-binding assays; voltage, membrane potential and conductance changes; 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.

[0137] Sensory testing (human or animal) may also be employed to determine whether a Myd (HTS) candidate polypeptide has sweet-taste modulation activity. Sensory evaluation is a scientific 41Atty Docket: 340391: 0640.49WO discipline that analyses and measures human responses to the composition of food and drink, e.g., appearance, touch, odor, texture, temperature and taste. Measurements using people as the instruments are sometimes necessary. Selection of an appropriate method to determine sweetening can be determined by one of skill in the art, and includes, e.g., discrimination tests or difference tests, designed to measure the likelihood that two products are perceptibly different. Responses from the evaluators are tallied for correctness and statistically analyzed to see if they are more correct than would be expected due to chance alone. The food industry had the first need to develop this measurement tool as the sensory characteristics of flavor and texture were obvious attributes that cannot be measured easily by instruments. For sweetness perception, for example, samples of, for example, one or more of 5% sucrose, 6% sucrose, 7% sucrose, 8% sucrose, 9% sucrose, 10% sucrose, and a test sample can be ranked by trained panelists in order of sweet taste intensity from low sweet to high sweet. In the instant invention, it should be understood that there are any number of ways one of skill in the art could measure the organoleptic properties (e.g., sensory differences). For example, organoleptic response can be measured using trained panelist who rate their responses on an accepted scaleor rating system, such as a hedonic rating system. Alternatively or in addition, instrumental methods are available or can be readily adapted when a subject’s (panelist’s) response to an organoleptic property can be linked to a property measured by the instrumental method. See, for example, Ray, S. (2221) “Sensory Properties of Foods and Their Measurement Methods.” In: Khan, M.S., Shafiur Rahman, M. (eds) Techniques to Measure Food Safety and Quality. Springer, Cham. https: / / doi.org / 10.1007 / 978-3-030-68636-9_15.

[0138] Brix measurement (or Brix scale) is a well-known application in the food and beverage industry that determines pure sucrose content in water: 1 degree Brix (°Bx) = 1g of sucrose / 100g of solution and represents the strength of the solution as percentage by mass.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 at 0.03 mg / ml by a trained sensory scientist (0.2 mL aliquot) and found to have a sweetness equivalent to 8° Bx (approximately 8% sucrose solution) (see Examples 4, 5, 9, and 10).

[0139] THERMAL STABILITY

[0140] Thermal stability of sweet-taste modifying polypeptides can impact the potential applications of the polypeptide, for example, for food applications at elevated temperature. In embodiments, HTS variants herein exhibit comparable thermal stability compared to the unmodified polypeptide of SEQ ID NO:3. Comparable thermal stability of a modified polypeptide, as described above, compared to the unmodified polypeptide of SEQ ID NO:3 is substantially the same which herein means less than or equal to a change in thermal stability of 4.5 % (including less than or equal to 42Atty Docket: 340391: 0640.49WO 1%, less than or equal to 2%, less than or equal to 3%, less than or equal to 4%) compared to the thermal stability of the unmodified polypeptide of SEQ ID NO:3.

[0141] In some embodiments, HTS variants herein exhibit enhanced thermal stability compared to the unmodified SEQ ID NO:3. In other embodiments, enhanced thermal stability of a modified polypeptide, as described above, compared to the unmodified polypeptide of SEQ ID NO:3 is enhanced greater than 4.5% (including, 4.5 to 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 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) compared to the thermal stability of the unmodified polypeptide of SEQ ID NO:3. Thermal stability is measured by any method known in the art for assessing thermal stability, and more particularly by a method for assessing thermal stability as described herein. In embodiments, sweet-taste modifying polypeptides herein exhibit comparable sweetness and comparable thermal stability compared to that of the unmodified polypeptide of SEQ ID NO:3. In embodiments, sweet-taste modifying polypeptides herein exhibit comparable sweetness and enhanced thermal stability compared to that of the unmodified polypeptide of SEQ ID NO:3. In embodiments, sweet-taste modifying polypeptides herein exhibit enhanced sweetness and comparable thermal stability compared to that of the unmodified polypeptide of SEQ ID NO:3. In embodiments, sweet-taste modifying polypeptides herein exhibit enhanced sweetness and enhanced thermal stability compared to that of the unmodified polypeptide of SEQ ID NO:3.

[0142] In some embodiments, the HTS variant herein exhibits comparable sweetness and comparable thermal stability compared to that of the unmodified polypeptide of SEQ ID NO:3. In some embodiments, the HTS variant herein exhibits enhanced sweetness and comparable thermal stability compared to that of the unmodified polypeptide of SEQ ID NO:3. In some embodiments, the HTS variant herein exhibits comparable sweetness and enhanced thermal stability compared to that of the unmodified polypeptide of SEQ ID NO:3. In some embodiments, the HTS variant herein exhibits enhanced sweetness and enhanced thermal stability compared to that of the unmodified polypeptide of SEQ ID NO:3.

[0143] Comparable thermal stability of a modified polypeptide, as described above, compared to the unmodified polypeptide of SEQ ID NO:3 is substantially the same, which herein means less than or 43Atty Docket: 340391: 0640.49WO equal to a change in thermal stability of 4.5 % (including less than or equal to 1%, less than or equal to 2%, less than or equal to 3%, less than or equal to 4%) compared to the thermal stability of the unmodified polypeptide of SEQ ID NO:3.

[0144] Enhanced thermal stability of a modified polypeptide, as described above, compared to the unmodified polypeptide of SEQ ID NO:3 is enhanced greater than 4.5% (including, 4.5 to 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 15% enhanced, greater than 5% up to 15% enhance, 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) compared to the thermal stability of the unmodified polypeptide of SEQ ID NO:3.

[0145] In embodiments, the polypeptide variant herein exhibits sweet-taste modulation activity. Non-limiting examples of sweet-taste modulation activity include providing a sweet taste.

[0146] In another aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by at least one mutation selected from those listed in Tables 8 and optionally one deletion, such as the deleition of Met at position 1. In another aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by at least one mutation up to 24 mutations at different amino acid positions selected from those listed in Tables 8.

[0147] In another aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by at least one mutation selected from those listed in Table 8 and at least one mutation selected from Table 7 and optionally one deletion such as the deleition of Met at position 1. In another aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by at least one mutation up to 24 mutations at different amino acid positions selected from those listed in Table 8 and from one to 24 mutations selected from Table 7.

[0148] In another aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by at least one mutation up to 24 selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In another aspect, the 44Atty Docket: 340391: 0640.49WO invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by at least one mutation up to 24 mutations at different amino acid positions selected from those listed in Table 8, Table 9 or Table 10.

[0149] In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by two mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by three mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by four mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by five mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet- taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by six mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1.

[0150] In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by seven mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by eight mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by nine mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one 45Atty Docket: 340391: 0640.49WO deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by ten mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet- taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by eleven mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by twelve mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1.

[0151] In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by thirteen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by fourteen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by fifteen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by sixteen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet- taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by seventeen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by eighteen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. 46Atty Docket: 340391: 0640.49WO

[0152] In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by nineteen mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by twenty mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by twenty-one mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by twenty-two mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by twenty-three mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In a related aspect, the invention provides a polypeptide having sweet-taste modulation activity (e.g., isolated polypeptide) comprising the amino acid sequence of SEQ ID NO:3 which is modified by twenty-four mutations at different positions selected from those listed in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. The invention also provides polynucleotides encoding the foregoing mutant polypeptides of SEQ ID NO:3 or SEQ ID NO:141. The invention further provides the foregoing mutant polypeptides which further comprise a protein tag and more specifically a histidine tag. The invention also provides polynucleotides encoding the foregoing mutant polypeptides of SEQ ID NO:3 or SEQ ID NO:141 which further comprise a protein tag and more specifically a histidine tag. In embodiments, HTS variants do not include any one or more of the mutations listed in Table 3, Table 6 or Table 7.

[0153] In some embodiments, the polypeptide having sweet-taste modulation activity also has enhanced thermal stability compared to the polypeptide of SEQ ID NO:3. In an embodiment, the polypeptide having sweet-taste modulation activity and enhanced thermal stability comprises a modified SEQ ID NO:3, wherein the polypeptide has at least 1 and up to 6 amino acid modifications as shown in Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In an embodiment, the polypeptide having sweet-taste modulation 47Atty Docket: 340391: 0640.49WO activity and enhanced thermal stability comprises a modified SEQ ID NO:3, wherein the polypeptide has at least 1 and up to 6 amino acid modifications as shown in Table 7 and optionally one deletion such as the deletion of Met at position 1. In an embodiment, the polypeptide having sweet-taste modulation activity and enhanced thermal stability comprises a modified SEQ ID NO:3, wherein the polypeptide has at least 1 and up to 6 amino acid modifications as shown in Table 8, Table 9 or Table 10 and further has 1-6 amino acid modifications as shown in Table 7 and optionally one deletion such as the deletion of Met at position 1. In further embodiments of the preceding embodiments, the polypeptide having sweet-taste modulation activity (including sweet taste) and enhanced thermal stability compared to the polypeptide of SEQ ID NO:141.

[0154] In another embodiment, the polypeptide having sweet-taste modulation activity (and particularly sweet taste) and enhanced thermal stability 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 modified polypeptide of SEQ ID NO:3 with at least 1 and up to 6 amino acid modifications as shown in Table 8, Table 9 or Table 10 and 1-6 amino acid modifications as shown in Table 7 and optionally one deletion such as the deletion of Met at position 1. In further embodiments of the preceding embodiments, the polypeptide has sweet-taste modulation activity (including sweet taste) and enhanced thermal stability compared to the polypeptide of SEQ ID NO:141.

[0155] In another embodiment, the polypeptide having sweet-taste modulation activity (and particularly sweet taste) and enhanced thermal stability compared to the polypeptide of SEQ ID NO:3 has both mutations I49C and S33C (compared to SEQ ID NO:3) and optionally 1, 2, 3, 4, 5 or 6 other mutations as shown in Table 8, Table 9, Table 10, Table 3, Table 6 or Table 7 and in particular optionally one deletion such as the deletion of Met at position 1. In a related embodiment, the polypeptide having sweet-taste modulation activity (and particularly sweet taste) and enhanced thermal stability compared to the polypeptide of SEQ ID NO:3 has both mutations I49C and S33C (compared to SEQ ID NO:3) and has at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to a modified polypeptide of SEQ ID NO:3. In further embodiments of the preceding embodiments, the polypeptide has sweet-taste modulation activity (including sweet taste) and enhanced thermal stability compared to the polypeptide of SEQ ID NO:141. 48Atty Docket: 340391: 0640.49WO

[0156] In another embodiment, the polypeptide having sweet-taste modulation activity (and particularly sweet taste) and enhanced thermal stability compared to the polypeptide of SEQ ID NO:3 has both mutations Y23C and Y61C (compared to SEQ ID NO:3) and optionally 1, 2, 3, 4, 5 or 6 other mutations as shown in Table 8, Table 9, Table 10, Table 3, Table 6 or Table 7 and optionally one deletion such as the deletion of Met at position 1. In a related embodiment, the polypeptide having sweet-taste modulation activity (and particularly sweet taste) and enhanced thermal stability compared to the polypeptide of SEQ ID NO:3 has both mutations I49C and S33C (compared to SEQ ID NO:3) and has at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to a modified polypeptide of SEQ ID NO:3. In further embodiments of the preceding embodiments, the polypeptide has sweet-taste modulation activity (including sweet taste) and enhanced thermal stability compared to the polypeptide of SEQ ID NO:141.

[0157] In an embodiment, the polypeptide having sweet-taste modulation activity and enhanced thermal stability comprises a modified SEQ ID NO:3 wherein the polypeptide has two modifications as shown in Table 7 and / or Table 8, Table 9 or Table 10 and optionally one deletion such as the deletion of Met at position 1. In an embodiment, the polypeptide having sweet-taste modulation activity and enhanced thermal stability comprises a modified SEQ ID NO:3 wherein the polypeptide has two modifications as shown in Table 7 and / or Table 8, Table 9 or Table 10 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 an embodiment, the polypeptide having sweet- taste modulation activity and enhanced thermal stability comprises a modified SEQ ID NO:3, wherein the polypeptide has at least two modifications as shown in Table 7 and / or Table 8, Table 9 or Table 10 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 preceding embodiments, the polypeptide has sweet-taste modulation activity (including sweet taste) and enhanced thermal stability compared to the polypeptide of SEQ ID NO:141.

[0158] In an embodiment, the polypeptide having sweet-taste modulation activity (and particularly sweet taste) and enhanced thermal stability 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, the polypeptide having sweet-taste modulation activity (and particularly sweet taste) and enhanced thermal stability compared to the polypeptide of SEQ ID NO:3 comprises the polypeptide of SEQ ID NO:142 or SEQ ID NO:145 49Atty Docket: 340391: 0640.49WO and in addition the methionine at position 1 is absent SEQ ID NO:146 and SEQ ID NO:147. In an embodiment, the polypeptide having sweet-taste modulation activity (and particularly sweet taste) and enhanced thermal stability compared to the polypeptide of SEQ ID NO:3 is the polypeptide of SEQ ID NO: 143. In an embodiment, the polypeptide having sweet-taste modulation activity (and particularly sweet taste) and enhanced thermal stability compared to the polypeptide of SEQ ID NO:3 is the polypeptide of SEQ ID NO:145. In an embodiment, the polypeptide having sweet-taste modulation activity (and particularly sweet taste) and enhanced thermal stability compared to the polypeptide of SEQ ID NO: 3 is the polypeptide of SEQ ID NO:146. In an embodiment, the polypeptide having sweet-taste modulation activity (and particularly sweet taste) and enhanced thermal stability compared to the polypeptide of SEQ ID NO: 3 is the polypeptide of SEQ ID NO: 147.

[0159] In some embodiments, polypeptides of the invention exhibit stability to low pH (<pH 7) that is enhanced compared to the polypeptide of SEQ ID NO:3. In some embodiments, polypeptides of the invention exhibit stability to high pH (> pH 7) that is enhanced compared to the polypeptide of SEQ ID NO:3. In an embodiment, polypeptides of the invention exhibit greater stability at pH of about 2 as compared to the polypeptide of SEQ ID NO:3. In another embodiment, polypeptides of the invention exhibit greater stability at pH of about 10 as compared to the polypeptide of SEQ ID NO:3. In an embodiment, polypeptides of the invention having methionine present at position 1 exhibit greater stability at pH of about 2 as compared to the polypeptide of SEQ ID NO:3. In another embodiment, polypeptides of the invention having methionine present at position 1 exhibit greater stability at pH of about 10 as compared to the polypeptide of SEQ ID NO:3.

[0160] The term "expression vector" or “expression cassette” refers to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cell. The term includes linear or circular expression systems. The term includes expression systems that remain episomal or integrate into the host cell genome. The expression systems can have the ability to self-replicate or not, i.e., drive only transient expression in a cell. The term includes recombinant expression cassettes which contain only the minimum elements needed for transcription of the recombinant nucleic acid.

[0161] A recent review of methods for expression of recombinant proteins is found in Tripathi & Shrivastava (2019) “Recent Developments in Bioprocessing of Recombinant Proteins; Expression Hosts and Process Development,” Frontiers in Bioeng. Biotech.7:420, doi:10.3389 / fbio.2019.00420. This reference is incorporated by reference herein in its entirety for details of host expression systems and methods for expression of recombinant proteins. 50Atty Docket: 340391: 0640.49WO

[0162] By "host cell" is meant a cell that contains an expression vector and supports the replication or expression of the expression vector. In an embodiment, the host cell is a prokaryotic cell. In an embodiment, the host cell is a eukaryotic cell. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa, HEK- 293, and the like, e.g., cultured cells, explants, and cells in vivo. In embodiments, the host cell is not a cell of the truffle Mattirolomyces terfezioides.

[0163] In an 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, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, Pseudomonas putida, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizopus oryzae, Yarrowia lipolytica, Candida albicans, Issatchenkia orientalis, Scheffersomyces stipitis, Yarrowia lipolytica, Ogataea polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, and Schwanniomyces occidentalis.

[0164] In an embodiment, the host cell is selected from the group consisting of Qualified Presumption of Safety (QPS) recommended biological agents. A list of such hosts is available on the website: efsa.europa.eu / efsajournal EFSA Journal 2021;19(7):6689. In an embodiment, the host organism is selected from the group consisting of Bacillus megaterium, Trichoderma reesei, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium Longum, Carnobacterium divergens, Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus alimentarius, Lactobacillus aviaries, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus collinoides, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus 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, Lactobacillus salivarius, Lactobacillus Sanfranciscensis, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc 51Atty Docket: 340391: 0640.49WO mesenteroides, Leuconostoc Pseudomesenteroides, Microbacterium imperial, Oenococcus oeni, Pasteuria nishizawae, Pediococcus acidilactici, Pediococcus parvulus, Pediococcus pentosaceus, Propionibacterium acidipropionic, Propionibacterium freudenreichii, Streptococcus thermophilus, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus flexus, Bacillus fusiformis, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus mojavensis, Bacillus paralicheniformis, Bacillus pumilus, Bacillus smithii, Bacillus subtilis, Bacillus vallismortis, Bacillus velezensis, Geobacillus stearothermophilus, Paenibacillus illinoisensis, Parageobacillus thermoglucosidasius, Gluconobacter oxydans, Komagataeibacter sucrofermentans, Xanthomonas campestris, Candida cylindracea, Cyberlindnera jadinii, Debaryomyces hansenii, Hanseniaspora uvarum, Kluyveromyces lactis, Kluyveromyces marxianus, Komagataella pastoris, Komagataella phaffi, Lindnera jadinii, Ogataea angusta, Saccharomyces bayanus, Schizosaccharomyces pombe, Wickerhamomyces anomalus, Xanthophyllomyces dendrorhous, or Zygosaccharomyces rouxii.

[0165] In another aspect, the host cell is selected from the group consisting of gram-positive non- spore forming bacteria, gram-positive spore forming bacteria, gram negative bacteria, yeast, and protists / algae. In other aspects, the host cell is selected from plant cells. In other aspects, the host cell is selected from insect cells. With respect to insect cells, Baculo virus insect expression systems are useful. Insect cells useful as hosts for production of recombinant protein include, among others, Spodoptera frugiperda cells (e.g., Sf9, Sf21), Drosophila cells (e.g., S2), Trichoplusia ni cells (e.g., Tn-368, High-Five™ (Thermo Fisher Scientific, Waltham, MA)). Various host cells are known in the art and available from commercial sources, among others.

[0166] Non-limiting examples of gram-positive non-spore forming bacteria include Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Carnobacterium divergens, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus alimentarius, Lactobacillus aviaries, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus collinoides, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus 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, 52Atty Docket: 340391: 0640.49WO Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactococcus lactis, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Microbacterium imperial, Oenococcus oeni, Pasteuria nishizawae, Pediococcus acidilactic, Pediococcus parvulus, Pediococcus pentosaceus, Propionibacterium acidipropioni, Propionibacterium freudenreichii, and Streptococcus thermophiles.

[0167] Non-limiting examples of gram-positive spore forming bacteria include Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus flexus, Bacillus fusiformis, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus mojavensis, Bacillus pumilus, Bacillus smithii, Bacillus subtilis, Bacillus vallismortis, Bacillus velezensis, Geobacillus stearothermophilus, Paenibacillus illinoisensis, and Parageobacillus thermoglucosidasius. Non-limiting examples of gram-negative bacteria include Cupriavidus necator, Gluconobacter oxydans, Komagataeibacter sucrofermentans, and Xanthomonas campestris.

[0168] Non-limiting examples of yeast include Candida cylindracea, Debaryomyces hansenii, Hanseniaspora uvarum, Kluyveromyces lactis, Kluyveromyces marxianus, Komagataella pastoris, Komagataella phaffi, Lindnera jadinii, Ogataea angusta, Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces pastorianus, Schizosaccharomyces pombe, Wickerhamomyces anomalus, Xanthophyllomyces dendrorhous, Yarrowia lipolytica, and Zygosaccharomyces rouxii.

[0169] Non-limiting examples of protists / algae include Aurantiochytrium limacinum, Euglena gracilis, and Tetraselmis chuii.

[0170] In certain embodiments, recombinant HTS is produced by a transgenic mammal, i.e., in milk.

[0171] The expression of HTS (or a variant thereof) may be stable or transient. In a stable expression system, the exogenous DNA is integrated into the chromosomes, or as an episome (a separate piece of nuclear DNA) and is passed on to future generations of the host cell.

[0172] The terms "mimetic" and "peptidomimetic" refer to a synthetic chemical compound that has substantially the same structural and / or functional characteristics of the polypeptides, e.g., translocation domains, ligand-binding domains, or chimeric receptors of the invention. The mimetic can be either entirely composed of synthetic, non-natural analogs of amino acids, or may be a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and / or activity. 53Atty Docket: 340391: 0640.49WO

[0173] As with polypeptides of the invention which are conservative variants, routine experimentation will determine whether a mimetic is within the scope of the invention, i.e., that its structure and / or function is not substantially altered. Polypeptide mimetic compositions can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like. A polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC). Linking groups that can be an alternative to the traditional amide bond ("peptide bond") linkages include, e.g., ketomethylene (e.g., --C(O)--CH2-- for --C(O)--NH--), 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)). A polypeptide can also be characterized as a mimetic 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 suite of tools available on the web to predict and analyze protein structure, function and mutations.

[0174] Examples of conservatively modified variations of Myd1 protein structure may be derived using homology-modelling algorithms: SWISS-MODEL, PHYRE2.0, and Jpred to identify a sequence-based consensus loop regions, as known in the art, see, e.g., Pechmann, S. & Frydman, J. Interplay between Chaperones and Protein Disorder Promotes the Evolution of Protein Networks. PLoS Computational Biology 10, e1003674 (2014).

[0175] Specific regions of the MYD / Myd nucleotide and amino acid sequences may be used to identify polymorphic variants, interspecies homologs, and alleles of MYD family members. This identification can be made in vitro, e.g., under stringent hybridization conditions or PCR (e.g., using primers encoding the Myd sequences identified herein), or by using the sequence information in a computer system for comparison with other nucleotide sequences. Different alleles of MYD genes within a single species population will also be useful in determining whether differences in allelic sequences correlate to differences in taste perception between members of the population. 54Atty Docket: 340391: 0640.49WO Classical PCR-type amplification and cloning techniques are useful for isolating orthologs, for example, where degenerate primers are sufficient for detecting related genes across species.

[0176] For instance, primers designed using the sequences disclosed herein can be used to amplify and clone MYD -related genes from different fungal genomes. In contrast, genes within a single species that are related to MYD are best identified using sequence pattern recognition software to look for related sequences. Typically, identification of polymorphic variants and alleles of MYD family members can be made by comparing an amino acid sequence of about 25 amino acids or more, e.g., 50-100 amino acids. Amino acid identity of approximately at least 35 to 50%, and optionally 60%, 70%, 75%, 80%, 85%, 90%, 95-99%, or above 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 bind specifically to Myd polypeptides or a conserved region thereof can also be used to identify alleles, interspecies homologs, and polymorphic variants.

[0177] In an embodiment, hybrid protein-coding sequences comprising nucleic acids encoding Myd variant fusion proteins may be constructed. These nucleic acid sequences can be operably linked to transcriptional or translational control elements, e.g., transcription and translation initiation sequences, promoters and enhancers, transcription and translation terminators, polyadenylation sequences, and other sequences useful for transcribing DNA into RNA. Fusion proteins may include C-terminal or N-terminal translocation sequences. Further, fusion proteins can comprise additional elements, e.g., for protein detection, purification, or other applications. Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts, histidine-tryptophan modules, or other domains that allow purification on immobilized metals; maltose binding protein; protein A domains that allow purification on immobilized immunoglobulin; or the domain utilized in the FLAGS extension / affinity purification system (Immunex Corp, Seattle Wash.).

[0178] In an embodiment, the fusion protein comprises a peptide or protein tag (e.g., for protein purification or detection). Protein / peptide tags are peptide sequences genetically grafted into a recombinant (e.g., fusion) protein. Peptide / protein tags are known in the art, such as those described in Johnson, “Protein / Peptide Tags,” DOI / / dx.doi.org / 10.13070 / mm.en.2.116 including but 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. 55Atty Docket: 340391: 0640.49WO

[0179] Affinity tags are a type of protein tag that is appended to proteins so that they can be purified from their crude biological source using an affinity technique. Affinity tags are known in the art, such as those described in Kimple et al. Curr Protoc Protein Sci.; 73: Unit–9.9, doi:10.1002 / 0471140864.ps0909s73. These include, among others, polyhistidine, GST, MBP, Calmodulin-binding peptide, intein-chitin binding domain, Streptavidin / Biotin-based tags, and His- Patch ThioFusion (thioredoxin). Affinity tags include small (e.g., 20 or less amino acid residues) or large affinity tags. Examples of small affinity tags include His, FLAG, Strep II, and S-peptide, and examples of large affinity tags include MBP, GST, cellulose binding domains, calmodulin binding peptide, and His-patch thioredoxin.

[0180] Protein / peptide tags include epitope tags and reporter tags. Reporter tags serve as reporters of protein expression and protein-protein interaction. Reporter tags include, but are not limited to, enzymes such as β-galactosidase (β-gal), alkaline phosphatase (AP), chloramphenicol acetyl transferase (CAT), and horseradish peroxidase (HRP).

[0181] Epitope tags include FLAG, hemagglutinin (HA), c-myc, T7, and Glu-Glu, which are used for the detection of fusion proteins in vitro and in cell culture. Their short, linear recognition motifs rarely affect the properties of the protein of interest and are usually very specific for their respective primary antibodies. If the anti-myc antibody is used, specificity can be increased by using an enzyme-linked secondary antibody to detect a conjugated anti-myc primary antibody instead of using an HRP- or AP-anti-myc conjugate alone.

[0182] Protein / peptide tags also include solubilization tags which are used to assist in the proper folding of proteins and keep them from aggregating in inclusion bodies. In embodiments, solubilization tags are employed for proteins expressed in E. coli. Solubilization tags include thioredoxin and poly(NANP), among others. Some affinity tags can also assist in solubilization, such as MBP and GST.

[0183] Protein / peptide tags can be at either end of the target protein. Some tags, such as FLAG, are often used in tandem to increase their desired features, or in combination with another tag, such as in the construct of His-Myc and His-V5.

[0184] Tandem affinity purification (TAP) is a dual-affinity purification method based on the fusion of two affinity tags to a protein of interest, which allows purification of a tagged protein and isolation of protein complexes interacting with the protein of interest. The use of TAP is encompassed within the present invention. 56Atty Docket: 340391: 0640.49WO

[0185] In an embodiment, the fusion protein comprises a histidine tag that comprises 2-10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) histidine residues. For example, the histidine tag can comprise 6 histidine residues.

[0186] The inclusion of a cleavable linker sequences such as Factor Xa (see, e.g., Ottavi, Biochimie 80:289-293 (1998)), subtilisin protease recognition motif (see, e.g., Polyak, Protein Eng.10:615- 619 (1997)); enterokinase (Invitrogen, San Diego, Calif.), and the like, between the translocation domain (for efficient plasma membrane expression) and the rest of the newly translated polypeptide may be useful to facilitate purification. For example, one construct can include a polypeptide encoding 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 C- terminal translocation domain. The histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the desired protein(s) from the remainder of the fusion protein. Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described in the scientific and patent literature, see, e.g., Kroll, DNA Cell. Biol.12:441-53 (1993).

[0187] The fusion protein can contain one or more linkers (e.g., flexible linkers, rigid linkers, and in vivo cleavable linkers). Besides the basic role in linking the functional domains together (as in flexible and rigid linkers) or releasing free functional domain in vivo (as in in vivo cleavable linkers), linkers offer many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles. Linkers are known in the art (see, e.g., Chen et al., Adv Drug Deliv Rev.65(10): 1357– 1369 (2013)).

[0188] Flexible linkers are used when the joined domains require a certain degree of movement or interaction. They are generally composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. The small size of these amino acids provides flexibility and allows for mobility of the connecting functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules and therefore reduces the unfavorable interaction between the linker and the protein moieties.

[0189] The most commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues (“GS” linker). An example of the most widely used flexible linker has the sequence of (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 the functional domains, or to maintain necessary inter-domain interactions. Besides the GS linkers, many other flexible linkers have been designed for recombinant fusion proteins. These flexible linkers are also rich in small or 57Atty Docket: 340391: 0640.49WO polar amino acids such as Gly and Ser but can contain additional amino acids such as Thr and Ala to maintain flexibility, as well as polar amino acids such as Lys and Glu to improve solubility.

[0190] Rigid linkers keep a fixed distance between the domains and maintain their independent functions. Examples of rigid linkers include alpha helix-forming linkers with the sequence of (EAAAK)n(SEQ ID NO:8) and linkers with a Pro-rich sequence, (XP)n, with X designating any amino acid, preferably Ala, Lys, or Glu.

[0191] The polypeptide of the present invention also can contain a signal peptide (i.e., a signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide), which is a short peptide present at the N-terminus or occasionally C- terminus of most newly synthesized proteins that are destined toward the secretory pathway. These proteins include those that reside either inside certain organelles (the endoplasmic reticulum, Golgi or endosomes), secreted from the cell, or inserted into most cellular membranes. Exemplary signal peptides are known in the art and a person of ordinary skill in the art would recognize how to select a particular signal peptide for use in the invention.

[0192] PROTEIN DERIVATIZATION

[0193] HTS proteins / polypeptide and sequence variants thereof can be further derivatized or modified other than by substitution of one or more amino acids. HTS wild-type (native) protein / polypeptide and HTS protein / polypeptide variants of this disclosure can be derivatized at the N-terminus, the C-terminus or at an amino acid side chain without loss of taste modulation activity. Derivatization, in general includes, acylation, esterification, glycosylation, oxidation, methylation, reductive alkylation, phosphorylation (as phospho-amino acids), sulfurylation, sulfonylation, or oxidation or reduction of side chain heteroatoms. Derivatization can be accomplished by chemical methods using reagents and methods that are well-known in the art. Alternatively, derivatization can be accomplished by biological methods, such as treatment with one or more enzymes, or by post-translational methods (post-translational modification). One of ordinary skill in the art can selected one or more enzymes to effect desired derivatization of HTS proteins / polypeptides or variants thereof. The HTS protein or polypeptide, preferably isolated, is treated with the selected enzyme to achieve the desired derivatization. Post-translational modifications (PTMs) are covalent modification to a protein / polypeptide by proteolytic cleavage (e.g., removal of N-terminal methionine) and / or addition of a modifying group, such as acetyl (more broadly acyl), a phosphoryl, a glycosyl and / or a methyl, to one or more amino acids. Other chemical modification of an amino acid of a protein / polypeptide (e.g., oxidation of the sulfur of a methionine group) can be achieved by both chemical and biological means to one skilled in the art. Post-translational modification can occur during native expression of a protein / polypeptide or post- 58Atty Docket: 340391: 0640.49WO translational modification can be controlled during protein expression in a non-native host using recombinant methods. Such recombinant methods rely on the use of specially constructed expression vectors including, for example, coding sequences for expression of one or more enzyme to actuate a selected post-translational modification. One of ordinary skill in the art, is aware of chemical or recombinant techniques to derivatize proteins at one or more position on a given protein. In an embodiment, preferred protein / polypepide derivatizations are those that do not significantly affect the sweet taste-modulation activity of the HTS protein / polypeptide variants of this disclosure. In an embodiment, preferred protein / polypeptide derivatizations are those that do not significantly detrimentally affect the sweet taste of the HTS protein / polypeptide variants of this disclosure (e.g., do not significantly decrease the sweet taste of the HTS variants herein). In embodiments, derivatization of the HTS variant can enhance flavor modification, sweet taste modification or sweet taste activity of HTS variants.

[0194] Derivatization of an HTS protein / polypeptide or variant, by any known method, can occur at the N-terminus, the C-terminus, or at one or more amino acid side group (e.g., a side-chain sulfur, a side chain amine or a side-chain carboxylic acid). In embodiments, a HTS protein / polypeptide or HTS variant of this disclosure can have 1, 2, 3, 4, 5 or 6 different derivations. Preferably, the HTS protein or HTS variant has a single derivatization. For example, the N-terminus of the HTS protein / polypeptide or variant can be derivatized by acylation and more specifically by acetylation. In more specific embodiments, the N-terminus of the protein / polypeptide or variant is a methionine that is N-acetylated. In embodiments, the derivatization is of the N-terminal amino acid or of an amine side chain of an amino acid of the HTS protein / polypeptide. The N-terminal amine acid or an amine side chain (e.g., a lysine side-chain) can be derivatized by acylation; glycosylation; methylation; reductive amination to form –NH-CH2-R, where R is an alkyl group (e.g., an alkyl group having 1-19 carbon atoms); phosphorylation of amino acid side chains such as serine or threonine (-OH) with kinases to form a phospho-polypeptides; reaction of sulfur in amino acid side chains with monooxygenases to produce sulfoxide derivatives. The N-terminal amine or an amine side-chain can be derivatized by chemical methods or by biological processes after the translation of the polypeptide, resulting in a post-translational modification of the nitrogen. Reactive side chains of other amino acids may be derivatized by chemical methods or biological processes after the translation of the polypeptide, resulting in post-translational modification of those side chains.

[0195] In specific embodiments, the derivatization is an addition, subtraction, or substitution of one or more of the following chemical moieties to an amine nitrogen: acyl (R-CO-, where R is a straight-chain or branched alkyl group having 1-20 carbon atoms); acetyl (CH3CO), formyl (HCO), 59Atty Docket: 340391: 0640.49WO glycosyl (e.g., C6H11O6-), hydroxyl (HO-), methyl (CH3-) or other alkyl group, a phosphatidyl (PO4), a phosphonyl (PO2), a sulfhydryl (SH-), or a sulfonyl (HSO2-).

[0196] In specific embodiments, wherein the first amino acid of the HTS protein / polypeptide is a methionine, the delta-position sulfur of the first amino acid is modified, by chemical methods, by enzymatic methods (in vitro or in vivo) or by biological processes after the translation of the polypeptide, resulting in a post-translational modification of the sulfur. In embodiments, the modification of the delta-position sulfur of the first amino acid is an addition, subtraction, or substitution of any of the following chemical moieties to the delta sulfur: acyl (R-CO-, where R is a straight-chain or branched alkyl group having 1-20 carbon atoms); acetyl (CH3CO), formyl (HCO), glycosyl (e.g., C6H11O6-), hydroxyl (HO-), methyl (CH3-) or other alkyl group, a phosphatidyl (PO4), a phosphonyl (PO2), a sulfhydryl (SH-),or a sulfonyl (HSO2-). In embodiments, the modification of the delta-position sulfur of the first amino acid is the oxidation of the sulfur, resulting in a sulfoxide derivative.

[0197] As used herein, "at least 80% identity" with reference to an amino acid sequence or a nucleotide sequence refers to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity.

[0198] As used herein, examples of "an amino acid sequence modified by deletion, insertion, substitution, or addition of one or more amino acids" include an amino acid sequence modified by deletion, insertion, substitution, or addition of 1 or more to 30 or less, preferably 20 or less, more preferably 10 or less, and further preferably 5 or less amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any ranges thereof). As used herein, examples of "a nucleotide sequence modified by deletion, insertion, substitution, or addition of one or more nucleotides" include a nucleotide sequence modified by deletion, insertion, substitution, or addition of 1 or more to 90 or less, preferably 60 or less, more preferably 30 or less, further preferably 15 or less, and further more preferably 10 or less nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or any ranges thereof).

[0199] For example, in sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, as described below for the BLASTN and BLASTP programs, or alternative parameters 60Atty Docket: 340391: 0640.49WO can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0200] A "comparison window," as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math.2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol. Biol.48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds.1995 supplement)).

[0201] A preferred example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul at al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J Mol. Biol.215:403-410 (1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word 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, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The 61Atty Docket: 340391: 0640.49WO BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5,N=-4, and a comparison of both strands.

[0202] Another example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a so-called "tree" or "dendogram" showing the clustering relationships used to create the alignment (see, e.g., FIG.1). PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J Mol. Evol.35:351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151- 153 (1989). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP 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) encoded by the genes were derived by conceptual translation of the corresponding open reading frames.

[0203] The polynucleotides encoding the polypeptides of the present invention can be synthesized chemically or by genetic engineering based on the amino acid sequence of a Myd. For example, the polynucleotide can be synthesized chemically based on the amino acid sequence of the polypeptides of the present invention or preprotein thereof. A contract synthesis service of nucleic acid (provided from, for example, Medical & Biological Laboratories Co., Ltd., Genscript etc.) can be used for the chemical synthesis of the polynucleotide. Further, the synthesized polynucleotide can be amplified by PCR and cloning etc.

[0204] The polypeptides of the present invention can be produced, for example, by expressing a gene encoding a Myd polypeptide variant of the present invention. Preferably, a Myd polypeptide 62Atty Docket: 340391: 0640.49WO variant of the present invention can be produced from a transformant in which the polynucleotide encoding a Myd polypeptide variant of the present invention is introduced. For example, a Myd polypeptide variant of the present invention is produced from a polynucleotide encoding a Myd polypeptide variant of the present invention introduced in a transformant after a polynucleotide encoding a Myd polypeptide variant of the present invention or a vector comprising it is introduced into a host to obtain a transformant and the transformant is cultured in an appropriate medium. The proteins of the present invention can be obtained by isolating or purifying the produced Myd polypeptide variant from the culture.

[0205] Therefore, the present invention further provides a polynucleotide encoding a Myd polypeptide variant of the present invention and a vector comprising it. The present invention further provides a method of manufacturing a transformant, comprising introducing a polynucleotide encoding a Myd polypeptide variant of the present invention or a vector comprising it into a host. The present invention further provides a transformant comprising a polynucleotide encoding a Myd polypeptide variant of the present invention or a vector comprising it introduced from the outside of a cell. The present invention further provides a method of manufacturing a Myd polypeptide variant of the present invention, comprising culturing the transformant.

[0206] The present invention also includes the polynucleotides of the invention, operably linked to a heterologous regulatory element. The invention may include an expression cassette or vector comprising the polynucleotides of the present invention, and a host cell transformed with a vector of the invention.

[0207] Alternatively, the polynucleotide encoding a Myd polypeptide variant of the present invention can be produced by introducing one or more mutation into the polynucleotide synthesized according to the procedure with known mutagenesis methods such as the ultraviolet irradiation and site-directed mutagenesis. For example, the polynucleotide encoding the polypeptides of the present invention can be obtained by introducing one or more mutation into the polynucleotide of SEQ ID NO:1 or SEQ ID NO:2 with a known method, expressing the obtained polynucleotide, investigating the expressed protein’s sweet-modification activity, and selecting a polynucleotide encoding the protein having desired sweet modification activity.

[0208] Site-directed mutagenesis of a polynucleotide can be performed with any methods such as, for example, inverse PCR and annealing (Muramatsu et al. edit., "Revised 4th edition New genetic engineering handbook", YODOSHA, p.82-88). A variety of commercially available kits for site- directed mutagenesis such as QuickChange II Site-Directed Mutagenesis Kit from Stratagene and QuickChange Multi Site-Directed Mutagenesis Kit can be used as needed. 63Atty Docket: 340391: 0640.49WO

[0209] Examples of the type of a vector comprising the polynucleotide encoding the polypeptides of the present invention include, without limitation, a vector usually used for gene cloning, for example, a plasmid, a cosmid, a phage, a virus, a YAC and a BAC. Examples of vectors include plasmids (e.g., DNA plasmids), yeast (e.g., Saccharomyces), and viral vectors, such as poxvirus, retrovirus, adenovirus, adeno-associated virus, herpes virus, polio virus, alphavirus, baculovirus, Sindbis virus, plant viruses (e.g., Alphaflexiviridae or Potyviridae), and insect viruses (e.g., Baculoviridae).

[0210] Among these, a plasmid vector is preferred and for example a commercially available plasmid vector for protein expression, for example, pUC19, pUC118, pUC119, pBR322 etc. (all of which are from TAKARA BIO INC.) can be used.

[0211] The vector can comprise a DNA region comprising a replication initiation region or a replication origin of DNA. Alternatively, a regulatory sequence such as a promoter region for initiating transcription of the gene, a terminator region or a secretory signal region for secreting an expressed protein to the outside of a cell can be operably linked to the upstream of the polynucleotide encoding the proteins of the present invention (i.e. the MYD gene of the present invention) in the vector. As used herein, a gene and a regulatory sequence being "operably liked" refers to a condition in which the gene and the regulatory region are positioned so that the gene can be expressed under the regulation by the regulatory region.

[0212] The type of the regulatory sequence of a promoter region, a terminator, and a secretory signal region etc. is not specifically limited, and a promoter and a secretory signal sequence usually used can be selected to use as appropriate depending on the host into which the sequence is introduced. For example, preferred examples of the regulatory sequence which can be incorporated to the vector of the present invention include the cbh1 promoter sequence derived from Trichoderma reesei (Curr, Genet, 1995, 28 (1): 71-79).

[0213] Alternatively, a marker gene to select a host into which the vector is appropriately introduced (for example, a resistance gene to an agent such as ampicillin, neomycin, kanamycin and chloramphenicol) can be further incorporated into the vector of the present invention. Alternatively, a gene encoding a synthase of a required nutrient can be incorporated into the vector as a marker gene, when an auxotrophic strain is used as a host. Alternatively, a related gene of the metabolism can be incorporated into the vector as a marker gene, when a selective medium requiring specific metabolism for growth is used. Examples of such a metabolism related gene include an acetamidase gene for using acetamide as a nitrogen source.

[0214] Ligation between the polynucleotide encoding a Myd polypeptide variant of the present invention and a regulatory sequence and a marker gene can be performed by a known method in the 64Atty Docket: 340391: 0640.49WO art such as SOE (splicing by overlap extension) -PCR (Gene, 1989, 77: 61-68). The procedure for introducing a ligated fragment into a vector is known in the art.

[0215] Examples of a host of a transformant into which the vector is introduced include a microorganism such as a bacterium or filamentous fungus. Examples of the bacterium include Escherichia coli and a bacterium belonging to Staphylococcus, Enterococcus, Listeria and Bacillus, of which Escherichia coli and Bacillus bacteria (for example, Bacillus subtilis or a mutant thereof) are preferred. Examples of the Bacillus subtilis mutant can include protease 9 double deficient strain KA8AX described in J. Biosci. Bioeng., 2007, 104 (2): 135-143 and a DBPA strain, a mutant from protease 8 double deficient strain described in Biotechnol. Lett., 2011, 33 (9): 1847-1852, of which protein folding efficiency is improved. Examples of the filamentous fungus include Trichoderma, Aspergillus and Rhizopus. Also, for example, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowia lipolytica, Schizosaccharomyces pombe, Kluyveromyces lactis are appropriate expression hosts. In embodiments, the host cell is a cell of a fungus other than Mattirolomyces terfezioides. In embodiments, the HTS protein variant can be expressed in mycelium.

[0216] In embodiments, the HTS protein variant can be expressed in a plant cell, a plant organ, a leaf, a root or in a whole plant.

[0217] 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 can be, for example, a mammalian cell (e.g., BHK, VERO, HT1080, 293, RD, COS-7, or CHO cells), an insect cell (e.g., Trichoplusia ni (Tn5) or Sf9), a bacterial cell, a plant cell, or a yeast cell.

[0218] In a particular embodiment, the HTS (or a variant thereof) is produced in a yeast expression system (i.e., yeast-derived HTS or variant thereof), for example, in the genera 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).

[0219] In another particular embodiment, the HTS (or a variant thereof) is produced in a bacterial expression system (i.e., bacteria-derived HTS or variant thereof), for example, in Escherichia coli or Bacillus subtilis. In an embodiment, the HTS (or a variant thereof) is not produced in E. coli. 65Atty Docket: 340391: 0640.49WO

[0220] In a further particular embodiment, the HTS (or a variant thereof) is produced in an insect expression system (i.e., insect-derived HTS or variant thereof), for example, in baculovirus infected or non-lytic insect cells (e.g., sf9, Sf21).

[0221] In another embodiment, the HTS (or a variant thereof) is produced in a fungal expression system (i.e., fungi-derived HTS or variant thereof), for example in the genera Chrysosporium, Thielavia, Talaromyces, Trichoderma, Thermomyces or Thermoascus.

[0222] In yet another embodiment, HTS (or a variant thereof) is produced in a mammalian expression system (i.e., mammalian-derived HTS or variant thereof), for example in Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK), COS and baby hamster kidney (BHK) cells. Alternatively, the HTS (or a variant thereof) may be produced in vitro using a cell-free expression system, such as an E. coli S30 extract.

[0223] PURIFICATION

[0224] The recombinantly expressed polypeptides from Myd-encoding expression cassettes are typically isolated from lysed cells or culture media. Purification can be carried out by methods known in the art including salt fractionation, ion exchange chromatography, gel filtration, size- exclusion chromatography, size-fractionation, affinity chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential precipitation or solubilization. Immunoaffinity chromatography can be employed using antibodies generated based on, for example, Gag antigens.

[0225] The invention provides a method of purifying a polypeptide having sweet-taste modulation activity comprising (a) obtaining a composition comprising the polypeptide, and (b) purifying the composition via hydrophobic interaction chromatography (HIC) followed by size exclusion chromatography (SEC).

[0226] One of skill in the art is familiar with the purification techniques of hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC), including the selection of appropriate columns, buffers, and eluting solutions. Exemplary HIC and SEC purification techniques are described herein in Example 11. In an exemplary aspect, the purity of the polypeptide following 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 ranges of values thereof.

[0227] In a cell-based system, the first step in the protein purification process extracts the protein from the cells by lysing or breaking them open. Any suitable cell lysis method may be used, for 66Atty Docket: 340391: 0640.49WO example, mechanical disruption, chemical breakdown, freeze-thaw cycles or enzymatic digestion. The protein can then by purified by any suitable protein purification method for example, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential precipitation or solubilization.

[0228] The yield of in vivo production of HTS (or a variant thereof) may vary. In a particular embodiment, HTS represents at least about 1% of total cellular proteins. In a particular embodiment, HTS represents between about 1% and about 5% of total cellular proteins. In another embodiment, HTS represents between about 5% and about 10% or total cellular proteins. In a further embodiment, HTS represents between 10% and about 20% of total cellular proteins. In certain embodiments, HTS represents more than 20% of total cellular proteins.

[0229] In another particular embodiment, HTS is purified to provide a yield between about 1 mg / mL and about 200 mg / mL, more particularly, between about 5 mg / mL and about 195 mg / mL, between about 10 mg / mL and about 190 mg / mL, between about 15 mg / mL and about 185 mg / mL, between about 20 mg / mL and about 180 mg / mL, between about 25 mg / mL and about 175 mg / mL, between about 30 mg / mL and about 170 mg / mL or about 35 mg / mL and about 165 mg / mL. In an embodiment, the HTS is purified to provide a yield of about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 75 mg / mL, about 100 mg / mL, about 125 mg / mL, about 150 mg / mL, about 175 mg / mL, or about 200 mg / L or more. Optionally, the HTS is produced as a fusion protein further comprising a tag and the yields described above reflect both purification and removal of the tag.

[0230] In a particular embodiment, the HTS (or a variant thereof) is substantially pure. In an embodiment, HTS (or a 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, HTS (or a 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.

[0231] PLANTS

[0232] The invention also contemplates a transgenic plant comprising a heterologous polynucleotide and / or heterologous polypeptide of the invention as described herein. The plant has an altered phenotype due to the expression of the heterologous nucleic acid sequence. The altered phenotype may include a phenotype with increased sweetness in any plant part, including fruits. The transgenic plant may contain an expression cassette as defined herein as a part of the plant, the cassette having been introduced by transformation of a plant with a vector of this invention. Such 67Atty Docket: 340391: 0640.49WO expression cassettes include regulatory sequences for expression of heterologous coding sequences in plants, including plant-expressible promoters and terminators. A transgenic plant can be any type of plant which can express the heterologous nucleic acid sequences described herein. The term “plant” includes whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same. The class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous (monocots) and dicotyledonous (dicots) plants. It includes plants of a variety of ploidy levels, including polyploid, diploid and haploid. For example, the transgenic plant can be an apple or strawberry. An HTS protein / polypeptide or variant of this disclosure can be produced in a plant or a plant culture.

[0233] Techniques for transforming a wide variety of plant species are well known in the art and described in the technical and scientific literature. See, for example, Weising et al. (1988) Ann. Rev. Genet., 22:421-477 and Joung et al. (2015) “Plant Transformation Methods and Applications,” in Current Technology in Plant Molecular Breeding, (Koh et al., eds) Springer Dordrecht Heidelberg New York London, Chapter 9, pages 297-344. Any method known in the art for transformation of plant cells, including plant protoplasts, or plant tissue can be employed for plant transformation. Specific methods for plant transformation include among others, bolistic methods (gene guns), electroporation, microinjection, protoplast fusion and Agrobacterium-mediated transformation. Agrobacterium-mediated transformation can, for example, employ binary vectors that replicate in Escherichia coli and Agrobacterium tumefaciens or other Agrobacterium strains. A variety of such binary vectors are known in the art and can be employed to introduce heterologous polynucleotides into plant cells and plant tissue. Plant expression vectors which include regulatory sequences for expression of heterologous coding sequences, including plant-expressible promoter sequences and other plant regulatory sequences, in plant cells and plant tissue are known in the art and can be employed to transform plants to express polypeptides as described herein.

[0234] A variety of plant-expressible promoters are known in the art and are available for use in heterologous constructs, vectors and transformed plant materials herein which contain polynucleotides encoding protein having sweet-taste modulation activity. Plant-expressible promoters can derive from natural plant sources, plant virus sources and from bacteria, such as Agrobacterium strains, having promoters that are plant-expressible. Plant-expressible promoters include, among others, Cauliflower Mosaic Virus promoter (CaMV 35S), octopine and nopaline synthase promoters (e.g., nos promoter), plant ubiquitin promoter (Ubi), rice actin promoter (Act-1), and maize alcohol dehydrogenase (Adh-1). Plant-expressible promoters include constitutive promoters, inducible promoters, tissue-specific promoters, developmental stage-specific promoters 68Atty Docket: 340391: 0640.49WO and examples of each type of promoter are known in the art. Tissue-specific promoters include, among others, those that direct expression in plant roots, plant leaves, fruit, flowers, pollen or cells engaged in active photosynthesis (e.g., phosphoenolpyruvate promoters (PEP)). Development stage-specific promoters include those that direct expression during fruit ripening, flowering or seed 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. & Kim W-C (2019) Frontiers in Plant Science, 10, article 1433).

[0235] Techniques for regeneration of plants from transformed protoplasts, plant cells, callus, or other plant tissue 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. Volume 1: Techniques for propagation and breeding (1983) Edited by D. A. Evans et al., Macmillan (New York); R.H. Smith, Plant Tissue Culture: Techniques and Experiments, 3rdEdition (2012) Academic Press (New York); M.R. Davey & P. Anthony, Plant Cell Culture: Essential Methods (2010) John Wiley & Sons (New York), particularly Chapters 3 and 9.

[0236] In embodiments, HTS (or a variant thereof) is produced in an algal expression system (i.e., algae-derived HTS or variant thereof).

[0237] In embodiments, HTS (or a variant thereof) is produced in a plant expression system (i.e., plant-derived HTS or variant thereof), for example, in maize, corn, tobacco, melon (e.g., watermelon), potatoes, strawberries, duckweed or sugarcane. In an embodiment, the plant expression system is a plant cell culture expression system.

[0238] In a particular embodiment, HTS (or a variant thereof) is produced in maize, and more particularly, the seeds of maize. In another particular embodiment, HTS (or a variant thereof) is produced in corn, and more particularly, the seeds of corn. According to these embodiments, HTS (or a variant thereof) may be utilized as HTS-containing germ flour.

[0239] METHODS OF PRODUCING A PROTEIN HAVING SWEET-TASTE MODULATION ACTIVITY (IN A HOST OR CELL-FREE EXPRESSION SYSTEM)

[0240] A method usually used in the field such as protoplast method and electroporation can be used as a method of introducing a vector into a host. A transformant of interest can be obtained by selecting a strain in which a vector is appropriately introduced using an index such as the expression of a marker gene and / or auxotrophy.

[0241] Alternatively, a fragment in which the polynucleotide encoding a Myd polypeptide variant of the present invention, a regulatory sequence and a marker gene are ligated can be directly 69Atty Docket: 340391: 0640.49WO introduced into the genome of a host. For example, the polynucleotide encoding a Myd polypeptide variant of the present invention is introduced into the genome of a host by constructing a DNA fragment added with a sequence complementary to the genome of the host at 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.

[0242] Culturing the thus obtained transformant, in which the polynucleotide encoding a Myd polypeptide variant of the present invention or a vector comprising is introduced, in an appropriate medium, results in the expression of the MYD cDNA on the vector, and then the production of a Myd polypeptide variant of the present invention. The medium used for the culture of such transformant can be selected depending on the type of microorganism of such transformant by those skilled in the art as appropriate.

[0243] Alternatively, a Myd polypeptide variant of the present invention can be expressed from the polynucleotide encoding a Myd polypeptide variant of the present invention or a transcription product thereof using a cell-free translation system. "Cell-free translation system" refers to an in vitro transcription-translation system or an in vitro translation system constructed by adding reagents such as amino acids required for the translation of a protein into a suspension obtained by mechanically destructing cells to be a host.

[0244] Cell-free systems that can be used to produce HTS for use in the compositions described herein include, but are not limited to, protein expression components from eukaryotic, prokaryotic, and / or viral sources. For example, cell-free systems as used herein can include mammalian and / or bacterial protein expression systems derived from mammalian and / or bacterial lysates.

[0245] A Myd polypeptide variant of the present invention produced in the culture or cell-free translation system can be isolated or purified, if desired, by using a general method used for the purification of a protein, for example, centrifugation, ammonium sulfate precipitation, gel chromatography, ion-exchange chromatography and affinity chromatography etc. alone or in combination as appropriate. Here, when the gene encoding a Myd polypeptide variant of the present invention and the secretory signal sequence are operably liked on the vector within the transformant, the produced Myd polypeptide variant can be collected more easily from the culture because the Myd polypeptide variant is secreted to the outside of a cell. The Myd polypeptide variant collected from the culture can be further purified by any known means.

[0246] In embodiments, the Myd protein is solubilized in a liquid solution, such as e.g., a buffered solution or any solution that readily dissolves the Myd protein into solution. In embodiments, the Myd protein contained in a liquid solution is lyophilized to form a powder. In embodiments, the Myd protein contained in a liquid solution is dried to form a powder, such as e.g., by using a spray 70Atty Docket: 340391: 0640.49WO dryer. In embodiments, spray drying includes the use of a carrier as is knownin the art. In embodiments, the carrier used for spray drying is maltodextrin, gum Arabic or whey protein concentrate,

[0247] The present invention also includes a method for producing a protein having sweet-taste modulation activity, comprising culturing the host cells of the invention in a medium under conditions that result in producing the protein having sweet-taste modulation activity, similar to a known sweet flavoring agent or compound.

[0248] SWEET COMPOSITIONS-FOODS, BEVERAGES, SUPPLEMENTS, MEDICINAL PRODUCTS

[0249] Disclosed herein are sweetener compositions and flavor modifying compositions, in each case, containing Myd (HTS) or variants thereof. In certain embodiments, the sweetener compositions and flavor modifying compositions change (e.g., improve) one or more sensory experiences of a subject who consumes the same. In a particular embodiment, the sweetener composition and flavor modifying compositions disclosed herein comprise a HTS variant. In an embodiment, the HTS variant differs from wild-type HTS at least one amino acid position and more particularly, at one, two, three or more amino acid positions.

[0250] As used herein, a “sweet flavoring agent,” “sweet compound” or "sweet receptor activating compound” refers to a composition that elicits a detectable sweet flavor in a subject, e.g., sucrose, fructose, glucose, and other known natural saccharide-based sweeteners, or known artificial sweeteners such as saccharine, cyclamate, aspartame, and the like as is further discussed herein, or a material that activates a T1R2 / T1R3 receptor in vitro. The subject may be a human or an animal.

[0251] A sweet flavoring agent or sweetening composition may be used in an effective amount, which refers to an amount of a sweetening composition of the invention that is sufficient to induce sweet taste in a subject when present in a product for oral administration.

[0252] An embodiment of the present invention includes a composition. In an embodiment, the composition comprises, consists essentially of, or consists of a combination of a product for oral administration and one or more sweetening composition comprising an isolated Myd polypeptide variant according to the invention, as described herein. In an embodiment, the combination has enhanced sweet taste compared to a product for oral administration lacking the Myd polypeptide variant (control). In an embodiment, the product for oral administration is not Mattirolomyces terfezioides truffle. The term “consisting essentially of” allows for the inclusion of components that are not essential to the function or activity of the product and do not materially affect the function or activity, such an anti-caking agent, filler, stabilizer (e.g., thermal stabilizer), and bulking agent 71Atty Docket: 340391: 0640.49WO (e.g., maltodextrose, gum acacia and the like). In an embodiment, the composition includes a plurality of isolated Myd polypeptides. In a particular embodiment, the composition includes a plurality of isolated Myd polypeptides which differ from each other to enhance taste. It should be appreciated that compositions comprising one or more Myd polypeptides of the invention are not limited by the form, shape, and means of administration, and encompass solids, liquids, powder, and other forms, either individually or in combinations of two or more thereof. Furthermore, the compositions may be administered or consumed orally, by injection, etc.

[0253] In another embodiment, compositions comprising an isolated Myd protein of the invention include formulations that provide enhanced functionality to the isolated Myd protein. For example, a composition may include a formulation that stabilizes the Myd protein against thermal, osmotic, pH, or other types of degradation. In an embodiment, the formulation stabilizes the Myd protein against thermal degradation. Exemplary compounds for stabilizing the Myd protein includes, for example, L-arginine glycine, L-proline, L-histidine, β-alanine, L-serine, L-arginine ethyl ester dihydrochloride, L-argininamide 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, guanidine hydrochloride, urea, N-methylurea, N-ethylurea, N- methylformamide, hypotaurine, TCEP hydrochloride, GSH (l-glutathione reduced), benzamidine hydrochloride, ethylenediaminetetraacetic acid disodium salt dihydrate, magnesium chloride hexahydrate, cadmium chloride hydrate, non-detergent sulfobetaine 195 (ndsb-195), non-detergent sulfobetaine 201 (NDSB-201), non-detergent sulfobetaine 211 (NDSB-211), non-detergent sulfobetaine 221 (NDSB-221), non-detergent sulfobetaine 256 (NDSB-256), taurine, acetamide, oxalic acid dihydrate, sodium malonate pH 7.0, succinic acid pH 7.0, tacsimate pH 7.0, tetraethylammonium bromide, choline acetate, 1-ethyl-3-methylimidazolium acetate, 1-butyl-3- methylimidazolium chloride, ethylammonium nitrate, ammonium sulfate, ammonium chloride, magnesium sulfate hydrate, potassium thiocyanate, gadolinium(III) chloride hexahydrate, cesium chloride, 4-aminobutyric acid (GABA), lithium nitrate, DL-malic acid pH 7.0, lithium citrate tribasic tetrahydrate, ammonium acetate, sodium benzenesulfonate, sodium p-toluenesulfonate, sodium chloride, potassium chloride, sodium phosphate monobasic monohydrate, sodium sulfate decahydrate, lithium chloride, sodium bromide, glycerol, ethylene glycol, polyethylene glycol 200, polyethylene glycol monomethyl ether 550, polyethylene glycol monomethyl ether 750, formamide, polyethylene glycol 400, pentaerythritol ethoxylate (15 / 4 EO / OH), 1,2-propanediol, polyethylene glycol monomethyl ether 1,900, polyethylene glycol 3,350, polyethylene glycol 8,000, 72Atty Docket: 340391: 0640.49WO polyvinylpyrrolidone k15, polyethylene glycol 20,000, (2-hydroxypropyl)-β-cyclodextrin, α- cyclodextrin, β-cyclodextrin, methyl-β-cyclodextrin.

[0254] In embodiments, the sweetening composition includes one or more Myd polypeptides described above. In an embodiment, the sweetening composition includes a plurality of Myd polypeptides described above. In a particular embodiment, the plurality of Myd polypeptides differ from each other.

[0255] 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, as described herein. In one aspect, the combination has enhanced sweet taste compared to a product for oral administration lacking the Myd polypeptide variant (control). In an embodiment, the product for oral administration is not Mattirolomyces terfezioides truffle.

[0256] The product for oral administration may be a food, a beverage, a dietary supplement composition, or a pharmaceutical composition. In additional embodiments, the product for oral administration is a dental hygiene product, for example, a toothpaste or a mouthwash.

[0257] The term “product for oral administration” may refer to a comestible product (herein designated consumable) such as a food product, a beverage product, a medicinal (pharmaceutical) product, or a dietary supplement product such as an herbal supplement. As used herein, the term “consumable” may be used interchangeably with the term “product(s) for oral administration”. As used herein, the term “medicinal product” includes both solids, pastes, gels and liquid compositions which are ingestible non-toxic materials which have medicinal value or comprise medicinally active agents such as cough syrups, cough drops, aspirin and chewable medicinal tablets. An oral hygiene or oral therapeutic product is also a product for oral administration and includes solids, pastes, gels and liquids such as toothpaste, tooth powder or mouthwash. Products for oral administration may include those that are not intended to actually be consumed or ingested after administration (termed “non-consumable” herein) but are intended to be expelled (spit out) or rinsed out of the mouth, e.g., toothpaste, mouthwash. In general terms, the present invention contemplates that food or beverage products may include an isolated sweet protein of the invention in an effective amount, e.g., in an amount of up to about 99% by weight relative to the total weight of the food or beverage product, for example in an amount from about 0.01% by weight to about 99% by weight. All intermediate weights (i.e., 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, ... 90%, 95%, 99%) by weight relative to the total weight of the food or beverage products are contemplated, as are all intermediate ranges based on these amounts. In embodiments, an effective amount of a sweet protein of this invention is that amount that provides a desired sweetness in the 73Atty Docket: 340391: 0640.49WO product for oral administration alone or in combination with one or more other sweeteners other than a MYD family protein. Compositions of the invention may include a “comestibly, biologically or medicinally acceptable carrier or excipient” which can include a solid or liquid medium and / or composition that is used to prepare a desired dosage form of a Myd polypeptide variant, in order to administer a Myd polypeptide variant in a dispersed / diluted form, so that the biological effectiveness of a Myd polypeptide variant is maximized.

[0258] A comestibly, biologically or medicinally acceptable carrier includes many common food ingredients, such as water at 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 substances in aqueous media, salts such as sodium chloride, wheat flours, solvents such as ethanol, solid edible diluents such as vegetable powders or flours, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents; thickening or emulsifying agents, preservatives, solid binders, lubricants and the like.

[0259] A medicinally acceptable carrier or excipient can include excipients which allow for microencapsulation of the Myd polypeptide variant to enhance functionality, such as protecting and extending the sweetness sensation. In fact, microencapsulation is known in the art to be a technology that can facilitate regular use in addition to creating many possible new uses for sweeteners. See, e.g., Favaro-Trindade, Carmen & Rocha-Selmi, Glaucia & dos Santos, Milla. (2015). Microencapsulation of Sweeteners.10.1016 / B978-0-12-800350-3.00022-4. In an embodiment, microencapsulation methods known in the art for stabilizing and / or modifying (e.g., extending) the sweetness release of Myd. For example, sugar-free chewing gums and chewable confections usually have encapsulated sweeteners in their formulations to prolong their sweet taste during chewing.

[0260] It should be appreciated that food or beverage products and compositions comprising one or more Myd polypeptides as described are not limited by the form and shape and encompass solids, liquids, powder, pastes, gels and other forms, either individually or in combinations of two or more thereof.

[0261] Examples of food or beverage products of the present invention include such as, but not limited to, baked goods; sweet bakery products, (including, 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 (including, but not limited to, fruit pie fillings and nut pie fillings such as pecan pie filling, as well as fillings for cookies, cakes, pastries, confectionary products and 74Atty Docket: 340391: 0640.49WO the like, such as fat-based cream fillings); desserts, gelatins and puddings; frozen desserts (including, but not limited to, frozen dairy desserts such as ice cream— including regular ice cream, soft serve ice cream and all other types of ice cream—and frozen non-dairy desserts such as non- dairy ice cream, sorbet and the like); carbonated beverages (including, but not limited to, soft carbonated beverages); non-carbonated beverages (including, but not limited to, soft non- carbonated beverages such as flavored waters and sweet tea or coffee based beverages); beverage concentrates (including, but not limited to, liquid concentrates and syrups as well as non-liquid concentrates, such as freeze-dried and / or powder preparations); yogurts (including, but not limited to, full fat, reduced fat and fat-free dairy yogurts, as well non-dairy and lactose-free yogurts and frozen equivalents of all of these); snack bars (including, but not limited to, cereal, nut, seed and / or fruit bars); bread products (including, but not limited to, leavened and unleavened breads, yeasted and un-yeasted breads such as soda breads, breads comprising any type of wheat flour, breads comprising any type of non-wheat flour (such as potato, rice and rye flours), gluten-free breads); pre-made bread mixes for preparing bread products; sauces, syrups and dressings; sweet spreads (including, but not limited to, jellies, jams, butters, nut spreads and other spreadable preserves, conserves and the like); confectionary products (including, but not limited to, jelly candies, soft candies, hard candies, chocolates, mints and gums); sweetened breakfast cereals (including, but not limited to, extruded (KIX type) breakfast cereals, flaked breakfast cereals and puffed breakfast cereals); and cereal coating compositions for use in preparing sweetened breakfast cereals. Other types of food and beverage products not mentioned here but which conventionally include one or more nutritive sweetener may also be contemplated in the context of the present invention.

[0262] In embodiments, the product for oral administration is a food product which is warmed or heated prior to eating or which is served for consumption warm or hot. In embodiments, polypeptide variants herein which exhibit enhanced thermal stability compared to the polypeptide of SEQ ID NO:3 are preferred for application in compositions for oral administration (e.g., food products) which are to be cooked, warmed or heated prior to administration or consumption or which are to be administered or consumed warm or hot.

[0263] As a consequence of the complete or partial replacement of nutritive sweeteners in the food or beverage products of the present invention, the food or beverage products of the present invention may be useful as low calorie or dietetic products, medical foods / products (including pills and tablets), and sports nutrition products, and may be particularly suitable for food or beverage products requiring a lower sweetness at a given soluble solids level.

[0264] In some embodiments, the sweetening composition of the invention can be supplemented with other nutritional or non-nutritional sweeteners to form a sweetener system. The sweetener 75Atty Docket: 340391: 0640.49WO system may comprise the sweetening composition of the invention, a bulking agent such as maltodextrose, gum acacia and the like, and at least one high intensity sweetener. The composition may be provided as liquid composition or a dried blend.

[0265] As used herein the term “high-intensity sweetener,” refers to any synthetic or semi-synthetic sweetener or sweetener found in nature. High-intensity sweeteners are compounds or mixtures of compounds which are sweeter than sucrose. High-intensity sweeteners are typically many times (e.g., 20 times and more, 30 times and more, 50 times and more or 100 times or more) sweeter than sucrose).

[0266] In an embodiment, the present invention includes a process for enhancing the sweet taste of a product for oral administration, comprising the addition of a Myd polypeptide variant (HTS peptide) of the invention. In embodiments, the HTS peptides is a peptide of SEQ ID NO:3 or SEQ ID NO:141 or a sequence variant thereof. Further embodiments include sequence variants having 1- 6, 1-3, 1-2, 2 or 1 amino acid variations (e.g., substitutions, deletions or additions) from the HTS peptide of SEQ ID NO:3 or SEQ ID NO:141. Additional embodiments include sequence variants having amino acid substitutions of the HTS peptide of SEQ ID NO:3 or SEQ ID No: 141. Additional embodiments include sequence variants having 1 or 2 amino acid substitutions of the HTS peptide of SEQ ID NO:3 or SEQ ID NO: 141. In embodiments, HTS peptides for use in enhancing the sweet taste of a product for oral administration are other than a naturally occurring Myd (mycodulcein) polypeptide of Mattirolomyces terfezioides.

[0267] In another embodiment, the methods of the invention include a method for improving the sweet flavor of a product for oral administration, comprising adding to the product for oral administration a sweetening composition made by the methods of the invention. Amounts of HTS peptide to add can be determined by methods known in the art, e.g., using sensory testing as a guide. In embodiments, the HTS peptides is a peptide of SEQ ID NO:3 or SEQ ID NO:141 or a sequence variant thereof. Further embodiments include sequence variants having 1-6, 1-3, 1-2, 2 or 1 amino acid variations (e.g., substitutions, deletions or additions) from the HTS peptide of SEQ ID NO:3 or SEQ ID NO:141. Additional embodiments include sequence variants having amino acid substitutions of the HTS peptide of SEQ ID NO:3 or SEQ ID No: 141. Additional embodiments include sequence variants having 1 or 2 amino acid substitutions of the HTS peptide of SEQ ID NO:3 or SEQ ID NO: 141. In embodiments, HTS peptides for use in improving the sweet flavor of a product for oral administration are other than a naturally occurring Myd (mycodulcein) polypeptide of Mattirolomyces terfezioides.

[0268] In another embodiment, the methods of the invention include methods for modifying the flavor of a product for oral administration, comprising adding to the product for oral administration 76Atty Docket: 340391: 0640.49WO a flavor modifying composition made by the methods of the invention. In embodiments, the flavor modifying composition comprises one or more MYD family proteins. In embodiments, the MYD family protein itself exhibits sweet taste. In embodiments, the MYD family protein does not itself exhibit sweet taste, but on addition to a product for oral administration enhances or improves the taste or at least one organoleptic property of the product for oral administration. The amount of MYD family protein effective for modification of flavor depends upon the components in the composition to which it is added. Such effective amounts can be determined by one of ordinary skill in the art using methods well known in the art, including methods described herein. Amounts to add can be determined by methods known in the art, e.g., using sensory testing as a guide. A flavor modifying composition may modify (e.g., enhance, inhibit or change) a taste, aroma and / or texture of a given composition, e.g., a consumable. In a particular embodiment, the flavor modifying composition modifies (e.g., enhances, inhibits or changes) a particular taste(s). 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(s) and texture. In embodiments, the HTS peptides is a peptide of SEQ ID NO:3 or SEQ ID NO:141 or a sequence variant thereof. Further embodiments include sequence variants having 1-6, 1-3, 1-2, 2 or 1 amino acid variations (e.g., substitutions, deletions or additions) from the HTS peptide of SEQ ID NO:3 or SEQ ID NO:141. Additional embodiments include sequence variants having amino acid substitutions of the HTS peptide of SEQ ID NO:3 or SEQ ID No: 141. Additional embodiments include sequence variants having 1 or 2 amino acid substitutions of the HTS peptide of SEQ ID NO:3 or SEQ ID NO: 141. In embodiments, HTS peptides for use in modifying the flavor of a product for oral administration are other than a naturally occurring Myd (mycodulcein) polypeptide of Mattirolomyces terfezioides.

[0269] A flavor modifying composition may be sweetened or unsweetened. Therefore, in some embodiments, the addition of a flavor modifying composition may serve both to add flavor modifiers and may further provide sweetness to a composition selected for taste adjustment. The addition of a sweetened flavor modifying composition may be used in addition to or alternatively to addition of another sweetening composition.

[0270] The sweetener compositions and flavor modifying compositions disclosed herein contain one or more MYD family protein (including Myd (HTS) and variants thereof). In certain embodiments, the MYD family protein (HTS or variant thereof) is the only sweet tasting component in the sweetener composition or flavor modifying composition. In certain embodiments, the sweetener composition or flavor modifying composition further comprises one or more additional sweet tasting components (i.e., additional sweetener or high-intensity sweetener, which 77Atty Docket: 340391: 0640.49WO in some cases is designated a first sweetener). In embodiments, the additional sweetener is a polypeptide or protein sweetener other than HTS or a variant thereof. In embodiments, the sweetener is a carbohydrate sweetener. In embodiments, the additional sweetener is a synthetic sweetener. In a particular embodiment, the one or more sweet tasting components include steviol glycosides (e.g., Reb M, Reb A) and high fructose corn syrup (HFCS).

[0271] 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 broken down into glucose by enzymes.

[0272] Steviol glycosides are compounds responsible for the sweet taste of leaves of the plant Stevia rebaudiana and several related plants. Certain steviol glycosides are the ingredients in or are precursors to ingredients in stevia sweeteners. Steviol glycoside may be a single compound or a mixture 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.

[0273] Mogrosides are glycosides of cucurbitane derivatives, including mogrol, associated with the sweet taste of extracts of Siraitia grosvenorii (monkfruit or luo han guo). Certain mogrosides are the ingredients in monkfruit sweeteners. Mogrosides include, among others, mogroside II A1, mogroside II B, 7-oxomogroside II E.11-oxomogroside A1,mogroside III A2,11-deoxymogroside III, 11-oxomogroside IV A, mogroside V, 7-oxomogroside V, 11-oxo-mogroside V, mogroside VI, siamenoside I and combinations thereof. Preferred mogrosides are mogroside V, mogroside VI and siamenoside.

[0274] In an embodiment, the one or more additional sweetener may be a carbohydrate sweetener. Non-limiting examples of suitable carbohydrate sweeteners include sucrose, fructose, glucose, erythritol, maltitol, lactitol, sorbitol, mannitol, xylitol, D-tagatose, trehalose, galactose, rhamnose, cyclodextrin (e.g., α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin), ribulose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, 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, glucono-lactone, abequose, galactosamine, xylo-oligosaccharides (xylotriose, xylobiose and the like), gentio-oligosaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), galacto-oligosaccharides, sorbose, ketotriose (dehydroxyacetone), aldotriose (glyceraldehyde), nigero-oligosaccharides, fructooligosaccharides (kestose, nystose and the like), maltotetraose, maltotriol, tetrasaccharides, mannan-oligosaccharides, malto-oligosaccharides 78Atty Docket: 340391: 0640.49WO (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), dextrins, lactulose, melibiose, raffinose, rhamnose, ribose, isomerized liquid sugars such as high fructose corn / starch syrup (HFCS / HFSS) (e.g., HFCS55, HFCS42, or HFCS90), coupling sugars, soybean oligosaccharides, glucose syrup and combinations thereof.

[0275] In an embodiment, the one or more additional sweetener may be honey. In an embodiment, the one or more additional sweetener may be agave extract. In an embodiment, the one or more additional sweetener may be tapioca syrup. In an embodiment, the one or more additional sweetener may be polyglycitol syrup. In an embodiment, the one or more additional sweetener may be dextrose.

[0276] In other embodiments, the at least one additional sweetener is a synthetic sweetener. As used herein, the phrase “synthetic sweetener” refers to any composition which is not found naturally in nature and characteristically has a sweetness potency greater than sucrose, fructose, or glucose, yet has less 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 glycosides (GSGs) and combinations thereof.

[0277] In other embodiments, the at least one additional sweetener is a protein sweetener (i.e., a polypeptide or protein that tastes sweet). Typically, such protein sweeteners are extracted from plants. Non-limiting examples of protein sweeteners are monellin, thaumatin, brazzein, curculin, pentadin, and mabinlin.

[0278] In a particular embodiment, the sweetener composition and flavor modifying compositions disclosed herein comprise a Myd (HTS) variant. In an embodiment, the HTS variant differs from wild-type HTS (with or without methionine at position 1) at least one amino acid position and more particularly, at one, two, three or more amino acid positions.

[0279] In an embodiment, the sweetener composition and flavor modifying composition disclosed herein comprises a HTS variant having a sweetness equal to or greater than wild-type HTS having an amino acid sequence of SEQ ID NO:3.

[0280] In an embodiment, the sweetener composition and flavor modifying composition disclosed herein comprise a HTS variant having a stability equal to or greater than wild-type HTS having an amino acid sequence of SEQ ID NO:3.

[0281] In an embodiment, the sweetener composition and flavor modifying composition disclosed herein comprises a HTS variant having a sweetness equal to or greater than wild-type HTS having an amino acid sequence of SEQ ID NO:3 without methionine at position 1. 79Atty Docket: 340391: 0640.49WO

[0282] In an embodiment, the sweetener composition and flavor modifying composition disclosed herein comprise a HTS variant having a stability equal to or greater than wild-type HTS having an amino acid sequence of SEQ ID NO:3 without methionine at position 1.

[0283] HTS (or variants thereof) suitable for use in compositions disclosed herein (e.g., sweetener compositions, flavor and / or taste modifying compositions, consumables) may be produced in any suitable manner as previously described herein. Representative methods of production include extraction, chemical synthesis (i.e., solid state synthesis) or recombinant production (i.e., in vivo production or in vitro production).

[0284] In an embodiment, HTS used in the compositions disclosed herein is isolated from the edible (i) mycelia truffle of family Terfeziaceae or an aqueous extract thereof or (ii) an aqueous extract of a fruiting body of truffle of family Terfeziaceae. In an embodiment, the HTS is isolated from the mycelia or fruiting body of a Mattirolomyces terfezioides truffle.

[0285] In another embodiment, HTS (or variant thereof) used in compositions disclosed herein is produced in vivo. In an embodiment, the nucleic acid coding sequence for HTS isolated from Mattirolomyces terfezioides truffle, optionally optimized, is introduced into a suitable vector, which is then cloned into a host cell in an appropriate growth system / environment—resulting in expression of the protein in recombinant fashion. Suitable host cells and expression systems are previously described herein.

[0286] The amount of HTS (or a variant thereof) in the sweetener composition and flavor modifying compositions disclosed herein may vary. In an embodiment, the HTS (or a variant thereof) is present in the sweetener composition above its sweetness threshold concentration.

[0287] In an embodiment, HTS (or a variant thereof) is present in the sweetener composition or flavor modifying composition in any amount to impart the desired sweetness when the sweetener composition or flavor modifying composition is added to a consumable (e.g., beverage), either alone or in combination with one or more additional sweet tasting components (e.g., steviol glycosides, HFCS) present in the sweetener composition or flavor modifying composition, i.e., before such compositions are added to the consumable.

[0288] In a particular embodiment, the desired sweetness of the consumable is isosweet to a sucrose-sweetened consumable having a sweetness from at least about 8 degrees Brix, such as, for example, 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.

[0289] In another embodiment, the desired sweetness of the consumable is isosweet to a sucrose- sweetened consumable having a sweetness from about 10 degrees Brix to about 15 degrees Brix, 80Atty Docket: 340391: 0640.49WO such as, for example, from about 10 degrees Brix to about 14 degrees Brix, from about 10 degrees Brix to about 13 degrees Brix, from about 10 degrees Brix to about 12 degrees Brix, from about 10 degrees Brix to about 11 degrees Brix, from about 11 degrees Brix to about 15 degrees Brix, from about 11 degrees Brix to about 14 degrees Brix, from about 11 degrees Brix to about 13 degrees Brix, from about 11 degrees Brix to about 12 degrees Brix, from about 12 degrees Brix to about 15 degrees Brix, from about 12 degrees Brix to about 14 degrees Brix, from about 12 degrees Brix to about 13 degrees Brix, from about 13 degrees Brix to about 15 degrees Brix, from about 13 degrees Brix to about 14 degrees Brix and from about 14 degrees Brix to about 15 degrees Brix.

[0290] In an embodiment, the Myd polypeptide (or variant thereof) is present in the sweetener composition or flavor modifying composition in an amount that enhances the sweetness of the consumable to which it is added by about 1.0% (w / v) sucrose equivalence (SE) or greater, either alone or in combination with one or more additional sweet tasting component (e.g. steviol glycosides, HFCS) present in the sweetener composition or flavor modifying composition, i.e., before such compositions are added to the consumable.

[0291] In a particular embodiment, the Myd polypeptide (or variant thereof) is present in the sweetener composition in an amount that enhances the sweetness of the consumable to which it is added from about 1.0% to about 3.0% (w / v) sucrose equivalence (SE), such as, for example, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9% or about 3.0% sucrose equivalence, either alone or in combination with one or more additional sweet tasting components (e.g., steviol glycosides, HFCS) present in the sweetener composition or flavor modifying composition, i.e., before such compositions are added to the consumable.

[0292] In another particular embodiment, the Myd polypeptide (or variant thereof) is present in the sweetener composition or flavor modifying composition in an amount that enhances the sweetness of the consumable to which it is added by about 3.0% to about 5% (w / v) sucrose equivalence (SE), for example, 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%, either alone or in combination with one or more additional sweet tasting components (e.g., steviol glycosides, HFCS) present in the sweetener composition or flavor modifying composition, i.e., before such compositions are added to the consumable.

[0293] 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 81Atty Docket: 340391: 0640.49WO Sweeteners,” G. E. DuBois, D. E. Walters, S. S. Schiffman, Z. S. Warwick, B. J. Booth, S. D. Pecore, K. Gibes, B. T. Carr, and L. M. Brands, in Sweeteners: Discovery, Molecular Design and Chemoreception, D. E. Walters, F. T. Orthoefer, and G. E. DuBois, Eds., American Chemical Society, Washington, D.C. (1991), pp 261-276.

[0294] The amount of sucrose in a reference solution may be described in degrees Brix (° Bx). One degrees Brix is 1 gram of sucrose in 100 grams of solution and represents the strength of the solution as percentage by weight (% w / w) (strictly speaking, by mass).

[0295] In an embodiment, a sweetener composition is provided that contains Myd polypeptide (or variant thereof) in an amount effective to provide sweetness equivalent from about 1 to about 12 degrees Brix of sugar when added to a consumable, such as, for example, from about 2 to about 9 degrees Brix, from about 3 to about 8 degrees Brix, from about 4 to about 7 degrees Brix, or about 5 degrees Brix, either alone or together with one or more sweet tasting components (e.g., steviol glycosides, HFCS) present in the sweetener composition or flavor modifying composition or consumable.

[0296] In another embodiment, Myd polypeptide (or variant thereof) is present in an amount effective to provide sweetness equivalent to about 10 degrees Brix when added to a sweetenable composition, either alone or in combination with one or more sweet tasting component (e.g., steviol glycosides, HFCS) present in the sweetener composition, flavor modifying composition or the consumable to which it is added.

[0297] The sweetness of a non-sucrose sweetener can also be measured against a sucrose reference by determining the non-sucrose sweetener's sucrose equivalence. Typically, taste panelists are trained to detect sweetness of reference sucrose solutions containing between 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 percent sucrose reference. For example, if a 1% solution of a sweetener is as sweet as a 10% sucrose solution, then the sweetener is said to be 10 times as potent as sucrose.

[0298] In an embodiment, the amount of Myd polypeptide (or variant thereof) present in the sweetener composition or flavor modifying composition disclosed herein is any amount that contributes to one or more improved organoleptic properties of the consumable (e.g., beverage) to which the sweetener composition or taste modifying composition is added. In a particular embodiment, the improved organoleptic property is associated with basic taste. In an embodiment, improving one or more organoleptic property results in the improvement of the taste profile. The overall taste profile of a composition is an interplay of several different tastes, such as sweetness, sourness, saltiness, bitterness, umami and the like. 82Atty Docket: 340391: 0640.49WO

[0299] As used herein, “organoleptic properties” are the aspects of food, water or other substances that create an individual experience via the senses—including taste, sight, smell, and touch. Organoleptic properties include, e.g., appearance, texture, color, odor, size, shape and flavor. It is the qualitative evaluation based on the study of morphological and sensory profiles of the food, water, or other substance (such as, e.g., sweetener compositions).

[0300] Examples of improved organoleptic properties can include, for example, a reduction in bitterness, a reduction in astringent and liquorice notes, slower onset of sweetness, a reduction in lingering sweetness, a reduction in lingering bitterness, a reduction in bitter aftertaste, a reduction in metallic aftertaste, a reduction in chemical and synthetic aftertaste, and a combination thereof. In a particular embodiment, the term “improved organoleptic properties” means that the sweetened or taste modified composition (e.g., a beverage) will have one or more improved organoleptic properties for the majority of users. The improvement may be expressed qualitatively or quantitatively, e.g. as a percentage improvement.

[0301] The improved organoleptic property may be measured by or using technical means such as a taste sensing system (TSS), a term referring to analytical sensory array units (e.g., electrochemical, gravimetrical, optic or biosensors) which can detect specific substances. Sliwi'nska, M et al. J. Agric. Food Chem. (2014), 62, 1423-1448.

[0302] In a particular embodiment, the Myd polypeptide (or variant thereof) is present in the sweetener composition or flavor modifying composition in any amount that reduces, suppresses or masks the bitterness of a consumable (e.g., a beverage) to which the sweetener or flavor modifying composition is added, either alone or together with one or more sweet tasting components (e.g., steviol glycosides, HFCS) in the sweetener composition or flavor modifying composition, i.e., before it is added to the consumable. The comparison is made to a consumable to which the sweetener or flavor modifying composition has not been added.

[0303] In a particular embodiment, the Myd polypeptide (or variant thereof) is present in the sweetener composition or flavor modifying composition in an amount that reduces the bitterness of 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, either alone or in combination with one or more sweet taste components (e.g., steviol glycosides, HFCS) in the sweetener composition or flavor modifying composition, i.e., before it is added to the consumable. In an embodiment, the reduction in bitterness is experienced by a majority of subjects. The comparison is made to a consumable to which the sweetener or flavor modifying composition has not been added.

[0304] In a particular embodiment, the Myd polypeptide (or variant thereof) is present in the sweetener composition or flavor modifying composition in any amount that reduces the bitter 83Atty Docket: 340391: 0640.49WO aftertaste of a consumable (e.g., a beverage) to which the sweetener or flavor modifying composition is added, either alone or in combination with one or more sweet taste components (e.g., steviol glycosides, HFCS) in the sweetener composition or flavor modifying composition, i.e., before it is added to the consumable. In a particular embodiment, the Myd polypeptide (or variant thereof) is present in the sweetener composition or flavor modifying composition in an amount that reduces bitter aftertaste of 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 an embodiment, the reduction in bitter aftertaste is experienced by a majority of subjects. The comparison is made to a consumable to which the sweetener or flavor modifying composition has not been added.

[0305] In another embodiment, the Myd polypeptide (or variant thereof) is present in the sweetener composition or taste modifying composition in any amount reduces the sweetness linger of a consumable (e.g., a beverage) to which the sweetener or taste modifying composition is added. Sucrose exhibits a sweet taste in which the maximal response is perceived quickly and where perceived sweetness disappears relatively quickly on swallowing a food or beverage. In contrast, the sweet tastes of essentially all high-potency sweeteners reach their maximal responses somewhat more slowly and they then decline in intensity more slowly than is the case for sucrose. This decline in sweetness is often referred to as “sweetness linger” and is a major limitation for high- potency sweeteners including NHPSs. Slow onset of sweetness also can be a problem. In general, however, sweetness linger is a more significant problem. And so, preferred embodiments of this invention exhibit significant reductions in sweetness linger.

[0306] In a particular embodiment, the Myd polypeptide (or variant thereof) is present in the sweetener composition or flavor modifying compositions in an amount that reduces the sweetness linger 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, either alone or in combination with one or more sweet tasting components (e.g., steviol glycosides, HFCS) in the sweetener composition or flavor modifying composition before it is added to the consumable. In an embodiment, the majority of subjects perceive the reduction in sweetness linger. In a particular embodiment, the comparison is made to a consumable to which the sweetener composition or flavor composition has not been added.

[0307] In a particular embodiment, the Myd polypeptide (or variant thereof) is present in the sweetener composition or flavor modifying compositions in an amount that results in at least one change / modification in organoleptic property in a consumable (e.g., beverage) compared to a consumable that does not contain the sweetener composition, wherein the organoleptic property is 84Atty Docket: 340391: 0640.49WO selected from the group consisting of aroma, flavor, basic taste (sweetness, sour, saltiness, bitterness or umami), aftertaste or linger, temporal profile, mouthfeel or a combination thereof. In this embodiment, a change or modification can be any perceived difference in the organoleptic property or properties that may or may not be considered an improvement. For example, if the Myd polypeptide being present in a consumable results in a change of a flavor from chocolate to caramel, this would be considered a “change” but not necessarily an “improvement”.

[0308] In certain embodiments, the Myd polypeptide (or variant thereof) can modify flavors selected from the group consisting of caramel, chocolate, citrus (lemon or lime), cola, cotton candy, cranberry, dairy, mango, mint, mixed berry, orange cream, strawberry, toffee, tomato, tropical fruit blends, vanilla or a combination thereof.

[0309] In certain embodiments, the sweetener composition or flavor and / or taste modifying compositions contain one or more additional sweeteners. In an embodiment, the additional sweetener is present above its sweetness threshold concentration. In certain embodiments, the sweetener composition containing Myd and the one or more additional sweeteners synergistically enhance the sweetness of the consumable to which the sweetener composition is added. In an embodiment, the sweetness of the consumable is enhanced in a manner that would be unexpected to one of skill in the art.

[0310] The additional sweetener can be any type of sweetener, for example, a natural, non-natural, or synthetic sweetener.

[0311] In at least one embodiment, the at least one additional sweetener is chosen from natural sweeteners other than Stevia sweeteners. In another embodiment, the at least one additional sweetener is chosen from synthetic high potency sweeteners (SHPS).

[0312] In a particular embodiment, the one or more additional sweetener may be a natural high potency sweetener (NHPS). Suitable natural high potency sweeteners include, but are not limited to, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, dulcoside A, dulcoside B, rubusoside, Stevia, stevioside, mogroside IV, mogroside V, Luo Han Guo sweetener, siamenoside, monatin and its salts (monatin SS, RR, RS, SR), curculin, glycyrrhizic acid and its salts, thaumatin, monellin, mabinlin, brazzein, hernandulcin, phyllodulcin, glycyphyllin, phloridzin, trilobatin, baiyunoside, osladin, polypodoside A, pterocaryoside A, pterocaryoside B, mukurozioside, phlomisoside I, periandrin I, abrusoside A, steviolbioside and cyclocarioside I. The natural high potency sweetener can be provided as a pure compound or, alternatively, as part of an extract. For example, rebaudioside A can be provided as a sole compound or as part of a Stevia extract. 85Atty Docket: 340391: 0640.49WO

[0313] In an embodiment, the one or more additional sweeteners is selected from the group consisting of rebaudioside M, rebaudioside A, siamenoside I and mogroside V.

[0314] In a particular embodiment, the sweetener composition and / or flavor modifying composition of the present invention comprises Myd polypeptide (or variant thereof) and siamenoside I.

[0315] In another particular embodiment, the sweetener composition and / or flavor modifying composition of the present invention comprises Myd polypeptide (or variant thereof) and mogroside V.

[0316] In another embodiment, the one or more additional sweeteners is selected from the group consisting of 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.

[0317] In a further embodiment, the one or more additional sweeteners is 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-α isomer of siamenoside I, monk fruit extract, and combinations thereof.

[0318] In a particular embodiment, the one or more additional sweeteners is rebaudioside M (13-[2- O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] Ent Kaur-16-end-19-oil acid-[2-O-β-D-glucopyranosyl-3-O-β-D-glycopyranosyl) ester having the formula of Formula (I): 86Atty Docket: 340391: 0640.49WO

[0319] Reb M may be provided in a purified or unpurified form, i.e., as part of a naturally occurring mixture that contains Reb M. In an embodiment, Reb M can be obtained from a Stevia extract by any suitable purification method. Suitable purification methods are known in the art, including, but not limited to, column chromatography, recrystallization, phase separation, extraction, high performance liquid chromatography and combinations thereof.

[0320] 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 of obtaining A95 are provided in WO 2017 / 059414, incorporated herein by reference.

[0321] The amount of Reb M in the sweeter composition or taste modifying composition may vary. In an embodiment, Reb M is present in a sweetener composition in any amount to impart the desired sweetness when the sweetener composition is added to a consumable (e.g., a beverage). In a particular embodiment, the desired sweetness of the consumable is greater than about 10 degrees Brix.

[0322] In an embodiment, the sweetener composition contains Reb M in an amount effective to provide sweetness equivalent from about 1 to 12 degrees Brix when added to a consumable (e.g., a beverage), such as, for example, from about 2 to about 9 degrees Brix, from about 3 to about 8 degrees Brix, from about 4 to about 7 degrees Brix, or about 5 degrees Brix. 87Atty Docket: 340391: 0640.49WO

[0323] In a particular embodiment, Reb M is present in an effective amount to provide a sucrose equivalence (SE) of about 8 or less, such as for example, about 7, about 6.5, about 6, about 5.5, or about 5 SE.

[0324] In another particular embodiment, Reb M is present in an effective amount to provide a sucrose equivalence of about 8 or greater, such as, for example, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5 or about 15.

[0325] In an embodiment, Reb M is present in the flavor modifying composition in any amount to impart the desired flavor when the flavor modifying composition is added to a flavor modifiable composition (e.g., a beverage). In a particular embodiment, the desired flavor is a more sugar-like temporal or taste profile.

[0326] In an embodiment, the Reb M and the Myd polypeptide (or variant thereof) produce a synergistic effect, e.g., synergistic sweetness, i.e., the sweetness of combination is greater than the sum of the individual sweeteners. In a particular embodiment, the Reb M and the Myd polypeptide (or variant thereof) produce an effect that would be unexpected by one of skill in the art.

[0327] 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 an embodiment, Reb X is provided as a component of a mixture. In a particular embodiment, the mixture is a Stevia extract. The Stevia extract may contain Reb M in an amount that ranges from about 5% to about 100% by weight on a dry basis, such as, for example, from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100% and from about 90% to about 100%. In still further embodiments, the Stevia extract contains Reb M in an amount greater than about 90% by weight on a dry basis, for example, 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%.

[0328] In an embodiment, Reb M is provided as a component of a steviol glycoside mixture, i.e., a mixture of steviol glycosides wherein the remainder of the non-Reb M portion of the mixture is comprised entirely of steviol glycosides. The identities of steviol glycosides are known in the art and include, but are not limited to, steviol, steviol monoside, rubososide, steviolbiocide, 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 from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to 88Atty Docket: 340391: 0640.49WO about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100% and from about 90% to about 100% Reb M by weight on a dry basis. In still further embodiments, the steviol glycoside mixture may contain greater than about 90%, for example, 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 by weight on a dry basis.

[0329] RebM80 refers to a Stevia extract or steviol glycoside composition having about 80% Reb M by weight.

[0330] In a particular embodiment, the one or more additional sweeteners is rebaudioside A.

[0331] Reb A may be provided in a purified or unpurified form, i.e., as part of a naturally occurring mixture that contains Reb a. In an embodiment, Reb A can be obtained from a Stevia extract by any suitable purification method. Suitable purification methods are known in the art, including, but not limited to, column chromatography, recrystallization, phase separation, extraction, high performance liquid chromatography and combinations thereof.

[0332] 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 an embodiment, Reb A is provided as a component of a mixture. In a particular embodiment, the mixture is a Stevia extract. The Stevia extract may contain Reb A in an amount that ranges from about 5% to about 100% by weight on a dry basis, such as, for example, from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100% and from about 90% to about 100%. In still further embodiments, the Stevia extract contains Reb A in an amount greater than about 90% by weight on a dry basis, for example, 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%.

[0333] In an embodiment, Reb A is provided as a component of a steviol glycoside mixture, i.e., a mixture of steviol glycosides wherein the remainder of the non-Reb A portion of the mixture is comprised entirely of steviol glycosides. The steviol glycoside mixture may contain from about 5% to about 100% Reb A weight on a dry basis. For example, a steviol glycoside mixture may contain from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100% and from about 90% to about 100% Reb A by weight on a dry basis. In still further embodiments, the steviol glycoside mixture may contain greater than about 90%, for example, greater than about 91%, greater than about 92%, 89Atty Docket: 340391: 0640.49WO greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98% and greater than about 99% Reb A by weight on a dry basis.

[0334] The amount of Reb A in the sweetener composition or taste modifying composition may vary. In an embodiment, Reb A is present in the sweetener composition in any amount to impart the desired sweetness when the sweetener composition is added to a sweetenable composition. In a particular embodiment, the desired sweetness of the sweetened composition is greater than about 10 degrees Brix.

[0335] In an embodiment, the Reb A and the Myd polypeptide (or variant thereof) produce a synergistic effect, e.g., synergistic sweetness, i.e., the sweetness of combination is greater than the sum of the individual sweeteners. In certain embodiments, the Reb A and the Myd polypeptide (or variant thereof) produce an effect that one of skill in the art would not have expected.

[0336] In a particular embodiment, Reb A is present in an effective amount to provide a sucrose equivalence (SE) of about 8 or less, such as for example, about 7, about 6.5, about 6, about 5.5, or about 5 SE.

[0337] In another particular embodiment, Reb A is present in an effective amount to provide a sucrose equivalence (SE) of about 8 or greater, such as, for example, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5 or about 15.

[0338] In another particular embodiment, Reb A is present in an effective amount to provide a sucrose equivalence of greater than about 8 SE, e.g., about 9 SE, about 9.5 SE, about 10 SE.

[0339] In an embodiment, Reb A is present in the flavor modifying composition in any amount to impart the desired taste when the taste modifying composition is added to a taste modifiable composition (e.g., a beverage). In a particular embodiment, the desired taste is a sugar-like taste.

[0340] In a particular embodiment, the Myd polypeptide (or variant thereof) and Reb A produce a synergistic effect. In an embodiment, the Myd polypeptide (or variant thereof) and Reb A produce an effect that would have been unexpected to one of skill in the art.

[0341] The sweetener compositions can be customized to obtain a desired calorie content. For example, sweetener compositions can be “high-calorie”, such that they impart the desired sweetness when added to a sweetenable composition (such as, for example, as beverage) and have about 120 calories per 8 oz serving. 90Atty Docket: 340391: 0640.49WO

[0342] The sweetener compositions can be customized to obtain a desired calorie content. For example, sweetener compositions can be “mid-calorie”, such that they impart the desired sweetness when added to a sweetenable composition (such as, for example, as beverage) and have about 80 calories per 8 oz serving.

[0343] For example, sweetener compositions can be “low-calorie”, such that they impart the desired sweetness when added to a sweetenable composition (such as, for example, as beverage) and have less than 40 calories per 8 oz serving.

[0344] In other embodiments, the sweetener compositions can be “zero-calorie”, such that they impart the desired sweetness when added to a sweetenable composition (such as, for example, a beverage) and have less than 5 calories per 8 oz. serving.

[0345] ADDITIVES

[0346] In addition to Myd (HTS) polypeptides (or variants thereof) and, optionally, one or more additional sweeteners (e.g., one or more steviol glycosides), the sweetener compositions or flavor modifying compositions disclosed herein can optionally include additional additives, detailed herein below. In some embodiments, the sweetener composition contains additives including, but not limited to, carbohydrates, polyols, amino acids and their corresponding salts, poly-amino 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 flavoring ingredients, 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 to provide a sweetener composition with a taste similar to sucrose.

[0347] In an embodiment, the sweetener compositions or flavor modifying compositions contain one or more polyols. The term “polyol”, as used herein, refers to a molecule that contains more than one hydroxyl group. A polyol may be a diol, triol, or a tetraol which contains 2, 3, and 4 hydroxyl groups respectively. A polyol also may contain more than 4 hydroxyl groups, such as a pentaol, hexaol, heptaol, or the like, which contain 5, 6, or 7 hydroxyl groups, respectively. Additionally, a polyol also may be a sugar alcohol, polyhydric alcohol, or polyalcohol which is a reduced form of carbohydrate, wherein the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group.

[0348] Non-limiting examples of polyols in some embodiments include erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, isomalt, propylene glycol, glycerol (glycerin), threitol, galactitol, palatinose, reduced isomalto-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio- 91Atty Docket: 340391: 0640.49WO oligosaccharides, reduced maltose syrup, reduced glucose syrup, and sugar alcohols or any other carbohydrates capable of being reduced which do not adversely affect taste.

[0349] 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. The sweet taste improving amino acid additives also may be in the D- or L- configuration and in the mono-, di-, or tri-form of the same or different amino acids. Additionally, the amino acids may be α-, β-, γ- and / or δ-isomers if appropriate. Combinations of the foregoing 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) also are suitable sweet taste improving additives in some embodiments. The amino acids may be natural or synthetic. The amino acids also may be modified. Modified amino acids refers to any amino acid wherein at least one atom has been added, removed, substituted, or combinations thereof (e.g., N-alkyl amino acid, N-acyl amino acid, or N-methyl amino acid). Non-limiting examples of modified amino acids include amino acid derivatives such as trimethyl glycine, N-methyl-glycine, and N-methyl-alanine. As used herein, amino acids encompass both modified and unmodified amino acids. As used herein, amino acids 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 salt forms thereof (e.g., calcium, potassium, sodium, or magnesium salts such as L-glutamic acid mono sodium salt). The sweet taste improving poly-amino acid additives also may be in the D- or L-configuration. Additionally, the poly-amino acids may be α-, β-, γ-,and ε-isomers if appropriate. Combinations of the foregoing poly-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) also are suitable sweet taste improving additives in some embodiments. The poly-amino acids described herein also may comprise co-polymers of different amino acids. The poly-amino acids may be natural or synthetic. The poly-amino acids also may be modified, such that at least one atom has been added, removed, substituted, or combinations thereof (e.g., N-alkyl poly-amino acid or N-acyl poly-amino acid). As used herein, poly-amino acids encompass both modified and unmodified poly-amino acids. For example, modified poly-amino acids include, but are not limited to, poly-amino acids of various 92Atty Docket: 340391: 0640.49WO molecular weights (MW), such as poly-L-α-lysine with a 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.

[0350] Suitable sugar acid additives include, but are not limited to, aldonic, uronic, aldaric, alginic, gluconic, glucuronic, glucaric, galactaric, galacturonic, and salts thereof (e.g., sodium, potassium, calcium, magnesium salts or other physiologically acceptable salts), and combinations thereof.

[0351] 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 also may comprise nucleotide-related additives, such as nucleosides or nucleic acid bases (e.g., guanine, cytosine, adenine, thymine, uracil). In particular embodiments, the nucleotide is present in the sweetener composition in an amount from about 5 ppm to about 1,000 ppm.

[0352] Suitable organic acid additives include any compound which comprises a —COOH moiety, such as, for example, C2-C30 carboxylic acids, substituted hydroxyl C2-C30 carboxylic acids, benzoic acid, substituted benzoic acids (e.g., 2,4-dihydroxybenzoic acid), substituted cinnamic acids, hydroxyacids, substituted hydroxybenzoic acids, substituted cyclohexyl carboxylic acids, tannic acid, lactic acid, tartaric acid, citric acid, gluconic acid, glucoheptonic acids, adipic acid, hydroxycitric acid, malic acid, fruitaric acid (a blend of malic, fumaric, and tartaric acids), fumaric acid, maleic acid, succinic acid, chlorogenic acid, salicylic acid, creatine, caffeic acid, bile acids, acetic acid, ascorbic acid, alginic acid, erythorbic acid, polyglutamic acid, glucono delta lactone, and their alkali or alkaline earth metal salt derivatives thereof. In addition, the organic acid additives also may be in either the D- or L-configuration.

[0353] Suitable organic acid additive salts include, but are not limited to, sodium, calcium, potassium, and magnesium salts of all organic acids, such as salts of citric acid, malic acid, tartaric acid, fumaric acid, lactic acid (e.g., sodium lactate), alginic acid (e.g., sodium alginate), ascorbic acid (e.g., sodium ascorbate), benzoic acid (e.g., sodium benzoate or potassium benzoate), and adipic acid. The examples of the sweet taste improving organic acid additives described optionally may be substituted with at least one group chosen 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, carboxalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, anhydride, oximino, hydrazino, carbamyl, phosphor or phosphonato. In particular embodiments, the 93Atty Docket: 340391: 0640.49WO organic acid additive is present in the sweetener composition in an amount from about 10 ppm to about 5,000 ppm.

[0354] 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).

[0355] Suitable bitter compound additives include, but are not limited to, caffeine, quinine, urea, bitter orange oil, naringin, quassia, and salts thereof.

[0356] Suitable flavorant and flavoring ingredient additives for include, but are not limited to, vanillin, vanilla extract, mango extract, cinnamon, Citrus, coconut, ginger, viridiflorol, almond, menthol (including menthol without mint), grape skin extract, and grape seed extract. “Flavorant” and “flavoring ingredient” are synonymous and can include natural or synthetic substances or combinations thereof. Flavorants also include any other substance which imparts flavor and may include natural or non-natural (synthetic) substances which are safe for humans or animals when used in a generally accepted range. Non-limiting examples of proprietary flavorants include Döhler™ Natural Flavoring Sweetness Enhancer K14323 (Döhler™, 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, N.J., U.S.A.), and Sucramask™ (Creative Research Management, Stockton, Calif., U.S.A.).

[0357] Suitable polymer additives include, but are not limited to, chitosan, pectin, pectic, pectinic, polyuronic, polygalacturonic acid, starch, food hydrocolloid or crude extracts thereof (e.g., gum acacia senegal (Fibergum™), gum acacia seyal, carageenan), poly-L-lysine (e.g., poly-L-α-lysine or poly-L-ε-lysine), poly-L-ornithine (e.g., poly-L-α-ornithine or poly-L-ε-ornithine), polypropylene glycol, polyethylene glycol, poly(ethylene glycol methyl ether), polyarginine, polyaspartic acid, polyglutamic acid, polyethylene imine, alginic acid, sodium alginate, propylene glycol alginate, and sodium polyethyleneglycolalginate, sodium hexametaphosphate and its salts, and other cationic polymers and anionic polymers.

[0358] Other suitable polymer additives that also provide gelling and thickening properties include conventional low methoxyl (LMC) pectins. 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 utilized for low-calorie jams and jellies since it relies on calcium instead of sugar to solidify. LMC pectin gets increasingly firmer as calcium is added until it hits a saturation point. At that time, the process reverses and it becomes less firm. In some embodiments, the products for oral 94Atty Docket: 340391: 0640.49WO administration described herein may comprise one or more 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. In an embodiment, the product for oral administration further comprises a low methoxyl pectin.

[0359] Suitable protein or protein hydrolysate additives include, but are not limited to, bovine serum albumin (BSA), whey protein (including fractions or concentrates thereof such as 90% instant whey protein isolate, 34% whey protein, 50% hydrolyzed whey protein, and 80% whey protein concentrate), soluble rice protein, soy protein, protein isolates, protein hydrolysates, 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, and the like), collagen (e.g., gelatin), partially hydrolyzed collagen (e.g., hydrolyzed fish collagen), and collagen hydrolysates (e.g., porcine collagen hydrolysate).

[0360] 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 sulfosuccinate sodium, 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, lecithins, sucrose oleate esters, sucrose stearate esters, sucrose palmitate esters, sucrose laurate esters, and other emulsifiers, and the like.

[0361] 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, rutins (e.g., enzyme modified rutin Sanmelin™ AO (San-fi Gen F.F.I., Inc., Osaka, Japan)), neohesperidin, naringin, neohesperidin dihydrochalcone, and the like.

[0362] Suitable alcohol additives include, but are not limited to, ethanol.

[0363] Suitable astringent compound additives include, but are not limited to, tannic acid, europium chloride (EuCl3), gadolinium chloride (GdCl3), terbium chloride (TbCl3), alum, tannic acid, and polyphenols (e.g., tea polyphenols). In particular embodiments, the astringent additive is present in an amount from about 10 ppm to about 5,000 ppm. 95Atty Docket: 340391: 0640.49WO

[0364] In particular embodiments, sweetener compositions or flavor modifying compositions comprise the Myd polypeptide (or variant thereof), optionally in combination with one or more steviol glycosides (e.g., Reb M, Reb A), a polyol selected from erythritol, maltitol, mannitol, xylitol, sorbitol, and combinations thereof; and optionally at least one additional sweetener and / or functional ingredient. In a particular embodiment, the polyol is erythritol. The steviol glycoside (e.g., Reb M, Reb A) can be provided as a pure compound or as part of a Stevia extract or steviol glycoside mixture, as described above. The steviol glycoside (e.g., Reb M, Reb A) can be present in an amount from about 5% to about 100% by weight on a dry basis in either a steviol glycoside mixture or a Stevia extract.

[0365] In particular embodiments, the sweetener compositions or flavor modifying compositions comprise the MYD family protein (e.g., Myd polypeptide or variant thereof), optionally in combination with one or more steviol glycosides (e.g., Reb M, Reb A); a carbohydrate sweetener selected from sucrose, fructose, glucose, maltose and combinations thereof; and optionally at least one additional sweetener and / or functional ingredient. The steviol glycoside (e.g., Reb M, Reb A) can be provided as a pure compound or as part of a Stevia extract or steviol glycoside mixture, as described above. The steviol glycoside (e.g., Reb M, Reb A) can be present in an amount from about 5% to about 100% by weight on a dry basis in either a steviol glycoside mixture or a Stevia extract.

[0366] In particular embodiments, the sweetener compositions or flavor modifying compositions comprise the MYD family protein (e.g., Myd polypeptide or variant thereof), optionally in combination with one or more steviol glycosides (e.g., Reb M, Reb A); an amino acid selected from glycine, alanine, proline and combinations thereof; and optionally at least one additional sweetener and / or functional ingredient. The steviol glycoside can be provided as a pure compound or as part of a Stevia extract or steviol glycoside mixture, as described above. The steviol glycoside (e.g., Reb M, Reb A) can be present in an amount from about 5% to about 100% by weight on a dry basis in either a steviol glycoside mixture or a Stevia extract.

[0367] In particular embodiments, sweetener compositions or flavor modifying compositions comprise the MYD family protein (Myd polypeptide or variant thereof), optionally in combination with one or more steviol glycosides (e.g., Reb M, Reb A), a salt selected from sodium chloride, magnesium chloride, potassium chloride, calcium chloride and combinations thereof; and optionally at least one additional sweetener and / or functional ingredient. The steviol glycoside (e.g., Reb M, Reb A) can be provided as a pure compound or as part of a Stevia extract or steviol glycoside mixture, as described above.

[0368] FUNCTIONAL INGREDIENTS 96Atty Docket: 340391: 0640.49WO

[0369] The sweetener compositions or flavor modifying compositions disclosed herein can also contain one or more functional ingredients, which provide a real or perceived health benefit to the composition. Functional ingredients include, but are not limited to, saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydration agents, probiotics, prebiotics, weight management agents, osteoporosis management agents, phytoestrogens, long chain primary aliphatic saturated alcohols, phytosterols and combinations thereof.

[0370] Examples of suitable antioxidants for embodiments of this invention include, but are not limited to, vitamins, vitamin cofactors, minerals, hormones, carotenoids, carotenoid terpenoids, non-carotenoid terpenoids, flavonoids, flavonoid polyphenolics (e.g., bioflavonoids), flavonols, flavones, phenols, polyphenols, esters of phenols, esters of polyphenols, nonflavonoid phenolics, isothiocyanates, and combinations thereof. In some embodiments, the antioxidant is vitamin A, vitamin C, vitamin E, ubiquinone, mineral selenium, manganese, melatonin, α-carotene, β-carotene, lycopene, lutein, zeanthin, crypoxanthin, reservatol, eugenol, quercetin, catechin, gossypol, hesperetin, curcumin, ferulic acid, thymol, hydroxytyrosol, tumeric, thyme, olive oil, lipoic acid, glutathinone, gutamine, oxalic acid, tocopherol-derived compounds, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediaminetetraacetic acid (EDTA), tert- butylhydroquinone, acetic acid, pectin, tocotrienol, tocopherol, coenzyme Q10, zeaxanthin, astaxanthin, canthaxantin, saponins, limonoids, kaempfedrol, myricetin, isorhamnetin, proanthocyanidins, quercetin, rutin, luteolin, apigenin, tangeritin, hesperetin, naringenin, erodictyol, flavan-3-ols (e.g., anthocyanidins), gallocatechins, epicatechin and its gallate forms, epigallocatechin and its gallate forms (ECGC) theaflavin and its gallate forms, thearubigins, isoflavone phytoestrogens, genistein, daidzein, glycitein, anythocyanins, cyaniding, delphinidin, malvidin, pelargonidin, peonidin, petunidin, ellagic acid, gallic acid, salicylic acid, rosmarinic 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, lignan, antinutrients, bilirubin, uric acid, R-α-lipoic acid, N-acetylcysteine, emblicanin, and phytic acid, or combinations thereof. In alternate embodiments, the antioxidant is a synthetic antioxidant such as butylated hydroxytolune or butylated hydroxyanisole, for example. Other sources of suitable antioxidants for embodiments of this invention include, but are not limited to, fruits, vegetables, tea, cocoa, chocolate, spices, herbs, rice, organ meats from livestock, yeast, whole grains, or cereal grains.

[0371] Particular antioxidants belong to the class of phytonutrients called polyphenols (also known as “polyphenolics”), which are a group of chemical substances found in plants, characterized by the 97Atty Docket: 340391: 0640.49WO presence of more than one phenol group per molecule. Suitable polyphenols for embodiments of this invention, include catechins, proanthocyanidins, procyanidins, anthocyanins, quercerin, rutin, reservatrol, isoflavones, curcumin, punicalagin, ellagitannin, hesperidin, naringin, Citrus flavonoids, chlorogenic acid, other similar materials, and combinations thereof.

[0372] In particular embodiments, the antioxidant is a catechin such as, for example, 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, grape seed, red grape skin, purple grape skin, red grape juice, purple grape juice, berries, pycnogenol, and red apple peel.

[0373] In some embodiments, the antioxidant is chosen 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, cacao beans, cranberry, apple peel, plum, blueberry, black currants, choke berry, green tea, sorghum, cinnamon, barley, red kidney bean, pinto bean, hops, almonds, hazelnuts, pecans, pistachio, pycnogenol, and colorful berries.

[0374] In particular embodiments, the antioxidant is an anthocyanin. Suitable sources of anthocyanins for embodiments of this invention include, but are not limited to, red berries, blueberries, bilberry, cranberry, raspberry, cherry, pomegranate, strawberry, elderberry, choke berry, red grape skin, purple grape skin, grape seed, red wine, black currant, red currant, cocoa, plum, apple peel, peach, red pear, red cabbage, red onion, red orange, and blackberries.

[0375] In some embodiments, the antioxidant is chosen from quercetin, rutin or combinations thereof. Suitable sources of quercetin and rutin for embodiments of this invention include, but are not limited to, red apples, onions, kale, bog whortleberry, lingonberry, chokeberry, cranberry, blackberry, blueberry, strawberry, raspberry, black currant, green tea, black tea, plum, apricot, parsley, leek, broccoli, chili pepper, berry wine, and ginkgo.

[0376] 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, cranberry, blueberry, bilberry, mulberry, Japanese Itadori tea, and red wine.

[0377] In particular embodiments, the antioxidant is an isoflavone. Suitable sources of isoflavones for embodiments of this invention include, but are not limited to, soybeans, soy products, legumes, alfalfa sprouts, chickpeas, peanuts, and red clover.

[0378] 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. 98Atty Docket: 340391: 0640.49WO

[0379] In particular embodiments, the antioxidant is chosen from punicalagin, ellagitannin or combinations thereof. Suitable sources of punicalagin and ellagitannin for embodiments of this invention include, but are not limited to, pomegranate, raspberry, strawberry, walnut, and oak-aged red wine.

[0380] 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, grapefruits, and Citrus juices.

[0381] In particular embodiments, the antioxidant is chlorogenic acid. Suitable sources of chlor...

Claims

Atty Docket: 340391: 0640.49WO WE CLAIM:

1. A product for oral administration, optionally a beverage or beverage product, comprising a sweetener composition, the sweetener composition comprising one or more protein of the mycoduclein (Myd) family of proteins and one or more additional sweetener other than a protein of the MYD family of proteins.

2. The product for oral administration of claim 1, wherein the one or more additional sweetener is selected from a steviol glycoside sweetener, a mogroside sweetener, sucrose, glucose, fructose, galactose, xylose, lactose, ribose, lactulose, maltose, trehalose, allulose, sucralose, a polyol, honey, aspartame, erythritol, xylitol, sorbitol, monk fruit extract, dextrose, maltitol, acesulfame potassium (Ace-K), agave extract, tapioca syrup, polyglycitol syrup, sodium saccharin, or high fructose corn syrup (HFCS).

3. The product for oral administration of claim 1, wherein the one or more additional sweetener comprises a steviol glycoside sweetener.

4. The product for oral administration of claim 1, wherein the one or more additional sweetener is selected from Rebaudioside M (“Reb M”), RebM80, Rebaudioside D (“Reb D”), RebA95 and Rebaudioside A (“Reb A”).

5. The product for oral administration of claim 1, wherein the one or more additional sweetener is Reb M with a purity of greater than about 95%.

6. The product for oral administration of claim 1, wherein the one or more additional sweetener is siamenoside I or mogroside V.

7. The product for oral administration of claim 1, wherein the one or more additional sweetener is mogroside V.

8. The product for oral administration of claim 1, wherein the one or more additional sweetener is not a protein.

9. The product for oral administration of claim 1, wherein the one or more additional sweetener is monellin, thaumatin or brazzein.

10. The product for oral administration of claim 1, wherein the one or more additional sweeteners comprises one or more nutritive sweeteners.

11. The product for oral administration of any one of claims 1-10, wherein the one or more MYD family protein is a polypeptide having the amino acid sequence of SEQ ID NO:3, the amino 240Atty Docket: 340391: 0640.49WO acid sequence of SEQ ID NO:141 or is a sequence variant thereof with 1-6 amino acid substitutions, deletions, or additions.

12. The product for oral administration of any one of claims 1-10, wherein the one or more MYD family protein is not a naturally-occurring Myd (mydodulcein) polypeptide of Mattirolomyces terfezioides.

13. The product for oral administration of any one of claims 1-10, wherein the one or more MYD family protein is a polypeptide having the amino acid sequence of SEQ ID NO:3 or the amino acid sequence of SEQ ID NO:

141.

14. The product for oral administration of any one of claims 1-10, wherein the one or more MYD family protein comprises: (i) a polypeptide sequence selected from amino acid sequences of any variant listed in Table 8, Table 9 or Table 10; (ii) a polypeptide sequence having at least 90% sequence identity to a polypeptide sequence selected from the group consisting of amino acid sequences SEQ ID NO:3, SEQ ID NO: 141 or of any variant listed in Table 8; Table 9 or Table 10; (iii) a polypeptide sequence containing at least one modification by deletion, insertion, substitution, or addition of no more than 24 amino acids, relative to a polypeptide sequence selected from the group consisting of an amino acid sequence of SSEQ ID NO:3, SEQ ID NO:141; or of any variant listed in Table 8, Table 9 or Table 10; or (iv) a polypeptide of any of (i)- (iii), wherein the methionine at position 1 is absent; and / or the polypeptide further comprises a protein / peptide tag, optionally an affinity tag, optionally a histidine tag or optionally a (His)6tag.

15. The product for oral administration of any one of claims 1-10, wherein the one or more MYD family proteins is a protein expressed in a host cell from a polynucleotide encoding the one or more MYD family protein, wherein the host cell is a cell other than a cell of the truffle Mattirolomyces terfezioides.

16. The product for oral administration of any one of claims 1-10, wherein the one or more MYD family proteins is a protein expressed in a host cell from the polynucleotide of SEQ ID NO:2, a polynucleotide having 90% sequence identity with the polynucleotide of SEQ ID NO:2, or variants thereof that are codon-optimized for expression in the host cell, wherein the host cell is a cell other than a cell of the truffle Mattirolomyces terfezioides. 241Atty Docket: 340391: 0640.49WO 17. The product for oral administration of any one of claims 1-10, wherein the one or more MYD family proteins are expressed in a host cell from the polynucleotide of SEQ ID NO:2 or a variant of SEQ ID NO:2 codon-optimized for expression in the host cell, wherein the host cell is a cell other than a cell of the truffle Mattirolomyces terfezioides.

18. The product for oral administration of any one of claims 15-17, wherein the host cell is a bacterium or yeast.

19. The product for oral administration of any one of claims 15-17, wherein the host cell is Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris.

20. The product for oral administration of any one of claims 1-20, wherein the one or more MYD family protein is present in an amount between about 1 ppm and about 50 ppm.

21. The product for oral administration of any one of claims 1-20, wherein the one or more MYD family protein is present in an amount between about 1 ppm and about 40 ppm.

22. The product for oral administration of any one of claims 1-20, wherein the polypeptide is present in an amount selected from an amount between about 1 ppm and about 30 ppm, about 1 ppm and about 25 ppm, about 1 ppm and about 20 ppm or about 1 ppm and about 15 ppm.

23. The product for oral administration of any one of claims 1-20, wherein the polypeptide is present in an amount less than about 9 ppm.

24. The product for oral administration of any one of claims 1-23, which has at least one improved organoleptic property compared to the product for oral administration without the one or more MYD family proteins, wherein the organoleptic property is selected from the group consisting of aroma, flavor, basic taste (sweetness, sour, saltiness, bitterness or umami), aftertaste or linger, temporal profile, mouthfeel or a combination thereof.

25. The product for oral administration of any one of claims 1-23, which has at least one improved organoleptic property compared to the product for oral administration without the one or more MYD family proteins, wherein the organoleptic property is a reduction in bitterness, aftertaste or an improvement in mouthfeel.

26. The product for oral administration of any one of claims 1-23, wherein the at least one improved organoleptic property is an improvement in mouthfeel and the amount of the polypeptide is between about 1 ppm and about 40 ppm. 242Atty Docket: 340391: 0640.49WO 27. The product for oral administration of claim 1, which is a beverage or beverage product selected from a low-calorie or no-calorie beverage or beverage product.

28. The product for oral administration of any one of claims 1-26, which is a beverage or beverage product selected from a low-calorie or no-calorie beverage or beverage product.

29. The product for oral administration of claim 1, which is a beverage or beverage product selected from the group consisting of cola, ginger-ale, soft drinks, root beer, fruit juice, fruit- flavored juice, vegetable juice, vegetable-flavored juice, sports drinks, energy drinks, plant protein beverage, near water drinks (e.g., water with natural or synthetic flavorants), tea type (e.g. black tea, green tea, red tea, oolong tea), coffee, cocoa drink, beverage containing milk components (e.g. milk beverages, coffee containing milk components, café au lait, milk tea, fruit milk beverages).

30. The product for oral administration of any one of claims 1-26, which is a beverage or beverage product selected from the group consisting of cola, ginger-ale, soft drinks, root beer, fruit juice, fruit-flavored juice, vegetable juice, vegetable-flavored juice, sports drinks, energy drinks, plant protein beverage, near water drinks (e.g., water with natural or synthetic flavorants), tea type (e.g. black tea, green tea, red tea, oolong tea), coffee, cocoa drink, beverage containing milk components (e.g. milk beverages, coffee containing milk components, café au lait, milk tea, fruit milk beverages).

31. The product for oral administration of claim 1, which is a mouthwash.

32. The product for oral administration of claim 1, which is a mint confectionary.

33. The product for oral administration of claim 1, which is chewing gum.

34. The product for oral administration of claim 1, which is a solid food product, optionally yogurt or ice cream.

35. The product for oral administration of claim 1, which is an oral hygiene product, optionally a toothpaste or tooth powder.

36. The product for oral administration of claim 1, which is a pharmaceutical.

37. The product for oral administration of any one of claims 1-26, which is a mouthwash, a mint confectionary, chewing gum, a solid food product, yogurt, ice cream, toothpaste or tooth powder, or a pharmaceutical 243Atty Docket: 340391: 0640.49WO 38. The product for oral administration of clam 1, wherein the product for oral administration is other than Mattirolomyces terfezioides 39. The product for oral admininstration of any one of claims 1-26, wherein the product for oral administration is other than Mattirolomyces terfezioides.

40. A method of modifying / changing at least one organoleptic property of a product for oral administration comprising adding a sweetener composition to a matrix thereby providing modified product for oral administration having at least one modified / changed organoleptic property, wherein the sweetener composition comprises one or more protein of the mycoduclein (MYD) family of proteins and one or more additional sweetener other than a protein of the MYD family of proteins.

41. The method of claim 40, wherein the one or more additional sweetener is selected from a steviol glycoside sweetener, a mogroside sweetener, sucrose, glucose, fructose, galactose, xylose, lactose, ribose, lactulose, maltose, trehalose, allulose, sucralose, a polyol, honey, aspartame, erythritol, xylitol, sorbitol, monk fruit extract, dextrose, maltitol, acesulfame potassium (Ace-K), agave extract, tapioca syrup, polyglycitol syrup, sodium saccharin, or high fructose corn syrup (HFCS).

42. The method of claim 40, wherein the one or more additional sweetener comprises a steviol glycoside sweetener.

43. The method of claim 40, wherein the one or more additional sweetener is selected from Rebaudioside M (“Reb M”), RebM80, Rebaudioside D (“Reb D”), RebA95 and Rebaudioside A (“Reb A”).

44. The method of claim 40, wherein the one or more additional sweetener is Reb M with a purity of greater than about 95%.

45. The method of claim 40, wherein the one or more additional sweetener is siamenoside I or mogroside V.

46. The method of claim 40, wherein the one or more additional sweetener is mogroside V.

47. The method of claim 40, wherein the one or more additional sweetener is not a protein.

48. The method of claim 40, wherein the one or more additional sweetener is monellin, thaumatin or brazzein. 244Atty Docket: 340391: 0640.49WO 49. The method of claim 40, wherein the one or more additional sweeteners comprises one or more nutritive sweeteners.

50. The method of any one of claims 40-49, wherein the one or more MYD family protein is a polypeptide having the amino acid sequence of SEQ ID NO:3, the amino acid sequence of SEQ ID NO:141 or is a sequence variant thereof with 1-6 amino acid substitutions, deletions, or additions.

51. The method of any one of claims 40-49, wherein the one or more MYD family protein is not a naturally-occurring Myd (mydodulcein) polypeptide of Mattirolomyces terfezioides.

52. The method of any one of claims 40-49, wherein the one or more MYD family protein is a polypeptide having the amino acid sequence of SEQ ID NO:3 or the amino acid sequence of SEQ ID NO:

141.

53. The method of any one of claims 40-49, wherein the one or more MYD family protein comprises: (i) a polypeptide sequence selected from amino acid sequences of any variant listed in Table 8, Table 9 or Table 10; (ii) a polypeptide sequence having at least 90% sequence identity to a polypeptide sequence selected from the group consisting of amino acid sequences SEQ ID NO:3, SEQ ID NO: 141 or of any variant listed in Table 8; Table 9 or Table 10; (iii) a polypeptide sequence containing at least one modification by deletion, insertion, substitution, or addition of no more than 24 amino acids, relative to a polypeptide sequence selected from the group consisting of an amino acid sequence of SSEQ ID NO:3, SEQ ID NO:141; or of any variant listed in Table 8, Table 9 or Table 10; or (iv) a polypeptide of any of (i)- (iii), wherein the methionine at position 1 is absent; and / or the polypeptide further comprises a protein / peptide tag, optionally an affinity tag, optionally a histidine tag or optionally a (His)6tag.

54. The method of claim 40, wherein the organoleptic property is selected from the group consisting of aroma, flavor, basic taste (sweetness, sour, saltiness, bitterness or umami), aftertaste or linger, temporal profile, mouthfeel or a combination thereof.

55. The method of claim 40, wherein the organoleptic property is flavor and the flavor is selected from the group consisting of caramel, chocolate, citrus (lemon or lime), cola, cotton candy, 245Atty Docket: 340391: 0640.49WO cranberry, dairy, mango, mint, mixed berry, orange cream, strawberry, toffee, tomato, tropical fruit blends, vanilla or a combination thereof.

56. A method for improving the sweet taste of a product for oral administration which product comprises one or more first sweetener, the method comprising adding one or more MYD family protein to the product for oral administration.

57. The method of claim 56, wherein the product for oral administration is a liquid or solid food product and optionally is a beverage.

58. The method of claim 56 or claim 57, wherein the one or more first sweetener comprises a steviol glycoside sweetener.

59. The method of claim 56 or 57, wherein the one or more first sweetener is selected from Rebaudioside M (“Reb M”), RebM80, Rebaudioside D (“Reb D”), RebA95 and Rebaudioside A (“Reb A”).

60. The method of claim 56 or 57, wherein the one or more first sweetener is Reb M with a purity of greater than about 95%.

61. The method of claim 56 or 57, wherein the one or more MYD family protein is added in an amount between about 1 ppm and about 50 ppm.

62. The method of claim 56 or 57, wherein the one or more MYD family protein is present in an amount less than about 9 ppm. 246

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