Sweet protein derived from truffles

Abstract

This paper describes a newly identified fungal sweet-modifying protein and a cDNA encoding the protein. Specifically, this paper describes a sweet-activating Myd protein and a cDNA encoding the same, as well as methods for isolating the cDNA and isolating and expressing the protein. This paper also describes the use of sweetening compositions containing the proteins of this invention, and methods for providing improved flavor to oral products.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 044245, filed June 25, 2020, which provisional patent application is incorporated by reference.

[0002] Incorporation by Reference of Electronically Submitted Materials

[0002] Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: one 86,678-byte ASCII (text) file named "338820_640_30A_WO_ST25.TXT" created on June 25, 2021. [Background technology]

[0003]

[0003] Excessive intake of nutritive sweeteners has long been associated with diet-related health problems such as obesity, heart disease, metabolic disorders, and dental disease. In response, consumers are increasingly seeking ways to reduce the amount of nutritive sweeteners in their diets. Manufacturers are responding to this demand by seeking to develop substitutes for nutritive sweeteners that can better mimic the desirable taste and functional properties of nutritive sweeteners.

[0004]

[0004] Zero- or low-calorie sweeteners, preferably derived from natural sources, are desirable to limit the adverse effects of high sugar consumption (e.g., diabetes and obesity, among others). Commonly known zero- or low-calorie sweeteners include aspartame, acesulfame potassium, monk fruit extract, neotame, saccharin, stevia, and sucralose. However, these sweeteners have taste deficiencies, such as bitterness.

[0005]

[0005] Truffles are the underground fruiting bodies of several ascomycete fungi, including genera belonging to the order Chamaecyparis of the class Chamaecyparis. Truffles are ectomycorrhizal fungi and are therefore usually found in close association with tree roots.

[0006]

[0006] Seven proteins that modify sweetness and taste perception have been identified to date: brazzein, thaumatin, monellin, curculin, mabinlin, miraculin, and pentadin. The key residues on the protein surface responsible for biological activity have yet to be reliably identified for any of these proteins. Monellin was found to be 100,000 times sweeter than sucrose on a molar basis, followed by brazzein and thaumatin, which are 500 and 3,000 times sweeter than sucrose on a gram basis, respectively. All of these proteins have been isolated from plants growing in tropical rainforests. While most of them share no sequence homology or structural similarity, thaumatin shares extensive similarity at the protein sequence level with certain nonsweet proteins found in other plants. No sweet-taste modifying proteins are known from fungi.

[0007]

[0007] There remains a need in the art to produce novel low- or zero-calorie sweeteners with improved taste from natural sources. There remains a need in the art to economically produce such sweetening compositions from their potential sources, particularly fungal species of the Ascomycetes. Summary of the Invention

[0008]

[0008] The present invention relates to a newly identified sweet-tasting protein from fungi, and the gene and cDNA encoding said protein, also referred to herein as MYD / Myd. More specifically, the present invention relates to a newly identified sweet-tasting protein, the gene and cDNA encoding said protein, and methods of using such protein, gene, and cDNA in modulating the taste of foods. The present invention provides, inter alia, DNA sequences encoding a novel sweet-tasting protein identified herein as MYD1 and the corresponding polypeptide, Myd1 (also referred to as mycodulcein). Myd1 is the first sweet-tasting protein identified from fungi. Myd1 reduces the sour, bitter, or astringent taste of foods and beverages, and furthermore, Myd1 has taste-enhancing, i.e., taste-modifying, activity in foods and beverages.

[0009]

[0009] The present invention provides a polynucleotide (e.g., an isolated polynucleotide) encoding a polypeptide having sweet taste modulating activity, wherein the polynucleotide sequence encodes a polypeptide selected from the group consisting of: (a) a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75; (b) a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; and (c) a polypeptide sequence modified from a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17 by deletion, insertion, substitution, or addition of 24 or less amino acids.

[0010] In one aspect, the polypeptide sequence of the polypeptide having sweet taste modulating activity is the polypeptide sequence set forth in SEQ ID NO: 3, or a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 3. In another aspect, the polypeptide encoding the polypeptide having sweet taste modulating activity is not the polypeptide of SEQ ID NO: 3.

[0011] In another aspect, the polypeptide sequence comprises amino acid residues 1-11, 17-32, 39, 40, 45-67, 73-100, and 110-121 of SEQ ID NO:3.

[0012]

[0012] The present invention also provides a polynucleotide (e.g., an isolated polynucleotide) selected from the group consisting of: (a) a polynucleotide comprising a nucleic acid sequence set forth in SEQ ID NO: 2 having at least one substitution modification; (b) a polynucleotide comprising a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 2, wherein the polynucleotide is not the polynucleotide of SEQ ID NO: 2; and (c) a polynucleotide comprising (i) the nucleic acid sequence set forth in SEQ ID NO: 2 and (ii) a nucleotide sequence encoding a histidine tag; wherein the polynucleotide encodes a polypeptide having sweet taste modulating activity.

[0013] In one aspect, the polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 75. In particular, the polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, or SEQ ID NO: 68.

[0014]

[0014] In certain aspects, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67.

[0015]

[0015] The polynucleotide encoding the polypeptide having sweet taste modulating activity is optionally operably linked to a heterologous regulatory element. Additionally or alternatively, the polynucleotide sequence further encodes a protein tag or label. The protein tag is optionally an affinity tag, and the protein is optionally a histidine tag.

[0016] In one aspect, the polynucleotide comprises SEQ ID NO:20, which corresponds to the coding sequence for His-tagged mycodulcein in E. coli (residues 364-381 correspond to the optional His-tag sequence). SEQ ID NO:20 is codon-optimized for expression in E. coli. In another aspect, the polynucleotide comprises SEQ ID NO:22, which corresponds to the coding sequence for His-tagged mycodulcein in S. cerevisiae (residues 364-381 correspond to the optional His-tag sequence). SEQ ID NO:22 is codon-optimized for expression in S. cerevisiae. The corresponding polypeptide of SEQ ID NO:21 corresponds to a His-tagged mycodulcein protein (residues 122-127 correspond to the optional His-tag sequence), and this polypeptide sequence is the same for expression in E. coli and S. cerevisiae.

[0017]

[0017] An expression cassette is provided, comprising a polynucleotide and a vector containing the polynucleotide, and a host cell transformed with the vector. Also provided is a method for producing a protein having sweet taste modulating activity, comprising culturing a host cell in a medium under conditions that result in the production of the protein having sweet taste modulating activity.

[0018]

[0018] The present invention encompasses a polypeptide (e.g., an isolated polypeptide) comprising a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17; wherein the polypeptide may contain at least one substitution or modification with respect to the polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, and the polypeptide may further comprise a protein tag, particularly a histidine tag, and the polypeptide has sweet taste modulating activity.

[0019] In one aspect, the polypeptide comprises SEQ ID NO: 3, or a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 3. In another aspect, the polypeptide is not the polypeptide of SEQ ID NO: 3.

[0020] In yet another aspect, the polypeptide comprises amino acid residues 1-11, 17-32, 39, 40, 45-67, 73-100, and 110-121 of SEQ ID NO:3.

[0021] In one aspect, a polypeptide (e.g., an isolated polypeptide) comprises a polypeptide sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75, wherein the polypeptide may not be SEQ ID NO:3. In particular, the polypeptide comprises the amino acid sequence of SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, or SEQ ID NO:68.

[0022] The present invention provides a composition comprising (a) a product for oral administration, wherein the product is not a Mattirolomyces terfezioides truffle, and (b) a sweetening composition comprising a polypeptide in combination, wherein the combination has an enhanced sweetness compared to the product for oral administration. In one aspect, the polypeptide comprises an amino acid sequence having at least 80% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. In another aspect, the polypeptide has a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; wherein the polypeptide may contain at least one substitution modification with respect to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, and the polypeptide may not be the polypeptide of SEQ ID NO:3, and the polypeptide may further comprise a histidine tag, and the polypeptide has sweet taste modulating activity. For example, the polypeptide in the sweetening composition may comprise the amino acid sequence of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75, and not consist of SEQ ID NO:3. In particular, the polypeptide comprises the amino acid sequence of SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66 or SEQ ID NO:68.

[0023]

[0023] In one aspect, the present invention provides a sweetening composition comprising a polypeptide comprising an amino acid sequence having at least 80% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, wherein the polypeptide may be a polypeptide other than the polypeptide having the amino acid sequence of SEQ ID NO:3.

[0024] The present invention provides a method for modifying the taste of an oral product, comprising combining the oral product with an effective amount of a sweetening composition comprising a polypeptide, wherein the oral product is not a Mattirolomyces terfezioides truffle, and the combination has an enhanced sweetness compared to the oral product. In one aspect, the polypeptide comprises an amino acid sequence having at least 80% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. In another aspect, the polypeptide has a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; wherein the polypeptide may contain at least one substitution modification with respect to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, the polypeptide may not be the polypeptide of SEQ ID NO:3, the polypeptide may further comprise a histidine tag, and the polypeptide has sweet taste modulating activity. For example, the polypeptide in the sweetening composition may comprise the amino acid sequence of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75, and may not consist of SEQ ID NO:3. In particular, the polypeptide comprises the amino acid sequence of SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66 or SEQ ID NO:68.

[0025]

[0025] The product for oral administration can be a food, a beverage, a dietary supplement composition, or a pharmaceutical composition. Examples of food products include, but are not limited to, baked goods; sweet bakery products, pre-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings, gelatin and pudding; frozen desserts; yogurt; snack bars; bread products; pre-made bread mixes for preparing bread products; sauces, syrups and dressings; sweet spreads; confectionery products; and sweet breakfast cereals. Examples of beverage products include, but are not limited to, carbonated beverages; non-carbonated beverages; and beverage concentrates.

[0026] The present invention also provides a method for purifying a polypeptide having sweet taste modulating activity, comprising: (a) obtaining a composition comprising the polypeptide; and (b) purifying the composition via hydrophobic interaction chromatography (HIC) followed by size exclusion chromatography (SEC). In one aspect, the polypeptide comprises an amino acid sequence having at least 80% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. In another aspect, the polypeptide has a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17; wherein the polypeptide may contain at least one substitution modification with respect to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, the polypeptide may not be the polypeptide of SEQ ID NO:3, the polypeptide may further comprise a histidine tag, and the polypeptide has sweet taste modulating activity. For example, the polypeptide may comprise the amino acid sequence of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75, and may not consist of SEQ ID NO:3. In particular, the polypeptide comprises the amino acid sequence of SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66 or SEQ ID NO:68.

[0027] Other aspects and embodiments of the present invention will become apparent upon review of the drawings, detailed description, and non-limiting examples herein. [Brief explanation of the drawings]

[0028] [Figure 1]

[0028] Figure 1 shows the predicted three-dimensional structure of Myd1 based on sequence data using the PHYRE2.0 protein fold prediction tool. [Figure 2]

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

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

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

[0032] Figure 5A shows a comparison of the predicted tertiary structure of mycodulcein with the known crystal structures (protein database) of thaumatin (PDB:1RQW), monellin (PDB:2O9U), brazzein (PDB:1BRZ), and hen egg white lysozyme (PDB:1LSN), demonstrating that mycodulcein has a predicted tertiary structure similar to other known sweet proteins, all of which contain an antiparallel β-sheet with an α-helix parallel to the β-sheet. [Figure 5B]

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

[0034] Figure 6 shows the results of comparing mutated His-tagged mycodulceins with each other and with His-tagged non-mutated mycodulcein equated to equivalent protein concentrations as measured by ELISA for sweetness intensity, onset time of sweetness perception, and duration of sweetness perception. [Figure 7A]

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

[0036] Figure 7B shows SDS-PAGE analysis, Coomassie stain, of two elution fractions collected during gradient elution from a HiScreen Capto Butyl column analyzed by SDS-PAGE. Lane 1 shows elution fraction 1, which does not contain mycodulcein, and lane 2 shows eluted mycodulcein. The purity of the elution fractions was determined to be approximately 86% by GelAnalyzer. The arrow indicates the mycodulcein band. [Figure 7C]

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

[0029]

[0038] Therefore, the present invention provides isolated nucleic acid molecules encoding proteins that can modulate sweetness, and the polypeptides they encode.As described herein, the polypeptides of the present invention mediate sweetness perception alone or in combination with foods, beverages, dietary supplements, or pharmaceuticals.The present invention also provides isolated polypeptides that can modify sweetness, and their compositions together with foods, beverages, dietary supplements, or pharmaceutical compositions, whereby the resulting combination has a sweet taste.The present invention also provides a method for modifying the sweetness of foods, beverages, dietary supplements, or pharmaceutical compositions by using the isolated polynucleotides and polypeptides of the present invention.

[0030]

[0039] In one aspect of the present invention, we provide a newly identified fungal sweet protein, designated herein as Myd1. The term "Myd polypeptide" is used herein to identify any polypeptide according to the present invention that has at least 80% sequence identity to, for example, SEQ ID NO:3 and also has sweet-tasting activity. Myd polypeptides also encompass peptides represented by SEQ ID NOs:8-17, which have sweet-tasting activity. A sweet, partially purified extract of M. terfezioides gleba was subjected to de novo amino acid sequencing to identify a 20-mer N-terminal sequence (SEQ ID NO:4). The Myd1 coding sequence (putatively derived from the MYD1 gene) was identified after de novo assembly of the entire transcriptome of M. terfeziodes gleba using RNA-seq reads. Screening the entire transcriptome of M. terfeziodes with the 20-mer N-terminal sequence identified a transcript predicted to encode a protein with 100% identity at the N-terminus. The identified transcript is predicted to encode a 121-amino acid protein. This method identified SEQ ID NO:1. The start and stop codons in the transcript were identified to identify the putative coding sequence of SEQ ID NO:2. SEQ ID NO:3 is a predicted protein of 121 amino acids. The identity between the predicted protein SEQ ID NO:3 and other protein sequences in GENBANK was 31% or less. The coding sequences of native mycodulceins codon-optimized for expression in E. coli and Saccharomyces cerevisiae correspond to the nucleic acid sequences of SEQ ID NO:20 and SEQ ID NO:22, respectively (which encode the amino acid sequence of SEQ ID NO:3 with an optional six-residue histidine tag, i.e., the amino acid sequence of SEQ ID NO:21).

[0031]

[0040] In one aspect, the "Myd polypeptide" herein has at least 10% or more (e.g., 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sweet taste-modifying activity compared to a naturally occurring Myd polypeptide isolated from M. terfeziodes gleba by extraction. In another aspect, the "Myd polypeptide" herein has at least 50% or more sweet taste-modifying activity compared to a naturally occurring Myd polypeptide isolated from M. terfeziodes gleba by extraction. In one aspect, the "Myd polypeptide" herein has at least 80% or more sweet taste-modifying activity compared to a naturally occurring Myd polypeptide isolated from M. terfeziodes gleba by extraction. Sweetness-modifying activity can be measured by any comparative method known in the art, particularly by any method described in the Examples herein, and more particularly using the sensory panel method described herein.

[0032]

[0041] Without wishing to be bound by any particular theory, Myd1 is thought to be involved in sweet taste activation, for example, as an agonist of taste receptor 1 member 2 (T1R2) and / or taste receptor 1 member 3 (T1R3). However, Myd1 may also agonize other taste receptors, such as bitter, umami, sour, and salty. Isolated or purified Myd polypeptides can then be used in the food and pharmaceutical industries to customize taste, for example, to adjust the sweetness of foods or drugs.

[0033]

[0042] In a first aspect, the present invention provides a polynucleotide (e.g., an isolated polynucleotide) encoding a polypeptide having sweetness modulating activity, wherein the polypeptide sequence is at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110% or more similar to the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17. and (c) an amino acid sequence modified from the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17 by deletion, insertion, substitution, or addition of 24 or fewer amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and any range thereof). In some embodiments, the amino acid sequence of the polypeptide having sweet taste modulating activity is the amino acid sequence set forth in SEQ ID NO: 3, an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 3, or an amino acid sequence modified from the amino acid sequence of SEQ ID NO: 3 by deletion, insertion, substitution, or addition of 24 or fewer amino acids.

[0034]

[0043] In a particular aspect, the present invention provides a polynucleotide (e.g., an isolated polynucleotide) encoding a polypeptide having sweet taste modulating activity, wherein the polynucleotide sequence encodes a polypeptide selected from the group consisting of: (a) a polypeptide sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; (b) a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; and (c) a polypeptide sequence modified from a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17 by a deletion, insertion, substitution, or addition of 24 or fewer amino acids; wherein the isolated polypeptide encoding the polypeptide having sweet taste modulating activity is not the polypeptide of SEQ ID NO:3.

[0035]

[0044] In another aspect, the invention provides a polynucleotide (e.g., an isolated polynucleotide), the polynucleotide comprising (a) a sequence selected from the group consisting of: (a) a sequence selected from the group consisting of: (i ... 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, and any ranges thereof; and (b) a polynucleotide comprising a nucleic acid sequence that may contain modifications (e.g., deletions, insertions, substitutions, or additions) of the following sequences relative to the nucleic acid sequence set forth in SEQ ID NO:2: The present invention encompasses polynucleotides selected from the group consisting of: a polynucleotide comprising a nucleic acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity, wherein the polynucleotide may not be the polynucleotide of SEQ ID NO:2. In one embodiment, the polynucleotide encodes a polypeptide having sweet taste modulating activity. In one embodiment, the amino acid sequence of the polypeptide having sweet taste modulating activity is the amino acid sequence set forth in SEQ ID NO:3.

[0036]

[0045] In one aspect, the invention provides a polynucleotide (e.g., an isolated polynucleotide), the polynucleotide sequence of which is (a) set forth in SEQ ID NO: 2 and at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 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, and any ranges therein); (b) a polynucleotide comprising a nucleic acid sequence having a substitution modification of SEQ ID NO: (c) a polynucleotide comprising a nucleic acid sequence having at least 90% (at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 2, wherein the polynucleotide may not be the polynucleotide of SEQ ID NO: 2; and (c) a polynucleotide comprising (i) the nucleic acid sequence set forth in SEQ ID NO: 2 and (ii) a nucleotide sequence encoding a histidine tag; wherein the polynucleotide encodes a polypeptide having sweet taste modulating activity.

[0037]

[0046] The polynucleotide encoding Myd of the present invention can be in the form of single-stranded or double-stranded DNA, RNA or an artificial nucleic acid, or can be an intron-free cDNA or chemically synthesized DNA.

[0038]

[0047] The terms "nucleic acid" or "nucleic acid sequence" refer to deoxyribonucleotide or ribonucleotide oligonucleotides in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides. The term also encompasses nucleic acid-like structures with synthetic backbones (e.g., Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, Oxford Univ. Press (1991); Antisense Strategies, Annals of the NY Academy of Sciences, Vol. 600, edited by 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)).

[0039]

[0048] Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved, for example, by generating sequences in which the third position of one or more selected codons is substituted with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0040]

[0049] The present invention also provides an expression cassette comprising a polynucleotide encoding a Myd polypeptide and a host cell transformed with the vector.

[0041]

[0050] In another aspect, the invention provides 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 polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. Peptides (e.g., isolated polypeptides) are provided, wherein the polypeptides comprise at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, The polypeptide may contain modifications (e.g., deletions, insertions, substitutions, or additions) of any of the following: 7, 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, and any ranges among these values; the polypeptide may further comprise a histidine tag; and the polypeptide has sweet taste modulating activity. The term "consisting essentially of" allows for the inclusion of ingredients that are not essential to and do not substantially affect the function or activity of the product, such as anti-caking agents, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltodextrose, gum acacia, etc.).

[0042]

[0051] The polypeptide comprises SEQ ID NO:3 or at least 80% sequence identity to SEQ ID NO:3. The polypeptide comprises SEQ ID NO:8 or at least 80% sequence identity to SEQ ID NO:8. The polypeptide comprises SEQ ID NO:9 or at least 80% sequence identity to SEQ ID NO:9. The polypeptide comprises SEQ ID NO:10 or at least 80% sequence identity to SEQ ID NO:10. The polypeptide comprises SEQ ID NO:11 or at least 80% sequence identity to SEQ ID NO:11. The polypeptide comprises SEQ ID NO:12 or at least 80% sequence identity to SEQ ID NO:12. The polypeptide comprises SEQ ID NO:13 or at least 80% sequence identity to SEQ ID NO:13. The polypeptide comprises SEQ ID NO:14 or at least 80% sequence identity to SEQ ID NO:14. The polypeptide comprises SEQ ID NO:15 or at least 80% sequence identity to SEQ ID NO:15. The polypeptide comprises SEQ ID NO:16 or at least 80% sequence identity to SEQ ID NO:16. The polypeptide comprises SEQ ID NO:17 or at least 80% sequence identity to SEQ ID NO:17.

[0043]

[0052] In one embodiment, the polypeptide sequence is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. In one embodiment, the polypeptide is not the polypeptide of SEQ ID NO: 3.

[0044]

[0053] In one aspect, the polypeptide comprises amino acid residues 1-11, 17-32, 39, 40, 45-67, 73-100, and 110-121 of SEQ ID NO: 3. In one aspect, the polypeptide comprises amino acid residues 1-11, 17-32, 39, 40, 45-67, 73-100, and 110-121 of SEQ ID NO: 3, but the polypeptide is not a polypeptide having the amino acid sequence of SEQ ID NO: 3.

[0045]

[0054] In another aspect, a polypeptide comprises amino acid residues 1-121 of SEQ ID NO:3, wherein there is at least one (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, or any range of values ​​therein) amino acid substitution, addition, insertion, or deletion at amino acid residues 12-16, 33-38, 41-44, 68-72, or 101-109 of the polypeptide sequence compared to SEQ ID NO:3 at the listed residues. In another aspect, the polypeptide sequence of the polypeptide having sweet taste modulating activity encoded by the polynucleotide is a polypeptide comprising amino acid residues 1-121 of SEQ ID NO:3, wherein there is at least one amino acid substitution, addition, insertion, or deletion at amino acid residues 12-16, 33-38, 41-44, 68-72, or 101-109 of the polypeptide sequence compared to SEQ ID NO:3 at the listed residues, and the polypeptide has at least 80% sequence identity to SEQ ID NO:3.

[0046]

[0055] In another aspect, the invention encompasses a recombinant polypeptide having sweet taste modulating activity, comprising, consisting essentially of, or consisting of 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 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17 fused to a heterologous signal or transit peptide. The term "consisting essentially of" allows for the inclusion of ingredients that are not essential to the function or activity of the product and do not substantially affect the function or activity, such as anti-caking agents, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltodextrose, gum acacia, etc.).

[0047]

[0056] In another aspect, the present invention encompasses polypeptides that have sweet taste modulating activity and comprise, consist essentially of, or consist of amino acids having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:3 (or SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17), and contain at least one substitution modification relative to SEQ ID NO:3 (or SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17). In some aspects, the polypeptides of the invention comprise 1-24 amino acid substitutions at positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 1 , 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or 121.The term "consisting essentially of" allows for the inclusion of ingredients that are not essential to the function or activity of the product and do not substantially affect the function or activity, such as anti-caking agents, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltodextrose, gum acacia, etc.).

[0048]

[0057] In particular aspects, the polypeptide sequence of the polypeptide encoded by the polynucleotide and having sweet taste modulating activity comprises the amino acid sequence of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75.

[0049]

[0058] SEQ ID NO:71 (consensus sequence 1) corresponds to SEQ ID NO:3, except that any amino acid can be at positions 3, 11-16, 26, 33, 34, 36-38, 41-43, 51, 57, 66, 68-72, 85, 86, 89, 97, 101-110, 117, and 120. The present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO:71.

[0050]

[0059] SEQ ID NO:72 (consensus sequence 2) corresponds to SEQ ID NO:3, except that positions 3, 11-16, 26, 33, 37, 38, 41, 43, 51, 57, 66, 68-70, 72, 85, 86, 89, 97, 101-103, 105-110, 117, and 120 are any amino acid (i.e., the prolines at positions 34, 36, 42, 71, and 104 of SEQ ID NO:3 are maintained). The present invention provides polypeptides comprising the amino acid sequence of SEQ ID NO:72.

[0051]

[0060] SEQ ID NO:73 (consensus sequence 3) and SEQ ID NO:74 (consensus sequence 4) correspond to SEQ ID NO:3, except that positions 3, 11-16, 26, 33, 37, 38, 41, 43, 51, 57, 66, 68-70, 72, 85, 86, 89, 97, 101-103, 105-110, 117, and 120 may include conservative modifications as described herein. The present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO:73. The present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO:74.

[0052]

[0061] SEQ ID NO:75 (consensus sequence 5) corresponds to SEQ ID NO:3, except that positions 3, 11, 26, 51, 57, 66, 69, 85, 86, 89, 97, 103, 106, 110, 117, and 120 may include conservative modifications as described herein. The present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO:75.

[0053]

[0062] In a particular aspect, the polypeptide sequence of a polypeptide having sweet taste modulating activity comprises SEQ ID NO: 3 with one or more modifications (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16) at residues 3, 11, 26, 51, 57, 66, 69, 85, 86, 89, 97, 103, 106, 110, 117, and 120 of SEQ ID NO: 3. Exemplary modifications for SEQ ID NO: 3 (for polypeptides) are described herein (see Example 8; Table 3). For example, the polypeptide sequence of a polypeptide having sweet taste modulating activity comprises SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, or SEQ ID NO:68, or an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, or SEQ ID NO:68.

[0054]

[0063] The polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO:24 (D3E) or at least 80% sequence identity to SEQ ID NO:24. The polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO:26 (K11R) or at least 80% sequence identity to SEQ ID NO:26. The polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO:30 (K26R) or at least 80% sequence identity to SEQ ID NO:30. The polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO:38 (K51R) or at least 80% sequence identity to SEQ ID NO:38. The polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO:42 (R57K) or at least 80% sequence identity to SEQ ID NO:42. The polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO:44 (R66K) or at least 80% sequence identity to SEQ ID NO:44. The polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO: 46 (D69E) or at least 80% sequence identity to SEQ ID NO: 46. The polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO: 50 (D85E) or at least 80% sequence identity to SEQ ID NO: 50. The polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO: 52 (E86D) or at least 80% sequence identity to SEQ ID NO: 52. The polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO: 54 (E89D) or at least 80% sequence identity to SEQ ID NO: 54. The polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO: 58 (D97E) or at least 80% sequence identity to SEQ ID NO: 58. The polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO: 60 (K103R) or at least 80% sequence identity to SEQ ID NO: 60. The polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO: 62 (R106K) or at least 80% sequence identity to SEQ ID NO: 62. The polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO: 64 (R110K) or at least 80% sequence identity to SEQ ID NO: 64.The polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO: 66 (E117D) or at least 80% sequence identity to SEQ ID NO: 66. The polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO: 68 (K120R) or at least 80% sequence identity to SEQ ID NO: 68.

[0055]

[0064] In another aspect, a polynucleotide encoding a polypeptide having sweet taste modulating activity comprises SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, or SEQ ID NO:67, or a nucleic acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, or SEQ ID NO:67.

[0056]

[0065] The polynucleotide comprises SEQ ID NO:23 (corresponding to D3E) or at least 80% sequence identity to SEQ ID NO:23. The polynucleotide comprises SEQ ID NO:25 (corresponding to K11R) or at least 80% sequence identity to SEQ ID NO:25. The polynucleotide comprises SEQ ID NO:29 (corresponding to K26R) or at least 80% sequence identity to SEQ ID NO:29. The polynucleotide comprises SEQ ID NO:37 (corresponding to K51R) or at least 80% sequence identity to SEQ ID NO:37. The polynucleotide comprises SEQ ID NO:41 (corresponding to R57K) or at least 80% sequence identity to SEQ ID NO:41. The polynucleotide comprises SEQ ID NO:43 (corresponding to R66K) or at least 80% sequence identity to SEQ ID NO:43. The polynucleotide comprises SEQ ID NO:45 (corresponding to D69E) or at least 80% sequence identity to SEQ ID NO:45. The polynucleotide comprises SEQ ID NO:49 (D85E) or at least 80% sequence identity to SEQ ID NO:49. The polynucleotide comprises SEQ ID NO:51 (corresponding to E86D) or at least 80% sequence identity to SEQ ID NO:51. The polynucleotide comprises SEQ ID NO:53 (corresponding to E89D) or at least 80% sequence identity to SEQ ID NO:53. The polynucleotide comprises SEQ ID NO:57 (corresponding to D97E) or at least 80% sequence identity to SEQ ID NO:57. The polynucleotide comprises SEQ ID NO:59 (corresponding to K103R) or at least 80% sequence identity to SEQ ID NO:59. The polynucleotide comprises SEQ ID NO:61 (corresponding to R106K) or at least 80% sequence identity to SEQ ID NO:61. The polynucleotide comprises SEQ ID NO:63 (R110K) or at least 80% sequence identity to SEQ ID NO:63. The polynucleotide comprises SEQ ID NO:65 (E117D) or SEQ ID NO:65. The polynucleotide comprises SEQ ID NO:67 (K120R) or at least 80% sequence identity to SEQ ID NO:67.

[0057]

[0066] In one aspect, the polynucleotide comprises SEQ ID NO:20, which corresponds to the coding sequence for His-tagged mycodulcein in E. coli (residues 364-381 correspond to the optional His-tag sequence). SEQ ID NO:20 is codon-optimized for expression in E. coli. In another aspect, the polynucleotide comprises SEQ ID NO:22, which corresponds to the coding sequence for His-tagged mycodulcein in S. cerevisiae (residues 364-381 correspond to the optional His-tag sequence). SEQ ID NO:22 is codon-optimized for expression in S. cerevisiae. The corresponding polypeptide of SEQ ID NO:21 corresponds to a His-tagged mycodulcein protein (residues 122-127 correspond to the optional His-tag sequence), and this polypeptide sequence is the same for expression in E. coli and S. cerevisiae. Accordingly, the present invention also provides a polypeptide comprising the amino acid sequence of SEQ ID NO:21.

[0058]

[0067] The present invention also provides polypeptides comprising the amino acid sequence of SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:48, or SEQ ID NO:56, or an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:48, or SEQ ID NO:56.

[0059]

[0068] The polypeptide comprises SEQ ID NO:28 (R20K) or at least 80% sequence identity to SEQ ID NO:28. The polypeptide comprises SEQ ID NO:32 (E35D) or at least 80% sequence identity to SEQ ID NO:32. The polypeptide comprises SEQ ID NO:34 (K44R) or at least 80% sequence identity to SEQ ID NO:34. The polypeptide comprises SEQ ID NO:36 (D46E) or at least 80% sequence identity to SEQ ID NO:36. The polypeptide comprises SEQ ID NO:40 (D52E) or at least 80% sequence identity to SEQ ID NO:40. The polypeptide comprises SEQ ID NO:48 (R75K) or at least 80% sequence identity to SEQ ID NO:48. The polypeptide comprises SEQ ID NO:56 (D94E) or at least 80% sequence identity to SEQ ID NO:56.

[0060]

[0069] In other aspects, the polynucleotide comprises SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:47, or SEQ ID NO:55, or a nucleic acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:47, or SEQ ID NO:55.

[0061]

[0070] The polynucleotide comprises SEQ ID NO:27 (corresponding to R20K) or at least 80% sequence identity to SEQ ID NO:27. The polynucleotide comprises SEQ ID NO:31 (corresponding to E35D) or at least 80% sequence identity to SEQ ID NO:31. The polynucleotide comprises SEQ ID NO:33 (corresponding to K44R) or at least 80% sequence identity to SEQ ID NO:33. The polynucleotide comprises SEQ ID NO:35 (corresponding to D46E) or at least 80% sequence identity to SEQ ID NO:35. The polynucleotide comprises SEQ ID NO:39 (corresponding to D52E) or at least 80% sequence identity to SEQ ID NO:39. The polynucleotide comprises SEQ ID NO:47 (corresponding to R75K) or at least 80% sequence identity to SEQ ID NO:47. The polynucleotide comprises SEQ ID NO:55 (corresponding to D94E) or at least 80% sequence identity to SEQ ID NO:55.

[0062]

[0071] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0063]

[0072] A polynucleotide or polypeptide can be naturally occurring or non-naturally occurring (e.g., synthetic, recombinant, modified, and / or variant product). In one aspect, the naturally occurring or non-naturally occurring product is isolated or purified.

[0064]

[0073] In one aspect, the term "isolated" encompasses products that have been removed from their biological environment (e.g., cells, tissues, culture media, body fluids, etc.) or otherwise enhanced in purity to any degree (e.g., isolated from a synthetic medium). Thus, an isolated product can be synthetic or naturally occurring.

[0065]

[0074] As used herein, the term "isolated," when referring to a nucleic acid or polypeptide, refers to a state of purification or concentration different from that found in nature. Any degree of purification or concentration greater than that found in nature, including (1) purification from other naturally occurring associated structures or compounds, or (2) association with structures or compounds not normally associated in the body, is within the meaning of "isolated" as used herein. The nucleic acids or polypeptides described herein can be isolated or associated with structures or compounds not normally associated in nature according to a variety of methods and processes known to those of skill in the art. In one embodiment, the polypeptides described herein contain up to 5% (e.g., up to 4%, up to 3%, up to 2%, up to 1%) by weight of other fungal proteins.

[0066]

[0075] As used herein, "recombinant" refers to a polynucleotide (e.g., a "recombinant polynucleotide") that has been synthesized or otherwise manipulated in vitro, a method of using a recombinant polynucleotide to produce a gene product in a cell or other biological system, or a polypeptide (a "recombinant protein") encoded by a recombinant polynucleotide. "Recombinant means" also encompasses the ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into an expression cassette or vector for inducible or constitutive expression, e.g., expression of a fusion protein comprising a translocation domain of the invention and a nucleic acid sequence amplified using the primers of the invention.

[0067]

[0076] A "modified" or "variant" product refers to a product (e.g., a polynucleotide or polypeptide) that is altered from the original (e.g., naturally occurring) structure. As described herein, variants include polynucleotides or polypeptides having one or more changes to the nucleic acid or amino acid sequence, respectively. Changes include modifications to the nucleic acid or amino acid sequence, such as additions, deletions, insertions, and substitutions. Modified or variant products can also include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation of the original structure, such as conjugation with a labeling component.

[0068]

[0077] As used herein, the terms "amplify" and "amplification" refer to the use of any suitable amplification methodology to generate or detect recombinant or naturally expressed nucleic acids, as described in detail below. For example, the present invention provides methods and reagents (e.g., specific degenerate oligonucleotide primer pairs) for amplifying in vivo or in vitro (e.g., by polymerase chain reaction, PCR) naturally expressed (e.g., genomic RNA or mRNA) or recombinant (e.g., cDNA) nucleic acids of the invention (e.g., taste stimulus binding sequences of the invention).

[0078] The term "library" refers to a preparation that is a mixture of different nucleic acid or polypeptide molecules, e.g., a library of recombinantly produced Myd-related polynucleotides generated by amplification of nucleic acids with degenerate primer pairs, or an isolated collection of vectors encompassing an amplified ligand-binding domain, or a mixture of cells each randomly transfected with at least one vector encoding MYD.

[0069]

[0079] As used herein, a "nucleic acid probe or oligonucleotide" is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bond, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe can include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe can be linked by linkages other than phosphodiester bonds, so long as they do not interfere with hybridization. Thus, for example, a probe can be a peptide nucleic acid in which the constituent bases are linked by peptide bonds rather than phosphodiester linkages. Those skilled in the art will understand that a probe can bind to a target sequence that lacks complete complementarity with the probe sequence, depending on the stringency of the hybridization conditions. The probe can be directly labeled, such as with an isotope, chromophore, lumiphore, chromogen, etc., or indirectly labeled, such as with biotin, to which a streptavidin complex can subsequently bind. The presence or absence of a selected sequence or subsequence can be detected by assaying for the presence or absence of the probe.

[0070]

[0080] The term "heterologous," when used with reference to a portion of a nucleic acid, indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced by recombinant techniques, having two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0071]

[0081] A "promoter" is defined as an array of nucleic acid sequences that directs transcription of a nucleic acid. As used herein, a promoter includes essential nucleic acid sequences near the start site of transcription, such as a TATA element in the case of a polymerase II type promoter. A promoter also optionally includes distal enhancer or repressor elements, which may be located as far away as several thousand base pairs from the start site of transcription. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to the functional linkage between a nucleic acid expression control sequence (e.g., a promoter, or an array of transcription factor binding sites) and a second nucleic acid sequence, where the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0072]

[0082] The term "MYD family" can refer to (1) naturally occurring alleles, mutants, alleles, and polymorphic variants, including interspecies homologs, that encode polypeptides having at least about 35-50% amino acid sequence identity, and 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, and optionally 50-100 amino acids.

[0073]

[0083] The term "expression vector" or "expression cassette" refers to any recombinant expression system intended for the constitutive or inducible expression of a nucleic acid sequence of the invention in vitro or in vivo in any cell, including prokaryotic, yeast, fungal, plant, insect, or mammalian cells. The term encompasses linear or circular expression systems. The term encompasses expression systems that remain episomal or that integrate into the host cell genome. Expression systems can be autonomously replicating or non-autoreplicating, i.e., capable of driving only transient expression in a cell. The term encompasses recombinant expression "cassettes" that contain only the minimal elements necessary for transcription of the recombinant nucleic acid.

[0074]

[0084] By "host cell" is meant a cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells can be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells, e.g., cultured cells, explants, and in vivo cells, such as CHO, HeLa, HEK-293, etc.

[0075]

[0085] In one embodiment, the host cell is Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, 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.

[0076]

[0086] In other aspects, the host cell is selected from the group consisting of gram-positive nonsporeforming bacteria, gram-positive sporeforming bacteria, gram-negative bacteria, yeast, and protists / algae.

[0077]

[0087] Non-limiting examples of gram-positive asporogenic 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 aviarieus, s 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、Lactobacillusparaplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, 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.

[0078]

[0088] 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.

[0079]

[0089] 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.

[0080]

[0090] Non-limiting examples of protists / algae include Aurantiochytrium limacinum, Euglena gracilis, and Tetraselmis chuii.

[0081]

[0091] The Myd proteins described herein also encompass "analogs" or "conservative variants" and "mimetics" ("peptidomimetics") that have structure and activity substantially corresponding to the representative sequences. Thus, the terms "conservative variant" or "analog" or "mimetics," as defined herein, refer to polypeptides having amino acid sequences modified such that the change(s) do not substantially alter the structure and / or activity of the polypeptide (conservative variants). These include conservatively modified variations of the amino acid sequence, i.e., amino acid substitutions, additions, or deletions of those residues that are not critical for protein activity, or substitutions of amino acids with residues having similar properties (e.g., acidic, basic, positively or negatively charged, polar or nonpolar, etc.), such that the structure and / or activity is not substantially altered even with substitutions of critical amino acids.

[0082]

[0092] More specifically, "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that encode identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.

[0083]

[0093] For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.

[0084]

[0094] Such nucleic acid variations are "silent variations", which are a type of conservatively modified variation.Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid.Those skilled in the art will recognize that each codon in the nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to obtain a functionally identical molecule.Therefore, every silent variation of the nucleic acid that encodes a polypeptide is implicit in each sequence described.

[0085]

[0095] Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, one representative guideline for selecting conservative substitutions includes the following (original residue, followed by the representative 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 representative 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 representative guideline uses the following six groups, in which proline is unique: 1) Gly (G), Ala (A), Val (V), Leu (L), Ile (I); 2) Ser (S), Cys (C), Thr (T), Met (M); 3) Pro (P); 4) Phe (F), Tyr (Y), Try (W); 5) His (H), Lys (K), Arg (R); and 6) Asp (D), Glu (E), Gln (N). Those skilled in the art will understand that the above substitutions are not the only possible conservative substitutions. For example, for some purposes, all charged amino acids, regardless of whether they are positive or negative, can be considered conservative substitutions for each other.Furthermore, individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a small percentage of amino acids in an encoded sequence can also be considered "conservatively modified variations." One of skill in the art will be familiar with codon choices in a given host expressing a protein of interest.

[0086]

[0096] The terms "mimetic" and "peptidomimetic" refer to a synthetic compound having substantially the same structural and / or functional characteristics of a polypeptide, e.g., a translocation domain, a ligand-binding domain, or a chimeric receptor of the invention. A mimetic can be composed entirely of synthetic, non-natural analogues of amino acids, or can be a chimeric molecule of partly natural peptide amino acids and partly non-natural amino acid analogs. A mimetic can also include any amount of conservative substitutions of natural amino acids as long as such substitutions do not substantially alter the mimetic's structure and / or activity.

[0087]

[0097] As with the polypeptides of the present invention that are conservative variants, routine experimentation will determine whether a mimetic falls within the scope of the present invention, i.e., whether its structure and / or function are substantially unchanged. Polypeptide mimetic compositions can contain any combination of non-natural structural components, typically derived from the following three structural groups: a) residue linkages other than natural amide bonds ("peptide bonds"); b) non-natural residues in place of naturally occurring amino acid residues; or c) residues that induce secondary structure mimicry, i.e., that induce or stabilize secondary structures, such as β-turns, γ-turns, β-sheets, α-helical conformations, etc. A polypeptide can be characterized as a mimetic if all or some of its residues are linked by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be linked by peptide bonds or other chemical bonds or coupling means, such as glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC), or N,N'-diisopropylcarbodiimide (DIC). Linking groups that can be used instead of the traditional amide bond ("peptide bond") linkage include, for example, ketomethylene (e.g., --C(O)--CH-- instead of --C(O)--NH--), aminomethylene (CH--NH), ethylene, olefin (CH=CH), ether (CH--O), thioether (CH--S), tetrazole (CN), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp. 267-357, "Peptide Backbone Modifications," Marcell Dekker, NY (1983)). Polypeptides can also be characterized as mimetics by containing all or some non-natural residues in place of naturally occurring amino acid residues; non-natural residues are well described in the scientific and patent literature.Phyre2 is a set of web-based tools for predicting and analyzing protein structure, function, and mutations.

[0088]

[0098] Protein folding analysis is performed using tools including PHYRE Protein Homology / Similarity Y Recognition Engine V2.0, a protein secondary structure prediction server, and JPred, which show that SEQ ID NO: 3 predicts a predominantly β-sheet globular protein with a significant portion of β-sheet and a small portion of α-helix. Specifically, the Jpred tool predicts putative β-sheets from about residues 5-11, 16-19, 28-29, 39-40, 61-67, 71-77, and 98-101 of SEQ ID NO:3, and α-helices from about residues 20-25, 45-55, and 111-119 of SEQ ID NO:3; and the PHYRE tool predicts β-sheets from about residues 5-12, 17-32, 38-40, 45-57, 62-66, 73-81, 98-104, and 112-116 of SEQ ID NO:3, and α-helices from about residues 84-89 and 116-118 of SEQ ID NO:3. See Figure 1.

[0089]

[0099] Examples of conservatively modified variations of Myd1 protein structure can be derived by identifying sequence-based consensus loop regions as known in the art using homology modeling algorithms: SWISS-MODEL, PHYRE2.0, and Jpred. See, for example, Pechmann, S. & Frydman, J. Interplay between Chaperones and Protein Disorder Promotes the Evolution of Protein Networks. PLoS Computational Biology 10, e1003674 (2014).

[0090]

[0100] Alternative protein sequences predicted to have similar structure and function to Myd1 are provided herein as SEQ ID NOs:8 to 17. To derive SEQ ID NO:17 from SEQ ID NO:8, the consensus loop region was selected as the mutation site. This is because most insertions and deletions are typically found in regions between secondary structural elements, where they can be more easily accommodated without causing significant distortions in the overall folding of the protein. The core of this protein has a higher degree of sequence conservation, as found within 4JOX.

[0091]

[0101] Of the 29 possible amino acid positions within the consensus loop region, 12 amino acids were replaced using conservative substitutions. When selecting for mutation, wild-type amino acids were given equal probability among conservative amino acid residues (Gly can be replaced equally by Ala, Cys, Asp, Glu, and Arg with a 20% probability).

[0092]

[0102] Specific regions of the MYD / Myd nucleotide and amino acid sequences can be used to identify polymorphic variants, interspecies homologs, and alleles of Myd family members. This identification can be performed in vitro, for example, under stringent hybridization conditions or PCR (e.g., using primers encoding the Myd sequences identified herein), or by using sequence information in a computer system to compare with other nucleotide sequences. Various alleles of the MYD gene within a single species population are also useful for determining whether differences in allele sequences correlate with differences in taste between members of the population. Classical PCR-based amplification and cloning techniques are useful for isolating orthologs, for example, when degenerate primers are sufficient to detect related genes across species.

[0093]

[0103] For example, primers designed using the sequences disclosed herein can be used to amplify and clone MYD-related genes from different fungal genomes. In contrast, genes related to MYD within a single species are best identified using sequence pattern recognition software to search for related sequences. Typically, identification of polymorphic variants and alleles of MYD family members can be achieved by comparing amino acid sequences of about 25 amino acids or more, e.g., 50-100 amino acids. Amino acid identities of generally at least 35-50%, and optionally 60%, 70%, 75%, 80%, 85%, 90%, 95-99% or more, typically indicate that a protein is a polymorphic variant, interspecies homolog, or allele of a MYD family member. Sequence comparison can be performed using any of the sequence comparison algorithms discussed below. Antibodies that specifically bind to Myd polypeptides or conserved regions thereof can also be used to identify alleles, interspecies homologs, and polymorphic variants.

[0094]

[0104] Nucleotide and amino acid sequence information for MYD family members can also be used to construct computer models of sweet taste-regulating polypeptides and how they interact with sweet taste receptors and their computer system models. The sweet taste receptor is composed of a heterodimer of taste receptor 1 member 2 (T1R2) and taste receptor 1 member 3 (T1R3). These models can then be used to identify Myd variants and mutations that can increase sweet taste receptor activation and identify more active versions of Myd.

[0095]

[0105] It is envisioned that various conservative mutations and substitutions are within the scope of the present invention.For example, it is within the level of those skilled in the art to carry out amino acid substitution using known protocols of recombinant gene technology, including PCR, gene cloning, cDNA site-directed mutagenesis, host cell transfection and in vitro transcription.Then, variants can be screened for taste receptor agonist functional activity.

[0096]

[0106] In one embodiment, hybrid protein-coding sequences can be constructed containing nucleic acids encoding Myds fusion proteins. These nucleic acid sequences can be operably linked to transcriptional or translational control elements, such as transcriptional and translational initiation sequences, promoters and enhancers, transcriptional and translational terminators, polyadenylation sequences, and other sequences useful for transcribing DNA into RNA. Fusion proteins can also include C- or N-terminal translocation sequences. Furthermore, fusion proteins can contain additional elements, for example, for protein detection, purification, or other uses. Domains that facilitate detection and purification include, for example, metal-chelating peptides, such as polyhistidine tracts, histidine-tryptophan modules, or other domains that allow purification on immobilized metals; maltose-binding proteins; protein A domains that allow purification on immobilized immunoglobulins; or domains utilized in the FLAGS extension / affinity purification system (Immunex Corp, Seattle, Wash.).

[0097]

[0107] In one embodiment, the fusion protein comprises a peptide or protein tag (e.g., for protein purification or detection). Peptide / protein tags are described in Johnson, "Protein / Peptide Tags," Tags are known to those skilled in the art, such as those described in "DOI / / dx.doi.org / 10.13070 / mm.en.2.116," and include, but are not limited to, green fluorescent protein (GFP), FLAG, Myc epitope, polyhistidine, glutathione-S-transferase (GST), HA, V5, 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.

[0098]

[0108] Affinity tags are a type of protein tag that can be attached to proteins, allowing them to be purified from their crude biological sources using affinity techniques. Affinity tags are known in the art and are described, for example, in Kimple et al. Curr Protoc Protein Sci.; 73: Unit-9.9. doi:10.1002 / 0471140864.ps0909s73. These include polyhistidine, GST, MBP, calmodulin-binding peptides, intein-chitin binding domains, streptavidin / biotin-based tags, and His-Patch ThioFusion (thioredoxin). Affinity tags include small (e.g., 20 or fewer amino acid residues) or large affinity tags. Examples of small affinity tags include His, FLAG, Strep II, and S-peptide, while examples of large affinity tags include MBP, GST, cellulose binding domain, calmodulin binding peptide, and His-patch thioredoxin.

[0099]

[0109] Affinity tags include epitope tags and reporter tags. Reporter tags act as reporters of protein expression and protein-protein interactions. Reporter tags include, but are not limited to, enzymes such as β-galactosidase (β-gal), alkaline phosphatase (AP), chloramphenicol acetyltransferase (CAT), and horseradish peroxidase (HRP).

[0100]

[0110] Epitope tags, including FLAG, hemagglutinin (HA), c-myc, T7, and Glu-Glu, are used to detect fusion proteins in vitro and in cell culture. Their short, linear recognition motifs have little effect on the properties of the protein of interest and are usually highly specific for their respective primary antibodies. When using anti-myc antibodies, specificity can be increased by using an enzyme-linked secondary antibody to detect the conjugated anti-myc primary antibody instead of using HRP- or AP-anti-myc conjugates alone.

[0101]

[0111] The tag can be present at either end of the target protein. Some epitope tags, such as FLAG, are often used in tandem or in combination with other tags, such as His-Myc and His-V5 constructs, to enhance their desirable characteristics.

[0102]

[0112] Tandem affinity purification (TAP) is a dual affinity purification method based on the fusion of two affinity tags to a protein of interest, allowing for the purification of the tagged protein and the isolation of protein complexes that interact with the protein of interest. The use of TAP is encompassed within the present invention.

[0103]

[0113] In one embodiment, the fusion protein comprises a histidine tag comprising 2 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) histidine residues. For example, the histidine tag can comprise 6 histidine residues.

[0104]

[0114] The inclusion of a cleavable linker sequence, such as Factor Xa (see, e.g., Ottavi, Biochimie 80:289-293 (1998)), a subtilisin protease recognition motif (see, e.g., Polyak, Protein Eng. 10:615-619 (1997)), or enterokinase (Invitrogen, San Diego, Calif.), between the translocation domain (for efficient plasma membrane expression) and the remainder of the newly translated polypeptide can be beneficial to facilitate purification. For example, one construct can include a nucleic acid sequence linked to six histidine residues, followed by a polypeptide encoding thioredoxin, an enterokinase cleavage site (see, e.g., Williams, Biochemistry 34:1787-1797 (1995)), and a C-terminal translocation domain. The 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. Techniques relating to vectors encoding fusion proteins and the application of fusion proteins are well described in the scientific and patent literature, see, e.g., Kroll, DNA Cell. Biol. 12:441-53 (1993).

[0105]

[0115] Fusion proteins can contain one or more linkers (e.g., flexible linkers, rigid linkers, and in vivo cleavable linkers). In addition to their essential role in linking functional domains together (such as in the case of flexible and rigid linkers) or releasing free functional domains in vivo (such as in the case of in vivo cleavable linkers), linkers offer many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yields, and achieving desirable pharmacokinetic profiles. Linkers are known in the art (see, for example, Chen et al., Adv Drug Deliv Rev. 65(10): 1357-1369 (2013)).

[0106]

[0116] Flexible linkers are used when the linked domains require some degree of movement or interaction. They are generally composed of small nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The small size of these amino acids provides flexibility, allowing mobility of the linked functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thus reducing unfavorable interactions between the linker and the protein moiety.

[0107]

[0117] The most commonly used flexible linkers have a sequence consisting primarily of a stretch of Gly and Ser residues ("GS" linker). An example of the most widely used flexible linker is (Gly-Gly-Gly-Gly-Ser). n (SEQ ID NO: 69). By adjusting the copy number "n", the length of this GS linker can be optimized to achieve proper separation of functional domains or maintain necessary inter-domain interactions. In addition to GS linkers, many other flexible linkers have been designed for recombinant fusion proteins. These flexible linkers are also rich in small or polar amino acids such as Gly and Ser, but can contain additional amino acids such as Thr and Ala to maintain flexibility, and polar amino acids such as Lys and Glu to improve solubility.

[0108]

[0118] Rigid linkers maintain a fixed distance between the domains, preserving their independent function. An example of a rigid linker is (EAAAK). n (SEQ ID NO: 70), and a linker having a Pro-rich sequence, (XP) n wherein X represents any amino acid, preferably Ala, Lys, or Glu.

[0109]

[0119] Polypeptides of the invention can also contain a signal peptide (i.e., 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 the C-terminus, of most newly synthesized proteins that are destined for the secretory pathway. These proteins include those that reside inside specific organelles (the endoplasmic reticulum, Golgi apparatus, or endosomes), those that are secreted from the cell, or those that are inserted into most cell membranes. Representative signal peptides are known in the art, and one of ordinary skill in the art would know how to select a particular signal peptide for use in the present invention.

[0110]

[0120] As used herein, "at least 80% identity" with respect to an amino acid sequence or a nucleotide sequence refers to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity.

[0111]

[0121] As used herein, examples of "an amino acid sequence modified by the deletion, insertion, substitution, or addition of one or more amino acids" include amino acid sequences modified by the deletion, insertion, substitution, or addition of 1 to 30, preferably 20 or fewer, more preferably 10 or fewer, and even more preferably 5 or fewer amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any range thereof). As used herein, examples of "nucleotide sequences modified by deletion, insertion, substitution, or addition of one or more nucleotides" include sequences that have been modified by deletion, insertion, substitution, or addition of 1 or more nucleotides to 90 or less, preferably 60 or less, preferably 30 or less, more preferably 15 or less, and even 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, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 1 , 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 range thereof).

[0112]

[0122] For example, in sequence comparison, typically one sequence serves as reference sequence, and test sequence is compared with it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and partial sequence coordinates are designated as needed, and sequence algorithm program parameters are designated.Default program parameters such as those described below for BLASTN and BLASTP program can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the sequence identity percentage of test sequence with reference sequence based on program parameters.

[0113]

[0123] As used herein, the term "comparison window" refers to any one segment of consecutive positions numbered 20 to 600, usually about 50 to about 200, and more usually about 100 to about 150. After optimally aligning two sequences, the sequence can be compared with a reference sequence at the same consecutive positions. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds., 1995, Supplement)).

[0114]

[0124] Preferred examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul at al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J Mol. Biol. 215:403-410 (1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that either match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J Mol. Biol. 215:403-410 (1990)). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction stops when the cumulative alignment score drops by an amount X from its maximum achieved value; when the accumulation of one or more negative-scoring residue alignments causes the cumulative score to fall below 0; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad Sci. USA 89:10915 (1989)). Alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0115]

[0125] 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 relatedness 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., Figure 2). PILEUP uses a simplified version of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method of use is similar to that described by Higgins & Sharp, CABIOS 5:151-153 (1989). The program can align up to 300 sequences, each with a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with a pairwise alignment of the two most similar sequences, generating a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by specifying specific sequences and their amino acid or nucleotide coordinates for the region of sequence comparison and specifying program parameters. PILEUP is used to compare a reference sequence to other test sequences to determine percent sequence identity using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, for example, version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395 (1984)). Gene-encoded sequences are derived by conceptual translation of the corresponding open reading frames.

[0116]

[0126] Polynucleotides encoding the polypeptides of the present invention can be synthesized chemically or by genetic engineering based on the amino acid sequence of Myd. For example, polynucleotides can be chemically synthesized based on the amino acid sequence of the polypeptides of the present invention or their preproteins. Chemical synthesis of polynucleotides can be achieved using nucleic acid custom synthesis services (e.g., those provided by Medical & Biological Laboratories Co., Ltd., Genscript, etc.). Synthesized polynucleotides can also be amplified by PCR, cloning, etc.

[0117]

[0127] The polypeptides of the present invention can be produced, for example, by expressing a gene encoding a Myd polypeptide of the present invention. Preferably, the Myd polypeptides of the present invention can be produced from a transformant into which a polynucleotide encoding a Myd polypeptide of the present invention has been introduced. For example, the Myd polypeptides of the present invention can be produced by introducing a polynucleotide encoding a Myd polypeptide of the present invention or a vector containing the same into a host to obtain a transformant, culturing the transformant in an appropriate medium, and then producing the Myd polypeptide from the polynucleotide encoding the Myd polypeptide of the present invention introduced into the transformant. The proteins of the present invention can be obtained by isolating or purifying the produced Myd polypeptide from the culture.

[0118]

[0128] Accordingly, the present invention further provides polynucleotides encoding the Myd polypeptides of the present invention and vectors comprising the same. The present invention further provides methods for producing transformants, which comprise introducing a polynucleotide encoding a Myd polypeptide of the present invention or a vector comprising the same into a host. The present invention further provides transformants comprising a polynucleotide encoding a Myd polypeptide of the present invention, or a vector comprising the same introduced from outside a cell. The present invention further provides methods for producing the Myd polypeptides of the present invention, which comprise culturing the transformants.

[0119]

[0129] The present invention also encompasses a polynucleotide of the present invention operably linked to a heterologous regulatory element. The present invention can include an expression cassette or vector comprising a polynucleotide of the present invention, and a host cell transformed with a vector of the present invention.

[0120]

[0130] Alternatively, polynucleotides encoding the Myd polypeptides of the present invention can be generated by introducing mutations into synthesized polynucleotides according to known mutagenesis procedures, such as ultraviolet irradiation and site-directed mutagenesis. For example, polynucleotides encoding the polypeptides of the present invention can be obtained by introducing mutations into the polynucleotide of SEQ ID NO: 1 or 2 by known methods, expressing the resulting polynucleotide, examining the sweetness-modifying activity of the expressed protein, and selecting polynucleotides encoding proteins with the desired sweetness-modifying activity.

[0121]

[0131] Site-directed mutagenesis of polynucleotides can be performed by any method, such as inverse PCR and annealing (Muramatsu et al., eds., "Revised 4th edition New genetic engineering handbook", YODOSHA, pp. 82-88). Various commercially available kits for site-directed mutagenesis, such as the QuickChange II Site-Directed Mutagenesis Kit and QuickChange Multi Site-Directed Mutagenesis Kit from Stratagene, can be used as needed.

[0122]

[0132] Examples of vector types containing a polynucleotide encoding a polypeptide of the present invention include, but are not limited to, vectors commonly used in gene cloning, such as plasmids, cosmids, phages, viruses, YACs, and BACs. Examples of vectors include plasmids (e.g., DNA plasmids), yeast (e.g., Saccharomyces), and viral vectors, such as poxviruses, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, polioviruses, alphaviruses, baculoviruses, Sindbis viruses, plant viruses (e.g., Alphaflexiviridae or Potyviridae), and insect viruses (e.g., Baculoviridae).

[0123]

[0133] Among these, plasmid vectors are preferred, and for example, commercially available protein expression plasmid vectors such as pUC19, pUC118, pUC119, and pBR322 (all manufactured by TAKARA BIO INC.) can be used.

[0124]

[0134] A vector can contain a DNA region containing a replication origin or DNA replication origin. Alternatively, a regulatory sequence, such as a promoter region for initiating gene transcription, a terminator region, or a secretion signal region for extracellular secretion of an expressed protein, can be operably linked upstream of a polynucleotide encoding a protein of the present invention (i.e., the MYD gene of the present invention) in a vector. As used herein, the phrase "operably linked" between a gene and a regulatory sequence refers to a state in which the gene and the regulatory region are positioned so that the gene can be expressed under the control of the regulatory region.

[0125]

[0135] The types of regulatory sequences such as promoter regions, terminators, and secretory signal regions are not particularly limited, and commonly used promoters and secretory signal sequences can be appropriately selected and used depending on the host into which the sequences are to be introduced. For example, a preferred example of a regulatory sequence that can be incorporated into the vector of the present invention is the cbh1 promoter sequence derived from Trichoderma reesei (Curr, Genet, 1995, 28 (1): 71-79).

[0126]

[0136] Alternatively, a marker gene (e.g., a resistance gene to drugs such as ampicillin, neomycin, kanamycin, and chloramphenicol) for selecting a host into which the vector has been appropriately introduced can be further incorporated into the vector of the present invention. Alternatively, when an auxotrophic strain is used as the host, a gene encoding a synthase of a necessary nutrient can be incorporated into the vector as a marker gene. Alternatively, when a selective medium requiring a specific metabolism for growth is used, a metabolically related gene can be incorporated into the vector as a marker gene. An example of such a metabolically related gene is the acetamidase gene for using acetamide as a nitrogen source.

[0127]

[0137] Polynucleotides encoding the Myd polypeptides of the present invention can be ligated to regulatory sequences and marker genes by methods known in the art, such as splicing by overlap extension (SOE)-PCR (Gene, 1989, 77:61-68). Procedures for introducing the ligated fragment into a vector are known in the art.

[0128]

[0138] Examples of hosts for transformants into which vectors can be introduced include microorganisms such as bacteria and filamentous fungi. Examples of bacteria include Escherichia coli and bacteria belonging to Staphylococcus, Enterococcus, Listeria, and Bacillus. Among these, Escherichia coli and Bacillus bacteria (e.g., Bacillus subtilis or mutants thereof) are preferred. Examples of Bacillus subtilis mutants include the protease 9 double-deficient strain KA8AX described in J. Biosci. Bioeng., 2007, 104 (2): 135-143, and the DBPA strain, a mutant derived from the protease 8 double-deficient strain described in Biotechnol. Lett., 2011, 33 (9): 1847-1852, which has improved protein folding efficiency. Examples of filamentous fungi include Trichoderma, Aspergillus, and Rhizopus. Also suitable expression hosts include, for example, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowia lipolytica, Schizosaccharomyces pombe, and Kluyveromyces lactis. In yet another aspect, the invention encompasses a host cell comprising one or more 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., a BHK, VERO, HT1080, 293, RD, COS-7, or CHO cell), an insect cell (e.g., a Trichoplusia ni (Tn5) or Sf9), a bacterial cell, a plant cell, or a yeast cell.

[0129]

[0139] Recombinantly expressed polypeptides from an expression cassette encoding Myd are typically isolated from lysed cells or culture medium. Purification can be achieved by methods known in the art, including salt fractionation, ion exchange chromatography, gel filtration, size exclusion chromatography, size fractionation, and affinity chromatography. Immunoaffinity chromatography can be used, for example, using antibodies raised against the Gag antigen.

[0130]

[0140] The present invention provides a method for purifying a polypeptide having sweet taste modulating activity, the method comprising: (a) obtaining a composition comprising the polypeptide; and (b) purifying the composition via hydrophobic interaction chromatography (HIC) followed by size exclusion chromatography (SEC). In one aspect, the polypeptide comprises an amino acid sequence having at least 80% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. In another aspect, the polypeptide has a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; wherein (a) the polypeptide contains at least one substitution modification relative to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, and the polypeptide is not the polypeptide of SEQ ID NO:3, or b) the polypeptide further comprises a histidine tag, and the polypeptide has sweet taste modulating activity.

[0131]

[0141] Those skilled in the art are familiar with hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC) purification techniques, including the selection of appropriate columns, buffers, and elution solutions. Exemplary HIC and SEC purification techniques are described in Example 11 herein. In exemplary aspects, the purity of the polypeptide after purification by HIC and SEC is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or any range of values ​​therein.

[0132]

[0142] The present invention also contemplates transgenic plants comprising the heterologous polynucleotides and / or heterologous polypeptides of the present invention described herein. The plants have an altered phenotype due to expression of the heterologous nucleic acid sequences. The altered phenotype can include a phenotype of increased sweetness in any plant part, including fruit. The transgenic plant can contain an expression cassette as defined herein as part of the plant, which cassette has been introduced by transforming the plant with a vector of the present invention. Such an expression cassette includes regulatory sequences for expression of the heterologous coding sequence in the plant, including a plant-expressible promoter and terminator. The transgenic plant can be any type of plant capable of expressing the heterologous nucleic acid sequences described herein. The term "plant" includes whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, and plant cells and their progeny. The class of plants that can be used in the methods of the present invention is as broad as the class of higher plants that are amenable to transformation techniques, generally including both monocotyledonous plants (monocots) and dicotyledonous plants (dicots). It encompasses plants of various ploidy levels, including polyploids, diploids, and haploids. For example, the transgenic plant can be an apple or strawberry.

[0133]

[0143] Techniques for transforming a wide variety of plant species are well known in the art and are described in technical and scientific literature.See, for example, Weising et al. (1988) Ann. Rev. Genet., 22:421-477 and Joung et al. (2015) "Plant Transformation Methods and Applications," Current Technology in Plant Molecular Breeding, (Koh et al., ed.) Springer Dordrecht Heidelberg New York London, Chapter 9, pp. 297-344.Any method known in the art for the transformation of plant cells or plant tissues, including plant protoplasts, can be used for plant transformation.Specific methods for plant transformation include, inter alia, bolistic methods (gene gun), electroporation, microinjection, protoplast fusion and Agrobacterium-mediated transformation. Agrobacterium-mediated transformation can use binary vectors that replicate, for example, in Escherichia coli and Agrobacterium tumefaciens or other Agrobacterium strains. A variety of such binary vectors are known in the art and can be used to introduce heterologous polynucleotides into plant cells and tissues. Plant expression vectors containing regulatory sequences for expression of heterologous coding sequences, including plant-expressible promoter sequences and other plant regulatory sequences, in plant cells and tissues are known in the art and can be used to transform plants to express the polypeptides described herein.

[0134]

[0144] A variety of plant-expressible promoters are known in the art and are available for use in the heterologous constructs, vectors, and transformed plant materials herein containing polynucleotides encoding proteins with sweet taste-modulating activity. Plant-expressible promoters can be derived from natural plant sources, plant viral sources, and bacteria, such as Agrobacterium strains, that have plant-expressible promoters. Plant-expressible promoters include, among others, the cauliflower mosaic virus promoter (CaMV 35S), octopine and nopaline synthase promoters (e.g., the 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, and developmental stage-specific promoters, and examples of each type of promoter are known in the art. Tissue-specific promoters include those that direct expression in plant roots, plant leaves, fruits, flowers, pollen, or cells involved in active photosynthesis (e.g., phosphoenolpyruvate promoter (PEP)). Developmental stage-specific promoters include those that direct expression during fruit ripening, flowering, or fruiting. 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 WC (2019) Frontiers in Plant Science, 10, Article 1433).

[0135]

[0145] Techniques for regenerating plants from transformed protoplasts, plant cells, calli, or other plant tissues are well known in the art and can be used to regenerate whole plants and plant parts from such transformed plant material. Regeneration methods include organogenesis and embryogenesis. See Handbook of Plant Cell Culture. Volume 1: Techniques for Propagation and Breeding (1983) DA Evans et al. (eds.), Macmillan (New York); R.H. Smith, Plant Tissue Culture: Techniques and Experiments, 3rd Edition (2012) Academic Press (New York); M.R. Davey & P. ​​Anthony, Plant Cell Culture: Essential Methods (2010) John Wiley & Sons (New York), for details, see Chapters 3 and 9.

[0136]

[0146] Methods commonly used in the field, such as the protoplast method and electroporation, can be used to introduce the vector into the host. The desired transformant can be obtained by selecting a strain into which the vector has been appropriately introduced using indicators such as the expression of a marker gene and / or auxotrophy.

[0137]

[0147] Alternatively, a fragment containing a polynucleotide encoding a Myd polypeptide of the present invention, a regulatory sequence, and a marker gene ligated thereto can be directly introduced into the genome of a host. For example, a polynucleotide encoding a Myd polypeptide of the present invention can be introduced into the genome of a host by constructing a DNA fragment in which sequences complementary to the host genome are added to both ends of the ligated fragment, introducing the fragment into a host, and inducing homologous recombination between the host genome and the DNA fragment by SOE-PCR.

[0138]

[0148] When the thus obtained transformant, into which a polynucleotide encoding the Myd polypeptide of the present invention or a vector containing the same has been introduced, is introduced into an appropriate medium and cultured, expression of the MYD cDNA on the vector and subsequent production of the Myd polypeptide of the present invention occur. Those skilled in the art can select an appropriate medium for culturing such a transformant depending on the type of microorganism used for the transformant.

[0139]

[0149] Alternatively, the Myd polypeptides of the present invention can be expressed from polynucleotides encoding the Myd polypeptides of the present invention or their transcription products using a cell-free translation system. The term "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, necessary for protein translation to a suspension obtained by mechanically disrupting host cells.

[0140]

[0150] The Myd polypeptide of the present invention produced in culture or in a cell-free translation system can be isolated or purified by common protein purification methods, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, and affinity chromatography, either alone or in appropriate combinations. When the gene encoding the Myd polypeptide of the present invention and a secretory signal sequence are operably linked on a vector in a transformant, the Myd polypeptide is secreted extracellularly, making it easier to collect the produced Myd polypeptide from the culture. The Myd polypeptide collected from the culture can be further purified using known means.

[0141]

[0151] The present invention also includes a method for producing a protein having sweet taste modulating activity, the method comprising culturing a host cell of the present invention in a medium under conditions that result in the production of a protein having sweet taste modulating activity similar to a known sweet flavor or sweet compound.

[0142]

[0152] As used herein, "sweet flavoring agent," "sweet compound," or "sweet taste receptor-activating compound" refers to a composition that produces a detectable sweet flavor in a subject, such as sucrose, fructose, glucose, and other known natural sugar-based sweeteners, or known artificial sweeteners such as saccharin, cyclamate, aspartame, as further discussed herein, or a material that activates the T1R2 / T1R3 receptor in vitro. The subject can be a human or an animal.

[0143]

[0153] The sweet flavoring agent or sweetener composition can be used in an effective amount, which refers to an amount of the sweetener composition of the present invention sufficient to induce a sweet taste in a subject when present in a product for oral administration.

[0144]

[0154] In one embodiment, the sweet protein of the present invention can have sweet taste-modulating activity. The Myd polypeptide of the present invention can have functional, physical, and chemical effects on taste receptors, such as sweet taste receptors. "Sweet taste-modulating activity" can refer to the inhibitory, activating, e.g., agonist or antagonist properties of the polypeptide of the present invention, as identified using in vitro and in vivo assays related to taste transduction. Binding of a protein with inhibitory activity can result in partial or total blocking of stimulation, reduction, prevention, or delay of activation, inactivation, desensitization, or downregulation of taste transduction (e.g., antagonist). Binding of an activating polypeptide can result in stimulation, increase, release, activation, promotion, enhancement, sensitization, or upregulation of taste transduction, e.g., agonist. Activating polypeptides are preferred.

[0145]

[0155] Sweetness modulation also refers to enhancing the taste, such as sweetness, of a particular product for oral administration when administered in combination.

[0146]

[0156] Sweet taste regulating activity can be detected by methods known in the art, for example, by in vitro methods or in vivo by animal or human sensory testing.Without wishing to be bound by any particular theory, Myd is involved in sweet taste activation, for example, as an agonist of taste 1 receptor member 2 (T1R2) and / or taste 1 receptor member 3 (T1R3).However, Myd also agonizes other taste receptors, such as bitter, umami, sour and salty. Such functional effects can be measured by measuring binding to the taste receptor T1R via any means known to one of skill in the art, such as spectroscopic (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties, patch clamp techniques, voltage-sensitive dyes, whole-cell currents, radioisotope efflux, inducible markers, changes in transcriptional activation of T1R 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.

[0147]

[0157] Sensory testing (human or animal) can also be used to determine whether a Myd candidate polypeptide has sweet taste-modulating activity. Sensory evaluation is a scientific field that analyzes and measures human responses to food and beverage compositions, such as appearance, touch, smell, texture, temperature, and taste. Measurements using humans as instruments are sometimes necessary. The selection of an appropriate method for determining sweetness can be determined by those skilled in the art and includes, for example, discrimination tests or difference tests designed to measure the likelihood that two products are perceptually different. The responses from the evaluators are recorded for accuracy and statistically analyzed to determine whether they are more accurate than would be expected due to chance alone.

[0148]

[0158] Sensory evaluation is the scientific field that analyzes and measures human responses to food and beverage compositions, such as appearance, touch, smell, texture, temperature, and taste. Measurements using humans as instruments are sometimes necessary. Because the sensory attributes of flavor and texture are distinct attributes that cannot be easily measured by instruments, the food industry first needed to develop measurement tools. The selection of an appropriate method for determining organoleptic properties, such as the sweetness of the proteins disclosed in the present invention, can be determined by those skilled in the art and includes, for example, discrimination or differential tests designed to measure the potential for sensory differences between two products. For sweetness perception, for example, trained panelists can rank one or more samples containing 5% sucrose, 6% sucrose, 7% sucrose, 8% sucrose, 9% sucrose, and 10% sucrose, and a test sample, in order of sweetness intensity from low to high. It should be understood that there are several ways that those skilled in the art can measure sensory differences.

[0149]

[0159] Brix measurement (or Brix scale) is a well-known application in the food and beverage industry and determines the pure sucrose content in water: 1 degree Brix (°Bx) = 1 g sucrose / 100 g solution, expressing the strength of the solution as a mass percentage. 8°Bx is equivalent to approximately an 8% sugar solution. As described in the Examples, purified polypeptide corresponding to SEQ ID NO: 21 was tasted (0.2 mL aliquot) at 0.03 mg / mL by trained sensory scientists and found to have a sweetness equivalent to 8°Bx (approximately an 8% sugar solution) (see Examples 4, 5, 9, and 10).

[0150]

[0160] In some embodiments, Myd polypeptides of the invention include polypeptides that are at least as sweet (on a weight / weight basis) as sugar (e.g., 1X), or 2X, 5X, 10X, 50X, 100X, 200X, 400X, 600X, 800X, 1000X, 1500X, 2000X, 3000X, 5000X, 10000X, 20000X, or more sweeter than sugar, as measured by any of the methods described above or known in the art. In other embodiments, the Myd polypeptide is at least 1% (at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) as sweet as sugar.

[0151]

[0161] Food, beverages, nutritional supplements, and pharmaceuticals

[0162] In one embodiment, the present invention encompasses a composition comprising, consisting essentially of, or consisting of a combination of an oral product and a sweetening composition comprising an isolated Myd polypeptide according to the present invention as described herein. In one aspect, the combination has enhanced sweetness compared to an oral product lacking the Myd polypeptide (control). In one embodiment, the oral product is not a Mattirolomyces terfezioides truffle. The term "essentially consisting of" allows for the inclusion of ingredients that are not essential to the function or activity of the product and do not substantially affect the function or activity, such as anti-caking agents, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltodextrose, acacia gum, etc.).

[0152]

[0163] In one embodiment, a composition comprising an isolated Myd protein of the present invention can include a formulation that provides enhanced functionality to the isolated Myd protein. For example, the composition can include a formulation that stabilizes the Myd protein against heat, osmotic pressure, pH, or other types of degradation. In one embodiment, the formulation stabilizes the Myd protein against thermal degradation. Representative compounds for stabilizing Myd protein include, for example, L-arginine lysine, 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, trimethylsilyl methylcellulose, and methylcellulose. amine 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 form), benzamidine hydrochloride, ethylenediaminetetraacetic acid disodium salt dihydrate, methyl chloride Magnesium hexahydrate, cadmium chloride hydrate, non-surfactant sulfobetaine 195 (NDSB-195), non-surfactant sulfobetaine 201 (NDSB-201), non-surfactant sulfobetaine 211 (NDSB-211), non-surfactant sulfobetaine 221 (NDSB-221), non-surfactant sulfobetaine 256 (NDSB-256), taurine, acetamide, oxalic acid dihydrate, sodium malonate pH 7.0, succinic acid pH 7.0, Taximate pH 7.0, tetraethylammonium bromide, choline acetate, 1-ethyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazolium chloride, ethylammonium nitrate, ammonium sulfate, ammonium chloride, magnesium sulfate hydrate, potassium thiocyanate, gadolinium(III) chloride hexahydrate, cesium chloride, 4-aminobutyric acid (GABA), lithium 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 1900, polyethylene glycol 3350, polyethylene glycol 8000, polyvinylpyrrolidone K15, polyethylene glycol 20000, (2-hydroxypropyl)-β-cyclodextrin, α-cyclodextrin, β-cyclodextrin, methyl-β-cyclodextrin.

[0153]

[0164] In one aspect, the Myd polypeptide of the sweet tasting composition can comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO:3 (or SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17). In another aspect, the Myd polypeptide has a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; wherein (a) the polypeptide contains at least one substitution modification with respect to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, and the polypeptide is not the polypeptide of SEQ ID NO:3, or (b) the polypeptide further comprises a histidine tag, and the polypeptide has sweet taste modulating activity. For example, the polypeptide comprises the amino acid sequence of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75, and does not consist of SEQ ID NO:3. In particular aspects, the polypeptide comprises the amino acid sequence of SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, or SEQ ID NO:68.

[0154]

[0165] The present invention also encompasses a method for modulating the taste of an oral product, comprising combining the oral product with an effective amount of an isolated Myd polypeptide described herein. In one aspect, the combination has an enhanced sweetness compared to an oral product lacking the Myd polypeptide (control). In one embodiment, the oral product is not a Mattirolomyces terfezioides truffle.

[0155]

[0166] In one aspect, the Myd polypeptide of the sweet tasting composition can comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO:3 (or SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17). In another aspect, the Myd polypeptide has a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; wherein (a) the polypeptide contains at least one substitution modification with respect to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, and the polypeptide is not the polypeptide of SEQ ID NO:3, or (b) the polypeptide further comprises a histidine tag, and the polypeptide has sweet taste modulating activity.

[0156]

[0167] The product for oral administration can be a food, a beverage, a dietary supplement composition, or a pharmaceutical composition.

[0157]

[0168] The term "oral administration product" can refer to edible products such as food products, beverage products, medicinal (pharmaceutical) products, or dietary supplements such as herbal supplements. As used herein, the term "medicinal product" encompasses both solid and liquid compositions that are ingestible non-toxic materials that have pharmacological effects or contain pharmaceutically active agents, such as cough syrup, cough drops, aspirin, and medicinal chewable tablets. Oral hygiene products are also oral administration products, and include solids and liquids such as toothpaste or mouthwash.

[0158]

[0169] Generally, the present invention contemplates that a food or beverage product can include an isolated sweet protein of the present invention in an effective amount, e.g., up to about 99% by weight, e.g., about 0.01% to about 99% by weight, based on the total weight of the food or beverage product. All intermediate amounts based on the total weight of the food or beverage product (i.e., 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 90%, 95%, 99% by weight) are contemplated, as are all intermediate ranges based on these amounts.

[0159]

[0170] The compositions of the present invention may include "edible, biologically, or pharmaceutically acceptable carriers or excipients," which can include solid or liquid media and / or compositions used to prepare a desired dosage form of Myd polypeptide for administration of the Myd polypeptide in dispersed / diluted form so as to maximize the biological effectiveness of the Myd polypeptide. Edible, biologically, or pharmaceutically acceptable carriers include 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; dispersions or emulsions of fatty acids, low molecular weight oligomers of propylene glycol, glyceryl esters of fatty acids, and other hydrophobic substances in aqueous media; salts such as sodium chloride; flour; solvents such as ethanol; solid edible diluents such as vegetable powders or flours; or other liquid vehicles; dispersing or suspending aids; surfactants; isotonic agents; thickeners or emulsifiers; preservatives; solid binders; lubricants; and the like.

[0160]

[0171] Food or beverage products that may be contemplated in the context of the present invention include baked goods; sweet bakery products (such as, but not limited to, rolls, cakes, pies, pastries and cookies); pre-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings (such as, but not limited to, nut pie fillings such as fruit pie fillings and pecan pie fillings, as well as fillings for cookies, cakes, pastries, confectionery products and the like); desserts, gelatins and puddings; frozen desserts (such as, but not limited to, regular ice cream frozen dairy desserts such as ice cream, including ice cream, including soft serve ice cream and all other types of ice cream, as well as frozen non-dairy desserts such as non-dairy ice cream, sorbet, and the like; carbonated beverages (e.g., but not limited to, carbonated soft drinks); non-carbonated beverages (e.g., but not limited to, non-carbonated soft drinks such as flavored waters and tea- or coffee-based sweetened beverages); beverage concentrates (e.g., but not limited to, liquid concentrates and syrups, and non-liquid concentrates such as freeze-dried and / or powder preparations); yogurt (e.g., but not limited to, high-fat, low-fat, and non-fat dairy yogurt, as well as non-dairy yogurt and lactose-free yogurt) yogurt and all frozen equivalents thereof); snack bars (e.g., but not limited to, cereal, nut, seed, and / or fruit bars); bakery products (e.g., but not limited to, leavened and unleavened breads, yeasted and unleavened breads, e.g., soda bread, breads containing any type of wheat flour, breads containing any type of non-wheat flour (such as potato, rice, and rye flour), gluten-free breads); pre-made bread mixes for preparing bread products; sauces, syrups, and dressings; sweet spreads (e.g., but not limited to, jellies, jams, butters, nut spreads, and other spreadable preserves, conserves, etc.);Confectionery products (such as, but not limited to, jelly candies, soft candies, hard candies, chocolates, and gums); sweetened breakfast cereals (such as, 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. Although not mentioned herein, other types of food and beverage products that conventionally include one or more nutritive sweeteners can also be contemplated in the context of the present invention.

[0161]

[0172] As a result 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 diet products, medical foods / products (including pills and tablets), and sports nutrition products, and may be particularly suitable for food or beverage products requiring less sweetness at a given soluble solids level.

[0162]

[0173] In some embodiments, the sweetening composition of the present invention can be supplemented with other nutritive or non-nutritive sweeteners to form a sweetener system. The sweetener system can include the sweetening composition of the present invention, a bulking agent such as maltodextrose or acacia gum, and at least one high-intensity sweetener. The composition can be provided as a liquid composition or a dry blend.

[0163]

[0174] In one aspect, the invention encompasses a process for enhancing the sweetness of a product for oral administration, the process comprising the addition of a Myd polypeptide of the invention.

[0164]

[0175] In another aspect, the methods of the present invention include a method for improving the sweet flavor of an oral product, comprising adding to the oral product a sweetening composition made by the method of the present invention. The amount to be added can be determined by methods known in the art, for example, using sensory testing as a guide.

[0165]

[0176] The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting its scope. [Example]

[0166]

[0177] Example 1

[0178] Fresh Mattirolomyces terfezioides truffles were obtained in situ in their natural range using appropriate procedures and permits. Fresh samples (29 total) were shipped to the MycoTechnology, Inc. facility and gently washed in RO water before being frozen in liquid nitrogen and stored at -80°C. The average moisture content of the truffles was 83.6% ± 4.6%.

[0167]

[0179] Aqueous extraction of truffles was performed as follows: Eight different samples of truffles were ground to a powder using a pestle in liquid nitrogen, then a 5:1 volume / weight ratio of truffle to water at 4°C was added and allowed to incubate at 4°C for 30 minutes. The extracted material was then briefly centrifuged at low speed and the filtrate was tasted "neat". Sweetness intensity was rated from 0 for no sweetness to 10 for extremely sweet. For the samples, sweetness was scored as follows:

[0180] Table 1: Sweetness of different truffle samples.

[0168] [Table 1]

[0181] The aqueous extract was stored at 4° C. in sodium phosphate buffer at pH 7 and pH 2. Little or no change in sweetness was observed over 8 days.

[0169]

[0182] Example 2

[0183] Purification of the sweet protein Myd from M. terfezoides. Fresh Mattirolomyces terfezioides truffles were obtained in situ from their natural range using appropriate procedures and authorizations and stored at -80°C. A 16.3 g sample was removed from the freezer and ground in liquid nitrogen using a white ceramic mortar and pestle. Grinding was carried out for 15 minutes to thoroughly pulverize the tissue and obtain a fine, frozen powder. The powder was added to a 50 mL Falcon tube, and 20 mL of RO-HO was added. The tissue was mixed by vortexing until no ice crystals were observed. Fragments were broken down using a rotor-stator at setting 20 for 2 x 1 min at 4°C to create a homogenous solution (H1). The volume of the slurry was adjusted to 53 mL with RO-HO and centrifuged at 7500 x G for 30 min at 4°C. The supernatant from this step (S1) was collected in a 2 mL Eppendorf tube and centrifuged at 20,000 × g for 15 minutes at 4 °C in a 5417R Eppendorf centrifuge. The pellet (P1) was discarded. Sweetness (according to human sensory perception) was perceived in the supernatant. The supernatants from this step were collected and pooled. The supernatant was then filtered through a 25 mm 0.45 micron syringe filter (cellulose, VWR International) to obtain S1 + 0.22 μm filtration. The filtrate was then washed with hexane (2 times with 38 mL to 50 mL of hexane), and the aqueous phase was collected. S1FH is the S1F + hexane wash. The hexane phase was saved and dried. The aqueous phase was then precipitated with acetone (50 mL was added to the 33 mL fraction S1FH at -20 °C for 30 minutes). The sample was centrifuged at 3,000 × g, and the precipitate was collected. The precipitate was resuspended in 10 mM sodium phosphate, pH 6; the supernatant from this step was designated S2 and the precipitate was designated P2. Supernatant S2 was first applied to an AMICON centrifugal filter unit with a 100 kD molecular weight cutoff to yield a filtrate (flow-through) portion (designated 100F) and a retentate (designated 100R); 100F was then applied to a unit with a 30 kD molecular weight cutoff to yield a filtrate (30F) and a retentate (30R). The sweet fraction flowed through the 100 kD column, emerging in 100F, and was retained on the 30 kD column (30R).A band at approximately 13 kDa, indicated by an arrow, was observed in the SDS-PAGE in Figure 2. This band was excised and subjected to N-terminal sequence analysis by Edman degradation using standard detection methods, such as liquid chromatography and mass spectrometry, to identify the residues in each cycle. A polypeptide of SEQ ID NO: 4 was detected.

[0170]

[0184] Example 3 (RNA Identification)

[0185] Sample collection

[0186] Fresh Mattirolomyces terfezioides truffles were obtained in situ in their natural range using appropriate procedures and permits. A wild isolate (BDP2_18) of Mattirolomyces terfezioides truffle (Gleba) was sourced from the natural environment. BDP2_18 is the largest wild truffle in captivity, and this truffle possesses the following sweet characteristics: a sweeter upfront, more fungal, earthy, and less lingering sweetness.

[0187] Sample identification

[0188] A wild isolate of M. gleba was cryopreserved and transferred to GeneWiz for Internal Transcribed Spacer (ITS) Sequencing. The genomic loci sequenced were the ITS 1 and 2 regions. The resulting Sanger sequencing reads were then aligned and low-quality bases were trimmed. Each sequence was then subjected to an individual Basic Local Alignment Search Tool (BLAST) (Altschul, Gish, Miller, Myers, & Lipman, 1990) search to verify identity. BLASTn searches were performed using a nucleotide collection (nr / nt) that excluded sequences from unpublished samples. The entry with the highest percent identity to the wild isolate was Mattirolomyces terfezioides strain rib02.

[0171]

[0189] Sample preparation

[0190] The field isolate BPD2_18 was superficially washed with sterile water, cut into cubes weighing approximately 100 mg, flash frozen in liquid N2, and stored at -80°C. It was shipped on dry ice to GeneWiz.

[0172]

[0191] RNA sequencing

[0192] The following samples were submitted through GeneWiz for Standard RNASeq and probed on an Illumina HiSeq, 2x150bp, single index, approximately 350M raw paired-end reads per lane. This RNASeq study included polyA+ selection for transcriptome profiling.

[0173]

[0193] GeneWiz RNA was extracted using the Qiagen RNeasy Plus Universal mini kit according to the manufacturer's instructions. RNA library preparation was performed using the NEBNext Ultra RNA Library Prep Kit for Illumina. Illumina adapter sequences are outlined below.

[0174]

[0194] 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 18)

[0195] 5'-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC[i7 barcode]ATCTCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 19)

[0196] Fastq sequence files from an RNA-seq study of two strains of Mattirolomyces terfezioides (each replicated three times) were trimmed and cleaned using HTStream to remove the following contaminants: PhiX (a common sequencing contaminant), rRNA reads, sequencing adapters, low-quality and "N" bases, poly-a tracts, primers, and reads <50 bp. rnaSPAdes (Bushmanova E, Antipov D, Lapidus A, Prjibelski AD. rnaSPAdes: a de novo transcriptome assembler and its application to RNA-Seq data. Gigascience. 2019;8(9):giz100. doi:10.1093 / gigascience / giz100) was then used to de novo assemble transcriptomes for each M. terfezioides strain from the cleaned files.Bandage (Bioinformatics Application for Navigating De novo Assembly Easily) (Ryan R. Wick, Mark B. Schultz, Justin Zobel, Kathryn E. Holt, Bandage: interactive visualization of de novo genome assemblies, Bioinformatics, Volume 31, Issue 20, October 15, 2015, pp. 3350-3352) was used to visualize assemblies for the target peptide, and a tBLASTn search was performed (Gertz EM, Yu YK, Agarwala R, Schaeffer AA, Altschul SF. Composition-based statistics and translated nucleotide searches: improving the TBLASTN module of BLAST. BMC Biol. 2006;4:41. Published December 7, 2006). doi:10.1186 / 1741-7007-4-41) and compare the protein query sequence (PDLSSFITIKNNSNHVFTRT, SEQ ID NO: 4) to an assembled transcriptome sequence database dynamically translated in all six reading frames. Contigs containing a perfect match with the query protein sequence were analyzed for open reading frames (ORFs) and ultimately used to construct a full-length mRNA linked to the target peptide.

[0175]

[0197] An RNA transcript was identified, and its DNA copy has the sequence shown as SEQ ID NO: 1, of which SEQ ID NO: 2 is the predicted coding sequence based on the start and stop codons. A predicted protein with a predicted size of 13.3 kDa and 121 amino acids, SEQ ID NO: 3, is also provided. Length: 122 aa, molecular weight: 13.381 kDa, predicted isoelectric point: 8.64, and predicted charge at pH 7: 1.01.

[0176]

[0198] Blast analysis. The identity between the predicted protein of SEQ ID NO: 3 and other protein sequences in GENBANK was less than 31%. A hypothetical protein from Pisolithus tincturius (GenBank: KIN98154.1; SEQ ID NO: 7) was found to have approximately 31% homology to SEQ ID NO: 3 and was designated hypothetical protein M404DRAFT_1005519 [Pisolithus tinctorius Marx 270]. SEQ ID NO: 7 is also hypothesized to have sweet taste-modulating activity. The complete cDNA copy of the RNA transcript is SEQ ID NO: 5, and the coding sequence is given as SEQ ID NO: 6.

[0177]

[0199] Example 4 (Cloning and heterologous expression of mycodulcein in E. coli; confirmation of sweetness)

[0200] Based on the nucleotide sequence identified as SEQ ID NO:3, three expression vectors for expressing SEQ ID NO:20 containing a histidine tag were synthesized and cloned by Atum, Inc. (Newark, CA) into three different Atum vector backbones: pD454-SR (plasmid pMy_3000), pD454-MR (plasmid pMy_3001), and pD454-WR (plasmid pMy_3002), all of which are E. coli IPTG-inducible T7 promoter expression vectors with ampicillin resistance, lacl, Lac01, Ori_pUC, and medium (M), strong (S), and weak (W) ribosome binding sites on the plasmid. E. coli BL21 DE3 (Studier et al. (1986) J. Mol. Biol. 189:113-130) (New England Biolabs) was transformed with pMy_3000, pMy_3001, and pMy_3002 using the manufacturer's protocol. Briefly, previously frozen competent cells were thawed, mixed with 1 pg to 100 ng of plasmid DNA, and kept on ice for 30 minutes. The mixture was heat-shocked at 42°C for 45 seconds. Immediately after, the mixture was placed in an ice bath and left for 10 minutes. 950 μL of prewarmed LB medium was added, followed by shaking at 225 rpm for 1 hour at 37°C. Each transformation reaction was performed by diluting cells 1:10 and 1:100 and plating 100 μL onto antibiotic plates. After overnight growth at 37°C, colonies fully recovered and were visible. To confirm successful transformation, csPCR (colony screening PCR) was performed to examine the cDNA region of the plasmid, and expression was confirmed through SDS-PAGE of the lysate. Inducible expression at the shake flask scale was used to confirm heterologous expression in the E. coli host. This process yielded strains Z14CE, Z15CE, and Z16CE, containing plasmids pMy_3000, pMy_3001, and pMy_3002, respectively. Three strains (Z14CE, Z15CE, and Z16CE) were maintained on LB + ampicillin 100 μg / mL agar plates, while a negative control (Eco_0001) was maintained on LB agar plates.Overnight cultures for each strain were grown in 50 mL of LB liquid medium with the appropriate antibiotic in a 250 mL unbaffled culture shake flask at 37°C, shaking at 150 rpm overnight. The following day, each overnight culture was inoculated into 200 mL of TB liquid medium in a 1000 mL baffled culture shake flask at 37°C, shaking at 200 rpm and adjusted to an OD600 of 0.1 with the appropriate antibiotic. When the OD600 reached 0.8, a supplement of IPTG was added to the medium to a final concentration of 0.66 mM. Shaking was continued for an additional 5 hours at 37°C. After expression, the cells were centrifuged at 4000 g for 20 minutes. The supernatant was discarded, and the pellet was suspended in approximately 20 mL of wash buffer (10 mM sodium phosphate buffer, pH 7.0). Suspended cells were disrupted in a high-pressure homogenizer (C3 Emulsiflex, Avestin, Inc., Ottawa, ON, Canada) operated at a maximum of 2000 bar. The disrupted cells were centrifuged at 13000 g (30 min), the supernatant was collected, and the pellet was discarded. The supernatant containing the solubilized protein was filtered through a 0.22 μm PES membrane B unit (Millipore, Burlington, MA, USA). SDS-PAGE was performed, and expression was confirmed by observing a 13.1 kD band (Coomassie staining).

[0178]

[0201] Thermo Scientific TM HisPur TMThe his-tagged protein SEQ ID NO:20 was purified using an efficient immobilized metal affinity chromatography (IMAC) technique with Ni-NTA resin. SEQ ID NO:20 was purified using a nickel-charged nitrilotriacetic acid (NTA) chelate immobilized on a 6% cross-linked agarose resin. The lysate was loaded onto a prepared IMAC column, equilibrated with binding buffer: 20 mM monobasic sodium phosphate, 0.5 M sodium chloride, 0.1 M imidazole, pH 7.4, and eluted with elution buffer: 20 mM monobasic sodium phosphate, 0.5 M sodium chloride, 0.5 M imidazole. After washing the column three times with binding buffer, the his-tagged mycodulcein was eluted four times with elution buffer. The eluted fraction was then ultrafiltered using a 30 kDa MWCO filter, and the filtrate was passed through a 10 kDa filter at 4000 x G for 15 minutes. The retentate was diluted and spun again for the washing steps, which were repeated three times. Analysis of the purification steps by SDS-PAGE is shown in Figure 3.

[0179]

[0202] The purified fraction (shown in lane 8 of Figure 3, containing highly purified SEQ ID NO:21) was tasted (0.2 mL aliquot) at 0.03 mg / mL by a trained sensory scientist and found to have a sweetness equivalent to 8° brix (approximately 8% sugar solution), thereby confirming that the mycodulcein isolated from M. terfezioides is responsible for the sweetening activity observed in Examples 1 and 2. The sweetness was very noticeably sweet, with a "clean" sweetness (sugar-like taste), no other flavors, a slightly delayed sweetness onset, and a sweet aftertaste.

[0180]

[0203] Example 5 (Pilot-scale production of mycodulcein (his tag))

[0204] The E. coli Z14CE strain prepared in Example 4, containing the coding sequence of SEQ ID NO:20, was tested for performance during fermentation in a laboratory-scale bioreactor. Bioreactor cultivation was carried out in a 10.0 L Bioflo 320 round-bottom stirred fermenter (BioFlo / CelliGen 310, New Brunswick Scientific, Edison, NJ, USA). The fermenter was equipped with pH and dissolved oxygen sensors (Mettler Toledo, OH, USA). Temperature was controlled via a water-filled stainless steel base. Agitation was provided by two six-blade Rushton turbines spaced 47 mm apart, with the lowermost impeller positioned just above the bottom of the shaft. Aeration was provided through a perforated tube sparger ring. Dissolved oxygen (DO) was controlled at 20% air saturation using a continuous cascade of 500-800 rpm agitation and 5-8 L / min aeration with sparged air at high cell density. The pH was controlled at 7.0 using 5.0 M ammonium hydroxide. Antiform 204 (Sigma, St. Louis, MO, USA) was added automatically to control foaming. The latter was sensed using a conductivity probe mounted 10 cm above the culture level. The main fermentation medium contained (per liter) 24 g yeast extract, 12 g tryptone, 5.42 mL glycerol, and 100 mL phosphate buffer stock (0.17 M KH2PO4, 0.72 M K2HPO4). The medium was adjusted to pH 7.0 using 2 M HCl. When the initial glucose feed was depleted (indicated by a rise in pH), a feed medium (per liter) consisting of 200 g glucose, 21.1 g (NH4)2SO4, and 19.7 g MgSO4 was pumped into the fermenter at an initial flow rate of 1.00 mL / min. Unless otherwise noted, the initial volume of medium in the vessel was 4.0 L. The inoculum (200 mL) consisted of a culture grown for 16 h in LB starter medium in a 1 L baffled shake flask (37 °C, 200 rpm). The fermenter temperature was 37 °C. Fermentation was induced with 0.66 mM IPTG after reaching an optical density of 10-20 and continued for 24 h.Subsequently, after 24 hours of induction, the broth was centrifuged at 4000 g for 20 minutes. The supernatant was discarded, and the pellet was suspended in 1 L of wash buffer (10 mM sodium phosphate buffer, pH 7.0). The suspended cells were disrupted in a high-pressure homogenizer (C3 Emulsiflex, Avestin, Inc., Ottawa, ON, Canada) operated at a maximum pressure of 1500 bar. The disrupted cells were centrifuged at 13000 g (30 minutes), the supernatant was collected, and the pellet was discarded. The supernatant containing the solubilized proteins was filtered through a 0.22 μm PES membrane unit (Millipore, Burlington, MA, USA).

[0181]

[0205] The clarified supernatant prepared in this example was tasted (0.2 mL aliquots) at 0.03 mg / mL by a trained sensory scientist and found to have a sweetness equivalent to 8° brix (approximately 8% sugar solution). The sweetness was very pronounced, with a "clean" sweetness (sugar-like taste), no other flavors, a slightly delayed sweetness onset, and a sweet aftertaste.

[0182]

[0206] The supernatant was stored in aliquots at −20° C. and used for further experiments.

[0183]

[0207] Example 6 (ELISA quantification of mycodulcein from E. coli)

[0208] To quantify His-tagged mycodulcein (SEQ ID NO: 21), a direct ELISA was developed using a horseradish peroxidase (HRP) antibody conjugated to the 6XHis-Tag sequence on the carboxy terminus of SEQ ID NO: 21. The ELISA allows for the measurement of mycodulcein concentration in combined lysates and purified proteins. Recombinant 6XHis-tagged E. coli mycodulcein has a molecular weight of 14.2 kDa and a denaturation constant of 27960 M, calculated from the amino acid sequence by methods known in the art. -1 cm -1The molar extinction coefficient of 1000 mg / mL was 1000 mg / mL. Purity was assessed by SDS-PAGE to be ≥98%. Mycodulcein protein concentration was determined by absorbance at 280 nm using the Beer-Lambert law, and a standard curve was then generated using known concentrations of mycodulcein (μg / mL) for ELISA.

[0184]

[0209] ELISA procedure: Proteins were bound to the walls of a high-protein-binding 96-well plate in 50 mM carbonate buffer, pH 9.4, coating buffer for 30 minutes at room temperature. The plate was washed three times with phosphate-buffered saline (PBS), pH 7.4, containing 0.02% Tween-20. Nonspecific binding sites on the microplate were blocked with 5% BSA in PBS, pH 7.4, for 15 minutes at room temperature and washed three times with PBS 0.02% Tween-20. Primary antibodies were diluted (1:1000) in blocking buffer, and the microplate was incubated for 1 hour and washed three times with PBS 0.02% Tween-20. The HRP reaction was developed using the colorimetric substrate 3,3',5,5'-tetramethylbenzidine (TMB) for 8 minutes and stopped with 2N sulfuric acid.

[0185]

[0210] Example 7 (Quantitative characterization of mycodulcein concentration dependence)

[0211] Quantitative characterization of the concentration-dependent taste properties of purified His-tagged mycodulcein (SEQ ID NO: 21), obtained from E. coli as described in Example 5 and purified as described in Example 4, was performed by Opertech Bio (Philadelphia, PA). The sweetening potency and relative efficacy of mycodulcein were compared to sucrose and other sweeteners, thaumatin, rebaudioside A, and aspartame. The control was a solution of 200 mM sucrose, 100 mM NaCl, 0.5 mM quinine, and 10 mM citric acid. Figure 4 shows the concentration-response functions for the sweetness of mycodulcein, aspartame, thaumatin, and rebaudioside A. Data are plotted as the percentage (p) of the response produced on the 200 mM sucrose-related ("sweet") target. Each data point in the curves for mycodulcein, aspartame, thaumatin, and rebaudioside A was calculated as the average over 32 replicates, and for the sucrose curve it was averaged over 16 replicates; error bars represent standard deviation. Points for the water and sucrose controls were similarly calculated as the average over 128 and 64 replicates, respectively. Curves were fitted by nonlinear regression.

[0186]

[0212] The concentrations producing a half-maximal sweetness response (EC50, or potency) were derived from nonlinear regression. The EC50s (and 95% confidence intervals) for mycodulcein (MYC), sucrose (SUC), aspartame (ASP), rebaudioside A (REB), and thaumatin (THN) are shown in Table 2. Equivalency to sucrose on a molar and weight basis is also provided.

[0187]

[0213] Table 2.

[0188] [Table 2]

[0214] Evaluation of thaumatin, sucrose, and SEQ ID NO: 21 was performed using the CATA (click all that apply) of the sweetness sensation time-intensity of named analytes at 0.045 mg / mL in water relative to protein and 10% sucrose. Methodology: Time-Intensity Technique; Data Collection Software: EyeQuestion, recording responses every 2.57 seconds; Scaling Method: 15-point sweetness scale, e.g., score 5 = 5% sucrose, score 10 = 10% sucrose; Evaluation Protocol: Small-Volume Sip, Tilt, and Spit Test. Panelists ranged from 3 to 6 individuals with two replicates. All samples were blinded and presented with a randomized 3-digit code.

[0189]

[0215] Training strategy: Intensive training on a 15-point sweetness scale over 6 weeks to confidently assign sweetness values. Due to unique sweetness behavior patterns, training in the time-intensity principle over 3 weeks was necessary. Samples were tasted using a stopwatch to record time and help reach consensus. Number of panelists: 3-6, number of replicates: 2. All samples were blinded and presented with a randomized 3-digit code. Statistical analysis: Due to the small number of panelists, statistical analysis was not possible.

[0190]

[0216] The maximum intensity (Imax) of mycodulcein and thaumatin is approximately 1 point higher on a 15-point scale at the test dose compared to sucrose. Thaumatin and mycodulcein have flatter slopes, longer peak times, and a more gradual / longer decline. Sucrose approaches threshold sweetness (intensity <1) sooner than thaumatin and mycodulcein, at 162 seconds. While sucrose reaches threshold levels, thaumatin and mycodulcein are perceptible at low to moderate intensities. Mycodulcein and thaumatin appear to have similar potencies in this experiment, approximately 3,000 times sweeter than sucrose on a weight basis, or approximately 120,000 times sweeter than sucrose on a molar basis. These two experiments indicate that mycodulcein is a high-intensity sweet protein with potencies ranging from 400 times sweeter than sucrose on a weight basis to 3,000 times sweeter than sucrose.

[0191]

[0217] Example 8 (Production and testing of mycodulcein variants for sweetness and heat stability)

[0218] A method for identifying potentially important residues in mycodulcein. Sweet proteins do not share primary sequence identity, but their overall tertiary structures share a sweet finger motif (Tancredi, T., Pastore, A., Salvadori, S., Esposito, V. & Temussi, PA. Interaction of sweet proteins with their receptor: A conformational study of peptides corresponding to loops of brazzein, monellin, and thaumatin. European Journal of Biochemistry 271, (2004): 2231-2240). Sweet proteins have an antiparallel beta sheet with an alpha helix perpendicular to the beta sheet. The tertiary structures of the sweet proteins thaumatin, monellin, brazzein, and lysozyme were analyzed using PyMOL 2.0 (The PyMOL Molecular Graphics System, Version 2.0, Schroedinger, LLC) and compared with a model of mycodulcein generated with Phyre2 (Kelley LA et al. The Phyre2 web portal for protein modeling, prediction, and analysis Nature Protocols 10, (2015): 845-858) (Figure 5A). Twenty-three conservative single-point mutations were made in ionizable amino acid residues. Negatively charged glutamine and aspartate differ by an additional carbon in the aliphatic chain. While replacing positively charged lysine and arginine is considered a conservative substitution, the guanidinium of arginine can form additional interactions with amino acids, including hydrogen bonds, aromatic, and aliphatic contacts. The ionic amino acid mutations were lysine to arginine, arginine to lysine, aspartic acid to glutamic acid, and glutamic acid to aspartic acid.The relative positions of each mutant were modeled by PyMOL 2.0 and classified as the N-terminus, three loop regions, five β-sheets, one α-helix, and the C-terminus (see Figure 5B).

[0192]

[0219] Specifically, the following single mutants were generated, and their predicted locations are shown in Table 3. See also Figure 5B, which shows the predicted secondary structure of SEQ ID NO: 3 superimposed on the putative secondary structure motifs and a representation of the locations of the point mutations in Table 3 within each motif.

[0193]

[0220] Cloning: Eco_0001 aka E. coli BL21 DE3 (E. coli strain BF - ompT gal dcm lon hsdS B (r B - m B - )λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5])[malB + ] K-12 (λ S)) (obtained from New England Biolabs # C2527I) was transformed with 23 plasmids (pMy_3018 to pMy_3040) using the manufacturer's protocol. Briefly, previously frozen competent cells were thawed, mixed with 1 pg to 100 ng of plasmid DNA, and kept on ice for 30 minutes. The mixture was heat-shocked at 42°C for 45 seconds. Immediately after, the mixture was placed in an ice bath and left for 10 minutes. 950 μL of pre-warmed LB medium was added, followed by shaking at 225 rpm for 1 hour at 37°C. Each transformation reaction was performed by diluting the cells 1:10 and 1:100 and plating 100 μL onto antibiotic plates. After overnight growth at 37°C, colonies fully recovered and were visualized. This process yielded strains Z18CE to Z40CE, which sequentially contain plasmids pMy_3018 to pMy_3040. Induced expression at the shake flask scale was used to confirm heterologous expression in the E. coli host. After transformation, mutants were plated and maintained on LB + ampicillin 100 μg / mL agar plates.

[0194]

[0221] Plates were incubated at 37°C for 16 hours. Colony screening PCR was performed to confirm the genotype using colony screening primers designed to interrogate the cDNA region of the plasmid as well as flanking regions. Successful transformation resulted in DNA fragments of specific sizes, while negative and no-template controls did not yield PCR bands. Successful transformation was observed for all mutants.

[0195]

[0222] Induced expression at shake flask scale was used to confirm heterologous expression in the E. coli host.

[0196]

[0223] Twenty-three strains (Z38CE–Z60CE) were maintained on LB + ampicillin 100 μg / mL agar plates, while a negative control (Eco_0001) was maintained on an LB agar plate. Overnight cultures for each strain were grown in 50 mL of LB liquid medium in 250 mL unbaffled culture shake flasks at 37°C with the appropriate antibiotic, shaking at 150 rpm overnight. The next day, the cultures were grown to 0.1 OD with the appropriate antibiotic, shaking at 200 rpm. 600 Each overnight culture was inoculated into 200 mL of TB liquid medium in a baffled 1000 mL culture shake flask conditioned at 37°C. 600 When the pH reached 0.8, a supplement of IPTG was added to the medium to a final concentration of 0.66 mM. Shaking was continued for an additional 5 hours at 37°C. Cells were then harvested by centrifugation at 5,000 g for 5 minutes at 4°C. The E. coli cells were washed once with cold dH2O and centrifuged again at 5,000 g for 10 minutes at 4°C. To confirm successful expression, cell lysates were prepared using liquid nitrogen and a mortar and pestle. The cell pellet was resuspended in 10 mL of cold dH2O, and the crude lysate was spun at 20,000 g for 5 minutes at 4°C. Finally, the supernatant was aspirated, filtered through a 0.2 μm PES filter, and subjected to SDS-PAGE protein electrophoresis. The crude lysate was tasted to identify sweet-tasting mutants. Table 3 shows the test results.

[0197]

[0224] Table 3. Results of sweetness test for single point mutations in mycodulcein

[0198] [Table 3]

[0225] Sixteen variants (Z38CE, Z39CE, Z41CE, Z45CE, Z47CE, Z48CE, Z49CE, Z51CE, Z52CE, Z53CE, Z55CE, Z56CE, Z57CE, Z58CE, Z59CE, and Z60CE), all of which have a sweet taste, were further re-expressed using 200 mL of medium. After 24 hours of induction, the cells were centrifuged at 4000 g for 20 minutes. The supernatant was discarded, and the pellet was suspended in approximately 20 mL of wash buffer (10 mM sodium phosphate buffer, pH 7.0). The suspended cells were disrupted in a high-pressure homogenizer (C3 Emulsiflex, Avestin, Inc., Ottawa, ON, Canada) operated at a maximum of 2000 bar. The disrupted cells were centrifuged at 13000 g for 30 minutes, the supernatant was collected, and the pellet was discarded. The supernatant containing the solubilized protein was filtered through a 0.22 μm PES membrane unit (Millipore, Burlington, MA, USA), and the material was then isolated by IMAC purification as described in Example 4.

[0226] The purified samples were tasted by trained sensory scientists. Mycodulcein stocks were diluted to equivalent protein concentrations, as measured by ELISA. Subjects followed an institutional review board-approved bite and saliva testing protocol. 0.2 mL of each purified variant was placed on the tongue, and the intensity of sweetness perception, the time to onset of sweetness perception, and the duration of sweetness perception were recorded. The results are shown in Figure 6 and described below.

[0199]

[0227] Effect of conservative point mutations on the sweetness of mycodulcein

[0228] To correlate the effects of conservative single-point mutations with sweetness, published mutations in thaumatin, brazzein, monellin, and lysozyme were compared with mycodulcein. Because the goal was to match the time and intensity profile of mycodulcein with that of sucrose, we measured sucrose equivalence, sweetness onset, and total duration by sensory analysis. Sucrose has a fast sweetness onset, high intensity, and fast duration. Therefore, mutations that reduce sweetness onset and duration are desirable, as are mutations that match or improve sucrose equivalence. Mutations that increase sweetness onset and total duration are undesirable, as are mutations that reduce sucrose equivalence. See Figures 5B and 6.

[0200]

[0229] N-terminal-external-D3E

[0230] A conservative single mutation of D3E at the external N-terminus resulted in loss of sucrose affinity and duration; however, sweetness expression was only slightly reduced. These results suggest that the charge, size, and polarity of the N-terminus of sweet proteins are important for sweetness and protein stability.

[0201]

[0231] β sheet 1 -external-K11R

[0232] A conservative single mutation at K11R resulted in a slight increase in sucrose affinity and a moderate decrease in sweet taste expression; however, the duration of sweet taste was dramatically increased. These results suggest that K11 is a critical residue for binding to the sweet taste receptor T1R2 / T1R3 and may affect the off-rate of mycodulcein from the receptor.

[0202]

[0233] Region between β-sheets 2 and 3 - external - K26R - linker region

[0234] A conservative mutation at K26 resulted in a small reduction in sucrose equivalence, indicating that this conservative mutation does not have a major effect on protein functionality.

[0203]

[0235] Loop 2 region-external-K51R

[0236] Molecular modeling showed that all putative loop region mutations were solvent-exposed. Except for a moderate reduction in sweetness expression, sensory testing showed only a slight reduction in sucrose equivalent and total duration, indicating that this conservative mutation did not have a significant effect on protein functionality.

[0204]

[0237] Loop 2 region-external-R57K

[0238] The mutation R57K has a significant inhibitory effect on sweetness expression, sucrose equivalence, and total duration.

[0205]

[0239] β sheet 4 -external-R66K

[0240] The R66K mutant significantly reduces the sucrose equivalent and total duration, but slightly increases sweetness expression. The R66K mutation may be an important residue for the affinity and off-rate for the sweet receptor.

[0206]

[0241] Loop 3 region-external-D69E

[0242] Mutations at D69E have a slight decrease in sucrose equivalence, duration, and a slight increase in duration. This region is predicted to be relatively insensitive to conservative mutations.

[0207]

[0243] α-helical region - outer - D85E and D86E

[0244] Both D85E and D86E had similar effects on the organoleptic properties of mycodulcein. The sucrose equivalence and duration of D85E and D86E were reduced. Sweetness onset was similar to that of the control.

[0208]

[0245] C-terminal-external-D97E, K103R, R106K, and E117D had minimal effects compared to the control, suggesting that these regions are relatively insensitive to conservative mutations. However, R110K and K120R showed reduced sweetness and duration, but similar sweetness expression.

[0209]

[0246] As shown in Table 3, conservative single-point mutations at R20K, E35D, K44R, D46E, D52E, R75K, and D94E resulted in loss of protein expression. These data suggest that these residues may be involved in protein folding or expression within the E. coli host. The predicted tertiary structure model discussed above in this example supports protein misfolding due to these changes, as all of these residues are contained in predicted β-sheets.

[0210]

[0247] References:

[0248] Korz, DJ, Rinas, U., Hellmuth, K., Sanders, EA & Deckwer, W.-D. Simple fed-batch technique for high cell cultivation density of Escherichia coli. Journal of Biotechnology 39, 59-65 (1995).

[0211]

[0249] Norsyahida, A., Rahmah, N. & Ahmad, RMY Effects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coli. Letters in Applied Microbiology 49, 544-550 (2009).

[0212] Example 9.

[0213]

[0250] Thermal stability is TM A Thermal Shift Protein Stability Kit (Biotium, Inc., Fremont, CA) was used to measure thermal shifts on protein samples from the IMAC purification of the 16 sweet variants described in Example 8 above, standardized to 0.04 mg / mL, using the manufacturer's instructions. Briefly, each reaction for measuring thermal shifts relies on mixing the following: mycodulcein, 36 μg / mL in 25 mM sodium phosphate buffer, pH 7.4, with the reagents provided in the kit according to the manufacturer's instructions. Thermal shift measurements were performed on a Bio-Rad CFX96 Touch system using a BR Clear plate, scan mode SYBER / FAM only, 25°C for 30 seconds, melting curve 25°C to 95°C, 0.5°C increments in 10 seconds, and plate read. Tm was determined based on the midpoint determined for a curve fitted to the experimental data using a five-parameter equation using the technique described in Schulz, MN, Landstroem, J. & Hubbard, RE, MTSA-A Matlab program to fit thermal shift data. Analytical Biochemistry 433, 43-47 (2013). The results (Table 4) show that thermal stability is minimally affected by the amino acid changes in the mutants tested.

[0214] [Table 4]

[0251] Example 10 (Cloning and heterologous expression of mycodulcein in Saccharomyces cerevisiae; confirmation of sweetness)

[0252] Based on the nucleotide sequence identified as SEQ ID NO:3, two expression vectors expressing mycodulcein SEQ ID NO:21 (pMy_4003) (His-tagged) and SEQ ID NO:3 (pMy_4002) (native) were synthesized and cloned by Atum, Inc. (Newark, CA) into the Atum vector non-secretory backbone pD1234, which contains the URA3 marker and the strong constitutive promoter GPD. The transformation procedure involves generating electrocompetent cells followed by introducing the expression vector by electroporation. Briefly, electrocompetent cells are first generated by growing cells to between early and mid-log phase, with multiple washes to remove salt from the growth medium. After mixing 1–5 μg of expression vector, the sample was electroporated in a Gene Pulser II electroporator with the following settings: charging voltage: 1.5 kV, capacitance: 25 μF, resistance: 200 Ω. 1 mL of pre-warmed 30°C YPD was immediately added, and the suspension was incubated at 30°C for 1–2 hours with shaking at 200–250 rpm. The transformed mutants were plated and maintained on SC-ura agar plates. This process yielded strains Z19ES and Z20ES, which contained the plasmids pMy_4002 and pMy_4003, respectively. csPCR (colony screening PCR) was performed to examine the cDNA region of the plasmids. Therefore, successful transformation yielded a 303-bp DNA fragment, while the negative and no-template controls did not yield a PCR band, confirming expression.

[0215]

[0253] Two strains (Z19ES, Z20ES) were maintained on SC-ura agar plates, while a negative control was maintained on SC agar plates. Overnight cultures of each strain were grown in 50 mL of SC-ura / SC liquid medium in 250 mL unbaffled culture shake flasks at 37°C, shaking at 150 rpm overnight. Each overnight culture was inoculated into 200 mL of SC-ura / SC (O-RDL-R10_TB medium) liquid medium in 1000 mL baffled culture shake flasks at 30°C, shaken at 200 rpm and adjusted to an OD of 0.02. Shaking was continued for an additional 48 hours at 30°C. Cells were then harvested by centrifugation at 5000 g for 5 minutes at 4°C. S. cerevisiae cells were then washed with cold dH2O and centrifuged again at 5000 g for 5 minutes at 4°C. To confirm successful expression, cells were lysed using liquid nitrogen and a mortar and pestle. The cell pellet was resuspended in 10 mL of cold dH2O, and the lysate was spun at 20,000 g for 5 minutes at 4°C. The supernatant was filtered through a 0.2 μm PES filter. The filtrate (for both strains) was confirmed to have a sweet taste by the method described in Example 3.

[0216]

[0254] Thermo Scientific TM HisPur TM The his-tagged protein SEQ ID NO:20 was purified from S. cerevisiae using efficient immobilized metal affinity chromatography (IMAC) with Ni-NTA resin. SEQ ID NO:20 was purified using a nickel-charged nitrilotriacetic acid (NTA) chelate immobilized on 6% cross-linked agarose resin. The lysate was loaded onto a prepared IMAC column, and the column was washed three times with 0.02 M imidazole in PBS. The his-tagged mycodulcein was then eluted four times with 0.3 M imidazole in PBS. The eluted fractions were then ultrafiltered using a 50 kDa MWCO filter, concentrated, and desalted using a 3 kDa MWCO filter.

[0217]

[0255] Example 11 (Purification of native mycodulcein from Z19ES strain (S. cerevisiae))

[0256] Three chromatographic techniques were evaluated for their effectiveness in purifying native mycodulcein (SEQ ID NO: 3) expressed in S. cerevisiae: cation exchange (CIEX), hydrophobic interaction (HIC), and size exclusion chromatography (SEC).

[0218]

[0257] Cation exchange rating.

[0219]

[0258] The isoelectric point of native mycodulcein was determined to be approximately 9.5 by isoelectric focusing, suggesting that a cation exchange column could be used successfully to purify mycodulcein. The clarified cell lysate, prepared as described in Example 10, was mixed with 2x starting buffer to obtain a cell lysate in 50 mM sodium phosphate, 1 M ammonium sulfate, pH 7.0, which was stored at 4°C for later use. The purification procedure was performed on an AKTA Explorer 100 system (GE Healthcare, Sweden), and the eluted protein was monitored at 280 nm and 215 nm using a UV detector UV-900 (GE Healthcare, Sweden). PreDictor plates (GE Healthcare, Sweden) pre-packed with CIEX resin were used to screen binding conditions for native mycodulcein. The pre-packed plates contain three main resins: Capto S (strong CIEX), Capto MMC (weak CIEX), and SP Sepharose Fast Flow (strong CIEX). The lysate was dialyzed into 20 mM dibasic sodium phosphate. Different pH values ​​ranging from 4 to 9 were screened, and the optimal conditions were then scaled up using a HiScreen column. Equilibration was performed using 25 mM dibasic sodium phosphate at pH 5 at a flow rate of 3 mL / min. Bound proteins were eluted using 25 mM dibasic sodium phosphate, 1 M sodium chloride, pH 7, with an increasing sodium chloride gradient from 0 to 1 M. Various binding and elution conditions were screened, and Capto MMC, a weak cation exchanger, showed the best binding capacity at pH 5. However, due to the low purity (25%) after this step, an alternative purification step was required. Figure 7A shows a PAGE analysis of the elution fractions from Capto MMC. M: protein marker; lane 1: elution fraction, showing low purity after cation exchange. The arrow indicates the mycodulcein band.

[0220]

[0259] HIC evaluation.

[0221]

[0260] The cell lysate was also subjected to hydrophobic interaction chromatography (HIC) using a HiScreen CaptoButyl column (Cytiva, Sweden); equilibration was performed with five column volumes of 50 mM sodium phosphate, 1 M ammonium sulfate, pH 7.0. The cell lysate was then loaded onto the column at a flow rate of 3 mL / min. Bound proteins were eluted using a decreasing ammonium sulfate gradient from 1 to 0 M with 50 mM sodium phosphate, pH 7.0. All fractions were analyzed by SDS-PAGE.

[0222]

[0261] Figure 7B shows two elution fractions collected during gradient elution from a HiScreen Capto Butyl column analyzed by SDS-PAGE. Lane 1 shows elution fraction 1, which does not contain mycodulcein, and lane 2 shows eluted mycodulcein. The purity of the elution fractions was determined to be approximately 86% by GelAnalyzer.

[0223]

[0262] SEC evaluation.

[0224]

[0263] The eluted fractions containing native mycodulcein were then further purified using a size-exclusion chromatography (SEC) HiPrep 26 / 60 Sephacryl S-200HR column (Cytiva, Sweden) and eluted with a buffer containing 50 mM sodium phosphate and 150 mM NaCl, pH 7.0. The fractions were collected, concentrated, and desalted using a 3 kDa molecular weight cutoff (MWCO) centrifugal filter (Millipore-Sigma, Germany) before analysis by SDS-PAGE.

[0225]

[0264] Summary: Native mycodulcein binds well to the weak cation exchanger Capto MMC, but the relatively low purity of the eluted fractions makes CIEX a less preferred capture / intermediate purification step. On the other hand, fractions of higher purity were obtained from HIC and Capto Butyl columns. SDS-PAGE analysis indicated that the impurities had relatively large molecular weights, making SEC a viable option for obtaining highly pure native mycodulcein.

[0226]

[0265] The purity after HIC / SEC is approximately 98% by GelAnalyzer SDS-PAGE. Figure 7C shows the eluted protein from the HIC column after chromatography on HiPrep 26 / 60 Sephacryl S-200. Lane 1 shows purified His-tagged mycodulcein, and lane 2 shows purified native mycodulcein.

[0227]

[0266] The purified native protein from S. cerevisiae was tasted (0.2 mL aliquots) at 0.03 mg / mL by trained sensory scientists and found to have a sweetness equivalent to 8° brix (approximately 8% sugar solution). The sweetness was very pronounced, with a "clean" sweetness (sugar-like taste), no other flavor notes, a slightly delayed sweetness onset, and a sweet aftertaste.

[0228]

[0267] Example 12 (Application Data)

[0268] His-tagged mycodulcein, prepared as in Example 5 and purified as described in Example 5, was tested in a yogurt base, which had the following recipe (Table 5):

[0269] Table 5:

[0229] [Table 5]

[0270] Cane sugar was added as a carbon source for the yogurt cultures and was at least partially consumed by the cultures. Mycodulcein was added to approximate the sweetness of 8-10°Brix sugars. The final concentration in the yogurt base was 0.05 mg / mL. Taste tests were performed by trained sensory scientists and the yogurt was found to have a sweetness equivalent to 8°Brix (approximately 8% sugar solution). The sweetness was very pronounced, with a "clean" sweetness (sugar-like taste) and no other flavor notes. The sweetness onset was slightly delayed and had a sweet aftertaste.

[0230]

[0271] His-tagged mycodulcein, prepared as described in Example 5 and purified as described in Example 5, was tested in whole milk; non-dairy pea-based milk (93.75% water, 4.2% pea protein, 1.7% canola oil, 0.3% TIC Gum Blend Pro 181 AG (Acacia + Gellan), 0.05% sunflower lecithin); cold coffee; and water (control) at a final concentration of 0.04 mg / mL, predicted to produce a sugar sweetness level of 8°-10°Brix. Sweetness testing confirmed that the sweet protein achieved a sweetness level between 8°-10°Brix in all samples, and all samples had similar sweetness intensity, sweetness onset, and sweetness duration to the water control.

[0231]

[0272] All references, including publications, patent applications, and patents, cited in this specification are herein incorporated by reference to the same extent as if each individual reference were specifically and individually indicated to be incorporated by reference.

[0232]

[0273] In the context of describing the present invention (particularly in the context of the claims below), the use of "a," "an," "the," "at least one," and similar terms of reference are to be construed as covering both the singular and the plural, unless otherwise stated herein or clearly contradicted by context. The use of the term "at least one," followed by a list of one or more items (e.g., "at least one of A and B"), should be construed to mean one item selected from the listed items (A or B), or any combination of two or more of the listed items (A and B), unless otherwise stated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted. The term "consisting of" is to be construed as a close-ended term (i.e., excluding components or steps other than those listed). The term "consisting essentially of" allows for the inclusion of components or steps that are not essential to, and do not materially affect, the function or activity of, the product or process.

[0233]

[0274] The recitation of ranges of values ​​herein is intended merely to serve as a shorthand method for individually referring to each separate value falling within that range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples or representative language (e.g., "etc.") provided herein are intended merely to better illustrate the invention and do not pose limitations on the scope of the invention, unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0234]

[0275] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect that skilled artisans will employ such variations as appropriate, and the inventors intend that the invention be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, all combinations of the above-described elements in all possible variations thereof are encompassed by the invention unless otherwise indicated herein or clearly contradicted by context. In one aspect, the present invention may be as follows. [Aspect 1] An isolated polynucleotide encoding a polypeptide having sweetness modulating activity, the polynucleotide sequence being: (a) a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75; (b) a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; and (c) a polypeptide sequence modified by deletion, insertion, substitution, or addition of 24 or fewer amino acids from a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; The isolated polynucleotide encoding a polypeptide selected from the group consisting of: [Aspect 2] An isolated polynucleotide according to aspect 1, wherein the polypeptide sequence of the polypeptide having sweetness regulating activity is the polypeptide sequence shown in SEQ ID NO: 3 or a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 3. [Embodiment 3] An isolated polynucleotide according to embodiment 1 or 2, wherein the polypeptide sequence comprises amino acid residues 1 to 11, 17 to 32, 39, 40, 45 to 67, 73 to 100, and 110 to 121 of SEQ ID NO:3. [Aspect 4] The isolated polynucleotide according to aspect 1 or 2, wherein the polypeptide sequence of the polypeptide having sweetness modulating activity comprises SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 75. [Aspect 5] The isolated polynucleotide according to Aspect 4, wherein the polypeptide sequence of the polypeptide having sweetness modulating activity comprises SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, or SEQ ID NO:68. [Aspect 6] The isolated polynucleotide of Aspect 4 or 5, wherein the polynucleotide comprises SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, or SEQ ID NO:67. [Aspect 7] An isolated polynucleotide according to any one of Aspects 1 to 6, operably linked to a heterologous regulatory element. [Embodiment 8] The isolated polynucleotide according to any one of embodiments 1 to 7, wherein the polynucleotide sequence further encodes a histidine tag. [Aspect 9] An expression cassette comprising the isolated polynucleotide according to any one of Aspects 1 to 8. [Aspect 10] A vector comprising the isolated polynucleotide according to any one of Aspects 1 to 8. [Aspect 11] A host cell transformed with the vector according to aspect 10. [Aspect 12] A method for producing a protein having sweet taste modulating activity, the method comprising culturing the host cell of Aspect 11 in a medium under conditions that result in the production of the protein having sweet taste modulating activity. [Aspect 13] An isolated polypeptide comprising a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, the polypeptide may contain at least one substitution modification with respect to a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, or The polypeptide may further comprise a histidine tag, and The polypeptide has sweetness-modulating activity. The isolated polypeptide. [Embodiment 14] The isolated polypeptide according to embodiment 13, wherein the polypeptide comprises SEQ ID NO: 3 or a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 3. [Embodiment 15] An isolated polypeptide according to embodiment 13 or 14, wherein the polypeptide comprises amino acid residues 1 to 11, 17 to 32, 39, 40, 45 to 67, 73 to 100, and 110 to 121 of SEQ ID NO:3. [Embodiment 16] The isolated polypeptide of embodiment 13 or 14, wherein the polypeptide comprises SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 75. [Aspect 17] The isolated polypeptide of Aspect 16, wherein the polypeptide comprises SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, or SEQ ID NO:68. [Aspect 18] The polynucleotide sequence is (a) the nucleic acid sequence set forth in SEQ ID NO:2; and (b) a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:2; 1. An isolated polynucleotide comprising a polynucleotide selected from the group consisting of: [Aspect 19] The isolated polynucleotide according to Aspect 18, wherein the polypeptide sequence of the polypeptide having sweetness modulating activity is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75. [Aspect 20] The isolated polynucleotide of Aspect 18 or 19, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, or SEQ ID NO:67. [Aspect 21] An isolated polynucleotide according to any one of Aspects 18 to 20, operably linked to a heterologous regulatory element. [Aspect 22] The isolated polynucleotide according to any one of Aspects 18 to 21, wherein the polypeptides (a) and (b) further comprise a nucleotide sequence encoding a histidine tag. [Aspect 23] An expression cassette comprising the polynucleotide according to any one of Aspects 18 to 22. [Aspect 24] A vector comprising the polynucleotide according to Aspect 23. [Aspect 25] A host cell transformed with the vector according to aspect 24. [Aspect 26] A method for producing a protein having sweet taste modulating activity, the method comprising culturing the host cell of Aspect 25 in a medium under conditions that result in the production of the protein having sweet taste modulating activity. [Embodiment 27] A composition comprising the following combination: (a) a product for oral administration, the product is not a Mattirolomyces terfezioides truffle; and (b) a sweetening composition comprising an isolated polypeptide, wherein: (i) the polypeptide comprises an amino acid sequence having at least 80% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; or (ii) the polypeptide is a polypeptide according to any one of aspects 13 to 17; The combination has an enhanced sweetness compared to the oral product. [Embodiment 28] A method for modulating the taste of a product for oral administration, comprising combining the product for oral administration with an effective amount of a sweetening composition comprising an isolated polypeptide, wherein: (i) the polypeptide comprises an amino acid sequence having at least 80% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; or (ii) the polypeptide is a polypeptide according to any one of aspects 13 to 17; the product for oral administration is not a Mattirolomyces terfezioides truffle, and The combination has an enhanced sweetness compared to the oral product. [Aspect 29] The composition according to aspect 27 or the method according to aspect 28, wherein the product for oral administration is a food, a beverage, a dietary supplement composition, or a pharmaceutical composition. [Embodiment 30] The composition of embodiment 27, wherein the product for oral administration is a food product selected from the group consisting of baked goods; sweet bakery products, pre-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings, gelatins and puddings; frozen desserts; yogurt; snack bars; bread products; pre-made bread mixes for preparing bread products; sauces, syrups and dressings; sweet spreads; confectionery products; and sweetened breakfast cereals. [Aspect 31] The composition of aspect 27 or the method of aspect 28, wherein the product for oral administration is a beverage product selected from the group consisting of carbonated beverages; non-carbonated beverages; and beverage concentrates. [Aspect 32] A method for purifying a polypeptide having sweetness-modulating activity, comprising: (a) obtaining a composition comprising a polypeptide; (b) purifying the composition via hydrophobic interaction chromatography (HIC) followed by size exclusion chromatography (SEC); wherein: (i) the polypeptide comprises an amino acid sequence having at least 80% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; or (ii) The polypeptide is a polypeptide according to any one of aspects 13 to 17.

Claims

1. 1. An isolated polynucleotide encoding a polypeptide having sweet taste modulating activity, the polynucleotide sequence being: (a) a polypeptide having the polypeptide sequence set forth in SEQ ID NO:3; and (b) a polypeptide having at least 90% sequence identity to the polypeptide sequence set forth in SEQ ID NO:3; The isolated polynucleotide encoding a polypeptide selected from the group consisting of:

2. 2. The isolated polynucleotide of claim 1, wherein the polypeptide sequence is modified from the polypeptide sequence of SEQ ID NO: 3 by deletion, insertion, substitution or addition of no more than one amino acid.

3. 3. The isolated polynucleotide of claim 1 or 2, wherein the polypeptide sequence comprises amino acid residues 1-11, 17-32, 39, 40, 45-67, 73-100, and 110-121 of SEQ ID NO:

3.

4. The isolated polynucleotide of claim 1, wherein the polypeptide sequence of the polypeptide having sweet taste modulating activity comprises SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66 or SEQ ID NO:

68.

5. 2. The isolated polynucleotide of claim 1, wherein the polynucleotide comprises SEQ ID NO:2, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:

67.

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

7. The isolated polynucleotide of any one of claims 1 to 6, wherein the polynucleotide sequence further encodes a histidine tag.

8. An expression cassette comprising the isolated polynucleotide of any one of claims 1 to 7.

9. A vector comprising the isolated polynucleotide of any one of claims 1 to 7.

10. A host cell transformed with the vector of claim 9.

11. 11. A method for producing a protein having sweet taste modulating activity, comprising culturing the host cell of claim 10 in a medium under conditions that result in the production of a protein having sweet taste modulating activity.

12. 1. An isolated polypeptide comprising a polypeptide sequence having at least 90% sequence identity to the polypeptide sequence set forth in SEQ ID NO:3, The polypeptide may further comprise a histidine tag, and The polypeptide has sweetness-modulating activity. The isolated polypeptide.

13. 13. The isolated polypeptide of claim 12, wherein the polypeptide comprises the polypeptide sequence set forth in SEQ ID NO:

3.

14. 13. The isolated polypeptide of claim 12, wherein the polypeptide sequence is modified from the polypeptide sequence of SEQ ID NO: 3 by deletion, insertion, substitution or addition of no more than one amino acid.

15. 15. The isolated polypeptide of any one of claims 12 to 14, wherein the polypeptide comprises amino acid residues 1 to 11, 17 to 32, 39, 40, 45 to 67, 73 to 100, and 110 to 121 of SEQ ID NO:

3.

16. An isolated polypeptide described in claim 12, wherein the polypeptide comprises the N-terminal amino acid sequence of SEQ ID NO:

4.

17. An isolated polypeptide described in claim 14, wherein the polypeptide comprises the N-terminal amino acid sequence of SEQ ID NO:

4.

18. 13. The isolated polypeptide of claim 12, wherein the polypeptide comprises SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66 or SEQ ID NO:

68.

19. The polynucleotide sequence is (a) the nucleic acid sequence set forth in SEQ ID NO:2; and (b) a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:2; 1. An isolated polynucleotide comprising a polynucleotide selected from the group consisting of:

20. 20. The isolated polynucleotide of claim 19, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:

67.

21. A combination of (a) and (b) below: (a) a product for oral administration, the product not being a Mattiromyces terfezioides truffle; and (b) a sweetening composition comprising the isolated polypeptide; and is free of Mattiromyces terfezioides truffles, wherein: (i) the isolated polypeptide comprises an amino acid sequence having at least 90% sequence identity to the polypeptide sequence set forth in SEQ ID NO:3, and has sweet taste modulating activity; or (ii) the isolated polypeptide is a polypeptide according to any one of claims 12 to 18; The combination has an enhanced sweetness compared to the oral product. The above composition.

22. 1. A method for modifying the taste of a product for oral administration, comprising combining the product for oral administration with an effective amount of a sweetening composition comprising an isolated polypeptide, wherein: (i) the isolated polypeptide comprises an amino acid sequence having at least 90% sequence identity to the polypeptide sequence set forth in SEQ ID NO:3, and has sweet taste modulating activity; or (ii) the isolated polypeptide is a polypeptide according to any one of claims 12 to 18; The product for oral administration is not a Mattiromyces terfezioides truffle, the sweetening composition does not contain Mattiromyces terfezioides truffles; and The combination has an enhanced sweetness compared to the oral product. The above method.

23. 22. The composition of claim 21, wherein the product for oral administration is a food, beverage, dietary supplement composition, or pharmaceutical composition.

24. 23. The method of claim 22, wherein the product for oral administration is a food, beverage, dietary supplement composition, or pharmaceutical composition.

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

26. 23. The method of claim 22, wherein the product for oral administration is a food product selected from the group consisting of baked goods; sweet bakery products, pre-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings, gelatins and puddings; frozen desserts; yogurt; snack bars; bread products; pre-made bread mixes for preparing bread products; sauces, syrups and dressings; sweet spreads; confectionery products; and sweetened breakfast cereals.

27. 22. The composition of claim 21, wherein the product for oral administration is a beverage product selected from the group consisting of carbonated beverages; non-carbonated beverages; and beverage concentrates.

28. 23. The method of claim 22, wherein the product for oral administration is a beverage product selected from the group consisting of carbonated beverages; non-carbonated beverages; and beverage concentrates.

29. A method for purifying a polypeptide having sweet taste modulating activity, comprising: (a) obtaining a composition comprising a polypeptide; (b) purifying the composition via hydrophobic interaction chromatography (HIC) followed by size exclusion chromatography (SEC); wherein: (i) the polypeptide comprises an amino acid sequence having at least 90% sequence identity to the polypeptide sequence set forth in SEQ ID NO:3 and has sweet taste modulating activity; or (ii) The polypeptide is a polypeptide according to any one of claims 12 to 18.

30. A combination of: (a) products for oral administration, and (b) a sweetening composition comprising the isolated polypeptide; 1. A method for producing a composition comprising: The method includes combining the oral product with the sweetening composition; The isolated polypeptide is prepared by the method of claim 11, The product for oral administration is not a Mattiromyces terfezioides truffle, the sweetening composition does not contain Mattiromyces terfezioides truffles; The combination has an enhanced sweetness compared to the oral product. The above manufacturing method.

31. 1. A method for modulating the taste of a product for oral administration, comprising combining the product for oral administration with an effective amount of a sweetening composition comprising an isolated polypeptide, The isolated polypeptide is prepared by the method of claim 11, The product for oral administration is not a Mattiromyces terfezioides truffle, the sweetening composition does not contain Mattiromyces terfezioides truffles; and The combination has an enhanced sweetness compared to the oral product. The above method.

Images