Sweet protein and its polynucleotide, expression vector, preparation method and purification method

Abstract

The present invention relates to sweet proteins and their polynucleotides, expression vectors, preparation methods and purification methods. The present invention provides newly identified fungal sweet taste modifier proteins and cDNAs encoding the proteins. The present invention provides an isolated polynucleotide encoding a polypeptide, which includes a polypeptide having an amino acid sequence of SEQ ID NO:3; and a polypeptide having an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:3; wherein the polypeptide has sweet taste regulating activity, and wherein the isolated polynucleotide is operably linked to a heterologous regulatory element. Specifically, the present invention describes a Myd protein active in sweet taste activation and a cDNA encoding the same, together with a method for isolating the cDNA and for isolating and expressing the protein. Also disclosed are the use of a sweet taste composition comprising the protein of the present invention, and a method for providing improved flavor for products for oral administration.

Description

[0001] This application is a divisional application of the Chinese patent application with an application date of June 25, 2021, application number "202180045258.1", and invention name "Sweet protein from truffles". Technical Field

[0002] The present invention relates to the field of sweeteners, in particular to sweet protein and its polynucleotide, expression vector, preparation method and purification method. Background Art

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

[0004] It is desirable to preferably use zero-calorie or low-calorie sweeteners derived from natural sources to limit the negative effects of high sugar consumption (e.g., diabetes and obesity, etc.). Well-known zero-calorie or low-calorie sweeteners include aspartame, acesulfame potassium, monk fruit extract, neotame, saccharin, stevia, and sucralose. However, these sweeteners have taste defects such as bitterness.

[0005] Truffles are underground fruiting bodies of some ascomycete fungi, including genera belonging to the order Pezizales of the class Pezizomycetes. Truffles are ectomycorrhizal fungi, and as such are often found in close association with tree roots.

[0006] So far, seven sweet taste modifying proteins are known, namely brazzein, thaumatin, taurine, curculin, mabinin, miraculin and pentadin. For any of these proteins, the key residues responsible for biological activity on the protein surface have not yet been determined. Taurine was found to be 100,000 times sweeter than sucrose on a molar basis, followed by brazzein and thaumatin, which are 500 times and 3000 times sweeter than sucrose on a gram basis, respectively. All these proteins are isolated from plants grown in tropical rainforests. Although most of them do not have sequence homology or structural similarity, thaumatin has extensive similarity to some non-sweet proteins found in other plants at the protein sequence level. There is no known sweet taste modifying protein in fungi.

[0007] There remains a need in the art for producing new low or zero calorie sweeteners with improved taste from natural sources.There remains a need in the art for economically producing sweetening compositions from potential sources of such compositions, particularly from Ascomycete fungal species. Summary of the invention

[0008] The present invention relates to newly identified sweet proteins from fungi, as well as genes and cDNAs encoding the proteins, also referred to herein as MYD / Myd. More specifically, the present invention relates to newly identified sweet proteins, genes and cDNAs encoding the proteins, and methods of regulating the taste of food using the proteins, genes and cDNAs. The present invention particularly provides a DNA sequence encoding a novel sweet protein identified herein as MYD1 and a corresponding polypeptide Myd1 (also referred to as mycodulcein). Myd1 is the first sweet protein identified from fungi. Myd1 reduces the sourness, bitterness or astringency of food and beverages, and in addition Myd1 has the activity of enhancing the taste of food and beverages, i.e., taste modification activity.

[0009] The present invention provides polynucleotides (e.g., isolated polynucleotides) encoding polypeptides 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:75. NO:17; and (c) a polypeptide sequence modified by deletion, insertion, substitution or addition of no more than 24 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.

[0010] In one aspect, the polypeptide sequence of the polypeptide having sweet taste modulating activity is the polypeptide sequence shown in SEQ ID NO: 3, or a polypeptide sequence having at least 80% sequence identity with 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 includes amino acid residues 1-11, 17-32, 39, 40, 45-67, 73-100 and 110-121 of SEQ ID NO:3.

[0012] The present invention also provides a polynucleotide (e.g., an isolated polynucleotide) selected from the group consisting of: (a) a polynucleotide comprising the nucleic acid sequence shown 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 with the nucleic acid sequence shown 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 shown in SEQ ID NO:2 and (ii) a nucleotide sequence encoding a histidine tag, wherein the polynucleotide encodes a polypeptide having sweet taste regulating activity.

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

[0014] In specific aspects, the polypeptide 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] 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 also encodes a protein tag or marker. The protein tag is optionally an affinity tag. The protein is optionally a histidine tag.

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

[0017] Provided are expression cassettes comprising polynucleotides and vectors comprising polynucleotides, as well as host cells transformed with the vectors. Also provided are methods for producing a protein having sweet taste modulating activity, the method comprising culturing a host cell in a culture medium under conditions leading to the production of a protein having sweet taste modulating activity.

[0018] The present invention includes 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 optionally comprises at least one substitution or 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, wherein the polypeptide optionally further comprises a protein tag, particularly a histidine tag, and wherein 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 includes 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, the 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 is optionally not 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 combination of (a) a product for oral administration, wherein the product is not a Mattirolomyces terfezioides truffle, and (b) a sweetening composition comprising the polypeptide, 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 with a polypeptide selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. On the other hand, the polypeptide has a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17; wherein the polypeptide optionally comprises 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, wherein the polypeptide is optionally not the polypeptide of SEQ ID NO:3, wherein the polypeptide optionally also includes a histidine tag, wherein the polypeptide has sweet taste modulating activity. For example, the polypeptide in the sweetening composition 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 optionally does not consist of SEQ ID NO:3.In particular, the polypeptide comprises the following amino acid sequence: 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] 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 is optionally a polypeptide other than the polypeptide of the amino acid sequence of SEQ ID NO:3.

[0024] The present invention provides a method for adjusting the taste of a product for oral administration, comprising combining the product for oral administration with an effective amount of a sweetening composition comprising a polypeptide, wherein the product for oral administration is not a truffle of the mushroom-like truffle, and wherein the combination has an enhanced sweet taste compared to the product for oral administration. In one aspect, the polypeptide comprises an amino acid sequence having at least 80% sequence identity with a polypeptide selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. On the other hand, the polypeptide has a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17; wherein the polypeptide optionally comprises 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, wherein the polypeptide is optionally not the polypeptide of SEQ ID NO:3, wherein the polypeptide optionally also includes a histidine tag, wherein the polypeptide has sweet taste modulating activity. For example, the polypeptide in the sweetening composition 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 optionally does not consist of SEQ ID NO:3.In particular, the polypeptide comprises the following amino acid sequence: 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] Products for oral administration may be foods, beverages, dietary supplement compositions, or pharmaceutical compositions. Examples of foods include, but are not limited to, baked goods; baked goods; sweet baked products, pre-made sweet baked mixes for preparing sweet baked products; pie fillings and other sweet fillings, gelatin, and puddings; frozen desserts; yogurt; snacks; bread products; pre-made bread mixes for preparing bread products; sauces, syrups, and condiments; sweet sauces; confectionery products; and sweet breakfast cereals. Examples of beverage products include, but are not limited to, carbonated beverages; non-carbonated beverages; and concentrated beverages.

[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 with a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. On the other hand, the polypeptide has a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17; wherein the polypeptide optionally comprises 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, wherein the polypeptide is optionally not the polypeptide of SEQ ID NO:3, wherein the polypeptide optionally also includes a histidine tag, wherein 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 optionally does not consist of SEQ ID NO:3.In particular, the polypeptide comprises the following amino acid sequence: 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 be apparent upon review of the drawings, detailed description, and non-limiting examples herein. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0029] Figure 2 Shown is a Coomassie-stained SDS-PAGE gel of proteins obtained from fractions of partially purified sporangial tissue of M. agrocybe.

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

[0031] Figure 4 Shown are the concentration response functions of mycodulcein, aspartame, thaumatin, and rebaudioside A for sweet taste. Data are plotted as the proportion (p) of responses on a 200 mM sucrose-related ("sweet") target. Each data point in the curves for mycodulcein, aspartame, thaumatin, and rebaudioside A is calculated as the mean of 32 repetitions, and for the sucrose curve, it is calculated as the mean of 16 repetitions; error bars are SEM. The points of water and sucrose controls are similarly calculated as the mean of 128 and 64 repetitions, respectively. Curves are fitted by nonlinear regression.

[0032] Figure 5AComparison of the predicted tertiary structure of mycodulcein with the known crystal structures (Protein Data Bank) of thaumatin (PDB: 1RQW), methamin (PDB: 2O9U), brazilin (PDB: 1BRZ), and hen egg white lysozyme (PDB: 1LSN) is shown, showing that mycodulcein has a predicted tertiary structure similar to the other known methamins, all of which contain antiparallel β-sheets and α-helices parallel to the β-sheets.

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

[0034] Figure 6 Shown are the results of comparing the sweetness intensity, sweetness perception onset time, and sweetness perception duration of his-tagged mutant mycodulceins at equal protein concentrations with each other and with his-tagged non-mutant mycodulcein as measured by ELISA.

[0035] Fig. 7A Coomassie-stained SDS-PAGE analysis of elution fractions from Capto MMC is shown. M: protein marker; Lane 1: elution fraction showing low purity after cation exchange. Arrow indicates mycodulcein band.

[0036] Figure 7B Coomassie-stained SDS-PAGE analysis of two elution fractions collected during gradient elution from a HiScreen Capto Butyl column analyzed on SDS-PAGE is shown. Lane 1 shows elution fraction 1 without mycodulcein, and lane 2 shows eluted mycodulcein. The purity of the elution fractions was determined to be ~86% by GelAnalyzer. Arrows indicate mycodulcein bands.

[0037] Figure 7C Coomassie-stained SDS-PAGE analysis of proteins eluted from the HIC column after chromatography on HiPrep 26 / 60 Sephacryl S-200 is shown. Lane 1 shows purified his-tagged mycodulcein, and lane 2 shows purified native mycodulcein. The purity of the eluted fractions was determined to be ~98% by GelAnalyzer. Arrows indicate mycodulcein bands. DETAILED DESCRIPTION

[0038] Therefore, the present invention provides nucleic acid molecules encoding proteins capable of regulating sweet taste, and polypeptides encoded by them. As described herein, the polypeptide of the present invention mediates sweet taste perception alone or in combination with food, beverage, dietary supplement or medicine. The present invention also provides isolated polypeptides capable of modifying sweet taste, and compositions thereof together with food, beverage, dietary supplement or pharmaceutical composition, and the resulting combination has sweet taste. The present invention also provides methods for modifying the sweet taste of food, beverage, dietary supplement or pharmaceutical composition by using isolated polynucleotides and polypeptides of the present invention.

[0039] In one aspect of the present invention, a newly identified fungal sweet protein is provided, referred to herein as Myd1. The term "Myd polypeptide" is used herein to identify any polypeptide according to the present invention, which, for example, has at least 80% sequence identity with SEQ ID NO:3 and also has sweet taste modifying activity. Myd polypeptides also include peptides SEQ ID NO:8 to SEQ ID NO:17 having sweet taste modifying activity. De novo amino acid sequencing was performed on a purified extract of the sweet portion of the spore-forming tissue of the fungus-like Martyrium to identify a 20-mer N-terminal sequence (SEQ ID NO:4). After the entire transcriptome of the spore-forming tissue of the fungus-like Martyrium was assembled from scratch using RNAseq reads, the Myd1 coding sequence (presumed to be derived from the MYD1 gene) was identified. Screening the entire transcriptome of the fungus-like Martyrium using the 20-mer N-terminal sequence identified a transcript that is expected to encode a protein with 100% identity at the N-terminus. The identified transcript is expected to encode a protein of 121 amino acids. The method identified SEQ ID NO:1. Start and stop codons were identified in the transcript to identify the putative coding sequence SEQ ID NO:2. SEQ ID NO:3 is a putative protein, a 121 amino acid protein. The identity between the predicted protein SEQ ID NO:3 and other protein sequences in GENBANK is 31% or less. The coding sequence of the natural mycodulcein codon-optimized for expression in Escherichia coli and Saccharomyces cerevisiae corresponds to the nucleic acid sequences of SEQ ID NO:20 and SEQ ID NO:22, respectively (which encodes the amino acid sequence of SEQ ID NO:3 with an optional 6-residue histidine tag, i.e., the amino acid sequence of SEQ ID NO:21).

[0040] In one aspect, the "Myd polypeptides" herein have 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 spore-forming tissue of the fungus by extraction. In other aspects, the "Myd polypeptides" herein have at least 50% or more sweet taste modifying activity compared to a naturally occurring Myd polypeptide isolated from spore-forming tissue of the fungus by extraction. In one aspect, the "Myd polypeptides" herein have at least 80% or more sweet taste modifying activity compared to a naturally occurring Myd polypeptide isolated from spore-forming tissue of the fungus by extraction. Sweet taste modifying activity can be measured by any comparative method known in the art, and in particular by any of the methods described in the Examples herein, and more particularly employing a method using a sensory panel as described herein.

[0041] While not wishing to be bound by any particular theory, it is believed that Myd1 is involved in sweet taste activation, for example, being an agonist of taste 1 receptor member 2 (T1R2) and / or taste 1 receptor member 3 (T1R3). However, Myd1 may agonize other taste receptors, such as bitter, umami, sour, and salty. The isolated or purified Myd polypeptides may then be used in the food and pharmaceutical industries to customize taste, for example, to adjust the sweetness of food or medicine.

[0042] In a first aspect, the present invention includes polynucleotides (e.g., isolated polynucleotides) encoding polypeptides having sweet taste modulating activity, wherein the polypeptide sequence comprises, consists essentially of, or consists of a polypeptide selected from the group consisting of: (a) 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; (b) 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; NO:17 has at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity; and (c) 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; and (d) 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; and (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 shown in SEQ ID NO: 3, an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 3, or an amino acid modified from the amino acid sequence of SEQ ID NO: 3 by deletion, insertion, substitution or addition of no more than 24 amino acids.

[0043] In a specific 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:1 by deletion, insertion, substitution, or addition of no more than 24 amino acids. 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 isolated polypeptide encoding a polypeptide having sweet taste modulating activity is not the polypeptide of SEQ ID NO:3.

[0044] In another aspect, the invention includes a polynucleotide (eg, an isolated polynucleotide), wherein the polynucleotide is selected from the group consisting of: (a) comprising SEQ ID NO:2, wherein the polynucleotide comprises 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 range of these values) modification (e.g., deletion, insertion, substitution or addition); and (b) comprising a nucleic acid sequence shown in SEQ A polynucleotide having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the nucleic acid sequence shown in SEQ ID NO: 2, optionally wherein the polynucleotide is not the polynucleotide of SEQ ID NO: 2. In embodiments, the polynucleotide encodes a polypeptide having sweet taste modulating activity. In embodiments, the amino acid sequence of the polypeptide having sweet taste modulating activity is the amino acid sequence shown in SEQ ID NO: 3.

[0045] In one aspect, the invention provides polynucleotides (e.g., isolated polynucleotides), wherein the polynucleotide sequence comprises, consists essentially of, or consists of a polynucleotide selected from the group consisting of: (a) comprising SEQ ID 64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75 NO:2 has 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 shown in NO:2, wherein the polynucleotide is optionally not the polynucleotide of SEQ ID NO:2, and (c) a polynucleotide comprising (i) the nucleic acid sequence shown 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.

[0046] The polynucleotide encoding the Myd of the present invention may be in the form of single-stranded or double-stranded DNA, RNA or artificial nucleic acid, or may be cDNA or chemically synthesized DNA, which does not include any introns.

[0047] The term "nucleic acid" or "nucleic acid sequence" refers to a deoxyribonucleotide or ribonucleotide oligonucleotide in single- or double-stranded form. The term encompasses nucleic acids containing known analogs of natural nucleotides, i.e., oligonucleotides. The term also encompasses nucleic acid-like structures with synthetic backbones (see, e.g., Oligonucleotides and Analogues, a Practical Approach, ed. F. Eckstein, Oxford Univ. Press (1991); Antisense Strategies, Annals of the NY Academy of Sciences, Vol. 600, Eds. Baserga et al. (NYAS 1992); Milligan J. Med. Chem. 36: 1923-1937 (1993); Antisense Research and Applications (1993, CRC Press); WO 97 / 03211; WO 96 / 39154; Mata, Toxicol. Appl. Pharmacol. 144: 189-197 (1997); Strauss-Soukup, Biochemistry 36: 8692-8698 (1997); Samstag, Antisense Nucleic Acids (1993). AcidDrug Dev, 6:153-156(1996)).

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

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

[0050] In another aspect, the invention provides a polypeptide (e.g., an isolated polypeptide) comprising, consisting essentially of, or consisting of a polypeptide 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 sequences of the group consisting of NO: 17 have a polypeptide sequence with 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 polypeptide optionally comprises 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 ranges of any of these values) modifications (e.g., deletions, insertions, substitutions or additions), wherein the polypeptide optionally further comprises a histidine tag, and wherein the polypeptide has sweet taste modulating activity. The term "consisting essentially of" permits the inclusion of components that are not essential to the function or activity of the product and do not substantially affect the function or activity, such as anti-caking agents, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltodextrose, gum arabic, etc.).

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

[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.

[0053] In one aspect, the polypeptide includes 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 includes 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.

[0054] In another aspect, the polypeptide includes amino acid residues 1-121 of SEQ ID NO:3, wherein, among amino acid residues 12-16, 33-38, 41-44, 68-72 or 101-109 of the polypeptide sequence, there is at least 1 (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 ​​thereof) amino acid substitution, addition, insertion or deletion compared to SEQ ID NO:3 in the listed residues. On the other hand, the polypeptide sequence of the polypeptide with sweet taste regulating activity encoded by the polynucleotide is a polypeptide comprising amino acid residues 1-121 of SEQ ID NO:3, wherein, among amino acid residues 12-16, 33-38, 41-44, 68-72 or 101-109 of the polypeptide sequence, there is at least one amino acid substitution, addition, insertion or deletion in the listed residues compared with SEQ ID NO:3, and wherein the polypeptide has at least 80% sequence identity with SEQ ID NO:3.

[0055] In another aspect, the present invention includes 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 peptide or transit peptide. The term "consisting essentially of" permits the inclusion of components that are not essential to the function or activity of the product and do not substantially affect the function or activity, such as anti-caking agents, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltodextrose, gum arabic, etc.).

[0056] In another aspect, the present invention includes a polypeptide having sweet taste modulating activity, comprising, consisting essentially of, or consisting of: 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 comprising a polypeptide having sweet taste modulating activity comprising, consisting essentially of, or consisting of: 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, In some aspects, the polypeptides of the invention include 1 to 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 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" permits the inclusion of components that are not essential to the function or activity of the product and do not substantially affect the function or activity, such as anti-caking agents, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltose dextrose, gum arabic, etc.).

[0057] In specific aspects, the polypeptide sequence of the polypeptide having sweet taste modulating activity encoded by the polynucleotide 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.

[0058] SEQ ID NO:71 (consensus sequence 1) corresponds to SEQ ID NO:3, except that 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 are any amino acids. The present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO:71.

[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 acids (i.e., the proline at positions 34, 36, 42, 71 and 104 of SEQ ID NO:3 is maintained). The present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO:72.

[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 invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO:73. The invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO:74.

[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.

[0062] In a specific aspect, the polypeptide sequence of the polypeptide having sweet taste modulating activity includes SEQ ID NO: 3, which has 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 relative to SEQ ID NO: 3 (for polypeptides) are given herein (see Example 8; Table 3). For example, the polypeptide sequence of the polypeptide having sweet taste modulating activity includes SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66 or SEQ ID NO:68, or a polypeptide sequence that is complementary 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. NO:68 has an amino acid sequence with at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity.

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

[0064] In another aspect, the polynucleotide encoding the 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 polypeptide with 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. NO:67 has a nucleic acid sequence with at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity.

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

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

[0067] The present invention also provides a polypeptide comprising an 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.

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

[0069] In another aspect, 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.

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

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

[0072] The polynucleotide or polypeptide may be naturally occurring or non-naturally occurring (eg, synthetic, recombinant, modified and / or variant products). In one aspect, the naturally occurring or non-naturally occurring product is isolated or purified.

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

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

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

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

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

[0078] The term "library" means a preparation of a mixture of different nucleic acid or polypeptide molecules, such as a library of recombinantly produced Myd-related polynucleotides generated by amplifying nucleic acids using degenerate primer pairs, or a collection of separate vectors that have introduced amplified ligand binding domains, or a mixture of cells randomly transfected with at least one vector encoding MYD.

[0079] As used herein, "nucleic acid probe or oligonucleotide" is defined as a nucleic acid that can bind to a target nucleic acid of a complementary sequence by one or more types of chemical bonding, usually by complementary base pairing, usually by hydrogen bond formation. As used herein, the probe can include natural (i.e., A, G, C or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in the probe can be connected by a bond other than a phosphodiester bond, as long as it does not interfere with hybridization. Thus, for example, the probe can be a peptide nucleic acid, in which the constituent bases are connected by peptide bonds rather than phosphodiester bonds. It will be appreciated by those skilled in the art that, depending on the stringency of the hybridization conditions, the probe can bind to a target sequence that lacks complete complementarity with the probe sequence. The probe is optionally directly labeled with an isotope, a chromophore, a luminophore, a chromogen, or indirectly labeled such as with biotin, and the streptavidin complex can then bind to the biotin. By determining the presence or absence of the probe, the presence or absence of a selected sequence or subsequence can be detected.

[0080] The term "heterologous" when used to refer to portions of a nucleic acid means that the nucleic acid includes two or more subsequences that do not exist in the same relationship to each other in nature. For example, nucleic acids are typically recombinantly produced, with two or more sequences from unrelated genes that are arranged to produce a new functional nucleic acid, such as a promoter from one source and a coding region from another source. Similarly, a heterologous protein means that the protein includes two or more subsequences that do not exist in the same relationship to each other in nature (e.g., a fusion protein).

[0081] "Promoter" is defined as a series of nucleic acid sequences that guide the transcription of nucleic acids. As used herein, a promoter includes essential nucleic acid sequences near the transcription start site, such as, in the case of a polymerase II type promoter, a TATA element. The promoter also optionally includes a distal end enhancer or repressor element, which can be located up to several thousand base pairs from the transcription start site. 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 connected" refers to a functional connection between a nucleic acid expression control sequence (such as a promoter or a transcription factor binding site array) and a second nucleic acid sequence, wherein the expression control sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0082] The term "MYD family" may refer to polymorphic variants, including natural alleles, mutants, alleles and interspecies homologs encoding polypeptides, wherein: (1) having at least about 35% to 50% amino acid sequence identity, optionally about 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO:3 over a window of about 25 amino acids, optionally 50-100 amino acids.

[0083] The term "expression vector" or "expression cassette" refers to any recombinant expression system for the purpose of constitutively or inducibly expressing the nucleic acid sequence of the present invention in any cell (including prokaryotic, yeast, fungal, plant, insect or mammalian cells) in vitro or in vivo. The term includes linear or circular expression systems. The term includes expression systems that maintain free genes or integrate into the host cell genome. The expression system may have the ability to self-replicate, or not have this ability, i.e., only drive transient expression in the cell. The term includes a recombinant expression "cassette" that contains only the minimum elements required to transcribe the recombinant nucleic acid.

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

[0085] In one embodiment, the host cell is selected from the group consisting of: Escherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, acetobutylicum), Pseudomonas fluorescens, Pseudomonas putida, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizopus oryzae, Yarrowialipolytica, Candida albicans, Issatchenkia orientalis, Scheffersomyces stipitis, Yarrowia lipolytica, Ogataea polymorpha, Phaffia rhodozyma, Candida utilisutilis), Arxulaadeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, and Schwanniomyces occidentalis.

[0086] In another aspect, the host cell is selected from the group consisting of: Gram-positive non-spore-forming bacteria, Gram-positive spore-forming bacteria, Gram-negative bacteria, yeast, and protists / algae.

[0087] Non-limiting examples of Gram-positive non-spore-forming bacteria include Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Carnobacterium divergens, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus alimentarius, Lactobacillus avium, Lactobacillus spp. aviaries), Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus collinoides, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus serratus ...helveticus), Lactobacillus hilgardii, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus mucosae, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus parafarraginis, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus shak sakei), Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactococcus lactis, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Microbacterium imperial, Oenococcus oeni, Pasteuria nishizawae, Pediococcusacidilactic, Pediococcus parvulus, Pediococcus pentosaceus, Propionibacterium acidipropioni, Propionibacterium freudenreich freudenreichii and Streptococcus thermophiles.

[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 valerianus, Bacillus spp. vallismortis, Bacillus velezensis, Geobacillus stearothermophilus, Paenibacillus sillinoisensis, and Parageobacillus thermoglucosidasius. Non-limiting examples of gram-negative bacteria include Cupriavidus necator, Gluconobacter oxydans, Komagataeibacter sucrofermentans, and Xanthomonas campestris.

[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.

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

[0091] The Myd proteins described herein also include "analogs" or "conservative variants" and "mimetics" ("peptide mimetics") having structures and activities that substantially correspond to the exemplary sequences. Thus, the term "conservative variant" or "analog" or "mimetics" refers to a polypeptide having an amino acid sequence modified so that one or more changes do not substantially alter the structure and / or activity of the polypeptide (conservative variant) as defined herein. These include conservatively modified variations of the amino acid sequence, i.e., amino acid substitutions, additions or deletions of residues that are not important for the activity of the protein, or amino acid substitutions of residues with similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.), such that even substitutions of important amino acids do not significantly alter the structure and / or activity.

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

[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.

[0094] Such nucleic acid variations are "silent variations," which are conservatively modified variations. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variation of the nucleic acid. The skilled artisan will recognize that each codon in the nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce functionally identical molecules. Therefore, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

[0095] Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, an exemplary guide to selecting conservative substitutions includes (original residue followed by exemplary substitutions): ala / gly or ser; arg / lys; asn / gln or his; asp / glu; cys / ser; gln / asn; gly / asp; gly / ala or pro; his / asn or gln; ile / leu or val; leu / ile or val; lys / arg or gln or glu; met / leu or tyr or ile; phe / met or leu or tyr; ser / thr; thr / ser; trp / tyr; tyr / trp or phe; val / ile or leu. Exemplary guidelines for substitution use the following six groups, each group comprising amino acids that are conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (I); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W); (see also, e.g., Creighton, Proteins, WH Freeman and Company (1984); Schultz and Schimer, Principles of Protein Structure, Springer-Vrlag (1979)). Another alternative exemplary guide 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 appreciate that the above substitutions are not the only possible conservative substitutions. For example, for some purposes, one can consider all charged amino acids as conservative substitutions for each other, whether they are positive or negative. In addition, individual substitutions, deletions or additions that change, add or delete a single amino acid or a small percentage of amino acids in the coding sequence can also be considered "conservatively modified variations". Those skilled in the art are familiar with the codon usage in a given host expressing a protein of interest.

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

[0097] For polypeptides of the invention that are conservative variants, routine experimentation will determine whether the mimetic is within the scope of the invention, i.e., whether its structure and / or function has not been substantially altered. A polypeptide mimetic composition may comprise any combination of non-natural structural components, which are generally from three structural groups: a) residue linking groups other than natural amide bonds ("peptide bonds"); b) non-natural residues that replace naturally occurring amino acid residues; c) residues that induce secondary structure mimicry, i.e., induce or stabilize secondary structures, such as beta turns, gamma turns, beta sheets, alpha helical conformations, etc. When all or some of the residues of a polypeptide are linked by chemical means other than natural peptide bonds, the polypeptide may be characterized as a mimetic. Individual peptide mimetic residues may be linked by peptide bonds, other chemical bonds, or coupling means, such as, for example, glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC), or N,N'-diisopropylcarbodiimide (DIC). Linking groups that can replace traditional amide ("peptide bond") linkages include, for example, ketomethylene (e.g., --C(O)--CH2-- instead of --C(O)--NH--), aminomethylene (CH2--NH), ethylene, olefin (CH=CH), ether (CH2--O), thioether (CH--S), tetrazole (CN4), thiazole, retroamide, thioamide or ester (see, for example, Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol.7, pp267-357, "Peptide Backbone Modifications," Marcell Dekker, NY (1983)). Polypeptides can also be characterized as mimetics by including all or some non-natural residues in place of naturally occurring amino acid residues; non-natural residues are well described in the scientific and patent literature. Phyre2 is a set of tools available on the web for predicting and analyzing protein structure, function and mutations.

[0098] Protein folding analysis was performed using tools including PHYRE Protein Homology / analogY Recognition Engine V 2.0 and protein secondary structure prediction server JPred. These tools revealed that SEQ ID NO: 3 predicted a β-sheet-dominated globular protein with a large number of β-sheet parts and some short α-helical parts. Specifically, the Jpred tool predicts putative beta sheets for about residues 5-11, 16-19, 28-29, 39-40, 61-67, 71-77, and 98-101 of SEQ ID NO:3; and alpha helices for about residues 20-25, 45-55, 111-119; the PHYRE tool predicts beta sheets for about residues 5 to 12, 17-32, 38-40, 45-57, 62-66, 73-81, 98-104, and 112-116 of SEQ ID NO:3, and alpha helices for about residues 84 to 89 and 116-118. See Figure 1 .

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

[0100] Alternative protein sequences with putatively similar structures and functions to Myd1 are provided herein as SEQ ID NO: 8 to SEQ ID NO: 17. To derive SEQ ID NO: 8 to SEQ ID NO: 17, consensus loop regions were selected as mutation sites because most insertions and deletions typically occur in regions between secondary structural elements where they can be more easily accommodated without creating major distortions in the overall folding of the protein. The core of the protein has a higher degree of sequence conservation, as found in 4JOX.

[0101] Of the 29 possible amino acid positions within the consensus loop region, 12 amino acids were replaced using conservative substitutions. If selected for mutation, the wild-type amino acid has an equal probability in the conservative amino acid residues. (Gly can be replaced by Ala, Cys, Asp, Glu, Arg with an equal probability of 20%).

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

[0103] For example, primers designed using sequences disclosed herein can be used to amplify and clone MYD-related genes from different fungal genomes. In contrast, genes related to MYD in a single species are preferably identified using sequence pattern recognition software to find related sequences. Typically, polymorphic variants and alleles of MYD family members can be identified by comparing about 25 amino acids or more, such as 50-100 amino acid amino acid sequences. About at least 35 to 50% and optionally 60%, 70%, 75%, 80%, 85%, 90%, 95-99% or more amino acid identity generally indicates that the protein is a polymorphic variant, interspecies homologue or allele of a MYD family member. Any sequence comparison algorithm discussed below can be used to compare sequences. Antibodies that specifically bind to Myd polypeptides or their conserved regions can also be used to identify alleles, interspecies homologues and polymorphic variants.

[0104] The nucleotide and amino acid sequence information of the MYD family members can also be used to build in silico models of sweet taste modulating peptides and how they interact with the sweet taste receptors and their in silico models. The sweet taste receptors consist of a heterodimer of taste 1 receptor member 2 (T1R2) and taste 1 receptor member 3 (T1R3). These models can then be used to identify variants and mutations of Myd that can increase activation of the sweet taste receptors and identify more active versions of Myd.

[0105] Various conservative mutations and substitutions are contemplated within the scope of the present invention. For example, it will be within the technical level of the art to use known protocols of recombinant gene technology including PCR, gene cloning, site-directed mutagenesis of cDNA, transfection of host cells and in vitro transcription to carry out amino acid substitutions. The variants can then be screened for their functional activity as taste receptor agonists.

[0106] In one embodiment, a hybrid protein coding sequence comprising a nucleic acid encoding a Myd fusion protein can be constructed. These nucleic acid sequences can be operably connected to the following items: transcription or translation control elements, such as transcription and translation initiation sequences, promoters and enhancers, transcription and translation terminators, polyadenylation sequences and other sequences for transcribing DNA into RNA. The fusion protein can include a C-terminal or N-terminal translocation sequence. In addition, the fusion protein can include additional elements, for example, for protein detection, purification or other applications. Detection and purification promotion domains include, for example, metal chelating peptides, such as polyhistidine bundles, histidine-tryptophan modules, or other domains that allow purification on immobilized metals; maltose binding proteins; protein A domains that allow purification on immobilized immunoglobulins; or domains used in FLAGS extension / affinity purification systems (Immunex Corp, Seattle Wash.).

[0107] In one embodiment, the fusion protein includes a peptide or protein tag (eg, for protein purification or detection). Peptide / protein tags are known in the art, such as those described in Johnson, “Protein / Peptide Tags,” DOI / / dx.doi.org / 10.13070 / mm.en.2.116, including but not limited to green fluorescent protein (GFP), FLAG, Myc epitope, poly-histidine, glutathione-S-transferase (GST), HA, V5, ABDz1-tag, adenylate kinase (AK-tag), BC2-tag, calmodulin binding peptide, CusF, Fc, Fh8, halogen tag, heparin binding peptide (HB-tag), ketosteroid isomerase (KSI), maltose binding protein (MBP), thioredoxin, PA (NZ-1), Poly-Arg, Poly-Lys, S-tag, SBP / streptavidin binding peptide, SNAP, Strep-II (Twin-Strep), and SUMO / SUMO2.

[0108] Affinity tags are protein tags that are attached to proteins so that they can be purified from their original biological sources using affinity techniques. Affinity tags are known in the art, such as those described in Kimple et al. CurrProtoc 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 peptides, and examples of large affinity tags include MBP, GST, cellulose binding domains, calmodulin binding peptides, and His-patch thioredoxin.

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

[0110] Epitope tags include FLAG, hemagglutinin (HA), c-myc, T7, and Glu-Glu, which are used to detect fusion proteins in vitro and in cell culture. Their short, linear recognition motifs rarely affect the properties of the target protein and are usually very specific for their respective primary antibodies. If an anti-myc antibody is used, the specificity can be improved by detecting the conjugated anti-myc primary antibody with an enzyme-linked secondary antibody, rather than using HRP- or AP-anti-myc conjugates alone.

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

[0112] Tandem affinity purification (TAP) is a dual affinity purification method based on fusing two affinity tags to a protein of interest, which allows purification of the tagged protein as well as isolation of protein complexes interacting with the protein of interest. The use of TAP is encompassed by the present invention.

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

[0114] The newly translated polypeptide includes a cleavable linker sequence between the translocation domain (for efficient plasma membrane expression) and the rest, 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)); enterokinase (Invitrogen, San Diego, Calif.), etc., can be used to facilitate purification. For example, a construct can include a polypeptide encoding a nucleic acid sequence linked to six histidine residues, followed by thioredoxin, an enterokinase cleavage site (see, e.g., Williams, Biochemistry 34: 1787-1797 (1995)), and a C-terminal translocation domain. The histidine residues facilitate detection and purification, while the enterokinase cleavage site provides a method for purifying one or more desired proteins from the rest of the fusion protein. Technology relating to vectors encoding fusion proteins and the use of fusion proteins is well described in the scientific and patent literature; see, eg, Kroll, DNA Cell. Biol. 12:441-53 (1993).

[0115] The fusion protein may comprise one or more linkers (e.g., flexible linkers, rigid linkers, and in vivo cleavable linkers). In addition to the basic role of connecting functional domains together (e.g., in flexible and rigid linkers) or releasing free functional domains in vivo (e.g., in vivo cleavable linkers), linkers also provide many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yield, and achieving desired pharmacokinetic characteristics. Linkers are known in the art (see, e.g., Chen et al., Adv Drug Deliv Rev. 65(10): 1357–1369(2013)).

[0116] Flexible linkers are used when the domains of the connection require a certain degree of mobility or interaction. They are usually composed of small non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The small size of these amino acids provides flexibility and allows the mobility of the connected functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, and thereby reduce the unfavorable interactions between the linker and the protein portion.

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

[0118] Rigid linkers keep a fixed distance between domains and maintain their independent functions. Examples of rigid linkers include (EAAAK) n (SEQ ID NO: 70) sequence alpha helix forming linker and having a Pro-rich sequence (XP) n A linker of wherein X represents any amino acid, preferably Ala, Lys or Glu.

[0119] The polypeptides of the present invention may also comprise a signal peptide (i.e., a signal sequence, a targeting signal, a localization signal, a localization sequence, a transit peptide, a leader sequence or a leader peptide), which is a short peptide present at the N-terminus or occasionally the C-terminus of most newly synthesized proteins that are intended to enter the secretory pathway. These proteins include those that reside in certain organelles (endoplasmic reticulum, Golgi apparatus or endosomes), are secreted from cells or are inserted into most cell membranes. Exemplary signal peptides are known in the art and one of ordinary skill in the art will recognize how to select a particular signal peptide for use in the present invention.

[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.

[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 or more to 30 or less, preferably 20 or less, more preferably 10 or less, and further preferably 5 or less amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or any range thereof). As used herein, examples of “a nucleotide sequence modified by deletion, insertion, substitution or addition of one or more nucleotides” include a nucleotide sequence modified by deletion, insertion, substitution or addition of 1 or more nucleotides or less, preferably 60 or less, more preferably 30 or less, further preferably 15 or less, and further more preferably 10 or less nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 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, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135

[0122] For example, in sequence comparison, a sequence is usually used as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates (if necessary) are specified, and sequence algorithm program parameters are specified. Default program parameters can be used, as described below for BLASTN and BLASTP programs, or alternative parameters can be specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters.

[0123] As used herein, "comparison window" includes a segment referring to any one of a plurality of contiguous positions selected from the group consisting of 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

[0124] Preferred examples of algorithms suitable for determining percent sequence identity and sequence similarity are 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 analysis is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a certain positive threshold score T when compared to words of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., Altschul et al., Nuc. Acids Res. 25: 3389-3402 (1977) and Altschul et al., J Mol. Biol. 215: 403-410 (1990)). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. Word hits are extended in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (a reward score for a pair of matching residues; always > 0) and N (a penalty score for mismatching residues; always < 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extension hits of the word in each direction will stop when: the cumulative alignment score drops by a number X from its maximum achieved value; the cumulative score becomes zero or below due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both chains. For amino acid sequences, the BLASTP program uses as default a word length of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad Sci. USA 89:10915 (1989)) alignment (B) 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both chains.

[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 relationships and percentages of sequence identity. It also draws a so-called "tree" or "dendrogram" showing the clustering relationships used to create the alignment (see, e.g., Figure 2 ). PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J Mol. Evol. 35: 351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5: 151-153 (1989). The program can align up to 300 sequences, with a maximum length of 5,000 nucleotides or amino acids for each sequence. The multiple alignment program starts with a pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. The cluster is then aligned with the next most relevant sequence or aligned sequence cluster. The two sequence clusters are aligned by a simple extension of the pairwise alignment of the two individual sequences. The final alignment is achieved by a series of progressive pairwise alignments. The program is run by specifying specific sequences and their amino acid or nucleotide coordinates for the sequence comparison region and by specifying program parameters. Using PILEUP, the reference sequence is compared to other test sequences to determine the sequence identity percentage relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, such as version 7.0 (Devereaux et al., Nuc. Acids Res. 12: 387-395 (1984), and the gene encoded is derived by conceptual translation of the corresponding open reading frame.

[0126] The polynucleotide encoding the polypeptide of the present invention can be chemically synthesized based on the amino acid sequence of Myd or synthesized by genetic engineering. For example, the polynucleotide can be chemically synthesized based on the amino acid sequence of the polypeptide of the present invention or its precursor protein. Contract synthesis services for nucleic acids (for example, provided by Medical & Biological Laboratories Co., Ltd., Genscript, etc.) can be used for chemical synthesis of polynucleotides. In addition, the synthesized polynucleotide can be amplified by PCR and cloning.

[0127] The polypeptide of the present invention can be produced, for example, by expressing a gene encoding the Myd polypeptide of the present invention. Preferably, the Myd polypeptide of the present invention can be produced from a transformant into which a polynucleotide encoding the Myd polypeptide of the present invention is introduced. For example, after the polynucleotide encoding the Myd polypeptide of the present invention or a vector comprising it is introduced into a host to obtain a transformant and the transformant is cultured in a suitable culture medium, the Myd polypeptide of the present invention is produced from the polynucleotide encoding the Myd polypeptide of the present invention introduced into the transformant. The protein of the present invention can be obtained by isolating or purifying the produced Myd polypeptide from the culture.

[0128] Therefore, the present invention further provides a polynucleotide encoding a Myd polypeptide of the present invention and a vector comprising the same. The present invention further provides a method for producing a transformant, comprising introducing a polynucleotide encoding a Myd polypeptide of the present invention or a vector comprising the same into a host. The present invention further provides a transformant comprising a polynucleotide encoding a Myd polypeptide of the present invention or a vector comprising the polynucleotide introduced from outside a cell. The present invention further provides a method for producing a Myd polypeptide of the present invention, comprising culturing a transformant.

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

[0130] Alternatively, a polynucleotide encoding the Myd polypeptide of the present invention can be produced by introducing mutations into a synthetic polynucleotide according to a procedure using a known mutagenesis method (such as ultraviolet irradiation and site-directed mutagenesis). For example, a polynucleotide encoding the polypeptide of the present invention can be obtained by introducing mutations into the polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 2 using a known method, expressing the obtained polynucleotide, studying the sweet taste modification activity of the expressed protein, and selecting a polynucleotide encoding a protein having the desired sweet taste modification activity.

[0131] Site-directed mutagenesis of polynucleotides can be carried out by any method, such as, for example, inverse PCR and annealing (Muramatsu et al. edit., "Revised 4th edition New genetic engineering handbook", YODOSHA, p. 82-88). Various commercial kits for site-directed mutagenesis can be used as needed, such as the QuickChange II site-directed mutagenesis kit and the QuickChange Multi site-directed mutagenesis kit from Stratagene.

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

[0133] Among these, a plasmid vector is preferred, and, for example, commercially available plasmid vectors for protein expression such as pUC19, pUC118, pUC119, pBR322, etc. (all of these from TAKARA BIO INC.) can be used.

[0134] The vector may include a DNA region containing a replication initiation region or a replication origin of DNA. Alternatively, a regulatory sequence such as a promoter region for initiating gene transcription, a terminator region, or a secretion signal region for secreting the expressed protein into the extracellular space may be operably connected to the upstream of a polynucleotide encoding the protein of the present invention (i.e., the MYD gene of the present invention) in the vector. As used herein, a gene and a regulatory sequence are "operably connected" to refer to a condition in which the gene and the regulatory region are positioned so that the gene can be expressed under the regulation of the regulatory region.

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

[0136] Alternatively, a marker gene (e.g., a resistance gene to agents such as ampicillin, neomycin, kanamycin, and chloramphenicol) for selecting a host into which the vector is suitable for introduction can be additionally incorporated into the vector of the present invention. Alternatively, when an auxotrophic strain is used as a host, a gene encoding a synthase of the desired nutrient can be incorporated into the vector as a marker gene. Alternatively, when a selective medium that requires a specific metabolism for growth is used, a metabolic related gene can be incorporated into the vector as a marker gene. Examples of such metabolic related genes include an acetamidase gene for use as a nitrogen source using acetamide.

[0137] The polynucleotide encoding the Myd polypeptide of the present invention can be linked to the regulatory sequence and marker gene by methods known in the art, such as SOE (Splicing by Overlap Extension)-PCR (Gene, 1989, 77: 61-68). The procedure for introducing the linked fragments into the vector is known in the art.

[0138] The example of the host of the transformant of the introduction vector includes microorganisms such as bacteria and filamentous fungi. The example of bacteria includes Escherichia coli and bacteria belonging to Staphylococcus, Enterococcus, Listeria and Bacillus, wherein preferably Escherichia coli and Bacillus bacteria (e.g., Bacillus subtilis or its mutant). The example of Bacillus subtilis mutant can include J.Biosci.Bioeng., 2007, 104 (2): Protease 9 double defect strain KA8AX described in 135-143, and Biotechnol.Lett., 2011, 33 (9): Protease 8 double defect strain DBPA strain described in 1847-1852, which improves protein folding efficiency. The example of filamentous fungi includes Trichoderma, Aspergillus and Rhizopus. In addition, for example, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowia lipolytica, Schizosaccharomyces pombe, Kluyveromyces lactis are suitable expression hosts. On the other hand, the present invention includes the following host cells, which include one or more of the expression cassettes described herein operably connected to control elements compatible with expression in the cell. The cell can be, for example, a mammalian cell (e.g., BHK, VERO, HT1080, 293, RD, COS-7 or CHO cell), an insect cell (e.g., Trichoplusiani (Tn5) or Sf9), a bacterial cell, a plant cell or a yeast cell.

[0139] The recombinantly expressed polypeptide from the expression cassette encoding Myd is typically isolated from lysed cells or culture medium. Purification can be performed by methods known in the art, including salt fractionation, ion exchange chromatography, gel filtration, size exclusion chromatography, size fractionation, and affinity chromatography. Immunoaffinity chromatography can be employed using antibodies generated based on, for example, the Gag antigen.

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

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

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

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

[0144] A variety of plant expressible promoters are known in the art and can be used for heterologous constructs, vectors and transformed plant materials herein containing polynucleotides encoding proteins with sweet taste regulating activity. Plant expressible promoters can be derived from natural plant sources, plant virus sources and bacteria with plant expressible promoters, such as Agrobacterium strains. Plant expressible promoters especially include cauliflower mosaic virus promoter (CaMV 35S), octopine and nopaline synthase promoters (e.g., nos promoter), plant ubiquitin promoter (Ubi), rice actin promoter (Act-1) and corn alcohol dehydrogenase (Adh-1). Plant expressible promoters include constitutive promoters, inducible promoters, tissue-specific promoters, developmental stage-specific promoters, and examples of promoters of each type are known in the art. Tissue-specific promoters are especially included in promoters (e.g., phosphoenolpyruvate promoter (PEP)) directly expressed in plant roots, plant leaves, fruits, flowers, pollen or cells participating in active photosynthesis. Developmental stage specific promoters include those that direct expression during fruit ripening, flowering or seed setting.Synthetic plant promoters are also known in the art and can be used for heterologous constructs, vectors and transformed plant materials (see, e.g., Ali S. & Kim WC (2019) Frontiers in Plant Science, 10, article 1433).

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

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

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

[0148] The transformant thus obtained into which the polynucleotide encoding the Myd polypeptide of the present invention or the vector comprising it is introduced is cultured in a suitable culture medium, resulting in the expression of the MYD cDNA on the vector, and then producing the Myd polypeptide of the present invention. The culture medium for culturing such a transformant can be appropriately selected by those skilled in the art according to the type of microorganism of such a transformant.

[0149] Alternatively, the Myd polypeptide of the present invention can be expressed from a polynucleotide encoding the Myd polypeptide of the present invention or its transcription product using a cell-free translation system. A "cell-free translation system" refers to an in vitro transcription translation system or an in vitro translation system constructed by adding reagents required for protein translation, such as amino acids, to a suspension obtained by mechanically disrupting host cells.

[0150] The Myd polypeptide of the present invention produced in culture or a cell-free translation system can be separated or purified by using a general method for purifying proteins, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., alone or in appropriate combination. Here, when the gene encoding the Myd polypeptide of the present invention and the secretion signal sequence are operably linked on a vector in a transformant, the Myd polypeptide produced can be more easily collected from the culture because the Myd polypeptide is secreted to the outside of the cell. The Myd polypeptide collected from the culture can be further purified by a known method.

[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 culture medium under conditions that result in the production of a protein having sweet taste modulating activity similar to a known sweet flavoring agent or compound.

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

[0153] Sweet flavoring agents or sweetening compositions may be used in an effective amount, which refers to an amount of the sweetening composition of the present invention sufficient to induce a sweet taste in a subject when present in a product for oral administration.

[0154] In an embodiment, the instant sweet protein can have sweet taste regulating activity. The Myd polypeptide of the present invention can have, for example, functional, physical and chemical effects on taste receptors such as sweet taste receptors. "Sweet taste regulating activity" can refer to the inhibition, activation (e.g., agonist or antagonist) properties of the polypeptide of the present invention identified using in vitro and in vivo taste transduction assays. Proteins with inhibitory activity can bind, partially or completely block stimulation, reduce, prevent, delay activation, inactivate, desensitize or turn down taste transduction, such as antagonists. Activating polypeptides can bind, stimulate, increase, open, activate, promote, enhance activation, sensitize or turn up taste transduction, such as agonists. Activating polypeptides are preferred.

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

[0156] Sweet taste modulating activity can be detected by methods known in the art, such as in vitro methods, or in vivo methods by animal or human sensory testing. Although not wishing to be bound by any particular theory, Myd is involved in sweet taste activation, for example, being an agonist of taste 1 receptor member 2 (T1R2) and / or taste 1 receptor member 3 (T1R3). However, Myd stimulates other taste receptors, such as bitter, umami, sour and salty. Such functional effects can be measured by any means known to those skilled in the art, for example, by measuring binding to taste receptor T1R via changes in spectral characteristics (e.g., fluorescence, absorbance, refractive index), fluid dynamics (e.g., shape), chromatography, or solubility properties, patch clamp, voltage-sensitive dye, whole-cell current, radioisotope efflux, induction markers, 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, etc.

[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 discipline that analyzes and measures human responses to food and drink compositions, such as appearance, touch, smell, texture, temperature, and taste. Measurements using humans as instruments are sometimes necessary. The selection of a suitable method for determining sweetness can be determined by one skilled in the art, and includes, for example, discrimination tests or difference tests designed to measure the likelihood that two products can be perceived differently. The correctness of the evaluator's responses is calculated, and statistical analysis is performed to see if there are more correct results than would be expected by chance alone.

[0158] Sensory evaluation is a scientific discipline that analyzes and measures people's reactions to food and drink compositions, such as appearance, touch, smell, texture, temperature and taste. It is sometimes necessary to use people as instruments for measurement. The food industry first needs to develop such measurement tools because the sensory characteristics of flavor and texture are obvious attributes that cannot be easily measured by instruments. The selection of suitable methods for determining the sensory qualities (e.g., sweetness) of the proteins disclosed in the present invention can be determined by those skilled in the art, and includes, for example, discrimination tests or difference tests, which are designed to measure the possibility that two products can perceive different differences. For sweetness perception, for example, samples and test samples of one or more of, for example, 5% sucrose, 6% sucrose, 7% sucrose, 8% sucrose, 9% sucrose, 10% sucrose can be arranged in the order of sweetness intensity from low sweetness to high sweetness by trained panel members. In the present invention, it should be understood that those skilled in the art can measure sensory differences in a variety of ways.

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

[0160] In some embodiments, Myd polypeptides of the invention include polypeptides that are at least as sweet as sugar (on a w / w basis) (e.g., IX), or alternatively, 2X, 5X, 10X, 50X, 100X, 200X, 400X, 600X, 800X, 1000X, 1500X, 2000X, 3000X, 5000X, 10,000X, 20,000X or sweeter than sugar as measured by any of the methods described above or by methods known in the art. In other embodiments, the Myd polypeptide has a sweetness that is at least 1% (at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) of sugar.

[0161] Food, Beverages, Supplements, Pharmaceutical Products

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

[0163] In one embodiment, a composition comprising an isolated Myd protein of the present invention may include a formulation that provides enhanced functionality to the isolated Myd protein. For example, a composition may include a formulation that stabilizes the Myd protein against heat, osmotic, pH, or other types of degradation. In one embodiment, the formulation stabilizes the Myd protein against thermal degradation. Exemplary compounds for stabilizing the Myd protein include, for example, L-arginine glycine, L-proline, L-histidine, β-alanine, L-serine, L-arginine ethyl ester dihydrochloride, L-arginine amide dihydrochloride, 6-aminocaproic acid, gly-gly, gly-gly-gly, tryptone, betaine monohydrate, D-(+)-trehalose dihydrate, xylitol, D-sorbitol, sucrose, hydroxyectoine, trimethylamine n-oxide dihydrate, methyl-α-d-pyranoglucoside, triethylene glycol, spermine tetrahydrochloride, spermidine, 5-aminovaleric acid, glutaric acid, adipic acid, ethyl dihydrochloride, 6-aminocaproic acid, gly-gly, gly-gly-gly, tryptic acid ... diamine, guanidine hydrochloride, urea, N-methylurea, N-ethylurea, N-methylformamide, hypotaurine, TCEP hydrochloride, GSH (reduced l-glutathione), benzamidine hydrochloride, ethylenediaminetetraacetic acid disodium salt dihydrate, magnesium chloride hexahydrate, cadmium chloride hydrate, non-detergent sulfobetaine 195 (NDSB-195), non-detergent sulfobetaine 201 (NDSB-201), non-detergent sulfobetaine 211 (NDSB-211), non-detergent sulfobetaine 221 (NDSB-221), non-detergent sulfobetaine 256 (NDSB-256), taurine, acetamide, oxalic acid dihydrate, sodium malonate pH 7.0, succinic acid pH 7.0, tacsimate pH 7.0, tetraethylammonium bromide, choline acetate, 1-ethyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazolium chloride, ethylammonium nitrate, ammonium sulfate, ammonium chloride, hydrated magnesium sulfate, potassium thiocyanate, gadolinium (III) chloride hexahydrate, cesium chloride, 4-aminobutyric acid (GABA), lithium nitrate, DL-malic acid pH 7.0, lithium citrate tetrahydrate, ammonium acetate, sodium benzenesulfonate, sodium p-toluenesulfonate, sodium chloride, potassium chloride, sodium dihydrogen phosphate monohydrate, sodium sulfate decahydrate, lithium chloride, bromide Sodium, glycerol, ethylene glycol, polyethylene glycol 200, polyethylene glycol monomethyl ether 550, polyethylene glycol monomethyl ether 750, formamide, polyethylene glycol 400, pentaerythritol ethoxylate (15 / 4EO / OH), 1,2-propylene glycol, polyethylene glycol monomethyl ether 1,900, polyethylene glycol 3,350, polyethylene glycol 8,000, polyvinyl pyrrolidone k15, polyethylene glycol 20,000, (2-hydroxypropyl)-β-cyclodextrin, α-cyclodextrin, β-cyclodextrin, methyl-β-cyclodextrin.

[0164] In one aspect, the Myd polypeptide of the sweetening composition may include 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; (a) wherein the polypeptide comprises 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, wherein the polypeptide is not the polypeptide of SEQ ID NO:3, or (b) wherein the polypeptide further comprises a histidine tag, wherein 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 specific 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.

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

[0166] In one aspect, the Myd polypeptide of the sweetening composition may include 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; (a) wherein the polypeptide comprises 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, wherein the polypeptide is not the polypeptide of SEQ ID NO:3, or (b) wherein the polypeptide further comprises a histidine tag, wherein the polypeptide has sweet taste modulating activity.

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

[0168] The term "product for oral administration" may refer to edible products such as food, beverage products; pharmaceutical (drug) products, or dietary supplement products such as herbal supplements. As used herein, the term "pharmaceutical product" includes both solid and liquid compositions, which are ingestible non-toxic materials with medicinal value or include pharmaceutically active agents such as cough syrups, cough drops, aspirin, and chewable pharmaceutical tablets. Oral hygiene products are also products for oral administration and include solids and liquids such as toothpaste or mouthwash.

[0169] In general, the present invention contemplates that food or beverage products can include an effective amount of the isolated sweet protein of the present invention, for example, relative to the gross weight of a food or beverage product, at most about 99% by weight, for example, about 0.01% to about 99% by weight. All intermediate weights are contemplated, relative to the gross weight of a food or beverage product, by weight (i.e. 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, ... 90%, 95%, 99%), and all intermediate ranges based on these amounts.

[0170] The composition of the present invention may include an "edible, biologically or medically acceptable carrier or excipient", which may include a solid or liquid medium and / or composition for preparing the desired dosage form of the Myd polypeptide, so as to administer the Myd polypeptide in a dispersed / diluted form, thereby maximizing the biological efficacy of the Myd polypeptide. Edible, biologically or medically acceptable carriers include many common food ingredients, such as water at neutral, acidic or alkaline pH, fruit or vegetable juice, vinegar, marinade, beer, wine, natural water / fat emulsions such as milk or condensed milk, edible oils and shortenings, fatty acids, low molecular weight oligomers of propylene glycol, glycerides of fatty acids, and dispersions or emulsions of such hydrophobic substances in aqueous media, salts such as sodium chloride, wheat flour, solvents such as ethanol, solid edible diluents such as vegetable powder or powdered substances, or other liquid carriers, dispersion or suspension aids, surfactants, isotonic agents; thickeners or emulsifiers, preservatives, solid binders, lubricants, etc.

[0171] Food or beverage products that may be considered in the context of the present invention include baked goods; sweet baked products (including but not limited to rolls, cakes, pies, pastries and cookies); pre-made sweet baking mixes for preparing sweet baked products; pie fillings and other sweet fillings (including but not limited to fruit pie fillings and nut pie fillings, such as pecan pie fillings, and fillings for cookies, cakes, pastries, confectionery products, etc., such as fat-based cream fillings); desserts, gelatin and puddings; frozen desserts (including but not limited to frozen dairy desserts, such as ice cream - including regular ice cream, soft ice cream and all other types of ice cream - and frozen non-dairy desserts, such as non-dairy ice cream, sorbet, etc.); carbonated beverages (including but not limited to soft carbonated beverages); non-carbonated beverages (including but not limited to soft non-carbonated beverages, such as flavored waters and sweetened tea or coffee-based beverages); beverage concentrates (including but not limited to liquid concentrates and syrups and non-liquid concentrates condensed products, such as freeze-dried and / or powdered preparations); yogurt (including but not limited to whole fat, low fat and skim yogurt, as well as non-dairy and lactose-free yogurt and frozen equivalents of all of these yogurts); snacks (including but not limited to cereals, nuts, seeds and / or fruit bars); bread products (including but not limited to leavened and unleavened breads, yeast and yeast-free breads, such as soda bread, breads including any type of wheat flour, breads including any type of non-wheat flour (such as potato, rice and rye flour), gluten-free bread); pre-made bread mixes for preparing bread products; sauces, syrups and condiments; sweet spreads (including but not limited to jellies, jams, butters, nut spreads and other spreadable preserves, candied fruits, etc.); confectionery products (including but not limited to jelly candies, soft candies, hard candies, chocolates and chewing gums); sweet breakfast cereals (including but not limited to extruded (kix-type) breakfast cereals, flaked breakfast cereals and puffed breakfast cereals); cereal coating compositions for preparing sweet breakfast cereals. Other types of food and beverage products not mentioned here but which typically include one or more nutritive sweeteners may also be considered in the context of the present invention.

[0172] Due to the complete or partial replacement of nutritive sweeteners in the food or beverage products of the present invention, the food or beverage products of the present invention can be used as low-calorie or weight loss products, medical foods / products (including pills and tablets) and sports nutrition products, and may be particularly suitable for food or beverage products that require lower sweetness at a given soluble solids level.

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

[0174] In an embodiment, the invention includes a method for enhancing the sweetness of a product for oral administration comprising adding a Myd polypeptide of the invention.

[0175] In another embodiment, the method of the present invention includes a method for improving the sweetness of a product for oral administration, the method including adding a sweetening composition prepared by the method of the present invention to a product for oral administration. The amount added can be determined by methods known in the art, for example, using sensory testing as a guide.

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

[0177] Example

[0178] Example 1

[0179] Fresh Mycorrhizal 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, then frozen in liquid nitrogen and stored at -80°C. The average moisture content of the truffles was 83.6% plus or minus 4.6%.

[0180] Aqueous extraction of truffles was performed as follows. Eight different truffle samples were ground into powder in liquid nitrogen, then 5:1 v / w truffles to 4°C water was added and incubated at 4°C for 30 minutes. The extracted material was then briefly centrifuged at low speed, and the filtrate tasted "clean". Sweetness intensity was scored between 0 (no sweetness) and 10 (extremely sweet). The sweetness of the samples was rated as specified in Table 1 below:

[0181] Table 1. Sweetness of various truffle samples.

[0182] sample Sweetness intensity Notes 1 5 Sweetness at the end 2 8 Sweetness is at the front and strengthens and lasts in the middle. 3 7 The sweetness is up front and intensifies and persists in the mid-palate. 4 5 Sweetness at the front, lasting sweetness at the bottom 5 4 Weak sweetness 6 3 Low sweetness 7 6 Mild and clean sweetness

[0183] The aqueous extracts were stored in sodium phosphate buffer at pH 7 and pH 2 at 4 °C and little change in sweetness was observed over a period of 8 days.

[0184] Example 2

[0185] Sweet protein Myd is purified from the ground mushroom-like Mati fungus. Fresh ground mushroom-like Mati fungus truffles are obtained in situ using appropriate procedures and permission in its natural range, and stored at -80 ° C. Take out 16.3g sample from the refrigerator and grind with mortar and pestle (white ceramic) in liquid nitrogen. Grinding is carried out for 15 minutes to fully crush the tissue and obtain fine frozen powder. The powder is added to a 50mL Falcon tube, and 20mL RO-H2O is added, and vortexed to mix the tissue until ice crystals are not observed. Use a rotor-stator set to 20 to continue 2X 1 minute at 4 ° C to decompose fragments and prepare a homogenous solution (H1). With RO-H2O, the slurry volume is adjusted to 53mL, and at 4 ° C, with 7500x G centrifugation for 30 minutes. The supernatant (S1) from this step is collected in a 2mL Eppendorf tube, and at 4 ° C, with 20,000x G centrifugation for 15 minutes in a 5417R and Eppendorf centrifuge. The precipitate (P1) is discarded. Sweetness is perceived in the supernatant (via human sense organs). The supernatant from this step is collected and pooled. The supernatant is then filtered through a 0.45 micron syringe filter (Cellulose, VWR International), 25 mm, and is called S1+0.22um filtration. The filtrate is then washed 2 times with hexane (38 mL to 50 mL hexane), and the aqueous phase is collected. S1FH is S1F+ hexane washing. The hexane phase is stored and dried. The aqueous phase is then precipitated with acetone (50 mL is added to 33 ml fraction S1FH at -20 ° C and placed at -20 ° C for 30 minutes). The sample is centrifuged at 3,000 x g and the precipitate is collected. The precipitate is resuspended in 10 mM sodium phosphate pH 6, the supernatant from this step is called S2, and the precipitate is called P2. The supernatant S2 was first applied to an AMICON centrifugal filtration unit with a molecular weight cutoff of 100 kD to obtain a filtrate (flow-through) fraction (referred to as 100F) and a retentate (referred to as 100R); 100F was then applied to a unit with a molecular weight cutoff of 30 kD to produce a filtrate (30F) and a retentate (30R). The sweet fraction flowed through the 100 kD column and emerged on the 100F, and was retained on the 30 kD column (30R). Figure 2 A band of approximately 13 kDa was observed on SDS-PAGE of , as indicated by the arrow, and the band was excised and subjected to N-terminal sequence analysis by Edman degradation using standard detection methods (e.g., liquid chromatography and mass spectrometry) to identify the residues of each cycle. A polypeptide, SEQ ID NO: 4, was detected.

[0186] Example 3 (Identification of RNA)

[0187] Sample collection

[0188] Fresh Truffles of the species Martyrium were obtained in situ in their natural range using appropriate procedures and permits. Wild isolates (BDP2_18) of the species Martyrium (spore-forming tissue) were obtained from natural environments. BDP2_18 is the largest wild truffle being found and has a sweetness profile of "sweet front, more fungal and earthy, low sweetness lingering".

[0189] Sample identification

[0190] Wild isolates of sporulating tissue were shipped frozen to GeneWiz for internal transcribed spacer (ITS) sequencing. The genomic loci sequenced were the ITS 1 and 2 regions. The generated sanger sequencing reads were then aligned and low-quality bases removed. Each sequence was then subjected to a separate basic local alignment search tool (BLAST) (Altschul, Gish, Miller, Myers, & Lipman, 1990) search to verify identity. BLASTn searches were performed using nucleotide sets (nr / nt) to exclude unpublished sample sequences. The entry with the highest percent identity to the wild isolate was the rib02 strain of the fungus-like Matrigel.

[0191] Sample preparation

[0192] The wild isolate BPD2_18 was surface washed with sterile water and cut into cubes weighing ˜100 mg and snap frozen in liquid N 2 for storage at −80° C. For GeneWiz, shipping was done on dry ice.

[0193] RNA sequencing

[0194] The following samples were submitted via GeneWiz for standard RNASeq via Illumina HiSeq, 2x150 bp, single index, with ~350M raw paired-end reads per lane. This RNASeq study involved polyA+ selection for transcriptome analysis.

[0195] GeneWiz RNA was extracted using the Qiagen RNeasy Plus Universal Mini Kit following the manufacturer's instructions. RNA library preparation was done using the NEBNext Ultra RNA Library Prep Kit for Illumina. Illumina adapter sequences are summarized below.

[0196] 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCT CTTCCGATCT-3'(SEQID NO:18)

[0197] 5'-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC[i7barcode]ATCTCGTATGCCGTCTTCTGCTTG-3'(SEQ ID NO:19)

[0198] Fastq sequence files from RNA-seq studies of two strains of M. agrocybe (three replicates each) were trimmed and cleaned with HTStream to remove the following contaminants: PhiX (a common contaminant from sequencing), rRNA reads, sequencing adapters, low-quality and "N" bases, poly-a traces, primers, and reads <50 bp. rnaSPAdes (Bushmanova E, Antipov D, Lapidus A, Prjibelski AD. rnaSPAdes: a de novo transcriptome assemblerand its application to RNA-Seq data. Gigascience. 2019; 8(9): giz100. doi: 10.1093 / gigascience / giz100) was then used to perform de novo transcriptome assembly for each M. agrocybe strain from the clean files. Bandage(a Bioinformatics Application for Navigating De novo AssemblyGraphs Easily)(Ryan R.Wick,Mark B.Schultz,Justin Zobel,Kathryn E.Holt,Bandage: interactive visualization of de novo genome assemblies,Bioinformatics,Volume 31,Issue 20,15October 2015,Pages 3350–3352) for searching using tBLASTn (Gertz EM, Yu YK, Agarwala R, AA, Altschul SF. Composition-based statistics and translated nucleotide searches: improving the TBLASTNmodule of BLAST. BMC Biol. 2006; 4: 41. Published 2006 Dec 7. doi: 10.1186 / 1741-7007-4-41) To visualize and search for assembly of target peptides, tBLASTn searches compare the protein query sequence (PDLSSFITIKNNSNHVFTRT, SEQ ID NO: 4) to a database of assembled transcriptome sequences dynamically translated in all six reading frames. Contigs containing perfect matches to the query protein sequence are subjected to open reading frame (ORF) analysis and ultimately used to construct full-length mRNAs linked to the target peptide.

[0199] RNA transcripts were identified, the DNA copies of which had the sequence shown in SEQ ID NO: 1, wherein SEQ ID NO: 2 is the predicted coding sequence based on the start and stop codons. A predicted protein SEQ ID NO: 3 is also provided, which has 121 amino acids and an estimated size of 13.3 kD. Length: 122 aa, molecular weight: 13.381 kDa, predicted isoelectric point: 8.64, and predicted charge at pH 7: 1.01.

[0200] Blast analysis. The predicted protein SEQ ID NO:3 has an identity of 31% or less with other protein sequences in GENBANK. A hypothetical protein from Pisolithus tincturius (GenBank: KIN98154.1; SEQ ID NO:7) was found to have approximately 31% homology with SEQ ID NO:3; it is called hypothetical protein M404DRAFT_1005519 [Pisolithus tincturius Marx 270]. It is speculated that SEQ ID NO:7 may also have sweet taste regulating activity. The complete cDNA copy of the RNA transcript is SEQ ID No:5, with the coding sequence shown in SEQ ID NO:6.

[0201] Example 4 (Cloning and heterologous expression of mycodulcein in E. coli; confirmation of sweet taste.)

[0202] Based on the nucleotide sequence identified as SEQ ID NO:3, three expression vectors expressing SEQ ID NO:20 containing a histidine tag were synthesized and cloned into three different Atum vector backbones by Atum, Inc. (Newark, CA): pD454-SR (plasmid pMy_3000), pD454-MR (plasmid pMy_3001) and pD454-WR (plasmid pMy_3002), all of which are E. coli IPTG-inducible T7 promoter expression vectors with ampicillin-r, lacl, Lac01, Ori_pUC and medium (M), strong (S) and weak (W) ribosome binding sites on the plasmid. E. coli BL21 DE3 (Studier et al. (1986) J. Mol. Biol. 189: 113-130) (New England Biolabs) was transformed with pMy_3000, pMy_3001, pMy_3002 using the manufacturer's protocol. Briefly, previously frozen competent cells were thawed and mixed with 1 pg-100 ng of plasmid DNA and placed on ice for 30 minutes. A heat shock of 42°C was applied to the mixture for 45 seconds. The mixture was then immediately placed in an ice bath for 10 minutes. 950 μL of preheated LB medium was added and then shaken at 225 rpm for 1 hour at 37°C. Cell dilutions of 1:10 and 1:100 were performed for each transformation reaction and 100 μL were plated on antibiotic plates. Overnight growth at 37°C allowed the colonies to fully recover and become visible. To confirm successful transformation, csPCR (colony screening PCR) was performed to inquire about the cDNA region of the plasmid, and expression was confirmed by lysate SDS-PAGE. Shake flask-scale induced expression was used to confirm heterologous expression in E. coli hosts. The process produced bacterial strains Z14CE, Z15CE, and Z16CE comprising plasmids pMy_3000, pMy_3001, and pMy_3002, respectively. Three bacterial strains (Z14CE, Z15CE, and Z16CE) were maintained on LB+ ampicillin 100 μg / mL agar plates, while negative control (Eco_0001) was maintained on LB agar plates. In a 250mL culture shake flask without baffles, at 37°C with appropriate antibiotics, shaken overnight at 150rpm, the overnight culture of each bacterial strain was grown in 50mL LB liquid medium. Each overnight culture was then inoculated into 200 mL of TB liquid medium and inoculated into a 1000 mL culture shake flask with baffles the next day, shaken at 200 rpm at 37°C, and adjusted to 0.1 OD600 with appropriate antibiotics. Once OD600 reached 0.8, IPTG supplement was added to the medium to a final concentration of 0.66 mM. Shaking was continued at 37°C for an additional 5 hours. After expression, the cells were centrifuged at 4000 g for 20 min.The supernatant was discarded and the precipitate was suspended in 20 mL of washing buffer (10 mM sodium phosphate buffer pH 7.0). The suspended cells were broken in a high pressure homogenizer (C3 Emulsiflex, Avestin, Inc., Ottawa, ON, Canada) running at up to 2,000 bar. The broken cells were centrifuged at 13,000 g (30 min), the supernatant was collected, and the precipitate was discarded. The supernatant containing the dissolved protein was filtered through a 0.22 μm PES membrane unit (Millipore, Burlington, MA, USA). SDS PAGE was run and a 13.1 kD band (Coomassie stain) was observed to confirm expression.

[0203] Thermo Scientific TM HisPur TM Ni-NTA resin was used to purify the his-tagged protein SEQ ID NO: 20 using an efficient immobilized metal affinity chromatography (IMAC). 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 preparative IMAC column, equilibrated with binding buffer: 20 mM sodium dihydrogen phosphate, 0.5 M sodium chloride, 0.1 M imidazole, pH 7.4, and eluted using elution buffer: 20 mM sodium dihydrogen phosphate, 0.5 M sodium chloride, 0.5 M imidazole. The column was washed three times with binding buffer, followed by elution of the his-tagged mycodulcein four times with elution buffer, followed by ultrafiltration of the eluted fraction using a 30 kDa MWCO filter, followed by filtration of the filtrate through a 10 kD filter at 4000 x G for 15 minutes. The retentate was diluted and spun again for the washing step, repeated three times. Figure 3 The purification steps analyzed by SDS-PAGE are shown in FIG.

[0204] Trained sensory scientists tasted purified fractions at 0.03 mg / ml (0.2 mL aliquot) ( Figure 3 8, containing highly purified SEQ ID NO: 21), and was found to have a sweetness equivalent to 8° Brix (approximately 8% sugar solution), confirming that mycodulcein isolated from Agrocybe agrocybe is responsible for the sweetening activity observed in Examples 1 and 2. The sweetness was very distinctly sweet, with a "clean" sweetness (sugar-like taste), without other flavors, a slightly delayed onset, and a sweet aftertaste.

[0205] Example 5 (Pilot production of MYCODULCEIN (HIS-label))

[0206] The performance of the E. coli strain Z14CE comprising the coding sequence SEQ ID NO:20 prepared in Example 4 was tested in a laboratory-scale bioreactor during fermentation. The bioreactor culture 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). The temperature was controlled via a stainless steel base filled with water. Agitation was provided by two mounted six-blade Rushton turbines, with the turbines spaced 47 mm apart and the lowest impeller positioned just above the bottom of the shaft. Aeration occurred through a perforated tube distributor ring. Dissolved oxygen (DO) was controlled at 20% of air saturation by using a continuous cascade of 500 to 800 rpm agitation and aeration between 5 and 8 L / min, and sparging air at high cell density. pH was controlled at 7.0 using 5.0 M ammonium hydroxide. Defoamer 204 (Sigma, St Louis, MO, USA) was added automatically to control foaming. The latter was detected using a conductivity probe installed at 10 cm above the culture level. The main fermentation medium contained (per liter) 24 g yeast extract, 12 g tryptone, 5.42 mL glycerol, 100 mL phosphate buffer stock solution (0.17 M KH2PO4, 0.72 M K2HPO4). 2 M HCl was used to adjust the medium to pH 7.0. When the initial glucose supply was exhausted (by pH raising the signal), the feed medium (per liter) composed of 200 g glucose, 21.1 g (NH4)2SO4 and 19.7 g MgSO4 was pumped into the fermentor at an initial flow rate of 1.00 mL / min. Unless otherwise stated, the initial volume of the medium in the container was 4.0 L. Inoculum (200ml) is composed of a culture grown for 16h in LB starting medium in a 1L baffled shake flask (37°C, 200rpm). The fermentation tank temperature is 37°C. After the optical density reaches 10-20, the fermentation is induced with 0.66mM IPTG, and then the fermentation is continued for 24 hours. After the 24-hour induction period, the fermentation broth is then centrifuged for 20min at 4000g. The supernatant is discarded and the precipitate is suspended in 1L washing buffer (10mM sodium phosphate buffer pH 7.0). The suspended cells are broken in a high pressure homogenizer (C3 Emulsiflex, Avestin, Inc., Ottawa, ON, Canada) running at a maximum of up to 1,500 bar. The broken cells are centrifuged (30min) at 13,000g, the supernatant is collected, and the precipitate is discarded. The supernatant containing the solubilized protein was filtered through a 0.22 μm PES membrane unit (Millipore, Burlington, MA, USA).

[0207] The purified supernatant prepared in this example was tasted at 0.03 mg / ml by a trained sensory scientist (0.2 mL aliquot) and found to have a sweetness equivalent to 8° Brix (approximately 8% sugar solution). The sweetness was very distinctly sweet, with a "clean" sweetness (sugar-like taste), no other flavors, a slightly delayed onset, and a sweet aftertaste.

[0208] The supernatant was stored in aliquots at −20 °C and used for further experiments.

[0209] Example 6 (ELISA Quantification of Mycodulcein from Escherichia coli)

[0210] A direct ELISA was developed to quantify histidine-tagged mycodulcein (SEQ ID NO: 21) using a horseradish peroxidase (HRP)-conjugated antibody to the 6XHis-tag sequence on the carboxyl terminus of SEQ ID NO: 21. The ELISA is capable of measuring mycodulcein concentrations in complex lysates and purified proteins. Recombinant 6XHis-tagged E. coli mycodulcein has a molecular weight of 14.2 kDa and a mass of 27,960 M. -1 cm -1 The molar extinction coefficient was calculated from the amino acid sequence by methods known in the art. The purity was evaluated by SDS-PAGE to be ≥98%. The mycodulcein protein concentration was determined by the absorbance at 280 nm using the Beer-Lambert law, and then a standard curve for ELISA was generated using known concentrations of mycodulcein (μg / ml).

[0211] ELISA procedure: The proteins were bound to the wall of a high protein binding 96-well plate in 50 mM carbonate buffer pH 9.4 coating buffer at room temperature for 30 minutes. The plates were washed 3 times with phosphate buffered saline (PBS) pH 7.4 with 0.02% Tween-20. Nonspecific binding sites on the microplates were blocked with 5% BSA in PBS pH 7.4 for 15 minutes at room temperature and washed 3 times with PBS 0.02% Tween-20. The primary antibody was diluted in blocking buffer (1:1000) and the microplates were incubated for 1 hour and washed 3 times in PBS 0.02% Tween-20. The colorimetric substrate 3,3',5,5'-tetramethylbenzidine (TMB) was used to develop the HRP reaction for 8 minutes and stopped with 2N sulfuric acid.

[0212] Example 7 (Quantitative Characterization of Concentration Dependence of Mycodulcein)

[0213] Opertech Bio (Philadelphia, PA) quantitatively characterized the concentration dependence of the taste properties of purified his-tagged mycodulcein (SEQ ID NO: 21) obtained from E. coli as described in Example 5 and purified as described in Example 4. The sweetness potency and relative efficacy of mycodulcein were compared to sucrose and other sweeteners thaumatin, rebaudioside A, and aspartame. The control standard was a solution of: 200 mM sucrose, 100 mM NaCl, 0.5 mM quinine, and 10 mM citric acid. Figure 4 Shown are the concentration response functions of mycodulcein, aspartame, thaumatin, and rebaudioside A for sweet taste. Data are plotted as the proportion (p) of responses occurring on a 200 mM sucrose-related ("sweet") target. Each data point in the curve for mycodulcein, aspartame, thaumatin, and rebaudioside A is calculated as the mean of 32 repetitions, and for the sucrose curve, it is calculated as the mean of 16 repetitions; error bars are SEM. The points of water and sucrose controls are similarly calculated as the mean of 128 and 64 repetitions, respectively. Curves are fitted by nonlinear regression.

[0214] The concentration that elicited a half-maximal sweet taste response (EC50 or potency) was 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. Equivalence to sucrose on a molar and weight basis is also provided.

[0215] Table 2.

[0216]

[0217]

[0218] Evaluation of thaumatin, sucrose and SEQ ID NO:21 was performed at 0.045 mg / ml protein and 10% sucrose using CATA (click all that apply) time intensity of sweetness perception of the specified analyte in water. Method: Time intensity technique; Data acquisition software: EyeQuestion, responses recorded 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. There were 3-6 panelists and 2 replicates. All samples were blinded and randomly provided with a 3-digit code.

[0219] Training Strategy: 6 weeks of intensive training on the sweetness scale on a 15-point sweetness scale to confidently assign sweetness values. Due to the unique sweetness behavior pattern, a 3-week training time-intensity principle was necessary. Samples were tasted and a stopwatch was used to record times and help reach consensus. #Number of panelists: 3-6, #Number of replicates: 2. All samples were blinded and randomly given a 3-digit code. Statistical Analysis: No statistical analysis was possible due to the small #number of panelists.

[0220] Under the amount tested, the maximum intensity (Imax) of mycodulcein and thaumatin is shown to be about 1 point higher than sucrose in a 15-point scale. Thaumatin and mycodulcein have flatter slopes, indicating longer peak times and more gradual / longer declines. Sucrose approaches threshold sweetness (intensity <1) at 162 seconds, faster than thaumatin and mycodulcein. When sucrose reaches the threshold level, thaumatin and mycodulcein can be identified under low-medium intensity. Mycodulcein and thaumatin seem to have similar effectiveness in this experiment, which is 3000x sweet of sucrose on a weight basis, or is about 120,000x sweet of sucrose on a molar basis. From these two experiments, mycodulcein is proven to be a high-intensity sweet protein, and its effectiveness is between 400x sweet of sucrose and 3000x sweet of sucrose on a weight basis.

[0221] Example 8 (Production of mycodulcein variants and testing for sweetness and thermal stability)

[0222] Methods for identifying potential key residues in mycodulcein. Although sweet proteins have no primary sequence identity, the overall tertiary structure has a sweet finger motif (Tancredi, T., Pastore, A., Salvadori, S., Esposito, V. & Temussi, P.A. Interaction of sweet proteins with their receptor: A conformational study of peptides corresponding to loops of brazzein, monellin and thaumatin. European Journal of Biochemistry 271, (2004): 2231–2240.). Sweet proteins have antiparallel β-sheets and α-helices perpendicular to the β-sheets. PyMOL 2.0 (The PyMOL Molecular Graphics System, Version 2.0 LLC) analyzed the tertiary structures of the sweet proteins thaumatin, tamanin, brazilin, and lysozyme and compared them with the mycodulcein model generated using Phyre2 (Kelley LA et al. The Phyre2 web portal for protein modeling, prediction, and analysis Nature Protocols 10, (2015): 845-858) ( Figure 5A ). Twenty-three conservative single-point mutations of ionic amino acid residues were generated. Negatively charged glutamine and aspartic acid differ by the additional carbon in the aliphatic chain. Although substitutions of positively charged lysine and arginine are considered conservative substitutions, the guanidinium of arginine can form additional interactions with the amino acid, including hydrogen bonds, aromatic and aliphatic contacts. The ionic amino acid mutations were lysine to arginine, arginine to lysine, aspartic acid to glutamate, and glutamate to aspartic acid. The relative position of each mutant was modeled by PyMOL2.0 and classified into the N-terminus, 3 loop regions, 5 β-sheets, 1 α-helix, and the C-terminus (see Figure 5B ).

[0223] Specifically, the following single mutants were generated and their predicted positions are in Table 3. See also Figure 5B , which shows a representation of the predicted secondary structure of SEQ ID NO: 3 superimposed on the putative secondary structure motifs and the positions of the point mutations in Table 3 within each motif.

[0224] Cloning: Eco_0001aka E. coli BL21 DE3 (E. coli str. BF – ompT gal dcm lon hsdS B (r B – m B – )λ(DE3[lacI lacUV5-T7p07ind1 sam7 nin5])[malB + ] K-12 (λ S)) (obtained from New England Biolabs #C2527I) for transformation. Briefly, previously frozen competent cells were thawed and mixed with 1 pg-100 ng of plasmid DNA and placed on ice for 30 minutes. A heat shock of 42°C was applied to the mixture for 45 seconds. The mixture was then immediately placed in an ice bath for 10 minutes. 950 μL of preheated LB medium was added and then shaken at 225 rpm for 1 hour at 37°C. Cell dilutions of 1:10 and 1:100 were performed for each transformation reaction and 100 μL was plated on antibiotic plates. Overnight growth at 37°C allowed the colonies to fully recover and become visible. This process sequentially produced strains Z18CE to Z40CE containing plasmids pMy_3018 to pMy_3040. Inducible expression at a shake flask scale was used to confirm heterologous expression in the E. coli host. The transformed mutants were plated and maintained on LB+ampicillin 100 μg / mL agar plates.

[0225] The plates were incubated at 37°C for 16 hours. Colony screening PCR was performed to confirm the genotype using colony screening primers designed to interrogate the flanking regions along with the cDNA region of the plasmid. Successful transformation will produce DNA fragments of a certain size, while the negative control and no template control will not produce PCR bands. Successful transformation of all mutants was observed.

[0226] Inducible expression at shake flask scale was used to confirm heterologous expression in the E. coli host.

[0227] 23 strains (Z38CE to Z60CE) were maintained on LB + ampicillin 100 μg / mL agar plates, while negative controls (Eco_0001) were maintained on LB agar plates. Overnight cultures of each strain were grown in 50 mL LB liquid medium in a 250 mL culture shake flask without baffles, shaking at 150 rpm overnight at 37°C with appropriate antibiotics. Each overnight culture was then inoculated into 200 mL of TB liquid medium and inoculated into a 1000 mL culture shake flask with baffles the next day, shaking at 200 rpm at 37°C, and adjusted to 0.1 OD with appropriate antibiotics. 600 Once OD 600When the pH reached 0.8, IPTG supplement was added to the culture medium to a final concentration of 0.66 mM. Continue shaking for an additional 5 hours at 37 ° C. Then, the cells were collected by centrifugation at 5000g for 5 min at 4 ° C. The E. coli cells were washed once with cold dH2O and centrifuged again at 5000g for 10 minutes at 4 ° C. To confirm successful expression, cell lysates were produced using liquid nitrogen and a mortar and pestle. The cell pellet was resuspended in 10 mL of cold dH2O, and the crude lysate was centrifuged at 20,000g for 5 minutes at 4 ° C. Finally, the supernatant was aspirated and filtered through a 0.2 μm PES filter and run on SDS-PAGE protein electrophoresis. The crude lysate was tasted to identify the sweet mutants. Table 3 shows the test results.

[0228] Table 3. Sweetness test results of single-point mutations of mycodulcein

[0229]

[0230]

[0231] 200 mL of culture medium was used to re-express 16 variants (Z38CE, Z39CE, Z41CE, Z45CE, Z47CE, Z48CE, Z49CE, Z51CE, Z52CE, Z53CE, Z55CE, Z56CE, Z57CE, Z58CE, Z59CE, Z60CE), all of which have a sweet taste. After expression, cells were centrifuged at 4000 g for 20 min after a 24-hour induction period. The supernatant was discarded and the pellet was suspended in ~20 mL of wash buffer (10 mM sodium phosphate buffer pH 7.0). Suspended cells were broken in a high pressure homogenizer (C3 Emulsiflex, Avestin, Inc., Ottawa, ON, Canada) running at up to 2,000 bar. The broken cells were centrifuged at 13,000 g (30 min), the supernatant was collected, and the pellet was discarded. The supernatant containing the solubilized protein was filtered through a 0.22 μm PES membrane unit (Millipore, Burlington, MA, USA). The material was then isolated by IMAC purification as described in Example 4.

[0232] Purified samples were tasted by trained sensory scientists. Mycodulcein stock solutions were diluted to equal protein concentrations as measured by ELISA. Subjects followed an institutional review board approved sip and spit protocol. 0.2 ml of each purified mutant was placed on the tongue and sweetness perception intensity, sweetness perception onset time, and sweetness perception duration were recorded. Results are shown in Figure 6 and discussed below.

[0233] Effects of conservative point mutations on the sweetness of Mycodulcein

[0234] To correlate the effects of conservative single-point mutations on sweetness, published mutations in thaumatin, brazilin, tamoxifen, and lysozyme were compared to mycodulcein. Since the goal was to match the temporal and intensity profiles of mycodulcein to sucrose, we measured sucrose equivalence, onset, and total duration by sensory analysis. Sucrose has a rapid onset, high intensity, and fast duration. Therefore, mutations that decrease onset and duration are as desirable as mutations that match or improve sucrose equivalence. Mutations that increase onset and total duration are as undesirable as mutations that decrease sucrose equivalence. See. Figure 5B and 6 .

[0235] N-terminal-external-D3E

[0236] Conservative changes in a single mutation of D3E at the outer N-terminus resulted in a loss of sucrose equivalence and duration; however, onset was only slightly reduced. These results suggest that the charge, size, and polarity of the sweet protein N-terminus are important for sweet taste and protein stability.

[0237] β-sheet 1-external-K11R

[0238] The conservative change of a single mutation at K11R resulted in a small increase in sucrose equivalence and a modest decrease in the onset of action; however, the duration of sweetness was significantly increased. These results suggest that K11 is an important residue for binding to the sweet taste receptors T1R2 / T1R3 and may affect the dissociation rate of mycodulcein from the receptors.

[0239] Region between β sheets 2 and 3 - external - K26R – linker region

[0240] The conservative mutation at K26 resulted in a slight decrease in sucrose equivalence, indicating that this conservative mutation had no significant effect on protein function.

[0241] Ring 2 Area - External - K51R

[0242] Molecular modeling showed that all putative loop region mutations were solvent exposed. In addition to a modest decrease in onset, only a small decrease in sucrose equivalence and total duration was observed in sensory studies, indicating that this conservative mutation had no significant effect on protein function.

[0243] Ring 2 Area - External - R57K

[0244] Mutation R57K had a significant inhibitory effect on onset, sucrose equivalence, and total duration of action.

[0245] β-sheet 4-external-R66K

[0246] Mutant R66K has significantly reduced sucrose equivalence and total duration, while slightly increased onset. The R66K mutation may be a key residue for the affinity and dissociation rate for the sweet taste receptor.

[0247] Loop 3 region - outer - D69E

[0248] The mutation at D69E had sucrose equivalence, a small decrease in duration, and a small increase in duration. This region in this area is predicted to be relatively insensitive to conservative mutations.

[0249] α-helical region - outside - D85E and D86E

[0250] Both D85E and D86E had similar effects on mycodulcein organoleptic properties. Sucrose equivalence and duration were reduced for D85E and D86E. Onset of effect was similar to control.

[0251] C-terminal-external-D97E, K103R, R106K and E117D had minimal effects compared to controls, indicating that these regions are relatively insensitive to conservative mutations. However, R110K and K120R showed reduced sweetness and duration, but similar onset.

[0252] As shown in Table 3, conservative single point mutations at R20K, E35D, K44R, D46E, D52E, R75K and D94E resulted in loss of protein expression. These data suggest that these residues may be involved in protein folding or expression in the E. coli host. The predicted tertiary structure model discussed above in this example supports protein misfolding caused by these changes, as all of these residues are included in the predicted beta sheet.

[0253] References:

[0254] Korz, DJ, Rinas, U., Hellmuth, K., Sanders, EA & Deckwer, W.-D. Simple fed-batch technique for high cell density cultivation of Escherichia coli. Journal of Biotechnology 39, 59–65 (1995).

[0255] 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).

[0256] Example 9.

[0257] Using GloMelt TM Thermal Shift Protein Stability Kit (Biotium, Inc. Fremont, CA) was used to perform thermal stability on IMAC-purified protein samples of 16 sweet mutants as described in Example 8 above, normalized to 0.04 mg / mL, using the instructions provided by the manufacturer. Briefly, a separate reaction to measure thermal shifts relies on mixing the following items according to the manufacturer's instructions: 36 μg / ml mycodulcein in 25 mM sodium phosphate buffer pH 7.4, and the reagents provided in the kit. For thermal shift measurements, a Bio-Rad CFX96 Touch system was used, which used BR clear plates, scanning mode SYBER / FAM only, 30 seconds at 25°C, melting curve from 25°C to 95°C, increments of 10 seconds 0.5°C plus plate reading. Tm was determined based on the midpoint determined for the curve fitted to the experimental data, which was measured using Schulz, MN, J. & Hubbard, REMTSA—AMatlab program to fit thermal shift data. Analytical Biochemistry 433, 43–47 (2013) Five parameter equation for the technique described in. The results (Table 4) show that thermal stability is minimally affected by amino acid changes in the tested mutants.

[0258] Table 4. Thermal stability test results of mutants

[0259]

[0260]

[0261] Example 10 (Cloning and heterologous expression of mycodulcein in Saccharomyces cerevisiae; confirmation of sweet taste)

[0262] Based on the nucleotide sequence identified as SEQ ID NO:3, two expression vectors expressing EQ ID NO:21 (pMy_4003) (with his tag) and SEQ ID NO:3 (pMy_4002) (native) mycodulcein were synthesized and cloned into the Atum vector non-secreting backbone pD1234 containing the URA3 marker and the strong constitutive promoter GPD by Atum, Inc. (Newark, CA). The transformation procedure involves preparing electrocompetent cells and then introducing the expression vector by electroporation. In brief, electrocompetent cells are produced by first growing the cells to between early and mid-log phases and washing multiple times to remove salts in the growth medium. After mixing 1-5 μ g expression vector, the sample was set up as follows on the Gene Pulser II electroporator (charging voltage: 1.5 kV, capacitance: 25 μ F, resistance: 200 Ω), 1 mL of preheated 30 ° C YPD was added immediately, and the suspension was incubated at 30 ° C with 200-250 rpm shaking for 1-2 h. The transformed mutants were plated and maintained on SC-ura agar plates. The process produced strains Z19ES and Z20ES comprising plasmids pMy_4002 and pMy_4003, respectively. csPCR (colony screening PCR) was carried out to query the cDNA region of the plasmid. Therefore, successful transformation will produce a DNA fragment of 303 bp, while negative controls and template-free controls will result in no PCR bands, and expression is confirmed.

[0263] Two strains (Z19ES, Z20ES) are maintained on SC-ura agar plates, while negative controls are maintained on SC agar plates. In a 250mL culture shake flask without baffles, shake overnight at 37°C with 150rpm, and the overnight culture of each strain is grown in 50mL SC-ura / SC liquid culture medium. Each overnight culture is inoculated into 200mL SC-ura / SC (O-RDL-R10_TB culture medium) liquid culture medium in a 1000mL culture shake flask with baffles vibrated at 200rpm at 30°C, and adjusted to 0.02OD600. Vibration is continued for an additional 48 hours at 30°C. Then, cells are collected by centrifugation for 5min at 5000g at 4°C. Then, Saccharomyces cerevisiae cells are washed with cold dH2O, and centrifuged again for 5 minutes at 4°C with 5000g. In order to confirm successful expression, liquid nitrogen and mortar and pestle are used to lyse cells. The cell pellet was resuspended in 10 mL of cold dH2O and the lysate was centrifuged at 20,000 g for 5 min at 4°C. The supernatant was filtered using a 0.2 μm PES filter. The filtrate (both strains) was confirmed to be sweet by the method described in Example 3.

[0264] Thermo Scientific TMHisPur TM Ni-NTA resin was used to purify the histidine-tagged protein SEQ ID NO: 20 from Saccharomyces cerevisiae using an efficient immobilized metal affinity chromatography (IMAC). 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 preparative IMAC column, the column was washed three times with 0.02 M imidazole in PBS, and then the his-tagged mycodulcein was eluted four times with 0.3 M imidazole in PBS, and then the eluted fractions were ultrafiltered using a 50 kDa MWCO filter, followed by concentration and desalting using a 3 kDa MWCO filter.

[0265] Example 11 (Purification of natural mycodulcein from strain Z19ES (Saccharomyces cerevisiae))

[0266] Three chromatographic techniques were evaluated for their effectiveness in purifying native mycodulcein (SEQ ID NO: 3) expressed in Saccharomyces cerevisiae: cation exchange (CIEX), hydrophobic interaction (HIC), and size exclusion chromatography (SEC).

[0267] Cation Exchange Evaluation.

[0268] The isoelectric point of native mycodulcein was determined to be ∼9.5 ​​by isoelectric focusing, indicating that a cation exchange column could successfully purify mycodulcein. The clarified cell lysate prepared as described in Example 10 was mixed with 2× starting buffer to obtain a cell lysate in 50 mM sodium phosphate, 1 M ammonium sulfate, pH 7.0, and stored at 4°C for future use. The purification procedure was performed on The eluted protein was monitored on Explorer 100 system (GE Healthcare, Sweden) and on UV detector UV-900 (GE Healthcare, Sweden) at 280nm and 215nm. PreDictor plates (GE Healthcare, Sweden) pre-filled with CIEX resin are used to screen the binding conditions of natural mycodulcein. Pre-filled plates include three kinds of main resins: Capto S (strong CIEX), Capto MMC (weak CIEX) and SP Sepharose Fast Flow (strong CIEX). Lysate is dialyzed into 20mM disodium hydrogen phosphate, and the screening range is at different pH values ​​of 4 to 9, and then the HiScreen column is used to amplify the optimal conditions. The 25mM disodium hydrogen phosphate using pH 5 is balanced with a flow velocity of 3mL / min. 25mM disodium hydrogen phosphate, 1M sodium chloride using pH 7 are used to elute the bound protein by the sodium chloride gradient from 0 to 1M that increases continuously. Different binding and elution conditions were screened, with the weak cation exchanger Capto MMC showing the best binding capacity at pH 5. However, due to the low purity after this step (25%), an alternative purification step was sought. Fig. 7A PAGE analysis of elution fractions from CaptoMMC is shown. M: protein marker; Lane 1: elution fraction showing low purity after cation exchange. Arrow indicates mycodulcein band.

[0269] HIC assessment.

[0270] Cell lysates were also subjected to hydrophobic interaction chromatography (HIC) using HiScreen CaptoButyl columns (Cytiva, Sweden); 5 column volumes of 50 mM sodium phosphate, 1 M ammonium sulfate at pH 7.0 were used for balance. The cell lysates were then loaded into the column at a flow rate of 3 mL / min. Elution of bound proteins was performed using 50 mM sodium phosphate at pH 7.0 with a decreasing ammonium sulfate gradient from 1 to 0 M. All fractions obtained were analyzed by SDS-PAGE.

[0271] Figure 7B Two elution fractions collected during gradient elution from a HiScreen Capto Butyl column analyzed on SDS-PAGE are shown. Lane 1 shows elution fraction 1 without mycodulcein, and lane 2 shows eluted mycodulcein. The purity of the elution fractions was determined by GelAnalyzer to be -86%.

[0272] SEC assessment.

[0273] Then use size exclusion chromatography (SEC) HiPrep 26 / 60Sephacryl S-200 HR column (Cytiva, Sweden) to further purify the elution fractions containing native mycodulcein, and elute with the buffer solution containing 50mM sodium phosphate and 150mMNaCl of pH 7.0. Collect fractions, and then use 3kDa molecular weight cutoff (MWCO) centrifugal filter (Millipore-Sigma, Germany) to concentrate and desalt, and then analyze by SDS-PAGE.

[0274] Summary: Although native mycodulcein successfully bound to the weak cation exchanger Capto MMC, the relatively low purity of the eluted fractions made CIEX a less favorable capture / intermediate purification step. On the other hand, higher purity fractions were obtained from the HIC, CaptoButyl column. Based on SDS-PAGE analysis, the impurities appeared to have relatively high molecular weights, which made SEC an ideal choice for obtaining highly pure native mycodulcein.

[0275] The purity after HIC / SEC was about 98% by GelAnalyzer of SDS-PAGE. Figure 7C Shown are proteins eluted from a 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.

[0276] Purified native protein purified from Saccharomyces cerevisiae was tasted at 0.03 mg / ml by trained sensory scientists (0.2 mL aliquot) and found to have a sweetness equivalent to 8° Brix (approximately 8% sugar solution). The sweetness was very distinctly sweet, with a "clean" sweetness (sugar-like taste), no other flavors, a slightly delayed onset and a sweet aftertaste.

[0277] Example 12 (Application Data)

[0278] His-tagged mycodulcein prepared as in Example 5 and purified as described in Example 5 was tested in a yogurt base. The yogurt base had the following formulation (Table 5)

[0279] Table 5.

[0280]

[0281] Sucrose is added as a carbon source for the yogurt culture and is at least partially consumed by the culture. Mycodulcein is added to a sugar with a sweetness of about 8° to 10° Brix, with a final concentration of 0.05 mg / ml in the yogurt base. Taste testing was performed by trained sensory scientists and found that the yogurt had a sweetness equivalent to 8° Brix (about 8% sugar solution). The sweetness was very noticeably sweet, with a "clean" sweetness (sugar-like taste), no other flavors, a slightly delayed onset and a sweet aftertaste.

[0282] His-tagged mycodulcein prepared as described in Example 5 and purified as described in Example 5 was tested at a final concentration of 0.04 mg / ml in whole milk, non-dairy pea-based milk (water, 93.75%, pea protein, 4.2%, rapeseed oil, 1.7%, TIC Gum Blend Pro 181AG (gum arabic + gellan gum) 0.3%, sunflower lecithin 0.05%), cold coffee, and water (control), and was expected to provide a sweetness level of 8° to 10° brix of sugar. Taste testing confirmed that thaumatin provided a sweetness level of 8° to 10° brix in all samples, and all samples had a sweetness intensity, onset, and duration similar to the water control.

[0283] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0284] Unless otherwise stated herein or clearly contradicted by context, the use of the terms "a / an" and "the" and "at least one" and similar referents in the context of describing the invention (particularly in the context of the appended claims) should be interpreted to include both the singular and the plural. Unless otherwise stated herein or clearly contradicted by context, the use of the term "at least one" in conjunction with a list of one or more items (e.g., "at least one of A and B") should be interpreted to mean one item (A or B) selected from the listed items or any combination of two or more of the listed items (A and B). Unless otherwise stated, the terms "comprising," "having," "including," and "containing" should be interpreted as open-ended terms (i.e., meaning "including but not limited to"). The term "consisting of" is interpreted as a closed term (i.e., excluding unlisted components or steps). The term "consisting essentially of" allows for the inclusion of components or steps that are not critical to the function or activity of a product or method and do not substantially affect the function or activity.

[0285] Unless otherwise specified herein, the reference to the range of values ​​herein is intended only to be used as a method of shorthand expression for referring to each individual value falling within the range individually, and each individual value is incorporated into the specification as if it were quoted individually herein. Unless otherwise specified herein or clearly contradictory to the context, all methods described herein can be performed in any suitable order. Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and is not intended to limit the scope of the present invention. Any language in the specification should not be interpreted as indicating that any unclaimed element is essential for the practice of the present invention.

[0286] Preferred embodiments of the present invention are described herein, including the best mode known to the inventor for implementing the present invention. After reading the foregoing description, changes in those preferred embodiments will become apparent to those of ordinary skill in the art. The inventor expects that skilled technicians will appropriately adopt such changes, and the inventor intends to implement the present invention in a manner different from that specifically described herein. Therefore, the present invention includes all modifications and equivalents of the subject matter listed in the appended claims as allowed by applicable law. In addition, unless otherwise stated herein or clearly contradictory to the context, the present invention includes any combination of all possible variations of the above-mentioned elements.

Claims

1. An isolated polynucleotide encoding a polypeptide selected from the group consisting of: (a) the polypeptide shown in SEQ ID NO: 3; and (b) a polypeptide having one amino acid modification or substitution relative to the amino acid sequence shown in SEQ ID NO:3, which is a polypeptide shown in any one 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.

2. The isolated polynucleotide according to claim 1, which is the polynucleotide shown in SEQ ID NO: 2 or SEQ ID NO:

20.

3. The isolated polynucleotide according to claim 1, wherein The polynucleotide sequence also encodes a protein tag or marker.

4. The isolated polynucleotide according to claim 1, wherein The polynucleotide sequence also encodes a histidine tag.

5. The isolated polynucleotide according to claim 1, wherein The polynucleotide sequence also encodes a histidine tag, and wherein the polynucleotide has a sequence as described in any one 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.

6. The isolated polynucleotide according to any one of claims 1 to 5, wherein The isolated polynucleotide also encodes a heterologous regulatory element operably linked to the polynucleotide sequence encoding the polypeptide.

7. The isolated polynucleotide according to any one of claims 1 to 5, wherein The isolated polynucleotide also encodes a heterologous promoter operably linked to the polynucleotide sequence encoding the polypeptide.

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

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

10. A host cell transformed with a vector comprising the isolated polynucleotide according to any one of claims 1-7.

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

12. A composition comprising a combination of: (a) A product for oral administration, wherein The product is not a truffle of the order Mushrooms, and (b) a sweetening composition comprising an isolated protein produced by the method according to claim 11, wherein the combination has an enhanced sweetness compared to the product for oral administration.

13. A method for adjusting the taste of a product for oral administration, the method comprising: combining a product for oral administration with an effective amount of a sweetening composition comprising an isolated protein produced by the method according to claim 11, wherein the product for oral administration is not a Agaricus truffle, and wherein the combination has an enhanced sweetness compared to the product for oral administration.

14. The composition according to claim 12, wherein The product for oral administration is a food, a dietary supplement composition or a pharmaceutical composition.

15. The composition according to claim 12, wherein The product for oral administration is a beverage.

16. The composition according to claim 15, wherein The beverage is selected from the group consisting of: carbonated beverages; Still drinks; and concentrated beverages.

17. The composition according to claim 12, wherein The product for oral administration is a food product selected from the group consisting of: baked goods; pie fillings and other sweet fillings, gelatin and puddings; frozen desserts; yogurt; snacks; pre-made bread mixes for preparing bakery products; spreads, syrups and condiments; sweet sauces; confectionery products; and sweet breakfast cereals.

18. The composition according to claim 17, wherein The baked goods are sweet baked products, pre-made sweet baked mixes or bread products for preparing sweet baked products.

19. The method according to claim 13, wherein: The product for oral administration is a food, a dietary supplement composition or a pharmaceutical composition.

20. The method according to claim 13, wherein: The product for oral administration is a beverage.

21. The method according to claim 20, wherein: The beverage is selected from the group consisting of: a carbonated beverage; a non-carbonated beverage; and concentrated beverages.

22. The method according to claim 13, wherein: The product for oral administration is a food product selected from the group consisting of: baked goods; pie fillings and other sweet fillings, gelatin and puddings; frozen desserts; yogurt; snacks; pre-made bread mixes for preparing bakery products; spreads, syrups and condiments; sweet sauces; confectionery products; and sweet breakfast cereals.

23. The method according to claim 22, wherein: The baked goods are sweet baked products, pre-made sweet baked mixes or bread products for preparing sweet baked products.

24. A method for purifying a protein having sweet taste modulating activity, the method comprising: (a) producing the protein by the method of claim 11, (b) The protein was purified via hydrophobic interaction chromatography (HIC) followed by size exclusion chromatography (SEC).

25. An isolated sweet protein as shown in SEQ ID NO:3, 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.

26. A composition comprising the following combination: (a) A product for oral administration, wherein The product is not a truffle of the order Mushrooms, and (b) a sweetening composition comprising the isolated protein of claim 25, wherein the combination has an enhanced sweetness compared to the product for oral administration.

27. A method for modifying the taste of a product for oral administration, the method comprising: combining a product for oral administration with an effective amount of a sweetening composition comprising the isolated protein of claim 25, wherein the product for oral administration is not a Agaricus truffle, and wherein the combination has an enhanced sweetness compared to the product for oral administration.

Images