Truffle-derived sweet protein

Abstract

The present invention provides a method for economically producing novel low-calorie or zero-calorie sweeteners with improved taste from their potential sources, particularly fungal species of ascomycetes, and also provides sweet protein. [Solution] This paper describes a newly identified fungal sweetness-modifying protein and the cDNA encoding the said protein. Specifically, it describes the Myd protein active in sweetness activation and the cDNA encoding it, as well as a method for isolating such cDNA and a method for isolating and expressing such protein. It also discloses the use of a sweetness composition containing the protein of the present invention and a method for providing improved flavor to orally administered products.

Description

[Technical Field]

[0001] Cross-reference of related applications

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

[0002] Reference to electronically submitted materials

[0002] In this specification, the entirety of the computer-readable nucleotide / amino acid sequence list, submitted concurrently with this specification and identified as follows: one 86678-byte ASCII (text) file named “338820_640_30A_WO_ST25.TXT” created on June 25, 2021. [Background technology]

[0003]

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

[0004]

[0004] Preferably, zero-calorie or low-calorie sweeteners derived from natural sources are desirable to limit the adverse effects of high sugar consumption (e.g., diabetes and obesity). Commonly 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]

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

[0006]

[0006] To date, seven proteins that modify sweetness and taste have been identified: blazein, thaumatin, monellin, curculin, mavinrin, miraculin, and pentadine. The key residues on the protein surface involved in biological activity have not yet been definitively identified for any of these proteins. Monellin was found to be 100,000 times sweeter than sucrose on a molar basis, followed by blazein and thaumatin, which were found to be 500 and 3,000 times sweeter than sucrose on a gram basis. All of these proteins have been isolated from plants growing in tropical rainforests. Most of them do not share sequence homology or structural similarity, but thaumatin shares extensive similarities at the protein sequence level with certain non-sweet proteins found in other plants. No fungal sweetness-modifying proteins are known.

[0007]

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

[0008]

[0008] The present invention relates to a newly identified fungal sweet protein, and as described herein This invention relates to the gene and cDNA encoding the aforementioned protein, also known as MYD / Myd. More specifically, this invention relates to a newly identified sweet-tasting protein. The present invention relates to genes and cDNA encoding the aforementioned protein, and to methods for using such proteins, genes and cDNA in regulating the taste of food. In particular, the present invention provides a novel sweet protein identified herein as MYD1 and a DNA sequence encoding the corresponding polypeptide Myd1 (also called mycodulcein). Myd1 is the first sweet protein identified from fungi. Myd1 reduces the sourness, bitterness, or astringency of food and beverages, and furthermore, Myd1 has the activity to enhance the taste of food and beverages, i.e., taste-modifying activity.

[0009]

[0009] The present invention provides a polynucleotide (e.g., an isolated polynucleotide) that encodes a polypeptide having sweetness-regulating 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 NOs: 3, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 71, 72, 73, 74, and 75; (b) a polypeptide having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NOs: 3, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17; and (c) a polypeptide sequence modified by deletion, insertion, substitution, or addition of 24 or fewer amino acids from a polypeptide sequence selected from the group consisting of SEQ ID NOs: 3, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17.

[0010]

[0010] In one view, the polypeptide sequence of a polypeptide having sweetness-regulating activity is the polypeptide sequence shown in SEQ ID NO: 3, or a polypeptide sequence having at least 80% sequence identity with SEQ ID NO: 3. In another view, the polypeptide encoding a polypeptide having sweetness-regulating activity is not the polypeptide of SEQ ID NO: 3.

[0011]

[0011] In other words, 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]

[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 respect to 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 sweetness-regulating activity.

[0013]

[0013] In one view, the polypeptide sequence of the polypeptide having sweetness-regulating 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 sweetness-regulating 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]

[0014] From a particular viewpoint, polynucleotides are SEQ ID NOs: 23, 25, 29, 37, 41, 43, 45, 49, Includes the nucleic acid sequence of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, or SEQ ID NO: 67.

[0015]

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

[0016]

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

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

[0018]

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

[0019]

[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]

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

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

[0022]

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

[0023]

[0023] In one view, the present invention provides a sweet composition comprising a polypeptide having 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, wherein the polypeptide may be a polypeptide other than the polypeptide of the amino acid sequence of SEQ ID NO: 3.

[0024]

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

[0025]

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

[0026]

[0026] The present invention also provides a method for purifying a polypeptide having sweetness-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 view, the polypeptide comprises an amino acid sequence having at least 80% sequence identity with a polypeptide selected from the group consisting of SEQ ID NOs: 3, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17. In other words, 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; where the polypeptide may contain at least one substitution modification with respect to the polypeptide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, the polypeptide may not be the polypeptide of SEQ ID NO: 3, the polypeptide may further contain a histidine tag, and the polypeptide has sweetness-regulating activity. For example, the polypeptide may contain the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74 or SEQ ID NO: 75, and may not be the polypeptide of SEQ ID NO: 3. In particular, the polypeptide contains the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, or SEQ ID NO: 68.

[0027]

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

[0028] [Figure 1]

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

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

[0030] Figure 3 shows the Coomassi-stained SDS-PAGE gel at the purification stage of Sequence ID No. 21 expressed in E. coli. Lane 1: Molecular weight standard; Lane 3: Crude lysate; Lane 4: Flow-through fraction from HisPur™ Ni-NTA; Lane 5: Wash 1; Lane 6: Wash 2; Lane 7: Wash 3; Lane 8: Eluent fraction. [Figure 4]

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

[0032] Figure 5A shows a comparison of the predicted tertiary structure of mycodulcein with the known crystal structures (protein databases) of thaumatin (PDB:1RQW), monellin (PDB:2O9U), blazein (PDB:1BRZ), and chicken egg white lysozyme (PDB:1LSN). It shows that mycodulcein has a similar predicted tertiary structure to other known sweet proteins, and all contain antiparallel β-sheets with α-helices parallel to the β-sheets. [Figure 5B]

[0033] Figure 5B shows the predicted secondary structure of Sequence ID 3 superimposed on the estimated secondary structure motif, along with the location of point mutations within each motif. [Figure 6]

[0034] Figure 6 shows the results of comparing mutant his-tagged mycodulcein with his-tagged non-mutant mycodulcein, both with each other and with his-tagged non-mutant mycodulcein, which was equivalent to comparable protein concentrations measured by ELISA, in terms of sweetness intensity, onset time of sweetness perception, and duration of sweetness perception. [Figure 7A]

[0035] Figure 7A shows the SDS-PAGE analysis and Coomassy staining of the fraction eluted from Capto MMC. M: protein marker; Lane 1: eluted fraction, showing low purity after cation exchange. Arrows indicate the mycodulcein band. [Figure 7B]

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

[0037] Figure 7B shows SDS-PAGE analysis and Coomassie staining of eluted proteins from an HIC column after chromatography using HiPrep 26 / 60 Sephacryl S-200. Lane 1 shows purified his-tagged mycodulcein, and lane 2 shows purified undenatured (native) mycodulcein. The purity of the eluted fraction was determined to be approximately 98% by GelAnalyzer. Arrows indicate the mycodulcein band. [Modes for carrying out the invention]

[0029]

[0038] Accordingly, the present invention provides isolated nucleic acid molecules encoding proteins that can modulate sweetness, and polypeptides encoded therein. As described herein, the polypeptides of the present invention mediate sweetness perception, either alone or in combination with foods, beverages, dietary supplements, or pharmaceuticals. The present invention also provides isolated polypeptides that can modify sweetness, and compositions thereof with foods, beverages, dietary supplements, or pharmaceutical compositions, such that the resulting combination has sweetness. The present invention also provides methods for modifying the sweetness of foods, beverages, dietary supplements, or pharmaceutical compositions by using the isolated polynucleotides and polypeptides of the present invention.

[0030]

[0039] In one aspect of the present invention, a newly identified fungal sweet protein referred to herein as Myd1 is provided. In this specification, the term "Myd polypeptide" is used to identify any polypeptide according to the present invention that has at least 80% sequence identity to, for example, SEQ ID NO: 3, and also possesses sweetness-modifying activity. Myd polypeptide also encompasses the peptides SEQ ID NOs: 8 to 17, which possess sweetness-modifying activity. A sweet and partially refined extract of M. terfezioides gleba, The N-terminal sequence of the 20-mer (SEQ ID NO: 4) was identified by novo amino acid sequencing. The Myd1 coding sequence (presumably derived from the MYD1 gene) was identified after de novo assembly of the entire transcriptome of the M. terfeziodes gleba using RNA-seq reads. By screening the entire transcriptome of M. terfeziodes using the N-terminal sequence of the 20-mer, a transcript predicted to encode a protein with 100% identity at the N-terminus was identified. The identified transcript is predicted to encode a 121-amino acid protein. SEQ ID NO: 1 was identified using this method. The start and stop codons in the transcript were identified to identify the putative coding sequence of SEQ ID NO: 2. SEQ ID NO: 3 is a protein predicted to be a 121-amino acid protein. The identity between the predicted protein SEQ ID NO: 3 and other protein sequences in GENBANK was less than 31%. The coding sequences for codon-optimized native mycodulcein for expression in E. coli and Saccharomyces cerevisiae correspond to the nucleic acid sequences of SEQ ID NO: 20 and SEQ ID NO: 22, respectively (which encode the amino acid sequence of SEQ ID NO: 3, i.e., SEQ ID NO: 21, with a desired 6-residue histidine tag).

[0031]

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

[0032]

[0041] While we do not wish to be bound by any particular theory, Myd1 is thought to be involved in sweet taste activation, for example, as an agonist of taste receptor member 2 (T1R2) and / or taste receptor member 3 (T1R3). However, Myd1 may also agonize other taste receptors, such as bitter, umami, sour, and salty. Isolated or purified Myd polypeptides can then be used in the food and pharmaceutical industries to customize tastes, for example, to modulate the sweetness of foods or drugs.

[0033]

[0042] In a first aspect, the present invention relates to a polynucleotide (e.g., isolated polynucleotide) encoding a polypeptide having sweetness-regulating activity, wherein the polypeptide sequence is (a) the amino acid sequence shown 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) 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%) relative to the amino acid sequence shown 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 The polynucleotide comprises a polypeptide selected from the group consisting of (c) an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown 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, modified by deletion, insertion, substitution, or addition of 24 or fewer amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and any range thereof). In some embodiments, the amino acid sequence of the polypeptide having sweetness-regulating 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 amino acids modified from the amino acid sequence of SEQ ID NO: 3 by deletion, insertion, substitution, or addition of 24 or fewer amino acids.

[0034]

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

[0035]

[0044] In other words, the present invention relates to a polynucleotide (e.g., an isolated polynucleotide) wherein the polynucleotide (a) is shown in SEQ ID NO: 2 and contains 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, (b) For the nucleic acid sequences shown in SEQ ID NO: 2, a polynucleotide comprising a nucleic acid sequence which may contain modifications (e.g., deletions, insertions, substitutions, or additions) of 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; and (b) for the nucleic acid sequences shown in SEQ ID NO: 2 The polynucleotide is selected from the group consisting of: a polynucleotide comprising a nucleic acid sequence having sequence identity of at least 80% (for example, 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%), wherein the polynucleotide is not the polynucleotide of SEQ ID NO: 2. In one embodiment, the polynucleotide encodes a polypeptide having sweetness-regulating activity. In one embodiment, the amino acid sequence of the polypeptide having sweetness-regulating activity is the amino acid sequence shown in SEQ ID NO: 3.

[0036]

[0045] In one view, the present invention relates to a polynucleotide (e.g., an isolated polynucleotide) wherein the polynucleotide sequence is (a) shown in SEQ ID NO: 2, and at least one ( For example, a polynucleotide comprising a nucleic acid sequence having substitution modifications of 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; (b) a polynucleotide comprising a nucleic acid sequence having substitution modifications of at least 90% (at least 91%, at least) of the nucleic acid sequence shown in SEQ ID NO: 2. The present invention provides a polynucleotide comprising, essentially, or consisting of, selected from the group comprising: (c)(i) a nucleic acid sequence having sequence identity of 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, the polynucleotide which is not the polynucleotide of SEQ ID NO: 2; and a polynucleotide comprising (c)(i) a 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 sweetness-regulating activity.

[0037]

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

[0038]

[0047] The terms “nucleic acid” or “nucleic acid sequence” refer to deoxyribonucleotides or ribonucleotide oligonucleotides in either single-stranded or double-stranded form. This term encompasses nucleic acids, i.e., oligonucleotides, that contain known analogues of natural nucleotides. This term also encompasses nucleic acid-like structures with synthetic backings (e.g., Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, Oxford Univ. Press (1991); Antisense Strategies, Annals of the NY Academy of Sciences, Vol. 600, Edited by Baserga et al. (NYAS 1992); Milligan J. Med. Chem. 36:1923-1937 (1993); Antisense Research and Applications (1993, CRC Press), WO 97 / 03211; WO 96 / 39154; Mata, Toxicol. Appl. Pharmacol. 144:189-197 (1997); Strauss-Soukup, Biochemistry 36:8692-8698 (1997); Samstag, Antisense Nucleic Acid Drug Dev, 6:153-156 (1996)).

[0039]

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

[0040]

[0049] The present invention also provides polynucleotides and vectors encoding Myd polypeptides. —We also provide expression cassettes containing host cells transformed with this technology.

[0041]

[0050] In other words, the present invention includes, essentially, or comprises a polypeptide sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity 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. We provide peptides (e.g., isolated polypeptides), where the polypeptide 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, 4 The polypeptide may contain modifications (e.g., deletions, insertions, substitutions or additions) of 7, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and any range of these values), and the polypeptide may further contain a histidine tag, and the polypeptide has sweetness-modulating activity. The term "essentially from" 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 anticoagulants, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltodextrose, acacia gum, etc.).

[0042]

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

[0043]

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

[0044]

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

[0045]

[0054] From another perspective, the polypeptide contains amino acid residues 1-121 of SEQ ID NO: 3. Herein, amino acid residues 12-16, 33-38, 41-44, 68-72, or 101-109 of the polypeptide sequence have at least one amino acid substitution, addition, insertion, or deletion (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 their values) compared to SEQ ID NO: 3 in the listed residues. In other words, the polypeptide sequence of a polynucleotide-encoded polypeptide having sweetness-modulating activity is a polypeptide comprising amino acid residues 1-121 of SEQ ID NO: 3, where amino acid residues 12-16, 33-38, 41-44, 68-72, or 101-109 of the polypeptide sequence have at least one amino acid substitution, addition, insertion, or deletion compared to SEQ ID NO: 3 in the listed residues, and the polypeptide has at least 80% sequence identity with SEQ ID NO: 3.

[0046]

[0055] In other words, the present invention includes recombinant polypeptides having sweetness-modulating activity, comprising, essentially, or derived from 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 with respect 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 transport peptide. The term "essentially derived from" allows for the inclusion of ingredients that are not essential to the function or activity of the product and do not substantially affect its function or activity, such as anticoagulants, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltodextrose, acacia gum, etc.).

[0047]

[0056] In other words, the present invention comprises polypeptides having sweetness-modulating activity and containing, essentially, or comprising amino acids having sequence identity of 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%) with respect 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 containing at least one substitution modification with respect to SEQ ID NO: 3 (or SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17). In some respects, the polypeptide of the present invention has 1 to 24 amino acid substitutions compared to the amino acids of the corresponding SEQ ID NO: 3, position 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 , included in 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or 121. The term "essentially derived from" refers to ingredients that are not essential to the function or activity of a product and do not substantially affect its function or activity, such as anticoagulants, fillers, stabilizers (e.g., This allows for the inclusion of heat stabilizers and bulking agents (e.g., maltodextrose, acacia gum, etc.).

[0048]

[0057] In a specific respect, the polypeptide sequence of a polynucleotide-encoded polypeptide having sweetness-modulating activity includes the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 75.

[0049]

[0058] Sequence ID 71 (Consensus Sequence 1) corresponds to Sequence ID 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 arbitrary amino acids. The present invention provides a polypeptide comprising the amino acid sequence of Sequence ID 71.

[0050]

[0059] Sequence ID 72 (consensus sequence 2) corresponds to Sequence ID 3, except that positions 3, 11-16, 26, 33, 37, 38, 41, 43, 51, 57, 66, 68-70, 72, 85, 86, 89, 97, 101-103, 105-110, 117, and 120 are any amino acid (i.e., the proline at positions 34, 36, 42, 71, and 104 of Sequence ID 3 is maintained). The present invention provides a polypeptide comprising the amino acid sequence of Sequence ID 72.

[0051]

[0060] Sequence IDs 73 (consensus sequence 3) and 74 (consensus sequence 4) correspond to Sequence ID 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 the conservative modifications described herein. The present invention provides a polypeptide comprising the amino acid sequence of Sequence ID 73. The present invention provides a polypeptide comprising the amino acid sequence of Sequence ID 74.

[0052]

[0061] Sequence ID 75 (consensus sequence 5) corresponds to Sequence ID 3, except that positions 3, 11, 26, 51, 57, 66, 69, 85, 86, 89, 97, 103, 106, 110, 117, and 120 may include the conservative modifications described herein. The present invention provides a polypeptide comprising the amino acid sequence of Sequence ID 75.

[0053]

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

[0054]

[0063] The polypeptide sequence of the polypeptide having sweetness-regulating activity contains at least 80% sequence identity with SEQ ID NO: 24(D3E) or SEQ ID NO: 24. The polypeptide sequence of the polypeptide having sweetness-regulating activity contains at least 80% sequence identity with SEQ ID NO: 26(K11R) or SEQ ID NO: 2 The polypeptide sequence of the polypeptide having sweetness-regulating activity contains at least 80% sequence identity with SEQ ID NO: 30 (K26R) or SEQ ID NO: 30. The polypeptide sequence of the polypeptide having sweetness-regulating activity contains at least 80% sequence identity with SEQ ID NO: 38 (K51R) or SEQ ID NO: 38. The polypeptide sequence of the polypeptide having sweetness-regulating activity contains at least 80% sequence identity with SEQ ID NO: 42 (R57K) or SEQ ID NO: 42. The polypeptide sequence of the polypeptide having sweetness-regulating activity contains at least 80% sequence identity with SEQ ID NO: 44 (R66K) or SEQ ID NO: 44. The polypeptide sequence of the polypeptide having sweetness-regulating activity contains at least 80% sequence identity with SEQ ID NO: 46 (D69E) or SEQ ID NO: 46. The polypeptide sequence of the polypeptide having sweetness-regulating activity contains at least 80% sequence identity with SEQ ID NO: 50 (D85E) or SEQ ID NO: 50. The polypeptide sequence of the polypeptide having sweetness-regulating activity contains at least 80% sequence identity with SEQ ID NO: 52 (E86D) or SEQ ID NO: 52. The polypeptide sequence of the polypeptide having sweetness-regulating activity contains at least 80% sequence identity with SEQ ID NO: 54 (E89D) or SEQ ID NO: 54. The polypeptide sequence of the polypeptide having sweetness-regulating activity contains at least 80% sequence identity with SEQ ID NO: 58 (D97E) or SEQ ID NO: 58. The polypeptide sequence of the polypeptide having sweetness-regulating activity contains at least 80% sequence identity with SEQ ID NO: 60 (K103R) or SEQ ID NO: 60. The polypeptide sequence of the polypeptide having sweetness-regulating activity contains at least 80% sequence identity with SEQ ID NO: 62 (R106K) or SEQ ID NO: 62. The polypeptide sequence of the polypeptide having sweetness-regulating activity contains at least 80% sequence identity with SEQ ID NO: 64 (R110K) or SEQ ID NO: 64. The polypeptide sequence of the polypeptide having sweetness-regulating activity contains at least 80% sequence identity with SEQ ID NO: 66 (E117D) or SEQ ID NO: 66.The polypeptide sequence of the polypeptide having sweetness-regulating activity contains at least 80% sequence identity with SEQ ID NO: 68 (K120R) or SEQ ID NO: 68.

[0055]

[0064] In other words, polynucleotides encoding polypeptides having sweetness-regulating activity include nucleic acid sequences having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity 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.

[0056]

[0065] The polynucleotide contains at least 80% sequence identity with SEQ ID NO: 23 (corresponding to D3E) or SEQ ID NO: 23. The polynucleotide contains at least 80% sequence identity with SEQ ID NO: 25 (corresponding to K11R) or SEQ ID NO: 25. The polynucleotide contains at least 80% sequence identity with SEQ ID NO: 29 (corresponding to K26R) or SEQ ID NO: 29. The polynucleotide contains at least 80% sequence identity with SEQ ID NO: 37 (corresponding to K51R) or SEQ ID NO: 37. The polynucleotide contains at least 80% sequence identity with SEQ ID NO: 41 (corresponding to R57K) or SEQ ID NO: 41. The polynucleotide contains at least 80% sequence identity with SEQ ID NO: 43 (corresponding to R66K) or SEQ ID NO: 43. The polynucleotide contains at least 80% sequence identity with SEQ ID NO: 45 (corresponding to D69E) or SEQ ID NO: 45. The polynucleotide contains at least 80% sequence identity with SEQ ID NO: 49 (D85E) or SEQ ID NO: 49. Polynucleotides are represented by sequence number 51(E The polynucleotide contains at least 80% sequence identity to sequence number 86D (corresponding to E89D) or sequence number 51. The polynucleotide contains at least 80% sequence identity to sequence number 53 (corresponding to E89D) or sequence number 53. The polynucleotide contains at least 80% sequence identity to sequence number 57 (corresponding to D97E) or sequence number 57. The polynucleotide contains at least 80% sequence identity to sequence number 59 (corresponding to K103R) or sequence number 59. The polynucleotide contains at least 80% sequence identity to sequence number 61 (corresponding to R106K) or sequence number 61. The polynucleotide contains at least 80% sequence identity to sequence number 63 (R110K) or sequence number 63. The polynucleotide contains at least 80% sequence identity to sequence number 65 (E117D) or sequence number 65. The polynucleotide contains at least 80% sequence identity to sequence number 67 (K120R) or sequence number 67.

[0057]

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

[0058]

[0067] The present invention also provides polypeptides comprising amino acid sequences having at least 80% (for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequences 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.

[0059]

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

[0060]

[0069] From another perspective, polynucleotides are nucleic acid sequences that have at least 80% (for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 96%, 97%, 98%, or 99%) sequence identity with 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. include.

[0061]

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

[0062]

[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 mimics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0063]

[0072] Polynucleotides or polypeptides may be of natural or non-natural origin (e.g., synthetic, recombinant, modified, and / or variant products). In one respect, products of natural or non-natural origin are isolated or purified.

[0064]

[0073] In one view, the term “isolated” encompasses products that have been removed from a biological environment (e.g., cells, tissues, culture media, body fluids, etc.) or otherwise purified to any degree (e.g., isolated from synthetic media). Therefore, isolated products can be synthesized or occur naturally.

[0065]

[0074] As used herein, when referring to nucleic acids or polypeptides, the term “isolated” means a state of purification or concentration different from that of natural origin. Any degree of purification or concentration higher than that of natural origin, including (1) purification from structures or compounds associated with other natural origins, or (2) association with structures or compounds not normally associated in the body, falls within the meaning of “isolated” as used herein. The nucleic acids or polypeptides described herein can be isolated or associated with structures or compounds not normally associated in nature according to a variety of methods and processes known to those skilled in the art. In one embodiment, the polypeptides described herein contain other fungal proteins by weight up to 5% (e.g., up to 4%, up to 3%, up to 2%, up to 1%).

[0066]

[0075] As used herein, “recombinant” means a polynucleotide synthesized in vitro or otherwise manipulated (e.g., “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 (“recombinant protein”). “Recombinant means” also encompass the expression of a fusion protein comprising a nucleic acid sequence amplified using the translocation domain and primers of the present invention, for example, ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into an expression cassette or vector for inducible or constitutive expression.

[0067]

[0076] A “modified” or “variant” product refers to a product (e.g., a polynucleotide or polypeptide) that has been altered from its original (e.g., naturally occurring) structure. As described herein, a variant encompasses a polynucleotide or polypeptide having one or more changes to the nucleic acid or amino acid sequence. Changes include modifications to the nucleic acid or amino acid sequence, such as additions, deletions, insertions, and substitutions. Modified or variant products may also include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation on the original structure, such as conjugation with a labeling component.

[0068]

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

[0078] The term "library" means a preparation which is a mixture of various nucleic acid or polypeptide molecules, for example, a library of recombinantly generated Myd-related polynucleotides produced by amplification of nucleic acids with degenerate primer pairs, or an isolated collection of vectors containing amplified ligand-binding domains, or a mixture of cells randomly transfected with at least one vector encoding MYD.

[0069]

[0079] As used herein, “nucleic acid probe or oligonucleotide” is defined as a nucleic acid capable of binding to a target nucleic acid of a complementary sequence via one or more types of chemical bonds, usually via complementary base pairing, usually via hydrogen bond formation. As used herein, a probe may include natural (i.e., A, G, C, or T) or modified bases (e.g., 7-deazaguanosine, inosine). In addition, the bases in the probe may be linked by linkages other than phosphodiester bonds, as long as this does not hinder hybridization. For example, a probe may be a peptide nucleic acid in which the constituent bases are linked by peptide bonds rather than phosphodiester links. Those skilled in the art will understand that a probe may bind to a target sequence that lacks complete complementarity with the probe sequence, depending on the stringency of the hybridization conditions. The probe may be directly labeled with an isotope, chromophore, luminiphore, chromochromic agent, or streptavidin complex, as desired. The probe is indirectly labeled with biotin or other substances to which it can later bind. By assaying for the presence or absence of the probe, the presence or absence of a select sequence or a subset can be detected.

[0070]

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

[0071]

[0081] A “promoter” is defined as an array of nucleic acid sequences that direct the transcription of a nucleic acid. As used herein, a promoter includes essential nucleic acid sequences near the transcription start site, such as the TATA element in the case of a polymerase type II promoter. The promoter may also optionally include distal elements that may be located thousands of base pairs away from the transcription start site. This also includes hunter or repressor elements. 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 “operatably linked” refers to a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter or an array of transcription factor binding sites) and a second nucleic acid sequence, where the expression regulatory sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0072]

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

[0073]

[0083] The terms “expression vector” or “expression cassette” refer to any recombinant expression system for the purpose of constitutively or inductively expressing the nucleic acid sequence of the present invention in vitro or in vivo in any cell, including prokaryotic cells, yeast cells, fungal cells, plant cells, insect cells, or mammalian cells. This term encompasses linear or cyclic expression systems. This term encompasses expression systems that remain in the episome or are integrated into the host cell genome. Expression systems may self-replicate or not self-replicate, i.e., have the ability to drive transient expression only in cells. This term encompasses recombinant expression “cassettes” that contain only the minimal elements necessary for the transcription of recombinant nucleic acids.

[0074]

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

[0075]

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

[0076]

[0086] From another perspective, host cells include Gram-positive non-spore-forming bacteria, Gram-positive spore-forming bacteria, and Gram The group is selected from a group consisting of necrotic bacteria, yeasts, and protists / algae.

[0077]

[0087] A non-exclusive example of a Gram-positive, non-spore-forming bacterium is 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 aviarie, s Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus collinoides, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus mucosae, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus parafarraginis, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactococcus lactis, Leuconostoc lemon、Leuconostoc lactis、Leuconostoc mesenteroides、Leuconostoc pseudomesenteroides、Microbacteriumimperial, Oenococcus oeni, Pasteuria nishizawae, Pediococcus acidilactic, Pediococcus parvulus, Pediococcus pentosaceus, Propionibacterium acidipropioni, Propionibacterium freudenreichii, and Streptococcus thermophiles.

[0078]

[0088] Non-limiting examples of Gram-positive spore-forming bacteria include Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus flexus, Bacillus fusiformis, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus mojavensis, Bacillus pumilus, Bacillus smithii, Bacillus subtilis, Bacillus vallismortis, Bacil Examples include *Lus velezensis*, *Geobacillus stearothermophilus*, *Paenibacillus illinoisensis*, and *Parageobacillus thermoglucosidasius*. Non-specific examples of Gram-negative bacteria include *Cupriavidus necator*, *Gluconobacter oxydans*, *Komagataeibacter sucrofermentans*, and *Xanthomonas campestris*.

[0079]

[0089] Non-specific examples of yeast include Candida cylindracea, Debaryomyces hansenii, Hanseniaspora uvarum, Kluyveromyces lactis, Kluyveromyces marxianus, Komagataella pastoris, and Komagataella. Examples include phaffi, Lindnera jadinii, Ogataea angusta, Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces pastorianus, Schizosaccharomyces pombe, Wickerhamomyces anomalus, Xanthophyllomyces dendrorhous, Yarrowia lipolytica, and Zygosaccharomyces rouxii.

[0080]

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

[0081]

[0091] The Myd proteins described herein also include “analogs” or “conserved variants” and “mimetics” (“peptide mimetics”) that have structure and activity substantially corresponding to representative sequences. Therefore, the terms “conserved variant” or “analog” or “mimetic” refer to polypeptides having modified amino acid sequences such that changes (one or more) do not substantially alter the structure and / or activity of the polypeptide (conserved 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 to protein activity, or amino acid substitutions at residues with similar properties (e.g., acidic, basic, positively or negatively charged, polar or nonpolar), so that even substitutions of important amino acids do not substantially alter the structure and / or activity.

[0082]

[0092] More specifically, the term "conservatively modified variant" applies to both amino acid sequences and nucleic acid sequences. For a given nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that codes for the same or essentially the same amino acid sequence, or, if the nucleic acid does not code for an amino acid sequence, for an essentially identical sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given protein.

[0083]

[0093] For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at any position where alanine is identified by the codon, the codon can be changed to any of the corresponding codons listed without altering the encoded polypeptide.

[0084]

[0094] Such variations of nucleic acids are called "silent variations," and these are a type of conservatively modified variation. Every nucleic acid sequence in this specification encoding a polypeptide also includes all possible silent variations of that nucleic acid. Those skilled in the art will recognize that by modifying each codon in a nucleic acid (except for AUG, which is usually the sole codon of methionine, and TGG, which is usually the sole codon of tryptophan), functionally identical molecules can be obtained. Thus, each silent variation of the nucleic acid encoding a polypeptide is latent in each described sequence.

[0085]

[0095] Tables of conservative substitutions that provide functionally similar amino acids are well known in the art. For example, one representative guideline for selecting a conservative substitution includes (original residue, followed by representative substitution): ala / gly or ser; arg / lys; asn / gln or his; asp / glu; cys / ser; gln / asn; gly / asp; gly / ala or pro; his / asn or gln; ile / leu or val; leu / ile or val; lys / arg or gln or glu; met / leu or tyr or ile; phe / met or leu or tyr; ser / thr; thr / ser; trp / tyr; tyr / trp or phe; val / ile or leu. Alternative representative guidelines use the following six groups containing amino acids in which each is a conserved substitution: 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); (e.g., Creighton, See also Proteins, WH Freeman and Company (1984); Schultz and Schimer, Principles of Protein Structure, Springer-Vrlag (1979). Other alternative representative guidelines use the following six groups in which proline is unique: 1) Gly(G), Ala(A), Val(V), Leu(L), Ile(I); 2) Ser(S), Cys(C), Thr(T), Met(M); 3) Pro(P); 4) Phe(F), Tyr(Y), Try(W); 5) His(H), Lys(K), Arg(R); and 6) Asp(D), Glu(E), Gln(N). Those skilled in the art will understand that the above substitutions are not the only possible conserved substitutions. For example, for some purposes, all charged amino acids, whether positive or negative, can be considered conserved substitutions of each other. Furthermore, individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a few percent of amino acids in the encoded sequence can also be considered "conservatively modified variations." Those skilled in the art will be familiar with codon selection in a given host expressing the protein of interest.

[0086]

[0096] The terms “mimetic” and “peptide mimetic” refer to synthetic compounds having substantially the same structural and / or functional features as polypeptides, e.g., translocation domains, ligand-binding domains, or chimeric receptors as of the present invention. Mimics may consist entirely of synthetic non-natural analogs of amino acids, or they may be chimeric molecules of partially natural peptide amino acids and partially non-natural amino acid analogs. Mimics may also include any amount of conserved substitution of natural amino acids, provided that such substitutions do not substantially alter the structure and / or activity of the mimetic.

[0087]

[0097] Similar to the conservative variant of the polypeptide of the present invention, routine experiments will determine whether the mimetic is within the scope of the invention, i.e., whether its structure and / or function are substantially unchanged. Polypeptide mimetic compositions may contain any combination of non-natural structural components, which typically derive from three structural groups: a) residue linking groups other than natural amide bond ("peptide bond") linking; b) non-natural residues replacing naturally occurring amino acid residues; or c) components that induce secondary structure mimicry, i.e., induce secondary structures such as β-turns, γ-turns, β-sheets, α-helix conformations, etc. or stabilizing residues. A polypeptide can be characterized as a mimetic if all or some of its residues are linked by chemical means other than natural peptide bonds. Individual peptide mimetic residues can be linked by peptide bonds, other chemical bonds or coupling means, such as glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC), or N,N'-diisopropylcarbodiimide (DIC). Linking groups that can be alternatives to conventional amide bonds ("peptide bonds") include, for example, ketomethylene (e.g., --C(O)--NH-- instead of --C(O)--NH--), aminomethylene (CH2--NH), ethylene, olefin (CH=CH), ether (CH2 Examples include thioethers (CH--S), tetrazoles (CN4), thiazoles, retroamides, thioamides, or esters (see, for example, Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY (1983)). Polypeptides can also be characterized as mimics by containing all or some non-natural residues instead of naturally occurring amino acid residues; non-natural residues are well documented in scientific literature and patent literature. Phyre2 is a set of tools available on the web for predicting and analyzing protein structure, function, and mutation.

[0088]

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

[0089]

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

[0090]

[0100] Alternative protein formulations that are presumed to have a similar structure and function to Myd1 The sequences are provided herein as Sequence IDs 8 to 17. To derive Sequence ID 17 from Sequence ID 8, the consensus loop region was selected as the displacement site. This is because most insertions and deletions are typically found in regions between secondary structural elements, where they can be adapted more easily without causing significant distortion in the overall folding of the protein. The core of this protein exhibits a higher degree of sequence conservation, as found within 4JOX.

[0091]

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

[0092]

[0102] Using MYD / Myd nucleotides and specific regions of the amino acid sequence, M Polymorphic variants, interspecific homologs, and alleles of yd family members can be identified. This identification can be performed in vitro, for example, under stringent hybridization conditions or PCR (e.g., using primers encoding the Myd sequences identified herein), or by using sequence information in a computer system for comparison with other nucleotide sequences. The various alleles of the MYD gene within a single species population are also useful in determining whether allele sequence differences correlate with taste differences among members of the population. Classical PCR amplification and cloning techniques are useful for isolating orthologues, for example, if degenerate primers are sufficient to detect the relevant genes across the species.

[0093]

[0103] For example, using a primer designed using the sequence disclosed herein Therefore, MYD-related genes from different fungal genomes can be amplified and cloned. In contrast, genes within a single species associated with MYD are best identified using sequence pattern recognition software to search for relevant sequences. Typically, the identification of polymorphic variants and alleles of MYD family members can be done by comparing amino acid sequences of approximately 25 amino acids or more, e.g., 50–100 amino acids. Amino acid identity of at least 35–50%, and preferably 60%, 70%, 75%, 80%, 85%, 90%, 95–99% or more, typically indicates that a protein is a polymorphic variant, interspecific homolog, or allele of a MYD family member. Sequence comparison can be performed using one of the sequence comparison algorithms discussed below. Alleles, interspecific homologs, and polymorphic variants can also be identified using antibodies that specifically bind to the Myd polypeptide or its conserved region.

[0094]

[0104] Nucleotide and amino acid sequence information for MYD family members Furthermore, these models can be used to construct computer systems for sweetness-regulating polypeptides, as well as how they interact with sweetness receptors and their computer system models. Sweetness receptors are composed of heterodimers of gustatory receptor member 2 (T1R2) and gustatory receptor member 3 (T1R3). These models can then be used to identify variants and mutations of Myd that can increase sweetness receptor activation, and to identify more active versions of Myd.

[0095]

[0105] Various conservative mutations and substitutions are assumed to be within the scope of the present invention. For example Performing amino acid substitutions using known protocols of recombinant gene techniques, including PCR, gene cloning, site-directed mutagenesis of cDNA, transfection of host cells, and in vitro transcription, would be within the scope of the art. The variants can then be screened for taste receptor agonist activity.

[0096]

[0106] In one embodiment, a hybrid comprising nucleic acid encoding a Myds fusion protein A protein-coding sequence can be constructed. These nucleic acid sequences can be operably ligated to transcriptional or translational regulatory elements, such as transcriptional and translational initiation sequences, promoters and enhancers, transcriptional and translational terminators, polyadenylation sequences, and other sequences useful for transcribing DNA to RNA. The fusion protein may include a C-terminal or N-terminal translocation sequence. Furthermore, the fusion protein may contain additional elements for, for example, protein detection, purification, or other applications. Domains that facilitate purification include, for example, metal chelate peptides such as polyhistidine tracts, histidine-tryptophan modules, or other domains that enable purification on immobilized metals; maltose-binding proteins; protein A domains that enable purification on immobilized immunoglobulins; or domains used in the FLAGS elongation / affinity purification system (Immunex Corp, Seattle Wash).

[0097]

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

[0098]

[0108] Affinity tags are a type of protein tag attached to proteins. This allows proteins to be purified from their crude biological sources using affinity technology. Affinity tags are publicly known in the art, for example, see Kimple et al. Curr Protoc Protein Sci.; 73: Unit-9.9. doi:10.1002 / 0471140864.ps090 These are described in 9s73. These include polyhistidine, GST, MBP, calmodulin-binding peptide, intein-chitin-binding domain, streptavidin / biotin-based tags, and His-Patch ThioFusion (thioredoxin). Affinity tags include small (e.g., 20 or fewer amino acid residues) or large affinity tags. Examples of small affinity tags include His, FLAG, Strep II, and S-peptides, while examples of large affinity tags include MBP, GST, cellulose-binding domain, calmodulin-binding peptide, and His-Patch Thioredoxin.

[0099]

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

[0100]

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

[0101]

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

[0102]

[0112] Tandem affinity purification (TAP) involves two affinity tags and objectives. This is a dual affinity purification method based on fusion with proteins, enabling the purification of tagged proteins and the isolation of protein complexes that interact with the target protein. The use of TAP is included within the scope of this invention.

[0103]

[0113] In one embodiment, the fusion protein consists of 2 to 10 proteins (for example, 2, 3, 4, 5, It includes a histidine tag containing 6, 7, 8, 9, or 10 histidine residues. For example, the histidine tag may contain 6 histidine residues.

[0104]

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

[0105]

[0115] The fusion protein contains one or more linkers (e.g., flexible linkers). It may contain rigid linkers and linkers that can be cut in vivo. In addition to their fundamental role in linking functional domains together (such as flexible and rigid linkers) or releasing free functional domains in vivo (such as in vivo cleavable linkers), linkers offer many other advantages for the production of fusion proteins, such as improved biological activity, increased expression yield, and the achievement of desirable pharmacokinetic profiles. Linkers are well known in the art (see, for example, Chen et al., Adv Drug Deliv Rev. 65(10): 1357-1369 (2013)).

[0106]

[0116] The flexibility linker allows linked domains to move or interact to some extent. Linkers are used when necessary. They generally consist of small, nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The small size of these amino acids provides flexibility, allowing for the mobility of the functional domains they link. Incorporating Ser or Thr can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thus reducing undesirable interactions between the linker and the protein moiety.

[0107]

[0117] The most commonly used flexibility linkers are primarily made of a series of Gly and S It has a sequence consisting of er residues ("GS" linkers). The most widely used example of a flexible linker is (Gly-Gly-Gly-Gly-Ser). n (Sequence ID 69) It has a sequence. By adjusting the copy number "n", the length of this GS linker can be optimized to achieve proper separation of functional domains or maintain the necessary interdomain interactions. In addition to the GS linker, many other flexible linkers have been designed for recombinant fusion proteins. These flexible linkers are also rich in small or polar amino acids such as Gly and Ser, but 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.

[0108]

[0118] The rigid linker maintains a fixed distance between domains, preserving their independent functions. An example of a rigid linker is (EAAAK) n An α-helix-forming linker having the sequence (SEQ ID NO: 70), and a linker having a Pro-rich sequence, (XP) n Examples include [wherein X represents any amino acid, preferably Ala, Lys, or Glu].

[0109]

[0119] The polypeptide of the present invention is also a signal peptide (i.e., a signal sequence, The signal peptides may include target signals, localization signals, localization sequences, transport peptides, leader sequences, or leader peptides, which are short peptides present at the N-terminus or occasionally the C-terminus of most newly synthesized proteins destined for secretory pathways. These proteins include those located inside specific organelles (endoplasmic reticulum, Golgi apparatus, or endosomes), those secreted from cells, or those inserted into most cell membranes. Representative signal peptides are known in the art, and those skilled in the art will recognize how to select specific signal peptides for use in the present invention.

[0110]

[0120] When used herein, the terms relating to amino acid sequences or nucleotide sequences "At least 80% identity" means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity.

[0111]

[0121] As used herein, "deletion, insertion, substitution, or Examples of amino acid sequences modified by addition include amino acid sequences modified by deletion, insertion, substitution, or addition of 1 to 30 amino acids, preferably 20 or less, more preferably 10 or less, and even more preferably 5 or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any range thereof). As used herein, an example of a "nucleotide sequence modified by deletion, insertion, substitution, or addition of one or more nucleotides" is one to 90, preferably 60 or fewer, preferably 30 or fewer, more preferably 15 or fewer, and even more preferably 10 or fewer 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 Includes nucleic acid sequences modified by deletion, insertion, substitution, or addition of , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or any range thereof.

[0112]

[0122] For example, in array comparison, typically one array acts as the reference array. This is performed, and the test sequence is compared with it. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into the computer, and subsequence coordinates are specified as needed, and the sequence A Specify the algorithm program parameters. For BLASTN and BLASTP programs, you can use the default program parameters described below, or you can specify alternative parameters. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence to the reference sequence based on the program parameters.

[0113]

[0123] The "comparison window" used in this specification is 20-600, typically around 50- The association with any one segment of consecutive positions of numbers selected from a group of approximately 200, and usually approximately 100 to 150, is included, and after optimally aligning two sequences, the sequences can be compared with a reference sequence of the same consecutive positions. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48:443 (1970), or by the similarity search of Pearson & Lipman, Proc. Natl. Acad Sci. USA 85:2444 (1988), or by computer implementations of these algorithms (Wisconsin Genetics Software Package, This can be done by GAP, BESTFIT, FASTA, and TFASTA (as described in Genetics Computer Group, 575 Science Dr., Madison, WI) or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds., 1995, Supplement)).

[0114]

[0124] Algorithms suitable for determining sequence identity percentage and sequence similarity Preferred examples are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J Mol. Biol. 215:403-410 (1990), respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy a certain positive threshold score T when aligned with words of the same length in the database sequence. T is called the neighbor 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 first neighbor word hits act as seeds to initiate a search for longer HSPs that contain them. These word hits are extended bidirectionally along each sequence as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for matched residue pairs; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Word hit expansion in each direction stops if the cumulative alignment score decreases by amount X from its maximum achieved value; if the cumulative score falls below 0 due to the accumulation of one or more negative scoring residue alignments; or if the end of any sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) uses, by default, word length (W) 11, expected value (E) 10, M=5, N=-4, and comparison of both strands. For amino acid sequences. The BLASTP program uses, by default, a word length of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad Sci. USA 89:10915 (1989)) with alignment (B) of 50, an expected value (E) of 10, M=5, N=-4, and comparison of both strands.

[0115]

[0125] Another example of a useful algorithm is PILEUP. PILEUP is related to relevance. To show the sequence identity percentage, progressive, pairwise alignment is used to construct a multisequence alignment from the group of related sequences. It also plots a so-called "tree" or "dendogram" showing the clustering relationships used to construct the alignment (see, for example, Figure 2). Program P uses a simplified version of the progressive alignment method described in Feng & Doolittle, J Mol. Evol.35:351-360 (1987). Its usage is similar to that described by Higgins & Sharp, CABIOS 5:151-153 (1989). This program can align sequences up to 5000 nucleotides or 300 amino acids, each with a maximum length. The multiple alignment procedure begins with pairwise alignment of the two most similar sequences, generating a cluster of two aligned sequences. This cluster is then aligned to the next most relevant or aligned sequence cluster. The two clusters of sequences 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 executed by specifying specific sequences and their amino acid or nucleotide coordinates for the region of sequence comparison, and by specifying program parameters. Using PILEUP with the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gap Then, the sequence identity percentage relationship is determined by comparing the reference sequence with other test sequences. PILEUP can be obtained from the GCG sequence analysis software package, for example, version 7.0 encoded by the gene (Devereaux et al., Nuc. Acids Res. 12:387-395 (1984)) was derived by conceptual translation of the corresponding open reading frame.

[0116]

[0126] The polynucleotides encoding the polypeptide of the present invention are chemically, or M Polynucleotides can be synthesized by genetic engineering based on the amino acid sequence of yd. For example, polynucleotides can be chemically synthesized based on the amino acid sequence of the polypeptide or its preprotein of the present invention. Nucleic acid contract synthesis services (e.g., provided by Medical & Biological Laboratories Co., Ltd., Genscript, etc.) can be used for the chemical synthesis of polynucleotides. Furthermore, the synthesized polynucleotides can be amplified by PCR and cloning.

[0117]

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

[0118]

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

[0119]

[0129] The present invention also includes a polynucleus of the present invention operably connected to different adjustment elements. The present invention includes rheotides. The present invention may include expression cassettes or vectors containing the polynucleotides of the present invention, and host cells transformed with the vectors of the present invention.

[0120]

[0130] Alternatively, the polynucleotide encoding the Myd polypeptide of the present invention is purple. The polypeptides of the present invention can be produced by introducing mutations into polynucleotides synthesized according to known mutagenesis methods, such as external irradiation and site-directed mutagenesis. For example, the 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 resulting polynucleotide, examining the sweetness modification activity of the expressed protein, and selecting the polynucleotide encoding a protein with the desired sweetness modification activity.

[0121]

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

[0122]

[0132] Types of vectors containing polynucleotides that represent the polypeptides of the present invention Examples of vectors, though not limited to them, include vectors commonly used in gene cloning, such as plasmids, cosmids, phages, viruses, YACs, and BACs. Examples of vectors include plasmids (e.g., DNA plasmids), yeast (e.g., Saccharomyces), and viral vectors, such as poxviruses, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, polioviruses, alphaviruses, baculoviruses, Sindbisviruses, plant viruses (e.g., Alphaflexiviridae or Potyviridae), and insect viruses (e.g., Baculoviridae).

[0123]

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

[0124]

[0134] The vector contains a DNA region that includes the replication initiation region or the DNA replication origin. This is possible. Alternatively, a regulatory sequence, such as a promoter region for initiating gene transcription, a terminator region, or a secretion signaling region for secreting the expressed protein extracellularly, can be operably ligated upstream of the polynucleotide encoding the protein of the present invention in the vector (i.e., the MYD gene of the present invention). As used herein, the gene and regulatory sequence are "operably ligated". This refers to a state in which genes and regulatory regions are positioned so that the gene can be expressed under the regulation of the regulatory region.

[0125]

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

[0126]

[0136] Alternatively, a marker gene for selecting a host into which the vector has been properly introduced. The vector of the present invention may further incorporate genes for resistance to drugs such as ampicillin, neomycin, kanamycin, and chloramphenicol. Alternatively, if a nutrient-dependent strain is used as a host, a gene encoding a synthase of a necessary nutrient may be incorporated into the vector as a marker gene. Alternatively, if a selective medium requiring specific metabolism for growth is used, a gene related to metabolism may be incorporated into the vector as a marker gene. An example of such a metabolism-related gene is the acetamidase gene for using acetamide as a nitrogen source.

[0127]

[0137] The polynucleotide encoding the Myd polypeptide of the present invention, and the regulatory sequence and Ligation with the marker gene can be performed by methods known in the art, such as SOE (splicing by duplication)-PCR (Gene, 1989, 77: 61-68). The procedure for introducing the ligated fragment into the vector is also known in the art.

[0128]

[0138] Examples of hosts for transformed organisms into which vectors are introduced include bacteria and filamentous fungi. Examples of microorganisms include Escherichia coli, as well as bacteria belonging to Staphylococcus, Enterococcus, Listeria, and Bacillus, of which Escherichia coli and Bacillus bacteria (e.g., Bacillus subtilis or its variants) are preferred. Examples of Bacillus subtilis variants include the protease 9 double knockout strain KA8AX described in J. Biosci. Bioeng., 2007, 104 (2): 135-143, and the DBPA strain, a variant derived from the protease 8 double knockout strain described in Biotechnol. Lett., 2011, 33 (9): 1847-1852, which exhibit improved protein folding efficiency. Examples of filamentous fungi include Trichoderma, Aspergillus, and Rhizopus. Furthermore, for example, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowia lipolytica, Schizosaccharomyces pombe, and Kluyveromyces lactis are suitable expression hosts. In yet another view, the present invention encompasses host cells comprising one or more expression cassettes described herein, operably linked to a regulatory element adapted to expression in the cell. The cells may be, for example, mammalian cells (e.g., BHK, VERO, HT1080, 293, RD, COS-7, or CHO cells), insect cells (e.g., Trichoplusia ni(Tn5) or Sf9), bacterial cells, plant cells, or yeast cells.

[0129]

[0139] Recombinant expression polypeptides from the expression cassette encoding Myd are typically It is isolated from lysed cells or culture medium. Purification is performed by salt fractionation, ion exchange chromatography, gel filtration, size exclusion chromatography, size fractionation, and affinity chromatography. This can be carried out by methods known in the art, including immunochromatography. Immunoaffinity chromatography can be used, for example, with antibodies generated based on the Gag antigen.

[0130]

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

[0131]

[0141] Those skilled in the art will know how to select appropriate columns, buffers, and eluents, including hydrophobic interactions. We are proficient in action chromatography (HIC) and size exclusion chromatography (SEC) purification techniques. Representative HIC and SEC purification techniques are described in Example 11 herein. In a typical view, the purification of polypeptides after 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 those values.

[0132]

[0142] The present invention also includes heterogeneous polynucleotides and / or other polynucleotides of the present invention as described herein. The invention aims to create transgenic plants containing heterologous polypeptides. The plants have a phenotype altered by the expression of heterologous nucleic acid sequences. The altered phenotype may include a phenotype having increased sweetness in any part of the plant, including the fruit. The transgenic plants may contain an expression cassette as defined herein as part of the plant, which is introduced by transforming the plant with the vector of the invention. Such expression cassettes include plant-expressible promoters and terminators. The invention includes regulatory sequences for the expression of heterologous coding sequences in plants, including ter. Transgenic plants can be any type of plant capable of expressing the heterologous nucleic acid sequences described herein. The term "plant" includes whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, and plant cells and their offspring. The class of plants that can be used in the methods of the present invention is as broad as the class of higher plants suitable for transformation techniques, generally including both monocotyledonous plants (monocot) and dicotyledonous plants (dicot). It includes plants with various levels of ploidy, including polyploid, diploid and haploid. For example, a transgenic plant could be an apple or a strawberry.

[0133]

[0143] Techniques for transforming a wide variety of plant species are well known in this field, and This is documented in the literature and scientific literature. For example, Weising et al. (1988) Ann. Rev. Genet., 22:421-477 and Joung et al. (2015) “Plant Transformation Met hods and Applications,” Current Technology in Plant Molecular Breeding, (Koh et al. See (al., ed.) Springer Dordrecht Heidelberg, New York, London, Chapter 9, pp. 297–344. Any method known in the art for the transformation of plant cells or plant tissues, including plant protoplasts, can be used for plant transformation. Specific methods for plant transformation include, among others, bolistic methods (gene guns), electroporation, microinjection, protoplast fusion, and Agrobacterium-mediated transformation. Agrobacterium-mediated transformation can use, for example, binary vectors that replicate in Escherichia coli and Agrobacterium tumefaciens or other Agrobacterium strains. Various 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 containing regulatory sequences for the expression of heterologous coding sequences, including plant expression promoter sequences and other plant regulatory sequences, are known in the art and can be used to transform plants to express the polypeptides described herein.

[0134]

[0144] Various plant expression promoters are known in the art, and sweetness regulation activity Polynucleotides encoding proteins containing these are available for use in heterologous constructs, vectors, and transformed plant materials as described herein. Plant-expressing promoters can be derived from natural plant sources, plant virus sources, and bacteria having plant-expressing promoters, such as Agrobacterium strains. Examples of plant-expressing promoters include, among others, the cauliflower mosaic virus promoter (CaMV 35S), octopine and nopalin synthase promoters (e.g., the nos promoter), the plant ubiquitin promoter (Ubi), the rice actin promoter (Act-1), and the maize alcohol dehydrogenase promoter (Adh-1). Plant-expressing promoters include constitutive promoters, inductive promoters, tissue-specific promoters, and developmental stage-specific promoters, and examples of each type of promoter are known in the art. Examples of tissue-specific promoters include, among others, those that direct expression in plant roots, plant leaves, fruits, flowers, pollen, or cells involved in active photosynthesis (e.g., the phosphoenolpyruvate promoter (PEP)). Stage-specific promoters include those that direct expression during fruit ripening, flowering, or fruiting. Synthetic plant promoters are also known in the art and are useful in heterologous constructs, vectors, and transformed plant materials (see, for example, Ali S. & Kim WC (2019) Frontiers in Plant Science, 10, article 1433).

[0135]

[0145] From transformed protoplasts, plant cells, callus, or other plant tissues Plant regeneration techniques are well known in this field 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) DAEvans et al., Macmillan (New York); RH Smith, Plant Tissue Culture: Techniques and Experiments, 3rd edition (2012) Academic Press (New York); MR Davey & P. ​​Anthony, Plant Cell Culture: Essential Methods (2010) John Wiley & Sons (New York), Chapters 3 and 9 for details.

[0136]

[0146] Methods for introducing vectors into the host include the protoplast method and electrophoresis. Methods commonly used in fields such as electroporation can be used. The desired transformant can be obtained by selecting a strain in which the vector has been properly introduced, using indicators such as the expression of marker genes and / or nutrient requirements.

[0137]

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

[0138]

[0148] Polynucleus encoding the Myd polypeptide of the present invention obtained in this manner When a transformant into which a rheotide or a vector containing the rheotide has been introduced is cultured in a suitable medium, expression of MYD cDNA on the vector and subsequent production of the Myd polypeptide of the present invention are obtained. The medium used for culturing such transformants can be appropriately selected by those skilled in the art depending on the type of microorganism of the transformant.

[0139]

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

[0140]

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

[0141]

[0151] The present invention also relates to a method for producing a protein having sweetness-regulating activity, public The present invention also includes a method comprising culturing the host cells in a culture medium under conditions that result in the production of proteins having sweetness-regulating activity similar to that of known sweeteners or sweet compounds.

[0142]

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

[0143]

[0153] If a sweetener or sweetener composition is present in an orally administered product, it may be used in the test. The sweetener composition of the present invention can be used in an effective amount, which refers to an amount sufficient to induce sweetness in the body.

[0144]

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

[0145]

[0155] Sweetness adjustment is also necessary when administered as a combination, and specific for oral administration. This refers to enhancing the flavor, such as sweetness, of a product.

[0146]

[0156] The sweetness-regulating activity can be determined by methods known in the art, for example, by in vitro methods. It can be detected in vivo by sensory evaluation of animals or humans. While we do not wish to be bound by any particular theory, Myd is involved in sweet taste activation and is, for example, an agonist of taste receptor member 2 (T1R2) and / or taste receptor member 3 (T1R3). However, Myd also agonizes other taste receptors such as bitter, umami, sour, and salty. Such functional effects can be measured by measuring binding to the taste receptor T1R via any means known to those skilled in the art, such as spectroscopic features (e.g., fluorescence, absorbance, refractive index), hydrodynamics (e.g., shape), chromatographic or solubility characteristics, patch-clamp method, voltage-sensitive dyes, total cell current, radioisotope efflux, inducible markers, changes in transcriptional activation of the T1R gene; ligand binding assays; voltage, membrane potential and conductance changes; ion bundle assays; changes in intracellular second messengers such as cAMP, cGMP, and inositol triphosphate (IP3); changes in intracellular calcium levels; neurotransmitter release, etc.

[0147]

[0157] Using sensory evaluation (human or animal), Myd candidate polypeptides were found to be active in sweetness regulation. It is also possible to determine whether or not something has a taste. Sensory evaluation is the scientific field of analyzing and measuring human responses to the composition of food and beverages, including appearance, touch, smell, texture, temperature, and taste. Measurement using a person as an instrument is sometimes necessary. The selection of an appropriate method for determining sweetness can be determined by those skilled in the art and may include, for example, discrimination tests or difference tests designed to measure the likelihood that two products are perceptually different. The responses from evaluators are recorded for accuracy and statistically analyzed. To verify whether it is more accurate than what would be expected due to chance alone.

[0148]

[0158] Sensory evaluation involves aspects such as appearance, touch, smell, texture, temperature, and taste of food and beverages. This is the scientific field of analyzing and measuring human responses to compositions. Measurements using humans as instruments are sometimes necessary. Since the sensory properties of flavor and texture are obvious attributes that cannot be easily measured by instruments, the food industry first needed to develop these measurement tools. The selection of an appropriate method for determining sensory stimulating properties, such as the sweetness of the proteins disclosed in this invention, can be determined by those skilled in the art and may include, for example, discrimination tests or difference tests designed to measure the likelihood that two products are perceptually different. For sweetness perception, for example, one or more samples from 5% sucrose, 6% sucrose, 7% sucrose, 8% sucrose, 9% sucrose, and 10% sucrose, and test samples, can be ranked by trained panelists in order of sweetness intensity from low to high. It should be understood in this invention that there are several methods by which those skilled in the art can measure differences in sensation.

[0149]

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

[0150]

[0160] In some embodiments, the Myd polypeptide of the present invention is as described above or in the present technology The Myd polypeptide comprises polypeptides that, as measured by any method known in the field, are at least as sweet as sugar (on a weight / weight basis) (e.g., 1X), or 2X, 5X, 10X, 50X, 100X, 200X, 400X, 600X, 800X, 1000X, 1500X, 2000X, 3000X, 5000X, 10000X, 20000X, or sweeter than sugar. In other embodiments, Myd polypeptides are sweet to at least 1% of sugar (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%).

[0151]

[0161] Food, beverages, nutritional supplements, pharmaceuticals

[0162] In one embodiment, the present invention relates to a product for oral administration and to the present invention as described herein. The product includes, essentially consists of, or comprises a combination of a sweetening composition containing isolated Myd polypeptide. In one aspect, the combination has enhanced sweetness compared to an orally administered product (control) lacking Myd polypeptide. In one aspect, the orally administered product is not Mattirolomyces terfezioides truffle. The term "essentially consists of" allows for the inclusion of components that are not essential to the function or activity of the product and do not substantially affect its function or activity, such as anticoagulants, fillers, stabilizers (e.g., heat stabilizers), and bulking agents (e.g., maltodextrose, acacia gum, etc.).

[0152]

[0163] In one embodiment, a composition comprising the isolated Myd protein of the present invention is isolated Myd The formulation may include one that provides enhanced functionality to the protein. For example, the composition may include a formulation that stabilizes the Myd protein against heat, osmotic pressure, pH, or other types of degradation. In one embodiment, the formulation stabilizes the Myd protein against thermal degradation. Representative compounds for stabilizing the Myd protein include, for example, L-arginine lysine, L-histidine, β-alanine, L-serine, L-arginine ethyl ester dihydrochloride, L-arginine amide dihydrochloride, 6-aminohexanoic acid, gly-gly, gly-gly-gly, tryptone, betaine monohydrate, D-(+)-trehalose dihydrate, xylitol, D-sorbitol, sucrose, hydroxyectoin, trimethylamine N-oxide dihydrate, methyl-α-d-glucopyranoside, triethylene glycol, spermine tetrahydrochloride, spermidine, 5-aminovaleric acid, glutaric acid, and arginine. Pinic acid, ethylenediamine dihydrochloride, guanidine hydrochloride, urea, n-methylurea, N-ethylurea, N-methylformamide, hypotaurine, TCEP hydrochloride, GSH (reduced l-glutathione), benzamidine hydrochloride, ethylenediaminetetraacetate disodium salt dihydrate, magnesium chloride hexahydrate, cadmium chloride hydrate, non-surfactant sulfobetaine 195 (NDSB-195), non-surfactant sulfobetaine 201 (NDSB-201), non-surfactant sulfobetaine 211 (NDSB-211), non-surfactant sulfobetaine 221 (NDSB-221), non-surfactant sulfobetaine 256 (NDSB- 256) Taurine, acetamide, oxalic acid dihydrate, sodium malonate pH 7.0, succinate pH 7.0, taximate pH 7.0, tetraethylammonium Mubromide, choline acetate, 1-ethyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazolium chloride, ethylammonium nitrate, ammonium sulfate, ammonium chloride, magnesium sulfate hydrate, potassium thiocyanate, gadolinium(III) chloride hexahydrate, cesium chloride, 4-aminobutyric acid (GABA), lithium nitrate, DL-malic acid pH 7.0, lithium citrate tribasic tetrahydrate, ammonium acetate, sodium benzenesulfonate, sodium p-toluenesulfonate, sodium chloride, potassium chloride, sodium phosphate monobasic monohydrate, sodium sulfate decahydrate, lithium chloride, sodium bromide, glycerol, ethylene glycol, polyethylene glycol 200, polyethylene glycol monomethyl ether 550, polyethylene glycol monomethyl ether 750, formamide, polyethylene glycol 400, pentaerythritol ethoxylate (15 / 4 Examples include (EO / OH), 1,2-propanediol, polyethylene glycol monomethyl ether 1900, polyethylene glycol 3350, polyethylene glycol 8000, polyvinylpyrrolidone K15, polyethylene glycol 20000, (2-hydroxypropyl)-β-cyclodextrin, α-cyclodextrin, β-cyclodextrin, and methyl-β-cyclodextrin.

[0153]

[0164] From one perspective, the Myd polypeptide of the sweetening composition is sequence number 3 (or, The polypeptide may include an amino acid sequence having at least 80% sequence identity with 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 other words, the Myd 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; where (a) the polypeptide contains at least one substitution modification with respect to the polypeptide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and the polypeptide is not the polypeptide of SEQ ID NO: 3, or (b) the polypeptide further contains a histidine tag, and the polypeptide has sweetness-modulating activity. For example, the polypeptide contains 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, but does not consist of SEQ ID NO: 3. In a particular view, the polypeptide contains the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, or SEQ ID NO: 68.

[0154]

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

[0155]

[0166] From one perspective, the Myd polypeptide of the sweetening composition is sequence number 3 (or, It may contain amino acid sequences that have at least 80% sequence identity with SEQ ID NOs. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17. In other words, Myd polypeptide may contain amino acid sequences that have at least 80% sequence identity with SEQ ID NOs. 3, 8, 9, 10, The polypeptide has a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NOs: 11, SEQ ID NOs: 12, SEQ ID NOs: 13, SEQ ID NOs: 14, SEQ ID NOs: 15, SEQ ID NOs: 16 and SEQ ID NOs: 17; wherein (a) the polypeptide contains at least one substitution modification with respect to a polypeptide sequence selected from the group consisting of SEQ ID NOs: 3, SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, SEQ ID NOs: 12, SEQ ID NOs: 13, SEQ ID NOs: 14, SEQ ID NOs: 15, SEQ ID NOs: 16 and SEQ ID NOs: 17, and the polypeptide is not the polypeptide of SEQ ID NOs: 3, or (b) the polypeptide further contains a histidine tag, and the polypeptide has sweetness-modulating activity.

[0156]

[0167] Products for oral administration include foods, beverages, nutritional supplement compositions, or pharmaceutical compositions. It is possible.

[0157]

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

[0158]

[0169] Generally, the present invention relates to a food or beverage product in which the isolated sweet protein of the present invention The substance is intended to be included in an effective amount, for example, up to about 99% by weight with respect to the total weight of the food or beverage product, for example, in amounts ranging from about 0.01% to about 99% by weight. All intermediate weights with respect to the total weight of the food or beverage product (i.e., 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, ... 90%, 95%, 99% based on weight) are intended to be all intermediate ranges based on these amounts.

[0159]

[0170] The composition of the present invention is designed to maximize the biological efficacy of the Myd polypeptide. This may include "food-grade, bio-grade, or pharmaceutically acceptable carriers or excipients" that are used to prepare Myd polypeptides in desired dosage forms for administering Myd polypeptides in dispersed / diluted forms. Food-grade, bio-grade, or pharmaceutically acceptable carriers include many common food components, such as water of neutral, acidic, or basic pH, fruit or vegetable juices, vinegar, marinades, beer, wine, milk or condensed milk, natural water / fat emulsions, edible oils and shortenings, fatty acids, low molecular weight oligomers of propylene glycol, glyceryl esters of fatty acids, and dispersions or emulsions of such hydrophobic substances in aqueous media, salts such as sodium chloride, solvents such as flour and ethanol, solid food diluents such as vegetable powders or powders, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents; thickeners or emulsifiers, preservatives, solid binders, lubricants, etc.

[0160]

[0171] Food or beverage products that may be considered in the context of the present invention include baked goods. Sweet bakery products (e.g., but not limited to rolls, cakes, pies, pastries and cookies); pre-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings (e.g., but not limited to fruit pie fillings and pecan pie fillings, as well as nut pie fillings, and fillings for cookies, cakes, pastries and confectionery products); desserts, gelatin and puddings; frozen desserts (e.g., but not limited to regular ice cream, soft serve and other) Frozen dairy desserts like ice cream, including all types of ice cream. This includes non-dairy frozen desserts such as ice cream and sherbet; carbonated beverages (e.g., carbonated soft drinks, but not limited to these); non-carbonated beverages (e.g., non-carbonated soft drinks, but not limited to these, such as flavored water and tea or coffee-based sweetened beverages); beverage concentrates (e.g., non-liquid concentrates, such as liquid concentrates and syrups, as well as freeze-dried and / or powdered preparations, but not limited to these); yogurt (e.g., high-fat, low-fat and fat-free dairy yogurts, as well as non-dairy yogurts and lactose-free yogurts and all their frozen equivalents, but not limited to these); snack bars (e.g., cereal, nut, seed and / or fruit bars, but not limited to these); bread products (e.g., fermented and unfermented breads, yeast breads and unleavened breads, e.g., Examples include soda bread, bread containing any type of wheat flour, bread containing 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 dressings; sweet spreads (e.g., but not limited to jellies, jams, butters, nut spreads, and other spreadable preserves and conserves); confectionery products (e.g., but not limited to jelly candies, soft candies, hard candies, chocolates, and gums); sweetened breakfast cereals (e.g., but not limited to extruded (kix type) breakfast cereals, flake breakfast cereals, and puff breakfast cereals); and cereal coating compositions for use in the preparation of sweetened breakfast cereals. Although not mentioned herein, other types of food and beverage products that conventionally contain one or more nutritional sweeteners may also be considered in the context of the present invention.

[0161]

[0172] Complete or partial replacement of nutritional sweeteners in food or beverage products of the present invention As a result, the food or beverage product of the present invention can be useful as a low-calorie or diet product, a medical food / product (including pills and tablets), and a sports nutrition product, and can be particularly suitable for food or beverage products that require less sweetness at a given level of soluble solids.

[0162]

[0173] In some embodiments, the sweetening composition of the present invention contains other nutritional or non-nutritional sweeteners. The sweetener system can be formed by supplementing with other ingredients. The sweetener system may include the sweetener composition of the present invention, a bulking agent such as maltodextrose or acacia gum, and at least one high-strength sweetener. The composition can be provided as a liquid composition or a dry blend.

[0163]

[0174] In one embodiment, the present invention relates to a process for enhancing the sweetness of orally administered products. The present invention comprises a process including the addition of the Myd polypeptide.

[0164]

[0175] In another embodiment, the method of the present invention improves the sweetness and flavor of orally administered products. The present invention relates to a method for which a sweetening composition prepared by the present invention is added to an orally administered product. The amount to be added can be determined by methods known in the art, for example, using sensory evaluation as a guide.

[0165]

[0176] The following examples further illustrate the present invention, but needless to say, It should not be interpreted as limiting its scope. [Examples]

[0166]

[0177] Example 1

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

[0167]

[0179] Aqueous extraction of truffles was performed as follows: Eight different truffle samples were extracted in aqueous solution. The truffles were ground into a powder using a mortar and pestle in a nitrogenous environment, then 5:1 volume / weight of truffles in 4°C water were added and incubated at 4°C for 30 minutes. The extracted material was then subjected to low-speed, short-duration centrifugation, and the filtrate was tasted "neat." Sweetness intensity was scored on a scale of 0 (no sweetness) to 10 (extremely sweet). For each sample, sweetness was scored as follows:

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

[0168] [Table 1]

[0181] The aqueous extract was incubated at 4°C in sodium phosphate buffer at pH 7 and pH 2. It was stored. Little to no change in sweetness was observed over an 8-day period.

[0169]

[0182] Example 2

[0183] Purification of the sweet protein Myd from M. terfezoides. Truffles of *Titirolomyces terfezioides* were obtained in situ using appropriate procedures and permits within their natural range and stored at -80°C. 16.3 g of the sample was removed from the freezer and ground in liquid nitrogen using a mortar and pestle (white ceramic). Grinding was carried out for 15 minutes to completely pulverize the tissue and obtain a fine frozen powder. The powder was added to a 50 mL Falcon tube, 20 mL of RO-H2O was added, and the tissue was mixed by vortexing until no ice crystals were observed. The fragments were broken down using a rotor-stator at 2 × 1 min at 4°C with a setting of 20 to prepare a homogeneous solution (H1). The volume of the slurry was adjusted to 53 mL with RO-H2O and centrifuged at 7500 × G for 30 minutes at 4°C. The supernatant (S1) from this stage was collected in a 2 mL Eppendorf tube and centrifuged in a 5417R and Eppendorf centrifuge at 4°C and 20000 × G for 15 minutes. The pellet (P1) was discarded. Sweetness (by human senses) was perceived in the supernatant. The supernatant from this stage was collected and pooled. The supernatant was then filtered through a 25 mm 0.45 micron syringe filter (cellulose, VWR International) to obtain S1 + 0.22 μm filtration. The filtrate was then washed with hexane (twice with 38 mL to 50 mL of hexane) and the aqueous phase was collected. S1FH is S1F + hexane wash. The hexane phase was stored and dried. The aqueous phase was then precipitated with acetone (50 mL at -20°C was added to 33 mL of fraction S1FH and set to -20°C for 30 minutes). The sample was centrifuged at 3000 × g and the precipitate was collected. The precipitate was resuspended in 10 mM sodium phosphate at pH 6, and the supernatant from this stage was designated S2, while the precipitate was designated P2. The supernatant S2 was first applied to an AMICON centrifugal filter unit with a molecular weight cutoff of 100 kD to obtain the filtrate (flow-through) portion (designated as 100F) and the retaining solution (designated as 100R); then 100F was applied to a unit with a molecular weight cutoff of 30 kD to obtain the filtrate (30F) and the retaining solution (30R). The sweetness fraction was flowed through a 100 kD column. It appeared at 00F and was retained on the 30kD column (30R). In the SDS-PAGE shown in Figure 2, a band indicated by the arrow was observed at approximately 13kDa. This band was excised, and the N-terminal sequence analysis by Edman degradation was performed using standard detection methods, such as liquid chromatography and mass spectrometry, to identify the residues in each cycle. The polypeptide of SEQ ID NO: 4 was detected.

[0170]

[0184] Example 3 (Identification of RNA)

[0185] Sample collection

[0186] Fresh Mattirolomyces terfezioides truffles It was obtained in situ within its natural range using appropriate procedures and permits. A wild isolate (BDP2_18) of the truffle (gleba) Mattirolomyces terfezioides was procured from the natural environment. BDP2_18 is the largest wild truffle ever captured, and this truffle had sweetness characteristics described as "preceding sweetness, more fungal, earthy, and with little lingering sweetness."

[0187] Identification of the sample

[0188] Gleba wild isolates are used in GeneWiz for Internal Transplantation. The sequences were frozen and transported to scribed Spacer (ITS) Sequencing. The sequenced genomic loci were ITS 1 and 2 regions. The resulting Sanger sequencing reads were then aligned and low-quality bases were trimmed. Subsequently, each sequence was analyzed using the individual Basic Local Alignment Search Tool (BLAST) (Altschul, Gish, Miller, Myers, Identity was verified by searching for the original specimen (Lipman, 1990). A BLASTn search was employed using a nucleotide collection (nr / nt) excluding sequences from unpublished samples. The entry with the highest identity percentage relative to the wild-type isolate was Mattirolomyces terfezioides strain rib02.

[0171]

[0189] Sample preparation

[0190] Wild-type isolate BPD2_18 was surface-washed with sterile water, and approximately 100 mg of standing granules were prepared. The material was cut into cubes, rapidly frozen in liquid nitrogen, and stored at -80°C. It was then transported to GeneWiz on dry ice.

[0172]

[0191] RNA sequencing

[0192] The following samples were analyzed using GeneWiz for Standard RNASeq. The samples were submitted and examined using Illumina HiSeq, 2x150bp, single index, with approximately 350M of untreated paired-end reads per lane. This RNASeq analysis included polyA+ selection for transcriptome profiling.

[0173]

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

[0174]

[0194] 5'-AATGATACGGCGACCACCGAGATCTACACTCT TTCCCTACACGACGCTCTTCCGATCT-3'(Sequence ID 18)

[0195] 5'-GATCGGAAGAGCACACGTCTGAACTCCAGTCA C[i7 barcode]ATCTCGTATGCCGTCTTCTGCTTG-3'(Sequence ID 19)

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

[0175]

[0197] Identify the RNA transcript, and the DNA copy of that transcript is the sequence shown as Sequence ID No. 1. It has a predicted coding sequence, of which Sequence ID No. 2 is a predicted coding sequence based on the start and stop codons. Sequence ID No. 3 is also provided, a predicted protein with an estimated size of 13.3 kDa and 121 amino acids. Length: 122aa, Molecular weight: 13.381 kDa, Predicted isoelectric point: 8.64, and Predicted charge at pH 7: 1.01.

[0176]

[0198] Blast analysis. Predicted protein of SEQ ID NO: 3 and other proteins in GENBANK. The identity with the protein sequence was less than 31%. A virtual protein derived from Pisolithus tincturius (GenBank:KIN98154.1; SEQ ID NO: 7) was found to have approximately 31% homology to SEQ ID NO: 3 and was named virtual protein M404DRAFT_1005519 [Pisolithus tincturius Marx 270]. SEQ ID NO: 7 is also hypothesized to have sweetness-regulating activity. The complete cDNA copy of the RNA transcript is SEQ ID NO: 5, and the coding sequence is given as SEQ ID NO: 6.

[0177]

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

[0200] Based on the nucleotide sequence identified as Sequence ID No. 3, it contains a histidine tag. Three expression vectors were synthesized to express sequence number 20, and Atum, Inc. (N Three different Atum vector skeletons were cloned by ewark (CA): pD454-SR (plasmid pMy_3000), pD454-MR (plasmid pMy_3001), and pD454-WR (plasmid pMy_3002). All of these are E. coli IPTG-inducible T7 promoter expression vectors with ampicillin resistance, lacl, Lac01, Ori_pUC, and moderate (M), strong (S), and weak (W) ribosome binding sites on the plasmid. E. coli BL21 DE3 (Studier et al. (1986) J. Mol. Biol. 189:113-130) (New England Biolabs) was transformed with pMy_3000, pMy_3001, and pMy_3002 using the manufacturer's protocol. In short, previously frozen competent cells were thawed, mixed with 1 pg to 100 ng of plasmid DNA, and kept on ice for 30 minutes. This mixture was subjected to a 42°C heat shock for 45 seconds. Immediately afterward, the mixture was placed in an ice bath and left for 10 minutes. 950 μL of pre-warmed LB medium was added, and the mixture was grown at 225 rpm for 1 hour with shaking at 37°C. Cells were diluted 1:10 and 1:100, and 100 μL was plated onto antibiotic plates for each transformation reaction. After overnight growth at 37°C, the colonies were fully recovered and visible. To confirm the success of the transformation, csPCR (colony screening PCR) was performed to examine the cDNA region of the plasmid, and expression was confirmed by lysate SDS-PAGE. Heterogeneous expression in the E. coli host was confirmed using shaken flask-scale inducible expression. This process yielded strains Z14CE, Z15CE, and Z16CE, each containing plasmids pMy_3000, pMy_3001, and pMy_3002, respectively. The three strains (Z14CE, Z15CE, and Z16CE) were maintained on LB+ ampicillin 100 μg / mL agar plates, while the negative control (Eco_0001) was maintained on LB agar plates. Cultures of each strain were grown overnight in 50 mL of LB liquid medium in a 250 mL culture shaking flask without baffles, with appropriate antibiotics, shaking at 150 rpm.Subsequently, the following day, each overnight culture was seeded in 200 mL of TB liquid medium into a 1000 mL baffled culture shaking flask at 37°C, which had been shaken at 200 rpm and adjusted to 0.1 OD600 with appropriate antibiotics. When the OD600 reached 0.8, the IPTG capture was added to the medium to a final concentration of 0.66 mM. Shaking was continued at 37°C for a further 5 hours. After expression, the cells were centrifuged at 4000 g for 20 minutes. The supernatant was discarded, and the pellet was suspended in approximately 20 mL of washing buffer (10 mM sodium phosphate buffer pH 7.0). The suspended cells were disrupted in a high-pressure homogenizer (C3 Emulsiflex, Avestin, Inc., Ottawa, ON, Canada) operated at a maximum of 2000 bar. The disrupted cells were centrifuged at 13000 g (30 minutes), the supernatant was collected, and the pellet was discarded. The supernatant containing solubilized protein is processed using a 0.22 μm PES membrane B unit (Millipore, Burlington,...). The sample was filtered through MA (USA). SDS-PAGE was performed, and expression was confirmed by observing a 13.1kD band (Coomassie staining).

[0178]

[0201] Thermo Scientific TM HisPur TM Ni-NTA The his-tagged protein SEQ ID NO: 20 was purified using efficient immobilized metal affinity chromatography (IMAC) with a resin. SEQ ID NO: 20 was purified using nickel-charged nitrilotriacetic acid (NTA) chelate immobilized on 6% cross-linked agarose resin. The lysate was loaded onto a prepared IMAC column and equilibrated with binding buffer: 20 mM monobasic sodium phosphate, 0.5 M sodium chloride, 0.1 M imidazole, pH 7.4. Elution was performed using elution buffer: 20 mM monobasic sodium phosphate, 0.5 M sodium chloride, 0.5 M imidazole. After washing the column three times with binding buffer, his-tagged mycodulcein was eluted four times with elution buffer. Subsequently, the eluted fraction was ultrafiltered using a 30 kDa MWCO filter, and the filtrate was filtered through a 10 kD filter at 4000 x G for 15 minutes. The retention solution was diluted, rotated again for the washing step, and repeated three times. Figure 3 shows the analysis of the purification process using SDS-PAGE.

[0179]

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

[0180]

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

[0204] E. coli Z1 prepared in Example 4 containing the code sequence of Sequence ID No. 20 The 4CE strain was tested for its performance during fermentation in a laboratory-scale bioreactor. Bioreactor culture was performed in a 10.0 L Bioflo 320 round-bottom stirred fermenter (BioFlo / CelliGen 310, New Brunswick Scientific, Edison, NJ, USA). pH and dissolved oxygen sensors (Mettler Toledo, OH, USA) were attached to the fermenter. Temperature was controlled via a water-filled stainless steel base. Stirring was provided by two 6-blade Rushton turbines spaced 47 mm apart, with the lowest impeller positioned just above the bottom of the shaft. Aeration was performed through a porous tube sparger ring. Dissolved oxygen (DO) was controlled to 20% air saturation using a continuous cascade of stirring at 500–800 rpm and aeration at 5–8 L / min with air sprayed at high cell density. pH was controlled to 7.0 using 5.0 M ammonium hydroxide. Antiform Foaming was controlled by automatically adding 204 (Sigma, St. Louis, MO, USA). The latter was sensed using a conductivity probe mounted 10 cm above the culture level. The main fermentation medium contained 24 g of yeast extract, 12 g of tryptone, 5.42 mL of glycerol, and 100 mL of phosphate buffer stock (0.17 M KH2PO4, 0.72 M K2HPO4) (per liter). The medium was adjusted to pH 7.0 using 2 M HCl. When the initial glucose supply was drastically reduced (indicated by an increase in pH), a supply medium (per liter) consisting of 200 g of glucose, 21.1 g of (NH4)2SO4, and 19.7 g of MgSO4 was pumped into the fermenter at an initial flow rate of 1.00 mL / min. Unless otherwise specified, the initial volume of medium in the container was 4.0 L. The inoculum (200 mL) consisted of a culture grown for 16 hours in LB initiation medium in a 1 L baffled shaking flask (37°C, 200 rpm). The fermenter temperature was 37°C. Fermentation was induced with 0.66 mM IPTG after the optical density reached 10–20, and continued for 24 hours. Subsequently, after 24 hours of induction, the broth was centrifuged at 4000 g for 20 minutes. The supernatant was discarded, and the pellet was suspended in 1 L of washing buffer (10 mM sodium phosphate buffer, pH 7.0). The suspended cells were disrupted in a high-pressure homogenizer (C3 Emulsiflex, Avestin, Inc., Ottawa, ON, Canada) operated at a maximum pressure of 1500 bar or less. The disrupted cells were centrifuged at 13000 g (30 minutes), the supernatant was collected, and the pellet was discarded. The supernatant containing the solubilized protein was filtered through a 0.22 μm PES membrane unit (Millipore, Burlington, MA, USA).

[0181]

[0205] The purified supernatant prepared in this example was measured by a trained sensory scientist at 0.03 ml. It was tasted in g / mL (0.2mL aliquot) and was equivalent to 8° Brix (approximately 8% sugar solution). It was found to possess a sweetness. The sweetness was very pronounced, a "clean" sweetness (like sugar), with no other flavors, a slightly delayed onset of sweetness, and a sweet aftertaste.

[0182]

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

[0183]

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

[0208] To quantify mycodulcein (sequence number 21) tagged with his, A horseradish peroxidase (HRP) antibody was conjugated to the 6XHis-Tag sequence on the carboxyl terminus of SEQ ID NO: 21, and a direct ELISA was performed. The ELISA allows for the measurement of mycodulcein concentrations in the complex lysate and purified protein. Recombinant 6XHis-tagged E. coli mycodulcein was calculated from its amino acid sequence by methods known in the art to have a molecular weight of 14.2 kDa and 27960 M -1 cm -1 It has a molar extinction coefficient. Purity was evaluated as ≥98% by SDS-PAGE. The mycodulcein protein concentration was determined by the absorbance at 280 nm using the Beer-Lambert law, and then a standard curve was created for ELISA using known concentrations of mycodulcein (μg / mL).

[0184]

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

[0185]

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

[0211] As described in Example 5, it was obtained from E. coli, and as described in Example 4 The concentration-dependent characterization of the taste properties of purified his-tagged mycodulcein (SEQ ID NO: 21), purified to the specified degree, was quantitatively evaluated using Opertech Bio (Philadelphia, PA). The sweetness potency and relative effectiveness of mycodulcein were compared with 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 kinin, and 10 mM citrate. Figure 4 shows the concentration-response functions for sweetness of mycodulcein, aspartame, thaumatin, and rebaudioside A. Data are plotted as the percentage (p) of response occurring on a 200 mM sucrose-related ("sweetness") target. For the curves for mycodulcein, aspartame, thaumatin, and rebaudioside A, each data point was calculated as the average over 32 iterations, and for the sucrose curve, it was averaged over 16 iterations; error bars are the standard deviation. The points for water and sucrose controls were similarly calculated as the average over 128 and 64 iterations, respectively. The curves were fitted using nonlinear regression.

[0186]

[0212] The concentration that produces a sweetness response (EC50, or potency) that is half of the maximum value is defined as a nonlinear concentration. The EC50 (and 95% confidence intervals) for mycodulcein (MYC), sucrose (SUC), aspartame (ASP), rebaudioside A (REB), and thaumatin (THN) were derived from morphological regression. Table 2 shows these values. Equivalency for sucrose on a molar and weight basis is also provided.

[0187]

[0213] Table 2.

[0188] [Table 2]

[0214] The evaluation of thaumatin, sucrose, and SEQ ID NO: 21 is for protein and 1 The time-intensity of sweetness sensation in named samples of 0.045 mg / mL water with 0% sucrose was measured using CATA (click all relevant). Methodology: 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 tests. The panel consisted of 3 to 6 participants, and the study was repeated twice. All samples were blinded and presented with randomized 3-digit codes.

[0189]

[0215] Training Strategy: A 15-point sweetness scale to confidently assign sweetness values. Intensive training on the sweetness scale over 6 weeks in a tasting log. Due to a unique sweetness behavior pattern, training on the time-intensity principle was required over 3 weeks. Samples were tasted using a stopwatch to record time and help reach a consensus. Number of panelists: 3-6, number of repetitions: 2. All samples were blinded and presented with randomized 3-digit codes. Statistical analysis: Due to the small number of panelists, statistical analysis could not be performed.

[0190]

[0216] The maximum intensity (Imax) of mycodulcein and thaumatin is measured in the scrotum. Compared to sucrose, it shows a higher level of sweetness of approximately 1 point on a 15-point scale at the test dose. Thaumatin and mycodulcein have a flatter gradient, longer peak times, and a more gradual / longer decline. Sucrose approaches the threshold sweetness level (intensity < 1) faster than thaumatin and mycodulcein, at 162 seconds. When sucrose reaches the threshold level, thaumatin and mycodulcein are recognizable at low to moderate intensity. Mycodulcein and thaumatin appear to have similar potency in this experiment, which is approximately 3000 times sweeter than sucrose on a weight basis, or approximately 120,000 times sweeter than sucrose on a molar basis. These two experiments indicate that mycodulcein is a high-intensity sweetness protein with potency ranging from 400 times sweeter to 3000 times sweeter than sucrose on a weight basis.

[0191]

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

[0218] A method for identifying potentially important residues in mycodulcein. (Sweet protein) Although the proteins lack primary sequence identity, their overall tertiary structure possesses a sweet finger motif (Tancredi, T., Pastore, A., Salvadori, S., Esposito, V. & Temussi, PA Interaction of sweet proteins with their receptor: A conformational study of peptides corresponding to loops of brazzein, monellin and thaumatin. European Journal of Biochemistry 271, (2004): 2231-2240). Sweet protein The protein has an antiparallel β-sheet with an α-helix perpendicular to the β-sheet. The tertiary structures of the sweet proteins somatine, monellin, brazzein, and lysozyme were analyzed using PyMO L 2.0 (The PyMOL Molecular Graphics System, Version 2.0 Schroedinger, LLC) and compared with the model of mycodulcein generated by 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 occurred. Negatively charged glutamine and aspartic acid differ by an additional carbon in the aliphatic chain. Substituting positively charged lysine and arginine is considered a conservative substitution, but the guanidinium of arginine can form additional interactions with amino acids including hydrogen bonds, aromatic and aliphatic contacts. The ionic amino acid mutations were from lysine to arginine, arginine to lysine, aspartic acid to glutamic acid, and glutamic acid to aspartic acid. The relative positions of each mutant were modeled by PyMOL 2.0 and classified as the N-terminus, three loop regions, five β-sheets, one α-helix, and the C-terminus (see Figure 5B).

[0192]

[0219] Specifically, the following single mutants were prepared. Their predicted positions are shown in Table 3 See also Figure 5B. This shows the predicted secondary structure of SEQ ID NO: 3 superimposed on the putative secondary structure motif and the positions of the point mutations in Table 3 within each motif.

[0193]

[0220] Cloning: Eco_0001 aka E.coli BL21 DE3 (E.coli strain B F - ompT gal dcm lon hsdS B (rB - m B - )λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5])[malB + ] K-12 (λ S Cells (obtained from New England Biolabs# C2527I) were transformed with 23 plasmids (pMy_3018~pMy_3040) using the manufacturer's protocol. Briefly, previously frozen competent cells were thawed, mixed with 1 pg~100 ng of plasmid DNA, and kept on ice for 30 minutes. This mixture was subjected to a 42°C heat shock for 45 seconds. Immediately thereafter, the mixture was placed in an ice bath and left for 10 minutes. 950 μL of pre-warmed LB medium was added, and the mixture was grown at 225 rpm for 1 hour with shaking at 37°C. Cells were diluted 1:10 and 1:100, and 100 μL was plated onto antibiotic plates to perform each transformation reaction. After overnight growth at 37°C, the colonies were fully recovered and visible. This process yielded Z18CE~Z40CE strains containing plasmids pMy_3018~pMy_3040 sequentially. Heterologous expression in the E. coli host was confirmed using expression induced on a shaking flask scale. Post-transformation mutants were plated and maintained on LB + ampicillin 100 μg / mL agar plates.

[0194]

[0221] The plates were incubated at 37°C for 16 hours. Colony screening P CR was performed, and genotyping was confirmed using colony screening primers designed to examine the adjacent regions along with the cDNA region of the plasmid. Successful transformations resulted in DNA fragments of a specific size, while negative controls and untemplated controls did not yield PCR bands. Successful transformations were observed for all mutants.

[0195]

[0222] Using expression induced in a shaking flask scale, in the E. coli host... Heterogeneous expression was confirmed.

[0196]

[0223] 23 strains (Z38CE~Z60CE) were given LB + ampicillin 100 μg / mL in cold water. The strains were maintained on top plates, while the negative control (Eco_0001) was maintained on LB agar plates. Each strain was incubated overnight at 37°C in 50 mL of LB liquid medium in a 250 mL unbaffled culture flask with appropriate antibiotics, shaking at 150 rpm. The culture was grown. The following day, it was shaken at 200 rpm and treated with an appropriate antibiotic until it reached 0.1 OD. 600 Each overnight culture was seeded in 200 mL of TB liquid medium in a 1000 mL baffled culture shaking flask, which was adjusted to 37°C. 600 When the concentration reached 0.8, the IPTG capture was added to the culture medium to achieve a final concentration of 0.66 mM. Shaking was continued for a further 5 hours at 37°C. Then, cells were collected by centrifugation at 5000 g for 5 minutes at 4°C. E. coli cells were washed once with cold dH2O and centrifuged again at 5000 g for 10 minutes at 4°C. To confirm successful expression, cell lysates were prepared using liquid nitrogen and a mortar and pestle. The cell pellet was resuspended in 10 mL of cold dH2O, and the crude lysate was rotated at 20000 g for 5 minutes at 4°C. Finally, the supernatant was aspirated, filtered through a 0.2 μm PES filter, and subjected to SDS-PAGE protein electrophoresis. The crude lysate was tasted to identify sweet-tasting mutants. The test results are shown in Table 3.

[0197]

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

[0198] [Table 3]

[0225] All 16 variants have a sweet taste (Z38CE, Z39CE, Z41C E, Z45CE, Z47CE, Z48CE, Z49CE, Z51CE, Z52CE, Z53CE, Z55CE, Z56CE, Z57CE, Z58CE, Z59CE, Z60CE) were further reexpressed using 200 mL of culture medium. After expression, cells were centrifuged at 4000 g for 20 minutes after 24 hours of induction. The supernatant was discarded, and the pellet was suspended in approximately 20 mL of washing buffer (10 mM sodium phosphate buffer pH 7.0). The suspended cells were disrupted in a high-pressure homogenizer (C3 Emulsiflex, Avestin, Inc., Ottawa, ON, Canada) operated at a maximum of 2000 bar. Disrupted cells The mixture was centrifuged at 13000g (30 minutes), the supernatant was collected, and the pellet was discarded. The supernatant containing solubilized protein was then processed using 0.22 μm PES membrane units (Millipore, The material was filtered through Burlington, MA, USA. The material was then isolated by IMAC purification as described in Example 4.

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

[0199]

[0227] Effect of Conservative Point Mutation on Mycodulcein Sweetness

[0228] To correlate the effects of conservative single-point mutations with sweetness, we compared published mutations in thaumatin, blazein, monellin, and lysozyme with mycodulcein. Since the objective was to match the time and intensity profiles of mycodulcein with those of sucrose, we measured sucrose equivalence, sweetness onset, and total duration by sensory analysis. Sucrose is, It has fast sweet taste expression, high intensity, and fast duration. Therefore, similar to mutations that match or improve sucrose equivalence, mutations that reduce sweet taste expression and duration are desirable. Similar to mutations that reduce sucrose equivalence, mutations that increase sweet taste expression and total duration are not desirable. See FIGS. 5B and 6.

[0200]

[0229] N-terminal - external - D3E

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

[0201]

[0201]

[0231] β-sheet 1 - external - K11R

[0232] Conservative changes in the single mutation at K11R resulted in a slight increase in sucrose equivalence and a moderate reduction in sweet taste expression; however, the duration of sweetness increased dramatically. These results suggest that K11 is an important residue for binding to the sweet receptor T1R2 / T1R3 and may affect the off-rate of mycodulcein from the receptor.

[0202]

[0202]

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

[0234] Conservative mutations at K26 resulted in a slight reduction in sucrose equivalence, indicating that this conservative mutation has no significant effect on protein functionality.

[0203]

[0203]

[0235] Loop 2 region - external - K51R

[0236] Molecular modeling showed that all putative loop region mutations were solvent-exposed Aside from a moderate reduction in sweetness expression, sensory tests showed only slight reductions in sucrose equivalence and total duration, indicating that this conservative mutation does not have a significant effect on protein functionality.

[0204]

[0237] Loop 2 region - External - R57K

[0238] The R57K mutant is effective in sweetness expression, sucrose equivalence, and total duration. It has a significant inhibitory effect.

[0205]

[0239] β Sheet 4 - External - R66K

[0240] The mutant R66K shows significant reductions in sucrose equivalence and total duration. It possesses this property, but slightly increases sweetness expression. The R66K mutation may be a crucial residue for affinity to and off-rate of the sweetness receptor.

[0206]

[0241] Loop 3 region - External - D69E

[0242] The mutation in D69E results in a slight reduction in sucrose equivalence and duration. It has a slight increase in duration. This range in this region is predicted to be relatively insensitive to conservative mutations.

[0207]

[0243] α-helix region - external - D85E and D86E

[0244] Both D85E and D86E indicate the sensory stimulation of mycodulcein. It has a similar effect on quality. The sucrose equivalence and duration of D85E and D86E were reduced. Sweetness development was similar to the control.

[0208]

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

[0209]

[0246] As shown in Table 3, R20K, E35D, K44R, D46E, D52E, R Conservative single-point mutations at 75K and D94E resulted in loss of protein expression. These data suggest that these residues may be involved in protein folding or expression within the E. coli host. The predicted tertiary structure model discussed above in this example supports protein misfolding resulting from these changes, as all of these residues are included in the predicted β-sheet.

[0210]

[0247] References:

[0248] 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).

[0211]

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

[0212] Example 9.

[0213]

[0250] Thermal stability was determined for GloMelt TM Thermal Shift Prote using the Thermal Shift Prote in Stability Kit (Biotium, Inc., Fremont, CA) and following the manufacturer's instructions, on protein samples from the IMAC purification of the 16 sweet mutants described in Example 8 standardized to 0.04 mg / mL. Briefly, each reaction to measure the thermal shift relied on mixing: mycodulcein, at 36 μg / mL in 25 mM sodium phosphate buffer pH 7.4, and the reagents provided in the kit according to the manufacturer's instructions. For thermal shift measurements, a BR Clear plate, scan mode SYBER / FAM only, a Bio-Rad CFX96 Touch system was used at 25°C for 30 s, melting curve 25°C to 95°C, increment 0.5°C every 10 s, and plate read. Tm was determined based on the midpoint of the curve fitted to the experimental data using the 5-parameter equation described in Schulz, M. N., Landstroem, J. & Hubbard, R. E. MTSA-A Matlab program to fit th ermal shift data. Analytical Biochemistry 433, 43-47 (2013). The results (Table 4) show that thermal stability is minimally affected by the amino acid changes in the mutants tested.

[0214]

Table 4

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

[0252] Based on the nucleotide sequence identified as SEQ ID NO: 3, SEQ ID NO: 21 (pMy_ Two expression vectors expressing mycodulcein (4003) (his-tagged) and SEQ ID NO: 3 (pMy_4002) (undenatured) were synthesized and cloned by Atum, Inc. (Newark, CA) into the Atum vector non-secretory scaffold pD1234, which contains the URA3 marker and a strong constitutive promoter GPD. Transformation procedure This process involves generating electrocompetent cells and then introducing an expression vector by electroporation. In short, electrocompetent cells are first generated by growing the cells to the early to mid-log phase, while removing salt from the growth medium through multiple washes. After mixing 1-5 μg of expression vector, the sample is placed on a Gene Pulser II Electroporator with the following settings (charging voltage: 1.5 kV, capacitance: 25 μF, resistance: 200 Ω), 1 mL of pre-warmed 30°C YPD is immediately added, and the suspension is incubated at 30°C for 1-2 hours while shaking at 200-250 rpm. The transformed mutants were plated on SC-ura agar plates and maintained. This process yielded strains Z19ES and Z20ES, each containing plasmids pMy_4002 and pMy_4003, respectively. The cDNA region of the plasmids was examined by csPCR (colony screening PCR). Therefore, successful transformation resulted in a 303 bp DNA fragment, while negative controls and untemplated controls did not produce PCR bands and expression was confirmed.

[0215]

[0253] Two strains (Z19ES, Z20ES) were maintained on SC-ura agar plates. Meanwhile, negative controls were maintained on SC agar plates. Each strain was grown overnight in 50 mL of SC-ura / SC liquid medium in a 250 mL unbaffled culture shaking flask at 37°C, shaking at 150 rpm. Each overnight culture was then seeded in 200 mL of SC-ura / SC (O-RDL-R10_TB medium) liquid medium in a 1000 mL baffled culture shaking flask at 30°C, adjusted to 0.02 OD600, and shaken at 200 rpm. Shaking was continued at 30°C for a further 48 hours. Cells were then collected by centrifugation at 5000 g for 5 minutes at 4°C. S. cerevisiae cells were then washed with cold dH2O and again centrifuged at 5000 g for 5 minutes at 4°C. To confirm successful expression, cells were lysed using liquid nitrogen and a mortar and pestle. The cell pellet was resuspended in 10 mL of cold dH2O, and the lysate was rotated at 20,000 g for 5 minutes at 4°C. The supernatant was filtered through a 0.2 μm PES filter. The filtrate (both strains) was confirmed to be sweet by the method described in Example 3.

[0216]

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

[0217]

[0255] Example 11 (Unmodified myc from strain Z19ES (S. cerevisiae)) Purification of odulcein)

[0256] Three chromatography techniques: cation exchange (CIEX), hydrophobic interaction The effectiveness of purifying undenatured mycodulcein (SEQ ID NO: 3) expressed in S. cerevisiae was evaluated using HIC and size exclusion chromatography (SEC).

[0218]

[0257] Cation exchange evaluation.

[0219]

[0258] The isoelectric point of undenatured mycodulcein is approximately 9.5, determined by isoelectric focusing electrophoresis. This was determined, suggesting that a cation exchange column could successfully purify mycodulcein. The clarified cell lysate prepared as described in Example 10 was subjected to 2x starting buffer. The solution was mixed with the cell lysate in 50 mM sodium phosphate, 1 M ammonium sulfate, pH 7.0, and stored at 4°C for later use. AKTA Explorer The purification procedure was performed using a 100 system (GE Healthcare, Sweden), and the eluted protein was monitored at 280 nm and 215 nm using a UV-900 UV detector (GE Healthcare, Sweden). Binding conditions for undenatured mycodulcein were screened using PreDictor plates (GE Healthcare, Sweden) pre-packed with CIEX resin. The pre-packed plates contained three main resins: Capto S (strong CIEX), Capto MMC (weak CIEX), and SP Sepharose Fast Flow (strong CIEX). The lysate was dialyzed in 20 mM dibasic sodium phosphate, and after screening different pH values ​​in the range of 4–9, the optimal conditions were scaled up using a HiScreen column. Equilibrium was achieved using 25 mM dibasic sodium phosphate at pH 5 at a flow rate of 3 mL / min. Binding proteins were eluted using 25 mM dibasic sodium phosphate, 1 M sodium chloride, and pH 7, with a sodium chloride gradient increasing from 0 to 1 M. Various binding and elution conditions were screened, and Capto MMC, a weak cation exchanger, showed the best binding ability at pH 5. However, the purity after this step was low (25%), so an alternative purification step was needed. Figure 7A shows the PAGE analysis of the eluted fraction from Capto MMC. M: protein marker; Lane 1: eluted fraction, showing low purity after cation exchange. Arrows indicate the mycodulcein band.

[0220]

[0259] HIC rating.

[0221]

[0260] HiScreen CaptoButyl Column (Cytiva, Swee Cell lysates were also attached to hydrophobic interaction chromatography (HIC) using a den (DEN) column; equilibration was performed using 50 mM sodium phosphate, 1 M ammonium sulfate, pH 7.0 in five different column capacities. Then, the cell lysates were loaded into the column at a flow rate of 3 mL / min. Binding proteins were eluted by reducing the ammonium sulfate gradient from 1 to 0 M using 50 mM sodium phosphate at pH 7.0. All obtained fractions were analyzed by SDS-PAGE.

[0222]

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

[0223]

[0262] SEC evaluation.

[0224]

[0263] Next, the eluent fraction containing undenatured mycodulcein is subjected to size exclusion. The sample was further purified using a chromatographic (SEC) HiPrep 26 / 60 Sephacryl S-200HR column (Cytiva, Sweden) and eluted with a buffer containing 50 mM sodium phosphate and 150 mM NaCl, pH 7.0. The fraction was collected, then concentrated, desalted using a 3 kDa molecular weight cutoff (MWCO) centrifuge filter (Millipore-Sigma, Germany), and analyzed by SDS-PAGE.

[0225]

[0264] Summary: Unmodified mycodulcein is a weak cation exchanger Capto MM Although it binds well to C, the relatively low purity of the eluent fraction made CIEX an undesirable capture / intermediate purification step. On the other hand, higher purity fractions were obtained from HIC and Capto Butyl columns. SDS-PAGE analysis revealed relatively large impurities. As it appears to possess a molecular weight, SEC became an important candidate for obtaining high-purity, unmodified mycodulcein.

[0226]

[0265] Purity after HIC / SEC is determined by GelAnalyzer on SDS-PAGE. This is approximately 98%. Figure 7C shows the eluted proteins from the HIC column after chromatography with HiPrep 26 / 60 Sephacryl S-200. Lane 1 shows purified his-tagged mycodulcein, and lane 2 shows purified undenatured mycodulcein.

[0227]

[0266] Purified undenatured protein from S. cerevisiae was trained It was tasted by sensory scientists at a concentration of 0.03 mg / mL (0.2 mL aliquot) and found to have a sweetness equivalent to 8° Brix (approximately 8% sugar solution). The sweetness is very pronounced. It had a "clean" sweetness (like sugar), no other flavors, a slightly delayed onset of sweetness, and a sweet aftertaste.

[0228]

[0267] Example 12 (Application Data)

[0268] Prepared as in Example 5 and purified as described in Example 5, with His tag Mycodulcein was tested in a yogurt base. The yogurt base had the following recipe (Table 5):

[0269] Table 5:

[0229] [Table 5]

[0270] Sugarcane sugar is added as a carbon source for yogurt cultures, and at least It is also partially consumed by the culture. Mycodulcein was added to bring the sweetness closer to that of sugars with a Brix of 8°~10°. The final concentration in the yogurt base was 0.05 mg / mL. Taste tests were conducted by trained sensory scientists and it was found that the yogurt had a sweetness equivalent to 8° Brix (approximately 8% sugar solution). The sweetness was very pronounced. It had a "clean" sweetness (like sugar), no other flavors, a slightly delayed onset of sweetness, and a sweet aftertaste.

[0230]

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

[0231]

[0272] All documents, including publications, patent applications, and patents, cited herein are, When a particular document is specifically cited as reference, it is indicated, and its entire content is cited as such in this specification.

[0232]

[0273] In the context describing this invention (particularly in the context of the following claims), the use of the terms “one (a),” “one (an),” “it,” “at least one,” and similar directives shall be interpreted as referring to both singular and plural, unless otherwise stated herein or the context clearly contradicts this. The use of the term “at least one” and the subsequent list of one or more items (e.g., “at least one of A and B”) shall be interpreted as referring to one item selected from the enumerated items (A or B), or any combination of two or more enumerated items (A and B), unless otherwise stated herein or the context clearly contradicts this. The terms “compising,” “having,” “including,” and “containing” shall be interpreted as open-ended terms (i.e., “including but not limited to”) unless otherwise specified. The term “consisting of” This is interpreted as a rose end term (i.e., excluding any ingredients or steps other than those listed). The term "essentially derived from" allows for the inclusion of ingredients or steps that are not essential to the function or activity of the product or method and do not substantially affect the function or activity.

[0233]

[0274] The enumeration of value ranges in this specification does not necessarily reflect the ranges of values ​​unless otherwise noted herein. It is intended solely as a shorthand notation for referring to each distinct value within individually, and each distinct value is incorporated herein as if it were individually enumerated herein. All methods described herein may be performed in any preferred order unless otherwise indicated herein or unless it is clearly inconsistent with the context. Any and all examples or representative language provided herein (e.g., "etc.") are intended solely to better illustrate the invention unless otherwise specifically asserted and do not impose any limitations on the scope of the invention. The language herein should not be construed as indicating that any element not asserted is essential to the practice of the invention.

[0234]

[0275] Preferred embodiments of the present invention are known to the inventors of the best way to carry out the present invention. The modes described herein are included. Variations of these preferred embodiments may be apparent to those skilled in the art by reading the above description. The inventors expect that skilled persons will appropriately adopt such variations, and they intend that the invention may be carried out in ways other than those specifically described herein. Accordingly, the invention encompasses all modifications and equivalents of the subject matter listed in the claims appended herein, as permitted by applicable law. Furthermore, all combinations of all possible variations of the above elements are incorporated herein unless otherwise indicated herein or unless they are clearly inconsistent with the context.

Claims

1. An isolated polynucleotide encoding a polypeptide having sweetness-regulating activity, wherein the polynucleotide sequence is (a) Polypeptide sequences selected from the group consisting of SEQ ID NOs: 3, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 71, 72, 73, 74, and 75; (b) Polypeptides 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; and (c) A polypeptide sequence modified by deletion, insertion, substitution, or addition of 24 amino acids or less from a polypeptide sequence selected from the group consisting of SEQ ID NOs: 3, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17; The isolated polynucleotide encoding a polypeptide selected from the group consisting of the following.

2. The isolated polynucleotide according to claim 1, wherein the polypeptide sequence of the polypeptide having sweetness-regulating activity is the polypeptide sequence shown in SEQ ID NO: 3, or a polypeptide sequence having at least 80% sequence identity with SEQ ID NO:

3.

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

3.

4. The isolated polynucleotide according to claim 1 or 2, wherein the polypeptide sequence of the polypeptide having sweetness-regulating activity comprises SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO:

75.

5. The isolated polynucleotide according to claim 4, wherein the polypeptide sequence of the polypeptide having sweetness-regulating 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.

6. The isolated polynucleotide according to claim 4 or 5, wherein the polynucleotide comprises SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, or SEQ ID NO:

67.

7. An isolated polynucleotide according to any one of claims 1 to 6, operably connected to a heterogeneous regulatory element.

8. An isolated polynucleotide according to any one of claims 1 to 7, wherein the polynucleotide sequence further encodes a histidine tag.

9. An expression cassette comprising an isolated polynucleotide according to any one of claims 1 to 8.

10. A vector comprising an isolated polynucleotide according to any one of claims 1 to 8.

11. A host cell transformed with the vector described in claim 10.

12. A method for producing a protein having sweetness-regulating activity, comprising culturing the host cells described in claim 11 in a culture medium under conditions that result in the production of a protein having sweetness-regulating activity.

13. An isolated polypeptide comprising a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NOs: 3, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17, The polypeptide may contain at least one substitution modification with respect to a polypeptide sequence selected from the group consisting of SEQ ID NOs: 3, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17, or The polypeptide may further contain a histidine tag, and The polypeptide has sweetness-regulating activity. The isolated polypeptide.

14. The isolated polypeptide according to claim 13, wherein the polypeptide comprises SEQ ID NO: 3, or a polypeptide sequence having at least 80% sequence identity with respect to SEQ ID NO:

3.

15. The isolated polypeptide according to claim 13 or 14, wherein the polypeptide comprises amino acid residues 1-11, 17-32, 39, 40, 45-67, 73-100, and 110-121 of SEQ ID NO:

3.

16. The isolated polypeptide according to claim 13 or 14, wherein the polypeptide comprises SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO:

75.

17. The isolated polypeptide according to claim 16, wherein the polypeptide comprises SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, or SEQ ID NO:

68.

18. The polynucleotide sequence is (a) the nucleic acid sequence shown in Sequence ID No. 2; and (b) A nucleic acid sequence having at least 90% sequence identity with the nucleic acid sequence shown in Sequence ID No. 2; An isolated polynucleotide comprising a polynucleotide selected from the group consisting of the following, wherein the polynucleotide encodes a polypeptide having sweetness-regulating activity.

19. The isolated polynucleotide according to claim 18, wherein the polypeptide sequence of the polypeptide having sweetness-regulating activity is selected from the group consisting of SEQ ID NOs: 3, SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, SEQ ID NOs: 12, SEQ ID NOs: 13, SEQ ID NOs: 14, SEQ ID NOs: 15, SEQ ID NOs: 16, SEQ ID NOs: 17, SEQ ID NOs: 71, SEQ ID NOs: 72, SEQ ID NOs: 73, SEQ ID NOs: 74, and SEQ ID NOs:

75.

20. Polynucleotides are sequence numbers 23, 25, 29, 37, 41, 43, 45, 49, 51, and 53. The isolated polynucleotide according to claim 18 or 19, comprising the nucleic acid sequence of SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, or SEQ ID NO:

67.

21. An isolated polynucleotide according to any one of claims 18 to 20, operably connected to a heterogeneous regulatory element.

22. The isolated polynucleotide according to any one of claims 18 to 21, wherein the polypeptides of (a) and (b) further comprise a nucleotide sequence encoding a histidine tag.

23. An expression cassette comprising a polynucleotide according to any one of claims 18 to 22.

24. A vector comprising the polynucleotide described in claim 23.

25. A host cell transformed with the vector described in claim 24.

26. A method for producing a protein having sweetness-regulating activity, comprising culturing the host cells described in claim 25 in a culture medium under conditions that result in the production of a protein having sweetness-regulating activity.

27. Compositions containing the following combinations (a) Products for oral administration, said products are not Mattiromyces terrefizioides truffles, and (b) A sweetening composition comprising an isolated polypeptide, wherein, (i) The polypeptide comprises an amino acid sequence having at least 80% sequence identity with a polypeptide selected from the group consisting of SEQ ID NOs: 3, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17; or (ii) The polypeptide is the polypeptide according to any one of claims 13 to 17; This combination has an enhanced sweetness compared to products intended for oral administration.

28. A method for adjusting the taste of an orally administered product, comprising combining the orally administered product with an effective amount of a sweetening composition comprising an isolated polypeptide, wherein (i) The polypeptide comprises an amino acid sequence having at least 80% sequence identity with a polypeptide selected from the group consisting of SEQ ID NOs: 3, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17; or (ii) The polypeptide is the polypeptide according to any one of claims 13 to 17, The product for oral administration is not Mattiromyces terfezioides truffle, and This combination has an enhanced sweetness compared to products intended for oral administration.

29. The composition according to claim 27 or the method according to claim 28, wherein the product for oral administration is a food, beverage, nutritional supplement composition, or pharmaceutical composition.

30. Products for oral administration include: baked goods; sweet bakery products; pre-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings, gelatin and pudding; frozen desserts; yogurt; snack bars; bread products; pre-made bread mixes for preparing bread products; sauces, syrups and dressings. The composition according to claim 27, which is a food product selected from the group consisting of singe; sweet spread; confectionery product; and sweetened breakfast cereal.

31. The composition according to claim 27 or the method according to claim 28, wherein the product for oral administration is a beverage product selected from the group consisting of carbonated beverages; non-carbonated beverages; and beverage concentrates.

32. A method for purifying polypeptides having sweetness-regulating activity, (a) Obtain a composition containing a polypeptide, (b) The composition is purified via hydrophobic interaction chromatography (HIC), followed by size exclusion chromatography (SEC). The method, which includes the following, (i) The polypeptide comprises an amino acid sequence having at least 80% sequence identity with a polypeptide selected from the group consisting of SEQ ID NOs: 3, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17; or (ii) The polypeptide is the polypeptide according to any one of claims 13 to 17.

Images