Sweet protein analogue

Abstract

The present invention relates to analogues of the sweet protein brazzein. In particular, the invention relates to analogues of brazzeins, said analogues having increased thermostability, host cells and methods for producing same.

Description

[0001] P7131 PC00

[0002] Sweet protein analogue

[0003] Technical field

[0004] The present invention relates to analogues of the sweet protein brazzein. In particular, the invention relates to analogues of brazzeins, said analogues having increased thermostability, host cells and methods for producing the same.

[0005] Background

[0006] The consumption of excessive refined sugar can lead to prolonged elevation of blood glucose levels, potentially contributing to the development of type 2 diabetes. Furthermore, excessive sugar intake is associated with a range of other adverse health outcomes, including cardiovascular, hepatic, and renal diseases. With the current focus on reducing intake of free sugars, interest in non-sugar sweeteners (NSS) as a possible alternative has intensified. NSS are classified as artificial or natural. In general, public opinion is negative towards artificial NSS and people are more inclined towards natural food products.

[0007] Sweet proteins have a natural origin, being extracted from the fruit of different tropical plants. There are 8 well-known sweet proteins: thaumatin, monellin, brazzein, mabinlin, pentadin, curculin / neoculin, miraculin, lysozyme, and the newly discovered sweet protein from honey truffle. These proteins are known for being extremely sweet, i.e. displaying 100 to 3000 times the sweetness of sucrose on a per weight basis.

[0008] Consequently, minute amounts of protein suffice in food products to trigger the sweet sensation. Therefore, they neither add notable calories nor increase blood glucose levels.

[0009] Summary

[0010] Sweet proteins retain their ability to interact with sweet taste receptors only when they are correctly folded. Protein engineering has emerged as a pivotal tool to uphold the tertiary structure of sweet proteins under various physio-chemical conditions, including the high temperatures encountered during food processing. Recent advancements in protein structure prediction have propelled strategies to enhance thermostability, which encompass structural calculations coupled with saturation mutagenesis, as well as rational design. P7131 PC00

[0011] Ming and Hellekant determined the thermostability of brazzein extract to be 80°C (Ming and Hellekant, 1994). Thus, it is not amenable to processes involving temperatures above 80°C, including above 100°C, which is the boiling point of water. Temperatures above 100°C are often required in food processing, for example in baking and sterilization or ultra-pasteurization processes, however high temperature can also be a barrier in consumer food applications, such as in the use of sweet proteins as sweeteners for hot food and beverages. Hence, there is a need for further variants or analogues of brazzein having increased thermostability.

[0012] The present inventors have improved the thermostability of brazzein, such as to more than 107°C or even more than 110°C, through protein engineering. Surprisingly, as proven by the inventors, this great increase in melting temperature may be achieved through the substitution of a single amino acid. With these advancements, applications of brazzein can be extended to bakery products and pre-treatments like sterilization and ultra-pasteurization, thus greatly expanding the range of applications in which it can be utilised.

[0013] In addition to increased thermostability, other physio-chemical properties of the analogues described herein may also be altered as a result of said substitution. For example, properties such as the analogues’ taste, in addition to the thermostability, may be altered as a result of the protein engineering. Furthermore, properties relating to sweetness perception may also be altered, for example the intensity peak and / or the perception of sweetness over time may be altered.

[0014] The present disclosure provides novel analogues of brazzein having increased thermostability, as well as compositions comprising said analogues, which can be used in food and beverage applications, in particular as a sweetener. Furthermore, provided herein are host cells, expression systems, nucleic acids, methods and a kit of parts for producing the same.

[0015] It is a main aspect of the present disclosure to provide an analogue of a sweet protein, said analogue comprising: i. at least one substitution at one residue corresponding to residue 31 of Q-brazzein, wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant P7131 PC00 thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of Q-brazzein, or ii. at least one substitution to phenylalanine (F) at one residue corresponding to residue 8, one substitution to phenylalanine (F) at one residue corresponding to residue 11, and one substitution to alanine (A) at one residue corresponding to residue 35, of Q-brazzein, wherein said sweet protein is Q-brazzein, pyrE- brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 8, one substitution at one residue corresponding to residue 11, and one substitution at one residue corresponding to residue 35, of Q-brazzein, preferably wherein the Tm of said analogue is increased compared to the Tm of the corresponding sweet protein without said at least one substitution or said at least three substitutions.

[0016] Thus, a main aspect of the present disclosure is to provide an analogue of a sweet protein, said analogue comprising at least one substitution at one residue corresponding to residue 31 of Q-brazzein, wherein said substitution is a substitution to an asparagine (N), and wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of Q-brazzein.

[0017] It is also a main aspect of the present disclosure to provide an analogue of a sweet protein, said analogue comprising at least three substitutions, said at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of Q-brazzein, a second substitution to phenylalanine (F) at a residue corresponding to residue 11 of the same polypeptide, and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, P7131 PC00 wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least three substitutions.

[0018] The analogues are particularly useful in foods, feed and / or beverages, that are heated for example to temperatures between 80 and 120°C during preparation or prior to consumption, such as temperatures between 80 and 110°C, for example between 100 and 115°C, such as between 105 and 112°C.

[0019] It is also an aspect of the disclosure to provide a sweetener or sweetener composition comprising an analogue of a sweet protein, wherein said analogue or sweet protein are as described herein.

[0020] It is an aspect of this disclosure to provide a sweetener composition or a food, feed, or beverage product comprising an analogue of a sweet protein, obtained by a method as described herein.

[0021] It is also an aspect of the disclosure to provide a composition comprising an analogue of a sweet protein, wherein said analogue or sweet protein is as described herein, and wherein said composition is a food composition, a feed composition, or a beverage composition.

[0022] It is also an aspect of the disclosure to provide use of an analogue of a sweet protein as a sweet protein and / or sweetener, wherein said analogue and / or sweet protein are as described herein.

[0023] It is also an aspect of the disclosure to provide use of the sweetener or sweetener composition as described herein, as a food product, food material, food ingredient, feed product, feed material, feed ingredient, beverage material and / or beverage ingredient.

[0024] It is also an aspect of the disclosure to provide use of the composition as described herein, as a food product, food material, food ingredient, feed product, feed material, feed ingredient, beverage material and / or beverage ingredient. P7131 PC00

[0025] Another aspect of this disclosure is a host cell capable of producing an analogue of a sweet protein, said host cell expressing an analogue comprising at least one substitution at one residue corresponding to residue 31 of Q-brazzein, wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of Q-brazzein.

[0026] It is also an aspect of the disclosure to provide a host cell capable of producing an analogue of a sweet protein, said analogue comprising at least three substitutions, said at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of Q-brazzein, a second substitution to phenylalanine (F) at a residue corresponding to residue 11 of the same polypeptide, and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, wherein said sweet protein is Q-brazzein, pyrE- brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least three substitutions.

[0027] It is also an aspect of the present disclosure to provide methods for producing an analogue of a sweet protein, comprising: i. providing a host cell as described herein; ii. incubating and optionally propagating said host cell in a cultivation medium, thereby obtaining a fermentation liquid comprising said analogue, wherein said analogue or sweet protein are as described herein.

[0028] It is another aspect of the present disclosure to provide a nucleic acid encoding an analogue of a sweet protein, said analogue comprising at least one substitution at one residue corresponding to residue 31 of Q-brazzein, wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE- brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having P7131 PC00 at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of Q-brazzein.

[0029] It is also an aspect of the disclosure to provide a nucleic acid encoding an analogue of a sweet protein, said analogue comprising at least three substitutions, said at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of Q-brazzein, a second substitution to phenylalanine (F) at a residue corresponding to residue 11 of the same polypeptide, and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least three substitutions.

[0030] It is also an aspect of the present disclosure to provide an expression system for expression in a host cell, comprising a nucleic acid encoding an analogue of a sweet protein, said analogue comprising at least one substitution at one residue corresponding to residue 31 of Q-brazzein, wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des- pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of Q-brazzein.

[0031] It is also an aspect of the present disclosure to provide an expression system for expression in a host cell, comprising a nucleic acid encoding an analogue of a sweet protein, said analogue comprising at least three substitutions, said at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of Q-brazzein, a second substitution to phenylalanine (F) at a residue corresponding to residue 11 of the same polypeptide, and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, P7131 PC00 or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least three substitutions.

[0032] It is also an aspect of the present disclosure to provide a kit of parts, comprising: i. a host cell; and / or ii. at least one nucleic acid as described elsewhere herein; and / or iii. the expression system as described elsewhere herein; and iv. optionally instructions for use.

[0033] Preferably, the host cell of the above described kit and method is generally-regarded- as-safe (GRAS), has safe-to-consume status, and / or is non-pathogenic.

[0034] Description of Drawings

[0035] Figure 1. Thermostabilities of the sweet protein Q-brazzein and two thermostable analogues (variants). Fitted Tm was calculated with Nanotemper Software for Q- brazzein with H31 N (SEQ ID NO: 3) and Q-brazzein with Y8F / Y11 F / G35A (SEQ ID NO: 19), both being more thermostable than the native protein sequence (SEQ ID NO: 1). 1 mg / mL of purified protein was used in the measurement which was performed in technical duplicates. For simplicity, Q-brazzein is denoted “brazzein” in figure.

[0036] Figure 2. Fold increase of best-performing Q-brazzein variants H31N (SEQ ID NO: 3) and Y8F / Y11 F / G35A (SEQ ID NO: 19), with 0.8% acetonitrile compared to native Q- brazzein. Data points are two technical replicates and bars represent the standard deviation. For simplicity, Q-brazzein is denoted “brazzein” in figure.

[0037] Figure 3. Raw fluorescence profile of native Q-brazzein (SEQ ID NO: 1) with increasing temperature. Fitted Tm is calculated from Ratio First Derivative.

[0038] Figure 4. Raw fluorescence profile of Q-brazzein_H31 N (SEQ ID NO: 3) with increasing temperature. Fitted Tm is calculated from Ratio First Derivative.

[0039] Figure 5. Q-brazzein-receptor interaction modelling. AFsample placed Q-brazzein near the cysteine rich domain (CRD) of the receptor complex T 1 R2 / T 1 R3. Q-brazzein is shown in dark grey with the position of the substitution and various residues (17Q, E36, P7131 PC00

[0040] R33 and E53) that are important for sweetness highlighted. The lower image shows a zoom in of the relevant site.

[0041] Figure 6. Thermostabilities of Q-brazzein variants. Fitted Tm was calculated for brazzein using Nanotemper Software. 1mg / mL of purified protein was used in the measurement which was performed in technical duplicates. Combined* corresponds to a Q-brazzein variant with changes K5R, Y8F, Y11 F, V13T, A19S, H31 N, G35A. Data points are the mean of two technical replicates and bars represent the standard deviation.

[0042] Figure 7. Sweetness rating and ranking of Q-brazzein_H31N and native Q-brazzein. Sweetness perception was evaluated using both ranking and rating methods. Participants (n = 61) ranked four protein variants based on sweetness (1st to 4th) and also rated each variant on a 1-10 scale. The bar plot shows the average of sweetness ratings per subject. Individual data points are overlaid. A gradient from light to dark represents increasing ranking position (decreasing sweetness). Statistical comparisons of sweetness ratings between samples were performed using the Mann-Whitney II test. PC = positive control, NC = negative control, ***p = 8.899 x 10-7, ns, p = 0.2476. The difference between Q-brazzein_H31 N (denoted “H31N”) and native Q-brazzein (denoted “Native”) was not statistically significant (p = 0.2476) indicating that they are equally sweet.

[0043] Figure 8. Thermostability measurement for native Q-brazzein (denoted “Brazzein”), Q- brazzein_H31 R (denoted “H31 R”), Q-brazzein_H31A (denoted “H31A”), and Q- brazzein_H31N (denoted “H31 N”). Measurements were performed in phosphate buffered saline (PBS) measured using Differential Scanning Fluorimetry using the NanoTemper Prometheus Panta using a ramp speed of 1°C / min at different guanidinium hydrochloride concentrations. A) The melting point of native brazzein measured directly indicates a melting temperature of 106.4°C, whereas extrapolation indicates a melting temperature of 105.8°C. B) The melting point of brazzein_H31 N measured using extrapolation indicates a melting temperature (Tm) of 107.9°C, whereas direct measurements without guanidinium hydrochloride exceeded the boundaries of the instrumentation (110°C). C) The melting point of brazzein_H31 R measured directly indicates a melting temperature of 105.2°C, whereas extrapolation indicates a melting temperature of 105.4°C. D) The melting point of brazzein_H31A P7131 PC00 measured directly indicates a melting temperature of 96.0°C whereas, extrapolation indicates a melting temperature of 93.3°C.

[0044] Detailed description

[0045] Definitions

[0046] The term analogue herein refers to a polypeptide and / or protein that differs from a reference polypeptide and / or protein due to one or more substitutions. The terms analogue, sweet protein analogue, analogue of a sweet protein, protein analogue, sweet protein variant, variant of a sweet protein, protein variant, and variant may be used interchangeably herein. The term functional variant is defined herein below.

[0047] The terms protein, polypeptide, and amino acid sequence are used interchangeably herein throughout.

[0048] The term sweet protein herein refers to proteins that are sweet, taste sweet and / or are perceived as sweet upon ingestion. Sweet proteins are capable of interacting with sweetness receptors, such as the T1 R2 / T1R3 receptor, to produce a sensation of sweetness. The sweetness of a sweet protein may be determined by known methods, such as by sensory analysis by a consumer panel.

[0049] The term melting temperature (Tm) herein refers to the temperature at which a protein’s free energy change is equal to zero (AG = 0). Thus, the Tm is the temperature at which equal proportions of a protein population are folded and unfolded, respectively. In other words, it is the temperature at which the proportion of a protein population that is in the native state is equal to the proportion of the protein population that is in the denatured state. The Tm of a protein may be determined by methods known in the art, for example calorimetric methods, or differential scanning fluorimetry methods, such as DSF, nanoDSF, or Circular Dichroism (CD). The melting temperature (T m) may also be referred to as the denaturation midpoint or the denaturation temperature and the terms may be used interchangeably herein.

[0050] The term thermostability herein refers to a protein’s ability to resist irreversible change to its chemical and / or physical properties, such as by denaturation, at a high relative temperature. A protein’s thermostability is determined by its Tm, whereby the higher the Tm of a protein, the more thermostable that protein is. Analogues may be referred P7131 PC00 to as having “increased thermostability” or being “more thermostable” than the sweet proteins from which they are derived; this should be understood to mean that the analogue has a higher Tm than the sweet protein from which it was derived.

[0051] The term sweet is herein used to describe a taste similar to that of sugar, that is to say no salty or bitter. “Sweet” tasting products may cause a smooth and pleasant sensation on the palate, like that produced by honey or sugar. The term sweetness herein refers to the quality of being sweet.

[0052] The term sweeteners refers to substances that, when added to a food or beverage product, cause a sweet taste or sweet flavour to be perceived upon ingestion of said food or beverage product.

[0053] The term functional variant herein refers to a functional variant of a sweet protein, such as brazzein, which retains at least some of organoleptic properties of the parent sweet protein. It follows that a functional variant of a sweet protein is also sweet, although its sweetness intensity and / or sweet perception may be altered. This alteration in sweetness intensity and / or sweetness perception may result from changes in the interactions between the functional variant and the sweet taste receptor relative to the parent sweet protein.

[0054] The term homologue herein refers to a variant of a nucleic acid encoding the same polypeptide. Skilled person knows that different codons may encode the same amino acid, and it therefore follows that two different nucleic acid sequences may encode the same polypeptide. Skilled person knows how to derive one or more distinct DNA sequences from an amino acid sequence. Skilled person also knows how to derive an amino acid sequence from a DNA sequence. Software for performing reverse translations from an amino acid sequence to DNA sequences and translations from DNA sequences to amino acid sequences are available for performing this task.

[0055] The term identity, homology or similarity with respect to a nucleic acid sequence (polynucleotide, DNA, RNA) or polypeptide (protein), are defined herein as the percentage of nucleotides or amino acids, respectively, in the candidate sequence that are identical, homologous or similar, respectively, to the residues of a corresponding native (may be codon-optimised) nucleotide or amino acid sequence, respectively, after P7131 PC00 aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity I similarity, and considering any conservative substitutions according to the NCIIIB rules ([https: / / iubmb.qmul.ac.uk / misc / naseq.html; NC-llIB, Eur J Biochem (1985)]) as part of the sequence identity. In particular, the percentage of similarity refers to the percentage of residues conserved with similar physiochemical properties. Neither 5' or 3' extensions nor insertions (for nucleic acids) or N’ or C’ extensions nor insertions (for polypeptides) result in a reduction of identity, similarity or homology. Methods and computer programs for the alignments are well known in the art. Generally, a given identity between two sequences implies that the similarity between these sequences is at least equal to the identity; for example, if two sequences are 70% identical to one another, they cannot be less than 70% similar to one another - but could be sharing 80% similarity. Thus, throughout the present disclosure, it will be understood that any variant, such as a functional variant, or homologue said to have at least 70% sequence identity, similarity, or homology to a specified sequence (nucleic acid sequence (polynucleotide) or polypeptide) refers to a sequence having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity, similarity, or homology thereto.

[0056] The terms nucleic acid, nucleic acid sequence, nucleic acid construct, nucleic acid molecule oligonucleotide and polynucleotide as well as the plural versions thereof may be used interchangeably herein.

[0057] The term heterologous, when referring to a polypeptide (such as an enzyme) or to a polynucleotide (such as a gene, coding sequence of a gene or genetic element), shall herein be construed to refer to an amino acid sequence (polypeptide) or a nucleic acid sequence (polynucleotide), such as a gene, which is not naturally present in a wild-type cell. P7131 PC00

[0058] The term mutation, when used herein in the context of nucleic acids, refers to a change in nucleic acid compared to the parent nucleic acid. The term mutation covers single nucleotide mutations, but also insertions and deletions of multiple nucleotides, i.e. any change that leads to a different nucleic acid than the parent nucleic acid. The term mutation thus encompasses deletions, such as deletions of a whole gene or of a coding sequence of a gene, or a fragment / fraction of a gene or of a coding sequence of a gene. The term mutation when used herein in the context of polypeptides, such as sweet proteins, refers to a change in amino acid sequence compared to the parent amino acid sequence. The term mutation covers single amino acid mutations, but also insertions and deletions of multiple amino acids, i.e. any change that leads to a different amino acid sequence than the parent amino acid sequence. The term mutation thus encompasses amino acid substitutions, defined below.

[0059] The term substitution, when used herein to in the context of polypeptides, refers a particular class of mutation, wherein a single amino acid is exchanged for a different amino acid at the same position.

[0060] The terms residue corresponding to or corresponding residue herein refers to a residue that aligns to a specified residue in a reference polypeptide. The corresponding residue need not be at the same position in its polypeptide as the specified residue is in the reference polypeptide. The residue in the reference polypeptide and the corresponding residue may or may not be the same amino acid. Methods, algorithms and software for performing alignments of polypeptides are known in the art, for example for performing pairwise sequence alignments or multiple sequence alignments. When referring to a specific amino acid of a polypeptide (protein), such as of a reference polypeptide, the term residue may also be used herein. In other words and as an example, residue 104 of SEQ ID NO: 1, refers to the amino acid at position 104 in SEQ ID NO: 1.

[0061] The term corresponding protein herein refers to the reference protein from which an analogue is derived. Thus a corresponding protein is the protein from which an analogue is derived and / or a variant of the analogue comprising a specified difference. For example “corresponding sweet protein without said at least one substitution” should be read a protein identical to the analogue, with the exception that it does not comprise the specified at least one substitution. The terms reference polypeptide, parent protein, P7131 PC00 in from which it has been derived and corresponding protein without said at least one substitution are interchangeable and may be used interchangeably herein.

[0062] When ranges, such as Tm ranges, are written as “between X and Y”, the range is inclusive of values X and Y. As an example, if a protein is described as having a Tm between 105°C and 110°C, the Tm of said protein may be 105°C, 105.1 °C, 105.2°C, 105.3°C, 105.4°C, 105.5°C, 105.6°C, 105.7°C, 105.8°C, 105.9°C, 106°C, 106.1 °C, 106.2°C, 106.3°C, 106.4°C, 106.5°C, 106.6°C, 106.7°C, 106.8°C, 106.9°C, 107°C, 107.1 °C, 107.2°C, 107.3°C, 107.4°C, 107.5°C, 107.6°C, 107.7°C, 107.8°C, 107.9°C, 108°C, 108.1 °C, 108.2°C, 108.3°C, 108.4°C, 108.5°C, 108.6°C, 108.7°C, 108.8°C, 108.9°C, 109°C, 109.1 °C, 109.2°C, 109.3°C, 109.4°C, 109.5°C, 109.6°C, 109.7°C, 109.8°C, 109.9°C or 110°C.

[0063] Thermostable analogue

[0064] Provided herein are sweet protein analogues that are more thermostable than the sweet protein(s) they are derived from. In other words, the analogues described herein have a higher Tm than the sweet protein(s) they are derived from. The sweet protein from which the analogue is derived may be brazzein or functional variants thereof. The term “brazzein” includes, but is not limited to, all forms of brazzein, including Q- brazzein, pyrE-brazzein and des-pyrE-brazzein.

[0065] Brazzein is a sweet protein found in the West African plant Pentadi plandra brazzeana. It is both the most pH and thermostable of the natural sweet proteins discovered up until today, as well as being substantially sweeter than sucrose on a per weight basis.

[0066] Brazzein variants, Q-brazzein, pyrE-brazzein and des-pyrE-brazzein, vary in their N- terminal amino acid, wherein Q-brazzein has a glutamine residue, pyrE-brazzein has a modified glutamate residue and des-pyrE-brazzin lacks the initial N-terminal amino acid. The modified glutamate residue of pyrE-brazzein is a pyroglutamic acid, which is a post-translational modification of N-terminal glutamic acid, wherein the glutamic acid is cyclized to form a pyroglutamate residue. PyrE-brazzein and des-pyrE-brazzein are natural variants whereas Q-brazzein is engineered. Des-pyrE-brazzein and Q-brazzein are sweeter than pyrE-brazzein.

[0067] Herein disclosed is an analogue of a sweet protein, said analogue comprising: P7131 PC00 i. at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, or ii. at least one substitution to phenylalanine (F) at one residue corresponding to residue 8, one substitution to phenylalanine (F) at one residue corresponding to residue 11, and one substitution to alanine (A) at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1 , wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 8, one substitution at one residue corresponding to residue 11, and one substitution at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1.

[0068] Preferably the Tm of said analogue is increased compared to the Tm of the corresponding sweet protein without said at least one substitution or without said at least three substitutions.

[0069] In some embodiments of the present disclosure, the analogue of a sweet protein comprises at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, wherein said substitution is a substitution to an asparagine (N). Abbreviations for asparagine include “N” or “Asn”, and the terms are used interchangeably herein.

[0070] In some embodiments of the present disclosure, the analogue of a sweet protein comprises at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1, a second substitution to phenylalanine (F) at a residue corresponding to residue 11 of the same polypeptide, and a third substitution to alanine (A) at a P7131 PC00 residue corresponding to residue 35 of the same polypeptide. Abbreviations for phenylalanine include “F” or “Phe”, and the terms are used interchangeably herein. Abbreviations for alanine include “A” or “Ala”, and the terms are used interchangeably herein.

[0071] The sweet protein may be Q-brazzein as set forth in SEQ ID NO:1 , pyrE-brazzein as set forth in SEQ ID NO: 10, des-pyrE-brazzein as set forth in SEQ ID NO: 11 , or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, similarity, or homology to SEQ ID NO: 1 , 10 or 11 , respectively.

[0072] Hence, disclosed herein is an analogue of a sweet protein, comprising at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, des-pyrE-brazzein, or a functional variant thereof having at least 70%, such as at least 71 %, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at P7131 PC00 least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1.

[0073] Also disclosed herein is an analogue of a sweet protein, comprising at least three substitutions, said at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1, a second substitution to phenylalanine (F) at a residue corresponding to residue 11 of the same polypeptide, and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto and comprising said at least three substitutions. P7131 PC00

[0074] In some embodiments the analogue is a Q-brazzein analogue, comprising at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, wherein said substitution is a substitution to an asparagine (N).

[0075] In some embodiments the analogue is a pyrE-brazzein analogue, comprising at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 10, wherein said substitution is a substitution to an asparagine (N).

[0076] In some embodiments the analogue is a des-pyrE-brazzein analogue, comprising at least one substitution at one residue corresponding to residue 30 of the polypeptide set forth in SEQ ID NO: 11, wherein said substitution is a substitution to an asparagine (N).

[0077] In some embodiments said substitution is a substitution from a histidine (H) to an asparagine (N) substitution, such as H31 N. Abbreviations for histidine include “H” or “His”, and the terms are used interchangeably herein.

[0078] In some embodiments the analogue is a Q-brazzein analogue, comprising at least three substitutions, a first substitution at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1, a second substitution at a residue corresponding to residue 11 of the same polypeptide, and a third substitution at a residue corresponding to residue 35 of the same polypeptide, wherein said substitutions are substitutions to phenylalanine (F), phenylalanine (F) and alanine (A), respectively.

[0079] In some embodiments the analogue is a pyrE-brazzein analogue, comprising at least three substitutions, a first substitution at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1, a second substitution at a residue corresponding to residue 11 of the same polypeptide, and a third substitution at a residue corresponding to residue 35 of the same polypeptide, wherein said substitutions are substitutions to phenylalanine (F), phenylalanine (F) and alanine (A), respectively.

[0080] In some embodiments the analogue is a des-pyrE-brazzein analogue, comprising at least three substitutions, a first substitution at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution at a residue P7131 PC00 corresponding to residue 11 of the same polypeptide, and a third substitution at a residue corresponding to residue 35 of the same polypeptide, wherein said substitutions are substitutions to phenylalanine (F) , phenylalanine (F) and alanine (A), respectively.

[0081] In some embodiments, said first and second substitutions comprise or consist of a substitution from tyrosine (Y) to phenylalanine (F), such as Y8F and Y11 F, and the third substitution comprises or consists of a substitution from glycine (G) to alanine (A), such as G35A. Abbreviations for tyrosine include “Y” or “Tyr”, and the terms are used interchangeably herein. Abbreviations for glycine include “G” or “Gly”, and the terms are used interchangeably herein.

[0082] In preferred embodiments the analogue is an analogue of the sweet protein Q-brazzein (SEQ ID NO: 1), and the analogue comprises or consists of Q-brazzein_H31N as set forth in SEQ ID NO: 3 or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 3.

[0083] In other embodiments the analogue is an analogue of the sweet protein pyrE-brazzein (SEQ ID NO: 10), wherein the analogue comprises or consists of pyrE-brazzein_H31 N as set forth in SEQ ID NO: 15 or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least P7131 PC00

[0084] 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 15.

[0085] In further embodiments the analogue is an analogue of the sweet protein des-pyrE- brazzein (SEQ ID NO: 11), wherein the analogue comprises or consists of des-pyrE- brazzein_H30N as set forth in SEQ ID NO: 16 or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 16.

[0086] In some embodiments, the analogue is an analogue of the sweet protein Q-brazzein (SEQ ID NO: 1), and the analogue comprises or consists of Q- brazzein_Y8F / Y11F / G35A as set forth in SEQ ID NO: 19 or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 19.

[0087] In other embodiments, the analogue is an analogue of the sweet protein pyrE-brazzein (SEQ ID NO: 10), wherein the analogue comprises or consists of pyrE- brazzein_Y8F / Y11F / G35A as set forth in SEQ ID NO: 20 or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at P7131 PC00 least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 20.

[0088] In further embodiments, the analogue is an analogue of the sweet protein des-pyrE- brazzein (SEQ ID NO: 11), wherein the analogue comprises or consists of des-pyrE- brazzeinY7F / Y10F / G34A as set forth in SEQ ID NO: 21 or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 21.

[0089] The analogue may comprise or consist of a sequence having at least 70% identity, homology, or similarity to SEQ ID NO: 3, with the proviso that at the most 16 residues are mutated, such as 16 residues are mutated, such as 15 residues are mutated, such as 14 residues are mutated, such as 13 residues are mutated, such as 12 residues are mutated, such as 11 residues are mutated, such as 10 residues are mutated, such as 9 residues are mutated, such as 8 residues are mutated, such as 7 residues are mutated, such as 6 residues are mutated, such as 5 residues are mutated, or less.

[0090] The analogue may comprise or consist of a sequence having at least 70% identity, homology, or similarity to SEQ ID NO: 3, with the proviso that at the most 16 residues are mutated, for example between 16 and 10 residues are mutated, such as between 10 and 5 residues are mutated, such as between 5 and 2 residues are mutated, or less. It will be clear to the skilled person that the analogue will still comprise the mutation at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said mutation is a substitution to an asparagine (N). The analogue P7131 PC00 may however comprise further mutations, with the proviso that at the most 15 additional residues are mutated.

[0091] The analogue may comprise or consist of a sequence having at least 70% identity, homology, or similarity to SEQ ID NO: 19, with the proviso that at the most 16 residues are mutated, such as 16 residues are mutated, such as 15 residues are mutated, such as 14 residues are mutated, such as 13 residues are mutated, such as 12 residues are mutated, such as 11 residues are mutated, such as 10 residues are mutated, such as 9 residues are mutated, such as 8 residues are mutated, such as 7 residues are mutated, such as 6 residues are mutated, such as 5 residues are mutated, or less.

[0092] The analogue may comprise or consist of a sequence having at least 70% identity, homology, or similarity to SEQ ID NO: 19, with the proviso that at the most 16 residues are mutated, for example between 16 and 10 residues are mutated, such as between 10 and 5 residues are mutated, such as between 5 and 2 residues are mutated, or less. It will be clear to the skilled person that the analogue will still comprise the three mutations at residues corresponding to residue 8, 11 , and 35 of the polypeptide set forth in SEQ ID NO: 1, wherein said mutation are substitutions to a phenylalanine (F), phenylalanine (F) and alanine (A), respectively. The analogue may however comprise further mutations, with the proviso that at the most 13 additional residues are mutated.

[0093] Thus described herein are analogues of brazzein, said analogues comprising one or more modifications. Said one or more modifications may comprise or consists of at least one substitution, preferably at a residue corresponding to residue 31 , or at three residues corresponding to residues 8, 11 , and 35 of the polypeptide set forth in SEQ ID NO: 1. Furthermore, said at least one substitution is preferably a substitution from histidine to asparagine, and said at least three substitutions are two substitutions from a tyrosine to phenylalanine and one substitution from glycine to alanine. Optionally, the analogues described herein may have further modifications, such as comprising secretion and / or purification tags, as described elsewhere herein, for example in the section “Tags”.

[0094] Thermostability

[0095] The analogues of sweet proteins disclosed herein, as well as functional variants thereof, have increased thermostability compared to the sweet protein from which they P7131 PC00 are derived. In other words, the analogues are more thermostable than the sweet proteins that they are derived from. A higher temperature is thus required to denature a thermostable analogue than its corresponding sweet protein.

[0096] Without being bound by theory, the experimentally determined thermostability may depend on the technique by which the thermostability is measured and the conditions in which the thermostability is measured.

[0097] The thermostability of a protein is related to the melting temperature (Tm) of said protein. Thus, the thermostable analogues disclosed herein preferably have increased Tm compared to the proteins they are derived from. Preferably, the increased Tm is caused by one or more modifications, such as mutations, in the analogue compared to the sweet protein that it is derived from. Such one or more modifications may be one or more substitutions, preferably any of the substitutions disclosed elsewhere herein, for example in section “Thermostable analogue” herein above. In particular, analogues of brazzein, Q-brazzein, pyrE-brazzein, des-pyrE-brazzein or functional variants thereof, either comprise at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , or comprise three substitutions: a first substitution at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution at a residue corresponding to residue 11 of the same polypeptide, and a third substitution at a residue corresponding to residue 35 of the same polypeptide as described herein above.

[0098] In some embodiments, the analogue of a sweet protein has an increased melting temperature (Tm), such as a Tm equal to 110°C, compared to the corresponding sweet protein without said at least one substitution. Preferably, the analogue is sweet.

[0099] In some embodiments, the analogue of a sweet protein has an increased melting temperature (Tm) compared to the corresponding sweet protein without said at least one substitution, wherein the corresponding sweet protein without said at least one substitution has a Tm of 106°C and the analogue has a Tm greater than 106°C, such as a Tm of 107°C, such as a Tm of 107.1 °C, such as a Tm of 107.2°C, such as a Tm of 107.3°C, such as a Tm of 107.4°C, such as a Tm of 107.5°C, such as a Tm of 107.6°C, such as a Tm of 107.7°C, such as a Tm of 107.8°C, such as a Tm of 107.9°C, such as a Tm of 108°C, such as a Tm of 109°C, such as a Tm of 110°C, such as a Tm of P7131 PC00

[0100] 111 °C, such as a Tm of 112°C, such as a Tm of 113°C, such as a Tm of 114°C, such as a Tm of 115°C.

[0101] In some embodiments the Tm of the analogue is greater than 107°C. In some embodiments, the Tm of the analogue is between 107°C and 110°C. In some embodiments, the Tm of the analogue is between 108°C and 110°C. In some embodiments, the Tm of the analogue is between 109°C and 110°C. In some embodiments, the Tm of the analogue is 108°C. In some embodiments, the Tm of the analogue is 109°C. In some embodiments, the Tm of the analogue is 110°C. In other embodiments the Tm of the analogue is greater than 110°C.

[0102] In some embodiments, the Tm of said analogue is increased by at least 1°C compared to the Tm of the corresponding sweet protein without said at least one substitution, such as by at least 1.5°C, such as by at least 2°C, such as by at least 2.5°C, such as by at least 3°C compared to the Tm of the corresponding sweet protein without said at least one substitution, such as by at least 3.5°C, such as by at least 4°C, such as by at least 4.5°C, such as by at least 5°C, such as by at least 5.5°C, such as by at least 6°C, such as by at least 6.5°C, such as by at least 7°C, such as by at least 7.5°C, such as by at least 8°C, such as by at least 8.5°C, such as by at least 9°C, such as by at least 9.5°C, such as by at least 10°C, such as by at least 10.5°C, such as by at least 11 °C, such as by at least 11.5°C, such as by at least 12°C, such as by at least 12.5°C, such as by at least 13°C, such as by at least 13.5°C, such as by at least 14°C, such as by at least 14.5°C, such as by at least 15°C compared to the Tm of the corresponding sweet protein without said at least one substitution.

[0103] In some embodiments, the Tm of said analogue is between 106°C and 110°C, such as between 107°C and 110°C, such as between 108°C and 110°C, such as between 109°C and 110°C.

[0104] In some embodiments the Tm of said analogue is at least 107°C, such as at least 108°C, such as at least 109°C, or more. In some embodiments the Tm of said analogue is at least 110°C.

[0105] In some embodiments the Tm of said analogue is at least 0.75-fold the Tm of the corresponding sweet protein without said at least one substitution, such as equal to the P7131 PC00

[0106] Tm of the corresponding sweet protein, such as increased 1.01 -fold, such as increased 1.02 fold, such as increased 1.03-fold, such as increased 1.04-fold, such as increased 1.05-fold, such as increased 1.06-fold, such as increased 1.07-fold, such as increased 1.08-fold, such as increased 1.09-fold, such as increased 1.10-fold, such as increased at least 1.25-fold compared to the Tm of the sweet protein from which it was derived, such as increased at least 1.5-fold compared to the Tm of the sweet protein from which it was derived.

[0107] In some embodiments, the Tm of said analogue is increased between 0.75-fold and 1.5-fold compared to the Tm of the corresponding sweet protein without said at least one substitution, such as increased between 1-fold and 1.5-fold, such as increased between 1.25-fold and 1.5-fold, such as increased by 1.5-fold compared to the Tm of the corresponding sweet protein without said at least one substitution.

[0108] In some embodiments, the Tm of said analogue is increased at most 1.5-fold compared to the Tm of the corresponding sweet protein without said at least one substitution.

[0109] In some embodiments the analogue comprises or consists of Q-brazzein_H31N (SEQ ID NO: 3) or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 3, wherein the Tm of said analogue may be between 106°C and 110°C, such as between 107°C and 110°C, such as between 108°C and 110°C, such as 110°C.

[0110] In some embodiments the analogue comprises or consists of pyrE-brazzein_H31 N (SEQ ID NO: 15) or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such P7131 PC00 as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 15, wherein the Tm of said analogue may be between 106°C and 110°C, such as between 107°C and 110°C, such as between 108°C and 110°C, such as 110°C.

[0111] In some embodiments the analogue comprises or consists of des-pyrE-brazzein_H30N (SEQ ID NO: 16) or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 16, wherein the Tm of said analogue may be between 106°C and 110°C, such as between 107°C and 110°C, such as between 108°C and 110°C, such as 110°C.

[0112] In some embodiments, the analogue comprises or consists of Q- brazzein_Y8F / Y11 F / G35A (SEQ ID NO: 19) or a functional variant thereof having at least 70%, such as at least 71 %, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 19, wherein the Tm of said analogue may be P7131 PC00 between 106°C and 110°C, such as between 107°C and 110°C, such as between 108°C and 110°C, such as 110°C.

[0113] In some embodiments, the analogue comprises or consists of pyrE- brazzein_Y8F / Y11 F / G35A (SEQ ID NO: 20) or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 20, wherein the Tm of said analogue may be between 106°C and 110°C, such as between 107°C and 110°C, such as between 108°C and 110°C, such as 110°C.

[0114] In some embodiments, the analogue comprises or consists of des-pyrE- brazzein_Y7F / Y10F / G34A (SEQ ID NO: 21) or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 21, wherein the Tm of said analogue may be between 106°C and 110°C, such as between 107°C and 110°C, such as between 108°C and 110°C, such as 110°C.

[0115] The melting temperature (Tm) may be determined using nanoDSF, preferably wherein said nanoDSF is performed by increasing the temperature from 20°C to 110°C at a rate of 1.5°C per minute, detecting the fluorescence emitted at both 330 nm and 350 nm by tryptophan upon UV excitation at 280 nm using a dual-UV detector, and determining P7131 PC00 the Tm for example by using PR.ThermControl software. Alternatively, said nanoDSF may be performed by increasing the temperature from 20°C to 110°C at a rate of 1°C per minute.

[0116] Skilled person knows that if the protein has a Tm above the detection threshold of the nanoDSF device, nanoDSF may not be suitable for performing direct Tm measurements of a protein, such as an analogue of a sweet protein as disclosed herein. Skilled person knows how to derive the Tm of proteins using indirect measurements. Examples of how this may be achieved are provided in “Example 1; Protein production and thermostability measurement” and “Example 2; Thermostability measurements” herein. As an example, a protein may be contacted with a chaotropic agent, which without being bound by theory, may reduce the Tm of the analogue and allow for a Tm to be estimated using regression-based statistical means. Examples of such chaotropic agents include, but are not limited to acetonitrile and guanidinium chloride.

[0117] Thus in some embodiments, the Tm of said analogue is determined using nanoDSF while said analogue is contacted with acetonitrile. Said acetonitrile may be 0.8% acetonitrile. In other embodiments, the Tm of said analogue is determined using nanoDSF while said analogue is contacted with guanidinium chloride. Said guanidinium chloride may be in the range of 0.5 to 4 M, such as 0.5 M, 1 M, 1.5 M, 2 M, 3 M, or 4 M guanidinium chloride.

[0118] In some embodiments, melting temperature (Tm) is determined using nanoDSF, wherein said nanoDSF is for instance performed by increasing the temperature from 20°C to 110°C at a rate of 1 ,5°C per minute, detecting the fluorescence emitted at both 330 nm and 350 nm by tryptophan upon UV excitation at 280 nm using a dual-UV detector, and determining the Tm, wherein said analogue is contacted with acetonitrile, such as 0.8% acetonitrile. In some embodiments, melting temperature (Tm) is determined using nanoDSF, wherein said nanoDSF for instance is performed by increasing the temperature from 20°C to 110°C at a rate of 1°C per minute, detecting the fluorescence emitted at both 330 nm and 350 nm by tryptophan upon UV excitation at 280 nm using a dual-UV detector, and determining the Tm, wherein said analogue is contacted with acetonitrile, such as 0.8% acetonitrile. P7131 PC00

[0119] In some embodiments, melting temperature (Tm) is determined using nanoDSF, wherein said nanoDSF for instance is performed by increasing the temperature from 20°C to 110°C at a rate of 1 ,5°C per minute, detecting the fluorescence emitted at both 330 nm and 350 nm by tryptophan upon UV excitation at 280 nm using a dual-UV detector, and determining the Tm, wherein said analogue is contacted with guanidinium chloride, such as 0.5 to 4 M guanidinium chloride, for example 0.5 M, 1 M, 1.5 M, 2 M, 3 M, or 4 M guanidinium chloride. In some embodiments, melting temperature (Tm) is determined using nanoDSF, wherein said nanoDSF for instance is performed by increasing the temperature from 20°C to 110°C at a rate of 1°C per minute, detecting the fluorescence emitted at both 330 nm and 350 nm by tryptophan upon UV excitation at 280 nm using a dual-UV detector, and determining the Tm, wherein said analogue is contacted with guanidinium chloride, such as 0.5 to 4 M guanidinium chloride, for example 0.5 M, 1 M, 1.5 M, 2 M, 3 M, or 4 M guanidinium chloride.

[0120] The Tm may also be determined using other differential scanning fluorimetry methods, such as DSF, nanoDSF or circular dichroism (CD). The Tm may also be determined by calorimetric methods, such as differential scanning calorimetry (DSC).

[0121] In other words, described herein are analogues of sweet proteins, said analogues having increased melting temperatures, and therefore increased thermostability, relative to the sweet proteins from which said analogues are derived.

[0122] Without being bound by theory, sweet proteins and their analogues remain sweet until they are denatured, for example by high temperatures; thus the increased thermostability of the analogues described herein allows said analogues to retain their sweetness at higher temperatures than the sweet proteins from which they are derived.

[0123] Sweetness

[0124] The analogues of a sweet protein disclosed herein, as well as functional variants thereof, are sweet.

[0125] Relative sweetness can be determined using a sucrose equivalence test, wherein test participants qualitatively compare the sweetness of test substances to standardised sucrose solutions. Sweetness can then be plotted on a labelled magnitude scale and P7131 PC00 relative sweetness determined. Alternatively, a threshold test may be used to determine the sweetness, as performed by Poirier at el. (2012).

[0126] In some embodiments the analogue is at least 25% as sweet as the corresponding sweet protein without said at least one substitution, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, or such as at least 95%, such as at least 100%, such as at least 105%, such as at least 110%, such as at least 115%, such as at least 120%, such as at least 125%, such as at least 130%, such as at least 135%, such as at least 140%, such as at least 145%, such as at least 150%, such as at least 155%, such as at least 160%, such as at least 165%, such as at least 170%, such as at least 175%, such as at least 180%, such as at least 185%, such as at least 190%, such as at least 195%, such as at least 200%, such as at least 205%, such as at least 210%, such as at least 215%, such as at least 220%, such as at least 225%, such as at least 230%, such as at least 235%, such as at least 240%, such as at least 245%, such as at least 250%, such as at least 255%, such as at least 260%, such as at least 265%, such as at least 270%, such as at least 275%, such as at least 280%, such as at least 285%, such as at least 290%, such as at least 295%, such as at least 300%, such as at least 400%, such as at least 500%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900% or such as at least 1000% as sweet as the corresponding sweet protein without said at least one substitution.

[0127] In some embodiments the analogue is at least 0.2 times as sweet as the corresponding sweet protein without said at least one substitution, such as at least 0.3 times, such as at least 0.4 times, such as at least 0.5 times, such as at least 0.6 times, such as at least 0.7 times, such as at least 0.8 times, such as at least 0.9 times, such as at least 1.0 time, such as at least 1.25 times, such as at least 1.5 times, such as at least 1.75 times, such as at least 2.0 times, such as at least 2.5 times, such as at least 3.0 times, such as at least 3.5 times, such as at least 4.0 times, such as at least 5.0 times, such as at least 7.5 times, such as at least 10 times, such as at least 15 times, such as at least 20 times, such as at least 25 times, such as at least 30 times, such as at least 40 times, such as at least 50 times, such as at least 75 times, such as at least 100 times as sweet as the corresponding sweet protein without said at least one substitution. P7131 PC00

[0128] In some embodiments, the sweetness of the analogue is similar or identical to the sweetness of the corresponding sweet protein without said at least one substitution.

[0129] In some embodiments, the analogue is sweet at any temperature smaller than or equal to its Tm. In some embodiments, the analogue is sweet at any temperature lower than or equal to its Tm.

[0130] In some embodiments, the analogue maintains its sweetness at any temperature smaller than or equal to its Tm. In some embodiments, the analogue maintains its sweetness at any temperature lower than or equal to its Tm.

[0131] The sweetness of analogues is preferably determined using analogues that do not comprise tags, such as purification tags, or wherein said tags have been cleaved prior to determining the sweetness of the analogues.

[0132] Without being bound by theory, the host cell by which the sweet protein analogues are produced does not affect the sweetness nor the thermostability of said analogues.

[0133] Host cell

[0134] The host cells disclosed herein produce, or are capable of producing, the analogues of sweet proteins disclosed elsewhere herein, for example in section “Thermostable analogue” herein above.

[0135] The host cells may be referred to as production organisms, microbial factories, microbial production organisms, hosts, host cells, host organisms, production hosts, cell factories, and the like.

[0136] Various species may be useful according to the present disclosure. In some embodiments the host may be a yeast, such as a Saccharomyces, Komagataella or Yarrowia, such as Y. lipolytica. In other embodiments the host cell may be a filamentous fungi, for example an Aspergillus, such as A. niger or A. oryzae. In preferred embodiments, the host cell is S. cerevisiae or K. phaffii. P7131 PC00

[0137] Preferably, the host cell is generally-regarded-as-safe (GRAS), has safe-to-consume status, and / or is non-pathogenic.

[0138] Provided herein is a host cell capable of producing an analogue of a sweet protein, said analogue comprising: i. at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, or ii. at least one substitution to phenylalanine (F) at one residue corresponding to residue 8, one substitution to phenylalanine (F) at one residue corresponding to residue 11, and one substitution to alanine (A) at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1 , wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 8, one substitution at one residue corresponding to residue 11, and one substitution at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1.

[0139] Preferably the Tm of said analogue is increased compared to the Tm of the corresponding sweet protein without said at least one substitution or said at least three substitutions.

[0140] In some embodiments the host cell is capable of producing an analogue of a sweet protein, said host cell expressing an analogue comprising at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at P7131 PC00 least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1. In some embodiments, the host cell is a S. cerevisiae cell. In other embodiments, the host cell is a K. phaffii cell.

[0141] Preferably the substitution in the brazzein, Q-brazzein, pyrE-brazzein, or des-pyrE- brazzein analogue produced by the host cell is a substitution from a histidine (H) to an asparagine (N), such as H31N.

[0142] In preferred embodiments, the analogue produced by the host cell is derived from a sweet protein, wherein the sweet protein is Q-brazzein as set forth in SEQ ID NO: 1, pyrE-brazzein as set forth in SEQ ID NO: 10, or des-pyrE-brazzein as set forth in SEQ ID NO: 11 , or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least P7131 PC00

[0143] 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto.

[0144] In some embodiments, the host cell is capable of producing an analogue of a sweet protein, said host cell expressing an analogue comprising at least three substitutions, said at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1, a second substitution to phenylalanine (F) at a residue corresponding to residue 11 of the same polypeptide, and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto and comprising at least three substitutions. In some embodiments, the host cell is a S. cerevisiae cell. In other embodiments, the host cell is a K. phaffii cell.

[0145] Preferably, the substitution in the brazzein, Q-brazzein, pyrE-brazzein, or des-pyrE- brazzein analogue produced by the host cell is either one substitution from a histidine P7131 PC00

[0146] (H) to an asparagine (N), such as H31 N, or are two substitutions from tyrosine (Y) to phenylalanine (F) and one substititution from glycine (G) to alanine (A).

[0147] In some embodiments, the analogue produced by the host cell has an increased melting temperature (Tm) compared to the corresponding sweet protein without said at least one substitution, preferably wherein said analogue is sweet.

[0148] The host cell may comprise the expression system as described herein below, whereby said host cell produces and / or is capable of producing said analogue of a sweet protein.

[0149] Additional modifications in the host cell may aid the production of, or result in higher titres of said analogues of sweet proteins. Such modifications may include mutations in genes encoding proteases; deletions of genes encoding proteases; insertions in genes encoding proteases, and the like, said modifications resulting in reduced or no activity of the proteases encoded by said genes. In some embodiments the host cell has full deletions of one or more of the genes hda2, vps5, tda3, gos1 , and / or genes with at least 70% homology, identity, or similarity thereof. In some embodiments the host cell has partial deletions of one or more of HDA2, VPS5, TDA3, GOS1, and / or nucleic acid sequences with at least 70% homology, identity, or similarity thereto. In some embodiments the host cell has inactivating mutations in one or more of the following; HDA2, VPS5, TDA3, GOS, and / or nucleic acid sequences with at least 70% homology, identity, or similarity thereto.

[0150] Additional host cell modifications that may aid the production of, or result in higher titres of said analogues of sweet proteins may also be modifications that result in the overexpression of host genes or coding sequences, wherein the expression level of the overexpressed gene or coding sequence is greater in a host cell where it has been overexpressed, relative to the expression level of the same gene or coding sequence in an unmodified host cell. A person skilled in the art knows how to overexpress a gene or a coding sequence in a host cell. For example, host cell overexpression of COG5 may aid the production of, or result in higher titres of said analogues of sweet proteins. Host cell overexpression of PDI1 may aid the production of, or result in higher titres of said analogues of sweet proteins. Without being bound by theory, overexpression of disulphide isomerases may aid in the production of, or result in higher titres of sweet proteins and / or analogues thereof. P7131 PC00

[0151] Thus disclosed herein are host cells, which produce or are capable of producing analogues of sweet proteins, wherein said analogues comprise at least one substitution, preferably as described herein elsewhere, for example in the section “Thermostable analogue”, and said analogues are more thermostable than the sweet proteins from which they are derived. In preferred embodiments said analogue is an analogue of brazzein, Q-brazzein, pyrE-brazzein des-pyrE-brazzein, or functional variants thereof, comprising at least one substitution from a histidine (H) to an asparagine (N), such as H31N and the host cell is S. cerevisiae or K. phaffii. In other embodiments said analogue is an analogue of brazzein, Q-brazzein, pyrE-brazzein des-pyrE-brazzein, or functional variants thereof, comprising at least three substitutions, wherein two substitutions are from tyrosine (Y) to phenylalanine (F) and one substitution is from glycine (G) to alanine (A), such as Y7F, Y10F and G34A, and the host cell is S. cerevisiae or K. phaffii.

[0152] Methods

[0153] The present disclosure relates to methods for producing analogues of sweet proteins using host cells, preferably engineered cells, such as engineered yeast cells. Further disclosed are methods for producing a sweetener composition and / or a food, feed, or beverage comprising said analogues of sweet proteins.

[0154] Useful host cells for the methods described in this section are described elsewhere herein, particularly in the section “Host cell”. Preferably the host cell is a Saccharomyces, such as S. cerevisiae, or Komagataella, such as K. phaffii.

[0155] The analogue may be a brazzein analogue, i.e. an analogue of brazzein, Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein or of functional variants thereof, as described herein above, and in particular comprising either at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, or at least three substitutions, a first substitution at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution at a residue corresponding to residue 11 , and a third substitution at a residue corresponding to residue 35 of the same polypeptide.

[0156] Disclosed herein is a method of producing an analogue of a sweet protein, comprising: P7131 PC00 i. providing a host cell, preferably as described elsewhere herein; ii. incubating and optionally propagating said host cell in a cultivation medium, thereby obtaining a fermentation liquid comprising said analogue, wherein said analogue or sweet protein are preferably as described elsewhere herein.

[0157] The method described above may further comprise a step of recovering the analogue. The method may also comprise cleavage of any tag covalently linked to the analogue, such as a purification tag or secretion tag, as described herein above.

[0158] The step of recovering the analogue may comprise filtering, such as by ultrafiltration, and / or concentrating the analogue, for example by ion-exchange chromatography, from the fermentation liquid.

[0159] In other words, disclosed herein is a method for producing an analogue of a sweet protein, wherein said analogue is produced by a host cell upon incubation in a cultivation medium.

[0160] Disclosed are methods of producing a sweetener composition and / or a food, feed, or beverage product comprising an analogue of a sweet protein, comprising: i. providing a host cell, as described elsewhere herein; ii. incubating and optionally propagating said host cell in a cultivation medium, thereby obtaining a fermentation liquid; iii. recovering said host cell, fermentation liquid comprising said host cell and / or said analogue; iv. optionally recovering said analogue from said fermentation liquid and / or host cell; and v. converting said analogue, and / or fermentation liquid comprising said host cell and / or analogue into a sweetener composition and / or into a food, feed or beverage product, whereby said sweetener composition, and / or said food, feed, or beverage product is produced, and wherein said analogue or sweet protein are as described elsewhere herein, for example in the section “Thermostable analogue”.

[0161] In some embodiments the analogue of the method described above maintains its sweetness upon heating until at least its Tm. P7131 PC00

[0162] In some embodiments the analogue of the method described above remains sweet upon heating until at least its Tm.

[0163] In some embodiments the analogue of the method described above maintains its sweetness at any temperature until at least its Tm.

[0164] In some embodiments the analogue of the method described above remains sweet at any temperature until at least its Tm.

[0165] In other words, described above is a method for producing a sweetener composition, food, feed, and / or beverage product, wherein said sweetener composition, food, feed, and / or beverage product comprises an analogue of a sweet protein produced by a host cell, preferably wherein said analogue is as described in the section “Thermostable analogue” and the host cell is as described in the section “Host cell”.

[0166] The analogues of sweet proteins in the methods described above comprise modifications, preferably at least one substitution as described elsewhere herein, for example in the section “Thermostable analogue”.

[0167] Sweetener and compositions

[0168] The present disclosure relates to sweeteners, sweetener compositions, foods, feeds and beverages that comprise or consist of analogues of sweet proteins. Sweet proteins and their analogues may function or be used as sweeteners in comestible products and beverage products, whereby they endow said products with sweetness and / or a sweet taste, as described elsewhere herein.

[0169] Without being bound by theory, sweeteners and sweetener compositions comprising or consisting of the thermostable analogues of the present disclosure may be particularly suitable for sweetening hot beverages, as well as baked and par-baked products given the analogues’ improved thermostability, relative to the sweet proteins from which they are derived. Said improved thermostability allows the analogues to maintain or retain their ability to induce a sensation of sweetness, even when said analogues have been heated above the Tm of the sweet protein from which they were derived, until at least their own Tm. P7131 PC00

[0170] In some embodiments, the present disclosure relates to a sweetener, sweetener composition, food, feed, or beverage product comprising or consisting of an analogue of a sweet protein, obtained by a method disclosed herein above, for example in the section “Methods”.

[0171] The sweetener or sweetener composition may comprise or consist of an analogue of a sweet protein, wherein the analogue of a sweet protein is as described elsewhere above, for example in the section “Thermostable analogue”. In particular, the analogue may be a brazzein analogue, in particular an analogue of Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or of a functional variant thereof, comprising at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , as described herein above in detail.

[0172] In some embodiments, the present disclosure relates to a composition comprising an analogue of a sweet protein, wherein said analogue or sweet protein are as described herein, and wherein said composition is a food composition, a feed composition, or a beverage composition.

[0173] The composition may comprise an analogue of a sweet protein, wherein the analogue of a sweet protein is as described elsewhere above, for example in the section “Thermostable analogue”. In particular, the analogue may be a brazzein analogue, in particular an analogue of Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or of a functional variant thereof, either comprising at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , or comprising at least three substitutions: a first substitution at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution at a residue corresponding to residue 11 of the same polypeptide, and a third substitution at a residue corresponding to residue 35 of the same polypeptide, as described herein above in detail.

[0174] Said composition may be a dairy product or a drink, such as an alcoholic drink or a soft drink. The term dairy product includes, but is not limited to, products such as milk, condensed milk, flavoured milk, yogurt, kefir, ice cream, cream and custard. The term soft drink may refer to sodas, fizzy drinks and other non-alcoholic beverages. The term P7131 PC00 alcoholic drink may refer to beer, alcoholic beverages such as ready-to-consume cocktails and the like.

[0175] In some embodiments, said composition is a food or beverage product that undergoes heating during its preparation, such as a baked or par-baked product.

[0176] Preferably, the food or beverage product that undergoes heating during its preparation, such as a baked or par-baked product, has after final heating at least 25% of the sweetness as a control product prepared under similar conditions with the corresponding sweet protein without said at least one substitution, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 100%, such as at least 105%, such as at least 110%, such as at least 115%, such as at least 120%, such as at least 125%, such as at least 130%, such as at least 135%, such as at least 140%, such as at least 145%, such as at least 150%, such as at least 175%, such as at least 200%, such as at least 225%, such as at least 250%, such as at least 275%, such as at least 300%, such as at least 400%, such as at least 500%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900%, or such as at least 1000% of the sweetness as a control product prepared with the corresponding sweet protein without said at least one substitution.

[0177] In preferred embodiments, the composition is as described above, wherein the analogue comprises or consists of Q-brazzein_H31 N (SEQ ID NO: 3) or a functional variant thereof having at least 70%, such as at least 71 %, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 3 and comprising said at least one substitution at one residue corresponding to residue 31 of the P7131 PC00 polypeptide set forth in SEQ ID NO: 1 , wherein the food or beverage product has after final heating at least 25% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 1 (Q-brazzein), such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 100%, such as at least 105%, such as at least 110%, such as at least 115%, such as at least 120%, such as at least 125%, such as at least 130%, such as at least 135%, such as at least 140%, such as at least 145%, such as at least 150%, such as at least 175%, such as at least 200%, such as at least 225%, such as at least 250%, such as at least 275%, such as at least 300%, such as at least 400%, such as at least 500%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900%, or such as at least 1000% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 1 (Q-brazzein).

[0178] In other embodiments, the composition is as described above, wherein the analogue comprises or consists of pyrE-brazzein_H31N (SEQ ID NO: 15) or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 15 comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, wherein the food or beverage product has after final heating at least 25% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 10 (pyrE-brazzein), such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 100%, such as at least 105%, such as at least 110%, P7131 PC00 such as at least 115%, such as at least 120%, such as at least 125%, such as at least 130%, such as at least 135%, such as at least 140%, such as at least 145%, such as at least 150%, such as at least 175%, such as at least 200%, such as at least 225%, such as at least 250%, such as at least 275%, such as at least 300%, such as at least 400%, such as at least 500%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900%, or such as at least 1000% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 10 (pyrE-brazzein).

[0179] In other embodiments, the composition is as described above, wherein the analogue comprises or consists of des-pyrE-brazzein_H30N (SEQ ID NO: 16) or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 16 comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, wherein the food or beverage product has after final heating at least 25% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 11 (des-pyrE-brazzein), such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 100%, such as at least 105%, such as at least 110%, such as at least 115%, such as at least 120%, such as at least 125%, such as at least 130%, such as at least 135%, such as at least 140%, such as at least 145%, such as at least 150%, such as at least 175%, such as at least 200%, such as at least 225%, such as at least 250%, such as at least 275%, such as at least 300%, such as at least 400%, such as at least 500%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900%, or such as at least P7131 PC00

[0180] 1000% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 11 (des-pyrE-brazzein).

[0181] In preferred embodiments, the composition is as described above, wherein the analogue comprises or consists of Q-brazzein_Y8F / Y11F / G35A (SEQ ID NO: 19) or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 19 and comprising at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1, a second substitution to phenylalanine (F) at a residue corresponding to residue 11 of the same polypeptide, and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, wherein the food or beverage product has after final heating at least 25% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 1 (Q- brazzein), such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 100%, such as at least 105%, such as at least 110%, such as at least 115%, such as at least 120%, such as at least 125%, such as at least 130%, such as at least 135%, such as at least 140%, such as at least 145%, such as at least 150%, such as at least 175%, such as at least 200%, such as at least 225%, such as at least 250%, such as at least 275%, such as at least 300%, such as at least 400%, such as at least 500%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900%, or such as at least 1000% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 1 (Q-brazzein). P7131 PC00

[0182] In other embodiments, the composition is as described above, wherein the analogue comprises or consists of pyrE-brazzein_Y8F / Y11 F / G35A (SEQ ID NO: 20) or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 20 comprising at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1, a second substitution to phenylalanine (F) at a residue corresponding to residue 11 of the same polypeptide, and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, wherein the food or beverage product has after final heating at least 25% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 10 (pyrE- brazzein), such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 100%, such as at least 105%, such as at least 110%, such as at least 115%, such as at least 120%, such as at least 125%, such as at least 130%, such as at least 135%, such as at least 140%, such as at least 145%, such as at least 150%, such as at least 175%, such as at least 200%, such as at least 225%, such as at least 250%, such as at least 275%, such as at least 300%, such as at least 400%, such as at least 500%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900%, or such as at least 1000% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 10 (pyrE-brazzein).

[0183] In other embodiments, the composition is as described above, wherein the analogue comprises or consists of des-pyrE-brazzein_Y7F / Y10F / G34A (SEQ ID NO: 21) or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at P7131 PC00 least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 21 comprising at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1, a second substitution to phenylalanine (F) at a residue corresponding to residue 11 of the same polypeptide, and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, wherein the food or beverage product has after final heating at least 25% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 11 (des- pyrE-brazzein), such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 100%, such as at least 105%, such as at least 110%, such as at least 115%, such as at least 120%, such as at least 125%, such as at least 130%, such as at least 135%, such as at least 140%, such as at least 145%, such as at least 150%, such as at least 175%, such as at least 200%, such as at least 225%, such as at least 250%, such as at least 275%, such as at least 300%, such as at least 400%, such as at least 500%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900%, or such as at least 1000% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 11 (des-pyrE- brazzein).

[0184] Uses

[0185] Provided herein is the use of the analogues of sweet proteins described herein as sweeteners. Sweeteners are substances that, when added to a food, feed or beverage product, causes a sweet taste or flavour to be perceived upon ingestion of said food, feed or beverage product. P7131 PC00

[0186] Disclosed herein is the use of an analogue of a sweet protein as a sweet protein and / or as a sweetener, wherein said analogue and / or sweet protein are as described elsewhere herein, for example in the section “Thermostable analogue”. In particular, the analogue may be a brazzein analogue, in particular an analogue of Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or of a functional variant thereof, either comprising at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, or comprising at least three substitutions: a first substitution at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1, a second substitution at a residue corresponding to residue 11 of the same polypeptide, and a third substitution at a residue corresponding to residue 35 of the same polypeptide, as described herein above in detail.

[0187] Also disclosed herein is the use of the sweetener or sweetener composition as described above, as a food product, food material, food ingredient, feed product, feed material, feed ingredient, beverage material and / or beverage ingredient.

[0188] Also disclosed herein is the use of the composition as described in the section “Sweetener and compositions”, as a food product, food material, food ingredient, feed product, feed material, feed ingredient, beverage material and / or beverage ingredient.

[0189] In other words, disclosed herein is the use of analogues of sweet proteins as sweeteners, as well as components of compositions, wherein those compositions are related to or used as food and beverage products, materials and ingredients thereof. In particular, disclosed herein is the use of analogues of sweet proteins to impart sweetness to food and beverage compositions through its use as a sweetener, food product, food material, food ingredient, food additive, beverage product, beverage material, beverage ingredient and / or beverage additive.

[0190] 3D structure

[0191] In the field of bioinformatics, the "template modeling score" (TM-score) is a recognized metric used to assess the similarity between two protein structures. The TM-score quantitatively measures this similarity on a scale from 0 to 1. Typically, scores below 0.20 indicate randomly chosen, unrelated proteins, whereas scores above 0.5 suggest that the structures share roughly the same fold. A detailed methodology for calculating the TM-score is provided in the publication Zhang and Skolnick (2004). P7131 PC00

[0192] The TM-score can be calculated, for example, by uploading two three-dimensional structures in PDB format to the online resource available at https: / / zhanggroup.org / TM- score / . In situations where a pair of three-dimensional structures for comparison is not available, established methods for predicting the three-dimensional structure of a polypeptide are well known to those skilled in the art. For instance, a neural network trained for this specific task, such as AlphaFold, can be employed.

[0193] 3D structures of proteins and / or domains thereof are available through different sources. Thus, 3D structures to be used with the present disclosure may be available through various databases or can be predicted structures. AlphaFold3, referred to herein as AlphaFold, represents a state-of-the-art artificial intelligence (Al) system developed by DeepMind for predicting the three-dimensional (3D) structures of proteins from their amino acid sequences. This system is detailed in the publication Jumper et al. (2021).

[0194] AlphaFold DB is an online database which hosts over 200 million entries, encompassing the human proteome as well as the proteomes of 47 other key organisms relevant to research and global health. These entries are freely accessible at https: / / alphafold.ebi.ac.uk / . For example, the (predicted) structure of native Q- brazzein can be retrieved by inputting identifiers such as the UniProt accession number P56552.

[0195] Structures of polypeptides / proteins not included in the AlphaFold DB may, for example be predicted using the source code available at https: / / github.com / google- deepmind / alphafold, and / or a Colab notebook accessible at https: / / colab.research.google.com / github / deepmind / alphafold / blob / main / notebooks / Alp haFold.ipynb. For example, new variants, such as analogues, of known proteins are typically not included in the AlphaFold DB. To generate a 3D structure using the Colab notebook, the amino acid sequence of an analogue, such as that of Q-brazzein_H31 N (SEQ ID NO: 3), can be inserted.

[0196] In some embodiments, the analogue or functional variant has a template modelling score (TM)-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least P7131 PC00

[0197] 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of an analogue as set forth in SEQ ID NO: 3.

[0198] In some embodiments, the analogue or functional variant has a template modelling score (TM)-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of an analogue as set forth in SEQ ID NO: 15.

[0199] In some embodiments, the analogue or functional variant has a template modelling score (TM)-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of an analogue as set forth in SEQ ID NO: 16.

[0200] In some embodiments, the analogue or functional variant has a template modelling score (TM)-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of an analogue as set forth in SEQ ID NO: 19.

[0201] In some embodiments, the analogue or functional variant has a template modelling score (TM)-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of an analogue as set forth in SEQ ID NO: 20.

[0202] In some embodiments, the analogue or functional variant has a template modelling score (TM)-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least P7131 PC00

[0203] 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of an analogue as set forth in SEQ ID NO: 21 .

[0204] Tags

[0205] Provided herein are both host cells and methods for the production of the thermostable sweet protein analogues disclosed herein. Purifications tags may aid in the recovery of a protein from a host cell culture, whether the protein is secreted or maintained inside the host cell after being translated.

[0206] In particular, the analogue: i. comprises at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or is a functional variant thereof having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , or ii. comprises at least one substitution to phenylalanine (F) at one residue corresponding to residue 8, one substitution to phenylalanine (F) at one residue corresponding to residue 11 , and one substitution to alanine (A) at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1 , wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or is a functional variant thereof having at least 70% identity, homology or similarity thereto, or is a functional variant thereof having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 8, one substitution at one residue corresponding to residue 11 of the same polypeptide, and one substitution at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1.

[0207] Hence, in some embodiments said analogue of a sweet protein, preferably brazzein, is linked, such as covalently linked, to a purification tag. In some embodiments, said purification tag comprises or consists of TEV-his tag as set forth in SEQ ID NO: 13 or a functional variant thereof having at least 70%, such as at least 71%, such as at least P7131 PC00

[0208] 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 13.

[0209] Secretion tags may promote the transfer of a protein out of the host cell, which may in turn increase the recovery of the protein, in a method where the cell is not lysed prior to the step of protein recovery. In some embodiments, said analogue is linked, such as covalently linked, to a secretion tag, optionally wherein said secretion tag comprises or consists of Alpha factor leader as set forth in SEQ ID NO: 14 or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 14.

[0210] It may be desirable to cleave any tag covalently linked to the present analogues, in particular if said analogues are to be used in the preparation of foods or beverages as described herein below. The skilled person will have no difficulty in designing such cleavable purification tags or cleavable secretion tags.

[0211] Nucleic acid

[0212] Provided herein are nucleic acids useful for obtaining an analogue of a sweet protein as described herein. The nucleic acids disclosed herein may comprise several nucleic acid sequences as described in the sequence listing. Said nucleic acids may be useful for expression in, engineering and / or modification of a host cell. The nucleic acids disclosed herein may be introduced into said host cell by methods known in the art. P7131 PC00

[0213] The terms nucleic acids, nucleic acid sequences, nucleic acid constructs, nucleic acid molecules, oligonucleotides, and polynucleotides may be used interchangeably herein.

[0214] The present disclosure provides a nucleic acid encoding an analogue of a sweet protein, said analogue comprising: i. at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or said analogue being a functional variant of the above having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , or ii. at least one substitution to phenylalanine (F) at one residue corresponding to residue 8, one substitution to phenylalanine (F) at one residue corresponding to residue 11 , and one substitution to alanine (A) at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1 , wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant of the above having at least 70% identity, homology or similarity thereto, or said analogue being a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 8, one substitution at one residue corresponding to residue 11 , and one substitution at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1.

[0215] Preferably the Tm of said analogue is increased compared to the Tm of the corresponding sweet protein without said at least one substitution or said at least three substitutions.

[0216] In some embodiments, the present disclosure provides a nucleic acid encoding an analogue of a sweet protein, said analogue comprising at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said substitution is a substitution to an asparagine (N), wherein said sweet P7131 PC00 protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1.

[0217] In some embodiments, the substitution is a substitution from a histidine (H) to an asparagine (N) substitution, such as H31 N.

[0218] In some embodiments, the sweet protein is Q-brazzein as set forth in SEQ ID NO: 1, pyrE-brazzein as set forth in SEQ ID NO: 10, or des-pyrE-brazzein as set forth in SEQ ID NO: 11 , or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least P7131 PC00

[0219] 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto.

[0220] In some embodiments, the present disclosure provides a nucleic acid encoding an analogue of a sweet protein, said analogue comprising at least three substitutions, said at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1, a second substitution to phenylalanine (F) at a residue corresponding to residue 11 of the same polypeptide, and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto and comprising said at least three substitutions.

[0221] In some embodiments, said first and second substitutions comprise or consist of a substitution from tyrosine (Y) to phenylalanine (F), such as Y8F and Y11 F, and wherein said third substitution comprises or consists of a substitution from glycine (G) to alanine (A), such as G35A. P7131 PC00

[0222] In some embodiments, the sweet protein is Q-brazzein as set forth in SEQ ID NO: 1 , pyrE-brazzein as set forth in SEQ ID NO: 10, or des-pyrE-brazzein as set forth in SEQ ID NO: 11 , or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto.

[0223] In some embodiments, said analogue has an increased melting temperature (Tm) compared to the corresponding sweet protein without said at least one substitution, preferably wherein said analogue is sweet.

[0224] Said analogue, sweet protein, or functional variants thereof may be as described elsewhere herein, for example as in the section “Thermostable analogue”. In particular, analogues of brazzein, Q-brazzein, pyrE-brazzein, des-pyrE-brazzein or functional variants thereof, either comprise at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 or comprise at least three substitutions, a first substitution at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution at a residue corresponding to residue 11 of the same polypeptide, and a third substitution at a residue corresponding to residue 35 of the same polypeptide, as described herein above.

[0225] In some embodiments, Q-brazzein as set forth in SEQ ID NO: 1 is encoded by a nucleic acid comprising or consisting of SEQ ID NO: 2 (Q-brazzein), or a homologue thereof having at least 70%, such as at least 71 %, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least P7131 PC00

[0226] 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto.

[0227] In other embodiments, said analogue comprises or consists of Q-brazzein_H31 N as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto.

[0228] In further embodiments, the nucleic acid comprises or consists of Q-brazzein_H31N as set forth in SEQ ID NO: 4, or a homologue thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity or homology to SEQ ID NO: 4.

[0229] In some embodiments, said analogue comprises or consists of pyrE-brazzein_H31 N as set forth in SEQ ID NO: 15, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at P7131 PC00 least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto.

[0230] In some embodiments, the nucleic acid comprises or consists of pyrE-brazzein_H31 N as set forth in SEQ ID NO: 17, or a homologue thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity or homology to SEQ ID NO: 17.

[0231] In some embodiments, said analogue comprises or consists of des-pyrE- brazzein_H30N as set forth in SEQ ID NO: 16, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto.

[0232] In some embodiments, the nucleic acid comprises or consists of des-pyrE- brazzein_H30N as set forth in SEQ ID NO: 18, or a homologue thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such P7131 PC00 as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity or homology to SEQ ID NO: 18.

[0233] In other embodiments, the nucleic acid encodes an analogue comprising or consisting of Q-brazzein_Y8F / Y11 F / G35A as set forth in SEQ ID NO: 19, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto.

[0234] In some embodiments, the nucleic acid encodes an analogue comprising or consisting of pyrE-brazzein_Y8F / Y11 F / G35A as set forth in SEQ ID NO: 20, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto.

[0235] In some embodiments, the nucleic acid encodes an analogue comprising or consisting of des-pyrE-brazzein_Y8F / Y11 F / G35A as set forth in SEQ ID NO: 21 , or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, P7131 PC00 such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto.

[0236] In some embodiments, the nucleic acid comprises or consists of a nucleic acid encoding Q-brazzein_Y8F / Y11 F / G35A (SEQ ID NO: 19) or a functional variant thereof having at least 70% identity, homology or similarity thereto. In some embodiments, the nucleic acid comprises or consists of a nucleic acid encoding pyrE- brazzein_Y8F / Y11 F / G35A as set forth in SEQ ID NO: 20 or a functional variant thereof having at least 70% identity, homology or similarity thereto. In some embodiments, the nucleic acid comprises or consists of a nucleic acid encoding des-pyrE- brazzein_Y8F / Y11 F / G35A as set forth in SEQ ID NO: 21 or a functional variant thereof having at least 70% identity, homology or similarity thereto.

[0237] The skilled person knows how to derive a DNA sequence from an amino acid sequence and vice versa. Software for performing reverse translations from amino acid sequence to DNA sequences and translations from DNA sequences to amino acid sequences are available for performing this task.

[0238] The nucleic acid may further comprise at least one promoter, such as at least one constitutive promoter or at least one inducible promoter. The promoter may be a medium strength promoter, for example TPI1 p (SEQ ID NO: 12) or a homologue thereof having at least 70%, such as at least 71 %, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity or homology thereto, or a strong promoter. P7131 PC00

[0239] The skilled person knows whether a given promoter is classified as a medium or strong promoter and / or how to characterise the promoter strength of an uncharacterised promoter. For example, RNA levels can be measured using RNA sequencing, or protein levels can be quantified with a reporter protein, such as green-fluorescent- protein (GFP), and used for determining the strength of the promoter. A medium or strong promoter may either be a native promoter or a non-native promoter of the host cell that it is comprised within. A non-native promoter may be a promoter that is native to another organism, or it may be a synthetic promoter.

[0240] In some embodiments, the nucleic acid may further comprise a nucleic acid encoding a purification tag, preferably wherein said purification tag is linked, such as covalently linked, to said analogue, optionally wherein said purification tag is TEV-his tag as set forth in SEQ ID NO: 13 or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 13.

[0241] In further embodiments, the nucleic acid further comprises a nucleic acid encoding a secretion tag, preferably wherein said secretion tag is linked, such as covalently linked, to said analogue, optionally wherein said secretion tag is Alpha factor leader as set forth in SEQ ID NO: 14 or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such P7131 PC00 as at least 98%, such as at least 99%, such as 100% identity, homology or similarity to SEQ ID NO: 14.

[0242] In some embodiments the nucleic acid is codon-optimised. The nucleic acid may be codon-optimised for expression in the host cell as disclosed elsewhere herein, such as codon-optimised for expression in a S. cerevisiae cell, or codon-optimised for expression in a K. phaffii cell.

[0243] Expression system

[0244] Provided herein are expression systems for expression in a host cell, that may be useful for obtaining analogues of sweet proteins. The expression systems disclosed herein may be obtained by methods known in the art.

[0245] In some embodiments, the present disclosure provides an expression system for expression in a host cell, comprising a nucleic acid encoding an analogue of a sweet protein, said analogue comprising: i. at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or said analogue being a functional variant having at least 70% identity, homology or similarity to the above and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , or ii. at least one substitution to phenylalanine (F) at one residue corresponding to residue 8, one substitution to phenylalanine (F) at one residue corresponding to residue 11 of the same polypeptide, and one substitution to alanine (A) at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1 , wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or said analogue being a functional variant thereof having at least 70% identity, homology or similarity thereto, or said analogue being a functional variant of the above having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 8, one substitution at one P7131 PC00 residue corresponding to residue 11 , and one substitution at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1.

[0246] Preferably the Tm of said analogue is increased compared to the Tm of the corresponding sweet protein without said at least one substitution or said at least three substitutions.

[0247] Herein disclosed is an expression system for expression in a host cell, comprising a nucleic acid encoding an analogue of a sweet protein, said analogue comprising at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1.

[0248] In some embodiments, said substitution is a substitution from a histidine (H) to an asparagine (N) substitution, such as H31 N. P7131 PC00

[0249] Herein disclosed is an expression system for expression in a host cell, comprising a nucleic acid encoding an analogue of a sweet protein, said analogue comprising at least three substitutions, said at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1, a second substitution to phenylalanine (F) at a residue corresponding to residue 11 , and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, wherein said sweet protein is Q- brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto and comprising said at least three substitutions.

[0250] In some embodiments, said first and second substitutions comprise or consist of a substitution from tyrosine (Y) to phenylalanine (F), such as Y8F and Y11 F, and wherein said third substitution comprises or consists of a substitution from glycine (G) to alanine (A), such as G35A.

[0251] In some embodiments, the sweet protein is Q-brazzein as set forth in SEQ ID NO: 1, pyrE-brazzein as set forth in SEQ ID NO: 10, or des-pyrE-brazzein as set forth in SEQ P7131 PC00

[0252] ID NO: 11 or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto.

[0253] In some embodiments, the analogue of the expression system has an increased melting temperature (Tm) compared to the corresponding sweet protein without said at least one substitution, preferably wherein said analogue is sweet.

[0254] Said analogue, sweet protein, or functional variants thereof are as described herein, for example in the section “Thermostable analogue”.

[0255] The nucleic acid of the expression system comprises or consists of any one of the nucleic acids as described elsewhere herein, for example the section “Nucleic acid”.

[0256] In some embodiments, the analogue of the expression system comprises or consists of Q-brazzein_H31N as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1.

[0257] In further embodiments, the nucleic acid of the expression system comprises or consists of Q-brazzein_H31N as set forth in SEQ ID NO: 4, or a homologue thereof P7131 PC00 having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity or homology to SEQ ID NO: 4.

[0258] In some embodiments, the analogue of the expression system comprises or consists of pyrE-brazzein_H31 N as set forth in SEQ ID NO: 15, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 10.

[0259] In further embodiments, the nucleic acid of the expression system comprises or consists of pyrE-brazzein_H31 N as set forth in SEQ ID NO: 17, or a homologue thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity or homology to SEQ ID NO: 17. P7131 PC00

[0260] In some embodiments, the analogue of the expression system comprises or consists of des-pyrE-brazzein_H30N as set forth in SEQ ID NO: 16, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto comprising said at least one substitution at one residue corresponding to residue 30 of the polypeptide set forth in SEQ ID NO: 11.

[0261] In further embodiments, the nucleic acid of the expression system comprises or consists of des-pyrE-brazzein_H30N as set forth in SEQ ID NO: 18, or a homologue thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity or homology to SEQ ID NO: 18.

[0262] In some embodiments, the analogue of the expression system comprises or consists of Q-brazzein_Y8F / Y11 F / G35A as set forth in SEQ ID NO: 19, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such P7131 PC00 as 100% identity, homology or similarity thereto comprising at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution to phenylalanine (F) at a residue corresponding to residue 11, and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide.

[0263] In some embodiments, the analogue of the expression system comprises or consists of pyrE-brazzein_Y8F / Y11F / G35A as set forth in SEQ ID NO: 20, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto comprising at least three substitutions, comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution to phenylalanine (F) at a residue corresponding to residue 11 , and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide.

[0264] In some embodiments, the analogue of the expression system comprises or consists of des-pyrE-brazzein_Y7F / Y10F / G34A as set forth in SEQ ID NO: 21 , or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, homology or similarity thereto comprising at least three P7131 PC00 substitutions, comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution to phenylalanine (F) at a residue corresponding to residue 11 , and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide.

[0265] The host cell may be as described elsewhere herein, for example in the section “Host cell”. In some embodiments, the host cell is a yeast. In preferred embodiments the host cell is an S. cerevisiae cell. In other embodiments, the host cell is a K. phaffii cell.

[0266] Kit of parts

[0267] In some embodiments, the present disclosure relates to a kit of parts, comprising: i. a host cell; and / or ii. at least one nucleic acid as described elsewhere herein; and / or iii. the expression system as described elsewhere herein; and iv. optionally instructions for use, preferably wherein the host cell is generally-regarded-as-safe (GRAS), has safe-to- consume status, and / or is non-pathogenic, and / or wherein the host cell is a yeast.

[0268] Such kits are useful for modifying a host cell so that it can produce the sweet protein analogues described herein, in particular a brazzein analogue comprising either at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, or at least three substitutions, a first substitution at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution at a residue corresponding to residue 11, and a third substitution at a residue corresponding to residue 35 of the same polypeptide.

[0269] The sweet protein analogue may be as described herein above, in particular it may either comprise at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , preferably wherein said substitution is a substitution from a histidine to an asparagine, or comprise at least three substitutions, a first substitution at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1, a second substitution at a residue corresponding to residue 11 of the same polypeptide, and a third substitution at a residue corresponding to residue 35 of the same polypeptide, preferably wherein said substitutions are substitutions to P7131 PC00 phenylalanine (F), phenylalanine (F) and alanine (A), respectively, wherein said sweet protein is brazzein or a functional variant thereof, and the analogue is more thermostable than the sweet protein from which it is derived. Preferably said sweet protein analogue is also sweet. The host cell may be as described herein above, in particular a S. cerevisiae or K. phaffii cell.

[0270] The nucleic acid may be SEQ ID NO: 4, SEQ ID NO: 17 or SEQ ID NO: 18, or a nucleic acid encoding the polypeptide as set forth in SEQ ID NO: 3, SEQ ID NO. 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 or functional variants thereof having at least 70% identity, similarity or homology thereto.

[0271] Examples

[0272] Example 1

[0273] Unless stated otherwise, references to “brazzein” in this Example refer to Q-brazzein.

[0274] Materials and methods

[0275] Strains and cultivation conditions

[0276] The strains employed here are derived from the parent strain Saccharomyces cerevisiae CEN.PK 530-1CK303 (MATa URA3 HIS3 LEU2 TRP1 SUC2 MAL2-8c tpi 1 (41 -707)::loxP Ahda2 Avps5 Atda3 PGK1 p-COG5 Agosl : : amdSYM-TEF1 p- PDI147), which has been genetically engineered to carry a truncated tpi1 gene, rendering it incapable of utilizing glucose as its sole carbon source. All plasmids are derived from pAlphaAmyCPOT (2 pm, AmpR, TPI1p-alpha factor leader-amylase gene- TPI1t) (Huang et al. 2018), with the POT1 gene from Saccharomyces pombe serving as the selection marker. In these plasmids, the amylase gene was replaced with the sweet protein or different sweet protein variants to create expression plasmids. The detailed lists of strains, plasmids, BioBricks, and primers utilized in this work can be found in Tables 1-4.

[0277] The native sequence for Q-brazzein, with a C-terminal TEV-his tag (SEQ ID NO: 13), was obtained from ThermoFisher, and mutations were introduced through PCR. The codon-optimized versions of the DNA encoding native Q-brazzein and Q- brazzein_H31N is SEQ ID NO: 2 and SEQ ID NO: 4, respectively. The PCR-amplified products were cloned into the previously mentioned plasmid using USER cloning. All newly constructed plasmids were validated through Sanger sequencing by Eurofins P7131 PC00

[0278] Scientific SE. Yeast transformations were executed using a lithium acetate-based method, following an established protocol (Gietz and Schiestl 2007). The transformed yeast cells were selected on minimal medium plates and confirmed via colony PGR (cPCR). The DNA encoding Q-brazzein_Y8F / Y11F / G35A (SEQ ID NO: 19) and other brazzein variants, as well as plasmids and yeast strains expressing the variants were generated in the same manner as Q-brazzein_H31N.

[0279] Table 1. List of strains.

[0280] Table 2. List of plasmids. P7131 PC00

[0281] Table 3. List of BioBricks.

[0282] Table 4. List of primers. P7131 PC00

[0283] Standard cultivation of the parental strain S. cerevisiae was done at 30°C in yeast peptone dextrose (YPD) medium supplemented with ethanol and glycerol. This medium contained 10 g / L yeast extract, 20 g / L peptone, 20 g / L D-glucose, 20 g / L ethanol, and 30 g / L glycerol. The newly created strains harbouring the plasmid were grown in a minimal medium. This minimal medium (pH 6.0), as previously reported (Jensen et al. 2014), consisted of 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4 7H2O, 20 g / L D-glucose, 2 ml / L trace metals solution, and 1 ml / L vitamins.

[0284] Protein production and thermostability measurement

[0285] To assess protein production, individual colonies of each yeast strain were inoculated into 4 mL of pre-culture medium and incubated overnight at 30°C. The following day, 2- liter baffled shake flasks were inoculated to achieve an OD600 of 0.1, and these cultures were allowed to grow for 96 hours at 30°C. After the growth period, the final OD600 was recorded, cultures were harvested, and the supernatants were filtered through 0.2-micron vacuum filters. The supernatants were then concentrated using Amicon® Stirred Cells with a 3 kDa membrane. To further concentrate the supernatant, Amicon® Ultra-15 Centrifugal Filter Units were used, along with a wash buffer comprising 300 mM NaCI, 20 mM imidazole, and 10 mM HEPES at pH 7.5.

[0286] Protein purification was conducted using HisPur™ Ni-NTA Resin and HisTrap™ HP Columns according to the manufacturer's protocol. The supernatant was loaded onto the resin, and the beads were washed twice with the wash buffer. The bound proteins were then eluted using an elution buffer containing 50 mM HEPES (pH 7.4), 300 mM NaCI, and 300 mM imidazole. The eluted proteins underwent a buffer exchange with a storage buffer containing 20 mM sodium phosphate and 300 mM NaCI at pH 7.5. After buffer exchange, the proteins were further concentrated using Amicon Ultra-0.5 Ultracel-3 Membrane, and the final volume was measured to determine protein P7131 PC00 concentration, which was assessed by reading the absorbance at 280 nm with a NanoDropTM 2000 spectrophotometer (ThermoFisher Scientific).

[0287] Protein samples at a concentration of 1 mg / mL were examined using nanoDSF with the Prometheus Panta device (NanoTemper Technologies, Munich, Germany). Small volumes of 10 microliters were dispensed into nanoDSF-grade standard capillaries (NanoTemper Technologies GmbH, Munich, Germany). These samples were then subjected to thermal stress as the temperature increased from 20°C to 110°C at a rate of 1 ,5°C per minute. The fluorescence emitted by tryptophan upon UV excitation at 280 nm was detected at both 330 nm and 350 nm using a dual-UV detector. The melting temperature (Tm) for a specific region of interest was determined using PR.ThermControl software (NanoTemper Technologies, Munich, Germany).

[0288] AAG analysis

[0289] To computationally predict the variants’ effects on sweet protein stability, Rosetta CartesianDDG protocol was used to predict AAG caused by each variant. The PDB file 4HE7 was used for the Rosetta CartesianDDG protocol, and the PDB structures were first relaxed using a Rosetta Relax.

[0290] Q-brazzein-receptor interaction modelling

[0291] To see how the variants are located with regard to the potential binding interface between each sweet protein and the sweet taste receptor dimer (T 1 R2+T 1 R3), AFsample was used to generate 2,000 complex predictions, half of which were generated using templates. They were plotted into heatmaps to visualize the pairwise Root Mean Square Deviation RMSD and clustered into five clusters using pairwise RMSD. The largest cluster was then chosen, and the highest quality predictions were extracted from the largest clusters. The prediction with the highest prediction quality is visualised in Figure 5.

[0292] Sensory analysis for H31N variant

[0293] For the sensory analysis, filtered supernatants were upconcentrated using ultrafiltration using 50 cm23 kDa TFF minimate™ filter from Pall. To reduce the conductivity to below 5 mS / cm as recommended by the column manufacturer, supernatants were diluted with MQ water and pH adjusted to pH 4 using pure Acetic Acid. Brazzein was purified using AKTA pure™ cation exchange chromatography, and eluted with a linear P7131 PC00 gradient going from 100% 100 mM NaCI to 100% 1000mM NaCI in 10 column volume (CV) and buffer exchanged to 1 mM of sodium phosphate buffer.

[0294] Participants (n = 61) ranked the protein samples as well as a positive and negative control by sweetness and rated each one on a scale from 1 (not sweet) to 10 (very sweet). Ranking data were quantified using a numerical system (1 = most preferred, 4 = least preferred).

[0295] Results

[0296] Q-brazzein variants with improved thermostability

[0297] Upon expressing and purifying a total of 13 different Q-brazzein variants, we experimentally determined their melting temperatures (Tm). To assess their heat stability, we used nano-differential scanning fluorometry (nanoDSF), a technique that assesses the conformational and colloidal stability of proteins and determine the melting temperature (Tm) by monitoring the intrinsic fluorescence of tryptophan or tyrosine residues as the protein unfolds. The Tm values for native Q-brazzein aligned with previous studies, which measured thermostability through heating and PAGE analysis (Ming and Hellekant 1994). Notably, Q-brazzein variants H31 N (SEQ ID NO: 3) and Y8F / Y11 F / G35A (SEQ ID NO: 19) exhibited a Tm higher than that of native Q- brazzein (SEQ ID NO: 1). However, the method could only measure Tm values up to 110°C (Figure 1a). Since the variants exceeded that threshold, we used 0.8% acetonitrile as a chaotropic agent to lower the proteins' thermostability (Figure 1 b). Q- brazzein_H31 N (SEQ ID NO: 3) showed a 1.25-fold increase in Tm, suggesting that it could potentially have a higher melting point when analyzed with other techniques capable of handling higher temperatures, such as Differential Scanning Calorimetry.

[0298] Other brazzein variants had reduced thermostability relative to the native protein. Notably, despite Q-brazzein variant Y8F / Y11 F / G35A (SEQ ID NO: 19) exhibiting a higher Tm than the native protein, both Q-brazzein Y8F / Y11 F and Q-brazzein G35A variants were determined to have lower melting temperatures than native Q-brazzein (Figure 6).

[0299] AAG analysis

[0300] Q-brazzein variants with negative AG value relative to the native Q-brazzein polypeptide (i.e. negative AAG) should theoretically be more thermostable than the P7131 PC00 native Q-brazzein polypeptide. Several Q-brazzein variants that were computationally predicted to have negative AAG values were produced and validated experimentally. Strikingly, it was found that not all of the Q-brazzein variants predicted to have negative AAG values were more thermostable than the native Q-brazzein (Table 5), and that a negative AAG is not tantamount to the variant having increased thermostability.

[0301] Table 5. Predicted AAG and measured Tm for Q-brazzein and Q-brazzein variants.

[0302] Q-brazzein-receptor interaction modelling

[0303] Brazzein has been predicted to bind to the cysteine rich domain (CRD) of the T1R3 subunit of the T 1 R3 / T 1 R2 receptor complex, however our best prediction with AFsample suggests that brazzein binds to the CRD of both receptor subunits (Figure 5).

[0304] Amino acids with positive charges have been established to be crucial for the sweetness of sweet proteins. In brazzein, the positions E36, R43 and E53 are especially important for sweetness. The H31 N substitution of the thermostable variant Q-brazzein_H31N (SEQ ID NO: 3), is not close to any of the residues considered important for sweetness. In fact, a change from histidine at position 31 to another amino acid may lead to said variant having increased sweetness.

[0305] Sensory analysis

[0306] We performed a preliminary sensory analysis of brazzein_H31N, which was the most thermostable variant, across all variants tested. To evaluate the sweetness of P7131 PC00 brazzein_H31 N and native brazzein, participants (n = 61) ranked the protein samples and a positive and negative control by sweetness and rated each one on a scale from 1 (not sweet) to 10 (very sweet). Ranking data were quantified using a numerical system (1 = most preferred, 4 = least preferred).

[0307] Both averaged sweetness ratings and rankings (figure 4) revealed a trend where the positive control (PC) was ranked higher, followed by H31 N, native and negative control (NC) (Figure 7). However, the difference between H31 N and native was not statistically significant (p = 0.2476) indicating that they are equally sweet. These findings provide empirical confirmation that the brazzein_H31 N variant is perceived as sweet by humans.

[0308] Example 2 Unless stated otherwise, references to “brazzein” in this Example refer to Q-brazzein.

[0309] Materials and Methods

[0310] Brazzein H31 R and Brazzein H31 A expression and purification Brazzein_H31 R and brazzein_H31A were expressed as His6-Sumo-Brazzein fusion proteins in BL21 (DE3)pLysS Escherichia coli using autoinduction media at 30°C for 24 hours. Cells were harvested by centrifugation and lysed by incubation in BugBuster (Merck) at 37°C for 60 min. Lysate was loaded onto a HiTrap TALON crude (Cytiva) pre-equilibrated with wash buffer (50 mM HEPES pH 8, 100 mM NaCI, 10 mM Imidazole), washed 3 times with wash buffer and eluted with 50 mM HEPES pH 8, 100 mM NaCI, 120 mM Imidazole. SUMO protease was added to 15 U / ml and proteolysis was allowed at 30°C for 16 h. The proteolyzed sample was diluted 5 times with 10 mM citric acid pH 4. The diluted sample was loaded onto a HiPrep CM FF 16 / 10 (Cytiva) column pre-equilibrated with 20 mM citric acid, 20 mM NaCI pH 4. Brazzein was eluted with a gradient to 100% 20 mM citric acid, 200 mM NaCI pH 4 over 30 min. Fractions containing brazzein were pooled and concentrated to 13 ml in a 3 kDa MWCO centrifugal filter unit. The pooled and concentrated fractions were loaded onto a HiLoad 26 / 600 Superdex 75 pg (Cytiva) column pre-equilibrated with PBS where from brazzein was eluted. P7131 PC00

[0311] Native Brazzein and Brazzein H31N expression and purification

[0312] Native brazzein (SEQ ID NO: 1) and brazzein_H31 N (SEQ ID NO: 3) were expressed in Komagataella phaffii (CBS 2612) with a disrupted KU70 gene. An expression construct was synthesized comprising native brazzein or brazzein_H31N, the A0X1 promoter, the MFa signal sequence and the A0X1 terminator. To generate the expression strains of K. phaffii the expression constructs were co-introduced together with a Cas9 dual gRNA plasmid targeting A0X1. Expression strains were cultivated in buffered minimal medium (BMM) glycerol medium for 48 h at 1000 rpm 30°C. After the growth phase in BMM glycerol, freshly prepared BMM with 1% methanol was added and the cells were cultivated for an additional 72 h, at 30°C, shaking at 1000 rpm, humidity at 80%. The methanol was replenished every 24 h to maintain 0.5% v / v final concentration. BMM comprised of 100 mM Potassium phosphate buffer pH 6.0, 1.34% w / v Yeast nitrogen base, 40 ug / L biotin and 2% v / v glycerol or 0.5% v / v methanol. Culture supernatants obtained from K. phaffii transformants cultivated were harvested by centrifugation 4000 g for 20 min. Following the supernatant was diluted 5 times with 10 mM citric acid pH 4. The diluted supernatant was loaded onto a HiPrep CM FF 16 / 10 (Cytiva) column pre-equilibrated with 20 mM citric acid, 20 mM NaCI pH 4. Brazzein was eluted with a gradient to 100% 20 mM citric acid, 200 mM NaCI pH 4 over 30 min. Fractions containing brazzein were pooled and concentrated to 13 ml in a 3 kDa MWCO centrifugal filter unit. The pooled and concentrated fractions were loaded onto a HiLoad 26 / 600 Superdex 75 pg (Cytiva) column pre-equilibrated with PBS where from brazzein was eluted.

[0313] Thermostability measurements

[0314] Thermostability was measured using nanoDSF (Prometheus Panta, NanoTemper Technologies) with a ramp speed of 1°C / min from 20°C to 110°C in PBS including 0 M, 0.5 M, 1 M, 1.5 M, 2 M, 3 M, and 4 M guanidinium chloride.

[0315] Results

[0316] Figure 8 A-D shows the melting temperature of native brazzein, brazzein_H31N, brazzein_H31R and brazzein_H31A as a function of guanidinium chloride concentration. For native brazzein, brazzein_H31A and brazzein_H31R, the melting temperature was observable in the absence of guanidinium chloride with melting temperatures of 106°C, 96°C, and 105.2°C respectively. Nevertheless, the linear relationship between melting temperature and guanidinium chloride concentration was P7131 PC00 fitted with R2 values of 0.9972, 0.9837, and 0.9992, respectively and extrapolated melting temperatures of 105.8°C, 93.3°C, and 105.4°C, respectively, are more or less in agreement with the observed melting temperatures. For brazzein_H31N, the melting temperature was not directly observable, thus the linear relationship between melting temperature and guanidinium chloride concentration was fitted with an R2 value of 0.997 and an extrapolated melting temperature of 107.9°C.

[0317] Unlike native brazzein, brazzein_H31A and brazzein_H31R, the melting temperature of brazzein_H31N lies outside the boundaries of the Prometheus Panta (NanoTemper Technologies) for directly measuring melting temperature. The boundary is theoretically 110°C but technically a melting temperature slightly below is necessary to be able to observe peak formation. Extrapolated melting temperatures for native brazzein, brazzein_H31A and brazzein_H31R are mostly in agreement with directly measured melting temperatures, thus it is assumed that the extrapolated melting temperature of brazzein_H31N is a relatively accurate measurement of the actual brazzein_H31N melting temperature.

[0318] Sequence overview

[0319] SEQ ID NO: 2 and SEQ ID NO: 4 are codon-optimised. P7131 PC00

[0320] References

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[0330] Zhang Y and Skolnick J. Scoring function for automated assessment of protein structure template quality. Proteins 2004; 57 (4): 702-710.

[0331] Items

[0332] 1. An analogue of a sweet protein, said analogue comprising: i. at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q- brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or said analogue being a functional variant thereof having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, or P7131 PC00 ii. at least one substitution to phenylalanine (F) at one residue corresponding to residue 8, one substitution to phenylalanine (F) at one residue corresponding to residue 11 , and one substitution to alanine (A) at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1 , wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or said analogue being a functional variant thereof having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 8, one substitution at one residue corresponding to residue 11 , and one substitution at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1, preferably wherein the Tm of said analogue is increased compared to the Tm of the corresponding sweet protein without said at least one substitution or said at least three substitutions.

[0333] 2. An analogue of a sweet protein, said analogue comprising at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE- brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1.

[0334] 3. The analogue according to any one of items 1 or 2, wherein said substitution is a substitution from a histidine (H) to an asparagine (N) substitution, such as H31 N. P7131 PC00

[0335] 4. An analogue of a sweet protein, said analogue comprising at least three substitutions, said at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution to phenylalanine (F) at a residue corresponding to residue 11 of the same polypeptide, and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE- brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least three substitutions.

[0336] 5. The analogue according to any one of items 1 or 4, wherein said first and second substitutions comprise or consist of a substitution from tyrosine (Y) to phenylalanine (F), such as Y8F and Y11 F, and wherein said third substitution comprises or consists of a substitution from glycine (G) to alanine (A), such as G35A.

[0337] 6. The analogue thereof according to any one of the preceding items, wherein the sweet protein is Q-brazzein as set forth in SEQ ID NO: 1 , pyrE-brazzein as set forth in SEQ ID NO: 10, or des-pyrE-brazzein as set forth in SEQ ID NO: 11 , or a functional variant thereof having at least 70% identity, homology or similarity thereto.

[0338] 7. The analogue according to any one of the preceding items, wherein said analogue has an increased melting temperature (Tm) compared to the corresponding sweet protein without said at least one substitution, preferably wherein said analogue is sweet.

[0339] 8. The analogue according to any one of the preceding items, wherein the sweet protein is Q-brazzein (SEQ ID NO: 1), and wherein the analogue comprises or consists of Q-brazzein_H31 N as set forth in SEQ ID NO: 3 or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 3. P7131 PC00

[0340] 9. The analogue according to any one of the preceding items, wherein the sweet protein is pyrE-brazzein (SEQ ID NO: 10), and wherein the analogue comprises or consists of pyrE-brazzein_H31 N as set forth in SEQ ID NO: 15 or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 15.

[0341] 10. The analogue according to any one of the preceding items, wherein the sweet protein is des-pyrE-brazzein (SEQ ID NO: 11), and wherein the analogue comprises or consists of des-pyrE-brazzein_H30N as set forth in SEQ ID NO: 16 or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 16.

[0342] 11. The analogue according to any one of the preceding items, wherein the sweet protein is Q-brazzein (SEQ ID NO: 1), and wherein the analogue comprises or consists of Q-brazzein_Y8F / Y11 F / G35A as set forth in SEQ ID NO: 19 or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 19.

[0343] 12. The analogue according to any one of the preceding items, wherein the sweet protein is pyrE-brazzein (SEQ ID NO: 10), and wherein the analogue comprises or consists of pyrE-brazzein_Y8F / Y11 F / G35A as set forth in SEQ ID NO: 20 or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 20.

[0344] 13. The analogue according to any one of the preceding items, wherein the sweet protein is des-pyrE-brazzein (SEQ ID NO: 11), and wherein the analogue comprises or consists of des-pyrE-brazzein_Y7F / Y10F / G34A as set forth in SEQ ID NO: 21 or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 21.

[0345] 14. The analogue according to any one of the preceding items, wherein the Tm of said analogue is increased by at least 1°C compared to the Tm of the corresponding sweet protein without said at least one substitution, such as by at least 1 ,5°C, such as by at least 2°C, such as by at least 2.5°C, such as by at P7131 PC00 least 3.5°C, such as by at least 4°C, such as by at least 4.5°C, such as by at least 5°C, such as by at least 5.5°C, such as by at least 6°C, such as by at least 6.5°C, such as by at least 7°C, such as by at least 7.5°C, such as by at least 8°C, such as by at least 8.5°C, such as by at least 9°C, such as by at least 9.5°C, such as by at least 10°C, such as by at least 10.5°C, such as by at least 11 °C, such as by at least 11 ,5°C, such as by at least 12°C, such as by at least 12.5°C, such as by at least 13°C, such as by at least 13.5°C, such as by at least 14°C, such as by at least 14.5°C, such as by at least 15°C compared to the Tm of the corresponding sweet protein without said at least one substitution.

[0346] 15. The analogue according to any one of the preceding items, wherein the Tm of said analogue is between 106°C and 110°C, such as between 107°C and 110°C, such as between 108°C and 110°C, such as between 109°C and 110°C.

[0347] 16. The analogue according to any one of the preceding items, wherein the corresponding sweet protein without said at least one substitution has a Tm of 106°C and the analogue has a Tm greater than 106°C, such as a Tm of 107°C, such as a Tm of 107.1 °C, such as a Tm of 107.2°C, such as a Tm of 107.3°C, such as a Tm of 107.4°C, such as a Tm of 107.5°C, such as a Tm of 107.6°C, such as a Tm of 107.7°C, such as a Tm of 107.8°C, such as a Tm of 107.9°C, such as a Tm of 108°C, such as a Tm of 109°C, such as a Tm of 110°C, such as a Tm of 111 °C, such as a Tm of 112°C, such as a Tm of 113°C, such as a Tm of 114°C, such as a Tm of 115°C.

[0348] 17. The analogue according to any one of the preceding items, wherein the Tm of said analogue is at least 107°C.

[0349] 18. The analogue according to any one of the preceding items, wherein the Tm of said analogue is at least 110°C.

[0350] 19. The analogue according to any one of the preceding items, wherein the Tm of said analogue is increased at least 0.75-fold compared to the Tm of the corresponding sweet protein without said at least one substitution, such as increased at least 1-fold, such as increased 1.01 -fold, such as increased 1.02 fold, such as increased 1.03-fold, such as increased 1.04-fold, such as P7131 PC00 increased 1.05-fold, such as increased 1.06-fold, such as increased 1.07-fold, such as increased 1.08-fold, such as increased 1.09-fold, such as increased 1.10-fold, such as increased at least 1.25-fold, such as increased at least 1.5- fold compared to the Tm of the sweet protein from which it was derived.

[0351] 20. The analogue according to any one of the preceding items, wherein the Tm of said analogue is increased between 0.75-fold and 1.5-fold compared to the Tm of the corresponding sweet protein without said at least one substitution, such as increased between 1-fold and 1.5-fold, such as increased between 1.25-fold and! 5-fold, such as increased by 1.5-fold compared to the Tm of the corresponding sweet protein without said at least one substitution.

[0352] 21. The analogue according to any one of the preceding items, wherein the Tm of said analogue is increased at most 1.5-fold compared to the Tm of the corresponding sweet protein without said at least one substitution.

[0353] 22. The analogue according to any one of the preceding items, wherein the analogue comprises or consists of Q-brazzein_H31 N (SEQ ID NO: 3) or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 3, and wherein the Tm of said analogue is between 106°C and 110°C, such as between 107°C and 110°C, such as between 108°C and 110°C, such as 110°C.

[0354] 23. The analogue according to any one of the preceding items, wherein the analogue comprises or consists of pyrE-brazzein_H31 N (SEQ ID NO: 15) or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 15, and wherein the Tm of said analogue is between 106°C and 110°C, such as between 107°C and 110°C, such as between 108°C and 110°C, such as 110°C.

[0355] 24. The analogue according to any one of the preceding items, wherein the analogue comprises or consists of des-pyrE-brazzein_H30N (SEQ ID NO: 16) or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 16, and wherein the Tm of said analogue is between P7131 PC00

[0356] 106°C and 110°C, such as between 107°C and 110°C, such as between 108°C and 110°C, such as 110°C.

[0357] 25. The analogue according to any one of the preceding items, wherein the analogue comprises or consists of Q-brazzein_Y8F / Y11 F / G35A (SEQ ID NO: 19) or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 19, and wherein the Tm of said analogue is between 106°C and 110°C, such as between 107°C and 110°C, such as between 108°C and 110°C, such as 110°C.

[0358] 26. The analogue according to any one of the preceding items, wherein the analogue comprises or consists of pyrE-brazzein_Y8F / Y11 F / G35A (SEQ ID NO: 20) or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 20, and wherein the Tm of said analogue is between 106°C and 110°C, such as between 107°C and 110°C, such as between 108°C and 110°C, such as 110°C.

[0359] 27. The analogue according to any one of the preceding items, wherein the analogue comprises or consists of des-pyrE-brazzein_Y7F / Y10F / G34A (SEQ ID NO: 21) or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 21 , and wherein the Tm of said analogue is between 106°C and 110°C, such as between 107°C and 110°C, such as between 108°C and 110°C, such as 110°C.

[0360] 28. The analogue according to any one of the preceding items, wherein the polypeptide comprises or consists of a sequence having at least 70% identity, homology, or similarity to SEQ ID NO: 3, or SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 , with the proviso that at the most 16 residues are mutated.

[0361] 29. The analogue according to any one of the preceding items, wherein the polypeptide comprises or consists of a sequence having at least 70% identity, homology, or similarity to SEQ ID NO: 3, or SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 , with the proviso that at the P7131 PC00 most 16 residues are mutated, for example between 16 and 10 residues are mutated, such as between 5 and 10 residues are mutated, or less.

[0362] 30. The analogue according to any one of the preceding items, wherein the Tm is determined using nano differential scanning fluorimetry (nanoDSF).

[0363] 31. The analogue according to item 30, wherein said nanoDSF is performed by increasing the temperature from 20°C to 110°C at a rate of 1.5°C per minute, detecting the fluorescence emitted at both 330 nm and 350 nm by tryptophan upon UV excitation at 280 nm using a dual-UV detector, and determining the Tm for example by using PR.ThermControl software.

[0364] 32. The analogue according to item 30, wherein said nanoDSF is performed by increasing the temperature from 20°C to 110°C at a rate of 1°C per minute.

[0365] 33. The analogue according to any one of items 30 to 32, wherein said analogue is contacted with acetonitrile whilst performing said nanoDSF, preferably 0.8% acetonitrile,

[0366] 34. The analogue according to any one of items 30 to 32, wherein said analogue is contacted with guanidinium chloride whilst performing nanoDSF, preferably wherein said guanidinium chloride is in the range of 0.5 to 4 M.

[0367] 35. The analogue or functional variant according to any one of the preceding items, wherein the analogue has a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least

[0368] 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of an analogue as set forth in SEQ ID NO: 3, or SEQ ID NO: 15, or SEQ ID NO: 16 or SEQ ID NO: 19 or SEQ ID NO: 20 or SEQ ID NO: 21.

[0369] 36. The analogue according to any one of the preceding items, wherein said analogue is linked, such as covalently linked, to a purification tag, optionally wherein said purification tag comprises or consists of TEV-his tag as set forth in P7131 PC00

[0370] SEQ ID NO: 13 or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 13.

[0371] 37. The analogue according to any one of the preceding items, wherein said analogue is linked, such as covalently linked, to a secretion tag, optionally wherein said secretion tag comprises or consists of Alpha factor leader as set forth in SEQ ID NO: 14 or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 14.

[0372] 38. The analogue according to any one of the preceding items, wherein the analogue is at least 25% as sweet as the corresponding sweet protein without said at least one substitution, such as at least 50%, such as at least 75%, such as at least 100%, such as at least 200%, such as at least 300%, such as at least 400%, such as at least 500%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900% or such as at least 1000% as sweet as the corresponding sweet protein without said at least one substitution.

[0373] 39. The analogue according to any one of the preceding items, wherein the analogue is at least 0.2 times as sweet as the corresponding sweet protein without said at least one substitution, such as at least 0.5 times, such as at least 1.0 time, such as at least 2.0 times, such as at least 3.0 times, such as at least 3.5 times, such as at least 5.0 times, such as at least 10 times, such as at least 20 times, such as at least 30 times, such as at least 40 times, such as at least 50 times, such as at least 75 times, such as at least 100 times as sweet as the corresponding sweet protein without said at least one substitution

[0374] 40. The analogue according to any one of the preceding items, wherein the sweetness of the analogue is similar or identical to the sweetness of the corresponding sweet protein without said at least one substitution.

[0375] 41. The analogue according to any one of the preceding items, wherein the analogue is sweet at any temperature smaller than or equal to its Tm. P7131 PC00

[0376] 42. The analogue according to any one of the preceding items, wherein the analogue is sweet at any temperature lower than or equal to its Tm.

[0377] 43. The analogue according to any one of the preceding items, wherein the analogue maintains its sweetness at any temperature smaller than or equal to its Tm.

[0378] 44. The analogue according to any one of the preceding items, wherein the analogue maintains its sweetness at any temperature lower than or equal to its Tm.

[0379] 45. A host cell capable of producing an analogue of a sweet protein, said analogue comprising: i. at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q- brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, or ii. at least one substitution to phenylalanine (F) at one residue corresponding to residue 8, one substitution to phenylalanine (F) at one residue corresponding to residue 11 , and one substitution to alanine (A) at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1 , wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 8, one substitution at one residue corresponding to residue 11 , and P7131 PC00 one substitution at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1, preferably wherein the Tm of said analogue is increased compared to the Tm of the corresponding sweet protein without said at least one substitution or said at least three substitutions.

[0380] 46. A host cell capable of producing an analogue of a sweet protein, said host cell expressing an analogue comprising at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE- brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1.

[0381] 47. The host cell according to any one of items 45 or 46, wherein said substitution is a substitution from a histidine (H) to an asparagine (N) substitution, such as H31 N.

[0382] 48. A host cell capable of producing an analogue of a sweet protein, comprising at least three substitutions, said at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution to phenylalanine (F) at a residue corresponding to residue 11, and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE- brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least three substitutions. P7131 PC00

[0383] 49. The host cell according to item 45 or 48, wherein said first and second substitutions comprise or consist of a substitution from tyrosine (Y) to phenylalanine (F), such as Y8F and Y11 F, and wherein said third substitution comprises or consists of a substitution from glycine (G) to alanine (A), such as G35A.

[0384] 50. The host cell according to any one of items 45 to 49, wherein the sweet protein is Q-brazzein as set forth in SEQ ID NO: 1 , pyrE-brazzein as set forth in SEQ ID NO: 10, or des-pyrE-brazzein as set forth in SEQ ID NO: 11 , or a functional variant thereof having at least 70% identity, homology or similarity thereto.

[0385] 51 . The host cell according to any one of items 45 to 50, wherein the analogue has an increased melting temperature (Tm) compared to the corresponding sweet protein without said at least one substitution, preferably wherein said analogue is sweet.

[0386] 52. The host cell according to any one of items 45 to 51 , wherein said analogue, sweet protein, or functional variants thereof are as defined in any one of items 1 to 43.

[0387] 53. The host cell according to any one of items 46 to 52, wherein the host cell is general ly-regarded-as-safe (GRAS), has safe-to-consume status, and / or is non- pathogenic.

[0388] 54. The host cell according to any one of items 46 to 53, wherein the host cell is a fungal cell, such as a filamentous fungal cell, or a yeast cell, such as a yeast cell belonging to the genus of Saccharomyces, for example S. cerevisiae, or Komagataella., for example K. phaffii, preferably wherein the yeast cell is a S. cerevisiae cell.

[0389] 55. The host cell according to any one of items 45 to 54, wherein the host cell comprises additional modifications, preferably wherein said additional modifications comprise overexpression of a disulphide isomerase, such as PDI1. P7131 PC00

[0390] 56. A method of producing an analogue of a sweet protein, comprising: i. providing a host cell as defined in any one of items 45 to 55; ii. incubating and optionally propagating said host cell in a cultivation medium, thereby obtaining a fermentation liquid comprising said analogue, wherein said analogue or sweet protein are as defined in any one of items 1 to 43.

[0391] 57. The method according to item 56, further comprising a step of recovering the analogue.

[0392] 58. The method according to item 57, wherein the step of recovering the analogue comprises filtering such as by ultrafiltration, and / or concentrating the analogue, for example by ion-exchange chromatography, from the fermentation liquid.

[0393] 59. A method of producing a sweetener composition and / or a food, feed, or beverage product comprising an analogue of a sweet protein, comprising: i. providing a host cell according to any one of items 45 to 55; ii. incubating and optionally propagating said host cell in a cultivation medium, thereby obtaining a fermentation liquid; iii. recovering said host cell, fermentation liquid comprising said host cell and / or said analogue; iv. optionally recovering said analogue from said fermentation liquid and / or host cell; and v. converting said analogue, and / or fermentation liquid comprising said host cell and / or analogue into a sweetener composition and / or into a food, feed or beverage product, whereby said sweetener composition, and / or said food, feed, or beverage product is produced, and wherein said analogue or sweet protein are as defined in any one of items 1 to 43.

[0394] 60. The method according to item 59, wherein the analogue maintains its sweetness upon heating until at least its Tm. P7131 PC00

[0395] 61. The method according to any one of items 59 to 60, wherein the analogue remains sweet upon heating until at least its Tm.

[0396] 62. The method according to any one of items 59 to 61 , wherein the analogue maintains its sweetness at any temperature until at least its Tm.

[0397] 63. The method according to any one of items 59 to 62, wherein the analogue remains sweet at any temperature until at least its Tm.

[0398] 64. A nucleic acid encoding an analogue of a sweet protein, said analogue comprising: i. at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q- brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, or ii. at least one substitution to phenylalanine (F) at one residue corresponding to residue 8, one substitution to phenylalanine (F) at one residue corresponding to residue 11 , and one substitution to alanine (A) at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1 , wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 8, one substitution at one residue corresponding to residue 11 , and one substitution at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1, P7131 PC00 preferably wherein the Tm of said analogue is increased compared to the Tm of the corresponding sweet protein without said at least one substitution or said at least three substitutions.

[0399] 65. A nucleic acid encoding an analogue of a sweet protein, said analogue comprising at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE- brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1.

[0400] 66. The nucleic acid according to any one of items 64 or 65, wherein said substitution is a substitution from a histidine (H) to an asparagine (N) substitution, such as H31 N.

[0401] 67. A nucleic acid encoding an analogue of a sweet protein, said analogue comprising at least three substitutions, said at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution to phenylalanine (F) at a residue corresponding to residue 11 , and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE- brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least three substitutions.

[0402] 68. The nucleic acid according to any one of items 64 or 67, wherein said first and second substitutions comprise or consist of a substitution from tyrosine (Y) to phenylalanine (F), such as Y8F and Y11 F, and wherein said third substitution P7131 PC00 comprises or consists of a substitution from glycine (G) to alanine (A), such as G35A.

[0403] 69. The nucleic acid according to any one of items 64 to 68, wherein the sweet protein is Q-brazzein as set forth in SEQ ID NO: 1 , pyrE-brazzein as set forth in SEQ ID NO: 10, or des-pyrE-brazzein as set forth in SEQ ID NO: 11 , or a functional variant thereof having at least 70% identity, homology or similarity thereto.

[0404] 70. The nucleic acid according to any one of items 64 to 69, wherein said analogue has an increased melting temperature (Tm) compared to the corresponding sweet protein without said at least one substitution, preferably wherein said analogue is sweet.

[0405] 71 . The nucleic acid according to any one of items 64 to 70, wherein said analogue, sweet protein, or functional variants thereof are as defined in any one of items 1 to 43.

[0406] 72. The nucleic acid according to any one of items 64 to 70, wherein Q-brazzein as set forth in SEQ ID NO: 1 is encoded by a nucleic acid comprising or consisting of SEQ ID NO: 2 (Q-brazzein), or a homologue thereof having at least 70% identity, homology or similarity thereto.

[0407] 73. The nucleic acid according to any one of items 64 to 72, wherein said analogue comprises or consists of Q-brazzein_H31 N as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% identity, homology or similarity thereto.

[0408] 74. The nucleic acid according to any one of items 64 to 73, wherein the nucleic acid comprises or consists of Q-brazzein_H31N as set forth in SEQ ID NO: 4, or a homologue thereof having at least 70% sequence identity or homology to SEQ ID NO: 4 or to a nucleic acid encoding the polypeptide of SEQ ID NO: 3.

[0409] 75. The nucleic acid according to any one of items 64 to 71 , wherein said analogue comprises or consists of pyrE-brazzein_H31 N as set forth in SEQ ID NO: 15, or P7131 PC00 a functional variant thereof having at least 70% identity, homology or similarity thereto.

[0410] 76. The nucleic acid according to any one of items 64 to 71 , or 75, wherein the nucleic acid comprises or consists of pyrE-brazzein_H31 N as set forth in SEQ ID NO: 17, or a homologue thereof having at least 70% sequence identity or homology to a nucleic acid encoding the polypeptide of SEQ ID NO: 15.

[0411] 77. The nucleic acid according to any one of items 64 to 71 , wherein said analogue comprises or consists of des-pyrE-brazzein_H30N as set forth in SEQ ID NO: 16, or a functional variant thereof having at least 70% identity, homology or similarity thereto.

[0412] 78. The nucleic acid according to any one of items 64 to 71 , or 77, wherein the nucleic acid comprises or consists of des-pyrE-brazzein_H30N as set forth in SEQ ID NO: 18, or a homologue thereof having at least 70% sequence identity or homology to a nucleic acid encoding the polypeptide of SEQ ID NO: 16.

[0413] 79. The nucleic acid according to any one of items 64 to 72, wherein said analogue comprises or consists of Q-brazzein_Y8F / Y11 F / G35A as set forth in SEQ ID NO: 19, or a functional variant thereof having at least 70% identity, homology or similarity thereto.

[0414] 80. The nucleic acid according to any one of items 64 to 71 , wherein said analogue comprises or consists of pyrE-brazzein_Y8F / Y11 F / G35A as set forth in SEQ ID NO: 20, or a functional variant thereof having at least 70% identity, homology or similarity thereto.

[0415] 81. The nucleic acid according to any one of items 64 to 71 , wherein said analogue comprises or consists of des-pyrE-brazzein_Y7F / Y10F / G34A as set forth in SEQ ID NO: 21 , or a functional variant thereof having at least 70% identity, homology or similarity thereto. P7131 PC00

[0416] 82. The nucleic acid according to any one of items 64 to 81 , wherein the nucleic acid further comprises at least one promoter, such as at least one constitutive promoter or at least one inducible promoter.

[0417] 83. The nucleic acid of item 82, wherein the promoter is a medium strength promoter, for example TPI1 p (SEQ ID NO: 12) or a homologue thereof having at least 70% sequence identity or homology thereto, or a strong promoter.

[0418] 84. The nucleic acid according to any one of items 64 to 83, wherein said nucleic acid further comprises a nucleic acid encoding a purification tag, preferably wherein said purification tag is linked, such as covalently linked, to said analogue, optionally wherein said purification tag is TEV-his tag as set forth in SEQ ID NO: 13 or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 13.

[0419] 85. The nucleic acid according to any one of items 64 to 84, wherein said nucleic acid further comprises a nucleic acid encoding a secretion tag, preferably wherein said secretion tag is linked, such as covalently linked, to said analogue, optionally wherein said secretion tag is Alpha factor leader as set forth in SEQ ID NO: 14 or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 14.

[0420] 86. The nucleic acid of any of items 64 to 85, wherein the nucleic acid is codon- optimised.

[0421] 87. An expression system for expression in a host cell, comprising a nucleic acid encoding an analogue of a sweet protein, said analogue comprising: i. at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q- brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one P7131 PC00 substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, or ii. at least one substitution to phenylalanine (F) at one residue corresponding to residue 8, one substitution to phenylalanine (F) at one residue corresponding to residue 11 , and one substitution to alanine (A) at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1 , wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 8, one substitution at one residue corresponding to residue 11 , and one substitution at one residue corresponding to residue 35, of the polypeptide set forth in SEQ ID NO: 1, preferably wherein the Tm of said analogue is increased compared to the Tm of the corresponding sweet protein without said at least one substitution or said at least three substitutions.

[0422] 88. An expression system for expression in a host cell, comprising a nucleic acid encoding an analogue of a sweet protein, said analogue comprising at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE- brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1. P7131 PC00

[0423] 89. The expression system according to any one of items 87 to 88, wherein said substitution is a substitution from a histidine (H) to an asparagine (N) substitution, such as H31N.

[0424] 90. An expression system for expression in a host cell, comprising a nucleic acid encoding an analogue of a sweet protein, said analogue comprising at least three substitutions, said at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1, a second substitution to phenylalanine (F) at a residue corresponding to residue 11 , and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE- brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least three substitutions.

[0425] 91. The expression system according to any one of items 87 or 90, wherein said first and second substitutions comprise or consist of a substitution from tyrosine (Y) to phenylalanine (F), such as Y8F and Y11 F, and wherein said third substitution comprises or consists of a substitution from glycine (G) to alanine (A), such as G35A.

[0426] 92. The expression system according to any one of items 87 to 91 , wherein the sweet protein is Q-brazzein as set forth in SEQ ID NO: 1, pyrE-brazzein as set forth in SEQ ID NO: 10, or des-pyrE-brazzein as set forth in SEQ ID NO: 11 or a functional variant thereof having at least 70% identity, homology or similarity thereto.

[0427] 93. The expression system according to any one of items 87 to 92, wherein said analogue has an increased melting temperature (Tm) compared to the corresponding sweet protein without said at least one substitution, preferably wherein said analogue is sweet. P7131 PC00

[0428] 94. The expression system according to any one of items 87 to 93, wherein said analogue, sweet protein, or functional variants thereof are as defined in any one of items 1 to 43.

[0429] 95. The expression system according to any one of items 87 to 94, wherein the nucleic acid comprises or consists of any one of the nucleic acids as defined in items 64 to 86.

[0430] 96. The expression system according to any one of items 87 to 95, wherein said analogue comprises or consists of Q-brazzein_H31 N as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% identity, homology or similarity thereto comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1.

[0431] 97. The expression system according to any one of items 87 to 96, wherein the nucleic acid comprises or consists of Q-brazzein_H31N as set forth in SEQ ID NO: 4, or a homologue thereof having at least 70% sequence identity or homology to SEQ ID NO: 4.

[0432] 98. The expression system according to any one of items 87 to 95, wherein said analogue comprises or consists of pyrE-brazzein_H31 N as set forth in SEQ ID NO: 15, or a functional variant thereof having at least 70% identity, homology or similarity thereto comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 10.

[0433] 99. The expression system according to any one of items 87 to 95, or 98, wherein the nucleic acid comprises or consists of pyrE-brazzein_H31 N as set forth in SEQ ID NO: 17, or a homologue thereof having at least 70% sequence identity or homology to SEQ ID NO: 17.

[0434] 100. The expression system according to any one of items 87 to 95, wherein said analogue comprises or consists of des-pyrE-brazzein_H30N as set forth in SEQ ID NO: 16, or a functional variant thereof having at least 70% identity, homology or similarity thereto comprising said at least one substitution at one P7131 PC00 residue corresponding to residue 30 of the polypeptide set forth in SEQ ID NO: 11.

[0435] 101. The expression system according to any one of items 87 to 95, or 100, wherein the nucleic acid comprises or consists of des-pyrE-brazzein_H30N as set forth in SEQ ID NO: 18, or a homologue thereof having at least 70% sequence identity or homology to SEQ ID NO: 18.

[0436] 102. The expression system according to any one of items 87 to 95, wherein said analogue comprises or consists of Q-brazzein_Y8F / Y11 F / G35A as set forth in SEQ ID NO: 19, or a functional variant thereof having at least 70% identity, homology or similarity thereto comprising at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution to phenylalanine (F) at a residue corresponding to residue 11 , and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide.

[0437] 103. The expression system according to any one of items 87 to 95, wherein said analogue comprises or consists of pyrE-brazzein_Y8F / Y11 F / G35A as set forth in SEQ ID NO: 20, or a functional variant thereof having at least 70% identity, homology or similarity thereto comprising at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution to phenylalanine (F) at a residue corresponding to residue 11 , and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide.

[0438] 104. The expression system according to any one of items 87 to 95, wherein said analogue comprises or consists of des-pyrE-brazzein_Y7F / Y10F / G34A as set forth in SEQ ID NO: 21 , or a functional variant thereof having at least 70% identity, homology or similarity thereto comprising at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution to phenylalanine (F) at a residue corresponding to residue P7131 PC00

[0439] 11, and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide.

[0440] 105. The expression system according to any one of items 87 to 104, wherein the host cell as defined in any one of items 45 to 55.

[0441] 106. The host cell according to any one of items 45 to 55, said cell comprising the expression system according to any one of items 87 to 105, whereby said host cell produces and / or is capable of producing said analogue of a sweet protein.

[0442] 107. A kit of parts, comprising: i. a host cell; and / or ii. at least one nucleic acid according to any one of the items 64 to 86; and / or iii. the expression system according to any one of items 87 to 105; and iv. optionally instructions for use. preferably wherein the host cell is generally-regarded-as-safe (GRAS), has safe-to-consume status, and / or is non-pathogenic, and / or wherein the host cell is a yeast.

[0443] 108. A sweetener composition or a food, feed, or beverage product comprising an analogue of a sweet protein, obtained by the method according to any one of items 59 to 63.

[0444] 109. A sweetener or sweetener composition comprising an analogue of a sweet protein, wherein said analogue or sweet protein are as defined in any one of items 1 to 43.

[0445] 110. A composition comprising an analogue of a sweet protein, wherein said analogue or sweet protein are as defined in any one of items 1 to 43, and wherein said composition is a food composition, a feed composition, or a beverage composition. P7131 PC00

[0446] 111. The composition according to item 110, wherein the composition is a dairy product or soft drink.

[0447] 112. The composition according to item 110, where in the composition is a food or beverage product that undergoes heating during its preparation, such as a baked or par-baked product.

[0448] 113. The composition according to item 112, wherein the food or beverage product has after heating at least 25% of the sweetness as a control product prepared with the corresponding sweet protein without said at least one substitution, such as at least 50%, such as at least 75%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900%, or such as at least 1000% of the sweetness as a control product prepared with the corresponding sweet protein without said at least one substitution.

[0449] 114. The composition according to any one of items 112 to 113, wherein the analogue comprises or consists of Q-brazzein_H31 N (SEQ ID NO: 3) or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 3 and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, wherein the food or beverage product has after heating at least 25% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 1 (Q-brazzein), such as at least 50%, such as at least 75%, such as at least 100%, such as at least 150%, such as at least 200%, such as at least 300%, such as at least 400%, such as at least 500%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900%, or such as at least 1000% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 1 (Q- brazzein).

[0450] 115. The composition according to any one of items 112 to 113, wherein the analogue comprises or consists of pyrE-brazzein_H31 N (SEQ ID NO: 15) or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 15 comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, P7131 PC00 wherein the food or beverage product has after heating at least 25% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 10 (pyrE-brazzein), such as at least 50%, such as at least 75%, such as at least 100%, such as at least 150%, such as at least 200%, such as at least 300%, such as at least 400%, such as at least 500%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900%, or such as at least 1000% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 10 (pyrE- brazzein).

[0451] 116. The composition according to any one of items 112 to 113, wherein the analogue comprises or consists of des-pyrE-brazzein_H30N (SEQ ID NO: 16) or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 16 comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO:

[0452] I , wherein the food or beverage product has after heating has at least 25% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 11 (des-pyrE-brazzein), such as at least 50%, such as at least 75%, such as at least 100%, such as at least 150%, such as at least 200%, such as at least 300%, such as at least 400%, such as at least 500%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900%, or such as at least 1000% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 11 (des- pyrE-brazzein).

[0453] 117. The composition according to any one of items 112 to 113, wherein the analogue comprises or consists of Q-brazzein_Y8F / Y11 F / G35A (SEQ ID NO: 19) or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 19 and comprising at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution to phenylalanine (F) at a residue corresponding to residue

[0454] I I , and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, wherein the food or beverage product has after heating at least 25% of the sweetness as a control product prepared with a P7131 PC00 polypeptide comprising or consisting of SEQ ID NO: 1 (Q-brazzein), such as at least 50%, such as at least 75%, such as at least 100%, such as at least 150%, such as at least 200%, such as at least 300%, such as at least 400%, such as at least 500%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900%, or such as at least 1000% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 1 (Q-brazzein).

[0455] 118. The composition according to any one of items 112 to 113, wherein the analogue comprises or consists of pyrE-brazzein_Y8F / Y11 F / G35A (SEQ ID NO: 20) or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 20 comprising at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution to phenylalanine (F) at a residue corresponding to residue 11 , and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, wherein the food or beverage product has after heating at least 25% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 10 (pyrE-brazzein), such as at least 50%, such as at least 75%, such as at least 100%, such as at least 150%, such as at least 200%, such as at least 300%, such as at least 400%, such as at least 500%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900%, or such as at least 1000% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 10 (pyrE-brazzein).

[0456] 119. The composition according to any one of items 112 to 113, wherein the analogue comprises or consists of des-pyrE-brazzein_Y7F / Y10F / G34A (SEQ ID NO: 21) or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 21 comprising at least three substitutions comprising or consisting of a first substitution to phenylalanine (F) at one residue corresponding to residue 8 of the polypeptide set forth in SEQ ID NO: 1 , a second substitution to phenylalanine (F) at a residue corresponding to residue 11 , and a third substitution to alanine (A) at a residue corresponding to residue 35 of the same polypeptide, wherein the food or beverage product has after P7131 PC00 heating has at least 25% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 11 (des-pyrE-brazzein), such as at least 50%, such as at least 75%, such as at least 100%, such as at least 150%, such as at least 200%, such as at least 300%, such as at least 400%, such as at least 500%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900%, or such as at least 1000% of the sweetness as a control product prepared with a polypeptide comprising or consisting of SEQ ID NO: 11 (des-pyrE-brazzein).

[0457] 120. Use of an analogue of a sweet protein as a sweet protein and / or sweetener, wherein said analogue and / or sweet protein are as defined in any one of items 1 to 43.

[0458] 121 . Use of the sweetener or sweetener composition as defined in item 108 or 109, as a food product, food material, food ingredient, feed product, feed material, feed ingredient, beverage material and / or beverage ingredient.

[0459] 122. Use of the composition according to item 110, as a food product, food material, food ingredient, feed product, feed material, feed ingredient, beverage material and / or beverage ingredient.

Claims

P7131 PC00Claims1. An analogue of a sweet protein, said analogue: comprising at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or said analogue being a functional variant of the above having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1, preferably wherein the Tm of said analogue is increased compared to the Tm of the corresponding sweet protein without said at least one substitution or said at least three substitutions.

2. The analogue according to claim 1, wherein said substitution to an asparagine (N) is a substitution from a histidine (H) to an asparagine (N) substitution, such as H31 N.

3. The analogue thereof according to any one of the preceding claims, wherein the sweet protein is Q-brazzein as set forth in SEQ ID NO: 1, pyrE-brazzein as set forth in SEQ ID NO: 10, or des-pyrE-brazzein as set forth in SEQ ID NO: 11 , or a functional variant thereof having at least 70% identity, homology or similarity thereto.

4. The analogue according to any one of the preceding claims, wherein the sweet protein is Q-brazzein (SEQ ID NO: 1), and wherein the analogue comprises or consists of Q-brazzein_H31 N as set forth in SEQ ID NO: 3 or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 3; or wherein the sweet protein is pyrE-brazzein (SEQ ID NO: 10), and wherein the analogue comprises or consists of pyrE-brazzein_H31 N as set forth in SEQ ID NO: 15 or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 15; orP7131 PC00 wherein the sweet protein is des-pyrE-brazzein (SEQ ID NO: 11), and wherein the analogue comprises or consists of des-pyrE-brazzein_H30N as set forth in SEQ ID NO: 16 or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 16.

5. The analogue according to any one of the preceding claims, wherein the Tm of said analogue is increased by at least 1°C compared to the Tm of the corresponding sweet protein without said at least one substitution, such as by at least 1 ,5°C, such as by at least 2°C, such as by at least 2.5°C, such as by at least 3°C, such as by at least 3.5°C, such as by at least 4°C, such as by at least 4.5°C, such as by at least 5°C, such as by at least 5.5°C, such as by at least 6°C, such as by at least 6.5°C, such as by at least 7°C, such as by at least 7.5°C, such as by at least 8°C, such as by at least 8.5°C, such as by at least 9°C, such as by at least 9.5°C, such as by at least 10°C, such as by at least 10.5°C, such as by at least 11 °C, such as by at least 11 ,5°C, such as by at least 12°C, such as by at least 12.5°C, such as by at least 13°C, such as by at least 13.5°C, such as by at least 14°C, such as by at least 14.5°C, such as by at least 15°C compared to the Tm of the corresponding sweet protein without said at least one substitution.

6. The analogue according to any one of the preceding claims, wherein the corresponding sweet protein without said at least one substitution has a Tm of 106°C and the analogue has a Tm greater than 106°C, such as a Tm of 107°C, such as a Tm of 108°C, such as a Tm of 109°C, such as a Tm of 110°C, such as a Tm of 111°C, such as a Tm of 112°C, such as a Tm of 113°C, such as a Tm of 114°C, such as a Tm of 115°C.

7. The analogue according to any one of the preceding claims, wherein the analogue comprises or consists of Q-brazzein_H31 N (SEQ ID NO: 3) or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 3, and wherein the Tm of said analogue is between 106°C and 110°C, such as between 108°C and 110°C, such as 110°C; or wherein the analogue comprises or consists of pyrE-brazzein_H31N (SEQ ID NO: 15) or a functional variant thereof having at least 70% identity, homology or106P7131 PC00 similarity to SEQ ID NO: 15, and wherein the Tm of said analogue is between 106°C and 110°C, such as between 108°C and 110°C, such as 110°C; or wherein the analogue comprises or consists of des-pyrE-brazzein_H30N (SEQ ID NO: 16) or a functional variant thereof having at least 70% identity, homology or similarity to SEQ ID NO: 16, and wherein the Tm of said analogue is between 106°C and 110°C, such as between 107°C and 110°C, such as between 108°C and 110°C, such as 110°C.

8. A host cell capable of producing an analogue of a sweet protein or a functional variant thereof, said host cell expressing an analogue comprising at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE-brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, wherein the functional variant of said analogue has at least 70% identity, homology or similarity thereto and comprises said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 ; preferably wherein said substitution is a substitution from a histidine (H) to an asparagine (N) substitution, such as H31 N; and / or wherein the sweet protein is Q-brazzein as set forth in SEQ ID NO: 1 , pyrE- brazzein as set forth in SEQ ID NO: 10, or des-pyrE-brazzein as set forth in SEQ ID NO: 11 , or a functional variant thereof having at least 70% identity, homology or similarity thereto; and / or wherein the analogue has an increased melting temperature (Tm) compared to the corresponding sweet protein without said at least one substitution, preferably wherein said analogue is sweet.

9. The host cell according to claim 8, wherein the host cell is a fungal cell, such as a filamentous fungal cell, or a yeast cell, such as a yeast cell belonging to the genus of Saccharomyces, for example S. cerevisiae, or Komagataella., for example K. phaffii, preferably wherein the yeast cell is a S. cerevisiae cell.

10. A method of producing an analogue of a sweet protein, comprising:107P7131 PC00 i. providing a host cell as defined in any one of claims 8 to 9; ii. incubating and optionally propagating said host cell in a cultivation medium, thereby obtaining a fermentation liquid comprising said analogue, wherein said analogue or sweet protein are as defined in any one of claims 1 to 7, optionally the method comprises a further step of recovering the analogue, preferably wherein the step of recovering the analogue comprises filtering such as by ultrafiltration, and / or concentrating the analogue, for example by ionexchange chromatography, from the fermentation liquid.

11. A nucleic acid encoding an analogue of a sweet protein, said analogue comprising at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 , wherein said substitution is a substitution to an asparagine (N), wherein said sweet protein is Q-brazzein, pyrE-brazzein, or des-pyrE- brazzein, or a functional variant thereof having at least 70% identity, homology or similarity thereto, or a functional variant of said analogue having at least 70% identity, homology or similarity thereto and comprising said at least one substitution at one residue corresponding to residue 31 of the polypeptide set forth in SEQ ID NO: 1 ; preferably wherein said substitution is a substitution from a histidine (H) to an asparagine (N) substitution, such as H31 N; and / or wherein the sweet protein is Q-brazzein as set forth in SEQ ID NO: 1 , pyrE- brazzein as set forth in SEQ ID NO: 10, or des-pyrE-brazzein as set forth in SEQ ID NO: 11 , or a functional variant thereof having at least 70% identity, homology or similarity thereto; and / or wherein the analogue has an increased melting temperature (Tm) compared to the corresponding sweet protein without said at least one substitution, preferably wherein said analogue is sweet.

12. The nucleic acid according to claim 11 , wherein said analogue comprises or consists of Q-brazzein_H31 N as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% identity, homology or similarity thereto; or wherein the nucleic acid comprises or consists of Q-brazzein_H31N as set forth in SEQ ID NO: 4, or a homologue thereof having at least 70% sequence identity108P7131 PC00 or homology to SEQ ID NO: 4 or a nucleic acid encoding the polypeptide of SEQ ID NO: 3; or wherein said analogue comprises or consists of pyrE-brazzein_H31N as set forth in SEQ ID NO: 15, or a functional variant thereof having at least 70% identity, homology or similarity thereto; or wherein the nucleic acid comprises or consists of pyrE-brazzein_H31 N as set forth in SEQ ID NO: 17, or a homologue thereof having at least 70% sequence identity or homology to a nucleic acid encoding the polypeptide of SEQ ID NO: 15; or wherein said analogue comprises or consists of des-pyrE-brazzein_H30N as set forth in SEQ ID NO: 16, or a functional variant thereof having at least 70% identity, homology or similarity thereto; or wherein the nucleic acid comprises or consists of des-pyrE-brazzein_H30N as set forth in SEQ ID NO: 18, or a homologue thereof having at least 70% sequence identity or homology to a nucleic acid encoding the polypeptide of SEQ ID NO: 16.

13. A composition comprising an analogue of a sweet protein, wherein said analogue or sweet protein are as defined in any one of claims 1 to 7, and wherein said composition is a food composition, a feed composition, a sweetener composition, or a beverage composition; or a sweetener comprising or consisting of an analogue of a sweet protein, wherein said analogue or sweet protein are as defined in any one of claims 1 to 7.

14. The composition according to claim 13, wherein the food or beverage product has after heating at least 25% of the sweetness as a control product prepared with the corresponding sweet protein without said at least one substitution, such as at least 50%, such as at least 75%, such as at least 100%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900%, or such as at least 1000% of the sweetness as a control product prepared with the corresponding sweet protein without said at least one substitution.

15. Use of an analogue of a sweet protein as defined in any one of claims 1 to 7 as a sweet protein and / or sweetener; and / or109P7131 PC00 use of the sweetener and / or composition as defined in claim 13, as a food product, food material, food ingredient, feed product, feed material, feed ingredient, beverage material and / or beverage ingredient.

Images