Modified polypeptides and their use

Abstract

The present invention relates to a method for reducing lipid binding of brazzein and / or to method for improving digestibility of brazzein in a subject's digestive track as well as to a modified brazzein polypeptide and uses of it and to a nucleic acid encoding the same.

Description

[0001] MODIFIED POLYPEPTIDES AND THEIR USE

[0002] FIELD OF INVENTION

[0003] The present invention relates to a method for reducing lipid binding of brazzein, to a modified brazzein polypeptide for said method and to a nucleic acid encoding the same. Further, the present invention relates to method for improving digestibility of brazzein in a subject’s digestive track, to a modified brazzein polypeptide for said method and to a nucleic acid encoding the same. The present invention also relates to plant based zero calorie sweeteners and food, snacks, beverages, oral treatment products, and orally administered drugs.

[0004] BACKGROUND

[0005] The world is facing an obesity epidemic, with huge healthcare and economic consequences. Over the period 1990 to 2022 the worldwide prevalence of obesity more than doubled to 14% of men and 19% of women. In developed countries the statistics are often worse, with 43% of American adults being considered obese. This rise in obesity has been associated with a massive rise in related healthcare issues. For example, the prevalence of diabetes has risen from 108 million in 1980 to 537 million adults in 2021 and is predicted to continue rising to over 600 million by 2030.

[0006] The economic impact of obesity has been estimated in 2020 to be around $2 trillion a year or more than 2% of world GDP. Linked to this rise in obesity, sugar consumption continues to rise, with current annual global consumption of 178 million tons. There is an urgent need for a consumer accepted zero calorie alternative to sugar.

[0007] The rise in obesity has hastened the development of alternative sweeteners. There are already a number of sweeteners that are used in the food and beverage industries. These include cyclamate, saccharin, acesulfame, aspartame, neotame, sucralose, stevia extracts and monkfruit sweetener.

[0008] There is no perfect sugar replacement on the market. All artificial sweeteners have issues associated with them, both consumer perception issues (e.g. neotame is the sweetest and is chemically stable but has not dominated the market as the taste profile is problematic) and issues related to their physical properties (e.g. aspartame loses sweetness when heated). There are also consumer-related health concerns including cancer as well as increased rates of metabolic syndrome and type 2 diabetes being associated with the use of artificial sweeteners.

[0009] Natural sweeteners also have issues related to them. Stevia the plant is not approved as a food additive by the FDA, but rather steviol glycosides extracted from Stevia are. RebA, currently the most widely used steviol glycoside, has strong licorice aftertaste and there are consumer concerns regarding the extraction process which uses a range of organic solvents, e.g. acetone, methanol, acetonitrile and isopropanol, to purify these from the plant.

[0010] Brazzein was first described in 1994. It is a naturally occurring sweet protein found in the fruit of the Oubli shrub (Pentadiplandra brazzeana Bail Ion) that has been used as a natural sweetener in West Africa for centuries.

[0011] Brazzein is one of a number of proteins which taste sweet, others include curculin (from Curculingo latifolia), monellin (from Dioscoreophyllum cumminsii), pentadin (from Pentadiplandra brazzeana) and thaumatin (from Thaumatococcus danielli). Of these brazzein has the highest relative sweetness and the best taste profile. In most reported tests it tastes like sugar with no side tastes. This makes it unique.

[0012] Brazzein also has other important bioproperties that make it very amenable for use in the food and beverage industries. Specifically, it is highly soluble, highly thermostable and pH stable over a wide range, especially at acidic pH values. This is important especially for the carbonated drinks industry.

[0013] Brazzein does not refer to a single protein. The naturally occurring protein has three variants which differ at their N-terminus.

[0014] The major form (Q1-Y54, described here by SEQ ID NO: 2) has a glutamine (Q) as the N-terminal amino acid. This residue may cyclize to form pyroglutamic acid. These two states interconvert, with the pyroglutamic acid state being prevalent for brazzein.

[0015] In this context, unless otherwise stated, the amino acid residues refer to the respective residue of SEQ ID NO: 2.

[0016] The minor form of brazzein from the Oubli fruit is one amino acid shorter losing the glutamine and having aspartic acid (D) as its N-terminal amino acid (D2-Y54, described here by SEQ ID NO: 1 ). The different natural variants have been reported to have different relative sweetness. WO 2011 / 05841 discloses a recombinant expression vector for expressing brazzein in Kluyveromyces lactis.

[0017] Lim Jin-Kyung et al. 2015 (J. Sci Food Agric 2016; 96: 3202-3206) discloses Glu53Ala, Glu53Asp, Glu53Lys and Glu53Arg mutations on the des-pE1M-brazzein lacking the N-terminal pyroglutamate.

[0018] While brazzein has many very highly favourable properties, the use of brazzein in the food and beverage industry has issues which should be overcome.

[0019] Wild-type brazzein undergoes a conformational change switching between monomeric and oligomeric states. This conformational change alters the relative sweetness of the protein up to 4x. The conformational change is triggered by certain lipids.

[0020] Observed strong interaction between brazzein and certain lipids is of potential concern for consumers. This raises both safety concerns and concerns about the potential for variable sweetness of the protein in different products.

[0021] An indigestible brazzein polypeptide is more likely to trigger an unwanted immune response and to have a potential environmental impact and / or an impact on the flora and fauna of the gut (brazzein is a member of the plant defensin family and defensins are anti-microbial or anti-fungals). This may cause severe regulatory approval issues.

[0022] There is thus a need for brazzein polypeptide(s) with improved biophysical properties and non-com promised sweetness profile.

[0023] There is a need for a zero-calorie sweetener that has a clean sugar-like sweet taste with no side tastes or unpleasant aftertaste. The ideal sweetener would not require extra flavoring to be used to hide unwanted tastes. There is also a need for alleviating or even eliminating potentially harmful effects when consumed.

[0024] SUMMARY

[0025] The present invention meets or alleviates at least some of the needs.

[0026] The present invention relates to methods of preparing and producing brazzein polypeptides with improved biophysical properties and sweetness profile when compared to wild-type brazzein which make it highly amenable for use in the food and beverage industry. The present invention relates also to nucleic acids encoding modified brazzein polypeptides and sweeteners and food, such as a food product, a snack or a beverage, comprising such modified brazzein polypeptide.

[0027] The aspects of the present invention are illustrated in the appended claims.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1. Sequence alignment of the mature form of brazzein with reported Ptdlns(4,5)P2 binding members of the defensin family.

[0030] Figure 2. Lipid binding by recombinant wild-type brazzein by dot blot.

[0031] Figure 3. SDS-PAGE analysis of brazzein wild-type variants under non-reducing conditions. The proteins migrate with apparent molecular weights of an octamer (Q1-Y54) and hexamer (D2-Y54).

[0032] Figure 4. shows sweetness determining regions of brazzein. The underlined loop regions represent consensus sweetness determining regions. The numbers above and below the sequence represent the number of publications reporting an increase or decrease in relative sweetness of the protein at that position.

[0033] Figure 5. shows an SDS-PAGE analysis of N-terminal His-tagged brazzein under non-reducing conditions. Both the Q1-Y54 and D2-Y54 variants run at the apparent molecular weight of a dimer.

[0034] Figure 6. shows superposition of the X-ray structure of the Glu53Lys variant (dark grey) with the published (PDB: 4HE7) wild-type (wt) structure (light grey). The spheres represent bound sodium ions.

[0035] Figure 7. shows thermal stability of wild-type (wt) brazzein and the Glu53Lys variant, measured by thermofluor in 20 mM Tris, 150 mM NaCI. Angiopoietin-like 4 (Angptl4) with a lower thermal stability is shown for comparison.

[0036] Figure 8. shows lipid binding by recombinant wild-type brazzein and Glu53Lys variant by dot blot.

[0037] Figure 9. shows an SDS-PAGE analysis of a representative time-dependent SIF digestion assay using trypsin and chymotrypsin for His-tagged Q1-Y54 variants Y11 F / E53K, K27F / E53K, and E53K (positions marked with a double arrow). Bovine serum albumin (BSA, marked with an arrow) was used as a positive control. Figure 10. shows an SDS-PAGE analysis of semi-purified brazzein variants under non-reducing conditions.

[0038] DETAILED DESCRIPTION

[0039] The present disclosure provides modified brazzein polypeptides and a method for their preparation and production. The modified brazzein polypeptides may also be referred to as brazzein variants, or brazzein mutants. Said modified brazzeins have biophysical properties that make them amenable for use in the food and beverage industry. Their production methods will not cause health concerns, they are metabolized when consumed and perceived as being natural. In addition, they can be produced sustainably and economically.

[0040] The present brazzein variants exhibit brazzein-like properties while offering higher potency, delivering sweetness several thousand times greater than sugar on a weight basis. It is suitable for a broad range of food and beverage applications due to its high sweetness intensity, clean taste, and excellent thermal and pH stability. The present brazzein variants exhibit enhanced sweetness compared to wild-type brazzeins. The sweetener can be incorporated into carbonated soft drinks, flavored waters, juices, and functional beverages, where it remains stable under acidic and pasteurization conditions while providing sugar-like sweetness. In dairy and plantbased products such as flavored milks, yogurts, and fermented alternatives, it maintains sweetness through processing and storage. Its stability under baking temperatures also allows use in bakery and confectionery formulations, where it is typically combined with bulking agents to maintain desirable texture. The ingredient performs well in sauces, dressings, and other heat-processed or acidic systems. To achieve optimal sweetness quality and mouthfeel, it may be blended with other sweeteners such as stevia, monk fruit, or allulose, along with fibers or other bulking components that enhance body and overall sensory balance in reduced-sugar formulations.

[0041] Two members of the plant defensin family, NaD1 (from Nicotiana alata) and TPP3 (Solatium lycopersicum), are reported to bind phospholipids. The homology between brazzein and these two proteins is low (Fig. 1), except for the conserved cysteines which define the protein family. In particular, residues reported to be directly involved in phospholipid binding by NaD1, including His33 and Lys36 which form salt bridges with the phospho-group of a lipid, are conserved between NaD1 and TPP3 but not brazzein.

[0042] It was therefore unexpected that recombinant wild-type brazzein was found to bind to some lipids in a dot blot test (Fig. 2). Specifically, brazzein gave a strong signal with phosphatidic acid, Ptdlns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) and to a lesser extent with Ptdlns(3,4,5)P3 (phosphatidylinositol 3,4,5-triphosphate) and weak binding to Ptdlns(5)P, Ptdlns(3,4)P2 and Ptdlns(3,5)P2. No visible binding was seen with cardiolipin, cholesterol, diacylglycerol, lysophosphatidic acid, lysophospho-choline, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, Ptdlns, Ptdins(3)P, Ptdlns(4)P, sphingomyelin, sphingosine-1 -phosphate, triglyceride, or 3-sulfogalactosyl-ceramide. While both brazzein and NaD1 bind to Ptdlns(4,5)P2 and Ptdlns(3,4,5)P3, the lipid binding specificity of brazzein is different from that of NaD1 which shows strong binding to Ptdlns(3,5)P2 and weak binding to cardiolipin and phosphatidic acid. This suggests a different and unpredictable mode of lipid binding for brazzein, consistent with the sequence analysis (Fig. 1).

[0043] The cellular role of phosphatidic acid is difficult to study as it has a short half-life. It is reported that it plays a role in ion channel gating e.g. it modulates the function of voltage-gated potassium channel (Kv) and inward rectifier potassium channel (Kir2.2) and in cellular signaling by interaction with a wide range of proteins such as mammalian target of rapamycin (mTOR) and TWIK-related potassium channel (TREK-1).

[0044] Ptdlns(4,5)P2 is present at low concentrations in cell membranes, with its level being highly regulated by multiple mechanisms.

[0045] The cellular role of Ptdlns(4,5)P2 is well characterized. Ptdlns(4,5)P2 is a key intermediary in the inositol 1,4,5-trisphosphate / diacylglycerol (IP3 / DAG) signaling pathway. It modulates multiple cellular processes, including regulation of i) cytoskeleton dynamics by binding to F-actin regulatory proteins; ii) potassium and sodium channel function including the M-channel by direct interactions; iii) Class A G-protein-coupled receptors (GPCRs) by binding directly; and iv) G-protein-coupled receptor kinase 2 (GRK2), by recruiting it to the membrane. Ptdlns(4,5)P2 depletion severely impairs synaptic vesicle and large dense core vesicle secretion.

[0046] Ptdlns(3,4,5)P3 is also involved in signaling pathways, binding to the pleckstrin homology (PH) domain of proteins such as serine / threonine protein kinase Akt, phosphoinositide-dependent kinase-1 (PDK1) and ARF nucleotide-binding site opener (ARNO). Akt is a protein kinase which activates signaling pathways required for cell growth. In neurons, Ptdlns(3,4,5)P3 has been linked to synaptic strengthening and long-term potentiation, with Ptdlns(3,4,5)P3 depletion being linked to synaptic depression and impairment of memory consolidation. Thus, the wild-type brazzein binds to, among other, to phosphatidic acid, phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P2) and phosphatidylinositol (3,4,5)-trisphosphate (Ptdlns(3,4,5)P3) which all three are involved in cell signaling. Phosphatidic acid and Ptdlns(4,5)P2 are also involved in activation of some lipid gated ion channels. The cell signaling pathways that involve these include memory consolidation, apoptosis and cytokinesis.

[0047] Therefore this potentially strong interaction between brazzein and phosphatidic acid, PtdIns(4,5)P2 or PtdIns(3,4,5)P3 (Fig. 2) is a concern if brazzein is to be consumed in high amounts over a long time period as each of these three lipids are involved in intracellular signaling pathways.

[0048] In addition to the unexpected binding to lipids, the inventor has surprisingly found that wild-type brazzein variants oligomerize (Fig. 3) and that point mutations near the N- or C-terminus or around the loop Lys30-Arg34 may abolish phospholipid binding and / or oligomerization. Further, the inventor has surprisingly been able to produce brazzein mutants which do not bind to lipids in the way that the wild-type proteins do, while retaining the other necessary biophysical properties and sweetness of the wild-type proteins.

[0049] In this connection, term ‘genetically modified to have a reduced lipid binding’ means that the amino acid sequence of brazzein has been modified to decrease or prevent binding of the brazzein polypeptide to at least some lipids of the human body. Lipid binding of the genetically modified polypeptide is compared with the wild-type polypeptide which does not carry the defined genetic modifications. This can be examined by regular binding studies.

[0050] Brazzein is not digested in conditions simulating those in the human digestive tract and thus is likely to trigger an unwanted immune response, to have a potential environmental impact or an impact on the flora and fauna of the gut (brazzein is a member of the plant defensin family and defensins are anti-microbial or anti-fungals). There could be potentially severe multiple accumulating long-term effects due to the lack of digestion of brazzein. This may also cause severe regulatory approval issues, intervention requirements, and possible concerns on the use of brazzein proteins with the identified digestion issue including the wild-type protein.

[0051] The primary proteases of the digestive system are pepsin, trypsin and chymotrypsin. Pepsin has a very broad specificity but is said to favour aromatic amino acids (F, Y, W) at the PT position and to have a preference for F / L / M at the P1 position. Chymotrypsin also favours aromatic amino acids (or L), but at the P1 position. Trypsin favours basic amino acids (K, R) at the P1 position.

[0052] Based on the sequence of brazzein it would be expected to be cleaved in both simulated intestinal fluid (SIF) and simulated gastric fluid (SGF) as it contains 9 K / R amino acids and 7 Y / F / W amino acids. The fact that it is not cleaved in either of these most probably results from the high disulfide density (4 disulfides in a 53 or 54 amino acid protein) which prevent accessibility of the proteases to their cleavage sites. This was confirmed by successful digestion after reduction of the disulfide bonds in the brazzein. Brazzein’s sweetness depends upon its native structure and hence on its disulfide bonds so these cannot be removed to allow digestion.

[0053] The inventor has surprisingly been able to introduce substitutions that allow digestion in either SGF and / or SIF without compromising the beneficial biophysical properties of brazzein polypeptide. The brazzein polypeptide disclosed herein, wherein substitutions are presented, may be referred to as a brazzein variant or a modified brazzein polypeptide.

[0054] In this connection, term ‘genetically modified to be digestible’ means that the amino acid sequence of brazzein has been modified to increase digestibility in a subject’s digestive tract. This can be examined e.g., by using artificial fluid simulating assays such as SGF and / or SIF.

[0055] The present invention relates to a method for reducing lipid binding of brazzein polypeptide when compared to wild-type brazzein polypeptide without defined modifications. The method may be or comprise a method for preparing a brazzein which is safe for consumers, for example comprising alleviating and / or eliminating harmful effects when consumed, and / or improving metabolization of the brazzein. Said method comprises introducing a substitution to amino acid E53 of brazzein polypeptide defined by SEQ ID NO: 1, 2, 3 or 4 or a sequence having at least at least 90% identity to any of said sequences, wherein the substitution site corresponds the respective residue of said mature wild-type brazzein (SEQ ID NO: 2). Within the context of this disclosure the term “mature” in connection to protein refers to the functional form of a protein that has completed its maturation process, which involves folding and post-translational modifications in a cell in which it is being produced. Said brazzein polypeptide may have at least 93%, 95%, 97% or 98% identity to any of said sequences wherein the substitution site corresponds the respective residue of SEQ ID NO: 2. A E53K substitution may be introduced. Such modified brazzein polypeptide has a reduced lipid binding when compared to a wildtype polypeptide. The brazzein polypeptide described above may be further genetically modified by introducing one or more substitution selected from Y11F and K27F substitution(s). Such modified polypeptide has a reduced lipid binding and is digestible in a subject’s digestive tract.

[0056] Further, the substitution (substitutions) at position(s) Y51F or Y54F, or Y51F and Y54F may be introduced, wherein the substitution site corresponds the respective residue of SEQ ID NO: 2.

[0057] The present invention relates also to a brazzein polypeptide having SEQ ID NO: 1, 2, 3 or 4; or a sequence having at least at least 90% identity to any of said sequences characterized by having a substitution at amino acid E53, wherein the substitution site corresponds the respective residue of SEQ ID NO: 2. Said brazzein polypeptide may have at least 93%, 95%, 97% or 98% identity to any of said sequences characterized by having a substitution at amino acid E53, wherein the substitution site corresponds the respective residue of SEQ ID NO: 2. The substitution may be E53K.

[0058] Said polypeptide may further comprise at least one substitution at a position selected from Y11 and K27, especially Y11 F and K2F7, wherein the substitution site corresponds the respective residue of SEQ ID NO: 2. Still further said polypeptide may comprise substitution(s) Y51F or Y54F, or Y51F and Y54F, wherein the substitution site corresponds the respective residue of SEQ ID NO: 2.

[0059] Said polypeptide has a reduced lipid binding when compared to a wild-type polypeptide. The lipid may comprise one or more selected from the group consisting of cardiolipin, cholesterol, diacylglycerol, lysophophatidic acid, lysophosphocholine, phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, Ptdlns, Ptdins(3)P, Ptdlns(4)P, sphingomyelin, sphingosine-1 -phosphate, triglyceride, or 3-sulfogalactosyl-ceramide. The lipid may be phosphatidic acid, PtdIns(4,5)P2 or PtdIns(3,4,5)P3.

[0060] The invention relates also to a nucleic acid encoding a brazzein polypeptide operably linked into transcriptional regulatory sequences and encoding the polypeptide defined here i.e. by SEQ ID NO: 1, 2, 3, or 4 with one or more mutations E53 or E53K and optionally one of more of Y11F, K27, Y51F or Y54F, for lipid binding and Y11F and K27F and one or more of Y51F or Y54F and E53K for improved digestibility, wherein the substitution site corresponds the respective residue of SEQ ID NO: 2. The present invention relates also to a method for improving digestibility of brazzein in a subject’s digestive track, comprising introducing at least one, such as one or more, substitution selected from Y11 F and K27F to brazzein polypeptide defined by SEQ ID NO: 1, 2, 3 or 4 or a sequence having at least 90% identity, or at least 93%, 95%, 97% or 98% identity to any of said sequences, wherein the substitution site corresponds the respective residue of said mature wild-type brazzein (SEQ ID NO: 2).

[0061] Further, substitution(s) Y51F or Y54F, or Y51F and Y54F may be introduced, wherein the substitution site corresponds the respective residue of said mature wildtype brazzein (SEQ ID NO: 2).

[0062] Also E53K substitution, wherein the substitution site corresponds the respective residue of said mature wild-type brazzein (SEQ ID NO: 2) may be introduced.

[0063] The present invention relates also to a brazzein polypeptide having SEQ ID NO: 1, 2, 3 or 4; or a sequence having at least 90% identity, or at least 93%, 95%, 97% or 98% identity to any of said sequences, characterized by comprising at least one substitution (substitutions) selected from Y11F and K27F, wherein the substitution site corresponds (sites correspond) the respective residue(s) of said mature wildtype brazzein (SEQ ID NO: 2). Such modified polypeptide has an improved digestibility compared to wild-type brazzein in a subject’s digestive track.

[0064] The polypeptide may further comprise substitution(s) Y51F or Y54F, or Y51F and Y54F, wherein the substitution site corresponds the respective residue of said mature wild-type brazzein (SEQ ID NO: 2).

[0065] Further, the polypeptide may comprise substitutions Y11 F, K27F and Y51 F, wherein the substitution site corresponds the respective residue of said mature wild-type brazzein (SEQ ID NO: 2).

[0066] Still further the polypeptide may comprise E53K substitution, wherein the substitution site corresponds the respective residue of said mature wild-type brazzein (SEQ ID NO: 2).

[0067] In one embodiment the polypeptide comprises substitutions Y11 F, K27F, Y51 F and E53K, wherein the substitution site corresponds the respective residue of said mature wild-type brazzein (SEQ ID NO: 2).

[0068] Said polypeptide may have improved digestibility in a subject’s gastrointestinal tract when compared to corresponding non-modified i.e. wild-type brazzein. The present polypeptides may be provided in compositions and / or products comprising one or more of the polypeptides and one or more further agents and / or products, such as edible and / or drinkable substances and / or components of edible and / or drinkable products, and additives, active agents and / or the like.

[0069] The present invention relates also to a sweetener comprising the brazzein polypeptide or peptides described here, i.e., the brazzein variant(s). The sweetener may consist of the brazzein polypeptide or peptides described here. The sweetener may be provided as a dry product, which may be crystalline, or as an aqueous solution. The sweetener may comprise one or more additional agents (additives), such as filler(s), coloring agents, flavoring agent(s) and / or the like.

[0070] Further, the present invention relates also to a food, more particularly to a food product and to other edible or drinkable products, preferably for human consumption, such as a snack, a beverage, an orally administered drug i.e. a drug for oral administration, an oral care or a treatment product containing brazzein polypeptide or polypeptides described here. The brazzein polypeptide or polypeptides described here may be the only added sweetening agent in said food, snack or beverage.

[0071] The food or the food product may comprise any applicable food or food product, especially ones which require sweetening. The food product may be a snack, a candy, a cake, a biscuit, ice cream, a meal, a bread, yogurt, and / or the like edible and / or consumable products. Examples of snacks include bars such as chocolate bars, protein bars, energy bars, candy bars and / or combinations thereof and the like. Further examples of snacks include chips, such as potato chips, nuts, fruits, berries and vegetables, and / or products comprising thereof.

[0072] The beverage may be a non-alcoholic beverage or an alcoholic beverage. Examples of beverages include sodas, juices, ciders, alcoholic long drinks or mixers for alcoholic long drinks, and / or the like products.

[0073] The orally administrated / administrable drugs or pharmaceuticals may include lozenges, therapeutic mouthwashes and / or the like oral products, which may comprise one or more active pharmaceutical ingredients, flavoring agents, and / or the like agents commonly used in the art.

[0074] The oral care or treatment product may be or comprise the orally administrated drug, or the products may be non-therapeutic products, i.e. lacking active pharmaceutical ingredients. Examples of oral care or treatment products include mouthwashes and other rinsing oral products, toothcare products, such as toothpastes, and / or the like. In this connection term ‘added sweetening agent’ means sweetness not derived from the main constituents such as fruit, berries or grain. Evidently, traditional sweeteners such as sugar or syrup as well as low or zero calorie sweeteners can be used in varying combinations with modified brazzeins described here.

[0075] The sweetener, food, snack, beverage, orally administered drug, oral care or treatment product containing brazzein described here have the advantages of the brazzein polypeptide discussed here, reduced lipid binding characterized by comprising the brazzein polypeptide(s) described here or consisting of the polypeptide(s) described here.

[0076] Alternatively, the sweetener, food, snack, beverage, orally administered drug, oral care or treatment product containing brazzein described here have the advantages of the brazzein polypeptide discussed here, reduced digestibility when compared to respective non-mutated wild-type polypeptide characterized by comprising the brazzein polypeptide(s) described here or consisting of the polypeptide(s) described here.

[0077] The brazzein polypeptides described here are zero-calorie sweeteners having a good taste profile by being sweet without an unpleasant side taste. Due to the reduced lipid binding and / or digestibility (when compared to a wild-type brazzein polypeptide) they are also safe for use in human nutrition.

[0078] Sweetness:

[0079] Also, a method for increasing dimerization of brazzein comprising introducing a substitution to E53 position of brazzein polypeptide is described here. For use as a sweetener, any variant made in brazzein to abolish phospholipid binding should not reduce the sweetness of the protein or its thermostability. There are a large number of mutations in brazzein reported to either increase or decrease its relative sweetness. Despite this the evidence in the literature determinants in brazzein is contradictory. There are two consensus regions in the literature, in particular the loop Tyr39-Arg43 contains multiple sites whose mutation increases or decreases the sweetness of the protein (Fig. 4). The second consensus region is the loop Lys15-Ala19.

[0080] Sweetness determining regions of brazzein are shown in Fig. 4. The underlined loop regions represent consensus sweetness-determining regions. The numbers above and below the sequence represent the number of publications reporting an increase or decrease in relative sweetness of the protein at that position. The third region reported in the literature to modulate sweetness is on another face of the protein from the first two. It includes the N- and C-terminus and the loop Lys30-Arg34. Given that it is not juxtaposed to the other sweetness determining regions, the possible direct role of this region in sweetness has been questioned. One possible explanation for this is that this region may be involved in binding phospholipids. Binding of brazzein to phospholipids may reduce the effective concentration available to bind to the sweet receptor and elicit the sweet response. Similarly, potential oligomerization of brazzein in the presence of phospholipids may decrease the effective concentration of the sweet form of brazzein. This would be consistent with the clustering of positively charged residues Lys30 / His31 / Arg33 as well as potentially Lys6 whose side chain is close in the structure to that of Lys30, being involved in interacting with the negatively charged head group of the bound phospholipid.

[0081] In Fig. 1, the underlined residues interact with the bound ligand (PDB: 4CQK). While there is good conservation of ligand binding residues between NaD1 and TPP3, conservation is poor with brazzein. In particular, His33 in NaD1 (bolded) which forms an electrostatic attraction with the phospho-group is substituted by a negatively charged Glu36 in brazzein, which would be expected to inhibit binding of phosphogroups at this position. In addition, Lys36 in NaD1 (bolded) which also forms an electrostatic attraction with the bound ligand is replaced by an aromatic group in brazzein. Ile37 / Leu38 in NaD1 which lie close to another phospho-group in the bound ligand are replaced by negatively charged Asp40 / Glu41 in brazzein, which would again be predicted to prevent phospholipid binding.

[0082] Supporting evidence for this comes from SDS-PAGE analysis of the modified brazzein with an N-terminal His-tag. This extension results in the protein changing its mobility relative to the wild-type proteins (compare Fig. 5, to Fig. 3). Both the Q1 -Y54 and D2-Y54 His-tagged variants run at the apparent molecular weight of a dimer. More than 40 different mutations subsequently made in a N-terminal His-tagged background also ran at the apparent dimeric position (data not shown).

[0083] The brazzein polypeptide, such as the polypeptide defined by SEQ ID NO: 1, 2, 3 or 4, can be modified by C-terminal or N-terminal addition of His-tag to be used in purification. Lipid binding:

[0084] In the lipid-binding study, 64 brazzein variants containing single point mutations, including non-tagged and His-tagged variants, were screened for having the following desired properties:

[0085] 1 ) Good production yields

[0086] 2) High thermal stability

[0087] 3) The variant ran at the dimeric position in non-reducing SDS-PAGE.

[0088] 4) Sweetness at the level of wild-type protein or higher, with no side tastes (as per the wild-type protein)

[0089] As dodecyl sulfate (as found in SDS) is structurally similar to phospholipids, a mobility shift is observed in non-reducing SDS-PAGE upon SDS binding (mimicking lipid binding) to brazzein. This allowed for rapid screening of variants with decreased lipid binding, observed as dimeric states in SDS-PAGE, similar to the mobility shift observed with His-tagged wild-type brazzein (Fig. 5).

[0090] Many of the mutants screened failed on one or more of the four points above, with oligomeric states ranging from dimers and trimers through other higher states to octamers. However, in addition to extensions (His-tag) to the N- and C-terminus of the protein, five single point mutation variants without His-tag showed a dimeric state in non-reducing SDS-PAGE (Table 1). Of these five candidates, E53K variant (both in the Q1-Y54 and D2-Y54 backgrounds), i.e., a brazzein variant with E53K substitution, met all of the above-mentioned criteria.

[0091] Table 1: Properties of single point mutant brazzein variants as well as their double E53K mutant variants from the lipid-binding assay. Yields relative to wild-type proteins using CyDisCo™ system in the cytoplasm of E. coli as judged by SDS-PAGE analysis of lysates and oligomer status as visualized in non-reducing SDS-PAGE. Variants which aggregate after heating to at least 80°C for at least 20 minutes in the E. coli lysate are indicated. His-tagged variants of the point mutants were omitted from this table for clarity.

[0092] Estimated Construct Yield oligomeric form Brazzein Q1-Y54 ++++ multimer

[0093] Brazzein Q1-Y54, His-tagged ++++ dimer

[0094] +++, but not

[0095] Brazzein Q1-Y54 K5F E53K heat stable monomer

[0096]

[0097] Brazzein Q1-Y54 N10F + oligomer Brazzein Q1-Y54 N10F, E53K ++++ dimer Brazzein Q1-Y54Y11F ++++ tetramer Brazzein Q1-Y54 Y11F, E53K +++ dimer Brazzein Q1-Y54 K27F + oligomer Brazzein Q1-Y54 K27F, E53K ++++ dimer Brazzein Q1-Y54 K30A ++ monomer ++++, but not Brazzein Q1-Y54 K30F E53K heat stable monomer Brazzein Q1-Y54 H31A ++++ multimer Brazzein Q1-Y54 H31F ++++ multimer Brazzein Q1-Y54 H31F E53K ++++ monomer Brazzein Q1-Y54 R33A ++++ multimer Brazzein Q1-Y54 R33E +++ multimer Brazzein Q1-Y54 R33F ++++ multimer Brazzein Q1-Y54 R33F E53K ++ monomer Brazzein Q1-Y54 D40F +++ dimer Brazzein Q1-Y54 D40F, E53K + dimer Brazzein Q1-Y54 D50E +++ multimer ++++, but not Brazzein Q1-Y54 D50K heat stable dimer Brazzein Q1-Y54 E53A ++++ trimer Brazzein Q1-Y54 E53K ++++ dimer

[0098] Brazzein D2-Y54 ++++ multimer Brazzein D2-Y54, His-tagged ++++ dimer Brazzein D2-Y54 K3A ++++ multimer Brazzein D2-Y54 K3E ++++ multimer Brazzein D2-Y54 N10F +++ oligomer Brazzein D2-Y54 N10F, E53K ++++ dimer Brazzein D2-Y54 Y11F ++++ tetramer Brazzein D2-Y54 Y11F, E53K +++ dimer Brazzein D2-Y54 K27F +++ oligomer Brazzein D2-Y54 K27F, E53K +++ dimer Brazzein D2-Y54 D40F +++ dimer Brazzein D2-Y54 D40F E53K + dimer

[0099]

[0100] Brazzein D2-Y54 E53K ++++ dimer Subsequently, the crystal structure of the E53K variant was solved (Fig. 6). As expected, it showed the same overall structure (RMSD 0.794A) and thermal stability (Fig. 7) as the wild-type protein.

[0101] In parallel to this lipid binding using a dot blot array and an anti-brazzein antibody was screened (Fig. 8). Lipid binding by the wild-type protein to phosphatidic acid, Ptdlns(4,5)P2 and Ptdlns(3,4,5)P3, were found. The E53K variant showed no visible lipid binding in the dot blot.

[0102] To support these data, several variants, which were tetramers or in a higher oligomeric state in the mobility-shift-based lipid-binding assay, such as Y11F and K27F, shifted to a dimeric position when introducing additional E53K mutation (Table 1).

[0103] All in all, as E53K variant was also found to taste sweet (see “Example 3: taste panel” later in the document), these results indicate that the E53K variant provides a good solution to the lipid-binding problem.

[0104] Digestibility:

[0105] In the digestion study, 90 variants comprising single or multiple mutations were screened for having the desired properties:

[0106] 1) Digestibility in in vitro SIF and / or SGF

[0107] 2) Good production yields

[0108] 3) High thermal stability

[0109] 4) Sweetness akin to the wild-type protein and / or E53K variant

[0110] Most of the variants screened failed on one or more of the four points above. The variant E53K, which solved the lipid-binding problem and met all the other criteria, failed to digest in SIF and SGF. However, two variants with single point mutations met all four criteria. These were the Y11 F and K27F variants. The criteria were also met with having these point mutations in the E53K background, i.e., the brazzein polypeptide with E53K substitution. A representative time-dependent digestion reaction is shown in Fig. 9 with Y11 F / E53K and K27F / E53K variants digested over 70% within 3 hours.

[0111] In addition, some variants with multiple point mutations, e.g. Y11F / Y51F and Y11F / Y54F (in the E53K background) also met the four criteria. Other mutations such as N10F or D40F showed increased proteolysis compared with the wild-type protein in SIF and / or SGF but showed less desirable other biophysical properties than Y11 F or K27F, e.g. lower sweetness. Production:

[0112] For brazzein production, growth of Pentadiplandra brazzeana Bail Ion and extraction of brazzein is not economically viable on a large scale due to the low protein level in the fruit (0.36%).

[0113] A number of different recombinant approaches have previously been adopted. These include production in plants and by fermentation.

[0114] Production in maize was supposed to have been brought to market under the name “Cweet” in 200912010, but the economics of production resulted in a switch by the company to a “bio-fermentation” method, but this has also not come to market -presumably due to the economics of production. The yield from maize seed was 54 mg / kg of seed and with a protein purity of 70.7% (non-proteinaceous contaminants were not considered).

[0115] Brazzein has been made by a chemical route using the fluoren-9-yl-methoxycarbonyl solid-phase synthesis route. The material produced was identical to natural / wi Id-type brazzein including by taste evaluation, while the D enantiomer was essentially tasteless. While chemically synthesized long peptides have been accepted as being biosimilar to those produced recombinantly, the length, complexity of disulfide bonds and the necessary extensive purification procedure make this an uneconomical route for production of brazzein.

[0116] A variety of bacterial production systems have been reported with most focusing on E. coli production either in the periplasm, the cytoplasm, or as a fusion protein in the cytoplasm. The maximum yield reported for in vivo folded material is 35 mg / L, far below economic viability, and acutely toxic compounds (e.g. cyanogen bromide) are used in the preparation. Higher yields were obtained as fusion proteins in the cytoplasm, but the material required purification steps inappropriate for scale up and it was unclear how native disulfide bonds were formed. The product also had the incorrect molecular weight to be brazzein with disulfides intact.

[0117] Similarly, a variety of yeast production systems have been reported. These include production in Kluyveromyces lactis and Pichia pastoris. The production system patented by Tate & Lyle has the highest yields yet reported, with yields of 280 mg / L from 1000 L fermentations. This is below economically viable levels for the wild-type protein.

[0118] The present solution to the economic production of brazzein is to combine production in E. coli (the most cost-effective fermentation system in the market), with production in the cytoplasm (the largest compartment in the cell enabling higher yields), with patented CyDisCo™ technology (see e.g. WO 2010 / 139858 and WO 2012 / 104494) developed for the active formation of native disulfide bonds. It is to be understood that the terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0119] Protein synthesis in E. coli starts with a methionine residue. This initiating methionine is incorporated into the brazzein polypeptides (SEQ ID NO: 3 and 4) at the start of the synthesis but is subsequently cleaved off by endogenous methionine aminopeptidases to form the mature brazzein polypeptides (SEQ ID NO: 1 and 2).

[0120] The invention is illustrated below by the following non-limiting examples. It should be understood that the embodiments given in the description above and the examples are for illustrative purposes only, and that various changes and modifications are possible within the scope of the invention. Various features of the invention described here in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

[0121] EXAMPLES

[0122] Brazzein is a member of the plant defensin family of proteins. As the name suggests many defensins are involved in defense of the host organism and have antimicrobial, anti-fungal or anti-insect properties. However, other members of the defensin family have a wide range of other functions such as mediation of chemotaxis and anti-inflammatory activity.

[0123] The fruit of the Oubli bush has been used as a natural sweetener for centuries and no evidence exists that when consumed in small quantities brazzein has any deleterious effects to human health. However, the wider introduction of brazzein as a sweetener in the food and beverage industry may result in much higher consumption and throughout the year, rather than just when berries are in season.

[0124] As such the inventors wanted to know what the function of brazzein may be. Using high concentrations of purified recombinant brazzein we tested for anti-microbial, anti-fungal or anti-insect cell properties, with all tests being negative (data not shown). The range of tests undertaken was then expanded.

[0125] Example 1: Lipid binding

[0126] Lipid binding can be difficult to screen, but dodecyl sulfate (as found in SDS) has some structural similarities with phospholipids and so SDS binding may mimic phospholipid binding and binding may be seen by mobility shifts in non-reducing SDS-PAGE. This is what is observed, both wild-type variants of brazzein (Q1-Y54 and D2-Y54) show much slower mobility in non-reducing SDS-PAGE (Fig. 3).

[0127] Figure 3: SDS-PAGE analysis of brazzein wild-type variants under non-reducing conditions. The proteins migrate with apparent molecular weights of an octamer (Q1-Y54, lane 2) and hexamer (D2-Y54, lane 3). The monomeric form of brazzein is shown as comparison (lane 1).

[0128] Supporting evidence for this comes from SDS-PAGE analysis of brazzein with an N-terminal His-tag. This extension results in the protein changing its mobility (Fig.

[0129] 5).

[0130] Figure 5: SDS-PAGE analysis of wild-type brazzein containing an N-terminal His-tag. Both non-reduced His-tagged wild-type variants (Q1-Y54, lane 3; D2-Y54, lane 4) run at the apparent molecular weight of a dimer. Under reducing conditions (lanes 1-2, respectively), they run as monomers. Given proteins containing disulfide bonds can migrate at abnormal apparent molecular weights, this does not mean the protein is a dimer. Subsequent analysis by gel filtration and dynamic light scattering suggest all variants of brazzein tested are monomeric in solution in the absence of phospholipids or SDS (data not shown).

[0131] Example 2

[0132] Based on result shown in Fig. 5 it was hypothesized that making point mutations near the N- or C-terminus or around the loop Lys30-Arg34 could abolish phospholipid binding and / or oligomerization. Mutations were made and screened based on i) the ability to be made in good yields; ii) thermal stability (the protein remaining soluble when the cell lysate expressing the protein was heated to at least 80°C for at least 20 minutes; iii) the oligomerization state in non-reducing SDS-PAGE (Fig. 10; Table 1).

[0133] Fig. 10 shows the result of SDS-PAGE analysis of semi-purified brazzein variants under non-reducing conditions. Lane 1 = wt Q1-Y54; lane 2 = wt D2-Y54; lane 3 = Asp2Ala; lane 4 = Asp2Lys; lane 5 = Lys3Ala; lane 6 = Lys3Glu; lane 7 = Lys3Cys / Cys4Lys; lane 8 = Lys30Ala; lane 9 = Lys30Phe; lane 10 = Lys30Glu; Lane 11 = His31Ala; lane 12 = His31Phe; lane 13 = Arg33Ala; lane 14 = Arg33Glu; lane 15 = Arg33Phe; lane 16 = Asp50Glu; lane 17 = Tyr51Cys / Cys52Tyr; lane 18 = Tyr51Cys / Cys52Phe; lane 19 = Glu53Ala; lane 20 = Glu53Lys; lane 21 = wt Q1-Y54; lane 22 = wt D2-Y54; R = reducing sample showing the monomeric protein. Bands running near the monomer position in the non-reducing lanes include contaminating E.coli host proteins. The Glu53Lys variant in both the Q1-Y54 and D2-Y54 backgrounds did not form oligomers in non-reducing SDS-PAGE and ran at the apparent molecular weight of a dimer. To confirm that phospholipid binding was reduced by these variants a dot blot was undertaken. The results (Fig. 8) show that no binding was observed for the Glu53Lys variant (dimer in SDS-PAGE). This implies that the mutation Glu53Lys removes the potentially dangerous phospholipid binding characteristic of the native protein.

[0134] Example 3: Taste panel

[0135] Any mutations at or near the sweetness-determining regions (Fig. 4) may alter the clean, sugar-like taste profile of brazzein. Therefore, to be amenable for use in the food and beverage industry, several brazzein variants containing mutations to decrease lipid binding and increase digestibility were tested for sweetness.

[0136] Several variants tested for sweetness which ran at the monomeric position in nonreducing SDS-PAGE were not sweet or were less sweet than wild-type brazzein. It was assumed this is because the monomeric position in non-reducing SDS-PAGE may reflect incorrect folding, and a native state is essential for sweetness.

[0137] Taste evaluation of variants which were produced in good yields, ran at apparent dimeric positions, and contained mutations to decrease lipid binding (E53K background) and / or increase digestibility further revealed that their clean, sugar-like taste profile was not impacted negatively by the mutations. For example, variants E53K and N10F / E53K, solving the lipid-binding problem but not the digestibility problem, as well as variants Y11F / E53K, Y11F / Y51F / E53K, Y11 F / E53K / Y54F, K27F / E53K, D40F / E53K, which solved both problems, were all found to taste sweet.

Claims

Claims1. A method for reducing lipid binding of brazzein, comprising introducing a substitution to amino acid E53 of brazzein polypeptide defined by SEQ ID NO: 1, 2, 3 or 4 or a sequence having at least 90% identity, or at least 93%, 95%, 97% or 98% identity to any of said sequences, wherein the substitution site corresponds the respective residue of SEQ ID NO:

22. The method of claim 1, wherein a E53K substitution is introduced, wherein the substitution site corresponds the respective residue of SEQ ID NO: 2.

3. The method of claim 1 or 2, wherein said brazzein polypeptide is further genetically modified by introducing at least one substitution selected from Y11F and K27F substitution(s), wherein the substitution site corresponds the respective residue of SEQ ID NO: 2.4 The method of any of claim 1 to 3, wherein the substitution (substitutions) at position(s) Y51F or Y54F, or Y51F and Y54F is (are) introduced wherein the substitution site corresponds the respective residue of SEQ ID NO: 2.

5. Brazzein polypeptide having SEQ ID NO: 1, 2, 3 or 4; or a sequence having at least 90% identity, or at least 93%, 95%, 97% or 98% identity to any of said sequences, comprising a substitution at amino acid E53, wherein the substitution site corresponds the respective residue of SEQ ID NO: 2.

6. The polypeptide of claim 5, wherein the substitution is E53. wherein the substitution site corresponds the respective residue of SEQ ID NO: 2.

7. The polypeptide of any of claims 5 or 6, wherein said polypeptide further comprises at least one substitution at a position selected from Y11F and K27F, wherein the substitution site corresponds the respective residue of SEQ ID NO: 2.

8. The polypeptide of any one of claims 5 to 7, further comprising substitution(s) Y51 F or Y54F, or Y51 F and Y54F, wherein the substitution site corresponds the respective residue of SEQ ID NO: 2.

9. The polypeptide of any of claims 5 to 8, wherein said polypeptide has reduced lipid binding, when compared to a wild-type polypeptide.

10. The polypeptide of claim 9, wherein the lipid comprises one or more selected from the group consisting of phosphatidic acid, PtdIns(4,5)P2 or PtdIns(3,4,5)P3.

11. A nucleic acid encoding a brazzein polypeptide operably linked into transcriptional regulatory sequences, encoding the mature polypeptide of any of claims 5 to 10.

12. Use of brazzein polypeptide of any of claims 5 to 10 as a sweetening agent.

13. A sweetener comprising the brazzein polypeptide of any of claims 5 to 10.

14. A food product comprising the brazzein polypeptide of any of claims 5 to 10.

15. A beverage comprising the brazzein polypeptide of any of claims 5 to 10.

16. An oral treatment product comprising the brazzein polypeptide of any of claims 5 to 10.

17. An orally administered drug comprising the brazzein polypeptide of any of claims 5 to 10.

Images