Compositions and methods for stabilized brazzein polypeptides

Abstract

Described herein are methods of producing a stabilized brazzein-54 (i.e., pyroE-BRZ54), and compositions comprising the same.

Description

Attorney Docket No.54282-0014WO1 COMPOSITIONS AND METHODS FOR STABILIZED BRAZZEIN POLYPEPTIDES CLAIM OF PRIORITY

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 617,623, filed on January 4, 2024. The entire content of the foregoing are hereby incorporated by reference. TECHNOLOGY FIELD

[0002] The present disclosure relates to compositions and methods for stabilizing brazzein ingredient and food products containing stabilized brazzein. BACKGROUND

[0003] It is an important theme in the food industry to improve the performance as well as the consistency of food products, such as shelf life, temperature stability and taste improvement. In some cases, it can be challenging to use an ingredient, where under some conditions, the resulting product can change characteristics over time, such as during the time period between manufacturer and customer usage of the food product. Therefore, there is a need for compositions and methods that increase the likelihood that sweet proteins used in the food industry have consistent characteristics over time. SUMMARY

[0004] Sweet proteins are a series of proteins derived from a series of fruits primarily found in West Africa and other equatorial environments that approximate the taste of sugar when using very small quantities of the protein. They bind to and activate the same taste receptors as sugar, the Type 1 taste receptors (TAS1R, e.g., TAS1R2 and TAS1R3). Sweet proteins can have multiple forms (e.g., multiple forms of brazzein) that can have different sweetness levels and sweetness profiles. In some cases, one form of a sweet protein can be converted into a different form of the sweet protein. If a sweet protein is used in a food product, the sweetness level and / or profile can be affected by the interconversion of sweet protein forms. Disclosed herein areAttorney Docket No.54282-0014WO1 methods and compositions for stabilizing sweet proteins (e.g., brazzein-54), and limiting conversion among brazzein forms in a food product or a composition to be used in a food product.

[0005] Disclosed herein are compositions including recombinant brazzein-54 (BRZ54), wherein at least 70%, 75%, 80%, 85% or more than 85% of the recombinant brazzein-54 (BRZ54) in the composition is pyroE-BRZ54. In some cases, the recombinant BRZ54 is produced in a microbial host. In some cases, the microbial host is a yeast. In some cases, the yeast a Pichia sp. In some cases, the Pichia sp. is Komagataella phaffii (Pichia pastoris). In some cases, the BRZ54 comprises (a) an amino acid sequence selected from the group consisting of SEQ ID NOs.9-34 or (b) two or more amino acid variations selected from the variant positions of any of SEQ ID NOs. 10-34. In some cases, the composition does not contain brazzein-53 (BRZ53). In some cases, the composition comprises one or more additional sweet proteins. In some cases, the one or more additional sweet proteins is selected from the group consisting of brazzein-53, monellin, miraculin, pentadin, mabinlin, curculin, thaumatin, and mycodulcein protein.

[0006] Also disclosed herein are powders including any of the compositions described herein.

[0007] Also disclosed herein are liquids including any of the compositions described herein

[0008] Also disclosed herein are edible ingredients including any of the compositions described herein.

[0009] Also disclosed herein are edible products including any of the compositions disclosed herein. In some cases, the edible product is a beverage, a food, or a sweetener. In some cases, the composition is blended or mixed with one or more additional sweet protein. In some cases, the one or more additional sweet protein is selected from the group consisting of brazzein-53, monellin, miraculin, pentadin, mabinlin, curculin, thaumatin, and mycodulcein.

[0010] Also disclosed herein are methods of stabilizing a brazzein-54 (BRZ54) protein, including incubating the BRZ54 protein in a basic solution to produce a stabilized BRZ54. In some cases, the pH of the basic solution is at or at least pH 9.0 (e.g., between 9.0 and 14.0). In some cases, the basic solution is a phosphate buffer. In some cases, the phosphate buffer comprises at least 50 mM, at least 62.5 mM, at least 100 mM, at least 125 mM, at least 150 mm, at least 175 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, at leastAttorney Docket No.54282-0014WO1 400 mM, at least 450 mM, or at least 500 mM phosphate. In some cases, the solution is incubated for at least 0.5 hr., 1 hr., 2 hr., 3 hr., 4 hr., 5 hr., 6 hr., 7 hr., 8 hr., 12 hr., 16 hr., 20 hr., 24 hr., 30 hr., or 36 hr. In some cases, the temperature of the incubation is at or at least 27 ºC, at or at least 28 ºC, at or at least 29 ºC, at or at least 30 ºC, at or at least 31 ºC, at or at least 32 ºC, at or at least 33 ºC, at or at least 34 ºC, at or at least 35 ºC. In some cases, the BRZ54 protein is a recombinant BRZ54 protein.

[0011] Also disclosed herein are methods of stabilizing a brazzein-54 (BRZ54) protein, comprising incubating the BRZ54 protein in an acidic solution to produce a stabilized BRZ54. In some cases, the pH of the acidic solution is at or at most pH 6.0. In some cases, the acidic solution is a phosphate buffer. In some cases, the phosphate buffer comprises at least 50 mM, at least 62.5 mM, at least 100 mM, at least 125 mM, at least 150 mm, at least 175 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, at least 400 mM, at least 450 mM, or at least 500 mM phosphate. In some cases, the solution is incubated for at least 0.5 hr., 1 hr., 2 hr., 3 hr., 4 hr., 5 hr., 6 hr., 7 hr., 8 hr., 12 hr., 16 hr., 20 hr., 24 hr., 30 hr., or 36 hr. In some cases, the temperature of the incubation is at or at least 27 ºC, at or at least 28 ºC, at or at least 29 ºC, at or at least 30 ºC, at or at least 31 ºC, at or at least 32 ºC, at or at least 33 ºC, at or at least 34 ºC, at or at least 35 ºC. In some cases, the BRZ54 protein is a recombinant BRZ54 protein.

[0012] Also disclosed herein are BRZ54 compositions including the stabilized BRZ54 produced by any of the methods disclosed herein. In some cases, the BRZ54 composition comprises at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% pyroE-BRZ54.

[0013] Also disclosed herein are host cells including a first coding sequence, wherein the first coding sequence encodes a heterologous brazzein-54 (BRZ54) protein and wherein the first coding sequence is configured to provide an N-terminal glutamine on the BRZ54 and a second coding sequence encoding a cyclotransferase, wherein the cell produces BRZ54 protein. In some cases, the first coding sequence comprises a signal sequence fused in-frame to a coding sequence of BRZ54. In some cases, at least 40% of the BRZ54 protein is pyroE-BRZ54. In some cases, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the BRZ54 is pyroE- BRZ54. In some cases, the cell is a microbial cell. In some cases, the microbial cell is a yeast cell. In some cases, the yeast cell is a Pichia cell, optionally Komagataella phaffii (Pichia pastoris). In some cases, the cyclotransferase has the amino acid sequence selected from any oneAttorney Docket No.54282-0014WO1 of SEQ ID Nos.36, 38, 40, and 42 or wherein the cyclotransferase is a functional homolog of any one of SEQ ID Nos.36, 38, 40, and 42.

[0014] Also disclosed herein are methods of producing a stable recombinant BRZ54 in vivo, including co-expressing in a host cell a first coding sequence encoding a heterologous BRZ54 protein, wherein the first coding sequence is configured to provide an N-terminal glutamine on the BRZ54, and a second coding sequence encoding a cyclotransferase. In some cases, the host cell is a microbial cell. In some cases, the host cell is a yeast cell. In some cases, the host cell is a Pichia cell, optionally Komagataella phaffii (Pichia pastoris). In some cases, the first coding sequence encodes an amino acid sequence of any one of SEQ ID Nos.9-34. In some cases, the first coding sequence encodes a signal sequence fused in-frame to a coding sequence for the BRZ54 protein. In some cases, the cyclotransferase has the amino acid sequence selected from the group consisting of SEQ ID Nos. 36, 38, 40, and 42 or wherein the cyclotransferase is a functional homolog of any one of SEQ ID Nos.36, 38, 40, and 42.

[0015] Also disclosed herein are stabilized BRZ54 proteins produced by any of the methods disclosed herein.

[0016] Also disclosed herein are stabilized BRZ54 proteins produced by any of the host cells described herein.

[0017] Also disclosed herein are edible ingredients including any of the stabilized BRZ54 proteins described herein.

[0018] Also disclosed herein are edible products including any of the stabilized BRZ54 proteins disclosed herein. In some cases, any of the edible products disclosed herein also include one or more additional sweet proteins. In some cases, the one or more additional sweet protein is selected from the group consisting of brazzein-53, monellin, miraculin, pentadin, mabinlin, curculin, thaumatin, and mycodulcein. In some cases, the edible product is selected from a beverage, a food, or a sweetener. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG.1 depicts the stability of glutamine brazzein-54 (Q-BRZ54) as a percentage of pyroglutamine brazzein-54 (pyroE-BRZ54) over time under different pH and temperature conditions.Attorney Docket No.54282-0014WO1

[0020] FIG.2 depicts the stability of pyroE-BRZ54 over time under different pH and temperature conditions.

[0021] FIG.3 depicts the perceived sweetness over time for equally sweet solutions of sucrose, Q-BRZ54, and pyroE-BRZ54. DETAILED DESCRPTION I. BRAZZEIN SWEET PROTEINS A. Brazzein and Recombinant Brazzein

[0022] Brazzein is a small, sweet-tasting protein originally isolated from the fruit of a West African plant known as the Oubli plant (Pentadiplandra brazzeana Baillon). (See, for example, Ming D et al., FEES Lett. (1994) 20355:106-108, incorporated by reference in its entirety). It is a monomer protein with a molecular weight of 6.5 kDa. As a member of the Csβα fold family, it contains four disulfide bonds that lend a high degree of thermal and pH stability to its structure. Specifically, the sweet taste of brazzein remains after incubation at 98°C for 2 hours and at 80°C for 4.5 hours in the pH range of 2.5-8. It is a highly soluble protein (more than 50 g / L) with an isoelectric point of 5.4. Residues 29-33, 36, and 39-43, as well as the C-terminus of the protein, can be involved in the sweet taste of the protein. The charge of the protein can also play a role in its interaction with the sweet taste receptor.

[0023] On a weight basis, brazzein can taste 500 to 2000 times sweeter than sucrose, as compared to a 10% sucrose and a 2% sucrose solution, respectively, or for example about 700 times sweeter when compared to a 5% sucrose solution.

[0024] At least three forms of the brazzein protein are known to exist. One form is 54 amino acids long and has a glutamine at its N-terminus (referred to as “Q-BRZ54” herein; SEQ ID NO:9). One form is 54 amino acids long and has a pyroglutamate (pGlu or pyroE) residue at its N-terminus (referred to as “pyroE-BRZ54” herein). As used herein, brazzein-54 (also “BRZ54”) can refer to either the Q-BRZ54 form or the pyroE-BRZ54 form or a combination thereof, unless specified. The third form is 53 amino acids long (sometimes called des-pGlu-brazzein and referred to as “brazzein-53” or “BRZ53” herein) and lacks the N-terminal glutamine / pyroglutamate residue.

[0025] In the naturally occurring Oubli fruit, only two forms of brazzein are present. In the ripe fruit, about 20% of the brazzein is BRZ53 and about 80% is pyroE-BRZ54. However,Attorney Docket No.54282-0014WO1 surprisingly, when BRZ54 is produced in a recombinant host from a coding region encoding Q- BRZ54, two forms result, the Q-BRZ54, and pyroE-BRZ54. As further described herein, under certain in vitro and in vivo conditions, the Q-BRZ54 form of recombinant BRZ54 can be converted to pyroE-BRZ54. The pyroE-BRZ54 under similar conditions is stable and does not convert back to Q-BRZ54. When compared, surprisingly, as further described herein, the Q- BRZ54 and pyroE-BRZ54 proteins have different intensities of sweetness. For example, pyroE- BRZ54 is 2-fold less sweet than Q-BRZ54. B. Brazzein-54 variants

[0026] A BRZ54 protein can be, for example, a variant of BRZ54 that has one or more amino acid sequence modifications (e.g., substitutions, insertions, or deletions), as compared to SEQ ID NO: 9 (with the N-terminal glutamine or with the N-terminal pyroglutamate). Exemplary variants of BRZ54 can have one or more amino acid sequence substitutions (e.g., 1, 2, 3, 4, 5, or 6 or more substitutions) as compared to SEQ ID NO: 9 (with the N-terminal glutamine or with the N-terminal pyroglutamate). For example, a variant of BRZ54 can have one or more amino acid sequence insertions (e.g., 1, 2, 3, 4, 5, or 6 or more insertions) as compared to SEQ ID NO: 9 (with the N-terminal glutamine or with the N-terminal pyroglutamate). Exemplary variants of brazzein-54 include, but are not limited to, brazzein-54 polypeptides containing any of the following variant positions: Q1A (SEQ ID NO: 10), D2insGP (SEQ ID NO: 11), D2insII (SEQ ID NO: 12), D2E (SEQ ID NO: 13), V7R (SEQ ID NO: 14), E9G (SEQ ID NO: 15), E9K (SEQ ID NO: 16), A19K (SEQ ID NO: 17), D29A (SEQ ID NO: 18), D29K (SEQ ID NO: 19), D29N (SEQ ID NO: 20), H31A (SEQ ID NO: 21), H31R (SEQ ID NO: 22), E36D (SEQ ID NO: 23), D40A (SEQ ID NO: 24), D40K (SEQ ID NO: 25), E41A (SEQ ID NO: 26), E41K (SEQ ID NO: 27), E41Q (SEQ ID NO: 28), K42E (SEQ ID NO: 29), D50K (SEQ ID NO: 30), D50N (SEQ ID NO: 31), E53R (SEQ ID NO: 32), Y54W (SEQ ID NO: 33), and A19K,H31R,E36D,E41A (SEQ ID NO: 34). Brazzein-54 variants can include the N- terminal glutamine to be a variant of Q-BRZ54 or can include the N-terminal pyroglutamate to be a variant of pyroE-BRZ54. In some cases, a variant of BRZ54 can have two or more of the amino acid sequence variations present in SEQ ID Nos.10-34 (as compared to SEQ ID NO.9), and can include the N-terminal glutamine to be a variant of Q-BRZ54, or can include the N- terminal pyroglutamate to be a variant of pyroE-BRZ54.Attorney Docket No.54282-0014WO1 C. Production of Brazzein Proteins 1. Exemplary host cells for producing brazzein proteins

[0027] Any of the brazzein proteins (e.g., Q-BRZ54 and / or pyroE-BRZ54) described herein can be isolated from naturally occurring sources and / or produced recombinantly by a variety of protein expression systems, including, but not limited, cell-based expression systems, and cell- free expression systems. Brazzein proteins produced recombinantly are recombinant proteins.

[0028] As used herein “recombinant protein” is a protein produced from a recombinant nucleic acid. A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of nucleic acid sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids (e.g., by genetic engineering techniques). The term recombinant nucleic acids can include nucleic acids that have been altered by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid can include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid can be part of a vector that is used, for example, to transform a cell.

[0029] Non-limiting examples of protein expression systems useful in producing recombinant sweet polypeptides such as any of the brazzein polypeptides disclosed herein (e.g., Q-BRZ54 and / or pyroE-BRZ54) include prokaryotic cell-based expression systems (e.g., archaeal systems, bacterial systems) and eukaryotic cell-based expression systems (e.g., filamentous or non-filamentous fungal cells, yeast cells, insect cells, and mammalian cells). In some cases, recombinant brazzein protein is produced by a microorganism (e.g., a prokaryotic or eukaryotic microorganism). In some cases, recombinant brazzein protein is produced by a yeast. In some cases, recombinant sweet protein is produced by a Pichia species, such as Pichia pastoris (also known as Komagataella phaffii).

[0030] A host cell selected for expression of any of the brazzein proteins disclosed herein (e.g., Q-BRZ54 and / or pyroE-BRZ54) can be, e.g., a bacterial cell. Suitable bacterial host cells include, but are not limited to, Escherichia coli cells (e.g., E. coli strain BL21, E. coli strain DE3, E. coli strain M15, E. coli strain DH5u, E. coli strain DH103, E. coli strain HB101), B. subtilisAttorney Docket No.54282-0014WO1 cells, Pseudomonas fluorescens cells, and cyanobacterial cells such as Synechococcus elongates cells.

[0031] A host cell selected for expression of any of the brazzein proteins disclosed herein (e.g., Q-BRZ54 and / or pyroE-BRZ54) can also be a fungal cell. The fungal cell can be, e.g., a yeast cell or a filamentous fungal cell. A yeast or filamentous fungal cell can include, but is not limited to, Arxula spp., Arxula adeninivorans, Kluyveromyces spp., Kluyveromyces lactis, Pichia spp., Pichia angusta, Pichia pastoris (also known as Komagataella phaffii), Saccharomyces spp., Saccharomyces cerevisiae, Schizosaccharomyces spp., Schizosaccharomyces pombe, Tetrahymena sp., Hansenula sp., Blastobotrys sp., Candida sp., Zygosaccharomyces sp., Debaryomyces sp. Yarrowia spp., and Yarrowia lipolytica. Fungi can include, but are not limited to, Agaricus spp., Agaricus bisporus, Aspergillus spp., Aspergillus awamori, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Colletotrichum spp., Colletotrichum gloeosporiodes, Endothia spp., Endothia parasitica, Fusarium spp., Fusarium graminearum, Fusarium solani, Mucor spp., Mucor miehei, Mucor pusillus, Myceliophthora spp., Myceliophthora thermophila, Neurospora spp., Neurospora crassa, Penicillium spp., Penicillium camemberti, Penicillium canescens, Penicillium chrysogenum, Penicillium (Talaromyces) emersonii, Penicillium funiculo sum, Penicillium purpurogenum, Penicillium roqueforti, Pleurotus spp., Pleurotus ostreatus, Rhizomucor spp., Rhizomucor miehei, Rhizomucor pusillus, Rhizopus spp., Rhizopus arrhizus, Rhizopus oligosporus, Rhizopus oryzae, Trichoderma spp., Trichoderma altroviride, Trichoderma reesei, and Trichoderma vireus. An exemplary host cell for expression of recombinant brazzein-54 (e.g., Q-BRZ54, pyroE-BRZ54) is a Pichia spp., such as Pichia pastoris.

[0032] A host cell selected for expression of any of the brazzein proteins disclosed herein (e.g., Q-BRZ54 and / or pyroE-BRZ54) can also be an insect cell. Suitable insect host cells include, but are not limited to, Sf9 cells from Spodoptera frugiperda, Sf2l cells from Spodoptera frugiperda, Hi-Five cells, BTI-TN-5B1-4 Trichophusiani cells, and Schneider 2 (S2) cells and Schneider 3 (S3) cells from Drosophila melanogaster.

[0033] A host cell selected for expression of any of the brazzein proteins disclosed herein (e.g., Q-BRZ54 and / or pyroE-BRZ54) disclosed herein can also be a mammalian cell. Non- limiting examples of mammalian host cells include HEK293 cells, HeLa cells, CHO cells, COSAttorney Docket No.54282-0014WO1 cells, Jurkat cells, NSO hybridoma cells, baby hamster kidney (BHK) cells, MDCK cells, NIH- 3T3 fibroblast cells, and any other immortalized cell line derived from a mammalian cell.

[0034] Any of the brazzein proteins disclosed herein (e.g., Q-BRZ54 and / or pyroE-BRZ54) can be produced in in vitro translation systems. An in vitro translation system generally refers to a translation system which is a cell-free extract containing elements for translation of an RNA molecule into a protein. An in vitro translation system can include ribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexes involved in translation, e.g., eIF2, eIF3, the cap- binding (CB) complex, including the cap-binding protein (CBP) and eukaryotic initiation factor 4F (eIF4F). A variety of in vitro translation systems are available. Non-limiting examples of in vitro translation systems include eukaryotic lysates, such as rabbit reticulocyte lysates, rabbit oocyte lysates, human cell lysates, insect cell lysates, and wheat germ extracts.

[0035] A recombinant brazzein protein produced in any of the host cells described herein can be, e.g., a stabilized recombinant BRZ54 (i.e., recombinant pyroE-BRZ54). 2. Exemplary expression cassettes and components

[0036] To express any of the brazzein-54 proteins disclosed herein (e.g., Q-BRZ54, and / or pyroE-BRZ54) in a cell-based expression system or a host cell disclosed herein, a coding sequence encoding the the BRZ54 polypeptide (such as SEQ ID NO. 9 or a brazzein-54 variant such as SEQ ID NOs.10-34) can be introduced into a host cell of the expression system by, for example, an expression vector, which includes additional sequences for expression of the protein. A coding sequence encoding a BRZ54 polypeptide can be made by various methods, including molecular cloning and synthesis. Molecular cloning methods can involve mutagenesis (e.g., site-directed mutagenesis), restriction enzyme-mediated cloning (e.g., restriction enzyme digestion and ligation), polymerase chain reaction (PCR), and overlap extension. Synthesis can include chemical synthesis (e.g., gene synthesis). The gene sequence can be codon optimized for any desired expression system.

[0037] Vectors including a coding sequence encoding a BRZ54 polypeptide disclosed herein are also provided herein, e.g., vectors including a coding sequence encoding for the brazzein polypeptide (e.g., a coding sequence encoding a recombinant brazzein-54 polypeptide) or a variant thereof.Attorney Docket No.54282-0014WO1

[0038] The coding sequence can be inserted into a vector by a variety of procedures, including, but not limited to restriction enzyme digestion, ligation, and homologous recombination. The vector can be capable of replicating and expressing the polynucleotides in prokaryotic and / or eukaryotic host cells of expression systems. A vector can contain various components that can be adjusted and optimized for compatibility with the particular host cell. A cloning vector and / or expression vector can include additional nucleic acid sequences, including but not limited to, a signal sequence, an origin of replication, a marker gene (e.g., a selection marker such as an antibiotic resistance gene), an enhancer element, a promoter, a ribosome binding site, a signal sequence, and a transcription termination sequence.

[0039] An expression vector containing a coding sequence encoding a BRZ54 polypeptide as disclosed herein can further include a promoter to which the coding sequence encoding the BRZ54 is operably linked. Promoters can include, but are not limited to, constitutive promoters, inducible promoters, and hybrid promoters. Exemplary promoters can include, but are not limited to, acu-5, adhl+, alcohol dehydrogenase (ADH1, ADH2, ADH4), AHSB4m, AINV, alcA, a-amylase, alternative oxidase (AOD), alcohol oxidase I (AOX1), alcohol oxidase 2 (AOX2), AXDH, B2, CaMV, cellobiohydrolase I (cbhl), ccg-l, cDNAl, cellular filament polypeptide (cfp), cpc-2, ctr4+, CUP1, dihydroxyacetone synthase (DAS), enolase (ENO, ENOl), formaldehyde dehydrogenase (FLD1), FMD, formate dehydrogenase (FMDH), Gl, G6, GAA, GAL1, GAL2, GAL3, GAL4, GAL5, GAL6, GAL7, GAL8, GAL9, GAL10, GCW14, gdhA, gla-l, a-glucoamylase (glaA), glyceraldehyde-3- phosphate dehydrogenase (gpdA, GAP, GAPDH), phosphoglycerate mutase (GPM1), glycerol kinase (GUT1), HSP82, invl+, isocitrate lyase (ICL1), acetohydroxy acid isomeroreductase (ILV5), KAR2, KEX2, b-galactosidase (lac4), LEET2, melO, MET3, methanol oxidase (MOX), nmtl, NSP, pcbC, PET9, peroxin 8 (PEX8), phosphoglycerate kinase (PGK, PGK1), phol, PH05, PH089, phosphatidylinositol synthase (PIS1), PYK1, pyruvate kinase (pkil), RPS7, sorbitol dehydrogenase (SDH), 3-phospho serine aminotransferase (SER1), SSA4, SV40, TEF, translation elongation factor 1 alpha (TEF1), THI11, homoserine kinase (THR1), tpi, TPS1, triose phosphate isomerase (TPI1), XRP2, and YPT1.

[0040] Expression vectors containing a coding sequence encoding a BRZ54 polypeptide can further include a polynucleotide sequence encoding a signal peptide. A signal peptide, also known as a signal sequence, targeting signal, localization signal, localization sequence, secretionAttorney Docket No.54282-0014WO1 signal, transit peptide, leader sequence, or leader peptide, can support secretion of a protein or polynucleotide. Extracellular secretion of a recombinantly expressed protein from a host cell can facilitate protein purification. For example, recovery of a recombinant protein from a host cell culture supernatant can be preferable to lysing host cells to release a complex mixture of proteins, including intracellular proteins of the host cell. Secretion, in some cases, can reduce deleterious effects that intracellular overexpression of a heterologous protein can have on a host cell such as toxicity or decreased growth rate. Secretion, in some cases, can allow increased protein production compared to intracellular expression in a host cell of limited volume to store the synthesized proteins. Secretory production of a protein, in some cases, can facilitate post- translational modification or processing (e.g., protein folding, formation of disulfide bonds, and glycosylation). In some cases, the signal sequence is cleaved such that the mature secreted protein, e.g., a BRZ54 protein has a glutamine at its N-terminus (Q-BRZ54). 3. Recovery and isolation of brazzein protein

[0041] Following production of any of the brazzein-54 proteins disclosed herein (e.g., Q- BRZ54, and / or pyroE-BRZ54, and variants thereof) in a cell-based or other system, the brazzein protein can be recovered and / or isolated from supernatants and lysates by any of a variety of methods, including, but not limited to, chemical extraction, column chromatography, and filtration. The polypeptides can be purified using any of a variety of methods including liquid chromatography such as normal or reversed phase, high-performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), and the like; affinity chromatography such as with inorganic ligands, monoclonal antibodies (e.g., immunoaffinity), and ion exchange (e.g., anion exchange, cation exchange); hydrophobic interaction chromatography; size exclusion chromatography; immobilized metal chelate chromatography; gel electrophoresis; ethanol precipitation; and any combination thereof. In some cases, the polypeptides can be purified by centrifugation and / or filtration, including sterile filtration, depth filtration, tangential flow filtration, ultrafiltration (UF), diafiltration (DF), and ultrafiltration / diafiltration (UF / DF).

[0042] A recovered and / or isolated brazzein-54 polypeptide described herein (e.g., Q- BRZ54, and / or pyroE-BRZ54, and variants thereof) can be concentrated, dehydrated, dewatered, or dried by freeze drying, vacuum tray drying, spray drying, rotary drum drying, or any combination thereof. Drying can be done in a desiccator, vacuum dryer, conical dryer, sprayAttorney Docket No.54282-0014WO1 dryer, fluid bed or any method known in the art. Preferably, methods are chosen that yield a dried product (e.g., a powder) with the greatest sweetness. The dried sweet protein can be optionally blended, pestled, milled, conched, pulverized, or other methods of processing known in the art. The resulting concentrated liquid or solid powder can be stored, diluted, rehydrated, or used in the formulation of food and beverage products. In some cases, the concentrated liquid or solid powder can be stored at -20°C, 4°C, or room temperature without significant loss in sweetness. For use, the concentrated liquid can be diluted, e.g., using water and the solid powder can be rehydrated as a paste or a liquid using water or another liquid in which the solid powder is soluble. II. METHODS OF STABILIZING BRAZZEIN-54 IN VITRO

[0043] Provided herein are methods for stabilizing BRZ54 protein. In some cases, the methods of stabilization include incubating the BRZ54 protein, such as a recombinant BRZ54 protein, under one or more conditions under which a majority of the Q-BRZ54 is converted to pyroE-BRZ54 (e.g., at least 60%, 70%, 80% or more of the BRZ54 is pyroE-BRZ54). In vitro methods of stabilizing BRZ54 can include incubation at a specific temperature, pH, and / or for a specific time. A. Temperature

[0044] A BRZ54 protein, such as a recombinant BRZ54 protein, can be incubated at a temperature between 25 ºC – 40 ºC for a time sufficient to produce a stabilized BRZ54 (i.e., pyroE-BRZ54). For example, the temperature of the incubation is at or at least 25 ºC, at or at least 26 ºC, at or at least 27 ºC, at or at least 28 ºC, at or at least 29 ºC, at or at least 30 ºC, at or at least 31 ºC, at or at least 32 ºC, at or at least 33 ºC, at or at least 34 ºC, at or at least 35 ºC, at or at least 36 ºC, at or at least 37 ºC, at or at least 38 ºC, at or at least 39 ºC, or at or at least (or at most) 40 ºC. B. pH

[0045] A BRZ54 protein, such as a recombinant BRZ54 protein, can be incubated in a basic solution having a pH greater than 7.0 (e.g., 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0) for a time sufficient to produce a stabilized BRZ54 (i.e., pyroE-BRZ54). For example, the pH of the basicAttorney Docket No.54282-0014WO1 solution is at or at least pH 9.0. Exemplary basic solutions include, but are not limited to, a phosphate buffer. For example, a basic solution can be a phosphate buffer having at least 50 mM, at least 62.5 mM, at least 100 mM, at least 125 mM, at least 150 mm, at least 175 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, at least 400 mM, at least 450 mM, or at least 500 mM phosphate.

[0046] Alternatively, BRZ54 protein, such as a recombinant BRZ54 protein, can be incubated in an acidic solution having a pH less than 7.0 (e.g., 6.0, 5.0, 4.0, 3.0, 2.0, 1.0) for a time sufficient to produce a stabilized BRZ54 (i.e., pyroE-BRZ54). For example, the pH of the acidic solution is at or at most pH 6.0. Exemplary acidic solutions include, but are not limited to, a phosphate buffer. For example, an acidic solution can be a phosphate buffer having at least 50 mM, at least 62.5 mM, at least 100 mM, at least 125 mM, at least 150 mm, at least 175 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, at least 400 mM, at least 450 mM, or at least 500 mM phosphate. C. Time

[0047] A solution comprising BRZ54 protein, such as a recombinant BRZ54 protein, can be incubated for a time sufficient to produce a stabilized BRZ54 (i.e., pyroE-BRZ54). For example, the time sufficient to produce a stabilized BRZ54 (i.e., pyroE-BRZ54) is at least 0.5 hours (hr.), 1 hr., 2 hrs., 3 hrs., 4 hrs., 5 hrs., 6 hrs., 7 hrs., 8 hrs., 12 hrs., 16 hrs., 20 hrs., 24 hrs., 30 hrs., or 36 hrs. III. HOST CELLS AND METHODS OF STABILIZING BRAZZEIN-54 IN VIVO

[0048] Provided herein are methods for producing and stabilizing a BRZ54 protein in vivo. In some cases, the methods include expressing a recombinant BRZ54 protein in the presence of a cyclase in a host cell, whereby a majority of the Q-BRZ54 is converted to pyroE-BRZ54 (e.g., at least 60%, 70%, 80% or more of the BRZ54 is pyroE-BRZ54).

[0049] Host cells can also express, e.g., are co-transformed with, a glutaminyl-peptide cyclotransferase (also referred to herein as cyclotransferase or cyclase), e.g., a heterologous cyclotransferase or an exogenous cyclotransferase, in addition to a coding sequence encoding a heterologous BRZ54 to promote cyclization of the BRZ54 polypeptide. Glutaminyl-peptide cyclotransferases are enzymes that belong to the family of cyclotransferases, specifically theAttorney Docket No.54282-0014WO1 aminoacyltransferases, and catalyze the chemical reaction to convert an N-terminal L- glutaminyl-peptide, or L-glutamyl-peptide, to 5-oxoprolyl-peptide and NH3, or 5-oxoprolyl- peptide and H2O, respectively. The enzyme effectively converts an N-terminal glutamine (Q) to an N-terminal 5-oxoproline residue, commonly known as pyroglutamic acid (pyroE). These cyclotransferase enzymes can also be called L-glutaminyl-peptide gamma-glutamyltransferase (cyclizing), glutaminyl-tRNA cyclotransferase, glutaminyl cyclase, and glutaminyl-transfer ribonucleate cyclotransferase. A. Host cells producing stabilized BRZ54

[0050] A stabilized BRZ54 (i.e., pyroE-BRZ54) can be produced in any cell-based expression system described herein, (e.g., by utilizing an expression system, wherein the expression system includes a coding sequence encoding the polypeptide, and wherein the expression system is introduced into any of the host cells described herein).

[0051] Expression systems useful in the present methods can include an expression vector. Exemplary expression vectors can include a first coding sequence, wherein the first coding sequence encodes a heterologous BRZ54 protein or an exogenous BRZ54 protein and wherein the first coding sequence is configured to provide an N-terminal glutamine on the BRZ54 (i.e., produce Q-BRZ54). Host cells can include additional genetic elements configured to convert Q- BRZ54 into pryoE-BRZ54. For example, in some instances, the host cell includes a second coding sequence encoding a cyclotransferase (e.g., as described below). The second coding sequence encoding a cyclotransferase can be, e.g., on an expression vector operably linked to a promoter. For example, the second coding sequence encoding a cyclotransferase can be, e.g., on the same expression vector as the first coding sequence encoding a heterologous BRZ54 protein or an exogenous BRZ54 protein or on a different expression vector (e.g., a second expression vector) as the first coding sequence encoding a heterologous BRZ54. In some embodiments, the host cell expresses the cyclotransferase at levels above those of any endogenous cyclotransferase; alternatively, the host cells can be engineered to overexpress an endogenous cyclotransferase, e.g., by addition of a strong promoter to drive an endogenous cyclotransferase.

[0052] The host cells described herein can comprise a first coding sequence encoding a BRZ54 protein (e.g., a heterologous BRZ54 protein or an exogenous BRZ54 protein), e.g., a BRZ54 protein comprising an N-terminal glutamine on the BRZ54 (i.e., Q-BRZ54), and aAttorney Docket No.54282-0014WO1 second coding sequence encoding any of the cyclotransferases disclosed herein such that pryoE- BRZ54 is produced. The amount of pyroE-BRZ54 can be from 45% to 100% of the total amount of produced BRZ54. For example, the host cell can produce at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% pyroE-BRZ54 of the total BRZ54. B. Exemplary cyclotransferases

[0053] Glutaminyl-peptide cyclotransferases belong to the family of aminoacyltransferases. Enzymes of this family catalyze a post-translational chemical reaction in proteins or peptides that converts N-terminal glutamine or glutamate residues into N-terminal pyroglutamate (pyroE) by releasing ammonia or a water molecule, respectively. The N-terminal pyroE modification protects the substrate of the enzyme against proteolytic degradation by aminopeptidases and increases protein stability. Exemplary glutaminyl-peptide cyclotransferases derived from plants include those put forth in Table 1. Table 1. Exemplary glutaminyl-peptide cyclotransferases.Attorney Docket No.54282-0014WO1Attorney Docket No.54282-0014WO1Attorney Docket No.54282-0014WO1Attorney Docket No.54282-0014WO1Attorney Docket No.54282-0014WO1Attorney Docket No.54282-0014WO1Attorney Docket No.54282-0014WO1Attorney Docket No.54282-0014WO1

[0054] Suitable glutaminyl-peptide cyclotransferases can include any of those put forth in Table 1, or a functional homolog thereof. For example, suitable glutaminyl-peptide cyclotransferases can include: Arabidopsis thaliana (AT) glutaminyl-peptide cyclotransferase, Arabidopsis thaliana (AT) glutaminyl-peptide cyclotransferase-HDEL, Saccharomyces cerevisiae (SC) glutaminyl-peptide cyclotransferase, Saccharomyces cerevisiae (SC) glutaminyl- peptide cyclotransferase-HDEL (nucleotide and amino acid sequences of which are provided in the “Sequences” Table below, see, SEQ ID NOS: 35-42). 1. Features of Exemplary Cyclotransferases – Endoplasmic retention signal

[0055] In some embodiments, the encoded cyclotransferase contains an endoplasmic retention signal that prevents the cyclotransferase from being secreted from the endoplasmic reticulum (ER) and facilitates its return if it is exported. In some embodiments, the cyclotransferase contains an ER retention signal at the C-terminal end of the cyclotransferase. Yeast recognize the amino acid sequence HDEL (SEQ ID NO: 43) as an endoplasmic retention signal. Mammalian cells recognize KDEL (SEQ ID NO: 44) as an ER retention signal. Plant cells recognize both the amino acid sequence HDEL (SEQ ID NO: 43) and the amino acid sequence KDEL (SEQ ID NO: 44) as ER retention signals. In some of the embodiments provided herein, the cyclotransferase has an ER retention signal that is or comprises the amino acid sequence HDEL (SEQ ID NO: 43). C. Production and purification of stabilized BRZ54

[0056] To produce stabilized BRZ54 (i.e., pyroE-BRZ54) in vivo, any of the host cells configured to produce pryoE-BRZ54 described herein can be incubated, fermented, or cultured in an appropriate medium to produce pyroE-BRZ54. In some cases, a host cell including an expression vector including i) a coding sequence encoding an exogenous BRZ54 protein or a heterologous BRZ54 protein and ii) a second coding sequence encoding a cyclotransferase is incubated in an appropriate medium to produce Q-BRZ54, and the cyclotransferase converts the Q-BRZ54 to stabilized BRZ54 (i.e., pyroE-BRZ54). For example, a host cell including i) an expression vector including a coding sequence encoding an exogenous BRZ54 protein or a heterologous BRZ54 protein and ii) a second expression vector including a coding sequenceAttorney Docket No.54282-0014WO1 encoding a cyclotransferase can be incubated in an appropriate medium to produce Q-BRZ54, and the cyclotransferase converts the Q-BRZ54 to stabilized BRZ54 (i.e., pyroE-BRZ54).

[0057] After producing stabilized BRZ54 (i.e., pyroE-BRZ54), the stabilized BRZ54 can be recovered and / or isolated. For example, the stabilized BRZ54 (i.e., pyroE-BRZ54) can be recovered and / or isolated from supernatants and lysates by any of a variety of methods, including, but not limited to, chemical extraction, column chromatography, and filtration. A recovered and / or isolated stabilized BRZ54 (i.e., pyroE-BRZ54) can be concentrated, dehydrated, dewatered, or dried by freeze drying, vacuum tray drying, spray drying, rotary drum drying, or any combination thereof. The resulting concentrated liquid or solid powder can be stored, diluted, rehydrated, or used in the formulation of food and beverage products. The concentrated liquid or solid powder can be stored at -20°C, 4°C, or room temperature without significant loss in sweetness. Before use, the concentrated liquid can be, e.g., diluted using water and the solid powder can be, e.g., rehydrated as a paste or a liquid using water or another liquid in which the solid powder is soluble. IV. COMPOSITIONS AND PRODUCTS CONTAINING pyroE-BRZ54

[0058] Disclosed herein are compositions, formulations, and products intended for human consumption, e.g., compositions, powders, formulations liquids, edible and drinkable ingredients, and edible and drinkable products. For example, these can include various types of foods, beverages, condiments, sweeteners, confectionary products, dairy products like creamers, etc. Any of the stabilized BRZ54 (i.e., pryoE-BRZ54) proteins described herein can be included in any of the compositions or products described herein. In some embodiments, inclusion of stabilized BRZ54 protein results in a more stable sweetness profile over time or has a smaller magnitude of change in a sweetness profile over time. For example, a composition, formulation, or product that includes stabilized BRZ54 protein has a more stable sweetness profile over time or has a smaller magnitude of change in a sweetness profile over time when stored as compared to the sweetness profile over time of a composition, formulation, or product that includes a non- stabilized BRZ54 protein (e.g., Q-BRZ54), or lacks a stabilized BRZ54 protein.Attorney Docket No.54282-0014WO1 Beverage Products

[0059] Non-limiting examples of a beverage product include a tea, a lemonade, or a tea and lemonade combination beverage. Any of the beverage products described herein can further include an additional ingredient such as one or more of a low glycemic natural sweetener, an acid, a bulking agent, a fat, an emulsifier, and / or an additional flavoring agent.

[0060] A tea can include a flavored tea, a sweet tea, a black tea, a green tea, a white tea, an herbal tea, an iced tea, an unsweetened tea, a bubble tea. A flavored tea can include a lemon tea.

[0061] A lemonade can include a lemon-flavored lemonade, a lime-flavored lemonade, or a strawberry lemonade. In some cases, any of the lemonades described herein can include a flavored lemonade (e.g., a fruit-flavored lemonade). In some case, a lemonade includes some amount of tea (e.g., between 10-90% tea, such as 40-60% tea). Frozen confections

[0062] Non-limiting examples of a frozen confection include an ice cream, a sorbet, a frozen popsicle, and a shaved ice. Any of the frozen confections described herein can further include an additional ingredient such as one or more of a low glycemic natural sweetener, cocoa, a bulking agent, a fat, an emulsifier, and / or an additional flavoring agent.

[0063] In some cases, the frozen confection is a frozen popsicle. Frozen popsicles may be stored at freezing temperature, or at room temperature and frozen later by the consumer.

[0064] In a particular aspect, a frozen popsicle as described herein may include one of more of the following ingredients: fruit puree (e.g., mango puree, passion fruit puree, strawberry puree, pineapple puree, etc.), water, salt, orange flower water, rose water, juice (e.g., lime juice, lemon juice), natural flavors, and any of the sweet proteins in any combination described herein. Dairy Products

[0065] Non-limiting examples of a dairy product include a yogurt, a yogurt drink, a kefir, a dairy creamer, or a non-dairy creamer. Any of the dairy products described herein can further include an additional ingredient such as one or more of a low glycemic natural sweetener, cocoa, a bulking agent, a fat, an emulsifier, and / or an additional flavoring agent. Any of the dairy products described herein can include a dairy ingredient. Non-limiting examples of dairy ingredients include milk or cream, or non-dairy based versions thereof. Milk can include skimAttorney Docket No.54282-0014WO1 milk, 1% milk, 2% milk, whole milk. Cream can include heavy cream, half and half cream, and whipping cream. Milk or cream components can be filtered or ultra-filtered.

[0066] In some cases, the dairy product is a yogurt drink. In some cases, any of the sweet proteins described herein can be added direct to or mixed into the yogurt drink.

[0067] In some cases, the dairy product is a creamer. In some cases, the creamer is a dairy creamer (i.e., contains milk or a milk-derived component). In some cases, the creamer is a non- dairy creamer. Non-dairy creamer can, for example, include plant-based milk-like components, such as oat-based, nut-based, soy-based, or rice-based. In some cases, the creamer is a coffee creamer. A coffee creamer can include a dairy coffee creamer or a non-dairy coffee creamer. Any of the coffee creamers described herein can include an additional flavoring agent. Non limiting examples include a vanilla flavor, a hazelnut flavor, and / or a caramel flavor. Sweetener

[0068] Non-limiting examples of a sweetener include a beverage sweetener, a food sweetener, or both a beverage and food sweetener. The sweetener can be similar to a sugar solution or honey. The sweetener can be a liquid, a solid, or a powder. The sweetener can be added to beverages or foods to produce a sweetened taste compared to the beverage or food without the added sweetener. Exemplary components included in any of the above-described compositions, formulations, products Sweet Proteins

[0069] In some embodiments, any of the compositions, formulations, or products described above include a total amount of BRZ54. Of the total amount of BRZ54 present in compositions, formulations, products, described above at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% is pyroE-BRZ54, with the remaining being Q-BRZ54.

[0070] In some embodiments, the BRZ54 compositions herein are combined with one or more additional sweet proteins. Exemplary sweet proteins include brazzein-53 (BRZ53), monellin, miraculin, pentadin, mabinlin, curculin, thaumatin, and mycodulcein. In some cases, the one or more additional sweet protein(s) are recombinant sweet proteins. In some embodiments, any of the compositions, formulations, or products described above also includeAttorney Docket No.54282-0014WO1 BRZ53. In some embodiments, any of the compositions, formulations, or products described above do not include BRZ53. V. DEFINITIONS

[0071] The term “promoter” is a nucleic acid sequence that is operably linked to a nucleic acid sequence encoding a polypeptide (e.g., a recombinant peptide) that can increase the transcription of the nucleic acid sequence encoding the polypeptide (e.g., a peptide described herein). In some cases, a promoter is constitutive. In other cases, a promoter is inducible. Non- limiting examples of promoters are described herein. Additional examples of promoters are known in the art.

[0072] The term “culturing” refers to growing a population of cells, e.g., microbial cells, under suitable conditions for growth, in a liquid or solid medium.

[0073] The term “isolating” means a step performed to separate a recombinant peptide from one or more other impurities (e.g., bulk impurities) or components present in a fluid containing a recombinant peptide (e.g., liquid culture medium polypeptides or one or more other components (e.g., DNA, RNA, other polypeptides, endotoxins, viruses, etc.) present in or secreted from a mammalian cell).

[0074] The terms “isolated”, “purified”, “separated”, and “recovered” as used herein refer to a material (e.g., a polypeptide, nucleic acid, or cell) that is removed from at least one component with which it is naturally associated. For example, these terms can refer to a material which is substantially or essentially free from components which normally accompany it as found in its native state, such as, for example, an intact biological system, or is substantially or essentially free from other proteins in the system from which it is expressed.

[0075] As used herein, the term “host cell” refers to a cell or cell line into which a recombinant expression vector (e.g., a nucleic acid construct) can be introduced for expression of the polypeptide (such as the BRZ54 proteins) in the host cell. A host cell including a recombinant vector can be referred to as a “recombinant host cell.”

[0076] The term “"ppm”", as used herein, means parts-per-million and is a weight relative parameter. A part-per-million is a microgram per gram, such that a component that is present at 10 ppm is present at 10 micrograms of the specific component per 1 gram of the aggregate mixture.Attorney Docket No.54282-0014WO1

[0077] Reference to the term “about” refers herein to a value of plus or minus 10% of the provided value. For example, “about 20” means or includes amounts from 18 to and including 22.

[0078] Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. As used herein, the singular form “a”, “an”, and “the” include plural references unless indicated otherwise. For example, “an” excipient includes one or more excipients. VI. EXAMPLES

[0079] Various aspects of the disclosure are further illustrated by the following non-limiting examples. Example 1: Brazzein-54 production A. Construct Assembly

[0080] DNA fragments were de novo synthesized (Twist Biosciences) and assembled into cloning vectors by Type IIS restriction enzymes (New England Biolabs). Each vector contained an expression cassette containing: (1) a PGCW14 promoter (SEQ ID NO: 1); (2) an EPX1 signal peptide (SEQ ID NO: 5 (nucleotide sequence (nt)); SEQ ID NO: 6 (amino acid (aa))); (3) a brazzein-54 (BRZ54) coding sequence (SEQ ID NO: 8 (nt); SEQ ID NO: 9 (aa)); and (4) the AOX1 transcription terminator sequence (SEQ ID NO: 6). B. Brazzein-54 Expression under Constitutive Promoter Regulation

[0081] The expression cassette described above was amplified using Q5 Polymerase (New England Biolabs), transformed into K. phaffii strain BG10 (ATUM Biosciences), and integrated into the genome by homologous recombination. Positive transformants were cultured in Basal Salts media in 2L Bioreactors (Sartorius). The Fed-batch cultures were allowed to grow for 96 hours before harvesting.

[0082] Cells were removed from spent fermentation broth by high-speed centrifugation. The remaining cells were then removed by microfiltration. The resulting Q-BRZ54-containing filtrate was then purified by cation exchange chromatography, desalted by ultrafiltration / diafiltration, analyzed by HPLC, and lyophilized to form a powder.Attorney Docket No.54282-0014WO1 Example 2: Stability of Brazzein-54 products

[0083] Brazzein-54, produced by fermentation, contains an N-terminal glutamine (Q- BRZ54). The N-terminal glutamine of Q-BRZ54 can be cyclized to form pyroglutamate-BRZ54 (pyroE-BRZ54). A. Stability of Q-BRZ54

[0084] The starting Q-BRZ54 powder as measured by HPLC contains about 71-72% Q- BRZ54 and about 28-29% pyroE-BRZ54. To assess the stability of the N-terminal Q of recombinant Q-BRZ54 over time, Q-BRZ54 powder was resuspended in water, at a concentration of 236 mg / L total BRZ54 protein. The Q-BRZ54 powder was adjusted to pH 3, 7, or 8 with citric acid or sodium bicarbonate and incubated for 0 (control starting material), 3, 7, or 15 days, at 4 C, 25 C, or 35 C. Levels of Q-BRZ54 and pyroE-BRZ54 were measured and the percentage of pyroE-BRZ54 was calculated.

[0085] As shown in FIG 1, increasing amounts of Q-BRZ54 were converted to pyroE- BRZ54 over time under each set of conditions. Q-BRZ54 remained most stable at 4 C. The stability over time deceased with increasing temperature. The stability of Q-BRZ54 was also pH sensitive, with the most conversion occurring at pH3 and the most stability at pH7. B. Stability of pyroE-BRZ54

[0086] To assess the stability of the cyclized BRZ54 (pyroE-BRZ54), the experiment from part A above was repeated using pyroE-BRZ54 instead of Q-BRZ54. As shown in FIG. 2, pyroE-BRZ54 was stable under all conditions tested, without converting back to Q-BRZ54. Example 3: In Vitro Cyclization of Brazzein-54 (BRZ54)

[0087] To test conditions for cyclizing Q-BRZ54 to from pyroE-BRZ54 in vitro, Q-BRZ54 was subjected to various conditions as shown in Table E1.

[0088] BRZ54 powder was resuspended in sodium phosphate (Millipore Sigma) buffers of various concentrations and pHs. The solutions were incubated at constant temperatures, for differing amounts of time. After incubation, the concentrations of pyroE-BRZ54 and Q-BRZ54 were measured by HPLC-MS. The percent pyroE-BRZ54 was calculated for each set of conditions. The results are provided in Table E1. Under the conditions tested, 38%-90% of the BRZ54 was converted to pyroE-BRZ54.Attorney Docket No.54282-0014WO1 Table E1. In vitro cyclization of BRZ54Example 4: In Vivo Cyclization of Brazzein-54 (BRZ54)

[0089] To generate cyclized BRZ54 in vivo, K. phaffii were transformed with genes encoding BRZ54 and a glutaminyl cyclase. A. Strain generation

[0090] DNA fragments were de novo synthesized (Twist Biosciences) and assembled into cloning vectors by Type IIS restriction enzymes (New England Biolabs). Glutaminyl cyclase vectors contained an expression cassette containing: (1) a native GAP promoter (SEQ ID NO: 2), a G1 promoter (SEQ ID NO: 3), or a G7 promoter (SEQ ID NO: 4); (2) a glutaminyl cyclase gene (from A. thaliana or S. cerevisiae), codon optimized for Komagataella phaffii expression with or without an HDEL endoplasmic retention sequence(SEQ ID NOS: 35, 37, 39, 41 (nt),Attorney Docket No.54282-0014WO1 encoding SEQ ID NOS: 36, 38, 40, 42 (aa), respectively); and (3) an AOX1 transcription terminator sequence (SEQ ID NO:6). BRZ54 vectors were constructed as described in Example 1.

[0091] The glutaminyl cyclase and BRZ54 expression cassettes were amplified using Q5 Polymerase (New England Biolabs), transformed into K. phaffii strain BG10 (ATUM Biosciences), and integrated into the genome by homologous recombination. B. Expression under Constitutive Promoter Regulation

[0092] Positive transformants, containing the genes encoding the cyclase and BRZ54, were cultured in 14-mL culture tubes containing CBMD (Citrate Buffered Minimal Dextrose) media for 48 hours at 275 rpm and 30 °C in a shaking incubator. The concentration of Q-BRZ54 and pyroE-BRZ54 were measured by HPLC. All strains tested produced 100% pyroE-BRZ54, compared to a base strain, not expressing the cyclase, which produced about 50% pyroE-BRZ54. C. Production and Purification of Brazzein-54 in bioreactors

[0093] Four strains from part B, containing BRZ54 expression regulated by the PGCW14 promoter, were grown in bioreactors to confirm BRZ54 conversion consistency and to produce material for sensory analysis.

[0094] The Pichia pastoris strains produced above were cultured in Basal Salts media in 2L Bioreactors (Sartorius). The Fed-batch cultures were allowed to grow for 96 hours before harvesting.

[0095] Cells were removed from spent fermentation broth by high-speed centrifugation. The remaining cells were then removed by microfiltration. The resulting filtrate was then purified by cation exchange chromatography and analyzed by HPLC. The cyclase-expressing strains produced 84%, 86%, 92%, and 95% pyroE-BRZ54, while a control strain, not expressing the cyclase, produced only 17% pyroE-BRZ54. The total amount of BRZ54 was similar among all strains examined (cyclase-expressing and control). These results indicate co-expressing a cyclotransferase facilitated the conversion of the N-terminal glutamine to pyroglutamate without affecting the overall recombinant protein production. Example 5: Sensory Assessment

[0096] The purified cyclized BRZ54 (approximately 94% of the BRZ54 is pyroE-BRZ as measured by HPLC) and control BRZ54 (approximately 16% of the BRZ54 is pyroE-BRZ54 asAttorney Docket No.54282-0014WO1 measured by HPLC) from Example 3 were dialyzed using ultrafiltration and lyophilized. The lyophilized BRZ54 was used for sensory analysis. A. Sweetness Potency

[0097] Each isoform of brazzein (Q-BRZ54, pyroE-BRZ54, and brazzein 53 (BRZ53), lacking the N-terminal Q or pyroE) was resuspended in water at a concentration of between 0.024 ppm and 0.2 ppm. Panelists were instructed to taste each sample and rank the sweetness as compared to a 5% sucrose reference on a scale between 1 and 200, where 100 would be equivalent to the reference solution and 200 is double the sweetness of the reference solution. Panelists’ responses were used to calculate sweetness potency as compared to sucrose. The results are provided in Table E2 and show that Q-BRZ54 is about 2 times the sweetness potency of pyroE-BRZ54. Table E2. Sweetness Potency of Brazzein Solutions.B. Temporal Profile

[0098] From the calculated sweetness potency above, 5% sucrose equivalent solutions were prepared. Panelists were asked to taste each sample for three seconds before expectorating. Panelists used a sliding scale to record sweetness intensity over a period of 60 seconds. As shown in FIG. 3, pyroE-BRZ54 and Q-BRZ54, adjusted for equivalent sweetness, have similar temporal profiles in terms of sweetness onset and linger, as compared to 5% sucrose and to each other.

[0099] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from theAttorney Docket No.54282-0014WO1 true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.Attorney Docket No.54282-0014WO1 SEQUENCESAttorney Docket No.54282-0014WO1Attorney Docket No.54282-0014WO1Attorney Docket No.54282-0014WO1Attorney Docket No.54282-0014WO1Attorney Docket No.54282-0014WO1Attorney Docket No.54282-0014WO1

Claims

Attorney Docket No.54282-0014WO1 WHAT IS CLAIMED IS:

1. A composition comprising recombinant brazzein-54 (BRZ54), wherein at least 70%, 75%, 80%, 85% or more than 85% of the recombinant BRZ54 in the composition is pyroE- BRZ54.

2. The composition of claim 1, wherein the recombinant BRZ54 is produced in a microbial host.

3. The composition of claim 2, wherein the microbial host is a yeast.

4. The composition of claim 3, wherein the yeast a Pichia sp.

5. The composition of claim 4, wherein the Pichia sp. is Komagataella phaffii (Pichia pastoris).

6. The composition of any one of claims 1-5, wherein the BRZ54 comprises (a) an amino acid sequence selected from the group consisting of SEQ ID NOs. 9-34 or (b) two or more amino acid variations selected from the variant positions of any of SEQ ID NOs.10-34.

7. The composition of any one of claims 1-6, wherein the composition does not contain brazzein-53 (BRZ53).

8. The composition of any one of claims 1-6, wherein the composition comprises one or more additional sweet proteins.

9. The composition of claim 8, wherein the one or more additional sweet proteins is selected from the group consisting of brazzein-53, monellin, miraculin, pentadin, mabinlin, curculin, thaumatin, and mycodulcein protein.

10. A powder comprising the composition of any one of claims 1-9.Attorney Docket No.54282-0014WO1 11. A liquid comprising the composition of any one of claims 1-9.

12. An edible ingredient comprising the composition of any one of claims 1-9.

13. An edible product comprising the composition of any one of claims 1-9 or the edible ingredient of claim 12.

14. The edible product of claim 13, wherein the edible product is selected from a beverage, a food, or a sweetener.

15. The edible ingredient of claim 12 or the edible product of claim 13 or claim 14, wherein the composition is blended or mixed with one or more additional sweet protein.

16. The edible ingredient or the edible product of claim 15, wherein the one or more additional sweet protein is selected from the group consisting of brazzein-53, monellin, miraculin, pentadin, mabinlin, curculin, thaumatin, and mycodulcein.

17. A method of stabilizing a brazzein-54 (BRZ54) protein, comprising incubating the BRZ54 protein in a basic solution to produce a stabilized BRZ54.

18. The method of claim 17, wherein the pH of the basic solution is at or at least pH 9.0 (e.g., between 9.0 and 14.0).

19. The method of claim 17 or claim 18, wherein the basic solution is a phosphate buffer.

20. A method of stabilizing a brazzein-54 (BRZ54) protein, comprising incubating the BRZ54 protein in an acidic solution to produce a stabilized BRZ54.Attorney Docket No.54282-0014WO1 21. The method of claim 20, wherein the pH of the acidic solution is at or at most pH 6.

0.

22. The method of any of claim 20 or claim 21, wherein the acidic solution is a phosphate buffer.

23. The method of claims 19 or 22, wherein the phosphate buffer comprises at least 50 mM, at least 62.5 mM, at least 100 mM, at least 125 mM, at least 150 mm, at least 175 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, at least 400 mM, at least 450 mM, or at least 500 mM phosphate.

24. The method of any one of claims 17-23, wherein the solution is incubated for at least 0.5 hr., 1 hr., 2 hrs., 3 hrs., 4 hrs., 5 hrs., 6 hrs., 7 hrs., 8 hrs., 12 hrs., 16 hrs., 20 hrs., 24 hrs., 30 hrs., or 36 hrs.

25. The method of any one of claims 17-24, wherein the temperature of the incubation is at or at least 27 ºC, at or at least 28 ºC, at or at least 29 ºC, at or at least 30 ºC, at or at least 31 ºC, at or at least 32 ºC, at or at least 33 ºC, at or at least 34 ºC, at or at least 35 ºC.

26. The method of any one of claims 17-25, wherein the BRZ54 protein is a recombinant BRZ54 protein.

27. A BRZ54 composition comprising the stabilized BRZ54 produced by the method of any one of claims 17-26.

28. The BRZ54 composition of claim 27, wherein the BRZ54 composition comprises at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% pyroE-BRZ54.

29. A host cell comprising a first coding sequence, wherein the first coding sequence encodes a heterologous brazzein-54 (BRZ54) protein and wherein the first coding sequence isAttorney Docket No.54282-0014WO1 configured to provide an N-terminal glutamine on the BRZ54 and a second coding sequence encoding a cyclotransferase, wherein the cell produces BRZ54 protein.

30. The cell of claim 29, wherein the first coding sequence comprises a signal sequence fused in-frame to a coding sequence of BRZ54.

31. The cell of claim 29 or claim 30, wherein at least 40% of the BRZ54 protein is pyroE-BRZ54.

32. The cell of claim 29 or claim 30, wherein at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the BRZ54 is pyroE-BRZ54.

33. The cell of any one of claims 29-32, wherein the cell is a microbial cell.

34. The cell of claim 33, wherein the microbial cell is a yeast cell.

35. The cell of claim 34, wherein the yeast cell is a Pichia cell, optionally Komagataella phaffii (Pichia pastoris).

36. The cell of any one of claims 29-35, wherein the cyclotransferase has the amino acid sequence selected from any one of SEQ ID Nos. 36, 38, 40, and 42 or wherein the cyclotransferase is a functional homolog of any one of SEQ ID Nos.36, 38, 40, and 42.

37. A method of producing a stable recombinant BRZ54 in vivo, comprising, co- expressing in a host cell a first coding sequence encoding a heterologous BRZ54 protein, wherein the first coding sequence is configured to provide an N-terminal glutamine on the BRZ54, and a second coding sequence encoding a cyclotransferase.

38. The method of claim 37, wherein the host cell is a microbial cell.

39. The method of claim 37, wherein the host cell is a yeast cell.Attorney Docket No.54282-0014WO1 40. The method of claim 37, wherein the host cell is a Pichia cell, optionally Komagataella phaffii (Pichia pastoris).

41. The method of any one of claims 37-40, wherein the first coding sequence encodes an amino acid sequence of any one of SEQ ID Nos. 9-34.

42. The method of any one of claims 37-40, wherein the first coding sequence encodes a signal sequence fused in-frame to a coding sequence for the BRZ54 protein.

43. The method of any one of claims 37-41, wherein the cyclotransferase has the amino acid sequence selected from the group consisting of SEQ ID Nos.36, 38, 40, and 42 or wherein the cyclotransferase is a functional homolog of any one of SEQ ID Nos.36, 38, 40, and 42.

44. A stabilized BRZ54 protein produced by the method of any one of claims 37-43.

45. A stabilized BRZ54 protein produced by the cell of any one of claims 29-36.

46. An edible ingredient comprising the stabilized BRZ54 protein of claim 44 or claim 45.

47. An edible product comprising the stabilized BRZ54 protein of claim 44 or claim 45.

48. The edible product of claim 47, further comprising one or more additional sweet proteins.

49. The edible product of claim 48, wherein the one or more additional sweet protein is selected from the group consisting of brazzein-53, monellin, miraculin, pentadin, mabinlin, curculin, thaumatin, and mycodulcein.Attorney Docket No.54282-0014WO1 50. The edible product of any one of claims 47-49, wherein the edible product is selected from a beverage, a food, or a sweetener.

Images