Bronaceae sweet protein variants

Abstract

There is provided a Bronstinan protein variant comprising X1 X2 KCKX6 X7 YX9 NYPX13 X14 KCX17 X18 AX20 QCNYDCKLDKHARX34 GECFYDEX42 RNX45X46 CX48 CDYCX53YX55 (SEQ ID NO: 1) wherein X1, X20 and X47 are each independently an amino acid having a polar uncharged side chain, X2 is an amino acid having a negatively charged side chain, X6 is glutamine (E) or an amino acid having a positively charged side chain, X7, X13, X18, X45 and X48 are each independently an amino acid having a hydrophobic side chain, X7, X13, X18, X45 and X48 are each independently an amino acid having a hydrophobic side chain, X7, X13, X18 X17 is lysine (K) or an amino acid having a polar uncharged side chain, X14 is arginine (R) or an amino acid having a polar uncharged side chain, X9 is arginine (R) or an amino acid having a negatively charged side chain, X42 is an amino acid having a positively charged side chain, X34 is alanine or an amino acid having a polar uncharged side chain, X17 is lysine (K) or an amino acid having a polar uncharged side chain, X14 is arginine (R) or an amino acid having a polar uncharged side chain. X46 is an amino acid having a polar uncharged side chain, X53 is glutamic acid, glutamine or lysine, and X55 is proline, arginine, threonine or no residue. Also disclosed are polynucleotides encoding the bristinin variants, cells expressing the bristinin variants, and methods of making the bristinin variants.

Description

Technical Field

[0001] This disclosure generally relates to protein variants. In particular, this disclosure relates to the brazzein variant. Background Technology

[0002] The dramatic rise in obesity and diabetes in recent decades has led to the widespread use of artificial sweeteners in food and beverages as sugar substitutes. These artificial sweeteners regulate blood sugar levels and provide a means of reducing calorie expenditure while maintaining palatability through sweetness. However, new evidence suggests an increased risk of cancer and cardiovascular disease, and these sweeteners have been shown to cause other harmful health effects such as gastrointestinal problems. This underscores the need for alternative sweeteners.

[0003] Due to their strong sweetness and low-risk safety profile, naturally derived sweet proteins have the potential to replace artificial sweeteners. Unlike sucrose, sweet proteins do not trigger insulin requirements in diabetics. To date, seven distinct sweet proteins have been identified from rainforest plants. These proteins are bunazhenin, thaumatin, monelin, curculin, mabinlin, miraculin, and pentadine.

[0004] Bunajin is a relatively small sweet-tasting protein composed of 54 amino acids, possessing an intense sweetness 500 to 2000 times stronger than sucrose. Originally isolated from the fruit of the West African plant *Pentadiplandra brazzeana* Bailon, its thermal and pH stability makes it an ideal system for applications in the biotechnology and food processing industries. Recent findings indicate that bunajin interacts with the human sweet taste receptors TAS1R2 / TAS1R3, providing the perception of sweetness in humans, and this is unrelated to harmful health effects.

[0005] While punazhenin is a promising alternative to sugars and artificial sweeteners, its natural preparation can be expensive. Therefore, to meet the demand for alternatives to sugars and artificial sweeteners, punazhenin variants are needed. Summary of the Invention

[0006] On the one hand, a variant of the puerarin protein is provided, which contains

[0007]

[0008] Where X1, X 20 and X 47Each of these is an amino acid with a polar, uncharged side chain; X2 is an amino acid with a negatively charged side chain; X6 is glutamine (E) or an amino acid with a positively charged side chain; X7, X... 13 X 18 X 45 and X 48 Each is an amino acid with a hydrophobic side chain, X 17 X is lysine (K) or an amino acid with a polar, uncharged side chain. 14 X1 is arginine (R) or an amino acid with a polar, uncharged side chain; X2 is arginine (R) or an amino acid with a negatively charged side chain; X3 is... 42 X is an amino acid with a positively charged side chain. 34 X is alanine or an amino acid with a polar, uncharged side chain. 46 X is an amino acid with a polar, uncharged side chain. 53 It is glutamic acid, glutamine, or lysine, and X 55 It may contain proline, arginine, threonine, or no residue.

[0009] In some instances, the variants share at least 85% sequence identity with wild-type bunazhensin.

[0010] In some instances, the variants differ from wild-type bujnazensin by 4 to 8 amino acids.

[0011] In some instances, the variants retain essentially the same structure as wild-type bujnazen.

[0012] In some instances, the variants have the same secondary structure as wild-type bujnazen.

[0013] In some instances, the variants are able to bind to taste receptors.

[0014] In some instances, the variants are able to bind to human sweet taste receptors.

[0015] In some instances, the variant contains

[0016] X1, which is either glutamine (Q) or serine (S),

[0017] X2, which is either aspartic acid (D) or glutamic acid (E),

[0018] X6 is glutamine (Q), lysine (K), or arginine (R).

[0019] X7 is either valine (V), methionine (M), or isoleucine (I).

[0020] X9 is either glutamic acid (E) or arginine (R).

[0021] X 13 It is either isoleucine (I) or valine (V).

[0022] X 14 It is asparagine (N), serine (S), or arginine (R).

[0023] X 17 It is either lysine (K) or glutamine (Q).

[0024] X 18 It is either leucine (L) or isoleucine (I).

[0025] X 20 It is either serine (S) or asparagine (N).

[0026] X 34 It is either serine (S), alanine (A), or threonine (T).

[0027] X 42 It is either arginine (R) or lysine (K).

[0028] X 45 It is methionine (M), leucine (L), or valine (V).

[0029] X 46 It is either glutamine (Q) or threonine (T).

[0030] X 48 It is isoleucine (I), valine (V), or leucine (L).

[0031] X 53 It can be independently glutamic acid, glutamine, or lysine, and

[0032] X 55 It can be independently proline, arginine, threonine, or without residues.

[0033] In some instances, the variant includes one or more permutations selected from K6Q, E9R, S14R, Q17K, S34A, E53Q, or E53K.

[0034] In some instances, the variant does not contain a residue at position 55.

[0035] In some instances, the variant contains

[0036] i.K6Q and Q17K,

[0037] ii.E9R,

[0038] iii. S34A and E53Q,

[0039] iv. The addition of S14R and 55R, or

[0040] The addition of v.E53K and 55T.

[0041] In some instances, the variants contain sequences selected from the following

[0042]

[0043] On the other hand, polynucleotides and / or vectors encoding bunazhensin proteins as described herein are provided.

[0044] On another front, a cell comprising a carrier is provided, the carrier containing the polynucleotides described herein.

[0045] In another aspect, a method for preparing bunazhensin is provided, the method comprising expressing, in an expression system, a polynucleotide and / or a vector encoding the bunazhensin as described herein. Detailed Implementation

[0046] Bunazine protein is a promising alternative to sugars and artificial sweeteners.

[0047] The inventors have demonstrated the applicability of protein language models for designing novel and diverse bunazhensin homologs, yielding thermostable and potentially sweeter homologs. The wild-type bunazhensin sequence is projected into the embedding space, followed by a random walk to explore sequences surrounding the neighboring regions of the wild-type protein. Representative sequences from the local embedding space of the wild-type bunazhensin are obtained and expressed.

[0048] Therefore, exemplary, non-limiting embodiments of a sweeter puerarin protein variant are disclosed below.

[0049] On the one hand, a variant of the puerarin protein is provided, which contains

[0050]

[0051] in

[0052] X1, X 20 and X 47 Each is an amino acid with a polar, uncharged side chain.

[0053] X2 is an amino acid with a negatively charged side chain.

[0054] X6 is glutamine (E) or an amino acid with a positively charged side chain.

[0055] X7, X 13 X 18 X 45 and X 48 Each is an amino acid with a hydrophobic side chain.

[0056] X 17 It is lysine (K) or an amino acid with a polar, uncharged side chain.

[0057] X 14 It is arginine (R) or an amino acid with a polar, uncharged side chain.

[0058] X9 is arginine (R) or an amino acid with a negatively charged side chain.

[0059] X 42 It is an amino acid with a positively charged side chain.

[0060] X 34 It is alanine or an amino acid with a polar, uncharged side chain.

[0061] X 46 It consists of amino acids with polar, uncharged side chains.

[0062] X 53 It is glutamic acid, glutamine, or lysine, and

[0063] X 55 It may contain proline, arginine, threonine, or no residue.

[0064] As used herein, the term "variant" and the term "homologous protein" are used interchangeably to refer to a protein that retains the same function and structure as its wild-type protein but has a different amino acid sequence. In some instances, variants may have improved function and / or properties. For example, a bunazhensin protein variant or bunazhensin protein homolog refers to a bunazhensin protein having a different amino acid sequence than the wild-type protein. Bunazhensin protein variants are capable of interacting with sweet taste receptors and may have improved binding and / or thermal stability. In some instances, the variant is not a naturally occurring variant. In some instances, the variant is an isolated variant. In some instances, the variant is a synthetic variant that is not naturally occurring.

[0065] In some instances, variants may include naturally occurring amino acids such as arginine (arg, R), histidine (his, H), lysine (lys, K), aspartic acid (asp, D), glutamic acid (glu, E), serine (ser, S), threonine (thr, T), asparagine (asn, N), glutamine (gln, Q), cysteine ​​(cys, C), glycine (gly, G), proline (pro, P), alanine (ala, A), valine (val, V), isoleucine (ile, L), leucine (leu, L), methionine (met, M), phenylalanine (phe, F), tyrosine (try, Y), and tryptophan (trp, W).

[0066] In some instances, amino acids with polar, uncharged side chains may include, but are not limited to, serine (ser, S), threonine (thr, T), asparagine (asn, N), and glutamine (gln, Q).

[0067] In some instances, amino acids with charged side chains may include amino acids with negatively charged side chains and / or amino acids with positively charged side chains.

[0068] In some instances, amino acids with positively charged side chains may include arginine (arg, R), histidine (his, H), and lysine (lys, K).

[0069] In some instances, amino acids with negatively charged side chains may include aspartic acid (asp, D) and glutamic acid (Glu, E).

[0070] In some instances, the amino acid can be a specific amino acid, such as cysteine ​​(cys, C), selenocysteine ​​(sec, U), glycine (gly, G), and proline (pro, P).

[0071] In some instances, the amino acid can be a hydrophobic side-chain amino acid, such as, but not limited to, alanine (ala, A), valine (val, V), isoleucine (ile, I), leucine (leu, L), methionine (met, M), phenylalanine (phe, F), tyrosine (tyr, Y), and tryptophan (trp, W).

[0072] As is known in the art, wild-type bunazhensin contains a core sequence.

[0073]

[0074] The amino acid sequence and structure of bubrazine are described in detail in Caldwell, J., Abildgaard, F., et al. Solution structure of the thermostable sweet-tasting protein brazzein. Nat Struct Mol Biol 5, 427–431 (1998). https: / / doi.org / 10.1038 / nsb0698-427, the contents of which are incorporated herein by reference.

[0075] In some instances, the bunazhensin protein may contain one or more mutations at the following positions: position 1, position 2, position 6, position 7, position 9, position 13, position 14, position 17, position 18, position 20, position 34, position 42, position 45, position 46, position 48, position 53 and / or position 55.

[0076] As used herein, the term "mutation" refers to an alteration of an amino acid (or the gene sequence encoding that amino acid sequence). A mutation may include one or more alterations known in the art. In some instances, a mutation may be a substitution, deletion, or insertion / addition. In some instances, a mutation is a substitution and / or deletion.

[0077] In some instances, the variant may have at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the wild-type protein (such as wild-type bunazhensin). In some instances, the variant may contain at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence described herein. In some instances, the variant has at least 85% sequence identity with wild-type bunazhensin.

[0078] In some instances, the variant may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids different from wild-type proteins (such as wild-type bunajunsin) and / or sequences as described herein. In some instances, the variant differs from wild-type bunajunsin by 4 to 8 amino acids. In some instances, the variant differs from wild-type bunajunsin by 8 amino acids (e.g., V23). In some instances, the variant differs from wild-type bunajunsin by 6 amino acids (e.g., V22). In some instances, the variant differs from wild-type bunajunsin by 4 amino acids (e.g., V21 and V24). In some instances, the variant differs from wild-type bunajunsin by 5 amino acids (e.g., V25).

[0079] In some instances, a variant may contain one mutation, two mutations, three mutations, four mutations, five mutations, six mutations, seven mutations, eight mutations, nine mutations, ten mutations, eleven mutations, twelve mutations, thirteen mutations, fourteen mutations, fifteen mutations, sixteen mutations, seventeen mutations, eighteen mutations, nineteen mutations, or more than nineteen mutations.

[0080] In some instances, the variant may contain mutations at the following positions: position 1, position 2, position 6, position 7, position 9, position 13, position 14, position 17, position 18, position 20, position 34, position 42, position 45, position 46, position 48, position 53 and / or position 55.

[0081] In some instances, the variant may contain mutations in the substitutions at the following positions: position 1, position 2, position 6, position 7, position 9, position 13, position 14, position 17, position 18, position 20, position 34, position 42, position 45, position 46, position 48, position 53 and / or position 55.

[0082] In some instances, variants may also include mutations at the following positions: position 1, position 2, position 7, position 13, position 18, position 20, position 42, position 45, position 46, and position 48. In some instances, mutations at positions 1, 2, 6, 7, 9, 13, 14, 17, 18, 20, 34, 42, 45, 46, and 48 are natural amino acids that replace amino acid residues with the same groups as the amino acid residues in the wild-type protein.

[0083] As used herein, variants can be indicated by specifying the position of the substituted amino acid that characterizes the variant. In this paper, substitutions are indicated by providing the wild-type amino acid residue, followed by its position number, and then the substituted amino acid residue to be substituted.

[0084] In some instances, the mutation is a substitution with a naturally occurring amino acid known in the art. In some instances, the substitution may be a substitution with a synthetic amino acid known in the art.

[0085] In some instances, the variant may also contain mutations known in the art that support increased thermal stability.

[0086] In some instances, variants may also include one or more mutations at the binding site of the sweet taste receptor.

[0087] In some instances, the variants retain essentially the same structure as wild-type bujnazen.

[0088] In some instances, the variants have secondary, tertiary, and / or quaternary structures that are substantially the same as (or similar to) those of wild-type bunazhensin, which gives the variants the same (or improved) biological functions as wild-type bunazhensin.

[0089] In some instances, the variants have the same secondary structure as wild-type bujnazen.

[0090] In some instances, the variant may contain β-folded sheets at positions 5 through 7, 34 through 39, and 45 through 50. In some instances, the variant may contain α-helical structures at positions 12 through 17 and 20 through 31.

[0091] In some instances, the variants are able to bind to taste receptors.

[0092] In some instances, the variant is able to bind to (or bind to) taste receptors. In some instances, the taste receptors are mammalian taste receptors. In some instances, the taste receptors are human taste receptors. In some instances, the taste receptors are sweet taste receptors. In some instances, the taste receptors are human sweet taste receptors, such as TAS1R2 / TAS1R3.

[0093] In some instances, the variants are able to bind to human sweet taste receptors.

[0094] In some instances, variants may include

[0095] X1, which is either glutamine (Q) or serine (S),

[0096] X2, which is either aspartic acid (D) or glutamic acid (E),

[0097] X6 is glutamine (Q), lysine (K), or arginine (R).

[0098] X7 is either valine (V), methionine (M), or isoleucine (I).

[0099] X9 is either glutamic acid (E) or arginine (R).

[0100] X 13 It is either isoleucine (I) or valine (V).

[0101] X 14 It is asparagine (N), serine (S), or arginine (R).

[0102] X 17 It is either lysine (K) or glutamine (Q).

[0103] X 18 It is either leucine (L) or isoleucine (I).

[0104] X 20 It is either serine (S) or asparagine (N).

[0105] X 34 It is either serine (S), alanine (A), or threonine (T).

[0106] X 42 It is either arginine (R) or lysine (K).

[0107] X 45 It is methionine (M), leucine (L), or valine (V).

[0108] X 46 It is either glutamine (Q) or threonine (T).

[0109] X 48 It is isoleucine (I), valine (V), or leucine (L).

[0110] X 53 It can be independently glutamic acid, glutamine, or lysine, and

[0111] X 55 It can be independently proline, arginine, threonine, or without residues.

[0112] In some instances, the variant contains one or more permutations selected from K6Q, E9R, S14R, Q17K, S34A, E53Q, or E53K.

[0113] In some instances, the variants include the addition of 55P, 55R, or 55T.

[0114] In some instances, the variant contains

[0115] i.K6Q and Q17K,

[0116] ii.E9R,

[0117] iii. S34A and E53Q,

[0118] iv. The addition of S14R and 55R, or

[0119] The addition of v.E53K and 55T.

[0120] In some instances, the variants contain sequences selected from the following

[0121]

[0122] Not wanting to be bound by theory, the inventors of this disclosure have discovered that amino acid residues at positions 29 to 33, 36, 39 to 43, and the C-terminus are associated with sweetness.

[0123] In some instances, the bunazhensin protein variant may also contain other mutations known in the art. For example, the bunazhensin protein variants disclosed herein may also contain other mutations, such as, but not limited to, D29, H31, K5, D2, E41, etc.

[0124] On the other hand, polynucleotides and / or vectors encoding variants of the punacin sweet protein as described herein are provided.

[0125] On another front, a cell comprising a carrier is provided, the carrier containing the polynucleotides described herein.

[0126] In some instances, the host cell is a bacterial cell, yeast cell, and / or mammalian cell. In some instances, the host cell is a bacterium generally considered safe. In some instances, the host cell is *Lactococcus lactis*.

[0127] To increase yield, the inventors also provide a faster and significantly higher-yield method for purifying buna cinnamic acid protein from generally considered safe (GRAS) bacteria such as Lactococcus lactis.

[0128] In this disclosure, protein folding algorithms are used to design puna tinsus protein variants for preparation in generally considered safe (GRAS) host organisms, including sweeter and more thermostable bio-similar puna tinsus protein variants. To improve yield, the inventors have also optimized the biochemical preparation and purification methods for expressing puna tinsus proteins in GRAS microorganisms.

[0129] Therefore, in another aspect, methods for preparing punatin protein variants are provided, including expressing polynucleotides and / or vectors encoding punatin protein variants as described herein in an expression system.

[0130] In some instances, the expression system may be one or more expression systems, including bacterial expression systems, yeast expression systems, and / or fungal expression systems. In some instances, the expression system may be a bacterial expression system, such as, but not limited to, GRAS (Generally Recognized As Safe) bacterial systems. In some instances, the expression system may be a yeast expression system, such as, but not limited to, *Saccharomyces cerevisiae*, *Pichia pastoris*, *Hansulae polymopha*, and *Kluyveromyces lactis*. In some instances, the expression system may be a fungal expression system (e.g., filamentous fungi), such as, but not limited to, *Aspergillus* and *Trichoderma*.

[0131] In some instances, the expression system is a bacterial expression system, Lactococcus lactis.

[0132] In some instances, the method further includes the step of extracting the expressed bunajunte protein variant. In some instances, the extraction of the expressed bunajunte protein variant can be performed by sonication or by heatlysis. Advantageously, the inventors of this disclosure have found that heatlysis provides better yields than sonication. Therefore, samples can be processed solely by heatlysis instead of sonication. In some instances, heatlysis may include boiling at about 90°C to 100°C, or about 91°C, about 92°C, about 93°C, about 94°C, about 95°C, about 96°C, about 97°C, about 98°C, about 99°C, 100°C, about 101°C, about 102°C, about 103°C, about 104°C, about 105°C, about 106°C, about 107°C, about 108°C, about 109°C, or about 110°C. In some instances, pyrolysis can be carried out for approximately 5 to 30 minutes, or approximately 5 minutes, approximately 10 minutes, approximately 15 minutes, approximately 10 minutes, approximately 20 minutes, approximately 25 minutes, or approximately 30 minutes.

[0133] In some instances, the method also includes boiling the sample for 1 to 6 hours, or about 3.5 hours, 4 hours, 4.5 hours, or 5 hours.

[0134] In some instances, methods for sweetening products are provided, including providing a puerarin protein variant as described herein.

[0135] The term “and / or”, such as “X and / or Y”, is understood to mean “X and Y” or “X or Y”, and should be used to provide clear support for both meanings or either meaning.

[0136] Furthermore, throughout this specification, the word “substantially” is understood to include, but is not limited to, “wholly” or “completely”. Additionally, terms such as “comprising” and “including” are intended as non-restrictive descriptive language, as they broadly encompass the elements / components described following these terms, as well as other components not explicitly described. For example, when “comprising” or “including” is used, a reference to “one” feature is also intended to refer to “at least one” of that feature. Terms such as “comprising” can be considered, in appropriate context, as a subset of terms such as “comprising” or “including”. Therefore, in embodiments disclosed herein that use terms such as “comprising” or “including”, it should be understood that these embodiments provide instruction for corresponding embodiments using terms such as “comprising”. Furthermore, terms such as “about,” “approximately,” and “approximately” generally imply reasonable variations, such as a variation of + / -5% of the disclosed value, or a variation of 4% of the disclosed value, or a variation of 3% of the disclosed value, or a variation of 2% of the disclosed value, or a variation of 1% of the disclosed value.

[0137] Furthermore, certain values ​​may be disclosed within a range in this specification. The values ​​shown at the endpoints of a range are intended to illustrate preferred ranges. Whenever a range is described, it means that the range covers and teaches all possible subranges as well as individual values ​​within that range. That is, the endpoints of a range should not be interpreted as immutable limitations. For example, a description of a range of 1% to 5% is intended to specifically disclose subranges such as 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, etc., and individual values ​​within that range, such as 1%, 2%, 3%, 4%, and 5%. It should be understood that individual values ​​within a range also include integers, fractions, and decimals. Furthermore, whenever a range is described, it also means that the range covers and teaches values ​​with up to two additional decimal places or significant figures (if appropriate) starting from the endpoint of the indicated value. For example, the description of a range of 1% to 5% is intended to specifically disclose ranges of 1.00% to 5.00% and 1.0% to 5.0%, as well as all intermediate values ​​across these ranges (e.g., 1.01%, 1.02%...4.98%, 4.99%, 5.00% and 1.1%, 1.2%...4.8%, 4.9%, 5.0%, etc.). The above specific disclosure is intended to apply to ranges of any depth / width.

[0138] Furthermore, when describing some embodiments, this disclosure may have disclosed methods and / or processes as a specific order of steps. However, unless otherwise required, it should be understood that the method or process should not be limited to the specific order of steps disclosed. Other orders of steps are also possible. The specific order of steps disclosed herein should not be construed as an undue limitation. Unless otherwise required, the methods and / or processes disclosed herein should not be limited to steps performed in the order they are written. The order of steps may be changed, and still remain within the scope of this disclosure.

[0139] Furthermore, it should be understood that while this disclosure provides embodiments having one or more features / characteristics discussed herein, one or more of these features / characteristics may be omitted in other alternative embodiments, and this disclosure provides support for these omissions and these related alternative embodiments. Attached Figure Description

[0140] For those skilled in the art, exemplary embodiments of this disclosure will be better understood and apparent from the following discussion, and, where applicable, in conjunction with the accompanying drawings. It should be understood that other modifications may be made to the variations without departing from the scope of the invention. The exemplary embodiments are not necessarily mutually exclusive, as some embodiments may be combined with one or more embodiments to form new exemplary embodiments. The exemplary embodiments should not be construed as limiting the scope of this disclosure.

[0141] Figure 1 This paper demonstrates a general strategy for designing proteins by exploring the potential space of large language models trained on protein sequences. The process of generating a library of bio-like proteins begins with the selection of initial natural proteins with desired sweetness characteristics.

[0142] Figure 2 The sequences of mutants derived from AI and data on the sequences are shown. (A) Sequence alignments of mutants / variants (V21 to V25) relative to wild-type are shown. Identical residues are highlighted in gray. Similar residues are labeled in gray, and dissimilar residues are circled. The structural elements of wild-type bunazhensin (PDB:4HE7) are also shown. Numbering begins with Q after the N-terminus M. (B) Comparison of sweet receptor responses to calcium mobilization using luminescence-based readout sweet receptor assays with various mutants and wild-type bunazhensin expressed in *E. coli* is shown. The percentage of calcium mobilization was calculated relative to the maximum assay response from 15 mM sucralose. Data were interpolated and averaged based on a nonlinear fit of experimentally sourced data from at least two independent assay runs at a normalized protein concentration of 0.1 mg / mL. Error bars are SD.

[0143] Figure 3 The expression and processing of bunazhensin from L. lactis, which is generally considered safe (GRAS), are shown. (A) The purification workflow is shown. (B) A representative protein gel of purified wild-type bunazhensin is shown, where L: Novex pre-stained ladder, lane 1: sonicated. Figure 3 Method A, Lane 2: Thermal pyrolysis (95℃ for 10 minutes). Figure 3 Method A, Lane 3: Thermal lysis, purification, and boiling at 95°C for 4 hours. Figure 3 Method A (3) and its equivalent V23 (lanes 4 to 6). Invitrogen Novex 16% Tricine gel was used with Tricine SDS electrophoresis buffer. The gel was stained with Coomassie blue and imaged. (C) shows the use of two lysis methods ( Figure 3 Total protein concentrations of His-tagged purified buna cinnamic acid protein were determined using Methods 1 and 2. Data were obtained from three replicates of three independent runs. Error bars are SD. Paired t-tests of protein yield between the two lysis methods showed statistical significance (p < 0.05).

[0144] Figure 4 The sweetness potency of wild-type bumarone protein and the V23 mutant expressed in *L. lactis* and subjected to two different cleavage methods and high-temperature heat treatment at 95°C for 4 hours is shown. Fluorescence-based sweetness receptor assays were used to test the samples. The scale of semathymidine for sweetness receptor assays at different concentrations is shown. Data were interpolated and averaged at a single protein concentration point of 68 μg / mL from at least six experimental replicates, based on a nonlinear fit of the experimentally sourced data. Error bars are SD. One asterisk (*) indicates p = 0.0138 (one-way ANOVA). Four asterisks (****) indicate p < 0.0001.

[0145] Experimental data

[0146] Materials and methods

[0147] plasmid construction

[0148] Codon-optimized DNA sequences of *E. coli* (E. coli) for the His-tagged buna cinnamic acid protein construct were synthesized from Twist Biosciences (Singapore) in the pET24a(+) vector. The constructs were transformed into *E. coli* Omnimax for sequencing and into *E. coli* BL21(DE3) for protein expression. Codon-optimized DNA sequences of *L. lactis* for the His-tagged buna cinnamic acid protein construct were also synthesized from Twist Biosciences. The fragments were cloned into the pNZ8148 vector using Gibson assembly. The constructs were transformed into *L. lactis* NZ9000 for sequencing and protein expression. The amino acid sequences of the final constructs are shown in Table S1.

[0149] Table 1. EC5 values ​​of wild-type bujnazentine and various mutants after treatment with three different cleavage methods. 50 Values, expressed in mg / mL and μM. EC values ​​of wild-type bujnazenta protein and AI-designed V23 mutants treated with three different cleavage methods. 50 Values, expressed in μM. Samples were tested using fluorescence-based assays.

[0150]

[0151] The EC was generated using the four-parameter logarithmic regression equation in Prism 8 (GraphPad) software. 50 The value was determined with the following constraints applied: bottom asymptote > 0.25; 0 < top asymptote < 2.

[0152] E. coli BL21(DE3) protein expression and purification

[0153] Single colonies of transformed E. coli BL21(DE3) were inoculated into LB broth containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking (200 rpm). The overnight culture was transferred to 300 mL of Terrific broth containing 50 μg / mL kanamycin at 37°C and 200 rpm. Protein expression was induced with 1 mM isopropyl β-D-1-thiogalactoside (IPTG) when the optical density (OD600) at 600 nm reached 0.4 to 0.6, and incubated overnight at 30°C and 200 rpm. The culture was then harvested by centrifugation at 8000 g (10 min) at 4°C, and the precipitate was resuspended in BugBuster protein extraction reagent (Merck, catalog number 70584) and incubated at room temperature (15 min). The resulting lysate was then centrifuged at 18000 g (20 min) at 4°C. The supernatant was incubated with PureCube 100 INDIGO Ni-Agarose resin (Cube-biotech, catalog number 75110) at room temperature for 1 hour, and the protein-bound resin was washed with 50 mM sodium phosphate buffer, 500 mM sodium chloride, and 20 mM imidazole at pH 7.4. The bound proteins were then eluted with 50 mM sodium phosphate buffer, 500 mM sodium chloride, and 500 mM imidazole at pH 7.4. The eluent was buffer-displaced and concentrated with Hank's balanced salt solution (HBSS) containing 20 mM HEPES at pH 7.0.

[0154] L. lactis NZ9000 protein expression and purification

[0155] Single colonies of transformed *L. lactis* NZ9000 were inoculated into M17 broth (0.5% glucose, 10 μg / mL chloramphenicol) and incubated overnight at 30°C without shaking. The overnight culture was then inoculated into 2 L of 2x M17 broth (2% glucose, 10 μg / mL chloramphenicol) to OD0.05. 600 The value was 0.1, and it was incubated at 30°C. When the OD... 600 When the expression level reached 1.0, protein expression was induced for 3 hours at 30°C with 50 ng / mL nisin.

[0156] The culture was centrifuged at 8000g for 10 minutes at 4°C. The resulting precipitate was freeze-thawed and resuspended in 50mM sodium phosphate buffer, 300mM sodium chloride, 10mM imidazole, and 0.03% Triton X-100 at pH 7.4, followed by incubation at room temperature for 15 minutes. The resuspended precipitate was sonicated four times at 10-second intervals on ice, or boiled at 95°C for 10 minutes. The resulting lysate was then centrifuged at 18000g for 20 minutes at 4°C. The supernatant was incubated with PureCube 100 INDIGO Ni-agarose resin at room temperature for 1 hour. The protein-bound resin was washed with 50mM sodium phosphate buffer, 500mM sodium chloride, and 20mM imidazole at pH 7.4. The bound protein was eluted with 50mM sodium phosphate buffer, 500mM sodium chloride, and 500mM imidazole at pH 7.4. The eluent was buffer-displaced and concentrated using HBSS containing 20 mM HEPES at pH 7.0. For thermal stability testing ( Figure 2 A-3), boil the sample in HBSS-HEPES buffer at 95°C for 4 hours, then rapidly cool to 4°C.

[0157] Sweetness receptor luminescence measurement

[0158] HEK 293T (ATCC) cells were maintained at 37°C in humid air with 5% CO2 and cultured in Duchenne Modified Eagle Medium (DMEM; Gibco) supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS; Biowest) and 1% (v / v) penicillin-streptomycin (Gibco) at high glucose. White 384-well Greiner tissue culture plates were coated with poly-D-lysine (PDL; Sigma) to a final concentration of 1 mg / mL. 293T cells were then seeded at a density of 20,000 cells / well into the coated plates and incubated overnight.

[0159] Cells were transiently transfected using two plasmids: a multi-gene CMV promoter-based expression vector containing genes for sweet receptors (TAS1R2 / TAS1R3) and a chimeric Gα16-gust44 gene; and a second plasmid expression vector containing genes for apophotoprotein and mitochondrial targeting (mt)-clytin II. Both plasmids were transfected using ViaFect (Promega) at a ratio of 20 ng:20 ng per well, with a transfection reagent to plasmid ratio of 3:1 μL:μg. Six hours after transfection, the culture medium was completely replaced with low-glucose DMEM (Gibco) supplemented with 10% (v / v) heat-inactivated FBS (Biowest) and 1% (v / v) penicillin-streptomycin (Gibco).

[0160] After overnight incubation under standard cell culture conditions, transfected cells were loaded with 10 μM coelentrin F (AATBioquest) in low glucose medium and assay buffer (1×HBSS assay buffer containing 20 mM HEPES, pH 7.0). The assay plate was then incubated in the dark at 27°C for 4 hours. Assays were performed using a fluorescence imaging plate reader (FLIPRTETRA, Molecular Devices) controlled by ScreenWorks software (version 4.0.0.30). During the run, baseline readings were captured for 10 seconds before dispensing 25 μL of the test ligand, prepared at twice the concentration in assay buffer, from the source plate to the assay plate. Kinetic data were then acquired for 100 seconds to record the response of each well to the added test sample. The response of each well was exported as the area under the curve (AUC) value, and the data were plotted using a four-parameter logarithmic regression equation using Prism 8 (GraphPad) software. Unless otherwise stated, the reported data are derived from at least two independent replicate experiments. For this study, two reference sweeteners, sucralose and semathymidine, were used for comparison with our bujnine test samples.

[0161] Fluorescence measurement of sweet taste receptors

[0162] Adenovirus 293AD (Cell Biolabs, Inc.) cells were maintained under cell culture conditions similar to those for 293T cells. Cells were seeded at a density of 12,000 cells per well in black 384-well Greiner tissue culture plates and allowed to grow overnight.

[0163] Using Viafect reagent, 25 ng per well was transiently transfected into 293AD cells with an expression vector based on a multi-gene CMV promoter containing genes for sweet receptors (TAS1R2 / TAS1R3) and a chimeric Gα16-gust44. Six hours after transfection, the growth medium was removed and replaced with low-glucose DMEM (Gibco) supplemented with 10% (v / v) heat-inactivated FBS (Biowest) and 1% (v / v) penicillin-streptomycin (Gibco). The next day, the transfected cells were loaded with Calcium 6 (Molecular Devices) fluorescent dye. The assay plates were first incubated at 37°C in a humidified incubator containing 5% CO2 for 2 hours, followed by a further 30 minutes of incubation at room temperature on a laboratory bench to reach equilibration.

[0164] Measurements were performed using the FLIPR-TETRA fluorescence mode. Fluorescence intensity was directly correlated with the amount of intracellular calcium released into the cytoplasm in response to ligand-mediated activation of sweet receptors, which is considered a measure of receptor activation. The change in calcium membrane potential over time was measured using excitation light from 470 nm to 495 nm and emission light from 515 nm to 575 nm. Baseline measurements were taken once per second for 10 seconds before the addition of sweetener or test sample, followed by subsequent measurements for 310 seconds.

[0165] The emission fluorescence values ​​were converted to a response (maximum) value compared to a baseline (minimum) value using ScreenWorks software (version 4.0.0.30, Molecular Devices), and the data were plotted using a four-parameter logarithmic regression equation using Prism 8 (GraphPad) software. The potency of the compound's sweet response to sweet receptors can be evaluated, denoted as EC. 50 , which is the molecular concentration required to produce a half-maximal response in sweetness receptor assays.

[0166] Protein library sequence characterization

[0167] The protein library contains five orthogonal sequences, which use bunazhensin as a natural wild-type reference to capture different variations (Table 2).

[0168] Table 2. Representative sequences captured from the sucralose library

[0169]

[0170] Table 3. EC50 of purified variants of L. lactis based on fluorescence assay of sweet receptors (TAS1R2 / R3) 50 * .

[0171] ID <![CDATA[EC 50 ]]> WT Bunazon Sweet Protein 52.2μM V23 39.1μM V23 H31R / E41A 24.2μM V23 31.9μM V23 H31R / E41A 16.8μM

[0172] The variants were purified by boiling and lysing the cells, but these variants were not subjected to further heat treatment. * EC 50 This refers to the molecular concentration required to produce a half-maximal response in sweet taste receptors.

[0173] The H31R / E41A mutant is derived from Lee, J.-W., Cha, J.-E., Jo, H.-J., & Kong, K.-H. (2013). Multiple mutations of the critical amino acid residues for the sweetness of the sweet-tasting protein, brazzein. Food Chemistry, 138(2-3), 1370-1373. https: / / doi.org / 10.1016 / j.foodchem.2012.10.140.

[0174] result

[0175] The generation of computable folded sweet protein variants

[0176] Using pre-trained language models (SeqVec, UniRep, CPCprot), the protein sequence of bunajinsin was embedded into the latent space of these models. It has been previously shown that the latent space near the target protein contains sequences that retain the structural and functional properties of the original protein. Leveraging this property, the inventors of this disclosure explored the latent space surrounding bunajinsin using a simple adaptive walk, which introduces mutations within the latent space, simultaneously optimizing several desired properties: 1) sequence heterogeneity, 2) thermostability, and 3) solubility. Directed evolution of the sequence along different paths in the latent space allows the inventors to construct libraries of high-confidence sequences as precursors for downstream characterization. Figure 1 ).

[0177] Scaling up increases the throughput of sweetness detection and comparison.

[0178] In this study, the inventors employed a cell-based sweet receptor assay to rapidly and systematically evaluate the relative sweetness of bunazhensin variants. Similar receptor-based assays have been routinely used in studies of sweet taste reception and sweetener molecule optimization (Riedel et al., 2017). Human sweet receptors and their signaling components are heterologously expressed in cultured mammalian HEK cells and have shown responses to a variety of sweeteners. This method measures calcium mobilization in response to sweetener activation of sweet receptors, including sweet proteins such as bunazhensin, carbohydrate sweeteners such as sucrose, and natural and synthetic sweetener molecules such as sucralose and steviol. This allows for rapid and increased screening throughput that would otherwise be impossible with human sensory groups.

[0179] Five variants of the bunazhenisin protein were generated through calculations, each with 5 to 8 mutations, including deletions. Figure 2 A). As an initial assay, prior to screening for sweet receptor responses, variants were expressed in *E. coli*, purified via affinity tag pull-down, and compared with wild-type bujnana protein as a control. Because optimization of bujnana protein purification was not achieved in this initial screening experiment, the samples expressed in *E. coli* had a high percentage of impurities (Figure 5), which contributed to non-specific signals in the assay readout. Therefore, the inventors could not fully attribute the cause to a specific sweet response for accurate comparison between the *E. coli*-expressed samples. This initial screening mutant dataset showed a potential tendency for the V23 variant to be sweeter than WT. Figure 2 B). To conduct further research, the inventors focused on preparing high-purity V23 variant samples and WT bunazhensin.

[0180] Bioprocessing optimization in L. lactis

[0181] To optimize the expression and purity of buñazin, the inventors turned to GRAS L. lactis NZ9000. Although buñazin expression from L. lactis was sufficient to improve purity, the yield was significantly low; <0.1 mg / L. Subsequently, the inventors also developed a heat-based purification protocol utilizing the thermal stability of buñazin. While both thermal lysis and sonication are effective in rupturing bacterial cells to release intracellular proteins, thermal lysis is useful when dealing with heat-stable proteins. Furthermore, heating can denature all heat-labile proteins, thereby increasing purity. Increased yield can also potentially be achieved through the thermal denaturation of proteases, which would otherwise degrade the target protein. Others have previously used a two-hour heat treatment at 80°C as a second purification step after ammonium precipitation, successfully increasing the purity of buñazin expressed in transgenic tobacco. Therefore, the inventors hypothesized that heating could be used instead of mechanical lysis (sonication) to lyse and purify expressed buñazin. Heat-based lysis of the cell pellet was performed by boiling the cell pellet at 95°C for 10 minutes. Figure 3 A). In our observations, using a thermal pyrolysis protocol increased the purity of the samples. Figure 3 B). More importantly, the yield of bunotecan protein also increased significantly by 10 times between the two methods ( Figure 3 C). Although the study did not explore further, the inventors anticipate that by optimizing fermentation conditions, yields can be further increased compared to thermal pyrolysis-mediated purification.

[0182] Representation of the V23 variant derived from AI

[0183] Using products derived from bioprocessing, the inventors examined the sweetness response of sonicated and thermally pyrolyzed products of the WT and V23 variants. Thermal stability was further tested by reboiling the puerarin protein samples at 95°C for 4 hours. Figure 3 A – Method 3). The V23 equivalent was more sensitive in sweetness receptor assays compared to the products of sonication, thermal pyrolysis, and boiling of WT. Figure 4 (Table 1). In general, the heat-treated products were less sensitive in taste receptor assays compared to the sonicated products, which is not surprising, as protein folding is expected to be disrupted in the presence of heat. However, in all three observations, V23 was more sensitive than WT, indicating that V23 is sweeter than wild-type bunazhenin, and this potency was maintained by heat treatment. Surprisingly, the sample boiled for 4 hours showed a slightly higher calcium response compared to lysis for 10 minutes, suggesting that further heating may have removed residual heat-labile proteins, including slightly misfolded bunazhenin. Although the sweetness potency of the products appears to decrease with both the 10-minute thermal lysis and sonication protocols, previous examples of heat treatment under mild conditions, such as 80°C for 2 hours, suggest that further refinements to heat treatment can be used to minimize the impact on sweetness potency while maintaining yield.

[0184] The inventors here demonstrate the application of zero-shot computational design, which creates a thermostable and potentially sweeter puerarin homolog, V23. To characterize this, the inventors established a workflow that includes quality control assays for quantitative protein sweetness, allows for accurate characterization and comparison of puerarin mutants, and a higher-yield puerarin purification protocol from GRAS L. lactis.

[0185] AI-driven protein design allows inventors to rapidly evaluate large numbers of protein sequences, which in turn allows them to identify highly optimized candidates that are difficult to discover using conventional methods. Without pre-input data on the specificity of bunazhensin, inventors were able to design high-order mutants (5 to 8 mutations), with screening of 5 mutants revealing one that was superior to wild-type bunazhensin. Previous studies have investigated mutants based on wild-type bunazhensin to identify key regions important for sweetness. The results of these experiments indicate that residues 29 to 33 and 39 to 43, plus residue 36 between these segments, and the C-terminus are involved in sweetness, and that charge plays a significant role in its interaction with sweetness receptors. Unexpectedly, the computationally generated mutations in this study were located far from previously observed regions of interest, suggesting that there is greater potential beyond conventionally probed regions to further enhance the sweetness of bunazhensin, and conversely, computationally designed variants can be further optimized using combinations of known mutations.

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[0219] application

[0220] The embodiments of the buna ginsenoside homologs or variants disclosed herein provide alternative sweeteners.

[0221] Advantageously, the bunazhenin homologs or variants described herein have superior thermal stability, wherein boiling of purified bunazhenin samples does not significantly reduce or weaken their ability to activate sweet receptors.

[0222] More advantageously, the bunazhenin homologs or variants described herein possess the desired properties: 1) sequence differences, 2) thermal stability, and / or 3) solubility. In some instances, bunazhenin variants or homologs have been found to be sweeter than wild-type bunazhenin.

[0223] Even more advantageously, homologs or variants of the bunazhensin protein described herein can be prepared at low cost.

[0224] This disclosure also provides an improved method for the preparation and purification of buna cinnamic acid protein for expression.

[0225] Those skilled in the art will understand that other changes and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of this disclosure, which is broadly described. For example, features of different exemplary embodiments may be mixed, combined, interchanged, combined, adopted, modified, incorporated, etc., in different exemplary embodiments. Therefore, the present embodiments are to be considered illustrative rather than restrictive in all respects.

Claims

1. A variant of bunotecanin, comprising X1X2KCKX6X7YX9NYPX 13 X 14 KCX 17 X 18 AX 20 QCNYDCKLDKHARX 34 GECFYDEX 42 RNX 45 X 46 CX 48 CDYCX 53 YX 55 (SEQ ID NO:1), in X1, X 20 and X 47 Each is an amino acid with a polar, uncharged side chain. X2 is an amino acid with a negatively charged side chain. X6 is glutamine (E) or an amino acid with a positively charged side chain. X7, X 13 X 18 X 45 and X 48 Each is an amino acid with a hydrophobic side chain. X 17 It is lysine (K) or an amino acid with a polar, uncharged side chain. X 14 It is arginine (R) or an amino acid with a polar, uncharged side chain. X9 is arginine (R) or an amino acid with a negatively charged side chain. X 42 It is an amino acid with a positively charged side chain. X 34 It is alanine or an amino acid with a polar, uncharged side chain. X 46 It consists of amino acids with polar, uncharged side chains. X 53 It is glutamic acid, glutamine, or lysine, and X 55 It may contain proline, arginine, threonine, or no residue.

2. The bunazhensin variant of claim 1, wherein the variant has at least 85% sequence identity with wild-type bunazhensin.

3. The puna cinnamic acid variant according to claim 1 or claim 2, wherein the variant differs from wild-type puna cinnamic acid by 4 to 8 amino acids.

4. A variant of bunazhenin according to any one of the preceding claims, wherein the variant retains substantially the same structure as wild-type bunazhenin.

5. A variant of bunazhenin according to any one of the preceding claims, wherein the variant has the same secondary structure as wild-type bunazhenin.

6. A variant of the puerarin protein according to any one of the preceding claims, wherein the variant is capable of binding to taste receptors.

7. A variant of the bunazhenin protein according to any one of the preceding claims, wherein the variant is capable of binding to human sweet taste receptors.

8. A variant of the puerarin protein according to any one of the preceding claims, wherein the variant comprises X1, which is either glutamine (Q) or serine (S), X2, which is either aspartic acid (D) or glutamic acid (E), X6 is glutamine (Q), lysine (K), or arginine (R). X7 is either valine (V), methionine (M), or isoleucine (I). X9 is either glutamic acid (E) or arginine (R). X 13 It is either isoleucine (I) or valine (V). X 14 It is asparagine (N), serine (S), or arginine (R). X 17 It is either lysine (K) or glutamine (Q). X 18 It is either leucine (L) or isoleucine (I). X 20 It is either serine (S) or asparagine (N). X 34 It is either serine (S), alanine (A), or threonine (T). X 42 It is either arginine (R) or lysine (K). X 45 It is methionine (M), leucine (L), or valine (V). X 46 It is either glutamine (Q) or threonine (T). X 48 It is isoleucine (I), valine (V), or leucine (L). X 53 It can be independently glutamic acid, glutamine, or lysine, and X 55 It can be independently proline, arginine, threonine, or without residues.

9. A variant of the buna cinnamic acid protein according to any one of the preceding claims, wherein the variant comprises one or more substitutions selected from K6Q, E9R, S14R, Q17K, S34A, E53Q or E53K.

10. A variant of the bunazhente protein according to any one of the preceding claims, wherein the variant does not contain a residue at position 55.

11. A variant of puerarin according to any one of the preceding claims, wherein the variant comprises i.K6Q and Q17K, ii.E9R, iii. S34A and E53Q, iv. The addition of S14R and 55R, or The addition of v.E53K and 55T.

12. A variant of the bunazhenin protein according to any one of the preceding claims, wherein the variant comprises a sequence selected from: and 13. A polynucleotide and / or vector encoding a variant of the buna cinnamic acid protein according to any one of the preceding claims.

14. A cell comprising a carrier, said carrier comprising the polynucleotide according to claim 13.

15. A method for preparing a variant of bunazhenin protein, the method comprising: The expression system expresses a polynucleotide and / or a vector that encodes a punacin sweet protein variant according to claims 1 to 14.

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