Fusion protein with sweet taste as well as preparation method and application thereof

Abstract

The invention provides a fusion protein with sweetness as well as a preparation method and application thereof, and relates to the field of synthetic biological and recombinant proteins. According to the fusion protein with the sweetness, the sweetness of the fusion protein after heterologous expression is improved by fusing other fragments at the nitrogen terminal and / or the carbon terminal of Brazilian sweet protein; the amino acid sequence of the brazitame protein is as shown in SEQ ID NO.1 or SEQ ID NO.25, and other fragments mainly contain histidine. Compared with natural Brazilian sweet protein, the fusion protein has higher sweetness, can realize heterologous expression, and lays a technical foundation for large-scale production.

Description

[0001] This application claims the priority of a Chinese patent application with an application date of December 19, 2023 (Application No.: 2023117586160, Invention Title: Sweet Fusion Protein and Its Preparation Method and Application), and part of the content of this Chinese patent application is incorporated into this application by reference in its entirety. Technical Field

[0002] The present invention relates to the fields of synthetic biology and recombinant proteins, and in particular to a sweet fusion protein and its preparation method and application. Background Art

[0003] In modern food industry, sweeteners such as sucrose, maltose, fructose, and glucose have always been indispensable food additives; however, due to the long-term excessive intake of such traditional sweeteners, more and more people have suffered from various diseases, including but not limited to high blood pressure, high blood sugar, high blood lipids, obesity, dental caries, etc. Therefore, there is an urgent need for a new type of sweet substance that has both high sweetness and low calories, and is different from the potential health hazards of artificial sweeteners, to meet people's living and health needs.

[0004] As is well known, most proteins in nature are tasteless, while some plant proteins have a sweet taste or can convert sour taste into sweet taste, collectively known as plant sweet proteins. They are not only natural and non-toxic, but also have high sweetness, low calories, and the degradation products after digestion are natural amino acids required by the human body, with good safety, and are one of the best substitutes for traditional and artificial sweeteners.

[0005] So far, 8 sweet proteins have been found in plants, including Thaumatin, Mabinlin, Pentadin, Curculin, Brazzein, Monellin, Miraculin & Neoculin. Among them, Brazzein is a sweet protein isolated and purified by Ding et al. in 1994 from the fruit of the wild plant Pentadiplandra Brazzeana Baillon in western Africa; it can cause a sweet taste by itself, has no sweetening modification effect, and its sweetness is 500 - 2000 times that of sucrose. Compared with other sweet proteins, Brazzein has the smallest molecular weight, the best water solubility, and its aqueous solution still maintains sweetness after heat treatment at 80 °C for 4 h, with good thermal stability and pH stability, and is suitable for various food processing technologies. However, the application of Brazzein in the prior art still has the following defects:

[0006] First, brazzein is obtained from a tropical plant that is unique to Africa. However, it is difficult for this tropical plant to bear fruit in other environments, which limits its development and application. In addition, the natural content of brazzein in the plant is low, making it difficult to extract and produce on a large scale. Second, although many studies have confirmed that the gene sequence of the target protein can be transferred into various expression hosts through protein engineering techniques, most of them only stay in the laboratory stage and there are still some problems to be solved. For example, brazzein has been heterologously expressed in microorganisms (such as Escherichia coli, yeast, and lactic acid bacteria) and plants as vectors. However, the sweet protein has a reduced or even disappeared sweetness due to factors such as incorrect folding or disulfide bond mismatch during heterologous expression, which limits the large-scale commercial production and application of the sweet protein. Therefore, how to improve brazzein so that it can maintain its sweetness during heterologous expression is a problem to be solved.

[0007] In view of this, the present invention is specifically proposed. Summary of the Invention

[0008] The object of the present invention is to provide a fusion protein with sweetness, which alleviates the problem of insufficient sweetness of brazzein after heterologous expression in the prior art.

[0009] Another object of the present invention is to provide polynucleotides, vectors, and cells related to the fusion protein with sweetness and their applications.

[0010] Another object of the present invention is to provide a preparation method of the fusion protein with sweetness.

[0011] To solve the above technical problems, the present invention specifically adopts the following technical solutions:

[0012] In the first aspect, a fusion protein with sweetness is provided.

[0013] In an optional embodiment, the amino acid sequence of the fusion protein contains a first unit and a second unit, and the carboxyl terminus of the first unit is connected to the amino terminus of the second unit;

[0014] The amino acid sequence of the first unit is as shown in SEQ ID NO.1 or SEQ ID NO.25;

[0015] The second unit contains a fragment composed of 2 to 22 histidines.

[0016] In an optional embodiment, the amino acid sequence of the fusion protein contains a first unit and a third unit, and the amino terminus of the first unit is connected to the carboxyl terminus of the third unit;

[0017] The amino acid sequence of the first unit is as shown in SEQ ID NO.1 or SEQ ID NO.25;

[0018] The third unit contains a fragment consisting of 2 to 22 histidines.

[0019] In an alternative embodiment, the amino acid sequence of the fusion protein contains a first unit, a second unit, and a third unit;

[0020] The amino acid sequence of the first unit is as shown in SEQ ID NO.1 or SEQ ID NO.25;

[0021] The carboxyl terminus of the first unit is connected to the amino terminus of the second unit, and the second unit contains a fragment consisting of 2 to 22 histidines;

[0022] The amino terminus of the first unit is connected to the carboxyl terminus of the third unit, and the third unit contains a fragment consisting of 2 to 22 histidines.

[0023] In a second aspect, a polynucleotide is provided, which contains a fragment encoding the sweet fusion protein of the first aspect.

[0024] In a third aspect, a vector is provided, and the vector carries the polynucleotide of the second aspect.

[0025] In a fourth aspect, a cell is provided, which expresses the sweet fusion protein of the first aspect, or carries the polynucleotide of the second aspect, or contains the vector of the third aspect.

[0026] In a fifth aspect, a method for preparing the sweet fusion protein of the first aspect is further provided, including expressing the sweet fusion protein in a cell.

[0027] In a sixth aspect, the sweet fusion protein of the first aspect, or the polynucleotide of the second aspect, or the vector of the third aspect, or the cell of the fourth aspect, or the preparation method of the fifth aspect is further provided for use in the preparation of a drug, feed, or food.

[0028] In a seventh aspect, a sweetener, drug, feed, or food is further provided, any one of which contains the sweet fusion protein of the first aspect.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The sweet fusion protein provided by the present invention is based on brazzein, and other fragments are fused to its amino terminus and / or carboxyl terminus. Its sweetness is increased by at least 3.5 to 16 times compared with the native brazzein before fusion, and the sweet taste delay of native brazzein is shortened, achieving unexpected technical effects. The fusion protein provided by the present invention not only significantly improves the sweetness of the original brazzein, but also improves the taste of this protein, shortens the time to perceive sweetness when entering the mouth, and lays a technical foundation for future industrial production. Brief Description of the Drawings

[0031] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0032] Figure 1 It is the plasmid map of the natural brazzein expression plasmid Brz in Example 1;

[0033] Figure 2 It is the plasmid map of the fusion protein expression plasmid Brz-C1 in Example 1;

[0034] Figure 3 It is the plasmid map of the Bxb-1 integrase in Example 2. Detailed Description of the Embodiments

[0035] The following will clearly and completely describe the technical solutions of the present invention in combination with the embodiments. Obviously, the described embodiments are some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0036] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, and polynucleotides include ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemical or biochemical modifications, unnatural or derived nucleobases. The polynucleotide encodes the above-mentioned SS-B binding protein, and the encoding can optionally be for the sense strand or the antisense strand. The polynucleotide can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" can be used interchangeably herein.

[0037] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector enables the protein encoded by the inserted polynucleotide to be expressed, the vector is called an expression vector. The vector can be introduced into cells by transformation, transduction or transfection, so that the genetic element carried by it can be expressed in the cells.

[0038] The vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses. In some embodiments, the vectors of the present invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRESs) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly(U) sequences, etc.).

[0039] As used herein, the terms "cell", "cell line" and "cell culture" are used interchangeably and all such names include progeny. The progeny may not be identical to the parental cell due to natural, accidental or deliberate mutations and may differ from the parental cell morphologically and / or in genomic DNA.

[0040] As used herein, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and their modified amino acids. Common naturally occurring amino acids are, for example: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V). The Chinese names, three-letter abbreviations and single-letter abbreviations of amino acids are used interchangeably herein.

[0041] In a first aspect, there is provided a fusion protein having a sweet taste, and the sweetness of the fusion protein after heterologous expression is enhanced by fusing other fragments to the N-terminus and / or C-terminus of brazzein.

[0042] In an alternative embodiment, the amino acid sequence of the sweet fusion protein contains a first unit and a second unit, with the C-terminus of the first unit connected to the N-terminus of the second unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.1 or SEQ ID NO.25, and the second unit contains a fragment consisting of 2 to 22 histidines. The second unit can be, for example but not limited to, a fragment consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 histidines.

[0043] In an alternative embodiment, the second unit is a fragment consisting of 2 to 16 histidines.

[0044] In an alternative embodiment, the second unit is a fragment consisting of 2, 4, 6, 8, 10, 12 or 16 histidines.

[0045] In an alternative embodiment, the second unit is a fragment consisting of 6 histidines.

[0046] In an alternative embodiment, the amino acid sequence of the fusion protein contains a first unit and a third unit, with the N-terminus of the first unit connected to the C-terminus of the third unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.1 or SEQ ID NO.25, and the third unit contains a fragment consisting of 2 to 22 histidines. The third unit can be, for example but not limited to, a fragment consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 histidines.

[0047] In an alternative embodiment, the third unit is a fragment consisting of 2 to 16 histidines.

[0048] In an alternative embodiment, the third unit is a fragment consisting of 2, 8 or 16 histidines.

[0049] In an alternative embodiment, the amino acid sequence of the fusion protein comprises a first unit, a second unit, and a third unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.1 or SEQ ID NO.25. The carboxyl terminus of the first unit is linked to the amino terminus of the second unit. The second unit comprises a fragment consisting of 2 to 22 histidines. The second unit may be, for example but not limited to, a fragment consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 histidines. The amino terminus of the first unit is linked to the carboxyl terminus of the third unit. The third unit comprises a fragment consisting of 2 to 22 histidines. The third unit may be, for example but not limited to, a fragment consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 histidines.

[0050] In an alternative embodiment, both the second unit and the third unit are fragments consisting of 8 histidines.

[0051] In an alternative embodiment, the sweet-tasting fusion protein consists of a first unit and a second unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.1, and the amino acid sequence of the second unit is as shown in SEQ ID NO.50, i.e., a protein sequence HHHHHHHH is added to the carboxyl terminus of the thaumatin sequence of SEQ ID NO.1. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.3. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.4.

[0052] In an alternative embodiment, the sweet-tasting fusion protein consists of a first unit and a second unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.1, and the amino acid sequence of the second unit is HH, i.e., a protein sequence HH is added to the carboxyl terminus of the thaumatin sequence of SEQ ID NO.1. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.5. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.6.

[0053] In an alternative embodiment, the sweet-tasting fusion protein consists of a first unit and a second unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.1, and the amino acid sequence of the second unit is as shown in SEQ ID NO.51, i.e., a protein sequence HHHH is added to the carboxyl terminus of the thaumatin sequence of SEQ ID NO.1. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.7. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.8.

[0054] In an alternative embodiment, the sweet fusion protein consists of a first unit and a second unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.1, and the amino acid sequence of the second unit is as shown in SEQ ID NO.52, that is, a protein sequence HHHHHH is added to the C-terminus of the thaumatin sequence of SEQ ID NO.1. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.9. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.10.

[0055] In an alternative embodiment, the sweet fusion protein consists of a first unit and a second unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.1, and the amino acid sequence of the second unit is as shown in SEQ ID NO.72, that is, a protein sequence HHHHHHHHHH is added to the C-terminus of the thaumatin sequence of SEQ ID NO.1. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.60. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.61.

[0056] In an alternative embodiment, the sweet fusion protein consists of a first unit and a second unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.1, and the amino acid sequence of the second unit is as shown in SEQ ID NO.73, that is, a protein sequence HHHHHHHHHHHH is added to the C-terminus of the thaumatin sequence of SEQ ID NO.1. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.62. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.63.

[0057] In an alternative embodiment, the sweet fusion protein consists of a first unit and a second unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.1, and the amino acid sequence of the second unit is as shown in SEQ ID NO.56, that is, a protein sequence HHHHHHHHHHHHHHH is added to the C-terminus of the thaumatin sequence of SEQ ID NO.1. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.64. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.65.

[0058] In an alternative embodiment, the sweet fusion protein consists of a first unit and a third unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.1, and the amino acid sequence of the third unit is HH, that is, a protein sequence HH is added to the N-terminus of the thaumatin protein sequence of SEQ ID NO.1. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.17. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.18.

[0059] In an alternative embodiment, the sweet fusion protein consists of a first unit and a third unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.1, and the amino acid sequence of the third unit is as shown in SEQ ID NO.50, that is, a protein sequence HHHHHHHH is added to the N-terminus of the thaumatin protein sequence of SEQ ID NO.1. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.19. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.20.

[0060] In an alternative embodiment, the sweet fusion protein consists of a first unit and a third unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.1, and the amino acid sequence of the third unit is as shown in SEQ ID NO.56, that is, a protein sequence HHHHHHHHHHHHHHHH is added to the N-terminus of the thaumatin protein sequence of SEQ ID NO.1. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.21. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.22.

[0061] In an alternative embodiment, the sweet fusion protein consists of a first unit, a second unit and a third unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.1, and the amino acid sequences of the second unit and the third unit are both as shown in SEQ ID NO.50, that is, a protein sequence HHHHHHHH is added to the N-terminus and the C-terminus of the thaumatin protein sequence of SEQ ID NO.1 respectively. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.23. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.24.

[0062] In an alternative embodiment, the sweet fusion protein consists of a first unit and a second unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.25, and the amino acid sequence of the second unit is as shown in SEQ ID NO.50, that is, a protein sequence HHHHHHHH is added to the C-terminus of the thaumatin sequence of SEQ ID NO.25. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.27. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.28.

[0063] In an alternative embodiment, the sweet fusion protein consists of a first unit and a second unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.25, and the amino acid sequence of the second unit is HH, that is, a protein sequence HH is added to the C-terminus of the thaumatin sequence of SEQ ID NO.25. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.29. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.30.

[0064] In an alternative embodiment, the sweet fusion protein consists of a first unit and a second unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.25, and the amino acid sequence of the second unit is as shown in SEQ ID NO.51, that is, a protein sequence HHHH is added to the C-terminus of the thaumatin sequence of SEQ ID NO.25. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.31. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.32.

[0065] In an alternative embodiment, the sweet fusion protein consists of a first unit and a second unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.25, and the amino acid sequence of the second unit is as shown in SEQ ID NO.52, that is, a protein sequence HHHHHH is added to the C-terminus of the thaumatin sequence of SEQ ID NO.25. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.33. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.34.

[0066] In an alternative embodiment, the sweet fusion protein is composed of a first unit and a second unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.25, and the amino acid sequence of the second unit is as shown in SEQ ID NO.72, that is, a protein sequence HHHHHHHHHH is added to the C-terminus of the brazzein sequence of SEQ ID NO.1. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.66. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.67.

[0067] In an alternative embodiment, the sweet fusion protein is composed of a first unit and a second unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.25, and the amino acid sequence of the second unit is as shown in SEQ ID NO.73, that is, a protein sequence HHHHHHHHHHHH is added to the C-terminus of the brazzein sequence of SEQ ID NO.1. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.68. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.69.

[0068] In an alternative embodiment, the sweet fusion protein is composed of a first unit and a second unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.25, and the amino acid sequence of the second unit is as shown in SEQ ID NO.56, that is, a protein sequence HHHHHHHHHHHHHH is added to the C-terminus of the brazzein sequence of SEQ ID NO.1. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.70. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.71.

[0069] In an alternative embodiment, the sweet fusion protein is composed of a first unit and a third unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.25, and the amino acid sequence of the third unit is HH, that is, a protein sequence HH is added to the N-terminus of the brazzein sequence of SEQ ID NO.25. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.41. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.42.

[0070] In an alternative embodiment, the sweet-tasting fusion protein is composed of a first unit and a third unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.25, and the amino acid sequence of the third unit is as shown in SEQ ID NO.50, that is, a protein sequence HHHHHHHH is added to the N-terminus of the brazzein sequence of SEQ ID NO.25. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.43. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.44.

[0071] In an alternative embodiment, the sweet-tasting fusion protein is composed of a first unit and a third unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.25, and the amino acid sequence of the third unit is as shown in SEQ ID NO.56, that is, a protein sequence HHHHHHHHHHHHHHHH is added to the N-terminus of the brazzein sequence of SEQ ID NO.25. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.45. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.46.

[0072] In an alternative embodiment, the sweet-tasting fusion protein is composed of a first unit, a second unit and a third unit. The amino acid sequence of the first unit is as shown in SEQ ID NO.25, and the amino acid sequences of the second unit and the third unit are both as shown in SEQ ID NO.50, that is, a protein sequence HHHHHHHH is added to the N-terminus and the C-terminus of the brazzein sequence of SEQ ID NO.25 respectively. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.47. In an alternative embodiment, the nucleotide sequence encoding this fusion protein is as shown in SEQ ID NO.48.

[0073] In an alternative embodiment, the fusion protein in any of the above embodiments is expressed by mammalian cells, preferably expressed by CHO cells (Chinese hamster ovary cells). CHO cells can correctly fold the spatial structure of the fusion protein. After the fusion protein is expressed in CHO cells, its sweetness is increased by at least 3.5 to 16 times compared with the natural brazzein protein without any fragment fusion, and the sweetness delay of natural brazzein is shortened.

[0074] In an alternative embodiment, the fusion protein in any of the above embodiments is endogenously expressed by CHO cells.

[0075] In an alternative embodiment, the sweet-tasting fusion protein is a protein with an amino acid sequence as shown in SEQ ID NO.9 endogenously expressed by CHO cells.

[0076] In a second aspect, there is also provided a polynucleotide encoding the sweet fusion protein of the first aspect.

[0077] In an optional embodiment, the nucleotide sequence encoding thaumatin shown in SEQ ID NO.1 in the polynucleotide is as shown in SEQ ID NO.2.

[0078] In an optional embodiment, the nucleotide sequence encoding thaumatin shown in SEQ ID NO.25 in the polynucleotide is as shown in SEQ ID NO.26.

[0079] In an optional embodiment, the polynucleotide contains at least one fragment shown in SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.42, SEQ ID NO.44, SEQ ID NO.46, SEQ ID NO.48, SEQ ID NO.61, SEQ ID NO.63, SEQ ID NO.65, SEQ ID NO.67, SEQ ID NO.69 and SEQ ID NO.71.

[0080] In an optional embodiment, the polynucleotide further contains a homologous arm sequence for homologous recombination with the genome of the cell expressing the fusion protein.

[0081] In a third aspect, there is also provided a vector carrying the polynucleotide of the second aspect.

[0082] In an optional embodiment, the vector includes a plasmid.

[0083] In a fourth aspect, there is also provided a cell that expresses the sweet fusion protein of the first aspect, or carries the polynucleotide of the second aspect, or contains the vector of the third aspect.

[0084] In an optional embodiment, the polynucleotide of the second aspect is integrated into the genome of the cell, that is, the polynucleotide expressing the fusion protein is integrated to achieve endogenous expression of the fusion protein in the cell.

[0085] In an optional embodiment, the cell includes mammalian cells.

[0086] In an optional embodiment, the mammalian cells include CHO cells.

[0087] In an alternative embodiment, the cell is a CHO cell, and a polynucleotide encoding the fusion protein is integrated into the genome of the CHO cell, and the insertion position of the polynucleotide is NW_003616785.1:83044.

[0088] In an alternative embodiment, a nucleotide sequence shown in SEQ ID NO.4 is integrated into the genome of the CHO cell.

[0089] In a fifth aspect, a method for preparing the fusion protein having a sweet taste according to the first aspect is further provided, and the preparation method includes expressing the fusion protein having a sweet taste in a cell.

[0090] In an alternative embodiment, the cell includes the cell according to the fourth aspect.

[0091] In an alternative embodiment, the fusion protein having a sweet taste is endogenously expressed or exogenously expressed in the cell.

[0092] In an alternative embodiment, the cell includes a CHO cell, and a polynucleotide encoding the fusion protein is integrated into the genome of the CHO cell to endogenously express a heterologous protein. The fusion protein can be extracellularly secreted in the CHO cell, and the structure and function of the protein are normal; it can be used after simple purification by downstream processes, which is extremely beneficial to the realization of large-scale production.

[0093] In an alternative embodiment, the preparation method includes integrating a polynucleotide encoding the fusion protein into the genome of a cell for expressing the fusion protein to achieve endogenous expression of the fusion protein in the cell. Any method known and conventional in the art can be used to integrate the polynucleotide encoding the fusion protein into the genome of the cell. Exemplary methods include but are not limited to using a gene editing system, such as the crispr / cas9 system; using a transposon system, or using an integrase system, etc.

[0094] In an alternative embodiment, a polynucleotide encoding the fusion protein is integrated into the genome of a cell using Bxb-1 integrase.

[0095] In an alternative embodiment, the cell for expressing the fusion protein contains a marker gene when a polynucleotide encoding the fusion protein is not integrated into the cell genome, and the marker gene is knocked out after the polynucleotide is integrated into the cell genome. The marker gene can be any marker gene known in the art. Exemplary marker genes include fluorescent protein genes, such as EGFP, TagGFP, AcGFP, EBFP, ECFP, CyPet, EYFP, mOrange, mRuby or mApple; exemplary marker genes can also include resistance genes.

[0096] In an alternative embodiment, the preparation method further includes screening cells with positive expression of the fusion protein by detecting whether a marker gene in the cells is expressed.

[0097] In an alternative embodiment, the cells used for constructing the cells capable of expressing the fusion protein are CHO cells expressing green fluorescent protein. The integration site of the green fluorescent protein gene (EGFP) in the CHO cells and the insertion site of the polynucleotide encoding the fusion protein in the CHO cells are both NW_003616785.1:83044. When the polynucleotide encoding the fusion protein is integrated into the CHO cells expressing green fluorescent protein, EGFP is knocked out, and the CHO cells that do not express fluorescence are the CHO cells with positive expression of the fusion protein.

[0098] In a sixth aspect, there is also provided the use of the sweet fusion protein of the first aspect, or the polynucleotide of the second aspect, or the vector of the third aspect, or the cells of the fourth aspect, or the preparation method of the fifth aspect in the preparation of drugs, feeds or foods.

[0099] In a seventh aspect, there is also provided a sweetener, a drug, a feed or a food, wherein the drug, the feed or the food contains the sweet fusion protein of the first aspect.

[0100] The present invention will be further described below through specific examples. However, it should be understood that these examples are only for more detailed description and should not be construed as limiting the present invention in any form.

[0101] The main biological materials and reagents used in the following examples are as follows:

[0102] 1. Strains and vectors: The site-specific integration CHO cell line and the vector pDonor1.0 were constructed and preserved by the applicant; Escherichia coli DH5α and Hygromycin B were purchased from Thermo Fisher.

[0103] 2. Enzymes and kits: Restriction endonucleases were purchased from NEB, and the plasmid extraction kit and the gel purification and recovery kit were purchased from TIANGEN.

[0104] 3. Media:

[0105] Escherichia coli medium (LB medium): 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0; LB + Amp medium: LB medium supplemented with 100 μg / mL ampicillin;

[0106] Ex-cell Advanced CHO Fed-batch medium and Glucose solution were purchased from Sigma. Cell Boost7a and CellBoost7b were purchased from Hyclone. Glutamax (100X) was purchased from Gibco. D-(+)-Glucose solution was purchased from Sigma.

[0107] 4. Preparation of stable high-fluorescence cell GBB003: Specific consumables involved: Neon Resuspension Buffer R (ThermoFisher), a special resuspension buffer for cell electroporator; E1 Buffer (ThermoFisher), the electroporation solution; the recovery medium was 80% (v / v) EX-CELL CHO Cloning Medium (Sigma-Aldrich) and 20% (v / v) EX-CELL Advanced CHO Fed-batch Medium (Sigma-Aldrich) supplemented with 1% GlutaMAX (ThermoFisher); the amplification medium and the passage medium were both EX-CELL Advanced CHO Fed-batch Medium supplemented with 1% GlutaMAX (ThermoFisher); the pressure medium 1 was the passage medium supplemented with G418 at a final concentration of 800 μg / ml; the pressure medium 2 was the passage medium supplemented with hygromycin at a final concentration of 250 μg / ml. The conditioned medium was the supernatant obtained by sterile filtration after inoculating CHO-K1 in the passage medium and culturing for 1 day. The cloning medium was 75% (v / v) EX-CELL CHO Cloning Medium, 20% (v / v) conditioned medium, and 5% (v / v) ClonaCell-CHO ACF Supplement supplemented with 1% GlutaMAX; the basal medium in the fed-batch medium was EX-CELL Advanced CHO Fed-batch Medium supplemented with 1% GlutaMAX (ThermoFisher), and the feeding medium was CellBoost7a / 7b (HyClone).

[0108] 5. Preparation of BCA reagent:

[0109] ① Reagent A, 1 L: Weigh 10 g of BCA (1%), 20 g of Na2CO3·H2O (2%), 1.6 g of Na2C4H4O6·H2O (0.16%), 4 g of NaOH (0.4%), and 9.5 g of NaHCO3 (0.95%) respectively. Add water to 1 L and adjust the pH value to 11.25 with NaOH or solid NaHCO3.

[0110] ② Reagent B, 50 ml: Take 2 g of CuSO4·5H2O (4%) and add distilled water to 50 mL.

[0111] ③ BCA reagent: Mix 50 parts of reagent A and 1 part of reagent B evenly. This reagent can be stable for one week.

[0112] Example 1

[0113] 1. Gene synthesis of Brazzein:

[0114] Search the gene sequences in GenBank, and find a brazzein gene in Pentadiplandra brazzeana with the GenBank number P56552.1. The whole gene was synthesized by Nanjing Genscript Corporation and named Braz-pUC57, with a full length of 159 bp.

[0115] 2. Construction of Brazzein Brz fusion protein:

[0116] Using the Braz-pUC57 gene as a template, perform PCR amplification with primer F and primer R, primer R-C respectively. The primers were synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0117] Primer F (SEQ ID NO.57):

[0118] agcctccgggatccGCCACCATGAAGACTCTAGTGCTAC;

[0119] Primer R (SEQ ID NO.58):

[0120] aagttTTAgaattcTCAGTACTCGCAGTAGTCGC;

[0121] Primer R-C (SEQ ID NO.59):

[0122] aagttTTAgaattcTCAatgatggtgatgatggtgatgatgGTACTCGCAGTAGTCGCAGATG.

[0123] The PCR conditions were as follows: denaturation at 98°C for 2 min; then denaturation at 98°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 30 s. After 30 cycles, incubation at 72°C for 5 min. The amplification product of primer F and primer R was named Brz, with a full-length gene of 159 bp; the Brazilian sweet fusion protein with a modified carbon terminus of the sequence was named Brz-C1 (SEQ ID NO.3), which was the amplification product of primer F and primer R-C, and the gene length of Brz-C1 was 183 bp.

[0124] The PCR products were recovered by gel extraction, digested with EcoRI and BamHI, and then ligated to the pDonor1.0 vector digested with the same enzymes. The ligation product was transformed into Escherichia coli DH5α, and the transformants were picked for sequencing verification. The transformants with correct sequencing verification were transferred to LB + Amp liquid medium and cultured overnight at 37°C. Two kinds of plasmids were extracted respectively, namely the natural Brazilian sweet protein expression plasmid Brz. The amino acid sequence of the natural Brazilian sweet protein was SEQ ID NO.1, and the nucleotide sequence encoding it was SEQ ID NO.2. The plasmid map was as Figure 1 shown; the other was the fusion protein expression plasmid Brz-C1. The amino acid sequence of the fusion protein Brz-C1 was SEQ ID NO.3, and the encoding nucleotide sequence was SEQ ID NO.4. The plasmid map was as Figure 2 shown.

[0125] Example 2

[0126] Construction of CHO cell line expressing Brazilian sweet fusion protein:

[0127] 1. Plasmid construction:

[0128] The plasmid prepared in Example 1 was linearized with PvuI-HF restriction endonuclease, and the product was purified by ethanol precipitation.

[0129] The linearized plasmid was co-transfected into the stable high-fluorescence cell GBB003 with Bxb-1 integrase by electroporation. The plasmid map of Bxb-1 integrase was as Figure 3 shown. The Brazilian sweet protein and the Brazilian sweet fusion protein were integrated into the position NW_003616785.1:83044 of the GBB003 cell genome. The green fluorescent protein gene (EGFP) was integrated at the position NW_003616785.1:83044 of the GBB003 cell. When the Brazilian sweet protein or the Brazilian sweet fusion protein was integrated into this position of the GBB003 cell, the green fluorescent protein gene was knocked out, enabling the screening of positive cells expressing the Brazilian sweet protein or the Brazilian sweet fusion protein through fluorescence changes.

[0130] 2. Cell line construction:

[0131] After 24 to 48 hours of electroporation, the cells were subjected to pressure screening in a 96-well plate. The pressure medium was the subculture medium supplemented with hygromycin at a final concentration of 200 to 300 μg / mL.

[0132] After 10 to 14 days, a fluorescence microscope was used to select and enrich non-fluorescent cell clusters; then, amplification culture was carried out to obtain multiple cell pools. The containers for cell amplification culture were, in sequence: 96-well plate → 24-well plate → 6-well plate → 50 mL shake tube → 125 mL shake flask. The culture conditions were 37°C, 5% CO2, saturated humidity. The rotation speed of the shake tube was 160 rpm, and that of the shake flask was 120 rpm.

[0133] Select 4 to 6 cell pools with a cell growth and proliferation rate of 2.1 to 2.3 times / 24 h and the highest non-fluorescence rate for fed-batch culture. The initial inoculation density was 5×10 5 cells / mL, and the cell viability was >95%. The inoculation day was counted as D0. On the 3rd day of culture (D3), cell counting was started and nutrients (3% Cell Boost 7a and 0.3% Cell Boost 7b) were supplemented; thereafter, the above operations were performed every other day, that is, sampling and counting were carried out on D5 (the 5th day), D7 (the 7th day), D9 (the 9th day), and D11 (the 11th day), and 5% Cell Boost 7a and 0.5% Cell Boost 7b were supplemented; during the entire fed-batch culture period, the glucose concentration was always controlled at the level of 2 - 8 g / L. Depending on the cell condition (viability ≥ 80%), the culture was terminated on D13 (the 13th day) to D16 (the 16th day). The cell supernatant was obtained by stepwise centrifugation, and after downstream purification of the supernatant, an aqueous solution of the Brazilian sweet fusion protein was obtained.

[0134] The preparation of the stable high-fluorescence cell GBB003 used in this example mainly includes the following steps:

[0135] (1) Using the enhanced green fluorescent protein gene (EGFP) as the screening marker gene and the attP sequence as the homologous arm, a recombinant plasmid containing RMCE was constructed; after linearizing the constructed plasmid, the linear DNA was purified and recovered; CHO K1 cells were taken, centrifuged, and the supernatant was discarded; the cells were resuspended with 100 μL of the special resuspension buffer R Buffer for the cell electroporator; electroporation was performed 3 times.

[0136] (2) Applying pressure, the pressure reagent was G418, and the pressure concentration was 800 μg / ml. It was cultured in minipool (i.e., 96-well plate).

[0137] (3) Checking the plate after ten days. When a large number of fluorescent cell clusters were observed, enrichment could be carried out, and the one-hole-to-one-hole principle was followed during enrichment.

[0138] (4) Observe the growth status of the cells after enrichment at any time. After the coverage rate reaches more than 50%, expand the cells.

[0139] (5) Expand the cells to a shake flask and passage them three times to form a stable cell pool. The formation of monoclonal cells and the screening of stable fluorescence are achieved through artificial intelligence. The specific steps are shown as a - h below.

[0140] a) Dilute the cells in the stable cell pool to a certain concentration and inoculate them into a culture dish with semi - solid medium. During the inoculation process, ensure that the cells are approximately evenly distributed at various positions in the culture dish, and let it stand for about half an hour to wait for the cells to settle to the bottom of the culture dish.

[0141] b) Transfer the culture dish onto the stage of an electron microscope, and the electron microscope performs high - throughput scanning on the cells in the culture dish.

[0142] c) Upload the images scanned by the electron microscope to the server for artificial intelligence image analysis. The analysis process includes monoclonal cell line detection, prediction of the protein expression level of monoclonal cell lines, sorting of protein expression levels, and coding and localization of the selected high - protein - expressing cell lines. The prediction of the protein expression level of monoclonal cell lines can be based on fluorescence or not. The following step c is for the prediction of protein expression level without fluorescence.

[0143] For the target detection of monoclonal cell lines in step c based on image processing technology, in this embodiment, the YOLOv8 target detection algorithm is used, and the actual detection effect reaches mAP 94.2%. The target detection model for monoclonal cell lines in step c is the same as the common deep - learning target detection models, so it is not described in detail. Specifically, it is necessary to first annotate the cell images. To improve the prediction accuracy of the algorithm, the bounding boxes of all monoclonal cell lines and adherent cell lines are annotated as the true target bounding boxes (ground truth) for loss calculation of the model output. After algorithm training, the model learns the ability to extract the bounding box information of monoclonal cell lines. In the actual application scenario of step c, the trained model can predict the bounding boxes of monoclonal cell lines in the image.

[0144] For the prediction of the fluorescence protein expression level of monoclonal cell images in step c, in this embodiment, the SqueezeNet deep - learning network and the MSE Loss loss function are used. Since the prediction of the protein expression level and sorting in this project, and the sorting result is the ultimate goal of the algorithm, the evaluation standard NDCG of the sorting algorithm is used here. The specific calculation formula is as follows:

[0145]

[0146] Among them, IDCG = the best-ranked DCG. Specifically in this embodiment, the cell image is used to predict the expression level, and the cells are sorted according to the predicted expression level and compared with the sorting result of the true fluorescence value. NDCG = 0.89. The expression level prediction model in this embodiment is described in detail in CN112037862B (application number CN202311058132.5, invention name "A cell transfer method and a cell transfer system").

[0147] d) The coding and positioning information of the selected protein-high-expressing cell lines are returned to the robot control software;

[0148] In this embodiment, the cell coding and positions returned in step d are the top 100 cells sorted by the predicted expression level. The coding is from 1 to 100 in sequence, and the position information includes the coordinates in the plane coordinate system relative to the center position of the microscope (temporarily not considering the depth of the culture medium because all cells to be selected have settled to the bottom of the culture dish. Additionally, during the photographing process, cells that have not settled to the bottom of the culture dish will be out of focus, resulting in blurred cells, and these blurred cells will also be excluded).

[0149] e) The robot control software automatically operates (or with human assistance) the robotic arm to aspirate the selected monoclonal cell lines and transfer them to the designated well plate, and this process is repeated until all high-expressing cell lines are transferred. The aspiration and transfer processes are described in CN113821287B (application number: CN202111040555..5, invention name "Method, device, equipment, and medium for processing cell operation tasks based on a robot"), CN113403431B (application number: CN202110735660.4, invention name "Cell liquid extraction control method, device, equipment, and storage medium based on a robot"), CN113771030A (application number: CN202111040562.5, invention name "Control method, device, equipment, and storage medium for a cell operation robot"), CN113733087B (application number: CN202111039293.0, invention name "Control information configuration method, device, equipment, and storage medium for a cell operation robot")

[0150] f) After culturing the cell clones in the well plate to a certain number, they are transferred to a larger well plate for culture, and finally expanded to shake flask culture, and the cell expression level is detected to determine that the cell lines selected by the artificial intelligence are high-yield cell lines; in this embodiment, step f) is to transfer to a 96-well plate for scale-up culture and then transfer to shake flask culture.

[0151] g) The selected high-yield cell lines are subcultured. Before subculturing each generation of cell lines, dilute part of the sample and place it in a culture dish to take images with an electron microscope, and continuously take images of several generations of cell lines; currently, the expression of the selected cells can be predicted through a model learned from cell morphology by taking pictures, or the expression of the selected cells can be selected using fluorescence labeling.

[0152] Step g) Photographing monoclonal cells In this embodiment, a Thermo Fisher Scientific M7000 electron microscope is used for photographing.

[0153] h) Input the images of several generations of the collected cell lines into an artificial intelligence algorithm, predict the subculture stability of the cell lines according to the fluorescence intensity or / and protein expression level, and select the cell lines with high protein expression characteristics that can be stably subcultured as the final candidate cell lines; the prediction of the subculture stability of the cell lines can be based on fluorescence, or it can be based on cell morphology instead of fluorescence. The following step h) is the prediction of protein expression level based on cell morphology instead of fluorescence.

[0154] Step h) Predicting the subculture stability of cell lines includes, but is not limited to, traditional image recognition algorithms using histograms to extract image features and make predictions, using deep learning neural networks to automatically extract image features and make predictions, and other image-based techniques for prediction. In this embodiment, a deep learning-based method is used to automatically extract the image features of stable and unstable cell lines and predict the stability, and the accuracy rate of the prediction results for different cell lines reaches 84%. See CN114417582A (Application No.: CN202210010493.1, Invention Title "Cell Line Stability Prediction Method, Device, Computer Equipment and Storage Medium")

[0155] Among them, steps d) and e) can be completed manually or automatically by a robotic arm. In this embodiment, it is completed automatically by a robotic arm.

[0156] (6) Using CHO-K1 cells as a control, the inoculation density is 5×10 5 cells / ml, the inoculation system is 30 ml, and the cells are counted on the day of inoculation, the 3rd day, the 5th day, the 7th day, the 9th day, the 11th day, the 13th day, and the 14th day of culture, and monoclonal cells with a growth rate lower than that of CHO-K1 are screened out and eliminated.

[0157] (7) Resuscitate the remaining monoclonal cells, and perform stable subculture after recovery. The subculture is carried out once every 3 or 4 days. The cell density for subculture every 4 days is 3×10 5 cells / ml, and the cell density for subculture every 3 days is 5×10 5cells / ml. During subculture, the fluorescence values of the cell lines were regularly monitored. The above-mentioned high-fluorescence cell lines were subjected to a subculture stability study for about 90 days using subculture medium, and the GBB003 cells with relatively small fluorescence changes and stable cell line growth were identified as stable high-fluorescence cells. The integration sites of this cell line were sequenced by NGS, and the sequence of the highly expressed fragment where the integration site was located was SEQ ID NO.49. Specifically, the annotation information of the integration site on CHO was: NW_003616785.1:83044.

[0158] Example 3

[0159] Detection of thaumatin and thaumatin fusion protein contents by BCA method

[0160] 1. Standard protein solution: Weigh 0.5 g of bovine serum albumin, dissolve it in distilled water and make up to 100 ml to prepare a 5 mg / mL solution. Dilute it ten times before use.

[0161] 2. Preparation of standard curve:

[0162] Take a 96-well enzyme-linked immunosorbent assay (ELISA) plate and add reagents according to Table 1.

[0163] Table 1

[0164] Tube number 1 2 3 4 5 6 7 8 Standard protein solution (μL) 0 1 2 4 8 12 16 20 Distilled water (μL) 20 19 18 6 12 8 4 0 BCA reagent (μL) 200 200 200 200 200 200 200 200 Protein concentration (mg / mL) 0 0.025 0.05 0.1 0.2 0.3 0.4 0.5

[0165] 3. Determination of samples

[0166] After adding the above reagents, accurately pipette 20 μl of the sample solution into the ELISA wells, add 200 μL of BCA reagent, gently shake, incubate at 37 °C for 30 - 60 min, cool to room temperature, and then, using the blank as a control, measure the absorbance at 562 nm on an ELISA reader. With the bovine serum albumin content as the abscissa and the absorbance value as the ordinate, draw a standard curve. Using the standard curve blank as a control, according to the absorbance value of the sample, find out the protein content of the sample from the standard curve: Brz is 0.841 mg / mL, Brz-C1 is 0.624 mg / mL, and the details are shown in Table 2.

[0167] Table 2

[0168]

[0169] Example 4

[0170] Detection method for the sweetness of natural thaumatin and thaumatin fusion protein

[0171] The sweetness identification of thaumatin was carried out by a blind test experiment. In the control group of the blind test experiment, sucrose aqueous solutions with different concentration gradients were used and numbered. The samples to be tested of brazzein (the natural brazzein protein and brazzein fusion protein prepared in Example 2) were formulated into protein aqueous solutions with a concentration of 0.08 mg / mL to 0.02 mg / mL. A panel of 7-8 people tasted the sucrose aqueous solutions with different concentrations and the samples successively and compared them pairwise until the numbers of the sucrose aqueous solutions with the same or similar sweetness as the brazzein aqueous solution (the natural brazzein protein or brazzein fusion protein prepared in Example 2) in different concentration gradients were found, so as to calculate the sweetness of the samples.

[0172] The formula is: relative sucrose sweetness multiple = sucrose concentration ÷ concentration of brazzein (the natural brazzein protein or brazzein fusion protein prepared in Example 2)

[0173] Preparation of sucrose samples with different concentration gradients: First, weigh 100 g of sucrose (CAS No.: 57-50-1, Sigma) and dilute it with distilled water to 200 ml to obtain a 500 mg / mL sucrose solution; then measure a certain volume of the stock solution and add different volumes of distilled water to further dilute it into 12 groups of sucrose aqueous solutions with different concentrations for testing, with a volume of 30 mL each. The details are shown in Table 3. The sensory test results of brazzein proteins with different configurations are shown in Table 4.

[0174] Table 3 Preparation of gradient sucrose aqueous solutions and relative sucrose sweetness multiples of samples

[0175]

[0176] Table 4 Test results of the sweetness of brazzein fusion proteins with different configurations

[0177]

[0178] Example 5

[0179] Using the gene of the natural brazzein protein expression plasmid Brz as a template, GenScript Nanjing was commissioned to perform total gene synthesis. A total of 6 groups of C-terminal optimized fusion proteins were constructed, named Brz-C2, Brz-C3, Brz-C4, Brz-C5, Brz-C6, and Brz-C7 respectively. The fusion proteins were all composed of the natural brazzein protein (the first unit) with the amino acid sequence of SEQ ID NO.1 and the corresponding second unit in Table 5. The C-terminal of the first unit was connected to the N-terminal of the second unit. As shown in Table 5:

[0180] Table 5 Structures of C-terminal optimized configurations of brazzein fusion proteins

[0181]

[0182] The construction of the above 6 groups of CHO cell lines expressing fusion proteins was completed respectively according to the method described in Example 2. After fed-batch culture of each cell line, protein aqueous solutions with certain concentrations were obtained. The sensory test results showed that the sweetness of Brz-C2 was about 9,700 times that of the standard sucrose, Brz-C3 was about 14,500 times, the sweetness of Brz-C4 was the highest, about equal to 16,800 times that of the same mass of sucrose; the sweetness of the remaining Brz-C5, Brz-C6 and Brz-C7 was about 10,500 times, 10,300 times and 9,700 times that of sucrose respectively, as shown in Table 6 in detail. It was 9.5 - 16.5 times higher than the natural brazzein before optimization, achieving unexpected technical effects.

[0183] Table 6 Sweetness test results of brazzein fusion proteins with optimized C-terminal configurations

[0184]

[0185]

[0186] Example 6

[0187] Using the gene of the natural brazzein expression plasmid Brz as a template, GenScript Corporation in Nanjing carried out total gene synthesis, and a total of 3 groups of N-terminal optimized fusion proteins were constructed, named N1-Brz, N2-Brz and N3-Brz respectively. The fusion proteins were all composed of the natural brazzein (the first unit) with the amino acid sequence of SEQ ID NO.1 and the corresponding third unit in Table 7. The N-terminus of the first unit was connected to the C-terminus of the third unit. As shown in Table 7:

[0188] Table 7 Structures of brazzein fusion proteins with optimized N-terminal configurations

[0189]

[0190] The construction of the CHO cell lines of the above 3 groups of fusion proteins was completed respectively according to the method described in Example 2. After the fed-batch culture of the 3 groups of cells was completed, sweet protein aqueous solutions with different concentrations were obtained through downstream purification processes. Through human sensory tests, the sweetness of N1-Brz, N2-Brz and N3-Brz was about 4,100 times, 7,100 times and 3,700 times that of the same mass of standard sucrose respectively, as shown in Table 8 in detail. It was 3.7 - 7.1 times higher than the natural brazzein Brz, and the additive effect of sweetness was not as good as that of the C-terminal optimization group described in Example 5.

[0191] Table 8 Sweetness test results of brazzein fusion proteins with optimized N-terminal and double-terminal configurations

[0192]

[0193] Example 7

[0194] Using the gene of the natural brazzein expression plasmid Brz as a template, GenScript Corporation in Nanjing performed total gene synthesis to construct a fusion protein optimized at both ends (the N-terminus and the C-terminus), named N-Brz-C. The fusion protein N-Brz-C consists of the natural brazzein (the first unit) with the amino acid sequence of SEQ ID NO.1 and the corresponding second and third units shown in Table 9. The C-terminus of the first unit is connected to the N-terminus of the second unit, and the N-terminus of the first unit is connected to the C-terminus of the third unit. As shown in Table 9. According to the method described in Example 2, a CHO cell line expressing this protein was constructed. The supernatant obtained from cell culture was subjected to sensory testing on the protein aqueous solution after purification and recovery. The results showed that the sweetness of N-Brz-C was approximately 10,600 times that of the sucrose standard of equal mass. See Table 10 for details. The sweetness was increased by 10.5 times compared to the natural brazzein Brz, and it was the median value of the sweetness of Brz-C1 and N2-Brz.

[0195] Table 9 Structure of the brazzein fusion protein with optimized dual-terminal configuration

[0196]

[0197] Table 10 Sweetness test results of the brazzein fusion protein with optimized dual-terminal configuration

[0198]

[0199]

[0200] Comparative Example 1

[0201] Using the gene of the natural brazzein expression plasmid Brz as a template, GenScript Corporation in Nanjing performed total gene synthesis to construct three groups of fusion proteins, named Brz-K1, Brz-K2, and Brz-K3, respectively. Each fusion protein consists of the natural brazzein (the first unit) with the amino acid sequence of SEQ ID NO.1 and the corresponding second unit shown in Table 11. The second unit is composed of aspartic acid (Asp) and lysine (Lys) in addition to histidine (His). The C-terminus of the first unit is connected to the N-terminus of the second unit. As shown in Table 11:

[0202] Table 11 Structure of the brazzein fusion protein with optimized C-terminal configuration

[0203]

[0204] According to the method described in Example 2, CHO cell lines expressing these three groups of proteins were constructed respectively. The supernatant obtained from cell culture was subjected to sensory testing on the protein aqueous solution after purification and recovery. The results showed that the sweetness of the three groups of fusion proteins was about 600 - 1000 times that of sucrose of the same mass as the standard product, and the sweetness was reduced to varying degrees compared with that of natural brazzein Brz. See Table 12 for details. Facts have shown that adding histidine (His) with different lengths at both ends of the natural brazzein sequence can increase the sweetness of the sweet protein to varying degrees; but on this basis, adding other amino acids (such as Asp and Lys), the enhanced sweetness may disappear or even be weaker than that before optimization.

[0205] Table 12 Sweetness test results of three configurations of brazzein fusion proteins in Example 8

[0206]

[0207] In the above examples, Chinese hamster ovary (CHO) cells were used as the protein expression host. The optimized brazzein gene sequence was cloned into the vector pDonor1.0, and 6 - 8 histidines were modified at the C - terminus of the sequence to form Brz - C1 (brazzein fusion protein); experiments have proved that regardless of whether the C - terminus of the sweet protein sequence was optimized, the spatial structure of the brazzein protein secreted by CHO cells could be correctly folded and function normally, with sweetness. Through sweet - taste sensory evaluation, it was determined that the novel brazzein fusion protein Brz - C1 with an optimized C - terminus had a sweetness at least 3.5 - 16 times higher than that of natural brazzein protein, and significantly improved the sweet - taste delay when entering the mouth.

[0208] Since there is a threshold for the senses of testers in the sensory experiment and the sweetness of the brazzein fusion protein is too high, in this example, by adjusting the mass concentration of natural brazzein protein to be the same as that of sucrose and the mass concentration of the brazzein fusion protein to be the same as that of sucrose, the relative sweetness multiple X of brazzein to sucrose was measured. Moreover, the fragment composed of histidine in the fusion protein can be used for affinity purification, which is beneficial to large - scale production.

[0209] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present invention, not to limit it; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing examples, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fusion protein with sweet taste, characterized in that: The amino acid sequence of the fusion protein comprises a first unit and a second unit, wherein the carbon end of the first unit is connected to the nitrogen end of the second unit; The amino acid sequence of the first unit is shown in SEQ ID NO.1 or SEQ ID NO.25; The second unit comprises a fragment consisting of 2 to 22 histidines.

2. The fusion protein according to claim 1, characterized in that The second unit is a fragment consisting of 2 to 16 histidines; Optionally, the second unit is a fragment consisting of 2, 4, 6, 8, 10, 12 or 16 histidines; Optionally, the second unit is a fragment consisting of 6 histidines; Optionally, the amino acid sequence of the fusion protein is selected from SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.31, SEQ ID NO.33, SEQ ID NO.60, SEQ ID NO.62, SEQ ID NO.64, SEQ ID NO.66, SEQ ID NO.68 or SEQ ID NO.

70.

3. A fusion protein with sweet taste, characterized in that: The amino acid sequence of the fusion protein contains a first unit and a third unit, and the nitrogen end of the first unit is connected to the carbon end of the third unit; The amino acid sequence of the first unit is shown in SEQ ID NO.1 or SEQ ID NO.25; The third unit comprises a fragment consisting of 2 to 22 histidines.

4. The fusion protein according to claim 3, characterized in that The third unit is a fragment consisting of 2 to 16 histidines; Optionally, the third unit is a fragment consisting of 2, 8 or 16 histidines; Optionally, the amino acid sequence of the fusion protein is selected from SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.41, SEQ ID NO.43 or SEQ ID NO.

45.

5. A fusion protein with sweet taste, characterized in that The amino acid sequence of the fusion protein contains a first unit, a second unit and a third unit; The amino acid sequence of the first unit is shown in SEQ ID NO.1 or SEQ ID NO.25; The carbon end of the first unit is connected to the nitrogen end of the second unit, and the second unit comprises a fragment consisting of 2 to 22 histidines; The nitrogen end of the first unit is connected to the carbon end of the third unit, and the third unit comprises a fragment consisting of 2 to 22 histidines.

6. The fusion protein according to claim 5, characterized in that The second unit and the third unit are both fragments consisting of 8 histidines; Optionally, the amino acid sequence of the fusion protein is selected from SEQ ID NO.23 or SEQ ID NO.

47.

7. The fusion protein according to any one of claims 1 to 6, characterized in that The sweet fusion protein is expressed by mammalian cells; Optionally, the sweet fusion protein is expressed by CHO cells.

8. A polynucleotide, characterized in that A fragment comprising a fragment encoding the sweet fusion protein according to any one of claims 1 to 7; Optionally, the polynucleotide contains at least one fragment as shown in SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, SEQ ID NO.46, SEQ ID NO.48, SEQ ID NO.61, SEQ ID NO.63, SEQ ID NO.65, SEQ ID NO.67, SEQ ID NO.69 and SEQID NO.

71.

9. A carrier, characterized in that The vector carries the polynucleotide of claim 8; Optionally, the vector comprises a plasmid.

10. A cell, characterized in that The cell expresses the sweet fusion protein according to any one of claims 1 to 7, or carries the polynucleotide according to claim 8, or contains the vector according to claim 9; Optionally, the polynucleotide of claim 8 is integrated into the genome of the cell; Optionally, the cells include mammalian cells, and further preferably include CHO cells; Optionally, the cell is a CHO cell, the polynucleotide of claim 8 is integrated into the genome of the CHO cell, and the insertion position of the polynucleotide is NW_003616785.1:83044.

11. The method for preparing a sweet fusion protein according to any one of claims 1 to 7, characterized in that: The method comprises expressing the fusion protein having a sweet taste in a cell; Optionally, the cell comprises the cell of claim 10; Optionally, the preparation method comprises integrating the polynucleotide of claim 8 into the genome of a cell; Optionally, the cell used to express the fusion protein contains a marker gene when the polynucleotide encoding the fusion protein is not integrated into the cell genome, and the marker gene is knocked out after the polynucleotide is integrated into the cell genome; Optionally, the cell is a CHO cell expressing green fluorescent protein, and the integration site of the green fluorescent protein gene in the CHO cell and the insertion site of the polynucleotide encoding the fusion protein in the CHO cell are both NW_003616785.1:83044.

12. Use of the sweet fusion protein according to any one of claims 1 to 7, or the polynucleotide according to claim 8, or the vector according to claim 9, or the cell according to claim 10, or the preparation method according to claim 11 in the preparation of medicines, feeds or foods.

13. A sweetener, a medicine, a feed or a food, characterized in that: A fusion protein having a sweet taste comprising the sweet fusion protein according to any one of claims 1 to 7.

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