CN117088958A - Sweet protein Monellin mutant with high sweetness and preparation method thereof - Google Patents

Abstract

Provided herein are monellin protein mutants comprising a deletion of amino acid 1. The monellin protein mutant has increased sweetness relative to wild single-chain monellin protein MNEI, and can be used as sweetener of edible products.

Description

Sweet protein monellin mutant with high sweetness and preparation method thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application CN202210553207.6 filed on May 20, 2022, the entirety of which is hereby incorporated by reference.

Technical Field

The present invention relates to monellin protein mutants, in particular monellin protein mutants comprising a deletion of amino acid 1 relative to wild-type single-chain monellin protein (MNEI). The present invention also relates to methods for preparing monellin protein mutants and uses thereof as sweeteners for edible products.

Background Art

Monellin is a sweet protein and a natural sweetener extracted from the West African plant Dioscoreophyllum cumminsii. Monellin has a strong sweet taste, and its sweetness is about 800 to 2000 times that of the same mass of sucrose under the same conditions. As a high-sweetness and low-calorie non-sugar protein sweetener, monellin can be used in the processing of sugar-free foods, as a sweetening additive for patients with epidemic diseases related to sugar intake such as obesity, diabetes and dental caries, and can also be used in children's food, with huge market potential. Natural monellin protein is composed of two different peptide chains (i.e., A chain and B chain) bound together by covalent bonds. Studies have found that the sweetness of natural monellin protein will be completely lost at a temperature ≥50°C. In 1989, Kim et al. combined two peptide chains into one protein chain through genetic engineering methods to create a single-chain monellin protein, in which the two natural chains were connected by a Gly-Phe dipeptide linker, which improved its thermal stability while not changing its sweetness much (Kim et al., Redesigning a sweet protein: increased stability and renaturability. Protein Eng. 1989 Aug; 2(8): 571-5).

Single-chain monellin protein has also been successfully expressed in other organisms using genetic engineering methods, such as Escherichia coli expression system, plant expression system, Bacillus subtilis expression system and yeast expression system. However, due to the problems of yield and toxic metabolites in these expression systems, monellin protein cannot be fermented and produced on a large scale. One strategy to reduce the production cost of monellin protein is to reduce the sweetness threshold of the protein as much as possible, that is, to enhance the sweetness so that a good sweetness effect can be achieved with extremely small additions.

Due to the huge market potential of monellin sweet protein, domestic and foreign scholars and research institutions have studied mutants of single-chain monellin protein with high sweetness. Chinese patent CN105566471A discloses a single-chain monellin protein mutant, which site-directedly mutates the second amino acid glutamic acid of the single-chain monellin protein to asparagine. AMAI's Chinese patent CN112313244A discloses a single-chain monellin protein mutant with modified taste and flavor, especially the DM09 mutant (E2N/E23A/Y65R). Zheng et al. conducted an in-depth study on the mutation of the second amino acid of the single-chain monellin protein, and the results showed that the second amino acid mediated the sweetness of monellin (Zheng et al., Expression, purification and characterization of a novel double-sites mutant of the single-chain sweet-tasting protein monellin (MNEI) with both improved sweetness and stability. Protein Expr Purif. 2018 Mar; 143: 52-56).

Summary of the invention

In one aspect, provided herein is a monellin protein mutant comprising a deletion of amino acid position 1 relative to a wild-type monellin protein.

In some embodiments, the wild-type monellin protein is a wild-type single-chain monellin protein.

In some embodiments, the monellin protein mutant has a sweetness higher than that of the wild-type single-chain monellin protein, preferably at least 5 times, at least 10 times, at least 15 times, or at least 20 times the sweetness of the wild-type single-chain monellin protein.

In some embodiments, the sweetness of the monellin protein mutant decreases by no more than 5% after being kept at 65° C. for 30 min.

In some embodiments, the monellin protein mutant comprises one or more amino acid substitutions selected from the group consisting of: 2, 23, 41, 65, and 76.

In some embodiments, the monellin protein mutant comprises an amino acid substitution E2N or E2A.

In some embodiments, the monellin protein mutant comprises the amino acid substitution E23A.

In some embodiments, the monellin protein mutant further comprises amino acid substitutions C41A, Y65R, S76Y, or any combination thereof.

In some embodiments, the monellin protein mutant comprises any combination of amino acid substitutions selected from the following combinations:

1) E2N/E23A/Y65R;

2) E2A/E23A/Y65R;

3) E2N/E23A/C41A;

4) E2N/E23A/C41A/Y65R;

5) E2N/E23A/C41A/S76Y;

6) E2N/E23A/C41A/Y65R/S76Y;

7) E2A/E23A/C41A;

8) E2A/E23A/C41A/Y65R;

9) E2A/E23A/C41A/S76Y; and

10)E2A/E23A/C41A/Y65R/S76Y.

In some embodiments, the wild-type single-chain monellin protein has the amino acid sequence shown in SEQ ID NO:2.

In some embodiments, the monellin protein mutant comprises an amino acid sequence as shown in any one of SEQ ID NOs: 24-34, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs: 24-34.

In some embodiments, the monellin protein mutant has an amino acid sequence as shown in any one of SEQ ID NOs: 24-34.

In another aspect, the present invention provides the use of the above-mentioned monellin protein mutant as a food additive, a beverage additive or a drug additive.

In another aspect, provided herein is an edible product comprising the monellin protein mutant described above.

In some embodiments, the edible product is a food, a beverage, or a medicine.

In some embodiments, the edible product further comprises a sweetener other than the monellin protein mutant.

In some embodiments, the sweetener is sucrose.

In some embodiments, the beverage is a yogurt drink.

In another aspect, provided herein is a nucleic acid molecule encoding the above-mentioned monellin protein mutant.

In another aspect, provided herein is an expression vector comprising the nucleic acid molecule.

In some embodiments, the expression vector is the expression vector PGAPZαA or a linearized product thereof.

In another aspect, provided herein is a host cell, the above-mentioned expression vector thereof.

In some embodiments, the host cell is Pichia pastoris.

In some embodiments, the host cell is Pichia pastoris X-33.

The monellin protein mutants provided herein have increased sweetness relative to the wild-type single-chain monellin protein MNEI and can be used as sweeteners for edible products.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a SDS-PAGE detection diagram of single-chain monellin and its mutant proteins. In the figure, M represents Marker, and lanes 1 to 22 are MNEI, MNEI-ΔG1, DM09, DM09-ΔG1, BZ01, BZ01-ΔG1, BZ02, BZ02-ΔG1, BZ03, BZ03-ΔG1, BZ04, BZ04-ΔG1, BZ05, BZ05-ΔG1, BZ06, BZ06-ΔG1, BZ07, BZ07-ΔG1, BZ08, BZ08-ΔG1, BZ09, and BZ09-ΔG1 proteins, respectively.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

"Sweet protein" refers herein to a protein with a sweet taste, which is suitable for sweetening food, beverages and/or pharmaceutical products for human consumption. By weight, the sweetness of the sweet protein of the present invention may be at least 1000 times, preferably at least 2000 times, more preferably at least 5000 times, and even more preferably 10000 times that of sucrose when compared to a 1% sucrose solution. For the purposes of the present invention, the comparison of sweetness can be determined by testing the human perception of the sweetness of the protein against a known control, using different concentrations of protein diluted in water, or in an aqueous solution to avoid surface adsorption, such as 20% skim milk.

"Sweetness" or "sweetness potency" herein refers to the sweetening ability of a sweetener, which can be measured or compared by the sweetness thresholds of different sweeteners. The sweetness or sweetness potency of a sweetener can be reported relative to sucrose.

"Sweetness threshold" herein refers to the lowest concentration of a sweetener at which a sample (eg, a food or beverage (eg, pure water) containing the sweetener) is recognized by a taster as having a sweet taste.

"Monellin protein" herein refers to any protein comprising the A chain and the B chain of the monellin protein, including a double-chain protein in which the A chain and the B chain are bound together by a covalent bond, and a single-chain protein formed by linking the A chain and the B chain together.

"Single-chain monellin protein" herein refers to a single-chain protein formed by linking the A chain and B chain of natural monellin protein through a short peptide linker molecule. In a single-chain monellin protein, from N-terminus to C-terminus, it is usually B chain-linker molecule-A chain.

"Wild type" in this article is not limited to proteins naturally produced in nature. It can refer to the A chain and B chain of the monellin protein that have the same amino acid sequence as the A chain and B chain of the naturally occurring monellin protein, including double-chain monellin protein (i.e., naturally occurring monellin protein) and also includes a single-chain protein formed by connecting the A chain and B chain of the naturally occurring monellin protein.

"Wild-type single-chain monellin protein (MNEI)" herein refers to the single-chain monellin protein formed by connecting with a Gly-Phe dipeptide linker reported by Kim et al., which has the amino acid sequence shown in SEQ ID NO: 2. The modified term "wild-type" used herein does not mean that it is naturally produced in nature, but means that the single-chain monellin protein is used as a reference for determining the mutation type (such as amino acid deletion or substitution) and mutation position of the monellin protein mutant provided herein, or it can be considered as the parent protein of the monellin protein mutant provided herein. In addition, in some cases, the wild-type single-chain monellin protein is also used as a reference for measuring the sweetness of the monellin protein mutant provided herein.

"Monellin protein mutant" herein refers to a protein that has a difference in amino acid sequence relative to the wild-type single-chain monellin protein (i.e., the parent protein). This difference can be manifested in the presence of one or more amino acid substitutions, deletions, or insertions at one or more positions in the amino acid sequence. Preferably, the monellin protein mutant is also in a single-chain form.

"Amino acid substitution", also known as "amino acid replacement", refers to the substitution of an amino acid (called the original amino acid) at a specific position in the amino acid sequence by another amino acid (the substituted amino acid) in this article. For example, the second amino acid of the parent protein is a glutamic acid residue (E), and the corresponding position of the mutant protein is an alanine residue (A), then it can be considered that there is an amino acid substitution in the mutant protein: alanine replaces glutamic acid. For amino acid substitution, the following nomenclature is used herein: original amino acid, position and substituted amino acid, and the amino acid name uses the single-letter abbreviation of amino acids specified by IUPAC. For the example described above, it can be expressed as E2A. "Amino acid substitution combination" refers to the presence of two or more positions of amino acid substitution in the mutant protein in this article. For example, the second amino acid of the parent protein is a glutamic acid residue (E), and the corresponding position of the mutant protein is an asparagine residue (N); at the same time, the 23rd amino acid of the parent protein is a glutamic acid residue (E), and the corresponding position of the mutant protein is an alanine residue (A), then it can be considered that there is an amino acid substitution combination in the mutant protein: E2N and E23A. When multiple amino acid substitutions exist simultaneously in a mutant, the multiple mutations are separated by “/”, for example, “E2N/E23A/Y65R” represents that glutamic acid is replaced by asparagine, glutamic acid is replaced by alanine, and tyrosine is replaced by arginine at positions 2, 23 and 65, respectively.

"Amino acid deletion", also known as "amino acid deletion", refers to the lack of an amino acid corresponding to the amino acid at that position in the parent protein in a certain position in the mutant protein compared to the parent protein. When compared with the parent protein, the position may appear as a gap. Amino acid deletion can be represented by "Δ". For example, when the amino acid glycine (G) at position 1 is missing in the mutant protein, it can be counted as ΔG1 (or G1Δ). Therefore, ΔG1/E2N/E23A/Y65R can be used to indicate that glycine at position 1 is missing, glutamic acid at position 2 is replaced by asparagine, glutamic acid at position 23 is replaced by alanine, and tyrosine at position 65 is replaced by arginine in the mutant protein.

"Amino acid insertion", also known as "amino acid addition", refers to an amino acid at a certain position in a mutant protein that has no corresponding amino acid in the parent protein compared to the parent protein. In other words, amino acid insertion refers to one or more extra amino acids when compared to the parent protein.

"Coding sequence" refers to a polynucleotide sequence in this article that directly determines the amino acid sequence of its protein product. The boundaries of the coding sequence are usually determined by an open reading frame, which usually starts with the ATG start codon or alternative start codons such as GTG and TTG, and ends with stop codons such as TAA, TAG and TGA. The coding sequence can be a DNA, cDNA, RNA, synthetic or recombinant nucleotide sequence.

"Expression" herein may include any step involved in the production of the monellin protein mutants provided herein, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

An "expression vector" herein refers to a linear or circular DNA molecule comprising a polynucleotide encoding a protein, such as a monellin protein mutant provided herein, and operably linked to additional nucleotides that provide for its expression.

"Host cell" refers herein to a cell that can be used to produce the monellin protein mutants provided herein. Host cells are generally suitable for the introduction of exogenous nucleic acid molecules (such as expression vectors) and have a series of enzymes that can be used to express exogenous proteins (including enzymes related to transcription, post-transcriptional processing, translation, and post-translational modification). Available host cells include prokaryotic cells and eukaryotic cells, such as Escherichia coli, yeast, mammalian cells, etc. In a preferred embodiment, Pichia pastoris (such as Pichia pastoris X-33) is used as a host cell to express the monellin protein mutants provided herein. Host cells also include the offspring of a single host cell, and the offspring may not necessarily be completely consistent with the original parent cell (in terms of morphology or genomic DNA) due to natural, accidental or deliberate mutations. The host cell can be an isolated cell or cell line.

As used herein, "polypeptide", "protein" and "peptide" are synonymous and are used interchangeably. When referring to the amino acid sequence of a protein, the conventional one-letter or three-letter symbols for the amino acid residues are used, wherein the amino acid sequence is presented in the standard amino to carboxyl terminal orientation (i.e., N→C).

When referring to amino acid sequences, the term "sequence identity" (also referred to as "sequence identity") refers to the amount of consistency between two amino acid sequences (e.g., a query sequence and a reference sequence), generally expressed as a percentage. Typically, before calculating the percentage of consistency between two amino acid sequences, a sequence alignment is performed and gaps (if any) are introduced. If the amino acid residues or bases in the two sequences are the same at a certain alignment position, the two sequences are considered to be consistent or matched at that position; if the amino acid residues or bases in the two sequences are different, they are considered to be inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence consistency. In other algorithms, the number of gaps and/or the length of the gaps are also taken into account. Commonly used sequence comparison algorithms or software include EMBOSS, DANMAN, CLUSTALW, MAFFT, BLAST, MUSCLE, etc. For the purposes of the present invention, the CLUSTALW algorithm is used in some embodiments for amino acid sequence alignment. The preset parameters of the CLUSTALW algorithm may be: the missing count is the residue that is different from the reference sequence, including the missing that occurs at any end. For example, a variant 500 amino acid residue polypeptide lacking the five amino acid residues at the C-terminus has a sequence identity percentage of 99% (495/500 identical residues x 100) relative to the parent polypeptide. Such variants are encompassed by the language "variants having at least 99% sequence identity to the parent".

As used herein, the term "about", "around" or the symbol "~" indicates that the numerical value may vary from the numerical value mentioned by up to 1%, up to 5%, up to 10%, up to 15%, and in some cases up to 20%. The range of deviation includes integer values and, if applicable, non-integer values to form a continuous range.

The term "or" refers to a single element of the listed optional elements, unless the context clearly indicates otherwise. The term "and/or" refers to any one, any two, any three, any more or all of the listed optional elements.

The terms "comprise", "contain", "have", "include" and similar expressions used herein do not exclude unrecited elements. These terms also include the situation consisting of only the listed elements.

In view of the fact that the second amino acid of the single-chain monellin protein mediates the sweetness of monellin, the inventors of the present application speculated based on protein structure simulation that the first amino acid of the single-chain monellin protein may form a steric hindrance to the second amino acid, thereby affecting the sweetness. Removing the first amino acid can fully expose the second amino acid, which may further increase the sweetness. The inventors of the present application verified through experiments that the single-chain monellin mutant with the first amino acid deleted did have a significant improvement in sweetness. The inventors of the present application have successfully achieved some results different from the prior art in the research on the modification of the single-chain monellin protein.

One of the purposes of the present invention is to provide a single-chain monellin protein mutant with high sweetness in order to reduce the production cost of single-chain monellin protein. The present invention performs site-directed mutagenesis on single-chain monellin protein to obtain a mutant with high sweetness and thermal stability, and successfully expresses it in Pichia pastoris. By increasing the sweetness of the protein, a good sweetening effect can be achieved with extremely small amounts of addition, thereby effectively reducing the production cost of monellin protein and laying a solid foundation for the large-scale industrial production of the sweet protein. The use of the PGAPZαA plasmid can directly secrete the target protein outside the cell, which is convenient for subsequent protein purification and reduces the purification cost. Due to the use of a self-inducing promoter, there is no need to add inducers such as methanol during the fermentation process, which reduces the risk of fermentation contamination, and does not produce toxic metabolites, thereby ensuring the safety of the protein as a food additive.

In the first aspect of the present invention, a single-chain monellin protein mutant is provided, the protein comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity with any one of the amino acids of SEQ ID NOs: 24-34. The single-chain monellin protein mutant with high sweetness provided by the present invention and the technical scheme thereof are as follows:

1) A single-chain monellin protein mutant, wherein the first amino acid glycine (Gly) of the wild-type single-chain monellin protein (MNEI) is site-specifically deleted.

2) A single-chain monellin protein mutant, wherein the first amino acid glycine (Gly) of the monellin protein DM09 (E2N/E23A/Y65R) is site-specifically deleted.

3) A single-chain monellin protein mutant, wherein the first amino acid glycine (Gly) of the monellin protein BZ01 (E2A/E23A/Y65R) is site-specifically deleted.

4) A single-chain monellin protein mutant, wherein the first amino acid glycine (Gly) of the monellin protein BZ02 (E2N/E23A/C41A) is site-specifically deleted.

5) A single-chain monellin protein mutant, wherein the first amino acid glycine (Gly) of the monellin protein BZ03 (E2N/E23A/C41A/Y65R) is site-specifically deleted.

6) A single-chain monellin protein mutant, wherein the first amino acid glycine (Gly) of the monellin protein BZ04 (E2N/E23A/C41A/S76Y) is site-specifically deleted.

7) A single-chain monellin protein mutant, wherein the first amino acid glycine (Gly) of the monellin protein BZ05 (E2N/E23A/C41A/Y65R/S76Y) is site-specifically deleted.

8) A single-chain monellin protein mutant, wherein the first amino acid glycine (Gly) of the monellin protein BZ06 (E2A/E23A/C41A) is site-specifically deleted.

9) A single-chain monellin protein mutant, wherein the first amino acid glycine (Gly) of the monellin protein BZ07 (E2A/E23A/C41A/Y65R) is site-specifically deleted.

10) A single-chain monellin protein mutant, wherein the first amino acid glycine (Gly) of the monellin protein BZ08 (E2A/E23A/C41A/S76Y) is site-specifically deleted.

11) A single-chain monellin protein mutant, wherein the first amino acid glycine (Gly) of the monellin protein BZ09 (E2A/E23A/C41A/Y65R/S76Y) is site-specifically deleted.

Among them, the sweetness of the mutant MNEI-ΔG1 in Scheme 1) is increased by 6.7 times compared with the wild single-chain monellin protein (MNEI). The sweetness of the mutant DM09-ΔG1 in Scheme 2) is increased by 7.6 times compared with the monellin protein DM09. The sweetness of the mutant BZ01-ΔG1 in Scheme 3) is increased by 7.2 times compared with the monellin protein BZ01. The sweetness of the mutant BZ02-ΔG1 in Scheme 4) is increased by 7.5 times compared with the monellin protein BZ02. The sweetness of the mutant BZ03-ΔG1 in Scheme 5) is increased by 7.3 times compared with the monellin protein BZ03. The sweetness of the mutant BZ04-ΔG1 in Scheme 6) is increased by 7.0 times compared with the monellin protein BZ04. The sweetness of the mutant BZ05-ΔG1 in Scheme 7) is increased by 6.8 times compared with the monellin protein BZ05. Scheme 8) The sweetness of the mutant BZ06-ΔG1 is increased by 7.4 times relative to the monellin protein BZ06. Scheme 9) The sweetness of the mutant BZ07-ΔG1 is increased by 7.4 times relative to the monellin protein BZ07. Scheme 10) The sweetness of the mutant BZ08-ΔG1 is increased by 7.3 times relative to the monellin protein BZ08. Scheme 11) The sweetness of the mutant BZ09-ΔG1 is increased by 6.6 times relative to the monellin protein BZ09. The sweetness of the mutants described in Schemes 1) to 11) is increased by about 15.5 to 24.4 times relative to the single-chain monellin protein (MNEI).

Based on these mutants provided herein, those skilled in the art can further introduce other amino acid deletions, substitutions or insertions, and detect the sweetness of the mutant products obtained, thereby obtaining other mutants that still have sweet taste. These mutants are also included in the scope of the present invention.

In some embodiments, these other mutants can be obtained by amino acid replacement. It is expected that the monellin protein mutants provided herein may further include other conservative amino acid substitutions. Conservative amino acid substitutions can generally be described as a substitution of an amino acid residue by another amino acid residue of similar chemical structure, and have little or substantially no effect on the function, activity or other biological properties of the polypeptide. Conservative amino acid substitutions are well known in the art. Conservative substitutions may, for example, be substitutions of one amino acid in the following (a)-(e) groups by another amino acid in the same group: (a) small aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar positively charged residues: His, Arg and Lys; (d) large aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

In some embodiments, the used linker molecule can also be replaced. Available linker molecules can include 1 or more amino acid residues, such as 1-100 amino acid residues, 1-50 amino acid residues, 1-20 amino acid residues or 1-10 amino acid residues. Kim et al. (Kim et al., Redesigning a sweet protein: increased stability and renaturability. Protein Eng. 1989 Aug; 2 (8): 571-5) described a method for connecting two peptide chains of natural monellin protein. Those skilled in the art can try to use other linker molecules for connection in a similar manner, and evaluate the performance of the connected single-chain monellin protein, such as sweetness, to obtain other mutants that are different from the present invention only in the linker molecule. In addition, Chinese patent application CN109627307A discloses the use of a hairpin protein domain to connect the A chain and the B chain of monellin protein, and obtains a single-chain monellin protein with improved heat resistance and substantially unchanged sweetness. The inventors expect that by replacing the linker molecules of the monellin protein mutants provided herein as described above, other monellin protein mutants with increased sweetness relative to the wild-type single-chain monellin protein mutants can also be obtained, and these other monellin protein mutants should also be included in the scope of the present invention. In addition, the inventors also expect that based on the wild-type double-chain monellin protein, the first amino acid glycine of its B chain is deleted, and the obtained mutant may also have increased sweetness.

In a second aspect of the present invention, a method for preparing the high-sweetness single-chain monellin protein mutant is provided, comprising the following steps:

1) Construction of expression vectors for wild-type single-chain monellin protein MNEI and mutants DM09 and BZ01-BZ09;

2) Design primers for deleting the first amino acid glycine of wild-type single-chain monellin MNEI and mutants DM09 and BZ01-BZ09, perform site-directed mutagenesis on the MNEI and mutant sequences of step 1), obtain gene fragments with the first amino acid deleted and use them for vector construction, and select positive transformants for sequencing verification;

3) transforming the plasmid sequenced correctly in step 2) into Pichia pastoris, and screening out the successfully transformed Pichia pastoris;

4) Induce protein expression in YPD medium for 3 days;

5) Purify the protein expressed in step 4).

Step 1) constructing an expression plasmid containing the single-chain monellin protein MNEI and the coding genes of mutants DM09 and BZ01-BZ09, namely PGAPZαA.

Step 2) Mutation site-specific primers: BZ06-R (SEQ ID NO: 16): 5'-ATAAGAATGCGGCCGCTTATGGTGGTGGAACTGGACC-3'; MNEI-ΔG1-F (SEQ ID NO: 17): 5'-CCGCTCGAGGAGTGGGAGATCATTGACATCG-3'; BZ01-ΔG1-F (SEQ ID NO: 18): 5'-CCGCTCGAGGCTTGGGAGATCATTGACATCG-3'; BZ02-ΔG1-F (SEQ ID NO: 19): 5'-CCGCTCGAGAACTGGGAGATCATTGACATCG-3'.

In steps 1) and 2), the PCR product was digested with Xhol and NotI endonucleases and ligated with the PGAPZαA empty vector after enzyme digestion, transformed into Escherichia coli DH5α competent cells, spread on LB solid medium containing 25 μg/ml Zeocin, cultured overnight, picked positive single colonies and extracted plasmids, sequenced to verify the results, and constructed the correct expression plasmid for future use.

The Pichia pastoris described in step 3) is Pichia pastoris X-33.

In step 4), the transformants were inoculated into a shake flask containing 50 ml of YPD medium and cultured at 30°C for 3 days. Trichloroacetic acid was added to the fermentation supernatant and mixed, and the mixture was kept at 4°C overnight. The mixture was centrifuged, washed with acetone, and the expression of the target protein was detected by SDS-PAGE. The positive transformants with the highest expression were selected and inoculated into a shake flask containing 200 ml of YPD medium and induced at 30°C for 3 days.

Purification method in step 5): collect the fermentation supernatant, put it into a dialysis bag with a molecular weight cutoff of 3500 and dialyze it at 4°C for 24 hours, and the dialysis buffer is 10mM sodium phosphate buffer (pH7.0). The dialyzed sample is loaded on the ion exchange chromatography column Sephedex CM-50, and the target protein is linearly eluted with 10mM sodium phosphate buffer (pH7.0) containing 0-0.4M sodium chloride. The collected target protein is placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed at 4°C for 24 hours, and the dialysis is repeated 3 times. The purified protein is detected by SDS-PAGE and the concentration of the target protein is determined by the Coomassie Brilliant Blue method.

The high-sweetness single-chain monellin protein mutant and the preparation method thereof of the present invention have the following beneficial effects: site-directed mutation is performed on the single-chain monellin protein to obtain a mutant with high sweetness and thermal stability. The production cost of the monellin protein is effectively reduced by increasing the sweetness of the protein, laying a solid foundation for the large-scale industrial production of the protein. The method of directly secreting the target protein into the extracellular space is adopted to effectively reduce the purification cost of the protein. At the same time, the self-inducing promoter is adopted to ensure that no inducing agent such as methanol is added during the fermentation process, and no toxic metabolites are generated while reducing the risk of fermentation contamination, thereby ensuring the safety of the protein as a food additive.

The present invention also relates to a method for increasing the sweetness of edible products such as food, beverages or pharmaceutical products for human consumption, comprising the steps of adding a sufficient amount of the monellin protein mutant provided herein to the above-mentioned food, beverage or pharmaceutical product so that they have increased sweetness.

Also provided herein are edible products for human consumption, such as food, beverages or pharmaceutical products, comprising the monellin protein mutants provided herein.

In some embodiments, the monellin protein mutants provided herein can be used in combination with other sweeteners. In one example, the monellin protein mutants provided herein and sucrose can be added to a yogurt drink. The presence of sucrose can improve the "delayed sweetness perception" of the monellin protein mutants.

Additional aspects and advantages of the present invention will be given in the following examples or will become apparent from the following examples. These examples are not intended to limit the scope of the present invention in any way. It is particularly noted that the reagents mentioned herein are commercially available unless otherwise specified.

Experimental materials and reagents

1. Strains and vectors: The expression host Pichia pastoris X-33 (Invitrogen) and the expression plasmid vector PGAPZαA (Invitrogen) were preserved in our laboratory.

2. Enzymes and other biochemical reagents: Endonucleases were purchased from Fermentas, ligases were purchased from Promaga, and DNA polymerases were purchased from Beijing Quanshijin Biological. Other reagents were domestic analytical grade reagents (all available from ordinary biochemical reagent companies).

3. Culture medium:

LB solid medium: 0.5% yeast extract, 1% peptone, 1% NaCl, 1% agar powder, pH 7.0.

YPD medium: 1% yeast extract, 2% peptone, 2% glucose.

Example 1 Construction of expression vectors for wild-type single-chain monellin protein MNEI and its mutants

The wild-type single-chain monellin protein MNEI and its mutants DM09 (E2N/E23A/Y65R), BZ01 (E2A/E23A/Y65R), BZ02 (E2N/E23A/C41A), BZ03 (E2N/E23A/C41A/Y65R), BZ04 (E2N/E23A/C41A/S76Y) and BZ05 (E2N/E23A/C41A/Y65R/S76Y) gene fragments were synthesized by Nanjing GenScript Biotechnology Co., Ltd., and two restriction endonuclease sites, XhoI and NotI, were introduced at both ends. After digestion of the target gene fragment and the PGAPZαA empty plasmid, they were ligated with T4 DNA ligase at 37°C overnight, transformed into DH5α Escherichia coli competent cells, and plated on LB solid medium containing 25 μg/ml Zeocin. Positive transformants were picked from overnight cultures, and plasmids were extracted using a plasmid extraction kit for sequencing verification. The wild-type single-chain monellin protein expression vector PGAPZαA-MNEI and its mutant expression vectors PGAPZαA-DM09, PGAPZαA-BZ01, PGAPZαA-BZ02, PGAPZαA-BZ03, PGAPZαA-BZ04 and PGAPZαA-BZ05 were successfully constructed. The coding sequences and amino acid sequences of the wild-type single-chain monellin protein MNEI and its mutant genes DM09, BZ01, BZ02, BZ03, BZ04 and BZ05 are shown in Table 1.

Table 1 Wild-type single-chain monellin protein MNEI and its mutant gene coding sequence and amino acid sequence

Primers for the single-chain monellin mutants BZ06 (E2A/E23A/C41A), BZ07 (E2A/E23A/C41A/Y65R), BZ08 (E2A/E23A/C41A/S76Y), and BZ09 (E2A/E23A/C41A/Y65R/S76Y) were designed. Since the primer sequences were the same, they were named BZ06-F and BZ06-R. The primer sequences are shown in Table 2. The gene fragments of BZ02 (E2N/E23A/C41A), BZ03 (E2N/E23A/C41A/Y65R), BZ04 (E2N/E23A/C41A/S76Y) and BZ05 (E2N/E23A/C41A/Y65R/S76Y) were used as templates, and the primer pair BZ06-F/BZ06-R was used for PCR amplification. The PCR reaction system is shown in Table 3. After the PCR was completed, it was detected by 1% agarose gel electrophoresis, and the gene fragments of single-chain monellin mutants BZ06, BZ07, BZ08 and BZ09 were recovered by gel recovery. The PCR product was treated with two restriction endonucleases, XhoI and NotI, for 6 hours and then connected with the open plasmid vector recovered by gel using T4 DNA ligase at 37°C overnight. The enzyme digestion and connection system are shown in Table 3. The ligation product was transformed into DH5α Escherichia coli competent cells and spread on LB solid medium containing 25 μg/ml Zeocin. Positive transformants were picked from overnight cultures, and plasmids were extracted and sequenced using a plasmid extraction kit to successfully construct single-chain monellin mutant vectors PGAPZαA-BZ06, PGAPZαA-BZ07, PGAPZαA-BZ08, and PGAPZαA-BZ09. The amino acid sequences of single-chain monellin mutants BZ06, BZ07, BZ08, and BZ09 are shown in Table 4.

Table 2 Primer sequences used in this article

Table 3 Reaction system

Table 4 Single-chain monellin mutant amino acid sequences

Primers were designed to delete the first amino acid glycine of the wild-type single-chain monellin protein MNEI and its mutants DM09, BZ01, BZ02, BZ03, BZ04, BZ05, BZ06, BZ07, BZ08 and BZ09. Since the primer sequences of some mutants were the same, the primers were named MNEI-ΔG1-F, BZ01-ΔG1-F and BZ02-ΔG1-F, respectively. The primers are shown in Table 2. Using the MNEI gene fragment as a template, PCR amplification was performed using the primer pair MNEI-ΔG1-F/BZ06-R to obtain the MNEI-ΔG1 gene fragment. PCR amplification was performed using primer pair BZ01-ΔG1-F/BZ06-R with BZ01, BZ06, BZ07, BZ08 and BZ09 gene fragments as templates, respectively. After PCR, 1% agarose gel electrophoresis was used for detection, and a gel recovery kit was used to obtain BZ01-ΔG1, BZ06-ΔG1, BZ07-ΔG1, BZ08-ΔG1 and BZ09-ΔG1 gene fragments. DM09, BZ02, BZ03, BZ04 and BZ05 gene fragments were used as templates, and primers BZ02-ΔG1-F/BZ06-R were used for PCR amplification. After the PCR was completed, 1% agarose gel electrophoresis was used to detect, and the DM09-ΔG1, BZ02-ΔG1, BZ03-ΔG1, BZ04-ΔG1 and BZ05-ΔG1 gene fragments were obtained using a gel recovery kit. The PCR reaction system is shown in Table 3. The PCR product was treated with two restriction endonucleases, XhoI and NotI, for 6 hours, and the open plasmid vector recovered from the gel was connected with T4 DNA ligase at 37°C overnight. The enzyme digestion and connection system are shown in Table 3. The ligation product was transformed into DH5α Escherichia coli competent cells and coated on LB solid medium containing 25μg/ml Zeocin. Positive transformants were picked from overnight cultures, and plasmids were extracted using a plasmid extraction kit for sequencing verification, and single-chain monellin mutant vectors PGAPZαA-MNEI-ΔG1, PGAPZαA-DM09-ΔG1, PGAPZαA-BZ01-ΔG1, PGAPZαA-BZ02-ΔG1, PGAPZαA-BZ03-ΔG1, PGAPZαA-BZ04-ΔG1, PGAPZαA-BZ05-ΔG1, and PGAPZαA-BZ06-ΔG1 were successfully constructed. 06-ΔG1, PGAPZαA-BZ07-ΔG1, PGAPZαA-BZ08-ΔG1, and PGAPZαA-BZ09-ΔG1, and the amino acid sequences of the single-chain monellin mutants MNEI-ΔG1, DM09-ΔG1, BZ01-ΔG1, BZ02-ΔG1, BZ03-ΔG1, BZ04-ΔG1, BZ05-ΔG1, BZ06-ΔG1, BZ07-ΔG1, BZ08-ΔG1, and BZ09-ΔG1 are shown in Table 4 .

Example 2 Preparation of wild-type single-chain monellin and its mutant proteins

(1) Transformation of the expression vector into Pichia pastoris X-33

The wild-type single-chain monellin and its mutant expression vectors constructed in Example 1 were linearized by restriction endonuclease AvrII, the linearized DNA was concentrated by ethanol precipitation DNA method, and detected by agarose gel electrophoresis.

Preparation of Pichia pastoris X-33 competent cells: 1) Inoculate a single colony of Pichia pastoris X-33 to 5 ml YPD medium, and culture at 30°C, 250rpm/min for 24h; 2) Take 1ml of the culture solution and inoculate it into a 500ml shake flask containing 100ml YPD medium and culture it for 12-16h, until OD600 = 0.8-1.2, transfer the bacterial solution into a 50ml pre-cooled centrifuge tube, centrifuge at 4°C, 5000g for 5min, and discard the supernatant; 3) Resuspend the bacteria with 25ml pre-cooled sterile water, centrifuge at 4°C, 5000g for 5min, discard the supernatant, and repeat this step 2-3 times; 4) Resuspend the bacteria with 5ml pre-cooled 1mol/L sorbitol in each tube, centrifuge at 4°C, 5000g for 5min, discard the supernatant, and repeat this step 2-3 times; 5) Finally, resuspend with 200μL sorbitol and divide into 1.5ml centrifuge tubes, 80μL per tube.

Transformation screening: 1) Take a competent cell and add 10 μL of linearized and concentrated DNA, mix gently, and transfer to a pre-cooled 0.2 cm electrode cup and place on ice for 5 minutes; 2) Perform electric shock at the electric shock parameters of 1.5Kv, 25μF, 200Ω. After the electric shock is completed, quickly add 1 ml of pre-cooled 1 mol/L sorbitol, and then transfer the mixed solution to a 1.5 ml centrifuge tube and resuscitate at 30°C for 1 hour; 3) Take 200 μL and spread it on YPDS solid culture medium containing 100 μg/ml Zeocin, culture the plate at 30°C for 3 days, observe positive transformants, use PCR to identify positive single colonies, and send PCR-positive colonies for sequencing verification.

(2) Expression verification and purification of target protein

The positive transformants verified by PCR and sequencing were inoculated into 250ml shake flasks containing 50ml YPD medium and cultured at 30℃ and 250rpm/min for 3 days. The fermentation liquid was centrifuged at 4℃ and 12000rpm, 900μL of supernatant was added with 100μL trichloroacetic acid and mixed, and kept overnight at 4℃. The mixture was centrifuged at 10000rpm/min, the supernatant was discarded, and the precipitate was washed with acetone 2-3 times. The expression results of the target protein were detected by SDS-PAGE.

The positive transformants with brighter target protein bands were selected and inoculated into 1L shaking flasks containing 200ml YPD medium, and induced at 30℃ and 250rpm for 3 days. The fermentation supernatant was collected by low-temperature centrifugation and placed in a dialysis bag with a molecular weight cutoff of 3500 for dialysis at 4℃ for 24h. The dialysis buffer was 10mM sodium phosphate buffer (pH7.0). The dialyzed sample was filtered through a 0.22μm filter and loaded on an ion exchange chromatography column Sephedex CM-50. The column was balanced with 10mM sodium phosphate buffer (pH7.0), and the target protein was linearly eluted with 10mM sodium phosphate buffer (pH7.0) containing 0-0.4M sodium chloride. The collected target protein was placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed at 4℃ for 24h. The dialysis buffer was 10mM sodium phosphate buffer (pH5.0), and the solution was changed 3 times during the period. The purified protein was detected by SDS-PAGE, and the results are shown in Figure 1. The concentration of the target protein was determined by the Coomassie brilliant blue method, and the sweet taste activity of the protein was preliminarily determined by tasting the dialyzed solution.

Example 3 Determination of the sweet taste threshold of wild-type single-chain monellin and its mutant proteins

The sweetness potency of the wild-type single-chain monellin MNEI described in this article relative to sucrose is generally in the range of 1:700 to 1:16,000. Therefore, for comparison, the MNEI and its mutant protein solutions were first diluted to 1:1000 and then further diluted as needed. For most people, the sweetness threshold of sugar in soft drinks is 0.32%-1.0%. In order to evaluate the sweetness threshold of each solution, a double-blind taste analysis was performed. The tested samples included MNEI and its mutants, sucrose and mineral water. The protein was diluted into a gradient from 0.01 to 1.5 μg/ml by diluting the protein stock solution with mineral water (pH 6.9). 10 healthy assessors participated in this evaluation, 5 males and 5 females, aged between 30 and 50 years old, of which 5 were trained wine tasters. Taste from the lowest concentration, increase the concentration by 0.1 μg/ml each time, and when the sweetness is felt, use this concentration as the benchmark, and then reduce the concentration by 0.01 μg/ml each time until the sweetness is no longer felt. In this way, the concentration gradient is gradually reduced to determine the sweetness threshold of the sample. Compared with the sugar solution, 20 ml of the sample is randomly tested. Before each analysis, the assessors are required to rinse their mouths with water and eat neutral-tasting crackers until there is no residual taste. Keep the test solution in the mouth for at least 10 seconds. Afterwards, the assessors score the samples according to their answers between 0 and 10; 0-no sweet feeling, 10-very sweet. The final result of each protein is the average of the tasting results of 10 people, and the results of the determination of the sweetness threshold of different proteins are shown in Table 5. According to the results, we can see that the deletion of the first amino acid glycine in the single-chain monellin MNEI can increase the sweetness by about 6.7 times, while the deletion of the first amino acid glycine in the mutant DM09 can increase the sweetness by about 7.6 times, and the sweetness of DM09-ΔG1 is about 20 times higher than that of MNEI. Deletion of the first amino acid glycine in mutant BZ01 can increase sweetness by about 7.2 times, and the sweetness of BZ01-ΔG1 is about 18.5 times higher than that of MNEI. Deletion of the first amino acid glycine in mutant BZ02 can increase sweetness by about 7.5 times, and the sweetness of BZ02-ΔG1 is about 22.7 times higher than that of MNEI. Deletion of the first amino acid glycine in mutant BZ03 can increase sweetness by about 7.3 times, and the sweetness of BZ03-ΔG1 is about 24.4 times higher than that of MNEI. Deletion of the first amino acid glycine in mutant BZ04 can increase sweetness by about 7.0 times, and the sweetness of BZ04-ΔG1 is about 22.7 times higher than that of MNEI. Deletion of the first amino acid glycine in mutant BZ05 can increase sweetness by about 6.8 times, and the sweetness of BZ05-ΔG1 is about 16.7 times higher than that of MNEI. The deletion of the first amino acid glycine in the mutant BZ06 can increase the sweetness by about 7.4 times, and the sweetness of BZ06-ΔG1 is about 21.7 times higher than that of MNEI. The deletion of the first amino acid glycine in the mutant BZ07 can increase the sweetness by about 7.4 times, and the sweetness of BZ07-ΔG1 is about 23.3 times higher than that of MNEI. The deletion of the first amino acid glycine in the mutant BZ08 can increase the sweetness by about 7.3 times, and the sweetness of BZ08-ΔG1 is about 22.2 times higher than that of MNEI. The deletion of the first amino acid glycine in the mutant BZ09 can increase the sweetness by about 6.6 times, and the sweetness of BZ09-ΔG1 is about 15.4 times higher than that of MNEI. These data prove the inventor's speculation that the removal of the first amino acid glycine of the single-chain monellin can fully expose the second glutamic acid, thereby further increasing the sweetness.

Table 5 Determination results of sweetness threshold of different mutant proteins

A patent reported that the method for determining the stability of sweet protein is to compare the protein samples before and after heating by protein electrophoresis, and judge the stability of the protein according to the gel map. In fact, this method is not very accurate, and even if the electrophoresis of the heated sample shows that the protein is not degraded, we do not know whether the sweet function of the protein is affected. In order to more accurately determine the stability of sweet protein, we use protein samples before and after heating to determine the sweetness threshold, and determine its stability according to the sweetness threshold deviation of the protein samples before and after heating. We divided the obtained single-chain monellin and its mutant protein into two groups, one group was not heated, and the other group was heated in a 65℃ water bath for 30min. The sweetness threshold of the two groups of samples was determined, and the results showed that the sweetness threshold deviation of the protein samples before and after heating was within 5%. One of the traditional methods of high-temperature pasteurization is to heat the sample to 62℃-65℃ and keep it for 30min. Our single-chain monellin mutant protein meets the corresponding requirements.

Example 4: Substituting sucrose in yogurt drinks

A taste test panel consisting of 10 people was set up to evaluate the sweetness of the single-chain monellin mutant BZ03-ΔG1 in the Jane Love zero sucrose plain yogurt beverage. Using a standard sample group of 0%, 2%, 4%, 6%, 8%, 10%, 12%, 14% and 16% sucrose and 23% Jane Love zero sucrose plain yogurt (the standard sample group refers to the addition of different percentages of sucrose (0-16%) to 23% Jane Love zero sucrose plain yogurt. 23% refers to the diluted plain yogurt, the ratio is: take 23g of plain yogurt and dilute it to 100ml with pure water), it was found that the sweetness of 0.35mg/l single-chain monellin mutant BZ03-ΔG1 in 23% Jane Love zero sucrose plain yogurt corresponds to an average of 7% sucrose ± 1% (SD) in 23% Jane Love zero sucrose plain yogurt. Therefore, under existing conditions, in 23% Jane Love zero sucrose plain yogurt, this single-chain monellin mutant batch is 200,000 times sweeter by weight than sucrose. In addition, the sweetness of single-chain monellin mutant and sucrose are additive, because in 23% Jane Love zero sucrose plain yogurt, 6% sucrose + 0.35mg/l single-chain monellin mutant BZ03-ΔG1 tastes like 12.5% ± 0.6% (SD) sucrose. The presence of 6% sucrose almost eliminates the "delayed perception of sweetness" from single-chain monellin mutant.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily being directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (24)

1. A mutant of a monellin protein comprising a deletion of amino acid 1 relative to a wild-type monellin protein.

2. The monellin protein mutant of claim 1, wherein said wild-type monellin protein is a wild-type single-chain monellin protein.

3. The monellin protein mutant as defined in claim 1 or 2, having a sweetness that is higher than that of the wild-type single-chain monellin protein, preferably at least 5-fold, at least 10-fold, at least 15-fold or at least 20-fold that of the wild-type single-chain monellin protein.

4. A monellin protein mutant as defined in any one of claims 1-3, which exhibits a sweetness reduction of no more than 5% after 30min at 65 ℃.

5. The monellin protein mutant of any one of claims 1-4, comprising one or more amino acid substitutions selected from the group consisting of: 2. 23, 41, 65 and 76.

6. The monellin protein mutant of any of claims 1-5, comprising an amino acid substitution E2N or E2A.

7. The monellin protein mutant of any of claims 1-6, comprising an amino acid substitution E23A.

8. The monellin protein mutant of any of claims 1-7, further comprising an amino acid substitution C41A, Y65R, S Y or any combination thereof.

9. The monellin protein mutant of any one of claims 1-8, comprising any one combination selected from the group consisting of amino acid substitution combinations of:

1)E2N/E23A/Y65R；

2)E2A/E23A/Y65R；

3)E2N/E23A/C41A；

4)E2N/E23A/C41A/Y65R；

5)E2N/E23A/C41A/S76Y；

6)E2N/E23A/C41A/Y65R/S76Y；

7)E2A/E23A/C41A；

8)E2A/E23A/C41A/Y65R；

9) E2A/E23A/C41A/S76Y; and

10)E2A/E23A/C41A/Y65R/S76Y。

10. the monellin protein mutant of any one of claims 1-9, wherein said wild-type single-chain monellin protein has the amino acid sequence of SEQ ID NO:2, and a polypeptide having the amino acid sequence shown in 2.

11. The monellin protein mutant of any of claims 1-10, comprising the amino acid sequence of SEQ ID NO:24-34 or an amino acid sequence as set forth in any one of SEQ ID NOs: 24-34, having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% sequence identity.

12. The monellin protein mutant of any one of claims 1-11 having the amino acid sequence of SEQ ID NO: 24-34.

13. Use of the monellin protein mutant of any one of claims 1-12 as a food additive, a beverage additive or a pharmaceutical additive.

14. An edible product comprising the monellin protein mutant of any one of claims 1-12.

15. The edible product of claim 14, which is a food, beverage or pharmaceutical.

16. The edible product of claim 14 or 15, further comprising a sweetener different from the monellin protein mutant.

17. The edible product of any one of claims 14-16, wherein the sweetener is sucrose.

18. The edible product as in any one of claims 14-17, wherein the beverage is a yoghurt beverage.

19. A nucleic acid molecule encoding the monellin protein mutant of any one of claims 1-12.

20. An expression vector comprising the nucleic acid molecule of claim 19.

21. The expression vector of claim 20, which is the expression vector pgapzαa or a linearization product thereof.

22. A host cell comprising the expression vector of claim 20 or 21.

23. The host cell of claim 22, which is pichia pastoris.

24. The host cell of claim 22 or 23, which is pichia pastoris X-33.