WO2023222111A1 - Sweet protein monellin mutant having high sweetness and method for preparing same - Google Patents

Abstract

Provided is a monellin protein mutant, comprising a deletion at amino acid position 1. The monellin protein mutant has an increased sweetness relative to the wild-type single-stranded monellin protein MNEI, and can be used as a sweetener for edible products.

Description

Sweet protein Monellin mutant with high sweetness and preparation method thereof

Cross-references to related applications

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

Technical field

This article relates to monellin protein mutants, particularly monellin protein mutants including a deletion of the first amino acid relative to wild-type single-chain monellin protein (MNEI). This article also relates to methods for the preparation of monellin protein mutants and their use as sweeteners for edible products.

Background technique

Monellin is a sweet protein and a natural sweetener extracted from the West African plant Dioscoreophyllum cumminsii. Monelin has a strong sweet taste, with the sweetness ranging from about 800 to 2000 times that of the same mass of sucrose under the same conditions. As a high-intensity and low-calorie non-carbohydrate protein sweetener, monellin can be used in the processing of sugar-free foods and as a sweetening additive for patients with epidemics related to sugar intake such as obesity, diabetes, and dental caries. , can also be used in children's food and has 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 natural monellin protein will completely lose its sweetness when the temperature is ≥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. The two natural chains were connected through a Gly-Phe dipeptide linker to improve its thermal stability. , while its sweetness has not changed much (Kim et al., Redesigning a sweet protein: increased stability and renaturability. Protein Eng. 1989Aug; 2(8):571-5).

Single-chain monellin protein has also been successfully expressed in other organisms using genetic engineering methods, such as E. coli expression system, plant expression system, Bacillus subtilis expression system and yeast expression system. However, due to problems with the yield and toxic metabolites of these expression systems, monellin protein cannot be produced by large-scale fermentation. One strategy to reduce the production cost of monellin protein is to lower 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 an extremely small amount of addition.

Due to the huge market potential of monellin protein, domestic and foreign scholars and research institutions are conducting research on mutants of highly sweet single-chain monellin protein. Chinese patent CN105566471A discloses a single-chain monellin protein mutant, in which glutamic acid, the second amino acid of the single-chain monellin protein, is site-directedly mutated into asparagine. AMAI Company's Chinese patent CN112313244A discloses single-chain monellin protein mutants 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 single-chain monellin protein, and the results showed that the second amino acid mediates 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).

Contents of the invention

In one aspect, provided herein are monellin protein mutants that include a deletion of the amino acid at position 1 relative to the wild-type monellin protein.

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

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

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

In some embodiments, the monellin protein mutants include one or more amino acid substitutions selected from the following positions: 2, 23, 41, 65, and 76.

In some embodiments, the monellin protein mutants include the amino acid substitutions E2N or E2A.

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

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

In some embodiments, the monellin protein mutant includes any combination selected from the following amino acid substitution 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 set forth in SEQ ID NO: 2.

In some embodiments, the monellin protein mutant includes the amino acid sequence shown in any one of SEQ ID NO: 24-34 or is at least 70%, An amino acid sequence with 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.

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

In another aspect, this article provides the use of the above-mentioned monellin protein mutants as food additives, beverage additives or pharmaceutical additives.

In another aspect, provided herein are edible products that include the above-described monellin protein mutants.

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

In some embodiments, the edible product further includes a sweetener that is different from the monellin protein mutant.

In some embodiments, the sweetener is sucrose.

In some embodiments, the beverage is a yogurt beverage.

In another aspect, provided herein are nucleic acid molecules encoding the above-described monellin protein mutants.

In another aspect, provided herein are expression vectors comprising the nucleic acid molecules.

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

In another aspect, provided herein are host cells and expression vectors as described above.

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.

Description of the drawings

Figure 1 shows the SDS-PAGE detection chart 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, and BZ05 respectively. , BZ05-ΔG1, BZ06, BZ06-ΔG1, BZ07, BZ07-ΔG1, BZ08, BZ08-ΔG1, BZ09, BZ09-ΔG1 proteins.

Detailed ways

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

"Sweet protein" as used herein refers to a protein having a sweet taste suitable for sweetening food, beverages and/or pharmaceutical products for human consumption. By weight, the sweetness of the sweet protein of the present invention can 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 this invention, comparison of sweetness can be determined by testing the human perception of sweetness of the protein against a known control using an aqueous solution diluted in water, or to avoid surface adsorption, such as 20% skim milk. were carried out with different concentrations of protein.

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

"Sweetness threshold" as used herein refers to the minimum sweetener concentration at which a taster would identify a sample, such as a food or beverage containing a sweetener, such as pure water, as having a sweet taste.

"Monellin protein" as used herein refers to any protein that contains the A and B chains of a monellin protein, including double-chain proteins in which the A and B chains are covalently bonded together, and the A and B chains are Single-chain proteins linked together.

"Single-chain monellin protein" as used herein refers to a single-chain protein formed by connecting the A chain and B chain of native monellin protein together through a short peptide linker molecule. In single-chain monellin proteins from N-terminus to C-terminus is usually B chain-linker molecule-A chain.

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

"Wild-type single-chain monellin protein (MNEI)" in this article refers to the single-chain monellin protein reported by Kim et al. to be connected through a Gly-Phe dipeptide linker, which has the amino acid sequence shown in SEQ ID NO: 2. The modified word "wild type" used here does not mean that it is naturally occurring in nature, but refers to the single-chain monellin protein used herein as a mutation that determines the mutations included in the monellin protein mutants provided herein. A reference to the type of mutation (eg, amino acid deletion or substitution) and position of the mutation, or may be considered to be the parent protein of the monellin protein mutants provided herein. Additionally, in some cases, wild-type single-chain monellin protein is also used as a reference for measuring the sweetness of the monellin protein mutants provided herein.

"Monellin protein mutant" as used herein refers to a protein that differs in amino acid sequence relative to a wild-type single-chain monellin protein (i.e., the parent protein). Such differences may be manifested by the substitution, deletion or insertion of one or more amino acids 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 substitution", in this article refers to the replacement of an amino acid (called the original amino acid) at a specific position in the amino acid sequence by another amino acid (replacement amino acid). For example, if 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), it can be considered that there is an amino acid substitution in the mutant protein. :Alanine replaces glutamic acid. For amino acid substitutions, the following nomenclature is used in this article: original amino acid, position, and substituted amino acid, and the one-letter amino acid abbreviation specified by IUPAC is used for the amino acid name. For the example described above, this can be represented as E2A. "Amino acid substitution combination" herein refers to the presence of amino acid substitutions at two or more positions in the mutant protein. For example, the 2nd 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. If the amino acid residue (E) is an alanine residue (A) in the mutant protein, it can be considered that there is an amino acid substitution combination: E2N and E23A in the mutant protein. When there are multiple amino acid substitutions in a mutant at the same time, the multiple mutations are separated by "/", for example, "E2N/E23A/Y65R" represents glutamic acid at positions 2, 23 and 65 respectively replaced by asparagine. substitutions, glutamate by alanine and tyrosine by arginine.

"Amino acid deletion", also known as "amino acid deletion", here refers to the lack of an amino acid corresponding to the amino acid at that position of the parent protein at a certain position in the mutant protein compared with the parent protein. When compared to the parent protein, This location can appear as a gap. Amino acid deletion can be expressed by "Δ". For example, when the first amino acid glycine (G) is deleted in the mutant protein, it can be counted as ΔG1 (or G1Δ). Therefore, ΔG1/E2N/E23A/Y65R can be used to represent the simultaneous deletion of glycine at position 1, substitution of glutamate with asparagine at position 2, substitution of glutamate with alanine at position 23, and tyrosine at position 65 in the mutant protein. Replaced by arginine.

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

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

"Expression" as used 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.

"Expression vector" as used herein refers to a linear or circular DNA molecule comprising a polynucleotide encoding a protein, such as a monellin protein mutant provided herein, together with additional nucleosides provided for its expression. The acid is operably connected.

"Host cell" as used herein refers to a cell that can be used to produce the monellin protein mutants provided herein. Host cells may generally be suitable for the introduction of exogenous nucleic acid molecules (eg, expression vectors) and have a range of enzymes (including enzymes related to transcription, post-transcriptional processing, translation, and post-translational modification) that can be used to express exogenous proteins. Useful host cells include prokaryotic and eukaryotic cells, such as E. coli, yeast, mammalian cells, etc. In a preferred embodiment, Pichia pastoris (eg, Pichia pastoris X-33) is used as a host cell to express the monellin protein mutants provided herein. Host cells also include the progeny of a single host cell, and progeny may not necessarily be identical (in terms of morphology or genomic DNA) to the original parent cell 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 used interchangeably. When representing the amino acid sequence of a protein, conventional one-letter or three-letter symbols for amino acid residues are used, with the amino acid sequence presented in the standard amino to carboxyl terminal orientation (ie, N→C).

When referring to an amino acid sequence, the term "sequence identity" (also called "sequence identity") refers to the quantity, typically expressed as a percentage, of the degree of identity between two amino acid sequences (such as a query sequence and a reference sequence). express. Usually, before calculating the percent identity between two amino acid sequences, the sequences are aligned and gaps (if any) are introduced. If at a certain alignment position, the amino acid residues or bases in the two sequences are the same, the two sequences are considered to be consistent or matching at that position; if the amino acid residues or bases in the two sequences are different, the two sequences are considered to be inconsistent or matching at that position. mismatch. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence identity. In other algorithms, the number of gaps and/or the gap length is also taken into account. Commonly used sequence comparison algorithms or software include EMBOSS, DANMAN, CLUSTALW, MAFFT, BLAST, MUSCLE, etc. Out of instinct For clarity purposes, in some embodiments the CLUSTALW algorithm is used to perform amino acid sequence alignment. The preset parameters for the CLUSTALW algorithm can be: Deletions are counted as residues that are not identical to the reference sequence, including deletions occurring at any terminus. For example, a variant 500 amino acid residue polypeptide that deletes five amino acid residues from the C-terminus has a percent sequence identity of 99% (495/500 identical residues x 100) relative to the parent polypeptide. Such variants are covered by the language "variants having at least 99% sequence identity with the parent".

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

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

As used herein, the terms "comprises," "contains," "having," "includes," and similar expressions mean that non-recited elements are not excluded. These terms also include instances that consist solely of the recited elements.

In view of the fact that the second amino acid of the single-chain monellin protein mediates the sweetness of monellin, the inventor of the present application speculated based on protein structure simulation that the first amino acid of the single-chain monellin protein may form steric hindrance to the second amino acid. This affects the sweetness. Removing the first amino acid can fully expose the second amino acid, which may further increase the sweetness. The inventor of the present application found through experimental verification that the sweetness of the single-chain monellin mutant in which the first amino acid was deleted did significantly improve. The inventor of the present application has successfully achieved some results that are different from the prior art in the research on the transformation of single-chain monellin protein.

One of the objects 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 mutation on the single-chain monellin protein to obtain a mutant with high sweetness and thermal stability, which is successfully expressed in Pichia pastoris. By increasing the sweetness of the protein, a good sweetness effect can be achieved with an extremely small amount of addition, thereby effectively reducing the production cost of monellin protein and laying a solid foundation for the large-scale industrial production of this sweet protein. . The use of PGAPZαA plasmid can directly secrete the target protein outside the cell, which facilitates subsequent protein purification and reduces purification costs. Due to the use of an auto-inducible promoter, there is no need to add inducers such as methanol during the fermentation process, which reduces the risk of fermentation contamination. At the same time, toxic metabolites will not be produced, ensuring the safety of the protein as a food additive.

In a first aspect of the invention, a single chain monellin protein mutant is provided, said protein comprising at least 70%, at least 75%, or at least 80% of any one of the amino acids of SEQ ID NO: 24-34. , an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical. The single-chain monellin protein mutant with high sweetness provided by the invention and its technical solution are as follows:

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

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

3) A single-chain monellin protein mutant in which the amino acid glycine (Gly) at position 1 of monellin protein BZ01 (E2A/E23A/Y65R) is site-specifically deleted.

4) A single-chain monellin protein mutant in which the amino acid glycine (Gly) at position 1 of monellin protein BZ02 (E2N/E23A/C41A) is site-specifically deleted.

5) A single-chain monellin protein mutant in which the amino acid glycine (Gly) at position 1 of monellin protein BZ03 (E2N/E23A/C41A/Y65R) is site-specifically deleted.

6) A single-chain monellin protein mutant in which the amino acid glycine (Gly) at position 1 of monellin protein BZ04 (E2N/E23A/C41A/S76Y) is site-specifically deleted.

7) A single-chain monellin protein mutant in which the amino acid glycine (Gly) at position 1 of monellin protein BZ05 (E2N/E23A/C41A/Y65R/S76Y) is site-specifically deleted.

8) A single-chain monellin protein mutant in which the amino acid glycine (Gly) at position 1 of monellin protein BZ06 (E2A/E23A/C41A) is site-specifically deleted.

9) A single-chain monellin protein mutant in which the amino acid glycine (Gly) at position 1 of monellin protein BZ07 (E2A/E23A/C41A/Y65R) is site-specifically deleted.

10) A single-chain monellin protein mutant in which the amino acid glycine (Gly) at position 1 of monellin protein BZ08 (E2A/E23A/C41A/S76Y) is site-specifically deleted.

11) A single-chain monellin protein mutant in which the amino acid glycine (Gly) at position 1 of monellin protein BZ09 (E2A/E23A/C41A/Y65R/S76Y) is site-specifically deleted.

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

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

In some embodiments, these other mutants can be obtained by amino acid substitutions. It is contemplated that the monellin protein mutants provided herein may further include other conservative amino acid substitutions. Conservative amino acid substitutions can generally be described as the replacement of one amino acid residue by another amino acid residue of similar chemical structure and that has an adverse effect on the function, activity, or function of the polypeptide. Other biological properties have little or no effect. Conservative amino acid substitutions are well known in the art. Conservative substitutions may, for example, be the substitution of one amino acid in the following groups (a)-(e) 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 linker molecules used may also be substituted. Useful linker molecules may 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 to connect the two peptide chains of the natural monellin protein. This paper Those skilled in the art can use similar methods to try to use other linker molecules for connection, and evaluate the properties 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 hairpin structural protein domains to connect the A chain and B chain of monellin protein, thereby obtaining a single-chain monellin protein with improved heat resistance and basically unchanged sweetness. The inventors anticipate that by replacing the linker molecules of the monellin protein mutants provided herein as described above, other monellin protein mutations with increased sweetness relative to wild-type single-chain monellin protein mutants can also be obtained mutants, these other monellin protein mutants are also intended to be included within the scope of the present invention. In addition, the inventor also expects that, based on the wild-type double-chain monellin protein, deleting the first amino acid glycine of its B chain, the mutant obtained 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, which includes the following steps:

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

2) Design primers for wild-type single-chain monellin MNEI and mutants DM09 and BZ01-BZ09 to delete the first amino acid glycine, and conduct site-directed mutagenesis of the MNEI and mutant sequences in step 1) to obtain genes with the first amino acid deleted. The fragments are used for vector construction, and positive transformants are selected for sequencing and verification;

3) Transform the correctly sequenced plasmid in step 2) into Pichia pastoris, and screen 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) Construct an expression plasmid containing single-chain monellin protein MNEI and mutant DM09 and BZ01-BZ09 encoding genes as 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), digest the PCR product with Xhol and NotI endonucleases and connect it with the digested PGAPZαA empty vector, transform it into E. coli DH5α competent cells, and spread it on LB solid containing 25 μg/ml Zeocin medium After culturing overnight, select positive single colonies and extract the plasmid, verify the results by sequencing, and construct the correct expression plasmid for later use.

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

In Step 4), the transformants were inoculated into shake flasks containing 50 ml YPD medium and cultured at 30°C for 3 days. Add trichloroacetic acid to the supernatant of the fermentation broth and mix, keep at 4°C overnight, centrifuge the mixture, wash with acetone, and detect the expression of the target protein using SDS-PAGE. Select the positive transformant with the highest expression level and inoculate it into a shake flask containing 200 ml YPD medium, and induce and culture it 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 at 4°C for 24 hours. The dialysis buffer is 10mM sodium phosphate buffer (pH7.0). The dialyzed sample was loaded onto the ion exchange chromatography column Sephedex CM-50, 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°C for 24 hours, and the dialysis was repeated three times. The purified protein was detected by SDS-PAGE and the concentration of the target protein was determined using the Coomassie Brilliant Blue method.

The beneficial effects of the high-sweetness single-chain monellin protein mutant and its preparation method of the present invention are: site-directed mutation is performed on the single-chain monellin protein to obtain a mutant with high sweetness and thermal stability. By increasing the sweetness of the protein, the production cost of monellin protein is effectively reduced, laying a solid foundation for large-scale industrial production of the protein. The method of directly secreting the target protein out of the cell effectively reduces the cost of protein purification. At the same time, it uses an auto-inducible promoter to ensure that there is no need to add inducers such as methanol during the fermentation process, while reducing the risk of fermentation contamination. No toxic metabolites will be produced, ensuring the safety of the protein as a food additive.

This article also relates to a method of increasing the sweetness of an edible product for human consumption, such as a food, beverage or pharmaceutical product, comprising the steps of: adding a sufficient amount of the monellin protein mutant provided herein to the above food, beverage or pharmaceutical product products, giving them an increased sweetness.

This article also relates to edible products for human consumption, such as foods, beverages, or pharmaceutical products, containing 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 improves the "delayed sweetness sensation" of monellin protein mutants.

Additional aspects and advantages of the invention will be set forth in the Examples section below, or will become apparent from the following description of the Examples. These examples do not limit the scope of the invention in any way. Special note: The reagents mentioned in this article are all commercially available unless otherwise specified.

Test materials and reagents

1. Strain and vector: The expression host Pichia pastoris X-33 (Invitrogen), and the expression plasmid vector PGAPZαA (Invitrogen) are preserved in this laboratory.

2. Enzymes and other biochemical reagents: Endonuclease was purchased from Fermentas Company, ligase was purchased from Promaga Company, and DNA polymerase was purchased from Beijing Quanjin Biotechnology. Others are domestic analytically pure reagents (all can be purchased 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

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 restrictions, XhoI and NotI, were introduced at both ends. Endonuclease site. After enzymatic 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 E. coli competent cells, and spread on LB solid medium containing 25 μg/ml Zeocin. Positive transformants were picked after overnight culture, and plasmids were extracted and verified using a plasmid extraction kit, and the wild-type single-chain monellin protein expression vector PGAPZαA-MNEI and its mutant expression vectors PGAPZαA-DM09, PGAPZαA-BZ01, and PGAPZαA-BZ02 were successfully constructed. , PGAPZαA-BZ03, PGAPZαA-BZ04 and PGAPZαA-BZ05. The coding and amino acid sequences of 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

Design of 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) ), because the primer sequences are the same, they are named BZ06-F and BZ06-R. The primer sequences are shown in Table 2. Using BZ02 (E2N/E23A/C41A), BZ03 (E2N/E23A/C41A/Y65R), BZ04 (E2N/E23A/C41A/S76Y) and BZ05 (E2N/E23A/C41A/Y65R/S76Y) gene fragments as templates, respectively, Use primers to perform PCR amplification of BZ06-F/BZ06-R. The PCR reaction system is shown in Table 3. After PCR, 1% agarose gel electrophoresis was used for detection, and the gene fragments of single-chain monellin mutants BZ06, BZ07, BZ08 and BZ09 were recovered from the gel. The PCR product was treated with XhoI and NotI restriction enzymes for 6 hours and then ligated with the open-circular plasmid vector recovered from the gel using T4 DNA ligase at 37°C overnight. The enzyme digestion and ligation system are shown in Table 3. The ligation product was transformed into DH5α E. coli competent cells and spread on LB solid medium containing 25 μg/ml Zeocin. Positive transformants were picked after overnight culture, and plasmids were extracted using a plasmid extraction kit and verified by sequencing. Single-chain monellin mutant vectors PGAPZαA-BZ06, PGAPZαA-BZ07, PGAPZαA-BZ08, and PGAPZαA-BZ09 were successfully constructed. 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 Amino acid sequence of single chain monellin mutants

Design primers for the wild-type single-chain monellin protein MNEI and its mutants DM09, BZ01, BZ02, BZ03, BZ04, BZ05, BZ06, BZ07, BZ08 and BZ09 to delete the first amino acid glycine. Since the primer sequences of some mutants are 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 of MNEI-ΔG1-F/BZ06-R was performed using primers to obtain the MNEI-ΔG1 gene fragment. Using BZ01, BZ06, BZ07, BZ08 and BZ09 gene fragments as templates, PCR amplification of BZ01-ΔG1-F/BZ06-R was performed using primers. After PCR was completed, 1% agarose gel electrophoresis was used for detection and gel recovery reagent was used. The cassette obtains 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 were used to perform PCR amplification of BZ02-ΔG1-F/BZ06-R. After PCR, 1% agarose gel electrophoresis was used for detection, and gel recovery reagents were used. The cassette obtained DM09-ΔG1, BZ02-ΔG1, BZ03-ΔG1, BZ04-ΔG1 and BZ05-ΔG1 gene fragments. The PCR reaction system is shown in Table 3. The PCR product was treated with XhoI and NotI restriction enzymes for 6 hours and then ligated with the open-circular plasmid vector recovered from the gel using T4 DNA ligase at 37°C overnight. The enzyme digestion and ligation system are shown in Table 3. The ligation product was transformed into DH5α E. coli competent cells and spread on LB solid medium containing 25 μg/ml Zeocin. Positive transformants were picked after overnight culture, and plasmids were extracted and verified using a plasmid extraction kit, and single-chain monellin mutant vectors PGAPZαA-MNEI-ΔG1, PGAPZαA-DM09-ΔG1, PGAPZαA-BZ01-ΔG1, and PGAPZαA-BZ02- were successfully constructed. ΔG1, PGAPZαA-BZ03-ΔG1, PGAPZαA-BZ04-ΔG1, PGAPZαA-BZ05-ΔG1, PGAPZαA-BZ06-ΔG1, PGAPZαA-BZ07-ΔG1, PGAPZαA-BZ08-ΔG1, and PGAPZαA-BZ09-ΔG1, single-chain monellin mutations The amino acid sequences of 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 expression vector into Pichia pastoris X-33

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

Preparation of competent cells of Pichia pastoris Culture the culture medium in a 500ml shake flask 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 5 minutes, discard the supernatant; 3) Use 25ml pre-cooled centrifuge tube Resuspend the bacterial cells in cold sterile water, centrifuge at 4°C and 5000g for 5 minutes, discard the supernatant, and repeat this step 2-3 times; 4) Resuspend the bacterial cells with 5 ml of pre-cooled 1mol/L sorbitol in each tube. Centrifuge at 4°C and 5000g for 5 minutes, discard the supernatant, and repeat this step 2-3 times; 5) Finally resuspend in 200 μL sorbitol and dispense into 1.5 ml centrifuge tubes, 80 μL 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 0.2cm electrode cup for ice bath for 5 minutes; 2) When the electroporation parameter is 1.5 Kv, 25μF, 200Ω for electric shock. After the electric shock is completed, quickly add 1ml of pre-cooled 1mol/L sorbitol, then transfer the mixture into a 1.5ml centrifuge tube, and recover at 30°C for 1 hour; 3) Draw 200μL and apply it to the solution containing On the YPDS solid medium with 100 μg/ml Zeocin, the plate was cultured at 30°C for 3 days. Positive transformants were observed and positive single colonies were identified by PCR. The PCR-positive colonies were sent for sequencing verification.

(2) Expression verification and purification of target protein

Inoculate the correct positive transformants verified by PCR and sequencing into a 250ml shake flask containing 50ml YPD medium, and culture at 30°C and 250rpm/min for 3 days. Centrifuge the fermentation broth at 4°C and 12,000 rpm. Add 900 μL of the supernatant to 100 μL of trichloroacetic acid and mix. Keep it overnight at 4°C. Centrifuge the mixture at 10,000 rpm/min. Discard the supernatant and wash the precipitate 2-3 times with acetone. Use SDS-PAGE to detect Target protein expression results.

Select the positive transformants with brighter target protein bands and inoculate them into 1L shake flasks containing 200 ml YPD medium, and induce and culture them at 30°C and 250 rpm for 3 days. Collect the fermentation supernatant by low-temperature centrifugation and put it into a dialysis bag with a molecular weight cutoff of 3500 for dialysis at 4°C for 24 hours. The dialysis buffer is 10mM sodium phosphate buffer (pH7.0). The dialyzed sample was filtered through a 0.22μm filter and loaded onto an ion exchange chromatography column Sephedex CM-50. The column was equilibrated with 10mM sodium phosphate buffer (pH7.0) and 10mM phosphoric acid containing 0-0.4M sodium chloride. Sodium buffer (pH7.0) linearly elutes the target protein. The collected target protein was put into a dialysis bag with a molecular weight cutoff of 3500 and dialyzed at 4°C for 24 hours. The dialysis buffer was 10mM sodium phosphate buffer (pH 5.0), and the medium was changed three times during this period. The purified protein was subjected to SDS-PAGE detection, and the results are shown in Figure 1. The concentration of the target protein was determined using the Coomassie Brilliant Blue method and the sweetening activity of the protein was preliminarily determined by tasting the dialyzed solution.

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

The sweetening potency of wild-type single-chain monellin MNEI described herein generally ranges from 1:700 to 1:16,000 relative to sucrose. Therefore, for comparison, MNEI and its mutant protein solutions were first diluted to 1:1000 and then further diluted as needed. For most people, the sweetness threshold for sugar in soft drinks is 0.32%-1.0%. To evaluate the sweetness threshold of each solution, a double-blind taste analysis was performed. Samples tested included MNEI and its mutants, sucrose and mineral water. By diluting the protein stock solution with mineral water (pH 6.9), the protein was diluted into a gradient from 0.01 to 1.5 μg/ml. 10 healthy evaluators participated in this evaluation, 5 men and 5 women, aged between 30 and 50 years old, 5 of whom were trained sommeliers. Start tasting from the lowest concentration, and increase the concentration by 0.1μg/ml each time until you feel the sweetness. Use this concentration as the benchmark. Then reduce the concentration by 0.01μg/ml each time until you can no longer feel the sweetness. Follow this method. Gradually reduce the concentration gradient to determine the sweetness threshold of the sample. Compared to the sugar solution, 20 ml samples were randomly tested. Before each analysis, assessors were asked to rinse their mouths with water and eat neutral-flavored crackers until no residual taste remained. Keep the test solution in the mouth for at least 10 seconds. The evaluators then rated the samples based on their responses between 0 and 10; 0 - no sweet taste, 10 - very sweet. The final result of each protein is the average of the tasting results of 10 people. The measurement results of the sweetness thresholds of different proteins are shown in Table 5. According to the results, we can see that deleting the first amino acid glycine in single-chain monellin MNEI can increase the sweetness by about 6.7 times, while the mutant DM09 deleting the first amino acid glycine can increase the sweetness by about 7.6 times. DM09-ΔG1 The sweetness is about 20 times higher than MNEI. The deletion of the first amino acid glycine in the mutant BZ01 can increase the sweetness by about 7.2 times, and the sweetness of BZ01-ΔG1 is about 18.5 times higher than that of MNEI. The deletion of the first amino acid glycine in the mutant BZ02 can increase the sweetness by about 7.5 times, and the sweetness of BZ02-ΔG1 is about 22.7 times higher than that of MNEI. The deletion of the first amino acid glycine in mutant BZ03 can increase the sweetness by about 7.3 times, and the sweetness of BZ03-ΔG1 is about 24.4 times higher than that of MNEI. The deletion of the first amino acid glycine in the mutant BZ04 can increase the sweetness by about 7.0 times, and the sweetness of BZ04-ΔG1 is about 22.7 times higher than that of MNEI. The deletion of the first amino acid glycine in the mutant BZ05 can increase the 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 removing the glycine at position 1 of single-chain monellin can fully expose glutamic acid at position 2, thereby further increasing sweetness.

Table 5 Determination results of sweet taste threshold of different mutant proteins

There is a patent report that the method for measuring 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 based on the gel pattern. In fact, this method is not very accurate, and even if the heated sample shows that the protein is not degraded by electrophoresis, we do not know whether the sweetening function of the protein is affected. In order to more accurately judge the stability of sweet proteins, we use protein samples before and after heating to measure the sweetness threshold, and determine their stability based on the sweetness threshold deviation of the protein samples before and after heating. We divided the obtained single-chain monellin and its mutant proteins into two groups, one group was not heated, and the other group was heated in a water bath at 65°C for 30 minutes. The sweetness threshold of the two groups of samples was measured, and the results showed that the deviation of the sweetness threshold of the protein samples before and after heating was within 5%. One of the traditional high-temperature pasteurization methods is to heat the sample to 62°C-65°C and keep it for 30 minutes. Our single-chain monellin mutant protein meets the corresponding requirements.

Example 4 Substituting sucrose in yogurt drinks

A taste test group of 10 people was established to evaluate the sweetness of the single-chain monellin mutant in the Jane Eyre zero sucrose original yogurt drink. Standard groups using 0%, 2%, 4%, 6%, 8%, 10%, 12%, 14% and 16% sucrose and 23% Jane Eyre zero sucrose original yogurt (the standard group refers to the 23% Different percentages of sucrose (0-16%) are added to the Jane Eyre zero sucrose original yogurt. 23% refers to the diluted original yogurt, the ratio is: take 23g of original yogurt and add pure water to dilute to 100ml), and found that 23% Jane Eyre The sweetness of the 0.35 mg/l single chain monellin mutant BZ03-ΔG1 in zero sucrose plain yogurt corresponds to an average of 7% sucrose ± 1% (SD) in 23% Jane Eyre zero sucrose plain yogurt. Therefore, under current conditions in 23% Jane Eyre Zero Sucrose Original Yogurt, this single chain monellin mutant batch is 200,000 times sweeter by weight than sucrose. In addition, the sweetness of single-chain monellin mutants and sucrose is additive, because in 23% Jane Eyre Zero Sucrose Original Yogurt, 6% sucrose + 0.35 mg/l single-chain The monellin mutant BZ03-ΔG1 tastes like 12.5% ± 0.6% (SD) sucrose. The presence of 6% sucrose nearly removed the "delayed sweetness sensation" from single-chain monellin mutants.

In the description of this specification, reference to the terms "one embodiment," "some embodiments," "an example," "specific examples," or "some examples" or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the invention. In this specification, schematic expressions of the above terms should not be understood as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may join and combine the different embodiments or examples described in this specification.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are illustrative and should not be construed as limitations of the present invention. Those of ordinary skill in the art can make modifications to the above-mentioned embodiments within the scope of the present invention. The embodiments are subject to changes, modifications, substitutions and variations.

Claims (24)

1. A monellin protein mutant includes a deletion of the first amino acid relative to the wild-type monellin protein.
2. The monellin protein mutant according to claim 1, wherein the wild-type monellin protein is a wild-type single-chain monellin protein.
3. The monellin protein mutant according to claim 1 or 2 has a sweetness higher than that of the wild-type single-chain monellin protein, preferably a sweetness equal to that of the wild-type single-chain monellin protein. At least 5 times, at least 10 times, at least 15 times, or at least 20 times.
4. The monellin protein mutant according to any one of claims 1 to 3, whose sweetness decreases by no more than 5% after being maintained at 65°C for 30 minutes.
5. The monellin protein mutant according to any one of claims 1 to 4, which includes one or more amino acid substitutions selected from the following positions: 2, 23, 41, 65 and 76.
6. The monellin protein mutant according to any one of claims 1 to 5, which includes amino acid substitution E2N or E2A.
7. The monellin protein mutant according to any one of claims 1 to 6, which includes amino acid substitution E23A.
8. The monellin protein mutant according to any one of claims 1 to 7, further comprising amino acid substitutions C41A, Y65R, S76Y or any combination thereof.
9. The monellin protein mutant according to any one of claims 1 to 8, which includes any combination selected from the following amino acid substitution 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.
10. The monellin protein mutant according to any one of claims 1-9, wherein the wild-type single-chain monellin protein has the amino acid sequence shown in SEQ ID NO: 2.
11. The monellin protein mutant according to any one of claims 1-10, which includes the amino acid sequence shown in any one of SEQ ID NO: 24-34 or is identical to the amino acid sequence shown in any one of SEQ ID NO: 24-34 The sequence has 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 to the amino acid sequence.
12. The monellin protein mutant according to any one of claims 1-11, which has the amino acid sequence shown in any one of SEQ ID NO: 24-34.
13. Use of the monellin protein mutant according to any one of claims 1 to 12 as a food additive, beverage additive or pharmaceutical additive.
14. Edible products include the monellin protein mutant according to any one of claims 1-12.
15. The edible product of claim 14, which is food, beverage or medicine.
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 of any one of claims 14-17, wherein the beverage is a yogurt beverage.
19. Nucleic acid molecule encoding the monellin protein mutant according to any one of claims 1-12.
20. An expression vector comprising the nucleic acid molecule of claim 19.
21. The expression vector according to claim 20, which is the expression vector PGAPZαA or its linearized product.
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 according to claim 22 or 23, which is Pichia pastoris X-33.