CN117088958A

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

Info

Publication number: CN117088958A
Application number: CN202310569096.2A
Authority: CN
Inventors: 张国秀; 卞芙蓉; 胡菲菲; 张玲艳; 李娟�; 白挨玺
Original assignee: Nanjing Bestzyme Bio Engineering Co ltd
Current assignee: Nanjing Bestzyme Bio Engineering Co ltd
Priority date: 2022-05-20
Filing date: 2023-05-19
Publication date: 2023-11-21
Also published as: WO2023222111A1

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

The present application claims priority from chinese patent application CN202210553207.6 filed 5/20 in 2022, which is incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates to mutant monellin proteins, and in particular, to mutants of monellin proteins that include an amino acid deletion at position 1 relative to wild-type single chain monellin protein (MNEI). The present disclosure also relates to methods of making the monellin protein mutants and uses thereof as sweeteners for edible products.

Background

Monellin (Monellin) is a sweet protein, and is a natural sweetener extracted from western non-plant Dioscoreophyllum cumminsii. Monellin has intense sweetness, and the sweetness is about 800 to 2000 times that of sucrose with the same mass under the same condition. The monellin as a high-sweetness and low-calorie non-sugar protein sweetener can be used for processing sugar-free foods, is used as a sweet additive for epidemic patients related to sugar intake such as obesity, diabetes and decayed tooth, can also be used for children foods, and has great market potential. The natural monellin protein is formed by combining two different peptide chains (namely an A chain and a B chain) together through covalent bonds, and researches show that the sweetness of the natural monellin protein can be completely lost after the temperature is more than or equal to 50 ℃. Kim et al genetically engineered two peptide chains into one protein chain, creating a single chain monellin protein in which the two native chains are linked by Gly-Phe dipeptide linker, resulting in improved thermostability without much change in sweetness (Kim et al Redesigning a sweet protein: increased stability and renaturation ability protein Eng.1989 Aug;2 (8): 571-5).

Single-chain monellin proteins are also successfully expressed in other organisms, such as E.coli expression systems, plant expression systems, B.subtilis expression systems, and yeast expression systems, using genetic engineering means. However, the production of monellin is not amenable to large scale fermentative production due to the problems of these expression systems and toxic metabolites. One strategy to reduce the cost of monellin production is to reduce the sweetness threshold of the protein as much as possible, i.e. to increase the sweetness, so that a good sweetness effect is achieved with very little addition.

Because of the great market potential of monellin protein, scholars and research institutions at home and abroad are researching mutants of single-chain monellin protein with high sweetness. Chinese patent CN105566471a discloses a single-chain monellin protein mutant, in which the glutamic acid at amino acid position 2 of the single-chain monellin protein is site-directed mutated to asparagine. Chinese patent CN112313244A of AMAI company discloses taste and flavor modified single chain Monilin protein mutants, especially DM09 mutants (E2N/E23A/Y65R). The mutation of amino acid 2 of single chain Monilin protein was studied intensively, and the result shows that amino acid 2 mediates the sweetness of Monilin (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 outer Purif.2018Mar; 143:52-56).

Disclosure of Invention

In one aspect, provided herein are monellin protein mutants that include a deletion of amino acid 1 relative to 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 at least 5-fold, at least 10-fold, at least 15-fold, or at least 20-fold greater than the sweetness of the wild-type single-chain monellin protein, preferably.

In some embodiments, the monellin protein mutant does not decrease in sweetness by more than 5% after 30min at 65 ℃.

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 an amino acid substitution E23A.

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

In some embodiments, the monellin protein mutant comprises any one combination selected from the group consisting 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。

in some embodiments, the 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.

In some embodiments, the monellin protein mutant comprises 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.

In some embodiments, the monellin protein mutant has the amino acid sequence of SEQ ID NO: 24-34.

In another aspect, provided herein is the use of the above-described monellin protein mutant as a food additive, a beverage additive, or a pharmaceutical additive.

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

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

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

In some embodiments, the sweetener is sucrose.

In some embodiments, the beverage is a yoghurt 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 linearization product thereof.

In another aspect, provided herein are host cells, the expression vectors 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 wild-type single chain monellin MNEI and are useful as sweeteners for edible products.

Drawings

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

Detailed Description

Unless otherwise defined, 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 that is suitable for sweetening foods, beverages, and/or medical products for human consumption. The sweet protein of the invention may have a sweetness of at least 1000 times, preferably at least 2000 times, more preferably at least 5000 times, and even more preferably 10000 times, by weight, as compared to a 1% sucrose solution. For the purposes of the present invention, the comparison of sweetness can be determined by artificial perception (human tolerance) of the sweetness of the protein against known control tests using proteins at different concentrations diluted in water or in aqueous solutions avoiding surface adsorption, such as 20% skim milk.

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

"sweetness threshold" herein refers to the minimum concentration of sweetener at which a sample, such as a food or beverage containing sweetener (e.g., pure water), is identified by a taster as having sweetness.

"Monilin protein" herein refers to any protein comprising the A and B chains of a Monilin protein, including double-stranded proteins in which the A and B chains are joined together by a covalent bond, and single-stranded proteins in which the A and B chains are joined together.

"Single chain monellin" herein refers to a single chain protein formed by linking together the A and B chains of a native monellin protein via a short peptide linker molecule. In single chain monellin proteins, the N-terminus to the C-terminus is typically the B-chain-linker molecule-the A-chain.

"wild-type" is not limited herein to naturally occurring proteins, and may refer to a single chain protein formed by joining together the A and B chains of a monellin protein, including double-stranded monellin proteins (i.e., naturally occurring monellin proteins), as well as to a single chain protein formed by joining together the A and B chains of a naturally occurring monellin protein, with the amino acid sequences of the A and B chains of a naturally occurring monellin protein.

"wild-type single chain monellin protein (MNEI)" herein refers to a single chain monellin protein reported by Kim et al as formed by a Gly-Phe dipeptide linker linkage having the amino acid sequence of SEQ ID NO:2, and a polypeptide having the amino acid sequence shown in 2. The term "wild-type" as used herein does not refer to the single chain monellin protein as a reference for determining the mutation type (e.g., amino acid deletion or substitution) and mutation position of the mutation included in the monellin protein mutants provided herein, or it may be considered as a parent protein of the monellin protein mutants provided herein, rather than being naturally occurring in nature. In addition, in some cases, wild-type single-chain monellin protein is also used as a reference for measuring sweetness of the monellin protein mutants provided herein.

"Monilin protein mutant" herein refers to a protein that differs in amino acid sequence relative to a wild-type single-chain Monilin protein (i.e., the parent protein). Such differences may be manifested by 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 single-chain form.

"amino acid substitution", which may also be referred to as "amino acid substitution", refers herein to the substitution of one amino acid (referred to as the original amino acid) at a particular position in an amino acid sequence with another amino acid (substituted amino acid). For example, amino acid substitution at position 2 of the parent protein is glutamic acid residue (E) and alanine residue (a) at the corresponding position of the mutant protein can be considered to be present in the mutant protein: alanine replaces glutamic acid. For amino acid substitutions, the following nomenclature is used herein: original amino acids, positions and substituted amino acids, and the IUPAC specified single letter abbreviations for amino acids are used for amino acid names. For the example described above, it may be denoted as E2A. "amino acid substitution combination" herein refers to a mutant protein in which the positions of amino acid substitutions are two or more. For example, the amino acid at position 2 of the parent protein is glutamic acid residue (E), and the corresponding position of the mutant protein is asparagine residue (N); meanwhile, the 23 rd amino acid of the parent protein is glutamic acid residue (E), and the corresponding position of the mutant protein is alanine residue (A), so that the mutant protein can be considered to have amino acid substitution combination: E2N and E23A. When multiple amino acid substitutions are present simultaneously in a mutant, the multiple mutations are separated by "/", e.g. "E2N/E23A/Y65R" represents substitution of glutamic acid with asparagine, glutamic acid with alanine and tyrosine with arginine at positions 2, 23 and 65, respectively.

"amino acid deletion", which may also be referred to as "amino acid deletion", refers herein to the absence of an amino acid at a position in the mutant protein corresponding to the amino acid at that position in the parent protein as compared to the parent protein. This position may appear as a notch when aligned with the parent protein. Amino acid deletions may be represented by "Δ", e.g., when glycine (G) is the 1 st amino acid in the mutant protein, it may be counted as Δg1 (or g1Δ). Thus, ΔG1/E2N/E23A/Y65R can be used to indicate the simultaneous presence of a glycine deletion at position 1, substitution of glutamic acid at position 2 with asparagine, substitution of glutamic acid at position 23 with alanine, and substitution of tyrosine at position 65 with arginine in a mutant protein.

"amino acid insertion", which may also be referred to as "amino acid addition", refers herein to the fact that an amino acid at a position in a mutant protein has no amino acid in the parent protein to which it corresponds as compared to the parent protein. In other words, amino acid insertion refers to one or more amino acids more than when aligned relative to the parent protein.

"coding sequence" herein refers to a polynucleotide sequence that directly determines the amino acid sequence of its protein product. The boundaries of the coding sequence are typically determined by open reading frames that typically begin with ATG start codons or alternative start codons such as GTG and TTG and end with stop codons such as TAA, TAG and TGA. The coding sequence may be a DNA, cDNA, RNA, synthetic or recombinant nucleotide sequence.

"expression" herein may include any step involving the production of a mutant of a monellin protein 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 mutant of a monellin protein as provided herein, operably linked to additional nucleotides providing for its expression.

"host cell" refers herein to a cell that can be used to produce the monellin protein mutants provided herein. The host cell may generally be suitable for introducing an exogenous nucleic acid molecule (e.g., an expression vector) and has a range of enzymes (including enzymes associated with transcription, post-transcriptional processing, translation, and post-translational modification) that can be used to express the exogenous protein. Useful host cells include prokaryotic and eukaryotic cells, such as E.coli, yeast, mammalian cells, and the like. In a preferred embodiment, pichia pastoris (e.g., pichia X-33) is used as a host cell for expression of the monellin protein mutants provided herein. Host cells also include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA) to the original parent cell, due to natural, accidental, or deliberate mutation. The host cell may be an isolated cell or cell line.

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

When referring to amino acid sequences, the term "sequence identity (sequence identity)" (also referred to as "sequence identity") refers to the amount of degree of identity between two amino acid sequences (e.g., a query sequence and a reference sequence), typically expressed as a percentage. Typically, sequence alignment (alignment) is performed and gaps (gaps), if any, introduced prior to calculation of the percent identity between two amino acid sequences. If at a certain alignment the amino acid residues or bases in the two sequences are identical, then the two sequences are considered to be identical or matched at that position; amino acid residues or bases in the two sequences differ, and 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 identity. In other algorithms, the number of gaps and/or the gap length are also considered. Common sequence alignment algorithms or software include EMBOSS, DANMAN, CLUSTALW, MAFFT, BLAST, MUSCLE, etc. For the purposes of the present invention, amino acid sequence alignment is performed using the CLUSTALW algorithm in some embodiments. The preset parameters of the CLUSTALW algorithm may be: deletion counts are residues that are not identical compared to the reference sequence, including deletions occurring at either end. For example, a variant 500 amino acid residue polypeptide lacking five amino acid residues at the C-terminus has a percentage of sequence identity 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", "left and right" or the symbol "-" means a value that may deviate from the mentioned value by at most 1%, at most 5%, at most 10%, at most 15%, and in some cases at most 20%. The deviation range includes integer values and, if applicable, non-integer values, constituting a continuous range.

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

The terms "comprising," "including," "having," "including," and similar referents used herein do not exclude the presence of unrecited elements. These terms also include cases consisting of only the recited elements.

In view of the sweetness of single-chain monellin protein mediated by amino acid 2, the present inventors speculated from the protein structure simulation that amino acid 1 of single-chain monellin protein may form steric hindrance to amino acid 2 to affect sweetness, and removal of amino acid 1 may fully expose amino acid 2, possibly further increasing sweetness. The inventor of the present application has found through experimental verification that the single-chain monellin mutant deleted the first amino acid has indeed a significant improvement in sweetness. The inventor succeeds in achieving a few achievements different from the prior art in the engineering research of single-chain monellin protein.

It is an object of the present invention to provide a single-chain monellin protein mutant having high sweetness aiming at reducing the production cost of single-chain monellin protein. The invention carries out site-directed mutagenesis on single-chain monellin protein to obtain mutant with high sweetness and heat stability, and successfully expresses the mutant in pichia pastoris. By increasing the sweetness of the protein, the sweet effect can be achieved under the condition of extremely small addition, so that the production cost of the monellin protein is effectively reduced, and a solid foundation is laid for large-scale industrial production of the sweet protein. The PGAPZ alpha A plasmid can directly secrete target protein outside cells, so that the subsequent protein purification is facilitated, the purification cost is reduced, and the self-induction promoter is adopted, so that the addition of inducers such as methanol and the like is not needed in the fermentation process, the risk of fermentation and bacterial contamination is reduced, toxic metabolic substances are not produced, and the safety of the protein serving as a food additive is ensured.

In a first aspect of the invention there is provided a single chain monellin protein mutant, said protein comprising a sequence identical to SEQ ID NO:24-34 has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical. The single-chain monellin protein mutant with high sweetness provided by the invention has the following technical scheme:

1) A single-chain monellin mutant is prepared by deleting glycine (Gly) at position 1 of wild single-chain monellin protein (MNEI) at fixed point.

2) A single-chain monellin mutant deletes the glycine (Gly) site of the 1 st amino acid of monellin DM09 (E2N/E23A/Y65R).

3) A single-chain monellin protein mutant deletes the glycine (Gly) site of the 1 st amino acid of monellin protein BZ01 (E2A/E23A/Y65R).

4) A single-chain monellin protein mutant deletes the glycine (Gly) site of the 1 st amino acid of monellin protein BZ02 (E2N/E23A/C41A).

5) A single-chain monellin protein mutant deletes glycine (Gly) at position 1 of monellin protein BZ03 (E2N/E23A/C41A/Y65R) in a fixed point mode.

6) A single-chain monellin protein mutant deletes glycine (Gly) at position 1 of monellin protein BZ04 (E2N/E23A/C41A/S76Y) in a fixed point mode.

7) A single-chain monellin protein mutant deletes glycine (Gly) at position 1 of monellin protein BZ05 (E2N/E23A/C41A/Y65R/S76Y) at fixed point.

8) A single-chain monellin protein mutant deletes the glycine (Gly) site of the 1 st amino acid of monellin protein BZ06 (E2A/E23A/C41A).

9) A single-chain monellin protein mutant deletes glycine (Gly) at position 1 of monellin protein BZ07 (E2A/E23A/C41A/Y65R) in a fixed point mode.

10 Single-chain monellin mutant, deletion of glycine (Gly) at amino acid 1 of monellin BZ08 (E2A/E23A/C41A/S76Y) is performed.

11 Single-chain monellin mutant, deletion of glycine (Gly) at amino acid 1 st site of monellin BZ09 (E2A/E23A/C41A/Y65R/S76Y).

Wherein the sweetness of the mutant MNEI-delta G1 in scheme 1) is improved by 6.7 times relative to that of the wild single-chain monellin protein (MNEI). Scheme 2) the mutant DM09- Δg1 has a sweetness that is enhanced by a factor of 7.6 relative to the monellin protein DM 09. Scheme 3) the sweetness of the mutant BZ 01-delta G1 is improved by 7.2 times relative to that of the monellin protein BZ 01. Scheme 4) the sweetness of the mutant BZ 02-delta G1 is improved by 7.5 times relative to that of the monellin protein BZ 02. Scheme 5) the sweetness of the mutant BZ 03-delta G1 is improved by 7.3 times relative to that of the monellin protein BZ 03. Scheme 6) the sweetness of the mutant BZ 04-delta G1 is improved by 7.0 times relative to that of the monellin protein BZ 04. Scheme 7) the sweetness of the mutant BZ 05-delta G1 is improved by 6.8 times relative to that of the monellin protein BZ 05. Scheme 8) the sweetness of the mutant BZ 06-delta G1 is improved by 7.4 times relative to that of the monellin protein BZ 06. Scheme 9) the sweetness of the mutant BZ07- ΔG1 is improved by 7.4 times relative to that of the monellin protein BZ 07. Scheme 10) the sweetness of the mutant BZ 08-delta G1 is improved by 7.3 times relative to that of the monellin protein BZ 08. Scheme 11) the sweetness of the mutant BZ09- ΔG1 is improved by 6.6 times relative to that of the monellin protein BZ 09. The sweetness of the mutant in the schemes 1) to 11) is improved by about 15.5-24.4 times compared with that of single-chain monellin protein (MNEI).

Based on these mutants provided herein, one of skill in the art can further introduce other amino acid deletions, substitutions or insertions and examine the sweetness of the resulting mutant product to obtain other mutants that still have sweetness. Such mutants are also included within the scope of the present invention.

In some embodiments, these other mutants may be obtained by amino acid substitutions. It is contemplated that the monellin protein mutants provided herein may further comprise other conservative amino acid substitutions. Conservative amino acid substitutions can generally be described as the substitution of one amino acid residue for another amino acid residue of similar chemical structure, with little or 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 be, for example, the substitution of one amino acid in the following groups (a) - (e) with another amino acid within the same group: (a) small aliphatic nonpolar or low polar residues: ala, ser, thr, pro and Gly; (b) Polar negatively charged residues and (uncharged) amides: asp, asn, glu and Gln; (c) a polar positively charged residue: his, arg and Lys; (d) large aliphatic nonpolar residues: met, leu, ile, val and Cys; and (e) an aromatic residue: phe, tyr and Trp.

In some embodiments, the linker molecules used may also be replaced. Useful linker molecules may include 1 or more amino acid residues, for example 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 renaturation ability protein Eng 1989Aug;2 (8): 571-5) describe a method of ligating two peptide chains of a native monellin protein, and one skilled in the art could use similar methods to attempt ligation using other linker molecules and evaluate the properties, such as sweetness, of the single chain monellin protein after ligation, thereby obtaining other mutants differing only in linker molecules from the present invention. In addition, chinese patent application CN109627307a discloses that single chain monellin proteins with improved heat resistance and substantially unchanged sweetness are obtained by using hairpin structural protein domains to join the a and B chains of monellin proteins. The inventors contemplate that substitution of the linker molecules of the monellin protein mutants provided herein as described above may also result in other monellin protein mutants having increased sweetness relative to the wild-type single chain monellin protein mutants, and such other monellin protein mutants are also included within the scope of the present invention. In addition, the inventors also expected that, on the basis of the wild-type double-stranded monellin protein, deletion of glycine, the first amino acid in the B chain, the obtained mutant may also have increased sweetness.

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

1) Constructing wild single-chain monellin MNEI and mutant DM09, BZ01-BZ09 expression vectors;

2) Designing primers for deleting the glycine of the first amino acid of the wild single-chain monellin MNEI and mutants DM09 and BZ01-BZ09, carrying out site-directed mutagenesis on the MNEI and mutant sequences in the step 1), obtaining a gene fragment for deleting the first amino acid, constructing a vector, and selecting a positive transformant for sequencing verification;

3) Transforming the plasmid with correct sequence in the step 2) into pichia pastoris, and screening successfully transformed pichia pastoris;

4) Inducing expression of the protein in YPD medium for 3 days;

5) Purifying the protein expressed in step 4).

Step 1) constructing an expression plasmid containing single-chain monellin MNEI and mutant DM09 and BZ01-BZ09 coding genes as PGAPZ alpha A.

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'; BZ 01-. DELTA.G1-F (SEQ ID NO: 18): 5'-CCGCTCGAGGCTTGGGAGATCATTGACATCG-3'; BZ 02-. DELTA.G1-F (SEQ ID NO: 19): 5'-CCGCTCGAGAACTGGGAGATCATTGACATCG-3'.

In the steps 1) and 2), the PCR product is digested by Xhol and NotI endonucleases and connected with the digested PGAPZalpha A empty vector, the PCR product is transformed into E.coli DH5 alpha competent cells, the E.coli DH5 alpha competent cells are coated on LB solid medium containing 25 mug/ml Zeocin, positive single colony is selected and extracted by overnight culture, the plasmid is extracted, the result is verified by sequencing, and the correct expression plasmid is constructed and stored for standby.

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

The transformant in step 4) was inoculated into a shake flask containing 50ml of YPD medium and cultured at 30℃for 3 days. Adding trichloroacetic acid into the supernatant of the fermentation liquor, mixing, centrifuging the mixed liquor at 4 ℃ overnight, washing and precipitating with acetone, and detecting the target protein expression result by SDS-PAGE. The positive transformant with the highest expression level is selected and inoculated into a shake flask containing 200ml of YPD culture medium, and induced culture is carried out for 3 days at 30 ℃.

The purification method in step 5): the fermentation supernatant was collected and placed in a dialysis bag having a molecular weight cut-off of 3500 and dialyzed at 4℃for 24 hours, and the dialysis buffer was 10mM sodium phosphate buffer (pH 7.0). The dialyzed sample was applied to an ion exchange chromatography column Sephadex CM-50 and the target protein was eluted linearly with 10mM sodium phosphate buffer (pH 7.0) containing 0-0.4M sodium chloride. The collected target protein is put into a dialysis bag with 3500 molecular weight cutoff for dialysis at 4 ℃ for 24 hours, and the dialysis is repeated for 3 times. Purified proteins were subjected to SDS-PAGE and the concentration of the protein of interest was determined using Coomassie Brilliant blue.

The high-sweetness single-chain monellin protein mutant and the preparation method thereof have the beneficial effects that: site-directed mutagenesis is carried out on the single-chain monellin protein to obtain a mutant with high sweetness and heat stability. The production cost of the monellin protein is effectively reduced by increasing the sweetness of the protein, and a solid foundation is laid for the large-scale industrialized production of the protein. The method of directly secreting the target protein out of the cell is adopted, so that the purification cost of the protein is effectively reduced, meanwhile, the self-induction type promoter is adopted, the addition of inducers such as methanol and the like is not required in the fermentation process, the risk of fermentation and bacterial contamination is reduced, toxic metabolic substances are not produced, and the safety of the protein serving as a food additive is ensured.

Also described herein are methods of increasing the sweetness of an edible product for human consumption, such as a food, beverage, or pharmaceutical product, comprising the steps of: sufficient amounts of the monellin protein mutants provided herein are added to the aforementioned food, beverage or pharmaceutical products such that they have increased sweetness.

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

In some embodiments, the monellin protein mutants provided herein may be used in combination with other sweeteners. In one example, the monellin protein mutants provided herein and sucrose may be added to a yogurt beverage. The presence of sucrose can improve the "sweet taste delayed feel" of the monellin protein mutant.

Additional aspects and advantages of the invention will be set forth in, or will be apparent from, the description of the embodiments that follows. These examples are not intended to limit the scope of the invention in any way. Specifically described are: the reagents mentioned herein are commercially available unless otherwise specified.

Test materials and reagents

1. Strains and vectors: expression hosts Pichia pastoris X-33 (Invitrogen), expression plasmid vector PGAPZαA (Invitrogen) was maintained for this laboratory.

2. Enzymes and other biochemical reagents: endonucleases were purchased from Fermentas, ligase from Promega, and DNA polymerase from Beijing holomorpha. The others are all domestic analytically pure reagents (all available from the common biochemistry reagent company).

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 wild-type single-chain monellin protein MNEI and its mutant expression vector

The wild-type single-chain monellin protein MNEI and its mutant 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 Jinsri biotechnology Co., ltd, and two restriction endonuclease sites of XhoI and NotI were introduced at both ends, respectively. The target gene fragment and PGAPZ alpha A empty plasmid are subjected to enzyme digestion, are connected with T4 DNA ligase at 37 ℃ overnight, are transformed into DH5 alpha escherichia coli competent cells, and are coated on LB solid medium containing 25 mug/ml Zeocin. Positive transformants were selected overnight, and plasmid sequencing was performed using a plasmid extraction kit to verify that wild single-chain monellin protein expression vectors PGAPZa-MNEI and mutant expression vectors PGAPZa-DM 09, PGAPZa-BZ 01, PGAPZa-BZ 02, PGAPZa-BZ 03, PGAPZa-BZ 04 and PGAPZa-BZ 05 were successfully constructed. The coding sequences and amino acid sequences of wild single-chain monellin protein MNEI and mutant genes DM09, BZ01, BZ02, BZ03, BZ04 and BZ05 thereof are shown in Table 1.

TABLE 1 wild type single chain monellin MNEI and its mutant gene coding sequence and amino acid sequence

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Primers were designed for single-stranded Monilin mutants BZ06 (E2A/E23A/C41A), BZ07 (E2A/E23A/C41A/Y65R), BZ08 (E2A/E23A/C41A/S76Y) and BZ09 (E2A/E23A/C41A/Y65R/S76Y), and were designated BZ06-F and BZ06-R due to the identical primer sequences, the primer sequences are shown in Table 2. The PCR amplification was performed 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, using primer pairs BZ06-F/BZ06-R, and the PCR reaction system was as shown in Table 3. After the PCR is finished, the gene fragments of single-chain monellin mutants BZ06, BZ07, BZ08 and BZ09 are obtained by gel recovery through 1% agarose gel electrophoresis detection. The PCR products were treated with XhoI and NotI restriction enzymes for 6 hours and ligated overnight with gel recovered open-loop plasmid vectors using T4DNA ligase at 37℃and the cleavage and ligation systems are shown in Table 3. The ligation product was transformed into DH 5. Alpha. E.coli competent cells and plated on LB solid medium containing 25. Mu.g/ml Zeocin. Positive transformants were picked up by overnight culture, and plasmid sequencing verification was performed by using a plasmid extraction kit to successfully construct single-chain monellin mutant vectors PGAPZa-BZ 06, PGAPZa-BZ 07, PGAPZa-BZ 08, and PGAPZa-BZ 09. The amino acid sequences of single-chain monellin mutants BZ06, BZ07, BZ08 and BZ09 are shown in Table 4.

TABLE 2 primer sequences used herein

TABLE 3 reaction system

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TABLE 4 Single chain Monilin mutant amino acid sequence

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Primers for deleting the glycine at the first amino acid position of the wild single-chain monellin protein MNEI and mutants DM09, BZ01, BZ02, BZ03, BZ04, BZ05, BZ06, BZ07, BZ08 and BZ09 thereof are designed, and are named MNEI-delta G1-F, BZ 01-delta G1-F and BZ 02-delta G1-F respectively because partial mutant primers have the same sequence, wherein the primers are shown in Table 2. And (3) taking the MNEI gene fragment as a template, and carrying out PCR amplification by using a primer pair MNEI-delta G1-F/BZ06-R to obtain the MNEI-delta G1 gene fragment. BZ01, BZ06, BZ07, BZ08 and BZ09 gene fragments are respectively used as templates, the primer pair BZ 01-delta G1-F/BZ06-R is used for PCR amplification, 1% agarose gel electrophoresis detection is carried out after the PCR is finished, and BZ 01-delta G1, BZ 06-delta G1, BZ 07-delta G1, BZ 08-delta G1 and BZ 09-delta G1 gene fragments are obtained by using a gel recovery kit. The DM 09-DeltaG 1, BZ 02-DeltaG 1, BZ 03-DeltaG 1, BZ 04-DeltaG 1 and BZ 05-DeltaG 1 gene fragments are obtained by using the gel recovery kit and the PCR reaction system is shown in Table 3. The PCR products were treated with XhoI and NotI restriction enzymes for 6 hours and ligated overnight with gel recovered open-loop plasmid vectors using T4DNA ligase at 37℃and the cleavage and ligation systems are shown in Table 3. The ligation product was transformed into DH 5. Alpha. E.coli competent cells and plated on LB solid medium containing 25. Mu.g/ml Zeocin. Positive transformants were selected overnight and plasmid sequencing was performed using a plasmid extraction kit to verify that single-stranded monellin mutant vectors PGAPZαA-MNEI- ΔG1, PGAPZαA-DM09- ΔG1, PGAPZαA-BZ01- ΔG1, PGAPZαA-BZ02- ΔG1, PGAPZαA-BZ03- ΔG1, PGAPZαA-BZ04- ΔG1, PGAPZαA-BZ05- ΔG1, PGAPZαA-BZ06- ΔG1, PGAPZαA-BZ07- ΔG1, PGAPZαA-BZ08- ΔG1, and PGAPZαA-BZ09- ΔG1 were successfully constructed, and single-stranded monellin mutants MNEI- ΔG1, DM09- ΔG1, BZ01- ΔG1, BZ02- ΔG1, BZ03- ΔG1, BZ04- ΔG1, BZ05- ΔG1, BZ 1, BZ06- ΔG1 and BZ 1 were shown in the tables of FIGS.

EXAMPLE 2 preparation of wild type single chain Monolin and mutant proteins thereof

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

The wild single-stranded monellin constructed in example 1 and its mutant expression vector were linearized with the restriction enzyme avril, and the linearized DNA was concentrated by ethanol precipitation DNA method and detected by agarose gel electrophoresis.

Pichia pastoris X-33 competent cell preparation: 1) Inoculating Pichia X-33 single colony into 5ml YPD culture medium, and culturing at 30deg.C and 250rpm/min for 24 hr; 2) Inoculating 1ml of culture solution into 500ml shake flask containing 100ml YPD culture medium, culturing for 12-16 hr until OD600 = 0.8-1.2, transferring the bacterial solution into 50ml precooled centrifuge tube, centrifuging at 4deg.C and 5000g for 5min, and discarding supernatant; 3) Re-suspending the thallus with 25ml pre-cooled sterile water, centrifuging at 4deg.C and 5000g for 5min, discarding supernatant, and repeating the steps for 2-3 times; 4) Re-suspending the thallus with 5ml pre-cooled 1mol/L sorbitol for each tube, centrifuging at 4deg.C and 5000g for 5min, discarding supernatant, and repeating this step for 2-3 times; 5) Finally, 200. Mu.L sorbitol was used for resuspension, and the suspension was dispensed into 1.5ml centrifuge tubes, each tube having 80. Mu.L.

Transformation screening: 1) Taking one competent cell, adding 10 mu L of linearized and concentrated DNA, gently mixing, and transferring into a precooled electrode cup with the specification of 0.2cm for 5min in an ice bath; 2) Electric shock is carried out under the conditions that the electric shock parameter is 1.5Kv,25 mu F and 200Ω, 1ml of precooled 1mol/L sorbitol is rapidly added after the electric shock is finished, and then the mixed solution is transferred into a 1.5ml centrifuge tube, and is resuscitated for 1h at 30 ℃; 3) 200. Mu.L of the mixture was plated on YPDS solid medium containing 100. Mu.g/ml Zeocin, the plate was incubated at 30℃for 3 days, positive transformants were observed, positive single colonies were identified by PCR, and PCR positive colonies were confirmed by sequencing.

(2) Expression verification and purification of target proteins

Positive transformants, which were confirmed to be correct by PCR and sequencing, were inoculated into 250ml shake flasks containing 50ml YPD medium and cultured at 30℃for 3 days at 250 rpm/min. The fermentation broth was centrifuged at 12000rpm at 4℃to obtain 900. Mu.L of supernatant, 100. Mu.L of trichloroacetic acid was added to the supernatant and mixed, the mixture was centrifuged at 10000rpm/min at 4℃overnight, the supernatant was discarded, the precipitate was washed 2-3 times with acetone, and the result of expression of the target protein was detected by SDS-PAGE.

Positive transformants with a bright band of the target protein were selected and inoculated into 1L shake flasks containing 200ml YPD medium and induced to culture at 30℃and 250rpm for 3 days. The fermentation supernatant was collected by low temperature centrifugation and dialyzed in a dialysis bag having a molecular weight cut-off of 3500 at 4℃for 24 hours with a dialysis buffer of 10mM sodium phosphate buffer (pH 7.0). The dialyzed sample was filtered through a 0.22 μm filter and applied to an ion exchange chromatography column Sephadex CM-50, the column was equilibrated with 10mM sodium phosphate buffer (pH 7.0), and the target protein was eluted linearly with 10mM sodium phosphate buffer (pH 7.0) containing 0-0.4M sodium chloride. The collected target protein was placed in a dialysis bag having a molecular weight cut-off of 3500 and dialyzed at 4℃for 24 hours, and the dialysis buffer was 10mM sodium phosphate buffer (pH 5.0) with 3 exchanges. The purified proteins were subjected to SDS-PAGE and the results are shown in FIG. 1. The concentration of the protein of interest was determined using coomassie brilliant blue and the sweetening activity of the protein was initially determined by tasting the dialyzed solution.

Example 3 determination of sweet taste threshold of wild type single chain monellin and its mutant proteins

The sweetening potency of wild-type single-chain monellin MNEI described herein relative to sucrose is typically 1:700 to 1: in the range of 16,000. Thus, for comparison, the MNEI and its mutant protein solutions were first diluted to 1:1000, followed by further dilution 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 mutants thereof, sucrose and mineral water. The protein was diluted to a gradient from 0.01 to 1.5. Mu.g/ml by diluting the protein stock with mineral water (pH 6.9). 10 healthy evaluators participated in the evaluation, 5 men and 5 women, respectively, aged between 30 and 50 years, of which 5 were trained wine tasters. Starting from the lowest concentration, tasting, increasing the concentration by 0.1 mug/ml each time, when sweetness is sensed, and then reducing the concentration by 0.01 mug/ml each time based on the concentration until sweetness is not sensed, and gradually reducing the concentration gradient to determine the sweetness threshold of the sample. Samples of 20ml were tested randomly compared to the sugar solution. Before each analysis, the evaluator was asked to rinse with water and eat a neutral tasting cracker until no residual taste was present. The test solution is held in the mouth for at least 10 seconds. Thereafter, the evaluator scored the sample according to their answer between 0 and 10; 0-no sweet sensation, 10-very sweet. The final results for each protein were averaged over 10 persons and the results for the measurement of the sweetness threshold for the different proteins are shown in table 5. According to the results, we can see that deletion of the first amino acid glycine of single-chain monellin MNEI can increase sweetness by about 6.7 times, while deletion of the first amino acid glycine of mutant DM09 can increase sweetness by about 7.6 times, and the sweetness of DM 09-delta G1 is improved by about 20 times compared with MNEI. Deletion of the first amino acid glycine of the mutant BZ01 can increase the sweetness by about 7.2 times, and the sweetness of BZ 01-delta G1 is improved by about 18.5 times compared with MNEI. Deletion of the first amino acid glycine in mutant BZ02 can increase the sweetness by about 7.5 times, and the sweetness of BZ 02-delta G1 is improved by about 22.7 times compared with MNEI. Deletion of the first amino acid glycine in mutant BZ03 can increase the sweetness by about 7.3 times, and the sweetness of BZ 03-delta G1 is improved by about 24.4 times compared with MNEI. Deletion of glycine, the first amino acid, of mutant BZ04 can increase sweetness by about 7.0 times, and the sweetness of BZ 04-delta G1 is improved by about 22.7 times compared with MNEI. Deletion of the first amino acid glycine of the mutant BZ05 can increase the sweetness by about 6.8 times, and the sweetness of the BZ 05-delta G1 is improved by about 16.7 times compared with that of MNEI. Deletion of the first amino acid glycine of the mutant BZ06 can increase the sweetness by about 7.4 times, and the sweetness of the BZ 06-delta G1 is improved by about 21.7 times compared with that of MNEI. Deletion of the glycine which is the first amino acid in the mutant BZ07 can increase the sweetness by about 7.4 times, and the sweetness of the BZ 07-delta G1 is improved by about 23.3 times compared with that of MNEI. Deletion of the glycine which is the first amino acid in the mutant BZ08 can increase the sweetness by about 7.3 times, and the sweetness of the BZ 08-delta G1 is improved by about 22.2 times compared with that of MNEI. Deletion of the glycine which is the first amino acid in the mutant BZ09 can increase the sweetness by about 6.6 times, and the sweetness of the BZ 09-delta G1 is improved by about 15.4 times compared with that of MNEI. These data demonstrate the inventors' hypothesis that removal of glycine, an amino acid at position 1 of single chain monellin, may allow for sufficient exposure of glutamic acid at position 2 to further increase sweetness.

TABLE 5 determination of sweetness threshold for different mutant proteins

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The patent reports that the method for measuring the stability of sweet protein is to compare protein samples before and after heating by protein electrophoresis, and judge the stability of the protein according to a gel diagram. In fact, this method is less accurate and it is not known whether the sweet taste function of the protein is affected even though the heated sample shows no degradation of the protein by electrophoresis. In order to accurately judge the stability of sweet protein, the sweet taste threshold value of protein samples before and after heating is measured, and the stability of the sweet taste threshold value is determined according to the sweet taste threshold deviation of the protein samples before and after heating. We split the single chain monellin and its mutant proteins obtained into two groups, one group without heating and one group with water bath heating at 65 ℃ for 30min. The sweetness threshold was measured for both samples, 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 pasteurization is to heat the sample to 62-65 ℃ for 30min, and our single-chain monellin mutant proteins meet the corresponding requirements.

Example 4 substitution of sucrose in yogurt beverages

A taste test group of 10 persons was established to evaluate the sweetness of the single-chain monellin mutant in a curtailed zero sucrose original taste yoghurt drink. The standard groups of 0%, 2%, 4%, 6%, 8%, 10%, 12%, 14% and 16% sucrose and 23% reduced zero sucrose glycogen taste yoghurt (standard group means that different percentages of sucrose (0-16%) are added to 23% reduced zero sucrose glycogen taste yoghurt.) 23% means diluted original yoghurt, the proportion is that 23G original yoghurt is diluted to 100ml by adding pure water, and the sweetness of 0.35mg/l single-chain monellin mutant BZ 03-delta G1 in 23% reduced zero sucrose glycogen taste yoghurt is found to correspond to an average of 7% sucrose + -1% (SD) in 23% reduced zero sucrose glycogen taste yoghurt. Thus, in 23% cursory zero sucrose flavored yogurt under existing conditions, the single chain monellin mutant batch had 20 ten thousand times the sweetness by weight of sucrose. Furthermore, the sweetness of the single-stranded monellin mutant and sucrose was additive, as in 23% reduced zero sucrose glycogen flavored yoghurt, 6% sucrose+0.35 mg/l single-stranded monellin mutant BZ03- Δg1 had a taste similar to 12.5% ± 0.6% (SD) sucrose. The presence of 6% sucrose almost removed the "sweet taste delayed feel" from the single stranded monellin mutant.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms should not be understood as necessarily being directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Further, one skilled in the art can engage and combine the different embodiments or examples described in this specification.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

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.