Monellin and its recombinant gene

Abstract

The application discloses a monellin protein and a recombinant gene thereof. The monellin protein after the improvement has a significant improvement in sweetness and stability, and the sweetness is more than 1.5 times of the initial sweetness, and the sweetness intensity is obviously enhanced. Meanwhile, most of the food requiring the addition of a sweetener needs to be subjected to pasteurization, and after being heated at 72 DEG C for 15 seconds, the sweetness of the initial monellin protein is obviously lost, which will affect the quality of the food. The improved monellin protein has excellent tolerance, and the sweetness is basically unchanged after being subjected to the same heating and sterilization, the feature not only ensures the flavor quality of the terminal product, but also provides a reliable guarantee for the standardization of product control in industrial production, and effectively expands the application boundary of the monellin protein as a sweetener in food.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more specifically to a monetin protein and its recombinant gene. Background Technology

[0002] Monellin is a plant from the tropical rainforests of West Africa. Dioscoreophyllum cumminsii The naturally occurring high-intensity sweet protein found in fruit was first isolated and identified in 1969. Its sweetness is approximately 3000 times that of sucrose, and it possesses excellent properties such as pure sweetness, extremely low calories, and non-participation in glucose metabolism, showing broad application prospects in food development for diabetic and obese individuals. However, the poor thermal stability of natural monetarin protein severely restricts its commercial application: in its natural state, this protein exists as a heterodimer, with two polypeptide chains linked by non-covalent bonds. It is prone to depolymerization at temperatures exceeding 50°C, leading to loss of sweetness activity. Under conventional food processing heat treatment conditions (such as pasteurization at 72°C / 15 seconds), monetarin protein undergoes irreversible structural damage and sweetness loss, failing to meet the processing requirements of liquid dairy products, fruit juice beverages, and other products requiring heat sterilization. Therefore, improving the thermal stability of monetarin protein and developing functional sweet proteins that can withstand the heat processing conditions of the food industry has become a key technical problem urgently needing to be solved in this field. Summary of the Invention

[0003] To address the shortcomings of existing technologies, one of the objectives of this invention is to provide a mutant of monetarin protein with increased thermal stability, which can be mass-produced using a bio-fermentation method.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a monetarin protein, the initial sequence of which is shown in SEQ ID NO.1. This initial monetarin protein sequence is derived from the protein sequence library GenBank: AFF58925.1. Using the Lésign platform, the initial monetarin protein sequence was designed, ultimately yielding a computationally optimal enzyme variant.

[0005] A monetarin protein, wherein the monetarin protein is mutated using the initial monetarin protein described in SEQ ID NO:1 as the parent material, and the following mutation set is used: E55K+N15E+Q29K+I39V, to obtain the monetarin protein mutant shown in SEQ ID NO:2.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a monetarin protein, wherein the monetarin protein is based on the initial monetarin protein described in SEQ ID NO:1, and is mutated using the following mutation set: E55K+E24H+R32K+R40K, to obtain the monetarin protein mutant shown in SEQ ID NO:3.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a monetarin protein, wherein the monetarin protein is based on the initial monetarin protein described in SEQ ID NO:1, and is mutated using the following mutation set: E55K+M43I+F53E+Q62F, to obtain the monetarin protein mutant shown in SEQ ID NO:4.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A monetarin protein, wherein the monetarin protein is based on the initial monetarin protein described in SEQ ID NO:1, and is mutated using the following mutation set: E55K+E49K+Y66E+R73E, to obtain the monetarin protein mutant shown in SEQ ID NO:5.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a monetarin protein, wherein the monetarin protein is based on the initial monetarin protein described in SEQ ID NO:1, and is mutated using the following mutation set: E55K+D75V+S77F, to obtain the monetarin protein mutant shown in SEQ ID NO:6.

[0010] To achieve the above objectives, the present invention provides the following technical solution: a monetarin protein, wherein the monetarin protein is based on the initial monetarin protein described in SEQ ID NO:1, and is mutated using the following mutation set: E55K+I27K+I56V+R83K, to obtain the monetarin protein mutant shown in SEQ ID NO:7.

[0011] The second objective of this invention is to provide a DNA or RNA capable of expressing the above-mentioned Monelin protein mutant.

[0012] To achieve the above objectives, the present invention provides the following technical solution: a recombinant genetic material of monetarin protein, capable of expressing the DNA or RNA of monetarin protein as described in any one of the above claims.

[0013] The third objective of this invention is to provide a strain capable of producing the aforementioned monetarin protein.

[0014] To achieve the above objectives, the present invention provides the following technical solution: a strain for producing monetarin protein, containing the above-mentioned recombinant genetic material.

[0015] Preferably, the chassis cells of the above-mentioned production strain are Aspergillus niger.

[0016] Compared with existing technologies, the advantages of this invention are as follows: The modified monetarin protein of this invention achieves significant improvements in both sweetness and stability, with its sweetness reaching more than 1.5 times that of the initial product, and its sweetness intensity is significantly enhanced. Meanwhile, most foods requiring added sweeteners undergo pasteurization. After heating at 72°C for 15 seconds, the initial monetarin protein experiences a significant loss of sweetness, which affects food quality. However, the modified monetarin protein exhibits excellent tolerance; its sweetness remains essentially unchanged after heat sterilization in the same manner. This characteristic not only ensures the flavor quality of the final product but also provides a reliable guarantee for standardized quality control in industrial production, effectively expanding the application boundaries of monetarin protein as a sweetener in food. Detailed Implementation

[0017] The term "recombinant gene" refers to DNA or RNA capable of expressing the monetarin protein of the present invention. Typically, the recombinant gene is initially synthesized in vitro via solid-phase phosphoramidite triester synthesis, TdT biosynthesis, or other suitable techniques known in the art. Once a template sequence is available, it can be amplified by PCR or other suitable techniques known in the art. With a recombinant bacterial strain, further large-scale amplification can be achieved by culturing the strain. In some embodiments, the recombinant gene may also include residual restriction enzyme sites, other accessory elements such as control elements (e.g., promoters), labeling substances (e.g., fluorescent labels), and other sequences that do not affect the expression of the target gene.

[0018] The term "clonal scar" refers to the promoter sequence of transcription, which is dependent on the initiation messenger ribonucleotide (mRNA) for protein expression, followed by the ribosome-binding site (RBS) that attracts the translation machinery, and then the signal peptide sequence that facilitates protein transport to the periplasm. Mature proteins are typically cloned after the signal peptide, cleaved from it by a signal peptidase as they cross the membrane. However, in cloning constructs after the signal peptide, restriction endonucleases often require specific sequences to cut the DNA, leaving a clonal scar following the signal peptide sequence.

[0019] The term "signal peptide" refers to a short peptide (typically 16-30 amino acids long) located at the N-terminus of most newly synthesized proteins, which are destined for the secretion pathway. It can also be called a signal sequence, targeting signal, localization signal, localization sequence, transport peptide, leader sequence, or leader peptide. Signal peptides are usually cleaved from proteins by signal peptidases.

[0020] Whether it is a cloned scar, signal peptide, or other elements in a recombinant gene, it does not affect the realization of the function of the monetarin protein. Therefore, if the difference between the amino acid sequence of the final obtained protein and the amino acid sequence disclosed in this invention is only the amino acid sequence corresponding to the above-mentioned DNA sequence, it still falls within the protection scope of this invention.

[0021] The term "signal peptide cleavage site" refers to a dipeptide between which a signal peptidase cleaves the signal peptide from the mature protein. In most (but not all) cases, the dipeptide is Ala-Ala.

[0022] The term "promoter" refers to a region of DNA that initiates the transcription (writing to mRNA) of a specific gene. Promoters are typically located near the transcription start site of a gene, on the same strand of the DNA and upstream of it (pointing to the 5' region of the sense strand). Promoters can be inducible, meaning that the expression of a gene operatively linked to the promoter can be activated in the presence of an inducing agent. Alternatively, promoters can be constitutive, meaning they are not regulated by any inducing agent.

[0023] The abbreviation "RBS" stands for ribosome-binding site, or ribosome binding site. This is the sequence of nucleotides upstream of the start codon in mRNA transcripts, responsible for recruiting ribosomes during the initiation of protein translation.

[0024] The term "expression" refers to the process of DNA being transcribed into messenger RNA (mRNA) and then translated into protein. To achieve successful expression and screening of monetarin protein, the aforementioned signal peptide, promoter, and RBS may be introduced into the recombinant gene. Therefore, some corresponding peptide segments may remain on the expressed monetarin protein. These peptide segments do not affect the function of the monetarin protein; therefore, even if the product contains additional peptide segments, as long as the amino acid sequence of the main component is identical to the sequence of this invention, the product is still an infringing product.

[0025] The present invention will be further described in detail below with reference to the embodiments. Example

[0026] Proteins are the material basis of life and essential components of human cells and tissues. All vital components of the human body require protein participation, playing a crucial role in cellular and biological life activities. It can be said that without protein, there is no life. There are many types of proteins in the human body, each with different functions. Some constitute human tissues, some provide energy, some participate in metabolism and transport, and some promote growth and development and regulate immune function. Different proteins perform different duties and roles, and their functions are determined by their structure. The 3D structure of a protein is determined by its amino acid sequence. Therefore, protein design depends on the correspondence between structure and sequence; designing proteins with specific functions requires designing sequences that conform to that functional structure. Understanding and designing proteins is of great significance for promoting innovation and progress in biology and medicine.

[0027] Designing protein sequences for a specific function is extremely difficult, as the final structure and function of the designed sequence are unpredictable. Furthermore, the sample space for fixed-length protein sequences is enormous. To address these challenges, Lésign, a protein design platform based on deep learning algorithms, was developed. This platform enables protein structure prediction, sequence design, and result evaluation. The various functional modules collaborate through interfaces, forming a comprehensive computational pipeline integrating prediction, design, and evaluation.

[0028] Using the Lésign platform, the initial monetarin protein (amino acid sequence as shown in SEQ ID NO.1) was sequenced, and the computationally optimal enzyme variant was finally obtained.

[0029] 1. Preparation of Monelin Protein 1.1 Construction of the production strain: The substrate cells were *Aspergillus niger* (CICC 41796, available for purchase on the CICC website). Based on the codon bias of *Aspergillus niger*, the monetarin protein gene was optimized to obtain the *des-pGlu1-monellin* gene. Using pUC57-simple as the vector backbone, the following functional elements were inserted: the glaA promoter, the glaA signal peptide, the artificially synthesized *des-pGlu1-monellin* gene, the trpC terminator for transcription termination, and the hygromycin B phosphotransferase gene *hph*, resulting in a plasmid. The DNA fragment was amplified using high-fidelity DNA polymerase, and then fused with protoplasts (*Aspergillus niger* was inoculated into a medium containing hygromycin B, cultured, and lysed, then the protoplasts were collected by filtration) to ultimately integrate the monetarin gene into the *Aspergillus niger* genome. Colonies growing on selection plates containing 100 μg / mL hygromycin B were considered positive transformants. After selecting positive strains, the *hph* gene was knocked out to obtain the production strain. There are many other available Aspergillus niger strains and corresponding production strain construction methods in the prior art. This embodiment only provides a specific solution.

[0030] 1.2 Expression and purification of monetarin protein: The shake-flask batch fermentation medium formulation was as follows: 10% maltodextrin (DE value 15-20) as carbon source, 3% yeast extract as nitrogen source, and inorganic salts including 0.3% potassium dihydrogen phosphate, 0.15% magnesium sulfate heptahydrate, 0.01% calcium chloride, and 0.001% ferrous sulfate heptahydrate. The initial pH was 5.5, automatically adjusted using 2 mol / L sodium hydroxide or hydrochloric acid. Operating conditions were: 100 mL of medium in a 500 mL Erlenmeyer flask, 10% inoculum (v / v), temperature 30°C, rotation speed 220 rpm, and a cycle of 7 days. Biomass dry weight, reducing sugar, protein concentration, and monetarin protein expression were measured every 24 hours.

[0031] The parameters for horizontal fed-batch fermentation in a 1000-liter fermenter are controlled as follows: temperature is automatically controlled at 30 ± 0.5 degrees Celsius; pH is automatically adjusted to 5.5 ± 0.2 using 2 mol / L sodium hydroxide or hydrochloric acid; dissolved oxygen is controlled to be greater than 20% by stirring speed of 200 to 800 rpm and aeration rate of 1 to 3 volumes per minute; and tank pressure is automatically adjusted from 0.05 to 0.1 MPa. The feeding strategy is to initially use a maltodextrin concentration of 5%, and when the reducing sugar concentration drops to 1%, add 50% maltodextrin solution to maintain the reducing sugar concentration at 2% to 5%. The feeding rate is dynamically adjusted according to the sugar consumption rate. Since the glaA promoter is used, maltodextrin itself is the inducer, and no additional inducer is needed. Many other methods for fermentation expression of production strains exist in the prior art; this embodiment only provides one specific scheme.

[0032] 1.3. The fermentation broth was filtered through 8 layers of gauze to remove mycelium. The filtrate was centrifuged at 10,000 g for 15 minutes to collect the supernatant. The supernatant was then filtered through a 0.45-micron filter membrane to obtain crude enzyme solution. The fermentation broth was further clarified by microfiltration or the addition of diatomaceous earth filter aid, and then the pH was adjusted to near the isoelectric point of Monelin to facilitate subsequent separation. The pre-treated solution was concentrated using a 10 kDa ultrafiltration membrane to remove small molecule impurities, followed by fractional precipitation with 30-60% saturated ammonium sulfate to achieve initial protein enrichment. After reconstitution of the precipitate, it was subjected to ion exchange chromatography (CM-Sepharose cation exchange column or DEAE-Sepharose anion exchange column selected according to the isoelectric point of Monelin), hydrophobic chromatography (Phenyl-Sepharose), and gel filtration chromatography (Superdex 75 or Sephacryl S-100) in sequence. Through a combination of multiple chromatographic steps, impurities, aggregates, and degradation fragments were finely removed, and high-purity monomeric proteins were finally obtained. The purified product was identified as having a purity greater than 95% by SDS-PAGE and RP-HPLC, and was further purified using MALDI-TOF. The molecular weight was verified by MS, and the biological activity was confirmed by sensory evaluation or receptor binding assay. After passing the assay, the protein was transferred to PBS or citrate buffer, and a protective agent such as trehalose was added. The mixture was then freeze-dried into a powder formulation and stored at -20°C or 4°C. Many other methods for purifying monetarine protein exist in the prior art; this embodiment only provides one specific method.

[0033] The initial monetarin protein (MNL, SEQ ID NO:1) and the monetarin protein variant were heterologously expressed and purified according to the above-described method for preparing monetarin protein.

[0034] 2. Sweetness determination: Sweetness was determined using a sensory evaluation method: the reference solution was a 5% (w / v) sucrose aqueous solution with a sweetness benchmark of 1; the purified monetarin protein was diluted with ultrapure water to different concentrations from 0.0005% to 0.0025% (the concentration gradient interval between groups was set at 0.0001%). Ten trained evaluators were recruited to use a three-point test to compare with the reference solution and determine the sample concentration C that was equivalent to the sweetness of 5% sucrose, thereby calculating the sweetness multiple of the monetarin protein. The sweetness multiple of the monetarin protein at room temperature was also determined.

[0035] Table 1. Sweetness multiples of monetarin protein at room temperature Brazilian sweet sequence Sweetness multiplier MNL-0 (SEQ ID NO:1) 2778 MNL-1 (SEQ ID NO:2) 4167 MNL-2 (SEQ ID NO:3) 5000 MNL-3 (SEQ ID NO:4) 4545 MNL-4 (SEQ ID NO:5) 4167 MNL-5 (SEQ ID NO:6) 5556 MNL-6 (SEQ ID NO:7) 6250 The purified monetarine protein mutant was diluted with ultrapure water to different concentrations from 0.0005% to 0.0025% (the concentration gradient interval between groups was set at 0.0001%). The solutions were rapidly heated to 72°C for 15 seconds (simulating pasteurization), and the sweetness factor of the monetarine protein mutant was measured after cooling to room temperature. The purified initial monetarine protein was diluted with ultrapure water to different concentrations from 0.001% to 0.008% (the concentration gradient interval between groups was set at 0.0005%) and heated in the same manner as the monetarine protein mutant solution. Ten trained evaluators were recruited to use the three-point test method to compare with the reference solution to determine the sample concentration C equivalent to the sweetness of 5% sucrose, thereby calculating the sweetness factor of the monetarine protein.

[0036] Table 2. Sweetness multiples of monetarin protein after pasteurization at 72℃ for 15 seconds and cooling to room temperature. Brazilian sweet sequence Sweetness multiplier MNL-0 (SEQ ID NO:1) 1667 MNL-1 (SEQ ID NO:2) 4167 MNL-2 (SEQ ID NO:3) 5556 MNL-3 (SEQ ID NO:4) 4545 MNL-4 (SEQ ID NO:5) 3846 MNL-5 (SEQ ID NO:6) 5556 MNL-6 (SEQ ID NO:7) 6250 As shown in Tables 1 and 2, the sweetness multiple of the modified monetarine protein is 1.5 to 2.25 times that of the original, indicating a significant improvement in sweetness. Furthermore, most foods requiring added sweeteners need to undergo pasteurization. After heating at 72°C for 15 seconds, the initial monetarine protein shows a significant loss of sweetness, which can affect food quality. However, the sweetness multiple of the modified monetarine protein remains essentially unchanged, which is beneficial for product quality control.

[0037] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A monetin protein, characterized in that... The monetin protein described in SEQ ID NO:1 was used as the parent protein, and the following mutation set was used for mutation: E55K+N15E+Q29K+I39V.

2. A monetin protein, characterized in that... The monetin protein described in SEQ ID NO:1 was used as the parent material and mutated using the following mutation set: E55K+E24H+R32K+R40K.

3. A monetin protein, characterized in that... The monetarin protein described in SEQ ID NO:1 was used as the parent protein, and the following mutation set was used for mutation: E55K+M43I+F53E+Q62F.

4. A monetin protein, characterized in that... The monetin protein described in SEQ ID NO:1 was used as the parent material and mutated using the following mutation set: E55K+E49K+Y66E+R73E.

5. A monetin protein, characterized in that... The monetin protein described in SEQ ID NO:1 was used as the parent material and mutated using the following mutation set: E55K+D75V+S77F.

6. A monetin protein, characterized in that... The monetin protein described in SEQ ID NO:1 was used as the parent protein, and the following mutation set was used for mutation: E55K+I27K+I56V+R83K.

7. A recombinant genetic material of a monetine protein, characterized in that... DNA or RNA capable of expressing the monetin protein as described in any one of claims 1 to 6.

8. A strain for producing monetarin protein, characterized in that... It contains the recombinant genetic material as described in claim 7.

9. The strain for producing monetarin protein according to claim 8, characterized in that... The chassis cells of the production strain are Aspergillus niger.