High stability monellin protein and its recombinant gene

Abstract

The application discloses a high-stability monellin protein and a recombinant gene thereof. The modified monellin protein realizes significant improvement in sweetness and stability, the sweetness reaches more than 1.5 times of the initial sweetness, and the sweetness intensity is obviously enhanced. Meanwhile, the matrix environment of different foods (such as the pH of high-acidity fruit juice is less than 4.5) and the sterilization conditions (such as some foods need to be subjected to ultra-pasteurization) are different. After being heated at 125 DEG C for 5 seconds in the pH 4.5 environment, the sweetness of the initial monellin protein is lost by half, which seriously affects the quality of the food. The modified monellin protein exhibits excellent tolerance, and the sweetness basically remains unchanged after being subjected to the same heating sterilization, the characteristic not only ensures the flavor quality of the terminal product, but also provides reliable guarantee for the standardization of product control in industrialized production, and effectively expands the application boundary of the monellin protein as a sweetener in foods.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more specifically to a highly stable 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, possessing a pure sweetness and extremely low calories. 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 the loss of its sweetness activity. Under conventional heat treatment conditions required for food processing (such as pasteurization at 72°C for 15 seconds), monetarin protein undergoes irreversible structural damage and sweetness loss, failing to meet the processing requirements of liquid dairy products, fruit juices, and other products requiring heat sterilization. Therefore, improving the thermal stability of monetarin protein and developing functional sweet proteins capable of withstanding the heat processing conditions of the food industry has become a critical 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 highly stable 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 highly stable 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: G84K+F12H+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: G84K+R32K+R40K+M43I, 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: G84K+F53E+Q62F+Y66E, 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: G84K+E49K+R54K+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: G84K+D75V+S77F+D79E, 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: G84K+N15E+E60I+R85K, 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 Pichia pastoris.

[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 a markedly enhanced sweetness intensity. Simultaneously, different foods exhibit varying matrix environments (e.g., the pH of high-acid fruit juice is less than 4.5) and sterilization conditions (e.g., some foods require ultrapasteurization). After heating at 125°C for 5 seconds in a pH 4.5 environment, the initial monetarin protein loses most of its sweetness, which severely affects food quality. However, the modified monetarin protein exhibits excellent tolerance; its sweetness remains essentially unchanged after heating and 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. The signal peptide cleavage site can be calculated using algorithms such as SignalP4.1.

[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.

[0026] Example 1

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 1. Preparation of Monelin Protein 1.1 Construction of the production strain: The chassis cells were Pichia pastoris (SMD1168H, available from Beyotime's official website). Based on the codon preference of Pichia pastoris, the entire gene of the monetarin protein sequence was synthesized, the natural signal peptide coding sequence was removed, and the α-mating factor (α-MF) signal peptide was fused to the N-terminus to guide secretory expression. A 6×His tag was optionally added to the C-terminus for subsequent purification. XhoI and XbaI restriction sites were introduced at both ends of the gene for targeted cloning. The pPICZα vector backbone was selected, and the original PAOX1 promoter was replaced with the PGAP promoter. The PGAP fragment (approximately 500 bp) was amplified by PCR, and the pPICZα vector was digested with appropriate restriction endonucleases (such as SacI and XhoI) to remove PAOX1. The PGAP fragment was then directionally inserted into the promoter position. Subsequently, the synthesized Brazil nut gene was digested with XhoI and XbaI and inserted into the multiple cloning site downstream of the PGAP, constructing the pGAPZα-monellin recombinant plasmid. This vector carries the hph gene as an antibiotic resistance selection marker, conferring hygromycin B resistance to transformants, while retaining the AOX1 terminator to ensure effective transcription termination. The ligation product was transformed into E. coli DH5α competent cells, plated on LB plates containing 25 μg / mL Zeocin, and positive clones were selected. Single colonies were picked for colony PCR and restriction enzyme digestion verification. Finally, sequencing confirmed that the sequences of the PGAP promoter, Brazil nut gene, and fusion signal peptide were correct.

[0031] The verified recombinant plasmid was linearized using restriction endonucleases such as SacI or BstXI. The cleavage site was located in the AOX1 transcription termination region or inside the His4 gene. Linearization can promote homologous recombination between the plasmid and the host chromosome, improving integration efficiency and stability. After enzyme digestion, complete linearization was confirmed by agarose gel electrophoresis. The linearized DNA was extracted with phenol-chloroform or purified using a kit, quantified, and stored at -20℃ for later use.

[0032] SMD1168H strain was inoculated into YPD medium and cultured at 30°C until the logarithmic growth phase. After collecting the cells, they were washed sequentially with ice-cold sterile water and sorbitol solution, and finally resuspended in 1 M sorbitol to prepare electrotransformation competent cells. 5-10 μg of linearized recombinant plasmid was mixed with 80 μL of competent cells and transferred to a pre-cooled 0.2 cm electrotransformation cuvette. Pulsed electroporation was performed in an electroporator with a voltage of 1500-2000 V, a capacitance of 25 μF, and a resistance of 200 Ω. Immediately after electroporation, 1 mL of ice-cold sorbitol solution was added and mixed well. After 1 hour of recovery, the cells were plated on YPDS plates containing 100 μg / mL hygromycin B (containing 1 M sorbitol) and cultured at 30°C for 3-5 days until transformant colonies appeared.

[0033] Since SMD1168H is his4 protrophic, HIS4 complementation screening is unnecessary; initial screening is performed directly using the hygromycin B resistance of the hph gene. Single colonies are transferred to YPD gradient plates containing different concentrations of hygromycin B (100-2000 μg / mL), gradually increasing the antibiotic pressure to screen for multi-copy integration clones. High-resistance clones typically contain more copies of the target gene. Colony PCR is performed on high-resistance clones for verification, using PGAP promoter-specific upstream primers and Brazil nut gene downstream primers to confirm that the target gene has been integrated into the host genome. Further analysis of the integration copy number using Southern blot or real-time quantitative PCR is conducted to screen for high-expression candidate clones containing 3-10 copies. After identifying positive strains, the hph gene is knocked out to obtain the production strain. Many other available chassis cells and corresponding production strain construction methods exist in the prior art; this embodiment only provides one specific approach.

[0034] 1.2 Expression and purification of monetarin protein: Positive clones were inoculated into 10-20 mL of BMGY medium (containing yeast extract, peptone, YNB, biotin, and glycerol) and cultured at 30°C and 250 rpm for 24 hours. They were then transferred to BMDY medium containing glucose and cultured for another 48-72 hours. Samples were taken every 12 hours, and the supernatant was collected by centrifugation. Protein expression bands were detected by SDS-PAGE. Western blot analysis was performed using anti-His-tagged antibodies or carnauba-specific antibodies to confirm the target protein. Sensory evaluation or electronic tongue assays were used to determine sweetness activity. Many other methods for fermentation expression of production strains exist in the prior art; this embodiment only provides one specific approach.

[0035] 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 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.

[0036] 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.

[0037] 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.

[0038] 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) 4167 MNL-3 (SEQ ID NO:4) 5556 MNL-4 (SEQ ID NO:5) 6250 MNL-5 (SEQ ID NO:6) 5000 MNL-6 (SEQ ID NO:7) 5556 The purified monetarine protein mutant was diluted with ultrapure water to different concentrations from 0.0005% to 0.0025% (with a concentration gradient interval of 0.0001%), and the pH was adjusted to 4.5 with hydrochloric acid. The solution was rapidly heated to 125°C for 5 seconds (simulating ultrapasteurization), and the sweetness multiple 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% (with a concentration gradient interval of 0.0005%), and subjected to the same heating and pH adjustment as the monetarine protein mutant solution. Ten trained evaluators were recruited to use a three-point test to compare the sample concentration C with the reference solution to determine the sweetness multiple of 5% sucrose, thereby calculating the sweetness multiple of the monetarine protein.

[0039] Table 2. Sweetness multiples of monetarin protein after ultrapasteurization at 125℃ for 5 seconds and cooling to room temperature. Brazilian sweet sequence Sweetness multiplier MNL-0 (SEQ ID NO:1) 769 MNL-1 (SEQ ID NO:2) 4545 MNL-2 (SEQ ID NO:3) 4167 MNL-3 (SEQ ID NO:4) 5000 MNL-4 (SEQ ID NO:5) 5556 MNL-5 (SEQ ID NO:6) 5000 MNL-6 (SEQ ID NO:7) 5556 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 initial protein, indicating a significant improvement in sweetness. Furthermore, the matrix environment (e.g., pH less than 4.5 for high-acid fruit juices) and sterilization conditions (e.g., some foods require ultrapasteurization) vary among different foods. To broaden the application range of monetarine protein, its stability needs to be improved. Heating at 125°C for 5 seconds at pH 4.5 resulted in a significant loss of sweetness in the initial monetarine protein, which can negatively impact food quality. However, the sweetness multiple of the modified monetarine protein remained essentially unchanged, which is beneficial for product quality control.

[0040] 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 highly stable 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: G84K+F12H+Q29K+I39V.

2. A highly stable monetarin 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: G84K+R32K+R40K+M43I.

3. A highly stable 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: G84K+F53E+Q62F+Y66E.

4. A highly stable monetarin 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: G84K+E49K+R54K+R73E.

5. A highly stable monetarin 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: G84K+D75V+S77F+D79E.

6. A highly stable monetarin 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: G84K+N15E+E60I+R85K.

7. A recombinant genetic material of a highly stable monetin 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 highly stable monetarin protein, characterized in that... It contains the recombinant genetic material as described in claim 7.

9. The strain for producing highly stable monetarin protein according to claim 8, characterized in that... The chassis cells of the production strain are Pichia pastoris.