High potency brazzein and recombinant genes thereof

Abstract

This invention discloses a high-concentration brassinoprotein and its recombinant gene. The modified brassinoprotein of this invention achieves significant improvements in both sweetness and stability, with its sweetness reaching more than 1.9 times that of the initial protein, and its sweetness intensity is significantly enhanced. At the same time, to meet the diverse needs of different food systems—such as the low pH environment (<4.5) and heat treatment processes such as ultra-pasteurization in juice products—the modified protein exhibits excellent tolerance. After heating at 125°C for 5 seconds at pH 3.0, its sweetness retention rate is 100%, while the initial sweetness shows a loss. 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 brassinoprotein in complex food matrices.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more specifically to a high-concentration brassinoprotein and its recombinant gene. Background Technology

[0002] In 1994, American scientists discovered it in the Congo Basin of West Africa, a perennial shrub. Pentadiplandra brazzeana The fruit (often called "forget-me-not" or "brazier") was first isolated from the red berries of the plant, which is endemic to the region. The fruit is sweet and sour, with the sweetness mainly coming from Brazilian sweet protein in the pulp.

[0003] Brazil sweetener contains four pairs of intramolecular disulfide bonds (Cys3-Cys54, Cys16-Cys37, Cys20-Cys44, Cys24-Cys48), making it the most stable natural sweetener (far exceeding semathy and monetarin). Its core characteristics are: Thermal stability: It retains more than 90% of its sweetness after heating at 80℃ for 4 hours, and there is no significant loss of sweetness after heating at 90℃ for 10 minutes. It is suitable for food processing processes such as high-temperature sterilization. Enzymatic resistance: It has a certain tolerance to pepsin and trypsin, and is not easily broken down in the human digestive tract, with no risk of gastrointestinal irritation. Solubility: Easily soluble in water and dilute alcohol solutions, leaving no precipitate or odor after dissolution, and does not affect the texture and appearance of food base materials.

[0004] Wild Brazier wood is scarce, and only a trace amount of Brazilin can be extracted from each kilogram of berries. Natural extraction is extremely costly and cannot be industrialized. Summary of the Invention

[0005] To address the shortcomings of existing technologies, one of the objectives of this invention is to provide a Brazilian sweet protein mutant with increased sweetness, which can be mass-produced using a bio-fermentation method.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a high-concentration carbapenem protein, the initial sequence of which is shown in SEQ ID NO.1. This initial carbapenem protein sequence is derived from the protein sequence library PDB: 2KGQ_A. Using the Lésign platform, the initial carbapenem protein sequence was designed, ultimately yielding a computationally optimal enzyme variant.

[0007] A high-concentration brassinoprotein, wherein the brassinoprotein is mutated using the initial brassinoprotein described in SEQ ID NO:1 as the parent material, and the following mutation set is used: L17I+K2E+Y7I, to obtain the brassinoprotein mutant shown in SEQ ID NO:2.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a high-concentration brassinoprotein, wherein the brassinoprotein is based on the initial brassinoprotein described in SEQ ID NO:1, and is mutated using the following mutation set: L17I+V12A+Q16L, to obtain the brassinoprotein mutant shown in SEQ ID NO:3.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a high-concentration brassinoprotein, wherein the brassinoprotein is based on the initial brassinoprotein described in SEQ ID NO:1, and is mutated using the following mutation set: L17I+A18E+N19G+Y23K, to obtain the brassinoprotein mutant shown in SEQ ID NO:4.

[0010] To achieve the above objectives, the present invention provides the following technical solution: A high-concentration brassinoprotein, wherein the brassinoprotein is based on the initial brassinoprotein described in SEQ ID NO:1, and is mutated using the following mutation set: L17I+D28N+R32K+F37R, to obtain the brassinoprotein mutant shown in SEQ ID NO:5.

[0011] To achieve the above objectives, the present invention provides the following technical solution: a high-concentration brassinoprotein, wherein the brassinoprotein is based on the initial brassinoprotein described in SEQ ID NO:1, and is mutated using the following mutation set: L17I+K41N+R42N, to obtain the brassinoprotein mutant shown in SEQ ID NO:6.

[0012] To achieve the above objectives, the present invention provides the following technical solution: a high-concentration brassinoprotein, wherein the brassinoprotein is based on the initial brassinoprotein described in SEQ ID NO:1, and is mutated using the following mutation set: L17I+Q45R+D49N, to obtain the brassinoprotein mutant shown in SEQ ID NO:7.

[0013] The second objective of this invention is to provide a DNA or RNA capable of expressing the above-mentioned high-concentration Brazilin mutant.

[0014] To achieve the above objectives, the present invention provides the following technical solution: a recombinant genetic material of high-concentration brassinoprotein, capable of expressing the DNA or RNA of brassinoprotein as described in any one of the above-mentioned embodiments.

[0015] The third objective of this invention is to provide a production strain capable of producing the aforementioned high-concentration Brazilian sweet protein.

[0016] To achieve the above objectives, the present invention provides the following technical solution: a high-concentration brassinoprotein production strain containing the above-mentioned recombinant genetic material.

[0017] Preferably, the chassis cells of the above-mentioned production strain are Pichia pastoris.

[0018] Compared with existing technologies, the advantages of this invention are as follows: the modified Brazil protein of this invention achieves significant improvements in both sweetness and stability, with its sweetness reaching more than 1.9 times that of the initial protein, and the sweetness intensity is significantly enhanced. At the same time, in response to the diverse needs of different food systems—such as the low pH environment (<4.5) and heat treatment processes such as ultra-pasteurization in juice products—the modified protein exhibits excellent tolerance. After heating at 125°C for 5 seconds at pH 3.0, its sweetness retention rate is 100%, while the initial Brazil protein shows a loss of sweetness. 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 Brazil protein in complex food matrices. Detailed Implementation

[0019] The term "recombinant gene" refers to DNA or RNA capable of expressing the Brazil protein of the present invention. Typically, the recombinant gene is initially synthesized in vitro via solid-phase phosphoramidite 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.

[0020] 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.

[0021] 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.

[0022] Whether it is a cloned scar, signal peptide, or other elements in a recombinant gene, it does not affect the function of Brazil protein. Therefore, if the amino acid sequence of the final protein differs from the amino acid sequence disclosed in this invention only in the amino acid sequence corresponding to the above-mentioned DNA sequence, it still falls within the protection scope of this invention.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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 Brazil 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 Brazil protein. These peptide segments do not affect the function of Brazil 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.

[0027] The present invention will be further described in detail below with reference to the embodiments.

[0028] Example 1

[0029] 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.

[0030] 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.

[0031] Using the Lésign platform, the initial Brazil protein (amino acid sequence shown in SEQ ID NO.1) was sequenced, and the computationally optimal enzyme variant was finally obtained.

[0032] 1. Preparation of Brazil Berry 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 whole gene of the Brazil glycoprotein 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 to facilitate 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 brassica gene was digested with XhoI and XbaI and inserted into the multiple cloning site downstream of the PGAP to construct the pGAPZα-Brazzein recombinant plasmid. This vector carries the hph gene (hygromycin B phosphotransferase gene) as a 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 to select positive clones, and single colonies were picked for colony PCR and restriction enzyme digestion verification. Finally, sequencing confirmed that the sequences of the PGAP promoter, brassica gene, and fusion signal peptide were correct.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 1.2 Expression and purification of Brazilin: Positive clones were inoculated into 10-20 mL of BMGY medium (containing 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 Brazil syrup-specific antibodies to confirm the target protein. Sensory evaluation or electronic tongue assay was 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.

[0037] 1.3 The solid-liquid separation steps are as follows: the fermentation broth is filtered through 8 layers of gauze to remove mycelium, the filtrate is centrifuged at 10000 g for 15 minutes to collect the supernatant, and the supernatant is filtered through a 0.45-micron filter membrane to obtain crude enzyme solution.

[0038] Heat treatment purification utilizes the excellent thermal stability of Brazil sweet protein: the crude enzyme solution is placed in an 80°C water bath for 2 hours, centrifuged at 12000 x g for 20 minutes to remove denatured impurities, and the supernatant is collected as the heat-treated sample.

[0039] Ammonium sulfate fractionation precipitation procedure: Slowly add ammonium sulfate powder to the heat-treated sample until 30% saturation, let stand at 4°C for 2 hours, centrifuge at 10000 x g for 15 minutes and discard the precipitate; continue adding ammonium sulfate to the supernatant until 80% saturation, let stand at 4°C overnight, centrifuge at 12000 x g for 20 minutes and collect the precipitate; dissolve the precipitate in 20 mmol / L PBS pH 7.0 and dialyze to remove salts.

[0040] DEAE-Sepharose Fast Flow Ion Exchange Chromatography: The chromatography column was an XK 16 / 20 with a bed volume of 50 mL. The equilibration buffer was 20 mmol / L Tris-HCl at pH 8.0. The sample was the dialyzed sample. Elution was performed using a linear gradient of 0 to 0.5 mol / L sodium chloride at a flow rate of 2 mL / min. The absorbance at 280 nm was monitored to collect the main peak component.

[0041] Optional purification steps use CM-Sepharose Fast Flow chromatography: adjust the DEAE eluent to pH 4.5, load the sample onto a CM column, equilibrate with 20 mmol / L sodium acetate at pH 4.5, elute with a linear gradient of 0 to 0.3 mol / L sodium chloride, and collect the main peak of brassinoprotein.

[0042] Concentration and drying: The purified sample was concentrated to 5 to 10 mg / mL using a 10 kDalton ultrafiltration membrane, pre-frozen at -80°C, and then freeze-dried under vacuum for 24 hours. After drying, the sample was stored at -20°C. Many other methods for purifying Brazil glycosides are available in the prior art; this embodiment only provides one specific method.

[0043] The initial brassinolide (BZ, SEQ ID NO:1) and its variants were heterologously expressed and purified according to the above-described preparation method. The obtained brassinolide and mutants were verified by SDS-PAGE to confirm that their sizes were consistent with expectations.

[0044] 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; purified carbapenem was diluted with ultrapure water to different concentrations from 0.001% to 0.0035% (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 carbapenem. The sweetness multiple of carbapenem was measured at room temperature (without pH adjustment) and after being heated at 125°C for 5 seconds (pH environment 3) and then cooled to room temperature.

[0045] Table 1. Sweetness multiples of Brazil protein at room temperature Brazilian sweet sequence Sweetness multiplier BZ-0 (SEQ ID NO:1) 1852 BZ-1 (SEQ ID NO:2) 4167 BZ-2 (SEQ ID NO:3) 3846 BZ-3 (SEQ ID NO:4) 4167 BZ-4 (SEQ ID NO:5) 3571 BZ-5 (SEQ ID NO:6) 5000 BZ-6 (SEQ ID NO:7) 4167 Table 2. Sweetness ratio of Brazilian macaroni after ultrapasteurization at 125 degrees Celsius for 5 seconds and cooling to room temperature. Brazilian sweet sequence Sweetness multiplier BZ-0 (SEQ ID NO:1) 1613 BZ-1 (SEQ ID NO:2) 4167 BZ-2 (SEQ ID NO:3) 4167 BZ-3 (SEQ ID NO:4) 4545 BZ-4 (SEQ ID NO:5) 3571 BZ-5 (SEQ ID NO:6) 5000 BZ-6 (SEQ ID NO:7) 3846 As shown in Tables 1 and 2, the sweetness multiple of the modified carrageenan is 1.9 to 2.7 times that of the initial product, indicating a significant improvement in sweetness. Furthermore, the matrix environment of different foods (e.g., the pH of high-acid fruit juice is less than 4.5) and sterilization conditions (e.g., some foods require ultrapasteurization) vary. To broaden the application range of carrageenan, its stability needs to be improved. Heating at 125°C for 5 seconds at pH 3 resulted in some loss of sweetness in the initial carrageenan, which could affect food quality. However, the sweetness multiple of the modified carrageenan remained essentially unchanged, which is beneficial for product quality control.

[0046] 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 high-concentration Brazilin protein, characterized in that... The brassinoprotein is based on the brassinoprotein described in SEQ ID NO:1, and the following mutation set is used for mutation: L17I+K2E+Y7I.

2. A high-concentration Brazilin protein, characterized in that... The brassinoprotein is based on the brassinoprotein described in SEQ ID NO:1, and the following mutation set is used for mutation: L17I+V12A+Q16L.

3. A high-concentration Brazilin protein, characterized in that... The brassinoprotein is based on the brassinoprotein described in SEQ ID NO:1, and the following mutation set is used for mutation: L17I+A18E+N19G+Y23K.

4. A high-concentration Brazilin protein, characterized in that... The brassinoprotein is based on the brassinoprotein described in SEQ ID NO:1, and the following mutation set is used for mutation: L17I+D28N+R32K+F37R.

5. A high-concentration Brazilin protein, characterized in that... The brassinoprotein is based on the brassinoprotein described in SEQ ID NO:1, and the following mutation set is used for mutation: L17I+K41N+R42N.

6. A high-concentration Brazilin protein, characterized in that... The brassinoprotein is based on the brassinoprotein described in SEQ ID NO:1, and the following mutation set is used for mutation: L17I+Q45R+D49N.

7. A recombinant genetic material of high-concentration brassinoprotein, characterized in that... DNA or RNA capable of expressing the Brazilian sweet protein as described in any one of claims 1 to 6.

8. A strain for producing high-concentration brassinolide, characterized in that... It contains the recombinant genetic material as described in claim 7.

9. The high-concentration brassinolide production strain according to claim 8, characterized in that... The chassis cells of the production strain are Pichia pastoris.