New method for realizing biosynthesis of hexapeptide-8 and engineering strain thereof
Through genetic engineering, the interval sequence of the hexapeptide-8 repeat sequence was designed, and acetyl hexapeptide-8 was synthesized by microbial fermentation, which solved the high cost and environmentally unfriendly problems of chemical synthesis methods, and achieved low-cost, environmentally friendly and efficient production.
Patent Information
- Application Number
- CN202510180057.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-19
- Publication Date
- 2025-05-30
AI Technical Summary
The existing chemical synthesis methods of acetyl hexapeptide-8 are costly, unfriendly in the environment, and have low yields in the production process and difficult purification.
Through genetic engineering, hexapeptide-8 repeat sequences were designed, and hexapeptide-8 was synthesized by microbial fermentation, and directed enzymatic lysis was used to achieve efficient synthesis.
It greatly reduces the production cost of hexapeptide-8, simplifies the process, improves the yield, and is environmentally friendly, promoting its promotion and application in the cosmetics industry.
Smart Images
Figure CN120060306A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of genetic engineering, and particularly relates to a method for biosynthesizing an important active small peptide, hexapeptide-8 (amino acid sequence: EEMQRR, glutamic acid-glutamic acid-methionine-glutamine-arginine-arginine) in cosmetic raw materials. The new method has lower production costs compared to chemical synthesis methods and is environmentally friendly. Background Art
[0002] Acetyl hexapeptide-8 is a synthetic peptide commonly used in anti-aging skin care products. It reduces muscle contraction by inhibiting the excessive release of neurotransmitters, thereby fading dynamic wrinkles such as crow's feet and forehead lines. Its effect is similar to that of botulinum toxin, but it is milder and non-invasive. In addition, it can promote collagen production, improve skin elasticity, and make the skin smoother and firmer. Due to its high efficiency and low irritation, acetyl hexapeptide-8 is widely used in high-end skin care products. Currently, acetyl hexapeptide-8 is prepared by chemical synthesis methods, with high prices, limited room for cost reduction, low production yields, difficult purification during the production process, and being not environmentally friendly enough. In recent years, synthetic biology has developed rapidly, and many products originally synthesized chemically have been transformed into biological synthesis methods. By using genetic engineering means to synthesize hexapeptide-8 through microbial fermentation, it is expected to significantly reduce its production costs. For the biosynthesis of the main-chain hexapeptide of acetyl hexapeptide-8, the present invention ingeniously designs the spacer sequence of the hexapeptide-8 repeat sequence, thus opening up the biosynthetic pathway of hexapeptide-8. The present invention can greatly reduce the production cost of hexapeptide-8 and is of great significance for promoting the application of acetyl hexapeptide-8. Summary of the Invention
[0003] The purpose of the present invention is to biosynthesize the short peptide hexapeptide-8 by a biological method using a genetically engineered strain. The biosynthetic pathway of hexapeptide-8 is opened up by designing an artificial repeat sequence of hexapeptide-8. The key to the design of the hexapeptide-8 repeat sequence is to creatively insert a specific spacer sequence into the repeat sequence, thereby achieving the efficient synthesis of hexapeptide-8 through directed enzymatic hydrolysis.
[0004] The invention process of the biosynthetic method of the hexapeptide-8 is as follows:
[0005] (1) Design an artificial amino acid sequence containing the hexapeptide-8 repeat sequence, named the hexapeptide-8 multiple sequence, which is characterized by the presence of multiple repeated EEMQRR sequences, and a specific spacer sequence KXF or DXF (amino acid abbreviation correspondence: D, aspartic acid; K, lysine; X, any amino acid type and its quantity) is added between each repeat unit;
[0006] (2) Entrust a gene synthesis company to synthesize the DNA sequence of the designed hexapeptide-8 multiple sequence and integrate it into a microbial expression plasmid, and preferably select the Escherichia coli pET series as the expression plasmid;
[0007] (3) Introduce the expression plasmid into the engineered strain, and ferment to express the polypeptide molecule of the hexa-peptide-8 multiple sequence. Preferably, Escherichia coli is selected as the engineered strain;
[0008] (4) Collect the polypeptide molecule expressed by the hexa-peptide-8 multiple sequence, add chymotrypsin, LysN enzyme or AspN enzyme for hydrolysis, and obtain the hexa-peptide-8 short peptide through ultrafiltration;
[0009] (5) Confirm the synthesis of the hexa-peptide-8 product through liquid chromatography-mass spectrometry analysis.
[0010] The beneficial effects of the present invention are as follows:
[0011] The invented biosynthesis method of hexa-peptide-8, compared with the chemical method, does not require high-purity amino acids and chemical substances such as catalysts as synthesis raw materials. The fermentation medium used is inexpensive, the fermentation preparation process is simple, the production process is green and environmentally friendly, and it is expected to greatly reduce the production cost in the future, thereby promoting the popularization and application of hexa-peptide-8 in the cosmetics industry. Description of the Drawings
[0012] Figure 1 , HPLC-ESI / Q-TOF liquid chromatography-mass spectrometry analysis of the product obtained by protease hydrolysis of the polypeptide recombinantly expressed by the EEMQRR-1 sequence. The upper figure is the extracted ion chromatogram of hexa-peptide-8, and the lower figure is its mass spectrum.
[0013] Figure 2 , HPLC-ESI / Q-TOF liquid chromatography-mass spectrometry analysis of the product obtained by protease hydrolysis of the polypeptide recombinantly expressed by the EEMQRR-2 sequence. The upper figure is the extracted ion chromatogram of hexa-peptide-8, and the lower figure is its mass spectrum. Detailed Embodiments
[0014] The technical content of the present invention will be further described below in conjunction with the embodiments. However, the present invention is not limited to these embodiments, and the protection scope of the present invention cannot be limited by the following embodiments.
[0015] Example 1: Engineered Strain and Biosynthesis of Hexa-peptide-8
[0016] 1. Design of the hexa-peptide-8 multiple sequence
[0017] The EEMQRR multiple sequence EEMQRR-1 was designed, which contains 15 EEMQRR units, and the DPF spacer sequence was inserted between the units. The specific sequence is as follows.
[0018] EEMQRR-1 polypeptide sequence (Sequence Listing SEQ ID NO.1):
[0019] DPFEEMQRRDPFEEMQRRDPFEEMQRRDPFEEMQRRDPFEEMQRRDPFEEMQRRDPF EEMQRRDPFEEMQRRDPFEEMQRRDPFEEMQRR
[0020] 2. Entrust a gene synthesis company to synthesize the DNA sequence of EEMQRR-1 and insert it into the pET28a(+) plasmid.
[0021] 3. Transform the constructed plasmid into Escherichia coli BL21(DE3).
[0022] (1) Take out the competent cells (80 μL) from -80 °C and thaw them on ice.
[0023] (2) Add 10 μL of the ligation product or 2 - 5 μL of the plasmid to be transformed into the competent cells, mix gently, and place on ice for 30 min.
[0024] (3) Heat shock in a water bath at 42 °C for 30 s and then place on ice for 2 min.
[0025] (4) Add 900 μL of LB medium preheated at 37 °C and culture with shaking at 220 r / min at 37 °C for 1 hour.
[0026] (5) Centrifuge at 4000 r / min for 5 min to collect the bacterial cells, discard part of the supernatant, resuspend the bacterial cells, take an appropriate amount and spread it on an LB plate with Amp (ampicillin) resistance, and place the plate in an incubator at 37 °C for overnight culture for 12 - 16 h.
[0027] 4. Ferment and induce the cultured strain after plasmid transformation.
[0028] (1) Transfer the bacterial liquid to an LB liquid medium with Amp (ampicillin) resistance (the final concentration of Amp is 100 μg / mL), and culture at 37 °C with shaking at 220 r / min until the OD 600 reaches 0.6 - 0.8.
[0029] (2) Add IPTG (final concentration 0.5 mmol / L) and induce culture at 16 °C with shaking at 120 r / min.
[0030] 4. Treatment of the fermented bacterial liquid
[0031] Centrifuge the fermented bacterial liquid at 12000 r / min for 30 min to collect the bacterial cells. The cell pellet is disrupted by ultrasonic disruption or a bead mill.
[0032] 5. Obtain hexa - peptide - 8 by protease hydrolysis.
[0033] Add an appropriate amount of chymotrypsin and AspN enzyme to the broken bacterial solution, hydrolyze at 37 °C for 3 h, then perform ultrafiltration treatment, and collect the filtrate.
[0034] 7. Detect the hydrolyzed polypeptide by mass spectrometry.
[0035] Use an Agilent liquid chromatography electrospray quadrupole time-of-flight mass spectrometer (HPLC-ESI / Q-TOF) to detect the hydrolysis product. The chromatographic column model is XBridge peptide BEH C18 (2.1 mm x 150 mm, 3.5 μm). The mobile phase is a 5% acetonitrile solution added with 0.1% formic acid, and the flow rate is 0.20 ml / min. The parameters of the ESI / Q-TOF mass detector are as follows: positive ion mode, Gas Temp (°C): 325, Gas flow (L / min): 13, molecular weight scanning range 300 - 1300.
[0036] The mass-to-charge ratio of the [M+H]+ ion of hexa-peptide-8 + is 848.4043. The HPLC-ESI / Q-TOF analysis result proves that the engineered strain constructed using the EEMQRR-1 sequence fermented and synthesized hexa-peptide-8, and the results are as shown in the appendix Figure 1 shown. The quantitative result by the external standard method shows that the shake flask fermentation level of hexa-peptide-8 reached about 0.2 mg / mL.
[0037] 8. Preparation of acetylated hexa-peptide-8 by acetylation modification
[0038] Dilute acetic anhydride to 800 mM with dioxane, then add it to the hexa-peptide-8 solution at a ratio of 5 mL / L. The pH is stabilized to 6.5 with 0.5 M sodium hydroxide, and the acetylation reaction is started with stirring for 30 min. Then, it is concentrated and the solvent is removed by rotary evaporation. Finally, dry the powder under vacuum to obtain acetylated hexa-peptide-8 dry powder.
[0039] Example 2: Engineered strain and biosynthesis of hexa-peptide-8
[0040] 1. Design of the hexa-peptide-8 multiple sequence
[0041] Designed the EEMQRR multiple sequence EEMQRR-2, which contains 30 EEMQRR units with a KPF spacer sequence inserted between the units. The specific sequence is as follows.
[0042] EEMQRR-2 polypeptide sequence (Sequence Listing SEQ ID NO.2):
[0043] KPFEEMQRRKPFEEMQRRKPFEEMQRRKPFEEMQRRKPFEEMQRRKPFEEMQRRKPFEEMQRRKPFEEMQRRKPFEEMQRRKPFEEMQRRKPFEEMQRRKPFEEMQRRKPFEEMQRRKPFEEMQRRKPFEEMQRRKPFEEMQRRKPFEEMQRRKPFEEMQRRKPFEEMQRR
[0044] 2. Entrust a gene synthesis company to synthesize the DNA sequence of EEMQRR-2 and insert it into the pET22a(+) plasmid.
[0045] 3. Transform the constructed plasmid into Escherichia coli BL21(DE3).
[0046] (1) Take out the competent cells (80 μL) from -80 °C and thaw them on ice.
[0047] (2) Add 10 μL of the ligation product or 2 - 5 μL of the plasmid to be transformed into the competent cells, gently mix, and place on ice for 30 min.
[0048] (3) Heat shock in a water bath at 42 °C for 30 s and then place on ice for 2 min.
[0049] (4) Add 900 μL of pre-warmed LB medium at 37 °C and incubate with shaking at 220 r / min for 1 hour.
[0050] (5) Centrifuge at 4000 r / min for 5 min to collect the bacteria, discard part of the supernatant, resuspend the bacteria, take an appropriate amount and spread it on an LB plate with Amp (ampicillin) resistance, and place the plate in an incubator at 37 °C for overnight culture for 12 - 16 h.
[0051] 4. Ferment and induce the culture of the strain into which the plasmid has been transformed.
[0052] (1) Transfer the bacterial solution to an LB liquid medium with Amp (ampicillin) resistance (the final concentration of Amp is 100 μg / mL), and culture at 37 °C and 220 r / min until the OD of the bacterial solution 600 reaches 0.6 - 0.8.
[0053] (2) Add IPTG (final concentration 0.5 mmol / L) and induce culture at 16 °C and 120 r / min.
[0054] 5. Treatment of the fermented bacterial solution
[0055] The fermented bacterial liquid was centrifuged at 12,000 r / min for 30 min to collect the bacterial cells. The cell precipitate was disrupted by ultrasonic disruption or a bead mill.
[0056] 6. Protease hydrolysis to obtain hexa-peptide-8.
[0057] An appropriate amount of chymotrypsin and LysN enzyme were added to the disrupted bacterial liquid, and hydrolysis was carried out at 37 °C for 3 h. The filtrate was collected by ultrafiltration.
[0058] 7. Perform mass spectrometry detection on the hydrolyzed polypeptide.
[0059] The hydrolyzate was detected using an Agilent HPLC-ESI / Q-TOF chromatograph-mass spectrometer. The chromatographic column model was XBridge peptide BEH C18 (2.1 mm x 150 mm, 3.5 μm). The mobile phase was a 5% acetonitrile solution containing 0.1% formic acid, and the flow rate was 0.20 ml / min. The parameters of the ESI / Q-TOF mass detector were as follows: positive ion mode, Gas Temp (°C): 325, Gasflow (L / min): 13, and the molecular weight scanning range was 300 - 1300.
[0060] The HPLC-ESI / Q-TOF analysis results proved that the engineered strain constructed using the EEMQRR-2 sequence also fermented and synthesized hexa-peptide-8, and the results are as shown in the appendix Figure 2 shown. The quantitative results by the external standard method showed that the preparation level of hexa-peptide-8 in shake flask fermentation reached 0.15 mg / mL.
[0061] 8. Acetylation modification to prepare acetylated hexa-peptide-8
[0062] Acetic anhydride was diluted to 800 mM with dioxane and then added to the hexa-peptide-8 solution at a ratio of 5 mL / L. The pH was stabilized to 6.5 with 0.5 M sodium hydroxide, and stirring was started for the acetylation reaction for 30 min. Then, it was concentrated and the solvent was removed by rotary evaporation. Finally, acetylated hexa-peptide-8 dry powder was obtained by vacuum drying.
Claims
1. A novel method for efficiently biosynthesizing hexapeptide-8 (amino acid sequence of glutamic acid-glutamic acid-methionine-glutamine-arginine-arginine, abbreviated as EEMQRR) by recombinant expression of artificial sequences and directed enzymatic hydrolysis, characterized in that: Artificially designed hexapeptide-8 multiple sequences (DXFEEMQRR) n or (KXFEEMQRR) n， A specific amino acid spacer sequence DXF or KXF (D, aspartic acid; K, lysine; X, any amino acid type and quantity) was added between each EEMQRR repeating unit. The polypeptide product of the sequence was expressed by microbial recombination, and then the efficient biosynthesis of hexapeptide-8 was achieved through directed enzymatic hydrolysis by protease.
2. A recombinant vector and strain (preferably a pET plasmid and an Escherichia coli strain) comprising the artificial sequence encoding gene according to claim 1.
3. The protease-directed enzymolysis method according to claim 1, characterized in that: The protease used is chymotrypsin, LysN enzyme or AspN enzyme.
4. A method for preparing acetyl hexapeptide-8, characterized in that: The hexapeptide-8 product synthesized by the method described in claims 1-3 is further prepared by N-terminal acetylation modification to synthesize acetyl hexapeptide-8.