Recombinant bacterium capable of producing ectoine, L-theanine and collagen III at high yield and application of recombinant bacterium

Abstract

The invention relates to a recombinant bacterium capable of producing ectoine, L-theanine and collagen III at high yield and application of the recombinant bacterium, and belongs to the technical field of genetic engineering. The recombinant bacteria obtained by introducing plasmids containing ectoine biosynthetase genes and plasmids containing gamma-glutamine synthetase genes and III type collagen genes into corynebacterium glutamicum ATCC 13032 can be used for simultaneously producing ectoine, L-theanine and III type collagen at high yield. Meanwhile, an aspartate kinase gene and an aspartate semialdehyde dehydrogenase gene are connected to a plasmid containing an ectoine biosynthase gene, so that the yield of ectoine is increased. The recombinant bacteria obtained by the transformation can be used for simultaneously synthesizing 25.1 g / L of ectoine, 9.82 g / L of L-theanine and 356 mg / L of III type collagen after being subjected to fermentation culture.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to a recombinant bacterium capable of highly producing ectoine, L-theanine and type III collagen and its application. Background Art

[0002] Ectoin is an organic compound with important physiological functions and a wide range of applications. In the cosmetics industry, ectoin is used in moisturizing products. This is because it binds to water molecules, enhancing the skin's ability to retain moisture, improving the skin's barrier function, reducing water loss, and providing long-lasting hydration. In the pharmaceutical and biotechnology fields, it can act as a protective agent for cells and enzymes, helping to maintain the stability of bioactive substances. It plays a role in drug development, gene therapy, and other processes, ensuring the stability and success rate of related technologies.

[0003] L-theanine is a non-protein amino acid found in high concentrations in tea. As a moisturizer and antioxidant, L-theanine enhances skin cell activity and promotes collagen production, thereby maintaining skin elasticity and anti-wrinkle properties. Furthermore, L-theanine has antibacterial properties, inhibiting the growth of Propionibacterium acnes, helping to reduce acne and control oiliness.

[0004] Type III collagen is a key member of the human collagen family. Structurally, it forms a triple helix consisting of three α-chains. These chains are tightly intertwined, forming a stable molecular structure with numerous repetitive amino acid sequences. In the human body, it is primarily found in tissues such as the skin, blood vessel walls, and internal organs. Type III collagen plays a prominent role in maintaining tissue elasticity, preventing skin sagging and wrinkles, and maintaining a youthful appearance. It has a wide range of applications in medicine and cosmetics. In medicine, it is used in tissue engineering and organ repair, such as constructing artificial skin and blood vessels. In cosmetics, type III collagen can be used in anti-wrinkle and moisturizing skincare products and cosmetic injections to improve skin condition. In the cosmetic and pharmaceutical fields, ectoine, L-theanine, and type III collagen are often used in combination. However, in actual production, ectoine, L-theanine, and type III collagen are typically synthesized separately by fermentation using three recombinant bacteria. This process is time-consuming and labor-intensive, necessitating a recombinant bacterium that can simultaneously synthesize ectoine, L-theanine, and type III collagen. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art of lacking a recombinant bacterium capable of simultaneously synthesizing ectoine, L-theanine and type III collagen.

[0006] To address the above technical problems, the present invention provides a recombinant bacterium that can produce high levels of ectoine, L-theanine, and type III collagen, and its use. The present invention discovered that introducing a plasmid containing an ectoine biosynthetic enzyme gene and a plasmid containing a γ-glutamine synthetase gene and a type III collagen gene into Corynebacterium glutamicum ATCC 13032 can simultaneously produce high levels of ectoine, L-theanine, and type III collagen. Furthermore, the present invention also linked an aspartate kinase gene and an aspartate semialdehyde dehydrogenase gene to the plasmid containing the ectoine biosynthetic enzyme gene to increase ectoine production. After fermentation, the recombinant bacterium can simultaneously synthesize 25.1 g / L of ectoine, 9.82 g / L of L-theanine, and 356 mg / L of type III collagen.

[0007] The first object of the present invention is to provide a recombinant bacterium that produces high levels of ectoine, L-theanine, and type III collagen. The recombinant bacterium is obtained by modifying Corynebacterium glutamicum to overexpress ectoine biosynthetic enzyme, γ-glutamine synthetase, and type III collagen, wherein the ectoine biosynthetic enzyme, γ-glutamine synthetase, and type III collagen are expressed via a dual-plasmid expression system, and γ-glutamine synthetase and type III collagen are expressed via the same plasmid.

[0008] Furthermore, since the synthesis of type III collagen itself is completed by translation and does not require the participation of enzymes, the synthesis of type III collagen can be achieved by directly connecting the gene sequence of type III collagen to the plasmid.

[0009] Furthermore, the recombinant bacteria also overexpresses aspartate kinase and aspartate semialdehyde dehydrogenase, and the aspartate kinase and the aspartate semialdehyde dehydrogenase are expressed with the tetrahydropyrimidine biosynthetic enzyme via the same plasmid.

[0010] Furthermore, the amino acid sequence of the tetrahydropyrimidine biosynthetic enzyme is shown in SEQ ID NO.1, the amino acid sequence of the γ-glutamine synthetase is shown in SEQ ID NO.4, and the amino acid sequence of the type III collagen is shown in SEQ ID NO.5.

[0011] Further, SEQ ID NO.5:

[0012]

[0013] Furthermore, the amino acid sequence of the aspartate kinase is shown in SEQ ID NO.2, and the amino acid sequence of the aspartate semialdehyde dehydrogenase is shown in SEQ ID NO.3.

[0014] Furthermore, the dual-plasmid expression system is a combination of two of the pXMJ19 plasmid, pECXK99E plasmid, pETM6R1 plasmid, pET28a plasmid, PRSF plasmid, pCOR plasmid, pCDF plasmid, and PACYC plasmid. Preferably, the aspartate kinase gene, the aspartate semialdehyde dehydrogenase gene, and the ectoine biosynthetic enzyme gene are expressed via the pXMJ19 plasmid, while the γ-glutamine synthetase gene and type III collagen gene are expressed via the pECXK99E plasmid. When plasmids pXMJ19 and pECXK99E are selected as the dual-plasmid expression system, since plasmid pXMJ19 itself can express antitoxins and plasmid pECXK99E itself can express toxins, the introduction of the dual-plasmid toxin-antitoxin system enables the recombinant bacteria to efficiently and stably co-produce ectoine, L-theanine, and type III collagen without the addition of antibiotics.

[0015] The second object of the present invention is to provide a method for high production of ectoine, L-theanine and type III collagen, which comprises inoculating the above-mentioned recombinant bacteria into a culture medium for fermentation and culture.

[0016] Furthermore, the fermentation culture temperature is 25-40°C.

[0017] Furthermore, the pH of the fermentation culture is 6-8.

[0018] Furthermore, the inoculation amount of the recombinant bacteria is 5-15%.

[0019] Furthermore, the culture medium includes a carbon source, a nitrogen source, an inorganic salt and metal ions.

[0020] Beneficial effects of the present invention:

[0021] The present invention discovered that introducing a plasmid containing an ectoine biosynthetic enzyme gene and a plasmid containing a γ-glutamine synthetase gene and a type III collagen gene into Corynebacterium glutamicum ATCC 13032 resulted in a recombinant bacterium capable of simultaneously producing high levels of ectoine, L-theanine, and type III collagen. Furthermore, the present invention also linked an aspartate kinase gene and an aspartate semialdehyde dehydrogenase gene to the plasmid containing the ectoine biosynthetic enzyme gene to increase ectoine production. After fermentation, the recombinant bacterium of the present invention was able to simultaneously synthesize 25.1 g / L of ectoine, 9.82 g / L of L-theanine, and 356 mg / L of type III collagen. This invention achieves the simultaneous synthesis of ectoine, L-theanine, and type III collagen, saving on fermentation technology and promising broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0023] Figure 1 It is the secondary structure of γ-glutamylmethylamide synthetase;

[0024] Figure 2 It is the secondary structure of tetrahydropyrimidine biosynthetic enzyme;

[0025] Figure 3 It is the secondary structure of type III collagen;

[0026] Figure 4 It is the yield graph of fed-batch fermentation;

[0027] Figure 5 It is the liquid phase diagram of tetrahydropyrimidine;

[0028] Figure 6 The effect of single plasmid expression system on yield. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0030] Strain: Corynebacterium glutamicum ATCC 13032 (C. glutamicum ATCC 13032) was purchased;

[0031] Plasmids: pXMJ19, pECXK99E;

[0032] BHI medium: brain heart infusion 37 g / L, sorbitol 91 g / L;

[0033] Fermentation medium: tryptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 40 g / L, K2HPO4 1.0 g / L, MgSO4·7H2O 0.2 g / L, MnSO4·H2O 0.05 g / L, FeSO4·7H2O 0.05 g / L, CaCO3 20 g / L, (NH4)2SO4 10 g / L, urea 7.0 g / L;

[0034] Preparation of competent bacteria of Corynebacterium glutamicum: Activation of bacteria: Take out the Corynebacterium glutamicum strain stored in -80℃ freezer, streak inoculate on BHI solid medium, and culture at 30℃ for 24-48 hours until obvious colonies appear. Seed culture: Pick a single colony from the plate and inoculate into 5mL BHI liquid medium, shake and culture at 30℃ and 220rpm for 8-12 hours until the OD 600 Expand the culture: inoculate 100 mL of BHI liquid medium with an initial OD of 0.2, and culture at 30°C and 220 rpm until the OD reaches 0.8-1.0. 600 Reach 0.8-1.0. Collect bacteria by centrifugation: Transfer the culture medium to a centrifuge tube, centrifuge at 4°C and 4000rpm for 10 minutes, and discard the supernatant. Washing: Resuspend the bacteria with pre-cooled 10% glycerol solution to a final volume of about 1 / 10 of the original culture volume. After gently mixing, centrifuge at 4°C and 4000rpm for 10 minutes, and discard the supernatant. Resuspension and aliquoting: Resuspend the bacteria again with pre-cooled 10% glycerol solution to a final volume of about 1 / 100 of the original culture volume. After gently mixing, aliquot into sterile 1.5mL centrifuge tubes, about 100μL per tube.

[0035] Purification and content determination of ectoine: 1 mL of fermentation broth was centrifuged (8000 rpm for 2 min). The supernatant was diluted with ultrapure water to an appropriate multiple (concentration within the standard curve range), filtered through a 0.22 μm aqueous microporous membrane, and then transferred to a liquid chromatography flask. Eectoine was determined using high-performance liquid chromatography (HPLC). The injection volume was 5 μL. The chromatographic column was a Thermo HypersilTM ODS-2C18 (4.6 × 250 mm, S um) column (or a Thermo HypersilTM GOLD aQ column). The column temperature was 30°C, and the mobile phase was pure acetonitrile:purified water (2:98) at a flow rate of 0.6 mL / min. A UV detector (VWD) was used at a wavelength of 210 nm.

[0036] Purification and content determination of L-theanine: 1 mL of fermentation broth was centrifuged (8000 rpm for 2 min). The supernatant was diluted with ultrapure water to an appropriate multiple (concentration within the standard curve range), filtered through a 0.22 μm water-based microporous filter, and transferred to a liquid chromatography flask. L-theanine was determined using high-performance liquid chromatography (HPLC). The column used was a Prevail C18 (250 mm × 4.6 mm i.d., 5 μm). The mobile phase consisted of a 0.15% trifluoroacetic acid (containing 0.06% heptafluorobutyric acid) aqueous solution and acetonitrile (88:12). The flow rate was 0.8 mL / min. The detector was an ELSD 2000. The drift tube temperature was 111°C, the gas flow rate was 2.8 L / min, and the injection volume was 25 μL.

[0037] Purification and content determination of type III collagen: 1 mL of fermentation broth was centrifuged (8000 rpm for 2 min). The supernatant was diluted with ultrapure water to an appropriate multiple (concentration within the standard curve range), filtered through a 0.22 μm water-based microporous filter, and then transferred to a liquid chromatography flask. Type III collagen was determined using high-performance liquid chromatography. Column: Prevail C18 (250 mm × 4.6 mm ID, 5 μm); Column temperature: 40°C; Mobile phase: Methanol: Tetrahydrofuran-methanol (0.05 mol / L): Sodium acetate = 1:15:84 (V / V); Gradient elution, flow rate: 1 mL / min; Column temperature: 40°C; Detection wavelength: 254 nm; Injection volume: 20 μL.

[0038] Example 1: Construction of an Efficient Tetrahydropyrimidine-Producing Strain

[0039] (1) Introduction of ectoine synthesis operon genes

[0040] Based on the genomic sequence of the ectoine biosynthetic enzyme from Pseudomonas stutzeri, the amino acid sequence of the gene cluster ectABC was synthesized by GENEWIZ (Suzhou) as shown in SEQ ID NO. 1. Primers were designed based on the ectoine biosynthetic enzyme gene sequence. Using the synthesized ectABC gene sequence as a template, primers ectABC-F and ectABC-R were designed for PCR amplification. The ectABC gene sequence was then ligated into the plasmid pXMJ19 using the T5 exonuclease-dependent assembly (TEDA) method to construct the recombinant expression plasmid pXMJ19-ectABC.

[0041] Table 1 Primers used to construct the recombinant expression plasmid pXMJ19-ectABC

[0042]

[0043] (2) Overexpression of the aspartate kinase gene lysC and the aspartate semialdehyde dehydrogenase gene asd

[0044] The amino acid sequence of the lysC gene, synthesized by GENEWIZ (Suzhou) using the C. glutamicum ATCC 13032 genome sequence, is shown in SEQ ID NO. 2. The amino acid sequence of the asd gene, synthesized by GENEWIZ using the E. coli BL21 genome sequence, is shown in SEQ ID NO. 3. Using the synthesized lysC and asd genes as templates, primers CglysC-F, CglysC-R, Ecasd-F, and Ecasd-R were designed for PCR amplification, respectively. The lysC and asd gene sequences were then ligated into the plasmid pXMJ19-ectABC using the T5 exonuclease-dependent assembly (TEDA) method to construct the recombinant expression plasmid pXMJ19-ectABC-CglysC-Ecasd, which was then transformed into Corynebacterium glutamicum ATCC 13032 to obtain the recombinant strain CG01.

[0045] Table 2 Primers used in the construction of recombinant strain CG01

[0046]

[0047] Example 2: Construction of a high-yield L-theanine plasmid

[0048] Based on the genomic sequence of γ-glutamine synthetase from Paracoccus aminovorans, the amino acid sequence of the gene PaGMAS was synthesized by GENEWIZ (Suzhou) as shown in SEQ ID NO. 4. Using the synthesized PaGMAS gene sequence as a template, primers PaGMAS-F and PaGMAS-R were designed for PCR amplification. The PaGMAS gene was then ligated into the plasmid pECXK99E using the TEDA method to construct the recombinant expression plasmid pECXK99E-PaGMAS.

[0049] Table 3 Primers used in the construction of recombinant expression plasmid pECXK99E-PaGMAS

[0050]

[0051] Example 3: Optimization of gene sequence truncation and plasmid construction for synthesis of type III collagen

[0052] According to the gene sequence of human collagen III α1 chain, the amino acid of gene col synthesized by GENEWIZ (Suzhou) is shown in SEQ ID NO.5. According to the secondary structure of type III collagen (such as Figure 3As shown in the figure, primers were designed to truncate type III collagen. Type III collagen selected a partial sequence of the human α1 chain. Type III collagen is a homotrimer composed of three identical α1 chains. This sequence was selected to ensure its structural integrity. At the same time, the sequence truncation was optimized, retaining the functional region and omitting the non-essential region to increase the expression efficiency and solubility of collagen.

[0053] Using the truncated col gene sequence as a template, primers col-F and col-R were designed for PCR amplification. The truncated col gene sequence was then ligated into the plasmid pECXK99E-PaGMAS by the T5 exonuclease-dependent assembly (TEDA) method to construct the recombinant expression plasmid pECXK99E-PaGMAS-col, which was then transformed into the recombinant Corynebacterium glutamicum CG01 to obtain CG02.

[0054] Table 4 Primers used in the construction of recombinant expression plasmid pECXK99E-PaGMAS-col

[0055]

[0056] Example 4: Fed-batch fermentation of recombinant Corynebacterium glutamicum in a 5 L fermentor

[0057] Fermentation production in a 5L fermenter: The recombinant Corynebacterium glutamicum CG02 constructed in Example 3 was inoculated into 5mL BHI medium and cultured overnight at 220rpm and 30℃. 600 =0.1 was transferred to a 25mL baffled Erlenmeyer flask containing fermentation medium. After incubation at 220rpm and 30℃ for 10h, 10% of the inoculum was inoculated into a 5L fermentor. The initial temperature was set at 30℃ and the rotation speed was 3000r / min. After 4h of fermentation, IPTG was added to a final concentration of 0.2mM to induce gene expression. During the fermentation process, the pH of the fermentation liquid was controlled at around 7 with 14% ammonia water. Glucose was fed to maintain the pH in the fermentor at around 10g / L. Figure 4 The recombinant strain Corynebacterium glutamicum was fermented in a 5L fermenter to produce 25.1 g / L of ectoine, 9.82 g / L of L-theanine, and 356 mg / L of type III collagen.

[0058] Comparative Example: Effect of Single Plasmid Expression System on Yield

[0059] The recombinant expression plasmid pECXK99E-PaGMAS obtained in Example 2 was introduced into Corynebacterium glutamicum ATCC 13032 to obtain recombinant bacteria CG03. The truncated col gene sequence in Example 3 was used as a template, and primers col-F and col-R were designed for PCR amplification. The truncated col gene sequence was then connected to the plasmid pECXK99E by the T5 exonuclease-dependent assembly (TEDA) method to construct a recombinant expression plasmid pECXK99E-col. The recombinant plasmid pECXK99E-col was introduced into Corynebacterium glutamicum ATCC 13032 to obtain recombinant bacteria CG04. CG01, CG03 and CG04 were inoculated into a fermenter for fermentation and culture at the same time. Figure 6 As shown, the final ectoine yield was 18.6 g / L, theanine yield was 6.68 g / L, and type III collagen yield was 241 mg / L.

[0060] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A recombinant bacterium that produces high levels of ectoine, L-theanine, and type III collagen, characterized in that: The recombinant bacteria are obtained by modifying Corynebacterium glutamicum in the following ways: at least overexpressing ectoine biosynthetic enzyme, γ-glutamine synthetase and type III collagen; The ectoine biosynthetic enzyme, γ-glutamine synthetase and type III collagen are expressed through a dual-plasmid expression system, and γ-glutamine synthetase and type III collagen are expressed through the same plasmid.

2. The recombinant bacterium according to claim 1, characterized in that The recombinant bacteria also overexpresses aspartate kinase and aspartate semialdehyde dehydrogenase, and the aspartate kinase and the aspartate semialdehyde dehydrogenase are expressed with the tetrahydropyrimidine biosynthetic enzyme via the same plasmid.

3. The recombinant bacterium according to claim 1, characterized in that The amino acid sequence of the tetrahydropyrimidine biosynthetic enzyme is shown in SEQ ID NO.1, the amino acid sequence of the γ-glutamine synthetase is shown in SEQ ID NO.4, and the amino acid sequence of the type III collagen is shown in SEQ ID NO.

5.

4. The recombinant bacterium according to claim 2, characterized in that The amino acid sequence of the aspartate kinase is shown in SEQ ID NO.2, and the amino acid sequence of the aspartate semialdehyde dehydrogenase is shown in SEQ ID NO.

3.

5. The recombinant bacterium according to claim 1, characterized in that The dual-plasmid expression system comprises any two plasmids selected from the group consisting of pXMJ19 plasmid, pECXK99E plasmid, pETM6R1 plasmid, pET28a plasmid, PRSF plasmid, pCOR plasmid, pCDF plasmid and PACYC plasmid.

6. A method for high production of ectoine, L-theanine and type III collagen, characterized in that: The method comprises inoculating the recombinant bacteria according to any one of claims 1 to 5 into a culture medium for fermentation and culture.

7. The method according to claim 6, characterized in that The fermentation culture temperature is 25-40°C.

8. The method according to claim 6, characterized in that The pH of the fermentation culture is 6-8.

9. The method according to claim 6, characterized in that The inoculation amount of the recombinant bacteria is 5-15%.

10. The method according to claim 6, characterized in that The culture medium includes a carbon source, a nitrogen source, inorganic salts and metal ions.

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