Recombinant thallus, construction method thereof and application of recombinant thallus in production of xanthoxylin WGX-50

Abstract

The invention relates to a recombinant thallus, a construction method of the recombinant thallus and application of the recombinant thallus in production of xanthoxylin WGX-50, and relates to the technical field of bioengineering. A recombinant plasmid containing a phenylalanine ammonialyase gene PAL with a nucleotide sequence as shown in SEQ ID NO.2 and a cinnamic acid coenzyme A ligase gene CNL with a nucleotide sequence as shown in SEQ ID NO.1 and a recombinant plasmid containing a tyramine N-hydroxycinnamoyl transferase gene THT with a nucleotide sequence as shown in SEQ ID NO.3 are introduced into the recombinant plasmid. A phenylalanine ammonialyase gene PAL, a cinnamic acid coenzyme A ligase gene CNL and a tyramine N-hydroxycinnamoyl transferase gene THT are overexpressed in escherichia coli, so that a biological synthesis path of xanthoxylin WGX-50 is formed by taking 3, 4-dioxomethyl phenylethylamine as a substrate for the first time, a foundation is laid for large-scale industrial production, and the method has a wide application prospect. The method has important economic value and social benefit.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a recombinant bacterial cell and a construction method thereof and an application thereof in the production of pepper WGX-50. Background Art

[0002] Huajiao WGX-50 is a natural ingredient found in the natural condiment Sichuan pepper, which has biological functions such as moisturizing and anti-oxidation. WGX-50 also has a specific gene regulatory effect, which can effectively inhibit the aggregation of Aβ amyloid protein in the human brain (the aggregation of Aβ amyloid protein in the brain is considered to be an important pathological feature of Alzheimer's disease) and the expression of lipid cell apoptosis protein, and can effectively protect nerve cells. Therefore, Huajiao WGX-50 has potential use in the preparation of anti-Alzheimer's disease drugs.

[0003] Huajiaosu WGX-50 has the following characteristics: English name Lemairamin, chemical name 2-Propenamide, N-[2-(3,4-dimethoxyphenyl)ethyl]-3-phenyl-, molecular formula C 19 H 21 NO3, molecular weight 311.1521, CAS number 29946-61-0, structural formula Figure 1 shown.

[0004] The traditional synthesis methods of pepper WGX-50 mainly include plant extraction and chemical synthesis. The plant extraction method is to extract pepper directly from pepper, but the extraction method is expensive and has low yield. The chemical synthesis method converts aromatic aldehydes and anhydrides into aromatic acrylic acid through the Perkin reaction, and then the aromatic acrylic acid intermediate is converted into aromatic acryloyl chloride through acyl chloride reaction. Finally, aromatic acryloyl chloride and aromatic amine are reacted by aminolysis to obtain the target product pepper WGX-50. However, this synthesis method will produce harmful gases such as hydrogen chloride gas and sulfur dioxide gas, and because the purity and yield of the product are difficult to control, its synthesis efficiency is not high and it is not environmentally friendly. Therefore, in view of the problems existing in the current production of pepper WGX-50, it is of great significance to study the biosynthesis method of pepper WGX-50. Summary of the invention

[0005] The purpose of the present invention is to provide a recombinant bacterial cell and a construction method thereof and an application thereof in the production of pepper WGX-50 in order to solve the above problems.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] The present invention provides a recombinant bacterial cell, which can simultaneously express several enzymes required for synthesizing xanthoxylum bungeanum WGX-50, including phenylalanine ammonia lyase, cinnamic acid coenzyme A ligase, and tyramine N-hydroxycinnamoyl transferase;

[0008] The recombinant bacteria uses Escherichia coli as a host strain, and is introduced with a recombinant plasmid containing a phenylalanine ammonia lyase gene PAL and a cinnamate coenzyme A ligase gene CNL, as well as a recombinant plasmid containing a tyramine N-hydroxycinnamoyltransferase gene THT;

[0009] The nucleotide sequence of the cinnamate-CoA ligase gene CNL is shown in SEQ ID NO.1;

[0010] The nucleotide sequence of the phenylalanine ammonia lyase gene PAL is shown in SEQ ID NO.2;

[0011] The nucleotide sequence of the tyramine N-hydroxycinnamoyltransferase gene is shown in SEQ ID NO.3.

[0012] As a further optimization scheme of the present invention, the host strain is Escherichia coli BL21 (DE3).

[0013] As a further optimization scheme of the present invention, the cinnamate coenzyme A ligase gene CNL is derived from Hypericum perilla;

[0014] The phenylalanine ammonia lyase gene PAL is derived from Arabidopsis thaliana;

[0015] The tyramine N-hydroxycinnamoyltransferase gene THT is derived from pepper.

[0016] The present invention also provides an application of the recombinant bacterial cell in producing peppermint WGX-50.

[0017] The present invention also provides a method for constructing a recombinant bacterial cell, the specific steps of which are as follows:

[0018] (1) ligating the phenylalanine ammonia lyase gene PAL and the cinnamate coenzyme A ligase gene CNL to the plasmid vector pRSFDuet-1 to obtain the recombinant plasmid pRSFDuet-CNL-PAL;

[0019] (2) connecting the tyramine N-hydroxycinnamoyltransferase gene THT to the plasmid vector pACYCDuet-1 to obtain the recombinant plasmid pACYCDuet-THT;

[0020] (3) The recombinant plasmid pRSFDuet-CNL-PAL and the recombinant plasmid pACYCDuet-THT obtained in step (1) and step (2) are introduced into the host strain to construct a recombinant bacterial cell.

[0021] The recombinant bacteria also contain three genes corresponding to the relevant enzymes in the biosynthesis pathway of zanthoxylum bungeanum WGX-50: phenylalanine ammonia lyase gene PAL, cinnamate coenzyme A ligase gene CNL and tyramine N-hydroxycinnamoyltransferase gene THT; Figure 2-3 As shown, the three genes construct the key biosynthetic pathway of pepper WGX-50 in the recombinant bacteria. Through a simple fermentation process, the purpose of producing pepper WGX-50 can be achieved using glucose as the carbon source and cheap 3,4-dioxymethylphenethylamine as the raw material.

[0022] The present invention also provides a method for preparing peppermint WGX-50 using recombinant bacteria, and the specific steps are as follows:

[0023] (1) inoculating the recombinant bacteria into a seed culture medium to obtain a seed solution;

[0024] (2) inoculating the seed solution obtained in step (1) into a fermentation medium for fermentation, adding IPTG to induce the expression of the target protein, and collecting bacterial cells;

[0025] (3) The bacterial cells obtained in step (2) are biotransformed using 3,4-dimethoxyphenylethylamine as a substrate to produce zanthoxylum bungeanum WGX-50.

[0026] The beneficial effects of the present invention are:

[0027] 1) The present invention provides a new biosynthetic method of pepper WGX-50 by overexpressing the phenylalanine ammonia lyase gene PAL of Arabidopsis thaliana and the cinnamate coenzyme A ligase gene CNL of Hypericum perforatum in Escherichia coli; and overexpressing the tyramine N-hydroxycinnamoyltransferase gene THT of pepper, and then using 3,4-dioxymethylphenethylamine as a substrate, thereby forming a biosynthetic pathway of pepper WGX-50. This method lays a foundation for large-scale industrial production and has important economic value and social benefits.

[0028] 2) In the present invention, Escherichia coli is used as a model organism, which has a clear background, grows quickly, is easy to ferment and culture on a large scale, and has low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the structural formula of Zanthoxylum bungeanum WGX-50.

[0030] Figure 2 Schematic diagram of the synthesis of zanthoxylum bungeanum WGX-50 using glucose as the carbon source and 3,4-dimethoxyphenylethylamine as the substrate.

[0031] Figure 3Schematic diagram of the metabolic pathway for synthesizing zanthoxylum bungeanum WGX-50 using 3,4-dimethoxyphenylethylamine and phenylalanine metabolism.

[0032] Figure 4 The HPLC analysis graphs of the biotransformation extracts (1 is the HPLC result graph of the standard product; 2 is the HPLC result graph of the biotransformation extracts of the recombinant bacteria).

[0033] Figure 5 This is the MS analysis spectrum of the biotransformation extract.

[0034] Figure 6 This is the MS analysis spectrum of Zanthoxylum bungeanum WGX-50 standard. DETAILED DESCRIPTION

[0035] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0036] 1. Reagents and Materials

[0037] 1. Both E. coli DH5α and E. coli BL21 (DE3) are commercially available. E. coli DH5α is used for cloning all genes in the present invention, and E. coli BL21 (DE3) is used for gene protein expression and biotransformation production of pepper WGX-50 in the present invention. E. coli DH5α and E. coli BL21 (DE3) competent cells are prepared according to conventional methods. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional conditions, such as the conditions described in "Molecular Cloning: Laboratory Manual", or according to the conditions recommended by the manufacturer of the corresponding biological reagent.

[0038] 2. Culture medium formula:

[0039] 1) Seed solution and protein expression medium

[0040] LB medium: 5 g / L yeast extract, 10 g / L NaCl, 10 g / L tryptone, the rest is water, sterilized at 121°C for 20 min.

[0041] 2) Biotransformation medium

[0042] M9 medium: 0.1mM 3,4-dimethoxyphenethylamine, 4.78g / L Na2HPO4, 3g / LKH2PO4, 0.5g / L NaCl, 0.5g / LNH4Cl, 0.12g / L MgSO4, 4.35g / L yeast extract, 20g / Lglucose.

[0043] In the actual culture process, a certain concentration of antibiotics can be added to the above culture medium to maintain the stability of the plasmid, such as 50 mg / L kanamycin and 25 mg / L chloramphenicol.

[0044] The methods used in this example are conventional methods known to those skilled in the art unless otherwise specified, and the reagents and other materials used are commercially available products unless otherwise specified.

[0045] 2. Methods

[0046] 1. Gene synthesis and construction of recombinant plasmids

[0047] The coding gene PAL of phenylalanine ammonia lyase from Arabidopsis thaliana (NCBI accession number: NP_190894.1), the coding gene CNL of cinnamate coenzyme A ligase from Hypericum perforatum (NCBI accession number: AFS60176.1), and the coding gene THT of tyramine N-hydroxycinnamoyltransferase from Capsicum annuum (NCBI accession number: NP_001311493.1) were codon optimized and then entrusted to General Bio (Anhui) Co., Ltd. for gene synthesis. The optimized gene sequences are shown in SEQ ID NO.1-3 (coding genes of cinnamate coenzyme A ligase, phenylalanine ammonia lyase, and tyramine N-hydroxycinnamoyltransferase, respectively);

[0048] The gene PAL was ligated between the restriction enzyme sites NdeI and BglII of the plasmid pRSFDuet-1 and named pRSFDuet-PAL;

[0049] The gene CNL was ligated between the restriction enzyme sites NcoI and BamHI of the plasmid pRSFDuet-1 and named pRSFDuet-CNL;

[0050] The gene THT was ligated between the restriction enzyme sites NcoI and BamHI of the plasmid pACYCDuet-1 and named pACYCDuet-THT;

[0051] Construction of recombinant plasmid pRSFDuet-CNL-PAL: The pRSFDuet-PAL and pRSFDuet-CNL obtained above were double-digested with NdeI and BglII, and the target fragment PAL and vector pRSFDuet-CNL after digestion were recovered using a recovery kit, and then ligated. The ligation product was transformed into E. coli DH5α, and positive clones were screened to obtain the recombinant plasmid pRSFDuet-CNL-PAL.

[0052] 2. Construction of recombinant strains

[0053] E. coli BL21 (DE3) competent cells were prepared according to conventional methods, and the recombinant plasmids pRSFDuet-CNL-PAL and pACYCDuet-THT were transformed into the host strain E. coli BL21 (DE3) competent cells by electroporation to obtain the recombinant strain. The specific method is as follows: 100 μL of E. coli BL21 (DE3) competent cells were taken, and about 1 μL of the above expression plasmids were added respectively, and the mixture was fully mixed and placed in an electric rotating cup that had been ice-bathed for 5 minutes, and then electroporated. After the electroporation, 1 mL of liquid LB medium was added, and the mixture was incubated at 37°C for 45 minutes on a shaking table, and then coated on a LB solid medium plate containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and cultured at 37°C overnight. After a single colony was grown, the protein induction expression in the next step was performed.

[0054] 3. Fermentation and culture to prepare bacteria for biotransformation

[0055] The preparation method of biotransformation bacteria is to induce protein expression in the recombinant Escherichia coli containing the expression plasmid obtained in step 2 above to obtain bacteria for biotransformation. The specific method is: pick a single clone and inoculate it in a test tube containing LB liquid culture medium, and culture it overnight in a shaker at 37°C and 220r / min. Then, transfer the overnight bacteria to LB liquid culture medium at a 2% inoculation amount, and culture it in a shaker at 37°C and 220r / min until the bacteria concentration OD 600 To about 0.6, add IPTG with a final concentration of 0.1-5mM, and culture in a shaker at 16-30°C and 220r / min for 18-24h to induce the expression of the target protein. After the induction process is completed, the fermentation culture is centrifuged to collect bacterial cells to obtain biotransformation bacteria.

[0056] 4. Establish a bioconversion process to produce peppermint WGX-50

[0057] The biotransformed bacteria obtained in step 3 above were resuspended in an equal volume of M9 medium to establish a biopreparation process for pepper WGX-50. Figure 2-3As shown, the specific method is: a 50mL whole cell transformation system is established using a triangular flask, 3,4-dimethoxyphenylethylamine with a final concentration of 0.1mM is added to 50mL of bacterial solution, and the catalytic reaction is carried out in a shaking table at 30°C and 220r / min for 24h. After the reaction is completed, the reaction components are analyzed by liquid chromatography.

[0058] 5. Detection of Zanthoxylum bungeanum WGX-50

[0059] 1) HPLC detection of products: The supernatant of biotransformation fermentation was extracted with an equal volume of ethyl acetate, dried on a rotary evaporator, dissolved in methanol, and determined by high performance liquid chromatography. The chromatographic conditions were: a C18 column was used as the analytical column, methanol-water was the mobile phase, the flow rate was 0.5 mL / min, and the detection wavelength was 277 nm.

[0060] The results are as follows Figure 4 As shown, HPLC analysis showed that after 24 hours of bioconversion reaction, peppermint WGX-50 was produced in the fermentation broth.

[0061] 2) MS analysis of the product: The product peak in step 1) was subjected to MS analysis, wherein the conditions for the MS analysis included: APCI positive ion source, and a molecular weight scanning range of 50-1000. The scanning results are shown in Figure 5 , the MS spectrum shows the MS characteristic peak 312.1594 of pepper WGX-50 (the MS spectrum of pepper WGX-50 standard is as follows Figure 6 ), indicating that peppermint WGX-50 was produced in the fermentation broth.

[0062] 3) H NMR analysis results of the product: The product in step 1) was subjected to H NMR analysis, and the result was 1H NMR (400 MHz, CDCl3): δ2.84 (2H, t, J = 6.9 Hz,), 3.64 (2H, q, J = 6.8 Hz), 3.87 (s, 6H, -OCH3), 6.31 (1H, d, J = 15.6 Hz), 7.62 (1H, d, J = 15.6 Hz, CH = CH-), 6.86–6.68 (3H, m, arom H), 7.51–7.32 (5H, m, arom H), which was consistent with the published H NMR data of Zanthoxylum bungeanum WGX-50, indicating that Zanthoxylum bungeanum WGX-50 was produced in the fermentation broth.

[0063] In summary, the present invention provides a new biosynthetic pathway for producing peppermint WGX-50, lays a foundation for the large-scale industrial production of peppermint WGX-50, and has important economic value and social benefits.

[0064] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A recombinant bacterial cell, characterized in that: The recombinant bacteria can simultaneously express several enzymes required for synthesizing the peppermint WGX-50, including phenylalanine ammonia lyase, cinnamic acid coenzyme A ligase, and tyramine N-hydroxycinnamoyl transferase; The recombinant bacteria uses Escherichia coli as a host strain, and is introduced with a recombinant plasmid containing a phenylalanine ammonia lyase gene PAL and a cinnamate coenzyme A ligase gene CNL, as well as a recombinant plasmid containing a tyramine N-hydroxycinnamoyltransferase gene THT; The nucleotide sequence of the cinnamate-CoA ligase gene CNL is shown in SEQ ID NO.1; The nucleotide sequence of the phenylalanine ammonia lyase gene PAL is shown in SEQ ID NO.2; The nucleotide sequence of the tyramine N-hydroxycinnamoyltransferase gene is shown in SEQ ID NO.

3.

2. The recombinant bacterial cell according to claim 1, characterized in that The host strain is Escherichia coli BL21 (DE3).

3. The recombinant bacterial cell according to claim 1, characterized in that The cinnamate coenzyme A ligase gene CNL is derived from Hypericum perforatum; The phenylalanine ammonia lyase gene PAL is derived from Arabidopsis thaliana; The tyramine N-hydroxycinnamoyltransferase gene THT is derived from pepper.

4. Use of the recombinant bacterial cell according to any one of claims 1 to 3 in the production of xanthoxylum bungeanum WGX-50.

5. A method for constructing a recombinant bacterial cell according to any one of claims 1 to 3, characterized in that: The specific steps are as follows: (1) ligating the phenylalanine ammonia lyase gene PAL and the cinnamate coenzyme A ligase gene CNL to the plasmid vector pRSFDuet-1 to obtain the recombinant plasmid pRSFDuet-CNL-PAL; (2) connecting the tyramine N-hydroxycinnamoyltransferase gene THT to the plasmid vector pACYCDuet-1 to obtain the recombinant plasmid pACYCDuet-THT; (3) The recombinant plasmid pRSFDuet-CNL-PAL and the recombinant plasmid pACYCDuet-THT obtained in step (1) and step (2) are introduced into the host strain to construct a recombinant bacterial cell.

6. A method for preparing Zanthoxylum bungeanum WGX-50 using the recombinant bacteria according to any one of claims 1 to 3, characterized in that: The specific steps are as follows: (1) inoculating the recombinant bacteria into a seed culture medium to obtain a seed solution; (2) inoculating the seed solution obtained in step (1) into a fermentation medium for fermentation, adding IPTG to induce the expression of the target protein, and collecting bacterial cells; (3) The bacterial cells obtained in step (2) are biotransformed using 3,4-dimethoxyphenylethylamine as a substrate to produce zanthoxylum bungeanum WGX-50.

Images