Method for constructing methoxy eugenol engineering strain and application thereof

Abstract

The present application relates to a kind of mustard alcohol acyltransferase gene and the use of protein coded by it, the key enzyme gene ElBAHD10 of methoxy eugenol biosynthesis in Elaeagnus pungens is identified and obtained in the present application in Elaeagnus pungens.The present application constructs the engineering bacteria of escherichia coli of methoxy eugenol biosynthesis by biological transformation using ElBAHD10 gene, and methoxy eugenol is obtained by using engineering bacteria biological transformation, and methoxy eugenol is obtained without being extracted by plant or being synthesized by chemistry.The ElBAHD10 gene provided in the present application can be greenly produced methoxy eugenol by bioengineering strain fermentation, and have great application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and biological transformation, and particularly relates to construction of an engineering strain expressing a key enzyme for biosynthesis of methoxyeugenol and application of the engineering strain to preparation of methoxyeugenol. BACKGROUND

[0002] Methoxyeugenol is a phenylpropene compound extracted from plants and has multiple biological activities. It is applied in food, spices and medicine and has effects of anti-pneumonia, anti-hepatitis and anti-cancer cell proliferation. In addition, methoxyeugenol has been proved to have an antifungal effect as a plant-derived defense compound. However, the yield of methoxyeugenol isolated from plants such as nutmeg is only 0.00061%, and although eugenol acid can be chemically synthesized into methoxyeugenol in one step, there are problems of low yield and high pollution risk of raw materials and reagents. With the increasing demand for methoxyeugenol, it is necessary to find a new sustainable and practical method to produce this kind of compound. The production problem of methoxyeugenol can be effectively solved by biological transformation. However, the biosynthesis pathway of methoxyeugenol needs to be analyzed, especially the key step of forming fat from the intermediate sinapyl alcohol, which affects the research and application of methoxyeugenol biosynthesis. SUMMARY

[0003] The present application aims to provide a sinapyl alcohol acyltransferase gene and its coding gene and application in preparation of methoxyeugenol, which can efficiently biosynthesize methoxyeugenol from sinapyl alcohol in microorganisms, providing a new way for mass production of methoxyeugenol.

[0004] To achieve the above-mentioned application purposes, the technical solutions of the present application are as follows:

[0005] In the above-mentioned engineering strain, the method for overexpressing the sinapyl alcohol acyltransferase gene ELBAHD10 is to place the entire sequence of the acetyltransferase ELBAHD10 gene after the promoter of an exogenous expression plasmid for expression.

[0006] The fragment is introduced by a recombinant expression vector;

[0007] The expression vector is specifically a recombinant expression plasmid pMAL-ELBAHD10 constructed by inserting the ELBAHD10 fragment into the EcoR I / Sal I enzyme cutting site of pMAL-c4x.

[0008] The nucleotide and amino acid sequences of the sinapyl alcohol acyltransferase ELBAHD10 are shown in SEQ ID No. 1 and SEQ ID No. 2.

[0009] The chassis strain of the engineering bacteria described above is Escherichia coli E. coli BL21(DE3).

[0010] The method for preparing methoxy eugenol catalyzed by the engineering bacteria described in the application has the synthetic route as shown below: the key esterification intermediate is generated by using the ELBAHD10 engineering strain to catalyze sinenol, and the compound methoxy eugenol is generated by using the EGS gene of the Escherichia coli itself to catalyze the key intermediate.

[0011] Based on the functional annotation information of the transcriptome of Elaeagnus umbellata, the candidate genes of acyltransferase are screened in the sequencing annotation results, sequence BLAST analysis is adopted, and one acyltransferase gene is found in the transcriptome of Elaeagnus umbellata. After the preparation of the cDNA of Elaeagnus umbellata, the amplification, cloning, protein expression, in-vitro enzyme activity reaction and HPLC-LC / MS detection of the candidate genes are performed, the ELBAHD10 gene catalyzing the acylation reaction of sinenol is finally identified. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 The schematic diagram of the synthesis of methoxy eugenol by the biological transformation of the engineering strain ElBAHD10.

[0013] Figure 2 The fragment map of the ElBAHD10 gene.

[0014] Figure 3 The protein expression results of ElBAHD10. 1, marker; 2, crude protein of ElBAHD10; 3, pure protein of ElBAHD10.

[0015] Figure 4 The results of the in-vitro analysis of the activity of ElBAHD10 by LC-MS. A, control group; B, catalysis results of the crude enzyme of ElBAHD10 and sinenol; C is the mass spectrum results of the product methoxy eugenol.

[0016] Figure 5 The nuclear magnetic carbon spectrum results of the conversion product methoxy eugenol.

[0017] Figure 6 The nuclear magnetic hydrogen spectrum results of the conversion product methoxy eugenol. DETAILED DESCRIPTION

[0018] The application is further explained according to the embodiments, and those skilled in the art can easily understand that the contents described in the embodiments are only used to illustrate the application, and should not and will not limit the application described in detail in the claims. The source of the biological material in the application is as follows:

[0019] The vector pMAL-c4x is a commercial vector purchased from Novagen

[0020] DNA polymerase Phanta was purchased from Vazyme (Nanjing Novozyme Biosearch Co., Ltd.)

[0021] The experimental methods used in the following examples are conventional methods unless otherwise specified

[0022] The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0023] Example 1, construction of methoxy eugenol-producing engineering strain

[0024] One, Construction of pMAL-ELAT1 plasmid

[0025] (1) Using the cDNA of Elaeagnus pungens as a template, using polymerase DNA polymerase Phanta, and using P1 and P2 as primers, PCR amplification was performed. The PCR program was: 95°C pre-denaturation for 1 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 90 s, 30 cycles, to obtain a 1359 bp ELAT1 gene fragment, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0026] ElBAHD10-F: agggaaggatttcagaattcATGGGATCCGAATCAGAAATTTT

[0027] ElBAHD10-R: aagcttgcctgcaggtcgacCTAGATGAGTTTTAGATAATCGCTAGTAAGC

[0028] (lowercase bases are homologous arms of the vector pMAL-c4x)

[0029] Homologous recombination was performed using the Vazyme One Step Cloning Kit to construct the plasmid. 3 μL Dpn I endonuclease (to remove circular template plasmid) was added to the reverse PCR amplified vector pMAL-c4x, and incubated at 37°C for 2 h. Single fragment homologous recombination reaction: optimal cloning vector addition amount = (0.02 x cloning vector base number) ng; optimal insert fragment addition amount = (0.04 x cloning vector base number) ng.

[0030] (2) According to the formula in (1), calculate the required DNA amount, mix the vector and the target fragment (operate on ice, the addition amount of each component is not less than 1 μL), add ddH2O to 5 μL, add 5 μL 2 x Clon Express Mix, use a pipette to gently blow and mix. Centrifuge at 3000 rpm for 30 s.

[0031] (3) Put into PCR instrument, 50 ℃, 5 min; take out, immediately place on ice to cool 2 min.

[0032] The recombinant liquid was introduced into E. coli DH5a, and positive transformants were screened by ampicillin-resistant plate, and verified by sequencing to obtain recombinant plasmid, named pMAL-ElBAHD10.

[0033] II. Transformation of plasmid into E. coli BL21(DE3)

[0034] Melt the competent cells in ice bath, 50 μL of competent cells, 5 μL of plasmid, gently and quickly add, flick with fingers, ice bath for 30 min, 42 ℃ heat shock for 45 seconds, ice bath for 2 min, add 500 μL of LB medium, 37 ℃, 220 rpm shaking for 45 min. 5000 rpm centrifugation for 1 min, remove 300 μL of supernatant, the remaining 200 μL, resuspend, all plate. After 30 min, invert the culture plate and incubate overnight, pick 10 single colonies, transfer to LB liquid medium containing ampicillin (pMAL-ElBAHD10), colony PCR to verify positive colonies, pick one correct colony for standby.

[0035] Example 2 ElBAHD10 activity verification

[0036] Strain: recombinant engineering bacteria E. coli BL21-pMAL-ELAT1;

[0037] Fermentation medium (g / L): peptone 10; yeast extract 5; NaCl 10; ampicillin 100 mg / mL.

[0038] Seed culture: glycerol-preserved strain was transferred to fresh LB liquid medium, 37 ℃, 200 rpm for 16 h; 1 ring of activated strain was streaked on LB solid medium with corresponding antibiotic, 37 ℃ for 12 h; a single colony was picked from fresh plate to a 250 mL flask containing 50 mL of seed medium, 37 ℃, 200 rpm for 12 h as the first level seed; the first level seed liquid was inoculated into another 50 mL of seed medium at a 1% inoculation amount, 37 ℃, 200 rpm for 12 h as the second level seed.

[0039] Fermentation culture and enzyme induction expression:

[0040] The secondary seed culture was inoculated at a rate of 1% into 50 mL of fermentation expression medium and cultured with shaking at 37°C and 220 rpm for 3 hours. After 3 hours, IPTG was added to a final concentration of 0.1 mM, and expression was induced at 16°C and 200 rpm. Expression was induced for 16 hours, and the cells were harvested, cell walls were broken, and protein supernatant was obtained after high-speed centrifugation. SDS-PAGE protein electrophoresis was then performed for detection. The results are shown below. Figure 3 .

[0041] Enzyme activity verification reaction

[0042] The activity of ElBAHD10 was determined by the synthesis of the sinaponic acid esterified product via acetylation. The reaction was carried out in 1.5 mL centrifuge tubes, and the reaction system contained: 10 mM acetyl-CoA, 100 μM sinaponic acid, 50 mM Tri-HCl (pH 7.5), and 10 μg purified ElBAHD10, with a total reaction volume of 100 μL. The reaction was carried out at 30 °C for 2 h. The reaction mixture was then extracted with an equal volume of methanol by sonication, centrifuged at 12000 rpm for 10 min, and the supernatant was collected for analysis of the reaction product by HPLC-MS.

[0043] The transformation products were analyzed using HPLC-TOF-MS-MS, and the analytical methods are as follows:

[0044] Chromatographic column: Agilent Poroshell 120 Ec-C 18 Column (2.1 mm × 150 mm, 2.7 μm), guard column (2.1 mm × 5 mm, 2.7 μm); mobile phase: water (A, containing 0.1% formic acid); methanol (B); gradient elution (B): 30-55% 0-8.0 min; 55% 8.0-12 min; 55-100% 12-22 min; 100% 22-30 min; 100-30% 30-30.1 min; 30% 30.1-40 min. Flow rate: 0.5 mL·min -1 Column temperature 28 ℃; injection volume 5 μL; detection wavelength 280 nm.

[0045] Example 3: Bioconversion for the production of methoxyeugenol

[0046] To 100 mL of the engineering strain ElBAHD10, ITPG was added to a final concentration of 0.1 mM, and the protein expression was induced at 16 °C, 200 rpm for 16 h. Then 100 mg of the substrate eugenol was added to the culture medium, and the biotransformation was carried out at 30 °C, 200 rpm for 12 h. The product methoxyeugenol was obtained by adding 200 mL of ethyl acetate to the fermentation broth for extraction, and then purified by thin layer chromatography. The structure of the methoxyeugenol product was confirmed by NMR analysis and UPLC-QTOF-MS / MS.

[0047] 1. ElBAHD10 base sequence

[0048] SEQ ID No. 1, ElBAHD10, 1374 bp

[0049]

[0050] 2. ElBAHD10 amino acid sequence

[0051] SEQ ID No. 2, ElBAHD10, 457aa

[0052] MGSESEIFRVNMNKKESISAALPLQEHWLQLSNLDLLLPPVDVGVFFCYNSNNSKKKMSFGSMVSVLKNSLAQVLVSYYAFAGEVVSNSAGEPEILCNNRGVDFFEAFADVCLRDVDFHDPDYSVEGKLVPKKKDGVLAVQATEMKCGGVVVGCTFDHRIADAYSTNMFLVAWAEIAQSKQISLLPSFRRSLLNPRRVDVDYSLDNMYIPVSQLKPDINITPDDEDYLISRIYYVKVDQLNHLQSLATPKNSPKPRTKLESFCALLWQLVAKSAKRDDYSYTKMGIVVDGRSRLGGESAAMDAYFGNVLSIPYGGRKIEELTENSISWVAQEVHNFLETAVTKEHFLGLIDWVEAHRPEPALAKIYSSGNEDGPAFVVSSGQRFPVEKVDFGWGVPVFGSYHFPWGGRAGYVMPMPSPSGNGDWVVYMHLLRSQMEYIETEASSIFKPLTSDYLKLI

Claims

1. The key enzyme gene ElBAHD10 for the biosynthesis of methoxyeugenol, the nucleotide sequence of which is shown in SEQ ID No.

1.

2. The protein encoded by the key enzyme gene ElBAHD10 as described in claim 1, the amino acid sequence of which is shown in SEQ ID No.

2.

3. A recombinant vector containing the key enzyme gene ElBAHD10 as described in claim 1.

4. A transgenic strain expressing the protein as described in claim 2, characterized in that: The expression strain was Escherichia coli BL21(DE3).

5. The application of the transgenic engineered strain according to claim 4 in the preparation of methoxyeugenol.

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