4-ethylene guaiacol oxygenase mutant and engineering strain for synthesizing vanillin in vitro and application of 4-ethylene guaiacol oxygenase mutant and engineering strain

Abstract

The invention relates to a 4-ethylene guaiacol oxygenase mutant for synthesizing vanillin in vitro, an engineering strain and application of the 4-ethylene guaiacol oxygenase mutant and the engineering strain. The amino acid sequence of the 4-ethylene guaiacol oxygenase mutant is obtained by mutation on the basis of a sequence shown in SEQ ID NO.1; the mutation site of the mutation comprises any one of W335, I404, V409 or Y413 or a combination of at least two of W335, I404, V409 or Y413. The enzyme activity of the obtained 4-ethene guaiacol oxygenase mutant is 1.4-6 times that before mutation, 16 g / L vanillin can be accumulated within 24 h in vitro by utilizing the mutant constructed by the invention, the conversion rate exceeds 90%, and the application of in-vitro synthesis of natural vanillin is realized.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and more particularly to a 4-vinylguaiacol oxygenase mutant and engineered strain for the in vitro synthesis of vanillin and its applications. Background Technology

[0002] Vanillin, also known as vanillin, has the chemical name 3-methoxy-4-hydroxybenzaldehyde, with the molecular formula C8H8O3 and a relative molecular weight of 152. Pure vanillin is a white or pale yellow powder or needle-like crystal, possessing the aroma of vanilla beans and a strong milky fragrance. Due to its unique aroma characteristics that cannot be artificially synthesized and its wide range of applications, it is known as the "Queen of Flavors" and is widely used in the food, pharmaceutical, feed, condiment, and cosmetic industries.

[0003] Currently, vanillin on the market includes natural vanillin and synthetic vanillin. Natural vanillin, extracted from plants, has a very limited yield and is expensive due to the long growing cycle and complex extraction process. Synthetic vanillin, obtained through chemical methods, has a large yield and low price, but its use in the food and pharmaceutical industries is strictly limited.

[0004] Microbial transformation, using natural substances as substrates and employing biotransformation, yields vanillin that is considered "natural vanillin," an ideal substitute for naturally sourced vanillin. Through the discovery and identification of related enzymes and genes, as well as research into metabolic pathways and fermentation conditions, more options and opportunities are provided for genetic engineering to increase vanillin yield and for large-scale production of vanillin via microbial transformation.

[0005] CN106754802A mutated the amino acid of isoeugenol monooxygenase from Pseudomonasputida E27, but the activity was only increased by 2-fold, and the highest vanillin concentration was less than 1.2 g / L. CN118389469A also mutated the amino acid of isoeugenol monooxygenase, but the enzyme activity was only twice that of the parent enzyme, and the highest yield was 13.44 g / L.

[0006] In summary, providing a method for the production of vanillin that is safe, has high enzyme activity, high conversion rate, and is suitable for large-scale production has become one of the urgent problems to be solved in this field. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a 4-ethyleneguaiacol oxygenase mutant and engineered strain for the in vitro synthesis of vanillin, along with their applications. This invention mutates the pocket center site of the 4-ethyleneguaiacol oxygenase Vgo. The Vgo mutant provided by this invention can efficiently catalyze the production of vanillin from 4-ethyleneguaiacol, demonstrating promising prospects for industrial applications.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a 4-ethylene guaiacol oxygenase mutant for the in vitro synthesis of vanillin, wherein the amino acid sequence of the 4-ethylene guaiacol oxygenase mutant is an amino acid sequence obtained by mutation based on the sequence shown in SEQ ID NO.1, and the mutation site includes any one or a combination of at least two of W335, I404, V409 or Y413.

[0010] SEQ ID NO.1:

[0011] .

[0012] This invention mutates the pocket center site of 4-vinylguaiacol oxygenase Vgo. The enzyme activities of the mutants W335F and W335F / I404D / V409W are 4.5 and 6 times that of the original mutants, respectively. They co-catalyze the conversion of ferulic acid to vanillin with ferulic acid decarboxylase, with a vanillin conversion rate of 90% and a yield of 17 g / L.

[0013] Preferably, the mutation includes:

[0014] Any one or a combination of at least two of the following: W335F, I404C, I404D, I404E, I404K, I404L, V409F, V409K, V409W, V409Y, Y413A, Y413C, Y413D, Y413E, Y413F, or Y413M.

[0015] In this invention, for ease of writing, "the amino acid at position 335 is mutated from tryptophan W to phenylalanine F" is abbreviated as "W335F", and the other point mutations are written in the same way.

[0016] Preferably, the combination of mutations includes any one of the following combinations:

[0017] (1) Combination of W335F and V409F; (2) Combination of W335F and V409Y; (3) Combination of W335F and V409W; (4) Combination of W335F, I404C and V409W; (5) Combination of W335F, I404D and V409W; (6) Combination of W335F, I404L and V409W; (7) Combination of W335F, I404E and Y413E; (8) Combination of W335F, I404E and Y413F; (9) Combination of W335F, V409W and Y413C; (10) Combination of W335F, V409W and Y413D; (11) Combination of W335F, V409Y and Y413M.

[0018] In this invention, for ease of writing, the point mutation combination "combination of W335F and V409F" is written as "W335F / V409F", and subsequent combinations follow the same pattern.

[0019] In a second aspect, the present invention provides a nucleic acid molecule that encodes the 4-vinylguaiacol oxygenase mutant described in the first aspect.

[0020] Thirdly, the present invention provides a recombinant vector containing the nucleic acid molecules described in the second aspect.

[0021] Preferably, the starting plasmid of the recombinant vector includes pETDuet-1.

[0022] Fourthly, the present invention provides a genetically engineered strain containing the nucleic acid molecule described in the second aspect or the recombinant vector described in the third aspect.

[0023] This invention obtains 4-ethyleneguaiacol oxygenase with higher enzyme activity by heterologous overexpression of the Vgo mutant, which can then be used for the in vitro synthesis of vanillin.

[0024] Preferably, the starting strain of the genetically engineered strain includes Escherichia coli.

[0025] Fifthly, the present invention provides a genetically engineered preparation containing any one or a combination of at least two of the following: a culture of the genetically engineered strain described in the fourth aspect, a culture extract, cell fragments, bacterial cells, fermentation broth, fermentation broth precipitate, or lyophilized powder.

[0026] In a sixth aspect, the present invention provides the use of the 4-vinylguaiacol oxygenase mutant as described in the first aspect, the nucleic acid molecule as described in the second aspect, the recombinant vector as described in the third aspect, the genetically engineered strain as described in the fourth aspect, or the genetically engineered preparation as described in the fifth aspect in the synthesis of vanillin.

[0027] In a seventh aspect, the present invention provides a method for synthesizing vanillin, the method comprising: mixing 4-vinylguaiacol and the 4-vinylguaiacol oxygenase mutant described in the first aspect to obtain the vanillin;

[0028] Alternatively, the method for synthesizing vanillin may include: mixing ferulic acid, ferulic acid decarboxylase, and the 4-vinylguaiacol oxygenase mutant described in the first aspect to obtain the vanillin.

[0029] Preferably, the mixing temperature is 45~55℃ (e.g., 45℃, 47℃, 49℃, 50℃, 51℃, 53℃ or 55℃, etc.).

[0030] Preferably, the method for synthesizing vanillin specifically includes:

[0031] S1. After activating the genetically engineered strain, seed culture was carried out to obtain seed solution;

[0032] S2. Inoculate the seed culture into the fermentation medium and ferment.

[0033] S3. Centrifuge to collect the genetically engineered strain, break the resuspended cell liquid, centrifuge again to collect the supernatant, and obtain the crude enzyme solution of the genetically engineered strain;

[0034] S4. Using 4-vinylguaiacol as a substrate, the crude enzyme solution of the genetically engineered strain from step S3 was added to react and vanillin was prepared.

[0035] S5. Using ferulic acid as a substrate, ferulic acid decarboxylase and crude enzyme solution of the genetically engineered strain from step S3 were added to react and vanillin was prepared.

[0036] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] This invention mutates the pocket center site of 4-vinylguaiacol oxygenase Vgo, resulting in enzyme activities (W335F, I404E, I404K, V409K, Y413A, W335F / V409F, W335F / V409W, W335F / V409Y, W335F / I404C / V409W, W335F / I404L / V409W, W335F / V409W / Y413C, W335F / V409Y / Y413M) that are 1.4-6 times higher than before the mutation. The mutant W335F / I404C / V409W achieves a 90% conversion rate of vanillin, yielding 17 g / L. The Vgo mutant provided by this invention can efficiently catalyze the conversion of 4-vinylguaiacol to vanillin, demonstrating promising industrial application prospects. Attached Figure Description

[0039] Figure 1 The HPLC chromatograms show the detection of vanillin, 4-vinylguaiacol, and ferulic acid.

[0040] Figure 2 Electrophoresis image of Vgo protein.

[0041] Figure 3 Electrophoresis diagram of Vgo mutant protein.

[0042] Figure 4 This is an electrophoresis image of Pad protein.

[0043] Figure 5 This is a diagram illustrating the reaction process of ferulic acid to vanillin.

[0044] Figure 6 The figure shows the yield of vanillin produced by the Vgo mutant and Pad enzyme. Detailed Implementation

[0045] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0046] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0047] The culture medium formulation involved in this invention is as follows:

[0048] LB liquid medium (1 L): 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, diluted with pure water to 1 L, autoclaved at 121°C for 15 min.

[0049] LB solid medium (1 L): Add 15 g of agar to the LB liquid medium formula.

[0050] Antibiotic-containing liquid culture medium: Ampicillin was added to the LB liquid culture medium formula to a final concentration of 100 μg / mL.

[0051] Vanillin detection method: Vanillin, ferulic acid, and 4-vinylguaiacol standards were dissolved in 100% methanol solution, and the contents of vanillin and 4-vinylguaiacol were detected by high performance liquid chromatography (HPLC).

[0052] The HPLC instrument was an Agilent 1260, the column was a Hypersil C18 (4.6 mm × 150 mm, 5 μm), and the mobile phase was water (containing 0.5% glacial acetic acid) and methanol gradient elution. The elution program is shown in Table 1.

[0053] Table 1

[0054]

[0055] A successful HPLC method for ferulic acid, 4-vinylguaiacol, and vanillin was established. Figure 1 As shown, the peak positions of vanillin, ferulic acid, and 4-vinylguaiacol were 9.01 min, 11.695 min, and 16.451 min, respectively. They were all single strong absorption peaks. The peak positions of the tested sample and the standard were consistent. The vanillin content of the sample was then calculated based on the standard curve.

[0056] Example 1: Construction of the pETDuet1-Vgo mutant

[0057] This embodiment first constructs an engineered strain of *Ecoli* BL21(DE3)-pETDuet1-Vgo, including the following steps: The recombinant plasmid *Ecoli* BL21(DE3)-pETDuet1-Vgo was synthesized by Tianjin Zhonghe Biotechnology Co., Ltd., and chemically transformed into competent *E. coli* cells. 5 μL of the recombinant plasmid was added to 50 μL of competent *E. coli* cells, gently resuspended and mixed, placed on ice for 30 minutes, heat-shocked at 42°C for 30 seconds, placed on ice for 2 minutes, and then 550 μL of liquid LB was added. The cells were incubated at 37°C and 150 rpm for 1 hour. The bacterial culture was then spread onto solid LB containing Amp. The 4-vinylguaiacol oxygenase Vgo has the amino acid sequence shown in SEQ ID NO. 1 and can stably catalyze the conversion of 4-vinylguaiacol to vanillin at 50°C. The nucleic acid sequence encoding the Vgo nucleic acid is shown in SEQ ID NO. 2, and the nucleic acid sequence of the plasmid vector pETDuet1 is shown in SEQ ID NO. 3.

[0058] SEQ ID NO.2:

[0059]

[0060] SEQ ID NO.3:

[0061]

[0062] Using the recombinant plasmid pETDuet1-Vgo as a template, in vitro mutation amplification was performed by PCR. The primer sequences used for site-directed mutagenesis are shown in Tables 2 and 3, where F represents the forward primer and R represents the reverse primer.

[0063] Table 2

[0064]

[0065] Table 3

[0066]

[0067] After PCR, 10 μL of PCR product was added to 1 μL of restriction endonuclease DpnI to digest the template plasmid. After reacting at 37℃ for 2 h, 5 μL of the product was transferred into 50 μL of E. coli BL21(DE3) competent cells, placed on ice for 0.5 h, heat-shocked at 42℃ for 30 s, and then incubated in 550 μL of LB liquid at 30℃ for 2 h. 100 μL of the product was plated on LB agar plates (containing 100 μg / mL Amp) and incubated overnight at 37℃. Five transformants were picked from each plate and cultured in LB liquid medium (containing 100 μg / mL Amp) for 12 h. The transformed strains were then sent for sequencing, and the correctly sequenced recombinant strains were obtained.

[0068] Example 2: Protein expression and purification of 4-ethylene guaiacol oxygenase Vgo and its mutants

[0069] 1. Protein expression

[0070] The recombinant strain E. coli BL21(DE3)-pETDuet-Vgo and its mutants were inoculated into 100 mL LB medium (containing 100 μg / mL Amp) and cultured at 37℃ and 220 rpm in a shaker until the bacterial concentration reached OD500. 600 The concentration was 0.6-0.8, and IPTG and FeCl2 were added to a final concentration of 1 mmol / L and 0.2 mmol / L, respectively. Expression was induced at 25℃ for 12-16 h.

[0071] After centrifuging the bacterial culture at 8000 rpm for 5 min, the supernatant was discarded. The cells were washed with 5 mL of 50 mM phosphate buffer (pH=7.4), centrifuged again, and the supernatant was discarded. The cells were then resuspended in Tri-HCl buffer, and the OD was measured using a UV spectrophotometer. 600The results were recorded. Cells were disrupted on ice using a cell disruptor (40 kHz, sonication for 3 s, pause for 5 s), centrifuged at 8000 rpm for 25 min, and the supernatant was collected. The precipitate was resuspended with an equal volume of Tri-HCl buffer, and the protein samples were subjected to SDS-PAGE gel electrophoresis to verify protein expression.

[0072] The protein expression of recombinant strain E. coli BL21(DE3)-pETDuet-Vgo is as follows: Figure 2 As shown, M is the marker, 1 is the supernatant of the Ecoli BL21(DE3) strain lysate, 2 is the precipitate of the Ecoli BL21(DE3) strain lysate, 3 is the supernatant of the Ecoli BL21(DE3)-Vgo strain lysate, and 4 is the precipitate of the Ecoli BL21(DE3)-Vgo strain lysate.

[0073] Protein expression in the recombinant E. coli BL21(DE3)-pETDuet-Vgo mutant is as follows: Figure 3As shown, M is the marker, 1 is the supernatant of the Ecoli BL21(DE3) strain lysate, 2 is the precipitate of the Ecoli BL21(DE3) strain lysate, 3 is the supernatant of the mutant I404E lysate, 4 is the precipitate of the mutant I404E lysate, 5 is the supernatant of the mutant I404K lysate, 6 is the precipitate of the mutant I404K lysate, 7 is the supernatant of the mutant V409K lysate, 8 is the supernatant of the mutant Y413A lysate, 9 is the precipitate of the mutant V409K lysate, 10 is the precipitate of the mutant Y413A lysate, 11 is the supernatant of the mutant W335F lysate, 12 is the precipitate of the mutant W335F lysate, and 13 is the supernatant of the mutant W335F / V404K lysate. 9 is the supernatant of the lysing fluid; 14 is the precipitate of the lysing fluid of mutant W335F / V409W; 15 is the supernatant of the lysing fluid of mutant W335F / V409F; 16 is the precipitate of the lysing fluid of mutant W335F / V409F; 17 is the supernatant of the lysing fluid of mutant W335F / V409Y; 18 is the precipitate of the lysing fluid of mutant W335F / V409Y; 19 is the supernatant of the lysing fluid of mutant W335F / I404C / V409W; 20 is the precipitate of the lysing fluid of mutant W335F / I404C / V409W; 21 is the supernatant of the lysing fluid of mutant W335F / I409Y. 4D / V409W lysis fluid supernatant, 22 is mutant W335F / I404D / V409W lysis fluid precipitate, 23 is mutant W335F / I404L / V409W lysis fluid supernatant, 24 is mutant W335F / I404L / V409W lysis fluid precipitate, 25 is mutant W335F / I404E / Y413E lysis fluid supernatant, 26 is mutant W335F / I404E / Y413E lysis fluid precipitate, 27 is mutant W335F / I404E / Y413F lysis fluid supernatant, 28 is mutant W 335F / I404E / Y413F lysate, 29 is the supernatant of the lysate of mutant W335F / V409W / Y413C, 30 is the lysate of the lysate of mutant W335F / V409W / Y413C, 31 is the supernatant of the lysate of mutant W335F / V409W / Y413D, 32 is the lysate of the lysate of mutant W335F / V409W / Y413D, 33 is the supernatant of the lysate of mutant W335F / V409Y / Y413M, and 34 is the lysate of the lysate of mutant W335F / V409Y / Y413M.

[0074] 2. Protein purification

[0075] The crude protein enzyme solution was separated and purified by nickel column affinity chromatography, using Hispur nickel column packing material. TMNi-NTAResin allows target proteins with a His tag to specifically bind to the resin. After washing away contaminating proteins with wash buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM imidazole; pH 7.5), the protein is eluted with elution buffer (50 mM Tris-HCl, 150 mM NaCl, 200 mM imidazole; pH 7.5). The resulting protein eluent is concentrated by centrifugation using an Amino Ultra-15 (30 kDa) ultrafiltration tube, and residual imidazole from the protein purification process is washed away with desalination buffer (50 mM Tris-HCl, 150 mM NaCl; pH 7.5). Glycerol is added to the resulting protein solution to a final concentration of 10%, and the solution is stored at -20°C.

[0076] Example 3 Comparison of catalytic activity of mutant and parental pure enzyme in the synthesis of vanillin

[0077] 1. Strains culture and preparation of crude enzyme solution

[0078] The bacterial strain culture and crude enzyme solution preparation were carried out in accordance with the protein expression method in Experiment Example 2.

[0079] The reaction of 2,4-vinylguaiacol to vanillin

[0080] A 2 g / L 4-vinylguaiacol solution was prepared using phosphate buffer (pH=7.4) containing 20% ​​glycerol. 990 μL of the 4-vinylguaiacol solution was added to 10 μL (0.46 mg / mL) of purified enzyme solution. The reaction temperature was 50℃, and the buffer pH was 9. After the reaction was complete, 1 mL of methanol was added to terminate the reaction. The sample was analyzed by HPLC. The relative activity of the mutant (%) = mutant vanillin yield (mM) / parental enzyme Vgo vanillin yield (mM) × 100%. After 15 min of reaction, the results of the mutant assay compared with wild-type Vgo are shown in Table 4.

[0081] Table 4

[0082]

[0083] Example 4: Application of mutant and ferulic acid decarboxylase Pad in the catalysis of ferulic acid to vanillin

[0084] 1. Construction of recombinant plasmid pETduet1-Pad

[0085] Pad, a ferulic acid decarboxylase derived from *Thermus thermophilus*, has the function of decarboxylating ferulic acid to 4-vinylguaiacol. Its NCBI accession number is WP_017550974.1. The encoding nucleic acid of Pad, after codon optimization, was synthesized by Zhonghe Gene Biotechnology Co., Ltd. A histidine tag was added to facilitate protein purification. The nucleic acid sequence is shown in SEQ ID NO. 36, and the amino acid sequence is shown in SEQ ID NO. 37. The nucleic acid sequence of the plasmid vector pETduet1 is shown in SEQ ID NO. 3.

[0086] SEQ ID NO.36:

[0087] .

[0088] SEQ ID NO.37:

[0089] MKTLEEFLGTHMIYTYENGWEYEFYVKNQNTVDYRIHSGMVGGRWVRGQKADIVKITDGVFKVSWTEPTGTDVSLNFMPDDKRMHGVIFFPKWVHEHPEITVCYQNDHIDLMEESREKYETYPKYVVPEFADITYIKNEGINNEKVISEAPYATMADDIRSGKLKFSHHHHHH.

[0090] 2. Strains culture and preparation of crude enzyme solution

[0091] The bacterial strain culture and crude enzyme solution preparation were performed according to the protein expression method in Example 2. Protein samples were subjected to SDS-PAGE gel electrophoresis to verify protein expression; Pad expression was as follows. Figure 4 As shown, M is the Marker, 1 is the supernatant of Ecoli BL21(DE3) lysate, 2 is the supernatant of Ecoli BL21(DE3)-Pad lysate, 3 is the precipitate of Ecoli BL21(DE3) lysate, and 4 is the precipitate of Ecoli BL21(DE3)-Pad lysate.

[0092] Prepare a 25 g / L ferulic acid solution using phosphate buffer (pH=9). Add 18.5 mL of ferulic acid solution, 1 mL of crude enzyme solution of the Vgo mutant W335F / I404L / V409W, and 500 μL of crude enzyme solution of Pad. The reaction temperature is 50℃. The conversion of ferulic acid to vanillin under the action of Pad and Vgo is as follows... Figure 5 As shown.

[0093] At 6 h, 8 h, 12 h, and 24 h, 10 μL of sample solution was taken and 900 μL of methanol was added to terminate the reaction. The samples were then analyzed by HPLC. Vanillin yield (g / L) = vanillin molar concentration (mM) × 152 (valine molecular weight), vanillin conversion rate (%) = final vanillin molar concentration (mM) / initial ferulic acid molar concentration (mM) × 100%.

[0094] Test results as follows Figure 6 As shown, 16 g / L of vanillin can be accumulated in vitro within 24 h, with a conversion rate of over 90%. This invention realizes the application of in vitro synthesis of natural vanillin.

[0095] In summary, this invention mutates the pocket center site of 4-vinylguaiacol oxygenase Vgo, and the resulting Vgo mutant enzyme has an activity that is 1.4-6 times higher than that before the mutation. It can efficiently catalyze the production of vanillin from 4-vinylguaiacol and has good prospects for industrial application.

[0096] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A 4-vinylguaiacol oxygenase mutant for in vitro synthesis of vanillin, characterized in that, The amino acid sequence of the 4-vinylguaiacol oxygenase mutant is the amino acid sequence obtained by mutating the sequence shown in SEQ ID NO.1, and the mutation sites of the mutation include any one or a combination of at least two of W335, I404, V409 or Y413.

2. The 4-vinylguaiacol oxygenase mutant according to claim 1, characterized in that, The mutation includes: any one or a combination of at least two of W335F, I404C or I404D or I404E or I404K or I404L, V409F or V409K or V409W or V409Y, Y413A or Y413C or Y413D or Y413E or Y413F or Y413M.

3. The 4-vinylguaiacol oxygenase mutant according to claim 1, characterized in that, The combination of the mutations includes any one of the following combinations: (1) The combination of W335F and V409F; (2) The combination of W335F and V409Y; (3) The combination of W335F and V409W; (4) The combination of W335F, I404C and V409W; (5) The combination of W335F, I404D and V409W; (6) The combination of W335F, I404L and V409W; (7) The combination of W335F, I404E and Y413E; (8) The combination of W335F, I404E and Y413F; (9) The combination of W335F, V409W and Y413C; (10) The combination of W335F, V409W and Y413D; (11) The combination of W335F, V409Y and Y413M.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the 4-vinylguaiacol oxygenase mutant according to any one of claims 1 to 3.

5. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule according to claim 4.

6. The recombinant vector according to claim 5, characterized in that, The starting plasmid of the recombinant vector includes pETDuet-1.

7. A genetically engineered strain, characterized in that, The genetically engineered strain contains the nucleic acid molecule according to claim 4 or the recombinant vector according to claim 5 or 6.

8. A genetic engineering preparation, characterized in that, The genetically engineered preparation contains any one or a combination of at least two of the culture, culture extract, cell lysate, bacterial cells, fermentation broth, fermentation broth precipitate or lyophilized powder of the genetically engineered strain according to claim 7.

9. Use of the 4-vinylguaiacol oxygenase mutant according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5 or 6, the genetically engineered strain according to claim 7 or the genetically engineered preparation according to claim 8 in the synthesis of vanillin.

10. A method for synthesizing vanillin, characterized in that, The method for synthesizing vanillin includes: mixing 4-vinylguaiacol and the 4-vinylguaiacol oxygenase mutant according to any one of claims 1 to 3 to obtain the vanillin; Alternatively, the method for synthesizing vanillin includes: mixing ferulic acid, ferulic acid decarboxylase and the 4-vinylguaiacol oxygenase mutant according to any one of claims 1 to 3 to obtain the vanillin.

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