A 4-vinylguaiacol oxygenase mutant and an engineering strain for synthesizing vanillin in vitro and application thereof

Abstract

The present application relates to a kind of 4-vinyl guaiacol oxygenase mutant and engineering strain for synthesizing vanilla in vitro and application thereof, the amino acid sequence of the 4-vinyl guaiacol oxygenase mutant is the amino acid sequence obtained by mutation based on the sequence shown in SEQ ID NO.1, the mutation site of the mutation includes any one or at least two combinations of W335, I404, V409 or Y413.The enzyme activity of the 4-vinyl guaiacol oxygenase mutant obtained by the present application is 1.4-6 times that before mutation, the mutant constructed by the present application can accumulate 16 g / L of vanilla within 24 h in vitro, and the conversion rate is more than 90%, which realizes the application of in vitro synthesis of natural vanilla.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, and in particular to a 4-vinylguaiacol oxygenase mutant and an engineered strain for synthesizing vanillin in vitro and application thereof. BACKGROUND

[0002] Vanillin, also known as vanillin, has a chemical name of 3-methoxy-4-hydroxybenzaldehyde, a molecular formula of C8H8O3, and a relative molecular weight of 152. Vanillin is a white or light yellow powder or needle-like crystal, has a fragrance of vanilla beans and a strong milk fragrance, is known as the queen of spices due to its unique fragrance characteristics that cannot be artificially compounded and a wide range of applications, and is widely used in food, medicine, feed, condiments, and cosmetic industries.

[0003] Currently, vanillin on the market includes natural vanillin and synthetic vanillin. Natural vanillin obtained by extraction from plants has a long planting cycle, a complex extraction process, extremely limited yield, and high price. Synthetic vanillin obtained by chemical methods has a large yield and low price, but the use of chemically synthesized vanillin in the food and pharmaceutical industries is strictly limited.

[0004] Microbial transformation method uses natural substances as substrates and biological transformation, and the obtained vanillin is considered to be "natural vanillin", which is an ideal substitute for natural vanillin. Through the exploration and identification of related enzymes and genes, as well as the research on metabolic pathways and fermentation conditions, more choices and opportunities are provided for genetically engineering to improve the yield of vanillin and for large-scale production of vanillin by microbial transformation method.

[0005] CN106754802A performs amino acid mutation on isoeugenol monooxygenase from Pseudomonas putida E27, but the activity is only increased by 2 times, and the highest vanillin concentration is less than 1.2 g / L. CN118389469A also performs amino acid mutation on isoeugenol monooxygenase, and the enzyme activity is only 2 times that of the parent enzyme, and the highest yield is 13.44 g / L.

[0006] In summary, it has become one of the problems to be solved in the field to provide a method for producing safe, high-enzyme activity, high-conversion rate, and large-scale vanillin production. SUMMARY

[0007] To solve the above technical problems, the application provides a 4-vinyl guaiacol oxygenase mutant and an engineering strain for synthesizing vanillin in vitro and application thereof, the pocket center site of 4-vinyl guaiacol oxygenase Vgo is mutated, and the Vgo mutant provided by the application can efficiently catalyze 4-vinyl guaiacol to generate vanillin, and has good industrial application prospect.

[0008] To achieve the purpose, the application adopts the following technical solutions:

[0009] In a first aspect, the application provides a 4-vinyl guaiacol oxygenase mutant for synthesizing vanillin in vitro, the amino acid sequence of the 4-vinyl 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 of the mutation includes any one or a combination of at least two of W335, I404, V409 or Y413.

[0010] SEQ ID NO. 1:

[0011] MAHIHELAAEISNYANNRLTDPSKVRFPRTPVFASMNKPSRFEGDVFDLEISGTIPADIDGTFFRVQPDHRFPPLFEDDIHFNGDGSVTAVRISGGHADLKQRYVHTDRYVHETRARRSLFGRYRNPWTDNESVKGVIRTASNTNVVFWRGVLLALKEDGPPFAMDPVTLETLGRYDFEGQILSPTFTAHPKFDPDTGEMVCFAYEAGGDGSDCSVDVAVWTIDRDGKKTEECWYKAPFAGMIHDCGISKNWLVLALTPIKMDLERMKRGGNKFAWDPNEDQWYAVVPRRGGKSEDITWFRADNGFHGHVAGCYELPSGEVVFDLTVADGNVFFWFPPDENVTPPDGFAKRNRLTSPTVRWVFDPKAKKSAIRTPGAGDADVWVADERVKPALTWLTNGEFSRIDDRFVTKPYRHFWQAVVDPSRPYDFEKCGPPAGGLFNCLGHYTWSEEHYHAGGSVQNGAAEQRDLKGKFGLEDVYFAGPTMTFQEPTFIPREGGAEGEGYLIALLNHLDELRNDVVIFDAQHLAKGPLAVIHLPLKLKLGLHGNWVDHRDIDAWKQRRAEGGDVGPLKVATEPLPWQKKLAQQNGTNGTRA.

[0012] The present application mutates the pocket center site of 4-vinylguaiacol oxygenase Vgo, and the enzyme activities of the mutants W335F, W335F / I404D / V409W are 4.5 and 6 times of that before mutation, respectively. The 4-vinylguaiacol oxygenase mutants catalyze together with ferulic acid decarboxylase to generate vanillin from ferulic acid, and the conversion rate of vanillin is 90% and the yield reaches 17 g / L.

[0013] Preferably, the mutation comprises:

[0014] any one 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 or a combination of at least two thereof.

[0015] In the present application, for the convenience of writing, "the amino acid at the 335th position is mutated from tryptophan W to phenylalanine F" is simply written as "W335F", and the rest of the point mutation is written in the same way.

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

[0017] (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.

[0018] In the present application, for the convenience of writing, the combination of point mutations "the combination of W335F and V409F" is written in the form of "W335F / V409F", and the subsequent combinations are written in the same way.

[0019] In a second aspect, the present application provides a nucleic acid molecule encoding the 4-vinylguaiacol oxygenase mutant of the first aspect.

[0020] In a third aspect, the present application provides a recombinant vector containing the nucleic acid molecule of the second aspect.

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

[0022] In a fourth aspect, the present application provides a genetically engineered strain, which comprises the nucleic acid molecule of the second aspect or the recombinant vector of the third aspect.

[0023] The present application obtains 4-vinylguaiacol oxygenase with higher enzyme activity by heterologous overexpression of Vgo mutant, thereby being used for in vitro synthesis of vanillin.

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

[0025] In a fifth aspect, the present application provides a genetically engineered preparation, which comprises any one of the culture, culture extract, cell disruption product, cell body, fermentation broth, fermentation broth precipitate or freeze-dried powder of the genetically engineered strain of the fourth aspect or a combination of at least two thereof.

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

[0027] In a seventh aspect, the present application provides a method for synthesizing vanillin, which comprises mixing 4-vinylguaiacol and the 4-vinylguaiacol oxygenase mutant of the first aspect to obtain the vanillin.

[0028] Alternatively, the method for synthesizing vanillin comprises mixing ferulic acid, ferulic acid decarboxylase and the 4-vinylguaiacol oxygenase mutant of the first aspect to obtain the vanillin.

[0029] Preferably, the temperature during the mixing is 45-55℃ (for example, it can be 45℃, 47℃, 49℃, 50℃, 51℃, 53℃ or 55℃, etc.).

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

[0031] S1. Activating the genetically engineered strain and then performing seed culture to obtain seed liquid;

[0032] S2. Inoculating the seed liquid into fermentation medium and performing fermentation culture;

[0033] S3. Centrifuging to collect the genetically engineered strain, disrupting the resuspended cell liquid, centrifuging again to collect the supernatant, and obtaining crude enzyme liquid of the genetically engineered strain;

[0034] S4. With 4-vinylguaiacol as a substrate, adding the crude enzyme solution of the genetically engineered strain of step S3 to carry out the reaction, vanillin is prepared;

[0035] S5. With ferulic acid as a substrate, adding ferulic acid decarboxylase and the crude enzyme solution of the genetically engineered strain of step S3 to carry out the reaction, vanillin is prepared.

[0036] Other specific point values in the above numerical range can be selected, which will not be repeated here.

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

[0038] The present application mutates the pocket center site of 4-vinylguaiacol oxygenase Vgo, and the enzyme activity of W335F, I404E, I404K, V409K, Y413A, W335F / V409F, W335F / V409W, W335F / V409Y, W335F / I404C / V409W, W335F / I404L / V409W, W335F / V409W / Y413C, W335F / V409Y / Y413M is 1.4-6 times that before mutation. The conversion rate of the mutant W335F / I404C / V409W to the substrate vanillin is 90%, and the yield reaches 17g / L. The Vgo mutant provided by the present application can efficiently catalyze 4-vinylguaiacol to generate vanillin, and has good industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

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

[0040] Figure 2 The Vgo protein electrophoresis chart.

[0041] Figure 3 The Vgo mutant protein electrophoresis chart.

[0042] Figure 4 The Pad protein electrophoresis chart.

[0043] Figure 5 The reaction process chart of ferulic acid converted into vanillin.

[0044] Figure 6 The yield result chart of vanillin generated by Vgo mutant and Pad enzyme. DETAILED DESCRIPTION

[0045] For further elucidation of the technical means adopted by the present application and its effects, the present application is further described below in conjunction with examples and drawings. It can be understood that the specific embodiments described herein are merely for the purpose of explaining the present application, but not limiting the present application.

[0046] Unless otherwise specified in the examples, the techniques or conditions are in accordance with those described in the literature in the art, or in accordance with the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be purchased through regular channels.

[0047] The medium formula involved in the present application is as follows:

[0048] LB liquid medium (1 L): Tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, and pure water to 1 L, sterilized by 121℃ high-pressure steam for 15 min.

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

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

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

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

[0053] Table 1

[0054]

[0055] The HPLC method for ferulic acid, 4-vinyl guaiacol, and vanillin was successfully established, as shown in Table 1, the peak positions of vanillin, ferulic acid, and 4-vinyl guaiacol were 9.01 min, 11.695 min, and 16.451 min, respectively, which were all single strong absorption peaks. The peak positions of the detected sample and the standard sample were consistent, and then the content of vanillin in the sample was calculated according to the standard curve. Figure 1

[0056] Example 1 Construction of pETDuet1-Vgo Mutant

[0057] ​The present embodiment first constructs an Ecoli BL21(DE3)-pETDuet1-Vgo engineering strain, including the following steps: Ecoli BL21(DE3)-pETDuet1-Vgo recombinant plasmid is synthesized by Tianjin Zhonghe Bioengineering Company, and is transformed into E. coli competent cells by a chemical method. 5 μL of the recombinant plasmid is taken into 50 μL of the E. coli competent cells, and is mixed uniformly by blowing gently, and is placed on ice for 30 minutes, and is heat-shocked at 42°C for 30 seconds, and is placed on ice for 2 minutes, and 550 μL of liquid LB is added, and is cultured at 37°C, 150 rpm for 1 h, and the bacterial liquid is taken and spread on solid LB containing Amp. The 4-vinyl guaiacol oxygenase Vgo has an amino acid sequence as shown in SEQ ID NO. 1, and can stably catalyze 4-vinyl guaiacol to generate vanillin at 50°C. The nucleic acid sequence of the Vgo coding 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] PCR was used to amplify the in vitro mutation with recombinant plasmid pETDuet1-Vgo as template. The sequences of the primers used for site-directed mutagenesis are shown in Table 2 and Table 3, in which F represents forward primer and R represents reverse primer.

[0063] Table 2

[0064]

[0065] Table 3

[0066]

[0067] After the PCR, 10 μL of the PCR product was added to 1 μL of restriction endonuclease DpnI to digest the template plasmid. After 37°C reaction for 2 h, 5 μL was transferred to 50 μL of E. coli BL21(DE3) competent cells, which were placed on ice for 0.5 h, heated at 42°C for 30 s, and then 550 μL of liquid LB was added and incubated at 30°C for 2 h. 100 μL was spread on an LB solid plate (containing 100 μg / mL Amp) and incubated at 37°C overnight. After 12 h of culture in LB liquid medium (containing 100 μg / mL Amp), five transformants were selected for sequencing, and the recombinant strain with correct sequencing was obtained.

[0068] Example 2 Protein expression and purification of 4-vinylguaiacol 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 of LB medium (containing 100 μg / mL Amp) and incubated in a 37°C, 220 rpm shaker until the bacterial concentration reached OD 600 600.8. The final concentration of IPTG was 1 mmol / L and the final concentration of FeCl2was 0.2 mmol / L. The expression was induced at 25°C for 12-16 h.

[0071] After centrifugation of the bacterial solution at 8000 rpm for 5 min, the supernatant was removed, the cells were washed with 5 mL of 50 mM phosphate buffer (pH=7.4), and the supernatant was removed after centrifugation. The cells were suspended in Tri-HCl buffer and the OD 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 3Figure 1 shows the results of a Western blot analysis of the supernatant and pellet of the lysate of the E. coli BL21 (DE3) strain and the supernatant and pellet of the lysate of the mutants I404E, I404K, V409K, Y413A, W335F, W335F / V409W, W335F / V409F, W335F / V409Y, W335F / I404C / V409W, W335F / I404D / V409W, W335F / I404L / V409W, W335F / I404E / Y413E, W335F / I404E / Y413F, W335F / V409W / Y413C, W335F / V409W / Y413D, W335F / V409Y / Y413M. M is Marker, 1 is the supernatant of the lysate of the E. coli BL21 (DE3) strain, 2 is the pellet of the lysate of the E. coli BL21 (DE3) strain, 3 is the supernatant of the lysate of the mutant I404E, 4 is the pellet of the lysate of the mutant I404E, 5 is the supernatant of the lysate of the mutant I404K, 6 is the pellet of the lysate of the mutant I404K, 7 is the supernatant of the lysate of the mutant V409K, 8 is the supernatant of the lysate of the mutant Y413A, 9 is the pellet of the lysate of the mutant V409K, 10 is the pellet of the lysate of the mutant Y413A, 11 is the supernatant of the lysate of the mutant W335F, 12 is the pellet of the lysate of the mutant W335F, 13 is the supernatant of the lysate of the mutant W335F / V409W, 14 is the pellet of the lysate of the mutant W335F / V409W, 15 is the supernatant of the lysate of the mutant W335F / V409F, 16 is the pellet of the lysate of the mutant W335F / V409F, 17 is the supernatant of the lysate of the mutant W335F / V409Y, 18 is the pellet of the lysate of the mutant W335F / V409Y, 19 is the supernatant of the lysate of the mutant W335F / I404C / V409W, 20 is the pellet of the lysate of the mutant W335F / I404C / V409W, 21 is the supernatant of the lysate of the mutant W335F / I404D / V409W, 22 is the pellet of the lysate of the mutant W335F / I404D / V409W, 23 is the supernatant of the lysate of the mutant W335F / I404L / V409W, 24 is the pellet of the lysate of the mutant W335F / I404L / V409W, 25 is the supernatant of the lysate of the mutant W335F / I404E / Y413E, 26 is the pellet of the lysate of the mutant W335F / I404E / Y413E, 27 is the supernatant of the lysate of the mutant W335F / I404E / Y413F, 28 is the pellet of the lysate of the mutant W335F / I404E / Y413F, 29 is the supernatant of the lysate of the mutant W335F / V409W / Y413C, 30 is the pellet of the lysate of the mutant W335F / V409W / Y413C, 31 is the supernatant of the lysate of the mutant W335F / V409W / Y413D, 32 is the pellet of the lysate of the mutant W335F / V409W / Y413D, 33 is the supernatant of the lysate of the mutant W335F / V409Y / Y413M, 34 is the pellet of the lysate of the mutant W335F / V409Y / Y413M.

[0074] 2. Purification of the protein

[0075] The crude protein solution was purified by nickel column affinity chromatography. The filler of the nickel column was Hispur TMNi-NTA resin, the target protein with His tag, will specifically bind to the resin. After washing away the impurities using Wash buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM imidazole; pH 7.5), the protein is eluted using Elution buffer (50 mM Tris-HCl, 150 mM NaCl, 200 mM imidazole; pH 7.5). The protein eluate obtained above is concentrated by centrifugation using Amino Ultra-15 (30 kDa) ultrafiltration tubes, and the remaining imidazole from the protein purification process is washed away using 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 mutants and parent pure enzyme in synthesizing vanillin

[0077] 1. Strain cultivation and preparation of crude enzyme solution

[0078] The strain cultivation and preparation of crude enzyme solution were performed according to the protein expression method in Experimental Example 2.

[0079] 2. Reaction of 4-vinylguaiacol to vanillin

[0080] A 2 g / L solution of 4-vinylguaiacol was prepared using a phosphate buffer containing 20% glycerol as the solvent (pH = 7.4). 990 μL of the 4-vinylguaiacol solution was taken, and 10 μL (0.46 mg / mL) of the pure enzyme solution was added. The reaction temperature was 50°C, and the buffer pH was 9. After the reaction was completed, 1 mL of methanol was added to terminate the reaction, and the sample was subjected to HPLC detection. The relative activity of the mutant (%) = (vanillin yield of the mutant (mM) / vanillin yield of the parent enzyme Vgo (mM)) x 100%. After 15 min of reaction, the results of the mutants were compared with those of the wild-type Vgo, as shown in Table 4.

[0081] Table 4

[0082]

[0083] Example 4 Application of mutants and ferulic acid decarboxylase Pad in catalyzing ferulic acid to vanillin

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

[0085] Ferulic acid decarboxylase Pad from Thermus thermophilus has the function of decarboxylating ferulic acid to generate 4-vinylguaiacol, and its NCBI accession number is WP_017550974.1. The coding nucleic acid of Pad was codon-optimized and sent to Zhejiang Hisgen Biotech Co., Ltd. for synthesis. A histidine tag was added for protein purification. The nucleic acid sequence is shown as SEQ ID NO. 36, and the amino acid sequence is shown as SEQ ID NO. 37. The nucleic acid sequence of the plasmid vector pETduet1 is shown as SEQ ID NO. 3.

[0086] SEQ ID NO. 36:

[0087] ATGAAAACCCTGGAAGAATTCCTGGGTACCCACATGATCTACACCTACGAAAACGGTTGGGAATACGAATTCTACGTTAAAAACCAGAACACCGTTGACTACCGTATCCACTCTGGTATGGTTGGTGGTCGTTGGGTTCGTGGTCAGAAAGCTGACATCGTTAAAATCACAGACGGTGTGTTCAAAGTGTCTTGGACCGAACCGACCGGTACCGACGTTTCTCTGAACTTCATGCCGGACGACAAACGTATGCACGGTGTTATCTTCTTCCCGAAATGGGTTCACGAACACCCGGAAATCACCGTTTGCTACCAGAACGACCACATCGACCTGATGGAAGAATCTCGTGAAAAATACGAAACCTACCCGAAATACGTTGTTCCGGAATTCGCTGACATCACCTACATCAAAAACGAAGGTATCAACAACGAAAAAGTTATCTCTGAAGCTCCGTACGCTACCATGGCTGACGACATCCGTTCTGGTAAACTGAAATTCTCTCACCACCACCACCACCACTAA.

[0088] SEQ ID NO. 37:

[0089] MKTLEEFLGTHMIYTYENGWEYEFYVKNQNTVDYRIHSGMVGGRWVRGQKADIVKITDGVFKVSWTEPTGTDVSLNFMPDDKRMHGVIFFPKWVHEHPEITVCYQNDHIDLMEESREKYETYPKYVVPEFADITYIKNEGINNEKVISEAPYATMADDIRSGKLKFSHHHHHH.

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

[0091] The strain culture and preparation of crude enzyme solution were carried out according to the protein expression method in Experimental Example 2. The protein samples were subjected to SDS-PAGE gel electrophoresis to verify the protein expression, and the Pad expression is shown in Figure 4 , wherein M is Marker, 1 is Ecoli BL21 (DE3) supernatant, 2 is Ecoli BL21 (DE3)-Pad supernatant, 3 is Ecoli BL21 (DE3) precipitate, and 4 is Ecoli BL21 (DE3)-Pad precipitate.

[0092] A 25 g / L solution of ferulic acid was prepared, and the solvent was phosphate buffer (pH=9). 18.5 mL of ferulic acid solution, 1 mL of Vgo mutant W335F / I404L / V409W crude enzyme solution and 500 μL of Pad crude enzyme solution were used, and the reaction temperature was 50℃. The process of converting ferulic acid into vanillin under the action of Pad and Vgo is shown in Figure 5 .

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

[0094] The detection results are shown in Figure 6 , which can accumulate 16 g / L of vanillin within 24 h in vitro, and the conversion rate is more than 90%. The application realizes the application of in vitro synthesis of natural vanillin.

[0095] In summary, the pocket center site of 4-vinyl guaiacol oxygenase Vgo is mutated, and the Vgo mutant enzyme activity is 1.4-6 times that before mutation, which can efficiently catalyze 4-vinyl guaiacol to generate vanillin, and has good industrial application prospect.

[0096] The applicant states that the above description is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and it should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily thought out by those skilled in the art, and all of them fall within the protection scope and disclosure scope of the present application.

Claims

1. A 4-ethyleneguaiacol oxygenase mutant for the in vitro synthesis of vanillin, characterized in that, The amino acid sequence of the 4-vinylguaiacol oxygenase mutant is an amino acid sequence obtained by mutation based on the sequence shown in SEQ ID NO.1; The mutation is a combination of W335F, I404L and V409W.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the 4-ethyleneguaiacol oxygenase mutant of claim 1.

3. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 2.

4. The recombinant vector according to claim 3, characterized in that, The starting plasmid of the recombinant vector includes pETDuet-1.

5. A genetically engineered strain, characterized in that, The genetically engineered strain contains the nucleic acid molecule as described in claim 2 or the recombinant vector as described in claim 3 or 4.

6. A genetically engineered preparation, characterized in that, The genetically engineered preparation contains the bacterial cells of the genetically engineered strain described in claim 5.

7. The use of the 4-ethyleneguaiacol oxygenase mutant of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3 or 4, the genetically engineered strain of claim 5, or the genetically engineered preparation of claim 6 in the synthesis of vanillin.

8. 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 claim 1 to obtain the vanillin; Alternatively, the method for synthesizing vanillin may include: mixing ferulic acid, ferulic acid decarboxylase, and the 4-vinylguaiacol oxygenase mutant of claim 1 to obtain the vanillin.

Images