4-vinylguaiacol oxygenase mutant for synthesizing vanillin, and engineered bacterium thereof

Abstract

The present invention provides a 4-vinylguaiacol oxygenase mutant for synthesizing vanillin, and an engineered bacterium thereof. The 4-vinylguaiacol oxygenase mutant is an enzyme having the sequence as shown in SEQ ID NO. 1, which exhibits a double-site mutation of D27P / T216M, D27P / M351F, D27P / Y156D, M351F / Y156D, T216M / Y156D or T216M / M351F, or a single-site mutation of T216M, T216L, S280M, G307P or M351F, and can be used for catalyzing 4-vinylguaiacol to generate vanillin.

Description

4-vinyl guaiacol oxygenase mutant for synthesizing vanillin and its engineered bacteria

[0001] The present application claims priority to the Chinese patent application No. 202410652461.0, filed on May 23, 2024, and titled "4-vinyl guaiacol oxygenase mutant and genetically engineered bacteria and application thereof". The entire content of the application is incorporated herein by reference.

[0002] The present application claims priority to the Chinese patent application No. 202410650535.7, filed on May 23, 2024, and titled "4-vinyl guaiacol oxygenase mutant for synthesizing vanillin and its engineered bacteria". The entire content of the application is incorporated herein by reference. TECHNICAL FIELD

[0003] The present application belongs to the field of genetic engineering, and specifically relates to 4-vinyl guaiacol oxygenase mutant for synthesizing vanillin and its engineered bacteria. BACKGROUND

[0004] Vanillin has a vanilla bean aroma and a rich milk aroma, and plays a role in flavoring and fixing. It has been widely used in food, tobacco, pharmaceuticals, cosmetics and other industries. Vanillin is an aromatic aldehyde and an important precursor of thermosetting resins / thermoplastic plastics. As the application field of vanillin becomes more and more extensive, the current production methods such as natural extraction and chemical synthesis cannot meet the market demand.

[0005] Plant extraction method cannot meet the market demand due to the influence of plant development cycle, growth environment, processing cost, etc. Chemical synthesis method is low in price, but it produces a large amount of waste, has high organic matter content, especially high ammonia nitrogen content which is difficult to be treated by biochemical method, and the quality of vanillin produced by chemical synthesis is relatively poor, contains more heavy metals, etc. Therefore, it is prohibited to be used in food and pharmaceutical industries.

[0006] Unlike the former two methods, biological transformation method can convert some natural substrates such as eugenol, isoeugenol and ferulic acid into "natural vanillin", which has advantages in price and quality compared with chemically synthesized vanillin, and the reaction conditions are mild and the pollution is less, which has a very broad prospect in providing an effective alternative route. In addition, using different renewable substrates such as ferulic acid and isoeugenol, vanillin can be produced by microorganisms in a green and environmentally friendly way, which has made great progress.

[0007] Some researchers heterologously expressed phenolic acid decarboxylase (Pad) from Bacillus coagulans DSM1 and phenol monooxygenase (Ado) from Thermothelomyces thermophila in Escherichia coli, and 13.3 g / L vanillin was generated in 1L reaction system in 18h. Patent CN115948315A discloses heterologous co-expression of feruloyl-CoA synthase and feruloyl-CoA hydrolase / aldehyde synthase in Escherichia coli, and 10.695 g / L vanillin is produced in 10.5h by whole cell catalysis. However, coenzyme A is needed in the reaction process of the above studies. Patent CN116590161A uses eugenol as a substrate to produce vanillin, and the concentration of vanillin in the culture obtained after fermentation of the recombinant bacteria can reach 9.34 g / L, and the conversion rate reaches 84.68%. In a 5L fermenter, the concentration of vanillin can reach 20.2 g / L, which is significantly higher than the concentration of vanillin of the same type. However, after the strain culture is completed, the fermentation time after adding the substrate is as long as 72 hours. At present, most of the research on vanillin biosynthesis is focused on whole cell fermentation method, and the research on enzyme method is less.

[0008] Therefore, seeking a cheap and efficient method for producing natural vanillin to meet people's quality and quantity requirements for "natural" flavor substances is the current research hotspot. SUMMARY

[0009] In view of the above problems, the present application provides a 4-vinyl guaiacol oxygenase mutant and its engineering bacteria for synthesizing vanillin. Any one of the 216th, 280th, 307th and 351st sites of the 4-vinyl guaiacol oxygenase gene Iso is mutated, or any two or more sites of the 27th, 156th, 216th and 351st sites of the 4-vinyl guaiacol oxygenase gene Iso are mutated, and the 4-vinyl guaiacol oxygenase gene Iso has the sequence shown in SEQ ID NO. 1. The Iso mutant provided by the present application can efficiently catalyze 4-vinyl guaiacol to generate vanillin, and has good industrial application prospect.

[0010] Terms:

[0011] In the present application, the term "D27P" represents that the 27th aspartic acid D is mutated to proline P.

[0012] In the present application, the term "Y156D" represents that the 156th tyrosine Y is mutated to aspartic acid D.

[0013] In the present application, the term "T216M" represents that the threonine T at position 216 is mutated to methionine M.

[0014] In the present application, the term "M351F" represents that the methionine M at position 351 is mutated to phenylalanine F.

[0015] In the present application, the term "V58W" represents that the valine V at position 58 is mutated to tryptophan W.

[0016] In the present application, the term "T216L" represents that the threonine T at position 216 is mutated to leucine L.

[0017] In the present application, the term "S280M" represents that the serine S at position 280 is mutated to methionine M.

[0018] In the present application, the term "G307P" represents that the glycine G at position 307 is mutated to proline P.

[0019] The technical solution of the present application is:

[0020] In a first aspect, the present application provides a mutant of 4-vinylguaiacol oxygenase for synthesizing vanillin, wherein the mutant comprises mutations at any two or more of positions 27, 156, 216, and 351 of SEQ ID NO. 1; or a mutation at any one of positions 216, 280, 307, and 351 of SEQ ID NO. 1.

[0021] Specifically, the mutant comprises:

[0022] (1) a mutant D27P / T216M in which aspartic acid D at position 27 is mutated to phenylalanine F and threonine T at position 216 is mutated to methionine M; or

[0023] (2) a mutant D27P / M351F in which aspartic acid D at position 27 is mutated to phenylalanine F and methionine M at position 351 is mutated to phenylalanine F; or

[0024] (3) a mutant D27P / Y156D in which aspartic acid D at position 27 is mutated to phenylalanine F and tyrosine Y at position 156 is mutated to aspartic acid D; or

[0025] (4) a mutant M351F / Y156D in which tyrosine Y at position 156 is mutated to aspartic acid D and methionine M at position 351 is mutated to phenylalanine F; or

[0026] (5) the mutant T216M / Y156D in which the tyrosine Y at position 156 is mutated into aspartic acid D and the threonine T at position 216 is mutated into methionine M; or

[0027] (6) the mutant T216M / M351 in which the threonine T at position 216 is mutated into methionine M and the methionine M at position 351 is mutated into phenylalanine F; or

[0028] (7) the mutant T216M in which the threonine T at position 216 is mutated into methionine M; or

[0029] (8) the mutant T216L in which the threonine T at position 216 is mutated into leucine L; or

[0030] (9) the mutant S280M in which the serine S at position 280 is mutated into methionine M; or

[0031] (10) the mutant G307P in which the glycine G at position 307 is mutated into proline P; or

[0032] (11) the mutant M351F in which the methionine M at position 351 is mutated into phenylalanine F.

[0033] Preferably, the mutant has an amino acid sequence as shown in SEQ ID NO. 2-12.

[0034] The mutant D27P / T216M has an amino acid sequence as shown in SEQ ID NO. 2.

[0035] The mutant D27P / M351F has an amino acid sequence as shown in SEQ ID NO. 3.

[0036] The mutant D27P / Y156D has an amino acid sequence as shown in SEQ ID NO. 4.

[0037] The mutant M351F / Y156D has an amino acid sequence as shown in SEQ ID NO. 5.

[0038] The mutant T216M / Y156D has an amino acid sequence as shown in SEQ ID NO. 6.

[0039] The mutant T216M / M351F has an amino acid sequence as shown in SEQ ID NO. 7.

[0040] The mutant T216M has an amino acid sequence as shown in SEQ ID NO. 8.

[0041] The mutant T216L has an amino acid sequence as shown in SEQ ID NO. 9.

[0042] The mutant S280M has an amino acid sequence as shown in SEQ ID NO. 10.

[0043] The mutant G307P has an amino acid sequence as shown in SEQ ID NO. 11.

[0044] The mutant M351F has an amino acid sequence as shown in SEQ ID NO. 12.

[0045] In some embodiments, the mutant V58W has an amino acid sequence as shown in SEQ ID NO. 13.

[0046] In a second aspect, the present application provides a mutant genetically engineered strain for synthesizing vanillin, wherein the mutant genetically engineered strain heterologously overexpresses the mutant described above.

[0047] Specifically, the genetically engineered strain is obtained by using primers to amplify the recombinant plasmid pET28a-Iso in vitro and then transforming the amplified product into E. coli.

[0048] Preferably, the genetically engineered strain comprises BL21-pET28a-Iso T216M, BL21-pET28a-Iso T216L, BL21-pET28a-Iso S280M, BL21-pET28a-Iso G307P, BL21-pET28a-Iso M351F, BL21-pET28a-Iso D27P / T216M, BL21-pET28a-Iso D27P / M351F, BL21-pET28a-Iso D27P / Y156D, BL21-pET28a-Iso M351F / Y156D, BL21-pET28a-Iso T216M / Y156D, or BL21-pET28a-Iso T216M / M351F.

[0049] In a third aspect, the present application provides a method for synthesizing vanillin, which comprises the following steps:

[0050] S1. Activating the genetically engineered strain and then performing seed culture to obtain a seed liquid;

[0051] S2. Inoculating the seed liquid into a fermentation medium and then performing fermentation culture;

[0052] S3. Centrifuging to collect the genetically engineered strain, disrupting the resuspended cell liquid, centrifuging again to collect the supernatant, and then obtaining a crude enzyme liquid of the genetically engineered strain;

[0053] S4, taking 4-vinylguaiacol as a substrate, adding the crude enzyme liquid of the genetically engineered strain of step S3 to perform a reaction, to prepare vanillin;

[0054] Or the method comprises the following steps:

[0055] (1) The genetically engineered strain is activated and then seed culture is performed to obtain a seed liquid;

[0056] (2) The seed liquid is inoculated into a fermentation medium to perform fermentation culture;

[0057] (3) The genetically engineered strain is collected by centrifugation, the resuspended cell liquid is broken, the supernatant is collected again by centrifugation, and a crude enzyme liquid of the genetically engineered strain is obtained;

[0058] (4) Taking ferulic acid as a substrate, adding a buffer and a crude enzyme liquid of a ferulic acid decarboxylase genetically engineered strain to perform a conversion;

[0059] (5) After the conversion is completed, a crude enzyme liquid of a mutant genetically engineered strain is added to perform a reaction to obtain a reaction liquid, and vanillin is obtained by purification.

[0060] The ferulic acid decarboxylase in the ferulic acid decarboxylase genetically engineered strain is a Bpf amino acid sequence as shown in SEQ ID NO. 14.

[0061] Further specifically, the genetically engineered strain in step (1) comprises a ferulic acid decarboxylase genetically engineered strain and an Iso mutant genetically engineered strain.

[0062] Preferably, the ferulic acid decarboxylase genetically engineered strain is BL21-pET28a-Bpf.

[0063] Preferably, the Iso mutant genetically engineered strain comprises BL21-pET28a-Iso D27P / T216M, BL21-pET28a-Iso D27P / M351F, BL21-pET28a-Iso D27P / Y156D, BL21-pET28a-Iso M351F / Y156D, BL21-pET28a-Iso T216M / Y156D, or BL21-pET28a-Iso T216M / M351F.

[0064] Further specifically, the culture in step (1) comprises the following steps: the activated genetically engineered strain is inoculated into a culture medium to culture to OD 600 = 1.8-2.2 to obtain a primary seed liquid; and the primary seed liquid is inoculated into a culture medium to culture to OD 600 = 3.8-4.2 to obtain a seed liquid.

[0065] Preferably, the activated genetically engineered strain is inoculated into the culture medium to culture to OD 600 = 2.0 to obtain a primary seed liquid.

[0066] Preferably, the primary seed liquid is inoculated into the culture medium to culture to OD 600 = 4.0 to obtain a seed liquid.

[0067] Further specifically, the inoculation amount of the genetically engineered strain or the primary seed liquid in step (1) is 0.5%-2%.

[0068] Preferably, the inoculation amount of the genetically engineered strain or the primary seed liquid in step (1) is 1%.

[0069] Further specifically, the culture medium in step (1) includes LB medium, SOB medium, SOC medium, TB medium or SB medium.

[0070] Still further specifically, the culture medium in step (1) is LB medium.

[0071] Preferably, the LB medium contains an antibiotic.

[0072] Still further preferably, the antibiotic includes but is not limited to one or more of ampicillin, kanamycin sulfate or chloramphenicol.

[0073] More preferably, the antibiotic is kanamycin sulfate.

[0074] Still further preferably, the concentration of the antibiotic is 40-60 μg / mL.

[0075] More preferably, the concentration of the antibiotic is 50 μg / mL.

[0076] Preferably, the temperature of the culture in step (1) is 35-37°C and the rotation speed is 200-220 rpm.

[0077] Specifically, the fermentation culture in step (2) includes the following steps: inoculating the seed liquid into a primary seed tank containing a fermentation culture medium, a base sugar and an antibiotic to culture to OD 600 = 5.8-6.2, transferring into a secondary seed tank containing a fermentation culture medium, a base sugar and an antibiotic, and fermenting to OD 600 = 15-20, starting to reduce the temperature to 22-25°C, adding an inducer to perform induction.

[0078] Further specifically, the inoculation amount of the seed liquid in step (2) is 8%-12%.

[0079] Preferably, the inoculation amount of the seed liquid in step (2) is 10%.

[0080] Further particularly, the base sugar in step (2) is glucose.

[0081] Further particularly, after the base sugar in step (2) is consumed, a feeding flow is started to maintain the glucose mass concentration in the fermentation liquid at 5-10 g / L.

[0082] Further particularly, the antibiotic in step (2) is kanamycin sulfate.

[0083] Preferably, the concentration of the kanamycin is 40-60 μg / mL.

[0084] Further preferably, the concentration of the antibiotic is 50 μg / mL.

[0085] Further particularly, in the primary seed tank or the secondary seed tank in step (2), the tank pressure is maintained at 0.03-0.04 Mpa, the dissolved oxygen is controlled at 40%-60%, the aeration amount is 1.5 times the medium volume / min, and the pH is regulated at 6.8-7.2.

[0086] Further particularly, the inducer in step (2) is IPTG.

[0087] Preferably, the concentration of the IPTG is 0.5-1.5 mmol / L.

[0088] Further preferably, the concentration of the IPTG is 1.0 mmol / L.

[0089] Further particularly, the induction time in step (2) is 12-16 h.

[0090] Particularly, the concentration of the ferulic acid in step (4) is 100-150 mmol / L.

[0091] Preferably, the concentration of the ferulic acid in step (4) is 130 mmol / L.

[0092] Particularly, the buffer in step (4) is a Glycine-NaOH buffer with pH 9.5.

[0093] Particularly, the addition amount of the buffer in step (4) is 0.3-0.7 mol / L.

[0094] Preferably, the addition amount of the buffer in step (4) is 0.5 mol / L.

[0095] Specifically, the conversion in step (4) is carried out at pH 6.5-7.5, a reaction temperature of 23-27℃, and a conversion time of 30 min-60 min.

[0096] Preferably, the conversion in step (4) is carried out at pH 7.0, a reaction temperature of 25℃.

[0097] Specifically, the reaction in step (5) is carried out at pH 9.0-9.5, a reaction temperature of 23-27℃, and a reaction time of 4-5 h.

[0098] Preferably, the reaction in step (5) is carried out at pH 9.5, a reaction temperature of 25℃.

[0099] The present application has the following advantages:

[0100] 1. The present application mines two water-soluble enzymes, ferulic acid decarboxylase Bpf and 4-vinyl guaiacol oxygenase Iso, which are expressed heterologously in E. coli, and 100 mmol / L substrate concentration can catalyze ferulic acid to produce vanillin up to 9.66 g / L within 5 h.

[0101] 2. The present application mutates 4-vinyl guaiacol oxygenase, and the enzyme activity of the mutant D27P / T216M, D27P / M351F, D27P / Y156D, M351F / Y156D, T216M / Y156D, T216M / M351F is 2.49-3.1 times that before mutation. The mutant strain can catalyze vanillin production up to 19.58 g / L within 5 h in the enzyme reaction system, and the E. coli engineering strain can stably express ferulic acid decarboxylase and 4-vinyl guaiacol oxygenase after being passed to 1000 generations, and the enzyme activity is not affected.

[0102] 3. The present application mutates 4-vinyl guaiacol oxygenase gene Iso at a single site to obtain 4-vinyl guaiacol oxygenase mutants T216M, T216L, S280M, G307P, M351F, and the enzyme activity is 1.04-2.29 times that before mutation. The conversion rate of vanillin is 67.9%-88.44% and the yield is 10.32-13.44 g / L at a substrate concentration of 100 mmol / L.

[0103] 4. The present application explores the production process, which is suitable for industrial production of vanillin and has good industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0104] Figure 1 is the HPLC detection spectrum of ferulic acid, vanillin and 4-vinyl guaiacol.

[0105] Figure 2 is a Bpf protein expression diagram; M represents Marker; 1 represents Lane 1 as pET28a empty load; 2 represents Lane 2 as Ecoli-Bpf total protein; 3 represents Lane 3 as Ecoli-Bpf supernatant protein; 4 represents Lane 4 as Ecoli-Bpf precipitated protein.

[0106] Figure 3 is an Iso protein expression diagram; M represents Marker; 1 represents Lane 1 as pET28a empty load; 2 represents Lane 2 as Ecoli-Iso total protein; 3 represents Lane 3 as Ecoli-Iso supernatant protein; 4 represents Lane 4 as Ecoli-Iso precipitated protein.

[0107] Figure 4 is an Iso and its mutant protein electrophoresis diagram (pure enzyme); M represents Marker; 1 represents Lane 1 as parent Iso of 4-vinyl guaiacol oxygenase; 2 represents Lane 2 as mutant D27P / T216M; 3 represents Lane 3 as mutant D27P / M351F; 4 represents Lane 4 as mutant D27P / Y156D; 5 represents Lane 5 as mutant M351F / Y156D; 6 represents Lane 6 as mutant T216M / Y156D; 7 represents Lane 7 as mutant T216M / M351F.

[0108] Figure 5 is an Iso and its mutant protein electrophoresis diagram (pure enzyme); M represents Marker; 1 represents Lane 1 as parent Iso of 4-vinyl guaiacol oxygenase; 2 represents Lane 2 as mutant V58W; 3 represents Lane 3 as mutant T216M; 4 represents Lane 4 as mutant T216L; 5 represents Lane 5 as mutant S280M; 6 represents Lane 6 as mutant G307P; 7 represents Lane 7 as mutant M351F. DETAILED DESCRIPTION

[0109] The present application will be further clarified by the following examples, which are intended to be purely exemplary of the present application. The following examples are only a part of the present application, and are not intended to limit the present application, but only to illustrate the present application. The experimental methods used in the following examples are all conventional experiments, and the materials, reagents, etc. used in the following examples are all commercially available, unless otherwise specified.

[0110] The culture medium formula involved in the present application is as follows:

[0111] 1. 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.

[0112] 2. LB solid medium (1 L): 15 g agar is added to the LB liquid medium formula.

[0113] 3. 2YT medium (1 L): Tryptone 16 g, yeast extract 10 g, sodium chloride 5 g, purified water to 1 L, sterilized by autoclaving at 121 °C for 15 min.

[0114] 4. The formula of the fermentation medium includes, by percentage: glucose 0.7-1.0%, potassium dihydrogen phosphate 1.34-1.5%, citric acid 0.18-0.25%, ammonium sulfate 0.4-1%, magnesium sulfate 0.05-0.1%, trace element solution 1-2%, and water as the solvent; the formula of the trace element solution (100x) includes, by mass percentage: EDTA 0.08-1.2%, zinc acetate 0.1-0.3%, sodium molybdate 0.01-0.03%, boric acid 0.03-0.05%, copper chloride 0.01-0.03%, cobalt chloride 0.03-0.05%, manganese chloride 0.08-0.12%, ammonium ferric citrate 1-2%, and water as the solvent.

[0115] 5. The formula of the fermentation process feed includes, by percentage: glucose 50-60%, magnesium sulfate 0.07-0.1%, trace elements 1-2%, and water as the solvent.

[0116] Basic Experimental Example 1: Vanillin detection method establishment

[0117] Vanillin, ferulic acid, and 4-vinyl guaiacol standard samples were dissolved in 100% methanol solution, and the contents of vanillin, ferulic acid, and 4-vinyl guaiacol were detected by high performance liquid chromatography (HPLC).

[0118] The HPLC detector was Waters Alliance e2695; the chromatographic column was Hypersil BDS C18 (4.6 mm x 150 mm; 5 μm); the mobile phase was water (containing 0.5% glacial acetic acid) and methanol gradient elution, 0-15 min methanol 15%-100%, 15-17 min methanol 100%-15%; the flow rate was 1.0 mL / min; the detection wavelength was 280 nm; the column temperature was set to 30 °C; and the injection volume was 10 μL.

[0119] The HPLC detection method for vanillin, ferulic acid, and 4-vinyl guaiacol was successfully established, as shown in Figure 1, the peak positions of vanillin, ferulic acid, and 4-vinyl guaiacol were 8.679 min, 9.491 min, and 12.768 min, respectively, all being single strong absorption peaks, and the peak positions of the detection sample and the standard sample were consistent, and then the content of vanillin in the sample was calculated according to the standard curve.

[0120] Basic Experimental Example 2: Enzyme activity determination of vinyl guaiacol oxygenase

[0121] The enzyme activity reaction system was (1 mL): 100 μg of pure enzyme was mixed with 0.5 mol / L glycine-NaOH buffer at pH 9.5, and then 10 mmol / L substrate 4-vinyl guaiacol was added. After reaction at 25°C for 10 min, 2 μL of the reaction solution was taken into a 96-well plate, 20 μL of 2,4-dinitrophenylhydrazine solution, 200 μL of 1 mol / L NaOH solution were added, and mixed. Distilled water was used as a blank control, and the absorbance at 520 nm was determined using an enzyme-labeled plate.

[0122] Enzyme activity definition: the amount of enzyme required to produce 1 μmol of product vanillin in 1 min at 25°C and pH 9.5 is defined as one unit (U).

[0123] Example 3 Construction of pET28a-Bpf for the expression vector of the gene related to the vanillin synthesis pathway

[0124] 1. Construction of pET28a-Bpf vector

[0125] The ferulic acid decarboxylase Bpf (NCBI accession number ACO50701.1) derived from Bacillus has an amino acid sequence as shown in SEQ ID NO. 14, which is optimized according to the codons of Escherichia coli. The codon-optimized sequence was synthesized by Universal Biological (Anhui) Co., Ltd., and PCR amplification was performed using primers P1 (Bpf-F, Bpf-R) (PrimeSTAR GXL DNA Polymerase, Takara Code No. R050A) to obtain a linear fragment Bpf (483 bp) having a nucleotide sequence as shown in SEQ ID NO. 15.

[0126] In the amplification of the linearized pET28a vector, the nucleotide sequence of pET28a was amplified by PCR using primers P2 (pET28a-F, pET28a-R), and a linearized pET28a vector (5308 bp) was obtained.

[0127] The primer sequences used are shown in Table 1:

[0128] Table 1 Primer P1 and P2 sequence information

[0129] In Table 1, F and R are amplification primers, wherein: F represents a forward primer, and R represents a reverse primer.

[0130] The linearized pET28a vector and fragment Bpf were connected by using the recombination cloning kit (CloneExpress II One Step Cloning Kit, Novagen CAT#: C112-01), and transformed into the competent cells E. coli DH5α (Weidi Biology, CAT#: DL1001) by using the calcium chloride chemical transformation method. The positive clones were picked, and the plasmid was extracted for sequencing. The obtained recombinant plasmid with correct sequence was recorded as pET28a-Bpf. The obtained correct plasmid pET28a-Bpf was transformed into E. coli BL21 (DE3) (Weidi Biology, CAT#: EE1002) by using the calcium chloride chemical transformation method. The obtained recombinant bacteria were recorded as BL21-pET28a-Bpf, the promoter of the target gene was T7 promoter, and the selection marker was kanamycin sulfate (Kana).

[0131] 2. Culture and induced expression of the recombinant strain

[0132] The recombinant strain BL21-pET28a-Bpf was inoculated in 2YT medium (containing 50 μg / mL Kana) with a liquid volume of 1L / 5L, and was placed in a 37°C, 220 rpm shaker for shaking culture until the bacterial concentration OD 600 = 0.6-0.8. Then, 1 mmol / L IPTG was added, and the expression was induced at 25°C for 12-16 h.

[0133] 50 OD BL21-pET28a-Bpf culture solution was taken into a centrifuge bottle, and the bacterial body was collected by centrifugation at 4500 rpm for 20 min. The supernatant was discarded, and 5 mL of 50 mmol / L phosphate buffer (pH 7.5) was added to resuspend the bacterial body. The bacterial body was broken on ice by using an ultrasonic disrupter for 10 min (70 kHz, ultrasonic for 3 s, pause for 7 s). The cell broken solution was obtained, and the supernatant was obtained by centrifugation at 12000 rpm for 30 min. The supernatant, precipitate and broken solution were collected for SDS-PAGE electrophoresis to detect protein expression.

[0134] The expression of the recombinant strain BL21-pET28a-Bpf enzyme is shown in FIG. 2. M represents Marker, lane 1 represents pET28a empty vector, lane 2 represents Ecoli-Bpf total protein, lane 3 represents Ecoli-Bpf supernatant protein, and lane 4 represents Ecoli-Bpf precipitate protein.

[0135] Construction of pET28a-Iso related to the vanillin synthesis pathway expression vector

[0136] 1. Construction of pET28a-Iso vector

[0137] Using the BLAST tool, search for all amino acid sequences with a similarity higher than 30% to the Cso2 sequence in the Uniprot database (set the amino acid length between 450-550), construct all similar sequences by AlphaFold2-based structure modeling method, and then construct a structure-based phylogenetic tree, select 5 enzymes with certain representative as the key starting enzymes for subsequent functional characterization. Among them, the 4-vinyl guaiacol oxygenase Iso from Sphingobium sp. GW456-12-10-14-TSB1 has better activity.

[0138] The 4-vinyl guaiacol oxygenase Iso (NCBI accession number OUC52898.1) derived from Sphingobium sp. has an amino acid sequence as shown in SEQ ID NO. 1. The codon-optimized sequence is constructed into a vector according to the method described in Basic Experiment Example 3. The primer P3 (Iso-F, Iso-R) is used for PCR amplification, and a linear fragment Iso (1482 bp) is obtained, which has a nucleotide sequence as shown in SEQ ID NO. 20. The primer sequences used are shown in Table 2:

[0139] Table 2 Primer P3 sequence information

[0140] In Table 2, F and R are amplification primers, wherein: F represents a forward primer, and R represents a reverse primer.

[0141] The obtained recombinant bacteria are recorded as BL21-pET28a-Iso, the promoter of the target gene is T7 promoter, and the selection marker is kanamycin sulfate (Kana).

[0142] 2. Culture and induction expression of recombinant strain

[0143] The protein expression and detection steps of the recombinant strain BL21-pET28a-Iso are the same as those described in "2. Culture and induction expression of recombinant strain" in Basic Experiment Example 3. The expression of the enzyme of the recombinant strain BL21-pET28a-Iso is shown in Figure 3. M represents Marker, lane 1 represents pET28a empty load, lane 2 represents Ecoli-Iso total protein, lane 3 represents Ecoli-Iso supernatant protein, and lane 4 represents Ecoli-Iso precipitated protein.

[0144] Example 1 Transformation of ferulic acid to 4-vinyl guaiacol by recombinant strain BL21-pET28a-Bpf crude enzyme

[0145] 1. Strain culture and preparation of crude enzyme solution

[0146] Strain cultivation and crude enzyme preparation refer to "2. Cultivation of recombinant strain and induction of expression" described in Basic Experimental Example 3.

[0147] 2. Reaction of converting ferulic acid into 4-vinylguaiacol

[0148] S1 reaction: In a 2L conical flask, 50mmol / L ferulic acid as substrate, 10 OD of BL21-pET28a-Bpf crude enzyme solution, 0.5mol / L pH 9.5 Glycine-NaOH buffer solution were added, and the reaction was carried out at pH 6.0-8.5 and reaction temperature 15-35℃ for 30min-60min, and HPLC detection analysis was performed.

[0149] 4-vinylguaiacol molar conversion rate (%) = 4-vinylguaiacol molar concentration (mmol / L) / ferulic acid initial concentration (mmol / L) x 100%.

[0150] 4-vinylguaiacol yield (g / L) = 4-vinylguaiacol molar concentration (mmol / L) x 150. (The relative molecular mass of 4-vinylguaiacol is 150).

[0151] The results are shown in Table 3 as follows:

[0152] Table 3 4-vinylguaiacol yield and conversion rate

[0153] The results show that the optimal conditions for recombinant strain BL21-pET28a-Bpf crude enzyme to convert ferulic acid into 4-vinylguaiacol are pH 7.0 and optimal temperature 25℃.

[0154] Example 2 Recombinant strain BL21-pET28a-Iso crude enzyme converts 4-vinylguaiacol into vanillin

[0155] 1. Strain cultivation and crude enzyme preparation

[0156] Strain cultivation and crude enzyme preparation refer to "2. Cultivation of recombinant strain and induction of expression" described in Basic Experimental Example 3.

[0157] 2. Reaction of converting ferulic acid into vanillin

[0158] S1 reaction: In a 2L conical flask, 50mmol / L ferulic acid as substrate, 10 OD of BL21-pET28a-Bpf crude enzyme solution, 0.5mol / L pH 9.5 Glycine-NaOH buffer solution were added, and the reaction was carried out at pH 7.0 and reaction temperature 25℃ for 30min-60min, and HPLC detection was performed after ferulic acid was completely converted into 4-vinylguaiacol.

[0159] S2 reaction: 40 OD of BL21-pET28a-Iso crude enzyme solution was added to the S1 reaction solution, and conversion was carried out at a reaction pH of 8-11 and a reaction temperature of 15-35°C for 4-5 h, and HPLC detection analysis was performed.

[0160] Vanillin molar conversion rate (%) = vanillin molar concentration (mmol / L) / ferulic acid initial concentration (mmol / L) x 100%.

[0161] Vanillin yield (g / L) = vanillin molar concentration (mmol / L) x 152. (The relative molecular mass of vanillin is 152).

[0162] The results are shown in Table 4 below:

[0163] Table 4 Vanillin yield and conversion rate

[0164] The results show that the optimal conditions for the recombinant strain BL21-pET28a-Iso crude enzyme to convert 4-vinylguaiacol to produce vanillin are a pH of 9.5 and an optimal temperature of 25°C.

[0165] Example 3 Whole-cell conversion of ferulic acid to produce vanillin by recombinant bacteria

[0166] 1. Strain culture and preparation of crude enzyme solution

[0167] The strain culture and preparation of the crude enzyme solution were performed according to the "2. Culture and induction of the recombinant strain" described in the basic experimental example 3, except that the centrifuged bacterial cells were resuspended without ultrasonic disruption, and then subjected to whole-cell catalytic reaction.

[0168] 2. Whole-cell catalytic reaction

[0169] S1 reaction: In a 2L conical flask, 50 mmol / L of substrate ferulic acid, 10 OD of BL21-pET28a-Bpf whole cells, and 0.5 mol / L pH 9.5 glycine-NaOH buffer were added, and conversion was carried out at a reaction pH of 7.0 and a reaction temperature of 25°C for 30-60 min, and HPLC detection was performed after ferulic acid was completely converted to 4-vinylguaiacol to perform S2 reaction.

[0170] S2 reaction: 40 OD of BL21-pET28a-Iso whole cells (60 mL) was added to the S1 reaction solution, and the total reaction volume was 1 L, and conversion was carried out at a reaction pH of 9.5 and a reaction temperature of 25°C for 4-5 h, and HPLC detection analysis was performed.

[0171] The results show that the vanillin yield is 6.18 g / L, and the 4-vinylguaiacol remaining is 2.81 g / L, and the conversion rate is 81.3%. Under the same reaction conditions, the conversion effect of the crude enzyme is better than that of the whole cell.

[0172] Example 4 Construction of 4-vinylguaiacol oxygenase mutant based on whole plasmid PCR method

[0173] The recombinant plasmid pET28a-Iso (Example 2) was used as a template, and PCR (PrimeSTAR HS DNA Polymerase, Takara Code No. R010A) was used for in vitro mutation amplification. The primer sequences for site-directed mutagenesis are shown in Table 5.

[0174] Table 5 Primer sequence information for site-directed mutagenesis

[0175] In Table 5, F and R are amplification primers, wherein: F represents a forward primer, and R represents a reverse primer.

[0176] After PCR, 10 μL of the PCR product was added to 1 μL of restriction endonuclease Dpn I to digest the template plasmid, and after 2 h of reaction at 37°C, the digested product was transferred into E. coli BL21 (DE3) (Weidi Biology, CAT#: EE1002) according to the method described in Example 1.1, and plated on LB solid plates (containing 50 μg / mL Kana) and incubated at 37°C overnight. Four transformants were picked from each and cultured in LB liquid medium (containing 50 μg / mL Kana) for 6 h, and then sent for sequencing. BL21-pET28a-Iso D27P, BL21-pET28a-Iso D27P, BL21-pET28a-Iso Y156D, BL21-pET28a-Iso V58W, BL21-pET28a-Iso T216M, BL21-pET28a-Iso T216L, BL21-pET28a-Iso S280M, BL21-pET28a-Iso G307P, and BL21-pET28a-Iso M351F were obtained with correct sequencing.

[0177] Using the BL21-pET28a-Iso D27P plasmid with correct sequencing as a template, and T216M-F / T216M-R or M351F-F / M351F-R as primers, after whole plasmid PCR, transformation, and plating, double-site mutant strains BL21-pET28a-Iso D27P / T216M or BL21-pET28a-Iso D27P / M351F were obtained.

[0178] With the sequencing correct BL21-pET28a-Iso Y156D plasmid as template, with D27P-F / D27P-R or M351F-F / M351F-R as primer, after whole plasmid PCR, transformation, plating, double site mutant strains BL21-pET28a-Iso D27P / Y156D or BL21-pET28a-Iso M351F / Y156D were obtained.

[0179] With the sequencing correct BL21-pET28a-Iso T216M plasmid as template, with Y156D-F / Y156D-R or M351F-F / M351F-R as primer, after whole plasmid PCR, transformation, plating, double site mutant strains BL21-pET28a-Iso T216M / Y156D or BL21-pET28a-Iso T216M / M351F were obtained.

[0180] Example 54-Protein expression and purification of vinyl guaiacol oxygenase and its mutants

[0181] 1. Protein expression

[0182] The recombinant strain BL21-pET28a-Iso and its mutants were inoculated in 2YT medium (containing 50 μg / mL Kana) with liquid volume of 1L / 5L, and placed in 37℃, 220 rpm shaker for culture until the bacterial concentration OD 600 = 0.6-0.8, and then 1 mmol / L IPTG and 1 mmol / L FeCl2 were added, and expression was induced at 25℃ for 12-16h.

[0183] 50 OD BL21-pET28a-Iso and its mutant culture solution was taken into centrifuge bottle, and the bacterial body was collected by centrifugation at 4500 rpm for 20 min, and the supernatant was discarded, and 5 mL 50 mmol / L phosphate buffer (pH 7.5) was added to resuspend the bacterial body. The cell broken liquid was obtained by ultrasonic crusher on ice for 10 min (70 kHz, ultrasonic 3 s, pause 7 s), and the supernatant was centrifuged at 12000 rpm for 30 min, and the supernatant was the crude enzyme solution of BL21-pET28a-Iso and its mutants.

[0184] 2. Protein purification

[0185] The crude enzyme solution was purified by nickel column affinity chromatography. The filler of the nickel column was Hispur™ Ni-NTA Resin, and the target protein with His tag was specifically bound 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 was eluted using Elution buffer (50 mM Tris-HCl, 150 mM NaCl, 300 mM imidazole; pH 7.5). The protein eluate obtained above was concentrated by centrifugation using Amino Ultra-15 (30 kDa) ultrafiltration tubes, and the residual imidazole from the protein purification process was washed away using Desalination buffer (50 mM Tris-HCl, 150 mM NaCl; pH 7.5). Glycerol was added to the obtained protein solution to a final concentration of 10%, and the solution was stored at -80°C.

[0186] After purification, the same protein content and the same volume of pure enzyme were loaded for SDS-PAGE verification. The results are shown in FIGS. 4-5. In FIG. 4, M represents Marker, lane 1 represents the parent Iso of 4-vinylguaiacol oxygenase, lane 2 represents mutant D27P / T216M, lane 3 represents mutant D27P / M351F, lane 4 represents mutant D27P / Y156D, lane 5 represents mutant M351F / Y156D, lane 6 represents mutant T216M / Y156D, and lane 7 represents mutant T216M / M351F. In FIG. 5, M represents Marker, lane 1 represents the parent Iso of 4-vinylguaiacol oxygenase, lane 2 represents mutant V58W, lane 3 represents mutant T216M, lane 4 represents mutant T216L, lane 5 represents mutant S280M, lane 6 represents mutant G307P, and lane 7 represents mutant M351F.

[0187] Example 6 Comparison of the activities of different 4-vinylguaiacol oxygenase mutants and the parent enzyme

[0188] The concentrated pure enzyme was diluted, and the protein concentration was determined by the Bradford method. The enzyme activity was determined according to the basic experimental example 2. The specific activity of the parent 4-vinylguaiacol oxygenase Iso was 2.80 U / mg, and the specific activities of the mutants D27P / T216M, D27P / M351F, D27P / Y156D, M351F / Y156D, T216M / Y156D, T216M / M351F, V58W, T216M, T216L, S280M, G307P, and M351F were 8.13 U / mg, 7.72 U / mg, 6.96 U / mg, 7.40 U / mg, 8.45 U / mg, 8.68 U / mg, 2.16 U / mg, 6.40 U / mg, 3.07 U / mg, 2.93 U / mg, 3.14 U / mg, and 5.40 U / mg, respectively. Among them, T216M / M351F showed the best specific activity, which was 3.1 times higher than that of the parent. The determination results are shown in Table 6.

[0189] Table 6 Comparison of activities of different 4-vinylguaiacol oxygenase mutants and parent enzyme

[0190] Example 7 Comparison of conversion rates of 4-vinylguaiacol oxygenase mutant and parent crude enzyme catalyzing ferulic acid to synthesize vanillin

[0191] 1. Strain cultivation and preparation of crude enzyme solution

[0192] The strain cultivation and preparation of the crude enzyme solution were performed according to the “2. Cultivation and induced expression of the recombinant strain” described in the basic experimental example 3.

[0193] 2. Ferulic acid conversion to generate vanillin reaction

[0194] S1 reaction: In a 2L conical flask, 100 mmol / L of substrate ferulic acid, 10 OD of BL21-pET28a-Bpf crude enzyme solution, and 0.5 mol / L of pH 9.5 glycine-NaOH buffer were added, and the reaction was performed at pH 7.0 and 25°C for 30 min-60 min. After HPLC detection of the complete conversion of ferulic acid to 4-vinylguaiacol, the S2 reaction was performed.

[0195] S2 reaction: In the S1 reaction solution, 40 OD of BL21-pET28a-Iso and its mutant crude enzyme solution was added, and the reaction was performed at pH 9.5 and 25°C for 4-5 h, and HPLC detection analysis was performed.

[0196] The results show that after 5h of reaction, the conversion rate of the parent Iso is 63.53%, and the yield is 9.66g / L. The conversion rates of the mutants D27P / T216M, D27P / M351F, D27P / Y156D, M351F / Y156D, T216M / Y156D, and T216M / M351F are 94.87%, 92.15%, 89.95%, 91.40%, 95.23%, and 97.28%, respectively, and the yields are 14.42g / L, 14.01g / L, 13.67g / L, 13.89g / L, 14.47g / L, and 14.79g / L, respectively. The results are shown in Table 7.

[0197] Table 7 Comparison of conversion rate and yield

[0198] 3. Conversion of 4-vinylguaiacol to vanillin

[0199] In a 2L conical flask, 100mmol / L of 4-vinylguaiacol was added as substrate, and 40OD of the crude enzyme solution of BL21-pET28a-Iso or its mutants was added. The reaction was carried out at pH 9.5 and 25°C for 4-5h, and HPLC was used for analysis.

[0200] The results show that after 5h of reaction, the conversion rates of the mutants V58W, T216M, T216L, S280M, G307P, and M351F are 38.94%, 88.44%, 68.76%, 67.9%, 70.52%, and 84.72%, respectively, and the yields are 5.92g / L, 13.44g / L, 10.45g / L, 10.32g / L, 10.72g / L, and 12.88g / L, respectively. The results are shown in Table 8.

[0201] Table 8 Comparison of conversion rate and yield

[0202] Example 8 Comparison of the ability of T216M / M351F to convert vanillin

[0203] T216M / M351F shows the best specific activity and conversion rate, so in this example, the ability of the 4-vinylguaiacol oxygenase mutant T216M / M351F to convert vanillin was compared.

[0204] 1. Strain culture and preparation of crude enzyme solution

[0205] The strain culture and preparation of crude enzyme solution were performed according to the “2. Culture of recombinant strain and induction of expression” described in the Basic Experimental Example 3.

[0206] 2. Ferulic acid conversion to vanillin reaction

[0207] S1 reaction: in a 2L conical flask, add substrate 50-400 mmol / L ferulic acid, 10 OD of BL21-pET28a-Bpf crude enzyme solution, 0.5 mol / L pH 9.5 glycine-NaOH buffer, at reaction pH 7.0, reaction temperature 25°C, convert for 30 min-60 min, HPLC detection of ferulic acid is completely converted to 4-vinyl guaiacol, then S2 reaction.

[0208] S2 reaction: add 40 OD of BL21-pET28a-Iso T216M / M351F crude enzyme solution to the S1 reaction solution, at reaction pH 9.5, reaction temperature 25°C, convert for 4-5 h, HPLC detection analysis.

[0209] The results are shown in Table 9 as follows:

[0210] Table 9 Comparison of the ability of T216M / M351F to convert vanillin

[0211] The experimental results show that: when the concentration of ferulic acid is in the range of 100-150 mmol / L, within a certain reaction time, the substrate conversion rate and the amount of product obtained are optimal, suitable for industrial production.

[0212] Example 9 Stability detection of E. coli engineering strain

[0213] To verify the stability of the strain, the enzyme activity stability of BL21-pET28a-Iso mutant strain T216M / M351F in continuous passage was monitored.

[0214] Continuous passage culture of the strain and calculation of culture generations: take 12h as one passage cycle, continuously pass and culture at 2% (volume fraction) inoculation amount. After one single cell of the strain proliferates n times, the number of bacteria is 2n. In each passage cycle (12h), the bacteria proliferate approximately in geometric progression (2n). n In each passage cycle (12h), the bacteria proliferate approximately in geometric progression (2n). n Through determination of the growth curve of the strain, it can be known that when the inoculation amount is 2%, the growth generations (i.e. the number of times of bacterial division) in each cycle is 12 generations.

[0215] The 0th generation strain is continuously passaged for 1000 generations. Among them, the initial strain is recorded as DP0, the 200th generation is recorded as DP1, the 400th generation is recorded as DP2, the 600th generation is recorded as DP3, the 800th generation is recorded as DP4, and the 1000th generation is recorded as DP5.

[0216] The BL21-pET28a-Iso mutant T216M / M351F was continuously subcultured, and the enzyme activity of DP0-DP5 was determined, with reference to the enzyme activity determination of 4-vinylguaiacol oxygenase.

[0217] The determination results are shown in Table 10, and the enzyme activity of the BL21-pET28a-Iso mutant T216M / M351F engineering strain does not change significantly during continuous subculture.

[0218] Table 10 Enzyme activity determination during continuous subculture of the strain

[0219] The enzyme activity of the BL21-pET28a-Iso mutant T216M / M351F does not change significantly during continuous subculture, and the strain is stable and can be used for continuous industrial production of vanillin.

[0220] The above detailed description is a specific description of one of the feasible embodiments of the present application, and is not used to limit the patent scope of the present application. It should be noted that any equivalent implementation or change made without departing from the present application should be included in the scope of the technical solutions of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A mutant of 4-vinylguaiacol oxygenase for synthesizing vanillin, characterized in that, The mutant includes mutation of any two or more of the 27th, 156th, 216th, 351st positions of SEQ ID NO. 1; or mutation of any one of the 216th, 280th, 307th, 351st positions of SEQ ID NO.

1.

2. The mutant according to claim 1, wherein The mutant includes: (1) mutant D27P / T216M in which aspartic acid D at the 27th position is mutated to phenylalanine F and threonine T at the 216th position is mutated to methionine M; or (2) mutant D27P / M351F in which aspartic acid D at the 27th position is mutated to phenylalanine F and methionine M at the 351st position is mutated to phenylalanine F; or (3) mutant D27P / Y156D in which aspartic acid D at the 27th position is mutated to phenylalanine F and tyrosine Y at the 156th position is mutated to aspartic acid D; or (4) mutant M351F / Y156D in which methionine M at the 351st position is mutated to phenylalanine F and tyrosine Y at the 156th position is mutated to aspartic acid D; or (5) mutant T216M / Y156D in which threonine T at the 216th position is mutated to methionine M and tyrosine Y at the 156th position is mutated to aspartic acid D; or (6) mutant T216M / M351F in which threonine T at the 216th position is mutated to methionine M and methionine M at the 351st position is mutated to phenylalanine F; or (7) mutant T216M in which threonine T at the 216th position is mutated to methionine M; or (8) mutant T216L in which threonine T at the 216th position is mutated to leucine L; or (9) mutant S280M in which serine S at the 280th position is mutated to methionine M; or (10) mutant G307P in which glycine G at the 307th position is mutated to proline P; or (11) mutant M351F in which methionine M at the 351st position is mutated to phenylalanine F.

3. The mutant of claim 1, wherein The mutant has an amino acid sequence as shown in SEQ ID NO. 2-12.

4. The mutant of claim 1, wherein The mutant D27P / T216M has an amino acid sequence as shown in SEQ ID NO. 2; or The mutant D27P / M351F has an amino acid sequence as shown in SEQ ID NO. 3; or The mutant D27P / Y156D has an amino acid sequence as shown in SEQ ID NO. 4; or The mutant M351F / Y156D has an amino acid sequence as shown in SEQ ID NO. 5; or The mutant T216M / Y156D has an amino acid sequence as shown in SEQ ID NO. 6; or The mutant T216M / M351F has an amino acid sequence as shown in SEQ ID NO. 7; or The mutant T216M has an amino acid sequence as shown in SEQ ID NO. 8; or The mutant T216L has an amino acid sequence as shown in SEQ ID NO. 9; or The mutant S280M has an amino acid sequence as shown in SEQ ID NO. 10; or The mutant G307P has an amino acid sequence as shown in SEQ ID NO. 11; or The mutant M351F has an amino acid sequence as shown in SEQ ID NO.

12. The mutant S280M has an amino acid sequence as shown in SEQ ID NO. 10; or The mutant G307P has an amino acid sequence as shown in SEQ ID NO. 11; or The mutant M351F has an amino acid sequence as shown in SEQ ID NO.

12.

5. Mutant genetically engineered bacterial strain for the synthesis of vanillin, characterized in that, The mutant genetically engineered strain heterologously overexpresses the mutant of any one of claims 1-3.

6. A method of synthesizing vanillin, characterized by, The method comprises using a genetically engineered strain; the genetically engineered strain comprises a mutant genetically engineered strain; the mutant genetically engineered strain overexpresses the mutant of any one of claims 1-3.

7. The method of claim 6, wherein, The method comprises the following steps: S1, after activation, the seed culture of the genetically engineered strain is carried out to obtain a seed liquid; S2, the seed liquid is inoculated into a fermentation medium for fermentation culture; S3, the genetically engineered strain is collected by centrifugation, the resuspended cell liquid is broken, and the supernatant is collected again by centrifugation to obtain a crude enzyme liquid of the genetically engineered strain; S4, 4-vinyl guaiacol is used as a substrate, and the crude enzyme liquid of the genetically engineered strain of step S3 is added for reaction to prepare vanillin; Or the method comprises the following steps: (1) after activation, the seed culture of the genetically engineered strain is carried out to obtain a seed liquid; (2) the seed liquid is inoculated into a fermentation medium for fermentation culture; (3) the genetically engineered strain is collected by centrifugation, the resuspended cell liquid is broken, and the supernatant is collected again by centrifugation to obtain a crude enzyme liquid of the genetically engineered strain; (4) ferulic acid is used as a substrate, and a buffer and the crude enzyme liquid of the ferulic acid decarboxylase genetically engineered strain are added for conversion; (5) after the conversion is completed, the crude enzyme liquid of the mutant genetically engineered strain is added for reaction to obtain a reaction liquid, and vanillin is purified; The ferulic acid decarboxylase in the ferulic acid decarboxylase genetically engineered strain has a Bpf amino acid sequence as shown in SEQ ID NO.

14.

8. The method of claim 7, wherein, The concentration of the ferulic acid in step (4) is 100-150 mmol / L.

9. The method of claim 7, wherein, The conversion conditions in step (4) are: pH 6.5-7.5, reaction temperature 23-27℃, and conversion time 30 min-60 min.

10. The method of claim 7, wherein, The reaction conditions in step (5) are: pH 9.0-9.5, reaction temperature 23-27℃, and reaction time 4-5 h.

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