4-vinylguaiacol oxygenase mutant and its engineered bacteria for the synthesis of vanillin

Abstract

The present invention provides a 4-vinylguaiacol oxygenase mutant and an engineering bacterium for synthesizing vanillin, and belongs to the field of genetic engineering technology. The present invention mutates any two or more sites in the 27th, 156th, 216th, and 351st positions of the 4-vinylguaiacol oxygenase gene Iso to obtain a 4-vinylguaiacol oxygenase mutant. The present invention successfully constructs a genetically engineered strain capable of efficiently catalyzing ferulic acid to generate vanillin by a two-step enzymatic method, and can catalyze the production of vanillin up to 19.58 g / L within 5 hours in an enzyme reaction system, and can stably express ferulic acid decarboxylase and 4-vinylguaiacol oxygenase after 1000 generations, and the enzyme activity is not affected, so as to realize the industrial production of natural vanillin and meet the market demand for vanillin.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and particularly relates to a 4-vinylguaiacol oxygenase mutant and an engineering bacterium thereof for synthesizing vanillin. Background Art

[0002] Vanillin has the aroma of vanilla beans and rich milky aroma, and plays a role in flavor enhancement and fixation. It is now widely used in food, tobacco, medicine, cosmetics and other industries. Vanillin is an aromatic aldehyde and an important precursor of thermosetting resins / thermoplastics. As the application of vanillin becomes more and more extensive, the current production methods such as natural extraction and chemical synthesis can no longer meet market demand.

[0003] Plant extraction cannot meet market demand because its process is greatly affected by plant development cycle, growth environment, processing cost, etc. Although the chemical synthesis method is cheap, it produces a large amount of three wastes and a high content of organic matter, especially ammonia nitrogen, which is difficult to biochemically treat. In addition, the quality of chemically synthesized vanillin products is relatively poor and contains more heavy metals, so it is banned in the food and pharmaceutical industries.

[0004] Different from the first two methods, the biotransformation method can transform some natural substrates such as eugenol, isoeugenol and ferulic acid into "natural vanillin", which has advantages over chemically synthesized vanillin in terms of price and quality. In addition, the reaction conditions are mild and less polluting, so it has a very broad prospect in providing an effective alternative route. In addition, great progress has been made in the production of vanillin by microorganisms using different renewable substrates such as ferulic acid and isoeugenol in a green and environmentally friendly way.

[0005] Some researchers heterologously expressed phenolic acid decarboxylase (Pad) from Bacillus coagulans DSM1 and phenol monooxygenase (Ado) from Thermothelomyces thermophila in Escherichia coli, and produced 13.3 g / L vanillin in 18 hours in a 1L reaction system. Patent CN115948315A disclosed heterologous co-expression of feruloyl-CoA synthase and feruloyl-CoA hydrolase / aldolase in Escherichia coli, and produced 10.695 g / L vanillin in 10.5 hours through whole-cell catalysis. However, coenzyme A is required in the reaction process of the above studies. Patent CN116590161A ferments vanillin with eugenol as substrate. The concentration of vanillin in the culture obtained after fermentation of the recombinant bacteria can reach 9.34g / L, and the conversion rate reaches 84.68%. The concentration of vanillin can be 20.2g / L in a 5L fermenter, which is significantly higher than the concentration of vanillin of the same type. However, after the strain culture is completed, the fermentation time is as long as 72 hours after adding the substrate. At present, most of the research on the biosynthesis of vanillin is concentrated on the whole cell fermentation method, and the research on the application of enzyme method is less.

[0006] Therefore, seeking a cheap and efficient method to produce natural vanillin that meets people's requirements for the quality and quantity of "natural" flavor substances is a hot topic in current research. Summary of the invention

[0007] In view of the above shortcomings, the present invention provides a 4-vinylguaiacol oxygenase mutant for synthesizing vanillin and an engineered bacterium thereof. The present invention mutates any two or more sites among the 27th, 156th, 216th and 351st positions of the 4-vinylguaiacol oxygenase gene Iso to obtain 4-vinylguaiacol oxygenase mutants D27P / T216M, D27P / M351F, D27P / Y156D, M351F / Y156D, T216M / Y156D and T216M / M351F. The present invention successfully constructs a genetically engineered strain capable of efficiently catalyzing ferulic acid to generate vanillin through a two-step enzymatic method, can catalyze the production of vanillin up to 19.58 g / L in an enzyme reaction system within 5 hours, can stably express ferulic acid decarboxylase and 4-vinylguaiacol oxygenase after being propagated to 1000 generations, and has no effect on enzyme activity, thereby realizing the industrialized production of natural vanillin and meeting the market demand for vanillin.

[0008]

[0009] the term:

[0010] In the present invention, the term "D27P" represents mutation of aspartic acid D at position 27 to proline P.

[0011] In the present invention, the term "Y156D" represents mutation of tyrosine Y at position 156 to aspartic acid D.

[0012] In the present invention, the term "T216M" represents mutation of threonine T at position 216 to methionine M.

[0013] In the present invention, the term "M351F" represents mutation of methionine M at position 351 to phenylalanine F.

[0014] The technical solution of the present invention is:

[0015] On the one hand, the present invention provides a mutant of 4-vinylguaiacol oxygenase for synthesizing vanillin, wherein the mutant comprises mutations at any two or more sites among positions 27, 156, 216, and 351 of SEQ ID NO.1.

[0016] SEQ ID NO.1:

[0017] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRN PFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLC LSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA.

[0018] Specifically, the mutation includes mutating aspartic acid D at position 27 to phenylalanine F.

[0019] Specifically, the mutation includes mutating the tyrosine Y at position 156 to aspartic acid D.

[0020] Specifically, the mutation includes mutating the threonine T at position 216 to methionine M.

[0021] Specifically, the mutation includes mutating the methionine M at position 351 to phenylalanine F.

[0022] Specifically, the mutants include:

[0023] (1) D27P / T216M, in which the aspartic acid D at position 27 is mutated to phenylalanine F, and the threonine T at position 216 is mutated to methionine M; or

[0024] (2) D27P / M351F, a mutant in which the aspartic acid D at position 27 is mutated to phenylalanine F and the methionine M at position 351 is mutated to phenylalanine F; or

[0025] (3) D27P / Y156D, a mutant in which the aspartic acid D at position 27 is mutated to phenylalanine F, and the tyrosine Y at position 156 is mutated to aspartic acid D; or

[0026] (4) a mutant M351F / Y156D in which the tyrosine Y at position 156 is mutated to aspartic acid D and the methionine M at position 351 is mutated to phenylalanine F; or

[0027] (5) a mutant T216M / Y156D in which the tyrosine Y at position 156 is mutated to aspartic acid D and the threonine T at position 216 is mutated to methionine M; or

[0028] (6) The mutant T216M / M351F was formed by mutating the threonine T at position 216 to methionine M and the methionine M at position 351 to phenylalanine F.

[0029] Preferably, the mutant has an amino acid sequence as shown in SEQ ID NO.2-7.

[0030] The mutant D27P / T216M has an amino acid sequence as shown in SEQ ID NO.2.

[0031] SEQ ID NO.2:

[0032] MPNYPQTIHFVGLNTPVGIEWSARNLPVIGTIPAEIDGAFFRAVPDPAHPP MFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSMIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

[0033] The mutant D27P / M351F has the amino acid sequence shown in SEQ ID NO.3.

[0034] SEQ ID NO.3:

[0035] MPNYPQTIHFVGLNTPVGIEWSARNLPVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDFPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

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

[0037] SEQ ID NO.4:

[0038] MPNYPQTIHFVGLNTPVGIEWSARNLPVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYDGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMRE AGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

[0039] The mutant M351F / Y156D has the amino acid sequence shown in SEQ ID NO.5.

[0040] SEQ ID NO.5:

[0041] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYDGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDFPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

[0042] The mutant T216M / Y156D has the amino acid sequence shown in SEQ ID NO.6.

[0043] SEQ ID NO.6:

[0044] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYDGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSMIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

[0045] The mutant T216M / M351F has the amino acid sequence shown in SEQ ID NO.7.

[0046] SEQ ID NO.7:

[0047] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTP VWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSMIHDFAITEHYAIFPIFPTTADLDRLKAGGAH WAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDFPRLRDADQGRPYRAAWYLSM NPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPPMMRAQVHGAWVSARQLANARQNVDSEAA.

[0048] In yet another aspect, the present invention provides a method for synthesizing vanillin, wherein the method comprises using a genetically engineered strain.

[0049] Specifically, the genetically engineered strain includes a mutant genetically engineered strain; the mutant genetically engineered strain overexpresses the mutant.

[0050] Specifically, the genetically engineered strain also includes a ferulic acid decarboxylase genetically engineered strain, and the ferulic acid decarboxylase is Bpf, having an amino acid sequence as shown in SEQ ID NO.8.

[0051] SEQ ID NO.8:

[0052] MDQFVGLHMIYTYENGWEYEIYIKNDHTIDYRIHSGMVGGRWVRDQEVNIVKLTKGVYKVSWTEPTGTDVSLNFMPEEKRMHGVIFFPKWVHERPDITVCYQNDYIDLMKESREKYETYPKYVVPEFADITYIHHAGVNDETIIAEAPYEGMTDEIRAGRK.

[0053] Specifically, the method comprises the following steps:

[0054] S1, activating the genetically engineered strain and then culturing the strain to obtain a seed solution;

[0055] S2, inoculating the seed liquid into a fermentation medium for fermentation and culturing;

[0056] S3, collecting the genetically engineered strain by centrifugation, breaking the resuspended cell solution, and collecting the supernatant by centrifugation again to obtain a crude enzyme solution of the genetically engineered strain;

[0057] S4, using ferulic acid as substrate, adding buffer and crude enzyme solution of ferulic acid decarboxylase genetic engineering strain, and transforming;

[0058] S5. After the transformation is completed, the crude enzyme solution of the Iso mutant genetic engineering strain is added to react to obtain a reaction solution, which is purified to obtain vanillin.

[0059] More specifically, the genetically engineered strains described in step S1 include ferulic acid decarboxylase genetically engineered strains and Iso mutant genetically engineered strains.

[0060] Preferably, the ferulic acid decarboxylase genetic engineering strain is BL21-pET28a-Bpf.

[0061] Preferably, the Iso mutant genetically engineered strain includes 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.

[0062] Specifically, the culture described in step S1 includes the following steps: inoculating the activated genetically engineered strain into the culture medium and culturing until the OD 600 =1.8-2.2 to obtain the first-level seed solution; inoculate the first-level seed solution into the culture medium and culture until OD 600 =3.8-4.2 to obtain seed solution.

[0063] Preferably, the activated genetically engineered strain is inoculated into the culture medium and cultured until OD 600 =2.0 to obtain the first-level seed solution.

[0064] Preferably, the first-level seed solution is inoculated into the culture medium and cultured until OD 600 =4.0 to obtain seed solution.

[0065] More specifically, the inoculation amount of the genetically engineered strain or the primary seed solution in step S1 is 0.5%-2%.

[0066] Preferably, the inoculation amount of the genetically engineered strain or the primary seed solution in step S1 is 1%.

[0067] More specifically, the culture medium in step S1 includes LB culture medium, SOB culture medium, SOC culture medium, TB culture medium or SB culture medium.

[0068] More specifically, the culture medium in step S1 is LB culture medium.

[0069] Preferably, the LB medium contains antibiotics.

[0070] Still more preferably, the antibiotics include but are not limited to one or more of ampicillin, kanamycin sulfate or chloramphenicol.

[0071] More preferably, the antibiotic is kanamycin sulfate.

[0072] Still more preferably, the concentration of the antibiotic is 40-60 μg / mL.

[0073] More preferably, the concentration of the antibiotic is 50 μg / mL.

[0074] Preferably, the culture temperature in step S1 is 35-37°C and the rotation speed is 200-220 rpm.

[0075] Specifically, the fermentation culture described in step S2 includes the following steps: inoculating the seed liquid into a primary seed tank containing a fermentation medium, a substrate sugar and an antibiotic, and culturing until OD 600 =5.8-6.2, transfer to the secondary seed tank containing fermentation medium, substrate sugar and antibiotics, and ferment until OD 600 =15-20, start cooling down, and when it drops to 22-25, add the inducer for induction.

[0076] More specifically, the inoculation amount of the seed solution in step S2 is 8%-12%.

[0077] Preferably, the inoculation amount of the seed solution in step S2 is 10%.

[0078] More specifically, the base sugar in step S2 is glucose.

[0079] More specifically, after the base sugar in step S2 is consumed, the feed flow is started to maintain the glucose mass concentration in the fermentation broth at 5-10 g / L.

[0080] More specifically, the antibiotic in step S2 is kanamycin sulfate.

[0081] Preferably, the concentration of kanamycin is 40-60 μg / mL.

[0082] More preferably, the concentration of the antibiotic is 50 μg / mL.

[0083] More specifically, in the primary seed tank or the secondary seed tank described in step S2, the tank pressure is maintained at 0.03-0.04 MPa, the dissolved oxygen is controlled at 40%-60%, the ventilation volume is 1.5 times the culture medium volume / min, and the pH is regulated at 6.8-7.2.

[0084] More specifically, the inducing agent in step S2 is IPTG.

[0085] Preferably, the concentration of IPTG is 0.5-1.5 mmol / L.

[0086] More preferably, the concentration of IPTG is 1.0 mmol / L.

[0087] More specifically, the induction time in step S2 is 12-16 hours.

[0088] Specifically, the concentration of ferulic acid in step S4 is 100-150 mmol / L.

[0089] Preferably, the concentration of ferulic acid in step S4 is 130 mmol / L.

[0090] Specifically, the buffer solution in step S4 is a Glycine-NaOH buffer solution with a pH of 9.5.

[0091] Specifically, the amount of the buffer added in step S4 is 0.3-0.7 mol / L.

[0092] Preferably, the amount of the buffer added in step S4 is 0.5 mol / L.

[0093] Specifically, the conditions for the conversion in step S4 are: pH 6.5-7.5, reaction temperature 23-27° C., and conversion time 30 min-60 min.

[0094] Preferably, the conditions for the conversion in step S4 are: pH 7.0, reaction temperature 25°C.

[0095] Specifically, the reaction conditions described in step S5 are: pH 9.0-9.5, reaction temperature 23-27° C., and reaction time 4-5 h.

[0096] Preferably, the reaction conditions in step S5 are: pH 9.5 and reaction temperature of 25°C.

[0097] The beneficial effects of the present invention are:

[0098] 1. The present invention has discovered that two water-soluble enzymes, ferulic acid decarboxylase Bpf and 4-vinylguaiacol oxygenase Iso, are heterologously expressed in Escherichia coli. At a substrate concentration of 100 mmol / L, they can catalyze ferulic acid to produce vanillin to a concentration of 9.66 g / L within 5 hours.

[0099] 2. The present invention mutates 4-vinylguaiacol oxygenase, and the enzyme activity of the mutants D27P / T216M, D27P / M351F, D27P / Y156D, M351F / Y156D, T216M / Y156D, and T216M / M351F is 2.49-3.1 times that before the mutation. The mutant strain can catalyze the production of vanillin up to 19.58 g / L within 5 hours in the enzyme reaction system, and the Escherichia coli engineered strain can still stably express ferulic acid decarboxylase and 4-vinylguaiacol oxygenase after 1000 generations, and the activity of the enzyme is not affected.

[0100] 3. The present invention explores a production process, which is suitable for industrial production of vanillin and has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 This is the HPLC detection spectrum of ferulic acid, vanillin and 4-vinylguaiacol.

[0102] Figure 2 It is the expression diagram of Bpf protein; M in the figure represents Marker; 1 represents lane 1 is pET28a empty load; 2 represents lane 2 is Ecoli-Bpf total protein; 3 represents lane 3 is Ecoli-Bpf supernatant protein; 4 represents lane 4 is Ecoli-Bpf precipitated protein.

[0103] Figure 3 It is the expression diagram of Iso protein; M in the figure represents Marker; 1 represents lane 1 is pET28a empty load; 2 represents lane 2 is Ecoli-Iso total protein; 3 represents lane 3 is Ecoli-Iso supernatant protein; 4 represents lane 4 is Ecoli-Iso precipitated protein.

[0104] Figure 4The figure is the electrophoresis diagram of Iso and its mutant proteins (pure enzyme); in the figure, M represents Marker; 1 represents lane 1, which is the parent Iso of 4-vinylguaiacol oxygenase; 2 represents lane 2, which is the mutant D27P / T216M; 3 represents lane 3, which is the mutant D27P / M351F; 4 represents lane 4, which is the mutant D27P / Y156D; 5 represents lane 5, which is the mutant M351F / Y156D; 6 represents lane 6, which is the mutant T216M / Y156D; 7 represents lane 7, which is the mutant T216M / M351F.

[0105] Figure 5 These are photos of the colony morphology of the BL21-pET28a-Bpf strains passaged at different times, DP0-DP5 (up to 1000 generations).

[0106] Figure 6 These are photos of the colony morphology of the BL21-pET28a-Iso mutant strain T216M / M351F at different passage times DP0-DP5 (the highest passage was 1000). DETAILED DESCRIPTION

[0107] The present invention will be further described in detail below by way of examples. The following examples are only a part of the present invention and are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are all routine experiments unless otherwise specified, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0108] The culture medium formula involved in the present invention is as follows:

[0109] 1. LB liquid medium (1 L): 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, dilute to 1 L with pure water, and sterilize with high-pressure steam at 121°C for 15 min.

[0110] 2. LB solid medium (1 L): Add 15 g agar to the LB liquid medium formula.

[0111] 3. 2YT medium (1 L): 16 g tryptone, 10 g yeast extract, 5 g sodium chloride, dilute to 1 L with pure water, and sterilize with high-pressure steam at 121°C for 15 min.

[0112] 4. The formula of the fermentation medium includes, by percentage, the following: 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 the solvent is water; the formula of the trace element solution (100×) includes, by mass percentage, the following: EDTA 0.08-1.2%, zinc acetate 0.1-0.3%, sodium molybdate 0.01-0.03%, boric acid 0.03-0.05%, cupric chloride 0.01-0.03%, cobalt chloride 0.03-0.05%, manganese chloride 0.08-0.12%, ammonium ferric citrate 1-2%, and the solvent is water.

[0113] 5. The feed formula for the fermentation process includes, by percentage, 50-60% glucose, 0.07-0.1% magnesium sulfate, 1-2% trace elements, and water as the solvent.

[0114] Basic Experimental Example 1 Establishment of Vanillin Detection Method

[0115] Vanillin, ferulic acid and 4-vinylguaiacol standard samples were dissolved in 100% methanol solution, and the contents of vanillin, ferulic acid and 4-vinylguaiacol were detected by high performance liquid chromatography (HPLC).

[0116] The HPLC detector was Waters Alliance e2695; the chromatographic column was Hypersil BDS C18 (4.6 mm × 150 mm; 5 μm); the mobile phase was water (containing 0.5% glacial acetic acid) and methanol in a 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 at 30°C; and the injection volume was 10 μL.

[0117] The HPLC detection method for vanillin, ferulic acid and 4-vinylguaiacol was successfully established. Figure 1 As shown, the elution positions of vanillin, ferulic acid and 4-vinylguaiacol are 8.679min, 9.491min and 12.768min respectively, all of which are single strong absorption peaks. The elution positions of the test sample and the standard are consistent. The vanillin content of the sample is then calculated based on the standard curve.

[0118] Basic Experimental Example 2 Determination of Enzyme Activity of Vinylguaiacol Oxygenase

[0119] The reaction system for determining enzyme activity was as follows (1 mL): 100 μg of pure enzyme was mixed with 0.5 mol / L pH 9.5 Glycine-NaOH buffer, and then with 10 mmol / L substrate 4-vinylguaiacol. After reacting at 25°C for 10 min, 2 μL of the reaction solution was taken into a 96-well plate, and 20 μL of 2,4-dinitrophenylhydrazine solution and 200 μL of 1 mol / L NaOH solution were added. The mixture was mixed well, and distilled water was used as a blank control. The absorbance at 520 nm was measured using an ELISA plate.

[0120] Enzyme activity definition: Under the conditions of 25°C and pH 9.5, the amount of enzyme required to produce 1 μmol of product vanillin within 1 minute is defined as one unit U.

[0121] Basic Experimental Example 3 Construction of pET28a-Bpf, an expression vector of genes related to the vanillin synthesis pathway

[0122] 1. Construction of pET28a-Bpf vector

[0123] The ferulic acid decarboxylase Bpf (NCBI accession number ACO50701.1) from Bacillus has an amino acid sequence as shown in SEQ ID NO.8, and is optimized according to the Escherichia coli codon. The codon-optimized sequence is sent to General Biotechnology (Anhui) Co., Ltd. for synthesis, and PCR amplification (PrimeSTAR GXLDNA Polymerase, Takara Code No. R050A) is performed using primers P1 (Bpf-F, Bpf-R) to amplify the linear fragment Bpf (483 bp) having the nucleotide sequence as shown in SEQ ID NO.9.

[0124] SEQ ID NO.9:

[0125] ATGGACCAGTTCGTTGGTCTGCACATGATCTACACCTACGAAAACGGTTGGGAATACGAAATCTACATCAAAAACGATCACACCATCGACTACCGTATCCACTCTGGTATGGTTGGTGGTCGTTGGGTTCGCGATCAGGAAGTTAACATCGTTAAACTGACCAAAGGTGTTTACAAAGTTTCCTGGACCGAACCGACCGGCACCGACGTTTCTCTCAACTTCATGCCAGAAGAAAAACGTAT GCACGGTGTTATCTTCTTCCCGAAGTGGTTTCACGAACGTCCAGACATTACCGTTTGCTACCAGAACGACTACATTGACCTGATGAAAGAATCTCGTGAAAAATACGAGACCTACCCGAAATACGTTGTTCCAGAGTTCGCGGACATCACCTACATCCACCACGCGGGTGTTAACGACGAAACCATCATCGCGGAAGCGCCGTACGAAGGTATGACCGACGAGATCCGTGCGGGTCGTAAA.

[0126] When amplifying the linearized pET28a vector, the nucleotide sequence of pET28a (as shown in SEQ ID NO. 10) was amplified by PCR using primers P2 (pET28a-F, pET28a-R) to obtain the linearized pET28a vector (5308 bp).

[0127] SEQ ID NO.10:

[0128]

[0129] The primer sequences used are shown in Table 1:

[0130] Table 1 Sequence information of primers P1 and P2

[0131]

[0132] In Table 1, F and R are amplification primers, wherein: F represents a forward primer, and R represents a reverse primer.

[0133] The linearized pET28a vector and fragment Bpf were connected using a recombination cloning kit (CloneExpressⅡOne Step Cloning Kit, Norwegian CAT#: C112-01), and transformed into competent cells E. coli DH5α (Weidi Biotechnology, CAT#: DL1001) using the calcium chloride chemical transformation method. Positive clones were picked, plasmids were extracted for sequencing, and the recombinant plasmid with the correct sequence was recorded as pET28a-Bpf. The correct plasmid pET28a-Bpf was transformed into E. coli BL21 (DE3) (Weidi Biotechnology, CAT#: EE1002) by the calcium chloride chemical transformation method, and the recombinant bacteria were recorded as BL21-pET28a-Bpf, the target gene promoter was T7 promoter, and the screening marker was kanamycin sulfate (Kana).

[0134] 2. Cultivation and induced expression of recombinant strains

[0135] The recombinant strain BL21-pET28a-Bpf was inoculated into 2YT medium (containing 50 μg / mL Kana) with a volume of 1 L / 5 L and placed in a shaker at 37 °C and 220 rpm to a bacterial concentration of OD 600 =0.6-0.8, add IPTG at a final concentration of 1mmol / L, and induce expression at 25℃ for 12-16h.

[0136] Take 50OD BL21-pET28a-Bpf culture solution to a centrifuge bottle, centrifuge at 4500rpm for 20min to collect the bacteria, discard the supernatant, add 5mL 50mmol / L phosphate buffer (pH 7.5) to the bacterial precipitate to resuspend the bacteria. Use an ultrasonic crusher to crush on ice for 10min (70kHz, ultrasound 3s, pause 7s) to obtain cell crushing liquid, centrifuge at 12000rpm for 30min, and the supernatant is the Ecoli-Bpf crude enzyme. After the crushing is completed, collect the supernatant, precipitate and crushing liquid for SDS-PAGE electrophoresis to detect protein expression.

[0137] The expression of the recombinant strain BL21-pET28a-Bpf enzyme is shown in Figure 2M represents Marker, lane 1 represents pET28a empty load, lane 2 represents Ecoli-Bpf total protein, lane 3 represents Ecoli-Bpf supernatant protein, and lane 4 represents Ecoli-Bpf precipitated protein.

[0138] Basic Experimental Example 4 Construction of pET28a-Iso Expression Vector of Genes Related to Vanillin Synthesis Pathway

[0139] 1. Construction of pET28a-Iso vector

[0140] Using the BLAST tool, we searched for all amino acid sequences with a similarity of more than 30% to the Cso2 sequence in the Uniprot database (with an amino acid length of 450-550), constructed all similar sequences by the AlphaFold2-based structural modeling method, and then constructed a structure-based evolutionary tree. We selected five enzymes with certain representativeness as key starting enzymes for subsequent functional characterization. Among them, the 4-vinylguaiacol oxygenase Iso from Sphingobium sp.GW456-12-10-14-TSB1 has good activity.

[0141] 4-vinylguaiacol oxygenase Iso from Sphingomonas (NCBI accession number OUC52898.1) has an amino acid sequence as shown in SEQ ID NO. 1. The codon-optimized sequence was used to construct a vector according to the method described in Basic Experimental Example 3. PCR amplification was performed using primers P3 (Iso-F, Iso-R) to amplify a linear fragment Iso (1482 bp) having a nucleotide sequence as shown in SEQ ID NO. 15.

[0142] SEQ ID NO.15:

[0143]

[0144] The primer sequences used are shown in Table 2:

[0145] Table 2 Primer P3 sequence information

[0146]

[0147] In Table 2, F and R are amplification primers, wherein: F represents a forward primer, and R represents a reverse primer.

[0148] The obtained recombinant bacteria were recorded as BL21-pET28a-Iso, the target gene promoter was T7 promoter, and the screening marker was kanamycin sulfate (Kana).

[0149] 2. Cultivation and induced expression of recombinant strains

[0150] The protein expression and detection steps of the recombinant strain BL21-pET28a-Iso are the same as those described in "2. Cultivation and induced expression of recombinant strains" in Basic Experimental Example 3. The expression of the recombinant strain BL21-pET28a-Iso enzyme 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.

[0151] Example 1: Recombinant strain BL21-pET28a-Bpf crude enzyme converts ferulic acid to produce 4-vinylguaiacol

[0152] 1. Strain culture and crude enzyme solution preparation

[0153] The strain culture and crude enzyme solution preparation refer to "2. Cultivation of recombinant strains and induced expression" in Basic Experimental Example 3.

[0154] 2. Conversion of ferulic acid to 4-vinylguaiacol

[0155] S1 reaction: In a 2L conical flask, add substrate 50mmol / L ferulic acid, 10OD BL21-pET28a-Bpf crude enzyme solution, and 0.5mol / L pH 9.5 Glycine-NaOH buffer, respectively, at a reaction pH of 6.0-8.5 and a reaction temperature of 15-35°C for 30min-60min, and analyze by HPLC.

[0156] 4-vinylguaiacol molar conversion rate (%) = 4-vinylguaiacol molar concentration (mmol / L) / initial ferulic acid concentration (mmol / L) × 100%.

[0157] 4-vinylguaiacol yield (g / L) = 4-vinylguaiacol molar concentration (mmol / L) × 150. (The relative molecular mass of 4-vinylguaiacol is 150).

[0158] The results are shown in Table 3 below:

[0159] Table 3 4-vinylguaiacol yield and conversion rate

[0160]

[0161] The results showed that the optimal conditions for the crude enzyme of the recombinant strain BL21-pET28a-Bpf to convert ferulic acid to 4-vinylguaiacol were pH 7.0 and temperature 25℃.

[0162] Example 2: Recombinant strain BL21-pET28a-Iso crude enzyme transforms 4-vinylguaiacol to produce vanillin

[0163] 1. Strain culture and crude enzyme solution preparation

[0164] The strain culture and crude enzyme solution preparation refer to "2. Cultivation of recombinant strains and induced expression" in Basic Experimental Example 3.

[0165] 2. Conversion of ferulic acid to vanillin

[0166] S1 reaction: In a 2L conical flask, add 50mmol / L ferulic acid as substrate, 10OD of BL21-pET28a-Bpf crude enzyme solution, and 0.5mol / L pH9.5 Glycine-NaOH buffer, and convert at pH 7.0 and 25℃ for 30min-60min. After HPLC detection, perform S2 reaction after all ferulic acid is converted into 4-vinylguaiacol.

[0167] S2 reaction: add 40OD of BL21-pET28a-Iso crude enzyme solution to the S1 reaction solution, convert at pH 8-11 and reaction temperature 15-35 for 4-5h, and analyze by HPLC.

[0168] Vanillin molar conversion rate (%) = vanillin molar concentration (mmol / L) / initial ferulic acid concentration (mmol / L) × 100%.

[0169] Vanillin yield (g / L) = vanillin molar concentration (mmol / L) × 152. (The relative molecular mass of vanillin is 152)

[0170] The results are shown in Table 4 below:

[0171] Table 4 Vanillin yield and conversion rate

[0172]

[0173] The results showed that the optimal conditions for the crude enzyme of the recombinant strain BL21-pET28a-Iso to convert 4-vinylguaiacol to produce vanillin were pH 9.5 and the optimal temperature was 25℃.

[0174] Example 3 Recombinant bacterial whole cell method to transform ferulic acid to produce vanillin

[0175] 1. Strain culture and crude enzyme solution preparation

[0176] The strain culture and crude enzyme solution preparation refer to "2. Cultivation and induced expression of recombinant strains" described in Basic Experimental Example 3, except that the cells collected by centrifugation are resuspended without ultrasonic disruption and the whole-cell catalytic reaction is directly carried out.

[0177] 2. Whole-cell catalytic reaction

[0178] S1 reaction: In a 2L conical flask, add substrate 50mmol / L ferulic acid, 10OD BL21-pET28a-Bpf whole cells, 0.5mol / L pH 9.5 Glycine-NaOH buffer, react at pH 7.0, reaction temperature 25℃ for 30min-60min, and perform S2 reaction after HPLC detection that ferulic acid is completely converted into 4-vinylguaiacol.

[0179] S2 reaction: 40 OD of BL21-pET28a-Iso whole cells (60 mL) were added to the S1 reaction solution. The total reaction volume was 1 L. The reaction was carried out at a pH of 9.5 and a reaction temperature of 25°C for 4-5 h, and then analyzed by HPLC.

[0180] The results showed that the yield of vanillin was 6.18 g / L, the remaining 4-vinylguaiacol was 2.81 g / L, and the conversion rate was 81.3%. Under the same reaction conditions, the conversion effect of crude enzyme was better than that of whole cell.

[0181] Example 4 Construction of 4-vinylguaiacol oxygenase mutants based on whole plasmid PCR

[0182] Using the recombinant plasmid pET28a-Iso (Example 2) as a template, PCR (PrimeSTAR HS DNA Polymerase, Takara Code No. R010A) was used for in vitro mutation amplification. The primer sequences used for site-directed mutagenesis are shown in Table 5 below.

[0183] Table 5 Primer sequence information for site-directed mutagenesis

[0184]

[0185] In Table 5, F and R are amplification primers, wherein: F represents a forward primer, and R represents a reverse primer.

[0186] After the PCR was completed, 10 μL of PCR product was added to 1 μL of restriction enzyme DpnⅠ to digest the template plasmid. After reacting at 37°C for 2 hours, the digested product was transferred into E. coli BL21 (DE3) (Weidi Biotechnology, CAT#: EE1002) according to the method described in Example 1.1, and spread on LB solid plate (containing 50 μg / mL Kana), cultured at 37°C overnight, and 4 transformants were selected and cultured in LB liquid medium (containing 50 μg / mL Kana) for 6 hours before being sent for sequencing. The correctly sequenced BL21-pET28a-IsoD27P, BL21-pET28a-Iso D27P, and BL21-pET28a-Iso Y156D were obtained.

[0187] Using the correctly sequenced BL21-pET28a-Iso D27P plasmid 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-IsoD27P / T216M or BL21-pET28a-Iso D27P / M351F, were obtained.

[0188] Using the correctly sequenced BL21-pET28a-Iso Y156D plasmid as a template and D27P-F / D27P-R or M351F-F / M351F-R as primers, after whole-plasmid PCR, transformation, and plating, the double-site mutant strains, BL21-pET28a-IsoD27P / Y156D or BL21-pET28a-Iso M351F / Y156D, were obtained.

[0189] Using the correctly sequenced BL21-pET28a-Iso T216M plasmid as a template and Y156D-F / Y156D-R or M351F-F / M351F-R as primers, after whole-plasmid PCR, transformation, and plating, double-site mutant strains, BL21-pET28a-IsoT216M / Y156D or BL21-pET28a-Iso T216M / M351F, were obtained.

[0190] Example 5 Protein expression and purification of 4-vinylguaiacol oxygenase and its mutants

[0191] 1. Protein expression

[0192] The recombinant strain BL21-pET28a-Iso and its mutants were inoculated into 2YT medium (containing 50 μg / mL Kana) with a volume of 1 L / 5 L and placed in a shaker at 37 °C and 220 rpm to culture until the bacterial concentration OD 600 =0.6-0.8, add IPTG and FeCl2 at a final concentration of 1mmol / L, and induce expression at 25℃ for 12-16h.

[0193] Take 50OD of BL21-pET28a-Iso and its mutant culture solution into a centrifuge bottle, centrifuge at 4500rpm for 20min to collect the cells, discard the supernatant, add 5mL 50mmol / L phosphate buffer (pH 7.5) to the cell pellet to resuspend the cells. Use an ultrasonic disruptor to disrupt on ice for 10min (70kHz, ultrasound for 3s, pause for 7s) to obtain cell disruption solution, centrifuge at 12000rpm for 30min, and the supernatant is the crude enzyme solution of BL21-pET28a-Iso and its mutants.

[0194] 2. Protein Purification

[0195] The crude protein enzyme solution was separated and purified by nickel column affinity chromatography. The filler of the nickel column was HispurTM Ni-NTAResin. The target protein with the His tag would specifically bind to the resin. Wash buffer (50mM Tris-HCl, 150mM NaCl, 10mM imidazole; pH 7.5) was used to wash away the impurities, and then the protein was eluted using Elution buffer (50mM Tris-HCl, 150mM NaCl, 300mM imidazole; pH 7.5). The protein eluate obtained above was concentrated by centrifugation using an Amino Ultra-15 (30kDa) ultrafiltration tube, and the imidazole left over during the protein purification process was washed away using Desalination buffer (50mM Tris-HCl, 150mM NaCl; pH 7.5). Glycerol was added to the resulting protein solution to a final concentration of 10% and stored at -80°C.

[0196] After purification, pure enzyme with the same protein content and volume was loaded for SDS-PAGE verification. The results are shown in the attached Figure 4 As shown, wherein 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.

[0197] Example 6 Comparison of the activities of different 4-vinylguaiacol oxygenase mutants and the parent enzyme

[0198] The concentrated pure enzyme was diluted, the protein concentration was determined by Bradford method, and the enzyme activity was determined according to Basic Experimental Example 2. The specific activity of the parent 4-vinylguaiacol oxygenase Iso was 2.80U / mg, and the specific activities of the mutants D27P / T216M, D27P / M351F, D27P / Y156D, M351F / Y156D, T216M / Y156D, and T216M / M351F were 8.13U / mg, 7.72U / mg, 6.96U / mg, 7.40U / mg, 8.45U / mg, and 8.68U / mg, respectively, among which 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.

[0199] Table 6 Comparison of the activities of different 4-vinylguaiacol oxygenase mutants and the parent enzyme

[0200] Enzymes Specific activity U / mg Iso 2.80 D27P / T216M 8.13 D27P / M351F 7.72 D27P / Y156D 6.96 M351F / Y156D 7.40 T216M / Y156D 8.45 T216M / M351F 8.68

[0201] Example 7 Comparison of the conversion rate of vanillin synthesized by 4-vinylguaiacol oxygenase mutant and parent crude enzyme

[0202] 1. Strain culture and crude enzyme solution preparation

[0203] The strain culture and crude enzyme solution preparation refer to "2. Cultivation of recombinant strains and induced expression" in Basic Experimental Example 3.

[0204] 2. Conversion of ferulic acid to vanillin

[0205] S1 reaction: In a 2L conical flask, add substrate 100mmol / L ferulic acid, 10OD BL21-pET28a-Bpf crude enzyme solution, 0.5mol / L pH 9.5 Glycine-NaOH buffer, react at pH 7.0, reaction temperature 25℃ for 30min-60min, and perform S2 reaction after HPLC detection that ferulic acid is completely converted into 4-vinylguaiacol.

[0206] S2 reaction: Add 40 OD of crude enzyme solution of BL21-pET28a-Iso and its mutants to the S1 reaction solution, convert at pH 9.5 and reaction temperature 25°C for 4-5 hours, and analyze by HPLC.

[0207] The results showed that after 5 hours of reaction, the vanillin conversion rate of the parent Iso was 63.53%, and the yield was 9.66 g / L. The conversion rates of the mutants D27P / T216M, D27P / M351F, D27P / Y156D, M351F / Y156D, T216M / Y156D, and T216M / M351F were 94.87%, 92.15%, 89.95%, 91.40%, 95.23%, and 97.28%, respectively, and the yields were 14.42 g / L, 14.01 g / L, 13.67 g / L, 13.89 g / L, 14.47 g / L, and 14.79 g / L, respectively. The measurement results are shown in Table 7.

[0208] Table 7 Comparison of conversion rate and yield

[0209] Enzymes Conversion Rate % Yield g / L Iso 63.53％ 9.66 D27P / T216M 94.87％ 14.42 D27P / M351F 92.15％ 14.01 D27P / Y156D 89.95％ 13.67 M351F / Y156D 91.40％ 13.89 T216M / Y156D 95.23％ 14.47 T216M / M351F 97.28％ 14.79

[0210] Example 8 Comparison of the ability of 4-vinylguaiacol oxygenase mutants T216M / M351F to convert vanillin

[0211] T216M / M351F showed the best specific activity and conversion rate, so in this example, the ability of 4-vinylguaiacol oxygenase mutants T216M / M351F to convert vanillin was compared.

[0212] 1. Strain culture and crude enzyme solution preparation

[0213] The strain culture and crude enzyme solution preparation refer to "2. Cultivation of recombinant strains and induced expression" in Basic Experimental Example 3.

[0214] 2. Conversion of ferulic acid to vanillin

[0215] S1 reaction: In a 2L conical flask, add 50-400mmol / L ferulic acid as substrate, 10OD of BL21-pET28a-Bpf crude enzyme solution, and 0.5mol / L pH9.5 Glycine-NaOH buffer solution. The reaction is carried out at pH 7.0 and 25°C for 30min-60min. After HPLC detection, the ferulic acid is completely converted into 4-vinylguaiacol, and then the S2 reaction is carried out.

[0216] S2 reaction: add 40OD of crude enzyme solution of BL21-pET28a-Iso T216M / M351F to the S1 reaction solution, convert at pH 9.5 and reaction temperature 25°C for 4-5h, and analyze by HPLC.

[0217] The results are shown in Table 8 below:

[0218] Table 8 Comparison of the ability of T216M / M351F to transform vanillin

[0219] Ferulic acid concentration (mmol / L) Vanillin yield (g / L) Vanillin conversion rate (%) 50 7.53 99.11 100 14.78 97.28 150 19.58 85.87 200 18.67 61.41 300 17.74 38.9 400 18.93 31.13

[0220] The experimental results show that when the concentration of ferulic acid is within the range of 100-150 mmol / L, within a certain reaction time, the substrate conversion rate and the amount of product obtained are optimal, which is suitable for industrial production.

[0221] Example 9 Stability test of Escherichia coli engineered strains

[0222] To verify the stability of the strains during continuous subculture, the BL21-pET28a-Bpf and BL21-pET28a-Iso mutant strains T216M / M351F were examined in terms of colony morphology, turbidity and enzyme activity, and the phenotypic characteristics and genetic information stability of the strains during continuous subculture were monitored.

[0223] Continuous subculture of the strain and calculation of the number of generations: 12 hours is one subculture cycle, and the inoculum is continuously subcultured at 2% (volume fraction). The number of bacteria after a single cell of the strain divides and multiplies n times is 2 n In each subculture cycle (12h), the number of cells increases in geometric progression (2 n ) proliferation. By measuring the growth curve of the strain, it can be seen that when the inoculation amount is 2%, the growth generation number (that is, the number of times the bacteria divide) in each cycle is 12 generations.

[0224] The 0th generation strain was cultured for a long time and continuously until the 1000th generation, wherein the initial strain was recorded as DP0, the 200th generation was recorded as DP1, the 400th generation was recorded as DP2, the 600th generation was recorded as DP3, the 800th generation was recorded as DP4, and the 1000th generation was recorded as DP5.

[0225] 1. Colony morphology observation

[0226] During the serial subculture process of BL21-pET28a-Bpf and BL21-pET28a-Iso mutant strains T216M / M351F, their DP0-DP5 were diluted to the appropriate gradient by 10-fold dilution method, spread on LB solid medium, and inverted cultured at 37°C for 48 h. The colony morphology, size, color and other characteristics were observed, photographed and recorded.

[0227] The colony morphology of BL21-pET28a-Bpf strain is shown in Figure 5 The colony morphology of the BL21-pET28a-Iso mutant strain T216M / M351F is shown in Figure 6 The results showed that during the continuous passage process, the colonies of the BL21-pET28a-Bpf strain and the BL21-pET28a-Iso mutant strain T216M / M351 had neat colony edges, smooth surfaces, a diameter of about 3 mm, and were milky white.

[0228] 2. Turbidity detection

[0229] The change in turbidity of the fermentation broth of the strain reflects the change in total biomass. During the continuous passage of BL21-pET28a-Bpf and BL21-pET28a-Iso mutant strains T216M / M351F, the turbidity of the culture broth was measured at DP0-DP5, and three parallel tests were performed (represented by C1, C2, and C3). The LB medium without bacteria was used as a blank control, and the absorbance of the culture broth at 600nm was detected using Nanodrop. The results are shown in Tables 9-10.

[0230] Table 9 BL21-pET28a-Bpf turbidity detection

[0231] Cultivating Algebra C1 C2 C3 average Bpf-DP0 4.4 3.6 4.2 4.07 Bpf-DP1 4.5 3.8 4.5 4.26 Bpf-DP2 4.3 4.9 4.7 4.63 Bpf-DP3 4.3 4.6 4.4 4.43 Bpf-DP4 4.2 3.9 3.6 3.9 Bpf-DP5 4 4.2 3.9 4.03

[0232] Table 10 Turbidity detection of BL21-pET28a-Iso mutant strain T216M / M351F

[0233]

[0234]

[0235] 3. Enzyme activity detection

[0236] During the continuous passage of BL21-pET28a-Iso mutant strain T216M / M351F, the enzyme activities of DP0-DP5 were determined respectively, referring to the enzyme activity determination of 4-vinylguaiacol oxygenase.

[0237] The test results are shown in Table 11, showing that the enzyme activity of the BL21-pET28a-Iso mutant strain T216M / M351F engineering strain had no significant change during the continuous passage process.

[0238] Table 11 Enzyme activity determination during continuous passage of strains

[0239] Cultivating Algebra Specific activity U / mg T216M / M351F-DP0 8.68 T216M / M351F-DP1 8.53 T216M / M351F-DP2 8.45 T216M / M351F-DP3 8.60 T216M / M351F-DP4 8.71 T216M / M351F-DP5 8.63

[0240] The results showed that the colony morphology, turbidity and enzyme activity of the BL21-pET28a-Iso mutant strain T216M / M351F did not change significantly during the continuous subculture process. The strain was stable and could be used for the continuous industrial production of vanillin.

[0241] Example 10 A process for producing vanillin using highly stable recombinant Escherichia coli

[0242] 1. Activation of recombinant E. coli strains

[0243] Use an inoculation loop to dip the bacterial solution (BL21-pET28a-Bpf and BL21-pET28a-Iso T216M / M351F) from the glycerol tube, streak it on a LB solid plate (containing 50μg / mL Kana), and incubate it upside down at 37°C in a constant temperature incubator for 12 hours. Then pick a single colony on the activated plate and inoculate it in 5mL LB liquid culture medium, and place it in a shaker at 37°C and 220rpm for 8-10 hours.

[0244] 2. Seed solution preparation

[0245] Preparation of primary seed solution: Take the activated bacteria and add 1% inoculum to 100 mL LB medium (containing 50 μg / mL Kana) and culture at 37°C and 220 rpm until OD 600 =about 2.

[0246] Preparation of secondary seed solution: Take the primary seed solution and add it to 1L LB medium (containing 50μg / mL Kana) at a 1% inoculum, and culture at 37°C and 220rpm until OD 600 =About 4.

[0247] 3. Fermentation tank expansion culture

[0248] The secondary seed liquid was transferred to the primary seed tank (50L) containing 30L fermentation medium at a 10% inoculation rate, and the substrate sugar and antibiotics (containing 50μg / mL Kana) were added. The pH was automatically adjusted to about 7 with 50% ammonia water by volume, the culture temperature was 37°C, the ventilation volume was 1.5 times the volume of the culture medium / min, the tank pressure was maintained at 0.03-0.04Mpa, and the dissolved oxygen was controlled at 40%-60%. When the initial glucose in the fermentation medium was consumed, the feed flow was started to maintain the glucose mass concentration in the fermentation liquid at 5-10g / L. Until OD 600 =6, it can be transferred to the secondary fermentation tank (about 7h).

[0249] The bacterial liquid from the first-stage seed tank was transferred to a 500L fermentation tank containing 300L fermentation medium at a 10% inoculation rate, and substrate sugar and antibiotics (containing 50μg / mL Kana) were added. The pH was automatically adjusted to 6.8-7.2 with 50% ammonia water by volume, the culture temperature was 37°C, the ventilation volume was 1.5 times the volume of the culture medium / min, the tank pressure was maintained at 0.03-0.04Mpa, and the dissolved oxygen was controlled at 40%-60%. When the initial glucose in the fermentation medium was consumed, the feed was started to control the specific growth rate of the genetically engineered bacteria between 0.2-0.5. OD 600=15-20℃, and then the temperature was lowered to 22-25℃. Then, 1mmol / L LIPTG (1mmol / L FeCl2 was required for BL21-pET28a-Iso T216M / M351F) was added for induction for about 12-16h.

[0250] 4. Preparation of crude enzyme solution

[0251] Take 10OD BL21-pET28a-Bpf culture solution and use a high-speed continuous flow centrifuge to centrifuge at 4500rpm for 30min to collect the bacteria, discard the supernatant, add 50mmol / L phosphate buffer (pH 7.5) to the bacterial pellet to resuspend the bacteria, use an ultra-high pressure cell homogenizer to disrupt the cells three times to obtain cell disruption solution, centrifuge at 12000rpm for 50min, and the supernatant is the BL21-pET28a-Bpf crude enzyme solution.

[0252] Another 40OD BL21-pET28a-Iso T216M / M351F culture medium was taken into a centrifuge bottle, centrifuged at 4500rpm for 30min to collect the bacteria, discarded the supernatant, added 50mmol / L phosphate buffer (pH 7.5) to the bacterial pellet to resuspend the bacteria, and disrupted them three times with an ultra-high pressure cell homogenizer to obtain cell disruption liquid, centrifuged at 12000rpm for 50min, and the supernatant was the BL21-pET28a-Iso T216M / M351F crude enzyme liquid.

[0253] 5. Conversion of ferulic acid to vanillin

[0254] S1 reaction: In a 500L reactor, add substrate 130mmol / L ferulic acid, 10OD BL21-pET28a-Bpf crude enzyme solution, 0.5mol / L pH9.5 Glycine-NaOH buffer, adjust the reaction pH to 6.5-7.5, maintain the reaction temperature at 25±2 for 30min-60min, and perform HPLC analysis.

[0255] S2 reaction: After all ferulic acid is converted into 4-vinylguaiacol, 40OD BL21-pET28a-IsoT216M / M351F crude enzyme solution is added, and the reaction is carried out at a pH of 9-9.5 and a reaction temperature of 25±2 for 4-5h, and HPLC detection and analysis is performed.

[0256] 6. Vanillin Purification

[0257] The reaction solution is adjusted to pH 3.0-5.0 for acidification, and ultrafiltration (ceramic membrane pore size 0.1 μm) is used to remove whole cells, cell fragments, macromolecular proteins, and macromolecules such as nucleic acids in the centrifugal supernatant. The vanillin content is detected by liquid chromatography. According to the vanillin content, 20-50g macroporous adsorption resin / 1g vanillin is added to the ultrafiltration supernatant. After adsorption, 3 times the volume of resin is used for elution at room temperature to obtain a crude vanillin ethanol solution. The crude vanillin solution is then put into a vacuum distillation device. Since vanillin is unstable, the temperature is maintained at 40-60°C and the pressure is less than -0.1MPa. When the vanillin content is about 25g / L, the concentration is stopped, and the cooling system is turned on to reduce the temperature to below 15°C for crystallization. The crystallization liquid is separated from the wet crystals by suction filtration, and the wet crystals are rinsed with a small amount of water while suction filtration. The crystals are dried at room temperature under reduced pressure to obtain the vanillin product. The filtrate is again put into the vacuum distillation device for concentration, crystallization, and drying.

[0258] The results showed that in a 500L reactor, the conversion rate of ferulic acid into vanillin was 92.6%. After further separation and purification, 6.6 kg of pure vanillin was obtained in the form of light yellow needle-shaped crystals with a purity of 99.8%, and it had a strong milky and vanilla flavor.

[0259] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A mutant of 4-vinylguaiacol oxygenase for synthesizing vanillin, characterized in that: The mutants include: (1) a mutant D27P / T216M in which the aspartic acid D at position 27 of SEQ ID NO.1 is mutated to phenylalanine F, and the threonine T at position 216 is mutated to methionine M; or (2) a mutant T216M / Y156D in which the tyrosine Y at position 156 of SEQ ID NO.1 is mutated to aspartic acid D, and the threonine T at position 216 is mutated to methionine M; or (3) A mutant T216M / M351F in which the threonine T at position 216 of SEQ ID NO. 1 is mutated to methionine M, and the methionine M at position 351 is mutated to phenylalanine F.

2. The mutant according to claim 1, characterized in that The amino acid sequence of the mutant is shown in SEQ ID NO.2, SEQ ID NO.6 or SEQ ID NO.

7.

3. A method for synthesizing vanillin, characterized in that: 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 according to any one of claims 1-2.

4. The method according to claim 3, characterized in that The genetically engineered strain also includes a ferulic acid decarboxylase genetically engineered strain, and the ferulic acid decarboxylase is a Bpf amino acid sequence as shown in SEQ ID NO.

8.

5. The method according to claim 3, characterized in that: The method comprises the following steps: S1, activating the genetically engineered strain and then culturing the strain to obtain a seed solution; S2, inoculating the seed liquid into a fermentation medium for fermentation and culturing; S3, collecting the genetically engineered strain by centrifugation, breaking the resuspended cell solution, and collecting the supernatant by centrifugation again to obtain a crude enzyme solution of the genetically engineered strain; S4, using ferulic acid as substrate, adding buffer and crude enzyme solution of ferulic acid decarboxylase genetic engineering strain, and transforming; S5. After the transformation is completed, the crude enzyme solution of the mutant genetic engineering strain is added to react to obtain a reaction solution, which is purified to obtain vanillin; The ferulic acid decarboxylase genetic engineering strain, the ferulic acid decarboxylase is Bpf amino acid sequence as shown in SEQ ID NO.

8.

6. The method according to claim 5, characterized in that The concentration of ferulic acid in step S4 is 100-150 mmol / L.

7. The method according to claim 5, characterized in that The conditions for the conversion in step S4 are: pH 6.5-7.5, reaction temperature 23-27° C., and conversion time 30 min-60 min.

8. The method according to claim 5, characterized in that The reaction conditions in step S5 are: pH 9.0-9.5, reaction temperature 23-27° C., and reaction time 4-5 h.

9. A mutant genetically engineered strain for synthesizing vanillin, characterized in that: The mutant genetically engineered strain heterologously overexpresses the mutant according to any one of claims 1-2.

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